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  • 戴丽霞,李亦蕾,余乐.硼替佐米联用obatoclax协同诱导人急性B淋巴细胞白血病细胞株Nalm-6细胞凋亡[J].第二军医大学学报,2020,41(2):151-160    [点击复制]
  • DAI Li-xia,LI Yi-lei,YU Le.Synergistic cytotoxic effect of bortezomib in combination with obatoclax on human acute B lymphoblastic leukemia cell line Nalm-6[J].Acad J Sec Mil Med Univ,2020,41(2):151-160   [点击复制]
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硼替佐米联用obatoclax协同诱导人急性B淋巴细胞白血病细胞株Nalm-6细胞凋亡
戴丽霞1,2,李亦蕾3*,余乐1*
0
(1. 南方医科大学药学院广东省新药筛选重点实验室, 器官衰竭防治国家重点实验室, 广州 510515;
2. 南方医科大学南方医院增城分院药学部, 广州 511300;
3. 南方医科大学南方医院药剂科, 广州 510515
*通信作者)
摘要:
目的 探讨硼替佐米分别与3种Bcl-2抑制剂(obatoclax、AT-101、ABT-199)联用诱导人急性B淋巴细胞株Nalm-6细胞凋亡的协同作用。方法 应用MTT法分别检测3种Bcl-2抑制剂单用或联用硼替佐米作用于Nalm-6细胞48 h后的细胞活力。通过流式细胞术和蛋白质印迹分析分别检测不同药物单独或联合作用于Nalm-6细胞后的细胞凋亡情况及Bcl-2家族蛋白、泛素、微管相关蛋白1轻链3B(LC3B)、p62、免疫球蛋白结合蛋白(Bip)、磷酸化p38、磷酸化JNK、C/EBP同源蛋白(CHOP)等蛋白的表达情况。同时利用qRT-PCR检测硼替佐米和obatoclax联用后细胞的内质网应激反应关键基因BipCHOP、活化转录因子(ATF4ATF6、肌醇需求酶1α(IRE1α)、X-盒结合蛋白1(XBP1)的表达水平。最后通过MTT和流式细胞术检测内质网应激反应抑制剂牛磺熊去氧胆酸(TUDCA)是否可以逆转两药联用诱导的细胞凋亡。结果 硼替佐米和3种Bcl-2抑制剂单用均可降低Nalm-6细胞的存活率。3组药物联用实验组中,仅有obatoclax+硼替佐米表现出协同细胞毒性作用,其余两组并无此现象。Obatoclax通过增加LC3B-Ⅱ、p62蛋白的表达水平抑制Nalm-6细胞的自噬活性。与单药相比,硼替佐米和obatoclax联用后泛素化蛋白的表达显著上调。硼替佐米联用obatoclax可同时抑制自噬和泛素蛋白酶体活性,造成大量蛋白蓄积,进而激活内质网应激反应,最终诱导Nalm-6细胞凋亡。内质网应激抑制剂TUDCA可削弱硼替佐米和obatoclax联用诱导的细胞凋亡。结论 硼替佐米联用obatoclax可同时抑制自噬和蛋白酶体活性,并激活内质网应激反应协同诱导人急性B淋巴细胞白血病细胞凋亡。
关键词:  急性B淋巴细胞白血病  硼替佐米  obatoclax  协同作用  自噬  内质网应激
DOI:10.16781/j.0258-879x.2020.02.0151
投稿时间:2019-10-21修订日期:2020-01-18
基金项目:国家自然科学基金(81773773),广州市科技计划项目(201607010350).
Synergistic cytotoxic effect of bortezomib in combination with obatoclax on human acute B lymphoblastic leukemia cell line Nalm-6
DAI Li-xia1,2,LI Yi-lei3*,YU Le1*
(1. State Key Laboratory of Organ Failure Research, Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, Guangdong, China;
2. Department of Pharmacy, Zengcheng Branch of Nanfang Hospital, Southern Medical University, Guangzhou 511330, Guangdong, China;
3. Department of Pharmacy, Nanfang Hospital, Southern Medical University, Guangzhou 510515, Guangdong, China
*Corresponding authors)
Abstract:
Objective To explore whether the combination of bortezomib and Bcl-2 inhibitor (obatoclax, AT-101, ABT-199) can synergistically induce the apoptosis of human acute B lymphoblastic leukemia cell line Nalm-6. Methods MTT assay was used to evaluate cell viability of Nalm-6 cells in response to Bcl-2 inhibitor alone or combined treatment for 48 h. Apoptosis was examined by flow cytometry and the expression of Bcl-2 family proteins, ubiquitin, microtubule-associated protein 1 light chain 3B (LC3B), p62, binding immunoglobulin protein (Bip), phosphorylated p38 (p-p38), phosphorylated c-Jun N-terminal kinase (p-JNK), and C/EBP homologous protein (CHOP) was detected by Western blotting after drug alone or combined treatment. The mRNA levels of critical factors of endoplasmic reticulum stress (ERS)response, including Bip, CHOP, activating transcription factor (ATF) 4, ATF6, inositol-requiring enzyme 1α (IRE1α) and X-box binding protein 1 (XBP1) were measured by qRT-PCR. Finally, MTT and flow cytometry were used to determine whether tauroursodeoxycholate acid (TUDCA, an ERS inhibitor) could reverse the apoptosis induced by the combination of the two drugs. Results The application of bortezomib, obatoclax, AT-101 and ABT-199 alone reduced the viability of Nalm-6 cells. Obatoclax potentiated the cytotoxicity of Nalm-6 cells in response to bortezomib, but not including AT-101 or ABT-199. Obatoclax blocked autophagy flux by upregulating the protein expression of LC3B-Ⅱ and p62. The accumulation of ubiquitin protein was observed after use of bortezomib or obatoclax alone, but the protein significantly increased after two drug combination. Bortezomib combined with obatoclax caused the dual blockade of autophagy and proteasome and a large amount of protein accumulation, leading to activated ERS, finally to cell apoptosis. TUDCA reduced the apoptosis induced by two drug combination. Conclusion Bortezomib in combination with obatoclax can simultaneously inhibit autophagy and protease activity, triggering ERS, finally inducing human acute B lymphoblastic leukemia cell apoptosis.
Key words:  acute B lymphoblastic leukemia  bortezomib  obatoclax  synergy  autophagy  endoplasmic reticulum stress