Prenylated Flavonoids from Roots of Glycyrrhiza uralensis Induce Differentiation of B16-F10 Melanoma Cells(Licorice)


Roots of Glycyrrhiza uralensis have been used as herbal medicine and natural sweetener. By activity-guided phytochemical investigation of the extracts from G. uralensis root, ten flavonoids, namely GF-1–GF-10, of which five were prenylated flavonoids, were found to show antiproliferative effects in melanoma B16-F10 cells. Three of the prenylated flavonoids, namely GF-1, GF-4 and GF-9, significantly induced the differentiation of B16-F10 cells; the inductions included increase of tyrosinase activity, tyrosinase protein, and melanin content. In GF-1 and GF-9 induced melanoma differentiation, the phosphorylation of p38 MAPK (mitogen activated potein kinase) was identified; while GF-4 could trigger the phosphorylation of PI3K/AKT (phosphatidylinositol 3-kinase/Protein Kinase B) signaling. However, application of GF-6 to the melanoma cells did not induce differentiation; but which promoted cell apoptotic signaling, i.e., increase levels of cleaved-PRAP, cleaved-caspase 3, and cleaved-caspase 9. These results suggested that different types of prenylated flavonoids from G. uralensis might have potential anticancer effects against melanoma cells by acting through different signaling pathways.

Keywords: Glycyrrhiza uralensis, prenylated flavonoids, antiproliferation, differentiation, melanoma cell

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1. Introduction

The roots of Glycyrrhiza uralensis Fisch (GlycyrrhizaeUralensis Radix; GUR), named as licorice or gancao, have been extensively used as a herbal drug in both Eastern and Western countries [1]. Clinical studies have shown GUR is highly effective in the treatment of respiratory, gastrointestinal, cardiovascular and genitourinary conditions [2]. According to traditional Chinese medicine (TCM) theory, GUR is described as a beneficial herb to enhance therapeutic effects; meanwhile, this herb is being employed commonly to detoxify potential adverse effects in many herbal mixtures during clinical application. Phytochemical studies revealed that triterpene saponins and flavonoid glycosides were two major active substances in GUR [3]. In the extractive of GUR, the nonpolar fraction of GUR is rich in various types of prenylated flavonoids, e.g., flavones, isoflavones, flavanones, chalcones, and coumestans [4]. These flavonoids are being considered to be active ingredients of GUR, and indeed which have been commonly used in food industries. In recent pharmacological studies, GUR flavonoids have been proposed to have anti-oxidation [5], anti-inflammatory [6], antiproliferative, and cytotoxic effects in various cells [7]. Despite the aforementioned proposed actions, the roles of GUR prenylated flavonoids in cancer cells have not been extensively investigated.

Malignant melanoma is a highly aggressive and invasive skin cancer with high metastatic potential and extraordinary resistance to cytotoxic agents [8]. The occurrence of melanoma is related to different factors, e.g., sun exposure, fair pigmentation, and genetic mutation [9]. In recent years, the incidence of melanoma is rapidly increasing throughout the world, especially in America and Europe [10]. Although drug therapy for this cancer has been developed rapidly, e.g., immunotherapies with PD-1 inhibition drugs (ipilimumab and nivolumab) and T-cell checkpoint blockade therapies [11,12], the usage of herbal products is still one of the alternative approaches for melanoma cancer treatment.

Considering the pathogenesis and clinical treatment of melanoma, the recent targets in cancer therapy is focusing on discovery of natural products that are able to suppress cancer cell proliferation and promote cell differentiation [13,14]. Here, the CH2Cl2 extract of GUR (GURCH2Cl2) was shown to exhibited potent effect in antiproliferation and inducing differentiation of cultured melanoma B16-F10 cells: these effects were significant higher than that deriving from water extract (GURwater) and ethanol extract of GUR (GUREtOH). To explore the possible underlying mechanism for anticancer effect of GUR, ten flavonoids (GF1-GF10) of which five were prenylated flavonoids, were isolated. The roles of these flavonoids in inducing the differentiation of cultures B16-F10 cells were illustrated, and subsequently the signaling cascades, triggered by various flavonoids, were revealed and compared.

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2. Results

2.1. G. uralensis Extracts in Proliferation and Differentiation of Melanoma Cells

Different extractives of GUR, i.e., GUwater, GUEtOH, and GUCH2Cl2, were subjected to HPLC analyses. As shown in HPLC chromatograms (Figure 1A), saponins and flavonoid glycosides were the major constituents in both GUwater (water extract) and GUEtOH (ethanol extract). Besides, a small amount of free flavonoids could be detected in GUEtOH. In comparison with GUwater and GUEtOH, GUCH2Cl2 (dichloromethane extract) exhibited a comparable enrichment of free flavonoids. To a certain extent, difference in chemicals might cause possible difference in their biological capacities.

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