Arch Pharm Res Vol 33, No 11, 1747-1751, 2010 DOI 10.1007/s12272-010-1106-4
Inhibitory Activities of Anthraquinones from Rubia akane on Phosphatase Regenerating Liver-3 Mi Kyeong Moon1,*, Young-Min Han2,*, Yu-Jin Lee2, Lan Hee Lee2, Jae Heon Yang1, Byoung-Mog Kwon2, and Dae Keun Kim1 1
College of Pharmacy, Woosuk University, Samrye 565-701, Korea and 2Korea Research Institute of Bioscience and Biotechnology, University of Science and Technology in Korea, Taejeon 305-333, Korea (Received June 6, 2010/Revised August 2, 2010/Accepted August 10, 2010)
The methanolic extract of the roots of Rubia akane (Rubiaceae) was found to show inhibitory activity on phosphatase of regenerating liver-3 (PRL-3). Bioassay-guided fractionation of the methanolic extract resulted in the isolation of two anthraquinone compounds, 2-methyl-1,3,6trihydroxy-9,10-anthraquinone-3-O-(6'-O-acetyl)-α-rhamnosyl(1→2)-β-glucoside and 2-methyl1,3,6-trihydroxy-9,10-anthraquinone, as inhibitors on PRL-3. These compounds inhibited PRL-3 in a dose-dependent manner with IC50 values of 5.2 and 1.3 µg/mL, respectively. Key words: Rubia akane, Rubiaceae, Anthraquinone, PRL-3
INTRODUCTION One of the characteristics of cancer cells is their high metastatic index. Metastasis is the neoplastic process responsible for most deaths from cancer because the primary tumors can usually be surgically removed. Many colorectal cancer patients suffer from the unexpected development of occult metastases, especially in the liver and lung, after the curative resection of their primary tumors (Weiss, 2000). Therefore, this has been studied to clarify the molecular mechanism involved in metastasis and to identify the specific biomarkers of colorectal cancer metastasis. Bardelli et al. (2003) reported that PRL-3 (phosphatase of regenerating liver-3) mRNA expression was elevated in nearly all metastatic lesions derived from colorectal cancer, regardless of the site of metastasis (liver, lung, brain, or ovary). Expression was found in neoplastic cells, although tumor endothelium also expressed the *These authors contributed equally to this work. Correspondence to: Dae Keun Kim, College of Pharmacy, Woosuk University, Samrye 565-701, Korea Tel: 82-63-290-1574, Fax: 82-63-290-1812 E-mail:
[email protected] Byoung-Mog Kwon, Korea Research Institute of Bioscience and Biotechnology, UST, Taejeon 305-333, Korea Tel: 82-42-860-4557, Fax: 82-42-861-2675 E-mail:
[email protected]
gene. In contrast, little or no PRL-3 expression was observed in normal colon, nonmetastatic primary cancers, or metastatic lesions derived from cancers other than those of the colon (pancreas, stomach, or esophagus). Consequently, PRL-3 is apparently expressed in colorectal cancer metastases to any organ but is not expressed in metastases of other cancers to the same organs or in nonmetastatic colorectal cancers (Saha et al., 2001; Bardelli et al., 2003). And Guo et al. reported that PRL-3 initiates tumor angiogenesis by recruiting endothelial cells in vitro and in vivo (Guo et al., 2006). Besides above reports, there were many reports about PRL-3 associated with the metastasis of cancer (Zeng et al., 2003; Kim et al., 2004; Rouleau et al., 2006; Yamashita et al., 2007). Recently, it was reported that aberrant overexpression of PRL-3 has been found in multiple solid tumor types. Ectopic expression of PRLs in cells induces transformation, increases mobility and invasiveness, and forms experimental metastases in mice (Daouti et al., 2008). These studies suggest that the catalytic domain of PRL-3 could serve as an ideal therapeutic target for drug development to block the spread of colorectal cancer (Guo et al., 2004). In continued searching for potential anticancer agents, we found that the methanol extracts of the roots of Rubia akane (Rubiaceae) inhibited the activity of PRL-3, which exhibited PRL-3 inhibitory activity 70% at 100 µg/mL. Rubia akane has been used as folk
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medicine in Korea, Japan and China to treat dysmenorrhea, epistaxis, uterine bleeding, hemoptysis and hepatitis (But et al., 1997). Anthraquinone, anthraquinone glycoside, naphthoquinone, cyclohexapeptide, and lignin compounds have been reported from Rubia species (Itokawa et al., 1983a, 1984a, 1984b, 1986; Han et al., 1990; Chung et al., 1994; Lee et al., 2008; Son et al., 2008). Among them several compounds including epoxymollugin and peptide alkaloids showed anticancer activity (Itokawa et al., 1983b; Lee et al., 2008; Son et al., 2008). This paper describes the purification, structural characterization, and the PRL-3 inhibitory activity of the isolated compounds.
