The Cytotoxic Effects of Morinda Citrifolia Extracts Through TLR4 in Human Breast Cancer Cells

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DigitalCommons@Robert W. Woodruff Library, Atlanta University Center ETD Collection for AUC Robert W. Woodruff Library

5-1-2009

The Cytotoxic Effects of Morinda Citrifolia Extracts Through TLR4 in Human Breast Cancer Cells Sabrenia M. Parker Clark Atlanta University

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THE CYTOTOXIC EFFECTS OF MORINDA CITRIFOLIA THROUGH TLR4 IN HUMAN BREAST CANCER CELLS

A DISSERTATION SUBMITTED TO THE FACULTY OF CLARK ATLANTA UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

BY SABRENIA M. PARKER

DEPARTMENT OF CHEMISTRY

ATLANTA, GEORGIA MAY 2009

© 2009 SABRENIA M. PARKER All Rights Reserved

Contents ACKNOWLEDGMENTS..

11

LIST OF FIGURES

x

LIST OF TABLES

xii

LIST OF ABBREVIATIONS

xlii

CHAPTER 1: INTRODUCTION 1.1 Background and Review of Literature

1

1.1.1 What is Complementary and Alternative Medicine9

1

1.1.2 What is Morinda citr~folia9

3

1.1.2.1 Origin

3

1.1.2.2 Traditional and Modern Uses

3

1.1.2.3 ProfihingM. citr~folia

6

1.1.2.4 Evidence of Bio logical Activity

12

1.1.3 Breast Cancer

15

1.1.3.1 Prevalence

15

1.1.3.2 Risk Factors

17

1.1.3.3 Etiology

17

1.1.4 Immunology: Innate and Adaptive Immunity

19

1.1.4.1 Immunity

19

1.1.4.2 Toll-like Receptors

20

1.1.4.2.1 Summary of the TLR Signaling Pathway. 1.1.4.2.2 Toll-like Receptor 4

.

.

22 25

1.1.5 Recent Patents in the TLR Pathway 1.1.5.1 Toll-like Receptor Patents.

28 .

28

.

1.1.5.2 TLR variants

28

1.1.5.3 Adaptor proteins

30

1.1.6 Cancer Cells and Immune Surveillance.

31

1.2 Specific Aims

33

CHAPTER 2: RESEARCH DESIGN AND METHODOLOGY 2.1 Subject Stock

37

2.1.1 Cell lines and culture

37

2.1.2 Morindacitrifolia

37

2.2 Empirical Data and Collection

38

2.2.1 Small-scale extractions of noni powder

38

2.2.2 Large-scale extractions of noni powder in BuOH

41

2.2.3 Analysis of cell cytotoxicity

41

2.2.3.1 Single treatment with noni extraction dilutions 2.2.3.2 Multiple treatments with noni extraction dilutions 2.2.3.3 Treatment with BuNoni dilutions plus LPS-RS

41 .

.

.

42 42

2.2.4 GeneChip Target Preparation and Hybridization

43

2.2.5 Microarray Data Analysis

44

2.2.6 Semiquantitative Reverse Transcription PCR

44

2.2.6.1 RT-PCR verification

47

2.2.6.2 Western blot analysis

48

vii

2.2.7 CytoTox~ONETM Homogeneous Membrane Integrity Assay.

.

.

50

2.2.8 Wound-healing Assay

51

2.2.9 Colony formation Assay

51

2.2.10 FACS analysis and Annexin V/PE Assay

52

2.2.10.1 Analysis of cellular DNA content by flow cytometry. 52 2.2.10.2 Assessment ofApoptosis 2.2.11 IC50 (Dose-response curve) Assay 2.3 Statistical Analysis

53 53 54

CHAPTER 3: RESULTS AND DISCUSSION 3.1. Noni Characterized

55

3.2 Results Observed

55

3.2.1 Extracts of Noni display cytotoxic effects

55

3.2.2 BuNoni’s IC50 value in cell proliferation

62

3.2.3 Gene expression profiles identify cytotoxic pathways

• 63

3.2.3.1 Microarray results were confirmed

78

3.2.3.2 Signaling pathway was analyzed

80

3.2.4 TLR4 agonist induces cell death in human BrCa cells 3.2.5 Viability assays used to show the cytotoxic effect of BuNoni.

• 86 91

3.2.5.1 BuNoni extract caused a decrease in LDH

91

3.2.5.2 FACS shows BuNoni extract caused apoptotic effect.

93

3.2.6 BuNoni inhibits cell migration and alters cell structure

viii

98

3.2.7 BuNoni prevents tumor formation

.



3.3 Discussion of Results

.





102



.



104

3.3.1 Effect of extraction on cell proliferation

• .111

3.3.2 Clinical Impact of the Research 3.4 Conclusion





.

.

.

.

112

.118

3.5 Future Directions and Impact

121

4.0 References

125

LIST OF FIGURES

Figure

Page

1.

Morinda citr~folia plant

4

2.

Structures of glycosidic sugars isolated from M. citrifolia

11

3.

Toll-like Receptor Pathway

24

4.

Multi-solvent Survival Curves

59

5.

BuOH Survival Curve with MDA-MB-231 cells

62

6.

Dose Response Curve of BuNoni on MDA-MB-23 1 cells

63

7.

RT-PCR Confirmations using BuNoniILPS

80

8.

Ingenuity Pathway

82

9.

RT-PCR Gene Confirmation

85

10.

TLR4 Expression on Breast Cancer Cell Line Panel

86

11.

Comparison of Glycosides from BuNoni extract

87

12.

Effect of BuNoni on Proliferation ofMDA-JvfB-231 Cells

89

13.

Effect of BuNoni on Proliferation of T47-D Cells

89

14.

Effect of LPS-RS on MDA-MB-231 Cell Survival

91

15.

Effect of LPS-RS on MDA-MB-23 1 Cells % Recovery

91

16.

Lactate Dehydrogenase Measurement

94

17.

Flow Cytometric Analysis

96

18.

IRAK-1 Analysis

97

19.

Caspase-7 Analysis

98

20.

PARP/IRAK-4/IRAK-2 Analysis

98

21.

Wound Healing ~a1ysis

~ 100

22.

Morphology Observation

103

23.

Colony Formation Assay Results

105

24.

TLR4 Pathway Leading to Apoptosis

111

25.

Toll-like Receptor Pathway with BuNoni

116

LIST OF TABLES

Table

Page

1.

List of Compounds Found in Morinda citr~folia.

2.

List of Toll-like Receptors and Their Mutants

29

3.

List of Adaptor Proteins

31

4.

List of Solvent Abbreviations

39

5.

Microscale Extraction Solvents

40

6.

List of RT-PCR Primers

45

7.

List of 1°Antibodies

49

8.

List of 2° Antibodies

50

9.

Microarray List of Regulated Genes

65

10.

List of Select Regulated Genes

77

11.

Description of Human BrCa Cell Lines.

xii

.

.

.

.

.

.

7

84

LIST OF ABBREVIATIONS AM

Alternative Medicine

AMPK

AMP-activated protein kinase

AP-l

Activator protein-i

BMI

Body Mass Index

BrCa

Breast Cancer

BuOH

Butanol

BuNoni

Butanol Noni extract

C. albicans

Candida albicans

CAM

Complementary and Alternative Medicine

CD14

Cluster of differentiation 14

cRNA

Copy ribonucleic acid

CHC13

Chloroform

CM

Complementary Medicine

CpG

Cytosine—phosphate-—-Guanine

DMSO

Dimethyl sulfoxide

DNA

Deoxyribonucleic acid

cDNA

Copy deoxyribonucleic acid

DPPH

1,1 -Diphenyl-2-Picrylhydrazyl

DTT

Dithiotbreitol

DXR

Doxorubicin

EDTA

Ethylenedinitrile tetra-acetic acid

EGFR

Epidermal growth factor receptor

xiii

EGTA

Ethylene glycol tetra-acetic acid

ER

Estrogen Receptor

Era

Estrogen receptor-a

EtBr

Ethidium bromide

EtOH

Ethanol

FAAD

Fas-Associated Death Domain Protein

FACS

Fluorescence-activated cell sorter

FBS

Fetal Bovine Serum

Fyn

Human gene ofprotein-tyrosine kinase oncogene family

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

GPR3O

G Protein-coupled Receptor 30

HC1

Hydrochloric acid

HepOH

Heptanol

HER2

Human epidermal growth factor receptor-2

HexOH

Hexanol

HIV

Human immunodeficiency virus

HRP

Horseradish peroxidase-conjugated

HRT

Hormone replacement therapy

1C50

Half maximal inhibitory concentration

IFN-~3

Interferon-Beta

IgG

Immunoglobulin G

IHC

Immunohisto chemistry

IKK

Inhibitor of Kappa Light Polypeptide Gene Enhancer in B Cells Kinase

xiv

IL-i

Interleukin- 1

IL-i R

Interleukin- 1 Receptor

IRAK

Interleukin (IL)- 1 Receptor—associated Kinase

IRF

Interferon regulatory factor

JNK

c-Jun N-terminal Kinase

Lck

Leukocyte-specific protein tyrosine kinase

LDH

Lactate dehydrogenase

LLC

Lewis Lung Carcinoma

LPS

Lipopolysaccharide

LPS-RS

Rhodobacter sphaeroides lipopolysaccaride

luc

Luciferase-positive

Lyn

V-yes-i Yamaguchi sarcoma viral related oncogene homolog

M. citrifolia

Morinda citrifolia

MAPK

Mitogen-activated protein kinases

MD-2

Lymphocyte antigen 96

MeOH

Methanol

MgC12

Magnesium Chloride

MKK

Mitogen-Activated Protein Kinase Kinase

mRNA

Messenger ribonucleic acid

MS

Mass Spectometry

MSDS

Material safety data sheet

MyD88

Myeloid differentiation primary response gene (88)

