Apoptosis And Pro-death Autophagy Induced By Spirostanol Saponin Isolated From Tupistra Chinensis Baker On HL-60 And HepG2 Cells

Background:Our previous study has revealed that the spirostanol saponins isolated from the rhizomes of Tupistra chinensis?Baker??Convallariaceae??a reputed folk medicine?exhibited potent antiproliferative activity against human tumor cells.However,the underlying mechanism of purified saponin remains unclear.More studies are necessary to assess the apoptosis and autophagy activities of the saponins from T.chinensis and clarify their antiproliferative mechanisms.Purpose:The present study certificated the potential antiproliferative activity and mechanism of 5?-spirost-25?27?-en-1?,3?-diol-1-O-?-L-rhamnopyranosyl-?1?2?-?-D-xylopyranosyl-3-O-?-L-rhamnopyranoside?SPD?,a spirostanol saponin from T.chinensis,against human acute promyelocytic leukemia cells?HL-60?and human hepatocellular carcinoma cells?HepG2?.Methods:MTT assay was employed to detect the antiproliferative activity of SPD in vitro and cis-dichlorodiammineplatinum?II?was used as a positive control.The autophagic activity was assessed using MDC staining and western blot,cell apoptosis inspection was detected by Annexin V-FITC/PI double staining and the mitochondrial membrane potential was detected by JC-1 fluorescence dye combined with flow cytometry.The potential mechanisms for protein levels of apoptosis and autophagy were evaluated by western blot.Results:Treatment of HL-60 and HepG2 cells with SPD resulted in growth inhibition with IC₅₀ value of 2.0±0.2?M and 7.4±0.2?M,after48 h treatment.Results from Annexin-V/propidium double-staining assay and mitochondrial membrane potential detection showed that apoptosis was happened after SPD treatment.After treatment with 4 and 8?M of SPD in HL-60 cells or 8 and 10?M in HepG2 cells,both early apoptotic cells and late apoptotic cells were obviously increased in a dose-dependent manner.??_m was determined using JC-1 staining and the result showed that SPD induced the collapse of mitochondrial membrane potential.The regulation of caspase-3,Bax,Bcl-2,PARP following SPD treatment contributed to the activate caspase-dependent apoptosis through mitochondrial-mediated pathways.These solid data confirmed that SPD can induce mitochondria-dependent apoptosis HL-60 cells and HepG2cells.Meanwhile,SPD exhibited potent effect on autophagy.Treatment with SPD could enhance dots of GFP-LC3 in HepG2 cells.MDC fluorescent intensity was markedly enhanced in SPD-treated cells.The regulation of autophagy markers LC3,p62 and Beclin-1 confirmed that SPD induced autophagy.After treatment with SPD,there was a significant decrease in the levels of phosphorylated Akt and mTOR,in a concentration and time-dependent manners compared with total normal levels in HL-60 cells and HepG2 cells.Together,these data suggested that SPD-triggered autophagymightmediatethroughinhibitionofthe?PI3K?/Akt/mTOR/p70S6K signaling pathway.Furthermore,blocking autophagy with Bafilomycin-A1 reduced the cytotoxicity of SPD in HL-60and HepG2 cells.The evidence that decreased the expression of cleaved-caspase 3,cleaved-PARP and Bax,but increased the Bcl-2protein level proved that SPD-induced autophagy promoted apoptosis in HL-60 and HepG2 cells.Conclusion:The antiproliferative,apoptosis and pro-death autophagy activities of SPD suggested that spirostanol saponins from T.chinensis would be a potential cytotoxic candidate against acute promyelocytic leukemia.