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GSK (glycogen synthase kinase 3β) Inhibitors

CHIR-99021(CT99021) is a glycogen synthase kinase 3β (GSK3β) inhibitor that has antiproliferative activity in vitro and in vivo. [1] CHIR-99021 inhibits GSK-3 with IC50 at 7 nM. In a series of carcinoma cell lines, the IC50 of CHIR99021 for proliferation is about 10μM. [1] CHIR-99201 does not exhibit cross-reactivity against cyclin-dependent kinases (CDKs) with 350-fold selectivity toward GSK-3β compared to CDKs . [1]

References on CHIR-99021 (CT99021):

[1] Tighe A et al.BMC Cell Biol. 2007 Aug 14;8:34 

Indirubin is a potent cyclin-dependent kinases and GSK-3β inhibitor with IC50 of median 75 and 28M, respectively. [1] Indirubin prevents CDK5- and GSK-3β-mediated tau phosphorylation, a process over-active in Alzheimer disease states. Indirubin also inhibits AMPK, LCK and SGK. Induces cell cycle arrest and inhibits cell proliferation. Indirubin inhibited tumor necrosis factor (TNF)-induced NF-κB activation in a dose- and time-dependent manner. Indirubin also suppressed the NF-κB activation triggered by various inflammatory molecules and carcinogens. Furthermore, indirubin blocked the phosphorylation and degradation of IκBα through the inhibition of activation of IκBα kinase and phosphorylation and nuclear translocation of p65. NF-κB reporter activity induced by TNFR1, TNF receptor-associated death domain, TRAF2, TAK1, NF-κB-inducing kinase, and IKKβ was inhibited by indirubin but not that induced by p65 transfection. [2]

Protocol:

Biochemical assay:
GSK-3β and CDKs were assayed using the GS-1 peptide or histone H1 as substrates, respectively, with 15 μm ATP and in the presence of increasing concentrations of indirubins.

Cell assay:
Sf9 cells were grown at 27 °C in monolayer culture Grace’s medium supplemented with 10% fetal bovine serum and 50 μg/ml gentamicin and 2.5 μg/ml amphotericin. BaculoGold was obtained from PharMingen, pVL1392 was obtained from Invitrogen.
To determine the effects of aminopurvalanol and indirubin-3′-monoxime on tau phosphorylation, Sf9 cells infected with baculovirus expressing htau23 protein were treated 36 h post-infection (when cells have already expressed levels of tau sufficient for the outgrowth of cell processes) with 20 μm inhibitors for 3 h before being harvested.

Animal study:
Adult mouse brain striatal slices were prepared using standard methodology. Following equilibration in Krebs’ bicarbonate buffer oxygenated with continuous aeration (95% O2/5% CO2), slices were treated with various concentrations of indirubin-3′-monoxime or 10 μm roscovitine for 60 min or were left in Krebs’ bicarbonate buffer for the same period of time. Slices were homogenized by sonication in boiling 1% SDS and 50 mm NaF. Protein concentrations were determined by the BCA method using a BSA standard curve. Equal amounts of protein (80 μg) were subjected to SDS-PAGE using a 15% acrylamide gel, electrophoretically transferred to nitrocellulose membrane, and immunoblotted with a phosphorylation state-specific antibody that selectively detects DARPP-32 phosphorylated at Thr-75. [1]

References on Indirubin:

[1] Leclerc S et al. Biol Chem. 2001 Jan 5;276(1):251-60.

[2] Benson JM et al. Toxicol Sci. 2011 Sep 26.

Protocol:

Biochemical assay:
GSK-3 kinase activity was measured, in the presence or absence of SB-216763 or SB-415286, in a reaction mixture containing final concentrations of: 1 nM human GSK-3α or rabbit GSK3α; 50 mM MOPS pH 7.0; 0.2 mM EDTA; 10 mM Mg-acetate; 7.5 mM β-mercaptoethanol; 5% (w/v) glycerol; 0.01% (w/v) Tween-20; 10% (v/v) DMSO; 28 μM GS-2 peptide substrate. The GS-2 peptide sequence corresponds to a region of glycogen synthase that is phosphorylated by GSK-3 as previously described. The assay was initiated by the addition of 0.34 μCi [33P]γ-ATP (IC50 determinations) or 2.7 μCi γ-ATP (Ki determinations). The total ATP concentration was 10 μM (IC50 determinations) or ranged from 0 to 45 μM (Ki determinations). Following 30 min incubation at room temperature the assay was stopped by the addition of one third assay volume of 2.5% (v/v) H3PO4 containing 21 mM ATP. Samples were spotted onto P30 phosphocellulose mats and these were washed six times in 0.5% (v/v) H3PO4. The filter mats were sealed into sample bags containing Wallac betaplate scintillation fluid. 33P incorporation into the substrate peptide was determined by counting the mats in a Wallac microbeta scintillation counter. [2]

