Geminin, a Replication Inhibitor Protein, Is Over-Expressed in Pancreatic Cancer Tissue and Apigenin Affects Replication by Down-Regulating Geminin and Cdc6, but Not CDT-1 in Pancreatic Cancer Cells

Introduction: In eukaryotic cells, replication is tightly controlled by several factors which ensure duplication of chromosomes only once in every cell cycle. Replication is initiated by binding of the origin replication complex (ORC) to DNA followed by Cdc6 and Cdt1 which recruit the minichromosome maintenance (MCM) proteins. After replication starts, geminin binds to Cdt1 and prevents another DNA replication within the same cell cycle. Apigenin is a flavonoid with anti-tumor effects. We have reported that apigenin causes growth arrest in pancreatic cancer cells, yet the molecular mechanism remains unclear. Microarray analysis showed that apigenin decreases geminin expression. Here we report that geminin is up-regulated in pancreatic cancer tissue and apigenin down-regulates both geminin and Cdc6 but not Cdt1. Methods: Immunohistochemistry with anti-geminin antibody was used to quantify expression in 15 each of human pancreatic tumor and respective control tissue samples. In CD18 and S2-013 human pancreatic cancer cells, geminin mRNA and protein expression were measured by real-time RT-PCR and western blot analysis. Nuclear localization of geminin was determined by confocal microscopy. Cdc6 and Cdt1 expression were evaluated by western blot analysis. Cdc6 and Cdt1 expression were evaluated by western blot analysis. Results: Geminin expression was significantly increased in human pancreatic tumor samples versus controls. Apigenin significantly decreases geminin expression at both the mRNA and protein levels and decreases the geminin protein localized to the nucleus. Apigenin also down-regulates Cdc6 protein expression but not Cdt1. Conclusions: Geminin is over-expressed in human pancreatic cancer. One of the mechanism of apigenin's anti-tumor effects may be through decreased geminin and Cdc6 expression but not Cdt1.