Edited by Ma JY
Published online Jul 15, 2003. doi: 10.3748/wjg.v9.i7.1474
Revised: December 4, 2002
Accepted: December 7, 2002
Published online: July 15, 2003
AIM: To study the antitumor and immunomodulatory activity of resveratrol on experimentally implanted tumor of H22 in Balb/c mice
METHODS: The cytotoxicity of peritoneal macrophages (MΦ) against H22 cells was measured by the radioactivity of [3H]TdR assay, mice with H22 tumor were injected with different concentrations of resveratrol, and the inhibitory rates were calculated and IgG contents were determined by single immunodiffusion method. the plaque forming cell (PFC) was measured by improved Cunningham method, the levels of serum tumor necrosis factor-α (TNF-α) were measured by cytotoxic assay against L929 cells.
RESULTS: Resveratrol 2.5 mg•l-1, 5.0 mg•l-1, 10.0 mg•l-1, 20.0 mg•l-1 (E:T = 10:1, 20:1) promoted the cytotoxicity of MΦagainst H22 cells. Resveratrol ip 500 mg•kg-1, 1000 mg•kg-1 and 1500 mg•kg-1 could curb the growth of the implanted tumor of H22 in mice. The inhibitory rates were 31.5%, 45.6% and 48.7%, respectively (P < 0.05), which could raise the level of serum IgG and PFC response to sheep red blood cell. Resveratrol 1000 mg•kg-1 and 1500 mg•kg-1 and BCG 200 mg•kg-1 ip could increase the production of serum TNF-α in mice H22 tumor. However, the effect of resveratrol was insignificant (P > 0.05).
CONCLUSION: Resveratrol could inhibit the growth of H22 tumor in Balb/c mice. The antitumor effect of resveratrol might be related to directly inhibiting the growth of H22 cells and indirectly inhibiting its potential effect on nonspecific host immunomodulatory activity.
- Citation: Liu HS, Pan CE, Yang W, Liu XM. Antitumor and immunomodulatory activity of resveratrol on experimentally implanted tumor of H22 in Balb/c mice. World J Gastroenterol 2003; 9(7): 1474-1476
- URL: https://www.wjgnet.com/1007-9327/full/v9/i7/1474.htm
- DOI: https://dx.doi.org/10.3748/wjg.v9.i7.1474
Recently, considerable attention has been focused on identifying naturally occurring chemopreventive substances capable of inhibiting, retarding, or reversing the multi-stage carcinogenesis. Resveratrol (3, 5, 4'-trihydroxy-trans-stilbene), a phytoalexin found in grapes and other dietary and medicinal plants, has been shown to have anti-inflammatory, antioxidant and antitumor activities[1-7]. Many of these beneficial effects of resveratrol require participation of the cells of the immune system. However, the effect of resveratrol on the development of immuological responses remains unknown.
In the present study, the antitumor activity and immunomodulatory actions of resveratrol, including MΦagainst H22 cells, serum IgG and the plaque forming cells and tumor necrosis factor (TNF-α) content in Balb/C mice with experimentally implanted tumor of H22 were investigated.
Resveratrol, MTT, IPS and dimethylsulfoxide (DMSO) were purchased from SGMA Co. Mouse hepatocellular carcinoma cells H22, L929 and sheep red blood cell (SRBC) were kindly supplied by Cheng Wei (Center of Molecular Biology, First Affiliated Hospital, Xi'an Jiaotong University). Cells were subcultured in RPMI 1640 (Gibco) which was supplemented with 10% fetal bovine serum, penicillin (100 IU•mL-1) and streptomycin (100 mg•l-1). Stock solution of resveratrol was made in dimethylsulfoxide (DMSO) at a concentration of 10 g•l-1. Working dilutions were directly made in the tissue culture medium. [3H]TdR was purchased from Shanghai Nuclear Research Institute. IL test kit and LPS were purchased from Gibco Co. Balb/C mice, 2.5 month old, weighing 202 g, were purchased from the Animal Center, Xi'an Jiaotong University.
Effect of resveratrol on cytotoxicity of peritoneal macrophages (MΦ) against H22 cells MΦ was collected from the peritoneal cavity of Balb/c mice 3 d after ip 10% sheep red blood cells (SRBC, 1 mL/mouse). The cells were washed twice and resuspended in RPMI 1640 culture medium. H22 cells were cultured for 12 h, and 100 μL MΦ suspension and different concentrations of resveratrol were added to each well of 96-well plates at a ratio of effectors: target (E:T) cell 10:1 or 25:1. After cultured for 24 h, each well was added with [3H]TdR (9.3 kBq/well), and then was incubated for another 6 h. Cells were placed onto the glass fiber filter and [3H]TdR incorporation was determined by liquid scintillation. The cytotoxicity was calculated with the following formula: the cytotoxicity of MΦ = (control-treatment)/control × 100% (dpm).
