DOCSLIB.ORG

Induction of Erythropoietin Increases the Cell Proliferation Rate in a Hypoxia‑Inducible Factor‑1‑Dependent and ‑Independent Manner in Renal Cell Carcinoma Cell Lines

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Induction of Erythropoietin Increases the Cell Proliferation Rate in a Hypoxia‑Inducible Factor‑1‑Dependent and ‑Independent Manner in Renal Cell Carcinoma Cell Lines ONCOLOGY LETTERS 5: 1765-1770, 2013 Induction of erythropoietin increases the cell proliferation rate in a hypoxia‑inducible factor‑1‑dependent and ‑independent manner in renal cell carcinoma cell lines YUTAKA FUJISUE1, TAKATOSHI NAKAGAWA2, KIYOSHI TAKAHARA1, TERUO INAMOTO1, SATOSHI KIYAMA1, HARUHITO AZUMA1 and MICHIO ASAHI2 Departments of 1Urology and 2Pharmacology, Faculty of Medicine, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan Received November 28, 2012; Accepted February 25, 2013 DOI: 10.3892/ol.2013.1283 Abstract. Erythropoietin (Epo) is a potent inducer of erythro- Introduction poiesis that is mainly produced in the kidney. Epo is expressed not only in the normal kidney, but also in renal cell carcinomas Erythropoietin (Epo) is a 30-kDa glycoprotein that functions (RCCs). The aim of the present study was to gain insights as an important cytokine in erythrocytes. Epo is usually into the roles of Epo and its receptor (EpoR) in RCC cells. produced by stromal cells of the adult kidney cortex or fetal The study used two RCC cell lines, Caki-1 and SKRC44, in liver and then released into the blood, with its production which Epo and EpoR are known to be highly expressed. The initially induced by hypoxia or hypotension (1-5). In the bone proliferation rate and expression level of hypoxia-inducible marrow, Epo binds to the erythropoietin receptor (EpoR) factor-1α (HIF-1α) were measured prior to and following expressed in erythroid progenitor cells or undifferentiated Epo treatment and under normoxic and hypoxic conditions. erythroblasts, which induces signal transduction mechanisms To examine whether HIF-1α or Epo were involved in cellular that protect the undifferentiated erythrocytes from apoptosis proliferation during hypoxia, these proteins were knocked and promote their proliferation and differentiation. down using small interfering RNA (siRNA) in Caki-1 and Previous studies have demonstrated that Epo and EpoR SKRC44 cells. The results demonstrated that Epo enhanced are not only produced or expressed in the kidneys, but the proliferation of the Caki-1 and SKRC44 cells. HIF-1α also in other cells and tissues, including macrophages (6), expression was increased upon the induction of hypoxia in the vascular endothelial cells (7), neurons (8), myoblasts (9), Caki-1 cells, but remained unaffected in the SKRC44 cells. the uterus, ovaries (10) and mammary glands (11), and in The proliferation rate was increased under hypoxic conditions various malignant tumors, including angioblastomas (12), in the Caki-1 cells, but was decreased in the SKRC44 cells. meningiomas (13), uterine or ovarian cancer (14) and breast Under hypoxic conditions, the proliferation of the Caki-1 cells cancer (15). The secretion of Epo due to hypoxia is upregu- was significantly reduced by the knock‑down of HIF‑1α or lated by the induction of Epo mRNA via hypoxia-inducible Epo, while the proliferation of the SKRC44 cells was signifi- factor (HIF)-1 signaling (16). cantly suppressed by the knock-down of Epo, but not HIF-1α. HIF-1 contains two subunits, HIF-1α and β. The expression In conclusion, these data suggest that the induction of Epo may of HIF-1α is altered by various conditions. HIF-1α is ubiqui- accelerate the proliferation of the RCC cell lines in either a tinated and proteolyzed in the proteasome under normoxic HIF-1α-dependent or -independent manner. conditions. With hypoxia, however, it is stabilized to bind with HIF-1β to form a heterodimer, which is internalized into the nucleus and binds with a hypoxia responsive element to induce Epo transcription. HIF-1β is constitutively expressed. HIF-1 contributes to hypoxia adaptation at the tissue or Correspondence to: Professor Michio Asahi, Department of