Na/K-ATPase Mimetic pNaKtide Peptide Inhibits the Growth of Human Cancer Cells

Abstract

Cells contain a large pool of nonpumping Na/K-ATPase that participates in signal transduction. Here, we show that the expression of ␣1 Na/K-ATPase is significantly reduced in human prostate carcinoma as well as in several human cancer cell lines. This down-regulation impairs the ability of Na/KATPase to regulate Src-related signaling processes. A supplement of pNaKtide, a peptide derived from ␣1 Na/K-ATPase, reduces the activities of Src and Src effectors. Consequently, these treatments stimulate apoptosis and inhibit growth in cultures of human cancer cells. Moreover, administration of pNaKtide inhibits angiogenesis and growth of tumor xenograft. Thus, the new findings demonstrate the in vivo effectiveness of pNaKtide and suggest that the defect in Na/K-ATPase-mediated signal transduction may be targeted for developing new anticancer therapeutics. Na/K-ATPase was originally discovered as an ion pump that is essential for cell vitality and provides a means for epithelium to secrete and/or absorb solutes and nutrients (1, 2). Recent studies have revealed that in addition to pumping ions across the cell membrane, Na/K-ATPase, specifically the ␣1 isoform, conducts many nonpumping functions, including scaffolding and signal transduction. As a signaling protein, it is involved in the formation of membrane structures such as tight junction and caveolae (3, 4). Moreover, a large fraction of cellular Na/KATPase is involved in tethering and regulating multiple protein and lipid kinases as well as membrane receptors (e.g. Src, human epidermal growth factor receptor, and PI3K) in a cellspecific manner (5, 6). Recently, the ␣1 Na/K-ATPase-Src receptor complex has been identified as one of the central components of ␣1 Na/K-ATPase-mediated signaling transduction (7). In this receptor complex, the Src SH2 domain binds to the second cytosolic domain, whereas the Src kinase domain interacts with the nucleotide binding (N) domain 4 of the ␣1 subunit. The latter interaction keeps Src in an inactive state (7). It is important to note that normal epithelial cells express approximately one million ␣1 Na/K-ATPase molecules (roughly five times the amount of Src). Thus, ␣1 Na/K-ATPase could provide at least two ways of regulating cellular Src activity. First, it could bind and keep Src in an inactive state. Consistently, when knock-out of one copy of the ␣1 gene caused a 20 -30% reduction in cellular ␣1 Na/K-ATPase, it produced a Ͼ2-fold increase in Src and ERK activities in tissues of ␣1 ϩ/Ϫ mice (8). Second, formation of the Na/K-ATPase-Src complex provides a functional receptor for endogenous cardiotonic steroids such as ouabain to regulate cellular signaling via Src and Src effectors (7, 9). Thus, changes in cellular ␣1 Na/K-ATPase would have a significant effect on cellular signaling events induced by either cardiotonic steroids or other growth factors through Src-related pathways.Based on the fact that the N domain of the ␣1 subunit binds and inhibits Src, we have recently made pNaKtide from the N domain of the human ␣1 subunit of...

Identification of a Mutant α1 Na/K-ATPase That Pumps but Is Defective in Signal Transduction

“…These new findings suggest an important role of the helical structure and amino acid residue Trp-418, Leu-419, and Arg-423 in the binding and regulation of Src. Because pNaKtide acts as an effective antagonist of receptor Na/KATPase⅐Src complex in vitro and in vivo (9,10), the new findings provide important structural information for directing the development of second generation of pNaKtide derivatives.…”

“…Confocal Fluorescence Microscopy-Immunostaining of ␣1 was done as previously described (10). Briefly, cells were cultured on coverslips in 6-well plates.…”

“…This peptide is a potent Src inhibitor in the in vitro and acts as a receptor antagonist by blocking the formation of functional Na/K-ATPase⅐Src complex when applied to cultured cells (9). Moreover, pNaKtide was effective in inducing tumor regression and inhibiting tumor growth in vivo (10). To understand the molecular basis of NaKtide-mediated Src regulation, we made several mutants of NaKtide and tested their effects on Src.…”

Expression of rat Na-K-ATPase α2 enables ion pumping but not ouabain-induced signaling in α1-deficient porcine renal epithelial cells

“…Studies from various laboratories have documented an important signaling function of the Na-K-ATPase (2, 27). For example, we and others have shown that Na-K-ATPase ␣1 is capable of keeping the Src kinase in an inactive state in many different types of cell (28,30). This interaction allows the formation of a functional receptor complex, which becomes a target for cardiotonic steroids such as ouabain to stimulate protein and lipid kinase cascades and subsequently regulate many cellular activities including cell growth and migration.…”

The Na/K-ATPase as an Amplifier for Oxidant Signals: Brief Historical Review and Possible Implications

“…To date, some agonists of the Na/K-ATPase signal cascade have been identified as have some antagonists. We have also specifically developed classes of peptides which serve as signal complex disrupters as well as signal terminators [17,20,[22][23][24][25]. It is our hope and expectation that pharmacological agents useful in the clinic will be developed from Professor Xie's insights which have reinvigorated the study of this important system.…”