Down-regulation of estrogen receptor-alpha and rearranged during transfection tyrosine kinase is associated with withaferin a-induced apoptosis in MCF-7 breast cancer cells.
Down-regulation of estrogen receptor-alpha and rearranged during transfection tyrosine kinase is associated with withaferin a-induced apoptosis in MCF-7 breast cancer cells.
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DOI：
10.1186/1472-6882-11-84
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发表时间：
2011-10-06
影响因子：
--
通讯作者：
Cohen MS
中科院分区：
文献类型：
--
作者：
Zhang X;Mukerji R;Samadi AK;Cohen MS
Withaferin A(Withaferin A，Withaferin A，Withaferin A)是一种天然存在的内酯类化合物，可诱导雌激素敏感型和非雌激素非依赖性乳腺癌MCF-7细胞株和非雌激素依赖性乳腺癌细胞株MCF-7细胞发生凋亡，但其作用机制尚不清楚。本研究的目的是观察水杨酸对人乳腺癌细胞株MCF7的抗癌作用，探讨水杨酸在体外对乳腺癌细胞雌激素受体α(ERα)及其相关分子表达的影响。用3-(4，5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium，MTS)比色法和台盼蓝拒染法检测Wa对MCF-7细胞活力和增殖的影响。Annexin V-异硫氰酸荧光素(FITC)/碘化丙啶(PI)流式细胞术和多聚ADP-核糖聚合酶(PARP)切割的Western印迹分析检测细胞凋亡。PI流式细胞仪分析细胞周期的影响。Western blotting还检测了ERα表达的变化以及与ERα功能相关的通路。Wa可抑制MCF-7细胞的生长，降低细胞存活率，IC50为576 nM，作用72 h，并引起剂量和时间依赖性的细胞凋亡和G2/M期细胞周期停滞。Wa诱导的细胞凋亡与ERα、转染期重排酪氨酸激酶和热休克因子1下调以及磷酸化p38丝裂原活化蛋白激酶、p53和p21蛋白表达上调有关。与蛋白质合成抑制剂放线菌酮或蛋白酶体抑制剂MG132共同处理表明，WA对ERα的耗竭是翻译后的，这是由于蛋白酶体依赖的ERα的降解。综上所述，下调ERα、RET、HSF1和上调磷酸化p38MAPK、P53、p21参与了Wa对乳腺癌MCF7细胞的促凋亡和生长抑制作用。WA对ERα蛋白水平的下调是由蛋白酶体依赖的ERα降解引起的。
Withaferin A (WA), a naturally occurring withanolide, induces apoptosis in both estrogen-responsive MCF-7 and estrogen-independent MDA-MB-231 breast cancer cell lines with higher sensitivity in MCF-7 cells, but the underlying mechanisms are not well defined. The purpose of this study was to determine the anti-cancer effects of WA in MCF-7 breast cancer cells and explore alterations in estrogen receptor alpha (ERα) and its associated molecules in vitro as novel mechanisms of WA action. The effects of WA on MCF-7 viability and proliferation were evaluated by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and trypan blue exclusion assays. Apoptosis was evaluated by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry and Western blot analysis of poly (ADP-ribose) polymerase (PARP) cleavage. Cell cycle effects were analyzed by PI flow cytometry. Western blotting was also conducted to examine alterations in the expression of ERα and pathways that are associated with ERα function. WA resulted in growth inhibition and decreased viability in MCF-7 cells with an IC50 of 576 nM for 72 h. It also caused a dose- and time-dependent apoptosis and G2/M cell cycle arrest. WA-induced apoptosis was associated with down-regulation of ERα, REarranged during Transfection (RET) tyrosine kinase, and heat shock factor-1 (HSF1), as well as up-regulation of phosphorylated p38 mitogen-activated protein kinase (phospho-p38 MAPK), p53 and p21 protein expression. Co-treatment with protein synthesis inhibitor cycloheximide or proteasome inhibitor MG132 revealed that depletion of ERα by WA is post-translational, due to proteasome-dependent ERα degradation. Taken together, down-regulation of ERα, RET, HSF1 and up-regulation of phospho-p38 MAPK, p53, p21 are involved in the pro-apoptotic and growth-inhibitory effects of WA in MCF-7 breast cancer cells in vitro. Down-regulation of ERα protein levels by WA is caused by proteasome-dependent ERα degradation.
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