背景:糖解通量受细胞的能量需求调节。因此,癌细胞中糖酵解的上调可能是由于对三磷酸腺苷(ATP)的需求增加所致。然而,目前还不清楚这种额外的ATP周转是用来做什么的。我们假设,由于癌细胞中钠离子稳态的改变,Na/K-ATPase(NKA)的ATP需求对癌细胞糖酵解通量的增加有重要贡献。
方法:在三种人乳腺癌细胞(MDA-MB-231,HCC1954,MCF-7)中进行细胞内钠[Na]i的活的全细胞测量,在小鼠乳腺癌细胞(4T1),和使用三量子过滤的23Na核磁共振(NMR)光谱控制人上皮细胞MCF-10A。通过2HNMR测量糖解通量,以监测基线时[6,6-2H2]D-葡萄糖向[2H]标记的L-乳酸的转化以及对乌巴因的NKA抑制的反应。使用具有不同[Na+]和[K+]的等渗缓冲液滴定细胞内[Na+]i,并使用离子载体小草菌素A引入人工Na+质膜渗漏。实验与细胞活力测定平行进行,细胞内和细胞外代谢物的1HNMR代谢组学,使用2-脱氧-2-[18F]氟葡萄糖(18F-FDG)正电子发射断层扫描(PET)在MDA-MB-231人异种移植小鼠模型中进行细胞外通量分析和体内测量。
结果:与对照MCF-10A细胞相比,人和鼠乳腺癌细胞中的细胞内[Na+]i升高。乌巴因对NKA的急性抑制导致所有三种乌巴因敏感的人类癌细胞中[Na]i升高和糖酵解通量的抑制,但在抗乌巴因的鼠细胞中却没有。用短链菌素A渗透细胞膜导致MDA-MB-231和4T1细胞中[Na]i的可滴定增加,糖酵解通量的Na依赖性增加。这在人细胞中但在鼠细胞中没有用哇巴因减毒。MDA-MB-231人异种移植小鼠模型中的18FDGPET成像记录了当用哇巴因治疗时18FDG肿瘤摄取较低,而鼠组织摄取不受影响。
结论:糖酵解通量与乳腺癌细胞中Na+驱动的NKA活性相关,在Warburg效应的机理基础上,为[Na+]i-NKA关系的中心性提供证据。
BACKGROUND: Glycolytic flux is regulated by the energy demands of the cell. Upregulated glycolysis in cancer cells may therefore result from increased demand for adenosine triphosphate (ATP), however it is unknown what this extra ATP turnover is used for. We hypothesise that an important contribution to the increased glycolytic flux in cancer cells results from the ATP demand of Na+/K+-ATPase (NKA) due to altered sodium ion homeostasis in cancer cells.
METHODS: Live whole-cell measurements of intracellular sodium [Na+]i were performed in three human breast cancer cells (MDA-MB-231, HCC1954, MCF-7), in murine breast cancer cells (4T1), and control human epithelial cells MCF-10A using triple quantum filtered 23Na nuclear magnetic resonance (NMR) spectroscopy. Glycolytic flux was measured by 2H NMR to monitor conversion of [6,6-2H2]D-glucose to [2H]-labelled L-lactate at baseline and in response to NKA inhibition with ouabain. Intracellular [Na+]i was titrated using isotonic buffers with varying [Na+] and [K+] and introducing an artificial Na+ plasma membrane leak using the ionophore gramicidin-A. Experiments were carried out in parallel with cell viability assays, 1H NMR metabolomics of intracellular and extracellular metabolites, extracellular flux analyses and in vivo measurements in a MDA-MB-231 human-xenograft mouse model using 2-deoxy-2-[18F]fluoroglucose (18F-FDG) positron emission tomography (PET).
RESULTS: Intracellular [Na+]i was elevated in human and murine breast cancer cells compared to control MCF-10A cells. Acute inhibition of NKA by ouabain resulted in elevated [Na+]i and inhibition of glycolytic flux in all three human cancer cells which are ouabain sensitive, but not in the murine cells which are ouabain resistant. Permeabilization of cell membranes with gramicidin-A led to a titratable increase of [Na+]i in MDA-MB-231 and 4T1 cells and a Na+-dependent increase in glycolytic flux. This was attenuated with ouabain in the human cells but not in the murine cells. 18FDG PET imaging in an MDA-MB-231 human-xenograft mouse model recorded lower 18FDG tumour uptake when treated with ouabain while murine tissue uptake was unaffected.
CONCLUSIONS: Glycolytic flux correlates with Na+-driven NKA activity in breast cancer cells, providing evidence for the \'centrality of the [Na+]i-NKA nexus\' in the mechanistic basis of the Warburg effect.