ABSTRACT
Various prostanoids and peroxisome proliferator-activated receptor γ (PPARγ) ligands play an important role in gastric cancer. Previously, we demonstrated that prostaglandin reductase 2 (PTGR2) catalyzes the reduction of the PPARγ ligand 15-keto-PGE(2) into 13,14-dihydro-15-keto-PGE(2). Here, we present functional data and clinical relevance for the role of PTGR2 in gastric cancer. Using lentiviral technology in AGS and SNU-16 gastric cancer cell lines, we either down-regulated or overexpressed PTGR2. In vitro analysis showed that PTGR2 knockdown resulted in decreased proliferation rate and colony formation, and in vivo xenograft models showed slower growth of tumors. Mechanistically, PTGR2 knockdown induced cell death, altered mitochondrial function, and increased reactive oxygen species production, which led to activation of ERK1/2 and caspase 3, with increased Bcl-2 and suppressed Bax expression. PTGR2 overexpression showed the opposite outcomes. Clinically, immunopathological staining showed strong PTGR2 expression in the gastric tumor portion, relative to nearby nontumor portions, and its expression negatively correlated with survival of patients with intestinal-type gastric cancer. Finally, in contrast to PTGR2-overexpressing cells, PTGR2-knockdown cells were more sensitive to cisplatin and 5-fluorouracil. Taken together, our findings not only provide functional and mechanistic evidence of the involvement of PTGR2 in gastric cancer, but also provide clinical observations affirming the significance of PTGR2 in gastric cancer and suggesting that PTGR2-target based therapy is worth further evaluation.
Subject(s)
Alcohol Dehydrogenase/metabolism , Cell Transformation, Neoplastic/pathology , Reactive Oxygen Species/metabolism , Stomach Neoplasms/mortality , Stomach Neoplasms/pathology , 15-Oxoprostaglandin 13-Reductase , Animals , Caspase 3/metabolism , Cell Death/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Transformation, Neoplastic/drug effects , Cisplatin/pharmacology , Cisplatin/therapeutic use , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Fluorouracil/pharmacology , Fluorouracil/therapeutic use , Gene Knockdown Techniques , Humans , Male , Mice , Mice, Inbred BALB C , Middle Aged , Mitochondria/drug effects , Mitochondria/metabolism , Proportional Hazards Models , Proto-Oncogene Proteins c-bcl-2/metabolism , Signal Transduction/drug effects , Stomach Neoplasms/drug therapy , Stomach Neoplasms/enzymology , Survival AnalysisABSTRACT
Filamentous phage display systems have been developed successfully to generate functional Fab antibody fragments. In this study, a recombinant Fab antibody fragment was successfully cloned from a murine monoclonal antibody 2H2 that can effectively neutralize Japanese encephalitis virus (JEV) in vitro. The recombinant Fab 2H2 antibody fragment expressed in Escherichia coli using the pComb3H phage vector resulted in a dose-dependent neutralization response using plaque reduction neutralization test. Molecular modeling of the Fab 2H2 indicated that the rational contact residues of the Fab 2H2 were targeted to the lateral surface of domain III of the JEV E protein. The combining sites of Fab 2H2 were mostly located at the variable region of the heavy chain genes. In vitro shuffling of the heavy-chain variable genes using pCom3H phage technology indicated that the sequence analysis of 10 high-affinity clones selected from the self-shuffling libraries presented no change in their amino acid sequences in 6CDRs, suggesting that the Fab 2H2 had evolved to be highly matured in the combining sites to the lateral surface of domain III. The information gained from this study may benefit the design of vaccines and therapeutic antibodies against JEV infection.