Cell assay:
Cells on 10 cm diameter dishes were treated with SB-216763, DMSO vehicle, LiCl or insulin for 60 (Chang cells) or 90 min (HEK293 cells). Incubation medium was removed and cells washed with 1×5 ml ice-cold PBS prior to lysis, on ice, in 450 μl 20 mM Tris–HCl pH 7.4, 50 mM NaF, 5 mM Na4P2O7, 2 mM Na3VO4, 10 mM β-glycerophosphate, 1 mM EDTA, 1 mM EGTA, 0.1% (v/v) β-mercaptoethanol, 1% (w/v) Triton X-100. Cell lysates were centrifuged at 15 000×g for 5 min at 4°C. Lysate supernatants were snap frozen on liquid nitrogen and stored at −80°C prior to assay. Lysates were assayed for glycogen synthase activity in buffer (67 mM Tris–HCl pH 7.5, 5 mM DTT, 6.7 mM EDTA, 13 mg/ml glycogen, 8.9 mM [14C]UDP-glucose) in the presence or absence of 20 mM glucose 6-phosphate. Data are expressed as fold increases in glycogen synthase activity ratios over those of control. Activity ratios of glycogen synthase in control cells were approximately 0.15 and 0.05 in Chang and HEK293 cells respectively.

Animal study:
Since SB is an ATP competitive GSK3 inhibitor, its impact on GSK3 inhibition is not effectively represented by changes in phospho-GSK3. Therefore we examined a downstream target phospho-glycogen synthase by Western analysis in the hippocampus. Besides examining glycogen histologically, we also quantified glycogen levels in dissected brain (thalamus), using methods previously described. Briefly, tissue was dissolved in 30% potassium hydroxide (KOH) and heated at 100 °C for 10 min. Anhydrous ethanol was added, and samples were centrifuged at 5700 rpm for 15 min. The pellet was re-suspended in 0.5 ml of H2O. Fre

References on SB 216763:

[1] Wang M et al. Bioorg Med Chem Lett. 2011 Jan 1;21(1):245-9.

[2] Coghlan MP et al. Chem Biol. 2000 Oct;7(10):793-803.

[3] Hu S et al. Neurobiol Dis. 2009 Feb;33(2):193-206. 

SB 415286 is a potent and selective cell-permeable, ATP-competitive GSK3α inhibitor with an IC50 and a Ki of 78 and 31 nM, respectively. SB 415286 competes with ATP. SB 415286 has minimal activity against 24 other protein kinases (IC50 > 10 μ M). SB 415286 stimulates glycogen synthesis, gene transcription and is neuroprotective. As a result of GSK3 inhibition, SB 415286 stimulates glycogen synthesis in the Chang human liver cell line with an EC50 value of 2.9 µM. SB 415286 also protects primary neurons from death induced by the PI3-kinase pathway. In L6 myotubes, SB 415286 induced a much greater activation of GS (6.8-fold) . In adipocytes, SB 415286 caused a comparable activation of GS despite a substantial differentiation-linked reduction in GSK3 expression ( approximately 85%) indicating that GSK3 remains an important determinant of GS activation in fat cells. [1] SB 415286 induced expression of a beta-catenin-LEF/TCF regulated reporter gene in HEK293 cells. [2]

References on SB 415286:

[1] MacAulay K et al. Eur J Biochem. 2003 Sep;270(18):3829-38.

[2] Coghlan MP et al. Chem Biol. 2000 Oct;7(10):793-803.
 

TWS119 is a glycogen synthase kinase-3β inhibitor with an IC50 of 30 nM. At 400 nM, it induces neuronal differentiation in pluripotent murine embryonal carcinoma cells and embryonic stem cells (ESCs). [1]

References on TWS119:

[1] Ding S et al. Proc Natl Acad Sci U S A. 2003 Jun 24;100(13):7632-7.