In vitro activity: Vorinostat inhibits the activities of HDAC1 and HDAC3 with IC50 of 10 nM and 20 nM, respectively. Vorinostat also results in a marked hyperacetylation of histone H4. Vorinostat inhibits the growth of three prostate cancer cell lines LNCaP, PC-3 and TSU-Pr1 at micromolar concentrations (2.5-7.5 μM), and induces dose-dependent cell death in LNCaP cells. Vorinostat treatment in MCF-7 cells inhibits cell proliferation at an IC50 of 0.75 μM resulting in the accumulation of cells in the G1 and G2-M phase of the cell cycle. Vorinostat also induces differentiation in the estrogen receptor-negative cell line SKBr-3 and the retinoblastoma-negative cell line MDA-468. Vorinostat treatment at 1 μM for 8 hours or more is sufficient to irreversibly induce apoptosis of human multiple myeloma (MM) cells. The gene expression profiles of Vorinostat treated MM cells are not hallmarked by global transcriptional activation, but by coordinated transcriptional changes of specific functional groups of genes such as cytokine-induced proliferative/survival signaling cascades, oncogenes-tumor suppressor genes, regulators of apoptosis, DNA synthesis-repair and cell cycle, and proteasome-ubiquitin function.

Kinase Assay: Immunoprecipitation-HDAC assays, the lysate of Jurkat cells is incubated for 1 hour on ice and cleared by centrifugation at 12,000 g for 10 minutes at 4 °C. Supernatants are precleared with 30 μL of 50% protein G-Sepharose slurry for 1 hour at 4 °C. Beads are pelleted by centrifugation and supernatants are incubated for 1 hour at 4 °C with 10 μg of IgG fraction from anti-HDAC1 or HDAC3 polyclonal antisera (preincubated 2 hours at room temperature with either the homologous or heterologous immunizing peptide). Both antisera are raised in rabbits against the carboxylterminal peptide of HDAC1 and HDAC3 by using synthetic peptides coupled to keyhole limpet hemocyanin. 30 μL of 50% protein G-Sepharose slurry is added for 1 hour at 4 °C. Immune complexes are pelleted by centrifugation and washed three times with 1 mL of lysis buffer. Beads are resuspended in 200 μL of HDAC buffer (20 mM Tris-HCl, pH 8.0/150 mM NaCl/10% glycerol), and the HDAC assay is performed with an 3H-acetylated peptide corresponding to amino acids 1-24 of histone H4. Released [3H]acetic acid is quantified by scintillation counting. For inhibitions studies, the immunoprecipitated complexes are preincubated with the different concentrations of Vorinostat for 30 minutes at 4 °C.

Cell Assay: Cells (LNCaP, PC-3, and TSU-Pr1) are exposed to various concentrations of Vorinostat for 1, 2, 3 and 4 days. Cell viability is assessed by trypan blue dye exclusion.

Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):3003-7; Cancer Res. 2000 Sep 15;60(18):5165-70; Proc Natl Acad Sci U S A. 2004 Jan 13;101(2):540-5.