Review Cardiotonic steroids on the road to anti-cancer...

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Review

Cardiotonic steroids on the road to anti-cancer therapy

Tatjana Mijatovic a, Eric Van Quaquebeke a, Bruno Delest a, Olivier Debeir b,Francis Darro a, Robert Kiss c,d,⁎

a Unibioscreen SA, 40 Avenue Joseph Wybran, 1070 Brussels, Belgiumb Department of Logical and Numerical Systems, Faculty of Applied Science, Free University of Brussels (ULB), Brussels, Belgium

c Laboratory of Toxicology, Institute of Pharmacy, Free University of Brussels, Campus de la Plaine CP205/1, Boulevard du Triomphe, 1050 Brussels, Belgiumd Belgian National Fund for Scientific Research (FNRS, Belgium), Belgium

Received 23 April 2007; received in revised form 19 June 2007; accepted 21 June 2007Available online 4 July 2007

Abstract

The sodium pump, Na+/K+-ATPase, could be an important target for the development of anti-cancer drugs as it serves as a versatile signaltransducer, it is a key player in cell adhesion and its aberrant expression and activity are implicated in the development and progression of differentcancers. Cardiotonic steroids, known ligands of the sodium pump have been widely used for the treatment of heart failure. However, earlyepidemiological evaluations and subsequent demonstration of anti-cancer activity in vitro and in vivo have indicated the possibility of developingthis class of compound as chemotherapeutic agents in oncology. Their development to date as anti-cancer agents has however been impaired by anarrow therapeutic margin resulting from their potential to induce cardiovascular side-effects. The review will thus discuss (i) sodium pumpstructure, function, expression in diverse cancers and its chemical targeting and that of its sub-units, (ii) reported in vitro and in vivo anti-canceractivity of cardiotonic steroids, (iii) managing the toxicity of these compounds and the limitations of existing preclinical models to adequatelypredict the cardiotoxic potential of new molecules in man and (iv) the potential of chemical modification to reduce the cardiovascular side-effectsand improve the anti-cancer activity of new molecules.© 2007 Elsevier B.V. All rights reserved.

Keywords: Cardiotonic steroid; Na+/K+-ATPase; New anti-cancer drug; Sodium pump targeting

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332. Cardiotonic steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

2.1. Chemical structures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332.2. Natural sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332.3. Traditional use of cardiotonic steroid-containing plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352.4. Ligands of Na+/K+-ATPase and their use in cardiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362.5. Diversity of cardiotonic steroid effects relating to interactions with the sodium pump . . . . . . . . . . . . . . . . . . . . 37

3. The sodium pump (Na+/K+-ATPase) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383.1. The structure of Na+/K+-ATPase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383.2. Na+/K+-ATPase subunit diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393.3. Na+/K+-ATPase expression and regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403.4. Pharmacological properties of Na+/K+-ATPase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413.5. Na+/K+-ATPase cardiotonic steroid binding site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433.6. Na+/K+-ATPase functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Biochimica et Biophysica Acta 1776 (2007) 32–57www.elsevier.com/locate/bbacan

⁎ Corresponding author. Laboratory of Toxicology, Institute of Pharmacy, Free University of Brussels, Campus de la Plaine CP205/1, Boulevard du Triomphe, 1050Brussels, Belgium. Tel.: +32 477 62 20 83; fax: +32 23 32 53 35.

E-mail address: [email protected] (R. Kiss).

0304-419X/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.bbcan.2007.06.002


3.6.1. The sodium pump as an ion transporter: establishing and maintaining the electrochemical gradient in cells . . . . . 453.6.2. The sodium pump as a signal transducer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453.6.3. Novel functional interactions of the sodium pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

4. The sodium pump as a new target in anti-cancer therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465. Cardiotonic steroids: Sodium pump ligands as new anti-cancer agents: From epidemiology studies to preclinical evaluation . . . . . 47

5.1. Epidemiological data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475.2. Cardiotonic steroid-mediated in vitro anti-tumour effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475.3. Cardiotonic steroids as radiosensitisers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505.4. Cardiotonic steroid-mediated anti-tumour activity in vivo. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505.5. How can cardiotonic steroid-related cardiotoxicity be circumvented? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

6. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

1. Introduction

Cardiotonic steroids (CS) or cardiotonic glycosides representa group of compounds that share the capacity to bind to theextra-cellular surface of the main ion transport protein in thecell, the membrane-inserted sodium pump (Na+/K+-ATPase)[1,2]. These compounds have long been and continue to be usedin the treatment of congestive heart failure as positiveinotropic agents [3]. Retrospective epidemiological studiesconducted during the late 20th century revealed intriguingresults: very few patients maintained on CS treatment forheart problems died from cancer [4]. Over the last 10 years,interest in developing CS as anti-cancer agents has grownprogressively. In addition, a hypothesis was postulated thatalterations in the metabolism of endogenous digitalis-likecompounds and changes in their interactions with Na+/K+-ATPase might be associated with the development of canceritself [5,6]. Furthermore, a number of publications emphasizedthe altered expression of sodium pump subunits in differentcancer types when compared to corresponding normal tissues[7–10].

In line with these considerations, the present review will putforward the case for developing cardiotonic steroids as anti-cancer agents and discuss the challenges to be overcome.

2. Cardiotonic steroids

2.1. Chemical structures

Chemically, glycosylated CSs are compounds presenting asteroid nucleus with a lactone moiety at position 17 and a sugarmoiety at position 3 (Fig. 1).

The aglycone moiety is composed of the steroid nucleus (inblue in Fig. 1) and the R group (lactone ring in green in Fig. 1) atposition 17 that defines the class of cardiac glycoside. Twoclasses have been observed: the cardenolides (with anunsaturated butyrolactone ring) and the bufadienolides (withan α-pyrone ring). The steroid nucleus has a unique set of fusedring systems that makes the aglycone moiety structurallydistinct from the other more common steroid ring systems.Generally, in CSs from Digitalis and Strophantus plant species

(like digoxin, digitoxin, etc. Table 1A) rings A/B and C/D arecis fused while rings B/C are trans fused. Such ring fusiongives the aglycone nucleus of these cardiac glycosides acharacteristic ‘U’ shape (Figs. 1, 2). In CSs produced by plantsfrom the milkweed family Asclepiadacea (like calactin,uscharin, 2″-oxovoruscharin, etc.; Table 1A) A/B rings aretrans fused thus resulting in rather flat structures (Figs. 1, 2).

The steroidal skeleton as indicated above can be substitutedat position 3 by the third structural component, a sugar moiety(glycoside) leading to the chemical classification of sub-families as glycosylated cardenolides or glycosylated bufadie-nolides (depending on the lactone moiety). Up to 4 sugarmolecules may be present in cardiac glycosides; attached inmany via the 3β-OH group. The sugars most commonly found/used include L-rhamnose, D-glucose, D-digitoxose, D-digitalose,D-diginose, D-sarmentose, L-vallarose and D-fructose. Thesesugars predominantly exist in the cardiac glycosides in the β-conformation. Whereas the cardiac glycosides from Digitalisand Strophantus species carry sugar units linked through the3β-OH of the steroid aglycone (single link), some of thoseproduced by plants from the milkweed family Asclepiadaceacontain a single sugar in a unique “dioxanoid” attachment(double link) [11–15].

It is important to note that there exists a great naturaldiversity in the chemical family of CSs. The substituents andtheir stereochemical orientation, notably on the steroidalskeleton and the lactone moiety may vary greatly [11–16].The structures of certain CSs are given in Fig. 1.

2.2. Natural sources

As far back as the ancient Egyptians, different cultures havelong been known to use medicinal plants containing CSs. Themost well-known plant containing cardiac steroids is thefoxglove (the common name for plants of the Digitalis species(Digitalis lanata, Digitalis purpurea, etc.)). It was first reportedin 1542, when the German physician and professor of botanyLeonard Fuchs compiled a herbarium of all plants known at thetime. He gave the plant its name (from digitulus meaning a“small finger”) because its flowers were similar to a thimble.Today the word “digitalis” or “digitalis glycoside” is often used

33T. Mijatovic et al. / Biochimica et Biophysica Acta 1776 (2007) 32–57


as a generic term for all CSs. Several plants (more particularlythose belonging to Asclepiadacea, Apocynaceae, Ranuncula-ceae and Scrophulariaceae families) are recognized to containCSs (Table 1) [11–16]. Among common decorative plants richin CSs are the Christmas rose, lily of the valley, water lily andoleander. CSs are also extensively found in animal species andoccur mainly in toads (species of the Bufo genera, Table 2)[11,12].

Several independent investigators have also found thatmammalian tissues and body fluids (including brain, adrenalglands, heart, blood plasma, cerebrospinal fluid and urine)contain digitalis-like compounds. In line with this: (i) ouabainwas identified in human plasma, hypothalamus and the adrenalgland [17–19], (ii) an ouabain isomer was detected in bovinehypothalamus [20], (iii) digoxin was shown to be present inhuman urine [21], (iv) 19-norbufalin and its peptide derivativewere identified in cataractous human lenses [22] and (v)dihydropyrone substituted bufadienolide was discovered inhuman placenta [23]. In addition, immunoreactivity ofmarinobufagenin-like [24] and proscillaridin A-like compounds[25], which both also belong to the bufadienolide family, hasbeen evidenced in human plasma and urine [24,26].

Furthermore, it has been demonstrated that cholesterol is asubstrate for the biosynthesis of endogenous CSs in mammaliansystems i.e. in the adrenal gland. This is effected via 3β-hydroxysteroid dehydrogenase/isomerase, which convertrespectively cholesterol into pregnenolone and progesterone;required for the initial step in the biosynthetic pathway ofendogenous CSs, namely cytochrome P450-cholesterol side-chain cleavage [27]. The bufadienolide marinobufagenin(Fig. 1) is produced in a pathway leading from acetate throughmevalonate to cholesterol, which at least partially overlaps thepathway by which all steroid hormones are synthesized in theabsence of exogenous cholesterol [28].

