Liver Cancer Open Access
Copyright ©The Author(s) 2002. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Feb 15, 2002; 8(1): 79-81
Published online Feb 15, 2002. doi: 10.3748/wjg.v8.i1.79
Anti-hepatoma activity of resveratrol in vitro
Zhong-Jie Sun, Cheng-En Pan, Hong-Shan Liu, Guo-Jun Wang, Department of Hepatobiliary Surgery, First Hospital of Xi’an Jiaotong University, Xi’an 710061, Shaanxi Province, China
Author contributions: All authors contributed equally to the work.
Correspondence to: Dr. Zhong-Jie SUN, Department of Hepatobiliary Surgery, First Hospital of Xi’an Jiaotong University Xi’an 710061, Shaanxi Province, China.
Telephone: +86-029-5260273 Fax: +86-029-5260273
Received: August 9, 2001
Revised: August 20, 2001
Accepted: August 23, 2001
Published online: February 15, 2002


AIM: To study the anti-tumor effect of resveratrol alone and the synergistic effects of resveratrol with 5-FU on the growth of H22 cells line in vitro

METHODS: The number of cells was measured by MTT method,the morphological changes of H22 cells were investigated under microscopy and electron microscopyq.

RESULTS: Resveratrol inhibited the growth of hepatoma cells line H22 in a dose- and time-dependent manner. IC50 of the resveratrol on H22 cells was 6.57 mg·L-1. The synergistic anti-tumor effects of resveratrol with 5-FU increased to a greater extent than for H22 cells treated with 5-FU alone (70.2% vs 28.4%)(P < 0.05). Under microscope and electron microscope, characteristics of apoptosis such as typical apoptotic bodies were commonly found in tumor cells in the drug-treated groups.

CONCLUSION: Resveratrol can suppresses the growth of H22 cells in vitro, its anti-tumor activity may occur through the induction of apoptosis.


Hepatoma is common in China[1-20],but only a few chemotherapeutic drugs hold a high place in the treatment of human primary hepatocellular carcinoma (PHC).Resveratrol, a phytoalexin found in grapes, fruits, and root extracts of the weed Polygonum cuspidatum, has been an important constituent of Japanese and Chinese folk medicine. Indirect evidence suggests that the presence of resveratrol in white and rose wine may explain for the reduced risk of coronary heart disease associated with moderate wine consumption.This effect has been attributed to the inhibition of platelet aggregation and coagulation, in addition to the antioxidant and anti-inflammatory activity of resveratrol[21-28].Moreover, a recent report shows that resveratrol is a potent cancer chemopreventive agent in assays representing three major stages of carcinogenesis[29-35].The ability to inhibit cellular events associated with tumor initiation, promotion, and progression has been attributed to the anticyclooxygenase activity (COX-1) of resveratrol[36]. We report here the results of our findings showing that resveratrol inhibited the growth of hepatoma cells line H22.


Resveratrol was kindly provided by Prof Li(Environment and Chemical Engineering School,Xi’an Jiaotong University)and dissolved in dimethylsulfoxide (DMSO);MTT was obtained from Sigma. PRMI1640 containing 100 mL·L-1 fetal bovine serum (FBS) was bought from Gibco. All other chemicals were standard commercial products of analytical grade.

Cell culture

H22 cells were obtained from Center of Molecular Biology of First Hospital, Xi’an Jiaotong University and routinely cultured in RPMI 1640 containing 100 mL·L-1 FBS at 37 °C in an atmosphere with 50 mL·L-1 CO2.

Assay of cell proliferation

H22 cells were plated in 96-well plates (2 × 104/well) for 24 h before the addition of resveratrol. Medium was then aspirated and replaced with fresh RPMI 1640 + 100 mL·L-1FBS containing resveratrol for 48 h. Different compositions to be tested were added according to designed groups: group A (cell control group) with nothing added, group B (DMSO control group) with DMSO 5 mL·L-1, group C1-5 with Resveratrol (1.25, 2.50, 5.0, 10.0 and 20.0 mg·L-1),group D 1-25-FU (2400 and 1200 mg·L-1), group E with Resveratrol 5.0 mg·L-1 + 5-FU 1200 mg·L-1 .Each group had 4 wells and was cultured for 48 h. The number of cells was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide) method as described in Sigma Technical Bulletin (Sigma, MO). Absorbance at 570 nm (A) was assayed at different time points.The A value was adjusted with the living cell number. Each sample was assayed three times. Inhibition rate(%) = (1-experimental A / control A) × 100%.

