Identification of 1,2,3,4,6-Penta-O-galloyl-β-d-glucopyranoside as a Glycine N-Methyltransferase Enhancer by High-Throughput Screening of Natural Products Inhibits Hepatocellular Carcinoma

Glycine N-methyltransferase (GNMT) expression is vastly downregulated in hepatocellular carcinomas (HCC). High rates of GNMT knockout mice developed HCC, while overexpression of GNMT prevented aflatoxin-induced carcinogenicity and inhibited liver cancer cell proliferation. Therefore, in this study, we aimed for the identification of a GNMT inducer for HCC therapy. We established a GNMT promoter-driven luciferase reporter assay as a drug screening platform. Screening of 324 pure compounds and 480 crude extracts from Chinese medicinal herbs resulted in the identification of Paeonia lactiflora Pall (PL) extract and the active component 1,2,3,4,6-penta-O-galloyl-β-d-glucopyranoside (PGG) as a GNMT inducer. Purified PL extract and PGG induced GNMT mRNA and protein expression in Huh7 human hepatoma cells and in xenograft tumors. PGG and PL extract had potent anti-HCC effects both in vitro and in vivo. Furthermore, PGG treatment induced apoptosis in Huh7 cells. Moreover, PGG treatment sensitized Huh7 cells to sorafenib treatment. Therefore, these results indicated that identifying a GNMT enhancer using the GNMT promoter-based assay might be a useful approach to find drugs for HCC. These data also suggested that PGG has therapeutic potential for the treatment of HCC.

. Flowchart of high-throughput screening. We used H7GPL cells to screen a traditional Chinese medicine drug library consisting of 324 pure compounds and 480 crude extracts from Chinese medicinal herbs. Hits of primary screening were sorted by Z score ≥ 1.5. There were 26 hits (13 pure compounds and 13 herb extracts) obtained and used for secondary screening. Hits of secondary screening were sorted by reporter activity (≥1.5-folds compared to DMSO solvent control), and 16 hits were identified and used for revalidation. After revalidation, 10 hits remained positive and tested for dose dependency. Among them, the extract from Paeonia lactiflora Pall (PL extract) was found to induce the highest increase in reporter activity and was chosen for further characterization.  (e) Figure S2. Chromatographs (a,b), mass spectrum (c) and nuclear magnetic resonance profiles (d,e) of 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranoside (PGG). (a,b) The UPLC chromatographs of the bioactive fraction of F3-6 (a) and 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranoside (PGG) (b). The analyses were performed on a Waters Acquity UPLC system including binary solvent manager, sampler manager, column compartment and Waters Photodiode Array Detector. The chromatographic data were collected and analyzed by Empower 2.0 software. The Thermo Syncronis C18 column (100 × 2.1 mm, 1.7 µm) was used. The column and sample temperature were maintained at 40 and 25 °C, respectively. The mobile phase consisted of water (0.1% phosphate buffer) (a) and acetonitrile (b) at a flow rate of 0.4 mL/min. The linear gradient was applied as following: 2% A, hold for 1 min; 2%-10% A, 1-2 min and hold for 2 min; 10%-25% A, 4-10 min; 25%-50% A, 10-14 min. The separation was followed by a 3-min washing procedure. The injection volume was 1 µL. The detection wavelength was set between 200 and 400 nm, and the chromatographs were extracted at 210 nm for purity check.

Supplementary Materials and Methods
Drug screening, extraction, isolation and identification of GNMT enhancer compounds, safety test in mice, diet and the Ames test:

Drug Screening
The traditional Chinese medicine library that contained 324 pure compounds (dissolved in DMSO at a concentration of 20 mg/mL) and 480 crude extracts (in DMSO at a concentration of 200 mg/mL) was provided from National Research Institute of Chinese Medicine. For primary screening, H7GPL cells seeded in 96-well plates were treated for 24 h with individual drugs at a concentration of 2 mg/mL for crude extracts and 0.2 mg/mL for pure compounds and then were lysed for the luciferase activity assay using the Luciferase Assay System (Promega, Madison, WI, USA). In each plate, six solvent control wells were treated with DMSO (the final concentration was 1%). Reporter activity for each well was transformed to the Z score by using data from all assay plates [35]. Then, hits of primary screening were sorted by Z score ≥1.5 and used for secondary screening. The same platform was used for secondary screening. Cells were treated with hits of the primary screen in duplicated plates for 20 h, and then alamarBlue ® reagent (AbD serotec, Oxford, UK) was added into assay plates and incubated for four additional hours. The cytotoxicity was measured according to the manufacturer's recommendation, then was used to normalize the reporter activity and presented as the relative luciferase activity fold to control. Drugs that induced GNMT promoter activity ≥1.5-fold were considered as hits of the secondary screen.

Extraction, Isolation and Identification of GNMT Enhancer Compounds
Paeoniae radix rubra was purchased from a local Chinese drug store (Taipei, Taiwan) in May 2012. It was identified as the roots of Paeonia lactiflora Pall. About 150 g of the ground material was reflux extracted twice with 0.6 L of 50% aqueous MeOH for 1 h each. The supernatant was filtered through a filter paper, combined and partitioned three times with 0.7 L ethyl acetate each. The initial fractionation of the ethyl acetate extract was conducted by using an MPLC system (300 × 30 mm, silica gel, 40-63 µm; Merck, Germany). Dichloromethane (A) and MeOH (B) were used as the mobile phase (gradient conditions: 100% A for 1 h, to 40% B in 20 min and then to 100% B in 20 min, flow rate: 18 mL/min). The collected fractions were assayed by TLC (silica gel 60 F254 plates; Merck, Germany), using a mixture of ethyl acetate, MeOH and 0.1% acetic acid (15:2:0.5) as the mobile phase. Results were evaluated at 254 nm and by spraying with vanillin/sulfuric acid reagent. The luciferase assay was used to identify the active fractions in PL extract as described in the drug screening. Further purification of the most active fraction (F3) was performed on a Sephadex LH-20 column using MeOH as the mobile phase and obtained the bioactive fraction of F3-6.
The fraction F3-6 was further purified by a HPLC system, Agilent 1100 series coupled with a photodiode array detector. An RP-18 column (Cosmosil, 250 × 10 mm, 5 µm; nacalai, Japan) was used. Water (A) and CH3CN (B), with 0.1% acetic acid each, were used as the mobile phase (gradient conditions: 5% B for 20 min, to 100% B, flow rate: 3 mL/min). Monitoring the separation at 203 nm led to the isolation of the effective compound, 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranoside (PGG). PGG was obtained as a pale brown, amorphous powder with UV maxima at 211, 231 and 278 nm. For MS analysis, PGG was diluted in MeOH and directly infused into a Finnigan MATLCQ. The mass spectra were recorded in the positive and negative ESI mode and showed an m/z value of 963.02 [M + Na] + and 939.03 [M − H] − , respectively. NMR spectra of the isolated compound in deuterated methanol (CD3OD) were recorded on a VNMRS 600 NMR spectrometer (Varian, Palo Alto, CA, USA). Identification was achieved by the comparison of the spectroscopic data obtained with those in the literature [36,37]. We used a 0.1-mg/mL concentration of purified fractions, F3-6 and PGG for most of the experiments, unless otherwise mentioned. PGG was dissolved in PBS for the cell-based assay.