Glioblastoma (GBM) is the most common and most agressive primary brain tumor,
characterized by high heterogenity, metabolic plasticity and resistance to therapy. A key role
in these properties is played by glioblastoma stem cells (GSCs), which mantain the ability
for self-renewal, multipotency and resistance to stress conditions. Their behaviour is closely
regulated by microenvironment, metabolic state and signaling pathways that control
proliferation, differentiation and survival. One of the molecules critically involved in these
processes is γ-enolase, a glycolytic enzyme that also exerts non-metabolic signaling
functions. Its activity is regulated by cathepsin X, a cysteine protease that cleaves the C
terminal region of γ-enolase and thereby modulates its functionality. Altered activity of both
proteins has been associated with neurodegenerative and tumor-related processes; however,
their mutual interaction is not yet fully understood.
In this master's thesis we investigated the effect of cathepsin X inhibiton on the expression
and function of γ-enolase and on GSC-associated stemness properties using the U87 cell
line. Cells were cultured in three different media: complete medium (CM), medium
supplemented with growth factors (NSM), and serum-free medium (SFM). A combination
of morphological, biochemical and cellular analyses was used to study spheroid formation,
expression of the surface marker CD133 and protein levels of α- and γ-enolase. To evaluate
the impact of cathepsin X inhibition, cells grown under different culture conditions were
additionally treated with the cathepsin X inhibitor AMS36.
The results showed that the microenvironment strongly influences cell growth and
phenotype. Differences between culture conditions reflect the metabolic adaptability of the
cells and highlight the importance of the active form of γ-enolase for GSC survival and
malignant potential. Inhibition of cathepsin X increased spheroid formation and CD133
expression, indicating the maintenance of a stem-like phenotype. Protein analyses revealed
increased levels of total γ-enolase and reduced levels of its active form, confirming the direct
role of cathepsin X in its proteolytic processing. Overall, the findings suggest that cathepsin
X and γ-enolase form a regulatory system that affects GSC metabolism and stemness and
may represent a novel therapeutic strategy in glioblastoma treatment.
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