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Objectives: The aim of this study was to investigate the effects of the activated P2X7 receptors on the proliferation and growth of human pancreatic cancer cells., Methods: Proliferation was measured by incorporating bromodeoxyuridine into pancreatic cancer cells, MIA PaCa-2 and HPAC. Expression of P2 receptors and signal molecules was examined using quantitative reverse transcription/polymerase chain reaction and/or Western blot. Proliferative effects of the P2X7 receptors in vivo were examined using a xenotransplant model of pancreatic cancer cell lines., Results: Incubating pancreatic cancer cells with adenosine triphosphate (ATP) and 2'(3')-O-(4-Benzoylbenzoyl)ATP resulted in a dose-dependent increase of cell proliferation. The P2 receptor antagonist, KN-62, and small interfering RNA against P2X7 receptors, significantly decreased the proliferative effects of ATP. The ATP-induced proliferation was mediated by protein kinase C, extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), and c-Jun N-terminal kinase (JNK); specifically, ATP increased the phosphorylation of ERK1/2 and JNK. The expression of inducible nitric oxide synthase was decreased by P2X7 receptor activation. In a xenotransplant model, applying ATP significantly increased the growth of induced tumors., Conclusions: The P2X7 receptor activation by extracellular nucleotides increased proliferation and growth of human pancreatic cancer cells via ERK1/2 and JNK. This supports the pathophysiological role of P2X7 receptors in pancreatic disease and recovery.

Objectives: Src is considered a rising therapeutic target for the treatment of solid tumors, and Src family kinases (SFKs) participate in cancer cell proliferation and survival. The role of SFK suppression was investigated in the proliferation, migration, and invasion of pancreatic cancer cells., Methods: Knockdown of the SFKs in pancreatic cancer cells was achieved by transfecting small interfering RNAs, and its effects were investigated using proliferation, wound, and invasion assays., Results: The SFK inhibitors suppressed proliferation and induced cell cycle arrest in pancreatic cancer cells. The SFK messenger RNA profiles showed that Yes1, Lyn, Fyn, Frk, Hck, and Src were expressed. Specific small interfering RNA transfection suppressed the messenger RNA expressions of Yes1, Lyn, Fyn, Frk, and Src, and the knockdown suppressed cell proliferation by 16.7% to 47.3% in PANC-1 cells. Knockdown of any of these 5 SFKs suppressed proliferation in other pancreatic cancer cell lines by 3.0% to 40.5%. The knockdowns significantly reduced pancreatic cancer cell migration by 24.9% to 66.7% and completely inhibited invasion., Conclusions: These results suggest that the knockdown of Yes1, Lyn, Fyn, Frk, or Src reduce human pancreatic cancer cell proliferation, migration, and invasion, and that SFKs should be viewed as critical therapeutic targets of pancreatic cancer.

Objectives: The aim of this study was to investigate the effect of uridine triphosphate (UTP) on the proliferation of human cancerous pancreatic duct epithelial cells., Methods: Proliferation was measured by immunoassay for bromodeoxyuridine incorporation into the pancreatic cell line PANC-1. Effect of UTP was assayed using selective P2 agonist and antagonist, small interfering RNA, intracellular signal inhibitors, and Western blot., Results: Incubation of PANC-1 cells with UTP or MRS2768, a selective P2Y2 receptor agonist, resulted in a dose- and time-dependent increase of proliferation. The messenger RNA transcript and protein of P2Y2 receptor were expressed in PANC-1 cells. P2 receptor antagonist suramin and small interfering RNA against P2Y2 receptor significantly decreased the proliferative effect of UTP and MRS2768. Activation of P2Y2 receptor by UTP transduced to phospholipase C, inositol 1,4,5-triphosphate (IP3), and protein kinase C. Uridine triphosphate-induced proliferation was mediated by protein kinase D, Src-family tyrosine kinase, Ca/calmodulin-dependent protein kinase II, phosphatidylinositol 3-kinase (PI3K), Akt, and phospholipase D. Uridine triphosphate increased phosphorylation of Akt through protein kinase C, Src-family tyrosine kinase, Ca/calmodulin-dependent protein kinase II, and PI3K., Conclusions: Uridine triphosphate increases proliferation of human pancreatic duct epithelial cells by activation of P2Y2 receptor and PI3K/Akt pathway. This could be helpful for discovering the long-term roles of P2Y2 receptor in pancreatic cells.

