UNIVERSITY OF NAIROBI

SCHOOL OF MEDICINE

DEPARTMENT OF BIOCHEMISTRY

EFFICACY OF NEEM (Azadirachta indica) EXTRACTS AS ANTICANCER AGENTS

USING A MONKEY MODEL

AMUKHALE MERCY RISPAH

H12/1251/2011

Research proposal submitted in partial fulfillment of the requirements of

HBC 305: Research Proposal

2014

ii

DECLARATION

iii

ACKNOWLEDGEMENT

I would like to thank God for giving me strength and keeping me in good health throughout the

writing of this proposal. Special appreciation goes to my parents (Mr. James Amukhale and Mrs.

Rachel Amukhale) for their unending support, commitment and encouragement during this

period. I thank God for my siblings (Joy, Brian and John Amukhale) and my dear nephew

Jeremy Muthomi for the ample time and peace they have given me during this time. I thank all

my classmates particularly (Allan Hai, Zaneta Kidiavai, Patience Kibet, Naomi Maina, Catherine

Mburu, Eve Wanjiku, Caroline Wanjiku and Grace Lekama) for the moral support and assistance

they have given in the development of important ideas pertaining this proposal. I also thank my

friends: Rebbea zubili, Joseph Otiende, Rachel Wanja, Janet Wamaitha and Ben Ruto for

supporting me. Special appreciation goes to my supervisor Ms. Caroline Wasonga for her

support and guidance through the writing of this proposal. I also thank my lecturers, particularly

Dr. Edward Muge (our class coordinator) and the entire Department of Biochemistry (University

of Nairobi) for the training they have given me in the pursuit of this course.

Table of Contents

DECLARATION......................................................................................................................................... ii

iv

ACKNOWLEDGEMENT ......................................................................................................................... iii

LIST OF ABBREVIATIONS .................................................................................................................. vii

ABSTRACT .............................................................................................................................................. viii

CHAPTER ONE ......................................................................................................................................... 1

1.0 INTRODUCTION ................................................................................................................................. 1

1.1 PROBLEM STATEMENT .................................................................................................................. 3

1.2 PROJECT JUSTIFICATION .............................................................................................................. 3

1.3 OBJECTIVES ....................................................................................................................................... 4

1.3.1 MAIN OBJECTIVES ........................................................................................................................ 4

1.3.2 SPECIFIC OBJECTIVES ................................................................................................................. 4

CHAPTER TWO ........................................................................................................................................ 5

2.0 LITERATURE REVIEW .................................................................................................................... 5

2.1 CANCER ............................................................................................................................................... 5

2.2 Characteristics of cancer cells .............................................................................................................. 5

2.3 Metabolism of cancer cells ................................................................................................................... 5

2.5 TYPES OF CANCERS ......................................................................................................................... 7

2.5.1 Digestive/Gastrointestinal Cancers .................................................................................................. 8

2.5.2 Colorectal cancer/colon cancer ......................................................................................................... 8

2.6 SIGNS AND SYMPTOMS OF COLORECTAL CANCER ............................................................. 9

2.6.1 Local colorectal cancer symptoms .................................................................................................... 9

2.6.2 Systemic colorectal cancer symptoms .............................................................................................. 9

2.7 TREATMENT OF COLORECTAL CANCER ................................................................................. 9

2.8 NEEM TREE ....................................................................................................................................... 10

2.8.1 Botanical description ....................................................................................................................... 11

2.9 MEDICINAL PROPERTIES OF THE NEEM TREE.................................................................... 11

2.10 CHEMISTRY .................................................................................................................................... 12

2.11 ANTI-CANCER PROPERTIES ...................................................................................................... 13

2.12 MOLECULAR MECHANISMS UNDERLYING ANTI-CANCER EFFECTS OF NEEM

EXTRACTS ............................................................................................................................................... 13

2.12.1 Carcinogen metabolism ................................................................................................................. 13

2.12.2 Antioxidants .................................................................................................................................... 14

2.12.3 Cell proliferation, cell cycle arrest, and DNA repair .................................................................. 14

2.12.4 Induction of apoptosis .................................................................................................................... 14

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2.12.5 Modulation of oncogenic Transcription Factors ......................................................................... 15

2.12.6 Modulation of intracellular signaling cascade ............................................................................. 15

2.12.7 Anti-inflammatory effects ............................................................................................................. 15

2.12.8 Immunomodulatory effects ........................................................................................................... 16

2.12.9 Epigenetic alterations .................................................................................................................... 16

2.13 TUMOR MARKERS ........................................................................................................................ 16

2.13.1 COLORECTAL CANCER TUMOUR MARKERS ................................................................... 17

CHAPTER THREE .................................................................................................................................. 19

3.0 MATERIALS ...................................................................................................................................... 19

3.1 METHODS AND PROCEDURES .................................................................................................... 19

3.1.1 Extract preparation ......................................................................................................................... 19

3.1.2 Induction of colorectal carcinogenesis. .......................................................................................... 19

3.1.3 Estimation of tumor markers.......................................................................................................... 19

3.1.4 Estimation of CEA and CA19-9 ...................................................................................................... 20

3.1.5 Determination of side effects ........................................................................................................... 20

3.1.6 Histological determination .............................................................................................................. 20

CHAPTER FOUR ..................................................................................................................................... 22

4.0 EXPECTED RESULTS ...................................................................................................................... 22

CHAPTER FIVE ...................................................................................................................................... 23

5.0 WORK PLAN ...................................................................................................................................... 23

CHAPTER SIX ......................................................................................................................................... 24

6.0 BUDGET .............................................................................................................................................. 24

Bibliography .............................................................................................................................................. 25

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LIST OF FIGURES

Figure 1: Diagram showing the characteristics of a cancer cell

Figure 2: Diagram showing causes of cancer

Figure 3: Diagram showing stages of colorectal cancer

Figure 4: Diagram showing molecular structure of azadirachtin

Figure 5: Diagram showing molecular structure of nimbolide

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LIST OF ABBREVIATIONS

WHO World Health Organization

DNA Deoxyribonucleic acid

HIV Human Immunodeficiency Virus

GSH Glutathione

MGMT Methylguanine- DNA methyltransferase

HUVECs Human umbilical vein endothelial cells

VEGF Vascular Endothelial Growth Factor

GIT Gastrointestinal tract

MMP Matrix metalloproteinase

ERK 1/ 2 Extracellular- signal- regulated kinases

NF-kB Nuclear factor kappa- light- chain enhancer of activated B cells.

