Delphinidin Attenuates Neoplastic Transformation in JB6 Cl41 Mouse Epidermal Cells by Blocking Raf/Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Signaling

Neoplastic transformation of cells is one of the major events that occur during the carcinogenic process. Accumulating evidence supports a strong link between inflammation and carcinogenesis (1, 2). Cyclooxygenase-2 (COX-2) expression and its product prostaglandin E₂ (PGE₂) have been linked to enhanced carcinogenesis, particularly skin cancer (3, 4). Moreover, tumor promoters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), epidermal growth factor (EGF), and UV irradiation, induce COX-2 protein expression in the epidermis of the skin and also in epidermal keratinocytes (5). Previous studies showing that COX-2 inhibitors suppress neoplastic transformation suggest that COX-2 overexpression is one of the mechanisms underlying the induction of neoplastic transformation (6, 7). Therefore, inhibiting neoplastic transformation and COX-2 expression might be an effective strategy for delaying carcinogenesis.

Activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) act as pivotal transcription factors in neoplastic transformation and cancer development (6, 8–10), and are regulated by signaling pathways, including the mitogen-activated protein kinase (MAPK) cascades. MAPKs are commonly up-regulated in various cancer cell types and are involved in cell transformation and proliferation (11). Among the components of the MAPK pathways, the MAPK kinase kinase (e.g., Raf)/MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK) cascade has been the focus of cancer chemotherapy because of its relevance in carcinogenesis. Various tumor promoters, including TPA and EGF, are known to induce transformation in various cell lines through the activation of the Raf/MEK/ERK pathway (8, 12, 13). This pathway was also identified as a key downstream effector of Ras, an oncogene that is mutated in 30% or more of human cancers (14, 15). The Raf/MEK/ERK pathway plays a critical role in linking extracellular signals associated with Ras activation to nuclear transcription events (16). Because aberrant activation of ERK was shown in various tumor types (17, 18), the targeted down-regulation of ERK through inhibition of upstream kinases, such as Raf or MEK, seems to be an effective method for intervening in carcinogenesis.

Epidemiologic studies suggest that a high consumption of fruits and vegetables can reduce the risk of cancer. Anthocyanins are naturally occurring polyphenolic compounds that provide intense color to fruits and vegetables, such as berries, red grapes, purple sweet potato, and red cabbages (19). Previous studies showed that anthocyanidins scavenge reactive oxygen species and suppress cell proliferation and migration, tumor cell invasion, and angiogenesis (20–22). Delphinidin (Fig. 1A), a representative dietary anthocyanidin, was shown to exert the strongest antitumor-promoting effects among the anthocyanidins tested, including delphinidin, cyanidin, petunidin, pelargonidin, peonidin, and malvidin, in TPA-promoted cell transformation of JB6 promotion-sensitive mouse skin epidermal (JB6 P+) cells (23). However, the molecular mechanisms and specific targets of the antitumorigenic effects of delphinidin remain unknown. The present study aimed to identify the molecular mechanisms and direct targets of the antitumorigenic effects of delphinidin in tumor promoter–induced neoplastic cell transformation. Here, we report that delphinidin is a potent inhibitor of Raf1 and MEK1. The inhibition of Raf1 and MEK1 corresponded with a suppression of the phosphorylation of ERK, p90 ribosomal S6 kinase (p90RSK), and mitogen- and stress-activated protein kinase (MSK), and the suppression of AP-1 and NF-κB activation, which subsequently resulted in an inhibition of neoplastic transformation and decreased COX-2 expression.

