Eugene A. Konorev

Doxorubicin-induced Apoptosis in Endothelial Cells and Cardiomyocytes Is Ameliorated by Nitrone Spin Traps and Ebselen ROLE OF REACTIVE OXYGEN AND NITROGEN SPECIES

Doxorubicin (DOX) is a broad spectrum anthracycline antibiotic used to treat a variety of cancers. Redox activation of DOX to form reactive oxygen species has been implicated in DOX-induced cardiotoxicity. In this work we investigated DOX-induced apoptosis in cultured bovine aortic endothelial cells and cardiomyocytes isolated from adult rat heart. Exposure of bovine aortic endothelial cells or myocytes to submicromolar levels of DOX induced significant apoptosis as measured by DNA fragmentation and terminal deoxynucleotidyltransferase-mediated nick-end labeling assays. Pretreatment of cells with 100 μm nitrone spin traps, N-tert-butyl-α-phenylnitrone (PBN) or α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) dramatically inhibited DOX-induced apoptosis. Ebselen (20–50 μm), a glutathione peroxidase mimetic, also significantly inhibited apoptosis. DOX (0.5–1 μm) inactivated mitochondrial complex I by a superoxide-dependent mechanism. PBN (100 μm), POBN (100 μm), and ebselen (50 μm) restored complex I activity. These compounds also inhibited DOX-induced caspase-3 activation and cytochrome crelease. PBN and ebselen also restored glutathione levels in DOX-treated cells. We conclude that nitrone spin traps and ebselen inhibit the DOX-induced apoptotic signaling mechanism and that this antiapoptotic mechanism may be linked in part to the inhibition in formation or scavenging of hydrogen peroxide. Therapeutic strategies to mitigate DOX cardiotoxicity should be reexamined in light of these emerging antiapoptotic mechanisms of antioxidants.

Doxorubicin-induced apoptosis: implications in cardiotoxicity.

In this review, we discuss the role of nitric oxide synthase in doxorubicin (DOX)-induced cardiomyopathy, a prominent side effect of DOX chemotherapy in cancer patients. It is becoming increasingly clear that apoptosis of myocardial cells plays a critical role in the onset of cardiomyopathy. DOX exposure to endothelial cells and cardiomyocytes caused apoptotic cell death at sub-micromolar concentrations. DOX-induced generation of H2O2 has been shown to be responsible for this drugs toxicity and apoptosis. H2O2 in turn enhanced endothelial nitric oxide synthase (eNOS) transcription in endothelial cells and myocytes. Antisense eNOS depressed DOX-induced oxidative stress and apoptosis. Redox-metal chelators inhibited DOX-induced apoptosis, clearly suggesting a role for reactive oxygen species in DOX-induced apoptosis. Here, we will focus on the role of eNOS expression, iron chelation, and iron signaling on DOX-mediated apoptosis.

Activation of nuclear factor-kappaB during doxorubicin-induced apoptosis in endothelial cells and myocytes is pro-apoptotic: the role of hydrogen peroxide.

Doxorubicin (DOX) is a widely used anti-tumour drug. Cardiotoxicity is a major toxic side effect of DOX therapy. Although recent studies implicated an apoptotic pathway in DOX-induced cardiotoxicity, the mechanism of DOX-induced apoptosis remains unclear. In the present study, we investigated the role of reactive oxygen species and the nuclear transcription factor nuclear factor kappaB (NF-kappaB) during apoptosis induced by DOX in bovine aortic endothelial cells (BAECs) and adult rat cardiomyocytes. DOX-induced NF-kappaB activation is both dose- and time-dependent, as demonstrated using electrophoretic mobility-shift assay and luciferase and p65 (Rel A) nuclear-translocation assays. Addition of a cell-permeant iron metalloporphyrin significantly suppressed NF-kappaB activation and apoptosis induced by DOX. Overexpression of glutathione peroxidase, which detoxifies cellular H(2)O(2), significantly decreased DOX-induced NF-kappaB activation and apoptosis. Inhibition of DOX-induced NF-kappaB activation by a cell-permeant peptide SN50 that blocks translocation of the NF-kappaB complex into the nucleus greatly diminished DOX-induced apoptosis. Apoptosis was inhibited when IkappaB mutant vector, another NF-kappaB inhibitor, was added to DOX-treated BAECs. These results suggest that NF-kappaB activation in DOX-treated endothelial cells and myocytes is pro-apoptotic, in contrast with DOX-treated cancer cells, where NF-kappaB activation is anti-apoptotic. Removal of intracellular H(2)O(2) protects endothelial cells and myocytes from DOX-induced apoptosis, possibly by inhibiting NF-kappaB activation. These findings suggest a novel mechanism for enhancing the therapeutic efficacy of DOX.

