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Pro-apoptotic and anti-adhesive effects of four African plant extracts on the breast cancer cell line MCF-7
© Engel et al.; licensee BioMed Central Ltd. 2014
Received: 6 December 2013
Accepted: 27 August 2014
Published: 9 September 2014
Jatropha curcas (JCP1), Pyrenacantha staudtii (PS), Picralima nitida (ZI) and Jatropha gossypifolia (JCP2) are plants used in the African folklore for the treatment of various cancers.
This study investigated the in vitro anticancer effects of the ethanol extracts against human epithelial MCF-7 breast cancer cells in a dose-dependent manner (1–50 μg/ml) by using cell cycle analysis, viability assay, annexin V/PI staining, TUNEL method and expression determination of apoptotic and adhesion relevant proteins. Adhesion processes were monitored by detachment via flow cytometry, β1-integrin expression and formation of the actin cytoskeleton.
The three extracts, termed PS, JCP1 and JCP2 at a concentration of 10 μg/ml induced cell death in MCF-7 breast cancer cells verified by high amounts of PI-positive cells in the cell cycle analysis, Annexin V/PI staining and DNA fragmentation measurements. In parallel cell detachment was accompanied by decreased β1- integrin expression and phosphorylation of the focal adhesion kinase at Tyr397. ZI extract was the exception by the increasing β1-integrin expression and strengthening the cortical actin cytoskeleton. However, all four plant extracts mediated strong anti-cancer properties with IC50 values between 23–38 μg/ml.
PS, JCP1 and JCP2 were found to be very active against MCF-7 cells by inducing anoikis and therefore possessing vast potential as medicinal drugs especially in estrogen receptor positive breast cancer treatment. ZI mediated their anti-cancer action by different signaling mechanisms which should be analyzed in future studies. Our results further supported the idea that medicinal plants can be promising sources of putative anticancer agents.
The use of natural products including medicinal plants has become more and more important in primary health care especially in developing countries. Many pharmacognostical and pharmacological investigations are carried out to identify new drugs or to find new lead structures to develop novel therapeutic agents for the treatment of human diseases such as cancer . In developing countries and particularly in Yemen, a large segment of the population still rely on folk medicine to treat serious diseases including infections, cancer and different types of inflammations.
Currently, there is insufficient scientific research on the plants from Nigeria. Previous studies described the anticancer investigations of some endemic and non-endemic plants from Nigeria . This study was carried out as a part of our continued exploration of Nigerian medicinal plants for interesting biological activities. Thus, the main aim of the present project was to carry out a phytochemical and cell biological investigation on selected plants from the southern part of Nigeria, especially on those that are endemic and those that find a use in traditional medicine as anticancer agents. In this study, four plants were collected for evaluation of their antitumor activities with respect to pro-apoptotic and anti-adhesive properties.
Picralima nitida (Stapf.) Th. & H. Durand (Flowering plant family: Apocynaceae) has widely varied applications in Nigeria folk medicine for antipyretic, antihypertensive, gastro-intestinal disorders, as an antimalarial, aphrodisiac, antitrypanocidal, and as a remedy against hyperglycemia [3–6]. The plant is used in Nigeria and West Africa as remedy against breast cancer .
Pyrenacantha staudtii Hutch and Dalz (Tropical forest tree family: Icacinaceae) is a medicinal plant widely used in tropical Africa for the treatment of various ailments such as stomach disorders, intestinal colic, menstrual disorders, and as anticancer and antiabortificient agents [8, 9].
Jatropha gossypifolia L (Euphorbiaceae) is widely distributed in many tropical countries [10, 11]. It has antibacterial, antiinflammatory, analgesic and anticancer activities . It was known in ancient medicine for its ethnomedicinal uses in the treatment of cancerous growth and for its pesticidal activity [13, 14].
Jatropha curcas Linnaeus (Euphorbiaceae) is a small tree or large shrub that can reach a height up to 5 m. It is used in traditional medicine as remedy against cough, cancer, and human immunodeficiency virus . The local populace in the eastern part of Nigeria uses the ethanol extract for the treatment of breast cancer. In some cases, traditional herbal practitioners use the aqueous decoction to cure cancer . The search for anticancer agents via activity directed identification and characterization lead to the present study with a view to validating the claimed ethno-medicinal property of these plants as anticancer remedy. Hence, this study is focused totally on the exposure of the ethanol root bark extracts of the plants to MCF-7 cancer cell line in a dose-dependent manner.
