Effect Of Proliferation And Apoptosis On Raji Cell By Celecoxib And Adriamycin And Its Underlying Mechanisms

Objective:Nonsteroidal anti-inflammatory drugs(NSAIDs)have been implicated in the chemoprevention of numerous malignanciesin Cluding colon,breast,and lung cancer.The effects of NSAIDs are thought to be through the cyclooxygenase(COXs)dependent pathway and COXs independent pathway.In this study,to elucidate the possible antitumor mechanism of NSAIDs,we used human Burkitt's lymphoma Raji cells to investigate the effects of proliferation and apoptosis in Raji cells treated by a selective COX-2 inhibitor celecoxib and in combination with adriamycin.The expression of caspase-9 would be examined too.Methods:Raji cells cultured in vitro were divided into control group, celecoxib group,adriamycin group,celecoxib+adriamycin group.Flow cytometry and microscopy were used to demonstrate apoptotic morphological changes in treated cells.Proliferation of Raji cells were evaluated by MTT assays,apoptosis percentage(AP)was assessed by flow cytometry(FCM).The expressions of caspase-9mRNA were detected by RT-PCR.Caspase-9 protein was detected by immunohistochemistry.All the statistical analyses were performed using SPSS 13.0 software.The data are expressed as means±+S.E.M.P＜0.05 was considered significant.Results:Celecoxib induced Raji cell towards apoptosis verified by celluar morphology.Cell viability for adriamycin(0.1μg/ml)at 24h was (93.41±3.16)%.Cell viability for celecoxib at diferent concentration(20, 40,60,80,100μM)at 24h was(93.56±2.08)%,(86.16±2.27)%, (63.92±3.17)%,(23.21±2.35)%,(18.27±1.36)%respectively,which decreased campared with control group(P＜0.05).After treated Raji cells with adriamycin(0.1μg/ml)in combination with celecoxib for 24h,cell viability was(80.58±1.95)%,(64.28±1.55)%,(41.62±1.79)%, (12.64±2.45)%,0%respectively.The experiment group of combination decreased cell viability significantly campared with used them alone (P＜0.05).In apoptosis rate detection experiment by flowcytometry,after Raji cells was treated with celecoxib at different concentration at 24h was (5.01±0.80)%,(14.00±1.68)%,(34.33±4.47)%,(57.63±2.27)%,(67.50±2.00)%,respectively,except the 20μM concentration group which increased compared with control group(3.50±1.18)%(P＜0.05).The apoptosis rate in adriamycin(0.1μg/ml)and celecoxib(60μM)+ adriamycin(0.1μg/ml)was(7.62±1.23)%,(48.07±2.32)%,respectively. The combination of celecoxib with adriamycin induced cell apoptosis to a greater degree than either agental one(P＜0.05).There is no caspase-9 mRNA expression in control group for 24 hours.The lever of caspase-9 mRNA of Raji cells treated with celecoxib at different concentrations was 0.13±0.04,0.23±0.02, 0.34±0.14,0.48±0.04,0.60±0.01,respectively,which was significantly higher than that of the control(P＜0.05).The lever of caspase-9 mRNA in cells treated with celecoxib(60μM)at 48h and 72h was 0.41±0.14, 0.51±0.01,respectively,which higher than that of 24h(P＜0.05).After Raji cells was treated with celecoxib(60μM)in combination with adriamycin(0.1μg/ml),the expression of caspase-9 mRNA was 0.47±0.02 higher than celecoxib(60μM)or adriamycin(0.1μg/ml)alone(P＜0.05).There is no caspase-9 protein expression in control group for 24 hours.The expression of caspase-9 protein of celecoxib group was (6.75±1.57)%,(19.00±1.41)%,(26.00±1.41)%,(38.50±2.12)%,(52.00± 2.83)%,respectively,each was higher than that of the control(P＜0.05). The caspase-9 protein expression in adriamycin group was(6.23±1.12) %.After Raji cells was treated with celecoxib(60μM)in combination with adriamycin(0.1μg/ml),the expression of caspase-9 protein was (37.50±3.54)%higher than celecoxib(60μM)or adriamycin(0.1μg/ml) alone(P＜0.05).Conclusions:1.Celecoxib could inhibit cell proliferation in a dose-dependent and time-dependent manner in Raji cells.2.Celecoxib could induce Raji cells apoptosis by up-regulating caspase-9 expression,and the effect was dose-dependent.3.Combination of celecoxib with adriamycin could enhance the effect of inhibiting cell proliferation and inducing cell apoptosis.