Anti-tumour effect of lapatinib in canine transitional cell carcinoma cell lines

2.1 Reagents and antibodies

Lapatinib used in the in vitro experiments and in vivo animal experiments were obtained from Selleck Chemicals (Houston, Texas) and LC Laboratories (Woburn, Massachusetts), respectively. Lapatinib was dissolved in dimethyl sulfoxide (DMSO). Recombinant human epidermal growth factor (EGF) was purchased from R&D Systems (Minneapolis, Minnesota). EGF was dissolved in phosphate buffered saline.

For western blotting, mouse primary antibodies against HER2 and Tyr 1112-phosphorylated HER2 were obtained from nanoTools (Teningen, Germany). Other mouse primary antibodies against Tyr 1068-phosphorylated EGFR and rabbit primary antibodies against EGFR, Erk 1/2, Tyr 202/Tyr 204-phosphorylated Erk 1/2, Akt, Ser 473-phosphorylated Akt, and β-Actin were obtained from Cell Signaling (Danvers, Massachusetts). IRDye 680RD donkey anti-mouse and IRDye 800CW donkey anti-rabbit secondary antibodies were purchased from M&S TechnoSystems Inc. (Osaka, Japan). All antibodies used for western blotting were diluted according to the manufacturer's recommendations. For immunohistochemistry (IHC), a rabbit primary antibody against HER2 and EnVision+System-HRP Labelled Polymer Anti-Rabbit were obtained from Dako (Glostrup, Denmark).

2.2 Cell culture

Five canine TCC cell lines (TCCUB, Love, Sora, LCTCC, and MCTCC) were used. TCCUB, Love, and Sora were developed in our previous study,21 whereas LCTCC and MCTCC were provided by Dr. Y. Hoshino, Hokkaido University.22 All cell lines were determined to be free of Mycoplasma using EZ-PCR Mycoplasma Test Kit (Biological Industries, Connecticut). Cells were maintained in RPMI 1640 medium (Sigma-Aldrich, St. Louis, Missouri) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, California) and 1% penicillin—streptomycin (Gibco) at 37°C with 5% CO₂.

2.3 Cell proliferation assay

Cell proliferation was determined by the methylthiazolyl tetrazolium (MTT) assay using Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan), according to the manufacturer's instructions. Briefly, canine TCC cells (TCCUB, Love, Sora, LCTCC, and MCTCC) were seeded in 96-well plates with 5 × 10³ cells per well. After 1 day, the cells were treated with various concentrations of lapatinib (0.001-10 μM) for 24, 48, or 72 hours. MTT solution was then added to each well. The survival fraction was determined based on the absorbance detected at 450 nm using the iMark Microplate Reader (Bio-Rad, Hercules, California). All the samples were examined in quadruplicate, and three independent experiments were performed.

2.4 Western blotting

Canine TCC cells (TCCUB, Love, Sora, LCTCC, and MCTCC) were cultured in petri dishes. When the cells reached 70% to 80% confluence, the cells were washed with Hanks’ Balanced Salt Solution (HBSS; Sigma-Aldrich) and were incubated in serum free medium with each concentration of lapatinib (0.1, 1, and 10 μM) for 1 hour. The cells were then treated with 10 ng/mL EGF for 30 minutes according to manufacturer's datasheet. After washing with HBSS, the cells were incubated with RIPA buffer (Cell Signaling) containing protease and phosphatase inhibitor cocktail tablets (Roche Diagnostics, Mannheim, Germany). The cell lysates were centrifuged at 14 000 rpm at 4°C for 10 minutes. Then, total protein concentrations were measured using a BCA Protein Assay kit (Thermo Scientific, Waltham, Massachusetts).

Equal sample concentrations (12 μg) were loaded in 7.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels for electrophoresis. After separation, proteins were transferred to nitrocellulose membranes and were blocked in Tris-buffered saline (TBS) containing 5% skim milk at room temperature for 1 hour. Primary antibodies were incubated at 4°C overnight. Then, the membranes were washed using TBS with 0.2% Tween 20 (Sigma-Aldrich) and were incubated with the appropriate secondary antibodies at room temperature for 1 hour. Proteins were detected using the ODYSSEY CLx (M&S TechnoSystems Inc.). Expression of β-Actin was used as a protein loading control. The cell lysate of a human ovarian cancer cell line (SKOV3; nanoTools) was used as a positive control according to the manufacture's protocol.

