8 - Nitric Oxide-Mediated Photobiological Responses
Meeting Room 2 10:00 - 12:00
|Chair(s): Al Girotti|
10:00 Nitric Oxide Derivatives and Skin Environmental Exposure to light: From Molecular Pathways to Therapeutic Opportunities C. V. Suschek*, Department of Trauma and Hand Surgery, Medical Faculty of the Heinrich-Heine-University Dusseldorf
Abstract: The physiological as well as pathophysiological role of cutaneous nitric oxide (NO) in human skin has been under investigation since first reports of nitric oxide synthase (NOS)-expression in human skin tissue in 1992. Already in the first years on NO research it became obvious that NO plays a pivotal role in the dermal response to environmental stimuli like ultraviolet radiation, heat, and cold. Additionally to enzymatically produced NO a range of non-enzymatic pathways for NO generation has been identified. Substantial quantities of NO radicals are continuously generated in the human skin, and formation of NO can be enhanced 3-5 fold by illumination with UVA light, especially in the outer skin. This phenomenon is attributed to the photolysis of photolabile nitroso compounds, which play an outstanding role in mammalian physiology. Previous work has shown that human skin is quite rich in these compounds, which attain local concentrations far higher than found in the blood circulation under normal conditions. Both are known to deliver free NO radicals upon UVA photolysis even at the modest UVA fluxes as found in sunlight at sea level. Accordingly, the degree of exposure of skin to ambient light will significantly affect the NO status of human skin, which plays an outstanding role in the regulation of local hemodynamic parameters, inflammation, infection, wound healing, and protection from the injurious effects of UV radiation.
10:30 Constitutive nitric oxide synthases as a chemopreventive target for UVB-induced skin carcinogenesis S Wu*, Ohio University
; L Tong, Ohio University
Abstract: Ultraviolet B light (UVB) is a hazardous environmental carcinogen that affects multiple oncogenic signaling pathways. Two of these signaling circuits are the reactive oxygen species/reactive nitrogen species (ROS/RNS)-mediated DNA damage and NF-kappa B-mediated anti-apoptotic pathways. In this presentation, we will provide evidences that constitutive nitric oxide synthases (cNOS), including both NOS1 (neuronal) and NOS3 (endothelial), mediates UVB-induced DNA damage (partially) and NF-kappa B activation (totally) via unique mechanisms. The activation and uncoupling of cNOS increases ROS/RNS production immediately after UVB irradiation and inhibition of cNOS can significantly reduce UVB-induced DNA damage. Meanwhile, the elevation of ROS/RNS also induces the phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2-alpha) and translational inhibition of I kappa B synthesis, which leads to the activation of NF-kappa B. The UVB-induced NF-kappa B activation is totally diminished in cNOS null HEK293T, and the inducibility of NF-kappa B can be restored by overexpressing cNOS in the cells. Finally we will demonstrate that inhibition of cNOS can completely suppress the transformation of keratinocytes induced by UVB-irradiation. Our results suggest that cNOS inhibitor could be a potential chemopreventive agent for UVB-induced skin cancer formation by reducing DNA damage-caused cell mutagenesis and suppressing NF-kappa B-mediated cell survival pathway.
11:00 ROLE OF ENDOGENOUS NITRIC OXIDE IN HYPER-AGGRESSIVENESS OF CANCER CELLS SURVIVING A PDT-LIKE OXIDATIVE CHALLENGE Johathan M. Fahey, Medical College of Wisconsin
; Albert W. Girotti*, Medical College of Wisconsin
Abstract: Nitric oxide (NO) is a pleiotropic free radical molecule generated naturally by enzymes of the nitric oxide synthase (NOS) family. Many tumors exploit low levels of endogenous NO for survival, growth, and invasive signaling and also to resist various anti-tumor therapies, including photodynamic therapy (PDT). Studies carried out in the laboratory ca. 6 years ago were the first to demonstrate that inducible NOS (iNOS) is rapidly and persistently elevated in several types of cancer cell subjected to a photodynamic challenge mimicking 5-aminolevulinic acid (ALA)-based PDT. In recent studies, we discovered that two human prostate cancer lines (PC3 and DU145) and a breast cancer line (MDA-MB-231) exploit iNOS/NO to not only resist photokilling, but also to promote aggressive expansion of surviving cells. For example, 24 h after an ALA/light challenge (fluence ~1 J/cm2), PC3 survivors proliferated (MTT assay), migrated (gap-closure assay), and invaded (trans-well assay) much more rapidly than dark controls. Each of these responses was suppressed by an iNOS inhibitor or NO scavenger, consistent with NO involvement. In agreement with accelerated invasion, surviving cells exhibited a marked NO-dependent activation of matrix metalloproteinase-9 (MMP-9, shown by in-gel zymography), down-regulation of MMP-9 inhibitor TIMP-1, and upregulation of alpha-6 and beta-1 integrins. Photostress-surviving MDA-MB-231 cells were also found to proliferate and migrate/invade more rapidly than dark controls in vitro. The significance of these findings was recently confirmed at the in vivo level. Using SCID mice bearing MDA-MB-231 tumor xenografts, ALA as a pro-sensitizer, and a 633 nm LED light source, we showed that ALA-PDT significantly reduced tumor expansion rate relative to light-only controls. Importantly, administered iNOS inhibitors further reduced this rate, implying that tumor iNOS/NO was reducing PDT efficacy, presumably by acting cytoprotectively as observed in vitro. (Supported by NIH/NCI grant CA70823)
11:30 Nitric Oxide-mediated Bystander Cell Responses in an Anti-tumor Photodynamic Therapy Model J Bazak, Jagiellonian University
; W Korytowski*, Medical College of Wisconsin and Jagiellonian University; JM Fahey, Medical College of Wisconsin; AW Girotti, Medical College of Wisconsin
Abstract: Non-ionizing photodynamic therapy (PDT) can induce a bystander effect, but far less is known about this than the ionizing radiation-induced counterpart. In the present study, we tested the hypothesis that photodynamically-stressed prostate cancer PC3 cells can elicit nitric oxide (NO)-mediated pro-growth/migration responses in non-stressed bystander cells. A novel approach was used whereby both cell populations existed on a culture dish, but made no physical contact with one other. Visible light irradiation of photosensitized (targeted) cells resulted in a large and prolonged upregulation of inducible NO synthase (iNOS) along with a slower, less pronounced upregulation in bystander cells. This was accompanied by post-irradiation appearance of NO-derived DAF-FM fluorescence, the level of which increased gradually in both cell compartments. Like targeted cells, bystanders exhibited a significant increase in growth and migration rate, both responses being strongly attenuated by an iNOS inhibitor (1400W) or NO scavenger (cPTIO). Incubating bystander cells with conditioned medium from targeted cells failed to stimulate growth/migration, ruling out involvement of relatively long-lived effectors. The pro-survival/pro-growth kinases Akt and ERK-1/2 exhibited progressive post-irradiation activation in bystander cells, NO again playing a key role. This is the first reported evidence for NO-enhanced bystander aggressiveness in the context of PDT and illustrates the need for pharmacologic iNOS inhibitors as PDT adjuvants. (Supported by NIH/NCI Grant CA70823)