Aim: To examine whether the cell growth inhibitory effect of the combination of baicalin and baicalein is related to apoptosis. demonstrated that this combination treatment had a synergistic effect. Moreover, apoptosis in MCF-7 cells was increased by 12% and 20% with the combination treatment at 24 h and 48 h, respectively. With the combination treatment in MCF-7 cells, cleaved caspase-3 and caspase-9 were observed, and the level of bcl-2 expression was decreased approximately 20% and 40% at 24 h and 48 h, respectively. The expression of bax and p53 were increased about 25% and 15% at 48 h, respectively. Moreover, the activation of caspase-3, -9 and the regulation of bcl-2, bax and p53 were related to ERK /p38 MAPK activation. Conclusion: In this study, apoptosis was enhanced by the combination treatment of baicalin and baicalein, which activated caspases-3 and caspase-9, downregulated the level of bcl-2 and upregulated the level of bax or p53 via the ERK/p38 MAPK pathway. Keywords: baicalin, baicalein, combination therapy, apoptosis, breast cancer cells Introduction Cancer is the second leading cause of death worldwide1. Breast cancer is the most common cancer among women, comprising 23% of all female cancers around the globe2. There is a lifetime risk of up to 12% and a risk of death of up to 5%3 in breast cancer. Although the incidence rates of breast cancer in the world have increased by about 0.5% annually since 1990, cancer registries in China recorded annual increases in incidence of 3%?4%4. There have been great advancements in the treatment and control of breast cancer, but significant deficiencies still remain. It is usually well known that cell homeostasis requires a balance between cell proliferation and cell death, including apoptosis5. Moreover, there is strong evidence that tumor growth is a result not only of uncontrolled proliferation but also of reduced apoptosis6. Therefore, induction of apoptosis has become an effective means of cancer therapy. Apoptosis has been defined as a discrete sequence of morphological changes resulting in cell death with extensive dsDNA cleavage accompanied by chromatin compaction and segregation along the nuclear membrane7. Many death and survival genes, such as bcl-28 or bax9, which are regulated by extracellular factors, are involved in apoptosis. Additionally, recent reports indicate that one of the molecular events of apoptosis is usually activation of the caspases by a signal transduction cascade10. Currently, chemotherapy using plant-derived, anti-cancer drugs such as paclitaxel11, vinorelbine12, or vincristine13 has been proven to be effective in Metoprolol tartrate manufacture clinical settings. It was shown that these products enhance cell growth inhibition in different cancer cell lines and are highly effective and safe in clinical trials. Baicalin and baicalein are components of Baikal Skullcap Root. It has been reported that either baicalin or baicalein has potential anti-tumor effects on liver14, prostate15, bladder16, and breast cancer cells17, 18. However, the anti-tumor effect of combination treatment with baicalin and baicalein on breast cancer cells is not clear. Mitogen-activated protein kinase (MAPK) pathways have been implicated in the response to chemotherapeutic drugs19. The proteins involved in MAPK cascades are expressed and respond to a wide variety of external cues and drugs. P38 MAPK has been implicated in the regulation of various cellular processes20. Previous reports have shown that MEK, which is usually activated by mitogenic stimuli, contributes to cell differentiation, proliferation, and survival21. Some responsiveness was regulated by ERK and MAPK in breast cancer cells22. It is of interest, therefore, to determine whether apoptosis is usually induced by the combination of baicalin and baicalein via the ERK/p38 MAPK signaling pathway. In this study, we demonstrate that this combination treatment of baicalin and baicalein enhances the growth-inhibitory effect NPM1 in human breast cancer cells. Furthermore, we report that this growth-inhibitory effect derives from the induction of apoptosis, which involves Metoprolol tartrate manufacture activation of caspase-3 and caspase-9, Metoprolol tartrate manufacture downregulation of bcl-2 expression and upregulation of bax or p53 expression via ERK/p38 MAPK pathway. Materials and methods Materials Baicalin and baicalein were obtained from National Institute for the Control of Pharmaceutical and Biological Products, China and dissolved in dimethyl sulfoxide (DMSO). Acridine orange (AO), Annexin V and propidium iodide (PI) were from Sigma (St Louis, MO, USA). The antibodies against caspase-9, caspase-3, bcl-2, bax, p-ERK, and p-p38 were obtained from Cell Signaling Technology (Boston, MA, USA). The anti-p53 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PD98059 and SB203580 were from Biomol (Philadelphia, PA, USA). Cell culture Human breast cancer MCF-7 and MDA-MB-231 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and were cultured in RPMI-1640 medium (Gibco, San Francisco, CA, USA) supplemented with 10% heat-inactivated (56 C, 30 min) fetal calf serum (PAA, A-4061, Pasching, Austria ), 0.01 mg/mL insulin (Sigma, St Louis, MO, USA), 2 mmol/L glutamine (Gibco, San Francisco, CA, USA), penicillin (100 U/mL) and streptomycin (100 g/mL). The cell culture was maintained at 37 C with.