In vitro activity: Gefitinib effectively inhibits all tyrosine phosphorylation sites on EGFR in both the high and low-EGFR-expressing cell lines including NR6, NR6M and NR6W cell lines. The phosphorylation sites Tyr1173 and Tyr992 are less sensitive requiring higher concentrations of Gefitinib for inhibition. Gefitinib effectively blocks the phosphorylation of PLC-γ, with IC50 of 27nM, in NR6W cells. The NR6wtEGFR and NR6M cell lines has low levels of PLC-γ phosphorylation but the level in the NR6M cell line is more resistant to inhibition by Gefitinib with IC50 of 43 nM and 369 nM, respectively. Gefitinib inhibits Akt phosphorylations, with IC50 of 220 and 263nM, in the low-EGFR- and -EGFRvIII-expressing cell lines, respectively. Gefitinib in the dose range from 0.1 to 0.5μM significantly facilitates, rather than abrogates, colony formation of NR6M cells. However, at a concentration of 2 μM Gefitinib completely blocks NR6M colony formation. Gefitinib rapidly and in a dose-dependent manner inhibits EGFR and ERK phosphorylation up to 72 hours after EGF stimulation in both the high- and low-EGFR-expressing cell lines. Gefitinib is the monolayer growth of these EGF-driven untransformed MCF10A cells with an IC50 of 20 NM. The combination of Gefitinib (0.2 μM and 0.5 μM) with irradiation lead to a significant growth inhibition in LoVo cells, compared with radiation alone.

Kinase Assay: Gefitinib hydrochloride is an inhibitor that specifically binds and inhibits the EGFR tyrosine kinase, with the IC50 value of 2-37 nM in NR6wtEGFR cells. Gefitinib effectively blocks the phosphorylation of PLC-γ, with IC50 of 27nM, in NR6W cells. The NR6wtEGFR and NR6M cell lines has low levels of PLC-γ phosphorylation but the level in the NR6M cell line is more resistant to inhibition by Gefitinib with IC50 of 43 nM and 369 nM, respectively. Gefitinib inhibits Akt phosphorylations, with IC50 of 220 and 263nM, in the low-EGFR- and -EGFRvIII-expressing cell lines, respectively.

Cell Assay: Exponentially growing cells including NR6, NR6M, NR6M and NR6W cells are seeded in sextuple in 96-well plates at a concentration of 2000 cells/well, allowed to adhere and subsequently washed in PBS and incubated overnight in medium containing 0.5% FCS. Cells are then treated with varying concentrations (0-2 μM) of Gefitinib or the solute control DMSO and EGF. The optimal EGF concentration for inducing proliferation of NR6wtEGFR and NR6W cells has previously been determined and hence NR6wtEGFR and NR6W cells are supplemented with 10 nM and 0.1 nM EGF, respectively. EGF is not added to NR6 and NR6M cells. After 72 hours the amount of cells are measured by performing a MTT proliferation assay.