- Email: [email protected]
Inhibition of Coix seed extract on fatty acid synthase, a novel target for anticancer activity
Journal of Ethnopharmacology 119 (2008) 252–258
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Inhibition of Coix seed extract on fatty acid synthase, a novel target for anticancer activity Fei Yu a,b , Jing Gao b , Yong Zeng b , Chang-Xiao Liu a,b,∗ a
Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China Tianjin State Key Laboratory of Pharmacokinetics and Pharmacodynamics, Tianjin Institute of Pharmaceutical Research, 308 An-Shan West Road, Tianjin 300193, China b
a r t i c l e
i n f o
Article history: Received 16 March 2008 Accepted 11 July 2008 Available online 23 July 2008 Keywords: Coix seed extract Fatty acid synthase Inhibition Lipometabolism Glycometabolism
a b s t r a c t Ethnopharmacological relevance: Coix seed has been traditionally used to treat cancers in folk medicine. Aim of the study: Study the anticancer action mechanism of Coix seed extract. Materials and methods: After the treatment with Coix seed extract (10 l/ml), the residual activity of fatty acid synthase (FAS) as overall reaction, -ketoacyl reduction, enoyl reduction, and acetyl acetyl coenzyme A (AcAcCoA) reduction was separately detected at 340 nm in the UV-190 spectrophotometer. After rats were administrated Coix seed extract (2.5, 5.0, and 10.0 ml/kg) intragastrically for 10 days consecutively, activities of FAS, malate dehydrogenase (MDH), lipid protein lipase (LPL), hepatic lipase (HL), triglyceride (TG), and glucose-6-phosphate dehydrogenase (G-6-PD) in the plasma, liver and fatty tissues were determined. Results: Experiments in vitro showed that the inhibition of Coix seed extract on FAS activity was significant and dose dependent, and two active sites inhibited were -ketoacyl reductases (KR) and enoyl reductase (ER). Experiments in vivo showed that Coix seed extract inhibited FAS activity in the liver, and elevated LPL and HL activity in the plasma, and effected G-6-PD activity. Conclusions: The study supports that FAS is a novel target for anticancer activity, and provides a theoretical foundation for the wide application of Coix seed extract in traditional medicine. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Ethnopharmacologically, the traditional use of Coix seed has lasted for thousands of years in Traditional Chinese Medicine (TCM) (Li, 1596). The herb is sweet and tasteless in flavour, slightly cold in nature. Coix seed is used to treat cancers in folk medicine (Normile, 2003). Epidemiologists have long suspected that the low cancer rates in Southeast China might be related to Coix lacryma-jobi L. subsp. Ma-yuen (Romanet) T. Koyama (family Poaceae) (Fig. 1), a grass-like relative of maize that is a dietary staple in the region, and a key ingredient of many traditional Chinese herbal medicines (Normile, 2003). Li extracted the anticancer compounds of the Coix seed with supercritical CO2 to produce an anticancer drug called Kanglaite injection (KLT), which showed anticancer activity in animal tumor models (Feng et al., 2004), and was applied clinically in China and Russia (Li, 2001; Qian et al., 2004).
∗ Corresponding author at: Tianjin State Key Laboratory of Pharmacokinetics and Pharmacodynamics, Tianjin Institute of Pharmaceutical Research, 308 An-Shan West Road, Tianjin 300193, China. Tel.: +86 22 23006863; fax: +86 22 23006860. E-mail address: [email protected] (C.-X. Liu). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.07.015
Coix seed extract from the seeds (Fig. 2) is an oily substance, and its main active ingredient is a triglyceride containing four fatty acids (Fig. 3) (Li et al., 1999a). Coix seed extract is formulated into an emulsion for injection, which has been proved to be an effective and safe TCM new preparation by preclinical antitumor studies, and pharmacokinetics and safety studies (Li et al., 2003a, 2005a; Yin and Jin, 2003; Li, 2006). Phases I, II and III clinical trials and tens of thousands of clinical applications proved that the preparation has adjunctive therapy effects to cancer patients (Li et al., 1999b, 2005b; Zhang et al., 1999; Li, 2001). When combined with chemotherapy, radiotherapy and surgery, it could improve the response rate, regulate the energy of advanced patients, and improve life quality so as to prolong survival time (Yang, 1998; Normile, 2003; Garin et al., 2004; Li, 2004, 2006). The USA Food and Drug Administration approved a Phase II clinical trial to test its efficacy in treating nonsmall-cell lung cancer in 2003 (Normile, 2003). It is the first drug derived from a traditional Chinese herbal remedy to go into clinical trials in the United States (Normile, 2003). As the key raw material for the synthesis of long-chain fatty acid in organisms, fatty acid synthase (FAS) extensively exists in fat tissues and the liver of humans and animals (Wakil et al., 1983). Fatty acids, the product of fatty acid synthesis, are the source of
F. Yu et al. / Journal of Ethnopharmacology 119 (2008) 252–258
253
Table 1 Triglyceride content of Coix seed oil
Fig. 1. Coix plant.
Fig. 2. Coix seeds.
Peak no.
Compound
Molecular formula
Molecular mass
Peak area (%)
1 2 3 4 5 6 7
1,2,3-Trilinoleylglycerol 1,2-Dilinoleyl-3-oleylglycerol 1,2-Dilinoleyl-3-palmitoylglycerol 2,3-Dioleoyl-1-linoleylglycerol 2-Linoleyl-3-oleyl-1-palmitoylglycerol 1,2,3-Trioleylglycerol 2,3-Dioleyl-1-palmitoylglycerol
C57 H98 O6 C57 H100 O6 C55 H98 O6 C57 H102 O6 C55 H100 O6 C57 H104 O6 C55 H102 O6
878.7 880.8 854.7 882.8 856.8 884.8 858.8
7.0 18.8 7.6 19.1 14.9 20.9 11.7
substance and energy for the proliferation of tumor cells. Compared with normal tissues, many tumor cells express FAS at high levels and undergo significant endogenous fatty-acid synthesis, and FAS inhibitors were proved to have antitumor activity, which made FAS a novel specific target in search for related anticancer drugs (Kuhajda, 2000; Loftus et al., 2000; Li et al., 2003b; Jenni et al., 2006; Maier et al., 2006). At present, most of the research on the inhibition of FAS has focused on the formation of fat in the body of animals especially on inhibition of the activity of FAS in poultry (Tian, 1994; Tian et al., 1996). Some researchers have studied the inhibitory effect of such plants as Polygonum Multiflorum Thunb. (Zhang et al., 2004a), Ginkgo Biloba L. (Zhang et al., 2004b) and green tea (Brusselmans et al., 2003) during the activity of FAS in human breast cancer cells and further proved beneficial effect through the inhibition of FAS. Although KLT injection has been successfully applied for the treatment of a variety of malignant tumors, such as carcinomas of the lung, liver, stomach, esophagus, colon, pancrease, kidney, ovaries, malignant lymphoma, leukemia for more than 200,000 cases (Li, 2006), it is uncertain that how it works. Studies in vitro about the action mechanism of the cytotoxic effects of Kanglaite injection indicated (Woo et al., 2007): Coix seed extract has a direct effect on PKC kinase activity; it inhibits NFB-mediated regulation of COX-2 expression; it inhibits the expression of MMP9, probably through the inhibition of NFB signaling, it inhibits matrigel invasion, it inhibits the tumor growth of human cancer xenografts and has apparent synergy with agents that inhibit fatty acid synthase. It was predicted that FAS may be a novel target for cancer. To further study whether Coix seed extract has an influence on FAS activity and its action mechanism, experiments in vitro were conducted to find out its capacity and possible action targets. Because the synthesis of fatty acid requires some apolipoprotein and glycometabolic enzymes (Zhou et al., 2004), the determination of their activities in blood and tissues could further demonstrate the regulation of FAS in the process of carcinogenesis. Changes of FAS, malate dehydrogenase (MDH), LPL, hepatic lipase (HL), triglyceride (TG), and G-6-PD were studied in animals after orally feeding Coix seed extract to search for the relationship between tumor cell growth and the activities of metabolic enzymes, and for new theoretical support for the use of Coix seed extract in the clinical treatment of cancer. 2. Materials and methods 2.1. Drug
Fig. 3. Chemical structures of the main active ingredients in Coix seeds.
Coix seed extract (Batch No.: 060309), provided by Zhejiang Kanglaite Pharmaceutical Co. Ltd. The fingerprint of Coix seed extract in which the main active ingredients, triglycerides, were assigned was obtained by high performance liquid chromatography (HPLC) (Fig. 4 and Table 1).
254
F. Yu et al. / Journal of Ethnopharmacology 119 (2008) 252–258
Fig. 4. Fingerprint of Coix seed extract by HPLC. Table 1 shows what every peak is and the relative content of every peak. HPLC conditions: the stationary phase was Agilent Zorbax Extend C18 column (4.6 mm × 250 mm, 5 m). The mobile phase was dichloromethane–acetonitrile (35:65), flow rate was 0.5 ml/min; column temperature was 35 ◦ C.