Collection and identification of plant materials
Properties of the four Nigerian plants used in this study
*Jatropha curcas Linn
cough, wound healing, HIV, cancer
*Pyrenacantha staudtii Hutch & Dalz
threatened abortion, malaria, GIT and cancer
*Picralima nitida Th. & H. Durand
malaria, hyperglyaceamia, antiseptic etc.
*Jatropha gossypifolia Linn
Preparation of plant extracts
The powdered plant samples (100 g) were each extracted by maceration, with ethanol (250 ml) at room temperature, and concentrated to dryness using a rotary evaporator at reduced pressure. The% yield (10, 23, 40 and 51 for JCP1, PS, ZI and JCP2, respectively) was obtained. Dried samples were stored at −20°C until further use. Finally, all plant extracts were dissolved in dimethylsulfoxide (DMSO) to give a desired stock solution of 50 mg/ml, which was aliquoted and stored at −80°C.
Phytochemical composition of extracts
The ethanol extracts were subjected to photochemical screening in order to identify the secondary metabolites and nature of the extracts. The method employed, was from Trease and Evans .
Human breast adenocarcinoma cell line MCF-7 (ATCC no. HTB-22) was obtained from the America Type Culture Collection (Manassas VA, USA). Cells were maintained at 37°C and in a 5% CO2 atmosphere in a monolayer in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Germany) with 10% fetal bovine serum (PAA Laboratories GmbH, Germany) and 1% gentamycin (Ratiopharm, Germany). Confluent cells were passaged by treating them with 0.05% trypsin/ 0.02% EDTA. The medium was changed every two days. MCF-7 cells were authenticated by morphology and growth rate and were mycoplasma free. Cultivation conditions were described previously .
Treatment with plant extracts
Treatment conditions were previously described . Treatments with the four plant extracts (final concentrations of 1, 10, 25, 50 μg/ml) were carried out for 48 h in assay medium. As negative control substance the vehicle dimethylsulfoxide (DMSO, 0.1%) was used in the same manner.
Cell cycle analysis
To determine proliferation and apoptosis alterations, the cell cycle analysis via flow cytometry (FACSCalibur, BD Biosciences) after propidium iodide staining (50 mg/ml) of the MCF-7 cells was carried out [17, 18]. For data acquisition, the software FlowJo version 7.6.5 (Tree Star; http://www.flowjo.com) was used. A minimum of 15,000 ungated events were recorded. For statistical analysis, the S-phase and G2/M-phase cells of the cell cycle were defined as proliferative cells and the sub-G1-peak of the histogram as apoptotic ones.
Annexin V/PI apoptosis detection
In this assay, Annexin-V detects the translocation of phosphatidylserine from the inner leaflets to the outer leaflets of the plasma membrane, which is a key feature of apoptotic cells, whereas PI detects necrotic cells with permeabilized plasma membrane. Labeling of early apoptotic and dead cells was performed according to the manufacturer's instructions from the Alexa Fluor488 Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher Seintific Inc., Germany). Cells were treated with 10 μg/ml plant extract for 48 h. After treatment detached as well as adherent cells were washed twice with cold PBS. The cell pellet was resuspended in 100 μl of annexin binding buffer at a density of 1 × 106 cells per ml and incubated with 5 μl of Alexa488-conjugated Annexin-V and 5 μl of PI for 15 min at room temperature in the dark. 400 μl of 1× binding buffer was added to each sample tube, and the samples were immediately analyzed by flow cytometry. Histograms and statistics were designed with the software FlowJo Version 7.6.5.
Calculation of cell detachment
400,000 MCF-7 cells were seeded in 6-well plates (Greiner, Germany). After treatment with the four plant extracts (10 μg/ml) and the DMSO control detached cells were counted by flow cytometry.
Measurement of integrin expression
Measurement and calculation of β1-integrin expression at the cell surface by flow cytometry (FACSCalibur) was described previously . Anti-integrin antibody β1 (CD29; Immunotech, 0.2 mg/ml, mouse anti-human 4B4, Isotype: IgG1) was secondarily labeled with fluorescein isothiocyanate-conjugated anti-mouse IgG (Fab2) fragment (Sigma). Ten thousand events were recorded for each measurement and each measurement was repeated three times.
