Conceived and designed the experiments: ARQ MAM. Performed the experiments: BMP. Analyzed the data: LV EB ARQ MAM. Contributed reagents/materials/analysis tools: LV EB. Wrote the paper: LV MAM.
EB is affilitated to Indena S.p.A. (Milan, Italy). Dicyclohexylammonium hyperforinate (hyperforin-DCHA), a stable form of hyperforin (compound 1), was provided by Indena S.p.A. (Milan, Italy). However, Indena S.p.A. was not a funder for this study and had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
We have previously shown that hyperforin, a phloroglucinol derivative found in St. John's wort, behaves as a potent anti-angiogenic compound. To identify the reactive group(s) mainly involved in this anti-angiogenic effect, we have investigated the anti-angiogenic properties of a series of stable derivatives obtained by oxidative modification of the natural product. In addition, in the present work we have studied the role of the four carbonyl groups present in hyperforin by investigating the potential of some other chemically stable derivatives.
The experimental procedures included the analysis of the effects of treatment of endothelial cells with these compounds in cell growth, cell viability, cell migration and zymographic assays, as well as the tube formation assay on Matrigel. Our study with hyperforin and eight derivatives shows that the enolized β-dicarbonyl system contained in the structure of hyperforin has a dominant role in its antiangiogenic activity. On the other hand, two of the tested hyperforin derivatives, namely, tetrahydrohyperforin and octahydrohyperforin, behave as potent inhibitors of angiogenesis. Additional characterization of these compounds included a cell specificity study of their effects on cell growth, as well as the
These observations could be useful for the rational design and chemical synthesis of more effective hyperforin derivatives as anti-angiogenic drugs. Altogether, the results indicate that octahydrohyperforin is a more specific and slightly more potent antiangiogenic compound than hyperforin.
St. John's wort (
Angiogenesis, the generation of new blood vessels from the existing vascular bed, has been described as one of the hallmarks of cancer, playing essential roles in tumor growth, invasion and metastasis
We have previously shown that hyperforin is able to inhibit angiogenesis in an
Hyperforin (
To overcome these issues, we have investigated the anti-angiogenic properties of a series of stable derivatives obtained by oxidative modification of the natural product. Our results throw light on the role of the enolized β-dicarbonyl system contained in the structure of hyperforin and identify two new promising antiangiogenic compounds, one of them even more potent than hyperforin.
Angiogenesis involves local proliferation of endothelial cells. We investigated the ability of hyperforin derivatives to inhibit the growth of bovine aorta endothelial cells (BAEC).
Compound | IC50 (µM) |
1 (hyperforin DCHA) | 2.1±0.7 |
2 | 12.4±1.5 |
3 | 12.4±3.1 |
4 | 4.5±2.0 |
5 | 84.4±12.7 |
6 | 5.3±1.7 |
7 | 8.0±2.0 |
8 | 1.7±0.1 |
IC50 values were calculated from dose-response curves as the concentration of compound yielding a 50% of control cell survival. They are expressed as means±S.D. of three different experiments with quadruplicate samples in each.
*Mean values are significantly higher than that of hyperforin (p<0.05, according to a Student's paired sample test).
Cell migration is another key step of angiogenesis. The wound assay is frequently used to assess the effects of tested compounds on the migratory potential of adherent cells. As previously described
Confluent monolayers of BAEC were wounded and a wound assay was carried out in the absence or presence of 10 µM of the tested compounds as described in
Angiogenesis involves the acquisition by endothelial cells of the capability to degrade the basement membrane and to remodel the extracellular matrix. Gelatin zymography of conditioned media and cell extracts of BAEC, untreated and treated for 24 h with hyperforin derivatives at concentrations in the range of their respective IC50 values in the MTT assay shows that only hyperforin and compound (
BAEC cells were treated in the presence of hyperforin derivatives at concentrations in the range of their respective IC50 values in the MTT assay for 24 h. Afterwards, conditioned media (for determination of secretion) and cell extracts (for determination of production) were normalized for equal cell density and used for gelatin zymography as indicated in
BAEC cells were treated in the presence of hyperforin derivatives at concentrations in the range of their respective IC50 values in the MTT assay for 24 h. Afterwards, conditioned media were normalized for equal cell density and used for the detection of urokinase by plasminogen zymography as indicated in
The final event during angiogenesis is the organization of endothelial cells in a three-dimensional network of tubes. In vitro, endothelial cells plated on Matrigel align themselves forming tubule-like structures.
