Effect Of α-hederin On The Apoptosis And Growth Inhibition Of Cisplatin–resistant Gastric Cancer Cells

Objective:To investigate the role of α-Hederin in regulating proliferation and apoptosis of human cisplatin–resistant gastric cancer cells and it may involve signalpathways.Methods:1.To construct human cisplatin–resistant gastric cancer cell line HGC27/DDP and CCK-8 assay was used to detect the effect of different concentration(0,0.5,1,2,4,8,16 μg/ml)cisplatin on the proliferation of human HGC27 cells and HGC27/DDP cells.Meanwhile,the effect of different concentrations(0,2.5,5,10,15,20,25 μmol/L)α-Hederin was evaluated by CCK-8 assay in 24 h and 48 h on the proliferation of HGC27/DDP cells,and the half effective inhibition concentration(IC50)was determined;Transwell invasion assay and Wound Healing assay to detect the effect of α-Hederin on invasion and migration of HGC27/DDP cells;Hoechst33258 staining and Flow Cytometry to detect the effect of α-Hederin on apoptosis of HGC27/DDP cells;DCFH-DA was used to detect intracellular reactive oxygen species(ROS)levels;JC-1 kit was used to detect mitochondrial membrane potential(MMP)changes of HGC27/DDP cells;GSH and GSSG detection kit was used to detect glutathione(GSH)content.2.The synthsis of intracellular GSH was intervented by buthionine sulfoximine(BSO)or N-acetylcysteine(NAC).Then the apoptosis of HGC27/DDP cells was evaluated using Hoechst 33258 staining,while the changes of ROS and MMP were further measured,and the expression of proteins related to mitochondrial apoptotic pathway were detected by Western Blot to analyze the potential mechanisms.3.An xneograft tumor model of HGC27/DDP cells was constructed.The nude mice were randomly grouped and injected intraperitoneally with different concentrations of α-Hederin to observe and record the growth of nude mice and tumor volume;HE staining was used to observe the histological characteristics of transplanted tumor;TUNEL method was used to detect the apoptosis of transplanted tumor somatic cells.Preliminary evaluation of α-Hederin safety through detection of liver and kidney function in nude mice.Results:1.The result of CCK-8 showed that HGC27 cells were more sensitive to cisplatin than HGC27/DDP cells and α-Hederin inhibited the proliferation of HGC27/DDP cells in a dose-and time-dependent manner.The IC50 of 24 h and 48 h was 9.339 ± 0.468 μM and 4.736 ± 0.342 μM,respectively. With the increase of α-Hederin concentration,the results of Transwell invasion assay and Wound Healing assay and Western Blot showed that the invasion and migration ability of HGC27/DDP cells decreased.The apoptotic morphological features,such as nuclear sequestration,nuclear fragmentation and chromatin condensation,could be observed by Hoechst 33258 staining.Flow Cytometry showed that the proportion of apoptosis of HGC27/DDP cells increased significantly compared with the control group(P<0.05).Meanwhile,α-Hederin induced the depletion of GSH(P<0.05),the accumulation of intracellular ROS(P<0.05)and changed the MMP(P<0.05).2.HGC27/DDP cells were pretreated with BSO(8m M)or NAC(12m M)before treated with α-Hederin(10 μM).The results showed that pretreatment with NAC significantly inhibited the apoptosis promotion of α-Hederin on HGC27/DDP cells,reduced ROS accumulation and increased MMP levels,whereas pretreatment with BSO had the opposite effect.Western Blot showed that α-Hederin increased the Bax、cleaved caspase-3、cleaved caspase-9、Apaf-1、AIF and Cyt C expression and decreased the protein level of Bcl-2 and PCNA(P<0.05).In addition,the pretreatment with NAC could attenuate the α-Hederin-induced decrease in protein impression levels and the with BSO could augment the tendency as mentioned above(P<0.05).3.Subcutaneous xenografts were successfully constructed in nude mice which treated with saline,α-Hederin 2mg/kg,α-Hederin 4mg/kg and α-Hederin 6mg/kg by intraperitoneal injection.Compared with the control group,the tumor weight and volum were gradually decreased(P<0.05).The results of HE staining to observe the histological morphological showed that the α-Hederin groups had a large nucleus,;TUNEL assay demonstrated that the proportion of apoptosis was apparent increased compared with the control group(P<0.05).In vivo,there was no significant defference in the function of the liver and renal in each group(P>0.05).Conclusions:α-Hederin inhibits the proliferation and induces the apoptosis of cisplatin–resistant gastric cancer cells via increasing levels of intracellular reactive oxygen species and triggering mitochondrial pathway activation.