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Hypericin in the Light and in the Dark: Two Sides of the Same Coin

Hypericin in the Light and in the Dark: Two Sides of the Same Coin REVIEW published: 06 May 2016 doi: 10.3389/fpls.2016.00560 Hypericin in the Light and in the Dark: Two Sides of the Same Coin Zuzana Jendželovská, Rastislav Jendželovský, Barbora Kuchárová and Peter Fedorocko ˇ * Department of Cellular Biology, Faculty of Science, Pavol Jozef Šafárik University in Košice, Košice, Slovakia ′ ′ ′ ′ Hypericin (4,5,7,4 ,5 ,7 -hexahydroxy-2,2 -dimethylnaphtodianthrone) is a naturally occurring chromophore found in some species of the genus Hypericum, especially Hypericum perforatum L. (St. John’s wort), and in some basidiomycetes (Dermocybe spp.) or endophytic fungi (Thielavia subthermophila). In recent decades, hypericin has been intensively studied for its broad pharmacological spectrum. Among its antidepressant and light-dependent antiviral actions, hypericin is a powerful natural photosensitizer that is applicable in the photodynamic therapy (PDT) of various Edited by: oncological diseases. As the accumulation of hypericin is significantly higher in Gregory Franklin, neoplastic tissue than in normal tissue, it can be used in photodynamic diagnosis Polish Academy of Sciences, Poland (PDD) as an effective fluorescence marker for tumor detection and visualization. In Reviewed by: Jirina ˇ Hofmanová, addition, light-activated hypericin acts as a strong pro-oxidant agent with antineoplastic Academy of Sciences of the Czech and antiangiogenic properties, since it effectively induces the apoptosis, necrosis or Republic, Czech Republic autophagy of cancer cells. Moreover, a strong affinity of hypericin for necrotic tissue Lester M. Davids, University of Cape Town, South Africa was discovered. Thus, hypericin and its radiolabeled derivatives have been recently Abhishek D. Garg, investigated as potential biomarkers for the non-invasive targeting of tissue necrosis in KU Leuven, Belgium numerous disorders, including solid tumors. On the other hand, several light-independent *Correspondence: Peter Fedorocko ˇ actions of hypericin have also been described, even though its effects in the dark have not peter.fedorocko@upjs.sk been studied as intensively as those of photoactivated hypericin. Various experimental studies have revealed no cytotoxicity of hypericin in the dark; however, it can serve Specialty section: This article was submitted to as a potential antimetastatic and antiangiogenic agent. On the contrary, hypericin can Plant Metabolism and Chemodiversity, induce the expression of some ABC transporters, which are often associated with the a section of the journal multidrug resistance (MDR) of cancer cells. Moreover, the hypericin-mediated attenuation Frontiers in Plant Science of the cytotoxicity of some chemotherapeutics was revealed. Therefore, hypericin Received: 12 February 2016 Accepted: 11 April 2016 might represent another St. John’s wort metabolite that is potentially responsible for Published: 06 May 2016 negative herb–drug interactions. The main aim of this review is to summarize the Citation: benefits of photoactivated and non-activated hypericin, mainly in preclinical and clinical Jendželovská Z, Jendželovský R, applications, and to uncover the “dark side” of this secondary metabolite, focusing on Kuchárová B and Fedorocko ˇ P (2016) Hypericin in the Light and in the Dark: MDR mechanisms. Two Sides of the Same Coin. Front. Plant Sci. 7:560. Keywords: hypericin, St. John’s wort, anticancer activities, photodynamic therapy, photodynamic diagnosis, drug doi: 10.3389/fpls.2016.00560 resistance Frontiers in Plant Science | www.frontiersin.org 1 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin INTRODUCTION to be an effective antiviral agent (Hudson et al., 1993; Prince et al., 2000). However, some clinical studies have revealed that ′ ′ ′ ′ Hypericin (4,5,7,4 ,5 ,7 -hexahydroxy-2,2 -dimethylnaphtodiant high doses of hypericin can induce phototoxic skin reactions hrone) is a naturally occurring compound synthesized by some without showing any detectable antiviral or antiretroviral activity species of the genus Hypericum. Hypericin was first isolated from in patients with viral infections (Gulick et al., 1999; Jacobson Hypericum perforatum L. (Brockmann et al., 1939), commonly et al., 2001). The controversy concerning the virucidal effect of known as St. John’s wort, which is one of the best characterized hypericin was summarized in detail by Kubin et al. (2005). and most important representatives of this genus, because of However, the potential use of this secondary metabolite in its broad pharmacological activity (antidepressant, antimicrobial, medicine might be broader than currently thought. Although anticancer, anti-inflammatory, wound healing, etc.) (reviewed hypericin has been extensively studied mainly because of its in Kasper et al., 2010; Wölfle et al., 2014). Hypericin and its photodynamic and photocytotoxic properties, it also possesses derivatives are accumulated in special morphological structures, various positive or negative biological activities without being so called dark nodules, occurring in the aerial parts of activated by light. hypericin-producing Hypericum species. The newest data on interspecific variation in localization of hypericins and spatial LIGHT-ACTIVATED HYPERICIN chemo-profiling of hypericin in some Hypericum species were published recently (Kusari et al., 2015; Kucharikova et al., Hypericin possesses several properties that make it a powerful 2016). fluorescent photosensitizer that is suitable for PDT and PDD— In addition to St. John’s wort, this secondary metabolite attractive applications for the treatment and detection of tumors. was found in several other Hypericum species (Kitanov, 2001; It possesses minimal or no toxicity in the dark (Thomas Ayan et al., 2004) and in some basidiomycetes (Dermocybe spp.) and Pardini, 1992; Vandenbogaerde et al., 1997; Miadokova (Dewick, 2002; Garnica et al., 2003) or endophytic fungi growing et al., 2010; Jendželovská et al., 2014; Feruszová et al., 2016), in Hypericum perforatum (Thielavia subthermophila) (Kusari accumulates preferentially in neoplastic tissues (Kamuhabwa et al., 2008, 2009). As hypericin is a bioactive compound that et al., 2002; Noell et al., 2011) and generates reactive oxygen is applicable in several medicinal approaches, its content has species (ROS) in the presence of light (at wavelengths around been evaluated in in vitro grown Hypericum perforatum and in 600 nm) and oxygen (Diwu and Lown, 1993). Thus, hypericin its transgenic clones (Cellárová et al., 1997; Košuth et al., 2003; represents a potent natural alternative to chemically synthesized Koperdáková et al., 2009), or in Hypericum cultures exposed photosensitizers. to various biotechnological applications that focused on their preservation or stimulation of secondary metabolite production Hypericin in Photodynamic Therapy (Urbanová et al., 2006; Brunˇáková et al., 2015; reviewed in: PDT represents a non-invasive therapeutic approach that is Cellárová, 2011). beneficial in the treatment of various cancerous (reviewed in Hypericin is well-known as a potent natural photosensitizing Agostinis et al., 2011) and even non-cancerous lesions and agent with great potential in anticancer photodynamic therapy disorders (reviewed in Kim et al., 2015). In general, it is (PDT) and photodynamic diagnosis (PDD). Besides its based on the combined action of a photosensitizer, light and antineoplastic action, light-dependent in vitro fungicidal molecular oxygen. PDT involves the administration of a non- (Rezusta et al., 2012; Paz-Cristobal et al., 2014) and bactericidal toxic photosensitizer that preferentially accumulates in the effects (Kashef et al., 2013; García et al., 2015) have also been target tissue, followed by its local illumination with harmless reported. In addition, light-activated hypericin is considered visible light of an appropriate wavelength, to activate and excite the photosensitizer. These photoreactions lead to the oxygen- dependent generation of cytotoxic ROS, resulting in cell death 1 64 64 Abbreviations: O , singlet oxygen; Cu-bis-DOTA-hypericin, Cu-Labeled bis- ′ ′ and tissue destruction. However, PDT is a multifactorial process 1,4,7,10-tetraazacyclododecane-N,N ,N,N -tetraacetic acid conjugated hypericin; ABC, ATP-binding cassette; AK, actinic keratosis; BCC, basal cell carcinoma; and the degree of cellular photodamage depends on many factors, BCRP, breast cancer resistance protein; BD, Bowen’s disease; CA4P, combretastatin including cell permeability, the subcellular localization of the A4 phosphate; Cdk4, cyclin-dependent kinase 4; CIS, carcinoma in situ; photosensitizer, the quantity of molecular oxygen, the light dose, CLE, confocal laser endomicroscopy; CYP3A4, cytochrome P450 3A4; DAMPs, the types of generated ROS and the attributes of cancer cells. damage-associated molecular patterns; DLI, drug–light interval; EGFR, epidermal The exact mechanisms of cellular hypericin uptake are still growth factor receptor; FE, fluorescence endoscopy; Hsp90, heat shock protein 90; HY-PDD, hypericin-mediated photodynamic diagnosis; HY-PDT, hypericin- unclear and require further investigation, but the results indicate mediated photodynamic therapy; ICD, immunogenic cell death; INF-α, interferon- that hypericin might be transported into or through cells via α; LIF, laser-induced fluorescence; MDR, multidrug resistance; MF, mycosis temperature-dependent diffusion (Thomas and Pardini, 1992; fungoides; MRP1, multidrug resistance-associated protein 1; NACA, necrosis-avid ›− Sattler et al., 1997), partitioning, pinocytosis or endocytosis contrast agent; O , superoxide anion; p53, phosphoprotein p53, tumor suppressor (Siboni et al., 2002). Concerning its subcellular redistribution, the p53, tumor protein p53; PDD, photodynamic diagnosis; PDT, photodynamic therapy; P-gp, P-glycoprotein; Plk, Polo-like kinase; PVP, polyvinylpyrrolidone; co-labeling of cancer cells with hypericin and fluorescent dyes Raf-1, serine/threonine kinase, Raf-1 proto-oncogene; ROS, reactive oxygen specific for cell organelles revealed that hypericin accumulates species; SCC, squamous cell carcinoma; Ser, serine; SN-38, 7-Ethyl-10-hydroxy- in the membranes of the endoplasmic reticulum, the Golgi camptothecin; TCC, transitional cell carcinoma; Thr, threonine; TNF-α, tumor apparatus, lysosomes and mitochondria (Agostinis et al., 2002; necrosis growth factor-α; TNT, tumor necrosis therapy; VEGF, vascular endothelial growth factor; WLE, white-light endoscopy. Ali and Olivo, 2002; Galanou et al., 2008; Mikeš et al., 2011). Frontiers in Plant Science | www.frontiersin.org 2 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin However, the cellular uptake and subcellular localization of patients) and found that HY-PDT was effective in the treatment hypericin might be affected by its lipophilicity, incubation of both skin disorders. A reduction in tumor size and the concentrations and/or interaction with serum lipoproteins generation of a new epithelium at the surface of lesions following (Crnolatac et al., 2005; Galanou et al., 2008; Kascakova et al., HY-PDT were observed. Moreover, as no necrosis or cell loss 2008). In brief, upon light-activation, hypericin is efficient was evident in the surrounding healthy tissues and no side primarily in the generation of singlet oxygen ( O ; type II effects were observed, with the exception of mild erythema in ›− mechanism) and superoxide anion (O ; type I mechanism) five cases (two patients with SCC, three patients with BCC), (Thomas et al., 1992; Diwu and Lown, 1993), which can HY-PDT-mediated tumor targeting was selective. The treatment ultimately lead to necrosis (Du et al., 2003b; Mikeš et al., resulted in a complete clinical response in one SCC patient 2007, 2009), apoptosis (Ali and Olivo, 2002; Mikeš et al., 2009), and two BCC patients, but in the remaining patients, only a autophagy-associated cell death (Buytaert et al., 2006; Rubio partial clinical response was observed. Thus, the efficacy of HY- et al., 2012) or even to immunogenic cell death (ICD) (Garg PDT appeared to be dependent on the initial lesion size, the et al., 2012a). As type II ICD inducer (Garg et al., 2015a), total dose of hypericin, or the frequency and duration of the HY-PDT represents a promising form of active immunotherapy therapy (Alecu et al., 1998). Several years later, the potential (Galluzzi et al., 2014) owing to spatiotemporally defined emission use of HY-PDT in the treatment of non-melanoma skin cancers of damage-associated molecular patterns (DAMPs) (Garg et al., was explored (Kacerovská et al., 2008). A complete clinical 2012b, 2015b, 2016; Zheng et al., 2016). response was observed in 50% of patients with actinic keratosis The photocytotoxicity of hypericin is strongly oxygen- (AK) 3 months after HY-PDT, and in 22% of patients with dependent, as no such effects are present in hypoxic conditions superficial BCC and 40% of patients with Bowen’s disease (BD) (Thomas and Pardini, 1992; Delaey et al., 2000). Nevertheless, 6 months after HY-PDT. However, in the case of AK, the the final response of hypericin-mediated PDT (HY-PDT) might percentage reduced to 29% 6 months after HY-PDT and only also be affected by the ability of cells to overcome oxidative partial remission was observed in patients with nodular BCC. stress through the activity of various cytoprotective mechanisms, On the other hand, complete histological remission was evident including cellular redox systems (Mikeš et al., 2011; Mikešová in 80% of patients with BD (Kacerovská et al., 2008). Only the et al., 2013). Furthermore, the light-dependent inhibitory effect partial response rate and suboptimal success of HY-PDT could of hypericin against various enzymes engaged in the regulation be caused by the limited penetration of the skin by hypericin of cell survival and proliferation (Ser/Thr kinases, tyrosine and by its low concentration in the final extraction product. In kinases, etc.) has been reported (reviewed in Kubin et al., 2005). the third clinical trial, Rook et al. (2010) tested HY-PDT as a These activities might also contribute to the cytotoxic and potentially well-tolerated and effective therapeutic modality for antiproliferative effects of HY-PDT. The exact mechanisms of the treatment of lymphocyte-mediated skin disorders: malignant action and the cellular aspects of HY-PDT have been outlined mycosis fungoides (MF; the most common type of cutaneous and summarized in several reviews (Agostinis et al., 2002; T-cell lymphoma) and non-cancerous autoimmune psoriasis. Theodossiou et al., 2009; Mikeš et al., 2013; Garg and Agostinis, The results were promising for both diseases. In the case of 2014). MF, HY-PDT led to an improvement in the treated lesions (a size reduction by at least 50%) in the majority of patients, Preclinical and Clinical Assessment of HY-PDT whereas the placebo was ineffective. Moreover, hypericin was Efficacy and Suitable Conditions well tolerated by the patients, with only mild to moderate Many in vitro studies have demonstrated the cytotoxicity of phototoxic skin reactions occurring after exposure to visible light. photoactivated hypericin in various cancer cell types (Xie et al., No serious adverse effects or events were observed (Rook et al., 2001; Head et al., 2006; Sacková et al., 2006; Mikeš et al., 2010). However, the authors themselves recommended a phase 2007; Koval et al., 2010; Mikešová et al., 2013; Kleemann et al., III study with a greater number of patients. All these clinical 2014). Moreover, recent in vivo, preclinical and clinical studies data indicate that topically applied hypericin, combined with have indicated that HY-PDT might be an effective and relevant its photoactivation, might be a promising and safe alternative approach in the treatment of some skin tumors, carcinomas for the treatment of some cancerous and non-cancerous skin and sarcomas. In general, the depth of tumor destruction after disorders. However, as the effectiveness of HY-PDT depends PDT commonly ranges from a few mm to 1 cm, due to limited on the hypericin concentration, its total dose, its rate of tissue photosensitizer and light penetration through the tissues. Thus, penetration, the frequency and duration of the therapy, or on the PDT is effective mostly against superficial lesions and small grade of malignancy, more clinical trials are necessary to define tumors. the optimal conditions for the whole procedure. Clinical studies to test HY-PDT efficacy Preclinical in vivo studies to test HY-PDT effects and To our knowledge, three clinical trials of HY-PDT applied to conditions various skin tumors have been published to date (Table 1). Many further studies to test HY-PDT efficacy have been In the first study, Alecu et al. (1998) tested the intralesional performed using mouse or rat animal models (Table 2). injection of hypericin with subsequent photoactivation with Several in vivo studies indicate that HY-PDT might be a visible light in the treatment of basal cell carcinoma (BCC) promising approach in the treatment of bladder carcinomas. (eleven patients) and squamous cell carcinoma (SCC) (eight Kamuhabwa et al. (2002) reported selective hypericin uptake Frontiers in Plant Science | www.frontiersin.org 3 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin TABLE 1 | Clinical studies to test HY-PDT efficacy. Disease/No. of Hypericin Hypericin dosage Light HY-PDT efficacy References patients administration dose/Fluence rate Squamous cell Intralesional 40–100 μg 3–5 times per week 86 J/cm /24 Reduction in tumor size, re-epithelization Alecu et al., 1998 carcinoma/8 injection for 2–4 weeks; mW/cm at the borders of the lesion, complete clinical remission in the case of one patient; Basal cell 40–200 μg 3–5 times per week Reduction in tumor size, complete clinical carcinoma/11 for 2–6 weeks remission in the case of two patients, no evident signs of tumor recurrence after 5 months Actinic keratosis/8 On the lesion Weekly for 6 weeks on average 75 J/cm 50% complete clinical response (AK) 28% Kacerovská et al., Basal cell complete clinical response (superficial 2008 carcinoma/21 BCC) 11% complete histological response (superficial BCC) 67% partial clinical response (nodular BCC) Bowen’s disease/5 40% complete clinical response (BD) 80% complete histological response (BD) 2 2 Mycosis fungoides On the lesion 0.005–0.025 mg/cm 8–20 J/cm 58.3% of responsive patients (reduction in Rook et al., 2010 (T-cell lymphoma)/12 twice-weekly for 6 weeks MF lesion size by 50% or more) Psoriasis/11 54.6% of responsive patients AK, actinic keratosis; BCC, basal cell carcinoma; BD, Bowen’s disease; MF, mycosis fungoides. in bladder tumors and subsequently, even HY-PDT-mediated shoulder tumors (91.2% ± 2.3%) and even pancreatic tumor tumor damage was observed without the destruction of normal nodules (42.2% ± 8.1%) was observed 4 weeks after HY-PDT, tissue (Kamuhabwa et al., 2003). In both studies, female Fisher indicating that intratumor hypericin and laser therapy might also rats with an orthotopic superficial transitional cell carcinoma be beneficial in the treatment of unresectable pancreatic cancer (TCC) were used as an experimental model and hypericin (Liu et al., 2000). was administered directly into the bladder via the catheter. However, instead of more clinically relevant orthotopic tumor The instilled hypericin accumulated selectively in the bladder models, more in vivo studies have been performed to test the urothelial tumors and the normal urothelium (in a ratio of 12:1), efficacy, conditions or responses of HY-PDT after the treatment, but no hypericin was detected in normal bladder submucosa and only in the murine or rat xenograft or allograft models of muscle layers, which is an important factor to avoid underlying subcutaneous carcinomas or sarcomas. Various positive effects of tissue damage. In addition, no hypericin was detected in plasma; HY-PDT involving the inhibition of tumor growth, a prolonged thus, systemic side-effects should not appear (Kamuhabwa et al., survival time of the treated animals, tumor necrosis, apoptosis 2002). or damage to the tumor vasculature were observed in mice Furthermore, photoactivated hypericin resulted in selective bearing human epidermoid carcinoma (Vandenbogaerde et al., urothelial tumor damage, with tumor cells shrinking and 1996), human prostate adenocarcinoma cells (Xie et al., 2001), detaching from the bladder wall, indicating that HY-PDT might human nasopharyngeal carcinoma cells (Du et al., 2003a,b; be beneficial in the treatment of superficial carcinomas and Thong et al., 2006), human squamous carcinoma cells (Head premalignant changes in the bladder. The HY-PDT that was et al., 2006), human bladder carcinoma cells (Bhuvaneswari performed under suitable light conditions had no significant et al., 2008), human rhabdomyosarcoma cells (Urla et al., 2015), effects on the other bladder layers; nevertheless, 2–5% of murine lymphoma cells (Chen and de Witte, 2000), murine tumor cells survived and were responsible for tumor regrowth colon adenocarcinoma cells (Blank et al., 2002; Sanovic et al., (Kamuhabwa et al., 2003). However, following the results of 2011), murine fibrosarcoma cells (Cavarga et al., 2001, 2005; in vitro study based on TCC-derived spheroids, the same Chen et al., 2001, 2002a,b; Bobrov et al., 2007) or murine Ehrlich authors suggested that hyperoxygenation could overcome this ascites carcinoma cells (Lukšiene˙ and De Witte, 2002) and in problem and might enhance the efficacy of HY-PDT (Huygens rats bearing rat bladder transitional bladder carcinoma (Zupkó et al., 2005). In addition to the orthotopic tumor model, et al., 2001) or rat pituitary adenoma cells (Cole et al., 2008) Liu et al. (2000) also used a xenograft model in their (Table 2). In addition, Blank et al. (2002) demonstrated the experiments. Human MiaPaCa-2 pancreatic adenocarcinoma dependence of HY-PDT efficacy on the irradiation conditions cells were injected subcutaneously and orthotopically into the (light dose and wavelength). Tumor necrosis was much more pancreatic bed of nude, athymic mice. To allow hypericin pronounced at 590 nm than at 550 nm and even increased photoactivation in orthotopic pancreatic tumor nodules, mice when the light dose was raised from 60 to 120 J/cm ; however, underwent a laparotomy that was necessary for the positioning of the maximum depth of tumor necrosis was 9.9 ± 0.8 mm the optical fiber. A significant decrease in growth of subcutaneous at 590 nm (Blank et al., 2002). Considering the relationship Frontiers in Plant Science | www.frontiersin.org 4 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin TABLE 2 | Preclinical in vivo studies to test HY-PDT effects and conditions. Experimental model/Type of Hypericin Hypericin dose Light dose/Fluence HY-PDT effects References tumor (cell line) administration rate Athymic nude mice/Epidermoid Intraperitoneal 2.5 mg/kg, 180 J/cm Tumor growth Vandenbogaerde et al., carcinoma (A431) injection 5 mg/kg inhibition, reduced 1996 tumor mass Athymic nude mice/Pancreatic Intratumoral Injection 10 μg/mouse 2 doses of 200 J Suppressed growth of Liu et al., 2000 carcinoma (MiaPaCa-2) subcutaneous and orthotopic tumors 2 2 DBA/2 mice/Lymphoma (P388) Intraperitoneal 2, 5 or 20 mg/kg 120 J/cm /100 mW/cm Reduced tumor mass Chen and de Witte, injection and tumor size, 2000 prolonged survival time Nude mice/Prostate carcinoma Oral 5 mg/kg 30 mW Tumor growth inhibition Xie et al., 2001 (LNCaP) 2 2 C3H/Km mice/Fibrosarcoma Intravenous injection 5 mg/kg 120 J/cm /100 mW/cm Tumor vasculature Chen et al., 2001, (RIF-1) damage after 0.5 h DLI 2002a,b PDT resulting in complete tumor cure, apoptosis as a main form of cell death Fischer CDF (F344)/CrlBR Intravenous injection 1 or 5 mg/kg 120 Reduced tumor size, Zupkó et al., 2001 2 2 rats/Bladder carcinoma (AY-27) J/cm /100 mW/cm no measurable tumor mass 9–10 days after 0.5 h DLI PDT C3H/DiSn mice/Fibrosarcoma Intratumoral or 5 mg/kg 180 J/cm /150 Reduced tumor Cavarga et al., 2001 (G5:1:13) intraperitoneal mW/cm volume, prolonged injection survival time, complete remission in smaller lesions (3 mm or less in size) 2 2 Intraperitoneal 1 × 5 mg/kg, 2 × 168 J/cm /70 mW/cm Higher efficiency of Cavarga et al., 2005; injection 2.5 mg/kg fractionated dose Bobrov et al., 2007 Vascular damage, formation of necrotic areas Balb/c mice/Colon carcinoma Intraperitoneal 5 mg/kg 60, 90 or Vascular damage, Blank et al., 2002 2 2 (C26) injection 120 J/cm /100 mW/cm tumor necrosis (the depth of tumor necrosis increased with increased light dose) Balb/c mice/Ehrlich ascites Intraperitoneal 40 mg/kg 50 mW/cm Prolonged survival time Lukšiene˙ and De Witte, carcinoma injection (75% of mice), no 2002 tumor recurrence (25% of survived mice) Fischer rats/Bladder carcinoma Instillation into the 30 μM 6–48 J/cm /25–50 Selective urothelial Kamuhabwa et al., 2003 (AY-27) bladder mW/cm tumor damage without destructive effects on detrusor musculature Balb/c nude Intravenous injection 2 mg/kg 120 J/cm /226 Inhibited tumor growth, Du et al., 2003a,b mice/Nasopharyngeal carcinoma mW/cm tumor shrinkage, (HK-1) necrosis as a main form of cell death 2 2 2 or 5 mg/kg 30 J/cm /25 mW/cm Increased apoptosis Thong et al., 2006 and lower serum levels of VEGF after 6 h DLI PDT Athymic nude mice/Squamous Intratumoral injection 10 μg per mg 0–60 J/cm Regression of smaller Head et al., 2006 carcinoma (SNU1) tumor tumors (under 400 mm ) (Continued) Frontiers in Plant Science | www.frontiersin.org 5 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin TABLE 2 | Continued Experimental model/Type of Hypericin Hypericin dose Light dose/Fluence HY-PDT effects References tumor (cell line) administration rate 2 2 Balb/c nude mice/Bladder Intravenous injection 5 mg/kg 120 J/cm /100 mW/cm Vascular damage after Bhuvaneswari et al., carcinoma (MGH) 0.5 h DLI PDT resulting 2008 in reduced tumor volume, increased expression of some angiogenic proteins after 6 h DLI PDT Wistar-Furth rats/Pituitary Intraperitoneal 4 × 1 mg/kg 105–130 J/m Inhibited growth of Cole et al., 2008 adenoma (GH4C1) injection smaller tumors (under 1 cm ), formation of apoptotic clusters 2 2 NMRI – HR-HR hairless Topical application 0.1% in gelcream 40 J/cm /20 mW/cm Full lesional necrosis Boiy et al., 2010 mice/UV-induced small skin resulting in total lesional tumors clearance (44%), replacement of atypical AK cells by normal keratinocytes Balb/c mice/Colon carcinoma Intravenous injection 2.5 or 10 mg/kg 14 or 60 J/cm /27 or Vascular damage after Sanovic et al., 2011 (CT26) 50 mW/cm “low power PDT” resulting in complete tumor regression, prevention of new tumor growth after the re-challenge of cured mice with CT26 cells NOD/LtSz-scid IL2Rγnull Intravenous injection 100 μg/mouse —- Induction of apoptosis Urla et al., 2015 mice/Rhabdomyosarcoma in tumor cells (Rh30) Balb/c mice/Colon carcinoma Subcutaneous 150 nM 2.70 J/cm Tumor-rejecting Garg et al., 2012a, (CT26) injection of HY-PDT anticancer vaccination 2015b, 2016 treated cells effect after the re-challenge of cured mice with CT26 cells Fischer 344 rats/Rat bladder Subcutaneous 150 nM 2.70 J/cm Absence of Garg et al., 2015b carcinoma (AY27) injection of HY-PDT tumor-rejecting treated cells anticancer vaccination effect after the re-challenge of cured rats with AY27 cells C57BL/6 mice/Lewis lung Subcutaneous 0.25 μM 1.85 J/cm Tumor-rejecting Zheng et al., 2016 carcinoma (LLC), Dendritic cells injection of HY-PDT anticancer vaccination (DC) co-cultured with PDT-LLCs treated cells effect after the re-challenge of cured mice with LLC-Luc cells AK, actinic keratosis; DLI, drug-light interval; VEGF, vascular endothelial growth factor; –, the parameter was not provided by the authors. between HY-PDT efficacy and tumor volume, similar results smaller tumors (3 mm or less in height), but the main aim of were obtained in other studies. Head et al. (2006) and Cole their study was to compare the impact of intraperitoneal and et al. (2008) observed a regression or reduction in tumor intratumoral hypericin injection on the effectiveness of HY-PDT. size only in tumors smaller than 0.4 or 1 cm , respectively, Both schedules of hypericin administration significantly reduced whereas larger tumors showed only a partial response followed tumor volume and increased the survival rate of animals. by their regrowth (Head et al., 2006), or did not respond to However, considering the complete response, a higher HY- the treatment (Cole et al., 2008). Thus, light penetration into PDT efficacy was observed for hypericin that was administered the tissue appeared to be a limiting factor. However, Cole intraperitoneally (44.4%) compared to intratumorally (33.3%) et al. (2008) also concluded that HY-PDT can be effective (Cavarga et al., 2001). Moreover, it was later demonstrated that in the elimination of small solid tumor residues. Cavarga a better therapeutic response was obtained after fractionated et al. (2001) also determined complete remission only in hypericin administration (two 2.5 mg/kg doses; 6 and 1 h before Frontiers in Plant Science | www.frontiersin.org 6 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin irradiation) than after a single hypericin dose (5 mg/kg; 1 or 6 h HY-PDT using a long DLI and a low fluence rate, which can before irradiation) (Cavarga et al., 2005). reduce the risk of new tumor vasculature formation (Thong et al., Furthermore, Chen et al. (2001) reported the correlation 2006). between hypericin biodistribution, HY-PDT efficacy and various As PDT-mediated tissue damage can lead to various cellular administration–irradiation time intervals (drug–light intervals— and molecular responses, Bhuvaneswari et al. (2008) examined DLIs). It was found that shortly (0.5 h) after the intravenous the potential anti-angiogenic vs. angiogenic properties of short administration of hypericin (5 mg/kg), the photosensitizer was (0.5 h) and long (6 h) DLI HY-PDT. Both HY-PDT scenarios led located preferentially within tumor blood vessels. At 6 h to a reduction in tumor volume, but the effect was much more after injection, maximum intratumoral hypericin content was pronounced for a short DLI. These findings agree with the above- evident and only poor fluorescence was detected in the tumor mentioned results (Chen et al., 2001, 2002a,b) and suggest the vasculature. Despite high tumor hypericin levels, no tumor destruction of the tumor vasculature after short DLI HY-PDT. cure was observed after longer DLI HY-PDT (6 h) treatment. However, in addition to its antitumor activities, cellular-targeted However, the efficacy of PDT was maximal (100% tumor long DLI HY-PDT induced the expression of some angiogenic cure) when irradiation was performed at 0.5 h after hypericin proteins in tumor tissue, including VEGF, tumor necrosis growth administration (short DLI), indicating strong HY-PDT-induced factor-α (TNF-α) and interferon-α (INF-α), which potentially damage to the tumor vasculature (Chen et al., 2001). Similar lead to the formation of new vessels (Bhuvaneswari et al., 2008). results were obtained by Zupkó et al. (2001). In TCC tumors, In subsequent studies, the efficacy of HY-PDT was enhanced the outcome of HY-PDT was highly dependent on DLI (0.5, using monoclonal antibodies against VEGF and the epidermal 6, or 24 h). The strongest effect, which resulted in no tumor growth factor receptor (EGFR) (Bhuvaneswari et al., 2010, 2011). regrowth in some rats, was evident after 0.5 h DLI PDT. At For short DLI HY-PDT, similar results were obtained by Sanovic the same time, the highest hypericin concentration was detected et al. (2011) in mice bearing CT26 colon carcinoma cells. in the plasma, indicating that PDT-mediated tumor vascular Complete tumor regression was observed after “low-power HY- damage was responsible for its antineoplastic action (Zupkó et al., PDT” (a hypericin dose of 2.5 mg/kg, a short DLI of 0.5 h, a 2 2 2001). The antivascular and strong antitumoral effects of short light dose of 14 J/cm and a fluence rate of 27 mW/cm ), as no DLI HY-PDT were also subsequently confirmed in the murine visible or palpable tumors were detected for at least 60 days fibrosarcoma model (Chen et al., 2002a,b). Damage to tumor after the treatment. In contrast, all mice exposed to “high-power vessels was also detected following long DLI HY-PDT treatments, HY-PDT” (a hypericin dose of 10 mg/kg, a short DLI of 1 h, a 2 2 but many viable tumor cells were present, especially at the tumor light dose of 60 J/cm and a fluence rate of 50 mW/cm ) died 2 periphery, indicating that this PDT modality only induced partial days after the treatment as a consequence of internal bleeding. vascular collapse (Chen et al., 2002a). In agreement with these Thus, both HY-PDT modalities with short DLIs appeared results, Bobrov et al. (2007) also observed primary vascular to preferentially target the vessels, but a higher hypericin damage after HY-PDT with either a single dose (5 mg/kg; 1 or concentration and light dose produced a much stronger response. 