Treatments with 0.5 µM hyperforin (1) and compound (8) were carried out as described in
Compound | MIC (µM) |
1 (hyperforin DCHA) | 0.5 |
2 | 10.0 |
3 | 5.0 |
4 | 5.0 |
5 | 100.0 |
6 | 10.0 |
7 | 25.0 |
8 | 0.5 |
Minimal inhibitory concentrations (MIC) were those inducing a clear inhibitory effect on the assay of tubule-like structure formation on Matrigel after 7 h of incubation. Each concentration was tested in duplicate, and two different observers evaluated the inhibition of tube formation.
Up to this moment, the results obtained altogether showed that only compound (
MDA-MB231 cells | NIH-3T3 cells | |
Compound | IC50 (µM) | IC50 (µM) |
1 (hyperforin DCHA) | 5±0 | 15±0 |
8 (tetrahydrohyperforin) | 2±0 | 13±3 |
9 (octahydrohyperforin) | 9±1 |
50±25 |
IC50 values were calculated from dose-response curves as the concentration of compound yielding a 50% of control cell survival. They are expressed as means±S.D. of two different experiments with quadruplicate samples in each.
*Mean values are significantly higher than that of hyperforin (p<0.05, according to a Student's paired sample test).
We have previously shown that hyperforin is a potent multi-target antiangiogenic compound
Hyperforin instability is due to the contemporary presence of fastly reacting functional groups: an enolized β-diketone moiety, apparently present in solution as 7-hydroxy, 9-keto tautomer due to the formation of a hydrogen bonding between the ketone in position 1 and the 7-hydroxy group, and the close proximity of this latter to the double bond of the 6- prenyl group. In addition, carbon 8 is strongly nucleophilic, and easily oxidized. Both these characteristics induce a fast reactivity toward oxidizing agents, including light, and lead to unexpected derivatives, some of which also accumulate in the extracts, like compounds (
Compounds (
The role of the four carbonyl groups, functionalities that could be involved in hydrogen bondings with enzymes active sites, was also investigated. The reaction of hyperforin with different reducing agents produced compounds (
The most relevant activities (equal or slightly more potent than those exhibited by hyperforin-DCHA) were observed on compound (
Altogether, the results discussed above indicate that only compound (
In conclusion, we can assert that the enolized β-dicarbonyl system is peculiar for the biological activity of hyperforin as an anti-angiogenic compound, whichever tautomer is present in solution, since the products devoid of this functionality are inactive or less active. Apparently the C1 and C10 carbonyl groups and the prenyl double bonds are not essential to maintain the activity, as shown by the behavior of compounds (
Dicyclohexylammonium hyperforinate (hyperforin-DCHA), a stable form of hyperforin (compound
Cell culture media were purchased from Gibco (Grand Island, NY, USA) and Cambrex (Walkersville, MD, USA). Fetal bovine serum (FBS) was a product of Harlan-Seralab (Belton, U.K.). Matrigel was purchased from Becton Dickinson (Bedford, MA, USA), and Calcein-AM was from Molecular Probes (Eugene, OR, USA). Supplements and other chemicals not listed in this section were obtained from Sigma-Aldrich. Plasticware for cell culture was supplied by NUNC (Roskilde, Denmark). Bovine aortic archs were isolated from calfs immediately after their sacrifice at the local slaughterhouse Famadesa (Málaga), transported to the lab immersed in PBS containing penicillin-streptomycin and amphotericin at standard cell culture concentrations, and used immediately upon arrival for isolation of primary bovine aortic endothelial cells (BAEC) by a collagenase treatment and maintained as previously described
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma Chemical Co., St. Louis, MO) dye reduction assay in 96-well microplates was used. The assay is dependent on the reduction of MTT by mitochondrial dehydrogenases of viable cell to a blue formazan product, which can be measured spectrophotometrically. BAE cells -and, in the second phase of this experimental work, also MDA-MB231 and NIH-3T3 cells- (3×103 cells in a total volume of 100 µL of complete medium) were incubated in each well with 1∶1 serial dilutions of compounds to be tested, beginning with 0.1 mM of the compound and down to concentrations in the submicromolar range. After 3 days of incubation (37°C, 5% CO2 in a humid atmosphere), 10 µl of MTT (5 mg/ml in PBS) were added to each well and the plate was incubated for a further 4 h (37°C). The resulting formazan was dissolved in 150 µl of 0.04 N HCl/2-propanol and read at 550 nm. All determinations were carried out in quadriplicate. IC50 values were calculated as those concentrations of the tested compounds yielding a 50% cell survival.
In order to check the viability of endothelial cells after the treatment with hyperforin derivatives in the “tubulogenesis”, migration assay and zymographies, BAE cells were incubated in 96-well plate with the tested compounds in the same conditions used for the aforementioned assays (that means, higher cell densities and shorter incubation times than those employed in the cell growth assay). After the maximum incubation time for these assays (4–24 h), cell viability in comparison to untreated control cells was determined by the addition of MTT as described for cell growth assay.
