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Cucurbitacin B purified from Ecballium elaterium (L.) A. Rich from Tunisia inhibits α5β1 integrin-mediated adhesion, migration, proliferation of human glioblastoma cell line and angiogenesis
Author’s Accepted Manuscript Cucurbitacin B purified from Ecballium elaterium (L.) A. Rich from Tunisia inhibits α5β1 integrinmediated adhesion, migration, proliferation of human glioblastoma cell line and angiogenesis Imen Touihri-Barakati, Olfa Kallech-Ziri, Wiem Ayadi, Hervé Kovacic, Belgacem Hanchi, Karim Hosni, José Luis
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S0014-2999(17)30007-9 http://dx.doi.org/10.1016/j.ejphar.2017.01.006 EJP71003
To appear in: European Journal of Pharmacology Received date: 31 August 2016 Revised date: 4 January 2017 Accepted date: 11 January 2017 Cite this article as: Imen Touihri-Barakati, Olfa Kallech-Ziri, Wiem Ayadi, Hervé Kovacic, Belgacem Hanchi, Karim Hosni and José Luis, Cucurbitacin B purified from Ecballium elaterium (L.) A. Rich from Tunisia inhibits α5β1 integrin-mediated adhesion, migration, proliferation of human glioblastoma cell line and angiogenesis, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2017.01.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cucurbitacin B purified from Ecballium elaterium (L.) A. Rich from Tunisia inhibits α5β1 integrin-mediated adhesion, migration, proliferation of human glioblastoma cell line and angiogenesis Imen Touihri-Barakatia, Olfa Kallech-Ziria, Wiem Ayadia, Hervé Kovacicb, Belgacem Hanchic, Karim Hosnia, José Luisb a
Laboratoire des Substances Naturelles (LR10INRAP02), Institut National de Recherche et
d’Analyse Physico-chimique, Sidi Thabet, 2020 Ariana, Tunisie b
Aix-Marseille Université, INSERM, Centre de Recherche en Oncologie Biologique et
Oncopharmacologie (CRO2) UMR 911, Faculté de Pharmacie, 13385 Marseille, France c
Faculté des Sciences de Tunis, Campus Universitaire, Tunis El Manar, 1000 Tunis, Tunisie
*
Corresponding author: Pr. José LUIS, CRO2 (INSERM UMR 911), Faculté de Pharmacie,
13385 Marseille, France;[email protected]
Abstract Integrins are essential protagonists in the complex multistep process of cancer progression and are thus attractive targets for the development of anticancer agents. Cucurbitacin B, a triterpenoid purified from the leaves of Tunisian Ecballium elaterium exhibited an anticancer effect and displayed anti-integrin activity on human glioblastoma U87 cells, without being cytotoxic at concentrations up to 500 nM. Here we show that cucurbitacin B affected the adhesion and migration of U87 cells to fibronectin in a dose-dependent manner with IC50 values of 86.2 nM and 84.6 nM, respectively. Time-lapse videomicroscopy showed
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that cucurbitacin B significantly reduced U87 cells motility and affected directional persistence. Cucurbitacin B also inhibited proliferation with IC50 value of 70.1 nM using Crystal Violet assay. Moreover, cucurbitacin B efficiently inhibited in vitro human microvascular endothelial cells (HMEC) angiogenesis with concentration up to 10 nM. Interestingly, we demonstrate for the first time that this effect was specifically mediated by α5β1 integrins. These findings reveal a novel mechanism of action for cucurbitacin B, which displays a potential interest as a specific anti-integrin drug. Keywords: Cucurbitacin; Cancer; Angiogenesis; Integrin; Glioblastoma; Migration
1. Introduction Cancer is the leading cause of death worldwide, accounting for 7.6 million deaths in 2008 (Ferlay et al., 2008). Glioblastoma is the most common malignant cancer of the central nervous system and remains a significant cause of death in young adults and in children. The medium survival of these patients is less than 12 months. Despite the multi-modality therapy integrating surgery, radiation therapy, and chemotherapy the prognosis remains very poor (Yin et al., 2008). Integrins are a large family of extracellular matrix (ECM) receptors, mediating the interaction of tumour cells with their microenvironment and playing an important role in glioma biology (Tabatabai et al., 2011). Integrins consist of two noncovalently bound α and β glycoprotein subunits. The combination of at least 18 α and 8 β subunits yields 24 distinct integrin dimers and determines the ligand specificity (Parsons et al., 2010; Delamarre et al., 2009). Importantly, major integrin heterodimers αvβ3 and αvβ5 (tenascin and vitronectin receptors), α5β1 (fibronectin receptor), α2β1 (collagens receptor), and α3β1, α6β4, and α6β1
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(laminins receptors), along with their ECM ligands, have been found to play specific role in human glioma cancer progression and metastasis (Delamarre et al., 2009). Research of selective antagonists of integrins is nowadays a strategy for the development of more efficient therapies to completely eliminate gliomas (Tabatabai et al., 2011). For this reason biochemists and biologists have been investigating a variety of purified compound from medicinal plants (Yin et al, 2008). Naturally occurring cucurbitacins constitute a group of tetracyclic triterpenes abundantly found in Cucurbitaceous species such as Ecballium elaterium (Salhab, 2013). Cucurbitacins are classified into cucurbitacin, B, D, E, I and L and their derivatives (Lee et al., 2010). Among these compounds, cucurbitacin B is the most abundant and active form (Chan et al., 2010). Ecballium elaterium (L.) A. Rich, a wild medicinal plant abundantly found in Tunisia, has long been used in Tunisian (Boukef, 1986), oriental and Mediterranean traditional medicine, for its anti-inflammatory (Greige-Gerges et al., 2007), anti-microbial (Adwan et al., 2011), and anti-cancer effects (Lavie, 1958). The major pharmacological and biological effects of E. elaterium plant have been attributed to cucurbitacin B (Adwan et al., 2011). This active compound has been reported to inhibit the growth of several types of cancers, including pancreatic (Thoennissen et al., 2009), laryngeal (Liu et al, 2008), breast (Gupta et al., 2014) and lung cancers (Zhang et al., 2014). Recently, cucurbitacin B has been reported to possess antiproliferative properties on several types of tumour cells and to be active against human glioblastoma multiform (Yin et al., 2008). In the present study, we investigate the inhibitory activities of cucurbitacin B purified from E elaterium leaves on several steps of cancer development. We show that this compound markedly inhibits adhesion, proliferation, migration of human glioma cancer cell lines and
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angiogenesis by interacting with α5β1 integrins. Results provide new evidence for the potent anti-integrin activity of cucurbitacin B on glioblastoma.
2.Materials and methods 2.1.Materials Dulbecco’s modified Eagle’s medium (DMEM), Eagle’s minimal essential medium (EMEM) and RPMI 1640 medium were purchased from GIBCO (Cergy-Pontoise, France) and fetal calf serum (FCS) from BioWhittaker (Fontenay-sous-Bois, France). Penicillin, streptomycin, human fibrinogen, human laminin and poly-L-lysine were from Sigma (St. Quentin Fallavier, France). Rat type I collagen was from Upstate (Lake Placid, NY, USA) and human fibronectin from Chemicon (Temecula, CA, USA). Human vitronectin was purified according to Yatogho et al, 1988. Mouse monoclonal antibodies (mAbs) LM609 (anti-αvβ3) and P1F6 (αvβ5) were purchased from Chemicon. Mouse mAbs Gi9 (anti-α2β1), SAM1 (antiα5β1) and C3VLA3 (anti-α3) and rat mAb GoH3 (anti-α6) were from Immunotech (Marseille, France). Rabbit anti-mouse and anti-rat IgG antibodies were purchased from Sigma. MatrigelTM was from BD Biosciences, Pont de Claix, France. Hexane was purchased from Fluka Chemical Co (Buchs, Swizerland).
2.2. Extract preparation and bioguided fractionation Leaves of Ecballium elaterium were collected in January 2012 from a region of Sidi Thabet, area of Ariana (Northern Tunisia, latitude 36°54′45.25′′N, longitude 10°06'02.10"E, altitude 30 m). Identification was made by Professor S. Ben Saad (Department of Botany,
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Faculty of Sciences of Tunis) according to the ‘‘Flora of Tunisia’’ handbook (Cuénod, 1954), and voucher specimens were deposited at the above-mentioned Laboratory of Natural Substances to serve as a future reference. The ground dried Ecballium elaterium leaves were extracted with methanol using a Soxhlet extraction for 4h. The solvent was removed via a rotary vacuum distillation at 40°C. The total extract was dissolved in H2O and subjected to a series of successive fractionations with hexane, dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and butanol (n-BuOH). The dichloromethane fraction exhibited a strong activity on U87 cell line. It was further concentrated and purified using preparative high performance liquid chromatography (HPLC). Two compounds C1 and C2 were isolated, the most active being C2. C2 was identified by LC-MS analysis by comparing of the retention times with authentic standards (Krepsky et al., 2009). 2.3. Semi-preparative liquid chromatography conditions Separation and isolation were carried out on a semi-preparative HPLC (Agilent Technologies, Palo Alto, CA, USA). The column was a Zorbax Eclipse XDB-C8 PrepHT (H) (21.2 µm * 150 mm, 5µm) from Agilent Technologies. Sample volume of 400 µL was kept with the help of a Rheodyne 77251 injector. Detection was carried out at 230 nm with 2996 photodiode array detector. The flow rate of the mobile phase was kept at 12 ml/min. The mobile phase was composed of 0.1 % acetic acid in water (A) and acetonitrile (B). The following multi-step solvent gradient was employed 0-5 min: 10% B, 5-20 min: 60% B, 2025min: 100% B, 25-27 min: 100% B, and 27-30 min: 10% B. Fractions were collected using a fraction collector (Agilent Technologies).
