Vitamin D enhances caspase-dependent and -independent TNFα-induced breast cancer cell death: The role of reactive oxygen species and mitochondria

Abstract

Calcitriol, the hormonal form of vitamin D1 potentiates the activity of some common anticancer drugs and agents of the anticancer immune system, including tumor necrosis factor α (TNFα). TNFα-induced cytotoxicity is due to both caspase-dependent and -independent pathways. Cotreatment with calcitriol enhanced both modes of TNFα-induced death in MCF-7 breast cancer cells. It increased caspase-3-like activity as assayed by the cleavage of poly-(ADP-ribose)polymerase and of the fluorogenic substrate ac-DEVD-AMC. It also enhanced TNFα-induced caspase-independent cytotoxicity in the presence of the pan-caspase inhibitor zD-2,6-dichlorobenzoyloxymethylketone. The antioxidants N-acetylcysteine, reduced glutathione, lipoic acid and ascorbic acid markedly reduced the enhancing effect of the hormone on TNFα-induced caspase activation. N-acetylcysteine and reduced glutathione also decreased caspase-independent cytotoxicity in the presence or absence of calcitriol, indicating that reactive oxygen species (ROS) have a key role in the cross talk between TNFα and calcitriol. Mitochondrial damage is common to both TNFα-induced caspase-dependent and -independent pathways and may underlie excessive production of ROS. Mitochondrial membrane potential (ΔΨ) was assessed by the specific potential-sensitive fluorescent probe JC-I. The hormone augmented the drop in ΔΨ and release of cytochrome c from mitochondria, induced by TNFα. The effect of calcitriol on ΔΨ was mimicked by rotenone, which increased both the drop in ΔΨ and caspase activation induced by TNFα. It is possible that the interaction of TNFα and calcitriol on the level of the mitochondria is the underlying mechanism responsible for the enhancement of TNFα-induced, ROS-mediated caspase-dependent and -independent cell death. © 2003 Wiley-Liss, Inc.