Phenylpropanoids isolated from Piper sarmentosum Roxb. induce apoptosis in breast cancer cells through reactive oxygen species and mitochondrial-dependent pathways

Accepted Manuscript Phenylpropanoids isolated from Piper sarmentosum Roxb. induce apoptosis in breast cancer cells through reactive oxygen species and mitochondrial-dependent pathways Arshia Hematpoor, Mohammadjavad Paydar, Sook Yee Liew, Yasodha Sivasothy, Nooshin Mohebali, Chung Yeng Looi, Won Fen Wong, Mohd Sofian Azirun, Khalijah Awang PII:

S0009-2797(16)30405-7

DOI:

10.1016/j.cbi.2017.11.014

Reference:

CBI 8150

To appear in:

Chemico-Biological Interactions

Received Date: 4 October 2016 Revised Date:

22 October 2017

Accepted Date: 21 November 2017

Please cite this article as: A. Hematpoor, M. Paydar, S.Y. Liew, Y. Sivasothy, N. Mohebali, C.Y. Looi, W.F. Wong, M.S. Azirun, K. Awang, Phenylpropanoids isolated from Piper sarmentosum Roxb. induce apoptosis in breast cancer cells through reactive oxygen species and mitochondrial-dependent pathways, Chemico-Biological Interactions (2017), doi: 10.1016/j.cbi.2017.11.014. 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 proof before it is published in its final 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.

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Phenylpropanoids isolated from Piper sarmentosum Roxb. induce apoptosis in breast cancer cells through reactive oxygen species and mitochondrial-dependent

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pathways

Arshia Hematpoora, Mohammadjavad Paydarb, Sook Yee Liewc,1, Yasodha Sivasothyd, Nooshin Mohebalib, Chung Yeng Looib,2, Won Fen Wonge, Mohd Sofian Aziruna,

Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603

Kuala Lumpur, Malaysia. b

Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur,

Malaysia. c

Centre for Natural Product and Drug Discovery (CENAR), University of Malaya,

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50603 Kuala Lumpur, Malaysia. d

Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur,

Malaysia.

Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala

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e

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a

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Khalijah Awangc,d*

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Lumpur, Malaysia.

*Corresponding author. Tel.: +603-7967-4064; Fax: +603-7967-4193. E-mail address: [email protected] Abstract

The aim of the present study is to isolate bioactive compounds from the roots of Piper sarmentosum and examine the mechanism of action using human breast cancer cell line 1 Present address: 1 Chemistry Division, Centre for Foundation Studies in Science, University of Malaya, 50603 Kuala Lumpur, Malaysia 2 School of Biosciences, Faculty of Health and Medical Sciences, Taylor’s University Lakeside Campus, 47500 Subang Jaya, Malaysia.

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(MDA-MB-231). Bioassay guided-fractionation of methanolic extract led to the isolation of asaricin (1) and isoasarone (2). Asaricin (1) and isoasarone (2) had significant cytotoxicity towards MDA-MB-231. MCF-10A (human normal breast epithelial cells)

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cells are less sensitive than MDA-MB-231, but they respond to the treatment with the same unit of measurement. Both compounds increase reactive oxygen species (ROS), decrease mitochondrial membrane potential (MMP) and enhance cytochrome c release in

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treated MDA-MB-231 cells. Isoasarone (2) markedly elevated caspase -8 and -3/7 activities and caused a decline in nuclear NF-κB translocation, suggesting extrinsic, death

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receptor-linked apoptosis pathway. Quantitative PCR results of MDA-MB-231 treated with asaricin (1) and isoasarone (2) showed altered expression of Bcl-2: Bax level. The inhibitory potency of these isolates may support the therapeutic uses of these compounds

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in breast cancer.

Keywords: Cytotoxicity, mitochondrial membrane, Piper sarmentosum, phenylpropanoid

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1. Introduction

Cancer is an emerging public health problem around the globe according to the

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World Health Organization (WHO) [1]. Breast cancer is the most common cancer among women in Malaysia and the leading cause of cancer death according to the Ministry of Health in Malaysia [2]. So far, the conventional treatments are surgical intervention, hormonal therapy, radiotherapy and chemotherapy. Although the emergence of new drugs such as Tamoxifen and Toremifene makes chemotherapy a viable choice for breast cancer patients, the development of drug resistance and severe side effects are unresolved

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problems in clinical oncology [3]. Therefore, the search for novel anti-cancer compounds with improved features is needed. In recent oncology research, different breast cancer cell lines have been applied by investigators for drug discovery purposes and among these

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cells estrogen non-dependant MDA-MB-231 is one of the most extensively used model [4].

