Effects Of 20(S)-Protopanaxadiol On Autophagy Of Human Non-small Cell Lung Cancer A549 Cells

In this study,20(S)-Protopanaxadiol(PPD)was used to induce autophagy in human nonsmall cell lung cancer A549 cells in vitro.Methods:Human lung cancer A549 cells were cultured in vitro and treated with different concentrations of PPD for 24 hours.Autophagy of A549 cells induced by PPD was detected by MDC staining,and autophagy inhibitors 3-methyladenine(3-MA)and chloroquine(CQ)were added respectively.The effect of PPD on autophagy and cell proliferation of A549 cells was detected by adding 3-methyladenine(3-MA)or chloroquine(CQ).The formation of autophagosomes was observed under fluorescence microscope by lysotracker staining.The expression of autophagy related proteins LC3 II/I,P62 and the PI3K/Akt/m TOR pathway were detected by Western blot.Results:1.MTT and plate cloning results showed that PPD could significantly inhibit the proliferation of A549 cells,and the IC50 of 20(s)-protopanaxadiol for 24 h was 30.46 μM.2.MDC staining showed that PPD could increase the number of autophagosomes in A549 cells in a dose-dependent manner.3.Western blot results showed that PPD increase LC3 II /I ratio and up-regulated P62 protein expression in A549 cells,it could also significantly down regulate the phosphorylation levels of PI3 K,Akt and m TOR.4.Autophagy inhibitor 3-methyladenine(3-MA)and chloroquine were added 1h before PPD.The results showed that 3-MA could block the increase of autophagosomes induced by PPD,and it enhanced the inhibition of PPD on the proliferation of A549 cells.CQ could enhanced the increase of autophagosomes induced by PPD.It also enhanced the inhibitory effect of PPD on the proliferation of A549 cells.5.Lysotracker Red staining results showed that PPD could significantly reduce the fluorescence intensity and florescence spots,suggesting that it could reduce the p H value of lysosomes and the amount of lysosomes.Further,Western blot showed that PPD could significantly inhibit the expression of LAMP-2 protein,indicating that 20(s)-protopanaxadiol could affect the function of lysosomes.Conclusion:This study shown that PPD can induce autophagy in human non-small cell lung cancer A549 cells,but inhibit the autophagy flow,which may be related to the inhibition of lysosome function.