Figure 1.
Chemical structures of the anthracyclines and the topoisomerase II catalytic inhibitors used in this study.
Anthracyclines doxorubicin (DOX) and daunorubicin (DAU) and topoisomerase II catalytic inhibitors dexrazoxane (DEX), sobuzoxane (SOB) and merbarone (MER) were used in this study.
Figure 2.
Protection of neonatal rat cardiomyocytes from toxicities induced by anthracyclines and hydrogen peroxide (H2O2).
Cells were pre-incubated with three concentrations of dexrazoxane for 3 h and then co-incubated with increasing concentrations of anthracyclines daunorubicin (DAU, A) or doxorubicin (DOX, B) for 3 h following a 48-h anthracycline-free period or for 48 h with H2O2 (C). Toxicity was assessed as the % of total lactate dehydrogenase (LDH) released from cardiomyocytes into the cell culture medium. Data obtained from ≥4 independent experiments are expressed as the mean ± SD, statistical significance: c – compared with drug-free control (DMSO); d – compared with DAU or DOX; p – compared with H2O2 (one-way ANOVA with Dunnett’s post-test, P≤0.05).
Figure 3.
Effects of the studied substances on the viability and glutathione levels in neonatal rat cardiomyocytes.
Neonatal rat cardiomyocytes were pre-incubated with dexrazoxane (DEX), sobuzoxane (SOB) or merbarone (MER) for 3 h and then incubated with anthracyclines daunorubicin (DAU, A) or doxorubicin (DOX, B) for 3 h following a 48-h anthracycline-free period or for 48 h with hydrogen peroxide (H2O2, C). DEX, SOB and MER individual toxicity after 48-h incubation is shown in panel D. Effects of DEX pre-treatment for 3 h with a 3-h DAU incubation and subsequent 48-h DAU-free period on cellular oxidised and reduced glutathione content are shown in panel E. Data obtained from ≥ 4 independent experiments are expressed as the mean ± SD, statistical significance: c – compared to control; d – compared to DAU or DOX (one-way ANOVA with Dunnett’s post-test, P≤0.05).
Figure 4.
Effects of dexrazoxane, sobuzoxane or merbarone and daunorubicin or doxorubicin on the proliferation of HL-60 cell line.
Cells were incubated either continuously with dexrazoxane (DEX, A), sobuzoxane (SOB, B) or merbarone (MER, C) and daunorubicin (DAU) or doxorubicin (DOX) for 72 h or pre-incubated with DEX (A), SOB (B) or MER (C) for 3 h or 6 h and then incubated for 72 h with all drugs at concentrations corresponding to their IC50 values and IC50 fractions and multiples (1/8; 1/4; 1/2; 1; 2; 4). Alternatively, cells were either co-incubated for 72 h or pre-incubated with DEX for 3 h and then co-incubated with DAU for 72 h at a ratio of 1:20 DAU:DEX (A). Computer simulations of combination index (CI) - cellular proliferation (fraction affected - Fa) dependences were obtained using CalcuSyn 2.0 software.
Figure 5.
Caspase activities in cardiomyocytes and the HL-60 cell line.
Neonatal rat cardiomyocytes (A - F) were pre-incubated with 30 µM dexrazoxane (DEX), sobuzoxane (SOB) and merbarone (MER) for 3 h and then co-incubated with 1.2 µM daunorubicin (DAU; A - C) or doxorubicin (DOX; D - F) for 3 h, followed by an anthracycline-free 48-h period. The HL-60 cell line (G - I) was co-incubated with 25 µM DEX, 38 µM SOB, 48 µM MER and 19 nM doxorubicin (DOX) for 72 h. The activity of caspases 3/7 (A, D, G), 8 (B, E, H) and 9 (C, F, I) was assessed with a chemiluminescence assay as described in the materials and methods, corrected for protein content and expressed as the percentage of untreated control cells (100%). Data from ≥4 experiments are expressed as the mean ± SD, statistical significance c – compared to control; d – compared to DAU or DOX; n.s. – not significant (one-way ANOVA with Dunnett’s post-test, P≤0.05).
Figure 6.
The human acute promyelocytic leukaemia HL-60 cell line was incubated with dexrazoxane (DEX), sobuzoxane (SOB), merbarone (MER) and doxorubicin (DOX) and combinations thereof in concentrations corresponding to their IC50 values for 72 h and then subjected to flow cytometry cell cycle analysis as described in the materials and methods. Data from 4 independent experiments are expressed as the mean ± SD, statistical significance: c – compared to untreated control cells; d – compared to DOX-treated cells (one-way ANOVA with Dunnett’s post-test, P≤0.05).
Figure 7.
Intracellular iron chelation properties.
The intracellular iron (Fe) chelation by dexrazoxane (DEX), sobuzoxane (SOB) and merbarone (MER) was measured as the rate of Fe displacement from the Fe-calcein complex in H9c2 cardiomyoblast cell line. The change of fluorescence of the intracellular-trapped calcein in the H9c2 cells loaded with 100 µM ferric-ammonium citrate was assessed after adding the studied substances (100 µM) as described in Materials and methods section. The experimental strong Fe chelator SIH was used as a positive control. Data from 4 independent experiments are expressed as mean ± SD. Statistical significance: Two-way ANOVA Dunnet’s post-hoc test, P≤0.05; c–compared to control.