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PerspectiVe Anthracycline Degradation in Cardiomyocytes: A Journey to Oxidative Survival Pierantonio Menna, Emanuela Salvatorelli, and Giorgio Minotti* UniVersity Campus Bio-Medico, Fondazione Alberto Sordi-Research Institute on Aging, Rome, Italy ReceiVed September 21, 2009
The clinical use of doxorubicin (DOX) and other quinone-hydroquinone antitumor anthracyclines is limited by dose-related cardiotoxicity. One-electron redox cycling of the quinone moiety has long been known to form reactive oxygen species (ROS) in excess of the limited antioxidant defenses of cardiomyocytes; therefore, anthracycline cardiotoxicity was perceived as a one-way process in which redox cycling of the quinone always primed cardiomyocytes to oxidant stress and death. The past few years witnessed a growing interest in an alternative process in which peroxidases and quinone-derived hydrogen peroxide were able to oxidize the hydroquinone moiety of anthracyclines. Such a process was initially thought to amplify the cardiotoxicity induced by anthracyclines. Here, we briefly review how oxyferrous myoglobin could be subsequently identified as the primary catalyst of anthracycline oxidation in cardiomyocytes and be shown to induce an anthracycline chemical degradation that diminished the cellular levels and toxicity of active parent compounds. Many aspects of anthracycline degradation remain obscure or only partially understood; nevertheless, it is not too naı¨ve to conclude that anthracyclines are degraded and inactivated as a result of ROS production from their own redox cycling. Anthracycline redox reactions might therefore be viewed as two-way processes in which oxidative stress mediated both the death and survival of cardiomyocytes. Contents Anthracyclines and the Heart: Quinone Reduction, Quinone Reoxidation, and Cell Death From Quinone Oxidation/Reoxidation and Cell Death to Hydroquinone Oxidation and Anthracycline Degradation Identifying Catalysts and Products of Anthracycline Degradation: Myoglobin and Phthalates N-(t-Butyloxycarbonyl)alanine to Explore the Role of Anthracycline Degradation in Cell Survival Missing Information and New Concepts From Chemical Toxicology to Patients’ Health
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Anthracyclines and the Heart: Quinone Reduction, Quinone Reoxidation, and Cell Death The anthracyclines doxorubicin (DOX1) and daunorubicin (DNR) are tetracyclic DNA intercalating agents and topoisomerase II inhibitors that enjoy a venerable tradition in the treatment of many human tumors; unfortunately, however, their clinical use is limited by the possible development of a severe dose-related cardiotoxicity. Second-generation anthracyclines * To whom correspondence should be addressed. University Campus Bio-Medico, CIR and Drug Sciences, Via Alvaro del Portillo, 21, 00128 Rome, Italy. Phone: 011-39-06-225419109. Fax: 011-39-06-22541456. E-mail:
[email protected]. 1 Abbreviations: DOX, doxorubicin; DNR, daunorubicin; epirubicin, 4′epidoxorubicin; idarubicin, 4-demethoxydaunorubicin; O2•-, superoxide anion; H2O2, hydrogen peroxide; ROS, reactive oxygen species; NO2-, nitrite; MbIII, metmyoglobin; MbIIO2, oxyferrous myoglobin; MbIVdO, myoglobin compound II; t-BA, N-(t-butyloxycarbonyl)alanine.
Figure 1. Quinone-hydroquinone chromophore of anthracyclines, R1, -OCH3 (DOX, DNR, and epirubicin), or -H (idarubicin); R2, -CH2OH (DOX, epirubicin), or -CH3 (DNR, idarubicin); sugar, 3-amino-2,3,6trideoxy-R-L-lyxo-hexopyranose (DOX, DNR, and idarubicin), or 3-amino-2,3,6-trideoxy-R-L-arabino-hexopyranose (epirubicin).
