University Code:
分 类 号：
Registration No.: 2012393028
密级：秘密
Master Thesis
IDENTIFICATION AND CHARACTERIZATION OF NATURAL
ANTI-BREAST CANCER COMPOUND: COSTUNOLIDE
By
HELDER OLIVEIRA
Supervised by
Dr. XIAOMENG LI
Professor,
Institute of Genetics and Cytology
School of Life Sciences
Northeast Normal University
School of Life Sciences, Northeast Normal University
Changchun, Jilin, P.R. China
APRIL, 2014
学校代码：
分 类 号：
研究生学号：2012393028
密级：秘密
Master Thesis
IDENTIFICATION AND CHARACTERIZATION OF NATURAL
ANTI-BREAST CANCER COMPOUND: COSTUNOLIDE
作者：HELDER
指导教师：Dr.
OLIVEIRA
Xiaomeng Li，Professor
学科专业：Biochemistry and Molecular Biology
研究方向：Cancer Biology
学位类型：Master’s Degree
东北师范大学学位评定委员会 2014年4月
2
CERTIFICATE
This is to certify that Mr. HELDER
OLIVEIRA have carried out research work
embodied in this thesis entitled “Identification
and Characterization of
Natural anti-Breast Cancer Compound: Costunolide”
in The Key
Laboratory of Molecular Epigenetic of MOE, Institute of Genetics and Cytology, School of Life
Sciences, Northeast Normal University, Changchun, Jilin, China. The work presented in this
thesis is creative and distinctive. His exposition is admirable of presentation to School of Life
Sciences, Northeast Normal University, Changchun, Jilin, China for the award of degree of
Master of Science in Biochemistry and Molecular biology.
Supervisor
Dr. Xiaomeng Li,
Professor,
The Key Laboratory of Molecular Epigenetic of MOE,
Institute of Genetics and Cytology,
School of Life Sciences,
Northeast Normal University,
Changchun, Jilin, China
3
ABSTRACT
Breast cancer is the most common cancer among women, 23% (1.3 million) of the total
of new cases and the second leading cause of cancer death in women exceeded only by lung
cancer. Natural medicines have been proven to be a central source of narrative agents with a
pharmaceutical potential. Costunolide is sesquiterpene lactones consisting of diverse plant
chemicals that exhibit anti cancer action through cytotoxic effects on various cancer cells. The
objectives of present study were to explore the effects of natural compounds on the proliferation
of MCF-7 cells and to determine the role of ROS in natural compounds-induced apoptosis in breast
cancer cells with a therapeutic potential. Results showed that costunolide screened, possess potent
anticancer properties against breast cancer MCF-7 cells, Costunolide was observed as strong
anti-proliferative agent with IC50 = 50µM. The anti-proliferative effect of costunolide on MCF
cells was confirmed by live/dead assay using fluorescent probes calcein AV/PI. The results
demonstrated that treatment of cells with costunolide decreased the viability of MCF-7 cells in a
dose-dependent manner.
To determine the costunolide-induced apoptosis, flow cytometric analysis was carried
out. The results showed that costunolide induced apoptosis in a dose-dependent manner in breast
cancer MCF-7cells. ROS are well known mediators of intracellular signaling of cascades. The
excessive generation of ROS can induce oxidative stress, loss of cell functioning, and apoptosis.
In the present study, we assumed that costunolide might arouse ROS level, which could be
involved in induction of apoptosis. Therefore, the intracellular ROS level was measured using
the ROS-detecting fluorescence dye 2, 7-dichlorofluorescein diacetate (DCF-DA). Interestingly
4
these effects were significantly abrogated when the cells were pretreated with N-acetyl- cysteine
(NAC), a specific ROS inhibitor.
Costunolide induces apoptosis through extrinsic pathway in MCF-7 breast cancer cells, In
order to examine whether costunolide suppresses cell growth inducing apoptotic cell death, we
analyzed DNA contents and apoptosis-related proteins expression level by flow cytometry and
western blot, respectively in MCF-7 breast cancer cells we investigated whether costunolide
activates extrinsic apoptotic pathway. We examined the expression levels of death receptor
signaling-related proteins, caspase-3, and PARP.
The results showed that procaspase-3 was cleaved to yield 17 and 20kDa fragments and
activation of PARP in treated cells with 25 and 50μM of costunolide. Costunolide induce
apoptosis through intrinsic mitochondria pathway in MCF-7 breast cancer Cells. We examined
the expression levels of mitochondrial apoptotic pathway related proteins such as anti-apoptotic
protein, B-cell lymphoma protein-2 (Bcl2), and pro-apoptotic protein Bax. Costunolide involved
in the down regulation of Bcl-2 and up regulation of Bax. These results suggest that costunolide
may have beneficial effects for the reduction of breast cancer growth, and new therapeutic
strategy for the treatment of human cancers.
Keywords: Breast cancer, costunolide, apoptosis, reactive oxygen species.
5
乳腺癌是女性最常见的恶性肿瘤之一，在新增癌症病例中占23%。乳腺癌在女性癌症
患者中的致死率位居第二位，仅次于肺癌。天然化合物与合成化合物相比具有低毒的特点
，是许多疾病的治疗药物生产的最初来源。木香烯内酯是一种倍半萜内酯，由多种植物化
学成分组成，通过细胞毒素作用对多种癌细胞发挥抗癌效应；但木香烯内酯在乳腺癌治疗
中的可能作用尚不明确。本研究旨在探索天然化合物对MCF7细胞增殖的影响，研究ROS
在由天然化合物诱导的乳腺癌细胞凋亡中潜在的治疗作用。
我们首先检测了木香烯内酯对乳腺癌MCF7细胞增殖的影响，并证明其具有高效的抗
癌能力。我们发现，木香烯内酯的IC50值为50µM，是强有力的细胞增殖抑制剂。利用荧
光探针钙绿化素AV/PI活体实验，我们发现木香烯内酯对MCF细胞的增殖抑制和凋亡诱导
效应，降低了MCF-7细胞在剂量依赖下的生存能力。
为了研究木香烯内酯诱导的细胞凋亡，我们采用了流式细胞检测技术。结果显示，在
剂量依赖下，木香烯内酯诱导乳腺癌MCF7细胞凋亡。由于ROS的过度表达会诱导氧化压力，导致细胞功能丧失和细胞凋亡。因此
，我们利用荧光染色的ROS追踪素－双氯荧光乙酸乙酯来检测细胞内ROS水平，并发现木
香烯内酯显著提高ROS水平。有趣的是，细胞被一种特殊的ROS抑制剂——
乙酰半胱氨酸预处理之后，ROS介导的细胞凋亡效应被消除。所以在本研究中，我们证明
木香烯内酯通过提高ROS水平，进而诱导细胞凋亡。
6
然后我们研究了木香烯内酯作为外源诱导剂对乳腺癌MCF7细胞凋亡的影响。通过流式细
胞检测技术和蛋白质免疫印记分析了DNA成分和细胞凋亡相关的蛋白质表达水平。在乳
腺癌MCF7细胞中，我们检测了木香烯内酯对凋亡外源途径（死亡受体途径）激活及对凋
亡效应蛋白质Caspase-3和PARP的活化作用。结果显示,
在25uM和50uM木香烯内酯处理的细胞中，蛋白质Caspase-3分裂为一个17kDa
和一个20kDa的片段，蛋白质PARP也被活化剪切。在此基础上，我们进一步检测了木香
烯内酯乳腺癌MCF7细胞凋亡过程中对凋亡内源途径（线粒体途径）的激活效应。我们检
测了线粒体凋亡途径中抗凋亡蛋白质BcL2和促凋亡蛋白Bax的表达水平，并证明木香烯内
酯可下调BcL2蛋白表达和上调Bax蛋白表达，从而证明木香烯内酯通过活化两种凋亡途径
从而诱导乳腺癌MCF7细胞产生凋亡。我们的研究结果表明，木香烯内酯对乳腺癌细胞具
有增殖抑制和凋亡促进效应，为人类癌症治疗提供了一种可能的新策略。
关键词: 乳腺癌，木香烯内酯，细胞凋亡，活性氧
7
Contents
ABSTRACT .................................................................................................................................... 4
摘 要...............................................................................................Error! Bookmark not defined.