Immunofluorescence and microscopy
Filamentous (F)-actin was selectively labeled with BODIPY® FL phallacidin emitting green fluorescence (Invitrogen, Germany). Nuclei were stained with Hoechst dye (Invitrogen, Germany). Bright field and all fluorescence images were obtained using Axio Scope A1 fluorescence microscope (Carl Zeiss, Germany). Individual fluorophores were imaged in black and white for maximum sensitivity and pseudocolored and overlaid using AxioVision Imaging Software Release 4.8.2. (Carl Zeiss, Germany).
Western blotting procedure
After treatment with the plant extracts for at least 48 h the cells were trypsinized, washed with PBS and lysed in ice-cold lysis buffer (Bio-Plex Cell Lysis Kit, Bio-Rad, USA). Cells were homogenized by brief sonification at 4°C and centrifuged at 10,000 g for 2 min at 4°C. Protein concentrations of the supernatants were estimated by Bradford protein assay so that equal amounts (10 μg) of total soluble protein could be separated by Criterion TGX Stain-Free precast gels (Bio-Rad, Germany) and blotted on PVDF membranes. After SDS-PAGE, protein content per lane as well separation quality was additionally controlled with the Criterion Stain FreeTM gel imaging system (Bio-Rad, Germany). After the protein transfer membranes were blocked with 5% skim milk in Tris buffered saline (TBS) and washed six times in TBS. For protein detection primary antibodies (β1 integrin: sc- 374429; PCNA: sc- sc-56; both from Santa Cruz, USA; caspase 7, 8, 9 from the Apoptosis sampler kit #9915; FAK antibodies within the sampler kit #9330; Akt #4691; pAkt (S473) #9271; p44/42 MAPK #9102; P-p44/42 MAPK (T202/204) #4377; β-Actin #4970: all from Cell Signaling, USA) were incubated overnight at 4°C followed by labeling with a horseradish peroxidase (HPR)-conjugated secondary antibody (Dako, Glostrup, Denmark) for 1 hour at room temperature. Protein signals were visualized by using SuperSignal West Femto Chemiluminescent Substrate (Pierce Biotechnology, Rockford, USA) for detection of peroxidase activity from HRP-conjugated antibodies (Thermo Fisher Scientific Inc., Rockford, USA). Band intensity was analyzed densitometrically with the Molecular Imager ChemiDoc XRS and Image Lab 3.0.1 software (Bio- Rad, USA). Protein detection was repeated at least three times with individual prepared cell lysates from independent passaged cells.
Apoptotic DNA degradation was stained using the terminal deoxynucleotidyl transferase (TdT)-mediated dUDP-biotin nick end labeling (TUNEL) method. In this study, the In Situ Cell Death Detection Kit, Fluorescein (Roche, USA) was used for this purpose according to the manufacturer’s protocol. Briefly, MCF-7 cells (1.5 × 106 cells/well) were cultured on cover glasses in 6-well plates. After exposure to the plant extracts, cells were washed with PBS, fixed with 4% paraformaldehyde solution for 1 h at 15–25°C, and incubated in permeabilisation solution for 2 min on ice. After washing with PBS cells were incubated with the TUNEL reaction mixture for 60 min at 37°C in a humidified atmosphere in the dark.
Fluorescence of the stained cells was observed using a Carl-Zeiss confocal laser scanning microscope (LSM 780, Jena, Germany) with an excitation wavelength of 488 nm.
Calculation of IC50 values
The half maximal inhibitory concentrations (IC50) values were calculated by colorimetric measurements of mitochondrial metabolic activity with the CellTiter MTS/PES assay following to the manufacturer’s instructions (Promega Corp., Madison, WI). MTS is a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] which is combined with an electron coupling reagent (phenazine ethosulfate; PES) to form a stable solution. The conversion to formazan is bioreduced by cells which is presumably accomplished by NADPH or NADH produced by dehydrogenase enzymes in metabolically active cells. The measured mitochondrial metabolic activity also reflects the cell cytotoxicity directly and the cell viability indirectly.
