The Role of Resveratrol in Cancer Therapy

Natural product compounds have recently attracted significant attention from the scientific community for their potent effects against inflammation-driven diseases, including cancer. A significant amount of research, including preclinical, clinical, and epidemiological studies, has indicated that dietary consumption of polyphenols, found at high levels in cereals, pulses, vegetables, and fruits, may prevent the evolution of an array of diseases, including cancer. Cancer development is a carefully orchestrated progression where normal cells acquires mutations in their genetic makeup, which cause the cells to continuously grow, colonize, and metastasize to other organs such as the liver, lungs, colon, and brain. Compounds that modulate these oncogenic processes can be considered as potential anti-cancer agents that may ultimately make it to clinical application. Resveratrol, a natural stilbene and a non-flavonoid polyphenol, is a phytoestrogen that possesses anti-oxidant, anti-inflammatory, cardioprotective, and anti-cancer properties. It has been reported that resveratrol can reverse multidrug resistance in cancer cells, and, when used in combination with clinically used drugs, it can sensitize cancer cells to standard chemotherapeutic agents. Several novel analogs of resveratrol have been developed with improved anti-cancer activity, bioavailability, and pharmacokinetic profile. The current focus of this review is resveratrol’s in vivo and in vitro effects in a variety of cancers, and intracellular molecular targets modulated by this polyphenol. This is also accompanied by a comprehensive update of the various clinical trials that have demonstrated it to be a promising therapeutic and chemopreventive agent.


Introduction
Cancer is one of the most commonly diagnosed diseases, and its related morbidity and mortality constitute a very significant health problem worldwide. Although great efforts have been made to discover a cure, cancer remains a very prominent cause of mortality in humans, and effective treatment remains a formidable challenge. An estimated 1.6 million new cancer diagnoses and approximately

In Vitro Pharmacological Properties and Anti-Cancer Effects of Resveratrol
It has been shown that resveratrol possesses multifaceted salubrious properties, e.g., anti-inflammatory, anti-oxidative, and anti-aging qualities [42][43][44]. Resveratrol is a constituent of red wine, and therefore it is often postulated that resveratrol is a significant element in the French Paradox, the reduced risk of cardiovascular disease in French populations despite the high intake of saturated fats; that has been associated with high red wine consumption [45]. After Jang et al. [46] found that resveratrol inhibited carcinogenesis in a mouse-skin cancer model in 1997, a wealth of publications followed. It has been shown that resveratrol has in vitro cytotoxic effects against a large range of human tumor cells, including myeloid and lymphoid cancer cells, and breast, skin, cervix, ovary, stomach, prostate, colon, liver, pancreas, and thyroid carcinoma cells [25,[47][48][49]. Resveratrol affects a variety of cancer stages from initiation and promotion to progression by affecting the diverse signal-transduction pathways that control cell growth and division, inflammation, apoptosis, metastasis, and angiogenesis.

Anti-Tumor-Initiation Activity
Neoplasia initiation concerns the alteration or mutation of genes resulting spontaneously from or caused by exposure to a carcinogenic agent, which finally results in mutagenesis [50]. Oxidative stress plays a dominant part in the causation of carcinogenesis [51]. Reactive oxygen species (ROS) can react with DNA in addition to chromatin proteins, resulting in several types of DNA damage [52,53]. In fact, chemical carcinogens cannot damage DNA until they are metabolized by phase-I biotransformation enzymes, especially cytochrome P450, in cells and converted to reactive electrophiles. In addition, carcinogen-DNA adduct formation gives rise to chemical carcinogenesis [54]. This initiation stage is irreversible but can be prevented by inhibiting the activity and expression of certain cytochrome P450 enzymes and augmenting the activity of phase-II detoxification enzymes, which transform carcinogens into less toxic and soluble products [55,56].
It has been found that resveratrol can inhibit events linked to the initiation of tumors. For instance, resveratrol treatment suppressed free radical formation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in human leukemia HL-60 cells [57]. The diverse anti-oxidant properties of resveratrol have already been described previously [58,59]. Resveratrol is an excellent scavenger of hydroxyls and superoxides, as well as radicals induced by metals/enzymes and generated by cells [59]. It also protects against lipid peroxidation within cell membranes and damage to DNA resulting from ROS [59]. Furthermore, resveratrol functions as an anti-mutagen, as shown by its inhibition of the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine in the Salmonella typhimurium strain TA100 [60]. It has been proposed that resveratrol can be a possible chemopreventive agent, and its anti-mutagenic and anti-carcinogenic properties have been demonstrated in several models [9,61,62].

In Vitro Pharmacological Properties and Anti-Cancer Effects of Resveratrol
It has been shown that resveratrol possesses multifaceted salubrious properties, e.g., anti-inflammatory, anti-oxidative, and anti-aging qualities [42][43][44]. Resveratrol is a constituent of red wine, and therefore it is often postulated that resveratrol is a significant element in the French Paradox, the reduced risk of cardiovascular disease in French populations despite the high intake of saturated fats; that has been associated with high red wine consumption [45]. After Jang et al. [46] found that resveratrol inhibited carcinogenesis in a mouse-skin cancer model in 1997, a wealth of publications followed. It has been shown that resveratrol has in vitro cytotoxic effects against a large range of human tumor cells, including myeloid and lymphoid cancer cells, and breast, skin, cervix, ovary, stomach, prostate, colon, liver, pancreas, and thyroid carcinoma cells [25,[47][48][49]. Resveratrol affects a variety of cancer stages from initiation and promotion to progression by affecting the diverse signal-transduction pathways that control cell growth and division, inflammation, apoptosis, metastasis, and angiogenesis.

