HCPT-induced Autophagy In Lung Cancer A549 Cells And Its Related Mechanismsl

Objective To explore the induction mechanism and specific mechanism of Hydroxycamptothecin(HCPT)on autophagy of human lung cancer A549 cells.Methods 1.Observe the effects of different concentrations of HCPT on the survival rate of human lung cancer A549 cells and the expression of autophagy-related proteins.Experimental grouping:(1)Control group: A549 cells were routinely cultured throughout;(2)50?M HCPT group;(3)200?M HCPT group;(4)400?M HCPT group(A549 cells were cultured with medium containing different concentrations(50?M,200?M,400?M)HCPT for 24h).The survival rate of A549 cells in each group was detected by CCK-8 method.Western blot was used to detect the effect of different concentrations of HCPT on the expression of autophagy-related proteins(P62,LC3,Beclin-1,BCL-2,PHB2)in each group of cells,and to determine the concentration of HCPT in subsequent experiments.2.Observe the effect of autophagy inhibitor 3-MA on HCPT-induced autophagy.Experimental grouping:(1)Control group: A549 cells were routinely cultured throughout;HCPT group: A549 cells were cultured with medium containing HCPT(200 ?M)for 24 hours;(3)Inhibitor(3-MA)+ HCPT group: A549 cells were added autophagy inhibitor 3-MA(5 m M)pre-treated for 1h,and then cultured with HCPT(200?M)containing medium for 24 h.(4)Inhibitor group: A549 cells were pre-treated with autophagy inhibitor 3-MA(5 m M)for 1 hour and then routinely cultured for 24 hours.Western blot was used to detect the expression of P62,LC3,Beclin-1,BCL-2,PHB2 and other proteins.Results 1.The results of CCK8 method showed that compared with the control group,the cell survival rate of HCPT(50?M,200?M,400?M)groups at different concentrations all decreased,and it was dose-dependent.0.001).2.Western blot detection of autophagyrelated proteins showed that the expression of p62 protein in different concentrations of HCPT(50 ?M,200 ?M,400 ?M)group was lower than that of the control group,the difference was statistically significant(P <0.05);,200?M,400?M)group LC3?/?ratio was higher than the control group,and the 200?M group was the most significant,the difference was statistically significant(P <0.05);200?M and 400?M HCPT group BCL-2 protein expression level was more obvious The difference was statistically significant(P <0.05);the expression of Beclin-1 protein in different concentrations of HCPT(50?M,200?M,400?M)groups was significantly higher than that of the control group,the difference was statistically significant(P <0.05);Different concentrations of HCPT(50?M,200?M,400?M)group PHB2 protein expression were higher than the control group,and the 200?M group was the most significant,the difference was statistically significant(P <0.05).3.The effects of HCPT(200?M)on reducing p62,BCL-2 and increasing LC3?/?,Beclin-1,PHB2 can be inhibited by the selective autophagy inhibitor 3-MA,the differences are statistically significant(P < 0.05).Conclusion HCTP can activate the BCL-2/Beclin-1 signaling pathway and increase PHB2 expression,promote cell autophagy,reduce the survival rate of A549 cells,and play an anti-tumor effect.Figure 7;Table 1;Reference 100...