Opioid growth factor (OGF) is an endogenous opioid peptide ([Met5]-enkephalin) that interacts with the OGF receptor (OGFr), and serves as a tonically active negative growth factor in neoplasia. Previous studies demonstrate that OGF targets cell proliferative pathways as a mechanism to inhibit the growth of human pancreatic cancer in vitro and in vivo. In the case of squamous carcinoma of the head and neck, OGF acts by way of the cyclin-dependent kinase inhibitor (CKI) p16. Because pancreatic cells have frequently lost p16 gene expression, yet these cells are repressed in cell replication by OGF, we assessed whether other CKIs might be playing a role in pancreatic cancer. BxPC-3 human pancreatic adenocarcinoma cell cultures treated with OGF and synchronized with nocodazole had 14% fewer cells exiting the G1 phase of the cell cycle in comparison to controls (p<0.05) using flow cytometry. Exposure to OGF decreased the phosphorylation of retinoblastoma protein (Rb) about 40% (p<0.05) from control values. Moreover, OGF treatment for 9 hours increased cyclin-dependent kinase inhibitor (CKI) p21 protein expression 2-fold in comparison to control (p<0.05), but had no effect on p27 or p57 protein expression. Blockade of the OGF-OGFr axis with the short-acting opioid antagonist, naloxone (NAL), revealed that the increased expression of p21 protein following OGF treatment was completely abolished by NAL. NAL alone had no effect on p21 protein expression suggesting that the OGF effects were OGFr mediated. Inhibition of p21 activation by p21 specific siRNA blocked OGF inhibitory action on the growth of BxPC3 cells. Scrambled siRNAs (negative controls) had no effect on p21 expression and did not block OGF’s growth inhibitory action. Collectively, these results indicate that the receptor-mediated, growth inhibitory effects of the OGF-OGFr axis on human pancreatic cancer are associated with induction of p21 expression that, in turn, repressed the Rb phosphorylation. These data reveal that OGF action is consistent in using the CKI pathway, but is responsive to cancer specific gene activity with respect to CKIs.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]