Various human melanoma cell lines were obtained from the following sources: CRL-1585 (also known as C32 cells), G-361, HMV-II and SK-MEL-28 from the Cell Resource Center for Biomedical Research, Tohoku University (Sendai, Japan); MeWo, SK-MEL-2 and SK-MEL-31 from the American Type Culture Collection (ATCC; Manassas, VA, USA); MM-AN were provided by Dr M. C. Mihm (Department of Dermatology, Harvard Medical School, Boston, MA, USA); GAK and HMY-1 from the Japanese Collection of Research Bioresources (Osaka, Japan). The murine melanoma B16BL6 cell line was also obtained from the RIKEN BioResource Center (Tsukuba, Japan). The human non-small cell lung cancer H460 cell line was also obtained from ATCC. The identities of cell lines used in this study were confirmed by a short tandem repeat analysis (data not shown). The cells were maintained at 37°C under 5% CO₂ in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (all from Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Silencer select siRNAs against NQO1 (cat. no. 4390824; IDs, s4089, s4090, and s4091), NRF2 (cat. no. 4392420; ID, s9491), and a negative control siRNA (cat. no. 4390844), Lipofectamine RNAiMAX transfection reagent and Opti-MEM were all obtained from Thermo Fisher Scientific, Inc. Silencer Select siRNAs were pre-designed and validated by the manufacturer. Cells at 50% confluence were treated for 72 h in prior to the subsequent experiments, with 10 nM siRNA, 10 µl Lipofectamine RNAiMAX transfection reagent, 1 ml Opti-MEM and 9 ml RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin in a 10-cm dish plate in accordance with the manufacturer's instructions.

β-lap, carnosic acid (CA), and a NQO1 inhibitor, ES936, were obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Stock solutions were prepared by dissolving the chemicals in dimethyl sulfoxide at 20 mM (β-lap), 100 mM (CA), and 10 mM (ES936).

The antibody directed against NRF2 (cat. no. ab-62352) was obtained from Abcam (Cambridge, MA, USA). The antibody against NQO1 (cat. no. 3187) was obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). An antibody against β-actin (cat. no. A2228) was obtained from Sigma-Aldrich; Merck KgaA. Anti-Rabbit IgG, HRP-Linked F(ab')₂ Fragment Donkey (cat. no. NA9340) and Anti-Mouse IgG, HRP-Linked Whole Ab Sheep (cat. no. NA931) were obtained from GE Healthcare (Chicago, IL USA). The fluorescent dyes against rabbit IgG (Alexa Fluor-488) was obtained from Thermo Fisher Scientific, Inc. The fluorescent dyes against mouse IgG (Alexa Fluor-594) was obtained from Thermo Fisher Scientific, Inc. DAPI was obtained from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan).

A Cell Counting kit-8 (Dojindo Molecular Technologies, Inc.), which utilizes water-soluble tetrazolium salts, was used to evaluate the proliferation of melanoma cells following drug treatment. All melanoma cell lines were seeded into 96-well plates (5,000 cells/well) and cultured for 24 h prior to treatment with β-lap and/or CA. Following the treatment, the medium in each well was replaced with 100 µl of drug-free fresh medium and 10 µl of Cell Counting kit-8 solution, incubated for an additional 1–2 h, and the absorbance of each well at 450 nm was measured using a Multiskan Spectrum spectrophotometer (Thermo Fisher Scientific, Inc.).

All melanoma cell lines at 80–90% confluence, which were maintained at 37°C under 5% CO₂ in RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin, were washed twice with ice-chilled PBS, treated with 10% trichloroacetic acid for 30 min on ice and then scraped off into a tube. The cell pellet was washed once with deionized water and lysed in 9 M urea, 2% Triton X-100, and 1% dithiothreitol (DTT). Protein concentration was measured using a BCA protein assay kit (EMD Millipore, Billerica, MA, USA) prior to the addition of DTT. Protein samples were separated using SDS-PAGE (10% gel) and then transferred onto polyvinylidene difluoride transfer membranes (Pall Corporation, Portsmouth, UK). All proteins were loaded 30 µg/lane.

