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Involvement of PI3K Pathway in Glioma Cell Resistance to Temozolomide Treatment
Adrian Zając1, Joanna Sumorek-Wiadro1, Ewa Langner2
1Department of Functional Anatomy and Cytobiology, Institute of Biological Sciences, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland.
Abstract:
The aim of the study was to investigate the anticancer potential of LY294002 (PI3K inhibitor) and temozolomide using glioblastoma multiforme (T98G) and anaplastic astrocytoma (MOGGCCM) cells. Apoptosis, autophagy, necrosis, and granules in the cytoplasm were identified microscopically (fluorescence and electron microscopes). The mitochondrial membrane potential was studied by flow cytometry. The activity of caspases 3, 8, and 9 and Akt was evaluated fluorometrically, while the expression of Beclin 1, PI3K, Akt, mTOR, caspase 12, and Hsp27 was determined by immunoblotting. SiRNA was used to block Hsp27 and PI3K expression. Cell migration and localization of Hsp27 were tested with the wound healing assay and immunocytochemistry, respectively. LY294002 effectively diminished the migratory potential and increased programmed death of T98G and MOGGCCM. Autophagy was dominant in MOGGCCM, while apoptosis was dominant in T98G. LY294002 with temozolomide did not potentiate cell death but redirected autophagy toward apoptosis, which was correlated with ER stress. A similar effect was observed after blocking PI3K expression with siRNA. Transfection with Hsp27 siRNA significantly increased apoptosis related to ER stress. Our results indicate that inhibition of the PI3K/Akt/mTOR pathway sensitizes glioma cells to apoptosis upon temozolomide treatment, which was correlated with ER stress. Hsp27 increases the resistance of glioma cells to cell death upon temozolomide treatment.
Insights
The PI3K/Akt/mTOR pathway inhibitor LY294002 enhances glioma cell apoptosis when combined with temozolomide, particularly through ER stress. Hsp27, however, confers resistance to this cell death.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- Glioblastoma multiforme (T98G) and anaplastic astrocytoma (MOGGCCM) are aggressive brain tumors.
- Investigating novel therapeutic strategies targeting these cells is crucial.
Purpose of the Study:
- To explore the anticancer effects of LY294002 (a PI3K inhibitor) and temozolomide on T98G and MOGGCCM cells.
- To elucidate the underlying mechanisms, including apoptosis, autophagy, and ER stress.
Main Methods:
- Microscopy (fluorescence and electron) for cell death analysis.
- Flow cytometry for mitochondrial membrane potential.
- Fluorometric and immunoblotting assays for enzyme activity and protein expression (PI3K, Akt, mTOR, caspases, Beclin 1, Hsp27).
- siRNA to inhibit PI3K and Hsp27 expression; wound healing assay for cell migration.
Main Results:
- LY294002 reduced migration and increased programmed cell death in both cell lines.
- Autophagy predominated in MOGGCCM, while apoptosis was more prominent in T98G.
- Combination therapy redirected autophagy toward apoptosis, linked to ER stress.
- Hsp27 siRNA enhanced ER stress-related apoptosis.
- PI3K inhibition via siRNA mimicked LY294002's effects.
Conclusions:
- Inhibiting the PI3K/Akt/mTOR pathway sensitizes glioma cells to temozolomide-induced apoptosis, associated with ER stress.
- Hsp27 expression confers resistance to temozolomide treatment in glioma cells.
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