Abstract
Increases in 1H nuclear magnetic resonance spectroscopy (NMR) visible lipids are a well-documented sign of treatment response in cancers. Lipids in cytoplasmic lipid droplets (LDs) are the main contributors to the NMR lipid signals. Two human primitive neuroectodermal tumour cell lines with different sensitivities to cisplatin treatment were studied. Increases in NMR visible saturated and unsaturated lipids in cisplatin treated DAOY cells were associated with the accumulation of LDs prior to DNA fragmentation due to apoptosis. An increase in unsaturated fatty acids (UFAs) was detected in isolated LDs from DAOY cells, in contrast to a slight decrease in UFAs in lipid extracts from whole cells. Oleic acid and linoleic acid were identified as the accumulating UFAs in LDs by heteronuclear single quantum coherence spectroscopy (HSQC). 1H NMR lipids in non-responding PFSK-1 cells were unchanged by exposure to 10 μM cisplatin. These findings support the potential of NMR detectable UFAs to serve as a non-invasive marker of tumour cell response to treatment.
Highlights
Oleic acid and linoleic acid were identified as the accumulating unsaturated fatty acids (UFAs) in lipid droplets (LDs) by heteronuclear single quantum coherence spectroscopy (HSQC). 1H nuclear magnetic resonance spectroscopy (NMR) lipids in non-responding PFSK-1 cells were unchanged by exposure to 10 lM cisplatin
The lipid signal intensities were taken as a ratio to the macromolecule peak area at 1.68 ppm as referencing to protein signal has been shown to be a good approach in NMR signal quantification (Bayet-Robert et al 2010) and renders the data from different cell lines comparable
The current study shows that unsaturated fatty acids detected by HR-MAS increase in whole DAOY cells undergoing cisplatin-induced cell death, consistent with studies on a glial tumour (Hakumaki et al 1999) and lymphoma cells (Schmitz et al 2005)
Summary
2.1 Cell cultureTwo neuronal brain tumour cell lines: DAOY, human medulloblastoma and PFSK-1, human supratentorial primitive neuroectodermal tumour were cultured in 75 cm flasks with filter-vented caps and maintained in 15 ml Dulbecco’s modified eagles medium (DMEM F:12) (Invitrogen, UK) supplemented with 10 % (v/v) foetal calf serum (PAA, UK), 1 % 200 mM L-glutamine (1009) and 1 % non-essential amino acid solution (1009) (Sigma, UK). 2 mM stock solution was made from cisplatin powder (Sigma Aldrich, UK) with DMEM stored at -80 °C in dark conditions. 10–100 lM cisplatin working solution was freshly made from the stock cisplatin with culture media before adding to the cell culture. 2.3 Alamar blue assay for cell viability. Alamar blue assay to estimate number of viable cells was performed as previously described (6). 40–60 9 106 cells were ground in 600 ll deionised water or D2O using a Dounce grinder and the homogenate was centrifuged at 20009g at 4 °C for 10 min. The supernatant was adjusted to 18.46 % sucrose, topped with the same volume of deionised water or D2O and centrifuged at 142,0009g for 120 min at 4 °C (Optima TLX Ultracentrifuge, Beckman). At least three independent preparations for each condition and cell type were used for isolation
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