Antiproliferative Effect and Induction of Apoptosis by Inula viscosa L. and Retama monosperma L. Extracts in Human Cervical Cancer Cells

L. Benbacer1, N. Merghoub1,2,3, H. El Btaouri3, S. Gmouh4, M. Attaleb1, H. Morjani3, S. Amzazi2 and M. El Mzibri1,* 1Unité de Biologie et Recherche Médicale, Centre National de l'Energie, des Sciences et Techniques Nucléaires (CNESTEN), Rabat 2Laboratoire de Biochimie-Immuniologie, Faculté des Sciences, Rabat 3MEDyC CNRS UMR 6237, UFR Sciences et UFR Pharmacie, Reims 4Unités d’Appui Technique à la Recherche Scientifique, Centre National pour la Recherche Scientifique et Technique, Rabat 1,2,4Morroco 3France


Introduction
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 . 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 Vinca Alkaloids; Taxus diterpenes; Camptotheca Alkaloids; and Podophyllum lignans. Currently, there are 16 new plant-derived compounds being tested in clinical trials and of these 13 are being tested in phase I or II, and 3 are in phase III. Among these compounds, flavopiridol, isolated from the Indian tree Dysoxylum binectariferum and mesoindigo, isolated from the Chinese plant Indigofera tinctoria, have been shown to exhibit anti-cancer effects with lesser toxicity than conventional drugs (Saklani A, 2008).
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 biologica l t e c h n i q u e s t o p r ovide 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: Inula viscosa L. (Ait.), Retama monosperma L. (Boiss.), Ormenis mixta L. (Dumont.), Ormenis eriolepis Coss., Rhamnus lycioides L., Berberis hispanica Bois. and Urginea maritima L. (Baker.) were collected and evaluated for the in vitro cytotoxic effect against SiHa and HeLa cell lines. The selection was made on the basis of their reputation as folk medicines and ethnobotanic informations to treat different illnesses and diseases. The selected plants have been described to exhibit several biological activities. However, the antiproliferative and apoptotic effects of these plants against cervical cancer cells have not yet been explored. The second part of this study, the most active plants were then selected and were evaluated for their potential antiproliferative effects against SiHa and HeLa cells. Furthers assays were used to elucidate its cytotoxic mechanism. www.intechopen.com

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.  (Bellakhdar, 1997) Anti-inflammatory effects (Hernandez, 2007;Máñez, 2007) Antimicrobial activity (Maoz., 1998) Antifungal activity (Cafarchia, 2002) Retama monosperma L. Bois (Fabaceae)

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. Inula viscosa L. and Retama monosperma L. were extracted successively in a Soxhlet with n-hexane and methanol. The resulting extracts were then evaporated by Rotavapor to give dried extracts. The methanol concentrated extract was dissolved in distilled water and was successively extracted with dichloromethane and ethyl acetate. The solvent was evaporated to obtain the crudes extracts, and kept in the dark at +4 °C until tested.

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% heatinactivated 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.

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. 10μL MTT (5mg/mL) was added to each well. After 4 hours incubation, 150μL DMSO were added to dissolve purple formazan crystals, and absorbance was then determined using a spectrophotometer at 590nm. Mitomycin C and vinblastin (~ 95 % HPLC, sigma-Aldrich) were used as a positive control.

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).

Mitochondrial membrane potential (∆Ψ m ) 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 I 590 nm/I 530 nm 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 = I 590 nm/I 530 nm.

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 x10 5 ) 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).

Gas chromatography/mass spectrometry (GC/MS) analysis
The identification of the compounds from I. viscosa hexanic fraction (IV-HE) and R. monosperma dichloromethane fraction (Rm-DF) was performed by (GC/MS) analysis using a Hewlett Packard 5890 II Gaz Chromatograph, equipped with a HP 5972 Mass selective detector and a VB5 ( 5% phenyl ; 95% methylpolisyloxane) capillary column (30 m, 0.25 mm, film thickness 0.25 µm). Injection volume was 1 µl with a splitless; the injector and detector temperatures was held constant at 250. For GC/MS detection an electron ionization system with ionization energy of 70 eV was used. Helium was used as the carrier gas with an inlet pressure of 10.48 psi, corresponding to a flow rate of 1.0 ml/min. The analytical conditions worked the following programme: oven temperature from 60 to 280°C at rate of 16°C min -1 , www.intechopen.com the final temperature of 300°C was held for 10 min. Tentative identification of the compounds was based on the comparison of their relative retention time and spectral mass with those of Nist and Wiley7 library data of the GC/MS system.

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 *p < 0.05; **p < 0.01; ***p < 0.001.

