Cardiac glycosides stimulate endocytosis of GLUT1 via intracellular Na⁺,K⁺-ATPase α3-isoform in human cancer cells

Glucose transporter GLUT1 plays a primary role in the glucose metabolism of cancer cells. Here, we found that cardiac glycosides (CGs) such as ouabain, oleandrin, and digoxin, which are Na⁺,K⁺-ATPase inhibitors, decreased the GLUT1 expression in the plasma membrane of human cancer cells (liver cancer HepG2, colon cancer HT-29, gastric cancer MKN45, and oral cancer KB cells). The effective concentration of ouabain was lower than that for inhibiting the activity of Na⁺,K⁺-ATPase α1-isoform (α1NaK) in the plasma membrane. The CGs also inhibited [³H]2-deoxy- d-glucose uptake, lactate secretion, and proliferation of the cancer cells. In intracellular vesicles of human cancer cells, Na⁺,K⁺-ATPase α3-isoform (α3NaK) is abnormally expressed. Here, a low concentration of ouabain inhibited the activity of α3NaK. Knockdown of α3NaK significantly inhibited the ouabain-decreased GLUT1 expression in HepG2 cells, while the α1NaK knockdown did not. Consistent with the results in human cancer cells, CGs had no effect on GLUT1 expression in rat liver cancer dRLh-84 cells where α3NaK was not endogenously expressed. Interestingly, CGs decreased GLUT expression in the dRLh-84 cells exogenously expressing α3NaK. In HepG2 cells, α3NaK was found to be colocalized with TPC1, a Ca²⁺-releasing channel activated by nicotinic acid adenine dinucleotide phosphate (NAADP). The CGs-decreased GLUT1 expression was significantly inhibited by a Ca²⁺ chelator, a Ca²⁺-ATPase inhibitor, and a NAADP antagonist. The GLUT1 decrease was also attenuated by inhibitors of dynamin and phosphatidylinositol-3 kinases (PI3Ks). In conclusion, the binding of CGs to intracellular α3NaK elicits the NAADP-mediated Ca²⁺ mobilization followed by the dynamin-dependent GLUT1 endocytosis in human cancer cells.