Cell culture and reagents

HUEhT-1 (JCRB1458) cells were purchased from Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan). They were cultured at 37 °C in a humidified atmosphere in 5% CO2 in air and maintained with endothelial cell growth medium supplemented with 0.02 ml/ml fetal calf serum, 5 ng/ml recombinant human epidermal growth factor, 10 ng/ml recombinant human basic fibroblast growth factor, 20 ng/ml insulin-like growth factor, 0.5 ng/ml recombinant human vascular growth factor-165, 1.0 µg/ml ascorbic acid, 22.5 µg/ml heparin, and 0.2 µg/ml hydrocortisone (Promo Cell, Heidelberg, Germany). Culture plates and dishes were coated with 0.5 µg/cm2 vitronectin (A14700, Gibco, Carlsbad, CA, USA) in advance of cell culture.

A human tongue squamous cell carcinoma cell line, SAS (JCRB0260) cells, were also purchased from JCRB Cell Bank. The cells were cultured at 37 °C in a humidified atmosphere in 5% CO2 in air and maintained with Dulbecco’s modified eagle medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 µg/mL streptomycin (Gibco).

An immortalized human oral keratinocyte cell line, RT7 cells, were established by transfection of hTERT and E7, as previously described (Fujimoto et al. 2004). The cells were cultured at 37 °C in a humidified atmosphere in 5% CO2 in air and maintained with KGM-Gold Bullet Kit (Lonza, Switzerland) culture medium.

MK-0429 was purchased from MedChemExpress (Monmouth Junction, NJ, USA). A stock solution of MK-0429 were reconstituted with dimethyl sulfoxide (DMSO) (Sigma-Aldrich). In vitro, the stock solution was diluted with culture medium prior to use.

Cell proliferation assay

The proliferation of culture cells was evaluated by determining the number of viable cells using Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. After incubation of cells for 24, 48, or 72 h in 96-well plates with the indicated various concentrations of MK-0429, kit reagent WST-8 was added to the medium and incubated for another 2 h. The absorbance of samples (450 nm) was determined using 800TS™ Absorbance Microplate Reader (BioTek Instruments Inc, Winooski, VT, USA).

Lactate dehydrogenase assay

The cytotoxicity of MK-0429 was evaluated by measuring the lactate dehydrogenase (LDH) activity using Lactate Dehydrogenase Activity Assay Kit (MAK066, Sigma-Aldrich). After incubation of HUEhT-1 cells for 24 h in 96-well plates with the indicated various concentrations of MK-0429, culture supernatant was harvested. Reagents were mixed to prepare samples following the manufacturer’s protocol. The absorbance of samples (490 nm) was determined using 800TS™ Absorbance Microplate Reader (BioTek Instruments Inc).

Wound healing assay

The cells were cultured in 12-well plates until reaching a confluent monolayer, when they were scratched with a 200 µl pipette tip. The cells were washed with phosphate buffered saline (PBS), and the indicated amount of MK-0429 was added to the medium and incubated for 24 h. The same section of the wound size of pre- and post-incubation were observed under a phase-contrast microscopy (BZ-9000; Keyence Corporation, Osaka, Japan), and the reduction rate of the wound area was calculated.

Adhesion assay

After harvesting more than 5.0 × 106 cells of each cell line, the cells were divided into four groups and pre-treated with the intended concentration of MK-0429 for one hour at 37 °C. Each cell suspension was adjusted in serum-free optimal medium for each cell line to 2.0 × 105 cells/500 µl in HUEhT-1 or 1.0 × 105 cells/500 µl in RT7 and SAS. The cells were seeded into 24-well plates precoated with vitronectin and allowed to stand for one hour at 37 °C in a humidified atmosphere in 5% CO2 in air. After rinsing the medium with PBS, adherent cells were fixed with 10% neutral buffered formalin solution and stained with methylene blue solution and measured under microscopic observation.

Western blotting

Following incubation with the culture media, HUEhT-1 cells were cultured in the presence of the indicated concentration of MK-0429 for two days. The cells were harvested, and their protein was extracted using homogenization in a radioimmunoprecipitation assay buffer (Nacalai Tesque, Kyoto, Japan). Western blotting was performed according to our previous study (Nakagawa et al. 2020). In brief, the protein concentration was determined using a Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). From each sample, 20 µg of protein was electrophoresed in 5–20% sodium dodecyl sulfate–polyacrylamide electrophoresis gradient gels (E-T520L, ATTO Corp, Tokyo, Japan) and transferred onto a polyvinylidene difluoride membrane. Non-specific binding was blocked in Tris-buffered saline (TBS) containing a chemical blocking reagent (Ez Block Chemi, ATTO Corp.) and 0.1% Tween-20 for one hour at room temperature. The membranes were incubated with the following primary antibodies: integrin alpha V rabbit mAb (ab179475; at 1: 1000), FAK rabbit mAb (ab40794; at 1: 1000), p-FAK (Y397) rabbit mAb (ab81298; at 1: 1000), (Abcam Inc., Cambridge, MA, USA), MEK 1/2 rabbit pAb (#9122; at 1: 1000), p-MEK 1/2 rabbit mAb (#9154; at 1: 1000), Erk 1/2 rabbit mAb (137F5; at 1: 1000), p-Erk 1/2 rabbit mAb (20G11; at 1: 1000) (Cell Signaling Technology, Danvers, MA, USA), and GAPDH mouse mAb (#MAB374 at 1:2000; Millipore, Billerica, MA, USA) at 4 °C overnight. Following washing with TBS-T, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (GE Healthcare Bio-Sciences) diluted in TBS-T with chemical blocking reagent described above for one hour at room temperature. The proteins of interest were then visualized using an ECL Advance Western Blotting Detection kit (GE Healthcare Bio-Sciences) on the LAS 4000 Mini-Imaging system (Fujifilm, Tokyo, Japan).