Anti-Tumor-Initiation Activity
Neoplasia initiation concerns the alteration or mutation of genes resulting spontaneously from or caused by exposure to a carcinogenic agent, which finally results in mutagenesis [50]. Oxidative stress plays a dominant part in the causation of carcinogenesis [51]. Reactive oxygen species (ROS) can react with DNA in addition to chromatin proteins, resulting in several types of DNA damage [52,53]. In fact, chemical carcinogens cannot damage DNA until they are metabolized by phase-I biotransformation enzymes, especially cytochrome P450, in cells and converted to reactive electrophiles. In addition, carcinogen-DNA adduct formation gives rise to chemical carcinogenesis [54]. This initiation stage is irreversible but can be prevented by inhibiting the activity and expression of certain cytochrome P450 enzymes and augmenting the activity of phase-II detoxification enzymes, which transform carcinogens into less toxic and soluble products [55,56].
It has been found that resveratrol can inhibit events linked to the initiation of tumors. For instance, resveratrol treatment suppressed free radical formation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in human leukemia HL-60 cells [57]. The diverse anti-oxidant properties of resveratrol have already been described previously [58,59]. Resveratrol is an excellent scavenger of hydroxyls and superoxides, as well as radicals induced by metals/enzymes and generated by cells [59]. It also protects against lipid peroxidation within cell membranes and damage to DNA resulting from ROS [59]. Furthermore, resveratrol functions as an anti-mutagen, as shown by its inhibition of the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine in the Salmonella typhimurium strain TA100 [60]. It has been proposed that resveratrol can be a possible chemopreventive agent, and its anti-mutagenic and anti-carcinogenic properties have been demonstrated in several models [9,61,62].

