Journal: International journal of cancer

Article Title: Hyperforin, a bio-active compound of St. John's Wort, is a new inhibitor of angiogenesis targeting several key steps of the process.

doi: 10.1002/ijc.21246

Figure Lengend Snippet: FIGURE 1 – Chemical structure of hyperforin.

Article Snippet: Highly purified hyperforin was supplied by ChromaDex (St. Ana, CA).

Techniques:

Journal: International journal of cancer

Article Title: Hyperforin, a bio-active compound of St. John's Wort, is a new inhibitor of angiogenesis targeting several key steps of the process.

doi: 10.1002/ijc.21246

Figure Lengend Snippet: FIGURE 2 – Hyperforin inhibits in vivo angiogenesis, as determined by the CAM assay. Experiments were carried out as described in Material and Methods. Methylcellulose discs containing the substance vehicle alone (control, a), 10 (b), 20 (c) and 30 (d) nmol hyperforin. Circles show the locations of the methyl cellulose discs. Arrows point to peripheral vessels avoiding the core of the treated area, asterisks indicate disorganization of pre-existing vessels and diamonds point to tissue inflammation.

Article Snippet: Highly purified hyperforin was supplied by ChromaDex (St. Ana, CA).

Techniques: In Vivo, Chick Chorioallantoic Membrane Assay, Control

Journal: International journal of cancer

Article Title: Hyperforin, a bio-active compound of St. John's Wort, is a new inhibitor of angiogenesis targeting several key steps of the process.

doi: 10.1002/ijc.21246

Figure Lengend Snippet: FIGURE 3 – Hyperforin inhibits the growth of BAE and non-endo- thelial cells. Experiments were carried out as described in Mate- rial and Methods. Survival curves for BAE (a), MDA-MB231 (b) and NIH-3T3 (c) cells. Data are means 6 SD of 3 independent experiments, each one with quad- ruplicate samples for each tested hyperforin concentration.

Article Snippet: Highly purified hyperforin was supplied by ChromaDex (St. Ana, CA).

Techniques: Concentration Assay

Journal: International journal of cancer

Article Title: Hyperforin, a bio-active compound of St. John's Wort, is a new inhibitor of angiogenesis targeting several key steps of the process.

doi: 10.1002/ijc.21246

Figure Lengend Snippet: FIGURE 4 – Hyperforin inhibits endothelial cell tubulogenesis in vitro. BAE cells seeded on Matrigel formed tubes (a), whereas tubulo- genesis was inhibited in the presence of 2.5 lM of hyperforin (b). Cells were photographed 7 hr after seeding under an inverted micro- scope (340).

Article Snippet: Highly purified hyperforin was supplied by ChromaDex (St. Ana, CA).

Techniques: In Vitro

Journal: International journal of cancer

Article Title: Hyperforin, a bio-active compound of St. John's Wort, is a new inhibitor of angiogenesis targeting several key steps of the process.

doi: 10.1002/ijc.21246

Figure Lengend Snippet: FIGURE 5 – Hyperforin inhibits the production and secretion of endothelial cell matrix metalloproteinase-2 but not its activity. (a) Conditioned media (c.m.) and cell extracts (c.e.) from BAE cells treated for 24 hr with 10 lM hyperforin and non-treated cells (control, C) were normalized for equal cell density and used for gelatin zymog- raphy as indicated in Material and Methods. (b) Conditioned media from non-treated cells were used for gelatin zymography, revealed with substrate buffer containing 10 lM hyperforin or not (control, C). Typical results are shown.

Article Snippet: Highly purified hyperforin was supplied by ChromaDex (St. Ana, CA).

Techniques: Activity Assay, Control, Zymography

Journal: International journal of cancer

Article Title: Hyperforin, a bio-active compound of St. John's Wort, is a new inhibitor of angiogenesis targeting several key steps of the process.

doi: 10.1002/ijc.21246

Figure Lengend Snippet: FIGURE 6 – Hyperforin inhibits the production and secretion of endothelial cell urokinase. Conditioned media from BAE cells treated for 24 hr with 1 or 10 lM hyperforin and non treated cells (control, C) were normalized for equal cell density and used for zymographic detection of urokinase as indicated in Material and Methods.

Article Snippet: Highly purified hyperforin was supplied by ChromaDex (St. Ana, CA).

