1,5-DiCQA was extracted from Vernonia anthelmintica (L.) Willd. Briefly, Vernonia anthelmintica seeds (1 kg) were pulverized. Ethanol (40%) was added and refluxed at 80°C three times, each time for 1 h. The extracts were combined and concentrated. This extract was subsequently purified by HPD-300 macroporous resin (diameter, 1:8; loading volume, 6 BV; Cangzhou Bon Adsorber Technology Co., Ltd., Cangzhou, China). Following drying, a 60 g powder was obtained. The powder was taken and dissolved in ethanol, and separated by medium pressure preparative chromatography. At last, the fraction was separated and purified by semi-preparative chromatography to obtain 1,5-diCQA, according to a previously described method (21).

The obtained 1,5-diCQA (white solid; purity, 98%) was dissolved in dimethyl sulfoxide (DMSO; 100 mM) and stored at −20 °C as a stock solution. ERK (cat. no. 4696), phosphorylated (p)-ERK (Thr202/Tyr204; cat. no. 9106), p38 (cat. no. 9212), p-P38 (Thr180/Tyr182; cat. no. 5140), CREB (cat. no. 9104), p-CREB (Ser133; cat. no. 9196), glycogen synthase kinase 3β (GSK-3β; cat. no. 9832), p-GSK-3β (cat. no. 9323), β-catenin (cat. no. 8480) and p-β-catenin (ser675; cat. no. 4176) antibodies were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA). p-MITF (cat. no. SAB4301514) antibody, phenyl methyl sulfonyl fluoride and the components of the whole cell lysis buffer for western blot analysis were obtained from Sigma-Aldrich, Merck KGaA (Darmstadt, Germany). β-actin antibody (cat. no. MA1115), rabbit anti-goat secondary antibody (cat. no. BA1060), goat anti-rabbit secondary antibody (cat. no. BA1054) and goat anti-mouse secondary antibody (cat. no. BA1050) were purchased from Wuhan Boster Biological Technology, Ltd., (Wuhan, China). TYR (cat. no. sc-7833), MITF (cat. no. sc-52938), TRP 1 (cat. no. sc-25543) and TRP 2 (cat. no. sc-25544) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The bicinchoninic acid (BCA) protein assay kit was purchased from Beijing Biomed Gene Technology Co., Ltd. (Beijing, China). The ERK (PD98059), JNK (SP600125) and p38 MAPK inhibitors (SB203580) were purchased from Beyotime Institute of Biotechnology (Haimen, China). The JNK inhibitor (SP600125) was obtained from EMD Biosciences (EMD Millipore, Billerica, MA, USA) and dissolved in DMSO. Deoxynucleotide triphosphates (dNTPs, cat. no. BH7201B) were purchased from TAKARA BIO INC. (Yokohama, Japan). The Recombinant RNasin Ribonuclease Inhibitor (cat. no. N2511) and 100 U Moloney murine leukemia virus (M-MLV) reverse transcriptase (cat. no. M170A) were purchased from Promega Corporation (Madison, WI, USA). Power SYBR™-Green PCR Master mix (cat. no. 4367659) was purchased from Applied Biosystems (Thermo Fisher Scientific, Inc., Waltham, MA, USA). 8-Methoxypsoralen (8-MOP, cat. no. M3501) was purchased from Sigma-Aldrich (Merck KGaA) and dissolved in DMSO (50 mM) for storage at −20°C as a stock solution.

B16 murine melanoma cells were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, (Beijing, China). Cells were grown in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 100 µg/ml streptomycin and 100 U/ml penicillin (Gibco; Thermo Fisher Scientific, Inc.; cat. no. 15140-122) in a humidified atmosphere with 5% CO₂ at 37°C.

