Vitexin Regulates Multidrug Resistance In Colorectal Cancer Through PI3K/Akt/mTOR Signaling Pathway

As one of the common tumors in the digestive tract,the incidence and mortality of Colorectal Cancer（CRC）,have been increasing year by year in the world,which has caused a huge health threat and economic burden to many families and society.Surgery and chemotherapy remain important therapeutic strategies for the treatment of colorectal cancer.However,to our disappointment,the emergence of Multidrug Resistance（MDR）in tumor cells has rendered chemotherapy ineffective.Therefore,overcoming MDR of colorectal cancer and improving the efficacy of chemotherapy are currently the top issues.MDR refers to the phenomenon that tumor cells are resistant to various chemotherapy drugs with different structures and different mechanisms due to continuous administration of chemotherapy drugs or short-term large-dose administration of chemotherapy drugs.The occurrence and development of MDR are the result of multi-factor intervention,and the activation of efflux drug transporter is the most classical mechanism.The transporters studied in this research mainly include P-glycoprotein（P-gp）.As a member of adenylate-dependent membrane transporters,P-gp expression level has a negative correlation with drug sensitivity and the degree of intracellular drug accumulation.P-gp pumps drug substrates out of cells through an ATP-dependent mechanism,preventing tumor cells from ingesting compounds including cancer therapeutic agents,thereby reducing the ability of a wide range of anti-tumor drugs and causing drug resistance.The PI3K/Akt/mTOR signaling pathway exists in a variety of tumor cells and regulates the occurrence and development of many cancers.It plays an important role in tumor cells.A large number of studies have found that PI3K/Akt/mTOR signaling pathway participates in drug resistance of tumors through cell apoptosis and autophagy,and it has also been found to be related to membrane transporter P-glycoprotein.Flavonoids play an important role in the reversal of multi-drug resistance in colorectal cancer,and vitexin,one of them,has received extensive attention.Vitexin is extracted from hawthorn,which has the function of invigorating spleen and promoting digestion,and can protect the digestive tract.Modern pharmaceutical research has found that vitexin has a wide range of anti-tumor effects.However,the relationship between vitexin and MDR and PI3K/Akt/mTOR signaling pathways in colorectal cancer is rarely reported.The purpose of this study was to investigate whether vitexin regulates MDR of colorectal cancer through PI3K/Akt/mTOR signaling pathway.Objective:To study Vitexin regulates multidrug resistance in CRC through PI3K/Akt/mTOR signaling pathwayMethod:1 Cultivated oxaliplatin-resistant HCT116/L-OHP cells in vitro,MTT method detected the inhibition rate of various concentrations of Vitexin on HCT116/L-OHP 24h,48h,72h and calculated the half inhibitory concentration of Vitexin(IC₅₀).2 Cultivated HCT116,HCT116/L-OHP,and HCT116/L-OHP cured with Vitexin in vitro（According to the results of the MTT experiment,the roots selected the inhibition rate of Vitexin within 10%as the non-toxic Vitexin concentration）.MTT detected the inhibition rate of various concentrations of L-OHP on the three groups of cells,and calculated the half inhibitory concentration(IC₅₀).and resistance index（RI）;Western blot detected the level of P-gp;q RT-PCR detected the level of MDR1 mRNA.3 Cultivated HCT116 and HCT116/L-OHP in vitro,and detected the levels of Akt,p-Akt,mTOR and p-mTOR in each group of cells by Western blot,and detected Akt and mTOR mRNA in each group of cells by q RT-PCR method mRNA expression.4 Cultivated HCT116/L-OHP in vitro,and HCT116/L-OHP cured with low,medium,and high concentrations of Vitexin（In order to ensure that Vitexin has no obvious cytotoxic effect on HCT116/L-OHP of CRC resistant cells,the inhibition rate of Vitexin is selected to be within 10%,so 30μmol/L,60μmol/L and 90μmol/L are selected as low,Medium and high concentration treatment group）.Western blot detected the levels of Akt,p-Akt,mTOR,p-mTOR and P-gp in each group of cells;q RT-PCR method detected changes in the levels of Akt,mTOR and MDR1 mRNA in each group of cells.5 Applied SPSS 25.0 for statistical analysis and draw experimental conclusions.Result:1 MTT indicated:0,30,60,90,120,150,180μmol/L Vitexin treatment of colorectal cancer drug-resistant HCT116/L-OHP after 24 h,48h,72h,the inhibition rate was correlated with the drug concentration（P<0.05）.IC₅₀ were 170.59±0.67μmol/L,162.81±1.05μmol/L,and147.04±2.32μmol/L respectively.2 MTT indicated:the IC₅₀ of oxaliplatin in the HCT116 group of CRC cells was 14.52±5.44μg/m L,and the IC₅₀ of the HCT116/L-OHP group of CRC cells was 108.53±7.58μg/m L,RI was 7.47,there was a significant difference between the two groups（P<0.05）.The IC₅₀ of oxaliplatin in the HCT116/L-OHP cell group treated with Vitexin decreased to52.85±1.85μg/m L,RI was 2.05（P<0.05）.3 Western blot and q RT-PCR indicated:the levels of P-gp and MDR1mRNA was low in the HCT116 group,and the levels of P-gp and MDR1mRNA was at a high level in the HCT116/L-OHP group.The levels of P-gp and MDR1 mRNA in the HCT116/L-OHP group cured with Vitexin were significantly reduced（P<0.05）.4 Western blot indicated:the levels of p-Akt and p-mTOR in HCT116/L-OHP group were significantly higher than HCT116 group（P<0.05）,the levels of Akt and mTOR were meaningless（P>0.05）;q RT-PCR indicated:the levels of Akt and mTOR mRNA in the HCT116 group and the HCT116/L-OHP group had no significant difference（P>0.05）.5 Western blot indicated:the levels of p-Akt,p-mTOR,and P-gp in the HCT116/L-OHP group cured with various concentrations of Vitexin were significantly reduced,and was relevant to concentration of Vitexin（P<0.05）the levels of Akt and mTOR were meaningless（P>0.05）;q RT-PCR indicated:the levels of Akt and mTOR mRNA in the HCT116/L-OHP group cured with various concentrations of Vitexin had no significant changes compared with the control group without Vitexin（P>0.05）,while the level of MDR1 mRNA was significantly reduced compared with the control group without Vitexin,and it was relevant to concentration of Vitexin（P<0.05）.Conclusion:1 Vitexin can regulate the expression of MDR1 mRNA and P-gp in HCT116/L-OHP drug-resistant colorectal cancer cells,and reverse the drug resistance of drug-resistant cells.2 Vitexin may be able to down-regulate the phosphorylation levels of Akt and mTOR in the PI3K/Akt/mTOR signaling pathway,regulate the expression of MDR1 mRNA and P-gp in HCT116/L-OHP in colorectal cancer resistant cells,and reverse the resistance of resistant cells.Medicinal properties.