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2.4. HPLC-PDA-MS analysis LC–ESI–MS analysis was conducted in negative mode electrospray ionisation on an Agilent 1100 series HPLC systems equipped with a photodiode array detector and a triple quadrupole mass spectrometer type Micromass Autospec Ultima Pt (Kelso, UK). The separation was performed on a reverse-phase Uptisphere C18 (Interchim) (2 mm × 100 mm, 5 μm particle size) column with a rate flow of 0.25 ml/min at 40°C. The samples (10 µl) were eluted through the column with a gradient mobile phase consisting of A (H2O 0.1% acetic acid) and B (acetonitrile). Starting at 10% of solvent B, the proportion was programmed to reach 60% at 20 min, 100% at 25 min, and solvent B was maintained at 100% for another 2 min. UV spectra were recorded from 190 to 800 nm and the mass spectra were recorded in negative ion mode, under the following operating conditions: capillary voltage, 3.2 kV; cone voltage, 40 V; probe temperature, 350°C; ion source temperature, 130°C. Mass range between 100 and 800 m/z. The purity of compounds was assessed using the peak purity analysis facilities of the diode array detection system of the MassLynx Software. 2.5. Cell culture The human glioblastoma U87 cell line was routinely cultured in Eagle’s minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS). Human cell lines derived from colonic fibrosarcoma (HT1080) and adenocarcinoma (HT29) were cultured in DMEM containing 10% FCS. Human leukemia (K562) cells were cultured in RPMI 1640 medium containing 10% FCS. Immortalized dermal Human Microvascular Endothelial Cells (HMEC-1) (Ades et al., 1992) were obtained from the Cell Culture Laboratory in the Hôpital
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de la Conception (Marseille, France) and were used between passages 3 and 12. Cells were routinely grown on 0.1% gelatin-coated flasks in MCDB-131 medium containing 10% heatinactivated FCS, 1% penicillin and streptomycin (Life Technologies, Paisley, UK), 1 mg/ml hydrocortisone (Pharmacia & Upjohn, St-Quentin-Yvelines, France) and 10 ng/ml epithelial growth factor (R&D Systems, Minneapolis, MN). Cell lines were maintained at 37°C in a humid atmosphere 5% CO2 in air.
2.6. Cytotoxicity assay Cytotoxicity was assessed by measuring the release of lactate dehydrogenase (LDH) activity into the culture medium upon damage of plasma membrane. Total release of LDH (100% toxicity) was obtained by adding 0.1% Triton-X100 in the incubation medium. The supernatants were collected, clarified by centrifugation 5 min at 600 g and 80 µl of supernatant were submitted to LDH-based cytotoxicity kit (Sigma) in accordance with the manufacturer’s instructions.
2.7. Cell Adhesion assay Adhesion assays were performed as previously described (Delamarre et al., 2009). Briefly, 96-well plates were coated with purified ECM protein solutions for 2 h at 37°C and blocked with 0.5% PBS/BSA. Cells in suspension were added to wells and allowed to adhere to the substrata for 1 h or 2 h depending on cell line at 37°C. After washing, adherent cells were fixed with 1% glutaraldehyde, stained with 0.1% crystal violet and lysed with 1% SDS. Absorbance was then measured at 600 nm. 7
For adhesion assay on antibodies, 96-well plates were coated with 50 µl of rabbit anti-mouse and anti-rat IgG antibodies (50 μg/ml), overnight at 4°C. Wells were washed once with PBS and 50 µl of blocking anti-integrin antibodies (10 μg/ml) were added for 5 h at 37°C. Then, wells were blocked with 0.5% PBS/BSA and adhesion assay was continued as above.
2.8. Cell migration assay In vitro cell migration assays were performed in modified Boyden chambers (NeuroProbe Inc., Bethesda, MD) with porous membranes pre-coated with 10 µg/ml of fibronectin or 50 µg/ml fibrinogen for 5 h at 37°C as previously described (Olfa et al., 2005; Rigot et al., 1998). In a second time, cell motility was recorded by time-lapse video-microscopy as previously described (Sadok et al., 2008). Briefly, human glioblastoma cells were trypsinized and then seeded on fibrinogen pre-coated 24-well plates at low confluence (104 cells/cm2) and allowed to adhere at 37◦C for 2 h. The plates were placed on an inverted Nikon microscope equipped with a heated stage and 5% CO2 supply. Three fields per well were imaged and followed at 5 min intervals over 3 h with a Coolsnap HQ camera (Photometrics, Tucson, AZ) operated by NIS-elements AR 2.30 software (Nikon). Manual single-cell tracking was performed by using Metamorph® image analysis software (Roper Scientific, Evry, France). Migration tracks were used to calculate total migration distance, distance to origin, velocity and directional persistence of cell migration. The distance to origin was determined as the net translocation between the initial and the final position. Velocity was calculated as the total migration distance divided by 180 min. Directional persistence was calculated as the ratio of the distance to origin to the total distance migration.
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2.9. Cell Proliferation assay Cells were seeded (5,000 cells/100 µl) in 96-wells plates. After 2h, cucurbitacin B was added at different concentrations (5, 10, 15, 20 and 25 µg/ml) and cells allowed to proliferate for 72 hours at 37°C. Cells were washed and then fixed by 1% glutaraldehyde and stained by 0.1% crystal violet. Cells were lysed with 1% SDS and the optical density was measured at 600 nm by a microplate reader.
2.10. Anti-angiogenesis assay on MatrigelTM 100 µl of 5.25 mg/ml MatrigelTM were added to 96-well plates and allowed to solidify for 1 h at 37°C. HMEC-1 cells were harvested, added to each well and incubated for 6 h at 37°C and 5% CO2. Cells were fixed with 4% formaldehyde and photographed using a DM-IRBE microscope (Leica, Rueil-Malmaison, France) coupled with a digital camera (CCD camera coolsnap FX, Princeton Instruments, Trenton, NJ). The formation of capillary networks was quantitatively evaluated by measuring the total capillary tube length in 5 view fields per well using Metamorph® image analysis software (Roper Scientific, Evry, France) as previously described previously (Pasquier et al., 2004).
2.11. Statistical analysis Otherwise indicated, all data were expressed as mean ± standard deviation of three measurements performed in triplicate and the difference between treated and untreated 9
(control) groups was determined by using one-way analysis of variance (ANOVA) followed by Tukey’s HSD post-hoc test (RStudio, Version 0.97, Boston, USA). Differences between means are considered statistically significant (*: P<0.05; **: P<0.01; *** P<0.001). 3. Results 3.1. Identification of the compound C2 Bioactivity-guided phytochemical study on dichloromethane fraction of dried leaves of Ecballium elaterium led to the isolation and purification of two compounds C1 and C2, the most active being C2. The identity and purity of this compound was assessed using High Performance Liquid Chromatography equipped with a Diode Array Detection, Mass Spectrometry (HPLC-DAD-MS). Chromatogram resulting from LC–ESI–MS analysis of Ecballium elaterium fraction is shown in Fig. 1A. The analysis of commercial cucurbitacin B shows a peak with the same retention time than C2 peak of E. elaterium fraction. Moreover, the UV spectra of the isolated C2 and commercial cucurbitacin B were quite similar (Fig. 1B), the maximum absorption was λMax=234. Besides, the MS fragmentation of the isolated C2 gave the same pattern than that of cucurbitacin B (Fig. 1C) and is consistent with reported values (Cordell and Shin, 1999; Shin and Van Breemen, 2001). The mass spectrum showed a molecular ion at m/z = 559 corresponding to [M+H]+. The exactly mass spectrum of cucurbitacin B shows a molecular ion at m/z = 558 corresponding to molecular formula C32H46O8. The peak at m/z = 499 was due to the loss of CO-CH3 and OH group. Taken together, these results highly suggest that C2 compound is equivalent to cucurbitacin B.
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The purity of the purified product, namely cucurbitacin B, was additionally confirmed by HPLC with UV-absorbance. Our HPLC analysis using MassLynx Software showed only one single peak at a retention time of 20.22 min (Fig. 1D), and peak purity was determined to be 98.5%. Our results intimated that cucurbitacin B with purity >98% could be obtained successfully by HPLC-preparative under the above-optimized conditions.
3.2. Cucurbitacin B inhibits cell adhesion of human glioma cancer cell line by interacting with α5β1 integrins Cytotoxicity of cucurbitacin B from E. elaterium was assessed using release of lactate dehydrogenase (LDH) as a marker for cell membrane integrity. Results show that cucurbitacin B up to 500 nM, did not significantly induce cytotoxicity on the human glioma cell line U87 (see Supplemental data, Fig. S1). Because cucurbitacin B purified from Ecballium elaterium leaves did not display any cytotoxicity, we tested its ability to inhibit cell adhesion of tumour cells on various purified ECM proteins. As illustrated in Fig. 2A, attachment of glioma cells U87 to fibronectin and laminin-1 was strongly decreased by 500 nM of cucurbitacin B, while no effect was observed on type I collagen and vitronectin. The inhibitory activity of cucurbitacin B on cell adhesion to
fibronectin and laminin-1 was dose-dependent, with a half-maximal inhibition (IC50) of 86 nM and 242 nM respectively (Fig. 2B). Moreover, no inhibition could be observed on the integrin-independent substratum polyL-lysine, suggesting that the effect of cucurbitacin B may involve the integrin family of adhesion receptors. To identify the targeted integrins, we checked cucurbitacin B effect on
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various cell/ECM protein pairs involving in each case a unique integrin, based on literature and/or personal data: α1β1 (PC12/type IV collagen), α2β1 (HT1080/type I collagen), α5β1 (K562/fibronectin), α6β4 (HT29- D4/laminin-1), αvβ3 (HT1080/fibrinogen), αvβ5 (HT29D4/vitronectin) and αvβ6 (HT29-D4/fibronectin). As illustrated in Fig. 3A, cucurbitacin B was not able to affect cell adhesion through α2β1, α1β1 or αvβ5 integrins, slightly affected αvβ6 integrin, but significantly reduced the adhesive function of α5β1, α6β4 and αvβ3 integrins. To confirm that cucurbitacin B could interact with α5β1 integrins, we performed adhesion assays to a panel of immobilized antibodies raised against several integrin subunits used as matrix: anti-α5β1 (mAb SAM1), anti-αvβ5 (mAb P1F6), anti-α3 (mAb C3VLa3), anti-α2 (mAb Gi 9), and anti-α6 (mAb GoH3). As illustrated in Fig. 3B, at 500 nM of cucurbitacin B, U87 cell attachment to anti-α5β1 antibodies was dramatically affected (over 97%) and slightly inhibited in the case of anti-α6 antibodies (over 64%), suggesting that the cucurbitacin B exert its effect on cell adhesion by inhibiting α5β1 integrin in human glioma cell lines. However, the cucurbitacin B did not modify U87 cells adhesion on α3 and αvβ5 antibodies, while adhesion on α2 was only slightly decreased.
