14hwang Phoebe R PDF

Anti-Carcinogenic Effects of Morinda citrifolia (Noni): Identifying Signal Transduction
Pathways using ER Positive and ER Negative Human Breast Cancer Cell Lines
A thesis submitted to the Graduate Division of the University of Hawai’i at Mānoa in
partial fulfillment of the requirements for the degree of
Master of Science
in
Molecular Biosciences and Bioengineering
August 2012
By
Phoebe Hwang
Thesis Committee:
Pratibha V. Nerurkar – Chairperson
Dulal Borthakur
Scot Nelson
i
Student: Phoebe W.N. Hwang
Student ID#: 6096491
Degree: MS
Field: Molecular Biosciences Bioengineering
Graduation date: May 2012
Title: Anti-carcinogenic effects of Morindacitrifolia(noni): Identifying signal

transduction pathways using ER positive and ER negative human breast cancer cell
lines
We certify that we have read this Thesis and that, in our opinion, it is satisfactory in
scope and quality as a Thesis for the degree of Master of Science in Molecular
Bioscience Bioengineering.
Thesis Committee:
Names Signatures
PratibhaNerurkar, Chairperson ____________________________
DulalBorthakur ____________________________
Scot Nelson ____________________________
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Dedication
To my brother, Andy Hwang.
iii
Acknowledgments
Thanks to Dr. Pratibha Nerurkar for all her support and guidance. I would never be able
to complete my project if she had not accepted me into her lab and taught me everything.
I would like to express my gratitude to Dr. Dulal Borthakur and Dr. Scot Nelson for their
counsel and guidance.
I would also like to thank our lab members for all their moral support. Especially my
colleagues who trained me.
Last but not least, thanks to my friends and family for all the emotional support and
understanding.
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Abstract
It is predicted that one in every eight women in the United States will be diagnosed with
breast cancer in her lifetime, making breast cancer the second most prevalent cancer in
the nation. Native Hawaiians have the highest incidence as well as mortality rates
compared to other ethnic groups in Hawaii. An increase in the use of alternative
medicine, specifically by breast cancer patients prompted us to study the anti-cancer
effects of Hawaiian traditional medicine, Morinda citritfolia (noni). Estrogen receptor
positive (ER +ve) cells, MCF-7 and estrogen receptor negative (ER –ve) cells, MDAMB-
231 were initially treated with fNJ at varying concentrations for up to 96h. 10% fNJ
demonstrated cell death in 30 – 40% of the cells and 15% fNJ demonstrated cell death in
50 – 60% of the cells. In addition, when non-carcinoma breast cells, MCF-10A
underwent the same treatment, 15% fNJ only demonstrated 10 – 15% cell death in MCF-
10A. Cell proliferation assays suggest that fNJ is toxic to breast cancer cell lines, MCF-7
and MDAMB-231 but not normal breast cell line, MCF-10A. Apoptosis array data have
shown a significant decrease of a class of proteins called inhibitors of apoptosis proteins
(IAPs) such as survivin, Bcl-2, cIAP-1, and cIAP-2 in fNJ treated MCF-7 and MDAMB-
231 cells. There was also a significant decrease in proteins that assist with cell
replication such as claspin and phospho-Rad17. This demonstrates that fNJ induces cell
selective apoptosis and inhibits cell proliferation in breast cancer cell lines, MCF-7 and
MDAMB-231. [Grants: NCCAM (R21AT003719), USDA-CREES (2004-34135-
15182)]
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Table of contents
Dedication .......................................................................................................................... iii
Acknowledgments.............................................................................................................. iv
Abstract ............................................................................................................................... v
Table of contents ................................................................................................................ vi
List of figures ................................................................................................................... viii
List of Tables ...................................................................................................................... x
List of Abbreviations ......................................................................................................... xi
Chapter 1: Introduction ....................................................................................................... 1
1.1 Breast Cancer ........................................................................................................... 1

1.1.1 Impact ............................................................................................................. 1
1.1.2 Factors and Causations ................................................................................... 1
1.1.3 Estrogen Receptor Negative vs. Estrogen Receptor Positive Breast Cancer
Cells ......................................................................................................................... 5
1.2 Complementary and Alternative Medicine .............................................................. 7

1.2.1 Usage in the United States ............................................................................. 7
1.2.2 Complementary and Alternative Medicine as Cancer Treatment ................... 8
1.3 Morinda citrofolia (Noni) ........................................................................................ 9

1.3.1 History............................................................................................................. 9
1.3.2 Traditional and Modern Uses........................................................................ 10
1.3.3 Fermented Noni Fruit Juice .......................................................................... 13
1.4 Noni and Cancer .................................................................................................. 14

1.4.1 In vitro studies.............................................................................................. 14
1.4.2 In vivo studies ............................................................................................... 15
1.4.3 Molecular Mechanisms ................................................................................. 19
Chapter 2: Hypothesis and Aims ...................................................................................... 21

2.1 Hypothesis............................................................................................................ 21
2.2 Specific Aims ....................................................................................................... 22
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Chapter 3: Materials and Methods .................................................................................... 24
3.1 Preparation of fNJ with endotoxins removed ........................................................ 24
3.2 Cell Culture ............................................................................................................ 24
3.3 MTT assay ............................................................................................................. 25
3.4 Human apoptosis antibody array ........................................................................... 25
Chapter 4: Results ............................................................................................................. 28

4.1 fNJ decreases cell viability in breast carcinoma cells ............................................ 28
4.2 Apoptotic proteins identified in fNJ treated breast carcinoma cells ...................... 29
4.3 fNJ inhibits survivin in MCF-7 (ER+ve) cells ....................................................... 29
4.4 fNJ up-regulates cytosolic levels of cytochrome c in MCF-7 (ER+ve) cells ........ 30
4.5 fNJ does not affect cytosolic and nuclear levels of nFκB in MCF-7 (ER+ve) cells30
4.6 fNJ inhibits survivin in MDAMB-231 (ER-ve) cells ............................................ 30
4.7 fNJ does not affect cytosolic levels of cytochrome c in MDAMB-231 (ER-ve)
cells ............................................................................................................................... 31
4.8 fNJ does not affect cytosolic and nuclear levels of nFκB in MDAM-231 (ER-ve)
cells ............................................................................................................................... 31
4.9 Figures of results .................................................................................................... 32
Chapter 5: Discussion ....................................................................................................... 46
Chapter 6: Conclusion....................................................................................................... 53
6.1 Significance............................................................................................................ 53
6.2 Future studies ......................................................................................................... 54
Reference: ......................................................................................................................... 55
vii
List of figures
Figure Page
Figure 1. Ductal and Lobular Carcinoma In Situ ................................................................ 2
Figure 2. Modifiable risk factors that affect cancer ............................................................ 4
Figure 3. General transcription mechanisms of estrogen receptor positive and estrogen

receptor negative breast cancer cells........................................................................... 6
Figure 4. Total out-of-pocket complementary and alternative medicine costs among