It is important to emphasize that endogenous ouabainisolated from mammals is identical to plant-derived ouabain[19]. Ouabain's release from adrenal glands is under thecontrol of epinephrine and angiotensin II and thus its bloodconcentration changes rapidly on physical exercise [29]. It isnot known whether there are storage vesicles which releaseouabain upon hormonal stimulus. It has also been demon-strated that endogenous digoxin evidently counteracts thehypertensive effect of endogenous ouabain. The plasmaconcentration of endogenous marinobufagenin is increased

Fig. 1. Classification and chemical structures of cardiotonic steroids.

34 T. Mijatovic et al. / Biochimica et Biophysica Acta 1776 (2007) 32–57


after cardiac infarction. It may show natriuretic propertiesbecause it inhibits the α1 isoform of Na+/K+-ATPase, the mainisoform in the kidney, much more potently than other sodiumpump isoforms. More precisely, the IC50 values for inhibition ofNa+/K+-ATPase by ouabain and marinobufagenin were2.6 nmol/L and 0.14 μmol/L in the neuronal plasmalemma(containing predominantly the α3 isoform), and 50 nmol/L and2.1 nmol/L in sarcolemma (containing predominantly the α1isoform) respectively [30]. Therefore, the resulting action of thedifferent endogenous cardiotonic steroids seems to be a co-operative effect in handling salt and water homeostasis [19].Apparently, Na+/K+-ATPase is used by these steroids as a signaltransducer to activate tissue proliferation, heart contractility,arterial hypertension and natriuresis via various intra-cellularsignalling pathways [29].

2.3. Traditional use of cardiotonic steroid-containing plants

Extracts of CS-containing plants and frogs have long beenused as arrow poisons [12,31,32]. These are prepared bytraditional methods for example from Acokanthera schimperi(containing acolongifloroside K as its major active principle, aswell as smaller amounts of ouabain and acovenoside A) in theMaasai plains of Kenya or from the ouabaio tree (containingouabain) by East African Somalis [31,32].

Ancient Greeks and Romans used the juice of the foxglovefor sprains and bruises [33]. Medieval witches grew foxglovesin their gardens to use as a potent ingredient in their potions andeven extracted the chemical digitalis for use as a rapid actionpoison. The foxglove was then discovered in the 1700s tostimulate the kidneys to release excess fluid. A tea brewed from

Table 1

(continued on next page)



the leaves was used to treat Dropsy, a condition in which wateraccumulates in the body and causes excessive swelling. From1785, when Sir William Withering published his textbook Onthe account of the foxglove [34], physicians have used digitalispreparations to treat edematous states, irregular heart beats andchronic heart failure. According to Withering, digitalis slowedthe heart rate in patients with irregular pulse and resulted indiuresis.

As early as the Middle Ages, extracts of the oleanderwere being used in the treatment of cancer. Oleander use inthe treatment of cancer has been documented for a numberof distinct geographical locations including Egypt, Vene-zuela, Cuba and India [35]. In Ireland and Turkey, aqueousoleander extracts have been used to treat terminally illpatients with various advanced cancers as well as patientswith AIDS [36].

2.4. Ligands of Na+/K+-ATPase and their use in cardiology

CSs and particularly digitalis are still widely used incardiology today (for reviews see [3,37]). However, modernunderstanding of digitalis therapy arose 50 years ago when itwas discovered that CSs are specific inhibitors of the sodiumpump [2] and that the digitalis receptor is the Na+/K+-ATPase ofplasma membranes [38]. This inhibition promotes sodium–calcium exchange, which increases the intra-cellular calciumconcentration that is available for the contractile proteins,resulting in an increase in the force of myocardial contraction[3,37,39,40]. Digitalis drugs (common preparations includedigitoxin and digoxin) are used in modern medicine to increasethe force of the systolic contractions and prolong duration of thediastolic phase in congestive heart failure [3,37,40]. Digitalisslows the pulse and the conduction of nerve impulses in the

Data from [11–15]; concerns Table 1A.Data from [11,12,16]; concerns Table 1B.

Table 1 (continued)



heart and increases the force of heart contractions and theamount of blood pumped per heart beat [3,37]. Digoxin isindicated in patients with heart failure and impaired systolicfunction who are in sinus rhythm and continue to have signs andsymptoms, despite standard therapy that includes angiotensin-converting enzyme inhibitors and beta-blockers [3,37].

The main reported CS-mediated toxicity is cardiotoxicity.Digoxin intoxication can cause arrhythmias and also gastro-intestinal and/or central nervous system abnormalities. Bene-ficial clinical digoxin-mediated effects rely on low serumconcentrations of b1 ng/mL [41]. A digoxin dose of 0.125 mgdaily results in a concentration of ∼0.8 ng/mL [42]. Higherdigoxin serum concentrations can provoke toxicity. Theincidence of digoxin-induced arrhythmia at a concentration of1.7 ng/mL is 10% and at 2.5 ng/mL is 50% and increases furtherwith increasing serum levels [37]. Therapeutic concentrations ofCSs produce partial inhibition of Na+/K+-ATPase (∼30%;[11]). When the concentration of digitalis compounds reaches

toxic levels, Na+/K+- ATPase inhibition is too high (N60%) anda sustained increase in [Na+]i and thus of [Ca2+]i, gives rise tothe toxic effects (i.e. arrhythmia) of these compounds [11,39].Despite the fact that the therapeutic doses of digitaliscompounds are dangerously close to the lethal doses they stillremain the most effective drugs available for the treatment ofheart failure caused by hypertension or arteriosclerosis.Approximately 1.7 million patients in the United States werereported to be receiving digoxin for heart failure and/or atrialfibrillation [3].

2.5. Diversity of cardiotonic steroid effects relating tointeractions with the sodium pump

There are an ever growing number of studies reportedconcerning the diverse effects of CSs in disease andtherapeutics. To date, these CSs-mediated effects have beenfound to result from their interaction with one specific receptor,

Fig. 2. CS binding to the sodium pump. CSs inhibit the sodium pump by binding to the extra-cellular surface of the pump; the binding “groove” being composedof multiple functional groups in the α subunit and to a lesser extent in the β subunit. Changes in amino acid composition and charge affect the conformation ofthe CS binding site. Mutagenesis studies have identified several amino acid residues found in the transmembrane TM1, TM4, TM7 and TM10 segments and inthe TM1–TM2, TM5–TM6 and TM7–TM8 loops, as important for the high affinity binding of ouabain. CSs are characterized by a great diversity of structurewhich can have an impact on their binding properties. One major structural difference concerning the steroid backbone is able to impact on CS binding features.This difference is delineated by “U shaped” CSs with cis–trans–cis fused steroid rings (as illustrated here with the oleandrin structure) versus “flat shape”cardenolides with trans–trans–cis fused steroid rings (as illustrated here with the structure of the 2″-oxovoruscharin derivative UNBS1450). The 3D structureswere obtained using the Chem Sketch software.



the sodium pump, to which this class of compound are reportedto bind. The actions of CSs are thus generally indissociablefrom their interaction with the sodium pump. Although Na+/K+-ATPase is obviously the major pharmacological target ofcardiac steroids, it should be noted that non-gastric H+/K+-ATPase activity can also be inhibited by relatively high ouabainconcentrations (μM to mM range depending on experimentalsystem used) [43–45].

3. The sodium pump (Na+/K+-ATPase)

The sodium pump is found in the cells of all highereukaryotes and is responsible for translocating sodium andpotassium ions across the cell membrane utilising ATP as the

driving force [46,47]. The existence of an ATP hydrolysingenzyme that requires the presence of sodium and potassium foractivity was first demonstrated conclusively in crab nerve byJens Christian Skou [38]. In 1997 Jens Christian Skou wasawarded the Nobel Prize for his outstanding contribution to thefield of Na+/K+-ATPase research.

3.1. The structure of Na+/K+-ATPase

The sodium pump is composed of two subunits in equimolarratios: (i) the α catalytic subunit which is a multipasstransmembrane protein containing the binding sites for Na+,K+, ATP and CSs, and (ii) the β regulatory subunit, a trans-membrane protein with several glycosylation sites, required for

Table 2Bufadienolide-containing toads

Data from [11,12].



the biogenesis and activity of the enzyme complex (Fig. 3).There are four different α and three β subunit isoformsidentified (the existence of two additional β subunits has beenreported: one found in human, pig, rat and mouse muscle (β-m)and the second in astrocytic glia) that are selectively expressedin various tissues [47–49]. All possible α,β combinations resultin catalytically competent enzymes, indicating that multipleNa+/K+-ATPase isozymes can operate in the cell [46,49–55].However, it is not known if in native tissues, the assembly ofparticular isoforms is regulated to favour formation of someisozymes over others. Additionally, in certain cells and tissues(Fig. 3), a third subunit type, has been found to be associatedwith functional Na+/K+-ATPase [56,57]. The members of thisthird type of subunit are regulatory single span (except forFXYD3: which is double span) type I transmembrane proteinsfrom the FXYD family, that are able to modify sodium pumptransport properties in a tissue- and isoform-specific manner(Fig. 4). To date seven members (FXYD1–7) of this familyhave been described. Li et al. [58] have shown that FXYD2

(first discovered and named the γ subunit) FXYD4 and FXYD7interact with α subunit TM9 segment (Fig. 4). Co-expressingFXYD5 with the α1 and β1 subunits of Na+/K+-ATPase inXenopus oocytes elicited a more than 2-fold increase in pumpactivity [59]. FXYD1, FXYD2 and FXYD3 decrease butFXYD4 increases sodium affinity, while FXYD2 increases ATPaffinity. Additionally, FXYD7 decreases the apparent K+

affinity of α1β1 and α2β1 but not of α3β1 isozymes [60].Increasing numbers of publications (mainly from the Geeringgroup) emphasize the role, structure and functions of theseproteins, revealing them to be fine regulators of sodium pumpfunctions.

