Cardiac glycosides induced toxicity in human cells expressing α1-, α2-, or α3-isoforms of Na-K-ATPase

Abstract

Cherniavsky Lev M, Karlish SJ, Garty H. Cardiac glycosides induced toxicity in human cells expressing ␣1-, ␣2-, or ␣3-isoforms of Na-K-ATPase. Am J Physiol Cell Physiol 309: C126 -C135, 2015. First published May 20, 2015 doi:10.1152/ajpcell.00089.2015.-The Na ϩ -K ϩ -ATPase is specifically inhibited by cardiac glycosides, some of which may also function as endogenous mammalian hormones. Previous studies using Xenopus oocytes, yeast cells, or purified isoforms demonstrated that affinities of various cardiac glycosides for three isoforms of the Na ϩ -K ϩ -ATPase (␣1-␣3␤1) may differ, a finding with potential clinical implication. The present study investigates isoform selectivity and effects of cardiac glycosides on cultured mammalian cells under more physiological conditions. H1299 cells (non-small cell lung carcinoma) were engineered to express only one ␣-isoform (␣1, ␣2, or ␣3) by combining stable transfection of isoforms and silencing endogenous ␣1. [21153][21154][21155][21156][21157][21158][21159][21160][21161][21162] 2014). Relative ␣2:␣1 selectivity of digoxin vs. ouabain was also manifested by enhanced internalization of ␣2 in response to digoxin. Cellular proliferation assays of H1299 cells confirmed the patterns of ␣2:␣1 selectivity for ouabain, digoxin, and a synthetic derivative and reveal a crucial role of surface pump density on sensitivity to cardiac glycosides. Because cardiac glycosides are being considered as drugs for treatment of cancer, effects of ouabain on proliferation of 12 cancer and noncancer cell lines, with variable plasma membrane expression of ␣1, have been tested. These demonstrated that sensitivity to ouabain indeed depends linearly on the plasma membrane surface density of Na ϩ -K ϩ -ATPase irrespective of status, malignant or nonmalignant. cardiac glycosides; cell death; isoforms; Na-K-ATPase THE NA ϩ -K ϩ -ATPASE (the Na ϩ pump) is a ubiquitously expressed P-type ATPase that actively transports 3Na ϩ ions out of cells in exchange for 2K ϩ ions per one molecule of ATP hydrolyzed (17). The pump consists of a catalytic ␣-subunit, ␤-subunit that modulates activity and stabilizes the protein, and an auxiliary FXYD polypeptide. Four ␣-isoforms (␣1-␣4), three ␤-isoforms (␤1-␤3), and seven FXYD isoforms exist in mammalian organisms, resulting in considerable tissue-specific variability in pump structure and kinetics (21).As can be expected from plasma membrane protein with such a vital function, the Na ϩ -K ϩ -ATPase is regulated by a number of independent mechanisms (25). One such mode of regulation involves inhibition by a family of inhibitors termed cardiac glycosides (CGs). Most CGs are derived from plants.However, several so-called endogenous CGs, including ouabain, marinobufagenin, and digoxin, have been identified in mammalian tissues and are apparently synthesized by the adrenal gland (1). CGs such as digoxin are also clinically relevant specific inhibitors of the Na ϩ -K ϩ -ATPase, used classically to treat heart failure (9).Relatively limited information exists regarding th...

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“…They demonstrated that apparent affinity of these isoforms for cardiac glycosides differs by less than three-fold, whereas their affinity for marinobufagenin is decreased compared to ouabain by 100–200-fold. More recently, this research team synthesized CTS derivatives whose selectivity for α2- vs. α1-isoform is increased by seven-fold [ 129 , 130 ]. The second hypothesis is consistent with our recent data obtained by isothermal titration microcalorimetry of purified α1β1-isozyme from duck salt glands [ 131 ].…”

Sodium chloride (NaCl) potentiates digoxin-induced anti-tumor activity in small cell lung cancer

“…Na + /K + -ATPase sub-unit, ATP1A1 is the target of digoxin. 12 Previous study has demonstrated that CGs killed cancer cell through inhibiting ATP1A1. 14 Eskiocak et al have reported that melanomas that expressed higher level of the ATP1A1 were more sensitive to CGs, compared with normal human melanocytes.…”

“…Higher extracellular NaCl concentration was associated with higher expression of ATP1A1 that directly contributed to SCLC's sensitivity towards digoxin ATP1A1 is a subunit of Na + /K + -ATPase. 12 In this study, we further analyzed the expression levels of ATP1A1 among a panel of SCLC cells. Western blotting demonstrated that ATP1A1 was expressed in almost all SCLC cell lines, except for H446 cell line ( Figure 7A).…”

“…Na + /K + -ATPase functions as the regulation of intracellular Na + level. 12,13 Next, we determined whether change of Na + concentration affected ATP1A1 expression. As shown in Figure 7B, the mRNA and protein expression of ATP1A1 were significantly up-regulated when SCLC cells were cultured in medium with increasing concentration of Na + .…”

Elimination of undifferentiated human embryonic stem cells by cardiac glycosides

“…Inhibiting the expression of the alpha1 subunit impaired proliferation and migration in NSCLC cell lines 43 . A recent study has shown that human NSCLC cells overexpressed the alpha1, alpha2, or alpha3 subunits of Na + /K + -ATPase and were induced cell death by cardiac glycosides (i.e., digoxin and ouabain) 44 . In our data, the expression levels of the alpha1 and alpha2 subunits of Na + /K + -ATPase were significantly higher in the hESCs than in the hMSCs or hESC-derived progeny (Figs 1a and 4e).…”