Effect Of Polyphyllin Ⅰ Inducing ROS Accumulation On Cell Cycle Arrest And Autophagy In Colon Cancer Cells

Purpose:To investigate the effect of Polyphyllin Ⅰ on human colon cancer cells HCT116 and to explore its possible mechanism.Methods:1.The inhibitory activity of Polyphyllin Ⅰ on HCT116 cells in vitro was determined by MTT assay.To observe the growth inhibitory effect of different concentrations ofPolyphyllin Ⅰon HCT116 cells,and the half inhibition rate(IC₅₀)was calculated;2.Hoechst33258 staining was used to observe the effect of gradient concentration of（0.5,1,2μg/ml）Polyphyllin Ⅰon nuclear morphology and the number of HCT116 cells;3.The effect of Polyphyllin Ⅰwith gradient concentration（0.5,1,2μg/ml）on the cell cycle of HCT116 cells was detected by flow cytometry;4.The effect of Polyphyllin Ⅰon the microtubules of HCT116 cells was detected by immunofluorescence and laser scanning confocal microscopy;5.The effect of Polyphyllin Ⅰon the Autophagy in HCT116 Cells were detected by double-labeled adenovirus（mRFP-GFP-LC3）Transfection;6.The effect of Polyphyllin Ⅰon reactive oxygen species was observed by flow cytometry and laser scanning confocal microscopy;7.The effect of autophagy inhibitor 3-MA or oxygen species inhibitor NAC combined with Polyphyllin Ⅰon cell viability was detected by CCK8 assay;8.The expression of cell cycle-related proteins p21,p27,cyclinB1,CDC2,CDC25C and P-CDC25C,the expression of autophagy-related proteins LC3,p62,p-AKT,AKT,p-mTOR and mTOR in HCT116 cells were detected by Immunoblotting（Western blot）.Result:1.Polyphyllin Ⅰ inhibited HCT116 cells proliferation in a dose-and-time-dependent manner with IC₅₀0 of 1.91±0.27,0.93±0.025,0.79±0.03μg/ml at 24h,48h and 72h;2.Hoechst 33258 staining showed that the number of cells decreased and the number of binucleated cells increased significantly after treatment with（0.5,1,and 2μg/ml）Polyphyllin Ⅰfor 12 h,24 h,and 36 h.The difference was statistically significant;3.It was found that the cell cycle was arrested in G2/M phase after treated with Polyphyllin Ⅰby flow cytometry.The proportion of G2/M phase in solvent group was（23.07±1.65）%,The proportion of the treated group（0.5,1,2μg/ml）was（30.7±2.05）%,（46.5±2.9）%and（51.6±3.45）%,The difference was statistically significant（P<0.05,P<0.01）.Polyphyllin Ⅰdid not inhibit the G2/M-related protein CDC2,CDC25C expression and activity,but on the contrary has a promoting effect;it could reduce cyclinB1 protein expression;4.Cells treated by2μg/ml Polyphyllin Ⅰwill lead to cell microtubule structure disorder compared with the solvent group;5.Polyphyllin Ⅰcould induce autophagy after treatment with2μg/ml Polyphyllin Ⅰby transfecting cells with mRFP-GFP-LC3 adenovirus.The expression of autophagy-related protein LC3B-II increased,p62 decreased,however,the expression of P-AKT and P-mTOR did not change obviously.The inhibitory rate of Polyphyllin Ⅰcombined with autophagy inhibitor 3-MA was significantly lower than that of Polyphyllin Ⅰalone treated group;6.Polyphyllin Ⅰcan promote the production of reactive oxygen species,Polyphyllin Ⅰand reactive oxygen scavenger NAC combination,the inhibition rate of cells was significantly lower than the same concentration of Polyphyllin Ⅰalone treatment group;7.Polyphyllin Ⅰinduced ROS accumulation in HCT116 cells,causing cycle arrest and autophagy.Conclusion:Polyphyllin Ⅰinhibited the growth of HCT116 cells in a dose-time manner and caused disorder of microtubule structure.It blocked cell cycle in G2/M phase and induced autophagy by inducing ROS accumulation.