3.3. Cucurbitacin B affects U87 cell migration Cell motility is a crucial step in the complex multistep process of cancer progression. We thus evaluated cell migration in the presence of cucurbitacin B by using haptotaxis assays towards fibronectin in modified Boyden chambers. Cucurbitacin B abolished cell migration to fibronectin at 500 nM (Fig. 4A). This inhibition was dose-dependent with a half-maximal inhibition (IC50) of 85 nM as shown in Fig. 4B. 12
In a second time, we performed a random two-dimensional motility assay by using timelapse videomicroscopy (Sadok et al., 2008). Representative migration paths (where the origin of each cell track has been set to the coordinates x=0, y=0) for 10 cells are shown in Fig. 5A. We observed that cucurbitacin B dramatically reduced U87 cell motility on fibronectin. As shown in Fig. 5B, analysis of the trajectory obtained from time-lapse recordings of each individual cell during 3h indicated that control untreated cells exhibited a high migration velocity (0.96±0.16 μm/min) (see Supplemental data, Video 1), similar to cells treated with 10 nM cucurbitacin B (0.92±0.14 μm/min). However, treatment with 500 nM of cucurbitacin B dramatically affected the velocity of migration (0.22±0.05 μm/min) (see Supplemental data, Video 2). When compared to control, cucurbitacin B also affected the mean distance to origin (8±0.5 μm versus 47±1 μm ) and directional persistence of migration (0.15±0.02 μm versus 0.49±0.04 μm). These effects of cucurbitacin B on cell velocity and directional persistence may be due to the alteration of protusion formation, as treated cells failed to extend any lamellipodium.
3.4. Cucurbitacin B inhibits proliferation of glioma cancer cells The effect of Cucurbitacin B on U87 cells growth was evaluated in the absence or in the presence of this compound. For all concentrations tested, the number of cells in the wells was reduced by the presence of cucurbitacin B purified from E. elaterium leaves (Fig. 6). This antiproliferative activity was dose-dependent with a half-maximal inhibition (IC50) of 70.14 nM.
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3.5. Cucurbitacin B displays anti-angiogenic activity Cucurbitacin B affects α5β1 integrin-mediated adhesion of U87 cells. As cell adhesion plays a central during angiogenesis, were performed angiogenesis assays using HMEC-1 cells to know whether cucurbitacin B modulates angiogenesis. As shown in Fig. 7A, cucurbitacin B indeed blocked in vitro tubulogenesis with a maximal activity at 500 nM. The inhibitory effect was dose-dependent with an half-maximal inhibition (IC50) of 68.85 nM of cucurbitacin B (Fig. 7B). In order to know whether the integrins inhibited by cucurbitacin B are involved in angiogenesis when using HMEC-1 cells, we performed angiogenesis assays in the presence of function-blocking antibodies against various integrins. Results demonstrated that angiogenesis is mainly supported by α5β1 and, to a lesser extend, by αv integrins (Fig. 7C), the two integrins being targeted by cucurbitacin B.
4. Discussion The cucurbitacins are of great interest because of the wide range of biological activity they exhibit. Several studies were published pointing out that these compounds exhibit anticancer activities on various human cancer cell lines and tumour xenografts (Tannin-Spitz et al., 2007; Wakimoto et al., 2008). The exact mechanism to explain the effect of cucurbitacin B on tumour growth is still unknown. Thus, cucurbitacin B induces apoptosis in laryngeal cancer (Liu et al., 2008). Some reports indicate that cucurbitacin B is a small selective inhibitor of Stat3 signaling pathway in laryngeal squamous cell carcinoma (Liu et al., 2010) and leukemia cell line K562 (Chan et al., 2010). Duabgmano et al. (2012) suggest that cucurbitacin B may inhibit proliferation of human breast cancer cells through disruption of the microtubule network. However, few works have been published on the anticancer effect 14
of the cucurbitacins B on brain cancer. Only Yin et al. (2008) evaluated the antiproliferative activity of cucurbitacin B extracted from Trichosanthes kirilowii Maximowicz in glioblastoma multiforme. The present study shows that cucurbitacin B inhibited cell proliferation of human glioblastoma cell line U87. Yin et al. (2008) also found that cucurbitacin B possessed strong antiproliferative effects on 5 GBM cell lines: U87, U118, U343, U373 and T98G. In our study, we shown using haptotaxis assays in modified Boyden chambers that migration of glioblastoma U87 cells to fibronectin was affected with cucurbitacin B isolated from Ecballium elaterium. Time-lapse videomicroscopy also show a decrease in glioma cell velocity and directional persistence. Migration of cells in higher organisms is mediated by adhesion (Palecek et al., 1997). Results showed that cucurbitacin B impaired adhesion of glioma cell line U87 specifically on fibronectin in a dose-dependent manner. This effect is not due to cytotoxicity, as cucurbitacin B, up to 1000 nM for 5h, did not significantly induce detectable lactate dehydrogenase (LDH) release by U87 cells (Supplemental data, Figure S1). Adhesion to the integrin-independent substratum was not affected, suggesting that the antiadhesive effect of cucurbitacin B was due to inhibition of integrins. This anti-adhesive activity appears to be specific as no effect was observed on the attachment of collagen I or vitronectin. In this study, we show that cucurbitacin B a natural molecule compound purified from Ecballium elaterium leaves, targeted α5β1 glioma integrin. Malignant gliomas are among the most vascularized tumours in humans. Hence it seems to be a logical consequence to research anti-angiogenic agents to prevent and treat malignant gliomas (Tuettenberg et al., 2006). The in vitro Matrigel™ tube formation assay was used with endothelial cells (HMEC-1) to evaluate the effect of cucurbitacin B on the angiogenic process. The present study shows that cucurbitacin B, used at non-toxic levels, inhibited
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angiogenesis at only 100 nM and that this effect was dose-dependent. Moreover, using function-blocking antibodies, we show that α5β1 and, to a lesser extend, αv integrins are involved in the angiogenic process. There are several oncogenic signaling pathways that are commonly involved in cancer cell proliferation and survival. Cucurbitacins seem to affect both cancer cells and normal macrophages through different mechanisms (Lee et al., 2010). In normal macrophages cucurbitacins work as inhibitors of the IKK/NF-κB pathway (Park et al., 2009 ; Escandell et al., 2007). However, in cancer cells, the JAK-STAT, Akt-PKB pathway and MAPK pathways are targets of the cucurbitacin family. It is still not clear how cucurbitacins can selectively choose their target pathway depending on the cell types. Cucurbitacin B has a prominent antiproliferative activity on glioblastoma cells and at least in part, the mode of action is by affecting the cytoskeleton, as well as, the JNK and MAPK pathway (Yin et al., 2008). Thoennissen et al. (2009) discovered that the anti-proliferative effect in pancreatic cancer cells exposed to cucurbitacin B could be explained as a result of inhibition of the JAK-STAT pathway. Cucurbitacin B inhibits human breast cancer cell proliferation through disruption of microtubule polymerization and nucleophosmin/B23 translocation (Duangmano et al., 2012). Although neglected for decades, cucurbitacin is once again attracting attention as a potential anticancer drug. Its role as a JAK/STAT inhibitor, a MAPK modulator, a cytoskeleton disruptor and finally integrin inhibitor qualify it as an excellent candidate for clinical investigation. Integrins have attracted increasing interest for their potential to act as tumour therapeutic targets (Schaffner et al., 2013). The critical role of α5β1 integrin in physiological tumorigenesis and angiogenesis has been recognized in recent years (Morjen et al., 2014).
