C.421CC done, from those bcl-2 family with the genotype. The AUC of fluvastatin and simvastatin lactone was 100% of HT29 and SW480 cells from American Type Culture Collection. The cells were cultured in Dulbecco modified Eagle medium with 10% f Fetal K Calf serum and 100 U / ml penicillin, maintained at 37 C in a humidified atmosphere re with 5% CO2. The irreversible EGFR inhibitor 324674, EGFR/ErbB2/ErbB4 inhibitor AG1478 and were purchased from Calbiochem. GW583340 was purchased from Sigma. The drugs were dissolved in dimethyl sulfoxide St, a 10 mM Stamml Solution was stored at 20 C. The final concentration of DMSO in all experiments was less than 0.1% in the culture medium. 2.2. The ability Lebensf Determine the cells to test IC50 cells were plated into each well of 96-well plates containingDMEM with 10% FBS.
When the cells reached 60% confluence, EGFR-kinase inhibitors were added to culture media at a final concentration of 0, 0.2, 0.6, 2.0, 6.0, or 20 lm. Media concentration was adjusted to 0.1% ofDMSOin. Seventy-two hours after the incubation, the number was lebensf HIGEN cells using 3 5 2 2H-tetrazolium, inner salt according to claim manufacturer’s protocol. Each test consists of six wells repeatedly with the same drug concentration. The IC50 was determined from the dose-response curve. 2.3. HT29 and SW480 cells apoptosis analysis were plated at 3105 cells per well in 6-well plates. Twenty-four hours after plating, the culture medium with fresh medium containing 10% FBS was replaced, were treated with or without 1 or 2 meters from the EGFR-inhibitor or inhibitors EGFR/ErbB2/ErbB4 324,674 and cells incubated for 48 h other.
Apoptosis was measured using annexin VF Staining kit of fluorescent manufacturer’s instructions. 2.4. Western blot cells were first Highest serum starved and then incubated overnight with various concentrations of different inhibitors of the EGFR kinase 3 h prior to stimulation of the epidermal growth factor, with a final concentration of 100 ng / ml for 15 min at 37 C After treatment, cells were incubated with 10% trichloroacetic acid lysis buffer were lysed and the lysates were clarified by centrifugation rt. The proteins Were separated by SDS-PAGE and transferred to 7.5% nitrocellulose membranes. After blocking with 5% nonfat milk, the membranes were incubated with the primary Ren Antique Body incubated at 4 C overnight.
Antique Body against EGFR was purchased from Santa Cruz Biotechnology. Antique Extracellular body against phosphorylated EGFR, total kinase Re signal and phosphorylated ERK, total AKT and phosphorylated AKT regulates actin and b were from Sigma Aldrich. After washing and incubation with secondary Rem Antique Body, develops protein signals and visualized using a chemiluminescent system. Third Results 3.1. Irreversible EGFR inhibitor inhibits 324,674 HT29 and SW480 cell proliferation initially Highest to study and compare the efficacy of four TKI inhibition of cell proliferation were, HT29 and SW480 cells in the presence of various concentrations of TKI cultured 0-20 lm. The relationship between the concentration and TKI inhibition of cell growth was determined by a CalcuSyn. As shown, although each TKI inhibit cell proliferation in both cell lines in a dose-dependent Ngigen inhibitory activity t of each was clear distinction