Cell culture and drugs administration
ATC cell lines 8305C and 8505C were kindly provided by Dr. Haixia Guan (The First Affiliated Hospital of China Medical University, Shenyang, P. R. China). C643 were obtained from Dr. Lei Ye (Ruijin Hospital, Shanghai, P. R. China). All the cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Invitrogen Technologies, Inc., CA) at 37°C. Cells were treated with lenvatinib (Selleck Chemicals, LLC) or/and doxorubicin (Selleck Chemicals, LLC) at the indicated concentrations and time points in some specific experiment. The lenvatinib and doxorubicin were dissolved in dimethylsulfoxide (DMSO), the same volume of DMSO was used as the vehicle control.
Cell viability assay
Cells (3000 to 4000/well) were seeded in 96-well plates. After a 24-h’s culture, cells were treated with indicated doses of doxorubicin or/and lenvatinib for the indicated period time. Then 20 μL of 5 mg/mL 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) was employed to assess the cell viability as described . IC50 values were calculated with the Reed-Muench method .
Colony formation assay
Monolayer culture was performed to measure colony formation. Cells (3000 to 4000/well) were seeded into 12-well plate, and cultured with various doses of doxorubicin and lenvatinib, individually or in combination. The medium was refreshed every 72 h. After 7-12 days of cultivation, colonies were fixed with 4 % paraformaldehyde, and then washed with PBS and stained with a crystal violet solution. Each assay was carried out in triplicate.
Cell apoptosis assay
Cells were cultured with indicated doses of doxorubicin and lenvatinib, individually or in combination, for 48-72 h. Then cells were stained with Annexin V-FITC/PI Apoptosis Detection Kit (Roche Applied Science, Penzberg, Germany) according to the manufacturer’s protocol. Apoptotic cells were measured by flow cytometer (BD Biosciences, NJ). Each experiment was carried out in triplicate.
Cell cycle analysis
Cells in the exponential growth phase were serum starved for 12 h. After individually or in combination co-culture with doxorubicin and lenvatinib for 48-72 h, cells were harvested, washed twice in PBS, and fixed in 70 % ethanol on ice for 30 min. Cells were then stained with PI solution (50 μg/mL PI, 50 μg/mL RNase A, 0.1% Triton-X, 0.1 mM EDTA). Cell cycles were analyzed based on DNA content using a flow cytometer (BD Biosciences, NJ).
8505C (5 × 106) were injected subcutaneously into the right armpit region of 5- to 6-week-old female nude mice purchased from SLAC laboratory Animal Co., Ltd. (Shanghai, China) to establish xenograft mouse model. Mice were then randomly divided into four groups when tumor volume grew to 50-80 mm3. Doxorubicin (3 mg per kg of body weight) was administered by intraperitoneal injection at a total volume of 0.2 mL, and/or lenvatinib (5 mg per kg of body weight) was administered via oral route. The treatment was daily administered for 3 weeks. All mice were sacrificed, and tumors were collected and weighted 5 h after the final dose of drugs. Animal experiments were was approved by the Institutional Review Board of Xi’an Jiaotong University Health Science Center.
Immunohistochemical (IHC) staining
Tumor tissues were embedded in paraffin, sectioned at 4 μm, then cell proliferation ability was assessed by quantification of Ki-67 staining (percentage of positive cells). In brief, antihuman Ki-67 antibody (BD Pharmingen, CAT 550609) was 1:300 diluted, and immunostaining was done according to a standard protocol using DAB Substrate Kit (ZSGB-BIO). To evaluate the effect of different treatments on animal hepatocytes, we performed hematoxylin and eosin (H&E) staining of liver, kidney and heart sections.
All the statistical analyses were performed with the SPSS statistical package (16.0, SPSS Inc. Chicago, IL). Unpaired student’s t test was used to compare the means of two groups of data. One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison test was used to compare differences between three or more groups. All values were expressed as the mean ± standard deviation (SD). P<0.05 was considering statistically significant differences.