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The Role of Nutraceuticals in Chemoprevention and Chemotherapy and Their Clinical Outcomes

The Role of Nutraceuticals in Chemoprevention and Chemotherapy and Their Clinical Outcomes Hindawi Publishing Corporation Journal of Oncology Volume 2012, Article ID 192464, 23 pages doi:10.1155/2012/192464 Review Article The Role of Nutraceuticals in Chemoprevention and Chemotherapy and Their Clinical Outcomes 1, 2 1, 3, 4, 5, 6 Sabita N. Saldanha and Trygve O. Tollefsbol Department of Biology, University of Alabama at Birmingham, 175A Campbell Hall, 1300 University Blvd, Birmingham, AL 35294-1170, USA Department of Math and Sciences, Alabama State University, P.O. Box 271, Montgomery, AL 36101-0271, USA Clinical Nutrition Research Center, 402 Webb Nutrition Sciences Building, 1675 University Blvd, University of Alabama at Birmingham, Birmingham, AL 35294-3360, USA Comprehensive Cancer Center, University of Alabama at Birmingham, 1802 6th Avenue South, North Pavilion 2500, Birmingham, AL 35294, USA Center for Aging, University of Alabama at Birmingham, 933 South 19th Street, Room 201, Community Health Services Building, Birmingham, AL 35294-2041, USA Nutrition Birmingham Obesity Research Center, University of Alabama at Birmingham, 402 Webb Nutrition Sciences Building, 1675 University Blvd, Birmingham, AL 35294-3360, USA Correspondence should be addressed to Trygve O. Tollefsbol, trygve@uab.edu Received 30 June 2011; Accepted 25 August 2011 Academic Editor: Julian J. Raffoul Copyright © 2012 S. N. Saldanha and T. O. Tollefsbol. 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. The genesis of cancer is often a slow process and the risk of developing cancer increases with age. Altering a diet that includes consumption of beneficial phytochemicals can influence the balance and availability of dietary chemopreventive agents. In chemopreventive approaches, foods containing chemicals that have anticancer properties can be supplemented in diets to prevent precancerous lesions from occurring. This necessitates further understanding of how phytochemicals can potently maintain healthy cells. Fortunately there is a plethora of plant-based phytochemicals although few of them are well studied in terms of their application as cancer chemopreventive and therapeutic agents. In this analysis we will examine phytochemicals that have strong chemopreventive and therapeutic properties in vitro as well as the design and modification of these bioactive compounds for preclinical and clinical applications. The increasing potential of combinational approaches using more than one bioactive dietary compound in chemoprevention or cancer therapy will also be evaluated. Many novel approaches to cancer prevention are on the horizon, several of which are showing great promise in saving lives in a cost-effective manner. 1. Introduction lung and bronchus, breast, colorectal, and prostate [4, 5]. These cancers are also more prevalent in the western parts The transformation of a normal cell into a cancerous pheno- of the world and are much lower in Asian countries. A well- type requires stages of initiation, progression, and promotion balanced diet that includes more of vegetables and fruits by altering specific genes [1–3]. Although predisposition with less fat/meat intake is in most cases a staple of many to cancer cannot be signaled out by a single factor, a Asian countries [4, 5]. Many hypotheses have supported that group of factors place some individuals at a higher risk diet and environment greatly influence cellular function and of acquiring the disease. Most of the high-risk cases may health [6]. have a genetic background, but in some instances dietary Phytochemicals are plant-based chemicals that mediate choices can dictate the outcome of health. As determined their positive health benefits directly, by affecting specific by population and epidemiological studies, the predominant molecular targets such as genes, or indirectly as stabilized forms of cancer and cancer-related deaths are those of the conjugates affecting metabolic pathways [7]. Many genes play 2 Journal of Oncology significant roles in the cell cycle pathway, and some of these (e.g., curcumin and resveratrol). Once tumor regression sets are altered in cancer cells [1, 2]. The aim of most studies is in, dietary composition of the molecule can be adjusted. to understand and formulate mechanistic pathways by which these naturally derived chemicals can alter the fate of a cell. 2. Nutraceuticals and Their Preventive and Foracancerouscelltosurvive,itshouldbeabletoproliferate, Therapeutic Roles obtain energy, and establish angiogenic pathways, in a tumor mass. Altering genes that affect these pathways can serve as 2.1. Genistein: A Potent Isoflavone. Many phytochemicals suitable tools to decrease tumor mass and also allow for are currently being investigated for their promising anti- tumor regression. In this paper, the key focus will be on carcinogenic properties. In vitro investigations have shown mechanistic pathways that are regulated by nutraceuticals to that some compounds exert their antitumor functions at bring about changes in the tumor environment and serve much higher concentrations and that dietary consumption as alternative approaches for cancer prevention and therapy is insufficient to achieve such effective concentrations at (Figure 1). the tumor site. Therefore, the mode of delivery is a very The study of phytochemicals and the classification of important factor that needs to be considered at clinical trials these compounds have been previously reviewed [8]. How- and during in vivo studies. The nontoxic properties of natural ever, in this paper only some of the most potent and prom- compounds are essential to the design of a formulated ising chemopreventive and therapeutic molecules will be therapy. However, evidence along several lines of treatment analyzed, with emphasis on combination therapy of these has shown that some compounds are preferentially more with other nutramolecules. Most phytochemicals derived potent in activity when administered early in life [9, 10]. For from dietary sources are classified under an umbrella of instance, soy-based prevention of breast cancer is thought to specific chemical compounds as detailed in Table 1. These be more successful when soy products and their derivatives molecules may not have a nutrient value but are germane are consumed in early development [9]. to the function of a cell. Various studies have shown that Isoflavones are a group of phytochemicals that are pre- these molecules can induce apoptosis, inhibit cellular prolif- dominant constituents of a soy-based diet [9, 10]. Among eration, affect angiogenesis, and affect cancer metabolism in isoflavones, the three major constituents that have been various cancers, all of which are hindrances to tumor growth shown to have remarkable influences in cancer prevention (Figure 1)[7]. and therapy are genistein, diadzein, and glycitin [11]. Several of the phytochemicals listed in Table 1 have been They are collectively grouped as phytoestrogens for their investigated in terms of their curative properties. However, weak estrogen-like activity and bind preferentially to ER- one must carefully interpret the observed results in vitro β receptors [12–15]. Evidence of antiproliferative activity and in vivo before testing the same in a clinical setting. The of genistein in vitro stems from its ability to inhibit the reasons for this are manyfold. Tests in culture are pure, in that there is only one cell type in the culture plate and all condi- tyrosine kinase enzyme that is most often upregulated in tions are controlled, including the bioactive compound. In cancer cells [16, 17]. As a chemopreventive agent, genistein is vivo, however, the scenario changes as there are a host of thought to influence the differentiation process of mammary other factors that need to be taken into account, including tissue. It is believed that early differentiation of mammary age, weight, diet, and metabolism of the compound. A tissue into terminal buds, as seen in rats, serves as a bioactive molecule in culture may be subjected to less chemopreventive strategy as it reduces the susceptibility of metabolic changes and may be presented to the cell in its the epithelial cells in the ducts to carcinogens or estrogen native form. However, in vivo the same compound may be and the ontogeny process [9]. Many aggressive cancers have presented differently, perhaps as a conjugate, and its mode of altered epidermal growth factor (EGF) receptors on their action may change amongst the multitude of other molecules cell surface allowing for a continuous downstream signaling in the host’s microenvironment. Many in vivo experiments pathway for cell division [18, 19]. This is interesting, as also control for the type of diet being administered to the genistein can serve as a two-fold approach molecule for organism, where the concentrations or plasma availability prevention and treatment. When EGF binds to its receptors, can be adjusted. Therefore, what may work well in vitro, tyrosine kinase activation results in the phoshorylation of may have no agonistic effects or even antagonistic effects in tyrosine residues of proteins involved in downstream cell vivo, and such discrepancies are often seen when comparing signaling pathways that trigger cell division. Though studies population and epidemiological studies in terms of chemical have shown that genistein increases the EGF transcript early efficacy. in development of mammary tissue, this perhaps is essential An effective nutraceutical is one that will have a low for differentiation and faster development of the breast nontoxic dose while creating a magnitude of change in tumor tissue. In the long run this is a positive preventive strategy dynamics. This means that at a low dose the compound of breast lesion formation in ducts [9]. However, as seen in should act fast on the tumor load. However, if the time older rats [9], EGF mRNA decreases. Therefore, a decrease taken to be effective is slow, the problems faced would be in EGF mRNA coupled with inhibition of tyrosine kinase by maintaining a tolerable dose and increasing bioavailability genistein would profoundly decrease tumor growth as cell and stability. A solution to such a problem would be to use signaling pathways are crucial to tumor maintenance. a combinatorial approach to therapy, a bioactive molecule Numerous studies have highlighted the antiproliferative with an effective synthetic drug or double-nutratherapy role of genistein in various cancers; however, there are some Journal of Oncology 3 Tumor spread • Inhibits tumor invasion Precancerous lesions • Antimetastasis • Activates anti-inflammatory Pathways modulators influenced by • Induce phaseIIenzymes • Inactivate phase I enzymes nutraceuticals Tumor growth Chemopreventive • Inhibits cell proliferation • Induce cell differentiation • Induce apoptosis Chemotherapeutic Figure 1: Cellular pathways affected by the activities of bioactive components in dietary sources. Of the natural compounds present in dietary sources, some are more involved in regulating chemopreventive pathways and some are more effective in influencing chemotherapeutic pathways. However, a few of the bioactive molecules found to date can impart both chemopreventive and therapeutic effects, such as EGCG and genistein. Compound combinations as discussed in the paper that can affect different pathways are shown and can have profound effects on tumor growth and inhibition. Table 1: Classification of nutrients as phytochemicals and their major food source availability. Phytochemical Bioactive compound Source Molecular formula Reference class Caffeine Cacao, tea, coffee C H N O 8 10 4 2 [20] Alkaloid Theophylline Cacao, tea, coffee C H N O 7 8 4 2 Citrus oils from orange, lemon, Monoterpenes Limonene C H [21] 10 16 mandarin, lime, and grapefruit Allicin Garlic C H OS [22–25] 6 10 2 Indole-3-carbinol Cabbage C H NO [26] 9 11 2 Organosulfides Isothiocyanates Broccoli CNS [27] Sulforaphane Broccoli C H NOS [28] 6 11 2 Carotenoids Beta-Carotene, lycopene Tomatoes C H [29] 40 56 Epigallocatechin-3-gallate Green tea C H O [30] 29 22 15 Flavonoids Quercetin Black tea C H O [31] 15 10 7 Curcumin Turmeric C H O [32] 21 20 6 Capsaicin Chilli peppers C H NO [33] 18 27 3 Phenolic Acids Ellagic acid Black berries, raspberry C H O [34, 35] 14 6 8 Gallic acid Pomegranate, nuts C H O [36, 37] 7 6 5 Pterostilbene Blueberries and grapes C H O [38] 16 16 3 Stilbenes Resveratrol Almonds, blueberries, grapes C H O [39] 14 12 3 Daidzein Soy C H O 15 10 4 Isoflavones [9, 40] Genistein Soy C H O 15 10 5 Molecular formulas obtained through the PUBCHEM COMPOUND Database. 4 Journal of Oncology studies indicating that genistein may increase cell prolifera- the conjugates in the tissue allows available free genis- tion [19, 47]. A key point to note is that nutraceuticals can tein delivery to the cells, as presented or tested in vitro. be effective based on the form of genistein or its dose given at For pharmaceutical companies, it is required to formulate the time of the study (Tables 2 and 3), especially with respect supplements with precise ratios of individual constituents to in vitro and in vivo models. Importantly, the downstream of the compound. Unless a very pure form, a capsule or targets of bioactive molecules under investigation need to supplement may contain a mixture of genistein, diadzein, be ascertained for each specific tissue, if overall health and glycetin (Tables 1, 2, 3,and 4). The percentage of each applications are an issue. The nutraceutical may not affect nutrient in the mixture will have a profound effect on the aspecific commonpathway fortumorsofdifferent origins. bioavailability of the compound after metabolism (Tables 2 For example, in breast tissue, EGF may be highly expressed, and 3). To design such a product is certainly not easy and but, in colon cells or pancreatic cells, genes that regulate is dependent on many factors, but the two essential factors cell division other than EGF may be affected [48]. Cell are the grade/stage of the tumor and the site or origin of culture experiments using plant-based nutrients depend on the tissue. Of the two isoflavones, diadzein has been shown the sensitivity of the cells that are being investigated. When to have a lesser apoptotic effect on prostate cancer cells cell lines are established, they are derived from cancerous but can inhibit neoplastic transformation [61]. Therefore, it tissues of specific organs and are, therefore, cell-type specific. would be advantageous to use supplements containing the This is drastically different in a clinical setting where the two bioactive nutrients as chemopreventive agents. molecule has to mediate its activity amongst a host of various Of the predominant high-risk cancers, genistein appears molecules and cell types. Therefore, the concentration of the to have a greater affect on prostate cancers [52–54]. Genis- phytonutrient in the supplemented diet will be crucial to its tein mediates the apoptosis of cancer cells by activating efficacy in the tumor environment. This can help explain the and/or inhibiting genes and/or enzymes germane to tumor discrepancies seen in clinical trials of genistein for different maintenance (Figure 1,Table 4). Some of these important tissues [47, 49, 50]. Outcomes of some in vitro studies suggest mechanisms are the inhibition of the activity of tyrosine that, like other bioactive compounds, genistein appears to kinase, nuclear factor kappa B (NF-κB), and vitamin D have a specific cut-off concentration at which this isoflavone 24-hydroxylase [86], activation of tumor suppressor genes, can exhibit anticarcinogenic activity (10 μMorevenhigher) and modulation of androgen-responsive gene expression, [48, 51], and it is, therefore, imperative to achieve such prostate-specific antigen (PSA), and the androgen receptor concentrations in vivo. (Table 4). Of the prominent isoflavonones in soy, diadzein Isoflavones, in particular, genistein, have been extensively is less effective in its action on prostate cancer, but, unlike studied as prospective antitumor molecules in the treatment genistein, it is metabolized to equol, an isoflavandiol which of prostate cancer [19, 52, 53]. There has been a well has a longer half-life than genistein [87]. The longer half- established line of evidence that genistein works against life of equol creates the possibility of using this chemical in prostate cancer, but a majority of studies are in vitro in combination with other available nutraceuticals, where the cultured cells [19, 52–56]. Limited clinical trials have tested net effect may be synergistic. However, prior preclinical tests the therapeutic efficacy of genistein in prostate cancer and are required to investigate this possibility. those that have revealed inconsistencies in cell proliferation Other dietary compounds are also of great interest and tumor growth [57–60]. Given the inconsistencies in in this regard. In vitro, vitamin D (Vit D) has potent some of the outcomes, emphasis should be on the dose tumor prevention ability and can induce differentiation and of the supplement and the form of the nutrient in the apoptosis in some of the most predominant cancers [48]. supplement at the time of administration to the patient in The use of nutrients as a possible treatment approach is clinical trials. The highest achievable plasma concentration based on the fact that chemicals occurring naturally will of isoflavones is 1 μM through orally administered food minimize side effects when applied to a biosystem. However, sources. From previous studies, this concentration is not the in vitro dose at which Vit D induces its antitumor sufficiently significant to bring about anticarcinogenic effects properties causes hypercalcemic conditions that can preclude on the tissue. However, there is ample evidence that genistein treatment in patients [49]. In prostate cancer, a leading cause and other isoflavones do exhibit anticancer properties and of cancer deaths in the western parts of the world, androgen inhibit cell proliferation and tumor growth. A clinical study ablation therapy is the choice of treatment. However, as the by Gardner et al. [61] showed that treatment of patients with cancer becomes aggressive, hormone ablation therapy fails, dietary supplements (82 mg/day aglycone equivalents) of and progression ensues via androgen-independent pathways. isoflavone yielded a higher concentration of total isoflavones Therefore, alternate therapies are very much in demand. in the prostatic tissues than in serum. Therefore, there is a Vitamin D is an alternate form of treatment in prostate possibility of increasing the concentration of isoflavones to cancer (PCA) and is shown to induce apoptosis in PCA cells anticarcinogenic levels in tissue. in vitro. However, all PCA cell lines in vitro are not equally An orally administered dose of isoflavones must with- receptive to the vitamin D treatment or genistein [88]. Cell stand the rigors of the alimentary canal and become lines such as DU145 prostate cancer cells are especially more metabolized before they can be made available to tissues. resistant as they express high levels of CYP24, an enzyme Most isoflavones exist as conjugates rather than in their free that catabolizes Vit D3 into less active metabolites [88]. To state. This conjugation is perhaps the best way to present circumvent this problem, a recent study showed that a dual the molecule to the cell in tissues, and the hydrolysis of combination therapy, of DU145 to genistein and Vit D3, Journal of Oncology 5 Table 2: Pharmacokinetic studies evaluating the bioavailability of phytochemicals at given doses. Phyto Experimental Maximum plasma Form Dose Model subject Half-life (h) Reference chemical setup concentration 15 g Human Soy beverage Diadzein : genistein 96.31 ng/mL 7.68 Diadzein postmenapausal (9.27 : 10.51 mg) Clinical [40] women Two soy Diadzein : genistein 96.02 ng/mL 6.67 capsules (7.79 : 22.57 mg) 15 g Diadzein : genistein Human Soy beverage 116.37 ng/mL 7.61 Genistein (9.27 : 10.51 mg) postmenapausal [40] Clinical women Two soy Diadzein : genistein 216.84 ng/mL 7.96 capsules (7.79 : 22.57 mg) Curcumoid powder form 10 g (n = 6) 2.04 ± 0.31 curcumin 6.77 ± 0.83 (75%), Healthy human Curcumin for total demethoxycur- Clinical subjects (5 men [41] glucoronide curcumin cumin (23%), and 7 women) 12 g (n = 6) conjugates 1.40 ± 0.74 and bisdemethoxy curcumin Curcumoid powder form curcumin 10 g (n = 6) 1.06 ± 0.40 6.77 ± 0.83 (75%), Healthy human Curcumin for total [41] demethoxycur- subjects (5 men Clinical sulfate curcumin cumin (23%), and 7 women) conjugates 0.87 ± 0.44 and 