Chemopreventive Effects of Lycopene and Other Various Carotenoids

Daniel Etter; A44073265

Dr. Bello-DeOcampo

Cancer Biology – ZOL 450

4/23/2015

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Table of Contents

Abstract…………………………………………………………………………………………………………3

Key Words……………………………………………………………………………………………………..3

Abbreviations………………………………………………………………………………………………...5

Objectives………………………………………………………………………………………………………6

Introduction…………………………………………………………………………………………………...7

Background……………………………...…………………………………………………………………….8

Reactive Oxygen Species and Oxidative Stress……………………………………….8

Antioxidants and Oxidative Stress………………………………………………………...9

Carotenoids…………………………………………………………………………………………10

Literature Review…………………………………………………………………………………………...11

-Carotene and -Carotene…………………………………………………………………..11

Lycopene…………………………………………………………….……………………………….15

-Cryptoxanthin………………………………………………………………………………......17

Lutein……………………….…………………………………………………………...…………….18

Discussion…………………………………………………………………………………………………..…...18

Dietary Risk Factors of Carcinogenesis………………………………………………….18

-Carotene and -Carotene…………………………………………………………………..20

Lycopene…………………………………………………………………………………………….22

-Cryptoxanthin………………………………………………………………………………….24

Lutein…………………………………………………………………………………………………24

Conclusions and Significance………………………………………………………………………….25

Figures………………………………………………………………………………………………………….27

References……………………………………………………………………………………………………..32

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Abstract

It is well documented that lifestyle factors such as an individual’s diet and smoking

are accountable for a large majority of cancers, so it is important to acknowledge this and

learn what you can do to improve your chances. Reactive oxygen species (ROS) are a group

of reactive molecules that are a byproduct of normal metabolism, but are commonly

overproduced in cells. ROS when overproduced have the ability to oxidize/damage

macromolecules such as deoxyribonucleic acid (DNA), lipids and proteins, which can

eventually lead to the development of cancer. They typical Western diet is high in refined

sugars and trans fatty acids, which employ multiple mechanisms that aid in the process of

carcinogenesis. Diets high in fruits and vegetables have been associated with a lower

incidence of cancer, and one group of molecules found in them that seem to possess

anticarcinogenic qualities are carotenoids. -Carotene showed to be approximately 10x

more potent of an inhibitor of proliferation than -carotene. Both and -carotene were

able to induce similar inhibition of invasion and migration, primarily through the decrease

in expression of matrix metalloproteinase 2/7/9 and the increase in the expression of the

tissue inhibitor of MMP (TIMP) 1 and 2. -Carotene has been shown to act as both a

procarcinogen and anticarcinogen, depending on whether or not the individual has a

history of smoking. Another carotenoid possessing potent in vitro anticarcinogenic

properties is lycopene, which studies have shown is able to inhibit proliferation/induce cell

cycle arrest, inhibit metastasis in a manner similar to and -carotene, induce the

antioxidant response element, induce apoptosis and improve cell-to-cell communication.

The carotenoids lutein and -cryptoxanthin have had much less research devoted to them,

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and although their consumption has been associated with lower cancer incidence future

research should take a closer examination into their specific mechanisms of action.

Key Words: Antioxidant, Carotenoid, Reactive Oxygen Species, Nuclear Factor E2-related

Factor 2 (Nrf-2), Growth Factor

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Abbreviations

ALU – Short interspersed element

AP-1 – Activator protein 1

ATBC - Alpha-Tocopherol, -Carotene Cancer Prevention Study

CARET - Beta Carotene and Retinol Efficacy Trial

CYP – Cytochrome P450

DNA – Deoxyribonucleic acid

DNMT – DNA methyltransferase

ERK – Extracellular signal-regulated kinase

FAK – Focal adhesion kinase

GCLC - Glutamate-cysteine ligase (catalytic subunit)

GCLM – Glutamate-cysteine ligase (modifier subunit)

GSR - Glutathione reductase

GSTs - Glutathione S-transferases

HDL – High-density lipoprotein

HO-1 - Heme oxygenase-1

ICAM – Intercellular adhesion molecule

JNK – c-Jun NH2-terminal kinase

LDH – Lactate dehydrogenase

LDL – Low-density lipoprotein

LINE-1 – Long interspersed element

MAPK – Mitogen activated protein kinase

mEH - Microsomal epoxide hydrolase 1

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MMP – Matrix metalloproteinase

