Nitric Oxide Inhibits the Proliferation and Invasion of Pancreatic Cancer Cells through Degradation of Insulin Receptor Substrate-1 Protein

Abstract

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-3β 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-3β, 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 COOHterminal 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.

NO donor and MEK inhibitor synergistically inhibit proliferation and invasion of cancer cells

“…NO inhibits IGF-IR and EGFR activity, as reported previously (29,30). Furthermore, NO induces the degradation of IRS-1 protein and the phosphorylation of IRS-1 (14,29,30). In addition, Yasukawa et al reported that NO directly modifies Akt protein and inhibits its activity (11).…”

“…In addition, Yasukawa et al reported that NO directly modifies Akt protein and inhibits its activity (11). Recent studies have shown that NO inhibits cell proliferation and induces apoptosis in various cells including cancer cells, in vitro and in vivo (14)(15)(16)(17)(18)(19)(20)(21).…”

“…Recent papers reported that endogenous NO promotes oncogenesis and angiogenesis in various cancers (12,13). In contrast, other studies have shown that NO inhibits cell proliferation and induces apoptosis in various cells including cancer cells, in vitro and in vivo (14)(15)(16)(17)(18)(19)(20)(21). These studies suggest that NO can act either as a tumor suppressor or as a tumor enhancer depending on cell type and the level of NO in the cells.…”

“…However, the molecular mechanism underlying the inhibitory effects of NO on cancer viability, remains unclear. We have reported that NO inhibits cancer cell proliferation and invasion through downregulation of IRS-1 protein, resulting in inhibition of the PI3K/Akt pathway (14). In contrast, NO enhances nitrosylation and activates N-Ras and H-Ras proteins (22).…”

Nitric oxide donor hybrid compounds as promising anticancer agents

“…Increasing research showed that NO donors were effective on various malignant tumors, such as myeloma, breast cancer, ovarian cancer, prostate cancer and pancreatic cancer (15)(16)(17). Moreover, it is worth noting that more and more NO-donor hybrids have been designed, synthesized and evaluated as antitumor agents (Table 1), which will be discussed in the following part of this review.…”