TAS102 selectively inhibits proliferation of α-thalassemia mental retardation X-linked gene-deficient glioma cells

Abstract

Objective To investigate the effect of TAS102 on the proliferation of α-thalassemia mental retardation X-linked (ATRX)-deficient U87 glioma cells in vitro and explore the mechanism. Methods Wild-type glioma U87 cells and ATRX knockout U87 cells (U87ATRX-/-) were both treated with TAS102, and the changes in the cell viability and cell cycle distribution were examined using CCK8 assay and flow cytometry, respectively. Beta-galactosidase staining was used to assess the cell senescence, and immunofluorescence staining and Western blotting were employed to detect the activation of DNA damage recognition reaction pathways and the expression of the related proteins. Results CCK-8 assay showed that TAS102 treatment for 72 h resulted in significantly lower cell viability in U87ATRX-/- cells than in wild-type U87 cells, and its IC50 value was significantly lower in U87ATRX-/- cells than in U87 cells (4.72±0.48 vs 10.50 ±1.52 mg/L, P < 0.01). TAS102 treatment resulted in more cells arrested in G2 phase in U87 cells [(23.04±3.57)% vs (17.86±2.12)%] and in U87ATRX-/- cells [(61.67±1.13)% vs (18.94±3.05)%], and the arrest was more obviously in the knockout cells than the wild-type ones(P < 0.01). Beta-galactosidase staining showed that the positive percentage was (7.73±0.71)% and (29.55±4.12)% in U87 cells and TAS102 treated cells, and (8.70±2.55)% and (53.69±7.03)% in control and treated knockout cells. There were more positive cells in the knockout than the wild-type cells (P < 0.01). Immunofluorescence assay showed that TAS102 treatment increased the percentage of 53BPI-positive cells in both of the cells [control and treated U87 cells: (14.5±7.39)% and (21.19±3.68)%; control and treated U87ATRK-/- A cells: (16.75±2.97)% and (54.27±8.38)%]. but the increment was significantly greater in U87ATRX-/- cells than in U87 cells (P < 0.05). In the phosphorylation levels of ATR, CHK1, ATM and CHK2 at different time points following TAS102 treatment, significant increment was seen in ATM/CHK2 levels in U87ATRX-/-cells. Conclusion TAS102 can selectively inhibit the proliferation of U87ATRX-/- cells by inducing cell senescence, activating ATM-CHK2 damage response pathways, and causing cell cycle arrest in G2 phase.

Keywords

• tas102
• α-thalassemia mental retardation x-linked genes
• glioma
• dna damage