WILLIAM BOYD MAYER, JR. The Effects of Iron and Quercetin on Apoptosis in a Human Hepatoma Cell Line (Under the Direction of JOAN G. FISCHER)

Quercetin, a flavonoid, is cytotoxic to human tumor cells at high concentrations.

The objective of the present study was to determine if cellular iron status influences

quercetin-stimulated cytotoxicity and apoptosis in a human hepatoblastoma cell line. To

test the hypothesis, cell viability, lipid peroxidation, and apoptosis were measured in

HepG2 cells following treatment with various levels of iron and quercetin. The data

suggest that Fe concentration may influence quercetin’s effect on cell viability and

apoptosis in HepG2 cells.

INDEX WORDS: Quercetin, Iron, Apoptosis, Liver cells, Oxidative stress,

Reactive oxygen species

THE EFFECTS OF IRON AND QUERCETIN ON APOPTOSIS IN A HUMAN

HEPATOMA CELL LINE

by

WILLIAM BOYD MAYER, JR.

B.S., Clemson University, 1995

A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial

Fulfillment of the Requirements for the Degree

MASTER OF SCIENCE

ATHENS, GEORGIA

2001

© 2001

William Boyd Mayer, Jr.

All Rights Reserved

THE EFFECTS OF IRON AND QUERCETIN ON APOPTOSIS IN A HUMAN

HEPATOMA CELL LINE

by

WILLIAM BOYD MAYER, JR.

Approved:

Major Professor: Joan G. Fischer Committee: Mary Ann Johnson Arthur Grider Electronic Version Approved:

Gordhan L.Patel Dean of the Graduate School The University of Georgia December 2001

ACKNOWLEDGEMENTS

I am truly grateful to the many individuals who contributed their time and efforts

to my development as a graduate student. First, I would like to express my appreciation

to Dr. Joan Fischer. As a mentor, she taught me with patience, always providing support

and direction. She was a perfect role model, both as a professional and as a person.

I would also like to thank my committee members, Drs. Johnson and Grider. For

my entire graduate career, they were instrumental in helping to improve my research and

analytical skills. As well, they opened their labs to me for use of much-needed

equipment.

In our lab, Joyce Power was my supervisor, life management consultant, and

mother-figure. Her friendship and kindness will always be a special part of my

experience at the University of Georgia.

Dr. Michael Mouat was an invaluable resource to my project. I am thankful not

only for his expert advice, but also for his hospitality and gracious attitude since the

beginning of my career.

I would also like to thank Dr. Barbara Grossman and Mrs. Linda Azain. Both

provided incredible examples as instructors, and gave me the opportunity to develop my

creativity and public speaking skills.

Thanks to Dr. Rick Lewis, Chris Modlesky, and Jean Edmonds. They provided

their time and lab equipment to help with my research.

iv

v

I would like to thank my entire family in both Mt. Pleasant and Augusta. They

provided me with the unconditional support and constant encouragement I needed to

achieve my goals.

Most of all, I am grateful to my wife, Mary Dickey. She has given so much to

support me in school and in my life.

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS............................................................................................... iv

LIST OF FIGURES ......................................................................................................... viii

LIST OF TABLES............................................................................................................. ix

CHAPTER

I INTRODUCTION...................................................................................................1

II LITERATURE REVIEW........................................................................................4

Cancer ..........................................................................................................4

Cell Proliferation/Division...........................................................................6

Apoptosis .....................................................................................................7

Measures of Apoptosis...............................................................................11

Reactive Oxygen Species...........................................................................15

Iron and Oxidative Stress...........................................................................19

Fruits and Vegetables and Cancer Prevention ...........................................21

Polyphenols................................................................................................23

Quercetin....................................................................................................30

III IRON STATUS OF THE CELL MAY ALTER QUERCETIN-INDUCED

APOPTOSIS OF HUMAN HEPATOMA CELLS ...............................................38

Abstract ......................................................................................................38

Introduction................................................................................................39

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vii

Methods .....................................................................................................41

Results........................................................................................................50

Discussion..................................................................................................52

Figures .......................................................................................................60

Tables.........................................................................................................64

IV SUMMARY

Major Findings...........................................................................................67

Implications ...............................................................................................68

Study Limitations.......................................................................................68

Further Research ........................................................................................69

REFERENCES ......................................................................................................71

LIST OF FIGURES

CHAPTER III

Figure 1: Cellular Iron Concentration Following 24 Hour Iron + 24 Hour Quercetin Treatments.........................................................60

Figure 2: Lipid Peroxidation......................................................................61

Figure 3: Cell Viability Measured by Trypan Blue Exclusion ..................62

Figure 4: Mean Percent Increase in Quercetin-Induced Apoptotic DNA Fragmentation Above Control Within Each Iron Treatment ....................................................................................63

viii

LIST OF TABLES

CHAPTER II

Table 1: Previous Studies Demonstrating the Effect of Flavonoids Alone or in Combination with Redox Metals on Cell Lines or Specific Cell Components ........................................................37

CHAPTER III

Table 2: Study Design - Assays and Treatment Groups............................47

Table 3: General Timeline for Assays .......................................................48

Table 4: DNA Fragmentation: Chi Square Analysis .................................64

Table 5: Percent Cells with Caspase Activity............................................65

Table 6: Percent of Cells with Apoptotic Morphology (Chromatin Condensation)............................................................66

ix

CHAPTER I

INTRODUCTION Iron is a redox active metal, meaning that it may readily alternate between ferrous

and ferric states, accepting or donating an electron to a wide range of biological

substances. Through this mechanism, iron catalyzes a variety of damaging reactions

within the cell (McCord 1998). Iron can catalyze the formation of the hydroxyl radical

from H2O2 as well as the decomposition of lipid hydroperoxides to alkoxyl, peroxyl, and

other free radicals (Ibrahim 1997). Therefore, iron is a key factor in the establishment of

a prooxidant status in the cell (Meneghini 1997). Previous studies in our laboratory and

others have suggested that iron status of a cell may alter apoptosis (Stimson 1998; Wang

et al. 1999; Whittaker et al. 1996). Further, studies by Cermak et al. (1993), proposed

that acute exposure of intestinal tumor cells to iron may increase their susceptibility to

oxidant-mediated lysis.

Apoptosis is described as a sort of cellular suicide (Samali 1996) in which a cell

takes an active role in its own demise. It consists of a preprogrammed cascade of

metabolic events that occurs in response to various environmental inducers or a

combination of intrinsic factors, ultimately leading to the disintegration of the cell

(Darzynkiewiez 1995). Studies have shown that cell death caused by apoptosis may be

the mechanism by which some chemotherapeutic agents function.

It has also been proposed that oxidative stress is a mediator of apoptosis in eukaryotic

cells via several different mechanisms (Buttke and Sandstrom 1994). Oxidative stress

1

2

may induce apoptosis by damaging DNA. This may occur via metal-catalyzed oxidation

with subsequent generation of reactive intermediates such as hydrogen peroxide and the

hydroxyl radical (Duthie et al. 1997). In addition, oxidative stress may lead to the

formation of oxidized lipids in cell membranes, which may induce apoptosis (Buttke and

Sandstrom, 1994).

Recent studies (Kuo 1996) have shown that apoptosis was the mechanism of cell

death when flavonoids were added to neoplastic cell lines. Some phenolic antioxidants,

such as flavonoids, can reduce ferric iron to ferrous iron, thereby facilitating free radical

damage to carbohydrates, lipids, proteins, and DNA (Smith et al. 1992). Free iron may

also be released from ferritin by an appropriate reducing agent such as quercetin (Cermak

1993). Sahu and Washington (1992) found that increasing quercetin levels in the

presence of iron significantly increased lipid peroxidation and nuclear DNA damage in

rat liver nuclei. Therefore, tumor cells exposed to high iron levels in the presence of a

reducing agent, such as quercetin, could theoretically undergo apoptosis.

The purpose of this study was to determine whether a flavonoid common to many

foods (quercetin) could induce apoptosis in the HepG2 cell line, a liver hepatoma cell

line, and whether cellular iron status alters apoptosis.

It was hypothesized that 1) cellular iron status would increase with higher media

iron concentrations, and that addition of quercetin to iron-loaded cells would have no

effect on cellular iron, 2) addition of quercetin to iron-loaded cells would increase lipid

peroxidation, 3) cellular iron concentration would influence the prooxidative ability of

quercetin to decrease cell viability, 4) cellular iron concentration would influence the pro-

oxidative ability of quercetin to induce specific apoptotic changes in the cell measured as

3

200-bp DNA nucleosome fragmentation (ladder effect), caspase activation, and

chromatin condensation.

Major findings were 1) cellular iron status increased with iron loading over a 24

hour incubation period, 2) addition of quercetin to iron-loaded cells had no effect on

cellular iron, 3) iron had a positive effect on lipid peroxidation, while quercetin did not,

4) 80 µM concentrations of quercetin tended to decrease cell viability in iron-loaded

cells, 5) an 80 µM concentration of quercetin was consistently associated with an

increase in apoptosis-induced DNA fragmentation, and increases were greater in iron-

loaded cells, but the iron effect was not significant, 6) chromatin condensation was

affected by quercetin, but not iron, and 7) caspase activity was significantly increased in

cells supplemented with 80 µM quercetin, with the greatest increases seen in iron-

loaded cells.

CHAPTER II

LITERATURE REVIEW

CANCER

More than 1 million new cases of cancer and over 500,000 deaths occur each

year in the U.S. due to cancer (Breslow 1997). Moreover, the number of Americans

ever diagnosed with any invasive (metastatic) cancer could be over 11 million by

2020. Finally, people diagnosed with certain cancers are at increased risk of

additional cancers (Polednak 1997). Overall, rates of cancer are quite varied among

U.S. racial and ethnic populations. Considering all American men, cancer rates are

highest among African Americans (560 cases per 100,000 people) and whites (469

cases per 100,000 people). Incidence was lower among women in every racial and

ethnic group than among the men (Breslow 1997). Cancer is a group of diseases

characterized by uncontrolled growth and proliferation of abnormal cells. It is the

result of both genetic and environmental factors, which may act together or in

sequence to initiate carcinogenesis (American Cancer Society 1998). In addition to

elucidating some of the genetic and environmental causes of cancer, current research

has also gone further to uncover the mechanisms by which carcinogenesis occurs.

Cancer is caused by aberrant DNA. First, genetic mutations in critical genes can lead

to tumors. In fact, in approximately one-half of human tumors, mutations exist in the

tumor-suppressor gene, p53. This is important because inactivation of the p53 gene

allows for uncontrolled cell division. Another mechanism involved in carcinogenesis

4

5

is DNA lesions. This refers to damaged bases or chromosome breaks that have a

certain probability of producing a mutation upon cell division. The “effectiveness” of

a particular lesion depends on the rate of excision by DNA repair enzymes, and on the

probability that a mutation will occur when the cell divides. Cellular apoptosis or

“programmed cell death” provides the body with a mechanism for removal of cells

with damaged DNA. The effect that a particular insult has on the cell is dependent on

the level of each of these defenses, which in turn are actually dependent on the past

history of exposure. These defenses can also be disabled by lack of micronutrients

such as antioxidants in the diet.

On a molecular level, antioxidant deficiency is considered to be a risk factor for

cancer, as it can lead to “unrepaired or misrepaired endogenous oxidative DNA

damage” (Sahu and Washington 1992) and membrane lipid peroxidation. Free

radicals may mediate many events in carcinogenesis by producing damage to DNA,

lipids, proteins, and carbohydrates. External sources of free radical damage include

polycyclic aromatic hydrocarbons in food, polluted air, cigarette smoke, background

radiation, and internal sources include oxidative transformations in prostaglandin

synthesis, redox cycling of quinones, and oxidative phosphorylation (Block 1992).

Still another mechanism that may underlie excess free radical production is

chronic infection and inflammation. Leukocytes and other phagocytic cells combat

bacteria, parasites, and virus-infected cells by destroying them with nitrogen oxide and

superoxide. Both of these compounds can lead to formation of peroxynitrite,

hypochlorite, and hydrogen peroxide, all of which are mutagenic oxidizing agents.

So, while these oxidants protect the body from immediate harm, they can cause

6

oxidative damage to DNA, DNA mutation, and chronic cell death with compensatory

cell division. All of these processes then contribute to cancer development.

Cancer, one of the three major diseases in the U.S, is becoming more common

in the Western world and also in many populations of developing countries. With

modern medicine, some treatments have met with success, but they have been very

expensive. It is becoming evident that the most cost-effective control of these diseases

is through prevention. This is mainly achieved through the diet and other lifestyle

changes (Argiles 1998). In addition, cancer chemoprevention is a new field that has

great potential to affect cancer incidence rates.

CELL PROLIFERATION / DIVISION

A major factor in mutagenesis, and hence cancer development, is cell division.

