Nitric oxide inhibits the proliferation and invasion of pancreatic cancer cells through degradation of insulin receptor substrate-1 protein.
Nitric oxide (NO), which plays a role in the posttranslational modification of proteins, exhibits tumoricidal activity. However, the mechanism remains largely unclear. We investigated whether the regulation of insulin receptor substrate (IRS)-1 protein expression and insulin/insulin-like growth factor (IGF) signaling by NO is involved in the proliferation and invasion of pancreatic cancer cells. NO donor inhibited insulin/IGF-I-stimulated phosphorylation of insulin receptor/IGF-I receptor, IRS-1, Akt/PKB, and glycogen synthase kinase-3beta along with decreased expression of IRS-1 protein in MIAPaCa-2 cells, whereas NO donor enhanced the phosphorylation of extracellular signal-regulated kinase-1/2. In contrast, a selective inducible nitric oxide synthase inhibitor, 1400W, upregulated the expression of IRS-1 protein and the phosphorylation of IRS-1, Akt/PKB, and glycogen synthase kinase-3beta, along with enhanced proliferation and invasion of Panc-1 cells expressing inducible nitric oxide synthase protein. NO donor induced IRS-1 protein reduction through increased ubiquitination and degradation. For the detection of the site responsible for NO-induced ubiquitination, IRS-1 deletion mutant genes were transfected and overexpressed in MIAPaCa-2 cells. The results indicate that the COOH terminus of the IRS-1 protein is required for NO donor-induced ubiquitination and protein degradation. Cells stably transfected with COOH-terminal deletion mutants of IRS-1 exhibited reduced IGF signaling and cell proliferation compared with vector alone-transfected cells, with no influence of NO on IGF signaling and invasion, although stable transfectants with full-length IRS-1 protein exhibited remarkable NO-induced reduction in IGF signaling, cell proliferation, and invasion. These findings indicate that NO inhibits the proliferation and invasion of pancreatic cancer cells, at least in part, through upregulation of IRS-1 protein degradation and resultant downregulation of the insulin/IGF-I-Akt pathway.
Sugita
Hiroki
H
Department of Gastroenterological Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan. sugitaf@ba2.so-net.ne.jp
Kaneki
Masao
M
Furuhashi
Satoshi
S
Hirota
Masahiko
M
Takamori
Hiroshi
H
Baba
Hideo
H
D015153:Blotting, Western; D002448:Cell Adhesion; D002465:Cell Movement; D049109:Cell Proliferation / Q000187:drug effects*; D016166:Free Radical Scavengers / Q000494:pharmacology*; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D047468:Immunoprecipitation; D007328:Insulin / Q000378:metabolism; D055504:Insulin Receptor Substrate Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D009361:Neoplasm Invasiveness; D009569:Nitric Oxide / Q000494:pharmacology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D058539:Phosphatidylinositol 3-Kinase / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination
D016166:Free Radical Scavengers; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D020030:Nitric Oxide Donors; D009573:Nitrites; D012333:RNA, Messenger; D025801:Ubiquitin; D009569:Nitric Oxide; D007334:Insulin-Like Growth Factor I; D052247:Nitric Oxide Synthase Type II; D058539:Phosphatidylinositol 3-Kinase; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3