Enhancement of 5-Fluorouracil-induced In Vitro and In Vivo Radiosensitization with MEK Inhibition

Abstract

Gastrointestinal cancers frequently exhibit mutational activation of the Ras/MAPK pathway, which is implicated in resistance to ionizing radiation (IR) and chemotherapy. Concurrent radiotherapy and 5-fluorouracil (5-FU) based chemotherapy is commonly used for treatment of gastrointestinal malignancies. We previously reported radiosensitization with selumetinib, an inhibitor of MEK1/2. The purpose of the current study was to evaluate if selumetinib could enhance radiosensitivity induced by 5-FU. Clonogenic survival assays were carried out with the HT29 (colorectal), HCT116 (colorectal), and MiaPaca-2 (pancreatic) cell lines using pre-IR treatment with selumetinib, 5-FU and 5-FU+selumetinib. Cell proliferation was determined using a tetrazolium conversion assay. Mitotic catastrophe and DNA repair were analyzed using immunocytochemistry. Flow cytometry was used to analyze cell cycle and apoptosis. Growth delay was used to determine effects of 5-FU+selumetinib on in vivo tumor radiosensitivity. Pre-IR treatment with 5-FU+selumetinib significantly decreased clonogenic survival compared with either agent alone. Dose modifying factors at a surviving fraction of 0.1 for 5-FU+selumetinib was 1.78, 1.52, and 1.3 for HT29, HCT116, and MiaPaca-2, respectively. Cell proliferation was decreased by treatment with selumetinib+5-FU as compared with single agent treatment regardless of treatment sequencing. Enhancement of 5-FU cytotoxicity and 5-FU mediated radiosensitization with selumetinib treatment was accompanied by an increase in mitotic catastrophe and apoptosis, and reductions in Stat3 phosphorylation and survivin expression. In vivo, an additive growth delay was observed with 5-FU+selumetinib+3Gy versus 5-FU+3Gy and selumetinib alone. These data suggest that selumetinib can be used with 5-FU to augment radiation response.