Quiescence and γH2AX in neuroblastoma are regulated by ouabain/Na,K-ATPase

Cardiac glycosides constitute a class of naturally derived compounds that bind to the ubiquitous sodium pump, Na,K-ATPase. For many years, members of this class (e.g. ouabain, digoxin, and digitoxin) have been in clinical use for the treatment of different heart diseases (Prassas and Diamandis, 2008). Interestingly, preclinical and retrospective patient data indicate that cardiac glycosides also can reduce the growth of various cancers, including breast, lung, prostate, and leukaemia (Stenkvist, 1999; Lopez-Lazaro, 2007; Mijatovic et al, 2007; Khan et al, 2009). Several signalling pathways have been proposed to account for this preferential cytotoxicity in cancer cells, including calcium (Ca2+) and Apo2L/TRAIL-induced apoptosis (McConkey et al, 2000; Frese et al, 2006). The recent interest in using cardiac glycosides to treat cancers has resulted in the initiation of a number of clinical trials (Vaklavas et al, 2011). The ability of cells to cycle and exit into senescence or quiescence is important for cell differentiation, tissue development, and prevention of tumourigenesis (Evan and Vousden, 2001; Liu et al, 2004; Lapenna and Giordano, 2009; Malumbres and Barbacid, 2009). In response to mitogens, cells overcome the G1 restriction point and commit to synthesise DNA and divide. The restriction point is regulated by the retinoblastoma protein (Rb) under the strict control of cyclin D-cyclin-dependent kinase (CDK)2 and cyclin E-CDK4 (Planas-Silva and Weinberg, 1997). These cyclin-CDK complexes phosphorylate Rb, thereby cancelling the growth-inhibitory function of Rb, to stimulate G1-S transition and S-phase progression. The CDK inhibitor p21Waf1/Cip1 (p21) binds to and inhibits the activity of cyclin-CDK2 or -CDK4 complexes, and causes G1 arrest in response to DNA damage (el-Deiry et al, 1994). p21 has also been reported to have a critical role in the transition out of the cell cycle and in maintaining cells in a quiescent state (Liu et al, 2009; Sang et al, 2008). Combinatorial therapies, in which cells are arrested in certain cell cycle phases thereby enhancing sensitivity to chemotherapy and reducing unwanted side effects, are becoming increasingly common in treating patients with cancer (Luo et al, 2009; Waldman et al, 1997). The cell signalling mechanisms that control how cells enter or exit from quiescence are not known. Slow proliferation rate and quiescence-like states in cancer cells are controlled by CDK inhibitors downstream of p53, for example p21. However, it was recently shown that a reduction in p21 per se was not sufficient to push arrested cells back into the cell cycle (Sang et al, 2008). This study identified the basic helix-loop-helix transcription factor hairy and enhancer of split1 (HES1) to be necessary for reversing the cell cycle arrest. Human neuroblastoma, the most common childhood solid tumour, is characterised by an extensive clinical heterogeneity ranging from spontaneous regression to extremely aggressive variants (Maris et al, 2007). The spontaneous regression is thought to take place through a constitutively active DNA-damage response (DDR) pathway, which is a negative regulator of cell cycle progression that may induce cellular senescence (Brodeur, 2003). Chemotherapy induces cellular responses that protect the cell from severe cellular damage, of which the activation of the DDR pathway is one such response (Downs, 2007; Bonner et al, 2008). DDR signal transduction senses genotoxic stress and coordinates the response into DNA repair, cell death, and/or growth arrest. The major regulators of the DDR pathway are the phosphoinositide 3-kinase (PI3K)-related protein kinases ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related), which phosphorylates histone H2AX on Ser 139 (γH2AX) (van Attikum and Gasser, 2009). The DDR pathway in cancer cells influences genome stability, cellular senescence and counteracts activated oncogenes and tumour progression (Bartek et al, 2007; Halazonetis et al, 2008). The inducers of replication stress in early tumours have not yet been identified. Intriguingly, embryonic stem cells have an elevated DDR pathway basal activity (Andang et al, 2008), similar to the early stages of cancer, as a result of increased ion channel activity. The ion homeostasis and electrochemical gradients are critically maintained in all eukaryotic cells. The gradient is established primarily by the Na,K-ATPase through which three intracellular Na+ ions and two extracellular K+ ions are exchanged for every molecule of ATP hydrolysed (Kaplan, 2002). The Na,K-ATPase is a heteromer of α- and β-subunits and serves as a functional receptor for the steroid hormone ouabain, forming a signalling complex (Kaplan, 2002; Aperia, 2007). Endogenous ouabain and ouabain-like compounds are synthesised in the adrenal cortex (Huang et al, 2006; Bagrov et al, 2009), the hypothalamus (Murrell et al, 2005) and the placenta, (Hilton et al, 1996) and can serve in a local niche or as a systemic signalling molecule. Several studies have demonstrated that the ouabain/Na,K-ATPase-complex triggers signalling cascades, involving Ca2+, PI3K/Akt, Ras/Raf, MAPK and/or Src (Schoner and Scheiner-Bobis, 2007). These signalling events have been shown to activate gene transcription, regulate cell growth, promote differentiation, and stimulate or protect against apoptosis (Kulikov et al, 2007; Desfrere et al, 2009; Tian et al, 2009; Li et al, 2010). Given these observations, we investigated in this study the in vivo and in vitro role of the endogenous cardiac glycoside ouabain in regulating the cell growth of malignant neuroblastoma cells through various reported (Schoner and Scheiner-Bobis, 2007) and unreported ouabain-mediated signalling pathways. We asked whether the aggressive neuroblastoma proliferation could be reversibly or irreversibly suppressed by the treatment of cells with physiological concentrations of ouabain.