Flumequine-Mediated Upregulation of p38 MAPK and JNK Results in Melanogenesis in B16F10 Cells and Zebrafish Larvae

Abstract

Flumequine is a well-known second generation quinolone antibiotic that induces phototoxicity. However, the effect of flumequine on skin melanogenesis is unclear. Therefore, we, for the first time, investigated whether flumequine regulates melanogenesis. The present study showed that flumequine slightly inhibited in vitro mushroom tyrosinase activity but significantly increased extracellular and intracellular melanin content in B16F10 cells and promoted the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. Additionally, flumequine remarkably increased melanin pigmentation in zebrafish larvae without any toxicity. We also found that flumequine stimulated p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation; inhibition of p38 MAPK and JNK resulted in significant downregulation of extracellular and intracellular melanin content in B16F10 cells and pigmentation of zebrafish larvae accompanied with suppression of MITF and tyrosinase expression, indicating that flumequine-mediated p38 and JNK promote melanogenesis in vitro and in vivo. According to the molecular docking prediction, flumequine targeted dual-specificity MAPK phosphatase 16 (DUSP16), which is a major negative regulator of p38 MAPK and JNK. Our findings demonstrate that flumequine induces an increase in melanin content in B16F10 cells and zebrafish larvae by activating p38 MAPK and JNK. These data show the potential of flumequine for use as an anti-vitiligo agent.

Keywords: flumequine; melanogenesis; mitogen-activated protein kinase.

Figure 1

Flumequine slightly upregulates mushroom tyrosinase activity in vitro at high concentrations. (A) Chemical structure of flumequine. (B) The effect of flumequine on in vitro mushroom tyrosinase activity. Tyrosinase activity was determined by oxidation of L-DOPA as a substrate. Briefly, flumequine (0–1000 µM), kojic acid (25 µM), and phenylthiourea (PTU) (250 nM) were loaded into a 96-well microplate. After incubation with mushroom tyrosinase at 37 °C for 30 min, the dopaquinone level was measured by spectrophotometry at 490 nm. The results are the average of the three independent experiments and are represented as the mean ± standard error median (SEM). ***, p < 0.001 and **, p < 0.01 vs. untreated control. V, vehicle control (0.1% DMSO).

Figure 2

High concentrations of flumequine slightly decrease the viability of B16F10 cells. B16F10 cells were treated with 0–1000 µM flumequine for 72 h. (A) A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to determine cell viability. Cell viability in each group was presented as the percentage of the values of the untreated control. (B) The cellular images were captured and analyzed. (C,D) B16F10 cells were treated with 0–400 µM flumequine for 72 h. Flow cytometric analysis was used to assess total cell count (left), cell viability (middle), and apoptotic cell population (right). (E) Cell cycle analysis carried out using the Muse™ cell cycle kit after treating with 0–400 µM flumequine for 72 h (top) and cell cycle distribution is represented (bottom). The results are the average of the three independent experiments; the data are expressed as the mean ± SEM: ***, p < 0.001, **, p < 0.01, and *, p < 0.05 vs. untreated control. V, vehicle control (0.1% DMSO).

Figure 3

Flumequine increases the extracellular and intracellular melanin production in B16F10 cells. B16F10 cells were exposed to flumequine (0–50 µM) for 72 h. (A) Images of the culture medium color were captured. Extracellular (B) and intracellular (C) melanin content was measured at 72 h. The percentage values in each group are relative to those in the untreated control. The results are the average of three independent experiments and are represented as the mean ± SEM. ***, p < 0.001 and **, p < 0.01 vs. untreated control. V, vehicle control (0.1% DMSO).

Figure 4

Flumequine stimulates microphthalmia-associated transcription (MITF) and tyrosinase expression in B16F10 cells. (A) B16F10 cells were exposed to flumequine (0–50 µM) for 48 h and the expression of MITF and tyrosinase was measured. (B) Under the same experimental condition, the protein expression of MITF and tyrosinase was measured by western blotting analysis at 72 h. The α-melanocyte stimulating hormone (α-MSH)-treated group was used as a positive control. The results are the average of three independent experiments and are represented as the mean ± SEM. ***, p < 0.001, **, p < 0.01, and *, p < 0.05 vs. untreated control.

Figure 5

Flumequine upregulates melanin pigmentation in zebrafish larvae. (A) The chorion of zebrafish larvae (n = 20) was removed at 1 dpf and they were treated with PTU (200 µM) for 24 h (by 2 dpf). Then, flumequine (0–20 µM) was added to 2 dpf zebrafish larvae for 72 h (by 5 dpf), and images were captured at 5 dpf under an Olympus microscope (40×). (B) Relative density was calculated using Image J software. (C) Average heart beat of zebrafish larvae (n = 20) was measured to assess the toxicity of flumequine. The results are the average of three independent experiments and are represented as the mean ± SEM. ***, p < 0.001 and *, p < 0.05 vs. untreated control.

Figure 6

Flumequine induces p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation, resulting in hypermelanogenesis. (A) B16F10 cells were exposed to flumequine (0–50 µM), and phosphorylation of p38 and JNK was measured by western blotting analysis at 72 h. In addition, B16F10 cells were pretreated with 10 μM SB203580 (B) or 10 μM SP600125 (C) for 1 h and were then treated with 50 µM flumequine for 72 h. Extracellular (left) and intracellular (right) melanin content was measured. The results are the average of three independent experiments and are represented as the mean ± SEM: ***, p < 0.001, **, p < 0.01, and *, p < 0.05 vs. untreated control, and ^##, p < 0.01 and ^#, p < 0.05 vs. flumequine-treated group.

Figure 7

p38 MAPK and JNK upregulate flumequine-mediated melanogenesis in B16F10 cells by activating MITF and tyrosinase. B16F10 cells were pretreated with (A) SB203580 (10 μM) or (B) SP600125 (10 μM) for 1 h, and then flumequine (50 µM) was treated for 72 h. Expression of p38, JNK, MITF, and tyrosinase was measured by western blotting analysis. Each relative density was normalized by the density of β-Actin. The results are the average of three independent experiments and are represented as the mean ± SEM. ***, p < 0.001, **, p < 0.01, and *, p < 0.05 vs. untreated control, and ^##, p < 0.01, ^#, p < 0.05 vs. flumequine-treated group.

Figure 8

p38 MAPK and JNK upregulate melanogenesis in zebrafish larvae. (A-a) The chorion of zebrafish larvae (n = 20) was removed at 1 dpf and treated with PTU (200 µM) for 24 h. Then, 2 dpf zebrafish larvae were treated with flumequine (20 µM) for 72 h followed by treatment with SB203580 (1 μM) or SP600125 (1 μM) for 2 h. (A-b,B-a) Images of 5 dpf zebrafish larvae were captured under a microscope (40×). (A-c,B-b) Relative density was calculated using the Image J software. (A-d,B-c) The average heart rate of zebrafish larvae (n = 20) was measured to assess the toxicity of flumequine. Data are reported as the mean ± SEM of three independent experiments (n = 3). ***, p < 0.001 vs. untreated control and ^###, p < 0.001 vs. flumequine-treated group preincubated with PTU.