The formation of cytosolic lipid droplets (LD) incorporating neutral lipids is a common adaptation to cellular stress triggered by factors such as redox imbalance, excessive free fatty acids or nutrient starvation [45,49]. conversion, LY2784544 (Gandotinib) tricarboxylic acid (TCA) cycle intensification, and hydrolysis of neutral lipids, while UA effects were much less pronounced. In MCF-10A cells, boosting of glucose metabolism by the two TAs was accompanied by diversion of glycolytic intermediates to the hexosamine biosynthetic pathway (HBP) and the synthesis of neutral lipids, possibly stored in detoxifying lipid droplets. Additionally, breast epithelial cells intensified pyruvate consumption and TCA cycle activity, possibly to compensate for oxidative impairment of pyruvate glycolytic production. This study provided novel insights into the metabolic effects of BA and UA in cancer and non-cancer breast cells, thus improving current understanding of the action of NNT1 these compounds at the molecular level. spp.) and across a wide range of plant families , have been widely studied LY2784544 (Gandotinib) for their anti-tumoral activity in multiple cancer models and were found to modulate diverse pathways involved in carcinogenic processes [14,15,16,17,18,19]. Open in a separate window Figure 1 Structural formula of (A) betulinic acid and (B) ursolic acid. Metabolic reprogramming is strongly linked to tumor-specific signaling pathways and supports tumor growth, invasion and immune escape . Several drugs targeting altered metabolic enzymes and pathways in cancer are currently under intense pre-clinical and clinical testing . The ability of plant-derived natural compounds to modulate tumor cell metabolism and, in this way, exert anticancer activity, has also started to be realized . A few works have addressed the impact of triterpenic acids (TAs) on tumor metabolism, namely on specific glycolytic enzymes and lactate production [22,23,24,25]. However, a more comprehensive picture of their impact on both tumor and non-tumor cell metabolism is still missing. The present work aims to assess the metabolic effects of BA and UA in MDA-MB-231 breast cancer cells (TNBC model), as well as in MCF-10A non-cancer breast epithelial cells. Identification and quantification of changes in the cells exo- and endometabolome were performed through 1H NMR analysis of cell culture medium supernatants, aqueous and organic cell extracts. This approach is expected to provide new insights into the involvement of metabolic reprogramming in cellular responses to these TAs, and will hopefully contribute to advance research on phytochemical-based therapy for TNBC. 2. Materials and Methods 2.1. Materials Dulbeccos modified Eagles medium (DMEM), DMEM/F12 medium and trypsin (5 g/L)-EDTA (2 g/L) were supplied by Biowest, (Nuaill, France). Fetal bovine serum (FBS) was from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Horse serum, human epidermal growth factor, human insulin, hydrocortisone and cholera toxin were obtained from Sigma-Aldrich (St. Louis, MO, USA). Betulinic acid (90% purity) and ursolic acid (98% purity) were purchased from Molekula GmbH (Munchen, Germany). Dimethylsulfoxide (DMSO, cell culture grade) was obtained from Applichem (Gatersleben, Germany). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and propidium iodide were purchased from Calbiochem (San Diego, CA, USA). Methanol was obtained from Merk (Darmstadt, Germany) and chloroform from Normapur (VWR, Radnor, USA). RNase was obtained from Sigma Chemicals Co. (Madrid, Spain). Deuterated water (D2O) containing 0.75% 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TSP-d4) and deuterated chloroform containing 0.03% (for 5 min at 4 C and resuspended in PBS, before being treated with RNase LY2784544 (Gandotinib) (50 g/mL) and propidium iodide staining solution (50 g/mL) and incubated, in the dark, for at least 20 min at room temperature. Propidium iodide-stained cells were analyzed on a Coulter EPICS XL (Beckman Coulter, Hialeah, FL, USA) flow cytometer. The results were acquired using the SYSTEM II software (version 3.0 Beckman-Coulter ?, Brea, CA, USA). Four replicates were performed for each treatment, and for each sample at least 5000 nuclei were acquired. Analysis of cell cycle distribution was performed using the FlowJo software (Tree Star, Ashland, USA). 2.5. Cell Exposure for Metabolomics Assays MDA-MB-231 and MCF-10A cells were seeded in 10-cm-diameter Petri dishes at a density of 6 105 cells/mL (10 mL per dish) and cultured for 24 h at 37 C. Cells were then incubated for 48 h with BA (5 and 15 M) or UA (10 and 20 M). Vehicle solvent control cells received DMSO (0.10% 0.05 significance level was identified by all pairwise multiple comparison procedures via the Tukeys test. 3. Results 3.1. Inhibitory Effects of Betulinic and Ursolic Acids on MDA-MB-231 and MCF-10A Cellular Viability MDA-MB-231 and MCF-10A cells were treated with various concentrations (0C50 M) of either BA or UA for 24 h, 48 h.
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