Doxorubicin

Doxorubicin: The Good, the Bad and the Ugly Effect

Cristina Carvalho1,2, Renato X. Santos1,2, Susana Cardoso1,2, Sónia Correia1,2, Paulo J. Oliveira1,2, Maria S. Santos1,2 and Paula I. Moreira*,1,3
1Center for Neuroscience and Cell Biology; 2Department of Zoology – Faculty of Sciences and Technology; 3Institute of Physiology – Faculty of Medicine, University of Coimbra, 3000-354 Coimbra, Portugal
Abstract: The anthracycline doxorubicin (DOX) is widely used in chemotherapy due to its efficacy in fighting a wide range of cancers such as carcinomas, sarcomas and hematological cancers. Despite extensive clinical utilization, the mechanisms of action of DOX remain under intense debate. A growing body of evidence supports the view that this drug can be a double-edge sword. Indeed, injury to nontargeted tissues often complicates cancer treatment by limiting therapeu- tic dosages of DOX and diminishing the quality of patients’ life during and after DOX treatment. The literature shows that the heart is a preferential target of DOX toxicity. However, this anticancer drug also affects other organs like the brain, kidney and liver. This review is mainly devoted to discuss the mechanisms underlying not only DOX beneficial effects but also its toxic outcomes. Additionally, clinical studies focusing the therapeutic efficacy and side effects of DOX treat- ment will be discussed. Finally, some potential strategies to attenuate DOX-induced toxicity will be debated.
Keywords: Brain, doxorubicin, heart, kidney, liver, therapeutic effects, side effects.

1. INTRODUCTION

The US Food and Drug Administration (FDA) has ap- proved 132 anticancer drugs, being anthracyclines the most widely used. Considered a mainstay of therapy for several decades, conventional anthracycline–containing regimens have demonstrated benefits in terms of response rate, time to disease progression, and overall survival [1].

The first two anthracyclines were isolated from the pig- ment-producing Streptomyces peucetius early in the 1960s and were named doxorubicin (DOX) and daunorubicin (DNR) Fig. (1) [2], both drugs possessing aglyconic and sugar moieties. The aglycone consists of a tetracyclic ring with adjacent quinone-hydroquinone groups, a methoxy sub- stituent and a short side chain with a carbonyl group. The sugar, called daunosamine, is attached by a glycosidic bond to one of the rings and consists of a 3-amino-2,3,6- trideoxy- L-fucosyl moiety. The only difference between these two molecules is the fact that the side chain of DOX terminates with a primary alcohol, whereas that of DNR terminates with a methyl group Fig. (1) [2]. Despite its widespread use, the cytotoxic effects of anthracyclines are multidirectional, car- diotoxicity being the most known side effect. In order to find a better anthracycline, about 2000 analogs were produced with several chemical modifications or substitutions and/or conjugations introduced in the tetracyclic ring, the side chain or the amino-sugar. For example, epirubicin (EPI) is a semi- synthetic derivative of DOX obtained by an axial-to- equatorial epimerization of the hydroxyl group in a dauno- samine carbon. This positional change has little effect on the mode of action and spectrum of antineoplastic activity of EPI compared with DOX but it introduces pharmacokinetic and metabolic changes such as increased volume of distribution and consequent enhanced total body clearance or shorter terminal half-life [3,4]. Yet, DOX replacement by EPI does not eliminate the risk of developing chronic cardiotoxicity.

Only two more anthracyclines have attained clinical ap- proval; idarubicin and valrubicin. Idarubicin hydrochloride is an analogue of daunorubicin that also intercalates into DNA, having an inhibitory effect on nucleic acid synthesis, and interacts with topoisomerase II. The absence of a methoxy group in the anthracycline structure gives the compound a high lipophilicity which results in an increased rate of cellu- lar uptake compared with other anthracyclines [5]. Valrubi- cin (N-trifluoroacetyladriamycin-14-valerate, Valstar) is a chemotherapeutic drug used to treat bladder cancer. It is a semisynthetic analog of DOX, and it is administered by di- rect infusion into the bladder. However, several side effects are associated with the use of valrubicin, including blood in urine, incontinence, painful or difficult urination [6].

Nevertheless, the studies focusing the activity and toxic- ity of the most commonly used anthracyclines suggest that a better anthracycline has yet to come, i.e., superior anti- neoplastic activity without cardiotoxicity. It is therefore not surprising that relatively old drugs like DOX and DNR re- main the focus of clinical and preclinical research aimed at improving our appraisal of their mechanisms of activity and/or toxicity and identifying new strategies for a saffer use in cancer patients.

2. DOXORUBICIN AS A THERAPEUTIC AGENT

DOX is one of the most potent antineoplastic drugs pre- scribed alone or in combination with other agents, remaining the compound of its class that has the widest spectrum of activity. Indeed, DOX is used in the treatment of solid tu- mours and hematological malignancies, including breast, bile ducts, prostate, uterus, ovary, oesophagus, stomach and liver tumours, childhood solid tumors, osteosarcomas and soft tissue sarcomas, Kaposi’s sarcoma, as well as acute mye- loblastic and lymphoblastic leukaemia and Wilms Tumor [4,7-11].
Many studies have attributed the antitumor activity of DOX to its ability to intercalate into the DNA helix and/or bind covalently to proteins involved in DNA replication and transcription Fig. (2) [12]. Such interactions result in inhibi- tion of DNA, RNA, and protein synthesis, leading ultimately to cell death [13,14]. Recently, Ashley and Poulton [15], using a novel method utilising the fluorescent DNA dye Pi- coGreen, found that anthracyclines intercalated not only into nuclear DNA but also mitochondrial DNA (mtDNA).

Fig. (1). Chemical structures of Doxorubicin (DOX) and Daunorubicin (DNR). These two anthracyclines possess aglyconic and sugar moieties. The aglycone consists of a tetracyclic ring with adjacent quinone-hydroquinone groups, a methoxy substituent and a short side chain with a carbonyl group. The sugar, termed daunosamine, is attached by a glycosidic bond to one of the rings and consists of a 3-amino- 2,3,6- trideoxy-L-fucosyl moiety. The only difference between these two molecules is the fact that the side chain of DOX terminates with a primary alcohol, whereas that of DNR terminates with a methyl group.

Fig. (2). Doxorubicin (DOX) mechanisms of action and toxicity. One major mechanism underlying the antineoplastic action of DOX con- cerns its ability to intercalate into the DNA helix and/or bind covalently to proteins involved in DNA replication and transcription. DOX enters cancer cells by simple diffusion and binds with high affinity to the proteasome in cytoplasm (step 1). Then DOX binds to the 20S pro- teasomal subunit, forming a DOX proteasome complex that translocates into the nucleus via nuclear pores (step 3). Finally, DOX dissociates from the proteasome and binds to DNA due to its higher affinity for DNA than for proteasome (step 3). DOX can also interact with mito- chondria and bind to cardiolipin blocking the binding of mitochondrial creatine kinase (MtCK) to mitochondrial membranes. Additionally, the increase in DOX redox cycling by complex I of the mitochondrial respiratory chain leads to an increase in reactive oxygen species (ROS) production. NADH (reduced nicotinamide adenine dinucleotide); NAD+ (oxidized nicotinamide adenine dinucleotide); ADP (adenosine diphosphate); ATP (adenosine triphosphate); Pi (inorganic phosphate).

