To Investigate The Inhibitory Effect Of Metformin On Human Colon Cancer HT29 Cells And Its Possible Mechanism

Objectives Explore metformin on colon cancer HT29 cell proliferation,migration,invasion,apoptosis and the possible mechanism of the influence of the thought of clinical treatment for colon cancer,add new experimental data.Methods Human colon cancer cell line HT29 was cultured in vitro and randomly divided into two groups.The metformin intervention concentrations in the experimental group were 5,10,20,and 40 mmol/L.The control group was added with the corresponding amount of PBS solution,and Detection of cell proliferation by MTT method after 24 hours,48 hours,and 72 hours.The concentration of metformin in the control group and experimental group was 5 and 40 mmol/L,and the scratch test was performed.The migration distance was measured after 48 hours.In the control group and the experimental group,the metformin intervention concentration was 5 and 40 mmol/L,transwell invasion experiments were performed,and the number of cells passing through each group was measured after 48 days.In another transwell invasion experiment,Metformin was administered at a concentration of 40 mmol/L to intervene human colon cancer cell line HT29.After 24 hours,48 hours,and 72 hours,the number of cells passed through each group was measured.Morphological changes were observed by optical microscope after intervention of 24 h with HT29 cell concentration,5,10,20,40 mmol/L Metformin.5,10,20,40 mmol/L metformin interfered with HT 29 cells 48 h,dyed anerxin V-FITC and PI,and analyzed the apoptosis by flow cytometry.The metformin intervention concentration in the experimental group was low(5 mmol/L)and high(40 mmol/L),and the control group was treated with the appropriate amount of PBS for 48 hours.The immunoimprinting test(Western Ink Method)was conducted to detect proteins PI3 K,AKT,expressing P-AKT(phosphorylated AKT)GSK-3?,P-GSK-3?(phosphorylated GSK-3?).Results Metformin inhibited the proliferation of colon cancer HT29 cells in a timeand dose-dependent manner.The difference in inhibition rates between the groups was statistically significant(P < 0.05).Scratch tests showed that the mean migration distance at 24 h and 48 h in the 40 mmol/L metformin-treated group was significantly reduced(P < 0.05).Transwell invasion experiments showed that the number of invasive cancer cells in the 40 mmol/L metformin-treated group was significantly reduced at 24 h and 48 h(P<0.05).Under light microscope,the number of cells decreased,from the polygon into a round,reduced in size,nuclear condensation,nuclear lysis and other apoptosis phenomenon,and with the increase in metformin apoptosis rate increased.The results of flow-through experiments showed that the apoptosis rate of the experimental group was greater than that of the negative control group(P<0.05).Western Blot assay showed that metformin could inhibit the expression of PI3 K,P-AKT and P-GSK-3? protein in HT29 cells,but had no significant effect on the expression of AKT and GSK-3? protein.The difference of the expression of PI3 K,P-AKT and P-GSK-3? protein in the low-concentration group compared with the control group and the high-concentration group was statistically significant(P<0.05),and was concentration-dependent.Conclusions 1 Metformin inhibits proliferation,migration,invasion,and apoptosis of human colon cancer HT29 cells.2 The mechanism may be related to the inhibition of PI3 K,PAKT,and P-GSK-3? protein expression in the PI3K/AKT/GSK-3? pathway.