To examine the effects of 1,5-diCQA on cell viability, a TransDetect Cell Counting kit (CCK) assay (cat. no. FC101; Beijing Transgen Biotech, Co., Ltd., Beijing, China) was performed according to the manufacturer's protocol. Briefly, B16 cells were seeded in 96-well plates (5x10³ cells/well) and allowed to adhere at 37°C for 12 h. Cells were then treated with 0, 5, 25, 50, 100, 200 or 400 µM 1,5-diCQA for 48 h. Following treatment, 10 µl CCK solution was added into each well and cells were incubated at 37°C for an additional 2 h. Optical absorbance was determined at 450 nm with a Spectra Max M5 plate reader (Molecular Devices, LLC, Sunnydale, CA, USA). The absorbance of cells without treatment was considered as 100% cell survival. Each treatment was performed in quintuplicate, and each experiment was repeated three times. To observe the cell morphology, B16 cells were seeded in 6-well plates (1x10⁴ cells/well) and allowed to adhere at 37°C for 12 h. Cells were subsequently treated with 0, 5, 50 or 100 µM 1,5-diCQA for 48 h. Cell morphology was observed and images were captured at room temperature using a LEICA DMI 8 fluorescence inversion microscope (magnification, x200; Leica Microsystems GmbH, Wetzlar, Germany).

The effect of 1,5-diCQA on melanogenesis in B16 cells was investigated according to a previously published protocol, with certain modifications (22). More specifically, intracellular melanin content was measured by using the NaOH dissolution method: B16 cells were seeded in a 6-well plate at a density of 2x10⁵ cells/well, and then allowed to attach for 12 h. Then, cells were treated with 1,5-diCQA at 0, 5, 50, 100 µM or 8-MOP at 50 µM for 48 h, and SB203580 (10 µM); PD98059 (1 µM); SP600125 (10 µM) for 2 h prior to 1,5-diCQA application. Cells were washed with PBS and harvested with 100 µl radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology) in 1.5 Eppendorf (Ep) tubes. Samples were centrifuged at −4°C and 12,000 × g for 20 min, and the supernatant was obtained to determine the protein concentrations. Sediment was dissolved in 150 µl 1M NaOH (with 10% DMSO) for 1 h at 80°C, and solubilized melanin was measured at 405 nm. The amount of melanin was calculated by normalizing the total melanin values with the protein content.

Tyrosinase activity was estimated by measuring the rate of L-DOPA oxidation as previously described, with certain modifications (23). B16 cells were seeded in a 6-well plate at a density of 3×10⁵ cells/well and allowed to attach for 12 h. Then, cells were treated with 1,5-diCQA at 0, 5, 50, 100 µM or 8-MOP at 50 µM for 24 h; SB203580 (10 µM); PD98059 (1 µM); or SP600125 (10 µM) for 2 h prior to 1,5-diCQA application. Cells were then washed with ice-cold PBS twice, 100 µl lysis buffer (1% sodium deoxycholate and 1% Triton X-100 in PBS) was added and cells were collected with a cell scraper into an Ep tube. All tubes were incubated at −20°C for 30 min, and then centrifuged at −4°C and 12,000 × g for 20 min. Following centrifugation, the supernatants were obtained for determining the protein concentrations and tyrosinase activity assay. Protein concentrations were determined using the BCA protein assay kit. A reaction mixture containing 90 µl cell lysate and 10 µl 10 mM L-DOPA was added to each well of a 96-well plate for each sample. Following a 20–60-min incubation (according to the content of dopachrome formation) at 37°C in the dark, the dopachrome product was detected at 490 nm by a multi-plate reader (Spectra Max M5/M5e), The tyrosinase activity of each sample was calculated and corrected for the concentrations of proteins, and normalized with protein content levels.

