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2-Methoxyestradiol and Its Combination with a Natural Compound, Ferulic Acid, Induces Melanoma Cell Death via Downregulation of Hsp60 and Hsp90

2-Methoxyestradiol and Its Combination with a Natural Compound, Ferulic Acid, Induces Melanoma... Hindawi Journal of Oncology Volume 2019, Article ID 9293416, 12 pages https://doi.org/10.1155/2019/9293416 Research Article 2-Methoxyestradiol and Its Combination with a Natural Compound, Ferulic Acid, Induces Melanoma Cell Death via Downregulation of Hsp60 and Hsp90 1 1 1 Anna Kamm, Paulina Przychodzen ´ , Alicja Kuban–Jankowska, 2 ,3 2 ,3 4 Antonella Marino Gammazza , Francesco Cappello, Agnieszka Daca, 5 1 1,2 ,6 Michał A. Zmijewski, Michał Woz ´niak, and Magdalena Go ´ rska–Ponikowska Department of Medical Chemistry, Medical University of Gdansk, Gdansk 80-211, Poland Euro-Mediterranean Institute of Science and Technology, Palermo, Italy Department of Biomedicine, Neurosciences and Advanced Diagnostics (BiND), University of Palermo, 90127 Palermo, Italy Department of Pathology and Rheumatology, Medical University of Gdansk, Gdansk 80-211, Poland Department of Histology, Medical University of Gdansk, Gdansk 80-211, Poland Department of Biophysics, Institute of Biomaterials and Biomolecular Systems, University of Stuttgart, Stuttgart, Germany Correspondence should be addressed to Magdalena Go´rska–Ponikowska; m.gorska@gumed.edu.pl Received 30 July 2019; Revised 28 August 2019; Accepted 4 September 2019; Published 2 October 2019 Guest Editor: Alessandro Pitruzzella Copyright © 2019 Anna Kamm et al. /is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Melanoma is an aggressive type of skin cancer with one of the highest mortality rates. Notably, its incidence in the last few decades has increased faster than any other cancer. /erefore, searching for novel anticancer therapies is of great clinical importance. In the present study, we investigated the anticancer potential of 2-methoxyestradiol, potent chemotherapeutic, in the A375 melanoma cellular model. In order to furthermore evaluate the anticancer efficacy of 2-methoxyestradiol, we have additionally combined the treatment with a naturally occurring polyphenol, ferulic acid. /e results were obtained using the melanoma A375 cellular model. In the study, we used MTT assay, flow cytometry, and western blot techniques. Herein, we have evidenced that the molecular mechanism of action of 2-methoxyestradiol and ferulic acid is partly related to the reduction of Hsp60 and Hsp90 levels and the induction of nitric oxide in the A375 melanoma cell model, while no changes were observed in Hsp70 expression after 2- methoxyestradiol and ferulic acid treatment separately or in combination. /is is especially important in case of chemoresistance mechanisms because the accumulation of Hsp70 reduces induction of cancer cell death, thus decreasing antitumour efficacy. recommended as it is not an independent prognostic factor. 1. Introduction Histological features such as tumour thickness, but also rate Melanoma is an agressive type of skin cancer with one of the of mitosis, are crucial for prognosis and determination of the highest mortality rates, while its incidence in the last few stage of melanoma [3]. /e evidence-based analysis that led decades has increased faster than any other cancer [1]. to the development of recommendations for the assessment Although there has been tremendous progress in the of melanoma progression was based on the updated database treatment of melanoma patients in recent years, and in over of the US Cancer Staging Manual (AJCC). /e Melanoma the last 7 years the US Food and Drug Administration (FDA) Evaluation Committee recommended that the mitotic rate has authorized many antimelanoma drugs, the ideal treat- should be determined by the “hotspot” method and expressed as the number of mitoses per square millimeter of ment is still not clearly defined and remains the subject of great debates [2]. the primary tumour [4]. /e Melanoma Evaluation Com- While being an integral evaluation criterion of gradu- mittee recommended that the mitotic rate should replace ation of melanoma for many years, Clark’s level is no longer Clark’s level as the main criterion for determining T1b 2 Journal of Oncology therapies [37–42]. /ese Hsps are also implicated in can- melanoma [5]. Moreover, it is enormously vital to constantly strive to explore knowledge about substances that can in- cerogenesis and further progression of melanoma [43–45]. /erefore, herein we address the question about the role of crease the effectiveness of cancer therapies. A larger un- derstanding of the molecular mechanisms of potential drugs major Hsps in efficacy of supportive anticancer treatment of can lead to creating new or developing existing therapies that FA separately and in combination with a potent, anticancer take into account the individual physiological profile of the agent, 2-ME, in the A375 melanoma cellular model. patient. Anticancer agent that may be effective in treatment of 2. Materials and Methods melanoma is 2-methoxyestradiol (2-ME), which is a natural 2.1. Cell Culture. Human melanoma A375 cells (CRL-1619) compound, a metabolite of 17β-estradiol, and a hormone of were purchased from the American Type Culture Collection both women and men [6]. 2-ME is a monomethyl ether of 2- (Manassas, VA, USA). /e cells were cultured in Dulbecco’s hydroxyestradiol formed in the reaction catalyzed by cate- modified Eagle’s medium (DMEM) supplemented with 10% chol-O-methyltransferase (COMT). Its physiological level in fetal bovine serum (FBS) (both Sigma-Aldrich; Merck the blood serum ranges from 30 pM up to 30 nM during KGaA) and 1% penicillin/streptomycin in an incubator with pregnancy [6], while pharmacological relevant concentra- 5% CO at 37 C. tions involve micromolar concentrations [7]. Induction of nitro-oxidative stress is involved in antitumour activity of 2- ME against various cancer cellular models. In our previous 2.2. Experimental Design: Cell Treatment. In the study we studies, we evidenced that 2-ME, at both physiological and used the A375 human melanoma cell model. First of all, the pharmacological relevant concentrations, increases the A375 cells were seeded in the standard medium at appro- nuclear fraction of neuronal nitric oxide synthase (nNOS) in priate densities on the plates according to the specific ex- osteosarcoma 143B cells. /us, we suggested nNOS as a perimental design 24 h before the treatment. /e treatments molecular messenger of 2-ME. Induction of nNOS via 2-ME were performed in DMEM containing 1% charcoal-stripped increased production of nitric oxide leading to DNA strand FBS and 1% antibiotic cocktail (Sigma-Aldrich, Poland). breaks and eventually cell death [8]. Another anticancer Charcoal-stripped FBS is used to elucidate the effects of mechanism of 2-ME revealed by our team is regulation of hormones in various in vitro systems. mitochondrial biogenesis and inhibition of the activity of Subsequently, the A375 cells were treated with 2-ME succinate dehydrogenase complex in osteosarcoma separately or in combination with FA for 24 hours or 8 hours 143B cells [9]. according to the experimental design. Based on previous /e effectiveness of 2-ME has been demonstrated in vitro research, 10 μM 2-ME was used [8, 46]. While, based on in many cancers, including lung cancer, breast cancer, co- MTT results, we chose 1 mM FA for further studies. Fol- lorectal cancer, and pancreatic cancer [10–16]. Currently, 2- lowing the incubation, the cells underwent procedure ME trade name PANZEM is in the second phase of clinical according to the specific experimental design described trials in the treatment of kidney, prostate, ovarian, and below. carcinoid tumours with high metastatic potential [7, 17–26]. In order to avoid the impact of the solvents, for the Notably, 2-ME seems to be cytotoxic towards melanoma further studies, control cells were treated with an equal cells in both in vitro and in vivo models [12, 27–29]. It is volume of the solvent used to prepare 2-ME and FA solu- further hypothesized that 2-ME specifically kills cancer cells tions. /e final concentration of solvents in the incubation without affecting normal cells [30]. medium was less than 0.1%. In the current study, we combined 2-ME with a natural compound, ferulic acid (FA). FA belongs to the group of hydroxycinnamic acids found in plant tissues [31] (Figure 1). 