Anti-tumor activity of resveratrol and its effect on serum antibody IgG, plaque forming cells (PFC) in Balb/C mice with implanted tumor of H22 Mouse ascites (including 2 × 105 cells) were injected into the right axilla of 40 Balb/c mice. On the second day, 40 Balb/c mice were divided into 4 groups randomly, and then were injected with resveratrol at a dose of 500 mg•kg-1, 1000 mg•kg-1, 1500 mg•kg-1 and normal saline for 10 d. Mice were sensitized to ip SRBC (3 × 107 cells). After 4 d, the mice were bled to obtain serum for IgG investigation. At the same time, spleens were excised for PFC counting. IgG contents were determined by single immunodiffusion method. PFC was measured by modified Cunningham method.
Effect of resveratrol on serum tumor necrosis factor alpha (TNF-α) production induced by LPS in Balb/c mice Ascites cells of 2 × 105 were injected into the Balb/c mice. Resveratrol at a dose a 500 mg•kg-1, 1000 mg•kg-1 and 1500 mg•kg-1 was injected for 10 d, and BCG of 200 mg•kg-1 as a positive control agent was injected ip on d 1.On d 11, 90 min after ip LPS of 0.1 mg•kg-1, the mice were exsanguinated. Blood was centrifuged (400 × g, 10 min). The levels of serum TNF-α were measured by cytotoxic assay against L929 cells as described previously. The TNF-α activity was calculated with the following formula: cytotoxicity = (Acontrol - Atest)/Acontrol× 100%.
Resveratrol at 2.5 mg•l-1 could decrease the cytotoxicity of MΦagainst H22 cells (P > 0.05). Resveratrol at 12.5 mg•l-1, 5.0 mg•l-1, 10.0 mg•l-1 could enhance insignificantly the cytotoxicity of MΦ against H22 cells (P > 0.05) concentration-dependently, However, resveratrol at 20.0 mg•l-1 could raise significantly the cytotoxicity of MΦ against H22 cells (P < 0.05) (Table 1).
Resveratrol ip at a dose 500 mg•kg-1, 1000 mg•kg-1 and 1500 mg•kg-1 could significantly curb the growth of implanted tumor of H22 in mice. The inhibitory rates were 31.5%, 45.6% and 48.7%, respectively (P < 0.05, Table 2).
The result also showed that the immunity of mice with tumor could be more significantly inhibited than that of normal mice, and resveratrol ip could raise the level of serum LgG and number of PFC in Balb/c mice with implanted tumor of H22 in vivo. Resveratrol, however, could insignificantly increase the immunity of mice with tumor (P > 0.05, Table 3).
The ability of TNF-α production of mice with H22 tumor was significantly stronger than that of normal mice. Furthermore, the group of control and BCG at 200 mg•kg-1 ip had an increase in the at production of serum TNF-α in mice with H22 tumor (P < 0.05), but resveratrol at a dose of 500 mg•kg-1, 1000 mg•kg-1 and 1500 mg•kg-1 had less effect on mice with H22 tumor (P > 0.05, Table 4).
Resveratrol is a phytopolyphenol isolated from the seeds and skin of grapes. Recent studies have indicated that resveratrol can block the process of multistage carcinogenesis, namely, tumor initiation, promotion and progression. Resveratrol can also reduce the risk of cardiovascular diseases in man. These activities have been identified by some authors[8-13]. Roberto et al have shown that PBMC exposure to resveratrol produced a biphasic effect on the anti-CD3/anti-CD28-induced development of both IFN-y-IL-2 and IL4-producing CD8+ and CD4+T cells, with stimulation at low resveratrol concentrations and suppression at high concentrations. Similarly, the compound was found to induce a significant enhancement at low concentrations and suppression at high concentrations of both CTL and NK cell cytotoxic activity. Gao et al found that mitogen-, IL-2- or alloantigen-induced proliferation of splenic lymphocytes, induction of cytotoxic T lymphocytes (CTLs) and lymphokine activated killer (LAK) cells, and production of the cytokine interferon (IFN)-y, interleukin (IL)-2, tumor necrosis factor(TNF)-α were significantly suppressed at 25-50 μM resveratrol, but in some cases mitogen/IL-2-induced production and CTL generation were enhanced following pretreatment of cells with resveratrol. The effects of resveratrol on immune function of mice in vivo have not been reported yet.
Our results indicate that resveratrol of 2.5 mg•l-1 could decrease the cytotoxicity of MΦagainst H22 cells (P > 0.05).Resveratrol of 2.5 mg•l-1, 5.0 mg•l-1 and 10.0 mg•l-1 could insignificantly enhance the cytotoxicity of MΦagainst H22 cells concentration-dependently (P > 0.05). However, resveratrol of 20.0 mg•l-1 could raise significantly the cytotoxicity of MΦagainst H22 cells (P < 0.05). So, resveratrol could alone affect the [3H]TDR uptake by H22 cells in vitro, suggesting that the antitumor action of resveratrol had a direct cytotoxic effect. This result is coincident with the previous studies[16-18]. Resveratrol ip could insignificantly increase the host nonspecific immunological defense of mice with H22 tumor, by raising the level of serum IgG and TNF-α and the number of PFC (P > 0.05). In vivo resveratrol could also augment the cytotoxicity of peritoneal macrophages against H22 cells, and there was an insignificant difference compared with the control group (P > 0.05). Therefore, resveratrol could inhibit the growth of H22 cells in vivo, but it could not significantly enhance the host immune defense against tumor. Based on the results of the present study, it can be suggested that the antitumor activity of resveratrol might be due to direct cytotoxic/antiproliferative activity against tumor cells, but not to the augmentation of immune response against tumors. It has demonstrated that resveratrol inhibits cell proliferation, cell-mediated cytotoxicity, and cytokine production, at least in part through the inhibition of NF-kappa B activation. But the molecular mechanism by which resveratrol imparts cancer chemopreventive effects has not been clearly defined and further studies are needed.
