Nuclear factor-kappaB (NF-kappaB) is a transcriptional regulator of inducible expression of genes including cyclooxygenase-2 (COX-2), regulating cell proliferation. NF-kappaB is kept silent in the cytoplasm via interaction with the inhibitory protein I kappaB alpha and transmigrated into the nucleus upon activation. However, constitutive NF-kappaB has been found in the nucleus of some cancer cells. We investigated the role of NF-kappaB 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-kappaB subunit p50, or they were transfected with a mutated I kappaB alpha 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 E-2. mRNA expression for COX-2, and protein levels for p50, I kappaB alpha, and COX-2 were determined by reverse transcription polymerase chain reaction and Western blot analysis. The NF-kappaB levels were examined by electrophoretic mobility shift assay. Thromboxane B-2 (TXB2) and 6-keto-prostaglandin F-1 alpha (6-keto-PGF(1 alpha)) levels were determined by enzyme-linked immunosorbent assay. Cell proliferation was assessed by viable cell counting, [H-3] thymidine incorporation, and colony formation. The nuclear level of p50 decreased in AGS cells treated with AS ODN. The I kappaB alpha mutant was observed in cells transfected with the mutated I kappaB alpha gene. NF-kappaB was inhibited in cells treated with AS ODN or transfected with the mutated I kappaB alpha 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-PGF(1 alpha) were inhibited in cells treated with AS ODN or transfected with the mutated I kappaB alpha gene, which had lower NF-kappaB 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-PGF(1 alpha), in a dose-dependant manner. Prostaglandin E, prevented the inhibition of proliferation in cells treated with AS ODN or transfected with the mutated I kappaB alpha gene. In conclusion, NF-kappaB mediates COX-2 expression, which may be related to cell proliferation, in human gastric cancer cells.
