The role of nitric oxide in anthracycline toxicity ... - The FASEB Journal

The role of nitric oxide in anthracycline toxicity and prospects for pharmacologic prevention of cardiac damage STEFANO FOGLI,1 PAOLA NIERI, AND MARIA CRISTINA BRESCHI Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Pisa, Italy Anthracycline antibiotics are potent antitumor agents whose activity is severely limited by a cumulative dose-dependent chronic cardiotoxicity that results from the summation of multiple biochemical pathways of cellular damage, which ultimately yields to disruption of myocardiocyte integrity and loss of cardiac function. Nitric oxide (NO) is a key molecule involved in the pathophysiology of heart; dysregulation of activity of NO synthases (NOSs) and of NO metabolism seems to be a common feature in various cardiac diseases. The contribution of NO to anthracycline cardiac damage is suggested by evidence demonstrating anthracycline-mediated induction of NOS expression and NO release in heart and the ability of NOSs to promote anthracycline redox cycling to produce reactive oxygen species (ROS), including O2ⴚ䡠 and H2O2. Overproduction of ROS and NO yields to reactive nitrogen species, particularly the powerful oxidant molecule peroxynitrite (ONOO–), which may produce the marked reduction of cardiac contractility. This review focuses on the anthracycline-mediated deregulation of NO network and presents an unifying viewpoint of the main molecular mechanisms involved in the pathogenesis of anthracycline cardiotoxicity, including iron, free radicals, and novel mechanistic notions on cardiac ceramide signaling and apoptosis. The data presented in the literature encourage the development of strategies of pharmacological manipulation of NO metabolism to be used as a novel approach to the prevention of cardiotoxicity induced by anthracyclines.OFogli, S., Nieri, P., Breschi, M. C. The role of nitric oxide in anthracycline toxicity and prospects for pharmacologic prevention of cardiac damage. FASEB J. 18, 664 – 675 (2004) ABSTRACT

Key Words: doxorubicin 䡠 NO 䡠 apoptosis 䡠 cardioprotection BACKGROUND Anthracycline antibiotics were introduced into clinical practice more than 20 years ago, and since then have been characterized by widespread clinical use that makes them one of the most successful drug categories. Unfortunately, cardiotoxicity represents a serious adverse effect of anthracyclines that limits their therapeu664

tic potential due to the development of irreversible, cumulative dose-dependent congestive cardiomyopathy (1). Synthesis of fewer cardiotoxic derivatives, drug carrier technology, and individual dose optimization represent some of the adopted strategies to improve the therapeutic index of this class of drugs (2, 3). However, notwithstanding the remarkable progress characterizing the past years, the problem of anthracycline cardiotoxicity is still unresolved. Dexrazoxane (ICRF-187), a nonpolar derivative of EDTA with a bis-ketopiperazine moiety, is the only FDA-approved drug for the prevention of cardiac dysfunction induced by anthracyclines (4). Although the drug proved to be effective in the prevention of early anthracycline cardiotoxicity, its efficacy on the development of late myocardial damage remains undetermined (5). Some concerns on the potential unfavorable effect of dexrazoxane on antineoplastic activity and hematological toxicity of anthracyclines (5) as well as on the carcinogenic activity on the cells of bone marrow and skin (6) were raised. The need of searching for novel cardioprotectants to be used in place of or in combination with dexrazoxane prompted us to review the available data in the literature to explore the consistency of novel molecular mechanisms of anthracycline cardiotoxicity. Anthracycline antibiotics are able to induce complex changes in cell homeostasis as a result of dysregulation of survival signals involved in the preservation of cardiomyocyte integrity. With these premises, a key molecule critically implicated in the pathophysiology of cardiac function could represent the prototype of anthracycline action. Nitric oxide (NO) may legitimately aspire to this role, because it regulates many aspects of cellular function in the normal heart as well as in ischemic and nonischemic heart failure, septic cardiomyopathy, cardiac allograft rejection, and myocarditis (7). The aim of this review is to provide an unifying viewpoint of the 1

Correspondence: Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Via Bonanno, 6, Pisa, PI 56126 Italy. E-mail: s.fogli@do. med.unipi.it doi: 10.1096/fj.03-0724rev 0892-6638/04/0018-0664 © FASEB

possible molecular mechanisms of drug damage, focusing on the deregulation of NO system, in order to devise novel pharmacological strategies against anthracycline cardiotoxicity.

CELLULAR BIOCHEMISTRY AND FUNCTIONS OF NO Aside from the well-defined role of NO as an endothelium-derived relaxant of vascular smooth muscle, much interest has focused on endogenous NO pathways as paracrine, autocrine and location-specific intracrine regulators of cardiac muscle function. Indeed, given the multiplicity of NO synthase (NOS) isoforms expressed in heart and of the molecular targets of NO, including aconitases, mitochondrial electron transport chain, cytoskeletal proteins and contractile myofilaments, tight regulation of NOS expression and activity appear to be needed to coordinate the many influences on signal transduction/␤-adrenergic stimulation, mitochondrial respiration/myocardial energetics, and calcium cycling (8 –10). In the heart, endogenous NO is generated by the action of a family of NOS: the neuronal (nNOS or NOS1) and endothelial (eNOS or NOS3) enzymes are constitutively expressed in sarcoplasmic reticulum of cardiac myocyte and in atrial and ventricular myocytes from human hearts, respectively, whereas the activity of the Ca2⫹-independent inducible isoform (iNOS or NOS2) is up-regulated by a variety of stimuli, including inflammatory cytokines and bacterial lipopolysaccharide; its activity has been detected in endocardial and endothelial cells, vascular smooth muscle cells, fibroblasts, as well as in neonatal and adult cardiac myocytes from several species (8, 11). All three NOS isoforms combine two functionally complementary portions: a carboxyl-terminal reductase domain, homologous to cytochrome P450 reductase, and an amino-terminal oxygenase domain containing binding sites for heme, l-arginine, and tetrahydrobiopterin (THB4), the two portions being connected by a calmodulin binding domain. Upon activation, the three isoforms presumably function as homodimers; within each monomer, electrons provided by NADPH are transferred from the flavins in the carboxyl-terminal portion of the molecule to heme iron, which is activated to bind O2 , and, in the presence of the substrate l-arginine, to catalyze the synthesis of NO and L-citrulline (12) (Fig. 1). The subcellular location of NO pathways is critical to the regulation of cardiovascular effects of NO; NO acts in cellular microdomains with site-specific signaling because NOS isoforms are restricted to specific cellular structures. As a matter of fact, compartmentalization of eNOS with ␤-adrenergic receptors and L-type Ca2⫹ channels into caveolae allows NO to modulate ␤-adrenergic-regulated cardiac inotropism whereas cardiac sarcoplasmic nNOS seems to have an opposite effect on contractility due to the NO stimulation of Ca2⫹ release via the ryanodine receptor (10). NO is a highly diffusNO AND ANTHRACYCLINE-INDUCED CARDIOTOXICITY

Figure 1. Structural representation of nitric oxide synthase homodimers. CaM, calmodulin; THB4 , tetrahydrobiopterin; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; NADPH, nicotinamide adenine dinucleotide phosphate. Adapted from Mungrue et al. (12).

ible molecule that functions by itself and/or through reactive nitrogen species (RNS) relatively far from the site of production; moreover, NO may directly or indirectly interact with a number of macromolecules including proteins, lipids, and nucleic acids. The direct effects are physiologically relevant and predominate at low concentrations of NO (⬍1 ␮M) that can be reached after activation of the constitutive isoforms eNOS and nNOS. For instance, low levels of NO most frequently target soluble guanylate cyclase, reversibly activating the cGMP-mediated signaling cascade, which results in various biological effects: modulation of Ca2⫹ influx and decreased myofilament response to Ca2⫹. Other targets of NO include cytochrome P450 and, to a lesser extent, nonheme iron- and zinc-containing proteins (8). On the other hand, when iNOS is induced, a large amount of NO (⬎1 ␮M) is available to interact with molecular oxygen (O2) and anion superoxide (O2⫺䡠), ultimately forming RNS, i.e., dinitrogen trioxide (N2O3) and peroxynitrite (OONO–). As a consequence, RNS-mediated indirect effects predominate, thus yielding to nitrosylation, oxidation, and nitration of amine, thiol, and hydroxyaromatic group and tyrosine residues (13). Such chemical modifications may substantially regulate molecular targets relevant to cardiovascular biology; for example, poly-Snitrosylation of up to 12 out of the available 84 cysteine residues leads to the reversible activation of the ryanodine receptor (14). Finally, protein nitration seems to be a critical component of NO biochemistry because it is involved in the negative feedback regulation of NO production whereas a putative denitrase activity has been demonstrated in several tissues, suggesting that substrate nitrosylation may be a reversible process (15). 665

