Inhibition of matrix metalloproteinase-2 secretion by chalcones from the twigs of Dorstenia barteri Bureau

Chalcones and their analogs were extracted from the twigs of Dorstenia barteri and investigated for their capacity to inhibit matrix metalloproteinase (MMP)-2 secretion from brain tumor-derived U87 glioblastoma cells. Among all tested compounds, potent inhibitory activities were recorded for chalcones 1 , 4 , 5


Introduction
It is well established that natural products constitute excellent sources of phytochemicals having a wide range of biological activities, including anticancer properties. 1 In the search for new antitumoral agents; several plant extracts have been investigated.This has opened new fields of investigations toward potential antitumor agents, some of which are already widely used in cancer chemotherapy. 2Flavonoids, a group of polyphenolic secondary metabolites present in a wide variety of plants, have been reported to display a large panel of biochemical properties, including antioxidant activity, inhibition of tyrosine kinases, cAMP phosphodiesterases, and induction of phase II metabolizing enzymes both in vivo and in vitro. 3These biological activities, have also been associated with their capacity to control cell growth or destroy pathogen organisms, such as fungi and viruses. 4,5Several prenylated (3-methylbut-2-enylated) flavonoids have shown interesting cytotoxic and/or antitumor properties, including flavones, 6 flavanones, 7 and chalcones. 7,8The cytotoxic activity of prenylated flavonoids have been reported and their structure-activity relationships discussed. 7,9However, from the compiled information on chalcones, it can be deduced that the presence of α,β-conjugated double bonds, the hydroxyl groups at C 2' and C 4' in ring A, and at C 4 of ring B, appear to be important for enhanced activity.Alternatively, little is known about the influence of substituents at other positions and their roles are not clear.Flavonoids, particularly 4-hydroxylonchocarpin (2) isolated from D. mannii, 10 are good chemopreventive molecules against ovarian cancer cell growth. 11Recently, isobavachalcone (1) and dorsmannin A (6) isolated from D. barteri Bureau 12 and D. mannii, 10 exhibited inhibitory effects on skin tumor promotion in an in vivo two-stage mouse skin carcinogenesis test. 13s part of a screening program on active ingredients isolated from Dorstenia species and other natural products having antitumor properties, the aim of the present investigation is to explore the potential inhibitory effect of chalcones isolated from the twigs of D. barteri var.multiradiata and of their semisynthetic analogs on matrix metalloproteinase (MMP)-2 secretion in the highly invasive human brain tumor-derived glioblastoma cell line U87.The inhibitory effects were compared to those of the phenolic compounds CHL and EGCg, both documented for their potential to inhibit MMP secretion. 14,15

Results and Discussion
Isolation, structure elucidation, and semisynthetic analogs The combined extract of CH 2 Cl 2 /MeOH (1:1) and MeOH of air-dried twigs of D. barteri Bureau var.multiradiata having inhibitory activities on MMP-2 secretion in glioblastoma cells was further fractionated by silica gel column chromatography, followed by size exclusion chromatography over Sephadex LH-20.The post-chlorophyll fractions were combined and subjected to silica gel column chromatography and preparative TLC successively to afford: isobavachalcone (1), 12 4-hydroxylonchocarpin (2), 10 kanzonol C (3), 16 paratocarpin C (4), 17 , stipulin (5), 6 , and dorsmannin A ( 6) 10 (Fig. 1).To further explore the structure-activity relationships of several modified chalcones about their capacity to inhibit MMP-2 secretion, kanzonol C (3) was converted into derivatives 7 and 8 under varied acid-catalyzed conditions 17 (Scheme 1).Hence, treatment of 3 in methanolic HCl at reflux provided a mixture of cyclized derivatives 7 (cycloglabrol) and 8 (isocycloglabrol; artoindonesianin J) in a ratio of 25% and 61%, respectively (Scheme 1).When boron trifluoridediethyl etherate was used as acid catalyst, 18 compound 8 19 was the only single isomer obtained in 83% yield (Scheme 1).Additionally, isobavachalcone 1 was transformed by hydrogenation into the dihydrochalcone derivative 9 20,21 in order to assess the importance of the double bonds on their biological activity (Scheme 2).The structures of the natural compounds (1-6) and their analogs (7-9) were determined on the basis of spectroscopic data (UV, IR, MS, 1 H NMR, and 13 C NMR).The known compounds 1-6, 8 were found identical to those previously described. 6,10,12,16,17,19  Compound 9 has been synthesized previously as a testosterone 5α-reductase inhibitor. 21here are no known studies on natural isobavachalcone 1 hydrogenation reported in the literature.The hydrogenated product 9 was isolated here for the first time via hemisynthesis by transformation of natural product 1 as a starting material.The structural identities of analogs 7, 8, and 9 were fully established by 1 H-1 H COSY and HMBC experiments (Fig. 2 and 3).
The genus Dorstenia (Moraceae) is represented by about 170 species worldwide. 22It contains several species commonly used in folk medicine for the treatment and/or management of an array of human disorders, including: arthritis, rheumatism, gout, stomach pain, cough and headache. 23To the best of our knowledge, no pharmacological studies have yet been made on this plant.While a previous phytochemical investigation of the same taxon afforded: 5,7,4'trihydroxy-8-prenylflavone, isobavachalcone (1), stipulin (5) and bichalcone, 12 only isobavachalcone (1) and stipulin (5) were isolated again from this species in our study, and this variation observed in the chemical constitution may potentially be accounted for by the climatic condition, the period and area of plant collection.

