Mitochondrial Dysfunction Associated with Doxorubicin

Cancer prevalence is scaling up each year. Anthracycline groups are still the best chemotherapeutic agent. The most popular anticancer drug in the group is doxorubicin (DOX). Unfortunately, DOX has potent toxicity on noncancerous tissues, e.g., heart, kidneys, etc. However, it is well documented that the severest toxicity of the drug affects heart tissue. Of course, some reasons have been suggested why and/or how the heart is so vulnerable to toxicity. The primary mechanism responsible for DOX’s cardiospecific toxicity remains unidentified so far; however, mitochondrial dysfunction induced by DOX is now considered one of the leading reasons for DOX’s toxicities and undesired side effects. Mitochondrial reactive oxygen production in the heart is a significant contributor to developing mitochondrial dysfunction-exposed DOX based on a variety of evidence. The objective of this review chapter is to critically evaluate and highlight the role of mitochondria in the development of DOX-induced cardiotoxicity

Study of genetic factors in treatment-related complications in patients with childhood acute lymphoblastic leukemia and post transplantation of hematopoietic stem cells

La leucémie lymphoblastique aiguë (LLA) est le cancer le plus fréquent chez les enfants. Malgré le fait que plus de 80% des enfants atteints de LLA sont aujourd'hui guéris de leur maladie, ce succès a toutefois un prix élevé, car l’exposition aux médicaments cytotoxique et/ou à l’irradiation pendant une période vulnérable du développement de l’enfant peut entraîner des conséquences à long terme. En effet, environ 60% des enfants ayant survécu à une LLA devront vivre avec des problèmes de santé liés au traitement, également appelés effets indésirables tardifs (late-adverse effects, LAEs). Parmi ces derniers, on notera des problèmes métaboliques, l’ostéoporose, une altération des fonctions cognitives ou cardiaques, ainsi que la dépression et l’anxiété. Si certains survivants ne présentent aucune de ces complications, d'autres peuvent en avoir plusieurs. Différents facteurs peuvent contribuer à cette variabilité, notamment le traitement reçu, les caractéristiques de la maladie, les habitudes de vie et, surtout, la constitution génétique du patient. Ce projet s'est concentré sur les biomarqueurs génétiques permettant d'identifier les individus les plus susceptibles de souffrir de LAEs. Récemment, une étude exhaustive (évaluations cliniques, psychosociales et biochimiques) s’est déroulée au CHU Sainte-Justine pour caractériser chacune de ces morbidités chez 250 survivants de la LLA de l'enfant (cohorte PETALE). De plus, on a obtenu le profil génétique de chaque participant. Nous avons utilisé cet ensemble de données et des outils statistiques et bio-informatiques pour réaliser des études d'association comparant la fréquence des variants génétiques chez les survivants ayant développé ou non des LAEs; en particulier, les complications cardiovasculaires et neurocognitives, ainsi que les troubles de l'humeur tels que l'anxiété et la dépression. D'autres facteurs de risque tels que les caractéristiques de traitement et/ou de la leucémie ont été pris en compte lors de l'analyse pour dériver les meilleurs prédicteurs génétiques. Ainsi, en utilisant l'approche des gènes candidats, nous avons identifié les variants communs des gènes MTR, PPARA, ABCC3, CALML5, CACNB2 et PCDHB10 qui étaient associés à des déficits de performance des tests neurocognitifs, tandis que les variants des gènes SLCO1B1 et EPHA5 étaient associés à l'anxiété et à la dépression. Deux variants, rs1805087 dans le gène MTR et rs58225473 dans le gène CACNB2 sont particulièrement intéressants, car ces associations ont été validées dans la cohorte de réplication SJLIFE (St. Jude Children's Research Hospital, Memphis, USA). Les analyses d'association ont été complémentées par une étude d'association à l'échelle de l'exome, qui a identifié plusieurs gènes supplémentaires comme des modulateurs potentiels du risque de développer des complications neurocognitives liées au traitement (gènes AK8 et ZNF382), ainsi que l'anxiété et la dépression (gènes PTPRZ1, MUC16, TNRC6C-AS1, APOL2, C6orf165, EXO5, CYP2W1 et PCMTD1). Le variant rs61732180 du gène ZNF382 a ensuite été validé dans la cohorte de réplication SJLIFE. Également, nous avons effectué des analyses d’association concernant les complications cardiaques liées au traitement qui ont identifié plusieurs nouveaux marqueurs associés à ces complications dans les gènes TTN, NOS1, ABCG2, CBR1, ABCC5, AKR1C3, NOD2 et ZNF267. De plus, nous avons résumé les connaissances actuelles sur les marqueurs pharmacogénomiques qui ont été associés aux effets de cardiotoxicités, induites par les anthracyclines, qui affectent les patients atteints de cancer pédiatrique. Nous avons également inclus un aperçu de l'applicabilité des résultats rapportés, notamment ceux qui ont été validés dans la cohorte PETALE. Par ailleurs, nous nous sommes intéressés aux complications qui surviennent après une greffe de cellules souches hématopoïétiques. Nous avons appliqué des approches bio-informatiques et statistiques similaires pour obtenir un profil plus complet de la composante génétique derrière ces complications potentiellement mortelles. Ainsi, une étude d'association à l'échelle de