Autophagy (but not metabolism) is a key event in mitoxantrone-induced cytotoxicity in differentiated AC16 cardiac cells.

Mitoxantrone (MTX) is an antineoplastic agent used to treat advanced breast cancer, prostate cancer, acute leukemia, lymphoma and multiple sclerosis. Although it is known to cause cumulative dose-related cardiotoxicity, the underlying mechanisms are still poorly understood. This study aims to compare the cardiotoxicity of MTX and its' pharmacologically active metabolite naphthoquinoxaline (NAPHT) in an in vitro cardiac model, human-differentiated AC16 cells, and determine the role of metabolism in the cardiotoxic effects. Concentration-dependent cytotoxicity was observed after MTX exposure, affecting mitochondrial function and lysosome uptake. On the other hand, the metabolite NAPHT only caused concentration-dependent cytotoxicity in the MTT reduction assay. When assessing the effect of different inhibitors/inducers of metabolism, it was observed that metyrapone (a cytochrome P450 inhibitor) and phenobarbital (a cytochrome P450 inducer) slightly increased MTX cytotoxicity, while 1-aminobenzotriazole (a suicide cytochrome P450 inhibitor) decreased fairly the MTX-triggered cytotoxicity in differentiated AC16 cells. When focusing in autophagy, the mTOR inhibitor rapamycin and the autophagy inhibitor 3-methyladenine exacerbated the cytotoxicity caused by MTX and NAPHT, while the autophagy blocker, chloroquine, partially reduced the cytotoxicity of MTX. In addition, we observed a decrease in p62, beclin-1, and ATG5 levels and an increase in LC3-II levels in MTX-incubated cells. In conclusion, in our in vitro model, neither metabolism nor exogenously given NAPHT are major contributors to MTX toxicity as seen by the residual influence of metabolism modulators used on the observed cytotoxicity and by NAPHT's low cytotoxicity profile. Conversely, autophagy is involved in MTX-induced cytotoxicity and MTX seems to act as an autophagy inducer, possibly through p62/LC3-II involvement.

Chemobrain: mitoxantrone-induced oxidative stress, apoptotic and autophagic neuronal death in adult CD-1 mice.

Mitoxantrone (MTX) is a topoisomerase II inhibitor used to treat a wide range of tumors and multiple sclerosis but associated with potential neurotoxic effects mediated by hitherto poorly understood mechanisms. In adult male CD-1 mice, the underlying neurotoxic pathways of a clinically relevant cumulative dose of 6 mg/kg MTX was evaluated after biweekly administration for 3 weeks and sacrifice 1 week after the last administration was undertaken. Oxidative stress, neuronal damage, apoptosis, and autophagy were analyzed in whole brain, while coronal brain sections were used for a closer look in the hippocampal formation (HF) and the prefrontal cortex (PFC), as these areas have been signaled out as the most affected in 'chemobrain'. In the whole brain, MTX-induced redox imbalance shown as increased endothelial nitric oxide synthase and reduced manganese superoxide dismutase expression, as well as a tendency to a decrease in glutathione levels. MTX also caused diminished ATP synthase ß expression, increased autophagic protein LC3 II and tended to decrease p62 expression. Postsynaptic density protein 95 expression decreased in the whole brain, while hyperphosphorylation of Tau was seen in PFC. A reduction in volume was observed in the dentate gyrus (DG) and CA1 region of the HF, while GFAP-ir astrocytes increased in all regions of the HF except in the DG. Apoptotic marker Bax increased in the PFC and in the CA3 region, whereas p53 decreased in all brain areas evaluated. MTX causes damage in the brain of adult CD-1 mice in a clinically relevant cumulative dose in areas involved in memory and cognition.

An application of p-sulfonatocalix[6]arenes to attenuate cardiotoxicity of mitoxantrone in vitro: preparation, characterization and evaluation.

OBJECTIVES: In this study, p-sulfonatocalix[6]arenes (SCA6) was proposed to construct a host-guest complexation to carry mitoxantrone (MIT) to maintain its anti-proliferation effect on HepG2 cells as well as to attenuate cardiotoxicity on H9C2 cells as a nano-size drug delivery system. METHODS: SCA6 binding to MIT evidenced through competitive fluorescence titration method. The complex was characterized using UV-visible, Fourier transform infrared, and proton nuclear magnetic resonance (1H-NMR) spectroscopies and differential scanning calorimetry analysis. The cytotoxicity was examined by a cell counting kit-8 assay on six cells. High content analysis, cell apoptosis and cell cycle experiments were measured to investigate the mechanism of detoxification in H9C2. KEY FINDINGS: The host-guest complexation was formed with a stoichiometry ratio of 1:1. Cytotoxicity study demonstrated that MIT/SCA6 complex could improve the cell viability on H9C2, MCF-7, A549, Hek293 and L02 cells and remained cytotoxicity effect on HepG2 cells. High content analysis showed that MIT/SCA6 complex could enhance the cell viability, mitochondrial mass and mitochondrial membrane potential and ameliorate the nuclear swelling on H9C2 cells. Moreover, the complex were arrested in G0/G1 phase of the cell cycle and same with MIT, while the detoxication was attributed to reducing early apoptosis. CONCLUSIONS: The host-guest complexation between SCA6 and MIT had the ability to attenuate cardiotoxicity and provided a potential strategy for the application of soluble calixarenes in chemotherapy.

Exploring the aging effect of the anticancer drugs doxorubicin and mitoxantrone on cardiac mitochondrial proteome using a murine model.

Current cancer therapies are successfully increasing the lifespan of cancer patients. Nevertheless, cardiotoxicity is a serious chemotherapy-induced adverse side effect. Doxorubicin (DOX) and mitoxantrone (MTX) are cardiotoxic anticancer agents, whose toxicological mechanisms are still to be identified. This study focused on DOX and MTX's cardiac mitochondrial damage and their molecular mechanisms. As a hypothesis, we also sought to compare the cardiac modulation caused by 9 mg/kg of DOX or 6 mg/kg of MTX in young adult mice (3 months old) with old control mice (aged control, 18-20 months old) to determine if DOX- and MTX-induced damage had common links with the aging process. Cardiac homogenates and enriched mitochondrial fractions were prepared from treated and control animals and analyzed by immunoblotting and enzymatic assays. Enriched mitochondrial fractions were also characterized by mass spectrometry-based proteomics. Data obtained showed a decrease in mitochondrial density in young adults treated with DOX or MTX and aged control, as assessed by citrate synthase (CS) activity. Furthermore, aged control had increased expression of the peroxisome proliferator-activated receptor γ coactivator 1 α (PGC1α) and manganese superoxide dismutase (MnSOD). Regarding the enriched mitochondrial fractions, DOX and MTX led to downregulation of proteins related to oxidative phosphorylation, fatty acid oxidation, amino acid metabolic process, and tricarboxylic acid cycle. MTX had a greater impact on malate dehydrogenase (MDH2) and pyruvate dehydrogenase E1 component subunit α (PDHA1). No significant proteomic changes were observed in the enriched mitochondrial fractions of aged control when compared to young control. To conclude, DOX and MTX promoted changes in several mitochondrial-related proteins in young adult mice, but none resembling the aged phenotype.

Estrone-targeted liposomes for mitoxantrone delivery via estrogen receptor: In vivo targeting efficacy, antitumor activity, acute toxicity and pharmacokinetics.

Estrogen receptor (ER) is a potential target receptor for ER-positive cancer therapy including breast cancers, gastric cancers, and human acute myeloblastic leukaemia. In order to reduce the side-effects of mitoxantrone (MTO), estrone-targeted liposomes for MTO delivery via ER were designed for selectively targeting cancer cells. In previous studies, MTO-loaded estrogen receptor targeted and sterically stabilized liposome (ES-SSL-MTO; ES: estrone, is known to bind the ER) had been synthesized and showed a very high antiproliferative effect with IC50 value of 0.7 ng/mL. Based on these, further studies including in vivo targeting efficacy and antitumor activity, acute toxicity and pharmacokinetics of MTO liposomes were carried out. The results showed SSL (sterically stabilized liposome, PEGylated liposome, PEG: Polyethylene Glycol) could reduce drug metabolism, improve the stability of liposomes, prolong in vivo circulation time of drugs, reduce the toxicity of MTO. But SSL could not be enriched in tumor tissues. However, estrone (ES)-targeted liposomes could be delivered to tumor sites. ES-SSL could effectively enter into ER-expressing tumor cellsand be accumulated, prolong the circulation time in vivo, reduce side effects of drug. ES-SSL-MTO could provide higher bioavailability than MTO, enhance the anti-tumor effect and the safety of MTO, reduce the toxicity and side effects of MTO and improve the therapeutic effect of MTO. These facts proved ES-SSL is a useful tumor-targeting drug delivery system for MTO.

