Cláudia M. Deus

Stimulating basal mitochondrial respiration decreases doxorubicin apoptotic signaling in H9c2 cardiomyoblasts

Doxorubicin (DOX) is currently used in cancer chemotherapy, however, its use often results in adverse effects highlighted by the development of cardiomyopathy and ultimately heart failure. Interestingly, DOX cardiotoxicity is decreased by resveratrol or by physical activity, suggesting that increased mitochondrial activity may be protective. Conversely, recent studies showed that troglitazone, a PPARγ agonist, increases the cytotoxicity of DOX against breast cancer cells by up-regulating mitochondrial biogenesis. The hypothesis for the current investigation was that DOX cytotoxicity in H9c2 cardiomyoblasts is decreased when mitochondrial capacity is increased. We focused on several end-points for DOX cytotoxicity, including loss of cell mass, apoptotic signaling and alterations of autophagic-related proteins. Our results show that a galactose-based, modified cell culture medium increased H9c2 basal mitochondrial respiration, protein content, and mtDNA copy number without increasing maximal or spare respiratory capacity. H9c2 cardiomyoblasts cultured in the galactose-modified media showed lower DOX-induced activation of the apoptotic pathway, measured by decreased caspase-3 and -9 activation, and lower p53 expression, although ultimately loss of cells was not prevented. Treatment with the PPARγ agonist troglitazone had no effect on DOX toxicity in this cardiac cell line, which agrees with the fact that troglitazone did not increase mitochondrial DNA content or capacity at the concentrations and duration of exposure used in this investigation. Our results show that mitochondrial remodeling caused by stimulating basal rates of oxidative phosphorylation decreased DOX-induced apoptotic signaling and increased DOX-induced autophagy in H9c2 cardiomyoblasts. The differential effect on cytotoxicity in cardiac versus breast cancer cell lines suggests a possible overall improvement in the clinical efficacy for doxorubicin in treating cancer.

Sirtuin 1-dependent resveratrol cytotoxicity and pro-differentiation activity on breast cancer cells

Sirtuins regulate several processes associated with tumor development. Resveratrol was shown to stimulate sirtuin 1 and 3 (SIRT1/3) activities and to result in cytotoxicity for some tumor types. The relationship between modulation of sirtuin activities, cellular metabolic remodeling and resveratrol cytotoxicity mechanism on breast cancer cells is still an open question. Here, we evaluated whether sirtuin 1 and 3 are involved in resveratrol toxicity and whether resveratrol leads to a metabolic remodeling and cell differentiation. Results using the Extracellular Flux Analyzer indicated that resveratrol inhibits mitochondrial respiration in breast cancer cells. We also demonstrated here for the first time that resveratrol cytotoxic effects on breast cancer cells were modulated by SIRT1 and also involved mitochondrial complex I inhibition. Importantly, we also demonstrated that resveratrol reduced the pool of breast cancer cells with stemness markers through a SIRT1-dependent mechanism. Our data highlights the role of SIRT1 in regulating resveratrol induced differentiation and/or toxicity in breast cancer cells.

Targeting mitochondrial function for the treatment of breast cancer

There are many approaches used to control breast cancer, although the most efficient strategy is the reactivation of apoptosis. Since mitochondria play an important role in cellular metabolism and homeostasis, as well as in the regulation of cell death pathways, we focus here on metabolic remodeling and mitochondrial alterations present in breast tumor cells. We review strategies including classes of compounds and delivery systems that target metabolic and specific mitochondrial alterations to kill tumor cells without affecting their normal counterparts. We present here the arguments for the improvement of already existent molecules and the design of novel promising anticancer drug candidates that target breast cancer mitochondria.

Berberine-induced cardioprotection and Sirt3 modulation in doxorubicin-treated H9c2 cardiomyoblasts

Doxorubicin (DOX) is one of the most widely used anti-neoplastic agents. However, treatment with DOX is associated with cumulative cardiotoxicity inducing progressive cardiomyocyte death. Sirtuin 3 (Sirt3), a mitochondrial deacetylase, regulates the activity of proteins involved in apoptosis, autophagy and metabolism. Our hypothesis is that pharmacological modulation by berberine (BER) pre-conditioning of Sirt3 protein levels decreases DOX-induced cardiotoxicity. Our results showed that DOX induces cell death in all experimental groups. Increase in Sirt3 content by transfection-mediated overexpression decreased DOX cytotoxicity, mostly by maintaining mitochondrial network integrity and reducing oxidative stress. p53 was upregulated by DOX, and appeared to be a direct target of Sirt3, suggesting that Sirt3-mediated protection against cell death could be related to this protein. BER pre-treatment increased Sirt3 and Sirt1 protein levels in the presence of DOX and inhibited DOX-induced caspase 9 and 3-like activation. Moreover, BER modulated autophagy in DOX-treated H9c2 cardiomyoblasts. Interestingly, mitochondrial biogenesis markers were upregulated in in BER/DOX-treated cells. Sirt3 over-expression contributes to decrease DOX cytotoxicity on H9c2 cardiomyoblasts, while BER can be used as a modulator of Sirtuin function and cell quality control pathways to decrease DOX toxicity.

