Inhibitors of histone deacetylase (HDAC) restore the p53 pathway in neuroblastoma cells

Abstract

Background and purpose: Inhibitors of histone deacetylase (HDAC) are emerging as a promising class of anti-cancer drugs, but a generic deregulation of transcription in neoplastic cells cannot fully explain their therapeutic effects. In this study we evaluated alternative molecular mechanisms by which HDAC inhibitors could affect neuroblastoma viability.

Experimental approach: Effects of HDAC inhibitors on survival of the I-type SK-N-BE and the N-type NB SH-SY5Y neuroblastoma cell lines were assessed by the MTT assay. Molecular pathways leading to this were examined by western blot, confocal microscopy and cytofluorometry. The mRNA levels of apoptotic mediators were assessed semi-quantitatively by RT-PCR. Tumour-suppressor p53 trans activity was assessed in EMSA experiments. HDAC inhibitors were also studied in cells subjected to plasmid-based p53 interference (p53i).

Key results: HDAC inhibitors induced cell death via the mitochondrial pathway of apoptosis with recruitment of Bcl-2 family members. Bcl-2 overexpression rendered neuroblastoma cells resistant to HDAC inhibitor treatment. Low concentrations of HDAC inhibitors (0.9 mM) caused a G(2) cell-cycle arrest and a marked upregulation of the p21/Waf1/Cip1 protein. HDAC inhibitors also activate the p53 protein via hyper-acetylation and nuclear re-localization, without affecting its protein expression. Accordingly, HDAC inhibitor-induced cell-killing and p21/Waf1/Cip1 upregulation is impaired in p53i-cells.

Conclusions and implications: In neuroblastoma cells, HDAC inhibitors may overcome the resistance to classical chemotherapeutic drugs by restoring the p53 tumour-repressor function via its hyper-acetylation and nuclear migration, events usually impaired in such tumours. In neuroblastoma cells, HDAC inhibitors are not able to induce p21/Waf1/Cip1 in the absence of a functional p53.

Figure 1

Characterization of the effects of HDAC inhibitors on human neuroblastoma cell lines. (a) Effect of 24 h treatment with VPM, butyrate (Bu) or VPA on SH-SY5Y and SK-N-BE neuroblastoma cell viability. Results (expressed as % surviving cells over respective control) represent the means±s.e. of three different experiments performed in quadruplicate. (b) Butyrate and VPA, but not VPM, treatment enhanced the level of acetylated histone H3 protein in neuroblastoma cells. Cells were exposed for 4 h to HDAC inhibitors at final concentrations ranging between 0.3 and 3 mM. After treatment, proteins were extracted in acid conditions to improve nuclear fraction as described in Materials and methods, and 25 μg per lane was resolved on a polyacrylamide gel (15%). (b) VPM did not induce histone acetylation after 24 h exposure to the highest concentration tested (10 mM). (c) HDAC inhibitors did not affect neuroblastoma cell differentiation. GAP-43 or β-tubulin III (βTubIII) was chosen as molecular markers of differentiation. Retinoic acid (RA, 10 μM) treatment (48 h) was used as positive control of differentiation. (b, c) Western blot analysis of SH-SY5Y and SK-N-BE cells gave similar results.

Figure 2

HDAC inhibitors induced a typical apoptotic death. (A) Nuclear fragmentation of neuroblastoma cells (SH-SY5Y) was assessed by fluorescent DNA staining. DRAQ5 nuclear dye showed the presence of fragmented, indented or condensed (arrows) nuclei only with the higher (b) butyrate or (c) VPA concentration (3 mM) used ((a) untreated control cells after 24 h in culture). (B) Confocal microscopy of cytochrome c (cyt c) mitochondrial de-localization (arrows) in SH-SY5Y following 12 h treatment with 3 mM (b) butyrate (Bu) or (c) VPA treatment and (a) control cells. (C) Western blot analysis of cytochrome c localization following subcellular fractionation (cytosol: soluble cytosolic fraction, 25 μg per lane; HM: heavy membrane fraction, 10 μg per lane) of SH-SY5Y neuroblastoma cells treated with Bu or VPA (12 h incubation). Heat-shock protein-90 (HSP-90) and cytochrome oxidase subunit IV (COX-IV) were evaluated as housekeeping proteins for cytosolic (HSP-90) or mitochondrial (COX-IV) fractions. Experiments on SK-N-BE cells gave similar results.

