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Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Pisa, Italy
1Correspondence: Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Via Bonanno, 6, Pisa, PI 56126 Italy. E-mail: s.fogli{at}do.med.unipi.it
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ABSTRACT |
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 TOP ABSTRACT BACKGROUNDCELLULAR BIOCHEMISTRY AND...THE POTENTIAL ROLE OF...ANTHRACYCLINE-INDUCED RELEASE OF...INTEGRATION OF NO WITH...PERSPECTIVES OF PHARMACOLOGIC...CONCLUSIONSREFERENCES |
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Key Words: doxorubicin • NO • apoptosis • cardioprotection
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BACKGROUND |
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TOPABSTRACTBACKGROUNDCELLULAR BIOCHEMISTRY AND...THE POTENTIAL ROLE OF...ANTHRACYCLINE-INDUCED RELEASE OF...INTEGRATION OF NO WITH...PERSPECTIVES OF PHARMACOLOGIC...CONCLUSIONSREFERENCES |
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.
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 possible molecular mechanisms of drug damage, focusing on the deregulation of NO system, in order to devise novel pharmacological strategies against anthracycline cardiotoxicity.
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CELLULAR BIOCHEMISTRY AND FUNCTIONS OF NO |
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TOPABSTRACTBACKGROUNDCELLULAR BIOCHEMISTRY AND...THE POTENTIAL ROLE OF...ANTHRACYCLINE-INDUCED RELEASE OF...INTEGRATION OF NO WITH...PERSPECTIVES OF PHARMACOLOGIC...CONCLUSIONSREFERENCES |
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9
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
).
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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 ß-adrenergic receptors and L-type Ca2+ channels into caveolae allows NO to modulate ß-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 diffusible 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 µM) 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 µM) is available to interact with molecular oxygen (O2) and anion superoxide (O2–·), 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-S-nitrosylation 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)
.
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THE POTENTIAL ROLE OF NO IN HEART PATHOLOGY |
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TOPABSTRACTBACKGROUNDCELLULAR BIOCHEMISTRY AND...THE POTENTIAL ROLE OF...ANTHRACYCLINE-INDUCED RELEASE OF...INTEGRATION OF NO WITH...PERSPECTIVES OF PHARMACOLOGIC...CONCLUSIONSREFERENCES |
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, 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–·) (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·) (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–· 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 contribution 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
23
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
27
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.
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ANTHRACYCLINE-INDUCED RELEASE OF NO IN CARDIAC CELLS |
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TOPABSTRACTBACKGROUNDCELLULAR BIOCHEMISTRY AND...THE POTENTIAL ROLE OF...ANTHRACYCLINE-INDUCED RELEASE OF...INTEGRATION OF NO WITH...PERSPECTIVES OF PHARMACOLOGIC...CONCLUSIONSREFERENCES |
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. 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 µM 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–·). O2–· 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–· 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)
.
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Regarding chronic cardiotoxicity, prolonged anthracycline exposure may induce a large synthesis of by-products 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–· 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–· 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 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)
.
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INTEGRATION OF NO WITH OTHER MECHANISMS OF HEART DAMAGE BY ANTHRACYCLINES |
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TOPABSTRACTBACKGROUNDCELLULAR BIOCHEMISTRY AND...THE POTENTIAL ROLE OF...ANTHRACYCLINE-INDUCED RELEASE OF...INTEGRATION OF NO WITH...PERSPECTIVES OF PHARMACOLOGIC...CONCLUSIONSREFERENCES |
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). Here, we examine the points of contact between NO and the major mechanisms of heart damage induced by anthracycline.
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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-in-duced 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· and converts it into O2–· (49)
; such an increase in O2–· 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 evidence suggest that apoptosis plays an important role in the loss of myocardial function due to anthracycline treatment (52
53
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 mitochondria-dependent 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 µM (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 p53-independent pathway activates the transcription of the Bax proapoptotic gene. Finally, in neonatal Bcl-xl-overexpressing rat cardiac myocytes treated with 1 µM 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 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 µM 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 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 µM 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 Ca2+ 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 µM) 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· 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
81
82
83)
(Fig. 4)
. 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 IRP-RNA binding and affects cellular iron metabolism.
The peroxynitrite ONOO– and the hydroxyl radical OH· 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 µM 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 µM for 80 min or 17.2 µM for 24 h, no change in COX-1 expression was detected whereas COX-2 and its 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 endocardial-derived paracrine factor neuregulin-1ß (NRG-1ß), 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)
, highlighting the pivotal role of this biochemical pathway in preserving cardiomyocyte integrity. At variance with NRG-1ß, treatment of rat ventriculocytes with anti-ErbB-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. ErbB-2/ErbB-4 heterodimer transmits NRG-1ß 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)
.
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PERSPECTIVES OF PHARMACOLOGIC MANIPULATION OF NO PATHWAY FOR CARDIOPROTECTION |
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TOPABSTRACTBACKGROUNDCELLULAR BIOCHEMISTRY AND...THE POTENTIAL ROLE OF...ANTHRACYCLINE-INDUCED RELEASE OF...INTEGRATION OF NO WITH...PERSPECTIVES OF PHARMACOLOGIC...CONCLUSIONSREFERENCES |
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, 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 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·, 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 doxorubicin-induced cardiotoxicity (108)
.
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TOPABSTRACTBACKGROUNDCELLULAR BIOCHEMISTRY AND...THE POTENTIAL ROLE OF...ANTHRACYCLINE-INDUCED RELEASE OF...INTEGRATION OF NO WITH...PERSPECTIVES OF PHARMACOLOGIC...CONCLUSIONSREFERENCES |
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; 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/nafamstat 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.
Received for publication July 15, 2003. Accepted for publication December 18, 2003.
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TOPABSTRACTBACKGROUNDCELLULAR BIOCHEMISTRY AND...THE POTENTIAL ROLE OF...ANTHRACYCLINE-INDUCED RELEASE OF...INTEGRATION OF NO WITH...PERSPECTIVES OF PHARMACOLOGIC...CONCLUSIONSREFERENCES |
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