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Therefore, specific α5β1 integrin antagonists may represent new therapeutic agents to fight defined subpopulations of particularly aggressive tumours (Maglott et al., 2006). However, few α5β1 integrin antagonists have been developed to date. Interestingly, our results suggest that cucurbitacin B purified from E. elaterium seed interferes with α5β1 integrin function on both U87 human glioblastoma and HMEC-1 cells. In agreement with our result, Morjen et al. (2013) showed that PIVL, a Kunitz-type serine protease inhibitor isolated from viper snake venom blocks α5β1 integrin function and inhibited adhesion, migration and invasion of human glioblastoma U87 cells. Maglott et al. (2006) demonstrated that SJ749, an α5β1 integrin antagonist, reduces proliferation, migration and clonogenicity of U87 cell lines and inhibits in vivo angiogenesis of endothelial cells. Zhu et al. (2010) reported that celastrol, a natural compound isolated from the plant Tripterygium wilfordii, inhibits adhesion and proliferation of murine melanoma cell line B16F10 and human lung cancer cell line 95-D. Celastrol targets β1 integrin, whose inhibition is likely responsible for its anti-adhesion effect to fibronectin, which may provide an understanding of its antimetastatic mechanisms. Previous studies show that salvicine, a novel diterpenoid quinone compound, significantly reduces the metastatic potential of MDA-MB435 cells both in vitro and in vivo. Salvicine inhibited the adhesion cells to fibronectin by down-regulating β1 integrin function (Zhou et al., 2008). 5. Conclusion In summary, we report in this paper that cucurbitacin B purified from Ecballium elaterium inhibited integrin-mediated cell adhesion, migration and proliferation of glioma tumour cell line U87 and angiogenesis. To our knowledge, this is the first study describing the inhibitory activities of this triterpenoid on integrin receptors. This effect was mediated by α5β1 17
integrins. Our results provide a new understanding of the anti-cancer activity of cucurbitacin B. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgement This research was supported by Ministry of Education and Scientific Research of Tunisia (MERST), the Institut National de la Santé et de la Recherche Médicale (INSERM) and grants SIRIC and ARCUS.
Fig. 1: (A) LC–PDA–TIC profiles of dichloromethane fraction from E. elaterium leaves and standard cucurbitacin B. (B) UV spectrum of standard cucurbitacin B and compound C2 isolated from the leaves of E. elaterium. (C) Mass spectra of standard cucurbitacin B and compound C2. (D) The purity of cucurbitacin B isolated from Ecballium elaterium was confirmed using MassLynx Software.
Fig. 2: Cucurbitacin B purified from of E. elaterium inhibits tumour cell adhesion. (A) Glioma cells (U87) were preincubated without (black bars) or with cucurbitacin B (500 nM) (grey bars) for 30 min at room temperature. Cells were then added to 96-well microtiter plates coated with different ECM proteins: type I collagen (Col I), fibronectin (Fn), vitronectin (Vn),
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Laminin-1 (Ln), or with poly-L-lysine (PLL). (B) Dose–effect of cucurbitacin B on U87 cell adhesion to fibronectin and laminin-1. Data shown are means (±S.D.) from 3 experiments performed in triplicate and difference between treated and untreated (control) cells was considered statistically significant (*: P<0.05; **: P<0.01; *** P<0.001).
Fig. 3: Cucurbitacin B inhibits human glioblastoma cell adhesion through α5β1. U87 cells were preincubated without (black bars) or with 500 nM cucurbitacin B (grey bars). (A) Adhesion assays were performed with various cell/ECM protein pairs involving unique integrins: α1β1 (PC12/ type IV collagen), α2β1 (HT1080/type I collagen), α5β1 (K562/fibronectin), αvβ3 (HT1080/fibrinogen), αvβ5 (HT29/vitronectin), α6β4 (HT29/laminin) and αvβ6 (HT29/fibronectin). (B) Adhesion was assessed using antibodies: microtiter plates were coated with antibodies raised against the indicated integrin subunits as described in the experimental section. Data shown are means (±S.D.) from 3 experiments performed in triplicate and difference between treated and untreated (control) cells was considered statistically significant (*: P<0.05; **: P<0.01; *** P<0.001).
Fig. 4: The cucurbitacin B inhibits cell migration. (A) Cell motility was determined in a modified Boyden chamber using porous membrane precoated with 10 μg/ml fibronectin. After treatment without or with 500 nM cucurbitacin B for 30 min at room temperature, U87 cells were seeded into the upper reservoir and allowed to migrate through the filter towards 19
the lower reservoir for 5 h at 37°C. Cells that migrated to the underside of the filter were stained with 0.1% crystal violet. Metamorph imaging software was used to capture images of U87 cell that migrate through the filter. (B) Dose–effect of cucurbitacin on cell migration to fibronectin. Data shown are means (±S.D.) from 3 experiments performed in triplicate and difference between treated and untreated (control) cells was considered statistically significant (**: P<0.01; *** P<0.001).
Fig. 5: Evaluation of cell motility by time-lapse videomicroscopy. U87 cells were seeded at 104 cells/well without or with cucurbitacin B on fibronectin pre-coated 24-well plates. Pictures of four fields per well were taken at 5 min intervals during 3 h. The trajectories followed by cells during the 3 h of recording were plotted in (A) with the initial position of each track aligned at 0.0. (B) Cell velocity, directional persistence and mean distance to origin were calculated as described in the Materials section . Data shown are means (±S.D.) from 3 experiments performed in duplicate and difference between treated and untreated (control) cells was considered statistically significant (*: P<0.05; *** P<0.001).
Fig. 6: Cucurbitacin B inhibits U87 cancer cells proliferation. (a) Cells were seeded on 96well plates. After 2h different, concentrations of cucurbitacin B were added and cells were allowed to proliferate for 72h. Cells were quantified by staining with 0.1% crystal violet, solubilization with 1% SDS and measure of absorbance at 600 nm. Data shown are means (±S.D.) from 3 experiments performed in triplicate and difference between treated and untreated (control) cells was considered statistically significant (*: P<0.05; **: P<0.01; *** P<0.001). 20
Fig. 7: Cucurbitacin B blocks in vitro angiogenesis. (A) Representative visualization of tubulogenesis assay after pre-treatment of HMEC-1 cells without (control) or with 10, 100 or 500 nM cucurbitacin B at room temperature. Cells were then added to MatrigelTM and allowed to form capillary-like structures for 6 h at 37 °C. Scale bar: 70 µm. (B) Dose-effect of cucurbitacin B on tubulogenesis on MatrigelTM. (C) Tubulogenesis on MatrigelTM was performed as above in the absence (-) or in the presence of the indicated anti-integrin antibodies (20 µg/ml). Quantification of tubulogenesis was done as described in experimental section. Scale bar: 70 μm. Data shown are means (±S.D.) from 3 experiments performed in triplicate and difference between treated and untreated (control) cells was considered statistically significant (*: P<0.05; **: P<0.01; *** P<0.001).
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Morjen, M., Honoré, S., Bazaa, A., Abdelkafi-Koubaa, Z., Ellafi, A., Mabrouk, Kovacic, H., El Ayeb, M., Marrakchi, N., Luis, J., 2014. PIVL, a snake venom Kunitz-type serine protease inhibitor, inhibits in vitro and in vivo angiogenesis. Microvasc. Res. 95, 149-156. Morjen, M., Kallech-Ziri, O., Bazaa, A., Othman, H., Mabrouk, K., Zouari-Kessentini, R., Sanz, L., José, J.C., Srairi-Abid, N., El Ayeb, M., Luis, J and Marrakchi, N., 2013. PIVL, a new serine protease inhibitor from Macrovipera lebetina transmediterranea venom, impairs motility of human glioblastoma cells. Matrix. Biol. 32, 52-62. Olfa, K.Z., Luis, J., Daoud, S., Basaa, A., Srairi Abid, N., Nicolas, A., Maxime, L., Zouari, R., Mabrouk, K., Jacques, M., Jean-Marc, S., M., El Ayeb, M., Marrakchi, N., 2005. Lebestatin, a disintegrin from Macrovipera venom, inhibits integrin-mediated cell adhesion, migration and angiogenesis. Lab. Invest. 85, 1507–1516. Palecek, S.P., Loftus, J.C., Ginsberg, M.H., Lauffenburger, D.A., Horwitz, A.F., 1997. Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness. Nature. 385, 537-540. Park, C.S., Lim, K.J., Baek, S.H., Sohn, H.O., Lee, D.W., Yun, H.Y., Baek, K.J., Lee, H.S., and Kwon, N.S., 2004. Inhibition of nitric oxide generation by 23,24-dihydrocucurbitacin D in mouse peritoneal macrophages. J. Pharmacol. Exp. Ther. 309, 705–710. Parsons, J.T., Slack-Davis, J.K., Tilghman, R.W., Iwanicki, M and Martin, H.K., 2010. Integrin Signaling: Cell Migration, Proliferation, and Survival. Handbook of Cell Signaling, Three-Volume Set 2 ed. Pasquier, E., Carré, M., Pourroy, B., Camoin, L., Rebaï, O., Briand, C., Braguer, D., 2004. Antiangiogenic activity of paclitaxel is associated with its cytostatic effect, mediated by the 24
initiation but not completion of a mitochondrial apoptotic signaling pathway. Mol. cancer. ther. 3, 1301-1310. Rigot, V., Lehmann, M., André, F., Daemi, N., Marvaldi, J and Luis, J., 1998. Integrin ligation and PKC activation are required for migration of colon carcinoma cells. J. Cell. Sci. 111, 3119–3127. Sadok, A., Bourgarel-Rey, V., Gattacceca, F., Penel, C., Lehmann, M., Kovacic, H., 2008. Nox1-dependent superoxide production controls colon adenocarcinoma cell migration. Biochim. Biophys. Acta. 1783, 23–33. Salhab, A.S., 2013. Human Exposure to Ecballium elaterium Fruit Juice: Fatal Toxicity and Possible Remedy. J. Pharm. Pharmacol. 4, 447. Schaffner, F., Ray, A.M., Dontenwill, M., 2013. Integrin α5β1, the fibronectin receptor, as a pertinent therapeutic target in solid tumors. Cancers. 5, 27-47. Tabatabai G, Tonn JC, Stupp R, Weller M., 2011. The role of integrins in glioma biology and anti-glioma therapies. Curr. Pharm. Des. 17, 2402-10. Tannin-Spitz, T., Grossman, S., Dovrat, S., Gottlieb, H. E., Bergman, M., 2007. Growth inhibitory activity of cucurbitacin glucosides isolated from Citrullus colocynthis on human breast cancer cells. Biochem. Pharmacol. 73, 56-67. Thoennissen, N. H., Iwanski, G. B., Doan, N. B., Okamoto, R., Lin, P., Abbassi, S., Koeffler, H. P., 2009. Cucurbitacin B induces apoptosis by inhibition of the JAK/STAT pathway and potentiates antiproliferative effects of gemcitabine on pancreatic cancer cells. Cancer. Res. 69, 5876-5884.
