Mechanistic Studies Of The Growth-inhibitory Effects Of Berberine On Human Breast Cancer Cells

INTRODUCTIONBreast cancer is one of the most common cancers and a leading causeof cancer mortality among women, and it accounted for approximately23%of total new cases and14%of all cancer deaths in2008. Most Asiancountries, including China, the former low-incidence areas of breast cancer,but in recent years the incidence of breast cancer has increased year by year,especially in some economically developed regions and cities, and there isa tendency younger. Therefore, the prevention and treatment of breastcancer have far-reaching significance.With the increase of the molecular mechanisms of breast cancer, thecomprehensive treatment level of breast cancer has been greatly improved.The treatment of breast cancer at present involves surgery, chemotherapy,radiotherapy, endocrine therapy, molecular targeted agents as well as avariety of traditional Chinese medicine treatment of adjuvant therapy. Therapid development of treatment methods significantly improved the qualityof life of breast cancer patients, prolonged patients’ survival. Many traditional Chinese medicine or herbal extracts have anti-canceractivity, such as paclitaxel and vincristine herbal extracts have been widelyused in clinical anti-tumor therapy. Chinese traditional drugs have a longhistory, the toxicity and side effects were relatively smaller and the costwas lower. Therefore, development of safe and effective anti-tumor agentsfrom Chinese traditional drugs is a unique advantage.Berberine is a natural compound derived from the roots and rhizomesof numerous herbal plants, such as Berberis and Coptis chinensis. It is anisoquinoline alkaloid component. It has been shown to exhibit a variety ofpharmacological activities, including anti-diarrheas, antidiabetes,anti-inflammatory, and anti-tumor capabilities. In recent years,accumulating evidences have demonstrated that berberine has inhibitoryeffects on different types of cancer cells such as colon cancer, prostatecancer, liver cancer and breast cancer by inducing cell cycle arrest,suppressing DNA synthesis, activating caspases and inducing apoptosis.Recent researches suggested that berberine induced cell cycle arrest andcell apoptosis in breast cancer cells, but the underlying mechanisms remainunclear. In this study, we demonstrated the mechanisms of growthinhibition effects of berberine on breast cancer cells, and we also exploredwhether berberine improve the anticancer efficacy of tamoxifen. It revealedthe anti-cancer action targets of berberine on breast cancer cells; our studyprovided the theoretical basis of berberine for clinical application of breast cancer.PartⅠ Genomic screening for targets regulated by berberine in breastcancer cellsObjective: To investigate the effects of berberine on cell growth, colonyformation, cell cycle distribution and apoptosis, and to explore thegenome-wide expression profiling of berberine-treated cells using cDNAmicroarray.Methods:1. MCF-7and MDA-MB-231cells were treated with berberine for48h and the cytotoxicity of berberine measured by an MTS assay;2. MCF-7and MDA-MB-231cells were treated with berberine for48h and the colony formation ability of cells measured by colony formationassay;3. The effects of berbeirne on MCF-7and MDA-MB-231cells cycledistribution were analyzed by flow cytometry;4. The effects of berbeirne on MCF-7and MDA-MB-231cellsapoptosis were analyzed by flow cytometry;5. The effects of berberine on global gene expression profiles inMCF-7and MDA-MB-231cells were analyzed by using Agilent humanwhole genomic4×44K arrays;6. Verification of microarray results using Real-time PCR. Results:1. Berberine reduced the viability of MCF-7and MDA-MB-231cellsin a dose dependent manner;2. Berberine markedly suppressed the colony formation abilities of theMCF-7and MDA-MB-231cells;3. Berberine induced G1-phase arrest in MCF-7cells but not inMDA-MB-231cells;4. Berberine induced cell apoptosis in MCF-7and MDA-MB-231cells;5. The