Biosci. Biotechnol. Biochem., 71 (8), 2061–2064, 2007
Note
Inhibitory Activity of Linoleic Acid Isolated from Proso and Japanese Millet toward Histone Deacetylase Nobuhiro A BURAI,1 Yasuaki E SUMI,2 Hiroyuki K OSHINO,2 Naoyuki N ISHIZAWA,1 and Ken-ichi K IMURA1; y 1 2
Department of Agro-Bioscience, Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan RIKEN(The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako 351-0198, Japan
Received February 2, 2007; Accepted April 21, 2007; Online Publication, August 7, 2007 [doi:10.1271/bbb.70068]
Linoleic acid was isolated from both the methanol extracts of proso and Japanese millet as a histone deacetylase inhibitor. It showed uncompetitive inhibitory activity toward histone deacetylase (IC50 ¼ 0:51 mM) and potent cytotoxicity toward human leukemia K562 (IC50 ¼ 68 M) and prostate cancer LNCaP cells (IC50 ¼ 193 M). Millet containing linoleic acid might have anti-tumor activity. Key words:
histone deacetylase; linoleic acid; proso millet; Japanese millet; anti-tumor activity
Histones are small basic proteins formed by complexing with DNA to give the nucleosome core and "-amino groups of lysine in them are modified by histone acetyl transferases (HATs) and histone deacetylases (HDACs). They can be present in one of the two antagonist forms, acetylated and deacetylated, regulated by HATs and HDACs, and are involved in transcription regulation. HDAC inhibitors are emerging as potential therapeutic agents for cancer, because HDACs play a fundamental role in regulating gene expression and the chromatin assembly that induces growth arrest, differentiation and apoptosis of tumor cells.1,2) A saturated fatty acid of the short chain (C4), sodium butyrate, is known as the first HDAC inhibitor and is a differentiating agent for many tumor cells.3,4) In addition, butyric acid derived from the fermentation products of dietary fiber by anaerobic microflora within the lumen of the large intestine inhibits HDACs, and induces caspase-3 protease activity and apoptosis.5,6) Thus, HDAC inhibitors in a food ingredient may be a useful compound for preventing cancer.7) Millet is an important food source in Africa and Asia. It has been reported that feeding a protein concentrate of proso millet (Panicum miliaceum L.) elevated the plasma level of the high-density lipoprotein (HDL) y
cholesterol concentration in rats and mice.8) In addition, this protein has a preventive effect on liver injury.9) Choi et al. have also demonstrated that feeding a protein concentrate of Korean foxtail millet resulted in a significant rise in the plasma levels of HDL cholesterol and adiponectin, and a large reduction in the insulin concentration of genetic type 2 diabetic model mice, suggesting improved insulin resistance.10) These results suggest the potent beneficial effect of these types of millet on human health, although there has been no report on the anti-tumor effect of millet until now. In the course of our screening program to find inhibitors of HDAC by using six hundred kinds of MeOH extract of plants (a fifth of them are food ingredients) and the HDAC colorimetric assay/drug discovery kit (AK-501, BIOMOL), each MeOH extract of commercial proso millet (Mochikibi) and Japanese millet (Hie), which are traditional types of millet in the northern area of Japan, showed about 80% HDAC inhibitory activity at the final concentration of 400 mg/ ml, but not foxtail millet (Mochiawa). We describe the isolation, identification and biological activity of the inhibitors from these millet samples in this paper. Commercial proso millet and Japanese millet (174.3 g and 163.6 g, respectively) was separately ground in a mixer and extracted with MeOH. Each MeOH extract (3.69 g and 2.01 g) was diluted with an excess of water and extracted twice with EtOAc. After evaporating EtOAc, the organic layer (2.71 g and 1.28 g) was subjected to silica gel TLC (0.75 mm, 20 20 cm, Silicagel 70 PF254 (Wako Pure Chemical Industries)) developed with a CHCl3 :MeOH solvent (100:1). The active fraction (Rf ¼ 0:26{0:38) was scraped off and eluted from the silica gel with MeOH. Finally, the active compound in each (1.34 g and 0.61 g) was dissolved in MeOH, and the inhibitor purified by HPLC (CAPCELL PAK C18 (10 mm’ 250 mm) Shiseido, 3.0 ml/min)