such as 4′-epidoxorubicin (epirubicin) or 4-demethoxydaunorubicin (idarubicin) exhibit an improved cardiac tolerability, but neither can be said to have abated the risk of untoward cardiac effects. Chronic anthracycline administration sooner or later induces dilative cardiomyopathy and congestive heart failure that only transiently respond to cardiovascular medications. The mechanisms of anthracycline-related cardiotoxicity have been a matter of several investigations. Since the development and approval of these drugs, the vast majority of research was focused on the chemical reactivity of the quinone-hydroquinone chromophore of their tetracyclic ring system (Figure 1). Anthracyclines showed a unique propensity to undergo one-electron redox cycling in which the quinone was enzymatically reduced to a semiquinone, and the latter, regenerated its parent quinone by reducing oxygen to superoxide anion (O2·-) and hydrogen peroxide (H2O2). In the era of a booming interest in reactive oxygen species (ROS), anthracycline-induced cardiotoxicity became a shining star in the constellation of diseases and toxic reactions supposedly mediated by ROS and oxidative stress (1).
10.1021/tx9003424 2010 American Chemical Society Published on Web 12/02/2009
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Several lines of evidence made this picture more than plausible. Cardiomyocytes are exceptionally rich in mitochondria but relatively poor in ROS-detoxifying enzymes; thus, cardiomyocytes were seen as the perfect site for mitochondrial reductases to generate an anthracycline semiquinone and for the latter to generate ROS in excess of the cellular defense mechanisms. Moreover, little or no preclinical cardioxicity was seen with 5-iminodaunorubicin, an analogue in which the quinone oxygen had been replaced by an imino group. 5-Iminodaunorubicin never reached the stage of clinical development because of its untoward exacerbation of myelotoxicity, but at least, it provided a good example of how anthracyclines could be made unable to form ROS and cause toxicity to cardiomyocytes (2). In sum, biological and chemical reasonings led to the conclusion that anthracycline cardiotoxicity could be fully explained by a perfect string of quinone reduction, quinone reoxidation, and cell death. Whether the latter occurred by apoptosis, necrosis, or summation of sublethal dysfunctional damage would be beyond the aims of this brief perspective.
From Quinone Oxidation/Reoxidation and Cell Death to Hydroquinone Oxidation and Anthracycline Degradation As perfect as it could sound, the aforementioned string did not incorporate other possible mechanisms of chemical toxicity. For example, simple hydroquinone compounds had long been known to oxidize with peroxidases and form cytotoxic electrophilic quinones; whether this occurred with the hydroquinone of anthracyclines had not been explored or weighed against the toxic sequelae of quinone reduction/reoxidation. In Vitro studies showed that H2O2-activated horseradish peroxidases and other (pseudo)peroxidases did oxidize the hydroquinone moiety of anthracyclines, but this often required submillimolar nitrite (NO2-) to accelerate the catalytic turnover of peroxidases and form a radical (•NO2) that contributed on its own to oxidize anthracyclines; furthermore, anthracycline oxidation only occurred at neutral pH in the face of a preferred peroxidatic metabolism of NO2- at low pH values (3). These observations denoted that anthracycline oxidation was chemically feasible but not as facile as that of simple hydroquinones, presumably because the hydroquinone of anthracyclines was made oxidationresistant by the juxtaposed electrophilic quinone. Once these redox barriers had been surmounted through pH adjustment and NO2- addition, peroxidases could nonetheless induce a permanent loss of the optical and fluorescent properties of the quinone-hydroquinone chromophore, which was not rescued by canonical antioxidants (3). This was highly suggestive of irreversible oxidative modifications of anthracyclines. Was this the beginning of a new story? Were anthracyclines chemical targets of their own redox cycling and consequent H2O2 activation of cellular peroxidases? More importantly, was anthracycline oxidation bad or good for the heart? Very many technical problems made these questions difficult to answer. Much of the popularity of the quinone reduction, quinone reoxidation, and cell death hypothesis reflected on the ease with which the investigators could identify pertinent quinone reductases, visualize semiquinone formation, define the stoichiometry of quinone reoxidation/ROS formation, and develop techniques for measuring ROS-mediated damage. This was not the case for anthracycline oxidation. Which particular peroxidase would have been so good at oxidizing anthracyclines in cardiomyocytes without assistance from submillimolar NO2-? Did the anthracycline hydroquinone oxidize to a semiquinone liable to detection by available techniques? Finally, what was the fate
