Resveratrol in the chemoprevention and treatment of hepatocellular carcinoma

Cancer Treatment Reviews 36 (2010) 43–53

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Cancer Treatment Reviews

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ANTI-TUMOUR TREATMENT

Resveratrol in the chemoprevention and treatment of hepatocellular carcinoma

Anupam Bishayee *, Themos Politis, Altaf S. DarveshDepartment of Pharmaceutical Sciences, Northeastern Ohio Universities Colleges of Medicine and Pharmacy, 4209 State Route 44, Rootstown, OH 44272, USA

a r t i c l e i n f o s u m m a r y

Article history:Received 7 July 2009Received in revised form 8 October 2009Accepted 9 October 2009

Keywords:ChemopreventionChemotherapyClinical studyHepatocarcinogenesisHepatocellular carcinomaIn vitroIn vivoLiverPharmacokineticsResveratrol

0305-7372/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.ctrv.2009.10.002

* Corresponding author. Tel.: +1 330 325 6449; faxE-mail address: [email protected] (A. Bisha

Hepatocellular carcinoma (HCC) is one of the most common cancers and lethal diseases in the world.Although the majority of HCC cases occur in developing countries of Asia and Africa, the prevalence of livercancer has risen considerably in Japan, Western Europe as well as the United States. HCC most commonlydevelops in patients with chronic liver disease, the etiology of which includes viral hepatitis (B and C),alcohol, obesity, iron overload and dietary carcinogens, including aflatoxins and nitrosamines. The currenttreatment modalities, including surgical resection and liver transplantation, have been found to be mostlyineffective. Hence, there is an obvious critical need to develop alternative strategies for the chemopreven-tion and treatment of HCC. Oxidative stress as well as inflammation has been implicated in the develop-ment and progression of hepatic neoplasia. Using naturally occurring phytochemicals and dietarycompounds endowed with potent antioxidant and antiinflammatory properties is a novel approach to pre-vent and control HCC. One such compound, resveratrol, present in grapes, berries, peanuts as well as redwine, has emerged as a promising molecule that inhibits carcinogenesis with a pleiotropic mode of action.This review examines the current knowledge on mechanism-based in vitro and in vivo studies on thechemopreventive and chemotherapeutic potential of resveratrol in liver cancer. Pre-clinical and clinicaltoxicity studies as well as pharmacokinetic data of resveratrol have also been highlighted in this review.Future directions and challenges involved in the use of resveratrol for the prevention and treatment of HCCare also discussed.

� 2009 Elsevier Ltd. All rights reserved.

Introduction

Primary liver cancer, also known as hepatocellular carcinoma(HCC), happens to be the sixth most common cancer as well asthe third leading cause of cancer mortality in the world.1 HCChas a poor prognosis with the number of deaths almost equal tothe number of cases being diagnosed annually (about 600,000)and the 5-year survival rate reported below 9%.2 The incidence ofHCC is on the rise in multiple geographic areas, including Asia Pa-cific, sub-Saharan Africa, southern Europe as well as North Amer-ica. The occurrence of HCC in the United States has dramaticallyincreased by more than 70% over the last 25 years.3 It has beenestimated that there will be more than 22,000 new cases and about18,000 deaths in the United States in 2009 due to liver cancerwhich represents about 4% of cancer mortality in this country.4

The vast majority of HCC cases are attributable to underlying infec-tions caused by the hepatitis B and C viruses5; nevertheless severalother risk factors, namely alcoholism, obesity, iron overload, aswell as dietary carcinogens, such as aflatoxins and nitrosaminesare also involved in its etiology.6–9

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: +1 330 325 5936.yee).

Although surgical resection is currently considered to be themost optimal treatment approach, only 10–20% of HCC patientsare candidates for surgery because of tumor size, multifocality,vascular invasion, or hepatic decompensation. In addition, forthose undergoing resection, the recurrence rates can be as highas 50% within several years of surgery.10 While liver transplanta-tion has been successful for the treatment of early-stage liver can-cer, regrettably only a small number of HCC patients qualify fortransplantation. The potential of this option is limited due to donororgan shortage as well as the rapid and frequent recurrence of HCCin the transplanted liver. At present, there is no proven effectivesystemic chemotherapy for HCC. Sorafenib, a vascular endothelialgrowth factor receptor tyrosine kinase inhibitor, has been shownto prolong the median survival time by nearly three months in pa-tients with advanced HCC.11 Although sorafenib has been approvedby the United States Food and Drug Administration for the treat-ment of unresectable HCC, recent studies indicate severe adverseeffects including a significant risk of bleeding.12 Alternative treat-ment modalities including transcatheter arterial chemoemboliza-tion, targeted intra-arterial delivery of Yttrium-90 microspheres,percutaneous intratumor ethanol injection, and radiofrequencyablation are primarily for palliation and are applicable only to pa-tients with localized liver tumors. In view of the limited treatmentand grave prognosis of liver cancer, preventive control approaches,

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44 A. Bishayee et al. / Cancer Treatment Reviews 36 (2010) 43–53

notably chemoprevention, have been considered as one of the beststrategies in lowering the current morbidity and mortality associ-ated with HCC.13,14 A detailed understanding of the pathogenesis ofHCC holds the promise of finding an effective and novel strategyfor the chemoprevention and treatment of liver cancer.

Although the cellular mechanisms contributing to hepatocarci-nogenesis are relatively unknown, a connection between inflam-mation and liver cancer is beginning to be unraveled. Duringrecent years, compelling evidence has accumulated which providesan insight in the role of inflammation in initiation, promotion andprogression of HCC.15 Hepatic inflammation, due to exposure toinfectious agents (hepatotropic viruses) as well as toxic com-pounds, may represent an early step in the development of malig-nancy with genetic and epigenetic events occurring as a latermanifestation of a prolonged inflammatory process. Despite intrin-sic differences among etiological factors for HCC, a commondenominator of the genesis of malignancy happens to be the per-petuation of a wound-healing response triggered by parenchymalcell death, and the ensuing inflammatory reaction.16,17 It has beenshown that HCC almost always develops on a background ofchronic liver injury including chronic hepatitis and cirrhosis, whichare both considered to be preneoplastic stages of hepatocellular tu-mor development.18 An expanding body of evidence suggests thatinflammation-mediated processes, including the production ofcytokines, chemokines, reactive oxygen and nitrogen species, andmediators of the inflammatory pathways may contribute to hepa-tic neoplasia.19–22 Environmental insults, including chemical toxi-cants, act as tumor initiators and/or promoters by inducingsteady-state increase in the generation of reactive oxygen species(ROS).23 Oxidative stress, through generation of ROS including sin-glet oxygen, superoxide anion, hydrogen peroxide and hydroxylradical, acts as a predisposing factor to hepatocarcinogenesis andis the common driving force of HCC in chronic liver diseases.19,24

All these findings have led researchers to theorize that the under-lying mechanisms which are most pronounced in occurrence andprogression of HCC deal with oxidative stress and the accompany-ing inflammatory insults.

