Effects Of Metformin Combined With Oxaliplatin On Migration And Invasion Capacity In Human Colon Cancer Cells HCT-8

ObjectivesTo explore the effect of metformin combined with oxaliplatin on migration and invasion capacity in human colon cancer cells?HCT-8?and explore its possible mechanism.MethodsHuman colon cancer cell line HCT-8 was cultured in vitro.The experimental group was treated with metformin?5,10,20,40 mmol/L?at different concentrations,and the control group plus an equal amount of PBS solution,treating 24h,48h,and 72h respectively.IC50 was detected.The colon cancer cells?HCT-8?were divided into 4groups?control group,metformin group,oxaliplatin group,combined drug group?and the cells were treated with metformin,oxaliplatin and metformin+oxaliplatin at the optimum concentration,and the control group was given an equal amount of PBS solution.48 hours later,MTT method was used to detect cell proliferation ability.The cells were planted on a6-hole plate,and the cells were treated with different concentration of drugs.The cell migration distance was detected by Wound Healing.The Matrigel was spread on the membrane at the bottom of the Transcell chamber,and the refrigerator was refrigerated at4°C.Cells treated with different drugs were inoculated at the bottom of the chamber and the invasive ability of colon cancer cells was detected by Transwell.Different drug treated cells,and the expression of MMP-2,MMP-9,and VEGF protein were detected by Western blot.The expression levels of MMP-2 mRNA,MMP-9 mRNA of colon cancer cells were measured by real-time PCR,and the experimental data were collated and statistically analyzed.The cells in the logarithmic growth period were divided into two groups,one treated with 20 mmol/L metformin+20?g/mL oxaliplatin and the other cultured with a blank medium containing 10%FBS.Changes in expression of p65,p-65,MMP-9 and MMP-2 proteins in combined drug group cells were detected by Western blot experiment.ResultsMTT showed that different concentrations of metformin after 24,48,72 h had effects on the proliferation of HCT-8 cells,and the effects were dose-time dependent.In addition,there is a phenomenon that low concentration of metformin promotes the growth of colon cancer cells in a short time.20 mmol/L metformin intervenes 48 h and the cell survival rate is 50%.48 h IC50 is 20mmol/L.After 48 h,compared with the control group,different drug treatments had inhibitory effect on HCT-8 cell proliferation,and there was difference in proliferation rate between groups,control group?1.251±0.012?,metformin group?0.985±0.081?,oxaliplatin group?0.620±0.043?,combined drug group?0.477±0.048?,?F=158.872?and the difference was statistically significant?P<0.05?.Compared with the control group,metformin,oxaliplatin and metformin+oxaliplatin decreased significantly the migration ability of colon cancer cells,and the difference was statistically significant?P<0.05?^[1];The number of invasive cells in metformin+oxaliplatin group was significantly less than in control group,metformin group,and oxaliplatin group.The difference was statistically significant?P<0.05?.Compared with the control group,the expression of MMP-2,MMP-9 and VEGF proteins in different drug treatment groups were all reduced,and the difference was significant?P<0.05?;Compared with the control group,MMP-2 mRNAandMMP-9 mRNA expression in different drug treatment groups were all reduced,and the differences between groups were significant?P<0.05?.Compared with the control group,the expression of p-p65,MMP-2,and MMP-9 proteins in the combined group of two drugs was reduced,and the difference was significant?P<0.05?,the expression of p65 protein is not significant difference.Conclusions1 Metformin and metformin combined with oxaliplatin can inhibit the migration and invasion of colon cancer HCT-8 cells and reduce the expression of MMP-2,MMP-9,VEGF protein and MMP-2 mRNA,MMP-9 mRNA molecules,and the combination group was more significant.2 The effect of metformin and oxaliplatin on the migration and invasion of human colon cancer cells is related to the inhibition of p65phosphorylation,blocking the activation of the NF-?B pathway,and reducing the expression of MMP-2 and MMP-9 proteins.Figure6;Table11;Reference 144...