NF-kB – Nuclear (transcription) factor kappa-B

NQO1 - NADPH:quinone oxidoreductase 1

Nrf-2 – Nuclear factor-E2 related factor 2

PAI – Plasminogen activator inhibitor protein

RAR - Retinoic acid receptor

RARE – Retinoic acid response element

RNA – Ribonucleic acid

ROS – Reactive oxygen species

TIMP – Tissue inhibitor of matrix metalloproteinase

UGT1A6 - UDP glucuronosyltransferase 1 family, polypeptide A6

uPA – Urokinase plasminogen activator

VCAM – Vascular cell adhesion molecule

Objectives

The objectives of the present review are to attempt to illustrate just how big of an

effect (positive or negative) an individual’s diet can have on the multistage process of

carcinogenesis. It will discuss multiple pro-carcinogenic factors in the typical Western diet

as well as numerous anti-carcinogenic agents and some of their associated mechanisms of

action, which may prove vital in halting/reversing the damage already done in many of our

bodies.

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Introduction

Cancer is one of the most unfortunate realities in life, but a reality nonetheless.

Therefore, we must accept it for what it is and do what we can for those who fall victim to

its processes. To put the problem into perspective, there will be an estimated 1,658,370

new cancer diagnoses in the year 2015 in the United States alone (American Cancer Society

Facts and Figures, 2015). Fortunately, there are certain lifestyle choices an individual can

make that can aid in reducing the risk of developing cancer in one’s lifetime.

It is estimated that only about 5-10% of cancers are of genetic origin, or inherited

from the individual’s parents. This means that approximately 90-95% of cancers can be

attributed to lifestyle/environmental factors (Anand et al., 2008). Such factors include

tobacco consumption (smoked and smokeless), alcohol consumption, sun exposure,

environmental pollutants, infections, stress, physical inactivity, obesity and diet. Since my

generation was in elementary school we have heard of the dangers of tobacco, in particular

its carcinogenic capabilities, so many of us have steered clear. Unfortunately, this same

level of education was not given to us regarding the impact that our diet has on disease

prevention.

The typical Western diet is characterized by its caloric excess and its nutritional

deficits, due in part to the overconsumption of low cost, calorie dense food options. This

problem is especially prevalent among people in the younger demographic, with 57

percent of individuals 18-29 reporting that they consume fast food at least weekly (Gallup

Annual Consumption Poll, 2013). The problem with fast food, and countless other

foodstuffs in the American diet, includes but is not limited to the overabundance of

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saturated fats/hydrogenated oils and refined sugars. Implications of these excesses will be

examined further in the discussion section. In addition to the overconsumption of fast food

items, 37.7 percent of American adults reported consuming fruit less than one time per day

while 22.6 percent reported consuming vegetables less than one time per day (CDC, 2013).

This shortage of fruit and vegetable consumption is an important contributing factor

to carcinogenesis. Diets high in fruits and vegetables have been consistently associated

with a lower incidence of cancer and this is due in part to the fact that fruits and vegetables

are some of the best sources of antioxidants in our diets (Gonzalez, 2006). It is these

antioxidants that protect our body from oxidative damage by scavenging free radicals and

quenching reactive oxygen species (ROS), which have been linked to development of cancer

(Ahsan and Waris, 2006). Some such compounds found within fruits and vegetables that

possess such chemopreventive capabilities are a class of compounds known as carotenoids,

and will be the focus of this review of research.

Background

Reactive Oxygen Species and Oxidative Stress

Reactive oxygen species (ROS) are a group of chemically reactive molecules that

contain oxygen, such as oxygen ions and peroxides. ROS are usually produced through

normal metabolic pathways, however they can also be produced in response to certain

dietary and lifestyle choices. Some dietary factors that can cause an increase in ROS

production include excesses in either dietary sugar (Yu et al., 2011) or trans fats (Bryk et

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al., 2011). Additionally, a lifestyle factor such as smoking is well known to increase the

production of ROS (Lin et al., 2012).

When under normal homeostatic conditions, ROS are produced but are kept under

strict regulation where they play an important role in multiple cell signaling pathways such

as proliferation and apoptosis (Ahsan and Waris, 2006). Problems arise when regulatory

mechanisms of the cell dysfunction and ROS production is increased. These elevated levels

of ROS can then induce oxidative stress though oxidation of the individual’s

deoxyribonucleic acid (DNA), lipids or proteins. Such oxidative damage may result in

multiple DNA modifications, however in regards to cancer these mutations are typically

substitutions in the G-C base pairings, such as that seen in the most common mutation in

the p53 suppressor gene where there is a GT conversion (Ahsan and Waris, 2006).

Mutations in the DNA then lead to altered gene/protein expression, which is ultimately the

source of the observed and sustained phenotypic changes resulting in carcinogenesis.

Signal transduction pathways important in cell growth, such as activator protein 1 (AP-1)

and nuclear transcription factor kappa B (NF-kB), are among the common signaling

pathways induced by ROS (Valko et al., 2006).