American adults in 2007 ............................................................................................. 8
Figure 5. Noni fruit ............................................................................................................. 9
Figure 6. Encapsulated freeze-dried noni fruit ................................................................. 13
Figure 7. fNJ induces apoptosis through hypothesized pathways .................................... 21
Figure 8. Phase contrast photomicrographs of MCF-7 cells treated with fNJ .................. 32
Figure 9. Phase contrast photomicrographs of MDAMB-231 cells treated with fNJ) ..... 32
Figure 10. Effects of fNJ on MCF7 cell viability ............................................................. 33
Figure 11. Effects of fNJ on MDAMB-231 cell viability................................................. 34
Figure 12. Effects of fNJ on MCF-10A cell viability ....................................................... 35
Figure 13. Human apoptosis array overlay ....................................................................... 36
Figure 14. Effects of 15% fNJ (v/v) on apoptotic proteins in MCF-7 cells...................... 37
Figure 15. Effects of 15% fNJ (v/v) on apoptotic proteins in MDAMB-231 cells. ......... 37
Figure 16. fNJ reduced survivin levels in MCF-7 cells .................................................... 38
Figure 17. fNJ increases cytosolic cytochrome c in MCF-7 cells .................................... 39
Figure 18. fNJ did not affect changes in cytosolic nFkB levels in MCF-7 cells .............. 40
Figure 19. fNJ did not affect changes in nuclear nFkB in MCF-7 cells ........................... 41
viii
Figure 20. fNJ reduced survivin levels MDAMB231 ....................................................... 42
Figure 21. fNJ did not affect cytosolic levels of cytochrome c in MDAMB-231 cells .... 43
Figure 22. fNJ does not affect cytosolic nFkB in MDAMB-231 cells ............................. 44
Figure 23. fNJ did not affect nuclear nFkB levels in MDAMB-231 cells ........................ 45
Figure 24. Extrinsic and intrinsic pathways of apoptosis ................................................. 47
Figure 25. Roles of Bcl-2 .................................................................................................. 50
Figure 26.The role of NF-kB in apoptosis ........................................................................ 52
ix
List of Tables
Table 1. Traditional uses of the noni ................................................................................ 10
Table 2. Modern uses of noni .......................................................................................... 12
Table 3.In vitro studies on the effects of noni and cancer................................................ 17
Table 4. Summary of molecular mechanisms involved in the effects of noni against

cancer ........................................................................................................................ 20
x
List of Abbreviations
AP1 Activator protein 1
ATM Ataxia telangiecstasia mutated
Bcl-2 B-cell lymphoma 2
BMI Body mass index
BRCA1 Breast cancer type 1
CAM Complementary and alternative medicine
CDH1 Cadherin 1
CHEK2 Dysfunctional check point homolog 2
cIAP-1 Cellular inhibitor of apoptosis protein 1
DCIS Ductal carcinoma in situ
EGF Epidermal growth factor
eIF-2α Eukaryotic translation initiation factor-2α
ER Estrogen receptors
ER-ve Estrogen receptor negative
ER+ve Estrogen receptor positive
Fas/TNFRSF6 Fibroblast associated tumor necrosis factor receptor

super family member 6
fNJ Ferment noni fruit juice
HepG2 Human laryngeal carcinoma
HSP-60 Heat shock protein 60
IAP Inhibitors of apoptosis
IFN-γ Interferon-γ
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IL-1β Interleukin-1β
JNK-1 c-Jun N-terminal kinase-1
LAN5 Human neuroblastoma
LCIS Lobular carcinoma in situ
LLC Lewis lung carcinoma
MCF-7 Human breast ER+ve carcinoma
NAG-1 Nonsteroidal anti-inflammatory activated gene-1
NfκB Necrosis factor-kappa beta
NJ Noni fruit juice
NK Natural killer
NO Nitric oxide
Noni-ppt Noni fruit precipitate
phospho-Rad17 Phosphorylated Rad 17
PK-B Protein kinase B
TNF Tumor necrosis factor
TP53 Tumor protein 53
TNFR Tumor necrosis factor receptor
TPA 12-O-tedtradecanoylphorbol-13-acetate
TRAIL TNF related apoptosis-inducing ligand
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Chapter 1: Introduction
1.1 Breast Cancer
1.1.1 Impact
Breast cancer is the third highest cause of cancer-related mortality in Hawaii [1].
Coming in behind prostate cancer, it is the second most prevalent cancer in the United
States [2]. An estimated number of 226,870 women in the United States will be
diagnosed with breast cancer by the end of 2012. Of these women, 1,120 will be from
Hawaii [1]. Although breast cancer mortality seems to remain relatively constant for the
past 30 years, the incidence rates have been slowly increasing from 1975 – 2008 [3].
1.1.2 Factors and Causations

Cancer is characterized as uncontrolled growth of damaged or abnormal cells in the
body. Unlike normal cells that proliferate and die and under apoptosis, these malignant
(cancerous) cells do not undergo cell cycle arrest and will continue to rigorously grow in
its abnormal state (www.cancer.org). Sometimes the cancer metastasizes and travels to
distant organs. The cancer is named after the body part where the tumor originates [3].
Thus, breast cancer originates from the breast tissue. Some breast cancers are formed
within the ducts or lobules of breasts and are therefore referred to as ductal carcinoma in
situ (DCIS) or lobular carcinoma in situ (LCIS), respectively (figure 1). Most breast
cancers are invasive and travel from ducts and lobules to nearby breast tissues [3]. Many
1
factors including age, sex, family history, physical fitness, and diet influence the type of
breast.
Figure 1. Ductal and Lobular Carcinoma In Situ [source: www.virtualmedicinecentre.com]
Aside from being female, age is the biggest risk factor for breast cancer. Invasive
breast cancer is found in 1 out of 8 women under the age of 45, whereas it is found in 2
out of 3 women over the age of 55 [4]. A woman at the age of 30 has a 0.43% risk of
developing breast cancer, compared to a woman at the age of60 who has a 3.45% risk of
developing breast cancer [3]. This evidence provided by the American Cancer Society
suggests that the probability of developing breast cancer increases with age. It is
believed that the increased likelihood of developing breast cancer at an older age may be
due to a longer life expectancy inmodern times. Other factors such as menopausal
hormone use, changes in reproductive patterns, rising obesity rates, and improved
detection and screening methods, also contribute to this increase.
Family history and genetics influence a woman’s likelihood of developing breast
cancer. Studies have shown that 5 – 10% of diagnosed breast cancer is thought to be
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hereditary [4, 5]. The more closely related a woman is to a family member who has
breast cancer, the greater her chance of developing breast cancer [6]. Studies have shown
that a number of individuals with genes such as breast cancer type 1 (BRCA1) and
BRCA2 [7]and dysfunctional check point homolog 2 (CHEK2)[8], tumor protein 53
(TP53)[9], ataxia telangiecstasia mutated (ATM)[10], and cadherin 1 (CDH1)[11] genes
have a higher risk of developing breast cancer.
In light of current studies on lifestyle choices and cancer, obesity, physical fitness[12,
13], and nutrition have been implicated as risk factors (figure 2)[14, 15]. Garnet
Anderson’s study showed a 70% increase risk factor of breast cancer in obese
premenopausal women compared to normal weight premenopausal women [16]. This
suggests that women with higher than normal body mass index (BMI) have an increased
risk of developing breast cancer. Besides having a high BMI, women with higher than
normal central adiposity (measured by waist circumference) also have a higher risk of
developing breast cancer [17]. Obese individuals tend to have higher concentrations of
pro-inflammatory markers circulating in the blood causing the person to be in a chronic
inflammatory state [18-20]. An activated pro-inflammatory marker, necrosis factor-
kappa beta (NF-κB), has been observed in mammary adipose tissues obtained from high
fat diet fed mice [21] and obese women with breast cancer [22]. These findings show
that breast tissues with high adipocyte density have elevated levels of NF-κB.
Furthermore, the correlation between adipocyte size, BMI, and inflamed mammary
glands supports the idea that obesity is associated with an increased risk of breast cancer
on a cellular level. In 2010, 35.8% of the women in the United States were considered
obese (www.cdc.org), indicating that 40.6 million American women have a high risk of
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developing breast cancer.
Figure 2.Modifiable risk factors that affect cancer [source: Food, nutrition, physical activity and the
prevention of cancer: a global perspective. World Cancer Research Fund/American Institute for Cancer
Research, 2007: p. 30-46]
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Whether or not an individual’s diet affects their risk of developing breast cancer is
still a controversial topic. A number of studies carried out to discover the correlation
between foods and the onset of breast cancer have yielded conflicting results[3, 15].
Foods such as bitter melon [23], curcumin[24], resveratrol [25], and other foods high in
antioxidants and fiber [26, 27] have been shown to decrease the onset of breast cancer.
Whereas high temperature cooked meats [28], red meats[29], and high fat foods [21]
have been shown to increase the risks. More research is needed to accurately determine
the effects of dietary consumption on breast cancer. Other factors such as periods and
amount of food consumption, family history, food-food and food-drug interactions might
also play an important role in the development of breast cancer and need to be further
analyzed.
1.1.3 Estrogen Receptor Negative vs. Estrogen Receptor Positive Breast Cancer Cells
Estrogen is a steroid hormone that is commonly found in women to regulate
pregnancy, bone density, menstrual cycle, liver function, breast and uterine cancer.
Estrogen receptors (ER) are molecules that bind to estrogen hormones and initiate a
number of signaling pathways [30]. The presence of intracellular ER indicates the cell’s
receptiveness to estrogen hormones (figure 3) [30, 31]. Consequently, elevated levels of
circulating estrogen increases the risk of developing breast cancer for individuals with
estrogen receptor positive (ER+ve) breast cells[32-34].
5
Figure 3. General transcription mechanisms of estrogen receptor positive and estrogen receptor negative
breast cancer cells [source: Food, nutrition, physical activity and the prevention of cancer: a global
perspective. World Cancer Research Fund/American Institute for Cancer Research, 2007: p. 30-46
Due to the lack of estrogen receptors, estrogen receptor negative (ER-ve) breast cells
are unresponsive to selective hormonal therapy compared to ER+ve breast cells [35].
Consequently, studies have shown that since the amount of ER present on each cell
varies, not all ER+ve breast cells respond well to hormonal treatment. As the amount of
ER decreases, so does the efficacy of the treatment [19, 36]. This raises a problem
because 15% of the breast cancer patients in the United States are ER-, leaving
approximately 450,000 women incurable [37]. Unfortunately, only 30% of breast cancer
patients have overexpressing ER that is highly susceptible to selective treatment [35],
leaving the rest of the women to rely on other non-selective cancer treatments such as
chemical and radiation therapy. Side effects from these non-selective cancer treatments
such as hair loss, lowered immune system, fatigue, nausea and abnormal digestive
processes (www.cancercare.org) are incentives to further investigate novel selective
breast cancer treatments.
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1.2 Complementary and Alternative Medicine
1.2.1 Usage in the United States

