M cell cycle arrest and apoptosis through mitochondria-mediated pathway in MDA-MB-231 human breast cancer cell

Author’s Accepted Manuscript Phaleria macrocarpa (Boerl.) Fruit induce G0/G1 and G2/M cell cycle arrest and apoptosis through mitochondria-mediated pathway in MDA-MB-231 Human Breast Cancer Cell Nowroji Kavitha, Chern Ein Oon, Yeng Chen, Jagat R. Kanwar, Sreenivasan Sasidharan

PII: DOI: Reference:

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S0378-8741(16)32492-8 http://dx.doi.org/10.1016/j.jep.2017.02.041 JEP10745

To appear in: Journal of Ethnopharmacology Received date: 24 December 2016 Revised date: 22 February 2017 Accepted date: 25 February 2017 Cite this article as: Nowroji Kavitha, Chern Ein Oon, Yeng Chen, Jagat R. Kanwar and Sreenivasan Sasidharan, Phaleria macrocarpa (Boerl.) Fruit induce G0/G1 and G2/M cell cycle arrest and apoptosis through mitochondria-mediated pathway in MDA-MB-231 Human Breast Cancer Cell, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2017.02.041 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Phaleria macrocarpa (Boerl.) Fruit induce G0/G1 and G2/M cell cycle arrest and apoptosis through mitochondria-mediated pathway in MDA-MB-231 Human Breast Cancer Cell Nowroji Kavithaa, Chern Ein Oona, Yeng Chenb, Jagat R. Kanwarc and Sreenivasan Sasidharana,* a

Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, USM

11800, Pulau Pinang, Malaysia b

Dental Research & Training Unit, and Oral Cancer Research and Coordinating Centre

(OCRCC), Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia c

Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (LIMBR),

School of Medicine (SoM), Faculty of Health, Institute for Frontier Materials (IFM), Deakin University, Waurn Ponds, VIC 3217, Australia. *

Correspondence: Institute for Research in Molecular Medicine (INFORMM), Universiti

Sains Malaysia, USM 11800, Pulau Pinang, Malaysia. Tel: +604 653 4820. Fax: +604 653 4803. [email protected]

ABSTRACT Ethnopharmacological relevance Phaleria macrocarpa (Scheff) Boerl, is a well-known folk medicinal plant in Indonesia. Traditionally, P. macrocarpa has been used to control cancer, impotency, hemorrhoids,

diabetes mellitus, allergies, liver and hearth disease, kidney disorders, blood diseases, acne, stroke, migraine, and various skin diseases. Aim of the study The purpose of this study was to determine the in situ cytotoxicity effect P. macrocarpa fruit ethyl acetate fraction (PMEAF) and the underlying molecular mechanism of cell death. Materials and methods MDA-MB-231 cells were incubated with PMEAF for 24 hours. Cell cycle and viability were examined using flow cytometry analysis. Apoptosis was determined using the Annexin V assay and also by fluorescence microscopy. Apoptosis protein profiling was detected by RayBio® Human Apoptosis Array. Results The AO/PI staining and flow cytometric analysis of MDA-MB-231 cells treated with PMEAF were showed apoptotic cell death. The cell cycle analysis by flow cytometry analysis revealed that the accumulation of PMEAF treated MDA-MB-231 cells in G0/G1 and G2/M-phase of the cell cycle. Moreover, the PMEAF exert cytotoxicity by increased the ROS production in MDA-MB-231 cells consistently stimulated the loss of mitochondrial membrane potential (∆Ψm) and induced apoptosis cell death by activation of numerous signalling proteins. The results from apoptosis protein profiling array evidenced that PMEAF stimulated the expression of 9 pro-apoptotic proteins (Bax, Bid, caspase 3, caspase 8, cytochrome c, p21, p27, p53 and SMAC) and suppressed the 4 anti-apoptotic proteins (Bcl-2, Bcl-w, XIAP and survivin) in MDA-MB-231 cells.

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Conclusion The results indicated that PMEAF treatment induced apoptosis in MDA-MB-231 cells through intrinsic mitochondrial related pathway with the participation of pro and antiapoptotic proteins, caspases, G0/G1 and G2/M-phases cell cycle arrest by p53-mediated mechanism.

Graphical abstract

Keywords: cells cycle arrest; apoptosis; caspase, p53, cytochrome c

1. Introduction

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Breast cancer is the second greatest cancer and the fifth main reason for cancer-related deaths in females throughout the world. Continued usage of chemotherapeutic treatment against breast cancer is frequently compromised due to the development of resistance against the anticancer drug (Venkatadri et al., 2016; American Cancer Society, 2015). Therefore, finding alternative drugs is essential to decrease the death rate related to breast cancer worldwide. Numerous medicinal plants rich in naturally occurring phytochemicals have been found to possess a cytotoxic effect that induces apoptotic cancer cell death (Banerjee et al., 2016). One such medicinal plant that is known to have anticancer potential with various pharmacological activities is Phaleria macrocarpa (Scheff) Boerl of the family Thymelaceae. P. macrocarpa. It grows well in tropical areas, and is commonly identified as ‘God's crown’ or locally known as ‘Mahkota dewa’. The mature tree height generally ranges from 1 m to 18 m. The flowers of P. macrocarpa make a compound of 2-4, and vary in colour from green to maroon. The unripe elliptical shaped fruit of P. macrocarpa is green and becomes red upon ripening with a diameter of 3 cm. The seeds of the P. macrocarpa fruit commonly present as one to two seeds, which are brown, ovoid and anatropous (Atlaf et al., 2013). The unprocessed usage of this herb has been reported as poisonous and toxic (Yosie et al., 2011). In Asian folk medicine, P. macrocarpa has been used to control cancer, impotency, haemorrhoids, diabetes mellitus, allergies, liver and heart diseases, kidney disorders, blood diseases, acne, strokes, migraines, and various skin diseases (Anggraini and Lewandowsky, 2015). Moreover, P. macrocarpa is a medicinal plant rich with many phytochemicals and has been broadly studied for its anticancer activities in multiple cancer cell lines. Even though extensive in vitro anticancer studies have been carried out (Shwter et al., 2016; Lay et al., 2014a; Muchtaridi et al., 2014; Riwanto et al., 2011; Tandrasasmita et al., 2010 Lay et al., 2014b; Tandrasasmita et al., 2015; Yosie et al., 2011), there have been only limited attempts to explore the cytotoxicity

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mechanism of the P. macrocarpa extract in relation to its anticancer activity. With this background, this research was designed to study the in vitro cytotoxicity mechanism of P. macrocarpa fruit ethyl acetate fraction (PMEAF) in a breast cancer cell line, MDA-MB-231. The cytotoxicity of anticancer drugs should destroy the cancerous cell without adverse effects on normal cells, which is possible through the induction of apoptosis (Blagosklonny, 2004). Medicinal plants trigger apoptotic pathways that inhibit cell growth through various mechanisms in cancer cells (Safarzadeh et al., 2014). Furthermore, a cancer cell undergoing apoptosis triggers a series of molecular and biochemical events that lead to unique characteristics that might be determined by microscopy, biochemically, or flow cytometry techniques. The typical morphological features of apoptotic cells can be observed through microscopic studies (Saraste and Pulkki, 2000). A morphological observation study using Acridine Orange/Propidium Iodide (AO/PI) staining with a fluorescence microscope strongly revealed that PMEAF treatment induced higher apoptotic cell death in PMEAF treated MDAMB-231 cells compared to the non-treated control cells. Moreover, the majority of structural and biochemical events happening during cell death can be examined by flow cytometry. Therefore, it is important to further study the molecular mechanism of PMEAF apoptotic cell death activities against MDA-MB-231 cells. Hence, the current study was conducted to provide biochemical evidence concerning the apoptotic cell death activities of PMEAF in detail by multi-parametric flow cytometric analysis and biochemical assay of the functional events associated with cell apoptosis. The molecular mechanism of PMEAF responsible for such apoptotic cell death activities plays a significant role in determining the efficacy of specific treatments that lead to efficient cancer cell death (Hassan et al., 2014).

