Different BAG-1 isoforms have distinct functions in modulating chemotherapeutic-induced apoptosis in breast cancer cells

Abstract

Aim: BAG-1 is a multifunctional anti-apoptotic gene with four isoforms, and different BAG-1 isoforms have different antiapoptotic 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-α, 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. Conclusion: 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.

Modulation of BAG-1 Expression Alters the Sensitivity of Breast Cancer Cells to Tamoxifen

“…We have previously used BAG-1 siRNA to downregulate the expression of BAG-1 in the human lung adenocarcinoma cell line A549, and found a significantly enhanced sensitivity to cisplatin-induced apoptosis in the BAG-1 siRAN transfected cells [30]. Similar observations were made in the MCF-7 breast cancer cells transfected with the BAG-1 siRNA and treated with doxorubin, docetaxel and 5-FU [24,31] and in the human cervical cancer cell lines treated with cisplatin staurosporine, paclitaxel and doxorubicine [23]. Our paradoxical finding of BAG-1 being a positive prognostic yet negative predictive biomarker in breast cancer is important clinically.…”

“…Construction of plasmids containing the BAG-1 p50, p46, p33 was described previously [23,24], and all recombinant plasmids were confirmed by DNA sequencing. DNA transfection was performed using the Lipofactamine™ 2000 reagent (Invitrogen) according to the manufacturer's protocol.…”

“…BAG-1 p46 isoform resulted the most profound inhibition in apoptosis [23]. In addition, BAG-1 isoforms also had distinct function in modulation the chemotherapeutic-induced apoptosis in breast cancer cells [24]. The different anti-apoptotic function of BAG-1 isoforms may result from the unique N-tuminus of the BAG-1 p50, which was important in its nuclear localization and binding to hormone receptors, transcription factors and certain DNA promoters [33,34,35].…”

DNMT Inhibitors and HDAC Inhibitors Regulate E-Cadherin and Bcl-2 Expression in Endometrial Carcinoma in vitro and in vivo

“…Downregulation of Bcl-2 mRNA in leukemic patients using antisense technology resulted in increased apoptosis and potentiation of the effect of antineoplastic drugs in these patients [20]. Cell cycle arrest in G2/M or G0/G1 has been attributed to Bcl-2 [21].…”

Role of BCL2-Associated Athanogene 1 in Differential Sensitivity of Human Endothelial Cells to Glucocorticoids

“…49–55 Furthermore, BAG1L and BAG1M overexpression have shown strong antiapoptotic and cell mitotic effects in cancerous cells, by activating BCL2 and RAF1. 56 It has been reported that BAG1 clients compete with each other to sequester BAG1 proteins. 48,49 Because BAG1 protein levels are much lower than its clients, a small change in BAG1 isoform expression, or that of HSP70, RAF1, and BCL2, could alter the interaction of BAG1 with GRα, RAF1, BCL2, or HSP70 and, subsequently, alter vascular homeostasis.…”