2.3. Cell Viability/Cell Proliferation Assay (MTT Assay). FA is a phenolic compound that possesses three charac- A375 melanoma cells were seeded into a 96-well plate at a teristic structural domains that may contribute to the ability density of 10,000 cells per well. After 24 hours, the cell to reduce free radicals [32]. /e antioxidant properties of FA culture medium was removed and the cells were treated depend on its chemical structure [33]. FA, due to the with serial dilutions of FA within the concentration range phenolic structure and the unsaturated side chain, may between 1 mM and 31.25 μM. Based on the results, for easily form a resonant-stabilized phenoxyl radical, which is further studies 1 mM FA was chosen. Consequently, the responsible for its strong antioxidant activity [34]. /e cells were treated with 1 mM FA and 10 μM 2-ME sepa- health benefits of using phenolic compounds, such as FA, rately or in combination for 24 hours. Solvent-treated A375 attract the attention of many researchers due to their an- melanoma cells were considered as the control (100% of cell tioxidant potential. Antitumor activity of polyphenols in- viability). cludes antiproliferative and proapoptotic effects in tumour After the appropriate incubation time, 0.5 mg/ml of 3- cells [35]. Phenolic acids of plant origin, like FA with strong [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro- antioxidant activity, have received special attention as po- mide (MTT) was added (Sigma-Aldrich, Poland). /e plates tential tumour inhibitors [36]. were incubated at 37 C for 4 hours, and the supernatant was Notably, major heat shock proteins, such as Hsp90, Hsp70, removed after centrifugation (700 ×g for 10 min). Finally, and Hsp60, may be considered as biomarkers for cancer 100 μl of DMSO (Sigma-Aldrich, Poland) was added to diagnosis and prognosis, as well as efficacy of anticancer dissolve the formazan crystals. Absorbance at 570 nm was Journal of Oncology 3 COOH COOH NH Cinnamic acid Phenylalanine COOH COOH COOH COOH COOH NH OH OCH CH O OCH 3 3 OH OH OH OH OH Tyrosine γ-Coumaric acid Caffeic acid Ferulic acid Sinapic acid Figure 1: /e synthesis pathway of hydroxycinnamic acids in plants (Castelluccio i wsp., 1995). read using a microplate reader (BioTek Instruments, Inc., After 24 hours, the cells were treated with 1 mM FA and USA). /e data are presented as a percent of control. Each 10 μM 2-ME separately or in combination for 24 hours. /e experiment was carried out at least three times. cells were then trypsinased and then harvested by centri- fugation at 1200g for 7 minutes. /e samples were washed 3 times with ice-cold PBS. /e cells were then incubated with 2.4. Determination of the Nitro-Oxidative Stress Pool by Flow annexin V and PI for 15 minutes at room temperature. /e Cytometry. A375 cells were seeded into 6-well plates at the whole procedure except incubation with annexin V and PI density of 300,000 cells per well. Subsequently, A375 cells was carried out on ice. /e cells were then counted at 30,000, were treated with 1 mM FA and 10 μM 2-ME separately or in and the fluorescence signals of annexin V and PI conjugate combination for 8 hours. Eight hours incubation time for were detected in fluorescence intensity channels FL1 and nitro-oxidative stress was based on our previous results FL3 (BD FACScan). /e results were analyzed using Cyflogic [8, 46]. software, version 1.2.1. /e procedure was repeated at least 3 /e level of oxidative stress was determined by the times to ensure repeatability of results. fluorescence intensity of 2,7-dichlorofluorescein diacetate (DCF-DA), and DCF was added at 10 μM final concentra- tion 30 minutes before the end of incubation time. DCF does 2.6. Analysis of Hsp 70, 60, and 90 Protein Levels by Western not show fluorescent properties until the oxidation reaction Blot Technique. /e level of Hsp 70, 60, and 90 proteins and is carried out in presence of free radicals. β-actin were determined by western blot technique. After 24 /e cells were detached from the plates with trypsin, hours, the cells were treated with 1 mM FA and 10 μM 2-ME collected, and centrifuged (1200g for 5 minutes). Washed separately or in combination for 24 hours. /en, the cells twice with phosphate buffered saline (PBS; 137 mM NaCl, were harvested and centrifuged. /e pellets were washed 3 2.7 mM KCl, and 4.3 mM Na2HPO4, pH 7.4), suspended in times with PBS (Sigma-Aldrich, Poland) and then sus- PBS, and then analyzed by flow cytometry. /e nitric oxide pended in RIPA buffer (Sigma-Aldrich, Poland) and a level was determined using a 4,5-diaminofluorescein diac- cocktail of protease inhibitors (Calbiochem, Germany). etate (DAF-DA) detector. A LSR II flow cytometer (Becton Protein concentration was determined using the Bradford Dickinson, USA) equipped with FACSDiva software was reagent [Bradford, 1976]. Afterwards, samples containing used. /e entire procedure was carried out on ice. /en 100 μg of protein were mixed with Laemmli loading buffer 30,000 cells were counted and analyzed by flow cytometry (Sigma-Aldrich, Poland) and incubated at 95 C for 10 min. (BD FACScan) with a dye spectrum filter (excitation l � 495 /e proteins were separated on a 7–20% gradient of poly- and l � 530). /e results were analyzed using Cyflogic acrylamide gel (GE Healthcare, Poland) by electrophoresis. software, version 1.2.1. /e procedure was repeated at least 3 /e separated proteins were transferred to a methanol- times to ensure repeatability of results. activated PVDF membrane in TBE buffer (90 mM Tris, 90 mM boric acid, and 1 mM EDTA, pH 8) using a semi-dry 2.5. Analysis of Apoptosis and Necrosis by Flow Cytometry. transfer device (250 mA, 63 V, and 45 minutes) (GE Analysis of the level of apoptosis and necrosis was per- Healthcare, Poland). /en, after 1 hour of blocking in 5% formed by means of flow cytometry. Briefly, A375 cells were nonfat milk in TBS-T (0.5% Tween20, 20 mM Tris-HCl, pH seeded in 6-well plates at a density of 300,000 cells per well. 7.4, and 0.5 M NaCl), the membranes were incubated with 4 Journal of Oncology primary antibodies overnight at 4 C. /e Hsp90 beta anti- between 2-ME and FA in A375 cells, we next determined the body (catalog number ab80159) and Hsp70 antibody impact of the compounds on the induction of cell death. As demonstrated in Figure 3, 24-hour treatment with [EP1007Y] purchased from Abcam (catalog number ab45133), and Hsp60 antibody (H-1) (catalog number sc- 10 μM 2-ME or 1 mM FA did not significantly increase the 13115) and beta-actin (catalog number sc-47778) purchased number of early and late apoptotic cells, while increased the from Santa Cruz Biotechnology were used in the study. After number of necrosis up to 9% and 4%, respectively. Notably, incubation time, the membranes were washed 3 times for 5 combined treatment with 2-ME and FA induced both ap- minutes in TBS-T and then incubated with horseradish optosis and necrosis in A375-treated cells. We observed peroxidase (HRP) conjugated secondary antibodies (1 : approximately 10% apoptotic cells and 25% necrotic cells 50,000 dilution in TBS-T) for 1 hour at room temperature. after 24-hour combined treatment with 10 μM 2-ME and Visualization was performed using chemiluminescence 1 mM FA as compared with control cells (0.6% apoptotic enhanced with a luminol reagent (chemiluminescence cells, 3% necrotic cells, respectively) (Figure 3). blotting, GE Healthcare, Poland) according to the manu- facturer’s protocol. /e signal was read using ImageQuant 3.3. Nitro-Oxidative Stress Is Involved in Anticancer Mecha- LAS 500 (GE Healthcare, Poland). Protein levels were nisms of 2-ME and FA in the Melanoma A375 Cellular Model. quantified using densitometry analysis by the Quantity One Due to the fact that both 2-ME and FA may regulate the level program. /e results were normalized to β-actin. Each of reactive oxygen (ROS) and nitrogen species (RNS) in experiment was carried out at least three times. cancer cells [47–50], we evaluated the effect of the com- pounds on pool of nitro-oxidative stress in the melanoma A375 cellular model by means of flow cytometry. First of all, 2.7. Statistical Analysis of the Obtained Results. /e results we performed the experiments using DCF-DA staining to are represented by the mean± SD of at least three in- determine the level of reactive oxygen species [51]. dependent experiments. Differences between control and As demonstrated in Figure 4(a), 8-hour treatment with treated samples were assessed by means of one-way analysis 1 mM FA reduced the level of DCF-DA-stained cells which of variance (ANOVA) with a post hoc test using Tukey’s confirms its antioxidant properties. On the other hand, multiple comparison test. A p value less than 0.01 was separate 8-hour treatment with 10 μM 2-ME increased the considered to be equivalent to statistical significance. Data level of oxidative stress in melanoma A375 