Several studies classified DOX as a topoisomerase II poi- son. The topoisomerase family of enzymes modify the topology of DNA without altering its structure and sequence and catalyze the unwinding of DNA for transcription and replica- tion, involving the process of cleavage of one strand of DNA duplex and passing a second duplex through this transient cleavage. The intermediate that is formed is termed the ‘‘cleavable complex’’ [16]. DOX poisons the cleavable complex, inhibiting the re-ligation of the cleaved duplex, a lesion that results in a DNA double-strand break (DSB) Fig. (2) [17,18]. Failure to repair DNA DSB results in an apop- totic response.

In the last few years it has been suggested that the protea- some modulates anthracyclines activity [2]. It has been shown that DOX enters cancer cells by simple diffusion and binds with high affinity to the proteasome in cytoplasm. DOX then binds to the 20S proteasomal subunit, forming a DOX proteasome complex that translocates into the nucleus via nuclear pores in an ATP-dependent process facilitated by nuclear localization signals. Finally, DOX dissociates from the proteasome and binds to DNA due to its higher affinity for DNA than for the proteasome Fig. (2) [19].

Interestingly, metabolic activation of drugs can also oc- cur inside tumour cells. It is known that intracellular NADPH cytochrome P450 reductase (CPR) expression can be modulated in cells by many internal factors such as oxy- gen deficiency, intracellular pH changes and by malignant transformation [20,21]. DOX can suffer a one-electron re- duction by a range of cellular oxidoreductases, including NADH dehydrogenase, NADPH cytochrome P450 reductase (CPR), xanthine oxidase and nitric oxide synthase [22-25]. The process comprises the one-electron transfer from re- duced nucleotides, which converts the anthracycline mole- cule to a semiquinone radical form. Subsequent nonenzy- matic semiquinone radical re-oxidation by molecular oxygen (O2) can form superoxide (O2●-) and hydrogen peroxide (H2O2) that interact with various macromolecules [26,27].The clinical use of DOX soon proved to be hampered by serious problems such as the development of resistance in tumor cells or toxicity in healthy tissues.

3. DOXORUBICIN-MEDIATED TOXICITY

In the earliest years of DOX clinical use, several phase II and III clinical studies were made to evaluate its benefits versus side effects. The most common side effects associated with its use are acute nausea and vomiting [28,29], stomatitis [30], gastrointestinal disturbances, alopecia [28,29] baldness [30], neurologic disturbances (hallucinations, vertigo, dizzi- ness) [29], cumulative cardiotoxicity [31,32] and bone- marrow aplasia [30]. The bone marrow depressant effects of DOX may result in an increased incidence of microbial in- fection, delayed healing and gingival bleeding [30,33]. In this regard, the major dose-limiting side effect is the myelo- suppression with leukopenia (principally granulocytopenia), neutropenia, thrombocytopenia [34] and anemia [35], usually reaching a lowest point during the second week of therapy [36] but the severity of the occurrence depends on the dose of the drug and on the regenerative capacity of the bone mar- row. Hypersensitivity (fever, chills, urticaria), hyperpigmen- tation of the nails, lacrimation and conjunctivitis can also occur [37].

Extravasation of DOX can produce severe local tissue necrosis, as well as possible cellulitis, vesication, thrombo- phlebitis, lymphangitis, or painful induration resulting in limitation of mobility of the adjacent joints [36]. Studies also reported that DOX can lead to the occurrence of phlebosclerosis especially when administered into small vein or repeat- edly into a single vein [36].
DOX-induced apoptosis is a common mechanism in can- cer cells and it is thought that toxicity in healthy cells may occur also through this effect, although via different routes.

Data has emerged suggesting that DOX interacts with mitochondria by the disruption of major mitochondrial func- tions [38]. The mitochondrial specificity of DOX is notewor- thy and has been correlated to its high affinity to cardiolipin, an inner mitochondrial membrane-specific phospholipid [39]. The association of DOX with cardiolipin blocks creatine kinase binding to the inner mitochondrial membrane Fig. (2) [40] and decreases the activity of critical mitochon- drial enzymes, which are dependent on cardiolipin [40]. It has been suggested that DOX accumulation in mitochondria leads to a redox cycling by complex I of the mitochondrial respiratory chain, where single electrons are transfered to DOX [41-43]. DOX enters mitochondria and reacts with mitochondrial complex I [43] to form semiquinone radical intermediates. The semiquinone form of DOX is a toxic short-lived metabolite [44], which in turn can react with O2 producing reactive oxygen species (ROS) [43]. ROS can then react with mitochondrial biomolecules in the vicinity, which include lipids, proteins and nuclei acids. DOX is known to react with mitochondrial DNA (mtDNA), forming adducts that interfere with normal mitochondrial function, proteins expression and lipid oxidation [45]. It is known that the oxidation of specific thiol residues in mitochondrial pro- teins is a critical regulator of the permeability transition pore (PTP) induction [46]. The PTP is a non-selective, high- conductance channel that spans the inner and outer mito- chondrial membranes [47-49], being modulated by several physiological factors [50,51]. When opened, the PTP causes a non-selective permeabilization of the inner mitochondrial membrane typically promoted by the accumulation of excessive Ca2+ in the matrix [48]. Although Ca2+ is considered to be the most important inducer, matrix pH, transmembrane electrical potential (ψm), Mg2+, Pi, cyclophilin D, oxidative stress and adenine nucleotides are also effective regulators [51-53]. Due to the pro-oxidant nature of DOX, it would be expected a relationship between DOX toxicity and PTP in- duction. In fact, according to Oliveira and co-workers [54], DOX treatment induces an increase in the amount of oxi- dized thiol residues in proteins of the PTP complex. The authors also observed a decrease in vitamin E and reduced gluthathione (GSH) levels, two key cellular antioxidants, which leads to a decreased protection against ROS produced during DOX redox cycling [54,55]. Interestigly, DOX also causes alterations in cell apoptotic signalling. An increase in the levels of Bax (pro-apoptotic protein) and a reduction in Bcl2 (anti-apoptotic protein) were observed in DOX treated animals [56]. Alterations in Bcl2/Bax ratio modulate the release of cytochrome c from mitochondria, with the increase of this ratio favoring cytochrome c release. DOX, as many other genotoxic agents, activates p53-DNA binding, a proc- ess mediated by nuclear factor-kB (NF-kB) activation [57]. NF-kB is a dimeric transcription factor that regulates genes associated with the stress response such as inflammation, oxidative stress, and apoptosis [58]. It has been reported that p53 induces apoptotic cell death, due to caspases cascade activation by two pathways: 1) p53 activated by DOX-mediated ROS production [59] mediates Bcl2 down- regulation and, consequently, leads to mitochondrial cyto- chrome c release [60]; 2) p53 accumulation induces the ex- pression of CD95/Fas/APO-1 receptor gene, a death receptor member of tumor necrosis factor (TNF)/nerve growth factor (NGF) receptor family [61-63]. When CD95L binds to its receptor, it triggers a potent apoptotic signal that activates the caspases cascade culminating in cell death [64]. Sardão et al. [65] demonstrated that H9c2 myoblasts exposed to DOX have increased p53 levels, which result in mitochon- drial dysfunction. The toxicity of DOX in the heart, brain, liver and kidney will be now described in detail.

