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Purpose : To search for effective antagonist peptide of CC chemokine receptor 1 (CCR1), and evaluate the potential role and mechanism of peptide C18P derived from viral macrophage inflammatory protein II (vMIP-II). Methods: Alignment, simulated peptide-cut, bioinformatics and protease digestion were used to screen and prepare antagonist peptide. Interactions between C18P and CCR1 were determined by radioligand binding assays and [ 35 S]GTPγS binding experiment. Chemotaxis assay was utilized to assess the potency for inducing or inhibiting peripheral blood mononuclear cells (PBMCs) migration. Ligandinduced intracellular calcium mobilization was tested by flow cytometry. Results: A peptide containing 18 amino acids (C18P) was screened. C18P bound to CCR1 with a Kd of 5.7 ng/ml and displaced 125 I-labeled MIP-1α and 125 I-labeled RANTES on human CCR1-transfected HEK293 cells (IC 50 = 11.2 and 9.6 ng/ml, respectively) in radioligand binding studies. C18P lacked intrinsic agonist activity but strongly inhibited HCC-1 (100 nM) induced [ 35 S]GTPγS binding (IC 50 = 3.7 ug/ml), chemotaxis induced by HCC-1, MIP-1α and RANTES (IC 50 = 23, 25 and 13.1 ng/ml, respectively), and intracellular calcium mobilization. Conclusion: These results demonstrate that bioinformatics and protease digestion are feasible to screen and prepare C18P, and that C18P is a novel and specific small molecule peptide antagonist of CCR1 with therapeutic potential for preventing cell migration. Keywords: CC Chemokine receptor 1; Simulated peptide-cut; Antagonist peptide; Viral macrophage inflammatory protein II; Bioinformatics; Protease digestion; HEK293 cells; Radioligand binding

Purpose : To evaluate the effect of synthetic polypeptide (N15P) derived from viral macrophage inflammatory protein II (vMIP-II) on the secretion of vascular endothelial growth factor (VEGF) as well as investigate the signaling pathways involved in stromal cell-derived factor-1α(SDF-1α)/CXC Chemokin Receptor 4 (CXCR4) axis-induced VEGF in glioblastoma U87 cells. Methods : Glioblastoma U87 cells were exposed to SDF-1a, N15P with various concentrations. The expression of CXCR4, SDF-1α and VEGF mRNA were assessed by RT-PCR, while expression level of VEGF was tested by ELISA and protein kinase B (Akt) phosphorylation detected by Western blot. Results : The results showed that CXCR4, SDF-1α, VEGF are expressed in human glioblastoma U87 cell lines. SDF-1α caused a dose-dependent sensitivity of cell proliferation with a maximum effect at 15 µmole/ml, while N15P decreased cell viability in U87 cells in a dose-dependent manner. SDF-1α stimulated the activation of VEGF, and N15P inhibited the activation of VEGF with or without SDF-1α stimulation. VEGF production in U87 cells was associated with Akt pathway. These changes in intracellular processes were blocked by N15P in a dose-dependent manner. Conclusion : The results suggest that N15P suppress SDF-1α/CXCR4 Mediated VEGF production through Akt signaling pathway and this may be a potent therapeutic strategy in glioblastoma. Keywords : Viral macrophage, Inflammatory protein II, Glioblastoma, CXC chemokin receptor 4, Stromal cell-derived factor-1α, Protein kinase B