Abstract A71: Effect of oxysterols in adipose tissue-derived mesenchymal stem cell
Authors: Levy Debora , Silva Suelen , Melo Thatiana , Ruiz Jorge , Isaac Cesar , Fidelis Maíra , Rodrigues Alessandro , Bydlowski Sergio 
Published: May 2019

Abstract
Introduction: Mesenchymal stem cells (MSCs) are multipotent cells characterized by self-renewal and cellular differentiation capabilities. Oxysterols comprise a very heterogeneous group derived from cholesterol through enzymatic and nonenzymatic oxidation. Potent effects in cell death processes, including cytoxicity and apoptosis induction, were described in several cell lines. Very little is known about the effects of oxysterols in MSCs. Here, we describe the short-term (24 h) cytotoxic effects of several oxysterols on MSCs derived from human adipose tissue.
Methods: MSCs were isolated from adipose tissue obtained from three young, healthy women. Oxysterols were synthetized from cholesterol. Cell viability were tested by hoerchst 33342/PI assay. The Annexin V: FITC Apoptosis Detection Kit I (556420-BD Biosciences, NJ, USA) was used to determine the percentage of apoptotic cells. Caspase-3/7 activity was measured using the NucView 488 Caspase-3 Assay kit for live cells. Ki67, LC3b and cleaved caspase -3 by indirect immunofluorescence. Cleaved caspase 3 and LC3b were detected by Western blotting. TRME staining was ued to measure transmembrane mitochondrial potential. Hoerchst 33342 evaluated cell cycle and changes in actin organization were investigated using Alexa Fluor 568 phalloidin. Results are expressed as mean ± SEM from at least three independent experiments of each sample. Means were compared using ANOVA followed by Bonferronipos hoc test GraphPad Prism.
Results: Cells isolated from adipose tissue adhered to plastic with fibroblast-like morphology. Besides, they were characterized as mesenchymal stem cells. 3,5 cholestan-7-one and 7-oxocholest-5-en-3-beta-yl acetate did not promoted cell death or affect cell proliferation. IC50 of cholestan-3α-5β-6α-triol (triol) and 3α-5β-6α)-cholestane-3,6-diol (diol) was, respectively, 38.68 μM and 76.95 μM. Cell death promoted by 5β-6β epoxy-cholesterol (epoxy) at a concentration of 100 μM was approximately 10%; the calculated IC50 for this oxysterol was 258.62 μM. Triol and diol promote apoptosis at higher concentration (50 and 100 μM) with activation of caspase 3/7 and presence of cleaved caspase 3. Triol and diol lead to an enhancement of the autophagic flux activation with the presence of autophagosome; cell cycle was not affected by any of the treatments. Diol did not change mitochondrial while at 50 and 100 μM of triol it led to mitochondrial depolarization. Cells treated with triol showed a cytoplasmatic structure modification, with brighter edges and structures resembling lamellipodia and the cytoplasma area increased with enhanced fluorescence in the membrane edges. Diol also changed cell morphology—the cytoplasmic area increased and cells lost the fibroblast-like morphology.
Conclusion: Cytotoxic oxysterols led to a complex mode of cell death that could include apoptosis, necrosis, and autophagy, depending on the type of oxysterol and concentration. Cholestan-3α-5β-6α-triol was the most effective.
Citation Format: Debora Levy, Suelen Silva, Thatiana Melo, Jorge Ruiz, Cesar Isaac, Maíra Fidelis, Alessandro Rodrigues, Sergio Bydlowski. Effect of oxysterols in adipose tissue-derived mesenchymal stem cell [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A71.