SEMINAR REPORT

On

THE ROLE OF FRUCTOSE IN PANCREATIC CANCER CELLS

Submitted by

ADJEKUKOR, UFUOMA CYNTHIA

13CP015803

BIOCHEMISTRY PROGRAMME

To the

DEPARTMENT OF BIOLOGICAL SCIENCES

COLLEGE OF SCIENCE AND TECHNOLOGY

COVENANT UNIVERSITY

IN FULFILLMENT OF THE REQUIREMENT FOR THE SEMINAR COURSE

(BCH 418)

Supervised by

DR T.M. DOKUNMU

Signature

SEPTEMBER 2016

i

CERTIFICATION

This is to certify that Adjekukor, Ufuoma Cynthia with matriculation number 13CP015803, of the

Department of Biological Sciences, College of Science and Technology, Covenant University did this

seminar report titled ‘The Role of Fructose in Pancreatic Cancer Cells’, under the supervision of Dr

T.M. Dokunmu.

DR T.M. DOKUNMU DR T.M. DOKUNMU

(SUPERVISOR) (SEMINAR COORDINATOR)

DR E.O. OMOTOSHO PROF A.A. AJAYI

(PROGRAMME COORDINATOR) (HOD, BIOLOGICAL SCIENCE)

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DEDICATION

I dedicate this work to God Almighty, my parents, Mr & Mrs I.O. Adjekukor, my brothers: Davis,

Christian and Cyril for their unending love and support throughout the preparation of this seminar report.

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ACKNOWLEDGEMENTS

I acknowledge God Almighty, my parents Mr and Mrs Adjekukor, my siblings, and my ever-able

supervisor Dr T.M. Dokunmu for her time and commitment. I also acknowledge the head of the

department Prof A.A. AJAYI, biochemistry programme coordinator Dr E.O. Omotosho and biochemistry

seminar coordinator Dr T.M. Dokunmu.

