Mechanism Of Artesunate Regulating Autophagy In Hepatoma Cells And Its Influence On Cell Growth

Objective:To study the antitumor effect of artesunate which artemisinin derivatives,to know the mechanism of artesunate induced autophagy,to explore the fact that the artesunate enhanced autophagy through IRGM receptor and mechanism of inhibiting tumor proliferation.It can be used to provide a new direction for the new chemotherapy treatment,improve the anti-tumor efficacy and reduce drug resistance.Methods:1.The inhibition rate of ART on SK cells was determined by MTT assay,and the IC50 value was worked out by Graphpad Prism.2.The cells were divided into groups according to the measured values of IC50.The morphology of SK cells was monitored by inverted microscope,hochest staining was used to observe the changes of cell nuclei,and the cell cycle and apoptosis of SK cells were detected by flow cytometry(150uM).3.The LC3,P62 and PARP1 proteins of SK cells that ART treated were analyzed by Western blot.And the number of GFP-LC3 fluorescence spots was observed by inverted fluorescence microscope.4.The autophagy inhibitor 3-MA and ART respectively and combinedly treated SK cells.Western blot was used to detected the protein levels of P62,PARP1,LC3,PI3K,P-MTOR,and IRGM in cells.The number of GFP-LC3 spots in the cell was observed by inverted fluorescence microscope.5.The expression of LC3,P62,PARP1 and IRGM in the cells were analyzed by using Western blot when siRNA interference technique was to knock down the expression of IRGM receptor.Results:1.Plot therate curve that was the time and the drug concentration dependent inhibition of ART treated cells,calculating according to the Graphpad Prism of the ART treated cells with IC50 of 24h was 150uM.2.SK cells was divided into ART treatment group and control group.The experimental group showed that the pseudopodium of the cells disappeared,the cells shrank,the contents of the cells increased,and the cell nucleus became pyknotic.The cell cycle was arrested in G1 phase and GO phase and early apoptosis increased significantly in ART treatment group.3.ART increased the expression of LC3?(P<0.05)and P62 decreased(P<0.05),and the GFP-LC3 spots increased significantly(P<0.01).4.The expression of LC3II,PI3K and PARP1 in the 3-MA treated group decreased(P<0.05),p-MTOR,P62 incresed(P<0.05),and there was no statistically difference of IRGM.3-MA and ART combined group showed no significant change of protein level,but IRGM was incresed(P<0.05),and the change of GFP-LC3 was not statistically significant.5.The expression of LC3II,IRGM,PARP1 decreased(P<0.05)and P62 incresed in the cells transfected with SilRGM(P<0.05).There was no difference in LC3?,IRGM,PARP1 and P62 protein in cells transfected with SiIRGM combined ART.Conclusion:The study found that artesunate can inhibit the proliferation of hepatoma cells through different signaling pathways regulating autophagy,the regulation of artesunate on PI3K and MTOR pathways has been more widely.Therefore,the main purpose of this paper is to study the anti-tumor effect of artesunate by inducing IRGM receptor to activate autophagy,which provide a new target for the study of anti-tumor mechanism.