Techniques: Control

Journal: International journal of cancer

Article Title: Hyperforin, a bio-active compound of St. John's Wort, is a new inhibitor of angiogenesis targeting several key steps of the process.

doi: 10.1002/ijc.21246

Figure Lengend Snippet: FIGURE 7 – Hyperforin slightly inhibits the migration of BAE cells. Confluent monolayers were wounded and a wound assay was carried out as described in Material and Methods. Confluent monolayers were wounded and washed. Fresh culture medium was added, either in the absence (control, C) or presence of 10 lM hyperforin. Photographs were taken at the beginning of the assay and after 4 hr of incubation.

Article Snippet: Highly purified hyperforin was supplied by ChromaDex (St. Ana, CA).

Techniques: Migration, Control, Incubation

Journal: International journal of cancer

Article Title: Hyperforin, a bio-active compound of St. John's Wort, is a new inhibitor of angiogenesis targeting several key steps of the process.

doi: 10.1002/ijc.21246

Figure Lengend Snippet: FIGURE 8 – Hyperforin inhibits BAE cell invasion. The invasion assay was carried out as described in Material and Methods. Relative velocities of invasion were determined for both control and treated (10 lM hyperforin) cells. Data are given in arbitrary units and are means 6 SD of triplicate samples in 2 independent experiments.

Article Snippet: Highly purified hyperforin was supplied by ChromaDex (St. Ana, CA).

Techniques: Invasion Assay, Control

Journal: Molecular Psychiatry

Article Title: Analysis of hyperforin (St. John’s wort) action at TRPC6 channel leads to the development of a new class of antidepressant drugs

doi: 10.1038/s41380-022-01804-3

Figure Lengend Snippet: A–C Whole-cell current-clamp recording of granule cells show effects of hyperforin (3 µM) on evoked APs ( A , C ) and on membrane potential ( B ). Dashed lines indicate −70 mV, depolarizing ramp was 0–70 pA for WT granule cell and 0–100 pA for TRPC6 KO cell. The hyperforin-induced biphasic response in wt slices was preserved after blocking fast synaptic transmission with kynurenic acid (KA) and picrotoxin (PTX) ( C ). D , E Voltage-clamp recordings (held at −70 mV) illustrate loss of hyperforin-induced initial inward current in neurons from TRPC6 KO mice ( D ). The remaining outward current involves K + channels, as indicated by the loss of current with CsGlu-filled pipette ( E ). *** p < 0.001.

Article Snippet: Hyperforin with different concentration was added freshly to the mixture and the spectra were recorded using a Fluoromax-4 (Horiba Scientific) fluorimeter.

Techniques: Blocking Assay, Transmission Assay, Transferring

Journal: Molecular Psychiatry

Article Title: Analysis of hyperforin (St. John’s wort) action at TRPC6 channel leads to the development of a new class of antidepressant drugs

