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 ¹H-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 potential metabolic targets for cardioprotection: energy metabolism, redox balance, as well as phospholipids and proteins metabolism.

Human papilloma virus (HPV) infection has been well-established as a risk factor in head and neck squamous cell carcinoma (HNSCC). The carcinogenic effect of HPV is mainly due to the E6 and E7 oncoproteins, which inhibit the functions of p53 and pRB, respectively. These oncoproteins could also play a role in the Warburg effect, thus favoring tumor immune escape. Here, we demonstrated that the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) is expressed at higher levels in HPV-negative patients than in HPV-positive patients. However, the secretion of MIF is higher in HPV-positive human HNSCC cell lines, than in HPV-negative cell lines. In-HPV positive cells, the half inhibitory concentration (IC₅₀) of MIF inhibitor (4-iodo-6-phenylpyrimidine (4-IPP)) is higher than that in HPV-negative cells. This result was confirmed in vitro and in vivo by the use of murine SCCVII cell lines expressing either E6 or E7, or both E6 and E7. Finally, to examine the mechanism of MIF secretion, we conducted proton nuclear magnetic resonance (¹H-NMR) experiments, and observed that lactate production is increased in both the intracellular and conditioned media of HPV-positive cells. In conclusion, our data suggest that the stimulation of enzymes participating in the Warburg effect by E6 and E7 oncoproteins increases lactate production and hypoxia inducible factor 1α (HIF-1α) expression, and finally induces MIF secretion.

Treatments of metastatic melanoma underwent an impressive development over the past few years, with the emergence of small molecule inhibitors targeting mutated proteins, such as BRAF, NRAS, or cKIT. However, since a significant proportion of patients acquire resistance to these therapies, new strategies are currently being considered to overcome this issue. For this purpose, melanoma cell lines with mutant BRAF, NRAS, or cKIT and with acquired resistances to BRAF, MEK, or cKIT inhibitors, respectively, were investigated using both ¹H-NMR-based metabonomic and protein microarrays. The ¹H-NMR profiles highlighted a similar go and return pattern in the metabolism of the BRAF, NRAS, and cKIT mutated cell lines. Indeed, melanoma cells exposed to mutation-specific inhibitors underwent metabolic disruptions following acute exposure but partially recovered their basal metabolism in long-term exposure, most likely acquiring resistance skills. The protein microarrays inquired about the potential cellular mechanisms used by the resistant cells to escape drug treatment, by showing decreased levels of proteins linked to the drug efficacy, especially in the downstream part of the MAPK signaling pathway. Integrating metabonomic and proteomic findings revealed some metabolic pathways (i.e., glutaminolysis, choline metabolism, glutathione production, glycolysis, oxidative phosphorylation) and key proteins (i.e., EPHA2, DUSP4, and HIF-1A) as potential targets to discard drug resistance.

Characteristic metabolic adaptations are recognized as a cancer hallmark. Breast cancer, like other cancer types, displays cellular respiratory switches—in particular, the Warburg effect—and important fluctuations in the glutamine and choline metabolisms. This cancer remains a world health issue mainly due to the side effects associated with chemotherapy, which force a reduction in the administered dose or even a complete discontinuation of the treatment. For example, Doxorubicin is efficient to treat breast cancer but unfortunately induces severe cardiotoxicity. In the present in vitro study, selected metabolic inhibitors were evaluated alone or in combination as potential treatments against breast cancer. In addition, the same inhibitors were used to possibly potentiate the effects of Doxorubicin. As a result, the combination of CB-839 (glutaminase inhibitor) and Oxamate (lactate dehydrogenase inhibitor) and the combination of CB-839/Oxamate/D609 (a phosphatidylcholine-specific phospholipase C inhibitor) caused significant cell mortality in both MDA-MB-231 and MCF-7, two breast cancer cell lines. Furthermore, all inhibitors were able to improve the efficacy of Doxorubicin on the same cell lines. Those findings are quite encouraging with respect to the clinical goal of reducing the exposure of patients to Doxorubicin and, subsequently, the severity of the associated cardiotoxicity, while keeping the same treatment efficacy.

Doxorubicin (DOX) is an anticancer drug widely used in oncology, especially for breast cancer. The main limitation of DOX treatment is its cardiotoxicity due to the cumulative dose. Clinically, DOX-induced cardiomyopathy develops as a progressive heart failure caused by a progressive cardiomyocyte’s death. For long, the oxidative stress induced by DOX was considered as the main toxic mechanism responsible for heart damage, but it is now controverted, and other processes are investigated to develop cardioprotective strategies. Previously, we studied DOX-induced cardiotoxicity and dexrazoxane (DEX), the only cardioprotective compound authorized by the FDA, by ¹H-NMR metabonomics in H9C2 cells. We observed an increased succinate secretion in the extracellular fluid of DEX-exposed cardiomyocytes, a finding that led us to the hypothesis of a possible protective role of this agonist of the GPR91 receptor. The objective of the present work was to study the effect of succinate (SUC) and cis-epoxysuccinate (cis-ES), two agonists of the GPR91 receptor, on DOX-induced cardiotoxicity to H9C2 cells. To this purpose, several toxicity parameters, including cell viability, oxidative stress and apoptosis, as well as the GPR91 expression, were measured to assess the effects of DEX, SUC and cis-ES either alone or in combination with DOX in H9C2 cells. A ¹H-NMR-based metabonomic study was carried out on cellular fluids collected after 24 h to highlight the metabolic changes induced by those protective compounds. Moreover, the effects of each agonist given either alone or in combination with DOX were evaluated on MCF-7 breast cancer cells. GPR91 expression was confirmed in H9C2 cells, while no expression was found in MCF-7 cells. Under such experimental conditions, both SUC and cis-ES decreased partially the cellular mortality, the oxidative stress and the apoptosis induced by DOX. The SUC protective effect was similar to the DEX effect, but the protective effect of cis-ES was higher on oxidative stress and apoptosis. In addition, the metabonomics findings pointed out several metabolic pathways involved in the cardioprotective effects of both GPR91 agonists: the stimulation of aerobic metabolism with glucose as the main fuel, redox balance and phospholipids synthesis. Finally, none of the GPR91 agonists jeopardized the pharmacological effects of DOX on MCF-7 breast cancer cells.