12 g (n = 6) bisdemethoxy curcumin Quercetin Quercetin 500 500 mg of quercetin 15.4 ng/mL 3.47 aglycone plus capsule Healthy human subjects (6 Not given for Clinical [42] Quercetin Quercetin 500 plasma level, but males and 4 500 mg of quercetin 336 ng/mL conjugates plus capsule female) renal clearance is 0.835 500 mg resveratrol/caplet 0.5 g ∗ ∗ 72.6 (48.9) ng/mL 2.85 Uncoated Healthy human Phase I immediate- Resveratol [43, 44] 1.0 g subjects clinical test release 117.0 (73.1) ng/mL 8.87 (91.1) caplets 2.5 g 268.0 (55.3) ng/mL 4.22 (51.6) 5.0 g 538.8 (72.5) ng/mL 8.52 (95.8) @ @ Broccoli raw 200 g 103 ± 31 ,nM 3.8 ± 0.8 Sulforaphane Healthy adult Broccoli [45] Clinical @ @ 200 g male subjects 31 ± 19 nM 4.6 ± 0.8 cooked −1 Per dose (L ) Beverage Healthy human −3 EGCG 112 mg Clinical 0.51 × 10 ± 3.2 ± 2.1 [46] 200 mL subjects −3 0.08 × 10 D-Limonene oxygenated 30–40 ounces 447–596 mg Healthy human Clinical 2.08–13.98 μM 12–24 [21] metabolite of lemonade D-limonene subjects perillic acid 6 Journal of Oncology Table 2: Continued. Phyto Experimental Maximum plasma Form Dose Model subject Half-life (h) Reference chemical setup concentration Range between Lycopene with Healthy adult 4.03 and Range between Lycopene up to 250 mL 10–120 mg Clinical [29] male subjects 11.27 μg/dL 28.1 61.6 h water (0.075–0.210 μM) coefficient of variation; SD—standard deviation. increased the sensitivity of the cells to Vit D3 by decreasing alone. In addition, NF-κB was transcriptionally inhibited in CYP24 expression. What is interesting to note is that the the combined treatment [90]. combination approach not only lowered the effective dose, From a number of investigations, a common thread of but was able to abrogate cell proliferation as well. This evidence seems to emerge that considerable variation in the lowered concentration of genistein at 100 nM is achieveable efficacy of bionutrients in cancer treatment exists and differs in vivo through dietary sources, and clinical studies would be even among the same cell lines tested. The reasons for this required to determine the localization of genistein and Vit are manyfold (Table 5). Cell lines derived from the same D3 in prostatic tissues. tissue hypothetically should be sensitive to the same dose or An in vivo study for colorectal cancer has demonstrated chemical class of the phytonutrients, but such is not always a similar effect [89], but in this case the mice were given a the case. Alternate medicinal approaches have an important single gavage of 250 μg of genistein. This mode of nutrient task to identify crucial factors that change the sensitivity administration is useful for a preclinical test and probably of the chemical and determine chemical modifications that has applications as a chemopreventive supplement. However, would be necessary to modulate more synchronized results in terms of a clinical setting, patients are often exposed to across several cell lines expressing similar genotypic and a host of other nutrients or isoflavones in their diet, and; phenotypic signatures. therefore, an in vivo model replicating such an environment with various percentages of isoflavones will allow for a 2.2. Epigallocatechin-3-gallate (EGCG): A Potent Flavanol. better understanding of concentration and bioavailability of Of the major food-derived phytochemical constituents genistein that can mediate an apoptotic effect and reduce that are extensively studied for their chemopreventive and CYP24 expression in colonic tissues in the presence of chemotherapeutic use, EGCG and genistein are by far the vitamin D. most investigated. EGCG has been shown to have numerous The antimetastatic properties of genistein are mediated anticancer properties which include antiangiogenic activ- by altering the expression of NF-κB, and inhibiting the itybyaffecting the transcriptional expression of vascular tyrosine kinase enzyme [17, 90]. Non-small-cell lung cancer endothelial growth factor (VEGF) [91], inhibiting tumor (NSCLC) is a highly aggressive form of lung cancer with a initiation and promotion by inhibiting signal transduction poor prognosis. Therefore, alternate approaches that drasti- pathways via [phosphatidyloinositol 3-kinase-Akt kinase- cally reduce tumor growth are of utmost importance. Acti- NF-κB] [92–94], inhibiting EGFR [95], inhibiting Her- vation of epidermal growth factor receptor tyrosine kinase 2 receptor phophorylation in breast carcinoma cells that (EGFR-TK) enhances the cell signaling pathways allowing constitutively expresses Her-2/neu receptor [95], inducing tumor growth. The use of drugs that inhibit EGFR-TK and apoptosis in estrogen receptor-(ER-) independent breast affect NF-κB, a gene whose transcribed products are essential cancer cells [96], causing antimetastatic activity [97], inhibit- for invasion and metastasis, can induce a more aggressive ing proteasome formation [98], inhibiting glucose-regulated approach of reducing tumor size and the spread of the protein (GRP78) activity [99]; inhibiting insulin-like growth disease. A clinical therapy should be aimed at reducing tumor factor-I receptor (IGF-IR) [100], and preventing invasion of growth and spread by inhibiting mechanisms that contribute tumors by inducing HMG-box transcription factor 1 (HBP1) to the activation of metastasis. In NSCLC, genistein remark- transcriptional repressor, an inhibitor of the Wnt signaling ably enhances the effects of EGFR-TK inhibitors, such as pathway crucial for tumor-invasive property [101]. erlotinib and gefitinib, when used in combination with each The serum level concentrations of EGCG are impor- of them, respectively. This effect was seen to be mediated tant to ensure that an effective response is seen without by a marked reduction in NF-κB and others, such as EGFR, adverse or even tumor-promoting functions. Studies have pAkt, COX-2, and PGE(2), essential for regulating genes that shown that high doses of catechins that include a higher control division, proliferation and metastasis [90]. A few concentration of two prominent compounds, epicatechin studies have shown how a combined approach can lower gallate (ECG) and EGCG, induce hypoxia-inducible factor 1 the effective dose concentration even of chemotherapeutic which is responsible for activating genes related to hypoxia drugs, minimizing potential side effects. A study conducted conditions. This allows tumor cell proliferation through on breast and pancreatic cells showed that, when the cells alternate survival pathway mechanisms [102]. Most breast were primed with genistein, lower concentrations of the cancers are ER dependent; however, for breast cancers and chemotherapeutic drugs were needed to significantly bring others that are ER independent, EGCG inhibits the growth about growth inhibition and apoptosis than with the drugs of tumor cells through the process of apoptosis [96, 103]. Journal of Oncology 7 Table 3: Single-dose clinical studies evaluating the bioavailability of phytochemicals or their conjugated or active metabolites. Route of Bioavailability area Phytochemical Form AUC values Reference administration under the curve (AUC) Adjusted to the Diadzein Oral Soy beverage 107 ± 49.16 ng·h/mL [40] dose Soy extract Adjusted to the 142.61 ± 43.94 ng·h/mL [40] capsules dose Adjusted to the Geistein Oral Soy beverage 121.48 ± 70.98 ng·h/mL [40] dose Soy extract Adjusted to the 131.04 ± 60.79 ng·h/mL [40] capsules dose Curcuminoid powder extract Oral 35.33 ± 3.78 μg·h/mL Relative [41] capsule form Curcumin (10 g) conjugates (glucoronide + Curcuminoid sulfate) powder extract Oral 26.57 ± 2.97 μg·h/mL Relative [41] capsule form (12 g) Capsule Quercetin aglycone Oral 62.5 ng·h/mL Relative [42] (500 mg) Quercetin- Capsule conjugated Oral 2000 ng·h/mL Relative [42] (500 mg) metabolites Caplet ranging from 0.5 g 223.7 ng·h/mL ∗ [43] Oral Relative for all Rersveratrol 1.0 g 544.8 (57.2) ng·h/mL 2.5 g 78.6 (36.2) ng·h/mL 5.0 g 1319 (59.1) ng·h/mL 200 g broccoli ◦ [45] Sulforaphane Oral Relative Raw 495 ± 40 nM·h Cooked 286 ± 139 nM·h Average 200 mL EGCG Oral AUC nd [46] beverage D-Limonene (perillic acid a 40 oz of major active Oral 5.07 to 32.59 μM·hRelative [62] Lemonade metabolite of d-Limonene) Liquid form (AUC) 0−96 (tomato paste) 10 mg 214 ± 124.8 μg·h/dL 30 mg 416.4 ± 183.9 μg·h/dL Lycopene Oral Relative [29] 60 mg 421.7 ± 59.3 μg.h/dL 90 mg 598.9 ± 396.8 μg·h/dL 120 mg 655 ± 298.6 μg·h/dL AUC value measured for resveratrol was AUC infinity with the coefficient of variance denoted in the brackets against the mean value. For the lowest dose of resveratrol AUC infinity value n = 1. AUC value measured for sulforaphane was AUC0-∞. Based on the reference paper a list of various AUC values was given for different single doses as experimentally performed by different laboratories. Since the sample numbers were different, an average AUC value has not been given for this compound. nd—not determined. 8 Journal of Oncology Table 4: Assessment of the chemotherapeutic and chemopreventive effects of nutraceuticals in combination studies. Combination of Pathways affected or Phase of Dose used Organ of study Model of study Reference nutraceutical mechanistic action study 50 μM/L + Inactivation of NF-κB Human breast Curcumin + 10–50 μM/L based and other metastatic In vitro cancer cells paclitaxel on the gene genes. MDA-MB-435 assessed [63] Breast Human breast Curcumin + 2% w/w Inhibition of In vivo cancer xenograft paclitaxel 10 mg/kg metastasis model Synergistic effect in Human breast Curcumin + Induction of the range from 5 to Breast In vitro MDA-MB-231 [64] xanthorrhizol apoptosis 20 μg/mL cancer cells Inhibition of proliferation, more Ratio of DHA to synergistic in one of CCM the 5 cell lines tested. MCF-7 Enhanced uptake of Human breast 55 : 30 μM curcumin by the cells. cancer cells MCF10A Upregulated genes SK-BR-3, Curcumin + 95 : 45 μM involved in cell cycle Breast In vitro MDA-MB-231, [65] docosahexenic acid MDA-MB arrest, apoptosis, MDA-MB-361, 35 : 35 μM inhibition of MCF-7, and SK-BR-3 metastasis, and cell MCF10AT 60 : 40 μM adhesion. MDA-MB Downregulated genes 50 : 25 μM involved in metstasis and invasion. 10 μM+25 μM T47D and Change in cell Curcumin + [66] morphology and Breast In vitro 10 μM+25 μMMCF-7 genistein growth inhibition 11 μM+25 μM MDA-MB-231 Alter multidrug resistance genes. Alters the cell cycle Curcumin + [67] 15 μM+10 μM Lung In vitro NCI-H460/R with cells inhibited sulphinosine primarily in the S G2/M phase of the cycle Inhibition of cell proliferation and HT-29 induction of apoptosis. IEC-18-K-ras Curcumin + 10–15 μM/L + [68] Colon In vitro (Cox-2, high celecoxib 5 μM/L Possible inhibition of levels) Caco-2 Cox-2 pathways or (COX-2, low through non-Cox-2 levels), and pathways SW-480 (no COX-2) Inhibition of tumor Coltect only 20 μM growth by induction In vitro HT-29 cells of apoptosis. Coltect + Chemical [69] 5-aminosalicylic Colon induction of acid (ASA) 150 mg/kg + Inhibits abnormal tumors by In vivo 50 mg/kg crypt formation 1,2-dimethyl hydrazine (DMH) model in rats. Journal of Oncology 9 Table 4: Continued. Combination of Pathways affected or Phase of Dose used Organ of study Model of study Reference nutraceutical mechanistic action study Additive effectives in 25 μM+10 μM the induction of In vitro PC-3 C4 cell line [27] apoptosis. Curcumin + NCr- Prostate PEITC immunodeficient Inhibition of tumor (nu/nu) mice growth through 3 μM+2.5 μM In vivo bearing s.c. [70] inhibition of Akt and xenografts of PC-3 NF-κBpathways. human prostate cancer cells Pure 3—curcumin Inhibit growth by PC-3, LnCaP and +resveratrol + inhibiting hedgehog In vitro mouse cell line EGCG; signaling pathways. TRAMP-C2 Pure 4—apigenin + baicalein + Individual genistein + Prostate [71] compounds, quercetin; Percentage Pure 7—Pure 3 + Transgenic composition in the Pure 4; Reduce or delay the adenocarcinoma of In vivo diet not defined Crude 7—soy + onsetoftumors. the mouse prostate sencha leaves + (TRAMP) mice turmeric + yucca roots + saw palmetto + chamomile flowers + gingko Induction of Human prostate apoptosis by the carcinoma DU-145 D-Limonene + regulation of proteins 0.2 mM+1.9 nM Prostate In vitro and normal [72] docetaxcel involved in prostate epithelial mitochondrial PZ-HPV-7 cells apoptotic pathways 11 nM of lycopene per g of diet and broccoli powder, Reduction of tumor 1.6 μMof Tomato powder + growth mediated by Dunning R3327-H glucoraphanin, broccoli powder reduced cell prostate 5.9 μMof Prostate In vivo [73] (10 : 10) g/100 g of proliferation and adenocarcinoma glucobrassicin, diet induction of model 3.9 μMof apoptosis gluconasturtiin, and 2.1 μMof neoglucobrassicin. Synergistic effect in inhibiting cell proliferation 1 μM/L + 2 mM/L mediated processes. In vitro Mat-Lylu rat cells Lycopene + Antioxidant activity ketosamine to prevent initiation Prostate [74] (fructose/amino of tumors. acid Fru/His) Subcutaneous injections of 20 μM/L + Reduce tumor growth In vivo Mat-Lylu cells in 5.6 mM/L and volume. male Copenhagen rats 10 Journal of Oncology Table 4: Continued. Combination of Pathways affected or Phase of Dose used Organ of study Model of study Reference nutraceutical mechanistic action study Synergistically Human PC-3, enhances the 1 μM+1 nM In vitro LnCaP, DU145 antiproliferative cells effects of docetaxel. Reduced tumor volume and growth Lycopene + Prostate [75] by affecting the levels docetaxel of IG-FR receptor that Xenograft of 15 mg/kg lycopene is highly expressed in DU145 cells in +10 mg/kg a majority of prostate In vivo NCR-nu/nu docetaxel tumors. Inhibited Akt (nude) mice signaling and suppressed surviving necessary for tumor growth Quercetin chalcone Reduction in the (QC) and a 1.6 mg/mL + growth of solid Colon In vivo Balb/c mice [76] pH-modified citrus 1.6 mg/mL primary tumors pectin (MCP) Inhibits the self renewal capacity of prostate cancer stem cells (PCSCs) by synergistically Prostate cancer Quercetin + EGCG 20 μM+0–60 μM inducing apoptosis Prostate In vitro [31] stem cells (PCSCs) decreasing cell viability in spheroids, cell migration, invasion and colony formation Antagonistic Resveratrol + estrogenic effects in Human MCF-7 10 μM+1 nM Breast In vitro [39] estrogen (E2) suppression of cells progesterone receptor Synergistically Either all at 0.5 μM inhibited cell Human and In vitro proliferation and MDA-MB-231 cells 5 μM, or 20 μM induced apoptosis. Inhibited cell cycle [77] Resveratrol + Breast progression with quercetin + predominat cell cycle catechin arrest in the G2 phase 0.5, 5, and Reduced primary Breast cancer 25 mg/kg body tumor growth and, In vivo xenografts in weight in a 100-μL therefore, inhibit mouse models volume tumor progression Inhibit cell proliferation via capase mediated cytotoxicity. Resveratrol + 50 μM+5 mM Enhanced Breast In vitro MCF-7 [78] cyclophosphamide proapoptotic genes Bax, Fas and suppressed anti apoptoticgeneBcl-2 Journal of Oncology 11 Table 4: Continued. Combination of Pathways affected or Phase of Dose used Organ of study Model of study Reference nutraceutical mechanistic action study Inhibited cell proliferation and induced differentiation. Resveratrol + 50 μM+2 mM/L Attentuated p27 Colon In vitro Caco-2 [79] n-Butyrate (Kip1) levels but enhanced p21 (Waf1/Cip1) expression. Inhibited cell proliferation and Resveratrol + 200 μM+IC HCT116 p53+/+ induced apoptosis by Colon In vitro [80] 5-Fluorouracil 800 μM and p53−/− increase in capase 6 activity Suppressed prostate Simian Virus-40 cancer development T-antigen-(SV-40 and mediated Tag-) targeted apoptosis by affecting probasin promoter Resveratrol + 250 mg/kg each in the expression of Prostate In vivo rat model, a [81] genistein the AIN-76 diet steroid-receptor transgenic model coactivor-3 and of spontaneously insulin-like growth developing factor-1 prostate cancer. Affected DNA methyltransferase activity and reversed thegeneexpression of promoter hypermethylated Genistein + genes of retinoic acid 5 μM/L + 15 μM/L Esophagous In vitro KYSE 510 cells [82] sulforaphane receptor h (RARb), RARB, p16INK4a p16 and O6-methylguanine methyltransferase enhanced growth inhibitory effects Changed cell morphology and inhibited cell Sulforaphane + proliferation. benzylisothio- 10 μM+10 μM Reduced cell viability Pancreas In vitro PANC-1 cells [83] cyanite that correlated with reduced pSTAT3 levels and an increase in PARP Cleavage Synergistically induced phase II enzyme UDP-glucoronyl transferases Sulforaphane + 10 μM+10 μM (UGT1A1) transcript Colon In vitro CaCo-2 [84] apigenin but to a lesser effect the protein level. Mediates this action by the induction of NF-κB 12 Journal of Oncology Table 4: Continued. Combination of Pathways affected or Phase of Dose used Organ of study Model of study Reference nutraceutical mechanistic action study Has an antagonistic effectatlow Human colon 2.5 μM+20 μM concentration on cell cancer 40–16 cell Sulforaphane + growth. line randomly [85] 3,3 - Colon In vitro At cytotoxic derived from diindolylmethane Total concentrations of the HCT116 clone (DIM) concentration compounds has 40 μM synergistic effects on growth inhibition AIN-76A diet Sulforaphane + supplemented with Blocked colon tumor min+ dibenzoymethane Colon In vivo Male Apc/ mice [28] 300 ppm SFN and development (DMB) 0.5% DMB Table 5: Factors conducive to the anticarcinogenic efficacy of EGCG with the drug taxol, which is commonly used to nutraceuticals. treat breast carcinomas, lowered the effective dose of EGCG, ranging from 0.1–1.0 μg/mL which is a serum obtainable Possible effects on the bioactive Factors level through metabolism. This same group showed that components in the dietary supplement higher doses (30–40 μg) of EGCG were required to mediate a Metabolism similar effect when used alone [95]. Bioavailability Time taken to achieve maximum EGCG can be exploited as a chemopreventive agent if plasma concentration it prevents cancerous lesions from occurring at lower dose Maximum plasma concentration, concentrations and for prolonged periods of time. Most half-life in vitro studies have used relatively high doses of EGCG Oral and such doses may prove to be more tumor promotive Method of Intraperitoneal than preventive in longer exposure time periods. In a study ingestion Subcutaneous designed by Pianetti et al. [92], contradictory results on the effects of EGCG on Her-2/neu overexpressed receptor in Powder/capsule NF639 breast cancer cells was observed. At short exposure Liquid Form of ingestion times, EGCG was very effective in reducing cell proliferation, Cooked (solid) but at prolonged exposure cells became resistant to EGCG Raw (solid) with increased levels of NF-κB. This observed change in Ratio of pure to the compound drug-induced resistance was related to the activation of Formulation conjugates mitogen-activated protein kinase. It appears that single doses Preference for an acidic or basic or one specific chemical constituent is mostly insufficient to Stability environment (pH) induce tumor suppression or regression. Such in vitro data Direct via receptors on the cell surface outcomes emphasize that a dual-drug treatment approach is Mechanism of or into the nuclear region via channels necessary to treat the disease. This also signals that the timing action Indirect conjugated metabolites of the nutradrug that is administered is important. Perhaps affecting parts of metabolic pathways EGCG should be administered early in treatment, but later other phytochemicals or drugs, in conjunction with EGCG, may need to be administered in the treatment regimen. In As seen in MDA-MB-468 ER-negative cells, cellular apoptosis their dual-drug treatment of NF639 Her-2/neu breast cancer is mediated by inducing p53 and Bax proteins that enhance cells, Yang et al. found that treating the cells initially with apoptotic functions in cells [96]. Such observations have EGCG lowered cell proliferation and the later introduction been corroborated by in vivo studies using animal models of the MAPK inhibitor, U0126, reduced invasive phenotype [97]. [93]. Most studies have shown that anticancer properties of Most studies determining the anticancer drug properties EGCG are mediated at higher doses. However, such doses of EGCG are preclinical. For better understanding of specific may be irrelevant to clinical applications as they may be EGCG effects, clinical trials should be carefully designed physiologically unachievable through dietary consumption. to include parameters that influence EGCG effectiveness. Therefore, clinical trials should be aimed at achieving EGCG has different roles in ER-dependent versus ER- desired anticancer preventive or tumor functions at much independent receptors, and, therefore, the type of diet lowered doses. Such outcomes are possible with a dual-drug needed to emulate in vitro doses need to be clearly under- approach. One study [95] demonstrated