过氧化麦角甾醇衍生物对人三阴性乳腺癌细胞增殖、迁移和侵袭的影响

Effect of ergosterol peroxide derivatives on the proliferation,migration and invasion of human triple negative breast cancer cell

目的研究过氧化麦角甾醇衍生物EP-3P对人三阴性乳腺癌细胞MDA-MB-231增殖、迁移和侵袭的影响,为乳腺癌治疗相关药物的开发提供参考。方法采用MTT法检测0(空白对照)、1.25、2.5、5、10、20、40μmol/L EP-3P作用于MDA-MB-231细胞24、48、72 h后细胞的增殖情况。以0(空白对照)、5、10、20μmol/L EP-3P作用于MDA-MB-231细胞24 h后,分别采用细胞划痕法、Transwell小室法检测细胞迁移和侵袭能力,采用流式细胞术分别测定细胞凋亡和周期分布情况,并采用Western blot法检测细胞中B淋巴细胞瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、胱天蛋白酶3(caspase-3)、活化的caspase-3(cleaved-caspase-3)、细胞色素C(Cyt-C)、基质金属蛋白酶2(MMP-2)、MMP-9蛋白的表达水平。结果与空白对照比较,2.5、5、10、20、40μmol/L EP-3P均可显著升高细胞的增殖抑制率(P<0.05或P<0.01),且呈浓度和时间依赖趋势。10、20μmol/L EP-3P作用24 h后,均可显著降低/减少细胞迁移愈合率和侵袭细胞数(P<0.01),将细胞周期阻滞于G_(2)/M期(P<0.05或P<0.01),显著升高细胞的凋亡率(P<0.05),并且可显著上调细胞中Bax、Cyt-C、cleaved-caspase-3蛋白表达和下调细胞中Bcl-2、caspase-3、MMP-2、MMP-9蛋白表达(P<0.01)。结论EP-3P可通过线粒体介导的内源性caspase途径抑制人乳腺癌细胞MDA-MB-231的增殖、迁移和侵袭,并可诱导细胞发生凋亡。

OBJECTIVE To study the effects of ergosterol peroxide derivatives EP-3P on the proliferation,migration and invasion of human tripe negative breast cancer cell MDA-MB-231,and to provide reference for the development of breast cancer related drugs.METHODS MTT assay was adopted to detect the proliferation of MDA-MB-231 cells after treated with 0(blank control),1.25,2.5,5,10,20,40μmol/L EP-3P for 24,48 and 72 h.Wound healing assay and Transwell chamber method were adopted to detect the migration and invasion ability of MDA-MB-231 cells after treated with 0(blank control),5,10,20 EP-3P for 24 h.The apoptosis and cell cycle distribution were detected by flow cytometry.Western blot assay was used to detect the expressions of B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),caspase-3,cleaved-caspase-3,cytochrome C(Cyt-C),matrix metalloproteinase-2(MMP-2)and MMP-9.RESULTS Compared with blank control group,2.5,5,10,20,40μmol/L EP-3P could significantly increase the inhibitory rate of cell proliferation(P<0.05 or P<0.01)in a dose and timedependent manner.After 24 h treatment of EP-3P(10,20μmol/L),the rate of cell migration and the number of invasive cells were decreased significantly(P<0.01),and cell was arrested at G_(2)/M stage(P<0.05 or P<0.01);the apoptotic rate was increased significantly(P<0.05);the protein expressions of Bax,Cyt-C and cleaved-caspase-3 were upregulated significantly,while those of Bcl-2,caspase-3,MMP-2 and MMP-9 were downregulated significantly(P<0.01).CONCLUSIONS EP-3P can inhibit the proliferation,migration and invasion of human tripe negative breast cancer cells MDA-MB-231 through mitochondrial mediated endogenous caspase pathway,and induce the apoptosis of cells.