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 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. 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. Upon termination of treatment, the mice were weighed and sacrificed, and their tumors were excised. The mean tumor weight per group was calculated.