Identification of pY654-ß-catenin as a critical co-factor in hypoxia-inducible factor-1α signaling and tumor responses to hypoxia.

Hypoxia is linked to epithelial-mesenchymal transition (EMT) and tumor progression in numerous carcinomas. Responses to hypoxia are thought to operate via hypoxia-inducible factors (HIFs), but the importance of co-factors that regulate HIF signaling within tumors is not well understood. Here, we elucidate a signaling pathway that physically and functionally couples tyrosine phosphorylation of ß-catenin to HIF1α signaling and HIF1α-mediated tumor EMT. Primary human lung adenocarcinomas accumulate pY654-ß-catenin and HIF1α. All pY654-ß-catenin, and only the tyrosine phosphorylated form, was found complexed with HIF1α and active Src, both within the human tumors and in lung tumor cell lines exposed to hypoxia. Phosphorylation of Y654, generated by hypoxia mediated, reactive oxygen species (ROS)-dependent Src kinase activation, was required for ß-catenin to interact with HIF1α and Src, to promote HIF1α transcriptional activity, and for hypoxia-induced EMT. Mice bearing hypoxic pancreatic islet adenomas, generated by treatment with anti-vascular endothelial growth factor antibodies, accumulate HIF1α/pY654-ß-catenin complexes and develop an invasive phenotype. Concurrent administration of the ROS inhibitor N-acetylcysteine abrogated ß-catenin/HIF pathway activity and restored adenoma architecture. Collectively, the findings implicate accumulation of pY654-ß-catenin specifically complexed to HIF1α and Src kinase as critically involved in HIF1α signaling and tumor invasion. The findings also suggest that targeting ROS-dependent aspects of the pY654-ß-catenin/ HIF1α pathway may attenuate untoward biological effects of anti-angiogenic agents and tumor hypoxia.

[Effects of MRI-assayed microvascular permeability on the accumulation of vinorelbine in xenograft tumors]. / Einfluss der mittels MRT bestimmten mikrovaskulären Permeabilität auf die Akkumulation von Vinorelbine in experimentellen Xenograft-Tumoren.

PURPOSE: To determine the effects of MRI-assayed vascular leakiness on the delivery of macromolecular therapeutics to tumors. MATERIALS AND METHODS: MDA-MB 435 tumors, subcutaneously implanted into nude rats were treated with a single dose of bevacizumab at levels of 0.1 mg (n = 5) or 1.0 mg (n = 10) or received saline (control animals, n = 8). After 24 hours, albumin-(Gd-DTPA) (30)-enhanced MRI was performed. Just prior to MRI, the cytotoxic drug vinorelbine was administered intravenously. Upon completion of the MR experiment, tumor vinorelbine concentrations were quantified by high performance liquid chromatography (HPLC). Vascular leakiness (K (PS)) was calculated based on the MRI data using a pharmacokinetic model. RESULTS: K (PS) was calculated as 3.70 +/- 1.12 (control tumors), 1.95 +/- 0.70 (0.1 mg group) and 0.75 +/- 0.46 microl min (-1)cm (-3) (1.0 mg group). K (PS) was significantly higher in the control group compared to the 1.0 mg bevacizumab group. Vinorelbine concentrations were measured as 409.4 +/- 109.7 (control tumors), 387.5 +/- 47.5 (0.1 mg group) and 250.7 +/- 71.9 (1.0 mg group). These differences were not significant. A moderate and significant correlation was found between K (PS) and Vinorelbine concentrations in tumors (r = 0.49, p < 0.05). CONCLUSION: MRI-assayed K (PS) based on dynamic MRI enhanced by albumin-(Gd-DTPA) (30) correlated significantly with vinorelbine accumulation in experimental xenograft tumors under angiogenesis inhibition. Thus, the MRI technique applied in our study could potentially help to predict accumulation of macromolecular cytotoxic drugs and to optimize individual therapeutic regimes in tumors.

Modulation of tumor angiogenesis by conditional expression of fibroblast growth factor-2 affects early but not established tumors.