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CONTENTS

TITLE PAGE………………………………………………………………….........................i

CERTIFICATION………………………………………………............................................ii

DEDICATION……………………………………………………………………………....iii

ACKNOWLEDGEMENT……………………………………………...................................iv

CONTENTS……………………………………………….....................................................v

LIST OF TABLES AND FIGURES………………………………………………...……..viii

SUMMARY……………………………………………….....................................................ix

CHAPTER ONE

1.0 Introduction.………………………………………………. ……………………….…...01

1.1 Background……………………………………………………………………………...01

1.2 Prevalence and Morbidity………………………………………………………….……02

CHAPTER TWO

2.0 Overview of Pancreatic Cancer……………………………………………………..…..04

2.1 The Pancreas…………………………………………………………………...………..04

2.1.1 Exocrine gland……………………………………………………………..……04

2.1.2 Endocrine gland…………………………………………………………....……04

2.2 Types of Pancreatic Cancer…………………………….………………………..……...04

2.2.1 Pre-cancerous growth in the pancreas………………....……..….……………...04

2.2.2 Pancreatic Exocrine Tumours……………………......…………………....……05

2.2.3 Pancreatic Endocrine Tumours…………………………………………............05

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CHAPTER THREE

3.0 Fructose Metabolism and Pancreatic Cancer……………………………………...…….09

3.1 Chemical Structure……………………………………………….……………………..09

3.2 Metabolism…………………………………………………………………………...…09

3.3 Glycolytic Pathway and Pancreatic Cancer…..................................................................13

3.4 The Non-oxidative Pentose Phosphate Pathway…………...…………………………...15

3.5 Uric Acid Production in Pancreatic Cancer……………………………………...……...15

3.6 Effect of Fructose in other cancer cells………………………………………….….......19

3.6.1 Liver Cancer………………………………………………………..………..….19

3.6.2 Breast Cancer……………………………………………………………....…...19

3.6.3 Colon Cancer…………………………………………………………..……..…19

3.6.4 Cancer of the Small Intestine…………………………………….……………..19

CHAPTER FOUR

4.0 Fructose Mechanism in Pancreatic Cancer Cells……………………………….…….....20

4.1 Cancer Mechanisms…………………………………………………………………......20

4.2 Insulin Relation to Pancreatic Cancer Cells………………………………………….....21

4.3 Oxidative Stress, Insulin Resistance and Inflammation in pancreatic cancer………..…21

4.4 Pancreatic Cancer and Diabetes Mellitus….....................................................................26

4.5 Pancreatic Cancer and Obesity……………………………………...……………..……26

4.6 Body Mass Index and Physical Activity in Relation to Pancreatic Cancer…………......27

4.7 Recent Advances in the Role of Fructose Metabolism and Mechanisms in Pancreatic

Cancer Cells………………………………………………...…………………………29

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CHAPTER FIVE

5.0 Dietary Factors in Relation to Pancreatic Cancer………………………………...……..31

5.1 High Fructose Corn Syrup (HFCS) in Relation to Pancreatic Cancer……….………….31

5.2 Fruit and Fruit Juices in Relation to Pancreatic Cancer………………………………...32

5.3 Phytochemicals and Antioxidants in Fruits in Relation to Pancreatic Cancer………….32

5.4 Glucose in Relation to Pancreatic Cancer………………………………………………34

5.5 Other Dietary Factors and Pancreatic Cancer…………………………………………...34

5.5.1 Vegetables………………………………………………………………….…...37

5.5.2 Dietary Meat and Fat…………………………………………..………………..37

5.6 Conclusion……………………………………………………………………………....37

REFERENCES………………………………………………………………………….......39

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LIST OF TABLES AND FIGURES

TABLE

Table 2.1 Differences between benign and malignant tumours…………………………….07

FIGURES

Fig 2.1 Parts of a human Pancreas…………………………………………………………..08

Fig 3.1 Different chemical structures of glucose and fructose……………………………...10

Fig 3.2 Glucose and fructose metabolic pathways in the liver……………………………...11

Fig 3.3 Fructose metabolism in the liver……………………………………………………12

Fig 3.4 Differences in the metabolic pathways of glucose [A] and fructose [B] in pancreatic

cancer cells………………………………………………………………………......16

Fig 3.5 Fructose breakdown in pancreatic cells………………………………………..……17

Fig 3.6 Fructose metabolism to uric acid……………............................................................18

Fig 4.1 The mechanism showing how fructose promotes carcinogenesis and cancer

growth…………………………………………………………………………..…...22

Fig 4.2 The mechanism showing how fructose supports the up regulation of fatty acid

synthesis and the triglyceride synthesis……………………………………...……...24

Fig 4.3 Fructose causes Hepatic Insulin Resistance from the synthesis of triglycerides…...25

Fig 4.4 Potential mechanisms for fructose induced insulin resistance………………….…..28

Fig 5.1 Effect of natural antioxidants on pancreatic cancer………………………………...35

Fig 5.2 Metabolic pathways that are altered by the oncogene Kras………………………...36

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SUMMARY

Pancreatic cancer occurs when abnormal cells grow out of control in the pancreas, it is the fourth

leading cause of cancer deaths in the United States. Over the past years, the consumption of

fructose especially in its principal form, High-Fructose Corn Syrup has drastically increased along

as the same time as nutrition-linked chronic diseases. Fructose has been linked to carcinogenesis

and cancer growth of the pancreas. This occurs by the up-regulation of de novo lipogenesis,

reactive oxygen species generation, hepatic insulin resistance, chronic inflammation and cellular

oxidative stress, which can lead to the promotion of deoxy ribonucleic acid damage. Due to the

differences in chemical structure of fructose and glucose, they both exhibit distinct metabolic

properties. Fructose is preferentially used to glucose in the non-oxidative pentose phosphate

pathway that produces ¿85% of ribose for deoxy ribonucleic acid synthesis in cancer cells.

Fructose has similar effects in proliferating human breast cancer, liver cancer cells and other

cancers. Michaud and colleagues reported that fructose may also increase pancreatic cancer risk

in obese or overweight individuals, with a high body mass index, low physical activity and in

susceptible individuals. Although fruits contain high levels of fructose, it is believed that fruits

possess natural antioxidants and phytochemicals, which are thought to inhibit the deleterious

effects of fructose in carcinogenesis. The biochemical mechanisms and the roles of high

consumption of refined fructose in the progression of pancreatic cancers are discussed.

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CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND

The word ‘cancer’ is used to describe any disease in which the cells are abnormal, grow out of control,

and can spread. Thus, pancreatic cancer occurs when abnormal cells grow out of control in the tissue of

the pancreas and form tumour. Tumours are mass of tissues formed from the build up of extra cells.

Tumours may be benign or malignant and just as other living cells they possess the potential for

proliferation, differentiation, cell cycle arrest and apoptosis. Due to this, macromolecule synthesis

pathways directly determine the survival of these cells by providing energy and substrates necessary for

cells to function under different pathophysiologic condition (Boros et al., 2002).

