losartan-potassium and thiazolyl-blue

Erythropoietin (EPO) is a hormone that induces red blood cell production. In its recombinant form, EPO is the one of most prescribed drugs to treat anemia, including that arising in cancer patients. In randomized trials, EPO administration to cancer patients has been associated with decreased survival. Here, we investigated the impact of EPO modulation on tumorigenesis. Using genetically engineered mouse models of breast cancer, we found that EPO promoted tumorigenesis by activating JAK/STAT signaling in breast tumor-initiating cells (TICs) and promoted TIC self renewal. We determined that EPO was induced by hypoxia in breast cancer cell lines, but not in human mammary epithelial cells. Additionally, we demonstrated that high levels of endogenous EPO gene expression correlated with shortened relapse-free survival and that pharmacologic JAK2 inhibition was synergistic with chemotherapy for tumor growth inhibition in vivo. These data define an active role for endogenous EPO in breast cancer progression and breast TIC self-renewal and reveal a potential application of EPO pathway inhibition in breast cancer therapy.

Topics: Animals; Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Disease Progression; Disease-Free Survival; Endothelial Cells; Erythropoietin; Female; Gene Expression Regulation; Humans; Hypoxia; Mammary Neoplasms, Experimental; Mice; Neoplasm Transplantation; Neoplastic Stem Cells; Recurrence; Signal Transduction; Tetrazolium Salts; Thiazoles; Time Factors

Podocytes are typically cultured on collagen I; however, collagen I is absent from healthy glomerular basement membranes. Erythropoietin (EPO) is thought to protect podocytes in vivo. Here, we studied how various types of extracellular matrix (ECM) proteins and EPO affect podocytes in culture.. Primary rat podocytes were replated on collagen I, collagen IV, whole ECM extract, laminin, or bare plastic. Cellular adhesion (8 hours after plating), proliferation (5 days, 10 % serum), and resistance to serum deprivation (3 days, 0.5 % serum) were assessed. BrdU incorporation and expression of podocyte-specific markers were employed as measures of cellular proliferation and differentiation, respectively. qPCR was used to verify expression of EPO receptor in cultured podocytes.. Cellular adhesion was similar on all ECM proteins and unaffected by EPO. Proliferation was accelerated by laminin and the ECM extract, but the final cell density was similar on all ECM surfaces. Collagen IV supported the serum-deprived cells better than the other ECM proteins. EPO (2-20 ng/ml) improved viability of serum-deprived podocytes on collagen I, collagen IV, and ECM, but not on laminin or bare plastic. The cells expressed mRNA for EPO receptor.. The physiological ECM proteins are more supportive of primary podocytic cultures compared with collagen I. The protective effects of EPO during serum deprivation are modulated by the cultivation surface.

Topics: Animals; Cells, Cultured; Coloring Agents; Erythropoietin; Extracellular Matrix; Extracellular Matrix Proteins; Kidney Glomerulus; Podocytes; Primary Cell Culture; Rats; Receptors, Erythropoietin; Recombinant Proteins; Tetrazolium Salts; Thiazoles

Diabetic retinopathy (DR) is a chronic, low-grade inflammatory disease. We aimed to investigate the regulatory effects of erythropoietin (EPO) on the inflammatory cytokine production by Muller cells under the condition of DR. The expression levels of TNF-alpha, IL-1beta, IL-6 and VEGF in cultured rat Muller cells were enhanced by 1 mM glyoxal. The elevated TNF-alpha and IL-1beta, but not IL-6 and VEGF, were decreased by 2 U/ml EPO as detected by real-time PCR and ELISA. Moreover, the activity of AP-1 but not NF-kappaB was modulated by glyoxal and EPO. Intravitreal injection of EPO performed 24 h prior to sacrifice significantly reduced TNF-alpha and IL-1beta production while moderately attenuating IL-6 and VEGF in the retinas of streptozotocin-induced diabetic rats. Furthermore, Muller cells were identified as the main source of IL-1beta production as indicated by co-localization of IL-1beta and CRALBP in situ. These findings implicate therapeutic potential of EPO in the amelioration of inflammation in diabetic retinas.