that combining stood through clinical trials and careful pharmacokinetic Journal of Oncology 13 Table 6: Surface receptors expressed by breast cancer cells that alter their sensitivity to treatment. Receptors on the surface Cancer type Phenotype Reference ER PR Her2 Breast cancer cell line SK-BR-3 Negative Negative Positive Adenocarcinoma Invasive [65] MDA-MB-231 Negative Negative negative Adenocarcinoma Invasive [65] MDA-MB-361 Positive Negative Positive Adenocarcinoma Metstasis [65] MCF-7 Positive Positive Negative Adenocarcinoma Invasive [65] Premalignant Isoform B of MCF10AT Positive Variable model for cancer Premalignant [65] PR and not A development studies of these doses in healthy individuals, ER-positive 3.1. Curcumin and Taxol (See [63]). Primary breast cancer cells are commonly treated with the drug taxol. Sus- breast cancer patients, and ER-independent tumors. In testing phytochemicals of the same or different class it tained chemotherapeutic treatment with this drug has often resulted in drug resistance and tumor progression. is rather uncertain which markers are necessary to determine Many chemotherapeutic drugs induce the expression of the comparable dosage values for in vitro versus in vivo efficacies. metastatic gene NF-κB which encourages tumor progression. Formulation of a diet is one of the major deciding factors in Interestingly, natural-based compounds that are pharmaco- the functional efficacy of a chemical constituent. It defines logicallysafehavebeenshown to inactivateNF-κBexpres- the concentration of the dose that will be available in vivo, sion. Taxol is a powerful drug in the treatment of cancer after metabolism, and determines the diet that needs to be therefore, in order to prevent metastasis, a combination of given to achieve such an outcome. Even though single-dose Taxol with curcumin has been shown to downregulate the individual or mixed phytochemical treatments are currently expression of NF-κB and induce apoptosis. available to cancer patients, they are relatively new and much more research in this direction is warranted. One such 3.2. Curcumin and Xanthorrhizol (See [64]). Astudy con- therapy that is rapidly gaining importance and holds promise for future cancer treatments is combination therapies using ducted on an invasive breast tumor cell line, MDA-MB-231, has shown how and when compounds added to the cells plant-based chemical compounds known as nutraceuticals. determine the overall efficacy of the treatment. A sequen- tial addition of curcumin and xanthorrhizol (a rhizomal 3. Combinatorial Therapy: A Promise of sesquiterpenoid of Curcuma xanthorrhiza) in culture resulted in additive and antagonistic effects depending on which theFuture(SeeTable 3) compound was added first to the culture. However, simul- In prevention or treatment, combinatorial approaches can taneous addition of the compounds resulted in synergistic be of the following types: a phytonutrient and an effective effects at lower concentrations and agonistic effects at higher drug, two or more phytonutrients, a synthetic phytonutrient concentrations. Such experiments provide evidence that the and an effective drug, or a synthetic phytonutrient and efficacy of a drug is dependent on dose, time, and how it a natural nutrient. Studies in the last few decades have is presented to the cells. Therefore, results obtained might focused attention on unraveling the protective properties be contradictory if doses used are simply antagonist or and mechanistic actions of many phytochemicals. Still the additive. For a successful combination therapy or prevention, pharmacokinetics of quite a few of these phytochemicals synergistic doses are more relevant to mediate downstream are not known, and, for a few that are known, there is effects, as lower concentrations of the test biomolecules will much variability based on mode and form of delivery, be required. dose, and the model organism of study (Tables 2, 3,and 4). Another interesting approach to enhancing curative 3.3. Curcumin and Docosahexaenoic Acid (DHA) (See [65]). and preventive properties of these nutrients is combination DHA is a dietary compound present in fish oil that has been therapies. The therapy is based on the factual information shown to have potent chemopreventive affects against cancer. available at hand and using the potent properties of one with Chemotherapeutic effects of compounds are often analyzed that of another to enhance synergistic or additive actions using in vitro models. However, what is most often observed (Figure 1). In this paper, groups that have worked with is that all cells from the same tissue sample do not react different phytomolecules belonging to a different or the the same way to the test compound. It is essential to have same chemical class of compounds have been analyzed for a chemopreventive or therapeutic agent that can induce its their antitumorigenic activities, and the overall results of the effects on a wide range of cancerous cells arising from the experiments for each group are described in Table 4. same tissue. In this study, the authors analyzed five cell lines 14 Journal of Oncology expressing different cell surface receptors (Table 6)which in cell cultures has been shown to inhibit cell growth. make them susceptible to chemotherapeutic compounds but This observed cytotoxicity is enhanced several folds when in different ways and to different degrees. The combinatorial low doses of the natural compound, curcumin, are used synergistic doses for each cell line were different, as shown in combination, which are otherwise ineffective unless very in the Table 4. In particular, one breast cancer cell line, high concentrations are used. These compounds mediate a SK-BR-3, which is ER-negative exhibited a higher uptake of synergistic effect in regulating the cell cycle phases and down- curcumin in the presence of DHA. DHA does not directly regulate MDR genes, thereby, enhancing tumor regression contribute to cell inhibition, but the combination of this phenotypes even in the presence of mutated p53 molecules. compound with curcumin greatly enhances the uptake of curcumin by the cells. This compound, DHA, can reach a 3.6. Curcumin and Celecoxib (See [68]). Cyclooxygenase- plasma concentration level of 200 μM. Although the focus 2 (COX-2) expression is central to the carcinogenesis of of this study was entirely based on the SK-BR-3 cell line, colorectal cancers. Compounds that regulate the expression the effects of reduced synergy on other cell lines in terms or activity of COX-2 in cells may be instrumental in of transcriptome effects need to be investigated. Mammary mediating chemotherapeutic effects on the tissue or cells. tumors may contain a heterogenous population of cells Celecoxib is a potent inhibitor of COX-2 and is presumed exhibiting different surface receptors. Using combination to target itsactivesite. However, prolongedexposureto therapy should be aimed at reducing the populations of celecoxib results in cardiovascular problems. It appears that all these cell types within the tumor site to truly exhibit monotherapy regimes are very effective in inhibiting cancer antitumor potency with minimal side effects. growth, proliferation, metastasis, and invasion, as seen in numerous in vitro and in vivo models. However, prolonged 3.4. Curcumin and Genistein (See [66]): A Preventive Strategy. exposures at concentrations relatively higher than what can The aim to use natural compounds in diets is to render be achieved with combination doses may result in unwanted the chemopreventive properties of the compounds to the side effects. Testing the efficacy of celecoxib with cucumin tissues. Numerous studies have shown that single dosage showed that at lower doses of celecoxib it was possible of compounds used alone is effective for chemoprevention. to enforce synergistic inhibitory growth effects on colon The problem faced is the inability to achieve high serum cells which expressed various levels of COX-2. Like many concentrations in vivo. Although combination studies are other in vitro investigations, this study emphasizes the fact just beginning to surface as more prominent approaches in that combining powerful drugs with naturally available clinical treatment, studies, though limited, have shown that potent compounds can reduce the dose needed to mediate synergistic effects of the compounds are able to be achieved potent anticarcinogenic effects with minimal side effects. at much lower doses than when compounds are used alone. Clinically, such studies are relevant as the doses used or Especially in cancers that are hormonally regulated, the needed are within the physiologically dose range. With colon tissues are often exposed to external or internal hormonal cancer having such a high incidence rate in the western stimulation, like estrogen, as in the case of breast tissue. Envi- populations, such therapies can be taken as advantage, ronmental agents that mimic estrogen-like activity can often and biomolecules having preventive potential against the stimulate or initiate the carcinogenic process. Curcumin, a formation of precancerous lesions need to be supplemented curcuminoid, and genistein, an isoflavone, are derived from in diets of patients at high-risk. two different chemical classes, yet they have been known to inhibit a variety of tumor types in vitro and in vivo. 3.7. Coltect and 5-Aminosalicylic Acid (5-ASA) (See [69]). Clinical trials of these compounds individually have been Coltect is a novel chemotherapeutic dietary drug with a tested [19, 60, 104, 105]. The mechanistic action of the formulation of curcumin, a turmeric extract (95% curcum- individual compounds in many different cancers has been inoids) mixed with turmeric powder 1 : 1, green tea (60% investigated as well. However, using these compounds in polyphenols and 25% EGCG) in a 2 : 1 ratio, and 0.1 mg/mL combination drastically affects the development of tumors of L-selenomethionine. 5-ASA is an anti-inflammatory drug, by mediating changes in shape and growth inhibition. Such which has been shown to have a preventive role in polyp for- changes were observed both in ER-positive and ER-negative mation that is thought to occur via the inflammation process cells, indicative of the dual use of such a combination in in conditions like inflammatory bowl disorder. Coltect has prevention and therapy. been effective against HT-29 human colon adenocarcinoma grade II cells in vitro, and this nutraceutical complex in 3.5. Curcumin and Sulfinosine (SF) (See [67]). The inef- combination with 5-ASA has been shown to inhibit the fectiveness of certain drugs in prolonged chemotherapy formation or growth of chemically induced aberrant crypt stems from the resistance that some cancers develop with foci (ACF) in rat models. The molecular mechanism by time. This is one of the major obstacles in cancer therapy, which this inhibition is mediated is via the inhibition of especially in cancers that are multidrug resistant (MDR). The COX-2pathwaysinHT-29 cells, whichhas been supported problem in finding a suitable cure for non-small-cell lung by in vitro studies of other groups [106, 107]. However, cancers is the MDR phenotype it exhibits. Treating MDR growth inhibition can be affected via COX-2-independent cells such as non-small-cell lung carcinoma NCI-H460/R pathways possibly through mechanisms that are regulated by cells with a commonly employed drug, SF (obtained by the the functional polyphenol complex in coltect. Such complex amination and subsequent oxidation of 6-thioguanosine), mixtures are of clinical significance as many different control Journal of Oncology 15 mechanisms can be regulated by the presence of individual into colon cancer. In colonic crypts that are chemically constituents of the polyphenols which are a part of the induced, limonene asserts its effect by inhibiting the activity of ornithine decarboxylase, an enzyme essential for the formulated mixture of coltect. polyamine biosynthesis pathway. This pathway regulates the cell cycle, and D-limonene-dependent inhibition of 3.8. Phenylethylisothiocynate (PEITC) and Curcumin (See ornithine decarboxylase (ODC) encourages an antiprolifer- [27]). Most prostate cancers begin as a hormone-dependent ative activity. If aberrant crypt foci are the initial markers tumor, and the hormone is primarily androgen. However, for colon carcinogenesis, and D-Limonene and its derivatives the more aggressive forms of prostate cancer are androgen- assert their roles against initiation and promotion phases of independent and hormonal therapies fail to be effective. cancer, then a diet rich in citrus foods can prevent crypt for- Alternate therapies are, therefore, necessary to treat such mation. Therefore, D-limonene appears to have potential as aggressive forms. Most cancerous cells express various sur- a chemopreventive agent in colon carcinogenesis. However, face receptors that propagate cellular growth. Targeting such in vivo studies often do not correlate with results in vitro receptors can be an effective chemotherapeutic approach. for many of the reasons discussed earlier. Once the intake Curcumin, obtained from Curcumin longa, has been shown of a compound is deemed safe for human consumption, to inhibit the phosphorylation of EGFR, inhibit the Akt it is imperative to analyze and study the mechanistic and signaling pathway, and negatively regulate NF-κB. It is an metabolic functions in human subjects to determine the effective molecule against prostate cancer. Phenylethylisoth- efficacy of the nutrient in question. As in the case of under- iocyanate, a phytochemical in cruciferous vegetables, has standing limonene protection against colonic carcinogenesis, been shown to inhibit prostate cancer cell growth in vitro the studies were performed on rats and for shorter exposure and this observation has been supported by epidemiological time to the compound or its derivatives. Therefore, further studies showing that consumption of cruciferous vegetables in vivo models are required to determine the toxicity of has an inverse effect on prostate cancer risk. When two the treatment for longer periods of time, as D-limonene is bioactive molecules with similar effects are used in treating nontoxic but its alcohol derivatives could be toxic. hormonally independent tumors in affecting receptor medi- ated signaling, the effects are more pronounced than when 4.1. D-Limonene and Docetaxcel (See [72]). Many combina- used as individual compounds. With PEITC and curcumin, tion studies are underway to determine an effective approach the observed effect was more additive than synergistic, in treating advanced and aggressive prostate cancers. Doc- but cell growth inhibition was profoundly affected by the etaxel, a synthetic derivative of taxol, is primarily used to inhibition of NF-κB pathways and Akt signaling pathways. inhibit the microtubular structures in cancerous cells that Such responses were seen at lower physiological achievable support cell division. In addition to its role as a microtubule doses. These results were corroborated by in vivo studies disruptive molecule, it has a host of inhibitory actions in mice using human PC-3 prostate cancer cells [70]. Since on genes which regulate cell proliferation, mitotic spindle EGCG has similar effects on prostate cancer cells, EGCG formation, transcription factors, and oncogenesis. It also could also possibly serve as a substitute in place of curcumin upregulates genes involved in apoptosis and cell cycle pro- for such a treatment strategy. gression in prostate cancer. D-Limonene, discussed earlier has been shown to have anti-prostate carcinogenic effects at low dose concentrations. Logically; therefore, combining the 4. D-Limonene and Its Combination Therapies two compounds may have a plethora of positive antitumor (See Table 3) functionalities. In a study by Rabi and Bishayee [72], the Although a few studies have shown that D-limonene, an combined treatment enhanced the sensitivity of DU145 abundant monoterpene in citrus oils, exhibits antimitogenic prostate cancer cells that are known to be apoptotic resistant. activity, its alcohol-derivated perillyl alchohol (PA) has a This enhanced sensitivity was thought to be mediated by greater inhibitory effect on cell migration in cancerous reactive oxygen species (ROS) generation and activation of cells [108]. A study by Reddy et al. [108] used subtoxic caspase 3 and 9. Such a positive in vitro outcome warrants doses of PA to determine this effect. Further preclinical further investigations in vivo, in models that mimic the studies are necessary to determine the effective yet nontoxic progression of the disease, before it can be used in dietary serum/tissue concentration that can be achieved from a diet supplements for therapy. rich in citrus intake, in conjunction with phytonutrients of the same class or a differentclass. Notmuchisknown about 4.2. Lycopene and Fru/His (See [74, 111–114]). Serum the percentage composition of D-limonene and its metab- lycopene (a carotenoid) levels have been shown to have olized constituents that are required to achieve an effective an inverse correlation with prostate cancer risk. A diet- monterpene anticarcinogenic activity. In comparison to its based population study showed that, of all the carotenoids oxygenated derivatives, limonene has the least cytotoxic assessed, high serum lycopene levels showed a statistically effect on both noncancerous and cancerous breast cell lines significant lower prostate cancer risk. Further analysis of and, therefore, can be applicable in chemoprevention [109]. their data revealed that lower serum lycopene levels in D-limonene appears to be more effective against chem- conjunction with β-carotene supplements were effective ically induced colonic crypt foci [110]. These foci are against lowering the risk of prostate cancer, suggestive for preneoplastic lesions and are biomarkers for the progression a combinatorial therapy [111]. Certain dietary compounds 16 Journal of Oncology can be the source of cancer formation as seen with prostate compound are still not known, the combination of the two cancer. It is believed that the nonfat portion of milk and seemed to preferentially localize in the prostate more than in excess calcium are some main factors in prostate cancer other tissues that were tested [74]. risk [112]. Numerous in vitro studies have shown that Occasionally, a combination may fail to incite anticar- carotenoids have a greater influence in reducing tumors of cinogenic effects as was seen by Mossine et al. [114]. Their the prostate origin, and lycopene and 1,2-dihydroxyvitamin experiments were conducted on the prostate adenocarci- D3 are at the forefront as risk reduction factors. In addition noma rat model that was used by other groups, and their data to their role as potent inhibitors of prostate cancer growth, had contradictory results to the effective action of lycopene they are biologically safe and cheaper forms of treatment. itself and in conjunction with other micronutrients. Their 1,2-dihydroxyvitamin D3 and lycopene have physiologically study revealed that lycopene was not able to inhibit or reduce different roles, but combined they modulate pathways to tumor load alone or in combination and that selenium synergistically inhibit proliferation and differentiation at alone in the mixture was able to induce antitumorigenic much lower concentrations [113] and bear additive effects effects. Such outcomes are important as they open up on cell cycle progression. more questionsastowhy amoleculethataffects a given The assessments that lycopene is a safe dietary molecule pathway behaves differently when tested within the same with anticancer properties is supported by a number of experimental model. Is it always dose or concentration or population epidemiological and cohort-based studies [112]. does molecule preparation and delivery impart effects on the However, it is important to ensure that the statistical models efficacy of a drug? used are able to adjust for many parameters for a true 4.3. Lycopene and Docetaxel (See [75]). Docetaxelisapotent significant outcome. Regardless of the statistical model chemotherapeutic drug that is clinically used to treat patients employed in these assessment studies, lycopene has emerged with advanced metastatic prostate cancers. Although the as a potent risk-reducing factor of prostate cancer and drug extends survival, it is for a very limited time period has been even supported by a study that was carried out and with a poor prognosis. Lycopene, a natural compound, across 28 countries. Intervention combination studies have has been shown to have strong cancer inhibitory properties not yet been performed. However, in vivo-based studies