Cell and animal models

Melanoma cell lines: A375, A375R, A2058, WM-115, SK-MEL-28, WM-3152, WM-2664, WM-1789, SK-MEL-2; colorectal cancer cell lines: HT-29, Colo-205, Colo-205R, NCI-H508, LoVo; lung cancer cell lines: HCC364, HCC364R, NCI-H1666, NCI-H1755, NCI-H2087, NCI-H460, HCC827. Among them, HCC364 cells were purchased from Sur biological (Shanghai), and those resistant to Vemurafenib and HCC364R and Colo-205R were constructed by the laboratory in the Department of Pharmacy of Shanghai Changzheng Hospital, and the rest were purchased from the Cell Bank of the Institute of Life Sciences of the Chinese Academy of Sciences (Shanghai).

Balb/c nude mouse (Nanjing University-Nanjing Institute of Biomedical Research, Nanjing, license number: SCXK (Su) 2015-0001). The animal experiment was approved by Ethics Committee of Changzheng Hospital of Shanghai. Throughout the experiment, all operations were based on the corresponding requirements of AAALAC.

Standards and reagents

Apatinib (purity ≥ 98%, Jiangsu Hengrui Pharmaceutical Co., Ltd.), vemurafenib (purity ≥ 98%, Selleckchem, USA), vemurafenib raw materials (purity ≥ 98%, Biedue, Shanghai). RPMI-1640 culture medium (Hyclone, USA), fetal bovine serum (Invitrogen, USA), double antibody (Invitrogen, USA), 0.25% trypsin (Invitrogen, USA). CCK8 Assay Kit (Tongren Chemical, Japan), Annexin V-FITC/PI Apoptosis Detection Kit (BD, USA), DMSO (Sigma, USA). BCA Protein Assay Kit (Pierce, USA), M-PER Cell Extraction Reagent (Pierce, USA). MEK/p-MEK antibody, ERK/p-ERK antibody (Abeam, USA), hypersensitive ECL chemiluminescence detection kit (Thermo, USA), polyvinylidene difluoride membrane (PVDF membrane) (Millipore, USA), Matrigel (BD, USA). sodium carboxymethyl cellulose (CMC-Na) (Sinopharm Group, Beijing), hydroxypropyl cellulose (Klucel LF) (Changwei Medicine, Shanghai), DMSO (GmbH, Germany), Tris-Tris (Hydroxymethyl) aminomethane, hydrazone red S, Tween-20, and β-mercaptoethanol were all chemical analysis reagents purchased from Amresco, USA.

Kinase screening

LanthaScreen kinase activity assay and Z’-LYTETM platform of Life Technologies Corporation were used to screen the inhibitory activity of 100 nM apatinib against 138 protein kinases, including BRAF and AKT. The inhibitory rates higher than 40% were considered as good inhibitory activity.

Cell culturing conditions

Corresponding culture medium of each cell line was selected, in which secondary-antibody (100 U/mL penicillin and 100 pg/mL streptomycin), and the culturing conditions were 37 °C, 5% CO2 and 95% relative humidity. When the cell density reached 70%, the subculture was carried out by trypsinization. Fresh complete culture medium was adopted for replacement every other day.

The effect of apatinib on proliferation rates of A375, HCC364, Colo-205 cell lines

This experiment was divided into one control group and 7 drug-administered groups with different concentrations (25, 50, 100, 200, 400, 800 and 1600 nM). The control group was treated with an equal volume of DMSO. The activity test was carried out 48 h later by using the CCK-8 kit with triplicate analyses for each cell line. Each analysis was averaged from measurement at different locations of the culturing well. The inhibitory rate (IC50) of each cell line was calculated based on absorbance of 450 nm light by Graphpad (San Diego, CA, US).

The effect of apatinib on cell cycle and apoptosis of A375, HCC364, Colo-205 cell lines

This experiment consisted of four groups: a blank control group, a vehicle control group, a control group with 20 nM vemurafenib and a treatment group with 20 nM apatinib. Cell cycle and apoptosis analyses were both completed 48 h after administration of drugs or vehicle solution, and they were completed by published method and Annexin V-FITC/PI kit respectively. Triplicate analyses were done for each cell line.

The effect of apatinib on cell migration of A375, HCC364, Colo-205 cell lines

A375, HCC364 and Colo-205 cells in the logarithmic phase were prepared, cell suspension was prepared by trypsinization, cell pellet/precipitation was collected by centrifugation, and cell density was adjusted to 1 × 105/mL cells with RPMI-1640 complete culture medium containing 10% FBS. These cells were inoculated into a 24-well cell culture plate (50 μl of Matrigel at a concentration of 10 ug/mL was pre-added, and it was gently shaken to make cells evenly distributed without bubble emerging, overnight in the refrigerator at 4 °C, and 500 μL of cell suspension was added to each well. The plate was gently shaken to make cells evenly distributed. When these cells were cultured for 24 h (37 °C and 5% CO2), cell scratch wound assay was conducted with a sterile 200 μL tip, then these cells were washed twice with serum-free medium, and finally 2 mL of 1% serum was added. Drugs or vehicle solution were/was added to each cell culture medium as required and they were grouped; these cells were cultured under normal conditions, and photographed by microscope after 72 h. This experiment was performed with 3 biological replicates (n = 3).