Antioxidants and Oxidative Stress

As the name might suggest, an antioxidant’s activity is to inhibit the damage done to

cellular structure and/or function via ROS. The body has multiple naturally occurring

enzymes that possess antioxidant capabilities such as NADPH, but due to the poor dietary

habits of many Westerners this balance between oxidants formed by normal aerobic

respiration and antioxidants is weighted in favor of oxidants (Ahsan and Waris, 2006). This

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imbalance is what eventually leads to the oxidative damage to various cellular

macromolecules as illustrated in Figure 1. Because many of us are constantly consuming

foods that are seen to increase levels of ROS in the body, it is important that we also

consume adequate amounts of foods high in antioxidants, such as fruits and vegetables.

One such group of antioxidants is known as carotenoids.

Carotenoids

Carotenoids are a group of more than 600 naturally occurring, fat-soluble pigments

that give plants their bright coloration (Higdon, 2004). They are a group of molecules called

tetraterpenoids, which are hydrocarbon chains consisting of 40 carbon atoms. There are

two main classifications of carotenoids; xanthophylls are those containing oxygen in their

otherwise hydrocarbon structure and carotenes are those that are pure hydrocarbon

molecules.

Xanthophylls (and carotenes) are not produced endogenously by humans and must

therefore be consumed in the diet by eating fruits and vegetables. Xanthophylls serve an

important role in protecting plants from high-intensity light-induced oxidative stress. They

include the molecules -cryptoxanthin and lutein, and function to absorb high blue

wavelengths, thus protecting the chloroplast from over excitation and subsequent

production of ROS through the action of the xanthophyll cycle (Latowski et al., 2011). With

sufficient dietary intake, humans preferentially sequester lutein and zeaxanthin in the

macula lutea of the retina, where it too protects the cells from ROS damage. Additionally,

recent research has elicited the positive effects of lutein and zeaxanthin on not only vision,

but on cognition as well (Johnson, E.J. 2014).

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Carotenes are carotenoids that possess purely hydrocarbon structures. These

molecules, such as -carotene, -carotene and Lycopene, function during photosynthesis

by transmitting its absorbed light energy to chlorophyll as well as absorbing the energy

from singlet oxygen. Two of the main dietary carotenes, -carotene and -carotene, are

considered provitamin A carotenoids, meaning that the body is able to metabolize these

molecules into vitamin A or its metabolites (Tanaka et al., 2012). It is believed that this

provitamin A activity is responsible for some, if not most, of the anticarcinogenic effects of

these provitamin A carotenoids.

Literature Review

-Carotene and -Carotene

-Carotene is one of the major dietary sources of vitamin A and some foods

containing the highest concentrations of this carotenoid include carrots, sweet potatoes

and winter squash. This carotenoid has been shown to exhibit significant antiproliferative

effects in vitro on the human neuroblastoma cell line GOTO in both a dose and time-

dependent manner (Murakoshi et al., 1989). The N-myc protein is highly expressed and

necessary for fetal development due to its ability to stimulate cell growth and proliferation,

but in cancer the gene amplification of this proto-oncogene results in activation and an

over-expression of the protein by 5-1000 times that of normal (DeOcampo, 2015).

Densitometry autoradiographs were used to determine the expression of N-myc messenger

ribonucleic acid (RNA) and -carotene was shown to significantly suppress the expression

of this messenger RNA in a concentration and time dependent manner, roughly ten times

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more effectively than did -carotene. In an additional experiment varying concentrations of

-carotene and -carotene were added to cultures containing the GOTO cell line and the

results showed that both possessed antiproliferative properties, with -carotene being

effective at a ten times lesser concentration (Murakoshi et al., 1989). -Carotene’s effect on

cell cycle progression was then determined by measuring the relative amounts of DNA

within the cell via flow cytometry of the control and the cells treated with -carotene.

Results indicated the cells treated with -carotene to be accumulating in the G0-G1 phase of

the cell cycle (Murakoshi et al., 1989). Additional in-vivo mice trials have shown that -

carotene is approximately 10x more potent of an inhibitor of liver, lung and skin

carcinogenesis than -carotene (Murakoshi et al., 1992).

Recent in-vitro and in-vivo studies have sought to indicate whether or not -

carotene (and -carotene) may possess any antimetastatic properties in the highly invasive

Lewis lung carcinoma (LLC). Cell culture containing the LLC cells were treated with -

carotene, -carotene and control and data showed that (at similar concentrations) and -

carotene induced comparable inhibition of invasion and migration of the LCC cells (Liu et

al., 2015). There was no effect on proliferation, however. Their effect (if any) on matrix

metalloproteinase (MMP) 9, MMP 2 and urokinase plasminogen activator (uPA) was then

examined in culture. and -carotene each significantly reduced the activity of MMP 9/2,

but only -carotene was shown to reduce the activity of uPA (Liu et al., 2015).

