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. 2017 Feb 28;8(9):14666-14679.
doi: 10.18632/oncotarget.14716.

Valproic acid inhibits glioblastoma multiforme cell growth via paraoxonase 2 expression

Jen-Ho Tseng  1   2 , Cheng-Yi Chen  3 , Pei-Chun Chen  3 , Sheng-Huang Hsiao  1   4 , Chi-Chen Fan  5 , Yu-Chih Liang  6 , Chie-Pein Chen  3   7
Affiliations

Valproic acid inhibits glioblastoma multiforme cell growth via paraoxonase 2 expression

Jen-Ho Tseng et al. Oncotarget. .

Abstract

We studied the potential mechanisms of valproic acid (VPA) in the treatment of glioblastoma multiforme (GBM). Using the human U87, GBM8401, and DBTRG-05MG GBM-derived cell lines, VPA at concentrations of 5 to 20 mM induced G2/M cell cycle arrest and increased the production of reactive oxygen species (ROS). Stress-related molecules such as paraoxonase 2 (PON2), cyclin B1, cdc2, and Bcl-xL were downregulated, but p27, p21 and Bim were upregulated by VPA treatment. VPA response element on the PON2 promoter was localized at position -400/-1. PON2 protein expression was increased in GBM cells compared with normal brain tissue and there was a negative correlation between the expression of PON2 and Bim. These findings were confirmed by the public Bredel GBM microarray (Gene Expression Omnibus accession: GSE2223) and the Cancer Genome Atlas GBM microarray datasets. Overexpression of PON2 in GBM cells significantly decreased intracellular ROS levels, and PON2 expression was decreased after VPA stimulation compared with controls. Bim expression was significantly induced by VPA in GBM cells with PON2 silencing. These observations were further shown in the subcutaneous GBM8401 cell xenograft of BALB/c nude mice. Our results suggest that VPA reduces PON2 expression in GBM cells, which in turn increases ROS production and induces Bim production that inhibits cancer progression via the PON2-Bim cascade.

Keywords: cell growth; glioblastoma multiforme; histone deacetylase; paraoxonase 2; valproic acid.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no potential conflicts of interest.

