Irradiated C6 glioma cells induce angiogenesis in vivo and activate endothelial cells in vitro

Abstract

Malignant gliomas are angiogenesis dependent and present a remarkable degree of resistance to radiotherapy. In the present work, we studied the effect of irradiation of C6 glioma cells on their proliferation and activation in vitro and on glioma cell-induced angiogenesis in vivo and in vitro. Irradiation of C6 glioma cells decreased cell proliferation in a dosedependent manner. Interestingly, metalloproteinase-2 and -9 expression and secretion, as well as integrin ␣ v expression, increased with elevated doses of X rays 48 hr after irradiation and was mostly evident at the higher doses used. When pre-irradiated C6 cells were implanted on nonirradiated chicken embryo chorioallantoic membranes (CAMs), there was a significant dose-dependent increase in tumor induced angiogenesis, compared to angiogenesis induced by nonirradiated cells. Similar results were obtained when C6 cells were irradiated 48 hr after their inocculation onto nonirradiated CAMs. In the same line, conditioned medium from irradiated C6 cells significantly increased endothelial cell proliferation and migration in vitro, in a manner dependent on the dose of X rays. These results explain at least in part the low effectiveness of radiation therapy of malignant gliomas and support the notion that inhibition of angiogenesis in parallel with radiotherapy may represent a new therapeutic approach. © 2004 Wiley-Liss, Inc. Key words: angiogenesis; endothelial cells; glioma cells; metalloproteinase-2; migration; proliferation; X raysMalignant gliomas, the most common type of brain tumors in adults, present a remarkable degree of neovascularisation and invasiveness into surrounding tissues. 1 These features account for their poor prognosis, and although combinations of surgery, radiotherapy and chemotherapy are used, more than 90% of the patients experience local recurrence and their survival is extremely low. These facts stress the need for more effective therapeutic strategies. 2 Rapid cell proliferation and high invasive capacity of glioma cells are crucial for tumor development and progression. Moreover, induction of angiogenesis is an essential prerequisite for tumor growth and spread, and precedes malignant tumor formation. 3 Angiogenesis is a fundamental process by which new blood vessels are formed from pre-existing ones. It is a very important step in reproduction, development and wound healing. Furthermore, angiogenesis is very important in several pathological mechanisms like carcinogenesis, tumor metastasis and inflammation. 4 Radiation is considered to inhibit angiogenesis through damage to the endothelium. 5,6 However, in vivo studies suggest that ionizing radiation induces vascular endothelial growth factor (VEGF) production 7 and enhances both the formation of new blood vessels 8 -11 and the secondary development of tumors. 11-13 We have previously shown in the in vivo system of the chicken embryo chorioallantoic membrane (CAM) that X-rays enhance angiogenesis induced by C6 glioma cells inoculated onto the tissue immediately after irradiation...

Carbon Ion Radiation Inhibits Glioma and Endothelial Cell Migration Induced by Secreted VEGF

“…In accordance with earlier studies [20], [24]–[25], the present data showed that X-ray radiation enhanced the secretion of VEGF , a potent angiogenic growth factor [26], as well as that of MMP - 2 and - 9 , which degrade the basement membrane and extracellular matrix during tumor cell invasion and metastasis [27]. In contrast, VEGF and MMP - 2 and - 9 levels were not significantly increased by carbon ion radiation; indeed, VEGF secretion was reduced at 4.0 Gy (Fig.…”

“…Carbon ion radiation has been shown to suppress migration and the formation of capillary-like tube structures in ECV304 and human umbilical vascular endothelial cells at a dose of 0.1 Gy [19]. In contrast, conditioned media from tumor cells exposed to X-ray radiation stimulated cell migration and tube formation, consistent with previous findings [20]. The migration and invasion of glioma cells into surrounding normal brain tissue and blood vessels contributes to the limited responsiveness of gliomas to conventional radiation treatments [21]–[22].…”

The chicken chorioallantoic membrane model in biology, medicine and bioengineering

“…The authors noticed a dose dependent vacuolization of endothelial cells as well as growth retardation, resulting in a remarkable reduction in the vascular endpoint density. Finally, several preclinical studies in the CAM have used combination therapy and demonstrated an increased effectiveness when radiotherapy is combined with an anti-angiogenic treatment [156,157]. …”

Antiangiogenic therapy in brain tumors

“…Malignant glioma cells are inherently chemo- [161] and radioresistant [162]. Also, as radiation of glioma cells may potentiate the expression of proangiogenic molecules [163], combining radiotherapy with antiangiogenic therapy may also prove to be clinically useful and effective. By way of the CSC vascular niche hypothesis, the future of therapeutic strategy will continue to require a multidimensional approach to tumor eradication.…”