Protective Effect of Green Tea (Camellia sinensis (L.) Kuntze) against Prostate Cancer: From In Vitro Data to Algerian Patients

Green tea (GT) has been studied for its effects as antioxidant and cancer-preventive agent. Epidemiological studies showed that GT consumption decreases the risk for prostate cancer (PC). To investigate whether erythrocyte oxidative stress (OS) is associated with PC and whether daily consumption of GT improves the oxidative phenotype, we performed a study in a group of Algerian PC patients, preceded by an in vitro study to characterize composition and antioxidant/antiproliferative activities of the GT used. This contained a high content of phenolic and flavonoid compounds, demonstrating in vitro antioxidant activity and significant antiproliferative effect on human prostate cancer PC-3 cell line. Seventy PC patients and 120 age-matched healthy subjects participated in the study, with glutathione (GSH), malondialdehyde (MDA), and catalase activity evaluated before and after GT consumption. The results showed a reduced GSH and catalase activity and a high level of MDA in erythrocytes from PC patients. The consumption of 2-3 cups per day of GT during 6 months significantly increased GSH concentration and catalase activity and decreased MDA concentration. In conclusion, GT significantly decreased OS in Algerian PC patients. Regular consumption of GT for a long period may prevent men from developing PC or at least delay its progression.

Diet, Lifestyles, Family History, and Prostate Cancer Incidence in an East Algerian Patient Group.

Prostate cancer (PC) is the fourth most common cancer in men and the sixth leading cause of death in Algeria. To examine the relationship between lifestyle factors, including diet, and family history and PC risk, a case-control study was performed in an eastern Algerian population, comprising 90 patients with histologically confirmed PC and 190 controls. Data collection was carried out through a structured questionnaire and statistical analysis was performed to evaluate the different variables. The data showed that consumption of lamb and beef meat and high intake of animal fat and dairy products increased PC risk. Seven to thirteen vegetables servings per week and fourteen or more servings decreased PC risk by 62% and 96%, respectively. Seven to fourteen fruit servings per week decrease PC risk by 98%. Green tea consumption reduced the risk of PC but the results were statistically borderline. Increased risk was observed for individuals with family history of PC in first and in second degree. A positive strong association was also found for alcohol and smoking intake and a dose-response relationship existed for quantity and history of smoking. This study suggests that dietary habits, lifestyle factors, and family history have influence on the development of PC in Algerian population.

Mitochondria: Targeting mitochondrial reactive oxygen species with mitochondriotropic polyphenolic-based antioxidants

Mitochondrial function and regulation of redox balance is fundamental in controlling cellular life and death pathways. Antioxidants have been used to counteract disruption of redox networks, normally associated with progressive loss of cell homeostasis and disease pathophysiology, although therapeutic success is limited mainly due to pharmacokinetic drawbacks. Attempts to improve mitochondrial function in a range of diseases spurred active drug discovery efforts. Currently, the most effective strategy to deliver drugs to mitochondria is the covalent link of lipophilic cations to the bioactive compound. Although targeting mitochondrial oxidative stress with antioxidants has been demonstrated, clinical use has been hampered by several challenges, with no FDA-approved drug so far. Development of new mitochondriotropic antioxidant agents based on dietary polyphenols has recently gained momentum. Due to their nature, mitochondria-targeted multi-functional antioxidants can trigger stress responses and contribute to tissue protection through hormesis mechanisms, inhibiting excessive mitochondrial ROS production and associated diseases.

Caffeine Cardiovascular Toxicity: Too Much of a Good Thing

Caffeine is most likely one of the most consumed drug-like molecules in the world. Although a large range of positive effects is associated with caffeine consumption, there is also another side of the coin. Caffeine consumption, especially in susceptible individuals, can trigger cardiovascular events, which can be fatal in some cases. Acute caffeine overconsumption, for example, can increase the incidence of acute myocardial infarction. Also, it has been described that caffeine consumption during pregnancy can alter the fetal cardiovascular system, as well as cause long-lasting alterations in the renin-angiotensin system, which can predispose the offspring for cardiovascular problems later in life. In this chapter we focus not only on the physiological consequences of caffeine toxicity, presenting what is recognized to be the safe limit for it consumption, but also the molecular and cellular (including mediated by the sarcoplasmic reticulum and mitochondria) targets through which caffeine may exert its toxicity.Abstract Caffeine is most likely one of the most consumed drug-like molecules in the world. Although a large range of positive effects is associated with caffeine consumption, there is also another side of the coin. Caffeine consumption, especially in susceptible individuals, can trigger cardiovascular events, which can be fatal in some cases. Acute caffeine overconsumption, for example, can increase the incidence of acute myocardial infarction. Also, it has been described that caffeine consumption during pregnancy can alter the fetal cardiovascular system, as well as cause long-lasting alterations in the renin-angiotensin system, which can predispose the offspring for cardiovascular problems later in life. In this chapter we focus not only on the physiological consequences of caffeine toxicity, presenting what is recognized to be the safe limit for it consumption, but also the molecular and cellular (including mediated by the sarcoplasmic reticulum and mitochondria) targets through which caffeine may exert its toxicity.

