Detection Of Functional FPR Expression By Human Glioma Cells And The Therapeutic Significance

Previous studies on chemokine receptors mainly focused on their functions mediating inflammatory processes. In recent years, these receptors have been found to play an extensive and complicated role in carcinogenesis. Many chemokines have been detected in malignant tumors, and multiple chemokine receptors have also been demonstrated to be expressed by tumor cells. Some of these receptors suppress tumor growth through chemotaxis, activation of immune cells and suppression of angiogenesis, while others promote tumor growth and metastasis through enhancing tumor cell mobility, attracting tumor cell invasion, promoting vascular proliferation and degradation of extracellular matrix.We previously detected the expression of formylpeptide receptor (FPR), a G-protein -coupled receptor, in glioblastoma tissues, and such expression was found to be associated with the malignancy level of glioma and the density of microvessels. The agonists of FPR promote chemotaxis and Ca2+ mobilization. Necrotic glioblastoma cells produce FPR agonists that activate FPR of live tumor cells. Glioma is the most common malignant neoplasm of the central nervous system, and the characteristics of glioma include rapid growth and invasion, and abundant de novo blood vessels. The effect of FPR expressed by gliomas on these characteristics, especially the therapeutic significance of FPR, is worthy of investigation. The studies on the expression and function of FPR we previously carried out were with cells line and tissues; however, whether primary cells of glioma maintain functional expression of FPR during cell culture is still unclear.Nordihydroguaiaretic acid (NDGA), a natural product of an evergreen shrub Larrea, is a potent lipoxygenase inhibitor. In recent years, NDGA has been found to effectively inhibit the growth and induce the differentiation of human glioma. In addition, it suppresses the incidence of experimental glioma and induces the differentiation of xenograft tumors. The new drug Nordy, researched and developed by our laboratory on the basis of the structure of natural NDGA, shows sound efficacy in inducing cell differentiation and suppressing angiogenesis.Since the functional significance of FPR in glioma is to be further defined and whether the anti-tumor mechanism of Nordy correlates to the functional expression of FPR is to be determined, we addressed these issues in this study. The main results and conclusions are as follows:1. Considering that primary-cultured cells maintain their in vivo growth and functional state for a certain period of time, we first utilized primary cultures of glioma cells to explore the relationship between the FPR expression by glioma cells and angiogenesis. We cultured fresh specimens of glioma by tissue mass culture and trypsin digestion. Nine specimens were successfully cultured and all of them were then identified.2. We investigated the FPR expression in human glioma primary cells by immunofluorescence and confocal laser microscopy. With these methods, we detected positive signals in 5 highly malignant (WHO grade III or IV) primary glioma specimens, and the positive signals were linear and discontinuous fluorescence along tumor cell membrane, while other lowly malignant (WHO grade I or II) glioma cells showed no FPR expression. These results indicate that the FPR expression in human glioma tissues and cells is associated with the malignancy level of the tumor.3. There were no obvious morphological changes of the tumor cells after FPR bound to its ligand; however, the cell density was increased and cells grew more rapidly. Moreover, as the time went by, this effect became more and more conspicuous, which indicates that FPR expressed by glioma cells is responsive to its ligand fMLF, and that activated FPR promotes proliferation of tumor cells in vitro. We investigated VEGF production following activation of glioma cells by FPR using ELISA, and the results showed that the VEGF protein production of the tumor cells was significantly increased as compared with the control group; furthermore, such increases were dependent on the concentration of fMLF. These results demonstrate that the FPR expression and functional activation of glioma cells are closely associated with VEGF production and, thus, may play an important role in angiogenesis of gliomas.4. Treatment of primary-cultured glioma cells with Nordy caused a decrease of cell proliferation and VEGF protein secretion. This indicates that Nordy significantly suppresses the expression of FPR and its functional effects, a finding further revealing the anti-tumor mechanism of Nordy.