cells. Notably, FA were analyzed using GraphPad Prism (GraphPad Software, scavenged the 2-ME-generated oxidative stress in our ex- Inc., version 6, USA). perimental model (Figure 4(a)). We have previously evidenced that one of the anticancer 3. Results modes of 2-ME is associated with a selective increase in the nitric oxide level [8, 9, 46, 47]. /erefore, in the next part of 3.1. Antiproliferative Effect of FA and 2-ME in the Melanoma the study, we aimed to determine the impact of compounds A375 Cellular Model. First of all, we addressed the question on changes within the level of nitric oxide in melanoma about antiproliferative efficacy of FA in A375 cells by means A375 cells via DAF-DA staining [8, 52, 53]. Notably, herein, of MTT assay. /e antiproliferative potential of FA was we evidenced that induction of nitric oxide after 8-hour evaluated by 24-hour treatment of A375 cells with serial treatment with 10 μM 2-ME can be also extended to the dilutions of FA within the concentration range between melanoma A375 cellular model (Figure 4(b)). We further 1 mM and 31.25 μM (Figure 2(a)). /e percentage of viable evaluated that separate 8-hour treatment with 1 mM FA cells in samples was calculated in comparison to control either increased the level of nitric oxide in the established A375 cells, which viability was assumed to be 100%. Based experimental model. Interestingly, combined 8-hour treat- on the survival curves obtained by the GraphPad Prism ment with 2-ME and FA significantly increases the level of Software, the calculated EC50 (50% decrease in the viability free radical as compared to separate treatment with both of the treated cells) concentration was equal to 701.9 μM. compounds (Figure 4(b)). /is result may suggest an ob- /erefore, for further studies, a representative concen- served synergistic effect between 2-ME and FA in melanoma tration of 1 mM FA was chosen. /e next goal of the study cells. was to determine the efficacy of combined treatment of FA with a potent anticancer agent, 2-ME, in the melanoma A375 3.4. Effect of 2-ME and FA on the Level of Major Hsps: Hsp60, cellular model. /e concentration of 10 μM of 2-ME was Hsp70, and Hsp90 in the Melanoma A375 Cellular Model. chosen as representative, corresponding to the pharmaco- Due to the fact that Hsps may be considered as the indicators logical concentration range, based on previous studies and biomarkers of nitro-oxidative stress, we determined the [8, 46]. As presented in Figure 2(b), we did observe statistical impact of both 2-ME and FA on the level of Hsp60, Hsp70, significant correlation between combined treatment of FA and Hsp90 by means of western blotting analyses. and 2-ME as compared to separate treatments. At the outset, we evaluated the influence of the 24-hour treatment with 10 μM 2-ME and 1 mM FA, separately or in 3.2. Effect of Combined and Separate Treatment with FA and combination, on the Hsp60 protein level. As presented in Figure 5(a), western blot analyses of Hsp60 indicate a de- 2-ME on Induction of Melanoma A375 Cell Death. In order to further explore the anticancer efficacy and interaction crease in Hsp60 protein level by 35% and 65% relative to the Journal of Oncology 5 150 150 100 100 ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ 0 1 2 3 4 010 10 10 10 10 FA concentration (μM) FA (μM) + 2ME (μM) (a) (b) Figure 2: (a) /e A375 cell viability graph after incubation with FA within the concentration range between 1 mM and 31.25 μM for 24 hours. (b) /e viability of melanoma A375 cells is inhibited after treatment with 10 μM 2-ME, 1 mM FA, and combination of both for 24 hours. /e cell viability was determined by MTT assay. Values are the mean ± SE of six independent experiments (N � 6 repeats). p< 0.01 ∗∗∗∗ and p< 0.00001 vs. control. Statistical significance was determined by a one-way ANOVA analyses followed by Tukey’s multiple comparison test and unpaired t test. control, after separate treatment with 10 μM 2-ME and after activity of 2-ME in melanoma cells. It is suggested that combined treatment with 10 μM 2 ME and 1 mM FA in phenolic compounds generally maintain normal homeo- melanoma A375 cells, respectively. /e Hsp60 protein level stasis by inducing apoptosis in various tumour cells [55]. was not significantly changed after combined treatment with Many studies investigated cytotoxic and proapoptotic effects separate treatment with 1 mM FA. of polyphenols in various cancers [36, 50, 56, 57]. In con- Subsequently, the changes within Hsp70 protein level sistency with our outcomes, Park et al. established the an- ticancer potential of FA in the mouse B16F10 melanoma were investigated in the melanoma A375 cellular model (Figure 5(b)). Notably, the obtained results indicate no cells [58]. Furthermore, Khanduja et al. proved that phenolic changes in Hsp70 protein level as compared to the control compounds, such as FA, significantly reduce apoptosis in after 24-hour treatment with both 10 μM 2-ME and 1 mM normal peripheral blood mononuclear cells, which suggests FA separately or in combination. limited cytotoxicity of FA [59]. Even more importantly, the Furthermore, our western blot analyses indicate a de- significant role of FA in the prevention of skin cancer was crease in Hsp90 protein expression in the A375 cell line by also proved [57]. 13%, 29%, and 69% relative to the control, after 24-hour /e cytotoxic activity of both compounds seems to be treatment with 1 mM FA and 10 μM 2 ME separately and in strictly associated with induction of nitro-oxidative stress. In combination, respectively (Figure 5(c)). our previous studies, we evidenced that 2-ME selectively upregulates neuronal nitric oxide synthase which results in generation of nitric oxide in cancer cells [8]. Herein, indeed 4. Discussion we observed increased level of nitric oxide both after treat- ment with FA and 2-ME. /is effect was even enhanced after In the current study, we presented the anticancer potential of combined treatment with the compounds. /e mechanism of 2-ME in the melanoma cellular model. Previously, the efficacy induction of nitric oxide by FA in cancer cells still needs to be of 2-ME towards melanoma cells was investigated in both in evaluated. Nonetheless, FA was reported to generate nitric vitro and in vivo studies [12, 27, 28, 54]. 2-ME has pleiotropic oxide through upregulation of argininosuccinate synthase in activity in cancer cells. Interestingly, 2-ME suppresses the inflammatory human endothelial cells [60]. On the other glycolytic state of melanoma 435R cells [27]. Moreover, 2-ME hand, FA inhibits nitric oxide production and inducible nitric treatment decreases pRb and cyclin B1 expression, increases oxide synthase expression in rat primary astrocytes [61]. p21/Cip1 expression, and induces G2/M cell cycle arrest in Notably, in contrast to altered nitric oxide induction, FA both 2D and 3D melanoma cellular models [12]. scavenged ROS in our melanoma experimental model. /e Notably, employed in our studies a natural compound, compounds were also able to reverse 2-ME induction of FA, has anticancer potential and even enhanced anticancer Cell viability (% of control) Cell viability (% of control) Control FA 1 mM 2ME 10 μM FA 1 mM + 2ME 10 μM 6 Journal of Oncology 3 ∗∗ 15 ∗∗∗ ∗∗ ∗∗ ∗∗∗ 2 10 1 5 0 0 FA (μM) + 2ME (μM) FA (μM) + 2ME (μM) (a) (b) ∗∗∗∗ 30 ∗∗∗∗ ∗∗∗∗ ∗∗ ∗∗ FA (μM) + 2ME (μM) (c) Figure 3: (a) Total cell levels in the early apoptosis phase after 24 hours incubation of A375 line cells with 10 μM 2-ME and 1 mM FA separately or in combination. (b) Total cell level in the late phase of apoptosis after 24 hours incubation of A375 line cells with 10 μM 2-ME and 1 mM FA separately or in combination. (c) Total cell level in the necrotic phase after 24 hours incubation of A375 with 10 μM 2-ME and 1 mM FA separately or in combination. Values are the mean± SE from three independent experiments. No error bar means the thickness of the line is greater than the error. p< 0.01 compared with the vehicle. /e data were analyzed using GraphPad Prism Software version 6.02, ∗ ∗∗ ∗∗∗ performing one-way ANOVA analyses followed by Tukey’s multiple comparison test. p< 0.01, p< 0.001, p< 0.0001, and ∗∗∗∗ p< 0.00001 vs. control. ROS. /ese results confirm antioxidant properties of FA. Herein, we presented the involvement of major Hsps However, the observed contradictory effect of 2-ME and FA namely Hsp60, Hsp70, and Hsp90 in the modes of action of on ROS may result in protective role of FA against cyto- 2-ME and FA in the melanoma A375 cellular model. No- toxicity of 2-ME. Indeed, the protective role of FA against tably, these Hsps seem to be also responsible for the cisplatin-induced ototoxicity was previously demonstrated mechanism of interaction between both compounds. To this [62]. FA was also reported to protect against methotrexate date, there are only a few studies considering the role of Hsps nephrotoxicity [63]. in anticancer mechanism of action of 2-ME [64–67], while % early apoptotic cells Control 1 mM FA % necrotic cells 10 μM 2ME Control 1 mM FA + 10μM 2ME 1 mM FA % late apoptotic cells 10 μM 2ME Control 1 mM FA + 10μM 2ME 1 mM FA 10 μM 2ME 1 mMFA + 10μM 2ME Journal of Oncology 7 300 1000 ∗∗∗∗ ∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗ ∗∗∗ 0 0 FA (μM) + 2ME (μM) FA (μM) + 2ME (μM) (a) (b) Figure 4: (a) Mean fluorescence intensity of ROS positive cells after 8 hours incubation of A375 line cells with 10 μM 2-ME and 1 mM FA separately or in combination. (b) Mean fluorescence intensity of RNS after 8 hours incubation of A375 line cells with 10 μM 2-ME and 1 mM FA separately or in combination. Values are the mean± SE from three independent experiments. No error bar means the thickness of the line is greater than the error. p< 0.01 compared with the vehicle. /e data were analyzed using GraphPad Prism Software version 6.02, ∗ ∗∗ ∗∗∗ performing one-way ANOVA analyses followed by Tukey’s multiple comparison test. p< 0.01, p< 0.001, p< 0.0001, and ∗∗∗∗ p< 0.00001 vs. Control. 