Evidence Supporting a Role for Calcium in Apoptosis Induction by the Synthetic Triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic Acid (CDDO)

The synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) is a novel anticancer agent that induces apoptosis in tumor cells. The cytotoxic stress underpinning CDDO-induced apoptosis has not been established. This study compared and contrasted the effects of CDDO on COLO 16 human skin cancer cells and their respiration-deficient (Ρ⁰) clones to elucidate the stress signal responsible for initiating apoptosis. CDDO promoted apoptosis in COLO 16 cells in a dose- and time-dependent manner. The Ρ ⁰ clones appeared to be more sensitive to CDDO-induced apoptosis implying that the disruption of mitochondrial respiration was not directly associated with triggering cell death. After a 4-h exposure to CDDO, mitochondrial inner transmembrane potential-sensitive dyes revealed mitochondrial hyperpolarization in the COLO 16 cells and mitochondrial depolarization in the Ρ⁰ clones. Electron microscopy illustrated that this exposure also promoted mitochondrial condensation, endoplasmic reticulum dilation, and chromatin condensation in the COLO 16 cells. Endoplasmic reticulum dilation and chromatin condensation were also observed in the ρ⁰ clones, but the mitochondria in these cells were markedly swollen implying that the disruption of intracellular Ca²⁺ homeostasis was associated with cell death. A Ca²⁺-sensitive dye confirmed that CDDO increased cytoplasmic free Ca²⁺ in the COLO 16 cells, their ρ⁰ clones, as well as in malignant breast and lung epithelial cells. A cell-permeant Ca²⁺ chelator reduced the CDDO-induced increase in cytoplasmic free Ca²⁺, and inhibited caspase activation, the development of apoptotic morphology, and DNA fragmentation in the COLO 16 cells, implying that Ca²⁺ played a pivotal role in signaling the initiation of apoptosis.