Iron chelation-afforded cardioprotection against chronic anthracycline cardiotoxicity: A study of salicylaldehyde isonicotinoyl hydrazone (SIH)
Introduction
Cardiotoxicity ranks among the most serious and hardly manageable toxic effects of anticancer chemotherapy. It is particularly dangerous due to the significant vulnerability of the cardiomyocytes to the oxidative damage, a very limited regenerative potential of the myocardial tissue, and the cardinal physiological importance of the heart. Besides some novel anticancer drugs, which have been only recently recognized as potentially cardiotoxic (e.g., imatinib, trastuzumab) (Ewer et al., 2005, Kerkela et al., 2006), anthracyclines (e.g., doxorubicin and daunorubicin) have remained for more than 40 years the main threat regarding the cardiac safety of the patients undergoing cancer treatment (Minotti et al., 2004, Jones et al., 2006).
From the clinical point of view, it is the chronic type of anthracycline cardiotoxicity which represents the major problem. Its risk is known to be closely related to the cumulative dose of anthracyclines (Von Hoff et al., 1979). The anthracycline-induced myocardial injury is typically characterized by a progressive myofibrillar loss and degeneration of the left ventricular (LV) cardiomyocytes ultimately resulting in dilated cardiomyopathy and congestive heart failure (Herman et al., 1997). In addition, the cardiac injury may manifest itself even after a considerable delay (4–20 years) and therefore it can compromise the cardiac function of survivors of childhood cancers even during their productive adulthood (Lipshultz et al., 1991).
Despite extensive scientific efforts, the molecular basis of anthracycline cardiotoxicity remains enigmatic, which significantly hampers the rational development of both less cardiotoxic anthracycline derivates as well as effective pharmacological cardioprotectants. Albeit many theories of anthracycline cardiotoxicity has been proposed (Minotti et al., 2004), the most widely accepted is the “ROS and iron hypothesis” (Keizer et al., 1990). The anthracyclines are known to generate ROS by a redox-cycling of the anthracycline aglycon as well as through their complexes with iron (Keizer et al., 1990, Minotti et al., 2004). The quinone part of the anthracycline aglycon can be converted into a semiquinone form by the one electron acquisition. In the further step of cyclic reaction, the superoxide anion radicals are formed by the interaction of semiquinone with molecular oxygen. Superoxide can further dismutate, either spontaneously or enzymatically (by superoxide dismutase) forming the hydrogen peroxide which is in turn converted in to the extremely reactive hydroxyl radicals through iron-catalyzed Haber–Weiss reaction. Furthermore, iron is involved in the anthracycline-induced ROS generation as part of Fe3+-anthracycline complex. In the presence of the reducing systems (e.g., NADH cytochrom P-450 reductase or glutathione), Fe3+-anthracycline complexes can be reduced and the products (Fe2+-anthracycline complexes) are promptly oxidized back via the interaction with molecular oxygen yielding again superoxide radicals. In addition, hydrogen peroxide can also participate in the latter reaction which results into the direct formation of hydroxyl radicals (Keizer et al., 1990). Alternatively, an internal electron shift within the iron–anthracycline complex may give reduction of iron, which subsequently delivers this electron to molecular oxygen, yielding further superoxide anions. In the excess of NO, the superoxide radicals can also react with NO and thus nitrogen reactive species (e.g., peroxynitrite) are formed (Weinstein et al., 2000; Fogli et al., 2004; Pacher et al., 2007).
The only cardioprotective drug with well-evidenced efficacy in both experimental and clinical settings is dexrazoxane – ICRF-187 (Cvetkovic and Scott, 2005). Today it is generally believed that dexrazoxane acts as a pro-drug which is bioactivated to the metal chelating compound ADR-925. The latter agent has been shown to chelate free or loosely bound iron within the cardiac cells and/or displace iron from its complexes with anthracyclines and thus it prevents the formation of reactive oxygen species, particularly the extremely toxic hydroxyl radicals (Hasinoff et al., 1998, Kwok and Richardson, 2000). Unfortunately, the clinical effectiveness of dexrazoxane is partially counterbalanced by its myelotoxicity which may aggravate the myelosuppression induced by anthracyclines and by its higher costs (Cvetkovic and Scott, 2005). Furthermore, there was a suspicion of decreased tumor response in dexrazoxane co-treated patients in one arm of a single clinical study (Swain et al., 1997), although other studies did not confirm this (Swain and Vici, 2004). All the arguments given above may be responsible for the recent estimation that in Europe cardioprotection with dexrazoxane is employed only in 6–7% of anthracycline-treated patients (Swain and Vici, 2004).