cellular level. At the tissue level, it upregulates the expres- Pharmacology, Faculty of Medicine, Osaka Medical College, 2-7 sion levels of Epo and vascular endothelial growth factor Daigaku-cho, Takatsuki, Osaka 569-8686, Japan (VEGF), which promote hematopoiesis and angiogenesis. At E-mail: [email protected] the cellular level, HIF-1 regulates cellular metabolism by the Abbreviations: Epo, erythropoietin; EpoR, erythropoietin receptor; upregulation of glycolytic enzymes and glucose transporters, RCC, renal cell carcinoma; HIF-1α, hypoxia-inducible factor-1α; which shifts the energy metabolism to oxygen-independent VHL, von Hippel-Lindau glycolysis. Dysfunction in these pathways may be associated with the onset or progression of cancer (17), e.g., the progres- Key words: erythropoietin, renal cell carcinoma, hypoxia-inducible sion of tumor angiogenesis by VEGF overexpression may be factor-1 correlated with tumor infiltration or metastasis (18). Numerous anti-angiogenesis therapies against HIF-1 or VEGF have been developed and tested (19). 1766 FUJISUE et al: INDUCTION OF ERYTHROPOIETIN IN RENAL CELL CARCINOMA CELL LINES Epo binds to EpoR, resulting in the activation of the and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) janus kinase-signal transducer and activator of transcription expression was quantified for normalization of the amount (JAK-STAT) signal transduction pathway (20). JAK2 phos- of cDNA in each sample. The specificity of the amplified phorylates STAT5, which is internalized into the nucleus to product was monitored using its melting curve. The primers induce the transcription of specific genes (21). The proliferation used in the present study are as follows: Epo, forward primer, signaling pathways, including the mitogen-activated protein 5'-CCCTGTTGGTCAACTCTTCC-3' and reverse primer, kinase (MAPK) and phosphatidylinositol 3-kinase-Ak-thymoma 5'-GTGTACAGCTTCAGCTTTCC-3'; EpoR, forward primer, (PI3K-AKT) pathways, which are involved in tumor prolifera- 5'-GCACCGAGTGTGTGCTGAGCAA-3' and reverse tion, are also activated (22). A previous study demonstrated that primer, 5'-GGTCAGCAGCACCAGGATGAC-3'; GAPDH, EpoR was expressed in kidney tissues removed by nephrectomy forward primer, 5'-ATTGCCCTCAACGACCACTT-3' and or in renal cell carcinoma (RCC) cell lines, and that the prolif- reverse primer, 5'-AGGTCCACCACCCTGTTGCT-3' (26,27) eration rate of these cells increased with the addition of Epo in a dose-dependent manner (23). Furthermore, RCC patients Cell proliferation assay [water‑soluble tetrazolium salt‑1 with high expression levels of EpoR in the kidney tissues and (WST‑1) assay]. Cell proliferation under normoxic or hypoxic high serum Epo (s-Epo) concentrations (>30 mU/ml) had better conditions was measured using Cell Counting Kit-8 (Wako 5-year survival rates than those with low EpoR levels and s-Epo Pure Chemicals, Osaka, Japan). Briefly, the cells were seeded concentrations (24). at a density of 1x104 per well in 96-well plates and allowed In the present study, five RCC cell lines were character- to attach overnight in appropriate serum-containing media. ized and two cell lines in which Epo and EpoR were highly The cells were rendered quiescent by starvation in serum-free expressed were identified. The comparative study of these two media for 24 h. FBS was used as a positive control at 10% of cell lines revealed that the induction of Epo may accelerate the total media volume. Various Epo doses (0.1, 0.33, 1, 3.3 and cellular proliferation in either a HIF-1α-dependent or -inde- 10 ng/ml) were then added and incubation was continued for a pendent manner. The possibility of using Epo as a promising further 24 h (28). The cellular proliferation was measured by target for anti-RCC drugs was also addressed. adding a 1/10 volume of WST-1. Following a 1-h incubation with WST-1, the absorbance at 450 nm was recorded using a Material and methods plate reader (Bio-Rad, Hercules, CA, USA). Materials. The monoclonal anti-HIF-1α antibody was purchased HIF‑1α induction. For HIF-1α induction, the cells were incu- from Invitrogen (Carlsbad, CA, USA) and the anti-Epo antibody bated