1.0 1.0 0.6 0.3 1.0 0.9 1.0 0.9 Hsp60 Hsp70 Control 1mM 10μM 1 mM FA + Control 1mM 10μM 1 mM FA + FA 2ME 10μM 2ME FA 2ME 10μM 2ME (a) (b) 1.0 0.8 0.7 0.3 Hsp90 Control 1mM 10μM 1 mM FA + FA 2ME 10μM 2ME β‐actin (c) Figure 5: (a) Impact of separate and combined 24-hour treatments with 10 μM 2-ME and 1 mM FA on Hsp60 protein expression in A375 cells evaluated by western blotting. (b) Impact of separate and combined 24-hour treatments with 10 μM 2-ME and 1 mM FA on Hsp70 protein expression in A375 cells evaluated by western blotting. (c) Impact of separate and combined 24-hour treatments with 10 μM 2-ME and 1 mM FA on Hsp90 protein expression in A375 cells evaluated by western blotting. Densitometric analysis of HSP/beta-actin was performed using Quantity One 4.5.2 software. /e representative images are shown. no one conducted on the melanoma experimental model. known as a mitochondrial chaperonin protein. However, Similarly, there are only limited data investigating Hsps in accumulating data support that it is localized in extra- FA mechanism [68, 69]. Depending on their localization and mitochondrial compartments as well [70–75]. As a primary expression Hsps may have a dichotomal effect in cancer mitochondrial chaperone, Hsp60 is essential for mito- biology. /e 60 kDa heat shock protein (Hsp60) is classically chondrial protein homeostasis [76]. However, it is also Average fluorescence intensity of ROS positive cells Control FA 1 mM 2ME 10 μM FA 1 mM + 2ME 10μM Average fluorescence intensity of RNS positive cells Control FA 1 mM 2ME 10 μM FA 1 mM + 2ME 10μM 8 Journal of Oncology [91]. /erefore, pronounced expression of Hsp90 has been implicated in the cell survival and apoptosis signaling pathways [41]. Increased protein level of Hsp60 has been detected in almost all types of cancers, including melanoma [44, 92, 93]. Hsp90 expression is higher in metastatic detected in various malignant cells including colon [77], cervix [78], prostate [79], or melanoma [80]. In many of the melanoma and associated with malignant features as cases examined, higher expression was correlated with Clarke’s level in cutaneous melanoma and larger tumour size poorer prognosis [81–83]. in uveal melanoma [44]. Herein, we evidenced that both FA In consistency with these studies, our obtained outcomes and 2-ME downregulate the Hsp90 expression, this effect is indicate that 2-ME decreased Hsp90 protein level in mela- even enhanced after combined treatment with the com- noma cells. To this date, there are no data considering the role pounds. /ese data are consistent with increased anticancer efficacy of combination of compounds in relation to separate of FA in regulation of Hsp60 protein level. Although FA itself does not affect the Hsp60 protein level in melanoma cells, it treatments. Up to date, there are only few data about the role of Hsp90 in anticancer mode of action of 2-ME. Chauhan enhances the activity of 2-ME to decrease Hsp60 expression. /ese data indeed are strictly associated with the level of and coworkers evidenced that downregulation of Hsp90 gene expression via 2-ME is a mechanism of overcoming the apoptotic and necrotic cells as well as concentration of nitric oxide in melanoma 2-ME and FA-treated cells. On the other chemoresistance [94]. On the other side, Kim et al. estab- hand, 2-ME was reported to increase Hsp60 protein level in lished upregulation of Hsp90 alpha in breast cancer MCF-7 estrogen-positive breast adenocarcinoma MCF-7 cells [66]. adenocarcinoma cells [66]. To this date, there are no studies /us, the role of expression of Hsp60 seems not to be clear about the role of Hsp90 in anticancer mode of action of FA. and to depend on an experimental model, i.e., type of cancer Nonetheless, it was hypothesized that antidepressant-like cells. Indeed, higher expression of Hsp60 was observed in effect of FA is associated with activation of MAPK kinases pathway and Hsp90 [95, 96]. /ese contradictory results early-stage ovarian cancer than advanced-stage in one other report [84]. It was further investigated that increased ex- may be explained by different experimental models (cancer and nontransformed cells) as well as experimental condi- pression of Hsp60 is correlated with higher susceptibility of melanoma cells to immune chemotherapy [85]. tions, i.e., time of incubation. Targeting Hsp70, beyond Hsp60, is a new therapeutic approach. Most compounds are active Hsp90/Hsp70 in- 5. Conclusions hibitors and induce cancer cell death [86]. Hsp70 directly or Herein, we presented a synergism between a potent anti- indirectly modulates the intrinsic and extrinsic apoptotic cancer compound, 2-ME, and a naturally occurring poly- pathways. Inhibition or knockdown of Hsp70 increases phenol, FA. /e molecular mechanism of observed sensitivity of cells to apoptosis [87, 88]. Human cells produce interaction is at least partially associated with down- high levels of Hsp70, constitutively expressed as Hsc70, regulation of Hsp60 and Hsp90 and induction of nitric oxide mitochondrial Hsp75, and GRP78, which are found in the in the melanoma A375 cellular model. Furthermore, scav- endoplasmic reticulum [86]. Under nonstressed conditions, enging of 2-ME-induced ROS by FA may be a protective cells express constitutive levels of Hsp70. However, their mechanism against enhanced toxicity of 2-ME. /erefore, enhanced expression, a feature of cancerous or stressed cells, further investigation of sources of nitro-oxidative stress in 2- increases survival of these cells. Clinical studies indicate that ME and FA-treated cells is still needed. Nonetheless, the increased expression of Hsp70 is associated with tumori- obtained data strongly support the anticancer effect of 2-ME genesis, poor prognosis, and chemoresistance of numerous and FA and their potential role in adjuvant chemotherapy. malignancies, including melanoma [86, 89, 90]. Notably, in our melanoma experimental model, changes in Hsp70 were Data Availability not observed after treatment with 2-ME and FA separately or in combination. It is important, especially for mechanisms of /e data used to support the findings of this study are chemoresistance, as accumulation of Hsp70 reduces the available from the corresponding author upon request. induction of cancer cell death, thus decreasing the anti- tumour efficacy [86]. Disclosure Due to the fact that Hsp90 forms a chaperone machinery with Hsp70, we have established the impact of 2-ME and FA /e funders had no role in study design, data collection and on this protein. Hsp90 is an interesting target for cancer analysis, decision to publish, or preparation of the article. therapy because it is involved in folding and stabilization of numerous proteins, including those that contribute to the Conflicts of Interest development of cancer. In mammals, Hsp90 chaperones include Hsp90 alpha and Hsp90 beta, GRP94 (94 kDa glu- /e authors declare that there are no conflicts of interest cose-regulated protein), and TRAP-1 (tumour necrosis regarding the publication of this paper. factor receptor-associated protein 1) localized in the cyto- plasm, ER, and mitochondria, respectively [91]. Hsp90 is Acknowledgments implicated in the pathogenesis of numerous diseases, in- cluding cancer. 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2-Methoxyestradiol and Its Combination with a Natural Compound, Ferulic Acid, Induces Melanoma Cell Death via Downregulation of Hsp60 and Hsp90

