Through hypoxia-inducible factor 1 (HIF-1), hypoxia regulates the expression of numerous genes and is a potent inducer of angiogenesis. However, interleukin-8 (IL-8), an important angiogenic mediator, has been reported to be downregulated by HIF-1, although the mechanisms have not been elucidated. HIF-1 was induced in human endothelial cells by hypoxia and dimethyloxaloylglycine (DMOG). Interestingly, both hypoxia and DMOG attenuated IL-8 expression, and a similar effect has been obtained by adenoviral overexpression of the stable form of HIF-1alpha. Heme oxygenase-1 (HO-1) expression was also downregulated by HIF-1 induction. This suggests similar mechanisms of regulation of IL-8 and HO-1, indicating the involvement of Nrf2, a transcription factor previously linked to hypoxia-mediated inhibition of HO-1. Indeed, HIF-1-mediated downregulation of both IL-8 and HO-1 was associated with both lowered Nrf2 expression and induction of Bach1, a repressor of Nrf2 transcriptional activity. Accordingly, overexpression of Nrf2 reversed the inhibitory effect of HIF-1 on IL-8 and HO-1 expression. However, neither overexpression of HO-1 nor HO-1 inhibition affected IL-8 synthesis. The data indicate that HIF-1-dependent inhibition of IL-8 expression is caused by downregulation of Nrf2. However, expression of IL-8 is independent of HO-1. Cross-talk between HIF-1 and Nrf2 may influence the outcome of anti-angiogenic therapies aimed at targeting HIF-1. Antioxid.

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Protamine, the only registered antidote of unfractionated heparin (UFH), may produce a number of adverse effects, such as anaphylactic shock or serious hypotension. We aimed to develop an alternative UFH antidote as efficient as protamine, but safer and easier to produce. As a starting material, we have chosen generally non-toxic, biocompatible, widely available, inexpensive, and easy to functionalize polysaccharides. Our approach was to synthesize, purify and characterize cationic derivatives of dextran, hydroxypropylcellulose, pullulan and γ-cyclodextrin, then to screen them for potential heparin-reversal activity using an in vitro assay and finally examine efficacy and safety of the most active polymers in Wistar rat and BALB/c mouse models of experimentally induced arterial and venous thrombosis. Efficacy studies included the measurement of thrombus formation, activated partial thromboplastin time, bleeding time, and anti-factor Xa activity; safety studies included the measurement of hemodynamic, hematologic and immunologic parameters. Linear, high molecular weight dextran substituted with glycidyltrimethylammonium chloride groups at a ratio of 0.65 per glucose unit (Dex40-GTMAC3) is the most potent and the safest UFH inhibitor showing activity comparable to that of protamine while possessing lower immunogenicity. Cationic polysaccharides of various structures neutralize UFH. Dex40-GTMAC3 is a promising and potentially better UFH antidote than protamine.

ADAM17 (a disintegrin and metalloprotease 17) is a major sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules and is often overexpressed in malignant cells. It is generally accepted that ADAM17 promotes tumor development via activating growth factors from the EGF family, thus facilitating autocrine stimulation of tumor cell proliferation and migration. Here we show, using MC38CEA murine colon carcinoma model, that ADAM17 also regulates tumor angiogenesis and cytokine profile. When ADAM17 was silenced in MC38CEA cells, in vivo tumor growth and in vitro cell motility were significantly diminished, but no effect was seen on in vitro cell proliferation. ADAM17-silencing was accompanied by decreased in vitro expression of vascular endothelial growth factor-A and matrix metalloprotease-9, which was consistent with the limited angiogenesis and slower growth seen in ADAM17-silenced tumors. Among the growth factors susceptible to shedding by ADAM17, neuregulin-1 was the only candidate to mediate the effects of ADAM17 on MC38CEA motility and tumor angiogenesis. Concentrations of TNF and IFNγ, cytokines that synergistically induced proapoptotic effects on MC38CEA cells, were significantly elevated in the lysates of ADAM17-silenced tumors compared to mock transfected controls, suggesting a possible role for ADAM17 in host immune suppression. These results introduce new, complex roles of ADAM17 in tumor progression, including its impact on the anti-tumor immune response.