Rapid and irreversible inhibition of creatine kinase by peroxynitrite

We examined the ability of peroxynitrite and other ⋅NO‐derived oxidants to inhibit creatine kinase (CK). Peroxynitrite potently inhibited CK activity and depleted protein thiols. The rate constant for this reaction was 8.85×105 M−1 s−1. Glutathione did not reactivate CK activity nor did it regenerate protein thiol content. In contrast, glutathione reactivated CK, and regenerated protein thiols, after inhibition by either ⋅NO or oxidized glutathione (GSSG). Peroxynitrite did not irreversibly inhibit CK after it had been treated with GSSG to block protein thiols. We conclude that thiol oxidation is a critical event leading to inactivation of CK by peroxynitrite.

Doxorubicin Inactivates Myocardial Cytochrome c Oxidase in Rats: Cardioprotection by Mito-Q

Doxorubicin (DOX) is used for treating various cancers. Its clinical use is, however, limited by its dose-limiting cardiomyopathy. The exact mechanism of DOX-induced cardiomyopathy still remains unknown. The goals were to investigate the molecular mechanism of DOX-induced cardiomyopathy and cardioprotection by mitoquinone (Mito-Q), a triphenylphosphonium-conjugated analog of coenzyme Q, using a rat model. Rats were treated with DOX, Mito-Q, and DOX plus Mito-Q for 12 weeks. The left ventricular function as measured by two-dimensional echocardiography decreased in DOX-treated rats but was preserved during Mito-Q plus DOX treatment. Using low-temperature ex vivo electron paramagnetic resonance (EPR), a time-dependent decrease in heme signal was detected in heart tissues isolated from rats administered with a cumulative dose of DOX. DOX attenuated the EPR signals characteristic of the exchange interaction between cytochrome c oxidase (CcO)-Fe(III) heme a3 and CuB. DOX and Mito-Q together restored these EPR signals and the CcO activity in heart tissues. DOX strongly downregulated the stable expression of the CcO subunits II and Va and had a slight inhibitory effect on CcO subunit I gene expression. Mito-Q restored CcO subunit II and Va expressions in DOX-treated rats. These results suggest a novel cardioprotection mechanism by Mito-Q during DOX-induced cardiomyopathy involving CcO.

Stretch-induced hypertrophy activates NFkB-mediated VEGF secretion in adult cardiomyocytes.

Hypertension and myocardial infarction are associated with the onset of hypertrophy. Hypertrophy is a compensatory response mechanism to increases in mechanical load due to pressure or volume overload. It is characterized by extracellular matrix remodeling and hypertrophic growth of adult cardiomyocytes. Production of Vascular Endothelial Growth Factor (VEGF), which acts as an angiogenic factor and a modulator of cardiomyocyte function, is regulated by mechanical stretch. Mechanical stretch promotes VEGF secretion in neonatal cardiomyocytes. Whether this effect is retained in adult cells and the molecular mechanism mediating stretch-induced VEGF secretion has not been elucidated. Our objective was to investigate whether cyclic mechanical stretch induces VEGF secretion in adult cardiomyocytes and to identify the molecular mechanism mediating VEGF secretion in these cells. Isolated primary adult rat cardiomyocytes (ARCMs) were subjected to cyclic mechanical stretch at an extension level of 10% at 30 cycles/min that induces hypertrophic responses. Cyclic mechanical stretch induced a 3-fold increase in VEGF secretion in ARCMs compared to non-stretch controls. This increase in stretch-induced VEGF secretion correlated with NFkB activation. Cyclic mechanical stretch-mediated VEGF secretion was blocked by an NFkB peptide inhibitor and expression of a dominant negative mutant IkBα, but not by inhibitors of the MAPK/ERK1/2 or PI3K pathways. Chromatin immunoprecipitation assays demonstrated an interaction of NFkB with the VEGF promoter in stretched primary cardiomyocytes. Moreover, VEGF secretion is increased in the stretched myocardium during pressure overload-induced hypertrophy. These findings are the first to demonstrate that NFkB activation plays a role in mediating VEGF secretion upon cyclic mechanical stretch in adult cardiomyocytes. Signaling by NFkB initiated in response to cyclic mechanical stretch may therefore coordinate the hypertrophic response in adult cardiomyocytes. Elucidation of this novel mechanism may provide a target for developing future pharmacotherapy to treat hypertension and heart disease.

