Conventional anticancer drugs show non-specific vascular toxicity, and using anticancer drugs as angiogenesis inhibitors was suggested. However, our previous study suggested that vascular endothelial growth factor (VEGF) protected endothelial cells against chemotherapy drugs in vitro. To further test whether the vascular toxicity of anticancer drugs is active in vivo, epirubicin was i.p. injected into nude mice with s.c. xenografts of human nasopharyngeal carcinoma CNE-2 once (one-day schedule) or once a day from day 1 to day 7 (seven-day schedule). At 48 hours after the single injection or the 7th injection tumors were removed for detection of apoptosis of vascular endothelial cells vessels and the content of VEGF in tumor tissues. The results showed that epirubicin damaged tumor microvessels when the drug was given as a single dose, whereas epirubicin lost its vascular toxicity when the drug was given continuously for seven days, accompanied by higher levels of VEGF in tumor tissues. These results suggest the sensitivity of endothelial cells lining tumor vessels is variable during chemotherapy, and the protective effect of VEGF on endothelial cells might be related to the schedule of administration.
Background
Abundant aspartyl-asparaginyl-β-hydroxylase (ASPH) expression supports robust neuronal migration during development, and reduced ASPH expression and function, as occur in fetal alcohol spectrum disorder, impair cerebellar neuron migration. ASPH mediates its effects on cell migration via hydroxylation-dependent activation of Notch signaling networks. Insulin and Insulin-like growth factor (IGF-1) stimulate ASPH mRNA transcription and enhance ASPH protein expression by inhibiting Glycogen Synthase Kinase–3β (GSK-3β). This study examines the role of direct GSK-3β phosphorylation as a modulator of ASPH protein expression and function in human cerebellar-derived PNET2 cells.
Methods
Predicted phosphorylation sites encoded by human ASPH were ablated by S/T→A site-directed mutagenesis of an N-Myc-tagged wildtype (WT) cDNA regulated by a CMV promoter. Phenotypic and functional features were assessed in transiently transfected PNET2 cells.
Results
Cells transfected with WT ASPH had increased ASPH protein expression, directional motility, Notch-1 and Jagged-1 expression, and catalytic activity relative to control. Although most single- and multi-point ASPH mutants also had increased ASPH protein expression, their effects on Notch and Jagged expression, directional motility and adhesion, and catalytic activity varied such that only a few of the cDNA constructs conferred functional advantages over WT. Immunofluorescence studies showed that ASPH phosphorylation site deletions can alter the subcellular distribution of ASPH and therefore its potential interactions with Notch/Jagged at the cell surface.
Conclusions
Inhibition of ASPH phosphorylation enhances ASPH protein expression, but attendant alterations in intra-cellular trafficking may govern the functional consequences in relation to neuronal migration, adhesion and Notch activated signaling.
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