requires an estrogen receptor beta (ERB)-mediated induction of the mitochondrial Mn-superoxide dismutase (MnSOD). The MnSOD induction and active mitochondrial respiration are both required for the growth inhibitory effect to be realized, as no effect on growth is observed in MnSOD deficient or rho0 cells. However, the molecular target(s) of MnSOD that mediate the growth

activity is the dismutation of superoxide to hydrogen peroxide, it is possible that the target(s) is redox-modifiable. Here we provide evidence that hypoxia inducible factor-1 (HIF-1), which is redox-regulated via its HIF-1alpha subunit, plays a critical role in the anti-growth effect of either RES or the ERB agonist DPN. The cell growth inhibitory effect of RES and DPN is particularly pronounced under hypoxic conditions, but has no effect in hypoxia in MnSOD-null mouse embryonic fibroblasts. In addition, neither RES nor DPN have any effect on cell growth in normoxia when HIF-1 is stabilized by CoCl2 (a hypoxia mimetic) or IOX2 (a prolyl hydroxylase inhibitor). Both RES and DPN suppress the induction of HIF-1 targets that occur during hypoxia and indeed are required for the metabolic adaptation to hypoxic conditions, such as lactate dehydrogenase and glucose-6-phosphate dehydrogenase, but these effects are reduced or absent in cells where HIF-1 has been stabilized by CoCl2 or IOX2. HIF-1 is increasingly recognized for its role in cancer cell growth in both normoxic and hypoxic conditions, via its role in regulating the glycolytic and mitochondrial metabolic machinery. Our data suggest that RES and DPN affect cellular metabolism and growth via MnSOD-mediated effects on HIF-1. This detailed knowledge of the architecture of this intracellular signaling pathway is important in fully exploiting the stilbenes as cancer cell growth inhibitors.

294 Site-Specific Nitrated Hsp90 Is a Target for Drug Development in Cancer Maria Clara Franco1, Karina C. Ricart2, Analia S. Gonzalez3, Cassandra N. Dennys1, Pascal A. Nelson1, Michael S. Janes4, Ryan A. Mehl5, Aimee Landar2, and Alvaro G. Estévez1 1Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, USA, 2Department of Pathology, University of Alabama at Birmingham, USA, 3Laboratory of Oxygen Metabolism, University Hospital, University of Buenos Aires, Argentina, 4ThermoFisher Scientific, Eugene, Oregon, USA, 5Department of Biochemistry and Biophysics, Oregon State University, USA Peroxynitrite production and nitrotyrosine formation occurs in several pathologies, including cancer. However, whether tyrosine nitration plays a role in cancer metabolism remains unknown. Here we show that site-specific nitrated Hsp90 regulates mitochondrial metabolism in cancer cells. Endogenously nitrated Hsp90 (NO2Hsp90) colocalized with mitochondria in high-density human MDA-MB-231 breast cancer cells and human breast cancer tumors. The nitrated chaperone was also associated with mitochondria in peroxynitrite-treated PC12 cells. NO2Hsp90 was not detected in low-density MDA-MB-231 cells or untreated PC12 cells. The intracellular delivery of NO2Hsp90 to PC12 cells significantly decreased the mitochondrial ATP-coupled oxygen consumption rate and the maximal and spare respiratory capacities six hours post-delivery without affecting mitochondrial content. These results suggest down-regulation of the mitochondrial electron transport chain activity. Accordingly, incubation of PC12 cell homogenates with NO2Hsp90 decreased mitochondrial membrane potential and ATP production. Replacement of tyrosine 33 by nitrotyrosine as the sole

modification on Hsp90 mimicked the effects of NO2Hsp90 on mitochondrial activity. Similar results were observed in cell homogenates from MDA-MB-231 cultured at low-density. NO2Hsp90 did not affect mitochondrial activity in homogenates from high-density cells, where endogenously nitrated Hsp90 was present. Although Hsp90 inhibitors are undergoing clinical trials in cancer patients, why cancer cells are more susceptible to Hsp90 inhibition is still unknown. Our preliminary results show that the Hsp90 inhibitor geldanamycin had higher affinity for nitrated than for unmodified Hsp90. Nitration of Hsp90 may be key in regulating mitochondrial metabolism in cancer cells, down-regulating mitochondrial activity and thereby increasing their resistance to hypoxia. Supported by NINDS Grant (AGE) and a Goldsmith Fellowship (MCF).

295 Redox-Modulating Agents Inhibit NOX2-Dependent IKK Oncogenic Kinase Expression in Breast Cancer Cell Lines. Espérance Mukawera1, Stefany Chartier1, Virginie Williams1,2, and Nathalie Grandvaux1,2 1CRCHUM, Canada, 2Department of Biochemistry and Molecular Medicine, Université de Montréal, Canada Purpose: The IKBKE in a number of plays a key role in breast cancer cell proliferation and invasiveness, and in the resistance to Tamoxifen. Persistent oxidative stress is observed in several cancers, including breast carcinomas, promoting pro-oncogenic, but also anti-tumorigenic, responses. Therefore the appropriate usage of redox modulating compounds in therapeutic strategies requires a better understanding of their molecular impact on cancer cells. Experimental design: The effect of in vitro treatment with the redox modulating triphenylmethane dyes, FDA-approved Gentian Violet and Brilliant Green, and nitroxide Tempol on IKK expression and cell proliferation was assessed in the human breast cancer epithelial cell lines MCF-7 and ZR75.1 exhibiting amplification of IKK . Gentian Violet and Brilliant Green were showed to inhibit superoxide-producing NADPH oxidases. Inhibition of NOX expression was achieved using siRNA. Results: We show that Gentian Violet, Brilliant Green and Tempol significantly decrease intracellular superoxide levels and inhibit IKK kinase and downstream cyclin D1 expression in MCF-7 and ZR75.1 cells. Cell viability was impaired in both cell lines in response to each of the compounds. While Gentian Violet and Brilliant Green induced caspase 3 and/or caspase 7 activation in both cell lines, Tempol triggered caspase activation only in MCF-7 cells. We also show that knock down of NOX2 results in significant decrease of IKK expression. Conclusions: Taken together our data show that treatment of breast cancer cells exhibiting amplification of the IKK kinase with redox modulating compounds, either triphenylmethane dyes, Gentian Violet and Brilliant Green, or nitroxide Tempol that target at least in part NOX2 inhibit oncogenic IKK expression and cell growth.

S125SFRBM 2014

doi: 10.1016/j.freeradbiomed.2014.10.190

doi: 10.1016/j.freeradbiomed.2014.10.192

doi: 10.1016/j.freeradbiomed.2014.10.191