Ajwa Date (Phoenix dactylifera L.) Extract Inhibits Human Breast Adenocarcinoma (MCF7) Cells In Vitro by Inducing Apoptosis and Cell Cycle Arrest

Introduction Phoenix dactylifera L (Date palm) is a native plant of the Kingdom of Saudi Arabia (KSA) and other Middle Eastern countries. Ajwa date has been described in the traditional and alternative medicine to provide several health benefits including anticholesteremic, antioxidant, hepatoprotective and anticancer effects, but most remains to be scientifically validated. Herein, we evaluated the anticancer effects of the Methanolic Extract of Ajwa Date (MEAD) on human breast adenocarcinoma (MCF7) cells in vitro. Methods MCF7 cells were treated with various concentrations (5, 10, 15, 20 and 25 mg/ml) of MEAD for 24, 48 and 72 h and changes in cell morphology, cell cycle, apoptosis related protein and gene expression were studied. Results Phase contrast microscopy showed various morphological changes such as cell shrinkage, vacuolation, blebbing and fragmentation. MTT (2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay demonstrated statistically significant dose-dependent inhibitions of MCF7 cell proliferation from 35% to 95%. Annexin V-FITC and TUNEL assays showed positive staining for apoptosis of MCF7 cells treated with MEAD (15 mg and 25 mg for 48 h). Flow cytometric analyses of MCF7 cells with MEAD (15 mg/ml and 20 mg/ml) for 24 h demonstrated cell cycle arrest at 'S' phase; increased p53, Bax protein expression; caspase 3activation and decreased the mitochondrial membrane potential (MMP). Quantitative real time PCR (qRT-PCR) analysis showed up-regulation of p53, Bax, Fas, and FasL and down-regulation of Bcl-2. Conclusions MEAD inhibited MCF7 cells in vitro by the inducing cell cycle arrest and apoptosis. Our results indicate the anticancer effects of Ajwa dates, which therefore may be used as an adjunct therapy with conventional chemotherapeutics to achieve a synergistic effect against breast cancer.

KSA. MEAD was prepared using previously described protocol with slight modification [17]. Briefly, the edible part of date fruit was manually separated, freeze dried and coarsely powdered using pestle and mortar. The powdered contents were then extracted in methanol (1:3 ratio, weight to volume) on a shaking incubator (Human Lab, Gyeonggi-Do, Korea) at 25°C for 48 h. The resultant extract was filtered using Whatman No. 1 filter paper and centrifuged at 4000 g for 15 min and vaporized under low pressure at 45°C using Rotavapor 1 R-300 (Buchi, Flawil Switzerland). The supernatant was separated and freeze dried using a lyophilizer (ilShin Biobase Europe, The Netherlands). The freeze dried powder was aliquoted and stored at -80°C until use in the experiments.

Cell culture
The human breast adenocarcinoma cell line (MCF7) was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Institutional ethical approval was obtained for use of cancer cells in this study. The MCF7 cells were maintained Dulbecco's minimal essential medium (DMEM), low glucose (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and 0.01 mg/ml bovine insulin and 1% penicillin-streptomycin, (Invitrogen, Carlsbad, CA) at 37°C in a humidified atmosphere of 95% air and 5% CO 2 .

Cell imaging
MCF7 cells were cultured in 24-well tissue culture plates (Beckton, Dickinson, Franklin Lanes, NJ) at a seeding density of 2×10 4 cells/well. After overnight attachment, the cells were treated with different concentrations (5,10,15,20 and 25 mg/ml) of MEAD and the untreated cells served as control (S1 Methods). The cells were cultured for up to 72 h under standard culture conditions of 37°C in a humidified atmosphere of 95% air and 5% CO 2 . Changes in cell morphology were imaged using inverted phase contrast microscope (Nikon Instruments, Tokyo Japan).

Cell proliferation assay
The effects of MEAD on MCF7 proliferation/inhibition was analyzed using MTT reagent [18]. Briefly, 2×10 4 cells/well were seeded in 24-well plates and allowed to attach overnight. The cells were then treated with different concentrations (5,10,15,20 and 25 mg/ml) of MEAD and incubated at 37°C in a humidified atmosphere with 5% CO 2 for up to 72 h. Following the treatment period, MTT reagent (5 mg/ml) in 100 μl of fresh culture medium was added to each well and incubated for 4 h. The formazan crystals formed with MTT treatment were solubilized with DMSO and the optical density was measured at 570 nm with a reference wavelength of 630 nm using a microplate ELISA reader (SpectraMax 1 i3x, Molecular Devices, Sunnyvale, CA). Percentage of inhibition was calculated as control-test/control Ã 100 [19] and IC50 was estimated (S2 Methods).