Although iron chelation seems to be a plausible mechanism of cardioprotective intervention against anthracycline cardiotoxicity, the potential of this approach was not appropriately appraised yet. So far, only several iron chelating agents have been examined (Link et al., 1996, Saad et al., 2001, Barnabe et al., 2002, Hasinoff et al., 2003, Xu et al., 2006) and, importantly, only very few in vivo studies on the relevant chronic models have been performed (Herman et al., 1994, Herman et al., 1997, Bruynzeel et al., 2006). Interestingly, the well-known iron chelator deferoxamine failed to protect against chronic anthracycline treatment, which was explained by the hydrophilicity of this compound preventing its sufficient distribution into the heart cells (Herman et al., 1994). On the other hand, we have recently shown that the lipophilic aroylhydrazone iron chelator pyridoxal 2-chlorbenzoylhydrazone (o-108) has a significant potential to decrease the toxicity of anthracyclines on the validated model of chronic daunorubicin cardiotoxicity in rabbits (Sterba et al., 2006). However, surprisingly, the cardioprotection disappeared with a higher dose of o-108 which raised certain controversy concerning both the latter agent as well as other pyridoxal-derived aroylhydrazones, including the PIH—a “mother compound” of this class of iron chelators (Simunek et al., 2005a).
In the present study, we have focused on a salicylaldehyde-derived chelator, salicylaldehyde isonicotinoyl hydrazone (SIH, Fig. 1), which has been recently shown to have very strong and concentration-dependent antioxidant properties on the in vitro model of hydrogen peroxide-induced oxidative stress in cardiac cells (Simunek et al., 2005b). The main aim of the present study is therefore to establish whether SIH is able to elicit significant cardioprotection against anthracycline cardiotoxicity both in vitro, using isolated rat ventricular cardiomyocytes, and in vivo, employing the rabbit model of chronic daunorubicin-induced cardiomyopathy (Gersl and Hrdina, 1994, Simunek et al., 2004).
Section snippets
Animals
Adult Chinchilla male rabbits (n = 64) of an average initial weight of 3.52 ± 0.05 kg were housed under a 12 h light cycle, constant temperature and humidity. The animals had free access to water and a standard laboratory pellet diet. Before experimental procedures the animals were fasting overnight. All experiments were performed under ketamine anesthesia (50 mg/kg, i.m.). Final invasive hemodynamic measurements were carried out under pentobarbital anesthesia (30 mg/kg, i.v.). All experiments were
Cardioprotection studies in vitro
Daunorubicin-treated cultured cardiomyocytes initially increased beating rate, which was followed by irregular activity, disruption of the cellular monolayer and eventually contractile arrest. NVCM generally enlarged cellular volume and the nuclear structure became distinctive. Cytoplasmic vacuolization and granulation were observed. Eventually, formation of cell debris was conspicuous (Fig. 2). Epifluorescence microscopy revealed marked accumulation of daunorubicin into the cardiomyocytes:
Discussion
Despite the decades of intensive research and development, dexrazoxane, a prodrug of the iron-chelating compound ADR-925, has remained the only drug with clearly proven ability to significantly decrease the risk and severity of chronic anthracycline cardiotoxicity. Interestingly, although numerous antioxidants were reported as encouraging in the models of acute toxicity (Fujita et al., 1982, Yoda et al., 1986, Tesoriere et al., 1994), they were shown to have only weak, partial or no
Acknowledgements
The authors wish to thank Mrs. Ludmila Latýnová for her skilful technical assistance during the whole study and Assoc. Prof. Bohuslav Mánek for his kind review of English. This study was supported by Research Projects of the Czech Ministry of Education Youth and Sports MSM0021620820 and Czech Ministry of Health MZO 00179906 and a grant from the Czech Science Foundation (GACR 305/05/P156).
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