2.2. Animals Duck: body weight 2–2.5 kg, purchased from Tianjin farm product market. Wistar rat: body weight 170–210 g, provided by the animal house of Tianjin Institute of Pharmaceutical Research. 2.3. Reagents for experiments in vitro Acetyl coenzyme A (AcCoA), malonyl coenzyme A (MalCoA), reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH), acetyl acetyl coenzyme A (AcAcCoA), and dithiothreitol molecular biology reagent (DTT) were all purchased from Sigma Co., USA. Ethyl crotonate provided by Guan Hengye Fine Chemical Co. Ltd., ethyl acetoacetate provided by Tianjin Guangfu Fine Chemical Institute, and other reagents were analytical grade, FAS was separated and purified from duck liver. 2.4. Kits for tests in vivo Kits for the assay of glucose-6-phosphate dehydrogenase (G6-PD), malate dehydrogenase (MDH plasma, tissue), plasma total lipase (lipid protein lipase (LPL) and hepatic lipase (HL)), tissue total lipase (LPL and HL), and protein determination with coomessie brilliant blue were procured from Jiancheng Bioengineering Laboratory, Nanjing; the triglyceride assay kit was procured from Zhongsheng Beikong Biotech Co. Ltd., Beijing. 2.5. Instruments Beckman J2-21 high-speed freezing centrifuge from Beckman, USA; low-pressure column chromatograph from High-tech Application Laboratory, Beijing; UV-190 spectrophotometer from Shimadzu Corporation, Japan. 2.6. Separation and purification of FAS from duck liver Livers were taken out immediately from three ducks after bleeding to death and put in an ice bath for cooling and weigh-
ing. As much as of 1.8 ml/g precooled buffer (0.1 mol/l potassium phosphate buffer, containing 0.07 mol/l KHCO3 , 1 mmol/l DTT, and 1 mmol/l EDTA, pH 7.8–8.0) was added to the duck liver to make a homogenate in the ice bath. The resulting suspension was centrifuged at 8000 rpm for 30 min at 4 ◦ C, the supernatant fluid was filtered through gauze and centrifuged at 18,000 rpm for 60 min. The precipitate was discarded and the supernatant fluid was collected and stored at −75 ◦ C for further purification. The supernatant solution (70 ml) was defrozen at 4 ◦ C, saturated ammonium sulfate ((NH4 )2 SO4 ) was added slowly to reach a saturation level of 25%, and the solution was stood for 2 h at 4 ◦ C. The supernatant fluid collected after centrifugation was brought to a saturation level of 50% with saturated (NH4 )2 SO4 and stood at 4 ◦ C overnight. The precipitate after centrifugation was dissolved in 200 ml balanced buffer (5 mmol/l potassium phosphate buffer, containing 1 mmol/l DTT) and centrifuged again, the supernatant fluid was applied to the balanced DEAE-sepharose FF column (2.6 cm × 50 cm) at a flow rate of 1.4 ml/min, and the column was eluted until A280 < 0.01 with balanced buffer solution. Then the column was eluted with 5–0.3 mol/l potassium phosphate buffer solutions by linear gradient elution. The eluate was collected stepwise and the FAS activity was determined. The elutions with active peaks were combined and added to the same volume of saturated (NH4 )2 SO4 and stood overnight at 4 ◦ C, the precipitate after centrifugation was dissolved in an appropriate amount of buffer solution (0.1 mol/l potassium phosphate buffer, 1 mmol/l DTT and 20% glycerine, pH 7.0)and dialyzed overnight. The dialyzed solution was sub-packed and stored at −75 ◦ C after activity determination (Tian et al., 1985). 2.7. Assays for FAS activity According to the method of Tian (Li et al., 2003b), 0.1 mol/l potassium phosphate buffer (pH 7.0) containing 1 mmol/l EDTA and 1 mmol/l DTT, and enzyme, and water, to make a final volume of 2 ml were added to a colorimetric cylinder (1 cm optical path) at constant temperature (37 ◦ C) water bath for 5 min. The diluted enzyme solution (20 g) was added to initiate the reaction, and the absorbance change was continuously monitored at 340 nm wavelength in a UV-spectrophotometer. The absorbance changed since NADPH was catalyzed and oxidized by FAS into NADP. The substrate concentrations in the FAS overall reaction and the activity determination for each activity center are listed below: (1) Overall reaction: 6 mol/l AcCoA, 12 mol/l MalCoA, 40 mol/l NADPH. (2) -Ketoacyl reduction: 0.8 mmol/l ethyl acetoacetate, 40 mol/l NADPH. (3) Enoyl reduction: 45 mmol/l ethyl crotonate, 40 mol/l NADPH. (4) AcAcCoA reduction: 31 mol/l AcAcCoA, 40 mol/l NADPH. This determination includes reactions at active centers, including transacylation, ketoacyl reduction, dehydration and enoyl reduction, etc. (5) Calculation of FAS activity: the unit of FAS activity is defined as the enzyme quantity consumed to oxidize 14 nmol/l NADPH in a minute. In this way, the unit of enzyme activity in 1 ml can be calculated with the equation: activity (U/ml) = A340 × 106 × C/(ε × V)/14where A340 is the absorbance change per min at 340 nm wavelength; ε is the mmol extinction coefficient of NADPH at 340 nm wavelength which is 6.022 at optical diameter 1 cm; V is the volume of system (2 ml); C is the dilution folds (40); and 106 is the coefficient in conversion from mmol to nmol.
F. Yu et al. / Journal of Ethnopharmacology 119 (2008) 252–258
255
Table 2 Inhibition of Coix seed extract (10 l/ml) on FAS overall reaction and activity centers (X¯ ± S.D., n = 3) Time (h)
Residual activity (%) Overall reaction
0 0.25 0.5 0.75 1 1.5 2
100 82.8 44.3 29.9 20.5 18.9 8.2
± ± ± ± ± ± ±
14.7 5.7 11.1 17.6 7.5 5.7 1.4
Keto-acyl reduction 100 54.0 49.9 36.4 32.4 27.0 21.6
± ± ± ± ± ± ±
Enoyl reduction
8.4 8.4 10.2 8.1 16.2 4.7 8.4
100 84.8 82.5 80.1 53.4 52.3 39.5
± ± ± ± ± ± ±
AcAcCoA reduction
16.5 15.7 19.2 22.8 4.0 12.6 24.7
100 71.3 56.1 42.8 34.8 23.2 8.9
± ± ± ± ± ± ±
17.2 6.2 3.8 17.5 3.8 3.1 3.1
2.8. Assays for inhibitory effects of Coix seed extract on animal FAS in vitro Coix seed extract and enzyme solution were mixed for some time in the swing bed at 25 ◦ C and centrifuged immediately to separate the oil from the water, the activity of the enzyme solution was determined as A, and A0 was the activity when Coix seed extract was replaced by water. A/A0 multiplied by 100% was the residual activity at that moment. The residual activities at a series of time points were determined and a graph with activity to time was plotted. 2.9. Assays for effects of Coix seed extract on activities of some enzymes of lipometabolism and glycometabolism in rats Upon arrival, the rats were kept for 3 days to adapt to the environment. On the 4th day, 42 rats were divided into 4 groups: control group (10 rats), test group I with 2.5 ml/kg Coix seed extract (10 rats), test group II with 5.0 ml/kg Coix seed extract (10 rats) and test group III with 10.0 ml/kg Coix seed extract (12 rats), the male/female ratio was 1:1 in each group. The rats were given Coix seed extract intragastrically once a day for 10 consecutive days. After the last administration, the rats were anesthetized with ether, blood samples were taken from the abdominal aorta of the rats, and were centrifuged to obtain the plasma. The liver and fat were removed and prepared into tissue homogenate. Activities of FAS, MDH, LPL, HL, TG, and G-6-PD in the plasma and tissues were determined and compared with the control group to evaluate the effects of Coix seed extract on the activities of some enzymes of lipometabolism and glycometabolism in rats. The animal tests were conducted in accordance with the principles for laboratory animal use and care as found in the European Community guidelines (EEC Directive of 1986; 86/609/EEC). 2.10. Statistical analysis Student’s t-test was used for statistical comparison of the differences in data between the test groups. Values with p < 0.05 were considered significant. All data are represented as arithmetic means and standard deviation (X¯ ± S.D.).