l'exome a été réalisée dans une cohorte de patients pédiatriques subissant une greffe de cellules souches hématopoïétiques après un régime de conditionnement contenant du busulfan. Nous avons identifié de nouvelles variations génétiques conférant un risque plus élevé de syndrome d'obstruction sinusoïdale (notamment dans les gènes UGT2B10, BHLHE22, et KIAA1715) et de maladie aiguë du greffon contre l'hôte (dans les gènes ERC1, PLEK, NOP9 et SPRED1), qui pourraient être utiles pour des stratégies personnalisées de prévention et de traitement. Ces travaux contribuent à la compréhension de l'influence des facteurs génétiques sur le risque de développer des complications liées au traitement, tant au cours du traitement qu'à long terme. De plus, les marqueurs génétiques signalés ainsi que d'autres facteurs de risque connus peuvent conduire à des modèles de prédiction identifiant les patients à risque accru de ces complications.Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Even though more than 80% of children with ALL are now cured of their disease, this success comes at a high price as exposure to cytotoxic drugs and/or radiation during a vulnerable period of child development may have long-term consequences. In fact, approximately 60% of children who survive ALL will have to live with treatment-related health problems, also called late-adverse effects (LAEs). These include metabolic problems, osteoporosis, impaired cardiac or cognitive functions, as well as depression and anxiety. While some survivors do not have any of these complications, others may have more than one. Different factors can contribute to this variability, in particular, the treatment received, the characteristics of the disease, the lifestyle, and, above all, the genetic makeup of the patient. This project focused on genetic biomarkers capable of identifying the individuals most likely to suffer from LAEs. Recently, an exhaustive study (clinical, psychosocial, and biochemical evaluations) took place at Sainte-Justine University Health Center (Montreal, Canada), with the goal to characterize each of these morbidities in 250 survivors of childhood ALL (PETALE cohort). In addition, the genetic profile of each participant was obtained, and we used statistical and bioinformatics tools to perform association studies on this dataset in order to compare the frequency of genetic variants in survivors with or without LAEs. We evaluated cardiovascular and neurocognitive complications, as well as mood disorders such as anxiety and depression. Other risk factors, such as treatment and/or leukemia characteristics were also considered during the analysis to derive the best genetic predictors. Thus, using the candidate gene approach, we identified common variants in the MTR, PPARA, ABCC3, CALML5, CACNB2, and PCDHB10 genes that were associated with deficits in neurocognitive tests performance, whereas variants in the SLCO1B1 and EPHA5 genes were associated with anxiety and depression. Two variants, rs1805087 in the MTR gene and rs58225473 in the CACNB2 gene, are of particular interest since these associations were validated in an independent SJLIFE replication cohort (St. Jude Children's Research Hospital, Memphis, USA). The association analyses were complemented by an exome-wide association study, which identified several additional genes as potential modulators of the risk of developing treatment-related neurocognitive complications (genes AK8 and ZNF382), as well as anxiety and depression (genes PTPRZ1, MUC16, TNRC6C-AS1, APOL2, C6orf165, EXO5, CYP2W1, and PCMTD1). Variant rs61732180 in the ZNF382 gene was further validated in the replication SJLIFE cohort. To a great extent, we performed association analyses regarding treatment-related cardiac complications which identified several novel markers associated with these toxicities in the TTN, NOS1, ABCG2, CBR1, ABCC5, AKR1C3, NOD2, and ZNF267 genes in survivors of childhood ALL. In addition, we summarized the current knowledge on pharmacogenomic markers related to anthracycline-induced cardiotoxicity affecting pediatric cancer patients. We also included a brief overview of the applicability of reported findings to the PETALE cohort, validating several of them. Besides, we were interested in the complications that arise after a hematopoietic stem cell transplantation. We applied similar bioinformatics and statistical approaches to gain a more complete insight into the genetic component behind these life-threatening complications. Thus, an exome-wide association study was performed in a cohort of pediatric patients undergoing hematopoietic stem cell transplantation following a conditioning regimen containing busulfan. Our results identified new genetic variations conferring a higher risk of sinusoidal obstruction syndrome (notably in the UGT2B10, BHLHE22, and KIAA1715 genes) and acute graft-versus-host disease (ERC1, PLEK, NOP9, and SPRED1 genes), which could be useful for personalized prevention and treatment strategies. This work contributes to the understanding of the influence of genetic factors on the risk of developing treatment-related complications, both during treatment and in the long term. Furthermore, the reported genetic markers along with other known risk factors can lead to prediction models identifying patients at increased risk for these complications

