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PL101 Vitamin D and Photoprotection and/or necrotic cell death) and secondary effects (i.e., damage to PL101 the vasculature and inflammatory reaction ending in the systemic Vitamin D and photoprotection: progress to date immunity). In recent times, more and more efforts are addressed Katie Marie Dixon to recognize the better PS employing in cancer PDT. Rose Department of Anatomy and Histology, Bosch Institute, Bengal Acetate (RBAc), administrated for 60 minutes at 10-5M University of Sydney, Sydney, NSW, Australia 2 and activated with 1,6 J/cm green light, is a powerful PS. It is Vitamin D compounds are produced in the skin following able to initiate in HeLa cells several signaling processes leading exposure to ultraviolet radiation (UVR). We previously reported to rapid, independent, successive, long-lasting and time-related that vitamin D compounds protect against UVR-induced cell onset of apoptosis and autophagic cell death by signals death and DNA damage (cyclobutane pyrimidine dimers; CPD originating from or converging on almost all intracellular and 8-oxoguanine) in cultured human skin cells. We have further organelles, i.e. mitochondria, lysosomes, Golgi apparatus and evidence that these photoprotective effects are mediated by the ER, despite RBAc primary perinuclear localization. Particularly, non-genomic pathway for vitamin D. We used an in vivo model apoptosis occurs as early as 1h after PDT via activation of to investigate the photoprotective effects of topical 1,25- intrinsic pathway, followed by activation of extrinsic, caspase- dihydroxyvitamin D3 (1,25D) and low calcemic analogs 12-dependent and caspase-independent pathways. The clearance (including a non-genomic agonist) in Skh:hr-1 mice exposed to 3 of RBAc photokilled HeLa cells, in terms of recruitment, MED of UVR, showing significant reductions in apoptotic recognition and removal, is very efficient both in vitro and in sunburn cells (SBCs) and CPDs. Topical 1,25D and analogs also vivo. HeLa cells recruit phagocytes through release of fractalkine, significantly reduced UVR-induced immunosuppression in these and are recognized by changes in cell surface amount and mice. Moreover, topical application of 1,25D or low calcemic distribution of phosphatidylserine and glycans. Finally, non-genomic agonist JN proved to be protective in a macrophages internalize RBAc-treated HeLa cells and release photocarcinogenesis model in which mice were exposed to IL-10, TGF-β and TNF-α that along with translocation and chronic low dose UVR. The average number of tumours per secretion of HSP70 by dead cells suggest that RBAc-PDT can tumour-bearing mouse was reduced from 5.0±0.8 in vehicle elicit a positive immunomodulatory response. In conclusion, treated mice to 1.7±0.3 (p < 0.01) and 2.9±0.4 (p < 0.05) in RBAc-induced photodamage commits not only a potent cell 1,25D and JN treated mice respectively. The latency to tumour death induction but also influences immune system to counteract onset was significantly greater in 1,25D treated mice compared cancer cells. with vehicle treated mice (p < 0.05). At the conclusion of the 40 week study, squamous cell carcinoma incidence was reduced from 42% to 17% and 33% in 1,25D and JN groups respectively. OC103 In separate human studies, biopsies from subjects treated In vivo PDT with a novel sulfonamide bacteriochlorin: topically with 1,25D (240pmol/cm2) 24h prior to and treatment optimization and the role of the immune system immediately after UVR (2 MED) showed reductions in SBCs Luís B. Rocha1, Janusz M. Dąbrowski2, Luís G. Arnaut1, measured 24h after UVR from 4.5±1.7 per mm to 1.4±1.5 Mariette Pereira3, Sérgio Simões4 (p<0.05). CPD were reduced from 12.0±2.5% to 1.0±0.2% (p < 1Luzitin, SA, Coimbra, Portugal; 2Jagiellonian University, 0.001) in subjects treated with 1,25D. We recently showed Kraków, Poland; 3Chemistry Department - University of similar reductions in CPD and also 8-oxoguanine when 1,25D Coimbra, Coimbra, Portugal; 4Bluepharma - Indústria was applied to human subjects immediately after UVR only. The Farmacêutica SA, Coimbra, Portugal effects of topical 1,25D on UVR-induced suppression of immune Photodynamic Therapy (PDT) is a non-invasive, safe and response to a recall antigen were examined using the Mantoux clinically-approved procedure that in the recent years is model in humans. Although higher doses of topical 1,25D were 2 increasingly been recognized as a promising anticancer immunosuppressive, topical 1,25D (24 pmol/cm ) was not therapeutic strategy. The PDT reaction produces reactive oxygen immunosuppressive alone but had no effect on UVR-induced species that are directly responsible for tumour cell and tumour suppression of erythema to this recall antigen. These studies vasculature destruction. It is now widely accepted that some PDT indicate that 1,25D is photoprotective for a variety of endpoints protocols can also induce a systemic and tumour-specific in humans, mice and cultured cells, and resulted in the first immune response, which can contribute to the elimination of the comprehensive report of a protective role for vitamin D primary