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Post-radiation increase in VEGF enhances glioma cell motility in vitro

BACKGROUND: Glioblastoma multiforme (GBM) is among the most lethal of all human tumors, with frequent local recurrences after radiation therapy (RT). The mechanism accounting for such a recurrence pattern is unclear. It has classically been attributed to local recurrence of treatment-resistant cells...

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Autores principales: Kil, Whoon Jong, Tofilon, Philip J, Camphausen, Kevin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3307492/
https://www.ncbi.nlm.nih.gov/pubmed/22356893
http://dx.doi.org/10.1186/1748-717X-7-25
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author Kil, Whoon Jong
Tofilon, Philip J
Camphausen, Kevin
author_facet Kil, Whoon Jong
Tofilon, Philip J
Camphausen, Kevin
author_sort Kil, Whoon Jong
collection PubMed
description BACKGROUND: Glioblastoma multiforme (GBM) is among the most lethal of all human tumors, with frequent local recurrences after radiation therapy (RT). The mechanism accounting for such a recurrence pattern is unclear. It has classically been attributed to local recurrence of treatment-resistant cells. However, accumulating evidence suggests that additional mechanisms exist that involve the migration of tumor or tumor stem cells from other brain regions to tumor bed. VEGFs are well-known mitogens and can be up-regulated after RT. Here, we examine the effect of irradiation-induced VEGF on glioma cell motility. MATERIALS AND METHODS: U251 and LN18 cell lines were used to generate irradiated-conditioned medium (IR-CM). At 72 h after irradiation, the supernatants were harvested. VEGF level in IR-CM was quantified by ELISA, and expression levels for VEGF mRNA were detected by RT-PCR. In vitro cancer cell motility was measured in chambers coated with/without Matrigel and IR-CM as a cell motility enhancer and a VEGF antibody as a neutralizer of VEGF bioactivity. Immunoblots were performed to evaluate the activity of cell motility-related kinases. Proliferation of GBM cells after treatment was measured by flow cytometry. RESULTS: Irradiation increased the level of VEGF mRNA that was mitigated by pre-RT exposure to Actinomycin D. U251 glioma cell motility (migration and invasion) was enhanced by adding IR-CM to un-irradiated cells (174.9 ± 11.4% and 334.2 ± 46% of control, respectively). When we added VEGF antibody to IR-CM, this enhanced cell motility was negated (110.3 ± 12.0% and 105.7 ± 14.0% of control, respectively). Immunoblot analysis revealed that IR-CM increased phosphorylation of VEGF receptor-2 (VEGFR2) secondary to an increase in VEGF, with a concomitant increase of phosphorylation of the downstream targets (Src and FAK). Increased phosphorylation was mitigated by adding VEGF antibody to IR-CM. There was no difference in the mitotic index of GBM cells treated with and without IR-CM and VEGF. CONCLUSIONS: These results indicate that cell motility can be enhanced by conditioned medium from irradiated cells in vitro through stimulation of VEGFR2 signaling pathways and suggest that this effect involves the secretion of radiation-induced VEGF, leading to an increase in glioma cell motility.
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spelling pubmed-33074922012-03-20 Post-radiation increase in VEGF enhances glioma cell motility in vitro Kil, Whoon Jong Tofilon, Philip J Camphausen, Kevin Radiat Oncol Research BACKGROUND: Glioblastoma multiforme (GBM) is among the most lethal of all human tumors, with frequent local recurrences after radiation therapy (RT). The mechanism accounting for such a recurrence pattern is unclear. It has classically been attributed to local recurrence of treatment-resistant cells. However, accumulating evidence suggests that additional mechanisms exist that involve the migration of tumor or tumor stem cells from other brain regions to tumor bed. VEGFs are well-known mitogens and can be up-regulated after RT. Here, we examine the effect of irradiation-induced VEGF on glioma cell motility. MATERIALS AND METHODS: U251 and LN18 cell lines were used to generate irradiated-conditioned medium (IR-CM). At 72 h after irradiation, the supernatants were harvested. VEGF level in IR-CM was quantified by ELISA, and expression levels for VEGF mRNA were detected by RT-PCR. In vitro cancer cell motility was measured in chambers coated with/without Matrigel and IR-CM as a cell motility enhancer and a VEGF antibody as a neutralizer of VEGF bioactivity. Immunoblots were performed to evaluate the activity of cell motility-related kinases. Proliferation of GBM cells after treatment was measured by flow cytometry. RESULTS: Irradiation increased the level of VEGF mRNA that was mitigated by pre-RT exposure to Actinomycin D. U251 glioma cell motility (migration and invasion) was enhanced by adding IR-CM to un-irradiated cells (174.9 ± 11.4% and 334.2 ± 46% of control, respectively). When we added VEGF antibody to IR-CM, this enhanced cell motility was negated (110.3 ± 12.0% and 105.7 ± 14.0% of control, respectively). Immunoblot analysis revealed that IR-CM increased phosphorylation of VEGF receptor-2 (VEGFR2) secondary to an increase in VEGF, with a concomitant increase of phosphorylation of the downstream targets (Src and FAK). Increased phosphorylation was mitigated by adding VEGF antibody to IR-CM. There was no difference in the mitotic index of GBM cells treated with and without IR-CM and VEGF. CONCLUSIONS: These results indicate that cell motility can be enhanced by conditioned medium from irradiated cells in vitro through stimulation of VEGFR2 signaling pathways and suggest that this effect involves the secretion of radiation-induced VEGF, leading to an increase in glioma cell motility. BioMed Central 2012-02-22 /pmc/articles/PMC3307492/ /pubmed/22356893 http://dx.doi.org/10.1186/1748-717X-7-25 Text en Copyright ©2012 Kit et al; BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research
Kil, Whoon Jong
Tofilon, Philip J
Camphausen, Kevin
Post-radiation increase in VEGF enhances glioma cell motility in vitro
title Post-radiation increase in VEGF enhances glioma cell motility in vitro
title_full Post-radiation increase in VEGF enhances glioma cell motility in vitro
title_fullStr Post-radiation increase in VEGF enhances glioma cell motility in vitro
title_full_unstemmed Post-radiation increase in VEGF enhances glioma cell motility in vitro
title_short Post-radiation increase in VEGF enhances glioma cell motility in vitro
title_sort post-radiation increase in vegf enhances glioma cell motility in vitro
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3307492/
https://www.ncbi.nlm.nih.gov/pubmed/22356893
http://dx.doi.org/10.1186/1748-717X-7-25
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