Hyperforin, an Anti-Inflammatory Constituent from St. John's Wort, Inhibits Microsomal Prostaglandin E2 Synthase-1 and Suppresses Prostaglandin E2 Formation in vivo

The acylphloroglucinol hyperforin (Hyp) from St. John's wort possesses anti-inflammatory and anti-carcinogenic properties which were ascribed among others to the inhibition of 5-lipoxygenase. Here, we investigated whether Hyp also interferes with prostanoid generation in biological systems, particularly with key enzymes participating in prostaglandin (PG)E2 biosynthesis, i.e., cyclooxygenases (COX)-1/2 and microsomal PGE2 synthase (mPGES)-1 which play key roles in inflammation and tumorigenesis. Similar to the mPGES-1 inhibitors MK-886 and MD-52, Hyp significantly suppressed PGE2 formation in whole blood assays starting at 0.03–1 μM, whereas the concomitant generation of COX-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid, thromboxane B2, and 6-keto PGF1α was not significantly suppressed up to 30 μM. In cell-free assays, Hyp efficiently blocked the conversion of PGH2 to PGE2 mediated by mPGES-1 (IC50 = 1 μM), and isolated COX enzymes were not (COX-2) or hardly (COX-1) suppressed. Intraperitoneal (i.p.) administration of Hyp (4 mg kg−1) to rats impaired exudate volume and leukocyte numbers in carrageenan-induced pleurisy associated with reduced PGE2 levels, and Hyp (given i.p.) inhibited carrageenan-induced mouse paw edema formation (ED50 = 1 mg kg−1) being superior over indomethacin (ED50 = 5 mg kg−1). We conclude that the suppression of PGE2 biosynthesis in vitro and in vivo by acting on mPGES-1 critically contributes to the anti-inflammatory efficiency of Hyp.

ear edema in mice after topical application (Sosa et al., 2007), impaired acute neutrophil recruitment and enhanced resolution in a pulmonary bleomycin-induced inflammation model reducing consequent fibrosis (Dell'Aica et al., 2007), and suppressed carrageenan-induced rat pleurisy when given intraperitoneally (i.p.; Feisst et al., 2009). Also for humans, topical treatment of atopic dermatitis with a Hypericum cream, standardized to 1.5% Hyp, revealed significant therapeutic efficacy (Schempp et al., 2003). However, despite these well-recognized anti-carcinogenic and antiinflammatory effects, the underlying molecular mechanisms and targets of Hyp are incompletely understood.
The lipid mediator prostaglandin (PG)E 2 is a key player in inflammation, pain, fever, and cancer but is also known to regulate physiological functions in the gastrointestinal tract, in the kidney, and in the immune and nervous system (Sugimoto and Narumiya, 2007). PGE 2 is formed from arachidonic acid (AA) by cyclooxygenase (COX)-catalyzed synthesis of PGH 2 and further transformation by PGE 2 synthases (Samuelsson et al., 2007). The microsomal PGE 2 synthase (mPGES)-1 is induced by pro-inflammatory stimuli and is responsible for excessive PGE 2 generation connected to pathologies (Jakobsson et al., 1999). Co-expression studies indicate a preferred functional coupling between mPGES-1 and COX-2 (Murakami IntroductIon Hyperforin (Hyp), a polyprenylated acylphloroglucinol ( Figure  1A), is assumed to be one of the main active components of St. John's wort, which is frequently used for the treatment of mild to moderate depressions (Muller, 2003). Besides its antidepressant activity, Hyp also exerts potent anti-inflammatory and anti-tumoral effects (Medina et al., 2006). Hyp was shown to reduce tumor cell growth (Schempp et al., 2002;Hostanska et al., 2003), cancer invasion, metastasis (Dona et al., 2004), angiogenesis (Martinez-Poveda et al., 2005), and lymphatic capillary growth (Rothley et al., 2009). On the cellular level, Hyp decreases proliferation rates and causes apoptosis of leukemic cells (Schempp et al., 2002;Quiney et al., 2006) and various cancer cell lines (Schempp et al., 2002;Hostanska et al., 2003;Dona et al., 2004), and also inhibits proliferation of non-transformed T lymphocytes (Schempp et al., 2000). In regard of its anti-inflammatory potential, Hyp blocks several proinflammatory functions of leukocytes in vitro such as chemotaxis and chemoinvasion (Dell'Aica et al., 2007;Lorusso et al., 2009), suppresses receptor-mediated Ca 2+ -mobilization and eicosanoid release in leukocytes (Albert et al., 2002;Feisst and Werz, 2004), and down-regulates effector functions of activated T lymphocytes (Cabrelle et al., 2008). In vivo, Hyp reduced croton-oil-induced flasks. Cell viability was assessed using the colorimetric thiazolyl blue tetrazolium bromide dye reduction assay (MTT assay). A549 cells (4 × 10 4 cells per 100 μl medium) were plated into a 96-well microplate and incubated at 37°C and 5% CO 2 for 16 h. Then, Hyp (30 μM) was added, and the samples were incubated for another 5 h. Thiazolyl blue tetrazolium bromide (20 μl, 5 mg ml −1 ) was added and the incubations were continued for 4 h. The formazan product was solubilized with sodium dodecylsulfate (SDS, 10%, m v −1 in 20 mM HCl), and the absorbance was measured at 595 nm relative to the absorbance of vehicle (DMSO)-treated control cells using a multiwell scanning spectrophotometer (Victor 3 plate reader, PerkinElmer, Rodgau-Juegesheim, Germany).

