Induction of apoptosis and cell cycle arrest by ethyl acetate fraction of

Journal of Ethnopharmacology 218 (2018) 35–44

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Induction of apoptosis and cell cycle arrest by ethyl acetate fraction of Phoenix dactylifera L. (Ajwa dates) in prostate cancer cells

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Muqtadir Baig Mirzaa, Ayman I. Elkadya,b, , Atef M. Al-Attara, Fareeduddin Quadri Syeda, Furkhan Ahmed Mohammeda, Khalid Rehman Hakeema a b

Department of Biological Science, Faculty of Science, King Abdulaziz University (KAU), PO Box 80203, Jeddah, Saudi Arabia Zoology Department, Faculty of Science, Alexandria University, Alexandria, Egypt

A R T I C L E I N F O

A B S T R A C T

Keywords: Phoenix dactylifera L. Ajwa dates Herbal medicine Apoptosis Prostate cancer Anticancer

Ethnopharmacological relevance: Phoenix dactylifera L. (Ajwa date) has high nutritive value and are consumed in Arabian Peninsula as an essential diet. Phoenix dactylifera L. have been mentioned in folk remedies of traditional Egyptian medicine and alternative medicine, for numerous health benefits including cancer treatment. The aim of the study is to evaluate the anticancer effects of the extract of Ajwa Date on human Prostate cancer cell line (PC3). Materials and methods: Antiproliferative effect was measured using MTT assay. The long-term effect of EAFAD was determined using colony assay. Different stains like Giemsa and fluorescent stains (DAPI and acridine orange / Ethidium bromide) measured morphological changes. Loss of mitochondrial membrane potential and increased oxidative stress were measured using JC-1 and DCFH-DA dyes. DNA degradation was analyzed by comet assay. Cell cycle distribution was measured by flow cytometer. The apoptotic cell was quantified by annexin V-FITC and Propidium iodide dual staining using flow cytometer. Results: PC3 cell line was treated with ethyl acetate fractions of Ajwa dates (EAFAD) to study their morphological and cellular changes and induction of apoptosis. MTT assay showed the strong inhibitory effect of EAFAD on PC3 cells. Loss of mitochondrial membrane potential and increased oxidative stress were observed in EAFAD treated cells, which suggested mitochondrial involvement in apoptosis. Comet assay proved DNA fragmentation induced by EAFAD. Flow Cytometer results demonstrated that Annexin V-FITC and propidium iodide staining showed that EAFAD induced apoptosis and arrest the cell cycle in S phase. Conclusion: Our results suggested EAFAD has potential therapeutics properties for prostate cancer.

1. Introduction

subsists with family history and age was believed (Albright et al., 2015). In adoption studies, 4.8% environmental risk factor (Sundquist et al., 2015) have also been quantified for prostate cancer risk, apart from that consumption of polyunsaturated fatty acids in overcooked fish, vitamin D deficiency and intake of red meat are included (Szymanski et al., 2010; Wright et al., 2011). STDs (sexually transmitted diseases) and tobacco smoking also put an individual in jeopardy for prostate cancer adenocarcinoma (PCa) (Caini et al., 2014; Lewinger et al., 2014). Inflammatory factors have been associated with the development and progression of PCa, such as bacterial endotoxins and exogenous carcinogens like 2-amino-1-methyl-6-phenylimidazo (4, 5-b) pyridine or PhIP (Nakai et al., 2007; Sfanos and Marzo, 2014). Major treatment for PCa remains to be Androgen deprivation therapy (ADT). Though ADT effects only in tumor deterioration, this type of therapy remains to be noncompliant to most of these patients,

Until now, the major causes of death worldwide remains to be cancer and only modest improvement has been done in minimizing the morbidity and mortality of this disease (Siegel et al., 2017). Around 95% of cancers are linked to poor lifestyle, and it takes around 20–30 years to clinically diagnosed. The International Agency for Research on Cancer(IARC) and American Cancer Society estimate that around 14.1 million cancer cases were diagnosed in the year 2012 alone with 8.2 million death globally. They also predicted that these number would double in 2030 with 13 million cancer deaths in 21.7 million cases (American Cancer Society, 2017). In men, prostate cancer is second most common cancer after skin cancer, all cancer-related death due to metastasis from Prostate cancer accounts for 4–10%. Etiology of Prostate cancer remains a baffling issue until now. A strong relationship ⁎

Corresponding author at: Department of Biological Science, Faculty of Science, King Abdulaziz University (KAU), PO Box 80203, Jeddah, Saudi Arabia. E-mail addresses: [email protected] (M.B. Mirza), [email protected], [email protected] (A.I. Elkady), [email protected] (A.M. Al-Attar), [email protected] (F.Q. Syed), [email protected] (F.A. Mohammed), [email protected], [email protected] (K.R. Hakeem). https://doi.org/10.1016/j.jep.2018.02.030 Received 26 November 2017; Received in revised form 13 February 2018; Accepted 18 February 2018 Available online 21 February 2018 0378-8741/ © 2018 Elsevier B.V. All rights reserved.


