Valproic acid downregulates the expression of MGMT and sensitizes temozolomide-resistant glioma cells

Abstract

Temozolomide (TMZ) has become a key therapeutic agent in patients with malignant gliomas; however, its survival benefit remains unsatisfactory. Valproic acid (VPA) has emerged as an anticancer drug via inhibition of histone deacetylases (HDACs), but the therapeutic advantages of a combination with VPA and TMZ remain poorly understood. The main aim of the present study was to determine whether an antitumor effect could be potentiated by a combination of VPA and TMZ, especially in TMZ-resistant cell lines. A combination of VPA and TMZ had a significantly enhanced antitumor effect in TMZ-resistant malignant glioma cells (T98 and U138). This enhanced antitumor effect correlated with VPA-mediated reduced O6-methylguanine-DNA methyltransferase (MGMT) expression, which plays an important role in cellular resistance to alkylating agents. In vitro, the combination of these drugs enhanced the apoptotic and autophagic cell death, as well as suppressed the migratory activities in TMZ-resistant cell lines. Furthermore, in vivo efficacy experiment showed that treatment of combination of VPA and TMZ significantly inhibited tumor growth compared with the monotherapy groups of mice. These results suggest that the clinical efficacy of TMZ chemotherapy in TMZ-resistant malignant glioma may be improved by combination with VPA.

Figure 1

Sensitivity of four human glioma cell lines to TMZ and VPA. (a) The number of viable cells was counted at 72 h after the addition of VPA (0–16 mM) to the culture medium. (b) The viability of glioma cells was also analyzed via MTT assay 72 h after TMZ (0–1,000 μM) treatment. Points, mean; bars, SE. The results are representative of three independent experiments.

Figure 2

Antitumor effect of a combination of VPA with TMZ in glioma cell lines. Malignant glioma cell lines were treated with VPA (4 mM) or TMZ (50 μM), alone or in combination, for 72 h. (a) The combination of VPA with TMZ had combined antitumor effects in U87 and U251 cells. (b) Significant enhanced antitumor effects were observed in T98 and U138 cells (TMZ-resistant glioma cell lines). Columns, mean; bars, SE. *P < 0.05, ANOVA. The results are representative of three independent experiments.

Figure 3

Effect of VPA on the expression of MGMT in TMZ-resistant glioma cells. (a) Cell lysates of four human glioma cell lines were subjected to western blotting using an anti-MGMT antibody. This revealed an absence of MGMT expression in the U87 and U251 cell lines. In contrast, the other two cell lines, T98 and U138, exhibited MGMT expression at the protein level. (b) MGMT expression in the T98 and U138 TMZ-resistant cells treated with varying concentrations of VPA (0–8 mM). β-Actin was used as a loading control. (c) Cells were exposed to medium containing various concentrations of VPA (0–4 mM) with 50 μM TMZ for 72 h and were measured using MTT assay. Columns, mean; bars, SE. *P < 0.05, Student's t-test. The results are representative of three independent experiments.

Figure 4

Induction of apoptotic and autophagic cell death in TMZ-resistant glioma cells by VPA and TMZ combination. T98 and U138 cells were untreated or treated with VPA, TMZ, or their combination. (a) The expression of MGMT, Bcl-2, Bak, and caspase-3 was evaluated using western blotting. (b) Conversion of LC3-I to LC3-II was also determined by western blotting. β-actin was used as a loading control. The results are representative of three independent experiments.

Figure 5

Antimigration effect of a combination of VPA and TMZ in TMZ-resistant glioma cells. Transwell migration assay demonstrates the migration of TMZ-resistant glioma cells in response to 20% FBS. (a) Representative photomicrographs of stained filters showing migrating T98 and U138 (data not shown). Magnification, ×100. (b) T98 and U138 cells were pretreated with VPA (4 mM) or TMZ (50 μM), alone or in combination, for 72 h. Serum-induced T98 cell migration was significantly inhibited by VPA and TMZ combination. Serum-free medium (SFM) was used as a experimental control. Columns, mean; bars, SE. *P < 0.05, ANOVA. The results are representative of three independent experiments.

Figure 6

Inhibition of the growth of T98 xenografts treated with VPA and TMZ. BALB/c nude mice bearing T98 cells were distributed into four treatment groups: filled circle, PSB; open circle, VPA (300 mg/kg); filled triangle, TMZ (50 mg/kg); open triangle, VPA (300 mg/kg) + TMZ (50 mg/kg). Points, mean; bars, SE. **P < 0.01, ANOVA. PBS was used as a control. The results are representative of two independent experiments.