So that you can identify extra critical mol ecules involved in gefitinib resistance, we examined the sensitivity to gefitinib of 23 lung cancer cell lines working with the 3 two,5 diphenyltetrazolium bromide cell proliferation assay and recognized sen sitive, intermediate delicate, and resistant lung cancer cell lines. Some reports have advised that small cell lung cancer is responsive to gefitinib. Tanno et al reported that MAPK, a downstream effector with the EGFR was inhibited by gefitinib in SCLC cell lines that expressed the EGFR even at a very low level. SCLC cell lines were integrated in our series. We also analyzed the genomic sta tus of your EGFR gene mutation and EGFR gene amplifica tion, as well as the protein expression level of critical molecules while in the EGFR family. PI3K Akt and Ras MEK Erk pathways which act down stream of EGFR.
We correlated the cytotoxic activity of gefitinib in our 23 lung tumor cell lines to your correspond ing expression patterns of those proteins. Strategies Cell Lines Twenty three lung cancer cell lines were utilized in selleckchem this review. AZ-960 They comprised. ten adenocarcinoma cell lines. eight squamous cell carcinoma cell lines. and 5 compact cell carcinoma cell lines. The Lu135 cell line was offered by Y. Shimosato and T. Terasaki, plus the LCD and LCOK cell lines by S. Hirohashi. The NCI N231, A549, and NCI H69 cell lines have been obtained from the American Form Culture Assortment. The PC1, PC3, PC7, PC9, PC10, PC14, and QG56 cell lines have been obtained from IBL. The RERF LCKJ, SQ5, LC one sq, RERF LCAI, and MS one cell lines have been obtained from RIKEN Cell Bank. The ABC one, RERF LCMS, LK 2, EBC 1, and SBC3 cell lines have been obtained from your Health Science Investigate Assets Bank. To be able to identify the activation of your members downstream of EGFR with no ligand stimulation, all cell lines had been separately cultured in serum containing and serum totally free problems for 24 h.
Drugs and Growth Inhibition Assay Gefitinib was provided by AstraZeneca and dissolved in dimethyl sulfoxide for in vitro research. We made use of the colorimetric MTT assay to examine the activity of gefit inib towards all 23 lung cancer cell lines as previously reported. Cell suspensions have been seeded into 96 properly microtiter plates and 101 of drug solution added, at different concentrations. After incu bation for 72 h at 37 C, 201 of MTT remedy was additional to every properly and incubation then continued for any even further 4 h at 37 C. The IC50 value was defined because the concentration of gefitinib needed for any 50% reduction in absorbance based on cell development curves. Western Blot Analysis Western blot analysis was carried out as previously described. The membranes have been 1st incubated over night at 4 C with antibody specific for the following pri mary antibodies.