Inhibitory effects of onion (Allium cepa L.) extract on proliferation of cancer cells and adipocytes via inhibiting fatty acid synthase.

Onions (Allium cepa L.) are widely used in the food industry for its nutritional and aromatic properties. Our studies showed that ethyl acetate extract of onion (EEO) had potent inhibitory effects on animal fatty acid synthase (FAS), and could induce apoptosis in FAS over-expressing human breast cancer MDA-MB-231 cells. Furthermore, this apoptosis was accompanied by reduction of intracellular FAS activity and could be rescued by 25 mM or 50 mM exogenous palmitic acids, the final product of FAS catalyzed synthesis. These results suggest that the apoptosis induced by EEO occurs via inhibition of FAS. We also found that EEO could suppress lipid accumulation during the differentiation of 3T3-L1 adipocytes, which was also related to its inhibition of intracellular FAS activity. Since obesity is closely related to breast cancer and obese patients are at elevated risk of developing various cancers, these findings suggested that onion might be useful for preventing obesity-related malignancy.

Fatty acid synthase inhibitors from the hulls of Nephelium lappaceum L.

Natural products inhibiting fatty acid synthase (FAS) are appearing as potential therapeutic agents to treat cancer and obesity. The bioassay-guided chemical investigation of the hulls of Nephelium lappaceum L. resulted in the isolation of ten compounds (1-10) mainly including flavonoids and oleane-type triterpene oligoglycosides, in which all of the compounds were isolated from this plant for the first time. Additionally, compounds 8 and 9 were new hederagenin derivatives and were elucidated as hederagenin 3-O-(2,3-di-O-acetyl-α-l-arabinofuranosyl)-(1→3)-[α-l-rhamnopyranosyl(1→2)]-ß-l-arabinopyranoside and hederagenin 3-O-(3-O-acetyl-α-l-arabinofuranosyl)-(1→3)-[α-l-rhamnopyranosyl-(1→2)]-ß-l-arabinopyranoside, respectively. All these isolates were evaluated for inhibitory activities of FAS, which showed these isolates had inhibitory activity against FAS with IC(50) values ranging from 6.69 to 204.40 µM, comparable to the known FAS inhibitor EGCG (IC(50)=51.97 µM). The study indicates that the hulls of Nephelium lappaceum L. could be considered as potential sources of promising FAS inhibitors and the oleane-type triterpene oligoglycosides could be considered as another type of natural FAS inhibitors.

Suppression of fatty acid synthase, differentiation and lipid accumulation in adipocytes by curcumin.

Curcumin is a well-known component of the cook seasoning and traditional herb turmeric (Curcuma longa), which has been reported to prevent obesity. However, the mechanism still remains to be determined. In this study, curcumin is found to be an effective inhibitor of fatty acid synthase (FAS), and its effects on adipocytes are further evaluated. Curcumin shows both fast-binding and slow-binding inhibitions to FAS. Curcumin inhibits FAS with an IC50 value of 26.8 µM, noncompetitively with respect to NADPH, and partially competitively against both substrates acetyl-CoA and malonyl-CoA. This suggests that the malonyl/acetyl transferase domain of FAS possibly is the main target of curcumin. The time-dependent inactivation shows that curcumin inactivates FAS with two-step irreversible inhibition, a specific reversible binding followed by an irreversible modification by curcumin. Like other classic FAS inhibitors, curcumin prevents the differentiation of 3T3-L1 cells, and thus represses lipid accumulation. In the meantime, curcumin decreases the expression of FAS, down-regulates the mRNA level of PPARγ and CD36 during adipocyte differentiation. Curcumin is reported here as a novel FAS inhibitor, and it suppresses adipocyte differentiation and lipid accumulation, which is associated with its inhibition of FAS. Hence, curcumin is considered to be having potential application in the prevention of obesity.

Fatty acid synthase inhibitors of phenolic constituents isolated from Garcinia mangostana.

