RESUMO
15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), an endogenous ligand for PPARγ, has differential effects on cancer cell proliferationand survival depending on the dose and the type of cells. In the present study, we have investigated the effects of 15d-PGJ2on apoptosis of the Ha-ras transformed human breast epithelial (MCF10A-ras) cells. When MCF10A-ras cells were treated with15d-PGJ2 (10 μM) for 24 hours, they underwent apoptosis as evidenced by characteristic morphological features, an increasedproportion of sub-G0/G1 cell population, a typical pattern of annexin V/propidium iodide staining, perturbation of mitochondrial transmembranepotential (Δψm), and cleavage of caspase-3 and its substrate PARP. A pan-caspase inhibitor, Z-Val-Ala-Asp (OCH3)-fluoromethylketone attenuated cytotoxicity and proteolytic cleavage of caspase-3 induced by 15d-PGJ2. The 15d-PGJ2-induced apoptosiswas accompanied by enhanced intracellular accumulation of reactive oxygen species (ROS), which was abolished by theantioxidant N-acetyl-L-cysteine (NAC). 15d-PGJ2 inhibited the DNA binding activity of NF-κB which was associated with inhibitionof 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, toinhibit NF-κB DNA binding, and to induce apoptosis, suggesting that the electrophilic α,b-unsaturated carbonyl group of 15d-PGJ2is essential for its pro-apoptotic activity. 15d-PGJ2-induced inactivation of IKKβ was also attributable to its covalent thiol modificationat the cysteine 179 residue of IKKβ. Based on these findings, we propose that 15d-PGJ2 inactivates IKKβ–ΝF-κB signaling throughoxidative or covalent modification of IKKβ, thereby inducing apoptosis in Ha-ras transformed human breast epithelial cells.