Fibroblast growth factor-2 (FGF2) is a pleiotropic heparin-binding growth factor endowed with a potent angiogenic activity in vitro and in vivo. To investigate the impact of the modulation of FGF2 expression on the neovascularization at different stages of tumor growth, we generated stable transfectants (Tet-FGF2) from the human endometrial adenocarcinoma HEC-1-B cell line in which FGF2 expression is under the control of the tetracycline-responsive promoter (Tet-off system). After transfection, independent clones were obtained in which FGF2 mRNA and protein were up-regulated compared with parental cells. Also, the conditioned medium of Tet-FGF2 transfectants caused proliferation, urokinase-type plasminogen activator up-regulation, migration, and sprouting of cultured endothelial cells. A 3-day treatment of Tet-FGF2 cell cultures with tetracycline abolished FGF2 overexpression and the biological activity of the conditioned medium without affecting their proliferative capacity. Tet-FGF2 cells formed tumors when nude mice received s.c. injections. The administration of 2.0 mg/ml tetracycline in the drinking water before cell transplantation, continued throughout the whole experiment, inhibited FGF2 expression in Tet-FGF2 tumor lesions. This was paralleled by a significant decrease in the rate of tumor growth and vascularization to values similar to those observed in lesions generated by parental HEC-1-B cells. Tetracycline administration 20 days after tumor cell implant, although equally effective in reducing FGF2 expression and inhibiting tumor vascularity, only minimally impaired the growth of established Tet-FGF2 tumors. The results indicate that FGF2 expression deeply affects the initial tumor growth and neovascularization of HEC-1-B human endometrial adenocarcinoma in nude mice. On the contrary, the growth of established tumors appears to be independent of the inhibition of FGF2 expression and decreased vascular density. The possibility that a significant reduction of angiogenesis may not affect the progression of large tumors points to the use of antiangiogenic therapy in early tumor stage.

Thrombospondin-1/HIV-1 tat protein interaction: modulation of the biological activity of extracellular Tat.

Tat protein, a trans-activating factor of the human immunodeficiency virus type 1, acts also as an extracellular molecule modulating gene expression, cell survival, growth, transformation, and angiogenesis. Here we demonstrate that human thrombospondin-1 (TSP), a plasma glycoprotein and constituent of the extracellular matrix, binds to glutathione-S-transferase (GST)-Tat protein but not to GST. Scatchard plot analysis of the binding of free GST-Tat to immobilized TSP reveals a high-affinity interaction (Kd equal to 25 nM). Accordingly, TSP inhibits cell internalization and HIV-1 LTR trans-activating activity of extracellular Tat in HL3T1 cells with ID50 equal to 10-30 nM. Also, TSP inhibits cell interaction and mitogenic activity of extracellular Tat in T53 Tat-less cells. TSP is instead ineffective when administered after the interaction of Tat with cell surface heparan-sulfate proteoglycans has occurred, in keeping with its ability to prevent but not disrupt Tat/heparin interaction in vitro. Finally, TSP inhibits the autocrine loop of stimulation exerted by endogenous Tat in parental T53 cells. Accordingly, TSP overexpression inhibits cell proliferation, angiogenic activity, and tumorigenic capacity of stable T53 transfectants. Our data demonstrate the ability of TSP to bind to Tat protein and to affect its LTR trans-activating, mitogenic, angiogenic, and tumorigenic activity. These findings suggest that TSP may be implicated in the progression of AIDS and in AIDS-associated pathologies by modulating the bioavailability and biological activity of extracellular Tat.

Effect of organic cosolvents on the stability and activity of the beta-1,4-galactosyltransferase from bovine colostrum.

The influence of various organic cosolvents on the stability and activity of the beta-1,4-galactosyltransferase from bovine colostrum (GalT) and of its ancillary enzyme UDP-galactose-4'-epimerase has been investigated using the glucosylated alkaloid colchicoside (1) as a model substrate. It has been found that some cosolvents, such as Me2SO and MeOH, can be used up to 20% v/v without any influence on the performance of these enzymes, while others, such as tetrahydrofuran, rapidly inactivated GalT at concentrations as low as 5% v/v. These results have been exploited for the galactosylation of the poorly water soluble coumarinic glucoside fraxin (2).