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The proliferation marker Ki-67 as novel molecular target for PDT
Oral Abstracts O010 The proliferation marker Ki-67 as novel molecular target for PDT R. Rahmanzadeh 1,2 , J. Celli 1 , I. Rizvi 1 , J. Gerdes 3 , T. Hasan 1 1
Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States 2 Institute of Biomedical Optics, University of L3 beck, L3 beck, Germany 3 Department of Immunology and Cell Biology, Leibniz Research Center Borstel, Borstel, Germany Introduction: Ki-67 is highly expressed in all proliferating cells and antibodies against this protein are widely used as prognostic tools in tumor diagnosis. Photo inactivation studies showed a crucial role of Ki-67 in the synthesis of rRNA and with a novel photodynamic therapy (PDT) approach we show the delivery of antibodies to the nucleus and Ki-67 inactivation after laser irradiation. Materials and methods: Anti-Ki-67 antibodies were first conjugated to fluorescein-isothiocyanate (FITC) and then microinjected into cells or encapsulated into liposomes. Ongoing RNA-synthesis after microinjection and irradiation was pulse labeled with 5fluorouridine. The efficacy of Ki-67 targeting on cell viability after liposomal delivery and irradiation was shown in vitro ovarian cancer models — monolayer cultures as well as 3D cultures. Results: Photo inactivation after microinjection resulted in inhibition of nucleolar rRNA synthesis, while mRNA synthesis in the nucleoplasm seemed to be unaffected. After incubation with the liposomal constructs, localization of the FITC-labeled antibodies in the nucleoli of the cells was confirmed by confocal microscopy. Irradiation with a 488 nm laser led to a significant loss of cell viability — in monolayer and in the 3D culture model. The observed cell killing was epitope specific. Only treatment with the antibody TuBB-9, let to inhibition of rRNA synthesis and cell death, while treatment with the antibody MIB-1 showed no effect. Conclusions: Our findings showed the first evidence for the biological role of the Ki-67 protein in rRNA-synthesis and suggest that photo inactivation of pKi-67 is an attractive novel therapeutic approach for cancer treatment. doi:10.1016/j.pdpdt.2011.03.025 O011 Vitamin D pretreatment augments aminolevulinate photodynamic therapy-induced cell death by upregulation of tumor necrosis factor alpha and caspase-8 in mouse models of skin carcinoma S. Anand 1,2,3 , T. Hasan 3 , E. Maytin 1,2,3
1
Department of Biomedical Engineering, Cleveland Clinic, Cleveland, OH, USA 2 Department of Dermatology, Cleveland Clinic, Cleveland, OH, USA 3 Wellman Center for Photomedicine, Harvard Medical School, Boston, MA, USA 5-Aminolevulinate-based photodynamic therapy (ALA-PDT) is a well-accepted therapy for skin cancers and precancers. ALA is preferentially taken up by tumor cells and metabolically converted to a photosensitizing intermediate, protoporphyrin IX (PpIX) followed by targeted local irradiation. Since current ALA-PDT protocols are suboptimal for deep tumors, we are developing a new combination approach that combines Vitamin D3 (Vit D3) with ALA-PDT to improve clinical response. In this study, superficial and deep tumors were generated by chemical carcinogenesis (DMBA/TPA) and by subcutaneous implantation of A431 human SCC cells in mice, respectively. Superficial tumors were treated with topical Vit D3 (Vectical) for 3 days, then topical ALA (Levulan) applied. For A431 tumors, Vit D3 and ALA were given systemically. Following noninvasive PpIX
129 fluorimetry (for superficial tumors), tissue was harvested to measure PpIX levels by confocal microscopy and for analysis of tumor histology, apoptosis, cell proliferation and heme-synthetic enzymes by immunohistochemistry or western blot. In both models, a significant increase in levels of PpIX was observed following pretreatment with Vit D3, relative to no-pretreatment controls. Vit D3 pretreatment also resulted in increased differentiation, cell proliferation and cell death in tumors following ALA-PDT. Tumor necrosis factor alpha (TNF-␣) was preferentially increased by Vit D3, leading to a selective increase in expression and cleavage of Caspases-8 and 3 following ALA-PDT. These results suggest that Vit D3, used as a short-term combination therapy prior to ALA-PDT, can increase the production of PpIX photosensitizer, hence enhancing cell death and improving the therapeutic response of skin cancers. doi:10.1016/j.pdpdt.2011.03.026 O012 Combination therapy using methotrexate, 5-fluorouracil, and vitamin D as PpIX-elevating agents followed by ALA-PDT of skin tumors: Mechanisms of action E. Maytin 1 , S. Anand 1 , B. Ortel 2 , T. Hasan 3 1