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of any such semiquinone? Elegant studies by Reszka and colleagues shed some light in this black box by answering the second question: in a test tube, lactoperoxidase/H2O2/NO2mixtures oxidized DNR to a semiquinone that probably disproportionated to generate a diquinone while also recycling the parent anthracycline (3). Diquinones are potent electrophiles that in principle attack -SH groups and many other cellular nucleophiles. The observations that horseradish peroxidase/H2O2/ anthracycline mixtures inactivated -SH dependent enzymes such as creatine kinase and succinate dehydrogenase were in keeping with these premises (4, 5), but whether these processes occurred in the cell milieu was much harder to accept. The ease with which peroxidases dissipated the quinone-hydroquinone chromophore attested to the instability of anthracycline diquinones; hence, any diquinone that formed too far from nucleophiles would undergo ring-opening and degradation before it caused oxidative damage, unless the degradation products were longlived and reactive enough to compensate for the diffusionlimited reactivity of unstable diquinones. Alternatively, the hydroquinone-derived semiquinone should be able to reduce oxygen to O2•-, but attempts to implicate O2•- in oxidative damage induced by horseradish peroxidase/H2O2/anthracycline mixtures were unsuccessful (4, 5). Bringing anthracycline oxidation into the arena of cardiotoxicity, therefore, urged unambiguous identification of both peroxidatic catalyst(s) and anthracycline degradation products. Was this reasonably possible?
Identifying Catalysts and Products of Anthracycline Degradation: Myoglobin and Phthalates In exploring the identity of possible catalysts of anthracycline degradation, we adopted the least elegant but most pragmatic approach we could: we went back to biochemistry textbooks and searched for the heme protein known to be most abundantly expressed in the heart. Next, we asked whether such a protein was also known for its authentic or pseudoperoxidatic activity. Myoglobin was the answer. The heart had long been known to contain 100 µM myoglobin, and H2O2 activation of myoglobin had long been known to form oxidants formally identical with compound I or II of authentic peroxidases. We probed this information: in a test tube, H2O2/metmyoglobin (MbIII) mixtures oxidized anthracyclines and did so more efficiently than authentic peroxidases, without pH barriers or requirement for NO2- (6, 7). We noted that anthracycline degradation only occurred when both H2O2 and MbIII were reacted with DOX or DNR; moreover, H2O2-activated MbIII bleached the quinonehydroquinone chromophore but never caused the formation of aglycons or other modified anthracycline products that retained an intact chromophore (7). These observations denoted differences from previous studies in which concentrated H2O2 spared the quinone-hydroquinone chromophore of anthracyclines but caused Baeyer-Villiger type oxidations at C14 to yield aglycons and, in the case of DOX, a carboxylic acid at C9 (8). Biochemistry textbooks could not help us in the search for anthracycline degradation products, but chemical references could. We retrieved reports of 3-methoxyphthalic acid formation during permanganate oxidation of monomethylated aloe-emodin, a tricyclic anthraquinone occasionally exploited as a synthon for the regiospecific synthesis of DOX (9); in the opposite direction, we retrieved reports that 3-methoxyphthalic acid and other phthalates could be used as starting materials or detected as key intermediates in the synthesis of methoxy substituted or unsubstituted anthraquinones (10). These references identified (3-methoxy)phthalates as facile precursors or products of anthracycline assembly or disassembly; therefore, we searched
