Introduction

Skin cancer is the most common type of cancer in the USA, with 5.4 million new cases of basal and squamous cell skin cancers diagnosed in the USA in 2012 (1). Excluding basal and squamous cancers, it is estimated that there will be approximately 0.1 million new cases of skin cancer in 2019 (2). The incidence of skin cancer is rising steadily worldwide because humans are being exposed to increasing amounts of UV radiation and chemical carcinogens that initiate and promote skin neoplastic processes (1,3,4).

Many antioxidative dietary supplements, such as butylated hydroxytoluene, curcumin, silibinin, ursolic acid (5,6,7,8), and anthocyanins/anthocyanidins (9,10,11,12,13,14,15), have been shown to exhibit antioxidative, antiinflammatory, and antimutagenic potency against skin carcinogenesis. Anthocyanins are water-soluble flavonoids, which are the secondary metabolites derived from phenylalanine through the phenylpropanoid, flavonoid, and anthocyanin pathways (16). Different types of anthocyanin compounds are biosynthesized from dihydroflavonols through catalysis by dihydrokaempferol 4-reductase (DFR) and anthocyanidin synthase (ANS) and are modified by glycosylation and acylation (16).

While all types of anthocyanidins (aglycons of anthocyanin) demonstrate antioxidative, antiinflammatory, and apoptosis-inducing activity, delphinidin is one of the most potent compounds due to its rich abundance of hydroxyl groups on the B and C rings (Fig. 1a) (11,17). Delphinidin contributes to the intense blue colors of fruits; it is prevalent in berries and black currants (18) and can also be found in grapes, purple basil, perilla, plums, purple sage, black sorghum, corn husk, Canna indica flowers, purple black rice, black bean coats, and banana bracts (19,20,21,22,23,24,25). Delphinidin inhibits AP-1 transactivation and 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced cell transformation by blocking the ERK/JNK pathway in mouse skin JB6 P+ cells (26). In the two-stage 7,12-dimethylbenz[a]anthracene (DMBA)/TPA skin carcinogenesis mouse model, delphinidin and SOD have been shown to exert a synergistic effect to prevent skin tumor progression (26,27). In addition, delphinidin shows photochemoprevention activity in human HaCaT keratinocytes and SKH-1 mice by reducing UVB-mediated oxidative stress, DNA damage, and apoptosis (28).

Fig. 1
figure 1

a Chemical structure of delphinidin chloride. b Effect of delphinidin on the viability of JB6 P+ cells. JB6 P+ cells were treated with various concentrations of delphinidin for 1, 3, or 5 days as described in the “Material and Methods” section. Cell viability was determined with an MTS cell proliferation assay and is presented as the mean ± SEM

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Nrf2 is a leucine zipper (bZIP) transcription factor that regulates the expression of antioxidant response element (ARE)–dependent genes to modulate the physiological response to the imbalance between free radicals and antioxidants (29). During responses to oxidative and electrophilic stress, Nrf2 is released from the repressor protein Keap1 and thus escapes ubiquitin–proteasome degradation (30,31,32,33). Subsequently, Nrf2 translocates from the cytoplasm to the nucleus, dimerizing with Maf family proteins to activate the ARE pathway, which upregulates the transcription of a variety of cytoprotective genes, including Ho-1, Nqo1, Sod, Gclm, and Mrp1 (31,32,34). The antioxidative stress defense mechanism of the Nrf2-ARE pathway is a potential target for cancer prevention and therapy (32,35,36,37). It has been demonstrated that anthocyanins upregulate Nrf2 target antioxidative proteins and carcinogen-detoxifying enzymes to exert chemopreventive effects both in vitro (in rat liver clone 9 cells) (10) and in vivo (in a hepatocellular carcinoma rat model) (38), implying that delphinidin may be able to activate the Nrf2-ARE pathway. CpG demethylation within the Nrf2 gene promoter region associated with the induction of the Nrf2-ARE pathway has been shown to be an inhibitory mechanism of fucoxanthin against JB6 P+ skin cell transformation (39). The underlying mechanism by which antioxidants modulate the methylation patterns and the transcription of Nrf2 target genes in skin cells remains poorly understood. In this study, we investigated how delphinidin exerts antioxidative activity against skin cell neoplastic transformation by reducing CpG methylation in the Nrf2 promoter region as a candidate agent for chemoprevention.