The effect of and -carotene on the expression of the tissue inhibitor of MMP

(TIMP) -1, TIMP-2 and plasminogen activator inhibitor (PAI)-1 proteins was examined as

well. Data showed that both molecules markedly increased expression of all three proteins

in LLC cells in culture when at similar concentrations (Liu et al., 2015). The ability of -

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carotene to alter the expression of integrin -1 and subsequent integrin -1 mediated

signaling molecules, such as those in the focal adhesion kinase (FAK) and mitogen activated

protein kinase (MAPK) families, was then examined. Results showed that -1 integrin

expression was markedly reduced (Liu et al., 2015). The effects of -carotene on metastasis

in LLC mice are summarized in Figure 2. Data also showed that activation of multiple -1

integrin mediated downstream signaling molecules was also significantly reduced

following -carotene treatment. The phosphorylation status of extracellular signal-

regulated kinase (ERK)-1, ERK-2, FAK, c-Jun NH2-terminal kinase (JNK)-1, JNK-2, and p38

(of the MAPK family) were all significantly reduced (Liu et al., 2015). However, data elicited

that although altered phosphorylation caused a change in activation, the protein expression

of these molecules remained constant (Liu et al., 2015).

In-vivo mouse experiments on LLC mice studied the effects of an only -carotene

treatment, only taxol treatment, and a combination treatment on body weight, primary

tumor growth and lung metastasis. Data showed that -carotene treatment, taxol

treatment, and a combination treatment did not alter body weight of the LLC-bearing mice

(Liu et al., 2015). In addition, -carotene treatment alone did not alter primary tumor

growth, whereas taxol (and a combination) treatment was shown to significantly inhibit

the growth of the primary tumor (Liu et al., 2015). Data showed that both -carotene

treatment and taxol treatment both significantly reduced lung metastasis, with taxol being

the more potent inhibitor. Additionally, results indicated that combination treatment with

-carotene and taxol together was more successful than either of the treatments alone (Liu

et al., 2015).

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Similar in-vivo LLC mice studies examined the effects -carotene, taxol, and

combination treatment on the expression of integrin -1, TIMP-1/2, and PAI-1 in addition

to the phosphorylation of FAK. Treatment of LLC mice with -carotene and taxol alone

markedly reduced integrin -1 expression, while only taxol treatment inhibited the

phosphorylation of FAK. Combination treatment did not enhance these effects (Liu et al.,

2015). The results indicated that both -carotene and taxol significantly increased the

expression of TIMP-1 and PAI-1, but only taxol increased the expression of TIMP-2.

Combination treatment proved to enhance these effects (Liu et al., 2015).

Studies on -carotene seem to suggest that under normal circumstances it acts as an

antioxidant and enhances immune function, but in smokers and other high-risk individuals

it can actually act as a pro oxidant and aid in the process of carcinogenesis. Two large

intervention studies, the Alpha-Tocopherol, -Carotene Cancer Prevention Study (ATBC)

and the Beta Carotene and Retinol Efficacy Trial (CARET), showed a strong correlation

between high-dose -carotene supplementation and lung cancer incidence when compared

to the control (ATBC Study Group, 1994), (Omenn et al., 1994). Recent in-vitro analysis of

rat microsomal membranes studied the procarcinogenic effects of -carotene in the

“presence” of cigarette smoke (tar in this case) and -tocopherol (form of vitamin E). Data

showed that at low (nonphysiological) O2 pressures, -carotene acted as an antioxidant and

reduced the amount of lipid peroxidation caused by the tar in the presence and absence of

-tocopherol (Palozza et al., 2006). At physiological O2 pressure, -carotene significantly

increased the lipid peroxidation induced by the tar, and this effect was reduced in the

presence of -tocopherol (Palozza et al., 2006). In addition, the effect of varying O2

pressure on -carotene consumption and oxidation was examined and data showed that

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both consumption and oxidation of -carotene was significantly increased during an

increase in air pressure (Palozza et al., 2006). Another study that may provide insight into

-carotene’s role as a co-carcinogen looked into its effects on microsomal cytochrome p450

(CYP)-linked microoxygenase activity and ROS production in lung, liver, kidney and

intestinal tissues. Data showed that not only were a multitude of CYP-induced enzymes

increased, but the associated increase in ROS production was substantial (up to a 33-fold

increase in liver tissue) (Paolini et al., 2001).