Natural inhibitors of fatty acid synthase (FAS) are emerging as potential therapeutic agents to treat cancer and obesity. The bioassay-guided chemical investigation of the hulls of Garcinia mangostana led to the isolation of 13 phenolic compounds (1-13) mainly including xanthone and benzophenone, in which compounds 7, 8, 9, 10, and 11 were isolated from this plant for the first time and compound 9 was a new natural product. These isolates possess strong inhibitory activity of FAS with the IC(50) values ranging from 1.24 to 91.07 µM. The study indicates that two types of natural products, xanthones and benzophenones, could be considered as promising FAS inhibitors.

Substrate-induced changes in protease active site conformation impact on subsequent reactions with substrates.

Enzymatic catalysis of biochemical reactions is essential to all living systems. The "lock and key" and "induced fit" models were early contributions to our understanding of the mechanisms involved in the reaction between an enzyme and its substrate. However, whether a given substrate-induced conformation is rigid or remains flexible has not yet been determined. By measuring the enzyme activity and intrinsic fluorescence of a nonspecific Eisenia fetida protease-I with different chromogenic substrates, we show that in subsequent reactions of protease with substrates, both the "lock and key" and "induced fit" mechanisms are used depending on the degree of conformational change required. Chromozym-Th- or chromosym-Ch-induced protease conformations were unable to bind chromozym-U. The chromosym-U-induced protease conformation remained flexible and could be further induced by chromozym-Th and chromozym-Ch. When low concentrations of guanidine HCl were used to disturb the conformation of the enzyme, only small changes in intrinsic fluorescence of the chromozym-Th-induced protease were detected, in contrast to the native enzyme whose intrinsic fluorescence markedly increased. This indicates that the substrate-induced enzyme was relatively rigid compared with the native protease. Utilizing a lock and key mechanism for secondary substrate reactions may have adaptive value in that it facilitates high efficiency in enzymatic reactions.

Inhibitory effects of thioethers on fatty acid synthase and 3T3-L1 cells.

Thioethers are the main flavor compounds found in Liliaceae Allium vegetables and have been shown to have beneficial effects against several diseases correlated with metabolic syndrome. The inhibitory effects of six thioethers on fatty acid synthase (FAS) were investigated. Dose-dependent and time-dependent inhibitions of FAS by one trisulfide and two disulfides were revealed. Diallyl trisulfide (DATS, IC(50) = 8.37 microM) was the most active of these thioethers. Inhibition kinetics, substrate protection analysis, and stoichiometric assay revealed that DATS interacted with both essential sulfhydryl groups on the acyl-carrier protein and beta-ketoacyl synthase domain of FAS to inactivate the enzyme. The inactivation by DATS represented affinity-labeling kinetics. The active thioethers also inhibited the differentiation and lipid accumulation of 3T3-L1 preadipocytes, and the effect was related to their inhibition of FAS. It is suggested that the inhibition on FAS by thioethers and Allium vegetables is an important factor for their effects against metabolic syndrome.

Inhibitory effects on bacterial growth and beta-ketoacyl-ACP reductase by different species of maple leaf extracts and tannic acid.

It is important to develop new antibiotics aimed at novel targets. The investigation found that the leaf extracts from five maples (Acer platanoides, Acer campestre, Acer rubrum, Acer saccharum and Acer truncatum Bunge collected in Denmark, Canada and China) and their component tannic acid displayed antibacterial ability against 24 standard bacteria strains with the minimum inhibitory concentration of 0.3-8.0 mg/mL. Unlike the standard antibiotic levofloxacin (LFX), these samples inhibited Gram-positive bacteria more effectively than they inhibited Gram-negative bacteria. These samples effectively inhibited two antidrug bacterial strains. The results show that these samples inhibit bacteria by a different mechanism from LFX. These samples potently inhibited b-ketoacyl-ACP reductase (FabG), which is an important enzyme in bacterial fatty acid synthesis. Tannic acid showed the strongest inhibition on FabG with a half inhibition concentration of 0.78 microM (0.81 microg/mL). Furthermore, tannic acid and two maple leaf extracts showed time-dependent irreversible inhibition of FabG. These three samples also exhibited better inhibition on bacteria. It is suggested that FabG is the antibacteria target of maple leaf extracts and tannic acid, and both reversible and irreversible inhibitions of FabG are important for the antibacterial effect.