Differences in doxorubicin-induced apoptotic signaling in adult and immature cardiomyocytes

A proposed mechanism for the cardiotoxicity of doxorubicin (DOX) involves apoptosis in cardiomyocytes. In the study described here, we investigated the molecular basis for the differences in DOX-induced toxicity in adult rat cardiomyocytes (ARCM), neonatal rat cardiomyocytes (NRCM), and rat embryonic H9c2 cardiomyoblasts. Activation of caspase-9 and -3 was considerably lower in DOX-treated ARCM as compared with NRCM and H9c2 cardiomyoblasts. Addition of cytochrome c caused the activation of caspase-9 and -3 in permeabilized NRCM and H9c2 cardiomyoblasts but not in permeabilized ARCM. Expression of proapoptotic proteins, apoptotic protease activating factor-1 (Apaf1), and procaspase-9 was significantly lower, and abundance of antiapoptotic X-linked inhibitor of apoptosis protein (XIAP) was higher in ARCM, as compared with immature cardiac cells. Despite the abundance of XIAP in ARCM, its role in the inhibition of apoptosome function was dismissed, as second mitochondria-derived activator of caspases (Smac)-N7 peptide, had no effect on caspase activation in response to cytochrome c in these cells. Adenoviral expression of Apaf1 exacerbated the activation of caspase-9 and -3 in DOX-treated NRCM, but did not increase their activities in DOX-treated ARCM. This finding points to a major difference in the apoptotic signaling between immature and adult cardiomyocytes. The mitochondrial apoptotic pathway is limited in ARCM treated with DOX.

Oxidant-induced iron signaling in doxorubicin-mediated apoptosis

Publisher Summary This chapter discusses the methodological aspects linking Doxorubicin (DOX)-induced intracellular oxidative stress, iron signaling, and apoptosis. Pertinent redox parameters measured are the following: intracellular glutathione (GSH) levels, aconitase activity, IRP–IRE interaction, transferrin receptor (TfR) expression, cellular iron uptake, DCFH oxidation to DCF, and apoptosis. DOX or adriamycin, a quinone-containing anthracycline antibiotic, is a widely used chemotherapeutic drug for treating leukemia, breast cancer, Hodgkins disease, or sarcomas. The clinical efficacy of this drug is greatly restricted because of the development of a severe form of cardiomyopathy or congestive heart failure in cancer patients treated with this drug. Current evidence indicates that DOX-mediated cardiotoxicity may be caused by increased generation of reactive oxygen species (ROS) such as superoxide, hydrogen peroxide (H2O2), or hydroxyl radicals (·OH) through redox-activation of DOX. The proapoptotic effect of DOX in myocytes and endothelial cells is attributed to intracellular iron and H2O2 formation. Recently, it is showed that intracellular iron plays a critical role in initiating oxidant-induced apoptosis through upregulation of TfR. Transferrin receptor synthesis is regulated by interaction of the iron regulatory protein (IRP) with the iron-responsive element (IRE) present on the 30-untranslated region of TfR mRNA. The oxidant-induced iron signaling mechanism is a new perspective that should be more fully explored in DOX cardiotoxicity.

Paradoxical effects of metalloporphyrins on doxorubicin-induced apoptosis: scavenging of reactive oxygen species versus induction of heme oxygenase-1

The cytoprotective effects of redox-active metalloporphyrins (e.g., FeTBAP and MnTBAP) were generally attributed to their ability to scavenge reactive oxygen and nitrogen species. In this study, we evaluated the pro- and antiapoptotic potentials of different metalloporphyrins containing iron, cobalt, zinc, and manganese in adult rat cardiomyocytes exposed to doxorubicin (DOX), an anticancer drug that forms superoxide and hydrogen peroxide via redox-cycling of DOX semiquinone in the presence of molecular oxygen. We used electron spin resonance/spin trapping and cytochrome c reduction to assess the scavenging of superoxide anion by metalloporphyrins. Superoxide anion was effectively scavenged by FeTBAP and MnTBAP but not by CoTBAP and ZnTBAP. FeTBAP efficiently scavenged H(2)O(2). Both CoTBAP and FeTBAP inhibited DOX-induced cardiomyocyte apoptosis. These findings implicate that mechanisms other than oxy-radical scavenging may account for their antiapoptotic property. In addition, CoTBAP and FeTBAP induced heme oxygenase-1 more potently than did MnTBAP and ZnTBAP. Inhibition of heme oxygenase abolished the protective effect of CoTBAP and reduced the protection by FeTBAP against DOX-induced cardiomyocyte apoptosis. We propose that metalloporphyrins can inhibit apoptosis either by inducing heme oxygenase-1 and antiapoptotic protein signaling or by scavenging reactive oxygen species.

