Revealing the anti-tumoral effect of Algerian Glaucium flavum roots against human cancer cells.
Cell cycle arrest
Glaucium flavum (G. flavum) is a plant from the Papaveraceae family native to Algeria where it is used in local traditional medicine to treat warts. G. flavum root crude alkaloid extract inhibited breast cancer cell proliferation and induced G2/M phase cycle arrest and apoptosis without affecting normal cells, which is a highly awaited feature of potential anti-cancer agents. G. flavum significantly reduced growth and vascularization of human glioma tumors on chicken chorioallantoic membrane (CAM) in vivo. The chromatographic profile of the clichloromethane extract of G. flavum root showed the presence of different constituents including the isoquinoline alkaloid protopine, as the major compound. We report for the first time that G. flavum extract may represent a new promising agent for cancer chemotherapy.
[c] 2013 Elsevier GmbH. All rights reserved.
Breast cancer is one of the most prevalent malignancies in women in many countries worldwide (Jemal et al. 2011; Youlden et al. 2012). After the rapid expansion of the use of monoclonal antibodies and various synthetic inhibitors directed against matrix metalloproteases or protein kinases, natural products are regaining attention in the oncology field. Due to their wide range of biological activities and low toxicity in animal models, natural products have been used as alternative treatments for cancers including breast cancer. An analysis of new and approved drugs for cancer by the United States Food and Drug Administration (FDA) over the period 1981-2010 showed that more than half of cancer drugs were of natural origin (Newman and Cragg 2012).
Cell cycle deregulation resulting in uncontrolled cell proliferation is one of the most frequent alterations that occur during tumor development. For this reason, blockade of the cell cycle is regarded as an effective strategy for eliminating cancer (Lapenna and Giordano 2009; Williams and Stoeber 2012). Key regulator proteins are cyclin-dependent kinases which activity is specifically controlled by cyclins and cyclin-dependent kinase inhibitor (CDKI) at specific points of the cell cycle (Besson et al. 2008). The G2/1V1 checkpoint is the most conspicuous target for many anticancer drugs. P21, a member of the CDKI family and cyclin B1 are the central players of G2/M phase transition (Vermeulen et al. 2003). There is a tight relationship between the control of cell cycle checkpoints and the progression to apoptosis, a mechanism responsible for maintaining tissue homeostasis by mediating the equilibrium between cell proliferation and death. Defective apoptosis represents a major causative factor in the development and progression of cancer (Cotter 2009; Ricci and Zong 2006). In cancer therapy, induction of apoptosis cells is one of the strategies for anticancer drug development (Alam 2003; Fischer and Schulze-Osthoff 2005; Ocker and Hopfner 2012).
Several drugs currently used in chemotherapy were isolated from plant species.The best known are the Vinca alkaloids, vinblastine and vincristine, isolated from Catharanthus roseus, etoposide and teniposide, which are semi-synthetic derivatives of the natural product epipodophyllotoxin, Paclitaxel isolated from the bark of Taxus brevifolia, the semi-synthetic derivatives of camptothecin, irinotecan and topotecan, isolated from Cam ptotheca acuminata, among several others (Cragg et al. 1993).
G. flavum belongs to the family of Papaveraceae. The aerial part of this plant is very rich in isoquinoline alkaloids, especially in aporphine bases namely, didehydroglaucine, 6',7-dehydroglaucine,(+)-glaucine, (+)-isocorydine, (+)-corydine, (+)-cataline, 1,2,9,10-tetramethoxyoxoaporphine, cy-allocryptopine, corunnine, and isoboldine (Israilov et al. 1979; Daskalova et al. 1988). The latter are known for exhibiting promising pharmacological activities including anti-inflammatory, analgesic and antipyretic (Pinto et al. 1998), hypoglycemic (Cabo et al. 2006) and antioxidant activity (Tawaha et al. 2007).
Interestingly, a recent study demonstrated that Sardinian G. flavum contained a homogeneous alkaloid pattern of aporphine type, significantly different from those reported for populations from other parts of Europe (Petitto et al. 2010). In this study, we used G. flavum collected in Algeria where the root is widely used in local traditional medicine to treat warts and inflammatory diseases. To our knowledge, the potential anticancer activities of G. flavum have never been investigated. We decided to evaluate the effects of its alkaloid extract on human normal and malignant cells.
