A pro-apoptotic Cdk2/cyclin A inhibitor
SU 9516 is a selective and novel inhibitor of CDK2 with IC50 value of 22 nM. Also, it inhibits CDK1, CDK4, PKC, p38, PDGFR with IC50 value of 0.04, 0.2, >10.0, >10.0, 18.0 μM, respectively [1].
Cyclin-dependent kinase 2 (CDK2) is a member of the cyclin-dependent kinase family and is a catalytic subunit of the cyclin-dependent kinase complex Cyclin E/CDK2 or Cyclin A/CDK2, which play an important role in the G1-S phase of the cell cycle [1].
In RKO cells, SU 9516 (5 μM) decreased cdk2-specific phosphorylation of pRb by 52%. While, in SW480 cells, SU9516 (5 μM) inhibited both cdk2-specific and cdk4-specific phosphorylation of pRb by 64% and 49%, respectively. Also, SU 9516 (5 μM) resulted in G0-G1 or G2-M block and induced apoptosis in a dose-dependent way [1]. In HT-29, SW480 and RKO human colon cancer cells, SU9516 (5 μM) inhibited dissociation of pRb from E2F1 in a time-dependant way. Also, SU 9516 decreased Cyclin D1 and CDK2 by 10-60% [2]. In human leukemia cells, SU 9516 (5 μM) induced Bax mitochondrial translocation, cytochrome c release and apoptosis, which were associated with down-regulation of the antiapoptotic protein Mcl-1. Also, SU 9516 induced activation of caspase-3 and -8 [3].
参考文献:
[1].? Lane ME, Yu B, Rice A, et al. A novel cdk2-selective inhibitor, SU9516, induces apoptosis in colon carcinoma cells. Cancer Res, 2001, 61(16): 6170-6177.
[2].? Yu B, Lane ME, Wadler S. SU9516, a cyclin-dependent kinase 2 inhibitor, promotes accumulation of high molecular weight E2F complexes in human colon carcinoma cells. Biochem Pharmacol, 2002, 64(7): 1091-1100.
[3].? Gao N, Kramer L, Rahmani M, et al. The three-substituted indolinone cyclin-dependent kinase 2 inhibitor 3-[1-(3H-imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516) kills human leukemia cells via down-regulation of Mcl-1 through a transcriptional mechanism. Mol Pharmacol, 2006, 70(2): 645-655.
Kinase experiment: | Kinase assays are performed in 96-well polypropylene plates. Each reaction contains 2 μg of histone H1 at a final concentration of 10 μM [-33P]ATP (0.2 μCi/well), 10 mM MgCl2,1mM DTT, 0.01% Triton X-100, and 10% glycerol in a 40 μL volume. The reaction is initiated with the addition of 20 μL enzyme (6 ng cdk2/well resulting in a final concentration of 1.6 nM), which is previously diluted 1:50-1:200 in the same buffer, and allowed to proceed for 1 h at room temperature. Reaction is stopped by the addition of 0.01 mL 10% phosphoric acid, and 25 μL of reaction mixture is transferred to P30 phosphocellulose filter mat paper. The filter mat is washed three times with 1.0% phosphoric acid, air dried, and then counted for radioactivity in a liquid scintillation counter. |
Cell experiment: | RKO cells and SW480 cells are seeded in replicates (n = 6) in 96-well plates at 1×104 cells/well and allowed to attach overnight. SU9516 is added in concentrations from 0.05 μM to 50.00 μM for 24 h, the cells are then washed twice with PBS, and cells are replenished with complete media. The cells are fixed at 0, 4, and 7 days post-drug removal and assayed for protein levels using a modified SRB cytotoxicity assay. The cells are fixed in 10% trichloroacetic acid for 1 h, washed in distilled H2O, and stained in 0.4% SRB/acetic acid for 30 min. The cells are then washed in 0.1% acetic acid, solubilized in 10 mM Tris (pH 9), and analyzed on a Bio-Rad 360 microplate reader at 595 nm. All experiments are repeated at least three times. |
参考文献: [1]. Lane ME, et al. A novel cdk2-selective inhibitor, SU9516, induces apoptosis in colon carcinoma cells. Cancer Res. 2001 Aug 15;61(16):6170-7. | |
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