MAPK Mitogen activated protein kinase

PI3K Phosphoinositide 3-kinase

TNF Tumor necrosis factor

NLGP Neem leaf glycoproteins

CEA Carcinoembryonic antigen

DNMTs DNA methyltransferases

HATs Histone acetylases

HDACs Histone deacetylases

DMBA 7, 12 dimethylbenz[a]anthracene

HBP Hamster Buccal Pouch

AFP Alpha-fetoprotein

B2M Beta 2 microglobulin

HCG Human chorionic gonadotropin

CA 19-9 Carbohydrate antigen 19-9

ELISA Enzyme linked immunosorbent assay

AOM Azoxymethane

IP Intraperitoneal

viii

ABSTRACT

The development of anticancer drugs is of major interest in the treatment of cancer. The new

drug development process involves acquisition of potential compounds, drug screening and pre-

clinical pharmacology and clinical development.

Neem extracts have been shown to exert anti-cancer effects on a wide array of cancer cell lines.

They contain phytochemicals like azadirachtin and nimbolide that have been shown to have anti-

cancer effects through different molecular mechanisms like induction of apoptosis, antioxidant

effects, carcinogen metabolism, inhibition of tumor invasion and angiogenesis. Animal

toxicological studies of anticancer agents are aimed at predicting a safe starting dose and dosage

regimen for human clinical trials, toxicities of the compound and the likely severity and

reversibility of drug toxicity.

This proposal suggests the preclinical efficacy screening on higher animals, in this case monkey

models. Monkey models are non-human primates. They are in the same order with humans hence

share physiological and behavioral similarities with humans. The results could therefore give

better evidence as compared to rodent models.

Neem extracts will be extracted using the homogenate method. Colon carcinogenesis will then be

induced using azoxymethane, a carcinogen. A set of monkeys will be used as controls, another

will be induced carcinogenesis with oral doses of the neem extracts while another will be

induced carcinogenesis without oral doses of neem extracts. Estimation of tumor markers, in this

case CEA and CA19-9 will be done to study the levels of this biomarkers in the three sets of

monkey models. The side effects will be studied by observation to see the effects of

administration of neem extracts to the monkey models. Histological examination of the colons

will be done on all the three sets of monkeys to study the nature of the tumors induced in the

second and third set of monkeys in comparison to the first which is the control. It is expected that

the set of monkeys administered with neem extracts will have their colons almost similar with

the controls. The colons of the monkeys without oral doses of neem extracts are expected to have

aberrant crypt foci.

1

CHAPTER ONE

1.0 INTRODUCTION

Cancer is a major burden of disease worldwide (Ma & Yu, 2007). Each year, tens of millions of

people are diagnosed with cancer around the world, and more than half of the patients usually die

from it. In many countries cancer ranks the 2nd most common cause of death following

cardiovascular diseases (Ma & Yu, 2007). With significant improvement in treatment and

prevention of cardiovascular diseases, cancer has or will soon become the number one killer in

many parts of the world.

A recent statistic has shown that a person born in The US today has a 41% lifetime risk of being

diagnosed with cancer (Amin & Lowe, 2012). This alarming fact has urged the healthcare

community to identify effective methods of cancer prevention. Cancer cells exhibit deregulation

in multiple cellular signaling pathways, yet all cancers share a number of common hallmark

capabilities such as, genetic instability, self-sufficiency in growth signals, avoidance of

apoptosis, unlimited replication, sustained angiogenesis and tissue invasion and metastasis.

Therefore, targeting single pathways is a tactic that frequently fails in cancer therapy.

Combination therapy that targets a number of distinct molecular mechanisms is therefore

preferable and considered. (Amin & Lowe, 2012)

There are many types of cancers. Examples include (breast, colon, and cervical, skin, throat

cancers). Therefore a treatment that is effective in controlling one type of cancer may be

ineffective on another. (Patrick & Spencer, 2009). Surgery alone, and sometimes radiation alone,

is only likely to be highly successful when the tumour is localized and small in size.

Chemotherapy alone can be effective for a small number of cancers, such as hematological

neoplasms (leukemias and lymphomas), which can generally be considered to be widespread

from the outset. Combined modality therapy requires close collaboration among the entire cancer

care team. (WHO, 2014).

Colorectal cancer is the third most non-skin diagnosed cancer in men and women in the U.S.Its

also the second highest cause of cancer deaths. Still, it is highly curable when detected early

enough. (Santero & Dennis, 2005). For colon cancers that have not spread to distant sites,

surgery is usually the primary or first treatment. Adjuvant (additional) chemotherapy may also be

used.

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Globally, there is an increasing trend towards the use of natural products for medicinal purposes

owing to their chemical diversity, intrinsic biological activity, affordability, and lack of

substantial toxic effects.

Neem (Azadirachta indica) commonly known in Kenya as muarubaini is a medicinal treasure.

The term muarubaini means40 in Swahili. This locally means that it has 40 cures. The leaves

of the neem (muarubaini) tree have antibacterial, antifungal and antiviral properties, and are

often used in cosmetic and skin treatment preparations. Neem leaves are also effective insect

repellants, can be used to treat ringworm and other parasitic skin infections, and promote healing

of wounds. Muarubaini leaves have been used over many years to treat several body ailments

(Jitegemea Foundation, 2008).

