Expression Of FPR By Malignant Glioma Cells And Glioma Stem Cells Promotes Angiogenesis

Angiogenesis is crucial for the growth and metastasis of malignant solid tumors. Therefore, the mechanism of angiogenesis and antiangiogenesis has been one of the frontiers in cancer research. Microvessel density in the tumor typically rich of microvessels have been revealed to be related to behaviors such as invasiveness and recurrence. Thus, it is of great importance and necessary to explore novel targets for therapeutis for malignant glioma.Proangiogenic factors including VEGF and IL-8 are involved in the development and angiogenesis of cancer. Our previous studies have showed that formylpeptide receptor (FPR), a G-protein coupled rceptor, was expressed by malignant glioma and related to the degree of maligancy and microvessel density within glioma tissue. The significance of functional FPR in cancer therapy needs futher studies. Recent evidences have demonstrated that various cancers including breast cancer, prostatic carcinoma and brain tumor have a minority of tumor cells capable of renewal and forming new tumors. It is unkown whether tumor stem cells contribute to angiogenesis untill recent studies showed that hypoxia induced the production of VEGF by glioma stem cells. However, the mechanism remain unclear.For a better understanding of the possible role and mechanism of FPR in malignant glioma growth and angiogenesis, we investigated the expression of FPR in human malignant glioma cells and glioma stem cells among them, and examined their contribution to angiogenesis. Moreover, we also explored whether the FPR may be become the therapeutic target of angiogenesis in malignant glioma. Firstly, we detected the expression of FPR in U87 cells and its relationship with tumor cells growth and the production of angiogenic factors. Then, we explored the inhibitory effect of Nordy, a sythesized chiral compound based on the structure of natural nordihydroguaiaretic acid (NDGA) on the the response of U87 cells to FPR agonist fMLF. Furthemore, we isolated and identified the glioma stem cells from U87 cells. Finally, we studied the functional expression of FPR by glioma stem cells promoting angiogenesis. The main results and conclusions are as follows: 1. By immunofluorescence assay, we found that U87cells and FPR-mock-U87 cells (cells transfected with a scrambled siRNA) expressed the FPR, however in FPR-siRNA-U87 cells (cells transfected with a FPR siRNA) the majority did not express FPR, and only the mitotic cells expressed the FPR. Thus, we used the FPR-mock-U87 cells and FPR-siRNA U87 as control cells to explored the function of FPR. By MTT assay, ELISA and RT-PCR, we examined the responses of glioma cells to FPR agonist fMLF. The results indicated that: (1) 100nmol/LfMLF significantly promoted U87 cells and FPR-mock-U87 cells proliferation (P<0.05), but did not influence the FPR-siRNA U87 cells proliferation (P>0.05). (2) U87 and FPR-mock-U87 cells stimulated with fMLF upregulated levels of VEGF and IL-8 mRNA, and increased the secretion of VEGF and IL-8 proteins in supernatants. In FPR-siRNA-U87 cells, fMLF did not change the level of VEGF and IL-8 mRNA and the production of VEGF and IL-8 proteins in supernatants.2. Quantitative immunofluorescence assay, MTT assy, ELISA and RT-PCR were used to explore the the effect of Nordy on the expression of FPR and its function actived by FPR agonist fMLF. Our data suggested that: (1)The mean absorbance of Nordy group was significantly lower than that control group. (2) Nordy inhibited the mRNA expression and secretion of VEGF and IL-8 protein in surpernatants after activation of FPR.3. Glioma stem cells were isolated and identified from human malignant glioma cell line U87. (1) By immunostaning and flow cytometry assay, we found that U87 cells contained 0.5% CD133+ cells. (2) Using a Miltenyi Biotec CD133 Cell Isolation kit, we isolated CD133+ cells from U87 cells and CD133+ cells cultured in stem cell medium formed neurosphere-like spheroids. By immunofluorescence, the spheroids formed by the stem cells were found to be positive for CD133 and nestin, but no GFAP staining was detected within spheroids. Under differentiation conditions (medium containing serum), the spheroids differentiated into astrocytes and grew as a monolayer. (3) Comparing the capacity of CD133+ cells versus CD133- cells to initiate tumor growth in nude mice, we found that the CD133+ and CD133- cells differ in tumorigenicity in vivo, and the capacity of CD133+ cells was stronger than that of CD133- cells. Immunohistochemistry stained Ki67, a proliferation marker, and vimentin, a marker of glioma malignancy, the results suggested that the xenografts from CD133+ was labeled by higher Ki67 and vimentin than from CD133- xenografts. Immunostaining for nestin and GFAP in CD133+ and CD133- xenografts showed a few of cells derived from CD133+ xenografts expressed GFAP, however the majority expressed nestin, in contrast, a few of cells derived from CD133- xenografts expressed nestin, and the marjority expressed GFAP. (4) We exerted a serial passage in vivo to detect the self-renewal of CD133+ cells, the results demonstrated that these cells derived from U87 cells are capable of self-renewal in vivo.4. We examined the effect of glioma stem cells and functional expression of FPR of glioma stem cells on the angiogenesis, and found that: (1) The CD133+ cells produced higher levels of VEGF in supernatants. (2) Immunostaining for CD133 and FPR demonstrated these markers coexpressed in CD133+ spheriods. (3) Calcium mobilization was used to determine whether FPR expressed by CD133+ cells are functionally active. the results showed that in D-Hanks (without Ca2+ in the medium), stimulation of CD133+ cells with 10-4-10-7mM fMLF caused intracellular calcium increase in a dose-dependent manner, particually in 10-4mM fMLF a rapid and robust elevation in [Ca2+]i appeared. (4) By ELISA, we found that CD133+ cells significantly produced higher level of VEGF in response to FPR ligand, fMLF. (5) In xenografts formed by CD133+ glioma stem cells, more newly-formed vessels existed near the CD133+ cells labled by CFSE. The CD133+ cells-forming xenografts expressed higher levels of VEGF and higher microvessel densities labeled by CD105.In summary, the present results suggest that (1) FPR was expressed on the cytoplasmic membrane of U87 cells and activating FPR can promote the proliferation U87 cells and enhance the production of VEGF and IL-8. Nordy inhibite the FPR expression and the effect of promoting proliferation as well as production of VEGF and IL-8 after activation of FPR, showing Nordy may be has the potential therapeutic effect on the angiogenesis of glioma. (2) CD133+ cells in human glioblastoma cells have characteristics of tumor stem cells. CD133+ glioma stem cells and activation of G-coupled receptor FPR on these cells are capable of potential pro-angiogenic activity, which might be of significant importance in antiangiogenic therapy for cancer.