Ca2+ influx-mediated dilation of the endoplasmic reticulum and c-FLIPL downregulation trigger CDDO-Me-induced apoptosis in breast cancer cells

Abstract

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 Ca²⁺ levels, reflecting influx from the extracellular milieu, make a critical contribution to ER-derived vacuolation and subsequent cell death. In parallel with increasing Ca²⁺ levels, CDDO-Me markedly increased the generation of reactive oxygen species (ROS). Interestingly, there exists a reciprocal positive-regulatory loop between Ca²⁺ 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 Ca²⁺ 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.