We explored the potential inhibitory growth effect of the dichloromethane extract of G.flavum root on 3 human breast cancer cell lines: MDA-MB-435. MDA-MB-231 and Hs578T and non malignant human cells. Interestingly, G. flavum induced cell cycle arrest and apoptotic cell death in all breast cancer cells tested but not in MCF10A normal mammary epithelial cells. Based on our results in vitro, we decided to explore further the anti-tumoral effect of G. flavum extract using the in vivo tumor chorioallantoic membrane (CAM) model. We demonstrated that G. flavum extract treatment induced a significant decrease in tumor growth and affected tumor associated angiogenesis in vivo. The chemical characterization of the dichloromethane extract was evaluated using EIPLC analysis, which showed the presence of protopine as the major alkaloid.
Materials and methods
Plant material extraction
The root of flowering plant G. flavum was collected in littoral area and far from any contact with pollution in Tichy, province of Bejaia (Algeria) according to botanists (University of Bejaia) previous identification. The alkaloids were extracted as described by Suau et al. (Suau et al. 2004). Briefly, the extraction was undertaken with (10g) of powdered plant material and (100 ml) of methanol in a Soxhlet apparatus. The methanol was evaporated using a rotavapor and the residue was taken up in 2% hydrochloric acid (50 ml), neutral components being removed by filtration. The filtrate was adjusted to pH 8 with aqueous ammonia and extracted with dichloromethane (3 x 25 ml). The resulting extracts were dried with MgSO4 and the solvent evaporated to obtain the crude alkaloid extract. The solid extract was reconstituted in DMSO solvent (50 mg/ml stock solution) and then filtered using 0.22 p.m filters before storage at--20[degrees]C. During all experiments, DMSO dilutions of G. flavum extract were adjusted in the culture media to achieve the indicated final concentrations and control cells were treated at the maximum concentration used in the experiment, 0.1%.
High performance liquid chromatography (HPLC) profiling
The HPLC of dichloromethane root extract of G. flavum (stock solution 20 mg/50 ml) was carried out for identification. The extract was dissolved in methanol and filtered through Acrodisc PSF GXF/GHP 0.45 nm filter. An injection of 10[micro]l of this filtered extract was chromatographed with an Agilent 1100 HPLC with DAD (diode-array detector) detection. The working wavelength was 290 nm. The column was a Polaris amide C-18 column (5 p.m, 250 mm x 4.6 mm) operated at 25 C. The mobile phase was composed of solution A(trifluoroacetic acid 0.05% in water) and solution B (acetonitrile) with the following gradient: equilibration time 15 min at 100% A and linear gradient elution: 0 min 100% A; 1 min 97% A; 45 min 60% A; 55 min 40% A; 65 min 40% A and 66 min 100% A. The flow rate was 1 ml/mm. The structure of protopine was elucidated using NMR spectroscopy ((1) H, (13) C, COSY, HMBC, HSQC), mass spectrometry (MS), and UV spectroscopy.
MCF10A cells (CRL-10317, ATCC) were cultured in DMEM/HAM'S F-12 medium supplemented with 0.01 mg/ml of human insulin, 2.5[micro]M L-glutamine, 20 ng/ml of epidermal growth factor, 0.5 mg/ml of hydrocortisone, 5% horse serum, and 100 ng/ml of cholera toxin. HUVEC (Human Umbilical Vein Endothelial) and skin fibroblast were isolated and maintained in culture as described previously (Jaffe et al. 1973; Rittie and Fisher 2005). MDA-MB-231 (HTB-26, ATCC), MDA-MB-435 (HTB-129, ATCC) cells were grown in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% of fetal bovine serum and 1% L-glutamine. Hs578T cells (HTB-126, ATCC) were cultured in DMEM supplemented with 10 [micro]g/ml of bovine insulin, 1 mM sodium pyruvate and 10% of fetal bovine serum. Human glioma cells U87-MG (89081402, ATCC) were maintained in Minimum Essential Medium with 10% FBS, 2 mM L-glutarnine, 1% non essential amino acid, and 1 mM sodium pyruvate. All the cells were cultured at 37[degrees]C in a humidified atmosphere and 5% C[O.sub.2].
Cell viability was determined using the cell proliferation reagent WST-1 assay according to the manufacturer's instructions (Roche, Basel, Switzerland). All analyzed cells were seeded to obtain 50% of confluence after 24h of incubation in 96-well plates then treated with serial dilutions of the plant extract (0-40 [micro]g/ml). Cells were incubated with the WST-1 reagent for 4 h. After this incubation period, the formazan dye formed is quantified with a scanning multi-well spectrophotometer at 450 nm. The measured absorbance directly correlates to the number of viable cells. Percentages of cell survival were calculated as follows: % cell survival = (absorbance of treated cells/absorbance of cells with vehicle solvent) x 100. The half inhibitory concentration ([IC.sub.50]) was calculated from the dose-response curve obtained by plotting the percentage of cell survival versus the concentration of plant extract used.