Fig. 5. Inhibition of Coix seed extract (10 l/ml) on FAS overall reaction and activity centers.
inhibition of Coix seed extract on FAS overall reaction activity with different dosages of Coix seed extract (0, 1, 2.5, 5, 10 l/ml) at 25 ◦ C for 2 h (Table 3 and Fig. 6). The results demonstrated that the inhibition in the range 1–10 l/ml concentration was dose dependent. 3.1.2. Inhibition of Coix seed extract on ˇ-ketoacyl reduction Over 50% of the FAS activity was inhibited after 0.5 h of reaction with Coix seed extract (10 l/ml) at 25 ◦ C, and after 2 h of reaction the residual activity of -ketoacyl reduction was only 21.6% (Table 2 and Fig. 5). Inhibition results of Coix seed extract in the range of 1–10 l/ml at 25 ◦ C for 2 h (Table 3 and Fig. 6) demonstrated that the inhibition to -ketoacyl reduction reaction was dose dependent. 3.1.3. Inhibition of Coix seed extract on enoyl reduction The inhibition of Coix seed extract (10 l/ml) on enoyl reduction was comparatively weak, and the residual activity after 2 h of reaction at 25 ◦ C was 39.5% (Table 2 and Fig. 5). 3.1.4. Inhibition of Coix seed extract on AcAcCoA reduction The inhibition of Coix seed extract (10 l/ml) on AcAcCoA reduction catalyzed by FAS was comparatively strong, over 50% of the
3. Results 3.1. Part I: experiments in vitro 3.1.1. Inhibition of Coix seed extract on overall reaction Over 50% of the FAS activity was inhibited after 0.5 h of reaction with Coix seed extract (10 l/ml) at 25 ◦ C, and after 2 h of reaction the residual activity of overall reaction was only 8.2% (Table 2 and Fig. 5). To further evaluate the inhibition of Coix seed extract on the FAS overall reaction activity, experiments were continued on the
Table 3 Inhibition of Coix seed extract (1–10 l/ml) on FAS overall reaction and some active centers (X¯ ± S.D., n = 3) Dosage (l/ml)
Residual activity (%) Overall
0 1 2.5 5 10
100 53.6 29.5 20.6 8.2
± ± ± ± ±
Keto-acyl 13.2 25.9 14.9 1.5 1.4
100 83.0 51.3 39.1 20.2
± ± ± ± ±
31.2 23.8 30.0 12.9 12.1
Enoyl 100 76.3 73.1 61.3 60.2
± ± ± ± ±
AcAcCoA 8.5 32.3 15.9 12.9 11.3
100 71.3 67.7 48.1 10.7
± ± ± ± ±
12.3 16.3 35.6 24.5 10.7
256
F. Yu et al. / Journal of Ethnopharmacology 119 (2008) 252–258 Table 6 Effects of Coix seed extract on LPL activity in rats (X¯ ± S.D., n = 10) Dosage (ml/kg)
LPL activity (U/ml) Plasma
Control group 2.5 5.0 10.0
3.318 3.505 2.832 5.620
± ± ± ±
Liver 0.872 0.985 1.057 3.051*
0.537 0.418 0.510 0.525
Fat ± ± ± ±
0.183 0.063 0.183 0.205
3.713 4.574 5.714 5.645
± ± ± ±
1.815 1.910 2.604 2.414
± ± ± ±
2.005 4.851 0.935 2.901
Significant difference relative to control group, * p < 0.05.
Table 7 Effects of Coix seed extract on HL activity in rats (X¯ ± S.D., n = 10) Dosage (ml/kg)
HL activity (U/ml) Plasma
Fig. 6. Inhibition of Coix seed extract (1–10 l/ml) on FAS overall reaction and activity centers.
enzyme activity was inhibited at 25 ◦ C for 0.75 h, and after 2 h of reaction the residual activity was only 8.9% (Table 2 and Fig. 5). Inhibition results of Coix seed extract in the range of 1–10 l/ml at 25 ◦ C for 2 h (Table 3 and Fig. 6) demonstrated that the inhibition of AcAcCoA reduction was also dose dependent. 3.2. Part II: experiments in vivo Rats were fed with Coix seed extract (2.5, 5.0, and 10.0 ml/kg) intragastrically for 10 days consecutively. Compared with the control group, the dosage of 10 ml/kg had a significant effect on FAS activity in the liver (p < 0.05). No notable effects on FAS activity in fatty tissues were observed (Table 4). There were no significant effects on MDH activity in the plasma, liver and fatty tissues (Table 5). These dosages all significantly elevated HL activity in plasma (p < 0.05). The dosage of 10.0 ml/kg significantly elevated HL activity in the liver and LPL activity in the plasma (p < 0.05) (Tables 6 and 7). No significant effects on TG content in the plasma were observed (Table 8). The blood G-6-PD (methemoglobin reduction percentage) of rats in high, medium, and low-dosage groups were reduced to different extents (p < 0.01) (Table 8).
Table 4 Effects of Coix seed extract on FAS activity in rat tissues (X¯ ± S.D., n = 10) Dosage (ml/kg)
FAS activity (U/g tissue) Liver
Control group 2.5 5.0 10.0
13047.40 9752.60 11007.26 10082.08
Fat ± ± ± ±
3268.21 4681.41 2490.75 2621.14*
3510.94 3004.86 2894.15 2846.70
± ± ± ±
2693.94 1717.95 1729.63 1355.86
Significant difference relative to control group, * p < 0.05.
MDH activity (U/ml) Plasma
Control group 2.5 5.0 10.0
0.570 0.419 0.394 0.452
± ± ± ±
Liver 0.249 0.177 0.141 0.112
12.209 11.924 13.372 12.949
Fat ± ± ± ±
2.950 2.269 1.961 2.227
8.040 10.310 10.833 9.786
3.980 5.555 5.376 6.286
± ± ± ±
1.329 1.581* 1.188* 2.787*
0.548 0.749 0.677 0.812
Fat ± ± ± ±
0.106 0.325 0.266 0.294*
4.405 6.946 6.233 6.469
Significant difference relative to control group, * p < 0.05.
4. Discussion and conclusions The products from FAS are the sources of substance and energy for the proliferation of tumor cells. The search for drugs that can arrest the activity of FAS to cut off or inhibit the source of substance and energy for the growth of tumors is the new target for anticancer drug discovery (Li and Hou, 2003). Traditionally considered as an anabolic-energy-storage pathway, fatty-acid synthesis is now regarded as the key process for tumor cell growth and survival. The inhibition of FAS activity should inhibit tumor cell growth, and finally realize the objective of cancer therapy. Because of its role in fatty acid synthesis, FAS is a target for drug development against obesity and obesity-related diseases, including diabetes and cardiovascular disorders (Maier et al., 2006). FAS is overexpressed in many forms of cancer (Kuhajda et al., 1994), and FAS inhibitors have demonstrated antitumor activity (Kuhajda et al., 2000). The present experiment in vitro showed that Coix seed extract could significantly inhibit FAS activity, and experiments in rats also showed that Coix seed extract significantly inhibited FAS activity in liver. Therefore, the antitumor effects of Coix seed extract are probably related to the inhibition of FAS activity. The study supports that FAS is a novel target for cancer therapy. The other mechanism of action of Coix seed extract, as a Kanglaite injection (KLT), is as follows: (1) the drug inhibits the mitosis of tumor cells during the G2/M phases; (2) induces apoptosis of tumor cells; (3) affects the genetic expression of tumor cells by up-regulating FAS/Apo-1 gene expression and down-regulating Bc1-2 gene expression; (4) inhibits tumor angiogenesis; (5) counteracts the cachexia of cancers; and (6) reverses the multi-resistance of tumor cells to anticancer drugs and the resistance modification in some chemotherapeutic agents (Li, 2006). All of the above mechanism of action of Coix seed Table 8 Effects of Coix seed extract on TG content and G-6-PD activity (X¯ ± S.D., n = 10)
Table 5 Effects of Coix seed extract on MDH activity in rats (X¯ ± S.D., n = 10) Dosage (ml/kg)
Control group 2.5 5.0 10.0
Liver
± ± ± ±
2.380 4.127 5.491 2.321
Dosage (ml/kg)
TG content in plasma (mmol/l)
Control group 2.5 5.0 10.0
1.119 1.027 1.061 0.942
± ± ± ±
0.538 0.315 0.330 0.353
Significant difference relative to control group, ** p < 0.01.
Reduction percentage of methemoglobin (%) 76.542 64.746 60.475 69.266
± ± ± ±
7.477 6.089** 3.308** 2.254**
F. Yu et al. / Journal of Ethnopharmacology 119 (2008) 252–258
257
FAS activity was significant and dose dependent. Coix seed extract inhibited two active sites: KR and ER (Fig. 8). Continued studies are required to explain whether Coix seed extract inhibits other active sites. Experiments in vivo showed that Coix seed extract inhibits FAS activity in the liver, elevates LPL and HL activity in the plasma, and effects G-6-PD activity, thereby inhibiting fatty acid synthesis and reducing the components and forms of energy for tumor cell growth. The present study supports FAS as a novel target for cancer therapy, and provides a theoretical foundation for the clinical application of Coix seed extract in cancer therapy. Acknowledgements Fig. 7. FAS—a new target for the active component of Coix seed.
extract reflect the multiple targets of action of Coix seed extract (Fig. 7). Maier obtained the overall architecture of mammalian FAS by X-ray crystallography at intermediate resolution, providing a new structural basis for further experiments required for a detailed understanding of the complex action mechanism of mammalian FAS (Maier et al., 2006). Animal FAS is a compound enzyme with two subunits (Smith et al., 2003). The molecular weight of each subunit is about 2.7 million and it has 6 different active sites and 1 acyl carrier protein (ACP) in each subunit covalently linked to a phosphopantetheinyl prosthetic group (Smith, 1994). The six active sites are malonyl/acetyl transacylase (MAT), -acyl-synthetase (KS), ketoacyl reductases (KR), -hydroxylacyl dehydrase (DH), enoyl reductase (ER), and long-chain fatty acid thioesterase (TE) (Fig. 8) (Maier et al., 2006). If any active site is inhibited, the whole FAS activity will be inhibited. This is the first time to describe a new action mechanism of Coix seed extract through the inhibition of FAS. Further experiments in vitro showed that the inhibition of Coix seed extract on
Fig. 8. Catalytic cycle and domain organization of FAS.