In vitro pathway analyses of A375, HCC364, Colo-205 cell lines

The cells were divided into five groups: a control group (vehicle solution), a control group with vemurafenib (20 nM), and three treatment groups with apatinib (10 nM, 20 nM and 50 nM).

A375, HCC364 and Colo-205 cells in the logarithmic phase were prepared, cell suspension was prepared by trypsinization, and the viable cell count was performed by trypan blue dyeing. The cells were collected by centrifugation at 1000 × g for 2 min. The RPMI-1640 culture medium containing 10% FBS was used to resuspend the cells, and cell concentration was adjusted to 1 × 105 /mL. The cells were inoculated into a 6-well culture plate (2 mL per well), overnight at 37 °C and 5% CO2. 2 mL of fresh complete culture medium was adopted for replacement per well. According to the final concentration of drugs, vemurafenib and apatinib was added to two groups respectively. The control group was added with an equal volume of DMSO to the drug intervention groups. And then the cells were cultured for 48 h under normal conditions and corresponding phosphorylation levels of the proteins in the regulatory pathway was detected.

The assay was performed 48 h after administration. The total protein was extracted by using M-PER, quantified by BCA kit with15 μg added to each well, electrophoresed in 11% SDS-PAGE, and transferred to a nitrocellulose membrane. And then, it was blocked with BSA for 2 h; the PVDF membrane was shaken slowly on the shaking table at room temperature. The cells were washed twice by TBST to remove the excess BSA; and the primary antibody diluted in a certain ratio was added: MEK (1:300), p-MEK (1:500), ERK (1:600), p-ERK (1:350), VEGFR (1:300), p-IVEGFR (1:500), AKT (1:400) and p-AKT (1:400), overnight in the refrigerator at 4 °C. The membrane was washed three times with TBST, and the primary antibody was removed from the membrane surface, followed by the secondary antibody (rabbit anti-mouse, 1:2000) added, and they were reacted at room temperature for 2 h. The luminescent liquid was configured according to the ECL kit instructions, dropped onto the PVDF membrane, and cells were imaged with X-ray film. The target band was scanned, and its optical density was analyzed. The degree of protein phosphorylation was analyzed and the total protein content was used as reference. This experiment was performed with 3 biological replicates (n = 3).

Preparation of working solution and experimental animals

Apatinib working solution was prepared by slowly adding apatinib powder to a 0.5% CMC-Na solution and dispersing it to make solutions at concentrations of 1.6, 3.25 and 5 mg/mL. Vemurafenib working solution was prepared by slowly adding Vemurafenib powder to a 0.2% Klucel LF solution and uniformly dispersing it to make a solution at concentration of 6.25 mg/mL.

Female Balb/c nude mice (6 weeks old, 20 ± 2 g), reared for 7 days to adapt to the environment (temperature: 24 ± 2 °C, relative humidity: 60 ± 10%, day and night (light and dark) alternate time: 12 h/12 h, free diet, free drinking water) after purchase. Tumor cells in the logarithmic growth phase were amplified and collected, and cell suspension was prepared: Matrigel = 1:1 cell mixture; the tumor cells were inoculated subcutaneously into the right lower back of the mice, and the number of cells inoculated was 5.0 × 106 volume of 0.1 mL.

When the tumors were grown to 100–200 mm3, 30 animals were selected according to their body weight and tumor volume, which were randomly divided into 5 groups, each group with 6 rats, and administered by intragastric administration as follow: (1) 5%CMC-Na, qd, 2 mL/100 g; (2) 6.25 mg/mL Vemurafenib, bid, 2 mL/100 g; (3) 1.6 mg/mL Apatinib, qd, 2 mL/100 g; (4) 3.25 mg/mL Apatinib, qd, 2 mL/100 g; (5) 5 mg/mL Apatinib, qd, 2 mL/100 g. The HCC364 model was administered for 26 days, the A375 and Colo-205 models were administered for 21 days. All animals were euthanized within 2 h when the experiment ended.

Statistical analyses

Differences between groups were analyzed by SPSS 19.0, and one-way ANOVA was used in combination with Dunnett (when variance homogeneity) or Dunnett's T3 (when variance was not uniform). P < 0.05 was considered to indicate a significant difference, P < 0.01 was considered to indicate a highly significant difference.