doi: 10.1038/s41380-022-01804-3

Figure Lengend Snippet: A The topology model of human TRPC6 (hTRPC6) shows α-helices in cylinders and dashed lines describe region with not sufficient density in CryoEM structure PDB: 6uz8. Potential hyperforin bindings site is marked with a red star. B Sketch demonstrating that amino acids LLKL were mutated in hTRPC6 into the respective amino acids IMRI of hTRPC3 to block hyperforin-mediated TRPC6 activation. In a second step, the amino acids IMRI in hTRPC3 were mutated into the corresponding amino acids LLKL of hTRPC3 to induce a hyperforin-sensitive hTRPC3 channel. hTRPC6 (black), TRPC6mut = IMRI TRPC6mut (red), hTRPC3 (gray), TRPC3mut = LLKL TRPC3mut . C Single-cell Ca 2+ imaging was conducted in HEK293 cells transiently expressing pcDNA3.1 plasmid vector with DNA coding only for eYFP (ctl, white), hTRPC6 (black), hTRPC6mut (red), hTRPC3 (gray), or hTRPC3mut (blue) all expressed as C-terminal eYFP fusion proteins. Cells were stimulated with the solvent DMSO (0.1%), OAG (100 µM) or hyperforin (10 µM) and intracellular Ca 2+ alterations were detected using fura-2 AM ( n = 7–9 ± SEM, cells were selected according to their eYFP fluorescence and their OAG sensitivity; Statistical significance was analyzed by ANOVA with post hoc Dunnett’s test *** p < 0.001) C Whole-cell currents were recorded from HEK293 cells transiently expressing eYFP (ctl, white), hTRPC6 (black), hTRPC6mut (red), hTRPC3 (gray), or hTRPC3mut (blue) all expressed as C-terminal eYFP fusion proteins. Mean current density are depicted at +100 and −100 mV after application of hyperforin (10 µM). Currents were normalized to the basic currents before compound application were subtracted ( n = 3 ± SEM¸ Statistical significance was analyzed by ANOVA with post hoc Dunnett’s test *** p < 0.001). D Representative time traces were monitored in HEK293 ctl cells (dashed line), hTRPC6-expressing HEK293 cells (black) or hTRPC6mut (red) stimulated with OAG (100 µM) 60 s after starting the experiment and after 300 s hyperforin (10 µM) was applied. E Representative time traces were monitored in HEK293 ctl cells (dashed line), hTRPC3-expressing HEK293 cells (gray) or hTRPC3mut (blue) stimulated with OAG (100 µM) 60 s after starting the experiment and after 300 s hyperforin (10 µM) was applied. F Whole-cell currents recorded from HEK293 ctl cells (dashed line), hTRPC6-expressing HEK293 cells (black) or hTRPC6mut (red). Application of hyperforin (10 µM) resulted in an increase in outward and inward current in hTRPC6 expressing cells. This effect is lost in TRPC6mut expressing cells. G Whole-cell currents recorded from HEK293 ctl cells (dashed line), hTRPC3-expressing HEK293 cells (gray) or hTRPC3mut (blue). Application of hyperforin (10 µM) showed no effect in ctl and hTRPC3 expressing cells but resulted in an increase in outward and inward current in hTRPC3mut expressing cells. H The hyperforin binding site LLKL at human hTRPC6 differs in the last amino acid from rat and mouse TRPC6 LLKF. To test if this amino acid interferes with hyperforin binding to TRPC6, we compared hTRPC6 with hTRPC6 LLKF. Single-cell calcium imaging was conducted in HEK239 cells transiently expressing hTRPC6 or hTRPC6 LLKF. Cells were stimulated with hyperforin (10 µM) and Fura-2-AM 340/380 nm ratio changes were analyzed and afterward converted into intracellular Ca 2+ in nM. No significant differences were observed ( n = 3 ± SEM, cells were selected according to their eYFP fluorescence; statistical significance was calculated using unpaired t -test, not significant 0.0576).

Article Snippet: Hyperforin with different concentration was added freshly to the mixture and the spectra were recorded using a Fluoromax-4 (Horiba Scientific) fluorimeter.

Techniques: Blocking Assay, Activation Assay, Imaging, Expressing, Plasmid Preparation, Fluorescence, Binding Assay

Journal: Molecular Psychiatry

Article Title: Analysis of hyperforin (St. John’s wort) action at TRPC6 channel leads to the development of a new class of antidepressant drugs

doi: 10.1038/s41380-022-01804-3

Figure Lengend Snippet: CD spectra of wild-type TRPC6 peptides in the absence and presence of hyperforin ( A ). Laurdan fluorescence measurement to monitor membrane fluidity changes caused by hyperforin (white bars), TRPC6 peptide (black bars), and TRPC6mut peptide (red bars) ( B ). Amino acid sequences from TRPC6 and TRPC6mut are shown in ( C ). Differences between the two sequences are underlined. Tryptophan fluorescence using residue W782 as a reporter of TRPC6 ( D ) and TRPC6mut titrated with hyperforin ( E ). Fluorescence maxima (vertical black line in D and E ) were blotted against hyperforin concentration and normalized against fluorescence maxima without hyperforin ( F ).

Article Snippet: Hyperforin with different concentration was added freshly to the mixture and the spectra were recorded using a Fluoromax-4 (Horiba Scientific) fluorimeter.

