Our initial study revealed that the main component of CKI, oxymatrine, can decrease both MCF-7 cell viability and the size of the SP (by approximately 90%) by inhibiting β-catenin, the main component

of the Wnt signaling pathway, in a dose-dependent manner, while cisplatin (DDP) only inhibits find more non-SP cells and spares SP cells in vitro [28]. However, studies of CKI therapy on the regulation of SP cells have never been evaluated. So we studied the effects of CKI on the treatment of SP cells and its mechanism. Methods Cell culture Breast cancer cell line MCF-7 was kindly donated by Prof. Shuren Zhang (Department of Immunology, Cancer Institute, Peking Union Medical College and Chinese Academy of Medical Sciences). MCF-7 cells were maintained in RPMI1640 culture (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone), 100 units/ml penicillin G, and 100 μg/ml streptomycin. All cells were cultured at 37°C in a humidified atmosphere containing 5%

CO2. SP cell isolation Cells were detached from cell culture flasks with 0.25% trypsin, and viable cells were counted with trypan blue and collected for inoculation into NOD/SCID mice. The remaining cells were stained with the fluorescent dye Hoechst 33342 (Sigma) at a concentration of 5 μg/mL (37°C for 90 min) as described by Goodell et al.[29] Apoptosis inhibitor After washing with HBSS/2% FBS, the cells were incubated with 1 μg/ml propidium iodide to exclude dead cells, cell analysis and sorting were performed on a FACS Vantage SE (Becton Dickinson) by using a dual-wavelength analysis (blue, 420-470 nm; red, 660- 680 nm). We collected both MCF-7 SP and non-SP cells for the experiment. Tumor formation in an animal model and drug intervention For the tumor formation assay, the NOD/SCID female mice (5-6 weeks old) were purchased from the Animal Institute of Peking Union Medical

College and maintained under standard conditions according to the guidelines of the Institutional Animal Care and Use Committee of Peking University. oxyclozanide The mice were allowed to adapt to the new environment for one week. We first identified the tumorigenicity of SP cells. Unsorted, SP and non-SP cells were collected, and cells were resuspended in PBS/Matrigel (BD Biosciences) (1:1) ranging from 103 to 5 × 106 cells per 100 μl. Cells were then injected s.c. into the bilateral mammary pads of the mice. The mice were received an estradiol supplement (0.4 mg/kg s.c., Sigma) every 10 days until the end of the experiment after cell injection. The mice without Sorafenib research buy tumors were examined visually everyday. Throughout the study, mice were weighed and tumors were measured with a caliper twice a week. Tumor volumes were calculated using the formula (length×width2/2). When the xenograft tumors grew to proper size, the mice were euthanized and a portion of the s.c.