AnImAls
Male adult CD1 mice (25-35 g, Harlan, Milan, Italy) and Wistar Han rats (190)(191)(192)(193)(194)(195)(196)(197)(198)(199)(200)Harlan,Milan,Italy) were housed in a controlled environment and provided with standard rodent chow and water. Animal care complied with Italian regulations on protection of animals used for experimental and other scientific purpose (Ministerial Decree 116192) as well as with the European Economic Community regulations (Official Journal of E.C. L 358/1 12/18/1986). determInAtIon of PGe 2 , 6-keto PGf 1α , And tXB 2 formAtIon In lPs-stImulAted humAn whole Blood Peripheral blood from healthy adult volunteers, who had not received any medication for at least 2 weeks under informed consent, was obtained by venepuncture and collected in syringes containing heparin (20 U ml −1 ). For determination of PGE 2 and 6-keto PGF 1α , aliquots of whole blood (0.8 ml) were mixed with the TX synthase inhibitor CV4151 (1 μM) and with aspirin (50 μM) to establish experimental conditions where prostanoids are essentially produced via the COX-2 pathway (Koeberle et al., 2008). For the determination of TXB 2 , whole blood aliquots were not pretreated with aspirin and CV4151. A total volume of 1 ml was adjusted with sample buffer (10 mM potassium phosphate buffer pH 7.4, 3 mM KCl, 140 mM NaCl, and 6 mM d-glucose). After pre-incubation with the indicated compounds for 5 min at room temperature, the samples were stimulated with LPS (10 μg ml −1 ) for 5 h at 37°C. Prostanoid formation was stopped on ice, the samples were centrifuged (2300 × g, 10 min, 4°C), and 6-keto PGF 1α and TXB 2 were quantified in the supernatant using a 6-keto PGF 1α or TXB 2 high sensitivity EIA kit (Assay Designs, Ann Arbor, MI, USA) according to the manufacturer's protocol. PGE 2 was determined after solid phase extraction and HPLC separation using a PGE 2 high sensitivity EIA kit (Assay Designs, Ann Arbor, MI, USA) as described (Koeberle et al., 2008). The mPGES-1 inhibitors MK-886 (30 μM) and MD52 (6 μM), the COX-1/2 inhibitor indomethacin (50 μM) and the COX-2-selective inhibitor celecoxib (20 μM) were used as control. Care should be taken when interpreting the controls in respect to COX-1/2 isoenzyme specificity which was not further evaluated for our experimental settings.
We have previously shown that Hyp inhibits leukotriene biosynthesis in neutrophils (IC 50 = 1-2 μM) by interference with the C2-like domain of 5-lipoxygenase (5-LO), and we observed an inhibitory effect of Hyp also on COX-1 activity in human platelets (IC 50 = 3 μM; Albert et al., 2002;Feisst et al., 2009). However, COX-2 activity (measured as 6-keto PGF 1α synthesis) in monocytic Mono Mac 6 cells was not affected up to 30 μM Hyp. This is surprising because Hammer et al. (2007) recently demonstrated that Hyp inhibits the release of PGE 2 from lipopolysaccharide (LPS)stimulated murine (RAW264.7) macrophages, a process coupled to COX-2 as well. Here, we identified mPGES-1 as functional target of Hyp and suggest that interference with mPGES-1 suppresses PGE 2 biosynthesis in vivo and contributes to the anti-inflammatory effectiveness in related animal models.