Complementary and alternative medicine (CAM) covers a spectrum of medical
and health care practices that range from traditional to modern methods to prevent or cure
certain diseases and ailments [38, 39]. Complementary medicines are used alongside
with other conventional medicines. Whereas, alternative medicines are used to substitute
conventional medicines [39]. There are two types of CAM therapies: practitioner
therapies and self-care therapies [40]. Commonly used practitioner-type CAM therapies
include deep breathing, chiropractic treatments, massage therapy, acupuncture, and
hypnotism therapy. Commonly used self-care therapies include natural product
consumption, diet based therapy, meditation, and yoga [41, 42].
The use of CAM in the United States has been increasing over the past decade. In
2002, 36.0% of American adults reported that they used CAM. In 2007, this number
jumped to 38.3% [38, 42]. With over a third of the American adults and 11.8% of the
American children using CAM, the total out-of-pocket expenses spent on CAM healing
philosophies, therapies and products was $33.9 billion [40]. 44% of the out-of-pocket
costs were spent on non-vitamin, non-mineral products indicating the popularity of
natural products (figure 4) [40]. Unfortunately by definition, CAM is not considered a
conventional medicine due to the lack of evidence to prove how safe and effective they
are [43]. Due to the increasing popularity of natural products in the United States,
additional research is needed to inform the public of their effectiveness and side effects,
if any.
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Figure 4.Total out-of-pocket complementary and alternative medicine costs among American adults in
2007 [source: Nahin, Richard, Barnes, Patricia, Stussman, Barbara J., and Bloom, Barbara, Costs of
Complementary and Alternative Medicine (CAM) and Frequency of Visits to CAM Practitioners: United
States, 2007.National Health Statistics Report, 2009. 18.]
1.2.2 Complementary and Alternative Medicine as Cancer Treatment

CAM treatments have been used for certain diseases and conditions such as head
or chest cold, anxiety, digestive illnesses, high cholesterol, and cancer [41, 42]. Many
cancer patients find additional ways to improve their diseased state or to prevent side
effects caused by conventional medicine [44]. An international survey showed that
35.9% of cancer patients used CAM [45]. In a 2005 study conducted by Molassiotiset al,
44.7% of the breast cancer patients studied (n=956) used CAM to either treat the disease
or increase physical and emotional well-being [46]. For a multitude of reasons, some
cancer patients turn to natural CAM products for treatment. 40.1% of the cancer patients
that utilize CAM rely on natural products [45]. Therefore, a solid investigation into the
effectiveness of CAM against cancer will be of great benefit to patients who utilize it.
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1.3 Morindacitrofolia (Noni)
1.3.1 History
Morindacitrifolia(noni), also known as Indian Mulberry, nono, or cheese fruit, is
a small shrub that is native to South Asia and grows prominently in the tropics [47].
Noni shrub grows about two to six meters tall, with either rounded, elliptic or long leaves
and globular fruits that range from three to 20 cm long [48, 49]. Noni has been
traditionally used in Hawaii, Polynesia and Southeast Asia as either a dye, famine food,
[50] and is considered the most important medicinal plant based on reports of usage
across Polynesian cultures [48, 51, 52].
Figure 5.Noni fruit [source: www.resorthealth.com]
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1.3.2 Traditional and Modern Uses
Traditional healers from Hawaii, Samoa, Rotuma, and Fiji have been known to
utilize different parts of the plant to treat wounds, infections, menstrual cramps and
bowel irregularities[48, 53]. Table 1 summarizes the traditional uses of noni prior to the
1800s. Contrary to popular belief, traditional healers do not commonly utilize the ripe
fruits. The most commonly used part of the noni plant was the leaf. Traditional healers
would heat noni leaves over a small fire then use it as a poultice by wrapping it around
the wounded or infected area [54]. Yet the use of noni fruits is popular as a modern
dietary supplement [48].
Noni plant part Traditional treatment for
Leaves Topical burns, headaches, fevers, neonatal inability to

breathe, bone fractures, menstrual cramps, back pain*,
insect infestations, boils, rheumatic pain, ulcers, gout,
internal bleeding, ringworm[48, 50, 55-57]
Fruit Sores in mouth, peeling or cracking of toes, boils, pimples,

blood impurities*, kava intoxication, insect infestations,
blotchy skin*, heart trouble , stomach pain*, menstrual
cramps*, heartburns , sore throat [48, 55, 56, 58]
Bark Stonefish poisoning, topical infections , asthma*, [55, 57,

58]
Root Topical infections, stomach pain* [55]
Stem Hernia [49]
Seeds
Flower Sore eyes, topical burns [57]
Table 1.Traditional uses of the noni
10
In the 1980s, commercial noni fruit juice (NJ) gained popularity fueled by Ralph
Heinicke’s claim that it contains Proxeronine, a precursor for Xeronine. Heinicke
suggested that Xeronine is an enzyme that can modify dysfunctional proteins to its
appropriate conformation[48, 53, 59]. In a study conducted in 1999, noni ranked the
second most commonly used complementary medicine[57]. The Nutrition Business
Journal reported that noni juice is the number one sales of single herbs in American in
2005 (www.nutritionbusiness.com). Noni was commonly used topically to treat
ailments up until the 1900s, when people began consuming noni products for its
medicinal properties. Fermented noni juice is popularly known as the “traditional”
method of preparing noni. It is believed that the Chinese, which composed a significant
proportion of the population in Hawaii, influenced fermenting the fruit. Reports have
shown that in the 1930s fermented noni juice was commonly prepared and consumed by
households in Hawaii. Furthermore, studies show an increase of demand for noni fruit
from early to late 1990’s[57]. Since then, multimillion-dollar companies have found
ways to package and sell this plant.
Due to the rising popularity of noni, many studies have been conducted to test its
efficacy as a medicine and have shown its anti-cancerous[60-62], anti-diabetic[63], anti-
tuberculosis[64], anti-viral[65], and anti-bacterial[66-68] effects. These studies were
already reviewed by Pawlus, Wang, Blanco, and Brown[49, 53, 69, 70]. The up to date
modern uses of noni are summarized in Table 2.
11
Noni plant part Treatment for
Leaves Tuberculosis ,helmintic infections, oxidative stress ,
open wounds , hyperlipidemia [64, 71-73]
Fruit Bacterial infections , cancer , pain , memory

