OBJECTIVETo investigate the role of miR-181c in glycolysis of cancer-associated fibroblasts (CAFs) and explore the mechanism.

METHODSHuman lung CAFs and normal fibroblasts (NFs), isolated from fresh human lung adenocarcinoma tissue specimens by primary culture of tissue explants, were transfected with a miR -181c mimics, a miR-181c inhibitor, a siRNA siRNA-HK2 or the vector HK2-vector via Lipofectamine(TM) 2000. Quantitative real-time PCR was used to analyze the changes in miR-125b expression in the transfected cells; hexokinase-2 (HK2) protein expression in the cells was detected using Western blotting, and the cellular glucose uptake was assessed with 2-NBDG. Lactate production in the cells was examined and expression of HK2 mRNA was detected with dual luciferase reporter gene assay.

RESULTSNo obvious difference was found in the cell morphology between CAFs and NFs. Compared with the NFs, the CAFs showed obviously increased glucose uptake, lactate production and HK2 protein expression with decreased expressions of the miR-181 family (P<0.05). Transfection with the miR-181 inhibito- rsignificantly increased glucose uptake, lactate production and HK2 protein expression in the NFs. In CAFs, transfection with the miR-181 mimics caused significantly lowered glucose uptake, lactate production and HK2 protein expression of. Knockdown of endogenous HK2 by siRNA abolished miR-181 mimics-mediated decrease of glucose uptake and lactate production in CAFs, while transfection with miR-181 mimics suppressed HK2 overexpression-induced enhancement of glucose uptake and lactate production in NFs.

CONCLUSIONTransfection with miR-181 mimics can suppress glycolysis in CAFs by inhibiting HK2 expression.

4-Chloro-7-nitrobenzofurazan ; analogs & derivatives ; Adenocarcinoma ; pathology ; Deoxyglucose ; analogs & derivatives ; Fibroblasts ; drug effects ; Glycolysis ; Hexokinase ; antagonists & inhibitors ; Humans ; Lung Neoplasms ; pathology ; MicroRNAs ; pharmacology ; RNA, Messenger ; RNA, Small Interfering ; Real-Time Polymerase Chain Reaction ; Transfection ; Tumor Cells, Cultured

OBJECTIVEA highly sensitive and rapid high-performance liquid chromatography method with pre-column derivatization with 4-fluoro-7-nitrobenzofurazan was developed for determination of taurine in biological samples, including plasma, brain, and liver.

METHODSThe optimum derivatization reaction temperature was 70 °C, and at this temperature the reaction was complete within 3 min. The derivatized taurine was separated using phosphate buffer (0.02 mol/L, pH 6.0):acetonitrile (84:16, v/v) as the mobile phase at a flow rate of 1.0 mL/min, and a column temperature of 25 °C. The taurine derivatives were separated within 20 min (tR:14.5 min) and fluorometrically detected at 530 nm with excitation at 470 nm.

RESULTSThe intra- and the inter-day coefficients of variation for the method were 5.3% and 7.7%, respectively. The calibration curve was linear from 0.1 μmol/L to 30.0 μmol/L with a correlation coefficient of 0.9995.

CONCLUSIONThis method can be used to determine the taurine contents in plasma, brain, and liver from normal rats and human plasma.

4-Chloro-7-nitrobenzofurazan ; analogs & derivatives ; chemistry ; Acetonitriles ; chemistry ; Animals ; Brain Chemistry ; Chromatography, High Pressure Liquid ; Female ; Fluorescent Dyes ; chemistry ; Humans ; Hydrogen-Ion Concentration ; Liver ; chemistry ; Male ; Rats ; Rats, Wistar ; Solvents ; chemistry ; Taurine ; analysis ; blood ; Temperature

OBJECTIVEThe purpose of this study was to assess the feasibility of fluorescent 2-deoxyglucose analog, 2-[N-(7-nitrobenz-2-oxa-1, 3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG), that could be taken up by breast cancer cells highly expressing glucose transporter 1 (GLUT-1). The purpose of this study was to clarify if a fluorescent 2-deoxyglucose analog, 2-[N-(7-nitrobenz-2-oxa-1, 3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG), can be taken up by breast cancer cells highly expressing glucose transporter 1 (GLUT-1), and to assess whether it can be used as a targeting imaging agent.

