Endothelial cell-surface tissue transglutaminase inhibits neutrophil adhesion by binding and releasing nitric oxide.

Nitric oxide (NO) produced by endothelial cells in response to cytokines displays anti-inflammatory activity by preventing the adherence, migration and activation of neutrophils. The molecular mechanism by which NO operates at the blood-endothelium interface to exert anti-inflammatory properties is largely unknown. Here we show that on endothelial surfaces, NO is associated with the sulfhydryl-rich protein tissue transglutaminase (TG2), thereby endowing the membrane surfaces with anti-inflammatory properties. We find that tumor necrosis factor-α-stimulated neutrophil adherence is opposed by TG2 molecules that are bound to the endothelial surface. Alkylation of cysteine residues in TG2 or inhibition of endothelial NO synthesis renders the surface-bound TG2 inactive, whereas specific, high affinity binding of S-nitrosylated TG2 (SNO-TG2) to endothelial surfaces restores the anti-inflammatory properties of the endothelium, and reconstitutes the activity of endothelial-derived NO. We also show that SNO-TG2 is present in healthy tissues and that it forms on the membranes of shear-activated endothelial cells. Thus, the anti-inflammatory mechanism that prevents neutrophils from adhering to endothelial cells is identified with TG2 S-nitrosylation at the endothelial cell-blood interface.

Particle replication in nonwetting templates nanoparticles with tumor selective alkyl silyl ether docetaxel prodrug reduces toxicity.

Delivery systems designed to have triggered release after passively targeting the tumor may improve small molecule chemotherapeutic delivery. Particle replication in nonwetting templates was used to prepare nanoparticles to passively target solid tumors in an A549 subcutaneous xenograft model. An acid labile prodrug was delivered to minimize systemic free docetaxel concentrations and improve tolerability without compromising efficacy.

The metal-responsive transcription factor-1 protein is elevated in human tumors.

We previously identified metal-responsive transcription factor-1 (MTF-1) as a positive contributor to mouse fibrosarcoma growth through effects on cell survival, proliferation, tumor angiogenesis and extracellular matrix remodeling. In the present study, we investigated MTF-1 protein expression in human tissues by specific immunostaining of both normal and tumor tissue samples. Immunohistochemical (IHC) staining of a human tissue microarray (TMA), using a unique anti-human MTF-1 antibody, indicated constitutive MTF-1 expression in most normal tissues, with liver and testis displaying comparatively high levels of expression. Nevertheless, MTF-1 protein levels were found to be significantly elevated in diverse human tumor types, including breast, lung and cervical carcinomas. IHC analysis of a separate panel of full-size tissue sections of human breast cancers, including tumor and normal adjacent, surrounding tissue, confirmed and extended the results of the TMA analysis. Taken with our previous findings, this new study suggests a role for MTF-1 in human tumor development, growth or spread. Moreover, the study suggests that MTF-1 could be a novel therapeutic target that offers the opportunity to manipulate metal or redox homeostasis in tumor cells.

Erythropoietin and erythropoietin receptor expression in head and neck cancer: relationship to tumor hypoxia.

PURPOSE: Erythropoietin, an oxygen-regulated glycoprotein hormone, is a hematopoietic cytokine that stimulates erythropoiesis by binding to its cellular receptor [erythropoietin receptor (EPOR)]. The recombinant form of human erythropoietin is used to prevent or treat anemia in cancer patients. However, in a recent randomized, placebo-controlled trial involving patients receiving curative radiotherapy for squamous cell carcinoma of the head and neck, erythropoietin treatment was associated with poorer locoregional progression-free survival. The purpose of our study was to determine whether EPOR and its ligand erythropoietin are expressed in primary head and neck cancer. We also investigated the hypothesis that erythropoietin expression in malignant cells may be associated with the presence of tumor hypoxia, an important factor involved in resistance to radiation treatment, tumor aggressiveness, and poor prognosis. EXPERIMENTAL DESIGN: Twenty-one patients received an i.v. infusion of the hypoxia marker pimonidazole hydrochloride before multiple tumor biopsies. Contiguous sections from 74 biopsies were analyzed by immunohistochemistry for EPOR and erythropoietin expression and pimonidazole binding. RESULTS: EPOR expression was present in tumor cells in 97% of the biopsies. Coexpression of erythropoietin was observed in 90% of biopsies. Erythropoietin and pimonidazole adduct staining did not always colocalize within tumors, but there was a significant positive correlation between levels of microregional erythropoietin expression and pimonidazole binding. CONCLUSIONS: The coexpression of erythropoietin and EPOR in tumor cells suggests that erythropoietin may potentially function as an autocrine or paracrine factor in head and neck cancer. The expression of the hypoxia-inducible protein erythropoietin in tumor cells correlates with levels of tumor hypoxia.