Lycopene

Lycopene is a non-provitamin A carotenoid that is found in high concentrations in

guava, watermelon and tomatoes and has been the subject of much recent research. Many

of lycopene’s biological functions are due to its ability to activate the antioxidant response

element, which is a series of cellular oxidative stress defense enzymes (Solis et al., 2013).

The effects of lycopene’s metabolite apo-10’-lipopenoic acid on nuclear factor-E2 related

factor 2 (Nrf-2) and subsequent antioxidant enzyme expression was examined in the

human bronchial epithelial cell line BEAS-2B. Data showed that not only did apo-10’-

lipopenoic acid cause nuclear translocation of Nrf-2, but it also induced an increase in the

messenger RNA of multiple phase II antioxidant/detoxification enzymes including heme

oxygenase-1 (HO-1), NADPH:quinone oxidoreductase 1 (NQO1), glutathione S-transferases

(GSTs), glutathione reductase (GSR), the catalytic and modifier subunits of glutamate-

cysteine ligase (GCLC and GCLM, respectively), microsomal epoxide hydrolase 1 (mEH)

and Uridine diphosphate (UDP) glucuronosyltransferase 1 family, polypeptide A6

(UGT1A6) (Lian and Wang, 2008). Additionally, treatment with apo-10’-lipopenoic acid

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resulted in the increase of the endogenous antioxidant glutathione and a significant

decrease in intracellular ROS production (Lian and Wang, 2008). Apo-10’-lipopenoic acid

was also found to decrease H2O2-induced oxidative damage as noted by a reduction of

lactate dehydrogenase (LDH) release (Lian and Wang, 2008).

Multiple in vitro and in vivo studies on lycopene have illustrated its effects on

multiple growth factors and their related signaling pathways, which are summarized in

Figure 5. For example, lycopene has been shown to decrease levels of circulating insulin-

like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF), while also

inhibiting the autophosphorylation and activation of platelet derived growth factor (PDGF)

(Solis et al., 2013). Studies have also elicited the fact that lycopene has the ability to induce

apoptosis and/or cell cycle arrest, inhibit metastasis, and enhance cell-cell communication

(Solis et al., 2013). In vivo studies showed lycopene’s association with a reduction in the

average number of tumors per mouse in both lung and liver cancer, seen in Table 1

(Nishino et al., 2002).

Lycopene’s effects on the androgen-sensitive LNCaP prostate cell line and androgen-

independent PC3 prostate cell line have also been investigated. In the androgen sensitive

LNCaP cell line data showed an induction of cell cycle arrest at the G1/S phase transition as

well as an induction of apoptosis (Ivanov et al., 2007). The methylation status of the GSTP-1

promoter region, as well as the expression DNA methyltransferases (DNMT), was

unaffected following lycopene treatment in the LNCaP cell line (Fu et al., 2014). However,

LNCaP cells did experience a reduction in methylation of the long interspersed element

(LINE-1) and short interspersed element (ALU) (Fu et al., 2014). The androgen-

independent PC3 cell line also saw an induction of cell cycle arrest at the G1/S phase

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transition and although it did not experience an induction of apoptosis, it did experience a

reactivation of glutathione-S-transferase- (GSTP-1) (Ivanov et al., 2007). Additionally,

data showed that lycopene treatment of the PC3 cell line experienced a significant increase

in methylation of the GSTP-1 promoter and subsequent GSTP-1 expression (Fu et al., 2014).

Data also showed a reduction of PC3 cells’ DNMT 3A protein expression (Fu et al., 2014).

-Cryptoxanthin

-Cryptoxanthin is a provitamin A carotenoid and falls into the xanthophyll

category. Common sources include red peppers and citrus fruits. The correlation of serum

-cryptoxanthin levels and lung cancer incidence was examined in a cohort study of

Chinese men, since they have a relatively high dietary intake of citrus. Data showed that

high serum -cryptoxanthin levels were associated with a significant reduction in lung

cancer risk (Yuan et al., 2003). The effects of -cryptoxanthin on the non-small cell lung

cancer cell lines A549 and BEAS-2B have also been examined in vitro. Data indicated -

cryptoxanthin exposure resulted in a significant reduction in the total number of cells due

to the induction of cell cycle arrest at the G1/S phase transition (Lian et al., 2006). Data also

indicated that -cryptoxanthin increased the expression of messenger RNA and protein

levels of the retinoic acid receptor (RAR), which correlated with an increase in the RAR-

mediated response element (RARE) (Lian et al., 2006). In vivo trials on F334 rats have

studied the effects of -cryptoxanthin on colon cancer incidence, and found that -

cryptoxanthin reduced the incidence of N-methylnitrosourea induced colon cancer

(Narisawa et al., 1999).