Novel and potent inhibitors of fatty acid synthase derived from catechins and their inhibition on MCF-7 cells.

Fatty acid synthase (FAS) is a potential target for cancer, but potent inhibitors against FAS are scarce. In this study, we found that activities of catechins on inhibiting FAS increased greatly by heating them in acid. The enhancement was positively correlated to H(+) concentration. The inhibitory activities of the final products from different catechins were similar, all of which were less than 1 microg/mL. The product from (-)-epigallocatechin gallate (EGCG) was stable at room temperature, and its inhibitory kinetics and reacting sites on FAS were obviously different from the known FAS inhibitors. It also affected the viability of MCF-7 cells more obviously than EGCG. A putative route of the reaction progress was proposed and the effective inhibitors were deduced to be oligomers of 2-hydroxy-3-(3', 4', 5'-trihydroxyphenyl) propenoic acid by analysis of their spectra. The work affords new and potent FAS inhibitors that would be promising candidates for the treatment of cancer.

Parasitic loranthus from Loranthaceae rather than Viscaceae potently inhibits fatty acid synthase and reduces body weight in mice.

AIM OF THE STUDY: Our previous results have shown one species of parasitic loranthus (Taxillus chinensis (DC.) Dancer) exhibits potent inhibition on fatty acid synthase (FAS) that is proposed to be a potential therapeutic target for treatment of obesity. However, the medicinal parasitic loranthus come from tens of plants of two families, the Loranthaceae and the Viscaceae. This study was carried out to figure out whether these parasitic loranthus from the two families have similar inhibitory ability on FAS, and whether the parasitic loranthus with potent inhibitory ability on FAS significantly reduce body weight of animal. MATERIALS AND METHODS: CD-1 mice were used to test the effects of samples on their body weight and food intake in 20 days. The reversible and irreversible inhibition on FAS was assayed to study the inhibitory ability of sixteen different medicinal plants from these two families, which were collected in nature. RESULTS: The reversible inhibitory ability of the extracts from the Loranthaceae was nearly 400-fold stronger than that from the Viscaceae. The species from the genus Taxillus Tieghem showed the best effect on FAS in both reversible and irreversible inhibition. Moreover, the difference in host plants did not affect markedly on the inhibitory ability of parasitic loranthus. The medicinal herb with high inhibitory ability on FAS significantly reduced the body weight and food intake of mice by oral administration. CONCLUSIONS: The medicinal herbs from the family Loranthaceae, rather than those from the family Viscaceae, should be suitable to apply as botanical sources of parasitic loranthus for weight control. The herbs from genus Taxillus Tieghem are the best.

Antimicrobial effect by extracts of rhizome of Alpinia officinarum Hance may relate to its inhibition of beta-ketoacyl-ACP reductase.

Inhibitory effects on bacterial growth showed that 40% ethanol extract of galangal (rhizome of Alpinia officinarum Hance) can inhibit Staphylococcus aureus, alpha-Hemolytic streptococcus, beta-Hemolytic streptococcus and Streptococcus pneumoniae. beta-ketoacyl-ACP reductase (FabG, EC.1.1.1.100) is a key enzyme in type II fatty acid synthase system in bacteria and catalyzes beta-ketoacyl-ACP reduction. The galangal extracts inhibited FabG with an IC(50) value of only 4.47 +/- 0.10 microg/mL and is more potent than other previously published inhibitors. Kinetics studies showed that the inhibition consisted of both reversible and irreversible inhibition. The extracts of galangal inhibit FabG in a competitive pattern against NADPH. So far, no inhibitor has been reported to exhibit irreversible inhibition of FabG, whereas the galangal ethanol extract can inhibit FabG irreversibly. The irreversible inhibition presented two phases. It is probable that the galangal extract inhibit FabG, thereby displaying antibacterial ability.

