62 Dickinson, Franklin Lakes, NJ). Western blot analysis Cell lysates were prepared using a lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM ethylenediamine- tetraacetic acid (EDTA), 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1 mM sodium orthovanadate; 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μM leupeptin, and 1 μg/ml aprotinin). After centrifugation of the lysate at 15,000 g for 20 min, supernatants were recovered, and protein was quantified by the Bradford protein assay using a reagent kit from Bio-Rad (Hercules, CA). Protein samples (20–60 µg) were size-separated by electrophoresis on sodium dodecylsulfate-polyacrylamide gels under non-reducing conditions. Separated proteins were electroblotted onto nitrocellulose membranes. The blot was blocked by incuba- tion in a blocking buffer (5% skim milk, 10 mM Tris, pH 7.5, 10 mM NaCl and 0.1% Tween 20) for 1 h at 20°C, and then incubated with the primary antibody overnight at 4°C. Incubation with a horseradish peroxidase (HRP)- conjugated anti-mouse or rabbit secondary antibody was then carried out at room temperature (RT) for 4 h. Antibody- bound proteins were detected by the ECL Western blotting analysis system (Perkin Elmer Life and Analytical Science, Boston, MA). Transfection and dual luciferase assay KB cells were plated in 12-well plates and pWWP (0.25 g) were transfected by Lipofectamine 2000 reagent according to the manufacturer’s protocol when plates reached 50–60% confluence. After 6 h, cells were treated with complete media containing either vehicle (DMSO) or SFN for 20–22 h. Cells were then lysed with 100 μl of 1X reported lysis buffer and 30 μl of cell extract was used for luciferase assays. Results are expressed as means ± S.D. for at least three independent determinations for each treatment group. Immunohistochemistry For immunohistochemistry, an EnVisionTM Detection Kit System (Dako, Glostrup, Denmark), which is based on the peroxidase-catalysed deposition of biotinylated tyramide was used. Briefly, 4-μm-thick sections were cut from a paraffin- embedded tissue block. Sections were then deparaffinized and hydrated by sequential immersion in xylene and grade alcohol solution. For antigen retrieval, sections were im- mersed in a 0.1 M citrate buffer solution at pH 6.0. After boiling twice in a microwave oven for 5 min, the slides were cooled to RT and rinsed in phosphate buffered saline (pH 7.4). The endogenous peroxidase activity was quenched by treating the specimens with 3% hydrogen peroxide for 5 min at RT. The sections were incubated with primary anti- bodies for 1 h at 37°C and then incubated with the secondary J.-H. Kim et al. antibody for 30 min at RT. Staining was visualized using a diaminobenzidine (DAB) and counterstained with Mayer’s hematoxylin. The negative control was performed with the incubated antibody diluent as a substitute for the primary antibody. Nude mouse xenograft assay Female nude BALB/cAnNCrjBgi-nu mice (6-week-old) were purchased from Orient Ltd. (Seoul, South Korea), and maintained in accordance with Institutional Animal Care Use Committee guidelines. A xenograft was established by s.c. injection of in vitro cultured KB cells (106 cells/200 ul) into the flank of mice. Mice were randomized into two groups of 5 mice/group (2 tumors/mouse). Experimental animals were treated orally with SFN (50 μg/kg) 3 times/ week (Monday, Wednesday and Friday) for 21 days begin- ning the day of tumor cell implantation. Control mice received an equal volume of the vehicle. Tumors were measured along two diameter axis with calibers to permit calculation of tumor volume, V = π/6{(D + d) / 2}3, where D and d were the larger and smaller diameters, respectively. Statistical significant differences in tumor volume, wet tumor weight or body weight between control and treated mice was assessed by Student’s t tests. Statistical analysis Statistical significance was assessed using Student’s t tests. A value of p<0.05 compared with solvent control was considered statistically significant. Results Effect of sulforaphane on KB and YD-10B cell proliferation The growth-inhibitory effect of SFN was examined initially by assessing the proliferation efficacy on KB and YD-10B human oral carcinoma cells using cell counting. As shown in Fig. 1A and B, SFN significantly reduced the viability of both cell lines. Treatment with 40 μM SFN inhibited the cell proliferation of KB and YD-10B cells by approximately 58% and 46%, respectively. The results of the cell counting assay were confirmed by estimating the number of viable cells using an MTT cell proliferation assay. This assay also showed that SFN inhibits cell viability (Fig. 1C and D). Based on these results, we confirmed that SFN has a growth-inhibitory effect on cell proliferation in KB and YD-10B cells. Sulforaphane induces the G2/M phase cell cycle arrest Since SFN inhibits KB cell proliferation, we analyzed the cell cycle distribution under the SFN-induced growth- inhibitory conditions using flow cytometry analysis. When the cells were treated with SFN for 12 h, cell proliferation was arrested and accumulated mainly in the G2/M phase J. Clin. Biochem. Nutr.