Study On The Effects Of NS398 On CTL To Ovarian Cancinoma Cell Line CAOV3 In Vitro

ObjectiveTo investigate the effects of COX - 2 specific inhibitor NS398 on proliferation of ovarian carcinoma cell line CA0V3 and on the immune killing effect of CTL cells in vitro.Materials and MethodsMaterialsHuman ovarian serous carcinoma cell line CA0V3 provided by Laboratory of Cell Biology of China Medical University.ReagentsNS398 (Cayman Company, USA) , mitocin C, separating medium of lymphocyte , interleukin - 2 ( IL - 2 ) , Anti - CD3 mono - antibody labeled with FFTC and Anti - CD8 mono - antibody labeled with PE ( Zhongshan biological Company Limited, Beijing) , MTT, Trypan Blue and DMSO (Sigma Company, USA) , PGE₂ Radioimmunoassay kit ( provided by Hematological Institute of Suzhou Medical College).Methods1. The effect of NS398 on CA0V3 growth suppression: Detection of growth velocity of CAOV3 and CA0V3 treated by NS398 with cytometry, and draw cellular growth curves.2. Detect PGE₂ contents were detected in supernatant fluid of culture medium of CA0V3 and CAOV3 treated by NS398 by radioimmunoassay.3. Ovarian carcinoma CAOV3 cells were stimulated in vitro and inducedspecific Cytotoxic T Lymphocytes ( CTL). Phenotypes of CTL were analyzed with flow cytometer.4. Detection of CTL killing activity: killing activity of CTL cells were detected with MTT chromatometry.5. Observation of cell morphology.6. Statistical analysis: Experimental data were analyzed with SPSS 10. 0 software package. Data were expressed as x s. t text was used in comparison of two groups, and variance analysis and Dunnett t test were used in comparison a-mong multiple groups.Results1. Cell growth curve showed that cellular amount of experimental group was fewer than control group, the growth speed became slower prominently and live cells decreased with the increase of drug concentration and the extensive of drug reactive duration ( Fig. 1).2. Morphologic changes(1) Compared with control group, the density of CAOV3 cells of experimental group decreased obviously after treated with NS398, cellular volume became small and round, intercellular space widened and particles increased in the cells with obvious " vacuoles" structure seen in it . (picture 1 ²).(2) CTL cells formed after activation and differentiation of T lymphocytes were mainly large lymphocytes which were round in shape with hyaline cytoplasm and colony growing. ( picture 3).3. Radioimmunoassay showed that PGE2 content in CAOV3 culture medium of experimental group was lower than that in control group with statistical significance (p<0.05) (Fig.2).4. When IL -2 existed, peripheral blood mononuclear cells (PBMC) were induced to produce specific CTL successfully after repeatly stimulated by CA-0V3 cells or CAOV3 cells treated by NS398, whose percents of expressing of CD3 ⁺ CD8 + cells were 41% and 64% respectively, but only 25% in PBMC not received antigen stimulation ( Fig. 3).5. MTT chromatometry to detect CTL killing activity showed that CTL cells had prominent killing activity to CA0V3, and the killing activity of CTL to CA-OV3 in experimental group was higher than that of control group ( p <0.05 ).DiscussionCyclooxygenase is an important restriction enzyme, which has two isoenzymes at least in mammal; COX - 1 and COX — 2. COX - 1 is regarded as " house - keeping gene" which can expressed in most normal cells, while COX- 2 is regarded as " rapid reactive gene" and its expression is upregulated in pathologic state, and produce prostaglandins which can response to inflammation and mitogen stimuation. COX -1 keeps the normal physiological function of the body such as protection of gastric mucosa, keep blood flow of kidney, etc. COX- 2 is synthesized rapidly after various stimulations and involved in kinds of pathologic and physiological procedures including inflammation and tumor genesis.Immune surveillance exists in the body and plays an important role of anti- oncogenesis. Cellular immunity is a key to resistant tumor genesis. CTL can kill tumor cells with specificity. Tumor occur under the immune activity of the body because tumor cells can escape the immune surveillance. Studys showed that highly expressed COX - 2 can cause the infiltration of tumor cells in micro-environment , and the defect of phenotype and function of dendritic cells which lead to decrease of antigen presentation function and have an influence on specific CTL to tumor cells, contributing to the immune escape of tumor cells.We used specific COX - 2 inhibitor NS398 to treat ovarian carcinoma cells, PGE₂ secretion and tumor cell proliferation were inhibited. Killing activity of CTL induced by CA0V3 treated by NS398 enhanced prominently. Increase of secretion of PGE₂ by tumor cells could affect the function of APC antigen presentation and tumor immune of the body. These results suggested that COX - 2 inhibitor NS398 is of immune regulation and enhance the activity of CTL to tumor cells, which is useful to reverse tumor local immune escape partially. It was consistent with Yangs investigation, in which COX - 2 play roles through its me-...