Anti-Tumor-Promotion Activity
Tumor promotion involves clonally enlarging initiated cells to create a continuously proliferating, premalignant lesion. Tumor promoters generally alter gene expression, resulting in increased cell proliferation and decreased death of cells [76]. Studies conducted in vitro have discovered that resveratrol exerts an anti-proliferative activity by inducing apoptosis. Of these, resveratrol modifies the balance of cyclins as well as cyclin-dependent kinases (CDKs), resulting in cell cycle inhibition at G0/G1 phase. For example, a link has been found between the inhibition of cyclin D1/CDK4 by resveratrol and cell cycle arrest in the G0/G1 phase within different cancer cells [77][78][79][80]. Resveratrol was also shown to increase the levels of cyclin A and E, with cell cycle arrest in the G2/M and S phases [81,82]. Similar findings have indicated that resveratrol causes the arrest of cell cycles and activation of the p53-dependent pathway [83][84][85].
p53, a tumor-suppressor protein, is an element critically linked to transcription, and is closely connected to the regulation of apoptosis and cell proliferation; and also acts as a key mediator in the prevention of carcinogenesis [86]. p53 that has been activated binds DNA and stimulates the expression of certain genes, e.g., WAF1/CIP1 encoding for p21, which belongs to the group of CDK inhibitors that are vital to the inhibition of cell growth [87]. Resveratrol reduced the development of human skin cancer A431 cells by downregulating the expression of cyclin D1, cyclin D2, and cyclin E, inhibiting the activities and/or expression of CDK2, CDK4, and CDK6, and upregulating the expression of p21. Resveratrol also suppressed the proliferation of breast cancer MCF-7 and human prostate cancer DU-145 cells [88] via modulating CDK4 and cyclin D1 expression, which have been linked to the induction of p21 and p53. When used to treat A549 cells, resveratrol caused S phase arrest, reduced retinoblastoma protein (Rb) phosphorylation, and induced p21 and p53 protein expression [89]. It has also been demonstrated that resveratrol limits the expression of Rb, another tumor-suppressor protein involved in the G1/S transition in normal conditions [79,82,85].
It has also been shown that resveratrol's anti-proliferative activity involves the stimulation of apoptosis within cancer cells [90][91][92]; it has been proposed that apoptosis activation could be a probable mechanism for chemotherapeutic agents to destroy cancerous cells [93,94]. In many human tumors, apoptosis has been found to be impaired, which suggests that the disruption of apoptotic functions significantly contributes to a normal cell being transformed into a tumor cell. Apoptosis is cell death that has been programmed, and a genetically regulated physiological mechanism to eliminate damaged or abnormal cells. It is also significant as a physiological-growth-control regulator and a tissue-homeostasis moderator in embryonic, fetal, and adult tissues. Apoptotic cells can be identified by regular biochemical and morphological properties, including membrane blebbing, cell shrinkage, nuclear DNA fragmentation, chromatin condensation, and formation of apoptotic bodies [95].
Apoptosis can be activated via two major pathways: the mitochondria-apoptosome-mediated intrinsic pathway and the death receptor-induced extrinsic pathway. [96,97]. The triggering of death receptors in the tumor necrosis factor (TNF) receptor superfamily, e.g., Fas (CD95/APO-1), or of TNF-related apoptosis-inducing ligand (TRAIL) receptors causes the initiator caspase-8 to be activated, which can mediate the apoptosis signal via direct cleavage of downstream effector caspases such as caspase-3 [98]. Caspases are an ubiquitous family of cysteine proteases, and have critical functions in apoptosis as upstream initiators and downstream effectors [99]. The intrinsic pathway is triggered by the dispensation of apoptogenic factors such as Omi/HtrA2, Smac/DIABLO, cytochrome c, apoptosis-inducing factors (AIFs), endonuclease G, caspase-2, or caspase-9 from the mitochondrial intermembrane space [100]. The dissemination of cytochrome c into the cytosol activates caspase-3 via the creation of the cytochrome c/apoptotic protease-activating factor-1 (Apaf-1)/caspase-9-containing apoptosome complex; Omi/HtrA2 and Smac/DIABLO encourage caspase activation by neutralizing the effects of inhibitors of apoptotic proteins (IAPs) [100,101].
Crosstalk also occurs between the two apoptotic pathways. For instance, Fas is connected to the intrinsic pathway that is regulated via the activation of caspase-8 to cause cleavage of the BID protein, causing cytochrome c to be released from the mitochondria [102,103]. Various apoptotic cell-death mechanisms have been propounded [104,105]. One logical approach to reducing the incidence of cancer appears to be the targeting of critical parts of apoptosis regulatory pathways, including the IAPs (in particular XIAP, cIAP1, and cIAP2), the anti-apoptotic Bcl-2 family of proteins, nuclear factor-kappa B (NF-κB), survivin, tyrosine kinases, caspases, and critical signaling pathways (phosphoinositide 3-kinase (PI3K)/AKT, STAT3/5, and MAPK pathways) [7,13,20,[106][107][108][109][110][111][112]. Resveratrol prompts the death of tumor cells by modulating diverse signal transduction pathways via regulation of the levels of Fas and Fas-ligand (FasL) [113,114]. Resveratrol also enhances FasL expression in HL-60 cells, and the resveratrol-induced apoptosis is Fas signaling-dependent [113].
Similar outcomes have also been observed in breast [113] and colon cancer cells [114]. Mechanisms of cell death that are independent of Fas and caused by cytotoxic agents have also been propounded [115,116], and apoptosis induced by doxorubicin occurs through a Fas-independent pathway [116]. Likewise, it has been shown that resveratrol exhibits Fas-independent apoptosis in another leukemic THP-1 cell line [117]. It has also been observed that resveratrol induced the death of leukemia CEM-C7H2 cells in a Fas-independent manner, as demonstrated by the absence of apoptotic change in the presence of antibodies antagonistic to Fas or FasL [118]. Furthermore, resveratrol effectively triggered apoptosis in Fas-resistant Jurkat human leukemia cells [118].
It has been shown that resveratrol induces cell death in some cancer cells by changing the proteins of the Bcl-2 family [119]. The inhibition of anti-apoptotic proteins of the Bcl-2 family, and activation of pro-apoptotic proteins such as Bad, Bak or Bax, by resveratrol has also been shown to be a mechanism for caspase activation and cytochrome c release [120,121]. Interestingly, these effects may be correlated with p53 activation [122][123][124][125]. For instance, resveratrol increased the cytoplasmic concentration of calcium in human breast cancer MDA-MB-231 cells, which activated p53 and caused different pro-apoptotic genes to be transcribed [126].
It has been suggested that resveratrol interferes with the MAPK pathway. In cervical carcinoma cells, resveratrol inhibited the activation of p38, JNK1, and ERK2 [141]. Resveratrol activates ERK1/2 at low concentrations (1 pM-10 µM), but at higher concentrations (50-100 µM) can inhibit MAPK in human neuroblastoma SH-SY5Y cells [142]. In contrast, resveratrol activates ERK1/2 in prostate [143], breast [144,145], glial [146], head and neck [147], and ovarian cancer cells [148]. MAPKs in a constitutively active state are necessary to maintain the malignant state; however, short-term activation of MAPK may drive the cells to apoptosis [149]. It has also been reported that resveratrol causes activation of other kinases, like JNK and p38 [150]. Notably, it has been shown that the resveratrol's anti-tumor effects require p53 activation that is MAPK-induced, as well as the subsequent induction of apoptosis [151][152][153].
Resveratrol also causes inhibition of signal transducers and activators of transcription 3 (STAT3), which adds to its pro-apoptotic and anti-proliferative potential [121]. STAT3 is a critical element in inflammation-related tumorigenesis as it promotes the proliferation, survival, invasion, angiogenesis, and metastasis of tumor cells [112,161]. The activation of NF-κB also promotes inflammation, proliferation, and tumorigenesis [162]. STAT3 and NF-κB are two central transcriptional factors linking tumorigenesis and inflammation; both of them can be activated as a response to certain stimuli, such as cytokines, growth factors, and stress signals. Abnormal signaling of STAT3 or NF-κB in malignant cells is therefore a promising target of therapy. STAT3 and NF-κB are activated via distinct pathways, and move to the nucleus to effect transcriptional activity. STAT3 and NF-κB that are constitutively activated by acetylation and/or phosphorylation in tumor cells, have been closely linked to both cancer development and progression [163,164]. Kim et al. reported that resveratrol caused inhibition of the nuclear translocation of STAT3 in renal cell carcinoma [165].
Interestingly, Wen et al. showed that inhibiting NF-κB nuclear translocation caused apoptosis in resveratrol-treated medulloblastoma cells [166]. It has been suggested that cross-talk occurs between the STAT3 and NF-κB pathways, because of the release of IL-6 and other cytokines, and because of the activation of cytokine receptors. STAT3 and NF-κB actually co-regulate many inflammatory and oncogenic genes, like IL-1β, Bcl-xL, Myc, COX-2, and cyclin D1 [161]. By their possible functional interaction, STAT3 and NF-κB collaboratively promote the development of tumors via inducing the expression of pro-tumorigenic genes [167]. The dysregulation of these genes because of the constant activation of both STAT3 and NF-κB in tumors and the tumor microenvironment is critical to tumor progression. Inflammation can regulate angiogenesis and cellular proliferation, and inhibits apoptosis [168]. It has also been reported that resveratrol inhibits the processes of several inflammatory enzymes in vitro, e.g., COXs and lipoxygenases (LOXs) [169,170]. It was shown in a recent study that resveratrol could radiosensitize and block the STAT3 signaling pathway by inducing SOCS-1, thereby reducing STAT3 phosphorylation and proliferation in head and neck tumor cells [171].