with 125 µM of cobalt chloride (CoCl2) or were cultured was purchased from Santa Cruz Biotechnology, Inc. (Santa in a hypoxia chamber (Stemcell Technologies, Vancouver, Cruz, CA, USA). The siRNAs against HIF-1α or Epo were Canada), which was adjusted to 5% O2 and 5% CO2 for the purchased from Sigma-Aldrich (St. Louis, MO, USA). period of interest. Cell culture. The 769P (renal cell adenocarcinoma), 786O Western blot analysis. The cells were harvested in lysis (renal cell adenocarcinoma) and Ku19/20 cells (RCC) were buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, maintained in RPMI-1640. The SKRC44 cells (RCC) were 5 mM ethylenediaminetetraacetic acid (EDTA), 1% (w/v) maintained in Dulbecco's modified Eagle's medium (DMEM) Nonidet P‑40, 10% (w/v) glycerol, 5 mM sodium pyrophosphate, and the Caki-1 cells (RCC) were grown in minimal essential 10 mM sodium fluoride, 1 mM sodium orthovanadate, 10 mM medium (MEM) in a humidified atmosphere containing 5% β‑glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ CO2 in the air (normoxia) (25). Unless stated otherwise, the ml aprotinin, 5 µg/ml leupeptin, and 1 mM dithiothreitol. In total, media were supplemented with 10% fetal bovine serum (FBS), five micrograms of the cellular proteins (50 µg for Epo detec- 100 U/ml penicillin and 100 µg/ml streptomycin. The study tion) were electrophoresed and blotted onto a polyvinylidene was approved by the Ethics Committee of Osaka Medical difluouride (PVDF) membrane (FluoroTrans, Pall, Pensacola, College, Takatsuki, Osaka, Japan (569-0801). FL, USA). Subsequent to blocking with 5% skimmed milk in Tris-buffered saline (TBS; 25 mM Tris, 0.15 M NaCl, pH 7.4) Real‑time PCR. Total RNA was prepared from each of the containing 0.1% Tween‑20 (TBS‑T) for 1 h, the membrane was five cell lines using TRIzol reagent (Invitrogen), according to incubated with a 1:1000 dilution of each antibody for a further the manufacturer's instructions. Real-time PCR analyses were hour. The membrane was washed three times with TBS-T for performed using a Thermal Cycler Dice Real Time System 10 min each, then the second antibody was added at a dilution of (Takara Bio, Shiga, Japan).
Load more

Recommended publications
  • Targeted Erythropoietin Selectively Stimulates Red Blood Cell Expansion in Vivo
    Targeted erythropoietin selectively stimulates red blood cell expansion in vivo Devin R. Burrilla, Andyna Verneta, James J. Collinsa,b,c,d, Pamela A. Silvera,e,1, and Jeffrey C. Waya aWyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115; bSynthetic Biology Center, Massachusetts Institute of Technology, Cambridge, MA 02139; cInstitute for Medical Engineering & Science, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; dBroad Institute of MIT and Harvard, Cambridge, MA 02139; and eDepartment of Systems Biology, Harvard Medical School, Boston, MA 02115 Edited by Ronald A. DePinho, University of Texas MD Anderson Cancer Center, Houston, TX, and approved March 30, 2016 (received for review December 23, 2015) The design of cell-targeted protein therapeutics can be informed peptide linker that permits simultaneous binding of both elements by natural protein–protein interactions that use cooperative phys- to the same cell surface. The targeting element anchors the mu- ical contacts to achieve cell type specificity. Here we applied this tated activity element to the desired cell surface (Fig. 1A, Middle), approach in vivo to the anemia drug erythropoietin (EPO), to direct thereby creating a high local concentration and driving receptor its activity to EPO receptors (EPO-Rs) on red blood cell (RBC) pre- binding despite the mutation (Fig. 1A, Bottom). Off-target sig- cursors and prevent interaction with EPO-Rs on nonerythroid cells, naling should be minimal (Fig. 1B) and should decrease in pro- such as platelets. Our engineered EPO molecule was mutated to portion to the mutation strength. weaken its affinity for EPO-R, but its avidity for RBC precursors Here we tested the chimeric activator strategy in vivo using was rescued via tethering to an antibody fragment that specifi- erythropoietin (EPO) as the drug to be targeted.