cArrAGeenAn-Induced PAw edemA In mIce
Mice were divided into groups (n = 10 for each group) and lightly anesthetized with enflurane 4% mixed with O 2 , 0.5 l min −1 , N 2 O 0.5 l min −1 . Each group of animals received subplantar administration of saline (0.05 ml) or of λ-carrageenan type IV 1% (w v −1 , 0.05 ml) in saline. The paw was marked in order to immerge it always at the same extent in the measurement chamber. The volume was measured by using a hydroplethismometer, specially modified for small volumes (Ugo Basile, Milan, Italy), immediately before subplantar injection and 2, 4, and 6 h thereafter (Posadas et al., 2004). The assessment of paw volume was performed always in double blind and by the same operator. The increase in paw volume was calculated by subtracting the initial paw volume (basal) to the paw volume measured at each time point.

cArrAGeenAn-Induced PleurIsy In rAts
Rats were anesthetized with enflurane 4% mixed with O 2 , 0.5 l min −1 , N 2 O 0.5 l min −1 and submitted to a skin incision at the level of the left sixth intercostal space. The underlying muscle was dissected, and saline (0.2 ml) or λ-carrageenan type IV 1% (w v −1 , 0.2 ml) was injected into the pleural cavity. The skin incision was closed with a suture, and the animals were allowed to recover. At 4 h after the injection of carrageenan, the animals were killed by inhalation of CO 2 . The chest was carefully opened, and the pleural cavity was rinsed with 2 ml saline solution containing heparin (5 U ml −1 ). The exudate and washing solution were removed by aspiration, and the total volume was measured. Any exudate that was contaminated with blood was discarded. The amount of exudate was calculated by subtracting the volume injected (2 ml) from the total volume recovered. Leukocytes in the exudate were resuspended in phosphate buffered saline (PBS) and counted with an optical light microscope in a Burker's chamber after vital trypan blue staining.
The amount of PGE 2 in the supernatant of centrifuged exudate (800 × g for 10 min) was assayed by radioimmunoassay according to manufacturer's protocol. The results are expressed as nanograms per rat and represent the mean ± SE of 10 rats. stAtIstIcs Data are expressed as mean ± SE. IC 50 values were calculated by nonlinear regression using SigmaPlot 9.0 (Systat Software Inc., San Jose, USA) one site binding competition. The program Graphpad was added, and after pre-incubation with the indicated compounds for 10 min at 37°C, prostanoid formation was initiated by addition of 20 μM AA. In contrast to the LPS-stimulated whole blood assay described above, whole blood aliquots were not pretreated with aspirin (inhibition of COX-1 product formation) because both COX-1 and -2 are described to contribute to mPGES-1-dependent PGE 2 synthesis at high AA concentrations (Murakami et al., 2000). PGE 2 or 6-keto PGF 1α formation within 10 min was determined as described for LPS-stimulated whole blood. Calculated prostanoid levels were corrected by the amount of PGE 2 formed during prestimulation with LPS.

ActIvIty AssAys of IsolAted coX-1 And coX-2
Inhibition of the activities of isolated ovine COX-1 and human COX-2 was performed as described (Koeberle et al., 2008). Briefly, purified COX-1 (ovine, 50 units) or COX-2 (human recombinant, 20 units) were diluted in 1 ml reaction mixture containing 100 mM Tris buffer pH 8, 5 mM glutathione, 5 μM hemoglobin, and 100 μM EDTA at 4°C and pre-incubated with the test compounds for 5 min. Samples were pre-warmed for 60 s at 37°C, and AA (5 μM for COX-1, 2 μM for COX-2) was added to start the reaction. After 5 min at 37°C, the COX product 12-HHT was extracted and then analyzed by HPLC as described (Albert et al., 2002).

