HDAC6-selective inhibitors enhance anticancer effects of paclitaxel in ovarian cancer cells

Abstract

Histone deacetylase 6 (HDAC6)-selective inhibitors are potent anticancer agents that are gaining increasing attention and undergoing various developments. These have been approved or are under clinical trials for use with other anticancer agents, such as pomalidomide, anti-programmed death-ligand 1 antibody and paclitaxel, for various types of cancer, including solid tumors. In the present study, a second generation HDAC6-selective inhibitor, ACY-241 (citarinostat), and a novel inhibitor, A452, exhibited synergistic anticancer effects with paclitaxel in AT-rich interaction domain 1A-mutated ovarian cancer in vitro. Co-treatment of paclitaxel and the two HDAC6 inhibitors synergistically decreased cell growth and viability of TOV-21G. Furthermore, the protein expression levels of pro-apoptotic markers, such as poly(ADP-ribose) polymerase, cleaved caspase-3, Bak and Bax, were increased, whereas the expression levels of anti-apoptotic markers, such as Bcl-xL and Bcl-2, were decreased synergistically. Treatment with all drug combinations increased the portion of apoptotic cells in fluorescence-activated cell sorting analysis. These results demonstrated synergy between paclitaxel and HDAC6-selective inhibitors, providing further impetus for clinical trials of combination therapy using HDAC6-selective inhibitors, not only in ovarian cancer but also in other tumors.

Keywords: A452; ACY-241; citarinostat; combination therapy; histone deacetylase 6; ovarian cancer; paclitaxel.

Figure 1.

PTX, ACY-241 and A452 individually suppress the growth and viability of the AT-rich interaction domain 1A-null, p53-wild-type TOV-21G ovarian cancer cell line. (A and B) PTX (0, 0.1, 1, 3, 10, 30 and 100 nM), (C and D) ACY-241 (0, 0.1, 0.3, 1, 3, 10 and 30 µM) and (E and F) A452 (0, 0.1, 0.3, 1, 3, 10 and 30 µM) were used for treatment of TOV-21G cells for 24, 48 and 72 h at the respective concentrations. (A, C and E) Cell growth and (B, D and F) cell viability were measured using Cell Counting Kit-8 assays in 96-well plates. All three drugs inhibited the growth and decreased the viability of TOV-21G cells. Cell counts were estimated indirectly from a standard curve derived from absorbance of known cell numbers. The absorbance at each concentration was normalized to that of the negative control within the corresponding time interval. Data are presented as the mean ± SD (n=3). **P<0.01 and ***P<0.001 vs. the DMSO control (one-way ANOVA with Bonferroni's post hoc test; the significance levels apply to all samples under a line). PTX, paclitaxel.

Figure 2.

PTX and HDAC6-selective inhibitors synergistically decrease cell viability and proliferation in the short and long term. (A) TOV-21G cells were treated with 0.1% DMSO control, PTX, ACY-241 or a combination of these agents for 72 h. (B) TOV-21G cells were treated with 0.1% DMSO control, PTX, A452 or a combination of these agents for 72 h. PTX and both HDAC6-selective inhibitors synergistically reduced cell viability. (C) Combination treatment of PTX and ACY-241 synergistically inhibited the colony formation of TOV-21G cells. (D) Combination treatment of PTX and A452 synergistically inhibited the colony formation of TOV-21G cells. The drug concentrations used for colony formation assays were as follows: 0.1% DMSO, 4 nM PTX, 0.2 µM ACY-241 and 0.1 µM A452. Data are presented as the mean ± SD (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. DMSO control; ^$P<0.05, ^$$P<0.01 and ^$$$P<0.001 vs. PTX; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. ACY-241 or A452, using a two-way ANOVA test for (A) and (B), and one-way ANOVA tests were used for (C) and (D). ACY, ACY-241; Combi, combination treatment; Fa, fraction affected; CI, combination index; PTX, paclitaxel; CFU, colony-forming unit; Ctrl, control; HDAC6, histone deacetylase 6.

Figure 3.