Delphinidin was purchased from Indofine Chemical and EGF, TPA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and DMSO were from Sigma/Aldrich. Eagle's MEM, basal medium Eagle, gentamicin, and l-glutamine were from Life Technologies, and fetal bovine serum (FBS) was from Gemini Bio-Products. The antibodies against phosphorylated MEK (Ser^217/221), phosphorylated ERK (Thr²⁰²/Tyr²⁰⁴), total ERK, p90RSK, phosphorylated MSK (Thr⁵⁸¹), and total MSK were from Cell Signal Biotechnology. The antibodies against total MEK, Raf1, and COX-2 were from Santa Cruz Biotechnology. The Raf1, MEK1, ERK2, and c-Jun NH₂-terminal kinase (JNK) 1 kinase assay kits were obtained from Upstate Biotechnology. CNBr-Sepharose 4B, glutathione-Sepharose 4B, [γ-³²P]ATP, and a chemiluminescence detection kit were from Amersham Pharmacia Biotech, and the protein assay kit was from Bio-Rad Laboratories. A PGE₂ enzyme immunoassay kit was from Cayman Chemical and the cDNA cycle kit, G418, and the luciferase assay substrate were purchased from Promega. Trizol reagent was from Invitrogen.

JB6 P+ cells and H-Ras–transformed JB6 P+ mouse epidermal (H-Ras JB6 P+) cells were cultured in monolayers at 37°C in a 5% CO₂ incubator in MEM containing 5% FBS, 2 mmol/L l-glutamine, and 25 μg/mL gentamicin. JB6 P+ cells stably transfected with a COX-2 luciferase reporter plasmid were a kind gift from Dr. Chauanshu Huang (New York University School of Medicine, New York, NY) and were described in the previous study (24). JB6 P+ cells were stably transfected with a COX-2, AP-1, or NF-κB luciferase reporter plasmid and maintained in 5% FBS/MEM and 200 μg/mL G418.

Cell viability was measured using the MTT assay. Briefly, JB6 P+ cells (1 × 10⁴) were seeded into 96-well plates and cultured for 6 h. The cells were treated with delphinidin at various concentrations (0-40 μmol/L). The cells were cultured for different times at 37°C followed by incubation with MTT for 4 h. The absorbance of each well was measured at 570 nm using an ELISA reader (Emax, Molecular Devices) at 24-h intervals up to 120 h.

The effects of delphinidin on H-Ras–induced cell transformation or TPA- or EGF-induced cell transformation in JB6 cells were investigated. Basal medium Eagle agar (3.5 mL of 0.5%) containing 10% FBS with or without delphinidin was layered onto each well of six-well plates. JB6 (exposed to TPA or EGF) or H-Ras JB6 cells (8 × 10³/mL) treated or not treated with delphinidin were mixed with 1 mL of 0.33% basal medium Eagle agar containing 10% FBS and layered on top of the 0.5% agar layer. The separate cultures were maintained at 37°C in a 5% CO₂ incubator for 14 d, at which time the numbers of cell colonies were counted under a microscope with the aid of the Image-Pro Plus software program (version 4, Media Cybernetics).

JB6 P+ cells (1.5 × 10⁶) were plated in six-well dishes and grown to 80% confluence. The cells were then exposed to 20 ng/mL TPA for 16 h in the presence or absence of delphinidin. The amounts of PGE₂ released into the medium were measured using the PGE₂ enzyme immunoassay kit.

Reverse transcription-PCR was done in accordance with the instructions provided by Promega. In brief, Trizol reagent was used to extract total RNA from cells, and reverse transcription-PCR was done with a cDNA cycle kit. The sense and antisense primers for COX-2 mRNA were 5′-TGTTCTTGTAACATGACACTTAC-3′ and 5′-GAAGTCCTATGTCTTGACCTCATCA-3′, respectively, which yielded a 448-bp PCR product. The amplification conditions for COX-2 were 40 cycles of 95°C for 90 s, 63°C for 90 s, and 72°C for 150 s, followed by 1 cycle for 10 min at 72°C. The sense and antisense primers for glyceraldehyde-3-phosphate dehydrogenase mRNA were 5′-GGTGAAGGTCGGTGTGAACGGATTT-3′ and 5′-AATGCCAAAGTTGTCATGGATGACC-3′, respectively, which yielded a 448-bp PCR product with 40 cycles of 94°C for 90 s, 54°C for 90 s, and 72°C for 150 s.

