Abstract 2877: Perifosine alone and in combination with antimetabolites interferes with NF-kB pathway activation in colon cancer cell lines

Abstract

Abstract Background: Perifosine, a novel, first-in-class, oral Akt inhibitor, in combination with the antimetabolite Capecitabine is currently in Phase III clinical trials for the treatment of refractory advanced colorectal cancer. We have shown previously that Perifosine in combination with antimetabolites displayed synergistic cytotoxicity and apoptosis in colon cancer and other cell lines [1]. Constitutive NF-kB activation is frequently observed in colorectal cancer and, moreover, is associated with resistance to chemotherapy [2]. Hence we analysed, wether downregulation of the NF-kB pathway contributes to Perifosine mechanism of action and to the observed synergism with antimetabolites in colon cancer cell lines. Methods: Cellular NF-kB activation in nuclear extracts was measured by the TransAm NF-kB p65 ELISA (Active Motif, USA) monitoring binding of NF-kB to its response element. To allow for higher throughput of NF-kB pathway analysis a monoclonal SW620 colon cancer cell line ectopically expressing a NF-kB response element driven reporter gene (Promega, USA) was established. Cellular cytotoxicity and apoptosis assays as well as western blotting were performed according to standard procedures. The analysis of drug combinations was conducted by CalcuSyn software (Biosoft, UK) [3]. Results: Perifosine pretreatment decreased TNFα-induced NF-kB activation in SW620 cells in both independent assay sytems with IC50 values of about 7-10 µM. This effect seems to be specific, since the results were not mimicked by cytotoxic effects of Perifosine. Some of the antimetabolites alone upregulated NF-kB activity in the reporter gene assay. In the presence of these antimetabolites Perifosine was still capable to downregulate NF-kB activity. Conclusions: In summary, these results demonstrate that downregulation of the NF-kB pathway may contribute to the mechanism of action of Perifosine. Moreover, cotreatment with Perifosine has the potential to interfere with the development of antimetabolite resistance due to NF-kB activation in colon cancer. Further experiments addressing Perifosine's mechanism of action in combination with antimetabolites in gastric cancer cell lines are ongoing.