Cleveland Clinic, Dermatology & Biomedical Engineering, Cleveland, OH, USA 2 University of Chicago, Dermatology, Chicago, IL, USA 3 Massachusetts General Hospital, Wellman Center for Photomedicine, Boston, MA, USA Introduction: Photodynamic therapy (PDT), using 5aminolevulinic acid (ALA) to drive protoporphyrin IX (PpIX) synthesis, is highly effective for cutaneous precancerous lesions but less effective for deep tumors. To address this, our strategy is to search for pharmacological agents that can enhance PpIX production within epithelial carcinoma cells in a tumor-specific manner. Methotrexate (MTX; Anand et al. Clin Cancer Res 2009;15:3333—43), calcitriol (VitD), and 5-fluorouracil (5FU) were each shown to enhance PpIX accumulation. Here, we report on the mechanisms for these effects. Methods: Two skin tumor models in vivo, (i) chemically-induced papillomas in SKH-1 mice and (ii) human A431 malignant carcinoma cells injected subcutaneously in nude mice, were used. Tumorbearing mice were preconditioned for 3 days using topical VitD or 5FU, or with systemic MTX, VitD, or 5FU. After receiving 5-ALA for 4 h, tissues were harvested for PpIX analysis. Some mice were exposed to light for studies of tumor involution. Results: All pretreatments led to higher PpIX (3—10-fold) and coprophyrinogen oxidase (CPO) levels. Ferrochelatase decreased after Vit D. Tumor cell proliferation was inhibited by MTX and 5-FU, and stimulated by VitD. Cell death (TUNEL; H&E stain) following ALA-PDT was enhanced by all agents. For VitD, marked caspase3 and caspase-8 cleavage was observed, suggesting a mechanism involving membrane death receptors. TNF-alpha was subsequently shown to be elevated after VitD. Conclusion: Pretreatment with MTX, VitD, or 5FU enhances CPO and PpIX levels, which improves ALA-PDT killing of tumors. The agents differed in terms of antiproliferative activity and involvement of TNF-alpha. doi:10.1016/j.pdpdt.2011.03.027
Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States 2 Institute of Biomedical Optics, University of L3 beck, L3 beck, Germany 3 Department of Immunology and Cell Biology, Leibniz Research Center Borstel, Borstel, Germany Introduction: Ki-67 is highly expressed in all proliferating cells and antibodies against this protein are widely used as prognostic tools in tumor diagnosis. Photo inactivation studies showed a crucial role of Ki-67 in the synthesis of rRNA and with a novel photodynamic therapy (PDT) approach we show the delivery of antibodies to the nucleus and Ki-67 inactivation after laser irradiation. Materials and methods: Anti-Ki-67 antibodies were first conjugated to fluorescein-isothiocyanate (FITC) and then microinjected into cells or encapsulated into liposomes. Ongoing RNA-synthesis after microinjection and irradiation was pulse labeled with 5fluorouridine. The efficacy of Ki-67 targeting on cell viability after liposomal delivery and irradiation was shown in vitro ovarian cancer models — monolayer cultures as well as 3D cultures. Results: Photo inactivation after microinjection resulted in inhibition of nucleolar rRNA synthesis, while mRNA synthesis in the nucleoplasm seemed to be unaffected. After incubation with the liposomal constructs, localization of the FITC-labeled antibodies in the nucleoli of the cells was confirmed by confocal microscopy. Irradiation with a 488 nm laser led to a significant loss of cell viability — in monolayer and in the 3D culture model. The observed cell killing was epitope specific. Only treatment with the antibody TuBB-9, let to inhibition of rRNA synthesis and cell death, while treatment with the antibody MIB-1 showed no effect. Conclusions: Our findings showed the first evidence for the biological role of the Ki-67 protein in rRNA-synthesis and suggest that photo inactivation of pKi-67 is an attractive novel therapeutic approach for cancer treatment. doi:10.1016/j.pdpdt.2011.03.025 O011 Vitamin D pretreatment augments aminolevulinate photodynamic therapy-induced cell death by upregulation of tumor necrosis factor alpha and caspase-8 in mouse models of skin carcinoma S. Anand 1,2,3 , T. Hasan 3 , E. Maytin 1,2,3
