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Recently, a number of steps in the progression of metastatic disease have been shown to be regulated by redox signalling. Electrophilic lipids affect redox signalling through the post-translational modification of critical cysteine residues in proteins. However, the therapeutic potential as well as the precise mechanisms of action of electrophilic lipids in cancer cells is poorly understood. In the present study, we investigate the effect of the electrophilic prostaglandin 15d-PGJ2 (15-deoxy-Delta12,14-prostaglandin J2) on metastatic properties of breast cancer cells. 15d-PGJ2 was shown to decrease migration, stimulate focal-adhesion disassembly and cause extensive F-actin (filamentous actin) reorganization at low concentrations (0.03-0.3 microM). Importantly, these effects seem to be independent of PPARgamma (peroxisome-proliferator-activated receptor gamma) and modification of actin or Keap1 (Kelch-like ECH-associated protein 1), which are known protein targets of 15d-PGJ2 at higher concentrations. Interestingly, the p38 inhibitor SB203580 was able to prevent both 15d-PGJ2-induced F-actin reorganization and focal-adhesion disassembly. Taken together, the results of the present study suggest that electrophiles such as 15d-PGJ2 are potential anti-metastatic agents which exhibit specificity for migration and adhesion pathways at low concentrations where there are no observed effects on Keap1 or cytotoxicity.

Prototypical electrophiles such as the lipid 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) are well recognized for their therapeutic potential. Electrophiles modify signalling proteins in both the cytosol and mitochondrion, which results in diverse cellular responses, including cytoprotective effects and, at high doses, cell death. These findings led us to the hypothesis that targeting electrophiles to specific compartments in the cell could fine-tune their biological effects. To examine this, we synthesized a novel mitochondrially targeted analogue of 15d-PGJ2 (mito-15d-PGJ2) and tested its effects on redox cell signalling. Mito-15d-PGJ2 caused profound defects in mitochondrial bioenergetics and mitochondrial membrane depolarization when compared with 15d-PGJ2. We also found that mito-15d-PGJ2 modified different members of the electrophile-responsive proteome, was more potent at initiating intrinsic apoptotic cell death and was less effective than 15d-PGJ2 at up-regulating the expression of HO-1 (haem oxygenase-1) and glutathione. These results demonstrate the feasibility of modulating the biological effects of electrophiles by targeting the pharmacophore to mitochondria.

The COX (cyclo-oxygenase) pathway generates the reactive lipid electrophile 15d-PGJ2 (15-deoxy-Delta(12,14)-prostaglandin J2), which forms covalent protein adducts that modulate cell signalling pathways. It has been shown that this regulates important biological responses, including protection against oxidative stress, and supports the proposal that 15d-PGJ2 has pharmacological potential. Protective pathways activated by 15d-PGJ2 include those controlling the synthesis of the intracellular antioxidants GSH and the enzyme HO-1 (haem oxygenase-1). The induction of the synthesis of these intracellular antioxidants is, in large part, regulated by covalent modification of Keap1 (Kelchlike erythroid cell-derived protein with 'capn'collar homologyassociated protein 1) by the lipid and the subsequent activation of the EpRE (electrophile-response element). For the first time, we show that the potency of 15d-PGJ2 as a signalling molecule in endothelial cells is significantly enhanced by the accumulation of the covalent adduct with 15d-PGJ2 and endogenous Keap1 over the time of exposure to the prostaglandin. The consequence of this finding is that signalling initiated by electrophilic lipids differs from agonists that do not form covalent adducts with proteins because the constant generation of very lowconcentrations of 15d-PGJ2 can lead to induction of GSH or HO-1. In the course of these studies we also found that a substantial amount (97-99%) of exogenously added 15d-PGJ2 is inactivated in the medium and does not enter the cells to initiate cell signalling. In summary, we propose that the accumulation of covalent adduct formation with signalling proteins provides a mechanism through which endogenous intracellular formation of electrophilic lipids from COX can exert an anti-inflammatory effect in vivo.

Mitochondrial dysfunction is known to be a contributing factor to a number of diseases including chronic alcohol induced liver injury. While there is a detailed understanding of the metabolic pathways and proteins of the liver mitochondrion, little is known regarding how changes in the mitochondrial proteome may contribute to the development of hepatic pathologies. Emerging evidence indicates that reactive oxygen and nitrogen species disrupt mitochondrial function through post-translational modifications to the mitochondrial proteome. Indeed, various new affinity labeling reagents are available to test the hypothesis that post-translational modification of proteins by reactive species contributes to mitochondrial dysfunction and alcoholic fatty liver disease. Specialized proteomic techniques are also now available, which allow for identification of defects in the assembly of multi-protein complexes in mitochondria and the resolution of the highly hydrophobic proteins of the inner membrane. In this review knowledge gained from the study of changes to the mitochondrial proteome in alcoholic hepatotoxicity will be described and placed into a mechanistic framework to increase understanding of the role of mitochondrial dysfunction in liver disease.

The electrophilic lipid 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) is known to allow adaptation to oxidative stress in cells at low concentrations and apoptosis at high levels. The mechanisms leading to adaptation involve the covalent modification of regulatory proteins, such as Keap1, and augmentation of antioxidant defences in the cell. The targets leading to apoptosis are less well defined, but mitochondria have been indirectly implicated in the mechanisms of cell death mediated by electrophilic lipids. To determine the potential of electrophilic cyclopentenones to induce pro-apoptotic effects in the mitochondrion, we used isolated liver mitochondria and demonstrated that 15d-PGJ2 promotes Ca2+-induced mitochondrial swelling and cytochrome c release. The mechanisms involved are consistent with direct modification of protein thiols in the mitochondrion, rather than secondary formation of reactive oxygen species or lipid peroxidation. Using proteomic analysis in combination with biotinylated 15d-PGJ2, we were able to identify 17 potential targets of the electrophile-responsive proteome in isolated liver mitochondria. Taken together, these results suggest that electrophilic lipid oxidation products can target a sub-proteome in mitochondria, and this in turn results in the transduction of the electrophilic stimulus to the cell through cytochrome c release.