Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Hypoxia is a distinct feature in GBM and plays a significant role in tumor progression, resistance to treatment, and poor outcome. However, there is lack of studies relating type of cell death, status of Akt phosphorylation on Ser473, mitochondrial membrane potential, and morphological changes of tumor cells after hypoxia and reoxygenation. The rat glioma C6 cell line was exposed to oxygen deprivation (OD) in 5 % fetal bovine serum (FBS) or serum-free media followed by reoxygenation (RO). OD induced apoptosis on both 5 % FBS and serum-free groups. Overall, cells on serum-free media showed more profound morphological changes than cells on 5 % FBS. Moreover, our results suggest that OD combined with absence of serum provided a favorable environment for glioblastoma dedifferentiation to cancer stem cells, since nestin, and CD133 levels increased. Reoxygenation is present in hypoxic tumors through microvessel formation and cell migration to oxygenated areas. However, few studies approach these phenomena when analyzing hypoxia. We show that RO caused morphological alterations characteristic of cells undergoing a differentiation process due to increased GFAP. In the present study, we characterized an in vitro hypoxic microenvironment associated with GBM tumors, therefore contributing with new insights for the development of therapeutics for resistant glioblastoma.
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Oike T, Suzuki Y, K-i S, Shirai K, S-e N, Tamaki T, Nagaishi M, Yokoo H, Nakazato Y, Nakano T (2013) Radiotherapy plus concomitant adjuvant temozolomide for glioblastoma: Japanese mono-institutional results. PLoS One 8(11):e78943
Sengupta S, Marrinan J, Frishman C, Sampath P (2012) Impact of temozolomide on immune response during malignant glioma chemotherapy. Clin Dev Immunol 2012
Shen G, Shen F, Shi Z, Liu W, Hu W, Zheng X, Wen L, Yang X (2008) Identification of cancer stem-like cells in the C6 glioma cell line and the limitation of current identification methods. Vitro Cellular & Developmental Biology-Animal 44(7):280–289
Knizetova P, Ehrmann J, Hlobilkova A, Vancova I, Kalita O, Kolar Z, Bartek J (2008) Autocrine regulation of glioblastoma cell-cycle progression, viability and radioresistance through the VEGF-VEGFR2 (KDR) interplay. Cell Cycle 7(16):2553–2561
Bachelder RE, Wendt MA, Mercurio AM (2002) Vascular endothelial growth factor promotes breast carcinoma invasion in an autocrine manner by regulating the chemokine receptor CXCR4. Cancer Res 62(24):7203–7206
Hamerlik P, Lathia JD, Rasmussen R, Wu Q, Bartkova J, Lee M, Moudry P, Bartek J, Fischer W, Lukas J (2012) Autocrine VEGF–VEGFR2–Neuropilin-1 signaling promotes glioma stem-like cell viability and tumor growth. J Exp Med 209(3):507–520
Li P, Zhou C, Xu L, Xiao H (2013) Hypoxia enhances stemness of cancer stem cells in glioblastoma: an in vitro study. Int J Med Sci 10(4):399–407
Karamboulas C, Ailles L (2013) Developmental signaling pathways in cancer stem cells of solid tumors. Biochimica et Biophysica Acta (BBA)-General Subjects 1830(2):2481–2495
Swamydas M, Ricci K, Rego SL, Dréau D (2013) Mesenchymal stem cell-derived CCL-9 and CCL-5 promote mammary tumor cell invasion and the activation of matrix metalloproteinases. Cell Adhes Migr 7(3):315–324