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Tuettenberg, J., Friedel, C., Vajkoczy, P., 2006. Angiogenesis in malignant glioma—a target for antitumor therapy? Crit. Rev. Oncol. Hematol. 59, 181-193. Wakimoto, N., Yin, D., O’Kelly, J., Haritunians, T., Karlan, B., Said, J., Koeffler, H. P., 2008. Cucurbitacin B has a potent antiproliferative effect on breast cancer cells in vitro and in vivo. Cancer. Sci, 99, 1793-1797. Yatogho T, Izumi M, Kashiwagi H., 1998. Novel purification of vitronectin from human plasma by heparin affinity chromatography. Cell. Struct. Funct.13:281–292. Yin, D., Wakimoto, N., Xing, H., Lu, D., Huynh, T., Wang, X., Koeffler, H. P., 2008. Cucurbitacin B markedly inhibits growth and rapidly affects the cytoskeleton in glioblastoma multiforme. Int. J. Cancer. 123, 1364-1375. Zhang, M., Bian, Z. G., Zhang, Y., Wang, J. H., Kan, L., Wang, X., He, P., 2014. Cucurbitacin B inhibits proliferation and induces apoptosis via STAT3 pathway inhibition in A549 lung cancer cells. Mol. med. rep. 10, 2905-2911. Zhou, J., Chen, Y., Lang, J. Y., Lu, J. J., Ding, J., 2008. Salvicine inactivates β1 integrin and inhibits adhesion of MDA-MB-435 cells to fibronectin via reactive oxygen species signaling. Mol. Cancer. Res. 6, 194-204. Zhu, H., Liu, X. W., Cai, T. Y., Cao, J., Tu, C. X., Lu, W., Yang, B., 2010. Celastrol acts as a potent antimetastatic agent targeting β1 integrin and inhibiting cell-extracellular matrix adhesion, in part via the p38 mitogen-activated protein kinase pathway. J. Pharm. Exp. Ther. 334, 489-499.
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Supplementary video 1: Time-lapse videomicroscopy of untreated U87 cells U87 cells were seeded at 104 cells/well without cucurbitacin B on fibronectin pre-coated 24well plates. Pictures were taken at 5 min intervals during 3 h.
Supplementary video 2: Time-lapse videomicroscopy of U87 cells treated with 500 nM of cucurbitacin B U87 cells were seeded at 104 cells/well in the presence of 500 nM of cucurbitacin B on fibronectin pre-coated 24-well plates. Pictures were taken at 5 min intervals during 3 h.
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PII: DOI: Reference:
www.elsevier.com/locate/ejphar
S0014-2999(17)30007-9 http://dx.doi.org/10.1016/j.ejphar.2017.01.006 EJP71003
To appear in: European Journal of Pharmacology Received date: 31 August 2016 Revised date: 4 January 2017 Accepted date: 11 January 2017 Cite this article as: Imen Touihri-Barakati, Olfa Kallech-Ziri, Wiem Ayadi, Hervé Kovacic, Belgacem Hanchi, Karim Hosni and José Luis, Cucurbitacin B purified from Ecballium elaterium (L.) A. Rich from Tunisia inhibits α5β1 integrin-mediated adhesion, migration, proliferation of human glioblastoma cell line and angiogenesis, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2017.01.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Cucurbitacin B purified from Ecballium elaterium (L.) A. Rich from Tunisia inhibits α5β1 integrin-mediated adhesion, migration, proliferation of human glioblastoma cell line and angiogenesis Imen Touihri-Barakatia, Olfa Kallech-Ziria, Wiem Ayadia, Hervé Kovacicb, Belgacem Hanchic, Karim Hosnia, José Luisb a
Laboratoire des Substances Naturelles (LR10INRAP02), Institut National de Recherche et
d’Analyse Physico-chimique, Sidi Thabet, 2020 Ariana, Tunisie b
Aix-Marseille Université, INSERM, Centre de Recherche en Oncologie Biologique et
Oncopharmacologie (CRO2) UMR 911, Faculté de Pharmacie, 13385 Marseille, France c
Faculté des Sciences de Tunis, Campus Universitaire, Tunis El Manar, 1000 Tunis, Tunisie
*
Corresponding author: Pr. José LUIS, CRO2 (INSERM UMR 911), Faculté de Pharmacie,
13385 Marseille, France;[email protected]
Abstract Integrins are essential protagonists in the complex multistep process of cancer progression and are thus attractive targets for the development of anticancer agents. Cucurbitacin B, a triterpenoid purified from the leaves of Tunisian Ecballium elaterium exhibited an anticancer effect and displayed anti-integrin activity on human glioblastoma U87 cells, without being cytotoxic at concentrations up to 500 nM. Here we show that cucurbitacin B affected the adhesion and migration of U87 cells to fibronectin in a dose-dependent manner with IC50 values of 86.2 nM and 84.6 nM, respectively. Time-lapse videomicroscopy showed
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that cucurbitacin B significantly reduced U87 cells motility and affected directional persistence. Cucurbitacin B also inhibited proliferation with IC50 value of 70.1 nM using Crystal Violet assay. Moreover, cucurbitacin B efficiently inhibited in vitro human microvascular endothelial cells (HMEC) angiogenesis with concentration up to 10 nM. Interestingly, we demonstrate for the first time that this effect was specifically mediated by α5β1 integrins. These findings reveal a novel mechanism of action for cucurbitacin B, which displays a potential interest as a specific anti-integrin drug. Keywords: Cucurbitacin; Cancer; Angiogenesis; Integrin; Glioblastoma; Migration
1. Introduction Cancer is the leading cause of death worldwide, accounting for 7.6 million deaths in 2008 (Ferlay et al., 2008). Glioblastoma is the most common malignant cancer of the central nervous system and remains a significant cause of death in young adults and in children. The medium survival of these patients is less than 12 months. Despite the multi-modality therapy integrating surgery, radiation therapy, and chemotherapy the prognosis remains very poor (Yin et al., 2008). Integrins are a large family of extracellular matrix (ECM) receptors, mediating the interaction of tumour cells with their microenvironment and playing an important role in glioma biology (Tabatabai et al., 2011). Integrins consist of two noncovalently bound α and β glycoprotein subunits. The combination of at least 18 α and 8 β subunits yields 24 distinct integrin dimers and determines the ligand specificity (Parsons et al., 2010; Delamarre et al., 2009). Importantly, major integrin heterodimers αvβ3 and αvβ5 (tenascin and vitronectin receptors), α5β1 (fibronectin receptor), α2β1 (collagens receptor), and α3β1, α6β4, and α6β1
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(laminins receptors), along with their ECM ligands, have been found to play specific role in human glioma cancer progression and metastasis (Delamarre et al., 2009). Research of selective antagonists of integrins is nowadays a strategy for the development of more efficient therapies to completely eliminate gliomas (Tabatabai et al., 2011). For this reason biochemists and biologists have been investigating a variety of purified compound from medicinal plants (Yin et al, 2008). Naturally occurring cucurbitacins constitute a group of tetracyclic triterpenes abundantly found in Cucurbitaceous species such as Ecballium elaterium (Salhab, 2013). Cucurbitacins are classified into cucurbitacin, B, D, E, I and L and their derivatives (Lee et al., 2010). Among these compounds, cucurbitacin B is the most abundant and active form (Chan et al., 2010). Ecballium elaterium (L.) A. Rich, a wild medicinal plant abundantly found in Tunisia, has long been used in Tunisian (Boukef, 1986), oriental and Mediterranean traditional medicine, for its anti-inflammatory (Greige-Gerges et al., 2007), anti-microbial (Adwan et al., 2011), and anti-cancer effects (Lavie, 1958). The major pharmacological and biological effects of E. elaterium plant have been attributed to cucurbitacin B (Adwan et al., 2011). This active compound has been reported to inhibit the growth of several types of cancers, including pancreatic (Thoennissen et al., 2009), laryngeal (Liu et al, 2008), breast (Gupta et al., 2014) and lung cancers (Zhang et al., 2014). Recently, cucurbitacin B has been reported to possess antiproliferative properties on several types of tumour cells and to be active against human glioblastoma multiform (Yin et al., 2008). In the present study, we investigate the inhibitory activities of cucurbitacin B purified from E elaterium leaves on several steps of cancer development. We show that this compound markedly inhibits adhesion, proliferation, migration of human glioma cancer cell lines and
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angiogenesis by interacting with α5β1 integrins. Results provide new evidence for the potent anti-integrin activity of cucurbitacin B on glioblastoma.
2.Materials and methods 2.1.Materials Dulbecco’s modified Eagle’s medium (DMEM), Eagle’s minimal essential medium (EMEM) and RPMI 1640 medium were purchased from GIBCO (Cergy-Pontoise, France) and fetal calf serum (FCS) from BioWhittaker (Fontenay-sous-Bois, France). Penicillin, streptomycin, human fibrinogen, human laminin and poly-L-lysine were from Sigma (St. Quentin Fallavier, France). Rat type I collagen was from Upstate (Lake Placid, NY, USA) and human fibronectin from Chemicon (Temecula, CA, USA). Human vitronectin was purified according to Yatogho et al, 1988. Mouse monoclonal antibodies (mAbs) LM609 (anti-αvβ3) and P1F6 (αvβ5) were purchased from Chemicon. Mouse mAbs Gi9 (anti-α2β1), SAM1 (antiα5β1) and C3VLA3 (anti-α3) and rat mAb GoH3 (anti-α6) were from Immunotech (Marseille, France). Rabbit anti-mouse and anti-rat IgG antibodies were purchased from Sigma. MatrigelTM was from BD Biosciences, Pont de Claix, France. Hexane was purchased from Fluka Chemical Co (Buchs, Swizerland).