Inhibition of MMP-2 secretion in U87 glioblastoma cells
The natural chalcones (1-6) isolated from D. barteri Bureau extracts and their synthetic analogs (7-9) were assayed for their ability to inhibit MMP-2 secretion in U87 glioblastoma cells using a gelatin zymography assay.The extent of gelatin hydrolysis by the MMP-2 secreted into the conditioned media upon treatment with the compounds tested was compared to that of known reference naturally occurring MMP secretion inhibitors CHL and EGCg (Fig. 4A). 14,15While cells remained adherent, indicative of very low cytotoxic effects of the treatments, all the natural chalcones (1-6) isolated from D. barteri Bureau species had moderate (20%) to good (80%) MMP-2 secretion inhibition ranging from 0.025 µM to 250 µM (Fig. 4B).These effects showed comparable potency of some of the compounds tested (1, 4, 5, and 6) to that of CHL and EGCg (Fig. 4B).
Comparing with hemisynthetic derivatives (7-9), natural compounds 1, 4, 5, and 6 were found more active on MMP-2 secretion inhibition and the maximum inhibitory effects ranged from 60-80% at 250 µM.The inhibitory activity of chalcones 1 and 3 tends to decrease when the prenyl group was hydrogenated or cyclized into benzopyrans to provide the analogs (7-9) since their activity was less potent than that of the corresponding prenylated chalcone precursors.This fact suggests that the prenyl and the hydroxyl groups on chalcone A or B ring was required for this series of compounds to show potent inhibition against MMP-2 secretion.With regards to the effects of the α,β-unsaturated double bond of chalcone on activity, this conjugated system seems to enhance the activity of the chalcones.In fact, the hydrogenation of this double bond in compound 1 decreased the inhibition of MMP-2 secretion (Fig. 4B).Brain tumor development is associated with extensive invasion into the surrounding brain tissue, and this infiltrative phenotype hampers efficient therapeutic interventions.Since MMP-2 is upregulated in glioblastomas, where its expression directly correlates with the invasive character of brain tumor cells and where it assists in extracellular matrix degradation and cancer cell movement, 24,25 any elucidation of the anti-MMP secreting effects against brain tumor cells will help design and optimize current targeted therapeutic strategies. 25For instance, the MMP-2 secretion inhibitor green tea catechin EGCg has already been used to efficiently target the infiltrative and radioresistant phenotype of U87 glioblastoma cells. 26,27The present investigation demonstrates for the first time that prenylated chalcones isolated from the twigs of Dorstenia barteri Bureau appear to target MMP-2 secretion mechanisms from highly invasive brain tumorderived U87 glioblastoma cells.In fact, investigating the mechanisms involved in the inhibition of MMP-2 secretion will provide information that should allow antitumor evaluation of new synthetic derivatives of Dorstenia species.Table 1. 1 H (500 MHz) and 13 C NMR (125 MHz) spectral data, COSY, HMQC and important HMBC 2 J, 3 J-correlated carbons of kanzoflavanone (7)  Gelatin zymography.To assess the extent of MMP-2 secretion in the conditioned media, gelatin zymography was used as described previously. 15Briefly, serum-starved U87 glioblastoma cells were treated with the appropriate compounds (1-9) for 18 hrs at 37 o C and an aliquot (20 µl) of the culture medium was subjected to SDS-PAGE in gels containing 0.1 mg/ml gelatin.The gels were then incubated in 2.5%  1).Isocycloglabrol (8) (artoindonesianin J). 19 Yellow oil; ESIMS m/z (rel.int.): 393 [M+H] + (100), 305 (8), 261 (12), 217 (16), 173 (11); (calcd for C 25 H 28 O 4 , 392.48742); 1 H NMR spectral data (500 MHz, CDCl 3 ); 13 C (125 MHz, CDCl 3 ), COSY, HMQC and HMBC (Table 2).