Anti-Tumor-Progression Activity
Tumor progression involves several processes such as that lead to tumor metastasis. Several genes are mutated or deleted that sustain the development of aggressive tumors. The invasion and metastasis of cancer cells involve the destruction of the extracellular matrix (ECM) and basement membrane, by proteolytic enzymes, such as matrix metalloproteinases (MMPs). Of these enzymes, MMP-2 and MMP-9 are overexpressed within a variety of malignant tumors modulating cell invasion and metastasis [172]. Tissue inhibitor metalloproteinase proteins (TIMPs), on the other hand, are a protein group comprising TIMP-1, -2, -3, and -4 acting as natural MMP inhibitors [173]. To sustain their swift growth, invasive tumors also need to grow new blood vessels via a process called angiogenesis. During angiogenesis, endothelial cells can be stimulated by various growth factors, including fibroblast growth factor (FGF) and VEGF, and travel to where the new blood vessels are required. Blocking the development of new blood vessels causes the supply of nutrients and oxygen to be reduced and, as a result, the size of the tumor and metastasis may also be reduced.
It has been suggested that resveratrol plays a role in inhibiting the expression of MMP (mainly MMP-9) [174][175][176][177] and angiogenesis markers such as VEGF, EGFR or FGF-2 [79,178]. Resveratrol reduced the phorbo-12-myristate 13-acetate (PMA)-induced migration and invasion ability of liver cancer HepG2 and Hep3B cells. In HepG2 cells, resveratrol up-regulated TIMP-1 protein expression and down-regulated MMP-9 activity, while the activities of MMP-2 and MMP-9 were decreased, along with a rise in the protein-expression level of TIMP-2 in Hep3B cells [175]. HepG2 cells treated with TNF-α expressed a high level of MMP-9, which resveratrol suppressed considerably via down-regulating the expression of NF-κB, resulting in the expression of MMP-9 protein being suppressed and the invasive capability of HepG2 cells being diminished [174]. Resveratrol treatment of breast cancer MDA-MB231 cells caused inhibition of the epidermal growth factor (EGF)-induced elevation of cell migration, and of the expression of MMP-9. Resveratrol also reduced a subunit of the mammalian mediator complex for transcription (called MED28, and whose over-expression can increase migration), via the EGFR/PI3K signaling pathways [176]. Both VEGF and hypoxia-inducible factor-1α (HIF-1α) are over-expressed in several human tumors and their metastases, and are closely linked to a more aggressive tumor phenotype. It has been reported that resveratrol suppresses the expression of VEGF and HIF-1α in human ovarian cancer cells via abrogating the activation of the PI3K/Akt and MAPK signaling pathways [179]. Resveratrol caused inhibition of the expression of these molecules, which suggests that it could be part of an efficacious anti-cancer therapy for preventing cancer and its metastasis [180][181][182].
Malignant transformation may be linked to signaling pathways during tumorigenesis, thereby promoting epithelial-to-mesenchymal transition (EMT), which may in turn increase the invasiveness and motility of cancer cells, and trigger cancer metastasis [183,184]. Many studies have shown that resveratrol suppresses the development of tumor invasion and metastasis through inhibiting signaling pathways associated with EMT [185]. Transforming growth factor-beta (TGF-β) is a widely known cytokine that encourages invasion, proliferation, EMT, and angiogenesis of cancer cells, and the TGF-β/Smad signaling pathway can activate EMT during cancer metastasis [186,187]. Resveratrol (20 µM) inhibited TGF-β-induced EMT in A549 lung cancer cells by augmenting the expression of E-cadherin and attenuating the expression of vimentin and fibronectin, as well as the EMT-inducing transcription factors Slug and Snail [188]. Qing Ji et al. showed that resveratrol inhibited EMT induced by TGF-β, as well as the invasion and metastasis of colorectal cancer, via reducing Smad2/3 expression [189]. NF-κB can also promote EMT, in addition to cancer migration and invasion [190][191][192].
Several studies have shown that NF-κB is a significant EMT regulator for different types of cells [190][191][192][193][194]. The roles for NF-κB have been found to be linked to the expression of various genes related to EMT, such as ZEB1, Snail, E-cadherin, MMP-7, MMP-9, and MMP-13 [192,193,195,196]. NF-κB can also be activated through PI3K/Akt signaling pathway to drive EMT and cancer-cell metastasis. Resveratrol suppressed the metastatic potential of pancreatic cancer PANC-1 cells in vitro by regulating factors related to EMT (vimentin, E-cadherin, N-cadherin, MMP-2, and MMP-9) and modulating the activation of PI3K/Akt/NF-κB pathways [197].

Pre-Clinical Studies
Resveratrol has also been reported to possess a significant anti-cancer property in various preclinical animal models (Table 1).
Resveratrol prevented DMBA/TPA-induced skin cancer from developing in mice, and was effective at all stages of carcinogenesis.
Soleas et al. discovered that resveratrol was somewhat efficacious in reducing the rate of tumor formation and the number of animals that developed skin tumors induced by DMBA [200]. Resveratrol inhibited tumor promotion in the DMBA-TPA mouse-skin carcinogenesis model, possibly because (at least in part) of its anti-oxidant properties [199]. Resveratrol administration restored glutathione (GSH) levels, superoxide dismutase (SOD), GSH peroxidase, and catalase activities to control values (mice without UVB irradiation) [244]. Furthermore, resveratrol exerted an anti-oxidant effect with a reduction in H 2 O 2 and lipid peroxidation in the skin [202]. It has been shown that the anti-proliferative effects of this stilbene can be regulated by cell-cycle regulatory proteins such as the expression of CDK2, 4, and 6, cyclin D1 and D2, and proliferating cell nuclear antigen (PCNA), while the expression of p21 was increased [203].
Resveratrol effectively hindered the development of DMBA/TPA-induced mouse-skin tumors by inducing apoptosis, which was indicated by the induction of cytochrome c release, the expression of Bax, p53, and Apaf-1, and the inhibition of Bcl-2 [201]. Afaq et al. determined that resveratrol had the ability to reduce edema and inflammation resulting from short-term UVB exposure in the skin of SKH-1 hairless mice, possibly because of the inhibition of ornithine decarboxylase (ODC) [202]. Treatment with resveratrol both before and after exposure to UVB suppressed development of skin tumor [204]. Resveratrol's anti-tumor properties have also been linked to lower expression levels of TGF-β1 and augmented expression levels of E-cadherin [243]. Oral gavage of resveratrol hindered the development of a mouse melanoma (B16BL6 cell line) xenograft carried in mice, with decreased expression of Akt [245]. In a murine model of the human cutaneous skin squamous carcinoma A431 cell-line xenograft, resveratrol treatment reduced the volume of the tumor, raised the expression levels of ERK and p53, and lowered the expression level of survivin [198]. Nevertheless, resveratrol did not reduce the tumor growth of other melanoma cell lines, including A375, B16M, and DM738 xenografts in mice [246,247].
Resveratrol, in a xenograft animal model, inhibited the development of ER-β-positive MDA-MB-231 and estrogen receptor (ER)-α-negative tumor explants, raised apoptosis, and lowered angiogenesis in nude mice [208]. However, resveratrol did not affect the in vivo development and metastasis of transplanted ER-α-negative 4T1 murine mammary cancer cells in nude mice [251]. Bove et al. studied resveratrol's in vivo effect with doses of 1-5 mg/kg per day administered intraperitoneally, and proposed that this ineffectiveness may have been the result of an insufficient dose of resveratrol. In another study, oral resveratrol at 100 or 200 mg/kg inhibited the development of 4T1 cells and metastasis in mouse lungs [252]. These findings were linked to both the reduced activity and expression of MMP-9. These data suggest that resveratrol's effects on breast cancer hinge on the dose and route of administration.
With breast cancer cell-implanted fat-pad models employing cigarette smoke condensate-transformed MCF-10ATr cells [209] or SUM159 cells [253], resveratrol caused down-regulation of the expression of various proteins linked to survival and cell proliferation (cyclin D1, PI3K, PCNA, and β-catenin), proteins related to DNA repair (Fen-1, DNA-ligase-I, Pol-δ, and Pol-ε), and an anti-apoptotic protein (Bcl-xL). It also caused an up-regulation of the pro-apoptotic protein Bax and tumor-suppressor gene p21 in mouse mammary tissue [209,253]. When used to supplement drinking water, resveratrol delayed the growth of spontaneous mammary tumors in HER-2/neu transgenic mice, and lowered the mean size and number of mammary tumors by causing down-regulation of the HER-2/neu gene expression and raising apoptosis in the mammary glands of these mice [207].

