Abstract
Therapeutic radiation is widely used in cancer treatments. The success of radiation therapy depends not only on the radiosensitivity of tumor cells but also on the radiosensitivity of endothelial cells lining the tumor vasculature. Vascular endothelial growth factor (VEGF) plays a critical role in protecting endothelial cells against a number of antitumor agents including ionizing radiation. Strategies designed to overcome the survival advantage afforded to endothelial cells by VEGF might aid in enhancing the efficacy of radiation therapy. In this report we examined the signaling cascade(s) involved in VEGF-mediated protection of endothelial cells against gamma-irradiation. gamma-Irradiation-induced apoptosis of human dermal microvascular endothelial cells (HDMECs) was predominantly mediated through the p38 MAPK pathway as an inhibitor of p38 MAPK (PD169316), and dominant negative mutants of p38 MAPK could significantly enhance HDMEC survival against gamma-irradiation. Inhibition of the PI3K and MAPK pathways markedly up-regulated gamma-irradiation-mediated p38 MAPK activation resulting in enhanced HDMEC apoptosis. In contrast, VEGF-treated HDMECs were protected from gamma-irradiation-induced apoptosis predominantly through the PI3K/Akt pathway. Bcl-2 expression was markedly elevated in VEGF-treated HDMECs, and it was significantly inhibited by the PI3K inhibitor LY294002. HDMECs exposed to irradiation showed a significant decrease in Bcl-2 expression. In contrast, VEGF-stimulated HDMECs, when irradiated, maintained higher levels of Bcl-2 expression. Taken together our results suggest that gamma-irradiation induces endothelial cell apoptosis predominantly via the activation of p38 MAPK, and VEGF protects endothelial cells against gamma-irradiation predominantly via the PI3K-Akt-Bcl-2 signaling pathway.
Highlights
Therapeutic radiation is widely used in cancer treatments
Taken together our results suggest that ␥-irradiation induces endothelial cell apoptosis predominantly via the activation of p38 mitogen-activated protein kinases (MAPK), and Vascular endothelial growth factor (VEGF) protects endothelial cells against ␥-irradiation predominantly via the phosphoinositide 3-kinase (PI3K)-AktBcl-2 signaling pathway
As observed in earlier experiments, blocking of p38 MAPK with PD169316 significantly protected human dermal microvascular endothelial cells (HDMECs) from ␥-irradiation-mediated apoptosis (Fig. 3B). These results demonstrate the role of p38 MAPK in ␥-irradiation-induced HDMEC apoptosis
Summary
HDMECs, endothelial cell basal medium-2 (EBM-2), and growth supplements were purchased from BioWhittaker (Walkersville, MD). A detergent-compatible protein assay kit was purchased from Bio-Rad. Enhanced chemiluminescence plus (ECL-plus) Western blotting detection reagents, anti-mouse, and anti-rabbit horseradish peroxidaseconjugated antibodies were purchased from Amersham Biosciences. HDMECs were further incubated in growth factor-free EGM2-MV medium for 1 h prior to stimulation with 50 ng/ml VEGF. Plasmid Constructions and Transient Transfections—HDMECs were transiently co-transfected with pIRES-enhanced green fluorescent protein and pcDNA3 plasmid containing the dominant negative mutants of ERK1/2, Akt, p38 MAPK or c-Jun (a gift from Dr Cun-Yu Wang, University of Michigan, Ann Arbor) using Lipofectin as described previously [27]. The two solutions were gently mixed and further incubated for 15 min at room temperature to prepare the lipid-DNA complexes. HDMECs were washed twice with EBM, and the lipid-DNA complexes were overlaid onto the cells.
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