Inhibitory effect of the coffee diterpene kahweol on carrageenan-induced inflammation in rats

Previous studies reported that kahweol, a coffee-specific diterpene, inhibits cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression in cultured lipopolysaccharide-activated macrophages. The aim of this study was to confirm the anti-inflammatory effects of kahweol by examining its effect on the inflammatory response induced by carrageenan in a rat using an acute air pouch inflammation model. Kahweol significantly reduced the levels of the inflammatory process markers in the air pouch, such as the volume of exudates, the amount of protein and the number of leukocytes and neutrophils. The levels of nitrite, TNF-alpha and prostaglandin E2 (PGE2) were also markedly lower in the air pouch of the kahweol-treated animals than in the controls. Immunoblot analysis showed that kahweol reduced the COX-2 and iNOS expression level in the exudate cells. The histological examination showed that there was a lower inflammatory response in the pouch tissues from the kahweol-treated animals. In addition, kahweol significantly reduced the paw edema induced by carrageenan and also markedly reduced the level of PGE2 production in the inflamed paw. These results suggest that kahweol has significant anti-inflammatory effects in vivo, which might be due to the inhibition of iNOS and COX-2 expression in the inflammatory sites.     

The coffee diterpene kahweol sensitizes TRAIL-induced apoptosis in renal carcinoma Caki cells through down-regulation of Bcl-2 and c-FLIP

Kahweol, a coffee-specific diterpene, found in the beans of Coffea arabica, has potent anti-carcinogenic, anti-tumor, and anti-inflammatory properties. TRAIL is a potential anti-cancer compound that induces apoptosis in a wide variety of cancer cells, but not in most normal human cell types. In the present study, we show that kahweol sensitizes human renal cancer cells, but not normal human mesangial cells, to TRAIL-mediated apoptosis. Moreover, treatment with a combination of kahweol and TRAIL induces significant apoptosis in various cancer cell types, thus presenting an attractive novel strategy for cancer treatment. Our experiments show that treatment with a combination of kahweol and TRAIL-induced apoptosis, and stimulated of DEVDase activity, DNA fragmentation, and cleavage of PARP, which was prevented by pretreatment with z-VAD, indicative of cell death via a caspase-dependent pathway. Kahweol-induced down-regulation of Bcl-2 and ectopic expression of Bcl-2 led to attenuation of kahweol plus TRAIL-mediated apoptosis, indicative of Bcl-2 involvement in the apoptotic process. In addition, the c-FLIP and caspase signal pathways seem to play a crucial role in apoptosis triggered by the combination of kahweol and TRAIL in Caki cells. Our results collectively demonstrate that down-regulation of Bcl-2 and c-FLIP contributes to the sensitizing effect of kahweol on TRAIL-mediated apoptosis in cancer cells.     

Antiangiogenic properties of cafestol, a coffee diterpene, in human umbilical vein endothelial cells

As angiogenesis plays important roles in tumor growth and metastasis, searching for antiangiogenic compounds is a promising tactic for treating cancers. Cafestol, a diterpene found mainly in unfiltered coffee, provides benefit through varied biological activity, including antitumorigenic, antioxidative, and anti-inflammatory effects. This study aimed to investigate the effects of cafestol on angiogenesis and to uncover the associated mechanism. We show that cafestol inhibits angiogenesis of human umbilical vascular endothelial cells. This inhibition affects the following specific steps of the angiogenic process: proliferation, migration, and tube formation. The inhibitory effects of cafestol are accompanied by decreasing phosphorylation of FAK and Akt and by a decrease in nitric oxide production. Overall, cafestol inhibits angiogenesis by affecting the angiogenic signaling pathway.     

The coffee diterpene kahweol induces apoptosis in human leukemia U937 cells through down-regulation of Akt phosphorylation and activation of JNK

Kahweol, the coffee-specific diterpene, has been reported for its tumor cell growth inhibitory activity and anti-carcinogenic activity. The mechanism by which kahweol initiates apoptosis remains poorly understood. In the present study, we investigated the effect of kahweol on the apoptotic pathway in U937 human promonocytic cells. We show that kahweol induces apoptosis in association with the activation of caspase 3 and cytochrome c release from the mitochondria to the cytosol, as well as down-regulation of anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1 and XIAP). Kahweol altered the phosphorylation state of members of the MAPKs and Akt. Ectopic expression of Bcl-2 or constitutive active Akt (myr-Akt) in U937 cells attenuates kahweol-induced apoptosis. In addition, we have also shown that JNK and Akt signal pathway plays a crucial role in kahweol-induced apoptosis in U937 cells. Taken together, our results show the activity of kahweol to modulate multiple components in apoptotic response of human leukemia cells and raise the possibility a novel therapeutic strategy in hematological malignancies.     