3.2. Na+/K+-ATPase subunit diversity

Na+/K+-ATPase α subunits have similar primary structuresacross species. α1 and α2 share∼92% homology in their aminoacid content and for α3 this is N96%. The α4 subunits from rat,mouse and human have 90% similarity in their amino acid

Fig. 3. Schematic representation of the sodium pump. The sodium pump is composed of two subunits in equimolar ratios: catalytic α (10-pass transmembrane proteins;presented in yellow) and the regulatory β (transmembrane proteins containing several glycosylation sites; presented in green). In some cells and tissues, an additionalthird subunit (presented in red) has been found to be associated with functional Na+/K+-ATPase. This third type of subunit is regulatory and belongs to transmembraneproteins from the FXYD family. There are four different α and three β subunit isoforms cloned to date. Sodium pump subunits are selectively expressed in varioustissues and in a species-dependent manner. This figure summarizes α and β isoform expression patterns for normal human cells and tissues, as well as human FXYDdistribution. The first extra-cellular loop of the α subunit (amino acids 111–122) is the most important component of the CS binding site. The basis for the relativesensitivity/insensitivity of the different α isoforms to ouabain resides mostly in two amino acids in this first extra-cellular region of the α subunit. The mouse α2isoform, which is sensitive to ouabain, has a leucine at position 111 and an asparagine at position 122, in contrast to the ouabain-insensitive α1 isoform where arginineand aspartic acid appear at these sites. The wild-type human α1 subunit is highly sensitive to ouabain inhibition while the rat α1 subunit is essentially ouabaininsensitive (∼1000-fold less sensitivity than the human subunit). Two key residues, glutamine Q111 and asparagine N122 are invariant in the human subunit but arerespectively, mutated to arginine (R) and aspartic acid (D) in rat α1. The two key residues (Q111 and N122) are invariant in all four human α subunits, characterized bythe same affinity (nM range) towards CSs. This figure is based on data reviewed in [49,56,57,61].



sequence. However, comparison of different α isoforms withinthe same species reveals a lower degree of similarity (∼87% forα1, α2, and α3 while α4 shares only 76% to 78% identity withthe other isoforms). The highest sequence similarities acrossthese different α subunits correspond to (i) the transmembranehydrophobic regions, (ii) the cytoplasmic mid-region around thephosphorylation site (Asp369) and (iii) the C-terminus[49,50,61]. In contrast, the highest structural variability acrossthese different α subunits is found in (i) the N-terminal portionof the polypeptides, (ii) the extra-cellular loop betweentransmembrane segments 1 and 2 that forms part of the ouabainbinding site and (iii) the isoform-specific region, an 11 aminoacid sequence in the major loop between transmembranedomains 4 and 5 that comprises residues 489 to 499 of the rat α1isoform [49,50,62].

The β subunits are also highly conserved across species:94% to 96% amino acid similarity for β1 and β2, and 75% forβ3. Within the same species, β subunits are more divergent thanthe α ones: β2 and β3 share with β1 respectively only 34% and39% similarity, whereas β2 and β3 have 49% amino acididentity. The major structural similarities across β subunitsinclude the transmembrane region and the 6 cysteines of theextra-cellular domain that participate in the formation ofdisulfide bridges. The β subunits are also characterized byglycosylation sites, which vary in different tissues and species,

and in their location and number: three N-linked glycosylationsites are present in β1, whereas 2 to 8 have been predicted in β2and β3 (reviewed in [49,50,61]).

3.3. Na+/K+-ATPase expression and regulation

Na+/K+-ATPase isozyme/subunit expression in varioustissues has been extensively reviewed by other authorspreviously [49,50,61], thus only a limited summary of theirselective distribution in normal human cells and tissues is givenhere (Fig. 3). Besides this tissue-specific expression patternunder normal physiological conditions, sodium pump isoformexpression is specifically altered in a tissue-specific manner indiseases such as hyper- and hypothyroidism, hypokalemia,hypertension, heart failure [63–66] and also in cancer cells andtissues [7–10,67–70]. Additionally, certain FXYD proteins areexpressed or over-expressed in cancer. Of the sodium pumpisozymes, α1β1 is the most widely expressed, while other αand β subunit combinations exhibit a much more restrictedpattern of expression. It is also interesting to note the exclusiveexpression of α4 in testes and spermatozoids [48–50].

Overall, Na+/K+-ATPase is most abundantly expressed inion-transporting epithelia (e.g. kidney) and in excitable tissuessuch as the brain, skeletal muscle and cardiac muscle [71–76].Tissue-dependent variation in Na+/K+-ATPase expression can

Fig. 4. Interaction of FXYD family proteins with the sodium pump. Schematic representation of the sodium pump α subunit transmembrane segments and loops(in green) spatial distribution with respect to the interaction with FXYD proteins (third subunit type) (in red) and the portion of the extra-cellular loop involved inthe interaction with β subunit indicated in grey. The table summarizes the different known effects of the FXYD proteins on sodium pump transport and activity.This figure is based on data reported in [56–60].



reach differences of 160,000 fold (erythrocytes vs. braincortex), while vascular smooth muscle with 400,000–700,000pumps/cell has 100 times lower levels than heart muscle, asevaluated by means of 3H-ouabain binding [77–79].

Sodium pump isoform expression is also developmentallyregulated. Rat fœtal heart expresses α1 and α3, while ratadult heart expresses α1 and α2 [49,50,61]. It is interesting tonote that in vitro culture conditions can significantly influencesodium pump isoform expression. Rat cardiomyocytescultured in serum-free medium display a neonatal Na+/K+-ATPase isoform composition of α1 and α3. In contrast, thosecultured in serum-supplemented medium revealed a gradualdecline in α3 and the appearance of α2 Na+/K+-ATPaseisoforms (albeit at a relatively low level; [80,81]). Sharabani-Yosef et al. [81] reported that while freshly isolated skeletalmuscle from new born rats expressed α1 and α2 subunitproteins, primary cultures from day 1 after plating expressedonly α1. Further studies revealed mRNA expression for α1,α2, β1 and β2 both in freshly isolated muscle and afterplating of cells in culture. These findings indicate that thelack of α2 protein expression in primary muscle cell culturesreflects a form of post-transcriptional regulation.

Sodium pump subunit expression and activity are regulatedin a timely manner. Short-term regulation of Na+/K+-ATPasefunction can be achieved by changes in: (i) intra-cellular sodiumconcentration, (ii) the number of enzyme molecules in the cellplasma membrane (increases in plasma membrane sodiumpump proteins due to trafficking of heterodimers from intra-cellular pools), (iii) the catalytic property of the enzyme alreadypresent in the plasma membrane and (iv) the PKA and PKC-mediated phosphorylation of α subunits, as certain phosphor-ylations can inhibit enzyme activity by reducing transportactivity or by inducing clathrin-mediated enzyme internalisation[61,82–85]. Adrenergic agonists increase the pump's affinityfor Na+ and recruit sodium pump subunits to the basolateralplasma membrane from intra-cellular endosomal compartments[86]. In lung alveolar epithelial cells, activation of G protein-coupled receptors, via either dopaminergic or adrenergic stimuli,rapidly (30 s to 15 min) increases Na+/K+-ATPase activity byinsertion of sodium pump proteins from intra-cellular compart-ments into the plasma membrane [87–92]. These effects aredependent on a dynamic interaction between protein-transport-ing vessicles, microtubulae and the actin cytoskeleton, as pre-treatment with colchicine, brefeldine or phallacidin prevents thisrecruitment.

Long-term regulation of Na+/K+-ATPase occurs via tran-scriptional and post-transcriptional mechanisms, includingchanges in transcription rate and mRNA stability, translation,protein degradation, and in membrane enzyme-specific activity

(reviewed in [93–95]). Transcriptional regulation of sodiumpump subunit genes is an important component of themultifaceted response to hormonal stimulation, hyperoxia andcellular stress. Because the subunit genes are on differentchromosomes, transcription may be independently regulated.Increased transcription of the Na+/K+-ATPase subunit genes inthe lung may be mediated by compounds and hormones such asdexamethasone, insulin and aldosterone [82]. Aldosteroneincreases both transcription and plasma membrane insertionof pre-formed pump molecules [83,96]. Both functional enzymeactivity and gene transcription are increased by low intra-cellular K+ or high Na+ concentrations or by various hormones,including thyroid hormone [97]. A recent study reported thatadrenergic stimulation of serum-starved alveolar epithelial cellsregulated Na+/K+-ATPase translation via extra-cellular regu-lated kinase-rapamycin pathways independent of changes inNa+/K+-ATPase transcription [98]. Translation of Na+/K+-ATPase mRNA is an important locus of regulation in a varietyof settings. For example, similar increases in steady-state levelsof mRNA result in different activity levels of the sodium pump,indicating that post-transcriptional steps play a role in theregulation of Na+/K+-ATPase [98,99]. In vitro studies oftranslation demonstrated that untranslated mRNA regions(UTRs) could affect subunit translation. The mRNA for α1 istranslated less efficiently than that for β1 because of α1mRNA's 3′UTR region being extremely GC rich and folded ina complex fashion and because translational efficiency may bealtered by glucocorticoids [100].

Besides being the regulators of sodium pump activity, CSsare also potent regulators of sodium pump expression. Rosenet al. [101] reported that CSs lead to the appearance ofcytoplasmic vacuoles containing membrane components includ-ing sodium pumps, decreasing thus the number of pumps at thecell surface.

