Cycloartane triterpenoid (23R, 24E)-23-acetoxymangiferonic acid inhibited proliferation and migration in B16-F10 melanoma via MITF downregulation caused by inhibition of both β-catenin and c-Raf–MEK1–ERK signaling axis

We recently reported that (23R, 24E)-23-acetoxymangiferonic acid (23R-AMA), a cycloartane triterpenoid isolated by activity-guided separation from a methanol extract of Garcinia sp. bark, inhibited melanin production via inhibition of tyrosinase (TYR) expression in the B16-F10 melanoma cell line. Since 23R-AMA also inhibited microphthalmia-associated transcription factor (MITF) expression, an upstream factor of TYR, these features of 23R-AMA were thought to be appropriate for development of whitening cosmetics. However, 23R-AMA exhibited growth inhibition other than inhibition of melanin production in B16-F10 cells. Therefore, we investigated biological activities of 23R-AMA in detail, focused on its application as an anti-melanoma compound. In this study, we demonstrated that 23R-AMA inhibited cell proliferation and basic FGF (bFGF)-induced migration in B16-F10 cells. Furthermore, 23R-AMA promoted ser45/thr41 phosphorylation of β-catenin and suppressed its intranuclear accumulation, which was suggested to be related to inhibition of MITF expression. The transcriptional activity of MITF is known to be regulated by phosphorylation via activated ERK. Further investigation revealed that 23R-AMA inhibited phosphorylation of c-Raf, MEK-1, and ERK, and also that of upstream molecules including FAK and c-Src. These results suggested that 23R-AMA inhibited growth and migration of B16-F10 melanoma by regulating both MITF expression and its activity. The activities of 23R-AMA reported in this study are new aspects of cycloartane triterpenoids. Electronic supplementary material The online version of this article (10.1007/s11418-018-1233-7) contains supplementary material, which is available to authorized users.

In our search for new bioactive compounds [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31], we screened Malaysian plants extracts for anti-melanin deposition activity. The bark MeOH extract of Garcinia sp. showed a dose-dependent inhibition of melanin deposition activity from 12.5 µg/mL. The extract was then partitioned into n-hexane, EtOAc, n-BuOH and water fractions for bioassay-guided isolation purpose, and the nhexane and EtOAc fractions showed promising activity. Activityguided separation of both active fractions, led to the isolation of compound 1 (Figure 1) as the active compound (IC50 9.9 µg/mL = 19.4 µM). Based on 1D and 2D NMR data, compound 1 was deduced to be 23-acetoxy-mangiferonic acid. 23-acetoxy-mangiferonic acid was previously reported by Anjaneyulu et al. as a component of an acetylation product mixture of a mixture of 23-hydroxymangiferonic acid and 22-hydroxy-isomangiferolic acid [32]. Hence this is the first report of the isolation of 23-acetoxy-mangiferonic acid as a pure compound, and the first complete assignment of its 1 H and 13 C NMR spectra (Table 1). To determine the absolute configuration of C-23, 1 was hydrolyzed to obtain 2. 2 was then methylated to obtain its methyl ester derivative 3. The absolute configuration of C-23 in 3 was determined to be R by using the  advanced Mosher's method ( Figure 2). Hence, the absolute configuration of C-23 in 1 and 2 was deduced to be R. Thus, compound 1 was deduced to be (23R, 24E)-23-acetoxymangiferonic acid and compound 2 was deduced to be (23R, 24E)-23-hydroxy-mangiferonic acid.
23-hydroxy-mangiferonic acid with unknown C-23 configuration was isolated from G. cornea [33]. The 1 H and 13 C NMR data of 2 were identical to those of 23-hydroxy-mangiferonic acid isolated from G. cornea. In addition, it has been reported that the chemical shifts of C-22 to C-25, C-26 and C-28 of two C-23-epimers of 23hydroxy-mangiferolic acid were different [32]. Thus, the 23-  To examine the action of 1, tyrosinase protein expression was analyzed by western blotting. Tyrosinase protein expression was induced by treatment with IBMX. As shown in Figure 2, addition of 12.5 or 25 µg/mL of 1 potently inhibited the tyrosinase protein expression at 24 hours. Furthermore, 1 inhibited the tyrosinase protein expression even without the addition of IBMX. Next, tyrosinase gene expression was analyzed by reverse-transcription polymerase chain reaction (RT-PCR). As shown in Figure 3, tyrosinase mRNA expression was elevated after 24 hours of the induction, and was suppressed by the addition of 1 (12.5 µg/mL). These results suggest that the anti-melanin deposition activity of 1 was caused by the down regulation of tyrosinase gene expression.

Experimental
General experimental procedures: Optical rotations were measured on a JASCO DIP-1000 polarimeter, UV spectra on a Shimadzu UVmini-1240 spectrophotometer, and IR spectra on a JASCO FT/IR-4100 spectrophotometer. High-resolution ESI MS were obtained on a LTQ Orbitrap XL (Thermo Scientific). 1 H and 2D NMR spectra were measured on a 400 MHz spectrometer at 300K, and 13 C NMR spectra on a 100 MHz spectrometer. Residual solvent chemical shifts were used as internal standard, δH 7.26 and δC 77.0. Standard pulse sequences were used for the 2D NMR experiments.
[α]D 17  Alkaline hydrolysis and methylation of 1: To a solution of 1 (0.8 mg in 100 µL MeOH), 100 µL of 2M NaOH(aq) was added, and the resulting mixture was left overnight at r.t. The reaction mixture was then neutralized with 2M HCl(aq) and partitioned with CHCl3. The CHCl3 fraction was dried under N2 blow and the resulting residue (23-hydroxy-mangiferonic acid, 2, 0.7 mg) was then dissolved in 50 µL MeOH. To the MeOH solution, 20 µL of TMS-diazomethane (10% in n-hexane) was added and was left at r.t. After 10 min., the reaction mixture was dried under N2 blow and the resulting residue was subjected to SiO2 column chromatography (CHCl3) to obtain methyl 23-hydroxy-mangiferonate (3, 0.8 mg).

Modified Mosher's method of 3:
First, (R)-a-methoxy-a-(trifluoromethyl)phenylacetic (MTPA) chloride (2 µL) was added to a solution of 3 (0.4 mg) with a catalytic amount of 4-(dimethylamino)pyridine and 1 µL of triethylamine in 50 µL of CH2Cl2, and the solution was allowed to stand at room temperature overnight. The residue obtained under N2 blow was subjected to SiO2 column chromatography (CHCl3) to obtain the (S)-MTPA ester of 3 (4). The same procedure was used to obtain of the (R)-MTPA ester of 3 (5). Anti-melanin deposition activity and cytotoxicity: Cells were seeded on a 24 well plate at 1.0×10 5 cells/mL, and treated with 100 µM 3-Isobutyl-1-methylxanthine (IBMX, Wako, Osaka, Japan), 0.25 µM of a-melanocyte stimulating hormone (a-MSH, Sigma, St. Louis, MO, USA) and the samples for 72 hours. After this treatment, to quantify the melanin deposition, the cells were lysed by 1N NaOH at 95 o C, and the absorbance at 360 nm was measured. In addition, the samples cytotoxicity was evaluated via measurement of the total protein contents using Coomassie Protein Assay Reagent (Thermo scientific, Rockford, IL, USA), and measurement of the absorbance at 595 nm. 750 µM of Arbutin (Sigma, St. Louis, MO, USA) was used as positive control.