Prostate Cancer
Dietary resveratrol considerably lowered the incidence of prostatic adenocarcinoma in the transgenic adenocarcinoma mouse prostate (TRAMP) model [216]. Resveratrol suppressed prostate cancer growth via down-regulating the androgen receptor (AR) expression in the TRAMP model of prostate cancer. Additionally, besides down-regulating the AR expression, resveratrol also suppressed the mRNA level of androgen-responsive glandular kallikrein 11, which has been determined to be an ortholog of the human prostate specific antigen (PSA) [217]. In a xenograft model, resveratrol delayed the development of AR-positive LNCaP tumors and inhibited the expression of steroid hormone response markers [254].
With the use of AR-negative PC-3 human prostate cancer-cell xenografts in the flank regions of mice, post-treatment with oral resveratrol (30 mg/kg/day) decreased the volume of tumors, with lowered tumor-cell proliferation and neovascularization, and induced apoptosis [214]. Intraperitoneal post-treatment with resveratrol (25 mg/kg/day) also decreased the tumor volume of PC-3 cell xenografts in mouse prostates [255]. Additionally, intraperitoneal post-treatment of resveratrol (50 mg/kg/day) in the orthotopic DU-145 prostate cancer model decreased the growth, progression, local invasion, and spontaneous metastasis of tumors [215].
In models with genetically modified mice (e.g., Apc Min/+ mice [223,225,226]), and in mice with the APC locus knockout and activated KRAS [267], resveratrol supplementation inhibited the development of colon tumors [223,225,226,267,268] and occurrence of dysplasia [223].

Liver Cancer
The anti-cancer potential of resveratrol in liver carcinogenesis was exemplified by a decreased incidence and smaller numbers of nodules in models of animals employing chemical carcinogens [e.g., diethylnitrosamine (DENA) [269], DENA plus phenobarbital [234,270], and DENA plus 2-acetylaminofluorene (2-AAF) [271] or transgenic mice (e.g., hepatitis B virus X protein (HBx)-expressing transgenic mice) [272]. Additionally, resveratrol's anti-tumor effects have been reported in xenograft models using hepatoma cell lines (e.g., H22, AH-130, HepG2, and AH109A) [227][228][229]232]. Dietary resveratrol completely prevented DENA-induced lipid peroxidation and enhanced protein carbonyl formation, which indicates that it may also attenuate oxidative stress in the liver. Resveratrol also elevated the expression of hepatic Nrf2 and reduced the expression of iNOS. That study reported that the attenuation of oxidative and nitrosative stress and the alleviation of the inflammatory response could be mediated through the transcriptional and translational regulation of Nrf2 signaling [273]. Recent studies with Nrf2-deficient mice have shown that Nrf2 plays a role in protecting the liver from xenobiotic-initiated hepatocarcinogenesis [274].
Rajasekaran et al. have studied resveratrol's ability to prevent or treat hepatocellular carcinoma by administering resveratrol, starting at the time of DENA injection or for 15 days after the development of hepatocellular carcinoma [269]. Resveratrol treatment at both time points also reduced cell crowding and alteration in the cellular architecture, and decreased the liver size compared with control rats treated with DENA [269]. In the DENA-induced hepatocellular carcinoma model, administration of resveratrol inhibited the formation of hepatocyte nodules via down-regulating Hsp70 and COX-2 expression, through lowering the translocation of NF-κB from the cytoplasm to the nucleus [275]. Another study using the same administered dose of resveratrol also determined that the levels and expressions of hepatic TNF-α, IL-1β, and IL-6 induced by DENA can be reversed [276]. Resveratrol also exhibited a remarkable anti-angiogenic effect during the development of DENA-induced hepatocellular carcinogenesis, perhaps by blocking VEGF expression via the down-regulation of HIF-1α [277].
Resveratrol considerably lowered the cell count of a swiftly growing tumor (Yoshida AH-130 ascites hepatoma) injected into rats, thereby triggering apoptosis and cell accumulation in the G2/M phase [228]. It was further demonstrated that the inhibition of cell cycle progression involved reductions in the expression of p34cdc2 and cyclin B1 in murine transplantable liver tumors after resveratrol administration [230]. It has also been reported that resveratrol had anti-tumor-growth and anti-metastasis effects in Donryu rats that had an ascites AH109A hepatoma cell line subcutaneously implanted [227].
In another study, resveratrol inhibited tumor growth and angiogenesis in a hepatoma xenograft mouse model [278]. Salado et al. used B16 melanoma (B16M) cells to study the effects of resveratrol treatment on hepatic metastasis caused mainly by the production of pro-inflammatory cytokines [279]. Lin et al. investigated the effects of treatment with resveratrol on the precancerous stage of liver carcinogenesis in spontaneously induced hepatocellular carcinoma in HBx transgenic mice [272]. Resveratrol supplementation significantly reduced the incidence of hepatocellular carcinoma and increased the latency of tumor formation. Resveratrol inhibited hepatic lipogenesis and intracellular ROS, and the results from liver cancer models have been consistently positive, indicating the potential benefit of resveratrol in hepatocellular carcinoma prevention and/or therapy.