Total cellular RNA was prepared from B16 cells using TRIzol reagent^® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The quality of RNA samples was assessed using 1.5% agarose gel electrophoresis, at 100 V for 20 min and was subsequently analyzed using Quantity One version 3 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The concentration of total RNA was determined using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc., Wilmington DE, USA). cDNA was synthesized from 1.0 µg total RNA using the Reverse Transcriptase M-MLV according to the protocol of the manufacturer. The cDNA from each sample was used as a template in the RT-qPCR to detect the mRNA level of the 1,5-diCQA target genes. qPCR was conducted as previously described, with certain modifications (24). qPCR was performed in a 7300 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) in a final volume of 25 µl [forward and reverse primers, 0.25 mol/l each; Power SYBR^®-Green PCR Master mix (cat. no. 4367659); and a 1 µl cDNA sample]. The thermoycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. PCR primers were as follows: TYR forward, 5′GAG AAG CGA GTC TTG ATT AG3′ and reverse, 5′TGG TGC TTC ATG CGC AAA ATC3′; TRP 1 forward, 5′GGC CTC TGA GGT TCT TTA AT3′ and reverse, 5′AAT GAC AAA TTG AGG GTG AG3′; TRP 2 forward, 5′ATG AGA AAC TGC CAA CCT TA3′ and reverse, 5′AGG AGT GAG GCC AAG TTA TGA3′; MITF forward, 5′AGT ACA GGA GCT GGA GAT G3′ and reverse, 5′GTG AGA TCC AGA GTT GTC GT3′ (25); β-actin forward, 5′ATG AGA AGG AGA TCA CTG C3′ and reverse, 5′CTG CGC AAG TTA GGT TTT GT3′ (26). Relative expression was analyzed and determined by 2^−ΔΔCq method, normalizing the data to β-actin mRNA levels (27,28). Experiments were repeated in triplicate.

Protein samples that were collected during the melanin content assay was used for western blot analysis. The lysates were denatured in SDS-PAGE protein loading buffer 5X (cat. no. AR1112; Wuhan Boster Biological Technology, Ltd.) separated on 10% SDS-PAGE at 80 V, and transferred onto polyvinylidene fluoride membranes for 2 h at 400 A. Membrane blocking was performed with 5% skim milk dissolved in TBS with 1% Tween-20 (TBST) at room temperature for 1 h and the membrane was incubated with primary antibodies at dilutions of 1:1,000 at 4°C overnight. Subsequent to washing in TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at a dilution of 1:2,000 for 1 h at room temperature. The membranes were then washed with TBST. Proteins were visualized by enhanced chemiluminescent western blotting detection reagents (GE Healthcare, Chicago, IL, USA). Densitometry analysis was performed using Quantity One version 3 (Bio-Rad Laboratories, Inc.).

B16 melanoma cells were treated with 1,5-diCQA at 0, 5, 50 or 100 µM at 37°C for 12 h. Intracellular cAMP levels were measured using a cAMP ELISA kit (cat. no. STA-500; Cell Biolabs, Inc., San Diego, CA, USA) following the manufacturer's protocol.

All data are expressed as mean ± standard deviation. Statistical analysis was performed by one-way analysis of variance followed by Tukey's post-hoc test for multiple comparisons using SPSS 19.0 (IBM Corp., Armonk, NY, USA) and Graphpad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

The total melanin content in the skin is determined by the number of melanocytes and the amount of melanin synthesized by single cells (29). In order to avoid the possibility that effect of melanin synthesis was due to the cell viability, the cytotoxicity of 1,5-diCQA to B16 cells was first determined. B16 cells treated at various concentrations of 1,5-diCQA were incubated for 48 h and compared with the 1,5-diCQA untreated cells. Cell viability was measured using a CCK assay. The results indicated that 1,5-diCQA exhibited no cytotoxicity at concentration ranges of 0–400 µM (Fig. 3A). Concurrently, 1,5-diCQA did not induce any change in cell morphology when compared with the control cells at the concentration of 0–100 µM (Fig. 3B). Accordingly, it was determined that 1,5-diCQA is not cytotoxic to melanoma cells.

The effect of 1,5-diCQA on melanin production in B16 cells was determined by a melanin content assay. As indicated in Fig. 4A and B, intracellular melanin levels increased in response to 1,5-diCQA treatment in a dose- and time-dependent manner, which suggests that 1,5-diCQA may promote melanin synthesis in B16 cells. Fig. 4C denotes the cell precipitation following centrifugation and visual observation. It indicates that cells became darker following the addition of 1,5-diCQA. Studies investigating pigmentary disorders have primarily focused on tyrosinase activity, as it is the most important enzyme in melanin biosynthesis in the melanocytes (30). The results also suggested that 1,5-diCQA promotes intracellular Tyr activity in B16 cells in a dose-dependent manner after 24 h of treatment (Fig. 4D).