The genus Piper is an important source of secondary metabolites with several

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promising bioactivities such as inducing cell apoptosis through mitochondrial disruption or modulating HIF-2 transcription [5, 6]. Certain alkaloids and phenylpropanoids isolated

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from this genus have shown strong antioxidant and allelopathy activities [7]. Relatively there are several reports on extracts from Piper genus with anticancer activities as well [8, 9]. The crude extract of P. capense L.f. exhibited good activity with IC50 values of below or around 10 µg/mL against the sensitive leukemia CCRF-CEM cells, its

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adriamycin-resistant subline CEM/ADR5000 and also the human pancreatic cancer cell line MiaPaCa-2 [10]. The cytotoxic effect of the P. sarmentosum ethanolic extract on a human hepatoma cell line (HepG2) was previously reported [11].

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Piper sarmentosum Roxb. locally known as ‘Kadok’ is mainly distributed in tropical regions including Malaysia. It is a glabrous, creeping terrestrial herb of about 20

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cm in height, and has an aromatic odour and a pungent taste [12, 13]. In local cuisine, its leaves are consumed as vegetable (‘ulam’) or used as food wrapping [14]. It is used in the traditional treatment of a variety of ailments [15, 16]. According to folk medicine practice, P. sarmentosum possesses various medicinal values and, therefore, is used as a herbal remedy. There are many reports indicating the usage of the leaves, fruits and roots

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of P. sarmentosum in curing hypertension, diabetes, joint aches, muscle pain, coughs, influenza, toothaches and rheumatism in Malaysia, Thailand and Indonesia [17]. In our continuous effort to search for bioactive molecules in Malaysian medicinal

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plants, we have found that the methanol extract of P. sarmentosum exhibited potent cytotoxic activity towards human invasive breast cancer cell line. The extract was

subjected to further cytotoxicity guided isolation to purify the compounds that were

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responsible in imparting the cytotoxic activity which resulted in the identification of asaricin (1) and isoasarone (2). A study was performed to investigate the possible

sarmentosum.

2. Material and methods

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2.1 Plant material

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mechanisms involved in the anticarcinogenic effects of the compounds derived from P.

Piper sarmentosum was collected in the vicinity of University of Malaya in 2011 and was identified by Mr. Teo Leong Eng. A voucher specimen (KU 0110) was deposited

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in the University of Malaya’s herbarium.

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2.2 General experimental procedures Analytical and preparative for thin layer chromatography (TLC) was performed

on silica gel 60 F254 plates 20 x 20cm (absorbent thickness: 0.25 and 0.50 mm, respectively). Column chromatography was carried out using silica gel 60, (Merck 230400 mesh, 0.040-0.063mm, ASTM). 1D- and 2D-NMR spectra were recorded in chloroform CDCl3 (Merck, Germany) with tetramethylsilane as an internal standard,

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using JEOL ECA 400 MHz NMR spectrometer. The LCMS-IT-TOF spectra were recorded on a UFLC Shimadzu Liquid Chromatography with a SPD-M20A diode array detector coupled to a IT-TOF mass spectrometer. UV spectra were recorded using a

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Shimadzu 1650 PC UV-Vis Spectrophotometer. IR spectra were recorded using a PerkinElmer Spectrum 400 FT-IR Spectrometer. All solvents were of analytical grade and were distilled prior to use.

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Human invasive breast cancer cell line (MDA-MB-231) and MCF-10A human

normal breast epithelial cell line were purchased from American Type Culture Collection

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(ATCC, Manassas, VA). The MDA-MB-231 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies, Inc, Rockville, MD) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO), 1% penicillin and streptomycin. MCF-10A cells were grown in Ham’s F12:DMEM (50:50), 2.5 mM L-

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glutamine, 20 ng/mL epidermal growth factor (EGF) (Sigma), 0.1 µg/ml cholera toxin (CT) (Sigma), 10 µg/mL insulin (Sigma), 500 ng/mL hydrocortisone (Sigma) and 5% horse serum (Atlanta Biologicals, Georgia, USA, cat. no. S12150). Cells were cultured in

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tissue culture flasks (Corning, USA) and were kept in an incubator at 37°C in a humidified atmosphere with 5% CO2. For experimental purposes, cells in exponential

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growth phase (approximately 70-80% confluency) were used [18].