Confluent monolayers of JB6 P+ cells stably transfected with a COX-2, AP-1, or NF-κB luciferase reporter plasmid were trypsinized, and cells (8 × 10³) suspended in 100 μL of 5% FBS/MEM were added to each well of a 96-well plate. Plates were incubated at 37°C in a 5% CO₂ incubator. At 80% to 90% confluence, cells were starved by culturing them in 0.1% FBS/MEM for 24 h. The cells were then treated with delphinidin for 30 min and then exposed to 20 ng/mL TPA for an additional 24 h to assess COX-2 activity or 12 h to determine AP-1 or NF-κB activities. After treatment, cells were disrupted with 100 μL of lysis buffer [0.1 mol/L potassium phosphate buffer (pH 7.8), 1% Triton X-100, 1 mmol/L DTT, 2 mmol/L EDTA], and the luciferase activity was measured using a luminometer (MicroLumat Plus LB 96V, Berthold Technologies).

The reporter gene assay for firefly luciferase activity was done using lysates from transfected cells. In addition, the reporter gene vector pRL-SV40 (Promega) was cotransfected into each cell line, with the transfection efficiencies normalized to the Renilla luciferase activity generated by this vector. Cell lysates were prepared by first washing the transfected JB6 P+ cells once in PBS at 37°C. After removing the PBS completely, 500 μL of passive lysis buffer (Promega Dual Luciferase Reporter Assay System) were added, and the cells were incubated for 1 h with gentle shaking. The lysate was then transferred to a reaction tube and the cellular debris was removed by centrifugation. The supernatant fraction was used to measure the firefly and Renilla luciferase activities. Cell lysates (20 μL each) were mixed with 100 μL of Luciferase Assay II reagent, and the emitted firefly luciferase light was measured (Luminoskan Ascent, Thermo Electron). Subsequently, the coelenterazine reagent (100 μL) containing the substrate for the emission of Renilla luciferase light was added for normalizing the firefly luciferase data. The c-fos luciferase promoter (pFos-WT GL3) and constructs were kindly provided by Dr. Ron Prywes (Columbia University, New York, NY) and were described in the previous study (25, 26).

After the cells (1.5 × 10⁶) were cultured in a 10-cm dish for 48 h, they were starved in serum-free medium for an additional 24 h. The cells were then treated with delphinidin at concentrations from 0 to 20 μmol/L for the indicated durations before being exposed to 20 ng/mL TPA for another 30 min. Cells were disrupted, and the supernatant fractions were boiled for 5 min. The protein concentration was determined using a dye-binding protein assay kit (Bio-Rad Laboratories) as described in the manufacturer's manual. Lysate protein (20 μg) was subjected to 10% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane. After blocking, the membrane was incubated with the appropriate specific primary antibody at 4°C overnight. Protein bands were visualized by a chemiluminescence detection kit after hybridization with the appropriate horseradish peroxidase–conjugated secondary antibody. The relative amounts of proteins associated with specific antibodies were quantified using Scion Image (NIH, Bethesda, MD).

