220 Novel Therapeutics To Restrict the Redox-Dependent Expression of MMP-1 and Tumor Cell Invasion by Modulating the Senescence Associated Secretory Phenotype Nilay Patel1, Donald McCarthy1, and J. Andres Melendez1 1College of Nanoscale Science & Engineering of the University at Albany State University of New York The senescence associated secretory phenotype (SASP) is a characteristic of aged fibroblasts that alters the tissue microenvironment promoting tumor initiation, proliferation, and invasion through paracrine mechanisms. Matrix Metalloproteinase-1 (MMP-1, interstitial collagenase) is a SASP component responsible for matrix turnover and associated with many age-associated disease processes. SASP-associated MMP-1 gene expression is highly responsive to H2O2-dependent redox signaling and likely amenable to antioxidant-based therapeutics. Here we identify compounds that restrict redox-dependent MMP-1 expression and the SASP. Treatment of senescent IMR-90 fibroblasts with apple peel extract (APE), pomegranate extract, and Didox restrict Senescence-Associated (SA) MMP-1 expression. Inhibition of SA-MMP-1 expression was accompanied by a decrease in c-jun phosphorylation, a key component of redox-dependent MMP-1 transcription. in addition, conditioned media from senescent fibroblasts dramatically enhanced the invasive capacity of A375 malignant melanoma cells when compared to media from presenescent fibroblasts. These studies suggest that SASP can provide an environment that supports the metastatic phenotype and compounds that prevent redox-dependent expression of MMP-1 may prove useful in restricting tumor cell invasion and the SASP.

doi:10.1016/j.freeradbiomed.2011.10.459

221 Proteolytic Processing of Extracellular Superoxide Dismutase (EC-SOD) is Redox Regulated Randi Heidemann Gottfredsen1, Ulrike Gabriele Larsen1, and Steen Vang Petersen1 1Department of Biomedicine, University of Aarhus, Denmark Extracellular superoxide dismutase (EC-SOD) is a homotetrameric metalloprotein responsible for the enzymatic removal of superoxide generated in the extracellular space. the C-terminal region of the protein has the capacity to bind a number of extracellular ligands and is therefore essential for the immobilization of the protein in tissues. during post-translational processing, this region can be proteolytically removed producing EC-SOD tetramers with variable avidity for ligands within the extracellular matrix. This cleavage event is therefore responsible for modulating the distribution of the protein within the extracellular space. Since the ratio between intact and cleaved subunits differs between tissues, this suggests that the cleavage event may be subject to regulation. The C-terminal region encompasses a cysteine residue (Cys219) which is engaged in a disulfide bridge with the corresponding residue of another EC-SOD subunit thus establishing a disulfide-linked homodimer. Since this inter-subunit disulfide bridge play no role in neither ligand binding capacity nor structural integrity, we speculated that the proteolytic removal of the C-terminal region is affected by the redox status of this disulfide bridge. We have determined the redox potential of this disulfide bridge and show that the potential dictates the presence of both EC-SOD monomers and dimers within the intracellular space. Moreover, we show that the proteolytic removal of the C-terminal region in vitro is affected by the redox status, allowing for cleavage at

reducing conditions only. Moreover, we show that proteolysis in cells expressing recombinant EC-SOD is induced by oxidative stress and that Cys219 plays a central role in this event. Based on our findings, we propose a scenario in where the redox status of Cys219 is used to regulate the proteolysis of the EC-SOD subunit prior to secretion.

doi:10.1016/j.freeradbiomed.2011.10.460

222 Attenuation of Carrageenan-Induced Inflammation by Tempol and Hydrophobic Derivatives Relates To Nitroxide Partition Coefficient and Myeloperoxidase Levels Raphael F. Queiroz1, Alessandro K. Jordão2, Anna C. Cunha2, Vitor F. Ferreira2, Maisa R.P.L. Brigagão3, Alberto Malvezzi1,4, Antonia T.-do Amaral4, and Ohara Augusto1 1Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, 2Departamento de Química Orgânica, Instituto de Química, Universidade Federal Fluminense, 3Departamento de Ciência Exatas, Universidade Federal de Alfenas, 4Departamento Química Fundamental, Instituto de Química, Universidade de São Paulo Tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) is the most investigated nitroxide in vivo and has been shown to effectively reduce tissue injury in several models of inflammation. the mechanisms of protection are not completely understood, but have been mainly attributed to the ability of nitroxides to act as multifunctional antioxidants. Recently, the ability of nitroxides to down-regulated the expression of inducible nitric oxide synthase during leishmaniasis and to inhibit the chlorinating activity of myeloperoxidase (MPO) in vitro and in activated neutrophils has been described. To examine whether nitroxides (0.17 mmol/kg) inhibit MPO and MPO-mediated damage in experimental animals, we investigated the effects of tempol (logP=0.44) and three more hydrophobic derivatives (4-azido-tempo, logP=1.81; 4-benzenesulfonyl-tempo, logP=2.45; 4-(4-phenyl-1H-1,2,3-triazol-1-yl)-tempo (tritempo), logP=2.81) on carrageenan-induced acute inflammation by monitoring edema, protein oxidation and nitration, and MPO level and activity in the rat paws. in vitro, all tested nitroxides inhibited MPO chlorinating activity into a similar extent (IC50 varied from 1.3 to 1.8 μM). in vivo, there was a good correlation between the inhibitory effect of the nitroxide on paw edema and the log of its partition coefficient at all time-points, except for the case of tritempo at 6h. At this time, all nitroxides reduced the levels of nitrated and oxidized proteins and MPO levels in muscle paw homogenates to about 50%. Although MPO-chlorinating activity (about 66%) was inhibited more than MPO levels (about 56%), it is difficult to conclude that nitroxides are inhibiting MPO activity in vivo because of the intrinsic uncertainties of Western-blot quantification. the main effect of nitroxides on acute inflammation appears to be the inhibition of neutrophil chemotaxis by mechanisms that remain to be elucidated. Supported by: INCT-Redoxoma/CNPq and FAPESP.

doi:10.1016/j.freeradbiomed.2011.10.461

SFRBM 2011S94