Human HNSCC cells, FaDu and HSC-4, were employed in this study. FaDu cells were provided by the American Type Culture Collection (Manassas, VA, USA). HSC-4 cells were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 100 U/ml penicillin G and 100 μg/ml streptomycin and 250 ng/ml amphotericin B (Gibco™ Antibiotic-Antimycotic; Gibco). The cells were maintained in a 5% CO₂/95% air, humidified atmosphere at 37°C. Interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) were purchased from Roche Applied Science (Indianapolis, IN, USA). IL-1β, IL-8, IL-11, IL-13, IL-17, IL-22, hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), cardiotrophin, oncostatin M, interferon (IFN)-β, quercetin, 3,4′,5-trihydroxy-trans-stilbene (resveratrol), fisetin, genistein, chlorogenic acid, estradiol, bisphenol A, sodium butyrate were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Dexamethasone (Dx) and o-dianisidine were from MP Biomedicals (Santa Ana, CA, USA). Daidzein was purchased from Fujicco Co., Ltd. (Kobe, Japan), epigallocatechin gallate from Enzo Life Sciences, Inc. (Framingdale, NY, USA), curcumin from Nacalai Tesque, Inc. (Tokyo Japan), ciglitazone and troglitazone from Cayman Chemical Co. (Ann Arbor, MI, USA), trichostatin A from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), and phorbol 12-myristate 13-acetate from Sigma-Aldrich (St. Louis, MO, USA).

The reporter constructs were prepared by inserting the 5′-flanking regions of human REG III (−1,985 to +87 of REG III gene) (28) upstream of a luciferase reporter gene in pGL4.17 vector (luc2/Neo; Promega Corp., Madison, WI, USA). The REG III promoter vector was introduced into FaDu cells by electroporation using Gene Pulser Xcell™ (Bio-Rad, Hercules, CA, USA) (44), after which the stable transformants were selected in DMEM supplemented with 10% FBS and 500 μg/ml Geneticin^® (Invitrogen) for 3 weeks. Introduction of the REG III promoter/luciferase construct in the Geneticin^®-resistant cells was confirmed by PCR.

The REG III promoter vector was introduced into FaDu cells as described above. The stable transformants were seeded in 24-well plates at an initial density of 1×10⁵ cells/well. After a 24-h incubation, the cells were treated with 20 ng/ml of IL-1β, 20 ng/ml of IL-6, 10 nM of IL-8, 100 ng/ml of IL-11, 100 ng/ml of IL-13, 1 μg/ml of IL-17, 20 ng/ml of IL-22, 50 ng/ml of HGF, 10 nM of bFGF, 10 nM of EGF, 20 ng/ml of TNF-α, 20 ng/ml of cardiotrophin, 20 ng/ml of oncostatin M, 100 nM of Dx, 50 ng/ml of IFN-β, 50 μM of quercetin, 20 μM of resveratrol, 20 μM of fisetin, 50 μM of genistein, 60 μM of chlorogenic acid, 200 μM of o-dianisidine, 10 nM of okadaic acid, 10 μM of daidzein, 100 nM of epigallocatechin gallate, 1 μM of estradiol, 10 μM of bisphenol A, 12.5 μM of curcumin, 50 μM of ciglitazone, 30 μM of troglitazone, 1 μM of trichostatin A, 1 mM of sodium butyrate, or 50 nM of phorbol 12-myristate 13-acetate. After a 24-h treatment, the medium was aspirated from each well and washed with PBS, and the cells were harvested with 100 μl of lysis buffer (0.1 M potassium phosphate, pH 8.8, 0.2% Triton X-100). The cells were processed for luciferase assay using a Pica Gene luminescence kit (TOYO B-Net Co., Ltd., Tokyo, Japan) as described (45–50).

Total RNA was isolated using RNeasy Protect^® Cell Mini Kit (Qiagen, Hiden, Germany) from FaDu cells. cDNA was reverse transcribed from 0.5–2 μg samples of total RNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) as described (44,45,47–51). cDNA was subjected to PCR with the following primers, synthesized and prepared by Nihon Gene Research Laboratories, Inc. (NGRL; Sendai, Japan): β-actin (NM_001101) sense, 5′-GCGAGAAGATGACCCAGA-3′ and antisense, 5′-CAGAGGCGTACAGGGATA-3′; REG III (AB161037) sense 5′-GAATATTCTCCCCAAACTG-3′ and antisense, 5′-GAGAAAAGCCTGAAATGAAG-3′.

Real-time PCR was performed using KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems, Boston, MA, USA) and Thermal Cycler Dice Real-Time System (Takara Bio, Inc., Otsu, Japan) as described (43–45,47–51). PCR was performed with an initial step of 3 min at 95°C followed by 40 cycles of 3 sec at 95°C, 20 sec at 60°C. The level of target mRNA was normalized to the mRNA level of β-actin as an internal standard.

