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Abstract
Multiple anti-cancer drugs are often combined to increase their effectiveness. This study investigates the combination between an epidermal growth factor receptor (EGFR) inhibitor and gemcitabine, a commonly used chemotherapy agent that causes cell death by incorporation into DNA. Clinical trials combining EGFR inhibitors with gemcitabine-based chemotherapy in non-small cell lung cancer (NSCLC) have not produced a survival advantage. This may be caused by antagonism between the two drugs or mutations that promote such, possibly RAS mutation. Furthermore, ERK, a critical growth regulator downstream of RAS, may play a role. Therefore the aim of this study is to explore the cytotoxic interaction between gemcitabine and an EGFR inhibitor as well as the mechanisms underlying this interaction. The impact of KRAS mutation will also be investigated. Two NSCLC cell lines (A549 and H322) with divergent KRAS mutation status were treated with gemcitabine and the EGFR inhibitor AG1478. Cytotoxic interaction was determined with median effect analysis. Cell cycle alteration was assessed using flow cytometry while EGFR downstream signalling was assessed using immunoblotting and ELISA. ERK was inhibited using U0126 to explore the effect on cytotoxic interaction. The effect of KRAS was explored using siRNA-mediated knockdown in A549. Cytotoxic interaction was concentration and schedule dependent with antagonism in AG1478 pre-treatment and high concentration AG1478. G1 arrest, being observed with both high concentration AG1478 and high concentration gemcitabine, was inconsistently associated with antagonism. On the other hand, ERK phosphorylation was increased by gemcitabine and its suppression by AG1478 was related to antagonism particularly in H322. Antagonism caused by inhibition of ERK was further confirmed following U0126 treatment. Greater antagonism was observed in the KRAS mutant cell line and KRAS knockdown by siRNA resulted in increased sensitivity to AG1478 as well as combination treatment. In conclusion, ERK appears to be an important determinant of cytotoxic interaction with its phosphorylation favouring synergism. However, the impact of other EGFR downstream pathways warrants further investigation. Furthermore, KRAS mutation confers resistance to AG1478 and combination treatment. Both ERK and KRAS may potentially be important biomarkers for EGFR inhibitor combination therapy.