Abstract: 【Abstract】 Objective To investigate the effect of triptolide on the apoptosis of human melanoma A375 cells through the inositol-requiring enzyme 1 （IRE1）/c-Jun N-terminal kinase （JNK） signaling pathway, and to explore its possible mechanisms. Methods Cultured A375 cells were treated with triptolide at different concentrations of 0, 50, 100, 200 nmol/L （experimental control group, 50, 100, 200 nmol/L triptolide groups, respectively）, and a blank control group （DMEM high-glucose medium without cells） was set up. Methyl thiazol tetrazolium （MTT） assay was performed to evaluate the cell viability at 24, 48, and 72 hours after the start of treatment, flow cytometry to detect cell apoptosis at 24 hours after the start of treatment, and real-time fluorescence-based quantitative PCR （RT-qPCR） and Western blot analysis were conducted to determine mRNA and protein expression of IRE1, JNK, and c-Jun, respectively. After pretreatment with the JNK inhibitor SP600125 for 72 hours, some A375 cells were then treated with 100 nmol/L triptolide for 24 hours （SP600125 + 100 nmol/L triptolide group）, and the A375 cells only treated with 100 nmol/L triptolide served as control group （100 nmol/L triptolide group）. Effects of triptolide on the mRNA expression of IRE1, JNK, and c-Jun in A375 cells, as well as on cell apoptosis, were investigated. Statistical analysis was performed using two-way analysis of variance, one-way analysis of variance, and Dunnett′s test. Results After the treatment with different concentrations of triptolide for different durations, the cell viability was significantly lower in all triptolide groups than in the experimental control group （Ftriptolide concentration = 18.36, P = 0.002）, and gradually decreased over time （Ftime = 8.54, P = 0.018）. After 24-hour treatment, the apoptosis rate of A375 cells significantly differed among the 4 groups treated with different concentrations of triptolide （F = 5 234.97, P < 0.001）; additionally, the apoptosis rate was significantly higher in the 50, 100, and 200 nmol/L triptolide groups （16.99% ± 0.33%, 30.78% ± 0.40%, 38.91% ± 0.51%, respectively） than in the experimental control group （4.33% ± 0.02%, all P < 0.05）, and gradually increased with the rising concentrations of triptolide. The mRNA expression levels of IRE1, JNK, and c-Jun were all significantly higher in the 50, 100, and 200 nmol/L triptolide groups than in the experimental control group （all P < 0.05）, and gradually increased with the increase of triptolide concentration. Moreover, the protein expression levels of IRE1, JNK, c-Jun, p-JNK, and p-c-Jun in A375 cells in the triptolide groups also showed the same trend. After pretreatment with the JNK inhibitor SP600125 for 72 hours, the apoptosis rate was significantly lower in the SP600125 + 100 nmol/L triptolide group （21.88% ± 0.55%） than in the 100 nmol/L triptolide group without SP600125 pretreatment （30.78% ± 0.40%, t = -22.51, P < 0.001）, and the mRNA expression levels of IRE1, JNK, and c-Jun were also significantly decreased in the SP600125 + 100 nmol/L triptolide group compared with the 100 nmol/L triptolide group（all P < 0.05）. Conclusion Triptolide may induce apoptosis of human melanoma A375 cells by activating the IRE1/JNK signaling pathway.

Key words: Melanoma, Triptolide, Apoptosis, A375 cells, IRE1/JNK pathway