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for 3-methoxyphthalic acid or simple phthalates as biochemical markers of anthracycline oxidative degradation. This search was successful: in test tubes containing H2O2/MbIII/anthracycline mixtures, 3-methoxyphthalic acid or simple phthalate accounted for ∼10 to ∼20% of the anthracycline material degraded, and recovering simple or methoxy-substituted phthalates only depended on whether we used anthracyclines that lacked or contained a methoxy group in their D ring (7) (see also R1 in Figure 1). Adding cell culture media with low to high concentrations of purifed 3-methoxyphthalic acid never caused toxicity as compared with equimolar undegraded DOX, thus anticipating that in all likelihood anthracycline degradation products were biologically harmless (7). At a glance, this was the beginning of the long-sought new story. We had the reaction catalyst (H2O2-activated MbIII), the reaction intermediates (anthracycline semiquinone and diquinone), and an apparently nontoxic reaction product (3-methoxyphthalic acid, oxidized remnant of ring D); everything looked well set to move to elaborate models and monitor the kinetics and consequences of anthracycline degradation that occurred inside the cardiomyocytes. Regrettably this was not the case. 3-Methoxyphthalic acid was formed by H2O2-activated MbIII, formally identical with a peroxidase compound I characterized by the presence of two oxidizing equivalents: a long-lived ironoxo moiety (FeIVdO) and a short-lived porphyrin π-cation radical that dissipates in globin in the form of aminoacid radicals (11). In contrast, 3-methoxyphthalic acid was not formed by H2O2activated oxyferrous myoglobin (MbIIO2), formally identical with a peroxidase compound II characterized by FeIVdO only (11, 12). While it is certainly true that DOX could accelerate the autoxidation of some MbIIO2 to MbIII (13) and favor hydroquinone oxidation by H2O2/MbIII (7), the vast majority of DOX would undoubtedly oxidize with H2O2/MbIIO2 and give degradation products that escape identification. Oxidation of DOX with H2O2/MbIII only occurred if animals were given anthracycline dosages many times higher than those adopted in humans and capable of accelerating the autoxidation of MbIIO2 to MbIII (7, 12).
N-(t-Butyloxycarbonyl)alanine to Explore the Role of Anthracycline Degradation in Cell Survival Therefore, what do we do next for clarifying the role of anthracycline degradation in cardiac cells exposed to pharmacokinetically relevant concentrations of, for example, DOX? We considered using cardiomyocytes that lacked or overexpressed myoglobin, but either approach had been shown to render cardiomyocytes hypersensitive to stressor agents. We did not want this; we needed a model in which mechanisms and consequences of anthracycline degradation could be defined in a normal context and disentangled from other ongoing reactions. For analogous reasons, we rejected using heme poisons or ligands, or antioxidants that spuriously competed with the compound II of myoglobin while also scavenging ROS. With no help from chemical references or previously published reports, we began screening for compounds that could selectively impede DOX oxidation with H2O2/MbIIO2 without exposing cells to side effects (12). One may think it was easier said than done, but it worked. In probing several candidate molecules in a test tube or in cell cultures, we found that N-(t-butyloxycarbonyl)alanine (t-BA) was quite good at inhibiting DOX oxidation by H2O2/MbIIO2 but not H2O2/MbIII or H2O2-activated horseradish peroxidase, lactoperoxidase, hemin, or cytochrome c (12). We did not know how precisely t-BA worked, but some hints were nonetheless at hand. We considered that anthracy-
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clines are bulky molecules which would not approach the heme pocket that accommodates the hypervalent iron of myoglobin compound II (MbIVdO); anthracyclines probably interact with myoglobin sites at a distance from heme and oxidize with its compound II through electron tunneling mechanisms (14). Moreover, we noticed that t-BA not only prevented dissipation of the quinone-hydroquinone chromophore but also blocked the decay of MbIVdO to MbIII, which otherwise accompanied oxidation of the hydroquinone with compound II (12). We therefore concluded that t-BA neither decomposed H2O2 before it oxidized MbIIO2 to compound II nor reduced compound II before it oxidized DOX; t-BA probably acted by introducing sterical constraints that impeded reactions of DOX with MbIVdO. Regardless of the precise mechanisms and determinants of such sterical interferences, t-BA inhibition of DOX degradation always increased the steady-state levels of undegraded DOX in a test tube or isolated cardiomyocytes; in the latter, DOX accumulation was accompanied by the exacerbation of toxicity (12). These results provided plausible evidence that anthracycline degradation served a salvage pathway for diminishing the levels and toxicity of DOX in cardiomyocytes.