Cell cycle analysis
MCF7 cells were plated at a seeding density of 3×10 5 cells/T25 flask and allowed to attach overnight. The cells were treated with three different concentrations (15, 20 and 25 mg/ml) of MEAD. Untreated cells served as negative control and doxorubicin (DOX) 50 μM was used as a positive control. After 24 h of incubation, the cells were trypsinized and washed with Dulbecco's phosphate buffered saline (PBS) containing no calcium and magnesium. The pellet was resuspended into single cell suspension and fixed with chilled 70% ethanol and kept overnight at -20°C temperature. Before analysis, the cells were centrifuged at 300 g for 5 min and washed twice with PBS. Cells were resuspended in 400 μl staining solution containing 0.5 μg/ml RNase A and 50 μg/ml propidium iodide (PI). Following 15 min of incubation in the dark, the cells were analyzed using the FACS Aria III flow cytometer (BD Biosc iences, San Jose, CA).
Annexin V/PI assay MCF7 cells were cultured overnight at a seeding density of 3×10 5 cells/ T25 tissue culture flasks and treated with 15 mg/ml and 25 mg/ml MEAD for 48 h. Annexin V/PI assay was carried out according to the manufacturer's instructions. Briefly, the cells were harvested using 1% trypsin in PBS (Invitrogen, Carlsbad, CA), washed once with cold PBS and twice with 1X binding buffer. The final cell pellet was resuspended with 100 μl of freshly prepared PI (Sigma, St. Louis, MO) and annexin V-FITC solution (Trevigen Inc., Gaithersburg, MD) and incubated at room temperature in the dark for 15 min. Additionally 400 μl of binding buffer was added to the sample and analyzed within 30 min using flow cytometer (FACS Aria III, BD Biosciences, San Jose, CA). The percentage of early apoptotic, late apoptotic and necrotic cells was analyzed using BD FACS DIVA software.

TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay
APO-DIRECT™ Apoptosis Kit (Novus Biologicals, Littleton, CO) was used according to the manufacturer's instructions. MCF7 cells were cultured overnight at a seeding density of 3×10 5 cells/T25 flask (Greiner Bio-One GmbH, Germany) with 15 mg/ml and 25 mg/ml concentrations of MEAD for 48 h. Cells were trypsinized, washed with DPBS, fixed with 1% paraformaldehyde (Sigma, St. Louis, MO) for 60 min at 4°C, washed twice with DPBS and then resuspended in ice-cold 70% ethanol and stored overnight at -20°C. They were then centrifuged and washed twice with wash buffer and incubated overnight with 50 μl of DNA labeling solution at RT in the dark. The reaction was stopped by washing twice with rinse buffer and the cells were incubated with PI/RNase solution in the dark at RT for 30 min before being analyzed using flow cytometer (FACS Aria III, BD Biosciences, San Jose, CA).

Apoptotic protein expression analysis
MCF7 cells were seeded at a density of 3x10 5 cells/T25 flask and treated with 15 mg/ml and 20 mg/ml MEAD and Bcl-2 inhibitor ABT-737 (10 μM) for 24 h. Treated cells were trypsinized, washed with cold DPBS and fixed with 4% formaldehyde for 10 min at 37°C. The cells were then washed twice with cold DPBS, permeabilized with 90% pre-chilled methanol for 30 min at RT, washed with incubation buffer (0.5% BSA) and incubated with primary rabbit polyclonal antibodies for Bax, p53 (Santa Cruz, Los Angeles, CA) and goat monoclonal antibody for Bcl-2 (Sigma, St. Louis, MO) at RT for 90 min. The cells were then washed thrice with incubation buffer and incubated with appropriate secondary antibodies [FITC conjugated bovine anti-rabbit polyclonal and mouse anti-goat Dylight 405] for 45 min at RT in the dark (S3 Methods). Finally, the cells were washed with incubation buffer, centrifuged and the pellet was resuspended in PBS before being analyzed using FACS Aria III flow cytometer (BD Biosciences, San Jose, CA).

