Autophagy induced by valproic acid is associated with oxidative stress in glioma cell lines

Abstract

Autophagy represents an alternative tumor-suppressing mechanism that overcomes the dramatic resistance of malignant gliomas to radiotherapy and proapoptotic-related chemotherapy. This study reports that valproic acid (VPA), a widely used anti-epilepsy drug, induces autophagy in glioma cells. Autophagy, crucial for VPA-induced cell death, is independent of apoptosis, even though apoptotic machinery is proficient. Oxidative stress induced by VPA occurs upstream of autophagy. Oxidative stress also activates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, whereas blocking this pathway inhibits autophagy and induces apoptosis. VPA-induced autophagy cannot be alleviated by inositol, suggesting a mechanism different from that for lithium. Moreover, VPA potentiates autophagic cell death, but not apoptosis, when combined with other autophagy inducers such as rapamycin, Ly294002, and temozolomide in glioma cells both in vitro and in vivo, which may warrant further investigation toward possible clinical application in patients with malignant gliomas.

Fig. 1.

Effect of VPA on malignant glioma cells. (A) Cytotoxic effect of VPA on U87MG, SF295, and T98G glioma cells for 96 h. The viability of the untreated cells was regarded as 100%. Points, mean of three independent experiments; bars, SD. (B) Effect of VPA on cell cycle in three glioma cell lines. After serum deprivation for 36 h, malignant glioma cells treated with or without VPA (1.0 mM) for 96 h were collected and stained with propidium iodide and analyzed in the FACScan. The percentage of cells in different phases of the cell cycle was determined using Cell Quest software.

Fig. 2.

(A) Ultrastructural features of VPA-induced autophagy in glioma cells. The cells treated with (1.0 mM) or without VPA for 96 h were harvested and fixed, and the electron microscopic observation was performed. GFP-LC3 plasmid was transfected into glioma cells 24 h before VPA treatment. (a and c) Untreated U87MG cells and (b and d) VPA-treated U87MG cells. N indicates nucleus. The arrowhead indicates autophagosome including residual digested material. Bar, a, b, 2.5 µm; c, d, 10 µm. (B) Representative Western blot showing MAP1-LC3 and Beclin-1 in U87MG cells before or after VPA exposure for dose (96 h) or for time study (1.0 mM). (C and D) MDC staining in glioma cells before or after VPA exposure for dose (96 h) or for time study (1.0 mM VPA). *P < .05, as compared to noVPA control. (E) MDC-specific activities induced by VPA (1.0 mM, 96 h) can be suppressed by autophagy inhibitor 3-MA. *P < .05, when compared with VPA-treated group. (F) The percentage of GFP-LC3–labeled cells in VPA group (1.0 mM, 96 h) can be reduced by autophagy inhibitor 3-MA. *P < .05, when compared with VPA-treated group. Results shown are the means ± SD of three independent experiments.

Fig. 3.

Autophagy is crucial for VPA-induced cell death. Cells were seeded in 12-well plates at 2 × 10⁴ per mL. At 48 h after exposure to VPA (1.0 mM), 3-MA (2.5 mM), zVAD (100 µM), and PD98059 (10 µM) were added and the cells were cultured for an additional 48 h. (A) Quantification of cell death after exposure to VPA alone or in combination. Cell death was determined by Trypan blue exclusion assay. *P < .05, as compared to noVPA group, **P < .05, when compared with VPA-treated group. ^#P > .05, when compared with VPA-treated group. (B) Apoptosis detection in glioma cells treated with VPA alone or in combination with various drugs. After treatment, cells were labeled with annexin V-FITC, and counterstained with DAPI. Five hundred cells were analyzed for each sample. Cisplatin (5 µM) was used as drug control for apoptosis. *P < .05, when compared with VPA-treated group. Results were the mean ± SD of three independent experiments. (C) Representative Western blot showing cleaved caspase-3 (p17) in U87MG cells following exposure to 1.0 mM VPA alone or in combination with various drugs for 96 h. β-Tubulin was used as loading control.

Fig. 4.

Oxidative stress is an early event after exposure to VPA. (A) After pretreatment with NAC (5 mM) or 3-MA (2.5 mM) for 24 h in U87MG cells, VPA (1.0 mM) was added and cells were cultured for an additional 96 h. H₂O₂ (100 µM, 6 h) was used as positive control. Intracellular oxidative stress was measured with DCF fluorescence intensity. *P < .05, as compared to noVPA group. **P < .05, when compared with VPA-treated group. ^#P > .05, when compared with VPA-treated group. Data are presented as the mean ± SD of three independent experiments. (B) Effects of VPA on MMP assessed by the JC-1 fluorescent probe. U87MG cells were treated with VPA (0.25, 0.5, 1.0, 1.5 mM) for up to 96 h valinomycin (10 mM) was used as positive control. NAC (5 mM) was added 24 h before VPA (1.0 mM) treatment. *P < .05, when compared with the nonVPA group. **P < .05, when compared with VPA-treated group (1.0 mM). Values are expressed as mean ± SD of three independent experiments. (C) Mitochondrial ROS generation in glioma cells after exposure to VPA. Representative fluorescent staining showing ROS visualized by DCF fluorescence (green), and mitochondria labeled by mitotracker red CMXRos (red) in U87MG cells yellow color indicates the colocalization between ROS and mitochondria. Bar, 10 µm. (D) VPA treatment resulted in mitochondrial injuries. After exposure to VPA, U87MG cells were stained by mitotracker red CMXRos (MTR) or collected for electron microscopic analysis. Upper panel, bar, 1 µm; lower panel, bar, 10 µm.