impairment, reduced cerebral blood flow , diabetes ,
nausea , gastric ulcers , liver disease , low humoral
immunity , neuronal damage , stress-induced cognitive
impairment , helminthic infections , lung cancer,
oxidative stress, inflammation, post surgery nausea,
hyperglycemic, liver diseases [62-64, 66-68, 72, 74-88]
Bark
Root Viruses , hypotension , colorectal cancer , NF-kB
associated inflammation , hyperlipidemia , diabetes,
pain [62, 65, 71, 73, 89, 90]
Stem
Seeds Hyperlipidemia , oxidative stress , melanoma cancer
[91-93]
Flower
Table 2.Modern uses of noni
12
From skincare products to supplement capsules and tea, noni has been packaged
in multiple ways with claims to a better and healthier lifestyle. In 1992, a Maui massage
therapist encapsulated the dry powder form of noni fruit and sold it as supplement[57].
In 1996, John Wadsworth and Stephen Story introduced
Tahitian Noni Juice as a natural healthy supplement to the
United States (http://www.tahitian-juice.com). Other noni
juice companies such as, Virgin Noni Juice, Healing Noni,
and Noni Maui, sprouted since then with their own line of
noni juices. These bottles juices are sold either non-
fermented or fermented.
Figure 6.Encapsulated freeze-dried noni fruit
[source: www.estatenoni.com]
1.3.3 Fermented Noni Fruit Juice

To present date, there are two popular methods to prepare noni fruit juice:
fermented and non-fermented. In the fermented method, ripe yellow fruits are picked,
washed and left in glass air sealed containers for about two weeks to two months. The
juice is extracted and the pulp is removed. Some fermented juices are pasteurized.
Pasteurized noni has a distinctively different taste as well as different pH. To prepare
non-fermented noni juice, fresh ripe fruits are pressed through a juicer. The juice can be
consumed pure, or diluted with water or other fruit juices. In some juices, the starchy
13
pulp of the noni is mixed with either apple or grape fruit juice to increase palatability
[94].
Most noni fruits are consumed as a naturally fermented juice [95]. It is suggested
that in Hawaii, this type of noni preparation might have originated from the prominent
Chinese ethnic influence in the mid-1800s [57]. Based on local records, Hawaii residents
commonly picked, fermented, and consumed noni fruits before meals to treat diabetes,
heart trouble and blood pressure [96, 97]. Recent literature suggests that the probiotic
properties of most fermented foods are beneficial to your health [98, 99]. The beneficial
effects include improved lactose digestion, controlled gastrointestinal infection, reduced
cholesterol levels and stimulated immune system [100]. Besides the difference in biotic
cultures found in fermented foods compared to non-fermented foods, the chemical
constituents may vary as well. In 2007, Shu-Chuan Yang et al demonstrated that the
chemical composition of fermented noni juice (fNJ) differed from that of non-fermented
NJ. The fNJ yielded higher phenolic compounds, condensed tannins, flavonoids and
scopoletin than non-fermented NJ. Additional results from the study showed that fNJ has
higher anti-oxidative activity as compared to non-fermented NJ [101].
1.4 Noni and Cancer
1.4.1 In vitro studies

In 1999, Hiramizu and Furusawa tested the antitumor properties of a
polysaccharide-rich ethanol extract of noni fruit precipitate (noni-ppt). They found that
noni-pptsuppressed the growth of Lewis lung (LLC) peritoneal carcinoma cells by up
14
regulating several mediator and murine effector cells[61]. In 2006, Arpornsuwan and
Punjanon also conducted a study investigating the effects of noni fruit extract against
cancer cells. They observed cytotoxic effects against human laryngeal carcinoma
(HepG2), human breast ER+vecarcinoma (MCF7), and human neuroblastoma (LAN5)
cell lines[102]. An interesting study conducted in 2003 by Hornicket al. looked at the
anti-angiogenic effects of noni juice in human breast tumor explants by using a three-
dimensional fibrin clot matrix model. The commercial noni juice used demonstrated
anti-angiogenic and degenerative effects against the tumor explants[103].
Aside frominvestigating various noni fruit extracts, several studies have shown
the anti-cancerous effects of chemical extractions from other noni plant parts. In 1993,
Hiramatsuet al. demonstrated that the damnacathal and anthraquinones extracted from
noni root inhibited ras functions in rat kidney cells [104]. Later in 2011, Nualsanitet al.
also utilized damnacanthal and anthraquinone extracts from the root and found that the
extracts exhibited pro-apoptotic effects by inducing caspase activity in human colorectal
cancer cells [105]. New chemical compounds such as 6-O-(beta-D-glucopyranosyl)-1-O-
octanoyl-beta-D-glucopyranose and asperulosidic acid extracted from noni fruit juice also
displayed anti-cancerous properties against mouse epidermal cells[106].
1.4.2 In vivo studies