Techniques: Fluorescence, Concentration Assay

Journal: Molecular Psychiatry

Article Title: Analysis of hyperforin (St. John’s wort) action at TRPC6 channel leads to the development of a new class of antidepressant drugs

doi: 10.1038/s41380-022-01804-3

Figure Lengend Snippet: Chemical structure of hyperforin ( A ) and Hyp13 ( B ). The phloroglucinol core structure is highlighted in red. ( C ) Concentration dependent effect of Hyp13 in HEK293 cells expressing hTRPC6 channels in whole cell patch clamp experminents. D Hyp13 also interacts to the LLKL binding motif at TRPC6. Single-cell Ca 2+ imaging was conducted in HEK293 cells transiently expressing pcDNA3.1 plasmid vector with DNA coding only for eYFP (ctl, white), hTRPC6 (black), hTRPC6mut (red), hTRPC3 (gray), or hTRPC3mut (blue) all expressed as C-terminal eYFP fusion proteins. Cells were stimulated with the hyperforin (10 µM) or Hyp13 (10 µM) and intracellular Ca 2+ alterations were detected using fura-2 AM ( n = 7–9 ± SEM, cells were selected according to their eYFP fluorescence and their OAG sensitivity). E Representative time traces were monitored in HEK293 ctl cells (dashed line), hTRPC6-expressing HEK293 cells (black) or hTRPC6mut (red) stimulated with OAG (100 µM) 60 s after starting the experiment and after 300 s hyperforin (10 µM) was applied. F Representative time traces were monitored in HEK293 ctl cells (dashed line), hTRPC3-expressing HEK293 cells (gray) or hTRPC3mut (blue) stimulated with OAG (100 µM) 60 s after starting the experiment and after 300 s hyperforin (10 µM) was applied. G Whole-cell currents recorded from HEK293 ctl cells (dashed line), hTRPC6-expressing HEK293 cells (black) or hTRPC6mut (red). Application of Hyp13 (10 µM) resulted in an increase in outward and inward current in hTRPC6 expressing cells. This effect is lost in TRPC6mut expressing cells. H Whole-cell currents recorded from HEK293 ctl cells (dashed line), hTRPC3-expressing HEK293 cells (gray) or hTRPC3mut (blue). Application of Hyp13 (10 µM) showed no effect in ctl and hTRPC3 expressing cells but resulted in an increase in outward and inward current in hTRPC3mut expressing cells.

Article Snippet: Hyperforin with different concentration was added freshly to the mixture and the spectra were recorded using a Fluoromax-4 (Horiba Scientific) fluorimeter.

Techniques: Concentration Assay, Expressing, Patch Clamp, Binding Assay, Imaging, Plasmid Preparation, Fluorescence

Journal: Molecular Psychiatry

Article Title: Analysis of hyperforin (St. John’s wort) action at TRPC6 channel leads to the development of a new class of antidepressant drugs

doi: 10.1038/s41380-022-01804-3

Figure Lengend Snippet: Induction of PXR activity by hyperforin and its derivate in HepG2 cells ( A ). HepG2 cells were co-transfected with plasmids expressing GAL4-responsive UAS-driven firefly luciferase, human PXR -LBD fused to GAL4-DBD, and Renilla luciferase. The transfected cells were exposed to 10 µM of positive controls rifampicin and SR12813 or different concentrations of hyperforin and its derivate. After 24 h, cell lysates were assayed for firefly and Renilla luciferase activity. Firefly luciferase activity was normalized against Renilla luciferase activity and fold induction relative to the solvent control (SC 0.5% DMSO) was calculated. Data are presented as means ± SD of three independent experiments performed with six replicates each. B Gene expression analysis of CYP3A4 . Differentiated HepaRG cells were exposed to hyperforin and its derivate as well 10 µM Rifampicin (PC) for 24 h. Total mRNA was isolated and transcribed into cDNA and subsequently mRNA expression of CYP3A4 was analyzed by real-time qPCR. For relative quantification, Ct values were normalized to reference genes ( ACTB and GAPDH ) according to the ΔΔCT method. Log2 fold changes of 2 −ΔΔCT values were calculated and mRNA levels were expressed in relation to the solvent control (SC 0.5% DMSO). Data are presented as means ± SD of two to three independent experiments.

Article Snippet: Hyperforin with different concentration was added freshly to the mixture and the spectra were recorded using a Fluoromax-4 (Horiba Scientific) fluorimeter.

