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MicroRNA-200b (miR-200b) has emerged as a therapeutic option for reducing inflammation and airway dysfunction in asthma. miR-200b belongs to a family of miRNAs that regulate epithelial-to-mesenchymal (EMT) transition and IL-33 abundance. In asthma, miR-200b abundance is reduced in the airways and is correlated with disease severity. Additionally, prophylactic treatment with a miR-200b mimetic reduces airway inflammation and airway dysfunction in a mouse model. However, it is unclear whether miR-200b deficiency is sufficient to drive airway dysfunction and airway inflammation in asthma. Here, we show that male and female mice deficient in miR-200b do not display heightened airway inflammation or alterations in lung function that are characteristic of asthma. Following sensitization with house dust mite, female miR-200b knockout (KO) mice have elevated total lung resistance and male miR-200b KO have increased airway resistance. However, neither male nor female miR-200b mice display any changes in methacholine sensitivity or responsiveness and do not have enhanced HDM induced airway inflammation. Collectively, these findings suggest that loss of miR-200b does not drive airway inflammation and airway dysfunction in mice. Thus, although treatment with exogenous miR-200b may ameliorate inflammation in asthma, deficiency of miR-200b is not likely driving pathobiology in asthma.

Purpose: Here, we establish a tracheal occlusion (TO) model with rat lung explants in nitrofen-induced pulmonary hypoplasia in congenital diaphragmatic hernia (CDH). Methods: We extracted lungs from rats on embryonic day 18. We mimicked TO in the lung explants by tying the trachea. We assessed lung weight, morphometry, and abundance of Ki-67, Active caspase-3, and Prosurfactant Protein C (proSP-C) with immunofluorescence. Results: Lung weight was higher in TO+ than TO– on day 1. Abundance of Ki-67 was higher in TO+ than TO– (0.15 vs. 0.32, p=0.009 for day 1, 0.07 vs. 0.17, p=0.004 for day 2, 0.07 vs. 0.12, p=0.044 for day 3), and Active caspase-3 was higher in TO+ than TO– on day 2 and day 3 (0.04 vs. 0.03 p=0.669 for day 1, 0.03 vs. 0.13 p p=0.008 for day3). However, proSP-C protein abundance was lower in TO+ than TO– (67.9 vs. 59.1 p=0.033 for day 1, 73.5 vs. 51.6 p=0.038 for day 2, 83.1 vs. 56.4 p=0.009 for day 3). Conclusions: The TO model in lung explants mimics the outcomes of current surgical models of TO and further studies can reveal the cellular and molecular effects of TO in CDH lungs.

The pathogenesis of lung hypoplasia in congenital diaphragmatic hernia (CDH), a common birth defect, is poorly understood. The diaphragmatic defect can be repaired surgically, but the abnormal lung development contributes to a high mortality in these patients. To better understand the underlying pathobiology, we used the nitrofen rat model of CDH and characterized the proteome of hypoplastic CDH lungs at the alveolar stage (E21). Amongst the 218 significantly altered proteins between CDH and control lungs were Tenascin C, CREBBP, LYN and STAT3. We showed that Tenascin C was decreased around the distal airway branches in nitrofen and human fetal CDH lungs. In contrast, STAT3 was significantly increased in the airway epithelium of nitrofen lungs at E21. STAT3 inhibition after direct nitrofen exposure to fetal rat lung explants (E14.5) partially reversed the hypoplastic lung phenotype ex vivo by increasing peripheral lung budding. Moreover, we demonstrated that several STAT3 associated cytokines (IL-15, IL-9, IL-2) are increased in fetal tracheal aspirates of CDH survivors compared to non-survivors after fetoscopic tracheal occlusion. Using pathway analysis for significantly altered proteins in our proteomic analysis, we observed an enrichment in inflammatory response associated with Epstein Barr Virus and cytokine signaling in nitrofen CDH lungs. However, we were unable to detect EBV mRNA via in-situ Hybridization in human CDH lungs. With our unbiased proteomics approach, we show for the first time that inflammatory processes are likely underlying the pathogenesis of abnormal lung development in CDH.

Nanoparticles administered into the maternal circulation and across the placenta are a potential clinical therapy to treat congenital diseases. The mechanism by which nanoparticles can safely cross the placenta for targeted drug delivery to the fetus remains poorly understood. We demonstrate that the maternal-fetal transfer of passive immunity through the neonatal Fc Receptor (FcRn) can induce the transplacental transfer of chitosan nanoparticles modifed with IgG antibodies (414 ± 27 nm). The transfer of FITC-tagged IgG-modified chitosan nanoparticles was 2.8 times higher (

Congenital diaphragmatic hernia (CDH) and congenital pulmonary airway malformation (CPAM) are two inborn pathologies of the lung of unknown origin. Alterations of gene expression in airway epithelial cells are involved in the pathobiology of both diseases. We previously found decreased expression of the epithelial cell adhesion protein cadherin 26 (CDH26) in hypoplastic mice lungs. Here, our objective was to describe the expression and localization of CDH26 in hypoplastic CDH lungs and hyperproliferative CPAM tissues. After ethical approval, we used human lung tissues from CDH and CPAM cases and age-matched control samples from a previously established biobank. Furthermore, lungs from the nitrofen rat model of CDH were included in the study. We performed immunohistochemistry and western blot analysis with antibodies against CDH26 to examine protein localization and abundance. Statistical analysis was performed using Mann–Whitney U test with significance set at p

The anticancer drug dasatinib (Sprycel) is a BCR-ABL1-targeted tyrosine kinase inhibitor used in treating chronic myelogenous leukemia that has been shown in clinical trials to display cardiovascular toxicities. While dasatinib potently inhibits BCR-ABL1, it is not a highly selective kinase inhibitor and may have off-target effects. A neonatal rat cardiac myocyte model was used to investigate potential mechanisms by which dasatinib damaged myocytes. The anthracycline cardioprotective drug dexrazoxane was shown to be ineffective in preventing dasatinib-induced myocyte damage. Dasatinib treatment increased doxorubicin accumulation in myocytes and doxorubicin-induced myocyte damage, likely through its ability to bind to one or more ABC-type efflux transporters. Dasatinib induced myocyte damage either after a brief treatment that mimicked the clinical situation, or more potently after continuous treatment. Dasatinib slightly induced apoptosis in myocytes as evidenced by increases in caspase-3/7 activity. Dasatinib treatment reduced pERK levels in myocytes most likely through inhibition of RAF, which dasatinib strongly inhibits. Thus, inhibition of the RAF/MEK/ERK pro-survival pathway in the heart may be, in part, a mechanism by which dasatinib induces cardiovascular toxicity.

Congenital diaphragmatic hernia (CDH) is a birth defect associated with abnormal lung development. Yes-associated protein (YAP) is a core kinase of the Hippo pathway, which controls organ size during development. The absence of YAP protein during lung development results in hypoplastic lungs comparable to the lung phenotype in CDH (Mahoney, Dev Cell 30(2):137-150, 2014). We aimed to describe the expression of YAP during normal and nitrofen-induced abnormal lung development.Intra-gastric administration of dams with 100 mg of nitrofen was used to induce CDH and abnormal lung development in the embryos. Immunofluorescence was performed to visualize the localization of YAP and p-YAP during lung development (E15, E18, E21). Western Blotting was used to determine the abundance of YAP and p-YAP in E21 control and nitrofen-induced hypoplastic CDH lungs.Immunofluorescence demonstrated cytoplasmic localization of YAP protein in airway epithelial and mesenchymal cells of nitrofen-induced hypoplastic lungs compared to nuclear localization in control lungs. Western Blotting showed a decrease (p = 0.0188) in abundance of YAP (active form) and increase in p-YAP (inactive form) in hypoplastic lungs compared to control lungs.Our results demonstrate that YAP protein is mostly phosphorylated, inactive, and expressed in the cytoplasm at the later stages of nitrofen-induced hypoplastic lung development indicating that the alteration in regulation of YAP can be associated with the pathogenesis of abnormal lung development in experimental CDH.

