The Evaluation On Polyphyllin ?-induced Apoptosis In Human Hepatocellular Carcinoma HepG2 Cells

Polyphyllin ?(PPI),is a steroidal saponin extracted from the rhizoma of Paris polyphyllin,has been reported to inhibit the proliferation in various cancer types.Hepatocellular carcinoma(HCC)ranked as the fifth leading cause of cancer worldwide.The patients have short survival and high mortality,and the incidence of liver cancer has been increasing significantly in recent years,which is a serious threat to human health.The foremost anti-liver cancer mechanism of PPI induce apoptosis through mitochondria signaling pathway,while no studies regarding the possible role of ER stress and death receptor pathways in PPI regulating Hep G2 cells apoptosis.The CCK-8,Hoechst 33258 Staining,q RT-PCR and Western blot assay and so on were applied to identify the function of ER stress and death receptor signaling pathways in PPI inducing Hep G2 cells apoptosis,which provided research direction and theoretical basis for finding out the effect proteins in PPI inducing Hep G2 cells apoptosis,and developed a new therapeutic target.The details show as following:1.The effect of PPI on proliferation and apoptosis of Hep G2 cellsThe CCK-8 and Hoechst 33258 staining assay were applied to detect the effect of PPI on viability and the changes of nuclear morp Hology of Hep G2 cells.Western blot was used to test the protein levels of Cleaved-caspase3,Bax and Bcl-2.Results:(1)CCK-8 assay result showed that PPI could reduce the viability of Hep G2 cells in dose-and timedependent manners,with IC50 values of 4.38 ± 0.45,2.45 ± 0.12 and 2.32 ± 0.18 ?mol/L at 6,12 and 24 h respectively.(2)PPI induced apoptosis in dose-dependent manner,and PPI treated cells exhibited apoptotic characteristics,such as karyopyknosis and nuclear fragmentation,and stained with deep blue by Hoechst 33258.(3)Western blots indicated that PPI could down-regulate the level of the anti-apoptotic protein Bcl-2.On the contrary,the levels of pro-apoptotic proteins of Claeaved-Caspase-3 and Bax were markedly increased.All these results showed that the mechanism of inhibiting the proliferation of Hep G2 cells was induced apoptosis.2.The effect of PPI on ER stress pathway of Hep G2 cellsIn order to investigate the mechanism of regualation of PPI with ER stress pathway,q RT-PCR assay was applied to detect GRP78,ATF-6,IRE-1,PERK and CHOP m RNA levels;Western blot was applied to determine the protein levels of Cleaved-caspase12,Caspase-12,IRE-1and CHOP;the protein level of IRE-1 and the change of apoptosis rate were detected by Western blot and Annexin V-FITC/PI staining assay after pre-treated with 4-PBA at dose of 20 mmol/L,which determined the role of ER stress in PPI induced Hep G2 cell apoptosis.Results:(1)The levels of ATF-6 and PERK m RNA had no obvious change,but the levels of GRP78 and IRE-1 m RNA increased significantly,which suggested that the IRE-1 pathway may be activated specificlly.(2)The gene of CHOP no significant change in m RNA level and protein level,a key pro-apoptotic protein in ER stress pathway.(3)The protein of IRE-1 was substantially decreased,and the apoptosis rate was substantially increased after pre-treatment 4-PBA for 2h.Above results indicated that ER stress played a protective role in PPI-induced apoptosis.3.The effect of PPI on death receptor pathway of Hep G2 cellsIn order to investigate the mechanism of regualation of PPI with death receptor pathway,q RT-PCR assay was applied to detect Fas,Fas L,TNFR1 and TNF-? m RNA levels;Western blot was applied to determine the protein levels of Cleaved-caspase8,FADD,Fas and TNF-?;the protein levels of RIPK1 and RIPK3,and the change of cell viability were detected by Western blot and CCK-8 assay after pre-treated with Z-IETD-FMK and Nec-1 for 2h to verify whether Hep G2 cells had necroptosis.Results:(1)The levels of Fas,Fas L,TNFR1 and TNF-? m RNA all increased significantly,which suggested that the death receptor pathway may be activated;Western blot results showed that Fas,TNF-?,FADD and Cleaved-caspase8 proteins all significant up-regulation,which verified the result of q RT-PCR.(2)Hep G2 cells were smaller,and the cell junctions disappeared after pre-treatment Z-IETD-FMK for 2h;the necroptosis-related proteins of RIPK1 and RIPK3 had high expression,but no obvious change between the groups;it is suggested that the absence of caspase-8 may activate another apoptosis,named necroptosis pathway,which continues to induce the apoptosis of Hep G2 cells.(3)Cell viability increased significantly after pre-treatment Z-IETD-FMK and Necrostatin-1 compared with alone pre-treatment Z-IETD-FMK group,which verified necroptosis occurred in Hep G2 cells.Above results indicated that PPI activated the death receptor pathway in Hep G2 cells;the presence of Caspase8 inhibitor Z-IETD-FMK activated necroptosis,and enhanced the cell death.In conclusion,the adaptation system of ER stress was activated in the process of Hep G2 cell apoptosis induced by PPI,inhibited the apoptosis and promoteed cell survival,which indicated that the ER stress had negative regulatory role for the death of Hep G2 cells,thus,PPI and inhibitor drugs were combined for treatment in clinic,which was expected to enhance the death of cancer cells,so as to achieve the aim of eventually clear the cancer cells.In addition,death receptor signaling pathway promoted apoptosis of Hep G2 cell,while the inhibited of death receptor signaling pathway will induce necroptosis enhanceed the death of Hep G2 cells.The potential of PPI inducing apoptosis and necroptosis expected to become an effective anti-cancer drug used in clinical.