    [Show full text]
  • Immunohistochemical Expression of Erythropoietin in Invasive Breast Carcinoma with Metastasis to Lymph Nodes
    Review and Research on Cancer Treatment Volume 4, Issue 1 (2018) ISSN 2544-2147 Immunohistochemical expression of erythropoietin in invasive breast carcinoma with metastasis to lymph nodes Maksimiuk M.* Students' Scientific Organization at the Medical University of Warsaw, Oczki 1a, 02-007 Warsaw Sobiborowicz A. Students' Scientific Organization at the Medical University of Warsaw, Oczki 1a, 02-007 Warsaw Sobieraj M. Students' Scientific Organization at the Medical University of Warsaw, Oczki 1a, 02-007 Warsaw Liszcz A. Students' Scientific Organization at the Medical University of Warsaw, Oczki 1a, 02-007 Warsaw Sobol M. Department of Biophysics and Human Physiology, Medical University of Warsaw, Chałubińskiego 5, 02-004 Warsaw Patera J. Department of Pathomorphology, Military Institute of Health Services, Warsaw, Poland Badowska-Kozakiewicz A.M. Department of Biophysics and Human Physiology, Medical University of Warsaw, Chałubińskiego 5, 02-004 Warsaw *corresponding author: Marta Maksimiuk, Department of Biophysics and Human Physiology, Medical University of Warsaw, Chałubińskiego 5, 02-004 Warsaw, [email protected], tel: 691 – 230 – 268 Abstract: Introduction: Tumor characteristics, such as size, lymph node status, histological type of the neoplasm and its grade, are well known prognostic factors in breast cancer. The ongoing search for new prognostic factors include Bcl2, Bax, Cox-2 or HIF1- alpha, which plays a key role in phenomenon of tumor hypoxia and might induce transcription of the EPO gene. Erythropoietin may influence lymph node metastasis or stimulate tumor progression, thus it seemed interesting to determine its expression in invasive breast cancers with lymph node metastases presenting different basic immunohistochemical profiles (ER, PR, HER2). Aim: To evaluate the relationship between histological grade, tumor size, lymph node status, expression of ER, PR, HER2 and immunohistochemical expression of erythropoietin in invasive breast cancer with metastasis to lymph nodes.
    [Show full text]
  • The Thrombopoietin Receptor : Revisiting the Master Regulator of Platelet Production
    This is a repository copy of The thrombopoietin receptor : revisiting the master regulator of platelet production. White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/175234/ Version: Published Version Article: Hitchcock, Ian S orcid.org/0000-0001-7170-6703, Hafer, Maximillian, Sangkhae, Veena et al. (1 more author) (2021) The thrombopoietin receptor : revisiting the master regulator of platelet production. Platelets. pp. 1-9. ISSN 0953-7104 https://doi.org/10.1080/09537104.2021.1925102 Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. More information and the full terms of the licence here: https://creativecommons.org/licenses/ Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ Platelets ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/iplt20 The thrombopoietin receptor: revisiting the master regulator of platelet production Ian S. Hitchcock, Maximillian Hafer, Veena Sangkhae & Julie A. Tucker To cite this article: Ian S. Hitchcock, Maximillian Hafer, Veena Sangkhae & Julie A. Tucker (2021): The thrombopoietin receptor: revisiting the master regulator of platelet production, Platelets, DOI: 10.1080/09537104.2021.1925102 To link to this article: https://doi.org/10.1080/09537104.2021.1925102 © 2021 The Author(s).