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Lutein

Lutein is considered a non-provitamin A carotenoid of the xanthophyll class and is

found in high concentrations in kale, spinach and egg yolks. Recent research has studied

the effects in vivo of lutein treatment during the promoting stage of carcinogenesis. Data

showed that in both mouse lung and skin cancer lutein, when administered during the

same period as the tumor promoter, significantly reduced the average number of tumors

shown by the mice (Nishino et al., 2002). Additionally, lutein was shown in vivo to inhibit

the formation of aberrant crypt foci in the rat colon (Nishino et al., 2002).

Discussion

Dietary Risk Factors of Carcinogenesis

It is no longer a secret that improper dietary habits are one of the biggest risk

factors of carcinogenesis, with an estimated 30-35 percent of cancers being linked to an

individual’s diet (Anand et al., 2008). This statistic is comparable to that of tobacco, which

is known to be littered with thousands of chemicals. Of these chemicals, at least 60 of them

are known carcinogens (DeOcampo, 2015). There are many aspects of the modern Western

diet that can be considered a risk factor of carcinogenesis. Two of the more common risk

factors include excesses of refined sugar and partially hydrogenated oils/trans fats.

The problem associated with excess refined sugar is multifold. One side of the

equation is that increased carbohydrate intake is typically seen as one of the primary

causes of obesity, a well-known risk factor of carcinogenesis (Anand et al., 2008).

Intuitively, such an overconsumption of refined sugars can commonly result in chronic

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elevated blood glucose levels, or hyperglycemia. Prolonged hyperglycemic conditions may

result in multiple modified intracellular signaling pathways. Signaling pathways relevant to

carcinogenesis that may be modified following high glucose exposure include those

involved with cell-to-cell adhesion molecule gene expression, inflammatory gene

expression, growth factor production and ROS production/oxidative stress (Popov, D.,

2010). Such alterations to signaling pathways induces phenotypic changes characterizing

malignancy.

Throughout recent years much attention has been brought regarding the health

risks associated with consuming trans fat. Trans fatty acids can be difficult to avoid even

today because they are widely used in the production of fried foods like chips and French

fries (partially hydrogenated vegetable oils used in frying), baked goods (many shortenings

use these oils), and margarines. Trans fatty acids simultaneously increases the “bad

cholesterol” known as low-density lipoprotein (LDL) and decrease the “good” cholesterol

known as high-density lipoprotein (HDL). Initially shown to have implications on

cardiovascular health by aiding in the development of heart disease, recent studies have

suggested that trans fat consumption is also positively correlated with an increase risk of

colon cancer (Slattery et al., 2001) and breast cancer (Breastcancer.org, 2015).

Nearly 90 percent of all cancers fall into the carcinoma category, or cancers that

arise from mutations in epithelial cells (DeOcampo, 2015). One such theory on the origin of

cancer that supports this statistic is the chronic inflammation/irritation theory. Trans fatty

acids have been shown to induce an inflammatory response in human aortic endothelial

cells that is mediated by NF-B activation (Bryk et al., 2011). During the inflammatory

response endothelial cells activate NADPH oxidase, which results in an increase in

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intracellular ROS production. These ROS then function as second messengers, activating a

multitude of protein kinases and transcription factors, including NF-B (Bryk et al., 2011).

Once activated, NF-B translocates into the nucleus, binding to the promoter regions of the

inflammatory intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion

molecule-1 (VCAM-1) genes, inducing an upregulation of the expression of these cellular

adhesion molecules on the cell surface as illustrated in Figure 4 (Bryk et al., 2011). This

upregulation of surface proteins ICAM-1 and VCAM-1 is important in the process of

carcinogenesis because CAMs bind to or are associated with the cytoskeleton, linking

molecules, second messenger systems, growth factor receptors, oncogenes products and

transcription products (DeOcampo, 2015).

-Carotene and -Carotene

The -carotene and -carotene molecules are important dietary sources of

antioxidants, due in part to their provitamin A metabolic activities. Adequate intake of

these and other carotenoids are important as they function to inhibit the carcinogenic

effects of certain dietary elements, such as excess sugar consumption/uptake and trans

fatty acids consumption. The N-myc protein has the ability to push cell growth and

proliferation, and it appeared that the maximum inhibition of growth and proliferation,

marked by the relative amount of cells in the G1/G0 phase of the cell cycle, occurred while

the expression of N-myc messenger RNA was maximally suppressed. Additionally, N-myc

messenger RNA recovery was associated with a decreased number of cells in the G1/G0

phase and an increase in the number of cells in the S phase indicating that cell cycle arrest

may be mediated through the repression of the N-myc gene (Murakoshi et al., 1989).