Techniques: Activity Assay, Transfection, Expressing, Luciferase, Isolation

Journal:

Article Title: St. John's wort induces hepatic drug metabolism through activation of the pregnane X receptor

doi:

Figure Lengend Snippet: Hyperforin is a potent activator of PXR. (A) CV-1 cells were transfected with expression plasmids for human PXR and the (CYP3A1)2-tk-CAT reporter. Cells were treated with extracts prepared from three different commercial preparations of St. John's wort [extract 1, Nature's Way (9 μg/ml); extract 2, Nature's Plus (75 μg/ml); extract 3, Solaray (7 μg/ml)] or with 10 μM of the indicated pure compounds, except for hyperforin, which was tested at 1 μM. Hyperforin was toxic to cells at concentrations >1 μM. Cell extracts subsequently were assayed for CAT activity. Data represent the mean of assays performed in quadruplicate ± SE and are plotted as -fold activation relative to transfected cells treated with vehicle alone. (B) Chemical structure of hyperforin. (C) CV-1 cells were transfected as in A and treated with increasing concentrations of hyperforin (squares) or rifampicin (circles). Data points represent the mean of assays performed in triplicate.

Article Snippet: Chemicals and their sources are as follows: hyperforin (Apin Chemicals Limited, Abingdon, Oxon, U.K.); amentoflavone and isoquercitrin (Indofine Chemical Company, Belle Mead, NJ); hyperoside (Research Plus, Bayonne, NJ); kaempferol, luteolin, myricetin, quercetin, quercitrin, rifampicin, β-sitosterol (from soybeans), scopoletin, and umbelliferone (Sigma); rutin (Acros Organics, subsidiary of Fisher Scientific); hypericin and pseudohypericin (Calbiochem); and SR12813 was synthesized in house.

Techniques: Transfection, Expressing, Activity Assay, Activation Assay

Journal:

Article Title: St. John's wort induces hepatic drug metabolism through activation of the pregnane X receptor

doi:

Figure Lengend Snippet: Hyperforin binds to PXR with high affinity. Competition binding assays were performed with purified human PXR LBD and 10 nM of the high-affinity PXR radioligand [3H]SR12813 in the presence of the indicated concentrations of hyperforin (squares), SR12813 (circles), or umbelliferone (triangles). Each data point represents the mean of assays performed in triplicate. The Ki value of hyperforin was calculated to be 27 nM by nonlinear regression analysis.

Article Snippet: Chemicals and their sources are as follows: hyperforin (Apin Chemicals Limited, Abingdon, Oxon, U.K.); amentoflavone and isoquercitrin (Indofine Chemical Company, Belle Mead, NJ); hyperoside (Research Plus, Bayonne, NJ); kaempferol, luteolin, myricetin, quercetin, quercitrin, rifampicin, β-sitosterol (from soybeans), scopoletin, and umbelliferone (Sigma); rutin (Acros Organics, subsidiary of Fisher Scientific); hypericin and pseudohypericin (Calbiochem); and SR12813 was synthesized in house.

Techniques: Binding Assay, Purification

Journal:

Article Title: St. John's wort induces hepatic drug metabolism through activation of the pregnane X receptor

doi:

Figure Lengend Snippet: St. John's wort extracts and hyperforin induce CYP3A4 expression in human hepatocytes. Northern blot analysis was performed with total RNA (10 μg) prepared from primary cultures of human hepatocytes treated for 30 h with extracts prepared from three different commercial preparations of St. John's wort [extract 1, Nature's Way (9 μg/ml); extract 2, Nature's Plus (75 μg/ml); extract 3, Solaray (7 μg/ml)], 1 μM hyperforin, or vehicle alone (0.1% ethanol). The blot was probed sequentially with 32P-labeled fragments of CYP3A4 and β-actin.

Article Snippet: Chemicals and their sources are as follows: hyperforin (Apin Chemicals Limited, Abingdon, Oxon, U.K.); amentoflavone and isoquercitrin (Indofine Chemical Company, Belle Mead, NJ); hyperoside (Research Plus, Bayonne, NJ); kaempferol, luteolin, myricetin, quercetin, quercitrin, rifampicin, β-sitosterol (from soybeans), scopoletin, and umbelliferone (Sigma); rutin (Acros Organics, subsidiary of Fisher Scientific); hypericin and pseudohypericin (Calbiochem); and SR12813 was synthesized in house.

Techniques: Expressing, Northern Blot, Labeling