The Impact Of Insulin On Growth,Metastasis,and Sensitivity To Gemcitabine In Cholangiocarcinoma QBC939 Cells

Objective: To investigate the effect of insulin on the growth,metastasis and the sensitivity of chemotherapeutic drugs to gemcitabine in cholangiocarcinoma QBC939 cells.Method:(1)Treated with 0,0.01,0.1,1 μM concentrations of insulin in the period of logarithmic phase of cholangiocarcinoma QBC939 cells,the number of cells were observed by optical microscope;MTT assay was used to detect cell activity,in order to investigate the change of insulin on the proliferation of human cholangiocarcinoma cell line QBC939.(2)Treated with upon concentrations of insulin in the period of logarithmic phase of cholangiocarcinoma QBC939 cells,apoptosis rate was detected by flow cytometry(FCM).(3)Treated with upon concentrations of insulin in the period of logarithmic phase of cholangiocarcinoma QBC939 cells,scratch assay was used to detect the migration of tumor cells.(4)Treated with upon concentrations of insulin in the period of logarithmic phase of cholangiocarcinoma QBC939 cells,the expression of phosphatidylinositol 3-kinese(PI3K)protein in tumor cells was detected by Western Blot in order to further detect the mechanism of the effect of insulin on cholangiocarcinoma.(5)To examine the sensitivity of insulin to gemcitabine,a chemotherapy drug for cholangiocarcinoma,We used the MTT assay to test the activity of cholangiocarcinoma QBC939 cells gemcitabine affected,and calculate the half inhibitory concentration(IC50);gemcitabine and insulin are treated in cholangiocarcinoma QBC939 cells for 24 h,optical microscope and MTT assay are used to detected the changes of the number and activity of cells.(6)In order to further detect the mechanism of the influence of insulin on the drug resistance of cholangiocarcinoma QBC939 cells,the expression of P-glycoprotein was detected by Western Blot after the interaction of gemcitabine and insulin on cholangiocarcinoma cell line QBC939 for 24 h.Result:(1)The number and density of QBC939 cells were increased in the insulin group under the light microscope.The results of MTT assay also indicated that insulin could enhance the activity of tumor cells.(2)Flow cytometry was used to detect the effect of insulin on the apoptosis of cholangiocarcinoma QBC939 cells,and the results showed no significant difference in the effect of apoptosis.(3)The results of the scratch test showed that insulin could promote the migration of cholangiocarcinoma QBC939 cells,and the difference was statistically significant.(4)Western Blot indicated that PI3 K,which can regulate the activity and proliferation of tumor cells,increasesd in the protein expression level of cholangiocarcinoma QBC939 cells after adding the insulin.(5)MTT assay showed that gemcitabine significantly inhibited the activity of cholangiocarcinoma QBC939 cells,and the half inhibition rate(IC50)is 48.5ug/ml.Gemcitabine and different concentrations of insulin treated in cholangiocarcinoma QBC9393 cells for 24 h,optical microscope and MTT assay showed that the cell number and activity were increased in insulin group.(6)After gemcitabine and insulin together being treated in cholangiocarcinoma QBC939 cells for 24 h,Western Blot results suggested that the expression level of Pglycoprotein in the drug group increased compared with the blank group.Conclusion:(1)Insulin(0,0.01,0.1,1 μM)can promote the proliferation and invasion of cholangiocarcinoma QBC939 cells,but have no obvious effect on cell apoptosis.(2)Insulin promoted the growth and metastasis of cholangiocarcinoma QBC939 cells and it’s mechanism may be related with the increased expression of PI3 K.(3)Insulin can reduce the sensitivity of cholangiocarcinoma QBC939 cells to gemcitabine,and the mechanism may be associated the increased expression of Pglycoprotein.