Abstract
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 1 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) 1 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 1 H NMR spectroscopy. Moreover, the apparent diffusion rates of lipids in apoptotic cells measured by HRMAS 1 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 1 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.
Highlights
Lipid droplets (LDs) are cytosolic organelles composed of neutral lipids enclosed within a phospholipid monolayer
3.1 | LD concentration determined by high-resolution magic angle spinning NMR (NMR) spectroscopy and the link to apoptosis
Since LD size by microscopy and mobile lipid diffusion by NMR spectroscopy were highly correlated, we investigated if combining the high-resolution magic angle spinning (HRMAS) 1H NMR diffusion data and fluorescence microscopy measurements would lead to correct estimation of the cytoplasmic viscosity
Summary
Lipid droplets (LDs) are cytosolic organelles composed of neutral lipids enclosed within a phospholipid monolayer. NMR spectroscopy has been widely used to study the effects of apoptotic agents on the metabolic profiles of tissues and cell lines.[7,8,9] In addition, 1H MRS provides a non-invasive method for assessing tissue responses to cancer treatments in vivo.[10] Solution-state 1H NMR spectroscopy on cell or tissue extracts gives quantitative information on lipid concentration.[11,12] In this case, the 1H NMR spectra represent the collective signal from cytosolic LDs, lipids contained within cell organelles and membrane lipids. High-resolution magic angle spinning (HRMAS) 1H NMR spectroscopy provides improved spectral resolution of heterogeneous biological samples.[24] It has been used to study lipid metabolism affected by a variety of diseases, for example cancer,[25] diabetes,[26] and schizophrenia,[27] as well as cellular processes such as apoptosis.[28] the lipid signals observed in HRMAS spectra of intact cells or tissue are from cytosolic LDs only, rather than lipids associated with membranes. We illustrate the advantages of using HRMAS 1H NMR over solution-state 1H NMR spectroscopy for the monitoring of apoptosis-related LD formation
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