Zhou X, Wang X, Qu F, Zhong Y, Lu X, Zhao P, Wang D, Huang Q, Zhang L, Li X (2009) Detection of cancer stem cells from the C6 glioma cell line. J Int Med Res 37(2):503–510
Ye X-Q, Wang G-H, Huang G-J, Bian X-W, Qian G-S, Yu S-C (2011) Heterogeneity of mitochondrial membrane potential: a novel tool to isolate and identify cancer stem cells from a tumor mass? Stem Cell Rev Rep 7(1):153–160
Yao K, Gietema J, Shida S, Selvakumaran M, Fonrose X, Haas N, Testa J, O’Dwyer P (2005) In vitro hypoxia-conditioned colon cancer cell lines derived from HCT116 and HT29 exhibit altered apoptosis susceptibility and a more angiogenic profile in vivo. Br J Cancer 93(12):1356–1363
Strasser U, Fischer G (1995) Quantitative measurement of neuronal degeneration in organotypic hippocampal cultures after combined oxygen/glucose deprivation. J Neurosci Methods 57(2):177–186
Cimarosti H, Rodnight R, Tavares A, Paiva R, Valentim L, Rocha E, Salbego C (2001) An investigation of the neuroprotective effect of lithium in organotypic slice cultures of rat hippocampus exposed to oxygen and glucose deprivation. Neurosci Lett 315(1):33–36
Keith B, Simon MC (2007) Hypoxia-inducible factors, stem cells, and cancer. Cell 129(3):465–472
Li Z, Bao S, Wu Q, Wang H, Eyler C, Sathornsumetee S, Shi Q, Cao Y, Lathia J, McLendon RE (2009) Hypoxia-inducible factors regulate tumorigenic capacity of glioma stem cells. Cancer Cell 15(6):501–513
Zagzag D, Lukyanov Y, Lan L, Ali MA, Esencay M, Mendez O, Yee H, Voura EB, Newcomb EW (2006) Hypoxia-inducible factor 1 and VEGF upregulate CXCR4 in glioblastoma: implications for angiogenesis and glioma cell invasion. Lab Investig 86(12):1221–1232
Axelson H, Fredlund E, Ovenberger M, Landberg G, Påhlman S (2005) Hypoxia-induced dedifferentiation of tumor cells—a mechanism behind heterogeneity and aggressiveness of solid tumors. In: Seminars in cell & developmental biology, vol 4. Elsevier, pp. 554–563
Nagaraj NS, Vigneswaran N, Zacharias W (2004) Hypoxia-mediated apoptosis in oral carcinoma cells occurs via two independent pathways. Mol Cancer 3(1):1
Harris AL (2002) Hypoxia—a key regulatory factor in tumour growth. Nat Rev Cancer 2(1):38–47
Weinmann M, Jendrossek V, Handrick R, Güner D, Goecke B, Belka C (2004) Molecular ordering of hypoxia-induced apoptosis: critical involvement of the mitochondrial death pathway in a FADD/caspase-8 independent manner. Oncogene 23(21):3757–3769
Leszczynska KB, Foskolou IP, Abraham AG, Anbalagan S, Tellier C, Haider S, Span PN, O’Neill EE, Buffa FM, Hammond EM (2015) Hypoxia-induced p53 modulates both apoptosis and radiosensitivity via AKT. J Clin Invest 125(6):2385–2398
Asai A, Miyagi Y, Sugiyama A, Gamanuma M, Hong SI, Takamoto S, Nomura K, Matsutani M, Takakura K, Kuchino Y (1994) Negative effects of wild-type p53 and s-Myc on cellular growth and tumorigenicity of glioma cells. J Neuro-Oncol 19(3):259–268
Zenali MJ, Tan D, Li W, Dhingra S, Brown RE (2010) Stemness characteristics of fibrolamellar hepatocellular carcinoma: immunohistochemical analysis with comparisons to conventional hepatocellular carcinoma. Annals of Clinical & Laboratory Science 40(2):126–134
Li Q, Rycaj K, Chen X, Tang DG (2015) Cancer stem cells and cell size: a causal link? In: Seminars in cancer biology. Elsevier, pp. 191–199