Material and Methods

Chemicals, Reagents, and Antibodies

Delphinidin chloride with a purity of ≥ 97% as determined by HPLC analysis was purchased from Alkemist Labs (Costa Mesa, CA, USA). 5-Aza-deoxycytidine (5-aza), trichostatin A (TSA), Eagle’s basal medium (BME), and TPA were supplied by Sigma-Aldrich (St. Louis, MO, USA). A CellTiter 96 AQueous One Solution Cell Proliferation Assay System and a luciferase assay system were provided by Promega (Madison, WI, USA). A TOPO TA Cloning Kit and One Shot™ TOP10 Chemically Competent Escherichia coli were purchased from Invitrogen (Waltham, MA, USA).

Cell Culture and Treatment

The mouse epidermal JB6 P+ cell line was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The human hepatocellular HepG2-C8 cell line was established from HepG2 cells stably transfected with the pARE-TI-luciferase construct using the FuGENE 6 method (a gift from Dr. William Fahl, University of Wisconsin) (40). The HepG2-C8 cells were grown and maintained in DMEM supplemented with 10% FBS, and the JB6 P+ cells were maintained in MEM with 5% FBS as instructed by the ATCC. The cells were first seeded and grown in plates for 24 h. Then, the cells were treated with various concentrations of delphinidin, with 0.1% DMSO as a vehicle control, or cotreated with 5-aza and TSA as a positive control in medium with 1% FBS. The treatment medium was renewed every other day. For 5-aza and TSA cotreatment, TSA (50 nM) was only added to the medium 20 h prior to harvesting the cells.

Cell Proliferation Assay

JB6 P+ cells were placed into a 96-well plate at a density of 3 × 103 cells per well and grown for 24 h. Then, the cells were incubated with different concentrations of delphinidin (10, 20, 40, 60, 80, or 100 μM) and with 0.1% DMSO as a vehicle control for 1, 3, or 5 days. Similarly, 5 × 103 cells/well of HepG2-C8 cells were seeded in a 96-well plate and treated with various concentrations of delphinidin (20, 40, 80, or 160 μM) or with 0.1% DMSO for 1 day. The cell culture media were renewed every other day. Cell viability was measured with the CellTiter 96® AQueous One Solution Cell Proliferation Assay System (Promega) at 490-nm absorbance as instructed by the manufacturer.

Anchorage-Independent Cell Transformation Assay

TPA-induced JB6 cell neoplastic transformation was established as indicated in previous publications (41,42). The JB6 P+ cells were seeded in 6-well plates for 24 h and then treated with different concentrations of delphinidin (5, 10, or 20 μM) or 0.1% DMSO for 3 days. Then 8 × 103 pretreated cells were subjected to an anchorage-independent cell transformation assay in 6-well plates with 0.3% BME agar (bacteriological agar, Sigma-Aldrich) as the upper agar and 0.5% BME agar as the bottom agar. The 0.3% and 0.5% BME agar were made by mixing BME supplemented with 10% FBS with 0.6% and 1% agar, respectively, in a 1:1 proportion. Cells pretreated with delphinidin were maintained in the upper agar with 10 ng/mL TPA. Cells not pretreated with delphinidin were also seeded in upper agar with and without 10 ng/mL TPA as positive and vehicle controls. After 14 days of incubation, images of the cell colonies in the soft agar were captured by a microscope camera using Nikon ACT-1 software (Version 2.20, LEAD Technologies, Charlotte, NC, USA). The colonies were quantified with the ImageJ program (Version 1.51d, NIH, Bethesda, MD, USA).

