Neuronal Nitric Oxide Synthase (nNOS)/NO, An Accelerator of Melanoma Progression, is a Potential Target for Chemoprevention

peptide (Aβ) in the brain, one of the pathological features of AD, is considered to be a central disease-causing and disease- promoting event in AD. in this study, we show that feeding male APP/PS1 transgenic mice, a well-established mouse model of AD, with a diet containing phenolic antioxidant tert- butylhydroquinone (TBHQ) for 6 weeks increased the concentration of glutathione (GSH), an important antioxidant, and suppressed the expression of NADPH oxidase 2 (Nox2) as well as lipid peroxidation in the brain. Most importantly, we show that TBHQ diet dramatically reduced brain Aβ load, which was associated with inhibition of the expression of plasminogen activator inhibitor-1 (PAI-1), a redox sensitive protease inhibitor which plays a critical role in brain Aβ accumulation in AD. This was accompanied by increases in the activities of tissue type and urokinase type plasminogen activators (tPA and uPA) as well as plasmin. Moreover, we show that TBHQ diet increased the expression of low density lipoprotein related protein-1 (LRP-1), a multi ligand endocytotic receptor involved in transporting Aβ out of the brain, and plasma Aβ40 and Aβ42 levels. No significant effect of TBHQ diet on the amounts of alpha- and beta-C-terminal fragments or full-length APP was detected. Collectively, our data suggest that TBHQ may have therapeutic potential for AD through increasing brain antioxidant capacity/reducing oxidative stress level, which leads to inhibition of PAI-1 expression and thereby stimulation of Aβ degradation/clearance from the brain. doi:10.1016/j.freeradbiomed.2011.10.413 Melatonin Can Attenuate HOCl-Mediated Hemolysis, Free Iron Release and Heme Degradation From Hemoglobin Dhiman Maitra 1 , Michael P. Diamond 1 , Ghassan M. Saed 1 , and Husam M. Abu-Soud 1 Wayne State University In inflammatory diseases, where hypochlorous acid (HOCl) is elevated, iron homeostasis is disturbed, resulting in iron overload. Free iron is toxic since it can lead to generation of other free radicals through the Fenton reaction. Recently, we have shown that HOCl binds to the heme moiety of hemoglobin (Hb) and generates a transient ferric species in which ultimately leads to heme destruction, free iron release and protein aggregation. HOCl is generated by myeloperoxidase, utilizing chloride and hydrogen peroxide as substrates. Melatonin, a neurohormone, has been shown to act as an inhibitor for MPO and is also know to scavenge HOCl. Here we show that HOCl-mediated Hb heme destruction can be partially or completely prevented by pre- incubation of Hb with increasing concentrations of melatonin prior to the addition of HOCl, as judged by UV-Vis spectrophotometry and in-gel heme staining. Melatonin also prevented HOCl- mediated free iron release and protein aggregation. Similarly, pre- incubation of erythrocytes with increasing concentrations of melatonin prior to the treatment with HOCl prevented HOCl- mediated loss of erythrocyte viability, indicating the biological relevance of this finding. Melatonin prevents HOCl-mediated Hb heme destruction by direct scavenging of HOCl or through the destabilization of the higher Hb oxidative states intermediates, ferryl porphyrin radical cation Hb-Fe(IV)=O +π● (Compound I) and Hb-Fe(IV)=O (Compound II), that are formed through the reaction of HOCl with Hb. This work also indicates that Hb/melatonin system can function as a catalytic sink for HOCl, establishing a direct mechanistic link between melatonin and its protective effect in chronic inflammatory diseases. doi:10.1016/j.freeradbiomed.2011.10.414 Manganese Superoxide Dismutase Knockdown and Subsequent mtDNA Damage Akira Marine 1 , Nirmala Parajuli 1 , and Lee Ann MacMillan-Crow 1 University of Arkansas for Medical Sciences Oxidative stress caused by reactive oxygen species (ROS) can lead to many disease conditions such as cancer, atherosclerosis, renal ischemia, aging, and neurodegeneration. One source of endogenous ROS is the mitochondrial electron transport chain (ETC). Manganese Superoxide Dismutase (MnSOD) is the major mitochondrial antioxidant, and it catalyzes the transformation of superoxide into hydrogen peroxide which can then be further detoxified by other cellular enzymes. MnSOD is critical in maintaining the normal function of mitochondria. Multiple copies of mitochondrial DNA (mtDNA) are present in each mitochondrion and remain in close proximity to the ETC located in the inner mitochondrial membrane. mtDNA has limited repair ability and replication of cells with damaged mtDNA is not inhibited by cell- cycle check point control mechanisms. Thus, damage to mtDNA can result in accumulation of mutations and the production of dysfunctional ETC proteins, which in turn may produce more ROS and initiate a damaging cycle. the goal of this study was to determine the effect that MnSOD knockdown has on mitochondrial damage. Experimental models included a siRNA mediated MnSOD knockdown of mouse collecting duct cells and a kidney-specific MnSOD knockout mouse. Our preliminary data show that MnSOD knockdown causes a significant increase in mtDNA copy numbers in vivo and in vitro. We hypothesize that loss of MnSOD activity initates mtDNA damage, which leads to an increase in mtDNA copy number as well as accumulation of damaged mtDNA which further exacerbates the initial oxidative stress. Mitochondrial damage is assessed, using long range PCR, high resolution respiration, and 8-hydroxydeoxyguanosine immunohistochemistry, and electron microscopy. the long term goal of our studies is to determine the sequence of events following inactivation of MnSOD that lead to mitochondrial damage. doi:10.1016/j.freeradbiomed.2011.10.415 Neuronal Nitric Oxide Synthase (nNOS)/NO, An Accelerator of Melanoma Progression, is a Potential Target for Chemoprevention Frank L Meyskens 1,2 , Sun Yang 1,2 , Zheng Yang 3 , Haitao Ji 4 , Thomas l. Poulos 1 , and Richard B. Silverman 4 University of California, Irvine, Chao Family Comprehensive Cancer Center, Shandong Provincial Hospital, Northwestern University As an important environmental carcinogen, UVR not only generates ROS, but also produces a large amount of nitric oxide (NO) in human skin. Utilizing a NO-donor (DETA) to mimic NO stress, we demonstrated that melanoma proliferation and invasion potential were significantly stimulated. Notably, long-term exposure of DETA/NO stress resulted in over-growth of primary normal human melanocytes with formation of foci in vitro. NO is predominantly generated in an enzyme-dependent manner via NO synthase (NOS). As melanocytes originate from the neural crest, we propose that neural NOS (nNOS) might play an important role in NO production in melanoma. Immunohistochemistry study using biopsies demonstrated marked elevation of nNOS expressions in melanoma and further trend analysis revealed that the staining scores of lesions are significantly associated with disease stage. More progressive stage samples exhibited higher nNOS levels. Significant SFRBM 2011 S91