Suberoylanilide hydroxamic acid (vorinostat) represses androgen receptor expression and acts synergistically with an androgen receptor antagonist to inhibit prostate cancer cell proliferation

Effect of SAHA on cell cycle distribution in LNCaP cells. A, cells cultured with SAHA (0, 2.5, or 7.5 μmol/L) for 24 h were harvested by trypsinization, fixed in 70% ethanol, and stained with propidium iodide. Cell cycle distribution was determined by flow cytometry. B, cells cultured in the absence or presence of SAHA (7.5 μmol/L) for 24 h or 4 d were harvested and fixed as described above and stained with propidium iodide. The fraction of hypodiploid (sub-G₁) nuclei was measured by flow cytometry using standard histogram analysis.

Induction of LNCaP cell death by SAHA. A, LNCaP cells (2.5 × 10⁴ per well in 24-well plates) were cultured with SAHA (0, 2.5, or 7.5 μmol/L) with or without the z-VAD-fmk caspase inhibitor for 48 h and assayed for caspase-3 activity. B, LNCaP cells (2.5 × 10⁴) were cultured with SAHA (0, 2.5, or 7.5 μmol/L) with or without the z-VAD-fmk caspase inhibitor for 5 d. Cells were counted at day 5 using a hemocytometer, and cell viability was assessed by trypan blue exclusion. The number of dead cells expressed as a percentage of total cells was counted. Columns, mean of triplicate wells in a representative experiment; bars, SE. C, DAPI staining of cell nuclei. LNCaP cells were seeded onto plastic coverslips and cultured with vehicle alone (DMSO) or SAHA (7.5 μmol/L) for 24 h. Cells were fixed with methanol and incubated with DAPI, before washing in PBS and mounting on PBS/glycerin. DAPI staining was visualized by fluorescence microscopy. D, effect of SAHA on mitochondrial membrane potential in LNCaP cells. Cells were cultured with vehicle alone (DMSO) or SAHA (7.5 μmol/L) for 48 h. Cells were harvested by trypsinization, resuspended in RPMI 1640 containing 10% FCS, and incubated with the mitochondrial dye rhodamine 123 (Rho123; 2 μg/mL) for 20 min at 37°C. Cells were washed and incubated in PBS containing 2 μg/mL of the viability dye 7-aminoactinomycin D at room temperature for 10 min and then analyzed by flow cytometry. Results shown indicate the level of rhodamine 123 fluorescence in the 7-aminoactinomycin D–negative population.

Summary of gene expression changes in LNCaP cells following SAHA treatment. A, scatter plot of control intensity versus SAHA (2.5 μmol/L) intensity for all elements on the UniGEM version 2.0 human cDNA microarray, which contains 8,372 unique gene sequences. B, Venn diagrams depicting the number of genes with ≥2-fold changes in mRNA levels. Top, the number of genes induced; bottom, the number of genes repressed by 2.5 and/or 7.5 μmol/L SAHA. C, summary of SAHA-induced alterations in expression of genes involved in androgen signaling. D, real-time PCR analysis of AR, PSA, and kallikrein 2 (KLK2) mRNA levels 2 h following treatment with SAHA.

Protein expression changes in LNCaP cells following culture with SAHA. Lysates from cells cultured in the absence or presence of SAHA (2.5, 5, or 7.5 μmol/L) for 12, 24, or 48 h were analyzed by immunoblotting for expression of AR and PSA (A), Her2/neu (B), and cyclin D1 and p21^WAF1(C). For each immunoblot, detection of calnexin was used as a loading control (bottom).

Effect of SAHA in combination with androgen withdrawal on AR expression, cell proliferation, and cell death. A, LNCaP cells (5 × 10⁵ per well in six-well plates) were cultured for 24 h with SAHA (0, 2.5, or 7.5 μmol/L) in the presence or absence of 5α-dihydrotestosterone (DHT; 1 nmol/L), in either regular RPMI 1640 with 10% FCS (left) or phenol red–free RPMI 1640 with 10% charcoal-stripped FCS (right). Cells were lysed and analyzed by immunoblotting for expression of AR, and calnexin was used as a loading control. B and C, LNCaP cells (2.5 × 10⁴ per well in 24-well plates) were cultured in medium containing charcoal-stripped FCS in the absence or presence of SAHA for up to 7 d. B, cells were counted every 2nd day using a hemocytometer, and cell viability was assessed by trypan blue exclusion. C, the number of dead cells is expressed as a percentage of total cells counted. Points, mean of triplicate wells in a representative experiment; bars, SE.

Effect of SAHA in combination with the AR antagonist bicalutamide on LNCaP cell proliferation and cell death. A and B, cells (2.5 × 10⁴ per well in 24-well plates) were cultured in the absence or presence of 0.5 μmol/L SAHA and 1.25 μmol/L bicalutamide, either alone or in combination. A, cells were counted every 2nd day using a hemocytometer, and cell viability was assessed by trypan blue exclusion. B, the number of dead cells is expressed as a percentage of total cells counted. Points, mean of triplicate wells in a representative experiment; bars, SE. C and D, LNCaP cells (2.5 × 10⁴ per well in 24-well plates) were cultured with 0.5 μmol/L SAHA and 1.25 μmol/L bicalutamide, alone or in combination, in the presence or absence of (C) the z-VAD-fmk caspase inhibitor or (D) the androgen 5α-dihydrotestosterone (10 nmol/L) for 5 d. Cell viability was assessed as described above. E, lysates from untreated LNCaP cells (U) and cells cultured with 0.5 μmol/L SAHA (S), 1.25 μmol/L bicalutamide (B), or SAHA and bicalutamide (S+B) were analyzed by immunoblotting for expression of AR and PSA. Calnexin was used as a loading control (bottom).