Dexrazoxane is clinically used to reduce doxorubicin cardiotoxicity and anthracycline-induced extravasation injury. Dexrazoxane is a strong catalytic inhibitor of topoisomerase II. It can also undergo metabolism to form an iron-binding analog of EDTA. Dexrazoxane was originally thought to act by reducing iron-dependent doxorubicin-based oxidative stress. However, a competing hypothesis posits that dexrazoxane may be protective through its ability to inhibit and reduce topoisomerase IIβ protein levels in the heart. A primary neonatal rat myocyte model was used to study the mechanism by which dexrazoxane protects against doxorubicin-induced myocyte damage. This study characterized the kinetics of the rapid and nearly complete dexrazoxane-induced loss of topoisomerase IIβ protein from neonatal rat cardiac myocytes. Immunofluorescent staining of attached myocytes for topoisomerase IIβ revealed that most of the topoisomerase IIβ was localized to the nucleus, although it was also present in the cytoplasm. Dexrazoxane treatment resulted in an almost complete reduction of topoisomerase IIβ in the nucleus and a lesser reduction in the cytoplasm. The recovery of topoisomerase IIβ levels after a pulse topoisomerase IIβ inhibitory concentration of dexrazoxane occurred slowly, with partial recovery only occurring after 24 h. The ability of dexrazoxane to reduce doxorubicin-induced damage to myocytes was greatest when topoisomerase IIβ levels were at their lowest.

Epigenetic factors are involved in the pathogenesis of congenital diaphragmatic hernia (CDH). Circular RNAs (circRNAs) are epigenetic regulators amenable to biomarker profiling. Here, we aimed to develop a liquid biopsy protocol to detect pathognomonic circRNA changes in biofluids. Our protocol is adapted from the existing BaseScope™ in situ hybridization technique. Rat biofluids were fixed in a gelatin-coated 96-well plate with formalin. Probes were designed to target circRNAs with significant fold change in nitrofen-induced CDH. FastRED fluorescence was assessed using a plate reader and confirmed with confocal microscopy. We tested maternal serum and amniotic fluid samples from control and nitrofen-treated rats. We detected circRNAs in rat serum and amniotic fluid from control and CDH (nitrofen-treated) rats using fluorescent readout. CircRNA signal was observed in fixed biofluids as fluorescent punctate foci under confocal laser scanning microscopy. This was confirmed by comparison to BaseScope™ lung tissue sections. Signal was concentration dependent and DNase resistant. We successfully adapted BaseScope™ to detect circRNAs in rat biofluids: serum and amniotic fluid. We detected signal from probes targeted to circRNAs that are dysregulated in rat CDH. This work establishes the preliminary feasibility of circRNA detection in prenatal diagnostics.

RNA-binding proteins (RBPs) form complexes with RNA, changing how the RNA is processed and thereby regulating gene expression. RBPs are important sources of gene regulation during organogenesis, including the development of lungs. The RBP called Quaking (QK) is critical for embryogenesis, yet it has not been studied in the developing lung. Here, we show that QK is widely expressed during rat lung development and into adulthood. The QK isoforms QK5 and QK7 colocalize to the nuclei of nearly all lung cells. QK6 is present in the nuclei and cytoplasm of mesenchymal cells and is only present in the epithelium during branching morphogenesis. QK knockdown in embryonic lung explants caused a greater number of multiciliated cells to appear in the airways, at the expense of basal cells. The mRNA of multiciliated cell genes and the abundance of FOXJ1/SOX2+ cells increased after knockdown, whereas P63/SOX2+ cells decreased. The cytokine IL-6, a known regulator of multiciliated cell differentiation, had increased mRNA levels after QK knockdown, although protein levels remained unchanged. Further studies are necessary to confirm whether QK acts as a blocker for the IL-6-induced differentiation of basal cells into multiciliated cells, and a conditional QK knockout would likely lead to additional discoveries on QK’s role during lung development.

The cardiotoxicity caused by doxorubicin and extravasation injury caused by anthracyclines is reduced by the clinically approved bisdioxopiperazine drug dexrazoxane. Dexrazoxane is a rings-closed analog of EDTA and is hydrolyzed in vivo to a form that strongly binds iron. Its protective effects were originally thought to be due to the ability of its metabolite to remove iron from the iron-doxorubicin complex, thereby preventing oxygen radical damage to cellular components. More recently it has been suggested that dexrazoxane may exert its protective effects by inhibiting topoisomerase IIβ in the heart and inducing a reduction in its protein levels through induction of proteasomal degradation. The ability of dexrazoxane, other bisdioxopiperazines, and mitindomide to protect against doxorubicin-induced damage was determined in primary neonatal rat myocytes. This QSAR study showed that the protection that a series of bisdioxopiperazine analogs of dexrazoxane and the bisimide mitindomide offered against doxorubicin-induced myocyte damage was highly correlated with the ability of these compounds to catalytically inhibit the decatenation activity of topoisomerase II. The structural features of the dexrazoxane analogs that contribute to the binding and inhibition of topoisomerase II have been identified. These results suggest that the inhibition of topoisomerase II in myocytes by dexrazoxane is central to its role in its activity as an anthracycline cardioprotective agent. Additionally, sequence identity analysis of the amino acids surrounding the dexrazoxane binding site showed extremely high identity, not only between both invertebrate topoisomerase II isoforms, but also with yeast topoisomerase II as well.

The alcohol abuse drug disulfiram has also been shown to exhibit potent cell growth inhibitory and anticancer activity. While a number of cellular and animal studies have suggested that disulfiram exhibits its anticancer activity through interaction with the proteasome, direct evidence for inhibition of proteasome activity is lacking. In this study we show that disulfiram potently inhibits the chymotrypsin-like activity of purified human 20S proteasome at low micromolar pharmacological concentrations. The enzyme progress curves displayed characteristics of a slow-binding reaction, similar to that observed for the FDA-approved proteasomal-targeted anticancer drugs bortezomib and carfilzomib. The apparent second order rate constant for reaction with 20s proteasome that was derived from an analysis of the progress curves was about 250-fold smaller than for bortezomib and carfilzomib. The concentration dependence of the enzyme kinetics was consistent with partial noncompetitive inhibition, whereby the putative disulfiram-proteasome adduct retains, partial but decreased enzyme activity. Disulfiram, which is known to have a high affinity for protein thiols, likely reacted with a non-critical cysteine residue, and not at the proteasome substrate binding site.

Background Human tissue samples are an invaluable and little available source of information for translational studies of congenital lung diseases such as Congenital Diaphragmatic Hernia (CDH) or Congenital Pulmonary Airway Malformation (CPAM). Purpose We aimed to establish a human lung tissue biobank of CDH and CPAM patients together with age-matched controls, coupled with a clinical database. Methods Pathology records from autopsies or surgical specimens for CDH and CPAM cases between 1980 and 2017 were reviewed. For surviving individuals, clinical patient data was obtained from corresponding pediatric surgery reports. Formalin-fixed, paraffin-embedded tissues of patients and age-matched controls were systematically stored for further translational studies. RNA integrity was determined on selected CDH blocks. Results A total of 16 CDH and 18 CPAM and age-matched control lung tissue blocks were included in our biobank. Ages ranged from 22 to 41 weeks of gestation (GA) in CDH (33.9 ± 6.35 weeks) and 26 weeks (GA) and 12 years in CPAM (2.3 ± 3.7 y). RNA isolation from CDH and control blocks yielded good RNA quality (OD 260/280 ratio: 2.01–2.09, OD 260/230 ratio: 2.04–2.09). Conclusion We established a unique human biobank for CDH and CPAM tissues. The combination with clinical patient data will allow us to design future translational studies to improve our understanding of the disease pathogenesis of these congenital malformations.