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Hindawi Publishing Corporation
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Copyright © 2019 Anna Kamm et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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10.1155/2019/9293416
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Abstract

Hindawi Journal of Oncology Volume 2019, Article ID 9293416, 12 pages https://doi.org/10.1155/2019/9293416 Research Article 2-Methoxyestradiol and Its Combination with a Natural Compound, Ferulic Acid, Induces Melanoma Cell Death via Downregulation of Hsp60 and Hsp90 1 1 1 Anna Kamm, Paulina Przychodzen ´ , Alicja Kuban–Jankowska, 2 ,3 2 ,3 4 Antonella Marino Gammazza , Francesco Cappello, Agnieszka Daca, 5 1 1,2 ,6 Michał A. Zmijewski, Michał Woz ´niak, and Magdalena Go ´ rska–Ponikowska Department of Medical Chemistry, Medical University of Gdansk, Gdansk 80-211, Poland Euro-Mediterranean Institute of Science and Technology, Palermo, Italy Department of Biomedicine, Neurosciences and Advanced Diagnostics (BiND), University of Palermo, 90127 Palermo, Italy Department of Pathology and Rheumatology, Medical University of Gdansk, Gdansk 80-211, Poland Department of Histology, Medical University of Gdansk, Gdansk 80-211, Poland Department of Biophysics, Institute of Biomaterials and Biomolecular Systems, University of Stuttgart, Stuttgart, Germany Correspondence should be addressed to Magdalena Go´rska–Ponikowska; m.gorska@gumed.edu.pl Received 30 July 2019; Revised 28 August 2019; Accepted 4 September 2019; Published 2 October 2019 Guest Editor: Alessandro Pitruzzella Copyright © 2019 Anna Kamm et al. /is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Melanoma is an aggressive type of skin cancer with one of the highest mortality rates. Notably, its incidence in the last few decades has increased faster than any other cancer. /erefore, searching for novel anticancer therapies is of great clinical importance. In the present study, we investigated the anticancer potential of 2-methoxyestradiol, potent chemotherapeutic, in the A375 melanoma cellular model. In order to furthermore evaluate the anticancer efficacy of 2-methoxyestradiol, we have additionally combined the treatment with a naturally occurring polyphenol, ferulic acid. /e results were obtained using the melanoma A375 cellular model. In the study, we used MTT assay, flow cytometry, and western blot techniques. Herein, we have evidenced that the molecular mechanism of action of 2-methoxyestradiol and ferulic acid is partly related to the reduction of Hsp60 and Hsp90 levels and the induction of nitric oxide in the A375 melanoma cell model, while no changes were observed in Hsp70 expression after 2- methoxyestradiol and ferulic acid treatment separately or in combination. /is is especially important in case of chemoresistance mechanisms because the accumulation of Hsp70 reduces induction of cancer cell death, thus decreasing antitumour efficacy. recommended as it is not an independent prognostic factor. 1. Introduction Histological features such as tumour thickness, but also rate Melanoma is an agressive type of skin cancer with one of the of mitosis, are crucial for prognosis and determination of the highest mortality rates, while its incidence in the last few stage of melanoma [3]. /e evidence-based analysis that led decades has increased faster than any other cancer [1]. to the development of recommendations for the assessment Although there has been tremendous progress in the of melanoma progression was based on the updated database treatment of melanoma patients in recent years, and in over of the US Cancer Staging Manual (AJCC). /e Melanoma the last 7 years the US Food and Drug Administration (FDA) Evaluation Committee recommended that the mitotic rate has authorized many antimelanoma drugs, the ideal treat- should be determined by the “hotspot” method and expressed as the number of mitoses per square millimeter of ment is still not clearly defined and remains the subject of great debates [2]. the primary tumour [4]. /e Melanoma Evaluation Com- While being an integral evaluation criterion of gradu- mittee recommended that the mitotic rate should replace ation of melanoma for many years, Clark’s level is no longer Clark’s level as the main criterion for determining T1b 2 Journal of Oncology therapies [37–42]. /ese Hsps are also implicated in can- melanoma [5]. Moreover, it is enormously vital to constantly strive to explore knowledge about substances that can in- cerogenesis and further progression of melanoma [43–45]. /erefore, herein we address the question about the role of crease the effectiveness of cancer therapies. A larger un- derstanding of the molecular mechanisms of potential drugs major Hsps in efficacy of supportive anticancer treatment of can lead to creating new or developing existing therapies that FA separately and in combination with a potent, anticancer take into account the individual physiological profile of the agent, 2-ME, in the A375 melanoma cellular model. patient. Anticancer agent that may be effective in treatment of 2. Materials and Methods melanoma is 2-methoxyestradiol (2-ME), which is a natural 2.1. Cell Culture. Human melanoma A375 cells (CRL-1619) compound, a metabolite of 17β-estradiol, and a hormone of were purchased from the American Type Culture Collection both women and men [6]. 2-ME is a monomethyl ether of 2- (Manassas, VA, USA). /e cells were cultured in Dulbecco’s hydroxyestradiol formed in the reaction catalyzed by cate- modified Eagle’s medium (DMEM) supplemented with 10% chol-O-methyltransferase (COMT). Its physiological level in fetal bovine serum (FBS) (both Sigma-Aldrich; Merck the blood serum ranges from 30 pM up to 30 nM during KGaA) and 1% penicillin/streptomycin in an incubator with pregnancy [6], while pharmacological relevant concentra- 5% CO at 37 C. tions involve micromolar concentrations [7]. Induction of nitro-oxidative stress is involved in antitumour activity of 2- ME against various cancer cellular models. In our previous 2.2. Experimental Design: Cell Treatment. In the study we studies, we evidenced that 2-ME, at both physiological and used the A375 human melanoma cell model. First of all, the pharmacological relevant concentrations, increases the A375 cells were seeded in the standard medium at appro- nuclear fraction of neuronal nitric oxide synthase (nNOS) in priate densities on the plates according to the specific ex- osteosarcoma 143B cells. /us, we suggested nNOS as a perimental design 24 h before the treatment. /e treatments molecular messenger of 2-ME. Induction of nNOS via 2-ME were performed in DMEM containing 1% charcoal-stripped increased production of nitric oxide leading to DNA strand FBS and 1% antibiotic cocktail (Sigma-Aldrich, Poland). breaks and eventually cell death [8]. Another anticancer Charcoal-stripped FBS is used to elucidate the effects of mechanism of 2-ME revealed by our team is regulation of hormones in various in vitro systems. mitochondrial biogenesis and inhibition of the activity of Subsequently, the A375 cells were treated with 2-ME succinate dehydrogenase complex in osteosarcoma separately or in combination with FA for 24 hours or 8 hours 143B cells [9]. according to the experimental design. Based on previous /e effectiveness of 2-ME has been demonstrated in vitro research, 10 μM 2-ME was used [8, 46]. While, based on in many cancers, including lung cancer, breast cancer, co- MTT results, we chose 1 mM FA for further studies. Fol- lorectal cancer, and pancreatic cancer [10–16]. Currently, 2- lowing the incubation, the cells underwent procedure ME trade name PANZEM is in the second phase of clinical according to the specific experimental design described trials in the treatment of kidney, prostate, ovarian, and below. carcinoid tumours with high metastatic potential [7, 17–26]. In order to avoid the impact of the solvents, for the Notably, 2-ME seems to be cytotoxic towards melanoma further studies, control cells were treated with an equal cells in both in vitro and in vivo models [12, 27–29]. It is volume of the solvent used to prepare 2-ME and FA solu- further hypothesized that 2-ME specifically kills cancer cells tions. /e final concentration of solvents in the incubation without affecting normal cells [30]. medium was less than 0.1%. In the current study, we combined 2-ME with a natural compound, ferulic acid (FA). FA belongs to the group of hydroxycinnamic acids found in plant tissues [31] (Figure 1). 