Prevention Of Doxorubicin-Induced Cardiotoxicity With Galenia africana: A Mechanistic Study
Recent evidence suggests that the therapeutic benefits of flavonoids such as pinocembrin (Pin) and 5,7-dihydroxy-2’-methoxyflavanone (5,7-DH) can aid in alleviating the burden of Doxorubicin-induced cardiotoxicity (DIC). In a previous study, Pin alleviated cardiac dysfunction and remodeling during ventricular arrythmia, while 5,7-DH inhibited the activity of carbonyl reductase 1 (CBR1), which is an enzyme that mediates the reduction of Dox to its cardiotoxic metabolites. These findings support the screening of these flavonoids as possible adjuncts to Dox, for the prevention of DIC. Therefore, the aim of this study was to investigate the cardioprotective benefits of various flavonoids against DIC using in vitro and in vivo cardiac experimental models. Furthermore, since novel cardioprotectants have been previously shown to reduce the efficacy of chemotherapeutic agents, we also studied the efficacy of Dox when used in combination with these flavonoids using cancerous experimental models and Vivid® recombinant cytochrome P450 (CYPs) enzymatic assays, which predict possible drug interactions. Molecular determinants of DIC revealed impaired mitochondrial function and autophagy, and increased apoptosis in the H9c2 cells exposed to Dox. However, co-treatment with Dox plus Pin or 5,7-DH, or the flavonoid fractions B1 and B2, which were formulated using different compositions of Pin and 5,7-DH, revealed reduced signs of toxicity which was represented by the improved expression of autophagy (LC3B, Beclin-1 and p-mTOR) and mitochondrial function (PPARγ, PGC1-α, and pAMPK) related proteins, as well as decreased apoptotic and necrotic activity. The results further showed no significant changes in Dox-induced apoptosis in the MCF-7 cancer cells co-treated with Pin and 5,7-DH. However, co-treatment with 5,7-DH and B1 drastically decreased the efficacy of Dox. A comparative analysis on our in vitro findings revealed that Pin had a more efficient therapeutic outcome, as an adjunct to Dox, than 5,7-DH, B1 and B2. Therefore, Pin’s prophylactic benefits against DIC were then investigated in neoplastic and non-neoplastic mice. Corresponding to our in vitro results, co-treatment with Pin alleviated DIC by improving the neoplastic and non-neoplastic mice’s radial and circumferential strain profiles, left ventricular ejection fraction (> 60%) and fractional shortening (> 30%), preserving myocardial structural integrity whilst decreasing interstitial fibrosis. Interestingly, Pin on its own prevented neoplasia-induced myocardial dysfunction and fibrosis, which further confirmed its therapeutic benefits. Moreover, the use of Pin as a cardioprotectant against DIC was substantiated by the comparable rate of tumor regression in neoplastic mice co-treated with Dox + Pin versus those treated with Dox alone. These findings were then validated by Pin’s inhibitory activity on the enzymes, CYP 2D6 and 3A4, which are involved in Dox metabolism. Briefly, inhibition of CYP 2D6 and 3A4 prevents the reduction of Dox to its cardiotoxic-metabolites, allowing for more Dox that is readily available for cancer cytotoxicity. In conclusion, our findings suggest that Pin can be safely administered with Dox to protect the hearts of cancer patients from developing DIC. We also found that while 5,7-DH and B1 are cardioprotective, they should not be used as adjuncts to Dox for the prevention of cardiotoxicity.