| BackgroundGlioblastoma multiforme (GBM) is one of the most common and lethal tumors in the central nervous system. Radiotherapy remains one of the most effective agents in the treatment of GBM. Despite of the use of radiosensitizers, the combination with chemotherapy, and the advance in techniques for administering irradiation (IR), GBMs invariably recur, leading to the death of most patients within one year. clinical studies have demonstrated that the majority of GBM tumors relapse within2cm of the previous tumors’enhancing edges, or the resection cavities after either WBI or large volume IR. The "2-cm margin" is regarded as the interaction zone between tumor cells and irradiated normal brain tissues. The vascular endothelial growth factor family (VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placenta growth factor) and their receptors (VEGFR-1and VEGFR-2) are important mediators of glioma progression. VEGF-A, the most important member, is upregulated in GBM and regulates tumor cell proliferation, angiogenesis and migration primarily via VEGFR-2.Stromal cell-derived factor la (SDF-la) is the only known ligand for G-protein-coupled C-X-C chemokine receptor4(CXCR4). The SDF-1α/CXCR4signaling mediates many physiological processes including cell trafficking, vasculogenesis, and embryogenesis. It is involved in brain development, such as assembly, differentiation and function of neural precursors, neurons and glial cells. The SDF-la/CXCR4signaling also has a vital role in glioma invasion, as well as in glioma proliferation or vasculogenesis. Recent studies confirmed that VEGF binding to VEGFR induces SDF-la and CXCR4production in endothelial cells, as well as in neuronal cells. In turn, the interaction between SDF-1α and CXCR4in endothelial cells promotes VEGF secretion. Here there is a possible interaction between VEGF/VEGFR and SDF-1α/CXCR4signalings in GBM invasion stimulated by irradiated normal brain nssues. ObjectiveTo evaluate whether irradiation in normal brain tissues could stimulate invasion of glioblastoma through the VEGF/VEGFR and SDF-la/CXCR4signalings.Methods1. Six-to eight-week-old male C57BL/6mice were randomized into four experimental groups:(i) whole-brain irradiation (WBI) of15Gy in3fractions alone;(ii) injected i.c. with GL261cells at Day0;(iii) WBI (3fractions of5Gy) at Days0-2, followed by implantation of GL261cells i.c. at Day3;(iv) sham surgery as control. Each experiment consisted of8mice.2. The changes of VEGF and SDF-1α in C57BL/6mice after WBI of15Gy in3fractions were evaluated by real-time PCR and immunohistochemistry.3. Cell invasion assay were performed to study the synergistic effect of VEGF and SDF-1α.4. miRNAs were designed against HIF-1α. Then, the vector with HIF-1α-miRNA or control miRNA plasmids were transfected into the GL261cells using Lipofectamine-2000. Transfectants were selected by Blasticidin.5. GL261cells were treated with varying concentrations (0,10,100ng/mL) of VEGF and SDF-1α for48h. Supernatants were collected to assay SDF-la and VEGF protein levels by using ELISA kits.6. The statistical tests were performed in the statistical software package SPSS, version12.0. When multiple groups were evaluated, one-way ANOVA was used. When two groups were compared, the unpaired independent-samples Student’s t-test was used. P<0.05was considered statistically significant.Results1. The VEGF and SDF-1α protein levels in normal brain tissues of C57BL/6mice were increased72h after15Gy WBI. VEGF mRNA levels in normal brain tissues reached maximum level by7days. SDF-1α mRNA levels in normal brain tissues showed significantlyhigher48h after IR (P<0.05).2. The primary tumor area of GL261cells in WBI pretreated mice showed a trend toward an increase that did not reach significance, compared to unirradiated mice ((8.0±1.8)%vs (13.0±5.4)%. P>0.05). Meanwhile. WBI prior to tumor implantation of GL261cells significantly increased the satellite numbers (P<0.05).3. Tumor cells with HIF-1α knockdown in WBI pretreated mice still showed a significant increase in satellite formation. as compared with those tumors in the unirradiated mice (P<0.05).4.100ng/mL VEGF and/or100ng/mL SDF-1α significantly increased GL261cell invasion with or without high-dcse IR (15Gy in3fractions), with the highest response occurring in reaction to VEGF plus SDF-1α. CXCR4antagonist AMD3100(10μmol/L) could abrogate this synergistic effect.5. Exogenous VEGF (10,100ng/mL) induced SDF-1α secretion. In the same way, the protein levels of VEGF were also up-regulated by100ng/mL SDF-la in GL261glioma cells.Conclusions1. Irradiation can inhibit invasion of GBM, but irradiated normal brain tissues can upregulate VEGF and SDF-1a expression and those factors may contribute to normal brain tissues susceptible to tumor invasion.2. There was a positive feedback loop between VEGF and SDF-1α in glioma cells. |
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