Fig. 5.

VPA promotes autophagy through induction of oxidative stress. Cells were seeded in 12-well plates at 2 × 10⁴ per mL. At 48 h after exposure to VPA (1.0 mM), 3-MA (2.5 mM), NAC (5 mM), zVAD (100 µM), PD98059 (10 µM), or inositol (1 mM) was added and cells were cultured for an additional 48 h. (A) Quantification of cell death after exposure to VPA alone or in combination. Cell death was determined by trypan blue exclusion assay. *P < .05, as compared to the VPA-treated group. (B) Quantification of cells expressing LC3 aggregation after VPA treatment. The percentage of LC3 aggregated cells was quantitated by counting the number of the cells showing the punctate pattern of GFP-LC3 in 200 GFP-positive cells. Results shown are the means ± SD of three independent experiments. *P < .05, **P > .05, when compared with the VPA-treated group. (C) MDC staining in U87MG cells. Autophagosome was labeled by MDC (green punctuate), Magnification 400×. (D) Representative Western blot showing MAP1-LC3 in U87MG cells following exposure to 1.0 mM VPA alone or in combination with various drugs for 96 h. β-Tubulin was used as a loading control.

Fig. 6.

Role of the ERK pathway in VPA-induced autophagy. (A) Representative Western blot showing ERK 1/2 phosphorylation in U87MG cells after treatment without (control) or with VPA (1.0 mM) alone for 24–96 h or with PD98059 (10 µM) or NAC (5 mM). (B) Apoptosis measured using annexin V-FITC staining. Cells were treated with 1.0 mM VPA alone or with 10 µM PD98059 for 96 h. Data were the mean of triplicate experiments; error bars, SD *P < .05, as compared to the VPA-treated group. (C) Quantitation of cells with GFP-LC3 dots among cells treated with 1.0 mM VPA alone or 1.0 mM VPA and 10 µM PD98059. Cells were transiently transfected with the GFP-LC3 plasmid for 24 h and then treated for 96 h as described. The number of cells with GFP-LC3 dots was counted, and their percentage among the total number of cells expressing GFP was determined. Data were the mean of triplicate experiments; error bars, SD *P < .05, when compared with the VPA-treated group. (D) Representative immunostaining of p-ERK1/2 in U87MG cells with VPA (1.0 mM) alone or with PD98059 (10 µM) and NAC (5 mM) for 96 h. Bar, 10 µm.

Fig. 7.

VPA potentiates autophagic cell death when combined with other autophagy inducers. VPA potentiates autophagic cell death when combined with TMZ, rapamycin (RPA), or Ly294002 (Ly). Cells were treated with 1.0 mM VPA alone or in combination with TMZ (25 µM), RPA (10 nM), or Ly294002 (1 µM) for 96 h. (A) Trypan blue exclusion assay was used to evaluate cell death after drug treatment. *P < .05, as compared to TMZ, RPA, or Ly alone. (B) Apoptosis detection in U87MG and T98G glioma cells treated with VPA alone or in combination with TMZ, RPA, or Ly294002. After annexin V-FITC staining, 500 cells were analyzed for each sample. *P > .05, when compared with TMZ, RPA, or Ly294002 alone. (C) Quantitation of autophagic cells with MDC incorporation among cells treated with 1.0 mM VPA alone or in combination with TMZ, RPA, or Ly294002 for 96 h. *P < .05, when compared with TMZ, RPA, or Ly294002 alone. (D) VPA enhances TMZ-induced cytotoxicity. For coincubation experiments, a single concentration of VPA (0.5, 1.0, or 1.5 mM) and different concentrations of TMZ were added to cells and incubations carried out for 96 h. The viability of untreated cells was regarded as 100%. The IC₅₀ value was calculated with SPSS 10.0 statistical software. Data are the mean of triplicate experiments; error bars, SD *P < .05, when compared with TMZ alone (no VPA).

Fig. 8.

VPA induces autophagy-related death in U87MG intracerebellar xenografts. Mice bearing intracerebellar xenografts were killed at the end of drug treatment and compared with the untreated group. (A) The number of cells identified by immunohistochemical staining with MAP1-LC3 were counted in digitally captured images under high-power (10 × 40) magnification and graphed as the percentage of total cells. Columns, mean; bars, SE. *P < .05, as compared to the PBS group (n = 4); **P < .05, when compared with single drug (n = 4). (B) Induction of autophagy in necrotic cells after drug treatment. Necrotic cells were stained with MAP1-LC3 antibody. Black arrow head indicates the site of necrotic cells with MAP1-LC3 staining. Bar, 100 µm.