PrePArAtIon of crude mPGes-1 In mIcrosomes of A549 cells And determInAtIon of PGe 2 synthAse ActIvIty
Preparation of A549 cells and determination of mPGES-1 activity was performed as described previously (Koeberle et al., 2008). In brief, cells were incubated for 16 h at 37°C and 5% CO 2 , the medium was replaced, 1 ng ml −1 interleukin (IL)-1β was added, and cells were incubated for another 48 h. After sonication, the homogenate was subjected to differential centrifugation at 10,000 × g for 10 min and 174,000 × g for 1 h at 4°C. The pellet (microsomal fraction) was resuspended in 1 ml homogenization buffer (0.1 M potassium phosphate buffer pH 7.4, 1 mM phenylmethanesulfonyl fluoride, 60 μg ml −1 soybean trypsin inhibitor, 1 μg ml −1 leupeptin, 2.5 mM glutathione, and 250 mM sucrose), and the total protein concentration was determined. Microsomal membranes were diluted in potassium phosphate buffer (0.1 M, pH 7.4) containing 2.5 mM glutathione to a final protein concentration of approx. 70 μg ml −1 . Test compounds or vehicle were added, and after 15 min at 4°C, the reaction (100 μl total volume) was initiated by addition of PGH 2 (20 μM, final concentration). After 1 min at 4°C, the reaction was terminated using stop solution (100 μl; 40 mM FeCl 2 , 80 mM citric acid, and 10 μM of 11β-PGE 2 as internal standard). PGE 2 was separated by solid phase extraction and analyzed by HPLC as described (Koeberle et al., 2008). 30 μM) can be excluded as determined by an A549 cell viability assay measuring mitochondrial dehydrogenase activity (see Materials and Methods, data not shown). The selective mPGES-1 inhibitor MD-52 (Côté et al., 2007) and the combined 5-LO-activating protein (FLAP), COX-1 and mPGES-1 inhibitor MK-886 (Claveau et al., 2003;Koeberle et al., 2009b) were used as reference (see also Koeberle et al., 2008). PGE 2 levels were neither completely suppressed by the mPGES-1 inhibitors nor by Hyp, even not at high Hyp concentrations (30 μM) or prolonged incubation times (24 h, data not shown). In contrast, the COX-1/2 inhibitor indomethacin (50 μM) and the COX-2 selective celecoxib (20 μM) effectively reduced PGE 2 ( Figure 1B) almost to the level of the unstimulated control (PGE 2 : 14.2 ± 8.4%). Celecoxib and/or indomethacin also inhibited the formation of 6-keto PGF 1α (the stable metabolite of PGI 2 , Figure 1C) and TXB 2 ( Figure 1D) as expected, whereas Hyp (up to 30 μM), MD-52, and MK-886 failed to suppress the formation of 6-keto PGF 1α ( Figure 1C) and TXB 2 ( Figure 1D) under the Instat (Graphpad Software Inc., San Diego, CA, USA) was used for statistical comparisons. Statistical evaluation of the data was performed by one-way or two-way ANOVAs for independent or correlated samples followed by Tukey HSD post hoc tests. A P value < 0.05 (*) was considered significant.

Koeberle et al.
Hyperforin inhibits mPGES-1 same experimental conditions. Together, unlike COX inhibitors but as like mPGES-1 inhibitors, Hyp selectively suppresses PGE 2 formation but not the synthesis of other prostanoids such as 6-keto PGF 1α or TXB 2 . Hyperforin may suppress PGE 2 formation by interference with LPS-signaling, mPGES-1 expression or liberation of AA as substrate for COX enzymes. Thus, heparinized human whole blood was first incubated with LPS for 16 h to induce the expression of COX-2 and mPGES-1 (in contrast to constitutively expressed COX-1 and cPGES). Then, the blood was pre-incubated with Hyp for 10 min, and prostanoid formation was initiated by addition of 20 μM AA. After 10 min at 37°C, generation of PGE 2 and 6-keto PGF 1α was determined in plasma as described above. PGE 2 formation was potently inhibited by Hyp in a concentration-dependent manner with an IC 50 of 0.25 μM reaching significance at 30 nM (Figure 2A), whereas formation of 6-keto PGF 1α was not significantly affected up to 30 μM (Figure 2B), again indicating selective suppression of PGE 2 biosynthesis by Hyp. Note that Hyp (3-30 μM) was more efficient in suppressing PGE 2 synthesis after long-term pre-stimulation with LPS (induction of COX-2 and mPGES-1, Figure 2A) than during short term stimulation ( Figure 1B) supporting that Hyp interferes with the inducible COX-2/mPGES-1 pathway.
In order to assess the effects of Hyp on the activity of COX-1 in whole blood (constitutively expressed in platelets), freshly withdrawn human blood (no incubation with LPS) was pre-incubated with Hyp for 10 min at 37°C, treated with Ca 2+ -ionophore and 100 μM AA for another 10 min, and the formation of 12-HHT (mainly derived from platelet COX-1) was determined. Hyp (up to 30 μM) failed to suppress 12-HHT formation under these assay conditions, whereas indomethacin and aspirin almost completely blocked it ( Figure 2C). In conclusion, the data suggest that Hyp potently and selectively blocks PGE 2 formation in whole blood without markedly affecting COX-1/2.

