Cal 27 (ATCC^® CRL-2095), FaDu (ATCC^®, HTB-43), Het-1A (ATCC^®, CRL-2692) and NK-92 (ATCC^® CRL-2407) cells were purchased from the American Type Culture Collection (ATCC). PE/CA-PJ34 (clone C12) (ECACC, 97062513) was purchased from Sigma-Aldrich; Merck KGaA. HSC-2, HSC-3, and HSC-4 were obtained from the Health Science Research Resources Bank (Osaka, Japan). All HNSCC cell lines were cultured in DMEM, 10% FBS and 1% penicillin-streptomycin. NK-92 cells were cultured in Alpha Minimum Essential Medium without ribonucleosides and deoxyribonucleosides, but containing 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 12.5% horse serum, 12.5% FBS and 100 UI/ml IL-2. Cell lines were authenticated every 6 months during the course of experiments via short-tandem repeat testing (UC Berkeley DNA Sequencing Facility). Mycoplasma testing was also performed during the course of this study. NK-92 was free of mycoplasma, but other HNSCC cell lines were all mycoplasma positive. Cell lines were passaged for a period of 3 months (~24 passages) after thawing from liquid nitrogen.

R,S-sulforphane was purchased from LKT Laboratiories, Inc. Recombinant human IL-2 was obtained from PeproTech. IL-2 was reconstituted in 100 mM acetic acid and diluted in PBS containing 0.1% BSA.

HNSCC cells or Het-1A cells were plated in 6 cm dishes (2 million/dish) 24 h prior to treatment. The cells were then treated with either vehicle (0.1% DMSO) or 10 mM sulforaphane for 8 or 16 h for quantitative PCR or immunoblotting experiments, respectively.

Total RNAs from cultured cells and murine tissues were purified using miRNeasy^® Mini Kit (Qiagen). cDNAs were synthesized using Superscript III First-Strand cDNA Synthesis System (Life Technologies; Thermo Fisher Scientific, Inc.)] according to the manufacturer's instructions. PCR reactions were performed using SYBR Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) and a Bio-Rad CFX96 C1000 Touch™ Thermal Cycler. Quantification was performed using the 2^−∆∆Cq method (55). Gene-specific primers for NQO1, GCLC, and GAPDH were as previously described (41). Primers for AKR1C2, AKR1C18, AKR1C19, HMOX1 and HSPA1A were from Qiagen. Relative mRNA levels were standardized to the mRNA levels of GAPDH gene.

RNAs from cultured cells were purified as described above and cDNAs were synthesized by the RT² First Strand kit (Qiagen) and used to probe the Human Oxidative Stress Plus RT2 profiler PCR Array (Qiagen, cat. #330231), according to the manufacturer's recommendations. Gene expression data were analyzed using the Web-Based PCR Array Data Analysis from SABiociences.

C57BL/6 mice (5–6 weeks; 5 mice/group) were treated by vehicle (PBS) or sulforaphane (6 µmol/mouse) via oral gavage for 6 or 18 h. Following treatment, mice were sacrificed and tissues harvested.

Cells were washed with ice-cold PBS twice and then lysed with RIPA lysis buffer (150 mM Tris, pH 7.4, 100 mM NaF, 120 mM NaCl, 100 µM sodium orthovannadate, and 1X protease inhibitor cocktail and phosphatase inhibitor cocktail; Roche Diagnostics). Lysates (20 µg) were resolved by SDS-PAGE, transferred to PVDF Membranes (Bio-Rad, #1620177), and incubated with primary antibodies at 4°C overnight, followed by incubation with horse radish peroxidase-conjugated secondary antibodies (Bio-Rad, #170-6516)] for 1 h at room temperature. Immunoreactive bands were visualized by chemiluminescence (Santa Cruz Biotechnology, #SC2048 or Thermo Fisher Scientific, #1856194). Antibodies against NRF2 (#12721), β-tubulin (#2146), and GAPDH (#5174) were from Cell Signaling Technology. Antibodies against HMOX1 (#A11919) and HSPA1A (#A12948) were from ABclonal. Anti-MICA/B (#SC-2093) was from Santa Cruz Biotechnology.

Cells were seeded in 96-well plates and incubated overnight. The following day, cells were treated with different concentrations of sulforaphane for 48 h, then stained with crystal violet for 30 min. Crystal violet solution was removed from the wells and the plates were washed under tap water before being dried for 24 h. Crystal violet-stained material was dissolved with 100 mM sodium citrate solution and subsequently quantified using a colorimetric plate reader at OD₅₉₀.

