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

Abstract

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.

Two-pore channels: going with the flows

“…The molecular switches downstream of the immediate Ca 2+ -binding decoders are growing, and the GTPase, dynamin, has been linked to the NAADP/TPC axis during ‘inward’ trafficking at phagocytosis [ 42 ] and endocytosis of the glucose transporter, GLUT1 [ 94 ]. Regarding downstream phosphorylation, the MAP kinase, ERK1/2, mediates cell proliferation driven by TPC2 [ 95 ], although ERKs are not themselves Ca 2+ -binding proteins.…”

“…Reciprocally, TPC2-KO enhances mTOR activity [ 107 ]. Manipulation of TPC1 expression reveals a potential link to glucose and fat metabolism [ 108 ], and the net surface expression of GLUT1 [ 94 ] and GLUT4 [ 108 ] glucose transporters are under the control of endosomal TPC1, probably by regulating endocytosis. Deletion of TPC2 in mice exacerbates the effects of a high-fat diet by reducing cholesterol/triglyceride clearance [ 87 , 109 ], although this does not translate into weight gain [ 109 ], partly due to enhanced insulin sensitivity in the absence of TPC2 [ 109 ].…”

Negative regulation of thyroid adenoma-associated protein (THADA) in the cardiac glycoside-induced anti-cancer effect

“…Cardiac glycosides, such as digoxin and digitoxin, have been used for treating congestive heart failure and cardiac arrhythmia by selective inhibition of Na + ,K + -ATPase. On the other hand, many reports suggest that cardiac glycosides may be potential chemicals for cancer, because they inhibit cell proliferation through multiple pathways, including activation of Src/MAPK signaling, induction of intracellular Ca 2+ signaling, and inhibition of PI3K/AKT/mTOR, HIF-1, and Wnt/β-catenin signaling [ 19 – 21 ]. In this study, we revealed that downregulation of the THADA-LAT1 pathway is involved in the cardiac glycoside-induced anti-cancer effect.…”

“…In various types of human cancer cells, α3NaK is found to be aberrantly expressed in intracellular vesicles in which Rab10, a small GTPase, is localized [ 18 ]. Recently, we found that cardiac glycosides (ouabain, oleandrin, and digoxin) stimulate glucose transporter GLUT1 endocytosis in HepG2 and KB cells and inhibit glycolysis of the cells by targeting the intracellular α3NaK [ 21 ]. In the cells, the [ 3 H]-ouabain uptake was increased in a time- and temperature-dependent manner and reached a maximum at 10 min [ 18 , 21 ].…”

Natural compounds targeting glycolysis as promising therapeutics for gastric cancer: A review

“…Accumulating evidence shows that CGs have significant anti-cancer effects ( Shah et al, 2022 ). In vitro experiments have confirmed that CGs reduce the levels of GLUT1 in MKN45 cells, and induce dynamin-dependent GLUT1 endocytosis, resulting in subsequent GLUT1 degradation in lysosomes, which significantly affects the uptake of glucose by cancer cells, thereby inhibiting glycolysis in gastric cancer cells ( Fujii et al, 2022 ).…”