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Autotaxin and LPA receptors represent potential molecular targets for the radiosensitization of murine glioma through effects on tumor vasculature.

Schleicher SM, Thotala DK, Linkous AG, Hu R, Leahy KM, Yazlovitskaya EM, Hallahan DE - PLoS ONE (2011)

Bottom Line: In co-culture experiments using bEND.3 and mouse GL-261 glioma cells, treatment with BrP-LPA reduced Akt phosphorylation in both irradiated cell lines and decreased survival and migration of irradiated GL-261 cells.Using siRNA to knock down LPA receptors LPA1, LPA2 or LPA3 in HUVEC, we demonstrated that knockdown of LPA2 but neither LPA1 nor LPA3 led to increased viability and proliferation.However, knockdown of LPA1 and LPA3 but not LPA2 resulted in complete abrogation of tubule formation implying that LPA1 and LPA3 on endothelial cells are likely targets of BrP-LPA radiosensitizing effect.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Vanderbilt University, Nashville, Tennessee, United States of America.

ABSTRACT
Despite wide margins and high dose irradiation, unresectable malignant glioma (MG) is less responsive to radiation and is uniformly fatal. We previously found that cytosolic phospholipase A2 (cPLA(2)) is a molecular target for radiosensitizing cancer through the vascular endothelium. Autotaxin (ATX) and lysophosphatidic acid (LPA) receptors are downstream from cPLA(2) and highly expressed in MG. Using the ATX and LPA receptor inhibitor, α-bromomethylene phosphonate LPA (BrP-LPA), we studied ATX and LPA receptors as potential molecular targets for the radiosensitization of tumor vasculature in MG. Treatment of Human Umbilical Endothelial cells (HUVEC) and mouse brain microvascular cells bEND.3 with 5 µmol/L BrP-LPA and 3 Gy irradiation showed decreased clonogenic survival, tubule formation, and migration. Exogenous addition of LPA showed radioprotection that was abrogated in the presence of BrP-LPA. In co-culture experiments using bEND.3 and mouse GL-261 glioma cells, treatment with BrP-LPA reduced Akt phosphorylation in both irradiated cell lines and decreased survival and migration of irradiated GL-261 cells. Using siRNA to knock down LPA receptors LPA1, LPA2 or LPA3 in HUVEC, we demonstrated that knockdown of LPA2 but neither LPA1 nor LPA3 led to increased viability and proliferation. However, knockdown of LPA1 and LPA3 but not LPA2 resulted in complete abrogation of tubule formation implying that LPA1 and LPA3 on endothelial cells are likely targets of BrP-LPA radiosensitizing effect. Using heterotopic tumor models of GL-261, mice treated with BrP-LPA and irradiation showed a tumor growth delay of 6.8 days compared to mice treated with irradiation alone indicating that inhibition of ATX and LPA receptors may significantly improve malignant glioma response to radiation therapy. These findings identify ATX and LPA receptors as molecular targets for the development of radiosensitizers for MG.

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Inhibition of ATX and LPA receptors reduces irradiated vascular endothelial cell migration.(A) bEnd.3 or (B) HUVEC were plated on 60 mm plates and allowed to grow to 70% confluency. The semi-confluent cell layer was scraped using a sterile pipette tip to create a scratch devoid of cells. The remaining cells were treated with vehicle control or 5 µM BrP-LPA for 45 min prior to irradiation with 3 Gy. Migration was observed at 36 h. Cells were fixed with ethanol and stained with 1% methylene blue. Migrated cells were counted and normalized to surrounding cell density per HPF. Shown are representative photomicrographs and bar graphs representing the mean percentages of migrating cells relative to corresponding controls with SEM from three experiments, * p<0.05.
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pone-0022182-g003: Inhibition of ATX and LPA receptors reduces irradiated vascular endothelial cell migration.(A) bEnd.3 or (B) HUVEC were plated on 60 mm plates and allowed to grow to 70% confluency. The semi-confluent cell layer was scraped using a sterile pipette tip to create a scratch devoid of cells. The remaining cells were treated with vehicle control or 5 µM BrP-LPA for 45 min prior to irradiation with 3 Gy. Migration was observed at 36 h. Cells were fixed with ethanol and stained with 1% methylene blue. Migrated cells were counted and normalized to surrounding cell density per HPF. Shown are representative photomicrographs and bar graphs representing the mean percentages of migrating cells relative to corresponding controls with SEM from three experiments, * p<0.05.

Mentions: To determine whether inhibition ATX and LPA receptors results in reduced endothelial cell migration, a scratch assay was performed by treating HUVEC and bEnd3 cells with 5 µM BrP-LPA or H20 as vehicle control for 45 min prior to irradiation (3 Gy). Migrated cells ware counted and normalized relative to surrounding cell density at 24 h after irradiation. Combined treatment with BrP-LPA and 3 Gy significantly attenuated endothelial cell migration compared to radiation alone in both HUVEC (Fig. 3A, 70% versus 30%) and bEnd3 cells (Fig. 3B, 80% versus 9%). These results indicate that BrP-LPA is able to attenuate migration in both HUVEC and bEnd3 cells.