The proteasome inhibitors bortezomib, carfilzomib, and ixazomib, which are used in the treatment of multiple myeloma have greatly improved response rates. Several other proteasome inhibitors, including delanzomib and oprozomib, are in clinical trials. Carfilzomib and oprozomib are epoxyketones that form an irreversible bond with the 20S proteasome, whereas bortezomib, ixazomib, and delanzomib are boronic acids that form slowly reversible adducts. Several of the proteasome inhibitors have been shown to exhibit specific cardiac toxicities. A primary neonatal rat myocyte model was used to study the relative myocyte-damaging effects of five proteasome inhibitors with a view to identifying potential class differences and the effect of inhibitor binding kinetics. Bortezomib was shown to induce the most myocyte damage followed by delanzomib, ixazomib, oprozomib, and carfilzomib. The sensitivity of myocytes to proteasome inhibitors, which contain high levels of chymotrypsin-like proteasomal activity, may be due to inhibition of proteasomal-dependent ongoing sarcomeric protein turnover. All inhibitors inhibited the chymotrypsin-like proteasomal activity of myocyte lysate in the low nanomolar concentration range and exhibited time-dependent inhibition kinetics characteristic of slow-binding inhibitors. Progress curve analysis of the inhibitor concentration dependence of the slow-binding kinetics was used to measure second-order "on" rate constants for binding. The second-order rate constants varied by 90-fold, with ixazomib reacting the fastest, and oprozomib the slowest. As a group, the boronic acid drugs were more damaging to myocytes than the epoxyketone drugs. Overall, inhibitor-induced myocyte damage was positively, but not significantly, correlated with their second-order rate constants.

Pixantrone is a new noncardiotoxic aza-anthracenedione anticancer drug structurally related to anthracyclines and anthracenediones, such as doxorubicin and mitoxantrone. Pixantrone is approved in the European Union for the treatment of relapsed or refractory aggressive B cell non-Hodgkin lymphoma. This study was undertaken to investigate both the mechanism(s) of its anticancer activity and its relative lack of cardiotoxicity. Pixantrone targeted DNA topoisomerase IIα as evidenced by its ability to inhibit kinetoplast DNA decatenation; to produce linear double-strand DNA in a pBR322 DNA cleavage assay; to produce DNA double-strand breaks in a cellular phospho-histone γH2AX assay; to form covalent topoisomerase II-DNA complexes in a cellular immunodetection of complex of enzyme-to-DNA assay; and to display cross-resistance in etoposide-resistant K562 cells. Pixantrone produced semiquinone free radicals in an enzymatic reducing system, although not in a cellular system, most likely due to low cellular uptake. Pixantrone was 10- to 12-fold less damaging to neonatal rat myocytes than doxorubicin or mitoxantrone, as measured by lactate dehydrogenase release. Three factors potentially contribute to the reduced cardiotoxicity of pixantrone. First, its lack of binding to iron(III) makes it unable to induce iron-based oxidative stress. Second, its low cellular uptake may limit its ability to produce semiquinone free radicals and redox cycle. Finally, because the β isoform of topoisomerase II predominates in postmitotic cardiomyocytes, and pixantrone is demonstrated in this study to be selective for topoisomerase IIα in stabilizing enzyme-DNA covalent complexes, the attenuated cardiotoxicity of this agent may also be due to its selectivity for targeting topoisomerase IIα over topoisomerase IIβ.

Elesclomol is an anticancer drug that is currently undergoing clinical trials. Elesclomol forms a strong 1:1 complex with Cu(II) and may exert its anticancer activity through the induction of oxidative stress and/or its ability to transport copper into the cell. A UV-vis spectrophotometric titration showed that Cu(I) also formed a 1:1 complex with elesclomol. Ascorbic acid, but not glutathione or NADH, potently reduced the Cu(II)-elesclomol complex to produce hydrogen peroxide. Even though hydrogen peroxide mediated reoxidation of the copper(I) produced by ascorbic acid reduction has the potential to lead to hydroxyl radical formation, electron paramagnetic resonance spin trapping experiments, either with or without added hydrogen peroxide, showed that the ascorbic acid-reduced Cu(II)-elesclomol complex could not directly generate damaging hydroxyl radicals. Both Cu(II)-elesclomol and elesclomol potently oxidized dichlorofluorescin in K562 cells. The highly specific copper chelators tetrathiomolybdate and triethylenetetramine were found to greatly reduce the cytotoxicity of both elesclomol and Cu(II)-elesclomol complex towards erythroleukemic K562 cells, consistent with a role for copper in the cytotoxicity of elesclomol. The superoxide dismutating activity of Cu(II)-elesclomol was much lower than that of Cu(II). Depletion of glutathione levels in K562 cells by treatment with buthionine sulfoximine sensitized cells to both elesclomol and Cu(II)-elesclomol. In conclusion, these results showed that elesclomol indirectly inhibited cancer cell growth through Cu(II)-mediated oxidative stress.

Five clinically approved BCR-ABL1-targeted tyrosine kinase inhibitors (bosutinib, dasatinib, imatinib, nilotinib, and ponatinib) used for treating chronic myelogenous leukemia have been studied in a neonatal rat myocyte model for their relative ability to induce myocyte damage. This was done in order to determine if kinase inhibitor-induced myocyte damage was a consequence of inhibiting ABL1 (on-target effects), or due to a lack of kinase selectivity (off-target effects) since previous studies have come up with conflicting conclusions about whether imatinib-induced cardiotoxicity results directly from inhibition of ABL1. The most specific and least potent inhibitors, imatinib and nilotinib, induced the least myocyte damage, while the least specific and most potent inhibitors, ponatinib and dasatinib, induced the most damage. Inhibitor-induced myocyte damage also correlated with clinically observed cardiovascular toxicity. Growth inhibition of the erythroleukemic K562 human cell line with a constitutively active BCR-ABL1 kinase was negatively correlated with inhibitor-induced myocyte damage, which suggests that inhibition of ABL1 causes myocyte damage. Myocyte damage was also negatively correlated with inhibitor dissociation binding constants and with inhibition of enzymatic ABL1 kinase activity. Myocyte damage was also positively correlated with two measures of inhibitor selectivity, which suggests that a lack of inhibitor selectivity is responsible for myocyte damage. In conclusion, myocyte damage, and thus the cardiovascular toxicity of the BCR-ABL1-targeted tyrosine kinase inhibitors, is due to direct inhibition of ABL1 and/or their lack of inhibitor selectivity.

Bortezomib and carfilzomib are anticancer drugs that target the proteasome. However, these agents have been shown to exhibit some specific cardiac toxicities by as yet unknown mechanisms. Bortezomib and carfilzomib are also being used clinically in combination with doxorubicin, which is also cardiotoxic. A primary neonatal rat myocyte model was used to study these cardiotoxic mechanisms. Exposure to submicromolar concentrations of bortezomib and carfilzomib resulted in significant myocyte damage and induced apoptosis. Both bortezomib and carfilzomib inhibited the chymotrypsin-like proteasomal activity of myocyte lysate in the low nanomolar concentration range and exhibited time-dependent inhibition kinetics. The high sensitivity of myocytes, which were determined to contain high specific levels of chymotrypsin-like proteasomal activity, to the damaging effects of bortezomib and carfilzomib was likely due to the inhibition of proteasomal-dependent ongoing sarcomeric protein turnover. A brief preexposure of myocytes to non-toxic nanomolar concentrations of bortezomib or carfilzomib greatly increased doxorubicin-mediated damage, which suggests that the combination of doxorubicin with either bortezomib or carfilzomib may produce more than additive cardiotoxicity. The doxorubicin cardioprotective agent dexrazoxane partially protected myocytes from doxorubicin plus bortezomib or carfilzomib treatment, in spite of the fact that bortezomib and carfilzomib inhibited the dexrazoxane-induced decreases in topoisomerase IIβ protein levels in myocytes. These latter results suggest that the doxorubicin cardioprotective effects of dexrazoxane and the doxorubicin-mediated cardiotoxicity were not exclusively due to targeting of topoisomerase IIβ.

