千金藤素增敏多柔比星抑制三阴性乳腺癌细胞增殖活性的机制研究

Cepharanthine sensitizes triple-negative breast cancer cells to doxorubicin and inhibits cell proliferation

目的探讨千金藤素增敏多柔比星抑制三阴性乳腺癌细胞增殖活性的机制。方法 MTT比色法测定不同浓度单用千金藤素(2、4、6、8、10μmol/L)或多柔比星(0.2、0.4、0.6、0.8、1.0μmol/L)及两者联合用药(千金藤素固定4μmol/L,多柔比星固定0.5μmol/L)对三阴性乳腺癌细胞MDA-MB-231和MDA-MB-468增殖的影响;流式细胞仪检测单用千金藤素或多柔比星及联合用药对三阴性乳腺癌细胞MDA-MB-231和MDA-MB-468凋亡的影响,以及对MDA-MB-231细胞内活性氧产生的影响;Western blot检测联合用药对细胞凋亡关键蛋白表达的影响;JC-1探针检测药物对线粒体膜电位的影响,激光共聚焦显微镜和透射电镜观察联合用药对细胞内线粒体形态和线粒体自噬体的影响。结果与单用多柔比星或千金藤素相比,联用千金藤素和多柔比星可明显抑制MDA-MB-231和MDA-MB-468细胞增殖(P<0.01),并且呈现协同效应(协同系数小于1);明显诱导MDA-MB-231和MDA-MB-468细胞凋亡(P<0.01),PARP-1剪切激活,Caspase 3激活,细胞色素C释放到细胞质,并且导致线粒体膜电位下降和线粒体自噬体的大量堆积,以及细胞内活性氧的大量产生(P<0.01)。结论千金藤素与多柔比星联用可引起三阴性乳腺癌细胞内活性氧大量产生和线粒体自噬体的堆积,导致线粒体损伤,进而诱导细胞凋亡。

Objective To explore the molecular mechanism by which cepharanthine(CEP) sensitizes triple-negative breast cancer cells to doxorubicin(DOX) chemotherapy. Methods The triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 were treated with CEP(2, 4, 6, 8, or 10 μmol/L) alone, DOX(0.2, 0.4, 0.6, 0.8, or 1.0 μmol/L) alone, or the combination of CEP(4 μmol/L) and DOX(0.5 μmol/L). MTT assay was used to test the cell viability after the treatments, and flow cytometry was used to analyze the cell apoptosis and the production of reactive oxygen species(ROS);Western blotting was performed to detect the expression of PARP-1, cleaved caspase-3 and cytochrome C in the cells. Transmission electron microscopy(TEM) was used to observe the ultrastructural changes of MDA-MB-231 cells in response to the treatments;the mitochondrial membrane potential of the cells was detected using JC-1 probe, and the mitochondrial morphology and mitophagosomes were observed with confocal laser scanning microscopy and TEM. Results Compared with CEP or DOX treatment alone, the combined treatment with CEP and DOX significantly suppressed the proliferation of both MDA-MB-231 and MDA-MB-468 cells(P<0.01) with an obvious synergistic effect(synergy coefficient <1). The combined treatment induced obvious apoptosis in the 2 cell lines(P<0.01) and caused the degradation of PARP-1, activation of caspase-3, and the release of cytochrome C. The combined treatment also resulted in significantly decreased mitochondrial membrane potential, accumulation of mitophagosomes, and a massive production of ROS in MDA-MB-231 cells(P<0.01). Conclusion CEP can sensitize triple-negative breast cancer cells to DOX chemotherapy probably by inducing the accumulation of mitophagosomes and increasing ROS production to cause mitochondrial damage and cell apoptosis.