NT-S100A8 inhibits insulin release and activates AKT and NF_KB cell signalling in pancreatic cancer (PC) cells.

Objective To verify whether NT-S100A8, a peptide isolated from PC tissue of diabetic patients: 1) alters Akt and NF-kappa B signalling in PC cells; 2) interferes with insulin (Ins) signaling; and 3) alters Ins response to glucose stimulation. Methods PC cell lines (BxPC3, Capan1, MiaPaCa2) remained unstimulated or were stimulated with 50 mU Ins for 10 minutes with/without 50 and 500 nM NT-S100A8 for 5, 10, 15 and 30 min. Immunoblots of the cell lysates were performed with pI-kappa B-alpha, pAkt (Ser473 and Thr308), Akt, pPDK1 (Ser241), p-mTOR (Ser2448) and beta-actin antibodies. Beta-TC6 rat insulinoma cells were left untreated or treated 30 min daily for one week with 50, 200 and 500 nM NT-S100-A8 alone or with 20 mM glucose. Cells were then stimulated with 20 mM glucose and Ins was measured in the at 2, 3, 5, 10, 15 and 30 min. Results Both Ins and NT-S100A8 independently induced Akt Ser473 phosphorylation in BxPC3 and MiaPaCa2, not in Capan1. Akt Thr308 phosphorylation in all PC cell lines was induced by Ins, not by NT-S100A8. NT-S100A8 time and dose-dependently induced p-mTOR in BxPC3, but it did not induce pPDK1. To study NF-kappa B signalling we assessed the phosphorylation of its cytoplasmic inhibitor I-kappa B-alpha. In all cell lines I-kappa B-alpha was constitutively phosphorylated. Ins determined a significant reduction of pI-kappa B-alpha in Capan1 and MiaPaCa2 and an enhancement in BxPC3, effects not counteracted by NT-S100A8. In BxPC3 and Capan1, NT-S100A8 caused an increase in pI-kappa B-alpha after 5 min, followed by a reduction at 10 and 15 min and a recovery at 30 min. pI-kappa B-alpha in MiaPaCa2 cells was not modified. Glucose induced early (2 min) and late (15-30 min) Ins release in control beta-TC6 cells. The amount of Ins release was progressively reduced when cells were stimulated with glucose for one week and almost completely abolished when glucose was given with NT-S100A8 (repeated measures analysis of variance: P