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The efficacy of -carotene as an anticarcinogenic agent has been in question,

especially when talking about high-risk individuals like smokers. One reason smokers may

experience adverse effects of -carotene supplementation is because many of their cells,

especially those of the lungs, have already experienced one or more hits (mutations) to

their DNA, due to the high carcinogenicity of tobacco and the high concentration of ROS in

cigarette smoke. Oxidation of certain biomolecules such as DNA, lipids or proteins can lead

to the development of multiple chronic diseases, including cancer (Rao and Rao, 2007).

With regards to lipids, -carotene reduces lipid peroxidation at low O2 pressure, however

at physiological O2 pressure -carotene causes a significant increase in lipid peroxidation,

which may have carcinogenic implications (Palozza et al., 2006). Also having important

carcinogenic implications was -carotene’s induction of CYP and associated enzymes with

the subsequent significant overproduction of ROS.

-Carotene seemed to exhibit a more potent antimetastatic effect than that of -

carotene. Proteins of the MMP family function in the breakdown of extracellular matrix,

and are believed to play a role in malignancy/metastasis (Liu et al., 2015). Alternatively,

TIMP proteins function by inhibiting these MMP proteins. Although both reduced the

expression of MMP-9 and MMP-2 while also increasing expression of TIMP-1, TIMP-2 and

PAI-1, only -carotene reduced the expression of uPA, which aids in the activation of

plasminogen and formation of active MMPs (Liu et al., 2015). Additionally, -carotene was

shown to markedly reduce the expression of integrin -1 (and associated downstream

kinases), which has important carcinogenic implications because integrins serve as the

primary cell surface receptor for extracellular matrix proteins, such as MMPs, (DeOcampo,

2015).

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Lycopene

Lycopene has been shown to be the most potent in vitro antioxidant of all the

studied carotenoid molecules. Many of lycopene/lycopene metabolites’ protective effects

are thought to be mediated through its antioxidant activity, which is the result of the

upregulation of Nrf-2. Nrf-2 has important implications in carcinogenesis because it

mediates the activation of the antioxidant response element when dissociation from the

inhibitory Keap-1 protein and translocation to the nucleus occurs (Lian and Wang, 2008).

This antioxidant response element increases the expression of a multitude of antioxidant

enzymes, which can control ROS overproduction.

Multiple growth factor signaling pathways associated with carcinogenic activity

have also been shown to be inhibited by lycopene exposure. IGF-1 signaling, important in

proliferation and apoptosis, was reduced by lycopene’s ability to increase levels of

circulating IGF binding proteins 1 and 2 (Solis et al., 2013). Lycopene also decreases levels

of circulating VEGF, which upon binding to its receptor can induce proliferation, migration,

and angiogenesis among other things (Solis et al., 2013). A third growth factor that is

inhibited by lycopene is PDGF. The PDGF is activated after undergoing dimerization and

becoming a hetero- or homodimer, and it is this process that lycopene inhibits thus

inhibiting activation and downstream signaling (Solis et al., 2013).

In the body lycopene is preferentially sequestered in certain tissues, such as breast

tissue and tissue of the prostate gland, which may prove to be important in future

treatment options. Lycopene seems to have different effects on the androgen sensitive and

androgen-independent prostate cancer cell lines, LNCaP and PC3 cell lines, respectively.

Lycopene was able to induce cell cycle arrest in both cell lines, via downregulation of the

22

IGF-1 receptor and consequent signaling pathways (Ivanov et al., 2007). The tumor

suppressor retinoblastoma protein (Rb) therefore remains bound to E2F, avoiding

phosphorylation and cell cycle progression. The tumor suppressor p53 is upregulated as

well as the cyclin dependent kinase (Cdk) inhibitors p21 and p27, further suppressing

proliferation (Ivanov et al., 2007). Apoptosis was induced only in the androgen-sensitive

LNCaP cell line and was mediated by the upregulation of pro-apoptotic proteins such as

Bax and a downregulation of anti-apoptotic proteins such as Bcl-2, BclXL and survivin,

which suggests the androgen receptor’s part in activating the genes that code these

proteins (Ivanov et al., 2007).

