Progastrin (PG) exerts proliferative and antiapoptotic effects on intestinal epithelial and colon cancer cells via Annexin II (ANX-II). In here, we show that ANX-II similarly mediates proliferative and antiapoptotic effects of PG on a pancreatic cancer cell line, AR42J. The role of several signaling molecules was examined in delineating the biological activity of PG. PG (0.1-1.0 nmol/L) caused a significant increase (2- to 5-fold) in the phosphorylation of phosphatidylinositol 3-kinase (PI3K), Akt (Thr(308)), p38 mitogen-activated protein kinase (MAPK; Thr(180)/Tyr(182)), extracellular signal-regulated kinases (ERK; Thr(202)/Tyr(204)), IkappaB kinase alpha/beta (IKKalpha/beta; Ser(176)/(180)), IkappaBalpha (Ser(32)), and p65 nuclear factor-kappaB (NF-kappaB; Ser(536)). Inhibition of p44/42 ERKs (PD98059), p38 MAPK (SB203580), Akt, and PI3K (LY294002), individually or combined, partially reversed antiapoptotic effects of PG. The kinetics of phosphorylation of IKKalpha/beta in response to PG matched the kinetics of phosphorylation and degradation of IkappaBalpha and correlated with phosphorylation, nuclear translocation, and activation of p65 NF-kappaB. NF-kappaB essential modulator-binding domain peptide (an inhibitor of IKKalpha/beta) effectively blocked the activity of p65 NF-kappaB in response to PG. Activation of p65 NF-kappaB, in response to PG, was 70% to 80% dependent on phosphorylation of MAPK/ERK and PI3K/Akt molecules. Down-regulation of p65 NF-kappaB by specific small interfering RNA resulted in the loss of antiapoptotic effects of PG on AR42J cells. These studies show for the first time that the canonical pathway of activation of p65 NF-kappaB mediates antiapoptotic effects of PG. Therefore, targeting PG and/or p65 NF-kappaB may be useful for treating cancers, which are dependent on autocrine or circulating PGs for their growth.