Caspase activation assay
Cleaved caspase 3 was detected using FITC conjugated active caspase 3 antibody (BD Biosciences, San Jose, CA) according to the manufacturer's instructions. MCF7 cells were seeded at a density of 3x10 5 cells/T25 flask and treated with MEAD (15 mg/ml and 20 mg/ml) and Bcl-2 inhibitor ABT-737 (10 μM) for 24 h. The cells were trypsinized, washed twice with cold (1X) PBS and centrifuged at 300 g x 5 min. The cell pellet was resuspended in 0.5 ml BD Cytofix/ Cytoperm solution and incubated for 20 min on ice. The cells were then washed twice with BD Perm/Wash buffer (1X) and incubated with 20 μl of FITC conjugated rabbit anti-caspase 3 antibody. Finally the cells were washed with wash buffer and resuspended in a 0.5 ml buffer and analyzed using FACS Aria III flow cytometer (BD Biosciences, San Jose, CA).

Quantitative Real Time-PCR (qRT-PCR)
MCF7 cells were seeded at a density of 2×10 5 cells/T25 tissue culture flasks and allowed to attach overnight. They were then treated with 15 mg/ml and 20 mg/ml of MEAD and cultured for 48 h. The untreated cells served as a control. Total RNA was extracted from the experimental and control cells using Pure Link 1 RNA Mini Kit (Ambion™, ThermoFischer Scientific, Waltham, MA) which included DNase-I treatment protocol. RNA quantity and quality were measured using Nanodrop™ spectrophotometer (NanoDrop products, Wilmington, CA). Firststrand cDNA synthesis was performed using random hexamers (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems). Primer sequences (Table 1) used in the study were obtained from earlier published studies [20]. qRT-PCR analysis was performed using the ABI Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA) using SYBR green dye master mix and relative quantification was performed using the comparative 2 -ΔΔCt method.

Statistical analysis
The differences observed between the control and treated groups for cell proliferation, cell cycle, apoptosis, caspase activation and MMP were analyzed using either One-way ANOVA or unpaired Student t-test (two-tailed) using GraphPad Prism 6 (GraphPad Software, San Diego, USA). The results were expressed as the Mean ± SEM (standard error of mean) from three different replicates and a value of p<0.05 was considered statistically significant.

MEAD induced changes in cell morphology
Phase contrast microscopy of untreated MCF7 cells demonstrated their characteristic epithelial nature and prolific growth as a monolayer. The cells appeared ovoid with central large nucleus with one or two nucleoli. In contrast, the MEAD treated cells showed mild to moderate decrease in cell numbers (S1 Fig) that were both time and dose dependent resulting in the presence of very few live cells (Fig 1). Cell shrinkage was observed starting from 10 mg/ml of MEAD. Cytoplasmic condensation, membrane disruptions, detachment and cell death were most evident in MCF7 cells treated with 20 mg/ml and 25 mg/ml of MEAD (Fig 1).

MEAD inhibited MCF7 proliferation
MTT assay demonstrated MCF7 growth inhibition following treatment with MEAD in a dose and time-dependent manner (Fig 2A-2C). Moderate decrease in cell numbers by 41.85 ± 4.6 percent was observed following incubation with 15 mg/ml MEAD for 48 h (Fig 2B) compared to the control. There was a decrease in cell numbers at 20 mg/ml concentration at 48 h and 72 h that ranged from 78.20 ± 1.34 to 96 ± 2.03 percentages respectively (Fig 2C). The cell proliferation showed a significant decrease (99.12 ± 0.08) in MCF7 cells treated with 25 mg/ml of MEAD at 72 h. The IC50 was determined to be 18.20 mg/ml (S2 and S3 Figs) for the 48 h duration. The decrease in proliferations were statistically significant (p<0.001).

MEAD increased expression of apoptotic proteins
The expression levels of Bax, Bcl-2 and p53 were analyzed using flowcytometry (Fig 6).   and 64% (Fig 6B) upon treatment with 15 and 20 mg/ml MEAD respectively. Treatment with ABT-737 showed 6% reduction in the expression of Bcl-2.

MEAD increased Caspase 3 activity
Treatment of MCF7 cells with MEAD showed a dose dependent increase in active Caspase 3 expression by FACS analysis. There was an increase in active caspase 3 staining (Fig 7B and  7C) by 32% and 37% following treatment with 15 mg/ml and 20 mg/ml MEAD respectively compared to the control (Fig 7A). Treatment with ABT-737 (10 μM) also demonstrated an increase in active caspase 3 expression.