3.1. DOX-Mediated Toxicity in the Heart

DOX-induced cardiotoxicity has received growing atten- tion since DOX discovery in 1969 and the beginning of its clinical use in the early 1970s [66,67]. As described above, although DOX is recognized as a potent antineoplastic agent, its cardiotoxic effects are the main reason for the dose- limited administration Fig. (3). Clinical manifestations of DOX-induced cardiotoxicity can be acute and chronic. How- ever, there is a wide variation in the frequency of clinical DOX-induced cardiotoxicity. Lefrak and collaborators [68] reported that the acute effects in the heart can be clinically controlled and frequently reversible, occurring in ~11% of patients within a short period of time following the begin- ning of the therapy. The acute effects include arrhythmias, hypotension and several electrocardiographic alterations [68- 70], which disappear once the treatment ceases. von Hoff and co-workers [71] reported that the chronic effects of DOX occur only in 1.7% of the patients undergoing DOX therapy and the mortality index is 50%. The chronic cardiotoxic ef- fects induced by DOX are dose-dependent and culminates in congestive heart failure (CHF) [68,71] in >4% of the patients receiving cumulative doses of 500-550 mg/m2, in >18% of the patients receiving cumulative doses of 551-600 mg/m2 and in ~36% of the patients receiving higher cumulative doses than 601 mg/m2 [72]. Analyzing a group of 630 pa- tients with breast carcinoma and small-cell lung carcinoma, Swain and collaborators [73] reported that an estimated 26% of patients would experience DOX-related CHF at a cumula- tive dose of 550 mg/m2.

It is documented that chronic DOX administration to mice for 7 weeks resulted in cardiac hypertrophy [74] Fig. (3). These results were corroborated by in vitro studies showing that DOX induce cardiomyocyte hypertrophy in primary neonatal rat cardiomyocytes [75] and in the myoblastic cell line H9c2 [76], which are also related with cytoskeletal alterations [77] Fig. (3). Zordosky and El-Kadi [78] showed that DOX causes a significant induction of the atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) genes, two cardiac hypertrophy markers, as well as of cytochrome P (CYP) genes, a super-family of monooxy- genases responsible for the oxidative metabolism of xenobi- otics and endogenous substances Fig. (3). The induction of these genes may result in altered arachidonic acid metabo- lism, which has also been associated to cardiac hypertrophy and heart failure [79]. It has been shown that acute DOX administration induces ultrastructural alterations in rat car- diomyocytes, such as marked interstitial oedema, perinuclear vacuolation, disorganization and degeneration of the myo- cardium [80]. Also in mice cardiomyocytes, DOX-induced acute alterations in mitochondria were observed, such as vacuolization, myelin deposition, disruption of membrane and organelle degeneration [81]. Previous studies from our laboratory showed that the ultrastructural alterations induced by DOX in the myoblastic cell line H9c2 are dependent on drug concentration [77]. Low concentrations of DOX (0.5 to 1 HM) cause alterations in fibrous structural proteins includ- ing the nuclear lamina and sarcomeric cardiac myosin, as well as mitochondrial depolarization and fragmentation and plasma membrane blebbing leading to cell shape changes. High DOX concentrations (5 to 50 HM) promote more pro- found cellular alterations, including nuclear swelling associ- ated with disruption of nuclear membrane structure, mito- chondrial swelling, and extensive cytoplasm vacuolization [77]. Interestingly, patients undergoing DOX therapy suffer some of those alterations, including myofibrillar loss, dilata- tion of sarcoplasmic reticulum and swollen mitochondria [82].

Fig. (3). Doxorubicin (DOX)-mediated toxicity in the heart. The heart is particularly sensitive to DOX-induced toxicity. This anticancer drug induces ultrastructural alterations and the enlargement of cardiomyocytes, which is associated with an increased expression of several genes like the atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). DOX also affects cardiac mitochondria. Besides the al- teration in the expression of mitochondrial proteins, the redox cycling of DOX at NADH dehydrogenase potentiates the production of reac- tive oxygen species (ROS). Additionally, toll-like receptors (TLR4), in the presence of DOX, apparently modulate the levels of ROS, proba- bly through the increase in the levels of tumour necrosis factor a (TNF-a). The increase in ROS levels triggers the activation of the apoptotic cascade. Furthermore, the impairment of mitochondria and DNA damage induced by DOX are associated with the activation of the autophagic process. There is also evidence that DOX induces an increase in antioxidant defenses that may represent an adaptive response to DOX-induced oxidative stress (see text for more complete information).

There are several reasons that make the heart a particu- larly susceptible organ to DOX-induced toxicity. As previ- ously discussed, DOX complexes with cardiolipin, which is a major component of the mitochondrial inner membrane [83] and heart cells have an elevated density of mitochondria per unit volume when compared to most other tissues [84]. The controversial existence of a heart specific isoform of the enzyme NADH dehydrogenase (mitochondrial complex I), able to initiate DOX redox cycling and, consequently, pro- moting the formation of ROS [41], associated to the fact that the heart has low levels of antioxidant defenses when com- pared with other tissues [85,86], contribute for the high sus- ceptibility of the heart to DOX-induced toxicity. Further- more, recovery of heart upon DOX-induced damage is very difficult since cardiomyocytes are post-mitotic cells [87,88].

Despite the clinical manifestations of DOX-induced car- diomyopathy and the knowledge that DOX induces several cardiac ultrastructural changes, the mechanism(s) responsi- ble for DOX-induced cardiac toxicity remain(s) elusive. Nevertheless, several mechanisms have been proposed: oxi- dative stress, inhibition of nucleic acid and protein synthesis [89-91], release of vasoactive amines [92], alteration of adrenergic function and adenylate cyclase activity [93], lysosomal changes [94], modifications of sarcolemmal Ca2+ transport [95] and alterations in cellular iron metabolism [96], among others.

As described, the role of oxidative stress in DOX- induced cardiotoxicity has been largely discussed and seems to be an event that occurs both acutely [80, 86, 97 – 100] and chronically [74, 88] Fig. (3), although the origin of ROS may be different in both cases. DOX concentrations in the plasma can reach 0.5–1 HM and the concentrations within mitochon- dria can be approximately 100-fold higher, making the heart a site of redox reactivity [101]. Although in acute DOX- administration the major source of ROS/ reactive nitrogen species (RNS) is NADH dehydrogenase of the mitochondrial respiratory chain, ROS produced during long-term admini- stration of DOX seem to result from other mechanisms in- volving unbalanced mitochondrial protein expression. Zhou et al. [102], performed experiments with cardiomyocytes isolated from rats receiving repeated injections of DOX for 6 weeks; despite the fact that a recovery period of 1 week or 5 weeks existed between the last injection of the drug and the sacrifice of the rats, oxidative stress was maintained in cells. The authors suggested that the persistent oxidative stress results from DOX-induced mtDNA alterations and decreased cytochrome oxidase (COX) activity and subunit expression leading to a sustained production of O2•- [102]. Recently, Chandran and collaborators [103] reported that DOX strongly downregulates the stable expression of COX subunits II and Va and had a slight inhibitory effect on COX subunit I gene expression. However, mitoquinone (Mito-Q) restored COX subunit II and Va expressions in DOX-treated rats [103]. These results suggest a novel cardioprotection mechanism by Mito-Q during DOX-induced cardiomyopathy.