Luciferase Reporter Activity Assay

HepG2-C8 cells stably transfected with a pARE-TI-luciferase plasmid construct were used to examine ARE-driven luciferase activity, as established in our previous study (40). HepG2-C8 cells were seeded in 12-well plates at a density of 1 × 105 cells/well and then grown for 24 h. The cells were then treated with various concentrations of delphinidin or 0.1% DMSO in DMEM with 1% FBS for 24 h. ARE-luciferase activity was measured with a luciferase activity assay kit (Promega). First, the cells were harvested in reporter lysis buffer. Then, 50 μl of luciferase assay reagent was added to 10 μl of the cell lysate supernatant to catalyze the bioluminescent reaction. The relative luminescence units (RLUs) were quantified using a Sirius luminometer (Berthold Detection System GmbH, Pforzheim, Germany). The readouts were normalized to the protein concentration measured with a BCA protein assay (Pierce Biotech, Rockford, IL, USA). The results are presented as the fold change compared with those for the 0.1% DMSO vehicle control.

Western Blotting

JB6 P+ cells were seeded into 100-mm dishes at a density of 3 × 105 cells/dish and then grown for 24 h. Next, the cells were treated with delphinidin (5, 10, or 20 μM) or 0.1% DMSO in MEM supplemented with 1% FBS for 5 days. The cells were lysed, and the total proteins were extracted with RIPA lysis buffer containing a protease inhibitor cocktail using a sonicator (42). The protein concentrations were measured by the BCA method (Pierce Biotech). Next, 25 μg of protein from each sample was added to the wells of 4 to 15% SDS-PAGE gels (Bio-Rad, Hercules, CA, USA). After electrophoretic separation, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA), which was blocked with 5% BSA in TBST to prevent nonspecific binding. Next, the blotting membrane was incubated with different primary antibodies and horseradish peroxidase–conjugated secondary antibodies. Finally, the blot was visualized and photographed with the aid of SuperSignal West Femto–enhanced chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA, USA) using the Gel Documentation 2000 system (Bio-Rad). The intensities of the signal bands were analyzed with ImageJ software, and the relative protein expression was normalized to that of β-actin. Anti-goat, anti-rabbit, and anti-mouse secondary antibodies were provided by Santa Cruz Biotechnology (Dallas, TX, USA). A primary antibody against β-actin was provided by Santa Cruz Biotechnology, and primary antibodies against Nrf2, NQO1, and HO-1 were obtained from Abcam (Cambridge, MA, USA). Primary antibodies against histone deacetylases (HDACs) (HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, and HDAC7) were supplied by Cell Signaling Technology (Beverly, MA, USA), and primary antibodies against DNA methyltransferases (DNMTs) (DNMT1, DNMT3a, and DNMT3b) were provided by Novus Biologicals (Centennial, CO, USA).

Quantitative Real-Time Polymerase Chain Reaction

JB6 P+ cells were seeded into 100-mm dishes at a density of 3 × 105 cells/dish and then grown for 24 h. Next, the cells were treated with various concentrations of delphinidin (5, 10, or 20 μM) or 0.1% DMSO in MEM with 1% FBS for 5 days. RNA isolation was performed using a GeneJET RNA Purification Kit (Thermo Fisher Scientific). The mRNA expression levels of Nrf2 (Nfe2l2) and Nrf2 target genes (Nqo1, Hmox1, and Sod-1) were quantified with an Applied Biosystems 7900HT Fast Real-Time PCR System using the comparative CT (ΔΔCt) method. The relative quantification (RQ) values for the treatment groups were determined compared with the vehicle control (0.1% DMSO) value. The sequences of the forward and reverse primers are as follows: Nrf2: 5′-GGCTCAGCACCTTGTATCTT-3′ and 5′-CACATTGCCATCTCTGGTTTG-3′; NQO1: 5′-GAGAAGAGCCCTGATTGTACTG-3′ and 5′-ACCTCCCATCCTCTCTTCTT-3′; GAPDH: 5′-AACAGCAACTCCCACTCTTC-3′ and 5′-CCTGTTGCTGTAGCCGTATT-3′; HO-1: 5′-CTCCCTGTGTTTCCTTTCTCTC-3′ and 5′-GCTGCTGGTTTCAAAGTTCAG-3′; and SOD1: 5′-GGTTCCACGTCCATCAGTATG-3′ and 5′-GTCTCCAACATGCCTCTCTTC-3′.

