Erlotinib is a tyrosine kinase inhibitor which acts on the epidermal growth factor receptor (EGFR), inhibiting EGFR-
1.Pollack, V.A., Savage, D.M., Baker, D.A., et al.Inhibition of epidermal growth factor receptor-associated tyrosine phosphorylation in human carcinomas with CP-358,774: Dynamics of receptor inhibition in situ and antitumor effects in athymic miceJ. Pharmacol. Exp. Ther.291(2)739-748(1999) 2.Greulich, H., Chen, T.H., Feng, W., et al.Oncogenic transformation by inhibitor-sensitive and -resistant EGFR mutantsPLoS Med.2(11)(2005) 3.Yamasaki, F., Zhang, D., Bartholomeusz, C., et al.Sensitivity of breast cancer cells to erlotinib depends on cyclin-dependent kinase 2 activityMol. Cancer Ther.6(8)2168-2177(2007) 4.Li, Z., Xu, M., Xing, S., et al.Erlotinib effectively inhibits JAK2V617F activity and polycythemia vera cell growthJ. Biol. Chem.282(6)3428-3432(2007) 5.Herbst, R.S., and Bunn, P.A., Jr.Targeting the epidermal growth factor receptor in non-small cell lung cancerClin. Cancer Res.9(16)5813-5824(2003) 6.Gerber, D.E.EGFR inhibition in the treatment of non-small cell lung cancerDrug Dev. Res.69(6)359-372(2008)
Kinase experiment: | The kinase reaction is performed in 50 μL of 50 mM HEPES (pH 7.3), containing 125 mM NaCl, 24 mM MgCl2, 0.1 mM Na3VO4, 20 μM ATP, 1.6 μg/mL EGF, and 15 ng of EGFR, affinity purified from A431 cell membranes. The compound in DMSO is added to give a final DMSO concentration of 2.5%. Phosphorylation is initiated by addition of ATP and proceeded for 8 mm at room temperature, with constant shaking. The kinase reaction is terminated by aspiration of the reaction mixture and is washed 4 times with wash buffer. Phosphorylated PGT is measured by 25 mim of incubation with 50 μL per well HRP-conjugated PY54 antiphosphotyrosine antibody, diluted to 0.2 μg/mL in blocking buffer (3% BSA and 0.05% Tween 20 in PBS). Antibody is removed by aspiration, and the plate is washed 4 times with wash buffer. The colonmetric signal is developed by addition of TMB Microwell Peroxidase Substrate, 50 μL per well, and stopped by the addition of 0.09 M sulfuric acid, 50μL per well. Phosphotyrosine is estimated by measurement of absorbance at 450 nm. The signal for controls is typically 0.6-1.2 absorbance units, with essentially no back ground in wells without AlP, EGFR, or POT and is proportional to the time of incubation for 10 mm[1]. |
Cell experiment: | To test the viability of cells treated with B-DIM, Erlotinib, or the combination, BxPC-3 and MIAPaCa cells are plated (3,000-5,000 per well) in a 96-well plate and incubated overnight at 37°C. A range of concentrations for both B-DIM (10-50 μM) and Erlotinib (1-5 μM) is initially tested. Based on the initial results, the concentration of B-DIM (20 μM) and Erlotinib (2 μM) are chosen for all assays. The effects of B-DIM (20 μM), Erlotinib (2 μM), and the combination on BxPC-3 and MIAPaCa cells are determined by the standard MTT assay after 72 h and is repeated three times. The color intensity is measured by a Tecan microplate fluorometer at 595 nm. DMSO-treated cells are considered to be the untreated control and assigned a value of 100%. In addition to the above assay, we have also done clonogenic assay for assessing the effects of treatment[2]. |
Animal experiment: | Mice[3] Bcrp1/Mdr1a/1b-/- and WT mice are treated p.o. or i.p. with 5 mg/kg Erlotinib. The i.p. administration is chosen assuming good drug absorption and complete bioavailability. Sampling is done from the tip of the lateral tail vein in three series. During the first series, whole blood samples are collected at 15 min and 0.5, 1.5, 5, and 10 h after injection. Based on the results of this initial group, the sampling times of the two subsequent series are adapted to 5 and 15 min and 0.5, 1.5, 4, and 8 h after injection. After collection, the blood samples are immediately centrifuged and plasma is stored at -20°C until high-performance liquid chromatographic analysis took place. Rats[4] Seven-week-old male Crl:CD (SD) rats (244-297 g) are used. The animals are treated with Erlotinib (10 mg/kg and 20 mg/kg) orally by gavage. |
参考文献: [1]. Moyer JD, et al. Induction of apoptosis and cell cycle arrest by CP-358,774, an inhibitor of epidermal growth factor receptor tyrosine kinase. Cancer Res. 1997 Nov 1;57(21):4838-48. | |
动态评分
0.0