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Comparative Study
. 2019 Jun 22;20(12):3052.
doi: 10.3390/ijms20123052.

Class I-Histone Deacetylase (HDAC) Inhibition Is Superior to pan-HDAC Inhibition in Modulating Cisplatin Potency in High Grade Serous Ovarian Cancer Cell Lines

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Free PMC article
Comparative Study

Class I-Histone Deacetylase (HDAC) Inhibition Is Superior to pan-HDAC Inhibition in Modulating Cisplatin Potency in High Grade Serous Ovarian Cancer Cell Lines

Jan J Bandolik et al. Int J Mol Sci. .
Free PMC article

Abstract

High grade serous ovarian cancer (HGSOC) is the most common and aggressive ovarian cancer subtype with the worst clinical outcome due to intrinsic or acquired drug resistance. Standard treatment involves platinum compounds. Cancer development and chemoresistance is often associated with an increase in histone deacetylase (HDAC) activity. The purpose of this study was to examine the potential of HDAC inhibitors (HDACi) to increase platinum potency in HGSOC. Four HGSOC cell lines with different cisplatin sensitivity were treated with combinations of cisplatin and entinostat (class I HDACi), panobinostat (pan-HDACi), or nexturastat A (class IIb HDACi), respectively. Inhibition of class I HDACs by entinostat turned out superior in increasing cisplatin potency than pan-HDAC inhibition in cell viability assays (MTT), apoptosis induction (subG1), and caspase 3/7 activation. Entinostat was synergistic with cisplatin in all cell lines in MTT and caspase activation assays. MTT assays gave combination indices (CI values) < 0.9 indicating synergism. The effect of HDAC inhibitors could be attributed to the upregulation of pro-apoptotic genes (CDNK1A, APAF1, PUMA, BAK1) and downregulation of survivin. In conclusion, the combination of entinostat and cisplatin is synergistic in HGSOC and could be an effective strategy for the treatment of aggressive ovarian cancer.