NK-92 cell-mediated cytotoxicity was assessed using the CytoTox 96^® Non-Radioactive Cytoxicity Assay (Promega Corporation, #G1780), according to the manufacturer's protocol. HNSCC cells were pre-treated with vehicle (0.1% DMSO) or sulforaphane for 48 h, then washed with medium twice. NK-92 cells and pre-treated HSNCC cells were counted and plated in round-bottom 96-well plates at ratios of 2.5:1, 5:1, and 10:1. Wells containing NK-92 cells alone or HNSCC cells alone served as controls for spontaneous LDH release of effector cells and target cells, respectively. To assess the target cell maximum LDH release, lysis buffer was added for one hour to wells containing HNSCC cells alone, followed by harvesting of the supernatant. Prior to supernatant harvest, 96-well plates were centrifuged at 250 × g for 5 min then kept in a 37°C incubator for 5 h. Plates were then centrifuged again at 250 × g for 5 min and 50 µl of supernatant from each well was transferred into a new 96-well plate. 50 µl/well of reconstituted substrate mix was then added to the wells. Plates were subsequently incubated at room temperature in the dark for 20 to 30 min, followed by addition of 50 µl stop solution to each well and reading of absorbance at 490 nm. The cytotoxicity mediated by NK-92 cells was calculated as follows: % cytotoxicity=(ELR-ESR-TSR-MB)/(TMR-TSR-LBB), where ELR, experimental LDH release; ESR, effector spontaneous release; TSR, target spontaneous release; MB, medium background; TMR, target spontaneous release; and LBB, lysis buffer background.

Cells in 6-cm dishes were transfected with 25 pmol of siRNA oligonucleotides mixed with Lipofectamine RNAiMAX (Thermo Fisher Scientific, Inc., #13778500). NRF2 siRNA oligonucleotides and non-target siRNA (siNT) were obtained from Sigma-Aldrich; Merck KGaA. The target sequence for NRF2 siRNAs was: 5′-UGACAGAAGUUGACAAUUA-3′.

Statistical differences between sulforaphane and vehicle treatment groups were determined using ANOVA followed by adjustment for multiple comparison by Bonferroni's method. Error bars for all figures represent SD.

We first sought to identify potential biomarkers of sulforaphane activity in HNSCC cells, as well as normal mucosal epithelial cells. Two HNSCC cell lines, PE/CA-PJ34 and FaDu, and a putative normal, non-tumorigenic mucosal epithelial cell line, Het-1A (56), were treated for 8 h with vehicle or 10 µM sulforaphane, followed by preparation of cellular RNAs. The RNAs were converted to cDNAs, then used to probe Human Oxidative Stress Plus PCR Arrays. This array enables expression profiling of 84 different human genes related to oxidative stress. Comparison of RNA expression levels in sulforaphane-treated vs. vehicle-treated cells allowed identification of genes whose expression was induced greater than 2-fold by sulforaphane treatment. In both HNSCC cell lines we observed >2-fold induction of 11 genes (Fig. 1A and B). HMOX1, encoding heme oxygenase 1, and HSPA1A, encoding a member of the heat shock protein 70 family, were the most strongly induced genes in both HNSCC cell lines, with induction levels ranging from roughly 10- to 20-fold. In the normal epithelial cell line, Het-1A, 9 genes were found to be induced >2-fold by sulforaphane treatment, with HMOX1 again being the most potently upregulated (~18-fold; Fig. 1C). HSPA1A was the third most potently induced gene in Het-1A (~10-fold).

We next sought to validate the findings we obtained with the Oxidative Stress Array by performing RT-qPCR analyses. PE/CA-PJ34, FaDu, and Het-1A cells were again treated with vehicle or 10 µM sulforaphane for 8 h and RNAs were prepared. RT-qPCR was then performed for 4 genes (HMOX1, HSPA1A, AKR1C2, GCLC) that were found to be upregulated in the Array studies. In addition, RT-qPCR was used to assess expression of NQO1, a known downstream target of sulforaphane (45). As shown in Fig. 2A-C, HMOX1 and HSPA1A were strongly upregulated by sulforaphane in all three cell lines, with induction levels ranging from roughly 8-fold to 27-fold. Similar upregulation of HMOX1 and HSPA1A was observed in three additional HNSCC cell lines (Cal 27, HSC-2, HSC-3; Fig. S1). In the HNSCC cell lines, HMOX1 and HSPA1A were the most potently induced genes, whereas AKR1C2 (~29-fold induction) was the most upregulated in Het-1A. Surprisingly, NQO1 was only weakly upregulated by sulforaphane.

Collectively, these experiments suggest that RNAs for oxidative stress genes, particularly HMOX1 and HSPA1A, may represent valuable biomarkers of sulforaphane activity in HNSCC and normal epithelium of the upper aerodigestive tract.

