Adenosine A2b receptor promotes progression of human oral cancer

The Ethics Committee of the Graduate School of Medicine, Chiba University (approval number, 236) approved the study protocol, which was performed according to the tenets of the Declaration of Helsinki. All patients provided written informed consent.

Immortalized human OSCC-derived cell lines (HSC-2, HSC-3, HSC-4, Ca9-22, Sa3, HO-1-u-1, HO-1-N-1, KOSC2, and SAS) were obtained from the Human Science Research Resources Bank (Osaka, Japan) or the RIKEN BioResource Center (Ibaraki, Japan) through the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology (Tokyo, Japan). Short tandem repeat profiles confirmed cellular identity. All OSCC-derived cells were grown in Dulbecco’s modified Eagle medium/F-12 HAM (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10 % fetal bovine serum (Sigma-Aldrich) and 50 units/ml penicillin and streptomycin (Sigma-Aldrich). Isolated and primary-cultured human normal oral keratinocytes (HNOKs) were used as a normal tissue control [10-15]. They were isolated from healthy oral mucosal epithelial specimens from three young patients at Chiba University Hospital. The HNOKs were primary cultured and maintained in Keratinocyte-SFM (Invitrogen, Carlsbad, CA, USA) comprised of growth supplement (Invitrogen) and 50 units/ml penicillin and streptomycin (Sigma).

One hundred pairs of primary OSCC samples and corresponding normal oral epithelial tissues were obtained during surgeries at Chiba University Hospital. The resected tissues were fixed in 20 % buffered formaldehyde solution for pathologic diagnosis and immunohistochemistry (IHC). The Department of Pathology of Chiba University Hospital histopathologically diagnosed each OSCC sample according to the World Health Organization criteria [16]. Clinicopathologic staging was determined by the TNM (tumor-node-metastasis) classification of the International Union against Cancer [17]. All patients had OSCC that was confirmed histologically; the samples were checked to ensure that tumoral tissue was present in more than 80 % of the specimens.

Total RNA was isolated using Trizol Reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was generated using 5 μg total RNA from OSCC-derived cell lines using Ready-To-Go You-Prime First-Strand Beads (GE Healthcare, Buckinghamshire, UK) and oligo (dT) primer (Hokkaido System Science, Sapporo, Japan) according to the manufacturer’s instructions. The cells were washed twice with cold phosphate-buffered saline (PBS) and centrifuged briefly. The cell pellets were incubated at 4 °C for 30 minutes in a lysis buffer (7 M urea, 2 M thiourea, 4 % w/v CHAPS, and 10 mM Tris pH 7.4) with a proteinase inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). HNOKs proteins were extracted from the same samples obtained for RNA. The protein concentration was measured using the BCA Protein Assay Kit (Thermo, Rockford, IL, USA).

Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed using the LightCycler 480 apparatus (Roche Diagnostics). Primers and universal probes were designed using the Universal Probe Library (Roche Diagnostics), which specifies the most suitable set. The primer sequences used for qRT-PCR were: ADORA2B, forward, 5′-GGGCTTCTGCACTGACTTCT -3′; reverse, 5′-CCGTGACCAAACTTTTATACCTG -3′; and universal probe #34, and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) , forward, 5′-AGCCACATCGCTCAGACAC -3′; reverse, 5′-GCCCAATACGACCAAATCC -3′; and universal probe #60. The transcript amount for ADORA2B was estimated from the respective standard curves and normalized to the GAPDH transcript amount determined in corresponding samples. All samples were analyzed in triplicate, and three independent RNA preparations were analyzed from each cell line.