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2. Materials and methods 2.1. P. macrocarpa fruits collection and extraction The ripen red fresh fruits of P. macrocarpa were collected from labelled trees growing in ECO HUB, Pusat Repositori Kearifan Tempatan, Universiti Sains Malaysia (USM), Penang around March 2013. The red ripen fruits were washed, cut into pieces and dried in an oven at approximately 50°C for 7 days. The fruits were then ground into a fine powder form by using grinder. The dried powder (100 g) was macerated with 80% aqueous methanol (400 mL) for 72 hours before filtration (Whatman paper No1) and the extraction process was repeated for three times to obtained crude extract. The filtrate obtained was concentrated under reduced pressure of 6 × g at 37°C by rotary evaporator (Buchi Rotavapor R 210; Buchi Labortchnik, Falwil, Switzerland). This crude methanol extract was then further fractionated with ethyl acetate (90%) and water (10%) by using separation tunnel. The ethyl acetate fraction was filtered again and evaporated at 37°C to remove excessive solvents. Then poured in glass beaker and brought to dryness at 40°C oven. The thick paste-like mass was obtained then sealed with parafilm and covered with aluminium foil before placed it in dark cabinet at room temperature. Fractionated P. macrocarpa fruits ethyl acetate extracts was denoted as Phaleria Macrocarpa Fruit Ethyl Acetate Fraction and abbreviated as PMEAF. 2.2. Chemical Analysis of PMEAF using GC-MS analysis GC-MS analysis was conducted by using thermo gas chromatograph-mass spectrometer (Agilent 6890N / 5973) equipped with DB-5 capillary column (30 m long. 0.25 mm i.d., film thickness 0.25 µm). The column temperature was programmed initial temperature of 70°C for 2 minutes, with 20°C per minute increases reached to final 280°C which was maintained for

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20 minutes. Helium gas (99.99%) was a carrier gas with constant flow rate of 1 mL per minute with split 10:1. The mass spectrometry mode used was electron ionization (EI) mode with a current of 70 eV. The PMEAF was diluted with methanol solvent to produce a concentration of 7 mg/mL. The diluted sample volume of 1 μL was injected with a split mode at a ratio of 1: 20. The injection port temperature was set to 230°C and the detector/interface temperature was set to 250°C. The results were collected for 40 minutes. The percentage of chemical components was expressed as percentage by peak area normalization. The compounds were identified by GC/MSD ChemStation Software searches in the commercial libraries of the National Institute of Standard and Technology (NIST). 2.3. Determination of Total Phenolic Contents (TPC) A total phenolic content in PMEAF was determined using the Folin-Ciocalteu method with slight modification (Hossain and Shah, 2015). Briefly, gallic acid was prepared at different concentrations in methanol (10, 20, 40, 60, 80, 100, 200 µg/mL) was used as positive reference standard. Five mL of Follin-Ciocalteau reagent (1: 10 diluted with dH2O) added to 1 mL of each concentration of prepared gallic acid into centrifuge tube and vortex for 10 seconds. Each sample was prepared in triplicates. The mixture was incubated at room temperature for 5 minutes. Subsequently 4 mL of 7.5% sodium carbonate (Na2CO3) was added into each tube and vortex again for 10 seconds. The mixture was incubated at 2 hours in darkness at room temperature. The absorbance was measured at 765 nm using microplate reader system (Molecular Devices Inc., USA). Total phenolic content was determined using a standard curve of gallic acid. The method was repeated with PMEAF at 100 µg/mL concentration and absorbance reading taken for each sample by triplicates.The TPC in

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PMEAF was determined from the standard curve and was expressed as milligram (mg) of gallic acid equivalents (GAE) per gram. The formula below was used to calculate the TPC in concentration of PMEAF C = cV/m Where, C = TPC (mg GAE/g)

c = concentration of gallic acid obtained from the standard curve (mg/mL) V = volume of extract (mL) m = mass of extract (g)

2.4. Cell culture and treatment The Triple-negative breast cancer MDA-MB-231 cell line was obtained from American Type Culture Collection (ATCC, Va, USA). MDA-MB-231 cells were grown in DMEM supplemented with 10% FBS, 2 mM glutamine, 1% penicillin-streptomycin in a humidified incubator (5% CO2 in air at 37 °C). Cultures were harvested and monitored for cell number by counting cell suspensions with a hemocytometer. Cell morphology was examined using phase contrast microscopy (200× magnification). The PMEAF IC50 concentration of 18.10 µg/mL against MDA-MB-231 cells resulting from previous MTT and CyQUANT cytotoxicity assays was used in this study (Kavitha et al., 2017). PMEAF sample was solubilized in sterile filtered Dimethyl sulfoxide (DMSO) (0.2% in the culture medium) prior to addition to the culture media. Cell were then treated with PLME at the concentration of half ½ × IC50 (9.05 µg/mL), IC50 (18.10 µg/mL) and 2 × IC50 (36.20 µg/mL) (Kavitha et al., 2017). The negative control for all the assays was represented by the untreated medium containing vehicle DMSO (0.2%).

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2.5. Morphological detection of apoptosis using Acridine Orange/Propidium Iodide (AO/PI) staining

AO/PI staining method use inclusion and/or exclusion dyes to test the integrity of the plasma membrane (Bank, 1988) for accurate determination of live and dead nucleated cell of isolated single suspension cells using dual-fluorescence. In brief, MDA-MB-231 cells at density of 1.5 × 105 were seeded in 25 cm2 culture dishes and treated with vehicle 0.2% DMSO and ½ × IC50, IC50 and 2 × IC50 of PMEAF for 24 hours. After treatment time, cells were harvested via adding 1 ml of trypsin to obtain single cell suspension and centrifuged at 125 × g for 5 minutes. Then, the cells were mixed with 10 µl of AO (1 mg/mL) and 10 µl of PI (1 mg/mL); the total 20 µL of the mixture was aliquoted into slides and covered with a cover slip. Then the slides were viewed under fluorescence microscope (Carl Zeiss, Germany) to identify viable, apoptotic and necrotic cells based on morphological changes, including membrane blebbing, nuclear and cytoplasmic condensation. AO was binds to DNA of live cells to fluoresce bright green while PI can only enter dead cells to produces bright orange or red colour and no signal is generated from non- nucleated cells or debris. A minimum of 200 cells were counted per slides. 2.6. Detection of apoptosis using the Annexin V-FITC/PI assay by flow cytometry Distribution of early and late apoptotic cells after treatment with PMEAF was further investigated using FITC Annexin V Apoptosis Kit 1 (BD Biosciences, USA). In brief, MDAMB-231 cells at density of 1.5 × 105 were seeded in 25 cm2 culture dishes and treated with vehicle 0.2% DMSO and ½ × IC50, IC50 and 2 × IC50 of PMEAF for 24 hours. Subsequently, the cells were treated with 1 mL of trypsin before centrifuged at 125 × g for 5 minutes. The supernatant was discarded before washed twice with cold PBS and resuspended the cells in 1

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× binding buffer at density of 1 × 106 cells/mL. After that, 100 µL of the solution contains of 1 × 105 cells was transferred into a 5 mL round bottom tube particularly for flow cytometry use. Next, 5 µL of FITC conjugated Annexin V (Annexin V-FITC) with 5 µL PI was added and vortex the mixtures before incubated for 15 minutes at room temperature (25°C) in dark. Finally, 400 µL of 1 × binding buffer was added into each tube and immediately analysed by flow cytometry (BD FACSCanto II) within 1 hour. Data from 10000 cells were collected for each sample. Four different populations of cells were easily distinguished; unlabelled as viable cells, bound to Annexin V-FITC only as early apoptotic and stained with PI considered as necrotic whereas bound with both dyes Annexin V-FITC and PI as late apoptotic/necrotic cells. The fluorescence distribution was displayed as a dot plot analysis and the percentage of fluorescence cells in each quadrant was determined. 2.7. Cell cycle analysis by flow cytometry Changes in cell cycle distribution induced by PMEAF were analyzed using the CycleTEST™ PLUS DNA Reagent Kit (BD, Bioscience). In brief, MDA-MB-231 cells at density of 1.5 × 105 were seeded in 25 cm2 culture dishes and treated with vehicle 0.2% DMSO and ½ × IC50, IC50 and 2 × IC50 of PMEAF for 24 hours. After treatment period, cells were harvested via adding 1 mL of trypsin to obtain single cell suspension and transferred to 15 mL centrifuge tube before centrifugation at 300 × g for 5 minutes at room temperature (25°C). Harvested cells were washed with PBS and stained with PI using a CycleTEST Plus DNA reagent kit (BD BioSciences, San Jose, USA) according to manufacturer’s instructions. The linearity FACS CantoII (BD BioSciences, San Jose, USA) was tested using BD DNA QC Particles kit (BD BioSciences, San Jose, USA). Doublets, disintegrated nuclei and other cell debris were removed from analysis by gating forward and side scatter profile of samples. The gates were