6 h before irradiation) or fractionated hypericin administration These results suggest that “low-power HY-PDT” is strong enough (two 2.5 mg/kg doses; 6 and 1 h before irradiation), which to completely eliminate tumors by damaging their vasculature. subsequently progressed to tumor tissue necrosis. The preference Moreover, the re-challenge of cured mice with tumorigenic CT26 of HY-PDT-mediated vasculature or cellular damage appears cells did not result in new tumor growth, indicating the HY- to be dependent on the distribution and accumulation of the PDT-mediated induction of the antitumor immune response photosensitizer; thus, greater vascular destruction is expected (Sanovic et al., 2011). HY-PDT-mediated induction of anticancer after short DLI PDT and more direct killing of tumor cells is immunity was also observed in the studies utilizing different expected after long DLI PDT. However, because the damage in vivo experimental models. Immunization of BALB/c mice to tumor vessels can have devastating consequences for whole with “dying or dead” colon carcinoma CT26 cells prevented tumor mass, the targeting of the tumor vasculature via short the tumor growth at the rechallenge site treated with live CT26 DLI PDT might be more effective in the treatment of solid tumor cells. Approximately 70–85% of the mice immunized with tumors than a long DLI PDT. Moreover, apoptosis was the main HY-PDT treated CT26 cells efficiently rejected the formation form of cell death responsible for tumor eradication following of CT26-derived tumors at challenge site (Garg et al., 2012a, short DLI HY-PDT (Chen et al., 2002b). In contrast, Thong 2015b, 2016). Activation of adaptive immune system was also et al. (2006) observed significantly more apoptosis after long detected in immunocompetent C57BL/6 mice immunized with DLI PDT (6 h) compared to short DLI PDT (1 h). However, HY-PDT treated Lewis lung carcinoma (LLC) cells and LLC cells different irradiation conditions and tumor models were used co-cultured with dendritic cells (Zheng et al., 2016). The results in both studies: whereas Chen et al. (2002b) photoactivated of these three independent experimental groups suggest great hypericin with a light dose of 120 J/cm delivered at a fluence potential of HY-PDT in development of anticancer vaccines. rate of 100 mW/cm , a lower fluence rate HY-PDT (light dose Similar results as in the case of partial remission in patients 2 2 of 30 J/cm , fluence rate of 25 mW/cm ) was applied by Thong with nodular BCC (Kacerovská et al., 2008) were obtained in et al. (2006). The results indicate that long DLI PDT might also an in vivo study using hairless mice with UV-induced non- be effective in the induction of programmed cell death, but only melanoma skin tumors (AK, SCC) as an experimental model under low fluence rate conditions. Moreover, lower serum levels (Boiy et al., 2010). Photoactivated hypericin induced a total and of vascular endothelial growth factor (VEGF) were detected after partial response in 44 and 22% of lesions (diameter of 1–2 mm), Frontiers in Plant Science | www.frontiersin.org 7 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin respectively, with evident lesional necrosis and the replacement et al., 2000, 2002). Moreover, the results obtained by confocal of atypical AK cells for normal keratinocytes. However, 33% of microscopy, white-light endoscopy (WLE) and histopathology lesions were non-responsive to HY-PDT. Tumor penetration and revealed that the intensity of hypericin fluorescence increased the selectivity of topically applied hypericin was also limited by with the stage and grade of bladder cancer (normal bladder tissue mouse skin, as the accumulation of hypericin was highest in < inflammation in the bladder < grade 1 TCC < grade 2 TCC the outermost epidermal layer and lower hypericin levels were < CIS < grade 3 TCC) (Olivo et al., 2003b). Therefore, HY-PDD detected in the rest of the epidermis and the dermis (Boiy et al., could be used as a diagnostic aid to the histopathology of bladder 2010). tumors. Some preclinical and clinical results gave relatively Nowadays, bladder lesions are conventionally diagnosed disappointing results and in some cases, the efficacy of HY- under WLE followed by biopsies that are necessary for the PDT was not as high as expected. The above-mentioned studies histological examination of suspicious tissues. However, in contributed to a better understanding of HY-PDT-induced previous studies, some lesions were barely visible or were absent responses and highlighted the importance of optimizing the in white light (D’Hallewin et al., 2000, 2002), thus, WLE appears conditions, such as suitable hypericin administration, light not to be sensitive enough to reveal all CIS lesions and leads dose, fluence rate, or time intervals and implicated HY-PDT to a high risk of missing the tumor. Therefore, another clinical as a promising anticancer therapeutic approach; however, study compared the WLE and HY-PDD methods (Sim et al., more clinically relevant studies and trials are required for the 2005). Hypericin was instilled into the bladder (for 2 h) and implementation of HY-PDT into clinical practice. immediately after WLE, FE (violet light) was used to induce fluorescence emission in the same bladder regions. Despite the comparable specificity of both approaches (91% for HY-PDD, Hypericin in Photodynamic Diagnosis 98% for WLE), HY-PDD was more sensitive (82%) than WLE In addition to their therapeutic abilities, most photosensitizers (62%) (Sim et al., 2005), suggesting that hypericin might be are also potent diagnostic agents. The main principle and very potent in the labeling and early detection of flat superficial relevance of PDD is to enhance the contrast between neoplastic bladder tumors. Similar results were obtained by Kubin et al. and surrounding healthy tissue, which should contribute to the surgical clearance of the whole tumor mass or its small residues. (2008) using polyvinylpyrrolidone (PVP) bound to hypericin as a new water-soluble formula for the improvement of hypericin- Due to the fluorescent properties of hypericin and its specificity for neoplastic tissue, hypericin-mediated PDD (HY-PDD) is mediated bladder cancer detection and diagnosis. Hypericin- PVP was intravesically instilled 1–2 h prior to FE. The overall being tested for various clinical uses, including optical tumor imaging, and the targeting, monitoring or detection of tumor sensitivity of PDD with PVP-hypericin (95%) was significantly higher than WLE (85%). The maximum contrast in sensitivity stages and grades. To date, fluorescence diagnosis using hypericin was evident in the case of CIS (100% for PDD vs. 33% for WLE) has been clinically tested in bladder, head and neck cancers or and dysplasia (85% for PDD vs. 31% for WLE) (Kubin et al., gliomas (Table 3). 2008). HY-PDD in the Detection and Identification of Bladder As the PVP–hypericin complex represents a potent water- Cancer soluble PDD agent without the necessity of binding to serum In most published clinical studies, HY-PDD was applied to proteins, its biodistribution (Vandepitte et al., 2010) and optimal bladder tumors. As already mentioned in the HY-PDT section, dosage and instillation time were evaluated (Straub et al., 2015) Kamuhabwa et al. (2002) (Table 4) revealed selective hypericin in tumor-bearing rats and in patients with bladder cancer, uptake in rat bladder tumors using in situ laser-induced respectively. Vandepitte et al. (2010) (Table 4) demonstrated fluorescence (LIF) and fluorescence microscopy. The results the uniform distribution of instilled PVP–hypericin in all cell suggest that hypericin is very beneficial in visualization and layers of the malignant urothelium, whereas its penetration distinguishing the tumor mass from normal tissue using various into the normal bladder epithelium was very limited. Straub fluorescent techniques. Thus, hypericin could be used not only in et al. (2015) tested various combinations of PVP–hypericin PDT, but also in the PDD of superficial bladder tumors. dosage (75 and 225 μg) and instillation time (15, 30, 60, At about the same time, the first clinical studies were and 120 min) to identify the optimal PDD conditions. Even performed by D’Hallewin et al. (2000, 2002), who examined though the fluorescence of 225 μg PVP-hypericin instilled the fluorescence-based detection of flat bladder carcinomas in for 120 and 60 min was very strong, the shorter instillation situ (CIS) and in papillary non-invasive bladder tumors after time (30 min) for 225 μg PVP–hypericin was evaluated as the intravesical instillation of hypericin (at least 2 h). The optimal. A lower photosensitizer dose (75 μg) and 15 min fluorescence emission was induced using fluorescence endoscopy with a dose of 225 μg were insufficient to detect the lesions (FE) under blue-light illumination. Hypericin accumulated (Straub et al., 2015). The authors established the most suitable selectively in tumor cells and papillary and flat lesions showed dosage and instillation time of PDD with PVP–hypericin, but red fluorescence, whereas no fluorescence was evident in the they suggest a larger phase IIB study should be performed normal bladder tissue. Subsequently, biopsies were taken from to determine the sensitivity and specificity of these optimal fluorescent regions for microscopic analyses. The results of both conditions. clinical studies suggested that HY-PDD has a high sensitivity All the above-mentioned results indicate that HY-PDD and specificity for the detection of bladder cancer (D’Hallewin is highly sensitive in the detection of early bladder cancer Frontiers in Plant Science | www.frontiersin.org 8 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin TABLE 3 | Clinical studies to test HY-PDD efficacy, sensitivity and specificity. No. of patients Hypericin administration Hypericin dose Fluorescence excitation HY-PDD efficacy References BLADDER CANCER 40 Instillation into the bladder 8 μM (40 ml) FE/blue light 93% sensitivity and 98.5% D’Hallewin et al., specificity (for CIS) 2000 87 Instillation into the bladder 8 μM (40 ml) FE/blue light 94% sensitivity and 95% D’Hallewin et al., specificity (for CIS) 2002 30 Instillation into the bladder 8 μM (50 ml) FE/blue light Fluorescence intensity Olivo et al., 2003b increased with the stage and grade of cancer (normal < inflammation < grade 1 TCC < grade 2 TCC < CIS < grade 3 TCC) 41 Instillation into the bladder 8 μM (40 ml) FE/violet light Higher sensitivity (82%) Sim et al., 2005 compared to conventional WLE (62%) 57 Instillation into the bladder 0.25 mg HY + FE/blue light Higher overall sensitivity Kubin et al., 2008 25 mg PVP (50 ml) (95%) compared to conventional WLE (85%), fewer overlooked malignant lesions compared to WLE, sensitivity increased with the grade of cancer 40 Instillation into the bladder 75 or 225 μg FE/blue light very strong fluorescence Straub et al., 2015 PVP-hypericin of 225 μg PVP-hypericin (50 ml) (120 and 60 min), optimal fluorescence of 225 μg PVP-hypericin instilled for 30 min, insufficient fluorescence of 75 μg and 225 μg PVP-hypericin (15 min) 8 Instillation into the bladder 8 μM (40 ml) FM/380–425 nm Ex vivo urine fluorescence Pytel and cytology—fluorescence Schmeller, 2002 detected in all eight tumor cases 29 urine samples Ex vivo staining of sediment extracted Concentration not CFM/488 nm Argon laser Higher fluorescence Olivo et al., 2003a from voided urine given (1 ml) intensity in tumor cells than in cells from normal urine and in high-grade tumors than in low-grade tumors 21 Ex vivo staining of sediment extracted Concentration not CFM/488 nm Argon laser Higher fluorescence Fu et al., 2007 from voided urine given (1 ml) intensity in tumor cells than in cells from normal urine GLIOMA 5 Intravenous injection 0.1 mg/kg NM/blue light Tumor fluorescence Ritz et al., 2012 clearly distinguishable from normal brain tissue, high specificity (100 and 90%) and sensitivity (91 and 94%) HEAD AND NECK CANCER 23 Oral rinsing 8 μM (100 ml) FE/blue light Distinguishing between Thong et al., 2009 various types of oral cancer (red-to-blue ratio), 90% and higher specificity and sensitivity (red-to-blue ratio) (Continued) Frontiers in Plant Science | www.frontiersin.org 9 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin TABLE 3 | Continued No. of patients Hypericin administration Hypericin dose Fluorescence excitation HY-PDD efficacy References 2 Oral rinsing 8 μM (100 ml) CLE/488 nm Argon laser 3-D visualization of human Thong et al., 2012 buccal mucosa at the surface and approximately 15 μm below the surface 27 Ex vivo tissue staining 8 μM pCLE/568 nm laser diode longer time interval for Abbaci et al., 2015 sufficient ex vivo staining (at least 30 min), the anomalies of keratinization not stained CIS, carcinoma in situ; CFM, confocal fluorescence microscopy; CLE, confocal laser endomicroscopy; FE, fluorescence endoscopy; FM, fluorescence microscopy; HY, hypericin; NM, neurosurgical microscopy; pCLE, probe-based CLE; PVP, polyvinylpyrrolidone; TCC, transitional cell carcinoma; WLE, white-light endoscopy. TABLE 4 | Preliminary data for clinical HY-PDD applications and in vivo studies concerning hypericin accumulation. Experimental Hypericin Hypericin dose Fluorescence Hypericin accumulation and References model/Type of tumor administration excitation fluorescence (cell line) Fischer rats/Bladder Instillation into the 8 or 30 μM LIF/410 nm Krypton Intense fluorescence in tumor Kamuhabwa et al., 2002 carcinoma (AY-27) bladder laser FM/525/50 nm tissue and faint fluorescence in normal bladder tissue (ratio 12:1), no fluorescence in submucosa and muscle layers 30 μM FM/510–560 nm Higher accumulation (3.5-fold Vandepitte et al., 2010 PVP-hypericin more) in malignant tissue than in normal urothelium Wistar rats/Glioma (C6) Intravenous 5 mg/kg FM/510–550 nm Higher accumulation in glioma Noell et al., 2011 injection than in normal brain tissue and infiltration zone VMDk mice/Glioma Intravenous 2.5 mg/kg FM/510–550 nm Time-dependent accumulation in Noell et al., 2013 (SMA-560) injection FME/405 nm glioma cells (maximal uptake—6 h after administration), FME—fluorescence detection also in intracerebral and extracranial gliomas and in brain vessels NOD/LtSz-scid IL2Rγnull Intravenous 100 μg/mouse FL/blue light Tumor fluorescence clearly Urla et al., 2015 mice/Rhabdomyosarcoma injection distinguishable from normal (Rh30) healthy tissue CLE, confocal laser endomicroscopy; FL, fluorescence laparoscopy; FM, fluorescence microscopy; FME, fluorescence microendoscopy; LIF, laser-induced fluorescence technique. and could be routinely used as a diagnostic approach. et al., 2007). In the first study conducted by Pytel and Schmeller Moreover, no photobleaching during FE and resection or (2002), voided urine was analyzed in eight patients following side effects were detected (D’Hallewin et al., 2002; Olivo intravesically instilled hypericin (for at least 1 h). Even though et al., 2003b; Sim et al., 2005; Kubin et al., 2008). Kamuhabwa the number of patients was quite low, hypericin fluorescence was et al. (2005) also conclude that either photosensitization detected in all cases of bladder cancer. On the contrary, Olivo or systemic side-effects should not be expected in patients et al. (2003a) and Fu et al. (2007) performed HY-PDD-mediated after intravesical hypericin administration, as the hypericin urine cytology without intravesical instillation of hypericin. In concentration in plasma was below the detection limit both studies, sediments extracted from patient urine samples (<6 nM). were incubated with hypericin in the dark for 15 min and were Another common method used for bladder cancer diagnosis subsequently analyzed using confocal fluorescence microscopy. is ex vivo urine cytology, which microscopically analyzes the The overall fluorescence intensity of the urothelial cells was exfoliated bladder cells from voided urine. This diagnostic significantly higher in urine from early-grade TCC than in technique is non-invasive and less time-consuming than taking normal samples, which enabled the differentiation between biopsy specimens. However, its sensitivity to detect early-stage normal and early bladder cancer specimens (Olivo et al., 2003a). or low-grade cancer is relatively low. Thus, several teams have This finding was later confirmed through a diagnostic algorithm focused on a technique that combines HY-PDD and urine (Fu et al., 2007). Moreover, fluorescence was even higher in cytology (Pytel and Schmeller, 2002; Olivo et al., 2003a; Fu high-grade tumors than in low-grade tumors (Olivo et al., Frontiers in Plant Science | www.frontiersin.org 10 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin 2003a). The results indicate that ex vivo fluorescence cytology and in patients with various types of head and neck cancer using hypericin might be a promising diagnostic method for (Thong et al., 2009, 2012; Abbaci et al., 2015) (Tables 3, 4). the detection and identification of early and low-grade bladder Similarly to bladder cancer, lesions in the oral cavity cancer. are conventionally diagnosed using WLE and histopathology. However, the results obtained by Thong et al. (2009) demonstrate HY-PDD in the Detection of Gliomas the great potential of hypericin for the diagnosis of various It is well-known that malignant gliomas are tumors with a oral cancer types (hyperplasia, cellular pleomorphic adenoma very poor prognosis and their complete resection significantly of the palate, dysplasia, SCC). After oral rinsing with hypericin improves and extends the survival of patients. Thus, in solution (over 30 min), FE was performed and the captured these cases, the enhancement of the contrast between tumor images were analyzed using several parameters. Firstly, the and surrounding healthy tissue would be very beneficial for selective uptake of hypericin in tumor tissue was confirmed. surgeons. Moreover, an increase in the red-to-blue fluorescence intensity In the first in vivo study, Noell et al. (2011) (Table 4) ratio was evaluated from normal tissue (0.3) to hyperplasia investigated the accumulation of hypericin in tumors arising (1.0) to SCC (2.0), which makes this parameter suitable for from intracerebrally implanted C6 glioma cells, in the zones distinguishing between these tissue types with high specificity surrounding the tumors and in healthy brain tissue. Hypericin and sensitivity (over 90%) (Thong et al., 2009). Subsequently, the was injected intravenously and its uptake was maximal 24 h endomicroscopy imaging technique was improved by the same after injection. Considering tissue autofluorescence, the ratios of research team (Thong et al., 2012). Preliminary study already fluorescence intensities were as follows: tumor:infiltration suggested that confocal laser endomicroscopy (CLE) might be zone:normal tissue = 19.8:2.5:1.0. Because hypericin a potent approach for the surface and subsurface imaging of accumulation was significantly higher in the tumor than in oral cavity tissues using various fluorescent dyes in both animal normal tissue, it could be effectively used as a fluorescence and human models (Thong et al., 2007). Recently, a computing marker for glioma detection (Noell et al., 2011). According to system was interfaced to CLE, which enables the 3-D fluorescence these promising preliminary results, the hypericin-mediated visualization of the oral cavity in real-time (Thong et al., 2012). visualization of tumor tissue during its surgical resection This system could be integrated into current techniques for oral was examined in five patients with recurrent glioblastomas cancer diagnosis and might ultimately lead to better clinical (Ritz et al., 2012). Hypericin was injected intravenously 6 h outcomes. prior to the surgical procedure, which was performed using a The fluorescent properties of hypericin and four neurosurgical microscope under switchable white- and blue-light additional fluorescent dyes were also tested for their ability modes. Malignant tumor tissue (red fluorescence) was clearly to characterize normal and cancerous head and neck distinguishable from the healthy brain tissue (blue color) in all tissue; however, the staining procedure was performed patients and the margins of the tumors showed weaker pink ex vivo on fresh samples obtained from head and neck fluorescence. Moreover, specimens were taken for histological surgeries (glossectomy, pharyngolaryngectomy, laryngectomy, evaluation, which was carried out by two neuropathologists and etc.). Hypericin accumulated in the cytoplasm of normal showed 100 and 90% specificity and 91 and 94% sensitivity. and tumor cells, but was the only fluorescent dye that The obtained results suggest that HY-PDD is well-tolerated and did not stain the anomalies of keratinization. Thus, the represents a method that is sufficiently sensitive and specific for authors conclude that hypericin might not be a suitable the intraoperative visualization of malignant gliomas (Ritz et al., photosensitizer for use in such head and neck specimens 2012). (Abbaci et al., 2015). Furthermore, time-dependent hypericin uptake was However, more promising in vivo results were obtained investigated and observed in a subcutaneous glioma mouse for intra-operative HY-PDD of rhabdomyosarcoma. In model using microendoscopy, an approach that is not designed the preclinical study conducted by Urla et al. (2015), for microsurgical tumor resection, but is very useful for mice were injected intraperitoneally with human alveolar applications such as the visualization of different tissue rhabdomyosarcoma cells and 3 weeks later, hypericin was compartments, the identification of vessels or the detection administered intravenously. After 24 h, conventional and of optimal regions for biopsy. To verify the potential to fluorescence laparoscopy were performed and the tumors were detect intracerebral gliomas using microendoscopy, tumor surgically resected using hypericin-mediated red fluorescence cells were also implanted into the brain. After craniotomy, as guidance. Tumor specimens were processed for histological hypericin fluorescence was detected in intracerebral and analyses. Conventional laparoscopy revealed 24 tumors extracranial gliomas and also in the vessels located in the cortical (ranging in size from 1.6 to 13.5 mm) and 28 tumors were surface of the contralateral hemisphere (Noell et al., 2013) detected only by fluorescence laparoscopy (0.5–11 mm). (Table 4). The results indicate that intraoperative HY-PDD is more sensitive than conventional laparoscopy and can clearly HY-PDD in the Detection and Visualization of Other distinguish rhabdomyosarcoma from healthy tissue (Urla Types of Malignancies et al., 2015). Moreover, the authors inform about clinical In addition to the above-mentioned studies, HY-PDD was also trial that will be initiated in children with advanced-stage examined in mice bearing rhabdomyosarcoma (Urla et al., 2015) rhabdomyosarcoma. Frontiers in Plant Science | www.frontiersin.org 11 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin HYPERICIN IN DARK CONDITIONS OR situation after multiple fractionated hypericin dosages or during a long-term investigation might be completely different. THE EFFECTS OF HYPERICIN WITHOUT Firstly, Blank et al. (2001) demonstrated the cytotoxic and LIGHT-ACTIVATION antiproliferative effect of non-activated hypericin both in vitro and in vivo. Hypericin significantly decreased the viability Although hypericin has been extensively studied mainly because HI of highly metastatic murine breast adenocarcinoma (DA3 ) of its photodynamic and photocytotoxic properties, it also and SCC (SQ2) cells. Moreover, the anticancer potential of possesses various positive and negative biological activities HI hypericin was observed even in vivo in DA3 - and SQ2-derived without light-activation. tumors. Even though hypericin slightly accelerated death in mice As some in vitro studies revealed cytotoxic or growth- HI with DA3 -derived tumor development that was very rapid, inhibitory effects of non-activated hypericin (Blank et al., 2001, intraperitoneal hypericin administration (6 declining doses) in 2003; Berlanda et al., 2010; Besic Gyenge et al., 2012) and other animals led to the inhibition of tumor growth, which antiproliferative (Blank et al., 2001) and antimetastatic (Blank was accompanied by prolonged survival time. Moreover, the et al., 2004) activities in vivo and antiangiogenic actions in vitro hypericin-mediated improvement of survival was also evident (Martínez-Poveda et al., 2005) have been described, hypericin in mice with high-grade SCC tumors (Blank et al., 2001). might also show antitumor potential in the absence of light. Subsequently, Blank et al. (2004) examined the antimetastatic Moreover, one clinical study has demonstrated antiglioma potential of non-activated hypericin and evaluated its influence activity of hypericin in dark conditions (Couldwell et al., 2011). on long-term survival (up to 300 days). Again, mice with breast However, the potential use of this natural compound in medicine adenocarcinoma or SCC tumors, which develop metastases might be broader. Regarding its avidity to necrotic tissues, the 123 predominantly in the lungs, were used as experimental models. radiolabeled hypericin derivative ([ I]iodohypericin) can be To evaluate the impact of hypericin only on metastases, primary used for radio-imaging of numerous necrosis-related pathologies tumors were surgically excised at a stage when micrometastases (acute myocardial infarction, liver infarction) (Ni et al., 2006; already existed. Hypericin was administered intraperitoneally in Fonge et al., 2008), including solid tumors (Van de Putte et al., multiple declining dosages (up to 6 doses of hypericin at 5- 2012). day intervals). In both cases of tumor origin, hypericin therapy On the other hand, hypericin can be associated with a together with the resection of primary tumors resulted in a decrease in the chemosensitivity of cancer cells because of significant increase in long-term animal survival compared to its ability to induce the expression of some ATP-binding the untreated control or to those animals that received surgery cassette (ABC) transporters, which are well-known multidrug alone. Moreover, the complete destruction of several, but not resistance (MDR) components (Jendželovský et al., 2009; all lung metastases, was evident 72 h after hypericin treatment. Jendželovská et al., 2014; Kuchárová et al., 2015). In addition, The results indicate that a single hypericin dose was insufficient the hypericin-mediated attenuation of the cytotoxicity of some to prolong the survival of animals, but fractionated hypericin chemotherapeutic agents was demonstrated (Jendželovská et al., doses could prevent animal death when administered shortly 2014). after the resection of the primary tumor (Blank et al., 2004). Similarly, multiple hypericin doses were applied in a previous Non-Activated Hypericin and