Department of Medical Oncology, The First Hospital, China Medical University, Heping District, Shenyang, Liaoning Province, China.
Acta Oncol. 2011 Jun;50(5):693-9. doi: 10.3109/0284186X.2010.543144. Epub 2010 Dec 22.
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.
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.
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.
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.
他莫昔芬（TAM）是一种非甾体抗雌激素药物，通过其抗雌激素活性被广泛用于乳腺癌的治疗。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 阴性细胞均具有细胞毒性。然而，这种作用的分子机制尚不清楚。本研究探讨了 c-Src、ERK、AKT 和 c-Cbl 泛素连接酶在 TAM 诱导 MCF-7 细胞凋亡过程中的作用。
通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）测定法和流式细胞术分别测定 MCF-7 细胞增殖和凋亡。通过 Western 印迹法测定 c-Cbl 表达和 c-Src、ERK、AKT 的活性。通过瞬时转染编码 c-Cbl 和 70Z/Cbl 的质粒来实现 c-Cbl 的野生型和显性失活型（70Z/Cbl）的过表达，并通过 Western 印迹法进行确认。使用 t 检验进行统计学分析，p 值<0.05 被认为具有统计学意义。
高浓度 TAM（25 μM）诱导 MCF-7 细胞呈时间依赖性凋亡。在 TAM 诱导的凋亡过程中，ERK1/2 和 AKT 被激活。ERK1/2 抑制剂 PD98059、PI3K/Akt 抑制剂 LY294002 和 c-Src 抑制剂 PP2 均增强了 TAM 的作用。此外，在这个过程中，泛素连接酶 c-Cbl 被上调。c-Cbl 的过表达显著增强了 TAM 的促凋亡作用，而 70Z/Cbl 则抑制了 TAM 的促凋亡作用。进一步研究表明，c-Cbl 的过表达显著下调了 c-Src 蛋白水平和 TAM 诱导的 AKT 活性。但是，70Z/Cbl 显著上调了 TAM 诱导的 ERK 和 AKT 活性。
本研究表明，c-Src、ERK 和 AKT 在 TAM 诱导的凋亡过程中发挥了保护作用，而 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。
