Nuclear factor-κB regulates cyclooxyoenase-2 expression and cell proliferation in human gastric cancer cells

Abstract

Nuclear factor-κB (NF-κB) is a transcriptional regulator of inducible expression of genes including cyclooxygenase-2 (COX-2), regulating cell proliferation. NF-κB is kept silent in the cytoplasm via interaction with the inhibitory protein lκBα and transmigrated into the nucleus upon activation. However, constitutive NF-κB has been found in the nucleus of some cancer cells. We investigated the role of NF-κB in COX-2 expression and cell proliferation in human gastric cancer AGS cells. AGS cells were treated with antisense oligodeoxynucleotide (AS ODN) or sense oligodeoxynucleotide (S ODN) for the NF-κB subunit p50, or they were transfected with a mutated lκBα gene (MAD-3 mutant) or a control vector, pcDNA-3. AGS cells were treated with COX-2 inhibitors such as indomethacine and NS-398 or prostaglandin E2. mRNA expression for COX-2, and protein levels for p50, lκBα, and COX-2 were determined by reverse transcription polymerase chain reaction and Western blot analysis. The NF-κB levels were examined by electrophoretic mobility shift assay. Thromboxane B2 (TXB2) and 6-keto-prostaglandin F1α (6-keto-PGF1α) levels were determined by enzyme-linked immunosorbent assay. Cell proliferation was assessed by viable cell counting, [3H] thymidine incorporation, and colony formation. The nuclear level of p50 decreased in AGS cells treated with AS ODN. The lκBα mutant was observed in cells transfected with the mutated lκBα gene. NF-κB was inhibited in cells treated with AS ODN or transfected with the mutated lκBα gene, compared with the cells treated with S ODN or transfected with control vector. Cell proliferation, mRNA expression and protein level of COX-2, and production of TXB2 and 6-keto-PGF1α were inhibited in cells treated with AS ODN or transfected with the mutated lκBα gene, which had lower NF-κB levels than cells treated with S ODN or transfected with control vector. COX-2 inhibitors suppressed cell proliferation and production of TXB2 and 6-keto-PGF1α, in a dose-dependant manner. Prostaglandin E2 prevented the inhibition of proliferation in cells treated with AS ODN or transfected with the mutated lκBα gene. In conclusion, NF-κB mediates COX-2 expression, which may be related to cell proliferation, in human gastric cancer cells.