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  • 彭彬,宋瑜婷,张雪,王莹,曹帆帆,张登海.雷公藤红素诱导未折叠蛋白反应信号通路中真核细胞翻译起始因子2α活化抑制多发性骨髓瘤细胞生长[J].第二军医大学学报,2019,40(9):963-969    [点击复制]
  • PENG Bin,SONG Yu-ting,ZHANG Xue,WANG Ying,CAO Fan-fan,ZHANG Deng-hai.Celastrol inhibits multiple myeloma cell growth by activating eukaryotic initiation factor 2α in unfolded protein response[J].Acad J Sec Mil Med Univ,2019,40(9):963-969   [点击复制]
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雷公藤红素诱导未折叠蛋白反应信号通路中真核细胞翻译起始因子2α活化抑制多发性骨髓瘤细胞生长
彭彬1△,宋瑜婷1,2△,张雪1,王莹1,曹帆帆1,张登海1*
0
(1. 海军军医大学(第二军医大学)附属公利医院中心实验室, 上海 200135;
2. 宁夏医科大学研究生院, 银川 750000
共同第一作者
*通信作者)
摘要:
目的 研究雷公藤红素抑制多发性骨髓瘤细胞增殖与未折叠蛋白反应(UPR)的关系和分子机制,为多发性骨髓瘤的治疗提供新的药物靶点。方法 4种多发性骨髓瘤细胞株RPMI 8226、U266、SKO、KMS-11,经不同浓度雷公藤红素(增殖实验0.0~10.0 μmol/L、凋亡实验0.0~4.0 μmol/L、周期阻滞实验0.0~1.5 μmol/L)处理不同时间(增殖实验1~3 d、凋亡实验1 d、周期阻滞实验1 d)后,检测细胞增殖、凋亡、周期阻滞情况;用蛋白质印迹法检测肌醇需求酶1(IRE1)、蛋白激酶样内质网激酶(PERK)和活化转录因子6(ATF6)3条UPR信号通路中主要分子的表达,包括葡萄糖调节蛋白78(GRP78)、ATF6、PERK、真核细胞翻译起始因子2α(eIF2α)、磷酸化eIF2α(p-eIF2α)、C/EBP同源蛋白(CHOP)、IRE1、磷酸化IRE1(p-IRE1)。用慢病毒包被的含短发夹RNA(shRNA)载体对eIF2α表达进行干扰,观察雷公藤红素对干扰eIF2α表达后的RPMI 8226细胞UPR信号分子表达、凋亡和细胞周期的影响。结果 雷公藤红素以剂量和时间依赖方式抑制4种多发性骨髓瘤细胞增殖、诱导凋亡、使细胞周期阻滞在G0/G1期,其中RPMI 8226细胞对雷公藤红素最敏感。在RPMI 8226细胞,雷公藤红素处理浓度在0.5~2.0 μmol/L作用30 min~24 h时均能使UPR的PERK通路中p-eIF2α表达水平升高(P<0.05或P<0.01),其下游CHOP表达也相应增加(P<0.05或P<0.01),而对其他2条通路ATF6和IRE1影响不明显。用慢病毒干扰eIF2α表达后,雷公藤红素上调CHOP表达、诱导凋亡和周期阻滞的作用均减弱或消失。结论 雷公藤红素能抑制多种多发性骨髓瘤细胞增殖,活化UPR的PERK信号通路中eIF2α分子可能是其作用机制之一。
关键词:  雷公藤红素  多发性骨髓瘤  未折叠蛋白反应  真核细胞翻译起始因子2α
DOI:10.16781/j.0258-879x.2019.09.0963
投稿时间:2019-04-17修订日期:2019-06-26
基金项目:上海市浦东新区卫生和计划生育委员会学科带头人项目(PWRd2018-12),海军军医大学(第二军医大学)附属公利医院人才培养计划(GLRb2018-01).
Celastrol inhibits multiple myeloma cell growth by activating eukaryotic initiation factor 2α in unfolded protein response
PENG Bin1△,SONG Yu-ting1,2△,ZHANG Xue1,WANG Ying1,CAO Fan-fan1,ZHANG Deng-hai1*
(1. Central Laboratory, Gongli Hospital, Naval Medical University(Second Military Medical University), Shanghai 200135, China;
2. Graduate School, Ningxia Medical University, Yinchuan 750000, Ningxia Hui Autonomous Region, China
Co-first authors.
* Corresponding author)
Abstract:
Objective To investigate the relationship between celastrol inhibition against multiple myeloma cell growth and unfolded protein response (UPR) and the related molecular mechanism, so as to provide new drug targets for multiple myeloma treatment. Methods Four multiple myeloma cell lines RPMI 8226, U266, SKO and KMS-11 were treated with different concentrations (proliferation:0.0-10.0 μmol/L; apoptosis:0.0-4.0 μmol/L; cell cycle:0.0-1.5 μmol/L) of celastrol for different periods (proliferation:1-3 d; apoptosis:1 d; cell cycle:1 d), and cell proliferation, apoptosis and cell cycle were examined. Western blotting analysis was used to detect the main molecules in the inositol-requiring enzyme 1 (IRE1), PRKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) signaling pathways of UPR, which included glucose-regulated protein 78 (GRP78), ATF6, PERK, eukaryotic initiation factor 2α (eIF2α), phosphorylated-eIF2α (p-eIF2α), C/EBP homologous protein (CHOP), IRE1 and phosphorylated-IRE1 (p-IRE1). After the lentivirus vector containing short hairpin RNA (shRNA) was used to interfere with eIF2α expression in RPMI 8226 cells, the effects of celastrol were detected on signaling molecule expression, apoptosis and cell cycle. Results Celastrol inhibited the proliferation of 4 multiple myeloma cells, induced apoptosis, and arrested the cell cycle at G0/G1 phase in dose- and time-dependent manners. RPMI 8226 cells were most sensitive to celastrol. For RPMI 8226 cells, when 0.5-2.0 μmol/L of celastrol was used for 30 min to 24 h, the p-eIF2α levels in the PERK signaling pathway of UPR were significantly increased (P<0.05 or P<0.01), and the downstream CHOP expression was risen correspondingly (P<0.05 or P<0.01), but the other two pathways ATF6 and IRE1 were not affected obviously. After interference of eIF2α expression with lentivirus vector containing shRNA, the effects of celastrol to increase CHOP expression, induce apoptosis and arrest cell cycle were significantly attenuated or disappeared. Conclusion Celastrol can inhibit the growth of a variety of multiple myeloma cells, and the activated eIF2α molecule in the UPR PERK signaling pathway may be one of the mechanisms.
Key words:  celastrol  multiple myeloma  unfolded protein response  eukaryotic initiation factor 2α