ProstaSe Cancer Chemoprewention by Tea Poiypiieiiols: Concepts,.Overwiew and Introduction

Chapter-I

Prostate Cancer Chemopreveotion by Tea Polyphenols:

Concepts, Overview and Introduction

This chapter describes the basic concepts regarding i) prostate cancer (CaP), ii)

chemoprevention, and ill) chemopreventive agent of choice; polyphenols obtained

from tea plant Came/ia Sinensis. The detailed account of individual projects including

their own introduction, rationale, methods and discussion is provided later with

individual chapters.

1.1 introduction

Cancer is the uncontrolled growth and spread of cells that may affect almost

any tissue of the body. More than 10 million people are diagnosed with cancer

worldwide every year. It is estimated that there will be 15 million new cases by the

year 2020. Cancer causes 6 million deaths every year—or 12% of deaths worldwide.

Carcinogenesis is a multistep process driven by genetic and epigenetic alterations that

disrupts the regulatory pathways controlling cellular proliferation, programmed cell

death (apoptosis), angiogenesis and differentiation (Lippman and Hong, 2002;

Vogelstein and Kinzler, 2002; Jones and'Baylin, 2002). As a normal routine,

mammalian cells grow, divide, and die in an orderly fashion. However, sometimes the

normal regulation of growth, division and death of cell is disturbed that leads to a rapid

and uncontrolled proliferation of cells ultimately resulting into the development of

‘Cancer’.

Cancer has afflicted humans throughout recorded history. It is no surprise that

from the dawn of history, a lot has been written about cancer. Some of the earliest

evidence of cancer is found among fossilized bone tumors, human mummies in

ancient Egypt, and ancient manuscripts. Bone remains of mummies have revealed

growths suggestive of the bone cancer, osteosarcoma. In other cases, bony skull

destruction as seen in cancer of the head and neck has been found. Oldest

description of cancer (although the term cancer was not used) was discovered in

Egypt and dates back to approximately 1600 B.C. The Edwin Smith Papyrus, or

writing, describes 8 cases of tumors or ulcers of the breast that were treated by

cauterization, with a tool called "the fire drill". This writing stated about the disease,

"There is no treatment".

The origin of the word cancer is credited to the Greek physician Hippocrates

(460-370 B.C.), considered the "Father of Medicine," Hippocrates used the terms

carcinos and carcinoma to describe non-ulcer forming and ulcer-forming tumors. In

Greek these words refer to a crab, most likely applied to the disease because the

finger-like spreading projections from a cancer called to mind the shape of a crab.

Carcinoma is the most common type of cancer.

Specific genetic and epigenetic alterations may affect gene expression and/or

the structure and function of specific gene products associated with carcinogenesis.

The two major classes of such genes are Oncogene and tumor suppressor genes

(TSGs), both of which are essential components of many regulatory circuits.

Oncogenes, some of which are the human analogues of viral genes, promote

tumorigenesis upon activation by a single event, such as point mutation, chromosomal

translocation and/or amplification. Tumor suppressor genes are the genes whose

inactivation is associated with tumor development. In contrast to oncogenes, both

alleles of a TSG must be affected to promote tumerogenesis (Knudson, 2001).

Inactivation of TSG can occur through a combination of genetic and epigenetic

mechanisms. Activation of oncogenes and inactivation of TSGs may result in

uncontrolled cell growth and acquisition of tumorigenic phenotypes (Hanahan and

Weinberg, 2000),

Cell growth, proliferation, apoptosis (programmed cell death), angiogenesis

and differentiation are controlled by complex signal transduction pathways (regulatory

circuits) (Hanahan and Weinberg, 2000). These pathways are typically activated by

the binding of ligand(s) (an extracellular protein or a small molecule, such as steroid

hormone) to a specific cell surface or nuclear receptor. Activation and transmission of

signal frequently depends on serine or tyrosine kinases associated with the

cytoplasmic portion of the receptor. The signal is then relayed into the nucleus by

cytoplasmic proteins, resulting in a change in intracellular gene and protein expression

patterns. Alteration of critical proteins in these regulatory pathways may have an

oncogenic effect. For example, the genes encoding the epidermal growth factor

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receptor (EGFR) and the platelet-derived growth factor receptor (PDGFR) are known

Oncogenes that are over expressed or mutated in a number of human malignancies

(Yarden, 2001; Blume-Jensen and Hunter, 2001), Similarly, intracellular kinases that

are intermediate effectors in relaying intracellular signals are frequently targets for

oncogenic activation (Frame, 2002; Sawyers, 2002). inactivation of TSGs involved in

cell signaling may result in malignant transformation as well. For example, the

inactivation of phosphatase and tensin homologue (PTEN), which normally promotes

apoptosis by inhibiting the Akt cell survival signaling pathway, may promote

carcinogenesis in the prostate, breast, ovary, endometrium and brain (Backman et ai,

2002; Mills eta/., 2001; Liu eta/,, 2000).

