关键词: 黑色素瘤, 雷公藤内酯醇, 内质网应激, 细胞凋亡, A375细胞, JNK/c-Jun信号通路

Abstract: 【Abstract】 Objective To explore the role of c-Jun N-terminal kinase （JNK）/c-Jun signaling pathway mediated by endoplasmic reticulum stress in triptolide-induced apoptosis of melanoma A375 cells. Methods Nude mice were subcutaneously inoculated with melanoma A375 cells on the back to establish the animal model of melanoma. Tumor formation could be observed at approximately 3 weeks after inoculation, and then the mice were divided into 4 groups （4 mice in each group）: control group （injected with sodium chloride physiological solution via the tail vein）, 0.1-, 0.2-, and 0.4-mg/kg triptolide groups （injected with 0.1, 0.2, and 0.4 mg/kg triptolide via the tail vein, respectively）. Injections were performed twice a week. After 3 weeks of injections, tumors were resected, and their size and weight were measured. The apoptosis levels of tumor xenografts were detected by the terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling （TUNEL） assay. qPCR was conducted to determine the mRNA expression of inositol-requiring enzyme 1 （IRE1）, JNK, and c-Jun, and Western blot analysis to determine the protein expression of IRE1, JNK, c-Jun, phosphorylated-JNK （p-JNK）, and phosphorylated-c-Jun （p-c-Jun）. Comparisons among multiple groups were performed using one-way analysis of variance, and multiple comparisons were performed using Dunnett's test. Results Significant differences were observed in the tumor mass, volume and tumor suppression rate among the control group, 0.1-, 0.2-, and 0.4-mg/kg triptolide groups （all P < 0.05）; all the triptolide groups showed significantly decreased tumor masses and volumes （all P < 0.05）, but significantly increased tumor suppression rates compared with the control group （all P < 0.05）. The tumor apoptosis index significantly differed among the control group, 0.1-, 0.2-, and 0.4-mg/kg triptolide groups （7.67% ± 1.15%, 9.67% ± 3.21%, 62.00% ± 6.08%, and 85.67% ± 5.51%, respectively; F = 305.91, P < 0.01）, and the 0.2- and 0.4-mg/kg triptolide groups showed significantly increased tumor apoptosis indices compared with the control group （t = 17.56, 27.72, respectively, both P < 0.05）. qPCR and Western blot analysis revealed significant differences in the mRNA expression of IRE1, JNK, and c-Jun among the control group, 0.1-, 0.2-, and 0.4-mg/kg triptolide groups （F = 112.23, 27.51, 112.37, respectively, all P < 0.05）, as well as in the relative protein expression levels of IRE1, JNK, c-Jun, p-JNK, and p-c-Jun among the above 4 groups （all P < 0.05）. Additionally, the 0.4-mg/kg triptolide group showed significantly increased mRNA and protein expression of IRE1, JNK and c-Jun （including p-JNK, p-c-Jun） compared with the control group （all P < 0.05）. The mRNA and protein expression levels of IRE1, JNK, and c-Jun in the tumor tissues tended to increase with the rise in drug concentrations, and the protein expression levels of p-JNK and p-c-Jun showed the same trend. Conclusion Triptolide could activate the JNK/c-Jun signaling pathway mediated by the endoplasmic reticulum stress, and then induce apoptosis of melanoma A375 cells in mice.

Key words: Melanoma, Triptolide, Endoplasmic reticulum stress, Apoptosis, A375 cells, JNK/c-Jun signaling pathway