2.5 Cell cycle analyses

Canine TCC cells (Sora and TCCUB) were seeded in 6-well plates with 2 × 10⁵ cells per well. After 1 day, the cells were treated with 10 μM lapatinib or vehicle for 24 hours. The cells were washed with HBSS, centrifuged at 3000 rpm for 3 minutes after trypsinization, fixed in 75% alcohol and kept at −20°C until analyses. The samples were then washed with HBSS and incubated with a staining solution containing 50 μg/mL propidium iodide (Sigma-Aldrich), 0.1 mg/mL RNase A (QIAGEN, Hilden, Germany), and 0.05% Triton X-100 (Sigma-Aldrich) at 37°C for 40 minutes. The DNA contents of the cells were evaluated using a FACS flow cytometer (BD Biosciences, Tokyo, Japan). All the samples were examined in triplicate, and three independent experiments were performed.

2.6 Terminal deoxynucleotidyl transferase deoxyuridine Triphosphate (dUTP) nick end labeling staining

Canine TCC cells (Sora and TCCUB) were seeded on cover glasses with 2 × 10⁵ cells per cover glass. After 1 day, the cells were treated with 10 μM lapatinib or vehicle for 24, 48, or 72 hours. The cells were washed with HBSS and fixed in 4% paraformaldehyde at 4°C for 25 minutes. Transferase dUTP nick end labeling (TUNEL) staining was then performed using the DeadEnd Fluorometric TUNEL System (Promega, Madison, Wisconsin) according to the manufacturer's protocol. Cells treated with 1 U DNase I (Promega) were used as positive controls.

2.7 Xenograft mouse model

Female BALB/cSlc nude mice (4 weeks old) were purchased from Japan SLC, Inc. (Shizuoka, Japan). The mice were acclimated to our facilities for 2 weeks prior to the experiment. To develop tumour-engrafted mice, canine TCC cells (Sora) were subcutaneously injected with 2 × 10⁶ cells into the left side flank of each mouse. The greatest longitudinal and transverse diameters of tumours were measured using a vernier calliper every 2 days, and tumour volume was calculated based on the following formula: tumour volume = (long diameter) × (short diameter)² × 0.52. Six days after tumour cell inoculation, the mice were treated with lapatinib (100 mg/kg; n = 10) or vehicle control (saline containing 10% DMSO and 10% Tween 20; n = 9) daily via intraperitoneal administration for 14 days.23 Clinical signs such as decreased activity, anorexia, vomiting, diarrhoea, or weight loss were daily evaluated to assess toxicity of lapatinib. At the endpoint, the mice were euthanized, and the tumours were collected. After measurement of tumour weight, the tumours were fixed in 10% neutral buffered formalin and embedded in paraffin. All of the experimental procedures using mice were approved by the Animal Care Committee of the Graduate School of Agricultural and Life Sciences at the University of Tokyo (authorization number: P17-127).

2.8 Histopathology

Sections of 4 μm were cut from the formalin-fixed paraffin-embedded tumour tissues from the lapatinib and vehicle control groups, and haematoxylin and eosin staining was performed. The tumour tissues were photographed with a 2× objective and a 10× eyepiece to capture the tumours in whole. Using image analysis software (ImageJ, U.S. National Institutes of Health, Bethesda, MD),24 percentage of necrosis in each tumour tissue was calculated by a single evaluator (K.S).