Its Potential study to obtain positive anticancer therapeutic results (Blank Antitumor Activity et al., 2001). Hypericin-mediated photocytotoxic effects have always been Considering the potential of hypericin to destroy metastases a relevant and attractive issue for researchers in the field of (Blank et al., 2004), the results are consistent with previous in HI oncology; however, the abilities of hypericin in the absence of vitro findings, where a decrease in the viability of DA-3 and light-activation have not been studied as intensively. Although SQ2 cells was also evident 72 h after treatment (Blank et al., 2001). several studies indicate that hypericin might possess some However, no hypericin-mediated induction of apoptosis in the anticancer activities even in dark conditions (Table 5). dark was observed. The inhibition of DNA synthesis indicated It has been found that non-activated hypericin possesses no that the anticancer action of non-activated hypericin in these cytotoxicity toward various cancer cell lines at concentrations cell lines was more cytostatic than cytotoxic (Blank et al., 2001). sufficient for its photocytotoxic action (Thomas and Pardini, Nevertheless, another in vitro experiment conducted by the same 1992; Hadjur et al., 1996; Vandenbogaerde et al., 1997). However, research group established mitotic cell death as the mechanism some in vitro studies have shown that hypericin can act as a of hypericin-mediated cytotoxicity. Hypericin was responsible cytotoxic or antiproliferative agent even in the dark (Blank et al., for the enhanced ubiquitinylation of the Hsp90 chaperone, 2001, 2003; Berlanda et al., 2010; Besic Gyenge et al., 2012). The resulting in the destabilization of its client proteins engaged in presence or absence of these effects often strongly depends on the regulation of cell proliferation, including p53, Cdk4, Plk, the hypericin concentration (higher concentrations are required and Raf-1. Ultimately, cytostasis and a decrease in cell viability than for HY-PDT-mediated toxicity), treatment conditions, the with no apoptosis were observed. Mitotic cell death is generally applied experimental methods, as well as on the type, origin and characterized by cell-cycle arrest in the G2/M phase, increased sensitivity of cancer cells. Moreover, most in vitro studies have cell volume and multinucleation. All these phenotypes were tested the cytotoxicity of non-activated hypericin following its evident in DA3 and SQ2 cells and even in B16.F10 melanoma single dose or during relatively short time intervals; however, the cells after hypericin treatment (Blank et al., 2003). Thus, mitotic Frontiers in Plant Science | www.frontiersin.org 12 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin TABLE 5 | Anticancer effects of hypericin in dark conditions. Experimental model Hypericin doses and administration Effects of non-activated hypericin References IN VITRO STUDIES Murine breast adenocarcinoma 0.065–10 μM (24 h), 0.2–20 μM (72 h), Mild decrease in cell viability detected by MTT Blank et al., 2001 cell line (DA3) 0.6 and 6 μM assay (24 h, SQ2 cells), Significant decrease in Murine anaplastic SCC cell line cell viability detected by Hemacolor assay (SQ2) (72 h), Decrease in DNA synthesis detected by Murine melanoma cell line 3 H-thymidine incorporation (72 h) (B16.F10) 1–40 μM Cytostasis detected by BrdU incorporation Blank et al., 2003 assay (72 h; doses ≤ 10 μM), Reduced cell viability detected by Hemacolor assay (72 h; doses > 10 μM); G2/M cell cycle arrest, formation of enlarged polynucleated cells and no evidence of apoptosis indicating mitotic cell death; enhanced ubiquitinylation of Hsp90 resulting in increased destabilization of its client proteins (p53, Cdk4, Plk, Raf-1) Bovine aorta endothelial cells Range of concentrations, 5, 10 or 20 μM Inhibition of some key steps of angiogenesis Martínez-Poveda (BAE) (decrease in urokinase extracellular level; et al., 2005 inhibition of: endothelial cells proliferation, endothelial tube formation, migration and invasive capability of endothelial cells) Human epidermoid carcinoma Range of concentrations (up to 100 μM) Antiproliferative and/or cytotoxic effect Berlanda et al., 2010 cell line (A431) detected by MTT assay (concentrations higher than 3.13 μM) Human head and neck SCC 0.6–10 μg/ml Antiproliferative effect detected by BrdU cell Besic Gyenge et al., carcinoma cell lines (UMB-SCC proliferation assay (all applied concentrations), 2012 745, UMB-SCC 969) no influence on RNA integrity, initial DNA damage (recovered after 3 h) IN VIVO STUDIES Balb/c mice/Murine breast 200, 100 and 50 μM; intraperitoneal Reduced volume of DA3-derived tumors (66% Blank et al., 2001 carcinoma (DA3) injection at 20 days after beginning of treatment), Murine SCC (SQ2) prolonged survival time (both DA3 and SQ2 models) 5, 2.5, and 1.25 mg/kg, 10 mg/kg; Increase in long-term (300 days) animal survival Blank et al., 2004 intraperitoneal injection (together with surgery), complete destruction of several DA3-derived metastatic foci in lungs (10 mg/kg, 72 h) CLINICAL STUDIES Glioblastoma (35 patients) 0.05–0.50 mg/kg; oral administration Stabilization or slight reduction of tumor volume Couldwell et al., 2011 Anaplastic astrocytoma (7 of 42 patients = 17%), partial clinical (7 patients) response (> 50% tumor reduction; 2 of 42 patients = 2%), mild adverse effects (photosensitivity, erythema, vomiting, diarrhea, etc.) BrdU, 5-bromo-2 -deoxyuridine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SCC, squamous cell carcinoma. cell death might participate in the tumoricidal and antimetastatic of the patients to this treatment. Hypericin was administered functions of hypericin in the absence of light. orally at gradually increasing dosages ranging from 0.05 to Martínez-Poveda et al. (2005) described another mechanism 0.5 mg/kg (once each morning for up to 3 months) and was that might be implicated in the anticancer effects of hypericin well-tolerated (mean maximum tolerated daily dose of 0.40 ± in the dark. The results of several in vitro assays indicated that 0.098 mg/kg), although some mild skin or gastrointestinal side hypericin can inhibit several key steps of angiogenesis, including effects were observed. More importantly, hypericin stabilized or the proliferation, migration and invasion of endothelial cells, slightly reduced tumor volume (in seven out of 42 patients). In extracellular matrix-degrading urokinase or tubular formation addition, a partial response (>50% reduction of tumor volume) on Matrigel (Martínez-Poveda et al., 2005). All these effects might was observed in two patients (Couldwell et al., 2011). These be beneficial in the prevention of tumor neovascularization. results indicate that synthetic orally administered hypericin To our knowledge, one clinical trial has investigated the can be moderately effective as an adjuvant therapy in cases impact of hypericin on recurrent malignant gliomas (anaplastic of malignant glioma; however, further clinical studies are astrocytoma and glioblastoma) and has monitored the tolerance required. Frontiers in Plant Science | www.frontiersin.org 13 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin Hypericin as a Necrosis-Avid Agent in viable tumor tissue (up to 24 h) (Van de Putte et al., 2012), its radiolabeled derivatives could be also used for so-called Oncology tumor necrosis therapy (TNT). This therapy is based on the In addition to its preferential accumulation in tumors compared destruction of adjacent viable tumor cells by the deposition to in normal healthy tissue, hypericin also has a specific strong and accumulation of radiation energy. In other words, attached affinity toward necrotic tissue (Van de Putte et al., 2008a,b,c). radioactive iodine “bombards” the neighboring living tumor cells The mechanisms of this phenomenon have not yet been fully with radiation. Each successive treatment kills more tumor cells, elucidated; although some hypotheses already exist that consider thus, increasing the necrotic region, which allows higher efficacy the binding of hypericin to specific constituents in the necrotic with each treatment. As three injections of [ I]iodohypericin space. Several radiolabeled hypericin derivatives, particularly 123 131 reduced the volume of RIF-1-derived tumors, this radiolabeled [ I]iodohypericin (Fonge et al., 2007) and [ I]iodohypericin derivate might have potential in TNT (Van de Putte et al., 2012). (Li et al., 2011), possess similar necrosis avidity; thus, hypericin In addition, similar results, especially the intense retention of can be used as a potent necrosis-avid contrast agent (NACA) [ I]iodohypericin in necrotic tumor tissue (over 168 h) and for the non-invasive detection and imaging of various necrosis- inhibited tumor growth after a single dose, were obtained by Liu related pathologies and diseases or to assess tissue viability et al. (2015) in mice bearing hepatomas or sarcomas. and therapeutic responses. Moreover, iodohypericins as NACAs Moreover, the approach of a necrosis-based anticancer might also be effective in relatively new approaches that combine treatment has been expanded into a dual-targeting theranostic both tumor diagnosis and therapy, so-called “theranostic” strategy by administering the vascular-disrupting agent prior modalities (Table 6). to the hypericin iododerivate. Firstly, a vascular-disrupting In a few initial in vivo studies, hypericin was investigated agent, such as combretastatin A4 phosphate (CA4P), targets as a potential indicator of therapeutic responses, following the tumor microenvironment and subsequently, iodine various necrosis-inducing anticancer treatments. Mice bearing radioactivity kills residual cancer cells. Several preclinical intrahepatic fibrosarcoma tumors were used as an experimental studies have demonstrated that dual-targeting using CA4P model and hypericin was injected intravenously 1 h before, with [ I]iodohypericin was much more effective than single or 24 h after intratumoral ethanol injection (Van de Putte treatments. A reduced tumor volume, a prolonged tumor et al., 2008b) or radiofrequency ablation (Van de Putte et al., doubling time, an increase in radiation-induced cell death and 2008c), which both induced tumor necrosis. Fluoromacroscopic intratumoral necrosis or prolonged survival time were reported and fluoromicroscopic examinations confirmed that hypericin in rats bearing liver rhabdomyosarcomas (Li et al., 2011, 2012), accumulated preferentially in necrotic tissue. In the cases of in mice bearing fibrosarcomas (Li et al., 2013) and in rabbits with necrosis, mean fluorescence densities were about 4.5- and 5- liver and muscular VX2 tumors (Shao et al., 2015). Thus, the fold higher than in viable tumor tissue and 14- and 12-fold dual-targeting theranostic approach appears to be well tolerated higher than in normal liver tissue. These results demonstrate and can enhance the therapeutic response, encouraging further the ability of hypericin to enhance the imaging contrast between development for other preclinical and even clinical applications. necrotic and viable tissues and ultimately, its potential role in the early assessment of the therapeutic response (Van Non-Activated Hypericin and Its Potential de Putte et al., 2008b,c). At about the same time, tumor uptake of radiolabeled hypericin (mono-[ I]iodohypericin) Negative Impact on Cancer Treatment and protohypericin (mono-[ I]iodoprotohypericin) derivatives All the above-mentioned preclinical and clinical results suggest were tested and compared, because of the applicability of that hypericin offers great potential in tumor diagnosis as well tomographic imaging techniques instead of fluorescence-based as in anticancer therapy. However, this secondary metabolite techniques. Radioactivity was measured using a gamma counter. might also cause some other effects that would not be beneficial Both radiolabeled compounds were retained by the tumors, for therapeutic outcomes. It is well-known that the efficacy of but mono-[ I]iodohypericin appeared to be a more suitable commonly used anticancer treatment modalities is often limited tumor diagnostic agent, due to its faster clearance from healthy by intrinsic or acquired MDR—a multifactorial phenomenon of organs (Fonge et al., 2007). Another radiolabeled derivate, the increased tolerance of cancer cells to various tumoricidal Cu-bis-DOTA-hypericin, was also applicable in the early agents. A number of cellular mechanisms can contribute to determination of the therapeutic response as its accumulation MDR (reviewed in Stavrovskaya, 2000; Zahreddine and Borden, was significantly lower in non-treated tumors than in those 2013), including the increased elimination of anticancer drugs treated by photothermal ablation therapy, inducing necrosis by tumor cells, which is mostly linked to the elevated expression (Song et al., 2011). and/or activity of several ABC transporters. It has been shown As necrotic tissue represents 30–80% of the solid tumor that non-activated hypericin can modulate some of these mass and is rarely present in normal healthy tissue and organs, efflux pumps. In vitro experiments conducted by our research it is a suitable target not only for cancer diagnosis, but also group (Jendželovský et al., 2009; Jendželovská et al., 2014; for anticancer therapy. The strong avidity of iodohypericins Kuchárová et al., 2015) have revealed an increased expression for necrotic tissue makes these compounds very potent in the of multidrug resistance-associated protein 1 (MRP1) and breast imaging of tumor necrosis. Furthermore, as hypericin can persist cancer resistance protein (BCRP) in colorectal HT-29 cells or in in necrotic tumor areas much longer (up to 72 h) than in ovarian A2780 and A2780cis cells following hypericin treatment Frontiers in Plant Science | www.frontiersin.org 14 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin TABLE 6 | Preclinical in vivo studies to test hypericin as a necrosis-avid agent. Experimental model/Type Hypericin derivate Detection method Effects References of tumor (cell line) C3H mice/Fibrosarcoma [ I]MIH Gamma counter Retention by the tumors and rapid Fonge et al., (RIF-1) clearance from healthy organs (faster 2007 clearance than [ I]MIprotoH) C3H/Km mice/Fibrosarcoma hypericin FM imaging Accumulation in intratumoral necrosis (4 h Van de Putte (RIF-1) after administration) et al., 2008a UV and Tungsten light, FM Preferential accumulation in intratumoral Van de Putte imaging necrosis (intratumoral necrosis > viable et al., 2008b,c tumor > normal liver tissue), enhanced contrast between necrotic and viable tissue = early assessment of therapeutic response (diagnosis) Nude mice/Mammary cancer CuBDH PET, autoradiography Higher accumulation in treated than in Song et al., 2011 (BT474) non-treated tumors = assessment of therapeutic response (diagnosis) Balb/c mice/Fibrosarcoma hypericin, [ I]MIH, FM, PET, autoradiography, Longer persistence of tracers in necrotic Van de Putte (RIF-1) [ I]MIH scintigraphy than in viable tumor, stabilization of tumor et al., 2012 growth and reduced tumor volume (3 injections of [ I]MIH) = potential in TNT WAG/Rij [ I]MIH MRI, CT scan, scintigraphy, Gamma Reduced tumor volume, prolonged tumor Li et al., 2011 rats/Rhabdomyosarcoma (R1) counter, autoradiography doubling time and inhibited tumor regrowth (dual-targeting with CA4P) 123 131 [ I]MIH, [ I]MIH Accumulation in intratumoral necrosis Li et al., 2012 ([ I]MIH); reduced tumor volume, prolonged tumor doubling time and increased intratumoral necrosis = tumoricidal effect (dual-targeting - [ I]MIH with CA4P) SCID mice/Fibrosarcoma [ I]MIH MRI, scintiscan, autoradiography Accumulation in intratumoral necrosis (over Li et al., 2013 (RIF-1) 120 h); prolonged survival time, marked radiation-induced cell death, reduced tumor volume, prolonged tumor doubling time (dual-targeting with CA4P) New Zealand white [ I]MIH MRI, SPECT, autoradiography High targetability to tumor necrosis; Shao et al., 2015 rabbits/VX2 tumors reduced tumor growth and prolonged tumor doubling time (dual-targeting with CA4P) Kunming (KM) [ I]MIH FM, SPECT, autoradiography Prolonged retention by the tumors, limited Liu et al., 2015 mice/Hepatoma (H22) systemic toxicity, tumor growth delay = Sarcoma (S180) therapeutic efficacy 123 123 123 123 131 131 64 64 [ I]MIH, mono-[ I]iodohypericin; [ I]MIprotoH, mono-[ I]iodoprotohypericin; [ I]MIH, mono-[ I]iodohypericin; CuBDH, Cu-bis-DOTA-hypericin; CA4P, combretastatin A4 phosphate; CT, computed tomography; FM, fluorescence microscopy; MRI, magnetic resonance imaging; PET, positron emission tomography; SPECT, single-photon emission computed tomography; TNT, tumor necrosis therapy. in the dark. In A2780 and A2780cis cells, 0.5 μM hypericin Wada et al. (2002) evaluated the impact of hypericin on the elevated MRP1 protein levels already 6 h after the treatment action of several anticancer drugs, using the cervical HeLa cell (Jendželovská et al., 2014). For HT-29 cells, an even lower line and its resistant subline Hvr100-6 that overexpress another hypericin concentration (0.1 μM) was sufficient to increase MDR-related ABC transporter, P-glycoprotein (P-gp), but no MRP1 and BCRP expression (16 h after hypericin addition) effect was observed. Several studies suggest that hypericin can (Jendželovský et al., 2009; Kuchárová et al., 2015). Therefore, neither modulate P-gp expression nor its activity (Wada et al., because many chemotherapeutic agents and photosensitizers 2002; Tian et al., 2005; Jendželovský et al., 2009), which explains are substrates of the above-mentioned transporters (reviewed its poor ability to influence the cytotoxicity and transport of in Nies et al., 2007; Robey et al., 2007), the hypericin- P-gp substrates, such as paclitaxel, daunorubicin, doxorubicin mediated stimulation of efflux systems might lead to a decrease or vinblastin (Wada et al., 2002). On the other hand, 24 in the efficacy of these therapeutic approaches when they h pre-treatment with hypericin resulted in the attenuation are applied at the same time or shortly following hypericin of mitoxantrone cytotoxicity in HL-60 cells and cisplatin treatment. cytotoxicity in sensitive A2780 and resistant A2780cis cells Frontiers in Plant Science | www.frontiersin.org 15 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin (Jendželovská et al., 2014). However, this effect was probably SUMMARY not caused by modulation of the analyzed ABC transporters. In this review, we have summarized the medicinal applications However, the results suggest that hypericin in the dark might of light-activated and non-activated hypericin in the field of have a negative impact on the onset or progress of cell death oncology. We have preferentially highlighted the “primary induced by some anticancer agents, possibly by affecting some beneficial side” of this secondary metabolite concerning its other mechanisms. Further studies are required, to elucidate anticancer potential, but have also outlined its “second non- the specific mechanisms responsible for the above-mentioned beneficial side,” which was revealed by preliminary in vitro changes and in vivo studies will be necessary to verify the impact studies. of non-activated hypericin on the outcome of chemotherapy. The vast majority of the results summarized here suggest Moreover, some MDR mechanisms, including ABC that hypericin in light and dark conditions might be a very transporters, are also involved in drug pharmacokinetics. potent agent in cancer treatment and diagnosis. Besides the The modulation of these mechanisms can affect the absorption, well-known and intensively investigated HY-PDT and HY-PDD, distribution or clearance and ultimately, the action of the some new and promising approaches using hypericin as NACA administered xenobiotics, resulting in negative drug interactions. are becoming the focus of various research groups. Moreover, Several clinical trials have demonstrated the interactions some tested modalities, including dual-targeting, might even between some chemotherapeutic agents and St. John’s wort improve the clinical outcome of cancer treatments. However, extract, which is often taken by oncological patients as an in the dark, hypericin might be responsible for the limited antidepressant. In the first study, the enhanced metabolism efficacy of conventionally applied chemotherapy or even PDT, of irinotecan and consequently, decreased plasma levels of its due to its ability to induce the expression of some ABC active metabolite (SN-38) were evaluated in cancer patients transporters. Moreover, there is a suspicion that non-activated following St. John’s wort treatment (Mathijssen et al., 2002). hypericin possesses much broader biological activity. Thus, the Furthermore, Frye et al. (2004) and Smith et al. (2004) examined chronic usage of St. John’s wort extracts as an antidepressant by the effect of St. John’s wort extract on the pharmacokinetics oncological patients undergoing anticancer treatment should be of imatinib mesylate in healthy adult volunteers. In both avoided. studies, imatinib was administered before and after the treatment with St. John’s wort (300 mg three times daily AUTHOR CONTRIBUTIONS for 2 weeks) and its clearance and half-life was significantly increased or decreased, respectively, by the herb extract. Similar ZJ designed the concept and issue of the review, studied the results were obtained by Goey et al. (2014) using a similar literature, contributed to all chapters and summarized the bulk of experimental design. Besides the enhanced clearance and the text, revised the text after it was completed and also following decreased plasma concentrations of docetaxel in cancer patients, English revision and approved the final version. RJ discussed the St. John’s wort lowered the incidence of docetaxel-mediated concept of the review with first author, studied the literature and toxicities. Thus, due to the risk of potential undertreatment, contributed to the chapters about HY-PDT and non-activated combining anticancer therapeutic approaches with St. John’s hypericin, revised the text and approved the final version. BK wort extracts should not be recommended to oncological studied the literature and contributed to the chapters about HY- patients. PDT and non-activated hypericin, revised the text and approved The probable reason for these effects is the induction of the the final version. PF discussed the concept of the review with first metabolic enzyme (CYP3A4) and/or the P-gp transporter by author, revised the text and approved the final version. hyperforin (Komoroski et al., 2004; Tian et al., 2005), another St. John’s wort metabolite. However, considering the potential of hypericin to induce the expression of some ABC efflux pumps, ACKNOWLEDGMENTS this secondary metabolite might also contribute to negative drug interactions with St. John’s wort. Therefore, a much broader This work was supported by the Slovak Research and spectrum of antineoplastic drugs might exist, including various Development Agency under contract No. APVV-14-0154 and chemotherapeutic agents or photosensitizers, whose action might the Scientific Grant Agency of the Ministry of Education of the be altered due to the presence of hypericin. Slovak Republic under contract No. VEGA 1/0147/15. REFERENCES Agostinis, P., Vantieghem, A., Merlevede, W., and de Witte, P. A. (2002). Hypericin in cancer treatment: more light on the way. Int. J. Biochem. Cell Biol. 34, Abbaci, M., Casiraghi, O., Temam, S., Ferchiou, M., Bosq, J., Dartigues, P., et al. 221–241. doi: 10.1016/S1357-2725(01)00126-1 (2015). Red and far-red fluorescent dyes for the characterization of head Alecu, M., Ursaciuc, C., Hãlãlãu, F., Coman, G., Merlevede, W., Waelkens, E., and neck cancer at the cellular level. J. Oral Pathol. Med. 44, 831–841. doi: et al. (1998). Photodynamic treatment of basal cell carcinoma and squamous 10.1111/jop.12316 cell carcinoma with hypericin. Anticancer Res. 18, 4651–4654. Agostinis, P., Berg, K., Cengel, K. A., Foster, T. H., Girotti, A. W., Gollnick, S. O., Ali, S. M., and Olivo, M. (2002). Bio-distribution and subcellular localization of et al. (2011). Photodynamic therapy of cancer: an update. CA Cancer J. Clin. 61, Hypericin and its role in PDT induced apoptosis in cancer cells. Int. J. Oncol. 250–281. doi: 10.3322/caac.20114 21, 531–540. doi: 10.3892/ijo.21.3.531 Frontiers in Plant Science | www.frontiersin.org 16 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin Ayan, A. K., Cirak, C., Kevseroglu, K., and Ozen, T. (2004). Hypericin in Cellárová, E., Brutovská, R., Daxnerová, Z., Brunˇáková, K., and Weigel, some Hypericum species from Turkey. Asian J. Plant Sci. 3, 200–202. doi: R. C. (1997). Correlation between hypericin content and the ploidy of 10.3923/ajps.2004.200.202 somaclones of Hypericum perforatum L. Acta Biotechnol. 17, 83–90. doi: Berlanda, J., Kiesslich, T., Engelhardt, V., Krammer, B., and Plaetzer, K. (2010). 10.1002/abio.370170111 Comparative in vitro study on the characteristics of different photosensitizers Chen, B., and de Witte, P. A. (2000). Photodynamic therapy efficacy and tissue employed in PDT. J. Photochem. Photobiol. B. Biol. 100, 173–180. doi: distribution of hypericin in a mouse P388 lymphoma tumor model. Cancer Lett. 10.1016/j.jphotobiol.2010.06.004 150, 111–117. doi: 10.1016/S0304-3835(99)00381-X Besic Gyenge, E., Forny, P., Lüscher, D., Laass, A., Walt, H., and Maake, C. (2012). Chen, B., Roskams, T., and de Witte, P. A. (2002a). Antivascular tumor eradication Effects of hypericin and a chlorin based photosensitizer alone or in combination by hypericin-mediated photodynamic therapy. Photochem. Photobiol. 76, in squamous cell carcinoma cells in the dark. Photodiagnosis Photodyn. Ther. 9, 509–513. doi: 10.1562/0031-8655(2002)0760509ATEBHM2.0.CO2 321–331. doi: 10.1016/j.pdpdt.2012.03.006 Chen, B., Roskams, T., Xu, Y., Agostinis, P., and de Witte, P. A. (2002b). Bhuvaneswari, R., Gan, Y. Y., Lucky, S. S., Chin, W. W., Ali, S. M., Soo, K. Photodynamic therapy with hypericin induces vascular damage and apoptosis C., et al. (2008). Molecular profiling of angiogenesis in hypericin mediated in the RIF-1 mouse tumor model. Int. J. Cancer. 98, 284–290. doi: photodynamic therapy. Mol. Cancer. 7:56. doi: 10.1186/1476-4598-7-56 10.1002/ijc.10175 Bhuvaneswari, R., Thong, P. S., Gan, Y. Y., Soo, K. C., and Olivo, M. (2010). Chen, B., Xu, Y., Roskams, T., Delaey, E., Agostinis, P., Vandenheede, J. R., Evaluation of hypericin-mediated photodynamic therapy in combination et al. (2001). Efficacy of antitumoral photodynamic therapy with hypericin: with angiogenesis inhibitor bevacizumab using in vivo fluorescence confocal relationship between biodistribution and photodynamic effects in the RIF-1 endomicroscopy. J. Biomed. Opt. 15:011114. doi: 10.1117/1.3281671 mouse tumor model. Int. J. Cancer. 93, 275–282. doi: 10.1002/ijc.1324 Bhuvaneswari, R., Yuen, G. Y., Chee, S. K., and Olivo, M. (2011). Antiangiogenesis Cole, C. D., Liu, J. K., Sheng, X., Chin, S. S., Schmidt, M. H., Weiss, M. H., agents avastin and erbitux enhance the efficacy of photodynamic therapy et al. (2008). Hypericin-mediated photodynamic therapy of pituitary tumors: in a murine bladder tumor model. Lasers Surg. Med. 43, 651–662. doi: preclinical study in a GH4C1 rat tumor model. J. Neurooncol. 87, 255–261. doi: 10.1002/lsm.21109 10.1007/s11060-007-9514-0 Blank, M., Kostenich, G., Lavie, G., Kimel, S., Keisari, Y., and Orenstein, A. (2002). Couldwell, W. T., Surnock, A. A., Tobia, A. J., Cabana, B. E., Stillerman, C. Wavelength-dependent properties of photodynamic therapy using hypericin B., Forsyth, P. A., et al. (2011). A phase 1/2 study of orally administered in vitro and in an animal model. Photochem. Photobiol. 76, 335–340. doi: synthetic hypericin for treatment of recurrent malignant gliomas. Cancer 117, 10.1562/0031-8655(2002)0760335WDPOPT2.0.CO2 4905–4915. doi: 10.1002/cncr.26123 Blank, M., Lavie, G., Mandel, M., Hazan, S., Orenstein, A., Meruelo, D., et al. Crnolatac, I., Huygens, A., van Aerschot, A., Busson, R., Rozenski, J., and de (2004). Antimetastatic activity of the photodynamic agent hypericin in the dark. Witte, P. A. (2005). Synthesis, in vitro cellular uptake and photo-induced Int. J. Cancer. 111, 596–603. doi: 10.1002/ijc.20285 antiproliferative effects of lipophilic hypericin acid derivatives. Bioorg. Med. Blank, M., Mandel, M., Hazan, S., Keisari, Y., and Lavie, G. (2001). Anti-cancer Chem. 13, 6347–6353. doi: 10.1016/j.bmc.2005.09.003 activities of hypericin in the dark. Photochem. Photobiol. 74, 120–125. doi: Delaey, E., Vandenbogaerde, A., Merlevede, W., and de Witte, P. (2000). 10.1562/0031-8655(2001)0740120ACAOHI2.0.CO2 Photocytotoxicity of hypericin in normoxic and hypoxic conditions. J. Blank, M., Mandel, M., Keisari, Y., Meruelo, D., and Lavie, G. (2003). Enhanced Photochem. Photobiol. B. Biol. 56, 19–24. doi: 10.1016/S1011-1344(00)00051-8 ubiquitinylation of heat shock protein 90 as a potential mechanism for mitotic Dewick, P. M. (2002). Medicinal Natural Products: A Biosynthetic Approach, 2nd cell death in cancer cells induced with hypericin. Cancer Res. 63, 8241–8247. Edn. Chichester: John Wiley & Sons Ltd. Bobrov, N., Cavarga, I., Longauer, F., Rybárová, S., Fedorocko, P., Brezáni, D’Hallewin, M. A., De Witte, P. A., Waelkens, E., Merlevede, W., and Baert, P., et al. (2007). Histomorphological changes in murine fibrosarcoma after L. (2000). Fluorescence detection of flat bladder carcinoma in situ after hypericin-based photodynamic therapy. Phytomedicine 14, 172–178. doi: intravesical instillation of hypericin. J. Urol. 164, 349–351. doi: 10.1016/S0022- 10.1016/j.phymed.2006.09.017 5347(05)67357-0 Boiy, A., Roelandts, R., and de Witte, P. A. (2010). Photodynamic therapy using D’Hallewin, M. A., Kamuhabwa, A. R., Roskams, T., De Witte, P. A., and Baert, L. topically applied hypericin: comparative effect with methyl-aminolevulinic acid (2002). Hypericin-based fluorescence diagnosis of bladder carcinoma. BJU Int. on UV induced skin tumours. J. Photochem. Photobiol. B. Biol. 102, 123–131. 