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2. Materials and methods

owing to rise in androgen-independent mechanisms enhancing tumor cell growth (Karantanos et al., 2013). Growing occurrence of prostate cancer and its inadequate options for management demand the search for new preventive methods for the disease. Chemoprevention is one such approach by which, cancer can be slowed, reversed, or completely prevented by naturally occurring compounds in fruits and vegetables (Reddy et al., 2003; Riboli and Norat, 2003). Date palm (Phoenix dactylifera L.), belonging to the family Arecaceae, is an essential fruit in Arabian countries, where most of the production comes from the arid and semi-arid region of the World (> 80%). Date palm posses great importance from the economic and nutritional point of view. Historical and religious importance of date palm are well documented and used as anthropomorphic symbols in Mesopotamian and Egyptian civilization. Dates were preferred from Indian folklore to middle eastern traditional medicine including countries like India, Iran, Iraq Egypt and Algeria for the treatment of microbial infection, hypertension, diabetes, atherosclerosis, constipation, and cancer (Manickavasagan et al., 2012; Aboul-Enein et al., 2012). Date palm fruits or syrups were highly recommended for treatment of liver diseases and it is consumed by pregnant women before and after delivery. The hepatoprotective effects had shown in-vivo by an extract of date fruit (El Arem et al., 2014). Dates and its pollen are even considered to be very beneficial in reducing sterility caused by various sexual disorders, increasing sexual stamina, and decreasing sluggishness (Baharara et al., 2015; Farag El-Kott et al., 2014). The previous studies presented that date palm contains thirteen different pharmacological important flavonoids glycosides of quercetin, luteolin, and apigenin (Abu-Reidah et al., 2017). The in vitro study reported that the aqueous and methanolic extract of various date fruits has anti-inflammatory, antioxidative and antiproliferative properties (Zhang et al., 2017). Ajwa dates (Fig. 1a) homegrown in Al-Madina, Saudi Arabia are delightful berries, soft, and have black exocarp with white lines. Ajwa dates are favorite among other dates due to their high nutritional value and richness in dietary fibers, carbohydrates, minerals, proteins, fats, and vitamins (Assirey, 2015). It also contains abundant phytochemicals such as glycosides, flavonoids, polyphenols, and phytosterols (Saleh, 2011). These phytochemicals exhibit anti-inflammatory, antioxidant, cardioprotective, hypolipidemic and anti-apoptotic properties (AlYahya et al., 2016). According to a Hadith (Prophet's saying), Saud (R.A) narrated that I heard Prophet Mohammad (PBUH) saying, “If Somebody takes seven Ajwa dates in the morning, neither magic nor poison will hurt him that day” (Sahih al-Bukhari, 1997). Polyphenolrich and methanolic extracts of Ajwa dates were reported to inhibit colon cancer and breast cell line in vitro (Eid et al., 2014; Khan et al., 2016). However, the anticancerous effects of Ajwa date fruits on Human Prostate cancers remain unexplored. In the present study, the evaluation of Ajwa date extract has been carried out on a human prostate carcinoma (PC3 cell line) in vitro to check their efficiency against cancer treatment.

