Thiolutin, an inhibitor of HUVEC adhesion to vitronectin, reduces paxillin in HUVECs and suppresses tumor cell-induced angiogenesis

Abstract

Recent studies have shown that integrin ␣v␤3, a receptor for vitronectin, plays an important role in tumor-induced angiogenesis and tumor growth and that antagonists of ␣v␤3 inhibit angiogenic processes including endothelial cell adhesion and migration. On the other hand, most inhibitors of integrin ␣v␤3 are peptide antagonists that include the ArgGly-Asp (RGD) motif. We therefore reasoned that non-peptide inhibitors of endothelial cell adhesion to vitronectin might be useful for inhibition of tumor angiogenesis in vivo. We screened for low-molecular-weight natural products able to inhibit adhesion of human umbilical vein endothelial cells (HUVECs) to vitronectin, and pyrrothine group compounds including aureothricin, thioaurin and thiolutin were isolated from microbial culture broths. Of these compounds, thiolutin inhibited adhesion of HUVECs to vitronectin the most effectively (IC 50 , 0.83 M). In vivo experiments showed that thiolutin significantly suppressed angiogenesis induced by tumor cells (S-180), a pathological form of neovascularization, in a mouse dorsal air sac assay system. To explore the mechanism of inhibition of HUVEC adhesion to vitronectin by thiolutin, we examined the effect of this agent on intracellular cell adhesion signaling. We found that the amount of paxillin in Key words: thiolutin; paxillin; HUVEC; vitronectin; tumor angiogenesisThe growth of solid tumors has been shown to be dependent on the development of new blood vessels. 1 Growing solid tumors produce diffusible angiogenic factors that induce host capillary endothelial cells to proliferate, migrate and form new vessels in a process termed tumor-induced angiogenesis. 2-4 Once vascularized, tumors increase in size almost exponentially. 1 The dependence of solid tumor growth upon neovascularization provides a therapeutic opportunity to inhibit neoplastic growth by blocking tumor-induced angiogenesis. Angiogenesis is characterized by invasion, migration and proliferation of endothelial cells, processes that depend on cell interactions with extracellular matrix (ECM) components. In this context, integrin ␣v␤3, the endothelial adhesion receptor for vitronectin, was shown to be a key player, 5-7 by providing a vasculature-specific target for antiangiogenic treatment strategies. The requirement for vascular integrin ␣v␤3 in angiogenesis was demonstrated by several in vivo models in which the generation of new blood vessels by transplanted human tumors was completely inhibited by systemic administration of peptide antagonists of integrin ␣v␤3 or anti-␣v␤3 antibody. 5,6,8 Integrins are ␣/␤ heterodimeric cell-surface receptors that link the ECM with the cell cytoskeleton and generate intracellular signals to regulate cell survival and growth, cell differentiation and cell motility. Integrins themselves have no instrinsic enzymatic activity and appear to function by interacting with a number of other proteins. Integrin aggregation upon their binding to the substratum, which accompanies an assembly of several proteins at or near the cytopla...

Thiolutin inhibits endothelial cell adhesion by perturbing Hsp27 interactions with components of the actin and intermediate filament cytoskeleton

“…Consistent with the previously reported effect of thiolutin on endothelial cell adhesion on vitronectin (Minamiguchi et al 2001), we found that ≥0.4 μM thiolutin was sufficient to rapidly and completely disrupt adhesion of attached HUVEC monolayers (Fig. 3a).…”

“…1a) and its effects on endothelial cells in vitro (Minamiguchi et al 2001;Dai et al 2008), we investigated the effect of thiolutin on vertebrate development using the transparent zebrafish model system. In addition to obtaining toxicology data, the effect of thiolutin on developmental angiogenesis was assessed using the transgenic zebrafish line Tg(fli1:EGFP) y1 , which carries a 15-kb promoter of friend leukemia integration-1 transcription factor (fli1) that drives green fluorescent protein (GFP) expression in the developing vasculature (Lawson and Weinstein 2002).…”

“…Subsequently, thiolutin was shown to potently inhibit endothelial cell adhesion with an IC 50 <1 μM and to inhibit S180 tumor-induced angiogenesis in mice (Minamiguchi et al 2001). We recently found that thiolutin potently stimulates phosphorylation of HSPB1/Hsp27 in endothelial cells and that expression of Hsp27 is important for the anti-proliferative activity of thiolutin (Dai et al 2008).…”

“…The mechanism by which thiolutin inhibits endothelial cell adhesion is not clear, but two focal adhesion proteins were found to be affected by thiolutin. Thiolutin inhibits the phosphorylation of focal adhesion kinase (FAK) and reduces the expression of paxillin in human umbilical vein endothelial cells (HUVEC) plated on vitronectin (Minamiguchi et al 2001).…”

Synthesis of Dithiolopyrrolone Derivatives and Their Leukocyte-Increasing Activities

“…[8][9][10][11][12][13][14] Dithiolopyrrolones have also been reported to have anticancer activity, [15][16][17] and shown potent membrane stabilization and platelet anti-aggregate activities. 18 We first reported that ZL-004 ( Figure 1) exhibited antileukopenia activity.…”