LOSS OF MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN 1 (MRP1/ABCC1) POTENTIATES DOXORUBICIN-INDUCED CARDIOTOXICITY IN MICE

Doxorubicin (DOX) is a broad-spectrum and effective chemotherapeutic agent, but its use in oncologic practice is limited by dose-dependent cumulative cardiotoxicity. DOX-induced cardiotoxicity is in large part due to its ability to cause oxidative stress. Multidrug resistance associated protein 1 (MRP1/ABCC1) is a member of the ATP-binding cassette (ABC) transporter superfamily. By effluxing a wide variety of endogenous and exogenous substrates, Mrp1 plays important physiological roles in multiple tissues and also protects normal tissues against toxicants. However, the role of MRP1 in heart is largely unknown. The role of Mrp1 in DOX-induced cardiotoxicity was investigated in Mrp1 null (Mrp1-/-) and their C57BL (WT) littermates. Chronic DOX caused body weight loss and hemotoxicity, and these adverse effects were significantly exacerbated in Mrp1-/- vs WT mice. Importantly, loss of Mrp1 potentiated DOX-induced cardiotoxicity, presenting as worsened cardiac function and more cellular apoptosis in DOX treated Mrp1-/- mice. Mrp1 also protected neonatal mouse cardiomyocytes (CM) and cardiac fibroblasts (CF) culture against DOX cytotoxicity in vitro. This was demonstrated by the decreased cell survival, more apoptosis and more DNA damage in DOX treated Mrp1-/- vs WT cells. In addition, the effects of deletion of Mrp1 was studied on glutathione (GSH)/glutathione disulfide (GSSG) homeostasis, glutathione conjugate of 4-hydroxy-2-nonenal (GS-HNE) accumulation, protein oxidative damage and expression of antioxidant enzymes. Loss of Mrp1 led to significantly higher GSH and GSSG basal levels in heart. Following DOX treatment, Mrp1-/- CM and CF showed increased GSH and GSSG levels vs WT cells. Meanwhile, DOX increased expression of the GSH synthesis enzymes in Mrp1-/- but not WT cells. Thus, increased GSH synthesis may contribute to the further increase in the GSH pool in DOX-treated Mrp1-/- cells. DOX induced comparable increases of GS-HNE concentration in WT and Mrp1-/- mice hearts. Finally, expression of extracellular superoxide dismutase (ECSOD/SOD3) was significantly lower in Mrp1-/- vs. WT CM treated with either saline or DOX. In summary, this study is the first to document a protective role of Mrp1 in DOX-induced cardiotoxicity. It gives critical information regarding the potential adverse sequelae of introduction of MRP1 inhibitors as adjuncts to clinical chemotherapy of multidrug resistant tumors