2.1. Preparation of ethyl acetate fraction of Ajwa dates Fresh Ajwa dates (1 kg) of ‘Tamr’ stage (Full ripening stage), were purchased from local market of Al-Medina al-Munawarra, Saudi Arabia (Fig. 1b). This region is a hot desert surrounded by mountains, where the average temperature in summer measures about 43 °C in a daytime and 29 °C in the night, there is very little rainfall recorded yearly. Medina is located in the northwestern part of Saudi Arabia, on longitude 39° 36' E and latitude 24° 28' N. The Ajwa dates were identified and authenticated by Dr. Dhafer Ahmed Al-Zahrani, Plant Taxonomist and deposited the dates (Specimen voucher number: P. dactylifera L. #PD17569) at Herbarium of Department of Biology, Faculty of Science, King Abdulaziz University, Jeddah, KSA. EAFAD (Ethyl acetate fraction of Ajwa dates) was prepared following the previously described protocol with slight modifications (Abbas and Ateya, 2011). The edible part of Ajwa dates was macerated in 2 l of 70% ethanol for 6–7 days at room temperature. The macerated content was filtered with Whatman No. 1 filter paper and concentrated using vacuum rotatory evaporator (BUCHI, Switzerland). The concentrated residue was defatted by an equal amount of N-Hexane and separated in a separation funnel. The aqueous portion was fractionated again with the equal amount of ethyl acetate and separated. Ethyl acetate portion was collected and dried in a vacuum evaporator. This residue was then dissolved in 50% DMSO to make the concentration of 100 mg/ml working stock and stored at 4 °C. 2.2. Cell culture and treatments The human prostate cancer cell line (PC3) was used in this work. PC-3 cell line was established in 1979 from a 62-year-old Caucasian male, it is a high potential metastatic cell line and widely used in prostate cancer research. For the current study, this cell line was procured from American Type Culture Collection (ATCC, CRL-1435). The normal fibroblast skin cell line (BJ) (ATCC, CRL-2522) from humans were also used. The cells were grown in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS (Fetal Bovine Serum) and 1% penicillin-streptomycin antibiotics. The cells were grown in a CO2 incubator containing 5% CO2 and 95% air at 37 °C. The cells were subcultured at 3–4 days of regular interval and subjected to BM-Cyclin and Fungizone periodically. 2.3. Assaying cell viability by MTT assay MTT reagent was used to determine the antiproliferative effect of EAFAD on PC3 and BJ (normal cell line) cell lines (Mohammadi et al., 2016). It is a colorimetric assay in which MTT (3-[4,5-dimethylthiazol2-yl]− 2,5-diphenyltetrazolium bromide) is reduced to blue formazan by mitochondrial dehydrogenase, which reflects mitochondrial

Fig. 1. (a) Ajwa dates, (b) Ajwa date farm in Medina city, Saudi Arabia.

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functions and hence cell's viability. Briefly, 7 × 103 cells/well were seeded in 96 well plate in 100 µL cell culture medium and incubated for 24 h. After 24 h, cells were treated with various concentration of EAFAD (0.2, 0.3, 0.4, 0.5, and 0.6 mg/ml) for 24 and 48 h. Following the treatments, MTT reagent (5 mg/ml) was added along with fresh culture medium into each well and incubated for 5–6 h. The formazan product formed by MTT was solubilized by DMSO and absorbance was measured under microplate reader (Synergy/HTX multimode Reader, BIOTEK) at 540 nm wavelength.

attach overnight. Cells were treated various concentration of EAFAD for 24 h. After 24 h cells were washed, fixed and stained with a 4,6-diamidino-2-phenylindole solution for 10 min at room temperature. After staining, cells were washed with PBS and analyzed under fluorescence microscope for chromatin condensation and fragmentation (Elkady et al., 2014). 2.8. Measurement of intracellular reactive oxygen species Production of intracellular reactive oxygen species is the hallmark of early apoptosis. Production, and accumulation of reactive oxygen species (ROS) in a cell such as the superoxide, hydrogen peroxide, and oxygen, degrade the mitochondrial membrane and releases the cytochrome c into the cytosol, thereupon the process of apoptosis begins (Simon et al., 2000). Here, Intracellular ROS production was measured using 2, 7-dichlorofluorescein diacetate (DCFH-DA) (Cayman Chemicals, USA). When DCFH enters the cells react with ROS to form a fluorescent compound called dichlorofluorescein. Briefly, PC3 cells were treated with EAFAD and after 24 h of treatment, working solution of DCFH-DA was added to wells and incubated for 20–30 min. After incubation, fluorescence was measured in a microplate reader at 485 nm excitation and 528 nm of emission. The values were expressed as a percent of fluorescence intensity relative to control wells.

2.4. Assaying clonogenicity For establishing the long-term cytotoxic effect of EAFAD, the clonogenic assay was performed. The individual ability of the cell to grow into a colony or a clone is defined as clonogenicity. Briefly, PC3 cells were seeded at a density of 1 × 103 cells/well in a six-well plate and allowed to adhere overnight. Next day, cells were treated with various concentrations of EAFAD and kept under observation in an incubator at 37 °C and 5% CO2 until the colonies were clearly distinguished. The colonies were washed with Phosphate buffer saline (PBS) and fixed in 10% formalin and stained with 20 × Giemsa. Each viable cell proliferates into a colony and defined as grouped of ≥ 50 cells, which were counted by colony counter in ImageJ software and manually as well in the inverted microscope (Franken et al., 2006).