Missing Information and New Concepts The anthracycline degradation story clearly introduces new issues in both preclinical and clinical settings. t-BA came to our attention quite empirically, but the efficacy with which it aggravated cardiotoxicity raises concerns that many drugs administered in combination with DOX or DNR might well induce t-BA-like effects and expose the hearts of cancer patients to higher levels of anthracyclines. We do not know which particular drug would be able to do so, but oncologists know that multiagent therapies often induce more cardiotoxicity than one would expect on the basis of the cumulative dose of DOX administered to cancer patients (15). Whether cardiotoxicity from multiagent therapies is aggravated by an unrecognized inhibition of anthracycline degradation is an attractive hypothesis that needs to be explored in preclinical models and then in patients. For readers conversant in chemical toxicology, anthracycline degradation probably sounds more a curiosity than a paradigm. After so many ruminations, too many actors remain in the shadows: (i) catalysts and substrates have been identified, but reaction products often remain undefined; (ii) t-BA proved useful to modulate anthracycline degradation, but biomolecules or drugs with t-BA-like effects await identification; (iii) myoglobin is surprisingly good at degrading anthracyclines, but anthracycline-induced modifications of its structure and functions have not been explored; (iv) 3-methoxyphthalic acid formation by H2O2/MbIII but not H2O2/MbIIO2 clearly denotes that different oxidants operate different mechanisms of anthracycline degradation. Finally, little is known about anthracycline degradation in tumors. Many tumor cells secrete peroxidases or get surrounded by inflammatory cells that secrete peroxidases while also producing H2O2 and NO2-. Would H2O2, NO2-, and peroxidases reach levels high enough to degrade anthracyclines in interstitial fluids before they diffused in cancer cells (16, 17)? Would anthracycline degradation diminish both antitumor activity and cardiotoxicity, thereby attenuating both the beneficial and detrimental effects of these drugs in patients? The aforementioned considerations caution against generalizations. We only know that anthracycline degradation may occur inside cardiomyocytes and protects them from toxicity; we only see the top of the iceberg. This having been said, we would emphasize that anthracycline degradation provides a good
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cardiotoxicity, which is consistent with the limited preventative efficacy of antioxidants in some preclinical studies and clinical trials (21, 22). Free radical-independent mechanisms of toxicity, possibly mediated by the accumulation of undegraded anthracycline (23), may include chaotropic effects on mitochondria (24) and direct inactivation of energy metabolism enzymes (25) and changes in the cardiac-specific gene expression program (26).
From Chemical Toxicology to Patients’ Health Very many patients are at risk for anthracycline cardiotoxicity. In the United States, more than 2 million breast cancer survivors can be considered to carry a high probability of anthracycline exposure and a lifetime risk for anthracycline-related cardiac events (27). At the moment, the most successful approach to diminish cardiotoxicity but not antitumor activity consists of replacing free DOX with liposomal formulations that are small enough to cross the discontinuous endothelium of tumors but are too big to cross the endothelial lining of coronary vessels; thus, liposomal DOX gains access to cancer cells but not cardiomyocytes (19). As complicated or poorly characterized as it may sound, anthracycline degradation would be the only chemical toxicology-oriented strategy for diminishing free DOX once it has entered cardiomyocytes. We only need to identify means for accelerating DOX oxidation with H2O2/MbIIO2 or preventing concomitant drugs from blocking such an oxidation; alternatively, we only need to identify DOX analogues that formed sufficient H2O2 for activating MbIIO2 but showed a facilitated oxidation of their hydroquinone. Easier said than done or easier done than feared? We might ask textbooks or chemical references: they may be generous in suggestions.
