Selective Na+ intracellular increase kills murine hepatocarcinoma cells: a novel diagnostic cancer biomarker and a new anticancer strategy beside immunotherapy?

“Selective Na+ intracellular increase kills murine hepatocarcinoma cells: a novel diagnostic cancer biomarker and a new anticancer strategy beside immunotherapy?” Nausicaa Clemente, Simona Baroni, Francesco Tasso, Simone Fiorilla, Simone Reano, Chiara Borsotti, Simonetta Geninatti Crich and Rita Carini Introduction Limited or lack of responses to chemotherapy, biological therapy or immunotherapy make necessary exploring novel anticancer targets and diagnostic biomarkers. Intracellular alkaline pH is a hallmark of cancer cells and relies on the increased activity or expression of pH regulatory proteins and, among them, of Na+ transporters. In primary non-cancerous hepatocytes, following ATP depletion, an irreversible increase of intracellular Na+ is the final cause of the osmotic lysis and death of the cells. In cancer research, the significance of Na+ intracellular variations is under investigated. Early X ray microanalysis of rodent cells and recent MRI studies on human cancers, however, evidence an augmented intracellular concentration of Na+ in cancer cells in comparison to healthy cells. Moreover, "Field‐Cycling Relaxometry" studies show that there is a correlation between cellular water molecule efflux rate constant and the related intracellular water mole fraction, with cancer aggressiveness. This study explores the hypothesis that cancer cells, owing to a constitutively high intracellular Na+ concentration and high energy-consuming metabolism might be, unlike non-cancerous cells, energetically unable to compensate and survive to a further Na+ load induced by the cation ionophore Monensin. Methods The effects of Monensin treatment were evaluated in vitro and in vivo employing murine and human HCC cell lines (C1C7 and HepG2 cells) and primary mouse hepatocytes (cultured in DMEM and Krebs or Krebs without Na+ buffers) or HCC (C1C7 cells) implanted allografts in male NSG mice. Cell death, ATP, levels and glycolisis were evaluated with standard protocols. Mitochondrial O2 consumption rate fluxes were analyzed with an Oxygraph-2 K high-resolution respirometer. Na+ levels were monitored by fluorimetry, ICP-MS, 23Na-MRI acquisition on a 7 T Bruker Biospin Pharmascan 70/16 scanner equipped with a 1H/23Na transmit-receive surface coil and confocal or time-lapse imaging analysis with the THUNDER Imager 3D Live Cell. Water fluxes across cell membrane were determined by 1H NMR relaxometry. Hemopoiesis was evaluated by analysing bone marrow cells acquired on a Attune NxT Acoustic Focusing Cytometer. Tissue damage and proliferation were estimated by histological and immuno-histochemical analyses (H&E and Ki67 staining). Results Na+ levels of healthy hepatocytes and liver was lower compared to mouse and human HCC cells and tumour tissue. Monensin further increased Na+ levels in HCC cells and in HCC allograft but not in primary hepatocytes and in normal hepatic and extrahepatic tissue. Sodium increase was associated to ATP depletion, mitochondrial Na+ load, inhibition of mitochondrial O2 consumption and decrease of the water efflux rate constant and preceded the death of HCC cells. Prevention of intracellular Na+ increase by maintaining HCC cells in a Na+ free media protected energy and osmotic alterations and appearance of cell death. Monensin systemic treatment induced shrinkage and necrosis of the allograft tumor but did not display damaging effects nor affected the integrity of normal organs. Monensin did not evidence cytostatic effects in either healthy or transformed proliferating tissues nor altered hemopoiesis. Conclusions: These results show that a forced sodium influx selectively kills hepatocarcinoma cells without affecting normal cells or inhibiting proliferation of normal or transformed tissues. Such observation enlightens Na+ homeostasis as novel druggable target for HCC therapy and cancer in general. The capacity of Monensin to selectively increase Na+ in transformed tissue thereby inducing specific cancer cell death reveals a novel anticancer mechanism of Na+ ionophores and, additionally, suggests their potentiality as diagnostic tools to magnify the detection of the increased sodium levels associated with malignancy.