NaC1

Sodium Chloride

NF-icB

Nuclear Factor-Kappa B

NIH

National Institutes of Health

NMR

Nuclear magnetic resonance

OctOH

Octanol

OXL

Oxaliplatin

PBS

Phosphate buffered saline

PE

Phycoerythrin

PenOH

Pentanol

P1

Propidium iodide

PR

Progesterone Receptor

PrOH

Propanol

PVDF

Polyvinylidene Fluoride membrane

RNA

Ribonucleic acid

RPMI

Roswell Park Memorial Institute

RT

Reverse transcriptase

RTPCR

Reverse transcription-polymerase chain reaction

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Ser/Thr

Serine/Threonine

shRNA

Short hairpin RNA

Src

A family of proto-oncogenic tyrosine kinases

TBE

Tris-Borate-EDTA buffer

TBK-1

TANK-binding kinase 1

TBST

Tris-Buffered Saline Tween-20

xvi

TC

Cytotoxic T cell

TIR

Toll-interleukin 1 receptor

TIRAP

Toll-interleukin 1 receptor (TIR) domain-containing adaptor protein

TLR

Toll-like Receptor

TNF

Tumor necrosis factor

TNJ

Tahitian Noni Juice®

TRAF

TNF Receptor Associated Factor

TRAM

TRIF-related adaptor molecule

Treg

Thymus regulatory cells

TRIF

TIR-domain-containing adapter-inducing interferon-~3

Tris—HC1

Tris hydrochloric acid

UVB-induced Ultraviolet radiation-induced at wavelength 290 to 320 nm

xvii

ACKNOWLEDGMENTS

This is dedicated to Reagan and Riley and the future Parker children to come. I would like to thank my family and friends for their constant encouragement and support throughout the course of this dissertation when it seemed this day would never come. More directly, I would like to thank my children for their understanding and unconditional love during this time of hard work and for believing in me and praying that I achieved my education goals to leave a new legacy for generations to come. I would like to thank the National Institutes of Health (NIH), National Center for Complementary and Alternative Medicine (NCCAM) for support from grant #5R03AT003935-02. I would also like to thank the Minority Biomedical Research Support (MBRS) Research Initiative for Scientific Enhancement (RISE) Program-NIH! National Institute of General Medical Sciences (NIGMS) for support from grant #5R25GM060414. I would like to thank the laboratories at Emory Winship Cancer Center for working with me to ensure that I completed my task at hand. I would also like to thank my professors for their patience and explanations throughout my research and this dissertation writing process. And, I would like to especially thank Dr. Paul McGeady (deceased) for giving me this opportunity and encouraging me to finish the task he started.

CHAPTER 1 INTRODUCTION 1.1 Background and Review ofLiterature 1.1.1 What is Complementary and Alternative Medicine? Complementary and alternative medicine (CAM), as used in the modern western world, covers an extensive range of medical approaches that do not fall within the realm of conventional medicine. CAM is a broad domain of healing resources that encompasses diverse health care systems and medical treatments, healing therapies, and various nontraditional medical approaches, which includes those health care practices and products not currently considered a fundamental part of traditional medicineJ’31 CAM practices complement mainstream medicine and are described by its users as preventing or treating illness, and promoting health and well being.~3’4~ The use of CAM contributes to a common whole, whether by satisfying a therapeutic demand not met by conventional practices, or by diversifying the conceptual basis of medicine. Although the two are used interchangeably, the distinction between complementary and alternative therapies is crucial.~’1 Complementary medicine (CM) is a group of diagnostic and therapeutic disciplines used in conjunction with conventional medicine. For example with complementary aromatherapy, the scent of oils from plants and trees, once inhaled, purportedly promotes health and well-being and helps to lessen a patient’s discomfort following surgery. [591 CM is typically used to supplement

2 mainstream medicine while relieving the symptoms of a disease or disorder through noninvasive treatments that carry minimal or no side effects. Although CM is usually not taught or used in Western medical schools or hospitals, the integration of conventional medicine and CM therapies is of increasing interest.~’°’41 This is, in part, because CM addresses how disease affects the whole person; ergo, it is sometimes referred to as holistic medicine. CM also includes acupuncture, herbal medicine, massage, support groups, and yoga.~517~ Alternative medicine (AM), on the other hand, is used in place of conventional medicine. AM covers a broad range of unconventional healing philosophies combined with a rich array of techniques, modalities, and approaches that use natural substances such as herbs, botanicals, homeopathics, nutritional supplements, and whole foods.~’8’ 19] Case in point, instead of undergoing surgery, radiation, or chemotherapy, conventional doctors have recommended using a special diet to treat cancer, which is just one of many examples of an AM.~20’211 Chemoprevention is another promising AM emerging in cancer research, which administers therapeutic agents to inhibit, delay, or reverse the process of carcinogenesisJ221 Although a fair amount of what is labeled AM comes to us from other cultures or from ancient healing traditions, a great deal of what is now labeled AM originated in the United States. Alternative therapies include folk medicine, herbal medicine, diet fads, homeopathy, faith healing, new-age healing, chiropractic, acupuncture, naturopathy, and music therapy. [23~241 A small number of CAM therapies originally considered to be purely alternative approaches are finding a place in cancer treatment, though not as cures, but as complementary therapies that may help patients feel better and recover faster. Use of

3 alternative medicines, particularly herbal supplements, has increased considerably over the past two decades in the US. The American market for herbs and other botanical remedies was

$4.5 billion in 2007, and there is increasing media focus on the benefits

of these preparations.~’8’ 25-30] The total costs for conventional care when combined with CAM are observably lower than the average costs annually. However, an important gap exists between the knowledge-based statistical and mechanistic understandings of the effects of these supplements and the claims made by their distributors. No single determinant of the present popularity of CAM exists, but there is a correlation between signs of positive effects and the sales figures of commercial CAM products. Whether applied singly or in combination with conventional medicine, CAM offers maximum therapeutic benefit by incorporating plant, animal and mineral based medicines to treat, diagnose, and prevent illnesses and/or maintain well-beingJ31341

1.1.2 What is Morinda cit4folia? 1.1.2.1 Origin Identified in older botanical literature as Indian mulberry, Morinda citr~folia is a member of the family Rubiaceae and is known to modern-day users as noni It is indigenous to Southeast Asia, perhaps originating in Indonesia, and is a native tree (approximately 3



9 meters tall) found along the coastlines of many Pacific regions.~351

The noni plant grows well on any soil, including sandy shores and open rocky as well as shady forests. It is tolerant of saline soils, drought conditions, and secondary soils; hence it is found on volcanic loams, lava-strewn coasts, and clearings or limestone outcrops. Despite its indifference to growth terrains, this shrub reaches maturity in

4 about 18 months, standing heights of 15-20 feet and yielding between 4-8 kilograms (kg) of fruit every month throughout the year. The plant flowers and produces a small white flower, growing from a fleshy structure. The noni fruit is oval and medium sized of approximately 4-7 centimeters (cm) and has many seeds as shown in figure 1.[36401 At first, the fruit buds green, then turns light yellow or white when ripe; it has a pungent odor when ripening and is thus oddly known as vomit fruit. Because of its impartial ground preferences, the Pacific regions house the optimal source of climate, soil conditions and the ideal environment, which allows M. citr~folia to grow larger and more lush there.~37’38’411

0

Fig. 1. Morinda citifolia.