Combination treatments of PTX and histone deacetylase 6-selective inhibitors synergistically suppress the proliferation and migration of TOV-21G cells. (A) PTX (40 nM) and ACY-241 (4 µM) significantly inhibited the wound closure of TOV-21G cells compared with either drug alone. (B) PTX (40 nM) and A452 (2 µM) significantly inhibited the wound closure of TOV-21G cells compared with either drug treatment as monotherapy. Magnification, ×50. Data are presented as the mean ± SD (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. DMSO control; ^$$$P<0.001 vs. PTX; ^###P<0.001 vs. ACY-241 or A452 (one-way ANOVA). Ctrl, control; Combi, combination treatment; PTX, paclitaxel.

Figure 4.

PTX and histone deacetylase 6-selective inhibitors synergistically induce and reduce pro-/anti-apoptotic markers. (A) Combination treatment of PTX (40 nM) and ACY-241 (4 µM) significantly increased pro-apoptotic markers while decreasing anti-apoptotic markers. (B) Combination treatment of PTX (40 nM) and A452 (2 µM) significantly induced pro-apoptotic markers while reducing anti-apoptotic markers. Western blot analysis was performed using the indicated antibodies. The protein expression levels were semi-quantified relative to α-tub or GAPDH. The levels in the 0.1% DMSO group were set as 1. GAPDH was used as the loading control. Data are presented as the mean ± SD (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. DMSO control; ^$P<0.05, ^$$P<0.01 and ^$$$P<0.001 vs. PTX; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. ACY-241 or A452 (one-way ANOVA). Bak, Bcl-2 homologous antagonist/killer; Bcl, B-cell lymphoma; Bcl-xL, Bcl-extra large protein; Cas-3, Caspase-3; Ctrl, control; Combi, combination treatment; PARP, poly(ADP-ribose) polymerase; PTX, paclitaxel.

Figure 5.

Combination therapy of histone deacetylase 6-selective inhibitors and anticancer agent PTX triggers synergistic apoptosis. (A) TOV-21G cells were treated with 0.1% DMSO (control), PTX alone, ACY-241 alone and ACY-241 in combination with PTX for 48 h. Whole-cell lysates were prepared and stained with PI and Annexin V. (B) TOV-21G cells were treated with 0.1% DMSO (control), PTX alone, A452 alone and A452 in combination with PTX for 48 h. Percentages of apoptotic cells were calculated from summation of cells in Q2 and Q4. Data are presented as the mean ± SD (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. DMSO control; ^$$P<0.01 and ^$$$P<0.001 vs. PTX; ^#P<0.05 and ^###P<0.001 vs. ACY-241 or A452 (one-way ANOVA). Ctrl, control; Combi, combination treatment; PI, propidium iodide; PTX, paclitaxel.

Figure 6.

Combined treatment of histone deacetylase 6-selective inhibitors and anticancer agent PTX synergistically alters lysine acetylation modifications of p53. Western blot analysis was performed using the indicated antibodies. (A) Combination treatment of PTX (40 nM) and ACY-241 (4 µM) synergistically altered the acetylation modification of p53. (B) Combination treatment of PTX (40 nM) and A452 (2 µM) synergistically altered acetylation modification of p53. (C) Relative protein levels of acetylated p53 are shown for combination treatment of PTX (40 nM) and ACY-241 (4 µM). (D) Relative protein levels of acetylated p53 are shown for combination treatment of PTX (40 nM) and A452 (2 µM). p53 protein expression was semi-quantified relative to α-tub; acetylated and phosphorylated p53 proteins were semi-quantified relative to total p53 levels. The levels in the 0.1% DMSO group were set as 1. α-tub was used as a loading control. Data are presented as the mean ± SD (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. DMSO control; ^$$P<0.01 and ^$$$P<0.001 vs. PTX; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. ACY-241 or A452, using a one-way ANOVA test. α-tub, α-tubulin; Ac-α-tub, acetylated-α-tubulin; Ctrl, control; Combi, combination treatment; PTX, paclitaxel; p53K120Ac, p53 lysine 120 acetylation; p53K381Ac, p53 lysine 381 acetylation; p-p53S15, p53 serine 15 phosphorylation.