Figure 3

Expression of the Bcl-2 family proteins in SH-SY5Y neuroblastoma cells treated with butyrate (Bu) or VPA for 12 or 24 h. (a, c) Western blot analysis of whole extracts with the specified antibodies (25 μg per lane of proteins). β-Actin levels were assessed as a proof of equal loading for each panel (data not shown). (b) RT-PCR analysis of BAX, Bcl-2, NOXA and αPUMA mRNA levels after 12 h of butyrate or VPA treatment. β-Actin mRNA levels were evaluated to equalize the amount of cDNA amplified by PCR. Experiments on SK-N-BE cells gave similar results. (d) BAX, αPUMA and Bcl-2 subcellular localization after HDAC inhibitor treatment (cytosol: soluble cytosolic fraction, 25 μg per lane; HM: heavy membrane fraction, 10 μg per lane). Heat-shock protein-90 (HSP-90), which has an exclusive cytosolic localization, was assessed in the mitochondrial compartment (HM) as a control for good quality subcellular fractionation (data not shown).

Figure 4

Viability of wild-type (wt) and overexpressing Bcl-2 (BCL-2) SH-SY5Y and SK-N-BE cells treated for 24 h with different butyrate (Bu) or VPA concentrations. Results (expressed as % cell death vs untreated controls) represented the means±s.e. of three different experiments performed in quadruplicate.

Figure 5

Cell-cycle distribution and expression of p21/Waf1/Cip1 in SH-SY5Y neuroblastoma cells treated with butyrate (Bu) or VPA. (a) Cell-cycle distribution of HDAC inhibitor-treated neuroblastoma cells. Cells were synchronized by serum deprivation for 16 h before butyrate or VPA treatment (16 h). Values represent percentages of phase-specific populations. MI is the product of G₁/G₂ ratio. (b) RT-PCR analysis of relative mRNA levels after 6 h treatment (left) and western blot analysis (right) of protein expression (30 μg per lane of proteins) after 8 h in the presence of butyrate or VPA (0.9 mM). β-Actin mRNA and protein levels were evaluated as a proof of equal loading. All the experiments on SK-N-BE cells gave similar results.

Figure 6

Activation of the p53 pathway in SH-SY5Y neuroblastoma cells treated with butyrate (Bu) or VPA. (a) Western blot analysis of p53 levels in whole extracts (40 μg per lane of proteins) after 24 h treatment in the presence of low (0.3 mM) or high (3 mM) concentrations of HDAC inhibitor. (b) Cytosolic (cyt, 40 μg per lane of proteins) vs nuclear (nuc, 7.5 μg per lane) distribution of p53 after 4 h treatment with 3 mM butyrate or VPA (upper panel). Heat shock protein-90 (HSP-90; middle panel) and Lamin A/C (lower panel) were evaluated as a proof of purity and equal loading for cytosolic (HSP-90) or nuclear (Lamin A/C) extracts. (c) Electromobility shift assay experiment with p53-specific probe after butyrate and VPA treatment using 2 μg of nuclear extracts. The specificity of p53 binding was assessed by performing the experiment in duplicate with the labelled probe alone or by adding 20-fold excess of unlabelled probe (Exc. Unl.). (d) Western blot analysis of p53 lysine (373 and 382) acetylation in cytosolic (cyt, 40 μg per lane of proteins) vs nuclear (nuc, 7.5 μg per lane) subcellular fractions. Total levels of p53 in the cytosolic and nuclear compartments are displayed in (b). Experiments on SK-N-BE cells gave similar results.

Figure 7

p53 dependence of HDAC inhibitor-induced effects. (a) Densitometric analysis of p53 interference (p53i) vs SH-SY5Y and SK-N-BE parental cell lines. Absorbance values of western blot p53-reactive bands from p53i cells were corrected for the β-actin values (internal standard) compared with the values obtained from parental cell lines and expressed as % of reduction. (b) Cell death of wild-type (wt) and p53i SH-SY5Y and SK-N-BE cells treated for 24 h with different butyrate or VPA concentrations. Results (expressed as % cell death vs untreated controls) represent the means±s.e. of three different experiments performed in quadruplicate. (c) Western blot analysis of p21/Waf1/Cip1 expression in wt and p53i SH-SY5Y and SK-N-BE cells following HDAC inhibitor treatment (0.9 mM, 8 h). (d) Bcl-2 overexpression did not affect HDAC inhibitor-induced p53 nuclear translocation. Parental SH-SY5Y and SK-N-BE cell lines or their Bcl-2 overexpressing derivatives were treated with butyrate (0.9 mM) for 6 h and p53 subcellular localization was studied by confocal microscopy with an anti-p53-specific antibody. Similar results were obtained after VPA treatment (not shown).