1.1.2.2 Traditional and Modern Uses Practically all of the noni plant is usable, contributing to its popularity throughout the Pacific. It was domesticated and cultivated by the Polynesians and Tahitians nearly 2000 years ago and was eventually adopted by Hawaiians.~35’411 Oddly,

5 the fruit is pungent and is sometimes also called the starvation or vomit fruit; notwithstanding its strong smell and bitter taste, the early Polynesians consumed noni in times of famine. Traditionally, Australian aborigines consumed whole noni fruit as did the people in Burma, who ate the ripened fruit raw with salt and cooked unripe fruits in curries and other dishes.~411 The young leaves were also eaten as a dietary source, containing 4-6% of protein, and even the seeds are edible when roasted.~421 Today, cultivators focus primarily on the fruit and its juice, but the roots and bark contain pigments that were also used by Polynesian natives as dyes for cloths.~41~ Depending upon the regional location and the needs of the native people, the traditional medicinal uses of the noni plant varies as well. Research into the medical applications of noni indicate that virtually every part of the plant is used as some form of medicine, and native healers have exploited its therapeutic qualities for ages.~411 It is the second most popular plant used in herbal remedies of Pacific Islanders, especially Hawaiians; the leaves, flowers, fruit, and bark are used to treat eye disorders, skin wounds and abscesses, gum and throat problems, respiratory ailments, constipation, and fever. Noni is also used in India for its unique ability to improve physical conditions. In Malaysia, the ripe fruits are infused with water, and then gargled to relieve sore throats, and the heated leaves are applied to the chest to alleviate coughs, nausea, and colic. It has been noted for its curative effects in both low blood pressure and high blood pressure.~431 Hirazumi et al. (1999) reported that noni is used to treat cancer, infection, arthritis, diabetes, asthma, and hypertension.~441 Other conditions treated with noni, either alone or in combination with other botanicals, include fevers, heart disease, respiratory ailments, gastrointestinal, skin disease, menstrual or urinary problems,

6 diabetes, human immunodeficiency virus (HIV), and venereal diseases.~39’431 Noni users also report that benefits of using it include immediate energy and enhanced physical performance, and many producers and distributors now offer it as a “healthier” alternative to sugary energy drinics and sodas.~45~ The National Institutes of Health (NIH) has sponsored clinical trials using well-defined preparations of noni to characterize and determine the tolerated dose of noni fruit extracts, and anti-tumor and symptom control properties of the extract. [46]

1.1.2.3 Profiling M citr~folia Chemicals from plant sources that possess protective or disease preventive properties, but lack nutritional value, are called phytochemicals and interchangeably nutraceuticals. Most identified biologically active phytochemicals possess antioxidant activity, protect eukaryotic cells against oxidative damage, and reduce the risk of developing certain types of cancers.~47’481 The M citr~folia plant has enjoyed decades in the limelight as popular folk medicine, and a variety of phytochemical constituents have been identified in the leaves, bark, stem, flowers and fruits of the plant as shown in Table 1. [35, 37, 39,41,49-62] Various components of noni have been shown to display epidemiological effects on human health; currently, there are several ongoing scientific studies using noni to analyze its chemical effects, revealing a plethora of new insights into the biological and pharmacological benefits of its use. In particular, the efficacy of noni on human cancers with respect to antitumor activity is of great interest.~42’56’631 Presumably, the biomedical interest in noni has increased over the last decade primarily because of its effects on cell proliferation and activity.

7 Table 1 List of Compounds Found in Morinda citrifolia. Noni Plant organ(s) FRUIT and FRUIT JUICE

Compound(s)

Description

Oligo- and Polysaccharides

Long-chain sugar molecules that function as dietary fiber, yielding short chain fatty acids with numerous potential health properties.

Alkaloids

Naturally occurring amines from plants.

Enzyme activity and protein structure.

Vitamins and Minerals

Magnesium; iron; potassium; selenium; zinc; copper; sulfur; ascorbic acid (vitamin C).

The positive effects of the vitamins and minerals in noni juice are documented.

Scopoletin

LEAVES, STEMS and OTHER FOLIAGE

ROOTS

Proposed effects of compounds Immunostimulatory; immuno- modulatory; antibacterial; antitumor; anticancer.

Dilates vasculature and lowers blood pressure; histamineinhibiting; allergies; Alzheimer ‘s disease.

Anthraquinones

Damnacanthal

Antiseptic and antibacterial effects in digestive tract.

Glycosides

Noni juice is a source of vitamin C.

DPPH free radical scavenging activity; inhibition ofUVB induced Activator Protein-i activity in cell cultures.

Anthraquinones

Damnacanthal

Inhibits formation of lung carcinoma in mice.

Morindin and Morindone

Flavonol glycoside; iridoids and a citrifolinoside.

Dyes, yellow and red colorants used for tapa cloth; antibacterial.

8 Recent research has identified several active phytochemicals in the noni plant, which supports the idea that these chemicals influence human physiology. Hence, the widely marketed Tahitian Noni Juice (TNJ®), which claims usefulness as a curative or preventative in disease treatment, is currently sold with the promise to “support bodily systems, increase mental clarity, and optimize physical performance.”~45’64’ 65] Although there is little scientific evidence to either support or refute these claims, a number of investigators have isolated a wide range ofbioactive substances. Among documented nutraceuticals, noni contains a polysaccharide-rich substance, glycosides, anthraquinones, iridoids and a several other bioactive plant substancesJ36’55’661 Polysaccharides play important roles in living organisms. They are sugar polymers made up of many monosaccharides that are joined together by glycosidic bonds, which are formed by a condensation reaction, in essence making them relatively complex carbohydrates.~67’681 They tend to be amorphous and insoluble in water. Polysaccharides have a general formula of C~(H2O)~- 1, where n is usually a large number between 200 and 2500. Often, six-carbon monosaccharides make up the polymer backbone of repeating units; thus, (C6HioO5)~ can also represent the general formula, where n

=

{40...3000}. They are often classified on the basis of the number of

monosaccharide types present in the molecule, and are therefore very large, often branched, macromolecules.~671 Polysaccharides can be divided into two broad groups: (1) storage polysaccharides, such as starch and glycogen, and (2) structural polysaccharides, such as cellulose and chitin; pectins are chitin of a glucose derivative. [68]

9 An alcohol-precipitate of noni fruit juice (noni-ppt) has been chemically characterized and contains predominantly pectic polysaccharides (e.g., homogalacturonan, arabinan, type I arabinogalactan, and rhamnogalacturonan).~55~ Hirazumi et al. (1994) found that noni-ppt was capable of inciting the release of several immune system mediators from other murine effector cells, including interleukin-1~3 (1 L- 113), IL- 10, other interleukins, and nitric oxide. [69] Studies also revealed that noni ppt displayed substantial anticancer activity, inhibited tumor growth, and stimulated immune responses in lung carcinomas.~42’441 As a result, enhancement of the immune response is expected to mediate anti-tumor activity by triggering macrophages to release tumoricidal mediators, resulting in suppressed tumor growth. Glycosides also play diverse important roles in the biological processes of life. Many glycosides are present in flowers and fruit pigments, various medicines (including antibiotics), condiments, and dyes derived from plants. Not all chemicals are active in plants; hence, many plants store important chemicals in the form of inactive g1ycosides.~701 In chemistry, the sugar part of this molecule is bound to a hydroxy compound i.e. a non-sugar, qualifying it as a glycoside, thus excluding the polysaccharides. If the stored chemicals are needed, they can easily become available for use by the plant; accordingly, the glyco sides are brought in contact with water and an enzyme, and the sugar part is broken off. Whereas the sugar group is called the glycone of the molecule, the non-sugar group as the aglycone moiety of the glycoside.~70’ 71] Anthraquinone glycosides contain an aglycone group that is a derivative of an aromatic organic compound, anthraquinone. Chemical analysis of noni also revealed the presence of anthraquinones, which are compounds based on anthracenes.~511

