Valproic acid shows a potent antitumor effect with alteration of DNA methylation in neuroblastoma

Abstract

Epigenetic aberrations and a CpG island methylator phenotype are associated with poor outcome in children with neuroblastoma (NB). Previously, we have shown that valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, exerts antitumor effects in an NB xenograft model. However, the underlying antitumor molecular mechanisms are largely unknown. In this study, we examined the role of HDAC in cell proliferation, cell cycle progression, gene expression patterns, and epigenome in NB. Cell proliferation, cell cycle progression, caspase activity, RNA and protein expression, quantitative methylation, and global DNA methylation were examined in NBL-W-N and LA1-55n NB cell lines. Our studies showed that inhibition of HDAC decreased NB proliferation, and induced caspase activity and G1 growth arrest. Expression patterns of cancer-related genes were modulated by VPA. The expression of THBS1, CASP8, SPARC, CDKN1A, HIC1, CDKN1B, and HIN1 was upregulated, and that of MYCN and TIG1 was downregulated. HDAC inhibition decreased methylation levels of THBS1 and RASSF1A promoters. Inhibition of HDAC increased acetylation of histone 4 and overall DNA methylation levels. Our studies showed that inhibition of HDAC blocked cell proliferation and cell cycle progression in relation to alteration in cancer-related genes, increased overall DNA methylation, and decreased methylation of tumor suppressor genes. Further studies examining the antitumor effects of VPA in NB are warranted.

Figure 1

Effect of HDAC inhibition on NB growth and cell cycle in vitro. (A) In vitro cell proliferation was examined by MTS colorimetric assays. NBL-W-N cells were grown for 1–3 days in the presence or absence of VPA with varying concentrations as indicated. (B) In vitro cell proliferation was examined by MTS colorimetric assays. LA1-55n cells were grown for 1–3 days in the presence or absence of VPA with varying concentrations as indicated. (C) BrdU incorporation assay was performed in LA1-55n cells treated with various concentrations of VPA for 1 day. (D) BrdU incorporation assay was performed in LA1-55n cells treated with various concentrations of TSA for 1 day. * p<0.05 compared to control

Figure 2

Effect of HDAC inhibition on cell cycle and apoptosis. (A) NBL-W-N cell line was treated with either 2.5 mM of VPA or vehicle control for 2 days and analyzed by flow cytometry. Each column represents the mean of three independent experiments. (B) LA1-55n cell line was treated with either 2.5 mM of VPA or vehicle control for 2 days and analyzed by flow cytometry. (C) NBL-W-N cells were treated with 2.5 mM VPA for 1 day and subjected to caspase 3/7 activity assay. (D) LA1-55n cells were treated with 2.5 mM VPA for 1 day and caspase 3/7 activity between control and VPA-treated samples were measured. * p<0.05 compared to control

Figure 3

Relative RNA expression of control and VPA-treated NB cell lines. Each cell line was treated with either 2.5 mM of VPA or vehicle control for 2 days. Cells were then harvested and RNA isolated from each group. SYBR green real-time PCR reactions were performed to study alterations of gene expression in cells treated with VPA. Each column represents the mean of two independent experiments. (A) NBL-W-N; (B) LA1-55n. .* p<0.05 compared to control

Figure 4

Effect of HDAC inhibition on the level of acetyl histone 4. (A) Total lysates from vehicle- or VPA-treated NBL-W-N cells were subjected to western blot analysis using antibody against acetyl histone 4. NBL-W-N cells were treated with 1 and 2.5 mM VPA for 1 day. Tubulin was used as a loading control. (B) Total lysates from vehicle- or VPA treated LA1-55n cells were subjected to western blot analysis using antibody against acetyl histone 4. LA1-55n cells were treated with 1 and 2.5 mM VPA for 1 day. Tubulin was used as a loading control. TSA treated samples were used as a positive control of acetyl histone 4. * p<0.05 compared to control

Figure 5

Quantitative RASSF1A methylation detection by high-resolution melt curve analysis. (A) Normalized HRM standard curve of RASSF1A was generated. (B) HRM analysis of DNA methylation status of RASSF1A was performed in LA1-55n cells treated with vehicle and 1 mM VPA for 7 days.

Figure 6

Quantitative THBS1 methylation detection by high-resolution melt curve analysis. (A) Normalized HRM standard curve of THBS1 was generated. (B) HRM analysis of DNA methylation status of THBS1 was performed in LA1-55n cells treated with vehicle and 1 mM VPA for 7 days. .

Figure 7

Relative RNA expression of vehicle- and VPA-treated LA1-55n cells. (A) The RNA expression of THBS1 was examined in LA1-55n cells treated with vehicle or 1 mM VPA for 7 days. After total RNA isolation and cDNA synthesis, THBS1 expression was determined by SYBR green real-time PCR. (B) The RNA expression of Casp8 was examined by SYBR green real-time PCR in LA1-55n cells treated with vehicle or 1 mM VPA for 7 days. * p<0.05 compared to control

Figure 8

Global methylation level was altered in NB cell lines after treatment with VPA. (A) Stock solutions of 2’-deoxycytidine and 5-methyl-2’-deoxycytidine were prepared in water. An eight-point stock mixture of a standard was carefully prepared to give an exact known concentration ratio of 2’-deoxycytidine and 5-methyl-2’-deoxycytidine. (B) DNA methylation was determined by LC/MS. Genomic DNA was isolated from control and VPA-treated NBL-W-N cells. DNA was then subjected to digestion with nuclease P1, venom phosphodiesterase I, and alkaline phosphatase respectively. The concentration of 2’-deoxycytidine and 5-methyl-2’-deoxycytidine in each sample was calculated from the standard curve. Each DNA sample was analyzed in triplicate. (C) Total DNA methylation was determined by LC/MS in control and VPA-treated LA1-55n cells. * p<0.05 compared to control

Figure 9