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From Na+/K+-ATPase and cardiac glycosides to cytotoxicity and cancer treatment

The cardiac glycosides (CGs, also referred to as cardiac steroid glycosides) are a diverse family of naturally derived compounds, C23 or C24 steroidal glycosides that have been found in many angiosperms. The most important CG-containing plant families are Apocynaceae, incl. Asclepiadaceae (Adenium [1], Cerbera [2], Cryptostegia [3], Nerium [4, 5], Parepigynum [6, 7], Periploca [8-10], Strophanthus [11-15], Thevetia [16-22]), Brassicaceae (Erysimum [23-29], and Lepidium [30]), Celastraceae (Euonymus [31, 32], and Lophopetalum [33]), Convallariaceae (Convallaria [34-45]), Crassulaceae (Cotyledon [46], and Tylecodon [47, 48]), Hyacinthaceae (Schizobasis [49], and Urginea [50, 51]), Fabaceae (Coronilla [52, 53]), Malvaceae (Corchorus [54-56], and Mansonia [57, 58]), Moraceae (Antiaris [59-61], Castilla [62], Maquira [63-66], and Naucleopsis [65]), Ranunculaceae (Adonis [67-71], Eranthis [72], and Helleborus [73]), Scrophulariaceae s.s. (Digitalis [13, 74-91]), and Solanaceae (Nierembergia [92]). CGs have also be found in some animals, such as members of the genus Bufo [7, 8, 15]. Endogenous cardiac glycosides have also been discovered [93, 94], the most important among them being ouabain; digoxin; 19-norbufalin and its peptide derivative; 3ß-hydroxy-14? 20:21-bufenolide; proscillaridin A; marinobufagenin; and telocinobufagin (Fig. 1). They have been found in different human tissues, in some cases related to pathological processes. The structure of the CGs allow two classes of them to be distinguished: to the cardenolides with a five-member lactone ring at the C17 position and the bufadienolides with a six-member lactone ring [95]. The sugar moieties attached to the aglycone by a C-3, linkage are compounds consisting of from one to four units. These units include glucose, rhamnose, and such deoxysugars as digitoxose and cymarose, which have been found only in this group of secondary metabolites (Fig.1). Especially, the sugar moiety at the C3 position of the steroidal skeleton affects the pharmacological and pharmacokinetic properties of the cardiac glycosides.

Pharmacology and usage of cardiac glycosides in conventional therapy
The first plant introduced into Western medicine was foxglove (Digitalis purpurea L.), which was used by William Withering in 1785 to treat dropsy. The mechanism of action of the cardiac glycosides is based on binding and inhibiting Na+/K+-ATPase in the cardiac myocyte membrane. This increases the intracellular concentration of Na+ and subsequently reduces the extrusion of calcium [96-100]. An increased concentration of calcium in the cytoplasm increases the uptake of calcium by the sarcoplasmic reticulum (SERCA2 transporter), which can finally cause increased contraction [101, 102]. On the other hand, an elevated concentration of Na+ compromises the mitochondrial energetics and redox balance by blunting the mitochondrial accumulation of Ca2+, thereby contributing to a possible cytotoxic effect of the CGs [103]. Cardiac glycosides have been used clinically for many years to treat heart failure and atrial arrhythmias [104-111]. The medicinally most important cardiac glycosides, which have been or still are used therapeutically are digoxin, digitoxin, lanatoside A, lanatoside C (Digitalis lanata Ehrh., D. purpurea L.), and thevetin (Nerium oleander L.). However, CGs are known to increase the levels of reactive oxygen species (ROS), which contribute to arrythmogenesis through the redox modification of cardiac ryanodine receptors [112, 113]. ROS may play a role in the cytotoxicity of CGs [114]. Direct blocking of the cardiac potassium channel hERG by CGs is another pro-arrytmogenic factor [115, 116].

Na+/K+-ATPase
Na+/K+-ATPase is an integral membrane protein present in all mammalian cells (Fig. 2). It transports Na+ and K+ ions across the plasma membrane, and is necessary for maintaining the electrochemical gradient which is important in the processes of electrical excitation and the transport of other ions. Na+/K+-ATPase is a heterodimer composed of two subunits. The alpha subunit, a catalytic subunit with 10 trans-membrane segments, couples ATP hydrolysis with ion transport. The beta subunit, with one trans-membrane segment, is involved in the processes of the structural and functional maturation of the enzyme and in trafficking to the plasma membrane [117]. Na+/K+-ATPase usually also contains an auxiliary subunit of the FXYD protein family. The alpha subunit also contains a functional site for cardiac glycoside inhibitors. Four alpha and three beta isoforms are expressed and regulated in tissue- and development-specific manners [118]. Some factors have been identified as modulators of Na+/K+-ATPase activity (Fig. 2); the most important of these are the FXYD proteins. The distribution of the individual Na+/K+-ATPase subunits is probably regulated by hormones. Treatment of hypothyroid rats with T-3 increased the relative abundance of both alpha 1 and beta 1 subunits in the total membranes and led to a 1.9-fold increase in enzyme activity. Na+/K+-ATPase uses energy from the hydrolysis of ATP to drive the movement of K+ ions into cells and exchange them for Na+ ions. This process also transports other solutes, amino acids, sugars, and phosphates. The homeostasis of these ions is crucial in the processes of cell cycle regulation, cell proliferation, and apoptosis. On the other hand, some hormones are able to affect processes closely connected with the activity and conformation of Na+/K+-ATPase [119], e.g., aldosterone [120], thyroid hormones [119, 121], glucocorticoids [122], catecholamines [123], and insulin [124]. But cAMP [125] also affects the expression of Na+/K+-ATPase in different tissues. It regulates the promoter activity of the alpha 4 isoform [126, 127], which is of great importance in light of the recently found role of Na+/K+-ATPase expression in regulating cell growth and proliferation [128]. In addition, Na+/K+-ATPase serves as a signal transducer. The above-mentioned hormones are also responsible for the translocation of Na+/K+-ATPase. Moreover, the changes in some hormonal systems are quite rapid, reflect both the endogenous and exogenous conditions. These changes are also involved in modifying the function of Na+/K+-ATPase, its eventual translocation, and changes in the promoter activity. In addition to the above-stated facts, the endogenous cardiac glycosides that have been described and will be discussed with respect to the regulation of the cell cycle in the following subchapter, are also involved in regulating the Na+/K+-ATPase activity. The regulatory role of hormones probably consists not only in inhibiting this enzyme, but also regulating the activation of signaling pathways, possibly by changing the conformation of the enzyme. It has been established that ouabain binds the phosphorylated E2P conformation of Na+/K+-ATPase (E2P-ouabain) with great affinity. This is followed by phosphorylation of the thyrosine 418 of Src kinase, which is required if the full catalytic activity of Src kinase is to be obtained. This activated form can enter signaling pathways. ADP as well as reduced level of ATP proved to have an inhibitory effect on the phosphorylation of Src, so the ATP/ADP ratio determines the extend of Src activation [129]. In conclusion, Na+/K+-ATPase inhibits Src kinase, which suggests a possible role for endogenous cardiac glycosides in regulating the cell cycle.

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