Low concentration of condensed tannins from catechu significantly inhibits fatty acid synthase and growth of MCF-7 cells.

Tannins exist widely in plants, but because they precipitate proteins, scientists frequently ignore them in search of bioactive components. Catechu, a traditional astringent, is rich in tannins. In this study, we found that condensed tannins from catechu potently inhibited animal fatty acid synthase (FAS). Among them, trimeric condensed tannin showed the most potent inhibition with IC(50) of 0.47 microg/ml and it also exhibited strong time-dependent inhibition. Its inhibitory kinetics and reacting sites on FAS were obviously different from the known inhibitors of FAS. Furthermore, condensed tannins were found to suppress the growth of MCF-7 breast cancer cells, and the effect was related to their activity of FAS inhibition. The inhibition of both FAS activity and MCF-7 growth was exhibited by low concentrations of condensed tannins without FAS being precipitated. These results suggest tannins would be a valuable resource of bioactive substances.

The antibacterial efficacy of an aceraceous plant [Shantung maple (Acer truncatum Bunge)] may be related to inhibition of bacterial beta-oxoacyl-acyl carrier protein reductase (FabG).

Polyphenols, including flavonoids, are the major components of the extracts from aceraceous plants. They possess remarkable antibacterial and antitumour activity. Our study focused on whether the inhibition of the bacterial type II fatty acid synthesis system is the mechanism for the antibacterial effect of the related plant polyphenols. Extracts obtained from the fallen leaves of the Shantung maple (Acer truncatum Bunge) using different solvents, and the related pure compound PGG (1,2,3,4,6-penta-O-galloyl-beta-D-glucose), potently inhibited the FabG (beta-oxoacyl-ACP reductase) steps in the fatty-acid-elongation cycle with the IC(50) values between 0.9 and 7.2 microg/ml. An ethyl acetate extract appeared to inhibit FabG reductase in a mixed manner with NADPH, as did PGG with NADPH, demonstrating that they interfered with the binding of the cofactor to the enzyme. Gram-positive and Gram-negative bacteria and some fungi were used to evaluate the antibacterial abilities of different extract samples. The experiments showed that a higher polyphenol content of the extracts led to a more potent inhibitory capacity against FabG, thus enhancing the antibacterial efficacy.

A substitutive substrate for measurements of beta-ketoacyl reductases in two fatty acid synthase systems.

Bacterial beta-ketoacyl-ACP reductase (FabG) and the beta-ketoacyl reductase domain in mammalian fatty acid synthase (FAS) have the same function and both are rendered as the novel targets for drugs. Herein we developed a convenient method, using an available compound ethyl acetoacetate (EAA) as the substitutive substrate, to measure their activities by monitoring decrease of NADPH absorbance at 340 nm. In addition to the result, ethyl 3-hydroxybutyrate (EHB) was detected by HPLC analysis in the reaction system, indicating that EAA worked effectively as the substrate of FabG and FAS since its beta-keto group was reduced. Then, the detailed kinetic characteristics, such as optimal ionic strength, pH value and temperature, and kinetic parameters, for FabG and FAS with this substitutive substrate were determined. The Km and kcat values of FabG obtained for EAA were 127 mM and 0.30 s(-1), while those of this enzyme for NADPH were 10.0 microM and 0.59 s(-1), respectively. The corresponding Km and kcat values of FAS were 126 mM and 4.63 s(-1) for EAA; 8.7 microM and 4.09 s(-1) for NADPH. Additionally, the inhibitory kinetics of FabG and FAS, by a known inhibitor EGCG, was also studied.

Evaluation of the inhibitory activities of aceraceous plants on fatty acid synthase.