In vivo studies play a crucial role in determining the efficacy of the plant on
humans. Table 3is a table compiled by Brown that summarizes the in vivo studies
conducted since 1994 to determine the effects of noni fruit juice against cancer. For all
the studies mentioned in Table 3, subjects that received noni juice displayed a significant
15
amount of anti-cancerous effects. Taskinet al.’s Ehrlich ascites tumor injected Balb-c
mice showed a reduction in tumor size in mice that have received oral dosages of noni
juice as compared to contraol. Consequently, the same results can be seen in Stoner et
al.’s study done on rats. A reduction in tumor size has been observed in rats that received
noni juice as a part of their diet, compared to control.
16
Table 3. In vitro studies on the effects of noni and cancer
17
Table 3. (continued)
18
1.4.3 Molecular Mechanisms
Although there have been many studies conducted on the efficacy of noni against
cancer, many of them do not explain the molecular mechanisms behind the anti-
cancerous effects. Table 4 summarizes studies done thus far that demonstrate signal
transduction pathways caused by noni.In 2007, Takashima et al. studied the apoptotic
effects of noni leaf extract via tumor necrosis factor (TNF) and TNF related apoptosis-
inducing ligand (TRAIL)[107]. Other studies done by Hirazumi and Furusawa also
examined the apoptotic effects of noni through TNF. In addition, they also demonstrated
that increased levels of interleukin-1β (IL-1β), IL-10, IL-12, interferon-γ (IFN-γ), and
nitric oxide (NO) may have contributed to the anti-cancerous effects [61]. Other studies
demonstrate how noni induces apoptosis by affecting the immune system. In 2008, Li et
al. showed that fermented noni juice increased levels of granulocytes and natural killer
(NK) cells in the peripheral blood,peritoneum, and spleen therefore contributing to the
decrease in tumor size of mice[108]. Soon after, Stoner et al. demonstrated that noni fruit
powder inhibited tumorigenesis by reducing the levels of cytokines in rats [109].
Although many studies have demonstrated anti-cancerous effects of noni, most
fail to identify the molecular mechanisms involved. Thus far, only eight mechanisms
have been identified and additional studies are warranted. Identifying the proteins and
signal transduction pathways involved in noni’s anti-cancerous properties may aide in
determining the benefits of noni against cancer.
19
Type of Noni Extract Model used Molecules observed and measurable outcomes
Damnacathal and anthraquinone Ras transformed normal rat kidney Inhibited ras function [104]
extracted from noni roots using cells
chloroform
Noni-ppt Lewis lung carcinoma cells TNF-α, IL-1β, IL-10, IL-12, IFN-γ, and NO levels
subcutaneously injected in C57BL/6 increased [61]
mice
6-O-(beta-D-glucopyranosyl)-1-O- Mouse epidermal JB6 cells Suppressed 12-O-tedtradecanoylphorbol-13-acetate
octanoyl-beta-D-glucopyranose and (TPA) and epidermal growth factor (EGF) induced
asperulosidic acid extracted from activator protein 1 (AP1) transactivation[106]
noni fruit juice
Anthraquinone compound extracted Immortalized human T-lymphocyte Increased TRAIL induced cytotoxic activity [107]
from noni leaves using methanol Jurkat cells
Fermented noni exudate C57BL/6J mice Increased levels of granulocytes and NK cells in the
peripheral blood,peritoneum, and spleen[108]
Noni fruit powder Rats Inhibited N-nitrosomethylbenzylamine
(NMBA) induced tumorigenesis in the rat
esophagus and reduced cytokine, IL-5 and GRO/KC
(rat homologue for human IL-8 serum levels) [109]
Damnacanthal extracted from noni Human colorectal carcinoma cells Induced nonsteroidal anti-inflammatory activated
roots gene-1 (NAG-1), caspase, and transcription factor
CCAAT/enhancer binding protein β (C/EBPβ) [105]
Noni fruit juice Human lung carcinoma A549 cells Induced phosphorylation of protein kinase B (PKB),
extracellular regulated protein kinase 1/2 (ERK-1/2),
c-Jun N-terminal kinase-1 (JNK-1), and eukaryotic
translation initiation factor-2α (eIF-2α)[110]
Table 4.Summary ofmolecular mechanisms involved in the effects of noni against cancer
20
Chapter 2: Hypothesis and Aims
2.1 Hypothesis
Based on our preliminary data and literature review, we hypothesize that fermented noni
fruit juice (fNJ) will prevent proliferation and induce programmed cell death (apoptosis)
in MCF-7, and ER –veMDAMB-231cancer cells by modulating cell cycle arrest proteins
as well as inhibiting cell death proteins called “inhibitors of apoptosis” (IAP).
Figure 7.fNJ induces apoptosis through hypothesized pathways
21
2.2 Specific Aims
2.2.1 Specific Aim 1
To investigate the effects of fNJinnormal human breast, MCF10A and cancerous human
breast cells, MCF-7 and MDAMB-231.
Objective 1: Identify dosage of fNJ that are non-toxic in normal cells, but will
induce cell death in MCF-7and MDAMB-231 cells.
Approach: Toxicity of varying doses of fNJ will be measured in cells after 24, 48,
72 and 96 h by using commercial 96 well non-radioactive cell proliferation
(MTT) assay kit.
Objective 2: Identify the apoptotic proteins present infNJtreated human breast
cancer cells that affects proliferation or causes cell death in MCF-7 and
MDAMB-231.
Approach:Apoptosis will be measured using an apoptosis array in cells treated
with two doses of fNJ that will achieve about 60-80% cell death.
2.2.2 Specific Aim 2
To identify the anti-proliferative and apoptotic mechanisms inducedbyfNJin MCF-7
and MDAMB-231 cells.
Objective 1: Identify fNJ-induced apoptotic proteins in MCF-7 and the apoptotic
signaling mechanisms.
22
Approach: Proteins will be identified from apoptosis array and confirmed by
western blotting.
Objective 2:Identify fNJ-induced apoptotic proteins in MDAMB-231 and the
apoptotic signaling mechanisms.
Approach 1: Proteins will be identified by apoptosis array will be confirmed by
western blotting.
Approach 2: Nuclear and cytosolic proteins of fNJ treated MCF-7 and MDAMB-
231 cells will be extracted and be confirmed by western blotting.
23
Chapter 3: Materials and Methods
3.1 Preparation of fNJ with endotoxins removed

Morindacitrofolia(noni) fruits were obtained from noni trees on the University of Hawaii
ManoaMagoon facility, Honolulu, Hawaii. The fruits were rinsed, dried, and left to
ferment in airtight jars placed in indirect sunlight for 14 days. The fermented juice was
extracted and centrifuged at 3000 rpm at room temperature for 15 minutes. The
supernatant was filtered with a 0.45μm filter then a 0.20μm filter. The pH of the
fermented noni juice (fNJ) was adjusted to pH 7 using 10mM NaOH .
Endotoxins were removed from the fNJ using Detoxi-Gel Endotoxin Removing Columns
(Pierce Biotechnology, Rockford, IL, U.S.A) as per manufacturer’s instructions. The fNJ
with removed endotoxins were then aliquoted into 1.5 mL tubes and store in -80°C, then
thawed once for cell treatment.
3.2 Cell Culture

