Radiosensitivity of glioblastoma multiforme and astrocytic cell lines in cell signalling aspects

Abstract

Objectives: The aim of this study is to investigate the radiosensitivity of Glioblastoma multiforme (GBM; U87 MG) and astrocyte (SVG p12) cell lines in vitro through the signalling pathways.

Methods: GBM and astrocytes were treated with 2, 4, 6, and 8 gray of ionized radiation, followed by a clonogenic assay. The effective dose of radiation was determined as 2 gray. Immunofluorescence technics selected to analyse the macrophage migration inhibiting factor (MIF), nuclear factor of activated T-cells cytoplasmic 2 (NFATc2), osteopontin (OPN), mammalian target of rapamycin (mTOR) and stage-specific embryonic antigen-1 (SSEA-1). Additionally, p53 and cell cycle assays were performed.

Results: On day 1, astrocytes showed decreased expression of MIF, OPN and mTOR and increased expression of SSEA-1 in the test group after 2 gray radiation. GBM showed decreased expression of p53 and mTOR, but increased expression of NFATc2. The results of MIF expression were found higher in GBM compared to astrocytes on day 1. Interestingly, on day 12, increased expression of SSEA-1, OPN and p53 were observed in both cell lines’ test groups. Further analysis showed that all control groups of GBM and astrocytes were significantly accumulated in the S phase. After radiotherapy application, percentage of GBM in G0/G1 phases and especially in G2/M phases increased; conversely, in the S phase it decreased. Moreover, percentage of astrocytes increased in the S phase and decreased in G0/G1 phases and in G2/M phases.

Conclusions: This combination of findings suggests that as a result of the radiotherapy effect, GBM started to accumulate on check points. The central question in this study focused on changes in molecular protein expression in cancer cells after radiotherapy, particularly key signalling pathways of tumorigenesis and a new possible point of view for treating such diseases.

Keywords

• Glioblastoma multiforme
• cell cycle
• radiosensitivity
• cell signalling

References

1. 1. Van Meir EG, Hadjipanayis CG, Norden AD, Shu HK, Wen PY, Olson JJ. Exciting new advances in neuro-oncology: the avenue to a cure for malignant glioma. CA Cancer J Clin 2010;60:166-93.
2. 2. Louis DN, Perry A, Wesseling P, Brat DJ, Cree IA, Figarella-Branger D, et al. The 2021 WHO classification of tumors of the central nervous system: a summary. Neuro Oncol 2021;23:1231-51.
3. 3. Stupp R, Hegi ME, Mason WP, van der Bent MJ, Taphoorn MJB, Janzer RC, et al. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 2009;10:459-66.
4. 4. Lee YW, Cho HJ, Lee WH, Sonntag WE. Whole brain radiation-induced cognitive impairment: pathophysiological mechanisms and therapeutic targets. Biomol Ther (Seoul) 2012;20:357-70.
5. 5. Jackson M, Hassiotou F, Nowak A. Glioblastoma stem-like cells: at the root of tumor recurrence and a therapeutic target. Carcinogenesis 2015;36:177-85.
6. 6. Bagheri V, Razavi MS, Momtazi AA, Sahebkar A, Abbaszadegan MR, Gholamin M. Isolation, identification, and characterization of cancer stem cells: a review. J Cell Physiol 2017;232:2008-18.
7. 7. Pauklin S, Vallier L. The cell cycle state of stem cells determines cell fate propensity. Cell 2013;155:135-47.
8. 8. Koestenbauer S, Zech NH, Juch H, Vanderzwalmen P, Schoonjans L, Dohr G. Embryonic stem cells: similarities and differences between human and murine embryonic stem cells. Am J Reprod Immunol 2006;55:169-80.

