Effect of radiation on vascular endothelial growth factor expression in the C2 canine mastocytoma cell line

Ivana Sekis Division of Small Animal Internal Medicine, Department for Companion Animals and Horses, University of Veterinary Medicine, 1210 Vienna, Austria.

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Wilhelm Gerner Institute of Immunology, and the Department for Pathobiology, University of Veterinary Medicine, 1210 Vienna, Austria.

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Michael Willmann Division of Small Animal Internal Medicine, Department for Companion Animals and Horses, University of Veterinary Medicine, 1210 Vienna, Austria.

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Laura Rebuzzi Division of Small Animal Internal Medicine, Department for Companion Animals and Horses, University of Veterinary Medicine, 1210 Vienna, Austria.

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Alexander Tichy Institute of Medical Physics and Biostatistics, Department for Biomedical Sciences, University of Veterinary Medicine, 1210 Vienna, Austria.

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Martina Patzl Institute of Immunology, and the Department for Pathobiology, University of Veterinary Medicine, 1210 Vienna, Austria.

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Johann G. Thalhammer Division of Small Animal Internal Medicine, Department for Companion Animals and Horses, University of Veterinary Medicine, 1210 Vienna, Austria.

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Armin Saalmüller Institute of Immunology, and the Department for Pathobiology, University of Veterinary Medicine, 1210 Vienna, Austria.

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Miriam M. Kleiter Division of Small Animal Internal Medicine, Department for Companion Animals and Horses, University of Veterinary Medicine, 1210 Vienna, Austria.

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Abstract

Objective—To establish the radiosensitivity and effect of irradiation on vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) expression in the canine mastocytoma cell line C2.

Sample Population—Canine mastocytoma cell line C2.

Procedures—C2 cells were irradiated with single doses of 2, 4, 6, and 8 Gy. The 3-(4, 5-di-methyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide assay and proliferation assays with (methyl-hydrogen 3) thymidine were used for radiosensitivity experiments. Expression of VEGFR was determined via flow cytometry and apoptotic rate via annexin assay. Human and canine VEGF ELISA kits were evaluated in crossover assay experiments, and the canine kit was used thereafter.

Results—C2 cells secreted VEGF constitutively. Radiation did not induce a significant increase in VEGF secretion, regardless of radiation dose. Consistently, radiation did not up-regulate VEGFR. Cell survival rates decreased in a dose-dependent manner. The apoptotic cell fraction had a dose-dependent increase that reached its maximum 24 to 48 hours after radiation.

Conclusions and Clinical Relevance—The C2 cell line was radiosensitive, and a fraction (up to 40%) of cells died via apoptosis in a dose-dependent manner. In response to radiation, C2 cells did not upregulate VEGF production or VEGFR. Further studies are needed to determine whether tumor control could be improved by combining radiotherapy with VEGFR inhibitors or apoptosis-modulating agents.

Abstract

Objective—To establish the radiosensitivity and effect of irradiation on vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) expression in the canine mastocytoma cell line C2.

Sample Population—Canine mastocytoma cell line C2.

Procedures—C2 cells were irradiated with single doses of 2, 4, 6, and 8 Gy. The 3-(4, 5-di-methyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide assay and proliferation assays with (methyl-hydrogen 3) thymidine were used for radiosensitivity experiments. Expression of VEGFR was determined via flow cytometry and apoptotic rate via annexin assay. Human and canine VEGF ELISA kits were evaluated in crossover assay experiments, and the canine kit was used thereafter.

Results—C2 cells secreted VEGF constitutively. Radiation did not induce a significant increase in VEGF secretion, regardless of radiation dose. Consistently, radiation did not up-regulate VEGFR. Cell survival rates decreased in a dose-dependent manner. The apoptotic cell fraction had a dose-dependent increase that reached its maximum 24 to 48 hours after radiation.

Conclusions and Clinical Relevance—The C2 cell line was radiosensitive, and a fraction (up to 40%) of cells died via apoptosis in a dose-dependent manner. In response to radiation, C2 cells did not upregulate VEGF production or VEGFR. Further studies are needed to determine whether tumor control could be improved by combining radiotherapy with VEGFR inhibitors or apoptosis-modulating agents.

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