The in vitro kinase assays were done in accordance with the instructions provided by Upstate Biotechnology. Briefly, each reaction contained 20 μL of assay dilution buffer [20 mmol/L MOPS (pH 7.2), 25 mmol/L β-glycerolphosphate, 5 mmol/L EGTA, 1 mmol/L sodium orthovanadate (Na₃VO₄), 1 mmol/L DTT] and a magnesium-ATP cocktail buffer. For MEK1, 1 μg of inactive ERK2 substrate peptide was included; for Raf1, 0.4 μg of inactive MEK substrate peptide and 1 μg of inactive ERK2 substrate peptide were included; and for ERK2, 0.33 mg/mL MBP substrate peptide was included. A 4-μL aliquot was removed after the reaction mixture had incubated at 30°C for 30 min, to which 20 μg of the MBP substrate peptide and 10 μL of diluted [γ-³²P]ATP solution were added. This mixture was incubated for 10 min at 30°C, and then 25-μL aliquots were transferred onto p81 filter paper and washed thrice with 0.75% phosphoric acid for 5 min per wash and once with acetone for 2 min. For JNK1, the activating transcription factor 2 substrate peptide was included at 3 μmol/L. An active JNK1 protein (20 ng) and 10 μL of diluted [γ-³²P]ATP solution were incubated at 30°C for 10 min with the above assay buffer and substrate peptide, and then 15-μL aliquots were transferred onto p81 paper and washed thrice with 0.75% phosphoric acid for 5 min and once with acetone for 5 min. The radioactive incorporation was determined using a scintillation counter (LS6500, Beckman Coulter). Each experiment was done thrice.

JB6 P+ cells were cultured to 80% confluence and then starved in 0.1% FBS/MEM for 24 h at 37°C. Cells were treated or not treated with delphinidin for 1 h and then exposed to 20 ng/mL TPA for 30 min, disrupted with lysis buffer [20 mmol/L Tris-HCl (pH 7.4), 1 mmol/L EDTA, 150 mmol/L NaCl, 1 mmol/L EGTA, 1% Triton X-100, 1 mmol/L β-glycerophosphate, 1 mg/mL leupeptin, 1 mmol/L Na₃VO₄, 1 mmol/L phenylmethylsulfonyl fluoride], and centrifuged at 14,000 rpm for 10 min in a microcentrifuge. The lysates (500 μg protein each) were used for immunoprecipitation with an antibody against Raf1 or MEK1 and then incubated at 4°C overnight. Protein A/G Plus agarose beads were then added, and the mixture was continuously rotated for an additional 3 h at 4°C. The beads were washed thrice with kinase buffer [20 mmol/L MOPS (pH 7.2), 25 mmol/L β-glycerolphosphate, 5 mmol/L EGTA, 1 mmol/L Na₃VO₄, 1 mmol/L DTT] and then resuspended in 20 μL of 1× kinase buffer supplemented with 1 μg of inactive ERK2 (for MEK1) or 0.4 μg of inactive MEK1 and 1 μg of inactive ERK2 (for Raf1) and incubated for an additional 30 min at 30°C. MBP (20 μg) and 10 μL of diluted [γ-³²P]ATP solution were then added, and the mixture was incubated for 10 min at 30°C. A 20-μL aliquot was transferred onto p81 paper and washed thrice with 0.75% phosphoric acid for 5 min/wash and once with acetone for 2 min. The radioactive incorporation was determined using a scintillation counter. Each experiment was done thrice.

Recombinant Raf1 (or MEK1; 2 μg) or a JB6 P+ cellular supernatant fraction (500 μg protein) was incubated with delphinidin/Sepharose 4B beads (100 μL, 50% slurry) or Sepharose 4B beads (as a control) in reaction buffer [50 mmol/L Tris-HCl (pH 7.5), 5 mmol/L EDTA, 150 mmol/L NaCl, 1 mmol/L DTT, 0.01% NP40, 2 μg/mL bovine serum albumin, 0.02 mmol/L phenylmethylsulfonyl fluoride, 1× protease inhibitor mixture]. After incubation with gentle rocking overnight at 4°C, the beads were washed five times with buffer [50 mmol/L Tris-HCl (pH 7.5), 5 mmol/L EDTA, 150 mmol/L NaCl, 1 mmol/L DTT, 0.01% NP40, 0.02 mmol/L phenylmethylsulfonyl fluoride], and proteins bound to the beads were analyzed by immunoblotting.

