Famitinib (SHR1020) was a gift from Jiangsu Hengrui Medicine Co., Ltd. (Lianyungang, China), and its appearance was a yellow powder. The drug was stored at 4°C in the dark. For in vitro studies, famitinib was dissolved in dimethylsulfoxide at 20 mmol/l and stored at −20°C until use. For in vivo animal experiments, famitinib was formulated in physiological saline as a homogeneous suspension (10 mg/ml) and stored at 4°C protected from light. Injectable 5-florouracil (5-FU, 250 mg/10 ml) was purchased from Tianjin Jinyao Amino Acid Co., Ltd. (Tianjin, China). Cisplatin lyophilized powder (DDP, 10 mg) was purchased from Qilu Pharmaceutical Co., Ltd. (Jinan, China) and was formulated in physiological saline. Paclitaxel (PTX, 30 mg/5 ml) injection was purchased from Hainan Haiyao Co., Ltd. (Hainan, China) and was formulated in physiological saline.

Human gastric cancer cells BGC-823 and MGC-803 were provided by Professor Youyong Lv (Peking University Cancer Hospital and Institute, Beijing, China). Both cell lines were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and incubated in a humidified 37°C incubator with 5% CO₂.

Both cell lines were seeded at ~3,000–5,000 cells/well in a 96-well plate and incubated overnight in complete medium, followed by treatment with different concentrations of famitinib for 24, 48 and 72 h. Cell viability was measured using MTS tetrazolium substrate (CellTiter 96^® AQueous One Solution Cell Proliferation Assay; Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. The absorbance was measured at 490 nm using a spectrophotometer. All experiments were repeated three times with at least triplicates for each concentration.

Cell were treated with famitinib for 48 h, followed by harvesting and fixing in 70% cold ethanol for ≥12 h at 4°C. Cells were stained with 50 µg/ml propidium iodide (BD Biosciences, Franklin Lakes, NJ, USA) at room temperature for 30 min in the dark, and the cell cycle was analyzed using a FACSAria or a FACSCalibur (BD Biosciences). Data were analyzed by ModFit 3.0 software (BD Biosciences). All experiments were performed in triplicate.

Cell apoptosis was measured via TUNEL assay (catalog no., C1086; Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's protocol. Upon treatment of the cells with famitinib for 48 h, cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 10 min at room temperature. Cells were then stained with the corresponding reagents provided in the TUNEL assay kit. Upon overlaying the coverslips, slides were imaged under fluorescence microscopy (TCS SP5; Leica Microsystems GmbH, Wetzlar, Germany). Positive cells exhibited green fluorescence and were counted from three random microscopic fields.

Total proteins prior and subsequent to famitinib treatment were extracted from BGC-823 and MGC-803 cell pellets using CytoBuster Protein Extraction Reagent (Merck Millipore, Darmstadt, Germany). Proteins were quantified with a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.), and ~20 µg of protein was separated on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were then transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, Chalfont, UK), which was subsequently incubated with anti-cyclin B1 (dilution, 1:1,000; catalog no., AJ1208a; Abgent Inc., San Diego, CA, USA), rabbit polyclonal anti-B-cell lymphoma 2 (BCL2; dilution, 1:1,000; catalog no., 2872; Cell Signaling Technology, Inc., Danvers, MA, USA) and mouse monoclonal anti-β-actin (dilution, 1:3,000; catalog no., A5441; Sigma-Aldrich, St. Louis, MO, USA) antibodies at 4°C overnight. Secondary anti-rabbit and anti-mouse horseradish peroxidase-conjugated IgG antibodies (dilution, 1:3,000; catalog nos., 7074 and 7076, respectively; Cell Signaling Technology, Inc.) were applied and allowed to incubate at room temperature for 1 h. Proteins were visualized with ECL Plus Western Blotting Detection Reagent (GE Healthcare Life Sciences).

BGC-823 cells were suspended in PBS (1×10⁷ cells/ml), and 100 µl of the cell suspension was subcutaneously injected into the right axillary area of 18-20-g female BALB/c athymic nu/nu mice (n=40; age, 6–8 weeks; Vital River Laboratories Co., Ltd., Beijing, China). The temperature of the housing conditions was maintained at 23–25°C with a humidity of 50–60% and a 10/14 h light/dark cycle. Food and water were changed 3 times a week. When the tumor volume reached ~100 mm³, mice were randomized into treatment groups. Tumors and animal weights were measured twice weekly, and tumor volume was calculated using the following formula: V=LxW²x1/2 (where V represents tumor volume, L is the length of the tumor and W is the width of the tumor).