Phytochemicals are widely accepted as validated treatment op-tions for various conditions. Scientific probing into potential bene-fits of many of these compounds sufficiently defines them asbeneficial pharmacological agents. Natural dietary components,obtained from several fruits, vegetables, nuts and spices havedrawn a considerable amount of attention due to their demon-strated ability to suppress carcinogenesis in animal models withsome of these substances able to partially prevent or delay cancerformation in several high-risk populations.25 Recent evidence hasshown that dietary polyphenolic compounds including anthocy-anidins from berries, catechins from green tea, curcumin from tur-meric, genistein from soy, lycopene from tomatoes, and quercetinfrom red onions and apples are phytochemicals with significantanticancer properties.26–29 A variety of bioactive food componentshave been shown to modify molecular targets involved in inflam-mation and redox signaling,30,31 which are implicated in the devel-opment and progression of HCC. Supported by several in vitroassays and studies involving animal models as well as humans, evi-dence is emerging to support potential chemopreventive and che-motherapeutic effects of several phytochemicals in HCC.32 In thiscontext, resveratrol, a naturally occurring antioxidant and antiin-flammatory agent, has emerged as the lead promising molecule.

Resveratrol (3,40,5-trihydroxy-trans-stilbene, Fig. 1) is a phyto-chemical found in several dietary sources, such as grapes, berries,peanuts as well as red wine. In nature, it functions as a fungicideproduced by the plant itself to ward off potentially lethal organ-isms and counteract environmental stress.33 Its value as a com-pound beneficial to human health is well documented.34–36

Perhaps, it is best known as the compound widely considered to

be the agent responsible for the ‘‘French Paradox”, a phenomenonin which consumption of red wine is thought to reduce the inci-dence of heart disease.37 Recent studies have indicated that besidesred wine, rose and white wine could be cardioprotective as theyalso contain resveratrol albeit at lesser concentrations than thatof red wine.38 Resveratrol can prevent or slow the progression ofa wide variety of inflammation-related illnesses, including cancer,neurodegenerative diseases, cardiovascular ailments, ischemic in-jury, and viral infections, as well as enhance stress resistance andextend the life span of various organisms.34,35,39,40 An impressivebody of experimental findings reveals multiple cellular targets ofresveratrol affecting cellular proliferation and growth, apoptosis,inflammation, invasion, angiogenesis and metastasis.41,42 Duringthe past decade, the amount of research on this phytoalexin hassoared, and there exists strong evidence which supports resvera-trol as a potent chemopreventive and chemotherapeutic agent.

Jang et al.43 first demonstrated the chemopreventive effects ofresveratrol in inhibiting multi-stage carcinogenesis (e.g., initiation,promotion and progression). Subsequently, resveratrol has beenshown to suppress proliferation of a wide variety of human tumorcells in vitro,44–46 which have led to numerous pre-clinical animalstudies to evaluate the cancer chemopreventive and chemothera-peutic potential of resveratrol.47,48 Several clinical trials, includingone sponsored by the National Cancer Institute, are currentlyunderway to investigate the use of both resveratrol and resvera-trol-rich products, for prevention and treatment of colon cancer.48

A significant amount of resveratrol accumulates and is retained inthe liver.49–52 Resveratrol has been shown to inhibit the hepaticcarcinogen-activating enzymes, including cytochrome P450 1A1(CYP1A1) and CYP3A/2 and induce hepatic phase 2 conjugating en-zymes, namely NAD(P)H:quinine oxidoreductase, UDP-glucurono-syl transferase and glutathione S-transferase (GST) in vitro andin vivo.53–56 The resultant effects of these enzyme modulation byresveratrol could be the reduced exposure of cells to carcinogensdue to inhibition of carcinogen activation and/or elevated carcino-gen detoxification and elimination. The most fascinating propertyof resveratrol, with regards to liver cancer, is its strong antiinflam-matory57 and antioxidant properties,58 as both oxidative stress andinflammation have been strongly implicated in the occurrence andprogression of HCC.16,17,19,24 However, despite its great promise,the effects of resveratrol on liver cancer have not been systemati-cally studied until recently. This review critically examines the cur-rent knowledge on the mechanism-based chemopreventive andchemotherapeutic potential of resveratrol in in vitro as well aspre-clinical animal models of HCC.

Resveratrol and liver cancer

In vitro studies

There is growing in vitro evidence demonstrating the inhibitoryeffects of resveratrol on liver cancer. According to a study con-ducted by Delmas et al.59 the proliferation of rat hepatoma Faoand human hepatoblastoma HepG2 cells were negatively impactedby the addition of resveratrol to the culture medium in both dose-and time-dependent fashion. Fao cells were more sensitive thanHepG2 cells. Another interesting finding of this study was thatthe presence of ethanol potentiated the effects of resveratrol inboth cell lines. These results were attributed to the ability of resve-ratrol to prevent or delay the cells from entering mitosis andincreasing the number of cells arrested in the S and G2/M phase.Hepatic growth factor (HGF) has largely been implicated in theability of primary hepatic tumors to proliferate and invade adja-cent tissue. The effects of resveratrol on HGF-mediated invasionwere determined in HepG2 cells, in part, to understand the

Fig. 1. Chemical structures of resveratrol (1) and resveratrol conjugates, trans-resveratrol-3-O-glucuronide (2), trans-resveratrol-40-O-glucuronide (3), trans-resveratol-3-O-sulfate (4), trans-resveratol-40-O-sulfate (5), and trans-resveratol-3-O-40-O-disulfate (6).

