Effects Of Metformin On Biological Characteristics Of Pancreatic Cancer Cells

Background and ObjectiveMetformin is the first-line treatment for type 2 diabetes and the most commonly prescribed drug worldwide.In addition to hypoglycemic effect,it can also improve insulin resistance,improve obesity status,improve aging and other effects.Epidemiological studies have shown that metformin has antitumor properties,which has attracted wide attention in the world.Pancreatic cancer is one of the most common malignant tumors with poor prognosis,high metastasis rate and low sensitivity to chemoradiotherapy.Hypoxia is one of the characteristics of tumor microenvironment,especially in pancreatic cancer.Studies have shown that anoxia is significantly associated with angiogenesis,metabolic remodeling,and early invasion and metastasis of pancreatic cancer.HIF-1α(hypoxia-inducing factor)is highly expressed in pancreatic cancer,which reduces the sensitivity of tumor cells to radiotherapy and chemotherapy,and shows stronger proliferation and migration.HIF-1α also activates genes related to tumor cell metabolism,thus activating the glycolysis process.In this study,pancreatic cancer cell lines were cultured in vitro to study the effects of metformin on biological characteristics of pancreatic cancer cells,and then to further explore the relevant mechanisms.Methods1.Human pancreatic cancer cell lines PANC-1 and BXPC-3 were cultured in vitro and treated with metformin at 0,5,10,15,20,40mmol/l.The effect of metformin on cell proliferation was detected by CCK-8 proliferation assay after 24 h,48h and 72 h.Cell inhibition rate and Halfinhiconcentration(IC50)were obtained.2.Two kinds of cells were treated with 20,40mmol/l metformin medium,and those treated without metformin were used as blank control group.Scratch test and Transwell chamber were used to detect the effect of metformin on the migration ability of pancreatic cancer cells.3.Blank control group,low concentration group(20mmol/l metformin treatment group)and high concentration group(40mmol/l metformin treatment group)were set.The invasiveness of pancreatic cancer cells in vitro was measured by Transwell assay.4.Td T-medinted Dutp Nick-End labelmg(TUNEL)method was used to verify the apoptotic ability of PANC-1 and BXPC-3 cells treated with metformin.5.The effects of metformin on glucose consumption and lactate production of pancreatic cancer cells were detected by glucose oxidase method and MTT reduction method.6.Western blotting was used to detect the effect of metformin on HIF-1a,GLUT1(glucose transporter)and LDH(lactate dehydrogenase protein)in pancreatic cancer.Results1.Metformin can significantly inhibit the proliferation of PANC-1 and BXPC-3cells,and the inhibitory effect of high concentration of metformin on pancreatic cancer cells is significantly higher than that of low concentration group,that is,it is concentration-dependent.2.The cells of Pan-1 and BXPC-3 were treated with 20mmol/l and 40mmol/l metformin,and it was found that the cells in the non-metformin treated group healed faster at the scratch,and the scratch healed more than 50% after 24 h,while the wound healed slowly after drug treatment.The cells healed more slowly in the 40mmol/l group than in the 20mmo/l group.Transwell cell migration experiment showed that the number of transmembrane cells in the group without metformin treatment was significantly higher than that in the drug treatment group,and the number of transmembrane cells in the high concentration group was significantly lower than that in the low concentration group.Therefore,metformin can inhibit the migration ability of PANC-1 and BXPC-3 cells in a concentration-dependent manner.3.Invasion experiment results showed that the number of transmembrane cells in blank control group was significantly higher than that in drug treatment group,and the number of transmembrane cells in 40mmol/l drug treatment group was significantly lower than that in 20mmol/l metformin treatment group.Therefore,metformin significantly inhibited the invasion ability of PANC-1 and BXPC-3 cells in vitro in a concentration-dependent manner.4.Apoptosis experiment showed that PANC-1 and BXPC-3 cells underwent obvious apoptosis after being treated with metformin for 24 h,and the apoptotic capacity increased with the increase of metformin concentration.5.The results of glucose and lactic acid detection kit showed that the glucose intake and lactic acid production of pancreatic cancer cells were significantly reduced after the metformin treatment of PANC-1 and BXPC-3 cells,and the glucose intake and lactic acid production of pancreatic cancer cells treated with 40mmol/l metformin were significantly lower than those treated with 20mmol/l metformin group.It was demonstrated that metformin can inhibit the glycolysis of pancreatic cancer cells in a concentration-dependent manner.6.WB results showed that the expression level of HIF-1a,GLUT1(glucose transporter)and LDH(lactate dehydrogenase)in PANC-1 and BXPC-3 cells were significantly decreased after metformin treatment.The expression level of related proteins in high concentration metformin group was significantly lower than that in low concentration group.The results indicated that metformin can inhibit the expression of HIF-1a protein,GLUT1 glucose transporter and LDH lactate dehydrogenase protein.ConclusionMetformin inhibited the proliferation,migration and invasion of PANC-1 and BXPC-3 cells,and promoted apoptosis.Metformin affects the biological behavior of pancreatic cancer by down-regulating the expression of HIF-1a and affecting glycolysis.