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Vascular vs. cellular targeting of anti-tumor photodynamic therapy using bacteriochlorin derivative
Abstracts
strong neutrophil infiltration was seen immediately after the treatment. Conclusion: Changes in pO2, vessel structure, blood perfusion and vessel density demonstrate anti-vasculature effect of PDT with bacteriochlorin derivative as a photosensitizer. Reference [1] Chen, B., et al., 2005]. Int. J. Radiation Oncology Biol. Phys. 61 (4), 1216–1226. doi:10.1016/j.vph.2011.08.170
P.12.6 Vascular vs. cellular targeting of anti-tumor photodynamic therapy using bacteriochlorin derivative Agnieszka Bema, Martyna Krzykawskaa, Janusz M. Dabrowskib, Grażyna Stochelb, Luis G. Arnautc, Mariette M. Pereirac, Krystyna Urbanskaa, Martyna Elasa a Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland b Faculty of Chemistry, Jagiellonian University, Krakow, Poland c Chemistry Department, University of Coimbra, Coimbra, Portugal E-mail address: [email protected] (M. Krzykawska) Background: Photodynamic therapy (PDT) is a promising modality for the treatment of neoplastic and non-neoplastic diseases. It takes an advantage of the activation, in the presence of oxygen, and of photosensitizer that has been localized in target tissues or in the vasculature. As a result cytotoxic reactive oxygen species (ROS) are produced. They cause damage of target tissue or vascular damage inducing rapid local thrombus formation, vascular occlusion and tissue hypoxia. Under certain conditions antitumor effects are also mediated by indirect stimulation of inflammatory and immune response that includes rapid local infiltration of tumors by neutrophils and macrophages. The sulfonamide halogenated bacteriochlorin was used as a photosensitizer because it exhibits excellent physicochemical, as well as phototoxic properties. Methods: Levis Lung carcinoma tumors were grown in the right hind legs of C57BL mice. When the tumor reached 3 mm of mean diameter, 2 mg/kg BW of photosensitizer was administered i.v. into C57BL tail vein. Tumors were treated with 100 J/cm2 of light at λ = 750 nm after different drug-light interval: 15 min or 72 h. Immunohistochemistry was performed to visualize neutrophils, macrophages, vasculature, hypoxia and morphology of tumors 15 min and 3, 24, 48, 96 h after illumination. Structure and function of the vasculature were investigated with Laser Doppler Perfusion Imaging (LDPI) and VEVO 2100 ultrasonograph with Doppler mode. Results: Strong immune response and vasculature destruction were observed after both PDT protocols. However, different kinetics and intensity of the effects were seen. Changes in blood perfusion and loss of vessel density in the tumors treated with vessel targeting PDT are stronger than in cellular targeting PDT. The increase in perfusion observed 5–10 days after PDT in both protocols displays different intensity/kinetics and might result from PDT caused inflammation, observed in treated legs. Moreover, strong neutrophil infiltration was detected immediately after vessels targeting PDT and strong macrophage infiltration after anti-cellular treatment. Conclusion: Both anti-vasculature and anti-cellular PDT cause vasculature destruction and changes of blood perfusion. Applied protocols of PDT using bacteriochlorin lead to different kinetics of immune response. doi:10.1016/j.vph.2011.08.171
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P.12.7 HO-1 and miR-378 regulate tumor microenvironment and angiogenic potential of human lung cancer Klaudia Skrzypeka,b, Magdalena Tertila,b, Slawomir Goldaa, Kazimierz Weglarczykb, Halina Wasa, Maciej Cieslaa, Agnieszka Lobodaa, Alicja Jozkowicza, Claudine Kiedab, Jozef Dulaka a Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-387 Krakow, Poland b Centre de Biophysique Moleculaire, CNRS, Rue Charles-Sadron, 45071 Orleans, France E-mail addresses: [email protected] (K. Skrzypek), [email protected] (J. Dulak) Heme oxygenase-1, an enzyme degrading heme, may play an important role in vascularization of tumor. We investigated whether HO-1 can affect microRNAs contributing to angiogenesis in human lung cancer. In HO-1 overexpressing non-small cell lung cancer NCI-H292 cells, generated by transduction with retroviral vectors, miRCURY™ LNA array analysis revealed significantly diminished expression of angiomirs (miR-378, miR-17-92 cluster), miRNAs tuning tumor cells to alteration of oxygen concentration (miR-20b*, miR-210) and oncomirs (miR-17-92 cluster), whereas tumor suppressive microRNAs (miR-24, miR-193b, miR-181a) were upregulated. In vitro, HO-1 overexpression diminished proliferation and migration of lung cancer cells. Restoration of the level of the most potently downregulated angiomir and oncomir in HO-1 overexpressing cells – miR-378 – was achieved by transfection with miRNA precursors. This did not reverse those effects in vitro. However, high miR-378 overexpression in wild type cells transduced with lentiviral vectors diminished HO-1 expression, enhanced migration and proliferation and upregulated production of proangiogenic VEGF and IL-8. Moreover, NCI-miR-378 cells exported more miR-378 in exosomes in comparison to control cells, whereas HO-1 overexpression diminished its level not only in cells, but also in exosomes. Human lung microvascular endothelial cells (HLMEC) incubated with media collected from control cells proliferated more potently than those cultured with unconditioned media, evidencing proangiogenic properties of NCI-H292 cells. However, HO-1 or miR-378 overexpression did not modulate the effect. Angiogenic assay revealed that HLMEC treated with NCI-miR-378 cells conditioned media formed more tubules in Matrigel compared to NCI-ctrl cells conditioned media, whereas media from NCI-HO-1 cells exerted the opposite effect. To conclude, HO-1 diminishes, whereas miR-378 enhances tumorigenic and angiogenic potential of human lung cancer. Supported by grant 347/N-INCA/2008 and N N301 314837 from the Ministry of Science and Higher Education and CNRS-INCA-MSHE Polish-French conv. 2009-2011. doi:10.1016/j.vph.2011.08.172
P.12.8 A novel spheroid–plug model to study tumor angiogenesis and development Krzysztof Szadea, Guillaume Colletb, Witold Nowaka, Alicja Jozkowicza, Claudine Kiedab, Jozef Dulaka a Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland b Glycobiology of Cell Recognition, Centre de Biophysique Moléculaire, UPR 4301 CNRS, Orléans, France E-mail addresses: [email protected] (K. Szade), [email protected] (J. Dulak)
strong neutrophil infiltration was seen immediately after the treatment. Conclusion: Changes in pO2, vessel structure, blood perfusion and vessel density demonstrate anti-vasculature effect of PDT with bacteriochlorin derivative as a photosensitizer. Reference [1] Chen, B., et al., 2005]. Int. J. Radiation Oncology Biol. Phys. 61 (4), 1216–1226. doi:10.1016/j.vph.2011.08.170
P.12.6 Vascular vs. cellular targeting of anti-tumor photodynamic therapy using bacteriochlorin derivative Agnieszka Bema, Martyna Krzykawskaa, Janusz M. Dabrowskib, Grażyna Stochelb, Luis G. Arnautc, Mariette M. Pereirac, Krystyna Urbanskaa, Martyna Elasa a Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland b Faculty of Chemistry, Jagiellonian University, Krakow, Poland c Chemistry Department, University of Coimbra, Coimbra, Portugal E-mail address: [email protected] (M. Krzykawska) Background: Photodynamic therapy (PDT) is a promising modality for the treatment of neoplastic and non-neoplastic diseases. It takes an advantage of the activation, in the presence of oxygen, and of photosensitizer that has been localized in target tissues or in the vasculature. As a result cytotoxic reactive oxygen species (ROS) are produced. They cause damage of target tissue or vascular damage inducing rapid local thrombus formation, vascular occlusion and tissue hypoxia. Under certain conditions antitumor effects are also mediated by indirect stimulation of inflammatory and immune response that includes rapid local infiltration of tumors by neutrophils and macrophages. The sulfonamide halogenated bacteriochlorin was used as a photosensitizer because it exhibits excellent physicochemical, as well as phototoxic properties. Methods: Levis Lung carcinoma tumors were grown in the right hind legs of C57BL mice. When the tumor reached 3 mm of mean diameter, 2 mg/kg BW of photosensitizer was administered i.v. into C57BL tail vein. Tumors were treated with 100 J/cm2 of light at λ = 750 nm after different drug-light interval: 15 min or 72 h. Immunohistochemistry