3466

Elevated expression or activity of heme oxygenase-1 (HO-1), a ubiquitous stress-responsive enzyme, has been reported to stimulate proliferation and to accelerate angiogenesis and metastasis in several types of tumor cells. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), an endogenous ligand of peroxisome proliferator-activated receptor γ, has been known to induce HO-1 in certain cell lines. In the present work, we found that treatment of human breast cancer (MCF-7) cells with nontoxic doses of 15d-PGJ2 (3-30 μM) led to concentration- and time-dependent increases in the expression and activity of HO-1. The induction of HO-1 expression preceded the up-regulation of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-1 (MMP-1). The up-regulation of VEGF and MMP-1 expression by 15d-PGJ2 was abrogated by pharmacological inhibition with zinc protophophyrin (ZnPP) or si-RNA knock down of HO-1. Moreover, ZnPP decreased the in vitro capillary formation induced by 15d-PGJ2 in human umbilical vein endothelial cells. Likewise, ZnPP treatment inhibited the migrative phenotype of 15d-PGJ2-treated MCF-7 cells as determined by the wound migration assay. In another experiment using luciferase reporter constructs containing different segments of promoter, the 15d-PGJ2-induced expression of HO-1 was found to be primarily governed by the distal enhancers (E1 and E2), which harbour three canonical ARE-like stress-responsive elements (StRE). Nrf2, a basic-leucine zipper transcription factor, plays a key role in regulating the antioxidant response element (ARE)-driven up-regulation of antioxidant enzymes including HO-1. 15d-PGJ2 treatment elevated nuclear translocation and subsequent ARE binding of Nrf2. MCF-7 cells transfected with dominant negative Nrf2 and Nrf2 si-RNA were less responsive to 15d-PGJ2 in terms of inducing expression of HO-1 and VEGF. Biotinylated 15d-PGJ2 bound to Keap1, a cytoplasmic repressor of Nrf2, which was confirmed by avidin pull-down. In contrast to 15d-PGJ2, 9,10-dihydro-15d-PGJ2 lacking an electrophilic α,β-unsaturated carbonyl moiety failed to activate Nrf2 and induce HO-1. Taken together, these results suggest that 15d-PGJ2 containing an electrophilic cyclopentenone moiety can covalently modify Keap1, presumably at the critical cysteine residue, thereby facilitating the dissociation of Nrf2 from this inhibitory protein. 15d-PGJ2-induced expression of HO-1 via the Nrf2 signaling may be implicated in angiogenesis and metastasis in human breast cancer.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA