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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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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.

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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 IC 50 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., (Copyright © 2021. Published by Elsevier B.V.)

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.

CuO nanostructured thin films supported on silicon with 6.5 cm 2 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 H 2 O 2 , 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 H 2 O 2 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., (Copyright © 2018. Published by Elsevier B.V.)

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.

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.

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., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

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.

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.

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., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Two novel types of supramolecular nanocarriers fabricated by the amphiphilic host-guest inclusion complex formed from water-soluble pillar[6]arene (WP6) and azobenzene derivatives G1 or G2 have been developed, in which G1 is structurally similar to G2 but has an extra phenoxy group in its hydrophobic region. Supramolecular micelles can be initially formed by WP6 with G1, which gradually transform into layered structures with liquid-crystalline properties, whereas stable supramolecular vesicles are obtained from WP6 and G2, which exhibit dual photo- and pH-responsiveness. Notably, the resulting WP6⊃G2 vesicles can efficiently encapsulate anticancer drug mitoxantrone (MTZ) to achieve MTZ-loaded vesicles, which maintain good stability in a simulated normal physiological environment, whereas in an acid environment similar to that of tumor cells or with external UV irradiation, the encapsulated drug is promptly released. More importantly, cytotoxicity assay indicates that such vesicles have good biocompatibility and the MTZ-loaded vesicles exhibit comparable anticancer activity to free MTZ, especially with additional UV stimulus, whereas its cytotoxicity for normal cells was remarkably reduced. Flow cytometric analysis further confirms that the cancer cell death caused by MTZ-loaded vesicles is associated with apoptosis. Therefore, the dual pH- and UV-responsive supramolecular vesicles are a potential platform for controlled release and targeted anticancer drug delivery., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

In recent years the use of natural dietary antioxidants to minimize the cytotoxicity and the damage induced in normal tissues by antitumor agents is gaining consideration. In literature, it is reported that vitamin C exhibits some degree of antineoplastic activity whereas Mitoxantrone (MTZ) is a synthetic anti-cancer drug with significant clinical effectiveness in the treatment of human malignancies but with severe side effects. Therefore, we have investigated the effect of vitamin C alone or combined with MTZ on MDA-MB231 and MCF7 human breast cancer cell lines to analyze their dose-effect on the tumor cellular growth, cellular death, cell cycle and cell signaling. Our results have evidenced that there is a dose-dependence on the inhibition of the breast carcinoma cell lines, MCF7 and MDA-MB231, treated with vitamin C and MTZ. Moreover, their combination induces: i) a cytotoxic effect by apoptotic death, ii) a mild G2/M elongation and iii) H2AX and mild PI3K activation. Hence, the formulation of vitamin C with MTZ induces a higher cytotoxicity level on tumor cells compared to a disjointed treatment. We have also found that the vitamin C enhances the MTZ effect allowing the utilization of lower chemotherapic concentrations in comparison to the single treatments.

Mitoxantrone (MTX) is a chemotherapeutic agent, which presents late irreversible cardiotoxicity. This work aims to highlight the mechanisms involved in the MTX-induced cardiotoxicity, namely the effects toward mitochondria using in vivo and in vitro studies. Male Wistar rats were treated with 3 cycles of 2.5 mg/kg MTX at day 0, 10, and 20. One treated group was euthanized on day 22 (MTX22) to evaluate the early MTX cardiac toxic effects, while the other was euthanized on day 48 (MTX48), to allow the evaluation of MTX late cardiac effects. Cardiac mitochondria isolated from 4 adult untreated rats were also used to evaluate in vitro the MTX (10 nM, 100 nM, and 1 μM) direct effects upon mitochondria functionality. Two rats of MTX48 died on day 35, and MTX treatment caused a reduction in relative body weight gain in both treated groups with no significant changes in water and food intake. Decreased levels of plasma total creatine kinase and CK-MB were detected in the MTX22 group, and increased plasma levels of lactate were seen in MTX48. Increased cardiac relative mass and microscopic changes were evident in both treated groups. Considering mitochondrial effects, for the first time, it was evidenced that MTX induced an increase in the complex IV and complex V activities in MTX22 group, while a decrease in the complex V activity was accompanied by the reduction in ATP content in the MTX48 rats. No alterations in mitochondria transmembrane potential were found in isolated mitochondria from MTX48 rats or in isolated mitochondria directly incubated with MTX. This study highlights the relevance of the cumulative MTX-induced in vivo mitochondriopathy to the MTX cardiotoxicity.

Mitoxantrone (MTX) is an antineoplastic agent that can induce hepato- and haematotoxicity. This work aimed to investigate the occurrence of cumulative early and late MTX-induced hepatic and haematological disturbances in an vivo model. A control group and two groups treated with three cycles of 2.5 mg/kg MTX at days 0, 10 and 20 were formed. One of the treated groups suffered euthanasia on day 22 (MTX22) to evaluate early MTX toxic effects, while the other suffered euthanasia on day 48 (MTX48), to allow the evaluation of MTX late effects. An early immunosuppression with a drop in the IgG levels was observed, causing a slight decrease in the plasma total protein content. The early bone marrow depression was followed by signs of recovery in MTX48. The genotoxic potential of MTX was demonstrated by the presence of several micronuclei in MTX22 leucocytes. Increases in plasma iron and cholesterol levels in the MTX22 rats were observed, while in both groups increases in the unconjugated bilirubin, C4 complement, and decreases in the triglycerides, alanine aminotransferase, alkaline phosphatase and transferrin were found in plasma samples. On MTX 48, the liver histology showed more hepatotoxic signs, the hepatic levels of reduced and oxidized glutathione were increased, and ATP hepatic levels were decreased. However, the hepatic total protein levels were decreased only in the livers of MTX22 group. Results demonstrated the MTX genotoxic effects, haemato- and direct hepatotoxicity. While the haematological toxicity is ameliorated with time, the same was not observed in the hepatic injury., (© 2013 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Background and Purpose: Mitoxantrone (MTX) has been shown to reduce progression of disability and number of clinical exacerbations in patients with progressive multiple sclerosis (MS). Prolonged administration of MTX, however, is limited by the risk of cardiotoxicity. Cardiac monitoring in MTX-treated patients includes usually measurement of left ventricular ejection fraction (LVEF) by means of echocardiography. The N-terminal pro-brain natriuretic peptide (NT-proBNP) represents a novel diagnostic tool in the assessment of heart failure. This study was aimed to evaluate the usefulness of NT-proBNP for early detection of MTX-induced cardiotoxicity in MS patients., Materials and Methods: We measured the NT-proBNP plasma levels in 45 MS patients who completed 24-month MTX therapy and in 37 MS patients of control group., Results: The median NT-proBNP plasma value was 15.12pg/mL. In 12 MTX-treated patients (27%), NT-proBNP plasma values were elevated, though this subgroup of patients neither clinical showed evidence of myocardial damage nor had the LVEF value <50%. In five patients with normal NT-proBNP, we observed LVEF decline >10%. We did not observe correlations between the NT-proBNP levels and patient age, MS duration, relapses index, Extended Disability Status Scale (EDSS), MTX single dose and the total cumulative dose of MTX. In 8 patients (22%) from control group, NT-proBNP plasma levels were also elevated., Conclusions: The results of our study confirm that MTX therapy is safe for carefully selected and closely monitored MS patients. We believe that serial evaluation of NT-proBNP levels (before, during and after MTX therapy) can identify MS patients at high risk for MTX-induced cardiotoxicity., (Copyright © 2014 Polish Neurological Society. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Mitoxantrone (MTX) is an antitumor agent that causes cardiotoxicity in 18 % patients. The metabolic profile of MTX was assessed after incubation of 100 μM MTX with hepatic S9 fraction isolated from rats. The presence of MTX and its metabolites was also assessed in vivo through the analysis of liver and heart extracts of MTX-treated rats. The cytotoxic effects of MTX and MTX metabolites were evaluated in the H9c2 cells after 24-h incubation with MTX alone and MTX + metabolites. The influence of CYP450- and CYP2E1-mediated metabolism for the cytotoxicity of MTX was assessed after 96-h incubation with MTX (100 nM and 1 μM) in the presence/absence of CYP450 or CYP2E1 inhibitors. After 4-h incubation in supplemented S9 fraction, the MTX content was 35 % lower and 5 metabolites were identified: an acetoxy ester derivative (never described before), two glutathione conjugates, a monocarboxylic acid derivative, and the naphtoquinoxaline, the later commonly related to MTX pharmacological effects. The presence of MTX and naphtoquinoxaline metabolite was evidenced in vivo in liver and heart of MTX-treated rats. The cytotoxicity caused by MTX + metabolites was higher than that observed in the H9c2 cells incubated with non-metabolized MTX group. The co-incubation of MTX with CYP450 and CYP2E1 inhibitors partially prevented the cytotoxicity observed in the MTX groups incubated with H9c2 cells, highlighting that the metabolism of MTX is relevant for its undesirable effects. The naphtoquinoxaline metabolite is described in heart and liver in vivo, highlighting that this metabolite accumulates in these tissues. It was demonstrated that MTX P450-mediated metabolism contributed to MTX toxicity.

In the present study, selected advanced oxidation processes (AOPs)-namely, photo-Fenton (with Fe(2+), Fe(3+), and potassium ferrioxalate-FeOx-as iron sources), solar photo-Fenton, Fenton, and UV/H2O2-were investigated for degradation of the antineoplastic drug mitoxantrone (MTX), frequently used to treat metastatic breast cancer, skin cancer, and acute leukemia. The results showed that photo-Fenton processes employing Fe(III) and FeOx and the UV/H2O2 process were most efficient for mineralizing MTX, with 77, 82, and 90% of total organic carbon removal, respectively. MTX probably forms a complex with Fe(III), as demonstrated by voltammetric and spectrophotometric measurements. Spectrophotometric titrations suggested that the complex has a 2:1 Fe(3+):MTX stoichiometric ratio and a complexation constant (K) of 1.47 × 10(4) M(-1), indicating high MTX affinity for Fe(3+). Complexation partially inhibits the involvement of iron ions and hence the degradation of MTX during photo-Fenton. The UV/H2O2 process is usually slower than the photo-Fenton process, but, in this study, the UV/H2O2 process proved to be more efficient due to complexing of MTX with Fe(III). The drug exhibited no cytotoxicity against NIH/3T3 mouse embryonic fibroblast cells when oxidized by UV/H2O2 or by UV/H2O2/FeOx at the concentrations tested.

A series of indazolo[4,3-gh]isoquinolinones derivatives have been synthesized to decrease cardiotoxic side effects in comparison to Mitoxantrone. The antiproliferative effects of different side chains were investigated and tested on at least four different cell lines of cervix, ovarian, CNS, NSCLC (non-small-cell lung cancer) and colon carcinoma. In addition to antiproliferative activities, influence on cell cycle and intercalation behavior have been tested., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Recent insights into the regulation of the androgen receptor (AR) activity led to novel therapeutic targeting of AR function in prostate cancer patients. Docetaxel is an approved chemotherapy for treatment of castration-resistant prostate cancer; however, the mechanism underlying the action of this tubulin-targeting drug is not fully understood. This study investigates the contribution of microtubules and the cytoskeleton to androgen-mediated signaling and the consequences of their inhibition on AR activity in human prostate cancer. Tissue microarrays from docetaxel-treated and untreated prostate cancer patients were comparatively analyzed for prostate-specific antigen (PSA) and AR immunoreactivity. The AR transcriptional activity was determined in prostate cancer cells in vitro, based on PSA mRNA expression and the androgen response element reporter activity. The interaction of AR with tubulin was examined by immunoprecipitation and immunofluorescence. Treatment of prostate cancer patients with docetaxel led to a significant translocation of AR. In untreated specimens, 50% prostate tumor cells exhibited nuclear accumulation of AR, compared with docetaxel-treated tumors that had significantly depleted nuclear AR (38%), paralleled by an increase in cytosolic AR. AR nuclear localization correlated with PSA expression. In vitro, exposure of prostate cancer cells to paclitaxel (1 μmol/L) or nocodazole (5 μg/mL) inhibited androgen-dependent AR nuclear translocation by targeting AR association with tubulin. Introduction of a truncated AR indicated the requirement of the NH(2)-terminal domain for AR-tubulin interaction. Our findings show that in addition to blocking cell division, docetaxel impairs AR signaling, evidence that enables new insights into the therapeutic efficacy of microtubule-targeting drugs in prostate cancer., (©2010 AACR.)

Natural and synthetic compounds characterized by an anthraquinone nucleus represent an important class of anti-neoplastic agents, the mechanism of action of which is related to intercalation into DNA. Ametantrone (AM) is a synthetic 9,10-anthracenedione bearing two (hydroxyethylamino)ethylamino residues at positions 1 and 4; along with other anthraquinones and anthracyclines, it shares a polycyclic intercalating moiety and charged side chains that stabilize DNA binding. All these drugs elicit adverse side effects, which represent a challenge for antitumor chemotherapy. In the present work the structure of AM was augmented with appropriate groups that target well-defined base pairs in the major groove. These should endow AM with DNA sequence selectivity. We describe the rationale for the synthesis and the evaluation of activity of a new series of compounds in which the planar anthraquinone is conjugated at positions 1 and 4 through the side chains of AM or other bioisosteric linkers to appropriate dipeptides. The designed novel AM derivatives were shown to selectively stabilize two oligonucleotide duplexes that both have a palindromic GC-rich hexanucleotide core, but their stabilizing effects on a random DNA sequence was negligible. In the case of the most effective compound, the 1,4-bis-[Gly-(L-Lys)] derivative of AM, the experimental results confirm the predictions of earlier theoretical computations. In contrast, AM had equal stabilizing effects on all three sequences and showed no preferential binding. This novel peptide derivative can be classified as a strong binder regarding the sequences that it selectively targets, possibly opening the exploitation of less cytotoxic conjugates of AM to the targeted treatment of oncological and viral diseases.

Purpose: The objective of our study was to investigate the effect of single and multiple flavonoids on the accumulation and cytotoxicity of mitoxantrone in BCRP-overexpressing breast cancer cells and on the transport of mitoxantrone in BCRP-expressing normal cells., Methods: The effect of flavonoids on mitoxantrone accumulation and cytotoxicity was studied in the human breast cancer MCF-7 MX100 cell line. Mitoxantrone transport in the presence of flavonoids was studied in human and murine BCRP-transfected MDCK cell lines, and mitoxantrone concentrations were determined by HPLC., Results: Our results demonstrated that multiple flavonoid combinations act additively and exhibit strong BCRP inhibition for increasing mitoxantrone accumulation in breast cancer cells. Kaempferide, biochanin A, 5,7-dimethoxyflavone, and 8-methylflavone greatly increased the cytotoxicity of mitoxantrone in BCRP-overexpressing breast cancer cells. Additionally, the basolateral-to-apical membrane-directed transport of mitoxantrone in murine Bcrp1- and human BCRP-expressing MDCK cells, in the presence of 2.5 microM of these flavonoids, was also significantly decreased., Conclusion: The results indicate that flavonoids are potent BCRP inhibitors and that they exert additive effects when used in combination. Flavonoids demonstrate MDR-reversing effects, but also may influence the disposition of mitoxantrone and cause pharmacokinetic interactions.

DNA-damaging agents cause a multifaceted cellular stress response. Cells set in motion either repair mechanisms or programmed cell death pathways, depending on the extent of the damage and on their ability to withstand it. The RNA-binding protein (RBP) Sam68, which is up-regulated in prostate carcinoma, promotes prostate cancer cell survival to genotoxic stress. Herein, we have investigated the function of Sam68 in this cellular response. Mitoxantrone (MTX), a topoisomerase II inhibitor, induced relocalization of Sam68 from the nucleoplasm to nuclear granules, together with several other RBPs involved in alternative splicing, such as TIA-1, hnRNP A1 and the SR proteins SC35 and ASF/SF2. Sam68 accumulation in nuclear stress granules was independent of signal transduction pathways activated by DNA damage. Using BrU labelling and immunofluorescence, we demonstrate that MTX-induced nuclear stress granules are transcriptionally active foci where Sam68 and the phosphorylated form of RNA polymerase II accumulate. Finally, we show that MTX-induced relocalization of Sam68 correlates with changes in alternative splicing of its mRNA target CD44, and that MTX-induced CD44 splicing depends on Sam68 expression. These results strongly suggest that Sam68 is part of a RNA-mediated stress response of the cell that modulates alternative splicing in response to DNA damage.

We developed the synthesis of a series of thiophene-fused tetracyclic analogues of the antitumor drug ametantrone. The reactions included nucleophilic substitution of methoxy groups in 4,11-dimethoxyanthra[2,3-b]thiophene-5,10-diones with ethylenediamines, producing the derivatives of 4,11-diaminoanthra[2,3-b]thiophene-5,10-dione in good yields. Several compounds showed marked antiproliferative potency against doxorubicin-selected, P-glycoprotein-expressing tumor cells and p53(-/-) cells. The cytotoxicity of some novel compounds for P-glycoprotein-positive cells is highly dependent on N-substituent at the terminal amino group of ethylenediamine moiety. The cytotoxic potency of selected compounds correlated with their ability to attenuate the functions of topoisomerase I and telomerase, strongly suggesting that these enzymes are the major targets of antitumor activity of anthra[2,3-b]thiophene-5,10-dione derivatives.

Mitoxantrone was encapsulated into pegylated SUVs using ammonium sulfate gradient method. Four formulations (LM-s, LM-p, LM-m and LM-m-L) were prepared, which were made from different PCs and exhibited different PEG grafting density. In vitro release studies revealed that drug release rate increased with decreased T(m) of PCs, and reduced PEG polymer coverage. In circulation, the trend towards increased circulation time as T(m) of PCs and PEG lipid content are elevated is observed. However, it was found that the order of toxicity in balb/c mice was Lm-s

We reported previously that doxorubicin, an anticancer agent that has an anthracycline structure, alters Ca2+ releasing and uptake mechanisms in the sarcoplasmic reticulum of myocardial cells. These effects of doxorubicin are apparently related to its cardiotoxicity. Mitoxantrone is a similar anticancer agent with an anthracenedion structure that has been shown to be significantly less cardiotoxic. In the present study, the effects of mitoxantrone on the functions of the sarcoplasmic reticulum were examined in isolated muscle preparations obtained from the guinea-pig heart. In electrically-stimulated left atrial muscle preparations, incubation in vitro for 4 hr with 30 or 100 microM mitoxantrone significantly prolonged the time to the peak of twitch tension, markedly increased the developed tension observed at lower stimulation frequencies, thereby attenuating the slope of positive force-frequency relationships, and increased the postrest contraction observed after a 60-sec quiescent period. In myocytes isolated from ventricular muscles, 30 microM mitoxantrone increased the peak and the size of intracellular Ca2+ concentrations ([Ca2+] i), and prolonged the time to peak [Ca2+]i. In skinned muscle fiber preparations obtained from the left ventricular muscle, 30 muM mitoxantrone significantly increased the caffeine-induced contraction without affecting the Ca2+ sensitivity of contractile proteins. These results suggest that mitoxantrone enhances Ca2+ release from the sarcoplasmic reticulum in isolated atrial muscle preparations obtained from the guinea-pig heart. Apparent enhancement of the sarcoplasmic reticulum functions, in contrast to anthracyclines that has been shown to suppress these functions, seems to explain the relative lack of marked cardiotoxicity of mitoxantrone.

Background: Treatment of peritoneal recurrence following surgical resection of intra-abdominal sarcomas presents a significant challenge to clinicians. Historically, treatment with systemic chemotherapy has been ineffective and surgical resection alone has not been durable. We prospectively evaluated the feasibility of cytoreductive surgery followed by hyperthermic intraperitoneal chemotherapy (HIPEC) with cisplatin (CDDP) alone or in combination with mitoxantrone (MITOX) for the treatment of sarcomatosis., Methods: Two phase I trials of HIPEC were conducted (1998-2003). Eligible patients with evidence of sarcomatosis underwent cytoreductive surgery followed by HIPEC. In the first trial, CDDP dosing was established as 90 mg/m2 with a perfusate time of 90 minutes and temperature of 41 degrees C. In the second trial, MITOX (20 mg/m2) was instilled following perfusion with CDDP. Toxicity, efficacy, and quality of life (QOL) were evaluated., Results: A total of 28 patients were enrolled in the two trials. We noted a higher overall toxicity score and complication rate with combination CDDP/MITOX versus CDDP alone and shorter overall survival duration (5.5 months vs 16.9 months, respectively). In addition, local recurrence rates were similar in both groups (CDDP 79% vs CDDP/MITOX 68%). As expected, QOL scores at 6-8 weeks following HIPEC were 15-25% lower than the baseline scores; however, they returned to baseline at 3-6 months., Conclusions: Although the HIPEC technique is feasible for patients with sarcomatosis, it is associated with significant toxicity and limited clinical benefit. Combination CDDP/MITOX failed to demonstrate any benefit over CDDP alone; moreover, there was an increase in toxicity.

Anthracyclines and anthracenediones are important oncotherapeutics; however, their use is associated with irreversible and cumulative cardiotoxicity. A novel aza-anthracenedione, pixantrone (BBR 2778), was developed to reduce treatment-related cardiotoxicity while retaining efficacy. This study evaluates the cumulative cardiotoxic potential of pixantrone compared with equiactive doses of doxorubicin and mitoxantrone in both doxorubicin-pretreated and doxorubicin-naïve mice. CD1 female mice were given doxorubicin 7.5 mg/kg (once weekly for 3 weeks) followed 6 weeks later by either sterile 0.9% saline, doxorubicin 7.5 mg/kg, pixantrone 27 mg/kg, or mitoxantrone 3 mg/kg (once weekly for 3 weeks). A second group of CD1 female mice were given 2 cycles of either sterile 0.9% saline, pixantrone 27 mg/kg, doxorubicin 7.5 mg/kg, or mitoxantrone 3 mg/kg (once weekly for 3 weeks). Animals were sacrificed at different time points for histopathologic examination of the heart. In the doxorubicin-pretreated mice, further exposure to doxorubicin or mitoxantrone resulted in a significant worsening of pre-existing degenerative cardiomyopathy. In contrast, pixantrone did not worsen pre-existing cardiomyopathy in these animals. Only minimal cardiac changes were observed in mice given repeated cycles of pixantrone, while 2 cycles of doxorubicin or mitoxantrone resulted in marked or severe degenerative cardiomyopathy. These animal studies demonstrate the reduced cardiotoxic potential of pixantrone compared with doxorubicin and mitoxantrone. Exposure to pixantrone did not worsen pre-existing cardiomyopathy in doxorubicin-pretreated mice, suggesting that pixantrone may be useful in patients pretreated with anthracyclines.

Bovine serum albumin (BSA) and chitosan (CS) nanospheres of mitoxantrone (MTO) were comparatively evaluated in terms of tissue distribution, acute toxicity and therapeutic efficiency against breast cancer and its lymph node metastases. After local injection in rats, MTO nanospheres showed a slower elimination rate and a much higher drug concentration in lymph nodes compared with MTO solution, and a lower drug concentration in other tissues. There was no observed acute toxicity to the main tissues of Kunming mice after local injection of MTO-BSA-NS. Mild toxicity to liver and lung was observed for MTO-CS-NS, but, for MTO solution, severe toxicity to liver and lung and much lower number of white blood cells were observed. Human MCF-7 breast cancer in nude mice and animal model of P388 lymph node metastases in Kunming mice were applied to investigate the therapeutic efficiency. The inhibition rate of the nanospheres against breast cancer was much higher than that of MTO solution, and lymph node metastases were efficiently inhibited by the nanospheres, especially MTO-BSA-NS.

Objective: To explore the potential of dexrazoxane to suppress subclinical cardiotoxicity in MS patients receiving mitoxantrone., Methods: An open-label study was performed to evaluate possible subclinical cardiotoxicity in multiple sclerosis patients treated quarterly with mitoxantrone (48 mg/m(2) cumulative), with and without concomitant dexrazoxane, using blinded serial radionucleide ventriculography., Results: No patient experienced symptoms of heart failure. Patients receiving dexrazoxane, which is cardioprotective for anthracyclines, exhibited a significantly lesser decline in left ventricular ejection fraction (mean change, -3.80% vs -8.55%, p < 0.001)., Interpretation: These results support a cardioprotective effect of dexrazoxane in mitoxantrone treated multiple sclerosis patients.

Objective: [corrected] To make a comparison between mitoxantrone (DHAQ) and liver targeting drug delivery system mitoxantrone-polybutylcyanoacrylate-nanosphere (DHAQ-PBCA-NS) in respect to their antitumor activity against experimental liver tumor H22 in mice., Methods: Drugs were given intravenously on the 1st, 5th, 9th day after planting tumor respectively. Weight of tumor in mouse was determined and the results were compared with those of mitoxantrone (DHAQ)., Results: There was relationship of dose-effect for both DHAQ and DHAQ-PBCA-NS, and the median effective dose (ED50) of DHAQ and DHAQ-PBCA-NS was 1.04 mg/kg and 0.34 mg/kg respectively. The lethal dose to 50% of the population (LD50) of DHAQ and DHAQ-PBCA-NS i.v. in mice with the same administration schedule was 3.670 mg/kg and 4.225 mg/kg respectively. Therefore, the calculated value of therapeutic index was 3.53 for DHAQ and 12.43 for DHAQ-PBCA-NS. In addition, the antitumor activity of both drugs with different treatment schedules was reported. The results showed: the earlier the mice were treated, the higher the antitumor activity of the two drugs were seen. However, DHAQ-PBCA-NS presented higher activity than DHAQ did, when the same treatment schedule was followed., Conclusion: The results demonstrated that the antitumor activity of DHAQ-PBCA-NS is much higher than that of DHAQ, and DHAQ-PBCA-NS is possessed of liver targeting property.

MDR in human cancers is one of the major causes of failure of chemotherapy. A member of the superfamily of ABC transporters, BCRP, was demonstrated to confer an atypical MDR phenotype to tumor cells. To overcome the BCRP-mediated drug resistance, the fungal secondary metabolite TPS-A, a diketopiperazine, was analyzed with regard to its potency to reverse the BCRP-mediated drug-resistant phenotype. At concentrations of 10-50 microM, TPS-A reversed a mitoxantrone-resistant phenotype and inhibited the cellular BCRP-dependent mitoxantrone accumulation in the human gastric carcinoma cell line EPG85-257RNOV, the human breast cancer cell line MCF7/AdrVp (both exhibiting acquired BCRP-mediated MDR) and the BCRP cDNA-transfected breast cancer cell line MCF-7/BCRP clone 8. No cytotoxicity was seen at effective concentrations. These data indicate that TPS-A is a novel BCRP inhibitor., (Copyright 2003 Wiley-Liss, Inc.)

BRCA1 is implicated in cellular responses to DNA damage, thereby substantially contributing to maintenance of the genome integrity. Mutations in the BRCA1 gene occur in breast and ovarian cancer and mutations that disable p53 are frequently found in human cancers, often accompanied by mutations in additional genes, contributing to tumor progression or high-grade malignancy. Therefore, the role of BRCA1 in the sensitivity to anticancer agents in p53-deficient cells was investigated using p53-deficient mouse knockout cell lines either deficient or proficient in Brca1 function. We report that Brca1-deficiency in p53-null cells was associated with increased sensitivity to the topoisomerase I poisons camptothecin and topotecan, the topoisomerase II poisons doxorubicin, mitoxantrone and etoposide, and to the platinum compounds carboplatin and oxaliplatin, but not to the antimetabolites 5-fluorouracil and gemcitabine and the taxanes docetaxel and paclitaxel. The increased growth inhibition to doxorubicin after loss of Brca1 correlated with increased cell killing caused by increased apoptosis. The data presented here indicate that Brca1 modulates p53-independent DNA damage response pathways and they support the case of a role of Brca1 to protect cells from apoptosis-mediated cell death in p53-deficient cells. These results suggest a higher chemotherapy susceptibility of cells disabled in both functions and they foster the concept that functional inhibition of BRCA1 may be a valuable adjunct to anticancer agents to increase the efficacy of chemotherapy in the treatment of p53-mutated cancers.

The toxicity of most drugs is associated with their enzymatic conversion to toxic metabolites. Bioactivation reactions occur in a range of cellular organs and organelles, including mitochondria. We have investigated different effects (i.e. growth inhibition, mortality and genotoxicity) of doxorubicin, epirubicin and mitoxantrone on the D7 strain of Saccharomyces cerevisiae and on its petite (rho degrees ) respiratory-deficient mutant at various cellular concentrations of cytochrome P450 and glutathione (GSH). The data confirmed the importance of oxygen production for doxorubicin toxicity. The complete absence, or a very low level, of cytochrome oxidase subunit IV conferred some resistance to doxorubicin. Low GSH levels decreased resistance to doxorubicin in both strains, suggesting that thiol depletion could potentiate membrane lipid peroxidation. Doxorubicin induction of petite colonies suggests that the drug is able to select rather than induce respiratory-deficient mutants. Epirubicin induced levels of cytotoxicity similar to those of doxorubicin. The effects did not appear to be significantly dependent on mitochondrial function or GSH levels, whereas cells were strongly protected by cytochrome P450. GSH did not induce an evident alteration. Neither were genotoxic effects induced. Mitoxantrone had reduced levels of both growth inhibition and cytotoxicity in comparison to anthracyclines and induced convertants, revertants and aberrants. All the effects considered were amplified at high cytochrome P450 cellular concentrations, although the drug was also shown to act without previous metabolism via cytochrome P450. Anthracenedione effectiveness was increased by metabolism via cytochrome P450 and partially reduced by GSH. However, further mechanisms were suggested, which might implicate mitochondrial function and/or production of electrophilic cytotoxic and/or genotoxic intermediates by means of GSH conjugation. The biological effectiveness of doxorubicin, epirubicin and mitoxantrone on S.cerevisiae was shown to be strictly dependent on cell-specific physiological/biochemical conditions, such as a functional respiratory chain and levels of cytochrome P450 and GSH.

In this study, we evaluated the influence of protein kinase C zeta (PKC zeta) on topoisomerase II inhibitor-induced cytotoxicity in monocytic U937 cells. In U937-zeta J and U937-zeta B cells, enforced PKC zeta expression, conferred by stable transfection of PKC zeta cDNA, resulted in total inhibition of VP-16- and mitoxantrone-induced apoptosis and decreased drug-induced cytotoxicity, compared with U937-neo control cells. In PKC zeta-overexpressing cells, drug resistance correlated with decreased VP-16-induced DNA strand breaks and DNA protein cross-links measured by alkaline elution. Kinetoplast decatenation assay revealed that PKC zeta overexpression resulted in reduced global topoisomerase II activity. Moreover, in PKC zeta-overexpressing cells, we found that PKC zeta interacted with both alpha and beta isoforms of topoisomerase II, and these two enzymes were constitutively phosphorylated. However, when human recombinant PKC zeta (rH-PKC zeta) was incubated with purified topoisomerase II isoforms, rH-PKC zeta interacted with topoisomerase II beta but not with topoisomerase II alpha. PKC zeta/topoisomerase II beta interaction resulted in phosphorylation of this enzyme and in decrease of its catalytic activity. Finally, this report shows for the first time that topoisomerase II beta is a substrate for PKC zeta, and that PKC zeta may significantly influence topoisomerase II inhibitor-induced cytotoxicity by altering topoisomerase II beta activity through its kinase function.

Purpose: Drug-resistant phenotypes of cancer cells may be caused by complex multimodal mechanisms of resistance. In order to gain further insighte into these mechanisms, a P-glycoprotein-mediated multidrug-resistant phenotype induced by daunorubicin-selection and an alternative drug resistance due to treatment with mitoxantrone were investigated in pancreatic carcinoma-derived cells., Methods: For assessing cross-resistance against various drugs, cell proliferation assays were performed. Drug accumulation was measured by flow cytometry. Messenger RNA expression was analyzed by Northern blot and RT-PCR, whereas protein expression was determined by Western blot. Catalytic activity of DNA-topoisomerases (Topo) II was determined by the decatenation assay., Results: In mitoxantrone-selected EPP85-181RNOV cells a decreased accumulation of mitoxantrone and daunorubicin was observed in the absence of P-glycoprotein, multidrug resistance protein or breast cancer resistance protein over-expression. An approximately twofold decrease of DNA topoisomerase II catalytic activity could be observed in both drug-resistance-exhibiting cell lines. The reduction of Topo II catalytic activity was reflected by decreased expression of Topo IIalpha and IIbeta mRNAs and proteins., Conclusions: The decreased drug accumulation in EPP85-181RNOV cells indicates that alternative transport events are occurring. The decreased catalytic activity and expression of Topo II indicate that modulation of Topo II catalytic activity contributes to both drug-resistant phenotypes in pancreatic carcinoma cells.

Breast cancer resistance protein (BCRP), an adenosine triphosphate-binding cassette transporter, confers resistance to a series of anticancer reagents, including mitoxantrone, SN-38 and topotecan. In the present study, we found that estrone and 17beta-estradiol potentiated the cytotoxicity of mitoxantrone, SN-38 and topotecan in BCRP-transduced K562 cells (K562 / BCRP). These estrogens showed only a marginal effect, or none, in parental K562 cells. Estrone and 17beta-estradiol increased the cellular accumulation of topotecan in K562 / BCRP cells, but not in K562 cells, suggesting that these estrogens inhibit the BCRP-mediated drug efflux and overcome drug resistance.

This experimental study compares the toxic effect of the two cytotoxic drugs, doxorubicin and mitoxantrone, on cardiac muscle and on serum lipids. The cardiotoxicity of doxorubicin due to the cumulative effect of repeated administration is known. A relative compound, mitoxantrone, is a member of the androstenedione class of synthetic antitumor agents and its chemical structure, based on a quinone ring, is similar to that of doxorubicin. Doxorubicin has wide application in cancer medicine but its dose-limited cardiac effect creates the need for a substitute compound. Mitoxantrone, also an effective agent, may be able to cover this need. Three groups of 35 Wistar rats were used during this experimental study of 12 weeks' duration. Drugs at a certain calculated dose were administered once weekly. Group A animals were treated with doxorubicin, Group B with mitoxantrone and Group C, the controls, with normal saline. Six animals per group were autopsied after euthanasia in the 3rd, 6th, 8th, 10th and 12th weeks. Cardiac muscle, liver and other organs, plus blood, were removed for macro- and microscopical, and biochemical tests. Our results showed that there was a cumulative toxic effect of doxorubicin (adriamycin) on cardiac muscle starting in the 6th week which gradually increased to Grade III lesions by the 10th and 12th weeks. In parallel, an increase in serum lipids, mainly cholesterol and triglycerides was observed. Mitoxantrone-treated animals showed moderate cardiotoxic lesions (but not cumulative) and no increase in serum lipids. In vitro testing of oxygen-free radical production showed high production by doxorubicin and very low production by mitoxantrone. Thus, mitoxantrone appears to be safer than doxorubicin.

Purpose: To ascertain the maximum tolerated duration of infusion of gemcitabine at 10 mg/m(2)/min in combination with mitoxantrone at 12 mg/m(2) daily for 3 days in the treatment of acute leukemia., Patients and Methods: Thirty-four patients were enrolled. Stratum I consisted of 26 patients, median age 50 years (range, 25 to 71 years), with relapsed or refractory leukemia. Stratum II contained eight patients, median age 62.5 years (range, 38 to 83 years), who had received fewer than three cycles of myelotoxic therapy for chronic myeloid leukemia or myelodysplasia that had evolved into leukemia. Patients received mitoxantrone at 12 mg/m(2) daily for 3 days. After the first mitoxantrone dose, gemcitabine was provided intravenously at 10 mg/m(2)/min with the duration adjusted by following a continuous reassessment model., Results: Severe myelosuppression, and stomatitis or esophagitis were the most common hematologic and nonhematologic dose-limiting toxicities. Several patients developed febrile neutropenia, nausea, or vomiting. In both strata, the maximum recommended duration of infusion of gemcitabine was 12 hours (7,200 mg/m(2)). The mean steady-state concentration of gemcitabine was 24.72 micromol/L and varied over a fivefold range among patients. Overall response rates in this phase I trial for strata I and II were 42% and 63%, respectively., Conclusion: Prolonged-infusion gemcitabine at a fixed dose rate of 10 mg/m(2)/min for 12 hours with 12 mg/m(2)/d mitoxantrone for 3 days is a tolerable induction regimen and achieves plasma concentrations sufficient for maximal intracellular activation. Stomatitis or esophagitis should be anticipated; however, this regimen may induce significant responses in patients with difficult-to-treat leukemias.

Idarubicin is an anthracycline antibiotic used in cancer therapy. Mitoxantrone is an anthracycline analog with presumed better antineoplastic activity and lesser toxicity. Using the alkaline comet assaywe showed that the drugs at 0.01-10 microM induced DNA damage in normal human lymphocytes. The effect induced by idarubicin was more pronounced than by mitoxantrone (P < 0.001). The cells treated with mitoxantrone at 1 microM were able to repair damage to their DNA within a 30-min incubation, whereas the lymphocytes exposed to idarubicin needed 180 min. Since anthracyclines are known to produce free radicals, we checked whether reactive oxygen species might be involved in the observed DNA damage. Catalase, an enzyme inactivating hydrogen peroxide, decreased the extent of DNA damage induced by idarubicin, but did not affect the extent evoked by mitoxantrone. Lymphocytes exposed to the drugs and treated with endonuclease III or formamidopyrimidine-DNA glycosylase (Fpg), enzymes recognizing and nicking oxidized bases, displayed a higher level of DNA damage than the untreated ones. 3-Methyladenine-DNA glycosylase II (AlkA), an enzyme recognizing and nicking mainly methylated bases in DNA, increased the extent of DNA damage caused by idarubicin, but not that induced by mitoxantrone. Our results indicate that the induction of secondary malignancies should be taken into account as side effects of the two drugs. Direct strand breaks, oxidation and methylation of the DNA bases can underlie the DNA-damaging effect of idarubicin, whereas mitoxantrone can induce strand breaks and modification of the bases, including oxidation. The observed in normal lymphocytes much lesser genotoxicity of mitoxantrone compared to idarubicin should be taken into account in planning chemotherapeutic strategies.

We describe here the preclinical studies of a novel formulation of liposome-entrapped mitoxantrone (LEM). The liposome entrapment efficiency of mitoxantrone was 93.4 +/- 2.8%. In vitro cytotoxicity studies in HL60 cells comparing LEM with conventional mitoxantrone (MTO) showed IC50 values of 0.31 +/- 0.05 ng/ml and 0.48 +/- 0.06 ng/ml for LEM and MTO, respectively. In CD2F1 mice, LEM was significantly less toxic as compared with MTO. A single intravenous (i.v.) dose of 15 mg/kg MTO produced 100% mortality in CD2F1 mice by Day 10, whereas a single i.v. dose as high as 35 mg/kg LEM caused no mortality for at least up to Day 60 post-treatment. Multiple doses of MTO (i.v., 5.0 mg/kg, 1x daily, x5) caused 100% mortality by Day 10, whereas a similar dose regimen of LEM caused no mortality in CD2F1 mice. Clinical and histopathology evaluations indicated long-term normal tissue protection in mice treated with relatively high single dose (i.v., 35 mg/kg) or multiple doses of LEM (i.v., 5.0 mg/kg, 1x daily, x5). LEM also demonstrated favourable pharmacokinetic profiles. CD2F1 mice injected with 5 mg/kg i.v. dose of LEM showed plasma levels 51-fold higher than with an equivalent dose of MTO. The area under the plasma concentration-time curve was 200-fold greater with LEM as compared to MTO. The plasma half-lives were 0.96 hours and 0.11 hours for LEM and MTO, respectively. An altered tissue distribution was observed with LEM; cardiac tissue demonstrating at least 2.6-fold lower levels of mitoxantrone with LEM vs. MTO. LEM exhibited significant anti-tumor activity against murine ascitic L1210 leukemia in CD2F1 mice. Treatment with a single dose of 20.0 mg/kg LEM resulted in 100% long-term survivors. LEM 2.5 mg/kg (i.v., x4) had antitumor activity against a human hormone-independent prostate carcinoma (PC-3) grown in athymic mice, while a comparable dose of MTO was too toxic. A significant decrease in toxicity, altered pharmacokinetics, and enhanced efficacy of LEM suggest that LEM may provide a viable alternative to the clinical use of conventional mitoxantrone.

In this work three human cell lines with multidrug resistance (MDR) caused by a P-glycoprotein (PGP) overexpression, CEM VLB, HL60 DNR, LOVO DX and two cell lines with MDR associated with a multidrug related protein (MRP) or a lung resistance-related protein (LRP) overexpression named GLC4 ADR and SW1573/2R120 were tested for Amifostine protection against Daunorubicin, Doxorubicin, Idarubicin and Mitoxantrone toxicity. This class of anticancer agents was chosen because they are commonly used in the first line treatments of acute leukemias where a PGP, an LRP or an MRP overexpression often occurs even at onset. A 7-day incubation with escalating doses of anticancer agents with or without a 15 minute preincubation in Amifostine or its active metabolite WR-1065 were used. In conclusion, in none of the MDR positive and negative cell lines did Amifostine modify the toxicity of the anticancer drugs. The observation that even the WR-1065 metabolite gave no protection against Anthracyclines toxicity strengthened the data and provided confirmation for the further in vivo testing of the safety and efficacy of Amifostine in leukemias.

Cellular resistance to the antifolate methotrexate (MTX) is often caused by target amplification, uptake defects, or alterations in polyglutamylation. Here we have examined MTX cross-resistance in a human breast carcinoma cell line (MCF7/MX) selected in the presence of mitoxantrone, an anticancer agent associated with the multidrug resistance (MDR) phenotype. Examination of protein expression and enzyme activities showed that MCF7/MX cells displayed none of the classical mechanisms of MTX resistance. They did, however, exhibit an ATP-sensitive accumulation defect accompanied by reduced polyglutamylation. Although the kinetics of drug uptake was similar between parental and resistant cells, the resistant cells exhibited increased energy-dependent drug efflux. This suggested the involvement of an ATP-binding cassette (ABC) transporter. However, cells transfected with the breast cancer resistance protein (BCRP)-the ABC transporter known to be highly overexpressed in MCF7/MX cells and to confer mitoxantrone resistance (D. D. Ross et al., J. Natl. Cancer Inst. 91: 429-433, 1999)-were not MTX resistant, which suggested that this transporter is not involved in MTX cross-resistance. Moreover, members of the MRP protein family of transport proteins, which had previously been implicated in MTX resistance, were not found to be overexpressed in the MCF7/MX cells. Thus, our data suggest that a novel MTX-specific efflux pump may be involved in this unusual cross-resistance phenotype.

The mitoxantrone resistance (MXR) gene encodes a recently characterized ATP-binding cassette half-transporter that confers multidrug resistance. We studied resistance to the camptothecins in two sublines expressing high levels of MXR: S1-M1-80 cells derived from parental S1 colon cancer cells and MCF-7 AdVp3,000 isolated from parental MCF-7 breast cancer cells. Both cell lines were 400- to 1,000-fold more resistant to topotecan, 9-amino-20(S)-camptothecin, and the active metabolite of irinotecan, 7-ethyl-10-hydroxycamptothecin (SN-38), than their parental cell lines. The cell lines demonstrated much less resistance to camptothecin and to several camptothecin analogues. Reduced accumulation and energy-dependent efflux of topotecan was demonstrated by confocal microscopy. A significant reduction in cleavable complexes in the resistant cells could be observed after SN-38 treatment but not after camptothecin treatment. In addition to topotecan and SN-38, MXR-overexpressing cells are highly resistant to mitoxantrone and epirubicin. Because these compounds are susceptible to glucuronidation, we examined UDP-glucurono-syltransferase (UGT) activity in parental and resistant cells by TLC. Glucuronides were found at equal levels in both parental and resistant colon cancer cell lines for epirubicin and to a lesser extent for SN-38 and mitoxantrone. Low levels of glucuronidation could also be detected in the resistant breast cancer cells. These results were confirmed by analysis of the UGT1A family mRNAs. We thus conclude that colon and breast cancer cells have a capacity for glucuronidation that could contribute to intrinsic drug resistance in colon cancer cells and may be acquired in breast cancer cells. The lack of selection for higher levels of UGT capacity in the colon cells suggests that high levels of expression of MXR alone are sufficient to confer resistance to the camptothecins.

Acute toxic effects of the antineoplastic anthraquinones carminomycin, epirubicin, idarubicin and mitoxantrone were studied in primary cultures of cardiomyocytes, which were isolated from adult rats. Both time- and concentration-dependent changes of cell structure and viability (trypan blue exclusion) following incubation of myocytes with subclinical, clinical and toxic concentrations of the anthraquinones were examined by light microscopy. The area under the decay curve of viable and rod-shaped myocytes was used to express cytotoxicity of the drugs. Mitoxantrone was found to reduce cell viability and number of rod-shaped cells to the greatest extent, followed by carminomycin, idarubicin and epirubicin. A significantly lower accumulation in cardiomyocytes was obtained with epirubicin and idarubicin compared with carminomycin. An inhibitory effect on oxygen consumption by the cells occurred already at 0.1 microM with epirubicin, whereas inhibition caused by other anthraquinones was less pronounced. Our data indicate a weak association of net accumulation and the toxicity parameter IC50 for carminomycin and idarubicin. In contrast to these results, a more significant correlation of cytotoxicity and anthraquinone lipophilicity was found, which suggests that the lipophilic character of a particular anthraquinone may be an important factor in drug-induced acute cardiotoxicity.

Selection for in vitro drug resistance can result in a complex phenotype with more than one mechanism of resistance emerging concurrently or sequentially. We examined emerging mechanisms of drug resistance during selection with mitoxantrone in the human myeloma cell line 8226. A novel transport mechanism appeared early in the selection process that was associated with a 10-fold resistance to mitoxantrone in the 8226/MR4 cell line. The reduction in intracellular drug concentration was ATP-dependent and ouabain-insensitive. The 8226/MR4 cell line was 34-fold cross-resistant to the fluorescent aza-anthrapyrazole BBR 3390. The resistance to BBR 3390 coincided with a 50% reduction in intracellular drug concentration. Confocal microscopy using BBR 3390 revealed a 64% decrease in the nuclear:cytoplasmic ratio in the drug-resistant cell line. The reduction in intracellular drug concentration of both mitoxantrone and BBR 3390 was reversed by a novel chemosensitizing agent, fumitremorgin C. In contrast, fumitremorgin C had no effect on resistance to mitoxantrone or BBR 3390 in the P-glycoprotein-positive 8226/DOX6 cell line. Increasing the degree of resistance to mitoxantrone in the 8226 cell line from 10 to 37 times (8226/MR20) did not further reduce the intracellular drug concentration. However, the 8226/MR20 cell line exhibited 88 and 70% reductions in topoisomerase II beta and alpha expression, respectively, compared with the parental drug sensitive cell line. This decrease in topoisomerase expression and activity was not observed in the low-level drug-resistant, 8226/MR4 cell line. These data demonstrate that low-level mitoxantrone resistance is due to the presence of a novel, energy-dependent drug efflux pump similar to P-glycoprotein and multidrug resistance-associated protein. Reversal of resistance by blocking drug efflux with fumitremorgin C should allow for functional analysis of this novel transporter in cancer cell lines or clinical tumor samples. Increased resistance to mitoxantrone may result from reduced intracellular drug accumulation, altered nuclear/cytoplasmic drug distribution, and alterations in topoisomerase II activity.

Fifteen anthracene-9,10-dione ("anthraquinone") derivatives with (omega-aminoalkyl)carboxamido substituents at the 1-, 2-, 1,4-, or 2, 6-ring positions were tested for bacterial mutagenicity in reverse-mutation assays using Salmonella typhimurium frameshift strains TA1538, TA98, and TA97a, in the presence and absence of a metabolic activation system prepared from the livers of rats treated with Aroclor 1254. Six of the compounds were also tested in S. typhimurium TA100 and Escherichia coli WP2uvrApKM101 strains, which carry mutations particularly sensitive to reversion by DNA base-pair substitution. Two structurally related compounds, mitoxantrone and bisantrene, were tested in parallel as positive controls. Mitoxantrone was mutagenic to S. typhimurium TA1538 and TA98, whereas bisantrene was weakly mutagenic to both these strains but strongly mutagenic toward the TA97a variant. By contrast, although they are also DNA-binding intercalators, none of the amide-functionalized anthracene-9,10-diones of the present study showed significant mutagenic activity in any of the bacterial strains examined. Further, neither substituent position nor systematic alterations in the nature of attached side chains appeared to induce mutagenicity with these agents, although other studies have shown that such structural factors markedly influence their cytotoxic potencies toward mammalian cells in vitro.

A number of 1,4- and 2,6-difunctionalized amidoanthracene-9, 10-diones have been prepared. We have examined their in vitro cytotoxicity in several tumor cell lines and their ability to inhibit the telomere-addition function of the human telomerase enzyme together with their inhibition of the Taq polymerase enzyme. Compounds with -(CH2)2- side chains terminating in basic groups such as piperidine show inhibition of telomerase at telIC50 levels of 4-11 microM. These are thus among the most potent nonnucleoside telomerase inhibitors reported to date. Cytotoxicity levels in human tumor cell lines were at comparable levels for several compounds. Implications for amidoanthracene-9,10-dione telomerase inhibitors as potential anticancer agents are discussed.