Na+/K+-ATPase-associated FXYD3 Protein As An Intracellular Therapeutic Target in Cancer

BackgroundFXYD proteins associate closely with- and protect plasmalemmal Na+/K+-ATPase against oxidative inhibition. One of them, FXYD3, is often overexpressed in cancers, including those of breast and pancreas. Down-regulation of overexpression in MCF-7 breast cancer cells with siRNA augments doxorubicin-induced cytotoxicity. Because down-regulation with siRNA is not readily translated therapeutically, we developed a peptide as an alternative for suppression of FXYD3.MethodsA shortened peptide derivative of the wild-type (WT) FXYD3 protein, FXYD3-pep has the four cysteine residues in the WT protein replaced by serine, which eliminates the WT protein’s protection against oxidative Na+/K+-ATPase inhibition. We exposed human cancer cells to FXYD3-pep and measured cytotoxicity and caspase 3/7 activity with co-exposure to doxorubicin. We also measured effects of the peptide on expression glutathione-S-transferase π (GST-π), implicated in treatment resistance, and on expression of tumor suppressor p53. Selected experiments were performed with parallel FXYD3 suppression with siRNA or FXYD3-pep.ResultsExposure of cells to FXYD3-pep displaced WT FXYD3 from Na+/K+-ATPase. Exposure of MCF-7 breast or pancreatic BxPC-3 cancer cells that highly express FXYD3 to the peptide had little effect alone but combined with doxorubicin it significantly (P < 0.05) increased cytotoxicity. A peptide not mutated to eliminate FXYD3’s protective effect of Na+/K+-ATPase had no effect. FXYD3-pep did not augment doxorubicin’s cytotoxicity against MDA-MB-468 breast and Panc-1 pancreatic cancer cells that have low- or no FXYD3 expression. Cellular FXYD3 expressions was reflected by expression of the α1 Na+/K+-ATPase subunits but not by plasmalemmal Na+/K+-ATPase function. Signals from fluorescently labeled FXYD3-pep were detected in a perinuclear distribution in BxPC-3 cells as reported for overexpressed FXYD3, α- and β Na+/K+-ATPase subunits in cancer. Exposure to FXYD3-pep or to FXYD3 siRNA almost eliminated expression of GST-π. FXYD3-pep alone had no effect on p53 levels but significantly augmented a doxorubicin-induced increase, and, while the peptide alone had no effect on caspase 3/7 activity, it significantly augmented a doxorubicin-induced increase. ConclusionsOverexpressed FXYD3 has intracellular roles beyond its accepted modulation of plasmalemmal Na+/K+-ATPase. These roles can be countered with a membrane-permeable peptide derivative of FXYD3 that suppresses GST-π and enhances chemosensitivity of cancer cells overexpressing FXYD3.