Hypoxia differentially upregulates the expression of embryonic, fetal and adult hemoglobin in human glioblastoma cells

Emara,Marwan; Turner,A. Robert; Allalunis-Turner,Joan

doi:10.3892/ijo.2013.2239

Hypoxia differentially upregulates the expression of embryonic, fetal and adult hemoglobin in human glioblastoma cells

Authors:
- Marwan Emara
- A. Robert Turner
- Joan Allalunis-Turner
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Affiliations: Department of Oncology, University of Alberta and Alberta Health Services, Cross Cancer Institute, Edmonton, AΒ T6G 1Z2, Canada
Published online on: December 31, 2013 https://doi.org/10.3892/ijo.2013.2239
Pages: 950-958

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Abstract

Hemoglobin is produced mainly in erythroid cells. However, it has been reported in non-erythroid cells of human and rodents. We have shown previously that neuroglobin, cytoglobin and hemoglobin are expressed in human glioblastoma multiforme (GBM) cells. We sought to determine whether hemoglobin expression is upregulated by hypoxia, and whether its expression is restricted to the cancer stem cell populations in different GBM cell lines or GBM brain tumor initiating cells (BTICs). Flow cytometry, magnetic cell sorting and qRT-PCR were used to examine the hypoxic upregulation of hemoglobins as well as erythropoietin (EPO) and erythropoietin receptor (EPOR) in GBM cell lines (M006x, M059J, M059K, U87R and U87T) and GBM-BTICs. The data showed significantly increased expression in globins (α, β, γ, δ, ζ and ε), EPO and EPOR mRNA levels under hypoxia. Globin expression is not limited to the stem cell populations or GBM-BTICs but is a property of the entire GBM population. We assumed that the total expression of mRNA of different normalized globins (α, β, γ, δ, ζ and ε) at different time‑points for the same cell line is 100%. Under aerobic conditions, ε globin was predominantly expressed, and then decreased gradually with increasing time in hypoxia. This was coupled to a concomitant increase in α and γ globins. Our findings suggest that hypoxic upregulation of hemoglobin expression in GBM cells may be a part of a repertoire of active defence and adaptation mechanisms enabling these cells to acquire resistance to aggressive multimodality treatments of chemotherapy and radiotherapy. New therapeutic strategies to interfere with hemoglobin expression or function in GBM cells are required.

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1.	Egawa T and Yeh SR: Structural and functional properties of hemoglobins from unicellular organisms as revealed by resonance Raman spectroscopy. J Inorg Biochem. 99:72–96. 2005. View Article : Google Scholar : PubMed/NCBI
2.	Wajcman H, Kiger L and Marden MC: Structure and function evolution in the superfamily of globins. C R Biol. 332:273–282. 2009. View Article : Google Scholar : PubMed/NCBI
3.	Pesce A, Bolognesi M, Bocedi A, et al: Neuroglobin and cytoglobin Fresh blood for the vertebrate globin family. EMBO Rep. 3:1146–1151. 2002. View Article : Google Scholar : PubMed/NCBI
4.	Schelshorn DW, Schneider A, Kuschinsky W, et al: Expression of hemoglobin in rodent neurons. J Cereb Blood Flow Metab. 29:585–595. 2009. View Article : Google Scholar : PubMed/NCBI
5.	Ohyagi Y, Yamada T and Goto I: Hemoglobin as a novel protein developmentally regulated in neurons. Brain Res. 635:323–327. 1994. View Article : Google Scholar : PubMed/NCBI
6.	Biagioli M, Pinto M, Cesselli D, et al: Unexpected expression of alpha- and beta-globin in mesencephalic dopaminergic neurons and glial cells. Proc Natl Acad Sci USA. 106:15454–15459. 2009. View Article : Google Scholar : PubMed/NCBI
7.	Dassen H, Kamps R, Punyadeera C, et al: Haemoglobin expression in human endometrium. Hum Reprod. 23:635–641. 2008. View Article : Google Scholar
8.	Liu L, Zeng M and Stamler JS: Hemoglobin induction in mouse macrophages. Proc Natl Acad Sci USA. 96:6643–6647. 1999. View Article : Google Scholar : PubMed/NCBI
