OGF-OGFR AXIS AND ITS INHIBITORY ACTIONS ON CELL CYCLE PROGRESSION
Open Access
- Author:
- Cheng, Fan
- Graduate Program:
- Cell and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 14, 2006
- Committee Members:
- Patricia Mclaughlin, Committee Chair/Co-Chair
Ian Stuart Zagon, Committee Member
Michael Verderame, Committee Member
Xuwen Peng, Committee Member - Keywords:
- cell cycle
OGF - Abstract:
- 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 showed that OGF inhibits the growth of human head and neck squamous cell carcinoma (HNSCC) cells and pancreatic cancer cells in vitro and in vivo, and is targeted to cell proliferation. However, the mechanism by which OGF inhibits cell replication was not clear. The studies described within this thesis were directed towards defining the molecular mechanisms of OGF inhibitory action. A number of unique findings are presented here. In SCC1 HNSCC cell cultures synchronized with nocodazole, flow cytometry revealed that OGF treatment (10-6 M) resulted in fewer cells exiting the G1 phase of the cell cycle in comparison to controls (p<0.05). Subsequent studies showed that OGF decreased the phosphorylation of retinoblastoma protein (Rb) (p<0.05) without changing the total Rb expression. Reduced phosphorylated Rb was correlated with reduced cdk4 kinase activity while the total cdk4 expression did not change. Moreover, OGF treatment increased cyclin-dependent kinase inhibitor (CKI) p16 protein expression 2-fold in comparison to controls (p<0.05), but had no significant effect on p15, p18, p19, p21 or p27 protein expression. Western blot analysis did not detect p57 protein in the SCC1 cell line. Blockade of OGF-OGFr interactions with the short-acting opioid antagonist, naloxone (NAL), demonstrated that increased expression of p16 protein by OGF was completely abolished by concomitant administration of OGF and NAL. NAL alone had no effect on p16 expression suggesting that this regulation of p16 was an opioid receptor mediated event. Moreover, OGF treatment increased p16 protein expression in comparison to controls (p<0.05) in CAL27 and SCC4 HNSCC cell lines, demonstrating the ubiquitous nature of this observation. Inhibition of p16 (INK4a) activation by p16 specific siRNA blocked OGF inhibitory action on proliferation of SCC1, CAL-27 and SCC4 HNSCC cells. Cultures treated with negative control siRNA, which had no effect on p16 expression, did not block OGF’s growth inhibitory action. Collectively, these results indicate that the receptor-mediated, growth inhibitory effects of the OGF-OGFr axis in HNSCC cells are associated with induction of p16/cdk4 resulting in decreased Rb phosphorylation. These data revealed that the target of cell proliferative inhibitory action of OGF in human HNSCC cells is by the way of cyclin dependent kinase inhibitory pathways, specifically, the p16 pathway. Because many human HNSCC tumors retain p16, OGF may be a valuable therapeutic agent in this setting. Alternatively, pancreatic cancer cells have frequently lost p16 gene expression, and thus other CKI pathways were examined for their role with OGF inhibition of pancreatic cancer growth. BxPC-3 human pancreatic adenocarcinoma cell cultures were synchronized with nocodazole, and flow cytometry revealed that OGF treatment (10-6 M) resulted in fewer cells (p<0.05) exiting the G1 phase of the cell cycle in comparison to controls. The present studies focused on the role of OGF on regulatory pathways for the G1 to S phase transition. Studies using the BxPC-3 pancreatic adenocarcinoma cell line suggest that OGF decreased the phosphorylation of retinoblastoma protein (Rb) (p<0.05). This change was correlated with reduced cdk2 kinase activity. There was no change in total cdk2 expression. Moreover, OGF treatment for 9 hours increased p21 protein expression 2-fold in comparison to control (p<0.05), but had no effect on p27 protein expression. Blockade of the OGF-OGFr axis with the short-acting opioid antagonist naloxone revealed that the increased expression of p21 protein following OGF treatment was completely abolished by concomitant administration of OGF and NAL. NAL alone had no effect on p21 protein expression suggesting that the OGF effects were OGF receptor mediated. Moreover, OGF treatment increased CKI p21 protein expression in comparison to control (p<0.05) in Panc1 and Capan2 pancreatic cancer cell lines. Inhibition of p21 activation by p21 specific siRNA blocked the OGF inhibitory action on BxPC3, Panc1 and Capan2 cell proliferation. 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 in human pancreatic cancers are associated with induction of p21 expression and repression of the Rb phosphorylation. In summary, these studies demonstrate for the first time that the target of cell proliferative inhibitory action of OGF is cyclin dependent kinase inhibitory pathways.