Showing papers by "Thomas Simmet published in 2008"

Targeting XIAP Bypasses Bcl-2–Mediated Resistance to TRAIL and Cooperates with TRAIL to Suppress Pancreatic Cancer Growth In vitro and In vivo

Abstract: Resistance to apoptosis is a hallmark of pancreatic cancer, a leading cause of cancer deaths. Therefore, novel strategies are required to target apoptosis resistance. Here, we report that the combination of X-linked inhibitor of apoptosis (XIAP) inhibition and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an effective approach to trigger apoptosis despite Bcl-2 overexpression and to suppress pancreatic cancer growth in vitro and in vivo. Knockdown of XIAP by RNA interference cooperates with TRAIL to induce caspase activation, loss of mitochondrial membrane potential, cytochrome c release, and apoptosis in pancreatic carcinoma cells. Loss of mitochondrial membrane potential and cytochrome c release are extensively inhibited by a broad range or caspase-3 selective caspase inhibitor and by RNAi-mediated silencing of caspase-3, indicating that XIAP inhibition enhances TRAIL-induced mitochondrial damage in a caspase-3-dependent manner. XIAP inhibition combined with TRAIL even breaks Bcl-2-imposed resistance by converting type II cells that depend on the mitochondrial contribution to the death receptor pathway to type I cells in which TRAIL-induced activation of caspase-3 and caspase-9 and apoptosis proceeds irrespective of high Bcl-2 levels. Most importantly, XIAP inhibition potentiates TRAIL-induced antitumor activity in two preclinical models of pancreatic cancer in vivo. In the chicken chorioallantoic membrane model, XIAP inhibition significantly enhances TRAIL-mediated apoptosis and suppression of tumor growth. In a tumor regression model in xenograft-bearing mice, XIAP inhibition acts in concert with TRAIL to cause even regression of established pancreatic carcinoma. Thus, this combination of XIAP inhibition plus TRAIL is a promising strategy to overcome apoptosis resistance of pancreatic cancer that warrants further investigation.

Thrombin-induced expression of endothelial CX3CL1 potentiates monocyte CCL2 production and transendothelial migration.

Abstract: CX3CL1 (fractalkine, neurotactin) is the sole CX3C chemokine. It induces monocyte locomotion in its cleaved form, but in its membrane-anchored form, it also acts as an adhesion molecule. The expression of CX3CL1 is up-regulated in endothelial cells by proinflammatory cytokines such as IL-1 or TNF-alpha. Here, we studied the effect of the serine protease thrombin on endothelial CX3CL1 induction and its putative relevance for monocyte function. In HUVEC, thrombin triggered a time- and concentration-dependent expression of CX3CL1 at the mRNA and the protein level as shown by RT-PCR, Western immunoblotting, and flow cytometric analysis. Thrombin induced CX3CL1 by activating protease-activated receptor 1 (PAR1) as demonstrated by the use of PAR1-activating peptide and the PAR1-specific antagonist SCH 79797. The thrombin-induced CX3CL1 expression was NF-kappaB-dependent, as shown by EMSA, ELISA, and by inhibition of the NF-kappaB signaling pathway by the IkappaB kinase inhibitor acety-11-keto-beta-boswellic acid or by transient overexpression of a transdominant-negative form of IkappaBalpha. Upon cocultivation of human monocytes with HUVEC, the thrombin-dependent induction of membrane-anchored CX3CL1 in HUVEC triggered monocyte adhesion and an enhanced release of the MCP-1/CCL2 by monocytes and potentiated the monocyte transendothelial migration. Accordingly, the recombinant extracellular domain of CX3CL1 induced CCL2 release by monocytes. Thus, the thrombin-induced monocyte/endothelial cell cross-talk mediated by increased CX3CL1 expression potentiates the CCL2 chemokine generation that might contribute to the recruitment of monocytes into inflamed areas.

Mature dendritic cells express functional thrombin receptors triggering chemotaxis and CCL18/pulmonary and activation-regulated chemokine induction.

Abstract: Protease-activated receptors (PARs) are a family of G protein-coupled receptors that are activated by serine protease-mediated proteolytic cleavage of their extracellular domain. We have previously characterized the expression and function of PARs in human monocytes and macrophages, yet information about PARs in dendritic cells (DC) is scarce. Monocyte-derived immature DC do not express PARs. Upon maturation with LPS, but not with TNF-α or CD40 ligand, DC express PAR1 and PAR3, but not PAR2 or PAR4. Stimulation of DC with the serine protease thrombin or PAR1-activating peptide elicits actin polymerization and concentration-dependent chemotactic responses in LPS-, but not in TNF-α-matured DC. The thrombin-induced migration is a true chemotaxis with only negligible chemokinesis. Stimulation of PARs with thrombin or the respective receptor-activating peptides activates ERK1/2 and Rho kinase as well as subsequent phosphorylation of the regulatory myosin L chain 2. The ERK1/2- and Rho kinase 1-mediated phosphorylation of myosin L chain 2 was indispensable for the PAR-mediated chemotaxis as shown by pharmacological inhibitors. Additionally, thrombin stimulated the Rho-dependent release of the CC chemokine CCL18/pulmonary and activation-regulated chemokine, which induces chemotaxis of lymphocytes and immature DC as well as fibroblast proliferation. The colocalization of CD83+ DC with CCL18 in human atherosclerotic plaques revealed by immunofluorescence microscopy combined with the presence of functionally active thrombin receptors on mature DC point to a previously unrecognized functional role of thrombin in DC biology. The thrombin-induced stimulation of mature DC may be of particular relevance in atherosclerotic lesions, which harbor all components of this novel mechanism.