Carbohydrate metabolism through glycolysis and tricarboxylic acid (TCA) cycle is very important for

cancer growth and increased consumption of refined carbohydrate promotes cancer survival. Fructose

stimulates the up regulation of De novo Lipogenesis (DNL), which contributes to cancer risk by

increasing oxidant stress and com- promising cellular antioxidant defense mechanisms (Port et al., 2012).

It does this by increasing depleting NADPH, which is not only required in large amounts for fatty acid

synthesis but is also a major cofactor in maintaining the reduced state level of glutathione and

thioreductase (Port et al., 2012). This is associated with many negative health consequences,

including development of insulin resistance, non alcoholic fatty liver, diabetes, hypertension and kidney

disease, which could increase cancer risk by elevating levels of insulin, glucose, inflammation

and oxidant stress (Port et al., 2012).

Recently, fructose intake contributed directly to oxidative stress in hamster islet tumour cells. It plays a

vital role in the risk of pancreatic cancer and also may act as a marker of high-sugar diet, studies have

shown that pancreatic cancer was inhibited by the drug metformin, which reduces insulin resistance in a

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hamster pancreatic adenocarcinoma model (Michaud et al., 2002). Also, the studies demonstrated that the

pancreatic ductal cancer either arises from islet cells or from some common progenitor cells that could

give rise to both islet and duct cells (Pour, 1978). Peripheral insulin resistance is associated hyperactivity

and proliferation of islet cells because of this; fructose may be involved in promoting pancreatic cancer

(Michaud et al., 2002).

1.2 PREVALENCE AND MORBIDITY

Pancreatic cancer is the fourth leading cause of cancer related death in the United States (Siegel et al.,

2013). In 2012, it was the twelfth most common cancer worldwide (World Cancer Research Fund

International, 2016). In Japan it is the fifth most common cause of cancer death, preceded in males by

lung, stomach, liver and large bowel, and in females by stomach, large bowel, lung and breast (Lowenfels

and Maisonneuve, 2004). Also, Pancreatic cancer is the 9th most common cancer in Western Europe

and 19th in Middle Africa (World Cancer Research Fund International, 2016).

By 2030, it is expected to be the second leading cause of cancer death. Only 5% of individuals who

develops pancreatic adenocarcinoma survive five years after diagnosis, and most patients live for less

than 12 months (Wolpin et al., 2013). Fructose consumption has increased over the past years and this has

been linked to various diseases including obesity, diabetes, cardiovascular diseases and cancer. The third

National Health and Nutrition Examination Survey reported that over 10% of Americanapolis daily

calories come from fructose of which the largest part comes from sugar sweetened beverages (30%),

followed by grains (22%) and fruit or fruit juices (19%) (Vos et al., 2008). The principal form of this

fructose is the High Fructose Corn Syrup (HFCS, 10-53% glucose and 42-90% fructose) (Tappy et al.,

2010), which has been implicated in pancreatic tumour progression.

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This review elicits the roles of fructose in pancreatic cancers and other cancers, giving a biochemical

understanding, the implications and ways of managing pancreatic cancers in a developing world.

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CHAPTER TWO

OVERVIEW OF PANCREATIC CANCER

2.1 THE PANCREAS

The pancreas is an important digestive organ that is about 6 inches long, located deep in the abdomen

between the stomach and the spine (back bone). The liver, intestine, and other organs surround the

pancreas (Figure 2.1). The pancreas is made up of three parts: the head (is closest to the small intestine),

the body (middle section) and tail (the thinnest part).

2.1.1 Exocrine gland makes pancreatic juices, which contains enzymes that help in digestion of food and

releases them into the intestine.

2.1.2 Endocrine gland also known as islet of Langerhans make important hormones, such as insulin and

glucagon and release them directly into the bloodstream.

2.2 TYPES OF PANCREATIC GROWTH

2.2.1 Pre-cancerous growth in the pancreas

Serous cystic neoplasm (also known as Serous Cystadonomas): they are usually benign tumours that have

sacs (cyst) filled with watery fluid.

Mucinous cystic neoplasm (also known as Mucinous Cystadonomas): they are slow growing tumours in

the body or tail of the pancreas that have cyst filled with a jelly-like substance called mucin.