Matrigel (50 µL of about 10.5 mg/mL) at 4°C was used to coat each well of a 96-well plate and allowed to polymerise at 37°C for a minimum of 30 min. 5×104 BAE cells were added with 200 µL of DMEM. Finally, different amounts of hyperforin derivatives were added and incubated at 37°C in a humidified chamber with 5% CO2. After 7 h incubation, cultures were observed (40x magnifications) and photographed with a NIKON inverted microscope DIAPHOT-TMD (NIKON Corp., Tokyo, Japan). Each concentration was tested in duplicate, and two different observers evaluated the inhibition of tube formation. Only those assays where no tubular structure could be observed were evaluated as positive in the inhibition of morphogenesis of endothelial cells on Matrigel.
The migratory activity of BAEC was assessed using a wounded migration assay. Confluent monolayers in 6-well plates were wounded with pipette tips following two perpendicular diameters, giving rise to two acellular 1 mm-wide lanes per well. After washing, cells were supplied with 1.5 mL complete medium in the absence (controls) or presence of 10 µM hyperforin derivatives. Wounded areas were photographed. After additional 4 h of incubation, plates were observed under microscope and photos were taken from the same areas as those recorded at zero time. Acellular surface was determined by image analysis in both controls and treated wells and normalized respect to their respective values at zero time.
To prepare conditioned media and cell lysates, BAE cells were grown in 6-well plates. When the cells were at 75% confluency, medium was aspirated, cells were washed twice with phosphate-buffered saline (PBS) and each well received 1.5 mL of DMEM/0.1% BSA containing 200 KIU of aprotinin/mL. Additionally, some wells received hyperforin derivatives at the concentrations mentioned in
Assays of urokinase-type plasminogen activator (uPA) activity in gel were carried out as follows. Aliquots of cell lysates normalized for equal cell numbers were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 4°C under non-reducing conditions, with 5% stacking gel and 10% resolving gel. Gels were washed for 10 min twice with 50 mM Tris/HCl, pH 7.4, supplemented with 2% Triton X-100 and twice with 50 mM Tris/HCl, pH 7.4 and laid over a substrate gel prepared with agar (0.8%), plasminogen (40 µg/mL) and skimmed milk (1.5% in PBS). Gels were incubated under a moist atmosphere overnight at 4°C and then incubated at 37°C. After 4–8 h, bands of proteolysis due to uPA activity were photographed under dark field.
The gelatinolytic activity of matrix metalloproteinase-2 (MMP-2) delivered to the conditioned media or present in cell lysates was detected in gelatinograms. Aliquots of conditioned media and cell lysates normalized for equal cell numbers were subjected to non-reducing SDS/PAGE as above but with gelatin (1 mg/mL) added to the 10% resolving gel. After electrophoresis, gels were washed twice with 50 mM Tris/HCl, pH 7.4, supplemented with 2% Triton X-100, and twice with 50 mM Tris/HCl, pH 7.4. Each wash with continuous shaking lasted 10 min. After the washes, the gels were incubated overnight at 37°C and immersed in a substrate buffer (50 mM Tris/HCl, pH 7.4, supplemented with 1% Triton X-100, 5 mM CaCl2, and 0.02% Na3N). In some experiments, hyperforin derivatives at the concentrations mentioned in results were added to the substrate buffer. Finally, the gels were stained with Commassie blue R-250 and the bands of gelatinase activity could be detected as non-stained bands in a dark, stained background.
C57BL/6 female mice were injected s.c. near the abdominal midline, via a 23-gauge needle with 300 mL of Matrigel (Beckton-Dickinson) containing basic fibroblast growth factor (bFGF; 0.5 µg/mL) and 10 nmol of the corresponding compound. Positive control mice received the same volume of Matrigel with bFGF mixed with the same amount of vehicle (DMSO). Negative control mice were injected with Matrigel containing the corresponding dose of PBS and DMSO. After injection, the Matrigel rapidly formed a single, solid gel plug. After 8 days, mice were sacrificed and plugs were removed. Plugs were processed for cryoprotection with increased concentrations of sucrose, embedded in OCT and frozen in liquid nitrogen. Sections of 10 mm thickness were collected on poly-L-lysinated slides and fixed in three stepts of acetone, acetone-chloroform (1∶1) and acetone, keeping the samples at −20° while fixing. Immunodetection of CD31 and DAPI staining were performed and random fields of the sections were photographed under fluorescence microscope. CD31 positive areas were quantified using ImageJ software and all data was expressed as means ± SD of duplicate plugs normalized to the positive control (100% vascularization).
Statistical significance was determined by the Student's paired sample test. Values of p <0.05 were considered to be significant.