Studies On The Mechanism Of Berberine In Combination With Doxorubicin Against Tumor

The developing of tumor is affected by many factors, the main mechanism involved multi gene changes.Different antineoplastic agents influence the appropriate targets to achieve its anti-tumor effect through a specific mechanism of action. As one of the three means for the treatment of malignant tumor,chemotherapy treatment plays an important role in the course of cancer treatment. Anticancer drugs can inhibit or kill tumor cell directly by binding to DNA or interfering the synthesis of RNA or protein.In addition, anticancer drugs can change the environment of the body. Chemotherapy can make up for lack of radiotherapy and surgery to prevent tumor recurrence and metastasis. to control a wide range of advanced cancer metastasis and to prolong survival and improve quality of life.Chemotherapy drugs have a direct killing effect to the tumor cell, but the body’s normal cells will also be damaged at the same time, which will produce the body’s weakened defenses, such as fatigue, hair loss, liver and kidney failure and other common clinical side effects Therefore we can choose some traditional Chinese medicine with righting role to match the chemotherapy to achieve attenuated synergistic effect. It also will reduce patient’s pain which produced by the side effects of chemotherapy through complementary medicineThis paper includes the following methods:1. MTT assay:to study cells growth activity, examine the effect of berberine in combination with doxorubicin and screen the two drugs’optimal proportions and duration of action.2.Flow cytometry:to detect apoptosis with Annexin-PI double staining, using DCFH-DA fluorescent probe labeled cells detect ROS, PI staining to detect a single cell cycle, test the potential anti-tumor mechanism of the combination of berberine and doxorubicin.3. RT-PCR:to detect changes in gene expression, to examine the impact of berberine and doxorubicin combination of gene expression.4. western-blotting:to detect changes in protein expression, to examine the impact of berberine and doxorubicin combination of protein expression.The results indicate that:1, Berberine and doxorubicin have an effectively inhibition to the proliferation of tumor cell MCF-7and the effect is synergistic. In the48h of drug effects, the best synergistic effects of the two drugs appears the drugs’concentration ratio of5:1; when drug duration of action achieve24h,10:1concentration of both drugs work best.2, Berberine can increase the apoptosis rate of MCF-7cells induced by doxorubicin, the apoptosis rate of MCF-7cells is changed from16.64%produced by doxorubicin alone to32.16%induced by doxorubicin combined with berberine. The difference was significant (P <0.05),3.Berberine and doxorubicin can increase the generation of reactive oxygen species of MCF-7cells when combined, the combined group can significantly improve the generation of reactive oxygen species compared with single group.The difference was significant.4, Berberine and doxorubicin can affect the cycle of MCF-7cells, doxorubicin may arrest cells in S phase in the combination therapy, the proportion of S phase cells increased significantly by doxorubicin combined with berberine. The difference was significant (P <0.05).5.The impact of gene and protein in apoptosis pathway, the berberine promote the expression of caspase genes, and the effect of caspase9in a dose-dependent manner, caspase3and caspase9expression in berberine combination with doxorubicin were higher than alone group. Berberine and doxorubicin can promote p53and mdm2gene expression, and the effect of mdm2in a dose-dependent manner. The expression of p53and mdm2in combination group were higher than the expression of p53and mdm2in alone group Berberine or doxorubicin alone promote the expression of Bax and Bak gene in a dose-dependent manner,and inhibit the expression of Bcl-2which is no significant dose-dependent manner. Doxorubicin inhibits the expression of related gene.The expression of Bax, Bak, Bcl-2decreased when two drugs combined. Protein expression was no significant time-dependent (Figure3-4-1) When berberine and doxorubicin were combined, the protein expression of pro-caspase7, mdm2decreased, the protein expression of cleaved-caspase9, PARP, p-p53expression increased.The impact in terms of autophagy pathway genes and proteins expression related mTOR pathway.Test results showed that changes in gene pathway after the two drugs treated is not obvious. Berberine promote mTOR and4ebp gene expression, and the impact of4ebp in a dose-dependent manner. BBR inhibiting AKT, S6k gene expression in a dose-dependent manner. When combination with doxorubicin, the expression of S6K,4ebp is lower than BBR alone.The change isn’t significant. By western-blotting,we found that the activated form of AKT protein expression tended to decrease after the combination,4ebp protein expression was significantly increased. It shows that two drugs may play anti-tumor effect by inhibiting translation of messenger RNA and inhibit cell proliferation.The issue explore the combination effects of berberine and doxorubicin on tumor growth of MCF-7cells from the vitro experiments perspective, and explain its possible mechanism of action.It would make the foundation for the future research.