Jordanian Ducrosia flabellifolia inhibits proliferation of breast cancer cells by inducing apoptosis

The potential apoptosis inducing effect of Ducrosia flabellifolia ethanol extract was evaluated in this study. The antiproliferative acti vity was tested against three cell lines using MTT assay. The apoptosis induction ability wa s determined using TUNEL colorimetric assay and agarose gel electrophoresis was used to detect DNA fragmentation. Morphological changes associated wit h apoptosis were observed using scanning electron microscopy. LC/MS-MS analysis was used to determine the main flavonoids present in the plant extract. Ducrosia flabellifolia ethanol extract showed selective antiproliferative activity against MCF-7 cells, with IC50 values of 25.34, 98.01, and 87.50 μg/mL, against MCF-7, Hep-2, and Vero cell lines, re p ctively. The antiproliferative effect was exerted by inducing ap optosis as indicted by the presence of DNA fragmentation, nuclear condensation, and for mation of apoptotic bodies in treated cancer cells. LC/MS-MS analysis revealed th presence of five flavonoids (quercetin, fisetin, kaempferol, luteolin, and apig enin) and their derivatives in the extract. This is the first study reporting the anti proliferative effects of Ducrosia flabellifolia. The apoptosis inducing ability of Ducrosia flabellifolia ethanol extract validate the use of this plant in traditional medic ine to treat different ailments including cancer. The anticancer synergistic effect of Ducrosia flabellifolia compounds has broad implication for understanding t he anticancer potential of plant natural products in vivo, where different compounds may act in concert to r educe


Introduction
Cancer is the second cause of death after cardiovascular [1]. The majority of cancer deaths (above 70%) occur in countries with low and middle income [2]. Jordan is among these countries and recent estimates in Jordan reported 5000 cancer cases per year [3]. Commercially available anticancer agents are either synthetic compounds or natural products originating from different sources including plants.
Synthetic chemistry is dominating the field of new drug discovery. However, the potential of bioactive plants to provide new and novel products for disease treatment and prevention is still enormous [4]. The antitumor area has the greatest impact of plant derived drugs, where drugs like vinblastine, vincristine, taxol, and camptothecin have improved the chemotherapy of some cancers [5].
Ducrosia species are normal flora in different countries including Jordan, Iraq, Iran, Afghanistan, Pakistan and in the region along the Arabic Gulf [6]. Ducrosia belong to Apiaceae family which characterized by having different phytochemicals especially coumarins [7].
Ducrosia flabellifolia is a wild plant growing in the eastern desert of Jordan and considered as a rare plant [11]. No reports are available about the biological activities of Ducrosia flabellifolia.
Taking into account the use of Ducrosia flabellifolia in traditional medicine, and the lack of studies about its biological activities, this study was conducted to evaluate the antiproliferative and apoptosis inducing effects of Ducrosia flabellifolia against different cancer cell lines. For a better understanding of the chemical composition of this plant, the major constituents of the plant extract was identified using LC/MS-MS.

Plant material and extraction procedure
The plant was collected from Wadi Hassan in the eastern desert of Jordan. The taxonomic identity of the plant was authenticated by Prof. Dawud EL-Eisawi (Department of Biological Sciences, University of Jordan, Amman, Jordan). Voucher specimens were deposited in the Department of Biological Sciences, University of Jordan, Amman, Jordan. The air dried areal parts of Ducrosia flabellifolia were finely ground. Suitable amounts of the powdered plant materials were soaked in 95% ethanol (1L per 100g) for two weeks. The crude ethanol extract was obtained after the solvent was evaporated at 40°C to dryness under reduced pressure using rotary evaporator (Buchi R-215, Switzerland). The ethanol extract was further subjected to solvent-solvent partitioning between chloroform and water. All solvents were evaporated to dryness under reduced pressure to produce the crude extracts which were collected and stored at −20°C for further testing [12].

Cell lines and culture conditions
Hep-2 (larynx carcinoma), MCF-7 (breast epithelial adenocarcinoma), and Vero (African green monkey kidney) cell lines were kindly provided by the Department of Biological Sciences, University of Jordan. Cells were grown in Minimum Essential Medium Eagle (Gibco, UK) supplemented with 10% heat inactivated fetal bovine serum (Gibco, UK), 29 µg/ml L-glutamine, and 40 µg/ml Gentamicin. Cells were incubated in a humidified atmosphere of 5% CO2 at 37°C.

Antiproliferative Activity Assay
The antiproliferative activity of Ducrosia flabellifolia extracts was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay The antiproliferative effect of the tested extracts was determined by comparing the optical density of the treated cells against the optical density of the control (cells treated with media containing 0.1% DMSO).

Assessments of Apoptosis in Cell Culture
Apoptosis was detected using terminal deoxynucleotidyl transferase (TdT) mediated-16-deoxyuridine triphosphate (dUTP) Nick-End Labelling (TUNEL) system (Promega, Madison, WI, USA). MCF-7 cells cultured in 24 well plates were treated with 30 µg/mL Ducrosia flabellifolia ethanol extract for 28 h. The assay was conducted according to the manufacturer's instructions. Briefly, treated cells were fixed using 10% formalin followed by washing with phosphate buffer saline (PBS).
Biotinylated dUTP in rTdT reaction mixture was added to label the fragmented DNA at 37 °C for one hour, followed by blocking endogenous peroxidases using 0. After centrifugation, the supernatant containing the DNA was separated and mixed with 600 µl isopropanol followed by centrifugation and mixing pellets with 600 µl 70% ethanol followed by centrifugation. The pellets were incubated with100 µl of DNA rehydration solution at 65 o C for 1 hour. DNA bands were separated using 2.0% agarose gel electrophoresis containing ethidium bromide and visualized using UV transilluminator.