2.3. Cell Viability/Cell Proliferation Assay (MTT Assay). FA is a phenolic compound that possesses three charac- A375 melanoma cells were seeded into a 96-well plate at a teristic structural domains that may contribute to the ability density of 10,000 cells per well. After 24 hours, the cell to reduce free radicals [32]. /e antioxidant properties of FA culture medium was removed and the cells were treated depend on its chemical structure [33]. FA, due to the with serial dilutions of FA within the concentration range phenolic structure and the unsaturated side chain, may between 1 mM and 31.25 μM. Based on the results, for easily form a resonant-stabilized phenoxyl radical, which is further studies 1 mM FA was chosen. Consequently, the responsible for its strong antioxidant activity [34]. /e cells were treated with 1 mM FA and 10 μM 2-ME sepa- health benefits of using phenolic compounds, such as FA, rately or in combination for 24 hours. Solvent-treated A375 attract the attention of many researchers due to their an- melanoma cells were considered as the control (100% of cell tioxidant potential. Antitumor activity of polyphenols in- viability). cludes antiproliferative and proapoptotic effects in tumour After the appropriate incubation time, 0.5 mg/ml of 3- cells [35]. Phenolic acids of plant origin, like FA with strong [4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bro- antioxidant activity, have received special attention as po- mide (MTT) was added (Sigma-Aldrich, Poland). /e plates tential tumour inhibitors [36]. were incubated at 37 C for 4 hours, and the supernatant was Notably, major heat shock proteins, such as Hsp90, Hsp70, removed after centrifugation (700 ×g for 10 min). Finally, and Hsp60, may be considered as biomarkers for cancer 100 μl of DMSO (Sigma-Aldrich, Poland) was added to diagnosis and prognosis, as well as efficacy of anticancer dissolve the formazan crystals. Absorbance at 570 nm was Journal of Oncology 3 COOH COOH NH Cinnamic acid Phenylalanine COOH COOH COOH COOH COOH NH OH OCH CH O OCH 3 3 OH OH OH OH OH Tyrosine γ-Coumaric acid Caffeic acid Ferulic acid Sinapic acid Figure 1: /e synthesis pathway of hydroxycinnamic acids in plants (Castelluccio i wsp., 1995). read using a microplate reader (BioTek Instruments, Inc., After 24 hours, the cells were treated with 1 mM FA and USA). /e data are presented as a percent of control. Each 10 μM 2-ME separately or in combination for 24 hours. /e experiment was carried out at least three times. cells were then trypsinased and then harvested by centri- fugation at 1200g for 7 minutes. /e samples were washed 3 times with ice-cold PBS. /e cells were then incubated with 2.4. Determination of the Nitro-Oxidative Stress Pool by Flow annexin V and PI for 15 minutes at room temperature. /e Cytometry. A375 cells were seeded into 6-well plates at the whole procedure except incubation with annexin V and PI density of 300,000 cells per well. Subsequently, A375 cells was carried out on ice. /e cells were then counted at 30,000, were treated with 1 mM FA and 10 μM 2-ME separately or in and the fluorescence signals of annexin V and PI conjugate combination for 8 hours. Eight hours incubation time for were detected in fluorescence intensity channels FL1 and nitro-oxidative stress was based on our previous results FL3 (BD FACScan). /e results were analyzed using Cyflogic [8, 46]. software, version 1.2.1. /e procedure was repeated at least 3 /e level of oxidative stress was determined by the times to ensure repeatability of results. fluorescence intensity of 2,7-dichlorofluorescein diacetate (DCF-DA), and DCF was added at 10 μM final concentra- tion 30 minutes before the end of incubation time. DCF does 2.6. Analysis of Hsp 70, 60, and 90 Protein Levels by Western not show fluorescent properties until the oxidation reaction Blot Technique. /e level of Hsp 70, 60, and 90 proteins and is carried out in presence of free radicals. β-actin were determined by western blot technique. After 24 /e cells were detached from the plates with trypsin, hours, the cells were treated with 1 mM FA and 10 μM 2-ME collected, and centrifuged (1200g for 5 minutes). Washed separately or in combination for 24 hours. /en, the cells twice with phosphate buffered saline (PBS; 137 mM NaCl, were harvested and centrifuged. /e pellets were washed 3 2.7 mM KCl, and 4.3 mM Na2HPO4, pH 7.4), suspended in times with PBS (Sigma-Aldrich, Poland) and then sus- PBS, and then analyzed by flow cytometry. /e nitric oxide pended in RIPA buffer (Sigma-Aldrich, Poland) and a level was determined using a 4,5-diaminofluorescein diac- cocktail of protease inhibitors (Calbiochem, Germany). etate (DAF-DA) detector. A LSR II flow cytometer (Becton Protein concentration was determined using the Bradford Dickinson, USA) equipped with FACSDiva software was reagent [Bradford, 1976]. Afterwards, samples containing used. /e entire procedure was carried out on ice. /en 100 μg of protein were mixed with Laemmli loading buffer 30,000 cells were counted and analyzed by flow cytometry (Sigma-Aldrich, Poland) and incubated at 95 C for 10 min. (BD FACScan) with a dye spectrum filter (excitation l � 495 /e proteins were separated on a 7–20% gradient of poly- and l � 530). /e results were analyzed using Cyflogic acrylamide gel (GE Healthcare, Poland) by electrophoresis. software, version 1.2.1. /e procedure was repeated at least 3 /e separated proteins were transferred to a methanol- times to ensure repeatability of results. activated PVDF membrane in TBE buffer (90 mM Tris, 90 mM boric acid, and 1 mM EDTA, pH 8) using a semi-dry 2.5. Analysis of Apoptosis and Necrosis by Flow Cytometry. transfer device (250 mA, 63 V, and 45 minutes) (GE Analysis of the level of apoptosis and necrosis was per- Healthcare, Poland). /en, after 1 hour of blocking in 5% formed by means of flow cytometry. Briefly, A375 cells were nonfat milk in TBS-T (0.5% Tween20, 20 mM Tris-HCl, pH seeded in 6-well plates at a density of 300,000 cells per well. 7.4, and 0.5 M NaCl), the membranes were incubated with 4 Journal of Oncology primary antibodies overnight at 4 C. /e Hsp90 beta anti- between 2-ME and FA in A375 cells, we next determined the body (catalog number ab80159) and Hsp70 antibody impact of the compounds on the induction of cell death. As demonstrated in Figure 3, 24-hour treatment with [EP1007Y] purchased from Abcam (catalog number ab45133), and Hsp60 antibody (H-1) (catalog number sc- 10 μM 2-ME or 1 mM FA did not significantly increase the 13115) and beta-actin (catalog number sc-47778) purchased number of early and late apoptotic cells, while increased the from Santa Cruz Biotechnology were used in the study. After number of necrosis up to 9% and 4%, respectively. Notably, incubation time, the membranes were washed 3 times for 5 combined treatment with 2-ME and FA induced both ap- minutes in TBS-T and then incubated with horseradish optosis and necrosis in A375-treated cells. We observed peroxidase (HRP) conjugated secondary antibodies (1 : approximately 10% apoptotic cells and 25% necrotic cells 50,000 dilution in TBS-T) for 1 hour at room temperature. after 24-hour combined treatment with 10 μM 2-ME and Visualization was performed using chemiluminescence 1 mM FA as compared with control cells (0.6% apoptotic enhanced with a luminol reagent (chemiluminescence cells, 3% necrotic cells, respectively) (Figure 3). blotting, GE Healthcare, Poland) according to the manu- facturer’s protocol. /e signal was read using ImageQuant 3.3. Nitro-Oxidative Stress Is Involved in Anticancer Mecha- LAS 500 (GE Healthcare, Poland). Protein levels were nisms of 2-ME and FA in the Melanoma A375 Cellular Model. quantified using densitometry analysis by the Quantity One Due to the fact that both 2-ME and FA may regulate the level program. /e results were normalized to β-actin. Each of reactive oxygen (ROS) and nitrogen species (RNS) in experiment was carried out at least three times. cancer cells [47–50], we evaluated the effect of the com- pounds on pool of nitro-oxidative stress in the melanoma A375 cellular model by means of flow cytometry. First of all, 2.7. Statistical Analysis of the Obtained Results. /e results we performed the experiments using DCF-DA staining to are represented by the mean± SD of at least three in- determine the level of reactive oxygen species [51]. dependent experiments. Differences between control and As demonstrated in Figure 4(a), 8-hour treatment with treated samples were assessed by means of one-way analysis 1 mM FA reduced the level of DCF-DA-stained cells which of variance (ANOVA) with a post hoc test using Tukey’s confirms its antioxidant properties. On the other hand, multiple comparison test. A p value less than 0.01 was separate 8-hour treatment with 10 μM 2-ME increased the