    [Show full text]
  • As a Key Regulator of Erythropoiesis, Bone Remodeling and Endothelial
    cells Review Erythropoietin (EPO) as a Key Regulator of Erythropoiesis, Bone Remodeling and Endothelial Transdifferentiation of Multipotent Mesenchymal Stem Cells (MSCs): Implications in Regenerative Medicine Asterios S. Tsiftsoglou Laboratory of Pharmacology, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; [email protected] Abstract: Human erythropoietin (EPO) is an N-linked glycoprotein consisting of 166 aa that is pro- duced in the kidney during the adult life and acts both as a peptide hormone and hematopoietic growth factor (HGF), stimulating bone marrow erythropoiesis. EPO production is activated by hypoxia and is regulated via an oxygen-sensitive feedback loop. EPO acts via its homodimeric erythropoietin receptor (EPO-R) that increases cell survival and drives the terminal erythroid mat- uration of progenitors BFU-Es and CFU-Es to billions of mature RBCs. This pathway involves the activation of multiple erythroid transcription factors, such as GATA1, FOG1, TAL-1, EKLF and BCL11A, and leads to the overexpression of genes encoding enzymes involved in heme biosynthesis Citation: Tsiftsoglou, A.S. and the production of hemoglobin. The detection of a heterodimeric complex of EPO-R (consist- Erythropoietin (EPO) as a Key ing of one EPO-R chain and the CSF2RB β-chain, CD131) in several tissues (brain, heart, skeletal Regulator of Erythropoiesis, Bone muscle) explains the EPO pleotropic action as a protection factor for several cells, including the Remodeling and Endothelial multipotent MSCs as well as cells modulating the innate and adaptive immunity arms. EPO induces Transdifferentiation of Multipotent the osteogenic and endothelial transdifferentiation of the multipotent MSCs via the activation of Mesenchymal Stem Cells (MSCs): EPO-R signaling pathways, leading to bone remodeling, induction of angiogenesis and secretion Implications in Regenerative of a large number of trophic factors (secretome).
    [Show full text]
  • Correlation with Vasculogenic Mimicry and Poor Prognosis
    Int J Clin Exp Pathol 2015;8(4):4033-4043 www.ijcep.com /ISSN:1936-2625/IJCEP0006586 Original Article Erythropoietin and erythropoietin receptor in hepatocellular carcinoma: correlation with vasculogenic mimicry and poor prognosis Zhihong Yang1,2*, Baocun Sun1,2,3*, Xiulan Zhao1,2*, Bing Shao1, Jindan An1, Qiang Gu1,2, Yong Wang1, Xueyi Dong1, Yanhui Zhang1,3, Zhiqiang Qiu1,3 1Department of Pathology, Tianjin Medical University, Tianjin 300070, China; 2Department of Pathology, General Hospital of Tianjin Medical University, Tianjin 300052, China; 3Department of Pathology, Cancer Hospital of Tianjin Medical University, Tianjin 300060, China. *Equal contributors. Received February 1, 2015; Accepted March 30, 2015; Epub April 1, 2015; Published April 15, 2015 Abstract: To evaluate erythropoietin (Epo) and erythropoietin receptor (EpoR) expression, its relationship with vas- culogenic mimicry (VM) and its prognostic value in human hepatocellular carcinoma (HCC), we examined Epo/EpoR expression and VM formation using immunohistochemistry and CD31/PAS (periodic acid-Schiff) double staining on 92 HCC specimens. The correlation between Epo/EpoR expression and VM formation was analyzed using two-tailed Chi-square test and Spearman correlation analysis. Survival curves were generated using Kaplan-Meier method. Multivariate analysis was performed using Cox regression model to assess the prognostic values. Results showed positive correlation between Epo/EpoR expression and VM formation (P < 0.05). Patients with Epo or EpoR expres- sion exhibited poorer overall survival (OS) than Epo-negative or EpoR-negative patients (P < 0.05). Epo-positive/VM- positive and EpoR-positive/VM-positive patients had the worst OS (P < 0.05). In multivariate survival analysis, age, Epo and EpoR were independent prognostic factors related to OS.