CHAPTER I: LITERATURE REVIEW .................................................................................. 10
1.1 Introduction ......................................................................................................................... 10
1.2 Breast cancer ....................................................................................................................... 11
1.3 Types of breast cancer ......................................................................................................... 13
1.4 Epidiliology and risk factors ............................................................................................... 15
1.5 Apoptosis............................................................................................................................. 16
1.6 Reactive oxygen species...................................................................................................... 18
1.7 Cell cycle progression ............................................................................................................. 19
1.8 Mitochondria menbrane potential (ΔΨm) ........................................................................... 21
1.9 Western Bloting................................................................................................................... 23
1.10 Hypothesis of research work ............................................................................................. 25
1.11 Objectives of present study ............................................................................................... 25
CHAPTER II: NATURAL COMPOUNDS CHEMOPREVENTIVE AGENTS ................. 26
2.1 Medicinal Plants .................................................................................................................. 26
2.2 Alkaloids exert anti-proliferative activity on cancer cells .................................................. 27
2.3 Costunolide.......................................................................................................................... 29
2.4 Molecular targets of costunolide ......................................................................................... 30
CHAPTER III: MATERIAL AND METHODS ..................................................................... 32
3.1 Chemicals and Reagents...................................................................................................... 32
3.2 Cell Culture ......................................................................................................................... 32
8
3.3 Cell Proliferation Assay (MTT) .......................................................................................... 32
3.4 Flow Cytometric Analysis of Cell Cycle ............................................................................ 33
3.5 Flow Cytometric Determination of Apoptosis .................................................................... 33
3.6 Flow Cytometric Determination of Reactive Oxygen Species (ROS) ................................ 34
3.7 Flow Cytometric Determination of Mitochondrial Membrane Potential (ΔΨm) ............... 34
3.8 Western Blotting ................................................................................................................. 34
3.9 Statistical Analysis of Data ................................................................................................. 35
CHAPTER IV: RESULTS AND DISCUSSION ...................................................................... 36
4.1 Anti-proliferative effects of Costunolide on breast cancer cells ......................................... 36
4.2 Costunolide Induced G2/M Cell Cycle Arrest on breast cancer MCF7 Cells .................... 37
4.3 Costunolide Induced Apoptotic Cell Death in Breast Cancer MCF7 Cells ........................ 38
4.4 Costunolide Increased Generation of ROS in MCF7 Cells................................................. 40
4.5 Costunolide Decreased Mitochondrial Membrane Potential in MCF7 cells....................... 41
4.6 Costunolide Regulated Apoptosis-Related Proteins in MCF-7 Cells ................................. 42
4.7 CONCLUSION ................................................................................................................... 44
4.8 Novel findings and significance .......................................................................................... 44
5.3 List of abreviations .............................................................................................................. 46
References ................................................................................................................................. 47
ACKNOWLEDGEMENTS .......................................................................................................... 66
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Chapter I: Literature review
1.1 Introduction
Trillions of living cells formed in the human body grow, divide and die in an orderly
fashion. Normal people during the early years, cells divide faster to allow the person to grow.
When the person becomes an adult, most cells divide only to replace worn-out or dying cells or
to repair injuries. Cancer begins when cells in a part of the body start to grow out of control.
There are many kinds of cancer, but they all start because of out-of-control growth of abnormal
cells [1].
Cancer comprises at least 100 different diseases, and each is classified by the type of cell
that is initially affected. Breast cancer is a type of cancer originating from breast tissue, most
commonly from the inner lining of milk ducts or the lobules that supply the ducts with milk.
Cancers originating from ducts are known as ductal carcinomas, while those originating from
lobules are known as lobular carcinomas. Breast cancer occurs in humans and other mammals
[3].remainder of the breast is made up of fatty, connective, and lymphatic tissue. Most masses
are benign; that is, they are not cancerous, do not grow uncontrollably or spread, and are not lifethreatening [2].
Plants have been long since use in the treatment of cancer and there are higher than 3000
plant species that have served in the treatment of cancer [4]. Traditional Chinese medicine and a
practice that already takes 500 years of tradition, and still maintains an important attention in
primary health in rural areas of China and is also very apparent in urban areas, nowadays the
Chinese government has made enormous effort to modernize this practice investing capital in
scientific research and economic exploitation of the same. In the Western world, interest in TCM
10
is born of the hope that it might complement Western medicine. The history, philosophy, theory
and practice of TCM have been recently reviewed [5].
Regulation of apoptosis and cell cycle is an important process to preserve cell
homeostasis between cell death and cell proliferation [6], which means that the induction of
apoptosis and suppression of cell cycle is only an advantageous strategy for cancer therapy. In
addition, the maximum effects and minimize side effects at low concentrations of drugs represent
efficiency for cancer therapy.
The purpose of this study was to evaluate the effect of costunolide on cell growth, cell
cycle progression and induction of ROS-mediated apoptosis in MCF-7 breast cancer cells.
1.2 Breast cancer
Breast cancer is a malignant tumor that is initiated in the cells of the breast. A malignant
tumor is a collection of cancer cells that can grow into (invade) surrounding tissues or spread
(metastasize) to distant areas of the body. The disease occurs almost entirely in women, but men
can get it too [8].
Among females the most frequently diagnosed cancer and the leading of cancer death is
breast cancer, 23% (1.3million) of the total new cases and14% (458,400) of total cancer deaths in
2008 worldwide is second only to lung cancer. About half the breast cancer cases and 60% of the
deaths are estimated to occur in economically developing countries. In general, incidence rates
are high in Western and Northern Europe, Australia/New Zealand, and North America;
intermediate in South America, the Caribbean, and Northern Africa; and low in sub-Saharan
Africa and Asia figure 1 [2].
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Figure 1: Incidence rates for total cancers by gender and country. Source: GLOBOCAN
2008
The female breast is made up mainly of lobules (milk-producing glands), ducts (tiny
tubes that carry the milk from the lobules to the nipple), and stroma (fatty tissue and connective
tissue surrounding the ducts and lobules (figure 2), blood vessels, and lymphatic vessels). Most
breast cancers start in the cells that line the ducts (ductal cancers). Some begin in cells that line
the lobules (lobular cancers), while a small number start in other tissues [8,1].
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1.3 Types of breast cancer
The histological classification of breast cancer includes adenocarcinoma, cancers that
originate in the glandular tissue, which include the ducts and lobules and sarcomas, cancers that
originate in the connective tissue of muscle, fat or blood vessels. Carcinoma in situ (CIS) is an
early stage form of cancer where the tumor is confined to the layer of the cells where the cancer
began and it has not invaded deeper breast tissue or spread to other areas of the body [9]. There
are several types of breast cancer, but some of them are quite rare. In some cases a single breast
tumor can be a combination of these types or be a mixture of invasive and in situ cancer [10].
Ductal carcinoma in situ (DCIS) is the most common type of non-invasive cancer in
women, where cancer cells have not spread beyond the duct walls into surrounding breast tissue.
The prevalence of DCIS is strongly correlated with mammographic screening and in countries
such as the US, can be as high as 18% of all newly diagnosed [11] but in countries such as India,
represents a very low proportion of total disease since most cases present in late stage [12].
Invasive or infiltrating ductal carcinoma originates in the breast duct, has broken
through the wall of the duct into surrounding fatty tissue of the breast and is capable of
metastasizing to other organs of the body through the lymphatic system and bloodstream. This
represents about 80% of breast cancers [13].
Lobular carcinoma in situ (LCIS) is not cancer but is sometimes classified as a noninvasive breast cancer and women who have this condition are more likely to develop invasive
breast cancer in the future [14].
13
Invasive or infiltrating lobular carcinoma originates in the milk-producing glands or
lobules of the breast and can spread to other parts of the body. This is less common and
represents about 1 in 10 breast cancer diagnoses [15].