3.4. Pharmacological properties of Na+/K+-ATPase

Conspicuous kinetic differences exist between sodium pumpisozymes and across species in their interaction to CSs i.e.ouabain (Table 3) [49,50,55]. In the rat, inhibition sensitivity tothe cardiotonic steroids can vary markedly with Ki values forα1, α2 and α3 that are in the mM, μM and nM range,respectively [53]. The basis for the relative insensitivity of themouse and rat α1 isoforms to ouabain resides in two aminoacids in the first extra-cellular region of the α1 subunit (Fig. 3)[102]. The mouse α2 isoform, which is sensitive to ouabain, hasa leucine at position 111 and an asparagine at position 122, incontrast to the ouabain-insensitive α1 where arginine andaspartic acid appear at these sites (Fig. 3). The positive charge of

Table 3Determination of inhibitory constants (Ki values in M) for ouabain against human and rodent sodium pump isozymes

α1β1 α2β1 α2β2 α3β1 α3β2

Human isozymes 4.9±1.7×10−9 22.0±4.5×10−9 25.8±3.2×10−9 4.6±0.5×10−9 10.1±2.1×10−9

Rodent isozymes 4.3±1.9×10−5 1.70±0.1×10−7 1.5±0.2×10−7 3.1±0.3×10−8 4.7±0.4×10−8

These data are from [49,55,61].



the latter two amino acids appears to confer resistance toouabain [102]. This resistance seems to relate to evolutionaryadaptations, as suggested by studies made in cardenolide-resistant insects [103,104]. Insects specialized in feeding oncardenolide-rich plants have adapted to the presence of thesetoxic compounds and in certain cases actually accumulate themin their bodies to deter predators. This is particularly so in thecase of the monarch butterfly (Danaus plexippus) whosecaterpillars feed almost exclusively on cardenolide-rich milk-weeds, and two coleoptera species Chrysococus auratus andC. cobaltinus.

These three evolutionary distinct species carry a histidine atposition 122 of their α1 subunit whereas ouabain-sensitiveinsects and vertebrates carry an asparagine instead. Sheep,human and rat sequences for the α1 subunit of Na+/K+-ATPasediffer sequentially by only ∼3%, yet modest single or doubleresidue substitutions can result in an extraordinary variability inthe enzyme's sensitivity to ouabain inhibition. For example, thewild-type sheep and human α1 subunits are highly sensitive toouabain inhibition while the rat α1 subunit is essentiallyouabain insensitive (∼1000-fold less sensitive). Two keyresidues, glutamine Q111 and asparagine N122, are invariantin sheep and human subunits but are respectively mutated toarginine (R) and aspartic acid (D) in rat (Fig. 3). Incorporatingthe single mutation Q111R into the wild-type sheep α1 results ina ∼10-fold decrease in ouabain inhibition, while expression ofthe N122D or the double mutation Q111R, N122D diminishesouabain inhibition by ∼1000 fold [105,106].

The increased α2 subunit sensitivity to ouabain and the factthat rodent heart expresses the ouabain-resistant α1 andouabain-sensitive α2 subunits has led to the hypothesis thatα2 may be responsible for mediating the inotropic effect ofdigitalis in the heart and to the concept of different subunitsmediating inotropic and toxic effects. Several mutagenesisstudies have been undertaken to assess this. The importance ofboth α1 and α2 subunits for survival was demonstrated usingknockout mice. Animals lacking the α1 gene died duringembryogenesis (embryos failed to develop beyond the blas-tocyst stage) [107,108] while animals lacking the α2 gene wereborn but died immediately following birth [107]. In contrast toanimals lacking both copies of either the α1 or α2 gene, animalslacking only one copy of either gene survived and appearednormal. Whereas the ablation and reduction of α1 or α2 showedthe importance of these subunits, these studies failed toconclusively demonstrate their physiological function, mainlydue to long-term compensation that occurs during developmentin knockout animals. As already emphasized, sensitivity toouabain is mainly driven by two amino acids in the first extra-cellular loop. Mice modified by the introduction of two keymutations rendering α1 ouabain-sensitive and α2 ouabain-insensitive, in which each isoform can be individually inhibitedby ouabain and its function determined, demonstrated that bothα1 and α2 subunits are able to mediate the inotropic effect ofdigitalis [109,110]. Indeed both subunit types (i) are regulatorsof cardiac muscle contractility, (ii) co-localize with the Na/Caexchanger and (iii) can mediate ACTH-induced hypertensionand thus have physiological significance in the regulation of

blood pressure [109,110]. In summary, from the studiesinvolving sodium pumps from species with ouabain-sensitiveand ouabain-resistant isoforms, the inotropic effects are likely tobe mainly mediated by ouabain-sensitive isoforms (either bynaturally sensitive forms in wild type animals or by geneticallymodified isoforms rendering them sensitive to CS). In contrast,in the human heart, which contains not only α1 and α2 but alsoα3 subunits, all were found to be highly sensitive to ouabain,and digitalis inotropy and toxicity could not be attributed to thesole inhibitory action of one specific α sub-type. However, anα2 sub-type specific function in the heart might be supported bythe observations that: (i) ouabain binding to α1 but not to α2 isefficiently antagonized by K+ at physiological concentrationsand (ii) α2 has 5–10 times faster ouabain association anddissociation kinetics [55], and thus it could be an importantcomponent with respect to the circulating digitalis concentra-tions. However, by comparing pharmacological properties ofsodium pump isoforms from microsomes prepared from non-failing human heart with those separately expressed in Xenopusoocytes, Lelievre et al. [111] reported the existence of two highaffinity ouabain binding sites: the very high affinity site forouabain deemed to be the α1β1 dimer and the high affinity sitedefined as α2β1. This was further supported by Crambert et al.[55] reporting that α2-containing isozymes had slightly higherKd values (12–23 nM) than α1- or α3-containing isozymes(5–7 nM). Thus, in contrast to rodents and other speciescontaining both sensitive and insensitive isoforms, the conceptof inotropic and toxic isoforms based on different affinities fordigitalis, is not justified in humans, which may explain thenarrow therapeutic margin for digitalis-like compounds.

In conclusion, besides the presence of particular isoforms inhuman heart, several other factors need to be considered asmediating the inotropic and potentially toxic effects of CSs,such as (i) the relative proportion of each isoform (and thusdifferences in their kinetic properties, saturation and compen-satory mechanisms that can develop), (ii) the number ofexpressed pumps per cell, (iii) the presence of specificadditional polypeptides, like FXYD proteins or transportersmodifying sodium pump kinetic properties (Fig. 4), (iv) the K+

concentration (hypo- or hyperkalemia; affecting sensitivity toCSs, as discussed above), (v) the half-life of CSs (for digoxin inhumans this is quite long: 36 to 48 h in patients with normalrenal function and 3.5 to 5 days in anuric patients) contributingthus to cumulative toxicity, (vi) the production of endogenouscardiotonic steroids that may be synthesized in sufficientconcentrations to contribute to sodium pump inhibition/saturation, (vii) the effect of various hormones on sodiumpump isoform expression and (viii) the presence of other drugsor neuropeptides (like neurotensin, [112]) able to affect sodiumpump activity.

The effect of K+ on CS binding is particularly interestingbecause according to the results reported by Crambert et al.[55], at physiological K+ concentrations, digitalis at therapeuticdoses may predominantly bind to α2- and α3-containingisozymes present in human heart and to a lesser extent to α1-containing isozymes. Therefore in the light of similar ouabainaffinities for Na+/K+-ATPase isozymes, it is likely that in



humans, the distinct K+/ouabain antagonism of differentisozymes is at the origin of both beneficial and toxic effectsof these compounds. It would appear that at physiological K+

concentrations, this would favour inhibition of the α2 andα3-containing isozymes present in neurons and the heart andprotect the ubiquitous α1-containing isozymes. Small changesin the status of (i) plasma K+ concentrations (leading tohypokalemia), (ii) digitalis concentrations (elevated throughan overdose of medication or by the additive effects ofmedication and over-production of endogenous CSs) and (iii)Na+/K+-ATPase expression (resulting from hypo- or hyper-thyroidism, diabetes, etc.) may lead to toxic effects. In contrast,since some pathological states (different cancer types) arecharacterized by over-expression of different α and β subunits,the increased number of expressed pumps may consequentlydecrease the level of toxicity of administered CSs, thusincreasing overall tolerance.

When comparing sodium pump isoform inhibition by CSs, itis important to compare the fold differences and not the absolutevalues. One should also bear in mind that different experimentalsystems yield different Ki values, due to factors able to affectassay sensitivity, such as lipid composition in the case ofextracted enzymes, the presence of endogenous (in)sensitiveenzymes in case of expression systems, the proportional subunitcomposition in preparations, etc. The transport and kineticproperties of sodium pump isoforms, notably α subunits, havebeen assessed by different techniques including their hetero-logous expression in a range of cells: yeast, Xenopus oocytesand Sf-9 insect cells [50,55,113–117]. These studies revealedthat while all human α subunits have similar sensitivity toouabain (nM range), they differ in association/dissociationrates: α2 has 5–10 times fold faster ouabain association anddissociation kinetics. According to Crambert et al. [55], humanα,β complexes formed with α1 and α3 subunits have slowdissociation rate constants corresponding to half-lives (t1/2)between 30 and 80 min, whereas those formed with α2 haverapid dissociation kinetics with a t1/2 of about 4–5 min.Similarly, the association kinetics of ouabain to human Na+/K+-ATPase isozymes followed the order α2NNα3=α1, with timerequired to reach equilibrium binding in the range 10 min(α2,β) and 60 min (α1,β and α3,β). These characteristics arehighly important when considering the evaluation of potentialtoxic effects of digitalis compounds. The association rate ofouabain seems to be dependent on the steroid moiety, whereasthe dissociation rate depends both on steroid and sugar moieties.Several amino acids have been determined to be involved inouabain binding kinetics [55,106].

While ouabain binding to α1 can be achieved only in thephosphorylated state of the Na+/K+-ATPase dimer, its bindingto α2 is possible both in the phosphorylated and non-phosphorylated (K+ occluded) state [55,61,106]. Ouabainbinding to α1 but not to α2 is efficiently antagonized by K+

at physiological concentrations. More precisely, according toCrambert et al. [55] for human α1,β dimers, 5 mM K+ induceda larger increase in Kd values for ouabain (3–4 fold) than forα2,β and α3,β complexes (2–3 fold), with the exception ofα2β2 dimers for which the Kd value was not influenced by the

presence of K+. These isoform-specific differences in the K+/ouabain antagonism may protect α1 but not α2 or α3 fromdigitalis inhibition at physiological K+ concentrations. Co-operation of transmembrane domains M1–2 with M3–4 and theformation of a “gate-like structure” is possible in α1 (due to thepresence of hydroxyl groups and negatively charged aminoacids) but not in α2. With respect to human sodium pumpturnover rates, Crambert et al. [55] also established that theseare mainly dependent on the α subunit and follows the order:α1,βNα2,βNα3,β (slowest). In view of the variable environ-mental and experimental conditions used in different studies,these data on human isozymes cannot easily be compared withavailable data on Na+/K+-ATPase from other species.