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 Inula viscosa L. and Retama monosperma L. have been found to exhibit marked cytotoxic effect on both SiHa and HeLa cell lines. Their IC 50 values were 54±12 and 99±1 µg/ml in SiHa cells and 60±8 and 112±4 µg/ml in HeLa cells, respectively. The methanolic extract of Ormenis eriolepis Coss., Ormenis mixta L. and Berberis hispanica Boiss. have lower cytotoxic effect on the cancer cell lines tested (Table 2). However, Urginea maritime L. and Rhamnus lycioides L. had insignificant or no cytotoxic effects at tested concentration with IC 50 500µ/ml.
The cytotoxic effect of extracts from Inula viscosa L. and Retama monosperma L.
As evidenced by MTT assays, we found that hexanic (IV-HE) and dichloromethane (IV-DF) extracts from Inula viscosa were able to inhibit cell growth in dose-dependent manner after 72h of treatment, in both cell lines. The IC 50 values for IV-HE on SiHa and HeLa were 9.56±1.68 and 13.17±0.79 μg/ml, respectively. However, for IV-DF, the IC 50 values on SiHa and HeLa were respectively 6.54±1.46 and 22.04±3.31 μg/ml. Retama monosperma dichloromethane fraction (Rm-DF) was the most active extract, exhibiting also cytotoxic activity against both cells lines in dose-dependent manner. Values of IC 50 obtained were 14± and 21±µg/ml, in SiHa and HeLa cell lines respectively (Table 3). The American National Cancer Institute assigns a significant cytotoxic effect of promising anticancer product for future bio-guided studies if it exerts an IC 50 value 30 μg/ml (Suffnes, 1990 Table 3. Cytotoxic effect of extracts and fractions of Retama monosperma L. and Inula viscosa L. extracts against SiHa and HeLa cervical cancer cells. Cells were exposed to different concentrations of extracts for 72h. As determined by MTT assay. Data are expressed as IC 50 values (µg/ml) and are means ± SD of three experiments. Vinblastin was used as a positive control.

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).

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).

Expression of Pro-caspase, Bcl2 and PARP cleavage
Inula viscosa and Retama monosperma extracts were able to induce apoptosis in HeLa and SiHa cells as evidenced by western blot analysis. Activation of caspase-3 causes the cleavage of poly-(ADP-ribose)-polymerase (PARP), a hallmark of apoptosis, to produce an 85 kDa fragment during apoptosis (Tewari, 1995). The results shown here were from two or three representative experiments.
www.intechopen.com 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 antiapoptotic 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).

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 (ΔΨm), release of apoptogenic factors and loss of oxidative phosphorylation (Zimmermann, 2001). To characterize the effect of IV-HE, IV-DF and Rm-DF on the mitochondrial apoptotic pathway, we measured the mitochondrial membrane potential (∆Ψ m ) in SiHa and HeLa cells after treatment for 24h. As shown in (Figure.3), IV-HE, IV-DF and Rm-DF, induced a significant decrease in mitochondrial membrane potential (ΔΨm), in both SiHa and HeLa cells.

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.

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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 Inula viscosa and dichloromethane fractions of both Inula viscosa and Retama monosperma have cytotoxic effects against cervical cancer cell lines SiHa and Hela by inducing apoptotic process. Previous studies have showed that some plant extracts with pronounced cytotoxic in vitro had marked effects in vivo and showed promising potential to be used as an anticancer drugs. Indeed, sesquiterpene lactones, isolated from Carpesium rosulatum, have recently been largely studied for their pharmacological proprieties as anti-neoplastic agents (Ma G, 2009;Moon, 2011;Robinson, 2008;Taylor, 2008). Sesquiterpenes lactones, artemisinin, thapsigargin and parthenolide and many of their synthetic derivatives, are in advanced stage for clinical trials (Ghantous, 2010).
Phytochemicals contained in Inula viscosa L. extracts including like tomentosin and inuviscolide, as evidenced by CG/MS analysis, have been shown recently to possess an antiproliferative and apoptotic effects on human melanoma cell lines (Rozenblat, 2008).
Quinolizidine alkaloids are known to present in Retama monosperma as main active constituents. Quinolizidine alkaloids contained in the dichloromethane fraction of Retama monosperma L. extract, may act as potential in vitro cytotoxic agents against human cervical cancer cells through the induction of apoptosis. In fact, previous reports have shown that quinolizidine alkaloids have been found to elicit a range of biological activities, including antiviral (Ding, 2006), antihypoglycemic (Brukwicki, 2009) and anti-tumoral (Zhang, 2010) activities. www.intechopen.com

Conclusion
The hexanic extract of Inula viscosa and dichloromethane fractions of both Inula viscosa and Retama monosperma showed pronounced cytotoxic effects against cervical cancer cell lines through the inhibition of proliferation and induction of apoptosis caspase-dependent and involving a mitochondria-mediated signaling pathway. Our findings suggest that these extracts might provide compounds which could be potential sources of anticancer drug leads. Further investigation into the isolation, characterization and mechanism of cytotoxic compounds from the selected plants extracts and in vivo are necessary. Moreover it will be interesting to use some in vivo models to evaluate the anti-tumor activity of these plant extracts.