In this study, we investigated the influence of plateletactivating factor (PAF) on the induction of apoptosisregulating factors in B16F10 melanoma cells. PAF increased the expression of mRNA and the protein synthesis of antiapoptotic factors, such as Bcl-2 and Bcl-xL, but did not increase the expression of the proapoptotic factor, Bax. A selective nuclear factor-KB (NF-KB) inhibitor, parthenolide, inhibited the effects of PAF. Furthermore, PAF inhibited etoposide-induced increases in caspase-3, caspase-8, and caspase-9 activities, as well as cell death. p50/p65 heterodimer increased the mRNA expression of Bcl-2 and Bcl-xL and decreased etoposide-induced caspase activities and cell death.In an in vivo model in which Matrigel was injected s.c., PAF augmented the growth of B16F10 cells and attenuated etoposide-induced inhibition of B16F10 cells growth. These data indicate that PAF induces up-regulation of antiapoptotic factors in a NF-KB-dependent manner in a melanoma cell line, therefore suggesting that PAF may diminish the cytotoxic effect of chemotherapeutic agents. (Cancer Res 2006; 66(9): 4681-6)

Platelet-activating factor (PAF) augments angiogenesis by promoting the synthesis of various angiogenic factors, via the activation of NF-jB. In this study, we investigated the role of the matrix metalloproteinase (MMP)-9, in PAF-induced angiogenesis. PAF increased mRNA expression, protein synthesis, and MMP-9 activity in ECV304 cells, in a NF-jB-dependent manner. PAF increased MMP-9 promoter activity in ECV304, which was inhibited by WEB2107, and NF-jB inhibitors. Transfected NF-jB subunits, p65 or/and p50, increased luciferase activity in the reporter plasmid MMP-9, resulting in an increase not only of MMP-9 luciferase activity, but also of mRNA expression in MMP-9. MMP-9 or NF-jB inhibitors significantly inhibited PAF-induced angiogenesis, in a dose-dependent manner, in an in vivo mouse Matrigel implantation model. In a parallel to the Matrigel implantation study, MMP-9 or NF-jB inhibitors inhibited PAF-induced sprouting of porcine pulmonary arterial endothelial cells. These data indicate that NF-jB-dependent MMP-9 plays a key role in PAF-induced angiogenesis.

SummaryPlatelet-activating factor (PAF) promotes tumour metastasis via activation of the transcription factor nuclear factor-jB (NF-jB). We here investigated the role of the protein kinase CK2 (formerly Casein Kinase 2 or II) in PAF-induced NF-jB activation and tumour metastasis, given that PAF has been reported to increase CK2 activity, and that CK2 plays a key role in NF-jB activation. PAF increased CK2 activity, phosphorylation and protein expression in vivo as well as in vitro. CK2 inhibitors inhibited the PAF-mediated NF-jB activation and expression of NF-jB-dependent proinflammatory cytokines and anti-apoptotic factors. Pre-treatment with the antioxidant N-Acetyl-L-Cysteine (NAC) resulted in a significant inhibition in PAF-induced enhancement of CK2 activity, phosphorylation and protein expression in vivo as well as in vitro. H 2 O 2 and known reactive oxygen species inducers, lipopolysaccharide (LPS) and tumour necrosis factor-a (TNF-a) enhanced CK2 activity, phosphorylation and protein expression, which was again inhibited by antioxidant. PAF, LPS and TNFa induced increased CK2 activity, phosphorylationand protein expression, which were inhibited by p38 inhibitor. PAF, LPS or TNF-a increased pulmonary metastasis of B16F10, which was inhibited by antioxidants, CK2 inhibitor and p38 inhibitor. Our data suggest that (i) reactive oxygen species activate CK2 via p38, which, in turn, induces NF-jB activation, and (ii) PAF, LPS and TNF-a increase pulmonary tumour metastasis via the induction of the reactive oxygen species (ROS)/p38/CK2/NF-jB pathway.

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In this study, the roles of matrix metalloproteinase (MMP)-2 and MMP-9 in platelet-activating factor (PAF)-induced experimental pulmonary metastasis of the murine melanoma cell, B16F10, were investigated. An injection of PAF resulted in increases in mRNA expression, protein levels and the activities of both MMP-2 and MMP-9 in the lungs. The overall expression of MMP-9 was stronger than that of MMP-2. The increased MMP-9 expression was inhibited by both NF-jB and AP-1 inhibitors, whereas the increased MMP-2 expression was inhibited by only AP-1 inhibitors. Immunohistochemical analysis revealed that MMP-9 was expressed in bronchial epithelial cells as well as in the walls of blood vessels, whereas MMP-2 expression was observed only in bronchial epithelial cells. PAF significantly enhanced the pulmonary metastasis of B16F10, which was inhibited by both NF-jB and c-jun inhibitors. MMP-9 inhibitor, but not that of MMP-2, completely inhibited PAF-induced B16F10 metastasis. These data indicate that MMP-9, the expression of which was regulated by NF-jB and AP-1, plays a critical role in PAF-induced enhancement of pulmonary melanoma metastasis. ' 2006 Wiley-Liss, Inc.