The role of TAK1 inhibition in doxorubicin-induced cardiotoxicity: an in vitro study

Introduction Doxorubicin, a common chemotherapeutic agent used in the treatment of many cancers, has a limited lifetime cumulative dose because it results in cardiotoxicity, impacting patient prognosis and survival. Doxorubicin causes oxidative stress and myocyte death, both potent inducers of inflammation. Oxidative stress activates toll-like receptors, which activates transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1), a protein that regulates many physiological and pathological processes, including inflammation via activation of nuclear factor-kappa B (NF-κB). However, mechanistic depth on the role of TAK1 signaling in doxorubicin-induced cardiotoxicity is limited. Purpose The aim of this study was to evaluate the effect of TAK1 inhibition on the pathophysiology of doxorubicin-induced cardiotoxicity in H9c2 cells. Methods H9c2 cell lines (Rat BDIX heart myoblast; Sigma Aldrich) were pretreated or not with a TAK1 inhibitor, 5Z-7-oxozeaenol (10 µM), for 2 hours and cultivated in the presence or absence of 25 µM doxorubicin for 24 hours, constituting four treatment groups: control (C), doxorubicin (D), TAK1 inhibitor (I), and doxorubicin + TAK1 inhibitor (DI). The experiments were conducted in triplicate. Protein expression of TAK1, IκB-α, NF-κB, ERK, TNF-α, and IL-10 was assessed in cell extracts by Western blot. Statistical analysis: two-way ANOVA followed by Tukey post-hoc test. A significance level was set at 0.05. Results Doxorubicin caused approximately 27% of death in H9c2 cells, assessed by crystal violet cytotoxicity assay, with no protective effect of TAK1 inhibition. Western blot analysis revealed, as expected, a decrease in TAK1 expression in cells treated with its inhibitor, 5Z-7-oxozeaenol (C: 0.019 ± 0.011; I: 0.072 ± 0.042; D: 0.195 ± 0.113; DI: 0.063 ± 0.036 a.u.; p dox= 0.906; p inhibitor= 0.001; p inhibitor x dox= 0.339). The expression of IkB-α was lower in the I and DI groups compared to the C group, and higher in the DI group compared to the I and D groups (C: 0.876 ± 0.205; I: 0.095 ± 0.060; D: 0.139 ± 0.033; DI: 0.393 ± 0.070 a.u.; p doxo= 0.001; p inhibitor= 0.010; p inhibitor x dox= 0.004). No differences were observed in protein expression of NF-κB, TNF-α, IL-10, or ERK between the experimental groups (Figure). Conclusion Our data suggest that TAK1 does not appear to play a significant role in doxorubicin-induced cardiotoxicity.