A. Bishayee et al. / Cancer Treatment Reviews 36 (2010) 43–53 45

mechanisms of resveratrol’s anti-HCC property. Resveratrol wasfound to decrease HGF-induced scattering and invasion of livercancer cells with concurrent inhibition of cell proliferation possiblydue to a post-receptor mechanism rather than apoptosis.60 Kozukiet al.61 demonstrated that resveratrol inhibited both the prolifera-tion and invasion of AH109A rat ascites hepatoma cells at higherconcentrations but suppressed only the invasion at lower concen-trations, and that resveratrol-loaded rat serum restrained only theinvasion. Results of this study suggest that the antiinvasive activityof resveratrol is independent of its antiproliferative activity andlinked to antioxidative property. Subsequent studies from thesame group also confirmed the involvement of the antioxidantproperty of resveratrol as sera from rats orally given resveratrolwere found to suppress ROS-potentiated invasion of AH109Acells.62 Another proposed mechanism of resveratrol’s ability to cur-tail hepatoma cell invasion hails from a study conducted by Zhanget al.63 This study focused on factors involved in angiogenesis ofexpanding tumors. Though it had been known for some time thatresveratrol had antiangiogenic abilities, the researches helpedcharacterize the underlying molecular mechanisms. By inducinghypoxia in HepG2 cell line, researchers found that resveratrolhad an inhibitory effect on vascular endothelial growth factor geneexpression via hypoxia-inducible factor-1a inhibition.63 Kuoet al.64 examined the antiproliferative effects of resveratrol intwo human liver cancer cell lines, namely HepG2 and Hep3B. Theresults showed that resveratrol inhibited cell growth only in p53-positive HepG2 cells, which was a result of cellular apoptotic deathvia p53-dependent pathway. It was also shown that resveratrol-

treated cells were arrested in G1 phase and were associated withan increase in p21 and Bax expression. The cytotoxic effects of res-veratrol in HepG2 cells were confirmed by Kim et al.65, who iso-lated the stilbene from the seeds of Paeonia lactiflora, a plantwidely used in Chinese traditional medicine. Kocsis et al.66 have re-ported an interesting observation regarding concentration- andtime-dependent effects of resveratrol on cytotoxicity, cell prolifer-ation activity, and apoptosis in HepG2 cells. Cytotoxicity becameevident at resveratrol concentration of 50 or 100 lM at treatmentslonger than 48 h. Cell cycle analysis showed an increment of S-phase cells at low concentrations of resveratrol (10–50 lM) anda decrement at high concentrations (100–200 lM). The ratio ofapoptotic cells increased following resveratrol treatment at orabove 50 lM mostly after 48 h. In a separate study, resveratrolwas found to inhibit the growth of H22 hepatoma cells in a dose-and time-dependent manner. The synergistic antitumor effects ofresveratrol with current anticancer drug 5-fluorouracil (5-FU) in-creased to a greater extent than for H22 cells exposed to 5-FUalone. A direct evidence of apoptosis was presented as the mecha-nism of antihepatoma activity of resveratrol.67 It has been theo-rized that hepatoma cell lines are more likely to undergoapoptosis when exposed to resveratrol rather than necrosis duetheir ability to quickly metabolize the phytochemical. Experimen-tal support to this hypothesis was presented by Michels et al.68

who observed the killing of metabolically active H4IIE rat hepa-toma cells due to induction of apoptosis via caspase activation.Another study has found the time- and dose-dependent effects ofresveratrol on cell proliferation in HepG2 cell lines.69 Like previous


studies, cell cycle effects were observed with regards to mitoticinterference; however, this study produced evidence that HepG2cells lines treated with resveratrol for just 2 h did, in fact, show evi-dence of hindrance of DNA synthesis. Notas et al.70 again foundthat apoptosis through cell cycle arrest was implemented as themain mechanism by which resveratrol interferes with HepG2 cellproliferation. This study also showed that resveratrol elicited anti-oxidant effects and modulated the nitric oxide (NO)/nitric oxidesynthase (NOS) system by increasing expressions and activities ofinducible nitric oxide synthase (iNOS) and endothelial nitric oxidesynthase (eNOS) enzymes as well as NO production. All these ac-tions were found to be mediated at the nanomolar or picomolarlevels, compatible with the concentrations of free resveratrol inbiological fluids following ingestion of resveratrol-rich foods andbeverages. Yu et al.71 have shown that resveratrol inhibited tumornecrosis factor-a-mediated matrix metalloproteinase-9 expressionand invasion of HepG2 cells. The inhibitory effects of resveratrolwere also associated with the down-regulation of nuclear factor-jB signaling pathway. Caveolin-1 (CAV1), a member of caveolinfamily, may function as a tumor suppressor abolishing anchor-age-independent growth of transformed cells, and is found to bepoorly expressed in HCC.72 The possible role of CAV1 in the cyto-toxic and pro-apoptotic actions of resveratrol has been recentlystudied in HepG2 cells transfected with various CAV mutants.The results suggested that resveratrol can induce a dose- andtime-dependent death of HepG2 cells and over-expression ofCAV1 can enhance the cell killing and apoptosis-inducing effectsof resveratrol. Another intriguing finding of this study is the obser-vation that over-expression of CAV1 enhances the transport of res-veratrol into HepG2 cells through its cholesterol shuttle domainrather than the scaffolding domain, inhibiting cell proliferationand inducing apoptosis mediated through the p38MAPK pathwayand caspase-3 expression.73 As resveratrol derivatives are consid-ered to be superior antiproliferative agents than the parentmolecule, Colin et al.74 compared the antiproliferative effects oftrans-resveratrol, trans-e-viniferin and their respective acetatederivatives as well as a polyphenol mixture extracted from grape-vine shoots, known as vineatrol. Cell growth experiments revealedthat resveratrol triacetate showed a slightly better antiproliferativepotential than resveratrol. Vineatrol was found to be the mostpotent compound indicating a possible synergistic effect of bothresveratrol and e-viniferin. By using the in situ autofluorescencetechnique, the investigators observed that resveratrol and relatedcompounds induce cellular NADPH and green fluorescent cytoplasmicgranular structures which may indicate a mechanism involvinginduction of detoxifying enzymes.

Cholangiocarcinoma represents a tumor originating from theepithelial cells of the biliary tree and accounts for about 3% of allthe gastrointestinal neoplasia with an increasing incidence of theintrahepatic form.75 Recently, Roncoroni et al.76 evaluated the effi-cacy of resveratrol on SK-ChA-1 human cholangiocarcinoma cells,cultured in the classical two-dimensional model as well as in thethree-dimensional spheroids. Resveratrol treatment inhibited cellgrowth in both cell culture systems with a concomitant cell cycleperturbation characterized by an accumulation in the G1/S-phase.Additional studies showed resveratrol-induced increase of lactatedehydrogenase and alkaline phosphatase activities in the culturemedium as well as elevated transglutaminase activity in the cell ly-sates. This study indicates new therapeutic potential of resveratrolin unoperable human cholangiocarcinoma patients.

