Abstract

Resveratrol (RES), a natural plant polyphenol, has gained interest as a nontoxic chemopreventive agent capable of inducing tumor cell death in a variety of cancer types. However, the early molecular mechanisms of RES-induced apoptosis are not well defined. Using the human breast cancer cell lines MDA-MB-231 and MCF-7, we demonstrate that RES is antiproliferative and induces apoptosis in a concentration- and time-dependent manner. Preceding apoptosis, RES instigates a rapid dissipation of mitochondrial membrane potential by directly targeting mitochondria. This is followed by release of cytochrome c and second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI (Smac/DIABLO) into the cytoplasm and substantial increase in the activities of caspases-9 and -3 in MDA-MB-231 cells. In addition, live cell microscopy demonstrates that RES causes an early biphasic increase in the concentration of free intracellular calcium ([Ca²⁺]_i), probably resulting from depletion of the endoplasmic reticulum stores in breast cancer cells. In caspase-3–deficient MCF-7 cells, apoptosis is mediated by the Ca²⁺-activated protease, calpain, leading to the degradation of plasma membrane Ca²⁺-ATPase isoform 1 and fodrin; the degradation is attenuated by buffering [Ca²⁺]_i and blocked by calpain inhibitors. Mitochondrial permeability transition pore antagonists also blocked calpain activation. In vivo mouse xenograft studies demonstrate that RES treatment inhibits breast cancer growth with no systemic toxicities. Together, these results suggest a critical role for mitochondria not only in the intrinsic apoptotic pathway but also in the Ca²⁺ and calpain-dependent cell death initiated by RES. Thus, RES may prove useful as a nontoxic alternative for breast cancer treatment.