THE POTENTIAL ROLE OF NO IN HEART PATHOLOGY Unlike constitutively expressed NOS isoforms (nNOS and eNOS), iNOS is regulated primarily at the transcriptional level in response to inflammatory cytokines and endotoxins. Increased iNOS expression has been reported in human cardiac disease including dilated cardiomyopathy (16), heart failure (17), and contractile dysfunction of the left ventricle after heart transplantation (18). It is conceivable that the balance between the physiologic and pathologic production of NO may be critical in regulating cardiac function and preserving cardiomyocyte integrity whereas the pathological switch may be expected when there is an abnormal level of NO production, and/or when there is an excess of superoxide anion synthesis (O2–䡠) (Fig. 2). For example, excessive production of superoxide in reperfused myocardium can inactivate NO, and this may contribute to the shutdown of the cGMP pathway with the consequence of increasing neutrophil aggregation and adherence (19) and coronary vasoconstriction (20). On the other hand, excess superoxide reacts with NO enhancing the production of ONOO-, a potent oxidant that is able to oxidize lipids, nitrate tyrosine residues on proteins, and decompose to toxic products including the highly reactive hydroxyl radicals (OH䡠) (15). It has been proposed that any species capable of Fenton-type chemistry, such as peroxidases, free heme, or metal ions, will mediate tyrosine nitration, providing an alternative to the ONOO– reaction (21). Thus, the abundant synthesis of NO and/or O2–䡠 generates a free radical cascade that, in turn, may disrupt key cellular targets in the heart and represent a cause of cardiomyopathy (Fig. 2). In agreement with this hypothesis, evidence is available demonstrating a significant contri-

bution of increased RNS/ROS production and protein nitration in the progression of cardiovascular disease (15). Data from the literature indicate that, at variance with NO, ONOO– inactivates human aconitases and induces irreversible inhibition of mitochondrial respiration (22–24). Nitration of ␣-actinin, myofibrillar creatine kinases and prostacyclin synthase by ONOO– may have deleterious effects on the function of contractile myofilaments (15) whereas increased production of ONOO– caused by up-regulation of cardiac iNOS in transgenic mice is associated with cardiac fibrosis, hypertrophy, and dilatation (25). Finally, the relevance of mitochondria in the damaging effect of NO further support the role of ONOO– in NO-mediated apoptotic damage and cardiotoxicity (26 –28). iNOS up-regulation by cytokines increases apoptosis in cultured myocytes by a process that is independent of guanylate cyclase/cGMP pathway and associated with the shift in the cellular balance toward proapoptotic Bak vs. antiapoptotic Bcl-xL (22, 29). Thus, the available data demonstrate the multiplicity of NO molecular targets and point to the important role of the disarray of NO homeostasis in cardiac cell injury in various heart diseases.

ANTHRACYCLINE-INDUCED RELEASE OF NO IN CARDIAC CELLS The influence of anthracyclines on the NO signaling pathway has been studied in experimental models. In vivo echocardiographic measurements in CF-1 mice showed that the marked reduction in cardiac contractility in animals given doxorubicin at 20 mg/kg i.p., as assessed by fractional shortening and stroke volume 5 days postdose, was associated with a statistically signifi-

Figure 2. The pathophysiologic hypothesis of nitric oxide-mediated cardiac diseases. NOS, nitric oxide synthases (e, endothelial; i, inducible; n, neuronal); NO, nitric oxide radical; ONOO–, peroxynitrite anion; O2–䡠, superoxide anion; NO2 , nitrogen dioxide; OH䡠, hydroxyl radical; H2O2 , hydrogen peroxide.

666

Vol. 18

April 2004

The FASEB Journal

FOGLI ET AL.

cant increase in the immunopositivity of myocardial iNOS (33⫾18 vs. 9⫾2%) and 3-nitrotyrosine formation (56⫾24 vs. 0.3⫾0.4%) as compared with the control group (30). A decrease in ejection fraction and cardiac output matching an increase in cardiac nitrotyrosine synthesis have recently been observed in mice 5 days after the administration of doxorubicin at 25 mg/kg i.p. (31). These findings suggest that iNOS induction, the putative enzymatic source of NO in cardiac myocytes exposed to anthracyclines, and the formation of the damaging by-product ONOO- may represent a critical mechanism of drug-induced cell injury. Doxorubicin treatment at 5 ␮M for 24 h increased the amount of iNOS protein without affecting eNOS and nNOS expression in the H9C2 rat cardiac cells (32) whereas iNOS⫺/⫺ mice treated with doxorubicin displayed a better cardiac function than iNOS⫹/⫹ littermates (31). Finally, doxorubicin-induced hydrogen peroxide synthesis and oxidative stress appear to be responsible for the increase in eNOS expression and NO synthesis in bovine aortic endothelial cells (33). It is known that oxygen radical production in the heart by anthracycline analogs, such as doxorubicin and daunorubicin, is mediated by reductases that catalyze a one-electron addition to the quinone moiety of anthracyclines (Fig. 3). This results in the formation of a semiquinone free radical that readily regenerates the parent quinone by reducing molecular oxygen to superoxide anion (O2–䡠). O2–䡠 is then transformed to hydrogen peroxide (H2O2) by superoxide dismutase, resulting in excess production of oxidative compounds above the physiologic levels generated during normal aerobic metabolism (34, 35). There is evidence that purified recombinant NOSs are able to trigger doxorubicin redox cycling to produce reactive oxygen species, including O2–䡠 and H2O2 (36, 37); the maximum velocity of iNOS for doxorubicin (Kcat⫽14.23 s–1) was

⬃5 to 10-fold higher than that of nNOS and eNOS (37), thus confirming the important role of iNOS in the pathogenesis of anthracycline cardiotoxicity. On the other hand, one-electron reduction of anthracyclines may result in inhibition of NO generation, presumably by diverting electrons from the reductase domain of NOSs (36, 37). The reduction in NO levels may be enhanced by the production of superoxide by redox cycling, because superoxide is able to quench NO to form ONOO– (38). Owing to the importance of NO as a key regulator of vascular tone and an important mediator in the myocardial contractile response, the resulting acute change in NO homeostasis may account at least in part for the early electrocardiographic changes that occur upon administration of anthracyclines in rats (39) and mice (30). Regarding chronic cardiotoxicity, prolonged anthracycline exposure may induce a large synthesis of byproducts of the NOS-mediated anthracycline redox cycling, including ONOO-. This mechanism appears to be highly efficient because the superoxide excess produced by different reductases, including NOSs, rapidly consumes NO, resulting in a marked up-regulation of cardiac NOS expression and activity, a mechanism demonstrated in hypertensive rats (40). As a consequence, increased levels of NO and O2–䡠 favor the formation of ONOO-, which can rapidly react with manganese-superoxide dismutase (Mn-SOD), leading to a metal-catalyzed nitration of the critical Tyr34 residue and inactivation of the enzyme (27). The subsequent decrease in O2–䡠 dismutation rates will initiate a deleterious faulty mechanism that will favor further formation of ONOO– and other NO-derived reactive nitrogen species, therefore promoting cardiomyocyte damage (Fig. 3). This self-propagated cascade of events after long-term treatment with anthracyclines may yield to alterations in calcium homeostasis in mitochondria, a mechanism that renders myocytes susceptible to the

Figure 3. Putative fee radical cascade and cell death pathways by anthracycline-NO cross-talk in heart. NO, nitric oxide; ONOO–, peroxynitrite; O2–䡠, superoxide anion; OH䡠, hydroxyl radical; H2O2 , hydrogen peroxide; RNS, reactive nitrogen species; NO2 , nitrogen dioxide; FeIV ⫽ O, ferryl ions; A-Fe, anthracycline–iron complex; IRP-1; iron regulatory protein-1; Mn-SOD, manganesesuperoxide dismutase; Ox, oxidase domain; Red, reductase domain; PARP, poly(ADP-ribose) polymerase.

NO AND ANTHRACYCLINE-INDUCED CARDIOTOXICITY

667

increase in cytosolic calcium, which in turn may promote the disruption of mitochondrial function, depletion of ATP, and eventually cell death (41, 42). To further support the role of NO deregulation in anthracycline cardiotoxicity, ONOO– produce the collapse of mitochondrial energy metabolism by inhibition of glycolysis and depletion of ATP pools (26), all of which promote cell death. Alternatively, when respiratory or glycolytic ATP production is sufficient, permeability transition pore opening may evoke mitochondrial release of cytochrome c, caspase activation, and apoptosis (26), a signaling pathway demonstrated for anthracyclines in the heart (43, 44) (Fig. 3).

