Ethnobotanical data and some reported pharmacological activities of plants species used in this study.
Worldwide cervical cancer is the second most common malignancy in women with nearly a half million new cases diagnosed and 250,000 deaths each year Almost 80% of cases occur in low-income countries, where cervical cancer is the most common cancer in women (WHO, 2009). In spite of recent advances in the development of new anticancer agents, cancer continues to be one of the major causes of death worldwide. Resistance to chemotherapeutic agents remains a principal obstacle in the successful treatment of cancer. Therefore, development and search of novel and effective anticancer agents to overcome resistance have become very important issues.
As other cancers, radiotherapy and chemotherapy are the conventional cancer treatment used nowadays and remain the routine method for the treatment of cervical cancer. These approaches present sole limits related to the cost, problems of unstable efficiency and severe side effects whose reduce the quality of life and discourage patients to observe medication protocols which then lead to the progression of cancer and associated complications. In addition, many of these treatments present limited anti-cancer activities (Mans, 2000). Therefore, development and search of novel and effective anticancer agents to overcome resistance and without severe side effects have become very important issues.
During last decades, natural products have been an important source of chemotherapeutics, more than half of effective cancer drugs can be traced to natural origins (Ma, 2009). Approximately 60% of drugs currently used for cancer treatment have been isolated from natural products (Gordaliza, 2007; Newman, 2007). Currently, medicinal plants constitute a common alternative for cancer treatment in many countries around the world (Gerson-Cwilich, 2006; Tascilar, 2006).
Many candidate compounds that are able to arrest proliferation and induce apoptosis in neoplastic cells have been discovered. These include
In Morocco, medicinal plants have always been associated with cultural behaviour and traditional knowledge. Herbal remedies are frequently used to treat a large variety of ailments and symptoms, like a fever, inflammation, and pain (Gonzalez-Tejero, 2008). However, there is little information about their anti-cancer properties.
Drug discovery from natural sources involves a multidisciplinary approach combining ethnobotanical, phytochemical and biological techniques to provide new chemical compounds for the development of new drugs against various pharmacological targets, including cancer and related complications. Cytotoxic screening models provide important preliminary data to select plant extracts with potential antineoplastic properties. The initial screenings are cell-based assays using established cell lines, in which the toxic effects of plant extracts or isolated compounds can be measured. Most of the clinically used antitumor agents possess significant cytotoxic activity in cell culture systems (Cardellina, 1999).
In the course to contribute to development of new anticancer drugs against cervical cancer, the human cervical carcinoma SiHa and HeLa cell lines, has been used as a model system in this study for screening promising plant materials from folk Moroccan medicine possessing anticancer effect. Thus, seven medicinal plants:
2. Materials and methods
2.1. Plant species
Seven plant species were collected from different regions of Morocco and were identified by Dr. M. Fennane from the Scientific Institute of Rabat. Voucher specimens are kept in the herbarium of institute. Table 1 shows the ethnobotanical data of the investigated plant species, including botanical names, local names, ethnomedical uses, as well as the plant parts employed in this study.
|Plants species (Family)||Place of collection||Part plant collected||Traditional use||pharmacological activities|
|Inula viscosa L. Ait|
|Ain atik Temara||Leaves||Skin diseases, treats cutaneous abcesses, wound healing, Tuberculosis, bronchial infections (Bellakhdar, 1997)||Anti-inflammatory effects (Hernandez, 2007; Máñez, 2007)|
Antimicrobial activity (Maoz., 1998)
Antifungal activity (Cafarchia, 2002)
L. Bois (Fabaceae)
|Leaves||Purgative, vermifuge, antihelmintic, abortive and disinfectant (Benrahmoune, 2003)||No information available|
Bois and Reut.
