Post-radiation increase in VEGF enhances glioma cell motility in vitro

<p>Abstract</p> <p>Background</p> <p>Glioblastoma multiforme (GBM) is among the most lethal of all human tumors, with frequent local recurrences after radiation therapy (RT). The mechanism accounting for such a recurrence pattern is unclear. It has classically been attributed to local recurrence of treatment-resistant cells. However, accumulating evidence suggests that additional mechanisms exist that involve the migration of tumor or tumor stem cells from other brain regions to tumor bed. VEGFs are well-known mitogens and can be up-regulated after RT. Here, we examine the effect of irradiation-induced VEGF on glioma cell motility.</p> <p>Materials and methods</p> <p>U251 and LN18 cell lines were used to generate irradiated-conditioned medium (IR-CM). At 72 h after irradiation, the supernatants were harvested. VEGF level in IR-CM was quantified by ELISA, and expression levels for VEGF mRNA were detected by RT-PCR. <it>In vitro </it>cancer cell motility was measured in chambers coated with/without Matrigel and IR-CM as a cell motility enhancer and a VEGF antibody as a neutralizer of VEGF bioactivity. Immunoblots were performed to evaluate the activity of cell motility-related kinases. Proliferation of GBM cells after treatment was measured by flow cytometry.</p> <p>Results</p> <p>Irradiation increased the level of VEGF mRNA that was mitigated by pre-RT exposure to Actinomycin D. U251 glioma cell motility (migration and invasion) was enhanced by adding IR-CM to un-irradiated cells (174.9 ± 11.4% and 334.2 ± 46% of control, respectively). When we added VEGF antibody to IR-CM, this enhanced cell motility was negated (110.3 ± 12.0% and 105.7 ± 14.0% of control, respectively). Immunoblot analysis revealed that IR-CM increased phosphorylation of VEGF receptor-2 (VEGFR2) secondary to an increase in VEGF, with a concomitant increase of phosphorylation of the downstream targets (Src and FAK). Increased phosphorylation was mitigated by adding VEGF antibody to IR-CM. There was no difference in the mitotic index of GBM cells treated with and without IR-CM and VEGF.</p> <p>Conclusions</p> <p>These results indicate that cell motility can be enhanced by conditioned medium from irradiated cells <it>in vitro </it>through stimulation of VEGFR2 signaling pathways and suggest that this effect involves the secretion of radiation-induced VEGF, leading to an increase in glioma cell motility.</p

Salen Mn Complexes Mitigate Radiation Injury in Normal Tissues

Salen Mn complexes, including EUK-134, EUK-189 and a newer cyclized analog EUK-207, are synthetic SOD/catalase mimetics that have beneficial effects in many models of oxidative stress. As oxidative stress is implicated in some forms of delayed radiation injury, we are investigating whether these compounds can mitigate injury to normal tissues caused by ionizing radiation. This review describes some of this research, focusing on several tissues of therapeutic interest, namely kidney, lung, skin, and oral mucosa. These studies have demonstrated suppression of delayed radiation injury in animals treated with EUK-189 and/or EUK-207. While an antioxidant mechanism of action is postulated, it is likely that the mechanisms of radiation mitigation by these compounds in vivo are complex and may differ in the various target tissues. Indicators of oxidative stress are increased in lung and skin radiation injury models, and suppressed by salen Mn complexes. The role of oxidative stress in the renal injury model is unclear, though EUK-207 does mitigate. In certain experimental models, salen Mn complexes have shown “mito-protective” properties, that is, attenuating mitochondrial injury. Consistent with this, EUK-134 suppresses effects of ionizing radiation on mitochondrial function in rat astrocyte cultures. In summary, salen Mn complexes could be useful to mitigate delayed radiation injury to normal tissues following radiation therapy, accidental exposure, or radiological terrorism. Optimization of their mode of delivery and other key pharmaceutical properties, and increasing understanding of their mechanism(s) of action as radiation mitigators, are key issues for future study

High throughput evaluation of gamma-H2AX

The DNA double-strand break (DSB) is the primary lethal lesion after therapeutic radiation. Thus, the development of assays to detect and to quantitate these lesions could have broad preclinical and clinical impact. Phosphorylation of histone H2AX to form γ-H2AX is a known marker for irradiation-induced DNA DSBs. However, the first generation assay involves the use of immunofluorescent staining of γ-H2AX foci. This assay is time consuming, operator dependent and is not scalable for high throughput assay development. Thus, we sought to develop a new assay using a high throughput electrochemiluminescent platform from Mesoscale Discovery Systems to quantify γ-H2AX levels. The results show that our assay utilizes significantly less time and labor, has greater intra-assay reproducibility and has a greater dynamic range of γ-H2AX versus irradiation dose

Metabolic alterations: A biomarker for radiation-induced normal brain injury—an MR spectroscopy study