Recombinant Raf1 (or MEK1; 0.2 μg) was incubated with 100 μL of delphinidin/Sepharose 4B or 100 μL of Sepharose 4B in a reaction buffer (see in vitro and ex vivo pull-down assays) for 12 h at 4°C, and ATP was added at either 10 or 100 μmol/L to a final volume of 500 μL and incubated for 30 min. The samples were washed and then proteins were detected by Western blotting.

Insight II (Accelrys) was used for the docking study and structure analysis with the crystal coordinates of MEK1 (accession code 1S9J) and B-Raf (accession code 1UWH) available in the Protein Data Bank.⁵

As necessary, data are presented as means ± SD values, and the Student's t test was used for single statistical comparisons. A probability value of P < 0.01 was used as the criterion for statistical significance.

Delphinidin had no effect on the viability of JB6 P+ cells when applied at concentrations from 0 to 20 μmol/L at 1, 3, or 5 days after treatment (Fig. 1B). TPA is a typical tumor promoter that has been shown to induce two-stage skin carcinogenesis and potently stimulate COX-2 expression in various cell lines (27, 28). Treatment with delphinidin markedly inhibited TPA-promoted transformation of JB6 P+ cells in a dose-dependent manner (Fig. 1C) and at 10 μmol/L inhibited TPA-induced neoplastic transformation by 43% (Fig. 1D).

Delphinidin at 5 to 20 μmol/L inhibited TPA-induced COX-2 expression in JB6 P+ cells (Fig. 2A) and also suppressed TPA-induced COX-2 promoter activity in a dose-dependent manner in JB6 P+ cells stably transfected with a COX-2 luciferase plasmid (Fig. 2B). Delphinidin also attenuated TPA-induced COX-2 expression at the mRNA level (Fig. 2C). The production of PGE₂ peaked at 16 hours following exposure to TPA (data not shown), and delphinidin suppressed the TPA-induced PGE₂ production (Fig. 2D). These results indicated that delphinidin could effectively repress TPA-induced COX-2 expression and PGE₂ production in JB6 P+ cells.

We next measured AP-1 transactivation using JB6 P+ cells stably transfected with an AP-1 luciferase reporter plasmid. Consistent with the above results for cell transformation and COX-2 expression, delphinidin inhibited TPA-induced transactivation of AP-1 (Fig. 3A) and NF-κB (Fig. 3B) in a dose-dependent manner. Because TPA induces c-fos expression mainly through the ERK signaling pathway and subsequently induces AP-1 transactivation, we next investigated whether delphinidin could inhibit c-fos promoter activation using the reporter plasmid carrying the luc gene under the control of the c-fos promoter. Results indicated that delphinidin attenuated TPA-induced c-fos promoter activity (Fig. 3C).

We next found that delphinidin inhibited TPA-induced phosphorylation of MEK and also strongly suppressed the phosphorylation of the MEK downstream kinases, including ERK, p90RSK, and MSK (Fig. 4A). These results suggested that the inhibition of the ERK pathways by delphinidin leads to the suppression of AP-1 and NF-κB activity, resulting in decreased neoplastic transformation.

We investigated the effects of delphinidin on the kinase activities of Raf1 and MEK1, upstream kinases of ERK. Kinase assay data revealed that delphinidin strongly suppressed Raf1 and MEK1 activities in vitro (Fig. 4B, top right and top left). Delphinidin at 10 μmol/L blocked Raf1 and MEK1 activities by 67.2% and 64.3%, respectively, but either had no significant effect or only slightly inhibited ERK2 or JNK1 activities (Fig. 4B, bottom left and bottom right, respectively). Consistent with results from an in vitro kinase assay, an ex vivo kinase assay revealed that delphinidin inhibited TPA-induced Raf1 and MEK1 activities in JB6 P+ cells (Fig. 4C). Additionally, overexpression of Raf1 or MEK1 slightly attenuated delphinidin inhibition of TPA-induced AP-1 activation (Fig. 4D). These results indicated that Raf1 and MEK1 are important molecular targets of delphinidin for the inhibition of cell transformation.