Other (less common) types of breast cancer: inflammatory breast cancer (1-3% of all
breast cancers), triple negative breast cancers, mixed tumors, medullary carcinoma (3-5% of all
breast cancers), metaplastic carcinoma, mucinous carcinoma, paget disease of the nipple, tubular
carcinoma, papillary carcinoma, adenoid cystic carcinoma (adenocystic carcinoma), phyllodes
tumor and angiosarcoma [14,15]. Estrogen receptor (ER) and Progesterone receptor (PR) status
Confirmed carcinomas of the breast are subjected to a test to determine the estrogen receptor
(ER) and progesterone receptor (PR) status. Breast cancers that contain estrogen receptors on the
outside surface of their cells are ER-positive cancers while those with progesterone receptors are
called PR-positive cancers. Women who are positive for either or both receptors generally have a
better prognosis because they are more responsive to hormone therapy than without any of these
receptors [14].
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Figure 2: Normal breast with non–invasive ductal carcinoma in situ (DCIS) in an enlarged
cross–section of the duct. A = ducts, B = lobules, C = dilated section of duct to hold milk, D =
nipple, E = fat, F = pectoralis major muscle, G = chest wall/rib cage.
Enlargement: A = normal duct cells, B = basement membrane, C = lumen (center of duct)
1.4 Epidiliology and risk factors
The factors that contribute to the international variation in incidence rates largely stem
from differences in reproductive and hormonal factors and the availability of early detection
services [7]. Reproductive factors that increase risk include a long menstrual history, nulliparity,
recent use of postmenopausal hormone therapy or oral contraceptives, and late age at first birth
[16]. Alcohol consumption also increases the risk of breast cancer [17,18]. The breast cancer
incidence increases observed in many Western countries in the late 1980s and 1990sb likely
result from changes in reproductive factors (including the increased use of postmenopausal
hormone therapy) as well as an increased screening intensity [19]. Incidence rates in some of
these countries, including the United States, United Kingdom, France, and Australia, sharply
decreased from the beginning of the millennium, partly due to lower use of combined
postmenopausal hormone therapy [20,21].
In contrast, breast cancer death rates have been decreasing in North America and several
European countries over the past 25 years, largely as a result of early detection through
mammography and improved treatment [7,22,23]. In many African and Asian countries
however, including Uganda, South Korea, and India, incidence and mortality rates have been
rising [24;25], with changes in reproductive patterns, physical inactivity, and obesity being the
main contributory factors [7,26,27]; increases in breast cancer awareness and screening activity
may be partially responsible for the rising incidence in these populations. Maintaining a healthy
body weight, increasing physical activity, and minimizing alcohol intake are the best available
strategies to reduce the risk of developing breast cancer [28]. Early detection through
mammography has been shown to increase treatment options and save lives, although this
approach is cost prohibitive and not feasible in most economically developing countries [29].
Recommended early detection strategies in these countries include the promotion of awareness
of early signs and symptoms and screening by clinical breast examination [30].
15
1.5 Apoptosis
Extreme and synchronized way of cell death is defined by apoptosis. It is characterized
by distinct morphological features, including chromatin condensation and nuclear fragmentation
[31, 32]. The importance of signaling has been recognized in cell regulation during normal and
disease [33, 34]. Cancer, a complex genetic disease resulting from mutation of oncogenes or
tumor suppressor genes, can be developed due to alteration of signaling pathways; it has been
well known to have numerous links to PCD [35]. Programmed cell death (PCD) is regulated by
proteolytic enzymes called caspases; Bcl-2 and IAP protein families. Caspases activates
apoptosis by cleaving precise protein in cytoplasm and nucleus. In cells, caspases exist as
inactive precursors or pro-caspases, which are generally stimulated by cleavage by other
caspases, generating proteolytic cascade of caspases [36]. Bcl-2 and IAP protein families are
involved in activation of caspases. In the regulation of apoptosis and growth arrest p53, the
tumor suppressor protein, plays a critical role [37].
The process of apoptosis is activated by two major pathways: the so-called, receptormediated extrinsic and mitochondria-mediated intrinsic pathway [38]. The extrinsic apoptotic
pathway is induced by the binding of extracellular ligands (for example, FasL) to transmembrane
death receptors (for example, Fas) on the cell surface leading to activation of caspase-8 [39]. On
the other hand, the intrinsic apoptotic pathway is induced by changing the permeability of the
outer mitochondrial membrane, reducing mitochondrial membrane potential (Δψm), and
releasing mitochondrial pro-apoptotic factors including cytochrome c and Apaf-1 into the
cytoplasm leading to activation of caspase-9 [40,41]. Caspase-8 of extrinsic pathway and
caspase-9 of intrinsic pathway activate caspase-3 and cleave poly(ADP-ribose) polymerase
(PARP), thus resulting in apoptosis [42].
16
Inhibition of cell cycle progression of cancer cells is an important step in cancer therapy
because abnormal cell proliferation is observed in cancer cells [43]. Cell proliferation is
regulated by multiple checkpoints in cell cycle progression. The transition between G1-S and
G2-M phases checkpoints is activated after DNA damage in the mammalian cells leading to cell
cycle arrest and is regulated by cyclins, cylin-dependent kinase (CDKs), and CDK inhibitors
(CKIs) [44,45].
Figure
1:
Schematic representation of the main molecular pathways leading to apoptosis.
In the extrinsic pathway upon ligand binding to specific receptors the DISC complex is
formed and caspase-8 activated. In the intrinsic pathway release of cyt-c from the
mitochondria result in the formation of the apoptosome and activation of caspases-9. Caspase-8
and -9 then activate downstream caspases such as caspases-3 resulting in cell death. The two
pathways are connected via the cleavage of the BH3 only protein BID.
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1.6 Reactive oxygen species
Oxidative stress and cell-cycle regulation are two essential elements in the apoptosis
process. Reactive oxygen species are emerging as critical signaling molecules [46-47]. The term
reactive oxygen species (ROS) encompasses a wide range of molecules. Free radicals are
chemical species containing one or more unpaired electrons. Examples include the hydrogen
atom, with one unpaired electron, most transition metal ions, nitric oxide, and oxygen, which
have two unpaired electrons [46]. The unpaired electrons of oxygen react to form partially
reduced highly reactive species that are classified as ROS, including superoxide (O2 -), hydrogen
peroxide (H2O2), hydroxyl radical, and peroxynitrite. Various enzyme systems produce ROS,
including the mitochondrial electron transport chain, cytochrome P450, lipoxygenase,
cyclooxygenase, the NADPH oxidase complex, xanthine oxidase, and peroxisomes [48].
Mitochondrial oxygen metabolism is the dominant source of O2- that results from
incomplete coupling of electrons and H+ with oxygen in the electron transport chain. Under
normoxic conditions, ROS are maintained within narrow boundaries by scavenging systems, as
would be expected where fluxes of such species are involved in cell signaling [49, 50]. Redox
balance, the ratio between oxidizing and reducing species within the cell, plays a significant role
in the regulation of signaling pathways, including kinase and phosphatase activity and gene
expression through modulation of transcription factor function [51,52].
Redox balance is
achieved by various enzyme systems that neutralize toxic oxidants, such as ROS. Superoxide
dismutases (SOD) catalyze the conversion of O2- to H2O2, which can then be converted to water
by catalase or glutathione (GSH) peroxidase coupled with glutathione reductase. Other relevant
scavengers include thioredoxin coupled with thioredoxin reductase, and glutaredoxin, which uses
GSH as a substrate. GSH plays a central role in maintaining redox homeostasis, and the GSH to
18
oxidized glutathione ratio provides an estimate of cellular redox buffering capacity [53].
Apoptosis may be triggered by oxidative insults [54]. Reactive oxygen species (ROS) are
important chemical messengers in normal cells. They keep the balance with antioxidants in
healthy cells [55]. When the accumulation of ROS is more than what defense systems can
accommodate, ROS can induce damage to DNA, protein and lipids [56].
