Mechanisms involved in mitoquinone-mediated protection of H9C2 cells against anti-cancer drug doxorubicin-induced cardiotoxicity

INTRODUCTION: Due to cardiotoxicity, doxorubicin (DOX), a chemotherapeutic agent, has limited use. Our lab has shown that Mitoquinone (MitoQ) given as a pre-treatment exerted better cardioprotection against DOX-induced cell damage than co-treatment. OBJECTIVE: This study investigated the underlying mechanisms associated with pre-treating cardiomyocytes with MitoQ and why it demonstrated more potent protection against Doxorubicin-induced cardiotoxicity when compared to co-treatment. METHODS: Cultured H9c2 cells were dose-dependently treated with DOX alone, MitoQ alone, MitoQ given concurrently with DOX (i.e., co-treatment), or MitoQ given 24 hours prior to DOX (i.e., pre-treatment). Various factors were explored such as cell viability via CCK assay, DOX accumulation, mitochondrial superoxide anions levels measured by MitoSOX assay, and mitochondrial membrane potential (MMP) as measured by JC-10 assay. Western blotting was used to measure the expression of antioxidant enzymes heme oxygenase 1 and superoxide dismutase 1. RESULTS: DOX (.5-50 μM) dose-dependently increased mitochondrial superoxide levels, and reduced cell viability. DOX also reduced the MMP, but demonstrated a plateau effect (≥ 1μM). Furthermore, when compared to the non-treated control, MitoQ alone (0.005– 5μM) reduced mitochondrial superoxide levels with a maximum reduction of 52% ± 1% at 1 μM. Contrastingly, MitoQ (10 μM) became pro-oxidant. Additionally, higher doses of MitoQ (1-10 μM) depolarized the MMP. Pre-treating with MitoQ (≥ 1μM) showed maximal protection, with a significant reduction of DOX accumulation and superoxide anion levels by 22% ± 8% and 53% ± 7% relative to DOX alone, respectively (both p \u3c 0.05), which was better than that of co- treatment. Western blot also demonstrated that pre-treatment upregulated both antioxidant enzymes. CONCLUSION: The findings presented in this study suggested that MitoQ works as an antioxidant against DOX. Pre-treatment showed significantly better cardioprotection than co- treatment due to its more potent antioxidant effects and improved ability toward reducing intracellular DOX accumulation