毛钩藤碱通过线粒体途径诱导乳腺癌MDA-MB-231细胞凋亡
黄器伟¹, 翟娜娜¹, 黄涛^1,*, 李道明^2
1黄河科技学院，郑州 450099；²郑州大学第一附属医院病理科，郑州 450052
摘要
本研究旨在观察毛钩藤碱对人乳腺癌细胞的抑制作用，并探讨其分子机制。选取人正常乳腺上皮MCF-10A细胞、乳腺癌MCF-7细胞和MDA-MB-231细胞作为研究对象，采用CCK-8法检测细胞活性，采用流式细胞术检测细胞凋亡和线粒体膜电位(mitochondrial membrane potential, MMP)，采用Western blot检测Bcl-2、Bax、cleaved-caspase 9、cleaved-caspase 3以及胞浆中细胞色素C (cytochrome C, Cyt C)的蛋白水平。结果显示，毛钩藤碱可显著降低MCF-7细胞和MDA-MB-231细胞存活率，且该作用呈时间和剂量依赖性(P < 0.05)；毛钩藤碱作用MCF-7细胞和MDA-MB-231细胞48 h的IC50分别为447.79和179.06 μmol/L；毛钩藤碱可诱导MDA-MB-231细胞发生凋亡和MMP去极化(P < 0.05)，促进线粒体释放Cyt C (P < 0.05)，激活caspase 9和caspase 3 (P < 0.05)，这些作用均可被线粒体通透性转换孔(mitochondrial permeability transition pore, MPTP)特异性阻断剂环孢素A (cyclosporin A, CsA)显著抑制(P < 0.05)；此外，毛钩藤碱显著下调MDA-MB-231细胞Bcl-2蛋白水平并上调Bax蛋白水平(P < 0.05)。以上结果提示，毛钩藤碱可诱导MDA-MB-231细胞发生凋亡，这可能与其调低Bcl-2/Bax蛋白比值，从而引起MPTP持续开放和Cyt C释放，最终导致caspase 9和caspase 3活化有关。

关键词： 毛钩藤碱; 乳腺癌; 凋亡; 环孢素A; 线粒体膜电位
分类号：R737.9

Hirsutine induces apoptosis of human breast cancer MDA-MB-231 cells through mitochondrial pathway
HUANG Qi-Wei¹, ZHAI Na-Na¹, Huang Tao^1,*, LI Dao-Ming^2
1Huanghe Science and Technology College, Zhengzhou 450099, China；²Department of Pathology, Zhengzhou University First Affiliated Hospital, Zhengzhou 450052, China
Abstract
The aim of this study was to investigate the effects of hirsutine on apoptosis of breast cancer cells and its possible mechanism. The MCF-10A, MCF-7 and MDA-MB-231 cells were treated with hirsutine at different concentrations for 48 h or incubated with 160 μmol/L hirsutine for 24, 48, and 72 h. The MCF-10A cell line is a non-tumorigenic epithelial cell line, and the MCF-7 and MDA-MB-231 are human breast adenocarcinoma cell lines. CCK-8 assay was employed to detect the cell viability. Flow cytometry was used to assay the apoptosis and mitochondrial membrane potential (MMP). The protein expressions of Bcl-2, Bax, cleaved-caspase 9, cleaved-caspase 3 and cytochrome C (Cyt C) in the MDA-MB-231 cells were detected by Western blotting. The results showed that hirsutine remarkably reduced the viability of MCF-7 and MDA-MB-231 cells in a time- and dose-dependent manner (P < 0.05) with IC50 values of 447.79 and 179.06 μmol/L, respectively. In the MDA-MB-231 cells, hirsutine induced apoptosis and depolarization of MMP (P < 0.05), released Cyt C from mitochondria (P < 0.05), and activated caspase 9 and caspase 3 (P < 0.05). However, these effects induced by hirsutine were all inhibited by cyclosporin A (CsA) (P < 0.05), a specific inhibitor of mitochondrial permeability transition pore (MPTP). In addition, hirsutine down-regulated the protein level of Bcl-2 and up-regulated the protein level of Bax (P < 0.05). These results suggest that hirsutine may induce apoptosis of human breast cancer MDA-MB-231 cells through decreasing the ratio of Bcl-2 to Bax, opening MPTP, releasing Cyt C from mitochondria, and activating caspase 9 and caspase 3.

Key words: hirsutine;;;;

收稿日期：2017-06-14　　录用日期：2018-01-24
通讯作者：黄涛　　E-mail: hytzn@163.com
DOI: 10.13294/j.aps.2018.0010
引用本文：
黄器伟, 翟娜娜, 黄涛, 李道明. 毛钩藤碱通过线粒体途径诱导乳腺癌MDA-MB-231细胞凋亡[J]. 生理学报 2018; 70 (1): 40-46.
HUANG Qi-Wei, ZHAI Na-Na, Huang Tao, LI Dao-Ming. Hirsutine induces apoptosis of human breast cancer MDA-MB-231 cells through mitochondrial pathway. Acta Physiol Sin 2018; 70 (1): 40-46 (in Chinese with English abstract).



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