9.	Wride MA, Mansergh FC, Adams S, et al: Expression profiling and gene discovery in the mouse lens. Mol Vis. 9:360–396. 2003.PubMed/NCBI
10.	Nishi H, Inagi R, Kato H, et al: Hemoglobin is expressed by mesangial cells and reduces oxidant stress. J Am Soc Nephrol. 19:1500–1508. 2008. View Article : Google Scholar : PubMed/NCBI
11.	Bhaskaran M, Chen H, Chen Z and Liu L: Hemoglobin is expressed in alveolar epithelial type II cells. Biochem Biophys Res Commun. 333:1348–1352. 2005. View Article : Google Scholar : PubMed/NCBI
12.	Newton DA, Rao KM, Dluhy RA and Baatz JE: Hemoglobin is expressed by alveolar epithelial cells. J Biol Chem. 281:5668–5676. 2006. View Article : Google Scholar : PubMed/NCBI
13.	Gorr TA, Wichmann D, Pilarsky C, et al: Old proteins - new locations: myoglobin, haemoglobin, neuroglobin and cytoglobin in solid tumours and cancer cells. Acta Physiol (Oxf). 202:563–581. 2011. View Article : Google Scholar : PubMed/NCBI
14.	Hardison RC: Globin genes on the move. J Biol. 7:352008. View Article : Google Scholar : PubMed/NCBI
15.	Bunn HF and Forget BG: Hemoglobin: Molecular, Genetic and Clinical Aspects. W.B. Saunders Co; Philadelphia, PA: 1986
16.	Cosby K, Partovi KS, Crawford JH, et al: Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nat Med. 9:1498–1505. 2003. View Article : Google Scholar : PubMed/NCBI
17.	Huang Z, Shiva S, Kim-Shapiro DB, et al: Enzymatic function of hemoglobin as a nitrite reductase that produces NO under allosteric control. J Clin Invest. 115:2099–2107. 2005. View Article : Google Scholar : PubMed/NCBI
18.	Masuoka N, Kodama H, Abe T, Wang DH and Nakano T: Characterization of hydrogen peroxide removal reaction by hemoglobin in the presence of reduced pyridine nucleotides. Biochim Biophys Acta. 1637:46–54. 2003. View Article : Google Scholar : PubMed/NCBI
19.	Gross SS and Lane P: Physiological reactions of nitric oxide and hemoglobin: a radical rethink. Proc Natl Acad Sci USA. 96:9967–9969. 1999. View Article : Google Scholar : PubMed/NCBI
20.	Tsiftsoglou AS, Vizirianakis IS and Strouboulis J: Erythropoiesis: model systems, molecular regulators, and developmental programs. IUBMB Life. 61:800–830. 2009. View Article : Google Scholar : PubMed/NCBI
21.	Rogers HM, Yu X, Wen J, Smith R, Fibach E and Noguchi CT: Hypoxia alters progression of the erythroid program. Exp Hematol. 36:17–27. 2008. View Article : Google Scholar : PubMed/NCBI
22.	Sakanaka M, Wen TC, Matsuda S, et al: In vivo evidence that erythropoietin protects neurons from ischemic damage. Proc Natl Acad Sci USA. 95:4635–4640. 1998. View Article : Google Scholar : PubMed/NCBI
23.	Tan CC, Eckardt KU, Firth JD and Ratcliffe PJ: Feedback modulation of renal and hepatic erythropoietin mRNA in response to graded anemia and hypoxia. Am J Physiol. 263:F474–F481. 1992.PubMed/NCBI
24.	Tezel G, Yang X, Luo C, et al: Hemoglobin expression and regulation in glaucoma: insights into retinal ganglion cell oxygenation. Invest Ophthalmol Vis Sci. 51:907–919. 2010. View Article : Google Scholar : PubMed/NCBI
25.	Lim SK, Llaguno SR, McKay RM and Parada LF: Glioblastoma multiforme: a perspective on recent findings in human cancer and mouse models. BMB Rep. 44:158–164. 2011. View Article : Google Scholar : PubMed/NCBI
26.	Chaudhry NS, Shah AH, Ferraro N, et al: Predictors of long-term survival in patients with glioblastoma multiforme: advancements from the last quarter century. Cancer Invest. 31:287–308. 2013. View Article : Google Scholar : PubMed/NCBI
27.	Bar EE: Glioblastoma, cancer stem cells and hypoxia. Brain Pathol. 21:119–129. 2011. View Article : Google Scholar : PubMed/NCBI
28.	Sathornsumetee S, Cao Y, Marcello JE, et al: Tumor angiogenic and hypoxic profiles predict radiographic response and survival in malignant astrocytoma patients treated with bevacizumab and irinotecan. J Clin Oncol. 26:271–278. 2008. View Article : Google Scholar