Cells were seeded in 96-well plates at a density of 2000 cells/well in 100 μl medium and left to attach for 24 h. Treatment with plant extracts at final concentrations of 1, 10, 25, and 50 μg/ml was carried out as described previously. In parallel, control approaches were carried out with medium only and 0.1% of the solvent DMSO to calculate background absorbance. No background absorbance was obtained for the extracts and MTS in the absence of cells, as some extracts are capable of reducing the MTS. After an initial incubation for 24 h cells were assayed with MTS according to the manufacturer’s instructions (Promega Corp., Madison, WI). Colorimetric changes were measured at 490 nm and raw data was transferred to Microsoft Excel and analyzed. At least 8 replicates corrected with the background absorbance were performed. Reduction of cell viability at each concentration was plotted as a dose response curve. The IC50 of the active extracts were calculated using nonlinear regression to fit data to the dose–response.
Live/dead cell staining
Live/Dead cell staining was performed following the manufacturer's instructions (Live/Dead Cell Staining Kit II, PromoCell GmbH, Germany).
All data were analyzed by the Students t-test using Microsoft Excel 2010. Every experiment was done in triplicate with individual passaged cells and data sets were expressed as means ± standard deviations (SD). Statistical significance was represented as ***P < 0.001, **P < 0.01, *P < 0.05.
Results and discussion
Determined substance classes
Proliferation and apoptosis events after treatment with the plant extracts
All four plant extracts caused a significant influence on the proliferation phases of the MCF-7 cells, which turned out to be concentration dependent but not always linear. To quantify proliferation we defined the cell cycle phases S and G2/M as proliferative phases so that the sum of it describes the proliferation rate. In parallel, the apoptotic rates were measured by determination of the sub G1-peak showing DNA fragmentation events. Both results were displayed in Figure 1B. As negative control the DMSO treated cells were set to 100%. As positive controls genistein was used which results were previously reported . Only the PS extract mediated an almost linear reduction in proliferation. In contrast, the ZI extract showed a biphasic effect. Low concentrations (1–10 μg/ml) caused an increase in the proliferative phases and at a concentration >25 μg/ml, the proliferation was significantly reduced. JCP1 induced only a slight proliferation induction after an exposure of 25 μg/ml accompanied by a significant elevation of sub-G1 positive cells. This effect of proliferation and apoptosis induction at the same time is noted by a number of studies have which have shown that cell-cycle regulators could interconnect with proliferation and apoptosis . In contrast, JCP2 displayed a bi-phasic effect on the distribution of the MCF-7 cell cycle phases. At the lowest concentration of 1 μg/ml the content of the G2/M phase nearly doubled in comparison to the DMSO control. Analogously to the PS extract, a JCP2 concentration of 10 μg/ml caused also a significant reduction of G2/M phase and an arrest in G0/G1 phase.
But ultimately, all four extracts mediate a significant increase in sub-G1 phase. The extracts JCP1 and JCP2, already caused at the low concentration of 10 μg/ml a significant increase in the sub-G1 phase. In summary, all four plant extracts were able to influence the proliferation rates of the estrogen receptor-positive cell line MCF-7 depending on the concentrations used. This phenomenon is not uncommon for some effective anti-cancer extracts. For example, soy ingredients like genistein causes biphasic effects on hormone-dependent cancer cell lines . At low concentrations (1–10 μM), genistein stimulates cell proliferation whereas higher concentrations are able to induce a block in G2/M phase. This result suggests that the plant extracts contain substances that trigger similar biphasic effects, known as phytoestrogens such as genistein. In subsequent experiments, the exact ingredients of the extracts by high-performance liquid chromatography (HPLC) and gas chromatography–mass spectrometry (GC-MS) will be analyzed to obtain a deeper insight into the existing drug classes.
Plant extracts induced cell detachment mediated by decreased β1 integrin expression
Detachment from the extracellular matrix or lost contact with the neighbor cells is a clear indication of induction of an apoptotic process that is termed anoikis . This observation confirmed the results of the cell cycle measurements that showed a significant increase of apoptotic cells after treatment with 50 μg/ml of the four plant extracts. To confirm the induction of anoikis, which is accompanied by cell detachment, we performed live/dead staining of the cell after exposure to 10 μg/ml of the extracts. These staining demonstrated that only attached cells were viable (green fluorescence) while detachment by the extracts caused cell death (red fluorescence). Moreover, this test showed that the concentration of 10 μg/ml of each extract led to a different degree of cell detachment. Treatment with JCP1 and JCP2 resulted in the highest cell detachment rates (approximately 60 - 90%), so that nearly no viable cells were attached to the cell culture plates. The ZI extract exhibited the lowest rate of cell detachment and only a few dead cells, which correlated with the results of the cell cycle measurements where the ZI extract at a concentration of 10 μg/ml showed a significantly increased proliferative phase.