Discussion
In this study, we have elucidated the anticancer effects of MEAD on MCF7 cells. MEAD inhibited the growth and proliferation of MCF7 cells. It also induced MCF7 cell death in a dose and time dependent manner. Natural products and their secondary metabolites have been demonstrated to induce apoptosis and/or modulate apoptotic pathways [20]. Many of the phytochemicals that exist in natural products/extracts such as flavonoids, aglycones, terepenoids and polyphenolic compounds are reported to have cancer inhibitory properties. Most importantly, in the last two decades some of them were subjected to clinical studies for various cancers including colon, liver, prostate, lung and breast cancer [21]. Cancer cells develop mechanism to regulate signaling pathways in order to escape host immune surveillance and avoid cell death, facilitating their prolonged survival [22]. The proliferative advantage conferred on these cells in turn promote their uncontrolled metastasis. Inhibition of their proliferation by induction of cell cycle arrest or apoptosis would be an advantage. Interestingly, in the present study, MEAD showed various morphological changes   that were indicative of apoptosis. The cellular changes included membrane blebbing, cell contraction, cell fragmentation and loss of cell adherence leading on to cell death (Fig 1). In addition, both annexin V-FITC co-staining with PI as well as the TUNEL assays demonstrated annexin V-FITC and TUNEL positive cells following treatment with MEAD. These results are in support of our morphological findings and further point towards apoptosis of MCF7 cells by MEAD. Externalization of phosphatidylserine (PS) associated with loss of cell membrane asymmetry and fragmentation of nuclear DNA are considered to be the hallmarks of cells in later stages of apoptosis [23]. Our findings are similar to an earlier study where pancreatic cancer cells treated with myricetin showed an increase in annexin-V and TUNEL positive cells [24].
Mitochondria play a central role in both cell survival and cell death. Depolarization of mitochondrial membrane potential causes translocation of Bax from the cytosol to the outer mitochondrial membrane as well as the release of cytochrome c. These events then orchestrate apoptosome complex formation and activation of intrinsic apoptotic pathway [25][26]. In the present study, the MCF7 cells treated with MEAD, demonstrated mild MMP changes compared to the control (Fig 8) indicating that the apoptotic events are mediated through the intrinsic pathway. However, significant caspase 3 activation was also noted in MCF7 cells treated with MEAD indicating that additional pathways of apoptosis are involved. Activation of the caspases including caspase 8 and caspase 3 are implicated in ligand mediated extrinsic pathway of apoptosis [27]. Quercetin, apigenin luteolin and myricetin are some of the common constituents of Ajwa date extract [15]. Interestingly, these compounds are reported to cause cancer cell inhibition [28][29][30]. Independent studies have demonstrated the anticancer effects of quercetin, where human breast cancer cells (MDA-MB-231 and MCF7) were inhibited in a dose and time dependent manner [31]. Quercetin also caused cell cycle arrest, increased proapoptotic Bax protein, reduced MMP and activated caspase 3, 8 and 9 leading to apoptosis [31,32].
Cell cycle regulation and cell death signaling pathways play a very important role in the developmental biology [32]. Dysregulation of cell signaling pathways leads to cancerous transformation and therefore serve as the potential targets for cancer therapeutics [32]. The cell cycle analysis in our study demonstrated 'S' phase arrest and decreased 'G1' phase following treatment with MEAD indicating that the extract and its components can bring about cell cycle arrest and inhibit the proliferation of cancer cells. MCF7 cell proliferation in vitro was inhibited in a dose and time dependent manner (Fig 2). This was in line with an earlier report where the methanolic extract of date pollen was found to have anti-proliferative effects on MCF7, Caco-2 and Hela-1 cell lines [33].
In line with our other results, the gene expression showed a significant increase in p53, Bax, Fas and FasL indicative of apoptotic cell death. p53 on stabilization and accumulation causes transcriptional regulation of different processes like cell cycle, apoptosis and DNA repair. Bax, PUMA and NOXA are up-regulated in apoptosis, while Bcl-2 family genes are down-regulated [34]. p53 up-regulate the transcription of Fas and enhances its processing and release through the Golgi apparatus [35,36]. Probably, the increase in p53 led to the transcriptional activation of other dependent pro-apoptotic genes such as Fas, FasL, and Bax. Increased expression of Bax and p53 protein in our study further support that apoptotic cell death of MCF7 could be due to p53 dependent mechanism. In addition, there was a decrease in anti-apoptotic Bcl-2 and increase in the ratio of Bax/Bcl-2 transcription suggesting that MCF7 cell death following treatment with MEAD was activated by p53 mediated signaling leading to apoptosis. Quercetin, one of the metabolite of Ajwa date induces apoptosis by arresting cells in 'S' phase and 'G1' phase; expression of p53, P57 and Bax proteins; changes in mitochondrial membrane potential and activation of caspases in MCF7 cells [31]. This is in accordance with our results showing 'S' phase arrest and over expression of p53 and increased Bax/Bcl-2 ratio in MEAD treated MCF7 cells.
Plant extract have many different phytochemicals and metabolites, that may interact in a synergistic manner to exert their potential benefits to fight against the multifactorial and multistage carcinogenesis process [37]. Although it is important to purify the different components of the extract and identify their independent effects on both normal and cancer cell lines, at times the very process of purification may lead to the loss of these active compounds and hence their useful properties. Undue caution should therefore be exercised in isolation of individual components, as in some instances only the whole extract may actually be beneficial. This is emphasized by the fact that total extracts obtained from cranberry, pomegranate and Nigella sativa demonstrated much higher inhibitory effects in various cancers than their individual fractions [38][39][40]. Their beneficial effects were attributed to the synergistic effects of many individual components in the extract.

