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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 tubule formation in irradiated vascular endothelial cells.(A, B) HUVEC or (C) bEnd.3 cells were plated on matrigel-coated 96 well plates and treated with vehicle control or 5 µM BrP-LPA for 45 min prior to irradiation with 3 Gy. (A) Shown are the representative photomicrographs of tubule formation taken 16 h after irradiation. (B) Tubule formation was quantified as number of tubules per high power field (HPF). Shown are bar graphs of mean number of tubules per HPF relative to control and SEM from three experiments; * p<0.05.
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pone-0022182-g002: Inhibition of ATX and LPA receptors attenuates tubule formation in irradiated vascular endothelial cells.(A, B) HUVEC or (C) bEnd.3 cells were plated on matrigel-coated 96 well plates and treated with vehicle control or 5 µM BrP-LPA for 45 min prior to irradiation with 3 Gy. (A) Shown are the representative photomicrographs of tubule formation taken 16 h after irradiation. (B) Tubule formation was quantified as number of tubules per high power field (HPF). Shown are bar graphs of mean number of tubules per HPF relative to control and SEM from three experiments; * p<0.05.

Mentions: ATX and LPA receptors inhibition attenuates tubule formation in irradiated vascular endothelial cells. To determine if ATX and LPA receptors inhibition disrupts the ability of endothelial cells to form capillary-like tubule structures, HUVEC were plated on Matrigel-coated 96-well plates. Cells were treated with 5 µM BrP-LPA or H20 as vehicle control for 45 min before irradiation (3 Gy). Tubule formation was monitored and recorded at 16 h after irradiation. Treatment with 3 Gy or BrP-LPA alone resulted in a slight decrease in the ability of cells to form tubule structures (Fig. 2A and B). However, the combination of ATX and LPA receptor inhibition with irradiation resulted in a 53% attenuation of tubule formation compared to corresponding controls (Fig. 2B, p = 0.0006). Similar results were observed in bEnd.3 cells (Fig. 2C) in which ATX and LPA receptors inhibition produced a 50% reduction in the number of tubules formed compared to its corresponding control (p = 0.0039).


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 tubule formation in irradiated vascular endothelial cells.(A, B) HUVEC or (C) bEnd.3 cells were plated on matrigel-coated 96 well plates and treated with vehicle control or 5 µM BrP-LPA for 45 min prior to irradiation with 3 Gy. (A) Shown are the representative photomicrographs of tubule formation taken 16 h after irradiation. (B) Tubule formation was quantified as number of tubules per high power field (HPF). Shown are bar graphs of mean number of tubules per HPF relative to control 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-g002: Inhibition of ATX and LPA receptors attenuates tubule formation in irradiated vascular endothelial cells.(A, B) HUVEC or (C) bEnd.3 cells were plated on matrigel-coated 96 well plates and treated with vehicle control or 5 µM BrP-LPA for 45 min prior to irradiation with 3 Gy. (A) Shown are the representative photomicrographs of tubule formation taken 16 h after irradiation. (B) Tubule formation was quantified as number of tubules per high power field (HPF). Shown are bar graphs of mean number of tubules per HPF relative to control and SEM from three experiments; * p<0.05.
Mentions: ATX and LPA receptors inhibition attenuates tubule formation in irradiated vascular endothelial cells. To determine if ATX and LPA receptors inhibition disrupts the ability of endothelial cells to form capillary-like tubule structures, HUVEC were plated on Matrigel-coated 96-well plates. Cells were treated with 5 µM BrP-LPA or H20 as vehicle control for 45 min before irradiation (3 Gy). Tubule formation was monitored and recorded at 16 h after irradiation. Treatment with 3 Gy or BrP-LPA alone resulted in a slight decrease in the ability of cells to form tubule structures (Fig. 2A and B). However, the combination of ATX and LPA receptor inhibition with irradiation resulted in a 53% attenuation of tubule formation compared to corresponding controls (Fig. 2B, p = 0.0006). Similar results were observed in bEnd.3 cells (Fig. 2C) in which ATX and LPA receptors inhibition produced a 50% reduction in the number of tubules formed compared to its corresponding control (p = 0.0039).

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