Intrinsic mechanism of estradiol-induced apoptosis in breast cancer cells resistant to estrogen deprivation
Intrinsic mechanism of estradiol-induced apoptosis in breast cancer cells resistant to estrogen deprivation
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DOI：
10.1093/jnci/dji400
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发表时间：
2005-12-07
影响因子：
10.3
通讯作者：
Jordan, VC
中科院分区：
文献类型：
--
作者：
Lewis, JS;Meeke, K;Jordan, VC
背景。我们之前开发了一种雌激素受体（ER）阳性乳腺癌细胞系（MCF-7:5C），该细胞系对长期雌激素剥夺具有抵抗力，并在生理浓度的 17 β-雌二醇存在下经历快速且完全的细胞凋亡。在这里，我们研究了线粒体凋亡途径在此过程中的作用。方法：通过膜联蛋白 V-碘化丙啶双染色和 4',6-二脒基-2-苯基吲哚 (DAPI) 染色研究用雌二醇、氟维司群或媒介物（对照）处理的 MCF-7:5C 细胞的凋亡。还在用小干扰 RNA (siRNA) 瞬时转染凋亡途径成分的 MCF-7:5C 细胞中分析了细胞凋亡。通过蛋白质印迹分析测量细胞凋亡途径中间体的表达。通过罗丹明-123保留测定法测定线粒体跨膜电位(psi(m))。通过细胞色素 c 的释放和聚（ADP-核糖）聚合酶（PARP）蛋白的裂解来测定线粒体途径活性。在注射 MCF-7:5C 细胞的去势无胸腺小鼠中研究肿瘤发生。通过双样本t检验或单因素方差分析确定治疗组和对照组之间的差异。所有统计检验都是双面的。结果：与用氟维司群或媒介物处理的细胞相比，用雌二醇处理的 MCF-7:5C 细胞发生凋亡，并显示促凋亡蛋白表达增加、psi(m) 降低、细胞色素 c 释放增强和 PARP 裂解。相对于对照，通过 siRNA 阻断 Bax、Bim 和 p53 mRNA 表达，雌二醇诱导的细胞凋亡减少了 76% [95% 置信区间 (CI) = 73% 至 79%，P
Background. We previously developed an estrogen receptor (ER)-positive breast cancer cell line (MCF-7:5C) that is resistant to long-term estrogen deprivation and undergoes rapid and complete apoptosis in the presence of physiologic concentrations of 17 beta-estradiol. Here, we investigated the role of the mitochondrial apoptotic pathway in this process. Methods: Apoptosis in MCF-7:5C cells treated with estradiol, fulvestrant, or vehicle (control) was investigated by annexin V-propidium iodide double staining and 4',6-diamidino-2-phenylindole (DAPI) staining. Apoptosis was also analyzed in MCF-7:5C cells transiently transfected with small interfering RNAs (siRNAs) to apoptotic pathway components. Expression of apoptotic pathway intermediates was measured by western blot analysis. Mitochondrial transmembrane potential (psi(m)) was determined by rhodamine-123 retention assay. Mitochondrial pathway activity was determined by cytochrome c release and cleavage of poly(ADP-ribose) polymerase (PARP) protein. Tumorigenesis was studied in ovariectomized athymic mice that were injected with MCF-7:5C cells. Differences between the treatment groups and control group were determined by two-sample t test or one-factor analysis of variance. All statistical tests were two-sided. Results: MCF-7:5C cells treated with estradiol underwent apoptosis and showed increased expression of proapoptotic proteins, decreased psi(m), enhanced cytochrome c release, and PARP cleavage compared with cells treated with fulvestrant or vehicle. Blockade of Bax, Bim, and p53 mRNA expression by siRNA reduced estradiol-induced apoptosis relative to control by 76% [95% confidence interval (CI) = 73% to 79%, P