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Tao Y.-F.,Soochow University of China | Lu J.,Soochow University of China | Du X.-J.,The 5th Hospital of Chinese PLA | Sun L.-C.,Peking Union Medical College | And 12 more authors.
BMC Cancer

Background: Survivin, a member of the family of inhibitor of apoptosis proteins, functions as a key regulator of mitosis and programmed cell death. YM155, a novel molecular targeted agent, suppresses survivin, which is overexpressed in many tumor types. The aim of this study was to determine the antitumor activity of YM155 in SK-NEP-1 cells.Methods: SK-NEP-1 cell growth in vitro and in vivo was assessed by MTT and nude mice experiments. Annexin V/propidium iodide staining followed by flow cytometric analysis was used to detect apoptosis in cell culture. Then gene expression profile of tumor cells treated with YM155 was analyzed with real-time PCR arrays. We then analyzed the expression data with MEV (Multi Experiment View) cluster software. Datasets representing genes with altered expression profile derived from cluster analyses were imported into the Ingenuity Pathway Analysis tool.Results: YM155 treatment resulted in inhibition of cell proliferation of SK-NEP-1cells in a dose-dependent manner. Annexin V assay, cell cycle, and activation of caspase-3 demonstrates that YM155 induced apoptosis in SK-NEP-1 cells. YM155 significantly inhibited growth of SK-NEP-1 xenografts (YM155 5 mg/kg: 1.45 ± 0.77 cm3; YM155 10 mg/kg: 0.95 ± 0.55 cm3) compared to DMSO group (DMSO: 3.70 ± 2.4 cm3) or PBS group cells (PBS: 3.78 ± 2.20 cm3, ANOVA P < 0.01). YM155 treatment decreased weight of tumors (YM155 5 mg/kg: 1.05 ± 0.24 g; YM155 10 mg/kg: 0.72 ± 0.17 g) compared to DMSO group (DMSO: 2.06 ± 0.38 g) or PBS group cells (PBS: 2.36 ± 0.43 g, ANOVA P < 0.01). Real-time PCR array analysis showed between Test group and control group there are 32 genes significantly up-regulated and 54 genes were significantly down-regulated after YM155 treatment. Ingenuity pathway analysis (IPA) showed cell death was the highest rated network with 65 focus molecules and the significance score of 44. The IPA analysis also groups the differentially expressed genes into biological mechanisms that are related to cell death, cellular function maintenance, cell morphology, carbohydrate metabolism and cellular growth and proliferation. Death receptor signaling (3.87E-19), TNFR1 signaling, induction of apoptosis by HIV1, apoptosis signaling and molecular mechanisms of cancer came out to be the top four most significant pathways. IPA analysis also showed top molecules up-regulated were BBC3, BIRC3, BIRC8, BNIP1, CASP7, CASP9, CD5, CDKN1A, CEBPG and COL4A3, top molecules down-regulated were ZNF443, UTP11L, TP73, TNFSF10, TNFRSF1B, TNFRSF25, TIAF1, STK17A, SST and SPP1, upstream regulator were NR3C1, TP53, dexamethasone , TNF and Akt.Conclusions: The present study demonstrates that YM155 treatment resulted in apoptosis and inhibition of cell proliferation of SK-NEP-1cells. YM155 had significant role and little side effect in the treatment of SK-NEP-1 xenograft tumors. Real-time PCR array analysis firstly showed expression profile of genes dyes-regulated after YM155 treatment. IPA analysis also represents new molecule mechanism of YM155 treatment, such as NR3C1 and dexamethasone may be new target of YM155. And our results may provide new clues of molecular mechanism of apoptosis induced by YM155. © 2012 Tao et al.; licensee BioMed Central Ltd. Source

Liu Y.,Beijing University of Chinese Medicine | Zhu X.-Q.,Beijing University of Chinese Medicine | Li W.-D.,Beijing Institute for Drug Control | Wen H.,Beijing University of Chinese Medicine | Liu C.-S.,Beijing University of Chinese Medicine
Chinese Journal of Natural Medicines

The present study was designed to determine the effects of copy number variations (CNVs) of squalene synthase 1(SQS1) gene on the mevalonate (MVA) pathway. SQS1 gene from G. uralensis (GuSQS1) was cloned and over-expressed in Pichia pastoris GS115. Six recombinant P. pastoris strains containing different copy number of GuSQS1 were constructed. HPLC was used to assay the level of ergosterol in all transgenic P. pastoris strains containing GuSQS1. HPLC analysis showed that the contents of ergosterol in all of the transgenic P. pastoris containing GuSQS1 were higher than that in the negative control. And with the increase of copy number of GuSQS1, the content of ergosterol showed an increasing-decreasing-increasing pattern. The contents of ergosterol in 10-copy- GuSQS1 P. pastoris and 47-copy- GuSQS1 P. pastoris were significantly higher than that in the rest recombinant P. pastoris strains. In conclusion, the CNVs of GuSQS1 influence the content of secondary metabolites in the MVA pathway. The present study provides a basis for over-expressing GuSQS1 and increasing the content of glycyrrhizin in G. uralensis cultivars. © 2015 China Pharmaceutical University. Source

