Department of Biochemistry, University of Cambridge, Cambridge, UK.
Early Chemical Development, Pharmaceutical Sciences, R&D, AstraZeneca, Macclesfield, UK.
NMR Biomed. 2021 Mar;34(3):e4456. doi: 10.1002/nbm.4456. Epub 2021 Jan 4.
Apoptosis maintains an equilibrium between cell proliferation and cell death. Many diseases, including cancer, develop because of defects in apoptosis. A known metabolic marker of apoptosis is a notable increase in H NMR-observable resonances associated with lipids stored in lipid droplets. However, standard one-dimensional NMR experiments allow the quantification of lipid concentration only, without providing information about physical characteristics such as the size of lipid droplets, viscosity of the cytosol, or cytoskeletal rigidity. This additional information can improve monitoring of apoptosis-based cancer treatments in intact cells and provide us with mechanistic insight into why these changes occur. In this paper, we use high-resolution magic angle spinning (HRMAS) H NMR spectroscopy to monitor lipid concentrations and apparent diffusion coefficients of mobile lipid in intact cells treated with the apoptotic agents cisplatin or etoposide. We also use solution-state NMR spectroscopy to study changes in lipid profiles of organic solvent cell extracts. Both NMR techniques show an increase in the concentration of lipids but the relative changes are 10 times larger by HRMAS H NMR spectroscopy. Moreover, the apparent diffusion rates of lipids in apoptotic cells measured by HRMAS H NMR spectroscopy decrease significantly as compared with control cells. Slower diffusion rates of mobile lipids in apoptotic cells correlate well with the formation of larger lipid droplets as observed by microscopy. We also compared the mean lipid droplet displacement values calculated from the two methods. Both methods showed shorter displacements of lipid droplets in apoptotic cells. Our results demonstrate that the NMR-based diffusion experiments on intact cells discriminate between control and apoptotic cells. Apparent diffusion measurements in conjunction with H NMR spectroscopy-derived lipid signals provide a novel means of following apoptosis in intact cells. This method could have potential application in enhancing drug discovery by monitoring drug treatments in vitro, particularly for agents that cause portioning of lipids such as apoptosis.
细胞凋亡维持着细胞增殖和细胞死亡之间的平衡。许多疾病，包括癌症，都是由于凋亡缺陷而发展的。凋亡的一个已知代谢标志物是与储存在脂滴中的脂质相关的 1 H NMR 可观察到的共振的显著增加。然而，标准的一维 NMR 实验仅允许定量脂质浓度，而不提供关于物理特性的信息，例如脂滴的大小、细胞质的粘度或细胞骨架的刚性。这些附加信息可以改善对完整细胞中基于凋亡的癌症治疗的监测，并为我们提供关于为什么会发生这些变化的机制见解。在本文中，我们使用高分辨率魔角旋转（HRMAS）1 H NMR 光谱法来监测用凋亡剂顺铂或依托泊苷处理的完整细胞中的脂质浓度和可移动脂质的表观扩散系数。我们还使用溶液态 NMR 光谱法来研究有机溶剂细胞提取物中脂质谱的变化。两种 NMR 技术都显示出脂质浓度的增加，但 HRMAS 1 H NMR 光谱法的相对变化要大 10 倍。此外，与对照细胞相比，通过 HRMAS 1 H NMR 光谱法测量的凋亡细胞中脂质的表观扩散速率显着降低。凋亡细胞中可移动脂质的较慢扩散速率与显微镜观察到的较大脂质滴的形成很好地相关。我们还比较了这两种方法计算的平均脂质滴位移值。两种方法都显示出凋亡细胞中脂质滴的位移更短。我们的结果表明，基于 NMR 的完整细胞扩散实验可区分对照细胞和凋亡细胞。表观扩散测量与 HRMAS 光谱衍生的脂质信号相结合，为在完整细胞中跟踪凋亡提供了一种新方法。这种方法在体外监测药物治疗方面具有潜在的应用，特别是对于导致脂质分配（如凋亡）的药物。
