Effect Of Thalidomide On Cellular Growth And Angiogenesis Of Human Pancreatic Carcinoma Cell Line SW1990

Part One: The studies of thalidomide on cellular growth and angiogenesis of human pancreatic carcinoma cell line SW1990.Objective: To study the effect of thalidomide on cellular growth and angiogenesis of human pancreatic carcinoma cell line SW1990 in vitro,and to explore its possible mechanisms.Methods: After using different concentrations'thalidomide (3.125,6.25,12.5,25,50,100,200 and 400μg/ml) to deal with pancreatic carcinoma SW1990 cells,OD values were evaluated by MTT , and inhibition ratioes were calculated in all groups and suitable IC50 of thalidomide were worked out by calculatation. With using different concentrations'thalidomide (50,100 and 200μg/ml) to deal with SW1990, the apoptosis ratioes and changes of cell cycle were detected by flow cytometry. And the expression of Bcl-2-mRNA,Bax-mRNA and VEGF-mRNA were detected by RT-PCR. And the levels of Bcl-2,Bax and VEGF proteins were detected by Western Blot.Results:(1)Thalidomide could inhibit the growth of the human pancreatic carcinoma cell line SW1990, and the inhibitory effect had dose-and-time dependence(P<0.05). (2)The early stage apoptosis of SW1990 cells was detected by flow cytometry after they were incubated 48 hours with thalidomide.,The early stage apoptosis rate of SW1990 cells was (8.77±3.30)% when they were incubated with 50μg/ml thalidomide, and the rate was (29.43±2.25)% with 200μg/ml thalidomide.Apparently, apoptosis rate was positively correlated to the concentration of thalidomide. With the increase of the concentration of thalidomide, ratio of G0/G1 phase increased homologously, it was (46.55±2.44)% with 50μg/ml thalidomide and (58.83±2.33)% with 200μg/ml thalidomide. And S phase declined homologously(P<0.05).(3)The expression of Bcl-2-mRNA and VEGF-mRNA was downregulated and the expression of Bax-mRNA was upregulated significantly in dose dependence after SW1990 cells were incubated 48 hours with thalidomide, which was detected by RT-PCR(.4)The levels of Bcl-2 and VEGF proteins decreased and the level of Bax protein was upregulated significantly in dose dependence after SW1990 cells were incubated 48 hours with thalidomide, which was detected by Western Blot. Comparisons of all thalidomide groups and control group have significant difference(P<0.05). Conclusion:Thalidomide could inhabit the growth of human pancreatic carcinoma cell line SW1990 in a dose-and-time dependent manner, and its mechanisms may directly inhibit tumor cells proliferation, arrest cell cycle in stillness stage, induce early stage apoptosis, and inhibite angiogenesis.Part two: Effect of THD in combination with GEM on human pancreatic carcinoma cell line SW1990Objective: To investigate the effect and mechanism of action in pancreatic carcinoma cell line SW1990 treated by THD in combination with GEM.Methods: SW1990 cells were cultivated with 50μg/ml of thalidomide and 20μmol/L of gemcitabine. OD values were detected by MTT colormetric assay and their inhibition ratios were calculated and early apoptosis of them were detected by AnnexinV/PI double staining method and the expression of Bcl-2mRNA and BaxmRNA were detected by Semi-quantitative RT-PCR.Result: Both thalidomide and gemcitabine could inhibit effectivly SW1990 cells'proliferation. Thalidomide combined with gemcitabine had synergistic effect. The early stage apoptosis rate of SW1990 cells was (8.7±1.87)% when they were incubated with 50μg/ml thalidomide, it was (27.73±2.40)% when 20μmol/L of gemcitabine, it was(39.4±3.24)% when thalidomide combined with gemcitabine.The level of Bcl-2mRNA was downregulated, and the expression of Bax-mRNA was upregulated,which was detected by RT-PCR. Comparisons of treated groups and control group, Bcl-2mRNA or Bax-mRNA have respectively significant difference(P<0.05), and comparisons of single drug groups and combination group, they have also respectively significant difference(P<0.05). The level of Bcl-2 protein was downregulated, and the level of Bax protein was upregulated,which was detected by Western Blot. Comparisons of treated groups and control group, Bcl-2 or Bax protein have respectively significant difference(P<0.05), and comparisons of single drug groups and combination group, they have also respectively significant difference(P<0.05).Conclusion:The combination of thalidomide and gemcitabine could obviously inhibit the proliferation of human pancreatic carcinoma SW1990 cells, its mechanisms maybe promote early stage apoptosis.