Intraductal papillary mucinous neoplasm: are benign tumours, which make mucin that grow in pancreatic

ducts

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2.2.2 Pancreatic Exocrine Tumours

Exocrine cancer is the most common type of pancreatic cancer. More than nine out of ten people (95%)

have this type. They include:

Pancreatic adenocarcinoma: 95% of pancreatic cancer type is the pancreatic adenocarcinoma. They

usually begin in the ducts (gland cells) of the pancreas but sometimes they develop from the cells that

make the pancreatic enzymes in which case they are called acinar cell carcinomas.

Solid pseudopapillary neoplasm: these are rare, slow growing tumours that almost always occur in young

women.

Less common type of cancer: adenosqua carcinomas, squamous cell carcinomas, signet cell carcinomas,

different carcinomas and undifferentiated with giant cells.

Ampullary cancer (carcinoma of the ampulla of vater): this cancer starts at the ampulla of vater which is

where the bile ducts and pancreatic duct come together and empty into the small intestine.

2.2.3 Pancreatic Endocrine Tumours

They include:

a) Functioning tumours: they make hormones that are released into the blood and cause symptoms

Gastrinomas: about half of gastrinomas are cancers. It comes from cells that make gastrin.

Insulinomas: most insulinomas are benign. It comes from cells that make insulin.

Glucagonomas: most glucagonomas are cancers. It comes from cells that make glucagon.

Somatostatinomas: most somatostatinomas are cancerous. It comes from cells that make somatostatin.

VIPomas: most VIPomas are cancerous. It comes from cells that make Vasoactive Intestinal Peptide

(VIP).

PPomas: most PPomas are cancerous. It comes from cells that make Pancreatic Polypeptide (PP).

5

b) Non functioning tumours: they are more likely to be cancerous than functioning tumours because these

tumours does not make enough excess hormones to cause symptoms, they can grow quite large before

they are found.

c) Carcinoid tumours: they rarely start in the pancreas, although they are much common in other parts of

digestive system. This tumour often makes serotonin (also called 5-HT) or its precursor 5-HTP.

Note: metastasis is when cancer cells often travel to other parts of the body, where they begin to grow and

form new tumours that replace normal tissue. It happens when cancer cells get into the lymph nodes or

bloodstream of the body. Tumours can be either malignant or benign. Table 2.1 shows a comparison

between malignant and benign tumours.

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Figure 2.1 Parts of a human pancreas (National Cancer Institute, 2010).

7

Table 2.1. Differences between benign and malignant tumours

Benign Tumours (e.g. cyst) Malignant Tumours

They are usually not a threat to life. They may be a threat to life.

They can be removed and usually

don’t grow back.

They are removed sometimes, but can

grow back.

They don’t invade the tissues around

them.

They can invade and damage nearby

tissues and organ.

They don’t spread to other parts of

the body.

They can spread to other parts of the

body.

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CHAPTER THREE

FRUCTOSE METABOLISM AND PANCREATIC CANCER

3.1 CHEMICAL STRUCTURE

Although glucose and fructose have identical chemical formulas (C6H1206) (figure 3.1), the difference in

their chemical structure result in completely distinct absorptive and metabolic properties, which have

fundamental implications for cellular functions and disease (Varman, 2011).

3.2 METABOLISM

The disaccharide (i.e. sucrose) components of the food we consume are cleaved in the gut into smaller

glucose and fructose units. Fructose is absorbed in the small intestine by the fructose specific transporter,

glucose transporter 5 (GLUT5) while glucose is absorbed through the gut by sodium-dependent glucose

transporter (Bray, 2013). The liver is the major site of metabolism, which removes up to 70% of the portal

fructose, leaving the 30% for metabolism by other tissues (kidney, musculoskeletal, testes, fat and brain).

In contrast, the glucose is transported to hepatocytes and most other cell types using the glucose specific

insulin-dependent transporter, GLUT4 (figure 3.2). Once in the hepatocytes, some of the glucose is

absorbed while others goes into glycolysis and other metabolic pathways. During elevated concentration

of glucose the pancreatic hormone, insulin, is released from the beta cells to regulate the glucose levels in

the bloodstream. It should be noted that GLUT5 does not respond to insulin, thus leaving fructose uptake

uninhibited. Fructose is independent of insulin and uses GLUT5 transporter, while glucose is regulated by

insulin and uses the GLUT4 transporter (Charrez et al., 2015).

