Differential expression of PKD1 and PKD2 in gastric cancer and analysis of PKD1 and PKD2 function in the model system.

AIM: To study the differential expression of PKD1 and PKD2 in primary gastric cancer samples and to examine the role of PKD1 and PKD2 protein kinases in regulation of gastric tumor cell biology in the model system. METHODS: Tumor samples of different histological variants of primary gastric cancer were analyzed. PKD1 and PKD2 expression levels in tumor samples were accessed by Western blot analysis and quantitative polymerase chain reaction (Q-PCR). As a model system we have used gastric adenocarcinoma сell line AGS sublines constitutively transfected by pcDNA3.1 coding PKD1 or PKD2, or empty pcDNA3.1 vector. These cell lines were analyzed by Western blot, Q-PCR, MTT and proliferation assays, in vitro scratch and Transwell assays, clonogenic assay. RESULTS: It was found that primary gastric tumors possess different levels of PKD1 and PKD2 expression on mRNA and protein levels. Low level of PKD1 expression on protein and mRNA level was detected in low differentiated adenocarcinoma and ring cell gastric cancer - disorders with poor clinical prognosis. The high level of PKD2 expression was also found in gastric tumors with poor prognosis: low differentiated adenocarcinoma and adenogen cancer. To find out whether differential expression of PKD1 and PKD2 could affect biology of gastric tumor cells in vitro, we used a model system based on AGS cell line that constitutively expressed PKD1 or overexpressed PKD2. PKD1 transfection led to the inhibition of cell proliferation, migration and colony formation, in the meanwhile, the PKD2 overexpression enhanced proliferation, migration and colony formation capacities of AGS cells. CONCLUSIONS: Our data suggest that both downregulation of PKD1 or upregulation of PKD2 expression may determine the behavior of gastric tumor cells, which promotes invasive phenotype and could result in general poor prognosis.

Helicobacter pylori infection of gastric cancer cells elevates the level of expression and activation of protein kinase D2.

AIM: To test the hypothesis, whether H. pylori infection may affect the level of PKD2 expression and/or activation in gastric cancer cells. METHODS: Studies were performed on AGS human gastric adenocarcinoma cell line, gastric tissues samples from 36 cases of different histological variants of gastric cancer. Immunohistochemical, cell and molecular biology, bacteriological and biochemical approaches have been used in this study. RESULTS: H. pylori 16S rRNA gene was detected in 97% cases of gastric tumors, and in 83% of cases csmall a, CyrillicgA gene was detected. In all tested adenocarcinoma samples cagA+ H. pylori was revealed. These cases were characterized by high level of PKD1/2 expression and autophosphorylation. In adenogenic cancer samples the presence of cagA- H. pylori was identified. Carcinoid and nondifferentiated gastric cancers contain H. pylori, with very low numbers of cagA+ copies. All cases of gastric tumors with cagA- H. pylori had very low levels of PKD1/2 autophosphorylation. AGS cell line infection with cagA- and cagA+ H. small er, Cyrillicylori resulted in elevation of PKD2 expression levels in 3.29 and 3.66 times respectively (p < 0.001). In cells infected by cag+ H. small er, Cyrillicylori the level of PKD2 transphosphorylation was 1.39 higher than in cells infected by cagA- H. pylori. For PKD2 autophosphorylation this difference was even higher - 3.27 times (p < 0.001). CONCLUSION: H. pylori infection enhanced the level of protein kinase D2 expression, trans- and autophosphorylation. The level of PKD2 autophosphorylation/activation was higher in AGS cell line inoculated of with cag+ H. pylori than in AGS cells with cagA- H. pylori. These suggest that H. pylori induces activation of PKD1/2 and could exploit PKD2 mediated signaling pathways that may contribute to the pathogenesis of gastric cancer.