J Vet Sci.  2013 Jun;14(2):207-214. 10.4142/jvs.2013.14.2.207.

Radiation up-regulates the expression of VEGF in a canine oral melanoma cell line

• Flickinger I
• Rutgen BC
• Gerner W
• Calice I
• Tichy A
• Saalmuller A
• Kleiter M

• KMID: 1705523
• DOI: http://doi.org/10.4142/jvs.2013.14.2.207

Abstract

To evaluate radiosensitivity and the effects of radiation on the expression of vascular endothelial growth factor (VEGF) and VEGF receptors in the canine oral melanoma cell line, TLM 1, cells were irradiated with doses of 0, 2, 4, 6, 8 and 10 Gray (Gy). Survival rates were then determined by a MTT assay, while vascular endothelial growth factor receptor (VEGFR)-1 and -2 expression was measured by flow cytometry and apoptotic cell death rates were investigated using an Annexin assay. Additionally, a commercially available canine VEGF ELISA kit was used to measure VEGF. Radiosensitivity was detected in TLM 1 cells, and mitotic and apoptotic cell death was found to occur in a radiation dose dependent manner. VEGF was secreted constitutively and significant up-regulation was observed in the 8 and 10 Gy irradiated cells. In addition, a minor portion of TLM 1 cells expressed vascular endothelial growth factor receptor (VEGFR)-1 intracellularly. VEGFR-2 was detected in the cytoplasm and was down-regulated following radiation with increasing dosages. In TLM 1 cells, apoptosis plays an important role in radiation induced cell death. It has also been suggested that the significantly higher VEGF production in the 8 and 10 Gy group could lead to tumour resistance.

Keyword

cell line; radiation; TLM 1; vascular endothelial growth factor

MeSH Terms

Animals
Apoptosis/*radiation effects
Cell Line, Tumor/radiation effects
Dogs
Dose-Response Relationship, Radiation
Enzyme-Linked Immunosorbent Assay/veterinary
Melanoma/genetics/metabolism
Mouth Neoplasms/genetics/metabolism
Radiation Tolerance
Tetrazolium Salts/metabolism
Thiazoles/metabolism
Up-Regulation/*radiation effects
Vascular Endothelial Growth Factor A/genetics/metabolism/*radiation effects
Vascular Endothelial Growth Factor Receptor-1/genetics/metabolism/*radiation effects
Vascular Endothelial Growth Factor Receptor-2/genetics/metabolism/*radiation effects
Tetrazolium Salts
Thiazoles
Vascular Endothelial Growth Factor A
Vascular Endothelial Growth Factor Receptor-1
Vascular Endothelial Growth Factor Receptor-2

Figure

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  Fig. 1 Growth curves of irradiated TLM 1 cells. Dose dependent growth inhibition was observed after single doses of 0, 2, 4, 6, 8 and 10 Gy. Each data point represents the mean ± standard deviation. Gy: Gray.

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  Fig. 2 Survival curve of irradiated TLM 1 cells. Following irradiation with 0, 2, 4, 6, 8 and 10 Gy, cells were incubated for 5 days and then harvested to measure cell viability with the MTT assay. Each data point represents the mean of three experiments (samples of each in duplicate) ± standard deviation.

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  Fig. 3 (A) Concentration of VEGF normalized to the number of viable cells (pg/mL/105) in the cell lysate. (B) Cell free supernatant. Means ± standard deviations of three independent experiments analysed in duplicate are shown. VEGF: vascular endothelial growth factor.

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  Fig. 4 VEGF expression in relation to radiation dose in cell free supernatant 72 hours after radiation. Mean ± standard deviation of three independent experiments analysed in duplicate are shown.

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  Fig. 5 Radiation induced apoptosis of TLM 1 cells analyzed by FCM (annexin/PI iodide staining). Cells were gated according to FSC/SSC light scattering properties (not depicted). Results are displayed in contour plots. One representative sample of three is shown. FCM: flow cytometry, PI: propidium iodide, FSC/SSC: forward scatter/side scatter.

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  Fig. 6 FCM analysis of VEGFR-2 in cytoplasm of TLM 1 cells four days post radiation (0, 2, 4, 6, 8 and 10 Gy). Cells were stained with live/dead stain, after which they were fixed, permeabilzed and stained with antibodies against VEGFR-2 or irrelevant isotype control antibodies. Only live cells were gated and analyzed for VEGFR-2 expression. VEGFR-2 positive cells are highlighted in blue. The percentage of VEGFR-2 positive cells is shown in the upper right corner. VEGFR: vascular endothelial growth factor receptor.

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