Associated with their transport and pharmacological proper-ties, human Na+/K+-ATPases also show: (i) an apparent sodiumaffinity (dependent mainly on α isoforms: α1Nα2Nα3), (ii) anapparent potassium affinity (dependent on both α andβ subunits: α2β1NNα2β2) and (iii) a voltage dependency(resulting mainly from α isoforms: α1Nα2 with α3-containingisozymes nearly voltage independent). The presence of all threeα-containing isozymes in human cardiomyocytes indicates thation homeostasis is also finely regulated in these cells as it is inskeletal muscle and neurons; with the Na+/K+-ATPase isozymesformed with the three α subunits possibly acting in concert tomeet physiological needs. A particular feature of α2-containingisozymes which relates to their ouabain binding kinetics led tospeculation of a further pharmacological role during digitalistreatment. Based on the important mass of muscle tissue in thebody and the predominant expression of Na+/K+-ATPase α2isozymes in this tissue, some investigators have suggested thatthis isozyme might function as a store for digitalis during heartfailure treatment [118] and/or that it might also be an importantcomponent of the regulation of digitalis circulating concentra-tions by binding or releasing the compound in response tochanging plasma levels, due to increased digitalis administra-tion, metabolism or elimination [55].

3.5. Na+/K+-ATPase cardiotonic steroid binding site

Na+/K+-ATPase has an evolutionarily conserved cardiacglycoside binding site [109]. The discovery of endogenous CSsin mammals (ouabain and marinobufagenin) capable of bindingto the sodium pump and evidence that the CS binding site on theα subunit can also mediate ACTH-induced hypertension [109]have all served to demonstrate that the CS binding site of theNa+/K+-ATPase α subunit has a physiological function.

CSs inhibit the sodium pump by interacting with an extra-cellular surface binding “groove” composed of multiplefunctional groups in the α and to a lesser extent the β subunit[46,47]. Essentially, the most important part of the binding site isrepresented by the first extra-cellular loop of the α subunit(amino acids 111–122). Changes in amino acid composition andcharge affect the conformation of CS binding site. Mutagenesisstudies have identified several amino acid residues found in theTM1, TM4, TM7 and TM10 segments and in TM1–TM2,TM5–TM6 and TM7–TM8 loops, as important for the highaffinity binding of ouabain (reviewed in [106]). Fig. 5A outlines



(in red) the reported amino acids that are involved in interactionswith CSs. In order to further explore the CS-binding sites, thegroup of De Pont adopted an original strategy in which ouabain-insensitive (or weakly sensitive) members of the IIC subfamilyP-type ATPases (i.e. gastric and non-gastric H+/K+-ATPase)were used as templates for mutational elucidation of the ouabain-binding site. With this strategy, they were able to transfer thehigh affinity binding site for ouabain to the ouabain-insensitivegastric H+/K+-ATPase by mutation of only seven amino acids,originating from extra-cellularly located parts of M4, M5 and

M6 of Na+/K+-ATPase [119]. Furthermore, they demonstratedthat the low affinity binding site for ouabain in the rat non-gastricH+/K+-ATPase could be converted into a high affinity bindingsite by replacement of only five amino acids with their counter-parts present in the catalytic subunit of rat Na+/K+-ATPase[120]. These results revealed that Na+/K+-ATPase residuesGlu312, Gly319, Pro778, Leu795 and Cys802 are important for highaffinity ouabain binding.

By docking a series of cardiac glycosides (9 different,repeated 12 times) onto the modelled extra-cellular surface of

Fig. 5. CS binding to the sodium pump. (A) Sequence alignment of porcine α1, murine α1 and human α1, -2, -3 and -4 sodium pump subunits with the amino acidsinvolved in interactions with CSs outlined in red, transmembrane domains are underlined in blue and extra-cellular domains are underlined in green. (B) Schematicrepresentation (adapted from one proposed by Keenan et al. [106]) of the extra-cellular loops and the transmembrane domains of the sodium pump α subunit. Bindingorientations of CSs to the α subunit, indicated by black arrows, are demonstrated using as the model molecule UNBS1450 (shown as 2D and 3D-structures).



the α1 subunit, Keenan et al. [106] were able to pinpoint aconsensus drug-binding site that accommodates the lactone ringmoiety orientated towards the TM1–TM2 loop (Fig. 5B) andthe sugar moieties directed towards the TM9–TM10 loop (forcompounds with multiple (bis- and tri-) sugar units thesemoieties appear to interact with the TM7–TM8 loop), with twoto three likely hydrogen-bond interactions between the drug andreceptor. In line with this, a potential hydrogen bond locatedbetween the keto-group of the lactone ring and the side chain ofα1 residue Q111, is consistent with the ∼10-fold loss insensitivity to ouabain observed for the Q111R mutant versus thewild-type sheep enzyme, resulting from re-orientation of theglycoside's substituted lactone moiety from the TM1–TM2loop toward the TM3–TM4 loop. Additional structural modelsof the cardiac glycoside-insensitive rat enzyme α1 subunit andmutated variants of sheep α1 reveal the possible loss of one ortwo hydrogen-bond interactions present in normal sheep andhuman α1 subunit models that consequently lead to decreasedouabain sensitivity. This binding model also suggests that theside chain of glutamate E908 and the backbone nitrogen ofmethionine M973 interact with the sugar and are respectively,hydrogen bond acceptors and donors, and thus potentiallycontribute to ouabain's high binding affinity. While the hydro-phobic nature of the steroid moiety is crucial for drug binding,the model does not show any obvious aromatic groups locatedwithin the binding groove about the hydrophobic steroid. Aplausible explanation is that these results illustrate the inherentflexibility of this region of the inhibitor, allowing for theenzyme to proceed through a series of ligand-induced con-formational changes [106].

As already indicated, CSs are characterized by a greatdiversity of structure which can greatly impact on their bindingproperties. Besides the presence or absence of a glycosidemoiety and the lactone type, differences in the cardenolidesteroid backbone between those that have cis–trans–cis fusedrings (U-shaped) and those which have trans–trans–cis fusedrings (flat molecules) (Figs. 2 and 5B) also appear to impact onCS binding features. This is indicated by ∼6 to N200 greaterinhibition of rat sodium pump α1β1, α2β1 and α3β1 isoforms(expressed in Sf-9 insect cells; Table 7) in vitro by UNBS1450(a hemi-synthetic derivative of 2″-oxovoruscharin with trans–trans–cis steroid rings) compared to ouabain and digoxin(cis–trans–cis steroids) [10]. These structural differences whichinfluence likely binding properties also appear to consequentlyinfluence subsequent signalling cascades (see Section 5.2).

3.6. Na+/K+-ATPase functions

3.6.1. The sodium pump as an ion transporter: establishingand maintaining the electrochemical gradient in cells

The Na+/K+-ATPase takes its place within the family of theso-called P-type ATPases, enzymes that become autopho-sphorylated by the γ phosphate group of the ATP molecule thatthey hydrolyse. For every molecule of ATP hydrolysed, threeNa+ ions from the intra-cellular space and two K+ ions from theexternal medium are exchanged. The Na+/K+-ATPase has twoconformational states, E1 and E2. Na+/K+-ATPase binds Na+

and ATP in the E1 conformational state and is phosphorylated atan aspartate residue. This leads to the occlusion of three Na+

ions and then to their release on the extra-cellular side. Thisnew conformational state (E2-P) binds K+ with high affinity.Binding of K+ leads to dephosphorylation of the enzyme and tothe occlusion of two K+ cations. K+ is then released to thecytosol after ATP binds to the enzyme with low affinity. Thus,the sodium pump contributes substantially to the maintenanceof the membrane potential of the cell, provides the basis forneuronal communication and muscle contractility and con-tributes to the osmotic regulation of the cell volume. In addition,the electrochemical Na+ gradient is the driving force behindsecondary transport systems [46,121].

Na+/K+-ATPase under normal conditions operates at a lowpercentage of its maximum pumping capacity [122–124].Indeed several reports have suggested that basal Na+/K+-ATPase activity in intact cells is one third of its maximumcapacity [123]. In fact, α1 containing isozymes pump at halftheir maximum capacity [124] and α2 containing isozymespump at 1/20 of their maximum [122]. Therefore, the Na+/K+-ATPase appears to have an important reserve capacity thoughtto be sufficient to maintain normal ion transport even inpathological conditions associated with down-regulation of itsexpression and/or activity. Thus harnessing this reserve capacityrepresents a mechanism by which cellular Na+/K+-ATPaseactivity can be rapidly upregulated. It is also interesting to notethat CSs, normally potent sodium pump inhibitors, might servealso as sodium pump activators. Indeed, Gao et al. [125]demonstrated that exogenous cardiac glycosides, specificallyouabain, at low concentrations (nM) increase Na+/K+-ATPaseactivity in vitro.

3.6.2. The sodium pump as a signal transducerIn addition to transporting ions, the sodium pump interacts

with neighbouring membrane proteins (forming thus a “sodiumpump signalosome”) and precipitates cytosolic cascades ofsignalling proteins to send messages to the intra-cellularorganelles. Xie and Askari [1] proposed the existence of twopools of sodium pumps within the plasma membrane, with twodistinct functions: one being the classical pool of the enzyme asan energy transducing ion pump and the other the signaltransducing pool of the enzyme restricted to caveolae [1,126].The signalling pathways that are rapidly activated/elicited bythe interaction of CSs (like ouabain) with the sodium pump andwhich are independent of changes in intra-cellular Na+ and K+

concentrations, include activation of Src kinase, transactivationof the epidermal growth factor receptor (EGFR) by Src[127,128], modulation of the NF-κB activity [129–132],activation of Ras and P42/P44 mitogen-activated proteinkinases and increased generation of reactive oxygen speciesby mitochondria [1,126,133]. Several CSs provoke an increasein [Ca2+]i as the result of the activation of these downstreamsignalling pathways [126,134,135]. However, in vitro this wasmainly achieved using high CS concentrations. It is alsoimportant to note that these studies which aimed to elucidatesodium pump signalling were undertaken essentially in normalcells, with in general ouabain the only CS investigated.



