J Korean Neurosurg Soc.  2020 Sep;63(5):566-578. 10.3340/jkns.2019.0187.

Dexamethasone Interferes with Autophagy and Affects Cell Survival in Irradiated Malignant Glioma Cells

• Komakech A
• Im JH
• Gwak HS
• Lee KY
• Kim JH
• Yoo BC
• Cheong H
• Park JB
• Kwon JW
• Shin SH
• Yoo H

• KMID: 2506020
• DOI: http://doi.org/10.3340/jkns.2019.0187

Abstract

Objective
: Radiation is known to induce autophagy in malignant glioma cells whether it is cytocidal or cytoprotective. Dexamethasone is frequently used to reduce tumor-associated brain edema, especially during radiation therapy. The purpose of the study was to determine whether and how dexamethasone affects autophagy in irradiated malignant glioma cells and to identify possible intervening molecular pathways.
Methods
: We prepared p53 mutant U373 and LN229 glioma cell lines, which varied by phosphatase and tensin homolog (PTEN) mutational status and were used to make U373 stable transfected cells expressing GFP-LC3 protein. After performing cell survival assay after irradiation, the IC₅₀ radiation dose was determined. Dexamethasone dose (10 µM) was determined from the literature and added to the glioma cells 24 hours before the irradiation. The effect of adding dexamethasone was evaluated by cell survival assay or clonogenic assay and cell cycle analysis. Measurement of autophagy was visualized by western blot of LC3-I/LC3-II and quantified by the GFP-LC3 punctuated pattern under fluorescence microscopy and acridine orange staining for acidic vesicle organelles by flow cytometry.
Results
: Dexamethasone increased cell survival in both U373 and LN229 cells after irradiation. It interfered with autophagy after irradiation differently depending on the PTEN mutational status : the autophagy decreased in U373 (PTEN-mutated) cells but increased in LN229 (PTEN wild-type) cells. Inhibition of protein kinase B (AKT) phosphorylation after irradiation by LY294002 reversed the dexamethasone-induced decrease of autophagy and cell death in U373 cells but provoked no effect on both autophagy and cell survival in LN229 cells. After ATG5 knockdown, radiation-induced autophagy decreased and the effect of dexamethasone also diminished in both cell lines. The diminished autophagy resulted in a partial reversal of dexamethasone protection from cell death after irradiation in U373 cells; however, no significant change was observed in surviving fraction LN229 cells.
Conclusion
: Dexamethasone increased cell survival in p53 mutated malignant glioma cells and increased autophagy in PTENmutant malignant glioma cell but not in PTEN-wildtype cell. The difference of autophagy response could be mediated though the phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin signaling pathway.

Keyword

Autophagy; Dexamethasone; Glioma; PTEN; Radiation

Figure

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  Fig. 1. Dex increases survival in both U373 and LN229 glioma cell lines after irradiation. Malignant glioma cells were pretreated with 10 μM Dex and different doses of irradiation. both U373 (A, p=0.02) and LN229 (b, p=0.03) showed significant increases in survival. RT : radiation, Dex : dexamethasone.

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  Fig. 2. Dex interferes with the cell cycle change after irradiation in both U373 and LN229 cells. A : Flow cytometry at 3 days after irradiation (10 Gy) with or without Dex. b and C : Proportional analysis of cell cycle fraction revealed radiation-induced G2/M arrest and subG1 accumulation were interrupted significantly by the addition of Dex in both U373 and LN 229. *p<0.05. PI : propidium iodide, RT : radiation, Dex : dexamethasone.

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  Fig. 3. Measurement of acidic vesicular organelles (AVOs) as an indicator of autophagy after irradiation (10 Gy). A and C : Quantitative analysis of AVO revealed that dexamethasone (b) decreases autophagy in U373 cells (p=0.010) but (D) increases autophagy in LN229 cells (p=0.010). *p<0.05. RT : radiation, Dex : dexamethasone, FITC-A : fluorescein isothiocyanate.

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  Fig. 4. Illustration of Dex’s effect on radiation-induced autophagy in glioma cells. A : Dex inhibits autophagosome formation in U373 cells but promotes LC3-II conjugation in LN 229 cells on western blot. b : Fluorescence confocal microscope pictures (×2000) showing GFP-LC3 punctuated cells after irradiation (middle), which decreased with Dex (right) in U373 cells. C : Graphs indicating reduced number of GFP-LC3 punctuated cells with Dex (p=0.016) in U373 cells. Three independent experiments were carried out and the mean value is presented. *p<0.05. RT : radiation, Dex : dexamethasone, AVO : acidic vesicular organelle.

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  Fig. 5. Colony-forming assay after Dex and radiation exposure with or without phospho-AKT inhibitor (LY294002). A : Addition of Dex and LY294002 results in the reversal of decreased cell death with Dex in U373 cells (p=0.0052). b : It provoked no significant change (p>0.05) in LN229. The experiment was in triplicate and the mean value was presented. C : Photography of colony-forming assay plate of LN229 cells. *p<0.05. RT : radiation therapy, Dex : dexamethasone, LY : LY294002.

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  Fig. 6. Phospho-AKT inhibition affects autophagy following Dex interference in irradiated malignant glioma cells. A : In U373, AVO expression increases significantly (RT+Dex+Ly vs. RT+Dex, p=0.038), indicating increased autophagy. b : In LN229, no significant change of autophagy is seen after blockage of p-AKT. *p<0.05. AVO : acidic vesicular organelle, RT : radiation therapy, Dex : dexamethasone, LY : LY294002.

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  Fig. 7. After autophagy-related gene 5 (ATG5) knockdown, the Dex effect of increased colony-forming ability after irradiation is significantly reversed in PTEN-mt U373 cells but not in PTEN-wt LN229 cells. *p<0.05. RT : radiation therapy, Dex : dexamethasone, siRNA : small interfering RNA.

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