| In recent years, the roles of chemoattractants and their receptors in the processes of cancer growth, invasion and metastasis are getting more and more recognition. It is demonstrated that some chemoattractant receptors that are expressed on normal cells and related to certain physiological or pathophysiological courses are also expressed in cancer cells and take roles in the progression of the cancer, which is a complicated process involes multiple faces and stages of interplays between cancer cells and host tissues, among which the increased capacity of cell proliferation and motility, overexpressed protease and proangiogenic factors, and their dysregulation during the cancer -host interplay are the most important part of the research, and the related receptors may be potential therapeutic targets.FPR (formylpeptide receptor) is a G-protein coupled receptor that was originally discovered in phagocytes, which can be mobilized through the activation of the receptor by endogenic (motochondria protein derived) or exogenic (bacterial protein derived) ligand, N-formyl peptide fMLF (N-formylmethionyl-leucyl-phenylalanine), and can initiate chemotaxis and mediator release, which are among the important host immune responses. Most recently, this receptor was also found to be expressed in malignant glioma cells, and its activation in vitro increased the motility and VEGF production. Malignant gliomas are characterized by robust invasion, active entothelium proliferation and easily discerned necrosis. fMLF, which exists in necrotic substances, is actually FPR agonist with high affinity. The coexistence and interaction between them may be one important mechanism for the proliferation, invasion and proangiogenic action of malignant glioma cells.To investigate the significance and mechanisms of FPR in the growth, invasion and proangiogenic activity of malignant gliomas, this study, with the implication of GBM cell line U87 which was successfully interferenced by FPR-targeted siRNA and its control counterparts, involes three parts: (1)detection of the expression of VEGF, MMP-2 and -9 in FPR-siRNA interferenced U87 cells with or without the stimulation of the agonist fMLF; (2) observation on the morphological changes of the orthotopical xenografts of FPR-siRNA interferenced U87 cells in nude mice, which includes the tumour growth, differentiation, invasion and angiogenesis; (3) further detection of VEGF, MMP-2 and -9 expressed in xenografts of FPR-siRNA interferenced U87 cells, as well as the detection of glioma differentiation marker vimentin. The main results and conclusions are as follows:1. FPR-siRNA interferenced U87 cells (FPR-siRNA U87) and the control counterparts, wild type U87 cell line and mock transfected cell line (Mock) were cultured, with or without the stimulation of specific ligand at known optimal concentratioin (100 nmol/L) for 6 hours. The VEGF, MMP-2 and -9 proteins in supernatants were detected by ELISA. It was found that the secretion of VEGF in FPR-siRNA U87cells was significantly lower than that of the control cells, and unlike that of the control cells, it did not change significantly with the stimulation of fMLF. MMP-9 production in FPR-siRNA U87cell supernatant was lower than that of control cells, but like that of the control cells, it did not change with the stimulation of fMLF. MMP-2 production in FPR-siRNA U87 cell supernatant was higher than that of control cells without stimulation of fMLF, but that of stimulated Mock cells increased significantly. These results indicate that VEGF expression is closely related to FPR activation, and that MMP-2 and -9 are somehow FPR expression related, but may involve other factors.2. The VEGF and FPR expressions in FPR-siRNA U87 cells were immunocytochemically found to be much lower than those in control cells. The VEGF, MMP-2 and -9 mRNA in cells treated the way mentioned above were assessed by RT-PCR. The VEGF mRNA in FPR-siRNA U87 cells changed the way VEGF protein in supernatants did. But MMP-2 and -9 mRNA did not show differences between the cell lines. These results also indicate that VEGF activation is closely related to FPR activation, and that MMP-2 and -9 are somehow FPR related, but may involve other factors.3. Using wild type U87 and Mock cells as control, the FPR-siRNA U87 cells were orthotopically injected into the brains of nude mice, the sizes of the xenografts at various timepoints (1, 2, 4, 5, and 6 weeks) were compared, the morpological changes of the xenografts with respect to tumour growth, differentiation, invasion and angiogenesis were also compared. The xenografts of FPR-siRNA U87 cells grew much slowly, without the acceleration which was noticed in controls at 5 to 6 weeks. FPR-siRNA U87 xenografts showed higher differentiation, fewer mitotic figures and less invasiveness of tumour cells than controls, and showed apparently fewer microvessels, with normalized appearance and architecture. These results demonstrate that the expression and activation of FPR in vivo are closely related to the growth, differentiation, invasion and angiogenesis of malignant gliomas.4. The expressions of VEGF, MMP-2 and -9 proteins in xenografts were further detected by Western blotting and found to be down-regulated. IHC staining showed less VEGF and MMP-9 expression. Vimentin expression was also found to be significantly or apparently down-regulated. These results further indicate that the expression and activation of FPR mediates or influences VEGF, MMP-2 and -9 productions and therefore promote angiogenesis, and that FPR expression is related to the differentiation of malignant gliomas.This study demonstrate that silencing of FPR by siRNA in U87 cells may cause the improved cell differentiation, inhibited proliferation, and down-regulated VEGF, MMP-2 and -9 expressions and attenuated invasiveness and angiogenesis. This in reverse confirm the significance of FPR in the growth, invasion and angiogenesis of malignant gliomas, hence a promising therapeutic target.
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