Factors escalating or influencing cancer risks are varied, acting alone or in

combination, usually over a period of many years. More than half of all cancers are

due to variations in dietary habits, environmental exposures and life style factors

including tobacco use, alcohol consumption, overweight and obesity, lack of physical

activity etc. Besides, genetic predisposition and medical interventions, infectious

agents such as viruses, bacteria and parasites, reproductive and hormonal factors

may also account for increased cancer risk. However, most of the prevalent human

cancers can, to a significant extent, be prevented and many could be avoided by a

suitable choice of lifestyle and environment. Many specific causes of cancer are now

known, the most important being smoking, obesity and a few oncogenic viruses, but a

large proportion of global variation for common cancers such as breast, prostate,

colon and rectum remain unexplained.

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1.2 Prostate Cancer

Prostate cancer (CaP), the most common non-dermatological malignancy in

many developing nations as well as in developed countries such as the United States

of America, ranks as the leading cause of cancer death in men (Jemal et a/,, 2004),

Currently in India, CaP is the fourth most prevalent cancer in men ranking after lung,

stomach and colon. Compared with western countries the incidence of CaP in India

and other Asian countries is less however every 8 men out of 100 thousands will

develop clinical detectible CaP in their life time. According to the projections from

American Cancer Society, in year 2004, 230,110 new CaP cases will be diagnosed in

the US alone and approximately 29,900 CaP-related deaths are predicted in the USA

(Jemal et ai, 2004). In 15 years time, CaP is predicted to be the most common

cancer in men and thus has become a significant public health concern. The incidence

of CaP increases rapidly with advancing age, and multiple genetic and epigenetic

factors have been implicated in the oncogenesis of the disease. Despite recent

improvements in diagnostic and therapeutic techniques, survival rate of these patients

is poor because the majority of the patients are present with advanced stage of the

disease at the time of diagnosis.

Death from CaP is usually associated with the development of hormone-

refractory metastatic disease (Gittes, 1991; Epstein, 1998).The initiation and

progression of CaP involves a complex series of both exogenous and endogenous

factors. Although clinical CaP incidence and mortalities vary greatly among populations

(Jemal et al., 2004; Setchell et al., 1984), the frequency of latent CaP is evenly

distributed, This suggests that extemal factors are important in the transformation from

latent into more aggressive clinical cancers (Adlercreutz et al., 1993; Adlercreutz et al.,

1990). Geographic variations and increasing incidence in populations migrating to high

incidence areas, demonstrate increasing evidence that lifestyle factors such as diet,

physical activity, smoking and other environmental factors may be important in the

etiology of invasive CaP (Jemal et al., 2004, Carlstrom and Stege, 1997; Nomura and

Kolonel, 1991).

iVlost cancers have a latency period of 10 to 20 years, which provides ample

time for preventive measures (Kelloff et al., 1999). In most epithelial tissues, including

the prostate, genetic progression and loss of cellular control functions occur as the cell

and tissue phenotype changes from normal to dysplasia (prostatic intraepitheliai

neoplasia [PIN]), to increasingly severe dysplasia high-grade PIN (HGPIN), then to

superficial cancers, and finally to invasive disease. These changes occur over a long

time period as CaP is known to have a long latency period (Kelloff et al., 1999; Boone

et al., 1992). Specifically in the prostate, PIN develops over approximately 20 years.

Progression from PIN to HGPIN and early latent cancer may take 10 or more years,

and clinically significant carcinoma may not occur for another three to 15 years (Kelloff

et al., 1999; Boone et ai, 1992). The features of prostate cancer including high

prevalence, long latency, significant mortality and morbidity, provide the most

promising opportunities for chemoprevention. The reason is that CaP is a disease

with high latency period and is typically diagnosed in men over the age of 50, The

present life expectancy in the world is around 78 years and those who live up to an

Chapter™I

age of 65 will ,have an average of 13 more years thus increasing the incidence of the

disease.

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1.3 Cancer Chemoprevention

Centuries old proverb in English, “An ounce o f prevention is worth a pound of

cure", though not said for Chemoprevention, is probably the basis behind this

emerging concept in cancer management.

Dr, Michael Sporn coined the term Chemoprevention (pronounced as KEE-mo-

pre-VEN-shun) in the 1970s, as part of his pioneering effort to encourage research

into preventing cancer before it begins rather than treating tumors once they appear.

