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Regulation of the leucocyte chemoattractant receptor FPR in glioblastoma cells by cell differentiation.

ABSTRACT: The G protein-coupled formylpeptide receptor (FPR), known to mediate phagocytic leucocyte chemotaxis in response to bacterial- and host-derived agonists, was expressed by tumor cells in specimens of surgically removed more highly malignant human gliomas. In human glioblastoma cell lines, FPR activation increased cell motility, tumorigenicity and production of angiogenic factors. In studies of the mechanistic basis for the selective expression of FPR in more highly malignant gliomas, we found that the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (Aza), while promoting the differentiation of human glioblastoma cells, downregulated FPR expression. Aza also reduced the global methylation levels in glioblastoma cells and activated the pathway of p53 tumor suppressor. Methylation-specific polymerase chain reaction revealed that Aza treatment of tumor cells reduced the methylation of p53 promoter, which was accompanied by increased expression of p53 gene and protein. In addition, overexpression of p53 in glioblastoma cells mimicked the effect of Aza treatment as shown by increased cell differentiation but reduction in FPR expression, the capacity of tumor sphere formation in soft agar and tumorigenesis in nude mice. Furthermore, Aza treatment or overexpression of the wild-type p53 in glioblastoma cells increased the binding of p53 to FPR promoter region shown by chromatin immunoprecipitation. These results indicate that increased methylation of p53 gene retains human glioblastoma cells at a more poorly differentiated phase associated with the aberrant expression of FPR as a tumor-promoting cell surface receptor.

SUBMITTER: Huang J

PROVIDER: S-EPMC2639049 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Regulation of the leucocyte chemoattractant receptor FPR in glioblastoma cells by cell differentiation.

Huang Jian J Chen Keqiang K Huang Jiaqiang J Gong Wanghua W Dunlop Nancy M NM Howard O M Zack OM Bian Xiuwu X Gao Yuqi Y Wang Ji Ming JM

Carcinogenesis 20081126 2

The G protein-coupled formylpeptide receptor (FPR), known to mediate phagocytic leucocyte chemotaxis in response to bacterial- and host-derived agonists, was expressed by tumor cells in specimens of surgically removed more highly malignant human gliomas. In human glioblastoma cell lines, FPR activation increased cell motility, tumorigenicity and production of angiogenic factors. In studies of the mechanistic basis for the selective expression of FPR in more highly malignant gliomas, we found tha ...[more]

PMID: 19037090

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Project description:The N-formyl peptide receptors (FPRs) have been identified within neuronal tissues and may serve as yet undetermined functions within the nervous system. The FPRs have been implicated in the progression and invasiveness of neuroblastoma and other cancers. In this study the effects of the synthetic FPR agonist FPRa14, FPR antagonists and FPR knockdown using siRNA on mouse neuroblastoma neuro2a (N2a) cell differentiation plus toxicity were examined. The FPRa14 (1-10μM) was found to induce a significant dose-dependent differentiation response in mouse neuroblastoma N2a cells. Interestingly, three distinct differentiated morphologies were observed, with two non-archetypal forms observed at the higher FPRa14 concentrations. These three forms were also observed in the human neuroblastoma cell-lines IMR-32 and SH-SY5Y when exposed to 100μM FPRa14. In N2a cells combined knockdown of FPR1 and FPR2 using siRNA inhibited the differentiation response to FPRa14, suggesting involvement of both receptor subtypes. Pre-incubating N2a cultures with the FPR1 antagonists Boc-MLF and cyclosporin H significantly reduced FPRa14-induced differentiation to near baseline levels. Meanwhile, the FPR2 antagonist WRW4 had no significant effect on FPRa14-induced N2a differentiation. These results suggest that the N2a differentiation response observed has an FPR1-dependent component. Toxicity of FPRa14 was only observed at higher concentrations. All three antagonists used blocked FPRa14-induced toxicity, whilst only siRNA knockdown of FPR2 reduced toxicity. This suggests that the toxicity and differentiation involve different mechanisms. The demonstration of neuronal differentiation mediated via FPRs in this study represents a significant finding and suggests a role for FPRs in the CNS. This finding could potentially lead to novel therapies for a range of neurological conditions including neuroblastoma, Alzheimer's disease, Parkinson's disease and neuropathic pain. Furthermore, this could represent a potential avenue for neuronal regeneration therapies.

Project description:BackgroundThe corpus luteum (CL) is a transient endocrine gland and prostaglandin F2-alpha is considered to be the principal luteolysin in pigs. In this species, the in vivo administration of prostaglandin F2-alpha induces apoptosis in large vessels as early as 6 hours after administration. The presence of the prostaglandin F2-alpha receptor (FPr) on the microvascular endothelial cells (pCL-MVECs) of the porcine corpus luteum has not yet been defined. The aim of the study was to assess FPr expression in pCL-MVECs in the early and mid-luteal phases (EL-p, ML-p), and during pregnancy (P-p). Moreover, the effectiveness of prostaglandin F2-alpha treatment in inducing pCL-MVEC apoptosis was tested.MethodsPorcine CLs were collected in the EL and ML phases and during P-p. All CLs from each animal were minced together and the homogenates underwent enzymatic digestion. The pCL-MVECs were then positively selected by an immunomagnetic separation protocol using Dynabeads coated with anti-CD31 monoclonal antibody and seeded in flasks in the presence of EGM 2-MV (Microvascular Endothelial Cell Medium-2). After 4 days of culture, the cells underwent additional immunomagnetic selection and were seeded in flasks until the confluent stage.PCR Real time, western blot and immunodetection assays were utilized to assess the presence of FPr on pCL-MVEC primary cultures. Furthermore, the influence of culture time (freshly isolated, cultured overnight and at confluence) and hormonal treatment (P4 and E2) on FPr expression in pCL-MVECs was also investigated. Apoptosis was detected by TUNEL assay of pCL-MVECs exposed to prostaglandin F2-alpha.ResultsWe obtained primary cultures of pCL-MVECs from all animals. FPr mRNA and protein levels showed the highest value (ANOVA) in CL-MVECs derived from the early-luteal phase. Moreover, freshly isolated MVECs showed a higher FPr mRNA value than those cultured overnight and confluent cells (ANOVA). prostaglandin F2-alpha treatment failed to induce an apoptotic response in all the pCL-MVEC cultures.ConclusionOur data showing the presence of FPr on MVECs and the inability of prostaglandin F2-alpha to evoke an in vitro apoptotic response suggest that other molecules or mechanisms must be considered in order to explain the in vivo direct pro-apoptotic effect of prostaglandin F2-alpha at the endothelial level.

Dataset Information

Regulation of the leucocyte chemoattractant receptor FPR in glioblastoma cells by cell differentiation.

Publications

Regulation of the leucocyte chemoattractant receptor FPR in glioblastoma cells by cell differentiation.

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