Figure 3.3 shows that a small amount of fructose goes into gluconeogenesis for the immediate production

of glucose. The green pathway is related to the phosphorylation of fructose by fructokinase and the by-

product of this is uric acid. Down to the left is an aldehyde pathway, which is the concept, used for the

production of alcohol. The yellow pathway leads to the production of Insulin receptor substrate-1 (IRS-1),

9

Figure 3.1 Different chemical structures of glucose and fructose (A) The hemiacetal group of glucose is

substituted to a hemiketal group for fructose. (B) Representation of the open ring structure of glucose and

fructose (Charrez et al., 2015).

10

Figure 3.2 Glucose and fructose metabolic pathways in the liver (Charrez et al., 2015).

11

Figure 3.3 Fructose metabolism in the liver (http://www.nofructose.com/introduction/metabolism/

Sourced on 08/05/2016).

12

which is important in the chemical feedback of insulin and the pancreas. The blue pathway effectively

shows the conversion of fructose into Very Low Density Lipoproteins (VLDL’s).

3.3 GLYCOLYTIC PATHWAY AND PANCREATIC CANCER

Glycolysis makes available substrates needed for the nonoxidative pentose phosphate pathway and other

metabolic pathway in the body. It should be noted that the pyruvate kinase isoform, PKM2, that catalyse

the reaction of phosphoenol pyruvate to pyruvate is only expressed in tumour cells. Due to its low

enzymatic activity it slows down pyruvate formation thus leading to the accumulation of upstream

intermediates, thereby increasing substrate available for the non-oxidative pentose phosphate pathway.

Some biotechnologist recognised that as glucose utilization increases, so does lactate production from

anaerobic glycolysis, which limits growth as pH becomes increasingly acidic (Port et al., 2012). This is

the same challenge cancer cells face. Substituting fructose into culture media limits growth, but

simultaneously decreases lactate levels and into culture media limits growth, but simultaneously

decreases lactate levels and increases protein yields, suggesting that once inside cells, fructose

derived carbons are directed away from glycolysis and into pathways that generate metabolites used for

protein synthesis (Port et al., 2012).

3.4 THE NON-OXIDATIVE PENTOSE PHOSPHATE PATHWAY AND PANCREATIC

CANCER

The non-oxidative pentose phosphate pathway (PPP) which involves the conversion of a six carbon

glucose to a five carbon ribose for deoxy ribo nucleic acid (DNA) and ribo nucleic acid (RNA) synthesis

is of utmost importance for the proliferation process and produces >85% of the ribose recovered from

tumour nucleic acids. The non-oxidative PPP is controlled by transketolase (TK) enzyme reactions which

is encoded by three human TK genes, namely: TKT, TKTL1 and TKTL2. Clinically, there is a tendency

13

for patients with cancer burden to develop thiamine depletion, which is a cofactor for TK-mediated

reaction, thus emphasizing the importance of the non-oxidative Pentose Phosphate Pathway for tumour

growth (figure 3.4). There is a preferential use of fructose in the TK-dependent nonoxidative pentose

phosphate shunt, while glucose is used mainly for glycolysis, TCA cycle, glycogen synthesis, lipid

synthesis, amino acid synthesis (nonessential). Thus, leaving a small percentage of glucose for the

pentose phosphate shunt. (Liu et al., 2010).

From the study and data analysis made by Liu et al. (2010) the contribution of fructose to nucleic acid

synthesis is considerably greater than glucose. They studied the effect of protein synthesis in pancreatic

cancer cell lines (Panc-1, MiaPaCa-2, HPAF, CaPan-1) and immortalized pancreatic ductal cells

(HPDE6; Port et al., 2012), and later discovered that fructose is preferentially used in cancer calls through

transketolase (TKT)-mediated metabolism to synthesize additional nucleic acid to facilitate proliferating

capacity. In their study the transketolase-like protein 1 (TKTL-1) was overexpressed in pancreatic cancer

when compared to normal tissue, but the TKT mRNA was the predominant expressed TK gene in

pancreatic cancer cells. Figure 3.5 shows the breakdown of fructose, and that fructose treatment in

pancreatic cell lines preferentially increased nucleotide synthesis. It does this by up regulating expression

of transketolase (TKT) leading to increased flux in pentose phosphate shunt (Port et al., 2012).

Also, Fructose-1-6-phosphate allosterically activates PKM2 through inducing the tetramer formation of

PKM2 (Xu et al., 2016). This lead to the accumulation of Fructose-6-phosphate during TKT knockdown,

which later resulted in the activation of PKM2, leading to the decrease of aerobic glycolysis and a

reduction of lactate accumulation (Xu et al., 2016) (Figures 3.4 and 3.5).

