Role of adenosine on the resistance to an anti-tumor agent in glioblastoma cell lines
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1
Unidade de Farmacologia Clínica, Instituto de Medicina Molecular (IMM), Universidade de Lisboa, Portugal
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2
Faculty of Health Sciences, University of Beira Interior, Portugal
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3
Institute of Molecular Medicine, Faculty of Medicine, University of Lisbon, Portugal
Glioblastoma is a primary brain tumor of glial cells with a high incidence in adults and is one of the most abundant brain cancer pathologies. This type of tumor is associated with high genomic instability caused by hypomethylation of the DNA, that induces activation of oncogenes, loss of imprinting and increased genomic instability.
Hypoxia and stress regions are commonly found in these types of tumors, where there is an accumulation of intracellular adenosine which, by combining with homocysteine, leads to an increase of S-adenosylhomocysteine (SAH) levels and a decrease of the S-adenosylmethionine (SAM)/SAH ratio. SAH is a competitive inhibitor of SAM-dependent methyltransferases causing global hypomethylation of DNA.
Early mutations in glioblastoma frequently lead to hypermethylation in the O-6-methylguanine-DNA methyltransferase (MGMT) gene promoter, which causes gene silencing and lack of expression of the MGMT DNA repairing protein. It is known that expression of this tumor suppressor protein is responsible for the lack of glioblastoma´s sensitivity to available first-line treatment, the alkylating drug Temozolomide (TMZ). Thus, the aim of this study is to understand if there is a relationship between the increased intracellular adenosine, associated with a global hypomethylation of DNA, and the chemotherapeutic resistance to TMZ caused by an increased in a MGMT expression and activity.
Three glioblastoma cell lines, U87, U373 and SNB19 were used. Intracellular adenosine levels were increased by incubating cells for 72h with ABT 702 dihydrochloride (ABT, 15µM), an adenosine kinase inhibitor. TMZ (100 µM) was added for the last three hours of experiment. MGMT expression and localization were visualized by immunocytochemistry. Cell viability was assessed by Propidium iodide (PI, 15 µM) staining for 30 minutes, followed by fluorescence microscopy visualization.
A perinuclear localization of MGMT was observed in all cell lines. In the absence of the drugs (control cells) no cell death, assessed by PI staining, was observed, while an increase in cell death was observed in the TMZ-treated cells. Preliminary results show that increased endogenous adenosine levels have a protective effect against TMZ, since cells incubated with both ABT and TMZ present a lower cell death (similar to control) compared to TMZ-incubated cells.
These results suggest increased endogenous adenosine levels as the responsible for the lack of success of TMZ therapy.
Keywords:
Glioblastoma,
Adenosine,
temozolomide,
O-6-methylguanine-DNA methyltransferase (MGMT),
hypoxia
Conference:
XVI Meeting of the Portuguese Society for Neuroscience (SPN2019), Lisboa, Portugal, 30 May - 1 Jun, 2019.
Presentation Type:
Poster presentation
Topic:
Cellular and Molecular Neurosciences
Citation:
Nogueira
VS,
Sebastião
AM,
Cascalheira
JF and
Valente
CA
(2019). Role of adenosine on the resistance to an anti-tumor agent in glioblastoma cell lines.
Front. Cell. Neurosci.
Conference Abstract:
XVI Meeting of the Portuguese Society for Neuroscience (SPN2019).
doi: 10.3389/conf.fncel.2019.01.00009
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Received:
28 Feb 2019;
Published Online:
27 Sep 2019.
*
Correspondence:
Prof. Ana M Sebastião, Institute of Molecular Medicine, Faculty of Medicine, University of Lisbon, Lisbon, Lisbon, 1649-028, Portugal, anaseb@medicina.ulisboa.pt
PhD. José F Cascalheira, Faculty of Health Sciences, University of Beira Interior, Covilhã, Portugal, jfcascalheira@ubi.pt
PhD. Claudia A Valente, Institute of Molecular Medicine, Faculty of Medicine, University of Lisbon, Lisbon, Lisbon, 1649-028, Portugal, cvalentecastro@medicina.ulisboa.pt