|Tamahdit||Bark roots||Blood pressure, digestive, disorders, anorexia, urinary system, nephritic, liver and astrointestinal disorders, ocular affections, febrifuge, antileishmania, antitumoral (Bellakhdar, 1997)||Antimicrobial activity (Ai-Rong, 2007)|
Antitumor activities (Fukuda, 1999; Meenakshi, 2007)
|Ormenis eriolepis Coss.|
|Ouarzazat||Aerial part||Stomachic, anthelmintic and antidiabetic (Bellakhdar, 1997)||Antibactrial activities (El Hanbali, 2004)|
Antileishmania activities (El Hanbali, 2005)
Antifungic activity (Amani, 2008)
|Aerial part||Drain the buttons, healing wounds (Haddad, 2003)||Antimicrobial activity (Satrani, 2007)|
|Rhamnus lycioides ssp. Oleoides|
|Leaves||Laxative, diuretic and hepatic affections (Hmamouchi, 2001)||Hypotensive activity (Terencio, 1990)|
|Urginea maritima L. Baker|
|Bulbs||Cardiac failures, whooping-cough, pneumonia, abortive, vipers bites, aphrodisiac, cough, bronchitis and the jaundice, diuretic and internal tumours (Bellakhdar, 1997)||Cytotoxic and antimalarial activities (Sathiyamoorthy, 1999)|
2.2. Plant extracts preparation
The seven Plants were dried and ground finely. 20g of each powdered plant were extracted by absolute methanol (100 ml, three times) for 72 h at room temperature. The extracts were evaporated to dryness under reduced pressure at 40 C. A total of 40 mg of obtained extract were dissolved in dimethyl sulfoxide (DMSO) to give a solution stock to 40 mg/ml and conserved at -20 C until use.
In second part of the study, the most actives plants were submitted to extraction with solvents with different polarities.
2.3. Cell lines
Human cervical cancer SiHa and HeLa cell lines were used in this study. Cells were grown as monolayers in Minimum Essential Medium (MEM) supplemented with 10% heat-inactivated fetal calf serum and 1% Penicillin-Spreptomycin mixture. Cultures were maintained at 37 C in 5% CO2. SiHa and HeLa cell lines were kindly provided by Dr. P. Coursaget, INSERM U618, University François Rabelais, Tours, France.
2.4. Cytotoxicity assay
Cytotoxicity of the plant extracts was determined using the MTT Assay as described previously (Mosmann, 1983). Cells were seeded in 96-well microplates. After 24 h of culture, the cells were treated with different concentrations ranging from 15.6 to 500 μg/ml, in quadruplicate for 48h or 72h incubation. 10
2.5. Detection of the morphological changes associated with apoptosis
SiHa and HeLa cells were cultured on glass chamber slides in 2 well plates and were treated with the IV-HE, IV-DF and Rm-DF for 24h, 48h and 72h at a concentration of 20µg/ml. After incubation, cells were washed with PBS twice and fixed with (4% paraformaldehyde and 0.1% Triton X-100) for 5 min. The cells were then washed with PBS and incubated with Hoecsht 33342 (10µg/ml) (Sigma) at 37 C for 30min. The cells were visualized through fluorescence inverted microscope (Axiovert 200M Zeiss, Germany) equipped with an LD achroplan 40X objective. The images were collected with a CCD cooled camera (Coolsnap HQ, Ropper Scientific).
2.6. Mitochondrial membrane potential (∆Ψm ) measurement
Analysis of mitochondrial membrane potential was carried out using the lipophilic cationic probe, JC-1 (Molecular Probes, Eugene, OR) whose monomer emits at 530 nm (green) after excitation at 500 nm. Depending on the mitochondrial membrane potential, JC-1 is able to form J-aggregates respectively from green to yellow-orange fluorescence emission (590 nm) as mitochondrial membrane becomes more polarized. Therefore, the I590nm/I530nm emission ratio value allows observation of mitochondrial dysfunction. SiHa and HeLa cells were treated with the extract for 24 h or 48h. JC-1 reagent (10µM) was added for 20 min at 37 C in the dark. Cells were then washed with PBS and centrifuged at 1500 rpm, 4 C for 5 min. The pellet was resuspended in 1 ml ice-cold PBS and the measurements were performed using the Spectrofluorometer (RF-5301PC, Shimadzu, Tokyo, Japan). Residual mitochondrial potential as percentage of control was expressed as follows: (R treated/R control) x 100; R = I590 nm/I530 nm.
2.7. Reactive oxygene species (ROS) production
Production of ROS (reactive oxygen species) was monitored via oxidation of the carboxydichlorofluorescein analog probe, C2938. SiHa and HeLa cells (2 x105) were seeded into a 6-well plate and treated with the appropriate concentration of the extract for 24 h. Control and treated cells were washed and stained with 10 µM C2938 (30 min, 37˚C). Fluorescence emission from the oxidized probe was quantified with a Spectrofluorophotometer (RF-5301PC, Shimadzu) (excitation: 488±1 nm; emission: 518±1 nm).
