| Background:Gastric cancer(GC)is one of the most common tumors in the digestive system,and its mortality rate ranks the third in the world of cancer-related deaths.By now,chemotherapy is still the most commonly used treatment method,however,the treatment effect is not ideal because of its side effects and drug resistance.In recent years,20(S)-protopanaxadiol ginsenosides have attracted much attention due to their pronounced anti-tumor activities both in vivo and in vitro.Although it has been proved that the regulated action includes blocking cell cycle,inducing apoptosis and interfering autophagy,the exact mechanism and structure-activity relationship of 20(S)-protopanaxadiol ginseno sides in cancer therapies are still need to be further studiedObjective:In the present study,the anti-tumor activity and structure-activity relationship of 20(S)-protopanaxadiol ginsenosides(Rg3,Rh2 and PPD)in HGC-27 cells were evaluated in vitro.Apoptosis,autophage,the function of mitochondria and lysosome were detected.And the mechanism of theirs anti-tumor action was studied to provide a basis for the application and development of ginsenoside Rg3,Rh2 and PPD in future.Methods:In this study,HGC-27 cells were used to performe the following experiments:(1)SRB assay was used for detection of cell viability;(2)Giemsa staining was used to observe cytoplasmic vacuoles;(3)TUNEL staining was used to detect apoptosis;(4)The expression level of apoptosis-related protein caspase3,autophagy protein LC3B and p62 were measured by Western Blot;(5)Immunocytochemistry was used to analyzed the integrity of lysosome membrane and detect lysosomal pH;(6)High performance liquid chromatography(HPLC)was used to analysed the content of ATP,ADP and AMP;(7)MitoSOXTM were used to measure mitochondrial reactive oxygen species(ROS)formation.;(8)JC-1 signal was visualized by fluorescence microscope to detect mitochondrial membrane potential;(9)Transmission electron microscope(TEM)was used to observe ultrastructureResult:Through the above experimental detection and analysis,the following results are obtained:(1)20(S)-protopanaxadiol saponins Rg3,Rh2 and PPD induced cell death in a dose-dependent manner.Rh2 showed higher cytotoxicity(IC50=4.93±0.19 μM)than PPD(IC50=6.25±0.64 μM),and Rg3 displayed lowest toxity(IC50=33.31±6.91μM);Cytosolic vacuolation could be induced in HGC-27 cells after incubation with 9μM Rh2 or PPD for 6 h.(2)Few TUNEL-positive cells were founded in Rh2 and PPD groups.And the cleaved caspase-3 was not altered significantly in ginsenosides groups Moreover,the pan-caspase inhibitor did not induce improvement in cell viability.(3)Rh2-and PPD-treatment induced significant transition from LC3Ⅰ to LC3Ⅱ in HGC-27 cells(p<0.01 and p<0.05,respectively),while Rg3 did not.Moreover,the expression of p62 protein in Rh2-treated group increased significantly(p<0.05).(4)Co-localization between Gal3 and Lysosomal Associated Membrane Protein 1(LAMP1)were observed in both Rh2 and PPD treated groups,which indicated that lysosomal membrane rupture were induced;Rg3,Rh2 and PPD raised the pH of acidic vesicular organelles(AVOs);Rg3,Rh2 and PPD raised lysosomal pH,and the difference in Rh2 group has statistical sense(p<0.05).(5)Bafilomycin Al(BafAl),the V-ATPase inhibitor aggravated significantly the vacuolation rate induced by Rh2 and PPD(p<0.05 in the both of groups),which also aggravated significantly the cell death induced by Rh2 and PPD(p<0.01 and p<0.05,respectively).(6)Rh2 and PPD induced dysfunction in mitochondria by decreasing the ATP level significantly(p<0.01 in the both of groups);Rg3,Rh2 and PPD decreased mitochondrial energy charge in varying degrees.(7)The content of mitochondrial ROS in Rh2-treated and PPD-treated groups increased in a time-dependent manner;Rh2 and PPD induced significant decrease in mitochondrial membrane potential(MMP),p<0.01 in the both of the groups.(8)Rh2 and PPD induced damage in mitochondria and lysosomes structures.Moreover,Rh2 and PPD led to accumulation of enlarged lysosomes and autophagic structuresConclusion:To summarize,all the results demonstrated that Rh2 and PPD showed stronger cytotoxic activity than Rg3 in vitro,which might be due to the different number of sugar molecules at C-3 position of the ginsenosides.A possible mechanism of Rh2-and PPD-induced cell death in HGC-27 cells is as follows:Rh2 and PPD induced bursting of mitochondrial ROS after entering HGC-27 cells,subsequently depolarizing mitochondria and disrupting energy metabolism.The dysfunction in mitochondria led to disruption of lysosomal acidic environment and lysosomal membrane rupture.The damage in lysosomes blocked the autophagy flux and caused accumulation of autophagy substrates,,which eventually leads to cell vacuolation and necrosis in HGC-27 cells... |
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