Objective: To determine the citrate ion content in human coagulation factor VIII (FVIII) by high performance anion exchange chromatography (HPAEC). Methods: The condition for HPAEC was as follows: chromatographic column: IonPac AS11-HC(4 mm x 250 mm); mobile phase: 20 mmol/L sodium hydroxide solution; time for isocratic elution: 20 min; flow rate; 1.0 ml/min; inhibitor: ASRS 4 mm anion inhibitor; inhibition current: 120 mA; detector: electrical conductivity detector; detector cell temperature: 30°C; sample loading: 25 μl. The developed method was determined for linear range and detection limit, verified for accuracy and precision, and applied preliminarily. Results: The chromatographic peak area of reference showed good linear relationship to citrate ion content at a range of 0.1-2.0 μg/ml (r = 0.999 0). Serving the signal-to-noise (S/N) ratio as 3, the detection limit of the developed method was 0.01 μg/ml. The total mean recovery of citrate ion in accuracy test was 97.80%, with a RSD of 0.69%. The RSD of 6 test results of one sample of FVIII was 0.08%. The determination results of three batches of FVIII by HPAEC, colorimetry and cation exchange chromatography were basically in agreement, which met the requirements in Chinese Pharmacopoeia(Volume III, 2010 edition). Conclusion: HPAEC may be used for determination of citrate ion content in FVIII, which was simple and rapid, with high accuracy and precision. Source

Fang X.,Shandong Agricultural University | Fang X.,State Key Laboratory of Crop Biology | Wang J.,Shandong Agricultural University | Wang J.,State Key Laboratory of Crop Biology | And 3 more authors.
Journal of Separation Science

An optimized microwave-assisted extraction (MAE) method and an efficient HPLC analysis method were developed for fast extraction and simultaneous determination of oleanolic acid and ursolic acid in the fruit of Chaenomeles sinensis. The open vessel MAE process was optimized by using a central composite experimental design. The optimal conditions identified were microwave power 600 W, temperature 52°C, solvent to material ratio 32 mL/g and extraction time 7 min. The results showed that MAE is a more rapid extraction method with higher yield and lower solvent consumption. The HPLC-photodiode array detection analysis method was validated to have good linearity, precision, reproduction and accuracy. Compared with conventional extraction and analysis methods, MAE-HPLC-photodiode array detection is a faster, convenient and appropriate method for determination of oleanolic acid and ursolic acid in the fruits of C. sinensis. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA. Source

Liu Z.,Peking Union Medical College | Liu Z.,Beijing Institute for Drug Control | Feng Z.,Peking Union Medical College | Yang Y.,Peking Union Medical College | And 2 more authors.

Eleven new acyl quinic acid derivatives, 4-O-caffeoyl-3-O-syringoylquinic acid methyl ester (1), 4-O-caffeoyl-3-O-vanilloylquinic acid (2), 4-O-caffeoyl-3-O-vanilloylquinic acid methyl ester (3), 5-O-caffeoyl-3-O-vanilloylquinic acid (4), 5-O-caffeoyl-3-O-vanilloylquinic acid methyl ester (5), 5-O-caffeoyl-3-O-sinapoylquinic acid (6), 5-O-caffeoyl-4-O-vanilloylquinic acid (7), 4-O-(7S, 8R)-glycosmisoyl-5-O-caffeoylquinic acid methyl ester (8), 4-O-(7S, 8-glycosmisoyl-5-O-caffeoylquinic acid (9), 3-O-(7S, 8R)-glycosmisoyl-4-O-caffeoylquinic acid (10), and 3-O-(7S, 8R)-glycosmisoyl-4-O-caffeoylquinic acid methyl ester (11), have been isolated from the stems of Erycibe obtusifolia together with eight known compounds (12-19). Their structures were elucidated on the basis of spectroscopic data analysis (UV, IR, HRESIMS, CD, and 1D and 2D NMR) and chemical evidence. In in vitro assay, compounds 7 and 16-18 exhibited significant neuroprotective effects against rotenone induced PC12 cell damage at 10 μM. © 2014 Elsevier B.V. All rights reserved. Source

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