Establishment of somaclonal variants of Robusta coffee with reduced levels of cafestol and kahweol

Cafestol (caf) and kahweol (kah) are two diterpenes uniquely associated with the unsaponified lipid fraction of coffee brew and are reported to be responsible for an increase in serum cholesterol and triglyceride levels. The plant growth regulators (PGRs) indole-3-acetic acid (IAA), N ⁶-benzyladenine (BA), and thidiazuron (TDZ); the plant growth-promoting agents silver nitrate, triacontanol (TRIA), and coconut water; and some PGR antagonists such as lovastatin, paclobutrazol, and 2,3,5-triiodobenzoic acid (TIBA) were used to determine the variation of caf and kah in somatic embryos of Robusta coffee (Coffea canephora, CxR variety). Embryogenic (EG) medium was comprised of half-strength Murashige and Skoog basal components (½MS) supplemented with 2.85 μM IAA and 1.11 μM BA. After an 8-wk culture, somatic embryos were subjected to diterpene extraction and HPLC analysis of caf and kah profiles. TRIA-supplemented (5 μg L^?1) EG medium devoid of IAA reduced the levels of caf and kah by 18–24 and 48–55%, respectively, in coffee somatic embryos. Similarly, the combination of 2.85 μM IAA, 2.27 μM TDZ, and 5–10% coconut water in ½MS basal medium drastically reduced the caf and kah levels in somatic embryos. There was 60–75% reduction in both caf and kah in the presence of 5 μM TIBA, followed by 56–62% reduction in the presence of 10 μM silver nitrate. In contrast, there was 25–32% elevation of caf and kah in EG medium supplemented with 5 μM paclobutrazol. In this study, for the first time, somaclonal variants of C. canephora with reduced levels of diterpenes caf and kah were established. Furthermore, these lines exhibited consistency in their metabolite profiles when cultivated under greenhouse conditions. In-depth investigations at physiological level are warranted in order to elucidate the actual mechanism of these PGR inhibitors on alterations in endogenous pools of diterpenes in coffee somatic embryos.     

Anti-angiogenic and anti-inflammatory effects of SERPINA3K on corneal injury

SERPINA3K is a member of the serine proteinase inhibitor (SERPIN) family. Here we evaluated the therapeutic effects of SERPINA3K on neovascularization and inflammation in a rat cornea alkali burn model that is commonly employed to study corneal wounding. Topical treatment of the injured rat cornea with SERPINA3K (20 μg/eye/day) for 7 days significantly decreased the neovascular area, compared with the groups treated with BSA or PBS. The SERPINA3K treatment also ameliorated the corneal inflammation as evaluated by the inflammatory index. Furthermore, SERPINA3K enhanced the recovery of corneal epithelium after the alkali injury. Toward the mechanism of action, SERPINA3K down-regulated the expression of the pro-angiogenic and pro-inflammatory factors, vascular endothelial growth factor and tumor necrosis factor-α and up-regulated the expression of the anti-angiogenic factor, pigment epithelium-derived factor. SERPINA3K specifically inhibited growth of vascular endothelial cells. Meanwhile, SERPINA3K significantly up-regulated the expression of EGFR in the corneal epithelium. These findings suggest that SERPINA3K has therapeutic potential for corneal inflammation and NV.     

The coffee diterpene kahweol induces heme oxygenase-1 via the PI3K and p38/Nrf2 pathway to protect human dopaminergic neurons from 6-hydroxydopamine-derived oxidative stress

In this study, we investigated the mechanisms of kahweol protection of neuronal cells from cell death induced by the Parkinson's disease-related neurotoxin 6-hydroxydopamine (6-OHDA). Pretreatment of SH-SY5Y cells with kahweol significantly reduced 6-OHDA-induced generation of ROS, caspase-3 activation, and subsequent cell death. Kahweol also up-regulated heme oxygenase-1 (HO-1) expression, which conferred neuroprotection against 6-OHDA-induced oxidative injury. Moreover, kahweol induced PI3K and p38 activation, which are involved in the induction of Nrf2, HO-1 expression, and neuroprotection. These results suggest that regulation of the anti-oxidant enzyme HO-1 via the PI3K and p38/Nrf2 signaling pathways controls the intracellular levels of ROS.     