    [Show full text]
  • Investigation of the Protective Effect of Erythropoietin on Spinal Cord Injury in Rats
    EXPERIMENTAL AND THERAPEUTIC MEDICINE 2: 837-841, 2011 Investigation of the protective effect of erythropoietin on spinal cord injury in rats ZHENGHUA HONG, HUAXING HONG, HAIXIAO CHEN, ZHANGFU WANG and DUN HONG Department of Orthopedics, Taizhou Hospital of Zhejiang Province, Taizhou 317000, P.R. China Received May 2, 2011; Accepted June 6, 2011 DOI: 10.3892/etm.2011.285 Abstract. Erythropoietin (EPO) is a promising therapeutic cell apoptosis occurs at the lesion site, affecting both neurons agent used in a variety of spinal cord injuries. Therefore, iden- and astrocytes (1,2) It has long been believed that the central tifying the specific molecular pathway mediating the neuronal nervous system (CNS) is incapable of regenerating itself after protective effect of EPO after spinal cord injury (SCI) is of injury and during the repair process after SCI. Thus, an ideal great value to the patients concerned. Platelet-derived growth treatment would be one administered systemically but without factor (PDGF)-B is an important factor in the recovery of significant side effects. Erythropoietin( EPO), a potent inhibitor neurological function. We explored changes in the expression of apoptosis and a promising therapeutic agent, has been used of PDGF-B in spinal cord injury rats after receiving EPO treat- in a variety of neurological insults, including traumatic brain ment. We used a weight-drop contusion SCI model, and EPO injury, brain stroke and spinal cord injury (3,4). Therefore, treatment group rats received single doses ofEPO (1,000 U/ identifying the specific molecular pathway mediating the EPO kg i.p.) immediately after the operation. Seven days after the neuronal protective effects of EPO after SCI is of great value operation, the results revealed a more rapid recovery as noted to the patients concerned.
    [Show full text]
  • Erythropoietin and Cancer: the Unintended Consequences of Anemia Correction
    REVIEW ARTICLE published: 11 November 2014 doi: 10.3389/fimmu.2014.00563 Erythropoietin and cancer: the unintended consequences of anemia correction Nataša Debeljak 1*, Peter Solár 2 and Arthur J. Sytkowski 3 1 Faculty of Medicine, Institute of Biochemistry, University of Ljubljana, Ljubljana, Slovenia 2 Department of Cell and Molecular Biology, Institute of Biology and Ecology, Faculty of Sciences, Pavol Jozef Šafárik University, Košice, Slovakia 3 Oncology Therapeutic Area, Quintiles Transnational, Arlington, MA, USA Edited by: Until 1990, erythropoietin (EPO) was considered to have a single biological purpose and Pietro Ghezzi, Brighton and Sussex action, the stimulation of red blood cell growth and differentiation. Slowly, scientific and Medical School, UK medical opinion evolved, beginning with the discovery of an effect on endothelial cell Reviewed by: Ralf Schindler, Charité, Germany growth in vitro and the identification of EPO receptors (EPORs) on neuronal cells. We Domenico Ribatti, University of Bari now know that EPO is a pleiotropic growth factor that exhibits an anti-apoptotic action on Medical School, Italy numerous cells and tissues, including malignant ones. In this article, we present a short Chris Thiemermann, Queen Mary discussion of EPO, receptors involved in EPO signal transduction, and their action on non- University of London, UK hematopoietic cells. This is followed by a more detailed presentation of both pre-clinical *Correspondence: Nataša Debeljak, Faculty of Medicine, and clinical data that demonstrate EPO’s action on cancer cells, as well as tumor angio- Institute of Biochemistry, University genesis and lymphangiogenesis. Clinical trials with reported adverse effects of chronic of Ljubljana, Vrazov trg 2, SI-1000 erythropoiesis-stimulating agents (ESAs) treatment as well as clinical studies exploring Ljubljana, Slovenia the prognostic significance of EPO and EPOR expression in cancer patients are reviewed.