Cell cycle analysis
Cells were seeded and incubated overnight to attach, and exposed to DMSO (control) or desired concentrations of G. flavum for specified time periods. Both floating and adherent cells were collected, washed with phosphate buffered saline (PBS), and fixed in 70% ethanol. The cells were then treated with 50 fig/ml RNase A and 5011g/ml propidium iodide for 30 min and analyzed using a FACSCalibur II and the Cell ProQuest program.
Anti-p21 and anti-cyclin B1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-beta actin from Sigma (Saint Louis, Missouri, USA).
Desired cell line was seeded in 6 well plates, allowed to attach overnight and treated according to their respective [IC.sub.50] with G. flavum extract. Both floating and attached cells were collected and lysed into an SDS buffer (SDS 1%, Tris-HCI 40 mM (pH 7.5), EDTA 1 mM, protease inhibitor mixture). Protein concentration was determined using a BCA kit according to manufacturer's instructions (Pierce, Rockford, IL). Equal amounts of proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose or PVDF membrane (lnvitrogen). Membranes were probed with primary antibodies. followed by horseradish peroxidase (HRP)-conjugated secondary antibodies, and developed by chemiluminescence detection. Blots were stripped and re-probed with anti-actin to normalize. Scanned bands were quantified using Image J software Version 1.43 (National Institutes of Health, http://rsb.info.nih.gov/ij/).
DAPI staining method was used to observe apoptotic morphological changes (chromatin condensation and nuclear fragmentation) in treated cells. Cancer cells were treated with G. flavum extract at their respective [IC.sub.50] values and MCFlOA breast cells was treated at the highest [IC.sub.50] observed for cancer cells (15 [micro]/ml) during 24 h. Briefly, the cells were seeded in 6 well plates and treated with G. flavum or with nocodazole (3 [micro]M) used as a positive control of apoptosis inducer. Cells were fixed with paraformaldehyde 3% and incubated in Vectashield solution (Vector Laboratories) for 30min in the dark. Cells were then examined and photographed using a fluorescence microscope (EVOS, AMG).
Quantitation of apoptosis by flow cytometry
Apoptotic cells were determined by Annexin V-FITC (fluorescein isothiocyanate) and non-vital dye P1 staining using an FITC-Annexin V apoptosis detection kit I (BD Biosciences) according to the manufacturer's instructions. Flow cytometry was performed using a FACS Caliber II and samples were analyzed using the CellQuest software (BD Biosciences).
Tumor CAM assay
The implantation of human glioblastoma U87-MG cells on the chorio-allantoic membrane (CAM) of embryonic chicken was performed as we described previously (Lamour et al. 2010). On day 13, size-matched tumors were divided into control and treatment groups. G. flavum extract was deposited locally at 100[micro]g/ml per egg per day. Digital pictures were taken under a stereomicroscope (Leica). On day 17, tumor size was calculated based on tumor volume formula: V= ([d.sub.1]/2)*([d.sub.2]/2)*([d.sub.3]/2 *3.14*4/3, with [d.sub.1], [d.sub.2], [d.sub.3], corresponding three measures taken on the experimental tumors. Quantification of drug effects on tumor cell growth were determined in ten representative tumors per group. For histological studies. U87-MG experimental tumors were embedded in paraffin and cut into 51.1m sections. Tissue sections were stained with hematoxylin and eosin (H&E). Vessels were stained using fluorescein-coupled Sambucus nigra lectin SNA-1 (FL-1301: Vector Laboratories). Statistical comparison between the two groups was performed by using the Student's t test. A value of p <0.05 was considered significant.
Phytochemical profile of G. flavum extract
The HPLC chromatogram of G. flavum root extract (Fig. 1A) revealed several peaks with a significant peak eluting at 24.85 min (peak 1) which is the major compound of dichloromethane extract and was identified as protopine. Fig. 1B shows the structure of protopine.