Topics: Animals; Cell Line; Cytokines; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Gene Expression Regulation; In Situ Nick-End Labeling; Interleukin-1beta; Interleukin-6; Luciferases; NF-kappa B; Rats; Reverse Transcriptase Polymerase Chain Reaction; Tetrazolium Salts; Thiazoles; Transcription Factor AP-1; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Erythropoietin (EPO) exhibits diverse cellular functions, including neurotrophic, anti-oxidant, anti-apoptotic, and anti-inflammatory effects in non-hematopoietic tissues. This study evaluated whether bone marrow mesenchymal stromal cells (MSCs) transduced with the EPO gene (EPO-MSCs) promoted neural cell survival and improved neurological deficits caused by ischemic stroke. EPO-MSCs stably produced high levels of EPO (10IU/ml) without any alteration of their mesenchymal phenotype. Both EPO transduction and treatment with 10 international units (IU) of recombinant human EPO (rhEPO) provided protection from H(2)O(2)-induced oxidative injury in human bone marrow mesenchymal stromal cells and in SH-SY5Y cells. EPO-MSCs were more protected than were MSCs treated with 10IU rhEPO (10U-MSCs). We also found that the expression of the neurotrophic factors BDNF, PD-ECGF, HGF, SDF-1alpha, and TGF-1beta increased in EPO-MSCs, while only BDNF and TGF-1beta increased in 10U-MSCs. Implantation of EPO-MSCs in an animal model of ischemic stroke significantly improved neurological function and decreased infarct volumes without affecting hematocrit level. An evaluation of the brain tissue 21days after implantation showed that EPO and phosphorylated Akt (a downstream mediator of EPO) increased only in brains implanted with EPO-MSCs. Transduction of the EPO gene into MSCs induced secretion of EPO and various trophic factors that may provide excellent neuroprotective effects in both in vitro and in vivo models of ischemic stroke.

Topics: Animals; Antigens, CD; Brain Infarction; Caspase 3; Cell Death; Cell Line; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Flow Cytometry; Gene Expression Regulation; Green Fluorescent Proteins; Humans; Hydrogen Peroxide; Infarction, Middle Cerebral Artery; Lentivirus; Magnetic Resonance Imaging; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Nerve Growth Factors; Nervous System Diseases; Rats; Statistics, Nonparametric; Tetrazolium Salts; Thiazoles; Transduction, Genetic

The neuroprotective effect of erythropoietin (EPO) against 1-methyl-4-phenylpyridinium (MPP(+))-induced oxidative stress in cultured PC12 cells, as well as the underlying mechanism, were investigated.. PC12 cells impaired by MPP(+) were used as the cell model of Parkinson's disease. Methyl thiazolyl tetrazolium (MTT) was used to assay the viability of the PC12 cells exposed to gradient concentrations of EPO, and the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay was used to analyze the apoptosis ratio of PC12 cells. The expression of Bcl-2 and Bax in PC12 cells were examined by Western blot, and the reactive oxygen species (ROS), the mitochondrial transmembrane potential and the activity of caspase-3 in each group were detected by spectrofluorometer.. Treatment of PC12 cells with MPP(+) caused the loss of cell viability, which may be associated with the elevation in apoptotic rate, the formation of ROS and the disruption of mitochondrial transmembrane potential. It was also shown that MPP(+) significantly induced the upregulation of Bax/Bcl-2 ratio and the activation of caspase-3. In contrast, EPO significantly reversed these responses and had the maximum protective effect at 1 U/mL.. The inhibitive effect of EPO on the MPP(+)-induced cytotoxicity may be ascribed to its anti-oxidative property and anti-apoptotic activity, and EPO may provide a useful therapeutic strategy for treatment of neurodegenerative diseases such as Parkinson's disease.

Topics: 1-Methyl-4-phenylpyridinium; Analysis of Variance; Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Survival; Dose-Response Relationship, Drug; Drug Interactions; Erythropoietin; Flow Cytometry; Herbicides; In Situ Nick-End Labeling; Membrane Potential, Mitochondrial; Neuroprotective Agents; PC12 Cells; Proto-Oncogene Proteins c-bcl-2; Rats; Reactive Oxygen Species; Tetrazolium Salts; Thiazoles

Although erythropoietin (Epo) is a known stimulator of erythropoiesis, recent evidence suggests that its biological functions are not confined to hematopoietic cells. To elucidate the role of Epo and erythropoietin receptor (EpoR) in melanoma, we examined the expression and function of these proteins in melanocytes and melanoma cells. We found increased expression of Epo in melanoma cells compared to melanocyte in vitro. EpoR was also strongly expressed in all of the melanoma cell lines and two of the three melanocyte cell lines examined. Epo expression was significantly higher in melanoma than in benign nevi as determined by immunohistochemistry. Although melanoma cells secreted Epo in normoxic condition in vitro, hypoxia and CoCl(2) treatment increased Epo secretion. EpoR in melanoma cells was functional, because exogenous Epo increased melanoma resistance to hypoxic stress, pretreatment of melanoma cells with Epo significantly increased resistance to dacarbazine treatment, and Epo increased the phosphorylation of EpoR, RAF, and MEK. In conclusion, we demonstrated constitutive expression of Epo and EpoR as well as autonomous secretion of Epo by melanoma cells, indicating a novel autocrine loop of Epo in melanoma. The results suggest that the autocrine and paracrine functions of Epo might play a role in malignant transformation of melanocytes and in the survival of melanoma cells in hypoxia and other adverse conditions.