29.	Bertout JA, Patel SA and Simon MC: The impact of O₂ availability on human cancer. Nat Rev Cancer. 8:967–975. 2008.
30.	Alves TR, Lima FR, Kahn SA, et al: Glioblastoma cells: A heterogeneous and fatal tumor interacting with the parenchyma. Life Sci. 89:532–539. 2011. View Article : Google Scholar : PubMed/NCBI
31.	Emara M, Salloum N and Allalunis-Turner J: Expression and hypoxic up-regulation of neuroglobin in human glioblastoma cells. Mol Oncol. 3:45–53. 2009. View Article : Google Scholar : PubMed/NCBI
32.	Emara M, Turner AR and Allalunis-Turner J: Hypoxic regulation of cytoglobin and neuroglobin expression in human normal and tumor tissues. Cancer Cell Int. 10:332010. View Article : Google Scholar : PubMed/NCBI
33.	Fang J, Ma I and Allalunis-Turner J: Knockdown of cytoglobin expression sensitizes human glioma cells to radiation and oxidative stress. Radiat Res. 176:198–207. 2011. View Article : Google Scholar : PubMed/NCBI
34.	Emara M, Turner AR and Allalunis-Turner J: Adult, embryonic, and fetal hemoglobin are expressed in human glioblastoma cells. Int J Oncol. 44:514–520. 2014.PubMed/NCBI
35.	Allalunis-Turner MJ, Barron GM, Day RS III, Dobler KD and Mirzayans R: Isolation of two cell lines from a human malignant glioma specimen differing in sensitivity to radiation and chemotherapeutic drugs. Radiat Res. 134:349–354. 1993. View Article : Google Scholar : PubMed/NCBI
36.	Allalunis-Turner MJ, Barron GM, Day RS III, Fulton DS and Urtasun RC: Radiosensitivity testing of human primary brain tumor specimens. Int J Radiat Oncol Biol Phys. 23:339–343. 1992. View Article : Google Scholar : PubMed/NCBI
37.	Allalunis-Turner MJ, Franko AJ and Parliament MB: Modulation of oxygen consumption rate and vascular endothelial growth factor mRNA expression in human malignant glioma cells by hypoxia. Br J Cancer. 80:104–109. 1999. View Article : Google Scholar : PubMed/NCBI
38.	Parliament MB, Allalunis-Turner MJ, Franko AJ, et al: Vascular endothelial growth factor expression is independent of hypoxia in human malignant glioma spheroids and tumours. Br J Cancer. 82:635–641. 2000.PubMed/NCBI
39.	Johnston AL, Lun X, Rahn JJ, et al: The p75 neurotrophin receptor is a central regulator of glioma invasion. PLoS Biol. 5:e2122007. View Article : Google Scholar : PubMed/NCBI
40.	Koch CJ, Howell RL and Biaglow JE: Ascorbate anion potentiates cytotoxicity of nitro-aromatic compounds under hypoxic and anoxic conditions. Br J Cancer. 39:321–329. 1979. View Article : Google Scholar : PubMed/NCBI
41.	Kelly JJ, Stechishin O, Chojnacki A, et al: Proliferation of human glioblastoma stem cells occurs independently of exogenous mitogens. Stem Cells. 27:1722–1733. 2009. View Article : Google Scholar : PubMed/NCBI
42.	Mohyeldin A, Dalgard CL, Lu H, et al: Survival and invasiveness of astrocytomas promoted by erythropoietin. J Neurosurg. 106:338–350. 2007. View Article : Google Scholar : PubMed/NCBI
43.	Said HM, Hagemann C, Staab A, et al: Expression patterns of the hypoxia-related genes osteopontin, CA9, erythropoietin, VEGF and HIF-1alpha in human glioma in vitro and in vivo. Radiother Oncol. 83:398–405. 2007. View Article : Google Scholar : PubMed/NCBI