considered to be equivalent to statistical significance. Data level of oxidative stress in melanoma A375 cells. Notably, FA were analyzed using GraphPad Prism (GraphPad Software, scavenged the 2-ME-generated oxidative stress in our ex- Inc., version 6, USA). perimental model (Figure 4(a)). We have previously evidenced that one of the anticancer 3. Results modes of 2-ME is associated with a selective increase in the nitric oxide level [8, 9, 46, 47]. /erefore, in the next part of 3.1. Antiproliferative Effect of FA and 2-ME in the Melanoma the study, we aimed to determine the impact of compounds A375 Cellular Model. First of all, we addressed the question on changes within the level of nitric oxide in melanoma about antiproliferative efficacy of FA in A375 cells by means A375 cells via DAF-DA staining [8, 52, 53]. Notably, herein, of MTT assay. /e antiproliferative potential of FA was we evidenced that induction of nitric oxide after 8-hour evaluated by 24-hour treatment of A375 cells with serial treatment with 10 μM 2-ME can be also extended to the dilutions of FA within the concentration range between melanoma A375 cellular model (Figure 4(b)). We further 1 mM and 31.25 μM (Figure 2(a)). /e percentage of viable evaluated that separate 8-hour treatment with 1 mM FA cells in samples was calculated in comparison to control either increased the level of nitric oxide in the established A375 cells, which viability was assumed to be 100%. Based experimental model. Interestingly, combined 8-hour treat- on the survival curves obtained by the GraphPad Prism ment with 2-ME and FA significantly increases the level of Software, the calculated EC50 (50% decrease in the viability free radical as compared to separate treatment with both of the treated cells) concentration was equal to 701.9 μM. compounds (Figure 4(b)). /is result may suggest an ob- /erefore, for further studies, a representative concen- served synergistic effect between 2-ME and FA in melanoma tration of 1 mM FA was chosen. /e next goal of the study cells. was to determine the efficacy of combined treatment of FA with a potent anticancer agent, 2-ME, in the melanoma A375 3.4. Effect of 2-ME and FA on the Level of Major Hsps: Hsp60, cellular model. /e concentration of 10 μM of 2-ME was Hsp70, and Hsp90 in the Melanoma A375 Cellular Model. chosen as representative, corresponding to the pharmaco- Due to the fact that Hsps may be considered as the indicators logical concentration range, based on previous studies and biomarkers of nitro-oxidative stress, we determined the [8, 46]. As presented in Figure 2(b), we did observe statistical impact of both 2-ME and FA on the level of Hsp60, Hsp70, significant correlation between combined treatment of FA and Hsp90 by means of western blotting analyses. and 2-ME as compared to separate treatments. At the outset, we evaluated the influence of the 24-hour treatment with 10 μM 2-ME and 1 mM FA, separately or in 3.2. Effect of Combined and Separate Treatment with FA and combination, on the Hsp60 protein level. As presented in Figure 5(a), western blot analyses of Hsp60 indicate a de- 2-ME on Induction of Melanoma A375 Cell Death. In order to further explore the anticancer efficacy and interaction crease in Hsp60 protein level by 35% and 65% relative to the Journal of Oncology 5 150 150 100 100 ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ 0 1 2 3 4 010 10 10 10 10 FA concentration (μM) FA (μM) + 2ME (μM) (a) (b) Figure 2: (a) /e A375 cell viability graph after incubation with FA within the concentration range between 1 mM and 31.25 μM for 24 hours. (b) /e viability of melanoma A375 cells is inhibited after treatment with 10 μM 2-ME, 1 mM FA, and combination of both for 24 hours. /e cell viability was determined by MTT assay. Values are the mean ± SE of six independent experiments (N � 6 repeats). p< 0.01 ∗∗∗∗ and p< 0.00001 vs. control. Statistical significance was determined by a one-way ANOVA analyses followed by Tukey’s multiple comparison test and unpaired t test. control, after separate treatment with 10 μM 2-ME and after activity of 2-ME in melanoma cells. It is suggested that combined treatment with 10 μM 2 ME and 1 mM FA in phenolic compounds generally maintain normal homeo- melanoma A375 cells, respectively. /e Hsp60 protein level stasis by inducing apoptosis in various tumour cells [55]. was not significantly changed after combined treatment with Many studies investigated cytotoxic and proapoptotic effects separate treatment with 1 mM FA. of polyphenols in various cancers [36, 50, 56, 57]. In con- Subsequently, the changes within Hsp70 protein level sistency with our outcomes, Park et al. established the an- ticancer potential of FA in the mouse B16F10 melanoma were investigated in the melanoma A375 cellular model (Figure 5(b)). Notably, the obtained results indicate no cells [58]. Furthermore, Khanduja et al. proved that phenolic changes in Hsp70 protein level as compared to the control compounds, such as FA, significantly reduce apoptosis in after 24-hour treatment with both 10 μM 2-ME and 1 mM normal peripheral blood mononuclear cells, which suggests FA separately or in combination. limited cytotoxicity of FA [59]. Even more importantly, the Furthermore, our western blot analyses indicate a de- significant role of FA in the prevention of skin cancer was crease in Hsp90 protein expression in the A375 cell line by also proved [57]. 13%, 29%, and 69% relative to the control, after 24-hour /e cytotoxic activity of both compounds seems to be treatment with 1 mM FA and 10 μM 2 ME separately and in strictly associated with induction of nitro-oxidative stress. In combination, respectively (Figure 5(c)). our previous studies, we evidenced that 2-ME selectively upregulates neuronal nitric oxide synthase which results in generation of nitric oxide in cancer cells [8]. Herein, indeed 4. Discussion we observed increased level of nitric oxide both after treat- ment with FA and 2-ME. /is effect was even enhanced after In the current study, we presented the anticancer potential of combined treatment with the compounds. /e mechanism of 2-ME in the melanoma cellular model. Previously, the efficacy induction of nitric oxide by FA in cancer cells still needs to be of 2-ME towards melanoma cells was investigated in both in evaluated. Nonetheless, FA was reported to generate nitric vitro and in vivo studies [12, 27, 28, 54]. 2-ME has pleiotropic oxide through upregulation of argininosuccinate synthase in activity in cancer cells. Interestingly, 2-ME suppresses the inflammatory human endothelial cells [60]. On the other glycolytic state of melanoma 435R cells [27]. Moreover, 2-ME hand, FA inhibits nitric oxide production and inducible nitric treatment decreases pRb and cyclin B1 expression, increases oxide synthase expression in rat primary astrocytes [61]. p21/Cip1 expression, and induces G2/M cell cycle arrest in Notably, in contrast to altered nitric oxide induction, FA both 2D and 3D melanoma cellular models [12]. scavenged ROS in our melanoma experimental model. /e Notably, employed in our studies a natural compound, compounds were also able to reverse 2-ME induction of FA, has anticancer potential and even enhanced anticancer Cell viability (% of control) Cell viability (% of control) Control FA 1 mM 2ME 10 μM FA 1 mM + 2ME 10 μM 6 Journal of Oncology 3 ∗∗ 15 ∗∗∗ ∗∗ ∗∗ ∗∗∗ 2 10 1 5 0 0 FA (μM) + 2ME (μM) FA (μM) + 2ME (μM) (a) (b) ∗∗∗∗ 30 ∗∗∗∗ ∗∗∗∗ ∗∗ ∗∗ FA (μM) + 2ME (μM) (c) Figure 3: (a) Total cell levels in the early apoptosis phase after 24 hours incubation of A375 line cells with 10 μM 2-ME and 1 mM FA separately or in combination. (b) Total cell level in the late phase of apoptosis after 24 hours incubation of A375 line cells with 10 μM 2-ME and 1 mM FA separately or in combination. (c) Total cell level in the necrotic phase after 24 hours incubation of A375 with 10 μM 2-ME and 1 mM FA separately or in combination. Values are the mean± SE from three independent experiments. No error bar means the thickness of the line is greater than the error. p< 0.01 compared with the vehicle. /e data were analyzed using GraphPad Prism Software version 6.02, ∗ ∗∗ ∗∗∗ performing one-way ANOVA analyses followed by Tukey’s multiple comparison test. p< 0.01, p< 0.001, p< 0.0001, and ∗∗∗∗ p< 0.00001 vs. control. ROS. /ese results confirm antioxidant properties of FA. Herein, we presented the involvement of major Hsps However, the observed contradictory effect of 2-ME and FA namely Hsp60, Hsp70, and Hsp90 in the modes of action of on ROS may result in protective role of FA against cyto- 2-ME and FA in the melanoma A375 cellular model. No- toxicity of 2-ME. Indeed, the protective role of FA