When the cell divides, a DNA lesion can produce a point mutation, a single base

substitution, a deletion, or a translocation. Therefore, a critical factor in the mutagenic

effect of an agent is the increment of division that it produces in addition to cell

division in those cells that are important - the stem cells. Stem cells are not discarded,

as daughter cells are. An increase in the rate of cell division of stem cells increases

the mutation, leading to cancer. Increased cell division can be caused by a wide array

of agents, including increased levels of a particular hormone, excess calories, chronic

inflammation, or chemical exposure (Ames 1995). It is easy to see that without

continuous aid from antioxidants and radical scavengers, and endogenous defense

mechanisms, survival would cease (Block 1992). Another important protective

mechanism is cell cycle checkpoints. These checkpoints prevent cells with too many

DNA lesions from dividing (Darzynkiewicz 1995).

7

Most recently, the view of cancer as a disease of cell proliferation has been

challenged. Cell death studies have now shown how cell populations are maintained,

and how defects in cell regulation may contribute to the development of malignancy.

Therefore, focus has shifted to include cell death inducers and repressors. Defects in

either may produce deleterious results, especially considering the fact that increasing

the life span of a cell also increases the probability of acquiring mutations. When cell

proliferation exceeds cell death, researchers believe that this is 1) a result of

overexpression of a cell death repressor protein, or 2) resistance to conditions that

would normally kill the cell. Reduced cell death may also be caused by loss-of -

function mutations in cell death inducer proteins (Martin 1997).

APOPTOSIS

Apoptosis, or “programmed cell death,” is described as a sort of cellular suicide.

The term “apoptosis” comes from a Greek word that describes the process of leaves

falling from a tree, or petals from a flower (Samali 1996). It is well established that

during this process, a cell takes an active role in its own demise. A preprogrammed

cascade of metabolic events occurs in response to various environmental inducers or

by a combination of intrinsic factors, ultimately leading to the disintegration of the cell

(Darzynkiewicz 1995). As a mechanism, apoptosis is believed to be evolutionarily

conserved due to the fact that it occurs in both nematodes and vertebrates (Clutton

1997).

Apoptosis is also important in many physiologic processes, especially during

embryonic and fetal development (Kuo 1996). Some examples of this process relative

to development are regression of the Mullerian duct in male embryos, the removal of

8

interdigital webs, and amphibian tail regression during metamorphosis (Columbano

1995). Other processes that require apoptosis are: establishment of immune self-

tolerance, killing of cells by cytotoxic immune cells (King and Cidlowski 1995),

hormone-dependent atrophy, and even tumor growth. Apoptosis is the mechanism in

each of these processes that regulates cell number and destroys damaged cells (Kuo

1996).

A variety of stimuli can induce apoptosis, such as DNA damage, growth factor

withdrawal, calcium ion influx, and viral infection (King and Cidlowski 1995). In

vivo, apoptosis is detected primarily in proliferating tissues (Meikrantz and Schlegel

1995). Apoptotic bodies are accumulations of separate, intact pieces of the cell body

that result from the effect of specific destructive molecules which dismantle the cell’s

interior (Martin 1997). These compact bodies can be found anywhere in the body

where cells are dividing. Examples include intestinal crypts, the epithelium of the

adrenal cortex, in differentiating spermatogonium, and in the germinal centers of

lymph nodes (Meikrantz and Schlegel 1995). Apoptosis is also evident after periods

of rapid proliferation, such as in mammary tissue following weaning, in the

endometrium at estrus, and during ovarian follicular atresia. It is therefore expected

that apoptosis is a hallmark of malignancies, as it occurs at increased frequency in

preneoplastic tissues. In this case, apoptosis is thought to serve as a balance for an

increase in cell number due to proliferation with cell loss caused by death (Meikrantz

and Schlegel 1995).

Cells undergoing apoptosis share a few morphological features that are typically

characteristic of mitosis. Both apoptotic and mitotic cells are characterized by lack of

9

adhesion, rounded morphology, and reduced cell volume. Also, both mitotic and

apoptotic cells condense their chromatin in disassembling the nuclear lamina (King

and Cidlowski 1995). In spite of these similarities, several differences do exist. First,

apoptotic cells do not assemble a mitotic spindle. Also, apoptotic cells exhibit rapid

cytoplasmic blebbing – zeisosis, or “bubbling” of the cell membrane - while mitotic

cells do not. Finally, in most cases, apoptotic cells cleave their own DNA into

approximately 200 bp fragments (King and Cidlowski 1995). Overall, apoptosis plays

an opposing role to mitosis in the maintenance of cell populations. Therefore, cell

deletion occurs at an increased rate in rapidly dividing tissues (Columbano 1995).

It has been proposed that there are two phases in the process of apoptosis: an

activation phase and an execution phase. The activation phase is composed of the

intracellular pathway that activates apoptosis. The execution phase is composed of the

molecular mechanisms of apoptosis, which may involve proteases and nucleases.

Reactive oxygen species are most likely to induce the activation phase by acting as

cytoplasmic messengers in cell signal transduction (Rollet-Labelle 1998). The

specific mechanisms of apoptosis involves several key events, which include rapid cell

dehydration, increase in free calcium concentration, activation of an endonuclease

which has affinity to internucleosomal DNA sections, and activation of

transglutaminase activity. The biochemical events of apoptosis coincide with changes

in cell morphology. The loss of intracellular water is reflected by cell shrinkage and

condensation of the cytoplasm. Condensation of chromatin occurs, starting from the

periphery of the nucleus, followed by nuclear fragmentation. Next, apoptotic bodies,

or regions of cytoplasm that harbor intact and still functionally active organelles,

10

together with fragmentation of nuclei (wrapped in fragments of the plasma membrane)

detach from the cell. Finally, apoptotic bodies are incorporated in the tissue by

neighboring phagocytic cells. Again, there is no leakage of the cytoplasmic contents

into the intercellular space, so tissue inflammation or scarring does not occur

(Darzynkiewicz 1995). Overall, there is a marked increase in transglutaminase

mRNA, protein and enzyme activity, and protein-bound lysine. The transglutaminase

activity may stabilize the apoptotic cells and form a shell around the cell, thereby

inhibiting membrane leakage (Samali 1996).

As apoptosis is a programmed cell death, it is the result of activation of specific

mechanisms that systematically destroy integral cellular structures (Jacob 1997).

However, another form of cell death exists in response to an acute departure from

normal physiologic conditions (Martin 1997). This sudden cell death is called

necrosis, a result of any environmental stress that directly overwhelms cellular defense

mechanisms (Jacob 1997). Ultimately, it is the intensity of the insult that determines

whether apoptosis or necrosis occurs (Stoian 1996).

The main differences between apoptosis and necrosis are evident in the events

that occur in each respective process. In necrosis, the cell membrane is damaged; the

cell swells (due to an influx of water) and lyses (Rollet-Labelle 1998). Lysis exposes

intracellular contents to the extracellular environment, many of which are toxic when

not contained within the cell. As neighboring cells are damaged by these exposed

toxins, a chain reaction of death radiates from the original necrotic cell. To further

exacerbate this chain reaction, neutrophils and inflammatory macrophages are

attracted to the dying cells, and release even more damaging toxic enzymes. In

11

apoptosis, cell shrinkage, chromatin condensation, and chromatin DNA fragmentation

occur (Jiang 1996). The changes in the plasma membrane of the cell attract

phagocytes (Martin 1997), and the cell is subsequently engulfed by these phagocytes

(Rollet-Labelle 1998). Therefore, as opposed to necrotic cells, apoptotic cells do not

release their contents into the intracellular matrix, so the inflammation response that

accompanies necrosis does not occur. This is an important factor that distinguishes

apoptosis as an “altruistic” form of cell death. In other words, damaged or injured cells

commit suicide to allow adjacent cells to proliferate without being affected by the

otherwise necrotic death of neighboring cells (Samali 1996).

MEASURES OF APOPTOSIS

Nuclear Morphology

“The main component of apoptotic cells is the appearance of highly condensed

chromatin, segregated into sharply defined bodies within an intact nuclear envelope”

(Meikrantz and Schlegel 1995). Key structural proteins that maintain the integrity of

the nuclear matrix are a group of cellular substrates affected by caspases during

apoptosis. Selective proteolysis of these proteins results in the disintegration of the

nuclear envelope (Lockshin 1998). As the nuclear envelope breaks down, chromatin

strands become detached and condense into “crescentic caps or toroids at the

periphery of the nucleus” (Martin 1997). These variations in cellular morphology can

be seen with Hoechst 33342, a cell permeable bisbenzimide adenine-thymine specific

fluorescent dye that binds to the minor groove of DNA (Zhang 1999). It stains

chromatin, a chain of nucleosomes joined by linker DNA (naked DNA; Voet and Voet

12

1990) and allows for the quantification of cells with the abnormal nuclear morphology

(chromatin condensation) characteristic of apoptosis.

DNA fragmentation

During apoptosis, endonucleases digest DNA. Therefore, detection of DNA

fragments is a commonly used assay for apoptosis (Meikrantz and Schlegel 1995).

The characteristic ladder pattern of DNA produced during the late stages of apoptosis

is caused by cleavage of internucleosomal linker DNA (Samali 1996). In contrast,

necrosis is identified by a diffuse smear on the gel, indicating random DNA

breakdown (Columbano 1995). Specifically, three patterns of the DNA degradation

are indicated in apoptosis: single strand nicks, larger DNA fragmentation of 50-200

kbp, and nucleosome size fragments of 180-200 bp in size (Samali 1996). Standard

gel electrophoresis is one method of identifying DNA ladders. In situ, a terminal

deoxynucleotidyl transferase mediated dUTP nick end-labeling (TUNEL) assay can be

used to stain apoptotic cells. This technique labels 3’-OH ends of DNA that have been

exposed during apoptotic DNA cleavage (Allen 1997). ELISA, or Enzyme-Linked

ImmunoSorbent Assay also can be used to quantify the hallmark 180-200 bp

nucleosomes produced by the cleavage of internucleosomal linker DNA (DNA not

associated with histones; Voet and Voet, 1990). This quantification is achieved with a

variety of techniques, an example being the Direct Sandwich ELISA. This particular

ELISA involves the passive adsorption of a monoclonal anti-histone antibody to a

solid phase, usually a plastic microtiter plate module, followed by addition of an

antigen (the histone-linked nucleosomes). The bound antigen is then “sandwiched”

between the solid phase antibody and an enzyme-labeled antibody conjugate. The

13

amount of conjugate retained within the immunocomplex after a washing procedure is

determined photometrically after binding with an ABTS (2,2’-azino-di-[3-

ethylbenzthiazoline sulfonate]) substrate. A spectrophotometer passes a specific

wavelength of light through the retained immunocomplexes, measuring the amount of

absorption of light (Crowther 1995).

Caspase Detection

Caspases are a family of proteases that have recently received much attention,

due to their suspected roles in programmed cell death. Caspases are present in healthy

cells as zymogens, or inactive configurations (Martin 1997), and are activated through

limited proteolysis by the removal of a pro-peptide (Slee 1999). Caspase activity is

triggered by specific stimuli. These stimuli can be organized into three broad

categories including 1) cellular death receptors, 2) the cytotoxic contents of certain

lymphocyte cell granules, and 3) stimuli that provoke generalized cell damage. The

third category of caspase stimuli includes cytotoxic drugs, radiation, heat shock,

survival factor deprivation, and other cellular stresses (Slee 1999).

Once activated by a stimulus, caspases cleave other caspases in a cascade of

activation. Most notably, many substrate proteins within the cell are also cleaved

during the cascade, leaving the hallmark structural changes of apoptosis (Martin

1997). During apoptotic cell death, caspases cleave approximately 200 polypeptides

of the cellular proteome. Approximately 70 of these caspase targets have been

identified. Therefore, genomic disassembly and subsequent cleavage into

oligonucleosomal fragments are accepted evidence of apoptosis (Nicholson 1999).

14

To ensure their efficacy, caspases are also responsible for halting normal DNA

repair processes by inactivating two key proteins involved in the homeostatic

maintenance of the genomic integrity – poly(ADP-ribose) polymerase (PARP) and

DNA-PKcs (catalytic subunit of DNA-dependent protein kinase). Obviously, if these

proteins remain activated, they would counteract any proteolysis events in the genome.

Simultaneously, caspases mediate the disablement of an inhibitor of an apoptosis-

dedicated endonuclease. In summary, results of the cleavage events are to 1) disable

DNA maintenance and repair mechanisms, 2) stop cell cycle progression, 3) render

caspase inhibitors inactive, 4) identify the dying cell as apoptotic, with the

consequence of phagocytosis. Finally, through proteolysis, caspases are also involved

in the disassembly of the cytoskeleton and nuclear scaffold (Nicholson 1999).

