Inhibitive Effect Of Non-steroidal Anti-inflamma-tory Drugs On Human Ovarian Epithelial Adenocarcinoma Cell Line CAOV3

The incidence of orarian malignant tumor occupied the third among the malignant tumor of female genital organs. Due to its difficulty in early diagnosis and poor treatment effect of late stage , ovarian cancer's 5 - year survival rate remains 25% -30%. Therefore, there is increasing urgency to develop new and effective strategies to prevent and treat ovarian cancer.Epidemiological, clinical, animal and laboratory studies have all provided evidence for eicosanoids in the tumorigenesis and development. Cyclooxygenase (COX) is the key enzyme in the conversion of eicosanoids to prostaglandins (PGs). Two isoforms of COX have been identified, namely COX - 1 and COX -2. COX - 1 is the constitutive gene and regulate normal activity of human body, such as stomatch mucosa protection and platelet agglutination; whereas COX -2 is inducible gene. Cytokines, growth factors, oncogenes, stress stimulating and mitogen have been found to induce COX - 2 up - expression . The common site activated by those factors can activate another one or more MAPK route, which means COX - 2 route might be activated by MAPK route. Studies in recent years have founded that COX - 2 played a key role in tumourigenesis and development. PGE2, a major COX - 2 derived product, has been reported to stimulate the tumor cell growth and inhibit its apoptosis. There are considerable reasons to believe that it could be reduced the production of PGs through inhibiting COX in order to inhibit the tumor cell proliferation and promote the ap-optosis. Therefore, It is important to reduce the production of the PGs in prevention, treatment and prognosis.Non - steroidal and - inflammatory drugs ( NSAIDs ) are COX inhibitors, It can be divided into three categories based on the difference of inhibition role in COX isomorides: COX -1 selective inhibitors, COX - 2 selective inhibitors, COX non - selective inhibitors. Studies have showed the potential of the NSAIDs for anticancer . It can inhibit tumor cell proliferation and induce its apoptosis. Although the precise mechanism for the anticancer effects of NSAIDs are unknown , many studies have showed NSAIDs might play anticancer role by inducing cell cycle arrest and apoptosis and inhibiting the angiogenesis, tumor infiltration and metastasis through different signal conduct route.NS398 is selective inhibitor of COX - 2, which can inhibit the carcinogen-sis and development through inhibiting COX - 2 and avoid side effects caused by non - selective cox inhibitor by inhibiting cox - 1, therefore it has good fortune. Recent studies have showed NS398 inhibit tumor cell growth not only by cox route but also by other route. Our previous studies showed NS398 can inhibit o-varian cancer cell line CAOV3 proliferation and induce CAOV3 apoptosis, but the precise mechanism is unknown. So in our studies we investigate the inhibiting mechanism of selective COX - 2 inhibitor NS398 and non - selective cox inhibitor indomethacin to human ovarian cancer cell line CAOV3 and provide laboratory proof for NSAIDs treating ovarian cancer.Methods1. MTT assay: To observe the proliferation of CAOV3 cell under the treatment with NS398 and indomethacin.2. FCM Assay for Analysis of DNA content: To observe the change of CA-0V3 cell cycle under the treatment of NS398 and indomethacin.3. RT - PCR assay: To observe the expression of COX -2 mRNA before and after NS398 and PD98059 treatment by semiquantitative method4. Western - bloting assay :To observe the expression change of COX -2, cyclinDl,cyclinE,p34,bcl -2,bax,erk,erk -p proteins of CAOV3 cell beforeand after treatment with NS398 and indomethacin. To observe the change of expression of COX -2 induced by EGF treated by MAPK inhibitor PD98059.5. Radioimmunity assay: To observe the change of PGE2 releasing of CAOV 3 cell treated by NS 398.6. DNA ladder electrophoresis: To observe the apoptosis of CAOV3 cell treated by indomethacin.7. Statistical Methods.Statistical analysis was performed using SPSS for windows 10. 0 software. The results of human ovarian cancer cell line CAOV3 treated with NS398 and indomethacin was expressed as mean ± s. And comparisons of means were carried out by one way ANOVA - Dunnett t test; different with a value of P <0.05were considered statistically significant.ResultsThe results of MTT assay indicated that NS398 and indomethacin induced significant growth inhibition in CAOV3 cell line in a time - dependent and dose - dependent manner. In the 50 p,mol/L group of NS398 and 100 μmol/L group of indomethacin, a marked decrease in the cell numbers were observed after treatment for 48 hours and 24 hours respectively; and the inhibition rate was increasing along with the concentration increasing. Compared with the control group ( NS398 and indomethacin are 0 μmol/L) , at 48,72 hours in 50 μmol/L NS398 group and at 24,48,72 hours in the 100 μmol/L indomethacin group , the inhibition of cell proliferation is marked ( p <0.05 ) and appears good time -dependent and dose - dependent.2. The results of FCM assay for analysis of DNA content showed that the percentage of cells in G0/G1 stage increased significantly following 72 hours of treatment with 100 |xmol/L NS398 (p <0.05) companied with the concomitant decreasing of the cells in S, G2/M stage ( p < 0.05 ) and increase of hypodiploid cells (p <0.05). This indicated the G0/G1 arrest after treatment with NS398 and induced apoptosis. While the percentage of cells in G0/G1 stage increased more significantly following 72 hours of treatment with 100 μmol/L indomethacin( p <0.001) companied with the concomitant decreasing of the cells in S ( p < 0.001) and no hypodiploid cells peak. This indicated the G0/G1 arrest after treatment with indomethacin.3. RT - PCR semiquantitative analysis showed in human ovarian cancer cell line CAOV3, the COX -2 mRNA expression decreased under NS398. EGF can induce the expression of COX - 2 mRNA and PD98059 can inhibit the expression of COX -2 mRNA induced by EGF.4. The results of Western - bloting assay showed that following 72 hours of treatment with NS398(50,100 |xmol/L) ,the expression level of COX -2 protein and ERK which is main kinase in MAPK cell signal conduct route decreased in human ovarian cancer cell line CAOV3 compared with the control group, and so did phosphorylated ERK and main cell cycle regulatory protein cyclinD1,cy-clinE. The expression of apoptosis - related protein bcl - 2 deceased and bax increased apparently. EGF increased the expression of COX - 2 protein of CA-OV3. PD98059 can inhibit the expression of COX -2 protein of CAOV3 induced by EGF. That indicated that NS398 inhibit CAOV3 cell not only by COX - 2 route but also by MAPK route. And MAPK route might be upstream route of cox. Following 72 hours of treatment with indomethacin(50,100 jxmol/L) ,the expression level of COX - 2 protein and ERK which is main kinase in MAPK cell signal conduct route decreased compared with the control group, and so did phosphorylated ERK and main cell cycle regulatory protein cyclinD1,cyclinE, p34. The expression of apoptosis - related protein bcl - 2 and bax has no change.5. Radioimmunoassay result showed the PGE2 releasing amount decreased apparently along with the increasing concentration of NS398, and the difference is marked compared with the control group ( p <0.05 ). This indicated the activity of COX - 2 was inhibited.6. DNA ladder electrophoresis: The results of CAOV3 cell DNA electropho-resis revealed the non - specific ladder bands following 72 hours of treatment with indomethacin(100 μmol/L) .