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Allium willeanum Holmboe exerts anticancer activities on metastatic breast cancer cells MCF-7 and MDA-MB-231

Allium species are medically important plants, rich in bioactive molecules with antitumoral properties. In this study, we aimed to investigate the molecular composition and in vitro anticancer activities of Allium willeanum Holmboe, an endemic Allium species of the island of Cyprus. GC-MS analysis of ethanolic extracts of A. willeanum H. bulb (AWB) showed bioactive molecules, octadecanoic acid 2-hydroxy-1-(hydroxymethyl)ethyl ester (21.99 %), hexadecanoic acid (20.42 %), pentadecanoic acid (9.19 %), 1,2-benzenedicarboxylic acid, diethyl ester (8.79 %), with known anticancer activities. AWB exerted significant reduction in mitochondria dependent metabolic activity as a measure of cell growth on MCF-7 (weakly metastatic) and MDA-MB-231 (strongly metastatic) human breast cancer (BCa) cell lines with a more prominent effect on highly metastatic BCa cells. Both trypan blue and lactate dehydrogenase (LDH) cytotoxicity assays quantitatively revealed that exposure to AWB extract significantly reduced cancer cell viability in both tested cell types. Differential activation of caspases in the tested cell lines indicated faster apoptotic activity on MDA-MB-231 cells compared to MCF-7. A significant reduction in cell motility was demonstrated upon AWB exposure of MCF-7 and MDA-MB-231 cells. AWB bioactive molecules collectively act on cancer cells to exert significant cytotoxic, apoptotic activities on both MCF-7 and MDA-MB-231 cells and significantly reduce their lateral motility, elucidating AWB as a promising agent to further be utilized in anticancer studies.


Introduction
Breast cancer is among the most prevalent cancer types globally observed in women, causing the highest number of cancer related deaths worldwide [1]. Chemotherapy, the currently preferred method, used along with surgical procedures have severe side effects that can reduce patients' quality of life [2] emphasizing the need to develop novel, direct and integrative therapies. Such developments can help improve disease outcomes and patients' quality of life [3].
Genus Allium (including garlic) has been historically used as therapeutic plants because of their anticancer, antiinflammatory, antiobesity, antidiabetic, antioxidant, antibacterial, antifungal, neuroprotective, immunomodulatory effects and in prevention of cardiovascular diseases [4,5]. Specifically, Allium sativum is of interest due to its various therapeutic properties including its anticoagulant, antihistaminic, antiparasitic, antifungal, antiprotozoan and antiviral functions [6,7]. Allium sativum's anticancer properties have also been widely demonstrated, revealing that increased consumption of garlic decreases the risk of several cancer types such as colon, pancreas, breast cancer [8] and increased intake of Allium class vegetables decrease stomach cancer risk [9]. Further, Allium associated molecular activities are involved in control of several cancers like BCa, including suppression of deoxyribonucleic acid (DNA) adduct formation, activation of metabolizing enzymes responsible for detoxification of carcinogens and induction of apoptotic mechanisms [8,10].
Natural flora of the island of Cyprus reserves several plants with therapeutic value, all of which are understudied. A. willeanum H. is one of eight Allium species endemic to the island of Cyprus belonging to the Amaryllidaceae family [11]. A. willeanum H. belongs to the same section as Allium sativum, which already has well established antitumoral activities [6], leading us to hypothesize that A. willeanum H. also has anticancer properties. So, in this study we sought to determine the in vitro anticancer activities of AWB on MDA-MB-231 (strongly metastatic BCa cell line) and MCF-7 (weakly metastatic BCa cell line) and revealed cytotoxic, antimetastatic, and apoptotic properties.

Extraction of Allium willeanum Holmboe
Collected and separated AWB bulb were dried at room temperature. Dried plant material was then powdered in mixer grinder. Maceration was carried out by mixing powdered material with ethanol (95 %) with 1:10 w/v ratio. Mixture was macerated at room temperature for 24 h and filtered through Whatman No. 1 filter paper. Concentration process of filtrates was performed at 40 C by Rotary-evaporator (Heidolph, Germany). AWB yield was 0.634 %. Extract was labelled accordingly and stored at 4 C for further analysis. MDA-MB-231 and MCF-7 BCa cells which were obtained from Imperial College, London, UK were grown in 4 mmol/l L-glutamine and 10 % foetal bovine serum supplemented Dulbecco's Modified Eagle Medium (DMEM) (Gibco by Life Technologies™, USA). Cells were incubated at 37 C temperature and 100 % relative humidity with 5 % CO 2 [12].