Pancreatic Cancer
Several lines of evidence suggests that age, being overweight, pancreatitis and family history of pancreatic cancer are the major risk factor for the development of pancreatic cancer. Within a xenograft mouse model, resveratrol delayed or suppressed the promotion of pancreatic cancer via inhibiting the activity of leukotriene A4 hydrolase (LTA 4 H), which stimulates the generation of pro-inflammatory cytokines and mediators [280], and also stimulates cancer cell proliferation [281,282]. Resveratrol blocked the tumor development of PANC-1 cells orthotopically implanted in nude mice, with augmented expression of apoptosis/cell cycle arrest proteins including Bim, p27, and cleaved caspase-3, and reduced cell survival/proliferation markers including PCNA expression and the phosphorylation of PI3K, ERK, Akt, FOXO3a (Ser253), and p-FOXO1 (Ser256) in tumor tissues [283]. Resveratrol treatment inhibited the formation and development of pancreatic cancer in Kras G12D transgenic mice that spontaneously develop pancreatic tumors [284]. However, dietary resveratrol had no anti-carcinogenic effect on BOP (N-nitrosobis(2-oxopropyl)amine)-induced pancreatic carcinogenesis in hamsters [285]. Further studies are necessary for additional preclinical evaluation of the efficacy of resveratrol in treating pancreatic cancer.

Lung Cancer
In preclinical models, lung carcinogenesis is known to be induced by a variety of agents, including diethylnitrosamine (DEN), nitrosamine 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), uracil mustard, vinyl carbamate, urethane, MNU, and BP [11]. In the BP-induced mouse lung carcinogenesis model, resveratrol treatment lowered the level of BP diolepoxide (BPDE)-DNA adducts [286], improved the ultrahistoarchitecture [287], and reduced the size of tumor nodules by increasing pulmonary caspase-3 and -9 activity. It also abrogated glucose uptake/turnover, reduced the serum lactate dehydrogenase (LDH) activity (which is heightened in cancer cells), and lowered the p-p53 levels at Ser15 (the hyperphosphorylation of which can result in the inactivation of p53) [288]. In Lewis lung carcinoma cell xenograft models, treatment with resveratrol reduced the growth of tumors [218,221]. It has been also discovered that treatment with resveratrol reduced the development of A549 and MSTO-211H xenografts in mice [219,289,290].
Resveratrol's anti-tumor effects in A549 xenografts were reduced in Forkhead box protein C2 (FOXC2)-overexpressing A549 xenografts, which suggests that resveratrol possibly induces anti-tumor activity through FOXC2 [289]. Another study discovered that resveratrol did not affect the development of Lewis lung carcinoma implanted in mice, but demonstrated an evident anti-metastatic effect, decreasing both the weight and number of lung metastases [220]. However, resveratrol used to supplement the diet did not affect lung tumor multiplicity in BP plus NNK-induced lung carcinogenesis in A/J mice [291]. Similarly, in BP-induced lung carcinogenesis, resveratrol did not cause a change in the expression levels of BP-metabolizing genes (such as CYP1A1 and CYP1B1) and the number of B[a]P-protein adducts in lung tissues [292]. Another study found that both the natural Egr-1 promoter and the synthetic promoter triggered the expression of GADD45α when used with resveratrol, and then suppressed the proliferation of A549 lung cancer cells and induced apoptosis [293].

Other Cancers
Resveratrol provides considerable protection against the induction of cancer within the oral cavity [294] and the esophagus [295], among other tissues. Its cancer chemopreventive activity aside, resveratrol can also inhibit the development and/or induce the regression of established tumors in xenograft models for cancers of the ovaries [296], urinary bladder [79], stomach [297], and head and neck [298,299]. Resveratrol treatment effectively suppressed the growth rate of and augmented apoptosis in neuroblastoma; this was accompanied by the up-regulation of cyclin E and the down-regulation of p21 [300]. It has recently been demonstrated that resveratrol considerably reduced tumor growth via inducing apoptosis, which involved direct activation of the mitochondrial intrinsic apoptotic pathway in the NGP and SK-N-AS xenograft models of human neuroblastoma [301]. Resveratrol caused significant inhibition of cerebral tumors through inducing apoptosis and inhibiting angiogenesis induced by glioma [302]. Rats that had undergone resveratrol treatment had lower growth rates of glioma, which correlated with the blood flow of tumors (signified by the color Doppler vascularity index) and density of microvessels.
Resveratrol's anti-angiogenic effect has caused researchers to investigate if it could inhibit the development of a murine fibrosarcoma; water supplemented with resveratrol indeed significantly inhibited the development of T241fibrosarcoma in mice via suppressing angiogenesis [303]. Resveratrol's in vivo anti-cancer effects were studied in N-nitrosomethyl-benzylamine (NMBA)induced esophageal tumorigenesis in rats. Resveratrol suppressed both the size and number of NMBA-induced esophageal tumors per rat through targeting prostaglandin E2 and COXs [304]. In a gastric cancer xenograft nude mouse model, resveratrol inhibited the growth of tumors, with reductions in the expression of cyclin D1, Ki67, CDK4, and CDK6, and increases in the expression of p16, p21, and β-Gal [305]. Resveratrol considerably inhibited carcinoma development when it was injected in close proximity to the carcinoma in a tumor model created by transplanting human primary gastric cancer cells into the subcutaneous tissue of nude mice [297]. Resveratrol induced apoptosis in implanted tumor cells via down-regulation of the apoptosis-regulated gene Bcl-2 and up-regulation of the apoptosis-regulated gene Bax. For the anti-tumor effects in head and neck cancer, resveratrol suppressed tumor stemness via lowering the expression of mesenchymal-like protein (Vimentin) and stemness markers (Oct4 and Nestin), inducing epithelial protein expression (E-cadherin) [299], and increasing γ-histone 2AX (a DNA damage marker) and cleaved caspase-3 expression [298]. In an ovarian cancer model, resveratrol abrogated the development of NuTu-19 ovarian cancer cells in vitro. However, in vivo, when NuTu-19 cells were injected into the ovarian bursa of rats and the rats were fed with resveratrol (100 mg/kg) mixed in their diet for 28 days, the growth of the ovarian tumors was not significantly inhibited [306].