44.	Brescia P, Ortensi B, Fornasari L, Levi D, Broggi G and Pelicci G: CD133 is essential for glioblastoma stem cell maintenance. Stem Cells. 31:857–869. 2013. View Article : Google Scholar : PubMed/NCBI
45.	Singh SK, Clarke ID, Terasaki M, et al: Identification of a cancer stem cell in human brain tumors. Cancer Res. 63:5821–5828. 2003.PubMed/NCBI
46.	Liu G, Yuan X, Zeng Z, et al: Analysis of gene expression and chemoresistance of CD133⁺ cancer stem cells in glioblastoma. Mol Cancer. 5:672006. View Article : Google Scholar : PubMed/NCBI
47.	Pallini R, Ricci-Vitiani L, Montano N, et al: Expression of the stem cell marker CD133 in recurrent glioblastoma and its value for prognosis. Cancer. 117:162–174. 2011. View Article : Google Scholar : PubMed/NCBI
48.	He Y, Hua Y, Liu W, Hu H, Keep RF and Xi G: Effects of cerebral ischemia on neuronal hemoglobin. J Cereb Blood Flow Metab. 29:596–605. 2009. View Article : Google Scholar : PubMed/NCBI
49.	Rebetz J, Tian D, Persson A, et al: Glial progenitor-like phenotype in low-grade glioma and enhanced CD133-expression and neuronal lineage differentiation potential in high-grade glioma. PLoS One. 3:e19362008. View Article : Google Scholar : PubMed/NCBI
50.	Valtz NL, Hayes TE, Norregaard T, Liu SM and McKay RD: An embryonic origin for medulloblastoma. New Biol. 3:364–371. 1991.PubMed/NCBI
51.	Schechter AN: Hemoglobin research and the origins of molecular medicine. Blood. 112:3927–3938. 2008. View Article : Google Scholar : PubMed/NCBI
52.	Crawford MJ and Goldberg DE: Regulation of the Salmonella typhimurium flavohemoglobin gene. A new pathway for bacterial gene expression in response to nitric oxide. J Biol Chem. 273:34028–34032. 1998.
53.	Richter F, Meurers BH, Zhu C, Medvedeva VP and Chesselet MF: Neurons express hemoglobin alpha- and beta-chains in rat and human brains. J Comp Neurol. 515:538–547. 2009. View Article : Google Scholar : PubMed/NCBI
54.	Acs G, Acs P, Beckwith SM, et al: Erythropoietin and erythropoietin receptor expression in human cancer. Cancer Res. 61:3561–3565. 2001.PubMed/NCBI
55.	Batra S, Perelman N, Luck LR, Shimada H and Malik P: Pediatric tumor cells express erythropoietin and a functional erythropoietin receptor that promotes angiogenesis and tumor cell survival. Lab Invest. 83:1477–1487. 2003. View Article : Google Scholar : PubMed/NCBI
56.	Marti HH, Wenger RH, Rivas LA, et al: Erythropoietin gene expression in human, monkey and murine brain. Eur J Neurosci. 8:666–676. 1996. View Article : Google Scholar : PubMed/NCBI
57.	Olive PL, Trotter T, Banath JP, Jackson SM and Le Riche J: Heterogeneity in human tumour hypoxic fraction using the comet assay. Br J Cancer (Suppl). 27:S191–S195. 1996.PubMed/NCBI
58.	Vaupel P, Schlenger K, Knoop C and Hockel M: Oxygenation of human tumors: evaluation of tissue oxygen distribution in breast cancers by computerized O₂ tension measurements. Cancer Res. 51:3316–3322. 1991.PubMed/NCBI