Tumor-associated lymphatic vessels actively participate in tumor progression and dissemination. ADAM17, a sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules, is believed to promote tumor development, facilitating both tumor cell proliferation and migration, as well as tumor angiogenesis. In this work we addressed the issue of whether ADAM17 may also promote tumor lymphangiogenesis. First, we found that ADAM17 is important for the migratory potential of immortalized human dermal lymphatic endothelial cells (LEC). When ADAM17 was stably silenced in LEC, their proliferation was not affected, but: (i) single-cell motility, (ii) cell migration through a 3D Matrigel/collagen type I matrix, and (iii) their ability to form sprouts in a 3D matrix were significantly diminished. The differences in the cell motility between ADAM17-proficient and ADAM17-silenced cells were eliminated by inhibitors of EGFR and HER2, indicating that ADAM17-mediated shedding of growth factors accounts for LEC migratory potential. Interestingly, ADAM17 depletion affected the integrin surface expression/functionality in LEC. ADAM17-silenced cells adhered to plastic, type I collagen, and fibronectin faster than their ADAM17-proficient counterparts. The difference in adhesion to fibronectin was abolished by a cyclic RGD peptide, emphasizing the involvement of integrins in the process. Using a soluble receptor array, we identified BIG-H3 among several candidate proteins involved in the phenotypic and behavioral changes of LEC upon ADAM17 silencing. In additional assays, we confirmed the increased expression of BIG-H3, as well as TGFβ2 in ADAM17-silenced LEC. The antilymphangiogenic effects of ADAM17 silencing in lymphatic endothelial cells suggest further relevance of ADAM17 as a potential target in cancer therapy.

BackgroundTristetraprolin (TTP) is a key mediator of processes such as inflammation resolution, the inhibition of autoimmunity and in cancer. It carries out this role by the binding and degradation of mRNA transcripts, thereby decreasing their half-life. Transcripts modulated by TTP encode proteins such as cytokines, pro-inflammatory agents and immediate-early response proteins. TTP can also modulate neoplastic phenotypes in many cancers. TTP is induced and functionally regulated by a spectrum of both pro- and anti-inflammatory cytokines, mitogens and drugs in a MAPK-dependent manner. So far the contribution of p38 MAPK to the regulation of TTP expression and function has been best described.ResultsOur results demonstrate the induction of the gene coding TTP (ZFP36) by EGF through the ERK1/2-dependent pathway and implicates the transcription factor ELK-1 in this process. We show that ELK-1 regulates ZFP36 expression by two mechanisms: by binding the ZFP36 promoter directly through ETS-binding site (+ 883 to +905 bp) and by inducing expression of EGR-1, which in turn increases ZFP36 expression through sequences located between -111 and -103 bp.ConclusionsEGF activates TTP expression via ELK-1 and EGR-1 transcription factors.

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BackgroundCurcumin is a natural polyphenol with anti-inflammatory, chemopreventive and anticancer activity. However, its high hydrophobicity and poor bioavailability limit its medical application. The development of nanocarriers for curcumin delivery is an attractive approach to overcome its low bioavailability and fast metabolism in the liver. We synthesized a blood compatible alginate-curcumin conjugate, AA-Cur, which formed colloidally stable micelles of approximately 200 nm and, as previously shown, exerted strong cytotoxicity against mouse cancer cell lines. Here we analyze in vivo toxicity and antitumor activity of AA-Cur in two different mouse tumor models.MethodPotential toxicity of intravenously injected AA-Cur was evaluated by: i) analyses of blood parameters (morphology and biochemistry), ii) histology, iii) DNA integrity (comet assay), and iv) cytokine profiling (flow cytometry). Antitumor activity of AA-Cur was evaluated by measuring the growth of subcutaneously inoculated colon MC38-CEA- or orthotopically injected breast 4T1 tumor cells in control mice vs mice treated with AA-Cur.ResultsInjections of four doses of AA-Cur did not reveal any toxicity of the conjugate, thus indicating the safety of its use. AA-Cur elicited moderate anti-tumor activity toward colon MC38-CEA or breast 4T1 carcinomas.ConclusionThe tested conjugate of alginate and curcumin, AA-Cur, is non-toxic and safe, but exhibits limited anticancer activity.