The tripartite structure of caspases consists of a prodomain, which varies among

the 11 known human caspases. Additionally, one large and one small subunit

comprise the active form of the enzyme. These subunits are released from the

proenzyme by cleavage at Asp (P1)-X(P1) bonds. This cleavage site enables the

caspases to auto-activate or be activated by other caspases. The proteolytic

components of the caspases include active site Cys and His residues. The active site

of caspases recognizes a very specific tetrapeptide sequence within a target substrate

polypeptide, which facilitates inhibitor and synthetic substrate design. Irreversible

caspase inhibitors, such as fluouromethylketones, may be labeled with a fluorogenic

agent, such as carboxyfluorescein. This cell permeable inhibitor binds covalently to a

wide range of active caspases to allow detection of caspase activity by flow cytometry,

fluorescence microscopy, or fluorescence spectroscopy (Nicholson 1999).

15

REACTIVE OXYGEN SPECIES

“Thirst” for electrons by molecules causes formation of a variety of reactive

oxygen intermediates. Reactive oxygen intermediates are oxygen species that possess

unpaired electrons or the ability to abstract electrons from other molecules. Reactive

oxygen species easily react with cellular macromolecules. This reaction can cause

immediate damage, or it may initiate a chain reaction where the free radical is passed

from one macromolecule to another, damaging cellular structures (Buttke and

Sandstrom 1994).

Under normal physiological conditions, reactive oxygen species can be formed.

For example, superoxide and hydrogen peroxide are produced as by-products of the

catalytic action of oxidases. These may also be formed by the leakage of electrons

from cellular electron-transferring chains (de Groot and Rauen 1998). More

physiological situations that produce free radicals include ubiquinone oxidation in

mitochondria, cytochrome p450 oxidation in microsomal membranes, reaction of

superoxide with hydrogen peroxide in the presence of free iron, prostaglandin

synthesis, respiratory bursts of phagocytic cells, xanthine oxidase activity, and

arginine oxidation by nitric oxide synthesis (Stoian 1996). Clearly, reactive oxygen

species are present in abundance in the normal human body. They can also be created

by external factors such as ionizing radiation (de Groot and Rauen 1998).

Of course, cells provide their own protection against reactive oxygen species.

Organisms have developed a variety of defenses to protect themselves against reactive

oxygen species. Superoxide dismutase, catalase, and glutathione (GSH) peroxidase

are some enzymes that perform this function. Also, some non-enzymatic compounds

16

protect against reactive oxygen species. Examples are glutathione, ascorbic acid, lipid

soluble alpha tocopherol, and some phytochemicals (de Groot and Rauen 1998).

Common examples of reactive oxygen species include hydrogen peroxide, the

superoxide and hydroxyl radicals, nitric oxide, singlet oxygen, and hypochlorous acid.

Although these reactive molecules are capable of attacking all biological molecules,

they tend to differ in their reactivities with regards to cellular molecules. The

hydroxyl radical attacks almost all biological molecules, while others like hydrogen

peroxide, singlet oxygen, and nitric oxide are less reactive. However, it should be

noted that the “low” reactivity species can be converted to highly reactive species.

Particularly, reaction of hydrogen peroxide with the lower valence states of iron and

copper can lead to the formation of the hydroxyl radical, or species of similar

reactivity, such as Fe2+O (ferryl ion) or Cu(OH)2+ (de Groot and Rauen 1998). Of the

above mentioned species, the hydroxyl radical is definitely thought to be the most

potent. This radical can attack every class of biological macromolecule. It can

depolymerize polysaccharides, cause DNA strand breaks, inactivate enzymes, and

initiate lipid peroxidation. Most of the hydroxyl radicals generated in vivo come from

metal ion-catalyzed breakdown of hydrogen peroxide. The extremely reactive

hydroxyl radical, once formed, can then initiate the process of lipid peroxidation

through abstracting a hydrogen atom from an unsaturated aliphatic lipid side chain.

This gives rise, by an autocatalytic chain reaction, to a lipid hydroperoxide (Haslam

1996). In vivo, it is estimated that generation of a single hydroxyl radical creates a

chain of 10 to 15 lipid peroxidation cycles until the chain is terminated, usually by

vitamin E. In addition, the general model of DNA modifications indicates the

17

hydroxyl radical is a common attacking species (Meneghini 1997). Therefore, it is the

action of the hydroxyl radical that may produce the greatest physiological damage to

human body components (McCord 1998).

Another radical worthy of mention is the superoxide radical. This free radical is

derived from molecular oxygen by the addition of a single electron. A wide variety of

pathological situations can produce the superoxide radical, including all infectious

diseases, and all diseases involving ischemia and reperfusion. Many researchers

believe that the most deleterious effect produced by the superoxide radical is the fact

that it causes reductive release of iron from ferritin. It is hypothesized that the

superoxide radical enters the ferritin core through hydrophilic channels. This is

followed by reduction of Fe3+ to Fe2+. This, in turn, allows release of iron from the

ferritin core. The combined presence of the superoxide radical and redox-active iron

can prove devastating to the cell in terms of maintaining membrane structure,

function, and viability (McCord 1998).

It is widely theorized that these reactive species may contribute to an array of

diseases, including cancer, atheroslerosis, Parkinson’s disease, Alzheimer’s dementia,

and reperfusion injury (de Groot and Rauen 1998). Reactive oxygen species generally

cause damage by reacting with essential cell and tissue molecules such as proteins,

lipids, and DNA. Furthermore, a concurrent decrease in cellular antioxidant capacity

might allow even more damage (de Groot and Rauen 1998). With regards to DNA,

reactive oxygen species can initiate many types of modifications. The most common

of these are approximately 20 possible base modifications. Also, strand cleavage is

another critical DNA lesion produced by reactive oxygen species (Meneghini 1997).

18

It has been shown that reactive oxygen species play a role as mediation molecules.

For example, nitric oxide acts as a neurotransmitter and has been shown to be an

endothelium-derived relaxing factor responsible for vasodilation. The generation of

these compounds is also important in signal transduction and microbicidal pathways

(de Groot and Rauen 1998).

Recently, investigators have suggested that reactive oxygen species play a

critical role in regulating apoptosis. Researchers have demonstrated that well-known

pathways of apoptosis are associated with moderate accumulation of reactive oxygen

species (McConkey 1998). Four observations that support this relationship are: 1)

antioxidants can inhibit apoptosis - which is not beneficial in destruction of tumor

cells, 2) generation of hydrogen peroxide has been detected in cells undergoing

apoptosis, 3) extracellular addition of reactive oxygen intermediates can stimulate

apoptosis, and 4) downregulation of intracellular antioxidant levels can induce

apoptosis. In addition, it is also thought that higher concentrations of reactive oxygen

species can induce necrosis, while lower concentrations stimulate apoptosis (Higuchi

1998).

There are several proposed mechanisms by which reactive oxygen species might

facilitate activation-induced cell death. First, reactive oxygen intermediate-mediated

damage has been shown to activate poly-ADP ribose transferase, which is associated

with apoptosis. Apparently, the polymerization of ADP-ribose to proteins results in a

rapid decline in cellular NAD/NADH stores, and a decrease in ATP. Both of these

conditions can induce apoptosis. Another mechanism states that oxidative stress may

cause formation of oxidized lipids in cell membranes. Reactive oxygen intermediates

19

react with polyunsaturated fatty acids and cholesterol in cell membranes, thereby

inducing apoptosis. Finally, it may also be possible that intracellular reactive oxygen

intermediates may activate apoptosis genes, most likely via an oxidative stress-

responsive nuclear transcription factor (Buttke and Sandstrom 1994).

IRON AND OXIDATIVE STRESS

Physiologically, iron is essential, but it also can be dangerous if present in a free

state. Human beings have evolved mechanisms for absorbing iron with 10%

efficiency. In humans, iron homeostasis is controlled by the rate of erythropoiesis and

the size of the iron stores (Khumalo 1998). The average adult male contains 4.5 grams

of iron, absorbs approximately 1 mg per day from the diet, and excretes about 1 mg

per day when in iron balance. Plasma iron turnover equals about 35 mg per day, with

slight disturbances of iron metabolism leading to iron overload or deficiency (Haslam

1996). There is no homeostatic mechanism to eliminate excess iron. Iron is rarely

present as a free ion, but instead, is bound to an array of active proteins such as

hemoglobin, myoglobin, ferritin, and transferrin (Jacob 1997). Cells store any excess

iron in the proteins ferritin and hemosiderin, predominantly in the liver. Therefore, as

long as the body is healthy, stored iron poses no great threat. However, under disease

conditions, it produces multiple possibilities for harmful superoxide production. Once

iron is freed in the presence of superoxide and its dismutation product, hydrogen

peroxide, the hydroxyl radical may be formed by the Haber-Weiss reaction (McCord

1998).

Although the transport protein, transferrin, has an exceptionally strong affinity

for ferric iron, iron concentration can also be locally and transiently increased through

20

tissue and blood degradation when injury occurs (Jacob 1997). At this point, iron can

promote free radical production, lipid peroxidation, and can initiate inflammatory

processes (Kato 1996) in conditions such as organ transplantation, carcinogenesis,

and aging (Kawabata 1997).

Many studies have indicated that transition metals are a major factor in the

initiation and propagation of peroxidative damage induced by free radicals (Ibrahim

1997). Iron is a redox active metal, meaning that it may readily alternate between

ferrous and ferric states, accepting or donating an electron to a wide range of

biological substances. Through this mechanism, iron catalyzes a variety of damaging

reactions within the cell (McCord 1998). In fact, sampling of chronic wounds (venous

stasis ulcers) shows a high local concentration of free iron ions. This lends credibility

to the idea that blood cells from a hemorrhage decompose and liberate their iron.

Then, the local increase in iron concentrations increases cellular iron concentration,

leading to excess production of reactive oxygen species (Jacob 1997).

The reactions by which some reactive species are formed are quite well known,

and include the Haber-Weiss and the Fenton reactions:

O2.- + Fe3+ O2 + Fe2+

O2.- + 1 e- + 2H+ H2O2

H2O2 + Fe2+ OH. + OH- + Fe3+

Net: O2.- + H2O2 O2 + OH. + OH. (Haber-Weiss reaction)

(Halliwell and Gutteridge 1999). On a molecular level, iron can catalyze the

decomposition of lipid hydroperoxides to generate alkoxyl, peroxyl, and other free

radicals (Ibrahim 1997). Therefore, iron may be a key factor in the establishment of a

21

prooxidant status in the cell (Meneghini 1997). The transition metals (vanadium, iron,

manganese, copper, and cobalt) are distinguished from other metals found in living

systems in that they can act as pi electron donors, and are redox active (Haslam 1996).

FRUITS AND VEGETABLES AND CANCER PREVENTION

An increasing amount of evidence indicates that nutrition is one of the most

important factors affecting health. Five of the ten leading causes of death for

Americans are associated with diet: heart disease, some cancers, stroke, diabetes, and

atherosclerosis (Glanz 1997). It has been estimated that about 30-35% of all cancers

can be attributed to diet. Some of the major hypotheses in this area are: high fat

consumption is a risk factor for prostate and colorectal cancers; high alcohol intake is

linked to oral, breast, or liver cancers; and low fiber intake is correlated with elevated

colon cancer risk. Conversely, another well-supported hypothesis is that high

consumption of fruits and vegetables is protective against many cancers (Steinmetz

and Potter 1996).

Inadequate consumption of fruits and vegetables is cited in almost 200 studies in

the epidemiological literature as correlating with increased cancer incidence (Block

1992). Cohort studies, case control studies, and animal studies have all shown an

inverse relationship between fruit and vegetable intake and risk of cancer (Steinmetz

and Potter 1996). Adequate intakes of fruits and vegetables and other fiber-rich foods

impact the incidence of cancers of the breast, colon, rectum, lung, and prostate (Glanz

1997). This suggests that the amount of cancer that could be prevented by increasing

fruit and vegetable consumption may be quite large, due to the fact that low intake is

so widespread. Furthermore, the quarter of the U.S. population with the lowest fruit

22

and vegetable intake has almost two times the rate of cancer when compared to the

quarter of the population with high fruit and vegetable consumption (Ames 1995).

Knowledge of how compounds in fruits and vegetables may lower cancer risk,

or be chemotherapeutic, is critical to our understanding of the effect of nutrition in

cancer prevention and treatment. It is believed that many of the compounds found in

fruits and vegetables protect against cancer via an antioxidant mechanism.

Historically, eating certain fruits and vegetables was thought to prevent or cure illness

ranging from headaches to heart disease. Early medicine was centered in large part

around prescription of specific food concoctions (particularly plant foods) for certain

illnesses. In recent years, researchers have evaluated which specific fruits and

vegetables have most often been found to be inversely associated with risk of any

cancer. Eighty-five percent of studies have shown that raw vegetables play a very

important role in preventive nutrition. Seventy percent of studies have also shown a

beneficial effect for allium vegetables (onions and leeks), carrots, green vegetables,

cruciferous vegetables, and tomatoes. An incredible number of substances in fruits

and vegetables have been shown or postulated to have anticarcinogenic properties

(Steinmetz and Potter 1996). If scientists are able to identify specific protective

compounds in foods, they may vary with the stage of the cancer process, but they

should also differ by sex, age, and across differing genetic metabolic profiles. The

effects of these foods may vary by organ site (Potter 1996).

