15-Deoxy-Δ12,14-prostaglandin J2 Induces Apoptosis in Ha-ras-transformed Human Breast Epithelial Cells by Targeting IκB kinase-NF-κB Signaling.

Abstract

15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), an endogenous ligand for PPARγ, has differential effects on cancer cell proliferation and survival depending on the dose and the type of cells. In the present study, we have investigated the effects of 15d-PGJ2 on apoptosis of the Ha-ras transformed human breast epithelial (MCF10A-ras) cells. When MCF10A-ras cells were treated with 15d-PGJ2 (10 μM) for 24 hours, they underwent apoptosis as evidenced by characteristic morphological features, an increased proportion of sub-G0/G1 cell population, a typical pattern of annexin V/propidium iodide staining, perturbation of mitochondrial transmembrane potential (Δψm), and cleavage of caspase-3 and its substrate PARP. A pan-caspase inhibitor, Z-Val-Ala-Asp (OCH3)-fluoromethyl ketone attenuated cytotoxicity and proteolytic cleavage of caspase-3 induced by 15d-PGJ2. The 15d-PGJ2-induced apoptosis was accompanied by enhanced intracellular accumulation of reactive oxygen species (ROS), which was abolished by the antioxidant N-acetyl-L-cysteine (NAC). 15d-PGJ2 inhibited the DNA binding activity of NF-κB which was associated with inhibition of expression and catalytic activity of IκB kinase β (IKKβ). 15d-PGJ2-mediated inhibition of IKKβ and nuclear translocation of phospho-p65 was blocked by NAC treatment. 9,10-Dihydro-PGJ2, a non-electrophilic analogue of 15d-PGJ2, failed to produce ROS, to inhibit NF-κB DNA binding, and to induce apoptosis, suggesting that the electrophilic α,β-unsaturated carbonyl group of 15d-PGJ2 is essential for its pro-apoptotic activity. 15d-PGJ2-induced inactivation of IKKβ was also attributable to its covalent thiol modification at the cysteine 179 residue of IKKβ. Based on these findings, we propose that 15d-PGJ2 inactivates IKKβ–ΝF-κB signaling through oxidative or covalent modification of IKKβ, thereby inducing apoptosis in Ha-ras transformed human breast epithelial cells.