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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 attenuates migration and enhances cell death in irradiated GL261 cells.(A, B) Mouse glioma GL261 cells were plated on 60 mm plates and allowed to grow to 70% confluency. Plates were scraped with a pipette tip to create a scratch devoid of cells and treated with vehicle control or 5 µM BrP-LPA for 45 min before irradiation with 3 Gy. After 24 h, cells were fixed and stained with methylene blue. Migrated cells were counted and normalized to surrounding cell density per HPF. Shown are representative photomicrographs (A) and a bar graph (B) representing the mean percentages of migrating cells relative to corresponding controls with SEM from three experiments; * p<0.05. (C) For clonogenic survival assay, GL261 cells were plated and allowed to attach. After 6 h, cells were treated vehicle control or with 5 µM BrP-LPA for 45 min and irradiated with 0, 2, 4, and 6 Gy. After 10 days, surviving colonies (>50 cells) were counted and normalized for plating efficiency. Shown is the clonogenic survival curve and mean surviving fractions and SEM from three experiments; * p<0.05.
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pone-0022182-g004: Inhibition of ATX and LPA receptors attenuates migration and enhances cell death in irradiated GL261 cells.(A, B) Mouse glioma GL261 cells were plated on 60 mm plates and allowed to grow to 70% confluency. Plates were scraped with a pipette tip to create a scratch devoid of cells and treated with vehicle control or 5 µM BrP-LPA for 45 min before irradiation with 3 Gy. After 24 h, cells were fixed and stained with methylene blue. Migrated cells were counted and normalized to surrounding cell density per HPF. Shown are representative photomicrographs (A) and a bar graph (B) representing the mean percentages of migrating cells relative to corresponding controls with SEM from three experiments; * p<0.05. (C) For clonogenic survival assay, GL261 cells were plated and allowed to attach. After 6 h, cells were treated vehicle control or with 5 µM BrP-LPA for 45 min and irradiated with 0, 2, 4, and 6 Gy. After 10 days, surviving colonies (>50 cells) were counted and normalized for plating efficiency. Shown is the clonogenic survival curve and mean surviving fractions and SEM from three experiments; * p<0.05.

Mentions: Since glioma cells express high levels of ATX and multiple LPA receptors [8] , we investigated the effects of BrP-LPA on cell migration and colony formation in GL261 cells. Treatment of GL261 cells with either 5 µM BrP-LPA or 3 Gy alone resulted in a minor reduction in cell migration (81% and 98% of control, respectively; Fig. 4A and 4B). However, combined treatment with BrP-LPA and irradiation reduced GL261 migration to 65% of control (Fig. 4B, p = 0.018). In clonogenic survival studies, irradiated GL261 cells treated with 5 µM BrP-LPA showed a modest but significantly reduced survival at the radiation dose of 2 Gy compared to cells treated with radiation alone (Fig. 4C).


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 attenuates migration and enhances cell death in irradiated GL261 cells.(A, B) Mouse glioma GL261 cells were plated on 60 mm plates and allowed to grow to 70% confluency. Plates were scraped with a pipette tip to create a scratch devoid of cells and treated with vehicle control or 5 µM BrP-LPA for 45 min before irradiation with 3 Gy. After 24 h, cells were fixed and stained with methylene blue. Migrated cells were counted and normalized to surrounding cell density per HPF. Shown are representative photomicrographs (A) and a bar graph (B) representing the mean percentages of migrating cells relative to corresponding controls with SEM from three experiments; * p<0.05. (C) For clonogenic survival assay, GL261 cells were plated and allowed to attach. After 6 h, cells were treated vehicle control or with 5 µM BrP-LPA for 45 min and irradiated with 0, 2, 4, and 6 Gy. After 10 days, surviving colonies (>50 cells) were counted and normalized for plating efficiency. Shown is the clonogenic survival curve and mean surviving fractions and SEM from three experiments; * p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0022182-g004: Inhibition of ATX and LPA receptors attenuates migration and enhances cell death in irradiated GL261 cells.(A, B) Mouse glioma GL261 cells were plated on 60 mm plates and allowed to grow to 70% confluency. Plates were scraped with a pipette tip to create a scratch devoid of cells and treated with vehicle control or 5 µM BrP-LPA for 45 min before irradiation with 3 Gy. After 24 h, cells were fixed and stained with methylene blue. Migrated cells were counted and normalized to surrounding cell density per HPF. Shown are representative photomicrographs (A) and a bar graph (B) representing the mean percentages of migrating cells relative to corresponding controls with SEM from three experiments; * p<0.05. (C) For clonogenic survival assay, GL261 cells were plated and allowed to attach. After 6 h, cells were treated vehicle control or with 5 µM BrP-LPA for 45 min and irradiated with 0, 2, 4, and 6 Gy. After 10 days, surviving colonies (>50 cells) were counted and normalized for plating efficiency. Shown is the clonogenic survival curve and mean surviving fractions and SEM from three experiments; * p<0.05.
Mentions: Since glioma cells express high levels of ATX and multiple LPA receptors [8] , we investigated the effects of BrP-LPA on cell migration and colony formation in GL261 cells. Treatment of GL261 cells with either 5 µM BrP-LPA or 3 Gy alone resulted in a minor reduction in cell migration (81% and 98% of control, respectively; Fig. 4A and 4B). However, combined treatment with BrP-LPA and irradiation reduced GL261 migration to 65% of control (Fig. 4B, p = 0.018). In clonogenic survival studies, irradiated GL261 cells treated with 5 µM BrP-LPA showed a modest but significantly reduced survival at the radiation dose of 2 Gy compared to cells treated with radiation alone (Fig. 4C).

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