雷公藤红素通过ROS／JNK通路诱导骨肉瘤细胞周期阻滞、凋亡和自噬的研究  

Celastrol induces cycle arrest, apoptosis and autophagy via ROS/JNK pathway in osteosarcoma cells

作　　者：李恒元[1] 孙凌凌[1] 李冰皓[1] 朱汀[1] 王盛东[1] 任海勇[1] 谢涛[1] 林秾[1] 叶招明[1] 

机构地区：[1]浙江大学医学院附属第二医院骨科,杭州310009

出　　处：《中华骨科杂志》2016年第8期490-502,共13页Chinese Journal of Orthopaedics

基　　金：国家自然科学基金项目（81172547）

摘　　要：目的探讨雷公藤红素对骨肉瘤的增殖抑制作用及其机制。方法以HOS、MG-63、U2-OS和Saos-2骨肉瘤细胞系和原代骨肉瘤细胞株为研究对象，采用细胞增殖实验和平板克隆形成实验测定雷公藤红素抑制骨肉瘤细胞增殖的效果，流式细胞术和Western Blot检测药物对骨肉瘤细胞周期分布的影响以及相关周期调控蛋白的变化，Hoechst染色、透射电镜、流式细胞术和Western Blot检测药物诱导肿瘤细胞发生的凋亡和自噬现象。荧光探针和Western Blot检测上游通路活性氧（reactive oxygen species，ROS）和c-Jun氨基末端激酶（c—Jun N—terminal kinase，JNK）分子的改变。通过应用凋亡抑制剂、自噬抑制剂、ROS和JNK抑制剂以验证骨肉瘤细胞中上述通路被雷公藤红素激活。构建裸鼠骨肉瘤模型进行体内实验，并通过Western Blot分析动物实验结果。结果雷公藤红素通过引起肿瘤细胞G2／M期阻滞而对骨肉瘤细胞有较强的增殖抑制作用。同时药物激活caspase-3、-8和-9，通过内、外源两条途径诱导肿瘤细胞凋亡；雷公藤红素还引起自噬，自噬和凋产互相作用，共同促进了骨肉瘤细胞死亡。雷公藤红素还引起ROS增高，而后激活JNK信号通路。JNK抑制剂很大程度上抑制了雷公藤红素引起的凋亡和自噬，而ROS抑制剂则完全逆转了此种效应。同时ROS抑制剂也能阻止JNK通路的激活和G2／M期周期阻滞。雷公藤红素对原代骨肉瘤细胞株也具有抑制肿瘤增殖的效应。动物实验表明雷公藤红素体内良好的抑瘤能力和安全性。结论雷公藤红素通过ROS／JNK通路诱导G2／M周期阻滞、凋广和自噬性死亡的产生，具有显著地抑制骨肉瘤细胞增殖的作用。[Abstract] Objective To explore the effect of celastrol on human osteosarcoma and the underlying mechanisms. Methods Osteosareoma cell lines （HOS, MG-63, U-2OS and Saos-2） and primary cells were used for test. The anti-proliferative effect of celastrol on osteosarcoma cells was determined by the MTS kit and clone formation assay. Cell cycle was monitored by flow cytometry and Western Blot for related cell cycle proteins. Hoechst staining, transmission electron microscopy, flow cytometry and Western Blot were used for detection of apoptosis and autophagy induced by celastrol. The level of ROS and JNK were investigated by fluorescent probe and Western Blot assay. Meanwhile, corresponding inhibitors of apoptosis （z-VAD）, autophagy （3-MA）, ROS （NAC） and JNK （SP600125） were added for confirming the phenomenon above. Finally, osteosareoma xenograft experiment was performed, the result of which was determined by Western Blot assay. Results Celastrol could potently inhibit cell proliferation of osteosarcoma by causing G2/M phase arrest. Exposure to celastrol resulted in the activation of caspase-3, -8, and -9, indicating that eelastrol induced apoptosis through both extrinsic and intrinsic pathways. Autophagy also occurred in celastrol-treated cells as evidenced by formation of autophagosome and accumulation of LC3B-Ⅱ. There exist complex interplays between apoptosis and autophagy. Inhibition of apoptosis enhanced autophagy while suppression of autophagy diminished apoptosis. The celastrol-induced cell death was remarkably restored by the combination of autophagy and apoptosis inhibitors. Celastrol also induced JNK activation and ROS generation. JNK inhibitor significantly attenuated celastrol-triggered apoptosis and autophagy while ROS scavenger could completely reverse them. ROS scavenger also prevented G2/M phase arrest and phosphorylation of JNK. Importantly, celastrol had the similar effects on primary osteosarcoma cells and suppressed tumor growth in the mouse xenograft model with sound safet

关 键 词：雷公藤 骨肉瘤 细胞凋亡 自噬 活性氧 

分 类 号：R285[医药卫生—中药学]