INTEGRATION OF NO WITH OTHER MECHANISMS OF HEART DAMAGE BY ANTHRACYCLINES As a result of years of intense research revolving around the molecular nature of drug-induced cardiomyocyte damage, it appears that anthracyclines exert a multitargeted toxicologic effect affecting different mechanisms of cell function and survival, including iron homeostasis, calcium regulation, and gene transcription (Fig. 4). Here, we examine the points of contact between NO and the major mechanisms of heart damage induced by anthracycline. Free radical generation A prominent role in the development of anthracycline cardiotoxicity has been assigned to overcoming of the antioxidant defenses of cardiomyocytes by drug-induced free radical reactions (34, 35, 45). In line with such a hypothesis, transgenic mice overexpressing the

thiol-rich nonenzymatic protein metallothionein receiving doxorubicin at 20 mg/kg showed reduced cellular damage, including myocardial lipid peroxidation, compared with their wild-type counterpart (46). According to this, 5/6 doxorubicin-treated mice with high levels of the redox-regulating molecule thioredoxin-1 survived for ⬎8 wk, whereas all of wild-type mice died within 6 wk of drug injection (47). Data against the key role of lipid peroxidation in anthracycline-induced cardiotoxicity derive from experiments demonstrating that doxorubicin decreases the myocardial release of conjugated dienes and hydroperoxides in blood samples collected from the coronary sinus of cancer patients (48). Possible explanations of such paradoxical antioxidant properties are that anthracyclines form an excess of radicals that would act as chain terminators by extinguishing oxidative stress (49); it has been demonstrated in the cytosol of human myocardial biopsies that doxorubicin is able to reduce the oxo-ferryl moiety [Fe(IV)⫽O, Mb(IV)] of H2O2-activated myoglobin, a lipid oxidant likely to be formed in the heart during drug treatment (50). These contrasting observations on the well-documented role of doxorubicin on oxidative stress may be reconciled by including NO into the free radical hypothesis of cardiac damage. For instance, the redox coupling of doxorubicin-derived semiquinone free radicals with molecular oxygen and iron generates an excess of H2O2 , which interacts with OH䡠 and converts it into O2–䡠 (49); such an increase in O2–䡠 levels generates ONOO– by reacting with NO as well as other reactive nitrogen species that may ultimately cause myocyte cell death and cardiac failure. Induction of apoptosis The relevance of apoptosis has been recognized in a variety of cardiac diseases (51), whereas increasing

Figure 4. Interferences of anthracyclines with signaling pathways in cardiac cells. A, anthracycline molecule; IRP-1; iron regulatory protein-1; IRE, iron responsive elements; Fer, ferritin; TfR, transferrin receptor; RyR, cardiac-type ryanodine receptor; SERCA2, sarco(endo)plasmic reticulum Ca2⫹ATPase; ROS, reactive oxygen species; Cyt-c, cytochrome c; APAF-1, apoptotic protease-activating factor 1; SMase, sphingomyelinase; Akt (PKB), protein kinase B; PI3K, phosphatidylinositol 3-kinase; gp130, glycoprotein 130; FADD, Fas-associated death domain protein; COX-2, cyclooxygenase 2; iNOS, inducible nitric oxide synthase. Bcl2, Bax, and Bad are members of the Bcl2 family of apoptotic regulatory proteins. Solid and dashed lines indicate activation and inhibition of target molecules, respectively.

668

Vol. 18

April 2004

The FASEB Journal

FOGLI ET AL.

evidence suggest that apoptosis plays an important role in the loss of myocardial function due to anthracycline treatment (52–54). Two independent pathways led to the execution of apoptosis in cardiac myocytes: 1) the extrinsic apoptotic pathway, mediated by Fas ligand or TNF-␣, which bind to their receptors Fas and TNF-␣R1, respectively, leading to the activation of caspase-8/ caspase-3 system, and 2) the intrinsic mitochondriadependent pathway characterized by the release of cytochrome c, synthesis of the ternary complex cytochrome c/procaspase-9/apoptotic protease-activating factor-1, and activation of the executioner enzyme caspase-3 (55, 51). The contribution of the extrinsic pathway to anthracycline cardiotoxicity has been shown in vivo by the ability of doxorubicin to induce overexpression of Fas antigen and apoptosis in rat hearts (53). Moreover, the involvement of the intrinsic apoptotic pathway was demonstrated in primary cultures of cardiomyocytes isolated from the hearts of mice and treated with doxorubicin at the clinically achievable concentration of 1 ␮M (56). Doxorubicin markedly elevated mitochondrial ROS concentrations and activated cytochrome c/caspase-3 pathway and apoptosis whereas ROS production and programmed cell death were almost completely inhibited in cardiomyocytes from transgenic animals overexpressing metallothionein (56). In agreement with these data, the expression of the proapoptotic protein Bax and the incidence of apoptosis were significantly reduced by treatment with probucol, an antioxidant agent that significantly reduced the extent of cardiac damage in rats treated with doxorubicin at the cumulative dose of 15 mg/kg (57). Nakamura et al. (53) suggested that the apoptotic cell death of cardiomyocytes induced in rats by intravenous administration of doxorubicin at 2 mg/kg weekly for 8 wk is p53 independent, as it appears that a p53independent pathway activates the transcription of the Bax proapoptotic gene. Finally, in neonatal Bcl-xloverexpressing rat cardiac myocytes treated with 1 ␮M of doxorubicin for 6 h, drug-induced apoptosis was not inhibited via suppression of ROS generation but via inhibition of the events downstream from ROS that involve cytochrome c release from mitochondria and caspase-3 activation (43). Altogether, these observations led to the postulation of the in vitro sequence of the molecular events involved in anthracycline-activated intrinsic pathway of myocyte apoptosis (Fig. 4). The in vivo relationship between cytochrome c increase in the cytosol and caspase-3 activation after a single dose of doxorubicin at 20 mg/kg (44) further supports the key role of proapoptotic signals in the molecular pathogenesis of anthracycline cardiotoxicity. Evidence of NO-mediated regulation of apoptotic cell death derive from the study of molecular events associated with rejection in heterotopic mouse cardiac transplantation using iNOS deficient recipients. A comparison of grafts from iNOS ⫹/⫹ vs. iNOS ⫺/⫺ animals showed that the expression of p53 gene was significantly higher (0.62⫾0.09 vs. 0.30⫾0.04, relative NO AND ANTHRACYCLINE-INDUCED CARDIOTOXICITY

units normalized against glyceraldehyde 3-phosphate dehydrogenase; P⬍0.05), as in the case of Bcl-xl expression (0.41⫾0.04 vs. 0.23⫾0.02 relative units; P⬍0.005), whereas the ratio of Bcl-2/Bax was lower (0.18⫾0.02 vs. 0.38⫾0.06; P⬍0.005) in iNOS ⫹/⫹ mice, demonstrating that induction of iNOS during acute rejection may promote apoptosis, which eventually induces cardiac dysfunction (58). Synthesis of ceramide Ceramide is a sphingosine-based lipid signaling molecule that plays an important role in biochemical events leading to cell death and apoptosis (59, 60). Of interest is the existence of a functional ceramide pathway involved in the occurrence of apoptosis in cardiomyocytes (61, 62), particularly in doxorubicin-induced cardiomyopathy (63). Moreover, enhanced de novo synthesis of ceramide has been proposed for the mechanism of action of daunorubicin (64), and sphingomyelinase activation is responsible for the anthracycline-induced ceramide accumulation and apoptosis in rat cardiac myocytes (65). Cell treatment with doxorubicin at 0.5 ␮M for 1 h resulted in a 31% sphingomyelin decrease and a parallel increase of ceramide during 7 days of culture; after this, up to 1750 pmol of sphingomyelin/mg of protein were hydrolyzed while ceramide levels increased up to 1850 pmol/mg protein, supporting the involvement of sphingomyelin pathway in anthracycline-mediated apoptosis in cardiac cells (65). On the basis of these findings and the ability of ceramide to induce dephosphorylation of the antiapoptotic protein Bcl-2 by mitochondrial protein phosphatase 2A (66, 67), it is possible to hypothesize that anthracyclines may induce sphingomyelin hydrolysis, ceramide synthesis and apoptosis in cardiac cells by activation of the intrinsic pathway (Fig. 4). Such a mechanism of cell death induced by anthracyclines seems to be particularly efficient due to the positive feedback control on neutral sphingomyelinase by ceramide, which makes the anthracycline-activated sphingomyelin cycle sufficient for inducing autonomous production of ceramide. These apoptotic signals are likely to overwhelm cell survival mechanisms (60). Finally, taking into account the interference of ceramide with additional signaling systems in cardiac cells such as down-regulation of Akt/PKB anti-apoptotic pathway (68), ceramide may conceivably represent a key molecular messenger in anthracycline cardiotoxicity. To further support this hypothesis, preincubation of cardiac myocytes with C2-ceramide for 2 h induces the activation of B-type Ca2⫹ channels and the loss of mitochondrial membrane potential (69), a mechanism that has been proposed to be involved in the cumulative and irreversible deterioration of myocardial function in patients receiving doxorubicin chemotherapy (41, 42). A possible link between ceramide and the NO pathway is suggested by the finding that neonatal mouse cardiomyocytes in culture treated with the NO donors 669

