The Effect Of Artesunate On Lysosomal Function And Its Role In Inhibiting Drug Resistance Of Cancer Cells

Aim:To study the effect of artesunate on the proliferation of cancer cells and on the cell lysosomal function,and to explore the role of lysosome in enhancing susceptibility of drug resistant cells.Methods:CCK8 was used to detect the proliferation of cancer cells.Western blot was used to check the expression of cathepsin D,cathepsin L,and the level of PARP and caspase 3;JC-1 staining and cytochrome c level in cytoplasm were used to value the function of mitochondria;Electron microscopy was used to observe mitochondria,autophagy and lysosome;GFP-RFP-LC3II stable transfection cells were used to check autophagy flux.LysoTracker and LysoSensor were used to check the pH of lysosome and lysosomal function.TFEB overexpression and knockdown were used to change the lysosomal function.Three drug resistant cell lines were used to detect the effect of artesunate on drug resistance of cancer cells.Results:CCK8 results showed that artesunate could significantly inhibit the proliferation of several cancer cells in a concentration-dependent manner.The results of JC-1,electron microscopy and cytochrome c level showed that artesunate significantly reduced the mitochondrial membrane potential and increased the vacuolar mitochondria and elevated the release of cytochrome c from mitochondria to cytoplasm.In addition,the increased level of PARP and caspase 3 suggested that artesunate damaged mitochondria and induced apoptosis.The electron microscopy results showed that the number of autophagosomes increased and the autolysosomes decreased after the artesunate treatment.The results with GFP-RFP-LC3II stable transfection cells showed the similar phenomenon,which suggested that artesunate inhibited lysosomal function.Westernblot results showed that artesunate reduced the activation of lysosomal cathepsin D and cathepsin L.In the meanwhile,artesunate decreased the fluorescence of LysoTraker and LysoSensor significantly,which suggested that artesunate increased the pH of lysosome and inhibited lysosomal activity.TFEB overexpression and knockdown experiments showed that the cells with higher lysosomal activity had stronger drug-resistance.Artesunate showed the strongest inhibitory effect to the A549/TAX cells which had the highest lysosomal function in the three kinds drug-resistance cancer cells.Conclusion:Artesunate inhibits lysosomal function,inhibits the clearance of dysfunctional mitochondria,and induces cell apoptosis.Artesunate inhibits drug resistance of cancer cells through inhibiting the lysosomal function.Artesunate has higher inhibitory effect on the cells with higher activity of lysosomal cathepsins.