The authors thanks Zhejing Kanglaite Pharmaceutical Co. Ltd. for financial support, and Professor Wei-Xi Tian (Department of Biology, Graduate School of the Chinese Academy of Sciences, Beijing, China) for useful suggestions and discussions. References Brusselmans, K., De Schrijver, E., Heyns, W., Verhoeven, G., Swinnen, J.V., 2003. Epigallocatechin-3-gallate is a potent natural inhibitor of fatty acid synthase in intact cells and selectively induces apoptosis in prostate cancer cells. International Journal of Cancer 106, 856–862. Feng, G., Kong, Q.Z., Huang, D.S., Huang, T., Lu, H.D., Fei, Y., Feng, J.P., 2004. Experimental study on inhibition angiogenesis in mice S1 80 sarcoma by Coix lacryma-jobi injection. China Journal of Cancer Prevention and Treatment 31, 229–230. Garin, A.M., Tjulandin, S.A., Kadagidze, Z.G., Bulanov, A.A., Chitya, L.V., 2004. Clinical trial of kanglaite injection in patients with non-small cell lung cancer in Russia. In: Li, D.P. (Ed.), Progress of Clinical Studies of Kanglaite Injection in Foreign Countries (USA, RUS), pp. 67–70. Jenni, M., Leibungut, Maier, T., Ban, N., 2006. Architecture of mammalian fatty acid synthase at 5 Å resolution. Science 311, 1263–1567. Kuhajda, F.P., 2000. Fatty acid synthase and human cancer: new perspectives on its role in tumor biology. Nutrition 16, 202–208. Kuhajda, F.P., Jenner, K., Wood, F.D., Hennigar, R.A., Jacobs, L.B., Dick, J.D., Pasternack, G.R., 1994. Fatty acid synthesis: a potential selective target for antineoplastic therapy. Proceedings of the National Academy of Sciences of the United States of America 91, 6379–6383. Kuhajda, F.P., Pizer, E.S., Li, J.N., Mani, N.S., Frehywot, G.L., Townsend, C.A., 2000. Synthesis and antitumor activity of an inhibitor of fatty acid synthase. Proceedings of the National Academy of Sciences of the United States of America 97, 3450–3454. Li, S.Z., 1596. Compendium of materia medica. Part of Cereal 24, 1489. Li, D.P., 2001. Clinical applications of Kanglaite injection (KLT). Chinese Journal of Clinical Oncology 28, 300–302. Li, D.P., 2004. Progress of Clinical Studies of Kanglaite Injection in Foreign Countries (USA, RUS). Zhejiang Kanglaite Pharmaceutical Co. Ltd., Hangzhou, pp. 1–28. Li, D.P., 2006. Research advance on ethnopharmacology, pharmacodynamics, pharmacokinetics and clinical therapeutics of Coix seed and its preparation, Kanglaite injection. Asian Journal of Pharmacodynamics and Pharmacokinetics 6, 83–102. Li, X.H., Hou, Y.S., 2003. Fatty acid synthase: specific target for cancer therapy. Journal of Biochemistry and Biophysics 35, 1–5. Li, D.P., Huang, J.M., Su, W.A., Shen, K.K., Jiang, L.L., Shi, J.Y., 1999a. The analysis of chemical composition in the Coix lipid. Traditional Chinese Drug Research & Clinical Pharmacology 10, 35–37. Li, T.D., Liu, A.G., Qin, S.L., Xu, P.M., Zhang, W.F., Chen, L., Cheng, G.Y., Zhuang, J.S., Zhang, Y.H., Liu, B.K., 1999b. Clinical study of Kanglaite injection in cancer pain control and life quality improvement in patients with advanced cancer. Chinese Journal of Clinical Oncology 26, 372–376. Li, Z.H., Lü, J.F., Zhou, L.Z., Li, B., 2005b. Clinical study of combined Kanglaite injection with chemotherapy in treatment of NSCLC. China Journal of Cancer Prevention and Treatment 12, 1339–1340. Li, L., Shen, M.Y., Ma, L., 2003a. Observation on security of Kanglaite injection in treating senile patients with advanced tumor. China Journal of Cancer Prevention and Treatment 10, 539–540. Li, D.P., Shen, K.K., Ma, Y.M., 2005a. The pharmacokinetic and bioavailability study of Coix seed extract preparation. China Journal of Traditional Chinese Medicine and Pharmacy 20, 81–85. Li, L.C., Wu, X.D., Tian, W.X., 2003b. lnhibition to fatty acid synthase with extract of Tuber Fleeceflower root. Chinese Journal of Biochemistry and Molecular Biology 19, 297–304. Loftus, T.M., Jaworsky, D.E., Frehywot, G.L., Townsend, C.A., Ronnett, G.V., Lane, M.D., Kuhajda, F.P., 2000. Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors. Science 288, 2379–2381.
258
F. Yu et al. / Journal of Ethnopharmacology 119 (2008) 252–258
Maier, T., Jenni, S., Ban, N., 2006. Architecture of mammalian fatty acid synthase at 4.5 Å resolution. Science 311, 1258–1562. Normile, D., 2003. The new face of traditional Chinese medicine. Science 299, 188–190. Qian, J., Qin, S.L., Yang, L.Q., Chen, Y.X., Shao, Z.J., 2004. The clinical study of Gefitnib combination with Kanglaite injection in treatment of non-small-cell lung cancer. Chinese Clinical Oncology 9, 568–570. Smith, S., 1994. The animal fatty acid synthase: one gene, one polypeptide, seven enzymes. The FASEB Journal 8, 1248–1259. Smith, S., Witkowski, A., Joshi, A.K., 2003. Structural and functional organization of the animal fatty acid synthase. Progress in Lipid Research 42, 289–317. Tian, W.X., 1994. The body fat level and the regulation of fatty acid synthase activity in animals. Chemistry of Life 14, 1. Tian, W.X., Dong, Y., Quan, H., Chen, W.F., 1996. The dependence of fat level of hen on activity of fatty acid synthase in liver on different ages. Chinese Biochemistry Journal 12, 234–236. Tian, W.X., Hsu, R.Y., Wang, Y.S., 1985. Studies on the reactivity of the essential sulfhydryl groups as a conformational probe for the fatty acid synthase of chicken liver. Journal of Biological Chemistry 260, 11375–11387. Wakil, S.J., Stoops, J.K., Joshi, V.C., 1983. Fatty acid synthesis and its regulation. Annual Review of Biochemistry 52, 537–579.
