Investigation Of The Roles Played By COX-2 In Pathogenesis Of Gastric Cancer

The current studies were designed to clone the cDNA sequence of human COX-2 encoding gene and to construct its sense, antisense eukaryotic expression vectors. By using gene transfer, COX-2 expression level was to be changed over in human gastric cancer cells that previously had either high or low expression level of COX-2. Some specific mechanisms through which COX-2 played roles in tumorigenesis of gastric cancer were discussed by observing the changes of biological behaviors, corresponding signal moleculars and signal transduction pathways of the gastric cancer cells after gene transfer. The main methods and results are:1. Human COX-2 cDNA was cloned with RT-PCR strategy by using RNA extracted from gastric cancer cells with high expression of COX-2. The PCR product was testified by DNA sequencing.2. The sense, antisense eukaryotic expression vectors pcDNA3.1/hCOX2(+) and pcDNA3.1/hCOX2(-) were constructed by directional cloning the target gene into eukaryotic expression vector pcDNA3.1(+). The direction was confirmed by endonuclease digestion.3. Using liposome mediated method, the COX-2 highly exprssed humangastric cancer cell line SGC7901 was transfected with the antisense recominant vector and control plasmid. Stable clones 7901-AS and 7901-P were obtained after G418 screening. Immunocytochemical studies and RNA dot blotting assay testified that the protein and mRNA level were both down-regulated in the transfected 7901-AS cells.4. Cell proliferation of 7901-AS was inhibited compared with SGC7901 cells by using MTT assay.5. Tumorigenesis assay in vivo showed that: 30 days after implantation, themean tumor weight (mg) of each nude mice group were (x ?s): 826.67?77.67 (SGC7901); 776.67 ?300.06 (7901-P); 486.67 ? 15.28(7901-AS). Statistics showed a significant growth suppresion ofantisense vector transfected cells (p<0.01).6. PGE2 in supernatant were detected: PGE2 produced by the three cell strains were ( ug /L) : 18.23+0.85 (SGC7901); 17.97 + 1.04 (7901-P); 12.9 ?0.92 (7901-AS). Transfection with COX-2 antisense vector significatly (p<0.01) decreased the PGE2 level of SGC7901 cells.7. Detection of protein kinase activity showed: in per ug cell extract, the activities of total PKA and active PKA were 0.207, 0.116pmol/min (SGC7901) and 0.214, 0.024 pmol/min (7901-AS). The antisense RNA transfected cells had a similar total PKA and a significant lower active PKA activity than the untransfected control cells.8. Electrophysiological studies indicated that both SGC7901 and 7901-AS cells had a typical delayed rectifier K+ curent. In same conditions (held in -40m V and pulsed between -80- +80mV with 20mV step, pulse duration 800ms with Is interval, pulse frequence 0.2Hz), Ik wassignificant lower in transfected cells under each test potential (p<0.01).When SGC7901 cells were treated with indomethacin (10nM), Ik wasalso lowered and entirely recovered after washing. These implied that theexpression and activity of COX-2 significantly related to the delayedrectifier K+ channels/currents. 9. Both inhibitors of K+ channels TEA and 4-AP could dose-dependantlyinhibit the growth of SGC7901 cells when they were administrated to thecultured cells with different dosage.All these results indicated that: the abnormal high expression of COX-2 in human gastric cancer cell line SGC-7901 was related to the malignant biological behaviors of cancer cells. PGE2, and PKA might participate in the signal transduction of tumorigenesis of gastric cancer induced by COX-2. The expression and activity of COX-2 significantly related to the delayed rectifier K+ channels. Its catalysed product prostaglandins might modulate the activity of this cannel by stimulating PKA or PKC pathway through one or more second messenger and then affect the biological behaviors of cells. Inhibition of the activity of either COX-2 or delayed rectifier potassium channels could inhibit the proliferation of tumor cells. These results were help...