Ratiometric Delivery of Mitoxantrone and Berberine Co-encapsulated Liposomes to Improve Antitumor Efficiency and Decrease Cardiac Toxicity.

Combination therapy is one of the most common clinical practices in the treatment of malignancies. Synergistic effects, however, are produced only when optimal ratios of combined drugs were delivered to tumor cells. Thus, carriers co-encapsulating of multiple drugs are widely utilized for coordinated delivery. Herein, co-encapsulated pegylated liposomal formulation of mitoxantrone (MIT) and berberine (BER) at an optimal ratio has been developed (MBL) with high encapsulation efficiency (EE) and drug loading in order to achieve the purpose of ratiometric loading and delivery. MBL can not only extend blood circulation but also enhance tumor accumulation for both MIT and BER. More importantly, MBL can maintain the originally desired drug ratio in tumors within 48 h of intravenous injection for synergistic therapy. Compared with the liposomal formulation of MIT-treated group (ML), the progression of tumor growth was inhibited significantly in murine 4T1 breast tumor model after the treatment of MBL, as well as a lower cardiac toxicity. In addition, MBL evidently prolonged the survival of mice with L1210 ascitic tumor model. In summary, such a strategy of co-encapsulated liposomes could improve the clinical applications against multiple cancers.

Subchronic administration of mitoxantrone and the influence of enzyme inhibitors on its induced cardiotoxicity in mice: role of NRF-2/CYP2E1.

OBJECTIVE: Mitoxantrone (MTX)- induced cardiotoxicity is a clinical concern that is limiting its use. The aim of this paper, therefore, was to investigate the subchronic administration of MTX plus nonspecific/specific inhibitors of CYP450/2E1, to assess the extent of oxidative-induced injury by measuring levels of oxidative cardiac and injury biomarkers in mice and to evaluate the effects of CYP2E1 on caspase 3 activity and nuclear factor erythroid 2-related factor-2 (NRF-2). MATERIALS AND METHODS: Mice (n = 32) were divided into four treatment groups of eight: control, MTX, MTX + 4-methlypyrazole (4MP) and MTX + disulfiram (Disf). After 6 weeks of treatments, blood and heart samples were collected. RESULTS: Liquid chromatography-mass spectrometry (LCMS) analysis of MTX-treated plasma samples revealed several metabolites with different retention times. Cardiac antioxidant enzymes and creatine kinase (CK) levels were not significantly different among the groups. However, cardiac troponin and caspase 3 activity were significantly raised, with increased CYP2E1 expressions and reduced NRF-2 expression. Tissue damage was observed in all the treatment groups, including MTX, leading to the conclusion that MTX-induced cardiotoxicity was mediated by CYP2E1 activity, which initiated caspase 3 production, and decreased NRF-2 expression. CONCLUSIONS: Therefore, agents that inhibit CPY2E1 expression might attenuate MTX-induced cardiotoxicity by increasing NRF-2 expression.

Mitoxantrone impairs proteasome activity and prompts early energetic and proteomic changes in HL-1 cardiomyocytes at clinically relevant concentrations.

Mitoxantrone (MTX) is used to treat several types of cancers and to improve neurological disability in multiple sclerosis. Unfortunately, cardiotoxicity is a severe and common adverse effect in MTX-treated patients. Herein, we aimed to study early and late mechanisms of MTX-induced cardiotoxicity using murine HL-1 cardiomyocytes. Cells were exposed to MTX (0.1, 1 or 10 µM) during short (2, 4, 6, or 12 h) or longer incubation periods (24 or 48 h). At earlier time points, (6 and 12 h) cytotoxicity was already observed for 1 and 10 µM MTX. Proteomic analysis of total protein extracts found 14 proteins with higher expression and 26 with lower expression in the cells exposed for 12 h to MTX (pH gradients 4-7 and 6-11). Of note, the expression of the regulatory protein 14-3-3 protein epsilon was increased by a factor of two and three, after exposure to 1 and 10 µM MTX, respectively. At earlier time-points, 10 µM MTX increased intracellular ATP levels, while decreasing media lactate levels. At later stages (24 and 48 h), MTX-induced cytotoxicity was concentration and time-dependent, according to the MTT reduction and lactate dehydrogenase leakage assays, while caspase-9, -8 and -3 activities increased at 24 h. Regarding cellular redox status, total glutathione increased in 1 µM MTX (24 h), and that increase was dependent on gamma-glutamylcysteine synthetase activity. Meanwhile, for both 1 and 10 µM MTX, oxidized glutathione was significantly higher than control at 48 h. Moreover, MTX was able to significantly decrease proteasomal chymotrypsin-like activity in a concentration and time-independent manner. In summary, MTX significantly altered proteomic, energetic and oxidative stress homeostasis in cardiomyocytes at clinically relevant concentrations and our data clearly demonstrate that MTX causes early cardiotoxicity that needs further study.

H2O2-assisted photoelectrocatalytic degradation of Mitoxantrone using CuO nanostructured films: Identification of by-products and toxicity.

CuO nanostructured thin films supported on silicon with 6.5 cm2 area (geometric area greater than the studies reported in the literature) were synthesized by a chemical bath deposition technique. The electrodes were characterized by MEV, XRD, XPS, contact angle, cyclic voltammetry and electrochemical impedance spectroscopy analyses. To evaluate the photoelectrochemical properties of the CuO films, photocurrent-voltage measurements were performed using linear voltammetry. The catalytic activities of CuO nanostructures were evaluated by monitoring photodegradation of Mitoxantrone (MTX) under UV-A light irradiation. The method of photoelectrocatalysis (PEC), applying a voltage of 1.5 V and assisted by adding H2O2, was undertaken. To the best of our knowledge, no studies on the degradation of anticancer agents using PEC process have been found in the literature. For comparison purposes, experiments were performed under the same conditions by assisted photocatalysis (PC) with H2O2 and direct photolysis. CuO deposits consist of a needle-like morphology. The presence of CuO in the tenorite phase was evidenced by XRD and the XPS spectra showed the presence of copper(II) oxide. The increase in current under illumination shows that CuO exhibits photoactivity. The PEC system showed a 75% level of MTX degradation, while the level achieved using PC was 50%. Under UV-A light alone only 3% removal was obtained after 180 min. Up to 10 by-products were identified by chromatography-mass spectrometry (LC-MS) with m/z values ranging between 521 and 285 and a plausible degradation route has been proposed. It is worth mentioning that 9 by-products identified in this work, were not found in the literature in other studies of degradation or products generated as metabolites. The toxicity tests of MTX before and after PEC treatment with Artemia Salina and Allium cepa showed a decrease in the acute toxicity of the medium as the antineoplastic was degraded.

Influence of PARP-1 inhibition in the cardiotoxicity of the topoisomerase 2 inhibitors doxorubicin and mitoxantrone.

Doxorubicin (DOX) and Mitoxantrone (MTX) are very effective drugs for a range of tumors despite being highly cardiotoxic. DNA topoisomerase 2 beta (Top2ß) was revealed as key mediator of DOX-induced cardiotoxicity, although ROS generation is also an important mechanism. Oxidative stress is also an important issue in MTX-induced cardiotoxicity that is manifested by mitochondrial dysfunction. Studies have demonstrated the relationship between PARP-1 overactivation and cell viability in DOX-treated cardiomyocytes. In reference of MTX, data regarding PARP-1 overactivation as the mechanism responsible for cardiotoxicity is difficult to find. The aim of this study was to evaluate the influence of PARP-1 inhibitor DPQ on DOX- and MTX-mediated cardiotoxicity. Cells were exposed for 24 h to DOX or MTX in the presence or absence of DPQ. Viability, apoptosis, and genotoxicity assays were carried out. Immunofluorescence of phosphorylated histone H2AX was analyzed in H9c2 cells and cardiomyocytes from neonatal rats. Results demonstrated that DPQ co-treatment increases DOX-induced apoptosis in H9c2 cells. DPQ also prevents DOX and MTX-ROS generation in part by increasing SOD and CAT activities. Furthermore, DPQ co-treatment increased the generation of DNA strand breaks by DOX and MTX whilst also inducing phosphorylation of H2AX, MRE11, and ATM in H9c2 cells. Our results demonstrated that as well as increasing DNA damage and inducing apoptotic cell death, DPQ enhances DOX- and MTX-mediated cytotoxicity in H9c2.