To whom correspondence should be addressed. Fax: +81-19-621-6124; E-mail:
[email protected] Abbreviations: HAT, histone acetyl transferase; HDAC, histone deacetylase; HDL, high-density lipoprotein; LA, linoleic acid; SAR, structureactivity relationship; PUFA, polyunsaturated fatty acid; CYP17, 17-hydroxylase/C17;20 -lyase
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Table 1. Structure-Activity Relationship for the HDAC Inhibitory Activity in Saturated and Unsaturated Fatty Acids Various fatty acids were dissolved in MeOH and subjected to the HDAC reaction. The HDAC reaction was performed by using a kit according to manufacturer’s instructions (AK-501, Biomol) with a small modification. Fatty acid
HDAC inhibition (IC50 , mM
Structure O
C 4:0 Butyric acid
OH
C 6:0 Hexanoic acid
O
C10:0 Decanoic acid
O
OH
OH O
C14:0 Myristic acid
OH O
C18:0 Stearic acid
OH
9
C18:1 Oleic acid
O
0.45 — — — — 1.31
OH
9
OH
C18:1 Ricinoleic acid
O OH
12
C18:2 Linoleic acid (LA)
9
O
— 0.51
OH
12
C18:2 Linoleaidic acid
O
9
OH
11
C18:2 Conjugated isomer of LA
9
O
— 0.55
OH
15
C18:3 -Linolenic acid
12
9
O
0.46 OH
O
C18:3 -Linolenic acid 12
9
OH
6 O
C20:0 Docosanoic acid
OH
14
C20:4 Arachidonic acid
11
8
5
O
0.38 — 0.27
OH
17
C20:5 Eicosapentaenoic acid
14
11
8
5
O
0.40 OH
O
C22:6 Docosahexaenoic acid 19
16 13
10
7
4
OH
0.36
—, Inhibition was not recognized at 1 mM.
with MeOH–H2 O (85:15) to yield 56.7 mg and 25.8 mg, respectively. Both active compounds were similar, judging from this physico-chemical properties and biological activity. The molecular formula was C18 H32 O2 by HR-ESI-MS ((nega.) m=z: 279.24987 ðM HÞ ) (JMS-T100LC), and the UV spectrum showed end absorption. The 1 HNMR and 13 C-NMR spectra indicated the inhibitors to have a long alkyl chain with two double bonds and a carboxylic acid. The positions of two double bonds were determined to be at C9 and C12 in the alkyl chain by a fragmentation analysis (m=z ¼ 221, 181 and 127) of the FAB/MS/MS spectra (Jeol JMS-HX/HX110 A). These data indicated that the active compound was 9, 12octadecadienoic acid. Finally, linoleic acid (LA), i.e., (cis-9, cis-12)-octadecadienoic acid, was identified by a comparison of the retention time by HPLC and the activity of the standard sample (Wako Pure Chemical Industries). LA (IC50 ¼ 0:51 mM), an unsaturated fatty acid of the long-chain (C18) type, had almost the same HDAC
inhibitory activity as butyric acid (IC50 ¼ 0:45 mM, it was 2.6-fold stronger than sodium butyrate (IC50 ¼ 1:33 mM)) (Table 1). It inhibited HDAC uncompetitively, judging from the Lineweaver-Burk plot shown in Fig. 1. As butyric acid and LA are very different fatty acids in their structure, we investigated the structure-activity relationship (SAR) of the saturated and unsaturated fatty acids in respect of the HDAC inhibitory activity. The HDAC inhibitory activities of various fatty acids (Wako Pure Chemical Industries) are shown in Table 1. Arachidonic acid (IC50 ¼ 0:27 mM), which had 1.9-fold stronger inhibitory activity than LA, had the strongest HDAC inhibitory activity of the examined fatty acids. The activity strength of fatty acids having aliphatic chains of 18 carbons was in the order of -linolenic acid >-linolenic acid >LA >(cis-9, trans-11)-octadecadienoic acid (the conjugated isomer of LA) >oleic acid, whereas the saturated fatty acids, except for butyric acid, had no inhibitory activity even at 1 mM (Table 1). The length of the aliphatic chain and the presence of the double bond were critical for the HDAC inhibitory
Inhibitory Activity of Linoleic Acid toward Histone Deacetylase
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120
1/v
10 Cell viability (%)
100 80 60 40 20 0 0.001
-10
-5
0
5
Fig. 1. Lineweaver-Burk Plot of the Inhibition of HDAC by Linoleic Acid. Linoleic acid was incubated for 5 min at 37 C with the enzyme, and the reaction was started by adding the substrate as described in the manufacturer’s instructions (AK-501, Biomol). 1=v is defined as 1/A405 . , linoleic acid 0.5 mM; , 0.25 mM.