    [Show full text]
  • Red Blood Cell Microrheological Effects of Some Antitumor Chemotherapy Drugs: in Vitro Study
    Journal of Cellular Biotechnology 1 (2015/2016) 151–158 151 DOI 10.3233/JCB-15015 IOS Press Red blood cell microrheological effects of some antitumor chemotherapy drugs: In vitro study A.V. Muravyova,∗, I.A. Tikhomirovaa, N.V. Kislovb and A.S. Petrochenkoa aState Pedagogical University, Yaroslavl, Russia bRegional Oncological Hospital, Yaroslavl, Russia Abstract. In consequence of limitation of capillary dilatation reserve the role of erythrocyte membrane elasticity and their microrheological properties is very important for the tissue perfusion. The drugs that enter blood flow could be as signaling molecules and their action can lead to alteration of red blood cell aggregation and whole cell deformability. Therefore the subject of this study was to estimate the effect of some antitumor chemotherapy drugs on red blood cell aggregation (RBCA) and deformability (RBCD). It was found that an exposure of red blood cells (RBCs) to cisplatin and epoetin alfa led to significant positive changes in the RBC microrheological properties. It was shown that cisplatin could activate tyrosine protein kinase (TPK). Preincubation of RBCs with the inhibitor of EGF-R and Src kinase lavendustin A was accompanied by a significant decrease of the cisplatin effect. It was found RBC aggregation rise after cell incubation with 5-fluorouracil. This effect was removed with Ca2+ chelation with EGTA and also with pentoxifylline. The drug for the targeted antitumor therapy sunitinib raised RBCA markedly. Whereas RBC deformability index was altered only slightly. The combination of two chemicals: sunitinib and cisplatin led to an elimination of proaggregative effect of sunitinib. Examination of the effects of some antitumor drugs on red blood cell microrheological properties has enabled us to suggest that the observed changes in red cell aggregation and deformability may be partly related to the effects of tyrosine protein kinase activation.
    [Show full text]
  • Inactivation of Erythropoietin Receptor Function by Point Mutations in a Region Having Homology with Other Cytokine Receptors OSAMU MIURA,'T JOHN L
    MOLECULAR AND CELLULAR BIOLOGY, Mar. 1993, p. 1788-1795 Vol. 13, No. 3 0270-7306/93/031788-08$02.00/0 Copyright © 1993, American Society for Microbiology Inactivation of Erythropoietin Receptor Function by Point Mutations in a Region Having Homology with Other Cytokine Receptors OSAMU MIURA,'t JOHN L. CLEVELAND,' AND JAMES N. IHLEl,2* Department ofBiochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 31051,1 and Department ofBiochemistry, University of Tennessee, Memphis, Tennessee 3816332 Received 13 July 1992/Returned for modification 12 August 1992/Accepted 21 December 1992 The cytoplasmic domain of the erythropoietin receptor (EpoR) contains a region, proximal to the transmembrane domain, that is essential for function and has homology with other members of the cytokine receptor family. To explore the functional significance of this region and to identify critical residues, we introduced several amino acid substitutions and examined their effects on erythropoietin-induced mitogenesis, tyrosine phosphorylation, and expression of immediate-early (c-fos, c-myc, and egr-1) and early (ornithine decarboxylase and T-cell receptor 'y) genes in interleukin-3-dependent cell lines. Amino acid substitution of W-282, which is strictly conserved at the middle portion of the homology region, completely abolished all the functions of the EpoR. Point mutation at L-306 or E-307, both of which are in a conserved LEVL motif, drastically impaired the function of the receptor in all assays. Other point mutations, introduced into less conserved amino acid residues, did not significantly impair the function of the receptor. These results demonstrate that conserved amino acid residues in this domain of the EpoR are required for mitogenesis, stimulation of tyrosine phosphorylation, and induction of immediate-early and early genes.
    [Show full text]
  • Erythropoietin Promotes Breast Tumorigenesis Through Tumor-Initiating Cell Self-Renewal Bing Zhou,1 Jeffrey S
    Downloaded on January 20, 2014. The Journal of Clinical Investigation. More information at www.jci.org/articles/view/69804 Research article Erythropoietin promotes breast tumorigenesis through tumor-initiating cell self-renewal Bing Zhou,1 Jeffrey S. Damrauer,1 Sean T. Bailey,1 Tanja Hadzic,1 Youngtae Jeong,2 Kelly Clark,1 Cheng Fan,1 Laura Murphy,1 Cleo Y. Lee,2 Melissa A. Troester,3 C. Ryan Miller,4 Jian Jin,1,5 David Darr,1 Charles M. Perou,1 Ross L. Levine,6 Maximilian Diehn,2 and William Y. Kim1 1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. 2Department of Radiation Oncology, Cancer Institute, and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA. 3Department of Epidemiology and 4Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. 5Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, Chapel Hill, North Carolina, USA. 6Human Oncology and Pathogenesis Program, Leukemia Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. 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 investi- gated 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-initi- ating cells (TICs) and promoted TIC self renewal.