Murayama A, Matsuzaki Y, Kawaguchi A, Shimazaki T, Okano H (2002) Flow cytometric analysis of neural stem cells in the developing and adult mouse brain. J Neurosci Res 69(6):837–847
Rietze RL, Valcanis H, Brooker GF, Thomas T, Voss AK, Bartlett PF (2001) Purification of a pluripotent neural stem cell from the adult mouse brain. Nature 412(6848):736–739
Narayanan G, Poonepalli A, Chen J, Sankaran S, Hariharan S, Yu YH, Robson P, Yang H, Ahmed S (2012) Single-cell mRNA profiling identifies progenitor subclasses in neurospheres. Stem Cells Dev 21(18):3351–3362
J-j D, Qiu W, Xu S-l, Wang B, X-z Y, Y-f P, Zhang X, X-w B, Yu S-c (2013) Strategies for isolating and enriching cancer stem cells: well begun is half done. Stem Cells Dev 22(16):2221–2239
Zheng X, Shen G, Yang X, Liu W (2007) Most C6 cells are cancer stem cells: evidence from clonal and population analyses. Cancer Res 67(8):3691–3697
Michelakis E, Sutendra G, Dromparis P, Webster L, Haromy A, Niven E, Maguire C, Gammer T-L, Mackey J, Fulton D (2010) Metabolic modulation of glioblastoma with dichloroacetate. Sci Transl Med 2(31):31ra34
Schieke SM, Ma M, Cao L, McCoy JP, Liu C, Hensel NF, Barrett AJ, Boehm M, Finkel T (2008) Mitochondrial metabolism modulates differentiation and teratoma formation capacity in mouse embryonic stem cells. J Biol Chem 283(42):28506–28512
Seton-Rogers S (2011) Cancer stem cells: VEGF promotes stemness. Nat Rev Cancer 11(12):831–831
Yuan X, Curtin J, Xiong Y, Liu G, Waschsmann-Hogiu S, Farkas DL, Black KL, John SY (2004) Isolation of cancer stem cells from adult glioblastoma multiforme. Oncogene 23(58):9392–9400
Galli R, Binda E, Orfanelli U, Cipelletti B, Gritti A, De Vitis S, Fiocco R, Foroni C, Dimeco F, Vescovi A (2004) Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res 64(19):7011–7021
Sakaki T, Yamada K, Otsuki H, Yuguchi T, Kohmura E, Hayakawa T (1995) Brief exposure to hypoxia induces bFGF mRNA and protein and protects rat cortical neurons from prolonged hypoxic stress. Neurosci Res 23(3):289–296
Kyurkchiev D (2014) Cancer stem cells from glioblastoma multiforme: culturing and phenotype. Stem Cells 2(1):3
Freyhaus H, Dagnell M, Leuchs M, Vantler M, Berghausen EM, Caglayan E, Weissmann N, Dahal BK, Schermuly RT, Östman A (2011) Hypoxia enhances platelet-derived growth factor signaling in the pulmonary vasculature by down-regulation of protein tyrosine phosphatases. Am J Respir Crit Care Med 183(8):1092–1102
Segovia J, Lawless GM, Tillakaratne NJ, Brenner M, Tobin AJ (1994) Cyclic AMP decreases the expression of a neuronal marker (GAD67) and increases the expression of an astroglial marker (GFAP) in C6 cells. J Neurochem 63(4):1218–1225
This study was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS).
Mariana Maier Gaelzer and Mariana Silva dos Santos contributed equally to this work.
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Morphological changes of C6 glioma cells after oxygen-deprivation (OD) on serum-free medium and reoxygenation (RO) in 5 % fetal bovine serum. (PDF 19 kb)
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Gaelzer, M.M., Santos, M.S.d., Coelho, B.P. et al. Hypoxic and Reoxygenated Microenvironment: Stemness and Differentiation State in Glioblastoma. Mol Neurobiol 54, 6261–6272 (2017). https://doi.org/10.1007/s12035-016-0126-6
- Cancer stem cell