Block (1992) cites approximately 120 of 130 studies conducted that show a

statistically significant reduction of risk of cancers of the lung, larynx, esophagus, oral

cavity, pancreas, stomach, rectum, cervix, colon, ovary, endometrium, breast, and

23

bladder with increased antioxidant intake. As a result of data such as these, it has been

hypothesized that the food supply may provide sufficient antioxidants to prevent some

of the events of cancer initiation and propagation. To do this, antioxidants must be

present at levels in proportion to the level of the agent that is causing oxidative

damage. However, individuals, or even populations, vary in the level of such

stressors. Therefore, no individual level will be sufficient at all times and for persons.

Furthermore, an effective level may differ depending on the cancer site.

POLYPHENOLS

Many natural substances in fruits and vegetables have been identified which

affect the process of carcinogenesis (Ren and Lien 1997). In the search for

chemopreventive agents, hundreds of compounds have been isolated from plant foods.

Some of the better studied compounds are carotenoids, vitamins C and E, selenium,

phytoestrogens, saponins, organic sulfur compounds, indoles, unsaturated fatty acids,

and polyphenols (Ren and Lien 1997; Steinmetz and Potter 1996).

Scientists have been interested in plant polyphenols for several decades. Their

role in plant physiology is essential, as they are involved in growth, reproduction, and

resistance to pathogens and predators. Polyphenols are widespread in plant foods and

beverages. The levels of each compound may vary greatly between varieties.

Polyphenols contained in plant foods depend upon genetic factors and environmental

conditions. Germination, degree of ripeness, variety, processing, and storage are also

factors that can influence the levels of these compounds in plants. Polyphenols are

found in virtually all plant organs. Therefore, they are a significant part of the human

diet. In the past, much of the nutritional focus was on the ability of some polyphenols

24

to bind and precipitate macromolecules (dietary protein, carbohydrates, and digestive

enzymes), altering the digestibility of food (Bravo 1998). Recently, the protective

effects of polyphenols are believed to be closely related to their status as antioxidants

(Yang 1997; Haslam 1996). Epidemiological evidence shows that consumption of

polyphenol-rich items in the diet is associated with an increase in plasma antioxidant

potential, thereby increasing resistance to oxidative stress (Haslam 1996). In fact, a

substantial body of evidence now exists that suggests that plant foods are a rich source

of molecules that quench the damaging effects of reactive oxygen species (Fenech

1997).

Antioxidants can be classified according to their actions: free radical

terminators, chelators of metal ions capable of catalyzing lipid oxidation, or as oxygen

scavengers that react with oxygen in closed systems. Phenolic antioxidants generally

fall in the category of free radical terminators (Shahidi 1992). The phenoxy radical

that occurs during the free radical termination process involving polyphenols cannot

initiate a new free radical or be subject to rapid oxidation by a chain reaction.

Therefore, phenolic antioxidants are very good hydrogen and electron donors. Also,

their radical intermediates are relatively stable due to resonance delocalization and

lack of suitable sites for attack by molecular oxygen (Shahidi 1992).

In general, “phenolic compounds” describes a wide range of plant substances

that have one or more aromatic rings with one or more hydroxyl substituents.

Phenolic compounds are often found as glycosides (with a sugar molecule attached),

and therefore tend to be water-soluble (Croft 1998 ). They have a molecular weight

range of 500-4000, possess 12-16 different phenolic groups and 5-7 different aromatic

25

rings per 1000 relative molecular mass; the ability to precipitate some alkaloids,

gelatin, and other proteins from solution; and they are categorized under 2 main

structural themes: galloyl and hexahydroxydiphenoyl esters and their derivatives, and

condensed proanthocyanidins (Haslam 1996).

Some phenolic flavonoids are more protective in vitro than ascorbate or vitamin

E (Fenech 1997). Non-flavonoid phenolic acids may also be suitable as antioxidants.

In particular, those possessing a catechol-type structure, such as caffeic acid, are

implicated (Croft 1998). Additionally, recent studies have shown that simple cell-

derived phenolic acids, such as 3-hydroxyxanthranilic acid may act as co-antioxidants

for alpha-tocopherol, causing inhibition of lipoprotein and plasma lipid peroxidation

(Croft 1998). Most plant polyphenols are known to inhibit lipid peroxidation and

lipoxygenases in vitro, and they have shown an ability to scavenge hydroxyl,

superoxide, and peroxyl radicals, all of which are important factors in cellular

prooxidant states (Haslam 1996).

Some studies have suggested a protective role for polyphenols, but some

polyphenols are better studied than others. Specifically, tea has been shown to have

an inhibitory action against experimental carcinogenesis in many animal cancer

models such as the lung, skin, liver, esophagus, and stomach (Yang 1997). Green tea

polyphenols have been shown to prevent metabolic activation of carcinogens, inhibit

tumor promotion and cell proliferation, and act as agents that induce antioxidant or

phase II enzymes (Leanderson 1997). Green tea contains certain polyphenolic

compounds, commonly known as catechins, and theaflavins (Yang 1998). The

catechins have been shown to inhibit lung, esophageal, gastric, and skin tumorigenesis

26

in animals. However, the relationship between green tea polyphenols to the same

cancers in humans remains to be elucidated (Yang 1997).

Some studies have indicated other specific polyphenols as having a beneficial

effect. In a study by Yang (1998) epigallocatechin-3-gallate is a polyphenol that may

be an important antioxidant, and possibly a cancer preventive compound. This

conclusion was reached by the fact that the polyphenol under study possessed growth

inhibitory activities against human lung tumor cell lines. This same study also

indicated that theaflavins caused growth inhibition in lung cancer cells (Yang 1998).

Another study, by Kaul and Khanduja (1998), utilized topical application of

polyphenolic compounds to inhibit skin tumor promotion. The researchers

hypothesized that plant polyphenols may reduce tumorigenesis by halting arachidonic

acid metabolism, as well as superoxide radical generation by known tumor promoters.

Red wine phenolic compounds have also been implicated in preventing lipid

oxidation (Fenech 1997), and caffeic acid has also been shown to act in a prooxidative

manner with copper- induced oxidation of LDL in the propagation phase (Croft 1998).

Overall, epidemiological evidence points to a reduced risk of certain degenerative

diseases by the consumption of beverages containing polyphenols. Specifically, green

tea and red wine are rich sources of these compounds. However, more research is

needed to elucidate the absorption, penetration, and metabolism of plant polyphenols

in the human body. Until this occurs, the evidence for their beneficial effects remains

based upon epidemiological correlation rather than strict scientific observation

(Haslam 1996).

27

Flavonoids

Among polyphenols, flavonoids are some of the most prevalent and important

low molecular weight phenolics (Bravo 1998). They are abundant in our food supply,

being found in fruits, leafy vegetables, roots, tubers, bulbs, herbs, spices, legumes, tea,

coffee, red wine, and beer (Sahu and Washington 1992). In addition to

phenylpropanoids or hydroxycinnamic acid derivatives, flavonols are found in almost

every plant. The concentration of flavonoids in these plant products is much greater in

the leaves and peels as compared to the deeper tissues (Stavric and Matula 1992). In

plants, the purpose of flavonoids is to protect the plant against fungal parasites,

herbivores, pathogens, and oxidative cell injury. Flavonoids also produce the stimuli

to assist in pollination and guide insects to their food source (Cook and Samman

1996).

Flavonoids also contribute to the flavor and color of many fruits, vegetables, and

other products, such as wine and tea (Croft 1998). More than 4000 varieties of

flavonoid compounds have been identified in vascular plants. These vary in quality

and type according to variations in plant growth, conditions, and maturity. Only a

small number of plant species have been systematically examined for their flavonoid

content. So, as yet, the indentification and quantification of every flavonoid is still

incomplete (Cook and Samman 1996). Major classes of flavonoids include flavanols,

flavones, flavanones, catechins (or flavanols), anthocyanidins, isoflavones,

dihydroflavonols, and chalcones (Cook and Samman 1996). These compounds are all

structurally related to the parent compound, 2-phenyl-benzopyrene (Yang 1997). The

most commonly occurring flavones and flavonols are those with dihydroxylation in

28

the 3’ and 4’ positions of the B ring, as well as those with a single B ring hydroxyl

group in the 4’ position (Rice-Evans 1996). The flavanols, especially catechin and the

catechin gallate ester family, and the flavonols quercetin, kaempferol, and their

glycosides, are all found in green and black tea and red wine. Quercetin is also a

major component of onions and apples, while myricetin and quercetin are both found

in berries. The flavanones are mostly found in citrus fruits (Rice-Evans 1996). The

role of many polyphenolic compounds, including flavonoids, has yet to be elucidated

(Bravo 1998). It is vital to attempt to understand the bioavailability of polyphenols,

their mechanisms of action, and possible relationships with other components of the

diet.

Originally, studies suggested that Americans consume approximately 1 gram of

polyphenols in their diets on a daily basis. Of this 1 gram, approximately 170 mg are

4-oxo-flavonoids (i.e. flavones, flavanone, flavonols, and chalcones). In addition,

most of the 4-oxo-flavonoids are probably composed of flavones (apigenin glycoside

and luteolin glycosides); and flavonols (kaempferol glycosides and quercetin

glycosides) (Brown 1980). However, more recent evidence has suggested that this

estimate is probably too high. Hertog (1993) documented that Dutch intake of

flavonols and flavones was 23 mg/day (as aglycones). Also, estimates of 115 mg/day

as aglycones are given for the United States (Hollman and Katan 1997). Chronic

pharmacologic doses of flavonoids have been tested, with deleterious effects including

acute renal failure, hemolytic anemia, thrombocytopenia, hepatitis, fever, and skin

reactions (Cook and Samman 1996). It is important to note, though, that flavonoids

29

are unlikely to be consumed in toxic amounts as part of a balanced diet (Cook and

Samman 1996).

Flavonoids possess a variety of biological effects, including antibacterial,

antiviral, anti-inflammatory, antiallergenic, and vasodilatory actions. They are also

shown to inhibit platelet aggregation, and decrease capillary permeability and fragility

(Cook and Samman 1996). Concentrated forms of flavonoids have been used for

centuries to treat various human ailments including inflammation, allergy, headache,

cancer, viral infection, colds, bee stings, and gastric and duodenal ulcers (Cook and

Samman 1996). In conventional medicine, flavonoids have been used for over 40

years to treat peripheral circulation disorders. Also, more than 100 different

preparations such as cianidol, diosmetin, hesperidin, leucocianidin, rutin, and

troxerutin are produced and sold in France in Switzerland. However, it should be

noted that many of these preparations have yet to be tested in clinical investigations

(Cook and Samman 1996). Flavonoids and other plant phenolics have also been

shown to be inhibitors of certain enzyme systems under some conditions. Among

these are phospholipase A2, cyclooxygenase, lipoxygenase, glutathione reductase, and

xanthine oxidase (Rice-Evans 1996).

Flavonoids are low molecular weight polyphenolic substances based on the

flavan nucleus. The common structure of flavonoids is C6-C3-C6 (diphenylpropane):

two aromatic rings linked through three carbons to form an oxygenated heterocycle

(Bravo 1998). The basic three phenolic rings are referred to as A, B, and C (pyran)

rings (Cook and Samman 1996). The structure of flavonoids varies widely within

major classifications. Hydrogenation, hydroxylation, methylation, malonylation,

30

sulphation, and glycosation are all substitutions that can occur (Cook and Samman

1996). Flavonoids are reputed to be efficient radical scavengers. Researchers have

developed three criteria for effective radical scavenging among the flavonoid group.

These are 1) the o-dihydroxy structure in the B ring. This gives higher stability to the

radical form and participates in electron delocalization. 2) The 2,3 double bond in

conjugation with a 4-oxo function in the C ring is responsible for electron

delocalization from the B ring. Electron delocalization of the aromatic nucleus

confers antioxidative potency. 3) The 3- and 5- OH groups with 4-oxo function in the

A and C rings are required for maximum radical scavenging potential (Rice-Evans

1996). Glycosylation in flavonoids generally occurs in the 3 position and also in the 7

position. Glucose is usually the most common sugar residue, but others include

galactose, rhamnose, and xylose (Rice-Evans 1996). Within the flavonoid group,

flavones, flavanols, and their glycosides are the most common. Quercetin - one of the

most potent and widely examined flavonoids - is part of the flavonol group (Bravo

1998).

QUERCETIN

Quercetin absorption

Studies of the absorption of flavonoids in humans are not conclusive at this time.

There is evidence that flavonoids are absorbed in significant quantities and that they

can be absorbed as both free aglycone and glycoside forms. Both forms have been

found in blood and urine. Some research indicates that peak absorption may be 2-3

hours after ingestion (Manach 1998).

