Study On The Antiproliferative Action And Mechanism Of Thalidomide In Human Hepatocellular Carcinoma Cells

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world which causes 25 thousand people die every year. About 40 percent of the total occurs in China. The etiological factors and pathogenesis can not be completely elucidated now. HCC has a very poor prognosis. It is about 25% to 29% that patients could survive 5 years including those performed resection. And moreover, we haven't effective chemotherapeutics and precautions against HCC, especially available chemopreventions and treating targets.The growth and metastasis of malignant tumors are associated with angiogenesis. Many experiments and studies have proved that thalidomide plays an important role in angiogenesis, expression of molecules involved in transcribes and adhesion and regulating some cytokines. Therefore, thalidomide could inhibit the growth, infiltrating and metastasis of cancer cells.ObjectiveCurrent research focuses on agents targeting molecular pathways involved in tumor growth and angiogenesis. This study investigated the effects of thalidomide, an inhibitor of vascular endothelial growth factor (VEGF), on proliferation and apoptosis in human hepatoma cell lines HepG2 in vitro and the interactions between thalidomide and the most common chemotherapy agent—adriamycin to inhibit tumor growth and induce apoptosis. The possible molecular mechanisms of thalidomide and its synergetic effects with adriamycin were also analyzed..MethodsHuman hepatoma cell lines HepG2 were grown in RPMI1640 with 10% newborn calf serum and penicillin-streptomycin. Thalidomide and adriamycin, alone or in combination, were given in vitro to HepG2 which were randomly divided into 4 groups: untreated control group; adriamycin group (1.0 mg'L~-1); thalidomide group (50, 100, 200, 400μg/ml); drug combination group (thalidomide 200μg/ml + adriamycin 1.0 mg'L~-1). After HepG2 treated for 12, 24, 48, 72 hours as described above, the effects of treatments were studied by the evaluation of cytotoxicity with MTT-test, apoptosis and cell cycle with flow cytometry, modulation of gene expression of PPARγ, Survivin, COX-2 immunohistochemistry, as well as the expression of vascular endothelial growth factor by RT-PCR. Statistical analysis: All experiments were done in triplicate and repeated at least twice. Data were expressed as mean FSE and analyzed by Student's t test or ANOVA followed by the Tukey's multiple comparison. The level of significance was P＜0.05.ResultsThe results of MTl-test showed that thalidomide could inhibit the proliferation and induce apoptosis of HepG2 cells in vitro as an time-and-dose-dependent manner. The results of flow cytometry showed that the apoptotic index (AI) of thalidomide group on HepG2 cell line were significantly higher than that of adriamycin group in vitro. Thalidomide 200μg/ml group showed the best treating effect on the AI of HepG2 cell line after treating 48 hours. There was no statistical significance of the difference of the proliferation index (PI) among every two groups on HepG2 cell lines. The results of immunohistochemical technique showed that thalidomide could up-regulate the expression of PPARγ, and down-regulate the expression of COX-2, Survivin. Thalidomide 200μg/ml group showed the best treating effect on HepG2 cell lines after treating 48 hours. After treated with thalidomide for 48 hours, the HepG2 cells displaied down-regulated expression of VEGFmRNA detected with RT-PCR method. The effect showed the VEGFmRNA expression of thalidomide 100μg/ml group was higher than that of thalidomide 200μg/ml group. The inhibitive effect of thalidomide combined with adriamycin on HepG2 cell lines was significantly higher than that of thalidomide or adriamycin alone in vitro.Conclusions1,Thalidomide inhibits the proliferation and induces apoptosis of HepG2 cell lines in vitro as an dose-and-time-dependent manner in extent.2,Thalidomide acts synergistically with the most common chemotherapy agent-adriamycin to inhibit tumor growth and induct HepG2 cell lines apoptosis in vitro.3,The possible molecular mechanisms of thalidomide antitumor maybe increase PPARγ, expression, deregulate modulates gene expression including COX-2, Survivin,VEGF and induce apoptosis in HepG2 cell lines.