Trypan blue exclusion assay
Trypan blue exclusion assay was used for or a direct identification and enumeration of live and dead MDA-MB-231 and MCF-7 breast cancer cells. Cells were plated in 35mm cell culture dishes (Thermo Scientific Nunc Cell Culture Dishes; Thermo Scientific; USA) with 3 Â 10 4 /ml density and incubated overnight before the AWB extract treatment  cells in the assay was calculated from 20 randomly selected areas [13] upon investigation under inverted light microscope (Leica Microsystems, Germany).

2.4.2.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay MTT assay was used in order to determine reduction of a 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide into formazan product by the mitochondria of viable breast cancer cell lines tested. Growth rate of the tested cell lines were detected by evaluating the linear relationship between cellular activity (via mitochondrial activity) and absorbance for the quantitative determination of cell proliferation. The change in the MDA-MB-231 and MCF-7 cell numbers was determined by colorimetric MTT assay as previously described by Isbilen and coworkers [14]. Both BCa cell lines were plated with 3 Â 10 4 /ml density overnight and then treated with AWB extract (10000, 5000, 2500, 1250, 625, 312.5 and 156.25 μg/ml) for designated time periods (24, 48, 72 h). Control experiments received only cell culture media. Measurements were taken at 490 nm on a multiwell plate reader (Biotek ELx800, Labx; Canada) [14]. Excel was used to calculate IC 50 value for the extract concentration that killed 50 % of the cells.

LDH cytotoxicity assay
Lactate Dehydrogenase (LDH) is a cytosolic enzyme released as a result of damaged plasma membrane which is directly proportional to cellular cytotoxicity. Effect of AWB exposure (10000, 5000, 2500, 1250, 625, 312.5 and 156.25 μg/ml) on LDH release was quantitatively measured by incubating MCF-7 and MDA-MB-231 cells (0.5 Â 10 6 /ml) for 24 h and assessed by using LDH Cytotoxicity Assay Kit (Thermo Scientific, USA) according to the manufacturers' instructions. Control experiments received only cell culture media.

Wound healing assay
For wound healing assay, BCa cells were plated with 4 Â 10 5 /ml density and incubated at 37 C, 5 % CO 2 overnight. Wounds were formed by using 200μl Gilson pipette tips with 0.5-0.8 mm width. The media was replaced with fresh media containing Allium extract and wounds widths were measured (margin to margin) under inverted light microscope camera (Leica, Germany) via ImageJ software. After 24h of treatment with AWB extract, wound widths were re-measured and re-evaluated [14]. Motility index (MoI) was used for calculation of lateral motility of breast cancer cell lines.
which W 1 stands for the width of the wound at time t (24h) while W 0 is the starting wound width (time ¼ 0 h) [14]. Control experiments received only cell culture media.