Figures

Figure 1
Figure 1. Valproic acid (VPA) inhibits glioblastoma cell growth
Cell proliferation was determined in U87 (A, D), GBM8401 (B, E), and DBTRG-05MG (C, F) cells after 5–20 mM VPA stimulation for 24 to 72 h using the MTS (A–C) and Bromodeoxyuridine (BrdU) (D–F) assays. The cell proliferation is significantly decreased in GBM cells using VPA in different doses. The data shown are from three independent experiments performed in triplicate. Error bars: SD. Values are shown as absorbance of VPA-treated cells relative to controls (C; cells without VPA treatment).
Figure 2
Figure 2. Valproic acid (VPA) induces cell cycle arrest at G2/M phase and increases ROS production
The cell cycle was analyzed by flow cytometry in U87 (A), GBM8401 (B), and DBTRG-05MG (C) cells treated with 5 (GBM8401 and DBTRG-05MG) or 10 mM (U87) VPA for 24 to 48 h. The levels of S and G2/M phase are quantified. A significant number of GBM cells arrested at the G2/M phase of cell cycle in GBM cells treated by VPA. The ROS pattern was analyzed by flow cytometry in U87 (D), GBM8401 (E), and DBTRG-05MG (F) cells treated with 5 (GBM8401and DBTRG-05MG) or 10 (U87) mM VPA for 24 to 48 h. The ROS levels are quantified in right panel (2) of D, E, and F, respectively. The ROS level is significantly increased by VPA treatment for 24 to 48 h in GBM cells. The data shown are from three independent experiments. Error bars: SD. Values are shown as cell cycle (%) and relative fold (ROS) of VPA-treated cells relative to controls (cells without VPA treatment). Red line: control; blue line: VPA treatment. C: control.
Figure 3
Figure 3. Oxidative stress–related molecules are analyzed in glioblastoma cells
(A) Western blot to show the expression levels of oxidative stress–related molecules such as PON2, cdc2, Bcl-xL, Bim, p27, p21 and cyclin B1, which were determined after stimulation with valproic acid (VPA) at 24 h in U87, GBM8401, and DBTRG-05MG cell lines. The expression of PON2, cdc2, Bcl-xL and cyclin B1 is decreased, and Bim, p27 and p21 is increased by VPA stimulation in GBM cells. (B) Immunohistochemistry (IHC, right panel) to show PON2 protein expression in human GBM biopsies and the left panel is hematoxylin and eosin (HE) staining. The PON2 staining is stronger in tumor cells (arrowhead) compared with normal neuron cells (arrow; magnification: 200×). Large inset: Two-fold magnification of the small inset with arrowheads or arrows. (C, D) The PON2 mRNA and protein levels were determined after stimulation with 5 and 10 mM VPA for 24 to 72 h in U87, GBM8401, and DBTRG-05MG cell lines by RT-PCR (C) and Western blot (D). The decreased PON2 induced by VPA is in a dose-dependent manner. C: control, cells without VPA treatment.
Figure 4
Figure 4. Valproic acid (VPA) inhibits PON2 expression in vitro and in vivo
(A) U87 and GBM8401 cells were transfected with the luciferase reporter plasmid driven by the PON2 5′-flanking region (position -1001 to -1) with or without pA3TK-luc. Promoter activities were calculated, relative to 0 nM VPA (+VPA/−VPA), and further normalized to the pA3TK-luc control as well as β-galactosidase activity (VPA-induced changes were normalized to that of β-gal). VPA decreases PON2 expression at position -400 to -1. Columns, mean values obtained from at least three independent experiments performed in triplicate; error bars, SE. (B) The glioblastoma cells were pre-treated with a HDAC activator, 1-benzoyl-3-phenyl-2-thiourea (HA, 20 μM) for 1 h followed by VPA (U87, 10 mM; GBM8401 and DBTRG-05MG, 5 mM) treatment 24 h. The acetyl histone H3 expression, an acetylation histone protein regulated by HDAC, was not increased as that by VPA. The HA did not significantly alter the PON2 expression compared with control cells. The PON2 expression was downregulated by VPA, which was abrogated by HA in U87, GBM8401 and DBTRG-05MG cells. (C) The glioblastoma cells were treated with VPA (U87, 10 mM; GBM8401 and DBTRG-05MG, 5 mM) and temozolomide (TMZ, 40 μM) for 24 hours, which has synergistic effect with VPA on Bim upregulation. PON2 downregulation was not observed in TMZ treatment. TMZ did not have synergistic effect with VPA on PON2 regulation. (D, E) PON2 mRNA expression was analyzed in 30 GBM specimens and 4 normal subjects from the Oncomine public Bredel GBM microarray dataset (GEO accession: GSE2223 [38]) (D) and 473 specimens from The Cancer Genome Atlas (TCGA) GBM microarray database (E). The PON2 level is significantly higher in GBM patients compared with normal controls. (F) The nude mice received subcutaneous xenograft with GBM8401 cells (5 × 107) were intraperitoneal injections of PBS (n = 3) or VPA (400 mg/kg; n = 3) every two days for 60 days. The tumor sizes were measured up to 60 days after inoculation of tumor cells. VPA decreased glioblastoma cells proliferation significantly in vivo. (G, H) The PON2 and Bim levels were measured in tumors of PBS and VPA conditions with GBM8401 cells inoculation by Western blot (G) and immunohistochemistry (H). The PON2 was downregulated and Bim upregulated by VPA in GBM8401 cells-injected mice. C: control, cells without treatment. p: promoter.
Figure 5
Figure 5. ROS level is inhibited by PON2
Cells overexpressing PON2 (A, C) and PON2-silenced (E, G) cells were established in U87 (A, E) and GBM8401 (C, G) as shown by Western blot. The ROS levels in U87 (B, F) and GBM8401 (D, H) cells in both PON2-overexpressed (B, D) and PON2-silenced (F, H) cells were analyzed by flow cytometry. VPA (10 mM) increases the ROS production in vector-control cells, the effect is reduced in PON2-overexpressed cells (B, D). The ROS levels in PON2-silenced cells with VPA (10 mM) treatment are higher than that of vector-control cells with VPA treatment (F, H). Control: cells transfected with empty vector only; KD: knockdown.
Figure 6
Figure 6. PON2-Bim cascade is involved in VPA-increased ROS
The expression levels of PON2 and Bim were determined by Western blot after VPA stimulation in U87 (A, C) and GBM8401 (B, D) cells in both PON2-overexpressed (PON2) (A, B) and PON2-silenced (PON2 KD) (C, D) conditions. The expression of Bim in VPA treated PON2-overexpressed cells is lower than that of VPA treated vector-control cells (A, B; lanes 2 vs 4). The levels of Bim in VPA-treated PON2-silenced cells is higher than that in VPA-treated vector-control cells (C, D; lanes 2 vs 4). (E, F) The Bim expression and cell proliferation were determined by Western blot and MTS assay after VPA stimulation in U87 and GBM8401 cells with Bim-silenced (Bim KD) condition. The decreasing cell proliferation was abrogated in the Bim-silenced (Bim KD) condition compared with the vector-control cells in the VPA stimulation cells (G, H) The mRNA levels of PON2 and Bim were analyzed in 473 specimens from The Cancer Genome Atlas (TCGA) GBM microarray database [32]. The mRNA expression levels of PON2 and Bim in the individuals were significantly different in 473 GBM specimens. The PON2 level is inversely correlated with Bim (Pearson r = –0.236, p < 0.001). Control: cells transfected with empty vector only; C; cells without VPA treatment; KD: knockdown.
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