We next confirmed that sulforaphane induction of mRNAs for oxidative stress genes was accompanied by upregulation of the corresponding oxidative stress proteins. Five HNSCC cells lines (PE/CA-PJ34, FaDu, Cal 27, HSC-2, HSC-3) and Het-1A cells were treated with vehicle or sulforaphane (10 mM) for 16 h, followed by immunoblot detection of HMOX1, HSPA1A, or the control protein GAPDH (Fig. 3). As shown, sulforaphane treatment led to upregulation of HMOX1 and HSPA1A in all cell lines examined (Fig. 3), although the fold induction was less than was observed with mRNA induction for these proteins (Fig. 2). It should be noted that while we observed sulforaphane induction of HMOX1 in all cell lines, we did not detect nuclear translocation of the protein (data not shown).

We next determined whether the oxidative stress genes induced by sulforaphane in HNSCC cells and Het-1A cells are induced in vivo in wild-type C57BL/6 mice. Mice were treated by oral gavage (5 per group) with a single dose of vehicle (6 or 18 h) or a single dose (6 µmol) of sulforaphane (6 or 18 h). The dose of 6 µmol/mouse was chosen, as we have previously shown that this dose is well tolerated and prevents the development of carcinogen-induced HNSCC tumors in mice (41). Following treatment, mice were sacrificed and RNAs purified from liver or peripheral blood mononuclear cells (PBMCs) for analysis by RT-qPCR (Fig. 4A and B). Since the AKR1C2 gene is human-specific, we instead analyzed the related murine genes AKR1C18 and AKR1C19. In liver, sulforphane treatment, primarily at the 18-h time-point, led to >2-fold upregulation of all 5 genes analyzed (HMOX1, HSPA1A, AKRC18, AKRC19, GCLC). HSPA1A was unique in being induced only at the 6-h treatment time-point. In PBMCs, only HMOX1, AKRC19, and GCLC were upregulated >2-fold, and only at the 18-h time-point. Immunoblotting of protein lysates from liver tissue revealed elevated expression HMOX1 protein in 3 of 5 mice treated with sulforaphane (18 h; Fig. 4C), validating upregulation at the protein level.

We next examined the impact of sulforaphane on expression of genes encoding members of the NKG2D ligand family (MICA, MICB, ULBP1-6), as well as the DNAM-1 ligands CD112 and CD155. Both NKG2D ligands and DNAM-1 ligands stimulate NK cell cytotoxicity. Treatment of HNSCC cells with sulforaphane resulted in a modest upregulation of RNA for MICA, with little apparent effect on other members of the NKG2D ligand family, as assessed by RT-qPCR (Fig. 5A). Immunoblotting with an antibody that cross-reacts with both MICA and MICB was also performed (Fig. 5B). Consistent with findings at the RNA level, sulforaphane treatment of the HNSCC cell lines PE/CA-PJ34 and Cal 27 also induced upregulation of MICA/B protein. Modest induction of RNAs for CD112 and CD155 was seen after 12 and 48 h of sulforaphane treatment, but not at the 24-h time-point (Fig. 5A).

To determine whether sulforaphane induction of MICA/B was dependent on NRF2 transcription factor, we utilized siRNA directed against NRF2 mRNA to prevent upregulation of NRF2 protein following sulforaphane treatment (Fig. 5C). As shown, in cells treated with a non-targeting siRNA (siNT), sulforaphane treatment resulted in upregulation of NRF2 and MICA/B. Treatment with NRF2 siRNA (siNRF2) markedly reduced NRF2 RNA levels (Fig. 5C, left panel) and prevented sulforaphane induction of NRF2 protein (right panel). Importantly, siNRF2 treatment also blocked sulforaphane upregulation of MICA/B, indicating that sulforaphane effects on MICA/B expression are dependent on NRF2.

The ability of sulforaphane to upregulate MICA/B in HNSCC suggested that sulforaphane treatment may sensitize HNSCC cells to NK cell-mediated cytotoxicity. To test this, we first needed to identify a concentration of sulforaphane that would be only minimally toxic when used alone. Dose-response analyses were performed (Fig. 6) with PE/CA-PJ34 (IC₂₅=9.1 µM) and Cal 27 (IC₂₅=7.1 µM), and a sulforaphane concentration below the IC₂₅'s, 5 µM, was chosen for subsequent experiments. We then pre-treated 6 different HNSCC cell lines for 48 h with vehicle or 5 µM sulforaphane before co-culturing for 5 h with the NK cell line NK-92 at different target to effector ratios. LDH release cytotoxicity assays were then performed. As Fig. 7 illustrates, sulforaphane treatment resulted in statistically significant sensitization to NK-92-mediated cytotoxicity in only one of the six HNSCC cell lines, Cal 27 cells. These findings raise questions whether sensitization to NK-mediated cytotoxicity represents a general mechanism contributing to the chemopreventive activity of sulforaphane against HNSCC.