10 Anthraquinone occurs naturally in some plants (e.g. aloe, senna, and rhubarb), fungi, lichens, and serves as a basic skeleton for pigment in insects.~72’ 73] A number of anthraquinone derivatives, which include rubiadin, morindone, lucidin and damnacanthal (3 -Hydroxy- 1 -methoxyanthraquinone-2-aldehyde), have been isolated from various parts of noni as shown in figure 1 ~[721 Conventional chemotherapeutics such as doxorubicin (DXR) and mitoxanthrone belong to this family of compounds as well. Ghai et al. (US Patent 2003/0004116 Al) describes three active glycoside compounds found in the fruit pulp of noni using a butanol (BuOH) extraction method. Mass Spectrometry (MS) and multiple nuclear magnetic resonance (NMR) methods identified the glycosidic structures to be 6-O-(13-D-glucopyranosyl)-1-O-octanoyl-/3-Dglucopyranose, 6-O-Q3-D-glucopyranosyl)- 1 -O-hexanoyl-j3-D-glucopyranose and 3methylbut-3-enyl 6-O-~3-D-glucopyranosy1-f3-D-glucopyranoside as shown in figure ~ 751 In addition to the BuOH extract, several other noni extractions were used in cell proliferation and cytotoxicity assays on various cancer cell lines, where data indicated the BuOH fraction as showing the most cytotoxic effects after 24 hours of treatment. They determined that these three active compounds isolated from noni using the BuOH extraction method displayed potential anti-tumor and anti-proliferative activity as cancer prevention or treatment agents. Oral administration of these nutraceutical compositions include, but are not limited to, formulation as a food supplement, capsule or tablet as described in the U.S. Pharmacopeia, and liquid preparations in the form of syrups and dry products.~65’ 76-80]

HO

Fig. 2. Structures of glycosidic sugars found in M. citr~folia. (A.) 6-O-(~3-Dglucopyranosyl)- 1 -O-octanoy1-~3-D-g1ucopyranose, (B.) 6-O-(f3-D-glucopyranosyl)- 1-0hexanoyl-~3-D-glucopyranose, and (C.) 3-methylbut-3-enyl 6-O-/3-D-glucopyranosyl-f3D-glucopyranoside.

Iridoids are cyclopentanopyran monoterpenoids, often occurring as glycosides, found in a large number of plant families.~81’ 82] One of the first iridoids isolated from noni was asperuloside, and several others have been described more recently, including citrifolinoside extracted from noni 1eaves.~59’831 Several novel di- and tri-saccharide fatty acid esters have also been isolated from noni fruit. They contain one or two short chain fatty acids attached to glucose residues, and given their amphipathic character, may be responsible for the soapy character of the fruit.~841

12 1.1.2.4 Evidence ofBiological Activity The nutraceuticals in the M citr~folia extract have been shown to have favorable effects in cancer treatment. Different components in a botanical may possess protective or disease preventive properties; and in the noni plant, damnacanthal, iridoids, and disaccharide fatty acid esters were identified as pertinent to these preventive processes. The polysaccharide-rich substance (noni-ppt) may operate by modulating the responsiveness of immune cells, and might prevent the escape of cells with hyperactive Ras from immune surveillance. In 1994, researchers reported anticancer activity of noni-ppt on lung cancer in C57 B116 mice; they concluded that noni acts indirectly by enhancing the host immune system involving macrophages.~441

Researchers at the

University of Hawaii later showed mice implanted with Lewis Lung Carcinoma (LLC) treated with noni-ppt had a significantly prolonged life by up to 75% in comparison to the control group.~411 Berg and Furusawa further described how noni-ppt stimulated tumor necrosis factor (TNF) and interleukin 1 (IL-i) in mice.~851 Asahina et al. (1996) reported that an alcohol extract of the noni fruit hindered the production of tumour necrosis factor-a (TNF- a), an endogenous tumor promoter in macrophages.~41~ Noni ppt displayed some anticancer activity and inhibited tumor growth in syngeneic mice (meaning they are geneticallyidentical or closely related and immunologically compatible). Other reports suggest that the enhancement of anticancer drugs, in combination with noni-ppt, may be beneficial to cancer patients by allowing decreased chemotherapy and/or radiation doses.~42~ Noni-ppt has proven anti-tumor activity, improved the survival time with cancer, and it is believed to be a useful supplement in cancer treatments.

13 Those compounds in noni listed above shown to possess protective or disease preventive properties i.e., damnacanthal, iridoids, and disaccharide fatty acid esters, have been shown or hypothesized to block or inhibit the function of Ras-pathway components, which activates many signaling cascades relating to cell growth, proliferation, and survival.~86’871 Of 500 tested botanical extracts, Hiramatsu et al. (1993) found noni to be the most effective in inhibiting growth of Ras-transformed cells.~871 Ras or its homologs are present in virtually all living things, and fungi possess two Ras genes. Banerjee et al. (2006) reported that an extract of noni fruit interfered with the conversion of Candida albicans (serum-induced) from cellular yeast to a deadly filamentous form i.e., pathogenic.~881 Hence, noni is effective in combating C. albicans and it may have potential therapeutic value with regard to candidiasis, which is a fungal infection (mycosis) or yeast infection. These results were similar to those obtained in C. albicans with FPT Inhibitor III, a synthetic substrate analog inhibitor of Ras prenylation.~88’891 Moreover, the similar response to noni and a prenylation inhibitor as well as the isoprenoid character of biologically active noni components suggests that Noni may also inhibit protein prenylation. The body’s ability to balance an underactive immune system versus an active immune system is referred to as immunomodulation, and noni is thought to indirectly employ immunomodulation to suppress tumors. Researchers state that both prophylactic and therapeutic potentials against the immunomodulator sensitive Sarcoma 180 tumour system are found in the fruit juice ofM citr~folia. They reported that allogeneic mice (meaning they are genetically different although belonging to or obtained from the same species) showed a cure rate of25%-45% with respect to the antitumour activity of noni

14 ppt.~90~ In 1999, Hirazuma and Furusawa reported that the ethanol-precipitable, watersoluble form of the noni fruit juice extract illustrated antitumor and immunomodulatory activity.~44’911 While the noni-ppt effects may not directly involve Ras inactivation, in the organism they may provide adjuvant activity to inhibition of the Ras pathway by small-molecule components. The anthraquinone, damnacanthal, causes phenotypic reversion of K-Ras transformed rat kidney cells (K-Ras-NRK).~871 Damnacanthal has also been shown to be an inhibitor of the activity of certain tyrosine kinases, whose aberrant expression levels are known to be involved in oncogenic processes. Compelling inhibitory effects on tyrosine kinases, such as leukocyte-specific protein tyrosine kinase (Lck), V-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (Src), and V-yes-i Yamaguchi sarcoma viral related oncogene homolog (Lyn), as well as epidermal growth factor receptor (EGFR), were reported by Hiwasa et al.(1999).~921 Faltynek et al.(1995) showed damnacanthal was a potent mixed inhibitor of p5&clc with an IC50 of 17 nM and




0.95) between cell number and fluorescence (560

Ex/590 Em) using the LDH cytotoxicity kit was measured 48 hours after treatment. The values shown for each treatment are significantly different than the “zero” cell background fluorescence. However, the BuNoni treatments are remarkably different from all other treatments where the no treatment (No Rx) is a baseline of cells undergoing normal necrosis. There appears to be a drastic increase of LDH release in cells treated with LPS-RS alone in comparison to the control; reasons for the perceived necrotic stimulation in this assay are currently unknown since cell growth is very much apparent in the survival curves using LPS-RS treatment alone. However, 2 mg/mi BuNoni plus LPS-RS showed a decrease in LDH release in comparison to the control, but had a marked increase in LDH release in comparison to the 2 mg/ml BuNoni alone, which was highly expected, where the calculated p-value was 0.024. Ergo, the BuNoni treatments alone (2 and 5 mg/mi) show the necrosis rate to be well under 50% release in comparison to the blank and 18% and 27% of the control, respectively. This decrease in LDH release in the BuNoni treatments verifies that the cells are not dying by means of necrosis in figure 16, suggesting that LPS-RS protects cells from the effects of BuNoni, and necrosis is nil in the BuNoni treatments alone.

94 Nerosis Ass~v ~00 250-~----------------—--------—--------.— 200

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150

-

B~aiik

Nc, Rx

ETOH

LPS-RS

2mg/mi BuNoni

2mg BuNoni + LPS-RS

5mg/ml

BuNoni

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Fig. 16. Lactate dehydrogenase release by MDA-MB-23 1 cells exposed to BuNoni, where *p 0.024.

