The Expression Of EphB4 Receptor And Its Ligand EphrinB2 In Brain Gliomas And Its Significance

As one of endothelium-specific growth factors, ephrinB2 has been proved to be essential to angiogenesis. Recent reports showed that ephrinB2 and its receptor EphB4 could express in arterial and venous endothelia respectively, and the ephrinB2- positive endothelia could also be noticed in some of newly-formed microvessels of several kinds of tumors, which suggests that tumor microvessels have their own different characteristics. However, the expression pattern of ephrinB2 and its EphB4 receptor in gliomas and its clinico-pathological significance remain obscure.Our previous studies have shown that nordihydroguaiaretic acid (NDGA) has potential growth-inhibitory and differentiation-inducing effects on glioma cell lines. Nordy , a newly synthesized compound in our labratory based on the structure of NDGA, can not only inhibit proliferation of several lines of tumor cells in vitro, including cell lines from lung cancer, hepatocarcinoma, leukaemia and glioma, but also has antitumor effect in vivo. However, the effects of Nordy on the expression of EphB4 receptor and ephrinB2 ligand in glioma cell lines and its significance in the antitumor mechanism of Nordy still remain unclear.In the present study, RT-PCR was used to detect the mRNA expression of ephrinB2 and its EphB4 receptor in fresh astrocytoma tissues and human malignant glioma cell line,CHG-5. Western blotting, immunohistochemistry and double-labelling immunofluorescence combined with confocal laser scanning microscopy(CLSM)were used to detect the protein expression of EphB4 and ephrinB2 genes.Moreover,the effects of Nordy on expression of EphB4 and ephrinB2 were evaluated in CHG-5 glioma cell line. The main results and conclusions are as follows:1. The EphB4 and ephrinB2 mRNA level of tumor tissues were significantly elevated than that of corresponding surrounding brain tissues. The mRNA of the two genes and protein levels of gradeâ… andâ…¡ astrocytomas were significantly lower than that of grade â…¢ and â…£ astrocytoma specimens. Survival analysis showed that the 3-year survival rate of patients were lower in the cases with high EphB4 and ephrinB2 protein expression thanthose with weak staining. These results suggest that both EphB4 and ephrinB2 genes might be of great importance in initiation and progression of astrocytomas and might be one of useful markers to predict the prognosis of astrocytoma.2. The mRNA and protein expression of EphB4 and ephrinB2 in astrocytoma were coincident and significantly correlated. This means that the binding of ephrinB2 ligand with EphB4 receptor may be the principal form of EphB4 receptor activation in astrocytoma. The two proteins may interact with each other to exert their biological function.3. EphB4 and ephrinB2 protein expression were correlated significantly with MVD in astrocytoma. We also noticed that EphB4 or ephrinB2 protein was expressed in tumor microvessels, and the ephrinB2(+) vessels were almost thick-wall, or their endothelial cells were highly proliferated. Double-labelling immunofluorescence combined with CLSM observation showed that the two proteins expression in neovessels were mainly localized in endothelial cells. This implys that ephrinB2 and its EphB4 receptor may play an important role in tumor angiogenesis and there exists microvessels of different attribute (artery or vein; ephrinB2+or EphB4+ microvessels ) in astrocytoma. 4. EphB4 and ephrinB2 genes are highly expressed in CHG-5 glioma cell line, which indicates that glioma cells are likely to produce and release EphB4 and ephrinB2 proteins through an autocrine fashion. Both EphB4 and ephrinB2 not only participate in tumor angiogenesis process, but also play important roles in neoplastic cell's malignant proliferation and differentiation.5. After treated with Nordy of different concentrations, we noticed that the mRNA and protein expression of ephrinB2 and EphB4 genes in CHG-5 cell line were down-regulated significantly. It means that Nordy can inhibit tumor cells from secreting ephrinB2 and EphB4, important angiogenesis-r...