Lycopene’s inhibition of metastasis occurs in a dose dependent manner, seen in

Figure 4, by decreasing the expression of MMP-2/9, similarly to -carotene, as well MMP-7

(Solis et al., 2013). Lycopene and its metabolites also induce simultaneous upregulation of

TIMP-1/2, so this combined effect drastically reduces the cell’s ability to degrade the

surrounding extra-cellular matrix. Another antimetastatic property of lycopene was its

upregulation of the nm23-H1 gene. Increased expression of the nm23-H1 gene is

associated with a decrease in metastasis in liver, colon, breast, melanoma and gastric

carcinomas (Tee et al., 2006). This differs from thyroid carcinomas, neuroblastomas and

osteosarcomas, where an increase in nm23-H1 is actually associated with a more

aggressive/malignant phenotype, which may suggest tissue specific roles of nm23-H1 (Tee

et al., 2006). Lycopene treatment was also shown to decrease -catenin expression, an

important cellular adhesion and signal transduction molecule utilized during

carcinogenesis (Solis et al., 2013). Loss of cell-to-cell communication is believed to be a

factor in metastasis, so lycopene’s ability to increase cellular gap junction communication

23

via upregulation of connexin-43 (an important gap junction component) messenger RNA

may play a role in its antimetastatic properties (Solis et al., 2013).

-Cryptoxanthin

Citrus fruits are some of the best dietary sources of -cryptoxanthin, which is the

reason cohort study decided to use Chinese men near Shanghai (higher than average citrus

fruit consumption). Additionally, -cryptoxanthin supplemented rats showed a significant

reduction in the incidence of colon cancer. These preventative effects of -cryptoxanthin

may be mediated through its ability to induce cell cycle arrest by suppressing the

expression of cyclin D and cyclin E, which are necessary in cell cycle progression, and by

increasing the expression of the cyclin dependent kinase inhibitor p21 (Lian et al., 2006).

The increase in RAR messenger RNA and protein expression is significant because

retinoids, such as retinoic acid, are the biologically active form of vitamin A and therefore

increasing receptor expression should increase the efficacy of not only vitamin A, but of the

provitamin A molecules as well.

Lutein

In vivo studies on mice supplemented with lutein during the tumor-promoting

phase of the experiment suggest that its anticarcinogenic effects may occur during tumor

promotion, as evidenced by the reduction in the incidence of skin and lung tumors in the

lutein supplemented group. Lutein is preferentially sequestered in the human eye,

particularly in the macula, where it functions to absorb the retinal-damaging blue light.

24

Lutein is also the most abundant carotenoid in human brain tissue, where it is believed to

have antioxidant, anti-inflammatory and structural activities (Johnson, 2014).

Conclusions/Comments

This semester has given me a glimpse into just how complex the multistep process

of carcinogenesis really is. There are so many different mechanisms that interact that it can

be intimidating at times. This class was unique and I really enjoyed how it was structured

around in class lectures and individual research. The majority of classes I have taken over

the past five years have been primarily lectures with exams on memorized information, so

it was refreshing to have to research and integrate information on your own. It was also

interesting to see what topics students chose to present on, and the overall quality of the

majority of presentations was quite impressive.

The vast majority of cancers are attributed to lifestyle choices such as your diet and

smoking, which is why I was interested in our diet’s role in carcinogenesis. There are

numerous dietary factors that aid in cancer development, but two of particular concern in a

typical Western diet are excess refined sugars and the consumption of trans fatty acids. As

a country with millions of soda drinkers and fast food frequenters, it is important to know

the risks associated with such habits. Luckily, there are also some dietary choices you can

make that can inhibit the process/progression of carcinogenesis such as increasing your

intake of fruits and vegetables, which are rich in nutrients like antioxidants and

carotenoids.

With the exception of the aphid, animals do not possess the ability to produce

carotenoids endogenously, so we must make sure we have an adequate intake of fruits and

25

vegetables. Some carotenoids have proven to be potent in vitro and in vivo inhibitors of

several mechanisms and pathways associated with cancer development and progression.

These include mechanisms such as cell cycle arrest/inhibition of proliferation, inhibition of

various growth factor activity, induction of apoptosis, increased cell-to-cell communication

and the inhibition of metastasis. These effects seem to be intensified when working in

combination, so make sure to eat a variety of fruits and vegetables to capitalize on their

anticarcinogenic effects.

26

Figures

Figure 1: Simplified flowchart detailing the steps in disease progression after ROS

production and lack of repair. (Rao and Rao, 2007).

Figure 2: A summary of the antimetastatic effects of -carotene treated LLC cells in vitro.

(Liu et al., 2015).

27

Figure 3: Lycopene’s effects on cell invasion and migration occurs in a dose dependent

manner. (Huang et al., 2005).

28

Figure 4: The effects of trans fatty acid exposure on ICAM-1 and VCAM-1 expression in

endothelial cells. Graphs A and B are surface protein expression and graphs C and D are

messenger RNA expression. (Bryk et al., 2011).

29

Figure 5: Summary of the multiple growth factor signaling pathways effected by lycopene.

(Solis et al., 2013).

30

Table 1: Lycopene treatment on lung and liver carcinogenesis in mice, in vivo. (Nishino et

al., 2002).

31

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