Overproduction of ROS results in oxidative stress, which mostly results in cell apoptosis
[57]. In addition, cell-cycle arrest is tightly associated with apoptosis. Because cancer involves
deregulated cell proliferation and survival, inducing cell-cycle arrest is an available treatment to
forestall continued tumor proliferation [58]. Cell-cycle regulation and apoptosis share some
regulation proteins [59]. Many chemical compounds trigger apoptosis in tumor cells, with
accompanying cell-cycle arrest [60, 61]. This schematic depicts the varied caspase
pathways involved in apoptosis. The intrinsic or mitochondrial-mediating pathway involves loss
of mitochondrial membrane potential and cytochrome c release leading to activation of caspase
9 followed by downstream effecter caspase-3 activation and resultant cell death. The extrinsic
pathway involves stimulation of pathways such as Fas (CD95) leading to activation of upstream
caspase-8 with resultant stimulation of effecter caspases. Reactive oxygen species (ROS) may
act as an extracellular intermediate directly stimulating the mitochondria and/or Fas cell
death pathways [62].
1.7 Cell cycle progression
Animal development from a single-cell zygote to fertile adult requires many rounds of
cell division. During each division, cells complete an ordered series of events that collectively
make up the "cell cycle". This cycle includes two consecutive processes, mainly characterized by
19
DNA replication and segregation of replicated chromosomes into two separate cells. Originally,
cell division was divided into two stages: mitosis (M), i.e. the process of nuclear division; and
interphase, the interlude between two M phases. Stages of mitosis include prophase, metaphase,
anaphase and telophase. Interphase includes G1, S and G2 phases [63].
Replication of DNA occurs in a restricted part of the interphase called S phase. S phase is
preceded by a gap called G1 during which the cell is preparing for DNA synthesis and is
followed by a gap called G2 during which the cell is prepared for mitosis. Cells in G1 can, before
commitment to DNA replication, enter a resting state called G0. Cells in G0 account for the
major part of the non-growing, non-proliferating cells in the human body [64].
Figure 5: The stages of the cell cycle progression. Different proteins regulate transition from
one cell cycle phase to another in an orderly way. Cyclin-dependent kinases (CDK), a family of
serine/threonine protein kinases that are activated at specific points of the cell cycle, require
association with a cyclin subunit for their activation. Cyclin D1, cyclin D2, cyclin D3 is required
to CDK4 and to CDK6 and CDK-cyclin D complexes are essential for entry in G1 phase ,
Another G1 cyclin is cyclin E, Cyclin A binds with CDK2 and this complex is required during
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S phase, In late G2 and early M, cyclin A complexes with CDK1 to promote entry into M.
Mitosis is further regulated by cyclin B in complex with CDK1, CDK7-cyclin H complex, also
called CAK, all cell cycle phases.
In cancer, there are fundamental alterations in the genetic control of cell division,
resulting in an unrestrained cell proliferation. Mutations mainly take place in two classes of
genes: proto-oncogenes and tumor suppressor genes. In normal cells, the products of protooncogenes to act at different levels along the pathways that stimulate cell proliferation. Mutated
versions of proto-oncogenes or oncogenes can promote tumor growth. Inactivation of tumor
suppressor genes like pRb and p53 results in dysfunction of proteins that normally inhibit cell
cycle progression. Cell cycle deregulation associated with cancer occurs through mutation of
proteins important at different levels of the cell cycle. In cancer, mutations have been observed
in genes encoding CDK, cyclins, CDK-activating enzymes, CKI, CDK substrates, and
checkpoint proteins [86,87].
1.8 Mitochondria menbrane potential (ΔΨm)
Mitochondria have been described to play a key role and perhaps even a central role in
the apoptotic process due in part to the mitochondria being the ‘junction’ of at least two distinct
signaling pathways. Mitochondrial control of apoptosis has been described at several levels: (1)
maintenance of ATP production and (2) mitochondrial membrane potential and mitochondrial
membrane permeability for the release of certain apoptogenic factors from the intermembrane
space into the cytosol [88]. Mitochondria are very specialized organelles containing an outer
membrane (OM) separated from an inner membrane (IM) by an intermembrane space (IMS)
containing many proteins implicated in cell death induction following their release from
21
mitochondria. These IMS proteins include caspase-independent death effectors such as nucleases
and/or proteases, as well as caspase activators.
In addition, certain pro-apoptotic proteins seem to be sequestered in the mitochondrial
cristae [89], the pleiomorphic involutions of the IM that increase the surface area for the electron
transport chain. Cytochrome c, the first molecule shown to be released from mitochondria in the
induction of apoptosis, normally functions in energy production but, upon release from
mitochondria, complexes with apoptosis protease-activating factor 1 (Apaf-1), dATP, and
procaspase-9 to form the apoptosome [90,91].
Figure 6: Schematic representation of apoptogenic proteins from the mitochondrial inter-
membrane space mediating the mitochondrial death decision. Many cell death stimuli converge
at mitochondrial level to induce mitochondrial outer membrane permeabilization (MOMP) andsubsequent
release of pro-apoptotic proteins including AIF, cytochrome c, Endo G, Omi/HtrA2, and Smac/DIABLO.
However, the execution of apoptosis mainly involves activation of a distinct cascade of
cysteine-aspartic acid proteases (caspases) that are present as inactive zymogens, causing
22
selective degradation of cell components, and the activation of further caspases. Cell surface
death receptor (such as FasR, TRAIL, TNFα7,8) activation typically involves the cleavage and
activation of caspase-8 and/or -10, and the subsequent activation of effectors caspases like
caspase-3, which is the central caspase responsible for the proteolytic cascade leading to cell
death. The caspase cascade may induce the activation of more than just other caspases. Caspase3 can also be activated via a feedback loop involving the mitochondria, amplifying the caspase
signal, but how this occurs remains to be elucidated. Besides a direct proteolytic action on the
mitochondria, one possible mechanism to account for the mitochondrial amplification/activation
of caspases involves apoptotic signaling through Bcl-2 superfamily members: Bax, Bak and Bid,
or via changes in calcium homeostasis [92,93,94].
Instead of directly activating caspases, Bax, Bak and Bid induce caspase signaling
through an as yet unidentified mechanism involving the mitochondrial release of several proapoptotic proteins, namely cytochrome c, Smac/Diablo [95,96]. Cytochrome c released during
apoptosis binds to the adaptor molecule Apaf-1, and leads to the activation of caspase-9 through
the formation of the multimeric apoptosome complex [97,98]. Caspase-9 then activates the
central caspase in the apoptotic signaling machinery, caspase-3. Once caspase-3 is in action,
apoptotic cell death is inevitable [99].
1.9 Western Bloting
The term "blotting" refers to the transfer of biological samples from a gel to a membrane
and their subsequent detection on the surface of the membrane. Western blotting (also called
immunoblotting because an antibody is used to specifically detect its antigen) was introduced by
Towbin, et al. in 1979 and is now a routine technique for protein analysis. The specificity of the
antibody-antigen interaction enables a target protein to be identified in the midst of a complex
23
protein mixture. Western blotting can produce qualitative and semi-quantitative data about that
protein.[100].
Gel electrophoresis is a technique in which charged molecules, such as protein or DNA,
are separated according to physical properties as they are forced through a gel by an electrical
current. Proteins are commonly separated using polyacrylamide gel electrophoresis (PAGE) to
characterize individual proteins in a complex sample or to examine multiple proteins within a
single sample. When combined with Western blotting, PAGE is a powerful analytical tool
providing information on the mass, charge, purity or presence of a protein. Several forms of
PAGE exist and can provide different types of information about the protein [101].
Following electrophoresis, the protein must be transferred from the electrophoresis gel to
a membrane. There are a variety of methods that have been used for this process, including
diffusion transfer, capillary transfer, heat-accelerated convectional transfer, vacuum blotting
transfer and electro-elution. The transfer method that is most commonly used for proteins is
electro-elution or electrophoresis transfer because of its speed and transfer efficiency [102].
Therefore, after the transfer of the proteins from the gel, it is important to block the remaining
surface of the membrane to prevent nonspecific binding of the detection antibodies during
subsequent steps. A variety of blocking buffers ranging from milk or normal serum to highly
purified proteins have been used to block free sites on a membrane. The blocking buffer should
improve the sensitivity of the assay by reducing background interference and improving the
signal to noise ratio [103].
Western blotting consists of a series of incubations with different immunochemical
reagents separated by wash steps to remove unbound reagents and reduce background, thereby
increasing the signal: noise ratio. Occasionally, wash buffer formulations consist of only a
24
physiological buffer such as Tris buffered saline (TBS) or phosphate buffered saline (PBS)
without any additives [104].