Keywords: antitumor platinum agents; caspase activity; cisplatin; combination treatment; entinostat; high grade serous ovarian cancer (HGSOC); histone deacetylase inhibitors; nexturastat A; panobinostat.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Characterization of the ovarian cancer cell lines. (a) Cell viability was measured by MTT assay after 72 h incubation with different concentrations of cisplatin. Data shown are mean ± SD of at least three independent experiments each carried out in triplicates, calculated as percentage of control. The cell line specific IC50 values are shown in Table 1. (b) Gene expression profile for HDAC enzymes was obtained by RT-PCR. Data shown are normalized to endogenous control gene expression of HPRT1 (hypoxanthine-guanine phosphoribosyltransferase), TBP (TATA binding protein), and GUSB (beta-glucuronidase). (c) Representative immunoblot analysis of HDAC enzymes. One representative immunoblot with a protein molecular weight marker is shown in Figure S3.
Figure 2
Figure 2
Effect of HDACi on acetylation level of α-tubulin and histone H3. Representative immunoblot analysis of histone H3, ac-histone H3, α-tubulin, and ac-α-tubulin in the different human ovarian cancer cell lines. Cells were treated with the indicated concentrations of HDACi. Control cells were incubated with vehicle. One representative immunoblot with a protein molecular weight marker is shown in Figure S3.
Figure 3
Figure 3
HDACi pretreatment enhances the cytotoxic effects of cisplatin. A2780 (a), CaOV3 (b), HEY (c), Kuramochi (d), and OVSAHO (e) were pretreated with the indicated HDACi 48 h prior to cisplatin (cDDP) administration. After another 72 h, IC50-values were determined by MTT assay. Data shown are normalized to vehicle control and mean ± SD of at least three experiments each carried out in triplicates. The vertical arrows show the antiproliferative effects of HDACi in the absence of cisplatin and the horizontal arrows show the shifts of the IC50-values of cisplatin (absence and presence of HDACi). Statistical analysis was performed using t-test. Levels of significance: ns (p > 0.05); * (p ≤ 0.05); ** (p ≤ 0.01); *** (p ≤ 0.001).
Figure 4
Figure 4
HDACi pretreatment enhances cisplatin-induced apoptosis. A2780 (a), CaOV3 (b), HEY (c), Kuramochi (d), and OVSAHO (e) cells were preincubated with HDACi for 48 h. Cisplatin was added in an IC50 concentration for each cell line for a further incubation period of 24 h. Apoptosis was analyzed by determining the sub-G1 cell fractions by flow cytometry analysis. 100 µM cisplatin served as positive control for apoptosis induction. 0.2% DMSO was added as a control for vehicle treated cells. All experimental conditions were incubated for same time periods. Data are the mean ± SD, n ≥ 2. Statistical analysis to compare the apoptosis induction by cisplatin or HDACi alone and the combination of HDACi and cisplatin was performed using t-test. Levels of significance: ns (p > 0.05); * (p ≤ 0.05); ** (p ≤ 0.01); *** (p ≤ 0.001).
Figure 4
Figure 4
HDACi pretreatment enhances cisplatin-induced apoptosis. A2780 (a), CaOV3 (b), HEY (c), Kuramochi (d), and OVSAHO (e) cells were preincubated with HDACi for 48 h. Cisplatin was added in an IC50 concentration for each cell line for a further incubation period of 24 h. Apoptosis was analyzed by determining the sub-G1 cell fractions by flow cytometry analysis. 100 µM cisplatin served as positive control for apoptosis induction. 0.2% DMSO was added as a control for vehicle treated cells. All experimental conditions were incubated for same time periods. Data are the mean ± SD, n ≥ 2. Statistical analysis to compare the apoptosis induction by cisplatin or HDACi alone and the combination of HDACi and cisplatin was performed using t-test. Levels of significance: ns (p > 0.05); * (p ≤ 0.05); ** (p ≤ 0.01); *** (p ≤ 0.001).
Figure 5
Figure 5
Synergistic effects of the combination of HDACi and cisplatin on apoptosis induction. The sum of single treatment effects (HDACi, cisplatin) are shown in black bars. The extended white bars show the difference (superadditive (= synergistic) part) between the sum of single treatment effects and the effect of combination treatments. Vehicle treated control was subtracted. Data are the mean ± SD. Statistical analysis was performed using t-test. Levels of significance: ns (p > 0.05); * (p ≤ 0.05); ** (p ≤ 0.01); *** (p ≤ 0.001).
Figure 6
Figure 6
Representative fluorescent imaging pictures (10× magnification) are shown for each cell line for the treatment of cisplatin (IC50 concentration), entinostat, and the combination of cisplatin and entinostat. Cell nuclei were stained by Hoechst 33342 and appear blue while cells with activated caspases3/7 showed green fluorescence. Scale bar in upper left image is 100 µm and applies to all images.
Figure 7
Figure 7
A2780 (a), CaOV3 (b), HEY (c), Kuramochi (d), and OVSAHO (e) cells were preincubated with HDACi for 48 h. Cisplatin was added in an IC50 concentration for each cell line for a further incubation period of 24 h. Caspase3/7-activation was analyzed by incubation with CellEvent Caspase-3/7 green detection reagent (Thermo Scientific, Germany) and visualized by ArrayScan XTI. Cisplatin 100 µM (24 h) was added as positive control for caspase3/7-activation. 0.2% DMSO was added as a control for vehicle treated cells. All experimental conditions were incubated for same time periods. To verify the involvement of caspases in the observed effects, 20 µM QVD was preincubated for 30 min prior to compound addition. No caspase3/7-activation was obtained (data not shown). Data are the mean ± SD. Statistical analysis to compare the caspase3/7-activation by cisplatin or HDACi alone and the combination of HDACi and cisplatin was performed using t-test. Levels of significance: ns (p > 0.05); * (p ≤ 0.05); ** (p ≤ 0.01); *** (p ≤ 0.001).
Figure 8
Figure 8
Synergistic effects of caspase3/7-activation upon combination treatment of HDACi and cisplatin. The sum of single treatment effects (HDACi, cisplatin) are shown in black bars. The extended white bars show the difference (superadditive (= synergistic) part) between the sum of single treatment effects and the effect of combination treatments. Results are shown for treatments significantly enhancing caspase3/7-activation (Figure 7). Before calculation, values were normalized to the effect of 100 µM cDDP and vehicle treated control was subtracted. Data shown are mean ± SD. Statistical analysis was performed using t-test. Levels of significance: ns (p > 0.05); * (p ≤ 0.05); ** (p ≤ 0.01); *** (p ≤ 0.001).
Figure 9
Figure 9
Gene expression data were obtained by RT-PCR and analyzed according to Vandesompele [40]. Cells were pretreated with concentrations of HDACi shown in Table 4 for 48 h followed by treatment with cisplatin for 24 h in an IC50 concentration. Data shown are normalized to endogenous control gene expression of HPRT1 (hypoxanthine-guanine phosphoribosyltransferase), TBP (TATA binding protein), and GUSB (beta-glucuronidase) and rescaled to cell line specific control (72 h incubation with vehicle). Data are shown on a decadic logarithmic scale. Negative values marked in green show a lower expression compared to cell line specific control. Positive values marked in red show a higher expression compared to cell line specific control.

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References

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