2.2. Extract preparation and bioguided fractionation Leaves of Ecballium elaterium were collected in January 2012 from a region of Sidi Thabet, area of Ariana (Northern Tunisia, latitude 36°54′45.25′′N, longitude 10°06'02.10"E, altitude 30 m). Identification was made by Professor S. Ben Saad (Department of Botany,
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Faculty of Sciences of Tunis) according to the ‘‘Flora of Tunisia’’ handbook (Cuénod, 1954), and voucher specimens were deposited at the above-mentioned Laboratory of Natural Substances to serve as a future reference. The ground dried Ecballium elaterium leaves were extracted with methanol using a Soxhlet extraction for 4h. The solvent was removed via a rotary vacuum distillation at 40°C. The total extract was dissolved in H2O and subjected to a series of successive fractionations with hexane, dichloromethane (CH2Cl2), ethyl acetate (EtOAc), and butanol (n-BuOH). The dichloromethane fraction exhibited a strong activity on U87 cell line. It was further concentrated and purified using preparative high performance liquid chromatography (HPLC). Two compounds C1 and C2 were isolated, the most active being C2. C2 was identified by LC-MS analysis by comparing of the retention times with authentic standards (Krepsky et al., 2009). 2.3. Semi-preparative liquid chromatography conditions Separation and isolation were carried out on a semi-preparative HPLC (Agilent Technologies, Palo Alto, CA, USA). The column was a Zorbax Eclipse XDB-C8 PrepHT (H) (21.2 µm * 150 mm, 5µm) from Agilent Technologies. Sample volume of 400 µL was kept with the help of a Rheodyne 77251 injector. Detection was carried out at 230 nm with 2996 photodiode array detector. The flow rate of the mobile phase was kept at 12 ml/min. The mobile phase was composed of 0.1 % acetic acid in water (A) and acetonitrile (B). The following multi-step solvent gradient was employed 0-5 min: 10% B, 5-20 min: 60% B, 2025min: 100% B, 25-27 min: 100% B, and 27-30 min: 10% B. Fractions were collected using a fraction collector (Agilent Technologies).
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2.4. HPLC-PDA-MS analysis LC–ESI–MS analysis was conducted in negative mode electrospray ionisation on an Agilent 1100 series HPLC systems equipped with a photodiode array detector and a triple quadrupole mass spectrometer type Micromass Autospec Ultima Pt (Kelso, UK). The separation was performed on a reverse-phase Uptisphere C18 (Interchim) (2 mm × 100 mm, 5 μm particle size) column with a rate flow of 0.25 ml/min at 40°C. The samples (10 µl) were eluted through the column with a gradient mobile phase consisting of A (H2O 0.1% acetic acid) and B (acetonitrile). Starting at 10% of solvent B, the proportion was programmed to reach 60% at 20 min, 100% at 25 min, and solvent B was maintained at 100% for another 2 min. UV spectra were recorded from 190 to 800 nm and the mass spectra were recorded in negative ion mode, under the following operating conditions: capillary voltage, 3.2 kV; cone voltage, 40 V; probe temperature, 350°C; ion source temperature, 130°C. Mass range between 100 and 800 m/z. The purity of compounds was assessed using the peak purity analysis facilities of the diode array detection system of the MassLynx Software. 2.5. Cell culture The human glioblastoma U87 cell line was routinely cultured in Eagle’s minimum essential medium (EMEM) supplemented with 10% fetal calf serum (FCS). Human cell lines derived from colonic fibrosarcoma (HT1080) and adenocarcinoma (HT29) were cultured in DMEM containing 10% FCS. Human leukemia (K562) cells were cultured in RPMI 1640 medium containing 10% FCS. Immortalized dermal Human Microvascular Endothelial Cells (HMEC-1) (Ades et al., 1992) were obtained from the Cell Culture Laboratory in the Hôpital
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de la Conception (Marseille, France) and were used between passages 3 and 12. Cells were routinely grown on 0.1% gelatin-coated flasks in MCDB-131 medium containing 10% heatinactivated FCS, 1% penicillin and streptomycin (Life Technologies, Paisley, UK), 1 mg/ml hydrocortisone (Pharmacia & Upjohn, St-Quentin-Yvelines, France) and 10 ng/ml epithelial growth factor (R&D Systems, Minneapolis, MN). Cell lines were maintained at 37°C in a humid atmosphere 5% CO2 in air.
2.6. Cytotoxicity assay Cytotoxicity was assessed by measuring the release of lactate dehydrogenase (LDH) activity into the culture medium upon damage of plasma membrane. Total release of LDH (100% toxicity) was obtained by adding 0.1% Triton-X100 in the incubation medium. The supernatants were collected, clarified by centrifugation 5 min at 600 g and 80 µl of supernatant were submitted to LDH-based cytotoxicity kit (Sigma) in accordance with the manufacturer’s instructions.
2.7. Cell Adhesion assay Adhesion assays were performed as previously described (Delamarre et al., 2009). Briefly, 96-well plates were coated with purified ECM protein solutions for 2 h at 37°C and blocked with 0.5% PBS/BSA. Cells in suspension were added to wells and allowed to adhere to the substrata for 1 h or 2 h depending on cell line at 37°C. After washing, adherent cells were fixed with 1% glutaraldehyde, stained with 0.1% crystal violet and lysed with 1% SDS. Absorbance was then measured at 600 nm. 7
For adhesion assay on antibodies, 96-well plates were coated with 50 µl of rabbit anti-mouse and anti-rat IgG antibodies (50 μg/ml), overnight at 4°C. Wells were washed once with PBS and 50 µl of blocking anti-integrin antibodies (10 μg/ml) were added for 5 h at 37°C. Then, wells were blocked with 0.5% PBS/BSA and adhesion assay was continued as above.
2.8. Cell migration assay In vitro cell migration assays were performed in modified Boyden chambers (NeuroProbe Inc., Bethesda, MD) with porous membranes pre-coated with 10 µg/ml of fibronectin or 50 µg/ml fibrinogen for 5 h at 37°C as previously described (Olfa et al., 2005; Rigot et al., 1998). In a second time, cell motility was recorded by time-lapse video-microscopy as previously described (Sadok et al., 2008). Briefly, human glioblastoma cells were trypsinized and then seeded on fibrinogen pre-coated 24-well plates at low confluence (104 cells/cm2) and allowed to adhere at 37◦C for 2 h. The plates were placed on an inverted Nikon microscope equipped with a heated stage and 5% CO2 supply. Three fields per well were imaged and followed at 5 min intervals over 3 h with a Coolsnap HQ camera (Photometrics, Tucson, AZ) operated by NIS-elements AR 2.30 software (Nikon). Manual single-cell tracking was performed by using Metamorph® image analysis software (Roper Scientific, Evry, France). Migration tracks were used to calculate total migration distance, distance to origin, velocity and directional persistence of cell migration. The distance to origin was determined as the net translocation between the initial and the final position. Velocity was calculated as the total migration distance divided by 180 min. Directional persistence was calculated as the ratio of the distance to origin to the total distance migration.
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2.9. Cell Proliferation assay Cells were seeded (5,000 cells/100 µl) in 96-wells plates. After 2h, cucurbitacin B was added at different concentrations (5, 10, 15, 20 and 25 µg/ml) and cells allowed to proliferate for 72 hours at 37°C. Cells were washed and then fixed by 1% glutaraldehyde and stained by 0.1% crystal violet. Cells were lysed with 1% SDS and the optical density was measured at 600 nm by a microplate reader.
2.10. Anti-angiogenesis assay on MatrigelTM 100 µl of 5.25 mg/ml MatrigelTM were added to 96-well plates and allowed to solidify for 1 h at 37°C. HMEC-1 cells were harvested, added to each well and incubated for 6 h at 37°C and 5% CO2. Cells were fixed with 4% formaldehyde and photographed using a DM-IRBE microscope (Leica, Rueil-Malmaison, France) coupled with a digital camera (CCD camera coolsnap FX, Princeton Instruments, Trenton, NJ). The formation of capillary networks was quantitatively evaluated by measuring the total capillary tube length in 5 view fields per well using Metamorph® image analysis software (Roper Scientific, Evry, France) as previously described previously (Pasquier et al., 2004).
2.11. Statistical analysis Otherwise indicated, all data were expressed as mean ± standard deviation of three measurements performed in triplicate and the difference between treated and untreated 9
(control) groups was determined by using one-way analysis of variance (ANOVA) followed by Tukey’s HSD post-hoc test (RStudio, Version 0.97, Boston, USA). Differences between means are considered statistically significant (*: P<0.05; **: P<0.01; *** P<0.001). 3. Results 3.1. Identification of the compound C2 Bioactivity-guided phytochemical study on dichloromethane fraction of dried leaves of Ecballium elaterium led to the isolation and purification of two compounds C1 and C2, the most active being C2. The identity and purity of this compound was assessed using High Performance Liquid Chromatography equipped with a Diode Array Detection, Mass Spectrometry (HPLC-DAD-MS). Chromatogram resulting from LC–ESI–MS analysis of Ecballium elaterium fraction is shown in Fig. 1A. The analysis of commercial cucurbitacin B shows a peak with the same retention time than C2 peak of E. elaterium fraction. Moreover, the UV spectra of the isolated C2 and commercial cucurbitacin B were quite similar (Fig. 1B), the maximum absorption was λMax=234. Besides, the MS fragmentation of the isolated C2 gave the same pattern than that of cucurbitacin B (Fig. 1C) and is consistent with reported values (Cordell and Shin, 1999; Shin and Van Breemen, 2001). The mass spectrum showed a molecular ion at m/z = 559 corresponding to [M+H]+. The exactly mass spectrum of cucurbitacin B shows a molecular ion at m/z = 558 corresponding to molecular formula C32H46O8. The peak at m/z = 499 was due to the loss of CO-CH3 and OH group. Taken together, these results highly suggest that C2 compound is equivalent to cucurbitacin B.