2.3 Extraction and Isolation P. sarmentosum dried roots were prepared in 1 kg quantity and extracted two

times with 3 liters of hexane at room temperature the same procedure were repeated by dichloromethane and methanol. Hexane (9.79 g), dichloromethane (23.08 g) and

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methanol (16.42 g) extracts were obtained. In a preliminary screening of the cytotoxicity of the extracts towards human invasive breast cancer cell line, the high potential

methanolic extract was subjected to further investigation.

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cytotoxicity was observed for the methanolic extract (Fig. 1B & C). Therefore, only the

The methanol extract (ME) was fractionated over a silica gel column eluting with mixtures of hexane : DCM : MeOH (50:50:0 v/v → 0:100:0 v/v → 0:0:100 v/v) to yield

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eight main fractions (M1-M8). Fraction two (M2) which eluted with hexane : DCM

(40:60 v/v - 20:80 v/v) was found to be the most active (IC50 7.1 ± 1.1 µg/mL) and was

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further purified via preparative-TLC with hexane : DCM (60:40 v/v) to afford asaricin (1, 22 mg) (Rf = 0.92) and isoasarone (2, 67 mg) (Rf = 0.66).

2.4 Characterization of compounds

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The bioassay guided fractionation of the methanol extract resulted in the isolation of two phenylpropanoids 1 and 2. The structures of the two compounds were identified as asaricin 1 and isoasarone 2 upon comparison of their spectroscopic data with those

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reported in the literatures [19-21].

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2.5 MTT cell viability assay

MTT cell viability assay was performed, as previously described [22]. Briefly, 8.0 × 103 cells were seeded in a 96-well plate and incubated overnight at 37 °C with 5% CO2. The cells were treated with various concentrations of the extract/compounds in triplicates, and were incubated for indicated time-points. MTT solution (4,5-dimethylthiazol-2-yl-2,5diphenyltetrazoliumbromide) was added at 2 mg/mL for 2 hours, removed and replaced

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by DMSO. The plates were then read using a Chameleon Multitechnology microplate reader (Hidex, Turku, Finland) at an absorbance wavelength of 570 nm. The percentages of the cell viability was calculated as previously described [23]. Cell viability (in

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percentages, %) was showed as ratio of absorbance (A570 nm) in treated cells relative to absorbance in control cells (DMSO) (A570 nm). The IC50 value was defined as the

minimal concentration required to reduce the absorbance of the treated cells to 50% of

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2.6 LDH release assay

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the DMSO-treated control cells.

Cytotoxicity of the compounds was determined by lactate dehydrogenase (LDH) [22]. Briefly, cells were pretreated with different concentrations of the compounds for 48 hours. Cell supernatant was used to assess the LDH activity. The amount of formazan salt

2.7 Cell cycle analysis

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was measured in a colorimetric assay. Triton X-100 (2%) was added as a positive control.

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Cell cycle analysis was performed as previously described [18, 24]. Briefly, 1×104 MDA-MB-231 cells per well were seeded in a 96-well plate and incubated

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overnight at 37 °C with 5% CO2. Cells were treated with different concentrations of the compounds or DMSO (negative control) for 24 hours. BrdU and Phospho-Histone H3 dyes were added into live cells for 30 minutes. Cells were fixed and visualized using Cellomics ArrayScan high content screening (HCS) reader (Cellomics, PA, USA). Target activation bioapplication module was used to quantify the fluorescence intensities of the dyes.

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2.8 Reactive Oxygen Species (ROS) assay The ROS assays were carried out as previously [22]. Briefly, 1×104 cells per well

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were seeded into a 96-well plate and incubated overnight at 37 ºC with 5% CO2. The cells were then treated with different concentrations of the compounds for 24 hours and then the dihydroethidium (DHE) dye was added into the live culture for 30 minutes. Cells

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were fixed and washed with wash buffer. The fluorescence intensity was measured using a fluorescent plate reader at an excitation wavelength of 520 nm and an emission

the mean ± SD.

2.9 Multiple Cytotoxicity Assay

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wavelength of 620 nm. The samples were run in triplicate and values were represented as

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Multiple cytotoxicity assay was performed using Cellomics Multiparameter Cytotoxicity 3 Kit as previously described [25]. Briefly, 1×104 MDA-MB-231 cells per well were seeded in a 96-well plate and incubated overnight at 37 °C with 5% CO2. Cells

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were treated with different concentrations of the compounds or DMSO (negative control) for 24 hours. Then, the mitochondrial membrane potential (MMP) dye and the cell

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permeability dye were added to the live cells and incubated for 30 minutes at 37°C. Mitochondrial function is a key indicator of cell health and can be assessed by monitoring changes in mitochondrial membrane potential (MMP). MMP dye (Excitation 552/Emission 576) is a cationic fluorescent dye commonly used to assess MMP. After 30 minutes incubation, cells were fixed, permeabilized, blocked with 1× blocking buffer before probing with primary anti-cytochrome c antibody and secondary DyLight 649-

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conjugated goat anti-mouse IgG for 1 hour each. Hoechst 33342 was added to stain the nucleus. Plates with stained cells were analyzed using the ArrayScan (HCS) system

Data Acquisition and Data Viewer version 3.0 (Cellomics).

2.10 Bioluminescent Assays for Caspase-3/7,-8 and -9 Activities

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(Cellomics, PA, USA). Data were captured, extracted and analyzed with the ArrayScan II

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Caspase assay was carried out using Caspase-Glo® 3/7, 8 and 9 (Promega,

Madison, WI) as described [25]. A total of 1×104 MDA-MB-231 cells were seeded per

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well and incubated overnight. Cells were treated with compounds or DMSO (negative control) for 6, 12, 18, 24, 30 hours. Then, 100 µL caspase-Glo reagent was added and incubated at room temperature for 30 minutes. The caspase activities were measured using a Tecan Infinite®200 Pro microplate reader (Tecan, Männedorf, Switzerland) at

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absorbance 530nm.

2.11 NF-κB (Nuclear factor kappa B) Translocation

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NF-κB translocation assay was carried out using Cellomics nucleur factor-κB (NF-κB) activation kit (Thermo Scientific) as previously described [25]. Briefly, 1.0 ×

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104 MDA-MB-231 cells were seeded in a 96-well plate and incubated overnight at 37 ºC in 5% CO2. The cells were pretreated with different concentrations of the compounds for 3 hours and then stimulated with 1 ng/mL TNF-α for 30 minutes. The medium was removed and the cells were fixed and stained. Cytoplasmic and nuclear NF-κB intensity ratio was measured in an Array Scan HCS Reader (Cellomics) and analyzed using a

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Cytoplasm to Nucleus Translocation BioApplication software. The average intensity of 200 objects (cells) per well was quantified.

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2.12 Quantitative PCR analysis

The analysis was performed as previously described [24]. Total RNAs were

isolated with Zymo Research Quick-RNA™ MiniPrep kit. Complimentary DNAs were

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synthesized with an Applied Biosystems High Capacity RNA-to-cDNA™ Kit.

Quantitative PCR was performed with an Applied Biosystems TaqMan® Fast Advanced

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Master Mix and TaqMan® Gene Expression Assays and carried out on an Applied Biosystems StepOnePlus™ system. All data were then normalized to GAPDH. TaqMan® Gene Expression Assays used in this experiment were GAPDH (Assay ID: Hs02758991_g1), Bcl-2 (Assay ID: Hs00608023_m1) and Bax (Assay

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ID: Hs00180269_m1).

2.13 Statistical Analysis

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Experimental values were presented as the mean ± standard deviation (SD) of the number of experiments. Analysis of variance (ANOVA) was carried out using a

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GraphPad Prism 5 software. Statistical significance was defined when P < 0.05.

3. Results

3.1 Cytotoxic activity of P. sarmentosum methanol extracts (ME) on MDA-MB-231 human breast cancer cells

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The MDA-MB-231 human breast cancer cells were treated with P. sarmentosum ME extract for 24, 48, 72, 96 and 120 hour time points. At each time point, cell viability was determined by MTT assays. P. sarmentosum ME extract showed a dose- and time-

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dependent inhibitory effect on the growth of MDA-MB-231 human breast cancer cells (P

MB-231 cells was shown in Fig. 1A.

3.2 Bioassay guided isolation of compounds 1 and 2

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< 0.05). The result of cytotoxic activity of P. sarmentosum ME extracts against MDA-

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Based on the results obtained above, the methanol extract was subjected to bioassay guided fractionation which gave fraction two (M2) as the active fraction. Result of cytotoxicity potential of the active fraction against the human invasive breast cancer cell line was shown in Fig. 1B. The potent cytotoxicity of M2 led to the isolation and

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characterization of asaricin (1) and isoasarone (2) as the active constituents (Fig. 1C).

3.3 MTT cell viability assay

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The inhibitory effect of compound 1 and 2 on cell viability was evaluated using MTT assays and IC50 values of each compound were shown in Table 1. A time-

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dependent inhibitory effect was observed in the MDA-MB-231 cells. To investigate the selectivity of the compounds, we tested the two compounds on MCF-10A human normal breast cell line. Both compounds exhibited much higher IC50 values against MCF-10A cells, compared to MDA-MB-231 cells. The selectivity index (SI) values were calculated for different treatment time points. As presented in Table 1, the values were significantly higher for both compounds (SI > 2), indicating better selectivity of the two compounds

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on MDA-MB-231 human invasive breast cancer cells, compared to MCF-10A human normal breast cells.

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3.4 LDH release assay

Next, the cytotoxic effect of 1 and 2 was examined by lactate dehydrogenase

(LDH) release assay. MDA-MB-231 cells were treated with different concentrations of

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the compound for 48 hours. LDH release in the medium was due to the loss of membrane integrity as a result of apoptosis or necrosis. Compounds 1 and 2 caused cytotoxicity in a

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dose dependant manner and increased the release of LDH, as compared to the control cells (Fig. 1D & E), indicating compound 1 and 2 are cytotoxic agents.

3.5 Cell cycle analysis

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To determine whether the cells were arrested at the G0/G1, S or G2/M phases, we treated MDA-MB-231 cells with each compound and stained the cells using BrdU and Phospho-Histone H3 dyes. In this assay, BrdU incorporation and mitosis-specific histone

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H3 phosphorylation was directly measured using a high content screening machine which detect immunofluorescence in cells grown on a standard 96 well microplate. As presented

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in Fig. 2, Phospho-Histone H3 was inhibited significantly by 1, which could indicate cell cycle arrest at the G0/G1 or S phase. On the other hand, a decline in BrdU staining was observed in MDA-MB-231 cells treated with 2, suggesting that the G0/G1-phase arrest was induced (Fig. 2).

3.6 Reactive Oxygen Species (ROS) assay

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ROS play an important role in apoptosis induction. To determine the influence of the compound exposure on ROS production, MDA-MB-231 cells were pretreated with 1 or 2 for 24 hours and stained with DHE dye (a widely used small molecule fluorescent

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ROS probe specific for O2−). As shown in Fig. 3A & B, exposure to both compounds caused an increase in the ROS level of the treated MDA-MB-231 cells.

membrane potential (MMP) and Cytochrome c release

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3.7 Effects of 1 and 2 on nuclear morphology, membrane permeability, mitochondrial

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Mitochondria are the key regulators of mechanisms controlling the survival or death of cells since it is the main source of cellular ROS and adenosine triphosphate (ATP). Mitochondrial membrane potential (MMP) fluorescent probes was used to examine the function of mitochondria in treated and untreated MDA-MB-231 cells. The

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untreated cells were strongly stained with MMP dye in comparison to the cells treated with different concentrations of 1 and 2 for 24 hours as shown in Fig. 4A & B. Dosedependent reduction of MMP fluorescence intensity reflects that the MMP was destroyed

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in the treated cells while a significant increase in the cell membrane permeability and

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cytochrome c release was also observed in the treated cells (Fig. 4A & B).

3.8 Bioluminescent Assays for Caspase-3/7,-8 and -9 Activities Activation of downstream caspase molecules and consequently leading to

apoptotic cell death may due to excessive production of ROS from mitochondria and the collapse of MMP. In order to examine this, the bioluminescent intensities of caspase-3/7, -8, -9 activities of the MDA-MB-231 cells treated with 1 and 2 for various time-points

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were measured. Significant time-dependent increase in caspase -9 and -3/7 activity was detected in the 1-treated cells, while no marked changes was observed in caspase-8 between 1-treated and untreated cells (Fig. 4C). Hence, cell apoptosis induced by 1 in

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MDA-MB-231 cells could be mediated via the intrinsic, mitochondrial-caspase-9 pathway [26]. Meanwhile, markedly elevated caspase -8 and -3/7 activities were

observed in 2-treated MDA-MB-231 cells, suggesting 2 may induce extrinsic, death

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3.9 NF-κB Translocation

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receptor-linked caspase-8 (Fig. 4C).

Nuclear factor kappa B (NF-κB) is a transcription factor, critical for cytokine gene expression. Activation of NF-κB in response to inflammatory cytokines, such as Tumour Necrosis Factor-α (TNF-α), mediates nuclear migration to enable DNA-binding activity

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and facilitate target gene expression. In this study, the effect of 1 and 2 on the activation of NF-κB after TNF-α-stimulation was investigated. As presented in Fig. 5A, 1 exhibited no inhibitory effect against TNF-α-mediated nuclear NF-κB translocation, while 2

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partially blocked the translocation of NF-κB from cytoplasm to the nucleus. Moreover, statistical analysis confirmed the inhibitory activity of compound 2 against nuclear

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translocation of NF-κB in TNF-α-stimulated MDA-MB-231 cells (Fig. 5B).

3.10 Quantitative PCR analysis Quantitative PCR assay was performed to check the changes in the expression

level of Bcl-2 and Bax, in MDA-MB-231 cells treated with different concentrations of the two compounds. The quantitative PCR results of the cells treated with 1 or 2 indicated

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a marked elevation in the expression level of Bax compared to the control (DMSOtreated) MDA-MB-231 cells. Comparison of the Bcl-2 expression level between 1/2treated and control cells showed that 2 caused a drastic dose-dependent decline in the

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expression level of Bcl-2, while 1-treatment led to a moderate decrease in Bcl-2 expression level (Fig. 5C).

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4. Discussion

Both compounds; asaricin 1 and isoasarone 2, were evaluated for their ability to

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inhibit the growth of human invasive breast cancer cell line (MDA-MB-231) and human normal breast (MCF-10A) cells using the MTT cell viability assay. We observed that MDA-MB-231 cells were more sensitive to compound 1 and 2 compared to the normal MCF-10A cells, indicating the selectivity of these compounds towards cancer cells.

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MCF-10 A cells are less sensitive than MDA-MB-231, but they respond to treatment with the same unit of measurement. The cytotoxic effect of the compounds was also confirmed by the increased LDH release via the loss of membrane integrity in 1 and 2-treated breast

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cancer cells, suggesting the induction of apoptosis in these cells. In order to elucidate the mechanisms underlying the antiproliferative effect of the

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two compounds on the MDA-MB-231 cells, the cell cycle distribution was analyzed by BrdU and Phospho-Histone H3 staining. The BrdU dye can attach to the synthesized DNA of replicating cells during S phase of the cell cycle, while PhosphoHistone H3 dye stains the cells in different mitotic stages. According to the cell cycle result, fluorescence of Phospho-Histone H3 considerably declined in MDA-MB-231 cells treated with 1, suggesting G0/G1 or S-phase arrest induction. The results obtained from

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the MDA-MB-231 cells treated with 2 indicated a significant reduction of BrdU, which could be caused by cell cycle arrest at the G0/G1 phase. The intrinsic or mitochondrial-dependent signaling pathway involves different

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factors of non-receptor-mediated stimuli which induce intracellular signals. As shown in Fig. 4A & B, changes in MMP after treatment with both compounds have led to the

mitochondrial membrane depolarization, proved by the Rhodamine 123 release to the

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cytoplasm from the mitochondrial matrix. The result implies that the induction of

apoptosis by 1 and 2 may be associated with the mitochondrial pathway. One of the

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important signals of apoptosis to initiate the procedure is cytosolic cytochrome c. The release of cytochrome c into cytosol has been shown to occur as a result of MMP changes. As the result illustrated, both compounds 1 and 2 revealed an increase in the level of cytochrome c in the cytosol compared to the control.

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Downstream caspase molecules could be activated by enhanced ROS and declined MMP, which leads to apoptotic cell death through intrinsic, mitochondrialcaspase-9 pathway. Initiator caspase-8 is known to be activated through extrinsic

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pathway, whereas caspase-9 is activated in the intrinsic event triggered by mitochondrial cytochrome c leakage. Both initiator caspases can activate caspase-3 or -7, which commit

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cells to apoptosis. In the intrinsic pathway, after the binding of cytochrome c to apoptotic activating factor-1, caspase-9 is activated via apoptosome formation which leads to active caspase-3/7. In the extrinsic caspase pathway, FAS ligand interacts with the FAS receptor, leading to the activation of caspase-8, which subsequently cleaves and activates the downstream executioner caspase-3/7. In our study, 1 exhibited significant elevation in the caspase-9 activities compared to the control. Meanwhile, it showed no activation of

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the caspase-8, suggesting that the apoptosis induced by 1 in MDA-MB-231 cells may be mediated via the intrinsic mitochondrial-caspase-9 pathway [26-28]. On the other hand, we observed marked induction of caspase-8, while caspase-9 was not significantly

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changed in compound 2-treated MDA-MB-231 cells. These results implied that 2 induced apoptosis via extrinsic, death receptor-linked caspase-8 pathway [29]. Highly elevated

compounds was through caspase-dependent pathway.

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caspase-3/7 in both 1/2-treated cells confirmed that the apoptosis induced by both

Nuclear factor-kappaB (NF-κB) is a transcription factor that can be activated by a

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great variety of stimuli including TNF-α. In the absence of TNF-α stimulation [30], NFκB is associated with the inhibitor of kappa B (IκB) and remained in the cytoplasm [31]. TNF-α stimulation induced phosphorylation dependent ubiquitination and degradation of inhibitor of kappa B (IκB) proteins and the dissociated NF-κB can translocate to the

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nucleus and initiate gene transcription. In our study, we observed that 2 exhibited partial inhibition against TNF-α-stimulated NF-κB nuclear translocation in the treated MDAMB-231 cells, while 1 showed no inhibitory effect. This result indicated that compound 2

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has a distinctive mechanism by inducing cell apoptosis via extrinsic death receptor-linked caspase-8 pathway and NF-κB blockage in comparison to compound 1.

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Members of the Bcl-2 family include major cell survival and cell death regulators.

Bcl-2 acts as anti-apoptosis regulator in the cells, while Bax acts as a pro apoptosis factor that inhibits cell survival. Our data showed that treatment by both compounds, 1 and 2 caused a significant decline of pro-survival Bcl-2, accompanied by an increase of proapoptotic Bax in MDA-MB-231 cells. The importance of Bc1-2 for the protection of mitochondria during cell death process has been previously reported [32]. Excessive

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expression of Bax may form Mitochondrial Apoptosis-Induced Channel (MAC) and mediates the release of cytochrome c. Bcl-2 has the ability to block the MAC formation through inhibition of Bax and/or Bak. Decline of Bcl-2 expression leads to the loss of

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MMP which enhances apoptosis-associated release of cytochrome c from the

mitochondria [33]. Hence, 1 and 2–mediated dysregulation of Bax and Bcl-2 molecules

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may lead to MMP loss and cytochrome c release which subsequently cause apoptosis.

5. Conclusion

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In summary, we have demonstrated that asaricin 1 and isoasarone 2, isolated from the methanol extract of the roots of P. sarmentosum, induce cell cycle arrest and apoptosis in human invasive breast cancer cells, MDA-MB-231. Pharmacological investigation showed that 1 and 2 induce apoptosis as shown by increased LDH release,

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decreased mitochondria membrane potential and increased cytochrome c release. Our study provides important information regarding the mechanisms by which 1 mediate apoptosis through intrinsic pathway, whereas 2 suppress breast cancer growth through

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extrinsic factor. Thus, our data suggest that 1 and 2 are potential therapeutic agents in breast cancer growth suppression.

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Conflict of interest

The authors declare that they have no conflict of interests.

Acknowledgments

This work was supported by University Malaya Research Grants (PV085/2011A and RP001-2012A/B). The funding sources were not involved in the study design, collection,

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analysis, interpretation of data, writing of the paper or the decision to submit the paper

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Figure captions

Fig. 1. Inhibition of human breast cancer cell viability by P. sarmentosum. MDA-MB-

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231 cells were seeded onto 96-well plate at 8 × 103 cells/well and were treated with (A) P. sarmentosum methanol extracts ranging from 20 to 320 µg/ml (p < 0.05) and (B)

relative active fraction from 20 to 160 µg/ml (p < 0.05) and percentage of cell viability

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was determined by MTT assay after 24, 48, 72, 96 and 120 hours of treatment,

respectively. Results are mean values ± SD of independent experiments performed in

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triplicate. (C) Phenylpropanoids; asaricin (1) and isoasarone (2) isolated from the methanol extract of the roots of P. sarmentosum. LDH release assay revealed the significant cytotoxicity of compounds 1 (D) and 2 (E) on MDA-MB-231 cells.

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Fig. 2. (A) Effect of compounds 1 and 2 on cell cycle arrest in S/M phase. After incubation with DMSO or compound 1/2 for 24 h, MDA-MB-231 cells were stained with BrdU and Phospho-Histone H3 and subjected to Cellomics ArrayScan HCS reader for

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cell cycle analysis. (B) Representative bar charts indicating that compound 1 treatment caused no significant changes in BrdU but significantly decreased Phospho-Histone H3

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fluorescence intensities in treated MDA-MB-231 cells. Compound 2 caused a considerable decline in BrdU, but no significant effect on Phospho-Histone H3 fluorescence intensities. Data were mean ± SD of fluorescence intensity readings of three independent experiments.

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Fig. 3. Effect of compounds 1 and 2 on ROS production. After incubation with DMSO or compound 1/2 for 24 h, MDA-MB-231 cells were collected, stained with DHE and subjected to fluorescence microplate reader. Representative bar charts indicating dose-

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dependent increment of ROS after compound 1 (A) and 2 (B) treatment.

Fig. 4. (A) Representative images of compound 1/2-treated and untreated MDA-MB-231

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cells, stained with Hoechst 33342 for nuclear, cytochrome c, membrane permeability, MMP dyes. Compounds 1 and 2 induced a noteworthy elevation in membrane

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permeability and cytochrome c release and marked reduction in MMP (magnification: 200×). (B) Representative bar charts indicating dose-dependent increased cell permeability, reduced MMP and increased cytochrome c release in compound 1/2-treated MDA-MB-231 cells. (C) Relative luminescence signal of Caspase 3/7, 8 and 9 in MDA-

points.

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MB-231 cells treated with compounds 1 (6 µg/ml) and 2 (8 µg/ml) over various time-

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Fig. 5. (A) Photographs of intracellular targets of stained MDA-MB-231 cells, treated with compound 1/2 for 3 h and then stimulated for 30 min with 1 ng/ml TNF-α (NF-κB

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activation). (B) Representative bar chart indicating that compound 2 partially blocked the translocation of NF-κB from cytoplasm to the nucleus, while compound 1-treatment caused no changes in terms of NF-κB nuclear translocation in TNF- α-stimulated MDAMB-231 cells. (C) Compounds 1 and 2 induce apoptosis through downregulation of prosurvival molecule (Bcl-2), and upregulation of pro-apoptotic molecule (Bax). MDA-MB231 cells were treated with DMSO or different concentrations of compound 1/2 for 24 h.

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Quantitative PCR indicating the level of Bcl-2 and Bax molecules. GAPDH was used as

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Table 1. Inhibitory effects of asaricin (1) and isoasarone (2) on the viability of human normal and cancer cells. Cells were treated with

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various concentrations of asaricin (1) and isoasarone (2) for 24, 48, 72 hours. The IC50 values were analyzed by non-linear regression

Asaricin (1) Isoasarone (2)

24 hr 3.68 ± 0.59 5.82 ± 0.78

MCF-10A 72 hr 2.58 ± 0.43 3.73 ± 0.96

24 hr 9.41 ± 1.45 14.42 ± 2.31

Selectivity Index (SI)

48 hr 72 hr 24 hr 7.85 ± 2.09 7.34 ± 1.63 2.56 12.27 ± 1.78 11.59 ± 1.17 2.48

48 hr 2.52 2.69

72 hr 2.85 3.11

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48 hr 3.12 ± 1.06 4.57 ± 0.35

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Compounds

IC50 (µg/ml) MDA-MB-231

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Asaricin and isoasarone were isolated from the methanol extract of roots of Piper sarmentosum. Asaricin and isoasarone significantly inhibited growth of human breast cancer MDAMB-231. Asaricin induced apoptosis through intrinsic mitochondrial pathway. Isoasarone activates extrinsic death receptor pathway in MDA-MB-231. Both compounds altered ratio of Bcl-2 : Bax expression in MDA-MB-231.

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