against the prostate tissue. One study tested the possibility in mice models have shown that lycopene administered to use this combination of compounds to enhance survival in the form of tomato powder and broccoli powder in of patients that were detected with aggressive, androgen- a 10 : 10 ratio, increases its serum concentration to about independent tumors. As predicted, docetaxel inhibited 538 nM/g with about 0.4 nM/g concentrated in the prostate tumor growth in nude mice that bore tumor xenografts of tissue itself. Diet-based intervention studies are required human DU 145 cells. Analysis of molecular mechanisms to determine the formulated diet required to improve the revealed that the action of docetaxel was on regulating availability in the serum of patients and enhance the localized the insulin-like growth factor receptor (IGFR) pathway by concentration of the molecule in the tissue. Such a diet- suppressing IGF, and this effect was synergistically enhanced based treatment may serve as a suitable chemopreventive in the presence of lycopene. Together the molecules asserted approach against prostate cancer or with patients at high-risk negative downstream effects on Akt signaling pathways and of the disease. Even though bioactive molecules successfully suppressed survivin, products of which have been known work in administering their protective functions in vitro, to maintain tumor growth and enhance metastasis. Clinical it appears through in vivo studies that diet and availability trials using this combination may prove effective in treating crucially dictate outcomes. A critical question to be asked is patients that express high levels of IGFR in the prostate what factors constitute a perfect blend of bioactive mixtures. tumor and extend survival for a longer duration than what With the current research thus far, it is hard to address is possibly achieved by docetaxel alone, which is about 18–20 what the cut-off ratios are that need to be used in a diet months. that contain mixtures of potent nutraceuticals to coordinate similar effects clinically. Possibly a slight change in con- centration of even one of the effective biomolecules may 5. EGCG and Quercetin (See [31]) render the mixture ineffective in its function. It is rather an important task for pharmaceutical chemists and nutritionists EGCG exhibits strong chemopreventive and therapeutic to determine the ratios of effective biomolecules in a mixture activities as it influences many pathways as shown in and determine the pharmacokinetics and dynamics of that Figure 1. Some of the mechanistic pathways are involved mixture. in regulating the levels of Bcl2, survivin, and XIAP and Fru/His, a ketosamine, is also a derived product from activation of caspase-3/7 to induce apoptosis. EGCG is also tomatoes obtained by the reaction of a carbohydrate with an involved in inhibiting genes that are required for transition amino acid. This particular ketosamine has been found to from epithelial to mesenchymal cells and retards migration assert chemopreventive effects by synergistically enhancing and invasion which are primarily advantageous in terms the activity of lycopene, by neutralizing ROS species and of controlling aggressive tumors. EGCG mediates such inhibiting DNA damage. Therefore, the complex of these synergistic actions in conjunction with quercetin to retard two molecules may have a pivotal role in prostate cancer the self-renewal properties of cancer stem cells (CSCs), a prevention. Although a rat model was used to determine population that, if inhibited, can influence tumor regression. the results of the treatment and pharmacokinetics of the Quercetin, a polyphenol, downregulates the expression of the Journal of Oncology 17 heat shock protein (Hsp90) known to influence apoptosis of a mixture of three polyphenols, resveratrol, quercetin, and and growth inhibition of prostate tumors. Therefore, the catechin, albeit pharmacokinetics studies are warranted. combination of these molecules modulates their respective therapeutic effects to mediate synergistic growth retardation 6.2. Resveratrol and Cyclophosphamide (See [78]). Cyclo- of CSCs. The study by Tang et al. [31] used relatively phosphamide, a neoplastic drug, has a broad spectrum of higher concentrations of EGCG (60 μM) in the presence of activity on a variety of cancers, including breast cancers. The 20 μM quercetin. Probably concentrations of EGCG that can shortfall of the drug is its myriad of toxic effects on other mediate similar synergistic levels, albeit at lower doses, need systems. Dose reduction of the compound would be a means to be investigated, and the therapeutic potential across cancer of reducing its toxicity without compensating its anticar- stems cells of other origins need to be assessed if clinical cinogenic activity. Resveratrol has been shown to success- applications are to be considered. fully lower the effective dose of cyclophosphamide without altering its anticarcinogenic activity. Both of the compounds 6. Resveratrol and Estrogen (See [39]) together synergistically enhance caspase-mediated cytotoxic activity, as demonstrated in MCF-7 cells, an aggressive breast Selective estrogen receptor modulators that are used in cancer cell line (Table 4). The combination therapy resulted the clinical treatment of breast cancers display dual ago- in the upregulation of p53, proapoptotic genes, Bax and Fas, nist/antagonist effects in the tissues, especially in cancer initi- and downregulation of antiapoptotic gene Bcl-2, suggestive ation and progression. Drugs like tamoxifen emulate antag- of an apoptotic mechanism involved in cell death. onistic effects on estrogen to contain the tumor. Agonistic- estrogen-like activity can in some instances enhance tumor 6.3. Resveratrol and n-Butyrate (See [79]). n-Butyrate is a progression which is not desired in most clinical treatments. short chain fatty acid produced by bacterial fermentation of Resveratrol, a polyphenolic compound abundant in grape fiber in the colon. The compound is a known differentiating skin and grape products including wine, is known to have agent and induces an epithelial phenotype in certain cultured chemopreventive properties as supported by numerous in cells. n-butyrate is a potent histone deacetylase (HDAC) vitro and in vivo studies. However, based on the experimental inhibitor as well and one of its differentiation-inducing cell type, resveratrol induces either agonistic or antagonistic properties stems from its ability to inhibit HDACs. Resver- effects that can be weak or very pronounced. Resveratrol ago- atrol, discussed above, induces apoptosis through other nistic effects are totally reversed in the presence of estrogen, mechanistic pathways. The combination of two bioactive possibly mediated through estrogen receptor β. This reversal molecules influencing apoptosis via different mechanistic of effects is pertinent to prevention of breast cancer lesions pathways may associate to render an apoptotic phenotype in ducts that could become long-term neoplastic and can- in cancerous cells and inhibit tumor formation and pro- cerous. Of its many cancer protective functions, resveratrol gression. The 2 mM dose of n-butyrate used in the Wolter in combination with glucan are potent immunomodulators and Stein study [79] is probably much higher than what can by upregulating Cdc42 expression [115]. When natural be physiologically achieved. This dose is probably suitable compounds exhibit multi-chemopreventive properties, con- for treatment of colon cancers where higher molar doses jugation therapies are advantageous over monotherapies. of n-butyrate are possible. However, n-butyrate is highly Albeit not clinically tested, harnessing cancer preventive and unstable, and its serum concentrations are lower than 2 mM. immune modulating functions of nutraceuticals seems to be Since this molecule is a differentiating agent, its clinical use a plausible approach to targeting hormonally independent in treatment of other cancers is relevant. However, such aggressive tumors. therapies require absolute lower effective doses and can probably be achieved by combining with molecules other 6.1. Resveratrol, Quercetin and Catechin (See Table 4 and than resveratrol or modifying the compound to specific [77]). Theprotectivefunctions of polyphenolsare many- conjugates to reach serum concentration levels. fold. Numerous studies have analyzed their protective and therapeutic functions in vitro on tumor initiation that was chemically induced or in vivo via cellular implanted tumor 6.4. Resveratrol and 5-Fluorouracil (5-FU) (See [80]). 5- formation. Few studies have established the functions of fluorouracil inhibits thymidylate synthase, prevents DNA combined polyphenols on established tumors, as the major- proliferation, and induces DNA damage-related apoptosis in ity of investigations have focused on individual mechanistic colon cancer cells. Phase I clinical trials using a combination effects of the compounds. Dietary serum concentrations of resveratrol and grape powder have shown that resveratrol are influenced by the individual percentage of biomolecules at low doses inhibit Wnt,agenethatisupregulated in present in the diet. Therefore, individual protective assess- colon cancers. Taking advantage of therapeutic effects of ments of a compound show higher dose requirements, whilst nutraceuticals, combined therapy of aforementioned resver- mixtures may require lower doses to achieve the same effects. atrol with 5-FU surfaces as a principal strategy in treating Additive and synergistic effects of compounds occur if their colon cancers. When used in combination, the presumption individual functions are enhanced in the presence of other is that either additive or synergistic effects of the two could molecules, perhaps by reinforcing the serum stability and mediate tumor inhibition by modulating their individual availability of the various compounds in the mixture. Such apoptotic effects. The concern in using resveratrol is that observations were seen in both in vitro and in vivo testing higher concentrations of the doses are required in the 18 Journal of Oncology treatment which clinically may not be reached through 7.1. Benzylisothiocynate (BITC) and SFN (See [83]). BITC dietary consumption. and sulforaphane are ITCs derived from cruciferous vegeta- bles like broccoli. Individually both these molecules exert potent chemotherapeutic properties strongly supported by 6.5. Genistein and Resveratrol (See [81]). Genistein and numerous studies. Oddly, even though both are isothio- resveratrol as individual phytochemicals are very effective in cyanates, they exert their therapeutic effects by control- the treatment and prevention of prostate cancer progression ling different pathways involved in tumorigenic inhibition. in rodent studies. Poorly differentiated prostate cancers STAT3, a member of the STAT group of transcriptional often fail to respond to androgen-dependent treatments, factors, is required for early development and is dispensable and alternate treatments are required. Androgen receptors in adult tissues. However, there appears to be a correlation likewise have two functional roles, one as a tumor suppressor between the constitutive expression of STAT3 and tumor in normal prostate tissue and the other as an oncogene in development, indicative of its role as an oncogene. This neoplastic transformation, where it is altered either by muta- gene appears to have important roles in cell proliferation, tions or DNA damage. Genistein and resveratrol used in an in angiogenesis, and metastsis, a crucial requirement of tumor vivo rat-based study, modeled to understand the mechanistic survival. Both BITC and sulforaphane have cancer inhibitory action of combined treatments in the progression of prostate effects, affecting independent cell signaling pathways. How- cancer, showed that they had more pronounced effects, albeit ever, the sequential combination of the two has been shown not synergistic. The statistically significant additive functions to regulate the STAT3 gene and others (Table 4), thereby, of reducing cell proliferation through mechanisms that inducing apoptosis. How dietary molecules are presented regulate the androgen receptor levels and IGF-1, a biomarker to the cells in vitro is important to its cellular mechanistic found in patient serums with progressive and aggressive actions. In the study by Hutzen et al. [83], sequential prostate cancers were achieved in combined therapies over addition of BITC to the cells after sulforaphane treatment the monotherapy regimes. Interestingly, the combination of was performed, which enhanced the reduction of STAT3 genistein and resveratrol increased serum availability of both, levels; however, simultaneous additions were not performed. but higher concentrations of resveratrol were achievable as Simultaneous additions would be important for any com- compared to the single-dose regimen. Perhaps, absorption bination study to determine possible synergistic, additive, and stability of resveratrol were profoundly affected in a or antagonistic effects between the compounds. Preclinical combined environment, which is clinically a clear advantage. studies should include various combinatorial interactions of The doses used in the study are physiologically safe and the nutraceuticals being tested to determine the best way of achievable in vivo by consumption of a soy-based diet high using combined molecules in therapy. in the percentage of genistein. However, resveratrol is found in low levels in grape-based dietary products, and, therefore; 7.2. SFN and Apigenin (See [84]). Phase I and Phase II a pure supplement of the compound is necessary in case that enzymes are extremely important to cancer prevention. higher doses are required. Dietary foods are sometimes modified to produce car- cinogens through metabolism by the action of Phase I enzymes. Subsequently, the action of Phase II enzymes 7. Genistein and Sulforaphane (SFN) (See [82]) rapidly metabolizes these products to more soluble forms Previous studies have shown that EGCG, a major polyphenol that are eliminated as body waste. Phase II enzymes are more in green tea, can inhibit tumor growth through mechanisms concentrated in the duodenum and small intestine and less that alter DNA methylation activity, reversing the expression available in the colon. Increasing the availability of these of silenced genes involved in tumor inhibition in cancer enzymes in the colon can get rid of harmful carcinogens cells. Hypomethylation of the promoters that are CpG-rich reducing the incidence of colon cancers, and, therefore, is more likely to be transcribed, with an exception of few dietary supplements that induce Phase II enzymes would like hTERT, the regulatory gene of telomerase [82, 116–119]. be promising tools for colon cancer prevention. SFN, an Epigenetics is a mechanism that has been studied for decades, isothiocyanate, and apigenin, a flavanol, have independent and factors that regulate epigenetics are now believed to cancer preventive functions. SFN is a strong inducer of UDP- be very important as treatment possibilities in controlling 27 glucuronyltransferase (UGT1A1). UGT1A1 is a major tumors. DNA methylation and histone deacetylation are player in the detoxification process of carcinogens formed well known epigenetic mechanisms that regulate many of in the body and, therefore, is a potent Phase II enzyme. the genes involved in cancers of various origins. Genis- Treating nondifferentiated colon cells with a combination of tein combined with SFN, an histone deacetylase inhibitor SFN and apigenin was found to synergistically induce the (HDACI) has been successful in inducing the transcription expression of UGT1A1 suggesting a possible dietary tool for of genes involved in regulating cell cycle by reversing the colon cancer prevention. The in vitro dose of the individual hypermethylated states of their promoters. This change was compounds used in the study was at physiological safe levels and can be easily achieved in vivo. observed at low doses and was enhanced in the presence of sulforaphane more than that when genistein was used alone. However, in vivo studies of the same are warranted 7.3. SFN and 3,3 -Diindolylmethane (DIM) (See [85]). The to determine epigenetic behavior of the dietary compounds importance of investigating the roles of dose combinations before applications to human treatments are considered. on chemopreventive or therapeutic functions has been well Journal of Oncology 19 dissected in a study by Pappa et al. [85]. Lower doses of an urgent need for alternate or adjuvant therapies has arisen. SFN demonstrate antagonistic effects on cell proliferation Phytochemicals are relatively safe and abundantly available and higher doses of both compounds had synergistic effects. from dietary sources. Therefore, alternate medicine aims at Synergism of compounds is preferred if the outcome is harnessing the protective properties of these nonessential tumor regression, but in clinical treatments synergistic nutrients toward cancer prevention and treatment. A large actions should be mediated at safe lower concentrations database of studies supports the use of biomolecules in rather than at cytotoxic levels. Presumably, the choice of cancer treatment, albeit a majority of those are in vitro compounds used, based on the genetic or cellular function studies. Regardless of limited in vivo studies and clinical required, is imperative to the success of the treatment. trials, phytochemicals show great promise in cancer treat- Possibly, SFN can synergistically inhibit the proliferation of ment considering their safe use. Caution must be taken cancer cells with compounds other than DIM at much lower when addressing the efficacy of these molecules in clinical doses, which has been investigated in studies using SFN trials as many factors modulate their effects on cellular with flavanols. This clearly highlights the problems of using functions as detailed in Table 5. Combinatorial studies combined therapies, especially since dosage is of critical also show great promise, especially when lower nontoxic importance for the success of clinical trials. doses are required for prolonged periods to mediate potent chemotherapeutic functions with minimal side effects. Two 7.4. SFN and Dibenzoylmethane (DMB) (See [28]). When of the major problems currently faced are dosage and seeking for dietary molecules with potential chemoprotective delivery. To maintain a constant physiological serum dose and therapeutic properties, it is essential to understand how availability, it is imperative that the agent is concentrated and they mediate their combined action. Based on mechanistic stable in the tissue of concern. Combination technologies studies, only compounds that are able to achieve synergistic may be a solution to this problem. Nanotechnology is or additive inhibitory or inductive actions on cellular fast catching pace as the next level of technology in all genes, pathways, and/or phenotypes can then be chosen for spheres of science. Limited in vitro studies have shown treatment, even though their individual actions may be more that encapsulating dietary supplements in nanoparticles can pronounced. DMB is antimutagenic. Patients with aberrant effectively deliver the supplement and increase its stability polyp crypt (Apc) mutations are prone to spontaneously and availability. Perhaps research needs to focus on such form aberrant polyps in their intestinal tissue, which later possibilities as avenues of using combination therapies. can transform to colorectal cancers. Treatment with DMB foundinlicoricecan effectively inhibit such mutations in Conflicts of Interest Apc, thereby protecting individuals from aberrant polyp formations. This molecule, therefore, has potential in terms The authors declare that they have no conflicts of interest. of colon cancer prevention. SFNhas amyriadofchemopreventive functionsasseen Acknowledgments before in other studies and in various tissues. A combination of these two chemopreventive agents will have a profound This work was supported in part by grants from the National impact on individuals that are at high-risk or reduce the Cancer Institute (RO1 CA129415), the American Institute incidence of colon cancers through dietary supplementation. for Cancer Research, and the Norma Livingston Foundation. The study by Shen et al. [28] showed that dietary intake of SFN and DMB negatively influenced the incidence and number of tumors formed in the Apc mice. The combined References doses used were half that of the individual doses. However, [1] W. C. Hahn, S. K. Dessain, M. W. 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The Role of Nutraceuticals in Chemoprevention and Chemotherapy and Their Clinical Outcomes