3.2.5.2 FA CS shows BuNoni extract caused apoptotic effect Flow cytometric analysis allowed quantitative measurement of live, dead, and early and late apoptotic cells in untreated cells and in response to BuNoni treatment. MDA-MB-23 1 cells were cultured with medium (RPMJ 1640 medium containing 10% FBS) alone (A), with vehicle control (B) or BuNoni extract (final concentration of 2, 3, and 5mg/ml in RPMJ 1640 medium containing

1000

FBS) with (F, G) or without 10

~g/jil LPS-RS (C, D, and E) in RPMI 1640 medium for 48 hours. Respective cells were pretreated with 10 jig/~.il LPS-RS for 1 hour prior to adding a final concentration of 2 mg/ml BuNoni plus LPS-RS. The treatment was adjusted to maintain 10 p.g j.~l LPS-RS concentration and modified with necessary amount of BuNoni to compensate for the pretreatment in wells. The cells were stained with annexin-V FAAD/PE, then subjected

95 to flow cytometry and FAAD-labeled cells were visualized using the BD lysis II software. In figure 17, FACS sorting showed that the apoptotic rates for cells were exposed to LPS-RS in the presence or absence of 2 mg/mi BuNoni extract and yielded an apoptosis rate of2l .52% and 26.73%, respectively. The BuNoni extracts (C, D, and E) alone were 68.8%, 78.8%, and 98.2%, respectively; these results showed that the apoptotic cells were significantly increased in a concentration dependent manner in MDA-MB-23 1 cells, which were considerably higher than the no treatment (A) and vehicle control (B): 9.1% and 7.4%, respectively. Similar results were obtained at least in three independent experiments.

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Fig. 17. Flow Cytometric Analysis of MDA-MB-231 cells after exposure to BuNoni. ON

97 Furthermore, the modification of several IRAKs and caspase-regulated genes such as PARP has been reported to function by inducing the apoptosis pathway. [247~ 248] In figures 22 and 23, we used the 2 hour time point for in vitro studies that was synonymous with our transcripts to observe post-translational events such as IRAK undergoing autophosphorylation shortly after IL-i stimulation and the caspase cleavage after exposure to BuNoni. These figures show protein regulation with increase expression of phosphorylated IRAK-1 (fig. 18), using 1 mg/mi BuNoni for 60 minutes and 2 mg/mi BuNoni for 30 minutes, and with increased expression of cleaved caspase 7 (fig. 19) using BuNoni alone versus BuNoni plus LPS-RS. Figure 20 demonstrates PARP cleavage at 2 and 20 hours using 2 mg/mi BuNoni as well as the induction of IRAK2 phosphorylation. There’s also an increase in the expression of IRAK-4 at 4 hours and 20 hours as shown in figure 20. The increase of IRAK-4, -1, and -2, caspase 7, and PARP, in addition to the annexin-V assay results, suggests BuNoni induces cell death and verifies the TLR4 signaling event leading to apoptosis in human BrCa cells.

C.)

~

111111

I

fIRAK~1 P-ACtiIi

Fig. 18. MDA-MB-231 cells regulate IRAK-1 after exposure to BuNoni.

98

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I I

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Caspase-7 P-Actin

Fig. 19. MDA-MB-23 1 cells regulate Caspase-7 after exposure to BuNoni.

2 mWmlBuNoni

g gggIii 4c~’.

~

PARP

IRAK-2

I___ -~

LRAK-4

Fig. 20. MDA-MB-23 1 cells regulate PARP, IRAK-4, and IRAK-2 after exposure to 2 mg/ml BuNoni.

99 3.2.6 BuNoni inhibits cell migration and alters cell structure To investigate whether the migratory properties of BrCa in response to BuNoni treatment, MDA-MB-23 1 cells were allowed to attach and reach confluence, then treated with BuNoni extracts to yield a final concentration of 1, 2, and 3 mg/mi in RPMI 1640 medium in the presence or absence of 10 jig/jil LPS-RS and to a final concentration of 10 j~g/~il LPS in the presence or absence of 10 ~g/jil LPS-RS. Respective cells were pretreated with 10 ~ig/pJ LPS-RS for 1 hour prior to adding the final concentrations of 1, 2, and 3 mg/mi BuNoni plus LPS-RS and 10 ~ig/~ii LPS plus LPS-RS. Treatments were adjusted to maintain 10 j.tg/~il LPS-RS concentration and modified with necessary amount of BuNoni to compensate for the pretreatment in wells. Control cultures received either no treatment or 95% EtOH vehicle in RPMI 1640 medium equivalent to the highest concentration. At 0, 12, and 24 hours, the cells attached to the culture dish were photographed in figure 21. Cell migration was not inhibited in the cultures treated with 10 ig/j.il LPS-RS and LPS (C, D) as compared to controls (A, B); but migration was drastically inhibited in cultures treated with BuNoni extract (E, G, I). In simultaneous experiments, however, addition of LPS-RS to BuNoni extract resulted in decreased visible migration inhibition. From these observations we chose to use 2 mg/mi BuNoni extract for subsequent routine experimentation.

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102 To further assess the migration- and growth-inhibitory activity of the BuNoni, a MDA-MB-23 1 cell population was exposed to 5 mg/mi of BuNoni in figure 22. After cells were allowed to attach and reach confluence, cell cultures received BuNoni extract to yield a final concentration of 5 mg/mi in RPMI 1640 medium in the presence or absence of 10 jig/pJ LPS-RS, which was then added in equal volumes to the wells. Respective cells were pretreated with 10 ~ig/j~l LPS-RS for 1 hour prior to adding the fmal concentrations of 5 mg/mi BuNoni plus LPS-RS. Morphology change of the cells was observed with an inverted microscope and photographs of treated cells were taken at the indicated time points. It is apparent that the 5 mglml BuNoni concentration was highly effective in altering the structure of the cells, which caused them to have a rounded appearance, which is inconsistent with normally spindle morphology found in untreated cells as demonstrated in figure 18. Thus, this change in morphology was acquiescent to induced cell death by BuNoni extract. This change in morphology supports our previous findings that cells treated with BuNoni extract were undergoing apoptosis.

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2.7 BuNoni prevents tumorformation Cells that are able to form colonies also have the ability to form tumors, and the colony formation assay is a conventional method to observe the cloning efficiency of cells. In this assay, MDA-MB-23 1 BrCa cells are plated at low density and allowed to grow to form individual attached colonies in liquid media. We examined the cells after treatment with 1 and 2 mg/ml BuNoni in the absence and in the presence of 10 jig/pJ LPS-RS. Respective cells were pretreated with 10 ~ig/j.ii LPS-RS for 1 hour prior to adding the final concentrations of 1 and 2 mg/mi BuNoni plus LPS-RS. The medium was replaced with fresh medium containing the corresponding treatments every 72

104 hours. After 5 days, the cell colonies were stained with 0.005% crystal violet. Although colony numbers and sizes are usually analyzed with a cell counter, however, we visually observed the appearance of the wells to conclude cloning efficiency rather than quantifying the surviving colonies. As shown in figure 23, BuNoni treatment changes the cells’ sensitivity to cell death as evidence by a decreased number of colonies formed. In comparison to the control (A), the rate of colony formation of the LPS-RS treated cells (B) is quite similar. However, cells treated with BuNoni in the absence and in the presence of LPS-RS showed a considerable difference in colony formation than the control (A). At 1 and 2 mg/ml concentrations (C, E), BuNoni inhibited the cells from forming colonies (i.e., containing more than 50 cells per colony). Similarly, cells treated with BuNoni in conjunction with LPS-RS (D, F) showed a substantial variation from the control as well as the LPS-RS treatment (B) alone.

105

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Fig. 23. BuNoni prevents tumor formation in human BrCa cells.

3.3 Discussion ofResults

Noni has unequivocally been established as an anti-cancer agent in a plethora of experimental data. A crucial step in our investigations was the systematic selection of the optimal solvent to use in the isolation of bioactive constituents found in noni. One challenge was presented by the fact that several studies have been conducted using different alcohols and aqueous solutions, indicating diverse effects of treatment on disease, particularly cancer. Consequently, we extended our experiment to include not only water, basic alcohols and chloroform, but also combinations of assorted alcohols to

106 evaluate the variability of alcohol extracted noni, and compare the cytotoxic effects of these noni extractions on human BrCa cell lines. This, in part, was because of the differences in solvent classification and effects. To clarifS’, solvents are classified into two basic categories: polar and non-polar, which are governed by their dielectric constants. A dielectric constant of less than 15 generally grades a solvent as nonpolar, such as those used in our multi-solvent extractions (CHC13 at 4.8, 1- PenOH at 13.9, 3BuOH at 12.47, 3-HepOH at 6.7, 3-HexOH at 13.3, and 3-OctOH at 10.3), which can reduce the solute’s internal charge.~228’2491 Moreover, these individual solvents, with the exception of CHC13 and 3-BuOH, are longer-chain alcohols (five or more carbon atoms), whose water miscibility decreases sharply as the number of carbons increases. Consequently, the preliminary results of our initial growth assay revealed that neither of these was an ideal solvent for our crude noni extract. Interestingly, those extracts collected by using alcohols with greater dielectric constants (EtOH at 24.3, MeOH at 17.3, PrOH at 20.1, and BuOH at 17.6) appeared to have more favorable effects on the inhibition of cellular proliferation.~2281 They are all moderately miscible as their hydroxyl group makes them soluble according to their polarity: EtOH