Purpose To assess if interval changes in metabolic status in normal cerebral tissue after radiation therapy (RT) can be detected by 2D CSI (chemical shift imaging) proton spectroscopy. Materials and Methods Eleven patients with primary brain tumors undergoing cranial radiation therapy (RT) were included. 2D-CSI MRS was performed before, during, and after the course of RT with the following parameters: TE/TR 144/1500 ms, field of view (FOV) 24, thickness 10 mm, matrix 16 × 16. The metabolic ratios choline/creatine (Cho/Cr), N-acetylaspartate (NAA)/Cr, and NAA/Cho in normal brain tissue were calculated. Results NAA/Cr and Cho/Cr were significantly decreased at week 3 during RT and at 1 month and 6 months after RT compared to values prior to RT ( P < 0.01). The NAA/Cr ratio decreased by −0.19 ± 0.05 (mean ± standard error [SE]) at week 3 of RT, −0.14 ± 0.06 at the last week of RT, −0.14 ± 0.05 at 1 month after RT, and −0.30 ± 0.08 at 6 months after RT compared to the pre-RT value of 1.43 ± 0.04. The Cho/Cr ratio decreased by −0.27 ± 0.05 at week 3 of RT, −0.11 ± 0.05 at the last week of RT, −0.26 ± 0.05 at 1 month after RT and −0.25 ± 0.07 at 6 months after RT from the pre-RT value of 1.29 ± 0.03. Changes in Cho/Cr were correlated with the interaction of the radiation dose and dose-volume at week 3 of RT, during the last week of RT ( P < 0.005), and at 1 month after RT ( P = 0.017). Conclusion The results of this study suggest that MRS can detect early metabolic changes in normal irradiated brain tissue. J. Magn. Reson. Imaging 2009;29:291–297. © 2009 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/61530/1/21657_ftp.pd

Gamma-ray irradiation stimulates the expression of caveolin-1 and GFAP in rat spinal cord: a study of immunoblot and immunohistochemistry

We studied the expression of caveolin-1 in the spinal cords of rats using 60Co γ-ray irradiation (single dose of 8 Gray (Gy)) in order to determine the possible involvement of caveolin-1 in the tissues of the central nervous system after irradiation. Spinal cords sampled at days 1, 4, and 9 post-irradiation (PI) (n = 5 per each time point) were analyzed by Western blot and immunohistochemistry. Western blot analysis showed that the expression of caveolin-1 was significantly increased at day 1 PI (p < 0.05), and returned to the level of normal control rats on days 4 and 9 PI. Immunohistochemistry showed that caveolin-1 immunoreactivity was enhanced in some glial cells, vascular endothelial cells, and neurons in the spinal cords. The increased expression of glial fibrillary acidic protein (GFAP), a marker for an astroglial reaction, was consistent with that of caveolin-1. In addition, caveolin-1 was co-localized in hypertrophied GFAP-positive astrocytes. Taking all these facts into consideration, we postulate that irradiation induces the increased expression of caveolin-1 in cells of the central nervous system, and that its increased expression in astrocytes may contribute to hypertrophy of astrocytes in the spinal cord after irradiation. The precise role of caveolin-1 in the spinal cords should be studied further

Radiation Induces Acute Alterations in Neuronal Function

Every year, nearly 200,000 patients undergo radiation for brain tumors. For both patients and caregivers the most distressing adverse effect is impaired cognition. Efforts to protect against this debilitating effect have suffered from inadequate understanding of the cellular mechanisms of radiation damage. In the past it was accepted that radiation-induced normal tissue injury resulted from a progressive reduction in the survival of clonogenic cells. Moreover, because radiation-induced brain dysfunction is believed to evolve over months to years, most studies have focused on late changes in brain parenchyma. However, clinically, acute changes in cognition are also observed. Because neurons are fully differentiated post-mitotic cells, little information exists on the acute effects of radiation on synaptic function. The purpose of our study was to assess the potential acute effects of radiation on neuronal function utilizing ex vivo hippocampal brain slices. The cellular localization and functional status of excitatory and inhibitory neurotransmitter receptors was identified by immunoblotting. Electrophysiological recordings were obtained both for populations of neuronal cells and individual neurons. In the dentate gyrus region of isolated ex vivo slices, radiation led to early decreases in tyrosine phosphorylation and removal of excitatory N-methyl-D-aspartate receptors (NMDARs) from the cell surface while simultaneously increasing the surface expression of inhibitory gamma-aminobutyric acid receptors (GABAARs). These alterations in cellular localization corresponded with altered synaptic responses and inhibition of long-term potentiation. The non-competitive NMDAR antagonist memantine blocked these radiation-induced alterations in cellular distribution. These findings demonstrate acute effects of radiation on neuronal cells within isolated brain slices and open new avenues for study

SMART syndrome: a late reversible complication after radiation therapy for brain tumours

With intensified treatment leading to longer survival, complications of therapy for brain tumours are more frequently observed. Regarding radiation therapy, progressive and irreversible white matter disease with cognitive decline is most feared. We report on four patients with reversible clinical and radiological features occurring years after radiation for brain tumours, suggestive for the so called SMART syndrome (stroke-like migraine attacks after radiation therapy). All four patients (males, age 36–60 years) had been treated with focal brain radiation for a primary brain tumour or with whole-brain radiation therapy for brain metastases. Ranging from 2 to 10 years following radiation therapy patients presented with headache and focal neurological deficits, suggestive for tumour recurrence. Two patients also presented with focal seizures. MRI demonstrated typical cortical swelling and contrast enhancement, primarily in the parieto-occipital region. On follow-up both clinical and MRI features improved spontaneously. Three patients eventually proved to have tumour recurrence. The clinical and radiological picture of these patients is compatible with the SMART syndrome, a rare complication of radiation therapy which is probably under recognized in brain tumour patients. The pathophysiology of the SMART syndrome is poorly understood but bears similarities with the posterior reversible encephalopathy syndrome (PRES). These four cases underline that the SMART syndrome should be considered in patients formerly treated with radiation therapy for brain tumours, who present with new neurologic deficits. Before the diagnosis of SMART syndrome can be established other causes, such as local tumour recurrence, leptomeningeal disease or ischemic disease should be ruled out