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Copyright © 2012 Sabita N. Saldanha and Trygve O. Tollefsbol. 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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Hindawi Publishing Corporation Journal of Oncology Volume 2012, Article ID 192464, 23 pages doi:10.1155/2012/192464 Review Article The Role of Nutraceuticals in Chemoprevention and Chemotherapy and Their Clinical Outcomes 1, 2 1, 3, 4, 5, 6 Sabita N. Saldanha and Trygve O. Tollefsbol Department of Biology, University of Alabama at Birmingham, 175A Campbell Hall, 1300 University Blvd, Birmingham, AL 35294-1170, USA Department of Math and Sciences, Alabama State University, P.O. Box 271, Montgomery, AL 36101-0271, USA Clinical Nutrition Research Center, 402 Webb Nutrition Sciences Building, 1675 University Blvd, University of Alabama at Birmingham, Birmingham, AL 35294-3360, USA Comprehensive Cancer Center, University of Alabama at Birmingham, 1802 6th Avenue South, North Pavilion 2500, Birmingham, AL 35294, USA Center for Aging, University of Alabama at Birmingham, 933 South 19th Street, Room 201, Community Health Services Building, Birmingham, AL 35294-2041, USA Nutrition Birmingham Obesity Research Center, University of Alabama at Birmingham, 402 Webb Nutrition Sciences Building, 1675 University Blvd, Birmingham, AL 35294-3360, USA Correspondence should be addressed to Trygve O. Tollefsbol, trygve@uab.edu Received 30 June 2011; Accepted 25 August 2011 Academic Editor: Julian J. Raffoul Copyright © 2012 S. N. Saldanha and T. O. Tollefsbol. 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. The genesis of cancer is often a slow process and the risk of developing cancer increases with age. Altering a diet that includes consumption of beneficial phytochemicals can influence the balance and availability of dietary chemopreventive agents. In chemopreventive approaches, foods containing chemicals that have anticancer properties can be supplemented in diets to prevent precancerous lesions from occurring. This necessitates further understanding of how phytochemicals can potently maintain healthy cells. Fortunately there is a plethora of plant-based phytochemicals although few of them are well studied in terms of their application as cancer chemopreventive and therapeutic agents. In this analysis we will examine phytochemicals that have strong chemopreventive and therapeutic properties in vitro as well as the design and modification of these bioactive compounds for preclinical and clinical applications. The increasing potential of combinational approaches using more than one bioactive dietary compound in chemoprevention or cancer therapy will also be evaluated. Many novel approaches to cancer prevention are on the horizon, several of which are showing great promise in saving lives in a cost-effective manner. 1. Introduction lung and bronchus, breast, colorectal, and prostate [4, 5]. These cancers are also more prevalent in the western parts The transformation of a normal cell into a cancerous pheno- of the world and are much lower in Asian countries. A well- type requires stages of initiation, progression, and promotion balanced diet that includes more of vegetables and fruits by altering specific genes [1–3]. Although predisposition with less fat/meat intake is in most cases a staple of many to cancer cannot be signaled out by a single factor, a Asian countries [4, 5]. Many hypotheses have supported that group of factors place some individuals at a higher risk diet and environment greatly influence cellular function and of acquiring the disease. Most of the high-risk cases may health [6]. have a genetic background, but in some instances dietary Phytochemicals are plant-based chemicals that mediate choices can dictate the outcome of health. As determined their positive health benefits directly, by affecting specific by population and epidemiological studies, the predominant molecular targets such as genes, or indirectly as stabilized forms of cancer and cancer-related deaths are those of the conjugates affecting metabolic pathways [7]. Many genes play 2 Journal of Oncology significant roles in the cell cycle pathway, and some of these (e.g., curcumin and resveratrol). Once tumor regression sets are altered in cancer cells [1, 2]. The aim of most studies is in, dietary composition of the molecule can be adjusted. to understand and formulate mechanistic pathways by which these naturally derived chemicals can alter the fate of a cell. 2. Nutraceuticals and Their Preventive and Foracancerouscelltosurvive,itshouldbeabletoproliferate, Therapeutic Roles obtain energy, and establish angiogenic pathways, in a tumor mass. Altering genes that affect these pathways can serve as 2.1. Genistein: A Potent Isoflavone. Many phytochemicals suitable tools to decrease tumor mass and also allow for are currently being investigated for their promising anti- tumor regression. In this paper, the key focus will be on carcinogenic properties. In vitro investigations have shown mechanistic pathways that are regulated by nutraceuticals to that some compounds exert their antitumor functions at bring about changes in the tumor environment and serve much higher concentrations and that dietary consumption as alternative approaches for cancer prevention and therapy is insufficient to achieve such effective concentrations at (Figure 1). the tumor site. Therefore, the mode of delivery is a very The study of phytochemicals and the classification of important factor that needs to be considered at clinical trials these compounds have been previously reviewed [8]. How- and during in vivo studies. The nontoxic properties of natural ever, in this paper only some of the most potent and prom- compounds are essential to the design of a formulated ising chemopreventive and therapeutic molecules will be therapy. However, evidence along several lines of treatment analyzed, with emphasis on combination therapy of these has shown that some compounds are preferentially more with other nutramolecules. Most phytochemicals derived potent in activity when administered early in life [9, 10]. For from dietary sources are classified under an umbrella of instance, soy-based prevention of breast cancer is thought to specific chemical compounds as detailed in Table 1. These be more successful when soy products and their derivatives molecules may not have a nutrient value but are germane are consumed in early development [9]. to the function of a cell. Various studies have shown that Isoflavones are a group of phytochemicals that are pre- these molecules can induce apoptosis, inhibit cellular prolif- dominant constituents of a soy-based diet [9, 10]. Among eration, affect angiogenesis, and affect cancer metabolism in isoflavones, the three major constituents that have been various cancers, all of which are hindrances to tumor growth shown to have remarkable influences in cancer prevention (Figure 1)[7]. and therapy are genistein, diadzein, and glycitin [11]. Several of the phytochemicals listed in Table 1 have been They are collectively grouped as phytoestrogens for their investigated in terms of their curative properties. However, weak estrogen-like activity and bind preferentially to ER- one must carefully interpret the observed results in vitro β receptors [12–15]. Evidence of antiproliferative activity and in vivo before testing the same in a clinical setting. The of genistein in vitro stems from its ability to inhibit the reasons for this are manyfold. Tests in culture are pure, in that there is only one cell type in the culture plate and all condi- tyrosine kinase enzyme that is most often upregulated in tions are controlled, including the bioactive compound. In cancer cells [16, 17]. As a chemopreventive agent, genistein is vivo, however, the scenario changes as there are a host of thought to influence the differentiation process of mammary other factors that need to be taken into account, including tissue. It is believed that early differentiation of mammary age, weight, diet, and metabolism of the compound. A tissue into terminal buds, as seen in rats, serves as a bioactive molecule in culture may be subjected to less chemopreventive strategy as it reduces the susceptibility of metabolic changes and may be presented to the cell in its the epithelial cells in the ducts to carcinogens or estrogen native form. However, in vivo the same compound may be and the ontogeny process [9]. Many aggressive cancers have presented differently, perhaps as a conjugate, and its mode of altered epidermal growth factor (EGF) receptors on their action may change amongst the multitude of other molecules cell surface allowing for a continuous downstream signaling in the host’s microenvironment. Many in vivo experiments pathway for cell division [18, 19]. This is interesting, as also control for the type of diet being administered to the genistein can serve as a two-fold approach molecule for organism, where the concentrations or plasma availability prevention and treatment. When EGF binds to its receptors, can be adjusted. Therefore, what may work well in vitro, tyrosine kinase activation results in the phoshorylation of may have no agonistic effects or even antagonistic effects in tyrosine residues of proteins involved in downstream cell vivo, and such discrepancies are often seen when comparing signaling pathways that trigger cell division. Though studies population and epidemiological studies in terms of chemical have shown that genistein increases the EGF transcript early efficacy. in development of mammary tissue, this perhaps is essential An effective nutraceutical is one that will have a low for differentiation and faster development of the breast nontoxic dose while creating a magnitude of change in tumor tissue. In the long run this is a positive preventive strategy dynamics. This means that at a low dose the compound of breast lesion formation in ducts [9]. However, as seen in should act fast on the tumor load. However, if the time older rats [9], EGF mRNA decreases. Therefore, a decrease taken to be effective is slow, the problems faced would be in EGF mRNA coupled with inhibition of tyrosine kinase by maintaining a tolerable dose and increasing bioavailability genistein would profoundly decrease tumor growth as cell and stability. A solution to such a problem would be to use signaling pathways are crucial to tumor maintenance. a combinatorial approach to therapy, a bioactive molecule Numerous studies have highlighted the antiproliferative with an effective synthetic drug or double-nutratherapy role of genistein in various cancers; however, there are some Journal of Oncology 3 Tumor spread • Inhibits tumor invasion Precancerous lesions • Antimetastasis • Activates anti-inflammatory Pathways modulators influenced by • Induce phaseIIenzymes • Inactivate phase I enzymes nutraceuticals Tumor growth Chemopreventive • Inhibits cell proliferation • Induce cell differentiation • Induce apoptosis Chemotherapeutic Figure 1: Cellular pathways affected by the activities of bioactive components in dietary sources. Of the natural compounds present in dietary sources, some are more involved in regulating chemopreventive pathways and some are more effective in influencing chemotherapeutic pathways. However, a few of the bioactive molecules found to date can impart both chemopreventive and therapeutic effects, such as EGCG and genistein. Compound combinations as discussed in the paper that can affect different pathways are shown and can have profound effects on tumor growth and inhibition. Table 1: Classification of nutrients as phytochemicals and their major food source availability. Phytochemical Bioactive compound Source Molecular formula Reference class Caffeine Cacao, tea, coffee C H N O 8 10 4 2 [20] Alkaloid Theophylline Cacao, tea, coffee C H N O 7 8 4 2 Citrus oils from orange, lemon, Monoterpenes Limonene C H [21] 10 16 mandarin, lime, and grapefruit Allicin Garlic C H OS [22–25] 6 10 2 Indole-3-carbinol Cabbage C H NO [26] 9 11 2 Organosulfides Isothiocyanates Broccoli CNS [27] Sulforaphane Broccoli C H NOS [28] 6 11 2 Carotenoids Beta-Carotene, lycopene Tomatoes C H [29] 40 56 Epigallocatechin-3-gallate Green tea C H O [30] 29 22 15 Flavonoids Quercetin Black tea C H O [31] 15 10 7 Curcumin Turmeric C H O [32] 21 20 6 Capsaicin Chilli peppers C H NO [33] 18 27 3 Phenolic Acids Ellagic acid Black berries, raspberry C H O [34, 35] 14 6 8 Gallic acid Pomegranate, nuts C H O [36, 37] 7 6 5 Pterostilbene Blueberries and grapes C H O [38] 16 16 3 Stilbenes Resveratrol Almonds, blueberries, grapes C H O [39] 14 12 3 Daidzein Soy C H O 15 10 4 Isoflavones [9, 40] Genistein Soy C H O 15 10 5 Molecular formulas obtained through the PUBCHEM COMPOUND Database. 4 Journal of Oncology studies indicating that genistein may increase cell prolifera- the conjugates in the tissue allows available free genis- tion [19, 47]. A key point to note is that nutraceuticals can tein delivery to the cells, as presented or tested in vitro. be effective based on the form of genistein or its dose given at For pharmaceutical companies, it is required to formulate the time of the study (Tables 2 and 3), especially with respect supplements with precise ratios of individual constituents to in vitro and in vivo models. Importantly, the downstream of the compound. Unless a very pure form, a capsule or targets of bioactive molecules under investigation need to supplement may contain a mixture of genistein, diadzein, be ascertained for each specific tissue, if overall health and glycetin (Tables 1, 2, 3,and 4). The percentage of each applications are an issue. The nutraceutical may not affect nutrient in the mixture will have a profound effect on the aspecific commonpathway fortumorsofdifferent origins. bioavailability of the compound after metabolism (Tables 2 For example, in breast tissue, EGF may be highly expressed, and 3). To design such a product is certainly not easy and but, in colon cells or pancreatic cells, genes that regulate is dependent on many factors, but the two essential factors cell division other than EGF may be affected [48]. Cell are the grade/stage of the tumor and the site or origin of culture experiments using plant-based nutrients depend on the tissue. Of the two isoflavones, diadzein has been shown the sensitivity of the cells that are being investigated. When to have a lesser apoptotic effect on prostate cancer cells cell lines are established, they are derived from cancerous but can inhibit neoplastic transformation [61]. Therefore, it tissues of specific organs and are, therefore, cell-type specific. would be advantageous to use supplements containing the This is drastically different in a clinical setting where the two bioactive nutrients as chemopreventive agents. molecule has to mediate its activity amongst a host of various Of the predominant high-risk cancers, genistein appears molecules and cell types. Therefore, the concentration of the to have a greater affect on prostate cancers [52–54]. Genis- phytonutrient in the supplemented diet will be crucial to its tein mediates the apoptosis of cancer cells by activating efficacy in the tumor environment. This can help explain the and/or inhibiting genes and/or enzymes germane to tumor discrepancies seen in clinical trials of genistein for different maintenance (Figure 1,Table 4). Some of these important tissues [47, 49, 50]. Outcomes of some in vitro studies suggest mechanisms are the inhibition of the activity of tyrosine that, like other bioactive compounds, genistein appears to kinase, nuclear factor kappa B (NF-κB), and vitamin D have a specific cut-off concentration at which this isoflavone 24-hydroxylase [86], activation of tumor suppressor genes, can exhibit anticarcinogenic activity (10 μMorevenhigher) and modulation of androgen-responsive gene expression, [48, 51], and it is, therefore, imperative to achieve such prostate-specific antigen (PSA), and the androgen receptor concentrations in vivo. (Table 4). Of the prominent isoflavonones in soy, diadzein Isoflavones, in particular, genistein, have been extensively is less effective in its action on prostate cancer, but, unlike studied as prospective antitumor molecules in the treatment genistein, it is metabolized to equol, an isoflavandiol which of prostate cancer [19, 52, 53]. There has been a well has a longer half-life than genistein [87]. The longer half- established line of evidence that genistein works against life of equol creates the possibility of using this chemical in prostate cancer, but a majority of studies are in vitro in combination with other available nutraceuticals, where the cultured cells [19, 52–56]. Limited clinical trials have tested net effect may be synergistic. However, prior preclinical tests the therapeutic efficacy of genistein in prostate cancer and are required to investigate this possibility. those that have revealed inconsistencies in cell proliferation Other dietary compounds are also of great interest and tumor growth [57–60]. Given the inconsistencies in in this regard. In vitro, vitamin D (Vit D) has potent some of the outcomes, emphasis should be on the dose tumor prevention ability and can induce differentiation and of the supplement and the form of the nutrient in the apoptosis in some of the most predominant cancers [48]. supplement at the time of administration to the patient in The use of nutrients as a possible treatment approach is clinical trials. The highest achievable plasma concentration based on the fact that chemicals occurring naturally will of isoflavones is 1 μM through orally administered food minimize side effects when applied to a biosystem. However, sources. From previous studies, this concentration is not the in vitro dose at which Vit D induces its antitumor sufficiently significant to bring about anticarcinogenic effects properties causes hypercalcemic conditions that can preclude on the tissue. However, there is ample evidence that genistein treatment in patients [49]. In prostate cancer, a leading cause and other isoflavones do exhibit anticancer properties and of cancer deaths in the western parts of the world, androgen inhibit cell proliferation and tumor growth. A clinical study ablation therapy is the choice of treatment. However, as the by Gardner et al. [61] showed that treatment of patients with cancer becomes aggressive, hormone ablation therapy fails, dietary supplements (82 mg/day aglycone equivalents) of and progression ensues via androgen-independent pathways. isoflavone yielded a higher concentration of total isoflavones Therefore, alternate therapies are very much in demand. in the prostatic tissues than in serum. Therefore, there is a Vitamin D is an alternate form of treatment in prostate possibility of increasing the concentration of isoflavones to cancer (PCA) and is shown to induce apoptosis in PCA cells anticarcinogenic levels in tissue. in vitro. However, all PCA cell lines in vitro are not equally An orally administered dose of isoflavones must with- receptive to the vitamin D treatment or genistein [88]. Cell stand the rigors of the alimentary canal and become lines such as DU145 prostate cancer cells are especially more metabolized before they can be made available to tissues. resistant as they express high levels of CYP24, an enzyme Most isoflavones exist as conjugates rather than in their free that catabolizes Vit D3 into less active metabolites [88]. To state. This conjugation is perhaps the best way to present circumvent this problem, a recent study showed that a dual the molecule to the cell in tissues, and the hydrolysis of combination therapy, of DU145 to genistein and Vit D3, Journal of Oncology 5 Table 2: Pharmacokinetic studies evaluating the bioavailability of phytochemicals at given doses. Phyto Experimental Maximum plasma Form Dose Model subject Half-life (h) Reference chemical setup concentration 15 g Human Soy beverage Diadzein : genistein 96.31 ng/mL 7.68 Diadzein postmenapausal (9.27 : 10.51 mg) Clinical [40] women Two soy Diadzein : genistein 96.02 ng/mL 6.67 capsules (7.79 : 22.57 mg) 15 g Diadzein : genistein Human Soy beverage 116.37 ng/mL 7.61 Genistein (9.27 : 10.51 mg) postmenapausal [40] Clinical women Two soy Diadzein : genistein 216.84 ng/mL 7.96 capsules (7.79 : 22.57 mg) Curcumoid powder form 10 g (n = 6) 2.04 ± 0.31 curcumin 6.77 ± 0.83 (75%), Healthy human Curcumin for total demethoxycur- Clinical subjects (5 men [41] glucoronide curcumin cumin (23%), and 7 women) 12 g (n = 6) conjugates 1.40 ± 0.74 and bisdemethoxy curcumin Curcumoid powder form curcumin 10 g (n = 6) 1.06 ± 0.40 6.77 ± 0.83 (75%), Healthy human Curcumin for total [41] demethoxycur- subjects (5 men Clinical sulfate curcumin cumin (23%), and 