The anticancer drug lapatinib (Tykerb) is a dual tyrosine kinase inhibitor targeting the HER2 (ERBB2) and EGFR (ERBB1, HER1) pathways that have been shown in clinical trials to display some cardiotoxicity. Because trastuzumab also targets HER2 receptors, the lapatinib/doxorubicin combination provides a good model to probe the mechanism of the increased cardiotoxicity caused by the concurrent use of trastuzumab and doxorubicin. Using a neonatal rat cardiac myocyte model, we have investigated the ability of lapatinib alone and in combination with doxorubicin to damage myocytes. Lapatinib treatment alone only slightly induced myocyte damage. However, doxorubicin-induced myocyte damage was greatly potentiated by the addition of nanomolar lapatinib concentrations. Lapatinib alone treatment decreased phosphorylated ERK (MAPK), which may have, in part, contributed to the increased myocyte damage. As measured by flow cytometry, lapatinib-treated myocytes displayed an increased accumulation of doxorubicin. As lapatinib is a strong inhibitor of several ATP-dependent ABC-type efflux transporters, this likely occurred because lapatinib blocked doxorubicin efflux, thereby increasing intracellular doxorubicin concentrations and, thus, increasing myocyte damage. These results suggest that the clinical use of concurrent doxorubicin and lapatinib should be approached with care due to the possibility of lapatinib increasing doxorubicin cardiotoxicity.

Dexrazoxane (ICRF-187) is clinically used as a doxorubicin cardioprotective agent and to prevent anthracycline extravasation injury. It may act by preventing iron-based oxygen free radical damage through the iron-chelating ability of its metabolite N , N ′-[(1 S )-1-methyl-1,2-ethanediyl]bis[( N -(2-amino-2-oxoethyl)]glycine (ADR-925). Dexrazoxane undergoes an initial metabolism to its two one-ring open intermediates [ N -(2-amino-2-oxoethyl)- N -[(1 S )-2-(3,5-dioxo-1-piperazinyl)-1-methylethyl]glycine (B) and N -(2-amino-2-oxoethyl)- N -[(2 S )-2-(3,5-dioxo-1-piperazinyl)propyl]glycine ( C )] and is then further metabolized to its presumably active metal-chelating form ADR-925. We previously showed that the first ring opening reaction is catalyzed by dihydropyrimidinase and the second by dihydroorotase (DHOase), but not vice versa. To determine whether DHOase was important in the metabolism of dexrazoxane, its metabolism and that of B and C to ADR-925 were measured in rats that were pretreated with the DHOase inhibitor 5-aminoorotic acid. In rats pretreated with 5-aminoorotic acid the area-under-the-curve concentration of ADR-925 was reduced 5.3-fold. In rats treated with a mixture of B and C, the maximum concentration of ADR-925 in the plasma was significantly decreased in rats pretreated with 5-aminoorotic acid, which indicates that DHOase directly metabolized B and C. Both heart and liver tissue levels of ADR-925 in rats were also greatly reduced by pretreatment with 5-aminoorotic acid. Together these results indicate that the metabolism of dexrazoxane and of B and C is mediated by DHOase. These results provide a mechanistic basis for the antioxidant cardioprotective activity of dexrazoxane.

RH1 is a new bioreductive agent that is an excellent substrate for the two-electron reducing enzyme, NAD(P)H quinone oxidoreductase 1 (NQO1). RH1 may be an effective NQO1-directed antitumor agent for treatment of cancer cells having elevated NQO1 activity. As some studies have indicated that RH1 may also be a substrate for the one-electron reducing enzyme, NADPH cytochrome P450 reductase (P450 Red), P450 Red may contribute to the activation of RH1 where NQO1 activities are low and P450 Red activities are high. The mean P450 Red activity in the human tumor cell line panel used by NCI for evaluation of new anticancer agents is 14.8 nmol min(-1) mg prot(-1), while the mean NQO1 activity in these cell lines is 199.5 nmol min(-1) mg prot(-1). Thus, we investigated whether P450 Red could play a role in activating RH1.Reduction of RH1 by purified human P450 Red was investigated using electron paramagnetic resonance and spectroscopic assays. The ability of RH1 to produce DNA damage following reduction by P450 Red was studied using gel assays. To determine the role of P450 Red in activation of RH1 in cells, cell growth inhibition studies with inhibitors of P450 Red and NQO1 were carried out in T47D human breast cancer cells and T47D cells transfected with the human P450 Red gene (T47D-P450) that have P450 Red activities of 11.5 and 311.8 nmol min(-1) mg prot(-1), respectively.Reduction studies using purified P450 Red and NQO1 confirmed that RH1 can be reduced by both enzymes, but redox cycling was slower following reduction by NQO1. RH1 produced DNA strand breaks and crosslinks in isolated DNA after reduction by either P450 Red or NQO1. DPIC, an inhibitor of P450 Red, had no effect on cell growth inhibition by RH1 in T47D cells, and had only a small effect on cell growth inhibition by RH1 in the presence of the NQO1 inhibitor, dicoumarol, in T47D-P450 cells.These results demonstrated that P450 Red does not contribute to the activation of RH1 in cells with normal P450 Red activity and plays only a minor role in activating this agent in cells with high levels of this enzyme. These studies confirmed that P450 Red could activate RH1 and provided the first direct evidence that RH1 could produce both DNA strand breaks and DNA crosslinks after reduction by P450 Red. However, the results strongly suggest that P450 Red does not play a significant role in activating RH1 in cells with normal P450 Red activity.

Drugs that target DNA topoisomerase II, such as the epipodophyllotoxin etoposide, are a clinically important class of anticancer agents. A recently published X-ray structure of a ternary complex of etoposide, cleaved DNA and topoisomerase IIβ showed that the two intercalated etoposide molecules in the complex were separated by four DNA base pairs. Thus, using a structure-based design approach, a series of bis-epipodophyllotoxin etoposide analogs with piperazine-containing linkers was designed to simultaneously bind to these two sites. It was hypothesized that two-site binding would produce a more stable cleavage complex, and a more potent anticancer drug. The most potent bis-epipodophyllotoxin, which was 10-fold more growth inhibitory toward human erythroleukemic K562 cells than etoposide, contained a linker with eight methylene groups. All of the mono- and bis-epipodophyllotoxins, in a variety of assays, showed strong evidence that they targeted topoisomerase II. COMPARE analysis of NCI 60-cell GI50 endpoint data was also consistent with these compounds targeting topoisomerase II.