31

Naturally occurring flavonoids are usually glycosylated. This is important in the

absorptive process. Unabsorbed flavonoids from the small intestine, as well as

absorbed, conjugated flavonoids secreted from the gallbladder reach the colon. Here,

both groups of flavonoids undergo microbial degradation, stripping them of their sugar

moieties, glucuronic acids and sulfates. Their absorption (flavonoids) is determined

by the hydroxylation pattern. 5, 7, and 3’, 4’ hydroxylated compounds are susceptible

to hydrolysis and heterocyclic ring cleavage by microbiological degradation in the

colon (Croft 1998). Absorption is then possible due to the fact that the resulting

aglycones are less polar. As a result of this structure-dependent hydrolysis, biological

effects of flavonoids predicted from in vitro studies may be different in vivo,

depending on the structure of the parent compounds (Hollman and Katan 1997).

Manach et al. showed that quercetin and rutin are both absorbed by rats in a 1995

study. Rats were fed diets containing various levels of quercetin and rutin, the main

glycoside form of quercetin. Measurement of cecal contents indicated similar

quantities of quercetin and rutin in rats fed the same molar amounts of each flavonoid.

Further, quercetin and rutin diets led to similar levels of plasma metabolites. It was

therefore concluded that the small intestine was not an effective site for absorption

(Manach 1995). Quercetin showed a high affinity for albumin (Manach 1995). They

concluded that the strong binding of quercetin metabolites to albumin may affect

quercetin’s actions within a physiological context, and that any effects in vivo are due

to the quercetin-albumin complex, rather than unbound quercetin (Manach 1995).

In a subsequent rodent study, Manach et al. (1997) found that quercetin was

indeed absorbed in the small intestine, while rutin was not. They also found that

32

absorption of quercetin was less efficient in rats previously maintained on a quercetin-

containing diet. Therefore, a steady intake of quercetin could lower the rate of

digestive absorption, as is the case with other micronutrients (Manach 1997).

Moving to the human model, Manach et al. (1998), studied plasma

concentrations of healthy volunteers following a meal composed mainly of plant

products high in quercetin. They observed a significant increase in conjugated

quercetin derivatives in the blood at 3 and 7 hour intervals following the meal, which

returned to basal levels or below after about 20 hours of no further quercetin

consumption. The relatively rapid increase in absorption, evidenced by the plasma

concentrations, suggests that absorption probably occurred in the proximal intestine.

Within the same study, the conjugated derivatives found in plasma were measured for

antioxidant capacity. Although their activity was half that of the aglycone form, Cu2+-

induced oxidation of human LDL was still significantly delayed. Overall, the

investigators concluded that beneficial effects of quercetin and other flavonoids would

depend on regular consumption, and that any physiological effects in vivo may be due

to a synergism with other compounds in fruits and vegetables (Manach 1998).

In another human study, Hollman (1995) studied the absorption of flavonoids

from foods (mostly flavonoid glycosides) in 30 ileostomy patients. Logically, without

colonic microbial degradation, the flavonoids could reach the end of the small

intestine as glycosides. Results showed that quercetin glycosides from onions were

more readily absorbed (52% of ingested amounts) than the aglycone forms (24% of

ingested amount). Based on these results, Hollman concluded that glycosides may be

absorbed in humans without prior hydrolization by colonic microbes (Hollman 1995).

33

Manach et al. (1998) agreed with Hollman that the glycosylated forms may have been

more easily absorbed. Recent studies have confirmed that quercetin glycosides are

hydrolyzed and absorbed in the small intestine (Erland et al. 2000; Wallen et al. 2000).

Flavonoids are metabolized in two main body compartments: the liver and the colon.

The liver, where absorbed flavonoids and their absorbed colonic metabolites are

received, is considered to be the main site of quercetin metabolism (Hollman and

Katan 1997). Biotransformation enzymes produce methoxy-, gluco-, and sulfo-

conjugations of flavonoids. Glucuronides and sulfates of quercetin and quercetin

aglycone are found in plasma of human subjects fed either quercetin or quercetin

glycosides (Manach 1997). Following transformation, a large amount of flavonoid

metabolites are excreted in the urine (Hollman and Katan 1997).

Quercetin - an antioxidant and a prooxidant

Oxidative stress occurs when antioxidant defenses are unable to overcome the

production of free radicals and may contribute to many different conditions including

atherosclerosis, cancer, and chronic inflammation. Therefore, the antioxidant capacity

of flavonoids is of great interest (Croft 1998). Flavonoids act as antioxidants by

breaking the free radical chain reaction. An antioxidant, by definition, is a substance,

when present at low concentrations relative to an oxidizable substrate, can

significantly delay or prevent oxidation of the substrate. In addition, the resulting

radical must be stable enough to prevent it from acting as a chain-propagating radical

(Croft 1998). Flavonoids quench the hydroxyl and superoxide ions, which are highly

reactive species involved in lipid peroxidation (Morel 1993). It is also proposed that

flavonoids terminate the propagation phase of lipid peroxidation by donating hydrogen

34

atoms to the peroxy radical, thereby forming a flavonoid radical. The flavonoid

radical then reacts with free radicals and terminates the propagation chain (Cook and

Samman 1996).

Not only are flavonoids soluble, chain-breaking inhibitors of the peroxidation

process, they are also thought to be potent metal chelators (Morel 1993). Through this

action, they can inhibit the Fenton reaction, which is driven by the superoxide anion

(Cook and Samman 1996). In fact, Morel et al. (1993) demonstrated iron removal

from iron-loaded hepatocyte cultures by catechin, quercetin, and diosmetin.

Under certain conditions – such as high phenolic concentrations, high pH, or

presence of metal ions – phenolic antioxidants can autooxidize and behave as

prooxidants (Bravo 1998). Flavonoids have been shown to produce reactive oxygen

species in vitro in the presence of reduced metals such as iron and copper (Sahu 1994).

Some flavonoids are able to mobilize iron from ferritin, and are capable of reducing

ferric iron to the more reactive ferrous iron (Morel 1993). A recent study by Cao et al.

(1997) used three different oxidation systems. In these, flavonoids had strong

antioxidant activity against peroxyl radicals generated from AAPH and also against

hydroxyl radicals. However, they were pro-oxidative with respect to copper. It was

concluded that the flavonoids must be able to reduce Cu2+ to Cu+, thereby allowing the

formation of initiating radicals (Croft 1998). This prooxidative effect has been shown

in numerous studies with one flavonoid in particular. Quercetin has been found to be

mutagenic in almost every in vitro mutagenicity test (Canada 1990). Because

quercetin is mutagenic only under aerobic conditions, active oxygen species are

strongly implicated (Sahu and Washington 1992). In a study by Miura et al. (1998),

35

quercetin was the most potent in generating hydrogen peroxide at pH 7.4.

Polyphenols that are similar to quercetin have been shown to generate active oxygen

species by autooxidation. Examples are pyrogallol, which generates the superoxide

dical by its autooxidation. Hydroquinone and catechol – other examples of

polyphenols – produce hydrogen peroxide and the superoxide radical at physiological

pH. In the presence of iron, these two reactive oxygen species generate the hydroxyl

radical (Sahu and Washington 1991).

Many flavonoids may cause lipid peroxidation or may accelerate oxidative

damage to non-lipids, such as carbohydrates and DNA. The products of this oxidative

damage can be cytotoxic (Smith 1992). For example, quercetin has also been found to

induce single strand DNA breaks in isolated rat liver nuclei, in calf thymus DNA, and

in open plasmid DNA (Duthie 1997). In a lipid system, quercetin exhibited

prooxidative effects at higher doses (Stadler 1995). Finally, from the evidence shown

in Table 1, it is obvious that flavonoids cannot be simply classified as antioxidants.

They must be examined more thoroughly for their pro-oxidant properties (Laughton

1989). The prooxidative properties of flavonoids may be beneficial. Free radical

production may stimulate apoptosis of tumor cell lines under certain circumstances. It

is now well accepted that the majority of chemo- and radiotherapeutic agents destroy

malignant cells by initiating apoptosis (Martin 1997). Evidence is also accumulating

that the efficiency of anti-tumor agents is related to the ability of the target tumor cells

to respond by apoptosis. Recent studies by Kuo (1996) have shown that apoptosis was

the mechanism of cell death when flavonoids were added to the neoplastic cell lines

36

CaCo2 and HT29. Kuo also found that curcumin - a popular spice - exhibits anti-

tumor activity by initiating apoptosis in human tumor cells (Kuo 1996).

Cermak et al. (1993) suggests that acute exposure of intestinal tumor cells to

iron may increase their susceptibility to oxidant-mediated lysis. This study postulates

that free iron may be released from ferritin by an appropriate reducing agent (e.g.,

quercetin) (Cermak 1993). It is therefore reasonable to think that if tumor cells were

exposed to high iron levels and an appropriate reducing agent, such as quercetin, that

oxidative stress might induce apoptosis in these cells.

TABLE 1 Previous Studies Demonstrating the Effect of Flavonoids Alone or in Combination with Redox Metals on Cell Lines or Specific Cell Components Authors Cell type or cell constituent Amount of flavonoid administered Results Sahu and Gray 1997 Isolated rat liver nuclei 0, 10, 20, 40, 60, 80, 100 µM of morin and Morin and naringenin alone produced naringenin alone. Also 100 µM morin lipid peroxidation and DNA damage in and naringenin in the presence of 100 µM a concentration-dependent manner. FeCl3 and 100 µM CuCl2 These results were enhanced when the flavonoids were incubated with iron and copper ions. Duthie et al. 1997 HeLa (epithelial); HepG2 0-2500 µM of quercetin, myricetin, silymarin Quercetin induced DNA strand (liver); CaCo2 (colon); for 18h breakage in a dose-dependent manner normal human lymphocytes in HepG2 cells (100, 500, 1000 µM)

and human lymphocytes (25, 50, 100 µM). Quercetin especially induced breakage in lymphocytes at concentrations less than 100 µM. CaCo2 cells exhibited little susceptibility to any flavonoids except myricetin at 600-1000 µM. All flavonoids increased strand breakage in HeLa cells (250, 500, 1000 µM) Stadler et al. 1995 Ethyl linoleate and 2-deoxy- Quercetrin (20, 100, 200 µM) in the Hydroxylation of 2'-dG to 8-oxo 2'dG. Guanosine presence of H2O2 and Fe2+, Mn2+, Cu2+, Peroxidation of ethyl linoleate at 100 Zn2+, and CO2+ and 200 µM. Hydroxylation activity was greatly enhanced by Mn2+ and Cu2+ Smith et al. 1992 Deoxyribose Morin, quercetin, fisetin, gossypol (100 uM) Extent of deoxyribose degradation: added to deoxyribose in the presence morin = .51; quercetin = 2.00; fisetin = of ferric EDTA and H2O2 .68; gossypol = 2.46; myricetin = 2.70

37

CHAPTER III

IRON STATUS OF THE CELL MAY ALTER QUERCETIN-INDUCED APOPTOSIS

OF HUMAN HEPATOMA CELLS

ABSTRACT

Quercetin, a flavonoid, is cytotoxic to human tumor cells at high concentrations.

The objective of the present study was to determine if cellular iron status influences

quercetin-stimulated cytotoxicity and apoptosis in a human hepatoblastoma cell line.

HepG2 cells were incubated in cell culture media supplemented with 0, 10 or 25 µM Fe

(added as ferric citrate) for 24 hours to achieve varying Fe concentrations. Fe-

supplemented media was then removed and cells were washed. Cells were subsequently

incubated with media containing 0, 40 or 80 µM quercetin in 0.5% DMSO for an

additional 24 hours. Following incubation with quercetin, lipid peroxidation was

measured with thiobarbituric reactive substances assay, cell viability was assessed with

the Trypan Blue exclusion assay, and apoptosis was assessed by enzyme immunoassay,

chromatin condensation, and caspase activity. At least three replicates were obtained for

each assay. Supplementation with 10 and 25 µM Fe resulted in cellular Fe levels that

were 3- and 6- times those of control, as determined with atomic absorption

spectrophotometry (AAS). Subsequent incubation with quercetin did not alter the Fe

content of cells, as found with AAS. Lipid peroxidation was increased with increasing

media iron concentrations, with quercetin showing no contribution to this effect. Cell

38

39

viability was significantly reduced only after incubation with 25 µM Fe followed by 80

µM quercetin (69% vs 78%, respectively, for treated vs control). Cellular apoptosis was

not consistently enhanced by incubation with 40 µM quercetin. Increases in cellular

apoptosis after incubation with 80 µM quercetin were greatest in iron loaded cells (10

µM and 25 µM Fe levels). Apoptotic morphology, assessed as chromatin condensation,

was only affected by quercetin. No synergism between iron and quercetin was suggested.

Caspase activity was decreased by quercetin at control iron levels, but increased by

quercetin in cells treated with the highest levels of iron. Overall, the data suggest that Fe

concentration may influence quercetin's effect on cell viability and apoptosis in HepG2

cells.

