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  • Comparative study of the expression of enzymes involved in adenosine metabolism in human astrocytes and glioblastoma
    Publication . Santos, Beatriz Alves dos; Lopes, Helena Tomás Marcelino; Cascalheira, José Francisco
    Glioblastoma (GB) continues to be a significant challenge in neuro-oncology due to its aggressive nature and resistance to current therapies. This tumor develops mainly from astrocytes (AST) and neural stem cells and it is characterized by a diffuse, infiltrative and fast proliferation. Despite advancements in surgeries and chemotherapies, GB remains challenging to treat due to its heterogeneity. New targeted treatments are being studied, specifically ones that use molecules already present in the human biochemical processes, like adenosine (Ado). Ado is a purine nucleoside essential for cellular metabolism and its regulation is crucial for maintaining physiological balance. In GB, the role of Ado is intricate. In fact, the low-oxygen environment within tumors boosts extracellular Ado production and purinergic signaling, facilitating tumor growth through multiple mechanisms. Enzymes such as ecto-5'-nucleotidase CD73, ectonucleotidase triphosphate diphosphohydrolase CD39, and Prostatic Acid Phosphatase (PAP) are highly expressed in GB, enhancing estracellular Ado production and activating receptors like A2A and A3, which affects tumor growth and immune system evasion. Ado kinase (ADK), which is abundantly present in GB, is pivotal in tumor proliferation by keeping intracellular Ado levels low, since high intracellular Ado inhibits cell proliferation, while Ado deaminase (ADA) and nucleoside transporters further influence Ado's metabolism. The present work studied and compared the role of Ado on cell proliferation and migration, as well as its metabolism, in human astrocytes and GB cells. Two conditions were tested to explore high concentration intracellular Ado's role in GB: ABT-702, which blocks Ado kinase (ADK) and a Cocktail of Antagonists (CA), which blocks Ado receptors. These were applied to three GB cell lines (U87, U373, SNB19) and human astrocytes (HA). Cell viability assays showed that combining ABT-702 and CA resulted in the lowest GB cell progression. ABT-702 alone led to intracellular Ado accumulation, which resulted in maintenance of proliferation., while CA alone reduced cell viability by preventing extracellular Ado from binding to those same receptors. The scratch assay supported these findings, showing less tumor cell proliferation in wells treated with CA or CA + ABT-702, compared to vehicle. Interestingly, apoptosis assay revealed intact nuclei in all cells, suggesting that they entered a senescence state rather than undergoing apoptosis. This indicates that tumor cells may pause proliferation in response to drug-induced stress. HA exhibited similar trends with CA reducing cell viability, underscoring that extracellular Ado plays a significant role in cell proliferation. Western Blot analysis further suggested that ADK is undetectably low in HA but present in U373 and SNB19 cells, indicating that ADK in HA may be less active.
    2025-01-16Master thesis Embargoed access Show more