Cell proliferation activity was assessed by using a Cell Counting Kit-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) cleavage; Dojindo Laboratories, Kumamoto, Japan] as described (43,44,47,50,52). Cells were plated in 96-well plates and incubated for 24 h. Initial density of FaDu and HSC-4 cells was 3×10³ cells/well. After 24 h, 30 μM of resveratrol was added to each well and incubated for 24, 48 and 72 h, while dimethylsulfoxide (DMSO; Sigma-Aldrich) was added as a control. At 24 h of incubation, medium was changed and resveratrol was removed from each well. After addition of 10 μl of WST-8 solution, the cells were incubated for another 2 h. The absorbance of each well at 450 nm (reference wavelength at 620 nm) was read by using a Multiskan™ FC Microplate Photometer (Thermo Fisher Scientific, Waltham, MA, USA). Each measurement was repeated at least four times on each cell line.

Cells were exposed to 0, 5 and 10 Gy radiation using a MBR-1520R (Hitachi, Ltd., Tokyo, Japan) operating at 150 kV and 20 mA as described (44), which delivered the dose at 0.8 Gy/min. Chemotherapy involved the application of cisplatin (Nihon Kayaku Co., Tokyo, Japan) to the cultures at a concentration of 0, 1.0, 3.0 or 10 μM as described (44).

As for chemo- and radiosensitivity, the cell viability was assessed using WST-8 cleavage. Cells were plated in 96-well plates and incubated for 24 h. Initial density of FaDu cells was 3×10³ cells/well, and of HSC-4 cells 1×10³ cells/well. After 24 h, 30 μM of resveratrol were added to each well, while DMSO was added as a control, and incubated for 48 h. For radiotherapy, they were then irradiated at 0, 5, or 10 Gy respectively. For chemotherapy, cisplatin (0–10 μM) was added to cultures. Following incubation for additional 72 h, the absorbance of each well at 450 nm (reference wavelength at 620 nm) was read as described above. Each measurement was repeated at least four times on each cell line.

Invasion assays were performed using 24-well Matrigel-coated Transwells (BD Biosciences, Bedford, MA, USA). A total of 4×10⁴ cells of FaDu and HSC-4 were suspended in 200 μl of serum-free DMEM and placed in the top chambers, and 700 μl of DMEM containing 10% FBS were added to the bottom chambers. After 48 h of incubation at 37°C, non-invading cells were removed from the top of the Matrigel with a cotton swab, while invading cells on the bottom surface of the filter were fixed in 4% paraformaldehyde and stained with Giemsa (Sigma-Aldrich) for 10 min. The invading cells were then visualized at ×200 magnification and counted in five fields for each filter.

Data were expressed as means ± standard error (SE). Statistical significant differences between groups were determined by Student’s t-test using StatMate IV (Abacus Concepts, Berkeley, CA, USA). P<0.05 was considered significant.

To investigate activators of REG III gene expression, we tested various stimulative substances in two clones of FaDu cells to which the REG III promoter vector was stably introduced (Fig. 1). Among these various stimulators, polyphenols such as resveratrol, genistein, daidzein, and histone deacetylase inhibitors such as sodium butyrate, significantly increased the REG III promoter activity (Fig. 1). We also tested the concentration-response relationship of resveratrol, genistein, daidzein, sodium butyrate for REG III promoter activation, and found that the optimum concentration was 10 μM (resveratrol), 25 μM (genistein), 40 μM (daizein), and 2 mM (sodium butyrate) (data not shown).

We examined mRNA levels of intrinsic REG III in FaDu cells by using real-time RT-PCR, after treating cells with 10 μM resveratrol, 25 μM genistein, 40 μM daidzein, and 2 mM sodium butyrate, respectively. Resveratrol significantly increased the mRNA levels of REG III as compared with the others (Fig. 2). The combined addition of resveratrol with other candidates did not enhance, rather inhibited, the increment of REG III mRNA expression by resveratrol alone. It might be due to over-stimulation for the signaling pathway, and as a result, it might be higher concentrations for the effect, as reported by Liu et al (53). We also tested the concentration (0–100 μM)-response relationship of REG III mRNA expression by resveratrol and found that the optimum concentration of resveratrol for REG III expression was 30 μM (data not shown).

We sought to evaluate the effect of resveratrol on cell proliferation of HNSCC cells, FaDu and HSC-4. In the proliferation assay using WST-8 cleavage, HNSCC cells (both FaDu and HSC-4 cells) treated with 30 μM resveratrol showed a significant decrease in growth compared to untreated cells as a control. The resveratrol-induced growth inhibitory effect in HNSCC cells was time-dependent (Fig. 3).

We measured the chemo- and radiosensitivity by addition of resveratrol to HNSCC cells using WST-8 cleavage. We found that HNSCC cells treated with resveratrol showed a significant increase in chemosensitivity (3.0 and 10 μM of cisplatin) and radiosensitivity (5 and 10 Gy), as compared with cells treated with DMSO as a control (Fig. 4). Therefore, these results indicated that resveratrol enhances the chemo- and radiosensitivity of HNSCC cells.

We measured the invasive ability by addition of resveratrol to HNSCC cells using an invasion assay. We found that cells treated with resveratrol displayed significantly less invasive ability compared to that of the untreated cells as a control (Fig. 5).