2.9 IHC for HER2

The 4-μm sections were deparaffinized in xylene and hydrated in graded ethanol. Antigen retrieval was performed at 121°C for 10 minutes using 10 mM citrate buffer (pH 6.0). After cooling for an hour, the slides were treated with peroxidase-blocking solution (Dako) at room temperature for 10 minutes, followed by three washes in TBS with 0.1% Tween 20 (TBS-T). After blocking with TBS-T containing 5% skim milk (Wako, Osaka, Japan) for 1 hour, the sections were incubated with the anti-HER2 rabbit polyclonal antibody (diluted to 1:100 in TBS) at 37°C for 40 minutes. The sections were sequentially incubated with the secondary antibody at 37°C for 40 minutes. The peroxidase reaction was conducted with 3,3′-diaminobenzidine tetra-hydrochloride (Dako) for 10 minutes. The reaction was stopped by two washes in distilled water, and counterstaining using Mayer's haematoxylin was then performed. Incubation of sections with the antibody diluents served as negative controls. The IHC protocol used for canine TCC tissues was described in a previous study with a slight modification.17

2.10 Statistical analyses

Student's t tests were used for comparisons between two groups. Prism software version 5.0.1 (Graph Pad Software, San Diego, California) was used for the statistical analysis. Statistical significance was defined as P < 0.05.

3.1 Basal protein expressions of HER2, EGFR, and their phosphorylated forms

HER2 and EGFR protein expressions were observed in all canine TCC cell lines (Figure 1A). The HER2 protein level was high in Love and Sora, whereas that of EGFR was high in TCCUB and MCTCC (Table 1). Autophosphorylation of HER2 and EGFR was not observed in any of the canine TCC cell lines (Figure 1A).

3.2 Effects of lapatinib on cell proliferation

Lapatinib showed dose-dependent growth inhibitory activity in all of the canine TCC cell lines at 24 hours (Figure 1B). Love and Sora were more sensitive to lapatinib than TCCUB, LCTCC, and MCTCC. In particular, in Sora, cell viability decreased to 23.1% of the control after treatment with 10 μM lapatinib. The 50% inhibitory concentrations (IC₅₀) of lapatinib correlated with the protein levels of HER2 in the canine TCC cell lines, whereas the protein level of EGFR was not associated with the sensitivity to lapatinib (Table 1). Similar results were obtained at 48 or 72 hours (data not shown).

3.3 Effects of lapatinib on signalling pathways of HER2 and EGFR

HER2 and EGFR were phosphorylated in all of the canine TCC cell lines through treatment with EGF (Figure 2). Lapatinib inhibited the EGF-induced phosphorylation of HER2 and EGFR in a dose-dependent manner (Figure 2). The total protein expression of HER2 and EGFR did not alter with lapatinib treatment.

As shown in Figure 3A, phosphorylation of Erk 1/2 and Akt was observed in unstimulated Sora cells. EGF treatment mildly enhanced the phosphorylation of Erk 1/2 and Akt. Lapatinib inhibited the phosphorylation of Erk 1/2 and Akt in a dose-dependent manner. In particular, phosphorylation of Erk 1/2 was more strongly suppressed than that of Akt. The total protein expression of Erk 1/2 and Akt did not alter with lapatinib treatment. Cell cycle analyses using flow cytometry showed that the sub-G₁ and G₀/G₁ phase fractions were significantly increased, whereas the S and G₂/M phase fractions were significantly decreased in Sora cells after treatment with lapatinib compared with the vehicle control (Figure 3B and C). After the treatment with lapatinib for 24 hours, there were a few TUNEL-positive cells in Sora (Figure 3D). Similar results were obtained at 48 or 72 hours (data not shown). Results of western blotting, cell cycle analysis, and TUNEL staining in TCCUB were similar to those in Sora (Figure S1).

3.4 Effects of lapatinib on tumour growth in canine TCC-engrafted mice

Six to fourteen days after treatment, the tumour volume in the lapatinib group was significantly smaller than those in the vehicle control group (P < 0.01, Figure 4A). The tumour weight collected from the mice was significantly reduced in the lapatinib group compared with the vehicle control group (P < 0.001, Figure 4B and C). During the treatment, mild soft stools and weight loss (<5%) were observed in the lapatinib group. The mean ± SD of the body weight at the end of the study was 25.1 ± 1.1 g and 23.9 ± 0.8 g in the control and lapatinib groups, respectively. There was no significant difference in the body weight of the mice between the two groups. Histological analyses showed that lapatinib significantly increased the necrotic area in the tumour tissues (P < 0.05, Figure 4D). HER2 protein overexpression was observed on the membrane of tumour cells in both of the lapatinib treatment and vehicle control groups (Figure 4E).