    [Show full text]
  • Erythropoietin and Growth Factors Exhibit Differential Angiogenic Potential in Mouse Heart
    in vivo 22 : 587-592 (2008) Erythropoietin and Growth Factors Exhibit Differential Angiogenic Potential in Mouse Heart OLGA PAGONOPOULOU 1, ANNA EFTHIMIADOU 1, MARIA LAMBROPOULOU 2, NIKOS PAPADOPOULOS 2 and NIKOS K. NIKOLETTOS 1 Departments of 1Physiology and 2Histology – Embryology, Democritus University of Thrace, Medical School, Alexandroupolis, 68100, Greece Abstract. Background: The possible angiogenic effect of functions of EPO, angiogenesis, the process through which recombinant human erythropoietin (rHuEpo) and several new blood vessels arise from preexisting ones, has been possibly angiogenic cytokines such as basic fibroblast growth indicated (5). The potential role of EPO in angiogenesis may factor (bFGF), acidic fibroblast growth factor (aFGF) and be considered as a subset of its possible function in improving vascular endothelial growth factor (VEGF) was investigated overall tissue oxygenation and its antiapoptotic role (5). in mouse heart. Materials and Methods: Mice were divided Recombinant human erythropoietin (rHuEPO) induces a into five groups (n=7/group): A, control; B, rHuEpo-treated; proangiogenic phenotype in human endothelial cells, including C, (aFGF-treated); D, (VEGF-treated); E, (bFGF-treated). both early ( i.e. increase in cell proliferation and matrix The antibody mouse anti-human CD31 was used to evaluate metalloproteinase-2 production) and late (differentiation into the vessels present in histological preparations. Results: The vascular tubes) angiogenic events. In vivo , in the chick embryo results show a significant increase of the vessel number per CAM assay, the angiogenic activity of rHuEPO was similar to optical field in the rHuEpo-treated, the bFGF-treated and that exerted by basic fibroblast growth factor (bFGF), a well- the VEGF-treated animals compared to controls whereas known angiogenic cytokine and endothelial cells of the CAM aFGF did not show a ny significant angiogenic activity.
    [Show full text]
  • Leukemia Inhibitory Factor Inhibits Erythropoietin-Induced Myelin Gene
    Gyetvai et al. Molecular Medicine (2018) 24:51 Molecular Medicine https://doi.org/10.1186/s10020-018-0052-3 RESEARCHARTICLE Open Access Leukemia inhibitory factor inhibits erythropoietin-induced myelin gene expression in oligodendrocytes Georgina Gyetvai, Cieron Roe, Lamia Heikal, Pietro Ghezzi* and Manuela Mengozzi Abstract Background: The pro-myelinating effects of leukemia inhibitory factor (LIF) and other cytokines of the gp130 family, including oncostatin M (OSM) and ciliary neurotrophic factor (CNTF), have long been known, but controversial results have also been reported. We recently overexpressed erythropoietin receptor (EPOR) in rat central glia-4 (CG4) oligodendrocyte progenitor cells (OPCs) to study the mechanisms mediating the pro-myelinating effects of erythropoietin (EPO). In this study, we investigated the effect of co-treatment with EPO and LIF. Methods: Gene expression in undifferentiated and differentiating CG4 cells in response to EPO and LIF was analysed by DNA microarrays and by RT-qPCR. Experiments were performed in biological replicates of N ≥ 4. Functional annotation and biological term enrichment was performed using DAVID (Database for Annotation, Visualization and Integrated Discovery). The gene-gene interaction network was visualised using STRING (Search Tool for the Retrieval of Interacting Genes). Results: In CG4 cells treated with 10 ng/ml of EPO and 10 ng/ml of LIF, EPO-induced myelin oligodendrocyte glycoprotein (MOG) expression, measured at day 3 of differentiation, was inhibited ≥4-fold (N =5,P < 0.001). Inhibition of EPO-induced MOG was also observed with OSM and CNTF. Analysis of the gene expression profile of CG4 differentiating cells treated for 20 h with EPO and LIF revealed LIF inhibition of EPO-induced genes involved in lipid transport and metabolism, previously identified as positive regulators of myelination in this system.
    [Show full text]