In vivo studies

Several studies have investigated the antitumor potential of res-veratrol in animal models of liver cancer. In 1999, a study by Carbóet al.77 showed promise for the use of resveratrol not only as a che-

mopreventive agent, but also as a chemotherapeutic agent. In thisstudy, resveratrol administration to rats inoculated with fast grow-ing Yoshida AH-130 hepatoma cells was found to exhibit signifi-cant decrease in the tumor cell count. The observed antitumoreffects were associated with an increase in the number of cells inthe G2/M phase of cell cycle and apoptosis of tumor cell popula-tion.77 Another study subsequently revealed that dietary resvera-trol suppressed the growth and metastasis of AH109A asciteshepatoma cells implanted into Donryu rats.78 Interestingly, itwas observed that tumor growth not only ceased after 12 days inresveratrol exposed rats, but the tumors actually began to regress.Furthermore, it was found that resveratrol was protective againsthyperlipidemic states in hepatoma bearing rats. Although theresearchers did not characterize the exact mechanisms of resvera-trol action, the observed antioxidant properties (inhibition of ser-um lipid peroxidation) could be implicated in such a therapeuticeffect.78 Resveratrol was also shown to have inhibitory effects onthe growth of H22 tumor cells transplanted into mice possiblydue to its effects on nonspecific host immunomodulatory activ-ity.79 In another interesting study, the effects of resveratrol wereinvestigated against the growth of H22 tumors in liver.80 Theinvestigators first developed external tumors by injecting H22 cellsinto the groin of BALB/c mice. Subsequently, tumor tissue was xe-nografted into the liver and allowed to grow. Resveratrol treatmentwas found to curtail hepatic tumor growth through reducedexpression of cell cycle proteins, namely cyclin B1 andp34cdc2.80 The potency of resveratrol in combination with 5-FUwas tested against the above transplanted murine tumor model.As with the in vitro study discussed earlier, 5-FU’s ability to reversetumor growth was again increased when resveratrol was co-administered. Furthermore, the same study illustrated decreased5-FU toxicity in mice concurrently given resveratrol, and a drasticincrease in the number of cells arrested in mitosis.81 Resveratrol’sability to enhance the therapeutic efficacy of 5-FU may be of valuein the treatment of HCC. In a recent study, the activity of CAV1 mu-tants on the growth of HepG2 cells in nude mice subjected to res-veratrol treatment has been evaluated.73 Wild type HepG2 cells orsimilar cells expressing various CAV1 mutants were implanted inanimals that received intraperitoneal (i.p.) injections of resveratrolon every alternate day for 21 days, starting the treatment 10 daysfollowing tumor cell inoculation. Although resveratrol afforded asignificant inhibition action on the growth of all HepG2 cells, amaximum tumor regression was achieved against xenografts ofHepG2 cells expressing CAV1. The involvement of CAV1 in thecytotoxic and pro-apoptotic actions of resveratrol through modula-tion of cellular sensitivity of resveratrol may represent a novel ap-proach in overcoming multi-drug resistance in the therapy ofhuman liver cancer.

Several studies have also evaluated the chemopreventive po-tential of resveratrol or resveratrol-rich product against chemi-cally-induced hepatocarcinogenesis in rodents. Kweon et al.82

investigated the effects of dietary grape extract (known to containresveratrol) on the development of placental glutathione S-trans-ferase (GST-P)-positive preneoplastic hepatic foci induced in ratsby i.p. injection of diethylnitrosamine (DENA) followed by partialhepatectomy. The results showed a suppressive effect of long-termgrape diet on GST-P-positive foci formation. Accompanying studiesrevealed that the grape diet attenuated the generation of hepaticthiobarbituric acid reactive substances (TBARS, indication of lipidperoxidation) and activity of fatty acid synthase in liver, whichcould explain the observed chemopreventive action. Tharappelet al.83 used another rat hepatocarcinogenesis model employingDENA as the initiating carcinogen and 3,30,40,4-tetrachlorobiphenyl(a polychlorinated biphenyl, PCB-77) as the promoting agent andobserved that dietary resveratrol had no effect on the numberand volume of GST-P-positive foci. The lack of chemotherapeutic


effect of resveratrol on hepatocarcinogenesis may be due to thepresence of very low amounts of this antioxidant in the diet(0.005% w/w) resulting in an extremely low doses, as comparedto the other in vivo studies. In order to delineate the role of resve-ratrol on chemoprevention of liver cancer, our laboratory has initi-ated a series of experiments using the well-characterized andclinically relevant two-stage rat hepatocarcinogenesis initiatedwith DENA and promoted by phenobarbital (PB).84–86 Dietary res-veratrol treatment was started 4 weeks prior to the initiation andcontinued for 20 weeks. Resveratrol dose-dependently reducedthe incidence and total number of visible hepatocyte nodules, theprecursors of HCC. Another striking observation of this study wasresveratrol-mediated attenuation of average number of nodules/li-ver (nodule multiplicity) of experimental animals as illustrated inFig. 2. This study provides evidence for the first time that resvera-trol (at 100 or 300 mg/kg) exerts significant chemopreventive ef-fects on DENA-initiated hepatic tumorigenesis which, at leastpartly, is attributable to inhibition of hepatic cell proliferationand induction of apoptosis. Our mechanistic studies have revealedthat resveratrol-mediated apoptogenic signal during rat liver carci-nogenesis may be achieved through the down-regulation of Bcl-2and up-regulation of Bax.87 Over-expression of antiapoptotic Bcl-2 has been associated with elevated cyclooxygenase-2 (COX-2)expression88 and resveratrol was shown to suppress COX activityin the livers of mice treated with DENA.89 Hence, the inhibitory ef-fect of resveratrol on Bcl-2 as observed in our study could strength-en the possibility of resveratrol-mediated amelioration of elevatedCOX-2 expression during hepatocarcinogenesis. This underscoresthe hypothesis that COX-2 is a potential target for chemopreven-tive action of resveratrol in hepatic carcinoma. Although severalCOX-2 inhibitors are known to exert chemopreventive effects,not all of these agents are considered ideal candidates for chemo-prevention due to the risk of adverse cardiovascular events. Re-cently, we have evaluated the role of resveratrol (50–300 mg/kg)on cardiac performance using transthoracic echocardiography dur-ing experimental hepatocarcinogenesis initiated with DENA andpromoted by PB. Our results indicate that resveratrol do not exhibit