Qualitative phytochemical screening
Thin layer chromatography (TLC) was used for qualitative phytochemical screening of the ethanol extract of Ducrosia flabellifolia. Aliquots (50-75 µl) of the extract were applied 1cm from the base of the TLC plates (0.25 mm, Macherey-Nagel, Germany).
Serial mixtures of chloroform and methanol (from 0-100 %) were used as eluents.
Development of the chromatograms was performed in a closed tank in which the atmosphere had been saturated with the eluent vapor by lining the tank with filter paper wetted with the eluent. For flavonoids and terpenoids detection, plates were sprayed with p-anisaldehyde/sulfuric acid reagent and carefully heated at 105°C for color development [14]. For alkaloids detection, plates were sprayed with iodoplatinate acid and placed in the fume hood for drying.

LC/MS-MS triple quadrapole
Liquid chromatography with MS/MS triple quadrapole was used to identify flavonoids content of Ducrosia flabellifolia ethanol extract. For chromatographic separation and mass spectral analysis a Shimadzu LC-8030 MS system (degasser, binary gradient pump, autosampler, and mixer)was used coupled with Shimadzu 8030 triple quadrapole system equipped with ESI ion source interface. Separation was conducted on Shimpack ODS C18 column ( 250mm X 4.7 mm, 5µm) at 30 O C.
Linear gradient system was applied as solvent A (water), solvent B (methanol) and solvent C (acetonitrile with 0.2 acetic acid).

Statistical analyses
The results of the antiproliferative part are presented as means ± SEM of three independent experiments. Statistical differences among fractions were determined by one way ANOVA using Graph Pad Prism5 (GraphPad Software Inc., San Diego, USA). Differences were considered significant at p< 0.05.

Results and Discussion
Screening of plants and their products for their potential to induce apoptosis have become the major strategy in the search for new anticancer agents. The present study was conducted to evaluate the apoptosis induction ability of Ducrosia flabellifolia against MCF-7 cell line.
MTT dye was used in different studies to determine cell viability for many herbals and phytochemicals [15]. In the present study, the antiproliferative activity of three  [16][17][18]. All extracts were more active against MCF-7 cell line (Table 1). This selectivity could be the result of the sensitivity of the cell line to the compounds in the extract or to tissue specific response [19].
Apoptosis (programmed cell death) is the main mechanism of cell death that is essential for diverse processes ranging from cell development to stress response [15].
Apoptosis induction seems to be an attractive goal to kill cancer cells since many cancers inactivate apoptosis to survive [20]. The main features of apoptosis include chromatin condensation, cell shrinkage, DNA fragmentation, and formation of apoptotic bodies [21].
The ability of the ethanol extract of Ducrosia flabellifolia to induce apoptosis in MCF-7 cells was evaluated using a TUNEL colorimetric assay which detects DNA fragmentation during programmed cell death. Cell shrinkage and DNA fragmentation were clearly observed in cells treated with 30 µg/mL Ducrosia flabellifolia ethanol extract ( Figure 1).
Systemic cleavage of DNA to produce nucleosomal fragments of 200 bp (or its multiples) is considered as a clear characteristic of apoptosis [22]. For more confirmation of our results, fragmented DNA molecules were detected using agarose gel electrophoresis. Clear DNA fragmentation was observed in cells treated with 30, 50, and 100 µg/mL Ducrosia flabellifolia ethanol extract, whereas untreated cells showed no evident DNA fragmentation ( Figure 2). Previous studies have reported that large fragment of 50-300 kb can be detected in early stages of apoptosis while further fragmentation to 200 bp or its multiples was observed in late stages of apoptosis [23].
Morphological alterations during apoptosis include chromatin condensation, nuclear remodeling and membrane blebbing [24]. In order to gain more insight into programmed cell death, morphological changes associated with apoptosis were detected using scanning electron microscopy. Cytomorphological changes corresponding to a typical morphology of apoptosis were detected in MCF-7 cells  (Table 2). This is the first study to report the antiproliferative activity and detailed phytochemical screening of Ducrosia flabellifolia flavonoids.
Quercetin is one of the flavonoids that are widely distributed in different plants [27].
Many biological effects of quercetin have been reported, including anticancer, antiinflammatory, antibacterial and muscle relaxation [28]. The anticancer and antimicrobial activity of quercetin derivatives were also documented [29]. It seems that the presence of quercetin and some of its derivatives in Ducrosia flabellifolia ethanol extract participate directly in the antiproliferative activity of this plant. On the other hand, kaempferol is not as widely distributed as quercetin, but is present in some plants that have potential anticancer activity like broccoli and endive [30]. Previous studies showed the ability of kaempferol to induce apoptosis in different cancer cell lines including osteosarcoma [31] and glioblastoma cell lines [32]. The synergistic antiproliferative effect of kaempferol and quercetin against human gut and breast cancer cells was also reported [30]. For other flavonoids detected in Ducrosia flabellifolia, previous studies reported antiproliferative activity for apigenin [33][34][35], fisetin [36][37], and luteolin [38][39]. Furthermore, the synergistic antiproliferative activity of luteolin with quercetin [40] and luteolin with diosmetin [41] was also documented.
It is unlikely that the apoptosis induction ability of Ducrosia flabellifolia ethanol extract is due to the action of a single agent; it is more likely to be due to one or several synergistic effects of a combination of active flavonoids present in this plant.
Such synergistic effect has broad implication for understanding the anticancer potential of plant natural products in vivo, where different compounds may act in concert to reduce tumor burden.