nitroso-glutathione or sodium nitroprusside for 24 h display a concentration-dependent decrease in cell viability whereas pretreatment with the ceramide synthase inhibitor fumonisin B1 at 10 ␮M reduced cell death induced by either NO donors (70). Furthermore, the same study demonstrated that the effect of fumonisin was not mediated through inhibition of H2O2 nor was cell death associated with the intracellular rise in cGMP. Therefore, these data demonstrate a functional link between NO-induced cell death in cardiomyocytes and de novo synthesis of ceramide. Abnormalities in Ca2ⴙ homeostasis and myofibrillar network impairment The alteration of intracellular Ca2⫹ homeostasis is an additional key mechanism of anthracycline toxicity (Fig. 4). Anthracyclines induce Ca2⫹ overload in cardiomyocytes through the activation of the L-type of calcium channels (71) and sarcoplasmic reticulum cardiac-type 2 ryanodine receptor (RyR2) (72, 73). The main metabolite of doxorubicin, doxorubicinol, may contribute to the decrease in gene expression of the cardiac RyR2 in rabbits administered doxorubicin at 1 mg/kg i.v. twice weekly for 8 wk (74). In isolated guinea pig ventricular myocytes, doxorubicinol (10 ␮M) decreases the action potential duration (APD; –24.6%) and cell shortening (–31%) induced by current clamp at a frequency of 0.5 Hz; doxorubicin displays opposite effects on APD (⫹31.2%) and contractility (⫹26.3%) (75). Such different effects between parent drug and metabolite may amplify the dysregulation of intracellular Ca2⫹ homeostasis and the detrimental effects on contractile function. The pathophysiologic effects of NO are also mediated at least in part through alteration of Ca2⫹ intracellular turnover. Indeed, the inhibition of Na⫹/H⫹ and Na⫹/Ca2⫹ exchangers attenuates postischemic myocardial formation of NO and OH䡠 in Langendorff-perfused rat hearts subjected to 30 min ischemia and 30 min reperfusion, suggesting that prevention of Ca2⫹ overload is cardioprotective via these mechanisms (76). Furthermore, high levels of endogenous myocyte-derived NO blunted the sensitivity of myofilaments to Ca2⫹ and were associated with reduced basal contractility in isolated perfused hearts from transgenic mice overexpressing eNOS in cardiac myocytes (77). Finally, the in vivo demonstration that protein nitration by ONOO– selectively impairs myofibrillar creatine kinase during doxorubicin-induced cardiac damage (78) supports the relevant contribution of NO metabolism to anthracycline impairment of contractile function observed in humans (79). Intracellular iron turnover Anthracyclines have the potential to deregulate iron homeostasis in cardiac cells, thereby altering mitochondrial respiration and energy metabolism and contributing to doxorubicin-induced apoptotic death of myocardial cells and chronic heart damage (80 – 83) (Fig. 4). 670

Vol. 18

April 2004

Iron regulatory proteins (IRPs) play vital roles in regulating cellular iron metabolism via their mRNA binding activity. Several studies point to the dysregulation of myocardial cell iron metabolism by anthracyclines by affecting the RNA binding activity of IRPs. The mechanism by which anthracyclines interact with the cellular turnover of IRP-1/aconitase is still controversial. Data from Minotti et al. (82) suggest that IRP-1 may be inactivated by doxorubicinol and ROS whereas ROS alone may impair the activity of IRP-2. Data from Kwok and Richardson (83) indicate that doxorubicinol has no effect on IRP-RNA binding activity whereas formation of anthracycline–iron complexes decreases IRPRNA binding and affects cellular iron metabolism. The peroxynitrite ONOO– and the hydroxyl radical OH䡠 may participate in the alterations in intracellular iron homeostasis caused by anthracyclines. It has been demonstrated that the ONOO– generated after exposure of recombinant aconitase/IRP-1 and cellular extracts of J774A.1 mouse macrophages to the NO donor 3-morpholino-sydnonimin-hydrochloride (SIN-1) removes iron from the catalytic Fe-S cluster of aconitase, making this enzyme switch to the cluster-free IRP-1 (84). IRP-1 interacts with target sequences in both the 5⬘-untranslated region of ferritin mRNA to inhibit its translation and the 3⬘-end of transferrin receptor (TfR) mRNA to stabilize it and increase the synthesis of TfR (8, 13). Moreover, ONOO– is likely to contribute to the cellular iron overload caused by anthracyclines by the enhancement of oxidative burst. In line with this, doxorubicin-mediated free radical synthesis increases TfR expression, iron uptake through TfR, and apoptosis in vascular endothelial cells (85). The intracellular iron overload in rat cardiac cells after treatment with Fe-nitrilo-triacetic acid (FeNTA) at 30 ␮M for 24 h (231.01⫾2.26 vs. 19.26⫾6.83 ng Fe/mg proteins) reverts the expression of iNOS by doxorubicin and the increase of NO production (32), suggesting the presence of a physiologic iron-mediated feedback regulation of NO metabolism. However, the large excess of ROSs derived from one-electron reduction of anthracycline quinone and their ability to convert IRP-1 into a null protein neither able to bind to mRNA nor to switch back to aconitase (82), and the direct inhibition of IRP-mRNA binding activity by anthracycline–iron complex (83), may eventually disrupt iron homeostasis. Such deregulation of iron turnover may further enhance cardiac damage by anthracyclines, because protein nitration is mediated by heme and free metals through a Fenton-type chemistry (21). Cyclooxygenase-2 activity The expression of cyclooxygenase-2 (COX-2) has been detected in areas of myocardial infarction and in heart tissue of patients with dilated cardiomyopathy, whereas COX-2 is undetectable in normal hearts (86). In rat neonatal cardiac cells treated with doxorubicin at 172 ␮M for 80 min or 17.2 ␮M for 24 h, no change in COX-1 expression was detected whereas COX-2 and its

The FASEB Journal

FOGLI ET AL.

product 6-keto-PGF1␣ were significantly induced; such an effect was associated with free radical formation and was prevented by 200 U/mL of PEG-superoxide dismutase and catalase (87). COX-2 induction in cardiac tissue of adult rats 4 h after a single dose of doxorubicin at 15 mg/kg was associated with an elevation in plasma levels of cardiac troponin T, which increased further when doxorubicin was combined with the COX-2 inhibitor SC236, suggesting a potential protective role of COX-2 (88). In the ischemic-reperfused rabbit heart tissue, iNOS activity as well as the sum of nitrite and nitrate levels (NOx) increased by 153 and 58%, respectively, above control levels 24 h after ischemic preconditioning, whereas the activity of calcium-dependent NOSs (eNOS and nNOS) did not change significantly (89). The expression of COX-2 increased by threefold 24 h after the end of the ischemia/reperfusion cycles with respect to control tissue (P⬍0.05). The selective iNOS inhibitors SMT and 1400W completely abrogated the increase in iNOS activity and NOx release and inhibited the synthesis of PGE2 and 6-keto-PGF1␣, demonstrating the dependence of COX-2 activity on iNOS activity (89). In agreement, Gunther et al. (90) showed, by electron spin resonance spectroscopy, the nitrosylation of the catalytically active tyrosine residue of human COX-2 during prostaglandin formation. Despite the lack of direct evidence of a relationship between COX-2, anthracycline damage and NO-mediated signaling in cardiac tissue, the aforementioned evidence suggests that the induction of COX-2 may reflect an early protective response to drug toxicity whereas the large increase in intracellular levels of NO, as a consequence of the cumulative toxic effect of doxorubicin in cardiac muscle, may cause a reduction in COX-2 expression in parallel with chronic cardiac damage. Accordingly, the work of Vane et al. (91) on the role of the inducible isoforms of COX and NOS in a murine air pouch model of granulomatous inflammation suggested that low levels of NO may activate COX-2; in contrast, the release of large amounts of NO by iNOS may blunt the induction of COX-2 and suppress the formation of prostanoids. Modulation of cell receptor signaling pathways The severity of anthracycline cardiotoxicity is worsened by the combined administration of doxorubicin and the anti-HER-2/neu (ErbB-2) cytotoxic monoclonal antibody trastuzumab (92). Recent data demonstrate that in adult rat ventricular myocytes, the endocardialderived paracrine factor neuregulin-1␤ (NRG-1␤), an ErbB-2 ligand, significantly reduced doxorubicin-induced cellular injury by promoting Erk1/2-Akt phosphorylation and activating an anti-apoptotic pathway (93). In line with this, activation of glycoprotein 130 by leukemia inhibitory factor (LIF) mediates a cytoprotective effect against doxorubicin-induced apoptosis in cardiac myocytes due to the restoration of phosphatidylinositol (PI) 3-kinase/Akt activities (94), highlightNO AND ANTHRACYCLINE-INDUCED CARDIOTOXICITY

ing the pivotal role of this biochemical pathway in preserving cardiomyocyte integrity. At variance with NRG-1␤, treatment of rat ventriculocytes with antiErbB-2 antibody down-regulates ErbB2 receptors, has no effect on Erk1/2 or Akt phosphorylation and increases the number of myocytes showing cellular damage after doxorubicin treatment (93). In vitro findings on the increased susceptibility of myocardial cells to doxorubicin in the presence of ErbB-2 antibody are in line with the contractile dysfunction observed in patients receiving trastuzumab and anthracyclines. ErbB2/ErbB-4 heterodimer transmits NRG-1␤ signals in developing and adult hearts (95) and, despite the lack of direct evidence of ErbB-NO interaction in cardiomyocytes, heregulin post-transcriptionally enhances the activity of nNOS in rat cerebellar granule neurons via the ErbB-4 receptor-MAPK pathway (96). These findings, together with the localization of nNOS in the cardiac sarcoplasmic reticulum (11), suggest that the NO system may participate in the ErbB-2/anthracycline cross-talk in cardiac muscle. Drug treatment of patients with epidoxorubicin-induced dilated cardiomyopathy includes the administration of angiotensin-converting enzyme inhibitors (97). One potential benefit of inhibiting the formation of angiotensin (AT) II is the reduced stimulation of the AT-2 receptor pathway, which is associated with increased NO generation, intracellular concentrations of ceramide, and apoptosis (98).