The clinical use of bleomycin is limited by a dose-dependent pulmonary toxicity. Bleomycin is thought to be growth inhibitory by virtue of its ability to oxidatively damage DNA through its complex with iron. Our previous preclinical studies showed that bleomycin-induced pulmonary toxicity can be reduced by pretreatment with the doxorubicin cardioprotective agent dexrazoxane. Dexrazoxane is thought to protect against iron-based oxygen radical damage through the iron chelating ability of its hydrolyzed metabolite ADR-925, an analog of ethylenediaminetetraacetic acid (EDTA). ADR-925 quickly and effectively displaced either ferrous or ferric iron from its complex with bleomycin. This result suggests that dexrazoxane may have the potential to antagonize the iron-dependent growth inhibitory effects of bleomycin. A study was undertaken to determine if dexrazoxane could antagonize bleomycin-mediated cytotoxicity using a CHO-derived cell line (DZR) that was highly resistant to dexrazoxane through a threonine-48 to isoleucine mutation in topoisomerase IIα. Dexrazoxane is also a cell growth inhibitor that acts through its ability to inhibit the catalytic activity of topoisomerase II. Thus, the DZR cell line allowed us to examine the cell growth inhibitory effects of bleomycin in the presence of dexrazoxane without the confounding effect of dexrazoxane inhibiting cell growth. The cell growth inhibitory effects of bleomycin were unaffected by pretreating DZR cells with dexrazoxane. These results suggest that dexrazoxane may be clinically used in combination with bleomycin as a pulmonary protective agent without adversely affecting the antitumor activity of bleomycin.

The clinically approved antioxidant cardioprotective agent dexrazoxane (ICRF-187) was examined for its ability to protect neonatal rat cardiac myocytes from doxorubicin-induced damage. Doxorubicin is thought to induce oxidative stress on the heart muscle, both through reductive activation to its semiquinone form, and by the production of hydroxyl radicals mediated by its complex with iron. Hydrolyzed dexrazoxane metabolites prevent site-specific iron-based oxygen radical damage by displacing iron from doxorubicin and chelating free and loosely bound iron. The mitochondrial stain MitoTracker Green FM and doxorubicin were shown by epifluorescence microscopy to accumulate in the myocyte mitochondria. An epifluorescence microscopic image analysis method to measure mitochondrial damage was developed using the mitochondrial membrane potential sensing ratiometric dye JC-1. This method was used to show that dexrazoxane protected against doxorubicin-induced depolarization of the myocyte mitochondrial membrane. Dexrazoxane also attenuated doxorubicin-induced oxidation of intracellular dichlorofluorescin. Annexin V-FITC/propidium iodide staining of myocytes was used to demonstrate that, depending on the concentration, doxorubicin caused both apoptotic and necrotic damage. These results suggest that doxorubicin may be cardiotoxic by damaging the mitochondria and dexrazoxane may be protective by preventing iron-based oxidative damage.

Drugs that target DNA topoisomerase II isoforms and alkylate DNA represent two mechanistically distinct and clinically important classes of anticancer drugs. Guided by molecular modeling and docking a series of etoposide analog epipodophyllotoxin–N-mustard hybrid compounds were designed, synthesized and biologically characterized. These hybrids were designed to alkylate nucleophilic protein residues on topoisomerase II and thus produce inactive covalent adducts and to also alkylate DNA. The most potent hybrid had a mean GI 50 in the NCI-60 cell screen 17-fold lower than etoposide. Using a variety of in vitro and cell-based assays all of the hybrids tested were shown to target topoisomerase II. A COMPARE analysis indicated that the hybrids had NCI 60-cell growth inhibition profiles matching both etoposide and the N-mustard compounds from which they were derived. These results supported the conclusion that the hybrids displayed characteristics that were consistent with having targeted both topoisomerase II and DNA.

The bis(thiohydrazide) amide elesclomol has extremely potent antiproliferative activity and is currently in clinical trials as an anticancer agent. Elesclomol strongly binds copper and may be exerting its cell growth inhibitory effects by generating copper-mediated oxidative stress. Nickel(II) and platinum(II) complexes of elesclomol were synthesized and characterized in order to investigate if these biologically redox inactive metal complexes could also inhibit cell growth. The nickel(II)-elesclomol and platinum(II) elesclomol complexes were 34- and 1040-fold less potent than the copper(II)-elesclomol complex towards human leukemia K562 cells. These results support the conclusion that a redox active metal is required for elesclomol to exert its cell growth inhibitory activity. Copper(II)-elesclomol was also shown to efficiently oxidize ascorbic acid at physiological ascorbic acid concentrations. Reoxidation of the copper(I) thus produced would lead to production of damaging reactive oxygen species. An X-ray crystallographic structure determination of copper(II)-elesclomol showed that it formed a 1:1 neutral complex with a distorted square planar structure. The kinetics and equilibria of the competition reaction of the strong copper(II) chelator TRIEN with copper(II)-elesclomol were studied spectrophotometrically under physiological conditions. These results showed elesclomol bound copper(II) with a conditional stability constant 24-fold larger than TRIEN. A log stability constant of 24.2 was thus indirectly determined for the copper(II)-elesclomol complex.

Bortezomib and carfilzomib are anticancer drugs that target the proteasomal system. Case reports indicate that the use of these drugs can result in cardiotoxicity. These drugs may also be used in combination with doxorubicin, which is itself cardiotoxic. Thus, in order to determine the mechanism of this cardiotoxicity studies were undertaken using a neonatal rat myocyte primary cell model. Using LDH release as a measure of damage in the myocyte model we showed that even brief exposure (6 h) to submicromolar concentrations of bortezomib resulted in significant myocyte damage. Carfilzomib was slightly less toxic and displayed myocyte damage in the low micromolar concentration range. Utilizing a fluorogenic substrate both bortezomib and carfilzomib inhibited the chymotrypsin-like proteasomal activity of myocyte lysate in the submicromolar concentration range. The inhibition kinetics were consistent with formation of an essentially irreversible covalent complex at the active site. Bortezomib irreversibly inhibited proteasomal activity about four-fold faster than carfilzomib. Thus, the reduced toxicity of carfilzomib may be due to its slower inhibition kinetics. A brief pre-exposure (6 h) of myocytes to low non-toxic nanomolar concentrations of bortezomib greatly increased doxorubicin-mediated damage. The doxorubicin cardioprotective agent dexrazoxane partially protected the myocytes from doxorubicin treatment and from bortezomib plus doxorubicin treatment under these conditions. However, at slightly toxic bortezomib concentrations dexrazoxane offered little protection against bortezomib plus doxorubicin treatment. Bortezomib, at least over short times, did not induce oxidative damage in myocytes as measured with a fluorogenic DCF assay. Likewise, a 6 h bortezomib treatment did not affect the mitochondrial membrane potential of myocytes as measured in a JC-1 ratiometric assay. In conclusion both bortezomib and carfilzomib are potently toxic to myocytes at submicromolar concentrations, likely through their ability to irreversibly inhibit myocyte proteasomal activity. Support: CIHR; a Canada Research Chair in Drug Development. Citation Format: Brian B. Hasinoff, Xing Wu, Daywin Patel. Molecular mechanisms of the cardiotoxicity of the proteasomal-targeted anticancer drugs bortezomib and carfilzomib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3060.

Many new targeted small molecule anticancer kinase inhibitors are actively being developed. However, the clinical use of some kinase inhibitors has been shown to result in cardiotoxicity. In most cases the mechanisms by which they exert their cardiotoxicity are not well understood. We have used large scale profiling data on 8 FDA-approved tyrosine kinase inhibitors and 10 other kinase inhibitors to a panel of 317 kinases in order to correlate binding constants and kinase inhibitor binding selectivity scores with kinase inhibitor-induced damage to neonatal rat cardiac myocytes. The 18 kinase inhibitors that were the subject of this study were: canertinib, dasatinib, dovitinib, erlotinib, flavopiridol, gefitinib, imatinib, lapatinib, midostaurin, motesanib, pazopanib, sorafenib, staurosporine, sunitinib, tandutinib, tozasertib, vandetanib and vatalanib. The combined tyrosine kinase and serine-threonine kinase selectivity scores were highly correlated with the myocyte-damaging effects of the kinase inhibitors. This result suggests that myocyte damage was due to a lack of target selectivity to binding of both tyrosine kinases and serine-threonine kinases, and was not due to binding to either group specifically. Finally, the strength of kinase inhibitor binding for 290 kinases was examined for correlations with myocyte damage. Kinase inhibitor binding was significantly correlated with myocyte damage for 12 kinases. Thus, myocyte damage may be multifactorial in nature with the inhibition of a number of kinases involved in producing kinase inhibitor-induced myocyte damage.

