Experimental Study Of The Anti-tumor Effect And Its Mechanisms Of Metformin Combined With Gemcitabine On Human Pancreatic Cancer Cell Line CFPAC-1

Part I: Effect and mechanism of metformin on human pancreaticcancer cell line CFPAC-1Objective: To investigate the effect of metformin on the proliferation and apoptosis ofhuman pancreatic cancer cell line CFPAC-1in vitro, and to appraise its possiblemechanisms.Methods: Human pancreatic cancer cell line CFPAC-1were cultured in vitro, andwere treated with metformin at different concentrations (0,1,2.5,5,10,20,40,60mmol/L) andat different time points(24h,48h and72h). Cell proliferation was evaluated by CCK-8, andinhibition ratios were calculated in all groups and suitable IC50of metformin were workedout by calculation; with using different concentrations metformin (0,10,20,40mmol/L) todeal with CFPAC-1by48h, cell cycle was determined by flow cytometry, apoptosis wasdetermined by AnnexinV/PI double staining method; and the expression of FASmRNA,Cyclin D1mRNA,Bcl-xl mRNA,Bax mRNA,Survivin mRNA,Caspase-3mRNAwere detected by RT-PCR; and Western blot was used to detect the expression ofP-AMPK,FAS,Cyclin D1,Bcl-xl,Bax,Survivin and Caspase-3.Results：(1) Metformin could inhibit the proliferation of the human pancreatic cancercell line CFPAC-1in a time and dose dependent manner (P<0.05).(2) The early stageapoptosis and cyclomorphosis of CFPAC-1cells was detected by flow cytometry andAnnexinV/PI double staining method, after they were incubated48hours with metformin.With the increase of Met concentration, ratio of G0/G1phase increased from(42.89±1.02)%to (65.98±0.27)%（,P<0.05）. The early stage apoptosis rate of CFPAC-1cells was (13.77±1.31)%when they were incubated with10mmol/L Met and the rate was (32.97±3.19)%with40mmol/L Met. Appearantly, apoptosis rate was positively correlated to Metconcentration（P<0.05）.(3)The expression of FAS mRNA,Cyclin D1mRNA,Bcl-xlmRNA,Survivin mRNA decreased with concentration-dependence after CFPAC-1cellswere incubated48hours with Met, which was detected by RT-PCR. Otherwise, Theexpression of Bax mRNA and Caspase-3mRNA increased with concentration-dependence,comparisons of all Met groups and control group have significant difference（P<0.05）.(4)The expression of P-AMPK was up-regulated, the expression of FAS,CyclinD1,Bcl-xl,Survivin were down-regulated and Bax,Caspase-3were up-regulated afterCFPAC-1cells were incubated48hours with Met, which was detected by Western Blot.Conclusion：Metformin could inhabit the growth of human pancreatic cancer cell lineCFPAC-1in a dose-and-time dependent manner; and its anti-proliferation mechanismmainly by the activation of AMPK pathway, influencing the cell metabolism bydown-regulating FAS, blocking the cell cycle at G0/G1by down-regulating Cyclin D1; aswell as inducing early stage apoptosis. Part Ⅱ: Effect and mechanism of Met in combination with GEM onhuman pancreatic cancer cell line CFPAC-1Objective: To investigate the effect and mechanism of Met in combination with GEMon human pancreatic cancer cell line CFPAC-1.Methods: Met, GEM, were tested alone and in combination for their abilities toinhibit CFPAC-1cell line proliferation by CCK-8. The early apoptosis of CFPAC-1cellline were detected by AnnexinV/PI double staining method, and the expression ofBcl-xl,Bax,Survivin and Caspase-3mRNA were detected by Semi-quantitative RT-PCR. Inaddition, the content of Bcl-xl,Bax,Survivin and Caspase-3proteins were evaluated byWestern Blot.Result: The CFPAC-1cell’s proliferation could be effectivly inhibited by Met or GEM; and the inhibition effect were more significantly while treated by Met combinatedwith GEM. The early stage apoptosis rate of CFPAC-1cells was (24.53±2.18)%when theywere treated with20mmol/L Met at48hr, and was (22.37±1.61)%when treated with5μmol/L of GEM, and was(52.07±2.81)%when Met combined with GEM. The expressionof Bcl-xl,Survivin mRNA and proteins were down-regulated, and Bax,Caspase-3mRNAand proteins levels were up-regulated in either Met,GEM or combination group.Comparisons of treated groups and control group have significant difference (P<0.05) andcomparisons of combination groups and single drug group have also significant difference(P<0.05).Conclusion：Metformin could obviously inhibit human pancreatic cancer cell lineCFPAC-1proliferation in coordination with Gemcitabine. Its major mechanisms maybepromote apoptosis of cells.