Mechanisms of synthetic serine protease inhibitor (FUT-175)-mediated cell death

Abstract

BACKGROUND. Constitutive activation of nuclear factor-κB (NF-κB) is a frequent molecular alteration in pancreatic cancer and a number of studies have suggested that constitutive NF-κB activity plays a key role in the aggressive behavior of this disease. In an attempt to identify an effective therapeutic agent for pancreatic cancer, the authors studied the role of FUT-175, a synthetic serine protease inhibitor, in the inhibition of NF-κB activation and the induction of apoptotic responses. METHODS. To examine the effect of FUT-175 on the inhibition of NF-κB and the induction of apoptosis in pancreatic cancer cell lines, Western and Northern blot analyses, electromobility shift (EMSA), luciferase reporter gene, DNA fragmentation, immunoprecipitation, in vitro kinase, small interfering RNA (siRNA), and chromatin immunoprecipitation (ChIP) assays were performed. RESULTS. In a time-dependent and dose-dependent manner, FUT-175 inhibited IκBα: phosphorylation and NF-κB activation, thereby inhibiting the antiapoptotic activity of NF-κB. Simultaneously, FUT-175 up-regulated the expression of tumor necrosis factor receptor-1 (TNFR1), which in turn activated the proapoptotic caspase-8 and Bid pathways and induced apoptosis in pancreatic cancer cells. FUT-175-induced activation of Fas-associated death domain (FADD) and caspase-8 was suppressed by RNA interference-mediated inhibition of TNFR1 expression. Furthermore, expression of the transcription factor PEA3 was up-regulated by FUT-175 and was involved in FUT-175-mediated TNFR1 expression. CONCLUSIONS. These results suggested a possible mechanism by which FUT-175 may disrupt interconnected signaling pathways by both suppressing the NF-κB antiapoptotic activity and inducing TNFR-mediated apoptosis. Supported by this unique function as a NF-κB inhibitor and apoptosis inducer, this well-established synthetic serine protease inhibitor with as-of-yet poorly understood mechanisms of actions appears to be a potentially therapeutic agent for pancreatic cancer. © 2007 American Cancer Society.