Signalosome composition and protein partnerships couldtherefore be different in different cells and tissues, and moreparticularly in normal versus pathological states, like cancer.Thus, it is not surprising to evidence different CSs-mediatedmechanisms of action in different cell types. Indeed, thesignalling pathways affected by CS actions seem to be quitedifferent in normal and in cancer cells [1,126,132,135–141].The compound concentration used could also be a source ofdivergent observations. Also with regard to the structuraldiversity of CSs previously detailed, it is reasonable to expectthat different compounds will elicit different mechanisms ofaction, affecting multiple signalling pathways and targets. Insummary, it is important to consider the sodium pump as asignal transducer able to mediate CS-induced effects in acompound-, concentration- and cell type-specific manner.

3.6.3. Novel functional interactions of the sodium pumpGrowing numbers of studies outline the role of the sodium

pump in cell adhesion and motility (for reviews see [142–144]).The complex interaction involving ankyrin and foldrin has beendemonstrated between Na+/K+-ATPase and the cellular cytos-keleton [145–147]. Rajasekaran and Rajasekaran [143] havealso evidenced that in epithelial cells Na+/K+-ATPase isinvolved in the formation of tight junctions through RhoAGTPase and stress fibres, and that it plays a crucial role inE-cadherin-mediated development of epithelial polarity, and thesuppression of invasiveness and motility of carcinoma cells[142]. Therefore, CS interactions with the sodium pump couldmarkedly affect cell migration features.

4. The sodium pump as a new target in anti-cancer therapy

Numerous studies have dealt with changes in the trans-membrane transport of cations during the course of malignantcell transformation, due to increases in Na+/K+-ATPase activity[5,148,149]. There is evidence that these kinetic changes inNa+/K+-ATPase activity are already present at the very earlystages of tumorigenesis, even before morphological evidence oftumour appearance [150,151]. Interestingly, in contrast to theincrease in Na+/K+-ATPase activity in cancer cells referred to inseveral studies [5,148,149], Davies et al. [151] have reportedinhibition of Na+/K+-ATPase. During the development of largebowel cancer, Davies et al. [151] observed alterations in the iontransport of colon epithelial, some of which resulted in alteredintra-cellular ionic composition. They also demonstrated thatchanges occur in Na+/K+-ATPase activity in pre-malignantmucosa, months before gross tumours develop, and thesechanges may partially explain the altered levels of Na+ and K+

in the cytoplasm of pre-malignant and malignant colonocytes.These differences are furthermore emphasized by the presenceof specific cancer-related FXYD proteins affecting the functionof the sodium pump, e.g. FXYD3 (Mat-8: a mammary tumourmarker which mediates the decrease in apparent Na+ and K+

affinity of the sodium pump) which is highly up-regulated inbreast and prostate tumours and FXYD5 (related to ionchannels) which is expressed in several cancer tissues but inonly a few normal cell types [56,57].

Also, not only does the activity of Na+/K+-ATPase differbetween normal and malignant cells, but also their sensitivitytowards cardiotonic steroids. This may be due to an altereddensity of Na+/K+-ATPase at the plasma cell membrane oftumour cells, as well as differences in isozyme expression. Inline with this, it has been widely reported that the Na+/K+-ATPase β1 subunit is very frequently down-regulated in humanepithelial cancer cells [7,9,67–69] (Table 4). In normalepithelial tissues, cells are held together by the cadherin/cateninsystem. It has been demonstrated by the Rajasekaran group[7,9,67,68,142–144] that when these cells down-regulate theirβ1 subunit, they detach from each other, as this down-regulation induces in turn a marked reduction in cadherinexpression, a process in which the Snail transcription factorexerts a major role [67]. Thus, down-regulation of the β1subunit seems essential for epithelial cancer cells to be able tobecome individually invasive. In contrast, Na+/K+-ATPase αsubunits seem to be up-regulated in some malignant cells[8,10,70], a phenomenon which has been quite poorlyinvestigated to date. Except for bladder cancer, investigatedby means of tissue microarray involving samples from 167different patients [7] and NSCLC, investigated by means ofimmunohistochemistry involving 94 samples [10], otherreported studies have relied merely on the use of cell lines(usually one per type) or a very limited number of tissuesamples (Table 4). Sakai et al. [8] reported that the α3 subunitwas over-expressed in colon cancer cells compared to normalcolon cells, while α1 subunit expression was reduced. The dataobtained by our group [10] strongly suggest an up-regulation ofthe Na+/K+-ATPase α1 subunit in a large proportion of clinicalnon-small cell lung cancer (NSCLC) samples compared tonormal lung tissue, while the expression of α2 and α3 subunitsfailed to reveal significant differences in distribution betweennormal lung tissue and NSCLCs. Moreover, specific siRNAknockdown of the Na+/K+-ATPase α1 subunit in human A549NSCLC cells which markedly reduced their migration andproliferation, further indicated the potential usefulness of the α1subunit as a potential anti-tumour target in NSCLC [10]. Furtherlarge-scale investigation should bring more evidence in supportof the proposal that over-expressed sodium pump subunitscould be suitable new targets in anti-cancer therapeutics.

In a recent review, Chen at al. [152] put forward severalpieces of evidence to support the novel concept that Na+/K+-ATPase could be a potentially important target for thedevelopment of promising anti-breast cancer drugs: (i) thesodium pump is a key player in cell adhesion and is involved incancer progression, (ii) it serves as a versatile signal transducerand is a target for a number of hormones including oestrogensand (iii) its aberrant expression and activity are implicated in thedevelopment and progression of breast cancer.

Collectively, the data from the literature strongly suggest thattargeting Na+/K+-ATPase could represent a novel means tocombat a growing number of malignancies. The question is howto optimally target the sodium pump in order to combat cancer.Approaches targeting sodium pump de novo expression such asanti-sense technologies (oligonucleotides, siRNA) or aptamers,might be considered only if delivered in situ, in order to avoid



dramatic generalized impairment of cell, tissue and organismfunctioning. Thus targeting the over-expressed sodiumpumpmaybe a more appropriate approach. The successful use of therapeuticmonoclonal antibodies has already evidenced their utility in anti-cancer therapy [153,154]. However, this proposal has to beconsidered with respect to whether the antibody should block orinhibit the sodium pump (will blocking (i.e. just “occupying” agiven binding site) without inhibiting pumping and/or signallingfunctions have sufficient anti-cancer effects?). While severalpublications have reported the ability of different compoundsfrom diverse chemical classes to inhibit the sodium pump e.g.2-methoxy-3,8,9-trihydroxy coumestan [155] or iantherans [156],attempts to synthesize effective sodium pump inhibitors havefailed due to their lack of selectivity (they targeted several otherpumps) and their limited anti-tumour activity. Table 5 gives somecompounds (other than CSs) that display anti-Na+/K+-ATPaseactivity [155–165]. However, it should be borne in mind that CSsare the natural ligands and inhibitors of the sodium pump and thisfact supports the possibility of their potential development as anti-cancer agents targeting over-expressed Na+/K+-ATPase subunits,notably α subunits.

5. Cardiotonic steroids: Sodium pump ligands as newanti-cancer agents: From epidemiology studies topreclinical evaluation

5.1. Epidemiological data

Epidemiological data have for some time indicated lowermortality rates in cancer patients who were on digitalis at time offirst diagnosis, compared to patients not on digitalis therapy

(summarized in [166,167]). Stenkvist et al. [168,169] reportedthat the tumour cell populations from breast cancer patients ondigitalis medication for cardiac problems were characterized by anumber of cytometric features. Notably, they appeared to have alower proliferative capacity than tumour cells from patients noton digitalis treatment [168,169]. Stenkvist et al. [170] thenreported in a 5-year follow-up study that the recurrence rate ofbreast cancer in patients not on digitalis was 9.6 times higher thanin patients treated with digitalis. In the subsequent 20-yearfollow-up, Stenkvist [166] reported that the death rate from breastcarcinoma (excluding other causes of death and confoundingfactors) was 6% (2 of 32) among patients on digitalis comparedwith 34% (48 of 143) among patients not on digitalis (p=0.002).Goldin and Safa [171] confirmed Stenkvist's results byconducting a retrospective study of 127 cancer patients in theirrecords. Of a total of 21 deaths they found only one cancer deathamong those who had taken digitalis. Additionally, a study with9271 patients showed a relationship between high plasmaconcentrations of digitoxin and a lower risk for leukaemia/lymphoma [172]. It should be noted that these studies were notspecifically designed to explore the relationship between the CStreatment and cancer. Furthermore, the inherent high toxicity andpoor therapeutic margin of these commonly used CSs haveprevented their development as anti-cancer agents.

5.2. Cardiotonic steroid-mediated in vitro anti-tumour effects

There are actually 965 papers referenced in the PubMedlibrary investigating CSs in cancer. However, it seems that thewider scientific community is still not sufficiently intrigued bythe idea of CS use in oncology. In fact, there is a huge

Table 4Sodium pump isoform subunit expression in cancer cells and tissues

Sodium pump subunit α α1 α2 α3 α4 β1 β2 AMOG β3 Assay type Assessed level Cell/tissue Case numb Ref. no.