InhIBItIon of mPGes-1 ActIvIty By hyPerforIn
Because Hyp potently inhibited PGE 2 formation in whole blood from AA but failed to block COX-2, inhibition of PGE 2 synthesis downstream of COX-2 (i.e., interference with PGE 2 synthases) appeared reasonable. mPGES-1 is the major PGE 2 synthase under pathological conditions related to inflammation and cancer (Samuelsson et al., 2007), and its expression is strongly increased in blood upon LPS treatment, primarily contributing to PGE 2 formation in blood (Mosca et al., 2007). To assess the effects of Hyp on mPGES-1 activity, micro-

FigurE 2 | Effects of hyperforin on arachidonic acid-induced prostanoid formation in human whole blood. (A,B)
Heparinized human whole blood was treated with 10 μg ml −1 LPS for 16 h at 37°C and 5% CO 2 , supplemented with TX synthase inhibitor CV4151 (1 μM), and pre-incubated with Hyp or vehicle (DMSO) for 10 min at 37°C. (A) Then, PGE 2 formation was initiated with 20 μM AA, and PGE 2 formed within 10 min was separated by RP-HPLC and quantified by ELISA. The 100% value corresponds to PGE 2 levels in the range of 18-31 ng ml −1 in the individual experiments, respectively. (B) 6-keto PGF 1α was directly determined in the plasma by ELISA. The 100% value corresponds to 6-keto PGF 1α levels in the range of 4-7 ng ml −1 . Indomethacin (Indo, 50 μM) and celecoxib (Cele, 20 μM) were used as controls. (C) 12-HHT formation in whole blood. Heparinized whole blood was pre-incubated with Hyp or vehicle (DMSO) for 10 min, and AA (100 μM) and Ca 2+ -ionophore (30 μM) were added to induce 12-HHT product formation. After 10 min at 37°C, 12-HHT was extracted form the plasma by RP-18 solid phase extraction and analyzed by RP-HPLC as described. The 100% value corresponds to 1.5-2.4 μg ml −1 12-HHT. Indomethacin (Indo, 20 μM) and aspirin (ASA, 30 μM) were used as controls. Data are given as mean ± SE, n = 3-5, *p < 0.05, **p < 0.01 or ***p < 0.001 vs. vehicle (0.1% DMSO) control, ANOVA + Tukey HSD post hoc tests. cell line to study mPGES-1 functions), and also β-actin levels were slightly reduced at 10 μM ( Figure 5). Since previous studies showed that Hyp causes apoptosis of various cancer cell lines (Schempp et al., 2002;Hostanska et al., 2003;Dona et al., 2004), we reasoned that reduced expression of COX-2 and mPGES-1 in A549 cells by Hyp may be related to induction of apoptotic cell death. In fact, the decrease of COX-2 and mPGES-1 protein correlated with the induction of apoptosis as determined by the cleavage of PARP (Figure 5), and the cell viability was strongly reduced after 24 h (EC 50 ≈ 3 μM, not shown).

hyPerforIn suPPresses cArrAGeenAn-Induced mouse PAw edemA
The anti-inflammatory properties of Hyp were investigated in carrageenan-induced mouse paw edema in which PGE 2 (and also other mediators) critically contributes to the inflammatory response (Guay et al., 2004). Hyp (0.25, 1, 4 mg kg −1 ) was administered i.p. 30 min prior to injection of carrageenan into the mouse paw. The increase in paw volume was time-dependently assessed and reached a maximum at 4-h post-carrageenan treatment ( Figure  6A). In fact, in mice treated with 0.25, 1, and 4 mg kg −1 of Hyp, the peak of the response to carrageenan at 4 h was reduced by 35, 63, and 78%, respectively, whereas indomethacin (5 mg kg −1 ), used as reference, caused 57% inhibition. Moreover, comparison of the areas under the curves of each group, between 2 and 6 h after carrageenan injection, yielded similar degrees of inhibition confirming a more potent effect of Hyp (ED 50 = 1 mg kg −1 ) over indomethacin (ED 50 = 5 mg kg −1 ; Figure 6B).