The effect of 1,5-diCQA on the expression of melanogenic genes was additionally explored by western blot analysis and RT-qPCR. Western blot analysis indicated that 1,5-diCQA significantly increased the protein expression levels of melanogenic genes TYR, TRP 1, TRP 2 and transcription factor MITF in a dose- and time-dependent manner (Fig. 4E and F). In order to confirm whether the changes in these protein expressions were due to the changes in RNA levels, the effects of 1,5-diCQA on mRNA levels was also detected, and RNA levels of MITF and its downstream genes TYR, TRP 1, and TRP 2. The results demonstrated that the transcriptional levels of MITF, TYR, TRP 1, and TRP 2 were significantly increased in B16 cells in the presence of 1,5-diCQA in a dose-dependent manner (Fig. 5).

It has been demonstrated that the phosphorylation of MAPK and signaling cascades of ERK, JNK and p38 regulate melanin production (31). As the results of the present study indicated that 1,5-diCQA increased melanin production via the induction of the melanogenic enzyme and pigmentation-associated transcription factor MITF, the melanin-associated signal pathways involved were additionally explored. To investigate the involvement of the MAPK signal pathway in 1,5-diCQA-promoted melanin synthesis, western blot analysis was performed on B16 cells following 1,5-diCQA treatment. The results demonstrated that the phosphorylation levels of p38 and ERK, but not JNK, were significantly increased following 30 min treatment with different concentrations of 1,5-diCQA (Fig. 6). These data indicated that 1,5-diCQA may increase melanin synthesis by increasing the levels of p38 and ERK phosphorylation. Therefore, inhibitors of p38 (SB203580), ERK (PD98059) and JNK (SP600125) were applied to verify our prior hypothesis. B16 cells were pre-treated with different inhibitors for 2 h prior to the addition of 1,5-diCQA (100 µM). Then, melanin content and Tyr activities were measured. The results indicated that SB203580 and PD98059 may reverse the 1,5-diCQA effects on melanin content and TYR activities (Fig. 7A–D). However, no effects from the JNK inhibitor (SP600125) were observed (Fig. 7E and F), which was consistent with the western blotting results. In addition, MITF expression was measured by western blot analysis following treatment with SB203580, PD98059, SP600125 and 1,5-diCQA (100 µM) for 48 h (Fig. 8). The results also indicate that 1,5-diCQA increased melanin content through the phosphorylation of p38 and ERK, but not JNK.

The Wnt signal pathway was demonstrated to be closely associated with melanin synthesis (32). In order to investigate whether the Wnt signal pathway was involved in the effects of 1,5-diCQA on melanogenesis, the changes in β-catenin and GSK-3β in B16 cells following treatment with 1,5-diCQA were measured by western blot analysis. B16 cells were treated with 5, 50 and 100 µM 1,5-diCQA for 48 h, and total and phosphorylated forms of β-catenin and GSK-3β were measured. The results indicated that neither total nor phosphorylated β-catenin and GSK-3β were altered following 1,5-diCQA treatment (Fig. 6).

It is well-known that the PKA signaling pathway is involved in melanogenesis. Increased cellular cAMP levels may activate PKA. In turn, activated PKA may activate CREB, leading to the upregulation of MITF transcription (33). Therefore, to determine whether the effects of 1,5-diCQA on melanogenesis were also mediated by the PKA signal pathway, a range of experiments using the PKA inhibitor H89 were conducted to identify if PKA affected this process. B16 cells were pre-incubated with H89 (10 µM) for 2 h prior to the addition of 1,5-diCQA (100 µM), and then incubated for 48 h for the measurement of melanin content and expression of MITF, or incubated for 24 h for the measurement of TYR activity. The results indicated that the effects of 1,5-diCQA on melanogenesis were inhibited following pre-incubation with H89 (Fig. 9A and B). MITF expression was also decreased in the samples that were treated with H89 (10 µM) and 1,5-diCQA (100 µM) (Fig. 9F). In addition, cAMP content of the B16 cells following treatment with 1,5-diCQA was measured. The results confirmed that cAMP content was also significantly increased in response to 1,5-diCQA treatment in a dose-dependent manner (Fig. 9C). The levels of CREB induced by PKA, which may activate MITF transcription levels, were also investigated using western blot analysis. Phosphorylation of CREB was significantly increased following treatment with 1,5-diCQA in time- and dose-dependent manners. (Fig. 9D and E).