Hep

atic

nod

ule

mul

tiplic

ity

0

5

10

15

20

25

30

35

a

DENA Res 50+ DENA

Res 100+ DENA

Res 300+ DENA

a, b

Fig. 2. Resveratrol chemoprevention of liver tumorigenesis in Sprague–Dawleyrats. Initiation of hepatocarcinogenesis was performed by a single i.p. injection ofDENA (200 mg/kg), followed by promotion with PB (0.05% w/v) in drinking water.The rats had free access to food supplemented with resveratrol equivalent to 50,100 or 300 mg/kg body weight/day. Resveratrol treatment was started 4 weeksprior to the initiation and continued for 20 weeks. Visible hepatocyte nodules werecounted at the end of the study. Values represent the mean ± standard deviation(n = 4–10). aP < 0.001 compared with DENA control. bP < 0.001 compared withresveratrol (50 mg/kg) plus DENA group. Modified from Ref.87

any cardiotoxicity but rather improve the cardiac function in adose-responsive fashion.90 The primary focus of ongoing researchin our laboratory is the elucidation of the molecular mechanismsunderlying the resveratrol chemoprevention of hepatic neoplasia.

Toxicity and pharmacokinetic studies

As described above, an impressive number of studies performedusing in vitro as well as pre-clinical in vivo animal models indicatethat resveratrol may be an effective anticancer agent in humans forprevention and therapy of liver cancer. In order to successfullytranslate such promising observations into the clinic, studies onpotential toxicity as well as bioavailability, pharmacokinetics ofresveratrol are essential. Within the past decade, several researchgroups have evaluated the potential pre-clinical in vivo toxicityof resveratrol primarily in rodents. Juan et al.91 did not find anyevidence of systemic toxicity as well as hematologic and histopath-ologic changes in rats administered 20 mg/kg/day for 28 days. Cro-well et al.92 studied the effect of three incremental doses ofresveratrol administered in both male and female CD rats for28 days. Oral administration of a high dose of 3000 mg/kg/day pro-duced nephropathy and renal toxicity. Although this dose resultedin an increase in several clinical markers of liver metabolism suchas elevated blood urea nitrogen and creatinine levels, it did notproduce any hepatotoxicity. Low dose of 300 mg did not produceany adverse effects. Oral administration of resveratrol (1000,2000 and 4000 mg/kg/day) in C57BL/6 p53 knockout mice didnot produce malignant or benign neoplasm, demonstrating the ab-sence of any carcinogenic potential of resveratrol.93 Chronic long-term administration of resveratrol in drinking water producednephrotoxicity as evidenced by increased markers of oxidativestress in the kidney.94 Resveratrol did not produce any dermal orophthalmic irritation in albino rabbits. Resveratrol administrationwas devoid of any genotoxicity in Sprague–Dawley rats. Both sub-chronic and chronic administration of resveratrol demonstrated anabsence of systemic as well as reproductive organ toxicity in Wis-tar rats.95 In summation, a high 3000 mg/kg/day produced renaltoxicity in rodents. Resveratrol showed a complete absence ofgenotoxicity, oncogenicity as well as systemic and reproductivetoxicity. These aforementioned studies highlight the relative safetyof resveratrol in moderate doses.

Although a number of reports have described the pharmacoki-netics of resveratrol in animal model systems34 albeit confusing,conflicting and contradictory results, reports on similar studieson humans are limited till date. Table 3 summarizes the widelyvarying situations in which resveratrol has been investigated inhuman subjects following administration either as a purecompound, conjugate or as a constituent of wine and/or other bev-erages. Soleas et al.96 performed a preliminary study on bioavail-ability of resveratrol (25 mg/person) in human subjects andfound that this polyphenol was well absorbed following oraladministration. During the first 24 h, nearly 25% of administeredresveratrol recovered in the urine as free as well as conjugatedforms. Goldberg et al.97 investigated metabolism of resveratrol inhealthy volunteers who took resveratrol (25 mg/70 kg) mixed withwhite wine, grape or vegetable juice. According to this study, fol-lowing an oral dosing, the compound appeared in serum and urinepredominantly as glucuronide and sulfate conjugates (Fig. 1) andreached at peak concentrations (10–40 nM) around 30 min post-administration. The free polyphenols accounted for 1.7–1.9% ofthe peak serum concentrations and urinary 24-h resveratrol wasabout 17% of the administered dose. Walle and co-workers98

examined the absorption, bioavailability, and metabolism of14C-resveratrol after oral and intravenous (i.v.) doses. The absorp-tion of 25-mg oral dose was at least 70%, with peak plasma levels

Table 1In vitro effects of resveratrol on liver cancer cells and underlying mechanisms.

Effects Mechanisms Concentrations(lM)

References

Inhibited the proliferation of Fao and HepG2 cells ; ability to enter mitosis; " S and G2/M phase cells 1–150 Delmas et al.59

Decreased the proliferation and invasion of HepG2 cells Post-receptor mechanism 2.5–50 De Ledinghen et al.60

Suppressed the proliferation and invasion of AH109A cells ; oxidative stress 25–200 Kozuki et al.61

Miura et al.62

Restrained hypoxia-stimulated invasiveness of HepG2 cells ; HIF-1a; ; VEGF 5–100 Zhang et al.63

Inhibited the growth of HepG2 cells " apoptosis; cell arrest in G1 phase; " p53;" p21; " Bax

4.4 � 10�6–9 � 10�5

Kuo et al.64

Exhibited cytotoxic effects in HepG2 cells 5.2 � 10�5 (IC50) Kim et al.65

Increased cytotoxicity in HepG2 cells " apoptosis; cell cycle regulation 50, 100 Kocsis et al.66

Arrested H22 cell growth and acted synergistically with 5-FU " apoptosis 5.5–88 Sun et al.67

Caused cytotoxicity to H4IIE hepatoma cells " apoptosis; " caspase 2, 3, 8/10;" DNA fragmentation

5–350 Michels et al.68

Inhibited the proliferation of HepG2 cells " apoptosis; cell cycle regulation 2.5–320 Stervbo et al.69