DNA Damage Induces a Secretory Program in the Quiescent TME that Fosters Adverse Cancer Phenotypes.

Carcinomas develop in complex environments that include a diverse spectrum of cell types that influence tumor cell behavior. These microenvironments represent dynamic systems that contribute to pathologic processes. Damage to DNA is a notable inducer of both transient and permanent alterations in cellular phenotypes. Induction of a DNA damage secretory program is known to promote adverse tumor cell behaviors such as proliferation, invasion, metastasis, and treatment resistance. However, prior studies designed to identify genotoxic stress-induced factors evaluated actively proliferating in vitro cultures of cells such as fibroblasts as experimental models. Conversely, the vast majority of benign cells in a typical tumor microenvironment (TME) are not proliferating but rather exist in quiescent (i.e., G0) or in terminally differentiated states. In this study, the diversity and magnitude of transcriptional responses to genotoxic damage in quiescent prostate fibroblasts were assessed using gene expression profiling. The secretory damage response in quiescent cells was highly concordant with that of actively dividing cells. Quiescent human prostate stroma exposed to genotoxic agents (e.g., mitoxantrone) in vivo resulted in significant upregulation (2.7- to 5.7-fold; P ≤ 0.01) of growth factors and cytokines including IL1ß, MMP3, IL6, and IL8. The paracrine effects of damaged quiescent cells consistently increased the proliferation and invasion of prostate cancer cells and promoted cell survival and resistance to apoptosis following exposure to chemotherapy.Implications: Benign quiescent cells in the TME respond to genotoxic stress by inducing a secretory program capable of promoting therapy resistance. Developing approaches to suppress the secretory program may improve treatment responses. Mol Cancer Res; 15(7); 842-51. ©2017 AACR.

Naphthoquinoxaline metabolite of mitoxantrone is less cardiotoxic than the parent compound and it can be a more cardiosafe drug in anticancer therapy.

Mitoxantrone (MTX) is an antineoplastic agent used to treat several types of cancers and on multiple sclerosis, which shows a high incidence of cardiotoxicity. Still, the underlying mechanisms of MTX cardiotoxicity are poorly understood and the potential toxicity of its metabolites scarcely investigated. Therefore, this work aimed to synthesize the MTX-naphthoquinoxaline metabolite (NAPHT) and to compare its cytotoxicity to the parent compound in 7-day differentiated H9c2 cells using pharmacological relevant concentrations (0.01-5 µM). MTX was more toxic in equivalent concentrations in all cytotoxicity tests performed [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction, neutral red uptake, and lactate dehydrogenase release assays] and times tested (24 and 48 h). Both MTX and NAPHT significantly decreased mitochondrial membrane potential in 7-day differentiated H9c2 cells after a 12-h incubation. However, energetic pathways were affected in a different manner after MTX or NAPHT incubation. ATP increased and lactate levels decreased after a 24-h incubation with MTX, whereas for the same incubation time and concentrations, NAPHT did not cause any significant effect. The increased activity of ATP synthase seems responsible for MTX-induced increases in ATP levels, as oligomycin (an inhibitor of ATP synthase) abrogated this effect on 5 µM MTX-incubated cells. 3-Methyladenine (an autophagy inhibitor) was the only molecule to give a partial protection against the cytotoxicity produced by MTX or NAPHT. To the best of our knowledge, this was the first broad study on NAPHT cardiotoxicity, and it revealed that the parent drug, MTX, caused a higher disruption in the energetic pathways in a cardiac model in vitro, whereas autophagy is involved in the toxicity of both compounds. In conclusion, NAPHT is claimed to largely contribute to MTX-anticancer properties; therefore, this metabolite should be regarded as a good option for a safer anticancer therapy since it is less cardiotoxic than MTX.

Nanomedical strategy to prolong survival period, heighten cure rate, and lower systemic toxicity of S180 mice treated with MTX/MIT.

