ABSTRACT
Oncogenic transformation involves reprogramming of cell metabolism, whereby steady-state levels of intracellular NAD(+) and NADH can undergo dramatic changes while ATP concentration is generally well maintained. Altered expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of NAD(+)-salvage, accompanies the changes in NAD(H) during tumorigenesis. Here, we show by genetic and pharmacological inhibition of NAMPT in glioma cells that fluctuation in intracellular [NAD(H)] differentially affects cell growth and morphodynamics, with motility/invasion capacity showing the highest sensitivity to [NAD(H)] decrease. Extracellular supplementation of NAD(+) or re-expression of NAMPT abolished the effects. The effects of NAD(H) decrease on cell motility appeared parallel coupled with diminished pyruvate-lactate conversion by lactate dehydrogenase (LDH) and with changes in intracellular and extracellular pH. The addition of lactic acid rescued and knockdown of LDH-A replicated the effects of [NAD(H)] on motility. Combined, our observations demonstrate that [NAD(H)] is an important metabolic component of cancer cell motility. Nutrient or drug-mediated modulation of NAD(H) levels may therefore represent a new option for blocking the invasive behavior of tumors.
Subject(s)
Cell Movement/physiology , Gene Expression Regulation, Neoplastic/physiology , Glioma/physiopathology , NAD/metabolism , Neoplasm Invasiveness/physiopathology , Nicotinamide Phosphoribosyltransferase/antagonists & inhibitors , Blotting, Northern , Blotting, Western , Glioma/metabolism , Humans , Hydrogen-Ion Concentration , L-Lactate Dehydrogenase/metabolism , Lactic Acid/pharmacology , Nicotinamide Phosphoribosyltransferase/genetics , Nicotinamide Phosphoribosyltransferase/metabolism , Time-Lapse Imaging , Tumor Cells, CulturedABSTRACT
BACKGROUND: Nebivolol (NEB) is a [beta]1-receptor blocker with nitric oxide-dependent vasodilating properties. NEB-induced nitric oxide release is mediated through the estrogen receptor. METHOD: Here, we tested the hypothesis that NEB decreases endothelial cell stiffness and that these effects can be abolished by both endothelial nitric oxide synthase and estrogen receptor blockade. Human endothelial cells (EAHy-926) were incubated with vehicle, NEB 0.7 nmol/l, metoprolol 200 nmol/l, 17[beta]-estradiol (E2) 15 nmol/l, the estrogen receptor antagonists tamoxifen 100 nmol/l and ICI 182780 (ICI) 100 nmol/l, the nitric oxide synthase inhibitor N[omega]-nitro-L-arginine methyl ester 1 mmol/l and combinations of NEB and E2 with either tamoxifen, ICI or N[omega]-nitro-L-arginine methyl ester as well as metoprolol and ICI. Atomic force microscopy was performed to measure cellular stiffness, cell volume and apical surface. Presence of estrogen receptor protein in EAHy-926 was confirmed by western blot analysis; quantification of ER[alpha] and ER[beta] total RNA was performed by semiquantitative PCR. RESULTS: Both NEB as well as E2 decreased cellular stiffness to a similar extent (NEB: 0.83 +/- 0.03 pN/nm, E2: 0.87 +/- 0.03 pN/nm, vehicle: 2.19 +/- 0.07 pN/nm), whereas metoprolol had no effect on endothelial stiffness (2.07 +/- 0.04 pN/nm, all n = 60, P < 0.01). The decrease in stiffness occurred as soon as 5 min after starting NEB incubation. The effects are mediated through nongenomic ER[beta] pathways, as ER[alpha] is not translated into measurable protein levels in EAHy-926. Furthermore, NEB increased cell volume by 48 +/- 4% and apical surface by 34 +/- 3%. E2 had comparable effects. Tamoxifen, ICI and N[omega]-nitro-L-arginine methyl ester substantially diminished the effects of NEB and E2. CONCLUSION: NEB decreases cellular stiffness and causes endothelial cell growth. These effects are nitric oxide-dependent and mediated through nongenomic ER[beta] pathways. The morphological and functional alterations observed in endothelial cells may explain improved endothelial function with NEB treatment.