Tube formation assay

Tube formation assay was performed using Angiogenesis Assay Kit (Promo Cell) according to the manufacturer’s protocol. Extracellular Matrix Solution (Promo Cell) was applied to the EZVIEW Glass Bottom Assay Plate 96 well (AGC Techno Glass, Shizuoka, Japan). After harvesting more than 9.0 × 105 HUEhT-1 cells, the cells were resuspended in serum-free endothelial cell growth medium (Promo Cell) with 100 ng/ml VEGF165 (Peprotech, Rocky Hill, NJ, USA). After adding MK-0429 to achieve the indicated various concentrations, 2.0 × 104 cells/well were seeded. As an inhibitor control, suramin was administered to the reference cell at a final concentration of 10 µM. The plate was incubated at 37 °C, 5% CO2, and 95% humidity for 16 h. After the cells were washed, fluorescent staining dye was applied to each well, and each cell was examined with a fluorescent phase-contrast microscope (BZ-9000; Keyence Corporation) by bright-field and fluorescent (FTIC/eGFP) field. Tube formation was measured on the images using Image J software (Angiogenesis Analyzer for Image J; US National Institutes of Health, Bethesda, MD, USA). The number of junctions, number of meshes, number of segments, and total length of segments were calculated for each image.

Animal experiments

The animal experimental protocol was reviewed and approved by Review Board of Animal Experiment Committee of Hiroshima University (approval no. A20-158). Four-weeks-old female NOD/SCID mice (CLEA Japan, Inc. Tokyo, Japan) were housed in a temperature and humidity-controlled facility under 12 h of light: 12 h of dark cycle. Animals had ad libitum access to food and water. A xenograft model of human oral cancer was established by inoculating 1.0 × 107 cells of SAS subcutaneously in the posterior neck. MK-0429 was administrated by osmotic minipump. MK-0429 was formulated in 50% DMSO/50% distilled water at a concentration of 20 mg/mL. MK-0429 or a vehicle solution were filled in minipumps (Alzet, # 1004, flow rate 0.11 μL/h). Minipumps were placed in mice subcutaneously in a pocket on the back. The total amount of MK-0429 administered by osmotic pump for 28 days was calculated to be 100 mg/kg. The mice were killed 28 days after tumor inoculation, and tumors were extracted. Tumor weight was measured by a digital scale (Sartorius Entris 5201-1S; Göttingen, Germany), and tumor volume was calculated from the Eq. 4π/3 × (R1 / 2 + R2 / 2)3, where R1 = longitudinal radius, and R2 = transverse radius measured by a caliper.

Histological examinations

Tumor specimens of mouse OSCC xenografts were fixed in 10% buffered formalin and dehydrated in a graded alcohol series. Specimens were then embedded in paraffin and cut into 4 µm thick sections using a microtome. The sections were de-paraffinized with xylene and rehydrated in graded alcohols and stained with hematoxylin and eosin (HE) according to standard protocols. Immunohistochemistry (IHC) was performed with primary antibodies against Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) (rabbit mAb, 55B11; at 1:1000; Cell Signaling Technology), integrin alpha V rabbit mAb (ab179475; at 1: 500), CD31 rabbit mAb (ab76533; at 1: 500) (Abcam Inc.), α-Smooth Muscle Actin (αSMA) (rabbit mAb, ARG66381; at 1: 2000), Ki-67 (rabbit mAb, ARG66347; at 1: 200)(Arigo Biolaboratories Corp, Hsinchu City, Republic of China). The sections were incubated with the primary antibodies at 4 °C for 12 h and visualized with phase-contrast microscopy (BZ-9000; Keyence Corporation).

Statistical analysis

All experiments were repeated at least three times throughout the study. Statistical analysis was performed using Student’s t test using SPSS version 23.0 (IBM Corp., Armonk, NY, USA). Results were described as the mean ± SD. Differences were considered statistically significant at P < 0.05.