7 women) conjugates 0.87 ± 0.44 and 12 g (n = 6) bisdemethoxy curcumin Quercetin Quercetin 500 500 mg of quercetin 15.4 ng/mL 3.47 aglycone plus capsule Healthy human subjects (6 Not given for Clinical [42] Quercetin Quercetin 500 plasma level, but males and 4 500 mg of quercetin 336 ng/mL conjugates plus capsule female) renal clearance is 0.835 500 mg resveratrol/caplet 0.5 g ∗ ∗ 72.6 (48.9) ng/mL 2.85 Uncoated Healthy human Phase I immediate- Resveratol [43, 44] 1.0 g subjects clinical test release 117.0 (73.1) ng/mL 8.87 (91.1) caplets 2.5 g 268.0 (55.3) ng/mL 4.22 (51.6) 5.0 g 538.8 (72.5) ng/mL 8.52 (95.8) @ @ Broccoli raw 200 g 103 ± 31 ,nM 3.8 ± 0.8 Sulforaphane Healthy adult Broccoli [45] Clinical @ @ 200 g male subjects 31 ± 19 nM 4.6 ± 0.8 cooked −1 Per dose (L ) Beverage Healthy human −3 EGCG 112 mg Clinical 0.51 × 10 ± 3.2 ± 2.1 [46] 200 mL subjects −3 0.08 × 10 D-Limonene oxygenated 30–40 ounces 447–596 mg Healthy human Clinical 2.08–13.98 μM 12–24 [21] metabolite of lemonade D-limonene subjects perillic acid 6 Journal of Oncology Table 2: Continued. Phyto Experimental Maximum plasma Form Dose Model subject Half-life (h) Reference chemical setup concentration Range between Lycopene with Healthy adult 4.03 and Range between Lycopene up to 250 mL 10–120 mg Clinical [29] male subjects 11.27 μg/dL 28.1 61.6 h water (0.075–0.210 μM) coefficient of variation; SD—standard deviation. increased the sensitivity of the cells to Vit D3 by decreasing alone. In addition, NF-κB was transcriptionally inhibited in CYP24 expression. What is interesting to note is that the the combined treatment [90]. combination approach not only lowered the effective dose, From a number of investigations, a common thread of but was able to abrogate cell proliferation as well. This evidence seems to emerge that considerable variation in the lowered concentration of genistein at 100 nM is achieveable efficacy of bionutrients in cancer treatment exists and differs in vivo through dietary sources, and clinical studies would be even among the same cell lines tested. The reasons for this required to determine the localization of genistein and Vit are manyfold (Table 5). Cell lines derived from the same D3 in prostatic tissues. tissue hypothetically should be sensitive to the same dose or An in vivo study for colorectal cancer has demonstrated chemical class of the phytonutrients, but such is not always a similar effect [89], but in this case the mice were given a the case. Alternate medicinal approaches have an important single gavage of 250 μg of genistein. This mode of nutrient task to identify crucial factors that change the sensitivity administration is useful for a preclinical test and probably of the chemical and determine chemical modifications that has applications as a chemopreventive supplement. However, would be necessary to modulate more synchronized results in terms of a clinical setting, patients are often exposed to across several cell lines expressing similar genotypic and a host of other nutrients or isoflavones in their diet, and; phenotypic signatures. therefore, an in vivo model replicating such an environment with various percentages of isoflavones will allow for a 2.2. Epigallocatechin-3-gallate (EGCG): A Potent Flavanol. better understanding of concentration and bioavailability of Of the major food-derived phytochemical constituents genistein that can mediate an apoptotic effect and reduce that are extensively studied for their chemopreventive and CYP24 expression in colonic tissues in the presence of chemotherapeutic use, EGCG and genistein are by far the vitamin D. most investigated. EGCG has been shown to have numerous The antimetastatic properties of genistein are mediated anticancer properties which include antiangiogenic activ- by altering the expression of NF-κB, and inhibiting the itybyaffecting the transcriptional expression of vascular tyrosine kinase enzyme [17, 90]. Non-small-cell lung cancer endothelial growth factor (VEGF) [91], inhibiting tumor (NSCLC) is a highly aggressive form of lung cancer with a initiation and promotion by inhibiting signal transduction poor prognosis. Therefore, alternate approaches that drasti- pathways via [phosphatidyloinositol 3-kinase-Akt kinase- cally reduce tumor growth are of utmost importance. Acti- NF-κB] [92–94], inhibiting EGFR [95], inhibiting Her- vation of epidermal growth factor receptor tyrosine kinase 2 receptor phophorylation in breast carcinoma cells that (EGFR-TK) enhances the cell signaling pathways allowing constitutively expresses Her-2/neu receptor [95], inducing tumor growth. The use of drugs that inhibit EGFR-TK and apoptosis in estrogen receptor-(ER-) independent breast affect NF-κB, a gene whose transcribed products are essential cancer cells [96], causing antimetastatic activity [97], inhibit- for invasion and metastasis, can induce a more aggressive ing proteasome formation [98], inhibiting glucose-regulated approach of reducing tumor size and the spread of the protein (GRP78) activity [99]; inhibiting insulin-like growth disease. A clinical therapy should be aimed at reducing tumor factor-I receptor (IGF-IR) [100], and preventing invasion of growth and spread by inhibiting mechanisms that contribute tumors by inducing HMG-box transcription factor 1 (HBP1) to the activation of metastasis. In NSCLC, genistein remark- transcriptional repressor, an inhibitor of the Wnt signaling ably enhances the effects of EGFR-TK inhibitors, such as pathway crucial for tumor-invasive property [101]. erlotinib and gefitinib, when used in combination with each The serum level concentrations of EGCG are impor- of them, respectively. This effect was seen to be mediated tant to ensure that an effective response is seen without by a marked reduction in NF-κB and others, such as EGFR, adverse or even tumor-promoting functions. Studies have pAkt, COX-2, and PGE(2), essential for regulating genes that shown that high doses of catechins that include a higher control division, proliferation and metastasis [90]. A few concentration of two prominent compounds, epicatechin studies have shown how a combined approach can lower gallate (ECG) and EGCG, induce hypoxia-inducible factor 1 the effective dose concentration even of chemotherapeutic which is responsible for activating genes related to hypoxia drugs, minimizing potential side effects. A study conducted conditions. This allows tumor cell proliferation through on breast and pancreatic cells showed that, when the cells alternate survival pathway mechanisms [102]. Most breast were primed with genistein, lower concentrations of the cancers are ER dependent; however, for breast cancers and chemotherapeutic drugs were needed to significantly bring others that are ER independent, EGCG inhibits the growth about growth inhibition and apoptosis than with the drugs of tumor cells through the process of apoptosis [96, 103]. Journal of Oncology 7 Table 3: Single-dose clinical studies evaluating the bioavailability of phytochemicals or their conjugated or active metabolites. Route of Bioavailability area Phytochemical Form AUC values Reference administration under the curve (AUC) Adjusted to the Diadzein Oral Soy beverage 107 ± 49.16 ng·h/mL [40] dose Soy extract Adjusted to the 142.61 ± 43.94 ng·h/mL [40] capsules dose Adjusted to the Geistein Oral Soy beverage 121.48 ± 70.98 ng·h/mL [40] dose Soy extract Adjusted to the 131.04 ± 60.79 ng·h/mL [40] capsules dose Curcuminoid powder extract Oral 35.33 ± 3.78 μg·h/mL Relative [41] capsule form Curcumin (10 g) conjugates (glucoronide + Curcuminoid sulfate) powder extract Oral 26.57 ± 2.97 μg·h/mL Relative [41] capsule form (12 g) Capsule Quercetin aglycone Oral 62.5 ng·h/mL Relative [42] (500 mg) Quercetin- Capsule conjugated Oral 2000 ng·h/mL Relative [42] (500 mg) metabolites Caplet ranging from 0.5 g 223.7 ng·h/mL ∗ [43] Oral Relative for all Rersveratrol 1.0 g 544.8 (57.2) ng·h/mL 2.5 g 78.6 (36.2) ng·h/mL 5.0 g 1319 (59.1) ng·h/mL 200 g broccoli ◦ [45] Sulforaphane Oral Relative Raw 495 ± 40 nM·h Cooked 286 ± 139 nM·h Average 200 mL EGCG Oral AUC nd [46] beverage D-Limonene (perillic acid a 40 oz of major active Oral 5.07 to 32.59 μM·hRelative [62] Lemonade metabolite of d-Limonene) Liquid form (AUC) 0−96 (tomato paste) 10 mg 214 ± 124.8 μg·h/dL 30 mg 416.4 ± 183.9 μg·h/dL Lycopene Oral Relative [29] 60 mg 421.7 ± 59.3 μg.h/dL 90 mg 598.9 ± 396.8 μg·h/dL 120 mg 655 ± 298.6 μg·h/dL AUC value measured for resveratrol was AUC infinity with the coefficient of variance denoted in the brackets against the mean value. For the lowest dose of resveratrol AUC infinity value n = 1. AUC value measured for sulforaphane was AUC0-∞. Based on the reference paper a list of various AUC values was given for different single doses as experimentally performed by different laboratories. Since the sample numbers were different, an average AUC value has not been given for this compound. nd—not determined. 8 Journal of Oncology Table 4: Assessment of the chemotherapeutic and chemopreventive effects of nutraceuticals in combination studies. Combination of Pathways affected or Phase of Dose used Organ of study Model of study Reference nutraceutical mechanistic action study 50 μM/L + Inactivation of NF-κB Human breast Curcumin + 10–50 μM/L based and other metastatic In vitro cancer cells paclitaxel on the gene genes. MDA-MB-435 assessed [63] Breast Human breast Curcumin + 2% w/w Inhibition of In vivo cancer xenograft paclitaxel 10 mg/kg metastasis model Synergistic effect in Human breast Curcumin + Induction of the range from 5 to Breast In vitro MDA-MB-231 [64] xanthorrhizol apoptosis 20 μg/mL cancer cells Inhibition of proliferation, more Ratio of DHA to synergistic in one of CCM the 5 cell lines tested. MCF-7 Enhanced uptake of Human breast 55 : 30 μM curcumin by the cells. cancer cells MCF10A Upregulated genes SK-BR-3, Curcumin + 95 : 45 μM involved in cell cycle Breast In vitro MDA-MB-231, [65] docosahexenic acid MDA-MB arrest, apoptosis, MDA-MB-361, 35 : 35 μM inhibition of MCF-7, and SK-BR-3 metastasis, and cell MCF10AT 60 : 40 μM adhesion. MDA-MB Downregulated genes 50 : 25 μM involved in metstasis and invasion. 10 μM+25 μM T47D and Change in cell Curcumin + [66] morphology and Breast In vitro 10 μM+25 μMMCF-7 genistein growth inhibition 11 μM+25 μM MDA-MB-231 Alter multidrug resistance genes. Alters the cell cycle Curcumin + [67] 15 μM+10 μM Lung In vitro NCI-H460/R with cells inhibited sulphinosine primarily in the S G2/M phase of the cycle Inhibition of cell proliferation and HT-29 induction of apoptosis. IEC-18-K-ras Curcumin + 10–15 μM/L + [68] Colon In vitro (Cox-2, high celecoxib 5 μM/L Possible inhibition of levels) Caco-2 Cox-2 pathways or (COX-2, low through non-Cox-2 levels), and pathways SW-480 (no COX-2) Inhibition of tumor Coltect only 20 μM growth by induction In vitro HT-29 cells of apoptosis. Coltect + Chemical [69] 5-aminosalicylic Colon induction of acid (ASA) 150 mg/kg + Inhibits abnormal tumors by In vivo 50 mg/kg crypt formation 1,2-dimethyl hydrazine (DMH) model in rats. Journal of Oncology 9 Table 4: Continued. Combination of Pathways affected or Phase of Dose used Organ of study Model of study Reference nutraceutical mechanistic action study Additive effectives in 25 μM+10 μM the induction of In vitro PC-3 C4 cell line [27] apoptosis. Curcumin + NCr- Prostate PEITC immunodeficient Inhibition of tumor (nu/nu) mice growth through 3 μM+2.5 μM In vivo bearing s.c. [70] inhibition of Akt and xenografts of PC-3 NF-κBpathways. human prostate cancer cells Pure 3—curcumin Inhibit growth by PC-3, LnCaP and +resveratrol + inhibiting hedgehog In vitro mouse cell line EGCG; signaling pathways. TRAMP-C2 Pure 4—apigenin + baicalein + Individual genistein + Prostate [71] compounds, quercetin; Percentage Pure 7—Pure 3 + Transgenic composition in the Pure 4; Reduce or delay the adenocarcinoma of In vivo diet not defined Crude 7—soy + onsetoftumors. the mouse prostate sencha leaves + (TRAMP) mice turmeric + yucca roots + saw palmetto + chamomile flowers + gingko Induction of Human prostate apoptosis by the carcinoma DU-145 D-Limonene + regulation of proteins 0.2 mM+1.9 nM Prostate In vitro and normal [72] docetaxcel involved in prostate epithelial mitochondrial PZ-HPV-7 cells apoptotic pathways 11 nM of lycopene per g of diet and broccoli powder, Reduction of tumor 1.6 μMof Tomato powder + growth mediated by Dunning R3327-H glucoraphanin, broccoli powder reduced cell prostate 5.9 μMof Prostate In vivo [73] (10 : 10) g/100 g of proliferation and adenocarcinoma glucobrassicin, diet induction of model 3.9 μMof apoptosis gluconasturtiin, and 2.1 μMof neoglucobrassicin. Synergistic effect in inhibiting cell proliferation 1 μM/L + 2 mM/L mediated processes. In vitro Mat-Lylu rat cells Lycopene + Antioxidant activity ketosamine to prevent initiation Prostate [74] (fructose/amino of tumors. acid Fru/His) Subcutaneous injections of 20 μM/L + Reduce tumor growth In vivo Mat-Lylu cells in 5.6 mM/L and volume. male Copenhagen rats 10 Journal of Oncology Table 4: Continued. Combination of Pathways affected or Phase of Dose used Organ of study Model of study Reference nutraceutical mechanistic action study Synergistically Human PC-3, enhances the 1 μM+1 nM In vitro LnCaP, DU145 antiproliferative cells effects of docetaxel. Reduced tumor volume and growth Lycopene + Prostate [75] by affecting the levels docetaxel of IG-FR receptor that Xenograft of 15 mg/kg lycopene is highly expressed in DU145 cells in +10 mg/kg a majority of prostate In vivo NCR-nu/nu docetaxel tumors. Inhibited Akt (nude) mice signaling and suppressed surviving necessary for tumor growth Quercetin chalcone Reduction in the (QC) and a 1.6 mg/mL + growth of solid Colon In vivo Balb/c mice [76] pH-modified citrus 1.6 mg/mL primary tumors pectin (MCP) Inhibits the self renewal capacity of prostate cancer stem cells (PCSCs) by synergistically Prostate cancer Quercetin + EGCG 20 μM+0–60 μM inducing apoptosis Prostate In vitro [31] stem cells (PCSCs) decreasing cell viability in spheroids, cell migration, invasion and colony formation Antagonistic Resveratrol + estrogenic effects in Human MCF-7 10 μM+1 nM Breast In vitro [39] estrogen (E2) suppression of cells progesterone receptor Synergistically Either all at 0.5 μM inhibited cell Human and In vitro proliferation and MDA-MB-231 cells 5 μM, or 20 μM induced apoptosis. Inhibited cell cycle [77] Resveratrol + Breast progression with quercetin + predominat cell cycle catechin arrest in the G2 phase 0.5, 5, and Reduced primary Breast cancer 25 mg/kg body tumor growth and, In vivo xenografts in weight in a 100-μL therefore, inhibit mouse models volume tumor progression Inhibit cell proliferation via capase mediated cytotoxicity. Resveratrol + 50 μM+5 mM Enhanced Breast In vitro MCF-7 [78] cyclophosphamide proapoptotic genes Bax, Fas and suppressed anti apoptoticgeneBcl-2 Journal of Oncology 11 Table 4: Continued. Combination of Pathways affected or Phase of Dose used Organ of study Model of study Reference nutraceutical mechanistic action study Inhibited cell proliferation and induced differentiation. Resveratrol + 50 μM+2 mM/L Attentuated p27 Colon In vitro Caco-2 [79] n-Butyrate (Kip1) levels but enhanced p21 (Waf1/Cip1) expression. Inhibited cell proliferation and Resveratrol + 200 μM+IC HCT116 p53+/+ induced apoptosis by Colon In vitro [80] 5-Fluorouracil 800 μM and p53−/− increase in capase 6 activity Suppressed prostate Simian Virus-40 cancer development T-antigen-(SV-40 and mediated Tag-) targeted apoptosis by affecting probasin promoter Resveratrol + 250 mg/kg each in the expression of Prostate In vivo rat model, a [81] genistein the AIN-76 diet steroid-receptor transgenic model coactivor-3 and of spontaneously insulin-like growth developing factor-1 prostate cancer. Affected DNA methyltransferase activity and reversed thegeneexpression of promoter hypermethylated Genistein + genes of retinoic acid 5 μM/L + 15 μM/L Esophagous In vitro KYSE 510 cells [82] sulforaphane receptor h (RARb), RARB, p16INK4a p16 and O6-methylguanine methyltransferase enhanced growth inhibitory effects Changed cell morphology and inhibited cell Sulforaphane + proliferation. benzylisothio- 10 μM+10 μM Reduced cell viability Pancreas In vitro PANC-1 cells [83] cyanite that correlated with reduced pSTAT3 levels and an increase in PARP Cleavage Synergistically induced phase II enzyme UDP-glucoronyl transferases Sulforaphane + 10 μM+10 μM (UGT1A1) transcript Colon In vitro CaCo-2 [84] apigenin but to a lesser effect the protein level. Mediates this action by the induction of NF-κB 12 Journal of Oncology Table 4: Continued. Combination of Pathways affected or Phase of Dose used Organ of study Model of study Reference nutraceutical mechanistic action study Has an antagonistic effectatlow Human colon 2.5 μM+20 μM concentration on cell cancer 40–16 cell Sulforaphane + growth. line randomly [85] 3,3 - Colon In vitro At cytotoxic derived from diindolylmethane Total concentrations of the HCT116 clone (DIM) concentration compounds has 40 μM synergistic effects on growth inhibition AIN-76A diet Sulforaphane + supplemented with Blocked colon tumor min+ dibenzoymethane Colon In vivo Male Apc/ mice [28] 300 ppm SFN and development (DMB) 0.5% DMB Table 5: Factors conducive to the anticarcinogenic efficacy of EGCG with the drug taxol, which is commonly used to nutraceuticals. treat breast carcinomas, lowered the effective dose of EGCG, ranging from 0.1–1.0 μg/mL which is a serum obtainable Possible effects on the bioactive Factors level through metabolism. This same group showed that components in the dietary supplement higher doses (30–40 μg) of EGCG were required to mediate a Metabolism similar effect when used alone [95]. Bioavailability Time taken to achieve maximum EGCG can be exploited as a chemopreventive agent if plasma concentration it prevents cancerous lesions from occurring at lower dose Maximum plasma concentration, concentrations and for prolonged periods of time. Most half-life in vitro studies have used relatively high doses of EGCG Oral and such doses may prove to be more tumor promotive Method of Intraperitoneal than preventive in longer exposure time periods. In a study ingestion Subcutaneous designed by Pianetti et al. [92], contradictory results on the effects of EGCG on Her-2/neu overexpressed receptor in Powder/capsule NF639 breast cancer cells was observed. At short exposure Liquid Form of ingestion times, EGCG was very effective in reducing cell proliferation, Cooked (solid) but at prolonged exposure cells became resistant to EGCG Raw (solid) with increased levels of NF-κB. This observed change in Ratio of pure to the compound drug-induced resistance was related to the activation of Formulation conjugates mitogen-activated protein kinase. It appears that single doses Preference for an acidic or basic or one specific chemical constituent is mostly insufficient to Stability environment (pH) induce tumor suppression or regression. Such in vitro data Direct via receptors on the cell surface outcomes emphasize that a dual-drug treatment approach is Mechanism of or into the nuclear region via channels necessary to treat the disease. This also signals that the timing action Indirect conjugated metabolites of the nutradrug that is administered is important. Perhaps affecting parts of metabolic pathways EGCG should be administered early in treatment, but later other phytochemicals or drugs, in conjunction with EGCG, may need to be administered in the treatment regimen. In As seen in MDA-MB-468 ER-negative cells, cellular apoptosis their dual-drug treatment of NF639 Her-2/neu breast cancer is mediated by inducing p53 and Bax proteins that enhance cells, Yang et al. found that treating the cells initially with apoptotic functions in cells [96]. Such observations have EGCG lowered cell proliferation and the later introduction been corroborated by in vivo studies using animal models of the MAPK inhibitor, U0126, reduced invasive phenotype [97]. [93]. Most studies have shown that anticancer properties of Most studies determining the anticancer drug properties EGCG are mediated at higher doses. However, such doses of EGCG are preclinical. For better understanding of specific may be irrelevant to clinical applications as they may be EGCG effects, clinical trials should be carefully designed physiologically unachievable through dietary consumption. to include parameters that influence EGCG effectiveness. Therefore, clinical trials should be aimed at achieving EGCG has different roles in ER-dependent versus ER- desired anticancer preventive or tumor functions at much independent receptors, and, therefore, the type of diet lowered doses. Such outcomes are possible with a dual-drug needed to emulate in vitro doses need to be clearly