In spite of the usual combination form of methotrexate (MTX)/mitoxantrone (MIT) and various complex combination regimens of MTX/MIT with other anticancer drugs, the survival period, cure rate, and systemic toxicity still need to be improved. For this purpose, a nanostructured amino group-modified mesoporous silica nanoparticles (MSNN)-MTX/MIT was designed. In the preparation, the surface of mesoporous silica nanoparticles (MSNs) was modified with amino groups to form MSNN. The covalent modification of the amino groups on the surface of MSNN with MTX resulted in MSNN-MTX. The loading of MIT into the surface pores of MSNN-MTX produced nanostructured MSNN-MTX/MIT. Compared with the usual combination form (MTX/MIT), nanostructured MSNN-MTX/MIT increased the survival period greatly, heightened the cure rate to a great extent, and lowered the systemic toxicity of the treated S180 mice, significantly. These superior in vivo properties of nanostructured MSNN-MTX/MIT over the usual combination form (MTX/MIT) were correlated with the former selectively releasing MTX and MIT in tumor tissue and inside cancer cells in vitro. The chemical structure and the nanostructure of MSNN-MTX/MIT were characterized using infrared and differential scanning calorimeter spectra as well as transmission electron microscope images, respectively.

Toxicity of Mitoxantrone-loaded Superparamagnetic Iron Oxide Nanoparticles in a HT-29 Tumour Spheroid Model.

BACKGROUND/AIM: Cancer research is commonly carried out in two-dimensional (2D) cell cultures, which poorly reflect in vivo settings where the growing tumours are exposed to mechanical forces and biochemical gradients. In this study we established a HF-29 colon carcinoma tumor spheroid model to investigate the effect of free mitoxantrone (MTO) and its nanoparticle-bound form (SPION(MTO)) under 3D cell culture conditions. MATERIALS AND METHODS: Tumour spheroids were generated by seeding HT-29 colon carcinoma cells on agarose-coated cell culture wells. Growth of the spheroids was monitored daily by transmission microscopy upon treatment with free MTO, SPION(MTO) or unloaded SPION. RESULTS AND CONCLUSION: Unloaded SPION did not affect the spheroid size compared to untreated controls, while both free MTO and SPION(MTO) inhibited growth of the spheroids in a dose- and time-dependent manner. In comparison to free MTO, the effect of SPION(MTO) on spheroid growth was slightly delayed. Further analyses are necessary to investigate if MTO infiltrates spheroids in its nanoparticle-bound form or whether it is released from SPION before infiltration.

Pathways of cardiac toxicity: comparison between chemotherapeutic drugs doxorubicin and mitoxantrone.

Anthracyclines, e.g., doxorubicin (DOX), and anthracenediones, e.g., mitoxantrone (MTX), are drugs used in the chemotherapy of several cancer types, including solid and non-solid malignancies such as breast cancer, leukemia, lymphomas, and sarcomas. Although they are effective in tumor therapy, treatment with these two drugs may lead to side effects such as arrhythmia and heart failure. At the same clinically equivalent dose, MTX causes slightly reduced cardiotoxicity compared with DOX. These drugs interact with iron to generate reactive oxygen species (ROS), target topoisomerase 2 (Top2), and impair mitochondria. These are some of the mechanisms through which these drugs induce late cardiomyopathy. In this review, we compare the cardiotoxicities of these two chemotherapeutic drugs, DOX and MTX. As described here, even though they share similarities in their modes of toxicant action, DOX and MTX seem to differ in a key aspect. DOX is a more redox-interfering drug, while MTX induces energy imbalance. In addition, DOX toxicity can be explained by underlying mechanisms that include targeting of Top2 beta, mitochondrial impairment, and increases in ROS generation. These modes of action have not yet been demonstrated for MTX, and this knowledge gap needs to be filled.

Cardiac effects of mitoxanthrone therapy in patients with multiple sclerosis.

