Histone deacetylase (HDAC) inhibitors are compounds that increase acetylation level of core nucleosomal histones, thereby relaxing the structure of chromatin associated with genes involved in cell growth, differentiation and apoptosis, allowing these genes to be expressed. Many HDAC inhibitors can suppress tumor growth in vivo. Valproic acid (VPA), a commonly used anti-seizure medication, is also a HDAC inhibitor. To determine whether VPA has potential clinical applications for chemoprevention and therapy in patients with head and neck squamous cell carcinoma (HNSCC), we analyzed the effect of VPA on growth and apoptosis in nine HNSCC cell lines. VPA suppressed the growth of all nine cell lines with IC 50 ranging from 1.73 mM to 15.17 mM after a 3-day treatment. Subsequent study focused on the most sensitive cell line, 17B. Cell cycle analysis revealed an increased percentage of cells in G1 after 16 hrs incubation with VPA in a dose-dependent manner. TUNEL assay demonstrated that a 16-hr exposure to VPA at concentrations of 2.5, 5 and 10 mM resulted in 15%, 22.1% and 37.5% apoptotic cells, respectively. Western blot analysis showed that VPA indeed increased nuclear content of acetylated histones. To further investigate the mechanisms underlying the effect of growth inhibition and apoptosis induction of VPA, Northern and Western blot analyses were performed to study the expression of key genes in growth arrest and apoptosis pathways. The levels of p53 mRNA and protein decreased, while the levels of p21WAF1 increased after treatment with VPA, suggesting that VPA exerted the growth inhibition effect through a p21-dependent, but p53-independent pathway. The protein levels of pro-caspases 2, 3, 6 and 9 decreased, accompanied by simultaneous increased levels of cleaved caspases 3 and 9, and cleaved PARP. Addition of a caspase inhibitor Z-VAD-FMK to VPA-treated 17B cells abolished VPA-induced apoptosis, indicating that VPA induced apoptosis through a cascade of caspases. VPA treatment did not change the protein levels of Fas, Bax, Bcl-2 or caspase 8. Together, our data suggest that Valproic acid has growth inhibitory and apoptosis-inducing activity in HNSCC cells in vitro and should be further investigated for therapeutic use in patients with this malignancy.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]