under- approach. One study [95] demonstrated that combining stood through clinical trials and careful pharmacokinetic Journal of Oncology 13 Table 6: Surface receptors expressed by breast cancer cells that alter their sensitivity to treatment. Receptors on the surface Cancer type Phenotype Reference ER PR Her2 Breast cancer cell line SK-BR-3 Negative Negative Positive Adenocarcinoma Invasive [65] MDA-MB-231 Negative Negative negative Adenocarcinoma Invasive [65] MDA-MB-361 Positive Negative Positive Adenocarcinoma Metstasis [65] MCF-7 Positive Positive Negative Adenocarcinoma Invasive [65] Premalignant Isoform B of MCF10AT Positive Variable model for cancer Premalignant [65] PR and not A development studies of these doses in healthy individuals, ER-positive 3.1. Curcumin and Taxol (See [63]). Primary breast cancer cells are commonly treated with the drug taxol. Sus- breast cancer patients, and ER-independent tumors. In testing phytochemicals of the same or different class it tained chemotherapeutic treatment with this drug has often resulted in drug resistance and tumor progression. is rather uncertain which markers are necessary to determine Many chemotherapeutic drugs induce the expression of the comparable dosage values for in vitro versus in vivo efficacies. metastatic gene NF-κB which encourages tumor progression. Formulation of a diet is one of the major deciding factors in Interestingly, natural-based compounds that are pharmaco- the functional efficacy of a chemical constituent. It defines logicallysafehavebeenshown to inactivateNF-κBexpres- the concentration of the dose that will be available in vivo, sion. Taxol is a powerful drug in the treatment of cancer after metabolism, and determines the diet that needs to be therefore, in order to prevent metastasis, a combination of given to achieve such an outcome. Even though single-dose Taxol with curcumin has been shown to downregulate the individual or mixed phytochemical treatments are currently expression of NF-κB and induce apoptosis. available to cancer patients, they are relatively new and much more research in this direction is warranted. One such 3.2. Curcumin and Xanthorrhizol (See [64]). Astudy con- therapy that is rapidly gaining importance and holds promise for future cancer treatments is combination therapies using ducted on an invasive breast tumor cell line, MDA-MB-231, has shown how and when compounds added to the cells plant-based chemical compounds known as nutraceuticals. determine the overall efficacy of the treatment. A sequen- tial addition of curcumin and xanthorrhizol (a rhizomal 3. Combinatorial Therapy: A Promise of sesquiterpenoid of Curcuma xanthorrhiza) in culture resulted in additive and antagonistic effects depending on which theFuture(SeeTable 3) compound was added first to the culture. However, simul- In prevention or treatment, combinatorial approaches can taneous addition of the compounds resulted in synergistic be of the following types: a phytonutrient and an effective effects at lower concentrations and agonistic effects at higher drug, two or more phytonutrients, a synthetic phytonutrient concentrations. Such experiments provide evidence that the and an effective drug, or a synthetic phytonutrient and efficacy of a drug is dependent on dose, time, and how it a natural nutrient. Studies in the last few decades have is presented to the cells. Therefore, results obtained might focused attention on unraveling the protective properties be contradictory if doses used are simply antagonist or and mechanistic actions of many phytochemicals. Still the additive. For a successful combination therapy or prevention, pharmacokinetics of quite a few of these phytochemicals synergistic doses are more relevant to mediate downstream are not known, and, for a few that are known, there is effects, as lower concentrations of the test biomolecules will much variability based on mode and form of delivery, be required. dose, and the model organism of study (Tables 2, 3,and 4). Another interesting approach to enhancing curative 3.3. Curcumin and Docosahexaenoic Acid (DHA) (See [65]). and preventive properties of these nutrients is combination DHA is a dietary compound present in fish oil that has been therapies. The therapy is based on the factual information shown to have potent chemopreventive affects against cancer. available at hand and using the potent properties of one with Chemotherapeutic effects of compounds are often analyzed that of another to enhance synergistic or additive actions using in vitro models. However, what is most often observed (Figure 1). In this paper, groups that have worked with is that all cells from the same tissue sample do not react different phytomolecules belonging to a different or the the same way to the test compound. It is essential to have same chemical class of compounds have been analyzed for a chemopreventive or therapeutic agent that can induce its their antitumorigenic activities, and the overall results of the effects on a wide range of cancerous cells arising from the experiments for each group are described in Table 4. same tissue. In this study, the authors analyzed five cell lines 14 Journal of Oncology expressing different cell surface receptors (Table 6)which in cell cultures has been shown to inhibit cell growth. make them susceptible to chemotherapeutic compounds but This observed cytotoxicity is enhanced several folds when in different ways and to different degrees. The combinatorial low doses of the natural compound, curcumin, are used synergistic doses for each cell line were different, as shown in combination, which are otherwise ineffective unless very in the Table 4. In particular, one breast cancer cell line, high concentrations are used. These compounds mediate a SK-BR-3, which is ER-negative exhibited a higher uptake of synergistic effect in regulating the cell cycle phases and down- curcumin in the presence of DHA. DHA does not directly regulate MDR genes, thereby, enhancing tumor regression contribute to cell inhibition, but the combination of this phenotypes even in the presence of mutated p53 molecules. compound with curcumin greatly enhances the uptake of curcumin by the cells. This compound, DHA, can reach a 3.6. Curcumin and Celecoxib (See [68]). Cyclooxygenase- plasma concentration level of 200 μM. Although the focus 2 (COX-2) expression is central to the carcinogenesis of of this study was entirely based on the SK-BR-3 cell line, colorectal cancers. Compounds that regulate the expression the effects of reduced synergy on other cell lines in terms or activity of COX-2 in cells may be instrumental in of transcriptome effects need to be investigated. Mammary mediating chemotherapeutic effects on the tissue or cells. tumors may contain a heterogenous population of cells Celecoxib is a potent inhibitor of COX-2 and is presumed exhibiting different surface receptors. Using combination to target itsactivesite. However, prolongedexposureto therapy should be aimed at reducing the populations of celecoxib results in cardiovascular problems. It appears that all these cell types within the tumor site to truly exhibit monotherapy regimes are very effective in inhibiting cancer antitumor potency with minimal side effects. growth, proliferation, metastasis, and invasion, as seen in numerous in vitro and in vivo models. However, prolonged 3.4. Curcumin and Genistein (See [66]): A Preventive Strategy. exposures at concentrations relatively higher than what can The aim to use natural compounds in diets is to render be achieved with combination doses may result in unwanted the chemopreventive properties of the compounds to the side effects. Testing the efficacy of celecoxib with cucumin tissues. Numerous studies have shown that single dosage showed that at lower doses of celecoxib it was possible of compounds used alone is effective for chemoprevention. to enforce synergistic inhibitory growth effects on colon The problem faced is the inability to achieve high serum cells which expressed various levels of COX-2. Like many concentrations in vivo. Although combination studies are other in vitro investigations, this study emphasizes the fact just beginning to surface as more prominent approaches in that combining powerful drugs with naturally available clinical treatment, studies, though limited, have shown that potent compounds can reduce the dose needed to mediate synergistic effects of the compounds are able to be achieved potent anticarcinogenic effects with minimal side effects. at much lower doses than when compounds are used alone. Clinically, such studies are relevant as the doses used or Especially in cancers that are hormonally regulated, the needed are within the physiologically dose range. With colon tissues are often exposed to external or internal hormonal cancer having such a high incidence rate in the western stimulation, like estrogen, as in the case of breast tissue. Envi- populations, such therapies can be taken as advantage, ronmental agents that mimic estrogen-like activity can often and biomolecules having preventive potential against the stimulate or initiate the carcinogenic process. Curcumin, a formation of precancerous lesions need to be supplemented curcuminoid, and genistein, an isoflavone, are derived from in diets of patients at high-risk. two different chemical classes, yet they have been known to inhibit a variety of tumor types in vitro and in vivo. 3.7. Coltect and 5-Aminosalicylic Acid (5-ASA) (See [69]). Clinical trials of these compounds individually have been Coltect is a novel chemotherapeutic dietary drug with a tested [19, 60, 104, 105]. The mechanistic action of the formulation of curcumin, a turmeric extract (95% curcum- individual compounds in many different cancers has been inoids) mixed with turmeric powder 1 : 1, green tea (60% investigated as well. However, using these compounds in polyphenols and 25% EGCG) in a 2 : 1 ratio, and 0.1 mg/mL combination drastically affects the development of tumors of L-selenomethionine. 5-ASA is an anti-inflammatory drug, by mediating changes in shape and growth inhibition. Such which has been shown to have a preventive role in polyp for- changes were observed both in ER-positive and ER-negative mation that is thought to occur via the inflammation process cells, indicative of the dual use of such a combination in in conditions like inflammatory bowl disorder. Coltect has prevention and therapy. been effective against HT-29 human colon adenocarcinoma grade II cells in vitro, and this nutraceutical complex in 3.5. Curcumin and Sulfinosine (SF) (See [67]). The inef- combination with 5-ASA has been shown to inhibit the fectiveness of certain drugs in prolonged chemotherapy formation or growth of chemically induced aberrant crypt stems from the resistance that some cancers develop with foci (ACF) in rat models. The molecular mechanism by time. This is one of the major obstacles in cancer therapy, which this inhibition is mediated is via the inhibition of especially in cancers that are multidrug resistant (MDR). The COX-2pathwaysinHT-29 cells, whichhas been supported problem in finding a suitable cure for non-small-cell lung by in vitro studies of other groups [106, 107]. However, cancers is the MDR phenotype it exhibits. Treating MDR growth inhibition can be affected via COX-2-independent cells such as non-small-cell lung carcinoma NCI-H460/R pathways possibly through mechanisms that are regulated by cells with a commonly employed drug, SF (obtained by the the functional polyphenol complex in coltect. Such complex amination and subsequent oxidation of 6-thioguanosine), mixtures are of clinical significance as many different control Journal of Oncology 15 mechanisms can be regulated by the presence of individual into colon cancer. In colonic crypts that are chemically constituents of the polyphenols which are a part of the induced, limonene asserts its effect by inhibiting the activity of ornithine decarboxylase, an enzyme essential for the formulated mixture of coltect. polyamine biosynthesis pathway. This pathway regulates the cell cycle, and D-limonene-dependent inhibition of 3.8. Phenylethylisothiocynate (PEITC) and Curcumin (See ornithine decarboxylase (ODC) encourages an antiprolifer- [27]). Most prostate cancers begin as a hormone-dependent ative activity. If aberrant crypt foci are the initial markers tumor, and the hormone is primarily androgen. However, for colon carcinogenesis, and D-Limonene and its derivatives the more aggressive forms of prostate cancer are androgen- assert their roles against initiation and promotion phases of independent and hormonal therapies fail to be effective. cancer, then a diet rich in citrus foods can prevent crypt for- Alternate therapies are, therefore, necessary to treat such mation. Therefore, D-limonene appears to have potential as aggressive forms. Most cancerous cells express various sur- a chemopreventive agent in colon carcinogenesis. However, face receptors that propagate cellular growth. Targeting such in vivo studies often do not correlate with results in vitro receptors can be an effective chemotherapeutic approach. for many of the reasons discussed earlier. Once the intake Curcumin, obtained from Curcumin longa, has been shown of a compound is deemed safe for human consumption, to inhibit the phosphorylation of EGFR, inhibit the Akt it is imperative to analyze and study the mechanistic and signaling pathway, and negatively regulate NF-κB. It is an metabolic functions in human subjects to determine the effective molecule against prostate cancer. Phenylethylisoth- efficacy of the nutrient in question. As in the case of under- iocyanate, a phytochemical in cruciferous vegetables, has standing limonene protection against colonic carcinogenesis, been shown to inhibit prostate cancer cell growth in vitro the studies were performed on rats and for shorter exposure and this observation has been supported by epidemiological time to the compound or its derivatives. Therefore, further studies showing that consumption of cruciferous vegetables in vivo models are required to determine the toxicity of has an inverse effect on prostate cancer risk. When two the treatment for longer periods of time, as D-limonene is bioactive molecules with similar effects are used in treating nontoxic but its alcohol derivatives could be toxic. hormonally independent tumors in affecting receptor medi- ated signaling, the effects are more pronounced than when 4.1. D-Limonene and Docetaxcel (See [72]). Many combina- used as individual compounds. With PEITC and curcumin, tion studies are underway to determine an effective approach the observed effect was more additive than synergistic, in treating advanced and aggressive prostate cancers. Doc- but cell growth inhibition was profoundly affected by the etaxel, a synthetic derivative of taxol, is primarily used to inhibition of NF-κB pathways and Akt signaling pathways. inhibit the microtubular structures in cancerous cells that Such responses were seen at lower physiological achievable support cell division. In addition to its role as a microtubule doses. These results were corroborated by in vivo studies disruptive molecule, it has a host of inhibitory actions in mice using human PC-3 prostate cancer cells [70]. Since on genes which regulate cell proliferation, mitotic spindle EGCG has similar effects on prostate cancer cells, EGCG formation, transcription factors, and oncogenesis. It also could also possibly serve as a substitute in place of curcumin upregulates genes involved in apoptosis and cell cycle pro- for such a treatment strategy. gression in prostate cancer. D-Limonene, discussed earlier has been shown to have anti-prostate carcinogenic effects at low dose concentrations. Logically; therefore, combining the 4. D-Limonene and Its Combination Therapies two compounds may have a plethora of positive antitumor (See Table 3) functionalities. In a study by Rabi and Bishayee [72], the Although a few studies have shown that D-limonene, an combined treatment enhanced the sensitivity of DU145 abundant monoterpene in citrus oils, exhibits antimitogenic prostate cancer cells that are known to be apoptotic resistant. activity, its alcohol-derivated perillyl alchohol (PA) has a This enhanced sensitivity was thought to be mediated by greater inhibitory effect on cell migration in cancerous reactive oxygen species (ROS) generation and activation of cells [108]. A study by Reddy et al. [108] used subtoxic caspase 3 and 9. Such a positive in vitro outcome warrants doses of PA to determine this effect. Further preclinical further investigations in vivo, in models that mimic the studies are necessary to determine the effective yet nontoxic progression of the disease, before it can be used in dietary serum/tissue concentration that can be achieved from a diet supplements for therapy. rich in citrus intake, in conjunction with phytonutrients of the same class or a differentclass. Notmuchisknown about 4.2. Lycopene and Fru/His (See [74, 111–114]). Serum the percentage composition of D-limonene and its metab- lycopene (a carotenoid) levels have been shown to have olized constituents that are required to achieve an effective an inverse correlation with prostate cancer risk. A diet- monterpene anticarcinogenic activity. In comparison to its based population study showed that, of all the carotenoids oxygenated derivatives, limonene has the least cytotoxic assessed, high serum lycopene levels showed a statistically effect on both noncancerous and cancerous breast cell lines significant lower prostate cancer risk. Further analysis of and, therefore, can be applicable in chemoprevention [109]. their data revealed that lower serum lycopene levels in D-limonene appears to be more effective against chem- conjunction with β-carotene supplements were effective ically induced colonic crypt foci [110]. These foci are against lowering the risk of prostate cancer, suggestive for preneoplastic lesions and are biomarkers for the progression a combinatorial therapy [111]. Certain dietary compounds 16 Journal of Oncology can be the source of cancer formation as seen with prostate compound are still not known, the combination of the two cancer. It is believed that the nonfat portion of milk and seemed to preferentially localize in the prostate more than in excess calcium are some main factors in prostate cancer other tissues that were tested [74]. risk [112]. Numerous in vitro studies have shown that Occasionally, a combination may fail to incite anticar- carotenoids have a greater influence in reducing tumors of cinogenic effects as was seen by Mossine et al. [114]. Their the prostate origin, and lycopene and 1,2-dihydroxyvitamin experiments were conducted on the prostate adenocarci- D3 are at the forefront as risk reduction factors. In addition noma rat model that was used by other groups, and their data to their role as potent inhibitors of prostate cancer growth, had contradictory results to the effective action of lycopene they are biologically safe and cheaper forms of treatment. itself and in conjunction with other micronutrients. Their 1,2-dihydroxyvitamin D3 and lycopene have physiologically study revealed that lycopene was not able to inhibit or reduce different roles, but combined they modulate pathways to tumor load alone or in combination and that selenium synergistically inhibit proliferation and differentiation at alone in the mixture was able to induce antitumorigenic much lower concentrations [113] and bear additive effects effects. Such outcomes are important as they open up on cell cycle progression. more questionsastowhy amoleculethataffects a given The assessments that lycopene is a safe dietary molecule pathway behaves differently when tested within the same with anticancer properties is supported by a number of experimental model. Is it always dose or concentration or population epidemiological and cohort-based studies [112]. does molecule preparation and delivery impart effects on the However, it is important to ensure that the statistical models efficacy of a drug? used are able to adjust for many parameters for a true 4.3. Lycopene and Docetaxel (See [75]). Docetaxelisapotent significant outcome. Regardless of the statistical model chemotherapeutic drug that is clinically used to treat patients employed in these assessment studies, lycopene has emerged with advanced metastatic prostate cancers. Although the as a potent risk-reducing factor of prostate cancer and drug extends survival, it is for a very limited time period has been even supported by a study that was carried out and with a poor prognosis. Lycopene, a natural compound, across 28 countries. Intervention combination studies have has been shown to have strong cancer inhibitory properties not yet been