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uniformly maintained across all samples in each run. For each sample, 10000 or 20000 events thousand events (nuclei) were collected and the resulting histograms with percentages of cells in G0/G1, S and G2/M phases were analyzed using Modfit software Version 3.2 (Verify Software House, USA). 2.8. Reactive Oxygen Species (ROS) assay Reactive oxygen species (ROS) were measured on the basis of the intracellular peroxidedependent oxidation of DCFH-DA to form the fluorescent compound 2′,7′-dichlorofluorescein (DCF). The generations of intracellular oxygen species ROS in MDA-MB-231 cells after treatment with PMEAF was estimated using Intracellular Cell Biolabs’ OxiSelect™ Intracellular ROS Assay Kit (San Diego, USA). Cells were seeded on to 96-well plates at a density of 1.0 × 104 cells per well and cultured for 24 h. Then, 100 µL fresh DMEM medium without FBS containing of 1 × DCFH-DA was added and cells were incubated for 1 hour in dark at 37°C. After washing twice with PBS, fresh medium containing 0.2% DMSO (Negative control), ½ × IC50, IC50 and 2 × IC50 concentration of PMEAF were added and cells were incubated for 24 h. Positive control hydrogen peroxide (H2O2 at 200 mM) was added to DCFH-DA treated cell 1 hour prior to the detection of fluorescence intensity. The cells were washed twice with PBS, 200 µL of 2 × lysis buffer (diluted with DMEM 1:1) were added to each well and mixed thoroughly before incubated for 5 minutes at room temperature (25°C). The fluorescence intensity was determined with a standard fluorescence microplate reader with wavelength of excitation 480 nm and emission 530 nm. The ROS was determined by comparison with the predetermined DCF standard curve. 2.9. Mitochondrial membrane potential analysis by flow cytometry

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The Mitochondrial Membrane Potential Detection Kit (BD™ MitoScreen, BDbiosciences, USA) was used to measure the alteration of the mitochondria membrane potential (ΔΨm). In brief, 1.5 × 105 cell/mL of MDA-MB-231 cells were seeded into 25 cm2 culture flasks and treated with 0.2% DMSO (negative control), 1 mM of carbonyl cyanide mchlorophenylhydrazone (CCCP, Sigma Aldrich, St. Louis, USA, positive control), and ½ × IC50, IC50 and 2 × IC50 of PMEAF for 24 hours. The ∆Ψm was assessed using JC-1 from BD™ MitoScreen kit (BD Biosciences, San Jose, USA) according to manufacturer’s instructions. JC-1 is a cationic dye that easily penetrates and accumulates in mitochondrial polarized membranes, serving as a sensitive way to measure ∆Ψm. Cells were harvested and transferred into 15 mL Falcon tubes. The JC-1 dye was added to the cells (500 µL) in each tube and incubated at 37°C with 5% CO2 for 15 min. Following incubation, 2 mL of JC-1 Assay Buffer (BD Biosciences, San Jose, USA) was added and centrifuged at 400× g for 5 min before resuspending in 500 µL JC-1 Assay Buffer. The flow cytometry analysis was performed on BD FACS CantoII (BD BioSciences, San Jose, USA). A 50,000 to 100,000 events were collected per sample and data were analysed using FACS Diva Version 6.1.3 software (BD BioSciences, San Jose, USA). A scatter plot of red (intact ∆Ψm) versus green fluorescence (decreased ∆Ψm) was produced. The percentage of cells with green flourescene (JC-1 monomers) was measured as mitochondrial depolarized (ΔΨm) cells. 2.10. Bicinchoninate Protein Assay Protein Assay Bicinchoninate Kit (Nacalai Tesque, Inc., Japan) was used to perform Bicinchoninate modified Lowry method in this study with bovine serum albumin (BSA) as a standard. In brief, 1.5 × 105 cell/mL of MDA-MB-231 cells were seeded into 25 cm2 culture flasks and treated with vehicle 0.2% DMSO (negative control), and IC50 of PMEAF for 24

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hours. Then, cells pallet were harvested via adding trypsin and centrifuged at 125 × g for 5 minutes. Then, 25 µL of sample and BSA standard (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/mL) was added into each well in 96 well microplates. Deionized water was used as a blank. Subsequently, 200 µL of working solution was added into each well and mixed well by pipetting for 30 seconds before incubated at 37°C for 30 minutes. The microplate was restored at room temperature (25°C) for 5 minutes before the measurement was taken by using ELISA microplate reader at 562 nm. A standard curve was plotted with x–axis is protein concentration and y-axis is an absorbance to determine the protein concentration of samples. 2.11. Bioray-Apoptosis Related protein RayBio® Human Apoptosis Array G-series (RayBiotech, Inc.) was used for efficiently screening the expression levels of apoptosis-related proteins such as Bax, Bid, Bcl-2, Bcl-w, cytochrome c, caspase 3, 8, p21, p27, and p53. In brief, 2.0 × 106 cell/mL of MDA-MB-231 cells were seeded into 75 cm2 culture flasks and treated with vehicle 0.2% DMSO (negative control), and IC50 of PMEAF for 24 hours. Total protein was extracted from the cells and approximately 600 µg of protein were analyzed using a protein array with antibodies against 43 apoptosis-related proteins (Human Apoptosis Array Kit, RayBiotech Inc, USA). After incubation with a cocktail of biotinylated antibodies and labeled-streptavidin, the signal was detected by chemiluminescence using an Axon GenePix AGP 4000B using the Cy2 channel. Spot signal intensity was analyzed using the GenePix Pro 6.0 software and the RayBio® Antibody Array Analysis Tool. Intensities among different arrays were normalized using an internal control as suggested by manufacturer. Intensity values above the average intensities of the negative controls were taken as positive signal and signal intensities greater than 1.5fold or higher than untreated cells were considered to be a significant change. This experiment

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was repeated four times. Results were reported as mean ± SEM for at least three analyses for each sample. 2.12. Statistical analysis Data were expressed as mean ± standard deviation (SD) from at least three independent experiments (n = 3; for each experiment). The statistical analysis was performed based on Analysis of Variance (ANOVA) followed by Turkey’s multiple comparison test by using Graphpad Prism version 7.01 (Graphpad, San Deigo, CA, USA). A value of p < 0.05 was considered to be statistically significant.

3. Results 3.1. Plants extract yield percentage The percentage extraction yield for the ethyl acetate fraction of P. macrocarpa fruit was 6.4% of the overall dried weight of fruit powder of 31.87 g used for extraction in this study. 3.2. Gas Chromatography Profile of PMEAF using GC-MS analysis The PMEAF fractionact was characterised by GC-MS. The chromatographic analysis revealed five major components including phenol, 2,4-bis(1,1-dimethylethyl) (1 with retention time (RT): 8.38 and area: 3.60%), Hexadecanoic acid, methyl ester (2 with RT: 10.65 and area: 17.01%), 8,11-Octadecadienoic acid, methyl ester (3 with RT: 11.49 and area: 28.72%), 9-Octadecenoic acid, methyl ester (4 with RT: 11.51 and area: 39.60%), and Heptadecanoic acid, 16-methyl ester (5 with RT: 11.61 and area: 4.14%) (Fig. 1). 3.3. Total phenolic content (TPC)