Cancer typeColorectal cancer Down Up Western blot Protein Tissues 17 [8]Colon cancerpoorly

differentiated cellsDown Western blot Protein Cell lines 1 [67]

Northern blot RNAAndrogen-dependent

prostate cancerLow Northern blot RNA Xenografted

Cell lines3 [68]

Androgen-independentprostate cancer

High Northern blot RNA XenograftedCell lines

4 [68]

Kidney cancer Down Down PCR RNA Tissues 1 [69]Kidney cancer poorly

differentiated cellsDown Down Western blot Protein Cell lines 1 [67]

Northern blot RNARenal clear-cell carcinoma Down Western blot Protein Tissues 14 [9]Lung cancer Up Immunohisto-

chemistryProtein Tissues 94 [10]

Down Down PCR RNA Tissues 1 [69]Hepatic cancer Down PCR RNA Tissues 1 [69]Neuroblastoma neuroblast-like Up Down PCR RNA Cell lines 1 [69]Neuroblastoma fibroblast-like Up Down PCR RNA Cell line 1 [69]Pancreatic cancer Down Down Western blot Protein Cell lines 1 [69]

poorly differentiated cells Northern blot RNABreast cancer poorly

differentiated cellsDown Western blot Protein Cell lines 1 [69]

Northern blot RNABladder cancer Down Down Tissue microarray Protein Tissues 167 patients [7]Metastatic melanoma Up Rapid substraction

hybridizationRNA Cell lines 3 [70]



discrepancy between the number of reports dealing with theidentification and isolation of new CSs with in vitro anti-tumouractivity, and those concerned with the further investigation ofthese compounds.

Several CSs have already been shown to display markedanti-proliferative effects against human cancer cell lines in vitro[10,13,126,132,135,138–140,167,173–175]. Moreover, thestriking feature of CSs' action is their different effects onnormal and cancer cells. CSs are inactive or even pro-proliferative in normal cells but are able to selectively inducedeath in cancer cells [1,10,126,132,135–139,167,173–178].Their potent cytotoxicity towards human tumour cells has beenreported to result from formation of reactive oxygen species[126,179]. The mechanism of CS-mediated cell death has been

studied mainly in prostate [135,139,173–175] and in NSCLCcells [10,132,138]. It should be remembered that the therapeuticwindow of commonly used CSs is very narrow and that theconcentration range seen in the plasma of patients with cardiacdisease receiving for example digitoxin is 20–33 nM [180]. Inline with this, studies using nM concentration ranges of CSsonly produced anti-proliferative effects without induction ofapoptosis (a feature induced by CSs only when used at μMconcentrations), as evident in breast cancer cells treated withouabain at concentrations b100 nM [140], in prostate cancercells treated with ouabain at concentrations 10–100 nM [173] orin NSCLC cells treated with UNBS1450 [132,138]. Huang et al.[173] have also demonstrated that increasing ouabain concen-trations induced different effects in PC-3 prostate cancer cells.

Table 5Non-CS inhibitors of the sodium pump

These data are from references [155–165].



Low concentrations of ouabain induced an increase in Par-4expression which sensitised the cells to cytotoxicity, while highconcentrations induced a loss of deltapsim, sustained ROSproduction and apoptosis. Frese et al. [176] have shown thatCSs sensitise NSCLC cells to Apo2L/TRAIL-induced apopto-sis via up-regulation of Death Receptors 4 and 5. The studyof Bielawski et al. [181] further suggests that the stabilizationof DNA–topoisomerase II complexes is closely linked to themechanism of digoxin, ouabain and proscillaridin A cyto-toxicity in human MCF-7 breast cancer cells. Also, Watabe etal. [182] reported that the treatment of U937 cells withbufalin induced the translocation of casein kinase 2 andmodulated the activity of topoisomerase II prior to the inductionof apoptosis.

Johnson et al. [183] used a multiplex assay for six genesover-expressed in cancer cells to screen 9000 chemicals andknown drugs in the human prostate cancer cell line PC-3.Cardiac steroids digitoxin and ouabain were identified asinhibitors of four prostate cancer target genes includingtranscription factors Hoxb-13, hPSE/PDEF, hepatocyte nuclearfactor-3α and the inhibitor of apoptosis, survivin.

Pro-apoptotic drugs represent the main component of thecurrent chemotherapeutic arsenal against cancer. However,these compounds are ineffective in many cancers becausecertain cancer types are naturally resistant to apoptotic-relatedcell death (especially true of migrating cells as reviewed in[184]). In addition to mechanisms enabling them to resistapoptotic cell death, cancer cells are also able to resist variouscytotoxic insults because they possess a large set of intra-cellular signalling pathways that counteract administereddrugs. In line with this, current strategies for anti-cancerdrug development must focus on new compounds able toovercome the multiple anti-cell-death mechanisms which areat least partly responsible for the failure of existing chemo-therapeutic agents.

As already mentioned, by binding to the sodium pump, CSselicit several downstream signalling cascades affecting anumber of different targets. UNBS1450 [13] a potent sodiumpump inhibitor (Section 3.5) has been reported to dramaticallyimpair NSCLC cancer cell growth by de-activating thecytoprotective NF-kappaB pathway and inducing non-apopto-tic lysosomal membrane-permeabilization-related cell death[132,138]. In addition, UNBS1450 did not induce intra-cellularsodium or calcium increases (at concentrations which elicitanti-tumour effects, i.e. at 10 and 100 nM), but it disorganizedthe actin cytoskeleton affecting cytokinesis and impairing thusboth proliferation and migration of human cancer cells ofdiverse histological origin [10,13,132,138]. Van Quaquebekeet al. [13] in comparing a series derived from 2″-oxovor-uscharin in terms of inhibition of Na+/K+-ATPase and cancercell growth, found that compounds with poor activity againstthe enzyme (IC50N50 μM) were characterized by a completeloss of activity against cancer cell growth (IC50N10 μM).Thus, it seemed that the inhibition of Na+/K+-ATPase wasnecessary for the inhibition of cancer cell growth. However,this is not sufficient to explain the totality of the effectsobserved.

Cardiac glycosides can also regulate one of the most potentangiogenesis promoting substances, fibroblast growth factor-2(FGF-2) [185]. Indeed, Smith et al. [186] reported that non-toxic concentrations of Anvirzel and oleandrin decreased by∼50% the FGF-2 release from prostate cancer cells.

Chen et al. [152] further to suggesting the utility of thesodium pump as an oncology target (Section 4) have alsosuggested that it could be useful to develop CSs as anti-breastcancer drugs; both as Na+/K+-ATPase inhibitors and oestro-gen receptor (ER) antagonists. In fact, while anti-oestrogentherapy (used to treat ER-positive breast cancer) is initiallysuccessful, a major problem is that most tumours developresistance and the disease ultimately progresses. As alreadyemphasized, there are several lines of evidence indicating thatCSs possess potent anti-breast cancer activity. While it is notcompletely clear how the suggested anti-cancer activity ofthese drugs is achieved, several observations point to ouabainand digitalis additionally behaving as potential ER antagonists[152].

The bufadienolide class of CSs remains largely unexploreddespite their structural abundance and great natural profusion(Tables 1B, 2). Reports from Zhang et al. [187] and Numazawaet al. [188] suggested that bufalin might have potential as anagent in differentiation therapy for human myelogenousleukaemia. Furthermore, Daniel et al. [189] reported selectiveanti-tumour activity induced by hellebrin derivatives (thatapparently did not contain cardioactive groups) towardsmalignant T lymphoblasts in contrast to normal peripheralblood mononuclear cells. This further indicates that it might bepossible to obtain potent anti-cancer CSs with loweredcardiotoxicity.

Additionally and as indicated above, endogenous CSs havebeen identified in human tissues and Fedorova and Bagrov [30]have reported that one of these marinobufagenin, preferentiallyinhibits α1 subunits of the sodium pump. It could thus representa starting point for the development of anti-cancer CSspecifically targetting α1 subunits.

Finally, the most recently available data (American Associa-tion for Cancer Research Annual Meeting, April 14–18th 2007)further point to the rationale of developing CSs as anti-canceragents with respect to their interactions with kallikreins (KLKs).KLKs, the largest contiguous group of proteases within thehuman genome act as biomarkers for the screening, diagnosis,prognosis and monitoring of various cancers including those ofthe prostate, ovary, breast, testes and lung. Using highthroughput screening techniques to identify modulators of theexpression of KLKs, Prassas et al. [190] identified 3 differentclasses of compounds, of which the cardiotonic steroids werethe most potent inhibitors of KLKs expression. The pathwaysaffected by CS-induced KLKs inhibition and potential ther-apeutic applications are currently under investigation. Further-more, using a cell-based high throughput screen, Simpson et al.[191] demonstrated that inhibitors of Na+/K+-ATPase canrestore sensitivity to anoikis and decrease the metastatic poten-tial of cancer cells.

All these data emphasize the potential usefulness of develop-ing CSs as anti-cancer agents.



5.3. Cardiotonic steroids as radiosensitisers

Some reports have emphasized CSs as potential radio-sensitisers. In line with this, Verheye-Dua and Bohm [192,193]demonstrated that ouabain accentuates irradiation damage inhuman tumour but not normal cell lines. In 1988, Lawrence[194] reported that in contrast to normal human lung fibroblasts,A549 human lung adenocarcinoma cells were radiosensitisedby ouabain. Nasu et al. [195] then reported the radiosensitisingeffect of oleandrin on human prostate PC-3 cancer cells. Theconcentrations required to down-regulate the sub-lethaldamage/repair system (thus disabling reparative cellularmechanisms) appeared to fall within the range in which CSsare found clinically. These results suggest that it may bepossible to exploit differences between Na+/K+-ATPases ofnormal and tumour cells to improve the therapeutic index ofradiation. The potential use of these drugs in radiotherapy thusseems feasible.

5.4. Cardiotonic steroid-mediated anti-tumour activity in vivo

While in vitro anti-cancer properties of CSs have beenwidely studied, few reports have evidenced their activity in invivo models. Either these compounds demonstrated appreciablein vivo anti-tumour activity but were quite toxic (i.e. ouabain) orthey were found to be relatively devoid of anti-tumour activity(i.e. digoxin or as recently reported Anvirzel) at tolerated doselevels.

