Quercetin derivatives regulate melanosome transportation via EPI64 inhibition and elongate the cell shape of B16 melanoma cells

Abstract

4′-O-β-d-glucopyranosyl-quercetin-3-O-β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside (3C4′GQ), first isolated from Helminthostachys zeylanica root extract, was synthesized as a compound that stimulates intracellular melanogenesis. 3-O-methylquercetin (3MQ) and 3,4′,7-O-trimethylquercetin (34′7TMQ) were synthesized as compounds that enhance extracellular melanin formation. The formation of dendrites and the expression of EBP50-PDZ interactor of 64 kDa (EPI64) relating to melanin transportation were investigated using B16 melanoma cells treated with 3C4′GQ, 3MQ, or 34′7TMQ in order to understand the mechanism underlying the observed activities. The influence of 3C4′GQ on the increase of intracellular melanin contents enhanced the expression of EPI64, exhibited no dendrite elongation activity, and inhibited melanin transportation. On the other hand, the increase of extracellular melanin content by 3MQ and 34′7TMQ inhibited the expression of EPI64 and formed elongated cells to stimulate melanin transportation.

Introduction

Melanin, a human body pigment, is biosynthesized by the catalysis of tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 in melanosomes [1], [2]. Melanin plays an important role in preventing skin diseases caused by the damage of ultraviolet (UV) rays in the human body [3], [4]. The change of the overall level of melanogenesisby aging, stress, and UV ray damage induces gray hairs, sunburn, and mottling on the skin. Therefore, a number of studies have investigated novel pharmacological and herbal agents aimed at controlling melanogenesis for maintaining the good health and cosmetic appearance of the human body [5], [6], [7], [8], [9], [10], [11], [12], [13], [14].

Quercetin, a flavonoid present as a glycoside in various fruits and vegetables [15], [16], [17], exhibits a variety of pharmacological effects. For instance, quercetin is recognized to show antioxidant, anti-cancer, and melanogenesis-controlling activities [18], [19]. Quercetin is reported as a potent inhibitor of tyrosinase activity and on melanogenesis in mouse B16 melanoma cells [20]. However, in human melanoma cells, quercetin elicits the opposite effect and stimulates melanogenesis [21]. Furthermore, it has been reported that the melanogenesis-regulating activity of quercetin depends on its concentration [22]. A few papers have shown that quercetin derivatives also can regulate melanogenesis. Quercetin-3-O-β-d-glucoside enhances the expression of TRP-1 and TRP-2, which catalyze the biosynthesis of eumelanin [23]. In our previous study, the quercetin glycoside, 4′-O-β-d-glucopyranosyl-quercetin-3-O-β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside (3C4′GQ), shown in Fig. 1, was isolated from Helminthostachys zeylanica root extract as an intracellular melanogenesis stimulator [24]. 34′7TMQ and other distinct quercetin derivatives were synthesized in order to understand the structure-activity relationship of quercetin derivatives in our previous report [25], [26]. Among the synthesized compounds, 3-O-methylquercetin (3MQ) and 3,4′,7-O-trimethylquercetin (34′7TMQ) (Fig. 1) increased extracellular melanin content more potently than theophylline as the positive control, suggesting that 3MQ and 34′7TMQ may stimulate the transportation of melanosome.

Melanin is biosynthesized in the melanosome at the perinuclear region of the melanocyte. The melanin accumulated in the mature melanosome is transported to the periphery through the dendritic cells [27], [28], [29]. The melanosome is transferred further to the hair matrix or to the keratinocyte present above the melanocyte. The keratinocyte that receives the melanin becomes cornified to form skin pigmentation. Similarly, hair pigmentation occurs due to melanin released on the outside of the melanocyte. Despite the importance of the extracellular pigmentation and of the transportation of melanosomes, an evaluation of melanogenesis activity using melanin-controlling agents has been performed only on production of intracellular melanin in B16 melanoma cells thus far [30], [31], [32]. The present study focused on clarifying the mechanism of the extracellular melanogenesis-stimulatory activity from the perspectives of cell elongation and of the expression or distribution of proteins involved in the transportation of melanosomes of cells treated with 3MQ or 34′7TMQ using western blot analysis and immunofluorescence microscopy.