PERSPECTIVES OF PHARMACOLOGIC MANIPULATION OF NO PATHWAY FOR CARDIOPROTECTION The data presented in the literature support the importance of prolonged heart exposure to reactive nitrogen species generated by anthracyclines in the pathogenesis of cardiac damage (30, 31, 99). Therefore, inhibition of NOS activity and/or scavenging toxic NO decomposition products may represent two potential strategies for rational development of novel cardioprotective agents. In the first approach, selective iNOS targeting may also reduce possible side effects that could occur as a result of inhibition of constitutive NOS. Indeed, the specific inhibitor of iNOS, aminoguanidine, decreases mortality rate, improves the histopathologic picture of rat hearts treated with doxorubicin (31, 100), and, at variance with N-methyl-l-arginine, which increases peripheral resistance, does not change systemic arterial blood pressure (101). Finally, the absence of significant hemodynamic alterations of L-N6-(1-iminoethyl)lysine 5-tetrazole amide in normal and asthmatic subjects (102) suggests that selective iNOS inhibitors may have therapeutic potential for treatment of diseases mediated by the overproduction of NO, including anthracycline-induced heart disease. Besides inhibiting NOS, ONOO– scavenging and inhibition of death pathways triggered by ONOO– may offer the advantage of preserving the beneficial effect 671

of NO production while neutralizing toxic molecules generated inside myocardial cells. In agreement with this hypothesis, cytokine-induced nitrite accumulation and myocyte apoptosis are significantly attenuated by the superoxide dismutase mimetic and ONOO– scavenger Mn(III)tetrakis (4-benzoic acid) porphyrin (22). The novel metalloporphyrinic catalyst of ONOO– decomposition, FP15, in the range of 0.03–1 mg/kg was able to reduce mortality and improve cardiac function in mouse models of doxorubicin-induced acute or chronic heart failure with no influence on antitumor activity of doxorubicin in breast carcinoma growth (31). Melatonin generates a free radical scavenging cascade reaction that is able to neutralize, at least in part, the toxic activity of OH䡠, H2O2 , NO, and ONOO– (103), a mechanism of action that fits well with the present hypothesis of NO contribution to anthracycline cardiotoxicity. Additional studies of cardiac protection by pharmacological targeting of NO and ROS have been reported. ATP depletion and activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) represent an important mechanism of ONOO–-induced cell death (26). Trimetazidine, a 3-keto-acyl CoA-thiolase inhibitor that raises myocyte ATP content and prevents ROS damage, improved the acute signs and symptoms and increased the systolic function in a breast cancer patient treated with epidoxorubicin after failure of dexrazoxane (104). Moreover, PARP inhibitors exert protective effects against the development of severe cardiac complications associated with doxorubicin treatment (105) whereas the natural compound chinonin decreases the percentage of apoptotic cardiomyocytes by scavenging NO and ROS radicals, and modulating the expression of Bcl-2 and p53 (106). Flavonoids are scavengers of ONOO– (107), and the synthetic derivative frederine proved to be active against doxorubicininduced cardiotoxicity (108).

stat mesylate (FUT-175) (110). Such a methodological approach may contribute to improve the therapeutic index of anthracyclines by identifying specific signaling pathways of cardiotoxicity distinct from those underlying the antitumor effects of these drugs.

REFERENCES 1.

2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

CONCLUSIONS

14.

The data of the literature agree with the notion that anthracyclines are able to trigger a cascade of NO and ROS production in cardiac cells. In the present review, we suggest that a multilevel molecular targeting of highly regulated signaling network, including the NO system, may represent a conceivable pathogenetic framework of anthracycline cardiotoxicity. Most important, the availability of novel differential gene expression technologies such as cDNA microarray analysis and proteomic platforms provides new avenues to rapidly obtain information in the context of cardiac disease. For instance, cardiochip monitoring in rats after myocardial ischemia and reperfusion injury contributed to the discovery of novel genes involved in the mechanism of preconditioning (109); in the same model system, the 2-dimensional analysis of myocardial protein expression has been applied to investigate the effect of the synthetic serine protease inhibitor futhan/nafam672

Vol. 18

April 2004

15. 16.

17.

18.

19. 20.

Wojtacki, J., Lewicka-Nowak, E., and Lesniewski-Kmak, K. (2000) Anthracycline-induced cardiotoxicity: clinical course, risk factors, pathogenesis, detection and prevention–review of the literature. Med. Sci. Monit. 6, 411– 420 Monneret, C. (2001) Recent developments in the field of antitumour anthracyclines. Eur. J. Med. Chem. 36, 483– 493 Danesi, R., Fogli, S., Gennari, A., Conte, P., and Del Tacca, M. (2002) Pharmacokinetic-pharmacodynamic relationships of the anthracycline anticancer drugs. Clin. Pharmacokinet. 41, 431– 444 Pai, V. B., and Nahata, M. C. (2000) Cardiotoxicity of chemotherapeutic agents: incidence, treatment and prevention. Drug Safety 22, 263–302 Lipshultz, S. (1996) Dexrazoxane for protection against cardiotoxic effects of anthracyclines in children. Editorial. J. Clin. Oncol. 14, 332–333 Pedersen-Bjergaard, J. (1992) The dioxopiperazine derivates, their leukemogenic potential and other biological effects. Leuk. Res. 16, 1057–1059 Massion, P. B., Moniotte, S., and Balligand, J. L. (2001) Nitric oxide: does it play a role in the heart of the critically ill? Curr. Opin. Crit. Care 7, 323–336 Balligand, J. L., and Cannon, P. J. (1997) Nitric oxide synthases and cardiac muscle. Autocrine and paracrine influences. Arterioscler. Thromb. Vasc. Biol. 17, 1846 –1858 Kelly, R. A., Balligand, J. L., and Smith, T. W. (1996) Nitric oxide and cardiac function. Circ. Res. 79, 363–380 Champion, H. C., Skaf, M. W., and Hare, J. M. (2003) Role of nitric oxide in the pathophysiology of heart failure. Heart Fail. Rev. 8, 35– 46 Xu, K. Y., Huso, D. L., Dawson, T. M., Bredt, D. S., and Becker, L. C. (1999) Nitric oxide synthase in cardiac sarcoplasmic reticulum. Proc. Natl. Acad. Sci. USA 96, 657– 662 Mungrue, I. N., Husain, M., and Stewart, D. J. (2002) The role of NOS in heart failure: lessons from murine genetic models. Heart Fail. Rev. 7, 407– 422 Davis, K. L., Martin, E., Turko, I. V., and Murad, F. (2001) Novel effects of nitric oxide. Annu. Rev. Pharmacol. Toxicol. 41, 203–236 Xu, L., Eu, J. P., Meissner, G., and Stamler, J. S. (1998) Activation of the cardiac calcium release channel (ryanodine receptor) by poly-S-nitrosylation. Science 279, 234 –237 Turko, I. V., and Murad, F. (2002) Protein nitration in cardiovascular diseases. Pharmacol. Rev. 54, 619 – 634 de Belder, A. J., Radomski, M. W., Why, H. J., Richardson, P. J., Bucknall, C. A., Salas, E., Martin, J. F., and Moncada, S. (1993) Nitric oxide synthase activities in human myocardium. Lancet 341, 84 – 85 Haywood, G. A., Tsao, P. S., von der Leyen, H. E., Mann, M. J., Keeling, P. J., Trindade, P. T., Lewis, N. P., Byrne, C. D., Rickenbacher, P. R., Bishopric, N. H., et al. (1996) Expression of inducible nitric oxide synthase in human heart failure. Circulation 93, 1087–1094 Lewis, N. P., Tsao, P. S., Rickenbacher, P. R., Xue, C., Johns, R. A., Haywood, G. A., von der Leyen, H., Trindade, P. T., Cooke, J. P., Hunt, S. A., et al. (1996) Induction of nitric oxide synthase in the human cardiac allograft is associated with contractile dysfunction of the left ventricle. Circulation 93, 720 –729 Lefer, A. M., Tsao, P. S., Lefer, D. J., and Ma, X. L. (1991) Role of endothelial dysfunction in the pathogenesis of reperfusion injury after myocardial ischemia. FASEB J. 5, 2029 –2034 Laurindo, F. R., da Luz, P. L., Uint, L., Rocha, T. F., Jaeger, R. G., and Lopes, E. A. (1991) Evidence for superoxide

The FASEB Journal

FOGLI ET AL.