2.9. Measurement of mitochondrial membrane potential with JC-1 2.5. Giemsa staining for detection of morphological features of apoptosis Mitochondrial membrane potential was measured by JC-1 (5, 5′, 6, 6′-Tetrachloro-1, 1′, 3, 3′-tetraethylbenzimi-dazolylcarbocyanineiodide), a cationic, lyophilic, and cytofluorimetric dye (Cayman Chemicals, USA). JC-1 exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from red to green. In brief, PC-3 cells were seeded and allowed to grow overnight and treated with various concentration of EAFAD for 24 h. The treated cells were washed with PBS followed by another wash and incubated with the JC-1 cocktail solution (JC-1 stain, JC-1 buffer solution, and virgin DMEM media) for 10–20 min at 37 °C. Finally, fluorescence was measured at 485 nm excitation and 590 nm emission in a microplate reader (Synergy/HTX multimode Reader, BIOTEK) and fluorescent signals were captures through fluorescence microscope (Leica, Wetzlar, Germany).

Giemsa stain used to determine the morphological feature of cells, like blebbing, shrinking and nuclear condensation of cells. Briefly, PC3 cells were plated at 2 × 105 cells/well into a six-well chamber plate. PC3 cells after treatment with EAFAD for 24 h, were fixed with methanol (75%) for 10 min at room temperature. The fixed cells were stained with Giemsa (diluted with phosphate buffer), visualized under the microscope (40 × ) and analyzed. At least 1 × 103 cells were counted to assess apoptotic cell death (Hapon et al., 2014). 2.6. Acridine orange/Ethidium bromide staining for detection of early and late apoptotic cells DNA-binding dyes acridine orange (AO) and Ethidium bromide (EtBr), procured from Sigma, USA, were used for the morphological detection of viable, apoptotic and necrotic cells. AO is taken up by both non-viable and viable cells that emits green fluorescence when intercalated into DNA. EtBr is taken up only by nonviable cells whereas, it is excluded by viable cells and emits red fluorescence by intercalation into DNA (Liu et al., 2009). Briefly, PC3 cells were seeded in a chamber slide at a density of 5 × 104cells/chamber and allowed to grown up to a 60–70% confluence and treated with various concentration of EAFAD for 24 h. Later on, the cells were washed with cold PBS and then stained with a mixture of AO (100 μg/ml) and EtBr (100 μg/ml) at the room temperature for 10 min. The stained cells were washed twice with PBS and observed under the blue filter of the fluorescent microscope. The cells after staining with AO/EtBr were categorically divided into four types as follows: living cells (normal green nucleus), early apoptotic (bright green nucleus with condensed or fragmented chromatin), late apoptotic (orange-stained nuclei with chromatin condensation or fragmentation) and necrotic cells (uniformly orange-stained cell nuclei) (Ittiyavirah et al., 2014).

2.10. Single cell gel electrophoresis or comet assay for detecting DNA fragmentation DNA fragmentation is one of the major hallmarks of apoptotic cells. Comet assay or Single cell gel electrophoresis is used to determine the DNA degradation. In our study, comet assay was performed by using Oxiselect comet assay kit by Cell Biolabs (San Diego, CA, USA). Manufacturer's protocol was followed. Briefly, after treatment of PC3 cells with EAFAD, cells were isolated, washed with ice-cold PBS, centrifuged and resuspended in ice-cold PBS. Low melting agarose was heated at 90–95 °C for 20 min and transferred to the 37 °C in a water bath for 20 min. Cell sample and low melting agarose were mixed in 1:10 ratio. The sample, 75 ul, was pipetted out and loaded to a comet slide provided with the kit. The slide was transferred to 4 °C in the dark for 15 min. Slides were then placed in cold lysis buffer (NaCl, EDTA, 10 × lysis solution (provided), H2O, and PH.10) for 30–60 min at 40C in dark. The lysis solution was aspirated and placed in a cold alkaline solution and incubate in dark at 4 °C for 30 min. After incubation, the slide was placed horizontally in electrophoresis chamber and subjected to electrophoresis for 10–15 min at 1 V. The slide was immersed in prechilled H2O for 2 min and repeated twice. Finally, H2O was replaced with cold 70% ethanol for 5 min. Ethanol was removed and slide was kept for air drying. After complete drying, ethidium bromide was added to the slide and kept at room temperature for 15 min and the slide was focused under the fluorescence microscope for comet tail positive cells.

2.7. DAPI The nucleus undergoes a morphological change in an apoptotic cell, which was analyzed using DAPI. It is a fluorescent nuclear dye that binds to DNA and highlights the changes in the nucleus, fragmentation of DNA and condensation of chromatin. Briefly, PC3 cells were seeded in a chamber slide at a density of 5 × 104 cells/chamber and allowed to 37


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Tail length, percentage of DNA in comet tail, and “Tail Moment” (TM = tail length × tail DNA) was calculated (Choucroun et al., 2001).

treated with EAFAD as compared to control colonies. The number of colonies decreased as the concentration of EAFAD increased as shown in the Fig. 3(a). The cells recovered and proliferate into a colony in the concentration less than IC50 value. The cells reproduced and minimal effect of colonies inhibition was noticed in the concentration equal to IC50 value. Whereas, in concentrations greater than IC50 (0.4 and 0.5 mg/ml) value, a significant reduction in the number of colonies was observed. The complete inhibition of colonies was observed in 0.6 mg/ ml i.e. highest treatment as seen in the Fig. 3. The statically values of colonies were significant as seen in Fig. 3(b).