2.8. Western blot analysis
Cells were treated with 20 µg/ml of extracts for (24h, 48h and 72h), scrapped, washed with PBS and lysed in ice-cold lysis buffer (10 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 1mM dithiothreitol, 10µg/ml Leupeptin, 10µg/ml aprotinin, 10% glycerol, 1%Brij (v/v)), placed on ice for 20 and centrifuged at 14,000g for 15 min at 4 C. The amount of protein was determined using the Bio-Rad protein quantification kit. Equal amounts of proteins (25-30µg/ml) was subjected to electrophorese on SDS-polyacrylamide gels and, transferred to a Nitrocellulose membrane by electroblotting. After blocking non-specific sites, the membrane was incubated overnight with appropriate primary antibodies: Monoclonal anti- pro-Caspase 3 (1/700), Monoclonal anti-β actin (dilution 1/5000), Monoclonal anti-BCl2 (1/700) and polyclonal anti- PARP (1/1000). Horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG were used as secondary antibodies and proteins were detected using an enhanced chemiluminescence (ECL) kit.
2.9. Gas chromatography/mass spectrometry (GC/MS) analysis
The identification of the compounds from
2.10. Statistical analysis
Data are presented as means ± SD of at least triplicate or quadruplicate determinations of three different assays. The statistical analysis was performed by student’s-test with Microsoft excel software. Significant differences are indicated by *
3. Results and discussion
3.1. Cytotoxic effect of the medicinal plants extracts from Morocco
Crude extracts of selected plants were made by exhaustive methanol extraction. These plants extracts were tested for their potential cytotoxic effects, SiHa and HeLa cells were treated with plants extracts at different concentrations ranging from 15 to 500 μg/ml for 48h. The cells viability were determined by MTT assay. Among the 7 medicinal plant extracts, methanolic extract from
|Methanolic extracts||IC50 ± SD (μg/ml)|
|Inula viscosa L.||54 ± 12||60 ± 8|
|Ormenis eriolepis Coss.||94 ± 4||112 ± 4|
|Ormenis mixta L.||383 ± 26||311± 14|
|Berberis hispanica Boiss.||178 ± 5||224 ± 10|
|Retama Monosperma L.||99 ± 1||96 ± 4|
|Urginea maritime L.||› 500||› 500|
|Rhamnus lycioides L.||› 500||› 500|
|Mitomycin C||6 ± 1||1 ± 0.30|
Cells were exposed to different concentrations of extracts for 48h. Data are expressed as IC50 values (µg/ml) and are means ± SD of three experiments. Mitomycine was used as positive control.The cytotoxic effect of extracts from
As evidenced by MTT assays, we found that hexanic (IV-HE) and dichloromethane (IV-DF) extracts from
|Retama monosperma L. extracts|
|Hexane extract (Rm-HE)||› 80||› 80|
|Methanol extract (Rm-ME)||› 80||› 80|
|Dichloromethane fraction (Rm-DF)||14.57±4.15||21.33±7.88|
|Acetate ethyle fraction (Rm-AF)||27.54±5.64||77.47±2.25|
|Inula viscosa L. extracts|
|Hexane extract (IV-HE)||9.56 ± 1.68||13.17±0.79|
|Methanol extract (IV-ME)||52.83±3.28||› 80|
|Dichloromethane fraction (IV-DF)||6.54±1.46||22.04±3.31|
|Ethyl acetate fraction (IV-AF)||63.62±10.55||› 80|
Cells were exposed to different concentrations of extracts for 72h. As determined by MTT assay. Data are expressed as IC50 values (µg/ml) and are means ± SD of three experiments. Vinblastin was used as a positive control.