We determined whether delphinidin directly interacts with Raf1 or MEK1. In an in vitro pull-down assay, Raf1 and MEK1 were found in the delphinidin/Sepharose 4B beads but not in Sepharose 4B beads (Fig. 5A). We also found ex vivo binding between delphinidin and Raf1 or MEK1 in JB6 P+ cell lysates (Fig. 5B). Competition data revealed that ATP did not compete with delphinidin for binding with Raf1 or MEK1 (Fig. 5C). These results indicated that delphinidin interacts with Raf1 or MEK1 and results in down-regulation of their respective kinase activities noncompetitively with ATP.

To confirm that inhibition of the Raf/MEK/ERK signaling pathway by delphinidin leads to the suppression of neoplastic transformation, we examined whether delphinidin could inhibit H-Ras–induced or EGF-induced JB6 P+ cell transformation. Results indicate that delphinidin at 10 μmol/L blocked H-Ras–induced or EGF-induced cell transformations by 53.7% or 40.3%, respectively (Fig. 6A and B). These findings further supported the evidence showing that delphinidin suppresses cell transformation mainly by targeting Raf1 or MEK1 (Fig. 6C).

Epidemiologic investigations indicate that moderate consumption of anthocyanin-containing foodstuffs, such as red wine and bilberry extract, is associated with a lower risk of cancer (29, 30). A previous study showed that delphinidin exerted the strongest anticarcinogenic effects among six types of anthocyanidins, including delphinidin, cyanidin, petunidin, pelargonidin, peonidin, and malvidin (23), which is consistent with our finding that delphinidin suppressed TPA-induced neoplastic transformation. Evidence suggests that inflammation is associated with the transformation of normal cells into tumors (31). Overexpression of COX-2 was found to be a general feature of neoplasms, particularly those of epithelial origin, in both experimental animals and humans. Accumulating evidence suggests that high levels of COX-2 expression and PGE₂ production play a role in tumorigenesis, particularly in skin cancer (3, 4, 32). Previous studies also showed that COX-2 inhibitors suppress neoplastic transformation and that COX-2 overexpression induces neoplastic transformation (7, 33). Therefore, the suppression of prostaglandin synthesis by selectively inhibiting COX-2 was suggested as a promising strategy for developing chemopreventive agents. Our results showed that delphinidin suppresses TPA-induced COX-2 expression and PGE₂ production in JB6 P+ cells. These findings indicated that delphinidin exerts potent antitumor-promoting and anti-inflammatory effects.

The AP-1 and NF-κB signal transduction pathways are important in tumor progression, and both AP-1 and NF-κB are activated by tumor promoters (28, 34, 35). Recent studies showed that COX-2 expression is primarily regulated by AP-1, NF-κB, and their upstream kinases, MAPKs (13, 28, 34). The activated form of each MAPK phosphorylates and activates other kinases or transcription factors, thereby altering the expression of the target genes. The induction of neoplastic transformation and COX-2 expression was abolished by the pharmacologic inhibition or dominant-negative knockout of MEK, suggesting that the MEK pathway is partially responsible for cell transformation and COX-2 expression (9, 36). Therefore, inhibition of signal transduction involving MEK, AP-1, and NF-κB might be functionally linked to the antitumor promotion effects of delphinidin. The present study showed that delphinidin inhibits TPA-induced AP-1 and NF-κB transactivation and c-fos promoter activity in JB6 P+ cells. Further, we found that the MEK/ERK/p90RSK/MSK signaling cascade was blocked by delphinidin, suggesting that other upstream effectors serve as target(s) for delphinidin.