performed. However, in vivo-based studies against the prostate tissue. One study tested the possibility in mice models have shown that lycopene administered to use this combination of compounds to enhance survival in the form of tomato powder and broccoli powder in of patients that were detected with aggressive, androgen- a 10 : 10 ratio, increases its serum concentration to about independent tumors. As predicted, docetaxel inhibited 538 nM/g with about 0.4 nM/g concentrated in the prostate tumor growth in nude mice that bore tumor xenografts of tissue itself. Diet-based intervention studies are required human DU 145 cells. Analysis of molecular mechanisms to determine the formulated diet required to improve the revealed that the action of docetaxel was on regulating availability in the serum of patients and enhance the localized the insulin-like growth factor receptor (IGFR) pathway by concentration of the molecule in the tissue. Such a diet- suppressing IGF, and this effect was synergistically enhanced based treatment may serve as a suitable chemopreventive in the presence of lycopene. Together the molecules asserted approach against prostate cancer or with patients at high-risk negative downstream effects on Akt signaling pathways and of the disease. Even though bioactive molecules successfully suppressed survivin, products of which have been known work in administering their protective functions in vitro, to maintain tumor growth and enhance metastasis. Clinical it appears through in vivo studies that diet and availability trials using this combination may prove effective in treating crucially dictate outcomes. A critical question to be asked is patients that express high levels of IGFR in the prostate what factors constitute a perfect blend of bioactive mixtures. tumor and extend survival for a longer duration than what With the current research thus far, it is hard to address is possibly achieved by docetaxel alone, which is about 18–20 what the cut-off ratios are that need to be used in a diet months. that contain mixtures of potent nutraceuticals to coordinate similar effects clinically. Possibly a slight change in con- centration of even one of the effective biomolecules may 5. EGCG and Quercetin (See [31]) render the mixture ineffective in its function. It is rather an important task for pharmaceutical chemists and nutritionists EGCG exhibits strong chemopreventive and therapeutic to determine the ratios of effective biomolecules in a mixture activities as it influences many pathways as shown in and determine the pharmacokinetics and dynamics of that Figure 1. Some of the mechanistic pathways are involved mixture. in regulating the levels of Bcl2, survivin, and XIAP and Fru/His, a ketosamine, is also a derived product from activation of caspase-3/7 to induce apoptosis. EGCG is also tomatoes obtained by the reaction of a carbohydrate with an involved in inhibiting genes that are required for transition amino acid. This particular ketosamine has been found to from epithelial to mesenchymal cells and retards migration assert chemopreventive effects by synergistically enhancing and invasion which are primarily advantageous in terms the activity of lycopene, by neutralizing ROS species and of controlling aggressive tumors. EGCG mediates such inhibiting DNA damage. Therefore, the complex of these synergistic actions in conjunction with quercetin to retard two molecules may have a pivotal role in prostate cancer the self-renewal properties of cancer stem cells (CSCs), a prevention. Although a rat model was used to determine population that, if inhibited, can influence tumor regression. the results of the treatment and pharmacokinetics of the Quercetin, a polyphenol, downregulates the expression of the Journal of Oncology 17 heat shock protein (Hsp90) known to influence apoptosis of a mixture of three polyphenols, resveratrol, quercetin, and and growth inhibition of prostate tumors. Therefore, the catechin, albeit pharmacokinetics studies are warranted. combination of these molecules modulates their respective therapeutic effects to mediate synergistic growth retardation 6.2. Resveratrol and Cyclophosphamide (See [78]). Cyclo- of CSCs. The study by Tang et al. [31] used relatively phosphamide, a neoplastic drug, has a broad spectrum of higher concentrations of EGCG (60 μM) in the presence of activity on a variety of cancers, including breast cancers. The 20 μM quercetin. Probably concentrations of EGCG that can shortfall of the drug is its myriad of toxic effects on other mediate similar synergistic levels, albeit at lower doses, need systems. Dose reduction of the compound would be a means to be investigated, and the therapeutic potential across cancer of reducing its toxicity without compensating its anticar- stems cells of other origins need to be assessed if clinical cinogenic activity. Resveratrol has been shown to success- applications are to be considered. fully lower the effective dose of cyclophosphamide without altering its anticarcinogenic activity. Both of the compounds 6. Resveratrol and Estrogen (See [39]) together synergistically enhance caspase-mediated cytotoxic activity, as demonstrated in MCF-7 cells, an aggressive breast Selective estrogen receptor modulators that are used in cancer cell line (Table 4). The combination therapy resulted the clinical treatment of breast cancers display dual ago- in the upregulation of p53, proapoptotic genes, Bax and Fas, nist/antagonist effects in the tissues, especially in cancer initi- and downregulation of antiapoptotic gene Bcl-2, suggestive ation and progression. Drugs like tamoxifen emulate antag- of an apoptotic mechanism involved in cell death. onistic effects on estrogen to contain the tumor. Agonistic- estrogen-like activity can in some instances enhance tumor 6.3. Resveratrol and n-Butyrate (See [79]). n-Butyrate is a progression which is not desired in most clinical treatments. short chain fatty acid produced by bacterial fermentation of Resveratrol, a polyphenolic compound abundant in grape fiber in the colon. The compound is a known differentiating skin and grape products including wine, is known to have agent and induces an epithelial phenotype in certain cultured chemopreventive properties as supported by numerous in cells. n-butyrate is a potent histone deacetylase (HDAC) vitro and in vivo studies. However, based on the experimental inhibitor as well and one of its differentiation-inducing cell type, resveratrol induces either agonistic or antagonistic properties stems from its ability to inhibit HDACs. Resver- effects that can be weak or very pronounced. Resveratrol ago- atrol, discussed above, induces apoptosis through other nistic effects are totally reversed in the presence of estrogen, mechanistic pathways. The combination of two bioactive possibly mediated through estrogen receptor β. This reversal molecules influencing apoptosis via different mechanistic of effects is pertinent to prevention of breast cancer lesions pathways may associate to render an apoptotic phenotype in ducts that could become long-term neoplastic and can- in cancerous cells and inhibit tumor formation and pro- cerous. Of its many cancer protective functions, resveratrol gression. The 2 mM dose of n-butyrate used in the Wolter in combination with glucan are potent immunomodulators and Stein study [79] is probably much higher than what can by upregulating Cdc42 expression [115]. When natural be physiologically achieved. This dose is probably suitable compounds exhibit multi-chemopreventive properties, con- for treatment of colon cancers where higher molar doses jugation therapies are advantageous over monotherapies. of n-butyrate are possible. However, n-butyrate is highly Albeit not clinically tested, harnessing cancer preventive and unstable, and its serum concentrations are lower than 2 mM. immune modulating functions of nutraceuticals seems to be Since this molecule is a differentiating agent, its clinical use a plausible approach to targeting hormonally independent in treatment of other cancers is relevant. However, such aggressive tumors. therapies require absolute lower effective doses and can probably be achieved by combining with molecules other 6.1. Resveratrol, Quercetin and Catechin (See Table 4 and than resveratrol or modifying the compound to specific [77]). Theprotectivefunctions of polyphenolsare many- conjugates to reach serum concentration levels. fold. Numerous studies have analyzed their protective and therapeutic functions in vitro on tumor initiation that was chemically induced or in vivo via cellular implanted tumor 6.4. Resveratrol and 5-Fluorouracil (5-FU) (See [80]). 5- formation. Few studies have established the functions of fluorouracil inhibits thymidylate synthase, prevents DNA combined polyphenols on established tumors, as the major- proliferation, and induces DNA damage-related apoptosis in ity of investigations have focused on individual mechanistic colon cancer cells. Phase I clinical trials using a combination effects of the compounds. Dietary serum concentrations of resveratrol and grape powder have shown that resveratrol are influenced by the individual percentage of biomolecules at low doses inhibit Wnt,agenethatisupregulated in present in the diet. Therefore, individual protective assess- colon cancers. Taking advantage of therapeutic effects of ments of a compound show higher dose requirements, whilst nutraceuticals, combined therapy of aforementioned resver- mixtures may require lower doses to achieve the same effects. atrol with 5-FU surfaces as a principal strategy in treating Additive and synergistic effects of compounds occur if their colon cancers. When used in combination, the presumption individual functions are enhanced in the presence of other is that either additive or synergistic effects of the two could molecules, perhaps by reinforcing the serum stability and mediate tumor inhibition by modulating their individual availability of the various compounds in the mixture. Such apoptotic effects. The concern in using resveratrol is that observations were seen in both in vitro and in vivo testing higher concentrations of the doses are required in the 18 Journal of Oncology treatment which clinically may not be reached through 7.1. Benzylisothiocynate (BITC) and SFN (See [83]). BITC dietary consumption. and sulforaphane are ITCs derived from cruciferous vegeta- bles like broccoli. Individually both these molecules exert potent chemotherapeutic properties strongly supported by 6.5. Genistein and Resveratrol (See [81]). Genistein and numerous studies. Oddly, even though both are isothio- resveratrol as individual phytochemicals are very effective in cyanates, they exert their therapeutic effects by control- the treatment and prevention of prostate cancer progression ling different pathways involved in tumorigenic inhibition. in rodent studies. Poorly differentiated prostate cancers STAT3, a member of the STAT group of transcriptional often fail to respond to androgen-dependent treatments, factors, is required for early development and is dispensable and alternate treatments are required. Androgen receptors in adult tissues. However, there appears to be a correlation likewise have two functional roles, one as a tumor suppressor between the constitutive expression of STAT3 and tumor in normal prostate tissue and the other as an oncogene in development, indicative of its role as an oncogene. This neoplastic transformation, where it is altered either by muta- gene appears to have important roles in cell proliferation, tions or DNA damage. Genistein and resveratrol used in an in angiogenesis, and metastsis, a crucial requirement of tumor vivo rat-based study, modeled to understand the mechanistic survival. Both BITC and sulforaphane have cancer inhibitory action of combined treatments in the progression of prostate effects, affecting independent cell signaling pathways. How- cancer, showed that they had more pronounced effects, albeit ever, the sequential combination of the two has been shown not synergistic. The statistically significant additive functions to regulate the STAT3 gene and others (Table 4), thereby, of reducing cell proliferation through mechanisms that inducing apoptosis. How dietary molecules are presented regulate the androgen receptor levels and IGF-1, a biomarker to the cells in vitro is important to its cellular mechanistic found in patient serums with progressive and aggressive actions. In the study by Hutzen et al. [83], sequential prostate cancers were achieved in combined therapies over addition of BITC to the cells after sulforaphane treatment the monotherapy regimes. Interestingly, the combination of was performed, which enhanced the reduction of STAT3 genistein and resveratrol increased serum availability of both, levels; however, simultaneous additions were not performed. but higher concentrations of resveratrol were achievable as Simultaneous additions would be important for any com- compared to the single-dose regimen. Perhaps, absorption bination study to determine possible synergistic, additive, and stability of resveratrol were profoundly affected in a or antagonistic effects between the compounds. Preclinical combined environment, which is clinically a clear advantage. studies should include various combinatorial interactions of The doses used in the study are physiologically safe and the nutraceuticals being tested to determine the best way of achievable in vivo by consumption of a soy-based diet high using combined molecules in therapy. in the percentage of genistein. However, resveratrol is found in low levels in grape-based dietary products, and, therefore; 7.2. SFN and Apigenin (See [84]). Phase I and Phase II a pure supplement of the compound is necessary in case that enzymes are extremely important to cancer prevention. higher doses are required. Dietary foods are sometimes modified to produce car- cinogens through metabolism by the action of Phase I enzymes. Subsequently, the action of Phase II enzymes 7. Genistein and Sulforaphane (SFN) (See [82]) rapidly metabolizes these products to more soluble forms Previous studies have shown that EGCG, a major polyphenol that are eliminated as body waste. Phase II enzymes are more in green tea, can inhibit tumor growth through mechanisms concentrated in the duodenum and small intestine and less that alter DNA methylation activity, reversing the expression available in the colon. Increasing the availability of these of silenced genes involved in tumor inhibition in cancer enzymes in the colon can get rid of harmful carcinogens cells. Hypomethylation of the promoters that are CpG-rich reducing the incidence of colon cancers, and, therefore, is more likely to be transcribed, with an exception of few dietary supplements that induce Phase II enzymes would like hTERT, the regulatory gene of telomerase [82, 116–119]. be promising tools for colon cancer prevention. SFN, an Epigenetics is a mechanism that has been studied for decades, isothiocyanate, and apigenin, a flavanol, have independent and factors that regulate epigenetics are now believed to cancer preventive functions. SFN is a strong inducer of UDP- be very important as treatment possibilities in controlling 27 glucuronyltransferase (UGT1A1). UGT1A1 is a major tumors. DNA methylation and histone deacetylation are player in the detoxification process of carcinogens formed well known epigenetic mechanisms that regulate many of in the body and, therefore, is a potent Phase II enzyme. the genes involved in cancers of various origins. Genis- Treating nondifferentiated colon cells with a combination of tein combined with SFN, an histone deacetylase inhibitor SFN and apigenin was found to synergistically induce the (HDACI) has been successful in inducing the transcription expression of UGT1A1 suggesting a possible dietary tool for of genes involved in regulating cell cycle by reversing the colon cancer prevention. The in vitro dose of the individual hypermethylated states of their promoters. This change was compounds used in the study was at physiological safe levels and can be easily achieved in vivo. observed at low doses and was enhanced in the presence of sulforaphane more than that when genistein was used alone. However, in vivo studies of the same are warranted 7.3. SFN and 3,3 -Diindolylmethane (DIM) (See [85]). The to determine epigenetic behavior of the dietary compounds importance of investigating the roles of dose combinations before applications to human treatments are considered. on chemopreventive or therapeutic functions has been well Journal of Oncology 19 dissected in a study by Pappa et al. [85]. Lower doses of an urgent need for alternate or adjuvant therapies has arisen. SFN demonstrate antagonistic effects on cell proliferation Phytochemicals are relatively safe and abundantly available and higher doses of both compounds had synergistic effects. from dietary sources. Therefore, alternate medicine aims at Synergism of compounds is preferred if the outcome is harnessing the protective properties of these nonessential tumor regression, but in clinical treatments synergistic nutrients toward cancer prevention and treatment. A large actions should be mediated at safe lower concentrations database of studies supports the use of biomolecules in rather than at cytotoxic levels. Presumably, the choice of cancer treatment, albeit a majority of those are in vitro compounds used, based on the genetic or cellular function studies. Regardless of limited in vivo studies and clinical required, is imperative to the success of the treatment. trials, phytochemicals show great promise in cancer treat- Possibly, SFN can synergistically inhibit the proliferation of ment considering their safe use. Caution must be taken cancer cells with compounds other than DIM at much lower when addressing the efficacy of these molecules in clinical doses, which has been investigated in studies using SFN trials as many factors modulate their effects on cellular with flavanols. This clearly highlights the problems of using functions as detailed in Table 5. Combinatorial studies combined therapies, especially since dosage is of critical also show great promise, especially when lower nontoxic importance for the success of clinical trials. doses are required for prolonged periods to mediate potent chemotherapeutic functions with minimal side effects. Two 7.4. SFN and Dibenzoylmethane (DMB) (See [28]). When of the major problems currently faced are dosage and seeking for dietary molecules with potential chemoprotective delivery. To maintain a constant physiological serum dose and therapeutic properties, it is essential to understand how availability, it is imperative that the agent is concentrated and they mediate their combined action. Based on mechanistic stable in the tissue of concern. Combination technologies studies, only compounds that are able to achieve synergistic may be a solution to this problem. Nanotechnology is or additive inhibitory or inductive actions on cellular fast catching pace as the next level of technology in all genes, pathways, and/or phenotypes can then be chosen for spheres of science. Limited in vitro studies have shown treatment, even though their individual actions may be more that encapsulating dietary supplements in nanoparticles can pronounced. DMB is antimutagenic. Patients with aberrant effectively deliver the supplement and increase its stability polyp crypt (Apc) mutations are prone to spontaneously and availability. Perhaps research needs to focus on such form aberrant polyps in their intestinal tissue, which later possibilities as avenues of using combination therapies. can transform to colorectal cancers. Treatment with DMB foundinlicoricecan effectively inhibit such mutations in Conflicts of Interest Apc, thereby protecting individuals from aberrant polyp formations. This molecule, therefore, has potential in terms The authors declare that they have no conflicts of interest. of colon cancer prevention. SFNhas amyriadofchemopreventive functionsasseen Acknowledgments before in other studies and in various tissues. A combination of these two chemopreventive agents will have a profound This work was supported in part by grants from the National impact on individuals that are at high-risk or reduce the Cancer Institute (RO1 CA129415), the American Institute incidence of colon cancers through dietary supplementation. for Cancer Research, and the Norma Livingston Foundation. The study by Shen et al. [28] showed that dietary intake of SFN and DMB negatively influenced the incidence and number of tumors formed in the Apc mice. The combined References doses used were half that of the individual doses. However, [1] W. C. Hahn, S. K. Dessain, M. W. 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