G. flavum extract treatment specifically decreased the viability of human breast cancer cells We first investigated the effects of G. flavum extract on MDA-MB-231, MDA-MB-435 and Hs578T human breast cancer cell lines. WST1 viability assay was used for the determination of G. flavum extract [IC.sub.50] values in these cells thus establishing the starting point for the next experiments. Cells were treated during 24 h at the following concentrations: 0, 2.5, 5, 10, 20 and 40 [micro]g/ml (Fig. 2A). The treatment significantly affected the viability of all cancer cell lines tested and displayed low [IC.sub.50] values (<151.1.g/ml) after 24 h of treatment (Fig. 26). Following the standard National Cancer Institute (NCI) criteria, an [IC.sub.50] less than 30 [micro]g/ml of crude extract is considered as an active compound against cancer cells (Stiffness and Pezzuto 1990). Next, we tested the effect of G. flavum extract on non malignant human cells such as spontaneously immortalized breast epithelial cells (MCF10A), human umbilical vein endothelial cells (HUVEC) and skin fibroblasts (Fig. 2A). For these cells, the calculated [IC.sub.50] values were higher than 30 [micro]g/ml indicating that G. flavum extract mainly inhibited breast cancer cell viability without affecting normal cells (Fig. 2B).
Cells Malignant human cells Normal human cells MDA MDA Hs578T MCF10A Skin HUVEC -MB -MB fibroblasts -435 -231 [IC.sub.50] 7.9[+ 9.7[+ 13.6[+ > 40 36.5[+ or 33.4[ G. flavum or -] or -] or -] -]0.1 + or ([miceo]g/ml) 0.1 0.3 0.2 -]0.4 Fig. 2. G.flavum extract inhibited the viability of malignant human breast cancer cells (M DA-MB-435, MDA-MB-231. Hs578T) in a dose-dependent manner without affecting normal human cells (MCF1OA human normal mammary cells, human skin fibroblasts and HUVEC): (A) cells were treated with DMSO vehicle or the indicated concentrations of G. flavum extract for 2411. Cell viability was determined using Wst1 assay and expressed as means [+ or -] SD of at least two separate experiments and (B) [IC.sub.50] values of G.flavum extract were determined based on the dose-response curves shown in (A) (means [+ or -] SD of at least two separate experiments).
G. flavum extract treatment caused G2/M phase cell cycle arrest in human breast cancer cells
Next, we tested whether inhibitory effect of G. flavum on breast cancer cell viability was due to perturbations in cell cycle progression. Fig. 3 depicts flow cytometry histograms for cell cycle distribution in G. flavum-treated MDA-MB-231, H5578T and MCF10A cells. After 24 h, C. flavum treatment ([IC.sub.50]) resulted in statistically significant enrichment of G2/M phase cell population in MDA-MB-231 cells as compared with DMSO-treated control cells (44.4% and 19.5%, respectively). In these cells, G. flavum mediated G2/M phase cell cycle arrest accompanied by a significant decrease in GO/G1 (from 62.8% to 26.5%) and S phase cells (from 16.5% to 12.7%). With time, a major increase from 0.6% to 16.2% in subG1 population was observed and corresponded to cells that have lost some of their DNA in late stages of the apoptotic process following endonucleases activity (Fig. 3). Notably, G. flavum-treated Hs578T cells presented with similar cell cycle pattern (Fig. 3). Consistent with the cell viability experiment, G. flavum extract used at the highest cancer cells [IC.sub.50] value (15 [micro]g/ml) did not affect cell cycle distribution of MCF10A cells and stability was generally observed in all cell cycle subpopulations after 12 and 24h of treatment (Fig. 3).
G. flavum extract treatment altered the expression level of proteins involved in the regulation of G2/M transition in MDA-MB-231 cells
To gain insight into the mechanism of G2/M phase cell cycle arrest, we determined the effect of G. flavum treatment on the expression of proteins known to be involved in the regulation of G2/M transition. The level of p21 protein was increased after 12 h of treatment with G. flavum extract and was still high after 48 h. Interestingly, G. flavum treatment caused an early increase in the level of cyclin B1 that was sustained after 24 h and showed a slight decrease at 48 h (Fig. 4).
