内质网应激介导的JNK/c-Jun信号通路在雷公藤内酯醇诱导小鼠体内黑色素瘤A375细胞凋亡中的作用研究

Role of JNK/c-Jun signaling pathway mediated by endoplasmic reticulum stress in triptolide-induced apoptosis of melanoma A375 cells in mice

目的探讨雷公藤内酯醇(TP)通过内质网应激介导的JNK/c-Jun信号通路诱导黑色素瘤A375细胞凋亡的相关机制。方法裸鼠背部皮下注射接种黑色素瘤A375细胞, 建立黑色素瘤动物模型。肿瘤形成3周后, 将裸鼠随机分为0(对照组)、0.1、0.2、0.4 mg/kg浓度TP组, 每组4只, 每周注射相应浓度药物2次, 3周后摘除肿瘤, 测量瘤体大小及重量。用TUNEL法检测肿瘤组织的凋亡水平。采用qPCR检测肿瘤组织肌醇酶1(IRE-1)、c-Jun氨基酸末端激酶(JNK)、c-Jun的mRNA水平, 采用Western印迹检测IRE-1、JNK、c-Jun及磷酸化JNK、c-Jun(p-JNK、p-c-Jun)蛋白水平。多组间比较采用单因素方差分析, 采用Dunnett''s检验进行多重比较。结果对照组和0.1、0.2、0.4 mg/kg TP组肿瘤质量、体积及抑瘤率差异均有统计学意义(均P < 0.05), 各药物组肿瘤质量、体积均低于对照组(均P < 0.05), 抑瘤率均高于对照组(均P < 0.05)。对照组和0.1、0.2、0.4 mg/kg TP组肿瘤凋亡指数(7.67% ± 1.15%、9.67% ± 3.21%、62.00% ± 6.08%、85.67% ± 5.51)差异有统计学意义(F = 305.91, P < 0.001), 0.2、0.4 mg/kg TP组肿瘤凋亡指数高于对照组(t = 17.56、27.72, 均P < 0.05)。qPCR和Western印迹显示, 对照组、0.1 ~ 0.4 mg/kg TP组肿瘤IRE1、JNK、c-Jun mRNA水平差异均有统计学意义(F = 112.23、27.51、112.37, 均P < 0.05), IRE1、JNK、c-Jun、p-JNK、p-c-Jun相对表达水平差异均有统计学意义(均P < 0.05), 0.4 mg/kg TP组IRE1、JNK、c-Jun mRNA及蛋白(包括p-JNK、p-c-Jun)表达均高于对照组(均P < 0.05)。肿瘤组织中IRE1、JNK、c-Jun mRNA和蛋白表达水平均随药物作用浓度增加有升高趋势, p-JNK和p-c-Jun蛋白表达水平变化也呈相同的趋势。结论 TP可以激活内质网应激介导的JNK/c-jun信号通路并诱导黑色素瘤A375细胞凋亡。

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.