1
Department of Biomedical Engineering, Cleveland Clinic, Cleveland, OH, USA 2 Department of Dermatology, Cleveland Clinic, Cleveland, OH, USA 3 Wellman Center for Photomedicine, Harvard Medical School, Boston, MA, USA 5-Aminolevulinate-based photodynamic therapy (ALA-PDT) is a well-accepted therapy for skin cancers and precancers. ALA is preferentially taken up by tumor cells and metabolically converted to a photosensitizing intermediate, protoporphyrin IX (PpIX) followed by targeted local irradiation. Since current ALA-PDT protocols are suboptimal for deep tumors, we are developing a new combination approach that combines Vitamin D3 (Vit D3) with ALA-PDT to improve clinical response. In this study, superficial and deep tumors were generated by chemical carcinogenesis (DMBA/TPA) and by subcutaneous implantation of A431 human SCC cells in mice, respectively. Superficial tumors were treated with topical Vit D3 (Vectical) for 3 days, then topical ALA (Levulan) applied. For A431 tumors, Vit D3 and ALA were given systemically. Following noninvasive PpIX
129 fluorimetry (for superficial tumors), tissue was harvested to measure PpIX levels by confocal microscopy and for analysis of tumor histology, apoptosis, cell proliferation and heme-synthetic enzymes by immunohistochemistry or western blot. In both models, a significant increase in levels of PpIX was observed following pretreatment with Vit D3, relative to no-pretreatment controls. Vit D3 pretreatment also resulted in increased differentiation, cell proliferation and cell death in tumors following ALA-PDT. Tumor necrosis factor alpha (TNF-␣) was preferentially increased by Vit D3, leading to a selective increase in expression and cleavage of Caspases-8 and 3 following ALA-PDT. These results suggest that Vit D3, used as a short-term combination therapy prior to ALA-PDT, can increase the production of PpIX photosensitizer, hence enhancing cell death and improving the therapeutic response of skin cancers. doi:10.1016/j.pdpdt.2011.03.026 O012 Combination therapy using methotrexate, 5-fluorouracil, and vitamin D as PpIX-elevating agents followed by ALA-PDT of skin tumors: Mechanisms of action E. Maytin 1 , S. Anand 1 , B. Ortel 2 , T. Hasan 3 1
Cleveland Clinic, Dermatology & Biomedical Engineering, Cleveland, OH, USA 2 University of Chicago, Dermatology, Chicago, IL, USA 3 Massachusetts General Hospital, Wellman Center for Photomedicine, Boston, MA, USA Introduction: Photodynamic therapy (PDT), using 5aminolevulinic acid (ALA) to drive protoporphyrin IX (PpIX) synthesis, is highly effective for cutaneous precancerous lesions but less effective for deep tumors. To address this, our strategy is to search for pharmacological agents that can enhance PpIX production within epithelial carcinoma cells in a tumor-specific manner. Methotrexate (MTX; Anand et al. Clin Cancer Res 2009;15:3333—43), calcitriol (VitD), and 5-fluorouracil (5FU) were each shown to enhance PpIX accumulation. Here, we report on the mechanisms for these effects. Methods: Two skin tumor models in vivo, (i) chemically-induced papillomas in SKH-1 mice and (ii) human A431 malignant carcinoma cells injected subcutaneously in nude mice, were used. Tumorbearing mice were preconditioned for 3 days using topical VitD or 5FU, or with systemic MTX, VitD, or 5FU. After receiving 5-ALA for 4 h, tissues were harvested for PpIX analysis. Some mice were exposed to light for studies of tumor involution. Results: All pretreatments led to higher PpIX (3—10-fold) and coprophyrinogen oxidase (CPO) levels. Ferrochelatase decreased after Vit D. Tumor cell proliferation was inhibited by MTX and 5-FU, and stimulated by VitD. Cell death (TUNEL; H&E stain) following ALA-PDT was enhanced by all agents. For VitD, marked caspase3 and caspase-8 cleavage was observed, suggesting a mechanism involving membrane death receptors. TNF-alpha was subsequently shown to be elevated after VitD. Conclusion: Pretreatment with MTX, VitD, or 5FU enhances CPO and PpIX levels, which improves ALA-PDT killing of tumors. The agents differed in terms of antiproliferative activity and involvement of TNF-alpha. doi:10.1016/j.pdpdt.2011.03.027