G. flavum extract treatment induces apoptosis in breast cancer cells in vitro
Staining of cells with DAPI showed morphological features characteristic of apoptotic cells such as DNA fragmentation and condensation of chromatin in breast cancer cells treated with G. flavum extract (I[C.sub.50], 24h) that were comparable to nocodazole treated cells used as control (Fig. 5A). Untreated breast cancer cells and MCF10A cells treated with G. flavum extract exhibited a normal nuclear morphology characterized by large nuclei with distinguishable nucleoli and diffused chromatin structure (Fig. 5A). The quantification of apoptosis was next evaluated by annexin-V/propidium iodide (PI) staining. Dual staining with annexin-V and PI allowed clear discrimination between unaffected cells (annexin-V negative and PI negative), early apoptotic cells (annexin-V positive and PI negative) and late apoptotic cells (annexin-V positive and PI positive). As shown in Fig. 5B, G. flavum extract induced the apparition of an apoptotic sub population in MDA-MB-231 and Hs578T cell lines (24.3% compared to 5% in the control at 24 h and 30.8% compared to 3.4% in the control at 48 h for MDA-MB-231). In accordance with DAPI staining, only minimal cell death was observed with MCF10A cells, even after 48 h of treatment, thus confirming the specific effect of G. flavum on cancer cell viability.
G. flavum extract decreases glioma tumor growth in vivo
To further confirm the anti-tumoral effect of G. flavum extract in vivo, we used a robust and highly reproducible glioma progression model where U87-MG human glioma cells that are grafted onto the vascularized chicken CAM develop into a tumor within a short period of time (Hagedorn et al. 2005). Tumors were treated daily by a local deposition of G. flavum extract (100 [micro]g/ml) or vehicle from the second to the seventh day post-implantation. At the end of the experiment, treated tumors appeared clearly smaller and visibly less vascularized than the control tumors (Fig. 6A). Indeed, the volume of treated experimental glioma was reduced up to 70% when compared to control tumors (Fig. 6B). The hematoxylin and eosin staining of histological sections generally showed a massive necrosis and infiltration of immune cells in G. flavum treated tumors (Fig. 6C, see inserts). Finally, the selective lectin staining of tumoral vasculature demonstrated noticeable differences between treated and non-treated tumors. Treated tumors consistently showed smaller vessels presenting with reduced lumen when compared with vessels in untreated tumor (Fig. 6D).
Medicinal herbs and plants continue to play a significant role in drug discovery and development, particularly in cancer research. Previous phytochemical analysis of G. flavum has shown that it is rich in several aporphine alkaloids: glaucine, isocorydine, protopine and isoboldine (Yakhontova et al. 1973). More recent studies reported the presence of other minor alkaloids such as adihydrochelirubine, dihydrosanguinarine, norsanguinarine and dihydrochelerythrine. Several other plants of the genus Papaveraceae are nowadays used to treat human tumors including Chelidonium majus, Sanguinaria canadensis L., and Macleaya cordata (Ahmad et al. 2000; Chmura et al. 2000). To date no study has reported anti-neoplasic activity for G. flavum.
In this study, we present the first evidence that G. flavum alkaloid root extract exerts a tumor cell growth inhibitory activity by using in vitro and in vivo experimental models. We show that G. flavum extract decreased the viability of all breast cancer cell lines analyzed in this study in a dose specific manner, while it did not affect human normal cells including mammary epithelial cells, fibroblasts and endothelial cells. A previous study demonstrated that at low concentrations, sanguinarine strongly inhibited the growth of all tested tumor and normal cell lines. With normal human fibroblasts showing a similar sensitivity to that of cancer cells, and no differential cytotoxicity could be observed (Debiton et al. 2003). In contrast, G.flavum extract exhibited a selective effect on cancer cells suggesting that this effect could be attributed to the major alkaloids of this plant (aporphine alkaloids) rather than to the minor quaternary benzo[c]phenanthridine alkaloids (e.g. sanguinarine). Interestingly, the HPLC profile revealed that the main compound of the root of G. flavum is protopine. It has been recently reported that protopine exhibited an anti-proliferative effect by induction of tubulin polymerization and mitotic arrest on human hormone refractory prostate cancer cells (Chen et al. 2012). This evidence indicates that protopine might be responsible for the anticancer activity of G. flavum dichloromethane extract reported in this study.
Disturbance of the cancer cell cycle is one of the therapeutic targets for development of new anticancer drugs (Carnero 2002). We showed that G. flavum extract induces G2/M arrest on breast cancer cells without affecting the cell cycle distribution in MCF10A cells. We further demonstrated that the anti-proliferative effects of G. flavum extract are linked, at least in part, with the specific induction of p21 expression in breast cancer cells. Cyclin B1 plays an important role in the regulation of G2/M transition. Flow cytometry studies performed on cycling cells reported that the level of cyclin B1 protein accumulates substantially during G2 phase and before cells enter mitosis, peaks during metaphase, and declines rapidly as the cells proceed through anaphase (King et al. 1994; Widrow et al. 1997). We observed an accumulation of cyclin B1 after 12 h while its expression level tended to slightly decrease after 48 h of treatment. As such, cyclin B1 accumulation is a marker of cells stopped in G2 and/or M cell cycle phases.