Figure 2. Anthracycline redox reactions: life versus death. One-electron reduction of the quinone moiety to a semiquinone, as mediated by NAD(P)H oxidoreductases, is followed by the formation of ROS that may prime cardiomyocytes to death. Hydrogen peroxide activation of MbIIO2 to a compound II-like species (MbIVdO) causes oxidation of the hydroquinone moiety to a semiquinone whose disproportionation recycles the hydroquinone and generates a diquinone; degradation of this latter is a salvage pathway to life. Hydroquinone oxidation by MbIVdO is inhibited by N-(t-butyloxycarbonyl)alanine (t-BA). Shaded boxes indicate the quinone-derived semiquinone (I), the hydroquinonederived semiquinone (II), and the diquinone (III).
example of how of one size does not fit all. Anthracyclines have been plagued by their infamous avidity for redox cycling and ROS formation that is aggravated by the inactivation of iron regulatory proteins and possible upset of metabolic disturbances or oxidative damage mediated by iron (18, 19). Nevertheless, the story reported in this perspective shows that quinone reduction/reoxidation does not always mean death; it could also mean life if H2O2 were diverted to activate myoglobin and degrade anthracyclines by oxidative mechanisms that pave the road to cardiomyocyte survival (Figure 2). Studies of cardiomyocytes exposed to anthracyclines and t-BA also showed that accumulated undegraded anthracyclines were less prone to cause ROS formation and iron-mediated oxidative stress to cell constituents such as, for example, polyunsaturated fatty acids; however, accumulated undegraded anthracyclines still aggravated toxicity, as if a concentration threshold existed above which anthracyclines changed their intracellular distribution and/ or mechanisms of action (20). Exploring anthracycline degradation, therefore, uncovered oxidant-independent mechanisms of
Acknowledgment. Work in the authors’ laboratory was supported, in part, by Associazione Italiana per la Ricerca sul Cancro and University Campus Bio-Medico (Special Project Cardio-Oncology).
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(10) Kim, K. S., Vanotti, E., Suarato, A., and Johnson, F. (1979) Anthracyclines and related substances. 2. An efficient and regiospecific synthesis of DL-7,9dideoxydaunomycinone. J. Am. Chem. Soc. 101, 2483–2484. (11) Egawa, T., Shimada, H., and Ishimura, Y. (2000) Formation of compound I in the reaction of native myoglobins with hydrogen peroxide. J. Biol. Chem. 275, 34858–34866. (12) Menna, P., Salvatorelli, E., and Minotti, G. (2007) Doxorubicin degradation in cardiomyocytes. J. Pharmacol. Exp. Ther. 322, 408– 419. (13) Trost, L. C., and Wallace, K. B. (1994) Adriamycin-induced oxidation of myoglobin. Biochem. Biophys. Res. Commun. 204, 30–37. (14) Yackzan, K. S., and Wingo, W. J. (1982) Transport of fatty acids by myoglobin: A hypothesis. Med. Hypotheses 8, 613–618. (15) Menna, P., Salvatorelli, E., and Minotti, G. (2008) Cardiotoxicity of antitumor drugs. Chem. Res. Toxicol. 15, 1179–1189. (16) Reszka, K. J., Wagner, B. A., Teesch, L. M., Britigan, B. E., Spitz, D. R., and Burns, C. P. (2005) Inactivation of anthracyclines by cellular peroxidases. Cancer Res. 65, 6346–6353. (17) Wagner, B. A., Teesch, L. M., Buettner, G. R., Britigan, B. E., Burns, C. P., and Reszka, K. J. (2007) Inactivation of anthracyclines by serum heme proteins. Chem. Res. Toxicol. 20, 920–926. (18) Minotti, G., Recalcati, S., Menna, P., Salvatorelli, E., Corna, G., and Cairo, G. (2004) Doxorubicin cardiotoxicity and the control of iron metabolism: Quinone dependent and independent mechanisms. Methods Enzymol. 378, 340–361. (19) Minotti, G., Menna, P., Salvatorelli, E., Cairo, G., and Gianni, L. (2004) Anthracyclines: Molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol. ReV. 56, 185– 229.
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