hyPerforIn suPPresses cArrAGeenAn-Induced PleurIsy In rAts
Hyperforin has previously been shown to suppress carrageenaninduced pleurisy in rats by decreasing pleural LTB 4 levels (Feisst et al., 2009). Here, we further assessed the formation of PGE 2 during pleurisy in order to get some information about its ability to inhibit PGE 2 synthesis in vivo. Injection of carrageenan into the pleural cavity of rats (DMSO 4% group) elicited an acute inflammatory response characterized by the accumulation of fluid that contained large numbers of inflammatory cells ( Table 1). As observed in carrageenan-induced paw edema, Hyp (4 mg kg −1 i.p., 30 min before carrageenan) significantly inhibited the inflammatory response 4 h after carrageenan injection as demonstrated by the significant attenuation of exudate formation (64%) and cell infiltration (50%). Indomethacin (5 mg kg −1 ) also reduced exudate formation and cell infiltration (75 and 65%, respectively), and its anti-inflammatory action is not significantly different from Hyp (Table 1). In comparison with the corresponding exudates from DMSO-treated rats, exudates of Hyp-treated animals exhibited significantly decreased PGE 2 levels, and indomethacin almost completely suppressed PGE 2 formation as expected ( Table 1).

dIscussIon
Systematic investigations of Hyp's pharmacological action within the PGE 2 biosynthetic pathway revealed mPGES-1 as a putative molecular target of Hyp. Thus, Hyp potently and concentrationdependently inhibited the enzymatic conversion of PGH 2 to PGE 2 catalyzed by mPGES-1 in a defined cell-free assay, diminished the formation of PGE 2 in human whole blood without affecting the somal preparations of IL-1β-stimulated A549 cells, a rich source of mPGES-1 (Jakobsson et al., 1999), were used. Microsomes were pre-incubated with Hyp for 15 min, and then, PGE 2 formation was initiated by addition of 20 μM PGH 2 as substrate for mPGES-1. MK-886, used as reference compound, concentration-dependently inhibited PGE 2 formation with an IC 50 of 2.1 μM (data not shown), which is in agreement with the literature (Koeberle et al., 2008). As shown in Figure 4A, Hyp concentration-dependently suppressed PGE 2 formation with an IC 50 of 1 μM, and at 10 μM, 85 ± 2% inhibition was evident. Decreasing the PGH 2 concentration to 1 μM did not significantly alter the potency of Hyp (data not shown). Interestingly, octahydro-hyperforin was equally effective, whereas the acylphloroglucinol core or the closely related polyprenylated acylphloroglucinol humulone ( Figure 4B) failed to significantly inhibit PGE 2 formation up to 10 μM ( Figure 4A). These data suggest that defined structural arrangements are required for Hyp's inhibitory effect on mPGES-1. Finally, also an ethanolic extract (60% ethanol, v v −1 ) of St. John's wort proved to be efficient in suppressing PGE 2 formation with an ED 50 = 4 μg ml −1 (Figure 4C).
To investigate whether Hyp inhibits PGE 2 synthesis in a reversible manner, microsomal preparations of A549 cells were pre-incubated with Hyp and subjected to wash-out experiments. Hyp at 0.3 μM failed to efficiently block PGE 2 synthesis, whereas PGE 2 formation was efficiently blocked at 3 μM ( Figure 4D). mPGES-1 activity was restored by 10-fold dilution of the sample containing 3 μM Hyp ( Figure 4D) implying a reversible mode of inhibition.

effects of hyPerforIn on the eXPressIon of coX-2 And mPGes-1 And InductIon of APoPtotIc cell deAth
We investigated whether Hyp might suppress PGE 2 formation by interfering with COX-2 or mPGES-1 expression during prolonged incubation times (24 h). In fact, Hyp concentration-dependently inhibited the induction of COX-2 and mPGES-1 with an EC 50 of 1-3 μM in IL-1β-stimulated A549 cells (a model lung carcinoma were added to a COX reaction mix containing 5 mM glutathione. The COX enzymes were pre-incubated with the test compounds for 5 min, and then, the reaction was started with 5 μM (COX-1) or 2 μM (COX-2) AA. After 5 min at 37°C, the formation of 12-HHT was determined by RP-HPLC as described.