In general, the primary antibody which recognizes the target protein in a Western blot is
not directly detectable. Therefore, tagged secondary antibodies or other detection reagents are
used as the means of ultimately detecting the target antigen (indirect detection). A wide variety
of labeled secondary detection reagents can be used for Western blot detection. While there are
many different tags that can be conjugated to a secondary or primary antibody, the detection
method used will limit the choice of what can be used in a Western blotting assay [105].
1.10 Hypothesis of research work
To determine the role of ROS in natural compounds- costunolide induced apoptosis in breast
cancer cells with a therapeutic potential.
1.11 Objectives of present study

To screen natural compound from Chinese Traditional Medicine Herbs (CTMH)
library against breast cancer MCF7 cells.

To explore the effects of natural compounds on the proliferation of breast cancer
MCF-7 cells.

To find the novel anti-cancer agents for treatment of breast cancer through screening
of Chinese herbal compounds.
25
CHAPTER II: NATURAL COMPOUNDS CHEMOPREVENTIVE
AGENTS
2.1 Medicinal Plants
Drug resistance is a multi-factorial phenomenon; it involves the expression of defense
factors and, or detoxification mechanisms, in addition, it also alters drug target interactions and
cellular responses to specific cytotoxic lesions [106]. Recently the attention in medicinal plants
and their biologically dynamic derivatives has augmented in relation to the possible development
of narrative potential drugs for several pathologies of relevant social impact [107]. Indeed, it is
well known that medicinal plants are reported and used in prenatal care, gynecology, obstetrics,
skin disorders, respiratory disorders, cardiac diseases, muscular disorders, nervous and mental
health [108, 109]. The promising applications of medicines with respect to potential anti-tumor
activity for cancer prevention have been recently described [110].
All over the history of realm, as a primary source of medicine the humans have relied on
natural products. Herbal medicines have been proven to be a central source of narrative agents
with a pharmaceutical potential. The history of traditional Chinese medicine goes back to 2000
to 3000 years. Dating from the Sui Dynasty (610 AD), oral pseudo membranous candidacies was
recorded in infants in General Treatise on the Causes and Symptoms of Diseases, written by
Yuan-Fang Chao, who named the condition thrush [111].
Traditionally natural products provided a prosperous source of drugs for many diseases
including cancer, and plants are vital source of novel natural products [112]. In exploring new
anti-cancer drugs plants and plants-constitutes have always played a vital role [106]. Many
anticancer drugs in use are either natural products or derivates of natural products. Until now,
among all approved anticancer drugs, about 73% drugs are either products derived from natural
26
sources or developed based on knowledge prevailed from natural products [43]. Herbal
medicines, such as paclitaxel, vinca alkaloids, amptothecin, and etoposide hold great potential as
hopeful agents for the treatment of cancer [113].
In 2008, 225 anticancer drugs have been developed among these drugs 164 were from
natural sources, and 108 were derived from plants, 24 from animal sources, 25 from bacterial
sources and 7 were from fungal sources. Among 108 plant-based drugs, 46 were in preclinical
development, in phase I, 41 in phase II, 5 in phase III and 2 had already reached preregistration
stage Among 155 FDA-approved small molecule anti-cancer drugs, 47% were either natural
products or directly derived from natural [114].
2.2 Alkaloids exert anti-proliferative activity on cancer cells
Alkaloids are an extremely diverse group of compounds containing a ring structure and
nitrogen atom located inside the heterocyclic ring structure. Different categories of alkaloids
have been classified on the basis of their biosynthetic pathways [115]. Alkaloids have been
reported to exist in a wide distribution of higher plants, such as Ranunculaceae, Leguminosae,
Papaveraceae, Menispermaceae, and Loganiaceae kingdoms [115]. Furthermore, several
alkaloids demonstrate considerable biological activities, for instance the relieving action of
ephedrine for asthma, the analgesic action of morphine, and the anticancer effects of vinblastine
[115-116].
Indeed, alkaloids are the most dynamic components in natural herbs, and few of these
compounds have already been effectively developed into chemotherapeutic drugs [116,117]. The
alkaloids derived from natural sources for instance evodiamine; berberine, piperine, matrine,
sanguinarine, and tetrandrine have comparatively more anticancer studies [118]. Berberine an
27
isoquinoline alkaloid is an extensively prescribed Chinese herb, and is broadly distributed in
natural herbs, including Rhizoma Coptidis [119]. Berberine possesses an extensive array of
bioactivities, like anti-inflammatory, anti-diabetes, sedation, expansion of blood vessels,
hepatoprotective, anti-bacterial, anti-ulcer, protection of myocardial ischemia-reperfusion injury,
inhibition of platelet aggregation and neuroprotective effects [120-121].
Matrine, a major alkaloid found in several Sophora plants including Sophora flavescens
Ait. [122], exhibits a broad range of pharmacological properties such as antiviral, anti-bacterial,
anti-asthmatic, anti-inflammatory, anti-obesity, anti-arrhythmic, diuretic, choleretic, anticancer,
hepatoprotective, cardioprotective, and nephroprotective effects [123-124]. Piperine a piperidine
alkaloid found in famous spices that have been used for centuries, it is extracted from Piper
nigrum and Piper longum [125]. Piperine reveals anti-oxidant, anti-diarrheal, anti-convulsant,
anti-inflammatory, hypolipidemic, anti-mutagenic, endorsing bile secretion, and tumor inhibitory
activities [126, 127]. Sanguinarine a benzophenanthridine alkaloid extracted from the
Papaveracea family including Sanguinaria canadensis L. and Chelidonium majus L. [128, 129],
possesses antibacterial, anti-schistosomal, antifungal, anti-platelet, and anti-inflammatory
properties [130-131], and is used for schistosomiasis control.
Tetrandrine bisbenzylisoquinoline alkaloid isolated from the root of Stephania tetrandra
reveals a wide range of pharmacological activities, such as immunomodulating, antiinflammatory, anti-arrhythmic, anti-hepatofibrogenetic, anti-portal, anti-cancer, hypertension,
and neuroprotective activities [132]. The appearance of drug resistance with poor patient
compliance, undesirable side effects of chemotherapy and the higher cost of combination
therapy, reveals an extreme demand for a therapeutic regimen having the same or higher
beneficial properties of anticancer agents but with low side effects. The use of natural herbs is
28
one possible approach. Alkaloids demonstrate considerable biological activities and can be used
as potent anti-cancer agents. To identify a novel and specific inducer of ROS mediated apoptosis
in bladder cancer cells, evodiamine, an alkaloid, was screened in the presence or absence of
NAC, a specific ROS scavenger, using the MTT assay [133].
2.3 Costunolide
Costunolide, an illustrious sesquiterpene, is an active compound found in medicinal
plants and it is isolated from crude extract of Chinese Traditional medicinal herb Saussurea lappa
roots [134]. Accrued data shows that Costunolide was extracted from several plant species such
as [135], Magnolia kobus [136], Eupatorium lindleyanum [137], Magnolia ovata [138], Laurus
nobilis [139], Laurus novocanariensis [140], Cosmos pringlei [141], Magnolia grandiflora
[142], Magnolia sieboldii [143], Tsoongiodendron odorum Chun [144], Podachaenium eminens
[145], and Michelia floribunda [146].
In medication, costunolide is used as a popular herbal medicine, having antiinflammatory [147-148], anti-ulcer [149], anti-fungal [150, 151], anti-viral [134], and anti-cancer
activities [143, 152, 153]. Costunolide was also reported to have antipyretic, anti-mycobacterial
activities [154], pro-differentiation, hypolipidemic [155], and inhibitory effects against cellular
production of melanin [156]. For the first time, the anticancer property of costunolide was
demonstrated in azoxymethane induced rat intestinal carcinogenesis model and then supported
by consequent study using hamster buccal pouch carcinogenesis model induced by DMBA
[157]. After these two in vivo experiments extensive research was conducted to understand the
mechanism accountable for anti-cancer activity of costunolide. Additionally, costunolide has
29
been demonstrated to possess anti-cancer properties on diverse cancer types including leukemia
[158], intestinal [159], melanoma [160], and breast cancer [161].