Fatty acid synthase (FAS) is a very significant lipogenic enzyme participating in energy metabolism in vivo and has been reported as a potential new therapeutic target for cancer treatment. The extracts from sixteen Aceraceae were prepared to assay their inhibitory activities against duck liver FAS and their correlated antitumor bioactivity. Their inhibition of FAS was composed of a reversible fast-binding inhibition, by which 0.41 microg/mL of the A. campestre extract inhibits 50% FAS activity, and an irreversible slow-binding inhibition with inactivation rate constants, k(obs), ranging between 1.5 x 10(-3) and 10.6 x 10(-3) min(-1). Three Aceraceae extracts were selected from their smaller IC50 values to study different type of inhibitions against the three substrates in the FAS overall reaction. As compared with other reported FAS inhibitors including EGCG with regard to inhibition constant and IC50 value, the extracts appeared to be more efficient inhibitors, and exhibited a considerable inhibition against the growth of five types of cancer cells (China patent application number 200610088901.6), which may be related to the inhibition of lipogenesis in these cells.

Inactivation mechanism of the beta-ketoacyl-[acyl carrier protein] reductase of bacterial type-II fatty acid synthase by epigallocatechin gallate.

Epigallocatechin gallate (EGCG), a major compound from green tea, reversibly inhibits beta-ketoacyl-[acyl carrier protein] reductase (FabG) from Escherichia coli. In this study, we found that EGCG exhibited an atypical time-dependent inhibition of FabG, which possibly resulted from the EGCG-induced aggregation of FabG. It was observed that FabG inactivation and aggregation occurred nearly simultaneously, with a lag time that decreased with increasing EGCG concentration. These results suggest that some chemical reactions, required for aggregation and inactivation, occurred during the lag time. Since EGC was detected by HPLC after the incubation of EGCG with FabG, EGCG probably covalently modified FabG. These further results showed that 1 tetramer of FabG must be modified by several, possibly 4, EGCG molecules before the formation of FabG aggregates. FabG aggregation was a first-order reaction independent of protein concentration. Due to an initial lag time, the first-order rate of aggregation gradually increased, reaching a maximal and constant value. The effect of increasing concentration of EGCG on the first-order rate constant for aggregation indicated that EGCG bound to FabG by affinity labeling. Based on the results, we propose a mechanism for the interaction of EGCG with FabG:EGCG first binds reversibly to each subunit of FabG, followed by covalent modification and then aggregation of the 4 EGCG-modified subunits.

The extract of leaves of Acer truncatum Bunge: A natural inhibitor of fatty acid synthase with antitumor activity.

Fatty acid synthase (FAS) has been identified as a potential antitumor target. The extract from the leaves of Acer truncatum Bunge (Extr) was prepared to assay its inhibitory activity against FAS, which was isolated from duck liver, and the correlated antitumor bioactivity. Its inhibition of FAS is composed of reversible fast-binding inhibition, IC50 = 0.7 microg/ml, and irreversible slow-binding inhibition following saturation kinetics with a dissociation constant of 0.68 microg/ml and a limiting rate constant of 0.0288 min(-1). The Extr exhibited different type of inhibitions against the three substrates in the FAS overall reaction. Compared with EGCG in inhibition constant and IC50 value, the Extr appeared to be a more efficient inhibitor, and exhibited a considerable inhibition against the growth of four kinds of cancer cells (patent application number 200510068054.2). It was infered that the inhibitory activity is likely attributable to the co-operative effect of the components.

Inhibition of fatty acid synthase by polyphenols.

Recently, animal fatty acid synthase (FAS) is reported as a potential therapeutic target for obesity and cancer. Considerable interest has been developed in identifying novel inhibitors of the enzyme. It is found that tea polyphenols inhibit FAS in both reversible and irreversible manners. Epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) inhibit FAS with IC(50) values of 52 microM and 42 microM mainly by reacting on the beta-ketoacyl reductase (KR) domain of FAS. The inhibitory ability of catechin gallate (CG) is 15 and 12 folds higher than that of EGCG and ECG. Its major reacting site on FAS is not KR. All of these irreversibly inactivate FAS on the KR domain with similar rates. Mulliken population analysis suggests that the positive charge is distributed on the carbon atom of galloyl ester, and this carbon becomes more susceptible for a nucleophilic attack. 12 flavonoids inhibit FAS with IC(50) values ranging from 2 to 112 microM. SAR analysis shows that the flavonoids containing two hydroxyl groups in B ring and 5, 7-hydroxyl groups in A ring with C-2, 3 double bond are the most potent inhibitors. The inhibition kinetics shows that they inhibit FAS competitively with acetyl CoA and most likely react mainly on acyl transferase domain. Further studies show that C ring of flavonoids is not necessary for the inhibition. Resveratrol, phlorizin and NDGA contain two phenyl rings connected by 2 to 4 atom chains instead of C ring. Their IC(50) values range from 5 microM to 40 microM. From these results, a common model for polyphenol inhibitor of FAS is conceived.

