HDAC8-Selective Inhibition by PCI-34051 Enhances the Anticancer Effects of ACY-241 in Ovarian Cancer Cells

Abstract

HDAC6 is overexpressed in ovarian cancer and is known to be correlated with tumorigenesis. Accordingly, ACY-241, a selective HDAC6 inhibitor, is currently under clinical trial and has been tested in combination with various drugs. HDAC8, another member of the HDAC family, has recently gained attention as a novel target for cancer therapy. Here, we evaluated the synergistic anticancer effects of PCI-34051 and ACY-241 in ovarian cancer. Among various ovarian cancer cells, PCI-34051 effectively suppresses cell proliferation in wild-type p53 ovarian cancer cells compared with mutant p53 ovarian cancer cells. In ovarian cancer cells harboring wild-type p53, PCI-34051 in combination with ACY-241 synergistically represses cell proliferation, enhances apoptosis, and suppresses cell migration. The expression of pro-apoptotic proteins is synergistically upregulated, whereas the expressions of anti-apoptotic proteins and metastasis-associated proteins are significantly downregulated in combination treatment. Furthermore, the level of acetyl-p53 at K381 is synergistically upregulated upon combination treatment. Overall, co-inhibition of HDAC6 and HDAC8 through selective inhibitors synergistically suppresses cancer cell proliferation and metastasis in p53 wild-type ovarian cancer cells. These results suggest a novel approach to treating ovarian cancer patients and the therapeutic potential in developing HDAC6/8 dual inhibitors.

Keywords: ACY-241; HDAC6; HDAC8; PCI-34051; epigenetics; ovarian cancer.

Figure 1

PCI-34051 suppresses the cell growth and viability of wild-type p53 ovarian cancer cells. (A,C,E,G) Cell growth and (B,D,F,H) viability of wild-type p53 ovarian cancer cell lines: (A,B) TOV-21G and (C,D) A2780 and mutant p53 ovarian cancer cell lines: (E,F) COV318 and (G,H) COV362. Cells were cultured with 0.1% DMSO (control) or PCI-34051 at the indicated concentrations for 24, 48, and 72 h. Cell growth and viability were measured using the CCK-8 assay. Data are presented as the mean ± SD (n = 3). * p < 0.05, ** p < 0.01, or *** p < 0.001 vs. DMSO control.

Figure 2

ACY-241 and PCI-34051 treatment synergistically suppresses short-term and long-term cell proliferation. (A,B) Cell viability of TOV-21G and A2780 cells treated with ACY-241 and PCI-34051 alone or in combination at a ratio of 1:5 for 48 h. Combination index (CI) values were calculated to determine the synergism of ACY-241 and PCI-34051. CI values less than 1 indicate synergism. (C,D) Colony formation in TOV-21G and A2780 cells treated with ACY-241 and PCI-34051 alone or in combination. TOV-21G cells were treated with 0.3 µM ACY-241 and 2 µM PCI-34051 and incubated for 14 days. A2780 cells were treated with 0.6 µM ACY-241 and 4 µM PCI-34051 and incubated for 7 days. Data are presented as the mean ± SD (n = 3). * p < 0.05, ** p < 0.01, or *** p < 0.001 vs. DMSO control; ^## p < 0.01 or ^### p < 0.001 vs. ACY-241-treated group; ^$ p < 0.05, ^$$ p < 0.01, or ^$$$ p < 0.001 vs. PCI-34051-treated group.

Figure 3

ACY-241 and PCI-34051 treatment synergistically induces apoptosis. (A,B) Immunoblotting of pro-apoptotic markers and anti-apoptotic markers in TOV-21G and A2780 cells treated with 3 µM ACY-241 and 20 µM PCI-34051 alone or in combination for 24 h. Protein expression levels were semi-quantified relative to the loading control, α-tubulin. (C,D) Apoptosis analysis of ACY-241 and PCI-34051 in TOV-21G and A2780 cells. Cells treated with 0.2% DMSO, ACY-241 (3 μM), and PCI-34051 (20 μM) alone or in combination for 48 h were double−stained with Annexin V and PI and analyzed by flow cytometry. Data are presented as the mean ± SD (n = 3). * p < 0.05, ** p < 0.01, or *** p < 0.001 vs. DMSO control; ^# p < 0.05, ^## p < 0.01, or ^### p < 0.001 vs. ACY-241-treated group; ^$ p < 0.05, ^$$ p < 0.01, or ^$$$ p < 0.001 vs. PCI-34051-treated group.

Figure 4

ACY-241 and PCI-34051 treatment synergistically suppresses cell migration. (A,B) Immunoblotting of TWIST1, MMP-9, and ZEB1 in TOV-21G and A2780 cells treated with 3 µM ACY-241 and 20 µM PCI-34051 alone or in combination for 24 h. Protein expression levels were semi-quantified relative to the loading control, α-tubulin. (C,E) Transwell migration assay in TOV-21G and A2780. Cells were treated with 3 µM ACY-241 and 20 µM PCI-34051 alone or in combination for 48 h. Migrated cells were stained and photographed at 100× magnification. (D,F) Wound healing assay in TOV-21G and A2780. Cells were treated with 3 µM ACY-241 and 20 µM PCI-34051 alone or in combination for 24 h. The width of the scratch was calculated and photographed at 50× magnification. Data are presented as the mean ± SD (n = 3). * p < 0.05, ** p < 0.01, or *** p < 0.001 vs. DMSO control; ^# p < 0.05. ^## p < 0.01, or ^### p < 0.001 vs. ACY-241-treated group; ^$ p < 0.05, ^$$ p < 0.01, or ^$$$ p < 0.001 vs. PCI-34051-treated group.

Figure 5

ACY-241 and PCI-34051 treatment synergistically enhances p53 stability in p53 wild-type ovarian cancer cells. (A) Immunoblotting of acetyl-p53. Cells were treated with indicated doses of PCI-34051 for 24 h. (B,C) Immunoblotting of acetyl-p53 and p21. Cells were treated with 3 µM ACY-241 and 20 µM PCI-34051 alone or in combination for 24 h. Protein expression levels were semi-quantified relative to the loading control, α-tubulin. Relative protein levels of acetyl-p53 were semi-quantified relative to total p53 levels and relative protein levels of acetyl SMC3 were semi-quantified relative to total SMC3 levels. Data are presented as the mean ± SD (n = 3). * p < 0.05, ** p < 0.01, or *** p < 0.001 vs. DMSO control; ^## p < 0.01, or ^### p < 0.001 vs. ACY-241-treated group; ^$ p < 0.05, ^$$ p < 0.01, or ^$$$ p < 0.001 vs. PCI-34051-treated group.