Estrogen receptor positive (ER +ve) human breast cancer cells, MCF-7 were generously
donated by Dr. June Panee. MCF-7 cells were maintained in Dulbecco’s modified eagle
medium (DMEM) (ATCC, Manassas, VA, U.S.A.) with 10% heat inactivate fetal bovine
serum (FBS), 1% penicillin/streptomycin, and 10mg/ml insulin.
Estrogen receptor negative (ER -ve) MDA-MB-231 cells were generously donated by Dr.
June Panee. MDA-MB-231 cells were maintain in Minimal essential medium eagle
(MEME) (ATCC, Manassas, VA, U.S.A)) with 10% heat inactivate fetal bovine serum
(FBS) and 1% penicillin/streptomycin.
24
3.3 MTT assay
Cell viability was determined and analyzed by the CellTiter96 Non-Radioactive cell
proliferation assay (MTT) (Promega, Madison, WI, U.S.A.). MCF-7 and MDA-MB-231
cells were cultured and plated on to 96-well plates at a density 100,000 cells/ml for 48 h
prior to the first treatment. The cells were treated with 2.5%, 5%, 10%, 15%, and 20%
fNJ (v/v) for a total of 96 h. Dosages were calculated based on the recommended dose
made by commercial noni juice companies. An average adult is recommended to
consume two ounces twice a day, which equates to 59.14 milliliters twice a day. Since an
average adult has a blood volume of 5 liters, the average adult would be consuming
1.18% of its blood volume per serving and 4.72% over the course of two days.
3.4 Human apoptosis antibody array
The expression profile of apoptosis-related proteins was detected and analyzed using a
human apoptosis array kit (R&D Systems, Minneapolis, MN, U.S.A.) as per
manufacturer’s instructions. This array contains duplicate spots of 35 apoptosis-related
proteins. Briefly, the membrane containing immobilized apoptosis-related antibodies was
blocked with bovine serum albumin for 1 h on a rocking platform at room temperature.
The membrane was then incubated with MCF-7 or MDA-MB-231 cell lysate treated with
or without fNJ along with detection antibody cocktail overnight at 2°C to 8°C on a
rocking platform. The membranes were incubated with streptavidin-horseradish
peroxidase conjugate followed by chemiluminescent detection reagent. The membranes
25
were scanned and pixel density was presented by quantifying the mean spot densities
from two experiments.
3.5 Western Blot
After the in vitro experiments, protein extracts were prepared from the cells using special
solubilizing buffer (SSB) containing 25 mMTris-HCl (pH 7.4), 2 mM Na3VO4, 10
mMNaF, 10 mM Na4P2O7, 1 mM EGTA, 1 mM EDTA, and 1 mM PMSF. After protein
was extracted, its concentration was determined by the Bradford assay (Bio-Rad
Laboratories, Hercules, CA, U.S.A). Equal amounts of protein extracts were separated
on a 4–10% gradient polyacrylamide gel and thenelectrotransferred to polyvinyl
diflouride (PVDF) membrane using a Bio-Rad mini-transfer tank. PVDF membranes
were activated in 100% methanol for 1 minute. Membranes were incubated with primary
antibodies overnight. The protein bands were detected with HRP-conjugated secondary
antibodies and then imaged developed on a film using Pierce enhanced
chemiluminescence (ECL) western blotting substrate (Thermo Scientific, Rockford, IL,
CA, USA). Membranes were stripped with Re-blot (Millipore, Temecula, CA, USA) for
20min minutes at room temperature and reprobed with antibodies to β-tubulin to control
for differences in loading.
The primary antibodies used were survivin, claspin, cytochrome-c, and NFkB (Santa
Cruz Biotechnology, Santa Cruz, CA, U.S.A.). The secondary antibodies used was goat
anti-mouse from Santa Cruz (Santa Cruz Biotechnology,Santa Cruz, CA. U.S.A.).
3.6 Statistical Methods
26
All tabulated data is presented as mean ± SEM. Analysis of statistical significance and
graphing was done using GraphPad Prism 5. P-values of P< 0.05 were considered to be
statistically significant. Means were tested for statistical significance difference using
one-way ANOVA and Tukey’s post-test analysis.
27
Chapter 4: Results
4.1 fNJ decreases cell viability in breast carcinoma cells
MTT assays wereperformed on MCF-7 and MDA-MB-231 cells after treated once
with 2.5%, 5%, 10%, 15%, and 20%fNJ (v/v) over the course of 48 hours. No cell death
was observed in cells that received only one treatment. Therefore two treatments were
used over the course of 96 hours (figure 10 and 11). In both cell lines, cell viability has
been observed to be significantly lower in all the treatments except for 2.5% fNJ (v/v) as
compared to control. For both breast carcinoma cell lines, 40% - 60% cell death was
observed in the 15% fNJ treated as compared to control. 20% fNJ treated cells have been
observed to have 70% - 85% cell death as compared to control. Based on these results,
15% fNJ was chosen for further experiments since higher doses induced cell death by
80%, while lower doses were non-toxic. Although MTT assay results show significant
change in cell viability in treated as compared to control in both cell lines, visual
observation shows that the cell death in treated MDAMB-231 cells are more apparent
than treated MCF-7 cells. In order to determine the toxicity of fNJ against non-
carcinoma cells, MCF10A, a normal breast epithelial cell line was treated under the same
conditions as the breast carcinoma cell lines (figure 12). MTT assay results showed that
there is no significant decrease of cell viability in any of the treatments compared to
control in MCF-10A normal human breast epithelial cells.
28
4.2 Apoptotic proteins identified in fNJ treated breast carcinoma cells
An apoptosis array was used to determine the changes in apoptotic protein levels
in 15% fNJ (v/v) treated breast cancer cells compared to control. The apoptosis array
detects 48 different proteins that are related to the downstream or upstream down-
regulation or up-regulation of apoptosis (figure 13). In 15% fNJ treated MCF-7 cells, a
significant decrease of B-cell lymphoma 2 (Bcl-2), cellular inhibitor of apoptosis protein
1(cIAP-1),survivin, claspin, and phosphorylated Rad 17 (phospho-Rad17) has been
observed compared to control (figure 14). Survivin and claspin resulted in a 60%
decrease, which were the highest amount of decrease compared to other proteins
observed. In addition, a significant 70% increase of heat shock protein 60 (HSP60) has
been observed in treated compared to control.
In 15% fNJ treated MDA-MB231 cells, a significant decrease was observed in
cIAP-1, cellular inhibitor of apoptosis protein 2 (cIAP-2), survivin, claspin, and fibroblast
associated tumor necrosis factor receptor super family member 6 (Fas/TNFRSF6) as
compared to control (figure 15). Fas/TNFRSF6 and cIAP-2 resulted in an 85% and 70%
decrease, respectively, which were the highest amount of decrease compared to the other
proteins. Subsequently, a 20% increase of clusterin has been observed in the treated
samples compared to control.
4.3 fNJ inhibits survivinin MCF-7 (ER+ve) cells

Western blots were used to further verify the change in apoptotic proteins
identified from the Apoptosis Array. In the apoptosis array, 15% fNJ treated breast cells,
MCF-7 displayed significantly lower levels of survivin and cIAP-1 compared to control.
Both survivin and cIAP-1 belong to a class of proteins called the inhibitors of apoptosis
29
(IAP) proteins. Western blot analysis demonstrated that there was a 40 – 60% decrease
in survivin protein levels in 15% fNJ treated breast carcinoma cells as compared to
control (figure 16).
4.4 fNJ up-regulates cytosolic levels of cytochrome c in MCF-7 (ER+ve) cells

In the apoptosis array, Bcl-2 levels were observed to be significantly lowered in
the 15% treated MCF-7 cells compared to control. Since Bcl-2 is known to inhibit the
release of cytochrome c from the mitochrondria to the cytosol, western blots were used to
detect the levels of cytochrome c in cytosolic protein extractions of MCF-7 cells in 15%
fNJ treated and control. Western blot analysis demonstrated a significant 200% increase
in the treated compared to control (figure 17).
4.5 fNJ does not affect cytosolic and nuclear levels of nFκB in MCF-7 (ER+ve) cells
Western blot analysis was used to determine cytosolic and nuclear protein levels
of the pro-inflammatory and pro-survival transcription factor, nFκB (figures 18 and 19).
No significant changes were observed in the 15% fNJ treated MCF-7 cells compared to
control.
4.6 fNJ inhibits survivin in MDAMB-231 (ER-ve) cells

Western blots were used to further verify the change in apoptotic proteins
identified from the Apoptosis Array. In the apoptosis array, 15% fNJ treated breast cells,
MDAMB-231 displayed significantly lower levels of surviving, cIAP-1 and cIAP-2
compared to control. Survivin, cIAP-1 and cIAP-2, all belong to a class of proteins
called the inhibitors of apoptosis (IAP) proteins. Western blot analysis demonstrated that
30
there was a 40 – 50% decrease in survivin protein levels in 15% fNJ treated breast
carcinoma cells as compared to control (figure 20).
4.7 fNJ does not affect cytosolic levels of cytochrome c in MDAMB-231 (ER-ve)
cells
In the apoptosis array, Bcl-2 levels were observed to have no significant difference in the
15% treated MDAMB-231 cells compared to control. Since Bcl-2 is known to inhibit the
release of cytochrome c from the mitochrondria to the cytosol, western blots were used to
detect the levels of cytochrome c in cytosolic protein extractions of MDAMB-231 cells in
15% fNJ treated and control. Western blot analysis confirm the apoptosis array by
showing no significant changes in the treated MDAMB-231 cells compared to control
(figure 21).
4.8 fNJ does not affect cytosolic and nuclear levels of nFκB in MDAM-231 (ER-ve)
cells
Western blot analysis was used to determine cytosolic and nuclear protein levels of the
pro-inflammatory and pro-survival transcription factor, nFκB (figures 22 and 23). No
significant changes were observed in the 15% fNJ treated MDAMB-231 cells compared
to control.
31
4.9Figures of results
Figure 8. Phase contrast photomicrographs of MCF-7 cells treated with fNJ for 96 hours
(magnification x 200)
(A) Untreated control (B) 15% fNJ (v/v) (C) 20% fNJ (v/v)
Figure 9. Phase contrast photomicrographs of MDAMB-231 cells treated with fNJ for 96
hours (magnification x 200)
(A) Untreated control (B) 15% fNJ (v/v) (C) 20% fNJ (v/v)
32
Figure 10. Effects of fNJ on MCF7 cell viability
Two independent experiments were performed in replicates of seven. Data is represented
a, b
as percentages of control and as mean ± SE (n=14). Mean values with common letters
to control do not differ (p<0.05).
33
Figure 11. Effects of fNJ on MDAMB-231 cell viability
a, b
34
Figure 12. Effects of fNJ on MCF-10A cell viability
a, b
35
Figure 13. Human apoptosis array overlay
36
Figure 14. Effects of 15% fNJ (v/v) on apoptotic proteins in MCF-7 cells as compared to
control.
Two independent experiments were performed in triplicate. Values are mean ± SE (n=6).
a, b
Mean values with common letters do not differ (p<0.05).
Figure 15. Effects of 15% fNJ (v/v) on apoptotic proteins in MDAMB-231 cells as
compared to control.
Two independent experiments were performed in triplicate. Values are mean ± SE (n=6).
a, b
Mean values with common letters do not differ (p<0.05).
37
Figure 16. fNJ reduced survivin levels in MCF-7 cells
Figure 16A shows images of the blots.Figure 16B represents the relative intensity of the
bands measured by densitometry units. Two independent experiments were performed in

