The Anti-tumor Effect And Mechanisms Of Berberine On H22 Hepatoma Cell

Liver cancer is one of a common cause of cancer death,death rates in our country ranked third only to gastric cancer and lung cancer,and the incidence rate has trend to high.Currently,Radiotherapy and Chemotherapy remain the one of the principal means of liver cancer in clinical treatment ,but there have shortcomings of toxic andeasy to resistance.Therefore,developing new effective and low toxic drugs are important.Recent studies show that the key enzyme COX-2 in AA metabolic pathway is closely related with tumor development.Research reports,COX-2 has increased expression in liver cancer,colorectal cancer,pancreatic cancer,breast cancer,prostate cancer,lung cancer and so on, selective COX-2 inhibitors reduce PGE2 by inhibit COX-2 expression, inhibit tumor cell growth and reproduction. PGE2 can promote cell proliferation,promote angiogenesis and promote tumor cell metastasis. In addition to COX-2, cPLA2 is anther key enzyme in AA metabolic pathway.In the process of tumor,abnormal apoptosis is a key factor in tumor formation,mitochondria is an important place in apoptosis.Mitochondrial membrane permeability can mitochondrial Cyto c and AIF release.Cyto c activated caspase-3 and cutting a variety of structural protein and function of cell protein Leading to the disintegration of cells in the form of apoptosis,that is the classic way to induce apoptosis by caspeas dependent way.AIF is a apoptosis-inducing factor,that is present in the mitochondrial membrane space in flavoprotein.When the transfer of AIF from mitochondria to cytoplasm and enter nucleus cause chromatin and large fragment of DNA breakage,leading to apoptosis.Berberine is extracted from buttercup coptis rhizome that is a quinoline alkaloid,the main clinical and intestinal detoxification for heat treatment and infectious diseases.Berberine has been found to have significant activity against multiple tumor.Objective:By culturing the human hepatocarcinoma cell line of H22 in vitro and implanting it in mouse in vivo, the present study was designed to investigate the anticarcinogenic effect of Berbenine and its correlation of cPLA2,COX-2,caspase-3,caspase-9 and AIF; and furthermore to discuss the possible mechanisms and settle theoretical and methodological basis to the clinical application of Berbenine.Methods:Inhibition effects of Berbenine on growth of H22 cell line were evaluated by MTT; Effects of Berbenine on the weight and size of the solid tumors in the mice, which were implanted with H22 cell line, were observed; expressing level of cPLA2,COX-2,caspase-3,caspase-9 and AIF in H22 cells treated by celecoxib was evaluated by the Western Blot method.Results:1. Inhibition effects of Berbenine on the H22 cell line cultured in vitro: after injected H22 tumor cells In the subcutaneous,administration of each dose group in the signs are better than the control group mice,after the removal of solid tumor ,the average tumor weight of high,medium,and low dose group are lighter than model group.High-dose berberine group is greater than 84% inhibition rate have obvious effect.Mice after administration of 11d in the body can touch the grain size of the tumor ,on 12d,13d,14d berberine high,medium,low and positive control group were less than the control group of solid tumor ,in the 13d of the middle and high dose group, and 14d of each dose group compared with the control group there were significant differences.2 . Berberine in vitro proliferation of H22 tumor cells: berbenine inhibited the growth of the H22 cell lines and induced apoptosis in a time-and-concentration- dependent manner. The inhibition rate of berberine in 24 hours were 21%,28%, 31%,36%, That as the dose increased, enhanced anti-tumor effect.3 Berbenine promote H22 tumor cells apoptosis:3.1 Effect on expressing of cPLA2 and COX-2 in H22 cell lines: In the Berbenine of different treatment group(12.50μM,25μM,50μM,100μM) had conspicuous effect on expressing of cPLA2,the lower expressing level of these proteins was observed; In the Berbenine of different treatment group had conspicuous effect on expressing of COX-2, the higher expressing level of these proteins was observed.3.2 Effect on expressing of caspase-3,caspase-9 and AIF in H22 cell lines: In the Berbenine of different treatment group had no conspicuous effect on expressing of caspase-3 and caspase-9, but in Berbenine of different treatment group had conspicuous effect on expressing of AIF, the higher expressing level of these proteins was observed.Conclusion:Berbenine can inhibit the growth of human hepatocarcinoma cell line of H22 in dose-dependent manner, base on the datum from the tests in vitro and in vivo. The anticaicinogenic effect of Berbenine through reducing expressing of cPLA2 ;on the other hand the anticarcinogenic effect of Berbenine dose not depend on caspase ,its Inhibition effects of tumors by increasing expressing of AIF.