21.

22.

23. 24.

25.

26. 27. 28.

29. 30.

31.

32.

33.

34. 35. 36.

37.

38.

39.

radical-dependent coronary vasospasm after angioplasty in intact dogs. Circulation 83, 1705–1715 Thomas, D. D., Espey, M. G., Vitek, M. P., Miranda, K. M., and Wink, D. A. (2002) Protein nitration is mediated by heme and free metals through Fenton-type chemistry: an alternative to the NO/O2– reaction. Proc. Natl. Acad. Sci. USA 99, 12691– 12696 Arstall, M. A., Sawyer, D. B., Fukazawa, R., and Kelly, R. A. (1999) Cytokine-mediated apoptosis in cardiac myocytes: the role of inducible nitric oxide synthase induction and peroxynitrite generation. Circ. Res. 85, 829 – 840 Hausladen, A., and Fridovich, I. (1994) Superoxide and peroxynitrite inactivate aconitases, but nitric oxide does not. J. Biol. Chem. 269, 29405–29408 Lizasoain, I., Moro, M. A., Knowles, R. G., Darley-Usmar, V., and Moncada, S. (1996) Nitric oxide and peroxynitrite exert distinct effects on mitochondrial respiration which are differentially blocked by glutathione or glucose. Biochem. J. 314, 877– 880 Mungrue, I. N., Gros, R., You, X., Pirani, A., Azad, A., Csont, T., Schulz, R., Butany, J., Stewart, D. J., and Husain, M. (2002) Cardiomyocyte overexpression of iNOS in mice results in peroxynitrite generation, heart block, and sudden death. J. Clin. Invest. 109, 735–743 Brown, G. C., and Borutaite, V. (2002) Nitric oxide inhibition of mitochondrial respiration and its role in cell death. Free Radic. Biol. Med. 33, 1440 –1450 Radi, R., Cassina, A., Hodara, R., Quijano, C., and Castro, L. (2002) Peroxynitrite reactions and formation in mitochondria. Free Radic. Biol. Med. 33, 1451–1464 Ramachandran, A., Levonen, A. L., Brookes, P. S., Ceaser, E., Shiva, S., Barone, M. C., and Darley-Usmar, V. (2002) Mitochondria, nitric oxide, and cardiovascular dysfunction. Free Radic. Biol. Med. 33, 1465–1474 Ing, D. J., Zang, J., Dzau, V. J., Webster, K. A., and Bishopric, N. H. (1999) Modulation of cytokine-induced cardiac myocyte apoptosis by nitric oxide, Bak, and Bcl-x. Circ. Res. 84, 21–33 Weinstein, D. M., Mihm, M. J., and Bauer, J. A. (2000) Cardiac peroxynitrite formation and left ventricular dysfunction following doxorubicin treatment in mice. J. Pharmacol. Exp. Ther. 294, 396 – 401 Pacher, P., Liaudet, L., Bai, P., Mabley, J. G., Kaminski, P. M., Virag, L., Deb, A., Szabo, E., Ungvari, Z., Wolin, M. S., et al. (2003) Potent metalloporphyrin peroxynitrite decomposition catalyst protects against the development of doxorubicininduced cardiac dysfunction. Circulation 107, 896 –904 Aldieri, E., Bergandi, L., Riganti, C., Costamagna, C., Bosia, A., and Ghigo, D. (2002) Doxorubicin induces an increase of nitric oxide synthesis in rat cardiac cells that is inhibited by iron supplementation. Toxicol. Appl. Pharmacol. 185, 85–90 Kalivendi, S. V., Kotamraju, S., Zhao, H., Joseph, J., and Kalyanaraman, B. (2001) Doxorubicin-induced apoptosis is associated with increased transcription of endothelial nitricoxide synthase. Effect of antiapoptotic antioxidants and calcium. J. Biol. Chem. 276, 47266 – 47276 Doroshow, J. H. (1983) Effect of anthracycline antibiotics on oxygen radical formation in rat heart. Cancer Res. 43, 460 – 472 Powis, G. (1989) Free radical formation by antitumor quinones. Free Radic. Biol. Med. 6, 63–101 Va´ squez-Vivar, J., Martasek, P., Hogg, N., Masters, B. S. S., Pritchard, K. A., and Kalyanaraman, B. (1997) Endothelial nitric oxide synthase-dependent superoxide generation from Adriamycin. Biochemistry 36, 11293–11297 Garner, A. P., Paine, M. J., Rodriguez-Crespo, I., Chinje, E. C., Ortiz De Montellano, P., Stratford, I. J., Tew, D. G., and Wolf, C. R. (1999) Nitric oxide synthases catalyze the activation of redox cycling and bioreductive anticancer agents. Cancer Res. 59, 1929 –1934 Gutierrez, J. A., Clark, S. G., Giulumian, A. D., and Fuchs, L. C. (1997) Superoxide anions contribute to impaired regulation of blood pressure by nitric oxide during the development of cardiomyopathy. J. Pharmacol. Exp. Ther. 282, 1643–1649 Danesi, R., Bernardini, N., Agen, C., Costa, M., Zaccaro, L., Pieracci, D., Malvaldi, G., and Del Tacca, M. (1992) Reduced cardiotoxicity and increased cytotoxicity in a novel anthracycline analogue, 4'-amino-3⬘-hydroxy-doxorubicin. Cancer Chemother. Pharmacol. 29, 261–265


40.

41.

42.

43.

44.

45. 46. 47.

48.

49. 50.

51. 52.

53.

54.

55. 56.

57.

58.

Vaziri, N. D., Ni, Z., Oveisi, F., and Trnavsky-Hobbs, D. L. (2000) Effect of antioxidant therapy on blood pressure and NO synthase expression in hypertensive rats. Hypertension 36, 957–964 Zhou, S., Heller, L. J., and Wallace, K. B. (2001) Interference with calcium-dependent mitochondrial bioenergetics in cardiac myocytes isolated from doxorubicin-treated rats. Toxicol. Appl. Pharmacol. 175, 60 – 67 Zhou, S., Starkov, A., Froberg, M. K., Leino, R. L., and Wallace, K. B. (2001) Cumulative and irreversible cardiac mitochondrial dysfunction induced by doxorubicin. Cancer Res. 61, 771–777 Kunisada, K., Tone, E., Negoro, S., Nakaoka, Y., Oshima, Y., Osugi, T., Funamoto, M., Izumi, M., Fujio, Y., Hirota, H., et al. (2002) Bcl-xl reduces doxorubicin-induced myocardial damage but fails to control cardiac gene downregulation. Cardiovasc. Res. 53, 936 –943 Childs, A. C., Phaneuf, S. L., Dirks, A. J., Phillips, T., and Leeuwenburgh, C. (2002) Doxorubicin treatment in vivo causes cytochrome C release and cardiomyocyte apoptosis, as well as increased mitochondrial efficiency, superoxide dismutase activity, and Bcl-2:Bax ratio. Cancer Res. 62, 4592– 4598 Olson, R. D., and Mushlin, P. S. (1990) Doxorubicin cardiotoxicity: analysis of prevailing hypotheses. FASEB J. 4, 3076 – 3086 Sun, X., and Kang, Y. J. (2002) Prior increase in metallothionein levels is required to prevent doxorubicin cardiotoxicity. Exp. Biol. Med. 227, 652– 657 Shioji, K., Kishimoto, C., Nakamura, H., Masutani, H., Yuan, Z., Oka, S., and Yodoi, J. (2002) Overexpression of thioredoxin-1 in transgenic mice attenuates adriamycin-induced cardiotoxicity. Circulation 106, 1403–1409 Minotti, G., Mancuso, C., Frustaci, A., Mordente, A., Santini, S. A., Calafiore, A. M., Liberi, G., and Gentiloni, N. (1996) Paradoxical inhibition of cardiac lipid peroxidation in cancer patients treated with doxorubicin. Pharmacologic and molecular reappraisal of anthracycline cardiotoxicity. J. Clin. Invest. 98, 650 – 661 Minotti, G., Cairo, G., and Monti, E. (1999) Role of iron in anthracycline cardiotoxicity: new tunes for an old song? FASEB J. 13, 199 –212 Menna, P., Salvatorelli, E., Giampietro, R., Liberi, G., Teodori, G., Calafiore, A. M., and Minotti, G. (2002) Doxorubicindependent reduction of ferrylmyoglobin and inhibition of lipid peroxidation: implications for cardiotoxicity of anticancer anthracyclines. Chem. Res. Toxicol. 15, 1179 –1189 Neuss, M., Crow, M. T., Chesley, A., and Lakatta, E. G. (2001) Apoptosis in cardiac disease-what is it-how does it occur. Cardiovasc. Drugs Ther. 15, 507–523 Arola, O. J., Saraste, A., Pulkki, K., Kallajoki, M., Parvinen, M., and Voipio-Pulkki, L. M. (2000) Acute doxorubicin cardiotoxicity involves cardiomyocyte apoptosis. Cancer Res. 60, 1789 – 1792 Nakamura, T., Ueda, Y., Juan, Y., Katsuda, S., Takahashi, H., and Koh, E. (2000) Fas-mediated apoptosis in adriamycininduced cardiomyopathy in rats: in vivo study. Circulation 102, 572–578 Kang, Y. J., Zhou, Z. X., Wang, G. W., Buridi, A., and Klein, J. B. (2000) Suppression by metallothionein of doxorubicin-induced cardiomyocyte apoptosis through inhibition of p38 mitogen-activated protein kinases. J. Biol. Chem. 275, 13690 – 13698 Bishopric, N. H., Andreka, P., Slepak, T., and Webster, K. A. (2001) Molecular mechanisms of apoptosis in the cardiac myocyte. Curr. Opin. Pharmacol. 1, 141–150 Wang, G. W., Klein, J. B., and Kang, Y. J. (2001) Metallothionein inhibits doxorubicin-induced mitochondrial cytochrome c release and caspase-3 activation in cardiomyocytes. J. Pharmacol. Exp. Ther. 298, 461– 468 Kumar, D., Kirshenbaum, L. A., Li, T., Danelisen, I., and Singal, P. K. (2001) Apoptosis in adriamycin cardiomyopathy and its modulation by probucol. Antioxid. Redox Signal. 3, 135–145 Koglin, J., Granville, D. J., Glysing-Jensen, T., Mudgett, J. S., Carthy, C. M., McManus, B. M., and Russell, M. E. (1999) Attenuated acute cardiac rejection in NOS2 ⫺/⫺ recipients correlates with reduced apoptosis. Circulation 99, 836 – 842