INTRODUCTION

Iron is a redox active metal, meaning that it may readily alternate between ferrous

and ferric states, accepting or donating an electron to a wide range of biological

substances. Through this mechanism, iron catalyzes a variety of damaging reactions

within the cell (McCord 1998). Iron can catalyze the formation of the hydroxyl radical

from H2O2 as well as the decomposition of lipid hydroperoxides to alkoxyl, peroxyl, and

other free radicals (Ibrahim 1997). Therefore, iron is a key factor in the establishment of

a prooxidant status in the cell (Meneghini 1997). Previous studies in our laboratory and

others have suggested that iron status of a cell may alter apoptosis (Stimson 1998; Wang

et al. 1999; Whittaker et al. 1996). Further, studies by Cermak et al. (1993), proposed

that acute exposure of some tumor cells to iron may increase their susceptibility to

oxidant-mediated lysis.

40

Apoptosis is described as a sort of cellular suicide (Samali 1996) in which a cell

takes an active role in its own demise. It consists of a preprogrammed cascade of

metabolic events that occurs in response to various environmental inducers or a

combination of intrinsic factors, ultimately leading to the disintegration of the cell

(Darzynkiewiez 1995). Studies have shown that cell death caused by apoptosis may be

the mechanism by which some chemotherapeutic agents function.

It has also been proposed that oxidative stress is a mediator of apoptosis in

eukaryotic cells via several different mechanisms (Buttke and Sandstrom 1994).

Oxidative stress may induce apoptosis by damaging DNA. This may occur via metal-

catalyzed oxidation with subsequent generation of reactive intermediates such as

hydrogen peroxide and the hydroxyl radical (Duthie et al. 1997). In addition, oxidative

stress may lead to the formation of oxidized lipids in cell membranes, which may induce

apoptosis (Buttke and Sandstrom, 1994).

Recent studies (Kuo 1996) have shown that apoptosis was the mechanism of cell

death when flavonoids were added to neoplastic cell lines. It has been proposed that

some phenolic antioxidants, such as flavonoids, can reduce ferric iron to ferrous iron,

thereby facilitating free radical damage to carbohydrates, lipids, proteins, and DNA

(Smith 1992). Free iron may also be released from ferritin by an appropriate reducing

agent such as quercetin (Cermak 1993). Sahu and Washington (1992) found that

increasing quercetin levels in the presence of iron significantly increased lipid

peroxidation and nuclear DNA damage in rat liver nuclei. Therefore, tumor cells

exposed to high iron levels in the presence of a reducing agent, such as quercetin, could

theoretically undergo apoptosis.

41

The purpose of this study was to determine whether a flavonoid common to many

foods (quercetin) could induce apoptosis in the HepG2 cell line, a liver hepatoma cell

line, and whether cellular iron status alters apoptosis.

It was hypothesized that 1) cellular iron status would increase with higher media

iron concentrations, and that addition of quercetin to iron-loaded cells would have no

effect on cellular iron, 2) addition of quercetin to iron-loaded cells would increase lipid

peroxidation, 3) cellular iron concentration would influence the prooxidative ability of

quercetin to decrease cell viability, and 4) cellular iron concentration would influence the

pro-oxidative ability of quercetin to induce specific apoptotic changes in the cell

measured as: 200-bp DNA nucleosome fragmentation (ladder effect), caspase activation,

and chromatin condensation.

METHODS

Cell culture, media, and experimental design

Cells used in this study were human hepatoma cells (HepG2; ATCC, Rockville,

MD). Cells were grown in filtered Eagle’s minimum essential medium (MEM) with

2mM L-glutamine and Earle’s balanced saline solution (BSS; Sigma, St. Louis, MO)

with 1.5g/L sodium bicarbonate (Sigma, St. Louis, MO), 1.0 mM/L sodium pyruvate

(Sigma, St. Louis, MO), 0.1 mM/L nonessential amino acids (Sigma, St. Louis, MO), 10

mg streptomycin + 10,000 units penicillin/ml (Sigma, St. Louis, MO), with 10% fetal

bovine serum (FBS; Equitech-Bio, Inc., Kerrville, TX). All assays were completed using

FBS from the same lot. HepG2 cells were maintained in 75 cm2 canted-neck flasks in 15

ml of media with 1:4 passage occurring once per week. Cells were incubated at 37oC,

42

5% CO2 (Harris Model# HWO701T-ABA, Norwalk, CT). Studies were conducted on

passages 78-163.

When cells were approximately 100% confluent and four days after passage, cells

were incubated in 0, 10, 25, and 50 µM /L iron-loaded media. Following a 24-hour

incubation, cells were washed with Hanks BSS (.03% EDTA) to remove iron-loaded

media. Media with 0, 40, 80 µM quercetin in .2% DMSO was added to cells. Cells were

incubated for another 24 hours, and harvested for assays for cell viability and apoptosis.

Each batch of iron and quercetin loaded media was tested for iron content using a

Perkin Elmer Model# 5000 atomic absorption spectrophotometer (Perkin Elmer Corp,

Shelton, CT; percent recovery = 90%; n=16). Measured Fe content of media was 3.5,

5.6, 10.4 µM for 0, 10, 25 µM /L media, respectively; n = 6 replicates. Iron content of

media was consistent across media batches. It was determined that the 50 µM /L iron-

loaded media did not significantly increase cellular iron levels over the levels achieved

with 25 µM /L iron-loaded media. Therefore, the 25 µM /L iron-loaded media was

chosen as the highest level for experiments.

Cellular iron concentration

Harvested cells in solution were centrifuged (Beckman J2HS, JS-7.5 swinging

bucket rotor, Palo Alto, CA) at 750 X G for 5 min. The supernatant was discarded, and

the pellet was resuspended in 3 ml PBS. Centrifugation/resuspension were repeated

twice, with resuspension in 1 ml PBS after the final centrifugation. The 1 ml suspension

was centrifuged once more to pellet the cells. The supernatant was discarded. HCl (.17

ml) was added to each pellet, and vortexed. 1.5 ml dH2O was added to each tube. Iron

43

concentration was determined with an atomic absorption spectrophotometer and

expressed as umol per million cells; n = 3 replicates.

Cell viability

The Trypan Blue assay was used to measure cell viability. Five ml cell

suspensions of each treatment group were pipetted from each flask into a test tube. All

tubes were centrifuged at 750 X G for 5 min. The supernatant was discarded. Next, 3

mL Hanks BSS was added to each cell pellet. In a separate tube, .5 ml of .4% Trypan

Blue solution (Sigma, St. Louis, MO) and .3 mL of Hanks BSS were combined.

Immediately before counting, cell suspensions were aspirated to obtain an even

distribution of cells. .2 ml of the cell suspension was then added to the Trypan

Blue/Hanks BSS solution and was incubated for 5 minutes before counting to avoid

overexposure of the stain. Each tube was mixed thoroughly by gentle thumping until

most clumps disappeared. A 10 µL aliquot of the Trypan Blue/cell suspension was added

to a hemocytometer, and cells were counted under an Olympus IMT-2 Inverted Research

Microscope (Olympus America Inc., Melville, NY). Cell viability was calculated as total

viable cells (unstained)/total cells (stained and unstained) x 100; n = 4 replicates.

Lipid peroxidation

The thiobarbituric acid reactive substances (TBARS) assay was used to assess

lipid peroxidation (n = 3 replicates). TBARS was measured as described by Buege and

Aust (1978). Briefly, washed cell suspensions were treated with TBA-TCA-HCl

(thiobarbituric acid-trichloracetic acid-hydrochloride acid), placed in boiling water for 15

minutes and then centrifuged at for 15 minutes (4000 x g, Beckman J2HS, JS-7.5

swinging bucket rotor, Palo Alto, CA). The supernatant was collected and absorbance at

44

535 nm was read using a Beckman DU-650 spectrophotometer (Beckman Instruments,

Fullerton, CA). Protein concentration was determined as described by Lowry et al.

(1951).

DNA fragmentation

An Enzyme-Linked Immunosorbent Assay (ELISA) was used to assess apoptosis

(Cell Death Detection ELISA, Boehringer Mannheim, GmbH, Germany) using a

microplate reader (Dynex Technologies, Chantilly, VA). Cell suspensions from 3 parent

flasks (0,10, 25 µM Fe levels) were counted immediately following passage procedure

each week prior to the assay. Resulting cell numbers were used to determine dilution

factor for the subsequent ELISA. Cell suspensions containing 105 cells/ml were used for

the assay.

Cell suspensions were centrifuged, and the cell pellet was resuspended in 1 ml

culture medium. After a second centrifugation, each cell pellet was resuspended with

500 µL incubation buffer containing Tween 20 or 80 and incubated approximately 30

minutes at 4oC to induce lysis. The lysate was centrifuged at 15,000 rpm (20,000 x G)

for 10 min. 50 µL of the supernatant (cytoplasmic fraction) was carefully removed and

transferred to duplicate eppendorf tubes. The supernatant was stored at - 20oC until the

following day.

100 µL coating anti-histone (monoclonal mouse antibody) solution was pipetted

into each well of a microtiter plate (MTP). MTP were covered tightly with an adhesive

cover foil and incubated overnight at 4oC. Maximum incubation time was 18 hours.

The coating solution was then removed, and 200 µL incubation buffer containing

Tween 20 or 80 was then pipetted into each well of the MTP. The MTP were covered

45

tightly with the adhesive cover foil and incubated for 30 min at room temperature. The

coating solution was then removed, and the MTP was washed three times.

Samples were diluted to obtain 1 x 104 cell equivalents/ml. Each sample was

vortexed after dilution. 100 µL of each sample solution was pipetted into the MTP. For

determination of the background of the immunosassay, 100 µL of incubation buffer were

pipetted into 2 additional wells. MTP were covered tightly with adhesive foil and

incubated for 90 minutes at room temperature. The washing procedure was repeated for

all samples and blanks.

The conjugate solution - anti-DNA peroxidase (monoclonal mouse antibody)

(10:1 dilution) was added to each well of the MTP, except the blank positions. The MTP

were covered tightly with adhesive foil and incubated for 90 minutes at room

temperature. The washing procedure was repeated. A substrate solution containing 2,2’-

azino-di[3-ethylbenzthiazoline sulfonate] was pipetted into each well of the MTP and

incubated on a plate shaker at 250 rpm for 15 minutes until the color development was

sufficient for a photometric analysis on a microplate reader at 405 nm against the

substrate reaction solution as the blank; n = 4 replicates.

Nuclear morphology

Apoptosis assessed with Hoechst staining (Intergen Company, Purchase, NY)

was used to examine nuclear morphology and chromatin condensation in the 0 and 80

µM /L quercetin groups with 0 and 25 µM /L added iron. 100 µL cell suspensions were

diluted with 200 µL media for treatment with 1.5 µL Hoechst 33342 stain. Hoechst-

stained cells were counted using a 480 nm UV filter. Apoptotic morphology was

assessed using 100x objective. This allowed more detailed view of chromatin assembly,

46

while allowing a minimal path of light to hit cells, thereby avoiding fading before

morphology could be determined. Cells showing apoptotic morphology were expressed

as a percentage of total cells; n = 3 replicates.

Caspase activity

An Intergen Caspatag Fluorescein Caspase Activity Kit (Intergen Company,

Purchase, NY) kit was used to determine caspase activity in the 0 and 80 µM/L quercetin

groups with 0 and 25 µM/L added iron. 300 µL cell suspensions were incubated with a

carboxyfluourescein (FAM) derivative of benzyloxycarbonyl valylalanyl aspartic acid

flouromethyl ketone (zVAD-FMK) (FAM-VAD-FMK fluorescein labeled inhibitor).

Caspase activity was determined with fluorescence microscopy using a Nikon Contrast-2

(Nikon Instruments Inc., Melville, NY) with a 520 nm filter. Caspase-positive cells were

counted, followed by dead (propidium iodide-labeled) cells in a hemocytometer. A ratio

of caspase positive cells to total cells is presented; n = 5 replicates.

Statistical analysis

Treatment means, standard error of the mean, analysis of variance (ANOVA),

least significant difference tests, tests for normality, and non parametric analysis were

determined using the statistical package SAS (SAS version 6.10, SAS Institute, Cary,

NC). The overall effects and interactions of iron, and quercetin on cellular iron

concentration, lipid peroxidation, and quercetin-induced DNA fragmentation were

determined with ANOVA. Fisher’s Least Significance Difference test was used to assess

the difference between means for treatments. Differences among treatment groups were

considered significant if p < 0.05. The apoptosis data did not appear to be normally

47

distributed, and were analyzed using non-parametric methods. However, ANOVA and

Kruskall-Wallace tests produced similar results.