For all antibodies, the membranes were blocked with 5% non-fat dried milk in 0.1% Tween-20/PBS at room temperature for 1 h, then probed with an appropriate primary antibodies overnight at 4°C, which were diluted to 1:1,000, and with HRP-conjugated secondary antibodies for 1 h at room temperature, which were diluted to 1:5,000. Each antibody was diluted in 5% non-fat dried milk in 0.1% PBS-Tween-20. Signals were detected with ECL prime detection reagents (GE Healthcare) and ChemiDoc XRS (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Densitometric analysis of each protein signal was performed using ImageJ software (version 1.50; National Institutes of Health, Bethesda, MD, USA) (24).

The B16BL6/pAO1 cell line, conditionally expressing NQO1 under the control of the AID system, was previously established (25). B16BL6/pAO1 cells were treated with β-lap and/or 0.5 mM auxin (BioROIS Co., Ltd., Mishima, Japan) for 24 h in 96-well plates. Cell viability was measured using a CCK-8 (Dojindo Molecular Technologies, Inc.) accordng to the manufacturer's instructions. To detect NQO1 using immunoblotting, B16BL6/pAO1 cells were treated with or without 0.5 mM auxin for 24 h in 10-cm dish plates. Following this treatment, western blotting was performed as aforementioned.

Cells were washed with PBS and fixed with 4.0% formaldehyde and 0.5% Triton X-100 (Sigma-Aldrich; Merck KGaA) for 20 min at room temperature. The slides were then blocked using 5% FBS (Thermo Fisher Scientific, Inc.) and 0.5% Triton X-100 for 30 min at room temperature. Following three washes with PBS, the slides were incubated with a primary antibody (NRF2 or NQO1) in blocking buffer (PBS with 5% FBS and 0.5% Triton X-100) at 4°C, which were diluted to 1:500. The slides were incubated with secondary antibodies: Alexa Fluor-488 (cat. no. A11008) and Alexa Fluor-594 (cat. no. A11005) (1:250; both from Thermo Fisher Scientific, Inc.) for 3 h at 37°C. Cells were counterstained with DAPI (Dojindo Molecular Technologies, Inc.) for 30 min at room temperature and images were captured using a KEYENCE BZ9000 fluorescence microscope (Keyence Corporation, Osaka, Japan). Images were captured at magnification, ×10.

Pearson's correlation coefficient was used to assess the correlation between cell viability and relative NQO1 protein expression. In the cell viability assay, a t-test with the Bonferroni correction was conducted on raw data for the respective doses. P<0.05 was considered to indicate a statistically significant difference. Fitting to a sigmoid function was performed for estimating the half-maximal inhibitory concentration (IC₅₀). One-way analysis of variance followed by Tukey's honest significant difference post-hoc test was used to assess the effect of CA on NQO1 expression. All analyses were conducted using Microsoft Excel 2013 (Microsoft Corporation, Redmond, WA, USA) and RStudio Desktop version 1.0.136 (R Studio, Boston, MA, USA).

A previous study demonstrated that melanoma cell lines exhibit detectable endogenous expression of NQO1, with the level of expression varying among the cell lines (21). In the present study, the protein expression levels of NQO1 and NRF2 were verified (Fig. 1A). As it has been shown that NQO1 is required for the bio-activation of β-lap (14), it was hypothesized that melanoma cells may be sensitive to the anti-proliferative effect of β-lap. To confirm the effect of β-lap, cell viability was preliminarily measured following treatment of the melanoma SK-MEL-28 and GAK cell lines with various concentrations of β-lap. The two cell lines exhibited dose-dependent decreases of cell viability (Fig. 1B). The calculated IC₅₀ values for β-lap were 1.49 and 1.51 µM for SK-MEL-28 and GAK cells, respectively (Table I). The viabilities of the remaining six cell lines were also tested and the IC₅₀ values are presented in Table I. These corresponded to the values that had been reported previously for SK-MEL-28 and another cell line, G361 (26). The association between the basal expression levels of NQO1 and the IC₅₀ values of β-lap in eight melanoma cell lines were then compared. Differences in the basal NQO1 expression level were detected (Fig. 1C and Table I). However, no significant correlation between the basal NQO1 expression levels and IC₅₀ values of β-lap was observed (R=−0.468, P=0.243).