Bisulfite Genomic Sequencing

Genomic DNA was extracted from JB6 P+ cells treated with 5, 10, or 20 μM delphinidin or 0.1% DMSO using a GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific) or a QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA). A subset of cells was cotreated with 5-aza (250 nM) and TSA (50 nM) as a positive control group; the TSA was only added 20 h before the harvest of cells. The treatment medium was replaced every other day. Next, the isolated DNA was subjected to bisulfite conversion using an EZ DNA Methylation Gold Kit (Zymo Research, Irvine, CA, USA). The methylation level of the 15 CpG sites located between − 863 and − 1226 nt from the transcription start site in the mouse Nrf2 gene was used as an indicator of Nrf2-ARE pathway activation and cancer development as established in our previous publication (43). The converted DNA fragments were magnified by PCR using Platinum Taq DNA polymerase (Invitrogen). The sequences of the forward and reverse PCR primers are 5′-TTATAATTTATAGTAATAAAATTAGTTATG-3′ and 5′-CCCATTCAATTACTAATACTCAAACAC-3′. A TOPO TA Cloning Kit (Invitrogen) was used to clone the PCR amplicons into pCR4 TOPO vectors, which were then transformed into One Shot™ TOP10 Chemically Competent E. coli (Invitrogen). Finally, 12 colonies from each group were randomly selected and sequenced by GENEWIZ (South Plainfield, NJ, USA).

Statistical Analysis

The quantitative results are expressed as the mean ± SEM, and Student’s t test was applied to evaluate the significance of statistical analysis; * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

Results

Determination of Delphinidin Concentrations and Duration of Treatments

To determine the optimal concentration and treatment duration, the cytotoxicity of delphinidin was evaluated with an MTS cell proliferation assay. Delphinidin inhibited cell viability in a time- and dose-dependent manner (Fig. 1b). Determination of an ideal treatment duration and concentration involves a trade-off between toxicity and efficacy. The 3-day treatment chosen in this study has been shown to be optimal for observing colony number reductions in cell transformation assays (42,44,45,46,47,48,49,50,51,52). In addition, a 5-day treatment was chosen for the qPCR studies because this treatment led to more significant differences between the control and treatment groups than 6-h or 3-day treatment (data not shown). A 5-day treatment was used for the western blot studies due to the greater effect observed after 5-day treatment than after 3-day treatment (data not shown). Finally, a 5-day treatment was determined to be optimal for identifying changes in CpG methylation in the Nrf2 promoter in JB6 P+ cells for the bisulfite sequencing study (42,44,45,49). The cell viability after 1- and 3-day delphinidin treatments (< 20 μM) was above 50%, so treatment concentrations of 5, 10, and 20 μM were used in the subsequent mechanistic studies.

Delphinidin Prevents TPA-Mediated JB6 P+ Cell Transformation

The anchorage-independent growth ability of cells in soft agar colony formation assays reflects their neoplastic transformation. TPA, a well-established promoter of skin carcinogenesis both in vitro and in vivo (53,54), was applied to induce transformation in JB6 P+ cells and was shown to effectively increase colony formation by 7.8-fold compared with the vehicle (Fig. 2). Delphinidin pretreatment significantly attenuated the anchorage-independent growth by 69.4%, 74.4%, and 99.4% in the 5, 10, and 20 μM groups, respectively (Fig. 2). In addition, the number of transformed colonies in the 20 μM group was even less than that in the vehicle control group (Fig. 2). To conclude, the above result indicates that delphinidin can significantly inhibit the TPA-induced anchorage-independent growth of JB6 P+ cells.