against tably, these Hsps seem to be also responsible for the cisplatin-induced ototoxicity was previously demonstrated mechanism of interaction between both compounds. To this [62]. FA was also reported to protect against methotrexate date, there are only a few studies considering the role of Hsps nephrotoxicity [63]. in anticancer mechanism of action of 2-ME [64–67], while % early apoptotic cells Control 1 mM FA % necrotic cells 10 μM 2ME Control 1 mM FA + 10μM 2ME 1 mM FA % late apoptotic cells 10 μM 2ME Control 1 mM FA + 10μM 2ME 1 mM FA 10 μM 2ME 1 mMFA + 10μM 2ME Journal of Oncology 7 300 1000 ∗∗∗∗ ∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗ ∗∗∗ 0 0 FA (μM) + 2ME (μM) FA (μM) + 2ME (μM) (a) (b) Figure 4: (a) Mean fluorescence intensity of ROS positive cells after 8 hours incubation of A375 line cells with 10 μM 2-ME and 1 mM FA separately or in combination. (b) Mean fluorescence intensity of RNS after 8 hours incubation of A375 line cells with 10 μM 2-ME and 1 mM FA separately or in combination. Values are the mean± SE from three independent experiments. No error bar means the thickness of the line is greater than the error. p< 0.01 compared with the vehicle. /e data were analyzed using GraphPad Prism Software version 6.02, ∗ ∗∗ ∗∗∗ performing one-way ANOVA analyses followed by Tukey’s multiple comparison test. p< 0.01, p< 0.001, p< 0.0001, and ∗∗∗∗ p< 0.00001 vs. Control. 1.0 1.0 0.6 0.3 1.0 0.9 1.0 0.9 Hsp60 Hsp70 Control 1mM 10μM 1 mM FA + Control 1mM 10μM 1 mM FA + FA 2ME 10μM 2ME FA 2ME 10μM 2ME (a) (b) 1.0 0.8 0.7 0.3 Hsp90 Control 1mM 10μM 1 mM FA + FA 2ME 10μM 2ME β‐actin (c) Figure 5: (a) Impact of separate and combined 24-hour treatments with 10 μM 2-ME and 1 mM FA on Hsp60 protein expression in A375 cells evaluated by western blotting. (b) Impact of separate and combined 24-hour treatments with 10 μM 2-ME and 1 mM FA on Hsp70 protein expression in A375 cells evaluated by western blotting. (c) Impact of separate and combined 24-hour treatments with 10 μM 2-ME and 1 mM FA on Hsp90 protein expression in A375 cells evaluated by western blotting. Densitometric analysis of HSP/beta-actin was performed using Quantity One 4.5.2 software. /e representative images are shown. no one conducted on the melanoma experimental model. known as a mitochondrial chaperonin protein. However, Similarly, there are only limited data investigating Hsps in accumulating data support that it is localized in extra- FA mechanism [68, 69]. Depending on their localization and mitochondrial compartments as well [70–75]. As a primary expression Hsps may have a dichotomal effect in cancer mitochondrial chaperone, Hsp60 is essential for mito- biology. /e 60 kDa heat shock protein (Hsp60) is classically chondrial protein homeostasis [76]. However, it is also Average fluorescence intensity of ROS positive cells Control FA 1 mM 2ME 10 μM FA 1 mM + 2ME 10μM Average fluorescence intensity of RNS positive cells Control FA 1 mM 2ME 10 μM FA 1 mM + 2ME 10μM 8 Journal of Oncology [91]. /erefore, pronounced expression of Hsp90 has been implicated in the cell survival and apoptosis signaling pathways [41]. Increased protein level of Hsp60 has been detected in almost all types of cancers, including melanoma [44, 92, 93]. Hsp90 expression is higher in metastatic detected in various malignant cells including colon [77], cervix [78], prostate [79], or melanoma [80]. In many of the melanoma and associated with malignant features as cases examined, higher expression was correlated with Clarke’s level in cutaneous melanoma and larger tumour size poorer prognosis [81–83]. in uveal melanoma [44]. Herein, we evidenced that both FA In consistency with these studies, our obtained outcomes and 2-ME downregulate the Hsp90 expression, this effect is indicate that 2-ME decreased Hsp90 protein level in mela- even enhanced after combined treatment with the com- noma cells. To this date, there are no data considering the role pounds. /ese data are consistent with increased anticancer efficacy of combination of compounds in relation to separate of FA in regulation of Hsp60 protein level. Although FA itself does not affect the Hsp60 protein level in melanoma cells, it treatments. Up to date, there are only few data about the role of Hsp90 in anticancer mode of action of 2-ME. Chauhan enhances the activity of 2-ME to decrease Hsp60 expression. /ese data indeed are strictly associated with the level of and coworkers evidenced that downregulation of Hsp90 gene expression via 2-ME is a mechanism of overcoming the apoptotic and necrotic cells as well as concentration of nitric oxide in melanoma 2-ME and FA-treated cells. On the other chemoresistance [94]. On the other side, Kim et al. estab- hand, 2-ME was reported to increase Hsp60 protein level in lished upregulation of Hsp90 alpha in breast cancer MCF-7 estrogen-positive breast adenocarcinoma MCF-7 cells [66]. adenocarcinoma cells [66]. To this date, there are no studies /us, the role of expression of Hsp60 seems not to be clear about the role of Hsp90 in anticancer mode of action of FA. and to depend on an experimental model, i.e., type of cancer Nonetheless, it was hypothesized that antidepressant-like cells. Indeed, higher expression of Hsp60 was observed in effect of FA is associated with activation of MAPK kinases pathway and Hsp90 [95, 96]. /ese contradictory results early-stage ovarian cancer than advanced-stage in one other report [84]. It was further investigated that increased ex- may be explained by different experimental models (cancer and nontransformed cells) as well as experimental condi- pression of Hsp60 is correlated with higher susceptibility of melanoma cells to immune chemotherapy [85]. tions, i.e., time of incubation. Targeting Hsp70, beyond Hsp60, is a new therapeutic approach. Most compounds are active Hsp90/Hsp70 in- 5. Conclusions hibitors and induce cancer cell death [86]. Hsp70 directly or Herein, we presented a synergism between a potent anti- indirectly modulates the intrinsic and extrinsic apoptotic cancer compound, 2-ME, and a naturally occurring poly- pathways. Inhibition or knockdown of Hsp70 increases phenol, FA. /e molecular mechanism of observed sensitivity of cells to apoptosis [87, 88]. Human cells produce interaction is at least partially associated with down- high levels of Hsp70, constitutively expressed as Hsc70, regulation of Hsp60 and Hsp90 and induction of nitric oxide mitochondrial Hsp75, and GRP78, which are found in the in the melanoma A375 cellular model. Furthermore, scav- endoplasmic reticulum [86]. Under nonstressed conditions, enging of 2-ME-induced ROS by FA may be a protective cells express constitutive levels of Hsp70. However, their mechanism against enhanced toxicity of 2-ME. /erefore, enhanced expression, a feature of cancerous or stressed cells, further investigation of sources of nitro-oxidative stress in 2- increases survival of these cells. Clinical studies indicate that ME and FA-treated cells is still needed. Nonetheless, the increased expression of Hsp70 is associated with tumori- obtained data strongly support the anticancer effect of 2-ME genesis, poor prognosis, and chemoresistance of numerous and FA and their potential role in adjuvant chemotherapy. malignancies, including melanoma [86, 89, 90]. Notably, in our melanoma experimental model, changes in Hsp70 were Data Availability not observed after treatment with 2-ME and FA separately or in combination. It is important, especially for mechanisms of /e data used to support the findings of this study are chemoresistance, as accumulation of Hsp70 reduces the available from the corresponding author upon request. induction of cancer cell death, thus decreasing the anti- tumour efficacy [86]. Disclosure Due to the fact that Hsp90 forms a chaperone machinery with Hsp70, we have established the impact of 2-ME and FA /e funders had no role in study design, data collection and on this protein. Hsp90 is an interesting target for cancer analysis, decision to publish, or preparation of the article. therapy because it is involved in folding and stabilization of numerous proteins, including those that contribute to the Conflicts of Interest development of cancer. In mammals, Hsp90 chaperones include Hsp90 alpha and Hsp90 beta, GRP94 (94 kDa glu- /e authors declare that there are no conflicts of interest cose-regulated protein), and TRAP-1 (tumour necrosis regarding the publication of this paper. factor receptor-associated protein 1) localized in the cyto- plasm, ER, and mitochondria, respectively [91]. Hsp90 is Acknowledgments implicated in the pathogenesis of numerous diseases, in- cluding cancer. 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