To assess whether NQO1 is involved in β-lap-mediated toxicity in melanoma cell lines, a cell line that conditionally expresses NQO1 under the control of the AID system was used (25,27). In this system, addition of the plant hormone auxin induces the rapid ubiquitination of AID, followed by the proteosome-mediated degradation of ectopically expressed AID-fused NQO1 within 1 h (Fig. 2). As presented in Fig. 2B, β-lap-mediated toxicity in melanoma cells was decreased following auxin treatment in comparison with untreated controls, as assessed by a cell viability assay. For the experiment using siRNAs, CRL-1585 cells were selected as they have relatively higher endogenous expression of NQO1 compared with the other cell lines (Fig. 1A and C). The β-lap-mediated toxicity was decreased following treatment with siRNA against NQO1 (Fig. 2D). However, the effect was not as evident as that in the AID system (Fig. 2A and B), potentially because endogenous NQO1 expression was not completely abolished by transfection with the NQO1 siRNA (Fig. 2C). These results indicated that NQO1 expression was involved in the cytotoxicity of β-lap in melanoma cell lines.

CA is a potent activator of the KEAP1-NRF2 axis (23). In the absence of CA, NRF2 is degraded through the formation of a complex with its inhibitor protein, KEAP1, a member of the E3 ligase family (20). CA induces conformational changes by targeting the cysteine residues on KEAP1 proteins via thiol S-alkylation and the released NRF2 is stabilized by escaping from its degradation complex (23,28). NRF2 is then able to translocate into the nucleus and operate as a transcription factor, forming a heterodimer with one of the small Maf-family proteins, and binding to an antioxidant-responsive element to activate the transcription of target genes, for instance NQO1 (20). Therefore, using the melanoma SK-MEL-28 and GAK cell lines, whether CA treatment would stabilize NRF2 was assessed, leading to further induction of NQO1 expression. The two cell lines had low basal NQO1 and NRF2 expression (Fig. 1A) compared with the non-small cell lung cancer H460 cell line, which has been shown to harbor somatic mutations in the KEAP1 gene, resulting in the high expression of NRF2 (29). Concentrations of CA that did not affect cell viability were used (Fig. 3A and B). Notably, CA was less toxic compared with other KEAP1-NRF2 activators, including sulforaphane (Fig. 3A and B). The expression of NRF2 was associated with that of NQO1 in the two melanoma cell lines (Fig. 3C and D). Furthermore, to confirm the activation of the KEAP1-NRF2 axis by CA in melanoma cell lines, western blot analysis was performed to assess the amount of NRF2 and NQO1 in siRNA-transfected melanoma cell lines. As expected, it was revealed that the significant increase in NQO1 induced by CA was abolished in siNRF2-treated melanoma cell lines (Fig. 3C and D). Furthermore, as assessed by immunofluorescence staining, CA treatment resulted in further NRF2 stabilization and induction of NQO1 expression in the entire population of the two cell lines (Fig. 3E and F). These data indicate that CA may be used as an inducer of NQO1 in melanoma cell lines.

Next, whether the induction of the expression of NQO1 was able to increase the sensitivity of melanoma cell lines to β-lap-mediated toxicity was investigated. As presented in Table I, sensitivity to β-lap was increased by combination treatment with CA. This induction was CA concentration-dependent (Table I). Treatment with the specific NQO1 inhibitor ES936 decreased the sensitivity of all the cell lines to β-lap-mediated toxicity (Table I). The concentrations of ES936 used did not affect cell viability (data not shown). These results indicate that combined treatment with CA increases the sensitivity of melanoma cells to β-lap through induction of NQO1 expression.