Inhibitory activity of chlorogenic acid on enzymes involved in the fatty acid synthesis in animals and bacteria.

It was found that chlorogenic acid inhibited in vitro animal fatty acid synthase (FAS I) and the ss-ketoacyl-ACP reductase (FabG) from Escherichia coli in a concentration-dependent manner with respective IC50 of 94.8 and 88.1 microM. The results of Lineweaver-Burk plots indicated that chlorogenic acid inhibited competitively the binding of NADPH to FAS I, while left those of acetyl-CoA and malonyl-CoA unaffected. Further kinetic studies showed that chlorogenic acid blocked the activity of FAS I mainly by inhibiting the ss-ketoacyl reductase domain, which catalyzed the same reaction as that done by FabG in the fatty acid synthesis. The ss-ketoacyl reduction reactions accomplished by both FAS I and FabG required nucleotide cofactor, NADPH. Furthermore, the Lineweaver-Burk and Yonetani-Theorell analyses implicated that chlorogenic acid filled competitively in the binding-pocket of NADPH in the ss-ketoacyl reductase domain of FAS I. The similar results were also obtained from the inhibition of FabG by chlorogenic acid. As observed in these results, the inhibitions of FAS I and FabG by chlorogenic acid were highly related to the interference of the inhibitor with NADPH, which was possibly due to the similarity between chlorogenic acid and some portion of NADPH, maybe the section consisting of the two ribose groups.

Potent inhibition of fatty acid synthase by parasitic loranthus [Taxillus chinensis (dc.) danser] and its constituent avicularin.

The medicinal herb parasitic loranthus in a screen was found to inhibit fatty acid synthase (EC 2.3.1.85, FAS) and reduce body weight of rats in our previous study. Now we have determined the inhibitory characteristics and kinetic parameters of extracts of parasitic loranthus [Taxillus chinensis (DC.) Danser]. The parasitic loranthus extracts (PLE) inhibits FAS reversibly and irreversibly and with an IC50 value of 0.48 microg/ml, appears to be the most potent inhibitor reported to date. PLE contains various potent inhibitors and may react with different sites on FAS. The irreversible inhibition exhibits a time-dependent biphasic process including a speedy fast-phase during the initial several minutes. The fast-phase inhibition seems to be caused by some potent but low-concentration component(s) in the extracts. In addition, we have found that avicularin existing in this herb can potently inhibit FAS. This glycosylated flavonoid and quercetin play an effective role in inhibiting FAS by parasitic loranthus.

Keemun black tea extract contains potent fatty acid synthase inhibitors and reduces food intake and body weight of rats via oral administration.

The inhibitory effects of a black tea extract on fatty acid synthase were measured through inhibition kinetics. The Keemun black tea extract showed more potent inhibitory activity on fatty acid synthase than green tea extract. Additionally, the inhibitory ability of the black tea extract depended on the extracting solvent and the conditions used. Only 10-23% of the inhibitory activity from the black tea was extracted by the general method of boiling with water. The results suggested that the main fatty acid synthase inhibitors in black tea might be theaflavins. Which were more potent than epigallocatechin gallate or C75. The reaction site on the fatty acid synthase and the inhibition kinetics for the extract were different from those of epigallocatechin gallate or C75. In addition, Keemun black tea extract significantly reduced food intake, body weight and blood triglyceride of diet-induced obesity SD rats via oral administration.