TABLE 2

Study Design - Assays and Treatment Groups Iron incubation: 24 hr (followed by wash) Quercetin incubation: 24 hr Assays: Cellular Iron Status (n=3 replicates) Cell Viability - Trypan Blue Exclusion (n=3-4 replicates) Lipid Peroxidation - TBARS (n=3 replicates) Apoptosis:

- DNA Fragmentation ELISA (n=4 replicates) - Caspase Activity (n=5 replicates) - Nuclear Morphology (Hoechst 33342) (n=3 replicates)

0 µM Fe

0µM 40 µM 80 µM

Quercetin Quercetin Quercetin

10 µM

Fe

0 µM 40 µM 80 µM Quercetin Quercetin Quercetin

25 µM Fe

0 µM 40 µM 80 µM Quercetin Quercetin Quercetin

TABLE 3 General Timeline for Assays

Trypan Blue Assay / Cellular Iron Status Following 24h Quercetin Incubation Day 1 Day 5 Day 6 Pass cells 1:4 (1 parent flask produces Change media in parent flasks. Discard iron-loaded media. 4 daughter flasks) Add Fe (0, 10, 25 µM) to Wash each flask with PBS. experimental flasks Add quercetin-loaded media (0, 40, 80 µM) to each iron group Day 7 Trypan Blue Assay Cells suspended - appropriate aliquots obtained from each group for 1) Trypan Blue Assay 2) Cellular Iron Analysis Lipid Peroxidation – TBARS Assay Day 1 Day 5 Day 6 Pass cells 1:4 (1 parent flask produces Change media in parent flasks. Discard iron-loaded media. 4 daughter flasks) Add Fe (0, 10, 25 µM) to Wash each flask with PBS. experimental flasks Add quercetin-loaded media (0, 40, 80 µM) to each iron group Day 7 Lipid Peroxidation – TBARS Assay Cells suspended – appropriate aliquots obtained for assay

48

DNA Fragmentation ELISA Day 1 Day 5 Day 6 Pass cells 1:4 (1 parent flask produces Change media in parent flasks. Discard iron-loaded media. 4 daughter flasks). Obtain cell count Add Fe (0, 10, 25 µM) to Wash each flask with PBS. from parent flasks to calculate dilutions experimental flasks Add quercetin-loaded media for assay (0, 40, 80 µM) to each iron group Day 7 Day 1 DNA Fragmentation Working Procedure of ELISA. Samples (180-200 bp nucleosomes) Pass cells 1:4. Obtain cell count obtained and stored at –20oC for from parent flasks to calculate working procedure of assay (12-18h incubation) dilutions for next week’s ELISA Caspase Activity / Chromatin Condensation Assays Day 1 Day 5 Day 6 Pass cells 1:4 (1 parent flask produces Change media in parent flasks. Discard iron-loaded media. 4 daughter flasks) Add Fe (0, 10, 25 µM) to Wash each flask with PBS. experimental flasks Add quercetin-loaded media (0, 40, 80 µM) to each iron group Day 7 Caspase activity/Hoechst stain Appropriate aliquots obtained from each group. For caspase activity, aliquots were not diluted. For Hoechst stain, separate aliquots were obtained from same experimental flasks and diluted.

49

50

RESULTS Cellular iron concentration

Cellular iron status increased as the media iron concentration increased from 0 to

25 µM (P = 0.0001). Iron was increased 3-fold between the 0 and 10 µM group, and 6-

fold between the 0 and 25 µM group (for the 0, 10, and 25 µM groups, mean cellular Fe

post-treatment was .0047 µM/106 cells, .0129 µM/106 cells, and .028 µM/106 cells,

respectively). Quercetin treatment had no effect on cellular iron (Fig. 1).

Lipid peroxidation (TBARS)

Iron concentration had an overall positive effect on lipid peroxidation (P<0.03).

Among the treatment groups, as iron increased, lipid peroxidation - indicated by the

formation of thiobarbituric acid reactive substances – increased. However, quercetin had

no significant effect on lipid peroxidation, either alone, or in combination with iron (Fig.

2). When no iron was added to the media, TBARS concentration was 0.1012 nmol

MDA/mg protein. Adding 10 µM and 25 µM iron increased TBARS 34% and 96%,

respectively. Of note, the 25 µM Fe / 40 µM quercetin group resulted in the highest

TBARS concentration.

Cell viability

As determined with the Trypan Blue assay, quercetin and iron alone had no

significant effect on cell viability. However, there was a weak trend toward a quercetin

effect (P=.0984) and a strong trend towards an iron x quercetin interaction (P=.0563)

(Fig. 3). Within the 0 µM Fe treatment group, cell viability was slightly increased with

each increase in media quercetin concentration. In contrast, within the 10 and 25 µM Fe

treatment groups, mean cell viability decreased with increasing media quercetin

51

concentrations. The cells treated with 25 µM Fe and 80 µM quercetin was the only group

with a significantly lower mean cell viability than controls.

DNA fragmentation

The average percent increase in DNA fragmentation that occurred with the

addition of 40 and 80 µM quercetin is shown in Figure 4, and the median changes in

DNA fragmentation are shown in Table 4. Iron concentration did not have a significant

effect on quercetin-induced DNA fragmentation. Quercetin level had a significant effect

on apoptotic DNA fragmentation (p<0.02). However, it was apparent that the addition of

40 µM quercetin did not consistently increase DNA fragmentation above control levels,

regardless of iron concentration. Also, apoptotic DNA fragmentation in cells treated with

80 µM quercetin was consistently increased only in iron-loaded cells.

The DNA fragmentation assay suggested that 80 µM quercetin was required to

have a consistent effect on apoptosis. Therefore, only 80 µM quercetin was used in the

final two apoptotic assays. Also, only two iron concentrations (0 and 25 µM were chosen

to determine if there was an iron effect.

Chromatin condensation

Apoptosis was also assessed with light microscopy examination of chromatin

condensation (Table 6). Both ANOVA and Chi-Square analyses showed a weak trend

toward an effect of quercetin on apoptosis. No synergism between iron and quercetin

was indicated to increase apoptosis, which does not support the apoptotic pattern as

measured with DNA fragmentation.

52

Caspase activity

Caspase activity was only measured with two concentrations of iron (0 and 25

µM) and quercetin (0 and 80 µM). Data are presented in Table 5. Results in the 0 µM

quercetin treatment groups showed no significant difference in percent of cells with

caspase activity between 0 µM Fe addition and 25 µM Fe addition. The data suggests a

weak interaction between iron and quercetin. 80 µM quercetin decreased caspase activity

in cells with normal iron concentrations, but increased caspase activity in cells with high

iron concentrations (ANOVA: Fe x Q: P=.0362).

DISCUSSION

The present study was conducted to determine if cellular iron status influences

quercetin-stimulated cytotoxicity and apoptosis in a human hepatoblastoma cell line. The

main findings were that 1) high cellular iron elevated lipid peroxidation, but quercetin

had no effect on peroxidation alone or in combination with iron, 2) cell viability was

decreased in groups treated with both high levels of iron and high levels of quercetin, and

3) high levels of iron and quercetin together triggered apoptosis, evidenced by increased

apoptotic DNA fragmentation and caspase activity.

Atomic absorption spectrophotometry revealed that increasing media iron content

increased cellular iron in a dose dependent manner. Further, cellular iron status was not

effected by addition of quercetin (Fig. 1). Cellular iron status in cells treated with 10 and

25 µM iron was three and six times higher, respectively, than the controls. This is very

similar to the increases in liver iron achieved by increasing dietary iron to 650 and 1500

mg Fe/kg diet in previous rat studies in our laboratory (Yin 1999). In acute iron

overload, free iron may be more prevalent than in chronic iron overload because ferritin

53

and transferrin synthesis may not be sufficient to accommodate the rapid excess of iron

(Cermak 1993). However, free iron was not assessed in this study, so it was unclear if

iron was free or bound.

The effect of quercetin on cellular iron concentrations was also examined. It is

well established that polyphenols as a group, and hence flavonoids, exert antioxidant

activity, in part, through their ability to chelate metal ions known to induce Fenton-type

reactions, such as iron and copper (Bravo 1998; Croft 1998). Obviously, this property

could prevent some free quercetin from being absorbed by the HepG2 cells if iron-loaded

media were used concurrently with quercetin loading. Therefore, before addition of

quercetin-treated media to cells, iron-loaded media was removed from the culture flasks,

and cells were washed. Some research has suggested that extracellular quercetin may

have the ability to lower cellular iron concentrations (Morel 1993). In our laboratory,

Stimson (1998) found that diets with 1% quercetin supplementation significantly

decreased liver iron concentrations in rats fed medium (424 mg Fe/kg diet) and high iron

(664 mg/kg diet) diets. However, it was not clear if this action took place in the lumen of

the intestine at the cellular level. While cellular iron was not altered by quercetin in the

present study, it is possible that a 24 hour incubation with quercetin was not quite

sufficient to liberate enough iron from the cell. Perhaps a longer incubation period with

the quercetin would be necessary to induce a change in cellular iron status.

Many researchers (Cermak 1993; Morel 1993; Jacob 1997; McCord 1998;

Halliwell and Gutteridge 1999), as well as previous studies (Stimson 1998) in our

laboratory have indicated that iron status of a cell may contribute to oxidative stress.

Similarly, in the present study, increased cellular iron significantly increased lipid

54

peroxidation, seen as higher TBARS concentrations. This is in accordance with animal

studies in our lab, where increased dietary iron increased lipid peroxidation in rat liver

(Yin 1999). The results of the present study also coincide with the observations of

Halliwell and Gutteridge’s review (1999), where they discuss that free iron or low

molecular weight iron chelates have the ability to form oxygen radicals that can initiate

and propagate lipid peroxidation. Two possible modes of action theorized are 1)

formation of hydroxyl radicals through a Fenton reaction that stimulates lipid

peroxidation, and 2) ability to decompose lipid peroxides to form alkoxyl and peroxyl

radicals - propagating further lipid peroxidation.

I further hypothesized that addition of high concentrations of quercetin to iron-

loaded cells would increase oxidative stress, thereby increasing lipid peroxidation, which

is one trigger of apoptosis. This result was not observed in my study, as quercetin

increased apoptosis, but had no significant effect on lipid peroxidation. However, it is

interesting to note that the highest lipid peroxidation values - although non-significant -

were found to be associated with quercetin. In a related study, Laughton et al. (1989)

found that addition of the flavonoids quercetin and myricetin to Fe3+ (100 µM /L as ferric

chloride) or Fe3+ EDTA (100 µM) consistently accelerated bleomycin-dependent DNA

degradation at concentrations from 0-75 µM, but had no effect on microsomal lipid

peroxidation at concentrations of 0-50 µM. This may suggest that the prooxidative

actions of flavonoids in conjunction with iron may be site and model specific.

The Trypan Blue assay is a simple assay of cell viability. It does not discriminate

between cell death caused by apoptosis or necrosis. It does, however, provide an initial

insight into the effects of quercetin and iron on cell viability. It has been proposed that

55

flavonoids may be protective by increasing apoptosis in damaged cells (Kuo 1996). In

support of this, Kuo (1996) found that quercetin and other flavonoids decreased cell

viability through apoptosis in Caco-2, HT-29, and IEC-6 cells. Regarding iron’s effect

on cell viability, Cermak et al. (1993) suggested that acute exposure of intestinal tumor

cells to iron might increase their susceptibility to oxidant-mediated lysis. Taken together,

these experiments point to a possibility of both quercetin and iron reducing cell viability

in tumor cells. While our results were not statistically significant, we did notice a strong

trend (P=.0563) towards an iron and quercetin interaction in decreasing cell viability.

This did suggest that iron and quercetin may interact to damage cell components, but this

assay could not distinguish between apoptosis and necrosis.

I hypothesized that at high concentrations, quercetin could act as a prooxidant,

particularly in the presence of added iron, thus enhancing apoptosis of HepG2 cells. It is

known that reactive oxygen species can trigger measurable apoptotic changes within

cells. Indeed, a possible trigger of the observed apoptotic DNA fragmentation may

specifically be oxidant-induced DNA damage. DNA appears to be extremely susceptible

to attack by certain radical species. For example, due to its high reactivity, the hydroxyl

radical can attack sugars, purines, and pyrimidines of DNA (Halliwell and Gutteridge

1999). Studies by Sahu and Washington (1991) and Sahu and Gray (1997) suggest that

the autooxidation of flavonoids catalyzed by iron and copper ions can result in reactive

oxygen species formation, causing DNA damage. These researchers proposed that lipid

peroxidation is the first consequence of formation of the reactive oxygen species. DNA

is located in close proximity to the nuclear membrane - hence lipid peroxidation could

also effect the DNA inside the nuclear membrane, as the levels of lipid peroxides and

56

intermediate radicals are then amplified by a chain reaction (Sahu and Gray 1997). The

same researchers also proposed that reactive oxygen species may be produced in close

proximity to DNA-bound metal ions...“as iron is an essential trace element in cells and is

endogenously associated with DNA, agents capable of producing H2O2 inside the cell can

produce metal-catalyzed, site-specific oxidative DNA damage” (Sahu and Gray 1997).

Overall, their studies demonstrated that both free radicals and lipid peroxides can damage

DNA, regardless of the sequence of production.

Finally, to again reinforce the possibility of DNA damage as an apoptotic trigger,

the same study by Sahu and Washington (1991), indicated that quercetin at 0-100 µM

decreased double stranded DNA content in isolated rat liver nuclei in a concentration-

dependent manner after a 30-minute incubation. When iron and copper ions were added

at equal molar ratios with the same 30-minute incubation period, the loss of double

stranded DNA was even greater.