The use of the anticancer multikinase inhibitor sorafenib is associated with cardiac ischemia or infarction and an increase in hypertension. We investigated various mechanisms that might be responsible for its cardiotoxicity in a neonatal rat myocyte model. As measured by lactate dehydrogenase release, sorafenib treatment of myocytes caused dose-dependent damage at therapeutically relevant concentrations. It had been hypothesized that inhibition of RAF1 and BRAF kinases may be responsible for sorafenib-induced cardiotoxicity. However, because sorafenib treatment did not inhibit phosphorylation of ERK (extracellular signal-regulated kinase), it was concluded that sorafenib did not exert its damaging effects through RAF inhibition of the RAF/MEK/ERK kinase cascade. The clinically approved doxorubicin cardioprotective agent dexrazoxane did not protect myocytes from damage. At lower sorafenib concentrations, at least, these results are consistent with sorafenib not being able to induce significant oxidative damage. In conclusion, given the extreme lack of kinase selectivity that sorafenib exhibits, it is likely that inhibition of kinases other than RAF, or combinations of kinases, contributes to the cardiotoxic effects of sorafenib.

The iron chelating agent Dp44mT (di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone) and the clinically approved cardioprotective agent dexrazoxane (ICRF-187) were compared for their ability to protect neonatal rat cardiac myocytes from doxorubicin-induced damage. Doxorubicin is thought to induce oxidative stress on the heart muscle through iron-mediated oxygen radical damage. While dexrazoxane was able to protect myocytes from doxorubicin-induced lactate dehydrogenase release, in contrast Dp44mT synergistically increased doxorubicin-induced damage. This occurred in spite of the fact that Dp44mT quickly and efficiently removed iron(III) from its complex with doxorubicin and that Dp44mT also rapidly entered myocytes and displaced iron from a fluorescence-quenched trapped intracellular iron-calcein complex. Electron paramagnetic resonance spin trapping was used to show that iron complexes of Dp44mT were not able to generate hydroxyl radicals, suggesting that its cytotoxicity was not due to reactive oxygen species formation. In conclusion Dp44mT is unlikely to be useful as an anthracycline cardioprotective agent.

The anticancer tyrosine kinase inhibitor sunitinib has been shown recently to be cardiotoxic. Using a neonatal rat myocyte model, we investigated various mechanisms that might be responsible for its cardiotoxicity. Sunitinib potently inhibited the enzyme activity of both AMP-activated protein kinase (AMPK) and the ribosomal S6 kinase RSK1 at therapeutically relevant concentrations. Heart tissue with its high energy needs might be particularly sensitive to inhibition of AMPK because of its role as an energy sensor regulating ATP levels. As measured by lactate dehydrogenase release, sunitinib treatment of myocytes caused dose-dependent damage at therapeutic levels. Sunitinib treatment also caused a dose-dependent reduction in myocyte protein levels of the phosphorylated alpha and beta isoforms of the AMPK phosphorylation target acetyl-Coenzyme A carboxylase. However, myocytes were not protected from sunitinib treatment by pretreating them with the AMPK-activating antidiabetic drug metformin. Sunitinib treatment of myocytes also did not affect cellular ATP levels. Together, these last two results do not suggest a major role for inhibition of AMPK in sunitinib-induced myocyte damage. Dexrazoxane, which is a clinically approved doxorubicin cardioprotective agent, also did not protect myocytes from damage, which suggests that sunitinib did not induce oxidative damage. In conclusion, even though sunitinib potently inhibits AMPK and RSK1, given the extreme lack of kinase selectivity that sunitinib exhibits, it is likely that inhibition of other kinases or combinations of kinases are responsible for the cardiotoxic effects of sunitinib.

The bacterial metabolite kinamycin F, which is being investigated as a potent antitumor agent, contains an unusual and potentially reactive diazo group as well as a paraquinone and a phenol functional group. Kinamycin F reacted with glutathione (GSH) in a complex series of reactions which suggested that kinamycin F may have its cytotoxicity modulated by GSH. Consistent with this idea 2-oxo-4-thiazolidinecarboxylic acid treatment to increase cellular GSH levels and buthionine sulfoximine treatment to decrease GSH levels resulted in decreased and increased kinamycin F cytotoxicity, respectively, in K562 leukemia cells. Kinamycin F weakly bound to DNA and induced DNA damage in K562 cells that was independent of GSH levels. The GSH-promoted DNA nicking induced by kinamycin F in vitro was attenuated by deferoxamine, dimethyl sulfoxide, and by catalase, which indicated that DNA damage initiated by this agent occurred in an iron-, hydrogen peroxide- and hydroxyl radical-dependent manner. Electron paramagnetic resonance spectroscopy experiments showed that the GSH/kinamycin F system produced a semiquinone free radical and that the hydrogen peroxide/peroxidase/kinamycin F system generated a phenoxyl free radical. In conclusion, the results indicated that kinamycin F cytotoxicity may be due to reductive and/or peroxidative activation to produce DNA- and protein-damaging species.

Pixantrone is a new non-cardiotoxic aza-anthracenedione anticancer drug structurally related to anthracyclines and anthracenediones such as doxorubicin and mitoxantrone. Pixantrone is approved in the EU for the treatment of relapsed or refractory aggressive B-cell non-Hodgkin lymphoma. This study was undertaken to investigate both the mechanisms of its anticancer activity and its lack of cardiotoxicity. The reduced cardiotoxicity of pixantrone compared to doxorubicin may be due to the fact that it did not bind iron(II) or iron(III). Thus, unlike doxorubicin it would not be able to induce iron-based oxidative stress in the heart. Pixantrone did, however, form a weak complex with Cu(II). Pixantrone was 10-fold less damaging to neonatal rat myocytes than doxorubicin as measured by LDH release. As measured by EPR spectrometry pixantrone generated about two-fold higher levels of its semiquinone free radical than doxorubicin in the hypoxanthine-xanthine oxidase reducing system. Conversely, in intact K562 cells under nitrogen, doxorubicin produced much more semiquinone radical than pixantrone. The reduced myocyte toxicity and semiquinone-forming ability in K562 cells compared to doxorubicin may be due low cell accumulation of dicationic pixantrone. Multidrug resistant Pgp- (ABCB1) overexpressing MDCK/MDR cells were 40-fold cross resistant to pixantrone, compared to parental cells. Pixantrone bound to DNA much more strongly than doxorubicin as determined in a DNA melting point assay. Pixantrone targeted topoisomerase II as evidenced by its ability to inhibit kDNA decatenation, to produce linear double-stranded DNA in a pBR322 DNA cleavage assay, and to form covalent topoisomerase II-DNA complexes in a cellular ICE assay. Pixantrone was 3-fold cross-resistant to K562-derived etoposide-resistant cells. DNA double strand breaks produced by pixantrone assessed in a cellular γH2AX assay were also characteristic of a topoisomerase II-targeted drug. These results support the conclusion that pixantrone displays characteristics that are consistent with targeting topoisomerase II. Support: CIHR; a Canada Research Chair in Drug Development; R01 CA090787. Citation Format: Brian B. Hasinoff, Xing Wu, Daywin Patel, Jack C. Yalowich. The non-cardiotoxic anticancer drug pixantrone targets topoisomerase II. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1659. doi:10.1158/1538-7445.AM2015-1659

The oral iron chelating agent ICL670A (deferasirox) and the clinically approved cardioprotective agent dexrazoxane (ICRF-187) were compared for their ability to protect neonatal rat cardiac myocytes from doxorubicin-induced damage. Doxorubicin is thought to induce oxidative stress on the heart muscle through iron-mediated oxygen radical damage. While dexrazoxane was able to protect myocytes from doxorubicin-induced lactate dehydrogenase release, ICL670A, in contrast, depending upon the concentration, synergistically increased or did not affect the cytotoxicity of doxorubicin. This occurred in spite of the fact that ICL670A quickly and efficiently removed iron(III) from its complex with doxorubicin, and rapidly entered myocytes and displaced iron from a fluorescence-quenched trapped intracellular iron-calcein complex. Continuous exposure of ICL670A to either myocytes or Chinese hamster ovary (CHO) cells resulted in cytotoxicity while treatment of CHO cells with the ferric complex of ICL670A did not. These results suggest that ICL670A was cytotoxic either by removing or withholding iron from critical iron-containing proteins. Electron paramagnetic resonance spectroscopy was used to show that neither ICL670A nor its ferric complex were able to generate free radicals in either oxidizing or reducing systems suggesting that its cytotoxicity is not due to radical generation.