3.2. Chemical identification of plants extracts
Analyses of the most active extracts by gas chromatography (GC) coupled with and GC-mass spectrometry (MS) revealed the presence of a sesquiterpene acid: isocostic acid (46.05%) and two sesquiterpenes lactones: tomentosin (33.27%) and inuviscolide (13.04%), as major compounds in IV-HE extract (Table 4). In the fact,
|Phenanthrene, 7-ethenyl-1,2,3,4,4α,4β,5,6,7,8,10,10α- dodecahydro-4α,7-dimethyl-1-methylene-, [4αS-(4αα',4βα',7α',10αα')]-||42.74||0.69|
|6,9,12,15-Docosatetraenoic acid, methyl ester||46.82||0.37|
|IV-DF||Benzeneacetic acid, α',4-bis[(trimethylsilyl)oxy]-, trimethylsilyl ester||18 .91||10.44|
|10 hydroxy-1 ,4,5,8-Tetramethyl anthrone||44.84||2.54|
|2,4,7 Trimethyl-5 ,6-diphenyl-1H-isoindol-1 ,3(2H)-dione||47.01||9.12|
However, CG/MS analysis of Rm-DF (Table 5) revealed the presence of five known quinolizidine alkaloids as well as, sparteine (10.97%), L- methyl cytisine (9.11%), 17-oxosparteine (3.49%), lupanine (0.93%) and anagyrine (39.63%). The Retama species have been reported to contain alkaloids (Abdel Halim, 1997) and flavonoids (Kassem, 2000). Fifteen quinolizidine and 3 dipiperidine alkaloids were isolated from the leaves of flowering plants of
|Benzeneacetic acid, α,4-bis[(trimethylsilyl)oxy]-,||18.91||4.71|
|L methyl cytisine||44,23||9.11|
3.3. Molecular mechanisms of apoptosis signalling pathways
Induction of apoptosis constitute an important mechanism for anticancer effects of many naturally occurring and synthetic agents. Activation of apoptotic pathways seems to be an effective strategy against tumor progression (Brown, 2005). The caspase pathway plays a pivotal role in the induction, transduction and amplification of intracellular apoptotic signals. Among the caspase family proteins, capase-3 is responsible for the proteolytic cleavage of many key proteins such as PARP, which is considered as a marker of apoptosis (Kothakota, 1997; Wang, 2005).
3.4. IV-HE, IV-DF and Rm-DF induced apoptosis in SiHa and HeLa cells
In order to determine whether plant extracts induced cell death was due to apoptosis, we analyzed chromatin condensation and nuclear fragmentation by Hoechst 33342 staining and fluorescence microscopy (Kerr, 1994). SiHa and HeLa cells were treated with IV-HE, IV-DF and Rm-DF for 24h, 48h and 72h. As shown in Figure 1, the rate of apoptotic cells was increased significantly in a time-dependant manner after treatment with IV-HE, IV-DF and Rm-DF (Figure.1).
3.5. Expression of Pro-caspase, Bcl2 and PARP cleavage
suggests that apoptosis induced by IV-HE, IV-DF and Rm-DF could be associated with a caspase-dependent pathway.
The activation and function of caspases are regulated by various key of molecules, such as inhibitors of apoptosis protein, Bcl-2 protein family. Increased expression of the anti-apoptotic protein Bcl-2 causes resistance to chemotherapeutic drugs, while decreasing Bcl-2 expression may promote apoptotic responses to anticancer drugs (Reed J.C., 1994). Our investigations showed a significant decrease in Bcl-2 expression after 24h treatment with IV-HE, IV-DF and Rm-DF (Figure 2A, 2B).
3.6. Statut of mitochondrial membrane potential
Mitochondrial dysfunction has been shown to participate in the induction of apoptosis. Indeed, opening of the mitochondrial permeability transition pore has been demonstrated to induce depolarization of the transmembrane potential (
3.7. Measurement of ROS production
Mitochondria are a source of ROS during apoptosis and reduced mitochondria membrane potential leads to increased generation of ROS and apoptosis (Zamzami, 1995). We investigate whether the intracellular ROS are involved in the signal transduction pathways of apoptosis. ROS generation was measured after cells treatment with IV-HE, IV-DF and Rm-DF (20µg/ml) for 24h, using a ROS-sensitive fluorescent C2938 probe. Tested extracts showed a dose-dependent increase in the intracellular ROS production when compared to the control (Figure.4). This indicate that ROS generation induced by IV-HE, IV-DF and Rm-DF in SiHa and HeLa cells can contribute to apoptosis via the mitochondrial pathway.
Taken together, these results show clearly that the hexanic extract of
Phytochemicals contained in
Quinolizidine alkaloids are known to present in
The hexanic extract of