Thalidomide And Oxaliplatin And 5-fluorouracil For In HepG2 Cells Research VEGF, Caspase-3 Gene Expression In Vitro Tests

HCC(hepatocellular carcinoma, HCC) is the most common type of malignant tumor in china which greatly threatened human health. It is also one of the leading causes of cancer deaths worldwide. Patient’s mortality rate remains high due to its high grade malignancy, easy recurrence and metastasis, strong invasiveness, rapid progression and insensitive to radiotherapy or chemotherapy. 5-fluorouracil combined with oxaliplatin therapy is currently the best option, but the effect is still unsatisfying and the incidence of recent adverse events is high. Therefore, developing new antitumor therapy strategy according to the characteristics of liver tumor cells is becoming a hot spot. Thalidomide(thalidomide, ThD) can inhibit angiogenesis, thus it has been applied in a series of cancers treatment. Whether Thalidomide combined with oxaliplatin and 5-fluorouracil can inhibit the growth of liver tumor cells remain to be confirmed by basic research. Purpose:In this study, we built an in vitro cultured model of human hepatoma HepG2 cells and intervened with three different strategies. MTT assay was used to observe the effects of the drugs on the proliferation of liver tumor cells. FACS was used to detect the apoptosis of liver tumor cells. Western Blot assay was applied to detect the expression of apoptotic factor of VEGF, Caspase-3 by liver tumor cells in different groups. Hereby we can compare the efficacy of thalidomide combined with oxaliplatin and 5-fluorouracil, and to explore possible mechanisms. The results of this study may help to provide new theoretical supports and ideas for clinical application to the treatment of liver cancer. Method:1 Cell source: HepG2 human hepatoma cell line was purchased from Shanghai Biological Technology Co., Ltd. US2 Groups: control group, the thalidomide group, oxaliplatin combined with 5-fluorouracil group, thalidomide combined with oxaliplatin and 5-fluorouracil group.3 MTT assay was used to detect the proliferation activity of HepG2 human hepatoma cells in different groups.4 FACS were used to detect the effect of the drugs on the HepG2 human hepatoma cell cycle.5 Western blot assay was used to detect the expression of Caspase-3 protein by Hep G2 human hepatoma cells.6 Western blot assay was used to detect the expression of VEGF protein by Hep G2 human hepatoma cells.7 Statistical analysis: SPSS17.0 was used and all the data were represented by Mean ± SD. ANOVA analysis was used to compare the data from different group, and SNK analysis was used to compare the data between two groups. P<0.01 indicates the difference has statistical significance. Results:1 The effects of halidomide combined with oxaliplatin and 5-fluorouracil on the proliferation of HepG2 cells: Firstly, we screened IC50 of each drug on 48h: Thalidomide was 0.1mmol/L,5-fluorouracil was 40umol/L and oxaliplatin was 5umol/L. Compared with the control group, the OD values in three treatment groups were decreased and the proliferation of HepG2 cells were inhibited. The differences had statistical significance(P <0.01). In three treatment groups, the OD values in the group of thalidomide combined with 5-fluorouracil and the group of oxaliplatin were the lowest, which suggest the two strategies inhibited the proliferation rate of HepG2 cells effectively.(The comparison among the four groups, P <0.001, the differences have statistical significance).2 The effects of Thalidomide combined with oxaliplatin and 5-fluorouracil on the cycle of Hep G2 cells. The HepG2 cells were in G0 / G1 phase on 48 h after transfection in all four groups. The percentage of apoptotic increased follow the consequence of the control group, the thalidomide group, the oxaliplatin combined with 5-fluorouracil group, the thalidomide combined with oxaliplatin platinum and the 5-fluorouracil group(P <0.05),but there had no such inclination in the S and G2 / M phase, which suggesting that the combing use of Thalidomide with oxaliplatin and 5- fluorouracil gradually increased the apoptosis of tumor cells, and their cell cycles were stagnated in G0/G1 phase.3 The effects of thalidomide combined with oxaliplatin and 5-fluorouracil on the expression of Caspase-3 by HepG2 cells. Compared with the control group, the expression of Caspase-3 by HepG2 cells in three treatment groups were increased, especially the group of thalidomide combined with oxaliplatin and the group of 5-fluorouracil, which suggest the combing use of the drugs gradually improve the expression of Caspase-3(P <0.001, the differences have statistical significance).4 The effects of thalidomide combined with oxaliplatin and 5-fluorouracil on the expression of VEGF by HepG2 cells. Compared with the control group, the expression of VEGF by HepG2 cells in three treatment groups were decreased, especially the group of thalidomide combined with oxaliplatin and the group of 5-fluorouracil, which suggest the combing use of the drugs gradually prevent the expression of Caspase-3(P <0.001, the differences have statistical significance). Conclusion:Thalidomide combined with oxaliplatin and 5-fluorouracil effectively promotes the inhibition of the drugs to the proliferation of HepG2 cells. HepG2 cells’ growth was stagnated at the G1 phase. The apoptosis factor of Caspase-3 secreted by HepG2 cells were up-regulated and the secretion of VEGF by HepG2 cells were downregulated, hereby promoting the apoptosis of tumor cells.