Role of Neurokinin 3 Receptor Signaling in Oral Squamous Cell Carcinoma

Materials and Methods

Immunohistochemical analysis of surgically resected samples. Surgically resected lower gingival, tongue, mouth floor, and buccal squamous cell carcinoma samples were collected as part of routine care by the authors at Okayama University Hospital (Okayama, Japan) in the years 2000-2013. No patient had received chemotherapy or radiation therapy before surgery. The retrospective study was approved by the Ethical Committee of the Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences (Protocol No: 1949). Anti-NK-3R immunohistochemical analysis for NK-3R (anti-rabbit IgG, BIOSS, MA, USA) was performed as described previously (5). Thereafter, sections were viewed under a microscope, and the stained area was measured using imaging software (Lumina Vision, Mitani Corporation, Tokyo, Japan).

Cell lines and culture conditions. Human oral squamous cell carcinoma cell line HSC-2 was obtained from the RIKEN BioResource Center Cell Bank (Tsukuba, Japan). HSC-2 was incubated in Dulbecco's Modified Eagle Medium Nutrient Mixture F12 (DMEM-F12, Gibco, NY, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, MO, USA) and 1% penicillin/ streptomycin (Gibco, NY, USA), and cultured in an atmosphere of 10% CO₂ at 37°C.

Proliferation assay. HSC-2 cells were seeded on 6-well plates, and NKB (0, 0.01, 0.1, 1.0 μM, Peptide Institute Inc., Osaka, Japan) was added every 24 h for 1 week. Cells were collected and the number of cells was determined using a TC10™ automated cell counter (Bio-Rad, Hercules, CA, USA).

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Figure 1.

Comparison of the expression of NK-3R in normal epithelium and oral squamous cell carcinoma by site. A: Analysis of immunohistochemical staining concentration of NK-3R in normal gingiva and oral squamous cell carcinoma at the tongue, mouth floor, buccal mucosa, and gingiva. B: Comparison of immunohistochemical staining concentration of gingival oral squamous cell carcinoma at the oral side and bone invasion site (n=8). Significant differences between the indicated groups were defined as *p<0.05, **p<0.01.

Scratch assay. HSC-2 cells were grown to subconfluence in a 6-well cell culture plate, and a single scratch was then made using the tip of a 200 μL pipette. After washing with phosphate-buffered saline, complete medium containing NKB (0, 0.01, 0.1, 1.0 μM) was added. Serial photographs of the same scratched section were taken after 24 h, and the amount of decrease in the scratched part was measured.

Invasion assay. HSC-2 invasion was assayed in a cell culture insert for a 24-well plate with a pore size of 8.0 μm (Corning, NY, USA) according to the manufacturer's protocol. The upper wells were placed in serum-free DMEM-F12 containing 3.0×10³ HSC-2 cells; the lower wells were filled with DMEM-F12 containing NKB (0, 0.01, 0.1, 1.0 μM). After a 24-h incubation of 10% CO₂ at 37°C, the cells were stained with Diff-Quik (Sysmex Corp., Tokyo, Japan) and observed with a phase contrast microscope.

Real time-polymerase chain reaction (PCR) for NK-3R. HSC-2 cells were grown to subconfluence in a 6-well cell culture plate, and NKB (0, 0.01, 0.1, 1.0 μM) was added. Total RNA was purified using the RNeasy Mini Kit (Qiagen, MD, USA) after 12 h. Furthermore, cDNA was purified using an RNA PCR Kit (AMV) Ver. 3.0 (Takara Bio Inc., Shiga, Japan). The PCR was set up in a 10 μL reaction volume containing 5 μM SsoAdvanced Universal SYBR Green Supermix (BioRad, CA, USA), 10 μM forward NK-3R primer (5’-TTGCGGTGGACAGGTATATGG-3’), 10 μM reverse NK-3R primer (5’-GGCCATTGCACAAAGCAGAG-3’), 2.2 μM distilled water, and sample DNA. PCR was performed in a Chromo 4 Four-Color Realtime PCR System Brochure (BioRad, CA, USA) with an initial denaturation step at 95°C for 30 sec, followed by 35 cycles of 95°C for 5 sec and 62°C for 30 sec.

Immunoblot analysis. HSC-2 cells were rinsed once with ice-cold phosphate buffered saline (PBS) and lysed in an ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1% Triton X-100, 1% NP-40, 10 mM NaF, 100 mM leupeptin, 2 mg/ml aprotinin, and 1 mM phenylmethyl sulfonyl fluoride). Cell lysates containing 10 μg of total protein in a lysis buffer were electrophoresed in Mini-PROTEAN TGX Gels (BioRad, CA, USA) and the proteins were transferred to Immobilon Transfer Membranes (Merck Millipore, Darmstadt, Germany). After blocking, the membranes were incubated with anti-Akt rabbit monoclonal antibody (Cell Signaling Technology (CST), MA, USA), anti-phospho-Akt (p-Akt) rabbit monoclonal antibody (CST), anti-mammalian target of rapamycin (mTOR) rabbit polyclonal antibody (CST), anti-phospho-mTOR (p-mTOR) rabbit monoclonal antibody (CST) and anti-β-actin (β-actin) rabbit monoclonal antibody (CST) at 4°C overnight. The secondary antibody used was anti-rabbit IgG, HRP-linked antibody (CST), and the antibodies were detected using EzWestLumi plus (ATTO, Tokyo, Japan).

Animal experiments. Mouse models of bone invasion by human oral squamous cell carcinoma were established in 4-week-old male BALB/c nude mice (CLEA Japan, Inc, Tokyo, Japan) by inoculating 6.2×10⁵ HSC-2 cells into the bone marrow space of the left tibial metaphysis. SB222200 (Sigma-Aldrich, St. Louis, MO, USA), which is a selective antagonist of the NK-3R, was intraperitoneally administered twice a week over a period of 4 weeks beginning 10 days after the inoculation. For a sham therapy as a control, dimethyl sulfoxide (Sigma-Aldrich) was intraperitoneally administered with the same protocol as SB222200. After 4 weeks, osteolytic bone destruction was assessed, and immunohistochemical analysis for NK-3R (BIOSS) and Ki-67 (anti-rabbit IgG, abcam, Cambridge, UK) was performed as described previously (6). The protocols were approved by the Ethics Review Committee for Animal Experimentation of the Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences (OKU-2016342).

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Figure 2.

The effect of NKB on HSC-2 cell growth. A-C: The cell proliferation (A), migration (B), and chemotaxis (C) were determined by the cell count, migration assay, and Boyden chamber methods after treatment with the indicated concentration of NKB. D: NK-3R expression by treatment with the indicated amount of NKB. E: Detection of anti-pAkt, Akt, pmTOR, and mTOR in HSC-2 cells by immunoblotting after treatment with 0.1 μM of NKB. Significant differences between the indicated groups were defined as *p<0.05, **p<0.01.

Statistical analysis. Analysis of variance was used to immunohistochemically analyze clinical specimens. If there was overall significance, post hoc analysis was performed with Bonferroni's correction for multiple comparisons, simultaneously comparing all five parts: normal, tongue, mouth floor, buccal mucosa, and gingiva. We used unpaired Student's t-test for analysis of two groups. Differences were regarded as significant at a two-tailed p<0.05. The statistical software Excel and SPSS were used.