BACKGROUND: Mitoxanthrone (MTX) is a synthetic anthracycline antibiotic that has been used for several years in the treatment of patients with primary progressive, secondary progressive, and relapsing remitting multiple sclerosis (MS) who do not respond to other drugs. MTX has antineoplastic, immunomodulatory, and antibacterial properties. The most common adverse effects of MTX include nausea and vomiting, hair loss, increased risk of urinary and respiratory tract infections, and amenorrhea. Less frequent problems include leukopenia, thrombocytopenia, anaemia, and an increase in hepatic enzyme and bilirubin levels. Other severe sequelae of MTX treatment are drug cardiotoxicity and a potential to induce leukaemia. Drug toxicity results from its affinity to iron ions. The resulting complex strongly induces formation of free oxygen radicals and increases lipid peroxidation. Asymptomatic reduction in left ventricular ejection fraction (LVEF) by two-dimensional (2D) echocardiography, cardiomyopathy, and congestive heart failure have been observed in patients with MS at a rate of about 2.6-5%. Few studies evaluated cardiotoxicity of MTX in MS patients. Most previous studies were performed in small groups of cancer patients and cardiac evaluation was limited to physical examination. AIM: To evaluate the effect of MTX treatment on LVEF by 2D echocardiography. METHODS: We studied 72 MS patients aged 25-63 years who were treated with MTX in 2002-2014. The diagnosis of MS was made using the 2001 McDonald criteria updated in 2005. The study group included primary progressive MS in 40 (56%) patients, secondary progressive MS in 5 (7%) patients, and relapsing remitting MS in 27 (37%) patients. MTX was administered at 12 mg/m2 of body surface area every 3 months (up to the total dose of 140 mg/m2). MTX treatment was initiated in patients with no signs of heart failure on physical examination, normal electrocardiogram (ECG), normal LVEF by 2D echocardiography, and normal laboratory test findings including complete blood count and hepatic and renal function parameters. Each MTX administration was preceded by 2D echocardiography with LVEF measurement, ECG, and physical examination of the cardiovascular system. The effect of MTX treatment on LVEF was evaluated by comparing baseline LVEF with LVEF measurements before the last MTX dose. Statistical analysis was performed using the Student t test. RESULTS: The mean LVEF before administration of the first MTX dose was 65 ± 3.3%. The lowest LVEF at the final 2D echo-cardiographic examination was 60 ± 2.1%. We did not find a significant LVEF reduction during MTX treatment in MS patients compared to baseline values. Severe myocardial dysfunction manifesting with significant LVEF reduction by 2D echocardiography or clinical evidence of heart failure was not noted in any patient in the study group. CONCLUSIONS: Our study showed no significant LVEF reduction during MTX monotherapy in MS patients without a history of a cardiac disease and with normal echocardiographic findings at baseline. Long-term cardiac effects of MTX require further studies.

Mitoxantrone-loaded superparamagnetic iron oxide nanoparticles as drug carriers for cancer therapy: Uptake and toxicity in primary human tubular epithelial cells.

Superparamagnetic iron oxide nanoparticles (SPIONs) are in use for many clinical diagnostic and experimental therapeutic applications, for example, for targeted drug delivery. To analyze the cellular responses to mitoxantrone-carrying SPIONs (SPION-MTO), and to the drug released from SPIONs, we used an in vitro system that allows comparison of primary human cells with different endocytotic capacities, namely, epithelial cells from proximal and distal parts of the nephron. SPIONs were selectively and rapidly internalized by proximal tubular cells with high endocytotic potential, but not by distal tubular cells. Uptake did not affect cell viability or morphology. In both cell types, free MTO (10-100 nM) induced double-strand DNA breaks and senescence, cell hypertrophy and reduced cell proliferation. However, cadherin-mediated cell-cell adhesion, cytoskeletal structures or polarity of the cells were not affected. Interestingly, a comparable response was also observed upon treatment with SPION-MTO and was independent of uptake of the particles. The effect of SPION-MTO on cells which did not internalize particles was primarily related to the release of MTO from drug-coated particles upon incubation in serum-containing cell growth medium. In conclusion, we show that whereas the uptake of SPIONs does not affect cellular functions or viability, the toxicity of drug-loaded SPIONs depends essentially on the type of drug bound to nanoparticles. Due to the relatively low systemic toxicity of MTO, the effects of MTO-SPIONs on human tubular cells were moderate, but they may become clinically relevant when more nephrotoxic drugs are bound to SPIONs.

Subcellular Localization of Proteins Responding to Mitoxantrone-Induced DNA Damage in Leukaemic Cells.