Clinical Trials with Resveratrol
Although it is clear that resveratrol has shown excellent anti-cancer properties, most of the studies were performed in cell-culture and pre-clinical models. These physiological effects of resveratrol were also investigated in humans because it cannot be assumed that the results of tests in animal models will hold true for humans, because of differences in genetics and metabolism profile. The pharmacokinetics, metabolism, and toxicity of resveratrol have been assessed in healthy volunteers and cancer patients [307][308][309]. Resveratrol is metabolized swiftly, mainly into glucuronide and sulfate conjugates that are excreted via the urine. Because of the poor bioavailability of resveratrol due to its extensive metabolism, large doses (up to a maximum of 5 g/day) have been utilized by researchers. These studies have shown that resveratrol seems to be well tolerated and safe. However, adverse effects including diarrhea, nausea, and abdominal pain were observed in subjects taking more than 1 g of resveratrol daily [307]. Subsequent clinical trials are currently investigating this dose limit [307,310]. Resveratrol's poor bioavailability is a significant issue with regard to extrapolating its effects to humans, and various approaches have been created to enhance its bioavailability [311], including consuming it with various foods [312], using it in combination with an additional phytochemical piperine [313], and using a prodrug approach [314], micronized powders [315,316], or nanotechnological formulations [317][318][319].
The effect of resveratrol in cancer patients has been investigated in a few clinical trials ( Table 2). The first clinical trial dealing with resveratrol and cancer was performed by Nguyen et al. [320]. They examined the effects of freeze-dried grape powder (GP) (containing resveratrol and resveratrol derived from plants) on the Wnt signaling pathway, which is known to be involved in colon carcinogenesis [321], in regular colon cancer and colonic mucosa. GP administration (80 g/day containing 0.07 mg of resveratrol) for two weeks resulted in decreased Wnt target gene expression within regular mucosa, but had no effect on cancerous mucosa. This indicates that GP or resveratrol may play a beneficial part in the prevention of colon cancer, rather than in the treatment of established colon cancer. Patel et al. studied the effects of the administration of resveratrol at 0.5 or 1 g/day for eight days on proliferation marker Ki-67 expression in colorectal tissue, and reported a 5% decrease in the proliferation of tumor cells [322]. In colorectal cancer patients with hepatic metastasis, SRT501 (a micronized resveratrol formulation manufactured by Sirtris Pharmaceuticals, a GSK Company, Cambridge, MA, USA) supplementation at 5 g/day for two weeks increased the amount of cleaved caspase-3 within hepatic tissue, which suggests that there was increased apoptosis of cancerous tissue compared with subjects treated with a placebo [315].
In a muscadine grape skin extract phase 1 study with biochemically recurrent prostate cancer patients who were assigned to a high dose (4000 mg/patient) of pulverized muscadine grape (Vitis rotundifolia) skin that contains ellagic acid, quercetin, and resveratrol was found to be safe and warrants further investigation in dose-evaluating phase II trial [323]. In another randomized placebo controlled clinical study using two doses of resveratrol (150 mg or 1000 mg resveratrol daily) for 4 months was found to significantly lowered serum levels of androstenedione, dehydroepiandrosterone and dehydroepiandrosterone-sulphate, whereas prostate size was unaffected in benign prostate hyperplasia patients [324]. Micronized resveratrol (SRT5001, 5 g) for 14 days Detection of resveratrol in hepatic tissue and increased (39%) content of cleaved caspase-3 in malignant hepatic tissue. [315] Multiple myeloma patients (n = 24) Micronized resveratrol (SRT5001, 5 g) for 20 days in a 21 day cycle up to 12 cycles Unacceptable safety profile and minimal efficacy in patients with relapsed/refractory multiple myeloma highlighting the risks of novel drug development in such populations. [316] Biochemically recurrent prostate cancer patients (n = 14) Pulverized muscadine grape skin extract (MPX) 4000 mg/patient MPX was found to be safe and warrants further investigation in dose-evaluating phase II trial [323] Benign prostate hyperplasis patients (n = 66) Two doses of resveratrol (150 mg or 1000 mg resveratrol daily) for 4 months Significantly lowered the serum levels of androgens with no changes in prostate tumor growth. [324] Primary protein carbonylation has been found to be increased several folds in presence of high levels of reactive oxygen species (ROS) such as superoxide anion free radical (O 2 − ) and nitric oxide free radical (NO) and other reactive free radicals, such as hydrogen peroxide (H 2 O 2 ), hydroxyl radical (HO), and peroxynitrite anion (ONOO − ). There are several sources of ROS in the digestive tract and several microbes present in the colon produce a large amount of ROS inside the cells are by products of mitochondrial respiration in aerobic metabolism, and in chronic inflammation, a large amount of ROS is produced by neutrophil phagocytosis of bacteria, granular materials, or soluble irritants [325,326]. The oxidative decomposition of polyunsaturated fatty acids can initiate chain reactions that lead to the formation of a variety of carbonyl species (three to nine carbons in length), the most reactive and cytotoxic being α,β-unsaturated aldehydes also referred to as electrophilic carbonyls. These include acrolein, glyoxal, methylglyoxal, crotonaldehyde, malondialdehyde, and 4-hydroxynonenal. Reactive ketones or aldehydes that can be reacted by 2,4-dinitrophenylhydrazine (DNPH) to form 2,4-dinitrophenylhydrazone (DNP). Ulcerative colitis (UC) is a type of chronic inflammatory bowel disease (IBD) in which oxidative stress plays a critical role in its pathogenesis and malignant progression to colorectal cancer (CRC) [327,328]. Oxidative activation of transcription factors NF-κB stimulates expression of a variety of pro-inflammatory cytokines in the intestinal epithelial cells, such as TNF-α, IL-1, IL-8, and COX-2, and promotes inflammation and carcinogenesis. Oxidative stress also activates mitogen-activated protein (MAP) kinase (MAPK) signaling pathways. The human gastrointestinal tract is exposed to carbonyl threats such as consumption red meat, alcoholic beverages and smoking increases protein carbonylation, inflammation and initiation of tumor development. However, dietary intake of green leafy vegetables, fruits, fish and wine has shown to decrease protein carbonylation [329]. It has also been reported that resveratrol supplementation at 5 mg/day for six days increased the degree of protein carbonyl concentrations and cytoprotective enzyme NQO1 in colorectal mucosa tissues from patients with colorectal cancer, compared with their control subjects [330]. However, contrary to these positive findings, some evidence that resveratrol supplementation may have adverse effects in certain cancer patients also exist. In a phase II clinical trial involving multiple myeloma patients, SRT501 supplementation at 5 g/day caused several unexpected adverse effects, including nephrotoxicity, which may have led to the death of one patient [316]. However, this high dose of SRT501 was determined to be safe in other clinical trials involving several healthy and diseased populations [315,316]. There are very low amounts of human data regarding the efficacy of resveratrol in cancer treatment. Since most of these clinical trials have had a small patient sample size and used different doses and different routes of resveratrol, the data from human clinical studies have shown inconsistent outcomes of resveratrol administration.
In addition to the effects in subjects with cancer, the effect of resveratrol in subjects with a higher cancer risk has also been demonstrated. For instance, resveratrol supplementation at 50 mg two times per day for 12 weeks reduced the DNA methylation of the tumor-suppressor gene Ras association domain-containing protein 1 (RASSF1A) in the breasts of women with higher risk of breast cancer [331]. It has also been shown that resveratrol supplementation at 1 g/day for 12 weeks increases the concentrations of sex steroid hormone binding globulin (SHBG), which has been linked to a reduction in the risk of breast cancer [332], and has favorable effects on estrogen metabolism; thus, it can lower risk factors for breast cancer in obese and overweight postmenopausal women [333]. Another clinical study concentrated on resveratrol's effects on potential biomarkers for cancer risk reduction. Circulating concentrations of insulin-like growth factor (IGF-1) and IGF-binding protein 3 (IGFBP-3) are linked to a higher risk of common cancers [334]. Brown et al. showed that resveratrol administration at 2.5 g/day for 29 days resulted in a reduction of the circulating levels of IGF-1 and IGFBP-3 in healthy volunteers [335]. Their research suggests that resveratrol's ability to decrease circulating IGF-1 and IGFBP-3 in humans may constitute an anti-carcinogenic mechanism. In another study, Chow et al. found that resveratrol administration at 1 g/day for four weeks modulated phase I isoenzymes (cytochrome P450) and phase II detoxification enzymes involved in carcinogen activation and detoxification [310]. However, these beneficial effects are mostly minimal and sometimes controversial. Nevertheless, it seems that resveratrol has had some beneficial effects with regard to the prevention and treatment of cancer. Therefore, the efficacy and safety of resveratrol in human trials must be further investigated to better understand and develop its therapeutic potential for cancer patients.

