Effects of metformin on gemcitabine resistance and Hippo/YAP pathway in pancreatic cancer cells
Objective To analyze the impact of metformin(Met)on the chemotherapy sensitivity of gemcitabine(GEM)in CFPAC-1 cells of pancreatic cancer,and explore the possible mechanism.Methods Human pancreatic cancer CFPAC-1 cells and gemcitabine-resistant CFPAC-1/gemcitabine cells were cultured in vitro.The MTT method was used to detect the effects of different concentrations(0,1.0,2.5,5.0,10.0,20.0,40.0 mmol/L)of metformin and different concentrations(0,50,100,150,200,250 μmol/L)of gemcitabine on the survival rates of CFPAC-1 cells and CFPAC-1/gemcitabine cells.The metformin concentration without cytotoxicity was screened and the half inhibitory concentration(IC50)of gemcitabine was calculated.The MTT method was used to detect the inhibitory effect of 2.5 mmol/L metformin on the survival rate of CFPAC-1 cells and CFPAC-1/gemcitabine cells.The effects of metformin and gemcitabine on the survival rate of CFPAC-1 cells and CFPAC-1/gemcitabine cells were analyzed by flow cytometry.The IC50 of metformin and the reversal fold of drug resistance were calculated.The flow cytometry AnnexinV-FITC/PI double staining method was used to detect the effects of gemcitabine alone and 2.5 mmol/L metformin+gemcitabine on the apoptosis rate of CFPAC-1 cells and CFPAC-1/gemcitabine cells with the IC50 of gemcitabine as the drug concentration.The relative expression levels of Hippo/YAP pathway-related proteins(MST1/2,LAST1/2,YAP,p-YAP)were detected by immunoblotting(WB).Results Compared with 0 mmol/L Met,the survival rates of CFPAC-1 and CFPAC-1/GEM cells treated with 10.0,20.0 and 40.0 mmol/L Met were significantly decreased,the differences were statistically significant(F=15.632,20.958;all P＜0.001).With the increase of GEM concentration,the survival rate of CFPAC-1 and CFPAC-1/GEM cells decreased,the IC50 of CFPAC-1/GEM cells against GEM[(93.35±5.12)µmol/L vs.(217.87±11.34)μmol/L]was significantly higher than that of CFPAC-1 cells,the difference was statistically significant(t=24.514,P＜0.05),and the drug resistance index of CFPAC-1/GEM cells was 2.33.The IC50(76.53±3.65)µmol/L of CFPAC-1 cells treated with 2.5 mmol/L Met combined with different concentrations of GEM was lower than that treated with GEM alone(93.35±5.12)μmol/L(t=6.552,P＜0.05).The IC50 of CFPAC-1/GEM cells(171.65±6.89)μmol/L was lower than that of GEM alone(217.87±11.34)μmol/L(t=8.532,P＜0.05),and the reversion multiple of CFPAC-1/GEM cell to GEM was 1.26.Compared with the GEM group,the apoptosis rate of CFPAC-1/GEM cells,the expression of MST1/2,LAST1/2 and p-YAP/YAP were significantly increased,the differences were statistically significant(F=53.798,35.585,14.512,25.525;all P＜0.001).Conclusion Met could increase the chemotherapy sensitivity of CFP-1/GEM cells to GEM to some extent,which might be related to the activation of Hippo/YAP pathway.
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