Protein extracts (20 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in 4-12 % gel, transferred to nitrocellulose membranes, and blocked for 1 hour at room temperature in Blocking One (Nacalai Tesque, Tokyo, Japan). The membranes were incubated with rabbit anti-ADORA2B polyclonal antibody (Chemicon International, Temecula, CA, USA), mouse anti-α tubulin monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti- HIF-1α monoclonal antibody (Santa Cruz Biotechnology), rabbit anti-Akt monoclonal antibody (Santa Cruz Biotechnology), rabbit anti-pAkt monoclonal antibody (Santa Cruz Biotechnology) , rabbit anti-ERK1/2 monoclonal antibody (Santa Cruz Biotechnology), and rabbit anti-pERK1/2 monoclonal antibody (Santa Cruz Biotechnology) overnight at 4 °C. The membranes were washed with 0.1 % Tween-20 in Tris-buffered saline, incubated with secondary antibody and coupled to horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Promega, Madison, WI, USA) for 1 hour at room temperature. The membranes were detected using SuperSignal West Pico Chemiluminescent substrate (Thermo), and immunoblotting was visualized by exposing the membranes to ATTO Light-Capture II (ATTO, Tokyo, Japan). Signal intensities were quantitated using the CS Analyzer version 3.0 software (ATTO).

OSCC cellular lines (HSC-3 and -4) were transfected with ADORA2B shRNA (shADORA2B) or control shRNA (shMock) vectors (Santa Cruz Biotechnology) using Lipofectamine LTX and Plus Reagents (Invitrogen). After transfection, the stable transfectants were isolated by a culture medium containing 2 ng/mL Puromycin (Invitrogen). Two to three weeks after transfection, viable colonies were transferred to new dishes. shADORA2B and shMock cells were used for further experiments.

The transfectants were seeded in six-well plates at a density of 1 × 10⁴ viable cells/well. Cells were harvested for 168 hours and counted every 24 hours. We determined proliferation rates using the 3-(4, 5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega).

Apoptosis detection was performed in transfected and control cells by staining with the Annexin V-FITC Apoptosis Detection kit (Abcam, Cambridge, MA, USA). Cells were harvested at a density of 2×10⁵ cells/ml in 1 × binding buffer and stained with FITC-labeled Annexin V and propidium iodide (PI) for 5 min at room temperature. Samples were immediately analyzed using the FACScaliber flow cytometer (Becton-Dickinson, San Jose, CA, USA).

To create hypoxia, cells were placed for 12 to 48 hours in a modular incubator chamber (Astec, Fukuoka, Japan) and flushed with a gas mixture containing 1 % oxygen, 5 % carbon dioxide, and balance N2.

IHC was performed on 4-μm sections of paraffin-embedded specimens using rabbit anti-ADORA2B polyclonal antibody (Chemicon International) and mouse anti- HIF-1α monoclonal antibody (Santa Cruz Biotechnology). Briefly, after deparaffinization and hydration, the endogenous peroxidase activity was quenched by a 30-minute incubation in a mixture of 0.3 % hydrogen peroxide solution in 100 % methanol; the sections were blocked for 2 hours at room temperature with 1.5 % blocking serum (Santa Cruz Biotechnology) in PBS before reacting with anti-ADORA2B antibody overnight at 4 °C in a moist chamber. Upon incubation with the primary antibody, the specimens were washed three times in PBS and treated with Envision reagent (DAKO, Carpinteria, CA, USA) followed by color development in 3,3′-diaminobenzidine tetrahydrochloride (DAKO). The slides then were counterstained slightly with hematoxylin, dehydrated with ethanol, cleaned with xylene, and mounted. Nonspecific binding of an antibody to proteins other than the antigen sometimes occurred. As a negative control, triplicate sections were immunostained without exposure to primary antibodies, which confirmed the staining specificity. To quantify the status of ADORA2B protein expression in those components, we used the IHC scoring systems described previously [13, 15, 18-20]. The mean percentages of positive tumoral cells were determined in at least three random fields at 400× magnification in each section. The intensities of the ADORA2B immunoreactions were scored as follows: 0+, none; 1+, weak; 2+, moderate; and 3+, intense. The cellular number and staining intensity were multiplied to produce an ADORA2B IHC score. Cases with an ADORA2B IHC score exceeding 120.0 (+3 standard deviation [SD] score for normal tissue) were defined as ADORA2B-positive. The ±3-SD cutoff, which statistically is just 0.2 % of the measurement and would be expected to fall outside this range, was used because it was unlikely to be affected by random experimental error produced by sample manipulation [21]. Two independent pathologists from Chiba University Hospital, neither of whom had knowledge of the patients’ clinical status, made these judgments.