Arbutin, L-DOPA, L-tyrosine, N-phenylthiourea (PTU), 12-O-tetradecanoylphorbol-13-acetate (TPA), TYR, sodium hydroxide (NaOH), thiazolyl blue tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), rabeprazole, esomeprazole, lansoprazole and pantoprazole were obtained from Sigma-Aldrich (St. Louis, MO, USA). All other reagents and chemicals were of high-grade and are commercially available.

TYR activity was measured as described previously (6). The mushroom TYR (EC 1.14.18.1) reactions were performed in 96-well microplates (SPL Life Sciences Co., Ltd., Pocheon, Gyeonggi, Korea) and consisted of 150 µl of 0.1 M phosphate buffer (pH 6.5), 3 µl of test sample, 36 µl of 1.5 mM L-tyrosine and 7 µl of mushroom TYR [2,100 U/ml in 0.05 M phosphate buffer, (pH 6.5)]. The initial absorbance of each mixture and the absorbance after incubation at 37°C for 30 min, was measured at 490 nm using a microplate reader (PerkinElmer, Waltham, MA, USA). The inhibitory activity was subsequently calculated using the following formula: Inhibitory activity (%) = [(A - B) - (C - D)]/(A - B) × 100; where A refers to the absorbance of the control sample following the reaction, B is the absorbance of the control sample prior to the reaction, C is the absorbance of the treated sample following the reaction and D is the absorbance of the treated sample prior to the reaction.

The copper-chelating activity was determined using the pyrocatechol violet (PV) method as described previously, and with slight modifications (12). Samples were mixed with 1 ml of 50 mM sodium acetate buffer (pH 6.0) and 100 µl of 2 mM CuSO₄. After 10 min of incubation at room temperature, 100 µl of 2 mM PV was added. Solutions were incubated for 20 min at room temperature and the absorbance of each sample at 632 nm (A₆₃₂) was measured. DMSO and PTU were used as a negative control and as a standard metal chelator, respectively. The copper-chelating activity was calculated using the following formula: Copper-chelating activity (%) = [1 - (A - B)/(C - D)] × 100; where A is the A₆₃₂ of the sample + buffer + CuSO₄ + PV mixture, B is the A₆₃₂ of the sample + buffer + buffer + PV mixture, C is the A₆₃₂ nm of the buffer + buffer + CuSO₄ + PV mixture and D is the A₆₃₂ nm of the buffer + buffer + buffer + PV mixture.

The melan-a immortalized mouse melanocyte cell line, which was derived from B57BL6 mice (13), was purchased from Bennett et al (13) (St. George's University of London, London, UK). The cells were cultured in RPMI-1640 medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (HyClone), streptomycin-penicillin (100 µg/ml each; HyClone) and 200 nM TPA (a potent tumor promoter) at 37°C in 5% CO₂. Cells were passaged every 3 days up to a maximal passage number of 40. Confluent monolayers of melanocytes were harvested using a mixture of 0.05% trypsin and 0.53 mM EDTA (Lonza, Basel, Switzerland).

Cell viability was determined using an MTT assay (6). Several concentrations of test samples (25, 50, 100 and 200 µM) were added to the cells and incubated for 24 h. A total of 100 µl of MTT solution [5 mg/ml MTT in phosphate-buffered saline (PBS)] was added to each well and samples were incubated at 37°C for 1 h. After removal of the MTT solution, 1 ml of DMSO was added to each well with vigorous mixing. The absorbance of each well at 470 nm was determined using a microplate reader (PerkinElmer).

Cells were seeded in 24-well plates (BD Biosciences, Franklin Lakes, NJ, USA) at a density of 1×10⁵ cells/well and allowed to attach overnight. The supernatants were subsequently removed and replaced with fresh media containing various concentrations (25, 50, 100 and 200 µM) of the test compounds. Cells were cultured for 72 h and further incubated for 1 day. Following washing with PBS, the cells were lysed with 250 µl of 1 N NaOH and transferred to 96-well plates. The melanin content was estimated by measuring the absorbance at 405 nm using a microplate reader (PerkinElmer). Arbutin was used as a positive control (10).