>

MeOH> PrOH >BuOH, which is indicative of the predominate -OH

group on the first three and more of a balance between two opposing solubility trends respective to the butano1.~2491 In addition, the nonpolar end of these alcohols also allows them to dissolve nonpolar substances such as plant materials, which includes essential oils, flavoring, and medicinal agents. This aided us in deciding which alcohol and/or combinations of solvents would be ideal for extracting the nutraceuticals from noni and identifying the best extraction method for anti-tumor treatment, predictably yielding the

107 best possible results to induce cell cycle arrest and/or apoptosis in BrCa cell lines. Our results demonstrated the EtOH, MeOH, and BuOH extracts displaying a cytotoxic effect on the growth and proliferation of BrCa cells; BuOH, as seen, yielded the greatest lethality to the cells and was ultimately identified as the optimal solvent of choice. Whereas data substantiated inhibited activity of BrCa cell growth using BuNoni, we did not fractionate by means of column chromatography or medium-pressure liquid chromatography, high-pressure liquid chromatography, or gel permeation chromatography (Sephadex LH-20). A precept for CAM research is that NCCAM (National Center for Complementary and Alternative Medicine) is less concerned about individual plant constituents as oppose to maintaining the integrity of the plant as a whole to use as medicinal agents. Hence, there was no need to fractionate noni in accordance with the guidelines of a CAM project. Moreover, it is possible that the components act together to create their cytotoxic effect on cancer cells, so fractionating may have resulted in losing some phytochemical activity. Our decision not to fractionate was also in part because other studies had afready shown fractions of noni being used in cancer cell growth inhibition assays. The most convincing evidence came by way of our collaborators in Hawaii, where they isolated sugars thought to be the active components in the BuOH extraction of noni. Their procedure led to the isolation of pure compounds, and the chemical structures of the compounds isolated were established by means of mass spectroscopy. They were identified as: a) 6-O-(f3 f3-Dglycopyranosyl)- 1 -O-octanoyl-13-D-glucopyranose, b) 6-O-(13-D-glycopyranosyl)- 1-0hexanoyl-~3-D-glucopyranose, c) 3-methylbut-3 -enyl-6-O-~3-D- glycopyranosyl-~3-Dglucopyranoside, which all have glycosidic backbones (US Patent 2003/000411 6A1).

108 Lui et al. (2001) reported that of the three novel glycosides isolated, 6-O-(13-Dglycopyranosyl)-1-O-octanoyl-f3-D~g1ucopyranose showed inhibitory effects in the mouse epidermal JB6 cell line on AP-1 activity induced by EGF, which controls a number of cellular processes including differentiation, proliferation, and apoptosis.

[84]

The in vitro responses of tumor cells to the multiple solvent noni extractions were measured by means of cell growth inhibition assays. Many factors that maintain the normal phenotype of a breast cell are lost in breast cancer progression. For example, these BrCa cells lines differ by their hormone receptor statuses, which are important regulators of growth and differentiation. Estrogen receptor (ER), progesterone receptor (PR), and the human epidermal growth factor receptor-2 (HER2) are prognostic markers for BrCa Different combinations of the expression or loss of these hormones receptors make up the BrCa phenotypes; ergo, ER- and ER+ tumors display entirely different gene-expression phenotypes. Two prominent phenotypes are triple positive and triple negative. While T47D and MCF-7 BrCa.cells are ER+/PR+/ HER2+, MDA-MB-23 1 cells are ER-/PR-/HER2- and have lost the expression of E cadherin.~250’ 251] Current target therapies are geared towards ER+/HER2+ (antiestrogens and Herceptin), which consequentially makes MDA-MB-23 1 cells (triple negative) more difficult to treat.~252~ This panel of BrCa cells was chosen to analyze the effects of noni among diverse cell lines and observe the sensitivity of the cells due to the treatment of the extract. Three of seven alcohol extractions in Table 4 inhibited the cellular growth of T47D, MDA-MB-23 1, and MCF-7 human BrCa cells. The other solvents were not as potent as n-BuOH on this BrCa cell trio. While the effect of MeOH appeared to be the next most effective extraction on these three cell lines,

109 repeated assays did not yield similar results. The BuOH extract had proven to be the most consistent treatment, particularly with the triple negative phenotype cells. We therefore proceeded with subsequent experiments using the BuNoni as our extract of choice on primarily MDA-MB-23 1 cells (ER-/PR-/HER2-). Favorable results may be beneficial for chemotherapeutics in this population of cancer cells intrinsically resistant to treatment and benefit CAM research by use of nutraceuticals extracted from noni. Cell signaling governs basic cellular activity and operates as a complex system of communication to relay cellular information in response to the environment. Through use of microarray analysis of BrCa cells treated with BuNoni, we elucidated the plausible mechanism of action and were able to exam the regulation of genes using RNA expression data that suggested the innate immunity system was the target site for BuNoni’s toxic mode of action. Although multiple pathways could be interacting as shown by 185 genes, we were looking for the major pathway that would lead to cell death. Observation of the TLR signaling pathways enabled us to determine the manner of cell death induction whether by apoptosis, necrosis, or some other form of mortality. In cells treated with BuNoni, microarray analysis revealed that the genes coding for apoptosis-inducing and -supporting products were increased, such as CXCL2 and CXCL3. IFIT1, IFIF2, and IFIT3, whose mRNA levels increased, are also connected with immunologically defensive roles against microbial infections and are known for their antiproliferative and differentiative activities. Many of the genes regulated in MDA-MB-23 1 after treatment with BuNoni encode proteins involved in the regulation of the Toll-like receptors; thus, the observed cell death was indicative of the TLR4 pathway leading to apoptosis. Apoptosis aids in

110 establishing a natural balance between cell proliferation and cell death by destroying excess, damaged or abnormal cells. To further investigate this pathway, we used western blot analysis to evaluate the regulation of key proteins involved in programmed cell death. The results showed an increase in apoptotic events that contributed to the decrease in cellular proliferation. The activation of the TLR/TLR4 pathway sensitizes the cells to apoptosis induction as shown in figure 24. Sun et al. (2008) reported consistent results in the reduction of apoptosis of colon cancer cells by upregulating Bcl-xL, an anti-apoptotic protein, using DXR and oxaliplatin (OXL); moreover, they contend that the anti-tumor effects of rapamycin is exercised through the reversal of TLR4-induced apoptosis resistance of tumor cells to chemotherapy, thereby causing the cancer cells to be susceptible to anti-tumor chemical reagents.~2531

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Fig. 24. Toll-like Receptor Pathway identifying mechanism of action of BuNoni through TLR4 activation.

112 We found that treatment of BrCa cells with BuNoni altered their cellular phenotype, causing them to lose their spindle shape as shown in figure 19. After BuNoni treatment, the morphology of MDA-MB-23 1 human BrCa tumor cells gradually detached from the bottom of culture plates (12 48 hours). The structure -

change of cells was verified with an inverted microscope, where it showed severe attenuation by 24 hours and distorted cell shape by 48 hours. These changes include retracted bead-like cells with an appearance of having considerable shrinkage from the treatment; they were no longer spindle shaped like the untreated cells. These effects are consistent in both 3 mg/nil (not shown) and 5 mg/mi BuNoni, which were extremely effective in inducing cell death. These results corroborate the results of our previous studies by our Hawaiian collaborators (Katalin Czissar, personal communication) indicating that the growth inhibitory effect of noni is associated with activation of specific stress response pathways and apoptosis.

3.1 Effect ofextraction on cellproliferation As for the dose-response curve using LPS-RS in conjunction with BuNoni, the IC50 value was not attained in figures 14 and 15, in part because of mixed inhibition of the two drugs. In Michaelis-Menten competitive kinetic inhibition, the LPS-RS would represent the substrate, and BuNoni would be considered as the inhibitor. Thus, LPS RS would bind to CD14 which binds to MD-2, and ultimately binds to the TLR4 active site without causing a reaction such as change in cell growth, thereby preventing BuNoni from binding to the same site to prevent proliferation. However, the BuNoni concentration was constant at 2 mg/ml, and there were only concentration variations in

113 the antagonist, LSP-RS, which was added to the cells 1 hour prior to adding the combination of the two drugs; the inhibitor (BuNoni), consequently, was unable to prevent the substrate (LPS-RS) from binding to TLR4. This new arrangement circumvented the Michaelis-Menten kinetic theory of competitive inhibition, where instead of vying for the same position i.e., E a different complex: E

+

S



ES



+

S

+

ES

+

I

I



-->

ES

+

I, the two drugs formed

ESI in a slightly modified mixed

inhibition. Initially, the BuNoni treatment reduced cell growth by 50%. Yet, the doseresponse at the lowest concentration of LSP-RS (0.39 j.~gI~.tl) revealed the inhibitor beginning to lose its efficacy. As the substrate concentration overcame the presence of BuNoni, the survival rate increased to 64% with a drastic increase to 85% with the maximum substrate concentration (10 j.tg/~il) application. It is apparent that exposure to BuNoni was ineffective in maintaining a steady killing rate because LPS-RS saturated the TLR4 active binding site. There are two possible pathways for BuNoni to affect the cells through TLR4: (1) IRF and (2) apoptosis. We surmised that binding of LPS-RS to the CD14/MD-2 complex and then to the active site of TLR4 in this modified mixed inhibition altered the apoptotic effect of BuNoni, sending it down the IFR pathway, which aids in cellular growth.

