Role of CHOP-dependent endoplasmic reticulum stress signaling pathway in triptolide-induced apoptosis of A375 melanoma cells and its mechanisms / 中华皮肤科杂志

ABSTRACT

Objective To investigate the role of C/EBP homologous protein (CHOP)-dependent endoplasmic reticulum stress signaling pathway in triptolide-induced apoptosis of A375 melanoma cells,and to explore its mechanisms.Methods In vitro cultured human A375 melanoma cells were divided into several groupsexperimental groups treated with triptolide at different concentrations of 12.5,25,50,100 and 200 nmol/L,and negative control group receiving no treatment.After 24-hour treatment,changes in the morphology of A375 cells were observed under a light microscope.After 24-,48-and 72-hour treatment,cell counting kit 8 (CCK8) assay was performed to evaluate the inhibitory effect of triptolide on cell proliferation.Flow cytometry was conducted to detect the apoptosis of A375 cells after annexin V-fluorescein isocyanate/propidium iodide double-staining,and transmission electron microscopy to observe the changes in the morphology of the endoplasmic reticulum.Western blot analysis was performed to determine the protein expression of glucose-regulated protein GRP78,protein kinase R-like endoplasmic reticulum kinase (PERK),phosphorylated PERK (p-PERK) and CHOP after 24-hour treatment,as well as to observe the changes in protein expression of GRP78 after treatment over time.Real-time fluorescencebased quantitative PCR (qPCR) was conducted to measure the mRNA expression of GRP78,PERK and CHOP.Results After the treatment with triptolide,A375 cells became long and thin,appeared fusiform with less cytoplasm,and varied in size.Their shape was irregular,and there were many protuberances on the cell surface.CCK8 assay showed that triptolide at different concentrations had inhibitory effects on the proliferation of A375 cells after 24-,48-and 72-hour treatment,and the inhibitory effects varied with the concentrations of triptolide and the duration of treatment (all P ＜ 0.05).The 50％ inhibitory concentration (IC50) of triptolide at 24,48 and 72 hours were 308,83 and 55 nmol/L respectively.The apoptosis rate of A375 cells was significantly higher in the 12.5-,25-,50-,100-and 200-nmol/L triptolide groups (10.3％ ± 0.1％,14.6％ ± 0.8％,17.4％ ± 0.7％,21.1％ ± 1.0％ and 29.5％ ± 1.1％,respectively) than in the negative control group (3.3％ ± 0.4％,all P ＜ 0.05).After 24-hour treatment with 200 nmol/L triptolide,damaged endoplasmic reticula were observed by using transmission electron microscopy.After 24-hour treatment with triptolide at different concentrations,the protein expression of GRP78,p-PERK,PERK and CHOP all gradually increased with the increase of triptolide concentrations (P ＜ 0.05).However,after 24-,48-and 72-hour treatment,the protein expression of GRP78 gradually decreased over time (P ＜ 0.05).qPCR showed that the mRNA expression of GRP78,PERK and CHOP gradually increased with the increase of triptolide concentrations after 24-hour treatment.Compared with the negative control group,all the experimental groups showed significantly higher mRNA expression of GRP78,PERK and CHOP (P ＜0.05) except the 12.5-nmol/L triptolide group with similar mRNA expression of PERK.Conclusion Triptolide can induce endoplasmic reticulum stress,and the apoptosis of A431 cells was induced by CHOP-dependent endo-plasmic reticulum stress along with the increase of triptolide concentrations and treatment duration.