Morphologic observation

After the cellular culture for 48 h, cells in groups A,C and E were observed and photographed with an Olympus BH-I microscope and a Hitachi-600 electron microscope.

Growth inhibition of H22 cells

H22 cells at 2 × 104/well were incubated with different concentrations of resveratrol for 8-48 h and the effect of resveratrol on the cells growth was examined by MTT assay. The growth of H22 cells was markedly inhibited by resveratrol with the IC50 value of 6.57 mg·L-1. Moreover,the cytotoxicity of resveratrol was in concentration-dependent and time-dependent manners (Table 1). The inhibition ability of 5-FU was 49.2%(2400 mg·L-1),28.4% (1200 mg·L-1) respectively;The inhibiting ability of resveratrol (5.0 mg·L-1)combined with 5-FU(1200 mg·L-1) was higher than that of 5-FU alone(70.2% vs 28.4%,P < 0.05).

Table 1 Effect of various concentrations of resveratrol on the growth of hepatoma cells H22.
c(resveratrol)/mg·L-18 h12 h24 h48 h
Morphology observation

Apoptotic cells were found in cells incubated with resveratrol. Light microscopic observation showed that apoptotic cells were characterized with cytoplasmic condensation, vacant bubbles, and condensed nuclei(Figure 1). Under electron microscope, H22 cells exhibited the characteristics of apoptosis including cytoplasmic condensatin, pyknotic nuclei, condensed chromatin and apoptotic bodies(Figures 2 and 3). Compared with control groups, groupC and E had much more cells with the apoptotic characteristics.

Figure 1
Figure 1 Treatment with resveratrol for 48 h: apoptotic cell with condensed nuclei and cytoplasmic condensation ( × 200).
Figure 2
Figure 2 Cytoplasmic condensation with vacant bubbles, pyknotic nuclei, some with condensed chromatin inside ( × 5000).
Figure 3
Figure 3 Apoptotic body ( × 10000).

To date, only a few chemotherapeutic drugs hold a high place in the treatment of human primary hepatocellular carcinoma (PHC) and there is clearly a need for evaluation of new anti-hepatoma drugs. Resveratrol (3,5,4’-trihydroxystibene), a natural compound present in grapes and other food, has been shown to provide cancer chemopreventive effects in different systems based on its striking inhibition of diverse cellular events associated with tumor initiation, promotion, and progression[29-35,37]. At the molecular level, these effects were related to the inhibition of free radical formation and cyclooxygenase activity[36], as well as induction of differentiation. In addition, resveratrol was shown to be a remarkable inhibitor of ribonucleotide reductase and DNA synthesis with cellular arrest in the S phase or the S-G2 phase transition[38-40]. In the present study, MTT assay was used to observe the effect of resveratrol on the growth of H22 mouse hepatoma cells in vitro, indicating that the drug could inhibit the growth of hepatoma cells. Its concentration- and time-effect relationships were also significant. Compared with control groups, group C had much more cells with apoptotic characteristics. The plausible mechanisms that could account for the anti-tumor activity of resveratrol might be related to induce apoptosis of tumor cells[41-48].

Resveratrol combined with 5-FU inhibited the cell growth much more strongly than each agent used alone. At a certain concentration, resveratrol inhibits H22 cell growth with the same effect as using 5-FU alone. Combination of resveratrol and 5-FU could have a cooperative effect. Both drugs inhibit cell growth at different phases of the cell cycle, i.e, resveratrol mainly causes G2/M arrest[39-40] and 5-FU mainly inhibits DNA synthesis(S phase) which naturally decreases the cellular growth more significantly. Our study indicates that combined use of resveratrol and 5-FU at low concentration that is used to treat hepatoma may be more efficient than using a single drug at higher concentration.The side-effects produced by 5-FU at the high doses can be avoided by its combination at lowdoses. These results suggest that resveratrol, may be potentially useful as a biochemical modulator to enhance the therapeutic effects of 5-FU in cancer chemotherapy.