Woo, J.H., Li, D.P., Orita, H., Coulter, J., Tully, E., Kwon, T.K., Xu, S., Gabrielson, E., 2007. Coix seed extract, a commonly used treatment for cancer in China, inhibits NFkappaB and protein kinase C signaling. Cancer Biology & Therapy 6 (Epub ahead of print). Yang, H., 1998. Collection of the Studies of Kanglaite Injection against Tumors. Zhejiang University Press, pp. 107–118. Yin, L., Jin, X.F., 2003. Observation of the clinical therapeutic effect of Kanglaite (KLT) combined with chemotherapy on advanced cancer patients. China Pharmacy 14, 166–167. Zhang, H.Q., Li, S.Y., Fu, Y., Yang, S., Fu, T.H., Qin, M., Lü, F.D., Yan, D.J., 1999. Clinical and pathological changes of Kanglaite injection in treatment of primary lung cancer. Chinese Journal of Clinical Oncology 26, 78–79. Zhang, Y.Y., Zhang, F.Z., Sun, L.Y., Wang, X.Z., 2004a. Inhibition to fatty acid synthase from human breast cancer cell with extract of Tuber Fleeceflower root in vitro. Journal of Qiqihar Medical College 25, 1102–1104. Zhang, Y.Y., Zhang, F.Z., Sun, L.Y., Wang, X.Z., 2004b. Inhibition to fatty acid synthase from human breast cancer cell with extract of Ginkgo leaf in vitro. Journal of Taishan Medical College 25, 433–435. Zhou, C.H., Tanaka, Kei-ichi., 2004. Research on the fatty acid synthase activity and the fatty acid synthetic method of the meat-type ducks. Journal of Northeast Normal University 36, 79–84.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm
Inhibition of Coix seed extract on fatty acid synthase, a novel target for anticancer activity Fei Yu a,b , Jing Gao b , Yong Zeng b , Chang-Xiao Liu a,b,∗ a
Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China Tianjin State Key Laboratory of Pharmacokinetics and Pharmacodynamics, Tianjin Institute of Pharmaceutical Research, 308 An-Shan West Road, Tianjin 300193, China b
a r t i c l e
i n f o
Article history: Received 16 March 2008 Accepted 11 July 2008 Available online 23 July 2008 Keywords: Coix seed extract Fatty acid synthase Inhibition Lipometabolism Glycometabolism
a b s t r a c t Ethnopharmacological relevance: Coix seed has been traditionally used to treat cancers in folk medicine. Aim of the study: Study the anticancer action mechanism of Coix seed extract. Materials and methods: After the treatment with Coix seed extract (10 l/ml), the residual activity of fatty acid synthase (FAS) as overall reaction, -ketoacyl reduction, enoyl reduction, and acetyl acetyl coenzyme A (AcAcCoA) reduction was separately detected at 340 nm in the UV-190 spectrophotometer. After rats were administrated Coix seed extract (2.5, 5.0, and 10.0 ml/kg) intragastrically for 10 days consecutively, activities of FAS, malate dehydrogenase (MDH), lipid protein lipase (LPL), hepatic lipase (HL), triglyceride (TG), and glucose-6-phosphate dehydrogenase (G-6-PD) in the plasma, liver and fatty tissues were determined. Results: Experiments in vitro showed that the inhibition of Coix seed extract on FAS activity was significant and dose dependent, and two active sites inhibited were -ketoacyl reductases (KR) and enoyl reductase (ER). Experiments in vivo showed that Coix seed extract inhibited FAS activity in the liver, and elevated LPL and HL activity in the plasma, and effected G-6-PD activity. Conclusions: The study supports that FAS is a novel target for anticancer activity, and provides a theoretical foundation for the wide application of Coix seed extract in traditional medicine. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Ethnopharmacologically, the traditional use of Coix seed has lasted for thousands of years in Traditional Chinese Medicine (TCM) (Li, 1596). The herb is sweet and tasteless in flavour, slightly cold in nature. Coix seed is used to treat cancers in folk medicine (Normile, 2003). Epidemiologists have long suspected that the low cancer rates in Southeast China might be related to Coix lacryma-jobi L. subsp. Ma-yuen (Romanet) T. Koyama (family Poaceae) (Fig. 1), a grass-like relative of maize that is a dietary staple in the region, and a key ingredient of many traditional Chinese herbal medicines (Normile, 2003). Li extracted the anticancer compounds of the Coix seed with supercritical CO2 to produce an anticancer drug called Kanglaite injection (KLT), which showed anticancer activity in animal tumor models (Feng et al., 2004), and was applied clinically in China and Russia (Li, 2001; Qian et al., 2004).
∗ Corresponding author at: Tianjin State Key Laboratory of Pharmacokinetics and Pharmacodynamics, Tianjin Institute of Pharmaceutical Research, 308 An-Shan West Road, Tianjin 300193, China. Tel.: +86 22 23006863; fax: +86 22 23006860. E-mail address: [email protected] (C.-X. Liu). 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2008.07.015
Coix seed extract from the seeds (Fig. 2) is an oily substance, and its main active ingredient is a triglyceride containing four fatty acids (Fig. 3) (Li et al., 1999a). Coix seed extract is formulated into an emulsion for injection, which has been proved to be an effective and safe TCM new preparation by preclinical antitumor studies, and pharmacokinetics and safety studies (Li et al., 2003a, 2005a; Yin and Jin, 2003; Li, 2006). Phases I, II and III clinical trials and tens of thousands of clinical applications proved that the preparation has adjunctive therapy effects to cancer patients (Li et al., 1999b, 2005b; Zhang et al., 1999; Li, 2001). When combined with chemotherapy, radiotherapy and surgery, it could improve the response rate, regulate the energy of advanced patients, and improve life quality so as to prolong survival time (Yang, 1998; Normile, 2003; Garin et al., 2004; Li, 2004, 2006). The USA Food and Drug Administration approved a Phase II clinical trial to test its efficacy in treating nonsmall-cell lung cancer in 2003 (Normile, 2003). It is the first drug derived from a traditional Chinese herbal remedy to go into clinical trials in the United States (Normile, 2003). As the key raw material for the synthesis of long-chain fatty acid in organisms, fatty acid synthase (FAS) extensively exists in fat tissues and the liver of humans and animals (Wakil et al., 1983). Fatty acids, the product of fatty acid synthesis, are the source of
F. Yu et al. / Journal of Ethnopharmacology 119 (2008) 252–258
253
Table 1 Triglyceride content of Coix seed oil
Fig. 1. Coix plant.
Fig. 2. Coix seeds.
Peak no.
Compound
Molecular formula
Molecular mass
Peak area (%)
1 2 3 4 5 6 7
1,2,3-Trilinoleylglycerol 1,2-Dilinoleyl-3-oleylglycerol 1,2-Dilinoleyl-3-palmitoylglycerol 2,3-Dioleoyl-1-linoleylglycerol 2-Linoleyl-3-oleyl-1-palmitoylglycerol 1,2,3-Trioleylglycerol 2,3-Dioleyl-1-palmitoylglycerol
C57 H98 O6 C57 H100 O6 C55 H98 O6 C57 H102 O6 C55 H100 O6 C57 H104 O6 C55 H102 O6
878.7 880.8 854.7 882.8 856.8 884.8 858.8
7.0 18.8 7.6 19.1 14.9 20.9 11.7
substance and energy for the proliferation of tumor cells. Compared with normal tissues, many tumor cells express FAS at high levels and undergo significant endogenous fatty-acid synthesis, and FAS inhibitors were proved to have antitumor activity, which made FAS a novel specific target in search for related anticancer drugs (Kuhajda, 2000; Loftus et al., 2000; Li et al., 2003b; Jenni et al., 2006; Maier et al., 2006). At present, most of the research on the inhibition of FAS has focused on the formation of fat in the body of animals especially on inhibition of the activity of FAS in poultry (Tian, 1994; Tian et al., 1996). Some researchers have studied the inhibitory effect of such plants as Polygonum Multiflorum Thunb. (Zhang et al., 2004a), Ginkgo Biloba L. (Zhang et al., 2004b) and green tea (Brusselmans et al., 2003) during the activity of FAS in human breast cancer cells and further proved beneficial effect through the inhibition of FAS. Although KLT injection has been successfully applied for the treatment of a variety of malignant tumors, such as carcinomas of the lung, liver, stomach, esophagus, colon, pancrease, kidney, ovaries, malignant lymphoma, leukemia for more than 200,000 cases (Li, 2006), it is uncertain that how it works. Studies in vitro about the action mechanism of the cytotoxic effects of Kanglaite injection indicated (Woo et al., 2007): Coix seed extract has a direct effect on PKC kinase activity; it inhibits NFB-mediated regulation of COX-2 expression; it inhibits the expression of MMP9, probably through the inhibition of NFB signaling, it inhibits matrigel invasion, it inhibits the tumor growth of human cancer xenografts and has apparent synergy with agents that inhibit fatty acid synthase. It was predicted that FAS may be a novel target for cancer. To further study whether Coix seed extract has an influence on FAS activity and its action mechanism, experiments in vitro were conducted to find out its capacity and possible action targets. Because the synthesis of fatty acid requires some apolipoprotein and glycometabolic enzymes (Zhou et al., 2004), the determination of their activities in blood and tissues could further demonstrate the regulation of FAS in the process of carcinogenesis. Changes of FAS, malate dehydrogenase (MDH), LPL, hepatic lipase (HL), triglyceride (TG), and G-6-PD were studied in animals after orally feeding Coix seed extract to search for the relationship between tumor cell growth and the activities of metabolic enzymes, and for new theoretical support for the use of Coix seed extract in the clinical treatment of cancer. 2. Materials and methods 2.1. Drug
Fig. 3. Chemical structures of the main active ingredients in Coix seeds.
Coix seed extract (Batch No.: 060309), provided by Zhejiang Kanglaite Pharmaceutical Co. Ltd. The fingerprint of Coix seed extract in which the main active ingredients, triglycerides, were assigned was obtained by high performance liquid chromatography (HPLC) (Fig. 4 and Table 1).
254
F. Yu et al. / Journal of Ethnopharmacology 119 (2008) 252–258
Fig. 4. Fingerprint of Coix seed extract by HPLC. Table 1 shows what every peak is and the relative content of every peak. HPLC conditions: the stationary phase was Agilent Zorbax Extend C18 column (4.6 mm × 250 mm, 5 m). The mobile phase was dichloromethane–acetonitrile (35:65), flow rate was 0.5 ml/min; column temperature was 35 ◦ C.
