The effects of the histone deacetylase inhibitor romidepsin (FK228) are enhanced by aspirin (ASA) in COX-1 positive ovarian cancer cells through augmentation of p21

Abstract

Histone deacetylase (HDAC) inhibitors have shown preclinical efficacy in solid tumors, including ovarian cancers. Our group has published that the HDAC inhibitor, romidepsin (FK228) suppresses ovarian cancer cell growth at nanomolar concentrations in vitro. HDAC inhibitors appear to be even more effective when used in combination with other antitumor agents. However, it remains unclear which antitumor agents are best suited for combination therapy. A recent report suggested that aspirin (acetylsalicylic acid, ASA ) is synergistic with HDAC inhibitors in ovarian cancer cells. ASA is a relatively selective inhibitor of cyclooxygenase-1 (COX-1) and has anti-proliferative effects in ovarian cancer cells. The goal of this study was to investigate the impact of ASA on the activity of the HDAC inhibitor, FK228 in COX-1 positive (OVCAR-3) and COX-1 negative (SKOV-3) human ovarian cancer cell lines. The growth inhibitory effects of FK228 were enhanced by ASA in COX-1 positive ovarian cancer cells. In contrast, ASA had no influence on the results of FK228 treatment in COX-1 negative ovarian cancer cells. Upregulation of the cell cycle control protein p21 was induced robustly by FK228 in both cell lines. In the COX-1 positive cells, p21 expression was augmented by the addition of ASA to FK228 treatment. Furthermore, COX-1 siRNA attenuated the effects of combined ASA and FK228 on the levels of p21 expression and the amount of growth inhibition. The additional increase in p21 by ASA in FK228-treated cells was not observed at the promoter or transcriptional levels. However, a significant delay in p21 protein degradation in the presence of ASA and FK228 in COX-1 positive cells was associated with inhibition of proteasome activity. Our study provides a potential rationale for combining ASA with HDAC inhibitors in a subset of ovarian cancers.

Figure 1. Aspirin enhances the growth inhibitory effects of FK228 in COX-1 expressing OVCAR-3 ovarian cancer cells

(A) COX-1 and HDAC protein expression in the ovarian cancer cell lines OVCAR-3 and SKOV-3 by Western blot. (B) Combined effects of ASA and FK228 on cell proliferation in OVCAR-3 and SKOV-3 ovarian cancer cells. Cells were treated for 48 h with ASA alone (0.25, 0.5 and 1 mM), FK228 alone (4, 8 and 16 nM) and the combination in a dose-dependent manner. Experiments were performed in triplicate and all data are shown as mean ± SE. Different letters indicate significance (p ≤ 0.05) with ANOVA and Tukey’s pairwise analyses.

Figure 2. ASA, FK228 and the combination of drugs induce a G0/G1 cell cycle arrest and increase sub-diploid DNA content in OVCAR-3 ovarian cancer cells

Cells were treated for 48 h with ASA (1 mM), FK228 (4, 8 and 16 nM) or the combination. Flow cytometry assays were performed to determine the percentage of cells in each phase of the cell cycle and the percentage of cells with sub-diploid DNA content as a measure of apoptosis. Experiments were performed in triplicate and all data are shown as mean ± SE. Asterisks indicate significance (p ≤ 0.05) using the paired Student’s t-test.

Figure 3. ASA increases and prolongs FK228-induced p21 expression in OVCAR-3 ovarian cancer cells

OVCAR-3 and SKOV-3 cells were treated with FK228 (8 nM), ASA (1 mM) or both for 24 and 48 h. The expression levels of p21 were measured by Western blot.

Figure 4. Silencing COX-1 partially reverses the growth inhibitory effects of ASA on FK228-induced p21 expression and growth inhibition in OVCAR-3 ovarian cancer cells

(A) Effects of COX-1 siRNA on p21 induced by ASA and FK228. Gene silencing with siRNA strategies was performed as described in Materials and Methods, with control samples being incubated with non-targeted siRNA sequences. After 72h transfection, OVCAR-3 cells were treated with FK228 (8 nM) alone and the combination of ASA (1 mM) for 24 h. Western blots were performed using antibodies for COX-1, p21 and a loading control β-actin. These figures represent data from duplicate experiments. C = control; A = ASA; F = FK228; FA = FK228+ASA. (B) Effects of COX-1 siRNA on cell growth suppressed by ASA and FK228. After 72h transfection, OVCAR-3 cells were treated with FK228 (8 and 16 nM) alone and the combination of ASA (1 mM) for 48 h and the rate of cell proliferation was measured using MTT assays. Experiments were performed in triplicate and all data are shown as mean ± SE. Asterisks indicate significance (p ≤ 0.05) with the paired Student’s t-test after ASA treatment.

Figure 5. ASA is not associated with FK228-induced p21 expression at the promoter or transcriptional levels in ovarian cancer cells

(A) The effect of ASA on FK228-induced p21 promoter activity. Cells were transfected for 3 h with the p21 promoter (500 ng/ml). Where indicated, cells were incubated with FK228 (8 nM), ASA (1 mM) or both overnight. The luciferase activity was normalized to total protein concentrations and expressed as a fold change in comparison to control. A gray bar indicates significant increases (p ≤ 0.05) when calculated by ANOVA and Tukey’s pairwise comparisons. (B) Effects of ASA on FK228-induced p21 mRNA. OVCAR-3 cells were incubated with FK228 (FK, 8 nM), ASA (1 mM) or both for 24 h. After isolating total RNA, RT-PCR was performed using specific primers for p21 and GAPDH as a loading control with 30 cycles. A representative result is shown. The first lane is the molecular marker (M).

Figure 6. ASA increases FK228-induced p21 protein stability

(A) OVCAR-3 cells were incubated with FK228 (8 nM) alone and the combination of ASA (1 mM) for 24 h and added with cycloheximide (5 µM) for 1, 2, 4, 6 and 8 h. The signal intensities were quantified by densitometry using Quantity One (Biorad, Hercules, CA). Absolute values were obtained after determining the average density of each spot, the background with minimum value and the interquartile ranges. Experiments were performed in triplicate and a representative result is shown. (B) The effect of ASA on proteasome activity. OVCAR-3 cells were treated for 24 and 48 h with ASA (1 mM), FK228 (8 and 16 nM) or the combination. Cell-based proteasome assays were performed in triplicate and all data are shown as mean ± SE. Black bars indicate significance (p ≤ 0.05) with the paired Student’s t-test.