Fig. 2
figure 2

Inhibitory effects of delphinidin on TPA-induced JB6 P+ cell transformation. JB6 P+ cells (3 × 105/10-cm dish) pretreated with 5, 10, or 20 μM delphinidin for 3 days were transferred to soft agar containing TPA for another 14 days. Then, the colonies showing anchorage-independent growth on soft agar were measured under a microscope and analyzed using ImageJ software. a Representative image of transformed JB6 colonies. b The statistical data are presented as the mean ± SEM of three replicates from two independent studies. Significant differences were evaluated by Student’s t test (*P < 0.05; **P < 0.01)

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Delphinidin Enhances ARE-Driven Luciferase Activity

To understand the inhibitory mechanism of delphinidin in cell transformation, HepG2-C8 cells transfected with the pARE-Tl-luciferase reporter construct were used to examine the effect of delphinidin on ARE-driven luciferase activity. Delphinidin increased the normalized RLU in a dose-dependent manner (Fig. 3b). In addition, delphinidin significantly increased ARE-driven luciferase activity compared with the control beginning at 20 μM and exhibited a more potent effect than the positive control, sulforaphane (SFN), at 160 μM (Fig. 3b). The above results suggest that delphinidin may induce the transcription of ARE-dependent genes.

Fig. 3
figure 3

Effect of delphinidin treatment on cell viability and ARE-luciferase activity in HepG2-C8 cells. a The cell viability of HepG2-C8 cells was evaluated after treatment with different concentrations of delphinidin for 1 day. Cell viability was quantified with an MTS assay and is shown as the mean ± SEM. b The relative luminescence (in RLU) was normalized to the protein concentrations measured in the BCA protein assay. Sulforaphane (SFN, 5 μM) was used as a positive control. The data are presented as the fold change ± SEM compared with the vehicle control group (0.1% DMSO) value from three independent experiments. Asterisks indicate significant differences between the treatment groups and the vehicle control group, as evaluated by Student’s t test (*P < 0.05; **P < 0.01; ***P < 0.001)

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Delphinidin Upregulates the mRNA and Protein Expression of Nrf2 and Its Target Genes

Nrf2 binding to the ARE region can regulate the expression of the carcinogen-detoxifying phase 2 enzymes HO-1 and NQO1 and the reactive oxygen species (ROS) scavenger SOD1 (55). qPCR and western blotting were conducted to understand the activation of the Nrf2-ARE pathway at the mRNA and protein levels in JB6 P+ cells. Five-day delphinidin treatment upregulated the mRNA expression of Nrf2 (Nfe2l2) and its target genes (Hmox1, Nqo1, and Sod1) in a dose-dependent manner (Fig. 4a). The protein expression of HO-1 was significantly upregulated by 5-day delphinidin treatment (Fig. 4b), consistent with the mRNA expression results. Taken together, the results suggest that delphinidin can trigger Nrf2-ARE pathways by upregulating antioxidative and carcinogen-detoxifying proteins at the transcriptional and translational levels.

Fig. 4
figure 4

Effects of delphinidin on the relative endogenous mRNA expression and protein expression of Nrf2 (NFE2L2) and Nrf2-targeted ARE genes in JB6 P+ cells. a The mRNA expression of Nrf2 (NFE2L2), Nqo1, HO-1 (Hmox1), and Sod-1 was measured in JB6 P+ cells following 5-day treatment with different concentrations of delphinidin. The data are presented as the mean ± SEM of three replicates from two independent experiments, and the results for the treatment groups were compared with those of the vehicle control group (0.1% DMSO). b The protein expression of Nrf2 (NFE2L2), NQO1, and HO-1 was measured in JB6 P+ cells after 5 days of treatment with various concentrations of delphinidin, and β-actin was used as an internal standard. Densitometric analysis was performed with ImageJ software to quantify the protein expression of at least three independent replicates, which is expressed as the mean ± SEM. The levels in the treatment groups were compared with those in the vehicle control group (0.1% DMSO). Student’s t test was used to evaluate the significant differences (*P < 0.05; **P < 0.01; ***P < 0.001)

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Delphinidin Decreases CpG Methylation in the Nrf2 Promoter Region