Neem is also a medicinal treasure of the Indian sub-continent. Its considered as sarva roga

nivarini(the panacea for all diseases) and has been hailed as heal all, divine tree, natures

drug store (Puri, 1999).

Neem is a promising candidate for anti-cancer drug development. Neem extracts and the

constituent limonoids target multiple molecular and cellular pathways that are dysregulated in

cancer including xenobiotic metabolism, cell cycle, DNA repair, apoptosis, matrix invasion,

angiogenesis, immune surveillance, and intracellular signaling (Nagini & Priyadarsini,

Azadirachta indica (neem) and Neem Limonoids as Anticancer Agents: Molecular Mechanisms

and Targets., 2014).

The efficacy of natural products as chemopreventive agents for primary and tertiary cancer

prevention has not yet been established. Observational studies have suggested that various

vitamins, minerals, and dietary components reduce the risk of developing specific cancers.

Unfortunately, how well these compounds can work in the inhibition of cancer has yet to be

rigorously tested. There is also little known about the interactions of naturally occurring

chemical (phytochemical) with other drugs prescribed by physicians and used by patients for the

treatment of cancer or other diseases. Thus, our current knowledge has many gaps that need to be

resolved before such compounds can receive approval by regulatory agencies, broad acceptance

by the medical community and join other pharmaceuticals on drugstore shelves. (Amin & Lowe,

2012).

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1.1 PROBLEM STATEMENT

Colorectal cancer is the third most commonly diagnosed cancer and the third leading cause of

cancer death. The majority of these cancers and deaths could be prevented by applying existing

knowledge about cancer prevention. (Society, colorectal cancer:Facts and Figures, 2011-2013) .

Incidence and death rates for colorectal cancer increase with age. The present treatment which is

mainly surgery may have possible complications like bleeding, or scar tissue formation.

Traditional chemotherapy also has side effects like hair loss, nausea and vomiting, constipation,

blood disorders, pain and fatigue. The use of natural plant extracts is of interest in adjuvant

therapy as it has less toxic effects to the body and more biological activity. (Enger, Ross, &

Bailey, 2009).

1.2 PROJECT JUSTIFICATION

Neem has been proven to have a wide array of phytochemicals and a promising candidate for

anticancer drug development. Nimbolide has been shown to exert significant cytotoxic effects

against a panel of human cancer cell lines including143B TK osteosarcoma, HL- 60, U-937 and

THP-1 leukaemic,B16 melanoma, SMMC 7721, A-549, MCF-7 breast, HT-29, SW-620, SW-

480, HOP-62, A-549, PC-3, and OVCAR -5 cell lines (Nagini & Priyadarsini, Azadirachta indica

(neem) and Neem Limonoids as Anticancer Agents: Molecular Mechanisms and Targets., 2014).

Although neem limonoids have been extensively tested for cytotoxicity against a panel of human

cancer cell lines, evidence for the in vivo inhibition of tumor growth in animal models are rather

scanty. Six key elements are ideal for the ideal animal model for testing: the animal used should

bear relevance to human cancers, resemblance in genetic abnormalities of the lesions with

humans, the model should have relevant intermediate lesions that simulate or approximate the

human cancer process both histologically and molecularly; model should be capable of

producing a consistent tumor burden of greater than 80% lesions within a reasonable period of

time; the carcinogen or genetic defect used to produce cancer should bear relevance to that

encountered by humans; the predictive values and accuracy of the animal model for human

efficacy should be more than 80%. (Vernon, Lubet, & Moon, 2005)

Monkeys are genetically more similar to humans than to other animal laboratory species. They

resemble humans in most psychological behaviors. (Arthur, 2014). Carrying out preclinical tests

on this non-human primates to test the efficacy of neem extracts as anti-cancer agents can help

4

advance the use of these extracts in the development of anti-cancer drugs. The effects observed

and the response of these models to treatment may help in the management of cancer in humans.

This will eventually lead to translation of the neem trees beneficial effects from bench to

bedside. (Nagini & Priyadarsini, Azadirachta indica (neem) and Neem Limonoids as Anticancer

Agents: Molecular Mechanisms and Targets., 2014)

1.3 OBJECTIVES

1.3.1 MAIN OBJECTIVES

To investigate the effects of neem extracts on progression of chemically induced colon tumour in

monkeys.

1.3.2 SPECIFIC OBJECTIVES

I. To determine the levels of CEA and CA 19-9 activity in normal, carcinogen induced

monkeys with treatment and carcinogen induced monkeys with no treatment.

II. To carry out the histology of the colon of normal, carcinogen induced monkeys with

treatment and carcinogen induced monkeys with no treatment.

To study the side effects of neem extracts as anti-cancer agent.

5

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 CANCER

Cancer is the uncontrolled growth of abnormal cells in the body. Cancerous cells are also called

malignant cells. (Patrick & Spencer, 2009)

Cancer cells are formed when normal cells lose the normal regulatory mechanisms that control

growth and multiplication. They become rogue cells and often lose the specialized

characteristics that distinguish one type of cell from another (for example a liver cell from a

blood cell). Thus, loss of differentiation.

The term neoplasm means new growth and is a more accurate terminology of the disease. The

terms cancer and tumor, however, are more commonly accepted. A tumor is a local swelling. If

the cancer is localized, its said to be benign. If the cancer cells invade other parts of the body

and set up secondary tumours a process known as metastases- the cancer is defined as

malignant. Malignant cancer is life threatening. (Patrick & Spencer, 2009)

2.2 Characteristics of cancer cells

The cancer cells show cellular and nuclear pleomorphism, loss of normal arrangement of cells,

they develop changes in the cell membranes and organelles, and they exhibit abnormal mitoses

and chromosomal abnormalities. Increased motility of malignant cells may be associated with

increased amount of contractile proteins in their microfilaments, with loss of contact inhibition

(probably caused by alteration in calcium ion concentration in the malignant cell membrane).