The clinically approved cardioprotective agent dexrazoxane (ICRF-187) and two of its hydrolyzed metabolites (a one-ring open form of dexrazoxane and ADR-925) were examined for their ability to protect neonatal rat cardiac myocytes from doxorubicin-induced damage. Dexrazoxane may protect against doxorubicin-induced damage to myocytes through its strongly metal-chelating hydrolysis product ADR-925, which could act by displacing iron bound to doxorubicin or chelating free or loosely bound iron, thus preventing site-specific iron-based oxygen radical damage. The results of this study showed that whereas dexrazoxane was able to protect myocytes from doxorubicin-induced lactate dehydrogenase release, neither of the metabolites displayed any protective ability. Dexrazoxane also reduced apoptosis in doxorubicin-treated myocytes. The ability of dexrazoxane and its three metabolites to displace iron from a fluorescence-quenched trapped intracellular iron-calcein complex was also determined to see whether the metabolites were taken up by myocytes. Although ADR-925 was taken up in the absence of calcium in the medium, in the presence of calcium, its uptake was greatly slowed, presumably because it formed a complex with calcium. Both of the one-ring open metabolites were taken up by myocytes and displaced iron from its complex with calcein. These results suggest either that the anionic metabolites do not have the same access to iron pools in critical cellular compartments, that their uptake is slowed in the presence of calcium, or, less likely, that dexrazoxane protects by some other mechanism.

Disulfiram is used in the treatment of alcoholism. It has also shown promise as an anticancer drug. The disulfide bond in disulfiram is metabolized in vivo to diethyldithiocarbamate (DDTC) which forms an extremely strong complex with copper(II) through which it may exert its cytotoxicity. Both disulfiram and diethyldithiocarbamate inhibited K562 cell growth in the low nanomolar range. The addition of the copper(II) chelators Trien and ammonium tetrathiomolybdate to the cell culture medium significantly reduced the cytotoxicity of disulfiram and diethyldithiocarbamate towards K562 cells, which suggests that their cytotoxicity is mediated by uptake of trace amounts of copper from the culture medium. Disulfiram, but not DDTC, strongly inhibited the decatenation activity of topoisomerase IIα in the low micromolar concentration range. Disulfiram, but not DDTC, also was shown to be a topoisomerase IIα poison as evidenced by its ability to induce formation of linear DNA. However, the K/VP.5 cell line, a K562-derived etoposide-resistant cell line with reduced levels of topoisomerase II, was not cross-resistant to either disulfiram or DDTC. K562 cells depleted in GSH levels by treatment with buthionine sulfoximine did not show increased sensitivity to disulfiram or DDTC. Disulfiram and DDTC also did not increase dichlorofluorescein fluorescence in K562 cells. These latter two results suggest that copper-mediated oxidative stress may not be a significant factor in their cytotoxicity. In summary, these results showed that while disulfiram and DDTC inhibited topoisomerase IIα, this inhibition may be only partly responsible for their cytotoxicity. Support: CIHR, a Canada Research Chair in Drug Development to B.B.H., and a National Institutes of Health grant CA090787 to J.C.Y. Citation Format: Xing Wu, Jack C. Yalowich, Daywin Patel, Brian B. Hasinoff. Disulfiram is a potent inhibitor of topoisomerase II. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 830. doi:10.1158/1538-7445.AM2014-830

The connexin family of proteins (Cx) that form intercellular gap junctions in vertebrates is well represented in the mammalian central nervous system. Among these, Cx30 and Cx43 are present in gap junctions of astrocytes. Cx32 is expressed by oligodendrocytes and is present in heterologous gap junctions between oligodendrocytes and astrocytes as well as at autologous gap junctions between successive myelin layers. Cx36 mRNA has been identified in neurons, and Cx36 protein has been localized at ultrastructurally defined interneuronal gap junctions. Cx26 is also expressed in the CNS, primarily in the leptomeningeal linings, but is also reported in astrocytes and in neurons of developing brain and spinal cord. To establish further the regional, cellular, and subcellular localization of Cx26 in neural tissue, we investigated this connexin in adult mouse brain and in rat brain and spinal cord using biochemical and immunocytochemical methods. Northern blotting, western blotting, and immunofluorescence studies indicated widespread and heterogeneous Cx26 expression in numerous subcortical areas of both species. By confocal microscopy, Cx26 was colocalized with both Cx30 and Cx43 in leptomeninges as well as along blood vessels in cortical and subcortical structures. It was also localized at the surface of oligodendrocyte cell bodies, where it was coassociated with Cx32. Freeze-fracture replica immunogold labeling (FRIL) demonstrated Cx26 in most gap junctions between cells of the pia mater by postnatal day 4. By postnatal day 18 and thereafter, Cx26 was present at gap junctions between astrocytes and in the astrocyte side of most gap junctions between astrocytes and oligodendrocytes. In perinatal spinal cord and in five regions of adult brain and spinal cord examined by FRIL, no evidence was obtained for the presence of Cx26 in neuronal gap junctions. In addition to its established localization in leptomeningeal gap junctions, these results identify Cx26 as a third connexin (together with Cx30 and Cx43) within astrocytic gap junctions and suggest a further level of complexity to the heterotypic connexin channel combinations formed at these junctions. J. Comp. Neurol. 441:302–323, 2001. © 2001 Wiley-Liss, Inc.

The cyclooxygenase (COX)-2 inhibitors celecoxib and rofecoxib were studied for their effects on neonatal rat cardiac myocytes as a possible model for the adverse cardiovascular effects that this class of compounds have shown in their clinical use. Celecoxib, but not rofecoxib, as measured by lactate dehydrogenase release was toxic to myocytes in the low micromolar concentration range. This toxicity shown by celecoxib was also associated with a high degree of myofibrillar disruption similar to that caused by doxorubicin. As measured by induction of caspase-3/7 activity and by changes in nuclear morphology, neither celecoxib nor rofecoxib strongly induced apoptosis in myocytes. The stable prostacyclin analog iloprost was unable to reduce celecoxib-induced damage, which suggested that celecoxib exerted its cytotoxicity through prostacyclin-independent pathways. Celecoxib treatment did not increase intracellular oxidation of 2′,7′-dichlorofluorescin in myocytes, which suggested that its cytotoxicity was not due to reactive oxygen species generation. The evidence supports the conclusion that celecoxib exerts its cytotoxicity towards myocytes through COX-2-independent mediated pathways.