To further understand the epigenetic alteration mediated by delphinidin, bisulfite genomic DNA sequencing was performed to evaluate the CpG methylation levels in the Nrf2 promoter region. We previously reported that the 15 CpG sites within − 1226 and − 863 nt of the Nrf2 gene promoter are hypermethylated in prostate TRAMP tumors compared with normal prostate tissues in C57BL/6J mice (43). Moreover, the CpG methylation ratio of the Nrf2 promoter is inversely correlated with the expression of Nrf2 target genes (43). In this study, the vehicle control group had a methylation ratio of 90.37% for the 15 CpG sites in the Nrf2 promoter. Five-day treatment with delphinidin decreased the methylation ratio of these 15 CpG sites to 84.55, 82.78, and 82.78% in the 5 μM, 10 μM, and 20 μM groups, respectively (Fig. 5). The positive control group treated with 5-aza (an inhibitor of DNMTs) and TSA (an inhibitor of HDACs) reduced the methylation ratio to 78.89% (Fig. 5), which is consistent with the findings of our previous study (44). In total, the results demonstrate that delphinidin decreases the CpG methylation ratio in the Nrf2 promoter.

Fig. 5
figure 5

Effects of delphinidin on the CpG methylation of Nrf2 in JB6 P+ cells. JB6 P+ cells (3 × 105 cells/10-cm dish) were treated with delphinidin for 5 days, and genomic DNA was extracted. Cotreatment with 250 nM 5-azacytidine (5-aza) and 50 nM trichostatin A (TSA) was used as a positive control, but the TSA was only added 20 h before harvesting cells. The DNA methylation profiles of the first 15 CpG sites between nt − 1226 and − 863 in the Nrf2 promoter were determined by bisulfite genomic sequencing, and at least 12 colonies from each group were selected and sequenced using T7 primers. The CpG methylation ratio was evaluated as the percentage of methylated cytosines in the 15 CpG sites for three replicates from two independent experiments and is expressed as the mean ± SEM. The CpG methylation patterns of the sequences of ten colonies randomly chosen from each treatment group are presented. The solid circles indicate the methylated CpG sites, and the open circles represent the unmethylated CpG sites

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Delphinidin Downregulates the Protein Expression of DNMTs and HDACs

The activity of epigenetic modifying enzymes was evaluated to understand the mechanism of DNA demethylation in the Nrf2 promoter. Delphinidin downregulated the protein expression of DNMTs (DNMT1 and DNMT3a) in a dose-dependent manner in JB6 P+ cells (Fig. 6). The decrease in DNMT3b expression caused by delphinidin was not significant (data not shown). The protein expression of HDACs, which have been reported to regulate DNA methylation synergistically with DNMTs (56), was also evaluated in this study. Delphinidin suppressed class I and class II HDACs in a dose-dependent manner after 5 days of treatment. The repression of DNMTs and HDACs was consistent with the decreased CpG methylation in the Nrf2 promoter region, as shown in Fig. 5. These findings suggest that DNMT and HDAC inhibition is a possible molecular mechanism for demethylation of CpG sites in the Nrf2 promoter and reactivation of the Nrf2 pathway.

Fig. 6
figure 6

Effect of delphinidin on the protein expression of a DNMTs (DNMT1 and DNMT3a), b class I HDACs (HDAC1, HDAC2, and HDAC3) and c class II HDACs (HDAC4, HDAC5, and HDAC7) in JB6 P+ cells. Western blotting was conducted following 5-day delphinidin treatment, and β-actin was used as an internal standard. Densitometric analysis was performed with ImageJ software to quantify the protein expression from three independent experiments, which is expressed as the mean ± SEM. Student’s t test was used to evaluate the significant differences (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001)

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Discussion and Conclusions

The primary prevention of skin cancers involves avoidance of risk factors such as overexposure to UV radiation and chemical pollutants. In situations in which exposure is unavoidable and in individuals with weakened endogenous antioxidative systems due to aging, applying phytochemicals can be a promising strategy for early interference with skin neoplastic development, as these compounds restore homeostasis between the production of ROS and the activity of the antioxidative defense system (57). To understand the effects of phytochemicals and the mechanisms by which they exert their biological activity, systematic study of these compounds at various molecular levels is urgently needed.

