DMSO, dimethyl sulfoxide;

HCC, hepatocellular carcinoma; lupeol, Lup-20(29)-en-3β-ol; MTT, 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PTEN, phosphatase and tensin homolog; T-IC, tumor-initiating cell. The human HCC cell lines MHCC-LM3 (from Liver Cancer Institute, Fudan University, Shanghai, China), Huh-7 (Japanese Cancer Research Bank, Tokyo, Japan), and PLC-8024 (Institute of Virology, Chinese Academy of Medical Sciences, Beijing, China) were maintained in Dulbecco’s modified Eagle’s medium with high glucose (Gibco BRL, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (Gibco BRL), 100 mg/mL penicillin G, and 50 μg/mL streptomycin (Gibco BRL) at 37°C in a humidified atmosphere containing 5% CO2. MIHA was kindly provided by J. R. Chowdhury, Albert Einstein College of Medicine, New York.25 Liver tumor tissue specimens were collected from five patients who underwent hepatectomy click here for HCC between 2008 and 2009 in the Department of Surgery, Queen Mary Hospital, Hong Kong, with Institutional buy GSK1120212 Review Board approval. Tumor tissue from fresh tumors was minced into 1-mm3 cubes and incubated with Liberase TM Research Grade (Roche Diagnostics, Indianapolis, IN) for 5-10 minutes at 37°C. A single-cell suspension was obtained by filtering the supernatant through a 100-μm cell strainer (BD Biosciences, San Jose, CA). Removal of CD45+ cells from within the tumor was performed

with a CD45 depletion kit (Miltenyi Biotech, Bergisch Gladbach, 上海皓元 Germany). A stock solution of lupeol (30 mmol/L) (NW = 426.72) was resuspended in warm alcohol and diluted in dimethyl sulfoxide (DMSO) at a 1:1 ratio. For dose-dependent studies, cells (50% confluent) were treated with lupeol (1-200 μmol/L) for 72 hours in complete Dulbecco’s modified Eagle’s medium/high-glucose cell medium. For all treatment protocols, the final concentrations of DMSO and alcohol were 0.25% and 0.075%, respectively. For HCC cell lines, CD133+ tumor cells were sorted on a BD FACSVantage SE (BD Biosciences, San Jose, CA). For clinical HCC samples, CD133+ cells were isolated

by way of magnetic cell sorting. Clinical HCC tumor cells were labeled with CD133/1 microbeads and sorted using the Miltenyi Biotec CD133 Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Magnetic separation was performed twice to obtain purity greater than 95% for both CD133+ and CD133− populations. Aliquots of CD133+ and CD133− sorted cells were evaluated for purity with a FAT-ICalibur machine and CellQuest software (BD Biosciences, San Jose, CA) using the phycoerythrin-conjugated anti-human CD133/2 antibody (Miltenyi Biotec). For cell sorting using flow cytometry, cells were stained with the phycoerythrin-conjugated anti-human CD133/1 antibody (Miltenyi Biotec). Isotype-matched mouse immunoglobulins served as controls. Samples were analyzed and sorted on a BD FACSVantage SE (BD Biosciences).