Elesclomol is an anticancer drug that has received both fast track and orphan drug status from the FDA and is currently undergoing clinical trials. Elesclomol forms a strong 1:1 complex with Cu2+ and may exert its anticancer activity through the induction of oxidative stress or its ability to transport Cu2+ into the cell. The complexes of elesclomol with redox inactive Pt2+ and Ni2+ were much less cytotoxic than its Cu2+ complex which suggests a role for Cu2+-induced oxidative stress. Elesclomol and Cu2+-elesclomol inhibited the growth of human erythroleukemic K562 cells in the low nanomolar concentrations with even a brief 1.5 hr exposure. The cell growth inhibitory effects of elesclomol were decreased with an increase in cell density, likely due to Cu2+ depletion of the medium. Consistent with this, the addition of the Cu2+ chelator Trien to the medium decreased cytotoxicity. Cu2+-elesclomol induced caspase 3/7 in K562 cells, indicating it was apoptosis inducing. The Cu2+-elesclomol complex inhibited topoisomerase I activity and induced topoisomerase I-covalent complexes in K562 cells at a relatively high concentration of 50 μM. It did not, however, inhibit or poison topoisomerase II or produce topoisomerase II-covalent complexes in cells. Cu2+-elesclomol, but not elesclomol, was able to induce γH2AX formation in K562 cells, indicative of DNA double strand breaks. Ascorbic acid, GSH and NADH were all able to slowly reduce the Cu2+-elesclomol complex. However, EPR spin trapping experiments, either in the presence or absence of added H2O2, showed that the reduced copper complex could not directly generate damaging hydroxyl radicals. Depletion of GSH in K562 cells by treatment with BSO sensitized cells to elesclomol and its copper complex, indicating oxidative stress may be responsible for cytotoxicity. In conclusion, these results show that elesclomol may inhibit cell growth through Cu2+-mediated oxidative stress. Support: CIHR, a Canada Research Chair in Drug Development to B.B.H., and a National Institutes of Health grant CA090787 to J.C.Y. Citation Format: Brian B. Hasinoff, Jack C. Yalowich, Daywin Patel, Aron Yadav, Xing Wu. Molecular mechanisms of elesclomol cytotoxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5559. doi:10.1158/1538-7445.AM2013-5559

Lapatinib (Tykerb) is dual tyrosine kinase inhibitor targeting the HER2 (ERBB2) and EGFR (ERBB1, HER1) pathways. Lapatinib has been shown in a clinical trial to produce low levels of cardiotoxicity that is reversible. Using a neonatal rat cardiac myocyte model we have investigated the ability of lapatinib alone, and in combination with doxorubicin, to damage myocytes. Lapatinib alone at pharmacological concentrations only slightly induced myocyte damage. However, doxorubicin-induced myocyte damage was greatly potentiated by the addition of low nanomolar lapatinib concentrations. Because trastuzumab also targets ERBB2 receptors, the lapatinib/doxorubicin combination also provides a good model to probe the mechanism of the increased cardiotoxicity caused by the concurrent use of trastuzumab and doxorubicin. Lapatinib treatment alone had little or no effect on myocyte mitochondrial membrane potential, on oxidation of intracellular DCF (dichlorofluorescein), on intracellular ATP levels, or on caspase 3/7 induction. While the doxorubicin cardioprotective agent dexrazoxane was able to greatly reduce doxorubicin-induced myocyte damage, it could only partially reduce the increase in myocyte damage produced by the lapatinib/doxorubicin treatment. Lapatinib alone had little effect on the phosphorylation status of ACC (acetyl-coenzyme A carboxylase) and AKT. Lapatinib alone decreased phosphorylated ERK (MAPK), which may have, in part, contributed to the increased doxorubicin-mediated myocyte damage. As measured by flow cytometry lapatinib-treated myocytes demonstrated an increased accumulation of doxorubicin. As lapatinib is a strong inhibitor of several ATP-dependent ABC-type efflux transporters, this likely occurred because lapatinib blocked doxorubicin efflux, thereby increasing intracellular doxorubicin concentrations. These results suggest that the clinical use of concurrent doxorubicin and lapatinib should be approached with care due to the possibility of lapatinib increasing doxorubicin cardiotoxicity. Several other kinase inhibitors (imatinib, dasatinib, sorafenib, sunitinib, gefitinib, pazopanib, nilotinib, erlotinib, vandetanib and canertinib) were also examined for their ability to potentiate doxorubicin-induced myocyte damage and induce an increase in intracellular doxorubicin levels. Support: CIHR and a Canada Research Chair in Drug Development to BBH. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 935. doi:1538-7445.AM2012-935

Lapatinib (Tykerb) is dual tyrosine kinase inhibitor targeting the HER2 (ERBB2) and EGFR (ERBB1, HER1) pathways. It is used to treat ER+/EGFR+/HER2+ breast cancer patients and patients who have HER2-positive advanced breast cancer that has progressed after previous treatment with other chemotherapeutic agents. Lapatinib, which was initially rated as having no cardiotoxicity, has recently been shown in a clinical trial to produce low levels of cardiotoxicity that is reversible. Using a neonatal rat cardiac myocyte model we have investigated the ability of lapatinib alone and in combination with doxorubicin to damage myocytes. Lapatinib alone at pharmacological concentrations did not significantly cause myocyte damage. However, doxorubicin-induced myocyte damage was greatly increased by the addition of low nanomolar lapatinib concentrations. Previous clinical studies have shown that the combination of trastuzumab (Herceptin) and doxorubicin was highly cardiotoxic. Because trastuzumab also targets HER2 receptors, the lapatinib/doxorubicin combination provides a good model to probe the mechanism of this cardiotoxicity using a rat myocyte model. Lapatinib treatment alone had little or no effect on myocyte mitochondrial membrane potential, on oxidation of intracellular DCF (dichlorofluorescein), on intracellular ATP levels or on caspase 3/7 induction. The doxorubicin cardioprotective agent dexrazoxane was able to greatly reduce doxorubicin-induced myocyte damage. However, dexrazoxane was not able to reduce the increase in myocyte damage produced by adding lapatinib to the doxorubicin treatment. The effect of lapatinib on the change in phosphorylation of ACC (acetyl-coenzyme A carboxylase), which is a measure of a change in cellular energy status, was also determined. Doxorubicin-induced growth inhibitory effects on K562 cells, which contain very low levels of ERBB1/2 protein, were not affected by the addition of lapatinib. This result suggests that inhibition of ERBB1/2 is required for the synergistic toxic effects seen with doxorubicin in myocytes. In conclusion these results suggest that combination therapy employing doxorubicin and lapatinib may produce increased cardiotoxicity. Support: CIHR and a Canada Research Chair in Drug Development to BBH. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3576. doi:10.1158/1538-7445.AM2011-3576

The use of the new anticancer tyrosine kinase inhibitors (TKI) has revolutionized the treatment of certain cancers. However, the use of some of these results in cardiotoxicity. Large scale profiling data recently made available for the binding of 7 of the 9 FDA-approved tyrosine kinase inhibitors to a panel of 317 kinases has allowed us to correlate kinase inhibitor binding selectivity scores with TKI-induced damage to neonatal rat cardiac myocytes. The 7 TKIs that were the subject of this study were: imatinib (Gleevec), sunitinib (Sutent), dasatinib (Sprycel), sorafenib (Nexavar), lapatinib (Tykerb), gefitinib (Iressa), and erlotinib (Tarceva). The tyrosine kinase selectivity scores, but not the serine-threonine kinase scores, were highly correlated with the myocyte damaging effects of the TKIs. Additionally, we showed that damage to myocytes gave a good rank order correlation with clinical cardiotoxicity. Finally, strength of TKI binding to colony stimulating factor 1 receptor (CSF1R) was highly correlated with myocyte damage, thus possibly implicating this kinase in contributing to TKI-induced cardiotoxicity. Support: CIHR and a Canada Research Chair in Drug Development to BBH. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2602.