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.     

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 μg/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 × 10^{? 3} and 10.6 × 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.     

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.     

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 IC₅₀ of 0.47 μg/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.     

Matrix metalloproteinase inhibition by green tea catechins

We have investigated the effects of different biologically active components from natural products, including green tea polyphenols (GTP), resveratrol, genistein and organosulfur compounds from garlic, on matrix metalloproteinase (MMP)-2, MMP-9 and MMP-12 activities. GTP caused the strongest inhibition of the three enzymes, as measured by fluorescence assays using gelatin or elastin as substrates. The inhibition of MMP-2 and MMP-9 caused by GTP was confirmed by gelatin zymography and was observed for MMPs associated with both various rat tissues and human brain tumors (glioblastoma and pituitary tumors). The activities of MMPs were also measured in the presence of various catechins isolated from green tea including (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate(ECG), (-)-epigallocatechin (EGC), (-)-epicatechin (EC) and (+)-catechin (C). The most potent inhibitors of these activities, as measured by fluorescence and by gelatin or casein zymography, were EGCG and ECG. GTP and the different catechins had no effect on pancreatic elastase, suggesting that the effects of these molecules on MMP activities are specific. Furthermore, in vitro activation of proMMP-2 secreted from the glioblastomas cell line U-87 by the lectin concanavalin A was completely inhibited by GTP and specifically by EGCG. These results indicate that catechins from green tea inhibit MMP activities and proMMP-2 activation.     

Presence of Fatty Acid Synthase Inhibitors in the Rhizome of Alpinia officinarum Hance

The galangal (the rhizome of Alpinia officinarum, Hance) is popular in Asia as a traditional herbal medicine. The present study reports that the galangal extract (GE) can potently inhibit fatty-acid synthase (FAS, E.C.2.3.1.85). The inhibition consists of both reversible inhibition with an IC50 value of 1.73?μg?dried?GE/ml, and biphasic slow-binding inactivation. Subsequently the reversible inhibition and slow-binding inactivation to FAS were further studied. The inhibition of FAS by galangin, quercetin and kaempferol, which are the main flavonoids existing in the galangal, showed that quercetin and kaempferol had potent reversible inhibitory activity, but all three flavonoids had no obvious slow-binding inactivation. Analysis of the kinetic results led to the conclusion that the inhibitory mechanism of GE is totally different from that of some other previously reported inhibitors of FAS, such as cerulenin, EGCG (epigallocatechin gallate) and C75.     

Presence of fatty acid synthase inhibitors in the rhizome of Alpinia officinarum hance

The galangal (the rhizome of Alpinia officinarum, Hance) is popular in Asia as a traditional herbal medicine. The present study reports that the galangal extract (GE) can potently inhibit fatty-acid synthase (FAS, E.C.2.3.1.85). The inhibition consists of both reversible inhibition with an IC50 value of 1.73 microg dried GE/ml, and biphasic slow-binding inactivation. Subsequently the reversible inhibition and slow-binding inactivation to FAS were further studied. The inhibition of FAS by galangin, quercetin and kaempferol, which are the main flavonoids existing in the galangal, showed that quercetin and kaempferol had potent reversible inhibitory activity, but all three flavonoids had no obvious slow-binding inactivation. Analysis of the kinetic results led to the conclusion that the inhibitory mechanism of GE is totally different from that of some other previously reported inhibitors of FAS, such as cerulenin, EGCG (epigallocatechin gallate) and C75.     

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 μg/ml, and irreversible slow-binding inhibition following saturation kinetics with a dissociation constant of 0.68 μg/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.     

The impact of the 67kDa laminin receptor on both cell-surface binding and anti-allergic action of tea catechins

Here, we investigated the structure-activity relationship of major green tea catechins and their corresponding epimers on cell-surface binding and inhibitory effect on histamine release. Galloylated catechins; (−)-epigallocatechin-3-O-gallate (EGCG), (−)-gallocatechin-3-O-gallate (GCG), (−)-epicatechin-3-O-gallate (ECG), and (−)-catechin-3-O-gallate (CG) showed the cell-surface binding to the human basophilic KU812 cells by surface plasmon resonance analysis, but their non-galloylated forms did not. Binding activities of pyrogallol-type catechins (EGCG and GCG) were higher than those of catechol-type catechins (ECG and CG). These patterns were also observed in their inhibitory effects on histamine release. Previously, we have reported that biological activities of EGCG are mediated through the binding to the cell-surface 67 kDa laminin receptor (67LR). Downregulation of 67LR expression caused a reduction of both activities of galloylated catechins. These results suggest that both the galloyl moiety and the B-ring hydroxylation pattern contribute to the exertion of biological activities of tea catechins and their 67LR-dependencies.     

