Abstract 1672: Mitochondrial and bacterial peptides act on the formyl peptide receptor (FPR) to promote migration and proliferation in high grade glioblastoma cells

Abstract

Abstract Background: Recently several aberrant molecular pathways and underlying genetic defects are elucidated in astrocytomas, among which the role of the formylpeptide receptor (FPR) malignant behavior. In a highly malignant human astrocytoma cell line FPR promotes cell motility, growth, and angiogenesis by interacting with host-derived agonists originating from necrotic cell material. Until now the only known ligands of FPR are N-formyl peptides, which are cleavage products of bacterial proteins like N-formyl-methionyl-leucyl-phenylalanine (fMLF). In humans the only conserved N-formyl peptides are the cleavage products of mitochondrial proteins. This study aims to identify these peptides as responsible ligands for FPR expressing high grade glioma cells. Methods: We used FITC-fMLF and flow cytometry to confirm binding to FPR-expressing high grade human gliobastoma cell line U87. In addition, U937 cells (human promonocytic cell line) transfected with the FPR (U937FPR) and empty expression vectors were generated. The hallmark of chemokine receptor activation is a rapid and transient increase in the free intracellular Ca2+ level upon ligand binding. Formylated mitochondrial peptides fMLKLIV/fMLALV and FPR's natural peptide ligand fMLF were used to induce Ca2+ mobilization, chemotaxis as determined by the Neuro Probe® system and proliferation in U87 cells, analyzed by MTT assay. Results: FITC-fMLF bound to FPR expressing U87 and U937FPR cells. Increasing concentrations of fMLF (10−8 M-10−5 M) caused a dose dependent calcium mobilization in FPR expressing U87 cells. Increasing concentrations of fMLF/fMLKLIV/fMLALV (10−8 M- 10−5M) were tested in U937FPR cells, again showing dose dependent calcium mobilization. In the chemotaxis assay, respectively 22, 20 and 24% of U937FPR cells migrated towards fMLF/fMLKLIV/fMLALV as compared to 4% towards 0.05% FCS. A 2.5 fold increase in migration of U87 cells towards 10−7 M fMLF was found as compared to 0.05% FCS. Respectively 20, 21 and 15% increase in U87 cell proliferation was found when cells were stimulated with 10−6 M fMLF/fMLKLIV/fMLALV. Conclusions: These results indicate that mitochondrial peptides present in necrotic tumor material serve as ligands for FPR, expressed on high grade human gliobastoma cell line U87. Activation of FPR via N-formyl peptides causes increased cell growth and motility and is a potential target for anti-cancer therapy. Supported by grant RUG 4622 of the Dutch Cancer Society Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1672. doi:10.1158/1538-7445.AM2011-1672