The Ras/Raf/MEK/ERK signaling pathway is responsible for the coordination and regulation of cell growth and differentiation in response to extracellular stimulation (37–40). The ERK group is considered the classic MAPK signaling pathway and consists of a MAPK (e.g., ERK1 and ERK2), a MEK (e.g., MEK1 and MEK2), and a MAPK kinase kinase (e.g., Raf or MKKK). MEK1 and MEK2 are dual specificity protein kinases that phosphorylate the downstream target ERK on specific tyrosine and threonine residues. MEKs function as key components of this evolutionarily conserved signaling module and are activated by the phosphorylation of key serine residues on the catalytic domain by the upstream serine kinase Raf. Constitutively activated MEK or Raf results in neoplastic transformation (41). Our results clearly showed that delphinidin was effective at inhibiting Raf1 and MEK1 activities in vitro and ex vivo and resulted in the suppression of ERK phosphorylation. In vitro and ex vivo pull-down assays revealed that delphinidin directly bound Raf1 and MEK1 noncompetitively with ATP, which might account for reduced kinase activities of Raf1 and MEK1. Delphinidin also suppressed H-Ras–induced and EGF-induced neoplastic transformation, supporting the idea that delphinidin inhibits the Raf/MEK/ERK pathway irrespective of the type of inducers stimulating this pathway. Collectively, these findings indicated that the inhibition of cell transformation and COX-2 expression by delphinidin was mainly caused by the suppression of Raf1 or MEK1 activities. In addition to the ERK pathway, JNK also was reported to be involved in neoplastic transformation and COX-2 overexpression in JB6 P+ cells (7, 34). We found that delphinidin inhibited TPA-induced JNK phosphorylation (data not shown) and slightly inhibited JNK1 activity. Thus, delphinidin might have another potent molecular target, such as an upstream kinase of JNK.

Agents targeting multiple kinases might be more effective than highly selective kinase inhibitors administered as single agents. Based on the observations that delphinidin binds to both Raf1 and MEK1 in an ATP-noncompetitive manner, we modeled the binding structures of delphinidin to Raf1 and MEK1. Because the crystal structure of Raf1 is not available, a docking study was carried out using the crystal structure of B-Raf (80% homologous amino acid sequence with Raf1) in complex with BAY43-9006. In the hypothetical structure of the B-Raf/ATP/delphinidin ternary complex, delphinidin could be docked to the pocket separate from the ATP-binding site of B-Raf in contrast with BAY43-9006, which is an ATP-competitive inhibitor (Fig. 6D). Several hydrogen bonds and hydrophobic interactions are involved in the putative binding site of delphinidin. Especially, the C-ring moiety of delphinidin forms hydrogen bonds with the backbone carbonyl group of Gly⁵⁹⁵ and the side chain of Thr⁵⁹⁸ of the activation loop of Raf1 and thereby can stabilize the inactive conformation of the activation loop of B-Raf. Interestingly, delphinidin also could be docked to the pocket separate from but adjacent to the ATP-binding site of MEK1, which is similar to that observed for PD318088 in the crystal structure of the MEK1/PD318088 complex (Fig. 6D; ref. 42). Delphinidin can make several hydrogen bonds and hydrophobic interactions in the ATP-noncompetitive binding site of MEK1. Especially, the interactions by the C-ring moiety are critical for the holding of the activation loop of the inactive MEK1. The inactive conformation of the activation loop can be stabilized by the hydrophobic interactions of Val²¹¹ and Leu²¹⁵ and the hydrogen bond of the backbone amide group of Ser²¹² with the C-ring of delphinidin.

In summary, delphinidin inhibits tumor promoter–induced neoplastic transformation and COX-2 expression in JB6 P+ cells. This inhibition is mediated mainly by the blockage of the Raf/MEK/ERK pathway and subsequent suppression of AP-1 and NF-κB activities. Delphinidin binds with Raf1 and MEK1 and strongly inhibits their kinase activity. Collectively, these results suggested that Raf1 and MEK1 are the potent molecular targets that bind delphinidin for suppressing neoplastic transformation. These results provide insight into the biological actions of delphinidin and the molecular basis for the development of new chemoprotective agents.

No potential conflicts of interest were disclosed.

We thank Dr. C.J. Marshall for mutant Raf1 plasmid.