Recently, the involvement of Toll-like receptor 4 (TLR- 4) in DOX-induced cardiomyopathy in mice was investi- gated [99]. TLRs are part of the innate immune system and, particularly TLR4 is a receptor involved in the recognition of multiple bacterial antigens mediating their effects and play- ing an important function in the maturation of the phagosome [104]. Moreover, it has been reported an in- creased TLR4 expression in cardiomyocytes isolated from humans and animals with cardiomyopathies [105]. TLRs have a role in the development of DOX-induced cardiomy- opathy, as suggested by studies performed in TLR-2 defi- cient mice [106]. Additionally, it has been shown that TLR4- deficiency prevents DOX-induced lipid peroxidation and nitrotyrosine formation in isolated cardiomyocytes and in left ventricular tissue [99]. Apparently, the release of cytokines by the activation of the innate immune system is involved in the pathogenesis of DOX-induced cardiotoxicity [2,106, 107]. Therefore, it is likely that the increase in oxidative stress is in part modulated by the activation of TLR4 recep- tor and subsequent increase in tumour necrosis factor-a (TNF-a) levels, since there is a significant correlation be- tween decreased parameters of systolic left ventricular func- tion and cardiac TNF-a expression Fig. (3) [99].

Interestingly, the activity of several enzymatic antioxi- dant defenses were observed to increase significantly follow- ing DOX treatment in young Fischer rats but not in the old rats of the same strain suggesting an increase in DOX- induced oxidative damage with age [97]. Previous studies report a significant increase in copper-zinc (CuZnSOD) and manganese (MnSOD) superoxide dismutase activities and protein levels induced by acute DOX treatment [81, 86].

Other study showed a significant increase in catalase (CAT) activity and an increase in reduced glutathione (GSH) [80]. Altogether these studies show an upregulation of antioxidant defenses, suggesting an adaptive response of cells to oxida- tive imbalance promoted by DOX Fig. (3). Glutathione per- oxidase 1 (GPx1) is the major isoform of the enzyme GPx and is present in both mitochondria and the cytosol [108]. It has been suggested that GPx1 has an important role in de- toxifying ROS in cells [109, 110]. To test the hypothesis that GPx1 has a central role in the clearance of H2O2, Gao et al.[100] compared the effects of DOX in heart and mitochon- drial function, protein nitration and apoptosis in GPx1- deficient and wild-type mice. The authors observed that GPx1-deficient mice were more prone to DOX-induced damage than wild type mice. Indeed, it was observed that mitochondria isolated from the heart of GPx1-deficient mice presented an increased rate of NAD-linked state 4 respiration and a decline in P/O ratio relatively to wild-type mice cor- roborating the harmful effect of oxidative stress in heart mi- tochondrial function [100]. As discussed previously, heart mitochondria are a preferential target of DOX, accumulating the drug at relatively high concentrations. Therefore, it is not surprising that these organelles are especially prone to DOX- induced oxidative/nitrative damage, and at the same time, they are also important sources of DOX-induced ROS [111, 112].

Acute administration of DOX induces a decrease in heart mitochondrial state 3 respiration but no differences were observed in the mitochondrial state 4 respiration, suggesting no alterations in the mitochondrial proton leak with DOX treatment [86]. Other studies report several defects in the mitochondrial respiratory chain, including a decrease in COX activity and COX I expression [88] and reduced creatine-stimulated respiration [40]. It has been previously shown that sub-chronic DOX treatment promotes a small decrease in total mitochondrial adenine nucleotide transloca- tor (ANT) pool or its activity predisposing myocytes to a situation of energy deficit in cases of high energy demands [113]. Additionally, DOX treatment increases the suscepti- bility to the mitochondrial PTP opening in the heart [113- 115]. As already mentioned, one of the consequences of the PTP induction, besides the disturbance of cell and mitochon- drial calcium homeostasis, is the release of cytochrome c, enabling the initiation of the apoptotic cascade [112,116]. DOX-induced apoptosis of cardiomyocytes has been sug- gested to occur both acutely and chronically [117]. Curi- ously, an in vitro study in which isolated cardiac mitochon- dria were exposed to DOX and DOX-derived metabolites (i.e. doxorubicinol, doxorubicin aglycone and doxorubicinol aglycone) showed that the aglycone derivates induce a more pronounced release of cytochrome c than DOX itself, sug- gesting a role of DOX metabolites in the cardiotoxicity in- duced by this chemotherapeutic drug [118]. A previous in vivo study shows that acute DOX administration potentiates the release of cytochrome c to the cytosol of cardiomyocytes enhancing caspase-3 activity. However, an increase in the Bcl2/Bax ratio was also observed, possibly representing an adaptive response to protect the cells against mitochondrial mediated apoptosis [86].Riad et al. [99] demonstrated that TLR4 deficient mice are protected against DOX-induced apoptosis when compared with the wild-type mice, although Bax levels remained unchanged. One downstream effector of DOX-mediated tox- icity seems to be the GATA-4 transcription factor, since DOX induces the down-regulation of this transcription fac- tor, suggesting that the activation of TLR4 down-regulates GATA-4 aggravating DOX toxicity [99]. Other mechanism underlying DOX toxicity involves cardiac β-adrenergic re- ceptors (βAR), β1AR and β2AR, that differently modulate cardiac function and exert physiological responses by dis- tinct signal transduction pathways [119]. It has been shown that β1AR stimulates apoptosis in cardiomyocytes while β2AR protects against apoptosis [120]. Yano et al. [121] focused their attention on β2AR, and showed that the stimu- lation of this receptor protects against DOX-induced apopto- sis in H9c2 cells. ROS exert a regulatory effect in the mito- genic-activated protein kinase (MAPK) pathways, which comprises extracellular-regulated (ERKs), c-jun-NH2- terminal kinase (JNKs), p38 MAPK and the big MAPK-1 (BMAPK-1) these kinases being important in the process of carcinogenesis including cell proliferation, differentiation and apoptosis [122]. There is a link between MAPKs path- ways and heat-stress proteins (HSPs). HSPs possess a chap- erone-like activity and have a key role in the maintenance of normal cellular function and restoration after an insult [123, 124]. Šimončiková et al. [125] reported that chronic admini- stration of DOX to rats induces activation of ERKs, up- regulation of HSP60 and down-regulation of HSP70 in the cardiac tissue. ERKs activation usually confers a survival advantage to cells that associated with an increased expres- sion of HSP60 may reflect an adaptive response of the car- diac cells to the prolonged exposure to the drug. Indeed, a previous study demonstrated an association between the overexpression of HSP60 in cardiomyocytes and the reduc- tion of DOX-mediated induction of the pro-apoptotic protein Bad [126]. Another study suggests that Akt activation is a possible protective response against DOX-induced cardio- myocyte apoptosis in the left ventricle [127]. Despite evi- dence showing an association between DOX-induced oxida- tive stress and apoptosis, a recent study suggests that oxida- tive stress does not play a role in apoptotic H9c2 myocyte cell death exposed to clinical relevant concentrations of DOX [128]. The authors suggest that DOX may facilitate apoptosis of cardiomyocytes by inhibiting the anti-apoptotic enzyme heme-oxygenase (HO-1) [128]. The protective role of HO-1 (whose physiological function is to catabolise heme to biliverdin, carbon monoxide and iron) against apoptosis has been previously established [129, 130]. The multifacto- rial mechanism for DOX cardiotoxicity is demonstrated by the fact that antioxidants provided limited protection against H9c2 cell death caused by DOX [65].
Lu et al. [131] showed, for the first time, that autophagic death of cardiomyocytes plays an important role in DOX- induced heart failure in rats Fig. (3). The authors report that DOX promotes mitochondrial injury leading to energy deple- tion and beclin-1 gene expression, an essential protein to the initiation of the autophagic process [131]. Autophagy of mi- tochondria (mitophagy) may cause an energy deficit in the cardiomyocyte and lead to cell failure upon stress (viz. cal- cium accumulation or other). Recently, Muñoz-Gamez et al.[132] reported that poly ADP-ribose polymerase 1 (PARP-1) is involved in autophagy promoted by DOX-induced DNA damage.In summary, the heart is particularly susceptible to DOX- induced injury, the cardiotoxicity induced by this anticancer drug being a multi-factorial process that involves several mechanisms.