tumour and also to the destruction of distant metastasis. compounds in UVR-induced skin carcinogenesis. We are currently developing a novel sulfonamide bacteriochlorin (LUZ11) with properties approaching those of an ideal PDT photosensitizer: simple and affordable synthesis, high purity and IL102 stability, molar absorptivity of 137,000 M–1cm–1 at 744 nm, high Response to RBAc-induced photodamage to HeLa cells: only yields of singlet oxygen and hydroxyl radical formation, and cell death induction? solubility in biocompatible formulations. After in vitro drug Luciana Dini screening studies, a formulation for IV administration of LUZ11 Department of Biological and Environmental Sciences and was developed and optimized. The preliminary safety Technologies (Di.S.Te.B.A.) University of Salento, Lecce, Italy pharmacology studies revealed no signs of toxic reactions. The Resistance to cell death and overruling immunosurveillance proof-of-concept was demonstrated in a mouse tumour model in represent two fateful hallmarks acquired by tumour cells. Thus, which complete and long term tumour destruction was obtained the ideal cancer treatment should merge the direct cytotoxic in one single vascular-PDT session. action on tumour cells with potent immunostimulatory effects. In We now report the optimization of a vascular-PDT protocol. We this context, PhotoDynamic Therapy (PDT) is a promising cancer show that 1 mg/kg of LUZ11, 15 minutes drug-to-light interval treatment for its efficiency in i) cell death induction; ii) high and 6.5 minutes laser irradiation at 173 mW (67 J) is 100% safe selectivity for tumour cells; iii) ignition of the immunogenic cell and cures 87.5% of the mice ("cure" is defined as the absence of death; iiii) triggering immune response upon dead cells removal. palpable tumor 60 days after the treatment). A preliminary Cancer PDT is based on the cellular oxidative damage following evaluation of the antitumor immune response induced by LUZ11 ROS generation upon visible light activation of a cell-localized vascular-PDT showed a significant increase (p=0.03) in the PhotoSensitizer (PS). The interaction between light, cell or tissue median survival of mice with bilateral tumours (1 in each leg), in molecular oxygen and PS gets the photodynamic reaction. The which only one tumour was treated (when compared to non- PS subcellular localization dictates the primary site of damage treated control). This seems to indicate that PDT can have a and the consequent outcome of the treatment, implying direct cell systemic effect against non-treated tumors. Moreover, we show damage (i.e., cytotoxicity eliciting apoptotic and/or autophagic that 38% of the mice previously cured with LUZ11 vascular-PDT 43 were able to rejected a rechallenge with the same tumour cells (> aggregates and colocalizes with RIP1 following 3 months after PDT treatment), while the control group with photosensitization. We demonstrate that PDT-mediated singlet surgically removed tumors were unable to reject the rechallange. oxygen production is the cause of RIP3-dependent necrotic This suggests that PDT is able to induce a sustained antitumor pathway activation. We also prove that PDT induces the immune memory. formation of a pro-necrotic complex containing RIP3 and RIP1 but lacking caspase-8 and FADD, two proteins usually part of the necrosome when TNF-α is used as a stimulus. Thus, we IL104 hypothesize that PDT might lead to the formation of a different Pro-Survival Signaling Associated with Stress-Induced Nitric necrosome whose components, besides RIP1 and RIP3, are still Oxide in Photodynamically Challenged Tumor Cells unknown. The composition of the PDT-induced pro-necrotic Albert W Girotti, Reshma Bhowmick complex is currently under investigation in the lab. Medical College of Wisconsin, Milwaukee, WI, United States Furthermore, in most cases, glioblastoma are characterized by a Many tumors exploit nitric oxide (NO) as an anti-apoptotic/pro- constitutive activation of NF-κB. This factor is a key regulator of survival effector molecule. We recently discovered that two various processes such as inflammation, immune response, cell human breast cancer cell lines rapidly upregulated inducible growth or apoptosis. Its inhibition was shown to further sensitize nitric oxide synthase (iNOS) after being subjected to glioblastoma cells to PDT-induced necrosis, however, no photodynamic stress sensitized by 5-aminolevulinic acid (ALA)- difference in RIP3 upshift or aggregation could be observed generated protoporphyrin IX in mitochondria. Apoptotic cell when NF-κB was inhibited. photokilling was strongly enhanced by iNOS inhibition, iNOS knockdown, or NO scavenging, indicating that iNOS/NO was IL106 acting cytoprotectively. Key signaling events associated with this Optimizing Vascular Responses to Photodynamic Therapy response have been identified in human breast cancer COH-BR1 cells. Immunocytochemistry and Western analysis revealed a Theresa Busch University of Pennsylvania, Philadelphia, PA, United States cytosol-to-nucleus translocation of transcription factor NF-κB in photostressed cells.
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