To measure famitinib, three groups were randomized (n=5 mice/group) as follows: Control group (gavage, physiological saline, once daily for 3 weeks); low-dose famitinib group (gavage, 50 mg/kg, once daily for 3 weeks); and high-dose famitinib group (gavage, 100 mg/kg, once for 3 weeks). A dose of 50 mg/kg was used for the following experiments.

To compare famitinib with other drugs, animals were randomized (n=5 mice/group) as follows: Control group (gavage, physiological saline, once daily for 3 weeks); famitinib group (gavage, 50 mg/kg, once daily for 3 weeks); 5-FU group [10 mg/kg, intraperitoneal (ip), once every 2 days for 3 weeks]; DDP group (3 mg/kg, ip, once weekly for 3 weeks); and PTX group (10 mg/kg, ip, once a week for 3 weeks). Then, tumors and weight were quantified. All animal experiments were approved by the Institutional Ethics and Animal Care Committee and performed in accordance with the animal experimental guidelines of Peking University Cancer Hospital (Beijing, China).

Once the mice were sacrificed, xenografts were isolated, and formalin-fixed, paraffin-embedded (FFPE) tissue blocks were obtained. FFPE tumor sections (4-µm thick) were deparaffinized in xylene and hydrated in graded alcohol. Then, tumor sections were either stained using a H&E staining kit (catalog no., C0105; Beyotime Institute of Biotechnology), according to the manufacturer's protocol, or incubated with rabbit polyclonal anti-cluster of differentiation (CD)34 antibody (dilution, 1:2,500; catalog no., ab81289; Abcam, Cambridge, UK) upon antigen retrieval and endogenous peroxidase treatment. Signals were visualized using an immunoglobulin G-horseradish peroxidase polymer (Beijing CoWin Biotech Co., Ltd., Beijing, China) and 3,3′-diaminobenzidine substrate. Sections were scored by two independent pathologists as previously described (15).

SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA) was used to perform the statistical analysis. One-way analysis of variance was used for the in vivo experiments. P<0.05 was considered to indicate a statistically significant difference.

BGC-823 and MGC-803 cells were treated with famitinib (0, 0.6, 1.25, 2.5, 5.0, 10.0 and 20.0 µM) for 24, 48 and 72 h, followed by MTS assay. Famitinib inhibited cell growth in a dose-dependent manner (Fig. 1). The half maximal inhibitory concentration (IC₅₀) values of famitinib in BGC-823 and MGC-803 cells were 3.6 and 3.1 µM, respectively. Based on these results, the IC₅₀ and 1/2 IC₅₀ were used for in vitro experiments.

The cell cycle was next analyzed following famitinib treatment, and it was observed that the number of G2/M-phase cells increased in BGC-823 (27.98 vs. 14.25%) and MGC-803 (58.23 vs. 10.72%) cells compared with the control (P=0.04 and P=0.02, respectively; Fig. 2A and B). Cell cycle arrest at the G2/M phase was confirmed by increased expression of the cell metaphase-specific protein cyclin B1 (Fig. 2C).

Cell apoptosis is an important mechanism of cell growth inhibition (16); therefore, apoptosis was measured via TUNEL assay. Fig. 3 indicates that, compared with the control, famitinib increased apoptosis in BGC-823 and MGC-803 cell lines significantly (P<0.01), and downregulated BCL2.

To identify the optimal famitinib dose for the in vivo study, two doses were used, 50 and 100 mg/kg. Both doses exerted a similar inhibitory power, but greater toxicity was observed with the highest dose (data not shown).

Mice were sacrificed 21 days after treatment, and tumors were isolated. Famitinib inhibited BGC-823 xenograft growth (tumor volume, 395.2 vs. 2,690.5 mm³, P<0.01; Fig. 4A), and animal weights were similar between groups (21.6 vs. 18.7 g, P=0.17; Fig. 4B).

Famitinib is considered to inhibit angiogenesis; therefore, microvessel density was measured with CD34 staining. Upon famitinib treatment, xenografts had greater tissue necrosis (Fig. 4C) and exhibited significantly weaker CD34 staining than the controls (Fig. 4D). Thus, famitinib inhibits tumor vascularization.

In clinical practice, 5-FU, DDP and PTX are the most commonly used chemotherapeutic drugs for gastric cancer. Thus, the activity of famitinib alone was compared with that of these compounds, and it was observed that famitinib exerted better tumor inhibition than 5-FU, DDP and PTX (Fig. 4E and F). The mean tumor volumes of the control, 5-FU, DDP, PTX and famitinib groups were 1,973.0, 1,680.3, 987.3, 1,577.6 and 287.6 mm³, respectively; and the corresponding tumor inhibitory ratios were 0.0, 14.9, 49.9, 20.0 and 85.4%, respectively.