2.4 Molecular targets of costunolide
Reported that two main pathways are involved to initiate apoptosis, the intrinsic or
mitochondrial, and the extrinsic or death receptor pathway; both pathways eventually activate the
same effectors caspases and apoptosis effectors molecules. In cancer cells costunolide is a strong
apoptosis inducer via various pathways, it has been demonstrated that costunolide readily
diminish intracellular GSH and suspend the cellular redox balance [162].
The interaction between members of the Bcl-2 protein family regulates the apoptosis
through mitochondrial pathway. ROS can also be involved in the process of lipid peroxidatio
and/or the cross-linking of thiol groups in proteins; both of these processes can induce the
opening of the mitochondrial permeability transition pore (PTP) [163, 164]. Formerly, in human
leukemia cells, it has been reported that costunolide induces apoptosis by regulation of
mitochondria permeability transition and consequent cytochrome c release [165], and via
reactive oxygen species and Bcl-2-dependent mitochondrial transition [165, 162]. When Bcl-2
protein was over expressed it attenuated apoptosis induced by costunolide [147].
The caspases, a family of cysteine proteases, are one of the focal executors of the
apoptotic process via triggering of the death receptors and mitochondrial pathways to accomplish
the programmed cell death [61]. Caspases are present in the form of inactive zymogens that are
activated during apoptosis. Among them, caspase-3 is a frequently activated death protease,
catalyzing the specific cleavage of many key cellular proteins [166,167]. Several studies reveal
effects of coustunolide on expression of caspases [168,169], and coustunolide induces apoptosis
30
in bladder and gastric adenocarcinoma cells by activation of caspase- 3 and modulating the level
of Bcl-2 protein family [127,169].
Moreover, Lei et al. demonstrate that caspase-8 activation is associated with alteration of
Bid in liver cancer cells [51a]. Furthermore, the cleavage of specific substrates for caspase- 3,
poly (ADP-ribose) polymerase (PARP), is involved costunolide-induced apoptosis in various
cancer cells [127,168,176]. Protein Bax induces the release of cytochrome c from the
mitochondria. It is considered an important event that affects apoptosis mediated by
mitochondrial pathway [168].
Table 1. Molecular targets of costunolide in different cancer types
EC50/
Type of
cancer
Cell lines
Concent.
Targets
References
NFκB↓; ↑MMP09↓;PARP ↓;BCL2 ↑;BAX↓;
P53↓;TNFα↓;p27Kip1↓; CDK2↓;Fas, caspase8, caspase-3↑, and degradation of PARP;
Cdc2↓; cyclin B1↓; tubulin↓;McTN↓;ROS↑
[147,161,162,1
63,176,167]
MDA-MB436;231;157 and Bt549
46 10 μM, 25
μM;50 µg/mL
Bladder r
T24
46.5 µM
BAX ↑;BCL2↓;survivin↓;caspase-3↑;
PARP ↑; ↑; NAC(i); ROS↑
Ovarian
SKOV3, A2780,
MPSC1 cells
5 0 µM
caspase-3, -8, and -9↑; BCL2↓; BAX ↑;
human
hepatocellular
carcinoma
HA22T/VGH
100 µM
Chk2 (Thr 68) ↑;Cdc25c (Ser 216) ↑;Cdk1
(Tyr 15) ↑;cyclin B1↑
Prostate
LNCaP; (PC-3 and
DU-145
36 µM
p21↑;cyclin E↑; Ca(2+), cyclin dependent
kinase↑
Leukemia
U937 cells; HL-60
10, 40, 50 µM
Bcl-2↑;JNK(i); GSH↓
Breast r
[169]
[153,169
. [101]
[100]
[162, 158
160,101]
↓: Downregulation; ↑: Upregulation
31
CHAPTER III: MATERIAL AND METHODS
3.1 Chemicals and Reagents
Cell culture medium, Dulbecco's modified Eagle's medium (DMEM) and MTT (3′-(4, 5dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide), propidium iodide (PI), and dimethyl
sulfoxide (DMSO) were purchased from Sigma. Fetal bovine serum (FBS) was purchased from
the Hangzhou Sijiqing Biological Engineering Materials Co., Ltd. An annexin V-FITC apoptosis
detection kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Rabbit
polyclonal anti-human Bcl-2, Bax, cleaved caspase-3 and PARP antibodies were purchased from
Wuhan Boster Biological Technology Co., Ltd. (Wuhan, China). Mouse anti-β-actin and antirabbit antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Ponceou and cell lysis buffer for western blots and IP were purchased from Bio SS Beijing
(Beijing, China). Rhodamine 123 was purchased from Invitrogen (Eugene, OR, USA).
3.2 Cell Culture
MCF-7 cells were cultured in Dulbecco's modified Eagle's medium (Gibco, USA)
supplemented with 10% fetal bovine serum (Gibco, USA), and maintained in a humidified
atmosphere of 5% CO2 in air at 37 °C. Cells were seeded in 10 cm culture, the medium was
changed every 2 days, and cells were allowed to grow to approximately 70% confluence before
experimentation.
3.3 Cell Proliferation Assay (MTT)
MMT assay was used to determine the cytotoxic effects of the Costunolide on the MCF-7
breast cancer cells. Briefly, cells were seeded at a density of 1×104 cells per well in 96-well
microtiter plates and were allowed to grow overnight. Cells were incubated with 100µL of
32
complete culture medium containing Costunolide, after 24 hours incubation, cell growth was
measured by adding 10µL MTT (5mg/mL in phosphate buffered saline) to each well and
incubated for 4 hour at 37o C. After 4 hours incubation medium was removed and 150µL DMSO
was added to each well following shaking gently and carefully. The absorbance was read at a
wavelength of 490 nm in a plate reader (ELX 800, BIO-TEK Instruments Inc., Winooski, VT,
USA).
3.4 Flow Cytometric Analysis of Cell Cycle
For cell cycle analysis, Breast cancer cells were seeded in 6-well plates for overnight and
then treated with Costunolide, for 24 hours. After 24 h incubation MCF-7 cells were trypsinized
and fixed in Ice-cold ethanol (70%) at 4oC for 10 minutes. After incubation cell pellet was
washed and re-suspend in propidium iodide (PI) staining buffer and incubated at 37oC for 15
minutes and then the percentages of cells in the different phases of cell cycle were evaluated by
determining the PI stained DNA contents by flow cytometry (Beckman Coulter, Epics XL,
Miami, FL, USA).
3.5 Flow Cytometric Determination of Apoptosis
Apoptotic cell death was measured by double staining annexin V-FITC and PI using the
annexin V-FITC apoptosis detection kit (Beyotime Biotechnology, Shanghai, China) according
to the manufacturer’s instructions. For these cells were cultured in 12-well plates and allowed to
attach overnight following treatment with indicated concentration of Costunolide, for 24 hours.
After 24 hour apoptotic cells were collected, washed in cold PBS and 1×105 cells were resuspended in 100μL annexin-V binding buffer and 5μL annexin V-FITC for 10 minutes in dark
at room temperature, then 5μL PI was added. Flow cytometric analysis was performed
33
immediately after staining. Data acquisition and analysis were performed by flow cytometry
using the Cell Quest software.
3.6 Flow Cytometric Determination of Reactive Oxygen Species (ROS)
In order to determine the intracellular changes in ROS generation, MCF-7 cells were
stained with 2', 7'-dichlorofluorescein-diacetate (DCFH-DA). The fluorescent dye DCFH-DA is
cell membrane permeable and is converted into the cell membrane impermeable non-fluorescent
compound DCFH by intracellular esterases. Oxidation of DCFH by reactive oxygen species
produces highly fluorescent DCF. The fluorescence intensity of DCF inside the cells is
proportional to the amount of peroxide produced. Briefly, cells were treated with indicated
concentration of natural compounds for 24 hours. After treatment, cells were further incubated
with 10μM final concentration of DCFH-DA at 37 °C for 30 min. Consequently, cells were
collected, washed, re-suspended in PBS, filtered with 300 apertures and analyzed for 2',7'dichlorofluorescein (DCF) fluorescence by flow cytometry (Beckman Coulter, Epics XL).