Total RNA was extracted from melan-a cells using TRIzol reagent (Invitrogen Life Technologies, Waltham, MA, USA), according to the manufacturer's instructions, with slight modification (14). Total RNA (2 µg) was reverse transcribed with RT (MP Biomedicals, Santa Ana, CA, USA) and oligo (dT) primers. cDNA was amplified using a PCR Thermal Cycler Dice TP600 (Takara Bio, Inc., Shiga, Japan) and oligonucleotide primers specific for mouse transcripts (6). The resulting PCR products were visualized by ethidium bromide staining following agarose gel electrophoresis.

Data are expressed as the means ± standard deviation. Statistical significance was determined by the Student's t-test for independent means using Microsoft Excel software (Microsoft Corporation, Redmond, WA, USA). P<0.05 was considered to indicate a statistically significant difference.

The effect of PPIs on the activity of TYR, a crucial enzyme for melanin synthesis, was examined. As depicted in Fig. 1, rabeprazole treatment resulted in significant, dose-dependent inhibition of TYR activity and the 50% inhibitory concentration (IC₅₀) of this compound was 110.1 µM. The IC₅₀ of the arbutin positive control was ~212.3 µM (Fig. 1A). By contrast, the PPIs esomeprazole, lansoprazole and pantoprazole exerted no inhibitory effect on TYR activity.

To understand the role of metal chelation in PPI TYR inhibition, the copper-chelating activities of each PPI were examined. In general, each of the PPIs tested exhibited strong copper-chelation activities. The copper chelation values of the PPIs were higher compared with that of the PTU positive control (Fig. 1B). While esomeprazole, lansoprazole and pantoprazole did not affect TYR activity, these compounds did exhibit copper-chelating activity. Conversely, rabeprazole exhibited TYR inhibitory and copper-chelating activity, indicating that this compound may inhibit TYR by chelating copper.

To examine the effect of PPIs on melanin biosynthesis, melan-a cells were treated with various concentrations (25, 50, 100 and 200 µM) of PPIs, and melanin content and cell viability were subsequently assessed. No cytotoxicity was observed in the melanocytes treated with PPIs or arbutin at concentrations of ≤100 µM (Fig. 2A). Cytotoxicity in melan-a populations treated with 200 µM of rabeprazole, esomeprazole, lansoprazole and pantoprazole was observed as ~25.8±6.9, 19.5±3.9, 24.6±5.8 and 31.6±10.1%, respectively.

At a concentration of 100 µM, PPI treatment resulted in significant decreases in melanin content without inducing cell death. Specifically, treatment with rabeprazole, esomeprazole, lansoprazole and pantoprazole resulted in 40.2±0.2, 49.3±0.2, 44.6±0.0 and 37.1±3.8% decreases in melanin content, compared with that of the control cell population, respectively (Fig. 2B). These results indicate that treatment with PPIs decreased melanin synthesis without influencing melanocyte viability in vitro.

The influence of PPIs on the mRNA levels of the TYR, TRP-1, TRP-2 and MITF genes was examined using RT-PCR. Treatment with 100 µM rabeprazole suppressed the expression of the TYR, TRP-1, TRP-2 and MITF genes by 77.1, 48.3, 76.2 and 37.1%, respectively. Esomeprazole (100 µM) decreased the expression of these genes by 23.1, 47.9, 53.9 and 30.7%, respectively, and 100 µM lansoprazole inhibited the expression of the TRP-1, TRP-2 and MITF by 25.9, 44.9 and 52.8%, respectively. Treatment with 100 µM pantoprazole suppressed the mRNA expression of the TRP-1, TRP-2 and MITF genes by 22.3, 37.9 and 15.7%, respectively (Fig. 3). Taken together, these results demonstrate that rabeprazole treatment affected mRNA expression of each of the melanogenesis-associated genes.