Fuscoside E: a strong anti-inflammatory diterpene from Caribbean octocoral Eunicea fusca

The screen of 10 soft coral extracts collected from the Colombian Caribbean Sea in the TPA-induced ear edema model allowed us to identify Eunicea fusca extract among others as an interesting source of active compounds. The new diterpene, fuscoside E (1), along with the known fuscoside B (2), fuscol (3), (+)-germacrene D (4) and a mixture of six sterols (5-10), were isolated from this soft coral. Their structures were elucidated by 1D and 2D NMR spectroscopy techniques. Fuscoside E (1) absolute stereochemistry was determined by chiroptical methods. Fuscoside E (1) and B (2) showed strong anti-inflammatory in the above mentioned bioassay. Additionally, fuscoside E (1) and the sterol mixture (5-10) presented antifouling activity against bacterial strains involved in surface colonization.     

Possible mechanisms underlying the cholesterol-raising effect of the coffee diterpene cafestol

Cafestol, a coffee diterpene present in unfiltered coffee brews, potently raises serum lipids in humans. The mechanism through which this dietary compound influences liporotein metabolism is largely unknown. Unravelling the mechanism of action might lead to new insights into the regulation of serum cholesterol levels in humans. This review summaries ways in which cafestol may act on serum lipids.     

Natural diterpenes from coffee,cafestol and kahweol induce apoptosis through regulation of specificity protein 1 expression in human malignant pleural mesothelioma

Background

Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with a very poor prognosis. Several clinical studies such as immunotherapy, gene therapy and molecular targeting agents have been tried for treatment of malignant mesothelioma, however, there is no application for effective clinical treatment. Coffee has various biological functions such as anti-oxidant, anti-inflammatory, anti-mutagenic and anti-carcinogenic activities. The therapeutic activities of the bioactive compounds in coffee was sugested to influence intracellular signaling of MPM. Regarding to the cancer-related functions, In this study, suppression of Sp1 protein level followed by induction of MSTO-211H cell apoptosis by cafestol and kahweol were investigated in oreder to determine Sp1''s potential as a significant target for human MPM therapy as well.

Methods

Cells were treated separately with final concentration of cafestol and kahweol and the results were analyzed by MTS assay, DAPI staining, PI staining, luciferase assay, RT-PCR, and immunoblotting.

Results

Viability of MSTO-211H and H28 cells were decreased, and apoptotic cell death was increased in MSTO-211H as a result of cafestol and kahweol treatment. Cafestol and kahweol increased Sub-G₁ population and nuclear condensation in MSTO-211H cells. Roles of Sp1 in cell proliferation and apoptosis of the MSTO-211H cells by the Sp1 inhibitor of Mithramycin A were previously confirmed. Cafestol and kahweol significantly suppressed Sp1 protein levels. Kahweol slightly attenuated Sp1 mRNA, while Cafestol did not affect in MSTO-211H cells. Cafestol and kahweol modulated the promoter activity and protein expression level of the Sp1 regulatory genes including Cyclin D1, Mcl-1, and Survivin in mesothelioma cells. Apoptosis signaling cascade was activated by cleavages of Bid, Caspase-3, and PARP with cafestol and by upregulation of Bax, and downregulation of Bcl-_xl by kahweol.

Conclusions

Sp1 can be a novel molecular target of cafestol and kahweol in human MPM.     

Synthesis, isomer characterization, and anti-inflammatory properties of nitroarachidonate

Nitrated fatty acids (nitroalkenes) have been recently detected and quantified in cell membranes and human plasma. However, nitration of arachidonate (AA), that could redirect AA-dependent cell signaling pathways, has not been studied in detail. Herein, we synthesized and determined for the first time the isomer distribution of nitroarachidonate (AANO2) and demonstrate its ability to modulate inflammation. Synthesis of AANO2 was achieved by AA treatment with sodium nitrite in acidic conditions following HPLC separation. Mass spectrometry (MS) analysis showed the characteristic MS/MS transition of AANO2 (m/z 348/301). Moreover, the IR signal at 1378.3 cm(-1) and NMR studies confirmed the presence of mononitrated nitroalkenes. Positional isomer distribution was determined by NMR and MS fragmentation with lithium; four major isomers (9-, 12-, 14-, and 15-AANO2) were identified, which exhibited key anti-inflammatory properties. These include their ability to release biologically relevant amounts of nitric oxide, induce cGMP-dependent vasorelaxation, and down-regulate inducible nitric oxide synthase (NOS2) expression during macrophage activation, providing unique structural evidence and novel regulatory signaling properties of AANO2.     

