Inhibitory Effects Of Cyclooxygenase-2 Specific Inhibitor On Human Osteosarcoma, Lung Cancer And Breast Cancer Cell Lines

Rofecoxib and celecoxib are two kinds of non-steroid anti-inflammatory agent that specifically inhibits cyclooxygenase-2(Cox-2), a critical enzyme in the conversion of arachidonic acid(AA) to prostaglandin E2. These drugs have antineoplastic effect according to the recently study except analgesic and anti-inflammatory effects and also partly the chemopreventive effects. Epidemiology and animal experiment confirmed that cyclooxygenase-2 specific inhibitor can decrease incidence rate of tumor and prevent tumor. Moreover, Cox-2 inhibitor can significantly attenuate recurrence and systematic metastasis of lung cancer. In this study, we observe the growth of three kinds of tumor cell lines after using rofecoxib and celecoxib, then, detect the apoptosis rate by FCM, so as to approach the negative mechanisms.Objective:To investigate the selective Cox-2 inhibitor depress proliferation of human osteosarcoma, breast and lung cancer cell lines and the difference between rofecoxib and celecoxib through the study of antitumous effect and the influence of cell cycle and apoptosis rate, so as to provide theoretic basis for the clinical application for tumorous prevention and cure.Methods: 1. Cell culture Tumor cell lines hos-8603 was incubated in Dulbecco's modified Eagle's medium(DMEM)supplemented with 10%(v/v)fetal calf serum(FCS), 100units/ml of penicillin, and 10 mg/ml of streptomysin . A549 and MDA-MB-543S was incubated in RPMI1640 supplemented with 10%(v/v) newborn calf serum. All the cells were maintained in humidified, 5%CO2, 95% air incubator at 37℃. 2. Detect the inhibition effect of Cox-2 inhibitor by MTT assay. The cells were plated in 96-well dishes and cultured. After they adhered, various concentrations of rofecoxib and celecoxib were added to 96-well dishes. Cells were incubated and examined at 36,60 and 84h.A 10μl aliquot of the 5mg/ml stock solution of MTT was added to each well,and incubate 4-6h. The optical density at 570nm was measured using a microplate reader 96-well multiscanner. 3. Cell cycle analysis Cell were treated with 20uM celecoxib and rofecoxib for 84h. Treated and control untreated cells were released from the substratum using passage-ease and fixed in 70% ethanol. Propidium iodine(PI) was added for 60min at 4℃, then, the cell were analyzed using FCM.Result: Cyclooxygenase-2 specific inhibitor had a growth inhibitory effect on three kinds of tumor cell in vivro when the concentration is higher than 2x 10'2uM. The inhibition effects grow more and more with the increasing concentration. There is a significant difference between treated groups and control groups. Moreover, celecoxib was more inhibitorily higher than rofecoxib. Both celecoxib and rofecoxib exhibited significant growth inhibition in three tumor cells with 2 and 20uM at 36,60 and 84h incubation. The best inhibitory time is 84h. The cells treated with Cyclooxygenase-2 specific inhibitors showed G2/M arrest. Phenomenon of apoptosis is apparent in osteosarcima.Conclusion: Cyclooxygenase-2 specific inhibitors have significant effect on the proliferation of three kinds of cell line. The inhibition effects showed a dose-dependence and time—dependence manner. CI50 is constant between 20—2uM after 60 and 84h. The inducing-apoptosis effect is relevant with cell cycle G2/M arrest and caryocinesis inhibition. Celecoxib was more inhibitorily higher than rofecoxib. Cyclooxygenase-2 specific inhibitors maybe became one of the important medicines in the treatment and metastasis inhibition of tumor.