In vitro effects of erlotinib and gemcitabine in erlotinib-resistant cells

2147 Erlotinib (Tarceva) inhibits the epidermal growth factor receptor (EGFR) tyrosine kinase by competing with ATP to inhibit phosphorylation of the kinase. Erlotinib was recently approved for use in combination with gemcitabine for advanced pancreatic cancer. However, in vitro evidence to support the use of erlotinib with gemcitabine as anticancer therapy is lacking. To address this gap, we created 2 erlotinib-resistant pools of A-431 cells, an EGFR-overexpressing, erlotinib-sensitive epidermoid cancer cell line, and assessed them for dose-response and cell cycling in response to erlotinib and gemcitabine. The resistant pools were developed by continuously exposing A-431 cells to erlotinib (pool 1, 3 μM for 1 mo, 5 μM for 1 mo, and 10 μM for 4 mo; pool 2, 3 μM for 1 mo and 5 μM for 5 mo). Basal expression of EGFR was no different among the 3 cell types, and erlotinib blocked EGF signaling in both resistant pools (as evidenced by EGFR phosphorylation after EGF stimulation). Intracellular concentrations of erlotinib (measured by LC/MS/MS) after a 24-h exposure were 0.98 ± 0.04 ng/ml for parental cells, 3.03 ± 0.97 ng/ml for pool 1, and 1.88 ± 0.47 ng/ml for pool 2, confirming that erlotinib penetrated the cell membrane in both pools. Next, parental cells (IC50 1.67 μM) and erlotinib-resistant cells (IC50 17.6 or 13.2 μM) were treated with 2-fold serial dilutions of erlotinib (0.125-16 μM), gemcitabine (0.000436-0.056 μM), or both. Combination indices (CI) for the 2 drugs were derived by using Calcusyn (Biosoft, Cambridge, UK). Drug synergy is indicated by a CI 1.0. In the parental cells, synergy between erlotinib and gemcitabine was noted (CI = 0.68 at IC50); strong synergy was also maintained in the erlotinib-resistant pools (CI = 0.69 for pool 1 and 0.56 for pool 2 at their respective IC50s) despite their being resistant to erlotinib. This synergism was confirmed by trypan blue exclusion. Flow cytometric cell-cycle analysis showed substantial increases in proportions of subdiploid (apoptotic) cells. These results suggest that synergistic effects between erlotinib and gemcitabine in cancer cells may not require sensitivity to erlotinib as a single agent at the concentration used as long as EGFR phosphorylation is blocked by erlotinib. Further studies are warranted to explore the use of erlotinib as a gemcitabine chemosensitizer in cancer cells that overexpress EGFR.