The mechanism of cardioprotection by S-nitrosoglutathione monoethyl ester in rat isolated heart during cardioplegic ischaemic arrest.

1 This study was designed (i) to assess the effect of S‐nitrosoglutathione monoethyl ester (GSNO‐MEE), a membrane‐permeable analogue of S‐nitrosoglutathione (GSNO), on rat isolated heart during cardioplegic ischaemia, and (ii) to monitor the release of nitric oxide (·NO) from GSNO‐MEE in intact hearts using endogenous myoglobin as an intracellular ·NO trap and the hydrophilic N‐methyl glucamine dithiocarbamate‐iron (MGD‐Fe2+) complex as an extracellular ·NO trap. 2 During aerobic perfusion of rat isolated heart with GSNO‐MEE (20 μmol 1−1), there was an increase in cyclic GMP from 105 ± 11 to 955 ± 193 pmol g−1 dry wt. (P < 0.05), and a decrease in glycogen content from 119 ± 3 to 96 ± 2 μmol g−1 dry wt. P < 0.05), and glucose‐6‐phosphate concentration from 258 ± 22 in control to 185 ± 17 nmol g−1 dry wt. (P < 0.05). During induction of cardioplegia, GSNO‐MEE caused the accumulation of cyclic GMP (100 ± 6 in control vs. 929 ± 168 pmol g−1 dry wt. in GSNO‐MEE‐treated group, P < 0.05), and depletion of glycogen from 117 ± 3 to 103 ± 2 μmol g−1 dry wt. (P < 0.05) in myocardial tissue. 3 Inclusion of GSNO‐MEE (20 μmol 1−1) in the cardioplegic solution improved the recovery of developed pressure (46 ± 8 vs. 71 ± 3% of baseline, P < 0.05), and rate‐pressure product from 34 ± 6 to 63 ± 5% of baseline (P < 0.05), and reduced the diastolic pressure during reperfusion from 61 ± 7 in control to 35 ± 5 mmHg (P < 0.05) after 35 min ischaemic arrest. GSH‐MEE (20 μmol 1−1) in the cardioplegic solution did not elicit the protective effect. 4 During cardioplegic ischaemia, GSNO‐MEE (20–200 μmol 1−1) induced the formation of nitrosylmyoglobin (MbNO), which was detected by electron spin resonance (ESR) spectroscopy. Inclusion of MGD‐Fe2+ (50 μmol 1−1 Fe2+ and 500 μmol 1−1 MGD) in the cardioplegic solution along with GSNO‐MEE yielded an ESR signal characteristic of the MGD‐Fe2+‐NO adduct. However, the MGD‐Fe2+ trap did not prevent the formation of the intracellular MbNO complex in myocardial tissue. During aerobic reperfusion, denitrosylation of the MbNO complex slowly occurred as shown by the decrease in ESR spectral intensity. GSNO‐MEE treatment did not affect ubisemiquinone radical formation during reperfusion. 5 GSNO‐MEE (20 μl 1−1) treatment elevated the myocardial cyclic GMP during ischaemia (47 ± 3 in control vs. 153 ± 34 pmol g−1 dry wt. after 35 min ischaemia, P < 0.05). The cyclic GMP levels decreased in the control group during ischaemia from 100 ± 6 after induction of cardioplegia to 47 ± 3 pmol g−1 dry wt. at the end of ischaemic duration. 6 Glycogen levels were lower in GSNO‐MEE (20 μmol 1−1)‐treated hearts throughout the ischaemic duration (26.7 ± 3.1 in control vs. 19.7 ± 2.4 μmol g dry−1 wt. in GSNO‐MEE‐treated group at the end of ischaemic duration), because of rapid depletion of glycogen during induction of cardioplegia. During ischaemia, the amounts of glycogen consumed in both groups were similar. Equivalent amounts of lactate were produced in both groups (148 ± 4 in control vs. 141 ± 4 μmol g−1 dry wt. in GSNO‐MEE‐treated group after 35 min in ischaemia). 7 The mechanism(s) of myocardial protection by GSNO‐MEE against ischaemic injury may involve preischaemic glycogen reduction and/or elevated cyclic GMP levels in myocardial tissue during ischaemia.