a, b
triplicate. Values are mean ± SE (n=6). Mean values with common letters do not differ
(p<0.05).
38
Figure 17. fNJ increases cytosolic cytochrome c in MCF-7 cells
Figure 17A shows images of the blots. Figure 17B represents the relative intensity of the

a, b
(p<0.05).
39
Figure 18. fNJ did not affect changes in cytosolic nFkB levels in MCF-7 cells

a, b
(p<0.05).
40
Figure 19. fNJ did not affect changes in nuclear nFkB in MCF-7 cells
Figure 19A shows images of the blots. Figure19B represents the relative intensity of the

a, b
(p<0.05).
41
Figure 20. fNJ reduced survivin levels MDAMB231

a, b
(p<0.05).
42
Figure 21.fNJdid not affect cytosolic levels of cytochrome c in MDAMB-231 cells

a, b
(p<0.05).
43
Figure 22.fNJ does not affect cytosolic nFkB in MDAMB-231 cells

a, b
(p<0.05).
44
Figure 23. fNJ did not affect nuclear nFkB levels in MDAMB-231 cells

a, b
(p<0.05).
45
Chapter 5: Discussion
In both MCF-7 and MDAMB-231 breast carcinoma cell lines, cell death was
observed in fNJ treated cells. Normal cells grown on a culture exhibit a behavior called
contact inhibition where they limit themselves to grow in a single layer[1]. Cancer cells
do not exhibit this behavior. Instead, cancerous cells tend to grow uncontrollably and
overlap each other[1]. Visual observation of the control breast carcinoma cells confirms
that behavior.MCF-7 and MDAMB-231 cells that were treated with fNJ cease to grow
uncontrollably. Rounding of cells was observed in the fNJtreated group, indicating cell
death possibly via apoptosis. 15% fNJ dosage was chosen for further experimentation
because if caused a significant amount of cell death yet it was not severely toxic to the
cells. Since an average adult has about 5 liters of blood, a 15% fNJ treatment would
mean that an average adult would have to consume 375 mililiters of fNJ per day.
Previous studies conducted by West et al. demonstrated that noni juice is clinically safe
even when consumed in amounts as high as 750 mililiters per day[111].Resulting data
from MTT assay demonstrates that cell viability decreases as fNJ treatment increases,
which agrees with visual observations. This suggests that fNJ somehow causes cell death
in MCF-7 and MDAMB-231.
Apoptosis is a form of intrinsic programmed cell death that can be characterized
by several morphological features such as cell shrinkage, membrane blebbing, and
nuclear DNA fragmentation [112]. Apoptosis plays a crucial role in regulating cell
growth and tissue homeostasis. Therefore, an important factor to consider in anticancer
46
research, are the extrinsic and intrinsic signal transduction pathways of apoptosis. The
intrinsic pathway of apoptosis can be activated via the mitochondria or extrinsic receptors
such as TRAIL or FADD (figure 24)[113].The mitochrondrial apoptosis pathway initiates
apoptosis through the release of cytochrome c, apoptosis inducing factor (AIF), or
Smac/DIABLO. The release of these proapoptotic proteins into the cytosol activates cell
death via the caspase cascade [113-115]. In cancer, the uncontrolled growth of abnormal
cells may be induced by anti apoptotic proteins such as survivin, Bcl-2, XIAP, cIAP-1
and cIAP-2. By examining the effects of fNJ on a molecular level, a specific molecular
approach to developing anticancer therapies can be developed.
Figure 24. Extrinsic and intrinsic pathways of apoptosis [source: Fulda, S. and Debatian, K.M.,
Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy.Oncogene, 2006.25: p.
4798-4811.]
47
In order to investigate the apoptotic mechanisms involved in cell death, an
apoptosis array was used to view the changes of apoptotic protein levels between control
and 15% fNJ treated MCF-7 and MDAMB-231 cells. Inbreast cancer cells, significant
reductions in cIAP-1, cIAP-2, Bcl-2, survivin, and claspin were observed in the 15% fNJ
group. A reduction of claspin has been observed in treated cells compared to control.
Claspin is a protein involved cell cycle arrest [116]. When DNA damage occurs, the
cells will either repair the damage or induce cell cycle arrest. ATR phosphorylates
claspin then recruits Plx1 to claspin causing claspin to dissociate from the chromatin.
The dissociation of claspindownregulates Chk1 which inhibits mitosis [116]. Thus, the
reduction of claspin indicates the anti-proliferative effect of fNJ.
A common characteristic seen in cancer is the dysregulation of apoptosis[117].
Therefore proteins involved in pro or anti apoptotic signals are of interest when it comes
to cancer research. Survivin and cIAP-1 belong to a class of proteins call the Inhibitor of
Apoptosis (IAP) proteins, which are known to suppress apoptotic cell death[118]. An
overexpression of IAP proteins is commonly observed in human cancers and has been
associated with tumor progression and treatment failure [119]. Thus, IAP proteins are
potential targets for cancer therapy and drug development.Survivin is an IAP that is
commonly expressed in breast cancer tissues and plays a big role in cell survival and
chemoresistance by inhibiting caspases 3 and 9[120, 121]. Results show that MCF-7 and
MDAMB-231 cells treated with 15% fNJ have approximately 50% less survivin than the
non-treated control. This suggests that fNJ inhibits the expression of survivin in breast
cancer cells. Previous studies have shown that IAPs such as, cIAP-1, cIAP-2, survivin,
and XIAP, block apoptosis byinhibiting caspases 3 and 9 downstream of Bax, Bik, Bak,
48
and cytochrome c[115, 118, 122]. Thus suggesting that fNJ’s pro-apoptotic effects
contribute to the inhibition of IAPs, which in return activates caspases 3 and 9.
Bcl-2 is a protein that is located mainly in the nuclear membrane, endoplasmic
reticulum and mitochondrial membrane [123]. It is known to inhibit the release of
cytochrome c from the mitochondria to the cytosol and therefore inhibiting caspases 3
and 9 induced apoptosis[114, 122, 123]. Results show that MCF-7 cells treated with 15%
fNJ significantly reduces Bcl-2 levels by 40% compared to non-treated control. In
addition, western blot results show an increase in cytosolic cytochrome c levels by 300%
in treated compared to control. These results demonstrate the indirect correlation of Bcl-
2 and cytosolic cytochrome c published by Klucket al., indicating that fNJ inhibits Bcl-2
therefore allowing the release of cytosolic cytochrome c and activating apoptosis through
caspases 3 and 9. These findings suggest the fNJ induces apoptosis in MCF-7 ER+ve
cells through the mitochondrial apoptosis pathway. The same results were not found in
MDAMB231 ER-ve cells. There were no significant changes in cytosolic cytochrome c
levels in 15% fNJ treated MDAMB-231 cells compared to control, suggesting that fNJ
does not induce apoptosis in MDAMB-231 cells through the mitochondrial apoptosis
pathway.
49
Figure 25.Roles of Bcl-2[source: Hengartner, Michael O., The biochemistry of apoptosis. Nature
2000. 407: p. 770-776.]
The anti apoptotic proteins discusses earlier (Bcl-2, cIAP-1, cIAP-2, and survivin)
are regulated by a pro-inflammatory transcription factor, NfκB[124]. NfκB works
upstream and controls the gene expression of certain IAP proteins [118, 125]. Evidence
suggests that NfκBmediate desensitization, chemoresistance, and radio resistance [126].
Consequently, Hirazumiet al. demonstrated that noni-ppt increased levels of TNF-α,
which functionsextrinsically upstream of NfκB[61]. To further verify the role of NfκB,
our results have demonstrated a significant decrease in tumor necrosis factor receptors
(TNFR) in 15% fNJ treated MCF-7 and MDAMB-231 cells compared to control. Since
inactive NfκB remain in the cytosol of cells [124], changes in NfκB levels wereexpected
to be seen in nuclear and cytosolic protein extracts of fNJ treated MCF-7 and MDAMB-
231. However, no changes in cytosolic and nuclear NfκB levels were observed in both
breast carcinoma cells lines. Although previous studies have indicated the early
50
activating properties of NfκB[127, 128]. Since the proteins were extracted 96 hours after
treatment, perhaps NfκB has already activated transcription and left the cell’s nucleus.
Many active chemical constituents in noni have been isolated and identified.
Studies have been conducted to identify the anticancer signal transduction pathways
induced by chemicals found in noni such as anthraquinone, scopoletin and damnacanthal.
For example, damnacanthal isolated from noni have been demonstrated to induced
apoptosis by activating caspase [105]. Our data has indicated that naturally fermented
noni fruit juice as a whole is also as effective as the isolated components.
51
Figure 26. The role of NF-kB in apoptosis.[source: Aggarwal, Bharat B., Nuclear factor-
kB: the enemy within.Cancer Cell, 2006.6: p. 203-208.]
(A) NfκB is activated by inflammatory agents, carcinogens, infection, cytokines, and
tumor promoters. (B) NfκB activates the expression of genes related to cell survival,
proliferation, angiogenesis, and inflammation.
52
Chapter 6: Conclusion
Although several studies have shown the apoptotic effects of fNJ in in vivo
models, to our knowledge, no studies have been conducted to identify the molecular
mechanisms association with the anti-cancer effects of fNJ.Our studies have shown
thatfNJ induces apoptosis in human breast carcinoma, MCF-7 and MDAMB-231 cells
through various intrinsic pathways.In both cell lines, fNJ is able to inhibit anti-apoptotic
proteins thus allowing the breast cancer cells to undergo apoptosis via caspases 3 and 9.
In addition, fNJ inhibited anti-apoptotic protein, Bcl-2 in treated MCF-7 cells. The
inhibition of Bcl-2 allows the release of cytochrome c into the cytosol. High cytosolic
levels of cytochrome c induced apoptosis by activating the caspase cascade.
Studies on the molecular mechanisms of breast cancer offer different and possibly
more selective treatments. Although hormone treatments are available for women with
ER+ve breast carcinoma, women suffering from ER-ve breast carcinoma will have to
turn to non-selective treatments such as chemical or radiation therapy. fNJ delivers
promising results as a selective treatment for ER+ve and ER-ve breast cancer.
6.1 Significance
Currently, the popular methods of cancer treatment are surgery, chemotherapy,
radiotherapy and hormone therapy (www.breastcancer.org). Unfortunately each
treatment has a side effect. More importantly, these treatments, besides hormone therapy,
are systematic and will kill normal healthy cells therefore leaving the patients with a
number of side effects, such as hair loss, lowered immune system, nausea, headache, and
53
possiblychemoresistanceand radioresistance. fNJcan potentially offer a cost friendly and
selective way of battling breast cancer.
6.2 Future studies

