吡柔比星诱导人三阴性乳腺癌 MDA-MB-231 细胞周期阻滞和凋亡。
Pyoluteorin induces cell cycle arrest and apoptosis in human triple-negative breast cancer cells MDA-MB-231.
机构信息
State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, Shanghai, China.
Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
出版信息
J Pharm Pharmacol. 2020 Jul;72(7):969-978. doi: 10.1111/jphp.13262. Epub 2020 Apr 4.
OBJECTIVES
To screen the cytotoxic activity of six secondary metabolites isolated from soil fungus Aspergillus niger. Importantly, to investigate the mechanism that pyoluteorin induced human triple-negative breast cancer MDA-MB-231 cells apoptosis in vitro.
METHODS
The cell viability assay was tested with CTG assay. Cell cycle, apoptosis and intracellular reactive oxygen species (ROS) production assay were tested with flow cytometry. Additionally, intracellular ROS production assay and mitochondrial membrane potential assay were determined with laser scanning confocal microscopy. The expression of apoptosis-related proteins was determined with Western blot.
KEY FINDINGS
Pyoluteorin displayed significantly selective cytotoxicity against human triple-negative breast cancer MDA-MB-231 cells (IC = 0.97 µm) with low toxicity against human breast epithelial cell MCF-10A. It was found that pyoluteorin could arrest MDA-MB-231 cells cycle at G /M phase and induce cell apoptosis. Further experiments demonstrated that the apoptosis-inducing effect of pyoluteorin was related to reduction of mitochondrial membrane potential, accumulation of ROS and change of apoptosis-related protein expressions.
CONCLUSION
Our studies revealed that pyoluteorin had potent proliferation inhibition against MDA-MB-231 cells through arresting cell cycle at G /M phase and inducing caspase-3-dependent apoptosis by mitochondrial pathway, implying that pyoluteorin may be a potential lead compound for drug discovery of human triple-negative breast cancer.
目的
筛选从土壤真菌黑曲霉中分离得到的六种次生代谢产物的细胞毒性。重要的是，要研究吡咯并[2,1-f][1,2,4]三嗪-1,4-二酮（pyoluteorin）在体外诱导人三阴性乳腺癌 MDA-MB-231 细胞凋亡的机制。
方法
采用 CTG 法检测细胞活力。采用流式细胞术检测细胞周期、细胞凋亡和细胞内活性氧（ROS）产生。此外，还通过激光共聚焦扫描显微镜测定细胞内 ROS 产生和线粒体膜电位。采用 Western blot 测定凋亡相关蛋白的表达。
主要发现
吡咯并[2,1-f][1,2,4]三嗪-1,4-二酮对人三阴性乳腺癌 MDA-MB-231 细胞具有显著的选择性细胞毒性（IC50=0.97µM），对人乳腺上皮细胞 MCF-10A 的毒性较低。结果表明，吡咯并[2,1-f][1,2,4]三嗪-1,4-二酮可将 MDA-MB-231 细胞周期阻滞在 G/M 期，并诱导细胞凋亡。进一步的实验表明，吡咯并[2,1-f][1,2,4]三嗪-1,4-二酮诱导凋亡的作用与线粒体膜电位降低、ROS 积累和凋亡相关蛋白表达变化有关。
结论
我们的研究表明，吡咯并[2,1-f][1,2,4]三嗪-1,4-二酮通过阻滞细胞周期于 G/M 期并通过线粒体途径诱导 caspase-3 依赖性凋亡，对 MDA-MB-231 细胞具有很强的增殖抑制作用，这表明吡咯并[2,1-f][1,2,4]三嗪-1,4-二酮可能是治疗人三阴性乳腺癌的潜在药物先导化合物。
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