Isocarnosol,a diterpene from salvia lanigera

Isocarnosol, a novel diterpene from the leaves of Salvia lanigera, has been characterized.     

Functional analysis of eubacterial diterpene cyclases responsible for biosynthesis of a diterpene antibiotic,terpentecin

Eubacterial diterpene cyclase genes had previously been cloned from a diterpenoid antibiotic terpentecin producer (Dairi, T., Hamano, Y., Kuzuyama, T., Itoh, N., Furihata, K., and Seto, H. (2001) J. Bacteriol. 183, 6085-6094). Their products, open reading frame 11 (ORF11) and ORF12, were essential for the conversion of geranylgeranyl diphosphate (GGDP) into terpentetriene (TTE) that had the same basic skeleton as terpentecin. In this study, functional analyses of these two enzymes were performed by using purified recombinant enzymes. The ORF11 product converted GGDP into a cyclized intermediate, terpentedienol diphosphate (TDP), which was then transformed into TTE by the ORF12 product. Interestingly, the ORF12 product directly catalyzed the conversion of GGDP into three olefinic compounds. Moreover, the ORF12 product utilized farnesyl diphosphate as a substrate to give three olefinic compounds, which had the same structures as those formed from GGDP with the exception of the chain lengths. These results suggested that the ORF11 product with a DXDD motif converted GGDP into TDP by a protonation-initiated cyclization and that the ORF12 product with a DDXXD motif completed the transformation of TDP to the olefin, terpentetriene by an ionization-initiated reaction followed by deprotonation. The kinetics of the ORF12 product indicated that the affinity for TDP and GGDP were higher than that of farnesyl diphosphate and that the relative activity of the reaction converting TDP into TTE was highest among the reactions using TDP, GGDP, or farnesyl diphosphate as the substrate. These results suggested that an actual reaction catalyzed by the ORF12 was the conversion of TDP into TTE in vivo.     

Salvidorol, a nor-abietane diterpene with a rare carbon skeleton and two abietane diterpene derivatives from Salvia dorrii

Salvidorol (1), a irregular nor-abietane-type diterpene, was isolated from the aerial parts of Salvia dorrii, in addition to two epimeric abietane diterpenes (2 and 3). This is the first report of a nor-diterpene with an irregular skeleton. The structures were established by high-field NMR techniques ((1)H-(1)H COSY, DEPT, HMQC, HMBC, NOESY and HRMS) and in case of 2 was confirmed by X-ray analysis.     

The role of epoxidation and electrophile-responsive element-regulated gene transcription in the potentially beneficial and harmful effects of the coffee components cafestol and kahweol

Cafestol and kahweol are diterpene compounds present in unfiltered coffees. Cafestol is known as the most potent cholesterol-raising agent that may be present in the human diet. Remarkably, the mechanisms behind this effect have only been partly resolved so far. Even less is known about the metabolic fate of cafestol and kahweol. From the structure of cafestol, carrying a furan moiety, we hypothesized that epoxidation may not only be an important biotransformation route but that this also plays a role in its effects found. In bile duct-cannulated mice, dosed with cafestol, we were able to demonstrate the presence of epoxy-glutathione (GSH) conjugates, GSH conjugates and glucuronide conjugates. In addition, it was shown that cafestol was able to induce an electrophile-responsive element (EpRE). Using a murine hepatoma cell line with a luciferase reporter gene under control of an EpRE from the human NQO1 regulatory region, we also found that metabolic activation by CYP450 enzymes is needed for EpRE induction. Furthermore, raising intracellular GSH resulted in a decrease in EpRE-mediated gene induction, whereas lowering intracellular GSH levels increased EpRE-mediated gene induction. In conclusion, evidence suggests that cafestol induces EpRE, apparently via a bioactivation process that possibly involves epoxidation of the furan ring. The epoxides themselves appear subject to conjugation with GSH. The effects on EpRE can also explain the induction of GSH which seems to be involved in the reported beneficial effects of cafestol, for example, when administered with aflatoxin B1 or other toxic or carcinogenic compounds.     

Cordatin,a new diterpene from Aparisthmium cordatum

A new furano diterpene, cordatin, with a clerodane skeleton has been isolated from the bark of Aparisthmium cordatum. Its molecular structure has been investigated by spectroscopic means and by X-ray analysis.