Autotaxin and LPA receptors represent potential molecular targets for the radiosensitization of murine glioma through effects on tumor vasculature.

Schleicher SM, Thotala DK, Linkous AG, Hu R, Leahy KM, Yazlovitskaya EM, Hallahan DE - PLoS ONE (2011)

Inhibition of ATX and LPA receptors reduces irradiated vascular endothelial cell migration.(A) bEnd.3 or (B) HUVEC were plated on 60 mm plates and allowed to grow to 70% confluency. The semi-confluent cell layer was scraped using a sterile pipette tip to create a scratch devoid of cells. The remaining cells were treated with vehicle control or 5 µM BrP-LPA for 45 min prior to irradiation with 3 Gy. Migration was observed at 36 h. Cells were fixed with ethanol and stained with 1% methylene blue. Migrated cells were counted and normalized to surrounding cell density per HPF. Shown are representative photomicrographs and bar graphs representing the mean percentages of migrating cells relative to corresponding controls with SEM from three experiments, * p<0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3140496&req=5

pone-0022182-g003: Inhibition of ATX and LPA receptors reduces irradiated vascular endothelial cell migration.(A) bEnd.3 or (B) HUVEC were plated on 60 mm plates and allowed to grow to 70% confluency. The semi-confluent cell layer was scraped using a sterile pipette tip to create a scratch devoid of cells. The remaining cells were treated with vehicle control or 5 µM BrP-LPA for 45 min prior to irradiation with 3 Gy. Migration was observed at 36 h. Cells were fixed with ethanol and stained with 1% methylene blue. Migrated cells were counted and normalized to surrounding cell density per HPF. Shown are representative photomicrographs and bar graphs representing the mean percentages of migrating cells relative to corresponding controls with SEM from three experiments, * p<0.05.
Mentions: To determine whether inhibition ATX and LPA receptors results in reduced endothelial cell migration, a scratch assay was performed by treating HUVEC and bEnd3 cells with 5 µM BrP-LPA or H20 as vehicle control for 45 min prior to irradiation (3 Gy). Migrated cells ware counted and normalized relative to surrounding cell density at 24 h after irradiation. Combined treatment with BrP-LPA and 3 Gy significantly attenuated endothelial cell migration compared to radiation alone in both HUVEC (Fig. 3A, 70% versus 30%) and bEnd3 cells (Fig. 3B, 80% versus 9%). These results indicate that BrP-LPA is able to attenuate migration in both HUVEC and bEnd3 cells.

Bottom Line: In co-culture experiments using bEND.3 and mouse GL-261 glioma cells, treatment with BrP-LPA reduced Akt phosphorylation in both irradiated cell lines and decreased survival and migration of irradiated GL-261 cells.Using siRNA to knock down LPA receptors LPA1, LPA2 or LPA3 in HUVEC, we demonstrated that knockdown of LPA2 but neither LPA1 nor LPA3 led to increased viability and proliferation.However, knockdown of LPA1 and LPA3 but not LPA2 resulted in complete abrogation of tubule formation implying that LPA1 and LPA3 on endothelial cells are likely targets of BrP-LPA radiosensitizing effect.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine, Vanderbilt University, Nashville, Tennessee, United States of America.

ABSTRACT
Despite wide margins and high dose irradiation, unresectable malignant glioma (MG) is less responsive to radiation and is uniformly fatal. We previously found that cytosolic phospholipase A2 (cPLA(2)) is a molecular target for radiosensitizing cancer through the vascular endothelium. Autotaxin (ATX) and lysophosphatidic acid (LPA) receptors are downstream from cPLA(2) and highly expressed in MG. Using the ATX and LPA receptor inhibitor, α-bromomethylene phosphonate LPA (BrP-LPA), we studied ATX and LPA receptors as potential molecular targets for the radiosensitization of tumor vasculature in MG. Treatment of Human Umbilical Endothelial cells (HUVEC) and mouse brain microvascular cells bEND.3 with 5 µmol/L BrP-LPA and 3 Gy irradiation showed decreased clonogenic survival, tubule formation, and migration. Exogenous addition of LPA showed radioprotection that was abrogated in the presence of BrP-LPA. In co-culture experiments using bEND.3 and mouse GL-261 glioma cells, treatment with BrP-LPA reduced Akt phosphorylation in both irradiated cell lines and decreased survival and migration of irradiated GL-261 cells. Using siRNA to knock down LPA receptors LPA1, LPA2 or LPA3 in HUVEC, we demonstrated that knockdown of LPA2 but neither LPA1 nor LPA3 led to increased viability and proliferation. However, knockdown of LPA1 and LPA3 but not LPA2 resulted in complete abrogation of tubule formation implying that LPA1 and LPA3 on endothelial cells are likely targets of BrP-LPA radiosensitizing effect. Using heterotopic tumor models of GL-261, mice treated with BrP-LPA and irradiation showed a tumor growth delay of 6.8 days compared to mice treated with irradiation alone indicating that inhibition of ATX and LPA receptors may significantly improve malignant glioma response to radiation therapy. These findings identify ATX and LPA receptors as molecular targets for the development of radiosensitizers for MG.

Show MeSH
Related in: MedlinePlus