89, 760–763. doi: 10.1046/j.1464-410X.2002.02690.x doi: 10.1016/j.jphotobiol.2010.09.012 Diwu, Z., and Lown, J. W. (1993). Photosensitization with anticancer agents. 17. Brockmann, H., Haschad, M. N., Maier, K., and Pohl, F. (1939). Über EPR studies of photodynamic action of hypericin: formation of semiquinone das Hypericin, den photodynamisch wirksamen Farbstoff aus Hypericum radical and activated oxygen species on illumination. Free Radic. Biol. Med. 14, perforatum. Naturwissenschaften 27, 550. doi: 10.1007/BF01495453 209–215. doi: 10.1016/0891-5849(93)90012-J Brunˇáková, K., Petijová, L., Zámecˇník, J., Turecˇková, V., and Cellárová, E. (2015). Du, H. Y., Bay, B. H., and Olivo, M. (2003a). Biodistribution and photodynamic The role of ABA in the freezing injury avoidance in two Hypericum species therapy with hypericin in a human NPC murine tumor model. Int. J. Oncol. 22, differing in frost tolerance and potential to synthesize hypericins. Plant Cell 1019–1024. doi: 10.3892/ijo.22.5.1019 Tissue Organ Cult. 122, 45–56. doi: 10.1007/s11240-015-0748-9 Du, H. Y., Olivo, M., Tan, B. K., and Bay, B. H. (2003b). Hypericin- Buytaert, E., Callewaert, G., Hendrickx, N., Scorrano, L., Hartmann, D., Missiaen, mediated photodynamic therapy induces lipid peroxidation and necrosis L., et al. (2006). Role of endoplasmic reticulum depletion and multidomain in nasopharyngeal cancer. Int. J. Oncol. 23, 1401–1405. doi: 10.3892/ijo.23. proapoptotic BAX and BAK proteins in shaping cell death after hypericin- 5.1401 mediated photodynamic therapy. FASEB J. 20, 756–758. doi: 10.1096/fj.05- Feruszová, J., Imreová, P., Bodnárová, K., Ševcˇovicˇová, A., Kyzek, S., Chalupa, I., 4305fje et al. (2016). Photoactivated hypericin is not genotoxic. Gen. Physiol. Biophys. Cavarga, I., Brezáni, P., Cekanová-Figurová, M., Solár, P., and Fedorocko, P., 35, 223–230. doi: 10.4149/gpb_2015045 Miskovský, P. (2001). Photodynamic therapy of murine fibrosarcoma with Fonge, H., Van de Putte, M., Huyghe, D., Bormans, G., Ni, Y., de Witte, P., et al. topical and systemic administration of hypericin. Phytomedicine 8, 325–330. (2007). Evaluation of tumor affinity of mono-[(123)I]iodohypericin and mono- doi: 10.1078/0944-7113-00057 [(123)I]iodoprotohypericin in a mouse model with a RIF-1 tumor. Contrast Cavarga, I., Brezáni, P., Fedorocko, P., Miskovský, P., and Bobrov, N., Media Mol. Imaging. 2, 113–119. doi: 10.1002/cmmi.136 Longauer, F., et al. (2005). Photoinduced antitumour effect of hypericin Fonge, H., Vunckx, K., Wang, H., Feng, Y., Mortelmans, L., Nuyts, J., et al. (2008). can be enhanced by fractionated dosing. Phytomedicine 12, 680–683. doi: Non-invasive detection and quantification of acute myocardial infarction 10.1016/j.phymed.2004.02.011 in rabbits using mono-[123I]iodohypericin microSPECT. Eur. Heart J. 29, Cellárová, E. (2011). “Effect of exogenous morphogenetic signals on differentiation 260–269. doi: 10.1093/eurheartj/ehm588 in vitro and secondary metabolite formation in the genus Hypericum,” in Frye, R. F., Fitzgerald, S. M., Lagattuta, T. F., Hruska, M. W., and Egorin, M. J. Medicinal and Aromatic Plant Science and Biotechnology 5 (Special Issue 1), eds (2004). Effect of St John’s wort on imatinib mesylate pharmacokinetics. Clin. M. S. Odabas and C. Çırak (Ikenobe: Global Science Books), 62–69. Pharmacol. Ther. 76, 323–329. doi: 10.1016/j.clpt.2004.06.007 Frontiers in Plant Science | www.frontiersin.org 17 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin Fu, C. Y., Ng, B. K., Razul, S. G., Chin, W. W., Tan, P. H., Lau, W. K., et al. (2007). Antimicrob. Agents Chemother. 45, 517–524. doi: 10.1128/AAC.45.2.517- Fluorescence detection of bladder cancer using urine cytology. Int. J. Oncol. 31, 524.2001 525–530. doi: 10.3892/ijo.31.3.525 Jendželovská, Z., Jendželovský, R., Hil’ovská, L., Koval’, J., Mikeš, J., and Galanou, M. C., Theodossiou, T. A., Tsiourvas, D., Sideratou, Z., and Fedorocˇko, P. (2014). Single pre-treatment with hypericin, a St. John’s wort Paleos, C. M. (2008). Interactive transport, subcellular relocation and secondary metabolite, attenuates cisplatin- and mitoxantrone-induced cell enhanced phototoxicity of hypericin encapsulated in guanidinylated liposomes death in A2780, A2780cis and HL-60 cells. Toxicol. In vitro 28, 1259–1273. doi: via molecular recognition. Photochem. Photobiol. 84, 1073–1083. doi: 10.1016/j.tiv.2014.06.011 10.1111/j.1751-1097.2008.00392.x Jendželovský, R., Mikes, J., Koval,’, J., Soucek, K., Procházková, J., Kello, M., Galluzzi, L., Vacchelli, E., Bravo-San Pedro, J. M., Buqué, A., Senovilla, L., Baracco, et al. (2009). Drug efflux transporters, MRP1 and BCRP, affect the outcome E. E., et al. (2014). Classification of current anticancer immunotherapies. of hypericin-mediated photodynamic therapy in HT-29 adenocarcinoma Oncotarget 5, 12472–12508. doi: 10.18632/oncotarget.2998 cells. Photochem. Photobiol. Sci. 8, 1716–1723. doi: 10.1039/b9pp0 García, I., Ballesta, S., Gilaberte, Y., Rezusta, A., and Pascual, Á. (2015). 0086k Antimicrobial photodynamic activity of hypericin against methicillin- Kacerovská, D., Pizinger, K., Majer, F., and Smíd, F. (2008). Photodynamic susceptible and resistant Staphylococcus aureus biofilms. Future Microbiol. 10, therapy of nonmelanoma skin cancer with topical Hypericum perforatum 347–356. doi: 10.2217/fmb.14.114 extract–a pilot study. Photochem. Photobiol. 84, 779–785. doi: 10.1111/j.1751- Garg, A. D., and Agostinis, P. (2014). ER stress, autophagy and immunogenic 1097.2007.00260.x cell death in photodynamic therapy-induced anti-cancer immune responses. Kamuhabwa, A. A., Cosserat-Gerardin, I., Didelon, J., Notter, D., Guillemin, Photochem. Photobiol. Sci. 13, 474–487. doi: 10.1039/c3pp50333j F., Roskams, T., et al. (2002). Biodistribution of hypericin in orthotopic Garg, A. D., Elsen, S., Krysko, D. V., Vandenabeele, P., de Witte, P., and Agostinis, transitional cell carcinoma bladder tumors: implication for whole bladder wall P. (2015b). Resistance to anticancer vaccination effect is controlled by a cancer photodynamic therapy. Int. J. Cancer. 97, 253–260. doi: 10.1002/ijc.1594 cell-autonomous phenotype that disrupts immunogenic phagocytic removal. Kamuhabwa, A. A., Di Mavungu, J. D., Baert, L., D’Hallewin, M. A., Hoogmartens, Oncotarget 6, 26841–26860. doi: 10.18632/oncotarget.4754 J., and de Witte, P. A. (2005). Determination of hypericin in human plasma Garg, A. D., Galluzzi, L., Apetoh, L., Baert, T., Birge, R. B., Bravo-San by high-performance liquid chromatography after intravesical administration Pedro, J. M., et al. (2015a). Molecular and translational classifications in patients with transitional cell carcinoma of the bladder. Eur. J. Pharm. of DAMPs in immunogenic cell death. Front. Immunol. 6:588. doi: Biopharm. 59, 469–474. doi: 10.1016/j.ejpb.2004.09.013 10.3389/fimmu.2015.00588 Kamuhabwa, A. A., Roskams, T., D’Hallewin, M. A., Baert, L., Van Poppel, H., Garg, A. D., Krysko, D. V., Vandenabeele, P., and Agostinis, P. (2012b). Hypericin- and de Witte, P. A. (2003). Whole bladder wall photodynamic therapy of based photodynamic therapy induces surface exposure of damage-associated transitional cell carcinoma rat bladder tumors using intravesically administered molecular patterns like HSP70 and calreticulin. Cancer Immunol. Immunother. hypericin. Int. J. Cancer. 107, 460–467. doi: 10.1002/ijc.11396 61, 215–221. doi: 10.1007/s00262-011-1184-2 Kascakova, S., Nadova, Z., Mateasik, A., Mikes, J., Huntosova, V., Refregiers, M., Garg, A. D., Krysko, D. V., Vandenabeele, P., and Agostinis, P. (2016). et al. (2008). High level of low-density lipoprotein receptors enhance hypericin Extracellular ATP and P X receptor exert context-specific immunogenic uptake by U-87 MG cells in the presence of LDL. Photochem. Photobiol. 84, 2 7 effects after immunogenic cancer cell death. Cell Death Dis. 7, e2097. doi: 120–127. doi: 10.1111/j.1751-1097.2007.00207.x 10.1038/cddis.2015.411 Kashef, N., Borghei, Y. S., and Djavid, G. E. (2013). Photodynamic effect Garg, A. D., Krysko, D. V., Verfaillie, T., Kaczmarek, A., Ferreira, G. B., Marysael, of hypericin on the microorganisms and primary human fibroblasts. T., et al. (2012a). A novel pathway combining calreticulin exposure and ATP Photodiagnosis Photodyn. Ther. 10, 150–155. doi: 10.1016/j.pdpdt.2012. secretion in immunogenic cancer cell death. EMBO J. 31, 1062–1079. doi: 11.007 10.1038/emboj.2011.497 Kasper, S., Caraci, F., Forti, B., Drago, F., and Aguglia, E. (2010). Efficacy Garnica, S., Weiss, M., and Oberwinkler, F. (2003). Morphological and molecular and tolerability of Hypericum extract for the treatment of mild to phylogenetic studies in South American Cortinarius species. Mycol. Res. 107, moderate depression. Eur. Neuropsychopharmacol. 20, 747–765. doi: 1143–1156. doi: 10.1017/S0953756203008414 10.1016/j.euroneuro.2010.07.005 Goey, A. K., Meijerman, I., Rosing, H., Marchetti, S., Mergui-Roelvink, M., Kim, M., Jung, H. Y., and Park, H. J. (2015). Topical PDT in the treatment of benign Keessen, M., et al. (2014). The effect of St John’s wort on the pharmacokinetics skin diseases: principles and new applications. Int. J. Mol. Sci. 16, 23259–23278. of docetaxel. Clin. Pharmacokinet. 53, 103–110. doi: 10.1007/s40262-01 doi: 10.3390/ijms161023259 3-0102-5 Kitanov, G. M. (2001). Hypericin and pseudohypericin in some Hypericum species. Gulick, R. M., McAuliffe, V., Holden-Wiltse, J., Crumpacker, C., Liebes, L., Stein, Biochem. Syst. Ecol. 29, 171–178. doi: 10.1016/S0305-1978(00)00032-6 D. S., et al. (1999). Phase I studies of hypericin, the active compound in St. Kleemann, B., Loos, B., Scriba, T. J., Lang, D., and Davids, L. M. (2014). St John’s John’s Wort, as an antiretroviral agent in HIV-infected adults. AIDS Clinical Wort (Hypericum perforatum L.) photomedicine: hypericin-photodynamic Trials Group Protocols 150 and 258. Ann Intern Med. 130, 510–514. doi: therapy induces metastatic melanoma cell death. PLoS ONE 9:e103762. doi: 10.7326/0003-4819-130-6-199903160-00015 10.1371/journal.pone.0103762 Hadjur, C., Richard, M. J., Parat, M. O., Jardon, P., and Favier, A. (1996). Komoroski, B. J., Zhang, S., Cai, H., Hutzler, J. M., Frye, R., Tracy, T. S., et al. Photodynamic effects of hypericin on lipid peroxidation and antioxidant status (2004). Induction and inhibition of cytochromes P450 by the St. John’s wort in melanoma cells. Photochem. Photobiol. 64, 375–381. doi: 10.1111/j.1751- constituent hyperforin in human hepatocyte cultures. Drug Metab. Dispos. 32, 1097.1996.tb02474.x 512–518. doi: 10.1124/dmd.32.5.512 Head, C. S., Luu, Q., Sercarz, J., and Saxton, R. (2006). Photodynamic therapy and Koperdáková, J., Komarovská, H., Košuth, J., Giovannini, A., and Cellárová, E. tumor imaging of hypericin-treated squamous cell carcinoma. World J. Surg. (2009). Characterization of hairy root-phenotype in transgenic Hypericum Oncol. 4:87. doi: 10.1186/1477-7819-4-87 perforatum L. clones. Acta Physiol. Plant. 31, 351–358. doi: 10.1007/s11738- Hudson, J. B., Harris, L., and Towers, G. H. (1993). The importance of light in 008-0241-8 the anti-HIV effect of hypericin. Antiviral Res. 20, 173–178. doi: 10.1016/0166- Košuth, J., Koperdáková, J., Tolonen, A., Hohtola, A., and Cellárová, E. (2003). 3542(93)90006-5 The content of hypericins and phloroglucinols in Hypericum perforatum Huygens, A., Kamuhabwa, A. R., Van Laethem, A., Roskams, T., Van L. seedlings at early stage of development. Plant Sci. 165, 515–521. doi: Cleynenbreugel, B., Van Poppel, H., et al. (2005). Enhancing the photodynamic 10.1016/S0168-9452(03)00210-3 effect of hypericin in tumour spheroids by fractionated light delivery in Koval, J., Mikes, J., Jendzelovský, R., Kello, M., Solár, P., and Fedorocko, combination with hyperoxygenation. Int. J. Oncol. 26, 1691–1697. doi: P. (2010). Degradation of HER2 receptor through hypericin-mediated 10.3892/ijo.26.6.1691 photodynamic therapy. Photochem. Photobiol. 86, 200–205. doi: Jacobson, J. M., Feinman, L., Liebes, L., Ostrow, N., Koslowski, V., Tobia, A., et al. 10.1111/j.1751-1097.2009.00639.x (2001). Pharmacokinetics, safety, and antiviral effects of hypericin, a derivative Kubin, A., Meissner, P., Wierrani, F., Burner, U., Bodenteich, A., Pytel, A., of St. John’s wort plant, in patients with chronic hepatitis C virus infection. et al. (2008). Fluorescence diagnosis of bladder cancer with new water Frontiers in Plant Science | www.frontiersin.org 18 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin soluble hypericin bound to polyvinylpyrrolidone: PVP-hypericin. Photochem. Mikeš, J., Koval’, J., Jendzelovský, R., Sacková, V., Uhrinová, I., Kello, Photobiol. 84, 1560–1563. doi: 10.1111/j.1751-1097.2008.00384.x M., et al. (2009). The role of p53 in the efficiency of photodynamic Kubin, A., Wierrani, F., Burner, U., Alth, G., and Grünberger, W. (2005). therapy with hypericin and subsequent long-term survival of colon Hypericin–the facts about a controversial agent. Curr. Pharm. Des. 11, 233–253. cancer cells. Photochem. Photobiol. Sci. 8, 1558–1567. doi: 10.1039/b9pp0 doi: 10.2174/1381612053382287 0021f Kucharikova, A., Kimakova, K., and Janfelt, C., and Cellarova, E. (2016). Mikešová, L., Mikeš, J., Koval’, J., Gyurászová, K., Culka, L., Vargová, J., et al. Interspecific variation in localization of hypericins and phloroglucinols in (2013). Conjunction of glutathione level, NAD(P)H/FAD redox status and the genus Hypericum as revealed by Desorption Electrospray Ionization Mass hypericin content as a potential factor affecting colon cancer cell resistance Spectrometry imaging. Physiol Plant. 157, 2–12. doi: 10.1111/ppl.12422 to photodynamic therapy with hypericin. Photodiagnosis Photodyn. Ther. 10, Kuchárová, B., Mikeš, J., Jendželovský, R., Vargová, J., Mikešová, L., Jendželovská, 470–483. doi: 10.1016/j.pdpdt.2013.04.003 Z., et al. (2015). Potentiation of hypericin-mediated photodynamic therapy Ni, Y., Huyghe, D., Verbeke, K., de Witte, P. A., Nuyts, J., Mortelmans, L., cytotoxicity by MK-886: focus on ABC transporters, GDF-15 and redox status. et al. (2006). First preclinical evaluation of mono-[123I]iodohypericin as a Photodiagnosis Photodyn. Ther. 12, 490–503. doi: 10.1016/j.pdpdt.2015.04.008 necrosis-avid tracer agent. Eur. J. Nucl. Med. Mol. Imaging 33, 595–601. doi: Kusari, S., Lamshöft, M., Zühlke, S., and Spiteller, M. (2008). An endophytic fungus 10.1007/s00259-005-0013-2 from Hypericum perforatum that produces hypericin. J. Nat. Prod. 71, 159–162. Nies, A. T., Rius, M., and Keppler, D. (2007). “Multidrug resistance proteins of the doi: 10.1021/np070669k ABCC subfamily,” in Drug Transporters: Molecular Characterization and Role Kusari, S., Sezgin, S., Nigutová, K., Cellárová, E., and Spiteller, M. (2015). Spatial in Drug Disposition, eds G. You and M. E. Morris (New Jersey, NJ: John Wiley chemo-profiling of hypericin and related phytochemicals in Hypericum species & Sons, Inc.), 223–262. using MALDI-HRMS imaging. Anal. Bioanal. Chem. 407, 4779–4791. doi: Noell, S., Feigl, G. C., Serifi, D., Mayer, D., Naumann, U., Göbel, W., et al. (2013). 10.1007/s00216-015-8682-6 Microendoscopy for hypericin fluorescence tumor diagnosis in a subcutaneous Kusari, S., Zühlke, S., Košuth, J., Cellárová, E., and Spiteller, M. (2009). Light- glioma mouse model. Photodiagnosis Photodyn. Ther. 10, 552–560. doi: independent metabolomics of endophytic Thielavia subthermophila provides 10.1016/j.pdpdt.2013.06.001 insight into microbial hypericin biosynthesis. J. Nat. Prod. 72, 1825–1835. doi: Noell, S., Mayer, D., Strauss, W. S., Tatagiba, M. S., and Ritz, R. (2011). 10.1021/np9002977 Selective enrichment of hypericin in malignant glioma: pioneering Li, J., Cona, M. M., Chen, F., Feng, Y., Zhou, L., Yu, J., et al. (2012). Exploring in vivo results. Int. J. Oncol. 38, 1343–1348. doi: 10.3892/ijo.20 theranostic potentials of radioiodinated hypericin in rodent necrosis models. 11.968 Theranostics 2, 1010–1019. doi: 10.7150/thno.4924 Olivo, M., Lau, W., Manivasager, V., Bhuvaneswari, R., Wei, Z., Soo, K. C., Li, J., Cona, M. M., Chen, F., Feng, Y., Zhou, L., Zhang, G., et al. (2013). Sequential et al. (2003a). Novel photodynamic diagnosis of bladder cancer: ex vivo systemic administrations of combretastatin A4 Phosphate and radioiodinated fluorescence cytology using hypericin. Int. J. Oncol. 23, 1501–1504. doi: hypericin exert synergistic targeted theranostic effects with prolonged survival 10.3892/ijo.23.6.1501 on SCID mice carrying bifocal tumor xenografts. Theranostics 3, 127–137. doi: Olivo, M., Lau, W., Manivasager, V., Tan, P. H., Soo, K. C., and Cheng, C. (2003b). 10.7150/thno.5790 Macro-microscopic fluorescence of human bladder cancer using hypericin Li, J., Sun, Z., Zhang, J., Shao, H., Cona, M. M., Wang, H., et al. (2011). A dual- fluorescence cystoscopy and laser confocal microscopy. Int. J. Oncol. 23, targeting anticancer approach: soil and seed principle. Radiology 260, 799–807. 983–990. doi: 10.3892/ijo.23.4.983 doi: 10.1148/radiol.11102120 Paz-Cristobal, M. P., Gilaberte, Y., Alejandre, C., Pardo, J., Revillo, M. J., and Liu, C. D., Kwan, D., Saxton, R. E., and McFadden, D. W. (2000). Hypericin and Rezusta, A. (2014). In vitro fungicidal photodynamic effect of hypericin on photodynamic therapy decreases human pancreatic cancer in vitro and in vivo. Trichophyton spp. Mycopathologia 178, 221–225. doi: 10.1007/s11046-014- J. Surg. Res. 93, 137–143. doi: 10.1006/jsre.2000.5949 9797-6 Liu, W., Zhang, D., Feng, Y., Li, Y., Huang, D., Jiang, C., et al. (2015). Prince, A. M., Pascual, D., Meruelo, D., Liebes, L., Mazur, Y., Dubovi, E., Biodistribution and anti-tumor efficacy of intratumorally injected necrosis-avid et al. (2000). Strategies for evaluation of enveloped virus inactivation in red theranostic agent radioiodinated hypericin in rodent tumor models. J. Drug cell concentrates using hypericin. Photochem. Photobiol. 71, 188–195. doi: Target. 23, 371–379. doi: 10.3109/1061186X.2014.1000337 10.1562/0031-8655(2000)0710188SFEOEV2.0.CO2 Lukšiene˙, Ž., and De Witte, P. (2002). Hypericin-based photodynamic therapy: I. Pytel, A., and Schmeller, N. (2002). New aspect of photodynamic diagnosis Comparative antitumor activity uptake studies in Ehrlich ascite tumor. Acta of bladder tumors: fluorescence cytology. Urology 59, 216–219. doi: Med Litu. 9, 195–199. 10.1016/S0090-4295(01)01528-X Martínez-Poveda, B., Quesada, A. R., and Medina, M. A. (2005). Hypericin in the Rezusta, A., López-Chicón, P., Paz-Cristobal, M. P., Alemany-Ribes, M., Royo- dark inhibits key steps of angiogenesis in vitro. Eur. J. Pharmacol. 516, 97–103. Díez, D., Agut, M., et al. (2012). In vitro fungicidal photodynamic effect doi: 10.1016/j.ejphar.2005.03.047 of hypericin on Candida species. Photochem. Photobiol. 88, 613–619. doi: Mathijssen, R. H., Verweij, J., de Bruijn, P., Loos, W. J., and Sparreboom, A. (2002). 10.1111/j.1751-1097.2011.01053.x Effects of St. John’s wort on irinotecan metabolism. J. Natl. Cancer Inst. 94, Ritz, R., Daniels, R., Noell, S., Feigl, G. C., Schmidt, V., Bornemann, A., 1247–1249. doi: 10.1093/jnci/94.16.1247 et al. (2012). Hypericin for visualization of high grade gliomas: first Miadokova, E., Chalupa, I., Vlckova, V., Sevcovicova, A., Nadova, S., Kopaskova, clinical experience. Eur. J. Surg. Oncol. 38, 352–360. doi: 10.1016/j.ejso.2011. M., et al. (2010). Genotoxicity and antigenotoxicity evaluation of non- 12.021 photoactivated hypericin. Phytother. Res. 24, 90–95. doi: 10.1002/ptr.2901 Robey, R. W., Polgar, O., Deeken, J., To, K. K. W., and Bates, S. (2007). “Breast Mikeš, J., Hýžd’alová, M., Kocˇí, L., Jendželovský, R., Koval’, J., Vaculová, A., et al. cancer resistance protein,” in Drug Transporters: Molecular Characterization (2011). Lower sensitivity of FHC fetal colon epithelial cells to photodynamic and Role in Drug Disposition, eds G. You and M. E. Morris (New therapy compared to HT-29 colon adenocarcinoma cells despite higher Jersey, NJ: John Wiley & Sons, Inc.), 319–358. doi: 10.1002/9780470140 intracellular accumulation of hypericin. Photochem. Photobiol. Sci. 10, 626–632. 505.ch12 doi: 10.1039/c0pp00359j Rook, A. H., Wood, G. S., Duvic, M., Vonderheid, E. C., Tobia, A., and Cabana, Mikeš, J., Jendželovský, R., and Fedorocˇko, P. (2013). “Cellular aspects of B. (2010). A phase II placebo-controlled study of photodynamic therapy with photodynamic therapy with hypericin,” in Photodynamic Therapy: New topical hypericin and visible light irradiation in the treatment of cutaneous Research, ed M. L. T. Elsaie (New York, NY: Nova Science Publishers), 111–147. T-cell lymphoma and psoriasis. J. Am. Acad. Dermatol. 63, 984–990. doi: Mikeš, J., Kleban, J., Sacková, V., Horváth, V., Jamborová, E., Vaculová, 10.1016/j.jaad.2010.02.039 A., et al. (2007). Necrosis predominates in the cell death of human Rubio, N., Coupienne, I., Di Valentin, E., Heirman, I., Grooten, J., Piette, J., colon adenocarcinoma HT-29 cells treated under variable conditions of et al. (2012). Spatiotemporal autophagic degradation of oxidatively damaged photodynamic therapy with hypericin. Photochem. Photobiol. Sci. 6, 758–766. organelles after photodynamic stress is amplified by mitochondrial reactive doi: 10.1039/B700350A oxygen species. Autophagy 8, 1312–1324. doi: 10.4161/auto.20763 Frontiers in Plant Science | www.frontiersin.org 19 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin Sacková, V., Fedorocko, P., Szilárdiová, B., Mikes, J., and Kleban, J. (2006). Urbanová, M., Košuth, J., and Cellárová, E. (2006). Genetic and biochemical Hypericin-induced photocytotoxicity is connected with G2/M arrest in HT- analysis of Hypericum perforatum L. plants regenerated after cryopreservation. 29 and S-phase arrest in U937 cells. Photochem. Photobiol. 82, 1285–1291. doi: Plant Cell Rep. 25, 140–147. doi: 10.1007/s00299-005-0050-0 10.1562/2006-02-22-RA-806 Urla, C., Armeanu-Ebinger, S., Fuchs, J., and Seitz, G. (2015). Successful in Sanovic, R., Verwanger, T., Hartl, A., and Krammer, B. (2011). Low dose vivo tumor visualization using fluorescence laparoscopy in a mouse model of hypericin-PDT induces complete tumor regression in BALB/c mice bearing disseminated alveolar rhabdomyosarcoma. Surg. Endosc. 29, 1105–1114. doi: CT26 colon carcinoma. Photodiagnosis Photodyn. Ther. 8, 291–296. doi: 10.1007/s00464-014-3770-9 10.1016/j.pdpdt.2011.04.003 Vandenbogaerde, A. L., Cuveele, J. F., Proot, P., Himpens, B. E., Merlevede, Sattler, S., Schaefer, U., Schneider, W., Hoelzl, J., and Lehr, C. M. (1997). Binding, W. J., and de Witte, P. A. (1997). Differential cytotoxic effects induced after uptake, and transport of hypericin by Caco-2 cell monolayers. J. Pharm. Sci. 86, photosensitization by hypericin. J. Photochem. Photobiol. B. Biol. 38, 136–142. 1120–1126. doi: 10.1021/js970004a doi: 10.1016/S1011-1344(96)07446-5 Shao, H., Zhang, J., Sun, Z., Chen, F., Dai, X., Li, Y., et al. (2015). Necrosis targeted Vandenbogaerde, A. L., Geboes, K. R., Cuveele, J. F., Agostinis, P. M., Merlevede, radiotherapy with iodine-131-labeled hypericin to improve anticancer efficacy W. J., and De Witte, P. A. (1996). Antitumour activity of photosensitized of vascular disrupting treatment in rabbit VX2 tumor models. Oncotarget 6, hypericin on A431 cell xenografts. Anticancer Res. 16, 1619–1625. 14247–14259. doi: 10.18632/oncotarget.3679 Vandepitte, J., Van Cleynenbreugel, B., Hettinger, K., Van Poppel, H., and de Siboni, G., Weitman, H., Freeman, D., Mazur, Y., Malik, Z., and Ehrenberg, B. Witte, P. A. (2010). Biodistribution of PVP-hypericin and hexaminolevulinate- (2002). The correlation between hydrophilicity of hypericins and helianthrone: induced PpIX in normal and orthotopic tumor-bearing rat urinary bladder. internalization mechanisms, subcellular distribution and photodynamic action Cancer Chemother. Pharmacol. 67, 775–781. doi: 10.1007/s00280-010- in colon carcinoma cells. Photochem. Photobiol. Sci. 1, 483–491. doi: 1375-0 10.1039/b202884k Van de Putte, M., Marysael, T., Fonge, H., Roskams, T., Cona, M. M., Li, J., et al. Sim, H. G., Lau, W. K., Olivo, M., Tan, P. H., and Cheng, C. W. (2005). Is (2012). Radiolabeled iodohypericin as tumor necrosis avid tracer: diagnostic photodynamic diagnosis using hypericin better than white-light cystoscopy and therapeutic potential. Int. J. Cancer. 131, E129–E137. doi: 10.1002/ijc. for detecting superficial bladder carcinoma? BJU Int. 95, 1215–1218. doi: 26492 10.1111/j.1464-410X.2005.05508.x Van de Putte, M., Ni, Y., and De Witte, P. A. (2008a). Exploration of the mechanism Smith, P., Bullock, J. M., Booker, B. M., Haas, C. E., Berenson, C. S., and Jusko, underlying the tumor necrosis avidity of hypericin. Oncol. Rep. 19, 921–926. W. J. (2004). The influence of St. John’s wort on the pharmacokinetics and doi: 10.3892/or.19.4.921 protein binding of imatinib mesylate. Pharmacotherapy 24, 1508–1514. doi: Van de Putte, M., Wang, H., Chen, F., de Witte, P. A., and Ni, Y. (2008b). 10.1592/phco.24.16.1508.50958 Hypericin as a marker for determination of tissue viability after intratumoral Song, S., Xiong, C., Zhou, M., Lu, W., Huang, Q., Ku, G., et al. (2011). Small-animal ethanol injection in a murine liver tumor model. Acad. Radiol. 15, 107–113. PET of tumor damage induced by photothermal ablation with 64Cu-bis- doi: 10.1016/j.acra.2007.08.008 DOTA-hypericin. J. Nucl. Med. 52, 792–799. doi: 10.2967/jnumed.110.086116 Van de Putte, M., Wang, H., Chen, F., De Witte, P. A., and Ni, Y. (2008c). Hypericin Stavrovskaya, A. A. (2000). Cellular mechanisms of multidrug resistance of tumor as a marker for determination of tissue viability after radiofrequency ablation cells. Biochemistry Mosc. 65, 95–106. in a murine liver tumor model. Oncol. Rep. 19, 927–932. doi: 10.3892/or.19. Straub, M., Russ, D., Horn, T., Gschwend, J. E., and Abrahamsberg, C. (2015). 4.927 A phase IIA dose-finding study of PVP-hypericin fluorescence cystoscopy for Wada, A., Sakaeda, T., Takara, K., Hirai, M., Kimura, T., Ohmoto, N., et al. (2002). detection of nonmuscle-invasive bladder cancer. J. Endourol. 29, 216–222. doi: Effects of St John’s wort and hypericin on cytotoxicity of anticancer drugs. Drug 10.1089/end.2014.0282 Metab. Pharmacokinet. 17, 467–474. doi: 10.2133/dmpk.17.467 Theodossiou, T. A., Hothersall, J. S., De Witte, P. A., Pantos, A., and Agostinis, Wölfle, U., Seelinger, G., and Schempp, C. M. (2014). Topical application of St. P. (2009). The multifaceted photocytotoxic profile of hypericin. Mol. Pharm. 6, John’s wort (Hypericum perforatum). Planta Med. 80, 109–120. doi: 10.1055/s- 1775–1789. doi: 10.1021/mp900166q 0033-1351019 Thomas, C., MacGill, R. S., Miller, G. C., and Pardini, R. S. (1992). Xie, X., Hudson, J. B., and Guns, E. S. (2001). Tumor-specific and Photoactivation of hypericin generates singlet oxygen in mitochondria and photodependent cytotoxicity of hypericin in the human LNCaP inhibits succinoxidase. Photochem. Photobiol. 55, 47–53. doi: 10.1111/j.1751- prostate tumor model. Photochem. Photobiol. 74, 221–225. doi: 1097.1992.tb04208.x 10.1562/0031-8655(2001)0740221TSAPCO2.0.CO2 Thomas, C., and Pardini, R. S. (1992). Oxygen dependence of hypericin- Zahreddine, H., and Borden, K. L. (2013). Mechanisms and insights into drug induced phototoxicity to EMT6 mouse mammary carcinoma cells. Photochem. resistance in cancer. Front. Pharmacol. 4:28. doi: 10.3389/fphar.2013.00028 Photobiol. 55, 831–837. doi: 10.1111/j.1751-1097.1992.tb08531.x Zheng, Y., Yin, G., Le, V., Zhang, A., Chen, S., Liang, X., et al. (2016). Thong, P. S., Kho, K. W., Zheng, W., Harris, M., Soo, K. C., and Olivo, M. (2007). Photodynamic-therapy activates immune response by disrupting immunity Development of a laser confocal endomicroscope for in vivo fluorescence homeostasis of tumor cells, which generates vaccine for cancer therapy. Int. J. imaging. J. Mech. Med. Biol. 7, 11–18. doi: 10.1142/S0219519407002108 Biol. Sci. 12, 120–132. doi: 10.7150/ijbs.12852 Thong, P. S., Olivo, M., Chin, W. W., Bhuvaneswari, R., Mancer, K., and Soo, K. C. Zupkó, I., Kamuhabwa, A. R., D’Hallewin, M. A., Baert, L., and De (2009). Clinical application of fluorescence endoscopic imaging using hypericin Witte, P. A. (2001). In vivo photodynamic activity of hypericin in for the diagnosis of human oral cavity lesions. Br. J. Cancer. 101, 1580–1584. transitional cell carcinoma bladder tumors. Int. J. Oncol. 18, 1099–1105. doi: doi: 10.1038/sj.bjc.6605357 10.3892/ijo.18.5.1099 Thong, P. S., Tandjung, S. S., Movania, M. M., Chiew, W. M., Olivo, M., Bhuvaneswari, R., et al. (2012). Toward real-time virtual biopsy of oral lesions Conflict of Interest Statement: The authors declare that the research was using confocal laser endomicroscopy interfaced with embedded computing. J. conducted in the absence of any commercial or financial relationships that could Biomed. Opt. 17:056009. doi: 10.1117/1.JBO.17.5.056009 be construed as a potential conflict of interest. Thong, P. S., Watt, F., Ren, M. Q., Tan, P. H., Soo, K. C., and Olivo, M. (2006). Hypericin-photodynamic therapy (PDT) using an alternative treatment regime Copyright © 2016 Jendželovská, Jendželovský, Kuchárová and Fedoroˇcko. This suitable for multi-fraction PDT. J. Photochem. Photobiol. B. Biol. 82, 1–8. doi: is an open-access article distributed under the terms of the Creative Commons 10.1016/j.jphotobiol.2005.08.002 Attribution License (CC BY). The use, distribution or reproduction in other forums Tian, R., Koyabu, N., Morimoto, S., Shoyama, Y., Ohtani, H., and Sawada, Y. is permitted, provided the original author(s) or licensor are credited and that the (2005). Functional induction and de-induction of P-glycoprotein by St. John’s original publication in this journal is cited, in accordance with accepted academic wort and its ingredients in a human colon adenocarcinoma cell line. Drug practice. No use, distribution or reproduction is permitted which does not comply Metab. Dispos. 33, 547–554. doi: 10.1124/dmd.104.002485 with these terms. Frontiers in Plant Science | www.frontiersin.org 20 May 2016 | Volume 7 | Article 560 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Frontiers in Plant Science Pubmed Central

