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Pharmacological concentrations of mitoxantrone are able to activate caspases and dually modify glutathione pathways in HL-1 cardiomyocytes Background: Mitoxantrone (MTX) is an anticancer drug commonly used in metastatic breast cancer, lymphomas, prostatic cancer, and acute leukaemia. Recently, MTX has been also recommended in multiple sclerosis. The cardiac toxicity is clearly reported after therapy with MTX in cumulative doses as low as 100 mg/m2 and it can reach 18% of treated patients. Aim: Study the cardiotoxic mechanisms of MTX in murine HL-1 cardiomyocytes. Methods: Murine HL-1 cardiomyocytes were selected since they maintain several structural and functional features of the adult heart. HL-1 cultured cells were incubated with pharmacological concentrations of MTX (concentration range 0.1 to 10 µM) for 24 and 48h. At these time-points several assays were performed: mitochondrial dysfunction through the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, caspase 3, 8 and 9 activities based on colorimetric assays with their respective substrates and total glutathione measurement based on the DTNB–GSSG redutase recycling assay. For statistical analysis, one-way analysis of variance (ANOVA) was used for the results of MTT and glutathione assays since data passed normality tests. For caspases activity data, ANOVA on ranks was used. When reaching a significant p (p<0.05), a post-hoc test was made to compare all groups. Results are presented in mean ± standard deviation from at least 5 independent experiments. Results: MTX caused a time- and concentration-dependent decrease in mitochondrial activity, evaluated by the MTT assay. At 24h, 10µM MTX decreased to 77.8 ± 6.1%, 1µM MTX to 85.3 ± 5.8%, and 0.1µM MTX to 95.0 ± 7.3% of control values (100 ± 4.8%). At 48h, 10µM MTX decreased to 41.9 ± 3.8%, 1 µM MTX to 48.5 ± 4.2%, and 0.1µM MTX to 69.3 ± 4.4% of control values (100 ± 5.6%). The concentrations of 1 and 10µM MTX caused an early increase (24h) in caspase 9, 8, and 3 activities, whilst at 48h no significant differences were found in MTX-incubated cells when compared to control. At 24h, caspase 3 activity in control was 0.98 ± 0.43 optic density (OD)/mg protein, while in MTX groups, it significantly increased to 3.25 ± 0.94 OD/mg (10µM) and 2.60 ± 1.01 OD/mg (1µM). Meanwhile, caspase 8 activity in control was 1.07 ± 0.72 OD/mg, while a significant increase occurred in 10µM (1.92 ± 0.87 OD/mg) and 1µM (2.49 ± 1.37 OD/mg) MTX groups. Also, activity of caspase 9 in control was 0.45 ± 0.17 OD/mg, while it significantly increased in 10µM MTX (1.32 ± 0.88 OD/mg) and 1µM MTX (0.97 ± 0.19 OD/mg) groups. Furthermore, total cellular glutathione levels presented a biphasic response after MTX exposure. At 24h, total glutathione levels significantly increased at 1µM (11.35 ± 2.20 nmol/mg protein) when compared to control (8.08 ± 2.27 nmol/mg protein), while after a 48h incubation, the values of 1µM MTX were similar to control. In 10 µM-incubated cells, total glutathione levels significantly decreased after a 48h incubation (7.06 ± 1.61 nmol/mg protein) when compared to control (10.94 ± 2.34 nmol/mg protein), whilst at 24h, the values were similar to control. Conclusions: MTX alters several intracellular pathways in HL-1 cardiomyocytes. Our findings can highlight the cardiotoxicity observed in MTX-treated patients and reveal putative markers of this drug. These biomarkers can be targeted pharmacologically to prevent or treat patients affected by MTX-cardiotoxic events. Acknowledgments: This work was supported by “Fundação para a Ciência e Tecnologia” (FCT) (PTDC/SAU-FCF/102958/2008)-QREN initiative with EU/FEDER financing through COMPETE. VMC and JPC acknowledge FCT for their Post-doc grants (SFRH/BPD/63746/2009 and SFRH/BPD/30776/2006), respectively.
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