New Insights About Doxorubicin-Induced Toxicity to Cardiomyoblast-Derived H9C2 Cells and Dexrazoxane Cytoprotective Effect: Contribution of In Vitro1H-NMR Metabonomics

Abstract

Doxorubicin (DOX) is an anticancer drug widely used in oncology. The main limitation to DOX treatments though is due to the cumulative dose that may lead to cardiotoxicity. Clinically, DOX-induced cardiomyopathy develops as a progressive heart failure consecutive to a progressive loss in cardiomyocytes due to cell necrosis and apoptosis induced by DOX. For many years, the cardiac oxidative stress caused by DOX was considered as its main toxic mechanism. Therefore, several clinical trials were carried out to assess the efficacy of various antioxidants as a cardioprotective strategy. Only dexrazoxane (DEX), did significantly reduce DOX cardiotoxicity. However, since other antioxidants used later on to counteract DOX cardiotoxicity were not as successful as DEX, DOX-induced oxidative stress and DEX antioxidant activity are not considered as the main feature anymore and this led the scientific world to suspect other involved mechanisms which are still unknown. The objective of the present work was to study from a metabolic point of view the side effects of DOX and the protective properties of DEX. In vitro 1 H-NMR metabonomics was applied to the rat cardiomyoblastic H9C2 cell line. This strategy was used with the hope of unveiling possible new targets to cope with DOX cardiotoxicity. Another underlying goal was the validation of H9C2 in vitro model for metabolic investigations of DOX and DEX effects. For this purpose, several parameters, including oxidative stress, cell mortality, and apoptosis, were measured to assess the effects of DOX and DEX alone or in combination. The metabonomic study was carried out on cellular fluids collected after either 4 or 24 hours of DOX-exposure. Under such experimental conditions, both the major adverse effects reported in patients exposed to DOX and the protective effect of DEX were demonstrated in vitro, suggesting that the H9C2 in vitro model is relevant to investigate both DOX cardiotoxicity and putative cardioprotective strategies. In addition, the metabonomics findings highlighted several metabolic pathways involved in DOX cardiotoxicity and DEX cardioprotective effects as

GPR91 Receptor Mediates Protection against Doxorubicin-Induced Cardiotoxicity without Altering Its Anticancer Efficacy. An In Vitro Study on H9C2 Cardiomyoblasts and Breast Cancer-Derived MCF-7 Cells

“…The impacts of DOX, DEX, succinate (SUC) and cis -ES on the H9C2 cell viability were assessed by a crystal violet procedure in 96-well plates, as previously mentioned [ 17 ]. After cell exposure, the culture medium was removed, and cells were washed twice with PBS and incubated with 100 µL of glutaraldehyde 1% for 15 min at room temperature (RT).…”

“…The ability of DOX, DEX, SUC and cis -ES to induce or prevent apoptosis was indirectly assessed using the caspase 3 enzyme activity, as previously described [ 17 ]. H9C2 cells were randomly split in different conditions and were exposed to DOX 5 µM, DEX 50 µM, SUC 50 µM and cis -ES 50 µM in 96-well plates.…”

“…Samples were centrifuged at 10,000× g during 10 min. Fifty microliters of trimethylsilylpropanoic acid (TSP) 14 mM was added to the supernatant (650 µL) in NMR tubes [ 17 ].…”

“…We highlighted that exposing cells to DEX prior to DOX caused an oversecretion of succinate in the culture medium. This led us to hypothesize that the release of succinate in DEX-exposed cells could protect against DOX-induced cardiotoxicity in H9C2 cells by an autocrine activation of the GPR91 receptor and the subsequent triggering of cell survival pathways [ 17 ].…”

NMR as a “Gold Standard” Method in Drug Design and Discovery

“…For these reasons, NMR spectroscopy is highly sought after in drug development [ 37 , 38 , 39 , 40 , 41 ], for both molecule identification [ 11 , 13 , 14 , 18 , 42 , 43 , 44 , 45 , 46 ] and structural elucidation [ 15 , 16 , 17 , 45 , 47 , 48 , 49 , 50 , 51 ]. NMR has been successfully applied in stereochemistry [ 52 , 53 , 54 , 55 , 56 ] and isomer determination [ 57 , 58 , 59 , 60 , 61 ], in drug-protein interactions studies [ 62 , 63 , 64 ], and in the evaluation of drug toxicity [ 65 , 66 , 67 , 68 ].…”