Inhibition of Catalase by Tea Catechins in Free and Cellular State: A Biophysical Approach

Tea flavonoids bind to variety of enzymes and inhibit their activities. In the present study, binding and inhibition of catalase activity by catechins with respect to their structure-affinity relationship has been elucidated. Fluorimetrically determined binding constants for (−)-epigallocatechin gallate (EGCG) and (−)-epicatechin gallate (ECG) with catalase were observed to be 2.27×10⁶ M⁻¹ and 1.66×10⁶ M⁻¹, respectively. Thermodynamic parameters evidence exothermic and spontaneous interaction between catechins and catalase. Major forces of interaction are suggested to be through hydrogen bonding along with electrostatic contributions and conformational changes. Distinct loss of α-helical structure of catalase by interaction with EGCG was captured in circular dichroism (CD) spectra. Gallated catechins demonstrated higher binding constants and inhibition efficacy than non-gallated catechins. EGCG exhibited maximum inhibition of pure catalase. It also inhibited cellular catalase in K562 cancer cells with significant increase in cellular ROS and suppression of cell viability (IC₅₀ 54.5 µM). These results decipher the molecular mechanism by which tea catechins interact with catalase and highlight the potential of gallated catechin like EGCG as an anticancer drug. EGCG may have other non-specific targets in the cell, but its anticancer property is mainly defined by ROS accumulation due to catalase inhibition.     

Combined effect of epigallocatechin gallate and triclosan on enoyl-ACP reductase of Mycobacterium tuberculosis

Among the various inhibitors known for enoyl-acyl carrier protein (ACP) reductases, triclosan and green tea catechins are two promising candidates. In the present study, we show, for the first time that epigallocatechin gallate (EGCG), a major component of green tea catechins, inhibits InhA, the enoyl-ACP reductase of Mycobacterium tuberculosis with an IC50 of 17.4 μM. EGCG interferes with the binding of NADH to InhA. We also demonstrate that EGCG increased the inhibitory activity of triclosan towards InhA and vice versa. Direct binding assay using [³H]EGCG and fluorescence titration assay support the spectrophotometric/kinetic inhibition data. The biochemical data has been explained by docking simulation studies.     

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)]-β-l-arabinopyranoside and hederagenin 3-O-(3-O-acetyl-α-l-arabinofuranosyl)-(1→3)-[α-l-rhamnopyranosyl-(1→2)]-β-l-arabinopyranoside, respectively. All these isolates were evaluated for inhibitory activities of FAS, which showed these isolates had inhibitory activity against FAS with IC₅₀ values ranging from 6.69 to 204.40 μM, comparable to the known FAS inhibitor EGCG (IC₅₀ = 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.     

茶多酚及其儿茶素单体对过氧化氢诱导的线粒体通透性改变孔道开放的影响

线粒体是细胞内重要的细胞器,是生成ATP的主要场所.线粒体通透性改变孔道（PT孔道）的开放会引起线粒体许多功能的紊乱而导致细胞死亡.对茶多酚及其单体儿茶素对过氧化氢诱导的线粒体膨胀及膜电势变化过程中PT孔开放的影响进行了研究.实验结果表明茶多酚及其儿茶素单体对PT孔开放的影响显著不同：茶多酚及其主要成分EGCG和ECG能够有效地抑制PT孔道的开放;而ECG,（+）-C和EGC却加速PT孔道的开放过程.从总体效果来看,茶多酚及其单体EGCG和ECG对线粒体的保护作用占主导地位.     

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.     

Distinct effects of tea catechins on 6-hydroxydopamine-induced apoptosis in PC12 cells.