The aim of the present study was to investigate the subcellular localization of proteins participating in the double-strand break response pathway - p53, Mdm2, p21 and Chk2. MOLT-4 cells were pre-treated with mitoxantrone in concentrations 1 nmol/l and 5 nmol/l. The trypan blue technique was used to determine cell viability and proliferation. Western blotting was used to evaluate changes in p53, Mdm2 and Chk2 protein expression and sandwich ELISA was used to evaluate changes in the p21 protein amount. After 1 nmol/l mitoxantrone cells did not die, but their ability to proliferate was decreased. The p53 protein was activated and phosphorylated at serines 15 and 392 and accumulated in the nucleus after 24 and 48 h. The Mdm2 protein was present in the cytoplasm with its maximal level after 8 and 16 h. The p21 protein was detected in the nucleus after 24 and 48 h. Increased levels of phosphorylated Chk2 at threonine 68 were observed in the cytoplasmic fraction after 24 and 48 h of mitoxantrone treatment. We used mitoxantrone as an inducer of double-strand breaks to bring new data about the subcellular distribution of proteins responding to DNA damage. In MOLT-4 cells, the p53 protein was activated. p53 was phosphorylated at serines 15 and 392 and accumulated in the nucleus. The Mdm2 protein was activated in advance to p53 and occurred in the cytoplasm. The p21 protein was present in the nucleus. Chk2 kinase was activated by the phosphorylation at threonine 68 and we observed increased levels of this protein in the cytoplasmic fraction.

Chrysin attenuates cardiomyocyte apoptosis and loss of intermediate filaments in a mouse model of mitoxantrone cardiotoxicity.

Chrysin (CHR) is a natural flavonoid and is present in high concentration in honey, propolis and many plant extracts. The aim of the present study was to evaluate the effects of CHR to reduce cardiomyocyte apoptosis and loss of intermediate filaments in a mouse model of mitoxantrone cardiotoxicity. Morphology of the cardiomyocytes was determined by optic and transmission electron microscopy and biochemistry methods. The expression of Bcl-2, Bax and Caspase-3 were assessed by immunofluorecence. Tunel assay was used to assess apoptosis in cardiomyocytes. In addition, the distribution of desmin protein was evaluated using immunohistochemistry. Our results show that MTX treatment significantly increased serum levels of creatine kinase isoenzyme (CK-MB), indicator of cardiac injury and withdrawn under CHR protection. Expression levels of Bcl-2 decreased, while those of Bax and caspase-3 increased following MTX treatment. 50 mg/kg of daily CHR intake reduced Bax and caspase-3 immunopositivity and restored Bcl-2 levels to a value comparable to the control. TUNEL (+) cardiomyocyte nuclei of MTX group showed typical signs of apoptosis which almost completely disappeared in response to 50 mg/kg CHR treatment. In parallel, an irregular distribution and a weak expression of desmin is associated with MTX induced cardiotoxic effects which was also restored by CHR treatment. In conclusion chrysin inhibits MTX-triggered cardiomyocyte apoptosis via multiple pathways, including decrease of the Bax/Bcl-2 ratio and caspase-3 expression along with preservation of the desmin disarray.

The age factor for mitoxantrone's cardiotoxicity: multiple doses render the adult mouse heart more susceptible to injury.

Age is a known susceptibility factor for the cardiotoxicity of several anticancer drugs, including mitoxantrone (MTX). The impact of anticancer drugs in young patients is underestimated, thus we aimed to evaluate the cardiotoxicity of MTX in juvenile and adult animals. Juvenile (3 week-old) and adult (8-10 week-old) male CD-1 mice were used. Each group was treated with a 9.0mg/kg cumulative dose of MTX or saline; they were maintained in a drug-free period for 3-weeks after the last administration to allow the development of late toxicity (protocol 1), or sacrificed 24h after the last MTX administration to evaluate early cardiotoxicity (protocol 2). In protocol 1, no adult mice survived, while 2 of the juveniles reached the end of the protocol. High plasma aspartate aminotransferase/alanine aminotransferase ratio and a high cardiac reduced/oxidized glutathione ratio were found in the surviving MTX-treated juvenile mice. In protocol 2, a significant decrease in plasma creatine-kinase MB in juveniles was found 24h after the last MTX-administration. Cardiac histology showed that both MTX-treated populations had significant damage, although higher in adults. However, MTX-treated juveniles had a significant increase in fibrotic tissue. The MTX-treated adults had higher values of cardiac GSSG and protein carbonylation, but lower cardiac noradrenaline levels. For the first time, mature adult animals were shown to be more susceptible to MTX as evidenced by several biomarkers, while young animals appear to better adjust to the MTX-induced cardiac injury. Even so, the higher level of fibrotic tissue and the histological damage showed that MTX also causes cardiac damage in the juvenile population.