Doxorubicin triggers bioenergetic failure and p53 activation in mouse stem cell-derived cardiomyocytes

ABSTRACT Doxorubicin (DOX) is a widely used anticancer drug that could be even more effective if its clinical dosage was not limited because of delayed cardiotoxicity. Beating stem cell‐derived cardiomyocytes are a preferred in vitro model to further uncover the mechanisms of DOX‐induced cardiotoxicity. Our objective was to use cultured induced‐pluripotent stem cell(iPSC)‐derived mouse cardiomyocytes (Cor.At) to investigate the effects of DOX on cell and mitochondrial metabolism, as well as on stress responses. Non‐proliferating and beating Cor.At cells were treated with 0.5 or 1 &mgr;M DOX for 24 h, and morphological, functional and biochemical changes associated with mitochondrial bioenergetics, DNA‐damage response and apoptosis were measured. Both DOX concentrations decreased ATP levels and SOD2 protein levels and induced p53‐dependent caspase activation. However, differential effects were observed for the two DOX concentrations. The highest concentration induced a high degree of apoptosis, with increased nuclear apoptotic morphology, PARP‐1 cleavage and decrease of some OXPHOS protein subunits. At the lowest concentration, DOX increased the expression of p53 target transcripts associated with mitochondria‐dependent apoptosis and decreased transcripts related with DNA‐damage response and glycolysis. Interestingly, cells treated with 0.5 &mgr;M DOX presented an increase in PDK4 transcript levels, accompanied by an increase in phospho‐PDH and decreased PDH activity. This was accompanied by an apparent decrease in basal and maximal oxygen consumption rates (OCR) and in basal extracellular acidification rate (ECAR). Cells pre‐treated with the PDK inhibitor dichloroacetate (DCA), with the aim of restoring PDH activity, partially recovered OCR and ECAR. The results suggest that the higher DOX concentration mainly induces p53‐dependent apoptosis, whereas for the lower DOX concentration the cardiotoxic effects involve bioenergetic failure, unveiling PDH as a possible therapeutic target to decrease DOX cardiotoxicity. HighlightsDOX induced dose‐dependent morphological and functional changes.DOX induced p53‐related caspase activation and decreased SOD2 content.DOX affected p53 target transcripts associated with apoptosis and DNA‐damage.DOX induced PDK4 transcription and PDH inactivation resulting in bioenergetic failure.Bioenergetic dysfunction was ameliorated by pre‐treatment with DCA.

Metabolic and Phenotypic Characterization of Human Skin Fibroblasts After Forcing Oxidative Capacity

Human skin fibroblasts present technical advantages for the study of mitochondrial-induced toxicity, because those cells can be isolated from patients by lowly invasive methods and present specific cumulative cellular damage and mutations of particular conditions. Several drugs lead to organ toxicity, with some of these drugs having been already withdrawn from the market. Frequently, drug-induced toxicity is attributed to mitochondrial liabilities. One of the approaches to identify drug-induced mitochondrial toxicity is using glucose-free/galactose/glutamine/pyruvate-containing cell culture media that force cells to be more dependent on oxidative phosphorylation for energy production. However, the effects of this modified culture medium itself on the mitochondrial phenotype of human skin fibroblasts have not been explored in detail. Our objective was to assess the mitochondrial biology of human skin fibroblasts under standard or modified culture conditions so that system can be validated and used in a more reliable way to disclose mitochondrial liabilities of drug candidates or intrinsic metabolic differences in fibroblasts. Our results showed that forcing mitochondrial remodeling in human skin fibroblasts increased oxygen consumption rate, ATP levels, and mitochondria-related transcripts and proteins. Moreover, the metabolic remodeling increased cytotoxicity of mitochondrial poisons. In general, no alterations in gene expression related with differentiation status were observed in human skin fibroblasts, with exception of increased paxilin gene expression. Not only the current work highlights the importance of using human skin primary cells to study drug-induced mitochondrial toxicity, it also reinforces the use of this tool to detect specific mitochondrial defects in skin fibroblasts from patients.