Green tea polyphenols have aroused considerable attention in recent years for preventing oxidative stress related diseases including cancer, cardiovascular disease, and degenerative disease. Neurodegenerative diseases are cellular redox status dysfunction related diseases. The present study investigated the different effects of the five main components of green tea polyphenols on 6-hydroxydopamine (6-OHDA)-induced apoptosis in PC12 cells, the in vitro model of Parkinson's disease (PD). When the cells were treated with five catechins respectively for 30 min before exposure to 6-OHDA, (-)-epigallocatechins gallate (EGCG) and (-)-epicatechin gallate (ECG) in 50-200 microM had obvious concentration-dependent protective effects on cell viability, while (-)-epicatechin (EC), (+)-catechin ((+)-C), and (-)-epigallocatechin (EGC) had almost no protective effects. The five catechins also showed the same pattern described above of the different effects against 6-OHDA-induced cell apoptotic characteristics as analyzed by cell viability, fluorescence microscopy, flow cytometry, and DNA fragment electrophoresis methods. The present results indicated that 200 microM EGCG or ECG led to significant inhibition against typical apoptotic characteristics of PC12 cells, while other catechins had little protective effect against 6-OHDA-induced cell death. Therefore, the classified protective effects of the five catechins were in the order ECG> or = EGCG>EC> or = (+)-C>EGC. The antiapoptotic activities appear to be structurally related to the 3-gallate group of green tea polyphenols. The present data indicate that EGCG and ECG might be potent neuroprotective agents for PD.     

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.     

The Green Tea Catechin Epigallocatechin Gallate (EGCG) Blocks Cell Motility,Chemotaxis and Development in Dictyostelium discoideum

Catechins, flavanols found at high levels in green tea, have received significant attention due to their potential health benefits related to cancer, autoimmunity and metabolic disease, but little is known about the mechanisms by which these compounds affect cellular behavior. Here, we assess whether the model organism Dictyostelium discoideum is a useful tool with which to characterize the effects of catechins. Epigallocatechin gallate (EGCG), the most abundant and potent catechin in green tea, has significant effects on the Dictyostelium life cycle. In the presence of EGCG aggregation is delayed, cells do not stream and development is typically stalled at the loose aggregate stage. The developmental effects very likely result from defects in motility, as EGCG reduces both random movement and chemotaxis of Dictyostelium amoebae. These results suggest that catechins and their derivatives may be useful tools with which to better understand cell motility and development in Dictyostelium and that this organism is a useful model to further characterize the activities of catechins.     

Analysis of the mechanism of inhibition of human matrix metalloproteinase 7 (MMP-7) activity by green tea catechins

Green tea catechins inhibit human matrix metalloproteinase 7 (MMP-7) activity non-competitively, and the galloyl group is essential for potent inhibition (Oneda et al., J. Biochem., 133, 571-576 (2003)). In this study, we analyzed the mechanism of this inhibition. In the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2), the inhibitory effects of (-)-epigallocatechin-3-gallate (EGCG), (-)-gallocatechin-3-gallate (GCG), (-)-epicatechin-3-gallate (ECG), and (-)-catechin-3-gallate (CG) increased with increasing pH levels from 7.0 to 8.5. The inhibitory effects of EGCG and GCG were more potent than those of ECG and CG, and increased with increasing CaCl(2) concentrations from 10 to 50 mM. The fluorescence of EGCG and GCG decreased with increasing CaCl(2) concentrations and with the addition of MMP-7, while those of ECG and CG did not. Our results suggest that these differences result from that in the B ring, EGCG and GCG have phenol hydroxyl groups at the 3', 4', and 5' positions, while ECG and CG have them at the 3' and 4' positions.     

Structure-activity relationships of bergenin derivatives effect on α-glucosidase inhibition

The α-glucosidase inhibitory activities of bergenin derivatives were evaluated. Bergenin derivatives were synthesized from bergenin which is a characteristic compound of B. ligulata. A new bergenin derivative, 11-O-(3′,4′-dimethoxybenzoyl)-bergenin showed the highest potent inhibitory activity among those of bergenin derivatives. The presence of substituents at 3′,4′-position in bergenin derivatives altered the α-glucosidase inhibitory activity. 11-O-(3′,4′-dimethoxybenzoyl)-bergenin was noncompetitive inhibitor for α-glucosidase. The present study reveals that bergenin derivatives could be classified as a new group of α-glucosidase inhibitors.     

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 μg/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.