The application of autofluorescence lifetime metrology to the study of heart failure models and heart disease

Autofluorescence spectroscopy offers a promising label-free approach to characterise biological samples and has already shown diagnostic potential in a number of medical applications, although study of myocardium has been relatively limited. A number of myocardial molecules display autofluorescence, including those involved in energetics, e.g. NADH and flavoproteins, as well as structural molecules, e.g. collagen. This thesis discusses the application of a custom-built single point fibre-optic probe-based instrumentation for time-resolved spectrofluorometry utilising spectrally resolved time-correlated single photon counting detection (TCSPC) and white light reflectometry to the investigation of models of heart failure, both ex vivo and in vivo. Heart failure (HF) is a pathophysiological state in which an abnormality of cardiac function causes failure of the heart to pump blood at a rate commensurate with the requirements of the metabolising tissues. It affects 1-2% of the population rising to greater than 10% aged over 70 years. Despite recent therapeutic advances, annualized mortality can still approach 10%. HF results from a myocardial injury (e.g. myocardial infarction, chemotherapy) causing loss of myocytes, and maladaptive changes in surviving myocytes and extracellular matrix by ‘pathological remodelling’. That HF is characterized by structural and energetic changes was the principal motivation for the creation of an instrument to investigate changes in myocardial autofluorescence signature in disease states in vivo. If the signatures associated with known pathological diagnoses could be ascertained, such a technique could perform ‘virtual biopsy’ to aid diagnosis. This thesis describes the application of autofluorescence technique to an ex vivo Langendorff-heart to characterise the changes in autofluorescence signature with controlled insults of glucose deprivation and hypoxia. Additionally, it reports for the first-time the characterization of the autofluorescence lifetime signature in vivo at different time points in an established rat post-myocardial infarction heart failure. The thesis describes development of in vivo intravenous doxorubicin chemotherapy-cardiomyopathy heart failure model (DOX-HF) and subsequent characterization of in vivo autofluorescence signature. This investigation prompted development of a clinically viable instrument and the progress to date is described.Open Acces

The effects of anthracyclines on calcium handling and contractility in sheep ventricular myocytes; role of oxidative stress

