Wang Yu, Wang Xingang, Zhao Hui, Liang Bo, Du Qihang
Department of Breast Surgery, the Affiliated Hospital of Medical College, Qingdao University, China.
J Chemother. 2012 Dec;24(6):348-57. doi: 10.1179/1973947812Y.0000000049.
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.
分泌型簇集素（sClu）是一种抗凋亡蛋白，在保护细胞免受肿瘤坏死因子（TNF）-α诱导的凋亡中发挥作用。本研究的目的是探讨sClu对乳腺癌细胞中TNF-α诱导凋亡作用的分子机制。将表达野生型p53的MCF-7细胞系进行改造以使其过表达sClu（MCF-7/sClu），而将具有突变型p53的MDA-MB-231细胞系用sClu沉默小干扰RNA（MDA-MB-231/sClu siRNA）进行转染。在体外检测了簇集素过表达和下调对凋亡及对TNF-α敏感性的影响。我们的结果表明，TNF-α处理可增加乳腺癌细胞中Bcl-2的mRNA和蛋白水平，提示Bcl-2在响应TNF-α时受核因子-κB（NF-κB）直接调控。Bcl-2的诱导由NF-κB的p65亚基介导。小干扰RNA介导的Bcl-2沉默导致TNF-α诱导的凋亡显著增加。MDA-MB-231/sClu siRNA细胞中sClu的沉默消除了TNF-α介导的NF-κB激活和Bcl-2过表达，并使MDA-MB-231/sClu siRNA细胞对TNF-α介导的凋亡比亲本细胞显著更敏感。此外，MCF-7/sClu细胞中sClu的过表达促进了TNF-α介导的NF-κB活性和Bcl-2过表达，并使MCF-7/Clu细胞对TNF-α介导的凋亡显著更具抗性。抑制NF-κB活性或p65及Bcl-2表达可逆转这些效应。目前的结果表明，sClu通过NF-κB激活和Bcl-2过表达赋予乳腺癌细胞对TNF-α诱导凋亡的抗性。
