Dovitinib (CHIR-258, TKI258) [4-amino-5-fluoro-3-[6(4-methyl-1-piperazinyl)-1H- benzimidazol-2-yl]-2(1H)-quinolinone], with a molecular weight of 392.4, was provided by Novartis Pharma AG (Novartis Institutes for Biomedical Research, Basel, Switzerland).
Cells and cell culture
The human HCC cell lines MHCC-97H, QGY-7703, SMMC7721, Hep3B, and CRI2234, as well as a human bone marrow endothelial (HBME) line, were maintained in DMEM or RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Invitrogen) in a humidified incubator containing 5% CO2 at 37°C. Human umbilical vascular endothelial cells (HUVECs), human dermal microvascular endothelial cells (HMVECs), human umbilical artery endothelial cells (HUAECs), and human lung microvascular endothelial cells (HLMVECs) were maintained in Clonetics Endothelial Basal Medium-2 (EBM-2) supplemented with essential growth factor supplements EGM-2 SingleQuots or EGM-MV SingleQuots (Lonza). All the cell lines were used within 50 passages.
Cell viability assay
Cell viability was assessed using an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay kit (Sigma) with dye conversion at 490 nm, following the manufacturer’s instructions. Briefly, cells were seeded 3 × 103/well in a 96-well flat-bottomed plate and starved in no serum for 18 h, and were then treated with increasing concentrations of dovitinib and stimulated with 30 ng/mL recombinant human VEGF or PDGF-BB (Sigma V7259 or SRP3138). At 72 h, 20 μl of MTS was added to each well. After 1.5 h of incubation at 37°C, the results were analyzed by a plate reader at 490 nm. The sample data was normalized to background readings of medium only.
In vitro migration and invasion assays
For Transwell migration assays, 5.0 × 104 HCC cells or endothelial cells in 500 μl of serum-free DMEM or EBM were added to the cell culture inserts with an 8-μm microporous filter without an extracellular matrix coating (Becton Dickinson Labware). To the bottom chamber was added 800 μL of DMEM or EGM containing 10% FBS. After 24 h of incubation, the cells on the lower surface of the filter were fixed, stained, and counted under a microscope (×100 magnification). The number of migrated cells in five random optical fields from triplicate filters was averaged. For invasion assays, the inserts of the chambers to which the cells were seeded were coated with Matrigel (Becton Dickinson Labware). The number of invaded cells in five random optical fields (×100 magnification) was averaged from triplicate inserts.
For the wound healing assay, the cells were plated in 6-well plates (3 × 105 cells/well) and allowed to attach and reach confluence. A scratch was made using a sterile 100-μl pipette tip and detached cells were removed by washing with PBS. Phase contrast images were taken at specified time points. The scratched wound was evaluated at 18 h (endothelial cells) or at 48 h (HCC cells) after scratching.
Efficacy of dovitinib in an orthotopic HCC model
Male athymic mice between 4 and 5 weeks of age were purchased from Shanghai Institutes for Biological Sciences (Shanghai, China). All the animal studies were conducted in accordance with the principles and procedures outlined in the guidelines of the Institutional Animal Care and Use Committee at Sun Yat-sen University Cancer Center. Mice were anesthetized by continuous inhalation of isoflurane (Baxter Healthcare, NJ).
For orthotopic xenografts, an upper abdominal midline incision was made. MHCC-97H, SMMC7721 or QGY-7703 HCC cells (1 × 106) in 30 μl of culture medium with 33% Matrigel were injected into the left lobe of the liver using an insulin syringe with a 31-gauge needle (Becton Dickinson, NJ). Two weeks later, the nude mice were randomized into three groups of 20 mice each and were treated either with 0.9% sodium chloride or 25 or 50 mg/kg of TKI258 for 14 days. On day 30 after tumor cell inoculation, the animals were weighed, euthanized, and autopsied. The liver and lungs were weighed and sampled for tissue sectioning. To examine lung metastases, 100 sequential lung sections (4 μm) were cut from the lungs of each mouse and every tenth section was stained with hematoxylin and eosin (H&E).
Formalin-fixed and paraffin-embedded sections 4 μm thick were dewaxed in xylene and a gradient of alcohols, hydrated, and washed in PBS. After pretreatment in a microwave oven (30 min in citrate buffer, pH 6.0), endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide for 10 min, and the sections were incubated with 10% normal goat serum for 15 min. Primary antibodies—rabbit polyclonal anti-CD34 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal anti-Ki67 (Abcam, Cambridge, UK) and rabbit polyclonal anti-PARP (Abcam, Cambridge, UK)—were applied overnight in a moist chamber at 4°C. A standard avidin-biotin peroxidase technique (DAKO, Carpinteria, CA) was applied. Briefly, biotinylated goat anti-rabbit immunoglobulin, goat anti-rat immunoglobulin, and avidin-biotin peroxidase complex were applied for 30 min each, with 15-min washes in PBS. The reaction was finally developed using the DAKO Liquid DAB+ Substrate-Chromogen System. The methods for quantification of microvessel density (MVD), proliferation index, and apoptosis index were reported previously [25, 26]. Briefly, the largest section of each xenograft tumor was analyzed by randomly capturing images of microscopic fields at low magnification, and the microvessels or stained cells were counted and averaged. The final results were the mean value of each case from two independent referees.
Immunoprecipitation and immunoblotting
Cells were lysed in cold RIPA buffer (100 mM Tris HCl, 300 mM NaCl, 2% NP40, 0.5% sodium deoxycholate) supplemented with a proteinase inhibitor cocktail (Roche, Indianapolis, IN) and a phosphatase inhibitor cocktail (Merck, Darmstadt, Germany). Protein concentration was determined using a detergent-compatible protein assay according to the manufacturer’s instructions (Bio-Rad). Protein (1 mg) from each sample was immunoprecipitated overnight at 4°C with an anti-VEGFR-2, anti-PDGFR-β, or anti-FGFR-1 (Cell Signaling Technology) antibody plus protein G/A agarose beads (Pierce). Immune complexes were washed with cold RIPA buffer containing proteinase inhibitors and phosphatase inhibitor. Proteins were eluted by boiling in SDS sample buffer, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membrane (Millipore). Membranes were probed with an anti-phosphotyrosine antibody (Cell Signaling Technology) and then stripped with stripping buffer (Abcam). To detect total VEGFR-2, PDGFR-β, and FGFR-1 levels, membranes were re-probed with the same anti-VEGFR-2, anti-PDGFR-β, and anti-FGFR-1 antibody that was used for the immunoprecipitation. Immunoblotting of phospho- ERK1/2 and ERK1/2 (Cell Signaling Technology) was performed on whole-cell lysates (40 μg) with β-actin (Abcam) as a loading control.
Continuous data were expressed as median and range and were compared between groups using one-way ANOVA and Dunnett t test. Categorical variables were compared using the chi-square test, or Fisher’s exact test, where appropriate. All data were analyzed using the SPSS 13.0 computer program, and significant difference was defined as P < 0.05.