MATERIALS AND METHODS General procedure 1 H- and 13C-NMR spectra were determined on a JEOL JMN-EX 600 spectrometer. TLC was carried out on Merck precoated silica gel F254 plates, with Kiesel gel 60 (230-400 mesh, Merck) used as the silica gel. The column used for MPLC was Lobar A (10 × 24 cm). Plant materials The roots of Rubia akane were collected in October 2006 at Jeonbuk, Korea (1 kg). A voucher specimen was deposited in the herbarium of the college of pharmacy, Woosuk University (WSU-06-021). Extraction and isolation The shade dried plant material (1 kg) was extracted with MeOH (2 × 3 L) for 48 h at room temperature. The filtrate was evaporated in vacuo to give a dark brownish residue. The resultant methanol extract (110 g) was followed by successive solvent partitioning to give n-hexane (95 g), CHCl3 (15 g), EtOAc (4 g), nBuOH (13 g) and H2O soluble fractions. EtOAc soluble fraction showed the most significant PRL-3 inhibitory activity. 2 g of EtOAc soluble fraction was subjected to sephadex LH-20 column chromatography (7 × 30 cm) using MeOH to give 4 fraction (E1-4), based on TLC (CHCl3:MeOH = 3:1). Lobar A column chromatography of the E1 with CHCl3-MeOH (10:1) gave compound 1 (140 mg), and of the E4 with CHCl3-MeOH (15:1) gave compound 2 (50 mg). 2-Methyl-1,3,6-trihydroxy-9,10-anthraquinone-3O-(6'-O-acetyl)-α-rhamnosyl(1→2)-β-glucoside (1) Yellow powder (MeOH); 1H-NMR (600 MHz, CD3OD, δ ppm) : 7.95 (1H, d, J = 8.4 Hz, H-8), 7.34 (1H, d, J = 3.0 Hz, H-5), 7.28 (1H, s, H-4), 7.00 (1H, dd, J = 8.4, 3.0 Hz, H-7), 5.31 (1H, d, J = 1.8 Hz, H-1''), 4.37 (1H,
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Fig. 1. Structures of compounds 1 and 2
dd, J = 12.0, 1.8 Hz, H-6'a), 4.37 (1H, dd, J = 12.0, 7.2 Hz, H-6'b), 2.11 (3H, s, COCH3), 1.91 (3H, s, CH3), 1.10 (1H, d, J = 6.0 Hz, H-6''); 13C-NMR (125 MHz, CD3OD, δ ppm) : 187.9 (C-9), 183.5 (C-10), 173.0 (COCH3), 164.9 (C-1), 163.2 (C-6), 161.5 (C-3), 136.9 (C-4a), 133.4 (C10a), 130.7 (C-8), 126.5 (C-8a), 123.0 (C-7), 122.2 (C-2), 113.8 (C-5), 112.2 (C-9a), 106.6 (C-4), 102.3 (C-1''), 99.5 (C-1'), 79.4 (C-3'), 78.9 (C-2'), 75.6 (C-5'), 74.0 (C4''), 72.2 (C-2''), 72.0 (C-3''), 71.7 (C-4'), 70.2 (C-5''), 64.7 (C-6'), 20.7 (COCH3), 18.3 (C-6''), 9.2 (CH3). 2-Methyl-1,3,6-trihydroxy-9,10-anthraquinone (2) Yellow powder (MeOH); 1H-NMR (600 MHz, CD3OD, δ ppm) : 7.99 (1H, d, J = 8.4 Hz, H-8), 7.39 (1H, d, J = 3.0 Hz, H-5), 7.07 (1H, dd, J = 8.4, 3.0 Hz, H-7), 7.06 (1H, s, H-4), 2.05 (3H, s, CH3); 13C-NMR (125 MHz, CD3OD, δ ppm) : 187.4 (C-9), 183.8 (C-10), 164.4 (C-3), 164.0 (C-1), 163.4 (C-6), 133.5 (C-4a), 133.5 (C-10a), 130.4 (C-8), 126.8 (C-8a), 122.0 (C-7), 119.4 (C-2), 113.6 (C-5), 109.3 (C-9a), 108.0 (C-4), 8.2 (CH3).
Assay method phosphatase of regenerating liver-3 The cDNA molecule encoding PRL-3 was obtained from human brain Quick-Clone cDNA (Clontech) by a polymerase chain reaction, and was inserted into the NdeI-BamHI sites of pET28a. PRL-3 (residues 1-168) lacking the C-terminal farnesylation site was expressed in Escherichia coli BL21 (DE3) cell for the native protein at 30oC, after induction with 0.5 mM IPTG. Cell pellets were resuspended in a lysis buffer containing 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 5% (v/v) glycerol, 0.04% (v/v) b-mercaptoethanol, and 1 mM PMSF. After cell lysis by sonication, the His-tagged protein was purified by nickel affinity chromatography. His-tag was removed by thrombin digestion an the PRL-3 protein was further purified by ion exchange chromatography. PRL-3 assay was done using a DiFMUP assay (6,8difluoro-4-methylumbelliferyl phosphate). The assays were performed in 96-well plates using a reaction mixture containing 20 mM Tris-HCl, pH 8.0, 10 mM
Inhibitory Anthraquinones from Rubia akane on PRL-3
DTT, 0.01% Triton X-100 and 4 mM DiFMUP. A total volume of 200 µL was used in the assay. 10 µL of PRL3 purified protein was added to 190 µL of reaction mixture. The mixture was incubated for 1 h at room temperature and the reaction was stopped by addition of 2 mM sodium orthovanadate. The results were determined using a fluorimeter, at an excitation/emission wavelength of 355 nm/460 nm (Matter et al., 2001).
