title: Endothelial PKCα-MAPK/ERK-phospholipase A2 pathway activation as a response of glioma in a triple culture model. A new role for pericytes?
abstract: In view of understanding the molecular mechanisms through which angiogenic switch on happens in the early phases of reciprocal interaction between tumor and cells constituting microvessel, a triple culture model in which endothelial cells (EC), pericytes (PC) and glioma C6 cells were cultured together. In the present work, we observed that C6 enhanced EC proliferation. This effect was reduced by cytosolic and Ca(2+)-independent phospholipase A2 (cPLA2 and iPLA2), cyclooxygenase-2 (COX-2), PI3-K, MEK-1, and ERK1/2 inhibitors and by siRNAs against both PLA2s. In EC, C6 induced an increase in iPLA2, cPLA2 and COX-2 total protein expression. Moreover, the increase in endothelial cPLA2 phosphorylation was attenuated by kinase inhibitors. Both EC proliferation and signal protein phosphorylation were attenuated when PC were in triple culture. In EC/C6 supernatants, and, in a lesser extent, in EC/PC co-cultures, an enhancement in prostaglandins E2 (PGE2) was found. The presence of PC in triple-cultures caused a decrease in production of PGE2 respect to EC/C6 double-cultures. In all systems, AACOCF3 and BEL significantly reduced PGE2 secretion. In Matrigel-based assays, emerging branch points from EC cell bodies and tubule-like structures were observed. C6 conditioned EC/PC co-cultures in constituting poorly organized tubules. Transfection of EC with c- and iPLA2 siRNA strongly reduced in vitro tubulogenesis. Data here reported indicate that PKCα, ERK kinase phosphorylation, PLA2s and COX-2 activation, and PGE2 production in EC stimulated by tumor cells are coincident phenomena and could represent therapeutic targets in chemoprevention of glioma. Moreover, PC exhibited an important "modulating" role in the initial stages of angiogenesis driven by a brain tumor.
authors.affiliation: Dept. Biomedicina Clinica e Molecolare, University of Catania, Catania, Italy.
authors.forename: Carmelina Daniela
authors.initials: CD
authors.lastname: Anfuso
authors.affiliation: Dept. Biomedicina Clinica e Molecolare, University of Catania, Catania, Italy.
authors.forename: Carla
authors.initials: C
authors.lastname: Motta
authors.affiliation: Dept. Biomedicina Clinica e Molecolare, University of Catania, Catania, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Giurdanella
authors.affiliation: Dept. Biomedicina Clinica e Molecolare, University of Catania, Catania, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: Arena
authors.affiliation: Dept. Biomedicina Clinica e Molecolare, University of Catania, Catania, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Alberghina
authors.affiliation: Dept. Biomedicina Clinica e Molecolare, University of Catania, Catania, Italy. Electronic address: lupogab@unict.it.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Lupo
mesh_terms: D000818:Animals; D001812:Blood-Brain Barrier / Q000378:metabolism; D002199:Capillary Permeability; D002417:Cattle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018920:Coculture Techniques; D051545:Cyclooxygenase 1 / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000378:metabolism; D015232:Dinoprostone / Q000096:biosynthesis; D005910:Glioma; D020935:MAP Kinase Signaling System; D009389:Neovascularization, Pathologic / Q000201:enzymology*; D062793:Occludin / Q000378:metabolism; D020286:Pericytes / Q000201:enzymology*; D054467:Phospholipases A2 / Q000378:metabolism*; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D012969:Sodium Fluoride / Q000378:metabolism
chemical_list: D062793:Occludin; D012969:Sodium Fluoride; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; C496628:PRKCA protein, human; D051571:Protein Kinase C-alpha; D054467:Phospholipases A2; D015232:Dinoprostone
keywords: C6 cells; COX-1/-2; Cytosolic and calcium independent phospholipase A(2); ERK1/2; Matrigel assay; Microvascular endothelial cells; Microvascular pericytes; PGE(2); PKCα; Permeability; TEER