METHODSThe expressions of GLUT-1 mRNA and protein in breast cancer MDA-MB-231 cells were detected by RT-PCR and immunohistochemistry, respectively. The difference of GLUT-1 protein expression between breast cancer MDA-MB-231 cells and MCF-7 cells was compared by Western blot. Secondly, MDA-MB-231 cells which were grown in 6-well plates were incubated with 2-NBDG, and the result of 2-NBDG uptake was analyzed by fluorescence microscopy and flow cytometry. The difference of 2-NBDG absorption in MDA-MB-231 and MCF-7 cells was compared by flow cytometry.

RESULTSThe results of RT-PCR and immunohistochemistry confirmed that MDA-MB-231 cells highly expressed GLUT-1. Furthermore, Western blot revealed that GLUT-1 expression of MDA-MB-231 cells (0.946 ± 0.007) was higher than that in the MCF-7 cells (0.833 ± 0.010). Fluorescence microscopic and flow cytometric analysis showed that 2-NBDG was uptaken rapidly by MDA-MB-231 cells. Addition of 50 mmol/L D-glucose to the media with 2-NBDG reduced its uptake by 46.0%. Moreover, flow cytometry indicated that the fluorescence intensity of MDA-MB-231 cells (25.10 ± 0.57) was higher than that of MCF-7 cells (10.12 ± 0.62) when incubated with 2-NBDG for 20 minutes.

CONCLUSIONThe preliminary data clearly demonstrate that 2-NBDG is taken up and accumulated in breast cancer cells that highly express GLUT-1, and may be used as an optical probe for glucose uptake in hypermetabolic malignant cells.

4-Chloro-7-nitrobenzofurazan ; analogs & derivatives ; pharmacokinetics ; Blotting, Western ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Deoxyglucose ; analogs & derivatives ; pharmacokinetics ; Female ; Flow Cytometry ; Glucose Transporter Type 1 ; genetics ; metabolism ; Humans ; Immunohistochemistry ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo study the effect of the Chinese compound prescription Chaihu Shugan Tang (CHSGT) on the excitability in the cerebral cortex and hippocampus (different brain regions) of pentetrazole (PTZ)-kindled chronic epileptic rats.

METHODTo establish the model of chronic kindling rats intraperitoneal injected with pentylenetet. Fully kindled rats were randomized into control and experimental groups for intragastric administration of normal saline (control, model), Sodium Valproate and CHSGT at the high, medium and low doses for 4 consecutive weeks. The content of 2-NBDG, the glutamate (Glu) and the aspartate (Asp) in different brain regions of rats were detected by fluorescence imaging techniques and HPLC assay respectively.

RESULTCHSGT at the high, medium and low doses all significantly decreased the content of 2-NBDG, the Glu and the Asp in different brain regions of chronic epileptic rats (P < 0.01).

CONCLUSIONCHSGT can inhibit the excitability in different brain regions of PTZ-induced epileptic rats, by decreasing the level of excitatory neurotransmitter maybe one of its antiepileptic mechanisms.

4-Chloro-7-nitrobenzofurazan ; analogs & derivatives ; metabolism ; Animals ; Aspartic Acid ; metabolism ; Brain ; drug effects ; metabolism ; physiopathology ; Chronic Disease ; therapy ; Deoxyglucose ; analogs & derivatives ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Epilepsy ; chemically induced ; drug therapy ; metabolism ; physiopathology ; Glutamic Acid ; metabolism ; Hippocampus ; drug effects ; physiopathology ; Humans ; Kindling, Neurologic ; drug effects ; Male ; Pentylenetetrazole ; administration & dosage ; adverse effects ; Random Allocation ; Rats ; Valproic Acid ; metabolism