3.2. DOX-Mediated Toxicity in the Brain

The cognitive function has several domains, resulting from a healthy brain activity, such as attention and concen- tration, executive function, information-processing speed, language, motor function, visuospatial skill and learning and memory [for further information see 133].Wefel et al. [134] used for the first time the term chemo- brain to describe the decline of the cognitive function associ- ated with chemotherapy. In the last years growing evidence came to consolidate the fact that chemotherapy induces im- pairments in various domains of cognitive function [135- 145]. A recent longitudinal study performed by Jansen et al. [145], filled in some gaps not taken into account in previous studies, such as the assessment of changes in cognitive func- tion over time, the evaluation of potential relationships be- tween cognitive function and anxiety, depression, fatigue, hemoglobin level, menopausal status, and perception of cog- nitive function as well as the evaluation of cognitive function before chemotherapy. The preliminary results of this study support the idea that chemotherapy may have a negative im- pact in some domains of the cognitive function such as a decrease in visuospatial skill and total cognitive scores [145]. Interestingly, a study on the chronicity of chemotherapeutic– mediated cognitive impairment suggests that the negative impact of chemotherapy on cognitive function is recovered within 1 year after the ending of the treatment [144]. Data obtained by magnetic ressonance imaging (MRI) studies performed in monozygotic twins, in which one individual underwent chemotherapy and the other was the control, pro- vides evidence of increased cortical activity, possibly repre- senting the recruitment of a larger neuronal network in order to accomplish a cognitive function comparable to the control twin [142]. Other studies also showed a similar expanded activation in functional MRI studies in mild cognitive im- pairment [146,147].

Since DOX does not cross the blood brain barrier (BBB) [148,149], its harmful effects in the brain must be indirect. Indeed, Tangpong et al. [149] provided for the first time, direct biochemical evidence that DOX-induced toxicity in the brain is TNF-a-mediated Fig. (4). The same authors re- ported that an increase in TNF-a levels is observed in the cortex and hippocampus of DOX-treated mice [149, 150]. Circulating inflammatory cytokines can reach the brain by several pathways and stimulate microglial cells to produce more inflammatory cytokines Fig. (4) [151]. An increase in markers of oxidative damage was observed in several animal studies, namely 4-hydroxynonenal (HNE), malondialdehyde (MDA), thiobarbituric acid reactive substances (TBARS), protein carbonyl groups and a reduction of mitochondrial aconitase activity [55,97,152-155]. It has been shown that DOX-induced brain injury is decreased by the simultaneous administration of anti-TNF-a antibody, a process that pre- vented the increase of serum TNF-a [149]. These results suggest that oxidative stress in the brains of DOX-injected animals is an indirect effect of DOX-mediated increase in serum levels of TNF-a.

Fig. (4). Proposed mechanism of Doxorubicin (DOX)-induced brain damage. DOX induces the systemic production of tumour necrosis factor-a (TNF-a), which, in turn, stimulates the production of inflammatory cytokines by microglial cells in the brain. The increase in the levels of TNF-a enhances the expression of the inducible nitric oxide synthase (iNOS) potentially leading to the increase in the levels of reactive nitrogen species (RNS) and consequent nitration of proteins, such as manganese superoxide dismutase (MnSOD). As a consequence of MnSOD nitration, its activity decreases leading to the increase in reactive oxygen species (ROS) that can potentiate the opening of mito- chondrial permeability transition pore (PTP), triggering apoptosis through the release of cytochrome c from mitochondria thereby leading to apoptotic cell death.

Several studies have showed a decrease in MnSOD, glu- tathione-S-transferase (GST) activities and GSH levels in the brain [55,150,154,155]. Giving the role of MnSOD in de- toxifying cells from O2•-, Tangpong and colleagues [150] focused their attention in the mechanism of inactivation of this enzyme. The authors observed that MnSOD is inacti- vated by nitration, since DOX induced the overexpression of the inducible nitric oxide synthase (iNOS), enhancing the production of nitric oxide (NO) and, consequently, the for- mation of RNS Fig. (4). Others demonstrated that the inhibi- tion of NOS with aminoguanidine improves DOX-induced oxidative stress in the brain [156], reinforcing the idea that increased NO production is a key factor mediating DOX- induced injury Fig. (4).

Without surprise, Pritsos and Ma [97] showed that lipid peroxidation induced by DOX is more pronounced in old animals than in young animals this effect being associated with a significant decrease in the activities of antioxidant enzymes. These observations suggest that there is an increase in DOX-induced oxidative damage with age and this suscep- tibility could be due to basal and drug-induced differences in tissue antioxidant enzyme activities.

Some studies report a decrease in the mitochondrial res- piratory control ratio (RCR) in animals subjected to a single injection of DOX [149, 150]. Other studies show that DOX does not induce a significant effect in the mitochondrial respiratory chain [40, 55]. Studies from our laboratory show that a sub-chronic administration of DOX increases the sus- ceptibility of PTP opening [55]. Giving that PTP is a redox- sensitive channel, and that the increase in the formation of ROS/RNS potentiates the formation of this channel, our re- sults are in agreement with previous studies showing in- creased oxidative stress and apoptosis in DOX-treated ani- mals Fig. (4).