3.3.2 Clinicallmpact of the Research Breast cancer is currently the second leading cause of cancer-related deaths of women in the United States.~2541 Although there have been major improvements in the prognosis of cancer over the past 10 years, surgery, radiotherapy, and chemotherapy remain the three forms of cancer treatments used. The latter treatment, chemotherapy,

114 uses medication to kill or slow tumor growth, but the efficacy of the drug depends on its ability to halt cell division. There are two types of chemotherapeutic drugs: cell-cycle specific (kills cancer cells only when they are dividing) and cell-cycle non-specific (kill cancer cells when they are at rest). Thus, chemotherapy is typically given in cycles since the scheduling of administering the medications is not only based on the type of cells and the rate at which they divide, but also the time at which a given drug is likely to be beneficial.~2551 Chemotherapy is thought to be most effective when cells are rapidly dividing, but unfortunately the drugs cannot make a distinction between normal cells and cancer cells. The aim of cancer treatment is to kill all of the tumor tissue without killing the other normal cells in the body, and clinical trials are necessary to evaluate the effectiveness of new drugs or CAM therapy in conjunction with current cancer treatments. Our findings with BuNoni as a CAM therapeutic may have a great impact on clinical cancer care and is promising in improving cancer treatment regimens. For example, Taxol® is a chemotherapy drug that is currently given for BrCa cells and is a mitotic inhibitor. Taxol is a diterpene produced by a coniferous plant from the genus of yews called Taxus, which also includes taxanes such as paclitaxel and docetaxel. Since the cells grow by cell division (mitosis), Taxol’s mechanism of action is to prevent cancer cells from completing the mitosis process by sticking to them while they try to divide.~2561 Taxol mimics bacterial LPS, and its highly reactive substituent analogs have been utilized in lieu of its superior chemistry to recognize LPS-binding and signaling molecules. Kawasaki et at. (2001) demonstrated that MD-2 is a prerequisite for Taxol induced TLR4-mediated signaling. [257, 258] Likewise, recognition of LPS requires the

115 MD-2 protein, and TLR4 cannot be activated by stimuli without the presence of MD 2 [200,256,259] Thus, they concluded that in addition to sharing a TLR4/MyD88dependent pathway, Taxol and LPS also share TLR4-dependent/MyD88-independent pathway, where one leads to stimulated NF-icB while the other leads to apoptosis as shown in figure 25.[260,26h1 Ergo, TLR4 has an absolute requirement of MD-2 for cell immunity activation, and we have illustrated noni’s cytotoxic effect on the human BrCa cells in vitro, where BuNoni inhibits the growth of cancer cells by binding to the TLR4 receptor, ultimately leading to apoptosis. A novel drug combination of Taxol and BuNoni would attack cancer cells by preventing them from dividing and metastasizing and consequently going through programmed cell death (fig. 25). It may be worthwhile to evaluate the addition of targeted agents, such as BuOH, to chemotherapy regimens in clinical trials and determine if they could improve efficacy without compromising safety.

LPS

Anthracyclines: Dox, Adriamycin .—Taxols: Paclitaxel

~bo [IRAK4I

[IRAii]

_____

—b

[~K2]

—‘“---‘

Apoptosis

MAPKs NFkB IRF IRF3

JNK

Kim bro Laboratory, 2008

Fig. 25. Toll-like Receptor Pathway demonstrating mechanism of action of chemotherapeutics and BuNoni.

-A

-A

117 Another common chemotherapeutic agent used to treat BrCa cells is doxorubicin (DXR), an anthracycline antibiotic; however, its mechanism of action is complex and still somewhat unclear, though it is thought to interact with DNA by intercalation. [262] Several combinations of drugs with DXR are being used in an attempt to increase the drug’s efficacy and decrease its side effects, such as complete alopecia, heart arrhythmias, and decrease in white blood cells called neutropenia. DXR causes the unwinding of DNA for transcription by inhibiting the progression of the enzyme topoisomerase II, which cuts both strands of the DNA helix simultaneously in order to change the linking number of the molecule.~263~ Chua et a!. (2006) further deduced that up-regulation of several caspases i.e., -2, -3, -8, -9, and -12, as well as p53 in H9c2 induced apoptosis by the treatment of DXR.~2641 A proposed clinical study will be useful in examining the efficacy of DXR in combination with BuNoni to determine any adverse or beneficial effects. We hypothesize that BuNoni will not decrease the effectiveness of the drug, but rather enhance efficacy of the chemotherapy by decreasing cell growth and increasing levels of apoptosis. This may reduce the need for cumulatively increased doses of DXR and decrease the risks of developing cardiac and other side effects. Additional future studies will combine CAM with conventional therapies i.e., Doxorubin and Taxol, which bind TLR4 or accompanying molecules. On another note, cancer patients also attempt to self-treat cancer through the use of complementary and alternative medicine; this is done quite often without informing their healthcare providers. Unfortunately, the use of CAM agents may decrease the efficacy of their allopathic treatments, and result in unfavorable outcomes of the chemotherapy treatments. Case in point, a single nucleotide change can alter the

118 response of the innate cellular immune response. Although this change in responsiveness can be diagnosed with genetic analysis by clinicians, patients might haphazardly choose a self-prescribed course of treatment without considering the genetic change identified in their tumor cells or germline DNA. Furthermore, recent studies have suggested that mutations in human TLR4 correlate with the outcomes of anticancer chemotherapy and radiotherapy.~2651 Qureshi et al. (1999) found that an LPS hyporesponsiveness is created by a mutation in mouse TLR4, where in vitro observations allowed them to identify in inbred endotoxin-tolerant mice the same gene at the LPS locus in two different hyporesponsive strains. Their results confirmed the hypothesis that the endotoxin tolerance was due to perturbation of the TLR4 function.~2661 Therefore, a patient unaware of such evidence does realize the effects that CAM agents may have on the mechanism of cellular activation respective to TLR4 in host responses to LPS nor whether or not the herbal might increase cancer cell growth and decrease levels of apoptosis, which lessens the efficacy of the chemotherapy. Dysfunction in TLRs or associated gene products, such as restoration of lost or mutated TLRs, may result in inadequate or inappropriate immune response and ultimately affect the cell’s innate immune response changing the prognosis.~2672691 Through knowledge of the chromosomal position, novel single nucleotide changes in TLR and TLR-related genes, prognostic and diagnostic screens could better determine which gene may contribute to or be responsible for a patient’s disease and consequential proper treatment.

119 3.4 Conclusion Convincing evidence reported by investigators shows that tumorigenic growth of selective cancer depends on the disruption of the normal apoptotic process.~253’2701 In this study, we sought to determine whether BuNoni induces apoptosis of triple negative human BrCa cells, MDA-MB-231; we observed the regulation of apoptosis in addition to the regulation of genes involved in control of cell proliferation. Our results demonstrated that BuNoni inhibits cell growth via the TLR4 pathway and indicated the extract could sensitize BrCa cells for apoptotic events. Future animal studies will be required to determine whether BuNoni induces the apoptosis of human BrCa cells in vivo. Since the concentrations of BuNoni required to effectively induce apoptosis are low, the noni extract should be evaluated further to determine its potential as a chemopreventive or chemo-complimentive agent. Furthermore, apoptosis is triggered by the activation of caspases that cleave many cellular substrates.~2711 In this study, we observed an increase in the levels of caspases, particularly Caspase-7 and noted that BuNoni appeared to induce this activation in a time-dependent manner. Our results also revealed that BuNoni caused an increase in PARP cleavage, which facilitates irreversible cellular disassembly and serves as a marker of cells undergoing apoptosis.~2721 Preincubation with LPS-RS appeared to protect the cells from BuNoni’s cytotoxic effect; the cells treated with LPS singly or in combination with pretreatment of LPS-RS showed no visible change in the activation of apoptotic events. In addition to these results, we not only concluded that BuNoni treatments induced the activation of Caspase-7 and cleaved PARP, but also showed compelling evidence by way of an annexin-V assay that verified the cells were