Edited by Ma JY

1.  Tang ZY. Hepatocellular carcinoma--cause, treatment and metastasis. World J Gastroenterol. 2001;7:445-454.  [PubMed]  [DOI]  [Cited in This Article: ]
2.  Lin NF, Tang J, Ismael HS. Study on environmental etiology of high incidence areas of liver cancer in China. World J Gastroenterol. 2000;6:572-576.  [PubMed]  [DOI]  [Cited in This Article: ]
3.  Yang CS. Chinese diet in the causation and prevention of cancer. World J Gastroenterol. 1998;4:36-37.  [PubMed]  [DOI]  [Cited in This Article: ]
4.  Liu E, Zhang QN, Li WG. Effect of various drinking water on human micronucleus frequency in high risk population of PHC. World J Gastroenterol. 1998;4:183-184.  [PubMed]  [DOI]  [Cited in This Article: ]
5.  Wu MC. Clinical research advances in primary liver cancer. World J Gastroenterol. 1998;4:471-474.  [PubMed]  [DOI]  [Cited in This Article: ]
6.  Tang ZY. Advances in clinical research of hepatocellular carcinoma in China. World J Gastroenterol. 1998;4:4-7.  [PubMed]  [DOI]  [Cited in This Article: ]
7.  Wu MC, Shen F. Progress in research of liver surgery in China. World J Gastroenterol. 2000;6:773-776.  [PubMed]  [DOI]  [Cited in This Article: ]
8.  Jiang YF, Yang ZH, Hu JQ. Recurrence or metastasis of HCC: predictors, early detection and experimental antiangiogenic therapy. World J Gastroenterol. 2000;6:61-65.  [PubMed]  [DOI]  [Cited in This Article: ]
9.  Li JY, Huang Y, Lin MF. Clinical evaluation of several tumor markers in the diagnosis of primary hepatic cancer. World J Gastroenterol. 2000;6:39.  [PubMed]  [DOI]  [Cited in This Article: ]
10.  Wang CF, Shao YF, Zhang HZ. Surgical treatment for patients with stage IVa hepatic carcinoma and related studies. World J Gastroenterol. 2000;6:86.  [PubMed]  [DOI]  [Cited in This Article: ]
11.  Gu GW, Zhou HG. Traditional Chinese Medicine in prevention of liver cancer. Shijie Huaren Xiaohua Zazhi. 1999;7:80-81.  [PubMed]  [DOI]  [Cited in This Article: ]
12.  Liu WW. Etiological studies of hepatocellular carcinoma.. Shijie Huaren Xiaohua Zazhi. 1999;93-95.  [PubMed]  [DOI]  [Cited in This Article: ]
13.  Zhou XD. Prevention and treatment of recurrences and metastases of hepatocellular carcinoma. Shijie Huaren Xiaohua Zazhi. 1999;7:260-261.  [PubMed]  [DOI]  [Cited in This Article: ]
14.  Lu B, Dai YM. Abnormal cycle regulation of cells in the HCC. Shijie Huaren Xiaohua Zazhi. 2001;9:205-208.  [PubMed]  [DOI]  [Cited in This Article: ]
15.  Li L, Wu PH, Li JQ, Zhang WZ, Lin HG, Zhang YQ. Segmental transcatheter arterial embolization for primary hepatocellular carcinoma. World J Gastroenterol. 1998;4:511-512.  [PubMed]  [DOI]  [Cited in This Article: ]
16.  Huang FG, Li Y, Xie XD. Side effects and complcations of hepatic arterial infusion and embolization of liver carcinoma in aged patients and its management. World J Gastroenterol. 1998;4:67-68.  [PubMed]  [DOI]  [Cited in This Article: ]
17.  Wang JH, Lin G, Yan ZP, Wang XL, Cheng JM, Li MQ. Stage II surgical resection of hepatocellular carcinoma after TAE: a report of 38 cases. World J Gastroenterol. 1998;4:133-136.  [PubMed]  [DOI]  [Cited in This Article: ]
18.  Cai WX, Zheng H, Ye QL. Combined measurement of serum tumor markers in patients with hepatocellular carcinoma. World J Gastroenterol. 1998;4:181-182.  [PubMed]  [DOI]  [Cited in This Article: ]
19.  Deng ZL, Ma Y, Yuan L, Teng PK. The importance of hepatitis C as a risk factor for hepatocellular carcinoma in Guangxi. World J Gastroenterol. 2000;6:75.  [PubMed]  [DOI]  [Cited in This Article: ]
20.  Fan J, Wu ZQ, Tang ZY, Zhou J, Qiu SJ, Ma ZC, Zhou XD, Ye SL. Multimodality treatment in hepatocellular carcinoma patients with tumor thrombi in portal vein. World J Gastroenterol. 2001;7:28-32.  [PubMed]  [DOI]  [Cited in This Article: ]
21.  Huang KS, Lin M, Cheng GF. Anti-inflammatory tetramers of resveratrol from the roots of Vitis amurensis and the conformations of the seven-membered ring in some oligostilbenes. Phytochemistry. 2001;58:357-362.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 118]  [Cited by in F6Publishing: 88]  [Article Influence: 3.8]  [Reference Citation Analysis (0)]
