Figure 2.

Cetuximab-resistant cell lines are sensitive to the EGFR targeted TKI erlotinib. (A) HER family members and downstream signaling molecules AKT and MAPK are effectively inhibited with erlotinib treatment in cetuximab-resistant cell lines. Cetuximab-resistant clones (HC1, HC4, HC8) were treated with vehicle (DMSO), cetuximab (100 nM) or erlotinib (250 or 750 nM) for 24 h. Whole cell protein lysates were fractionated on SDS-PAGE followed by immunoblotting for the indicated proteins. α-Tubulin was used as a loading control. (B) Erlotinib inhibits the proliferation of cetuximab-resistant cell lines. Parental cells (HP) and cetuximab-resistant cell lines (HC1, HC4, HC8) were plated and allowed to adhere for 24 h prior to vehicle (DMSO), cetuximab (100 nM) or erlotinib treatment: 0.25 µM, 0.75 µM or 1 uM. Growth was measured at 72 h after drug treatment using the growth proliferation assay described in experimental methods and plotted as a percentage of growth relative to untreated control cells. Data points are represented as mean ± SEM (n = 4). *p ≤ 0.05, **p ≤ 0.001. (C) Erlotinib treatment induces G₁ cell cycle phase arrest in cetuximab-resistant cell lines. Cetuximab-resistant cell lines (HC1, HC4, HC8) were plated and grown for 24 h prior to treatment with vehicle (DMSO), cetuximab (100 nM) or erlotinib (250 or 750 nM) for 24 h. Cell cycle phase distribution was analyzed as described in the materials and methods. Data points are represented as mean ± SEM (n = 3). *p ≤ 0.05, **p ≤ 0.001.