Hypericin in the Light and in the Dark: Two Sides of the Same Coin

Frontiers in Plant Science , Volume 7 – May 6, 2016

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REVIEW published: 06 May 2016 doi: 10.3389/fpls.2016.00560 Hypericin in the Light and in the Dark: Two Sides of the Same Coin Zuzana Jendželovská, Rastislav Jendželovský, Barbora Kuchárová and Peter Fedorocko ˇ * Department of Cellular Biology, Faculty of Science, Pavol Jozef Šafárik University in Košice, Košice, Slovakia ′ ′ ′ ′ Hypericin (4,5,7,4 ,5 ,7 -hexahydroxy-2,2 -dimethylnaphtodianthrone) is a naturally occurring chromophore found in some species of the genus Hypericum, especially Hypericum perforatum L. (St. John’s wort), and in some basidiomycetes (Dermocybe spp.) or endophytic fungi (Thielavia subthermophila). In recent decades, hypericin has been intensively studied for its broad pharmacological spectrum. Among its antidepressant and light-dependent antiviral actions, hypericin is a powerful natural photosensitizer that is applicable in the photodynamic therapy (PDT) of various Edited by: oncological diseases. As the accumulation of hypericin is significantly higher in Gregory Franklin, neoplastic tissue than in normal tissue, it can be used in photodynamic diagnosis Polish Academy of Sciences, Poland (PDD) as an effective fluorescence marker for tumor detection and visualization. In Reviewed by: Jirina ˇ Hofmanová, addition, light-activated hypericin acts as a strong pro-oxidant agent with antineoplastic Academy of Sciences of the Czech and antiangiogenic properties, since it effectively induces the apoptosis, necrosis or Republic, Czech Republic autophagy of cancer cells. Moreover, a strong affinity of hypericin for necrotic tissue Lester M. Davids, University of Cape Town, South Africa was discovered. Thus, hypericin and its radiolabeled derivatives have been recently Abhishek D. Garg, investigated as potential biomarkers for the non-invasive targeting of tissue necrosis in KU Leuven, Belgium numerous disorders, including solid tumors. On the other hand, several light-independent *Correspondence: Peter Fedorocko ˇ actions of hypericin have also been described, even though its effects in the dark have not peter.fedorocko@upjs.sk been studied as intensively as those of photoactivated hypericin. Various experimental studies have revealed no cytotoxicity of hypericin in the dark; however, it can serve Specialty section: This article was submitted to as a potential antimetastatic and antiangiogenic agent. On the contrary, hypericin can Plant Metabolism and Chemodiversity, induce the expression of some ABC transporters, which are often associated with the a section of the journal multidrug resistance (MDR) of cancer cells. Moreover, the hypericin-mediated attenuation Frontiers in Plant Science of the cytotoxicity of some chemotherapeutics was revealed. Therefore, hypericin Received: 12 February 2016 Accepted: 11 April 2016 might represent another St. John’s wort metabolite that is potentially responsible for Published: 06 May 2016 negative herb–drug interactions. The main aim of this review is to summarize the Citation: benefits of photoactivated and non-activated hypericin, mainly in preclinical and clinical Jendželovská Z, Jendželovský R, applications, and to uncover the “dark side” of this secondary metabolite, focusing on Kuchárová B and Fedorocko ˇ P (2016) Hypericin in the Light and in the Dark: MDR mechanisms. Two Sides of the Same Coin. Front. Plant Sci. 7:560. Keywords: hypericin, St. John’s wort, anticancer activities, photodynamic therapy, photodynamic diagnosis, drug doi: 10.3389/fpls.2016.00560 resistance Frontiers in Plant Science | www.frontiersin.org 1 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin INTRODUCTION to be an effective antiviral agent (Hudson et al., 1993; Prince et al., 2000). However, some clinical studies have revealed that ′ ′ ′ ′ Hypericin (4,5,7,4 ,5 ,7 -hexahydroxy-2,2 -dimethylnaphtodiant high doses of hypericin can induce phototoxic skin reactions hrone) is a naturally occurring compound synthesized by some without showing any detectable antiviral or antiretroviral activity species of the genus Hypericum. Hypericin was first isolated from in patients with viral infections (Gulick et al., 1999; Jacobson Hypericum perforatum L. (Brockmann et al., 1939), commonly et al., 2001). The controversy concerning the virucidal effect of known as St. John’s wort, which is one of the best characterized hypericin was summarized in detail by Kubin et al. (2005). and most important representatives of this genus, because of However, the potential use of this secondary metabolite in its broad pharmacological activity (antidepressant, antimicrobial, medicine might be broader than currently thought. Although anticancer, anti-inflammatory, wound healing, etc.) (reviewed hypericin has been extensively studied mainly because of its in Kasper et al., 2010; Wölfle et al., 2014). Hypericin and its photodynamic and photocytotoxic properties, it also possesses derivatives are accumulated in special morphological structures, various positive or negative biological activities without being so called dark nodules, occurring in the aerial parts of activated by light. hypericin-producing Hypericum species. The newest data on interspecific variation in localization of hypericins and spatial LIGHT-ACTIVATED HYPERICIN chemo-profiling of hypericin in some Hypericum species were published recently (Kusari et al., 2015; Kucharikova et al., Hypericin possesses several properties that make it a powerful 2016). fluorescent photosensitizer that is suitable for PDT and PDD— In addition to St. John’s wort, this secondary metabolite attractive applications for the treatment and detection of tumors. was found in several other Hypericum species (Kitanov, 2001; It possesses minimal or no toxicity in the dark (Thomas Ayan et al., 2004) and in some basidiomycetes (Dermocybe spp.) and Pardini, 1992; Vandenbogaerde et al., 1997; Miadokova (Dewick, 2002; Garnica et al., 2003) or endophytic fungi growing et al., 2010; Jendželovská et al., 2014; Feruszová et al., 2016), in Hypericum perforatum (Thielavia subthermophila) (Kusari accumulates preferentially in neoplastic tissues (Kamuhabwa et al., 2008, 2009). As hypericin is a bioactive compound that et al., 2002; Noell et al., 2011) and generates reactive oxygen is applicable in several medicinal approaches, its content has species (ROS) in the presence of light (at wavelengths around been evaluated in in vitro grown Hypericum perforatum and in 600 nm) and oxygen (Diwu and Lown, 1993). Thus, hypericin its transgenic clones (Cellárová et al., 1997; Košuth et al., 2003; represents a potent natural alternative to chemically synthesized Koperdáková et al., 2009), or in Hypericum cultures exposed photosensitizers. to various biotechnological applications that focused on their preservation or stimulation of secondary metabolite production Hypericin in Photodynamic Therapy (Urbanová et al., 2006; Brunˇáková et al., 2015; reviewed in: PDT represents a non-invasive therapeutic approach that is Cellárová, 2011). beneficial in the treatment of various cancerous (reviewed in Hypericin is well-known as a potent natural photosensitizing Agostinis et al., 2011) and even non-cancerous lesions and agent with great potential in anticancer photodynamic therapy disorders (reviewed in Kim et al., 2015). In general, it is (PDT) and photodynamic diagnosis (PDD). Besides its based on the combined action of a photosensitizer, light and antineoplastic action, light-dependent in vitro fungicidal molecular oxygen. PDT involves the administration of a non- (Rezusta et al., 2012; Paz-Cristobal et al., 2014) and bactericidal toxic photosensitizer that preferentially accumulates in the effects (Kashef et al., 2013; García et al., 2015) have also been target tissue, followed by its local illumination with harmless reported. In addition, light-activated hypericin is considered visible light of an appropriate wavelength, to activate and excite the photosensitizer. These photoreactions lead to the oxygen- dependent generation of cytotoxic ROS, resulting in cell death 1 64 64 Abbreviations: O , singlet oxygen; Cu-bis-DOTA-hypericin, Cu-Labeled bis- ′ ′ and tissue destruction. However, PDT is a multifactorial process 1,4,7,10-tetraazacyclododecane-N,N ,N,N -tetraacetic acid conjugated hypericin; ABC, ATP-binding cassette; AK, actinic keratosis; BCC, basal cell carcinoma; and the degree of cellular photodamage depends on many factors, BCRP, breast cancer resistance protein; BD, Bowen’s disease; CA4P, combretastatin including cell permeability, the subcellular localization of the A4 phosphate; Cdk4, cyclin-dependent kinase 4; CIS, carcinoma in situ; photosensitizer, the quantity of molecular oxygen, the light dose, CLE, confocal laser endomicroscopy; CYP3A4, cytochrome P450 3A4; DAMPs, the types of generated ROS and the attributes of cancer cells. damage-associated molecular patterns; DLI, drug–light interval; EGFR, epidermal The exact mechanisms of cellular hypericin uptake are still growth factor receptor; FE, fluorescence endoscopy; Hsp90, heat shock protein 90; HY-PDD, hypericin-mediated photodynamic diagnosis; HY-PDT, hypericin- unclear and require further investigation, but the results indicate mediated photodynamic therapy; ICD, immunogenic cell death; INF-α, interferon- that hypericin might be transported into or through cells via α; LIF, laser-induced fluorescence; MDR, multidrug resistance; MF, mycosis temperature-dependent diffusion (Thomas and Pardini, 1992; fungoides; MRP1, multidrug resistance-associated protein 1; NACA, necrosis-avid ›− Sattler et al., 1997), partitioning, pinocytosis or endocytosis contrast agent; O , superoxide anion; p53, phosphoprotein p53, tumor suppressor (Siboni et al., 2002). Concerning its subcellular redistribution, the p53, tumor protein p53; PDD, photodynamic diagnosis; PDT, photodynamic therapy; P-gp, P-glycoprotein; Plk, Polo-like kinase; PVP, polyvinylpyrrolidone; co-labeling of cancer cells with hypericin and fluorescent dyes Raf-1, serine/threonine kinase, Raf-1 proto-oncogene; ROS, reactive oxygen specific for cell organelles revealed that hypericin accumulates species; SCC, squamous cell carcinoma; Ser, serine; SN-38, 7-Ethyl-10-hydroxy- in the membranes of the endoplasmic reticulum, the Golgi camptothecin; TCC, transitional cell carcinoma; Thr, threonine; TNF-α, tumor apparatus, lysosomes and mitochondria (Agostinis et al., 2002; necrosis growth factor-α; TNT, tumor necrosis therapy; VEGF, vascular endothelial growth factor; WLE, white-light endoscopy. Ali and Olivo, 2002; Galanou et al., 2008; Mikeš et al., 2011). Frontiers in Plant Science | www.frontiersin.org 2 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin However, the cellular uptake and subcellular localization of patients) and found that HY-PDT was effective in the treatment hypericin might be affected by its lipophilicity, incubation of both skin disorders. A reduction in tumor size and the concentrations and/or interaction with serum lipoproteins generation of a new epithelium at the surface of lesions following (Crnolatac et al., 2005; Galanou et al., 2008; Kascakova et al., HY-PDT were observed. Moreover, as no necrosis or cell loss 2008). In brief, upon light-activation, hypericin is efficient was evident in the surrounding healthy tissues and no side primarily in the generation of singlet oxygen ( O ; type II effects were observed, with the exception of mild erythema in ›− mechanism) and superoxide anion (O ; type I mechanism) five cases (two patients with SCC, three patients with BCC), (Thomas et al., 1992; Diwu and Lown, 1993), which can HY-PDT-mediated tumor targeting was selective. The treatment ultimately lead to necrosis (Du et al., 2003b; Mikeš et al., resulted in a complete clinical response in one SCC patient 2007, 2009), apoptosis (Ali and Olivo, 2002; Mikeš et al., 2009), and two BCC patients, but in the remaining patients, only a autophagy-associated cell death (Buytaert et al., 2006; Rubio partial clinical response was observed. Thus, the efficacy of HY- et al., 2012) or even to immunogenic cell death (ICD) (Garg PDT appeared to be dependent on the initial lesion size, the et al., 2012a). As type II ICD inducer (Garg et al., 2015a), total dose of hypericin, or the frequency and duration of the HY-PDT represents a promising form of active immunotherapy therapy (Alecu et al., 1998). Several years later, the potential (Galluzzi et al., 2014) owing to spatiotemporally defined emission use of HY-PDT in the treatment of non-melanoma skin cancers of damage-associated molecular patterns (DAMPs) (Garg et al., was explored (Kacerovská et al., 2008). A complete clinical 2012b, 2015b, 2016; Zheng et al., 2016). response was observed in 50% of patients with actinic keratosis The photocytotoxicity of hypericin is strongly oxygen- (AK) 3 months after HY-PDT, and in 22% of patients with dependent, as no such effects are present in hypoxic conditions superficial BCC and 40% of patients with Bowen’s disease (BD) (Thomas and Pardini, 1992; Delaey et al., 2000). Nevertheless, 6 months after HY-PDT. However, in the case of AK, the the final response of hypericin-mediated PDT (HY-PDT) might percentage reduced to 29% 6 months after HY-PDT and only also be affected by the ability of cells to overcome oxidative partial remission was observed in patients with nodular BCC. stress through the activity of various cytoprotective mechanisms, On the other hand, complete histological remission was evident including cellular redox systems (Mikeš et al., 2011; Mikešová in 80% of patients with BD (Kacerovská et al., 2008). Only the et al., 2013). Furthermore, the light-dependent inhibitory effect partial response rate and suboptimal success of HY-PDT could of hypericin against various enzymes engaged in the regulation be caused by the limited penetration of the skin by hypericin of cell survival and proliferation (Ser/Thr kinases, tyrosine and by its low concentration in the final extraction product. In kinases, etc.) has been reported (reviewed in Kubin et al., 2005). the third clinical trial, Rook et al. (2010) tested HY-PDT as a These activities might also contribute to the cytotoxic and potentially well-tolerated and effective therapeutic modality for antiproliferative effects of HY-PDT. The exact mechanisms of the treatment of lymphocyte-mediated skin disorders: malignant action and the cellular aspects of HY-PDT have been outlined mycosis fungoides (MF; the most common type of cutaneous and summarized in several reviews (Agostinis et al., 2002; T-cell lymphoma) and non-cancerous autoimmune psoriasis. Theodossiou et al., 2009; Mikeš et al., 2013; Garg and Agostinis, The results were promising for both diseases. In the case of 2014). MF, HY-PDT led to an improvement in the treated lesions (a size reduction by at least 50%) in the majority of patients, Preclinical and Clinical Assessment of HY-PDT whereas the placebo was ineffective. Moreover, hypericin was Efficacy and Suitable Conditions well tolerated by the patients, with only mild to moderate Many in vitro studies have demonstrated the cytotoxicity of phototoxic skin reactions occurring after exposure to visible light. photoactivated hypericin in various cancer cell types (Xie et al., No serious adverse effects or events were observed (Rook et al., 2001; Head et al., 2006; Sacková et al., 2006; Mikeš et al., 2010). However, the authors themselves recommended a phase 2007; Koval et al., 2010; Mikešová et al., 2013; Kleemann et al., III study with a greater number of patients. All these clinical 2014). Moreover, recent in vivo, preclinical and clinical studies data indicate that topically applied hypericin, combined with have indicated that HY-PDT might be an effective and relevant its photoactivation, might be a promising and safe alternative approach in the treatment of some skin tumors, carcinomas for the treatment of some cancerous and non-cancerous skin and sarcomas. In general, the depth of tumor destruction after disorders. However, as the effectiveness of HY-PDT depends PDT commonly ranges from a few mm to 1 cm, due to limited on the hypericin concentration, its total dose, its rate of tissue photosensitizer and light penetration through the tissues. Thus, penetration, the frequency and duration of the therapy, or on the PDT is effective mostly against superficial lesions and small grade of malignancy, more clinical trials are necessary to define tumors. the optimal conditions for the whole procedure. Clinical studies to test HY-PDT efficacy Preclinical in vivo studies to test HY-PDT effects and To our knowledge, three clinical trials of HY-PDT applied to conditions various skin tumors have been published to date (Table 1). Many further studies to test HY-PDT efficacy have been In the first study, Alecu et al. (1998) tested the intralesional performed using mouse or rat animal models (Table 2). injection of hypericin with subsequent photoactivation with Several in vivo studies indicate that HY-PDT might be a visible light in the treatment of basal cell carcinoma (BCC) promising approach in the treatment of bladder carcinomas. (eleven patients) and squamous cell carcinoma (SCC) (eight Kamuhabwa et al. (2002) reported selective hypericin uptake Frontiers in Plant Science | www.frontiersin.org 3 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin TABLE 1 | Clinical studies to test HY-PDT efficacy. Disease/No. of Hypericin Hypericin dosage Light HY-PDT efficacy References patients administration dose/Fluence rate Squamous cell Intralesional 40–100 μg 3–5 times per week 86 J/cm /24 Reduction in tumor size, re-epithelization Alecu et al., 1998 carcinoma/8 injection for 2–4 weeks; mW/cm at the borders of the lesion, complete clinical remission in the case of one patient; Basal cell 40–200 μg 3–5 times per week Reduction in tumor size, complete clinical carcinoma/11 for 2–6 weeks remission in the case of two patients, no evident signs of tumor recurrence after 5 months Actinic keratosis/8 On the lesion Weekly for 6 weeks on average 75 J/cm 50% complete clinical response (AK) 28% Kacerovská et al., Basal cell complete clinical response (superficial 2008 carcinoma/21 BCC) 11% complete histological response (superficial BCC) 67% partial clinical response (nodular BCC) Bowen’s disease/5 40% complete clinical response (BD) 80% complete histological response (BD) 2 2 Mycosis fungoides On the lesion 0.005–0.025 mg/cm 8–20 J/cm 58.3% of responsive patients (reduction in Rook et al., 2010 (T-cell lymphoma)/12 twice-weekly for 6 weeks MF lesion size by 50% or more) Psoriasis/11 54.6% of responsive patients AK, actinic keratosis; BCC, basal cell carcinoma; BD, Bowen’s disease; MF, mycosis fungoides. in bladder tumors and subsequently, even HY-PDT-mediated shoulder tumors (91.2% ± 2.3%) and even pancreatic tumor tumor damage was observed without the destruction of normal nodules (42.2% ± 8.1%) was observed 4 weeks after HY-PDT, tissue (Kamuhabwa et al., 2003). In both studies, female Fisher indicating that intratumor hypericin and laser therapy might also rats with an orthotopic superficial transitional cell carcinoma be beneficial in the treatment of unresectable pancreatic cancer (TCC) were used as an experimental model and hypericin (Liu et al., 2000). was administered directly into the bladder via the catheter. However, instead of more clinically relevant orthotopic tumor The instilled hypericin accumulated selectively in the bladder models, more in vivo studies have been performed to test the urothelial tumors and the normal urothelium (in a ratio of 12:1), efficacy, conditions or responses of HY-PDT after the treatment, but no hypericin was detected in normal bladder submucosa and only in the murine or rat xenograft or allograft models of muscle layers, which is an important factor to avoid underlying subcutaneous carcinomas or sarcomas. Various positive effects of tissue damage. In addition, no hypericin was detected in plasma; HY-PDT involving the inhibition of tumor growth, a prolonged thus, systemic side-effects should not appear (Kamuhabwa et al., survival time of the treated animals, tumor necrosis, apoptosis 2002). or damage to the tumor vasculature were observed in mice Furthermore, photoactivated hypericin resulted in selective bearing human epidermoid carcinoma (Vandenbogaerde et al., urothelial tumor damage, with tumor cells shrinking and 1996), human prostate adenocarcinoma cells (Xie et al., 2001), detaching from the bladder wall, indicating that HY-PDT might human nasopharyngeal carcinoma cells (Du et al., 2003a,b; be beneficial in the treatment of superficial carcinomas and Thong et al., 2006), human squamous carcinoma cells (Head premalignant changes in the bladder. The HY-PDT that was et al., 2006), human bladder carcinoma cells (Bhuvaneswari performed under suitable light conditions had no significant et al., 2008), human rhabdomyosarcoma cells (Urla et al., 2015), effects on the other bladder layers; nevertheless, 2–5% of murine lymphoma cells (Chen and de Witte, 2000), murine tumor cells survived and were responsible for tumor regrowth colon adenocarcinoma cells (Blank et al., 2002; Sanovic et al., (Kamuhabwa et al., 2003). However, following the results of 2011), murine fibrosarcoma cells (Cavarga et al., 2001, 2005; in vitro study based on TCC-derived spheroids, the same Chen et al., 2001, 2002a,b; Bobrov et al., 2007) or murine Ehrlich authors suggested that hyperoxygenation could overcome this ascites carcinoma cells (Lukšiene˙ and De Witte, 2002) and in problem and might enhance the efficacy of HY-PDT (Huygens rats bearing rat bladder transitional bladder carcinoma (Zupkó et al., 2005). In addition to the orthotopic tumor model, et al., 2001) or rat pituitary adenoma cells (Cole et al., 2008) Liu et al. (2000) also used a xenograft model in their (Table 2). In addition, Blank et al. (2002) demonstrated the experiments. Human MiaPaCa-2 pancreatic adenocarcinoma dependence of HY-PDT efficacy on the irradiation conditions cells were injected subcutaneously and orthotopically into the (light dose and wavelength). Tumor necrosis was much more pancreatic bed of nude, athymic mice. To allow hypericin pronounced at 590 nm than at 550 nm and even increased photoactivation in orthotopic pancreatic tumor nodules, mice when the light dose was raised from 60 to 120 J/cm ; however, underwent a laparotomy that was necessary for the positioning of the maximum depth of tumor necrosis was 9.9 ± 0.8 mm the optical fiber. A significant decrease in growth of subcutaneous at 590 nm (Blank et al., 2002). Considering the relationship Frontiers in Plant Science | www.frontiersin.org 4 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin TABLE 2 | Preclinical in vivo studies to test HY-PDT effects and conditions. Experimental model/Type of Hypericin Hypericin dose Light dose/Fluence HY-PDT effects References tumor (cell line) administration rate Athymic nude mice/Epidermoid Intraperitoneal 2.5 mg/kg, 180 J/cm Tumor growth Vandenbogaerde et al., carcinoma (A431) injection 5 mg/kg inhibition, reduced 1996 tumor mass Athymic nude mice/Pancreatic Intratumoral Injection 10 μg/mouse 2 doses of 200 J Suppressed growth of Liu et al., 2000 carcinoma (MiaPaCa-2) subcutaneous and orthotopic tumors 2 2 DBA/2 mice/Lymphoma (P388) Intraperitoneal 2, 5 or 20 mg/kg 120 J/cm /100 mW/cm Reduced tumor mass Chen and de Witte, injection and tumor size, 2000 prolonged survival time Nude mice/Prostate carcinoma Oral 5 mg/kg 30 mW Tumor growth inhibition Xie et al., 2001 (LNCaP) 2 2 C3H/Km mice/Fibrosarcoma Intravenous injection 5 mg/kg 120 J/cm /100 mW/cm Tumor vasculature Chen et al., 2001, (RIF-1) damage after 0.5 h DLI 2002a,b PDT resulting in complete tumor cure, apoptosis as a main form of cell death Fischer CDF (F344)/CrlBR Intravenous injection 1 or 5 mg/kg 120 Reduced tumor size, Zupkó et al., 2001 2 2 rats/Bladder carcinoma (AY-27) J/cm /100 mW/cm no measurable tumor mass 9–10 days after 0.5 h DLI PDT C3H/DiSn mice/Fibrosarcoma Intratumoral or 5 mg/kg 180 J/cm /150 Reduced tumor Cavarga et al., 2001 (G5:1:13) intraperitoneal mW/cm volume, prolonged injection survival time, complete remission in smaller lesions (3 mm or less in size) 2 2 Intraperitoneal 1 × 5 mg/kg, 2 × 168 J/cm /70 mW/cm Higher efficiency of Cavarga et al., 2005; injection 2.5 mg/kg fractionated dose Bobrov et al., 2007 Vascular damage, formation of necrotic areas Balb/c mice/Colon carcinoma Intraperitoneal 5 mg/kg 60, 90 or Vascular damage, Blank et al., 2002 2 2 (C26) injection 120 J/cm /100 mW/cm tumor necrosis (the depth of tumor necrosis increased with increased light dose) Balb/c mice/Ehrlich ascites Intraperitoneal 40 mg/kg 50 mW/cm Prolonged survival time Lukšiene˙ and De Witte, carcinoma injection (75% of mice), no 2002 tumor recurrence (25% of survived mice) Fischer rats/Bladder carcinoma Instillation into the 30 μM 6–48 J/cm /25–50 Selective urothelial Kamuhabwa et al., 2003 (AY-27) bladder mW/cm tumor damage without destructive effects on detrusor musculature Balb/c nude Intravenous injection 2 mg/kg 120 J/cm /226 Inhibited tumor growth, Du et al., 2003a,b mice/Nasopharyngeal carcinoma mW/cm tumor shrinkage, (HK-1) necrosis as a main form of cell death 2 2 2 or 5 mg/kg 30 J/cm /25 mW/cm Increased apoptosis Thong et al., 2006 and lower serum levels of VEGF after 6 h DLI PDT Athymic nude mice/Squamous Intratumoral injection 10 μg per mg 0–60 J/cm Regression of smaller Head et al., 2006 carcinoma (SNU1) tumor tumors (under 400 mm ) (Continued) Frontiers in Plant Science | www.frontiersin.org 5 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin TABLE 2 | Continued Experimental model/Type of Hypericin Hypericin dose Light dose/Fluence HY-PDT effects References tumor (cell line) administration rate 2 2 Balb/c nude mice/Bladder Intravenous injection 5 mg/kg 120 J/cm /100 mW/cm Vascular damage after Bhuvaneswari et al., carcinoma (MGH) 0.5 h DLI PDT resulting 2008 in reduced tumor volume, increased expression of some angiogenic proteins after 6 h DLI PDT Wistar-Furth rats/Pituitary Intraperitoneal 4 × 1 mg/kg 105–130 J/m Inhibited growth of Cole et al., 2008 adenoma (GH4C1) injection smaller tumors (under 1 cm ), formation of apoptotic clusters 2 2 NMRI – HR-HR hairless Topical application 0.1% in gelcream 40 J/cm /20 mW/cm Full lesional necrosis Boiy et al., 2010 mice/UV-induced small skin resulting in total lesional tumors clearance (44%), replacement of atypical AK cells by normal keratinocytes Balb/c mice/Colon carcinoma Intravenous injection 2.5 or 10 mg/kg 14 or 60 J/cm /27 or Vascular damage after Sanovic et al., 2011 (CT26) 50 mW/cm “low power PDT” resulting in complete tumor regression, prevention of new tumor growth after the re-challenge of cured mice with CT26 cells NOD/LtSz-scid IL2Rγnull Intravenous injection 100 μg/mouse —- Induction of apoptosis Urla et al., 2015 mice/Rhabdomyosarcoma in tumor cells (Rh30) Balb/c mice/Colon carcinoma Subcutaneous 150 nM 2.70 J/cm Tumor-rejecting Garg et al., 2012a, (CT26) injection of HY-PDT anticancer vaccination 2015b, 2016 treated cells effect after the re-challenge of cured mice with CT26 cells Fischer 344 rats/Rat bladder Subcutaneous 150 nM 2.70 J/cm Absence of Garg et al., 2015b carcinoma (AY27) injection of HY-PDT tumor-rejecting treated cells anticancer vaccination effect after the re-challenge of cured rats with AY27 cells C57BL/6 mice/Lewis lung Subcutaneous 0.25 μM 1.85 J/cm Tumor-rejecting Zheng et al., 2016 carcinoma (LLC), Dendritic cells injection of HY-PDT anticancer vaccination (DC) co-cultured with PDT-LLCs treated cells effect after the re-challenge of cured mice with LLC-Luc cells AK, actinic keratosis; DLI, drug-light interval; VEGF, vascular endothelial growth factor; –, the parameter was not provided by the authors. between HY-PDT efficacy and tumor volume, similar results smaller tumors (3 mm or less in height), but the main aim of were obtained in other studies. Head et al. (2006) and Cole their study was to compare the impact of intraperitoneal and et al. (2008) observed a regression or reduction in tumor intratumoral hypericin injection on the effectiveness of HY-PDT. size only in tumors smaller than 0.4 or 1 cm , respectively, Both schedules of hypericin administration significantly reduced whereas larger tumors showed only a partial response followed tumor volume and increased the survival rate of animals. by their regrowth (Head et al., 2006), or did not respond to However, considering the complete response, a higher HY- the treatment (Cole et al., 2008). Thus, light penetration into PDT efficacy was observed for hypericin that was administered the tissue appeared to be a limiting factor. However, Cole intraperitoneally (44.4%) compared to intratumorally (33.3%) et al. (2008) also concluded that HY-PDT can be effective (Cavarga et al., 2001). Moreover, it was later demonstrated that in the elimination of small solid tumor residues. Cavarga a better therapeutic response was obtained after fractionated et al. (2001) also determined complete remission only in hypericin administration (two 2.5 mg/kg doses; 6 and 1 h before Frontiers in Plant Science | www.frontiersin.org 6 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin irradiation) than after a single hypericin dose (5 mg/kg; 1 or 6 h HY-PDT using a long DLI and a low fluence rate, which can before irradiation) (Cavarga et al., 2005). reduce the risk of new tumor vasculature formation (Thong et al., Furthermore, Chen et al. (2001) reported the correlation 2006). between hypericin biodistribution, HY-PDT efficacy and various As PDT-mediated tissue damage can lead to various cellular administration–irradiation time intervals (drug–light intervals— and molecular responses, Bhuvaneswari et al. (2008) examined DLIs). It was found that shortly (0.5 h) after the intravenous the potential anti-angiogenic vs. angiogenic properties of short administration of hypericin (5 mg/kg), the photosensitizer was (0.5 h) and long (6 h) DLI HY-PDT. Both HY-PDT scenarios led located preferentially within tumor blood vessels. At 6 h to a reduction in tumor volume, but the effect was much more after injection, maximum intratumoral hypericin content was pronounced for a short DLI. These findings agree with the above- evident and only poor fluorescence was detected in the tumor mentioned results (Chen et al., 2001, 2002a,b) and suggest the vasculature. Despite high tumor hypericin levels, no tumor destruction of the tumor vasculature after short DLI HY-PDT. cure was observed after longer DLI HY-PDT (6 h) treatment. However, in addition to its antitumor activities, cellular-targeted However, the efficacy of PDT was maximal (100% tumor long DLI HY-PDT induced the expression of some angiogenic cure) when irradiation was performed at 0.5 h after hypericin proteins in tumor tissue, including VEGF, tumor necrosis growth administration (short DLI), indicating strong HY-PDT-induced factor-α (TNF-α) and interferon-α (INF-α), which potentially damage to the tumor vasculature (Chen et al., 2001). Similar lead to the formation of new vessels (Bhuvaneswari et al., 2008). results were obtained by Zupkó et al. (2001). In TCC tumors, In subsequent studies, the efficacy of HY-PDT was enhanced the outcome of HY-PDT was highly dependent on DLI (0.5, using monoclonal antibodies against VEGF and the epidermal 6, or 24 h). The strongest effect, which resulted in no tumor growth factor receptor (EGFR) (Bhuvaneswari et al., 2010, 2011). regrowth in some rats, was evident after 0.5 h DLI PDT. At For short DLI HY-PDT, similar results were obtained by Sanovic the same time, the highest hypericin concentration was detected et al. (2011) in mice bearing CT26 colon carcinoma cells. in the plasma, indicating that PDT-mediated tumor vascular Complete tumor regression was observed after “low-power HY- damage was responsible for its antineoplastic action (Zupkó et al., PDT” (a hypericin dose of 2.5 mg/kg, a short DLI of 0.5 h, a 2 2 2001). The antivascular and strong antitumoral effects of short light dose of 14 J/cm and a fluence rate of 27 mW/cm ), as no DLI HY-PDT were also subsequently confirmed in the murine visible or palpable tumors were detected for at least 60 days fibrosarcoma model (Chen et al., 2002a,b). Damage to tumor after the treatment. In contrast, all mice exposed to “high-power vessels was also detected following long DLI HY-PDT treatments, HY-PDT” (a hypericin dose of 10 mg/kg, a short DLI of 1 h, a 2 2 but many viable tumor cells were present, especially at the tumor light dose of 60 J/cm and a fluence rate of 50 mW/cm ) died 2 periphery, indicating that this PDT modality only induced partial days after the treatment as a consequence of internal bleeding. vascular collapse (Chen et al., 2002a). In agreement with these Thus, both HY-PDT modalities with short DLIs appeared results, Bobrov et al. (2007) also observed primary vascular to preferentially target the vessels, but a higher hypericin damage after HY-PDT with either a single dose (5 mg/kg; 1 or concentration and light dose produced a much stronger response. 