22.  Surh YJ, Chun KS, Cha HH, Han SS, Keum YS, Park KK, Lee SS. Molecular mechanisms underlying chemopreventive activities of anti-inflammatory phytochemicals: down-regulation of COX-2 and iNOS through suppression of NF-kappa B activation. Mutat Res. 2001;480-481:243-268.  [PubMed]  [DOI]  [Cited in This Article: ]
23.  Olas B, Wachowicz B, Saluk-Juszczak J, Zieliński T, Kaca W, Buczyński A. Antioxidant activity of resveratrol in endotoxin-stimulated blood platelets. Cell Biol Toxicol. 2001;17:117-125.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 46]  [Cited by in F6Publishing: 47]  [Article Influence: 2.1]  [Reference Citation Analysis (0)]
24.  Stojanović S, Sprinz H, Brede O. Efficiency and mechanism of the antioxidant action of trans-resveratrol and its analogues in the radical liposome oxidation. Arch Biochem Biophys. 2001;391:79-89.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 193]  [Cited by in F6Publishing: 180]  [Article Influence: 7.8]  [Reference Citation Analysis (0)]
25.  Wu JM, Wang ZR, Hsieh TC, Bruder JL, Zou JG, Huang YZ. Mechanism of cardioprotection by resveratrol, a phenolic antioxidant present in red wine (Review). Int J Mol Med. 2001;8:3-17.  [PubMed]  [DOI]  [Cited in This Article: ]
26.  Russo P, Tedesco I, Russo M, Russo GL, Venezia A, Cicala C. Effects of de-alcoholated red wine and its phenolic fractions on platelet aggregation. Nutr Metab Cardiovasc Dis. 2001;11:25-29.  [PubMed]  [DOI]  [Cited in This Article: ]
27.  Murcia MA, Martínez-Tomé M. Antioxidant activity of resveratrol compared with common food additives. J Food Prot. 2001;64:379-384.  [PubMed]  [DOI]  [Cited in This Article: ]
28.  Olas B, Zbikowska HM, Wachowicz B, Krajewski T, Buczyński A, Magnuszewska A. Inhibitory effect of resveratrol on free radical generation in blood platelets. Acta Biochim Pol. 1999;46:961-966.  [PubMed]  [DOI]  [Cited in This Article: ]
29.  Igura K, Ohta T, Kuroda Y, Kaji K. Resveratrol and quercetin inhibit angiogenesis in vitro. Cancer Lett. 2001;171:11-16.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 162]  [Cited by in F6Publishing: 167]  [Article Influence: 7.3]  [Reference Citation Analysis (0)]
30.  Gusman J, Malonne H, Atassi G. A reappraisal of the potential chemopreventive and chemotherapeutic properties of resveratrol. Carcinogenesis. 2001;22:1111-1117.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 288]  [Cited by in F6Publishing: 294]  [Article Influence: 12.8]  [Reference Citation Analysis (0)]
31.  Kimura Y, Okuda H. Resveratrol isolated from Polygonum cuspidatum root prevents tumor growth and metastasis to lung and tumor-induced neovascularization in Lewis lung carcinoma-bearing mice. J Nutr. 2001;131:1844-1849.  [PubMed]  [DOI]  [Cited in This Article: ]
32.  Yang CS, Landau JM, Huang MT, Newmark HL. Inhibition of carcinogenesis by dietary polyphenolic compounds. Annu Rev Nutr. 2001;21:381-406.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 866]  [Cited by in F6Publishing: 763]  [Article Influence: 33.2]  [Reference Citation Analysis (0)]
33.  Kozuki Y, Miura Y, Yagasaki K. Resveratrol suppresses hepatoma cell invasion independently of its anti-proliferative action. Cancer Lett. 2001;167:151-156.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 67]  [Cited by in F6Publishing: 73]  [Article Influence: 3.2]  [Reference Citation Analysis (0)]
34.  Nakagawa H, Kiyozuka Y, Uemura Y, Senzaki H, Shikata N, Hioki K, Tsubura A. Resveratrol inhibits human breast cancer cell growth and may mitigate the effect of linoleic acid, a potent breast cancer cell stimulator. J Cancer Res Clin Oncol. 2001;127:258-264.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 130]  [Cited by in F6Publishing: 133]  [Article Influence: 5.8]  [Reference Citation Analysis (0)]
35.  Mollerup S, Ovrebø S, Haugen A. Lung carcinogenesis: resveratrol modulates the expression of genes involved in the metabolism of PAH in human bronchial epithelial cells. Int J Cancer. 2001;92:18-25.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in F6Publishing: 2]  [Reference Citation Analysis (0)]