genes regulated by berberine in both cells were sorted intofunctional assay by GO database, genes related to regulation of cell cycle,cell apoptosis, cell migration and drug response were identified;6. The real-time PCR and microarray expression results matched well,although there was variation in fold change for genes expression.Conclusions:1. Berberine reduced the viability of MCF-7and MDA-MB-231cellsin a dose dependent manner.2. Berberine induced G1-phase arrest in MCF-7cells, and induced cellapoptosis in MCF-7and MDA-MB-231cells.3. Microarray technique was an effective high-flux method fordetecting targets of berberine. Alteration of genes involved in cell cycle,cell apoptosis and drug response may explain the inhibitory activity of berberine.Part Ⅱ Effects of berberine on CYP1A1and CYP1B1expression inMCF-7cellsObjective: To explore the effects of berberine on CYP1A1and CYP1B1expression in MCF-7cells.Methods:1. The effects of berberine on the mRNA expression of CYP1A1andCYP1B1in MCF-7cells were analyzed by real-time PCR assay.2. Effects of berberine on the protein expression of CYP1A1andCYP1B1in MCF-7cells were analyzed by western blot assay.Results:1. Treatment of MCF-7cells with berberine resulted in a dose-andtime-dependent increase in CYP1A1mRNA levels, significant elevationsin CYP1B1mRNA expression were observed after exposure to berberine.2. Treatment of MCF-7cells with berberine resulted in an increase inCYP1A1protein levels; however, CYP1B1protein levels were notchanged.3. Berberine treatment elevated the expression of CYP1A1tosignificantly higher levels than CYP1B1in MCF-7cells.Conclutions:1. Treatment of MCF-7cells with berberine resulted in an increase in CYP1A1mRNA and protein levels.2. Treatment of MCF-7cells with berberine resulted in an increase inCYP1B1mRNA levels; however, CYP1B1protein levels were notchanged.3. Berberine treatment elevated the expression of CYP1A1tosignificantly higher levels than CYP1B1in MCF-7cells.Part Ⅲ Cooperative antitumor effects and mechanisms ofberberine with tamoxifen on MCF-7and MCF-7/TAM cellsObjective: To observe the cooperative inhibitory effects of berberine withtamoxifen on MCF-7and MCF-7/TAM cells, study the anti-cancermechanisms of the co-treatment of berberine and tamoxifen.Methods:1. Sustained induction of tamoxifen-resistant cell line MCF-7/TAMbased on high-dose induction “two-stage screening method”, tamoxifenresistance of MCF-7and MCF-7/TAM cells were indentified by MTSassay.2. Cytotoxicity of berberine on MCF-7and MCF-7/TAM cells wasmeasured by MTS assay.3. Cytotoxicity of co-treatment of berberine and tamoxifen on MCF-7and MCF-7/TAM cells was measured by MTS assay.4. The effects of co-treatment of berberine and tamoxifen on MCF-7 and MCF-7/TAM cell cycle distribution were analyzed by flow cytometry.5. The effects of co-treatment of berberine and tamoxifen on MCF-7and MCF-7/TAM cell apoptosis were analyzed by flow cytometry.6. The effects of co-treatment of berberine and tamoxifen on MCF-7and MCF-7/TAM cells cycle and apoptosis related protein expression bywestern blot assay.Results:1. MCF-7/TAM cells were successfully constructed.2. Berberine reduced the viability of MCF-7and MCF-7/TAM cells ina dose-and time-dependent manner.3. Co-treatment of berberine and tamoxifen promoted the decrease ofcell viability in MCF-7and MCF-7/TAM cells.4. Co-treatment of berberine and tamoxifen induced G1phase arrest inMCF-7and MCF-7/TAM cells.5. Co-treatment of berberine and berberine induced cell apoptosis inMCF-7and MCF-7/TAM cells.6. Co-treatment of berberine and tamoxifen caused the expressionalterations of cell cycle and apoptosis related protein.Conclusions:1. Co-treatment of berberine and tamoxifen enhanced the growthinhibition in MCF-7and MCF-7/TAM cells.2. Co-treatment of berberine and tamoxifen induced G1phase arrest and cell apoptosis in MCF-7and MCF-7/TAM cells.3. Co-treatment of berberine and tamoxifen induced P21and Baxprotein expression, inhibited Bcl-2protein expression in MCF-7andMCF-7/TAM.