3.3. DOX-Mediated Toxicity in the Liver

The liver is a vital organ that plays a major role in me- tabolism and has a number of important functions in the body including glucose storage in the form of glycogen, plasma protein synthesis and detoxification [157]. During DOX therapy, the liver receives, accumulates and metabo- lizes high concentrations of DOX [158] hence, it is expect- able that the liver is one of the most affected organs by DOX therapy [159]. Indeed, almost 40% of patients suffered liver injury after DOX treatment [160]. The primary mechanism of DOX reduction in the liver is catalyzed by CPR [24, 25]. Other studies also revealed an important role for carbonyl reductase 1 (CBR1) in DOX reduction [161,162, 163]. That conclusion was based on Vm and Km values of DOX reduc- tion that are almost identical for CBR1 and human liver cy- tosol.
The liver is one of the few internal organs capable of natural regeneration of lost tissue with hepatocytes re- entering the cell cycle. However, it was already demonstrated that DOX is able to arrest cell cycle of hepatocytes inhibiting its self-regeneration [163]. Despite this effect, the most important cause of DOX toxicity in the liver seems to be oxidative stress Fig. (5). In the liver, oxidative stress caused by increased ROS production can result from two different ways, the most common occuring when the semiquinone form of DOX reacts with O2 producing O2●- and H2O2, an alternative way occur through NADPH- oxidases, the principal extra-mitochondrial producers of ROS in hepatocytes [163]. NADPH-oxidases are present in small levels but their levels increase in response to extracel- lular stimulus, such as DOX treatment [163]. The literature shows that DOX-induced ROS production leads to an in- crease in lipid peroxidation and SOD, CAT and glutathione peroxidase (GPx) activities, DNA damage, and a decrease in GSH and vitamin E levels, which confirm DOX hepatotoxic- ity Fig. (5) [85,164-171]. The impaired mitochondrial func- tion observed with DOX treatment may be the reason for alterations in the typical markers of oxidative stress observed in damaged liver tissue [172,173]. ROS production could also lead to the activation of IкB kinase (IKK) that phos- phorylates IкB inhibitors activating IкB that, in turn, acti- vates NF-kB that leads to the expression of pro- inflammatory cytokines culminating in cell death Fig. (5) [174]. DOX also causes pathological changes in hepatocytes such as an increase in mitochondrial vacuolization, swelling, weak pyknotic nucleus and dilatation of the intercellular space [166,172,175].

Fig. (5). Doxorubicin (DOX)-mediated toxicity in the liver. The liver is the primary organ responsible for drugs metabolism, including DOX. In the liver, DOX-induced reactive oxygen species (ROS) production leads to an increase in lipid peroxidation, superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) activities, DNA damage, and decrease in reduced glutathione (GSH) and vitamin E levels contributing to an oxidative imbalance and confirming DOX hepatotoxicity. ROS production could also lead to the activation of IкB kinase (IKK) that phosphorylates IкB inhibitors activating IкB that, in turn, activates the nuclear factor-kappaB (NF-kB) that leads to the expression of pro-inflammatory cytokines culminating in cell death. Finally, DOX treatment decreases adenosine triphosphate (ATP) and increases adenosine diphosphate (ADP), adenosine monophosphate (AMP) and inorganic phosphate (Pi) within cells and causes pathological changes in hepatocytes such as an increase in mitochondrial vacuolization.

DOX efflux through the cells is mediated by the ATP- binding cassette/ABC proteins such as P-glycoprotein, which maintain chemical homeostasis and decreases intracellular accumulation of drugs and are one of the first lines of de- fense of cells [176]. However, this mechanism demands high energy expenditure [176], which is limited in the presence of DOX since it decreases adenosine triphosphate (ATP) and increases adenosine diphosphate (ADP), adenosine mono- phosphate (AMP) and inorganic phosphate (Pi) within cells Fig. (5) [176]. Similarly the phosphorylation potential, a measure of the capacity of the cell for carrying out ATP- dependent processes decreases with DOX therapy [176].

3.4. DOX-Mediated Toxicity in the Kidney

Besides removing waste products, the kidney also re- moves normal components of the blood that are present in greater-than-normal concentrations. When an excess of water and ions are present, the excess is quickly eliminated in the urine. On the other hand, if the levels of these substances are too low in the blood, the kidney as the ability to recover them back to the blood. Thus, the kidneys continuously regu- late the chemical composition of the blood within narrow limits. For this reason, the kidneys are one of the major ho- meostatic devices of the body [177,178]. Since the regenera- tive capacity of kidneys is too low, they are very susceptible to damage. The epithelial degeneration seems to have a pri- mary role in the deterioration of the renal glomerulus [179], where the filtration of plasma occurs; in fact, the damage of the glomerulus is a hallmark of nephropathies and may lead to the development of glomerulosclerosis.
Several studies revealed that DOX interferes with glome- rular podocytes leading to their injury and, consequently, to nephropathy [152,179-183]. The most common event docu- mented in all the studies is the presence of a severe proteinu- ria Fig. (6). Exposure of renal tissue to the local passage of leaked proteins may evoke structural changes in the nephron leading to focal glomerulosclerosis [179,181,184,185], a glomerular disease characterized by marked proteinuria, steroid resistance, hypertension, and a high incidence of pro- gression to renal failure [186]. Proteinuria is also related with focal fusion of podocytes foot processes and swelling, extensive glomerular vacuolization and quick and progres- sive renal failure [152,179–181,183]. Other important ef- fects, although present in a smaller scale of intensity, are the presence of extensive glomerular lesions, tubular dilatation, interstitial fibrosis and inflammation [179,183,187], an in- crease in plasma creatinine levels and hipoalbuminemia [179,181,182], dyslipidemia [188-191], hypercoagulability [62], increase in kidneys size with a granular pale color sur- face [179,181] and glomerular capillary permeability Fig. (6) [192]. The mechanisms by which DOX induces toxicity are not fully understood but some studies suggest that they are most likely mediated by the formation of an iron– anthracycline complex that generates free radicals, which in turn, causes oxidative lesions on critical cellular components [44, 89,193-197]. Lebrecht and co-workers [184] suggested that DOX enters mitochondria leading to the production of ROS, which cause mtDNA damage causing mitochondrial dysfunction and, consequently, contributes to the fast pro- gression of nephron damage. This hypothesis support the studies of Okuda and collaborators [179] showing that DOX- induced glomerular injury was related with ROS formation. Indeed, the authors also demonstrated that DOX was respon- sible for a decrease in mitochondrial complexes I and IV activities, by an increase in citrate synthase activity and triglycerides Fig. (6), a common occurrence in situations characterized by the impairment of the respiratory chain [198] and an increase in O2●- levels, which can react with NO and induce the apoptotic process [199]. It has also been reported that DOX increases mtDNA mutations, which could be a result of topoisomerases II inhibition and/or due to an increase in oxidative stress [184].

Other studies suggest that lipid peroxidation and reduc- tion in natural antioxidant levels (vitamin E and GSH) Fig.(6) could be the reason for DOX-induced nephropathies [63] leading to Bowman’s capsule thickening, presence of multi- focal tubular casts and adhesion of the glomerular tuft to Bowman’s capsule [152]. According to Rook et al. [182], the tissue angiotensin-converting enzyme (ACE) is also involved in renal tissue damage induced by DOX treatment. The authors observed an increase in ACE activity, which is re- sponsible for the interstitial damage due to pro-inflammatory and pre-fibrotic effects that interfere with the kidney’s ability to autoregulate glomerular pressure and, consequently, glomerular filtration rate [200]. The same authors hypothe- size that the susceptibility of the nephrons to DOX-induced toxicity could be genetic and that genetically-determined individual differences in ACE activity, could be the reason for the different susceptibilities to DOX therapy. Indeed, the cases of proteinuria and nephropathy in humans are rare and may represent a primary genetic susceptibility [182,183, 201]. In this line, Zheng et al. [178] suggested that the sus- ceptibility to DOX-induced nephropathy is a defect in a gene with recessive inheritance.