Interrupted cellular adhesiveness (for the solid surface) and contact inhibition (among cells) may

result from changes in the cell surface glycoproteins and the poorly developed tight junctions and

desmosomes in malignant cells. (Hulin, 1997)

Changes in motility, adhesiveness and contact inhibition may promote invasion and subsequent

establishment of secondary malignant growth metastasis.

2.3 Metabolism of cancer cells

The cancer cells exhibit differences in metabolism as compared to normal cells. The

metabolism of malignant cells is usually more anaerobic than that of normal non-rapidly dividing

cells and is greatly accelerated .Malignant cells may be able to withstand hypoxic conditions.

6

They may have increased glucose and amino acid uptake. These cells have high levels of

hexokinase increasing their glucose utilization. The cancer cells loss capabilities to synthesize

specialized proteins typical for differentiated cells. Enzymes and other proteins produced by

cancer cells are needed for the tumor growth.

(McCance KL, 1990)

Figure 1: diagram showing the characteristics of a cancer cell

2.4 CAUSES OF CANCER

Cancer-causing substances are called carcinogens. A 'carcinogen' is an agent that can help to

cause cancer. This include: Tobacco smoke, diet, body weight and physical activity, genetic

7

factors, the sun and UV exposure, radiation exposure, carcinogens, viruses, bacterial infections

and the immune system. (Society, cancer: facts & figures, 2014)

Figure 2: diagram showing causes of cancer

2.5 TYPES OF CANCERS

Cancer is a broad term used to encompass several malignant diseases. Each type of cancer is

unique with its own causes, symptoms, and methods of treatment. Like with all groups of

disease, some types of cancer are more common than others. Cancer has the potential to affect

every organ in the body. The cells within malignant tumors have the ability to invade

neighboring tissues and organs, thus spreading the disease. It is also possible for cancerous cells

to break free from the tumor and enter the bloodstream, in turn spreading the disease to other

organs.

When cancer has metastasized and has affected other areas of the body, the disease is still

referred to the organ of origination. For instance, if cervical cancer spreads to the lungs, it is still

called cervical cancer, not lung cancer. ((IARC), 2004).

8

Types of cancers classified by body system include: skin cancers, respiratory cancers, head and

neck cancers, gynecologic cancers, genitourinary cancers, eye cancer, endocrine cancers,

digestive/gastrointestinal cancers, breast cancer, and blood cancer. ((IARC), 2004)

2.5.1 Digestive/Gastrointestinal Cancers

This is a broad category of cancer that affects the gastrointestinal tract from the esophagus to the

anus. Each type is specific and has its own symptoms, causes, and treatments. These are: anal

cancer, bile duct cancer (extra hepatic), carcinoid tumor (gastrointestinal), colon cancer,

esophageal cancer, gallbladder cancer, liver cancer (adulthood), liver cancer (childhood),

pancreatic cancer, rectal cancer, small intestine cancer, stomach (gastric) cancer. (Yamada, 2009)

2.5.2 Colorectal cancer/colon cancer

Colon cancer is the third most commonly diagnosed cancer among men and women in the

United States. (United States Cancer Statistics, 1999-2011) Colon cancer is the cancer of the

large intestine (colon), the lower part of the digestive system. Rectal cancer is cancer of the last

several inches of the colon. Together they are often referred to as colorectal cancer. (Santero &

Dennis, 2005)

Colorectal cancers often begin as polyps. These are benign growths on the interior surface of the

colon. The two most common types of intestinal polyps are adenomas and hyperplastic polyps.

They develop when there are errors in the way cells grow and repair the lining of the colon. Most

polyps remain benign, but some have the potential to turn cancerous. Removing them early

prevents colorectal cancer. (Santero & Dennis, 2005)

(Santero & Dennis, 2005)

Figure 3: diagram showing stages of colorectal cancer

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The risk factors of colorectal cancer include age, polyps, family history of colorectal cancer,

history of ovarian or breast cancer. This cannot be controlled. Other risk factors that can be

controlled include diet high in red or processed meats, being obese, exercising too little, smoking

or heavy alcohol use.

2.6 SIGNS AND SYMPTOMS OF COLORECTAL CANCER

There are usually no early warning signs and signals. As the disease progresses, colorectal cancer

signs and symptoms can be broken down into two general categories: local and systemic.

2.6.1 Local colorectal cancer symptoms

Local symptoms are those that have a direct effect on the colon or rectum. If you experience

symptoms of colorectal cancer for an extended period of time, it is important that you visit your

healthcare professional. Common local symptoms include: rectal bleeding, diarrhea, or

constipation collectively sometimes named change in bowel habit loss of weight,

abdominal pain, and anemia. (Astin, Griffin, Neal, Rose, & Hamilton, 2011)

2.6.2 Systemic colorectal cancer symptoms

Systemic colorectal cancer symptoms are those that affect your entire body. If you experience

any of these for any length of time, even for only a couple of days, it is important to have your

doctor or healthcare provider diagnose your symptoms. Common systemic symptoms of

colorectal cancer include: Unexplained weight loss, unexplained loss of appetite, nausea or

vomiting, anemia, jaundice, weakness or fatigue. (Cancer treatment centers of America).

2.7 TREATMENT OF COLORECTAL CANCER

There are three traditional approaches to the treatment of cancer. These are surgery, radiotherapy

and chemotherapy.

Once cancer has been detected it is possible to remove the tumor through surgical intervention.

Many cancers of the skin or breast are dealt with in this manner. However, in some cases like

leukemia where cancer cells spread throughout the body, surgery is impractical. Surgery is also

not useful when the tumor is located where it cant be removed without destroying healthy tissue.