Erythropoietin and erythropoietin receptor expression in human prostate cancer.

Erythropoietin is a hematopoietic cytokine that regulates the production of red blood cells. Erythropoietin is normally produced in the adult kidney in a hypoxia-inducible manner. The recombinant form of human erythropoietin is in clinical use for the prevention and treatment of anemia that is associated with cancer and its treatment with chemoradiation therapy. A series of recent studies from our laboratory and others have reported the expression of receptors for erythropoietin in several different types of human cancer cells. In the present study, we investigated the expression of erythropoietin receptor and its ligand erythropoietin in human prostate cancer. In clinical specimens of prostate cancer, we found abundant expression of erythropoietin receptor protein in all primary tumors examined using immunohistochemistry. Furthermore, we observed erythropoietin coexpression in prostate cancer cells by immunohistochemical analysis. To determine whether monolayer cultures of continuous cell lines derived from prostate cancer also express erythropoietin receptor and erythropoietin, we studied well-characterized hormone-responsive (LNCaP) and hormone-refractory (PC-3) prostate cancer cell lines. We performed reverse-transcription and polymerase chain reaction assays to detect erythropoietin receptor and erythropoietin mRNA transcripts, and also immunoprecipitation and immunoblotting to detect erythropoietin receptor protein expression in prostate cancer cells. These experiments revealed the expression of both erythropoietin receptor and erythropoietin in LNCaP and PC-3 cells suggesting that these prostate cancer cell lines may serve as useful experimental models for further studies of erythropoietin and erythropoietin receptor function in prostate cancer. The coexpression of erythropoietin receptor and its ligand erythropoietin in human prostate cancer cells suggests the potential for growth regulation by erythropoietin-erythropoietin receptor in an autocrine or paracrine manner.

Loss of metal transcription factor-1 suppresses tumor growth through enhanced matrix deposition.

Metal transcription factor-1 (MTF-1) is a ubiquitous transcriptional regulator and chromatin insulator with roles in cellular stress responses and embryonic development. The studies described herein establish for the first time the involvement of MTF-1 in tumor development. Genetically manipulated ras-transformed mouse embryonic fibroblasts (MEFs), wild-type (MTF-1+/+), or nullizygous for MTF-1 (MTF-1-/-) were used to develop fibrosarcoma tumors. Loss of MTF-1 resulted in delayed tumor growth associated with increased matrix collagen deposition and reductions in vasculature density. Molecular consequences of MTF-1 loss include increased expression and activation of the transforming growth factor-beta1 (TGF-beta1) and tissue transglutaminase (tTG), two proteins with documented roles in the production and stabilization of extracellular matrix (ECM). Our findings support the hypothesis that MTF-1 enhances the ability of the developing tumor mass to evade fibrosis and scarring of the tumor, a critical step in tumor cell proliferation.

The relationship between hypoxia and angiogenesis.

Recent studies have generated a large amount of data supporting the hypothesis that hypoxia drives tumor angiogenesis. The relationship between the two is often considered a matter of supply and demand: ineffectively-vascularized tumor tissue becomes hypoxic, stimulating neoangiogenesis to improve the influx of oxygen, thereby diminishing the angiogenic drive. Although this paradigm is logically pleasing, much of what is known about tumor biology argues against such a straightforward relationship. In fact, some preclinical data convincingly shows that tumor hypoxia and angiogenesis do not always go hand in hand. It is important to begin to explore means of reconciling these discrepancies. Although poor oxygenation is a strong stimulus for tumor angiogenesis, (1) the pathogenesis of tumor hypoxia is much more complicated than the supply-demand paradigm lets on and (2) hypoxia is not necessarily sufficient or necessary for neovascularization to occur. These subtleties may help to explain why so much data disagrees with the current hypoxia-angiogenesis model and may begin to build a better understanding of the role hypoxia plays in tumor vascularization. This article will review what is known about hypoxia and angiogenesis in nononcological processes and will apply these lessons to tumor biology to more deeply describe their relationship.

A novel role for erythropoietin during fibrin-induced wound-healing response.