6 h before irradiation) or fractionated hypericin administration These results suggest that “low-power HY-PDT” is strong enough (two 2.5 mg/kg doses; 6 and 1 h before irradiation), which to completely eliminate tumors by damaging their vasculature. subsequently progressed to tumor tissue necrosis. The preference Moreover, the re-challenge of cured mice with tumorigenic CT26 of HY-PDT-mediated vasculature or cellular damage appears cells did not result in new tumor growth, indicating the HY- to be dependent on the distribution and accumulation of the PDT-mediated induction of the antitumor immune response photosensitizer; thus, greater vascular destruction is expected (Sanovic et al., 2011). HY-PDT-mediated induction of anticancer after short DLI PDT and more direct killing of tumor cells is immunity was also observed in the studies utilizing different expected after long DLI PDT. However, because the damage in vivo experimental models. Immunization of BALB/c mice to tumor vessels can have devastating consequences for whole with “dying or dead” colon carcinoma CT26 cells prevented tumor mass, the targeting of the tumor vasculature via short the tumor growth at the rechallenge site treated with live CT26 DLI PDT might be more effective in the treatment of solid tumor cells. Approximately 70–85% of the mice immunized with tumors than a long DLI PDT. Moreover, apoptosis was the main HY-PDT treated CT26 cells efficiently rejected the formation form of cell death responsible for tumor eradication following of CT26-derived tumors at challenge site (Garg et al., 2012a, short DLI HY-PDT (Chen et al., 2002b). In contrast, Thong 2015b, 2016). Activation of adaptive immune system was also et al. (2006) observed significantly more apoptosis after long detected in immunocompetent C57BL/6 mice immunized with DLI PDT (6 h) compared to short DLI PDT (1 h). However, HY-PDT treated Lewis lung carcinoma (LLC) cells and LLC cells different irradiation conditions and tumor models were used co-cultured with dendritic cells (Zheng et al., 2016). The results in both studies: whereas Chen et al. (2002b) photoactivated of these three independent experimental groups suggest great hypericin with a light dose of 120 J/cm delivered at a fluence potential of HY-PDT in development of anticancer vaccines. rate of 100 mW/cm , a lower fluence rate HY-PDT (light dose Similar results as in the case of partial remission in patients 2 2 of 30 J/cm , fluence rate of 25 mW/cm ) was applied by Thong with nodular BCC (Kacerovská et al., 2008) were obtained in et al. (2006). The results indicate that long DLI PDT might also an in vivo study using hairless mice with UV-induced non- be effective in the induction of programmed cell death, but only melanoma skin tumors (AK, SCC) as an experimental model under low fluence rate conditions. Moreover, lower serum levels (Boiy et al., 2010). Photoactivated hypericin induced a total and of vascular endothelial growth factor (VEGF) were detected after partial response in 44 and 22% of lesions (diameter of 1–2 mm), Frontiers in Plant Science | www.frontiersin.org 7 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin respectively, with evident lesional necrosis and the replacement et al., 2000, 2002). Moreover, the results obtained by confocal of atypical AK cells for normal keratinocytes. However, 33% of microscopy, white-light endoscopy (WLE) and histopathology lesions were non-responsive to HY-PDT. Tumor penetration and revealed that the intensity of hypericin fluorescence increased the selectivity of topically applied hypericin was also limited by with the stage and grade of bladder cancer (normal bladder tissue mouse skin, as the accumulation of hypericin was highest in < inflammation in the bladder < grade 1 TCC < grade 2 TCC the outermost epidermal layer and lower hypericin levels were < CIS < grade 3 TCC) (Olivo et al., 2003b). Therefore, HY-PDD detected in the rest of the epidermis and the dermis (Boiy et al., could be used as a diagnostic aid to the histopathology of bladder 2010). tumors. Some preclinical and clinical results gave relatively Nowadays, bladder lesions are conventionally diagnosed disappointing results and in some cases, the efficacy of HY- under WLE followed by biopsies that are necessary for the PDT was not as high as expected. The above-mentioned studies histological examination of suspicious tissues. However, in contributed to a better understanding of HY-PDT-induced previous studies, some lesions were barely visible or were absent responses and highlighted the importance of optimizing the in white light (D’Hallewin et al., 2000, 2002), thus, WLE appears conditions, such as suitable hypericin administration, light not to be sensitive enough to reveal all CIS lesions and leads dose, fluence rate, or time intervals and implicated HY-PDT to a high risk of missing the tumor. Therefore, another clinical as a promising anticancer therapeutic approach; however, study compared the WLE and HY-PDD methods (Sim et al., more clinically relevant studies and trials are required for the 2005). Hypericin was instilled into the bladder (for 2 h) and implementation of HY-PDT into clinical practice. immediately after WLE, FE (violet light) was used to induce fluorescence emission in the same bladder regions. Despite the comparable specificity of both approaches (91% for HY-PDD, Hypericin in Photodynamic Diagnosis 98% for WLE), HY-PDD was more sensitive (82%) than WLE In addition to their therapeutic abilities, most photosensitizers (62%) (Sim et al., 2005), suggesting that hypericin might be are also potent diagnostic agents. The main principle and very potent in the labeling and early detection of flat superficial relevance of PDD is to enhance the contrast between neoplastic bladder tumors. Similar results were obtained by Kubin et al. and surrounding healthy tissue, which should contribute to the surgical clearance of the whole tumor mass or its small residues. (2008) using polyvinylpyrrolidone (PVP) bound to hypericin as a new water-soluble formula for the improvement of hypericin- Due to the fluorescent properties of hypericin and its specificity for neoplastic tissue, hypericin-mediated PDD (HY-PDD) is mediated bladder cancer detection and diagnosis. Hypericin- PVP was intravesically instilled 1–2 h prior to FE. The overall being tested for various clinical uses, including optical tumor imaging, and the targeting, monitoring or detection of tumor sensitivity of PDD with PVP-hypericin (95%) was significantly higher than WLE (85%). The maximum contrast in sensitivity stages and grades. To date, fluorescence diagnosis using hypericin was evident in the case of CIS (100% for PDD vs. 33% for WLE) has been clinically tested in bladder, head and neck cancers or and dysplasia (85% for PDD vs. 31% for WLE) (Kubin et al., gliomas (Table 3). 2008). HY-PDD in the Detection and Identification of Bladder As the PVP–hypericin complex represents a potent water- Cancer soluble PDD agent without the necessity of binding to serum In most published clinical studies, HY-PDD was applied to proteins, its biodistribution (Vandepitte et al., 2010) and optimal bladder tumors. As already mentioned in the HY-PDT section, dosage and instillation time were evaluated (Straub et al., 2015) Kamuhabwa et al. (2002) (Table 4) revealed selective hypericin in tumor-bearing rats and in patients with bladder cancer, uptake in rat bladder tumors using in situ laser-induced respectively. Vandepitte et al. (2010) (Table 4) demonstrated fluorescence (LIF) and fluorescence microscopy. The results the uniform distribution of instilled PVP–hypericin in all cell suggest that hypericin is very beneficial in visualization and layers of the malignant urothelium, whereas its penetration distinguishing the tumor mass from normal tissue using various into the normal bladder epithelium was very limited. Straub fluorescent techniques. Thus, hypericin could be used not only in et al. (2015) tested various combinations of PVP–hypericin PDT, but also in the PDD of superficial bladder tumors. dosage (75 and 225 μg) and instillation time (15, 30, 60, At about the same time, the first clinical studies were and 120 min) to identify the optimal PDD conditions. Even performed by D’Hallewin et al. (2000, 2002), who examined though the fluorescence of 225 μg PVP-hypericin instilled the fluorescence-based detection of flat bladder carcinomas in for 120 and 60 min was very strong, the shorter instillation situ (CIS) and in papillary non-invasive bladder tumors after time (30 min) for 225 μg PVP–hypericin was evaluated as the intravesical instillation of hypericin (at least 2 h). The optimal. A lower photosensitizer dose (75 μg) and 15 min fluorescence emission was induced using fluorescence endoscopy with a dose of 225 μg were insufficient to detect the lesions (FE) under blue-light illumination. Hypericin accumulated (Straub et al., 2015). The authors established the most suitable selectively in tumor cells and papillary and flat lesions showed dosage and instillation time of PDD with PVP–hypericin, but red fluorescence, whereas no fluorescence was evident in the they suggest a larger phase IIB study should be performed normal bladder tissue. Subsequently, biopsies were taken from to determine the sensitivity and specificity of these optimal fluorescent regions for microscopic analyses. The results of both conditions. clinical studies suggested that HY-PDD has a high sensitivity All the above-mentioned results indicate that HY-PDD and specificity for the detection of bladder cancer (D’Hallewin is highly sensitive in the detection of early bladder cancer Frontiers in Plant Science | www.frontiersin.org 8 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin TABLE 3 | Clinical studies to test HY-PDD efficacy, sensitivity and specificity. No. of patients Hypericin administration Hypericin dose Fluorescence excitation HY-PDD efficacy References BLADDER CANCER 40 Instillation into the bladder 8 μM (40 ml) FE/blue light 93% sensitivity and 98.5% D’Hallewin et al., specificity (for CIS) 2000 87 Instillation into the bladder 8 μM (40 ml) FE/blue light 94% sensitivity and 95% D’Hallewin et al., specificity (for CIS) 2002 30 Instillation into the bladder 8 μM (50 ml) FE/blue light Fluorescence intensity Olivo et al., 2003b increased with the stage and grade of cancer (normal < inflammation < grade 1 TCC < grade 2 TCC < CIS < grade 3 TCC) 41 Instillation into the bladder 8 μM (40 ml) FE/violet light Higher sensitivity (82%) Sim et al., 2005 compared to conventional WLE (62%) 57 Instillation into the bladder 0.25 mg HY + FE/blue light Higher overall sensitivity Kubin et al., 2008 25 mg PVP (50 ml) (95%) compared to conventional WLE (85%), fewer overlooked malignant lesions compared to WLE, sensitivity increased with the grade of cancer 40 Instillation into the bladder 75 or 225 μg FE/blue light very strong fluorescence Straub et al., 2015 PVP-hypericin of 225 μg PVP-hypericin (50 ml) (120 and 60 min), optimal fluorescence of 225 μg PVP-hypericin instilled for 30 min, insufficient fluorescence of 75 μg and 225 μg PVP-hypericin (15 min) 8 Instillation into the bladder 8 μM (40 ml) FM/380–425 nm Ex vivo urine fluorescence Pytel and cytology—fluorescence Schmeller, 2002 detected in all eight tumor cases 29 urine samples Ex vivo staining of sediment extracted Concentration not CFM/488 nm Argon laser Higher fluorescence Olivo et al., 2003a from voided urine given (1 ml) intensity in tumor cells than in cells from normal urine and in high-grade tumors than in low-grade tumors 21 Ex vivo staining of sediment extracted Concentration not CFM/488 nm Argon laser Higher fluorescence Fu et al., 2007 from voided urine given (1 ml) intensity in tumor cells than in cells from normal urine GLIOMA 5 Intravenous injection 0.1 mg/kg NM/blue light Tumor fluorescence Ritz et al., 2012 clearly distinguishable from normal brain tissue, high specificity (100 and 90%) and sensitivity (91 and 94%) HEAD AND NECK CANCER 23 Oral rinsing 8 μM (100 ml) FE/blue light Distinguishing between Thong et al., 2009 various types of oral cancer (red-to-blue ratio), 90% and higher specificity and sensitivity (red-to-blue ratio) (Continued) Frontiers in Plant Science | www.frontiersin.org 9 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin TABLE 3 | Continued No. of patients Hypericin administration Hypericin dose Fluorescence excitation HY-PDD efficacy References 2 Oral rinsing 8 μM (100 ml) CLE/488 nm Argon laser 3-D visualization of human Thong et al., 2012 buccal mucosa at the surface and approximately 15 μm below the surface 27 Ex vivo tissue staining 8 μM pCLE/568 nm laser diode longer time interval for Abbaci et al., 2015 sufficient ex vivo staining (at least 30 min), the anomalies of keratinization not stained CIS, carcinoma in situ; CFM, confocal fluorescence microscopy; CLE, confocal laser endomicroscopy; FE, fluorescence endoscopy; FM, fluorescence microscopy; HY, hypericin; NM, neurosurgical microscopy; pCLE, probe-based CLE; PVP, polyvinylpyrrolidone; TCC, transitional cell carcinoma; WLE, white-light endoscopy. TABLE 4 | Preliminary data for clinical HY-PDD applications and in vivo studies concerning hypericin accumulation. Experimental Hypericin Hypericin dose Fluorescence Hypericin accumulation and References model/Type of tumor administration excitation fluorescence (cell line) Fischer rats/Bladder Instillation into the 8 or 30 μM LIF/410 nm Krypton Intense fluorescence in tumor Kamuhabwa et al., 2002 carcinoma (AY-27) bladder laser FM/525/50 nm tissue and faint fluorescence in normal bladder tissue (ratio 12:1), no fluorescence in submucosa and muscle layers 30 μM FM/510–560 nm Higher accumulation (3.5-fold Vandepitte et al., 2010 PVP-hypericin more) in malignant tissue than in normal urothelium Wistar rats/Glioma (C6) Intravenous 5 mg/kg FM/510–550 nm Higher accumulation in glioma Noell et al., 2011 injection than in normal brain tissue and infiltration zone VMDk mice/Glioma Intravenous 2.5 mg/kg FM/510–550 nm Time-dependent accumulation in Noell et al., 2013 (SMA-560) injection FME/405 nm glioma cells (maximal uptake—6 h after administration), FME—fluorescence detection also in intracerebral and extracranial gliomas and in brain vessels NOD/LtSz-scid IL2Rγnull Intravenous 100 μg/mouse FL/blue light Tumor fluorescence clearly Urla et al., 2015 mice/Rhabdomyosarcoma injection distinguishable from normal (Rh30) healthy tissue CLE, confocal laser endomicroscopy; FL, fluorescence laparoscopy; FM, fluorescence microscopy; FME, fluorescence microendoscopy; LIF, laser-induced fluorescence technique. and could be routinely used as a diagnostic approach. et al., 2007). In the first study conducted by Pytel and Schmeller Moreover, no photobleaching during FE and resection or (2002), voided urine was analyzed in eight patients following side effects were detected (D’Hallewin et al., 2002; Olivo intravesically instilled hypericin (for at least 1 h). Even though et al., 2003b; Sim et al., 2005; Kubin et al., 2008). Kamuhabwa the number of patients was quite low, hypericin fluorescence was et al. (2005) also conclude that either photosensitization detected in all cases of bladder cancer. On the contrary, Olivo or systemic side-effects should not be expected in patients et al. (2003a) and Fu et al. (2007) performed HY-PDD-mediated after intravesical hypericin administration, as the hypericin urine cytology without intravesical instillation of hypericin. In concentration in plasma was below the detection limit both studies, sediments extracted from patient urine samples (<6 nM). were incubated with hypericin in the dark for 15 min and were Another common method used for bladder cancer diagnosis subsequently analyzed using confocal fluorescence microscopy. is ex vivo urine cytology, which microscopically analyzes the The overall fluorescence intensity of the urothelial cells was exfoliated bladder cells from voided urine. This diagnostic significantly higher in urine from early-grade TCC than in technique is non-invasive and less time-consuming than taking normal samples, which enabled the differentiation between biopsy specimens. However, its sensitivity to detect early-stage normal and early bladder cancer specimens (Olivo et al., 2003a). or low-grade cancer is relatively low. Thus, several teams have This finding was later confirmed through a diagnostic algorithm focused on a technique that combines HY-PDD and urine (Fu et al., 2007). Moreover, fluorescence was even higher in cytology (Pytel and Schmeller, 2002; Olivo et al., 2003a; Fu high-grade tumors than in low-grade tumors (Olivo et al., Frontiers in Plant Science | www.frontiersin.org 10 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin 2003a). The results indicate that ex vivo fluorescence cytology and in patients with various types of head and neck cancer using hypericin might be a promising diagnostic method for (Thong et al., 2009, 2012; Abbaci et al., 2015) (Tables 3, 4). the detection and identification of early and low-grade bladder Similarly to bladder cancer, lesions in the oral cavity cancer. are conventionally diagnosed using WLE and histopathology. However, the results obtained by Thong et al. (2009) demonstrate HY-PDD in the Detection of Gliomas the great potential of hypericin for the diagnosis of various It is well-known that malignant gliomas are tumors with a oral cancer types (hyperplasia, cellular pleomorphic adenoma very poor prognosis and their complete resection significantly of the palate, dysplasia, SCC). After oral rinsing with hypericin improves and extends the survival of patients. Thus, in solution (over 30 min), FE was performed and the captured these cases, the enhancement of the contrast between tumor images were analyzed using several parameters. Firstly, the and surrounding healthy tissue would be very beneficial for selective uptake of hypericin in tumor tissue was confirmed. surgeons. Moreover, an increase in the red-to-blue fluorescence intensity In the first in vivo study, Noell et al. (2011) (Table 4) ratio was evaluated from normal tissue (0.3) to hyperplasia investigated the accumulation of hypericin in tumors arising (1.0) to SCC (2.0), which makes this parameter suitable for from intracerebrally implanted C6 glioma cells, in the zones distinguishing between these tissue types with high specificity surrounding the tumors and in healthy brain tissue. Hypericin and sensitivity (over 90%) (Thong et al., 2009). Subsequently, the was injected intravenously and its uptake was maximal 24 h endomicroscopy imaging technique was improved by the same after injection. Considering tissue autofluorescence, the ratios of research team (Thong et al., 2012). Preliminary study already fluorescence intensities were as follows: tumor:infiltration suggested that confocal laser endomicroscopy (CLE) might be zone:normal tissue = 19.8:2.5:1.0. Because hypericin a potent approach for the surface and subsurface imaging of accumulation was significantly higher in the tumor than in oral cavity tissues using various fluorescent dyes in both animal normal tissue, it could be effectively used as a fluorescence and human models (Thong et al., 2007). Recently, a computing marker for glioma detection (Noell et al., 2011). According to system was interfaced to CLE, which enables the 3-D fluorescence these promising preliminary results, the hypericin-mediated visualization of the oral cavity in real-time (Thong et al., 2012). visualization of tumor tissue during its surgical resection This system could be integrated into current techniques for oral was examined in five patients with recurrent glioblastomas cancer diagnosis and might ultimately lead to better clinical (Ritz et al., 2012). Hypericin was injected intravenously 6 h outcomes. prior to the surgical procedure, which was performed using a The fluorescent properties of hypericin and four neurosurgical microscope under switchable white- and blue-light additional fluorescent dyes were also tested for their ability modes. Malignant tumor tissue (red fluorescence) was clearly to characterize normal and cancerous head and neck distinguishable from the healthy brain tissue (blue color) in all tissue; however, the staining procedure was performed patients and the margins of the tumors showed weaker pink ex vivo on fresh samples obtained from head and neck fluorescence. Moreover, specimens were taken for histological surgeries (glossectomy, pharyngolaryngectomy, laryngectomy, evaluation, which was carried out by two neuropathologists and etc.). Hypericin accumulated in the cytoplasm of normal showed 100 and 90% specificity and 91 and 94% sensitivity. and tumor cells, but was the only fluorescent dye that The obtained results suggest that HY-PDD is well-tolerated and did not stain the anomalies of keratinization. Thus, the represents a method that is sufficiently sensitive and specific for authors conclude that hypericin might not be a suitable the intraoperative visualization of malignant gliomas (Ritz et al., photosensitizer for use in such head and neck specimens 2012). (Abbaci et al., 2015). Furthermore, time-dependent hypericin uptake was However, more promising in vivo results were obtained investigated and observed in a subcutaneous glioma mouse for intra-operative HY-PDD of rhabdomyosarcoma. In model using microendoscopy, an approach that is not designed the preclinical study conducted by Urla et al. (2015), for microsurgical tumor resection, but is very useful for mice were injected intraperitoneally with human alveolar applications such as the visualization of different tissue rhabdomyosarcoma cells and 3 weeks later, hypericin was compartments, the identification of vessels or the detection administered intravenously. After 24 h, conventional and of optimal regions for biopsy. To verify the potential to fluorescence laparoscopy were performed and the tumors were detect intracerebral gliomas using microendoscopy, tumor surgically resected using hypericin-mediated red fluorescence cells were also implanted into the brain. After craniotomy, as guidance. Tumor specimens were processed for histological hypericin fluorescence was detected in intracerebral and analyses. Conventional laparoscopy revealed 24 tumors extracranial gliomas and also in the vessels located in the cortical (ranging in size from 1.6 to 13.5 mm) and 28 tumors were surface of the contralateral hemisphere (Noell et al., 2013) detected only by fluorescence laparoscopy (0.5–11 mm). (Table 4). The results indicate that intraoperative HY-PDD is more sensitive than conventional laparoscopy and can clearly HY-PDD in the Detection and Visualization of Other distinguish rhabdomyosarcoma from healthy tissue (Urla Types of Malignancies et al., 2015). Moreover, the authors inform about clinical In addition to the above-mentioned studies, HY-PDD was also trial that will be initiated in children with advanced-stage examined in mice bearing rhabdomyosarcoma (Urla et al., 2015) rhabdomyosarcoma. Frontiers in Plant Science | www.frontiersin.org 11 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin HYPERICIN IN DARK CONDITIONS OR situation after multiple fractionated hypericin dosages or during a long-term investigation might be completely different. THE EFFECTS OF HYPERICIN WITHOUT Firstly, Blank et al. (2001) demonstrated the cytotoxic and LIGHT-ACTIVATION antiproliferative effect of non-activated hypericin both in vitro and in vivo. Hypericin significantly decreased the viability Although hypericin has been extensively studied mainly because HI of highly metastatic murine breast adenocarcinoma (DA3 ) of its photodynamic and photocytotoxic properties, it also and SCC (SQ2) cells. Moreover, the anticancer potential of possesses various positive and negative biological activities HI hypericin was observed even in vivo in DA3 - and SQ2-derived without light-activation. tumors. Even though hypericin slightly accelerated death in mice As some in vitro studies revealed cytotoxic or growth- HI with DA3 -derived tumor development that was very rapid, inhibitory effects of non-activated hypericin (Blank et al., 2001, intraperitoneal hypericin administration (6 declining doses) in 2003; Berlanda et al., 2010; Besic Gyenge et al., 2012) and other animals led to the inhibition of tumor growth, which antiproliferative (Blank et al., 2001) and antimetastatic (Blank was accompanied by prolonged survival time. Moreover, the et al., 2004) activities in vivo and antiangiogenic actions in vitro hypericin-mediated improvement of survival was also evident (Martínez-Poveda et al., 2005) have been described, hypericin in mice with high-grade SCC tumors (Blank et al., 2001). might also show antitumor potential in the absence of light. Subsequently, Blank et al. (2004) examined the antimetastatic Moreover, one clinical study has demonstrated antiglioma potential of non-activated hypericin and evaluated its influence activity of hypericin in dark conditions (Couldwell et al., 2011). on long-term survival (up to 300 days). Again, mice with breast However, the potential use of this natural compound in medicine adenocarcinoma or SCC tumors, which develop metastases might be broader. Regarding its avidity to necrotic tissues, the 123 predominantly in the lungs, were used as experimental models. radiolabeled hypericin derivative ([ I]iodohypericin) can be To evaluate the impact of hypericin only on metastases, primary used for radio-imaging of numerous necrosis-related pathologies tumors were surgically excised at a stage when micrometastases (acute myocardial infarction, liver infarction) (Ni et al., 2006; already existed. Hypericin was administered intraperitoneally in Fonge et al., 2008), including solid tumors (Van de Putte et al., multiple declining dosages (up to 6 doses of hypericin at 5- 2012). day intervals). In both cases of tumor origin, hypericin therapy On the other hand, hypericin can be associated with a together with the resection of primary tumors resulted in a decrease in the chemosensitivity of cancer cells because of significant increase in long-term animal survival compared to its ability to induce the expression of some ATP-binding the untreated control or to those animals that received surgery cassette (ABC) transporters, which are well-known multidrug alone. Moreover, the complete destruction of several, but not resistance (MDR) components (Jendželovský et al., 2009; all lung metastases, was evident 72 h after hypericin treatment. Jendželovská et al., 2014; Kuchárová et al., 2015). In addition, The results indicate that a single hypericin dose was insufficient the hypericin-mediated attenuation of the cytotoxicity of some to prolong the survival of animals, but fractionated hypericin chemotherapeutic agents was demonstrated (Jendželovská et al., doses could prevent animal death when administered shortly 2014). after the resection of the primary tumor (Blank et al., 2004). Similarly, multiple hypericin doses were applied in a previous Non-Activated Hypericin and Its