The Study Of Chemoreventive Effect Of Celecoxib Combined With Metformin Against DEN-induced Hepatocellular Carcinoma In Rat Model

Objective Metformin and cox-2 inhibitor Celecoxib are commonly used clinical drugs,in recent years,it has been found that the two drugs have antitumor effect,but clinical studies have shown that use of one of the drugs alone can not effectively reduce the postoperative recurrence of liver cancer.The primary objective of this study was to observe the inhibitory effect of Metformin Combined with Celecoxib on Diethylnitrosamine(DEN)induced liver in rats,Meanwhile,detected the expression of p-mTOR and p-AMPK in rat and human liver cancer tissues,and to explore the anti-tumor effect and possible mechanism of Metformin and cox-2 inhibitor Celecoxib in liver cancer.Methods 1.human hepatocellular carcinoma cell line HCCLM3 and HepG2 were cultured and each cell line was divided into 3 groups: metformin group alone(treated with 500 ?mol / L metformin),celecoxib group alone(treated with 50 mmol/L celecoxib),the combination group(metformin 500 ?mol/L +celecoxib 50 mmol/L).After treated for 48 h,MTT assay was used for the detection of viability.2.Using DEN to establish the hepatocellular caicinoma model in rats: all rats received weekly intraperitoneal injections of 50 mg/kg body weight of DEN dissolved in 0.9% NaCl solution over a period of 18 weeks.Ninety-eight male Sprague Dawley rats were selected,10 rats were randomly selected as normal group,and no special treatment was taken during the experiment.The remaining eighty-eight rats were randomly divided into 4 groups:control group(DEN group,N=22),were intragastric administration with normal saline(3ml/d);DEN induced hepatocellular carcinoma+metformin group(Metformin group,N=22)were only intragastric administration with metformin(300 mg/kg/d);DEN induced hepatocellular carcinoma+celecoxib group(Celecoxib group,N=22)were only intragastric administration with celecoxib(100 mg/kg/d);DEN induced hepatocellular carcinoma+metformin+ celecoxib group(Combined treatment group,N=22)were intragastric administration with celecoxib(100 mg/kg/d)and metformin(300 mg/kg/d).Body weights and physiological status were monitored weekly through out the experimental period.After18 weeks of the treatments,All the rats were killed under anesthesia by pentobarbital.The livers were excised and prepared for hematoxylin-eosin staining(HE-staining)and immunohistochemistry.The protein expression of p-AMPK ? p-mTOR was examined by immunohistochemistry.3.We collect 35 cases of paraffin embedded specimens with radical hepatic resection of HCC patients in the Affiliated Tumor Hospital of Guangxi Medical University.The expression of p-AMPK and p-mTOR was determined in 35 specimens of hepatocellular carcinomas and 7 of the adjacent tissues by immunohistochemistry.Results 1.Compared with the single-drug group,the cell viability of the combination group was significantly decreased.2.After18 weeks of the treatments,in a total of 77 rats were sacrificed under anesthesia by pentobarbital:19 rats in DEN group,18 in celecoxib group,14 in metformin group,16 in combination group and 10 in normal group.After dissection,the liver tissue of normal group was bright red with soft texture and smooth surface.However,the rats in the experimental group showed that some of the livers were hard and narrow,with uneven surface and dark red color,a large single tumor or multiple tumors and some granular sclerosis nodules were visible.Combined the results of gross anatomy and HE staining,The final tumor formation rate of rats are as follows: control group(DEN)was 63.2%,the metformin group was 42.9%,celecoxib group was 38.9%,the combination group was 18.8%,the tumor formation rate of the combination group was the lowest,There was a significant difference in the difference between the groups(p < 0.05).3.The effect of combined medication on p-mTOR and p-AMPK in DEN induced liver cancer rats: the immunohistochemical results showed that the positive expression rates of p-mTOR in group DEN,celecoxib group,metformin group and combination group were 61.10%,53.2%,41.7% and 35.7% respectively,while the positive expression rates of p-AMPK were 33.3%,53.3%,66.70% and 71.4%.It could be seen that the positive expression rate of p-mTOR in liver tissue of combined group was the lowest,while that of p-AMPK was the highest.4.The immunohistochemical results of the cancer tissues and adjacent tissues of HCC patients showed that the positive rate of p-mTOR protein expression in the liver cancer was higher than that in the adjacent tissues,and the positive rate of the low differentiated hepatocellular carcinoma was higher than that of the high and medium differentiated hepatocellular carcinoma;The positive rate of p-AMPK protein in the liver cancer tissues was lower than that in the adjacent tissues,and the positive rate of the high and medium differentiated hepatocellular carcinoma was higher than that of the low differentiated hepatocellular carcinoma.Conclusion 1.COX-2 Inhibitor Celecoxib combined with metformin can inhibit the proliferation of hepatoma cells.2.COX-2 inhibitor,celecoxib combined with metformin,can inhibit the occurrence of DEN induced liver cancer in rats.The mechanism may be related to the activation of AMPK protein and the inhibition of mTOR protein.