(Enger, Ross, & Bailey, 2009)

Chemotherapy and radiation therapy take advantage of the cells ability to monitor cell division

at the cell cycle checkpoints. By damaging DNA or preventing its replication, they cause the

10

cells to die. Most common cancers have to be treated by a combination of chemotherapy and

radiotherapy. Radiation therapy utilizes powerful X-rays or gamma rays to damage the DNA of

cancerous cells. It can be used when surgery is impractical. Radiation can be applied from

outside the body and in this case, a beam of radiation is focused on the cancerous cells and

shields protect as much healthy tissue as possible. (Enger, Ross, & Bailey, 2009)

Unfortunately, chemotherapy and radiotherapy can have negative effects on normal cells.

Chemotherapy might expose all the bodys cells to toxic ingredients, weakening the bodys

normal defense mechanisms as the body cannot produce white blood cells through cell division.

This condition is referred to as immunosuppression. Thus antibiotics are administered alongside

to help patients defend themselves against dangerous bacteria that they may be exposed to. Other

side effects of chemotherapy include; hair loss and intestinal disorders.

The advantages of chemotherapy include increased efficiency of action, decreased toxicity, and

evasion of drug resistance. Their effectiveness therefore is dependent on them becoming more

concentrated in cancer cells than normal cells. This often turns out to be the case because cancer

cells are generally growing faster than normal cells. Anticancer drugs are therefore most

effective against cancers which are rapidly proliferating.

Radiation sickness is one side effect of radiotherapy. Symptoms include hair loss, bloody

vomiting and diarrhea, and a reduced white blood cell count. (Enger, Ross, & Bailey, 2009)

2.8 NEEM TREE

The neem tree is scientifically known as Azadirachta indica. Its local name here in Kenya is

Mkilifi, or Mwarubaini Kamili in Swahili. (Dharani, 2002). It is a hardy, fast growing,

medium-sized tree growing from 15-20 m in height, with a dense, leafy, oval shaped canopy;

evergreen except in the driest areas; drought resistant, flourishing in arid and semi-arid regions.

Its bark is pale, grey-brown and rough. Its leaves are shiny, green and are compound leaves. Its

flowers are small, scented, creamy white and hang down in low sprays. The fruits are oval,

yellow berries when ripe and they yield an aromatic oil. (Dharani, 2002). The neem tree is native

to India and The Indian Sub-Continent. Mwarubaini in Swahili means forty (40). This means tree

of the 40 cures. (Jitegemea Foundation, 2008)

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2.8.1 Botanical description

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order: Sapindales

Family: Meliaceae

Genus: Azdirachta

Species: Indica.

Scientific name: Azadirachta Indica

2.9 MEDICINAL PROPERTIES OF THE NEEM TREE

Neem has attracted increasing research attention because of the plethora of health benefits that it

confers. All parts of the neem tree- leaves, flowers, seeds, fruits, roots, and bark- have been used

in traditional systems of medicine. (Nagini & Priyadarsini, Azadirachta indica (neem) and Neem

Limonoids as Anticancer Agents: Molecular Mechanisms and Targets., 2014) .In India, the neem

tree is considered as sarva roga nivarini (the panacea for all diseases) and has been hailed as

heal all, village dispensary and natures drug store (Puri, 1999). Extracts of neem seeds

and leaves were shown to provide protection against various strains of malarial parasite and

human fungi including candid. The antibacterial effects of neem leaves, seeds, and bark have

been demonstrated against gram positive and gram negative bacteria. Neem and neem based

products exhibit anti viral activity against Chikungunya, herpes simplex virus -1, dengue virus

type 2, and human immunodeficiency virus (HIV). Alcoholic neem leaf extracts were found to

be effective against chronic skin disease such as eczema. Neem has been used in the treatment of

gingivitis, periodontitis, oral infections and inhibition of plaque growth. Both the aqueous and

alcoholic neem leaf extracts are reported to exhibit hepatoprotective effects against liver damage

induced by paracetamol and anti-tubercular drugs. Neem preparations were demonstrated to be

useful in the control of gastric hyperacidity and ulcer by blocking acid secretion. Studies have

also documented that oral administration of aqueous as well as alcoholic extract of neem leaf

decreased blood glucose level in experimentally induced diabetes by releasing endogenous

12

insulin. (Nagini & Priyadarsini, Azadirachta indica (neem) and Neem Limonoids as Anticancer

Agents: Molecular Mechanisms and Targets., 2014)

2.10 CHEMISTRY

Phytochemicals are chemical compounds that occur naturally in plants. Over 300 structurally

complex bioactive, organic compounds have been isolated and characterized from various parts

of the neem tree. These compounds are categorized into two major classes: isoprenoids or

terpenoids and non-isoprenoids. (Nagini & Priyadarsini, Azadirachta indica (neem) and Neem

Limonoids as Anticancer Agents: Molecular Mechanisms and Targets., 2014) The isoprenoids

include diterpenoids, triterpenoids, vilasinin type of compounds, and C-secomeliacins. The

triterpenoids are categorized into protolimonoids and mono- to nonanortriterpenoids. The

tetranortriterpenoids are also known as limonoids.