673

59.

60. 61. 62.

63.

64.

65.

66.

67.

68.

69.

70. 71.

72.

73. 74.

75. 76.

77.

78.

674

Maestre, N., Tritton, T. R., Laurent, G., and Jaffrezou, J. P. (2001) Cell surface-directed interaction of anthracyclines leads to cytotoxicity and nuclear factor kappaB activation but not apoptosis signaling. Cancer Res. 61, 2558 –2561 Laurent, G., and Jaffre´ zou, J. P. (2001) Signaling pathways activated by daunorubicin. Blood 98, 913–924 Di Paola, M., Cocco, T., and Lorusso, M. (2000) Ceramide interaction with the respiratory chain of heart mitochondria. Biochemistry 39, 6660 – 6668 Levade, T., Auge, N., Veldman, R. J., Cuvillier, O., NegreSalvayre, A., and Salvayre, R. (2001) Sphingolipid mediators in cardiovascular cell biology and pathology. Circ. Res. 89, 957– 968 Delpy, E., Hatem, S. N., Andrieu, N., de Vaumas, C., He´ naff, M., Rucker-Martin, C., Jaffrezou, J. P., Laurent, G., Levade, T., and Mercadier, J. J. (1999) Doxorubicin induces slow ceramide accumulation and late apoptosis in cultured adult rat ventricular myocytes. Cardiovasc. Res. 43, 398 – 407 Bose, R., Verheij, M., Haimovitz-Friedman, A., Scotto, K., Fuks, Z., and Kolesnick, R. (1995) Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals. Cell 82, 405– 414 Andrieu-Abadie, N., Jaffrezou, J. P., Hatem, S., Laurent, G., Levade, T., and Mercaider, J. J. (1999) L-Carnitine prevents doxorubicin-induced apoptosis of cardiac myocytes: role of inhibition of ceramide generation. FASEB J. 13, 1501–1510 Ruvolo, P. P., Deng, X., Ito, T., Carr, B. K., and May, W. S. (1999) Ceramide induces Bcl2 dephosphorylation via a mechanism involving mitochondrial PP2A. J. Biol. Chem. 274, 20296 – 20300 Ruvolo, P. P., Clark, W., Mumby, M., Gao, F., and May, W. S. (2002) A functional role for the B56 alpha-subunit of protein phosphatase 2A in ceramide-mediated regulation of Bcl2 phosphorylation status and function. J. Biol. Chem. 277, 22847– 22852 Martin, D., Salinas, M., Fujita, N., Tsuruo, T., and Cuadrado, A. (2002) Ceramide and reactive oxygen species generated by H2O2 induce caspase-3-independent degradation of Akt/protein kinase B. J. Biol. Chem. 277, 42943– 42952 He´ naff, M., Antoine, S., Mercadier, J. J., Coulombe, A., and Hatem, S. N. (2002) The voltage-independent B-type Ca2⫹ channel modulates apoptosis of cardiac myocytes. FASEB J. 16, 99 –101 Rabkin, S. W. (2002) Fumonisin blunts nitric oxide-induced and nitroprusside-induced cardiomyocyte death. Nitric Oxide 7, 229 –235 Keung, E. C., Toll, L., Ellis, M., and Jensen, R. A. (1991) L-type cardiac calcium channels in doxorubicin cardiomyopathy in rats morphological, biochemical, and functional correlations. J. Clin. Invest. 87, 2108 –2113 Shadle, S. E., Bammel, B. P., Cusack, B. J., Knighton, R. A., Olson, S. J., Mushlin, P. S., and Olson, R. D. (2000) Daunorubicin cardiotoxicity: evidence for the importance of the quinone moiety in a free-radical-independent mechanism. Biochem. Pharmacol. 60, 1435–1444 Saeki, K., Obi, I., Ogiku, N., Shigekawa, M., Imagawa, T., and Matsumoto, T. (2002) Doxorubicin directly binds to the cardiac-type ryanodine receptor. Life Sci. 70, 2377–2389 Gambliel, H. A., Burke, B. E., Cusack, B. J., Walsh, G. M., Zhang, Y. L., Mushlin, P. S., and Olson, R. D. (2002) Doxorubicin and C-13 deoxydoxorubicin effects on ryanodine receptor gene expression. Biochem. Biophys. Res. Commun. 291, 433– 438 Wang, G. X., Wang, Y. X., Zhou, X. B., and Korth, M. (2001) Effects of doxorubicinol on excitation-contraction coupling in guinea pig ventricular myocytes. Eur. J. Pharmacol. 423, 99 –107 Maczewski, M., and Beresewicz, A. (2003) Role of nitric oxide and free radicals in cardioprotection by blocking Na⫹/H⫹ and Na⫹/Ca2⫹ exchange in rat heart. Eur. J. Pharmacol. 461, 139 –147 Brunner, F., Andrew, P., Wolkart, G., Zechner, R., and Mayer, B. (2001) Myocardial contractile function and heart rate in mice with myocyte-specific overexpression of endothelial nitric oxide synthase. Circulation 104, 3097–3102 Mihm, M. J., Yu, F., Weinstein, D. M., Reiser, P. J., and Bauer, J. A. (2002) Intracellular distribution of peroxynitrite during doxorubicin cardiomyopathy: evidence for selective impair-

Vol. 18

April 2004

79.

80.