Chemoprevention, by definition, is a means of cancer control in which the occurrence

of the disease can be entirely prevented, slowed or reversed by the administration of

one or more naturally occurring and/or synthetic compounds (Wattenberg, 1990;

Ames, 1983; Morse and Stoner, 1993; Sporn, 1991; Ames and Gold, 1990; Kohlmeier

et at., 1997). The expanded definition of cancer chemoprevention also includes the

chemotherapy of precancerous lesions which are called preinvasive neoplasia,

dysplasia or intraepithelial neoplasia, depending on the organ system (Boone ef al.,

1990; Boone et al., 1992). Because of this expanded definition of chemoprevention

some degree of overlap between chemotherapy and chemoprevention also occur. For

example, vi/hen an established cancer is cured by chemotherapy, chemoprevention could

prevent its recurrence and any further development of new cancers. Thus, in recent

years the concept of chemoprevention has matured to an extent that it appears real to

achieve significant reversal and/or complete to partial suppression of pre-malignancy to

malignancy at several sites by chemopreventive agents.

Ideally chemopreventive compounds should have a) little or no toxic effects, b)

high efficacy in multiple sites, c) capability of oral consumption, d) a known

mechanism of action, e) low cost, and f) human acceptance, Chemoprevention of

cancer thus differs from cancer treatment in that the goal of this approach is to lower

the rate of cancer incidence. This approach is promising because the therapy and

surgery have not been fully effective against the high incidence or low survival rate of

most of the cancer types. Furthermore, this approach appears to have practical

implications in reducing cancer risk because unlike the carcinogenic environmental

factors that are difficult to control, individuals can make decisions to modify their

choice for the food and beverage they consume, in recent years, the naturally

occurring compounds, especially the antioxidants, present in the common diet and

beverages consumed by human population have gained considerable attention as

chemopreventive agents for potential human benefit (Wattenberg, 1990; Ames, 1983;

Morse and Stoner, 1993; Sporn, 1991; Ames and Gold, 1990; Kohlmeier et al., 1997),

Abundant epidemiological, experimental, and metabolic studies have provided

convincing evidence that nutrition plays an important causative role in the initiation,

promotion and progression stages of several types of human cancers (Wattenberg,

1990; Ames, 1983, Boone et al., 1992), It has become clear that, in addition to

substances that pose a cancer risk, the human diet also contains agents which are

capable of affording protection against some forms of cancer (Ames, 1983; Boone et

al., 1990; Surh, 2003; Kohlmeier et al., 1997). This collective information strongly

suggest that the occurrence of cancer can be prevented or slowed by dietary intake of

substances that have the capacity to afford protection against the occurrence of

cancer.

Some of the chemopreventive agents that demonstrate potential for cancer are

drugs, biologies, and micronutrients. For all cancer targets, successful chemopreventive

strategies require well-characterized agents, suitable cohorts, and reliable intermediate

biomarkers of cancer for evaluating chemopreventive efficacy. Agent requirements

include experimental or epidemiological data that show chemopreventive efficacy, safety

on chronic administration, and a mechanistic rationale for the observed chemopreventive

activity. According to conventional classification originally proposed by Lee Watenberg,

chemopreventive agents are subdivided into two main categories- blocking agents and

suppressive agents (Wattenberg, 1985). Blocking agents prevent carcinogens from

reaching specific target sites, from undergoing metabolic activation or from

subsequently interacting with crucial cellular macromolecules (DNA, RNA and

Proteins). Suppressing agents, on the other hand, inhibit the malignant transformation

of initiated cells in either the promotion or the progression stage. Chemopreventive

phytochemicals have an ability to block or reverse the premalignant stage (initiation

and promotion) of multi stage carcinogenesis.

in recent years, attention has been focused on the role of nutrients as

chemopreventive agents. The concept of using micronutrients for the chemoprevention

of cancer is based on the evidence from human epidemiology, the results of a few clinical

trials, and studies of animal carcinogenesis models for cancer-inhibiting potential of these

Chapter-I

ter~I

substances. Basic research has identified nutrients as agents that inhibit mutagenesis

and hyperproliferation, as well as those that induce apoptosis or differentiation as critical

characteristics for chemoprevention regardless of their specific molecular targets. Some

of the most promising nutrients identified as chemopreventive agents in prostate cancer

include phytoestrogens/isoflavones, vitamins D and E, dietary phytochemicals, selenium

and lycopene.

The research aimed towards the prevention of cancer development began in

mid 1950s corresponding to the time when the researchers were busy understanding

the process of carcinogenesis. However, the field of chemoprevention became more

prominent and focused in late 1960’s, when Lee W. Wattenberg, one of the founders

of the field, conceptualized this strategy and suggested a mechanistic framework to

the diverse array of agents identified (Wattenberg, 1966; 1985 and 1992), He initially

called the concept of cancer chemoprevention as ‘chemoprophylaxis of

carcinogenesis’ (Wattenberg, 1966).