120 indeed going through apoptosis. Figure 17 illustrated that compared to the control and the vehicle, the BuNoni treated cells were positive for annexin-V, which indicated that there was a loss of cell viability. The BuNoni treated cells had an overwhelming increase in apoptosis i.e., 68.8%, 78.8%, and 98.2%, respectively; uptake of the dye meant that they were either in early or late stage apoptosis as seen in the right upper and lower quadrants of the square. Also, the results of the necrosis assay in figure 16 showed that only small amounts of LDH was being released from the BuNoni treated cells as compared to the controls, yielding under 50% release of lactate dehydrogenase in comparison to the blank and 18% and 27% of the controls, respectively. Conclusively, these results present BuNoni as a potential cancer chemotherapeutic agent and further evaluations of its potential as an anti-carcinogenic agent in experimental animal models is warranted. Goto et a!. (2008) elucidated that cell migration was induced by specific ligand activation of TLR4.~273~ There is also supporting evidence that innate immune cells participate in neoplastic progression.~2741 In particular, the development of full-blown neoplasia requires that cancer cells have to overcome their autonomous immune pathways as well as those extrinsic immune barriers.~2751 As a result, TLRs are key participants in the mammalian innate immune system. To investigate whether BuNoni retards growth and migration of MDA-MB-23 1 human BrCa cells, we performed wound healing assays; we chose to use multiple BuNoni extract concentrations to further identify the minimum amount required to still deliver maximum effectiveness. We also decided to test the effects of BuNoni on cultures of BrCa cells to see if it would exhibit antitumor activity and prevent colony formation. Our results revealed that

121 BuNoni undeniably demonstrated antitumor activities against BrCa cells, in an interesting dose-dependent manner. Preincubation with LPS-RS blocked BuNoni from binding to the cells, thereby acting as a protector; cells treated with BuNoni in conjunction with pretreatment of LPS-RS showed no visible cell migration inhibition. However, it is also apparent that the 2 mg/mi BuNoni concentration was highly effective in inhibiting cell migration and/or killing cells at 24 hours. The controls exhibited continued migration; the BuNoni-treated cells both showed partial migration or no migration at all, mainly during the closing of the wound. Furthermore, when the biological defense system is impaired, cells carrying the abnormal genes are able to pass mutations from one cell generation to the next. It is the accumulation of these pathological abnormalities that leads to development of aberrant cell colony formations over time.~2761 The soft agar colony formation assay is classical and is more commonly used as an in vitro assay to measure anchorage-independent growth; however, we used the liquid media colony formation assay to visually observe cloning efficiency instead. In this assay, wells treated with as little as 1 mg/mi BuNoni showed impeded colony formation; the controls showed that colonies had formed in the wells, but no colonies were visible in the wells treated with BuNoni. This indicated that the extract was effective in preventing colony formations and would thus be successful in preventing tumor formation. It is worth highlighting that cloning efficiency determined by this liquid media assay is the visual effect of changes in the cells’ sensitivity and slower growth affected by BuNoni treatments. Additionally, manual quantitation of colonies in this format can be laborious and subjective, particularly for liquid media colonies, which perhaps lends to its infrequent application. This has

122 always been a complication to the extensive application of colony formation analyses altogether. Albeit, there are colony-counting machines for quantitating attached colonies, but there exists no similar reported quantitation of colonies in liquid suspension. Thus, our results visually explicate the functional activity of BuNoni on human BrCa cells, and substantiate the concept of noni extracts as potential therapeutic agents to control cancer tumor formation and progression.

Future Directions Over the past several years, cancer biologist have spent vast amounts of energy studying the tumor microenvironment and discovering relevant drug targets related to this unique environment. The Morinda citr~folia (noni) tree is a popular complementary and alternative medicine (CAM)-related botanical that has been used as a food supplement and herbal medicine throughout the Pacific for over 2000 years.~’55~ The nutraceuticals in noni extracts act as anti-tumor agents and may enhance current cancer treatments. We have shown the BuNoni extract to inhibit BrCa cell growth by inducing apoptosis through interaction with Toll-like Receptor 4 (TLR4), which is a precursor to immunological defense. This pathway may be stimulated by the interaction of noni constituents with TLR4, followed by a cascade signal via several adaptor molecules (e.g., MyD88) and kinases as shown in figure 3. Noni’s proposed interaction with TLR4 is significant since a common polymorphism in TLR4 (Asp299Gly) appears to contribute to the efficacy of certain chemotherapies (e.g., adriamycin and paclitaxel). [149, 1571 This suggests BrCa patients harboring the TLR4 299Asp allele may have higher responsiveness to noni combined with chemotherapeutic agents. As a

123 result, the identification and characterization of TLR genes associated with compromised immunity and/or cancer is critical for identifying new targets for treatment and may offer insights into the mechanisms of disease development. Moreover, with the long-term goal of improving clinical management of BrCa, the researchers may seek to explore the molecular mechanisms of noni on BrCa cells via TLR4. Such studies may elucidate the anti-tumor effect of noni respective to TLR4signaling mediation, which induces apoptosis in BrCa cells and increases the efficacy of adriamycin and paclitaxel. Two approaches may be used to analyze this hypothesis: (1) delineate the cytotoxic action of noni on BrCa cells and xenograft tumors treated with or without adriamycin or paclitaxel; and (2) determine the effect of noni on syngeneic breast tumor growth and recipient tumor-associated host cells in TLR4-deficient and wild-type mice. In order to implement approach one (1), studies will have to be done to analyze the molecular mechanisms and efficacy of noni to treat and prevent BrCa; then, researchers will be able to delineate the cytotoxic action of noni on BrCa cells and xenograft tumors treated with or without adriamycin or paclitaxel. They may quantify the extent of BrCa metastasis to the lungs, liver, and lymph nodes, and do a measurement of tumor burden in MDA-MB-23 1 -luciferase-positive (luc) as well as MCF-7-luc xenografts (by orthotopic mammary fat pad injection) before and after noni treatment using a Caliper/Xenogen IVIS Biophotonic Imaging System. Also, investigators might evaluate the impact of noni treatment on apoptosis and cell proliferation in excised breast tumors using immunohistochemistry (IHC) and morphologic analysis of respective biomarkers (e.g., caspase-3, Ki67) as well as TLR4-

124 signaling proteins (phospho-NFkB, IRAK2, IRAK4, and TBK). The molecular mechanisms of noni (plus chemotherapeutic)-mediated apoptosis may be revealed by quantifying and validating temporal mRNA and active protein expression of essential TLR4-signaling factors (e.g., IRAKs, p65, Akt) in the presence or absence of TLR4 or adaptor molecule agonists (LPS) and antagonists (LPS-RS, siRNA TLR-4, siRNA MyD88) treated BrCa cell lines. We believe that these studies will reveal the molecular mechanisms of noni-mediated apoptosis of BrCa cells. The second approach (2) will determine the effect of noni on syngeneic breast tumor growth and recipient tumor-associated host cells in TLR4-deficient and wild-type mice. These studies will disclose whether TLR4 over- or under-expression may reduce BrCa tumor growth and/or alter TLR4 signaling pathways, as outlined in approach 1, following treatment with noni or noni plus adriamycin or paclitaxel using a novel syngeneic BrCa model harboring a hyposensitive TLR4 (Pro7l2His) allele i.e., TLR4LPS-d BALB/cByJ mice. We anticipate synegenic mice orthotopically (breast fat pad challenged) with 4T1-luc cells over-expressing TLR-4 will have reduced tumor growth relative to control groups (i.e., wild-type 4T1-luc cells or 4T1-luc expressing shRNA TLR4 cells in TLR4-/- or wild-type BALB/cByJ mice. Shared genetic backgrounds between these mice and cell lines will allow for clarification of the function of TLR4 on tumor growth in the context of an intact (innate and adaptive) immune system. Cumulatively, the findings of these two approaches will: (1) provide proof of concept for use of noni in mainstream BrCa treatment protocols to improve the efficacy of hard to treat triple-negative BrCa cases; (2) eluidate the mechanism(s) of noni on

125 BrCa tumor burden, presumably by stimulating apoptosis and inhibiting cell proliferation; (3) determine whether the genetic pre-disposition of some patients (i.e., Asp299Gly TLR4) may enhance responsiveness to noni alone or together with conventional chemotherapies to reduce tumor burden; and (4) be the first of future studies to established “personalized” CAM approaches to put an end to BrCa.

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