Conclusion
MEAD induces cell cycle arrest and causes cell death via apoptosis by activating both intrinsic and extrinsic pathways in MCF7 cells. Unlike conventional chemotherapeutics, MEAD is observed to exert only mild to moderate beneficial effects against MCF7 cells. Purification of MEAD and the identification of individual components that are responsible for anti-cancer properties will pave way for the development of novel anticancer therapeutics. In addition, the aglycone metabolites of Ajwa dates such as Luteolin, myricetin, apigenin, quercetin and petunidin have been reported to induce apoptosis in cancers [41][42][43][44]. Given the folklore claims of cancer inhibitory properties of Ajwa date extract and our results on MCF7 cells, the date fruits could be added daily as a nutritional supplement for synergistic chemopreventive effects against breast cancer and other malignancies. The histogram represents increase in Annexin V staining inactive of increase in apoptotic cells compared to the control. These increases were statistically significant (p>0.05). (TIF) S6 Fig. P53 protein expression in MCF7 cells. MCF7 cell were treated with 15 mg/ml and 20 mg/ml MEAD for 24 h and immunocytochemistry was performed. Rabbit polyclonal primary antibody for P53 and FITC-labelled secondary antibody was used. Cells were additionally stained with DAPI nuclear stain. The upper row represent 15 mg/ml and lower row 20 mg/ml MEAD respectively. (TIF) S1 Methods. 3T3L1 cells imaging. 3T3L1 cells were cultured in 24-well tissue culture plates (Beckton, Dickinson, Franklin Lanes, NJ) at a seeding density of 2×10 4 cells/well. After overnight attachment, the cells were treated with MEAD at 0, 1, 5, 10, 15, 20, 25, 30, 50 and 100 mg/ml concentrations for 48 h. The cells were cultured under standard culture conditions of 37°C in a humidified atmosphere of 95% air and 5% CO 2 . Changes in cell morphology were imaged using inverted phase contrast microscope (Nikon Instruments, Tokyo Japan). (DOCX) S2 Methods. IC50 Calculation of MEAD on 3T3L1 and MCF7 cell lines. 3T3L1 and MCF7 cells were cultured in 96-well tissue culture plates (Beckton, Dickinson, Franklin Lanes, NJ) at a seeding density of 5×10 3 cells/well. After overnight attachment, the MCF7 cells were treated with MEAD at 0, 5, 10, 15, 20 and 25 mg/ml concentrations and the 3T3L1 cells were treated with MEAD at 0, 1, 5, 10, 15, 20, 25, 30, 50 and 100 mg/ml concentrations for 48 h and MTT assay was done. The optical densities were analyzed using log (inhibitor) vs. response-Variable slope (four parameters) analysis function with the help of Prism GraphPad 6.0 software to calculate the half maximal inhibitory concentration (IC50) value. (DOCX) S3 Methods. Immunocytochemistry. MCF7 cells were plated at a seeding density of 2×10 4 cells/well in a 24-well plate and allowed to attach overnight. Fresh medium containing 20 mg/ml and 25 mg/ml MEAD was added and the cells were incubated for 48 h. Following incubation the cells were washed twice with cold PBS and fixed with 4% formaldehyde for 20 min. The cells were then washed and permeabilized with 0.25% triton X-100 for 30 min at RT. The cells were washed twice with cold PBS and blocked with 1% BSA (bovine serum albumin), followed by overnight incubation with primary antibody P53 (Santa Cruz, Los Angeles, CA) at 1:200 dilution at 4°C. The cells were then washed and incubated with FITC labelled anti-rabbit polyclonal secondary antibody (1:500) (Santa Cruz, Los Angeles, CA) in dark for 1 h at RT. The cells were stained with 1 μg/ml DAPI for 1 min and observed under ZEISS LSM 780 Laser Scanning Microscope (Carl Zeiss, Oberkochen). (DOCX)