Fig. (6). Doxorubicin (DOX)-mediated toxicity in the kidney. The kidney is one of the major homeostatic devices of the body. DOX inter- feres with glomerular podocytes leading to their injury and, consequently, to nephropathy. The most common events promoted by DOX treatment is the presence of a severe proteinuria, an increase in plasma creatinine levels, hipoalbuminemia, dyslipidemia, hypercoagulability, increase in kidneys size and glomerular capillary permeability. DOX interferes with mitochondrial function leading to a decrease in mito- chondrial complexes I and IV activities, an increase in citrate synthase activity and triglycerides and superoxide (O2•-) levels. DOX can also induce lipid peroxidation and decrease the levels of antioxidant compounds, vitamin E and reduced glutathione (GSH), which can also be involved in DOX-induced nephropathy.

However, the bulk of the studies show that a direct expo- sure of cells to DOX is necessary to the development of nephropathy, independently of drug metabolism [62,183]. This was proved by the clipping of the renal artery during DOX injection, which prevented DOX-induced nephropathy, suggesting that initial exposure to this drug, rather than its metabolites, causes kidney damage [177,180]. It is also well established that DOX nephropathy has chronic and self- perpetuating characteristics of human progressive and chronic renal disease [202]. DOX-induced nephropathy in rats represents a good animal model to study kidney diseases [179] since glomerulosclerosis is the most common progres- sive glomerular disease in children and is the second leading cause of end-stage renal disease in this age group; focal and segmental glomerulosclerosis accounts for 20 to 25% of idiopathic nephrotic syndrome in adults [203,204].Altogether these studies show that ROS production and mitochondrial dysfunction induced by DOX are major causes of nephrons’ damage.

4. MANAGEMENT OF DOX-INDUCED TOXICITY

Although DOX-induced cardiotoxicity has been the driv- ing force for the design of strategies aimed at improving the outcome of the chemotherapeutic treatment, the management of the toxicity induced in other organs such as the brain, the liver or the kidney is also important. Several lines of investigation have been followed: the administration of compounds with antioxidant and/or anti-apoptotic activities and the de- velopment of efficacious delivery systems and DOX analogs.

N-Acetylcysteine (NAC) has been previously reported to have an important antioxidant role as a precursor of GSH synthesis and as stimulator of the cytosolic enzymes in- volved in GSH regeneration [205]. Park et al. [152] reported that both NAC and selenium, a component of GPx [206], are effective in reducing the levels of MDA induced by DOX in cultured astrocytes. Furthermore, in vivo studies showed that NAC and selenium are especially effective in the prevention of DOX-induced nephrotoxicity [152]. Moreover, the GSH precursor γ-glutamyl cysteine ethyl ester improved the levels of GSH and glutathione-s-transferase (GST) activity in the brain [154]. Several other compounds, such as melatonin [155], lycopene [80], phenylbutyrate [81] and coumarin [207] have proved to be efficient in protecting against DOX- induced oxidative imbalance in different organs. However, although efficient in cellular or acute animal experiments, antioxidants have failed to alleviate anthracycline toxicity in clinically relevant chronic animal models or clinical trials [for further information see 208].
In the last years, the heat-shock protein 20 (HSP20) emerged as a cardioprotector, particularly in situations of physiological stress [209-211]. Indeed, HSP20 proved to be efficient in protecting against DOX-induced apoptosis. Recently, it has been reported that HSP20 preserves Akt phos- phorylation/activity protecting against DOX-induced toxicity [212]. Additionally, erythropoietin has proved to protect against DOX-induced apoptotic cardiomyocyte death, appar- ently through the increase of phosphatidylinositol 3-kinase (PI3K)-dependent Akt phosphorylation [213]. A recent study showed that vincristine also activates the PI3K/Akt and MEK/ERK survival pathways, conferring protection against DOX-induced cardiotoxicity [214]. Han et al. [215, 216], reported that naringenin-7-O-glucoside, a phytocompound, modulates the expression of several apoptotic-related genes, such as Bcl-2, caspase-3 and caspase-9 and also induces the expression of several antioxidant enzymes through the phos- phorylation of ERK 1/2 and nuclear translocation of the tran- scription factor Nrf2.
Dexrazoxane (DZR) is a promising compound that be- longs to the class of bis(2,6-dioxopiperazines). The mecha- nism of action of this drug has been attributed to its hydro- lytic transformation into the iron-chelating metabolite ADR- 925, which may act by displacing iron from anthracycline- iron complexes or by chelating free or loosely bound cellular iron, thus preventing site-specific iron-catalyzed ROS dam- age. A multicenter randomized phase III trial showed that DZR is clinically effective in the reduction of anthracycline- induced cardiac damage in breast cancer patients undergoing chemotherapy [217]. DZR up-regulates Akt and ERK signal- ing pathways protecting against DOX-induced cardiomyopa- thy and that protection was showed to be sustained beyond the treatment period [218]. Cochrane meta-analysis [219, 220] revealed that DZR prevents heart damage and no evi- dence for a difference in response rate or survival between the DZR and control group was identified. Only for an ab- normal white blood cell count at nadir a clearly significant difference in favour of the control group was identified. The authors conclude that if the risk of cardiac damage is ex- pected to be high, it might be justified to use DZR in patients with cancer treated with anthracyclines [219, 220]. However, the American Society of Clinical Oncology 2008 [221] point that DZR is not recommended for routine use in breast can- cer in adjuvant setting or metastatic setting with initial DOX- based chemotherapy.