Since apoptosis is facilitated through extrinsic and intrinsic pathways [112], it is
important to examine all aspects of it. Therefore, our lab will be not only further
analyzing the intrinsic pathways by looking at the presence of caspase, but also the
extrinsic pathway by looking at the presence of TNF-α and TNFR. In addition, our lab
will be reexamining the pro-survival activites of early activating transcription factor,
nFκB by extracting proteins 1-4 hours post treatment.
Anti-proliferation is an important factor to consider when looking at cancer
therapies. Our studies contain preliminary data demonstrating the anti-proliferative
effects of fNJ. Further studies will be conducted to verify it by looking in other proteins
such as claspin, phosphorylated claspin, and chk1 in treated compared to control.
Many commercial noni fruit juice products are not fermented. Therefore a study
on the comparison of the fermented and non-fermented fruit juices will be beneficial to
their consumers. In addition, chemical fractionation and analysis is warranted to identify
the constituents responsible for the anti-cancerous effects of noni fruit juice.
54
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Anti-Carcinogenic Effects of Morinda citrifolia (Noni): Identifying Signal Transduction

A thesis submitted to the Graduate Division of the University of Hawai’i at Mānoa in

partial fulfillment of the requirements for the degree of

Molecular Biosciences and Bioengineering

Pratibha V. Nerurkar – Chairperson

Title: Anti-carcinogenic effects of Morindacitrifolia(noni): Identifying signal

PratibhaNerurkar, Chairperson ____________________________

Scot Nelson ____________________________

To my brother, Andy Hwang.

counsel and guidance.

colleagues who trained me.

compared to other ethnic groups in Hawaii. An increase in the use of alternative

medicine, specifically by breast cancer patients prompted us to study the anti-cancer

effects of Hawaiian traditional medicine, Morinda citritfolia (noni). Estrogen receptor

50 – 60% of the cells. In addition, when non-carcinoma breast cells, MCF-10A

shown a significant decrease of a class of proteins called inhibitors of apoptosis proteins

MDAMB-231. [Grants: NCCAM (R21AT003719), USDA-CREES (2004-34135-

Dedication .......................................................................................................................... iii

Table of contents ................................................................................................................ vi

List of figures ................................................................................................................... viii

List of Tables ...................................................................................................................... x

List of Abbreviations ......................................................................................................... xi

Chapter 1: Introduction ....................................................................................................... 1

1.1 Breast Cancer ........................................................................................................... 1

1.2 Complementary and Alternative Medicine .............................................................. 7

1.3 Morinda citrofolia (Noni) ........................................................................................ 9

1.4 Noni and Cancer .................................................................................................. 14

Chapter 2: Hypothesis and Aims ...................................................................................... 21

Chapter 4: Results ............................................................................................................. 28

Chapter 5: Discussion ....................................................................................................... 46

Figure 1. Ductal and Lobular Carcinoma In Situ ................................................................ 2

Figure 2. Modifiable risk factors that affect cancer ............................................................ 4

Figure 3. General transcription mechanisms of estrogen receptor positive and estrogen

Figure 4. Total out-of-pocket complementary and alternative medicine costs among

Figure 5. Noni fruit ............................................................................................................. 9

Figure 6. Encapsulated freeze-dried noni fruit ................................................................. 13

Figure 7. fNJ induces apoptosis through hypothesized pathways .................................... 21

Figure 10. Effects of fNJ on MCF7 cell viability ............................................................. 33

Figure 11. Effects of fNJ on MDAMB-231 cell viability................................................. 34

Figure 12. Effects of fNJ on MCF-10A cell viability ....................................................... 35

Figure 13. Human apoptosis array overlay ....................................................................... 36

Figure 16. fNJ reduced survivin levels in MCF-7 cells .................................................... 38

Figure 17. fNJ increases cytosolic cytochrome c in MCF-7 cells .................................... 39

Figure 24. Extrinsic and intrinsic pathways of apoptosis ................................................. 47

Figure 25. Roles of Bcl-2 .................................................................................................. 50

Figure 26.The role of NF-kB in apoptosis ........................................................................ 52

Table 1. Traditional uses of the noni ................................................................................ 10

Table 2. Modern uses of noni .......................................................................................... 12

Table 3.In vitro studies on the effects of noni and cancer................................................ 17

Table 4. Summary of molecular mechanisms involved in the effects of noni against

AP1 Activator protein 1

ATM Ataxia telangiecstasia mutated

Bcl-2 B-cell lymphoma 2

BMI Body mass index

BRCA1 Breast cancer type 1

CAM Complementary and alternative medicine

CHEK2 Dysfunctional check point homolog 2

cIAP-1 Cellular inhibitor of apoptosis protein 1

DCIS Ductal carcinoma in situ

EGF Epidermal growth factor

eIF-2α Eukaryotic translation initiation factor-2α

ER-ve Estrogen receptor negative

ER+ve Estrogen receptor positive