Mechanisms of synthetic serine protease inhibitor (FUT-175)-mediated cell death.
BACKGROUND
Constitutive activation of nuclear factor-kappaB (NF-kappaB) is a frequent molecular alteration in pancreatic cancer and a number of studies have suggested that constitutive NF-kappaB 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-kappaB activation and the induction of apoptotic responses.


METHODS
To examine the effect of FUT-175 on the inhibition of NF-kappaB 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 IkappaBalpha phosphorylation and NF-kappaB activation, thereby inhibiting the antiapoptotic activity of NF-kappaB. 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-kappaB antiapoptotic activity and inducing TNFR-mediated apoptosis. Supported by this unique function as a NF-kappaB 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.
Uwagawa
Tadashi
T
Department of Surgical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
Li
Zhongkui
Z
Chang
Zhe
Z
Xia
Qianghua
Q
Peng
Bailu
B
Sclabas
Guido M
GM
Ishiyama
Satoshi
S
Hung
Mien-Chie
MC
Evans
Douglas B
DB
Abbruzzese
James L
JL
Chiao
Paul J
PJ
D000230:Adenocarcinoma / Q000473:pathology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001550:Benzamidines; D053181:Caspase 8 / Q000378:metabolism; D045744:Cell Line, Tumor / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D053200:Fas-Associated Death Domain Protein / Q000378:metabolism; D006146:Guanidines / Q000494:pharmacology*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D016328:NF-kappa B / Q000009:adverse effects / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000473:pathology*; D010766:Phosphorylation / Q000187:drug effects; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D014157:Transcription Factors / Q000378:metabolism
D000970:Antineoplastic Agents; D001550:Benzamidines; D053200:Fas-Associated Death Domain Protein; D006146:Guanidines; C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D016328:NF-kappa B; D047888:Receptors, Tumor Necrosis Factor, Type I; D015842:Serine Proteinase Inhibitors; D014157:Transcription Factors; C065788:transcription factor PEA3; D053181:Caspase 8; C032855:nafamostat