The process of programmed cell death, or apoptosis is an important homeostatic mechanism that balances cell division and cell death to maintain appropriate cell number in tissues (Elmore 2007). Disturbance of apoptosis pathways is a common feature of cancer cells and thus represents one of the strategies for anticancer drug development. Our findings demonstrate that G. flavum extract significantly inhibited cell viability through the specific induction of apoptosis in breast cancer cells. An interesting finding in the present study is that alkaloid extract of G. flavum committed cells to apoptosis at a concentration that was below the concentration ranges reported for other plant extracts (Cheng et al. 2005). Altogether our results indicate that G. flavum treated MDA-MB-231 show an increased p21 expression, are arrested in G2/M and driven to apoptotic death. Further experiments are needed to dissect cell cycle events and all molecular players associated with G. flavum treatment.
Our in vitro findings urged us to test whether G. flavum extract has anti-tumoral effects in vivo. For this purpose, we used a CAM tumor glioma cell model which allows the evaluation of both tumor growth and tumor associated-angiogenesis in vivo. Interestingly, we observed a significant impact of G. flavum treatment on both processes. Treated experimental tumors were significantly smaller and less vascularized, as they appeared whiter than fully vascularized control tumors. These observations suggest for the first time that G. flavum extract possesses not only an anti-proliferative effect on cancer cells but may also affect endothelial cells and impede angiogenesis. Hematoxylin and eosin staining exhibited large zones of necrosis that could be associated with less vascularized regions in treated tumor sections when compared to untreated tumors. In light of these data, it is tempting to speculate that inhibition of tumor growth in vivo by G. flavum is associated with induction of apoptotic processes and/or limited neovessel formation inside the tumor. Ongoing and further studies will help define the potential anti-angiogenic activity of G. flavum.
In summary, we demonstrate for the first time that G. flavum root extract inhibits the growth of breast cancer cells by causing specific cell cycle arrest in G2/M phase and leading to apoptosis, without affecting normal breast cells. These anticancer effects of G. flavum extract could be attributed to the alkaloid protopine, which is the major compound of the root. This hypothesis remains nevertheless to be confirmed. Besides, additional studies are necessary to identify the possible correlation between the anticancer activity and the major alkaloids present in G. flavum extract to ensure the proper medicinal use of this natural wealth, which could lead to the potential development of an effective cancer chemotherapy agent.
L.B received an Averroes study grant (Erasmus Mundus program and University of Liege). A.B is a Senior Research Associate, P.P is a Televie post-doctoral fellow and A.G is a Televie fellow, all at the National Fund for Scientific Research (FNRS), Belgium. The authors acknowledge the expert technical guidance of Naima Maloujahmoum, Lola Vanoorschot, Pascale Heneaux and Vincent Hennequiere. The authors are thankful for the use of Cell Imaging and Flow Cytometry GIGA Technological Platform at the University of Liege.
* Corresponding author at: Metastasis Research Laboratory, GIGA-Cancer, University of Liege, 4000 Liege, Belgium. Tel.: +32 04 366 25 57; fax: +32 04 366 29 75.
E-mail address: firstname.lastname@example.org (A. Bellahcene).
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Lamine Bournine (a), (b), (c), Sihem Bensalem (a), Paul Peixoto (b), Arnaud Gonzalez (b), Fadila Maiza-Benabdesselam (a), Fatiha Bedjou (a), Jean-Noel Wauters (c), Monique Tits (c), Michel Frederich (c), Vincent Castronovo (b), Akeila Bellahcene (b), *
(a) Laboratory of Plant Biotechnology and Ethnobotany, Faculty of Natural Sciences and Life, University of Bejaia, Bejaia, Algeria
(b) Metastasis Research Laboratory, G1GA-Cancer, University of Liege, Liege, Belgium
(c) Laboratory of Pharmacognosy, C1RM, University of Liege, Liege. Belgium
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|Author:||Bourninea, Lamine; Bensalem, Sihem; Peixoto, Paul; Gonzalez, Arnaud; Maiza-Benabdesselama, Fadila; B|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Oct 15, 2013|
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