The non-isoprenoids include polysaccharides include polysaccharides, proteins, amino acids,

sulphur compounds, hydrocarbons, fatty acids, and their esters, tannins. And polyphenolics such

as flavonoids and coumarin. (Nagini & Priyadarsini, Medicinal properties of neem leaves, 2004,

2005). Only a few compounds have been screened for biological activity. The complex

limonoids constitute about one third of the phytochemical constituents of the neem tree. The

biological activity of neem has been largely attributed to these limonoids.

azadirachtin

Figure 4: diagram showing molecular structure of azadirachtin

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Figure 5: diagram showing molecular structure of nimbolide

2.11 ANTI-CANCER PROPERTIES

Neem extracts have been shown to exert potent anti-cancer effects attributed to the presence of

limonoids. Azadirachtin isolated from seed kernels, and nimbolide, abundant in neem leaves and

flowers are the most active neem limonoids that display anticancer effects. (Nagini &

Priyadarsini, Azadirachta indica (neem) and Neem Limonoids as Anticancer Agents: Molecular

Mechanisms and Targets., 2014)

Examples of neem compounds that exert potent anti- cancer effects are Azadirachtin, azadirone,

deacetyl nimbin, gedunin, nimbin, nimbolide, salannin and quercetin. Studies have revealed that

nimbolide is the most potent anti-cancer agent among the various neem limonoids examined.

2.12 MOLECULAR MECHANISMS UNDERLYING ANTI-CANCER EFFECTS OF

NEEM EXTRACTS

The molecular mechanisms underlying the anti-cancer effects of neem include: carcinogen

metabolism, antioxidants, cell proliferation, cell cycle and DNA repair, induction of apoptosis,

inhibition of tumor invasion and angiogenesis, modulation of oncogenic transcription factors,

immunomodulatory effects and epigenetic alterations.

2.12.1 Carcinogen metabolism

The neem limonoids modulate phase I and phase II xenobiotic metabolizing enzymes which play

a central role in xenobiotic/ drug metabolism. Phase I enzymes like cytochrome P450

monooexygenases, catalyze the biotransformation of procarcinogens to highly reactive

electrophilic intermediates that can damage cellular macromolecules. Phase II enzymes such as

Glutathione S-transferases, catalyze the neutralization of electrophilic intermediates generated in

phase I reactions, thereby resulting in reduced chemical reactivity and cellular damage (Iyanagi,

2007). Ethanolic neem leaf extracts, neem leaf fractions, and the limonoids azadirachtin and

14

nimbolide function as dual-acting agents. They constrain the activities of total cytochrome P450

as well and its isoforms. They then enhance the activities of phase II detoxification enzymes

(Manikandan et al. 2008, 2009; Vidya Priyadarsini et al. 2009).

2.12.2 Antioxidants

Neem leaves, fruits, flowers and stem bark exhibit antioxidative properties against free radicals

both in vitro and in vivo. Ethanolic neem leaf extracts significantly mitigate carcinogen- induced

genotoxicity and oxidative stress by augmenting GSH- dependent antioxidant defense

mechanisms (Nagini & Priyadarsini, Medicinal properties of neem leaves, 2004, 2005). Both

azadirachtin and nimbolide exhibit concentration-dependent ROS scavenging activity and

reductive potential in vitro and protected against oxidative DNA damage in vivo by up regulating

antioxidants (Vidya, Manikandan, Kumar, & Nagini, 2009).

2.12.3 Cell proliferation, cell cycle arrest, and DNA repair

Perturbation of cell cycle control with consequent uncontrolled cell proliferation is a major

hallmark of cancer. Azadirachtin exerts antimitotic effects by interfering with polymerization of

tubules and formation of the mitotic spindle, thereby preventing replication (Salehzadeh, et al.,

2003). The extracts reduce the expression of 06-alkylguanine lesions in human peripheral blood

lymphocytes by increasing the levels of 06-MGMT repair protein.

2.12.4 Induction of apoptosis

Apoptosis refers to programmed cell death. Defective apoptosis is also another hallmark of

cancer. Apoptosis is initiated via complex interactions between pro and anti-apoptotic members

of the Bc-2 family that dictate the integrity of the mitochondrial membrane. Neem limonoids

have been shown to induce apoptosis in various cancer cells by both the death receptor and

mitochondrial pathways.

Angiogenesis is the formation of new blood vessels from pre-existing blood vessels. Cancer cells

invasion and endothelial transmigration, critical events in tumor progression and metastasis,

depend on an intricate balance between proinvasive and proangiogenic factors and their

inhibitors. Ethanolic neem leaf extracts have been shown to exhibit antiangiogenic effects in

human umbilical vein endothelial cells (HUVECSs) by attenuating VEGF (Vascular endothelial

Growth Factor) stimulation. In a recent study, nimbolide was demonstrated to block tumor cell

invasion, migration, and angiogenesis in colon cancer cells in vitro by down regulating the

15

expression of MMP and VEGF via abrogation of ERK and NF-kB signaling. (Babykutty &

Priya, 2012).

2.12.5 Modulation of oncogenic Transcription Factors

An oncogene is a gene with the potential to cause cancer. They cause the cells designed for

apoptosis to survive and proliferate further. Neem extracts and its constituent limonoids are

reported to modulate various transcription factors associated with oncogenesis, chiefly nuclear

factor kappa B (NF-kB) and B-catenin. In the nucleus, NF-kB binds to the kB enhancer element

and transactivates over 500 target genes that are implicated in various processes such as cell

proliferation, cell survival, apoptosis evasion, invasion, metastases, and angiogenesis

(Chaturvedi, Sung, Yadav, Kannappan, & Aggarwal, 2011).

Laboratory studies have demonstrated that nimbolide inhibits both the constitutive as well as

tumour necrosis factor- a-induced NF-kB activation in NF-kB responsive luciferase reporter

plasmid transfected human hepatocarcinoma (HepG2) cells (Nagini & Priyadarsini, Azadirachta

indica (neem) and Neem Limonoids as Anticancer Agents: Molecular Mechanisms and Targets.,

2014).

2.12.6 Modulation of intracellular signaling cascade

Intracellular signaling cascade is characterized by intracellular kinases like protein kinase C,

phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinases (MAPKs).

Inappropriate regulation of these kinases transmit mitogenic signals to transcription factors, co-

activators, and co-repressors resulting in transcription of target genes that promote

carcinogenesis. Neem extracts and limonoids have been shown to target this intracellular kinases

and promote apoptosis induction and NF-kB abrogation in diverse malignant cell lines

(Gunadharini et al. 2011).