Figure 4 .
Figure 4. Effects of compounds (1-9) and of the crude twig extract (CE) of Dorstenia barteri bureau on MMP-2 secretion from U87 glioblastoma cells.A) Representative gelatin zymography of media from U87 glioblastoma cells treated with increasing concentrations of compounds (1-9) and of crude extract.B) The bar graphs present the relative inhibitory effect of the compounds tested.The extent of gelatin hydrolysis was quantified by densitometry.Each value indicates the mean ± SD from three experiments.

Table 2 .
13 CDCl 3 1 H (500 MHz) and13C NMR (125 MHz) spectral data, COSY, HMQC and important HMBC 2 J, 3 J-correlated carbons of compound 8 in CDCl 3 Optical rotation was recorded on a Perkin-Elmer 241 polarimeter.UV spectra were measured on Varian Cary 1E spectrometer. IRspectra were recorded on a Nicolet Avatar 370 spectrometer with CCl 4 solution. NR spectra were obtained with a Bruker AMX-500 or on Varian Gemini-300 spectrometers.ESIMS were recorded on a Micromass Quattro LC mass spectrometer.Precoated silica gel plates (Merck, Kieselgel 60 F-254, 0.5 mm) were used for PTLC.Thin layer chromatography (TLC) was performed on silica gel F254 (Merck) precoated aluminum sheets and spots were visualized under UV and by spraying with molybdenum solution and heating.Cell culture.The U87 glioblastoma cell line was purchased from American Type Culture Collection and maintained in Eagle's Minimum essential medium (MEM) containing 10% (v/v) fetal bovine serum (HyClone Laboratories, Logan, UT), 2 mM glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin.Cell culture was performed at 37 o C under a humidified atmosphere containing 5% CO 2 .
Triton X-100 and rinsed in nanopure distilled H 2 O. Gels were further incubated at 37 o C for 20 hrs in 20 mM NaCl, 5 mM CaCl 2 , 0.02% Brij-35, 50 mM Tris-HCl buffer, pH 7.6, then stained with 0.1% Coomassie Brilliant blue R-250 and destained in 10% acetic acid, 30% MeOH in H 2 O. Gelatinolytic activity was detected as unstained bands on a blue background.Plant material.The twigs of D. barteri Bureau var.multiradiata were collected in March 2003 from Kumba, Cameroon, and identified by Mr. Victor Nana of the National Herbarium in Yaoundé, Cameroon where a voucher specimen (No. 44016HNC) was deposited.To a solution of 3 (60 mg, 0.153 mmol) in dry CH 2 Cl 2 was added one drop of borontrifluoride diethyl etherate.The progress of the reaction was monitored by TLC.After 2 hours at rt, the reaction mixture was diluted with EtOAc and washed with saturated aq.NaHCO 3 (10 ml x 2).The organic phase was dried over Na 2 SO 4 and concentrated under reduced pressure and the residue was purified by flash column chromatography on silica gel with a gradient of Hexane-