3.7 Flow Cytometric Determination of Mitochondrial Membrane
Potential (ΔΨm)
To probe the changes in ΔΨm, MCF-7 cells were stained with rhodamine 123 (1μM)
after treatment with indicated concentration of costunolide, and control group for 24 hours. The
fluorescence of Rhodamine 123 was measured by flow cytometry with excitation and emission
wavelengths of 488 and 530 nm.
3.8 Western Blotting
Western Blot analysis was used to determine the mechanism of the apoptotic effect of
Costunolide on apoptotic associated proteins. Briefly, MCF-7 cells were incubated with
34
indicated concentration of Costunolide for 24 h. Cells were trypsinized, collected in 1.5mL
centrifuge tube and washed with PBS. The cell pellets were re-suspended in cell lysis buffer and
were lysed on ice for 30 min. After centrifugation for 15 min, the supernatant fluids were
collected and the protein content of the supernatant was measured by the Nano-Drop 1000
spectrophotometer (Thermo Scientific, Waltham, MA, USA). The protein lysates were separated
by electrophoresis on 10% SDS-polyacrylamide gel and transferred to a PVDF membrane
(Amersham Biosciences, Piscataway, NJ, USA). The membranes were soaked in blocking buffer
(5% skimmed milk) for 2 h. To probe for BAX, Bcl-2, cleaved caspase-3, PARP, and β-actin;
membranes were incubated overnight at 4°C with relevant antibodies, followed by appropriate
HRP conjugated secondary antibodies and ECL detection
3.9 Statistical Analysis of Data
For the statistical analysis of data, comparisons between results from different groups
were analyzed with SPSS for Window Version 15.0. Student’s t-test was employed to determine
the statistical significance of the difference between different experimental groups and control
group. p < 0.05 value was defined as statistically significant. All experiments were repeated at
least two times. Data was presented as mean ± standard deviation (S.D).
35
CHAPTER IV: RESULTS AND DISCUSSION
4.1 Anti-proliferative effects of Costunolide on breast cancer cells
We evaluated the effects of costunolide (fig 7) on the growth of MCF-7, we performed
cell viability assay. MCF-7 cell line was used to assess the anti-proliferative effects of
costunolide. The cells were treated with Costunolide for 24 h. MTT assay showed that
costunolide inhibited cell growth in a dose- and time-dependent manner. Previous results showed
that costunolide have antiproliferative effects on breast cancer cells [151,152,153] and in others
cancer cells such as colon, melanoma, ovary, lung, leukemia [164, 165], pancreatic carcinoma
cells, MK-1, HeLa and B16F10 cells [144]. Pretreatment with 5mM NAC restored the viability
of cells indicating that costunolide exerts cytotoxic effect on cell viability through ROS
generation. cytotoxic effects on various cancer cells such as human leukemia cell line [30, 31],
lung, ovary, colon, melanoma cancer cells[153], human bladder cancer [169], and human breast
cancer [,166,167,168].
36
Figure 7: Costunolide inhibited the cell growth and induced cell death. MCF-7 Cells were treated
with indicated doses of costunolide in the presence or absence of NAC for 24 h and cell viability was
measured by MTT assay. Data are expressed as Mean ± SD (n = 3). Columns not sharing the same
superscript letter differ significantly (p < 0.05).
4.2 Costunolide Induced G2/M Cell Cycle Arrest on breast cancer MCF7
Cells
Cell cycle arrest is one of the major causes of cell death, many studies show that cells
with malfunctions checkpoint are more vulnerable to anticancer agents [119]. Cells can arrest at
cell cycle checkpoints temporarily to allow for: (i) cellular damage to be repaired; (ii) the
dissipation of an exogenous cellular stress signal; or (iii) availability of essential growth factors,
hormones, or nutrients. Checkpoint signaling may also result in activation of pathways leading to
programmed cell death if cellular damage cannot be properly repaired. Defects in cell cycle
checkpoints can result in gene mutations, chromosome damage, and aneuploidy, all of which can
contribute to tumorigenesis. Many laboratories are now searching for compounds that interfere
with cell cycle checkpoints, in the hope that such agents will be more effective in anticancer
therapy [180].
The result showed that costunolide arrested cell cycle at G2/M phase and the percentage of
accumulation of cells in the G2/M phase was increased from 4.9% in control group to 20.4% and
25.9% in the cells treated with 25 and 50μM of costunolide for 24 hours respectively. This
increase was coupled with the decreased percentage of cells in G0/G1 phase as showed in Figure
8. Acumulated data reported that costunolide induced G1 phase cell cycle arrest in human
prostate cancer cells [151] and G2/M phase arrest in human hepatocellular carcinoma cells , Soft
tissue sarcomas (STS) [162], human breast cancer [156] Human bladder cancer T24 cells [161].
37
control
25µM
50µM
Figure 8: Flow cytometry analysis of cell cycle phase distribution. Flow cytometry analysis of cell
cycle phase distribution in MCF-7 cells treated with 25 and 50μM costunolide for 24 hours. Data are
expressed as Mean ± SD (n = 3). Columns not sharing the same superscript letter differ significantly (p <
0.05).
4.3 Costunolide Induced Apoptotic Cell Death in Breast Cancer MCF7 Cells
Apoptosis, autophagy, and necrosis are the major types of cell death [163]. Among the
three major pathways of cell death, apoptosis is most well planned and orderly mode of cell
death [13, 164]. More than 50% of neoplasms undergo aberrations in the apoptotic machinery
which leads to abnormal cell proliferation [155, 156]. The regulation of apoptosis is, therefore,
38
the most important in the treatment of cancer [126-168]. Accumulated evidences indicated that
the most of chemotherapeutic agents halt tumor cells proliferation via induction of apoptosis
[119, 190, 121].
We wanted to know if costunolide inhibits MCF7 cells through induction of apoptosis
Costunolide-induced apoptosis was determined by flow cytometric analysis. Cells were sown in
12 well plates. After incubation of cells with costunolide for 24 hours, cells were collected in
centrifuge tubes and stained with annexin V-FITC and PI double staining as described in the
Experimental section. The results of flow cytometric analysis showed that the rates of apoptosis
were 20.77 ± 1.36% and 52 ± 1.53% and in the cells treated with 25 and 50μM of costunolide
respectively for 24 hours as compared to the 4.68 ± 0.42% in control cells. Pretreatment with
NAC completely blocked the apoptotic effect of costunolide indicating that induction of
apoptosis is a ROS-dependent manner. Costunolide-induced apoptosis in breast cancer cells was
compatible with previously reported studies [139, 159,166, 147].
Figure 9: Apoptosis induced by costunolide in MCF-7 cells. (A) MCF-7 cells were treated with 25 and
50 μM of costunolide for 24 hours in the presence or absence of NAC. Then cells were stained with
39
FITC-conjugated Annexin V and PI for flow cytometric analysis. The flow cytometry profile represents
Annexin V-FITC staining in x axis and PI in y axis. (B) Data are expressed as Mean ± SD (n = 3).
Columns not sharing the same superscript letter differ significantly (p < 0.05).
4.4 Costunolide Increased Generation of ROS in MCF7 Cells
Oxidative stress is the cellular status associated with enhanced production of intracellular
ROS and/or impaired function of the cellular anti-oxidant defense system [162]. The intracellular
redox status is a precise balance between oxidative stress and endogenous thiol buffers present in
the cell [133]. It has been well-established that the intracellular redox status plays an important
role in cell survival and death [144]. Unbalanced intra-cellular redox states trigger downstream
cellular events, such as alterations of mitochondrial function and cell signaling pathways [195],
which lead to apoptotic cell death. Many cancer therapeutic drugs can induce apoptosis by
disrupting the redox balance. Formerly, in human leukemia cells, it has been reported that
costunolide induces apoptosis by regulation of mitochondria permeability transition and
consequent cytochrome c release [75], and via reactive oxygen species and Bcl-2-dependent
mitochondrial transition [166,158]. In our study, we addressed the question of whether or not
costunolide could induce apoptosis and increase ROS generation in breast cancer cells.