Inada et al. [196] reported that two-stage carcinogenesis ofmouse skin papillomas induced by 7,12-dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate(TPA), and mouse pulmonary tumours induced by 4-nitroquino-line-N-oxide (4NQO) and glycerol, were inhibited by digitoxin.Svensson et al. [197] reported digoxin-induced growth inhibi-tion of neuroblastoma cell grafts in mice (44% for SH-SY5Yand 19% for Neuro-2a grafts), in contrast to LS174T coloncancer cell xenografts or Lewis lung cancer cells syngraftswhich were not affected by digoxin treatment. Afaq et al. [198]reported inhibition of TPA-induced tumour promotion markersin CD-1 mouse skin by oleandrin. The potential therapeuticbenefit of UNBS1450 was evidenced in vivo in s.c. andorthotopic human NSCLC models in mice both by i.p. and p.o.administration [132,138]. Preliminary data are also veryencouraging with respect to orthotopic xenograft models ofhuman glioblastoma [199] and refractory prostate cancer [200].

The classic CSs appear to lack sufficient anti-tumour activityto be used as single anti-cancer agents at clinically acceptabledoses. Indeed, the recently completed Phase I clinical trial(investigating CSs as anti-cancer agents) with Anvirzel™ (aNerium oleander extract containing several CSs but particularlyenriched in oleandrin) demonstrated that it can be safelyadministered to patients with solid tumours but unfortunately noobjective tumour regressions were seen [36]. On the other hand,a combination of CSs with EGFR targeting antibodies mightimprove the therapeutic benefit of anti-EGFR therapy, in viewof the previously described close partnership between thesodium pump and EGFR in caveolae. Indeed, there is actually

ongoing patient recruitment for a Phase II clinical trial com-bining daily digoxin with Tarceva [201]. To the best of ourknowledge, the above clinical trials are the only two reported tobe directly investigating CS use in oncology.

5.5. How can cardiotonic steroid-related cardiotoxicity becircumvented?

The limited available data confirming in vivo CS-mediatedanti-cancer activity must be considered against the cardiotoxi-city these compounds provoke at comparatively low doses. Theearly evaluation of the maximum tolerated dose and pharma-cokinetics of novel compounds may contribute to the optimumselection of new anti-cancer CS with combined potent anti-tumour activity and decreased toxicity. However, there is a lackof reliable in vivo animal models able to effectively evaluatepotential CS-mediated toxicity. The model should ideallydisplay all the characteristics discussed in the previous chapterand more particularly have: (i) the same sodium pump subunitcomposition, in comparable proportions and with similarsensitivity to CSs as evident in the human heart, (ii) similartransport and kinetic properties as human isozymes, (iii) similarresponses to stimuli regulating sodium pump subunit expressionand (iv) similar numbers and quantities of the endogenousdigitalis-like compounds produced in human tissues. As shownin Table 6, there is no animal species expressing all three αsubunits in the heart, in sharp contrast to the human heart[49,202–204]. Furthermore, for all tested animal models, thereare obvious differences in CS sensitivity to expressed isoformscompared to human. Moreover, mouse and rat hearts are uniquecompared to other species in that their intra-cellular sodium ishigher and the distribution of intra-cellular calcium markedlydifferent (given that most of it is derived from the sarcoplasmicreticulum in contrast to other mammalian species). Importantlyupon stimulation, rat and mouse hearts accrue calcium whileother mammals extrude it [205]. Although bearing one CS-insensitive isozyme, another rodent the guinea pig seems to bethe least inappropriate species to be used. Indeed, Gao et al.[125] reported that (i) the sodium pump current in human atrialcells has similar properties to the α2-containing isozymes inguinea pig ventricles and (ii) human atrial cell sodiumpumps appear to share some functional properties with thesodium pump in guinea pig ventricles i.e. they are stimulated by

Table 6Sodium pump α subunit expression in the hearts of different species

Heart from species α1 α2 α3

Human + + +Monkey + − +Goat + − −Dog + − +Pig + − −Guinea pig + + −Rat (embryo) + − +Rat (adult) + + −Mouse + + −

These data are from references [49,202–204].



α-adrenergic but are unaffected by β-adrenergic activation.However, the potential use of guinea pig is season-restrictedsince it has been observed that in summer its ventricularmyocytes express extremely low levels of the α2 isoform [125].The dog is generally used to assess digitalis-induced effects[206–209]. However, like rodents, canine cardiac myocytescontain ouabain-resistant (α1) and ouabain-sensitive (α3) Na+/K+-ATPase catalytic subunits, the two forms are active anddiffer by a factor of 150 in their respective affinity for digitalis[210]. Finally, the pig is also not a suitable investigationalmodel. It expresses only α1 in the heart and this isoform is twotimes less sensitive than human α1. The different expression ofsodium pump isoforms in human versus porcine cardiac tissuessuggests that pig hearts may not be pharmacologically orendocrinologically compatible when used in humans. Thisprecise point was one of the major limitations restricting the useof pig hearts for human transplantation [203]. Thus with respectto the lack of a suitable experimental animal model for theevaluation of CS-mediated toxicity and regulatory requirementsfor drug development, the use of comparative studies involvingthe candidate drug and a fully characterized CS (such as digoxinor digitoxin) might be informative for defining the suitability ofthe new candidate, if it displays similar or lower cardiotoxicitythan the reference CS.

It is evident that the toxicity of naturally occurring CSs has tobe reduced by medicinal chemistry in order to increase tolerancewhile maintaining or increasing their anti-cancer activity. Thishas been applied to 2″-oxovoruscharin and has enabled themaximum tolerated dose to be increased 12 times (10 mg/kg for2″-oxovoruscharin versus 120 mg/kg intraperitoneally for itsderivative UNBS1450; [13,132,138]). Furthermore, sincedifferent cancer types are characterized by over-expression ofgiven α subunit [8,10,70], the development of specific CSs withselectivity towards one sodium pump β subunit sub-type mightincrease their anti-tumour effects while reducing potential toxiceffects. Thus structure–activity relationship (SAR)-directedchemistry against specific sodium pump isozymes or sub-units, together with early in vivo evaluation of activity versustoxicity, might be the way forward to discover and developnovel CSs in oncology, with their eventual clinical use relyingon previous characterization of the over-expressed sodiumpump subunits in the cancer type being treated. As indicatedabove, UNBS1450 demonstrates a much higher inhibition ofsodium pump isozymes than classic CSs; with a potency N200times greater than that of ouabain and ∼6 times greater thandigoxin in respect of α1β1 (Table 7) [10], which is of particularimportance given the over-expression of α1 subunits in anumber of different cancers. Through medicinal chemistry, itmight thus be possible to selectively target different isozymeswith improved anti-cancer activity without increased potentialtoxicity. It should be emphasized again that in contrast toouabain, digoxin and digitoxin [173,174], UNBS1450 at anti-proliferative concentrations does not induce apoptosis or intra-cellular Ca2+ increases [132,138,200] involved in CS-mediatedcardiotoxicity [40]. As mentioned previously, these features ofUNBS1450 may be explained by its structural uniqueness, as itcontains a double linked novel glycoside and a trans–trans–cis

fused steroid ring system which confers a markedly differentshape compared to classic CSs.

The sugar moiety as indicated previously also seems to bean important factor mediating CS-induced cancer cell cytotoxi-city. The role of the glycoside in mediating cytotoxicity wasanalyzed by Langenhan et al. [211] who grafted different sugarsonto the digitoxin skeleton and demonstrated the possibility ofincreasing the anti-cancer activity of CSs by neo-glycorando-misation. Van Quaquebeke et al. [13] in comparing compoundsderived from 2″-oxovoruscharin and its hemi-synthetic deriva-tive UNBS1450, concluded that the potency of inhibition ofboth cancer cell growth and Na+/K+-ATPase activity wasaffected when reactive groups were modified with bulkier lesspolar residues. Further studies are required to determine thedifferent inhibition potencies of existing CSs against differentsodium pump isozymes, in order to build up an understandingof their structural characteristics with respect to isozymeselectivity, to in turn build a rudimentary SAR, for furthermedicinal chemistry against specific isozyme subunits over-expressed in cancer.

6. Conclusions

Cardiotonic steroids or cardiotonic glycosides are char-acterized by their abundance in nature, diversity of structure,potential for chemical modification and wide use in car-diology for heart failure management. CSs act by binding tothe extra-cellular surface of Na+/K+-ATPase. This fact plusthe altered expression of sodium pump subunits in differentcancers strongly suggest that targeting Na+/K+-ATPase couldrepresent a novel means to combat a growing number ofmalignancies.

Epidemiological data along with reports based on in vitroand in vivo demonstration of CS-mediated anti-cancer activity,further emphasize the possibility of developing this class ofcompound as anti-tumour agents. However, CSs are character-ized by a narrow therapeutic window that has prevented theirdevelopment in the past as anti-cancer drugs. The use ofmedicinal chemistry to target CSs towards specific sodiumpump subunits over-expressed in certain cancers may improveboth anti-tumour activity and reduce cardiotoxicity, thelimiting toxicity for this class of compound. This strategyhas in part been demonstrated with the hemi-synthetic ana-logue UNBS1450 derived from the naturally occurring CS:2″-oxovoruscharin.

Adverse effects have always been the companions ofeffective chemotherapeutics in oncology and the benefits

Table 7Determination of the inhibition (Ki in nM) by UNBS1450, ouabain and digoxinof rat α1β1, α2β1 and α3β1 sodium pump isozymes transfected by means ofbaculovirus into Sf-9 insect cells

Molecule α1β1 α2β1 α3β1

UNBS1450 160±70 15±8 3.2±3.4Digoxin 930±410 190±90 40±15Ouabain 43,000±19,000 170±10 31±3

This table originally taken from [10].



contributed to patients have had to be counterbalanced bycumulative toxicity. In the absence of totally appropriatepreclinical cardiotoxic models, the search for totally cleannon-toxic anti-cancer CSs could prevent the development ofsome promising compounds.

Acknowledgements

We would like to acknowledge the scientific contributionand advice of Dr. V Facchini in the finalisation of this article.The authors would like to thank Dr. Laurent Ingrassia for hishelp with Fig. 1. The present work was supported by grantsawarded by the Fonds Yvonne Boël (Brussels, Belgium) and theRégion de Bruxelles-Capitale (Brussels, Belgium).

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