2.11. Detection of apoptosis by Annexin V-FITC& PI staining (flow cytometer) For detection of apoptosis in the PC3 cell line, Annexin V-FITC Apoptosis Detection kit (Sigma-Aldrich) was used. Cells were seeded at density of 30 × 104 and incubated overnight before treating with various concentrations of EAFAD for 24 h. This experiment was carried out as per the manufacturer's instruction. The treated cells were trypsinized and pelleted by centrifugation at 5000 RPM for 5 min. The pellet was resuspended in the binding buffer provided by the Kit. 10 µL of Propidium Iodide and 5 µL of Annexin V-FITC were added to the cell solution mixture and incubated for 20–30 min in dark at room temperature. The sample was analyzed using flow cytometer (FACS, Novocyte, Acea Bioscience, Inc. San Diego, CA.) and percentage of viable, early apoptotic, late apoptotic and necrotic cells were quantified by Novo Express 1.0.2 software.

3.3. EAFAD induced cellular morphological changes, visualized by Giemsa, acridine orange/Ethidium bromide (Dual staining) and DAPI In AO/EtBr dual staining, most of the cells were uniformly stained green with normal, round, intact nuclei and cytoplasm which indicates the viability of the cells in control. Whereas, in treated cells (0.3 mg/ ml) (Fig. 5), few early apoptotic cells were noticed with condensed nuclei and stained bright green along with normal green (viable) cells. Moreover, in highest concentration (0.6 mg/ml) (Fig. 4) most of the cells were visible in orange/red color that indicates late apoptosis and some of them were bright green indicates early apoptosis. Necrotic cells pose orange to red fluorescence with no signs of chromatin degradation. As shown in (Fig. 5), when cells stained with DAPI, it was evident that, as the concentration of EAFAD increased, the number of apoptotic cells with chromatin condensation and nuclear fragmentation was also increased, but no apoptotic cells were observed in control.

2.12. Cell cycle analysis by PI & RNase (flow cytometer) PC3 cells were seeded in 25 cm2 flask at a density of 20 × 104 and allowed to grow for 24 h. The cells were later treated with various concentration of EAFAD and kept for 24 h. After the treatment, cells were harvested by trypsin and pelleted by centrifugation and washed with PBS. Again, the cells were resuspended in PBS and fixed in ice-cold 70% ethanol for 30 min at 40C. After Fixation, cells were centrifuged and washed twice with PBS, then 1 ml Propodium Iodide master mix solution (40 µL PI, 10 µL RNase, and 950 µL PBS) were added and incubated at 37 °C for 30 min in dark. The cell cycle arrest was analyzed by flow cytometer (FACS, Novocyte, Acea Bioscience, Inc. San Diego, CA.). The percentage of cell populations in G1, S and G2/M phases were determined by using Novo express 1.0.2 software.

3.4. EAFAD induced oxidative stress As the concentration of EAFAD increased the intracellular ROS also increased when compared to control (Fig. 6a). In the highest treatment, ROS production was remarkably significant which indicates that the cells undergo early apoptosis caused by oxidative stress.

2.13. Statistical analysis The differences observed between the control and treated groups for cell proliferation, colony assay, ROS, and MMP were analyzed using Graph Pad Prism 6. Comparison between two groups were made using unpaired student t-test or one-way ANOVA. To assess the IC50 value, log (inhibitor concentration) vs response – variable slope (four parameter logistic) were used. The results were expressed as the Mean ± SEM (standard error of the mean) from three different replicates and a value of p < 0.05 was considered statistically significant.

3.5. EAFAD effects mitochondrial membrane potential in PC3 cells The significant damaged of mitochondrial membrane was observed in treated cells (Fig. 6b), compared to control. When fluorescence was measured at 485 nm excitation and 590 nm emission in a microplate reader, the reading was very high in control and decreased as the concentration of EAFAD increased, it indicates that membrane has been damaged in treated cells compared to control. The fluorescent shift from red to green was observed in EAFAD treated cells by fluorescence microscope (Fig. 6c).

3. Results 3.1. EAFAD has anti-proliferative effect on PC3

3.6. EAFAD induced DNA fragmentation in PC3 cells The demonstrated MTT assay inhibited the growth of PC3 cell lines after treatment with a various concentration of EAFAD in time-dependent manner (Fig. 2a and b). Cell number was gradually decreased, as the concentration of EAFAD increased in both 24(2a) and 48(2b) hours of incubation. EAFAD showed moderate inhibition in PC3 cells, when treated for 48 in comparison to control. Whereas, when treated for 24 h significant decreased in cell number was measured. The calculated IC50 value was 0.3887 and 0.4753 mg/ml for 24 and 48 h respectively. There was no cytotoxic effect of EAFAD were noticed on BJ cell line until 0.5 mg/ml (Fig. 2c). The calculated IC50 value for normal cell line is 0.6169 mg/ml.

Treatment with two concentrations, 0.3 and 0.6 mg/ml were done in compared to untreated cells. In both the treatments, cells with fragmented DNA formed a comet tail (Fig. 7) when stained with Ethidium Bromide and visualized under fluorescence microscope. The length of the comet tail indicated that the fragmented DNA leaking out of the nucleus. The round bright green colored nuclei were noticed without any comet tail in untreated cells (Fig. 7). The amount of DNA in comet tail, length of the tail and tail moment was measured by opencomet, ImageJ software. The average percentage of DNA in the head was 36.52871%, tail contains 63.47129% of DNA in treated cells. The mean tail length measured about 67.33333 whereas, the mean tail moment is measured about 44.4646 by opencomet, imajej software. Thus in both the treatments, comet tail positive cells clearly indicates that EAFAD induced DNA fragmentation which is one of the hallmarks of apoptosis.

3.2. Long-term effect of EAFAD on colony formation Colonies proliferate after 10 days of incubation in control wells. Significant inhibition was noticed in PC3 colony formation when 38


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EAFAD (mg/ml) Fig. 2. Growth inhibition of PC3 cell line. EAFAD inhibit the proliferation of PC3 cell line in a concentration-dependent (0.2, 0.3, 0.4, 0.5, and 0.6 mg/ml) fashion as performed by MTT assay for (a) 24 and (b) 48 h with respect to control. Whereas, EAFAD does not affect the viability of (c) normal cell line. The data was analyzed using log inhibition vs dose response variable slope (four parameters) in Prism GraphPad 6.0 software. The IC50 value of PC3 cell line was calculated as 0.3887 and 0.4753 mg/ml for 24 and 48 h respectively. The IC50 value of BJ cell line was calculated as 0.6169 mg/ml. The values were statistically significant, * ** *** indicates p < 0.05, P < 0.01 and P < 0.001 respectively. The values expressed as mean ± SEM of three different experiments in triplicates.

gradually increases to 35.60% and 37.13% in 0.2 and 0.4 mg/ml respectively (Fig. 9). Number of cell in G2 phase decreased from control 23.83% to 21.68%, and 19.39% in 0.2, and 0.4 mg/ml respectively. This signified that EAFAD arrested the cells in S phase.

3.7. Annexin V-FITC and PI staining EAFAD treated cells with three different concentration in comparison to control demonstrated a concentration-dependent increase in apoptotic cells. Early apoptotic cell increased in 0.2, 0.4, and 0.6 mg/ml in EAFAD treated cells by 23 ± 0.9, 57 ± 0.5 and 67 ± 0.14% respectively, as it was observed by flow cytometer analysis (Fig. 8). In the meantime, under the same treatment the number of viable cells reduced to 76 ± 0.1, 42 ± 0.5, and 32 ± 0.86 respectively. There was a significant increase of late apoptotic cells in highest concentration. These results can be concluded that EAFAD induced apoptosis in the PC3 cell line.

4. Discussion Dietary products have a long history as preventive agents against various cancers. Natural products like green tea, grape skin, pomegranate etc., have chemo-preventive effects (Zhou et al., 2016). Various recent studies proved that many plant-based natural compounds like taxol, vincristine, vinblastine etc., have strong anticancerous properties with low or no side effects (Liu et al., 2009). Ajwa dates are also among one of the naturally occurring fruits with active ingredients that have many pharmacological activities including anti-inflammatory, antioxidative, nephroprotective hepatoprotective and anticancer properties (Mallhi et al., 2014). The Phoenix dactlifera contains various phytochemicals including phenolic acids, carotenoids, flavonoids, anthocyanins, and tannins (Oni et al., 2015). In another study on phenolic content of ajwa dates showed the presence of three pharmocology important phenols that is rutin, catechin and caffeic acid (Saleh, 2011).

3.8. EAFAD arrested cell cycle in S phase PC3 cells after treating with various concentration of EAFAD such as 0.2 mg/ml, and 0.4 mg/ml showed an altered pattern of the cell cycle with respect to control. As the concentration of EAFAD increased, the percentage of cell in S phase also increased significantly, whereas there was drastically decreased in G2 phase in comparison to untreated cells. In control, the percentage of cell in S phase was 28.70%, which 39


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EAF FAD (mg//ml) Fig. 3. Long-term effect of EAFAD on the clonogenic assay. (a) Digital images of wells and (b) microscopic image of colonies at 100 × are depicted in the above pictures after treatment of PC3 cells with various concentration of EAFAD for 10 days in respect to control. (c) The statistical difference between control and treated cells as calculated by one way ANOVA were significant. The number of colonies decreased as the dose of EAFAD increased. The values were statistically significant * and *** p < 0.05 and p < 0.001.

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Fig. 4. Morphological changes in EAFAD treated cells. PC3 cells after treatment with the various concentration of EAFAD demonstrated morphological changes such as cell shrinkage, blebbing and detachment of cell. The images are taken at 100 × and 200 × after staining with Giemsa.

markers including cell shrinkage, loss of cytoskeletal structure, DNA fragmentation were noticed in EAFAD-treated cells. Previous studies had shown the anticancer activity of the methanolic and aqueous extract of ajwa dates on Caco-2 and MCF-7 cell line in vitro (Eid et al., 2014; Khan et al., 2016). However, this is the first study on ethyl acetate fraction of ajwa dates on prostate cancer cell line. MTT assay has shown strong antiproliferation activity of EAFAD in concentration and time-dependent manners, in comparison with untreated cells. The clonogenic assay was performed to confirm the preservative and reproductive ability of a single cell to proliferative into a colony. Therefore, cell survival curve is the relation between the amount of concentration used to produce insult and ability of the cell to reproduce

Rutin and Caffeic acid reported to possess anti-proliferative activity against HepG2 and HT-1080 cell line respectively (Alía et al., 2006; Rajendra Prasad et al., 2011). Cancer cells controlled the cell cycle regulation that helps in evading the host immune system and avoid the cell death and metastasize other tissues in the body (Su et al., 2015). Controlling the cell cycle and inducing apoptosis by the natural compound in cancer cells, would be helpful in treating the diseases in a safer way. The present study was conducted to evaluate the apoptotic effect of ethyl acetate fraction of ajwa dates (EAFAD) on prostate cancer cell line PC3 in vitro. Our study suggested that EAFAD induced feature that is indicative of apoptosis in the prostate cancer cell line. Apoptotic 40


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Fig. 5. Morphological and nuclear changes induced by EAFAD. PC3 cells after treatment with EAFAD induced various nuclear changes such as chromatin condensation, nuclei condensation, and nuclear degradation, as demonstrated by DAPI and acridine orange/Ethidium bromide staining at 400 × . Red arrows indicate early apoptotic and late apoptotic cells.

previous study on crude ethyl acetate extract of marine microalga, Chaetoceros calcitrans, induces Apoptosis in MDA-MB-231 breast cancer cells (Loh et al., 2014). DAPI stained the DNA of cells and gives a morphological feature of the nucleus. It is evident from our results that the EAFAD treated cells, increased the permeability of DAPI in the nucleus with the presence of nuclear apoptotic bodies and condensed chromatin which are the markers of apoptosis. Nuclear permeabilization of DAPI in apoptotic cells was confirmed in earlier studies (Afsar et al., 2016). High level of ROS disrupt the plasma membrane, effect cytoskeletal and finally damage the DNA of the cell and it is collectively called as oxidative stress. Many natural compounds that used anticancer agents are potent ROS generator and can be coined as logical, strategic candidate for killing the cancer cell by increasing oxidative stress (Simon et al., 2000). High level of ROS indicates the sign of early apoptosis.

(Munshi et al., 2005). Our study showed that EAFAD had induced the long-term damage on the PC3 cell line. In oncology, reducing the clonogenicity infers to reducing the number of cells, and dropping the metastatic potential by EAFAD (Hapon et al., 2014). Our result on clonogenicity assay goes in line with the previous study on the longterm effect of an extract of Prosopis strombulifera on HCT and MCF-7 cell lines (Hapon et al., 2014). Further, confirmation of apoptosis was done through morphological assessment of the cell. Fluorescent dyes like Acridine orange/Ethidium bromide dual staining and DAPI were used for morphological changes in cells. As the cells prepare to die, it undergoes numerous morphological changes, chromatin condensation, nuclear condensation and cytoplasm condensation, followed by degradation of nuclei into discrete particles. The results from AO/EtBr stain deduced that the treated cells undergo apoptosis but not necrosis. This result goes in line with the

(a)

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Fig. 6. ROS production and loss of mitochondrial membrane potential by EAFAD. (a) PC3 cells when treated with increasing concentration of EAFAD oxidative stress also increased after 24 h of treatment. Loss of mitochondrial membrane potential was measured using JC-1 dye. (b) Accumulation of JC-1 dye was statically measured by fluorescence in the microplate reader. The p-value was statically significant. (c) JC-1 intensity shift from red to green was measured by Fluorescence microscope. The red arrow indicates the green fluorescence in the treated cells.

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Fig. 7. EAFAD Induced DNA Fragmentation. DNA fragmentation was assessed by Comet assay. Cells were treated for 24 h with 0.3 and 0.6 mg/ml of EAFAD with respect to control. The images were taken by a fluorescent microscope (Leica, Wetzlar, Germany) at 400 × following staining with Ethidium bromide. The tail represents the fragmented DNA leaking out of the nucleus. The amount of DNA ± SEM was calculated in head and tail with respect to control. The average DNA in head and tail contain 36.52871% and 63.47129% respectively. The mean tail length is 67.33333. The mean tail moment was measured about 44.4646 in treated cells as calculated using opencomet, ImageJ software.

comparable with previous findings on induction of apoptosis in human leukemia K562 cells by tea (Chakraborty et al., 2006). For detection of apoptotic cells, Annexin V-FITC co-stain with propidium iodide was used mostly. Annexin V is a phosphatidylserine binding protein that binds specifically to phosphatidylserine residual that externalized to outer membrane in cells committing apoptosis. Our results confirmed the presence of annexin V-FITC and propidium iodide positive cells in EAFAD treated cells. Similar results were observed previously when Myricetin induced apoptosis in the pancreatic cell line (Phillips et al., 2011). Our result showed that EAFAD induced cell cycle arrest in the PC3 cell line. Dysregulation of cell cycle perhaps linked with the cancerous transformation of the cell and often serve as a therapeutic target in cancer biology (Evan and Vousden, 2001). Till now most of the anticancer agent arrests the cell in G0/G1 or G2/M phase, but in rare cases, S phase arrest was reported in previous studies (Pan et al., 2010). Results in our study concluded that the EAFAD arrested the cell in S phase and decreased the number of cells in G2 phase. These findings go in line with the previous study on ajwa dates where MCF-7 cells were arrested in S phase (Khan et al., 2016).

Our result confirmed that EAFAD stimulates ROS production in the PC3 cells that created the oxidative stress, destabilize mitochondria and consequently induced apoptosis as confirmed in the previous study (Looi et al., 2013). The mitochondria play a vital role in apoptosis by sending death signals that activate the caspases and eventually lead to cell death. As mitochondria losses it's membrane integrity, then cytochrome c released into the cytosol that ultimately leads to the formation of apoptosome and orchestrates the complete process of apoptosis. Accumulation of JC-1 inside mitochondria when mitochondrial integrity lost gives the clear difference between the apoptotic and viable cells. When PC3 cell lines were treated with EAFAD and stained using JC-1, it showed degradation of the mitochondrial membrane. Many anticancerous compounds have the ability to dissipate the mitochondrial membrane potential. Our results cope up with the previous investigation apoptotic inducing effect of estragole isolated from Fennel (Villarini et al., 2014). Cells with fragmented DNA is hard to be repaired, so it will undergo apoptosis. The massive amount of fragmented DNA in a single cell can be identified by comet assay. Comet assay showed distinct feature between apoptotic and necrotic cells also (Yasuhara et al., 2003). Comet assay was established for measuring DNA damage after exposure to genotoxic agents. However, comet assay images are different for apoptotic cells compare to DNA damaged induced by genotoxic agents (Chakraborty et al., 2006). After following the comet assay procedure, almost all the degraded DNA coming out of the head of apoptotic cells forming a comet-like appearance when treated with EAFAD. No comet tail formed in untreated cells. Our results on comet assay were

5. Conclusion In conclusion, the results of our study demonstrated that the EAFAD induced apoptosis in the human prostate cancer cell line (PC3). This clearly provides us a clue that the active compounds of Ajwa dates could be the potent natural anticancer agents. However, further studies are required to explore these active compounds in EAFAD that could

Fig. 8. The Dot plot representation of dual staining of annexin V-FITC and propidium iodide assay on the PC3 cell line. Annexin V positive and annexin V/propidium iodide positive cells are early and late apoptotic cells as confirmed by Flow cytometer. The EAFAD induced apoptosis in the PC3 cell line.

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Fig. 9. Effect of EAFAD on cell cycle arrest. Representation of cell cycle arrest induced by EAFAD after exposed to 24 h in the PC3 cell line. EAFAD induced cell cycle arrest in S phase as it is indicated and decreased in G2 phase.

lead to potent novel anticancer agents.

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