In this study, we investigated the role of the hematopoietic cytokine erythropoietin (EPO) during wound healing, the physiological response to tissue injury. We used an in vivo wound-healing assay (fibrin Z-chambers) consisting of fibrin-filled chambers implanted subcutaneously in rats. The fibrin inside the chambers is replaced by granulation tissue consisting of new blood vessels, macrophages and fibroblasts as part of the wound-healing response. Local, exogenous recombinant EPO administration into the fibrin matrix significantly increased granulation tissue formation in a dose-dependent manner. To investigate the physiological role of endogenous EPO during wound healing, we used soluble EPO receptor or anti-EPO monoclonal antibodies to neutralize EPO and observed dose-dependent inhibition of granulation tissue formation, consistent with an important role for EPO in the wound-healing cascade. The ability of recombinant EPO to promote wound healing was associated with a proangiogenic effect during granulation tissue formation. We also found abundant expression of EPO receptor protein in macrophages, cells that play a pivotal role during wound healing. Modulation of wound healing because of administration of recombinant EPO or inhibition of endogenous EPO-EPO receptor correlated with changes in levels of inducible nitric oxide synthase protein in granulation tissue. These data demonstrate a novel function for EPO by providing in vivo evidence for a physiological role during fibrin-induced wound healing.

Expression of erythropoietin receptor splice variants in human cancer.

Erythropoietin (EPO) regulates mammalian erythropoiesis by binding to its transmembrane receptor EPOR. Recent studies demonstrated functional EPOR expression in human cancer cells. Recombinant human EPO was reported to stimulate the proliferation of monolayer cultures of breast and renal carcinoma cells. Furthermore, administration of EPO-EPOR antagonists delayed the growth of uterine, ovarian, and mammary carcinoma cells in experimental animal models. In this study, we show EPOR transcript and protein expression in breast, colon, lung, ovary, and prostate cancer cells. Using reverse transcription-polymerase chain reaction, we isolated and characterized several novel cDNAs for EPOR splice variants expressed in cancer cells. Deduced amino acid sequences of the cDNAs revealed splice variants encoding soluble EPOR or membrane-bound EPOR peptides with intra-cytoplasmic, carboxy-terminal truncations. These findings indicate the expression of multiple EPOR isoforms in human cancer cells that may modulate the cellular effects of recombinant human EPO or EPO-EPOR antagonists.

Dietary glycine inhibits angiogenesis during wound healing and tumor growth.

In this study we investigated the effects of glycine on angiogenesis during embryogenesis, wound healing and tumor growth. In chorioallantoic membrane (CAM) assay, glycine (100 microM) inhibited angiogenesis by more than 50%. We studied dietary glycine's effect on fibrin induced wound healing response in a novel (Fibrin Z-chamber) assay. Fibrin within the chamber triggers the healing cascade leading to formation of granulation tissue (GT) rich in blood vessels and stroma. GT was reduced by more than 30% (p < 0.0001) in dietary Glycine groups as compared to control. We found that microvessel density dropped significantly (15%, p < 0.0003) with dietary glycine whereas the other components of GT were unaffected. We evaluated tumor growth delay utilizing Tumor Z-Chamber (fibrin with R3230 mammary adenocarcinoma cells) since tumors take advantage of angiogenesis and matrix formation. We observed that tumor growth decreased by 15% (p < 0.03) and tumor microvessel density dropped by 20% (p < 0.03) with dietary glycine compared to controls. We found that iNOS protein levels were decreased significantly in both GT (24%-57%) and tumor tissue (19-75%). In conclusion, we found that dietary glycine is a potent anti-angiogenic agent that can reduce wound healing and tumor growth through reduction of iNOS expression.

Effect of flaxseed supplementation on prostatic carcinoma in transgenic mice.