In this study, we applied a methodology based mainly on the central dogma of molecular biology. In this methodology, a key cytoprotective pathway involved in cancer development is first selected. Second, an in vitro cell transformation model is designed in which effective carcinogens are applied to initiate cell transformation. Third, the cytoprotective pathway is evaluated with luciferase report assays, qPCR, and western blot analyses to measure gene expression at the transcriptional and translational levels. Finally, epigenetic modifications can be examined by quantifying CpG methylation levels in promoter regions. This methodology can genetically and epigenetically characterize the cancer prevention mechanisms of bioactive compounds to fulfill the need for systematic phytochemical studies.

The ARE is a cis-acting enhancer sequence in the promoter region of various antioxidative genes. Nrf2 can interact with the ARE to activate a variety of antioxidative pathways (58). Nrf2 knockout mice are more susceptible than wild-type mice to chemical carcinogens in various types of cancer models, such as stomach, bladder, and skin cancer models (59). High numbers of skin tumors have been found in Nrf2 knockout mice after exposure to the carcinogens DMBA and TPA (59). Activation of Nrf2 and its target genes, such as Ho-1 and Nqo1, by phytochemicals can block skin cell neoplastic transformation in JB6 P+ cells (39,49). Similarly, in our study, we observed a correlation between inhibition of TPA-mediated anchorage-independent growth and genetic/epigenetic activation of the Nrf2 pathway in JB6 P+ cells. TPA-treated JB6 P+ cells exhibited 7.8-fold greater anchorage-independent growth than vehicle-treated control cells. Pretreatment with delphinidin attenuated the anchorage-independent growth by 69.4%, 74.4%, and 99.4% in the 5 μM, 10 μM, and 20 μM groups, respectively (Fig. 2). The inhibitory mechanism of delphinidin was elucidated by the observed upregulation of ARE-driven luciferase activity, implicating the transcriptional activation of ARE-dependent genes (Fig. 3b). The activation of the Nrf2-ARE pathway was also shown by the increased mRNA and protein expression of the Nrf2 target genes Ho-1, Nqo1, and Sod1 (Fig. 4).

We previously reported that hypermethylation in the Nrf2 promoter was associated with prostate cancer progression in 27 clinical prostate cancer samples and in LNCaP cells (60). Moreover, the inhibitory effect of SFN against TPA-induced neoplastic transformation has been found to be regulated by demethylation of the Nrf2 promoter (42). In this study, we examined whether the CpG methylation level in the Nrf2 promoter region was altered by delphinidin to understand the activation of the Nrf2-ARE antioxidative pathway. Five-day delphinidin treatment reduced the methylation levels at the 15 CpG sites between nt − 1226 and − 863 of the Nrf2 gene promoter in a dose-dependent manner in JB6 P+ cells (Fig. 5). Several previous studies have explored how delphinidin regulates histone acetyltransferase (HATs) and HDACs to exert antiinflammatory and apoptosis-inducing effects (61,62). It has been reported that delphinidin can repress inflammatory signaling by specifically inhibiting HATs to downregulate NF-κB acetylation in fibroblast-like synoviocyte MH7A cells (61). In addition, delphinidin could mediate p53-induced apoptosis by specifically inhibiting HDAC3 to activate p53 acetylation in human prostate cancer LNCaP cells (62). In our present study, we found that delphinidin significantly reduced the expression of DNMT1 and DNMT3a as well as class I HDACs (HDAC1, HDAC2, and HDAC3) and class II HDACs (HDAC4, HDAC5, and HDAC7) in JB6 P+ cells (Fig. 6). This result correlates with the observed demethylation of the Nrf2 promoter (Fig. 5). Further studies are needed to understand the overall chemopreventive effects of delphinidin mediated by inhibition of DNMT and HDAC, such as its effects on cell cycle arrest, differentiation, and cell death (63,64).

In conclusion, this study demonstrates that delphinidin, an epigenetic activator of the Nrf2-ARE pathway, inhibits JB6 P+ cell transformation by demethylating 15 CpG sites of the Nrf2 promoter and inhibiting DNMTs and HDACs. The results suggest that delphinidin has the potential to be applied as a skin cancer chemopreventive agent.