Potential study to obtain positive anticancer therapeutic results (Blank Antitumor Activity et al., 2001). Hypericin-mediated photocytotoxic effects have always been Considering the potential of hypericin to destroy metastases a relevant and attractive issue for researchers in the field of (Blank et al., 2004), the results are consistent with previous in HI oncology; however, the abilities of hypericin in the absence of vitro findings, where a decrease in the viability of DA-3 and light-activation have not been studied as intensively. Although SQ2 cells was also evident 72 h after treatment (Blank et al., 2001). several studies indicate that hypericin might possess some However, no hypericin-mediated induction of apoptosis in the anticancer activities even in dark conditions (Table 5). dark was observed. The inhibition of DNA synthesis indicated It has been found that non-activated hypericin possesses no that the anticancer action of non-activated hypericin in these cytotoxicity toward various cancer cell lines at concentrations cell lines was more cytostatic than cytotoxic (Blank et al., 2001). sufficient for its photocytotoxic action (Thomas and Pardini, Nevertheless, another in vitro experiment conducted by the same 1992; Hadjur et al., 1996; Vandenbogaerde et al., 1997). However, research group established mitotic cell death as the mechanism some in vitro studies have shown that hypericin can act as a of hypericin-mediated cytotoxicity. Hypericin was responsible cytotoxic or antiproliferative agent even in the dark (Blank et al., for the enhanced ubiquitinylation of the Hsp90 chaperone, 2001, 2003; Berlanda et al., 2010; Besic Gyenge et al., 2012). The resulting in the destabilization of its client proteins engaged in presence or absence of these effects often strongly depends on the regulation of cell proliferation, including p53, Cdk4, Plk, the hypericin concentration (higher concentrations are required and Raf-1. Ultimately, cytostasis and a decrease in cell viability than for HY-PDT-mediated toxicity), treatment conditions, the with no apoptosis were observed. Mitotic cell death is generally applied experimental methods, as well as on the type, origin and characterized by cell-cycle arrest in the G2/M phase, increased sensitivity of cancer cells. Moreover, most in vitro studies have cell volume and multinucleation. All these phenotypes were tested the cytotoxicity of non-activated hypericin following its evident in DA3 and SQ2 cells and even in B16.F10 melanoma single dose or during relatively short time intervals; however, the cells after hypericin treatment (Blank et al., 2003). Thus, mitotic Frontiers in Plant Science | www.frontiersin.org 12 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin TABLE 5 | Anticancer effects of hypericin in dark conditions. Experimental model Hypericin doses and administration Effects of non-activated hypericin References IN VITRO STUDIES Murine breast adenocarcinoma 0.065–10 μM (24 h), 0.2–20 μM (72 h), Mild decrease in cell viability detected by MTT Blank et al., 2001 cell line (DA3) 0.6 and 6 μM assay (24 h, SQ2 cells), Significant decrease in Murine anaplastic SCC cell line cell viability detected by Hemacolor assay (SQ2) (72 h), Decrease in DNA synthesis detected by Murine melanoma cell line 3 H-thymidine incorporation (72 h) (B16.F10) 1–40 μM Cytostasis detected by BrdU incorporation Blank et al., 2003 assay (72 h; doses ≤ 10 μM), Reduced cell viability detected by Hemacolor assay (72 h; doses > 10 μM); G2/M cell cycle arrest, formation of enlarged polynucleated cells and no evidence of apoptosis indicating mitotic cell death; enhanced ubiquitinylation of Hsp90 resulting in increased destabilization of its client proteins (p53, Cdk4, Plk, Raf-1) Bovine aorta endothelial cells Range of concentrations, 5, 10 or 20 μM Inhibition of some key steps of angiogenesis Martínez-Poveda (BAE) (decrease in urokinase extracellular level; et al., 2005 inhibition of: endothelial cells proliferation, endothelial tube formation, migration and invasive capability of endothelial cells) Human epidermoid carcinoma Range of concentrations (up to 100 μM) Antiproliferative and/or cytotoxic effect Berlanda et al., 2010 cell line (A431) detected by MTT assay (concentrations higher than 3.13 μM) Human head and neck SCC 0.6–10 μg/ml Antiproliferative effect detected by BrdU cell Besic Gyenge et al., carcinoma cell lines (UMB-SCC proliferation assay (all applied concentrations), 2012 745, UMB-SCC 969) no influence on RNA integrity, initial DNA damage (recovered after 3 h) IN VIVO STUDIES Balb/c mice/Murine breast 200, 100 and 50 μM; intraperitoneal Reduced volume of DA3-derived tumors (66% Blank et al., 2001 carcinoma (DA3) injection at 20 days after beginning of treatment), Murine SCC (SQ2) prolonged survival time (both DA3 and SQ2 models) 5, 2.5, and 1.25 mg/kg, 10 mg/kg; Increase in long-term (300 days) animal survival Blank et al., 2004 intraperitoneal injection (together with surgery), complete destruction of several DA3-derived metastatic foci in lungs (10 mg/kg, 72 h) CLINICAL STUDIES Glioblastoma (35 patients) 0.05–0.50 mg/kg; oral administration Stabilization or slight reduction of tumor volume Couldwell et al., 2011 Anaplastic astrocytoma (7 of 42 patients = 17%), partial clinical (7 patients) response (> 50% tumor reduction; 2 of 42 patients = 2%), mild adverse effects (photosensitivity, erythema, vomiting, diarrhea, etc.) BrdU, 5-bromo-2 -deoxyuridine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SCC, squamous cell carcinoma. cell death might participate in the tumoricidal and antimetastatic of the patients to this treatment. Hypericin was administered functions of hypericin in the absence of light. orally at gradually increasing dosages ranging from 0.05 to Martínez-Poveda et al. (2005) described another mechanism 0.5 mg/kg (once each morning for up to 3 months) and was that might be implicated in the anticancer effects of hypericin well-tolerated (mean maximum tolerated daily dose of 0.40 ± in the dark. The results of several in vitro assays indicated that 0.098 mg/kg), although some mild skin or gastrointestinal side hypericin can inhibit several key steps of angiogenesis, including effects were observed. More importantly, hypericin stabilized or the proliferation, migration and invasion of endothelial cells, slightly reduced tumor volume (in seven out of 42 patients). In extracellular matrix-degrading urokinase or tubular formation addition, a partial response (>50% reduction of tumor volume) on Matrigel (Martínez-Poveda et al., 2005). All these effects might was observed in two patients (Couldwell et al., 2011). These be beneficial in the prevention of tumor neovascularization. results indicate that synthetic orally administered hypericin To our knowledge, one clinical trial has investigated the can be moderately effective as an adjuvant therapy in cases impact of hypericin on recurrent malignant gliomas (anaplastic of malignant glioma; however, further clinical studies are astrocytoma and glioblastoma) and has monitored the tolerance required. Frontiers in Plant Science | www.frontiersin.org 13 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin Hypericin as a Necrosis-Avid Agent in viable tumor tissue (up to 24 h) (Van de Putte et al., 2012), its radiolabeled derivatives could be also used for so-called Oncology tumor necrosis therapy (TNT). This therapy is based on the In addition to its preferential accumulation in tumors compared destruction of adjacent viable tumor cells by the deposition to in normal healthy tissue, hypericin also has a specific strong and accumulation of radiation energy. In other words, attached affinity toward necrotic tissue (Van de Putte et al., 2008a,b,c). radioactive iodine “bombards” the neighboring living tumor cells The mechanisms of this phenomenon have not yet been fully with radiation. Each successive treatment kills more tumor cells, elucidated; although some hypotheses already exist that consider thus, increasing the necrotic region, which allows higher efficacy the binding of hypericin to specific constituents in the necrotic with each treatment. As three injections of [ I]iodohypericin space. Several radiolabeled hypericin derivatives, particularly 123 131 reduced the volume of RIF-1-derived tumors, this radiolabeled [ I]iodohypericin (Fonge et al., 2007) and [ I]iodohypericin derivate might have potential in TNT (Van de Putte et al., 2012). (Li et al., 2011), possess similar necrosis avidity; thus, hypericin In addition, similar results, especially the intense retention of can be used as a potent necrosis-avid contrast agent (NACA) [ I]iodohypericin in necrotic tumor tissue (over 168 h) and for the non-invasive detection and imaging of various necrosis- inhibited tumor growth after a single dose, were obtained by Liu related pathologies and diseases or to assess tissue viability et al. (2015) in mice bearing hepatomas or sarcomas. and therapeutic responses. Moreover, iodohypericins as NACAs Moreover, the approach of a necrosis-based anticancer might also be effective in relatively new approaches that combine treatment has been expanded into a dual-targeting theranostic both tumor diagnosis and therapy, so-called “theranostic” strategy by administering the vascular-disrupting agent prior modalities (Table 6). to the hypericin iododerivate. Firstly, a vascular-disrupting In a few initial in vivo studies, hypericin was investigated agent, such as combretastatin A4 phosphate (CA4P), targets as a potential indicator of therapeutic responses, following the tumor microenvironment and subsequently, iodine various necrosis-inducing anticancer treatments. Mice bearing radioactivity kills residual cancer cells. Several preclinical intrahepatic fibrosarcoma tumors were used as an experimental studies have demonstrated that dual-targeting using CA4P model and hypericin was injected intravenously 1 h before, with [ I]iodohypericin was much more effective than single or 24 h after intratumoral ethanol injection (Van de Putte treatments. A reduced tumor volume, a prolonged tumor et al., 2008b) or radiofrequency ablation (Van de Putte et al., doubling time, an increase in radiation-induced cell death and 2008c), which both induced tumor necrosis. Fluoromacroscopic intratumoral necrosis or prolonged survival time were reported and fluoromicroscopic examinations confirmed that hypericin in rats bearing liver rhabdomyosarcomas (Li et al., 2011, 2012), accumulated preferentially in necrotic tissue. In the cases of in mice bearing fibrosarcomas (Li et al., 2013) and in rabbits with necrosis, mean fluorescence densities were about 4.5- and 5- liver and muscular VX2 tumors (Shao et al., 2015). Thus, the fold higher than in viable tumor tissue and 14- and 12-fold dual-targeting theranostic approach appears to be well tolerated higher than in normal liver tissue. These results demonstrate and can enhance the therapeutic response, encouraging further the ability of hypericin to enhance the imaging contrast between development for other preclinical and even clinical applications. necrotic and viable tissues and ultimately, its potential role in the early assessment of the therapeutic response (Van Non-Activated Hypericin and Its Potential de Putte et al., 2008b,c). At about the same time, tumor uptake of radiolabeled hypericin (mono-[ I]iodohypericin) Negative Impact on Cancer Treatment and protohypericin (mono-[ I]iodoprotohypericin) derivatives All the above-mentioned preclinical and clinical results suggest were tested and compared, because of the applicability of that hypericin offers great potential in tumor diagnosis as well tomographic imaging techniques instead of fluorescence-based as in anticancer therapy. However, this secondary metabolite techniques. Radioactivity was measured using a gamma counter. might also cause some other effects that would not be beneficial Both radiolabeled compounds were retained by the tumors, for therapeutic outcomes. It is well-known that the efficacy of but mono-[ I]iodohypericin appeared to be a more suitable commonly used anticancer treatment modalities is often limited tumor diagnostic agent, due to its faster clearance from healthy by intrinsic or acquired MDR—a multifactorial phenomenon of organs (Fonge et al., 2007). Another radiolabeled derivate, the increased tolerance of cancer cells to various tumoricidal Cu-bis-DOTA-hypericin, was also applicable in the early agents. A number of cellular mechanisms can contribute to determination of the therapeutic response as its accumulation MDR (reviewed in Stavrovskaya, 2000; Zahreddine and Borden, was significantly lower in non-treated tumors than in those 2013), including the increased elimination of anticancer drugs treated by photothermal ablation therapy, inducing necrosis by tumor cells, which is mostly linked to the elevated expression (Song et al., 2011). and/or activity of several ABC transporters. It has been shown As necrotic tissue represents 30–80% of the solid tumor that non-activated hypericin can modulate some of these mass and is rarely present in normal healthy tissue and organs, efflux pumps. In vitro experiments conducted by our research it is a suitable target not only for cancer diagnosis, but also group (Jendželovský et al., 2009; Jendželovská et al., 2014; for anticancer therapy. The strong avidity of iodohypericins Kuchárová et al., 2015) have revealed an increased expression for necrotic tissue makes these compounds very potent in the of multidrug resistance-associated protein 1 (MRP1) and breast imaging of tumor necrosis. Furthermore, as hypericin can persist cancer resistance protein (BCRP) in colorectal HT-29 cells or in in necrotic tumor areas much longer (up to 72 h) than in ovarian A2780 and A2780cis cells following hypericin treatment Frontiers in Plant Science | www.frontiersin.org 14 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin TABLE 6 | Preclinical in vivo studies to test hypericin as a necrosis-avid agent. Experimental model/Type Hypericin derivate Detection method Effects References of tumor (cell line) C3H mice/Fibrosarcoma [ I]MIH Gamma counter Retention by the tumors and rapid Fonge et al., (RIF-1) clearance from healthy organs (faster 2007 clearance than [ I]MIprotoH) C3H/Km mice/Fibrosarcoma hypericin FM imaging Accumulation in intratumoral necrosis (4 h Van de Putte (RIF-1) after administration) et al., 2008a UV and Tungsten light, FM Preferential accumulation in intratumoral Van de Putte imaging necrosis (intratumoral necrosis > viable et al., 2008b,c tumor > normal liver tissue), enhanced contrast between necrotic and viable tissue = early assessment of therapeutic response (diagnosis) Nude mice/Mammary cancer CuBDH PET, autoradiography Higher accumulation in treated than in Song et al., 2011 (BT474) non-treated tumors = assessment of therapeutic response (diagnosis) Balb/c mice/Fibrosarcoma hypericin, [ I]MIH, FM, PET, autoradiography, Longer persistence of tracers in necrotic Van de Putte (RIF-1) [ I]MIH scintigraphy than in viable tumor, stabilization of tumor et al., 2012 growth and reduced tumor volume (3 injections of [ I]MIH) = potential in TNT WAG/Rij [ I]MIH MRI, CT scan, scintigraphy, Gamma Reduced tumor volume, prolonged tumor Li et al., 2011 rats/Rhabdomyosarcoma (R1) counter, autoradiography doubling time and inhibited tumor regrowth (dual-targeting with CA4P) 123 131 [ I]MIH, [ I]MIH Accumulation in intratumoral necrosis Li et al., 2012 ([ I]MIH); reduced tumor volume, prolonged tumor doubling time and increased intratumoral necrosis = tumoricidal effect (dual-targeting - [ I]MIH with CA4P) SCID mice/Fibrosarcoma [ I]MIH MRI, scintiscan, autoradiography Accumulation in intratumoral necrosis (over Li et al., 2013 (RIF-1) 120 h); prolonged survival time, marked radiation-induced cell death, reduced tumor volume, prolonged tumor doubling time (dual-targeting with CA4P) New Zealand white [ I]MIH MRI, SPECT, autoradiography High targetability to tumor necrosis; Shao et al., 2015 rabbits/VX2 tumors reduced tumor growth and prolonged tumor doubling time (dual-targeting with CA4P) Kunming (KM) [ I]MIH FM, SPECT, autoradiography Prolonged retention by the tumors, limited Liu et al., 2015 mice/Hepatoma (H22) systemic toxicity, tumor growth delay = Sarcoma (S180) therapeutic efficacy 123 123 123 123 131 131 64 64 [ I]MIH, mono-[ I]iodohypericin; [ I]MIprotoH, mono-[ I]iodoprotohypericin; [ I]MIH, mono-[ I]iodohypericin; CuBDH, Cu-bis-DOTA-hypericin; CA4P, combretastatin A4 phosphate; CT, computed tomography; FM, fluorescence microscopy; MRI, magnetic resonance imaging; PET, positron emission tomography; SPECT, single-photon emission computed tomography; TNT, tumor necrosis therapy. in the dark. In A2780 and A2780cis cells, 0.5 μM hypericin Wada et al. (2002) evaluated the impact of hypericin on the elevated MRP1 protein levels already 6 h after the treatment action of several anticancer drugs, using the cervical HeLa cell (Jendželovská et al., 2014). For HT-29 cells, an even lower line and its resistant subline Hvr100-6 that overexpress another hypericin concentration (0.1 μM) was sufficient to increase MDR-related ABC transporter, P-glycoprotein (P-gp), but no MRP1 and BCRP expression (16 h after hypericin addition) effect was observed. Several studies suggest that hypericin can (Jendželovský et al., 2009; Kuchárová et al., 2015). Therefore, neither modulate P-gp expression nor its activity (Wada et al., because many chemotherapeutic agents and photosensitizers 2002; Tian et al., 2005; Jendželovský et al., 2009), which explains are substrates of the above-mentioned transporters (reviewed its poor ability to influence the cytotoxicity and transport of in Nies et al., 2007; Robey et al., 2007), the hypericin- P-gp substrates, such as paclitaxel, daunorubicin, doxorubicin mediated stimulation of efflux systems might lead to a decrease or vinblastin (Wada et al., 2002). On the other hand, 24 in the efficacy of these therapeutic approaches when they h pre-treatment with hypericin resulted in the attenuation are applied at the same time or shortly following hypericin of mitoxantrone cytotoxicity in HL-60 cells and cisplatin treatment. cytotoxicity in sensitive A2780 and resistant A2780cis cells Frontiers in Plant Science | www.frontiersin.org 15 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin (Jendželovská et al., 2014). However, this effect was probably SUMMARY not caused by modulation of the analyzed ABC transporters. In this review, we have summarized the medicinal applications However, the results suggest that hypericin in the dark might of light-activated and non-activated hypericin in the field of have a negative impact on the onset or progress of cell death oncology. We have preferentially highlighted the “primary induced by some anticancer agents, possibly by affecting some beneficial side” of this secondary metabolite concerning its other mechanisms. Further studies are required, to elucidate anticancer potential, but have also outlined its “second non- the specific mechanisms responsible for the above-mentioned beneficial side,” which was revealed by preliminary in vitro changes and in vivo studies will be necessary to verify the impact studies. of non-activated hypericin on the outcome of chemotherapy. The vast majority of the results summarized here suggest Moreover, some MDR mechanisms, including ABC that hypericin in light and dark conditions might be a very transporters, are also involved in drug pharmacokinetics. potent agent in cancer treatment and diagnosis. Besides the The modulation of these mechanisms can affect the absorption, well-known and intensively investigated HY-PDT and HY-PDD, distribution or clearance and ultimately, the action of the some new and promising approaches using hypericin as NACA administered xenobiotics, resulting in negative drug interactions. are becoming the focus of various research groups. Moreover, Several clinical trials have demonstrated the interactions some tested modalities, including dual-targeting, might even between some chemotherapeutic agents and St. John’s wort improve the clinical outcome of cancer treatments. However, extract, which is often taken by oncological patients as an in the dark, hypericin might be responsible for the limited antidepressant. In the first study, the enhanced metabolism efficacy of conventionally applied chemotherapy or even PDT, of irinotecan and consequently, decreased plasma levels of its due to its ability to induce the expression of some ABC active metabolite (SN-38) were evaluated in cancer patients transporters. Moreover, there is a suspicion that non-activated following St. John’s wort treatment (Mathijssen et al., 2002). hypericin possesses much broader biological activity. Thus, the Furthermore, Frye et al. (2004) and Smith et al. (2004) examined chronic usage of St. John’s wort extracts as an antidepressant by the effect of St. John’s wort extract on the pharmacokinetics oncological patients undergoing anticancer treatment should be of imatinib mesylate in healthy adult volunteers. In both avoided. studies, imatinib was administered before and after the treatment with St. John’s wort (300 mg three times daily AUTHOR CONTRIBUTIONS for 2 weeks) and its clearance and half-life was significantly increased or decreased, respectively, by the herb extract. Similar ZJ designed the concept and issue of the review, studied the results were obtained by Goey et al. (2014) using a similar literature, contributed to all chapters and summarized the bulk of experimental design. Besides the enhanced clearance and the text, revised the text after it was completed and also following decreased plasma concentrations of docetaxel in cancer patients, English revision and approved the final version. RJ discussed the St. John’s wort lowered the incidence of docetaxel-mediated concept of the review with first author, studied the literature and toxicities. Thus, due to the risk of potential undertreatment, contributed to the chapters about HY-PDT and non-activated combining anticancer therapeutic approaches with St. John’s hypericin, revised the text and approved the final version. BK wort extracts should not be recommended to oncological studied the literature and contributed to the chapters about HY- patients. PDT and non-activated hypericin, revised the text and approved The probable reason for these effects is the induction of the the final version. PF discussed the concept of the review with first metabolic enzyme (CYP3A4) and/or the P-gp transporter by author, revised the text and approved the final version. hyperforin (Komoroski et al., 2004; Tian et al., 2005), another St. John’s wort metabolite. However, considering the potential of hypericin to induce the expression of some ABC efflux pumps, ACKNOWLEDGMENTS this secondary metabolite might also contribute to negative drug interactions with St. John’s wort. Therefore, a much broader This work was supported by the Slovak Research and spectrum of antineoplastic drugs might exist, including various Development Agency under contract No. APVV-14-0154 and chemotherapeutic agents or photosensitizers, whose action might the Scientific Grant Agency of the Ministry of Education of the be altered due to the presence of hypericin. Slovak Republic under contract No. VEGA 1/0147/15. REFERENCES Agostinis, P., Vantieghem, A., Merlevede, W., and de Witte, P. A. (2002). Hypericin in cancer treatment: more light on the way. Int. J. Biochem. Cell Biol. 34, Abbaci, M., Casiraghi, O., Temam, S., Ferchiou, M., Bosq, J., Dartigues, P., et al. 221–241. doi: 10.1016/S1357-2725(01)00126-1 (2015). Red and far-red fluorescent dyes for the characterization of head Alecu, M., Ursaciuc, C., Hãlãlãu, F., Coman, G., Merlevede, W., Waelkens, E., and neck cancer at the cellular level. J. Oral Pathol. Med. 44, 831–841. doi: et al. (1998). Photodynamic treatment of basal cell carcinoma and squamous 10.1111/jop.12316 cell carcinoma with hypericin. Anticancer Res. 18, 4651–4654. Agostinis, P., Berg, K., Cengel, K. A., Foster, T. H., Girotti, A. W., Gollnick, S. O., Ali, S. M., and Olivo, M. (2002). Bio-distribution and subcellular localization of et al. (2011). Photodynamic therapy of cancer: an update. CA Cancer J. Clin. 61, Hypericin and its role in PDT induced apoptosis in cancer cells. Int. J. Oncol. 250–281. doi: 10.3322/caac.20114 21, 531–540. doi: 10.3892/ijo.21.3.531 Frontiers in Plant Science | www.frontiersin.org 16 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin Ayan, A. K., Cirak, C., Kevseroglu, K., and Ozen, T. (2004). Hypericin in Cellárová, E., Brutovská, R., Daxnerová, Z., Brunˇáková, K., and Weigel, some Hypericum species from Turkey. Asian J. Plant Sci. 3, 200–202. doi: R. C. (1997). Correlation between hypericin content and the ploidy of 10.3923/ajps.2004.200.202 somaclones of Hypericum perforatum L. Acta Biotechnol. 17, 83–90. doi: Berlanda, J., Kiesslich, T., Engelhardt, V., Krammer, B., and Plaetzer, K. (2010). 10.1002/abio.370170111 Comparative in vitro study on the characteristics of different photosensitizers Chen, B., and de Witte, P. A. (2000). Photodynamic therapy efficacy and tissue employed in PDT. J. Photochem. Photobiol. B. Biol. 100, 173–180. doi: distribution of hypericin in a mouse P388 lymphoma tumor model. Cancer Lett. 10.1016/j.jphotobiol.2010.06.004 150, 111–117. doi: 10.1016/S0304-3835(99)00381-X Besic Gyenge, E., Forny, P., Lüscher, D., Laass, A., Walt, H., and Maake, C. (2012). Chen, B., Roskams, T., and de Witte, P. A. (2002a). Antivascular tumor eradication Effects of hypericin and a chlorin based photosensitizer alone or in combination by hypericin-mediated photodynamic therapy. Photochem. Photobiol. 76, in squamous cell carcinoma cells in the dark. Photodiagnosis Photodyn. Ther. 9, 509–513. doi: 10.1562/0031-8655(2002)0760509ATEBHM2.0.CO2 321–331. doi: 10.1016/j.pdpdt.2012.03.006 Chen, B., Roskams, T., Xu, Y., Agostinis, P., and de Witte, P. A. (2002b). Bhuvaneswari, R., Gan, Y. Y., Lucky, S. S., Chin, W. W., Ali, S. M., Soo, K. Photodynamic therapy with hypericin induces vascular damage and apoptosis C., et al. (2008). Molecular profiling of angiogenesis in hypericin mediated in the RIF-1 mouse tumor model. Int. J. Cancer. 98, 284–290. doi: photodynamic therapy. Mol. Cancer. 7:56. doi: 10.1186/1476-4598-7-56 10.1002/ijc.10175 Bhuvaneswari, R., Thong, P. S., Gan, Y. Y., Soo, K. C., and Olivo, M. (2010). Chen, B., Xu, Y., Roskams, T., Delaey, E., Agostinis, P., Vandenheede, J. R., Evaluation of hypericin-mediated photodynamic therapy in combination et al. (2001). Efficacy of antitumoral photodynamic therapy with hypericin: with angiogenesis inhibitor bevacizumab using in vivo fluorescence confocal relationship between biodistribution and photodynamic effects in the RIF-1 endomicroscopy. J. Biomed. Opt. 15:011114. doi: 10.1117/1.3281671 mouse tumor model. Int. J. Cancer. 93, 275–282. doi: 10.1002/ijc.1324 Bhuvaneswari, R., Yuen, G. Y., Chee, S. K., and Olivo, M. (2011). Antiangiogenesis Cole, C. D., Liu, J. K., Sheng, X., Chin, S. S., Schmidt, M. H., Weiss, M. H., agents avastin and erbitux enhance the efficacy of photodynamic therapy et al. (2008). Hypericin-mediated photodynamic therapy of pituitary tumors: in a murine bladder tumor model. Lasers Surg. Med. 43, 651–662. doi: preclinical study in a GH4C1 rat tumor model. J. Neurooncol. 87, 255–261. doi: 10.1002/lsm.21109 10.1007/s11060-007-9514-0 Blank, M., Kostenich, G., Lavie, G., Kimel, S., Keisari, Y., and Orenstein, A. (2002). Couldwell, W. T., Surnock, A. A., Tobia, A. J., Cabana, B. E., Stillerman, C. Wavelength-dependent properties of photodynamic therapy using hypericin B., Forsyth, P. A., et al. (2011). A phase 1/2 study of orally administered in vitro and in an animal model. Photochem. Photobiol. 76, 335–340. doi: synthetic hypericin for treatment of recurrent malignant gliomas. Cancer 117, 10.1562/0031-8655(2002)0760335WDPOPT2.0.CO2 4905–4915. doi: 10.1002/cncr.26123 Blank, M., Lavie, G., Mandel, M., Hazan, S., Orenstein, A., Meruelo, D., et al. Crnolatac, I., Huygens, A., van Aerschot, A., Busson, R., Rozenski, J., and de (2004). Antimetastatic activity of the photodynamic agent hypericin in the dark. Witte, P. A. (2005). Synthesis, in vitro cellular uptake and photo-induced Int. J. Cancer. 111, 596–603. doi: 10.1002/ijc.20285 antiproliferative effects of lipophilic hypericin acid derivatives. Bioorg. Med. Blank, M., Mandel, M., Hazan, S., Keisari, Y., and Lavie, G. (2001). Anti-cancer Chem. 13, 6347–6353. doi: 10.1016/j.bmc.2005.09.003 activities of hypericin in the dark. Photochem. Photobiol. 74, 120–125. doi: Delaey, E., Vandenbogaerde, A., Merlevede, W., and de Witte, P. (2000). 10.1562/0031-8655(2001)0740120ACAOHI2.0.CO2 Photocytotoxicity of hypericin in normoxic and hypoxic conditions. J. Blank, M., Mandel, M., Keisari, Y., Meruelo, D., and Lavie, G. (2003). Enhanced Photochem. Photobiol. B. Biol. 56, 19–24. doi: 10.1016/S1011-1344(00)00051-8 ubiquitinylation of heat shock protein 90 as a potential mechanism for mitotic Dewick, P. M. (2002). Medicinal Natural Products: A Biosynthetic Approach, 2nd cell death in cancer cells induced with hypericin. Cancer Res. 63, 8241–8247. Edn. Chichester: John Wiley & Sons Ltd. Bobrov, N., Cavarga, I., Longauer, F., Rybárová, S., Fedorocko, P., Brezáni, D’Hallewin, M. A., De Witte, P. A., Waelkens, E., Merlevede, W., and Baert, P., et al. (2007). Histomorphological changes in murine fibrosarcoma after L. (2000). Fluorescence detection of flat bladder carcinoma in situ after hypericin-based photodynamic therapy. Phytomedicine 14, 172–178. doi: intravesical instillation of hypericin. J. Urol. 164, 349–351. doi: 10.1016/S0022- 10.1016/j.phymed.2006.09.017 5347(05)67357-0 Boiy, A., Roelandts, R., and de Witte, P. A. (2010). Photodynamic therapy using D’Hallewin, M. A., Kamuhabwa, A. R., Roskams, T., De Witte, P. A., and Baert, L. topically applied hypericin: comparative effect with methyl-aminolevulinic acid (2002). Hypericin-based fluorescence diagnosis of bladder carcinoma. BJU Int. on UV induced skin tumours. J. Photochem. Photobiol. B. Biol. 102, 123–131. 