Cell culture and drugs administration
ATC cell lines 8305C, 8505C and human immortalized thyroid epithelial cell Hthy-ori3-1 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 of 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 [18]. IC50 values were calculated with the Reed-Muench method [19].
Colony formation assay
Monolayer culture was performed to measure colony formation. Cells (3000 for 8305C and 4000 for C643) were seeded into 12-well plate, and cultured with various doses of doxorubicin and lenvatinib, individually or in combination (high doses for C643 because of its higher cells’ density in one clone). 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 seeded at a concentration of 80-90% in 6 well plate, and cultured with indicated doses of doxorubicin and lenvatinib, individually or in combination for, 48 h to 8305C, 72 h to C643 and 60 h to 8505C. Next, the supernatant was exchanged to the normal medium without drugs for 4 h, and the apoptotic cells were collected by centrifuged. Then the cells were washed twice by PBS and 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 were seeded at a concentration of 60% in 6 well plate and serum starved for 12 h. After individually or in combination co-culture with doxorubicin and lenvatinib for, 48 h to 8305C, 72 h to C643 and 60 h to 8505C. Next, the supernatant was exchanged to the normal medium without drugs for 4 h, and then the cells were washed twice by 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).
Animal studies
8505C (5 × 106) cell lines 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 (3 mice each group) 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 every day, and/or lenvatinib (5 mg per kg of body weight) was administered via oral route every day. The treatment was administered for 3 weeks. All mice were sacrificed via cervical dislocation, and tumors were collected and weighted 5 h after the final dose of drugs. Animal experiments were 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 different organs, we performed hematoxylin and eosin (H&E) staining of liver, kidney and heart sections.
Statistical analysis
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.