RESUMO
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 groups:experimental 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.
RESUMO
Objective To estimate effects of AG490, a selective inhibitor of JAK2/STAT3 signaling pathway, on the biological behavior of human keloid?derived fibroblasts (HKFs), and to explore their possible mechanisms. Methods In vitro cultured human skin fibroblasts(HSFs)and HKFs were both divided into several groups to be treated with AG490 at different concentrations (12.5, 25, 50, 75, 100 μmol/L), with those receiving no treatment serving as the control group. Then, cell counting kit?8(CCK?8)assay was performed to evaluate cellular proliferative activity of HSFs and HKFs after 24?, 48?and 72?hour treatment, flow cytometry to estimate cell cycle distribution and apoptosis rate in HKFs after 24?hour treatment, reverse transcription(RT)?PCR to measure STAT3 and cyclin D1 mRNA expressions in treated HKFs as well as STAT3 mRNA expression in untreated HSFs and HKFs after 24?hour culture, and Western blot analysis to measure the protein expressions of STAT3 and p?STAT3 in HSFs and HKFs after 24?hour treatment. Results CCK?8 assay showed that the proliferation inhibition rates of both HSFs and HKFs gradually increased along with the increase in AG490 concentrations and treatment duration, and the inhibitory effects increased in both dose?and time?dependent manners(all P<0.05). Besides, when cells were treated with the same concentrations of AG490 for same durations, the proliferation of HKFs were inhibited to a greater extent than that of HSFs(all P<0.05). As flow cytometry revealed, along with the increase of AG490 concentrations, the proportion of HKFs in G1 phase and the apoptosis rate in HKFs both increased gradually(all P < 0.01), while the proportion of HKFs in G2 phase gradually decreased(all P < 0.01), and the proportion of HKFs in S phase remained insignificantly changed. RT?PCR showed that the mRNA expression of STAT3 was significantly higher in untreated HKFs than in untreated HSFs after 24?hour normal culture(P < 0.05). After 24?hour treatment with AG490, the mRNA expressions of STAT3 and cyclin D1 in HKFs gradually decreased with the increase of AG490 concentrations. Correlation analysis revealed that the mRNA expression of cyclin D1 was positively correlated with that of STAT3 in AG490?treated HKFs (r = 0.855, P < 0.01). Western blot analysis showed that the protein expressions of both STAT3 and p ? STAT3 gradually decreased in HKFs and HSFs along with the increase of AG490 concentrations(all P < 0.05), and were significantly lower in HKFs than in HSFs (both P < 0.05). Conclusion AG490 can effectively inhibit HKF proliferation by selectively blocking the JAK2/STAT3 signaling pathway.