Abstract 50: Comparison of NMR lipid profiles in mitotic arrest and apoptosis as indicators of drug resistance

Abstract

Abstract Aim: To compare NMR-visible lipids in paclitaxel exposed cells undergoing apoptosis or mitotic arrest in order to explore their utility as a biomarker of drug resistance. Methods: Cultured cervical cell lines (HeLa, C33A and Me180, ATCC, USA) were exposed to 1 µM paclitaxel (Sigma, UK) for 8, 16, 24, and 48 hours (n=3). Diffusion-weighted (DW) spectra were acquired using a 11.74T spectrometer (Avance Bruker BioSpin, Germany) using a stimulated echo sequence with bipolar gradients. Cellular morphology was assessed with transmission electron microscopy (TEM) using uranyl acetate followed by lead citrate staining. Following TOPRO-3 and nile red co-staining, flow cytometry and confocal microscopy verified cell size and viability, visualized cell cycle phase distribution, apoptotic features, and accumulation of cytoplasmic lipid droplets. Western blots were used to assess activation (by phosphorylation) of cytoplasmic phospholipase A2 (P-cPLA2) and expression of fatty acid synthase (FAS) at each stage. Results: 24 h after exposure to paclitaxel, all lines showed >65% mitotic arrest, and HeLa showed apoptosis as well. At 48 h HeLa cells progressed to apoptosis (due to mitotic catastrophe) while C33A and Me180 cells progressed beyond mitotic arrest to normal morphology or multinucleation indicating resistance. There was significant increase in saturated and unsaturated lipids at 24 h (mitotic arrest) followed by a further striking increase at 48 h in all lines, especially in the 5.3ppm peak (unsaturated) in HeLa at 48 h. Unsaturated lipids increased more than saturated lipids in all lines, triglycerides increase the most in HeLa cells. Resistant lines showed lower increased methylene / methyl (1.3 ppm / 0.9 ppm) ratio (from 1.3 to 2.8, 1.7 to 2.8 compared with 1.5 to 3.8 in HeLa cells at 48 h). The levels of P-cPLA2 and FAS were unchanged at 24 h followed by a drop at 48 h in HeLa cells. The percentage of cells displaying lipid droplets increased significantly at 24 and 48 h in all lines with increase in droplet size only in HeLa cells at 24 and 48 h. This implies that the larger droplets are associated with apoptosis but that smaller ones are less specific. Apoptotic cells showed 3-4 x the number of droplets compared to cells arrested in mitosis. Conclusion: After exposure of cells to paclitaxel, increase in lipids on NMR results from increased visibility not synthesis (more droplets, FAS not increased). In apoptosis predominant increases are in unsaturated fatty acids in larger droplets, whereas paclitaxel resistant lines accumulate smaller droplets with significantly less triglycerides. Acknowledgements: This work was funded by the EC FP6 Marie Curie Action: Early Stage Training (contact No. 020718). We also acknowledge the support received for the CRUK and EPSRC Cancer Imaging Centre in association with the MRC and Department of Health (England) (grant C1060/ A10334) and NHS Funding to the NIHR Biomedical Research Centre. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 50.