Anthracyclines such as doxorubicin (DOX) and daunorubicin (DAUN) are effective chemotherapeutics and contribute to improved cancer survival rates in children and adults. However, anthracyclines exhibit acute and chronic cardiotoxicity which can produce heart failure in cancer survivors. While the cellular basis remains unclear, limited previous studies show DOX perturbs certain aspects of excitation-contraction coupling and increases production of reactive oxygen species (ROS). Fewer studies have investigated the effects of DAUN and the effects of either anthracycline on excitation-contraction coupling (ECC) in a large animal model has yet to be demonstrated. Furthermore, the extent to which altered ECC is dependent on anthracycline-induced ROS production remains ambiguous. This is compounded by the fact that no studies have investigated whether elevated ROS production produces oxidative stress in cardiac myocytes. To address these gaps in our understanding, we performed the first integrative investigation of the effects of DOX and DAUN in sheep ventricular myocytes. We also measured the effect of DOX and DAUN on oxidative stress in these cells and further elucidated the underlying sources of ROS. Furthermore, we investigated the dependence of perturbed ECC on ROS thence oxidative stress elevations.Sheep ventricular myocytes were enzymatically isolated in accordance with the Animals (Scientific Procedures) Act, UK, 1986 and used for all experiments. Intracellular calcium and contractility dynamics were measured using epi-fluorescent photometry and video sarcomere detection simultaneously. Cells were field stimulated at 0.5 Hz then acutely exposed to 1 nM DOX or DAUN. Rapid application of 10 mM caffeine was used to measure SR Ca content. Oxidative stress was measured using CellROX red. Fluorescent images were captured using the cytation imaging system and cell fluorescence determined using ImageJ software. DOX reduced the activity of SERCA and increased the activity of NCX resulting in a reduction in SR Ca content. DAUN also reduced SR Ca content however due to an interaction with caffeine the mechanism could not be fully elucidated. The decrease in SR Ca content accounted for a decrease in systolic Ca which underpinned a decrease in systolic shortening. Both DOX and DAUN increased myofilament sensitivity to Ca, potentially offsetting the effect on contractility. DOX increased oxidative stress in a concentration and time-dependent manner. DAUN also increased oxidative stress, but only at relatively high concentrations (10 mM). Removal of oxidative stress by n-acetylcysteine (NAC) attenuated the effects of DOX on the majority of Ca handling and contractility parameters. For example, the effect of DOX on SR Ca content and Ca transient amplitude were reduced by approximately 50 %. Inhibition of the ROS producing enzymes NADPH oxidase (NOX) and xanthine oxidase (XO) reduced DOX-mediated oxidative stress by ~50 % and ~20 % respectively and attenuated the effects on ECC.In cells from sheep with heart failure, DOX reduced SR Ca content thence systolic Ca and contractility but had no effect on SERCA and NCX. These findings suggest that in a large animal model, DOX and DAUN decrease SR Ca content leading to a reduction in systolic Ca thence contractility. In the case of DOX, decreased SERCA and increased NCX activity likely contribute to the decrease of SR Ca content. However, that this isn’t the case in heart failure suggests a role for other mechanisms. These findings are also the first to show that DOX and DAUN increase intracellular oxidative stress in the heart and that NOX and XO are key enzymatic sources of ROS. Furthermore, these findings show this increase in oxidative stress is pathologically important as it accounts for approximately half of the effects of DOX on ECC. Collectively, these findings further elucidate the effects of anthracyclines on ECC and make important contributions to the understanding of the cellular basis of anthracycline-mediated cardiotoxicity. Furthermore, the dependence on and sources of oxidative stress reveal clinically relevant therapeutic targets

Supremacy of nanoparticles in the therapy of chronic myelogenous leukemia

Background and purpose The reciprocal translocation of the ABL gene from chromosome 9 to chromosome 22 near the BCR gene gives rise to chronic myelogenous leukemia (CML). The translocation results in forming the Philadelphia chromosome (BCR-ABL) tyrosine kinase. CML results in an increase in the number of white blood cells and alteration in tyrosine kinase expression. CML prognosis includes three stages, namely chronic, accelerated, and blast. The diagnosis method involves a CT scan, biopsy, and complete blood count. However, due to certain disadvantages, early diagnosis of CML is not possible by traditional methods. Nanotechnology offers many advantages in diagnosing and treating cancer. Experimental approach We searched PubMed, Scopus and Google Scholar using the keywords Philadelphia chromosome, bionanotechnology, tyrosine kinase pathway, half-life, passive targeting, and organic and inorganic nanoparticles. The relevant papers and the classical papers in this field were selected to write about in this review. Key results The sensitivity and specificity of an assay can be improved by nanoparticles. Utilizing this property, peptides, antibodies, aptamers, etc., in the form of nanoparticles, can be used to detect cancer at a much earlier stage. The half-life of the drug is also increased by nanoformulation. The nanoparticle-coated drugs can easily escape from the immune system. Conclusion Depending on their type, nanoparticles can be categorized into organic, inorganic and hybrid. Each type has its advantages. Organic nanoparticles have good biocompatibility, inorganic nanoparticles increase the half-life of the drugs. In this review, we highlight the nanoparticles involved in treating CML