81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

ment of myofibrillar creatine kinase. Br. J. Pharmacol. 135, 581–588 Leandro, J., Dyck, J., Poppe, D., Shore, R., Airhart, C., Greenberg, M., Gilday, D., Smallhorn, J., and Benson, L. (1994) Cardiac dysfunction late after cardiotoxic therapy for childhood cancer. Am. J. Cardiol. 74, 1152–1156 Minotti, G., Cavaliere, A. F., Mordente, A., Rossi, M., Schiavello, R., Zamparelli, R., and Possati, G. (1995) Secondary alcohol metabolites mediate iron delocalization in cytosolic fractions of myocardial biopsies exposed to anticancer anthracyclines. Novel linkage between anthracycline metabolism and iron-induced cardiotoxicity. J. Clin. Invest. 95, 1595–1605 Minotti, G., Recalcati, S., Mordente, A., Liberi, G., Calafiore, A. M., Mancuso, C., Preziosi, P., and Cairo, G. (1998) The secondary alcohol metabolite of doxorubicin irreversibly inactivates aconitase/iron regulatory protein-1 in cytosolic fractions from human myocardium. FASEB J. 12, 541–552 Minotti, G., Ronchi, R., Salvatorelli, E., Menna, P., and Cairo, G. (2001) Doxorubicin irreversibly inactivates iron regulatory proteins 1 and 2 in cardiomyocytes: evidence for distinct metabolic pathways and implications for iron-mediated cardiotoxicity of antitumor therapy. Cancer Res. 61, 8422– 8428 Kwok, J. C., and Richardson, D. R. (2002) Unexpected anthracycline-mediated alterations in iron-regulatory protein-RNAbinding activity: the iron and copper complexes of anthracyclines decrease RNA-binding activity. Mol. Pharmacol. 62, 888 – 900 Cairo, G., Ronchi, R., Recalcati, S., Campanella, A., and Minotti, G. (2002) Nitric oxide and peroxynitrite activate the iron regulatory protein-1 of J774A.1 macrophages by direct disassembly of the Fe-S cluster of cytoplasmic aconitase. Biochemistry 41, 7435–7442 Kotamraju, S., Chitambar, C. R., Kalivendi, S. V., Joseph, J., and Kalyanaraman, B. (2002) Transferrin receptor-dependent iron uptake is responsible for doxorubicin-mediated apoptosis in endothelial cells: role of oxidant-induced iron signaling in apoptosis. J. Biol. Chem. 277, 17179 –17187 Wong, S. C., Fukuchi, M., Melnyk, P., Rodger, I., and Giaid, A. (1998) Induction of cyclooxygenase-2 and activation of nuclear factor-kappaB in myocardium of patients with congestive heart failure. Circulation 98, 100 –103 Adderley, S. R., and Fitzgerald, D. J. (1999) Oxidative damage of cardiomyocytes is limited by extracellular regulated kinases 1/2-mediated induction of cyclooxygenase-2. J. Biol. Chem. 274, 5038 –5046 Dowd, N. P., Scully, M., Adderley, S. R., Cunningham, A. J., and Fitzgerald, D. J. (2001) Inhibition of cyclooxygenase-2 aggravates doxorubicin-mediated cardiac injury in vivo. J. Clin. Invest. 108, 585–590 Shinmura, K., Xuan, Y. T., Tang, X. L., Kodani, E., Han, H., Zhu, Y., and Bolli, R. (2002) Inducible nitric oxide synthase modulates cyclooxygenase-2 activity in the heart of conscious rabbits during the late phase of ischemic preconditioning. Circ. Res. 90, 602– 608 Gunther, M. R., Hsi, L. C., Curtis, J. F., Gierse, J. K., Marnett, L. J., Eling, T. E., and Mason, R. P. (1997) Nitric oxide trapping of the tyrosyl radical of prostaglandin H synthase-2 leads to tyrosine iminoxyl radical and nitrotyrosine formation. J. Biol. Chem. 272, 17086 –17090 Vane, J. R., Mitchell, J. A., Appleton, I., Tomlinson, A., Bishop-Bailey, D., Croxtall, J., and Willoughby, D. A. (1994) Inducible isoforms of cyclooxygenase and nitric-oxide synthase in inflammation. Proc. Natl. Acad. Sci. USA 91, 2046 –2050 Seidman, A., Hudis, C., Pierri, M. K., Shak, S., Paton, V., Ashby, M., Murphy, M., Stewart, S. J., and Keefe, D. (2002) Cardiac dysfunction in the trastuzumab clinical trials experience. J. Clin. Oncol. 20, 1215–1221 Sawyer, D. B., Zuppinger, C., Miller, T. A., Eppenberger, H. M., and Suter, T. M. (2002) Modulation of anthracycline-induced myofibrillar disarray in rat ventricular myocytes by neuregulin1beta and anti-erbB2: potential mechanism for trastuzumabinduced cardiotoxicity. Circulation 105, 1551–1554 Negoro, S., Oh, H., Tone, E., Kunisada, K., Fujio, Y., Walsh, K., Kishimoto, T., and Yamauchi-Takihara, K. (2001) Glycoprotein 130 regulates cardiac myocyte survival in doxorubicin-induced apoptosis through phosphatidylinositol 3-kinase/akt phos-

The FASEB Journal

FOGLI ET AL.

95. 96.

97. 98.

99.

100.

101.

102.

103.

phorylation and bcl-xl/caspase-3 interaction. Circulation 103, 555–561 ¨ zcelik, C., and Birchmeier, C. (2003) ErbB2 Garratt, A. N., O pathways in heart and neural diseases. Trends Cardiovasc. Med. 13, 80 – 84 Krainock, R., and Murphy, S. (2001) Regulation of functional nitric oxide synthase-1 expression in cerebellar granule neurons by heregulin is post-transcriptional, and involves mitogenactivated protein kinase. J. Neurochem. 78, 552–559 Jensen, B. V., Nielsen, S. L., and Skovsgaard, T. (1996) Treatment with angiotensin-converting enzyme inhibitor for epirubicin-induced dilated cardiomyopathy. Lancet 347, 297–299 Berry, C., Touyz, R., Dominiczak, A. F., Webb, R. C., and Johns, D. G. (2001) Angiotensin receptors: signaling, vascular pathophysiology, and interactions with ceramide. Am. J. Physiol. Heart Circ. Physiol. 281, 2337–2365 Sayed-Ahmed, M. M., Khattab, M. M., Gad, M. Z., and Osman, A. M. (2001) Increased plasma endothelin-1 and cardiac nitric oxide during doxorubicin-induced cardiomyopathy. Pharmacol. Toxicol. 89, 140 –144 Mostafa, A. M., Nagi, M. N., Al Rikabi, A. C., Al-Shabanah, O. A., and El-Kashef, H. A. (1999) Protective effect of aminoguanidine against cardiovascular toxicity of chronic doxorubicin treatment in rats. Res. Commun. Mol. Pathol. Pharmacol. 106, 193–202 Doursout, M. F., Hartley, C. J., and Chelly, J. E. (2001) Comparison of cardiac and regional hemodynamic responses to N-methyl-L-arginine and aminoguanidine infusions in conscious pigs. J. Cardiovasc. Pharmacol. 37, 349 –358 Hansel, T. T., Kharitonov, S. A., Donnelly, L. E., Erin, E. M., Currie, M. G., Moore, W. M., Manning, P. T., Recker, D. P., and Barnes, P. J. (2003) A selective inhibitor of inducible nitric oxide synthase inhibits exhaled breath nitric oxide in healthy volunteers and asthmatics. FASEB J. 17, 1298 –1300 Tan, D. X., Reiter, R. J., Manchester, L. C., Yan, M. T., El-Sawi, M., Sainz, R. M., Mayo, J. C., Kohen, R., Allegra, M., and


104.

105.

106.

107. 108.

109.

110.

Hardeland, R. (2002) Chemical and physical properties and potential mechanisms: melatonin as a broad spectrum antioxidant and free radical scavenger. Curr. Top. Med. Chem. 2, 181–197 Pascale, C., Fornengo, P., Epifani, G., Bosio, A., and Giacometto, F. (2002) Cardioprotection of trimetazidine and anthracycline-induced acute cardiotoxic effects. Lancet 359, 1153– 1154 Pacher, P., Liaudet, L., Bai, P., Virag, L., Mabley, J. G., Hasko, G., and Szabo, C. (2002) Activation of poly(ADP-ribose) polymerase contributes to development of doxorubicin-induced heart failure. J. Pharmacol. Exp. Ther. 300, 862– 867 Shen, J. G., Quo, X. S., Jiang, B., Li, M., Xin, W., and Zhao, B. L. (2000) Chinonin, a novel drug against cardiomyocyte apoptosis induced by hypoxia and reoxygenation. Biochim. Biophys. Acta 1500, 217–226 Haenen, G. R., Paquay, J. B., Korthouwer, R. E., and Bast, A. (1997) Peroxynitrite scavenging by flavonoids. Biochem. Biophys. Res. Commun. 236, 591–593 van Acker, F. A., Boven, E., Kramer, K., Haenen, G. R., Bast, A., and van der Vijgh, W. J. (2001) Frederine, a new and promising protector against doxorubicin-induced cardiotoxicity. Clin. Cancer Res. 7, 1378 –1384 Onody, A., Zvara, A., Hackler, L., Jr., Vigh, L., Ferdinandy, P., and Puskas, L. G. (2003) Effect of classic preconditioning on the gene expression pattern of rat hearts: a DNA microarray study. FEBS Lett. 536, 35– 40 Schwertz, H., Langin, T., Platsch, H., Richert, J., Bomm, S., Schmidt, M., Hillen, H., Blaschke, G., Meyer, J., Darius, H., et al. (2002) Two-dimensional analysis of myocardial protein expression after myocardial ischemia and reperfusion in rabbits. Proteomics 2, 988 –995 Received for publication July 15, 2003. Accepted for publication December 18, 2003.

675