Signaling pathways mediating conjugated linoleic acid-induced apoptosis
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Conjugated linoleic acid (CLA) is a powerful anticancer agent in a number of tumor model systems. CLA inhibits rat mammary carcinogenesis, in part by inducing apoptosis of preneoplastic and neoplastic mammary epithelium. The current study focused on understanding the mechanism of the anticancer effect of CLA. In cell culture, we found that t10,c12 CLA, a component of synthetic CLA supplements, induced apoptosis and G1 arrest of p53 mutant TM4t murine mammary tumor cells. Furthermore, t10,c12-CLA induced a time- and concentration-dependent cleavage of caspases-3 and -9, and release of cytochrome c from mitochondria to cytosol. Levels of Bcl-2 protein were decreased both in total cellular lysates and in mitochondria after t10,c12-CLA treatment; however, there was no significant change in Bax or Bak. Overexpression of Bcl-2 attenuated apoptosis in response to t10,c12-CLA treatment. These results demonstrate that t10,c12-CLA triggers apoptosis of p53 mutant murine mammary tumor cells through the mitochondrial pathway by targeting Bcl-2. In addition, using electron microscopy, we observed that t10,c12-CLA treatment resulted in marked dilatation of the lumen of the endoplasmic reticulum (ER) in TM4t mammary tumor cells, suggesting disruption of ER homeostasis and induction of ER stress. Indeed, t10,c12-CLA induced proapoptotic C/EBP-homologous protein (CHOP) concurrent with the cleavage of poly (ADP-ribose) polymerase (PARP). Knockdown of CHOP attenuated t10,c12-CLA-induced apoptosis. Furthermore, t10,c12-CLA induced cleavage of ER-resident caspase-12, and a selective inhibitor of caspase-12 significantly decreased t10,c12-CLA-induced apoptosis. Together, these data suggested that t10,c12-CLA induces apoptosis through ER stress. To further explore the ER stress pathway, we examined the expression of the following upstream ER stress signature markers in response to CLA treatment: XBPI mRNA (unspliced and spliced); phospho-eIF2α, ATF4 and BiP proteins. We found that t10,c12-CLA induced expression and splicing of XBPI mRNA, as well as phosphorylation of eIF2α. In contrast, ATF4 was induced modestly, but not significantly, and BiP was not altered. Our data demonstrate that apoptosis induced by t10,c12-CLA is mediated, at least in part, through an atypical ER stress response that culminates in the induction of CHOP and cleavage of caspase-12. Furthermore, we found that pre-feeding CLA to BALB/c mice significantly decreased TM4t mammary tumor latency and tumor growth. Both c9,t11 and t10,c12 isomers of CLA were equally effective in inducing apoptosis of TM4t mammary tumor cells in vivo, as determined by TUNEL immunohistochemistry. In summary, we found that CLA has the ability to induce apoptosis in TM4t tumor cells both in vitro and in vivo. The apoptotic effect of CLA on the tumor cells might be one potential mechanism by which CLA exerts its chemopreventive effects on breast cancer. Our findings will help to define an optimal CLA strategy for chemoprevention.