Suppressed the proliferation of HepG2 cells " apoptosis; cell cycle arrest in G1 and G2/M phase;; ROS; " iNOS;" eNOS; " NO

10�6–1 Notas et al.70

Inhibited TNF-a-mediated invasion of HepG2 cells ; MMP-9; ; NF-jB 50, 100 Yu et al.71

Prevented the proliferation of HepG2 cells; CAV1 overexpressingcells exhibited increased growth inhibition

" apoptosis; cell cycle regulation;" p38MAPK activity; " caspase-3

20–300 Yang et al.73

Attenuated the proliferation of HepG2 cells; analog and mixtureshowed better cell killing efficacy

" NADPH; " detoxifying enzymes 1–100 Colin et al.74

Inhibited the growth of SK-CHA-1 cells cultured in two-dimensional model and three-dimensional spheroids

" G1/S-phase cells; " LDH; " ALP;" TG

8–64 Roncoroni et al.76

ALP, alkaline phosphatase; IC50, half maximal inhibitory concentration; CAV1, caveolin-1; eNOS, endothelial nitric oxide synthase; 5-FU, 5-fIuorouracil; HIF-1a, hypoxia-inducible factor-1a; iNOS, inducible nitric oxide synthase; LDH, lactate dehydrogenase; MMP-9, matrix metalloproteinase-9; NF-jB, nuclear factor-jB; NO, nitric oxide; ROS,reactive oxygen species; TG, transglutaminase; TNF-a, tumor necrosis factor-a; VEGF, vascular endothelial growth factor.


of resveratrol and metabolites about 2 lM, and a plasma half-life9.2 h. However, only trace amounts of unchanged resveratrol couldbe detected in the plasma, as the extremely rapid sulfate conjuga-tion by the intestine/liver appeared to be the rate-limiting step inresveratrol’s bioavailability. Most of the oral dose (�53–85%) wasrecovered in urine, whereas the recovery in feces was highly vari-able (�0.3–38%). Following the i.v. administration, the recoveriesin urine were about 42–83% of the dose and nearly 0.6–23% foundin the feces. In another study, oral administration of resveratrol(0.5 or 1 mg/kg) to humans yielded detectable levels of resveratroland its derivatives in the plasma and urine. At a low dose (0.03 mg/kg), more than half of the ingested resveratrol was recovered in theurine in 24 h, whereas at a higher dose (1 mg/kg), only a quarter ofthe administered dose could be recovered during the same peri-od.99 Recently, Boocock and group100 identified 6 major conjugatedmetabolites in the plasma and urine of human volunteers afteradministration of a single oral dose of 1 g resveratrol. A Phase Idose escalation pharmacokinetic study by the same group thenindicated that resveratrol may be administered safely withoutany serious adverse effects in a single dose up to 5 g, resulting ina peak plasma level of 2.4 lM that occurred 1.5 h post-dose. Peaklevels of two monoglucuronides and resveratrol-3-sulfate were3- to 8-fold higher than the parent compound. Urinary excretionof resveratrol and its metabolites were rapid, with 77% of all uri-nary agent-derived species excreted within 4 h after ingestingthe lowest dose of 0.5 mg/kg.101 Another study was performed toinvestigate the effects of food on the bioavailability of resveratrol(400 mg) following oral administration in healthy subjects. Therate of absorption was significantly delayed in the presence of foodbut, the extent of absorption was not affected.102 A pharmacoki-netic and safety evaluation of multiple dose regimens of resvera-trol (150–900 mg/day) has recently been performed in healthyvolunteers.103 Peak plasma concentrations of resveratrol wereachieved at 0.8–1.5 h post-dose with higher bioavailability follow-ing morning administration. The repeated administration waswell-tolerated but produced relatively low plasma concentrationsof resveratrol despite the high doses and short dosing intervals.

In addition to the bioavailability studies following intake ofpure compound, resveratrol bioavailability from a moderate con-sumption of red wine in healthy volunteers has been studied. Inone study, the wine ingestion was associated with three differentdietary regimens: fasting, a standard meal, a meal with high andlow amount of lipids. Trace quantity of resveratrol was found insome serum samples collected 30 min after red wine consumption,while after longer times resveratrol glucuronides predominated.The bioavailability of resveratrol was shown to be independentfrom the meal or its lipid content.104 In another study, resveratrolmetabolites were evaluated as potential biomarkers of wine con-sumption in humans after moderate consumption of sparkling,white, or red wine. Although no resveratrol metabolites were de-tected in serum an average of 10 h after the consumption of wine,those were identified in urine, suggesting that these metabolitescan be used as a measure of compliance in interventional studiesas well as an objective measure of wine consumption in epidemi-ological studies.105 The metabolism of trans-resveratrol-3-O-b-D-glycoside (known as piceid), a prominent form of resveratrol infood as well as in red wine, has been studied in healthy volunteers.Several sulfate, glucuronide as well as two novel diglucuronidemetabolites of resveratrol were identified and quantified in plasmaand urine with resveratrol sulfate being the dominant conjugate. Itwas shown that up to 50% of the plasma resveratrol metaboliteswere bound to proteins and 13.6–35.7% administered piceid wereexcreted in urine.106

It is clear from the above clinical observations that resveratrol israpidly absorbed following oral administration and levels aredetectable in both plasma and urine with the maximum plasmaconcentrations being reached within an hour post-administration.The rapid and extensive phase 2 metabolism of resveratrol generat-ing glucuronide and sulfate conjugates may partly explain the lowcirculating levels of this polyphenol. However, the major limitationof the bioavailability studies in human seems to be the small num-ber of healthy volunteers involved. It has been shown that resvera-trol glucuronidation and sulfation are mediated by specific isoformsof UDP-glucuronosyltransferases and sulfotransferases,107,108

Table 2In vivo effects of resveratrol on development and growth of liver cancer with underlying mechanisms.