The chemoprevention research efforts of National Cancer Institute of the USA,

regarded as one of the best cancer research organizations, started in the early 1980s

and have grown considerably since that time. Currently, approximately 400

compounds are being studied as potential chemopreventive agents, mainly in

laboratory research. Over 40 of these compounds are being studied in clinical trials.

However the biggest challenge remains to find the answers for; a) which

chemopreventive agent(s), b) what form and route of administration, and c) which

population. Once we are able to find the satisfactory answers to these questions, the

reduction of cancer through chemoprevention could become a reality.

In recent years, naturally occurring compounds, especially the antioxidants,

present in the common diet and beverages consumed by human population have

gained considerable attention as chemopreventive agents for potential human benefit

(Wattenberg, 1990; Ames, 1983; Morse and Stoner, 1993; Sporn, 1991; Ames and Gold,

1990; Kohlmeier et al„ 1997). Abundant epidemiological, experimental, and metabolic

studies have provided convincing evidence that nutrition plays an important causative

role in the initiation, promotion and progression stages of several types of human

cancers (Wattenberg, 1990; Ames, 1983; Boone et a/., 1992). It has become clear that,

in addition to substances that pose a cancer risk, the human diet also contains agents

which are capable of affording protection against some forms of cancer (Ames, 1983;

Ames and Gold, 1990; Kohlmeier et a!., 1997). This collective information strongly

suggest that the occurrence of cancer can be prevented or slowed by dietary intake of

substances that have the capacity to afford protection against the occurrence of cancer.

Chapter-I

1.3.1 Prostate cancer chemoprevention

Evidences from geographic, epidemiological and in vitro and in vivo

experiments suggest that environmental carcinogenic factors and nutrition play

important causative role in the initiation, promotion and progression stages of CaP

(Hebert et a I., 1998; Rose, 1996; Hayward ef a/,, 1998). Thus, chemoprevention

appears to be a useful strategy for the management of CaP. This is well-supported by

the epidemiological observation that the Japanese and Chinese populations, which

are habitual drinker of several cups of tea, have one of the lowest rate of CaP in the

World (Wynder et al., 1994; Kohlmeier et a/., 1997).

CaP is believed to be an ideal candidate disease for chemoprevention

because of its high latency period and as it is typically diagnosed in men over the age

of 50. Thus, even a slight delay in the progression of this disease by chemoprevention

could result in a substantial reduction on the incidence of the disease and more

importantly, improve the quality of life of the patients with the disease (Klein &

Thompson, 2004; Wynder ef a/., 1994), The identification of promising agents (and

their molecular targets) for CaP prevention is guided by data derived from a variety of

sources, These evidence-based leads come from (a) epidemiological observations, (b)

prostate cancer treatment trials, (c) secondary analyses from large, randomized,

controlled cancer prevention trials, (d) an understanding of cancer biology and

prostate carcinogenesis, and (e) experimental animal models.

1.4 Chemopreventive Agents

Observations based on statistical and epidemiological data suggest that dietary

factors stimulate the development, growth and spread of tumors, and transform

normal cells into malignant ones. These are regarded as suspected carcinogens,

however there is also accumulating evidence from population as well as laboratory

studies to support an inverse relation between regular consumption of fruit and

vegetables and the risk of specific cancers. Certain foods, Including many vegetables,

fruits and grains, offer protection against various cancers. Cliemoprevention

researchers try to find substances--either in food or pharnnaceuticals--that can prevent

or halt carcinogenesis. Although it is said that there is no 'magic bullet’ that can

completely conquer cancer but using chemopreventive agents many types of the

disease could be avoided. It has been estimated that more than two-thirds of human

cancer could be prevented through appropriate lifestyle and dietary modifications

(Surh, 2003).

1.4,1 Dietary phytochemicals as chemopreventive agents

Phyto’ Is from the Greek word meaning plants and thus phytochemicals are non-nutritive

components in the plant-based diet that possess substantial anticarcinogenic and

antimutagenic properties. It is believed that people who eat five servings of fruits and

vegetables have half the risk of developing cancer than those who have three or less.

The NCI has identified about 35 plant-based foods that possess cancer-preventive

properties. These include tea, turmeric, garlic, soybeans, ginger, onion, tomato and

various cruciferous vegetables including cabbage, borocolli, cauliflower and Brussels

sprouts (Figure 1). Ongoing and performed studies have shown the validity of use of

these phytochemicals in cancer chemoprevention (Surh, 2003). Evidence suggests that

a diet high in fruits and vegetables may decrease the risk of chronic diseases, such as

cardiovascular disease and cancer, and phytochemicals including phenolics,

flavonoids and carotenoids from fruits and vegetables may play a key role in reducing

chronic disease risk (Surh, 2003; Ames, 1983). In the laboratory, phytochemicals have

been found to have very strong antioxidant activity, inhibit cancer ceil proliferation,

decrease lipid oxidation, and lower cholesterol. Phytochemicals, include quercetin,

catechin, phloridzin and chlorogenic acid, all of which are strong antioxidants.

