Effect Of Melatonin On Cellular Growth And Angiogenesis Of Human Pancreatic Carcinoma Cell Lines SW1990

Objective: To study for melatonin's effect on human pancreatic cancer cell line SW1990 cells'growth and angiogenesis in vitro,and to explore its possible mechanisms.Methods: After using different concentrations'melatonin (0.1,0.5,1.0,2.5,5.0mmol/L) to deal with pancreatic cancer cells SW1990,OD values were evaluated by MTT , and inhibition ratioes were calculated in all groups and suitable IC50 of melatonin were worked out by calculatation. With using different concentrations'melatonin (1.0,2.5,5.0mmol/L) to deal with SW1990, the apoptosis ratioes and changes of cell cycle were detected by flow cytometry and AnnexinV/PI fluos stainging, and the expression of Bcl-2 and Bax proteinum were detected by Western Blot in vitro,and the expression of VEGFmRNA were detected by RT-PCR,and the effect of melatonin on chorioallantoic membrane was observed.Results:(1)Melatonin 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 and AnnexinV/PI fluos stainging after they were incubated 48 hours with Melatonin.,The early stage apoptosis rate of SW1990 cells was (24.5±2.14)% when they were incubated with 1.0mmol/L Melatonin and the rate was (44.76±3.24)% with 5.0mmol/L Melatonin. Appearantly, apoptosis rate was positively correlated to MT concentration. With the increase of MT concentration, ratio of G0/G1 phase increased homologously and S phase declined homologously(P<0.05).(3)cell mainly arrested in stillness by flow cytometry and AnnexinV/PI (P<0.05).The expression of Bcl-2 was downregulated and the expression of bax was upregulated significantly and the ratio of Bcl-2/Bax decreased (P<0.05) after SW1990 cells were incubated 48 hours with Melatonin, which was detected by Western Blot.(4)The expression of VEGFmRNA decreased with concentration-dependence after SW1990 cells were incubated 48 hours with Melatonin, which was detected by RT-PCR. Comparisons of all Melatonin groups and control group have significant difference(P<0.05).(5)With the increase of Melatonin, the number of the second and the third grade blood vessel was decreased gradually in all Melatonin groups(1.0mmol/L,2.5mmol /L,5.0 mmol/L), which was detected in the study of chorioallantoic membrane ( CAM). Comparisons of the number of the third grade blood vessel in all Melatonin groups and control group have significant difference(P<0.01). The morphous of blood vessels and their branches were intact in the control group, but they became thinner after treated with melatonin,and the chorioallantoic membranes were hydropic.Conclusion:Melatonin could inhabit the growth of human pancreatic cancer cell line SW1990 in a dose-and-time dependent manner, and its mechanisms may directly inhibit tumor cells to proliferate and arrest cell cycle in stillness stage and induce early stage apoptosis and inhibite angiogenesis.Part two: Effect and mechanism of MT in combination with GEM on pancreatic cancer SW1990 cellsObjective: To investigate the effect and mechanism of action in pancreatic cancer cells treated by melatonin in combination with GEM.Methods: SW1990 cells were cultivated with 1.5mmol/L of melatonin 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 melatonin and gemcitabine could inhibit effectivly SW1990 cells'proliferation. Melatonin combination with gemcitabine had synergistic effect and their combination could increase both early stage apoptosis obviously and advanced stage apoptosis,and the expression of Bcl-2 was downragulated ,and the expression of Bax was upragulated. Comparisons of treated groups and control group have significant difference(P<0.05)and comparisons of single drug groups and combination group have also significant difference(P<0.05).Conclusion:The combination of Melatonin and Gemcitabine could obviously inhibit pancreatic cancer SW1990 cells proliferation, in a dose-and-time dependent manner. Its mechanisms maybe promote early stage apoptosis of cells.