Nevertheless, it is suggested that DZR can be considered for patients with metastatic breast cancer and other malignancies and that have received more than 300 mg/m2 DOX in the metastatic setting and who may benefit from continued DOX-containing therapy [221]. Testore and colleagues [222] analyzed the incidence of cardiac dysfunc- tion over a 10-year period in patients with breast cancer who were treated with anthracycline-based regimens with addi- tion of DZR, mainly in an adjuvant setting. No patient died of heart failure during the period analyzed. DZR was well tolerated, with no reports of adverse events associated with this drug. The authors concluded that DZR appears to have a cardioprotective effect in women with early-stage or ad- vanced breast cancer treated with anthracycline-based com- bination chemotherapy, mainly as an adjuvant treatment [222].Another line of investigation concerning the management of DOX-induced toxicity is the pursuit for an efficacious DOX delivery system that minimizes the damage to non- targeted tissues. According to Minotti et al. [2], liposomal delivery systems are the leading method to passively target anthracyclines to tumors. Liposomes must possess optimal features such as vesicle size, rate of plasma clearance and the stability of the liposome drug association in the blood and in the tumor. Tumors have characteristics that favor liposomal vesicle accumulation such the intense angiogenic process, permeable microvasculature of the newly formed vessels and an insufficient lymphatic drainage [2]. Once the liposomes reach the tumor, one objective is the complete release of the drug. Several mechanisms can contribute to that, including slightly acidic pH of the tumoral interstitial fluids, the re- lease of lipases and other enzymes and oxidizing agents by dying tumor cells and tumor-infiltrating inflammatory cells [223]. Indeed, it has been demonstrated in a randomized clinical trial, that liposomal DOX is an advantageous alterna- tive to conventional DOX administration and even to epiru- bicin administration, which is a less toxic DOX analogue [224]. Recently, a particular liposomal formulation has re- ceived growing attention, the polyethyleneglycol-coated (“pegylated”) liposomal doxorubicin (PLD). The PLD for- mulation has a much more favorable pharmacokinetics than DOX [225,226]. The PLD formulation induces less adverse side effects in subjects under anticancer therapy enhancing the quality of life of patients [227-229]. PLD administration at a minimum cumulative dosage of 500 mg/m2 and a maxi- mum of 1500 mg/m2 showed no evidence of CHF [227]. In the same line, Lyass et al. [230] reported no signs of car- diotoxicity for dosages at a maximum of 1500 mg/m2. In both studies, a minority of the patients showed impaired heart function, however these patients had previously un- derwent clinical interventions to fight cancer, which may have contributed to cardiac problems [227, 230]. More re- cently, a study performed in elderly breast cancer patients proved that the administration of PLD at a cumulative dose of 180 mg/m2 showed no signs of a decrease in left ventricu- lar ejection fraction or CHF [231]. A phase II clinical trial that consisted in the administration of 50 mg/m2 of PLD every 4 weeks to patients with Müllerian carcinoma, with a previous history of platinum-based chemotherapy, showed efficacy of the PLD formulation and manageable toxicity by dose modification or supportive care [232]. Several other lipidic formulations has been made in an attempt to improve the efficacy of these delivery systems, for example, with poly(ethylene oxide)-b-poly(e-cabrolactone-DOX) [PEO-b- P(CL-DOX)] that intended to reduce the premature release of DOX outside the tumor, such as the formulation made by Mahmud et al. [233], in which DOX can bind to free car- boxyl groups in the micellar structure by an amine bond. The release of DOX occurs through the endocytosis of the mi- celles by tumor cells and its dissociation in the acidic envi- ronment of the lysosomes [233]. To further improve the PEO-b-P(CL-DOX) formulation and provide selective intra- cellular delivery to tumor cells, thus avoiding healthy cells, Xiong et al. [234] coated the micelles with an internalizing antagonist peptide, GRGDS, for aVβ3 integrin. The litera- ture shows that tumor endothelial and metastatic cancer cells overexpress aVβ3 integrins on their membranes [235-237]. Indeed, the authors concluded that the formulation accumu- lated preferentially in metastatic cancer cells through endo- cytosis and showed an optimal release of DOX from the mi- celle at pH 5.0 [234]. Yadav et al. [238] used a different formulation, a copolymer of hyaluronic acid-polyethyl- eneglycol-polycaprolactone (HA-PEG-PCL) to deliver DOX.

This strategy is based in the fact that tumor cells overexpress the receptor CD44 for hyaluronic acid (HA), whose function involves cell motility and its capacity of internalize HA [239, 240]. The authors showed an efficient delivery of DOX in Ehrlich ascites tumor by receptor-mediated endocytosis, as well as enhanced permeability and retention effects [237].

The development of analogs of DOX is another possible strategy to deal with the toxicity induced by this che- motherapeutic drug. There are two strategies concerning the development of new anthracyclines, the nuclear-targeted versus the non-nuclear-targeted analogs [2]. Several DOX analogs have been developed, such as the methoxymor- pholinyl-doxorubicin (MMDX), which is a nuclear targeted anthracycline. MMDX contains a methoxymorpholinyl group at the 3’ position of the sugar group, being a highly lipophilic molecule. MMDX is non cardiotoxic at optimal anti-neoplastic doses [241]. The cytotoxicity of MMDX in cell cultures can be potentiated by pre-incubating this com- pound with liver microsomes and NADPH by a metabolic process that is inhibited by ketoconazole and triacetylolean- domycin, two inhibitors of the cytochrome P450 3A (CYP3A), suggesting that MMDX is a prodrug activated in the liver by the enzyme CYP3A [242-244]. Furthermore, it has been previously shown that the MMDX prodrug is me- tabolized by CYP3A and originates a potent cytotoxic me- tabolite that is formed rapidly and decays relatively slowly [174]. The authors proposed an interesting solution to mini- mize the toxic side-effects and further enhance the therapeu- tic effect of MMDX, which is based in the use of gene ther- apy vectors to increase the expression of CYP3A4 in tumors that not express naturally high levels of that enzyme [174]. Another nuclear-targeted DOX analog is the MEN 10755 (or sabarubicin), a demethoxydoxorubicin with an insertion of 2,6-dideoxy-L-fucose between the aglycone and daunosa- mine. In preclinical studies, MEN 10755 apparently pre- sented improved therapeutic efficacy over DOX, particularly in gynecological and lung cancers [245, 246]. Curiously, it was shown that MEN 10755 activates the transcription factor NF-kB, however this activation occurs earlier than DNA fragmentation [247]. More recently, Devalapally et al. [248] tested the β-galactoside prodrug of DOX in vivo and con- cluded that this prodrug had a better clearance and volume of distribution when compared to DOX. The rationale of those authors is that tumoral tissues express high levels of the en- zyme β-galactosidase and this enzyme is expressed at very low levels in the circulation, diminishing the risk of toxic side-effects [249, 250].
N-Benzyladriamycin-14-valerate (AD 198) is a non-activates PKC-γ, inducing the phosphorylation of phosphol- ipid scramblase 3 (PLS3) and apoptotic cell death [254]. It was previously demonstrated that PLS3 is involved in the regulation of mitochondrial structure, respiratory function and distribution of cardiolipin, therefore being critical in the regulation of apoptosis [255].Since DOX has a wide clinical application, it is essential to elucidate the mechanisms underlying DOX-induced toxic- ity in order to use that knowledge to develop new and more efficient therapeutic strategies.

CONCLUSION

DOX is perhaps the most widely used agent in the treat- ment of several types of cancers due to its wide spectrum of action. However, the dosage should be carefully considered from case to case since the use of this chemotherapeutic agent could lead to several adverse effects in different organs including the heart, brain, liver and kidney. Although the exact mechanisms of action of DOX are complex and still somewhat unclear, it is already known that this anticancer drug intercalates into DNA, inhibits topoisomerase II, im- pairs mitochondria and potentiates free radical formation and oxidative damage, among others. Several attempts have been made to decrease DOX side effects, such as the administra- tion of compounds with antioxidant and/or anti-apoptotic activity, the development of efficacious delivery systems and the production of DOX analogues. However, some of these strategies failed to alleviate anthracycline toxicity in clini- cally relevant animal models or clinical trials. Efforts should be employed in the search for more effective strategies against DOX toxicity while preserving or enhancing its therapeutic effects.

ACKNOWLEDGEMENTS
Work in the authors’ laboratory is funded by the Portu- guese Foundation for Science and Technology (FCT) (grant PTDC-SAU-OSM-64084-2006).

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