5-LO forms the basis of the anti-inflammatory effectiveness of Hyp and St. John's wort observed in vitro and in vivo
St. John's wort is frequently used for the treatment of mild and moderate depressions but also to intervene with inflammatory disorders, peptic ulcers, and skin wounds (Medina et al., 2006). Although the anti-depressive, anti-inflammatory, and anti-carcinogenic effects of St. John's wort extracts could be ascribed to Hyp (Muller, 2003;Medina et al., 2006), the underlying molecular mechanisms and molecular targets of Hyp are still elusive. Postulated anti-inflammatory mechanisms include down-regulation of the expression of the chemokine receptor CXCR3 on activated T cells (Cabrelle et al., 2008), down-modulation of matrix metalloproteinase 9, seemingly due to inhibition of leukocyte elastase, and reduced expression of the adhesion molecule CD11b in neutrophils synthesis of other COX-derived prostanoids (i.e., 6-keto PGF 1α , TXB 2 , and 12-HHT), and reduced PGE 2 levels in the pleurisy model in vivo. COX-2, phospholipases, or LPS-signaling components as possible point of attack were excluded based on cell-free and cellular assays of COX-2 activity and by experiments applying exogenous AA as substrate for COX enzymes. Discrepancies were observed in the potency of Hyp to inhibit COX-1 activity between whole blood ( Figure 2C) and cell-free assays (Figure 3A) or platelet COX-1 activity assays (Feisst and Werz, 2004) which apparently do not solely depend on plasma protein binding and are not readily understood. The pharmacological relevance of the inhibition of mPGES-1 and, as previously reported, 5-LO (Feisst and Werz, 2004) is supported by the beneficial effect of Hyp during eicosanoid (mainly PGE 2 )-driven acute inflammation in mouse paw edema and rat pleurisy. We suggest that the dual interference with mPGES-1 and Microsomal preparations of IL-1β-stimulated A549 cells were pre-incubated with 3 μM inhibitor for 15 min at 4°C. An aliquot was diluted 10-fold to obtain an inhibitor concentration of 0.3 μM. For comparison, microsomal preparations were pre-incubated for 15 min with 0.3 μM Hyp or with vehicle (DMSO), and then, 20 μM PGH 2 was added (no dilution). Then, all samples were incubated for 1 min on ice, and PGE 2 formation was analyzed as described by RP-HPLC. Data are given as mean ± SE, n = 3-4, **p < 0.01 vs. vehicle (0.1% DMSO) control, ANOVA + Tukey HSD post hoc tests. characterized based on cell-free assays and assays based on isolated cells, and thus, the pharmacological relevance of these postulates is unclear. These assays lack the influence of a physiologically relevant environment as compared to studies using whole blood or tissues, and important factors (plasma protein binding, cell-cell interactions, etc.) that may potentially modulate the efficacy are therefore neglected. Moreover, the effective concentrations of Hyp for intervention with certain targets or mechanisms are often substantially higher (>1 μM) than Hyp levels reached in vivo after oral administration of St. John's wort preparations. Thus, daily oral intake of 3 × 300 mg hypericum extract by human volunteers led to Hyp steady-state concentrations of 0.28 μM (Biber et al., 1998). In light of these facts, suppression of PGE 2 formation by Hyp was evident in human whole blood with significant suppressive effects at 0.03-1 μM, congruent with the ability of Hyp to directly interfere with mPGES-1 activity in the cell-free assay (IC 50 = 1 μM).
Hyperforin almost completely blocked mPGES-1 activity in the cell-free assay, but PGE 2 formation in LPS-stimulated whole blood was only partially inhibited (residual PGE 2 formation approx. 50% of vehicle control) as observed for mPGES-1 inhibitors such as MK-886 (this study and Koeberle et al., 2008), MD-52 (this study and Koeberle et al., 2009a), and others (Koeberle and Werz, 2009). In contrast, COX inhibitors (i.e., indomethacin or celecoxib) were markedly more effective to suppress PGE 2 formation in whole blood. Possibly, constitutively expressed homeostatic PGE synthases contribute to PGE 2 synthesis under these conditions and are not inhibited by Hyp. In fact, when whole blood was first pre-incubated with LPS to induce COX-2-and mPGES-1-expression (in contrast to the expression of constitutively expressed COX-1 or PGE 2 synthases like cPGES and mPGES-2, Murakami and Kudo, 2006), Hyp showed an increased efficiency to reduce PGE 2 formation. The contribution of PGE 2 synthases others than mPGES-1 may also explain the incomplete suppression of PGE 2 formation in the pleurisy model by Hyp as compared to indomethacin. Future studies will address the dissection of the different enzymatic pathways leading to PGE 2 biosynthesis in cellular assays and focus on the respective interference with Hyp.