9. 9. Son MJ, Woolard K, Nam D-H, Lee J, Fine HA. SSEA-1 Is an enrichment marker for tumor-initiating cells in human glioblastom Cell Stem Cell 2009;4:440-52.
10. 10. Campos B, Gal Z, Baader A, Schneider T, Sliwinski C, Gassel K, et al. Aberrant self-renewal and quiescence contribute to the aggressiveness of glioblastoma. J Pathol 2014;234:23-33.
11. 11. Behl C, Ziegler C. Cell Aging: Molecular Mechanisms and Implications for Disease. Berlin, Heidelberg: Springer Berlin Heidelberg, 2014.
12. 12. Pawlik TM, Keyomarsi K. Role of cell cycle in mediating sensitivity to radiotherapy. Int J Radiat Oncol 2004;59:928-42.
13. 13. Nagpal J, Jamoona A, Gulati ND, Mohan A, Braun A, Murali R, et al. Revisiting the role of p53 in primary and secondary glioblastomas. Anticancer Res 2006;26:4633-9.
14. 14. Mcllwrath AJ, Vasey PA, Ross GM, Brown R. Cell cycle arrests and radiosensitivity of human tumor cell lines: dependence on wild-type p53 for radiosensitivity. Cancer Res 1994;54:3718-22.
15. 15. Mitchell R, Bacher M, Bernhagen J, Pushkarskaya T, Seldin MF, Bucala R. Cloning and characterization of the gene for mouse macrophage migration inhibitory factor (MIF). J Immunol 1995;154:3863-70.
16. 16. Krockenberger M, Dombrowski Y, Weidler C, Ossadnik M, Hönig A, Hausler S, et al. Macrophage migration inhibitory factor contributes to the immune escape of ovarian cancer by down-regulating NKG2D. J Immunol 2008;180:7338-48.
17. 17. Yamate T, Mocharla H, Taguchi Y, Igietseme JU, Manolagas SC, Abe E. Osteopontin expression by osteoclast and osteoblast progenitors in the murine bone marrow: demonstration of its requirement for osteoclastogenesis and its increase after ovariectomy. Endocrinology 1997;138:3047-55.
18. 18. Hira VVV, Ploegmakers KJ, Grevers F, Verbovsek U, Silvestre-Roig C, Aronica E, et al. CD133 + and nestin + glioma stem-like cells reside around CD31 + arterioles in niches that express SDF-1α, CXCR4, osteopontin and cathepsin K. J Histochem Cytochem 2015;63:481-93.
19. 19. Henry A, Nokin M-J, Leroi N, Lallemand F, Lambert J, Goffart N, et al. New role of osteopontin in DNA repair and impact on human glioblastoma radiosensitivity. Oncotarget 2016;7:63708-21.
20. 20. Pan M-G, Xiong Y, Chen F. NFAT gene family in inflammation and cancer. Curr Mol Med 2013;13:543-54.
21. 21. Franken NAP, Rodermond HM, Stap J, Haveman J, Van Bree C. Clonogenic assay of cells in vitro. Nat Protoc 2006;1:2315-9.
22. 22. Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature 2000;408:307-10.
23. 23. Fernet M, Mégnin-Chanet F, Hall J, Favaudon V. Control of the G2/M checkpoints after exposure to low doses of ionising radiation: implications for hyper-radiosensitivity. DNA Repair (Amst) 2010;9:48-57.
24. 24. Daniele S, Costa B, Zappelli E, Da Pozza E, Sestito S, Nesi G, et al. Combined inhibition of AKT/mTOR and MDM2 enhances glioblastoma multiforme cell apoptosis and differentiation of cancer stem cells. Sci Rep 2015;5:9956.
25. 25. Chandrika G, Natesh K, Ranade D, Chugh A, Shastry P. Suppression of the invasive potential of Glioblastoma cells by mTOR inhibitors involves modulation of NFκB and PKC-α signaling. Sci Rep 2016;6:22455.
26. 26. Mittelbronn M, Platten M, Zeiner P, Dombrowski Y, Frank B, Zachskorn C, et al. Macrophage migration inhibitory factor (MIF) expression in human malignant gliomas contributes to immune escape and tumour progression. Acta Neuropathol 2011;122:353-65.
27. 27. Lamour V, Le Mercier M, Lefranc F, Hagedorn M, Javerzat S, Bikfalv A, et al. Selective osteopontin knockdown exerts anti‐tumoral activity in a human glioblastoma model. Int J Cancer 2010;126:1797-805.