Gas chromatography mass spectroscopy (GC-MS) analysis
Gas chromatography mass spectroscopy analysis was performed as previously described by Leikshmi and coworkers [15]. 1ml of A. willeanum H. bulb extract was analysed using a GCMS-QP2010 PlusSystem (Shimadzu, Japan). Helium was used as carrier gas in the constant flow mode at 1 ml/min. GC-MS oven initial temperature was 50 C (for 2 min) which was gradually increased by rate of 5 C/min up to 280 C and maintained for 9 min. Injection port temperature was ensured as 250 C and detector temperature was 300 C. The ionization voltage was 70eV. 30 m long RTS volatile column was used for separation of compounds. Quadrupole Mass Detector was employed to detect compounds when they were vented from the column. Using MS data library WILEY7.LIB the spectrum was analysed and compounds were identified [15].
Overall, AWB incubation resulted in a consistent, concentration dependent decrease in cell viability. Significant cytotoxic activity was shown at 625 μg/ml and onwards at 24 and 48h and 312.5 μg/ml and onwards at 72h of incubation with MDA-MB-231 cells (p < 0.05).
However, significant toxicity in MCF-7 cells was observed at 2500 μg/ml and onwards at 24 and 48h; 5000 μg/ml and onwards at 72h of incubation (p < 0.05, Figure 1). The increased cytotoxic activity of AWB on MDA-MB-231 cells at lower concentrations compared to MCF-7 cells reveals the cytotoxic efficiency of AWB on strongly metastatic cancer cells.  (Table 1). AWB cytotoxicity on cancer cells was quantitatively assessed using L-Lactate dehydrogenase (LDH) release. Upon cellular exposure to endogenous or exogenous damage, LDH is released from cytoplasm to the extracellular milieu [16]. Measuring LDH release can provide quantitative insight towards the damage exerted on the cancer cells by AWB. LDH cytotoxicity assay results revealed that incubation with AWB extract had no significant increase in LDH release in MCF-7 cells where MDA-MB-231 cells showed elevated levels of LDH through the 24 h incubation (Figure 3). This result is consistent with the higher levels of cytotoxicity observed in MDA-MB-231 with Trypan Blue Exclusion assay (Figure 1) emphasizing the increased toxicity exerted on the strongly metastatic cell line MDA-MB-231 compared to the weakly metastatic cell line MCF-7. Molecular investigation of AWB composition revealed 20.42 % hexadecanoic acid which was previously revealed to exhibit cytotoxic activities on MCF-7 and MDA-MB-231 breast cancer cells [17] likely contributing to the cytotoxic activities associated with AWB extract.

AWB extract induces Caspase-3 and -9 activities in MCF-7 and MDA-MB-231
Caspase-3 and -9 activation studies were carried out in order to gain insight towards the mechanism associated with the observed cell death. Caspases are a family of endonucleases that are responsible for sustaining homeostasis by regulating inflammation and apoptotic cell death [18]. Caspase family comprises of the initiator caspases (caspase-8 and -9) which are responsible for activation of the executioner caspases (caspases-3, -6, and -7) that subsequently organize their activities to demolish key structural proteins and activate other enzymes where this activation in the caspase cascade leads to programmed cell death mechanisms [18,19].
In order to investigate if cytotoxic properties associated with AWB are mechanistically based on apoptosis, caspase-3 and -9 activities were investigated. % relative caspase-3 activity was evaluated compared to the negative control for AWB extract on MCF-7 and MDA-MB-231 BCa cell lines. Control experiments were taken as 100 %. In comparison to untreated MDA-MB-231 BCa cells, AWB treatment caused significantly increased caspase-3 activity (24h: 290 AE 7.97 %; at 10000 μg/ml; p < 0.05; Figure 4A) for the 24h period. On the other hand, AWB treatments on MCF-7 cells did not show any significant increase in caspase-3 activity (24h: 99.38 AE 1.48 %; at 10000 μg/ml; p > 0.05; Figure 4A It was previously shown that octadecanoic acid and hexadecanoic acid incubations induced caspase-8 and caspase-3 mediated cell death in PC12 cells [20,21]. Incubation with AWB caused a significant increase (p < 0.05) in caspase-3 in MDA-MB-231 cells where no significant increase was observed in MCF-7 cells (p > 0.05) ( Figure 4A). Conversely, AWB incubation led to a higher induction in caspase-9 activation in MCF-7 cells compared to the MDA-MB-231 cells ( Figure 4B). This phenomenon suggest that presence of these bioactive molecules may activate caspase-dependent cell death mechanism in both MCF-7 and MDA-MB-231 breast cancer cell lines. Albeit a faster and more robust activation of apoptotic pathways in MDA-MB-231 cells was observed indicating that this cell line moved forward to the activation of executioner caspases like caspase-3 whereas MCF-7 cells were at the beginning stages of a lagging apoptotic process, only achieving the initiator caspase-9 activation at the time point tested. Morphological investigation of AWB extract treated MDA-MB-231 cells via microscopy also revealed presence of membrane blebs (data not shown) which are associated with apoptotic cascade activation [22]. Membrane blebbing observed during apoptosis depends on caspase-3 mediated cleavage of ROCK-I. Rho-activated serine/threonine kinase (ROCK-I) protein regulates actomyosin filament assembly as well as myosin contractility by inducing increased phosphorylation of the myosin regulatory light chain (MLC) [22]. Previous studies revealed that the activation of caspase-3 induces ROCK-I cleavage, leading to removal of its inhibitor domain. Cleavage of ROCK-I protein activates MLC phosphorylation along with cell contraction and membrane blebbing [22]. The differential activation of caspases by AWB, associated with a delay or inability to activate caspase-3 in MCF-7 cells may provide an explanation towards the decreased viability observed in weakly metastatic MCF-7 cell line.