The anti-inflammatory efficiency of Hyp in vivo was confirmed in carrageenan-induced rat pleurisy and further demonstrated for carrageenan-induced mouse paw edema. During paw edema formation, PGE 2 levels were significantly elevated (Harada et al., 1982;Guay et al., 2004), and COX inhibitors were shown to prevent the inflammatory response (Gemmell et al., 1979). Also in the pleurisy model, PGE 2 essentially contributes to the inflammatory (Dell'Aica et al., 2007), as well as suppression of Ca 2+ mobilization, leukocyte elastase release, and formation of reactive oxygen species in neutrophils (Feisst and Werz, 2004). In addition, we recently identified Hyp to inhibit 5-LO and COX-1 in pro-inflammatory eicosanoid biosynthesis in vitro (Albert et al., 2002) thereby exhibiting a unique molecular interference of 5-LO at its C2-like domain (Feisst et al., 2009). Nevertheless, most of these postulated antiinflammatory mechanisms have been essentially identified and FigurE 5 | Effects of hyperforin on apoptosis and the expression of COX-2 and mPgES-1. A549 cells, 60% confluent, were incubated with 2 ng ml −1 interleukin-1β together with vehicle (DMSO) or Hyp in cell culture medium containing 2% (v v −1 ) fetal calf serum. Cells were harvested after 24 h, total cell lysates were prepared and analyzed for the induction of COX-2 (72 kDa), mPGES-1 (16 kDa) and cleaved PARP (89 kDa) using SDS-PAGE and Western blotting. β-Actin (45 kDa) was used as loading control. Data are representatives of two to three independent experiments.
we could demonstrate that also human A549 lung carcinoma cells, constitutively expressing mPGES-1 (Jakobsson et al., 1999), undergo apoptosis in response to Hyp (EC 50 ≈ 3 μM) as monitored by the cleavage of PARP. It is tempting to speculate whether a functional correlation between the inhibition of PGE 2 formation and the induction of apoptosis by Hyp exists. Interestingly, also major depression represents an inflammatory process (Leonard, 2007), where PGE 2 levels are significantly elevated, and suppression of COX-2 by celecoxib showed clinical efficacy (Muller et al., 2006). A recent meta-analysis suggests significant effectiveness of St. John's wort in major depression (Linde et al., 2008) but whether inhibition of mPGES-1 contributes to the antidepressant activity of Hyp cannot be answered so far.
In conclusion, Hyp potently inhibits cellular and cell-free PGE 2 formation via interference with mPGES-1 at physiologically achievable plasma concentrations and shows anti-inflammatory effectiveness in vivo. Hyp also suppresses leukotriene formation by inhibition of 5-LO (Albert et al., 2002), which is also involved in inflammation and carcinogenesis (Werz and Steinhilber, 2006) like mPGES-1 (Murakami and Kudo, 2006). Dual inhibition of mPGES-1 and 5-LO might therefore provide a molecular basis for Hyp's anti-inflammatory and anti-carcinogenic properties. Such a pharmacological profile would be advantageous, particularly in view of the emerging link between chronic inflammation and cancer, where mPGES-1 and 5-LO may act as potential players. response following carrageenan stimulation (Harada et al., 1996;Kawamura et al., 2000). Hyp efficiently suppressed paw edema and was even more potent in this respect than indomethacin. Similarly, Hyp impaired PGE 2 levels in the exudates of rats during pleurisy and markedly attenuated the concomitant exudate formation and inflammatory cell infiltration. However, our data cannot exclude that inhibition of 5-LO (as previously reported in cell-free and cellular studies and for carrageenan-induced rat pleurisy; Albert et al., 2002;Feisst et al., 2009) contributes to the suppression of PGE 2 formation by reducing leukocyte recruitment. Taken together, both mPGES-1 and 5-LO seem to be relevant targets underlying the therapeutic efficiency of Hyp in eicosanoid-related disorders.
Besides inflammation, excessive PGE 2 formation is also associated with tumorigenesis, particularly of colon, breast, prostate, and lung carcinoma. Pharmacological intervention with PGE 2 formation (COX-2 selective inhibitors) has shown a chemopreventive potential (Rao and Reddy, 2004), and in vitro studies suggest induction of apoptosis as critical step (Jana, 2008). mPGES-1 is overexpressed in various cancers such as non-small cell lung cancer, invasive breast cancer, colorectal cancer, and gastric cancer (Samuelsson et al., 2007). Hyp induced apoptosis of various cancer cells such as mammary carcinoma, squamous cell carcinoma, malignant melanoma, and lymphoma cells (Schempp et al., 2002;Hostanska et al., 2003;Dona et al., 2004;Quiney et al., 2006), and