中华皮肤科杂志 ›› 2018, Vol. 51 ›› Issue (4): 288-293.doi: 10.3760/cma.j.issn.0412-4030.2018.04.010

• 论著 • 上一篇    下一篇

CHOP依赖内质网应激通路在雷公藤内酯醇诱导黑素瘤A375细胞凋亡中的作用机制

张亚美1,孙悦鑫1,陶玥2,包军3   

  1. 1. 南京大学医学院附属鼓楼医院
    2. 南京大学医学院附属鼓楼医院皮肤性病科
    3. 南京大学医学院附属鼓楼医院皮肤科
  • 收稿日期:2017-05-12 修回日期:2017-07-21 发布日期:2018-03-29
  • 通讯作者: 陶玥 E-mail:peachyue@126.com
  • 基金资助:
    江苏省自然科学基金

Role of CHOP-dependent endoplasmic reticulum stress signaling pathway in triptolide-induced apoptosis of A375 melanoma cells and its mechanisms

Ya-Mei ZHANG1,Yuexin SUN2,Yue TAOJun Bao   

  • Received:2017-05-12 Revised:2017-07-21 Published:2018-03-29
  • Contact: Yue TAO E-mail:peachyue@126.com
  • Supported by:
    National Natural Science Foundation of Jiangsu Province of China

PDF

31

摘要： 目的 探讨CHOP依赖的内质网应激通路在雷公藤内酯醇诱导黑素瘤A375细胞凋亡中的作用机制。方法 培养人黑素瘤A375细胞，用12.5、25、50、100、200 nmol/L雷公藤内酯醇处理，以不加雷公藤内酯醇处理的细胞作为阴性对照组。分别作用一定时间后，光镜观察A375细胞形态变化，CCK8法检测药物对细胞的增殖抑制作用，以双染色法流式细胞仪检测细胞凋亡，透射电镜观察内质网形态变化，Western印迹检测细胞GRP78、p?PERK、PERK及CHOP蛋白的表达及GRP78 蛋白在药物作用下随时间变化的趋势，qPCR检测GRP78、PERK、CHOP mRNA相对表达量。结果 雷公藤内酯醇作用后，A375细胞变为细长梭形，胞质变少，细胞大小不一，形状不规则，出现较多突起。CCK8实验显示，不同浓度雷公藤内酯醇作用A375细胞24、48、72 h均对A375有抑制增殖作用，且与时间、浓度呈依赖关系（P < 0.05），24、48、72 h时半数抑制浓度分别为308、83、55 nmol/L。12.5、25、50、100、200 nmol/L雷公藤内酯醇组细胞凋亡率分别为10.3% ± 0.1%、14.6% ± 0.8%、17.4% ± 0.7%、21.1% ± 1.0%、29.5% ± 1.1%，均高于阴性对照组（3.3% ± 0.4%），差异均有统计学意义（P < 0.05）。200 nmol/L 雷公藤内酯醇作用A375细胞24 h后透射电镜观察到内质网形态呈现损伤性改变。不同浓度雷公藤内酯醇作用A375细胞24 h后，GRP78、p?PERK、PERK、 CHOP蛋白表达逐渐增高（P < 0.05），而GRP78蛋白表达量随药物作用时间的延长逐渐减少（P < 0.05）。qPCR结果示，不同浓度雷公藤内酯醇作用A375细胞24 h后，GRP78、PERK、CHOP mRNA相对表达量随着药物浓度的增加而增加，除12.5 nmol/L浓度组PERK mRNA外，各浓度雷公藤内酯醇组GRP78、PERK、CHOP mRNA与阴性对照组比较，差异均有统计学意义（P < 0.05）。结论 雷公藤内酯醇能够诱导内质网发生应激，随着药物作用浓度的增加和时间的延长，A375细胞会启动CHOP依赖的内质网应激反应性凋亡途径。

关键词: 雷公藤内酯, 黑色素瘤, 实验性, 内质网应激, 细胞凋亡

Abstract: Zhang Yamei, Sun Yuexin, Tao Yue, Bao Jun Department of Dermatology and Venereology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing 210008, China Corresponding authors: Tao Yue, Email: peachyue@126.com; Bao Jun, Email: baojun1968@sina.com 【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 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 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 of GRP78 after treatment over time. Real-time fluorescence-based quantitative PCR （qPCR） was conducted to measure the mRNA 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 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 of GRP78 gradually decreased over time （P < 0.05）. qPCR showed that the mRNA 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 of GRP78, PERK and CHOP （P < 0.05） except the 12.5-nmol/L triptolide group with similar mRNA 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.

Key words: Triptolide, Melanoma, experimental, Endoplasmic reticulum stress, Apoptosis

引用本文

张亚美 孙悦鑫 陶玥 包军. CHOP依赖内质网应激通路在雷公藤内酯醇诱导黑素瘤A375细胞凋亡中的作用机制[J]. 中华皮肤科杂志, 2018,51(4):288-293. doi:10.3760/cma.j.issn.0412-4030.2018.04.010

Ya-Mei ZHANG Yuexin SUN Yue TAO Jun Bao. Role of CHOP-dependent endoplasmic reticulum stress signaling pathway in triptolide-induced apoptosis of A375 melanoma cells and its mechanisms[J]. Chinese Journal of Dermatology, 2018, 51(4): 288-293.doi:10.3760/cma.j.issn.0412-4030.2018.04.010