2.12.7 Anti-inflammatory effects

Neem leaf extracts and limonoids have been shown to exert potent anti-inflammatory effects by

inhibiting the activation of tumor necrosis factor (TNF)-a, a multifunctional proinflammatory

cytokine that plays a key role in inflammation through signaling and by inhibiting activation of

NF-kB. Inhibition of TNF activation by azadirachtin also reduces NF-kB activation and the

expression of NF-kB dependent pro-inflammatory mediator COX-2 (Thoh, Kumar, Nagarajaram,

& Manna, 2010)

16

2.12.8 Immunomodulatory effects

Increased cytokine secretion results to immunosuppression. Immunosuppression is a key

phenomenon in the growth and development of tumour cells. Aqueous extracts of neem were

also shown to enhance both humoral immunity and cell mediated immunity. (Ray, Barnerjee, &

Sen, 1996). Neem leaf glycoproteins (NLGP) and extracts exhibit potential to enhance

immunogenicity and block negative immunoregulatory host mechanisms in various tumours.

NLGP induces anti-tumour immunity by enhancing carcinoembryonic antigen (CEA)

presentation of dendritic cells to T and B cells (Sarkar, Goswami, Chakraborty, Bose, & Baral,

2010). NLGP exhibits anti-tumor activity in patients with head and neck squamous cell

carcinoma by activating cytotoxic T lymphocytes and natural killer cells.

2.12.9 Epigenetic alterations

Epigenetic modifications like DNA methylations and histone acetylation play a major role in

regulating the dynamics of gene expression. The key mediators of the epigenetic processes

include DNA methyltransferases (DNMTs), DNA demethylases, histone acetyltranferases

(HATs), and histone deacetylases (HDACs). The limonoids azadirachtin and nimbolide exhibit

the potential to inhibit the expression of HDAC-1 and restrict tumor invasion and angiogenesis

during DMBA- induced HBP carcinogenesis. This is of particular significance in the perspective

of the emerging interest in epigenetic reprogramming in cancer and the potential anticancer

effects of HDAC inhibitors (Carew, Giles, & Nawrocki, 2008)

These are the molecular targets of neem and its constituent phytochemicals.

2.13 TUMOR MARKERS

A tumor marker is a substance found in a person's blood, urine, or the tumor itself and is

produced by the tumor or the body in response to cancer, or a noncancerous condition, such as

inflammation. Doctors suggest tumor marker tests at various stages in the diagnosis and

treatment of cancer. Tumor markers for gastrointestinal cancers may be used for screening, to

find early evidence of cancer, staging and treatment planning (staging is a way of describing a

cancer: where it is located, if or where it has spread, and if it is affecting the functions of other

organs in the body), after surgery, to learn whether the cancer has returned or spread and to

check how the treatment administered is working, also known as prognosis. Clinical uses can be

broadly classified into 4 groups: screening and early detection, diagnostic confirmation,

17

prognosis and prediction of therapeutic response and monitoring disease and recurrence.

(Sharma, 2009)

If the tumor marker is being used to monitor whether treatment is working or whether there is a

recurrence, the markers level will be measured in multiple samples taken over time.

Serial measurements, which show whether the level of a marker is increasing, staying the

same, or decreasing, are more meaningful than a single measurement. Examples of tumor

markers include: alpha-fetoprotein (AFP), beta 2 microglobulin (B2M), Beta- human chorionic

gonadotropin (Beta-HCG), CA 19-9, CA-125, calcitonin, CEA, CD 20 and fibrin among others.

2.13.1 COLORECTAL CANCER TUMOUR MARKERS

The specific tumor markers used for colorectal cancer include carcinoembryonic antigen (CEA)

and CA 19-9, that are released into the bloodstream.

Carcinoembryonic antigen (CEA) is a tumor marker that is measured using a blood test. High

levels of CEA may indicate that cancer has spread; however, other medical conditions and some

treatments, including certain types of chemotherapy, may raise CEA levels.

CEA involves a glycoprotein belonging to the immunoglobulin gene super-family with at least

19 related molecules. It functions as an intercellular adhesion molecule that promotes the

aggregation of human colorectal carcinoma cells. In patients with metastatic colorectal cancer,

CEA can be high in up to 85% of all patients. In surveillance, it is used after curative resection

for detection of recurrence. After curative resection, the CEA level normally drops within a

month to normal levels, but in some cases this interval can be extended to four months. (B,

Riedel, & Lampert, 2001)

CEA is used to find out how treatment is working by estimating its levels every one to three

months in patients with metastatic cancer. (cancer.net).

Carbohydrate antigen 19-9 (CA 19-9) is a tumor marker that is also measured using a blood

test. High levels of CA 19-9 may indicate that cancer has spread, but other medical conditions

may cause high levels of CA 19-9.

18

CA 19-9 assays measure a tumour related mucin that contains the sialylated Lewis-a

pentasachharide epitope, lacto-N-fucopentaose II. Serum CA 19-9 is higher than normal with

20%-40% of patients with metastatic colorectal cancer. (B, Riedel, & Lampert, 2001).

19

CHAPTER THREE

3.0 MATERIALS

fresh neem leaves, 6 monkeys, blender, flask, distilled water, centrifuge, gloves, Azoxymethane,

syringes, CEA ELISA kit, CA19-9 ELISA kit, colorimeter, surgical blades, 3 % formaldehyde,

alcohol, xylol and toluol, hematoxylin and eosin, mounting medium, slides, cover slips, paraffin,

light microscope.