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The TPC of PMEAF was measured by the Folin-Ciocalteu method and expressed in terms of gallic acid equivalents (mg of GAE/g of fraction). Gallic acid was used as calibration for the standard curve graph, which was plotted in a range of concentrations from 10 to 200 µg/mL. The TPC was expressed as mg GAE/g of fraction using the standard curve linear regression equation: y = 0.0082x + 0.1425, R2 = 0.987, where y is absorbance at 765 nm, and x is the total phenolic content in the PMEAF. The total phenolic content of 1mg/mL of PMEAF was evaluated to be 14.91 ± 0.97 mg GAE/g of fraction. 3.4. Quantification of apoptosis using propidium iodide and acridine orange double staining

With the aim of quantifying the degree of apoptosis induced by PMEAF in MDA-MB-231 cells, propidium iodide (PI) and acridine orange (AO) double-staining was used in this study. The MDA-MB-231 cells were scored under a confocal microscope after treatment with PMEAF, to enumerate the number of cells that were categorised as viable, apoptotic and necrotic. A total of 200 MDA-MB-231 cells were used randomly and differentially, together with untreated negative control cells for counting. The results of this study showed that PMEAF caused morphological changes in the treated MDA-MB-231 cells that relate to the induction of apoptosis upon treatment in a dose-dependent manner at ½ × IC50, IC50 and 2 × IC50 (Figs. 2a, 2b, 2c and 2d). The untreated MDA-MB-231 control cells showed the normal morphology of a circular cell without prominent apoptosis and necrosis with a round and green intact nuclear structure (Fig. 2a). In addition, the MDA-MB-231 cells treated with ½ × IC50 of PMEAF for 24 hours showed slight changes in the morphology with chromatin condensation. The presence of more early apoptosis cells (AP) marked by crescent-shaped or granular yellowgreen (AO) nuclear staining is obvious compared to the late apoptotic cells (LA) (Fig. 2b) in 15

the MDA-MB-231 cells treated with ½ × IC50 of PMEAF. MDA-MB-231 cells treated with IC50 of PMEAF for 24 hours showed considerable changes in morphology with membrane blebbing and chromatin condensation. The presence of extra late apoptotic cells (LA) marked by a reddish-orange colour due to the binding of (AO) to denatured DNA is obvious compared to the apoptosis cells (AP) and necrosis cells (N) (Fig. 2c). However, the MDAMB-231 cells treated with 2 × IC50 of PMEAF for 24 hours showed a considerable change in the morphology with shrinking cells with a condensed fragmented nucleus and membrane blebbing. The presence of extra late apoptotic cells (LA) and necrosis cells (N) were obvious compared to apoptosis cells (AP) (Fig. 2d). The necrosis cells were marked by red cells. Besides the study of the morphological changes, the assessment of the frequency of viable, apoptotic and necrotic cell populations (200 cells) was also recorded and plotted as a histogram; Fig. 3. The differential scoring of PMEAF treated MDA-MB-231 cells showed that there is a significant difference (p < 0.05) in the apoptotic cell population, which indicates that PMEAF has a dose-dependent manner apoptogenic effect on MDA-MB-231 cells. Conversely, there was no statistically significant (p > 0.05) difference in the necrotic counts at ½ × IC50 and IC50 except at 2 × IC50 (Fig. 3). 3.5. Quantitative analysis of apoptotic cells by flow cytometry To determine whether the decrease in cell viability after PMEAF treatment is the result of apoptosis in the MDA-MB-231 cells, flow cytometry analysis was further conducted to detect the apoptotic cells. The flow cytometry analysis revealed the induction of apoptosis in MDAMB-231 cells after treatment with PMEAF at ½ × IC50, IC50 and 2 × IC50 concentrations for 24 hours. The untreated cells (Fig. 4a and Fig. 5) showed 87.57% viability (in Q3 quadrant), 5.1% in early apoptosis (in Q4 quadrant), 7.17% in late apoptosis (in Q2 quadrant) and 0.2% in necrosis (in Q1 quadrant). However, the treatment of MDA-MB-231 cells with PMEAF at 16

doses of ½ × IC50, IC50 and 2 × IC50 concentration for 24 hours resulted in a dose-dependent increase (p < 0.05) in the number of apoptotic cells (Fig. 4 and Fig. 5). For instance, after treatment for 24 hours with IC50 concentration of PMEAF, the MDA-MB-231 cells showed 49.37% viability, 14.83% in early apoptosis, 35.1% in late apoptosis, and 0.67% in necrosis. As the treatment doses increased from half IC50 to double IC50, the percentage of both early and late apoptotic cells continued to increase substantially (Fig. 4 and Fig. 5). Conclusively, the total percentage of apoptotic cells (top right Q2 quadrant + bottom right Q4 quadrant) after PMEAF treatment (from ½ × IC50, to 2 × IC50) was dose-dependently increased (Fig. 5). This result provided proof that treatment of MDAMB-231 cells with PMEAF showed the presence of early apoptosis, late apoptosis and necrosis in MDA-MB-231 cells as the concentration of treatment was increased from ½ × IC50 to 2 × IC50 for 24 hours.

3.6. Cell cycle analysis Flow cytometric analysis of the cell cycle and DNA content was accomplished to study the ability of PMEAF to induce cell cycle arrest and apoptosis in MDA-MB-231 cells treated with ½ × IC50, IC50 and 2 × IC50 concentration of PMEAF for 24 hours. The G0/G1 population increased significantly (p < 0.05) in a dose-dependent manner from 52.50% in the untreated cells to 62.19%, 69.73% and 73.22% in the ½ × IC50, IC50 and 2 × IC50 of PMEAF treated MDA-MB-231cells for 24 hours, respectively (Fig. 6 and Fig. 7). Similarly, the G2/M population increased significantly (p < 0.05) in a dose-dependent manner from 15.87% in the untreated cells to 17.18%, 25.80% and 29.65% in the ½ × IC50, IC50 and 2 × IC50 of PMEAF treated MDA-MB-231cells for 24 hours, respectively (Fig. 6 and Fig. 7). Moreover, the PMEAF treatment of MDA-MB-231 cells led to the increased accumulation (p < 0.05) of

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apoptotic sub G1 phase cells in a concentration-dependent manner (1.40% in the untreated cells to 2.72%, 5.48% and 8.18% in the ½ × IC50, IC50 and 2 × IC50, respectively) indicating that PMEAF is capable of inducing significant G0/G1 and G2/M phases arrest. However, the S phase population decreased significantly (p < 0.05) in a dose-dependent manner from 28.34% in the untreated cells to 24.65%, 10.77% and 9.77% in the ½ × IC50, IC50 and 2 × IC50 of PMEAF treated MDA-MB-231 cells for 24 hours, respectively (Fig. 6 and Fig. 7).

3.7. Intracellular ROS Intracellular reactive oxygen species (ROS) analysis was conducted to study the role of ROS induction by PMEAF to induce apoptosis in MDA-MB-231 cells treated with ½ × IC50, IC50 and 2 × IC50 concentration of PMEAF for 24 hours. In the presence of ROS, dichlorodihydrofluorescein is oxidized to produce a fluorescent product, namely, dichlorofluorescein (DCF). To determine whether the MDA-MB-231 cell death induced by PMEAF is reliant on the ROS levels, the DCF standard curve was plotted to interpret the intracellular ROS production (Fig. 8). Treatment with PMEAF resulted in the significantly (p < 0.05) increased production of ROS in MDA-MB-231 cells (p < 0.05) in a dosedependent manner from 1.97 of the relative fluorescence units in the untreated cells to 2.11, 4.01 and 4.80 of the relative fluorescence units in the ½ × IC50, IC50 and 2 × IC50 of PMEAF treated MDA-MB-231 cells for 24 hours, respectively (Fig. 9). Meanwhile, the positive control H2O2 exhibited the highest production of ROS with 5.99 of the relative fluorescence units. 3.8. Mitochondrial membrane potential analysis The mitochondria are an essential part of the apoptotic mechanism and the loss of mitochondrial membrane potential (MMP or ∆Ψm) is standard evidence for apoptosis induced 18

by ROS. An increase in ROS leads to mitochondrial depolarization during apoptosis. Hence, to study the alterations in the (∆Ψm) and the effects of intracellular ROS production by PMEAF, the ∆Ψm was determined using JC-1 dye staining and flow cytometry analysis of the fluorescence emission shift from green (~529 nm) to red (~590 nm) on untreated MDA-MB231 cells and MDA-MB-231 cells treated with ½ × IC50, IC50 and 2 × IC50 concentration of PMEAF for 24 hours. After PMEAF treatment for 24 hours, the green fluorescence (JC-1 as a monomer at low membrane potentials) and a red fluorescence (JC-1 as “J-aggregates” at higher membrane potentials) were measured by flow cytometry. As shown (Fig. 10 and Fig. 11), the untreated control cells exhibited strong red fluorescence with a high ∆Ψm with 94.2% of red fluorescence and 4.2% of green fluorescence (Fig. 10 and Fig. 11). The PMEAF treated MDA-MB-231 cells showed strong green fluorescence due to the presence of monomeric JC-1 in a dose-dependent manner with 9.8%, 37.87% and 79.63% green fluorescence in the ½ × IC50, IC50 and 2 × IC50 of PMEAF, respectively (Fig. 10 and Fig. 11). The results clearly showed that the bright green fluorescence intensity of PMEAF treated MDA-MB-231 cells increased as the treatment dose increased. The MDA-MB-231 cells treated with positive-control carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupling agent exhibited 26.5% of red fluorescence and 70.13% of green fluorescence (Fig. 10 and Fig. 11). Thus, the current findings indicate that PMEAF treatment decreases the ∆Ψm, leading to mitochondrial depolarization. 3.9. Protein array analysis MDA-MB-231 cells treated with PMEAF with IC50 concentration for 24 hours were evaluated for apoptotic protein markers. The BSA was used as calibration for the standard curve graph, which was plotted for a range of concentrations from 0.5 to 3.0 mg/mL. The protein

19

concentration was expressed as mg/mL using the standard curve linear regression equation: y = 0.7338x + 0.279, R2 = 0.9878, where y is absorbance and x is protein concentration in the cells. By analysis of the protein profile array, 13 proteins were found to be involved in apoptosis (Fig. 12 and Table 1). Of these proteins, 9 of the pro-apoptotic protein consisted of Bax, Bid, caspase 3, caspase 8, cytochrome c, p21, p27, p53 and SMAC were observed to be up-regulated, in MDA-MB-231 cells that had been treated with PMEAF with the IC50 concentration for 24 hours. Meanwhile, the expression levels of the remaining 4 anti-apoptotic proteins were downregulated when treated with PMEAF with IC50 concentration for 24 hours. The anti-apoptotic proteins were Bcl-2, Bcl-w, XIAP and survivin.

4. Discussion P. macrocarpa is a popular medicinal plant comprising many phytochemicals and has been extensively investigated for its anticancer activities in multiple cancer cell lines. Despite the extensive study on anticancer activities, there have been only limited attempts to explore the cytotoxicity mechanism of this plant in relation to its anticancer activity. The morphological observation study using Acridine Orange/Propidium Iodide (AO/PI) staining with fluorescence microscope strongly revealed that PMEAF treatment induced greater apoptotic cell death in PMEAF treated MDA-MB-231 cells in comparison to the non-treated control cells. Therefore, the current study was conducted to provide further biochemical proof concerning the apoptotic cell death activities of PMEAF in detail to elucidate the mechanism of apoptosis by PMEAF in MDA-MB-231 cells. 4.1. Quantification of apoptosis using propidium iodide and acridine orange double staining

20

Further fluorescence microscopic analysis of AO/PI stained MDA-MB-231 cells treated with PMEAF showed that the number of viable cells was reduced with an increase in the treatment dose. It was found that the number of cells undergoing apoptosis was greater at higher dosage. However, when the treatment dosage increased to 2 × IC50, the presence of necrosis amongst the treated MDA-MB-231 cells was evident. This is possible since treated MDA-MB-231 cells undergoing apoptosis may have progressed into necrosis due to the higher dosage of PMEAF. 4.2. Quantitative analysis of apoptotic cells by flow cytometry From the fluorescence microscopic analysis of AO/PI stained cell results, it can be deduced that PMEAF is a favourable inducer of cell apoptosis. Further proof of this finding was confirmed by flow cytometry analysis. The results from the Annexin V-FITC also point to the connection of apoptosis in PMEAF treated MDA-MB-231 cells. Analysis of apoptotic cells by flow cytometry suggested dose-dependent activation of apoptosis, which was evidenced by the accumulation of apoptotic cells in the top right Q2 quadrant and bottom right Q4 quadrant after PMEAF treatment (Fig. 4). In addition, the data obtained from Annexin VFITC was consistent with AO/PI, confirming PMEAF induced apoptosis in MDA-MB-231 cells. This finding suggests that PMEAF is a good candidate for green anti-cancer agents that induce apoptosis. In contrast to necrosis, apoptosis is an essential cell death mechanism that does not activate an inflammatory response that causes collateral damage to normal cells in the neighbouring microenvironment (Elmore, 2007). Therefore, anti-cancer agents that induce apoptosis, such as PMEAF, are an effective approach in green cancer chemotherapy (Lowe and Lin, 2000). 4.3. Cell cycle analysis

21

The cell cycle is a steady event by which eukaryotic cells reproduce themselves. However, the initiation of cell cycle arrest at a specific checkpoint, and, thus, inducing apoptosis, is the normal mechanism for the cytotoxic effects of cytotoxic agents in cancer cells (Li et al., 2016). The results of the cell cycle arrest analysis suggested that the PMEAF treated MDAMB-231 cells were arrested in the G0/G1 and G2/M-phase of the cell cycle together with the increase in sub-G1, which indicates that the mechanism by which PMEAF may act to prevent the proliferation of MDA-MB-231 cells is inhibition of the cell cycle progression. Cell cycle checkpoints play crucial roles in the preservation of genomic DNA against errors that may happen during DNA replication and chromosome segregation (Nyberg et al., 2002). The G0/G1 phase arrest leads cells to undergo repair or to follow the apoptosis pathway (Mantena et al., 2006). In the case of PMEAF treated MDA-MB-231 cells, the cells follow the apoptosis pathway and respond to the cytotoxic effects PMEAF. In addition, the mammalian cell cycle is accurately managed by cell cycle checkpoints that permit progress through the cell cycle or arrest the cells in the G2/M phase in reaction to DNA damage for DNA repair (Kang et al., 2010). Cell cycle deregulation and apoptosis are closely connected happenings, and disturbance of the cell cycle progress may ultimately lead to apoptotic death in the case of serious DNA damage (Kang et al., 2010). The current findings suggest that the DNA damage in the MDA-MB-231 cells triggered by PMEAF at 24 hours might be permanent (as observed in AO/PI fluorescence microscopy study that indicated that the MDA-MB-231 cells DNA underwent degradation and fragmentation), and, consequently, forced the arrested cells in the G2/M phase to apoptosis cell death. A small Sub-G1 peak (with the percentages 2.72%, 5.48% and 8.18% in the ½ × IC50, IC50 and 2 × IC50, respectively, in a dose-dependent manner) appeared to represent the apoptotic cells with reduced DNA content. These findings showed that treatment of PMEAF for 24 hours resulted in G2/M phase arrest, as well as induced

22

apoptosis (Apoptotic Sub-G1 area), which is associated with the MDA-MB-231 cell growth inhibition triggered by PMEAF. A similar result was reported by Lin et al. (2006) who found that indoloquinoline, IQDMA (N0-(11H-indolo[3,2-c]quinolin-6-yl)-N,N-dimethylethane-1, 2-diamine arrested K562 cells at the G2/M phase, which was accompanied by an increase in the sub G1 population and caused apoptotic cell death. 4.4. Effect of Intracellular ROS Reactive oxygen species (ROS) are recognised as a second messenger in the intracellular signal transduction pathway for a variety of cellular processes (Wang et al., 2016). It is extensively reported that ROS play an important role in triggering cell damage and that an increase in ROS is involved in various events in cancer, including cell cycle progression and apoptosis (Boonstra and Post, 2004). Therefore, analysis of the effects of ROS generation in the MDA-MB-231 cells treated with PMEAF was conducted. As expected, the results showed increased production of ROS in the MDA-MB-231 cells treated with PMEAF in a dose-dependent manner. These observations were consistent with the cell cycle analysis results which showed that increased production of ROS will arrest cells in all phases of the cell cycle, especially in the G0/G1 and G2/M-phases, and that the cells will undergo apoptosis (Boonstra and Post, 2004) by activation of numerous signalling proteins (Finkel and Holbrook, 2000). ROS are generally believed to be toxic, causing oxidation of DNA which leads to DNA damage. Thus, similar to when DNA is oxidatively damaged beyond repair, it can lead to the induction of cell cycle arrest and apoptotic death in a well-controlled manner (Kang et al., 2010), which was confirmed in this study. Moreover, the ability of ROSmediated oxidative stress during extensive cellular damage has been linked to the loss of mitochondrial membrane potential (ΔΨm), and the mitochondrial release of the pro-apoptotic

23

protein cytochrome c (Kim et al., 2004). This finding suggests that PMEAF exhibited its cytotoxic effects via the ROS-dependent pathway in the MDA-MB-231 cells. Further study was conducted to evaluate the role of ROS-mediated oxidative stress with the loss of mitochondrial membrane potential (ΔΨm) and the mitochondrial release of the pro-apoptotic protein cytochrome c. 4.5. Effect of mitochondrial membrane potential (MMP) Mitochondria have been identified as playing a significant central role in the apoptotic process. Mitochondria control apoptosis through the mitochondrial membrane potential and mitochondrial membrane permeability for the release of certain mitochondrial apoptogenic factors, such as cytochrome c from the mitochondrial intermembrane space into the cytosol (Ly et al., 2003). Furthermore, depolarization of the transmembrane potential can lead to the release of mitochondrial pro-apoptotic factors, such as cytochrome c (Christensen et al., 2013). The freed pro-apoptotic protein triggers pathways that are important for the execution of the morphological and biochemical changes started by various death stimuli (Chiu and Oleinick, 2001). As the important role of mitochondria in apoptosis is unavoidable, the effect of PMEAF treatment on ∆Ψm was evaluated in the MDA-MB-231 cell mitochondria. The finding of this study showed that PMEAF treatment collapsed the ∆Ψm in the MDA-MB-231 cells and was dose-dependent. The downfall of the ∆Ψm can facilitate the release of proapoptotic factors from the space between the outer and inner mitochondrial membranes into the cytosol, which activates the caspase cascade (Christensen et al., 2013). These findings showed that PMEAF treatment might trigger apoptosis through stimulating the intrinsic, mitochondria-mediated caspase pathway. Therefore, the increase in the mitochondria membrane permeability may be due to the up and down regulation of the apoptosis proteins

24

involved in the cell death mechanism. Dose-dependent manner intracellular ROS accumulation in MDA-MB-231 cells treated with PMEAF resulted in the loss of ∆Ψm, which might lead to the release of cytochrome c (Wang et al., 2016). Moreover, Fig. 4 clearly revealed that PMEAF induced apoptosis was simultaneous to the increased ROS generation (Fig. 9) in the MDA-MB-231 cells, indicating that PMEAF may exert cell cytotoxicity by increasing the production of ROS. Similar findings have been reported by Xie et al. (2015) who found that berberine increased the production of ROS, which triggered Δ Ψm depolarization leading to the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, and, ultimately, triggered apoptosis. 4.6. Role of anti-apoptotic and pro-apoptotic proteins

Apoptosis is closely controlled by anti-apoptotic and pro-apoptotic proteins and can be facilitated by numerous different pathways. Therefore, protein array analysis was conducted in this study to assess the role anti-apoptotic and pro-apoptotic proteins have in terms of the induction of apoptosis in MDA-MB-231 cells treated with PMEAF. The results of the current study showed that PMEAF stimulated the expression of pro-apoptotic proteins, such as Bax and Bid. Meanwhile, PMEAF suppressed the anti-apoptotic proteins, including the Bcl-2 (Bcell lymphoma 2) and Bcl-w (Bcl-2-like protein 2) proteins in the MDA-MB-231 cells, leading to apoptosis. This effect causes depolarization of the mitochondrial transmembrane potential, which leads to the release of cytochrome c into the cytoplasm (Ricci et al., 2004). Interestingly, the results of the current study showed that PMEAF stimulated the expression of cytochrome c. The release of cytochrome c into the cytoplasm is the key inducer of mitochondrial-associated apoptosis through the activation of caspase cascade during apoptosis (Jin et al., 2007). In the present study, the protein expression levels of caspase 3 and caspase 25

8 increased following PMEAF treatment in MDA-MB-231 cell. Active caspase 8 can activate caspase 3, or it can cleave the Bid that increases following PMEAF treatment, which enables the release of mitochondrial cytochrome c. Moreover, caspase 8 cleaves Bid into tBid, which initiates the mitochondrial apoptosis pathway causing the release of cytochrome c and SMAC/DIABLO from the mitochondria (Kantari and Walczak, 2011). The Bid (BH3 interacting-domain death agonist) gene is a pro-apoptotic protein in the Bcl-2 protein family (Wang et al., 1996). Bid works with Bax to insert the Bax protein into the outer mitochondrial membrane to form an oligomeric pore, which results in the release of cytochrome c and second mitochondria-derived activator of caspase, SMAC/DIABLO (Weinberg, 2007). The pro-apoptotic molecule SMAC/DIABLO, which is simultaneously released with cytochrome c from the mitochondria into the cytosol, eradicates the inhibitory role of inhibitor-of-apoptosis proteins, such as XIAP, and, consequently, promotes caspase activation (Saito et al., 2003). The freed cytochrome c with Apaf-1 forms the apoptosome, an activation stage for caspase 9. The SMAC/DIABLO offsets the inhibitory role of the X chromosome-linked inhibitor-of-apoptosis protein (XIAP), thus allowing for full activation of caspase 3 and 9, which leads to apoptosis (Kantari and Walczak, 2011). The inhibitor-ofapoptosis protein (IAP); namely, cIAP1, cIAP2, X chromosome–linked IAP (XIAP), NAIP, Livin/KIAP, BRUCE/Apollon, and survivin, negatively control the caspase stimulation (Saito et al., 2003; Silke and Vaux, 2001; Deveraux et al., 1999). The IAP proteins bind to and inhibit both the initiator caspases, such as caspase 9, and the effector caspases, such as caspases 3 and 7 (Martins et al., 2002). Between the above IAP proteins, XIAP is the strongest inhibitor of caspases and apoptosis (Deveraux et al., 1999). In the present study, the protein expression levels of SMAC/DIABLO were increased while the expression of XIAP decreased following PMEAF treatment in the MDA-MB-231 cells, which demonstrates cell

26

apoptosis. Expression of the surviving protein is stimulated at high concentrations in cancer cells to inhibit the apoptosis of cancerous cells by negatively controlling the caspase stimulation (Deveraux and Reed, 1999; Nordin et al., 2016). The PMEAF treated MDA-MB231 cells showed the down-regulation of surviving, thereby demonstrating cell apoptosis. This result showed that by inhibiting the action of survivin in the MDA-MB-231 cells, PMEAF promotes apoptosis. Other similar findings reported that the inhibition of survivin prior to irradiation leads to an increase in apoptosis and reduced tumour cell survival; therefore, it has been suggested that survivin is a therapeutic target for radiation sensitization in lung cancer (Hoffman et al., 2002). The tumour suppressor p53 is sustained at low levels in normal cells, but p53 protein will accumulate in the nucleus in reaction to various incitements, and begin to control gene expression (Giono and Manfredi, 2007). The p53 plays an important role as a cell nucleus phosphate protein that determines whether the cell reacts to various types of stimuli with apoptosis, cell cycle arrest, senescence, DNA repair, cell metabolism, or autophagy (Wu et al., 2013). Furthermore, the pro-apoptotic p53 protein can also induce apoptosis by repression of anti-apoptotic genes, such as survivin and Bcl-2, thus promoting caspase activation via activation of pro-apoptotic genes, such as Bax (Hoffman et al., 2002; Giono and Manfredi, 2007; Wu et al., 2013; Amaral et al., 2010). In the present study, it was found that PMEAF, which plays a role as an apoptosis trigger, suppressed the survivin and Bcl-2 expression through increasing p53 in the MDA-MB-231 cells. A previous study reported a similar finding where the gambogic acid acted as an efficient apoptosis inducer by repressing Bcl-2 expression via increasing p53 in the MCF-7 cells (Gu et al., 2009). In addition, the expression of pro-apoptotic

proteins,

such

as

Bax,

is

also

upregulated

by

the tumour

suppressor protein p53 to permeabilize mitochondria and trigger the apoptotic cell death 27

programme. In addition, Bax has been shown to be involved in p53-mediated apoptosis (Chipuk et al., 2004) as observed in this study where the Bax protein was upregulated via increasing the p53 protein in the MDA-MB-231 cells treated with PMEAF. The expression of the cyclin-dependent kinase (CDK) inhibitor p21 protein will be upregulated due to the accumulation of p53 protein. The CDK inhibitor p21 protein is a key mediator of cell cycle arrest at the G1 phase and facilitates G2 phase arrest (Giono and Manfredi, 2007). The advancement of the cell cycle in cancer cells is controlled by three main proteins; namely, cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors (CDKIs) (Schwartz and Shah, 2005). The presence of cell cycle inhibitory proteins, such as p21 and p 27 (CDK inhibitors), act as negative controllers of the cell cycle, and inhibit the cell from progressing to the next phase of the cell cycle (Schwartz and Shah, 2005; Du et al., 2015). Furthermore, it has been reported that the existence of a single p27 molecule should be enough to inhibit cdk 2 activities and cell cycle arrest at G1/S phase transition (Russo et al., 1996). Interestingly, in the present study, the results showed that p53, p21 and p27 expression was significantly increased in MDA-MB-231 cells treated with PMEAF, and was found to promote the cell cycle arrest in the G0/G1 and G2/M-phase. The cell cycle analysis in this study revealed that PMEAF might induce cell cycle arrest in the G0/G1 and G2/M phase by upregulating the expression levels of the CDK inhibitors, p21 and p27. A previous study also reported similar results in which the cell cycle analysis revealed that resveratrol, a non-flavone polyphenol compound, induced cell cycle arrest in the G0/G1 phase by downregulating the expression levels of cyclin D1, cyclin-dependent kinase CDK 4 and CDK

28

6, and upregulating the expression levels of the CDK inhibitors, p21 and p27 (Yuan et al., 2015).

5. Conclusion

In conclusion, the results of this study clearly demonstrated that PMEAF treatment induced apoptosis in the MDA-MB-231 cells through Bcl2/Bax signalling pathways with the participation of caspases 3 and 8, while the induced G0/G1 and G2/M-phases cell cycle arrest via the p53-mediated mechanism; as illustrated in Fig. 13. These findings are consistent with the morphological changes observed during the apoptosis process in MDA-MB-231 cells treated with PMEAF through fluorescence microscopy and thus suggest that PMEAF effectively induces apoptosis in MDA-MB-231 cells. The current findings also warrant further research on PMEAF as a novel chemotherapeutic agent for breast cancer including studies on the role of miRNAs in the induction of apoptosis in MDA-MB-231. Conflict of interest On behalf of all authors, the corresponding author states that there is no conflict of interest Authors’ contributions SS, CEO, YC, JRK and NK designed the study, SS, CEO, YC and JRK supervised all analyses and experiment, NK performed most of the experiments, SS and NK performed statistical analysis, SS and YC supplied materials and reagents, SS, CEO, YC, JRK and NK wrote the manuscript. All the authors have read and accepted the final version of the manuscript. Acknowledgment 29

Nowroji Kavitha was supported by the MyPhD fellowship from the Ministry of Higher Education, Government of Malaysia, Malaysia.

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Russo, A.A., Jeffrey, P.D., Patten, A.K., Massagué, J., Pavletich, N.P., 1996. Crystal structure of the p27Kip1 cyclin-dependent-kinase inibitor bound to the cyclin A-Cdk2 complex. Nature 382(6589), 325-331. Safarzadeh, E., Shotorbani, S.S., Baradaran, B., 2014. Herbal medicine as inducers of apoptosis in cancer treatment. Adv. Pharm. Bull. 4(1), 421-427. Saito, A., Hayashi, T., Okuno, S., Ferrand-Drake, M., Chan, P.H., 2003. Interaction between XIAP and Smac/DIABLO in the mouse brain after transient focal cerebral ischemia. J. Cereb. Blood Flow Metab. 23(9), 1010-1019. Saraste, A., Pulkki, K., 2000. Morphologic and biochemical hallmarks of apoptosis. Cardiovasc. Res. 45(3), 528-537.

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Fig. 1. GC-MS profile of major components in PMEAF with specified peak at retention time.

36

Fig. 2. Fluorescent micrograph of acridine orange and propidium iodide double-stained human breast cancer cells lines (MDA-MB-231) at 200 × magnifications. Untreated (a), ½ × IC50 (b), IC50 (c), and 2 × IC50 (d) concentration of Phaleria macrocarpa ethyl acetate fraction (PMEAF) treated MDA-MB-231cells. V: Viable cell, Apoptotic cells (AP), late apoptotic cells (LA), necrosis (N), membrane blebbing (MB) and chromatin condensation (CC) in enlarged apoptotic cells cell (400 × magnifications)

37

100

a V ia b le

C e ll P o p u la tio n (% )

80

A p o p to tic

j 60

N e c ro sis

40

d 20

h

0 U n tre a te d

½ × IC 50

IC 50

2 × IC 50

C o n c e n tr a tio n (µ g /m L )

g e

k 38

b

c,f

c

f

i

Fig. 3. Histogram of quantitative analysis of viable, apoptotic, and necrotic cells after Phaleria macrocarpa ethyl acetate fraction (PMEAF) treatment at ½ × IC50, IC50 and 2 × IC50 concentration for 24 hours. Values are expressed as means ± SD of triplicates. Different alphabets (a-k) indicate significant differences (p < 0.05) using One-way ANOVA followed by Turkey’s multiple comparison tests.

(b)

39

(d)

40

Fig. 4. Flow cytometric analysis of Annexin V in MDA-MB-231 cells which were treated with Phaleria macrocarpa ethyl acetate fraction (PMEAF) with ½ × IC50, IC50 and 2 × IC50 concentration for 24 hours. (a) Untreated, (b) ½ × IC50, (c) IC50, and (d) 2 × IC50. Q1 quadrant (An-, PI+) shows necrosis cells, Q2 quadrant (An+, PI+) shows late apoptotic cells, Q3 quadrant (An-, PI-) shows viable cells and Q4 quadrant (An+, PI-) shows early apoptotic cells.

Q1

100

Q2 90

Q3 Q4

A n n e x in V /P I c e ll p e r c e n ta g e (% )

80

70

60

50

40

30

20

10

0 U n t re a t e d

½ × IC

IC

50

50

C o n c e n tr a tio n (µ g /m L )

41

2 × IC

50

Fig. 5. Histogram of quantitative analysis of necrosis (Q1), late apoptotic (Q2), viable (Q3) and early apoptotic (Q4) MDA-MB-231 cells treated with Phaleria macrocarpa ethyl acetate fraction (PMEAF) with ½ × IC50, IC50 and 2 × IC50 concentration for 24 hours. Values are expressed as means ± SD of triplicates. Different alphabets (a-n) indicate significant differences (p < 0.05) using One-way ANOVA followed by Turkey’s multiple comparison test.

A

B

42

C

D

Fig. 6. Flow cytometric analysis of cell cycle distribution in MDA-MB-231 cells treated with Phaleria macrocarpa ethyl acetate fraction (PMEAF) with ½ × IC50, IC50 and 2 × IC50 concentration for 24 hours. (A) Untreated, (B) ½ × IC50, (C) IC50, and (D) 2 × IC50.

Fig. 7. Histogram of cell cycle distribution (%) in MDA-MB-231 cells treated with Phaleria macrocarpa ethyl acetate fraction (PMEAF) with ½ × IC50, IC50 and 2 × IC50 concentration for 24 hours. Values are expressed as means ± SD of triplicates. Different alphabets (a-m) Sub G 1 100 G 0 /G 1 90

l

C e ll P o p u la tio n (% )

S 80

i

70

f

60 50

G 2 /M

b

40 30 20 10 0

c

U n tre a te d

½ × IC 50

IC 50

g

2 × IC 50

k

C o n c e n tr a tio n (µ g /m L )

d (p < 0.05) using One-wayd ANOVA followed by Turkey’s indicate significant differences j multiple comparison test. e,h a a,e 43

h

Relative fluorescence unit

16 14 y = 0.0014x + 0.4025 R² = 0.999

12 10 8 6 4 2 0 0

2000

4000

6000 DCF (nM)

8000

10000

12000

Fig. 8. DCF (2',7'-dichlorofluorescein) standard curve was used to interpret intracellular ROS production of MDA-MB-231 cells treated with Phaleria macrocarpa ethyl acetate fraction (PMEAF) with ½ × IC50, IC50 and 2 × IC50 concentration for 24 hours. Fluorescence measurement was performed on SpectraMax Gemini XS Fluorometer (Molecular Devices) with a 485/538 nm filter set.

44

Relative fluorescence unit

7 d 6 c

5 b 4 3 a

a

2 1 0 control

Half IC50

IC50

2 x IC50

H2O2

Concentration (µg/mL)

Fig. 9. Histogram based on fold change of reactive oxygen species (ROS) production in MDA-MB-231 cells treated with Phaleria macrocarpa ethyl acetate fraction (PMEAF) with ½ × IC50, IC50 and 2 × IC50 concentration for 24 hours. Values are expressed as means ± SD of triplicates. Different alphabets (a-d) indicate significant differences (p < 0.05) using One-way ANOVA followed by Turkey’s multiple comparison tests.

45

A

C

B

D

E

Fig. 10. Density diagram of flow cytometry analysis showed the distribution of JC-1 aggregates (red) and JC-1 monomer (green) in the mitochondrial membrane of MDA-MB-231 cells treated with Phaleria macrocarpa ethyl acetate fraction (PMEAF) with ½ × IC50, IC50 and 2 × IC50 concentration for 24 hours. JC-1 dye shows the dissipation of mitochondrial membrane potential (MMP/∆Ψm) from red (P4; aggregates) to green (P5; monomer) by flow cytometry. (A) Positive control treated with Carbonyl cyanide m-chlorophenylhydrazone (CCCP), (B) Untreated, (C) ½ × IC50, (D) IC50, and (E) 2 × IC50. (A), (B), (C), (D) and € are composite contour/dot plots, in which areas of infrequent events are shown as individual dots and higher density areas are shown as concentric probability contours with each successive layer depicting an increased frequency of events. The figures are representative of three parallel experiments.

Depolarization of of MMP (%)

120 100 80

a

c h

60

j

e

40

20

f

0 Untreated

b

½ × IC50 IC50 2 g× IC50 d Concentration (µg/mL) JC 1-Red

i

CCCP (mM)

JC 1-Green

Fig. 11. Histogram presented the percentage of depolarization of mitochondrial membrane potential (MMP / ∆Ψm) in MDA-MB-231 cells treated with Phaleria macrocarpa ethyl acetate fraction (PMEAF) with ½ × IC50, IC50 and 2 × IC50 concentration for 24 hours. Values are expressed as means ± SD of triplicates. Different alphabets (a-j) indicate significant differences (p < 0.05) using One-way ANOVA followed by Turkey’s multiple comparison tests. 1 mM Carbonyl cyanide m-chlorophenylhydrazone (CCCP) as positive control.

47

24

(A)

22

18 16

(r e la tiv e e x p r e s sio n )

C h a n g e s r e la te d to c o n tr o l

20

14 12 10 8 6 4 2 0

Su

SM

AC

n iv i rv

AP XI

3

p2

p5

7

1 p2

c Cy

to

e8 as sp Ca

as sp Ca

Bc

e3

l-w

l-2

d Bi

Bc

x -4

Ba

-2

(C)

(B)

RayBio® Human Apoptosis Antibody Array

(D)

Detect 43 Apoptotic Markers in One Experiment 1

2

3

4

5

6

7

8

9

10

11

12

13

1

Pos 1

Pos 2

Pos 3

Neg

Neg

bad

bax

bcl-2

bcl-w

BID

BIM

Caspase3

Caspase8

2

Pos 1

Pos 2

Pos 3

Neg

Neg

bad

bax

bcl-2

bcl-w

BID

BIM

Caspase3

Caspase8

3

CD40

CD40L

cIAP-2

cytoC

DR6

Fas

FasL

neg

HSP27

HSP60

HSP70

HTRA

IGF-I

4

CD40

CD40L

cIAP-2

cytoC

DR6

Fas

FasL

neg

HSP27

HSP60

HSP70

HTRA

IGF-I

5

IGF-II

IGFBP-1

IGFBP-2

IGFBP-3

IGFBP-4

IGFBP-5

IGFBP-6

IGF-1sR

livin

p21

p27

p53

SMAC

6

IGF-II

IGFBP-1

IGFBP-2

IGFBP-3

IGFBP-4

IGFBP-5

IGFBP-6

IGF-1sR

livin

p21

p27

p53

SMAC

7

Survivin

sTNF-R1

sTNF-R2

TNF-alpha

TNF-beta

TRAILR-1

TRAILR-2

TRAILR-3

TRAILR-4

XIAP

Neg

Neg

Neg

XIAP

Neg

Neg

Neg

8

Survivin

sTNF-R1

sTNF-R2

TNF-alpha

TNF-beta

TRAILR-1

TRAILR-2

TRAILR-3

TRAILR-4

Fig. 12. Human apoptosis proteome profile array in MDA-MB-231 cells treated with Phaleria macrocarpa ethyl acetate fraction (PMEAF) with IC50 concentration for 24 hours. 48

(A) Histogram of quantitative analysis between untreated and treated cells, with the positive and negative fold changes indicating up-regulation and down-regulation of protein expression, respectively. (B) Expression of apoptotic proteins in untreated cells. (C) Expression of apoptotic proteins upon PMEAF treatment. (D) The exact protein name of each dot in the array. Values are expressed as means ± SD of triplicates

49

Fig. 13. Proposed model of Phaleria macrocarpa ethyl acetate fraction (PMEAF) mechanism of action for apoptosis in human breast cancer MDA-MB-231 cell lines.

Table 1. Normalized values of signal intensities from Analysis Tool Software for RayBio® Human Apoptosis Antibody Array in untreated MDA-MB-231 cells and MDA-MB-231 cells treated with Phaleria macrocarpa ethyl acetate fraction (PMEAF) with IC50 concentration for 24 hours. The relative expression of fold changed as comparable to control (untreated cell lines).

Apoptosis Proteins

Untreated

Treated

Fold Changed

Bax

27400 ± 4055.5

58201 ± 527

2.12 ± 0.292*

Bid

10332 ± 654.5

24316 ± 1416

2.35 ± 0.042*

Bcl-2

24353 ± 1049

12531 ± 849.5

-1.94 ± 0.034*

Bcl-w

31342 ± 631.5

18699 ± 760.5

-1.68 ± 0.146*

4361 ± 227.5016

27637 ± 2078

6.33 ± 0.037*

Caspase 3

50

55458 ± 2315.5

193188 ± 5524.5

3.48 ± 0.452*

Cytochrome c

13256 ± 1633

26330 ± 2640.5

1.99 ± 0.096*

p21

212510 ± 381

4778035 ± 50284.5

22.48 ± 0.234*

p27

11796 ± 668.5

31527 ± 972.5

2.67± 0.019*

p53

319133 ± 15722

576282 ± 11205

1.81 ± 0.054*

XIAP

153370 ± 6065.5

93968 ± 550.5

-1.63 ± 0.055*

Survivin

846806 ± 9528.5

436798 ± 2468

-1.94 ± 0.010*

87239 ± 853

176340 ± 9018

2.02 ± 0.084*

Caspase 8

SMAC

Values are expressed as means ± SD of triplicates. “*” indicates significant differences (p < 0.05) using T-test test.

Highlight 

The mode of PMEAF-induced cell death and the mechanisms involved were examined.



PMEAF induced mainly apoptotic death of breast cancer MDA-MB-231 cells.



PMEAF induced mitochondrial-mediated and caspase-dependent apoptosis.



p53-mediated pathways are involved in PMEAF-mediated apoptosis in the MDA-MB231 cells.

51