2.2. Animals Duck: body weight 2–2.5 kg, purchased from Tianjin farm product market. Wistar rat: body weight 170–210 g, provided by the animal house of Tianjin Institute of Pharmaceutical Research. 2.3. Reagents for experiments in vitro Acetyl coenzyme A (AcCoA), malonyl coenzyme A (MalCoA), reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH), acetyl acetyl coenzyme A (AcAcCoA), and dithiothreitol molecular biology reagent (DTT) were all purchased from Sigma Co., USA. Ethyl crotonate provided by Guan Hengye Fine Chemical Co. Ltd., ethyl acetoacetate provided by Tianjin Guangfu Fine Chemical Institute, and other reagents were analytical grade, FAS was separated and purified from duck liver. 2.4. Kits for tests in vivo Kits for the assay of glucose-6-phosphate dehydrogenase (G6-PD), malate dehydrogenase (MDH plasma, tissue), plasma total lipase (lipid protein lipase (LPL) and hepatic lipase (HL)), tissue total lipase (LPL and HL), and protein determination with coomessie brilliant blue were procured from Jiancheng Bioengineering Laboratory, Nanjing; the triglyceride assay kit was procured from Zhongsheng Beikong Biotech Co. Ltd., Beijing. 2.5. Instruments Beckman J2-21 high-speed freezing centrifuge from Beckman, USA; low-pressure column chromatograph from High-tech Application Laboratory, Beijing; UV-190 spectrophotometer from Shimadzu Corporation, Japan. 2.6. Separation and purification of FAS from duck liver Livers were taken out immediately from three ducks after bleeding to death and put in an ice bath for cooling and weigh-
ing. As much as of 1.8 ml/g precooled buffer (0.1 mol/l potassium phosphate buffer, containing 0.07 mol/l KHCO3 , 1 mmol/l DTT, and 1 mmol/l EDTA, pH 7.8–8.0) was added to the duck liver to make a homogenate in the ice bath. The resulting suspension was centrifuged at 8000 rpm for 30 min at 4 ◦ C, the supernatant fluid was filtered through gauze and centrifuged at 18,000 rpm for 60 min. The precipitate was discarded and the supernatant fluid was collected and stored at −75 ◦ C for further purification. The supernatant solution (70 ml) was defrozen at 4 ◦ C, saturated ammonium sulfate ((NH4 )2 SO4 ) was added slowly to reach a saturation level of 25%, and the solution was stood for 2 h at 4 ◦ C. The supernatant fluid collected after centrifugation was brought to a saturation level of 50% with saturated (NH4 )2 SO4 and stood at 4 ◦ C overnight. The precipitate after centrifugation was dissolved in 200 ml balanced buffer (5 mmol/l potassium phosphate buffer, containing 1 mmol/l DTT) and centrifuged again, the supernatant fluid was applied to the balanced DEAE-sepharose FF column (2.6 cm × 50 cm) at a flow rate of 1.4 ml/min, and the column was eluted until A280 < 0.01 with balanced buffer solution. Then the column was eluted with 5–0.3 mol/l potassium phosphate buffer solutions by linear gradient elution. The eluate was collected stepwise and the FAS activity was determined. The elutions with active peaks were combined and added to the same volume of saturated (NH4 )2 SO4 and stood overnight at 4 ◦ C, the precipitate after centrifugation was dissolved in an appropriate amount of buffer solution (0.1 mol/l potassium phosphate buffer, 1 mmol/l DTT and 20% glycerine, pH 7.0)and dialyzed overnight. The dialyzed solution was sub-packed and stored at −75 ◦ C after activity determination (Tian et al., 1985). 2.7. Assays for FAS activity According to the method of Tian (Li et al., 2003b), 0.1 mol/l potassium phosphate buffer (pH 7.0) containing 1 mmol/l EDTA and 1 mmol/l DTT, and enzyme, and water, to make a final volume of 2 ml were added to a colorimetric cylinder (1 cm optical path) at constant temperature (37 ◦ C) water bath for 5 min. The diluted enzyme solution (20 g) was added to initiate the reaction, and the absorbance change was continuously monitored at 340 nm wavelength in a UV-spectrophotometer. The absorbance changed since NADPH was catalyzed and oxidized by FAS into NADP. The substrate concentrations in the FAS overall reaction and the activity determination for each activity center are listed below: (1) Overall reaction: 6 mol/l AcCoA, 12 mol/l MalCoA, 40 mol/l NADPH. (2) -Ketoacyl reduction: 0.8 mmol/l ethyl acetoacetate, 40 mol/l NADPH. (3) Enoyl reduction: 45 mmol/l ethyl crotonate, 40 mol/l NADPH. (4) AcAcCoA reduction: 31 mol/l AcAcCoA, 40 mol/l NADPH. This determination includes reactions at active centers, including transacylation, ketoacyl reduction, dehydration and enoyl reduction, etc. (5) Calculation of FAS activity: the unit of FAS activity is defined as the enzyme quantity consumed to oxidize 14 nmol/l NADPH in a minute. In this way, the unit of enzyme activity in 1 ml can be calculated with the equation: activity (U/ml) = A340 × 106 × C/(ε × V)/14where A340 is the absorbance change per min at 340 nm wavelength; ε is the mmol extinction coefficient of NADPH at 340 nm wavelength which is 6.022 at optical diameter 1 cm; V is the volume of system (2 ml); C is the dilution folds (40); and 106 is the coefficient in conversion from mmol to nmol.
F. Yu et al. / Journal of Ethnopharmacology 119 (2008) 252–258
255
Table 2 Inhibition of Coix seed extract (10 l/ml) on FAS overall reaction and activity centers (X¯ ± S.D., n = 3) Time (h)
Residual activity (%) Overall reaction
0 0.25 0.5 0.75 1 1.5 2
100 82.8 44.3 29.9 20.5 18.9 8.2
± ± ± ± ± ± ±
14.7 5.7 11.1 17.6 7.5 5.7 1.4
Keto-acyl reduction 100 54.0 49.9 36.4 32.4 27.0 21.6
± ± ± ± ± ± ±
Enoyl reduction
8.4 8.4 10.2 8.1 16.2 4.7 8.4
100 84.8 82.5 80.1 53.4 52.3 39.5
± ± ± ± ± ± ±
AcAcCoA reduction
16.5 15.7 19.2 22.8 4.0 12.6 24.7
100 71.3 56.1 42.8 34.8 23.2 8.9
± ± ± ± ± ± ±
17.2 6.2 3.8 17.5 3.8 3.1 3.1
2.8. Assays for inhibitory effects of Coix seed extract on animal FAS in vitro Coix seed extract and enzyme solution were mixed for some time in the swing bed at 25 ◦ C and centrifuged immediately to separate the oil from the water, the activity of the enzyme solution was determined as A, and A0 was the activity when Coix seed extract was replaced by water. A/A0 multiplied by 100% was the residual activity at that moment. The residual activities at a series of time points were determined and a graph with activity to time was plotted. 2.9. Assays for effects of Coix seed extract on activities of some enzymes of lipometabolism and glycometabolism in rats Upon arrival, the rats were kept for 3 days to adapt to the environment. On the 4th day, 42 rats were divided into 4 groups: control group (10 rats), test group I with 2.5 ml/kg Coix seed extract (10 rats), test group II with 5.0 ml/kg Coix seed extract (10 rats) and test group III with 10.0 ml/kg Coix seed extract (12 rats), the male/female ratio was 1:1 in each group. The rats were given Coix seed extract intragastrically once a day for 10 consecutive days. After the last administration, the rats were anesthetized with ether, blood samples were taken from the abdominal aorta of the rats, and were centrifuged to obtain the plasma. The liver and fat were removed and prepared into tissue homogenate. Activities of FAS, MDH, LPL, HL, TG, and G-6-PD in the plasma and tissues were determined and compared with the control group to evaluate the effects of Coix seed extract on the activities of some enzymes of lipometabolism and glycometabolism in rats. The animal tests were conducted in accordance with the principles for laboratory animal use and care as found in the European Community guidelines (EEC Directive of 1986; 86/609/EEC). 2.10. Statistical analysis Student’s t-test was used for statistical comparison of the differences in data between the test groups. Values with p < 0.05 were considered significant. All data are represented as arithmetic means and standard deviation (X¯ ± S.D.).
