Histone deacetylase inhibitors, valproic acid and trichostatin-A induce apoptosis and affect acetylation status of p53 in ERG-positive prostate cancer cells

Abstract

An ETS family member, ETS Related Gene (ERG) is involved in the Ewing family of tumors as well as leukemias. Rearrangement of the ERG gene with the TMPRSS2 gene has been identified in the majority of prostate cancer patients. Additionally, overexpression of ERG is associated with unfavorable prognosis in prostate cancer patients similar to leukemia patients. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) regulate transcription as well as epigenetic status of genes through acetylation of both histones and transcription factors. Deregulation of HATs and HDACs is frequently seen in various cancers, including prostate cancer. Many cellular oncogenes as well as tumor viral proteins are known to target either or both HATs and HDACs. Several studies have demonstrated that there are alterations of HDAC activity in prostate cancer cells. Recently, we found that ERG binds and inhibits HATs, which suggests that ERG is involved in deregulation of protein acetylation. Additionally, it has been shown that ERG is associated with a higher expression of HDACs. In this study, we tested the effect of the HDAC inhibitors valproic acid (VPA) and trichostatin-A (TSA) on ERG-positive prostate cancer cells (VCaP). We found that VPA and TSA induce apoptosis, upregulate p21/Waf1/CIP1, repress TMPRSS2-ERG expression and affect acetylation status of p53 in VCaP cells. These results suggest that HDAC inhibitors might restore HAT activity through two different ways: by inhibiting HDAC activity and by repressing HAT targeting oncoproteins such as ERG.

Figure 1

Truncated-ERG is expressed in a prostate cancer cell line. Western blot analysis was performed with either COS-1 transfected with full-length ERG-2 expression vector (lane 1) or VCaP cell lysate (lane 2).

Figure 2

Both TSA and valproic acid impair cell viability of VCaP cells. (A) Cells were treated with variable amount of valproic acid (0.05–20 mM) or vehicle control for 24, 48, or 72 h. (B) Cells were treated with variable amount of trichostatin-A (10–200 nM) or vehicle control for 24, 48, or 72 h. Results are shown as the mean of percentage of control of three independent experiments with standard deviations.

Figure 3

Both TSA and valproic acid induce apoptosis in VCaP cells. Apoptosis of VCaP cells were analyzed using TUNEL reaction. (A) Cells were treated with variable amount of valproic acid (0.1–10 mM) or vehicle control for 24 h. (B) Cells were treated with variable amount of TSA (10–00 nM) or vehicle control for 24 h. Fluorescent signals after labeling with fluorescein-dUTP are shown in the top panels. Bottom panels show DAPI staining for nucleus. Scale bar, 50 μm.

Figure 4

Both valproic acid and TSA increase caspase 3/7 activity. (A) Cells were treated with valproic Acid (0.1–10 mM) or vehicle control for 12 or 24 h before the measurement of caspase 3/7 activity. (B) Cells were treated with TSA (5–1000 nM) or vehicle control (DMSO) for 12 or 24 h before the measurement of caspase 3/7 activity. Caspase 3/7 activity was determined using Caspase-Glo 3/7 assay reagent (Promega) according to manufacturer’s protocol. Values represent the mean of luminescence signals of three independent experiments with standard deviations. ^*P<0.01

Figure 5

Effect of TSA and valproic acid on p21 expression. (A) Western blot assay was performed after 12 or 24 h valproic acid treatments at the indicated concentrations. The blot with anti-p21 antibody is shown at the top. β-actin used as a loading control is shown at the bottom. (B) Western blot assay was performed after 12 or 24 h TSA treatments at the indicated concentrations. The blot with anti-p21 antibody is shown at the top. β-actin used as a loading control is shown at the bottom. (C) The relative expression level of p21 mRNA was determined with quantitative RT-PCR after 24 h treatment with 10 mM valproic acid. (D) The relative expression level of p21 mRNA was determined with quantitative RT-PCR after 24 h treatment with 1 μM TSA. In both cases, GAPDH was used to normalize samples. The results represent fold activation over control of three independent experiments with standard deviations. *P<0.05

Figure 6

Effect of TSA and valproic acid on TMPRSS2-ERG expression. (A) Western blot assay was performed after 12 or 24 h valproic acid treatments at the indicated concentrations. The blot with anti-ERG antibody is shown at the top. β-actin used as a loading control is shown at the bottom. (B) Western blot assay was performed after 12 or 24 h TSA treatments at the indicated concentrations. The blot with anti-ERG antibody is shown at the top. β-actin used as a loading control is shown at the bottom. (C) The relative expression level of TMPRSS2-ERG mRNA was determined with quantitative RT-PCR after 24 h treatment with 10 mM valproic acid. (D) The relative expression level of TMPRSS2-ERG mRNA was determined with quantitative RT-PCR after 24 h treatment with 1 μM TSA. In both cases, GAPDH was used to normalize samples. The results represent relative values against control (arbitrarily set to 1) of three independent experiments with standard deviations. * P<0.05

Figure 7

Both TSA and valproic acid induce acetylation of p53 in VCaP cells. (A) Western blot assays were performed on VCaP cells incubated for 12 or 24 h at the indicated concentrations of valproic acid. Blot with anti-acetylated p53-Lys 373 (top), anti-p53 (middle), or anti-β-actin (bottom) are shown. (B) Western blots were performed on VCaP cells incubated for 12 or 24 h at the indicated concentrations of TSA. Blot with anti-acetylated p53-Lys 373 (top), anti-p53 (middle), or anti-β-actin (bottom) are shown.

Figure 8

ERG interferes with acetylation of p53 in two ways. ERG binds CBP/p300 histone acetyltransferase and inhibits its activity of acetylation on p53. ERG also induces expression of HDACs which deacetylate p53.