1. Ubiquitin ligase c-Cbl is involved in tamoxifen-induced apoptosis of MCF-7 cells by downregulating the survival signals.
- Author
-
Yan, Shun-Chao, Liu, Yun-Peng, Zhang, Ling-Yun, Qu, Jing-Lei, Xu, Ling, Liu, Jing, Zhang, Ye, Hou, Ke-Zuo, Teng, Yue-E, and Qu, Xiu-Juan
- Subjects
ANALYSIS of variance ,APOPTOSIS ,BIOLOGICAL assay ,BREAST tumors ,CELL culture ,CHEMILUMINESCENCE assay ,COMPUTER software ,ENZYMES ,FLOW cytometry ,PROTEIN kinases ,PROTEINS ,REGRESSION analysis ,RESEARCH funding ,T-test (Statistics) ,TAMOXIFEN ,WESTERN immunoblotting ,DATA analysis ,PHARMACODYNAMICS - 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 μμM) 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. [ABSTRACT FROM AUTHOR]
- Published
- 2011
- Full Text
- View/download PDF