activity of fatty acids. The double bond might have been involved in the increased flexibility of the long aliphatic chain and/or the strong interaction in the active-site pocket. Trichostatin A, which is a potent HDAC inhibitor (IC50 ¼ 7:8 nM in our assay), isolated from Streptomyces hygroscopicus showed 30-fold stronger HDAC inhibitory activity than suberoylanilide hydroxamic acid without the double bond.11–13) A carbonyl group in the fatty acids might also have reacted with the active site, because sodium butyrate and valproic acid both have the carbonyl group in each molecule.7,14) In contrast, ricinoic acid with a hydroxyl group at the C12 position had less inhibitory activity than oleic acid (Table 1). The hydrophilic moiety might not have interacted with the hydrophobic portion of the active site. We investigated the cytotoxicity of LA against tumor cells by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrasodium bromide) assay (Chemicon International).15,16) LA showed cytotoxicity against cells of human chronic myelogenous leukemia K562 (ATCC CCL-243, IC50 ¼ 68 mM comparable to IC50 ¼ 13:4 mM of arachidonic acid) and human prostate carcinoma LNCaP (ATCC CRL-1740, IC50 of 193 mM) (Fig. 2). The cytotoxicity of LA was 2- to 4-fold stronger than the HDAC inhibitory concentration. The cytotoxicity against tumor cells by such unsaturated fatty acids as LA might have resulted from the HDAC inhibitory activity, in addition to other inhibitory activity such as lipid peroxidation, DNA polymerase17,18) and CYP17 (17-hydroxylase/C17;20 -lyase) (IC50 ¼ 0:13 mM (17hydroxylase reaction)/IC50 ¼ 0:17 mM (C17;20 -lyase reaction)). Proso and Japanese millet containing LA, which was an HDAC inhibitor, could be useful in preventing cancer like oleic acid of olive oil and polyunsaturated fatty acids (PUFAs) like -linoleic acid that had HDAC
0.1
1
Linoleic acid concentration (mM)
10
1 / s (mM -1)
0.01
Fig. 2. Anti-Tumor Activity of Linoleic Acid. K562 (5 104 cells/ml) and LNCaP (1 105 cells/ml) cells were treated with linoleic acid at various concentrations at 37 C in a humidified, 5% CO2 atmosphere for 4 days in an RPMI 1640 medium supplemented with 10% fetal bovine serum, 50 units/ml of penicillin and 50 mg/ml of streptomycin, and the cytotoxicity was determined by an MTT assay. The percentage of viable cells was calculated as the ratio of the A570 values of treated and control cells (treated with the 0.05% MeOH vehicle). Each value is the average of four independent experiments. , K562 cells; , LNCaP cells.
inhibitory activity in this study (Table 1).19,20) Proso and Japanese millet also included such other fatty acids as, oleic acid and -linoleic acid, the amount of LA in them being proportional to the inhibitory activity toward HDAC and cancer cell growth (data not shown). Although the anti-tumor activity of PUFAs including LA in vivo is contradictory, our results show the fascinating explanation that the anti-tumor effects of PUFAs might depend on this HDAC inhibitory activity and/or CYP17 inhibitory activity,21) since the HDAC inhibitory activity of various MeOH extracts of millet was correlated with the CYP17 inhibitory activity involved in the growth inhibition of prostate cancer for the lack of androgen (Kimura, K., et al., Biosci. Biotechnol. Biochem., in press). We plan to examine the anti-tumor activity of millet having a high content of LA against tumor-bearing mice.
Acknowledgment This work was partially supported by the Iijima Memorial Foundation for the Promotion of Food Science and Technology.
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