| Objective To investigate the effect of combined use of COX-2 Inhibitor Celecoxib and cisplatin and fluorouracil,a commonly used chemotherapeutic agent,on the biological behavior of gastric cancer cell line SGC-7901 and the possible molecular mechanisms involved.Methods Using immunohistochemical method(IHC)to detect expression of COX-2 in gastric cancer and paracancerous tissues;MTT was used to detect the effect of celecoxib(Ce)and cisplatin(DDP)and fluorouracil(5-Fu)effects of single or combination on the proliferation of gastric cancer cells;apoptosis by flow cytometry to detect the gastric cancer cells using AnnexinV-PE and 7-AAD staining;the expression of COX-2,Caspase-3,Survivin,Mcl-1,MDR1,mRNA between before and after qRT-PCR gastric cancer cells were detected.Results1.The expression of COX-2 1 in gastric cancer and paracancerous tissues:immunohistochemistry showed that in 46 cases of gastric cancer tissues and corresponding para carcinoma,the positive expression of COX-2 strong gastric cancer accounted for 10.87%(5/46),the positive expression was 21.74%(10/46),weakly positive expression accounted for 63.04%(29/46),negative expression accounted for 4.35%(2/46),and the corresponding paracancerous tissues and there were no positive and positive,weak positive expression was 21.74%(10/46),negative expression accounted for 78.26%(36/46).The expression of COX-in gastric cancer tissues was significantly higher than that in adjacent tissues,and the difference was statistically significant(P<0.05)2.The relationship between 2.COX-2 expression and clinicopathological features of gastric cancer: further analysis of the expression of COX-2 in gastric carcinoma,COX-2 expression and tumor differentiation,lymph node metastasis,TNM staging and tumor size;but the patients with gastric cancer by gender,age and tumor location,whether associated with vascular invasion and tumor invasion independent factors.3.The proliferation of gastric cancer cells in each group before and after the drug: MTT test results showed that different concentrations of Ce,DDP and5-Fu can inhibit the proliferation of gastric cancer cells,and the inhibition effect increases with the increase of drug concentration.The half inhibitory concentration(IC50)of Ce,DDP and 5-Fu were calculated to be92.04±4.93umol/L,10.43 ± 0.27mg/L and 156.56 ± 2.07mg/L.In further study,we average inhibitory concentration by half of the previously calculated dose for treatment of gastric cancer cells were showed in DDP+Ce group compared with DDP group,24 h,48h and 72 h cell proliferation inhibition rate were increased,respectively 59.66%vs46.88%,67.05%vs50.02% and 63.77%vs52.17%;5-Fu+Ce group compared with 5-Fu group,the inhibition of these at different timepoints were also significantly increased,respectively 55.58%vs39.23%,62.84%vs47.62% and 59.00%vs51.19%.Ce+DDP group and Ce+5-Fu group compared with the corresponding single drug group,the difference was statistically significant(P<0.05).4.The apoptosis of gastric cancer cells in each group before and after the drug flow cytometry found in half inhibition dose 48 h concentration in gastric cancer cells,apoptosis in blank control group,DDP group,5-Fu group,Ce+DDP group and Ce+5-Fu group respectively was 7.10% ± 0.30%,46.53% ±1.50%,39.17% ± 1.10%,66.93%±0.70% and 62.13% ± 1.60%,the drug group cells apoptosis rate was obviously higher than the control group,the difference was statistically significant(P<0.05),Ce+DDP group and Ce+5-Fu group compared with the corresponding single drug group,the apoptosis rate was significantly increased,the difference was statistically significant(P<0.05).5.Drug effects before and after gastric cancer cell groups COX-2,Caspase-3,survivin,Mcl-1 and MDR1 mRNA expression: qRT-PCR results showed that the drug dose effect of half inhibitory concentration of 48 h,compared with DDP group,5-FU group,Ce+DDP group and Ce+5-Fu group with the control group,the expression of COX-2,survivin,Mcl-1 and MDR1 mRNA were decreased,and the Caspase-3 was significantly increased,the difference was statistically significant(P<0.05),and Ce+DDP group and Ce+5-Fu group and single drug group(DDP group and 5-FU group)compared to the expression of COX-2,survivin,Mcl-1 and MDR1 mRNA were significantly decreased,the expression of Caspase-3 increased,the difference was statistically significant(P<0.05).Conclusion1.The high expression of COX-2 in gastric cancer tissues,and the expression of differentiation and lymph node strength and the metastasis of gastric cancer,TNM staging and tumor size;but the patients with gastric cancer by gender,age and tumor location,whether associated with vascular invasion and tumor invasion depth and other factors.2.Compared with chemotherapy,cisplatin and 5-fluorouracil combined with celecoxib can further promote gastric cancer cell apoptosis,inhibit cell proliferation,increase drug sensitivity,and then enhance the efficacy of chemotherapy drugs.The mechanism of increased apoptosis may be related to the downregulation of Caspase-3 expression and the downregulation of Survivin and Mcl-1 expression after COX-2 downregulation,while the increase of drug sensitivity may be related to the inhibition of MDR1 expression after down regulation of COX-2. |
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