OBJECTIVES: To investigate the effects of flaxseed supplementation on prostatic neoplasia in the transgenic adenocarcinoma mouse prostate (TRAMP) model. METHODS: A total of 135 male TRAMP mice 5 to 6 weeks old were randomized to a control group (AIN-76A diet) or an experimental group (AIN-76A diet plus 5% flaxseed by weight). One half of the mice in each group were treated for 20 weeks and the remainder for 30 weeks. At autopsy, urogenital tissues (four prostatic lobes, seminal vesicles, and emptied bladder), lungs, lymph nodes, and grossly abnormal tissues were collected for histologic evaluation. RESULTS: Of the control mice, 100% developed prostate cancer versus 97% of the mice in the flaxseed group. The tumor/urogenital weight was 3.6 +/- 0.4 g in the controls versus 1.9 +/- 0.2 g in the flaxseed-treated mice (P = 0.0005). At 20 weeks, no significant difference in tumor grade was seen between the two groups; however, at 30 weeks, the flaxseed-treated mice had significantly less aggressive tumors than did the controls (P = 0.01). The prevalence of lung and lymph node metastases was 13% and 16%, respectively, in the control mice versus 5% and 12%, respectively, in the experimental group (difference not significant). After 20 weeks of treatment, cellular proliferation (Ki-67) differed significantly between the control and experimental groups (38.1 +/- 2.03 versus 26.2 +/- 2.03; P <0.0001), and the apoptotic index (deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling) was 1.45 +/- 0.14 versus 3.3 +/- 0.31 (P <0.0001). Similar differences were seen after 30 weeks of treatment. CONCLUSIONS: A diet supplemented with 5% flaxseed inhibits the growth and development of prostate cancer in the TRAMP model.

SU5416 delays wound healing through inhibition of TGF-beta 1 activation.

Angiogenesis, development of new blood vessels, is essential for wound healing and tumor growth. A potentially important side effect of anti-angiogenic therapy can be delayed wound healing. In this study we address this issue by using a novel in vivo method utilizing fibrin containing dual porous plexiglass chambers (Fibrin Z-Chambers; F-ZC) to investigate wound healing in rats administered with SU5416 (inhibitor of Flk-1 and Flt-1, at 20 mg/kg i.p.). SU5416 treated F-ZCs developed 45% less granulation tissue (p = 0.0076) and showed a 10% reduction in microvessel density (p = 0.0009) than controls treated with drug carrier alone. The granulation tissue showed distinctly decreased collagen deposition (p = 0.0006) in SU5416 treated animals that was associated with 90% reduction in active TGF-beta 1 level. We found that tissue transglutaminase (TG), a cross-linking enzyme involved in TGF-beta 1 activation and matrix stabilization, was inhibited by SU5416. These results suggest that SU5416 delays wound healing by reducing matrix synthesis and stabilization through inhibition of TGF-beta 1 activation. This study was made feasible via the development of a unique method to study anti-angiogenic compounds that provides highly reproducible and quantitative results. Further studies are needed to evaluate in vivo whether anti-angiogenic agents alter wound healing.

Functional significance of erythropoietin receptor expression in breast cancer.

Erythropoietin (EPO) is the principal hematopoietic cytokine that regulates mammalian erythropoiesis by binding to its transmembrane receptor EpoR. Recent experimental evidence suggests that the biologic effects of EPO are not limited to the regulation of erythropoiesis. In studies focusing on nonhematopoietic effects of EpoR signaling, we found high levels of EpoR protein expression in human breast cancer cells. The purpose of the present study was to evaluate clinical breast cancer specimens for EPO and EpoR expression, characterize the relationship between EPO expression and tumor hypoxia in biopsies prelabeled with hypoxia marker pimonidazole, analyze breast cancer cell lines for EpoR expression, and study the functional significance of EpoR expression in breast cancer cells in vivo. Immunohistochemical analysis for EPO, EpoR expression, and pimonidazole adducts was performed on 26 tumor biopsies with contiguous sections from 10 patients with breast cancer. High levels of EpoR expression were found in cancer cells in 90% of tumors. EPO expression was found in 60% of tumors and EPO and EpoR colocalization in tumor cells was present in many cases. The expression pattern of EPO with respect to tumor hypoxia was variable, without consistent colocalization of EPO and hypoxia in tumor cells. Human and rat breast cancer tissue culture cells express EpoR mRNA and protein. To study the in vivo function of EpoR expression in breast cancer cells, we used rat syngeneic R3230Ac mammary adenocarcinoma cells in a tumor Z-chamber model (dual porous plexiglass chambers containing fibrin gel, cancer cells, and a putative anti-tumor compound implanted into the subcutaneous tissue of rats). Local, one-time administration of a neutralizing anti-EPO antibody, soluble EPO receptor, or an inhibitor of Jak2, a cytoplasmic tyrosine kinase essential for EPO-mediated mitogenesis, resulted in a delay in tumor growth with 45% reduction in maximal tumor depth in tumor Z-chambers in a dose-dependent manner. These studies demonstrate the expression of functional receptors for EPO in breast cancer cells.