Ubiquitin ligase c-Cbl is involved in tamoxifen-induced apoptosis of MCF-7 cells by downregulating the survival signals
Ubiquitin ligase c-Cbl is involved in tamoxifen-induced apoptosis of MCF-7 cells by downregulating the survival signals
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泛素连接酶 c-Cbl 通过下调生存信号参与他莫昔芬诱导的 MCF-7 细胞凋亡
DOI：
10.3109/0284186x.2010.543144
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发表时间：
2011-06-01
期刊：
影响因子：
3.1
通讯作者：
Qu, Xiu-Juan
中科院分区：
文献类型：
--
作者：
Yan, Shun-Chao;Liu, Yun-Peng;Qu, Xiu-Juan
抽象背景。他莫昔芬 (TAM) 是一种非甾体抗雌激素，通过其抗雌激素活性广泛用于治疗乳腺癌。最近的研究表明，TAM 通过诱导细胞凋亡对雌激素受体 (ER) 阳性和 ER 阴性细胞具有细胞毒性。然而，这种效应的分子机制尚不清楚。在本研究中，我们研究了 c-Src、ERK、AKT 和 c-Cbl 泛素连接酶在 TAM 诱导的 MCF-7 细胞凋亡过程中的作用。材料和方法。分别通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑(MTT)测定和流式细胞术测量MCF-7细胞增殖和凋亡。通过Western blotting检测c-Cbl的表达以及c-Src、ERK、AKT的活性。野生型和显性失活型c-Cbl (70Z/Cbl)的过表达是通过分别编码c-Cbl和70Z/Cbl的质粒瞬时转染实现的，并通过蛋白质印迹法证实。使用t检验进行统计分析，p值<0.05被认为具有统计显着性。结果。高浓度 TAM (25 μM) 诱导 MCF-7 细胞发生时间依赖性凋亡。 ERK1/2 和 AKT 在 TAM 诱导的细胞凋亡过程中被激活。 ERK1/2 抑制剂 PD98059、PI3K/Akt 抑制剂 LY294002 和 c-Src 抑制剂 PP2 均增强 TAM 作用。此外，泛素连接酶 c-Cbl 在此过程中上调。 c-Cbl的过表达显着增强了TAM的凋亡诱导作用，而70Z/Cbl则抑制了TAM的凋亡诱导作用。进一步研究表明，c-Cbl 的过表达显着下调 c-Src 蛋白水平和 TAM 诱导的 AKT 活性。但 70Z/Cbl 显着上调 TAM 诱导的 ERK 和 AKT 活性。结论。本研究表明，c-Src、ERK 和 AKT 在 TAM 诱导的细胞凋亡过程中发挥保护作用，并且 c-Cbl 通过调节 c-Src 的表达以及 TAM 诱导的 ERK 和 AKT 活性，使 MCF-7 细胞对 TAM 敏感。
Abstract Background. Tamoxifen (TAM) is a nonsteroidal antiestrogen that has been widely used in the treatment of breast cancer through its anti-estrogen activity. Recent studies show that TAM is cytotoxic to both estrogen receptor (ER)-positive and ER-negative cells via the induction of apoptosis. However, the molecular mechanisms of this effect are not well understood. In the present study, we investigated the roles of c-Src, ERK, AKT and c-Cbl ubiquitin ligases during TAM-induced apoptosis of MCF-7 cells. Material and methods. MCF-7 cell proliferation and apoptosis were measured by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and flow cytometry, respectively. c-Cbl expression, and the activity of c-Src, ERK, AKT were assayed by Western blotting. Overexpression of the wild and the dominant-negative type of c-Cbl (70Z/Cbl) were achieved by transient transfection of plasmids encoding c-Cbl and 70Z/Cbl, respectively, and were confirmed by Western blotting. Statistical analysis was performed using the t-test, and a p-value <0.05 was considered to be statistically significant. Results. A high concentration of TAM (25 μM) induced a time-dependent apoptosis of MCF-7 cells. ERK1/2 and AKT were activated during TAM-induced apoptosis. The ERK1/2 inhibitor PD98059, the PI3K/Akt inhibitor LY294002, and the c-Src inhibitor PP2 all enhanced TAM action. Moreover, the ubiquitin ligase c-Cbl was up-regulated during this process. Over-expression of c-Cbl significantly enhanced the apoptosis-inducing effects of TAM, while 70Z/Cbl suppressed the apoptosis-inducing effects of TAM. Further investigation revealed that, overexpression of c-Cbl significantly downregulated the c-Src protein levels and TAM-induced AKT activity. But 70Z/Cbl significantly upregulated TAM-induced ERK and AKT activity. Conclusions. This study demonstrates that c-Src, ERK, and AKT played a protective role during TAM-induced apoptosis, and that c-Cbl sensitized MCF-7 cells to TAM by modulating the expression of c-Src, and TAM-induced ERK and AKT activity.