Experimental Study On The Effect Of Ginsenoside Rh2 On Drug Resistance Of Colorectal Cancer Cell HCT15/5-FU

Objective:Observe the effect of ginsenoside Rh2（G-Rh2）combined with the chemotherapy drug 5-fluorouracil（5-FU）on the chemotherapy sensitivity of human colorectal cancer HCT15/5-FU multidrug resistant cells,and study its possible molecular mechanism.Method:1.Detect the chemosensitivity of 5-FU and L-OHP to HCT15 sensitive cell lines and HCT15/5-FU resistant cell lines by CCK-8 method,and verify the drug resistance of HCT15/5-FU cells.2.CCK-8 method,plate clone formation experiment to detect the growth inhibitory effect of G-Rh2 combined with 5-FU on drug-resistant cells.Flow cytometry was used to detect the effect of G-Rh2 combined with 5-FU on apoptosis.3.Cell scratch test and Transwell test to detect the effect of G-Rh2 combined with5-FU on cell migration and invasion.4.The effect of Western blotting G-Rh2 combined with 5-FU on the expression of copper transporters ATP7A,ATP7B,CTR1 and apoptosis-related proteins P53 and Caspase-3.Result:1.The results of the CCK-8 experiment showed that 5-FU and L-OHP chemotherapy drugs acted on HCT15 and HCT15/5-FU cells,respectively.The IC50 values of HCT15 cells were 3.13μg/mL,6.25μg/mL;The IC₅₀0 values of HCT15/5-FU cells were 21.36μg/mL and 14.27μg/mL,respectively.The drug resistance indexes were6.28 and 2.28,respectively.2.CCK-8,plate cloning experiment and flow cytometry results showed that after G-Rh2 combined with 5-FU,the IC₅₀0 value of HCT15/5-FU cells was 9.1μg/mL,the reversal factor was 2.35;The cloning rates of G-Rh2 group,5-FU group,G-Rh2+5-FU group were 40.4%,23.2%,15.8%,7.4%respectively,compared with5-FU group,The clonal formation rate in the G-Rh2+5-FU group was significantly reduced（P<0.001）;the total apoptosis rates in the control group,G-Rh2 group,5-FU group and G-Rh2+5-FU group were（1.13±0.20）%,（6.10±0.87）%,（11.80±2.26）%,（20.17±1.53）%,respectively.Compared with the 5-FU group,the total apoptosis rate in the 5-FU+G-Rh2 group increased significantly（P<0.01）.3.The results of cell scratch test and Transwell test show that the scratch area of?the control group,G-Rh2 group and 5-FU group is gradually decreasing at 24h and48h,and the migrating cells in the scratch are gradually increasing,compared with the control Group,G-Rh2 group and 5-FU group have weaker healing ability,but there is no significant difference between the two groups.The cell scratch area of the G-Rh2+5-FU group was not significantly reduced after drug treatment for 24h and 48h.Compared with the control group and 5-FU group,the scratch healing ability was significantly weakened.The number of drug-resistant cells passing through the microwell membrane of the Transwell cell was significantly higher than that of the parent cells.After drug-resistant cells were treated with drugs,compared with the control group,the number of microfiltration membranes passing through the small cells in the G-Rh2 group,5-FU group and G-Rh2+5-FU group were significantly reduced,and the degree of reduction gradually increase.Compared with the single-drug group,the G-Rh2+5-FU group had the weakest invasive ability and the least number of filters.4.ATP7A,ATP7B,and CTR1 are lowly expressed in HCT15 and highly expressed in HCT15/5-FU,while the expressions of P53 and Caspase3 are reversed.Compared with the control group,the expressions of ATP7A and ATP7B were reduced in the5-FU group and the G-Rh2+5-FU group,and the expressions of P53 and Caspase3were increased,but there was no significant difference in the G-Rh2 group.Compared with the 5-FU group,the protein expression changes in the G-Rh2+5-FU group were more significant.There was no significant change in the expression of CTR1 in each group.Conclusion:1.Human colorectal cancer cell HCT15/5-FU is a cross-resistant cell of 5-FU and L-OHP,with good drug resistance and stability.2.G-Rh2 can enhance the chemotherapy sensitivity of 5-FU to human colorectal cancer resistant cells HCT15/5-FU.Combined with 5-FU,it can significantly inhibit cell proliferation and promote apoptosis.3.G-Rh2 combined with 5-FU can significantly inhibit the migration and invasion of colorectal cancer resistant cell HCT15/5-FU.4.G-Rh2 combined with 5-FU can increase the chemotherapy sensitivity of colorectal cancer resistant cells HCT15/5-FU by down-regulating the expression of drug-resistant proteins ATP7A and ATP7B and up-regulating the expression of apoptosis-related protein P53/Caspase3.