Different BAG-1 isoforms have distinct functions in modulating chemotherapeutic-induced apoptosis in breast cancer cells.
OBJECTIVE
BAG-1 is a multifunctional anti-apoptotic gene with four isoforms, and different BAG-1 isoforms have different anti-apoptotic functions. In this study, we transfected BAG-1 isoforms into the human breast cancer cell lines Hs578T (ER negative) and MCF-7 (ER positive) to study their effect on apoptosis with or without estrogens.


METHODS
The constructed recombinant expression vectors carrying individual BAG-1 isoforms was used to transfect human breast cancer cell lines Hs578T (ER negative) and MCF-7 (ER positive). After stable cell lines were made, a variety of apoptosis-inducing agents, including doxorubicin, docetaxel, and 5-FU, was used to treat these cell lines with or without estrogen to test the role of BAG-1. The mechanism by which BAG-1 affected the function of Bcl-2 was exploredby using the cycloheximide chase assay.


RESULTS
The BAG-1 p50 and p46 isoforms significantly enhanced the resistance to apoptosis in both cell lines according to flow cytometry analysis. BAG-1 p33 and p29 failed to protect the transfected cells from apoptosis. The cell viability assay showed that only BAG-1 p50, but not p46, p33, or p29, increased estrogen-dependent function in ER-positive cell line MCF-7. Only BAG-1 p50 dramatically increased its anti-apoptotic ability in the presence of estrogen, while estrogen has very little effect on the anti-apoptotic ability of other BAG-1 isoforms. In the detection of the expression of K-ras, Hsp70, cytochrome c, Raf-1, ER-alpha, and Bcl-2 in MCF-7 cells by Western blot, only Bcl-2 protein expression was significantly increased in MCF-7 cells transfected with BAG-1 p50 and p46, respectively. Furthermore, the cycloheximide chase assay indicated that the degradation of Bcl-2 protein was extended in the BAG-1 p50 and p46 transfected MCF-7 cells.


CONCLUSIONS
Distinct isoforms of BAG-1 have different anti-apoptotic functions in breast cancer cells, and that the BAG-1 p50 isoform can potentiate the role of estrogen in ER-positive breast cancer.
Liu
Hong-Yu
HY
Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin, China.
Wang
Zhuo-Min
ZM
Bai
Yun
Y
Wang
Min
M
Li
Ying
Y
Wei
Sen
S
Zhou
Qing-Hua
QH
Chen
Jun
J
D000970:Antineoplastic Agents / Q000627:therapeutic use*; D017209:Apoptosis / Q000502:physiology*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000473:pathology; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004967:Estrogens / Q000378:metabolism; D005260:Female; D006801:Humans; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D000970:Antineoplastic Agents; C091732:BCL2-associated athanogene 1 protein; D004268:DNA-Binding Proteins; D004967:Estrogens; D020033:Protein Isoforms; D011960:Receptors, Estrogen; D014157:Transcription Factors