3.1 METHODS AND PROCEDURES

3.1.1 Extract preparation

A mixture of fresh leaves will be ground and blended by addition of distilled water. The

homogeneous mixture will be transferred into a clean flask. It will be left to stand in the

refrigerator for 24 hours to allow extraction. The mixture will then be centrifuged for 15 minutes

at 3000rpm at 4. The supernatant will then be collected and stored in the refrigerator at -20

for use in the subsequent steps. (Kwasi & Kofi, 2011)

3.1.2 Induction of colorectal carcinogenesis.

A set of 6 monkeys will be used. There will be three sets of animals. The first two monkeys will

be used as controls with no induced carcinogenesis, the second will be induced carcinogenesis

over a period of 37 weeks with no subsequent treatment while the third set will have monkeys

administered with the carcinogen over a period of time alongside oral administration of doses of

neem extracts through drinking water.

Carcinogenesis will be induced to the second set of monkeys by intra-peritoneal (I.P)

administration of AOM (azoxymethane) twice, over a period of 37 weeks. A dose of 15mg/kg

animal weight will be used as described by Bissonnette et al (2000).

The third set of monkeys will receive the same treatment of the chemicals, alongside oral doses

of up to 1000mg/kg body weight of the neem extracts (Biswas et al. 2002).

3.1.3 Estimation of tumor markers

Weekly estimation of CEA and CA 19-9 levels are carried out continuously.

20

Blood samples will be drawn in three sets, for the normal monkeys, carcinogen induced monkeys

with no treatment and carcinogen induced monkeys with treatment.

3.1.4 Estimation of CEA and CA19-9

5 ml serum samples will be taken through marginal ear vein puncture from the three sets of the

monkeys.

The serum will be centrifuged for ten minutes and separated from the centrifugate. The test

systems used to estimate the tumor markers will be CEA ELISA test kit and CA 19-9 ELISA test

kit. Both rely on the colorimetric detection method.

The microplates are coated with the specific anti-cancer antigen- antibody, specifically anti CEA

antibody, and anti CA19-9 antibody separately. The test samples will then be added in the plates.

Horse-radish peroxidase conjugated anti-cancer antigen- antibody will be added. The specific

substrate will then be added to develop color. The concentration of cancer antigen will be

directly proportional to color intensity measured at 450 nm.

3.1.5 Determination of side effects

The side effects of the neem extracts will be studied by observation. This will include presence

of hair loss, physiological behavior and general activity of the monkeys administered with neem

extracts over the period of the experiment.

3.1.6 Histological determination

After the 37 weeks, the three sets of monkeys will be sacrificed to obtain samples of their colons.

Their anatomy will be observed grossly and also histologically. For histological determination,

tissue samples will be obtained from the colons and taken through a procedure of tissue

processing. They will be fixed in 37% aqueous solution formaldehyde, washed and dehydrated in

a series of alcohol solutions of ascending concentrations till 100%. Organic solvents will be

cleared using xylol or toluol, then infiltration will be done with melted paraffin. A microtome

will be used to cut the sections into (5-15 micrometers) then mounted on glass slides using

mounting medium. The paraffin will be dissolved out using xylol or toluol and the slides

rehydrated through a series of solutions of descending alcohol concentrations. The tissues will

then be stained with hematoxylin in water. The specimen will be dehydrated through a series of

alcohol solutions of ascending concentration then stained with eosin in alcohol. The specimen

21

will then be passed through xylol to a non- aqueous mounting medium and covered with a

coverslip to obtain permanent preparations. (Ross & Wojciech, 2011)

The tissues will then be examined under a light microscope for the tumors. The set of monkeys

without induced carcinogenesis will be used as controls.

22

CHAPTER FOUR

4.0 EXPECTED RESULTS

It is expected that when carcinogenesis will be induced in the 2nd and 3rd set of monkeys, the

colons will develop aberrant crypt foci which are clusters of abnormal tube-like glands in the

lining of the colon and the rectum. The controls will remain with normal histology of the colons.

The 2nd set of monkeys is expected to have highly increasing levels of CEA and CA19-9 over the

37 weeks and show aberrant crypt foci with possible development of colorectal polyps when

observed histologically. The 3rd set of monkeys are expected to have highly increasing levels of

CEA and CA19-9 during the first few weeks of the test.However,it is expected that there will be

a significant decrease in the levels of this tumor markers as the experiment progresses. The

histological preparations are expected to have aberrant crypts but at lower levels as compared to

those in the 2nd set. They are expected to be those almost similar with in the 1st set of the

monkeys which will be used as controls.

23

CHAPTER FIVE

5.0 WORK PLAN

TIME (MONTHS) ACTIVITY

1 -2 Application of the research work permit and

license from the government regulatory body

and the University of Nairobi (Biochemistry

department) respectively.

Application of permit from the primate

research institute.

3-4 Preparation and purchase of laboratory

materials.

5-6 Extraction of the neem extracts and storage.

7-15 Induction of carcinogenesis and treatment

with neem extracts.

16-17 Estimation of tumor markers, determination

of side effects and histological study.

18-19 To do data analysis and report writing.

20 Project presentation to the board of

researchers of anticancer drug development.

24

CHAPTER SIX

6.0 BUDGET

STEP ITEM AMOUNT COST (KSH)

EXTRACTION Fresh neem leaves 500g 200

Flask 1 500

Gloves Box 500

INDUCING

CARCINOGENESIS

Monkeys 6 600,000

Azoxymethane 2000

Syringes Box 2000

ESTIMATION OF

TUMOR MARKERS

CEA ELISA kit

(sigma-Aldrich)

1 70,000

CA 19-9 ELISA

kit (Sigma-

Aldrich)

1 50,000

HISTOLOGY

PREPARATION

Surgical blades 10 500

Formaldehyde 1000

Alcohol 1000

Xylol 1000

Toluol 1000

Hematoxylin 1000

Eosin 1000

Mounting medium 1000

Slides 500

Cover slips 500

Paraffin 500

TOTALS 734,200

25

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