89, 760–763. doi: 10.1046/j.1464-410X.2002.02690.x doi: 10.1016/j.jphotobiol.2010.09.012 Diwu, Z., and Lown, J. W. (1993). Photosensitization with anticancer agents. 17. Brockmann, H., Haschad, M. N., Maier, K., and Pohl, F. (1939). Über EPR studies of photodynamic action of hypericin: formation of semiquinone das Hypericin, den photodynamisch wirksamen Farbstoff aus Hypericum radical and activated oxygen species on illumination. Free Radic. Biol. Med. 14, perforatum. Naturwissenschaften 27, 550. doi: 10.1007/BF01495453 209–215. doi: 10.1016/0891-5849(93)90012-J Brunˇáková, K., Petijová, L., Zámecˇník, J., Turecˇková, V., and Cellárová, E. (2015). Du, H. Y., Bay, B. H., and Olivo, M. (2003a). Biodistribution and photodynamic The role of ABA in the freezing injury avoidance in two Hypericum species therapy with hypericin in a human NPC murine tumor model. Int. J. Oncol. 22, differing in frost tolerance and potential to synthesize hypericins. Plant Cell 1019–1024. doi: 10.3892/ijo.22.5.1019 Tissue Organ Cult. 122, 45–56. doi: 10.1007/s11240-015-0748-9 Du, H. Y., Olivo, M., Tan, B. K., and Bay, B. H. (2003b). Hypericin- Buytaert, E., Callewaert, G., Hendrickx, N., Scorrano, L., Hartmann, D., Missiaen, mediated photodynamic therapy induces lipid peroxidation and necrosis L., et al. (2006). Role of endoplasmic reticulum depletion and multidomain in nasopharyngeal cancer. Int. J. Oncol. 23, 1401–1405. doi: 10.3892/ijo.23. proapoptotic BAX and BAK proteins in shaping cell death after hypericin- 5.1401 mediated photodynamic therapy. FASEB J. 20, 756–758. doi: 10.1096/fj.05- Feruszová, J., Imreová, P., Bodnárová, K., Ševcˇovicˇová, A., Kyzek, S., Chalupa, I., 4305fje et al. (2016). Photoactivated hypericin is not genotoxic. Gen. Physiol. Biophys. Cavarga, I., Brezáni, P., Cekanová-Figurová, M., Solár, P., and Fedorocko, P., 35, 223–230. doi: 10.4149/gpb_2015045 Miskovský, P. (2001). Photodynamic therapy of murine fibrosarcoma with Fonge, H., Van de Putte, M., Huyghe, D., Bormans, G., Ni, Y., de Witte, P., et al. topical and systemic administration of hypericin. Phytomedicine 8, 325–330. (2007). Evaluation of tumor affinity of mono-[(123)I]iodohypericin and mono- doi: 10.1078/0944-7113-00057 [(123)I]iodoprotohypericin in a mouse model with a RIF-1 tumor. Contrast Cavarga, I., Brezáni, P., Fedorocko, P., Miskovský, P., and Bobrov, N., Media Mol. Imaging. 2, 113–119. doi: 10.1002/cmmi.136 Longauer, F., et al. (2005). Photoinduced antitumour effect of hypericin Fonge, H., Vunckx, K., Wang, H., Feng, Y., Mortelmans, L., Nuyts, J., et al. (2008). can be enhanced by fractionated dosing. Phytomedicine 12, 680–683. doi: Non-invasive detection and quantification of acute myocardial infarction 10.1016/j.phymed.2004.02.011 in rabbits using mono-[123I]iodohypericin microSPECT. Eur. Heart J. 29, Cellárová, E. (2011). “Effect of exogenous morphogenetic signals on differentiation 260–269. doi: 10.1093/eurheartj/ehm588 in vitro and secondary metabolite formation in the genus Hypericum,” in Frye, R. F., Fitzgerald, S. M., Lagattuta, T. F., Hruska, M. W., and Egorin, M. J. Medicinal and Aromatic Plant Science and Biotechnology 5 (Special Issue 1), eds (2004). Effect of St John’s wort on imatinib mesylate pharmacokinetics. Clin. M. S. Odabas and C. Çırak (Ikenobe: Global Science Books), 62–69. Pharmacol. Ther. 76, 323–329. doi: 10.1016/j.clpt.2004.06.007 Frontiers in Plant Science | www.frontiersin.org 17 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin Fu, C. Y., Ng, B. K., Razul, S. G., Chin, W. W., Tan, P. H., Lau, W. K., et al. (2007). Antimicrob. Agents Chemother. 45, 517–524. doi: 10.1128/AAC.45.2.517- Fluorescence detection of bladder cancer using urine cytology. Int. J. Oncol. 31, 524.2001 525–530. doi: 10.3892/ijo.31.3.525 Jendželovská, Z., Jendželovský, R., Hil’ovská, L., Koval’, J., Mikeš, J., and Galanou, M. C., Theodossiou, T. A., Tsiourvas, D., Sideratou, Z., and Fedorocˇko, P. (2014). Single pre-treatment with hypericin, a St. John’s wort Paleos, C. M. (2008). Interactive transport, subcellular relocation and secondary metabolite, attenuates cisplatin- and mitoxantrone-induced cell enhanced phototoxicity of hypericin encapsulated in guanidinylated liposomes death in A2780, A2780cis and HL-60 cells. Toxicol. In vitro 28, 1259–1273. doi: via molecular recognition. Photochem. Photobiol. 84, 1073–1083. doi: 10.1016/j.tiv.2014.06.011 10.1111/j.1751-1097.2008.00392.x Jendželovský, R., Mikes, J., Koval,’, J., Soucek, K., Procházková, J., Kello, M., Galluzzi, L., Vacchelli, E., Bravo-San Pedro, J. M., Buqué, A., Senovilla, L., Baracco, et al. (2009). Drug efflux transporters, MRP1 and BCRP, affect the outcome E. E., et al. (2014). Classification of current anticancer immunotherapies. of hypericin-mediated photodynamic therapy in HT-29 adenocarcinoma Oncotarget 5, 12472–12508. doi: 10.18632/oncotarget.2998 cells. Photochem. Photobiol. Sci. 8, 1716–1723. doi: 10.1039/b9pp0 García, I., Ballesta, S., Gilaberte, Y., Rezusta, A., and Pascual, Á. (2015). 0086k Antimicrobial photodynamic activity of hypericin against methicillin- Kacerovská, D., Pizinger, K., Majer, F., and Smíd, F. (2008). Photodynamic susceptible and resistant Staphylococcus aureus biofilms. Future Microbiol. 10, therapy of nonmelanoma skin cancer with topical Hypericum perforatum 347–356. doi: 10.2217/fmb.14.114 extract–a pilot study. Photochem. Photobiol. 84, 779–785. doi: 10.1111/j.1751- Garg, A. D., and Agostinis, P. (2014). ER stress, autophagy and immunogenic 1097.2007.00260.x cell death in photodynamic therapy-induced anti-cancer immune responses. Kamuhabwa, A. A., Cosserat-Gerardin, I., Didelon, J., Notter, D., Guillemin, Photochem. Photobiol. Sci. 13, 474–487. doi: 10.1039/c3pp50333j F., Roskams, T., et al. (2002). Biodistribution of hypericin in orthotopic Garg, A. D., Elsen, S., Krysko, D. V., Vandenabeele, P., de Witte, P., and Agostinis, transitional cell carcinoma bladder tumors: implication for whole bladder wall P. (2015b). Resistance to anticancer vaccination effect is controlled by a cancer photodynamic therapy. Int. J. Cancer. 97, 253–260. doi: 10.1002/ijc.1594 cell-autonomous phenotype that disrupts immunogenic phagocytic removal. Kamuhabwa, A. A., Di Mavungu, J. D., Baert, L., D’Hallewin, M. A., Hoogmartens, Oncotarget 6, 26841–26860. doi: 10.18632/oncotarget.4754 J., and de Witte, P. A. (2005). Determination of hypericin in human plasma Garg, A. D., Galluzzi, L., Apetoh, L., Baert, T., Birge, R. B., Bravo-San by high-performance liquid chromatography after intravesical administration Pedro, J. M., et al. (2015a). Molecular and translational classifications in patients with transitional cell carcinoma of the bladder. Eur. J. Pharm. of DAMPs in immunogenic cell death. Front. Immunol. 6:588. doi: Biopharm. 59, 469–474. doi: 10.1016/j.ejpb.2004.09.013 10.3389/fimmu.2015.00588 Kamuhabwa, A. A., Roskams, T., D’Hallewin, M. A., Baert, L., Van Poppel, H., Garg, A. D., Krysko, D. V., Vandenabeele, P., and Agostinis, P. (2012b). Hypericin- and de Witte, P. A. (2003). Whole bladder wall photodynamic therapy of based photodynamic therapy induces surface exposure of damage-associated transitional cell carcinoma rat bladder tumors using intravesically administered molecular patterns like HSP70 and calreticulin. Cancer Immunol. Immunother. hypericin. Int. J. Cancer. 107, 460–467. doi: 10.1002/ijc.11396 61, 215–221. doi: 10.1007/s00262-011-1184-2 Kascakova, S., Nadova, Z., Mateasik, A., Mikes, J., Huntosova, V., Refregiers, M., Garg, A. D., Krysko, D. V., Vandenabeele, P., and Agostinis, P. (2016). et al. (2008). High level of low-density lipoprotein receptors enhance hypericin Extracellular ATP and P X receptor exert context-specific immunogenic uptake by U-87 MG cells in the presence of LDL. Photochem. Photobiol. 84, 2 7 effects after immunogenic cancer cell death. Cell Death Dis. 7, e2097. doi: 120–127. doi: 10.1111/j.1751-1097.2007.00207.x 10.1038/cddis.2015.411 Kashef, N., Borghei, Y. S., and Djavid, G. E. (2013). Photodynamic effect Garg, A. D., Krysko, D. V., Verfaillie, T., Kaczmarek, A., Ferreira, G. B., Marysael, of hypericin on the microorganisms and primary human fibroblasts. T., et al. (2012a). A novel pathway combining calreticulin exposure and ATP Photodiagnosis Photodyn. Ther. 10, 150–155. doi: 10.1016/j.pdpdt.2012. secretion in immunogenic cancer cell death. EMBO J. 31, 1062–1079. doi: 11.007 10.1038/emboj.2011.497 Kasper, S., Caraci, F., Forti, B., Drago, F., and Aguglia, E. (2010). Efficacy Garnica, S., Weiss, M., and Oberwinkler, F. (2003). Morphological and molecular and tolerability of Hypericum extract for the treatment of mild to phylogenetic studies in South American Cortinarius species. Mycol. Res. 107, moderate depression. Eur. Neuropsychopharmacol. 20, 747–765. doi: 1143–1156. doi: 10.1017/S0953756203008414 10.1016/j.euroneuro.2010.07.005 Goey, A. K., Meijerman, I., Rosing, H., Marchetti, S., Mergui-Roelvink, M., Kim, M., Jung, H. Y., and Park, H. J. (2015). Topical PDT in the treatment of benign Keessen, M., et al. (2014). The effect of St John’s wort on the pharmacokinetics skin diseases: principles and new applications. Int. J. Mol. Sci. 16, 23259–23278. of docetaxel. Clin. Pharmacokinet. 53, 103–110. doi: 10.1007/s40262-01 doi: 10.3390/ijms161023259 3-0102-5 Kitanov, G. M. (2001). Hypericin and pseudohypericin in some Hypericum species. Gulick, R. M., McAuliffe, V., Holden-Wiltse, J., Crumpacker, C., Liebes, L., Stein, Biochem. Syst. Ecol. 29, 171–178. doi: 10.1016/S0305-1978(00)00032-6 D. S., et al. (1999). Phase I studies of hypericin, the active compound in St. Kleemann, B., Loos, B., Scriba, T. J., Lang, D., and Davids, L. M. (2014). St John’s John’s Wort, as an antiretroviral agent in HIV-infected adults. AIDS Clinical Wort (Hypericum perforatum L.) photomedicine: hypericin-photodynamic Trials Group Protocols 150 and 258. Ann Intern Med. 130, 510–514. doi: therapy induces metastatic melanoma cell death. PLoS ONE 9:e103762. doi: 10.7326/0003-4819-130-6-199903160-00015 10.1371/journal.pone.0103762 Hadjur, C., Richard, M. J., Parat, M. O., Jardon, P., and Favier, A. (1996). Komoroski, B. J., Zhang, S., Cai, H., Hutzler, J. M., Frye, R., Tracy, T. S., et al. Photodynamic effects of hypericin on lipid peroxidation and antioxidant status (2004). Induction and inhibition of cytochromes P450 by the St. John’s wort in melanoma cells. Photochem. Photobiol. 64, 375–381. doi: 10.1111/j.1751- constituent hyperforin in human hepatocyte cultures. Drug Metab. Dispos. 32, 1097.1996.tb02474.x 512–518. doi: 10.1124/dmd.32.5.512 Head, C. S., Luu, Q., Sercarz, J., and Saxton, R. (2006). Photodynamic therapy and Koperdáková, J., Komarovská, H., Košuth, J., Giovannini, A., and Cellárová, E. tumor imaging of hypericin-treated squamous cell carcinoma. World J. Surg. (2009). Characterization of hairy root-phenotype in transgenic Hypericum Oncol. 4:87. doi: 10.1186/1477-7819-4-87 perforatum L. clones. Acta Physiol. Plant. 31, 351–358. doi: 10.1007/s11738- Hudson, J. B., Harris, L., and Towers, G. H. (1993). The importance of light in 008-0241-8 the anti-HIV effect of hypericin. Antiviral Res. 20, 173–178. doi: 10.1016/0166- Košuth, J., Koperdáková, J., Tolonen, A., Hohtola, A., and Cellárová, E. (2003). 3542(93)90006-5 The content of hypericins and phloroglucinols in Hypericum perforatum Huygens, A., Kamuhabwa, A. R., Van Laethem, A., Roskams, T., Van L. seedlings at early stage of development. Plant Sci. 165, 515–521. doi: Cleynenbreugel, B., Van Poppel, H., et al. (2005). Enhancing the photodynamic 10.1016/S0168-9452(03)00210-3 effect of hypericin in tumour spheroids by fractionated light delivery in Koval, J., Mikes, J., Jendzelovský, R., Kello, M., Solár, P., and Fedorocko, combination with hyperoxygenation. Int. J. Oncol. 26, 1691–1697. doi: P. (2010). Degradation of HER2 receptor through hypericin-mediated 10.3892/ijo.26.6.1691 photodynamic therapy. Photochem. Photobiol. 86, 200–205. doi: Jacobson, J. M., Feinman, L., Liebes, L., Ostrow, N., Koslowski, V., Tobia, A., et al. 10.1111/j.1751-1097.2009.00639.x (2001). Pharmacokinetics, safety, and antiviral effects of hypericin, a derivative Kubin, A., Meissner, P., Wierrani, F., Burner, U., Bodenteich, A., Pytel, A., of St. John’s wort plant, in patients with chronic hepatitis C virus infection. et al. (2008). Fluorescence diagnosis of bladder cancer with new water Frontiers in Plant Science | www.frontiersin.org 18 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin soluble hypericin bound to polyvinylpyrrolidone: PVP-hypericin. Photochem. Mikeš, J., Koval’, J., Jendzelovský, R., Sacková, V., Uhrinová, I., Kello, Photobiol. 84, 1560–1563. doi: 10.1111/j.1751-1097.2008.00384.x M., et al. (2009). The role of p53 in the efficiency of photodynamic Kubin, A., Wierrani, F., Burner, U., Alth, G., and Grünberger, W. (2005). therapy with hypericin and subsequent long-term survival of colon Hypericin–the facts about a controversial agent. Curr. Pharm. Des. 11, 233–253. cancer cells. Photochem. Photobiol. Sci. 8, 1558–1567. doi: 10.1039/b9pp0 doi: 10.2174/1381612053382287 0021f Kucharikova, A., Kimakova, K., and Janfelt, C., and Cellarova, E. (2016). Mikešová, L., Mikeš, J., Koval’, J., Gyurászová, K., Culka, L., Vargová, J., et al. Interspecific variation in localization of hypericins and phloroglucinols in (2013). Conjunction of glutathione level, NAD(P)H/FAD redox status and the genus Hypericum as revealed by Desorption Electrospray Ionization Mass hypericin content as a potential factor affecting colon cancer cell resistance Spectrometry imaging. Physiol Plant. 157, 2–12. doi: 10.1111/ppl.12422 to photodynamic therapy with hypericin. Photodiagnosis Photodyn. Ther. 10, Kuchárová, B., Mikeš, J., Jendželovský, R., Vargová, J., Mikešová, L., Jendželovská, 470–483. doi: 10.1016/j.pdpdt.2013.04.003 Z., et al. (2015). Potentiation of hypericin-mediated photodynamic therapy Ni, Y., Huyghe, D., Verbeke, K., de Witte, P. A., Nuyts, J., Mortelmans, L., cytotoxicity by MK-886: focus on ABC transporters, GDF-15 and redox status. et al. (2006). First preclinical evaluation of mono-[123I]iodohypericin as a Photodiagnosis Photodyn. Ther. 12, 490–503. doi: 10.1016/j.pdpdt.2015.04.008 necrosis-avid tracer agent. Eur. J. Nucl. Med. Mol. Imaging 33, 595–601. doi: Kusari, S., Lamshöft, M., Zühlke, S., and Spiteller, M. (2008). An endophytic fungus 10.1007/s00259-005-0013-2 from Hypericum perforatum that produces hypericin. J. Nat. Prod. 71, 159–162. Nies, A. T., Rius, M., and Keppler, D. (2007). “Multidrug resistance proteins of the doi: 10.1021/np070669k ABCC subfamily,” in Drug Transporters: Molecular Characterization and Role Kusari, S., Sezgin, S., Nigutová, K., Cellárová, E., and Spiteller, M. (2015). Spatial in Drug Disposition, eds G. You and M. E. Morris (New Jersey, NJ: John Wiley chemo-profiling of hypericin and related phytochemicals in Hypericum species & Sons, Inc.), 223–262. using MALDI-HRMS imaging. Anal. Bioanal. Chem. 407, 4779–4791. doi: Noell, S., Feigl, G. C., Serifi, D., Mayer, D., Naumann, U., Göbel, W., et al. (2013). 10.1007/s00216-015-8682-6 Microendoscopy for hypericin fluorescence tumor diagnosis in a subcutaneous Kusari, S., Zühlke, S., Košuth, J., Cellárová, E., and Spiteller, M. (2009). Light- glioma mouse model. Photodiagnosis Photodyn. Ther. 10, 552–560. doi: independent metabolomics of endophytic Thielavia subthermophila provides 10.1016/j.pdpdt.2013.06.001 insight into microbial hypericin biosynthesis. J. Nat. Prod. 72, 1825–1835. doi: Noell, S., Mayer, D., Strauss, W. S., Tatagiba, M. S., and Ritz, R. (2011). 10.1021/np9002977 Selective enrichment of hypericin in malignant glioma: pioneering Li, J., Cona, M. M., Chen, F., Feng, Y., Zhou, L., Yu, J., et al. (2012). Exploring in vivo results. Int. J. Oncol. 38, 1343–1348. doi: 10.3892/ijo.20 theranostic potentials of radioiodinated hypericin in rodent necrosis models. 11.968 Theranostics 2, 1010–1019. doi: 10.7150/thno.4924 Olivo, M., Lau, W., Manivasager, V., Bhuvaneswari, R., Wei, Z., Soo, K. C., Li, J., Cona, M. M., Chen, F., Feng, Y., Zhou, L., Zhang, G., et al. (2013). Sequential et al. (2003a). Novel photodynamic diagnosis of bladder cancer: ex vivo systemic administrations of combretastatin A4 Phosphate and radioiodinated fluorescence cytology using hypericin. Int. J. Oncol. 23, 1501–1504. doi: hypericin exert synergistic targeted theranostic effects with prolonged survival 10.3892/ijo.23.6.1501 on SCID mice carrying bifocal tumor xenografts. Theranostics 3, 127–137. doi: Olivo, M., Lau, W., Manivasager, V., Tan, P. H., Soo, K. C., and Cheng, C. (2003b). 10.7150/thno.5790 Macro-microscopic fluorescence of human bladder cancer using hypericin Li, J., Sun, Z., Zhang, J., Shao, H., Cona, M. M., Wang, H., et al. (2011). A dual- fluorescence cystoscopy and laser confocal microscopy. Int. J. Oncol. 23, targeting anticancer approach: soil and seed principle. Radiology 260, 799–807. 983–990. doi: 10.3892/ijo.23.4.983 doi: 10.1148/radiol.11102120 Paz-Cristobal, M. P., Gilaberte, Y., Alejandre, C., Pardo, J., Revillo, M. J., and Liu, C. D., Kwan, D., Saxton, R. E., and McFadden, D. W. (2000). Hypericin and Rezusta, A. (2014). In vitro fungicidal photodynamic effect of hypericin on photodynamic therapy decreases human pancreatic cancer in vitro and in vivo. Trichophyton spp. Mycopathologia 178, 221–225. doi: 10.1007/s11046-014- J. Surg. Res. 93, 137–143. doi: 10.1006/jsre.2000.5949 9797-6 Liu, W., Zhang, D., Feng, Y., Li, Y., Huang, D., Jiang, C., et al. (2015). Prince, A. M., Pascual, D., Meruelo, D., Liebes, L., Mazur, Y., Dubovi, E., Biodistribution and anti-tumor efficacy of intratumorally injected necrosis-avid et al. (2000). Strategies for evaluation of enveloped virus inactivation in red theranostic agent radioiodinated hypericin in rodent tumor models. J. Drug cell concentrates using hypericin. Photochem. Photobiol. 71, 188–195. doi: Target. 23, 371–379. doi: 10.3109/1061186X.2014.1000337 10.1562/0031-8655(2000)0710188SFEOEV2.0.CO2 Lukšiene˙, Ž., and De Witte, P. (2002). Hypericin-based photodynamic therapy: I. Pytel, A., and Schmeller, N. (2002). New aspect of photodynamic diagnosis Comparative antitumor activity uptake studies in Ehrlich ascite tumor. Acta of bladder tumors: fluorescence cytology. Urology 59, 216–219. doi: Med Litu. 9, 195–199. 10.1016/S0090-4295(01)01528-X Martínez-Poveda, B., Quesada, A. R., and Medina, M. A. (2005). Hypericin in the Rezusta, A., López-Chicón, P., Paz-Cristobal, M. P., Alemany-Ribes, M., Royo- dark inhibits key steps of angiogenesis in vitro. Eur. J. Pharmacol. 516, 97–103. Díez, D., Agut, M., et al. (2012). In vitro fungicidal photodynamic effect doi: 10.1016/j.ejphar.2005.03.047 of hypericin on Candida species. Photochem. Photobiol. 88, 613–619. doi: Mathijssen, R. H., Verweij, J., de Bruijn, P., Loos, W. J., and Sparreboom, A. (2002). 10.1111/j.1751-1097.2011.01053.x Effects of St. John’s wort on irinotecan metabolism. J. Natl. Cancer Inst. 94, Ritz, R., Daniels, R., Noell, S., Feigl, G. C., Schmidt, V., Bornemann, A., 1247–1249. doi: 10.1093/jnci/94.16.1247 et al. (2012). Hypericin for visualization of high grade gliomas: first Miadokova, E., Chalupa, I., Vlckova, V., Sevcovicova, A., Nadova, S., Kopaskova, clinical experience. Eur. J. Surg. Oncol. 38, 352–360. doi: 10.1016/j.ejso.2011. M., et al. (2010). Genotoxicity and antigenotoxicity evaluation of non- 12.021 photoactivated hypericin. Phytother. Res. 24, 90–95. doi: 10.1002/ptr.2901 Robey, R. W., Polgar, O., Deeken, J., To, K. K. W., and Bates, S. (2007). “Breast Mikeš, J., Hýžd’alová, M., Kocˇí, L., Jendželovský, R., Koval’, J., Vaculová, A., et al. cancer resistance protein,” in Drug Transporters: Molecular Characterization (2011). Lower sensitivity of FHC fetal colon epithelial cells to photodynamic and Role in Drug Disposition, eds G. You and M. E. Morris (New therapy compared to HT-29 colon adenocarcinoma cells despite higher Jersey, NJ: John Wiley & Sons, Inc.), 319–358. doi: 10.1002/9780470140 intracellular accumulation of hypericin. Photochem. Photobiol. Sci. 10, 626–632. 505.ch12 doi: 10.1039/c0pp00359j Rook, A. H., Wood, G. S., Duvic, M., Vonderheid, E. C., Tobia, A., and Cabana, Mikeš, J., Jendželovský, R., and Fedorocˇko, P. (2013). “Cellular aspects of B. (2010). A phase II placebo-controlled study of photodynamic therapy with photodynamic therapy with hypericin,” in Photodynamic Therapy: New topical hypericin and visible light irradiation in the treatment of cutaneous Research, ed M. L. T. Elsaie (New York, NY: Nova Science Publishers), 111–147. T-cell lymphoma and psoriasis. J. Am. Acad. Dermatol. 63, 984–990. doi: Mikeš, J., Kleban, J., Sacková, V., Horváth, V., Jamborová, E., Vaculová, 10.1016/j.jaad.2010.02.039 A., et al. (2007). Necrosis predominates in the cell death of human Rubio, N., Coupienne, I., Di Valentin, E., Heirman, I., Grooten, J., Piette, J., colon adenocarcinoma HT-29 cells treated under variable conditions of et al. (2012). Spatiotemporal autophagic degradation of oxidatively damaged photodynamic therapy with hypericin. Photochem. Photobiol. Sci. 6, 758–766. organelles after photodynamic stress is amplified by mitochondrial reactive doi: 10.1039/B700350A oxygen species. Autophagy 8, 1312–1324. doi: 10.4161/auto.20763 Frontiers in Plant Science | www.frontiersin.org 19 May 2016 | Volume 7 | Article 560 Jendželovská et al. Light-Activated and Non-Activated Hypericin Sacková, V., Fedorocko, P., Szilárdiová, B., Mikes, J., and Kleban, J. (2006). Urbanová, M., Košuth, J., and Cellárová, E. (2006). Genetic and biochemical Hypericin-induced photocytotoxicity is connected with G2/M arrest in HT- analysis of Hypericum perforatum L. plants regenerated after cryopreservation. 29 and S-phase arrest in U937 cells. Photochem. Photobiol. 82, 1285–1291. doi: Plant Cell Rep. 25, 140–147. doi: 10.1007/s00299-005-0050-0 10.1562/2006-02-22-RA-806 Urla, C., Armeanu-Ebinger, S., Fuchs, J., and Seitz, G. (2015). Successful in Sanovic, R., Verwanger, T., Hartl, A., and Krammer, B. (2011). Low dose vivo tumor visualization using fluorescence laparoscopy in a mouse model of hypericin-PDT induces complete tumor regression in BALB/c mice bearing disseminated alveolar rhabdomyosarcoma. Surg. Endosc. 29, 1105–1114. doi: CT26 colon carcinoma. Photodiagnosis Photodyn. Ther. 8, 291–296. doi: 10.1007/s00464-014-3770-9 10.1016/j.pdpdt.2011.04.003 Vandenbogaerde, A. L., Cuveele, J. F., Proot, P., Himpens, B. E., Merlevede, Sattler, S., Schaefer, U., Schneider, W., Hoelzl, J., and Lehr, C. M. (1997). Binding, W. J., and de Witte, P. A. (1997). Differential cytotoxic effects induced after uptake, and transport of hypericin by Caco-2 cell monolayers. J. Pharm. Sci. 86, photosensitization by hypericin. J. Photochem. Photobiol. B. Biol. 38, 136–142. 1120–1126. doi: 10.1021/js970004a doi: 10.1016/S1011-1344(96)07446-5 Shao, H., Zhang, J., Sun, Z., Chen, F., Dai, X., Li, Y., et al. (2015). Necrosis targeted Vandenbogaerde, A. L., Geboes, K. R., Cuveele, J. F., Agostinis, P. M., Merlevede, radiotherapy with iodine-131-labeled hypericin to improve anticancer efficacy W. J., and De Witte, P. A. (1996). Antitumour activity of photosensitized of vascular disrupting treatment in rabbit VX2 tumor models. Oncotarget 6, hypericin on A431 cell xenografts. Anticancer Res. 16, 1619–1625. 14247–14259. doi: 10.18632/oncotarget.3679 Vandepitte, J., Van Cleynenbreugel, B., Hettinger, K., Van Poppel, H., and de Siboni, G., Weitman, H., Freeman, D., Mazur, Y., Malik, Z., and Ehrenberg, B. Witte, P. A. (2010). Biodistribution of PVP-hypericin and hexaminolevulinate- (2002). The correlation between hydrophilicity of hypericins and helianthrone: induced PpIX in normal and orthotopic tumor-bearing rat urinary bladder. internalization mechanisms, subcellular distribution and photodynamic action Cancer Chemother. Pharmacol. 67, 775–781. doi: 10.1007/s00280-010- in colon carcinoma cells. Photochem. Photobiol. Sci. 1, 483–491. doi: 1375-0 10.1039/b202884k Van de Putte, M., Marysael, T., Fonge, H., Roskams, T., Cona, M. M., Li, J., et al. Sim, H. G., Lau, W. K., Olivo, M., Tan, P. H., and Cheng, C. W. (2005). Is (2012). Radiolabeled iodohypericin as tumor necrosis avid tracer: diagnostic photodynamic diagnosis using hypericin better than white-light cystoscopy and therapeutic potential. Int. J. Cancer. 131, E129–E137. doi: 10.1002/ijc. for detecting superficial bladder carcinoma? BJU Int. 95, 1215–1218. doi: 26492 10.1111/j.1464-410X.2005.05508.x Van de Putte, M., Ni, Y., and De Witte, P. A. (2008a). Exploration of the mechanism Smith, P., Bullock, J. M., Booker, B. M., Haas, C. E., Berenson, C. S., and Jusko, underlying the tumor necrosis avidity of hypericin. Oncol. Rep. 19, 921–926. W. J. (2004). The influence of St. John’s wort on the pharmacokinetics and doi: 10.3892/or.19.4.921 protein binding of imatinib mesylate. Pharmacotherapy 24, 1508–1514. doi: Van de Putte, M., Wang, H., Chen, F., de Witte, P. A., and Ni, Y. (2008b). 10.1592/phco.24.16.1508.50958 Hypericin as a marker for determination of tissue viability after intratumoral Song, S., Xiong, C., Zhou, M., Lu, W., Huang, Q., Ku, G., et al. (2011). Small-animal ethanol injection in a murine liver tumor model. Acad. Radiol. 15, 107–113. PET of tumor damage induced by photothermal ablation with 64Cu-bis- doi: 10.1016/j.acra.2007.08.008 DOTA-hypericin. J. Nucl. Med. 52, 792–799. doi: 10.2967/jnumed.110.086116 Van de Putte, M., Wang, H., Chen, F., De Witte, P. A., and Ni, Y. (2008c). Hypericin Stavrovskaya, A. A. (2000). Cellular mechanisms of multidrug resistance of tumor as a marker for determination of tissue viability after radiofrequency ablation cells. Biochemistry Mosc. 65, 95–106. in a murine liver tumor model. Oncol. Rep. 19, 927–932. doi: 10.3892/or.19. Straub, M., Russ, D., Horn, T., Gschwend, J. E., and Abrahamsberg, C. (2015). 4.927 A phase IIA dose-finding study of PVP-hypericin fluorescence cystoscopy for Wada, A., Sakaeda, T., Takara, K., Hirai, M., Kimura, T., Ohmoto, N., et al. (2002). detection of nonmuscle-invasive bladder cancer. J. Endourol. 29, 216–222. doi: Effects of St John’s wort and hypericin on cytotoxicity of anticancer drugs. Drug 10.1089/end.2014.0282 Metab. Pharmacokinet. 17, 467–474. doi: 10.2133/dmpk.17.467 Theodossiou, T. A., Hothersall, J. S., De Witte, P. A., Pantos, A., and Agostinis, Wölfle, U., Seelinger, G., and Schempp, C. M. (2014). Topical application of St. P. (2009). The multifaceted photocytotoxic profile of hypericin. Mol. Pharm. 6, John’s wort (Hypericum perforatum). Planta Med. 80, 109–120. doi: 10.1055/s- 1775–1789. doi: 10.1021/mp900166q 0033-1351019 Thomas, C., MacGill, R. S., Miller, G. C., and Pardini, R. S. (1992). Xie, X., Hudson, J. B., and Guns, E. S. (2001). Tumor-specific and Photoactivation of hypericin generates singlet oxygen in mitochondria and photodependent cytotoxicity of hypericin in the human LNCaP inhibits succinoxidase. Photochem. Photobiol. 55, 47–53. doi: 10.1111/j.1751- prostate tumor model. Photochem. Photobiol. 74, 221–225. doi: 1097.1992.tb04208.x 10.1562/0031-8655(2001)0740221TSAPCO2.0.CO2 Thomas, C., and Pardini, R. S. (1992). Oxygen dependence of hypericin- Zahreddine, H., and Borden, K. L. (2013). Mechanisms and insights into drug induced phototoxicity to EMT6 mouse mammary carcinoma cells. Photochem. resistance in cancer. Front. Pharmacol. 4:28. doi: 10.3389/fphar.2013.00028 Photobiol. 55, 831–837. doi: 10.1111/j.1751-1097.1992.tb08531.x Zheng, Y., Yin, G., Le, V., Zhang, A., Chen, S., Liang, X., et al. (2016). Thong, P. S., Kho, K. W., Zheng, W., Harris, M., Soo, K. C., and Olivo, M. (2007). Photodynamic-therapy activates immune response by disrupting immunity Development of a laser confocal endomicroscope for in vivo fluorescence homeostasis of tumor cells, which generates vaccine for cancer therapy. Int. J. imaging. J. Mech. Med. Biol. 7, 11–18. doi: 10.1142/S0219519407002108 Biol. Sci. 12, 120–132. doi: 10.7150/ijbs.12852 Thong, P. S., Olivo, M., Chin, W. W., Bhuvaneswari, R., Mancer, K., and Soo, K. C. Zupkó, I., Kamuhabwa, A. R., D’Hallewin, M. A., Baert, L., and De (2009). Clinical application of fluorescence endoscopic imaging using hypericin Witte, P. A. (2001). In vivo photodynamic activity of hypericin in for the diagnosis of human oral cavity lesions. Br. J. Cancer. 101, 1580–1584. transitional cell carcinoma bladder tumors. Int. J. Oncol. 18, 1099–1105. doi: doi: 10.1038/sj.bjc.6605357 10.3892/ijo.18.5.1099 Thong, P. S., Tandjung, S. S., Movania, M. M., Chiew, W. M., Olivo, M., Bhuvaneswari, R., et al. (2012). Toward real-time virtual biopsy of oral lesions Conflict of Interest Statement: The authors declare that the research was using confocal laser endomicroscopy interfaced with embedded computing. J. conducted in the absence of any commercial or financial relationships that could Biomed. Opt. 17:056009. doi: 10.1117/1.JBO.17.5.056009 be construed as a potential conflict of interest. Thong, P. S., Watt, F., Ren, M. Q., Tan, P. H., Soo, K. C., and Olivo, M. (2006). Hypericin-photodynamic therapy (PDT) using an alternative treatment regime Copyright © 2016 Jendželovská, Jendželovský, Kuchárová and Fedoroˇcko. This suitable for multi-fraction PDT. J. Photochem. Photobiol. B. Biol. 82, 1–8. doi: is an open-access article distributed under the terms of the Creative Commons 10.1016/j.jphotobiol.2005.08.002 Attribution License (CC BY). The use, distribution or reproduction in other forums Tian, R., Koyabu, N., Morimoto, S., Shoyama, Y., Ohtani, H., and Sawada, Y. is permitted, provided the original author(s) or licensor are credited and that the (2005). Functional induction and de-induction of P-glycoprotein by St. John’s original publication in this journal is cited, in accordance with accepted academic wort and its ingredients in a human colon adenocarcinoma cell line. Drug practice. No use, distribution or reproduction is permitted which does not comply Metab. Dispos. 33, 547–554. doi: 10.1124/dmd.104.002485 with these terms. Frontiers in Plant Science | www.frontiersin.org 20 May 2016 | Volume 7 | Article 560

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