Ca(2+) influx-mediated dilation of the endoplasmic reticulum and c-FLIP(L) downregulation trigger CDDO-Me–induced apoptosis in breast cancer cells

The synthetic triterpenoid 2-cyano-3, 12-dioxooleana-1, 9(11)-dien-C28-methyl ester (CDDO-Me) is considered a promising anti-tumorigenic compound. In this study, we show that treatment with CDDO-Me induces progressive endoplasmic reticulum (ER)-derived vacuolation in various breast cancer cells and ultimately kills these cells by inducing apoptosis. We found that CDDO-Me–induced increases in intracellular Ca(2+) levels, reflecting influx from the extracellular milieu, make a critical contribution to ER-derived vacuolation and subsequent cell death. In parallel with increasing Ca(2+) levels, CDDO-Me markedly increased the generation of reactive oxygen species (ROS). Interestingly, there exists a reciprocal positive-regulatory loop between Ca(2+) influx and ROS generation that triggers ER stress and ER dilation in response to CDDO-Me. In addition, CDDO-Me rapidly reduced the protein levels of c-FLIP(L) (cellular FLICE-inhibitory protein) and overexpression of c-FLIP(L) blocked CDDO-Me–induced cell death, but not vacuolation. These results suggest that c-FLIP(L) downregulation is a key contributor to CDDO-Me–induced apoptotic cell death, independent of ER-derived vacuolation. Taken together, our results show that ER-derived vacuolation via Ca(2+) influx and ROS generation as well as caspase activation via c-FLIP(L) downregulation are responsible for the potent anticancer effects of CDDO-Me on breast cancer cells.