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. 2015 Aug;194(2):547-55.
doi: 10.1016/j.juro.2015.02.2918. Epub 2015 Mar 3.

Synergy of Histone-Deacetylase Inhibitor AR-42 with Cisplatin in Bladder Cancer

David R Li  1 , Hanwei Zhang  2 , Elizabeth Peek  3 , Song Wang  4 , Lin Du  5 , Gang Li  6 , Arnold I Chin  7
Affiliations

Synergy of Histone-Deacetylase Inhibitor AR-42 with Cisplatin in Bladder Cancer

David R Li et al. J Urol. 2015 Aug.

Abstract

Purpose: Cisplatin based chemotherapy regimens form the basis of systemic bladder cancer treatment, although they show limited response rates and efficacy. Recent molecular analysis of bladder cancer revealed a high incidence of mutations in chromatin regulatory genes, suggesting a therapeutic avenue for histone deacetylase inhibitors. We investigated the ability of the novel histone deacetylase inhibitor AR-42 to synergize with cisplatin in preclinical models of bladder cancer.

Materials and methods: We assessed the ability of the pan-histone deacetylase inhibitor AR-42 with and without cisplatin to destroy bladder cancer cells by survival and apoptosis assays in vitro, and by growth and differentiation in an in vivo xenograft model. We also assessed the response to the bladder cancer stem cell population by examining the effect of AR-42 on the CD44(+)CD49f(+) population with and without cisplatin. Synergy was calculated using combination indexes.

Results: The AR-42 and cisplatin combination synergistically destroyed bladder cancer cells via apoptosis and it influenced tumor growth and differentiation in vivo. When tested in the CD44(+)CD49f(+) bladder cancer stem cell population, AR-42 showed greater efficacy with and without cisplatin.

Conclusions: AR-42 may be an attractive novel histone deacetylase inhibitor with activity against bladder cancer. Its efficacy in bladder cancer stem cells and synergy with cisplatin warrant further clinical investigation. Our in vitro and animal model studies provide preclinical evidence that AR-42 may be administered in conjunction with cisplatin based chemotherapy to improve the treatment of bladder cancer in patients.

Keywords: apoptosis; chromatin; cisplatin; histone deacetylase inhibitors; urinary bladder neoplasms.

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Figures

Figure 1.
Figure 1.
Viability according to MTT incorporation in SW780 and HT1376 cells treated for 48 hours with titrated doses of single cisplatin and HDAC inhibitor drugs (A). Dashed horizontal lines indicate IC50 (Conc). Data represent mean of triplicate preparations and represent 3 independent experiments. Bars indicate SD. Calculated IC50 concentrations (B). uM, μM.
Figure 2.
Figure 2.
Cisplatin synergized with HDAC inhibitors, including novel broad-spectrum classes I and 2b HDAC inhibitor AR-42. Cisplatin was combined with AR-42, NaB, VA and TSA at ratio of 5:1, 1:75, 1:75 and 80:1, respectively, as determined by each IC50 (Conc) (A). Viability was measured by MTT incorporation in SW780 and HT1376 cells treated for 48 hours. Green curves represent combined therapies. Blue curves represent cisplatin alone. Red curves indicate HDAC inhibitor alone. Data represent mean of triplicate presentations and represent 3 independent experiments. Bars indicate SD. Isobolograms created with CalcuSyn show relationship of cisplatin and each HDAC inhibitor in SW780 cells (B). Combination data points on diagonal, lower left and upper right indicate additive, synergistic and antagonistic effects, respectively.
Figure 3.
Figure 3.
Cisplatin and AR-42 combination decreased CD44+CD49f+ population. SW780 cells treated with indicated doses of cisplatin and AR-42 to achieve approximately IC75 doses were examined by flow cytometry to assess CD44 and CD49f expression in surviving population. Higher 2 μM AR-42 dose was combined with cisplatin to evaluate dose response. Data represent 3 independent experiments. MFI, median fluorescence intensity. Asterisk indicates CD44+CD49f+ cell group comparisons statistically significantly different (1-way ANOVA p <0.05).
Figure 4.
Figure 4.
In vivo combined cisplatin and AR-42 decreased tumor size relative to single treatment or untreated tumors in NSG mice implanted with unsorted (A) or CD44+CD49f+ enriched (B) SW780 cells mixed with fetal bladder mesenchymal cells in Matrigel suspension. Treatments began on day 15 after palpable tumor first presented. Curves indicate mean of 6 tumors. Bars indicate SEM. Asterisk indicates statistically significant (2-way ANOVA p <0.05). Representative unsorted and CD44+CD49f+ enriched tumors (C). CK5 and CK20 staining, reduced from ×400. Cis, cisplatin. Columns indicate mean of percent positive staining cells in 4 representative sections at 400× magnification. Bars indicate SD. Asterisk indicates statistically significant (1-way ANOVA p <0.05).
Figure 5.
Figure 5.
SW780 cells treated with cisplatin (Cis), AR-42 (AR) or cisplatin plus AR-42 for 24 hours showed enhanced apoptosis on annexin V and PI staining followed by flow cytometry (A). Annexin V+PI− cells were considered early apoptotic cells (blue bars). Annexin V+PI+ cells were considered late apoptotic cells (red bars). Live cells were unstained (green bars). Ctrl, control. Single asterisk indicates statistical significance between all populations and other treatment groups (1-way ANOVA p <0.05). Double asterisks indicate statistical significance between all populations and other treatment groups except early apoptotic cells between AR-42 and AR-42 plus cisplatin (1-way ANOVA p <0.05). SW780 cells were treated with varying cisplatin and AR-42 concentrations for 48 hours (B). Viability was assessed by MTT incorporation. Curves indicate mean of triplicate preparations and represent 2 independent experiments (not significant according to 1-way ANOVA p <0.05).
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