| Objective:Lymphoma is a kind of tumor of lymphoid stem cell prosoplasia and clonal proliferation.it is one of the common cancers in pediatrics with an increasing incidence.At present,traditional chemo is still the main method of therapy,and the drug resistance is one of the important factors to influence long term survival.Therefore,it is significant that exploring a more effective prevention and cure measure to improve the patient's prognosis and quality of life. Celecoxib, Nonsteroidal anti-inflammatory drugs(NSAIDs), is a selective inhibitor of COX-2 in cyclooxygenase(COXs), and it has been indicated by research that Celecoxib can inhibit numerous malignant cancers.It is common that the mechanisms of Celecoxib in resisting cancers through two pathway : COX-2 dependent pathway and COX-2 independent pathway, but the concrete mechanism was not illuminate completly yet.Recently, it has been founded that Survivin is one of the member of inhibitibor of apoptosis protein(IAP)family. As a tumor-suppressing gene, Survivin plays a control role in the apoptosis procedure of cancer cells.It has been confirmed by studies that Survivin can inhibit the apoptosis induced by some chemotherapeutics, which makes it have a hope to become the potential new target of tumor therapy. At present, little has been elucidated on the relationship between expression of Survivin and apoptosis induced by Celecoxib in lymphoma cells. In this study, in order to investigate the possible antitumor mechanism of Celecoxib in COX-2 independent pathway, we observed the effect of selective COX-2 inhibitor Celecoxib on proliferation, apoptosis, cell cycle distribution and Survivin expression in human malignant lymphoma cell line Raji.Thus, theory evidence of Celecoxib in clinical application will be provided.Methods:Human Burkitt's lymphoma cells Raji were cultivated in vitro and then treated with various concentrations of Celecoxib.Cell inhibitive rate was investigated by MTT assay.Apoptosis rate, cell cycle distribution and expression of Survivin protein in Raji cells were detected by Flow cytometry(FCM). Expression of Survivin mRNA in Raji cells was detected by RT-PCR.Results:1 MTT showed:Cell inhibitive rate of Raji after treated with various concentrations of Celecoxib for 24 hours were 9.44% , 23.08% , 55.53% , 73.00% respectively.Cell inhibitive rate of Raji after treated with various concentrations of Celecoxib for 48 hours were 29.10%,53.25%,71.86%,88.01% respectively. Cell inhibitive rate of Raji after treated with various concentrations of Celecoxib for 72 hours were 37.88%,62.92%,78.57%,93.87% respectively. Absorbance light degree in experiment groups after treated Raji cells for 24h,48h,72h were significantly different compared with control group(P<0.05), at the same time, there was a significant difference between concentration groups and time groups( P<0.05 ) .2 FCM showed:Apoptosis peak emerged significantly after Raji cells treated with Celecoxib for 48 hours.Apoptosis percentage ( AP ) were ( 9.20±0.19 ) % ,( 23.07±1.77 ) % ,( 35.66±1.05 ) % ,( 44.63±1.34 ) % respectively;there was no or only low apoptosis peak in control group,AP in control was(2.23±0.91)%. There was remarkable difference between experiment groups and control group by least significant difference test, AP were significant higher in experiment groups than that in control group. By one-way ANOVA , AP were significantly different among concentration groups(P<0.05).3 FCM showed : After Raji cells treated with various concentrations of Celecoxib for 48 hours, the cell percentage in G0/G1 phase of test groups were(58.53±1.12)%,( 64.97±0.60 ) % ,( 69.17±0.40 ) % ,( 73.83±0.35 ) % respectively;the cell percentage in S phase were(28.70±1.32)%,(23.87±0.59)%,(21.07±0.67)%,(17.17±0.49)% respectively ; the cell percentage in G2/M phase were(12.77±0.21)%,(11.17±0.35)%,(9.77±0.35)%,(9.0±0.26)% respectively. The cell percentage in G0/G1, S, G2/M phase of control group were ( 53.50±1.35 ) % ,( 32.80±1.61 ) % ,(13.70±0.40)% respectively.Compared with control group,cell cycle distribution in test groups of Celecoxib existed a significant difference(P<0.05). By one-way ANOVA, cell cycle distribution were significantly different among concentration groups(P<0.05).4 FCM showed : The FI of Survivin protein expression in Raji cells after treated with Celecoxib for 48 hours at the concentrations of 12.5μmol/L, 50μmol/L, 100μmol/L, 150μmol/L were 0.95±0.022,0.81±0.044,0.72±0.039,0.64±0.056 respectively.The FI of Survivin protein expression in control group was regarded as 1, there was no remarkable difference between 12.5μmol/L group and control group(P>0.05); however,the FI of Survivin protein expression in the other concentration groups of Celecoxib were significantly different compared with control group(P<0.05). The FI of Survivin protein expression was significantly different among concentration groups(P<0.05). 5 RT-PCR showed : The correspondent expression of Survivin mRNA in Raji cells after treated with Celecoxib for 48 hours at the concentrations of 12.5μmol/L, 50μmol/L, 100μmol/L, 150μmol/L were 0.73±0.033,0.67±0.038,0.61±0.013,0.54±0.039 respectively. The correspondent expression of Survivin mRNA in control group was 0.79±0.018, the correspondent expression of Survivin mRNA in experiment groups were significantly different compared with control group ( P<0.05 ) .The decreasing expression of Survivin mRNA existed significant difference among concentration groups(P<0.05).Conclusions:1 The survival of Raji cells was inhibited by Celecoxib in vitro with a concentration and time dependent manners from 12.5~150μmol/L.2 The number of Raji cells in G1 phase was increased,while those in S and G2 phase was decreased by the effect of Celecoxib.Celecoxib induced cell cycle arrest in G1 phase and inhibited the growth of Raji cells,which may be one of the mechanisms of COX-2 independent pathway.3 In this study, before the cells were interfered with drugs,survivin protein and survivin mRNA were highly expressed in Raji cells,which suggested that the expression of Survivin increased in Raji cells on both transcription and translation level.4 The expression of Survivin protein and Survivin mRNA in Raji cells can be downregulated by Celecoxib in vitro with a concentration dependent manner,and apoptosis in Raji cells can be induced by Celecoxib through the downregulation of Survivin expression,which may be another possible anticancer mechanism of Celecoxib in COX-2 independent pathway.
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