|1.||Kris-Etherton PM, Hecker KD, Bonanome A, Coval SM, Binkoski AE, Hilpert KF, Griel AE, Etherton TD. Bioactive compounds in foods: their role in the prevention of cardiovascular disease and cancer. Am J Med. 2002;113 Suppl 9B:71S-88S. [PubMed]|
|2.||Park EJ, Pezzuto JM. Botanicals in cancer chemoprevention. Cancer Metastasis Rev. 2002;21:231-255. [PubMed] [DOI]|
|3.||Olas B, Wachowicz B, Saluk-Juszczak J, Zieliński T. Effect of resveratrol, a natural polyphenolic compound, on platelet activation induced by endotoxin or thrombin. Thromb Res. 2002;107:141-145. [PubMed] [DOI]|
|4.||Martinez J, Moreno JJ. Effect of resveratrol, a natural polyphenolic compound, on reactive oxygen species and prostaglandin production. Biochem Pharmacol. 2000;59:865-870. [PubMed] [DOI]|
|5.||Latruffe N, Delmas D, Jannin B, Cherkaoui Malki M, Passilly-Degrace P, Berlot JP. Molecular analysis on the chemopreventive properties of resveratrol, a plant polyphenol microcomponent. Int J Mol Med. 2002;10:755-760. [PubMed] [DOI]|
|6.||Ding XZ, Adrian TE. Resveratrol inhibits proliferation and induces apoptosis in human pancreatic cancer cells. Pancreas. 2002;25:e71-e76. [PubMed] [DOI]|
|7.||Cal C, Garban H, Jazirehi A, Yeh C, Mizutani Y, Bonavida B. Resveratrol and cancer: chemoprevention, apoptosis, and chemo-immunosensitizing activities. Curr Med Chem Anticancer Agents. 2003;3:77-93. [PubMed] [DOI]|
|8.||Asensi M, Medina I, Ortega A, Carretero J, Baño MC, Obrador E, Estrela JM. Inhibition of cancer growth by resveratrol is related to its low bioavailability. Free Radic Biol Med. 2002;33:387-398. [PubMed] [DOI]|
|9.||De Lédinghen V, Monvoisin A, Neaud V, Krisa S, Payrastre B, Bedin C, Desmoulière A, Bioulac-Sage P, Rosenbaum J. Trans-resveratrol, a grapevine-derived polyphenol, blocks hepatocyte growth factor-induced invasion of hepatocellular carcinoma cells. Int J Oncol. 2001;19:83-88. [PubMed] [DOI]|
|10.||Tian XM, Zhang ZX. The anticancer activity of resveratrol on implanted tumor of HepG2 in nude mice. Shijie Huaren Xiaohua Zazhi. 2001;9:161-164.|
|11.||Liu HS, Pan CE, Qi Y, Liu QG, Liu XM. Effect of resveratrol with 5-FU on growth of implanted H22 tumor in mice. Shijie Huaren Xiaohua Zazhi. 2002;10:32-35.|
|12.||Sun ZJ, Pan CE, Liu HS, Wang GJ. Anti-hepatoma activity of resveratrol in vitro. World J Gastroenterol. 2002;8:79-81. [PubMed]|
|13.||Zhou HB, Yan Y, Sun YN, Zhu JR. Resveratrol induces apoptosis in human esophageal carcinoma cells. World J Gastroenterol. 2003;9:408-411. [PubMed]|
|14.||Falchetti R, Fuggetta MP, Lanzilli G, Tricarico M, Ravagnan G. Effects of resveratrol on human immune cell function. Life Sci. 2001;70:81-96. [PubMed] [DOI]|
|15.||Gao X, Xu YX, Janakiraman N, Chapman RA, Gautam SC. Immunomodulatory activity of resveratrol: suppression of lymphocyte proliferation, development of cell-mediated cytotoxicity, and cytokine production. Biochem Pharmacol. 2001;62:1299-1308. [PubMed] [DOI]|
|16.||Delmas D, Jannin B, Cherkaoui Malki M, Latruffe N. Inhibitory effect of resveratrol on the proliferation of human and rat hepatic derived cell lines. Oncol Rep. 2000;7:847-852. [PubMed] [DOI]|
|17.||Bråkenhielm E, Cao R, Cao Y. Suppression of angiogenesis, tumor growth, and wound healing by resveratrol, a natural compound in red wine and grapes. FASEB J. 2001;15:1798-1800. [PubMed] [DOI]|