MATERIALS AND METHODS Cell culture and reagents HepG2, Bel-7402, Hep-3B, HUVE-12 and L-02 cell lines, were purchased from the China Center for Type Culture Collection (CCTCC), were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin and 100 ��g/mL streptomycin (Life Technologies) in an incubator containing selleck kinase inhibitor 50 mL/L CO2 at 37��C. rVBMDMP was over-expressed in Escherichia coli with pGEX-4T-1-VBMDMP and purified as previously described[10] with a purity of over 95%. Synthetic peptide CNYYSNSYSFWLASLNPER (amino acid 185-203 of tumstatin, T4 peptide) and its rabbit polyclonal antibody were provided by Xi��an Huacheng Biotechnology Co., Ltd (China). Bevacizumab was purchased from Roche (Avastin?; Basel, Switzerland).

Mouse monoclonal antibodies against proliferating cell nuclear antigen (PCNA) and CD31, as well as peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA). MTT assay Cells were seeded in a 96-well plate at a density of 1000 cells/well as described previously[12]. Different concentrations of drugs were added to each well and cultured for 48 h, followed by incubation with 0.5 g/L MTT for 4 h. The supernatant was removed after centrifugation. Finally, 100 ��L DMSO was added and the adsorbance at 570 nm wavelength (A570) was measured with an enzyme-labeling instrument (ELX-800 type). Relative cell proliferation inhibition rate (IR) = (1-average A570 of the experimental group/average A570 of the control group) �� 100%.

The IR was analyzed using the Calcusyn program to determine the IC50. Tumor xenograft experiments Balb/c-nude female mice (Vital River Laboratory Animal Technology Co., Ltd), used in in vivo study, were housed in a sterile room at Institute of Cancer Research, University of South China, with free access to food and water. Tumors were generated by harvesting HepG2 cells from mid-log phase cultures using 0.25% trypsin (Life Technologies). Cells were resuspended in PBS to a final cell count of 2.5 �� 107/mL. A cell suspension (0.2 mL) was subcutaeously injected into the back of each mouse. The mice received a total of 10 injections of 1, 3, and 10 mg/kg body Dacomitinib weight rVBMDMP (i.p) every other day when their average tumor volume reached 200 mm3. Tumor dimensions and body weight were recorded every 5 d from the beginning of treatment. Tumor length and width were measured using a Vernier caliper, and tumor volume was calculated as described previously[13]. Upon termination of treatment, the mice were weighed and sacrificed, and their tumors were excised. The mean tumor weight per group was calculated.