Experimental Research On Oxymatrine Regulating Chemotherapeutic Drug Resistance Of Colon Cancer By Autophagy

Objective: To investigate the effect of oxymatrine on the level of autophagy in drug-resistant colorectal cancer cells,to explore the mechanism of reversal of chemoresistance by oxymatrine,and to provide a theoretical basis for clinical treatment of colorectal cancer.Methods:(1)The cell viability test kit(CCK-8)was used to treat human colon cancer cells(HCT-8)and vincristine resistant human colon cancer cell line(HCT-8 / VCR)with different concentrations of oxymatrine.After 24 hours of treatment,the viability of the two cell lines was detected to investigate the effect of oxymatrine on the viability of human colon cancer cell line(HCT-8)and vincristine resistant cell line(HCT-8 / VCR).(2)Cell autophagy assay kit was used to detect Dansylcadaverine(MDCN),and the intracellular autophagy production was detected after 24 hours of the treatment with oxymatrine.The expressions of autophagy-related gene P62,LC3-? / LC3-?,Beclin-1 protein were detected by Western Blotting assay,and the expressions of autophagy-related gene P62,LC3-? / LC3-?,Beclin-1 protein were detected by Western Blotting assay to investigate the effect of oxymatrine on autophagy of HCT-8 and HCT-8/VCR cells.(3)The autophagic inhibitor chloroquine and autophagy activator rapamycin pretreated HCT-8/VCR cells for 1 h,and the levels of autophagy-related protein P62,LC3-? / LC3-? and Beclin-1 in HCT-8/VCR cells were determined after 24 hours of treatment with oxymatrine to further study the effect of oxymatrine on autophagy of HCT-8/VCR cells.(4)The data were analyzed by SPSS 26.0statistical software.The mean±SD of data was used to show that the experimental results obtained from three separate experiments.The results of the experiment were analyzed bymeans of variance analysis.The comparisons between the groups were compared with the SNK-q method.The comparison between the groups was t-test with paired data,and the statistical test level took the double-sided a= 0.05.Result:(1)Effect of oxymatrine on HCT-8 cell viability: compared with the control group,1.00mg/ml~10.0mg/ml of oxymatrine significantly decreased the viability of HCT-8cells in a dose-dependent manner.The effect of oxymatrine on the viability of HCT-8/VCR cells: compared with the control group,1.00 mg / ml of oxymatrine significantly decreased the viability of HCT-8/VCR cells in a dose-dependent manner.The higher the dose of oxymatrine,the greater the activity of HCT-8 / VCR cells.Comparison of the effects of oxymatrine on the viability of HCT-8 and HCT-8/VCR cells: under the same dose of oxymatrine,the activity of HCT-8 / VCR cells decreased more than that of HCT-8,but there was no significant difference between them.(2)The effect of oxymatrine on autophagy formation of HCT-8 and HCT-8/VCR cells: after adding oxymatrine,autophagy production in HCT-8 / VCR cells decreased in a dose-dependent manner.The greater the dose of oxymatrine,the less autophagy in HCT-8 / VCR cells.Effects of oxymatrine on autophagy-related protein in HCT-8 and HCT-8/VCR cells: oxymatrine had no significant change on autophagy-related protein in HCT-8 cells,but it could increase the expression of P62 in HCT-8/VCR cells,decrease the expression of LC3-?/ LC3-?and Beclin-1,and inhibit autophagy in a dose-dependent manner.The higher the dose of oxymatrine,the higher the expression of P62 in HCT-8 / VCR cells and the lower the expression of Beclin-1 in LC3-? / LC3-?and Beclin-1.Effect of oxymatrine on the regulation of autophagy formation by autophagy inhibitors or activators in HCT-8/VCR cells: oxymatrine can enhance the effects of autophagy inhibitors and inhibit autophagy,which was characterized by a further decrease in autophagy production in HCT-8/VCR cells.Oxymatrine could reverse autophagy induced by autophagy activator,which showed that autophagy production increased first and then decreased in HCT-8/VCR cells.The effect of oxymatrine on the regulation of autophagy associated proteinin HCT-8/VCR cells by autophagy inhibitor or activator: oxymatrine could enhance the effect of autophagy inhibitor and inhibit autophagy.The effect of oxymatrine on autophagy induced by autophagy activator was reversed,which showed that P62 decreased and LC3-?/LC3-?increased the recovery of Beclin-1.Conclusion:(1)In a dose-dependent manner,the activity of HCT-8 / VCR cells was decreased in a dose-dependent manner by the use of more than 1.00 mg / ml of oxymatrine,and the down-regulation of the activity of HCT-8 / VCR cells in the same dose was greater than that of HCT-8,but there was no significant difference between the two groups.(2)Oxymatrine reduced the number of autophagy vesicles in HCT-8/VCR cells,inhibited the autophagy of colon cancer cell line HCT-8/VCR,and reversed chemotherapeutic resistance.The mechanism of oxymatrine reversing chemotherapeutic resistance of HCT-8/VCR cells might be the up-regulation of the expression of P62 in HCT-8/VCR cells and the down-regulation of the expression of LC3-?/ LC3-?and inhibition to autophagy.