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The purity of the purified product, namely cucurbitacin B, was additionally confirmed by HPLC with UV-absorbance. Our HPLC analysis using MassLynx Software showed only one single peak at a retention time of 20.22 min (Fig. 1D), and peak purity was determined to be 98.5%. Our results intimated that cucurbitacin B with purity >98% could be obtained successfully by HPLC-preparative under the above-optimized conditions.
3.2. Cucurbitacin B inhibits cell adhesion of human glioma cancer cell line by interacting with α5β1 integrins Cytotoxicity of cucurbitacin B from E. elaterium was assessed using release of lactate dehydrogenase (LDH) as a marker for cell membrane integrity. Results show that cucurbitacin B up to 500 nM, did not significantly induce cytotoxicity on the human glioma cell line U87 (see Supplemental data, Fig. S1). Because cucurbitacin B purified from Ecballium elaterium leaves did not display any cytotoxicity, we tested its ability to inhibit cell adhesion of tumour cells on various purified ECM proteins. As illustrated in Fig. 2A, attachment of glioma cells U87 to fibronectin and laminin-1 was strongly decreased by 500 nM of cucurbitacin B, while no effect was observed on type I collagen and vitronectin. The inhibitory activity of cucurbitacin B on cell adhesion to
fibronectin and laminin-1 was dose-dependent, with a half-maximal inhibition (IC50) of 86 nM and 242 nM respectively (Fig. 2B). Moreover, no inhibition could be observed on the integrin-independent substratum polyL-lysine, suggesting that the effect of cucurbitacin B may involve the integrin family of adhesion receptors. To identify the targeted integrins, we checked cucurbitacin B effect on
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various cell/ECM protein pairs involving in each case a unique integrin, based on literature and/or personal data: α1β1 (PC12/type IV collagen), α2β1 (HT1080/type I collagen), α5β1 (K562/fibronectin), α6β4 (HT29- D4/laminin-1), αvβ3 (HT1080/fibrinogen), αvβ5 (HT29D4/vitronectin) and αvβ6 (HT29-D4/fibronectin). As illustrated in Fig. 3A, cucurbitacin B was not able to affect cell adhesion through α2β1, α1β1 or αvβ5 integrins, slightly affected αvβ6 integrin, but significantly reduced the adhesive function of α5β1, α6β4 and αvβ3 integrins. To confirm that cucurbitacin B could interact with α5β1 integrins, we performed adhesion assays to a panel of immobilized antibodies raised against several integrin subunits used as matrix: anti-α5β1 (mAb SAM1), anti-αvβ5 (mAb P1F6), anti-α3 (mAb C3VLa3), anti-α2 (mAb Gi 9), and anti-α6 (mAb GoH3). As illustrated in Fig. 3B, at 500 nM of cucurbitacin B, U87 cell attachment to anti-α5β1 antibodies was dramatically affected (over 97%) and slightly inhibited in the case of anti-α6 antibodies (over 64%), suggesting that the cucurbitacin B exert its effect on cell adhesion by inhibiting α5β1 integrin in human glioma cell lines. However, the cucurbitacin B did not modify U87 cells adhesion on α3 and αvβ5 antibodies, while adhesion on α2 was only slightly decreased.
3.3. Cucurbitacin B affects U87 cell migration Cell motility is a crucial step in the complex multistep process of cancer progression. We thus evaluated cell migration in the presence of cucurbitacin B by using haptotaxis assays towards fibronectin in modified Boyden chambers. Cucurbitacin B abolished cell migration to fibronectin at 500 nM (Fig. 4A). This inhibition was dose-dependent with a half-maximal inhibition (IC50) of 85 nM as shown in Fig. 4B. 12
In a second time, we performed a random two-dimensional motility assay by using timelapse videomicroscopy (Sadok et al., 2008). Representative migration paths (where the origin of each cell track has been set to the coordinates x=0, y=0) for 10 cells are shown in Fig. 5A. We observed that cucurbitacin B dramatically reduced U87 cell motility on fibronectin. As shown in Fig. 5B, analysis of the trajectory obtained from time-lapse recordings of each individual cell during 3h indicated that control untreated cells exhibited a high migration velocity (0.96±0.16 μm/min) (see Supplemental data, Video 1), similar to cells treated with 10 nM cucurbitacin B (0.92±0.14 μm/min). However, treatment with 500 nM of cucurbitacin B dramatically affected the velocity of migration (0.22±0.05 μm/min) (see Supplemental data, Video 2). When compared to control, cucurbitacin B also affected the mean distance to origin (8±0.5 μm versus 47±1 μm ) and directional persistence of migration (0.15±0.02 μm versus 0.49±0.04 μm). These effects of cucurbitacin B on cell velocity and directional persistence may be due to the alteration of protusion formation, as treated cells failed to extend any lamellipodium.
3.4. Cucurbitacin B inhibits proliferation of glioma cancer cells The effect of Cucurbitacin B on U87 cells growth was evaluated in the absence or in the presence of this compound. For all concentrations tested, the number of cells in the wells was reduced by the presence of cucurbitacin B purified from E. elaterium leaves (Fig. 6). This antiproliferative activity was dose-dependent with a half-maximal inhibition (IC50) of 70.14 nM.
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3.5. Cucurbitacin B displays anti-angiogenic activity Cucurbitacin B affects α5β1 integrin-mediated adhesion of U87 cells. As cell adhesion plays a central during angiogenesis, were performed angiogenesis assays using HMEC-1 cells to know whether cucurbitacin B modulates angiogenesis. As shown in Fig. 7A, cucurbitacin B indeed blocked in vitro tubulogenesis with a maximal activity at 500 nM. The inhibitory effect was dose-dependent with an half-maximal inhibition (IC50) of 68.85 nM of cucurbitacin B (Fig. 7B). In order to know whether the integrins inhibited by cucurbitacin B are involved in angiogenesis when using HMEC-1 cells, we performed angiogenesis assays in the presence of function-blocking antibodies against various integrins. Results demonstrated that angiogenesis is mainly supported by α5β1 and, to a lesser extend, by αv integrins (Fig. 7C), the two integrins being targeted by cucurbitacin B.
4. Discussion The cucurbitacins are of great interest because of the wide range of biological activity they exhibit. Several studies were published pointing out that these compounds exhibit anticancer activities on various human cancer cell lines and tumour xenografts (Tannin-Spitz et al., 2007; Wakimoto et al., 2008). The exact mechanism to explain the effect of cucurbitacin B on tumour growth is still unknown. Thus, cucurbitacin B induces apoptosis in laryngeal cancer (Liu et al., 2008). Some reports indicate that cucurbitacin B is a small selective inhibitor of Stat3 signaling pathway in laryngeal squamous cell carcinoma (Liu et al., 2010) and leukemia cell line K562 (Chan et al., 2010). Duabgmano et al. (2012) suggest that cucurbitacin B may inhibit proliferation of human breast cancer cells through disruption of the microtubule network. However, few works have been published on the anticancer effect 14
of the cucurbitacins B on brain cancer. Only Yin et al. (2008) evaluated the antiproliferative activity of cucurbitacin B extracted from Trichosanthes kirilowii Maximowicz in glioblastoma multiforme. The present study shows that cucurbitacin B inhibited cell proliferation of human glioblastoma cell line U87. Yin et al. (2008) also found that cucurbitacin B possessed strong antiproliferative effects on 5 GBM cell lines: U87, U118, U343, U373 and T98G. In our study, we shown using haptotaxis assays in modified Boyden chambers that migration of glioblastoma U87 cells to fibronectin was affected with cucurbitacin B isolated from Ecballium elaterium. Time-lapse videomicroscopy also show a decrease in glioma cell velocity and directional persistence. Migration of cells in higher organisms is mediated by adhesion (Palecek et al., 1997). Results showed that cucurbitacin B impaired adhesion of glioma cell line U87 specifically on fibronectin in a dose-dependent manner. This effect is not due to cytotoxicity, as cucurbitacin B, up to 1000 nM for 5h, did not significantly induce detectable lactate dehydrogenase (LDH) release by U87 cells (Supplemental data, Figure S1). Adhesion to the integrin-independent substratum was not affected, suggesting that the antiadhesive effect of cucurbitacin B was due to inhibition of integrins. This anti-adhesive activity appears to be specific as no effect was observed on the attachment of collagen I or vitronectin. In this study, we show that cucurbitacin B a natural molecule compound purified from Ecballium elaterium leaves, targeted α5β1 glioma integrin. Malignant gliomas are among the most vascularized tumours in humans. Hence it seems to be a logical consequence to research anti-angiogenic agents to prevent and treat malignant gliomas (Tuettenberg et al., 2006). The in vitro Matrigel™ tube formation assay was used with endothelial cells (HMEC-1) to evaluate the effect of cucurbitacin B on the angiogenic process. The present study shows that cucurbitacin B, used at non-toxic levels, inhibited
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angiogenesis at only 100 nM and that this effect was dose-dependent. Moreover, using function-blocking antibodies, we show that α5β1 and, to a lesser extend, αv integrins are involved in the angiogenic process. There are several oncogenic signaling pathways that are commonly involved in cancer cell proliferation and survival. Cucurbitacins seem to affect both cancer cells and normal macrophages through different mechanisms (Lee et al., 2010). In normal macrophages cucurbitacins work as inhibitors of the IKK/NF-κB pathway (Park et al., 2009 ; Escandell et al., 2007). However, in cancer cells, the JAK-STAT, Akt-PKB pathway and MAPK pathways are targets of the cucurbitacin family. It is still not clear how cucurbitacins can selectively choose their target pathway depending on the cell types. Cucurbitacin B has a prominent antiproliferative activity on glioblastoma cells and at least in part, the mode of action is by affecting the cytoskeleton, as well as, the JNK and MAPK pathway (Yin et al., 2008). Thoennissen et al. (2009) discovered that the anti-proliferative effect in pancreatic cancer cells exposed to cucurbitacin B could be explained as a result of inhibition of the JAK-STAT pathway. Cucurbitacin B inhibits human breast cancer cell proliferation through disruption of microtubule polymerization and nucleophosmin/B23 translocation (Duangmano et al., 2012). Although neglected for decades, cucurbitacin is once again attracting attention as a potential anticancer drug. Its role as a JAK/STAT inhibitor, a MAPK modulator, a cytoskeleton disruptor and finally integrin inhibitor qualify it as an excellent candidate for clinical investigation. Integrins have attracted increasing interest for their potential to act as tumour therapeutic targets (Schaffner et al., 2013). The critical role of α5β1 integrin in physiological tumorigenesis and angiogenesis has been recognized in recent years (Morjen et al., 2014).
