药学学报, 2021, 56(7): 1911-1920
引用本文:
甄虹恒, 游方芳, 程凡, 邹坤, 陈重旭, 陈剑锋*. 吉祥草活性成分RCE-4与塞来昔布联合应用抗宫颈癌Ca Ski细胞增殖效果与机制研究[J]. 药学学报, 2021, 56(7): 1911-1920.
ZHEN Hong-heng, YOU Fang-fang, CHENG Fan, ZOU Kun, CHEN Chong-xu, CHEN Jian-feng*. The mechanism of RCE-4, an active ingredient of Reineckia carnea, in combination with celecoxib on the anti-proliferation of cervical cancer Ca Ski cells[J]. Acta Pharmaceutica Sinica, 2021, 56(7): 1911-1920.

吉祥草活性成分RCE-4与塞来昔布联合应用抗宫颈癌Ca Ski细胞增殖效果与机制研究
甄虹恒, 游方芳, 程凡, 邹坤, 陈重旭, 陈剑锋*
三峡大学生物与制药学院, 三峡大学天然产物研究与利用湖北省重点实验室, 湖北 宜昌 443002
摘要:
本研究旨在探讨RCE-4与非甾体抗炎药联用抗宫颈癌Ca Ski细胞增殖的协同作用及分子机制。采用MTT法和CalcuSyn V2.0软件检测细胞增殖并计算联合用药指数(combination index,CI);Western blot法分析蛋白表达;Jc-1染色检测线粒体膜电位;吖啶橙/溴化乙锭(acridine orange/ethidium bromide,AO/EB)染色检测细胞凋亡;免疫共沉淀法检测Bcl-2-Beclin 1复合体的相对含量。结果显示,塞来昔布与RCE-4的协同效果最好,最佳协同指数为0.32。二者联用能显著抑制环氧合酶-2(cyclooxygenase-2,COX-2)和核转录因子κB (nuclear transcription factor kappa B,NF-κB)的表达,促进非甾体抗炎药活化因子1(non-steroidal anti-inflammatory drugs activity gene-1,NAG-1)的表达。同时,联用也显著抑制了微管相关蛋白轻链3(microtubule-associated protein 3,LC3)-Ⅱ、Beclin 1、p62以及自噬相关基因(autophagy-related gene,ATG)3/4B/5/7/14的表达,促进了线粒体膜电位的去极化,显著抑制了B淋巴细胞瘤-2(B cell lymphoma-2,Bcl-2)和B淋巴细胞瘤-xl (B cell lymphoma-xl,Bcl-xl)的表达,促进了Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、Bcl-2/Bcl-xl相关死亡启动因子(Bcl-2/Bcl-xl-associated death promoter,Bad)以及活化的含半胱氨酸的天冬氨酸蛋白水解酶(cleaved cysteinyl aspartate specific proteinase,cleaved-caspase)3/7/9的表达。另外,二者联用也极显著地抑制了Bcl-2-Beclin 1复合体的形成。综上所述,RCE-4与塞来昔布联用可能通过抑制Bcl-2-Beclin 1复合体的形成,抑制RCE-4诱导的自噬并增强其凋亡,从而提高RCE-4对宫颈癌Ca Ski细胞的敏感性。
关键词:    RCE-4      塞来昔布      凋亡      自噬      Bcl-2-Beclin 1复合体     
The mechanism of RCE-4, an active ingredient of Reineckia carnea, in combination with celecoxib on the anti-proliferation of cervical cancer Ca Ski cells
ZHEN Hong-heng, YOU Fang-fang, CHENG Fan, ZOU Kun, CHEN Chong-xu, CHEN Jian-feng*
College of Biological and Pharmaceutical Sciences of China Three Gorges University, Hubei Key Laboratory of Natural Products Research and Development, Yichang 443002, China
Abstract:
This research explored the synergistic effects and the potential mechanisms of RCE-4 and various nonsteroidal anti-inflammatory drugs (NSAIDs) on the proliferation of cervical cancer Ca Ski cells. The MTT assay and CalcuSyn V2.0 software were used to detect cell proliferation and calculate the combination index (CI); the expression levels of various proteins were analyzed using Western blot assay; mitochondrial membrane potential (MMP) was assessed using JC-1 staining; acridine orange/ethidium bromide (AO/EB) double-fluorescence staining was used to detect the apoptosis of Ca Ski cells; a co-immunoprecipitation (Co-IP) assay was used to analyze the relative content of Bcl-2-Beclin 1 complex in Ca Ski cells. The results demonstrate that the combination of RCE-4 and NSAIDs increases the inhibition of Ca Ski cells compared to the single-RCE-4 group, and celecoxib provided the best synergistic effect among the four NSAIDs tested, with a CI of 0.32. The combination of RCE-4 and celecoxib significantly down-regulated the expression of cyclooxygenase-2 (COX-2) and nuclear transcription factor-κB (NF-κB), and promoted the expression of non-steroidal anti-inflammatory drugs activity gene-1 (NAG-1). In addition, autophagy induced by RCE-4 was markedly inhibited in combination with celecoxib, which was associated with down-regulation of the expression of microtubule-associated protein 3 (LC3)-Ⅱ, Beclin 1, p62 and autophagy-related gene (ATG) 3/4B/5/7/14. RCE-4-induced apoptosis was significantly enhanced by altering the depolarization of mitochondrial membrane potential and the expression of B cell lymphoma-2 (Bcl-2), B cell lymphoma-xl (Bcl-xl), Bcl-2 associated X protein (Bax), Bcl-2/Bcl-xl-associated death promoter (Bad) and cleaved cysteinyl aspartate specific proteinase (cleaved-caspase) 3/7/9. Furthermore, the formation of the Bcl-2-Beclin 1 complex was significantly inhibited in Ca Ski cells treated with RCE-4 in combination with celecoxib. Taken together, this research shows that the combination of RCE-4 and celecoxib has a significant synergistic effect on the proliferation of Ca Ski cells by promoting apoptosis, inhibiting autophagy and disturbing the formation of the Bcl-2-Beclin 1 complex, which may be a novel strategy to increase the sensitivity of anti-cervical cancer drugs.
Key words:    RCE-4    celecoxib    apoptosis    autophagy    Bcl-2-Beclin 1 complex   
收稿日期: 2021-01-01
DOI: 10.16438/j.0513-4870.2021-0004
基金项目: 国家自然科学基金资助项目(81773952).
通讯作者: 陈剑锋,Tel:15997621712,E-mail:chenjianfeng2003@126.com
Email: chenjianfeng2003@126.com
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