Abstract
All-trans retinoic acid (RA) causes differentiation of neuroblastoma cells, and retinoids have been used in clinical trials in children with advanced neuroblastoma. Combination of RA with histone deacetylase inhibitors (HDACi) could result in improved antitumorigenic activity. We have examined the effect of the HDACi trichostatin A (TSA), sodium butyrate, and suberoylanilide hydroxamic acid (SAHA), alone and in combination with RA in human neuroblastoma SH-SY5Y cells. At concentrations that cause sustained increase of histone H3 acetylation, HDACi produced extensive apoptotic cell death as shown by flow cytometry analysis and induction of poly(ADP-ribose) polymerase proteolysis. HDACi inhibited SH-SY5Y cell growth at a much larger extent than RA. This compound did not cause apoptosis and did not further increase HDACi-mediated cell death. In contrast, both types of drugs cooperated to inhibit cell growth, although synergistic effects were not found. In surviving cells, HDACi repressed cyclin D1 expression and increased the cyclin kinase inhibitors (CKI) p21(Waf1/Cip1) and p27(Kip1). Cyclin D1 was not affected by RA, but this retinoid also increased CKI levels. Induction of p21(Waf1/Cip1) and p27(Kip1) by HDACi was further enhanced in the presence of RA. This effect seems to be at least partially due to transcriptional stimulation of CKI gene expression because both types of drugs cooperated to increase CKI mRNA levels and to activate the CKI promoters in transient transfection assays. These results show the strong antitumorigenic effects of HDACi in neuroblastoma cells and reinforce the idea that combination therapy could be useful to inhibit tumor growth.
Highlights
Histone deacetylase inhibitors (HDACi) constitute a promising treatment for cancer therapy due to their low toxicity, and first-generation HDACi are currently being tested in phase I/II clinical trials [1, 2]
Incubation of SH-SY5Y cells during 6 h with trichostatin A (TSA), sodium butyrate, and suberoylanilide hydroxamic acid (SAHA) caused a dose-dependent increase of histone H3 acetylation
A detectable increase was observed with the lower dose of TSA used, 50 nmol/L, and 100 nmol/L TSA caused an acetylation at least as strong as that produced by 2 mmol/L sodium butyrate or 1 Amol/L SAHA (Fig. 1A)
Summary
Histone deacetylase inhibitors (HDACi) constitute a promising treatment for cancer therapy due to their low toxicity, and first-generation HDACi are currently being tested in phase I/II clinical trials [1, 2]. Histone hypoacetylation can result in the silencing of genes involved in regulation of cell growth, differentiation, and apoptosis. HDACi increase acetylation of histones and transcription factors [4], which can reverse gene silencing facilitating gene expression [5]. HDACi have been shown to induce G1-phase cell cycle arrest with upregulation of p21Waf1/Cip1 [6, 7], which was associated with an Sp1 site in the gene promoter (8 – 11). These changes can result in cell type – specific effects, including reduced proliferation and metabolic activity, induction of apoptosis, and differentiation (12 – 15)
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