Endothelial PKCα-MAPK/ERK-phospholipase A2 pathway activation as a response of glioma in a triple culture model. A new role for pericytes?

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 Ca2+-independent phospholipase A(2) (cPLA(2) and iPLA(2)), cyclooxygenase-2 (COX-2), P13-K, MEK-1, and ERK1/2 inhibitors and by siRNAs against both PLA(2)s. In EC, C6 induced an increase in iPLA(2), cPLA(2) and COX-2 total protein expression. Moreover, the increase in endothelial cPLA(2) 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 E-2 (PGE(2)) was found. The presence of PC in triple-cultures caused a decrease in production of PGE(2) respect to EC/C6 double-cultures. In all systems, AACOCF(3) and BEL significantly reduced PGE(2) 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 iPLA(2) siRNA strongly reduced in vitro tubulogenesis. Data here reported indicate that PKC alpha, ERR kinase phosphorylation, PLA(2)s and COX-2 activation, and PGE(2) 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. (C) 2013 Elsevier Masson SAS. All rights reserved.