Khalid Amin

5′-AMP-Activated Protein Kinase (AMPK) Is Induced by Low-Oxygen and Glucose Deprivation Conditions Found in Solid-Tumor Microenvironments

ABSTRACT Low oxygen gradients (hypoxia and anoxia) are important determinants of pathological conditions under which the tissue blood supply is deficient or defective, such as in solid tumors. We have been investigating the relationship between the activation of hypoxia-inducible factor 1 (HIF-1), the primary transcriptional regulator of the mammalian response to hypoxia, and 5′-AMP-activated protein kinase (AMPK), another regulatory system important for controlling cellular energy metabolism. In the present study, we used mouse embryo fibroblasts nullizygous for HIF-1α or AMPK expression to show that AMPK is rapidly activated in vitro by both physiological and pathophysiological low-oxygen conditions, independently of HIF-1 activity. These findings imply that HIF-1 and AMPK are components of a concerted cellular response to maintain energy homeostasis in low-oxygen or ischemic-tissue microenvironments. Finally, we used transformed derivatives of wild-type and HIF-1α- or AMPKα-null mouse embryo fibroblasts to determine whether AMPK is activated in vivo. We obtained evidence that AMPK is activated in authentic hypoxic tumor microenvironments and that this activity overlaps with regions of hypoxia detected by a chemical probe. We also showed that AMPK is important for the growth of this tumor model.

Erythropoietin receptor expression in adult rat cardiomyocytes is associated with an acute cardioprotective effect for recombinant erythropoietin during ischemia-reperfusion injury

Erythropoietin (EPO), the principal hematopoietic cytokine that regulates mammalian erythropoiesis, exhibits diverse cellular effects in non‐hematopoietic tissues. The physiologic functions of EPO are mediated by its specific cell‐surface receptor EPOR. In this study, we demonstrate EPOR expression in adult rat cardiac myocytes and examine the direct effects of EPO on the heart to investigate whether recombinant EPO may exert an acute cardioprotective effect during ischemia‐reperfusion injury. To determine whether EPO is cardioprotective, isolated rat hearts were perfused for 10 min in the Langendorff‐mode with Krebs‐Henseleit buffer in the absence or presence of brief recombinant EPO treatment while left‐ventricular‐developed pressure (LVDP) was measured continuously to assess contractile function. The hearts were then subjected to 20 min of normothermic global ischemia followed by 25 min of reperfusion. The post‐ischemic recovery of LVDP in the untreated control hearts was 26 ± 5% of their baseline LVDP, whereas hearts pretreated with EPO exhibited significantly improved post‐ischemic recovery to 57 ± 7%. We used 31P nuclear magnetic resonance (NMR) spectroscopy to determine whether modulation of intracellular pH and/or high‐energy phosphate levels during ischemia contributed to EPO‐mediated cardioprotection. These experiments revealed that the rapid cardioprotective effect of EPO during ischemia‐reperfusion injury was associated with preservation of ATP levels in the ischemic myocardium.

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.

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.

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.

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 μM) 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

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 in‐ sulator with roles in cellular stress responses and em‐ bryonic development. The studies described herein establish for the first time the involvement of MTF‐1 in tumor development. Genetically manipulated ras‐trans‐ formed 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 reduc‐ tions in vasculature density. Molecular consequences of MTF‐1 loss include increased expression and activation of the transforming growth factor–β1 (TGF‐β1) and tissue transglutaminase (tTG), two proteins with docu‐ mented 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 prolifera‐ tion.—Haroon, Z. A., Amin, K., Lichtlen, P., Sato, B., Huynh, N. T., Wang, Z., Schaffner, W., Murphy, B. J. Loss of metal transcription factor‐1 suppresses tumor growth through enhanced matrix deposition. FASEB J. 18, 1176 –1184 (2004)

Characterization of ANIT-Induced Toxicity using Precision-Cut Rat and Dog Liver Slices Cultured in a Dynamic Organ Roller System

This article describes the toxicity of α-naphthylisothiocyanate (ANIT), a compound known to induce dose-dependent hepatobiliary toxicity in vivo, using the slice model. Liver slices (200 μm thick) from male Sprague–Dawley rats and male beagle dogs were cultured for 7 days while exposed to a range of ANIT concentrations (1– 100 μM for rat and 4–320 μM for dog). Tissues (and medium for dog) were evaluated using a panel of clinically relevant biomarkers for liver and histological endpoints to assess viability and proliferation. ANIT increased slice levels of enzyme biomarkers corresponding to biliary markers. At high concentrations (80–100 μM for rat, 320 μM for dog) a diminution of tissue enzyme levels was observed, corresponding to severe hepatobiliary injury. By days 5 and 7, biochemical markers in the medium of dog slices indicated an elevation of hepatocellular and biliary markers. Histologically for both species, minimal hepatocellular injury was noted, but proliferation of biliary epithelial cells (BEC) was observed using 5-bromo-2-deoxyuridine (BrdU) immunostaining. In rat slices, ANIT increased the expression of inducible nitrous oxide synthase (iNOS) within 12 hrs of exposure. In summary, additional experimentation using slice culture may further demonstrate its value in screening compounds that cause hepatobiliary toxicity.

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.

Inspiratory contrast for in vivo optical imaging

We demonstrate the use of inspired oxygen and carbon dioxide as a possible route to increase contrast in optical imaging of cancerous tissue. Differential imaging in human xenograft rodent models of cancer exhibits significant variation in signal between normal and cancerous tissue. This differential cancer-specific contrast is stronger and more consistent than the conventional static contrast. This differential technique exploits the response of abnormal tumor vasculature to inhaled gases and could provide a promising alternative to supplement mainstream cancer imaging modalities such as x-rays and MRI.