Wortmannin

The inhibition of MLCK by Wortmannin is not affected by calmodulin or peptide substrat, while reduced by high concentration of ATP. This compound directly interacts with the catalytic domain of MLCK and leads to an irreversible loss of the enzyme activity. It has no inhibitory to cAMP-dependent protein kinase, cGMP-dependent protein kinase, and calmodulin-dependent protein kinase II, and has little effect on protein kinase C activity. ^[1]

This inhibitor inhibits N-formylmethionyl-leucylphenylalanine (fMLP)-stimulated PtdInsP3 (phosphatidylinositol 3,4,5-trisphosphate) formation with IC50 of 5 nM and this inhibition is completely abolished when pretreated with 100 nM of this compound in human neutrophils, with increased PtdInsP2 levels and no effects on cellular PtdInsP and PtdIns contents. It could develop oscillatory changes in F-actin content and does not inhibit fMLP-stimulated actin polymerization in neutrophils. ^[2]

This chemical irreversibly inhibits phosphatidylinositol 3-kinase (PI3-kinase) activity with binding to the 110-kDa protein (IC50 of 3 nM) and has no effect PI4-kinase in RBL-2H3 cells. It also inhibits leukotriene release, with no effect on the activation of the tyrosine kinase Lyn. ^[3]

This compound completely abolishes the induced hexose uptake in isolated rat adipocytes at 0.1 μM, without impairing stimulated lipolytic activity. ^[4]

It suppresses induced production of nitric oxide by 50% at 500 nM in human umbilical vein endothelial cells, which is in response to IGF-1. ^[5]

This chemical suppresses DNA double strand break (DSB) repair and has no effect on DSB levels or the kinetics of single strand break (SSB) repair in Chinese hamster ovary cells at 50 μM. It could potentiate ionizing radiation (IR)-induced cytotoxicity with no toxicity by itself. ^[6]

This inhibitor inhibits polo-like kinase (PLK1) activity IC50 of 24 nM in intact G2/M-arrested cells. ^[7]

It increases Toll-like receptor (TLR)-mediated accumulation of IL-6 in human macrophages with EC50 of 50 nM. Meanwhile this compound significantly enhances TLR-mediated inducible nitric-oxide synthase (iNOS) expression and nitrite accumulation in mouse macrphages. It activates the nuclear factor-κB and up-regulates the cytokine mRNA production. ^[8]

This chemical also inhibits Polo-like kinase (PlK) 1 and PlK3, which play important roles in mitosis. Its treatment could lead to a reduction in phosphorylation of p53 on serine 20 induced by DNA damage. ^[9]

It suppresses hyaluronan-induced Akt phosphorylation and cell motility/migration in SW1990 cells. ^[10]

Wortmannin inhibits peritoneal metastasis of SW1990 in mice at 1 mg/kg, without any weight loss. ^[10]

This compound inhibits phosphatidylinositide 3-kinase-protein kinase B (PKB)/Akt phosphorylation in both normal tissues (lung, heart and brain homogenates) and tumor tissue in mice, without mortality or acute toxicity at 0.7 mg/kg. Combination with LY188011, this chemical significantly increases apoptosis and inhibit tumor growth in orthotopic tumor, while both monotherapy could not. ^[11]

• MLCK assay

  MLCK activity is assayed with peptide substrate (KKRPQRATSNVFS-NH₂) or myosin light chain. The peptide substrate (24 μM) is phosphorylated in a reaction mixture containing 25 mM Tris-HC1 (pH 7.5), 0.5 mg/mL bovine serum albumin, 4 mM MgCl₂, 0.5 mM CaCl₂, 2.6 nM calmodulin, 1.5 nM MLCK, and 400 μM ATP in a final volume of 0.25 mL. After a 10-min preincubation at 28 ºC without ATP, the reaction is started by addition of ATP at 28 ºC and terminated by the addition of 0.1 mL of 10% (v/v) acetic acid after 30 min. The mixture is analyzed by high performance liquid chromatography: column, Unisil Pack 5C18 4.6 X 150 mm; solvent, 18% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid in water; flow rate, 1.0 mL/min; temperature, 40 ºC; detection, absorbance at 220 nm. Percent of reaction is calculated from the ratio of peak areas of phosphorylated form to those of unphosphorylated form. Specific activity measured under the conditions described above is 0.81 μmol/min/mg. Myosin light chain (108 μg/mL) is phosphorylated in a reaction mixture containing 25 mM Tris-HC1 (pH 7.5), 0.5 mg/mL bovine serum albumin, 4 mM MgCl₂, 0.5 mM CaCl₂, 4.2 nM calmodulin, 0.92 nM enzyme, and 10 μM[γ-³²P]ATP (100-900 cpm/pmol) in a final volume of 0.25 mL. After a 3-min preincubation at 30 ºC without ATP, the reaction is started by the addition of [γ-32P]ATP at 30 ºC and stopped by the addition of 0.125 mL of trichloroacetic acid after 5 min. The acid-precipitable materials are collected on a nitrocellulose membrane filter and washed with four 1-mL aliquots of 5% (v/v) trichloroacetic acid. The radioactivity on the filter is measured in a toluene scintillation fluid, using a Packard Tri-Carb liquid scintillation spectrometer Model 4530. The specific activity measured under the conditions is 1.23 μmol/min/mg. ^[1]

• Cell lines

  NK cells

• Concentrations

  1 μM

• Incubation Time

  1 h

• Method

  Primary NK cells were pretreated with wortmannin (1 μM) for 1 h, washed twice with RPMI 1640, and then treated with IL-15 (10 ng/mL) for 24 h. DMSO was used as control.

• Animal Models

  Human pancreatic adenocarcinoma cells PK1 are injected both s.c. and orthotopically into SCID mice.

• Dosages

  0.175, 0.35, and 0.7 mg/kg

• Administration

  Injected by i.v.