Rhein_478-43-3_UsaMSDS_18836_MedChemExpress.PDF
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1、 Inhibitors, Agonists, Screening Librarieswww.MedChemEData SheetBIOLOGICAL ACTIVITY: PD173955 is src familyselective tyrosine kinase inhibitor with IC50 of 22 nM for Src, Yes and Abl kinase; less potent for FGFR andno activity on InsR and PKC.IC50 value: 22 nMTarget: Src kinase inhibitorin vitro: PD
2、173955 inhibits the growth of MDAMB468 and MCF7 breast cancer cells with IC50s of 500 nM and 1 M, respectively,with an accumulation of suspended cells. Cells treated with PD173955 show a near complete redistribution to the G2M phase of thecell cycle in comparison with control cells, and quantitation
3、 of mitotic indices by immunofluorescence microscopy shows anaccompanying accumulation of mitotic cells.PD173955 shows antimitotic activity in breast cancer cells with high or low src and yeskinase activities, the antimitotic activity of PD173955 is independent of cell type or malignant transformati
4、on 1. PD173955 inhibitsboth the active and inactive forms of Abl. By contrast, Imatinib only inhibits the active form of the enzyme. In addition, the Ki forinhibition of Abl by PD173955 is very low, making it a more potent inhibitor of Abl and a more effective inhibitor of cancer cellproliferation t
5、han Imatinib 2. PD173955, a Src familyspecific tyrosine kinase inhibitor, increases the susceptibility of HT29 cells toanoikis in a dose and timedependent manner 3.PROTOCOL (Extracted from published papers and Only for reference) Kinase Assay(Src kinase) 1: For src kinase assays, total cellular lysa
6、tes were harvested in modified RIPA buffer 1% sodiumdeoxycholate, 1% NP40, 0.1% SDS, 150 mM NaCl, and 10 mM sodium phosphate (pH 7.2) supplemented with 10 M aprotinin, 10 Mleupeptin, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulfonyl fluoride. Total cellular lysates (300 g) were incubated with
7、antibodies specific for each of the src family kinases and immunoprecipitated using protein GSepharose (Pharmacia).Immunoprecipitates were washed twice in cold lysis buffer and once in kinase buffer and added to an in vitro kinase reactionconsisting of 50 mM PIPES (pH 7.0), 10 mM MnCl2, 10 mM DTT, 1
8、0 M ATP, 2 g of aciddenatured enolase, and 5 Ci of 32PATP.Reactions were allowed to proceed at 30C for 5 min and then stopped immediately by boiling in sample buffer, products separatedon a 10% SDSPAGE gel, transferred to membrane, and exposed to film. Monoclonal antibodies specific for src (m327; C
9、albiochem),cyes (1B7; Wako), lyn (LO5620; Transduction Laboratories), and polyclonal antifyn antibodies (SC16; Santa Cruz Biotechnology)were used in immunoprecipitations and in immunoblotting, using the appropriate secondary immunological reagents. Cell Cycle Assay1: All cell lines were obtained fro
10、m the American Type Culture Collection; maintained in a 1:1 mixture of DMEM:Hams F12supplemented with 100 units/ml penicillin, 100 g/ml streptomycin, 4 mM glutamine, and 10% heatinactivated fetal bovine serum;and incubated at 37C in 5% CO2. PD173955 was maintained as a 10 mM frozen stock solution in
11、 DMSO. For determination of cellcycle effects, cells were seeded at approximately 2 million cells/10cm dish and placed the next day in media containing 5 MPD173955 or an equal volume of DMSO. After 24 or 48 h, cells were harvested by trypsinization, taking care to preserve all suspendedand adherent
12、cell populations. After washing in cold PBS, cell nuclei were prepared by the method of Nusse et al. and cell cycleProduct Name: PD173955Cat. No.: HY-10395CAS No.: 260415-63-2Molecular Formula:C21H16Cl2N4OSMolecular Weight: 443.35Target: BcrAbl; SrcPathway: Protein Tyrosine Kinase/RTKSolubility: DMS
13、O: 32 mg/mLwww.MedChemEdistribution was determined by flow cytometric analysis of DNA content using red fluorescence of 488 nm excited ethidiumbromidestained nuclei. For determination of mitotic indices, aliquots of cells were fixed in 3% paraformaldehyde, stained in 24 g/mlbisbenzimide, and analyze
14、d under fluorescence microscopy. Mitotic cells were identified by their characteristic chromatincondensation. A total of 1000 cells were counted, and the percentage of cells in mitosis was calculated.References: 1. Moasser MM, et al. Inhibition of Src kinases by a selective tyrosine kinase inhibitor
15、 causes mitotic arrest. Cancer Res. 1999 Dec 15;59(24):614552.2. Kraus GA, et al. New effective inhibitors of the Abelson kinase. Bioorg Med Chem. 2010 Sep 1;18(17):631621.3. Windham TC, et al. Src activation regulates anoikis in human colon tumor cell lines. Oncogene. 2002 Nov 7;21(51):7797807.Caut
16、ion: Product has not been fully validated for medical applications. For research use only.Tel: 609-228-6898 Fax: 609-228-5909 E-mail: techMedChemEAddress: 1 Deer Park Dr, Suite Q, Monmouth Junction, NJ 08852, USAwww.MedChemE撠撠ORMATIONSARA 302 Components:No chemicals in this material are subject to t
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