Topics: Blotting, Western; Cell Line, Tumor; Cell Survival; Cell Transformation, Neoplastic; Cobalt; Coloring Agents; Culture Media, Conditioned; Disease Progression; Dose-Response Relationship, Drug; Erythropoietin; Humans; Hypoxia; Immunohistochemistry; MAP Kinase Signaling System; Melanocytes; Melanoma; Membrane Potentials; Mitochondria; Phosphorylation; Receptors, Erythropoietin; Tetrazolium Salts; Thiazoles; Trypan Blue

To examine the roles for NF-kappaB family proteins in hematopoiesis, we first expressed dominant negative Rel/NF-kappaB(IkappaBSR) in a factor-dependent cell line, Ba/F3. Although IkappaBSR neither affected thrombopoietin-dependent nor gp130-mediated growth, it suppressed interleukin-3- and erythropoietin-dependent growth at low concentrations. In addition, IkappaBSR enhanced factor-deprived apoptosis through the accumulation of reactive oxygen species (ROS). When expressed in normal hematopoietic stem/progenitor cells, IkappaBSR induced apoptosis even in the presence of appropriate cytokines by accumulating ROS. We also expressed IkappaBSR in an inducible fashion at various stages of hematopoiesis using the OP9 system, in which hematopoietic cells are induced to develop from embryonic stem cells. When IkappaBSR was expressed at the stage of Flk-1(+) cells (putative hemangioblasts), IkappaBSR inhibited the development of primitive hematopoietic progenitor cells by inducing apoptosis through the ROS accumulation. Furthermore, when IkappaBSR was expressed after the development of hematopoietic progenitor cells, it inhibited their terminal differentiation toward erythrocytes, megakaryocytes, and granulocytes by inducing apoptosis through the ROS accumulation. These results indicate that NF-kappaB is required for preventing apoptosis at multiple steps of hematopoiesis by eliminating ROS.

Topics: Animals; Apoptosis; Blotting, Northern; Cell Line; Cell Separation; Cell Survival; Cells, Cultured; Coloring Agents; Culture Media, Conditioned; Cytokines; Erythropoietin; Flow Cytometry; Glutathione; Granulocytes; Hematopoiesis; Hematopoietic Stem Cells; I-kappa B Proteins; Immunoblotting; Interleukin-3; Mice; Mice, Inbred BALB C; NF-kappa B; Plasmids; Reactive Oxygen Species; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; Tetracycline; Tetrazolium Salts; Thiazoles; Time Factors

Tetrahydrobiopterin (BH(4)) synthesis is reported to be stimulated by nerve growth factor (NGF) in PC12 cells, suggesting involvement of BH(4) in the trophic effect of NGF. We have recently reported that erythropoietin (EPO) and BH(4) enhance survival of PC12 cells. In the present study, we investigated involvement of BH(4) in the trophic effect of EPO on PC12 cells. Cellular BH(4) content was increased by EPO (10(-10) to 10(-8) M) in a dose- and time-related manner. EPO (10(-10) to 10(-8) M) increased the viable cell number of PC12 cells. In addition to EPO, BH(4) (1, 3, and 10 microM) increased the viable cell number of PC12 cells. Administration of 0.3 mM 2,4-diamino-6-hydroxypyrimidine, an inhibitor of BH(4) synthesis, blunted EPO-induced increases in BH(4) content and the viable cell number of PC12 cells. These results taken together suggest that BH(4) is involved in the trophic effects of EPO on PC12 cells.

Topics: Animals; Biopterins; Cell Line; Cell Survival; Coloring Agents; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Enzyme Inhibitors; Erythropoietin; Hypoxanthines; PC12 Cells; Protein Binding; Rats; Tetrazolium Salts; Thiazoles; Time Factors

The impairment of function of human T lymphocytes, leading to an inappropriate cytokine production, is partially responsible for defective immunological response in hemodialysis patients (HD). Recent data suggest that recombinant human erythropoietin (rhEPO) may exert immunological effects. The aim of this study was to find out whether rhEPO treatment of the HD patients may have an effect on the interleukin 2 (IL-2) production by their whole blood cell cultures. The study was carried out in 10 HD patients receiving rhEPO for 6 months. Compared with the levels seen before the treatment, the concentration of IL-2 increased in the phytohemagglutinin-stimulated whole blood cell cultures of 7 of 10 patients under study. Addition of rhEPO in vitro to the whole blood cell cultures of the HD patients before implementation of erythropoietin confirmed that rhEPO is able to directly stimulate IL-2 production. Our studies show that the therapy with rhEPO affects IL-2 secretion.

Topics: Adjuvants, Immunologic; Adult; Blood Cells; Cells, Cultured; Chromogenic Compounds; Chronic Disease; Colorimetry; Coloring Agents; Erythropoietin; Female; Glomerulonephritis; Humans; Interleukin-2; Leukocyte Count; Lymphocyte Activation; Male; Middle Aged; Phytohemagglutinins; Recombinant Proteins; Renal Dialysis; T-Lymphocytes; Tetrazolium Salts; Thiazoles