Clusterin confers resistance to TNF-alpha-induced apoptosis in breast cancer cells through NF-kappaB activation and Bcl-2 overexpression.

Secretory clusterin (sClu) is an anti-apoptotic protein that plays a role in protecting cells from Tumour-necrosis factor (TNF)-alpha-induced apoptosis. The aim of the present study was to investigate the molecular mechanisms underlying the effect of sClu on TNF-alpha-induced apoptosis in breast cancer cells. The wild-type p53 expressing MCF-7 cell line was engineered to overexpress sClu (MCF-7/sClu), whereas the MDA-MB-231 cell line with mutant p53 was transfected with a sClu silencing siRNA (MDA-MB-231/sClu siRNA). The effects of clusterin overexpression and downregulation on apoptosis and sensitivity to TNF-alpha were examined in vitro. Our results showed that TNF-alpha treatment increased Bcl-2 mRNA and protein levels in breast cancer cells, suggesting that Bcl-2 is directly regulated by nuclear factor-kappaB (NF-kappaB) in response to TNF-alpha. The induction of Bcl-2 was mediated by the p65 subunit of NF-kappaB. siRNA-mediated silencing of Bcl-2 led to a significant increase in TNF-alpha-induced apoptosis. Silencing of sClu in MDA-MB-231/sClu siRNA cells abrogated TNF-alpha-mediated NF-kappaB activation and Bcl-2 overexpression, and rendered the MDA-MB-231/sClu siRNA cells significantly more sensitive to TNF-alpha-mediated apoptosis than the parental cells. Furthermore, overexpression of sClu in MCF-7/sClu cells promoted TNF-alpha-mediated NF-kappaB activity and Bcl-2 overexpression, and rendered the MCF-7/Clu cells significantly more resistant to TNF-alpha-mediated apoptosis. Inhibition of NF-kappaB activity or p65 and Bcl-2 expression reversed these effects. The present results suggested that sClu confers breast cancer cells resistance to TNF-alpha-induced apoptosis through NF-kappaB activation and Bcl-2 overexpression.