was performed to visualize neutrophils, macrophages, vasculature, hypoxia and morphology of tumors 15 min and 3, 24, 48, 96 h after illumination. Structure and function of the vasculature were investigated with Laser Doppler Perfusion Imaging (LDPI) and VEVO 2100 ultrasonograph with Doppler mode. Results: Strong immune response and vasculature destruction were observed after both PDT protocols. However, different kinetics and intensity of the effects were seen. Changes in blood perfusion and loss of vessel density in the tumors treated with vessel targeting PDT are stronger than in cellular targeting PDT. The increase in perfusion observed 5–10 days after PDT in both protocols displays different intensity/kinetics and might result from PDT caused inflammation, observed in treated legs. Moreover, strong neutrophil infiltration was detected immediately after vessels targeting PDT and strong macrophage infiltration after anti-cellular treatment. Conclusion: Both anti-vasculature and anti-cellular PDT cause vasculature destruction and changes of blood perfusion. Applied protocols of PDT using bacteriochlorin lead to different kinetics of immune response. doi:10.1016/j.vph.2011.08.171
369
P.12.7 HO-1 and miR-378 regulate tumor microenvironment and angiogenic potential of human lung cancer Klaudia Skrzypeka,b, Magdalena Tertila,b, Slawomir Goldaa, Kazimierz Weglarczykb, Halina Wasa, Maciej Cieslaa, Agnieszka Lobodaa, Alicja Jozkowicza, Claudine Kiedab, Jozef Dulaka a Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-387 Krakow, Poland b Centre de Biophysique Moleculaire, CNRS, Rue Charles-Sadron, 45071 Orleans, France E-mail addresses: [email protected] (K. Skrzypek), [email protected] (J. Dulak) Heme oxygenase-1, an enzyme degrading heme, may play an important role in vascularization of tumor. We investigated whether HO-1 can affect microRNAs contributing to angiogenesis in human lung cancer. In HO-1 overexpressing non-small cell lung cancer NCI-H292 cells, generated by transduction with retroviral vectors, miRCURY™ LNA array analysis revealed significantly diminished expression of angiomirs (miR-378, miR-17-92 cluster), miRNAs tuning tumor cells to alteration of oxygen concentration (miR-20b*, miR-210) and oncomirs (miR-17-92 cluster), whereas tumor suppressive microRNAs (miR-24, miR-193b, miR-181a) were upregulated. In vitro, HO-1 overexpression diminished proliferation and migration of lung cancer cells. Restoration of the level of the most potently downregulated angiomir and oncomir in HO-1 overexpressing cells – miR-378 – was achieved by transfection with miRNA precursors. This did not reverse those effects in vitro. However, high miR-378 overexpression in wild type cells transduced with lentiviral vectors diminished HO-1 expression, enhanced migration and proliferation and upregulated production of proangiogenic VEGF and IL-8. Moreover, NCI-miR-378 cells exported more miR-378 in exosomes in comparison to control cells, whereas HO-1 overexpression diminished its level not only in cells, but also in exosomes. Human lung microvascular endothelial cells (HLMEC) incubated with media collected from control cells proliferated more potently than those cultured with unconditioned media, evidencing proangiogenic properties of NCI-H292 cells. However, HO-1 or miR-378 overexpression did not modulate the effect. Angiogenic assay revealed that HLMEC treated with NCI-miR-378 cells conditioned media formed more tubules in Matrigel compared to NCI-ctrl cells conditioned media, whereas media from NCI-HO-1 cells exerted the opposite effect. To conclude, HO-1 diminishes, whereas miR-378 enhances tumorigenic and angiogenic potential of human lung cancer. Supported by grant 347/N-INCA/2008 and N N301 314837 from the Ministry of Science and Higher Education and CNRS-INCA-MSHE Polish-French conv. 2009-2011. doi:10.1016/j.vph.2011.08.172
P.12.8 A novel spheroid–plug model to study tumor angiogenesis and development Krzysztof Szadea, Guillaume Colletb, Witold Nowaka, Alicja Jozkowicza, Claudine Kiedab, Jozef Dulaka a Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland b Glycobiology of Cell Recognition, Centre de Biophysique Moléculaire, UPR 4301 CNRS, Orléans, France E-mail addresses: [email protected] (K. Szade), [email protected] (J. Dulak)