Many population-based studies have highlighted the ability of macronutrients (for

example, carbohydrates, fat, proteins, fibres etc.) and micronutrients (vitamins and

trace metals etc.) that are present in the vegetables and fruits to reduce the incidence

of cancer. However, plants contain numerous chemical substances other than these

micronutrients that might also be useful in preventing cancer,

Chapter-I

Chapter-I

5 c) {\ }

•

^ 0■> /

”iV {

u/ \

T7

I ^ "1 > ci

0

)—ji, s

Figure 1: Representative chemopreventive phytochemicals and their dietary sources.

These micro- and macro- nutrients involve numerous cellular molecules and

events in their act of chemoprevention. The cellular and molecular events affected or

regulated include carcinogen activation/detoxification by xenobiotic metabolizing

enzymes, DNA repair, cell-cycle progression, cellular proliferation, differentiation and

apoptosis, expression and functional activation of oncogenes, angiogenesis and

metastasis, and hormonal and growth-factor activity (Surh, 2003; Mukhtar & Ahmad,

1999). Despite these remarkable advances, the identification of molecular and cellular

targets of chemopreventive phytochemicals is still incomplete. Many of the molecular

alterations that are associated with carcinogenesis occur in cell-signaling pathways

that regulate cell proliferation and differentiation.

1.5 The beverage Tea: an overview

Tea, made from the leaves of Camellia sinensis, an evergreen shrub of the

Theaceae family, is a beverage of choice in many countries around the world.

Consumption of tea can be traced back to 2737 B.C., according to a Chinese legend,

it was Shen Nung a legendary Emperor known as the Divine Healer, who discovered

the refreshing effects and fine taste of tea when he was out camping and a few

Camellia leaves chanced to fall into boiling water. The Chinese started cultivating this

plant and it subsequently spread to Japan, Java, India, Sri Lanka etc. as tea became

an increasingly popular beverage. Currently tea plant is being cultivated in

approximately 30 countries. According to an estimate of the Food and Agriculture

Organization of the United Nations, world tea production in 2002 reached 3 million

tons and the demand for tea has been growing ever since, For many thousand years,

the harvesting and processing of the leaves of the tea plant Camellia sinensis, has

become as an integral part of human society and traditional culture. Because of its

characteristic flavor and pharmacological properties, next to water, tea is the most

popular beverage consumed worldwide (Harbowy and Balentine, 1997). The per

capita consumption of tea in the United States of America is approximately 340 g.

Although the largest total consumption of tea is registered in India (540,000 metric

tons, 620 g per capita), Ireland has the largest per capita consumption of tea (3220 g).

Chapter-I

11

1.5.1 Consumption, Composition and Chemistry of Tea

1.5.11 Consumption

Tea plant originated in Southeast Asia and is presently cultivated in over 30

countries around the globe and currently tea beverage is consumed worldwide, although

at greatly varying levels. Consumption of tea is far from uniform. A large segment of the

world's population virtually consumes no tea. Not only does the tea consumption vary

from country to country, but also there is enormous variation in any given population.

This ranges from none to as many as 20 or more cups per day. Although firm data is not

available, it is generally accepted that next to water, tea is the most consumed beverage

in the world; with a per capita worldwide consumption of approximately 120 ml per day

(Katiyar and Mukhtar, 1996). Approximately 2.5 million metric tons of dried tea is

annually manufactured in the whole world of which, 78% is black tea, 20% is green tea

and less than 2% is oolong tea. Green tea is produced in relatively few countries, and is

mainly consumed in China, Japan, India, and a few countries in North Africa and Middle

East. About 78% of the worid's total tea consumed is black tea, wtnich is mainly

consumed in the western countries and some Asian countries. Oolong tea production

and consumption is confined to southeastern China, and Taiwan (Katiyar and Mukhtar,

1996),

Chapter™I

1.5.1.2 Composition

The composition of tea-leaf varies with climate, season, horticultural practices, variety of

the plant, and age of the leaf, i.e. the position of the leaf on the han^ested shoot. Three

main varieties of the commercial tea are available: Green (unfermented), Oolong

(partially fermented) and Black (fully fermented). Their composition varies according to

the manufacturing process which differs in the degree of 'enzymatic oxidation' or

fermentation. In Table 1 most important polyphenolic components present in a typical

green and black tea beverage is shown but variations may be considerable (Katiyar and

Mukhtar, 1996). Oolong tea composition in general fails in between green and black teas.