Conclusions and Future Perspectives
Using a variety of in vivo and in vitro models, it has been proven that resveratrol is capable of attenuating the various stages of carcinogenesis, some of which are briefly described in Figure 2. A vast body of experimental in vivo and in vitro studies and a few clinical trials has presented evidence of resveratrol's great potential as an anti-cancer agent, both for the prevention and therapy of a large range of cancers. Resveratrol has a very low toxicity, and, although it has multiple molecular targets, it acts on different protective and common pathways that are usually altered in a great number of tumors. This suggests that resveratrol may be more suitable for use as an anti-carcinogen and it can also effectively exert it antineoplastic effects in conjunction with diverse chemotherapeutics and targeted therapies. The ability to prevent carcinogenesis includes the inhibition of oxidative stress, inflammation, and cancer-cell proliferation, and the activation of tightly regulated cell-death mechanisms. Due to the complexity and number of cellular processes involved, however, more studies must be performed to completely understand how resveratrol could be used to prevent the development of cancer. Moreover, resveratrol's poor bioavailability in humans has been a critical concern with regard to the translation of basic research findings to the development of therapeutic agents. Although human clinical trials have produced positive findings, many conflicting results remain, which may be partly because of the dosing protocols employed. To augment resveratrol's bioavailability and as a potential adjuvant, active research should be focused on resveratrol delivery systems, formulations, and modulation of resveratrol metabolism, and resveratrol's possible interactions with other compounds, as well as the development of more bioavailable analogs of the compound.
A vast body of experimental in vivo and in vitro studies and a few clinical trials has presented evidence of resveratrol's great potential as an anti-cancer agent, both for the prevention and therapy of a large range of cancers. Resveratrol has a very low toxicity, and, although it has multiple molecular targets, it acts on different protective and common pathways that are usually altered in a great number of tumors. This suggests that resveratrol may be more suitable for use as an anti-carcinogen and it can also effectively exert it antineoplastic effects in conjunction with diverse chemotherapeutics and targeted therapies. The ability to prevent carcinogenesis includes the inhibition of oxidative stress, inflammation, and cancer-cell proliferation, and the activation of tightly regulated cell-death mechanisms. Due to the complexity and number of cellular processes involved, however, more studies must be performed to completely understand how resveratrol could be used to prevent the development of cancer. Moreover, resveratrol's poor bioavailability in humans has been a critical concern with regard to the translation of basic research findings to the development of therapeutic agents. Although human clinical trials have produced positive findings, many conflicting results remain, which may be partly because of the dosing protocols employed. To augment resveratrol's bioavailability and as a potential adjuvant, active research should be focused on resveratrol delivery systems, formulations, and modulation of resveratrol metabolism, and resveratrol's possible interactions with other compounds, as well as the development of more bioavailable analogs of the compound.