Effects Mechanisms of action Dose/duration Route References

Arrested tumor growth in male Wistar rats implanted with AH-130 hepatoma cells

" cells at G2/M; " apoptosis 1 mg/kg;7 days

i.p. Carbó et al.77

Hampered tumor growth and metastasis in male Donryu ratsimplanted with AH109A hepatoma cells

; lipid peroxidation; ; serum triglycerides;; VLDL; ; LDL

10, 50 ppm;20 days

Diet Miura et al.78

Inhibited tumor weights in BALB/c mice implanted with H22hepatoma cells

Immunomodulatory activity 500, 1000, 1500 mg/kg;10 days

abd Liu et al.79

Reduced tumor volumes in BALB/c mice implanted with H22hepatoma cells

; cyclin B1; ; p34cdc2; 5, 10, 15 mg/kg;10 days

abd Yu et al.80

Worked synergistically with 5-FU for cancer treatment in maleBALB/c mice transplanted with H22 cancer cells

\ S-phase 5, 10, 15 mg/kg;10 days

abd Wu et al.81

Inhibited the growth of CAV1-expressing HepG2 cellstransplanted in female BALB/c mice

; cell proliferation; " apoptosis;" caspase-3

15 mg/kg;every alternate day for21 days

i.p. Yang et al.73

Grape extract reduced DENA-initiated GST-P-positive hepaticpreneoplastic foci in male Sprague–Dawley rats

; lipid peroxidation; ; FAS 15% (w/w) grape extractin diet;11 weeks

Diet Kweon et al.82

Did not modify DENA-initiated and PCB-77-promotedhepatocarcinogenesis in female Sprague–Dawley rats

0.005% in diet;�10 weeks

Diet Tharappelet al.83

Suppressed DENA-initiated and PB-promoted hepatocyte noduleformation in female Sprague–Dawley rats

; cell proliferation; " apoptosis;" Bax; ; Bcl-2

50, 100, 300 mg/kg;20 weeks

Diet Bishayee andDhir87

abd, abdominal injection; DENA, diethylnitrosamine; FAS, fatty acid synthase; 5-FU, 5-fluorouracil; GST-P, placental glutathione S-transferase; i.p., intraperitoneal; LDL, low-density lipoprotein; PB, phenobarbital; PCB-77, polychlorinated biphenyl-77; VLDL, very low-density lipoprotein.


which exist in polymorphic states with interethnic variability.Hence, clinical studies with more volunteers are needed to betterunderstand the pharmacokinetics of resveratrol.

From the data presented in this review, it is very clear that theconcentration ranges of resveratrol at which cytotoxic effectshave been achieved against various liver cancer cells are indeedin excess of the maximum peak plasma levels observed in thepharmacokinetic studies cited. It has been indicated that the plas-ma concentration of resveratrol following daily intake of twoglasses of wine (�375 ml) in a normal person (70 kg) would bebetween 1 and 10 nM,109 which is obviously below the lM rangesof concentration that exert the antineoplastic effects in vitro inmost of the studies presented here (Table 1). Nevertheless, oneshould consider several facts while comparing the bioavailabilityof resveratrol with observed antitumor effects. First of all, most ofthe pharmacokinetic studies have focused on measuring onlytrans-resveratrol. It has been shown that the trans to cis isomeri-zation of resveratrol is facilitated by exposure to UV and evennatural light.110 Accordingly, it is possible to underestimate resve-ratrol concentration if only trans-resveratrol is detected andmeasured by analytical methods. Additionally, total plasma resve-ratrol content should also include resveratrol in plasma protein-and lipoprotein-bound fractions in addition to free resveratrol.Moreover, concern has been raised for possible underestimationof resveratrol concentrations in plasma as the efficiency of extrac-tion in biosamples was as low as 60%,100 indicating limitation ofexisting analytical procedures. Resveratrol metabolites (knownas well as yet unknown) should also be considered as they mayalso contribute to the anticancer activities (see below for details).Finally, resveratrol could be bound to cell membrane as well aslipophilic tissues that may not be accounted while measuringit’s the plasma concentration of this molecule. The undetectedcellular fraction of resveratrol could also contribute to its biolog-ical responses, and thus the overall pharmacological effects ofresveratrol should not be solely interpreted from its measuredplasma levels (see Table 2)

Conclusion and future directions

In recent years, epidemiological data have shown an alarmingtrend in an increased prevalence of hepatocellular carcinoma. Thistendency, compiled with the disease’s high rate of mortality due to

imperfect treatment methods, has forced researchers to examinepreventive approaches as well as alternate routes to treatment.Using naturally occurring compounds, including those derivedfrom fruits, vegetables and herbs, as potential cancer preventiveand therapeutic agents has become a fascinating strategy. Of thevarious phytochemicals tested for potential beneficial effectsagainst HCC, resveratrol stands out as the molecule with the mostpotential for stifling the disease’s growing incidence and conse-quent mortality rate. From numerous studies using various livercancer cell lines and chemically-induced tumors as well as im-planted cancers in animal models as described in this review, it be-comes apparent that resveratrol may play an important role notonly in the prevention but also in the therapy of metastatic diseaseof the liver. All these studies largely establish that resveratrol hasgreat promise for battling liver cancer. Considering the aggressivenature and consequent high levels of mortality associated withHCC, resveratrol’s anticancer properties are of great interest toresearchers and continue to be examined.

From a large number of studies discussed here, it is evident thatthe anti-HCC actions of resveratrol are largely due to inhibition ofabnormal cell proliferation and apoptosis through cell cycleregulation. Several investigations have indicated that resveratrolsuppresses the growth of HCC cells and prevents hepatocarcino-genesis by mitigating oxidative stress. Perhaps, future researchshould deal with further characterizing the exact mechanism bywhich resveratrol possesses its effects on the cell cycle, apoptosisand redox signaling. Though it is now known that resveratrolscavenges ROS and modulates activities of antioxidant enzymes,58

further research would be able to identify the underlying signalingpathways to improve the therapeutic modalities of this agent. Con-sidering the pleiotropic actions of resveratrol, it is reasonable tospeculate that the antihepatocarcinogenic effects of resveratrolmay be due to regulation of multiple molecular pathways includ-ing those involved in hepatic xenobiotic biotransformation andelimination,53–56 inflammation,30,111 gap-junction intercellularcommunication.112,113 Future studies should explore these andother possible mechanisms of resveratrol action to understandthe full potential of this dietary agent in the prevention and treat-ment of HCC.

Accumulating evidence suggests that even in the event of lackof efficacy for a single agent at low concentrations, combinationsof two or more compounds could be much more effective.

Table 3Clinical pharmacokinetic and safety studies of resveratrol and resveratrol containing food products.