Studies on the Protection Mechanisms Against Doxorubicin Toxicity in Resistant and Sensitive Human Tumour Cells

A rapid, sensitive and selective HPLC method for the quantitation of glutathione (GSH) at the cellular level was developed by the author. Glutathione is resolved isocratically by ion-pair high-performance liquid chromatography and detected by UV at 200 - 210 nm. The mobile phase consisted of an aqueous buffer of methanol containing 0.1% tetrabutyl ammonium hydroxide adjusted to pH 3.5 by 10% v/v orthophosphoric acid. This method is able to detect GSH in a small amount of cells and can be adapted for quantitative determination of biological thiols and some other cellular compounds of special interest such as amino acids at small tissue volumes. The non invasive nuclear magnetic resonance (NMR) technique which was developed for this study (1H spin echo NMR) is capable of detecting certain small molecules and structural entities in intact tumour cells. This method is specific and selective, providing information on the concentration and conformation of such molecules as glutathione, phosphorylcholine, lactate, mobile triglyceride, acetyl choline etc in the living cells. The technique has the advantage that it is non-invasive, providing detailed structural information on individual species present in the cell matrix. It has been used in this case to study the rate of energy consumption following the activation of the glycolytic pathway with glucose. The signals and patterns observed have been used in a preliminary way to study changes in glutathione metabolism and in lactate production when challenged by therapeutic agents. The effect of doxorubicin on the cellular biochemistry of Hela tumour cells using 1H spin echo NMR of the intact and viable cell in conjunction with the dual wave length HPLC of cell lysate is reported here. Dose-related changes were observed in lactate and reduced glutathione concentration. Doxorubicin induces a time-dependent depletion of the cytosolic pool of glutathione and a change in the glycolytic pattern of the cells. The glutathione depletion could be partially reversed by controlled pretreatment of the cells with N-acetylcysteine and cysteine, the protection being linked to the intracellular concentration of the thiol. Glutathione was also measured in other doxorubicin-sensitive cells from small cell carcinoma of lung (GLC4 210), and the levels compared with those in cells with acquired resistance and a line of resistant non-small-cell adenocarcinoma of lung A549 (alveolar type 2). The effect of different doxorubicin concentrations on GSH was measured using the HPLC method which has been shown to correlate with the NMR studies in live cells. 1H spin echo NMR of the leukaemia cell line (J111) is also reported here for studying cellular glycolysis. The concentration of cells in the NMR tube is high (approx. 10e9 cells in 0.4 ml) and as a result the available oxygen is restricted, making the NMR experiment a plausible in vitro tumour model in which kinetics in the living cell can be probed in a non-invasive manner. Treatment with pharmacological concentrations of doxorubicin produced immediate enhanced anaerobic glycolysis and eventual cell death. Glutathione-S-transferase (GST) activity in the different lung tumour cells was linked with their content of glutathione and the effect of doxorubicin on such activity was studied as well. High levels of lipid peroxidation were noticed in the two sensitive cell lines (Hela and GLC4 210 [S]) as doxorubicin readily passes into these cells and interacts with glutathione. N-acetylcysteine pre-treatment of Hela cells showed little protection from the effect of doxorubicin. The effect of doxorubicin on the viability of the different tumour cells was studied using MTT dye reduction by living but not dead cells. An increase in A549 sensitivity to doxorubicin was produced using Buthionine-S,R-Sulfuximine at a non-toxic concentration. The inherent resistant of A549 tumour cells toward doxorubicin was circumvented by using one of the antiarrythmic drugs (amiodarone) which trap the drug in the cells by decreasing its efflux. This was indicated by measuring intracellular doxorubicin and amiodarone after harvesting of the cells in two different ways (scraping and trypsinization). These results were confirmed by the HPLC measurement of GSH in these cells after amiodarone and doxorubicin treatment and by using a chemosensitivity assay