The largely undefined signal transduction mechanisms and cross-talk between human melanoma cell (HMC) lines and brain endothelial cells (ECs) involved in tumor cell interaction and adhesion were investigated. In immortalized rat brain GP8.3 EC cultures, conditioned media (CM) prepared from SK-MEL28 and OCM-1 melanoma cells significantly enhanced arachidonic acid release, cytosolic phospholipase A(2) (cPLA(2)) and Ca(+)-independent phospholipase A(2) (iPLA(2)) specific activities, and cell growth by 24 h. Inhibitors such as wortmannin and LY294002 (vs. PI3 kinase activity), AACOCF(3), (vs. cPLA(2) and iPLA(2)), PD98059 (vs. ERK1/2 activity) and NS-398 (vs. cyclooxygenase-2 activity, COX-2) were all able to block cell proliferation and motility determined using a scratch wound healing assay in melanoma CMs-stimulated EC monolayers. These media also support the enhanced cell proliferation of primary ECs derived from rat brain (BBEC). Electroporation of anti-cPLA(2) antibody into ECs markedly inhibited the EC proliferation in response to CMs. With both CMs, phosphorylation of cPLA(2), PKCalpha, ERK1/2, protein and mRNA expression of cPLA(2) and iPLA(2), and COX-2 protein expression were significantly stimulated after 24 h coincubation, and attenuated by specific inhibitors. By confocal microscopy, activation of cPLA(2), ERK1/2, PKCalpha and COX-2 in perinuclear and membrane regions of ECs grown in CM-stimulated cultures were clearly observed. Thus MEK-PKCalpha-ERK1/2 and PI3-K/Akt survival pathways are activated in EC cultures during the interaction with CM from both melanoma cell lines, providing new insight in understanding EC metabolism and signaling. These pathways represent potential therapeutic targets to inhibit or enhance tumor angiogenesis.