The intracellular ROS level of costunolide treated with MCF-7 cells was measured using
the ROS-detecting fluorescence dye 2, 7-dichlorofluorescein diacetate (DCF-DA). The level of
ROS was significantly increased after treating the cells with indicated dose of costunolide for 24
hours. As shown in Figure 10 A, B, the ratio of DCF-positive cells, treated with 25 and 50 μM
costunolide was significantly higher (19.52 ± 1.73 and 35.94 ± 1.83 vs. 1.07 ± 0.53 in control
group, p < 0.05). The findings evidenced that costunolide had enhanced the generation of ROS
in Breast cancer cells, which corroborated with a previous report about Breast cancer [126].
40
Figure 10: Flow cytometry analysis of ROS generation. (A) MCF-7 cells were treated with 25
and 50μM costunolide in the presence or absence of 5mM NAC for 24 h. (B) Data are expressed
as mean ± SD (n=3). Columns not sharing the same superscript letter differ significantly (p <
0.05).
4.5 Costunolide Decreased Mitochondrial Membrane Potential in MCF7 cells
Mitochondria have been described to play a key role and perhaps even a central role in
the apoptosis process due in part to the mitochondria being the ‘junction’ of at least two distinct
signaling pathways, which contains pro-apoptotic proteins (e.g., cytochrome c) [104]. It has been
elucidated that upon the depolarization of the mitochondrial membrane potential results in
mitochondrial swelling and subsequent release of cytochrome c from the intermitochondrial
membrane space into the cytosol [105]. It is becoming increasingly apparent that the
mitochondria play a key role in the processes leading to cell death [169].
The effects of costunolide on the mitochondrial membrane potential of MCF-7 cells were
determined by flow cytometry using Rhodamine 123 staining. The rates of depletion of
mitochondrial membrane potential were 80.1% and 71.3% in the cells treated with 25 and 50μM
41
of costunolide, respectively, for 24 h as compared to 94.6 % in the control group. To further
confirm the involvement of ROS in disruption of mitochondrial membrane potential, cells were
treated with 5mM NAC. Pretreatment with NAC completely prevented dissipation of
mitochondrial membrane potential, indicating that this was ROS-dependent as shown in figure
11
A
B
Figure 11: The effects of costunolide on mitochondrial membrane potential of MCF7 cells. The
effects of costunolide on mitochondrial membrane potential of MCF7 cells were determined by flow
cytometry. (A) The values indicates the percentages of Rhodamine 123 fluorescence in the MCF7 cells
treated without and with 25 and 50μM of costunolide for 24 hours in the presence or absence of NAC. (B)
Data are expressed as Mean ± SD (n = 3). Columns not sharing the same superscript letter differ
significantly (p < 0.05).
4.6 Costunolide Regulated Apoptosis-Related Proteins in MCF-7 Cells
Costunolide induces apoptosis through extrinsic pathway in MCF-7 breast cancer cells, In
order to examine whether costunolide suppresses cell growth inducing apoptotic cell death, we
analyzed DNA contents and apoptosis-related proteins expression level by flow cytometry and
42
western blot, respectively in MCF-7 breast cancer cells we investigated whether costunolide
activates extrinsic apoptotic pathway we examined the expression levels of death receptor
signaling-related proteins including caspase-3, and PARP. The results showed that procaspase-3
was cleaved .Da fragments and activation of PARP in treated cells with 25 and 50 μM of
costunolide after 24 hours as compared to that of control cells as shown in Figure 12B. To
determine whether costunolide induces intrinsic mitochondria apoptotic pathway in MCF-7
breast cancer cells, we examined the expression levels of mitochondrial apoptotic pathway
related proteins such as anti-apoptotic protein, B-cell lymphoma protein-2 (Bcl2), and proapoptotic protein Bax It was observed that costunolide involved in the down regulation of Bcl-2
in a dose-dependent manner, and up regulation of BAX as shown in Figure 11. These results are
similar with previously reported studies [162,116, 169]
A
B
Figure 12: The effect of costunolide on the expression of major apoptosis regulatory
proteins. MCF-7 cells were exposed to 25 and 50μM of costunolide for 24 hours. Equal amounts
of lysate protein were subjected to gel electrophoresis. (A,B) Expression levels of Bax, Bcl-2,
caspase-3 and PARP were monitored by western blot assay. β-actin was used as loading control
43
4.7 CONCLUSION
In this study we reported the anti-proliferative potential and apoptotic effect of
costunolide against human breast cancer MCF-7 cells. Ours results showed that costunolide is
effectively inhibits the proliferation of breast cancer cells through induction of ROS-mediated
apoptosis.
We also observed that costunolide treatment accumulated the cell population at G2/M
phase, costunolide induces apoptosis through extrinsic pathway in Breast cancer cell, This event
was confirmed with the appearance of cleaved caspase-3 and PARP and costunolide also induce
apoptosis through intrinsic mitochondria pathway due induced the up-regulation of BAX and the
parallel down-regulation Bcl-2 expression. Costunolide Decreased Mitochondrial Membrane
Potential. To further confirm the involvement of ROS in disruption of mitochondrial membrane
potential, cells were treated with 5mM NAC. Pretreatment with NAC completely prevented
dissipation of mitochondrial membrane potential, indicating that the mechanism was ROSdependent. These results suggest that costunolide may have beneficial effects for the reduction of
breast cancer growth
4.8 Novel findings and significance

In this study, we reported anti-proliferative activities of Costunolide in human breast
cancer MCF-7 cells.

We reported that Costunolide, induced apoptosis in dose dependent manner in human
breast cancer MCF-7 cells.
44

In this study, for the first time we reported that costunolide, exerts its growth inhibitory
effect through ROS generation, and G2/M phase cell cycle arrest human breast cancer
MCF-7 cells.

In this study, we reported the ROS-dependent induction of apoptosis by costunolide in
human breast cancer MCF-7 cells.

We reported that costunolide involved in the down regulation of anti-apoptotic proteins,
Bcl-2, and the activation of pro-apoptotic protein, PARP in a dose-dependent manner.

These findings provide the rationale for further molecular mechanistic and in vivo
investigation of costunolide, against human breast cancer MCF-7 cells.
45
5.3 List of abreviations
CDK- Cyclin-dependent kinases
CKIs -Cyclin-dependent Kinase Inhibitors
ATM - Ataxia-telangiectasia-mutated
ATR- Rad3-related kinase
DNA-PK -DNA protein kinase
DNA- Deoxyribonucleic acid
TCM-Traditional Chinese medicine
ROS-Reactive oxygen species
CIS - Carcinoma in situ
DCIS- Ductal carcinoma in situ
LCIS -Lobular carcinoma in situ
ER- Estrogen receptor
PR -Progesterone receptor
PCD- programmed cell death
PARP-poly(ADP-ribose) polymerase
H2O2--), hydrogen peroxide
SOD-Superoxide dismutases
IM- inner membrane
OM - outer membrane
IMS- intermembrane space
MTT-Cell Proliferation Assay
NAC- N-acetyl-L-cysteine
DMSO- Dimethyl sulfoxide
FBS -Fetal bovine serum
46
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ACKNOWLEDGEMENTS
It is an honor to express my sincerest thanks to my very kind scholarly and
compassionate supervisor Dr. Xiaomeng Li, Professor, The Key Laboratory of
Molecular Epigenetic of MOE, Institute of Genetics and Cytology, School of Life
Sciences, Northeast Normal University, for her precious encouragement, priceless
suggestions, instructions and competent guidance without this my project would
not be materialized.
I am also broadening my thanks to my Co-supervisor Dr. Azhar Rasul for
his guidance and support throughout the project, without of that this work would
not have been brought to end.
I am also grateful to express our sincere thanks to my all the lab-fellows and
teachers from Northeast Normal University for their honorable assistance.
I'd like to thank China Scholarship Council (CSC) and Ministry of
Education of CApe verde. for financial support.
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In the last but not least, I am also very thankful to my parents, mainly my
Mother Josefa Lopes, my brothers Osvaldina, Edelson, Oteldino, Alexandra Milco
and Tamires
and all other family members due to their full support,
encouragement and blessing during the study period.
HELDER OLIVEIRA
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