Fig. 5. Inhibition of Coix seed extract (10 l/ml) on FAS overall reaction and activity centers.
inhibition of Coix seed extract on FAS overall reaction activity with different dosages of Coix seed extract (0, 1, 2.5, 5, 10 l/ml) at 25 ◦ C for 2 h (Table 3 and Fig. 6). The results demonstrated that the inhibition in the range 1–10 l/ml concentration was dose dependent. 3.1.2. Inhibition of Coix seed extract on ˇ-ketoacyl reduction Over 50% of the FAS activity was inhibited after 0.5 h of reaction with Coix seed extract (10 l/ml) at 25 ◦ C, and after 2 h of reaction the residual activity of -ketoacyl reduction was only 21.6% (Table 2 and Fig. 5). Inhibition results of Coix seed extract in the range of 1–10 l/ml at 25 ◦ C for 2 h (Table 3 and Fig. 6) demonstrated that the inhibition to -ketoacyl reduction reaction was dose dependent. 3.1.3. Inhibition of Coix seed extract on enoyl reduction The inhibition of Coix seed extract (10 l/ml) on enoyl reduction was comparatively weak, and the residual activity after 2 h of reaction at 25 ◦ C was 39.5% (Table 2 and Fig. 5). 3.1.4. Inhibition of Coix seed extract on AcAcCoA reduction The inhibition of Coix seed extract (10 l/ml) on AcAcCoA reduction catalyzed by FAS was comparatively strong, over 50% of the
3. Results 3.1. Part I: experiments in vitro 3.1.1. Inhibition of Coix seed extract on overall reaction Over 50% of the FAS activity was inhibited after 0.5 h of reaction with Coix seed extract (10 l/ml) at 25 ◦ C, and after 2 h of reaction the residual activity of overall reaction was only 8.2% (Table 2 and Fig. 5). To further evaluate the inhibition of Coix seed extract on the FAS overall reaction activity, experiments were continued on the
Table 3 Inhibition of Coix seed extract (1–10 l/ml) on FAS overall reaction and some active centers (X¯ ± S.D., n = 3) Dosage (l/ml)
Residual activity (%) Overall
0 1 2.5 5 10
100 53.6 29.5 20.6 8.2
± ± ± ± ±
Keto-acyl 13.2 25.9 14.9 1.5 1.4
100 83.0 51.3 39.1 20.2
± ± ± ± ±
31.2 23.8 30.0 12.9 12.1
Enoyl 100 76.3 73.1 61.3 60.2
± ± ± ± ±
AcAcCoA 8.5 32.3 15.9 12.9 11.3
100 71.3 67.7 48.1 10.7
± ± ± ± ±
12.3 16.3 35.6 24.5 10.7
256
F. Yu et al. / Journal of Ethnopharmacology 119 (2008) 252–258 Table 6 Effects of Coix seed extract on LPL activity in rats (X¯ ± S.D., n = 10) Dosage (ml/kg)
LPL activity (U/ml) Plasma
Control group 2.5 5.0 10.0
3.318 3.505 2.832 5.620
± ± ± ±
Liver 0.872 0.985 1.057 3.051*
0.537 0.418 0.510 0.525
Fat ± ± ± ±
0.183 0.063 0.183 0.205
3.713 4.574 5.714 5.645
± ± ± ±
1.815 1.910 2.604 2.414
± ± ± ±
2.005 4.851 0.935 2.901
Significant difference relative to control group, * p < 0.05.
Table 7 Effects of Coix seed extract on HL activity in rats (X¯ ± S.D., n = 10) Dosage (ml/kg)
HL activity (U/ml) Plasma
Fig. 6. Inhibition of Coix seed extract (1–10 l/ml) on FAS overall reaction and activity centers.
enzyme activity was inhibited at 25 ◦ C for 0.75 h, and after 2 h of reaction the residual activity was only 8.9% (Table 2 and Fig. 5). Inhibition results of Coix seed extract in the range of 1–10 l/ml at 25 ◦ C for 2 h (Table 3 and Fig. 6) demonstrated that the inhibition of AcAcCoA reduction was also dose dependent. 3.2. Part II: experiments in vivo Rats were fed with Coix seed extract (2.5, 5.0, and 10.0 ml/kg) intragastrically for 10 days consecutively. Compared with the control group, the dosage of 10 ml/kg had a significant effect on FAS activity in the liver (p < 0.05). No notable effects on FAS activity in fatty tissues were observed (Table 4). There were no significant effects on MDH activity in the plasma, liver and fatty tissues (Table 5). These dosages all significantly elevated HL activity in plasma (p < 0.05). The dosage of 10.0 ml/kg significantly elevated HL activity in the liver and LPL activity in the plasma (p < 0.05) (Tables 6 and 7). No significant effects on TG content in the plasma were observed (Table 8). The blood G-6-PD (methemoglobin reduction percentage) of rats in high, medium, and low-dosage groups were reduced to different extents (p < 0.01) (Table 8).
Table 4 Effects of Coix seed extract on FAS activity in rat tissues (X¯ ± S.D., n = 10) Dosage (ml/kg)
FAS activity (U/g tissue) Liver
Control group 2.5 5.0 10.0
13047.40 9752.60 11007.26 10082.08
Fat ± ± ± ±
3268.21 4681.41 2490.75 2621.14*
3510.94 3004.86 2894.15 2846.70
± ± ± ±
2693.94 1717.95 1729.63 1355.86
Significant difference relative to control group, * p < 0.05.
MDH activity (U/ml) Plasma
Control group 2.5 5.0 10.0
0.570 0.419 0.394 0.452
± ± ± ±
Liver 0.249 0.177 0.141 0.112
12.209 11.924 13.372 12.949
Fat ± ± ± ±
2.950 2.269 1.961 2.227
8.040 10.310 10.833 9.786
3.980 5.555 5.376 6.286
± ± ± ±
1.329 1.581* 1.188* 2.787*
0.548 0.749 0.677 0.812
Fat ± ± ± ±
0.106 0.325 0.266 0.294*
4.405 6.946 6.233 6.469
Significant difference relative to control group, * p < 0.05.
4. Discussion and conclusions The products from FAS are the sources of substance and energy for the proliferation of tumor cells. The search for drugs that can arrest the activity of FAS to cut off or inhibit the source of substance and energy for the growth of tumors is the new target for anticancer drug discovery (Li and Hou, 2003). Traditionally considered as an anabolic-energy-storage pathway, fatty-acid synthesis is now regarded as the key process for tumor cell growth and survival. The inhibition of FAS activity should inhibit tumor cell growth, and finally realize the objective of cancer therapy. Because of its role in fatty acid synthesis, FAS is a target for drug development against obesity and obesity-related diseases, including diabetes and cardiovascular disorders (Maier et al., 2006). FAS is overexpressed in many forms of cancer (Kuhajda et al., 1994), and FAS inhibitors have demonstrated antitumor activity (Kuhajda et al., 2000). The present experiment in vitro showed that Coix seed extract could significantly inhibit FAS activity, and experiments in rats also showed that Coix seed extract significantly inhibited FAS activity in liver. Therefore, the antitumor effects of Coix seed extract are probably related to the inhibition of FAS activity. The study supports that FAS is a novel target for cancer therapy. The other mechanism of action of Coix seed extract, as a Kanglaite injection (KLT), is as follows: (1) the drug inhibits the mitosis of tumor cells during the G2/M phases; (2) induces apoptosis of tumor cells; (3) affects the genetic expression of tumor cells by up-regulating FAS/Apo-1 gene expression and down-regulating Bc1-2 gene expression; (4) inhibits tumor angiogenesis; (5) counteracts the cachexia of cancers; and (6) reverses the multi-resistance of tumor cells to anticancer drugs and the resistance modification in some chemotherapeutic agents (Li, 2006). All of the above mechanism of action of Coix seed Table 8 Effects of Coix seed extract on TG content and G-6-PD activity (X¯ ± S.D., n = 10)
Table 5 Effects of Coix seed extract on MDH activity in rats (X¯ ± S.D., n = 10) Dosage (ml/kg)
Control group 2.5 5.0 10.0
Liver
± ± ± ±
2.380 4.127 5.491 2.321
Dosage (ml/kg)
TG content in plasma (mmol/l)
Control group 2.5 5.0 10.0
1.119 1.027 1.061 0.942
± ± ± ±
0.538 0.315 0.330 0.353
Significant difference relative to control group, ** p < 0.01.
Reduction percentage of methemoglobin (%) 76.542 64.746 60.475 69.266
± ± ± ±
7.477 6.089** 3.308** 2.254**
F. Yu et al. / Journal of Ethnopharmacology 119 (2008) 252–258
257
FAS activity was significant and dose dependent. Coix seed extract inhibited two active sites: KR and ER (Fig. 8). Continued studies are required to explain whether Coix seed extract inhibits other active sites. Experiments in vivo showed that Coix seed extract inhibits FAS activity in the liver, elevates LPL and HL activity in the plasma, and effects G-6-PD activity, thereby inhibiting fatty acid synthesis and reducing the components and forms of energy for tumor cell growth. The present study supports FAS as a novel target for cancer therapy, and provides a theoretical foundation for the clinical application of Coix seed extract in cancer therapy. Acknowledgements Fig. 7. FAS—a new target for the active component of Coix seed.
extract reflect the multiple targets of action of Coix seed extract (Fig. 7). Maier obtained the overall architecture of mammalian FAS by X-ray crystallography at intermediate resolution, providing a new structural basis for further experiments required for a detailed understanding of the complex action mechanism of mammalian FAS (Maier et al., 2006). Animal FAS is a compound enzyme with two subunits (Smith et al., 2003). The molecular weight of each subunit is about 2.7 million and it has 6 different active sites and 1 acyl carrier protein (ACP) in each subunit covalently linked to a phosphopantetheinyl prosthetic group (Smith, 1994). The six active sites are malonyl/acetyl transacylase (MAT), -acyl-synthetase (KS), ketoacyl reductases (KR), -hydroxylacyl dehydrase (DH), enoyl reductase (ER), and long-chain fatty acid thioesterase (TE) (Fig. 8) (Maier et al., 2006). If any active site is inhibited, the whole FAS activity will be inhibited. This is the first time to describe a new action mechanism of Coix seed extract through the inhibition of FAS. Further experiments in vitro showed that the inhibition of Coix seed extract on
Fig. 8. Catalytic cycle and domain organization of FAS.
