Ti-obesity effects remains unclear. Therefore, the present study aimed to examine whether or not the anticancer activity of quercetin is associated with its anti-obesity effects. This study investigated the inhibitory impact of quercetin on human liver HepG2 cancer cells with overexpression of FASN. Materials and techniques Reagents and antibodies. Quercetin, acetyl-CoA, alonyl-CoA, dexamethasone, Hoechst 33258, insulin, NADPH, MTT dye, 3-isobutyl-1-methylxanthine, palmitic acid, EDTA and DTT had been bought from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum have been bought from Gibco-BRL (Gaithersburg, MD, USA) as well as the penicillin-streptomycin was bought from Oriental Bio-Technology Co., Ltd. (Beijing, China). Rabbit anti-human polyclonal FASN andCorrespondence to: Dr Yu Zhang, Health Management Center,Hangzhou Sanatorium of PLA, 5 Longjing Road, Hangzhou, Zhejiang 310007, P.R. China E mail: hz128jkgl@163Key words: quercetin, cancer, fatty acid synthase, HepG2 cells,apoptosisZHAO et al: QUERCETIN INHIBITS FATTY ACID SYNTHASEmouse anti-human monoclonal GAPDH antibodies have been purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Cell culture. Human liver cancer HepG2 cells were obtained in the Cell Bank in the Chinese Academy of Sciences (Shanghai, China). Cells had been incubated in DMEM (high-glucose), ten fetal bovine serum and 100 U/ml penicillin-streptomycin. MTT assay. HepG2 cells were seeded in a 96-well plate (5×103 cells/well) and after that treated with quercetin at unique concentrations for 24 h. Thereafter, 20 ml of MTT option [5 mg MTT/ml in phosphate-buffered saline (PBS)] was added into every single effectively of a microtiter plate and incubated for 4 h at 37 . The resultant formazan solution was dissolved in 200 ml dimethylsulfoxide/well, and its concentration was measured at 492 nm by a microplate reader (Model EL 307C; BioTek, Shanghai, China). Cell lysis and immunoblotting. Cells have been lysed as previously described (36) plus the cell lysates had been heated within a water bath to fully denature the proteins. The proteins had been then separated by SDS-PAGE [Bio-Rad Laboratories (Shanghai) Ltd., Shanghai, China] and transferred to polyvinylidene difluoride membranes (Immobilon; Millipore, Billerica, MA, USA). Immunoblotting was performed with antibodies against FASN and GAPDH, and visualized utilizing an enhanced chemiluminescence light detection kit (Amersham, Piscataway, NJ, USA). Cell apoptosis assay. HepG2 cells were seeded in 12-well culture dishes (5×10 four cells/well). Following experimental treatment with 25 and 50 quercetin for 24 h, cells had been washed twice with PBS, stained with Hoechst 33258 (five mg/ml) for five min inside the dark, after which washed extensively three times with PBS.Dihydroartemisinin Nuclear staining was examined beneath a fluorescence microscope (Nikon LH-M100CB; Jirui Co.Lornoxicam , Ltd.PMID:25016614 , Suzhou, China) and pictures have been captured making use of Image-Pro Plus application (MediaCybernetics, Silver Spring, MD, USA). Intracellular fatty acids assay. The quantity of intracellular fatty acid was determined by the Fatty Acid Assay kit (Lab-Bio Co., Ltd., Beijing, China). Briefly, HepG2 cells were seeded in 100-mm cell culture dishes. Following experimental therapy, cells had been washed twice with PBS after which extracted by homogenization with 200 chloroform-Triton X-100 (1 Triton X-100 in pure chloroform; Shanghai XiTang Biotechnology Co., Ltd., Shanghai, China) inside a microhomogenizer. Subsequently, the extract was centrifuged for.
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