Erlotinib Hydrochloride

DUSP6 is regulated by EGFR/ERK inhibition in NSCLC cell lines. Protein expression levels were assayed by immunoblot for phosphor-EGFR at indicated tyrosine sites, total-EGFR, phosphor-ERK, total-ERK, ETS1, DUSP6 and GAPDH demonstrating suppression of DUSP6 following inhibition of activated ERK (P-ERK) and ETS1 levels in the presence of appropriate drug for each of the following cell lines: (a) HCC827 cells treated with erlotinib; (b) H1975 cells treated with erlotinib or CL-387,785 (an irreversible EGFR inhibitor); H441 cells treated with (c) erlotinib or U0126 (a MEK1/2 inhibitor) and (d) erlotinib or AG1478 (specific EGFR inhibitor) (d). Cells were starved overnight with serum-free media, then treated with drug as indicated by the following abbreviations: (E) 100ng/ml EGF; (D) 0.01% DMSO control; (Er) 1uM erlotinib, (CL) 1μM CL-387,785, (U) 20μM U0126 or AG1478. Whole cell lysates were obtained using 10% TCA lysis buffer and immunoblotting was performed at indicated time points.

DUSP6 gene expression is inhibited by erlotinib using quantitative real-time PCR analysis. RNA was extracted from treated (a) HCC827 and (b) PC9 cells and relative expression level standardized against GAPDH at several time points.

Pressure-induced myogenic tone in coronary arterioles with and without EGFR tyrosine kinase inhibitors (AG1478 and Erlotinib, 1 μM) n=6, *Pb0.05 CTR vs. EGFR inhibitor

(B–C) LNCaP (B) and LNCaP-AI (C) cells were transiently transfected with sPLA2-IIa(-800)-Luc (0.5 lg). The cells were then treated with Erlotinib (20 lM), Gefitinib (20 lM), Lapatinib (20 lM), CI-1033 (8 lM), LY294002 (20 lM) and Bortezomib (20 lM) without or with EGF (100 ng/ml) for 24 h. Luciferase assay was performed according to a standard protocol with Renilla luciferase as an internal control. Data are presented as the mean (±SD) of duplicate values of a representative experiment that was independently repeated for five times.

Breast cancer cells were pretreated with 100ng/ml EGF for 15 min and then treated with the indicated concentrations of Erlotinib for 24 hours.

Immunoblots of MCF7-HER2 cells treated with DMSO (c), BEZ235 (B, 500 nM) and lapatinib (L, 500 nM) as well as with erlotinib (E, 500 nM) and the indicated combinations for 24 h.

Inhibition of anchorage-independent growth of lung tumor cell lines by selected inhibitors. Each selected cell line was treated with the indicated inhibitor at 0.1 μM and 1 μM concentrations for two weeks and cell colony size formation was scored under the Nikon inverted-phase microscope.

LNCaP-AI cells were starved in 1% stripped medium for 24 h. The cells were then treated with Erlotinib (20 lM), Gefitinib (20 lM), Lapatinib (20 lM), CI-1033 (8 lM), LY294002 (20 lM) and Bortezomib (20 lM) for 24 h. Cell culture medium was collected from each sample and subjected to ELISA for sPLA2-IIa. The condition medium samples were diluted 10 times for ELISA. Average of duplicate samples was converted to nanogram per milliliter against standard curve. The data represent one of five repeated experiments.