Conjugated linoleic acid (CLA) inhibits rat mammary carcinogenesis, in part by inducing apoptosis of preneoplastic and neoplastic mammary epithelium. The current study focused on the mechanism by which apoptosis is induced. Treatment of TM4t mouse mammary tumor cells with t10,c12-CLA induced proteolytic cleavageof poly(ADP-ribose) polymerase (PARP), which is a hallmark of apoptosis. Using electron microscopy, we observed that this treatment resulted in marked dilatation of the lumen of the endoplasmic reticulum (ER), suggesting disruption of ER homeostasis and induction of ER stress. To investigate this, we examined the effect of t10,c12-CLA on key components of the ER stress pathways, including proteins involved in both the initial cell survival response, as well as proteins which lead to apoptosis if the stress is prolonged or severe. t10,c12-CLA induced an ER stress response in both a time- and concentration-dependent manner, as indicated by the induction or activation of the following proteins: the ER resident chaperone BiP/GRP78; the proximal ER membrane transducer PERK; eIF2α, which in its active phosphorylated state inhibits general protein synthesis; and ATF4, a transcription factor which induces pro-apoptotic genes such as CEBP-homologous protein (CHOP). CLA also activated the ER stress response pathways initiated by the ER membrane transducers ATF6 and IRE1, as indicated by the increased synthesis and splicing of XBP1 mRNA, which encodes a transcription factor that aids survival by increasing cellular levels of chaperones. With prolonged CLA treatment, expression of the downstream proapoptotic protein CHOP and cleavage of ER-specific caspase 12 were induced, concurrent with detection of PARP cleavage. Furthermore, knockdown of CHOP attenuated CLA-induced PARP cleavage. Our data demonstrate that t10,c12-CLA induces apoptosis of mouse mammary tumor cells, and suggest that apoptosis is mediated, at least in part, through an ER stress response that culminates in the induction of CHOP, a critical effector of CLA-induced apoptosis. The above effects were specific to t10,c12-CLA, since the c9,t11 isomer of CLA did not appear to disrupt ER homeostasis or induce ER stress. Supported by NIH CA61763 and by pre-doctoral DOD Grant W81XWH-06-1-0288.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA