# Persea declinata (Bl.) Kosterm Bark Crude Extract Induces Apoptosis in MCF-7 Cells via  G0/G1 Cell Cycle Arrest, Bcl-2/Bax/Bcl-xl Signaling Pathways, and ROS Generation

Authors: Putri Narrima; Mohammadjavad Paydar; Chung Yeng Looi; Yi Li Wong; Hairin Taha; Won Fen Wong; Mustafa Ali Mohd; A. Hamid A. Hadi
Journal: Evidence-Based Complementary and Alternative Medicine
 (2014)

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## Abstract

Persea declinata (Bl.) Kosterm is a member of the Lauraceae family, widely distributed in Southeast Asia. It is from the same genus with avocado (Persea americana Mill), which is widely consumed as food and for medicinal purposes. In the present study, we examined the anticancer properties of Persea declinata (Bl.) Kosterm bark methanolic crude extract (PDM). PDM exhibited a potent antiproliferative effect in MCF-7 human breast cancer cells, with an IC50 value of 16.68 µg/mL after 48 h of treatment. We observed that PDM caused cell cycle arrest and subsequent apoptosis in MCF-7 cells, as exhibited by increased population at G0/G1 phase, higher lactate dehydrogenase (LDH) release, and DNA fragmentation. Mechanistic studies showed that PDM caused significant elevation in ROS production, leading to perturbation of mitochondrial membrane potential, cell permeability, and activation of caspases-3/7. On the other hand, real-time PCR and Western blot analysis showed that PDM treatment increased the expression of the proapoptotic molecule, Bax, but decreased the expression of prosurvival proteins, Bcl-2 and Bcl-xL, in a dose-dependent manner. These findings imply that PDM could inhibit proliferation in MCF-7 cells via cell cycle arrest and apoptosis induction, indicating its potential as a therapeutic agent worthy of further development.


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## Body

## 1. Introduction

Breast cancer is a heterogeneous disease which is counted as the second leading cause of cancer-related deaths in women worldwide. In recent years breast cancer has become a global public health concern due to the upward trend of its incidence at an annual rate of 3.1%, from 1.38 million women in 2008 to more than 1.6 million in 2010 [1, 2]. In 2007, the number of reported breast cancer cases in Malaysia was 3,242 women, which was 18.1% of the total reported cancer cases and 32.1% of the total cancer cases in women [3]. In 1970, an estrogen receptor-positive cell line, called MCF-7, was derived from a metastatic breast cancer patient at the Michigan Cancer Foundation in 1973 [4], which has become the most extensively used model of estrogen-positive breast cancer cell line for the study of breast cancer as it relates to the susceptibility of the cells to apoptosis [5].Historically, plants were of the main sources of pharmaceutical agents used in traditional medicine. The application of plant-derived drugs in modern medicine has undergone a dramatic upward trend during the last decades, and a large number of therapeutic compounds (such as vinblastine, taxotere, etoposide, and topotecan) have been discovered in medicinal plants and approved to be used as anticancer drugs [6, 7]. In many countries, medicinal plants are still collected from wild vegetation. But in response to the combined impact of dwindling supplies due to overexploitation of the natural resources and increasing demands by global population growth, medicinal plants are also being cultivated using modern farming systems. Malaysia is rich in biodiversity, which is believed to be 130 million years old, and is mostly covered with enormous forests that include an estimated 14,500 species of flowering plants. About 15% of these plants were claimed to have medicinal properties, of which only a handful have been studied for their potential bioactivities and are currently cultivated by various farming communities, but there are still many more plants to be discovered [8].Persea declinata (Bl.) Kosterm (common names: medang inai, medang tanah, kayu helah, huru manok, huru leu-ur, and meang telut) is a member of the Lauraceae family, which is widely distributed in Borneo, Java, Malaysia (Penang, Kelantan, Terengganu, Pahang, Selangor) and Singapore. It is an evergreen tree which can grow above 30 m tall, with smooth gray bark. The leaves are elliptic to lanceolate (long: from 5.5 to 13 cm, width: 2–3.5 cm), subcoriaces, cuneiform at the base, the apex acute, and the petiole glabrous (long: 5–15 mm) with globular fruits (diameter: 5–10 mm). It shares the same genus with the popularly investigatedPersea sp., avocado (Persea americana Mill). Avocado is widely consumed as food and for medicinal purposes. It possesses various potential cancer preventive phytochemicals [9] and other pharmacological properties including hepatoprotective [10], anticonvulsant [11], wound healing [12], analgesic, anti-inflammatory [13], hypoglycaemic, and hypocholesterolaemic [14].While countless studies have been done on avocado,Persea declinata (Bl.) Kosterm on the other hand has never been investigated for any pharmacological potential. Therefore, the present study will focus on the preliminary cytotoxic testing ofPersea declinata (Bl.) Kosterm bark methanolic crude extract on various cancer cell lines and the effective one is used as a model to further investigate the mechanistic action.


## 2. Materials and Methods

### 2.1. Chemical Reagents and Solvents

Chemical reagents and solvents used for extraction and assays were of analytical grade and were purchased from Fisher Scientific (Pittsburgh, PA).


### 2.2. Plant Source

The bark ofPersea declinata (Bl.) Kosterm was collected from Dungun, Terengganu, Malaysia. The plant species was identified by the university’s botanist with a Voucher specimen (no. KL 5068) and was deposited in the herbarium of the Chemistry Department, University of Malaya.


### 2.3. Cell Culture

Human colon adenocarcinoma cell line (HT29), human hepatocarcinoma cell line (HepG2), human breast cancer cell lines (T47D, MDA-MB-231), and human normal hepatic cell line (WRL-68) were purchased from American Type Culture Collection (ATCC, Manassas, VA). Human breast cancer cell line (MCF-7) was purchased from Cell Line Services (300273; Eppelheim, Germany), and oral carcinoma cell lines (H400, H413, and BICR31) were provided by Professor Ian Charles Paterson. HT29, HepG2, T47D, MDA-MB-231, and MCF-7 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, USA), 1% penicillin and streptomycin. WRL-68 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies, Inc., Rockville, MD) supplemented with 15% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, USA), 1% penicillin and streptomycin. H400, H413, and BICR31 cells were grown in Dulbecco’s Modified Eagle Medium/Ham’s F-12 (DMEM/F12) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, USA), 1% penicillin and streptomycin. Cells were cultured in 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 growth phase (approximately 70–80% confluency) were used.


### 2.4. Extraction

50 grams of grounded bark ofPersea declinata (Bl.) Kosterm was extracted with 500 mL of methanol. Approximately 5 g of polyvinylpyrrolidone (PVP, Sigma-Aldrich, USA) was added for detannification and the mixture was kept for 48 hours. After that, the crude extract (PDM) was filtered and evaporated to about 15 mL volume using a rotary evaporator and further frozen and freeze-dried for another 48 hours.


### 2.5. MTT Cell Viability Assay

The cytotoxic effect of PDM was assessed by MTT cell viability assay against different cancer cells [19]. 1.0 × 104 cells were seeded in a 96-well plate and incubated overnight at 37°C in 5% CO2. On the next day, the cells were treated with a two-fold dilution series of six concentrations of PDM, and then they were incubated at 37°C in 5% CO2 for 48 hours. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, USA) solution was added at 2 mg/mL and after 2 hours of incubation at 37°C in 5% CO2, DMSO was added to dissolve the formazan crystals. The plates were then read at 570 nm absorbance. The cell viability percentage after exposure to PDM for 48 hours was calculated by a previously described method [20]. The ratio of the absorbance of treated cells to the absorbance of DMSO-treated control cells was determined as percentage of cell viability. IC50 value was defined as the concentration of PDM required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. The experiment was carried out in triplicates.


### 2.6. Real-Time Cell Proliferation

In vitro proliferation of PDM-treated and untreated cells was surveyed using a Real-Time Cellular Analysis (JuLi Br) system. 2.0 × 105 cells were seeded in a 6-well plate and incubated overnight at 37°C in 5% CO2. The RTCA system monitored the proliferation of cells every 5 minutes for about 24 hours. During the log growth phase, the cells were treated with IC50 concentrations of the PDM (17 μg/mL) or left untreated and monitored continuously for another 24 hours.


### 2.7. LDH Release Assay

Measurement of lactate dehydrogenase (LDH) release is a biomarker that can determine the cytotoxicity of a compound or an extract. Briefly, MCF-7 cells were treated with different concentrations of PDM and Triton X-100 (positive control) for 24 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100μL of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro (Tecan, Männedorf, Switzerland) microplate reader. The amount of formazan salt and the intensity of red color in treated and untreated samples were represented as the LDH activity of cells.


### 2.8. Cell Cycle Analysis

1 × 104 cells per well were seeded in a 96-well plate and incubated overnight at 37°C in 5% CO2. Cells were treated with different concentrations of the PDM 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 stained as described by the manufacturer’s instruction. Stained cells were visualized and acquired using Cellomics ArrayScan HCS reader (Thermo Scientific). Target activation bioapplication module was used to quantify the fluorescence intensities of dyes.


### 2.9. Reactive Oxygen Species (ROS) Assays

1 × 104 cells per well were seeded onto a 96-well plate. Cells were treated with the PDM or DMSO (negative control) at indicated concentrations for 12 hours. Dihydroethidium (DHE) dye contained in Cellomics ROS kit was added into a live culture for 30 minutes. Cells were fixed and washed with wash buffer as described by the manufacturer’s instruction. Stained cells were visualized and acquired using Cellomics ArrayScan HCS reader (Thermo Scientific). Target activation bioapplication module was used to quantify the fluorescence intensities of DHE dye in the nucleus.


### 2.10. Nuclear Morphology, Membrane Permeability, and Mitochondrial Membrane Potential (MMP) Assays

Cellomics multiparameter cytotoxicity 3 kit (Thermo Scientific) was used as described previously [21]. 1×104 cells per well were plated in a 96-well plate and incubated overnight at 37°C in 5% CO2. The cells were treated with different concentrations of the PDM and further incubated at 37°C in 5% CO2 for 24 hours. MMP dye and the cell permeability dye were added to live cells and incubated for 1 hour. After fixing the cells, the nucleus was stained with Hoechst 33258. Stained cells were visualized and images were captured using Cellomics ArrayScan HCS reader (Thermo Scientific).


### 2.11. DNA Fragmentation Analysis by Acridine Orange

1 × 104 cells per well were plated on a 96-well plate and incubated overnight at 37°C in 5% CO2. PDM at various concentrations were added and further incubated for 24 hours. Acridine orange staining solution was added to live cells and incubated for 15 min. The cells were then fixed and visualized and images were captured using a camera connected to a fluorescent microscope.


### 2.12. Bioluminescent Assays for Caspase-3/7 Activities

A dose-dependent study of caspase-3/7 activity was performed in triplicates using assay kits Caspase-Glo 3/7 (Promega, Madison, WI) on a white 96-well microplate. A total of 1 × 104 cells were seeded per well and incubated with different concentrations of PDM for 24 hours. Caspase activities were investigated according to the manufacturer’s protocol. Briefly, 100 μL caspase-Glo reagent was added and incubated at room temperature for 30 minutes. Activated caspases cleaved the aminoluciferin-labeled synthetic tetrapeptide, leading to release of luciferase substrate. The caspase activities were measured using a Tecan 12 Infinite 200 Pro (Tecan, Männedorf, Switzerland) microplate reader.


### 2.13. NF-κB Translocation

Briefly, 1.0 × 104 cells were seeded in a 96-well plate and incubated overnight at 37°C in 5% CO2. The cells were pretreated with different concentrations of PDM for 3 h and then stimulated with 1 ng/mL TNF-α for 30 min. The medium was removed and the cells were fixed and stained with Cellomics nucleus factor-κB (NF-κB) activation kit from Thermo Scientific based on the manufacturer’s instructions. The plate was evaluated on Array Scan HCS Reader. The calculation of cytoplasmic and nuclear NF-κB intensity ration was carried out using Cytoplasm to Nucleus Translocation BioApplication software. The average intensity of 200 objects (cells) per well was quantified. The ratios were then compared among TNF-α-stimulated, treated, and untreated cells [21].


### 2.14. Western Blot Analysis

SDS-PAGE and Western blot analyses were done as described with slight modifications [22]. Briefly, 24 hours posttreatment, cells were lysed in RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with freshly added 10 mM β-glycerophosphate, 1 mM sodium orthovanadate, 10 mM NaF, and 1 mM phenylmethylsulfonyl fluoride and Protease Inhibitor Cocktail (Santa Cruz, CA) and loaded onto 10% polyacrylamide gel. Proteins were then transferred to microporous polyvinylidene difluoride (PVDF) membrane (Milipore). Membranes were incubated in 5% BSA (Sigma) blocking buffer for 1 h at room temperature. Incubations with primary antibody were carried out overnight at 4°C. Immunoblotting was performed with rabbit anti-Bcl-2, anti-Bcl-xl, and anti-Bax antibodies (1 : 200) (Cell Signaling Technology, Danvers, MA). Membranes were washed 3 times (10 min each) in Tween buffer before incubating with HRP-conjugated goat anti-mouse or rabbit secondary antibodies. To remove excess antibodies, membranes were washed 4 times before HRP activities were detected using ECL Plus Chemiluminescence Reagent (Amersham, Chalfont, UK) according to the protocol supplied with the kit.


### 2.15. Quantitative PCR Analysis

MCF-7 cells were treated with various concentrations of extract for 24 h. Total RNAs were isolated with Zymo Research Quick-RNA MiniPrep kit. Complimentary DNAs were synthesized with Applied Biosystems High Capacity RNA-to-cDNA Kit. Quantitative PCR was performed with Applied Biosystems TaqMan Fast Advanced Master Mix and TaqMan Gene Expression Assays and run on a real-time PCR machine (Applied Biosystems StepOnePlus system). All data were then normalized to GAPDH. The IDs for TaqMan Gene Expression Assays used in this experiment are listed in Table1.Table 1
IDs for TaqMan gene expression assays.
Target gene
Assay ID
GAPDH
Hs02758991_g1
Bcl-2
Hs00608023_m1
Bcl-xl
Hs00236329_m1
Bax
Hs00180269_m1


### 2.16. GC-TOFMS Identification and Chemical Analysis of Crude Extract

Gas chromatography time of flight mass spectrometry (GC-TOFMS) analysis of the crude extract was carried out on a Pegasus HT GC-TOFMS 7890A (LECO, USA) system. Separation was conducted on an RXI-5 MS column (30 m × 0.32 mm × 0.25μm), with helium as the carrier gas (flow rate of 1.0 mL/min). The injection volume was 1 μL in a split mode. The column temperature was initially held at 40°C for 5 minutes and then increased to 260°C at a rate of 10°C/min, then maintained at 260°C for 10 minutes. The temperatures of the injector and detector were 250°C and 280°C, respectively. Mass acquisition was performed in the range of 40–550 atomic mass units (a. m. u) using electron impact ionization at 70 eV. The major components in this sample were predicted by a spectral database matching against the library of National Institute of Standards and Technology (NIST21 and NISTWiley).


### 2.17. Statistical Analysis

Experimental values were presented as the means ± standard deviation (SD) of the number of experiments indicated in the legends. Analysis of variance (ANOVA) was performed using GraphPad Prism 5 software. Statistical significance was defined whenP<0.05.


## 2.1. Chemical Reagents and Solvents

Chemical reagents and solvents used for extraction and assays were of analytical grade and were purchased from Fisher Scientific (Pittsburgh, PA).


## 2.2. Plant Source

The bark ofPersea declinata (Bl.) Kosterm was collected from Dungun, Terengganu, Malaysia. The plant species was identified by the university’s botanist with a Voucher specimen (no. KL 5068) and was deposited in the herbarium of the Chemistry Department, University of Malaya.


## 2.3. Cell Culture

Human colon adenocarcinoma cell line (HT29), human hepatocarcinoma cell line (HepG2), human breast cancer cell lines (T47D, MDA-MB-231), and human normal hepatic cell line (WRL-68) were purchased from American Type Culture Collection (ATCC, Manassas, VA). Human breast cancer cell line (MCF-7) was purchased from Cell Line Services (300273; Eppelheim, Germany), and oral carcinoma cell lines (H400, H413, and BICR31) were provided by Professor Ian Charles Paterson. HT29, HepG2, T47D, MDA-MB-231, and MCF-7 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, USA), 1% penicillin and streptomycin. WRL-68 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies, Inc., Rockville, MD) supplemented with 15% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, USA), 1% penicillin and streptomycin. H400, H413, and BICR31 cells were grown in Dulbecco’s Modified Eagle Medium/Ham’s F-12 (DMEM/F12) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich, USA), 1% penicillin and streptomycin. Cells were cultured in 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 growth phase (approximately 70–80% confluency) were used.


## 2.4. Extraction

50 grams of grounded bark ofPersea declinata (Bl.) Kosterm was extracted with 500 mL of methanol. Approximately 5 g of polyvinylpyrrolidone (PVP, Sigma-Aldrich, USA) was added for detannification and the mixture was kept for 48 hours. After that, the crude extract (PDM) was filtered and evaporated to about 15 mL volume using a rotary evaporator and further frozen and freeze-dried for another 48 hours.


## 2.5. MTT Cell Viability Assay

The cytotoxic effect of PDM was assessed by MTT cell viability assay against different cancer cells [19]. 1.0 × 104 cells were seeded in a 96-well plate and incubated overnight at 37°C in 5% CO2. On the next day, the cells were treated with a two-fold dilution series of six concentrations of PDM, and then they were incubated at 37°C in 5% CO2 for 48 hours. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, USA) solution was added at 2 mg/mL and after 2 hours of incubation at 37°C in 5% CO2, DMSO was added to dissolve the formazan crystals. The plates were then read at 570 nm absorbance. The cell viability percentage after exposure to PDM for 48 hours was calculated by a previously described method [20]. The ratio of the absorbance of treated cells to the absorbance of DMSO-treated control cells was determined as percentage of cell viability. IC50 value was defined as the concentration of PDM required to reduce the absorbance of treated cells to 50% of the DMSO-treated control cells. The experiment was carried out in triplicates.


## 2.6. Real-Time Cell Proliferation

In vitro proliferation of PDM-treated and untreated cells was surveyed using a Real-Time Cellular Analysis (JuLi Br) system. 2.0 × 105 cells were seeded in a 6-well plate and incubated overnight at 37°C in 5% CO2. The RTCA system monitored the proliferation of cells every 5 minutes for about 24 hours. During the log growth phase, the cells were treated with IC50 concentrations of the PDM (17 μg/mL) or left untreated and monitored continuously for another 24 hours.


## 2.7. LDH Release Assay

Measurement of lactate dehydrogenase (LDH) release is a biomarker that can determine the cytotoxicity of a compound or an extract. Briefly, MCF-7 cells were treated with different concentrations of PDM and Triton X-100 (positive control) for 24 h, and the supernatants of the untreated and treated cells were transferred to a new 96-well plate for LDH activity analysis. Next, 100μL of LDH reaction solution was added to each well, the plate was incubated at room temperature for 30 min, and the absorbance was read at 490 nm using a Tecan Infinite 200 Pro (Tecan, Männedorf, Switzerland) microplate reader. The amount of formazan salt and the intensity of red color in treated and untreated samples were represented as the LDH activity of cells.


## 2.8. Cell Cycle Analysis

1 × 104 cells per well were seeded in a 96-well plate and incubated overnight at 37°C in 5% CO2. Cells were treated with different concentrations of the PDM 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 stained as described by the manufacturer’s instruction. Stained cells were visualized and acquired using Cellomics ArrayScan HCS reader (Thermo Scientific). Target activation bioapplication module was used to quantify the fluorescence intensities of dyes.


## 2.9. Reactive Oxygen Species (ROS) Assays

1 × 104 cells per well were seeded onto a 96-well plate. Cells were treated with the PDM or DMSO (negative control) at indicated concentrations for 12 hours. Dihydroethidium (DHE) dye contained in Cellomics ROS kit was added into a live culture for 30 minutes. Cells were fixed and washed with wash buffer as described by the manufacturer’s instruction. Stained cells were visualized and acquired using Cellomics ArrayScan HCS reader (Thermo Scientific). Target activation bioapplication module was used to quantify the fluorescence intensities of DHE dye in the nucleus.


## 2.10. Nuclear Morphology, Membrane Permeability, and Mitochondrial Membrane Potential (MMP) Assays

Cellomics multiparameter cytotoxicity 3 kit (Thermo Scientific) was used as described previously [21]. 1×104 cells per well were plated in a 96-well plate and incubated overnight at 37°C in 5% CO2. The cells were treated with different concentrations of the PDM and further incubated at 37°C in 5% CO2 for 24 hours. MMP dye and the cell permeability dye were added to live cells and incubated for 1 hour. After fixing the cells, the nucleus was stained with Hoechst 33258. Stained cells were visualized and images were captured using Cellomics ArrayScan HCS reader (Thermo Scientific).


## 2.11. DNA Fragmentation Analysis by Acridine Orange

1 × 104 cells per well were plated on a 96-well plate and incubated overnight at 37°C in 5% CO2. PDM at various concentrations were added and further incubated for 24 hours. Acridine orange staining solution was added to live cells and incubated for 15 min. The cells were then fixed and visualized and images were captured using a camera connected to a fluorescent microscope.


## 2.12. Bioluminescent Assays for Caspase-3/7 Activities

A dose-dependent study of caspase-3/7 activity was performed in triplicates using assay kits Caspase-Glo 3/7 (Promega, Madison, WI) on a white 96-well microplate. A total of 1 × 104 cells were seeded per well and incubated with different concentrations of PDM for 24 hours. Caspase activities were investigated according to the manufacturer’s protocol. Briefly, 100 μL caspase-Glo reagent was added and incubated at room temperature for 30 minutes. Activated caspases cleaved the aminoluciferin-labeled synthetic tetrapeptide, leading to release of luciferase substrate. The caspase activities were measured using a Tecan 12 Infinite 200 Pro (Tecan, Männedorf, Switzerland) microplate reader.


## 2.13. NF-κB Translocation

Briefly, 1.0 × 104 cells were seeded in a 96-well plate and incubated overnight at 37°C in 5% CO2. The cells were pretreated with different concentrations of PDM for 3 h and then stimulated with 1 ng/mL TNF-α for 30 min. The medium was removed and the cells were fixed and stained with Cellomics nucleus factor-κB (NF-κB) activation kit from Thermo Scientific based on the manufacturer’s instructions. The plate was evaluated on Array Scan HCS Reader. The calculation of cytoplasmic and nuclear NF-κB intensity ration was carried out using Cytoplasm to Nucleus Translocation BioApplication software. The average intensity of 200 objects (cells) per well was quantified. The ratios were then compared among TNF-α-stimulated, treated, and untreated cells [21].


## 2.14. Western Blot Analysis

SDS-PAGE and Western blot analyses were done as described with slight modifications [22]. Briefly, 24 hours posttreatment, cells were lysed in RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with freshly added 10 mM β-glycerophosphate, 1 mM sodium orthovanadate, 10 mM NaF, and 1 mM phenylmethylsulfonyl fluoride and Protease Inhibitor Cocktail (Santa Cruz, CA) and loaded onto 10% polyacrylamide gel. Proteins were then transferred to microporous polyvinylidene difluoride (PVDF) membrane (Milipore). Membranes were incubated in 5% BSA (Sigma) blocking buffer for 1 h at room temperature. Incubations with primary antibody were carried out overnight at 4°C. Immunoblotting was performed with rabbit anti-Bcl-2, anti-Bcl-xl, and anti-Bax antibodies (1 : 200) (Cell Signaling Technology, Danvers, MA). Membranes were washed 3 times (10 min each) in Tween buffer before incubating with HRP-conjugated goat anti-mouse or rabbit secondary antibodies. To remove excess antibodies, membranes were washed 4 times before HRP activities were detected using ECL Plus Chemiluminescence Reagent (Amersham, Chalfont, UK) according to the protocol supplied with the kit.


## 2.15. Quantitative PCR Analysis

MCF-7 cells were treated with various concentrations of extract for 24 h. Total RNAs were isolated with Zymo Research Quick-RNA MiniPrep kit. Complimentary DNAs were synthesized with Applied Biosystems High Capacity RNA-to-cDNA Kit. Quantitative PCR was performed with Applied Biosystems TaqMan Fast Advanced Master Mix and TaqMan Gene Expression Assays and run on a real-time PCR machine (Applied Biosystems StepOnePlus system). All data were then normalized to GAPDH. The IDs for TaqMan Gene Expression Assays used in this experiment are listed in Table1.Table 1
IDs for TaqMan gene expression assays.
Target gene
Assay ID
GAPDH
Hs02758991_g1
Bcl-2
Hs00608023_m1
Bcl-xl
Hs00236329_m1
Bax
Hs00180269_m1


## 2.16. GC-TOFMS Identification and Chemical Analysis of Crude Extract

Gas chromatography time of flight mass spectrometry (GC-TOFMS) analysis of the crude extract was carried out on a Pegasus HT GC-TOFMS 7890A (LECO, USA) system. Separation was conducted on an RXI-5 MS column (30 m × 0.32 mm × 0.25μm), with helium as the carrier gas (flow rate of 1.0 mL/min). The injection volume was 1 μL in a split mode. The column temperature was initially held at 40°C for 5 minutes and then increased to 260°C at a rate of 10°C/min, then maintained at 260°C for 10 minutes. The temperatures of the injector and detector were 250°C and 280°C, respectively. Mass acquisition was performed in the range of 40–550 atomic mass units (a. m. u) using electron impact ionization at 70 eV. The major components in this sample were predicted by a spectral database matching against the library of National Institute of Standards and Technology (NIST21 and NISTWiley).


## 2.17. Statistical Analysis

Experimental values were presented as the means ± standard deviation (SD) of the number of experiments indicated in the legends. Analysis of variance (ANOVA) was performed using GraphPad Prism 5 software. Statistical significance was defined whenP<0.05.


## 3. Results

### 3.1. Effect of PDM on Cell Viability

The cytotoxic effect of PDM was evaluated on HepG2, MDA-MB-231, MCF-7, T47D, H400, H413, BICR31, and WRL-68 cells using MTT assays. Table2 shows the IC50 values after 48 hours of treatment with PDM. Highest cytotoxicity was observed in MCF-7 breast cancer cells (IC50 = 16.68 ± 0.89). Moreover, PDM exhibited higher selectivity on human breast cancer MCF-7 cells, compared to human normal hepatic WRL-68 cells.Table 2
Effect ofPersea  declinata (Bl.) Kosterm bark crude extract on cells expressed as IC50 values in 48 hours MTT assay.
Cell line
IC50 (μg/mL)
Bicr31
27.68
±
2.14
H400
31.39
±
1.85
H413
24.16
±
1.44
MCF7
16.68
±
0.89
T47D
18.53
±
1.26
MDA-MB-231
21.80
±
1.71
HepG2
26.41
±
2.09
HT-29
28.59
±
1.93
WRL-68
97.90
±
5.73Next, real-time cell proliferation assay was carried out to monitor the morphological changes of MCF-7 cells treated with PDM. The results indicated significant reduction of cell number, cell shrinkage, and apoptotic body formation throughout the 24 hours of treatment (Figure1).PDM induced significant reduction of cell number, cell shrinkage, and apoptotic body formation in treated MCF-7 cells. (a) MCF-7 cells were treated with DMSO, doxorubicin, or different concentrations of PDM for 24 h. Live cell images indicating cell shrinkage and apoptosis in a dose-dependent manner. (b) MCF-7 cells were treated with DMSO or PDM (25μg/mL) and were screened for 24 h using a real-time cellular analysis (JuLi Br) system. Real-time cell proliferation throughout the 24 hours of treatment indicating significant reduction of cell number, cell shrinkage, and formation of apoptotic body in the MCF-7 cells treated with PDM.
(a)
(b)


### 3.2. PDM Induced Higher LDH Release in MCF-7 Cells

Lactate dehydrogenase (LDH) release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of PDM, as determined by the LDH release assay, was quantified on MCF-7 cells treated with various concentrations of the extract for 24 h. PDM induced a significant elevation in LDH release, demonstrating cytotoxicity at 25 and 50μg/mL concentrations compared to the control cells (Figure 2).Figure 2
The LDH release assay revealed the significant cytotoxicity of PDM on MCF-7 cells at 25 and 50μg/mL concentrations.


### 3.3. Effect of PDM on Cell Cycle

We investigated the cell cycle arrest of MCF-7 cells treated with different concentrations of PDM, using BrdU and Phospho-histone H3 dyes. Comparison of the images and intensity bar charts of PDM-treated and untreated MCF-7 cells indicated a decreased level of BrdU and Phospho-histone H3 intensities (Figures3(a) and 3(b)). The histogram plot on Hoechst total intensity also demonstrated a decreased level of cells in S and G2/M phases and an increased number of cells at G0/G1 in PDM-treated MCF-7 cells (Figure 3(c)). These results show that PDM induced G0/G1 arrest in MCF-7 after 24 hours.(a) Effect of PDM on cell cycle arrest. The MCF-7 cells were treated with DMSO, doxorubicin, or PDM for 24 h, stained with BrdU and Phospho-histone H3, and subjected to the Cellomics ArrayScan HCS reader for cell cycle analysis. (b) Representative bar charts indicating that PDM treatment caused no significant changes in BrdU or Phospho-histone H3 fluorescence intensities, and consequently induced no cell cycle arrest in the treated MCF-7 cells. Data were expressed as the mean ± SD of fluorescence intensity readings for three independent experiments. (c) Cells were stained with Hoechst and the distribution in G0/G1, S, and G2/M cell cycle phases was determined with Cellomics High Content Screening.
(a)
(b)
(c)


### 3.4. PDM Increased Reactive Oxygen Species (ROS) Production

ROS is produced as a byproduct of normal metabolism of oxygen. ROS formation may undergo a drastic increase under environmental or chemical stress. Enhanced levels of ROS may lead to apoptosis or cell cycle arrest. In this study, we stained PDM-treated (24 hours) or untreated MCF-7 live cells with DHE dye to assess whether the exposure of PDM promotes ROS production. As shown in Figure4, the levels of DHE increased significantly in MCF-7 cells treated with PDM, indicating higher ROS production.Effect of PDM on ROS production. (a) Representative images of MCF-7 cells treated with DMEM, doxorubicin, or PDM for 24 h and stained with DHE dye. PDM induced a noteworthy elevation in nuclear ROS in the PDM-treated MCF-7 cells (magnification: 200x). (b) Representative bar charts indicating dose-dependent increased ROS in the nucleus of the PDM-treated MCF-7 cells.
(a)
(b)


### 3.5. PDM Decreased Mitochondrial Membrane Potential (MMP) and Increased Cell Membrane Permeability

As the main source of cellular ROS and adenosine triphosphate (ATP), mitochondria are the key regulators of mechanisms controlling the survival or death of cells. We used mitochondrial membrane potential (MMP) fluorescent probes to examine the function of mitochondria in treated and untreated MCF-7 cells. As shown in Figure5, the untreated cells were strongly stained with MMP dye in comparison to PDM-treated cells. A dose-dependent reduction of MMP fluorescence intensity reflects that the MMP is gradually destroyed in response to higher PDM concentration (Figure 5). On the other hand, a significant increase in cell membrane permeability was also observed in PDM treated cells after 24 hours of treatment (Figure 5).(a) Representative images of MCF-7 cells treated with DMEM, doxorubicin, or PDM for 24 h and stained with Hoechst 33342 for nuclear, membrane permeability, and mitochondrial membrane potential (MMP) dyes. PDM induced a noteworthy elevation in membrane permeability and a marked reduction in MMP (magnification: 200x). (b) Representative bar charts indicating dose-dependent increased cell permeability and reduced MMP in the PDM-treated MCF-7 cells.
(a)
(b)


### 3.6. PDM Induced DNA Fragmentation

Acridine orange (AO) is used to determine DNA integrity. We stained MCF-7 16 cells with acridine orange dye after 24 hours of treatment. Figures6(a) and 6(b) showed a dose-dependent increase of nucleus restricted acridine orange dye in PDM-treated cells, indicating DNA fragmentation.(a) Acridine orange DNA fragmentation analysis. The cells were treated with DMEM or PDM for 24 h and stained with acridine orange dye. (b) Representative bar charts that show dose-dependent increased fluorescent intensity of acridine orange dye inside the nucleus of the PDM-treated MCF-7 cells, indicating DNA fragmentation.
(a)
(b)


### 3.7. PDM Increased Caspase-3/7 Activity

The excessive production of ROS from mitochondria and the collapse of MMP may activate downstream caspase molecules and consequently lead to apoptotic cell death. To examine this, we measured the caspase-3/7 activity using bioluminescent assays. As shown in Figure7, a significant dose-dependent increase in caspase-3/7 activity was detected in PDM-treated cells. Hence, the apoptosis induced by PDM in MCF-7 cells could be mediated through caspase activation.Figure 7
Relative luminescence expression of caspases 3/7 in MCF-7 cells treated with various concentrations of PDM.


### 3.8. Effect of PDM on NF-κB Activation

The nuclear factor kappa B (NF-κB) is a transcription factor, critical for cell proliferation and apoptosis. Activation of NF-κB is indicated by cytoplasm to nuclear translocation to enable DNA-binding activity and facilitate target gene expression. NF-κB remains in the cytoplasm in the absence of activation signal in MCF-7 (Figure 8(a)). In the presence of TNF-α, NF-κB localized mainly in the nucleus of most cells (Figure 8(a)). As shown in Figure 10, PDM treatment has no inhibitory effect on TNF-α-induced NF-κB translocation from cytoplasm to nucleus, compared to positive control curcumin (Figures 8(a) and 8(b)).(a) Photographs of the intracellular targets of stained MCF-7 cells that were treated with DMSO, PDM (25μg/mL), or curcumin (50 μM) for 3 h and then stimulated for 30 min with 1 ng/mL TNF-α (NF-κB activation). (b) Representative bar charts indicating that PDM treatment caused no changes in TNF-α-induced NF-κB nuclear translocation in MCF-7 cells.
(a)
(b)


### 3.9. PDM Modulated Expression of Bcl-2, Bcl-xl, and Bax

Bcl-2 includes a family of proteins that regulate Mitochondrial Outer Membrane Permeabilization (MOMP). They include antiapoptotic molecules such as Bcl-2 and Bcl-xl, which could preserve cell survival and proapoptotic molecules like Bax that inhibit cell survival. To examine if PDM initiated apoptosis by affecting the cellular level of these molecules, Western blot analysis was performed using untreated or extract-treated breast cancer cells. Our data showed that PDM dose-dependently upregulated Bax and downregulated the expression level of Bcl-2 and Bcl-xl in MCF-7 cells (Figure9).Figure 9
Induction of apoptosis by reducing the expression level of prosurvival molecules (Bcl-2, Bcl-xl) and increasing the expression of proapoptotic molecule (Bax). MCF-7 cells were treated with DMSO or different concentrations of PDM for 24 h. Western blot results showing the expression levels of Bcl-2, Bcl-xl, and Bax in PDM or DMSO-treated MCF-7 cells.β-Actin serves as a loading control.Figure 10
MCF-7 cells were treated with DMSO or different concentrations of PDM for 24 h. RNAs were isolated and converted to cDNA. Quantitative real-time PCR was performed to determine expression level of Bcl-2, Bcl-xl, and Bax genes. GAPDH was used as a housekeeping gene.Next, we performed quantitative PCR using a real-time PCR machine to examine whether the expression of these molecules was affected at the transcriptional level. Results indicated a marked increase in Bax expression but a decrease in the expression level of Bcl-2 and Bcl-xl in the PDM-treated MCF-7 cells, consistent with Western blotting data (Figure10).


### 3.10. GC-TOFMS Identification and Chemical Analysis of Crude Extract

To analyse the chemical constituent of PDM, the extract was subjected to GC-TOFMS analysis and the result is presented in Table3. A total of 4 main compounds were detected in PDM. The most abundant compound detected was 2-methoxy-4-propylphenol (4-propylguaiacol) (61.901%), followed by Caryophyllene (21.877%), α-Copaene (10.226%), and Iso-α-humulene (5.996%). The GC-TOFMS profile is depicted in Figure 11, whereas the mass spectra for all 4 peaks detected by the GC-TOFMS are shown in Figure 12.Table 3
Compounds tentatively identified inPersea  declinata (Bl.) Kosterm bark crude extract.
Peak number
RTa
Percentage of the peakb
Molecular weight
Molecular formula
Similarity index
Compound
1
17.025
10.226
204
C15H24
89.1
α-Copaene [15]
2
17.643
21.877
204
C15H24
90.6
Caryophyllene [16]
3
18.082
5.996
204
C15H24
92.2
Iso-α-humulene [17]
4
31.123
61.901
166
C10H14O2
87.3
2-Methoxy-4-propylphenol(4-propylguaiacol) [18]
Total
100.04
aRT: retention time (min).
bPeak area relative to the total peak area percentage.Figure 11
Pegasus HT GC-TOFMS chromatogram data forPersia declinata (Bl.) Kosterm crude methanol bark extract.Mass spectra of the 4 major peaks detected by the GC-TOFMS based on their similarity indexes.
(a)
(b)
(c)
(d)


## 3.1. Effect of PDM on Cell Viability

The cytotoxic effect of PDM was evaluated on HepG2, MDA-MB-231, MCF-7, T47D, H400, H413, BICR31, and WRL-68 cells using MTT assays. Table2 shows the IC50 values after 48 hours of treatment with PDM. Highest cytotoxicity was observed in MCF-7 breast cancer cells (IC50 = 16.68 ± 0.89). Moreover, PDM exhibited higher selectivity on human breast cancer MCF-7 cells, compared to human normal hepatic WRL-68 cells.Table 2
Effect ofPersea  declinata (Bl.) Kosterm bark crude extract on cells expressed as IC50 values in 48 hours MTT assay.
Cell line
IC50 (μg/mL)
Bicr31
27.68
±
2.14
H400
31.39
±
1.85
H413
24.16
±
1.44
MCF7
16.68
±
0.89
T47D
18.53
±
1.26
MDA-MB-231
21.80
±
1.71
HepG2
26.41
±
2.09
HT-29
28.59
±
1.93
WRL-68
97.90
±
5.73Next, real-time cell proliferation assay was carried out to monitor the morphological changes of MCF-7 cells treated with PDM. The results indicated significant reduction of cell number, cell shrinkage, and apoptotic body formation throughout the 24 hours of treatment (Figure1).PDM induced significant reduction of cell number, cell shrinkage, and apoptotic body formation in treated MCF-7 cells. (a) MCF-7 cells were treated with DMSO, doxorubicin, or different concentrations of PDM for 24 h. Live cell images indicating cell shrinkage and apoptosis in a dose-dependent manner. (b) MCF-7 cells were treated with DMSO or PDM (25μg/mL) and were screened for 24 h using a real-time cellular analysis (JuLi Br) system. Real-time cell proliferation throughout the 24 hours of treatment indicating significant reduction of cell number, cell shrinkage, and formation of apoptotic body in the MCF-7 cells treated with PDM.
(a)
(b)


## 3.2. PDM Induced Higher LDH Release in MCF-7 Cells

Lactate dehydrogenase (LDH) release in the medium is a marker that shows the loss of membrane integrity, apoptosis, or necrosis. The cytotoxicity of PDM, as determined by the LDH release assay, was quantified on MCF-7 cells treated with various concentrations of the extract for 24 h. PDM induced a significant elevation in LDH release, demonstrating cytotoxicity at 25 and 50μg/mL concentrations compared to the control cells (Figure 2).Figure 2
The LDH release assay revealed the significant cytotoxicity of PDM on MCF-7 cells at 25 and 50μg/mL concentrations.


## 3.3. Effect of PDM on Cell Cycle

We investigated the cell cycle arrest of MCF-7 cells treated with different concentrations of PDM, using BrdU and Phospho-histone H3 dyes. Comparison of the images and intensity bar charts of PDM-treated and untreated MCF-7 cells indicated a decreased level of BrdU and Phospho-histone H3 intensities (Figures3(a) and 3(b)). The histogram plot on Hoechst total intensity also demonstrated a decreased level of cells in S and G2/M phases and an increased number of cells at G0/G1 in PDM-treated MCF-7 cells (Figure 3(c)). These results show that PDM induced G0/G1 arrest in MCF-7 after 24 hours.(a) Effect of PDM on cell cycle arrest. The MCF-7 cells were treated with DMSO, doxorubicin, or PDM for 24 h, stained with BrdU and Phospho-histone H3, and subjected to the Cellomics ArrayScan HCS reader for cell cycle analysis. (b) Representative bar charts indicating that PDM treatment caused no significant changes in BrdU or Phospho-histone H3 fluorescence intensities, and consequently induced no cell cycle arrest in the treated MCF-7 cells. Data were expressed as the mean ± SD of fluorescence intensity readings for three independent experiments. (c) Cells were stained with Hoechst and the distribution in G0/G1, S, and G2/M cell cycle phases was determined with Cellomics High Content Screening.
(a)
(b)
(c)


## 3.4. PDM Increased Reactive Oxygen Species (ROS) Production

ROS is produced as a byproduct of normal metabolism of oxygen. ROS formation may undergo a drastic increase under environmental or chemical stress. Enhanced levels of ROS may lead to apoptosis or cell cycle arrest. In this study, we stained PDM-treated (24 hours) or untreated MCF-7 live cells with DHE dye to assess whether the exposure of PDM promotes ROS production. As shown in Figure4, the levels of DHE increased significantly in MCF-7 cells treated with PDM, indicating higher ROS production.Effect of PDM on ROS production. (a) Representative images of MCF-7 cells treated with DMEM, doxorubicin, or PDM for 24 h and stained with DHE dye. PDM induced a noteworthy elevation in nuclear ROS in the PDM-treated MCF-7 cells (magnification: 200x). (b) Representative bar charts indicating dose-dependent increased ROS in the nucleus of the PDM-treated MCF-7 cells.
(a)
(b)


## 3.5. PDM Decreased Mitochondrial Membrane Potential (MMP) and Increased Cell Membrane Permeability

As the main source of cellular ROS and adenosine triphosphate (ATP), mitochondria are the key regulators of mechanisms controlling the survival or death of cells. We used mitochondrial membrane potential (MMP) fluorescent probes to examine the function of mitochondria in treated and untreated MCF-7 cells. As shown in Figure5, the untreated cells were strongly stained with MMP dye in comparison to PDM-treated cells. A dose-dependent reduction of MMP fluorescence intensity reflects that the MMP is gradually destroyed in response to higher PDM concentration (Figure 5). On the other hand, a significant increase in cell membrane permeability was also observed in PDM treated cells after 24 hours of treatment (Figure 5).(a) Representative images of MCF-7 cells treated with DMEM, doxorubicin, or PDM for 24 h and stained with Hoechst 33342 for nuclear, membrane permeability, and mitochondrial membrane potential (MMP) dyes. PDM induced a noteworthy elevation in membrane permeability and a marked reduction in MMP (magnification: 200x). (b) Representative bar charts indicating dose-dependent increased cell permeability and reduced MMP in the PDM-treated MCF-7 cells.
(a)
(b)


## 3.6. PDM Induced DNA Fragmentation

Acridine orange (AO) is used to determine DNA integrity. We stained MCF-7 16 cells with acridine orange dye after 24 hours of treatment. Figures6(a) and 6(b) showed a dose-dependent increase of nucleus restricted acridine orange dye in PDM-treated cells, indicating DNA fragmentation.(a) Acridine orange DNA fragmentation analysis. The cells were treated with DMEM or PDM for 24 h and stained with acridine orange dye. (b) Representative bar charts that show dose-dependent increased fluorescent intensity of acridine orange dye inside the nucleus of the PDM-treated MCF-7 cells, indicating DNA fragmentation.
(a)
(b)


## 3.7. PDM Increased Caspase-3/7 Activity

The excessive production of ROS from mitochondria and the collapse of MMP may activate downstream caspase molecules and consequently lead to apoptotic cell death. To examine this, we measured the caspase-3/7 activity using bioluminescent assays. As shown in Figure7, a significant dose-dependent increase in caspase-3/7 activity was detected in PDM-treated cells. Hence, the apoptosis induced by PDM in MCF-7 cells could be mediated through caspase activation.Figure 7
Relative luminescence expression of caspases 3/7 in MCF-7 cells treated with various concentrations of PDM.


## 3.8. Effect of PDM on NF-κB Activation

The nuclear factor kappa B (NF-κB) is a transcription factor, critical for cell proliferation and apoptosis. Activation of NF-κB is indicated by cytoplasm to nuclear translocation to enable DNA-binding activity and facilitate target gene expression. NF-κB remains in the cytoplasm in the absence of activation signal in MCF-7 (Figure 8(a)). In the presence of TNF-α, NF-κB localized mainly in the nucleus of most cells (Figure 8(a)). As shown in Figure 10, PDM treatment has no inhibitory effect on TNF-α-induced NF-κB translocation from cytoplasm to nucleus, compared to positive control curcumin (Figures 8(a) and 8(b)).(a) Photographs of the intracellular targets of stained MCF-7 cells that were treated with DMSO, PDM (25μg/mL), or curcumin (50 μM) for 3 h and then stimulated for 30 min with 1 ng/mL TNF-α (NF-κB activation). (b) Representative bar charts indicating that PDM treatment caused no changes in TNF-α-induced NF-κB nuclear translocation in MCF-7 cells.
(a)
(b)


## 3.9. PDM Modulated Expression of Bcl-2, Bcl-xl, and Bax

Bcl-2 includes a family of proteins that regulate Mitochondrial Outer Membrane Permeabilization (MOMP). They include antiapoptotic molecules such as Bcl-2 and Bcl-xl, which could preserve cell survival and proapoptotic molecules like Bax that inhibit cell survival. To examine if PDM initiated apoptosis by affecting the cellular level of these molecules, Western blot analysis was performed using untreated or extract-treated breast cancer cells. Our data showed that PDM dose-dependently upregulated Bax and downregulated the expression level of Bcl-2 and Bcl-xl in MCF-7 cells (Figure9).Figure 9
Induction of apoptosis by reducing the expression level of prosurvival molecules (Bcl-2, Bcl-xl) and increasing the expression of proapoptotic molecule (Bax). MCF-7 cells were treated with DMSO or different concentrations of PDM for 24 h. Western blot results showing the expression levels of Bcl-2, Bcl-xl, and Bax in PDM or DMSO-treated MCF-7 cells.β-Actin serves as a loading control.Figure 10
MCF-7 cells were treated with DMSO or different concentrations of PDM for 24 h. RNAs were isolated and converted to cDNA. Quantitative real-time PCR was performed to determine expression level of Bcl-2, Bcl-xl, and Bax genes. GAPDH was used as a housekeeping gene.Next, we performed quantitative PCR using a real-time PCR machine to examine whether the expression of these molecules was affected at the transcriptional level. Results indicated a marked increase in Bax expression but a decrease in the expression level of Bcl-2 and Bcl-xl in the PDM-treated MCF-7 cells, consistent with Western blotting data (Figure10).


## 3.10. GC-TOFMS Identification and Chemical Analysis of Crude Extract

To analyse the chemical constituent of PDM, the extract was subjected to GC-TOFMS analysis and the result is presented in Table3. A total of 4 main compounds were detected in PDM. The most abundant compound detected was 2-methoxy-4-propylphenol (4-propylguaiacol) (61.901%), followed by Caryophyllene (21.877%), α-Copaene (10.226%), and Iso-α-humulene (5.996%). The GC-TOFMS profile is depicted in Figure 11, whereas the mass spectra for all 4 peaks detected by the GC-TOFMS are shown in Figure 12.Table 3
Compounds tentatively identified inPersea  declinata (Bl.) Kosterm bark crude extract.
Peak number
RTa
Percentage of the peakb
Molecular weight
Molecular formula
Similarity index
Compound
1
17.025
10.226
204
C15H24
89.1
α-Copaene [15]
2
17.643
21.877
204
C15H24
90.6
Caryophyllene [16]
3
18.082
5.996
204
C15H24
92.2
Iso-α-humulene [17]
4
31.123
61.901
166
C10H14O2
87.3
2-Methoxy-4-propylphenol(4-propylguaiacol) [18]
Total
100.04
aRT: retention time (min).
bPeak area relative to the total peak area percentage.Figure 11
Pegasus HT GC-TOFMS chromatogram data forPersia declinata (Bl.) Kosterm crude methanol bark extract.Mass spectra of the 4 major peaks detected by the GC-TOFMS based on their similarity indexes.
(a)
(b)
(c)
(d)


## 4. Discussion

Fruits and vegetables contain multiple anticancer phytochemicals, which have been extensively explored as a cancer prevention approach. Studies have shown that avocado fruit extracts exhibited antiproliferative effects in human cancer cell lines [23]. By studying the same avocado family, we found that PDM is active on various cancer cells, demonstrating antitumor potential for further development. In fact, PDM exhibited higher selectivity on MCF-7 breast cancer cells and was less cytotoxic towards WRL-68 normal hepatic cells. This is a good indication as some compounds have been shown to have side effects such as causing liver or kidney toxicity [24, 25].Excessive production of ROS, known as oxidative stress, may cause cell death by nonphysiological (necrotic) or regulated pathways (apoptotic). Previous studies indicated that death receptors, including the TNF receptor-1 (TNF-RI), are able to initiate caspase-independent cell death. This form of necrotic cell death appears to be dependent on the generation of ROS, through activation of poly(ADP-ribose) polymerase (PARP) [26, 27]. In this study, we observed a dose-dependent increase in ROS level after PDM treatment. PDM-induced ROS production could affect mitochondria’s function, which has been shown to play an indispensable role in cell survival. The decrease of MMP fluorescent intensity and the increase in cell membrane permeability in PDM-treated cells might be due to excessive generation of ROS.Increased level of ROS and MMP collapse may also activate cysteine proteases (caspases) which converged on caspase 3/7. Caspase 3/7 normally cleaves several target proteins such as PARP (the enzyme responsible for repairing DNA) and leads to apoptotic DNA fragmentation. Using acridine orange dye, DNA fragmentation and cleavage were detected in PDM-treated MCF-7 cells. These results suggest that PDM induced apoptosis via caspase 3/7 activation. Since MCF-7 is a cell line deficient in caspase-3 expression, it is possible that DNA fragmentation could be mediated by activation of caspase-7 and PARP cleavage, as shown previously by another study [28].Analysis of volatile nonpolar constituents of PDM by GC-TOFMS showed 4 major compounds, which exhibit various bioactive evidence. The sesquiterpene hydrocarbonα-copaene has been shown to exhibit antibacterial activities [29–31], whereas, α-humulene was discovered to be cytotoxic against MCF-7 cancer cells [32, 33]. Another study showed that this compound was active against human lung carcinoma A-549 and colon adenocarcinoma DLD-1 cell lines with GI50 values of 62 ± 2 and 71 ± 2 μM, respectively [34]. Legault and Pichette [35] reported that caryophyllene alone was unable to inhibit any of the cell lines tested. However, when combined with paclitaxel (an antitumor agent used clinically to treat breast, ovarian, and lung cancers), caryophyllene could increase growth inhibition by about 91% in DLD-1 cell lines. Another study by Okada et al. [36] demonstrated that 2-methoxy-4-propylphenol induces apoptosis and is a potent inhibitor of lipopolysaccharide (LPS)-induced cyclooxygenase-2 (COX-2) gene expression, in which, the overexpression of the COX-2 gene protects cancer cells from apoptosis.Members of the Bcl-2 family include major cell survival and cell death regulators. Bcl-2 and Bcl-xL act as apoptosis inhibitors in the cells. While Bax functions as a proapoptotic factor and inhibits cell survival. Our data showed that PDM treatment dose-dependently decreased the expression of prosurvival proteins Bcl-2 and Bcl-xl and increased the expression of the proapoptotic molecule, Bax. The importance of Bc1-2 and Bcl-xl for protection of mitochondria during cell death process has been previously studied [37]. Excessive expression of Bax may form Mitochondrial Apoptosis-Induced Channel (MAC) and mediate the release apoptotic factor. In contrast, Bcl-2 has the ability to block apoptosis through inhibition of Bax and/or Bak. The decline in Bcl-2 expression may lead to loss of MMP which may trigger downstream caspase activation [38]. In fact, interaction of Bcl-xL with Apaf1 has a principle role in cell survival through inhibition of Apaf1-dependent caspase-9 activation [39]. Hence, upregulation of Bax and downregulation of Bcl-2 and Bcl-xl molecules by PDM treatment may lead to MMP loss, caspase cascade activation, and subsequent DNA fragmentation.To come to a conclusion, the evidence of LDH release, MMP suppression, elevation in the level of cytochromec, and activation of caspases demonstrated the promising anticancer activity of PDM against MCF-7 human breast cancer cell line via cell cycle arrest and apoptosis induction.



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*Source: 248103-2014-04-07.xml*