Tetrazanbigen (TNBG) is a novel synthetic antitumor drug with significant antitumor effects on common solid tumors in vitro and in vivo. It may lead to death of cancer cells through a tumor-associated lipoidosis mechanism, and result in lipid droplets (LDs) accumulation at the cytoplasm. In this study, the effects of TNBG on protein expression in human hepatocellular carcinoma cell line QGY-7701 were studied for elucidating its antitumor mechanism. The proteins extracted from TNBG-treated human hepatocellular carcinoma cell line QGY-7701 were analyzed and compared with control cells by two-dimensional gel electrophoresis. The differential proteins were identified by matrix-associated laser desorption ionization time-of-flight mass (MALDI-TOF-MS) spectrometry. Two proteins of interest, the levels of which were significantly increased in TNBG-treated cells, were further characterized by Western blot analysis. The results showed a total of 846+/-23 spots in control cells and 853+/-30 spots in TNBG-treated cells. Twenty-six up-regulated or down-regulated proteins were found by analyzing differential proteomic 2-DE map. Eleven of them were identified by mass spectrometry. They were protein disulfide-isomerase precursor, 94 kD glucose-regulated protein, heat shock protein (HSP) 90-alpha, ATP-citrate lyase, HMG-CoA reductase, glucose-6-phosphate 1-dehydrogenase, very-long-chain specific acyl-CoA dehydrogenase, squalene synthetase, sterol regulatory element-binding protein 1, fructose-bisphosphate aldolase A, and peroxiredoxin-1. These up-regulated or down-regulated proteins are mostly related to lipid metabolism. The TNBG antitumor mechanism is probably to influence tumor lipid metabolism, resulting in accumulation of LDs in tumor cells.
Antineoplastic Agents/*pharmacology ; Azo Compounds/*pharmacology ; Carcinoma, Hepatocellular/*pathology ; Cell Line, Tumor ; Gonanes/*pharmacology ; Liver Neoplasms/*pathology ; Proteins/*metabolism ; Proteome

OBJECTIVETo establish a highly sensitive screening method for phytoestrogen active constituents and to primarily screen the phytoestrogenic active constituents from the chickpea extractions by the method.

METHODHuman ERalpha cDNA was cloned using MCF-7 total RNA as the template by RT-PCR and then was constructed into a pcDNA3 and named as pERalpha. The cell line MCF-7 was co-transfected with pERalpha and the reporter plasmid pERE-Luc which carrying the estrogen response element (ERE) plus the luciferase reporter gene. The luciferase activity was then assayed. The model was optimized by changing the ratio of two plasmids. The feasibility of the optimized model was further proved by the several known phytoestrogen compounds including fermononetin, biochanin A and genistein, et al. As an application of the model, the phytoestrogen activity of the extracts of the chickpea was assayed.

RESULTThe recombinant plasmid (pERalpha) can enhance luciferase activities of pERE-Luc transfected MCF-7 cells. The highest transfection efficiency and luciferase activity were found at the ratio of 10:1 (pERE-Luc: pERalpha), the luciferase activity was improved five times as high as the unique pERE-Luc transfection. The co-transfection screening model also indicated that fermononetin, biochanin A and genistein could induce ERE-driven luciferase activity and ICI 182,780 suppressed the induced transcription. As the application of the model, the results showed that the ethanol (70%) total extraction, the ethyl acetate extraction and the ligarine extraction of the chickpea can induce ERE-driven luciferase activity. Concurrent treatment with ICI 182,780 abolished the induced luciferase activity.

CONCLUSIONA phytoestrogen active constituent screening mode have been established based on co-transfection method. It is sensitive to assay the phytoestrogen active constituents and can be applied to screen the active component of phytoestrogens.

Cell Line, Tumor ; Cicer ; chemistry ; metabolism ; Drug Evaluation, Preclinical ; methods ; Estrogen Receptor alpha ; genetics ; metabolism ; Genes, Reporter ; drug effects ; Genetic Vectors ; metabolism ; Genistein ; chemistry ; pharmacology ; Humans ; Luciferases ; drug effects ; metabolism ; Phytoestrogens ; analysis ; pharmacology ; Plant Extracts ; chemistry ; metabolism ; pharmacology ; Plasmids ; drug effects ; metabolism ; Transfection ; methods

d per-oxiredoxin-1. These up-regulated or down-regulated proteins are mostly related to lipid metabolism. The TNBG antitumor mechanism is probably to influence tumor lipid metabolism, resulting in accu-mulation of LDs in tumor cells.

ObjectiveTo establish an ischemia-reperfusion model in cynomolgus macaques and to analyse the effects of edaravone intervention. MethodsA total of fifteen adult male cynomolgus macaques were randomly divided into three groups: sham operation (Sham group, n=3), ischemia-reperfusion model (Model group, n=6) and edaravone treatment (Edaravone group, n=6). Ischemic-reperfusion model of cynomolgus macaques was established by clamping the M1 branch of the left cerebral artery for 1 h. After 2 h of reperfusion, the animals in Edaravone group were injected with 0.5 mg/kg edaravone intravenously for intervention treatment, while the animals in Sham and Model groups were injected with an equal volume of normal saline intravenously, twice a day, from the 2nd to 7th day. The behavioral video recordings, clinical observations and neurological deficit scores of cynomolgus macaques were obtained, and brain edema volume and cerebral ischemia volume were statistically analyzed. ResultsCompared with the Sham group, the animals in Model group showed typical symptoms of ischemic stroke, with a significant increase in the neurological deficit score， the volumes of edema and infarct of brain tissue (all P＜0.01). Compared with Model group, the neurological deficit score, the volumes of edema and infarct of brain tissue were significantly reduced in Edaravone group (all P＜0.05). ConclusionAn animal model of ischemia-reperfusion in cynomolgus macaques was successfully established, and edaravone was confirmed to alleviate the damage caused by ischemia-reperfusion.