N-(4-Hydroxyphenyl)retinamide induced differentiation with repression of telomerase and cell cycle to increase interferon-gamma sensitivity for apoptosis in human glioblastoma cells

Abstract

Glioblastoma is the most malignant and prevalent brain tumor in humans. It is composed of heterogenic abnormal astroglial cells that avoid differentiation, maintain proliferation, and hardly commit apoptosis. N-(4-Hydroxyphenyl)retinamide (4-HPR) induced astrocytic differentiation and increased sensitivity to interferon-gamma (IFN-gamma) for apoptosis in human glioblastoma A172, LN18, and SNB19 cells. Combination of 4-HPR and IFN-gamma significantly inhibited human telomerase reverse transcriptase (hTERT), cyclin dependent kinase 2 (CDK2), and survivin to up-regulate caspase-8, caspase-9, and caspase-3 for increasing apoptosis in all glioblastoma cell lines. Hence, combination of 4-HPR and IFN-gamma should be considered for controlling growth of different human glioblastoma cells.

Fig. 1

Morphological features of astrocytic differentiation in human glioblastoma A172, LN18, and SNB19 cells. Treatment of cells treated with 0.5 μM 4-HPR for 5 days induced morphological features such as small and retracted cell bodies with thin, elongated, and branched processes, indicating the prominence of differentiation process in these cells.

Fig. 2

Western blotting for determination of changes in cytosolic levels of markers of astrocytic differentiation and cell cycle progression in glioblastoma A172, LN18, and SNB19 cells. Treatments: control, 0.5 μM 4-HPR (5 days), 200 units/ml IFN-γ (2 dads), and 0.5 μM 4-HPR (3 days) + 200 units/ml IFN-γ (2 days). Glioblastoma cells were grown to 70% confluency before being treated with 0.5 μM 4-HPR alone for 3 days and then in combination with 200 units/ml IFN-γ for 2 days. Representative Western blots (n ≥ 3) and bar graphs show levels of: (A) 51 kD GFAP, (B) 120 kD hTERT, (C) 35 kD CDK2, (D) 47 kD pRB, and (E) 17 kD survivin.

Fig. 3

In situ Wright staining for morphological features of apoptosis in glioblastoma A172, LN18, and SNB19 cells. Treatments: control, 0.5 μM 4-HPR (5 days), 200 units/ml IFN-γ (2 dads), and 0.5 μM 4-HPR (3 days) + 200 units/ml IFN-γ (2 days). Based on the morphological features such as cell volume shrinkage along with chromatin condensation, and membrane-bound apoptotic bodies, changes in percent apoptosis were determined in glioblastoma (A) A172, (B) LN18, and (C) SNB19 cells.

Fig. 4

Western blotting for examination of activation of extrinsic pathway of apoptosis in glioblastoma A172, LN18, and SNB19 cells. Treatments: control, 0.5 μM 4-HPR (5 days), 200 units/ml IFN-γ (2 dads), and 0.5 μM 4-HPR (3 days) + 200 units/ml IFN-γ (2 days). Representative Western blots (n ≥ 3) and bar graphs show changes in: (A) 20 kD active caspase-8 and (B) 15 tBid.

Fig. 5

Western blotting for determination of increase in Bax:Bcl-2 ratio leading to mitochondrial release of cytochrome c into the cytosol in glioblastoma A172, LN18, and SNB19 cells. Treatments: control, 0.5 μM 4-HPR (5 days), 200 units/ml IFN-γ (2 dads), and 0.5 μM 4-HPR (3 days) + 200 units/ml IFN-γ (2 days). Representative Western blots (n ≥ 3) and bar graphs show changes in: (A) Bax:Bcl-2 ratio, (B) 15 KD cytochrome c in mitochondria, and (C) 15 kD cytochrome c in cytosol.

Fig. 6

Western blotting for examination of activation of caspase-9 and caspase-3 and also formation of ICAD fragment in glioblastoma A172, LN18, and SNB19 cells. Treatments: control, 0.5 μM 4-HPR (5 days), 200 units/ml IFN-γ (2 dads), and 0.5 μM 4-HPR (3 days) + 200 units/ml IFN-γ (2 days). Representative Western blots (n ≥ 3) and bar graphs show changes in: (A) 35 kD active caspase-9, (B) 20 kD active caspase-3, and (C) 40 kD ICAD.

Fig. 7

Schematic presentation of molecular events leading to astrocytic differentiation and apoptosis in glioblastoma cells after treatment with combination of 4-HPR and IFN-γ. Treatment with 4-HPR could down regulate hTERT, CDK2 and pRb, which would most likely result in cell cycle arrest. Prolongation of cell cycle arrest would enhance sensitivity of the cells to IFNγ for activation of caspase cascades for apoptosis.