AWB significantly reduces the lateral motility of MCF-7 and MDA-MB-231 cells
Wound healing assay also known as lateral motility assay is classically performed in order to evaluate the closure of the manually created scratch on a monolayer of cells. This assay was used to determine the effect of AWB extract on lateral motility of breast cancer cell lines MCF This result is in line with the metastatic nature and motility of both cell lines. Since one of the prevailing mechanisms of cancer leading to high mortality and morbidity is metastasis, AWB extract interfering with lateral motility demonstrates its potential as a possible protective, antimetastatic agent, especially effective against nonaggressive cancers like weakly metastatic MCF-7 cells as well as highly aggressive cells like MDA-MB-231.
Phytochemical profiling of AWB carried out using GC-MS analysis provided insight towards its chemical composition and bioactive compounds associated with in vitro anticancer activities. The obtained results were in line with the observed inhibitory effect of the AWB on both breast cancer cell lines as 21.99 % octadecanoic acid was detected in its composition (Table 2). Octadecanoic acid (stearic acid) was previously shown to have inhibitory activities against viability of various BCa cells while triggering apoptosis of MDA-MB-361, MDA-MB-231 and MCF-7 human BCa cells by induction of apoptosis via caspase-3 activation [18,36]. The presence of a high percentage of octadecanoic acid has likely contributed to the observed apoptotic activities. GC-MS analysis also revealed the presence of hexadecanoic acid (20.42 %) as well as 9-octadecenamide (13.68 %) ( Table 2) in AWB extract, which were previously reported to have antimetastatic potential [36,37]. Hexadecanoic acid induces caspase-8 and caspase-3 mediated apoptosis [22], generation of reactive oxygen species (ROS) by interacting with Toll like receptor 4 (TLR4)/ROS/p53 pathway, causes alterations in structure and function of mitochondria [38], also prevents tumor cell invasion and metastasis [36]. Similarly, 9-octadecenamide was demonstrated to inhibit the spontaneous metastasis of BL6 skin melanoma cells [36].

Conclusion
Overall, our study reveals significant antitumoral potency against strongly metastatic BCa cells with minor effects on weakly metastatic cells in a concentration dependent manner. The cytotoxic effects associated with AWB suggest, a more potent activity of AWB on concentration-dependent reduction of cell viability via mitochondrial activity of highly metastatic BCa cells MDA-MB-231 compared to the weakly metastatic cells MCF-7. Moreover, lateral motility studies revealed the inhibitory effects of AWB on motility of BCa cells where a strong inhibition was observed on the motility of both cell lines with a prominent effect on MCF-7 cells. Caspase-3 and -9 activation revealed the apoptotic mechanism underlying the anticancer properties exerted by AWB with significantly high activity of caspase-3 and -9 on MDA-MB-231 cells whereas only significantly high caspase-9 activity on MCF-7 cells. Hence, AWB extract has been demonstrated to trigger a faster apoptotic activity at 24h incubations on MDA-MB-231 cells compared to the MCF-7 cells. Collectively, our results demonstrate anticancer properties of the never before studied endemic species A. willeanum H. revealing it as a potential natural product to be further studied against metastatic cancers.

Author contribution statement
Ovgu Isbilen: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.
Ender Volkan: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Funding statement
Work was supported by Cyprus International University Biotechnology Research Center.

Data availability statement
Data included in article/supplementary material/referenced in article.

Declaration of interests statement
The authors declare no conflict of interest.

Additional information
No additional information is available for this paper.