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Therefore, specific α5β1 integrin antagonists may represent new therapeutic agents to fight defined subpopulations of particularly aggressive tumours (Maglott et al., 2006). However, few α5β1 integrin antagonists have been developed to date. Interestingly, our results suggest that cucurbitacin B purified from E. elaterium seed interferes with α5β1 integrin function on both U87 human glioblastoma and HMEC-1 cells. In agreement with our result, Morjen et al. (2013) showed that PIVL, a Kunitz-type serine protease inhibitor isolated from viper snake venom blocks α5β1 integrin function and inhibited adhesion, migration and invasion of human glioblastoma U87 cells. Maglott et al. (2006) demonstrated that SJ749, an α5β1 integrin antagonist, reduces proliferation, migration and clonogenicity of U87 cell lines and inhibits in vivo angiogenesis of endothelial cells. Zhu et al. (2010) reported that celastrol, a natural compound isolated from the plant Tripterygium wilfordii, inhibits adhesion and proliferation of murine melanoma cell line B16F10 and human lung cancer cell line 95-D. Celastrol targets β1 integrin, whose inhibition is likely responsible for its anti-adhesion effect to fibronectin, which may provide an understanding of its antimetastatic mechanisms. Previous studies show that salvicine, a novel diterpenoid quinone compound, significantly reduces the metastatic potential of MDA-MB435 cells both in vitro and in vivo. Salvicine inhibited the adhesion cells to fibronectin by down-regulating β1 integrin function (Zhou et al., 2008). 5. Conclusion In summary, we report in this paper that cucurbitacin B purified from Ecballium elaterium inhibited integrin-mediated cell adhesion, migration and proliferation of glioma tumour cell line U87 and angiogenesis. To our knowledge, this is the first study describing the inhibitory activities of this triterpenoid on integrin receptors. This effect was mediated by α5β1 17
integrins. Our results provide a new understanding of the anti-cancer activity of cucurbitacin B. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgement This research was supported by Ministry of Education and Scientific Research of Tunisia (MERST), the Institut National de la Santé et de la Recherche Médicale (INSERM) and grants SIRIC and ARCUS.
Fig. 1: (A) LC–PDA–TIC profiles of dichloromethane fraction from E. elaterium leaves and standard cucurbitacin B. (B) UV spectrum of standard cucurbitacin B and compound C2 isolated from the leaves of E. elaterium. (C) Mass spectra of standard cucurbitacin B and compound C2. (D) The purity of cucurbitacin B isolated from Ecballium elaterium was confirmed using MassLynx Software.
Fig. 2: Cucurbitacin B purified from of E. elaterium inhibits tumour cell adhesion. (A) Glioma cells (U87) were preincubated without (black bars) or with cucurbitacin B (500 nM) (grey bars) for 30 min at room temperature. Cells were then added to 96-well microtiter plates coated with different ECM proteins: type I collagen (Col I), fibronectin (Fn), vitronectin (Vn),
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Laminin-1 (Ln), or with poly-L-lysine (PLL). (B) Dose–effect of cucurbitacin B on U87 cell adhesion to fibronectin and laminin-1. Data shown are means (±S.D.) from 3 experiments performed in triplicate and difference between treated and untreated (control) cells was considered statistically significant (*: P<0.05; **: P<0.01; *** P<0.001).
Fig. 3: Cucurbitacin B inhibits human glioblastoma cell adhesion through α5β1. U87 cells were preincubated without (black bars) or with 500 nM cucurbitacin B (grey bars). (A) Adhesion assays were performed with various cell/ECM protein pairs involving unique integrins: α1β1 (PC12/ type IV collagen), α2β1 (HT1080/type I collagen), α5β1 (K562/fibronectin), αvβ3 (HT1080/fibrinogen), αvβ5 (HT29/vitronectin), α6β4 (HT29/laminin) and αvβ6 (HT29/fibronectin). (B) Adhesion was assessed using antibodies: microtiter plates were coated with antibodies raised against the indicated integrin subunits as described in the experimental section. Data shown are means (±S.D.) from 3 experiments performed in triplicate and difference between treated and untreated (control) cells was considered statistically significant (*: P<0.05; **: P<0.01; *** P<0.001).
Fig. 4: The cucurbitacin B inhibits cell migration. (A) Cell motility was determined in a modified Boyden chamber using porous membrane precoated with 10 μg/ml fibronectin. After treatment without or with 500 nM cucurbitacin B for 30 min at room temperature, U87 cells were seeded into the upper reservoir and allowed to migrate through the filter towards 19
the lower reservoir for 5 h at 37°C. Cells that migrated to the underside of the filter were stained with 0.1% crystal violet. Metamorph imaging software was used to capture images of U87 cell that migrate through the filter. (B) Dose–effect of cucurbitacin on cell migration to fibronectin. Data shown are means (±S.D.) from 3 experiments performed in triplicate and difference between treated and untreated (control) cells was considered statistically significant (**: P<0.01; *** P<0.001).
Fig. 5: Evaluation of cell motility by time-lapse videomicroscopy. U87 cells were seeded at 104 cells/well without or with cucurbitacin B on fibronectin pre-coated 24-well plates. Pictures of four fields per well were taken at 5 min intervals during 3 h. The trajectories followed by cells during the 3 h of recording were plotted in (A) with the initial position of each track aligned at 0.0. (B) Cell velocity, directional persistence and mean distance to origin were calculated as described in the Materials section . Data shown are means (±S.D.) from 3 experiments performed in duplicate and difference between treated and untreated (control) cells was considered statistically significant (*: P<0.05; *** P<0.001).
Fig. 6: Cucurbitacin B inhibits U87 cancer cells proliferation. (a) Cells were seeded on 96well plates. After 2h different, concentrations of cucurbitacin B were added and cells were allowed to proliferate for 72h. Cells were quantified by staining with 0.1% crystal violet, solubilization with 1% SDS and measure of absorbance at 600 nm. Data shown are means (±S.D.) from 3 experiments performed in triplicate and difference between treated and untreated (control) cells was considered statistically significant (*: P<0.05; **: P<0.01; *** P<0.001). 20
Fig. 7: Cucurbitacin B blocks in vitro angiogenesis. (A) Representative visualization of tubulogenesis assay after pre-treatment of HMEC-1 cells without (control) or with 10, 100 or 500 nM cucurbitacin B at room temperature. Cells were then added to MatrigelTM and allowed to form capillary-like structures for 6 h at 37 °C. Scale bar: 70 µm. (B) Dose-effect of cucurbitacin B on tubulogenesis on MatrigelTM. (C) Tubulogenesis on MatrigelTM was performed as above in the absence (-) or in the presence of the indicated anti-integrin antibodies (20 µg/ml). Quantification of tubulogenesis was done as described in experimental section. Scale bar: 70 μm. Data shown are means (±S.D.) from 3 experiments performed in triplicate and difference between treated and untreated (control) cells was considered statistically significant (*: P<0.05; **: P<0.01; *** P<0.001).
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Supplementary video 1: Time-lapse videomicroscopy of untreated U87 cells U87 cells were seeded at 104 cells/well without cucurbitacin B on fibronectin pre-coated 24well plates. Pictures were taken at 5 min intervals during 3 h.
Supplementary video 2: Time-lapse videomicroscopy of U87 cells treated with 500 nM of cucurbitacin B U87 cells were seeded at 104 cells/well in the presence of 500 nM of cucurbitacin B on fibronectin pre-coated 24-well plates. Pictures were taken at 5 min intervals during 3 h.
Fig. 1 (A)
(B)
27
Fig. 1 (C)
28
Fig. 1 (D)
29
30
Fig. 2
(A)
(B)
Fig. 3 (A)
(B)
Fig. 4
31
(A) Control
500 nM cucurbitacin
(B)
Fig. 5
(A) Control
Cucurbitacin B (500 nM)
32
(B)
Fig. 6
33
Fig. 7
(A) Control
10 nM
100 nM
500 nM
(B)
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Fig. 7 (C)
35