The authors thanks Zhejing Kanglaite Pharmaceutical Co. Ltd. for financial support, and Professor Wei-Xi Tian (Department of Biology, Graduate School of the Chinese Academy of Sciences, Beijing, China) for useful suggestions and discussions. References Brusselmans, K., De Schrijver, E., Heyns, W., Verhoeven, G., Swinnen, J.V., 2003. Epigallocatechin-3-gallate is a potent natural inhibitor of fatty acid synthase in intact cells and selectively induces apoptosis in prostate cancer cells. International Journal of Cancer 106, 856–862. Feng, G., Kong, Q.Z., Huang, D.S., Huang, T., Lu, H.D., Fei, Y., Feng, J.P., 2004. Experimental study on inhibition angiogenesis in mice S1 80 sarcoma by Coix lacryma-jobi injection. China Journal of Cancer Prevention and Treatment 31, 229–230. Garin, A.M., Tjulandin, S.A., Kadagidze, Z.G., Bulanov, A.A., Chitya, L.V., 2004. Clinical trial of kanglaite injection in patients with non-small cell lung cancer in Russia. In: Li, D.P. (Ed.), Progress of Clinical Studies of Kanglaite Injection in Foreign Countries (USA, RUS), pp. 67–70. Jenni, M., Leibungut, Maier, T., Ban, N., 2006. Architecture of mammalian fatty acid synthase at 5 Å resolution. Science 311, 1263–1567. Kuhajda, F.P., 2000. Fatty acid synthase and human cancer: new perspectives on its role in tumor biology. Nutrition 16, 202–208. Kuhajda, F.P., Jenner, K., Wood, F.D., Hennigar, R.A., Jacobs, L.B., Dick, J.D., Pasternack, G.R., 1994. Fatty acid synthesis: a potential selective target for antineoplastic therapy. Proceedings of the National Academy of Sciences of the United States of America 91, 6379–6383. Kuhajda, F.P., Pizer, E.S., Li, J.N., Mani, N.S., Frehywot, G.L., Townsend, C.A., 2000. Synthesis and antitumor activity of an inhibitor of fatty acid synthase. Proceedings of the National Academy of Sciences of the United States of America 97, 3450–3454. Li, S.Z., 1596. Compendium of materia medica. Part of Cereal 24, 1489. Li, D.P., 2001. Clinical applications of Kanglaite injection (KLT). Chinese Journal of Clinical Oncology 28, 300–302. Li, D.P., 2004. Progress of Clinical Studies of Kanglaite Injection in Foreign Countries (USA, RUS). Zhejiang Kanglaite Pharmaceutical Co. Ltd., Hangzhou, pp. 1–28. Li, D.P., 2006. Research advance on ethnopharmacology, pharmacodynamics, pharmacokinetics and clinical therapeutics of Coix seed and its preparation, Kanglaite injection. Asian Journal of Pharmacodynamics and Pharmacokinetics 6, 83–102. Li, X.H., Hou, Y.S., 2003. Fatty acid synthase: specific target for cancer therapy. Journal of Biochemistry and Biophysics 35, 1–5. Li, D.P., Huang, J.M., Su, W.A., Shen, K.K., Jiang, L.L., Shi, J.Y., 1999a. The analysis of chemical composition in the Coix lipid. Traditional Chinese Drug Research & Clinical Pharmacology 10, 35–37. Li, T.D., Liu, A.G., Qin, S.L., Xu, P.M., Zhang, W.F., Chen, L., Cheng, G.Y., Zhuang, J.S., Zhang, Y.H., Liu, B.K., 1999b. Clinical study of Kanglaite injection in cancer pain control and life quality improvement in patients with advanced cancer. Chinese Journal of Clinical Oncology 26, 372–376. Li, Z.H., Lü, J.F., Zhou, L.Z., Li, B., 2005b. Clinical study of combined Kanglaite injection with chemotherapy in treatment of NSCLC. China Journal of Cancer Prevention and Treatment 12, 1339–1340. Li, L., Shen, M.Y., Ma, L., 2003a. Observation on security of Kanglaite injection in treating senile patients with advanced tumor. China Journal of Cancer Prevention and Treatment 10, 539–540. Li, D.P., Shen, K.K., Ma, Y.M., 2005a. The pharmacokinetic and bioavailability study of Coix seed extract preparation. China Journal of Traditional Chinese Medicine and Pharmacy 20, 81–85. Li, L.C., Wu, X.D., Tian, W.X., 2003b. lnhibition to fatty acid synthase with extract of Tuber Fleeceflower root. Chinese Journal of Biochemistry and Molecular Biology 19, 297–304. Loftus, T.M., Jaworsky, D.E., Frehywot, G.L., Townsend, C.A., Ronnett, G.V., Lane, M.D., Kuhajda, F.P., 2000. Reduced food intake and body weight in mice treated with fatty acid synthase inhibitors. Science 288, 2379–2381.
258
F. Yu et al. / Journal of Ethnopharmacology 119 (2008) 252–258
Maier, T., Jenni, S., Ban, N., 2006. Architecture of mammalian fatty acid synthase at 4.5 Å resolution. Science 311, 1258–1562. Normile, D., 2003. The new face of traditional Chinese medicine. Science 299, 188–190. Qian, J., Qin, S.L., Yang, L.Q., Chen, Y.X., Shao, Z.J., 2004. The clinical study of Gefitnib combination with Kanglaite injection in treatment of non-small-cell lung cancer. Chinese Clinical Oncology 9, 568–570. Smith, S., 1994. The animal fatty acid synthase: one gene, one polypeptide, seven enzymes. The FASEB Journal 8, 1248–1259. Smith, S., Witkowski, A., Joshi, A.K., 2003. Structural and functional organization of the animal fatty acid synthase. Progress in Lipid Research 42, 289–317. Tian, W.X., 1994. The body fat level and the regulation of fatty acid synthase activity in animals. Chemistry of Life 14, 1. Tian, W.X., Dong, Y., Quan, H., Chen, W.F., 1996. The dependence of fat level of hen on activity of fatty acid synthase in liver on different ages. Chinese Biochemistry Journal 12, 234–236. Tian, W.X., Hsu, R.Y., Wang, Y.S., 1985. Studies on the reactivity of the essential sulfhydryl groups as a conformational probe for the fatty acid synthase of chicken liver. Journal of Biological Chemistry 260, 11375–11387. Wakil, S.J., Stoops, J.K., Joshi, V.C., 1983. Fatty acid synthesis and its regulation. Annual Review of Biochemistry 52, 537–579.
Woo, J.H., Li, D.P., Orita, H., Coulter, J., Tully, E., Kwon, T.K., Xu, S., Gabrielson, E., 2007. Coix seed extract, a commonly used treatment for cancer in China, inhibits NFkappaB and protein kinase C signaling. Cancer Biology & Therapy 6 (Epub ahead of print). Yang, H., 1998. Collection of the Studies of Kanglaite Injection against Tumors. Zhejiang University Press, pp. 107–118. Yin, L., Jin, X.F., 2003. Observation of the clinical therapeutic effect of Kanglaite (KLT) combined with chemotherapy on advanced cancer patients. China Pharmacy 14, 166–167. Zhang, H.Q., Li, S.Y., Fu, Y., Yang, S., Fu, T.H., Qin, M., Lü, F.D., Yan, D.J., 1999. Clinical and pathological changes of Kanglaite injection in treatment of primary lung cancer. Chinese Journal of Clinical Oncology 26, 78–79. Zhang, Y.Y., Zhang, F.Z., Sun, L.Y., Wang, X.Z., 2004a. Inhibition to fatty acid synthase from human breast cancer cell with extract of Tuber Fleeceflower root in vitro. Journal of Qiqihar Medical College 25, 1102–1104. Zhang, Y.Y., Zhang, F.Z., Sun, L.Y., Wang, X.Z., 2004b. Inhibition to fatty acid synthase from human breast cancer cell with extract of Ginkgo leaf in vitro. Journal of Taishan Medical College 25, 433–435. Zhou, C.H., Tanaka, Kei-ichi., 2004. Research on the fatty acid synthase activity and the fatty acid synthetic method of the meat-type ducks. Journal of Northeast Normal University 36, 79–84.