59.	Foley RN: Erythropoietin: physiology and molecular mechanisms. Heart Fail Rev. 13:405–414. 2008. View Article : Google Scholar : PubMed/NCBI
60.	Jelkmann W, Bohlius J, Hallek M and Sytkowski AJ: The erythropoietin receptor in normal and cancer tissues. Crit Rev Oncol Hematol. 67:39–61. 2008. View Article : Google Scholar : PubMed/NCBI
61.	Milano M and Collomp R: Erythropoietin and neuroprotection: a therapeutic perspective. J Oncol Pharm Pract. 11:145–149. 2005. View Article : Google Scholar : PubMed/NCBI
62.	Yasuda Y, Fujita Y, Matsuo T, et al: Erythropoietin regulates tumour growth of human malignancies. Carcinogenesis. 24:1021–1029. 2003. View Article : Google Scholar : PubMed/NCBI
63.	Yin D, Kawabata H, Tcherniamtchouk O, Huynh T, Black KL and Koeffler HP: Glioblastoma multiforme cells: expression of erythropoietin receptor and response to erythropoietin. Int J Oncol. 31:1193–1198. 2007.PubMed/NCBI
64.	Hassouna I, Sperling S, Kim E, et al: Erythropoietin augments survival of glioma cells after radiation and temozolomide. Int J Radiat Oncol Biol Phys. 72:927–934. 2008. View Article : Google Scholar : PubMed/NCBI
65.	Wood WG: Haemoglobin synthesis during human fetal development. Br Med Bull. 32:282–287. 1976.PubMed/NCBI
66.	Allen DW and Jandl JH: Factors influencing relative rates of synthesis of adult and fetal hemoglobin in vitro. J Clin Invest. 39:1107–1113. 1960. View Article : Google Scholar : PubMed/NCBI
67.	Thomas ED, Lochte HL Jr, Greenough WB III and Wales M: In vitro synthesis of foetal and adult haemoglobin by foetal haematopoietic tissues. Nature. 185:396–397. 1960. View Article : Google Scholar : PubMed/NCBI
68.	Seidel S, Garvalov BK, Wirta V, et al: A hypoxic niche regulates glioblastoma stem cells through hypoxia inducible factor 2 alpha. Brain. 133:983–995. 2010. View Article : Google Scholar : PubMed/NCBI
69.	Soeda A, Park M, Lee D, et al: Hypoxia promotes expansion of the CD133-positive glioma stem cells through activation of HIF-1alpha. Oncogene. 28:3949–3959. 2009. View Article : Google Scholar : PubMed/NCBI
70.	Bao S, Wu Q, McLendon RE, et al: Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature. 444:756–760. 2006. View Article : Google Scholar : PubMed/NCBI
71.	Tamura K, Aoyagi M, Wakimoto H, et al: Accumulation of CD133-positive glioma cells after high-dose irradiation by Gamma Knife surgery plus external beam radiation. J Neurosurg. 113:310–318. 2010. View Article : Google Scholar : PubMed/NCBI
72.	Ma YH, Mentlein R, Knerlich F, Kruse ML, Mehdorn HM and Held-Feindt J: Expression of stem cell markers in human astrocytomas of different WHO grades. J Neurooncol. 86:31–45. 2008. View Article : Google Scholar : PubMed/NCBI
73.	Sankaran VG: Targeted therapeutic strategies for fetal hemoglobin induction. Hematology Am Soc Hematol Educ Program. 2011:459–465. 2011. View Article : Google Scholar : PubMed/NCBI
74.	Witt O, Monkemeyer S, Ronndahl G, et al: Induction of fetal hemoglobin expression by the histone deacetylase inhibitor apicidin. Blood. 101:2001–2007. 2003. View Article : Google Scholar : PubMed/NCBI