Regarding mechanism, flavonoids may increase free radical damage to DNA

through a reduction of Fe3+ to Fe2+ (Smith 1992). In particular, when flavonoids

gossypol, quercetin, or myricetin were added to reaction mixtures containing Fe3+-EDTA

and H2O2 at pH 7, they produced an increase in deoxyribose degradation. DNA

degradation was almost completely halted by catalase, and other hydroxyl radical

scavengers. Therefore, the deoxyribose degradation was attributed to hydroxyl radical

formation (Laughton 1989). However, it should be noted that inclusion of the same

flavonoids in reaction mixtures containing H2O2 and Fe3+-citrate had no effect on

increasing hydroxyl radical generation. It is possible that the form of iron or iron

complex may have an effect on its interaction with the flavonoids, and may explain

57

differences between our results and the results of others. Comparing these studies to ours

with DNA damage as the main possible trigger is speculative, as we did not include

assays that measured DNA damage as an endpoint. In the current study, while an iron

and quercetin interaction was not found to be significant, the greatest quercetin-induced

increases in DNA fragmentation were seen only in the iron-loaded cells. Clearly, our

study evaluated a different endpoint. However, as DNA damage is a known initiator of

apoptosis (King and Cidlowski 1995), and it could be the mechanism for quercetin-

induced apoptotic DNA fragmentation we found in iron loaded cells.

Caspase activity is an assay of early apoptotic changes within the cell. We

proposed that an iron and quercetin interaction would synergistically increase caspase

activity. Our results indicated that iron alone had no effect on caspase activity.

However, the effect of quercetin depended on cellular iron status. First, in cells that were

not iron-loaded, quercetin treatment had a protective effect, lowering caspase activity. In

the cells treated with both high levels of iron and quercetin, caspase activity was

increased. The redox status of the cell has a complex effect on apoptosis. Hampton

(1998) reviewed studies showing that the addition of exogenous oxidants induces

apoptotic caspase activity. It is possible that the levels of quercetin and iron used in the

current study functioned within this “apoptotic window” to induce a concentration of

reactive oxygen species that effectively increased caspase activity. The redox

environment of the cell may also explain why a protective effect was seen in the control

cells with quercetin loading. At a quercetin concentration of 80µM, without cellular iron

overload, quercetin may have acted as an antioxidant, scavenging radical species, thereby

58

preventing the reducing environment necessary to enhance reactive oxygen species

production.

Chromatin condensation, a result of the proteolytic action of caspases, is a late

stage indicator of apoptosis. It can be used to reinforce the evidence of caspase activity,

as theoretically, levels of chromatin condensation should be somewhat proportional to

caspase activity and show trends similar to those shown with the DNA fragmentation

assay. Our results from examining chromatin condensation were similar to the results

obtained in the DNA fragmentation assay in that only the cells loaded with quercetin

showed a trend towards increasing apoptosis. However, chromatin condensation did not

necessarily coincide with the results of our caspase activity assay and did not confirm an

iron and quercetin interaction toward increasing numbers of cells with apoptotic

morphology. Only a weak trend was noticed towards a positive quercetin effect

(P=.0773) on increasing numbers of cells with apoptotic morphology. One possible

explanation for a positive iron and quercetin interaction on caspase activity not being

reinforced by a proportional effect on apoptotic morphology comes from the fact that the

cells examined for chromatin condensation were coincubated with propidium iodide (PI),

an indicator of dead cells. While staining with propidium iodide may have allowed

detection of dead cells, it did not enable inference into the mode of cell death. It is

possible that a number of the PI-labeled cells that were already dead by the time of the

assay may have died by apoptosis. A possible way of discerning dead necrotic cells from

dead apoptotic cells would be to discriminate apoptotic cells by the cellular markers that

attract phagocytic macrophages. Overall, difference in timing between caspase activation

and physical manifestation of that activity (chromatin condensation) may be the reason

59

for the difference between results for the caspase activity assay and the Hoechst

procedure.

In conclusion, this study indicates that the level of quercetin needed to increase

apoptosis is well above levels that may be achieved by diet alone. Additionally, iron may

enhance the susceptibility of cells to quercetin-induced apoptosis.

60

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

uM Fe / uM Quercetin

ug F

e/10

6 cel

ls

Figure 1. Cellular iron concentration following 24 hr iron + 24 hr quercetin treatments. Cellular iron concentration increased as media iron concentration increased (Fe: P = 0.0001). Quercetin treatment had no effect on cellular iron.

0/0 0/80 10/0 10/40 10/80 25/0 25/40 25/800/40

61

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

uM Fe / uM Quercetin

nmol

MD

A/m

g pr

otei

n

Figure 2. Lipid Peroxidation. Thiobarbituric acid reactive substances measured as nmol malondialdehyde (MDA) per mg protein. As iron increased, lipid peroxidation increased (Fe: P<0.03). Quercetin had no significant effect.

0/0 0/40 0/80 10/0 10/40 10/80 25/0 25/40 25/80

62

0%5%

10%15%20%25%30%35%40%45%50%55%60%65%70%75%80%85%90%

uM Fe / uM Quercetin

% V

iabi

lity

Figure 3. Cell viability measured by Trypan Blue exclusion. Quercetin and iron alone had no significant effect on cell viability. However, there was a strong trend towards an iron and quercetin interaction (P=.0563). Cell viability = viable cells/total cells (viable + dead).

0/0 0/80 10/0 10/40 10/80 25/0 25/40 25/800/40

63

0

20

40

60

80

100

120

140

160

um Fe / uM Quercetin

% in

crea

se in

180

-200

bp

frag

men

tatio

n ab

ove

cont

rol

0/40 0/80 10/40 10/80 25/40 25/80

Figure 4. Apoptotic DNA fragmentation=180-200 bp nucleosomes above control. Iron did not have a significant effect on quercetin-induced DNA fragmentation. However, DNA fragmentation was consistently increased only by 80 uM quercetin (p<0.02).

64

TABLE 4 DNA Fragmentation: Chi-Square Analysis

Treatment Median (25,75% quantiles) 0 µM Fe 40 uM quercetin 1.24 (1.10 – 1.36) 80 uM quercetin 1.02 (0.76 – 1.91) 10 µM Fe 40 µM quercetin 0.97 (0.65 – 1.43) 80 µM quercetin 1.90 (1.42 – 2.55) 25 µM Fe 40 µM quercetin 0.96 (0.85 – 1.17) 80 µM quercetin 1.54 (1.31 – 2.05) Chi Square 0.139

65

TABLE 5 Percent Cells with Caspase Activity

Treatment Mean1 Median2 Range 0 µM Fe 0 µM quercetin 0.2662 + 0.0868a,b 0.3407 (0.1271-0.4178) (0-0.4454) 80 µM quercetin 0.0561 + 0.0345b 0 (0-0.1323) (0-0.1484) 25 µM Fe 0 µM quercetin 0.2350 + 0.0892a,b 0.1832 (0.1087-0.4367) (0-0.4464) 80 µM quercetin 0.5083 + 0.1673a 0.5175 (0.3125-0.7519) (0-0.9597) P-value Fe 0.0638 Quercetin 0.7686 Fe/quercetin 0.0362 Chi Square 0.1398

1Mean + SEM (n=5 trials) 2Median (25,75 quantiles)

66

TABLE 6

Percent of Cells with Apoptotic Morphology (Chromatin Condensation) Mean1 Median2 0 µM Fe 0 µM quercetin 24.18 + 1.63 22.86 (22.26 – 27.42) 80 µM quercetin 39.04 + 4.85 39.68 (30.34 – 47.09) 25 µM Fe 0 µM quercetin 34.96 + 5.73 31.63 (27.12 – 46.12) 80 µM quercetin 44.26 + 9.12 36.42 (33.91 – 62.44) P-value Fe 0.2164 quercetin 0.0773 Fe/quercetin 0.6536 Chi Square 0.1009 1Mean + SEM (n=3 trials) 2Median (25,75% quantile)

CHAPTER IV

SUMMARY

MAJOR FINDINGS

The purpose of this study was to determine whether a flavonoid common to many

foods (quercetin) could induce apoptosis in the HepG2 cell line, a liver hepatoma cell

line, and whether cellular iron status alters apoptosis. It was hypothesized that 1) cellular

iron status would increase with higher media iron concentrations, and that addition of

quercetin to iron-loaded cells would have no effect on cellular iron, 2) addition of

quercetin to iron-loaded cells would increase lipid peroxidation, 3) cellular iron

concentration would influence the prooxidative ability of quercetin to decrease cell

viability, 4) cellular iron concentration would influence the prooxidative ability of

quercetin to induce specific apoptotic changes in the cell measured as: 200-bp DNA

nucleosome fragmentation (ladder effect), caspase activation, and chromatin

condensation.

Major findings were 1) cellular iron status increased with iron loading over a 24

hour incubation period, 2) addition of quercetin to iron-loaded cells had no effect on

cellular iron, 3) iron had a positive effect on lipid peroxidation, while quercetin did not,

4) 80 µM concentrations of quercetin tended to decrease cell viability in iron-loaded

cells, 5) 80 µM concentration of quercetin was consistently associated with an increase in

apoptosis-induced DNA fragmentation, and increases were greater in iron-loaded cells,

67

68

but the iron effect was not significant, 6) chromatin condensation was affected by

quercetin, but not iron, and 7) finally, caspase activity was significantly increased in iron-

loaded cells supplemented with 80 µM quercetin.

IMPLICATIONS

This study suggested that the level of quercetin needed to increase apoptosis is

well above levels that may be achieved by diet alone. It also indicates that iron may

enhance the susceptibility of cells to quercetin-induced apoptosis. The majority of

chemo- and radiotherapeutic agents destroy malignant cells by initiating apoptosis

(Martin 1997). Apoptosis is an ideal way to reduce tumor cell growth, because it induces

damaged or injured cells to commit suicide, allowing adjacent cells to proliferate without

being affected by the otherwise necrotic death of neighboring cells (Samali 1996).

Some predictions indicate that the prevalence of Americans ever diagnosed with

invasive cancer could be over 11 million by 2020 (Polednak 1997). Regarding modern

medicine, cancer treatments have met with success, but it has been very expensive

(Argiles 1998). Therefore, more cost-effective therapies need to be researched and

implemented. This study used an inexpensive and readily available combination of

components to produce apoptosis - an effective and established method of chemotherapy.

STUDY LIMITATIONS

Cellular iron levels were measured to ensure consistent increases in cellular iron

status with increased iron media levels. As well, media iron concentrations were

determined to ensure consistency among media batches. Due to the scope of this study,

though, quercetin levels in the media and the cells - following supplementation - could

not be measured. Obviously, knowledge of cellular quercetin status post-

69

supplementation, as well as the prominent areas of quercetin accumulation within the

cells, would provide further insight into specific cellular components that may be affected

by this compound. Further, the aglycone form of quercetin was used in this study, while

quercetin metabolites are most prevalent in human tissues. Also, only one incubation

time was tested for both iron and quercetin. Longer incubation time with either iron or

quercetin, or both, could have produced different results. Another important limitation

again occurs due to the scope of this study. More replicates of each assay would have

provided a stronger basis for discussion of assay results. Finally, although our model was

a human cancer cell line, we could not duplicate the complex environment that occurs in

vivo. Also, the levels of quercetin used were pharmacologic - they are higher than any

level that could be achieved by diet alone. However, as a neoplastic human cell line, our

model provides a foundation for additional chemotherapy research in human studies.

FURTHER RESEARCH

This study builds on a growing area of research into the dual roles of flavonoids.

Where much prior research has focused on the antioxidant capabilities of flavonoids like

quercetin, in conjunction with redox metal-catalyzed oxidative stress, this study further

examined the prooxidative effects produced by combining specific amounts of flavonoids

and redox metals. First, longer incubation times should be tested. Before assaying for

apoptosis, it would be helpful to have additional measures of oxidative stress produced by

each respective treatment. Further, identification of the specific reactive oxygen species

produced by iron and quercetin interactions would provide more insight into the

mechanism behind apoptosis in this system.

70

This study used the endpoints of 180-200 bp DNA fragmentation, chromatin

condensation, and general caspase activity to specifically determine apoptosis. Further

investigation into the intervals at which these endpoints are produced might indicate a

better timepoint by which to conduct the respective assays. As well, other assays such as

the TUNEL and electrophoretic assays, could be utilized to further reinforce the presence

of cellular apoptosis.

This study also indicated that a high level of quercetin is necessary to induce

apoptosis - more than diet can provide. Building from the maximum dose used - 80 µM -

future investigation of lower dose levels could determine the safest, most effective

pharmacologic range of quercetin concentrations that could induce apoptosis in tumor

cells. Finally, as iron status of the cell may influence the efficacy of quercetin

supplementation, iron loading must be refined to specifically target tumor cells to avoid

affecting healthy cells.

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