Observations Dose/duration Route References

Plasma free and conjugates peaked in 30 min; nearly 25% recovered in urine over 24 h 25 mg/person Oral Soleas et al.96

Peak glucuronide and sulfate conjugates appeared in serum in 30 min; urinary 24-h excretionwas 16–17% of the dose

25 mg/70 kg Oral Goldberg et al.97

Absorption was 70% with plasma half-life of 9.2 h; mostly excreted in urine 25 mg/person Oral, i.v. Walle et al.98

Pure compound and its derivatives are detectable in plasma and urine; 25–50% resveratrol wasrecovered in urine during 24 h

0.03, 0.5, 1 mg/kg Oral Meng et al.99

Six major conjugate metabolites were detected in and separated from serum and urine 1 g/person Oral Boocock et al.100

Did not exhibit adverse effects; peak plasma levels occurred in 1.5 h; 77% of all urinary speciesexcreted in 24 h

0.5, 1, 2.5, 5g/person

Oral Boocock et al.101

Food delayed the absorption; did not affect the extent of absorption 400 mg/person Oral Vaz-da-Silva et al.102

Repeated dosing produced only mild adverse effects; peak plasma concentration between 0.8and 1.5 h

25, 50, 100, 150 mg; 6 times/day Oral Almeida et al.103

Pure or glucuronide conjugate was found in serum; meal did not affect bioavailability 3.4, 7.5, 33 lg/kg Oral Vitaglione et al.104

Increase in total metabolites, which may be used as biomarkers for clinical studies 0.36, 0.4, 2.6 mg/person Oral Zamora-Ros et al.105

Sulfate, glucuronide and C/O-diglucuronide conjugates appeared in plasma and urine; 50%plasma metabolites bound to proteins

85.5 mg/70 kg Oral Burkon and Somoza106

Fig. 3. Chemical structures of resveratrol analogs, 3,30 ,40 ,5-tetrahydroxy-trans-stilbene or piceatannol (1), 3,4,40-trihydroxy-trans-stilbene (2), 3,4-dihydroxy-trans-stilbene(3), 3,4,5-trihydroxy-trans-stilbene (4), 3,5-dimethoxy-40-hydroxy-trans-stilbene or pterostilbene (5), 3,4,5,40-tetramethoxy-trans-stilbene (6), 3,40 ,5-trimethoxy-30-hydroxy-cis-stilbene (7), and 3,40 ,5-trimethoxy-30-amino-cis-stilbene (8).


Combinations of several chemotherapeutic drugs also offer thepossibility of lowering their doses and consequently may reduceunwanted adverse effects. Resveratrol has been found to potentiatethe effects of chemotherapeutic agents and ionizing radiation.114

Several animal studies55,91–95 have clearly demonstrated that res-

veratrol is well-tolerated and pharmacologically safe molecule,which is supported by clinical studies as presented here. Consider-ing these advantages, resveratrol may be used in combinationwith other chemotherapeutic drugs and radiation therapy to enhancetheir therapeutic efficacy while limiting chemotherapy- and


radiotherapy-associated negative side effects. Additionally, it seemsto be logical to extend this approach to the field of chemopreven-tion, especially if one considers that dietary chemopreventiveagents are naturally present in the diet in combination.115 In viewof the observed in vitro synergistic effects of resveratrol with manyagents, it is reasonable to identify possible interactions with otherdietary factors and explore resveratrol treatment in combinationwith other agents in pre-clinical studies.

In spite of the anticancer efficacy of resveratrol in pre-clinicalmodels, its low bioavailability remains enigmatic and elusive. Inorder to explore whether resveratrol metabolites exert antitumorproperties, three major human sulfated conjugates have recentlybeen tested against human breast cancer cells. In contrast to resve-ratrol, its sulfated metabolites have exhibited poor cytotoxicity inhuman malignant breast cancer cells.116 Nevertheless, the in vitroactivity of these metabolites may not necessarily reflect theirin vivo efficacy. Considering the fact that in vivo concentrations ofindividual metabolites of resveratrol can be more than 10-foldhigher than those of the native compound, critical pharmacoki-netic studies are needed to clarify whether the metabolites act justthe less active or inactive forms of the molecule, represent as a poolfrom which free resveratrol can be released as ubiquitously excit-ing human sulfatases or b-glucuronidase could convert the metab-olites back to resveratrol, or themselves could mediate, at least inpart, the efficacy exerted by the parent molecule. Additionally, ac-tive research should also be aimed at novel drug formulation anddelivery systems, including liposomal, neosomal and nanoparticleformulations of resveratrol to enhance its hepatic concentrationand efficacy.

Another future research area would be designing novel resvera-trol analogs with cancer chemopreventive and/or chemotherapeu-tic activities superior to that of the parent compound withimproved pharmacokinetic and pharmacodynamic properties.The structural simplicity of the stilbene molecule accompaniedwith the presence of hydroxyl groups linked with its diverse bio-logical activities have inspired medicinal chemists to design novelresveratrol analogs with cancer chemopreventive and/or chemo-therapeutic activities superior to that of the parent compound. Anumber of cis- and trans-stilbenes similar to resveratrol with alter-ation of hydroxy and methoxy moieties have been synthesized(Fig. 3), and some of these novel compounds have been reportedto exhibit more cytotoxicity against various human cancer cellsin vitro.39 In vivo anticancer effects of novel resveratrol analogshave been explored.117–121 One of these studies shows that heyne-anol, a tetramer of resveratrol, has comparable or better antitumorefficacy than resveratrol in a mouse lung cancer model.118 Never-theless, more studies are expected to provide in vivo data onhead-to-head comparison of anticancer potential of resveratrolwith its analogs.

Resveratrol is currently investigated for the prevention andtreatment of human colon cancer.48 It is expected that additionalresearch would lay the foundation for clinical trials with resvera-trol in the prevention of HCC in high-risk patients predisposedwith viral hepatitis, other liver diseases and environmental carcin-ogens. Future research would also aid in the development of resve-ratrol as a clinically effective drug for the treatment of HCC. Thein vitro cell culture experiments, pre-clinical animal studies as wellas clinical findings described in this review probably suggest thatresveratrol is a promising agent in chemopreventive and chemo-therapeutic strategies to combat human HCC, which remains adevastating disease.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

The authors thank Werner J. Geldenhuys, Ph.D., for technicalassistance with the chemical structures. Our research on resvera-trol and liver cancer chemoprevention is supported by a ResearchIncentive Grant from the Ohio Board of Regents, State of Ohio, Uni-ted States.

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Resveratrol in the chemoprevention and treatment of hepatocellular carcinoma

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