Green tea. The manufacturing process of green tea involves rapid steaming or

pan frying of freshly harvested leaves to inactivate enzymes, prevent fermentation and

thereby producing a dry stable product. Green teas are generally produced in two

12

Chapter"I

Table 1. Major polyphenolic constituents present in tea leaves

(%wt/wt).

COMPGNEWTS, ' ‘ GREEN TEA ’ BLACK TEA

Catechins 30-42 3-10

Flavonols 5-10 6-8

Other flavanoids 2-4 _

Theogallin 2-3

Gallic acid 0.5

Quinic add 2.0

Theanine 4-6 “

Methylxanthines 7-9 8-11

Theaflavins 3-6

Thearubigens 12-18

13

different varieties; White tea and Yellow tea, the latter is less fermented because of

the increasing popularity of green tea, a wide variety of green tea products are coming in

the market. Epicatechins are the main constituent compounds in green tea, giving it the

characteristic color and flavor.

Chapter-I

Black tea and Oolong tea. When producing black and oolong tea, the fresh

leaves are aliowed to wither until the moisture content of leaves is reduced to about 55%

of the original leaf-weight that resuits in the concentration of poiyphenois in the leaves

and the deterioration of leaf-structurai integrity. This step gives the typical aroma to the

tea. The withered leaves are rolled and crushed, initiating fermentation of the

polyphenols. This process is known as maceration and the fermenting mass is known as

'dhool'. The process used to macerate the leaf plays an important role in the final grading

of tea. During these processes the catechins are converted to theaflavins and

thearubigins. Theaflavins are astringent compounds contributing importantly to the color

and taste of the black tea. The thearubigen fraction is a mixture of substances, with a

molecular weight distribution of 1,000-40,000 and account for 15% of dry weight solids of

black tea.

Oolong teas are prepared by firing the leaves shortly after rolling to terminate the

oxidation and dry the leaves. Normal oolong tea is considered to be about half fermented

compared to black tea. Oolong tea extracts contain catechins at a level of 8-20% of the

total dry matter. The fermentation process results in the oxidation of simple polyphenols

to more complex condensed polyphenols to give black and oolong teas their

characteristic colors and flavors (Harbowy and Balentine, 1997).

1.5.1.3 Tea Chemistry

The chemical composition of green tea is approximately similar to that of the

fresh leaf with regard to the major components. Green tea contains polyphenolic

■compounds, which include flavanols, flavandiols, flavonoids, and phenolic acids. These

compounds account up to 30% of the dry weight of green tea leaves. Most of the

polyphenols present in green tea are flavanols, commonly known as catechins. Some

major catechins present in green tea are (-)-epicatechin (EC), (-)-epicatechin-3-ga!late

(ECG), (-)-epigallocatechin (EGC), and (-)-epigallocatechin-3-gallate (EGCG). The

14

chemical structures of these compounds are given in Figure 2, In addition, caffeine,

theobromine, theophylline, and phenolic acids such as gallic acids are also present in

green tea (Table-1).

During the fermentation process involved in the manufacture of black tea, the

monomeric fiavan-3-ols undergo polyphenol oxidase-dependent oxidative polymerization

leading to the formation of bisflavanols, theaflavins, thearubigins, and some other

oligomers. Theaflavins (1-2%, on dry weight basis) contains benzotropolone rings with

dihydroxy or trihydroxy substitution systems. About 10-20% of the dry weight of black tea

is due to thearubigens, which are even more extensively oxidized and polymerized. The

structures of theaflavins and thearubigins are shown in Figure 2.

Oolong tea contains monomeric catechins, theaflavins, and thearubigins. In

addition, epigallocatechin esters, theasinensins, dimeric catechins, and dimeric

proanthocyanidins are also the characteristic components of oolong tea.

1.5.2 Health beneficial effects of tea

For many thousand years, the harvesting and processing of the leaves of

Camellia sinensis, popularly known as tea in the whole world, has become as an integral

part of the human society and culture. Next to water, tea is the most popular beverage

consumed worldwide preferably because of its characteristic flavor and pharmacological

properties (Harbowy and Balentine, 1997). Although the largest total consumption of tea

is registered in India (540,000 metric tons, 620 g per capita), Ireland has the largest per

capita consumption of tea (3220 g). The per capita consumption of tea in the USA is

approximately 340 g. Tea contains several poiyphenolic components, which are

antioxidant in nature, and many studies have shown that tea polyphenols possess the

ability to prevent oxidant-induced cellular damage (Harbowy and Balentine, 1997; Katiyar

and Mukhtar, 1996). In recent year studies, from many laboratories worldwide,

conducted in various organ specific animal bioassay systems have shown that tea and

the poiyphenolic constituents isolated from it, are capable of affording protection against

a variety of cancer types. Although majority of the studies is conducted with green tea, a

limited number of studies have also shown the anti-cancer efficacy of black tea.