Migration assay Cell migration assay was performed using 8.0 µm pore size transwell inserts (Falcon). Cells were harvested with trypsin-EDTA, and washed twice with serum-free RPMI 1640 medium. Cells were resuspended in serum-free medium and 8 × 104 cells in 0.2 mL Serum free RPMI 1640 medium were added to the upper chamber. Various concentrations of chemicals in 0.5 mL RPMI 1640 medium with 10% FBS were placed into the lower chamber. Cells were incubated for 12~24 h at 37oC in a humidified atmosphere of 5% CO2. At the end of the experiment, the cells on the upper surface of the membrane were removed using cotton tips. The migrant cells attached to the lower surface were stained with crystal violet and incubated for 30 min. the membrane was washed several times with phosphate buffered saline, and the cells has penetrated the filter were counted under a microscope. Cell proliferation assay Cells were harvested with trypsin-EDTA, and resuspended in RPMI 1640 with 10% FBS. 6 × 103 cells were seeded into 96 well plates in RPMI 1640 with 10% FBS. After 20~24 h, cells were replenished with
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fresh complete medium containing a compound. After incubation for 24 h, cells proliferation reagent cell counting kit-8 (Dojindo) was added to each well. It was measured at 450 nm using an ELISA Reader (BioRad).
RESULTS AND DISCUSSION In searching for anticancer agents from plants, we found that the methanol extracts of the roots of Rubia akane inhibited the activity of PRL-3 with 70% at 100 µg/mL concentration. The extracts were subjected to solvent fractionation to give n-hexane, CHCl3, EtOAc, n-BuOH and H2O soluble fractions. The major active EtOAc soluble fraction (data not shown) was chromatographed to sephadex LH-20 column and purified by Lobar A column to yield compounds 1 and 2. Struc-
Fig. 2. PRL-3 inhibitory effect of compounds 1 and 2
Fig. 3. Photographs of cell migration inhibitory activity of compound 2 on DLD-1-PRL-3 cells
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tures of compounds 1 and 2 were elucidated as 2methyl-1,3,6-trihydroxy-9,10-anthraquinone-3-O-(6'O-acetyl)-α-rhamnosyl(1→2)-β-glucoside (1) and 2methyl-1,3,6-trihydroxy-9,10-anthraquinone (2) by comparing of the NMR spectral data with that of published literatures (Itokawa et al., 1983a; Qiao et al., 1990). Quinones are found in bacteria, fungi, lichens, gymnosperms and angiosperms. The majority of quinines found in plants are relatively simple benzoquinones, naphthoquinones and anthraquinones. In higher plants, anthraquinones are found in the Rubiaceae, Leguminosae, Rhamnaceae, Polygonaceae, Liliaceae, Bignoniaceae, Verbenaceae and Scrophulariaceae (Dey and Harborne, 1989). There were little pharmacological reports on compounds 1 and 2. Compound 1 was reported to show antibacterial activities on Escherichia coli and Staphylococcus aureus (Qiao et al., 1990), and compound 2 was reported to show inhibitory effects on
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the release of β-hexosaminidase in RBL-2H3 cells (Tao et al., 2003). Compounds 1 and 2 inhibited PRL-3 with IC50 values of 5.2 and 1.3 µg/mL, respectively (Fig. 2). Ginkgetin, a PRL-3 inhibitor isolated from Taxus cuspidata showed an IC50 value of 16.2 µg/mL (positive control, Choi et al., 2006). To our best knowledge, this is first report on the PRL-3 inhibitory anthraquinone compounds. Compound 2 was more potent than 1. Compound 2 was evaluated for its ability to inhibit the migration of tumor cell as shown in Fig. 3. Compound 2 inhibited the migration of PRL-3 expressed tumor cells (Fig. 4). However compound 2 did not inhibited the cell proliferation up to 20 µg/mL. Compound 2 specifically inhibited the migration of PRL-3 expressed tumor cells through inhibition of phosphatase activity of PRL-3 without cytotoxicity. Therefore, compounds 1 and 2 may be useful as lead compounds for the development of antitumor drugs through the control of PRL-3mediated signal pathways.
ACKNOWLEDGEMENTS This work was supported by the Grant of the Korean Ministry of Education, Science and Technology (The Regional Core Research Program/Center for Healthcare Technology Development).
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Fig. 4. Cell migration inhibitory activity of compound 2 on DLD-1-PRL-3 cells
Fig. 5. Inhibitory effect of compound 2 on the proliferation of DLD-1-PRL-3 cells
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