These findings suggest that the plant extracts target different signaling pathways in the MCF-7 cells and thus initiate various mechanisms of cell detachment. Loss of integrin binding to extracellular matrix proteins results in impairment of cell spreading which depends on the integrity of the internal actin cytoskeleton formation . This assertion is supported by the fact that the formation of the actin cytoskeleton is affected differently by the plant extracts (Figure 5B). Under control conditions MCF-7 cells form a diffuse actin cytoskeleton without any visible stress fibers. The actin filaments are short and find to be enriched at sides of cell-cell contacts (Figure 5B; red arrows). JCP1, JCP2 and PS revealed a distinct alteration of the actin organization. No strong accumulation of actin in adjacent cells could be observed in comparison with controls. By treatment with the PS and JCP1 strong cellular contacts were diminished. Cells seem to be enlarged in their areas and express filopodia. Consistent with the increased β1-integrin expression by ZI increased formation of actin filaments was observed between neighboring cells (Figure 5B, red arrows). In addition, the western blot results confirm that after treatment with JCP1 and JCP2 the β-actin filaments were degraded: further evidence for the induction of anoikis (Figure 3C).However, the three plant extracts (JCP1, JCP2 and PS) harbor anticancer potential, which should be analyzed in further studies. Especially the signaling mechanisms should be identified because initial analysis of central signaling molecules like the serine/threonine-specific protein kinase Akt and mitogen-activated protein kinases (MAPKs) showed alterations in their expression levels (Figure 3C).
Plant extracts harbor IC50 values between 23 and 38 μg/ml
Anticancer screening experiments for bioactive agents from natural products are in focus of current research. But effective screening methods are rare and sometimes difficult to interpret. Therefore in this study standard methods were chosen for the interpretation of the influence on proliferation (cell cycle measurement, PCNA expression by Western blotting), apoptosis (cell cycle measurement, Annexin V/PI labeling, TUNEL assay, live-dead staining, caspase claevage), morphology (bright field imaging), metabolism (MTS assay) and adhesion (β1-integrin expression, F-actin staining, cell detachment). These methods allow an overview of the potential effectiveness of the extracts on the key mechanisms of cancer cells, but do not allow a clear identification of signaling pathways. All four plant extracts displayed distinct alterations on these major cellular mechanisms in the breast cancer cell line MCF-7. These results reveal an anti-tumorigenic potential of the four plant extract. But the dose response curves and calculated IC50 values also indicate a general cytotoxic activity. This could be due to the mixture of ingredients within the extracts. Accordingly, in subsequent experiments the active substance classes will be identified and fractionated. The overall goal for future work is to isolate the active compounds and their testing on different cancer cell lines.
Interestingly, the PS, JCP1 and JCP2 plant extracts induced cell rounding and cell detachment at concentrations ≥10 μg/ml. Cell detachment could be caused by several processes. One protein relevant for cancer cell progression and metastasis is the β1-integrin . This finding implicates that the ZI extracts influences different cellular pathway compared with other extracts. However, the four tested plant extracts exert cytotoxic and anticancer potential which should be investigated in further studies.
In conclusion, the results in the present study coincide to some extent with the traditional uses of the plants investigated. Our results further supported the idea that medicinal plants can be promising sources of potential anticancer, antimicrobial and antioxidants agents. The present results will form the basis for selection of plant species for further investigation in the potential discovery of new natural bioactive compounds. Studies aimed at the isolation and structure elucidation of anticancer chemical constituents are in progress.
We would like to thank Deutsche Krebshilfe (FKZ: 107821) and the habilitation scholarship program of University of Rostock for the funding of our work. Special thanks to the University of Benin for the facilities. Funding from DFG-TWAS 2010–12 is highly acknowledged.
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