Chapter-I

15

Chapter“I

O H

Catechin

OHA ^ oh

Y ^ ohOH

Gallocatechin

OH

Gallocatechin gailate

OH

Epicatechin

OH

HOv/v .0,Y Y ' Y ' V A qH

OHOH

Epigallocatechin Epicatechin gailate

OH

“PO:*-O H

Epigallocatechin gailate Thearublgins

O H

Theaflavins

Figure 2: Chemical structures of major Green and Black tea polyphenols. R« galio]

group.

16

Tea is consumed worldwide for different reasons ranging from improving blood flow,

combating cancer and cardiovascular disease, eliminating various toxins, and

improving resistance to various diseases. Supportive scientific evidences for these

claims, surfacing in recent times have lead to an increase in the consumption of green

tea. Much emphasis is being placed on events at the cellular level due to its strong

antioxidant activity. Several studies have suggested that the polyphenols, present in

green tea possess high antioxidant activities, which in turn, protects cells against the

adverse effects of damaging reactive oxygen species (ROS) that are constantly

produced in the body. ROS, such as superoxide radical, hydroxyl radical, singlet

oxygen, hydrogen peroxide, peroxynitrite, and alkoxyradicals, damage lipids, protein,

and nucleic acids, and cellular components such as ion channels, membranes, and

chromatin, this in turn leads to cellular injury and cellular dysfunctions. These ROS

are known to contribute to the etiology of many chronic health problems including

cardiovascular diseases, inflammatory diseases, diabetes, obesity and cancer

(Katiyar and Mukhtar, 1996; Liao et a!., 2001), Wei et al. (1996) have shown that the

polyphenolic constituents of tea can act as scavengers of ROS and thereby prevent

damage to cellular macromolecules. The scavenging activity of the specific catechin

molecules is related to the number of o-dihydroxy and o-hydroxyketo groups, solubility,

concentration, the accessibility of the active group to the oxidant, and the stability of

the reaction product (Liao eta!., 2001).

Some of the effects of tea polyphenols may also be due to the chelation of

metal ions. Tea manifests chelating activity in vivo as indicated by the fact that tea

consumption lowers absorption of dietary iron in controlled feeding studies and

decreases body iron balance (Liao et al., 2001). Also, this chelating activity is

important since it protects iron-loaded hepatocytes from lipid peroxidation by

removing iron-from these cells. The study has shown that EGCG may chelate the

cations, which may contribute to its ability to inhibit angiotensin-converting enzyme.

Polyphenols of tea chelate copper ions and this mechanism has also been

suggested to protect low-density lipoproteins from peroxidation. Because of its

chelating properties tea may additionally protect against toxicity due to heavy

metals (Liao et al., 2001). Catechins may also affect signal transduction pathways,

modulate many endocrine systems, and alter hormones and other physiological

17

Chapter"I

processes as a result of their binding tliese metals/enzyme co-factors (Kao et a/.,

2000). Tlie beneficial effects of tea consumption in health and the associated

mechanisms by which it provides health benefits are summarized in table 2

Table-2; Beneficial effects of tea consumption

Disease Evidence in Humans/ Animals

Possible Targets/iechanisms

Cardiovascular Strong/ Moderate Ability to inhibit the oxidation of LDL. Decrease the plasma phoshatidylcholine hydroperoxide level. Improve brachial artery dilation. Lower hypercholesterolemia to normal levels and reduce blood pressure.

Cancer Population dependent/ Strong

Prevent the activation of procarcinogens. Protect against DNA scissions & mutations. Antioxidant actions. Block activated protein-1 and inhibit mitotic signal transducers. Reduce the occurrence of chromosomal aberrations. Inhibit the formation of 5-a-reductase. Inhibition of inducible NO- synthase and generation of NO. Inhibition of urokinase. Induction of apoptosis and cell cycle arrest.

Diabetes Suggestive /Moderate Inhibit the formation of sugars. Repress glucose production and phoshoenolpyruvate carboxykinase and glucose-6- phosphatase gene expression, antihyperglycemic effects. Protect against cytokine- mediated damage of pancreatic p- cells.

Obesity Suggestive/ Moderate Decline in food intake. Lower the blood levels of glucose and insulin. Increase the 24-hour energy expenditure. Reduce high-fat diet-induced body weight gain.

Osteoporosis Some/ None Increase bone mineral density.

Arthritis Suggestive/ Some Reduction of inflammatory mediators, neutral endopeptidase activity and IgG levels.

Neurological Some/ None Inhibit tyrosinase. Inhibit COMT and Prolylendopeptidase. APP secretion and protection against Abeta toxicity.