36.  MacCarrone M, Lorenzon T, Guerrieri P, Agrò AF. Resveratrol prevents apoptosis in K562 cells by inhibiting lipoxygenase and cyclooxygenase activity. Eur J Biochem. 1999;265:27-34.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 151]  [Cited by in F6Publishing: 136]  [Article Influence: 5.4]  [Reference Citation Analysis (0)]
37.  Jang M, Cai L, Udeani GO, Slowing KV, Thomas CF, Beecher CW, Fong HH, Farnsworth NR, Kinghorn AD, Mehta RG. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science. 1997;275:218-220.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 3633]  [Cited by in F6Publishing: 3322]  [Article Influence: 123.0]  [Reference Citation Analysis (0)]
38.  Fontecave M, Lepoivre M, Elleingand E, Gerez C, Guittet O. Resveratrol, a remarkable inhibitor of ribonucleotide reductase. FEBS Lett. 1998;421:277-279.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 295]  [Cited by in F6Publishing: 295]  [Article Influence: 11.3]  [Reference Citation Analysis (0)]
39.  Park JW, Choi YJ, Jang MA, Lee YS, Jun DY, Suh SI, Baek WK, Suh MH, Jin IN, Kwon TK. Chemopreventive agent resveratrol, a natural product derived from grapes, reversibly inhibits progression through S and G2 phases of the cell cycle in U937 cells. Cancer Lett. 2001;163:43-49.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 95]  [Cited by in F6Publishing: 97]  [Article Influence: 4.2]  [Reference Citation Analysis (0)]
40.  Ragione FD, Cucciolla V, Borriello A, Pietra VD, Racioppi L, Soldati G, Manna C, Galletti P, Zappia V. Resveratrol arrests the cell division cycle at S/G2 phase transition. Biochem Biophys Res Commun. 1998;250:53-58.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 197]  [Cited by in F6Publishing: 203]  [Article Influence: 7.8]  [Reference Citation Analysis (0)]
41.  Pervaiz S. Resveratrol--from the bottle to the bedside. Leuk Lymphoma. 2001;40:491-498.  [PubMed]  [DOI]  [Cited in This Article: ]
42.  Huang C, Ma WY, Goranson A, Dong Z. Resveratrol suppresses cell transformation and induces apoptosis through a p53-dependent pathway. Carcinogenesis. 1999;20:237-242.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 261]  [Cited by in F6Publishing: 263]  [Article Influence: 10.5]  [Reference Citation Analysis (0)]
43.  Dörrie J, Gerauer H, Wachter Y, Zunino SJ. Resveratrol induces extensive apoptosis by depolarizing mitochondrial membranes and activating caspase-9 in acute lymphoblastic leukemia cells. Cancer Res. 2001;61:4731-4739.  [PubMed]  [DOI]  [Cited in This Article: ]
44.  She QB, Bode AM, Ma WY, Chen NY, Dong Z. Resveratrol-induced activation of p53 and apoptosis is mediated by extracellular-signal-regulated protein kinases and p38 kinase. Cancer Res. 2001;61:1604-1610.  [PubMed]  [DOI]  [Cited in This Article: ]
45.  Tsan MF, White JE, Maheshwari JG, Bremner TA, Sacco J. Resveratrol induces Fas signalling-independent apoptosis in THP-1 human monocytic leukaemia cells. Br J Haematol. 2000;109:405-412.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 70]  [Cited by in F6Publishing: 72]  [Article Influence: 3.0]  [Reference Citation Analysis (0)]
46.  Szende B, Tyihák E, Király-Véghely Z. Dose-dependent effect of resveratrol on proliferation and apoptosis in endothelial and tumor cell cultures. Exp Mol Med. 2000;32:88-92.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 90]  [Cited by in F6Publishing: 94]  [Article Influence: 3.9]  [Reference Citation Analysis (0)]
47.  Bernhard D, Tinhofer I, Tonko M, Hübl H, Ausserlechner MJ, Greil R, Kofler R, Csordas A. Resveratrol causes arrest in the S-phase prior to Fas-independent apoptosis in CEM-C7H2 acute leukemia cells. Cell Death Differ. 2000;7:834-842.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 127]  [Cited by in F6Publishing: 129]  [Article Influence: 5.4]  [Reference Citation Analysis (0)]
48.  Tian XM, Zhang ZX. The anticancer activity of resveratrol on im-plant ed tumor of HepG2 in nude mice. Shijie Huaren Xiaohua Zazhi. 2001;9:161-164.  [PubMed]  [DOI]  [Cited in This Article: ]