Objectives: The aim of this study was to investigate the effect of P2Y receptor activation on proliferation of human pancreatic duct epithelial cells., Methods: Proliferation was measured by immunoassay for bromodeoxyuridine incorporation into a pancreatic duct epithelial cell line, PANC-1. Expression of P2Y receptors was examined using quantitative reverse transcription-polymerase chain reaction and Western blot., Results: Extracellular nucleotides, adenosine diphosphate (ADP) and uridine diphosphate (UDP), stimulated proliferation of pancreatic duct cells in a concentration-dependent manner. The nucleotide efficacy order was ADP > UDP > uridine triphosphate (UTP) > adenosine triphosphate. P2Y(1) and P2Y(6) receptor blockers, MRS2500 and MRS2578, blocked the effect of ADP and UDP. The signal that transmitted the proliferative activity of ADP and UDP was transducted to phospholipase C, inositol 1,4,5-triphosphate receptor, and protein kinase C. These results indicate involvement of P2Y(1) and P2Y(6) receptors in ADP- and UDP-stimulated proliferation. Pancreatic duct cells expressed the messenger RNA transcripts of P2Y receptors, P2Y(1) , P2Y(2), and P2Y(6), and P2Y(1) and P2Y(6) receptor protein., Conclusions: Extracellular nucleotides increase proliferation of human pancreatic duct epithelial cells by activation of P2Y(1) and P2Y(6) receptors. This provides the basic model for the effect of P2Y receptors on the proliferation of pancreatic duct epithelial cells.

Objectives: This study aimed to identify proteins important for the primitive gut tube differentiation from human embryonic stem cells (hESCs) by derivation method for pancreatic cells., Methods: Proteins with altered expression levels in the process of differentiating to primitive gut tube from definitive endoderm of hESCs were investigated by comparative proteomic analysis using 2-dimensional gel electrophoresis and mass spectrometric analyses., Results: Differentiation to primitive gut tube from hESCs was analyzed using differentiation marker genes and proteins. Twenty-seven protein spots with significant changes in intensity were found by 2-dimensional gel electrophoresis, and 24 proteins were further identified. These proteins were functionally annotated based on gene ontology. The expression levels of 3 proteins, RREB1, PDE6B, and CD209, involved in signal transduction, were validated using quantitative reverse transcription-polymerase chain reaction and Western blot. Their mRNA and protein expression levels increased in primitive gut tube but not in definitive endoderm or embryonic body., Conclusions: The increase in expression of RREB1, PDE6B, and CD209 suggests that these proteins might play important roles in the differentiation of primitive gut tube cells from hESCs and in human primitive gut tube development into pancreas. Therefore, they could be developed as differentiation markers for identifying primitive gut tube cells.

Objectives: This study was aimed to investigate important proteins associated with endoderm differentiation by pancreatic derivation protocol from human embryonic stem cells (hESCs)., Methods: Comparative proteomic analysis of endoderm cells differentiated from hESCs by activin A and low serum was performed. Proteins with altered expression levels during endoderm differentiation were investigated by 2-dimensional gel electrophoresis (2-DE) with mass spectrometric analysis., Results: Thirty-four protein spots with significantly changed intensities were identified. These were functionally annotated based on gene ontology. The messenger RNA expression levels of 5 genes, APC, CCNB3, HSPA9, CCT2, and YWHAE, were correlated with 2-DE analysis. We further validated the protein expression levels of adenomatous polyposis coli (APC) and cyclin B3 (CCNB3) by using Western blot analysis and immunocytochemistry. They are involved in the regulation of cell cycle, thus, cyclins and cyclin-dependent kinases, which regulate the cell cycle, were examined. Cyclin A1, cyclin D2, and cyclin E2 were upregulated, and other cyclins and cyclin-dependent kinases were downregulated in endoderm cells., Conclusions: The increase in expression of APC and CCNB3 suggests that these proteins will be important markers for identifying endoderm cells differentiated from hESCs, and they can play important roles in the differentiation of endoderm cells from hESCs or in human endoderm development for pancreas.