Bacterial Suggestive/Some Modulation in composition of microflora. Inhibit the growth of Clostridia and promote bifidobacteria colonization. Decrease

ration of oxidants. Inhibiting the

18

Chapter-I

1.5.3 Tea and cancer chemoprevention

Cancer is believed not to be a single disease but rather a conglorneration of

several diseases. Most of the work on chemoprevention of cancer by tea has been

conducted using green tea or its individual polyphenolic constituents, especially

EGCG, which is a major constituent of green tea. Green tea catechins act as

antioxidants and inhibit the growth of cancer in experimental animal models. This

raises the possibility that consumption of green tea or its catechins may lower

cancer risk in humans. Several epidemiological studies conducted so far have

verified this suggestion. EGCG inhibits the action of enzymes to prevent the

activation of procarcinogens, resulting in their inactivation and finally excretion (Lin

etal., 1999), As shown by McArdle etal. (1999) consumption of both green tea and

black tea aqueous extracts influences the excretion of mutagens and promutagens

in the urine of animals. In animal studies, the polyphenolic fraction isolated from

green tea, the water extract of green tea, or individual polyphenolic antioxidants

present in green tea have been shown to afford protection against chemically

induced carcinogenesis in lung, liver, esophagus, pancreas, forestomach,

duodenum, colon, and breast (Ahmad etal., 1996, Katiyarand Mukhtar, 1996, Liao

et a!., 2001). From some recent studies conducted, it is now believed that much of

the cancer chemopreventive properties of green tea are mediated by EGCG, but

other polyphenolic constituents may also possess similar effects (Ahmad et al.,

1996; Katiyar and Mukhtar, 1996; Mukhtar and Ahmad, 1999). However, it is yet to

be decided whether the different polyphenolic constituents of green tea work

through similar or different mechanisms.

Outcome of several epidemiological studies have suggested that tea and its

associated compounds prevent certain types of cancers (Liao et al., 2001). This is

understandable as cancer is a complex disease with multiple etiologies, even for

one body site. Therefore, it seems to be a false hope that any single nutritional or

synthetic agent can prevent or treat all types of cancer. However, based on a large

volume of cell culture, animal studies, and human observational studies, there is a

hope that tea consumption can retard cancer development at selected sites in

some populations. The challenge is to find these populations that could reap the

benefit.

19

Chapter-I

Green tea users have an approximate 50% reduction in risk for both

esophageal cancer (Gao et al., 1994) and stomach cancer (Yu et al., 1995).

Inhabitants of tea-producing districts in Japan have a lower mortality due to

stomach cancer, possibly due to regular consumption of green tea (Oguni et al.,

1989). In addition to regular drinking of tea, the Japanese population consumes

green tea in all types of products, including candy, gums, bread, and many other

edible products. Green tea was found to be linked to a reduced risk of oral cancer

in northern Italians and a Chinese population; esophageal cancer in Chinese

women; gastric cancer in Swedish adolescents; pancreatic cancer in residents of a

retirement community in the USA; and colon cancer amongst retired male self-

defense officials in Japan (Kono et al., 1991; Liao et al., 2001; Schwarz et al., 1994).

Cohort studies suggest a protective effect of green tea for colon, urinary bladder,

stomach, pancreatic, and esophageal cancer (Bushman, 1998), In a Japanese

population survey, an overall protection together with a slowdown of the increase of

cancer incidence with age was reported (Imai et al., 1997). The effects were found

to be more pronounced when the consumption of tea was over 10 cups per day.

According to a study, consumption of seven or more cups per day of green tea

significantly decreased the risk of stomach cancer (by 31%) compared with no

green tea consumption (Inoue et a!., 1998). Regular drinkers of tea experienced a

12% and 53% lower incidence of cancer among males and females, respectively,

compared with non-tea drinkers (Ji et al., 1997). When the intake of tea exceeded

200 g/month (dry weight of tea prior to brewing), the risk reduction remained

unchanged among women, whereas the incidence of pancreatic cancer was further

decreased by 43% in men (Ji et al., 1997). Another case-contro! study from Poland

reported a significant reduction in risk of pancreatic cancer with increasing lifetime

consumption of tea (Zatonski et al., 1993). An increased consumption of green tea

was closely associated with a decreased number of axillary lymph node

metastases among premenopausal patients with stage I and II breast cancer and

overall decreased recurrence of stage I and II breast cancer (17% for Individuals

drinking more than five cups and 24% for those drinking less than four cups.

Recent studies have shown that green tea polyphenols inhibit the growth

and progression of prostate cancer in |ransgenic adenocarcinoma of mouse

20

C l ia L p t e r - I

grostate (TRAMP) model that mimics human disease (Gupta et al., 2001). Prostate

cancer is an ideal cancer type for prevention by green tea because the disease is

typically diagnosed in older men and thus even a modest delay in disease

development could produce a substantial benefit (Gupta et al., 2001).

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