Exploring the effects of topoisomerase II inhibitor XK469 on anthracycline cardiotoxicity and DNA damage

Figures

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  Figure 1.. Cytotoxicity/protection of dexrazoxane and XK469. Rat neonatal cardiomyocytes were treated with the range of concentrations of dexrazoxane (DEX) (A) and XK469 (B) for 3 h either alone or prior to 3 h incubation with 1.2 µM daunorubicin (DAU). After these 6 h, the drug-containing medium was removed, and cells were incubated in fresh media for the next 48 h. Toxicity was assessed by lactate dehydrogenase (LDH) assay. Structure of XK469 (C) and its analogs evaluated in this study. Statistical analyses: n = 4, mean ± SD, 1-way ANOVA, Holm-Sidak’s post hoc test, p ≤ .05, “c”—compared with untreated control cells, “d”—compared with daunorubicin.

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  Figure 2.. Antiproliferative effects of dexrazoxane (DEX) and XK469 and their influence on antiproliferative efficiency of daunorubicin (DAU). HL-60 leukemic cells were incubated with increasing concentrations of dexrazoxane (A), or XK469 (B) for 72 h and combined with daunorubicin in its IC50 concentration (15 nM). The multiples of the respective IC50 values of individual drugs were used in combination experiments according to Chou-Talalay with 48 h incubations (C, D; CI—combination index). Toxicity was assessed by MTT assay. Statistical analyses: n = 4, mean ± SD, 1-way ANOVA, Holm-Sidak’s post hoc test, p ≤ .05, “c”—compared with control, “d”—compared with daunorubicin.

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  Figure 3.. Long-term continuous cytotoxicity assessments of dexrazoxane, XK469 and their combinations with daunorubicin in rat neonatal cardiomyocytes. Cells were incubated with dexrazoxane (A), or XK469 (C) alone or in combinations with daunorubicin (B, D) for up to 48 h. Cytotoxicity was assessed by the Sytox Green. DMSO (0.1% final concentration) was present in all samples. The statistical analyses: n ≥ 4, mean ± SD, 1-way ANOVA, Dunn’s post hoc test, p ≤ .05, “c”—compared with control; “d”—compared with daunorubicin.

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  Figure 4.. TOP2 assays. TOP2 activity was analyzed by decatenation assay using purified TOP2 isoforms and catenated DNA. Upper panels show quantification of visualized gels of the inhibition by dexrazoxane (A) and XK469 (B). Lower panels show representative gels of the inhibition of TOP2A (C) and TOP2B (D). TARDIS assay of TOP2-DNA covalent complexes in neonatal cardiomyocytes (E) and wild-type HL-60 cells (F) were incubated with increasing concentrations of either dexrazoxane or XK469 for 2 h. About 100 µM ETO was used as the positive control. Data are expressed as median with an interquartile range of intensities of the individual images. Statistical analyses: n = 4, 1-way ANOVA, Holm-Sidak’s post hoc test, p ≤ .05, “e”—compared with a positive control (100 µM ETO).

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  Figure 5.. Cytotoxicity of dexrazoxane or XK469 in TOP2B+/+ and TOP2B−/− in HL-60 cells. The cells were incubated for 72 h with dexrazoxane (A) and XK469 (B). Statistical analyses: n = 4, mean ± SD, 1-way ANOVA, Holm-Sidak’s post hoc test, p ≤ .05.

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  Figure 6.. TOP2B proteasomal degradation in rat neonatal cardiomyocytes. Cells were treated with increasing concentrations of dexrazoxane (DEX) (A) or XK469 (B) for 24 h. Then, TOP2 content in cells was evaluated by immunodetection. Statistical analyses: n = 3–4, mean ± SD, 1-way ANOVA, Holm-Sidak’s post hoc test, p ≤ .05, “c”—compared with drug-free control.

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  Figure 7.. Activation of p53 (pSer392), expression of p21 and DNA damage analyses. Rat neonatal cardiomyocytes were incubated with dexrazoxane (DEX) (orange) and XK469 (blue) either alone or coincubated with 1.2 µM daunorubicin (DAU). The phosphorylated isoform of p53 protein (pSer392) (A, B), the expression of p21 mRNA (C, D), alkali-labile sites (E) and phosphorylation of gH2AX (F) were evaluated (see Supplementary Figures 5–8 for details). Statistical analyses: n = 4, mean ± SD, 1-way ANOVA, Holm-Sidak’s post hoc test, p ≤ .05, “c”—compared with control, “d”—compared with daunorubicin.

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  Figure 8.. Pilot in vivo experiments in rabbits. In acute experiments (single dose of XK469 [6 mg/kg], daunorubicin [3 mg/kg], and their combination, 6 h treatment), the level of p53 protein (A; see Supplementary Figure 9 for details) and expression of p21 gene (B) in the left ventricle were evaluated. In chronic settings (10 weekly treatments by XK469 [6 mg/kg], daunorubicin [3 mg/kg], and their combination) left ventricular fractional shortening (LVFS, C) and systolic function (LV dP/dtmax, D), and cardiac troponin T in plasma (cTnT, E) were evaluated. Statistical analyses: n = 5, mean ± SD, 1-way ANOVA, Holm-Sidak’s post hoc test, p ≤ .05, “c”—compared with control group, “d”—compared with daunorubicin.