In contrast, Xu et al. (2009) reported that the strong proliferation of tumour cells is governed by the

replication of DNA in the S phase where the conversion of glucose to ribose is controlled by non-

14

oxidative PPP. They discovered by flow cytometry that TKLT-1 showed slower growth rates when cell

cycle was arrested in G0/G1 phase.

3.5 URIC ACID PRODUCTION IN PANCREATIC CANCER

Excess fructose is hazardous for the body because it causes hyperuricaemia due to the accumulation of

adenosine diphosphate (ADP) and adenosine monophosphate (AMP). Dietary fructose when absorbed by

the body is the only sugar that raises the uric acid concentration in the blood (Nakagawa et al., 2006).

Ketohexokinase (KHK) is insulin and citrate independent which permits the phosphorylation of fructose

to Fructose-1-phosphate in the liver and this reaction occurs rapidly without any feedback inhibition (Vos

et al., 2013). The phosphate molecule required for this reaction comes from the depletion of adenosine

triphosphate (ATP) (Figure 3.6).

ATP is the molecule involved in energy transfer within cells, the phosphate group within it, is depleted

following the metabolism of Fructose by Hexoketokinase. The decrease in intracellular phosphate

stimulates adenosine monophosphate deaminase (AMP D) which catalyses the degradation of adenosine

monophosphate (AMP) to inosine monophosphate and eventually uric acid (Johnson et al., 2013).

Fructose promotes the production of uric acid, which is a by-product of nucleotide (purine) metabolism

through the non-oxidative PPP (Charrez et al., 2015). Uricase activity (which correlate closely with uric

acid production) that was measured in pancreatic cancer cells, they found out that fructose-treated cells

resulted in 20%, and 50% higher uricase activity in the cancer cells than glucose-treated cells. This study

shows that fructose is preferentially utilized for nucleic acid synthesis (Liu et al., 2011).

15

Figure 3.4. Differences in the metabolic pathways of glucose [A] and fructose [B] in pancreatic cancer

cells (Liu et al., 2010).

16

Figure 3.5. Fructose breakdown in pancreatic cells (Port et al., 2012).

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Figure 3.6 Fructose metabolism to uric acid. (http://www.nofructose.com/introduction/metabolism/

Sourced on 08/05/2016).

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3.6 EFFECTS OF FRUCTOSE IN OTHER CANCER CELLS

3.6.1 Liver Cancer

A study reported that liver tumour was found to be two fold greater with increased metastasis, and

reduced survival in rats treated with N-nitrosormorpholine for 7 weeks and subsequent fructose

frequently. Moreover, the authors also observed that the fructose increased the number of preneoplastic

lesions by 26% (Charrez et al., 2015). TkT enzyme in the non-oxidative pentose phosphate pathway was

found to be the most predominant enzyme in hepatocellular carcinoma (HCC). This enzyme was over

expressed, which lead to venous invasion, tumor microsatellite formation, tumor size, and absence

of tumor encapsulation. (Xu et al., 2016).

3.6.2 Breast Cancer

The GLUT 1 and GLUT 2 were found to be present in both normal and neo-plastic breast cancer

(Zamora-Léon et al., 1996). But GLUT 5, which is the fructose transporter, was found to be over

expressed human breast cancer and was absent from normal breast tissue (Zamora-Léon et al., 1996).

GLUT 5 transports fructose with high affinity while GLUT 2 does not. This shows that fructose may have

an effect in proliferating human breast cancer cells.

3.6.3 Colon Cancer

In a study, males with high intakes of dietary glycaemic load: fructose and sucrose resulted in a

significant 27% - 37% increase in colorectal cancer. However, for women, these factors did not lead to an

increase in colorectal cancer (Michaud et al., 2005).

3.6.4 Cancer of the Small Intestine

Banzzan et al. (2013) reported that fructose intake that is associated with obesity has been shown to be

used preferentially in the proliferation of cancer cells of the small intestine.

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CHAPTER FOUR

FRUCTOSE MECHANISM IN PANCREATIC CANCER CELLS

4.1 CANCER MECHANISMS

Cancer is not a single disease with a single aetiology, but is rather a general disease category that includes

many distinct diseases with distinct aetiologies (Terry et al., 2001). Carcinogenesis is a multistep process

and oxidative damage has been linked to the formation of tumours. Free radicals due to oxidative stress

can cause DNA damage, which, when left unrepaired, can lead to base mutation, single and double strand

breaks, DNA cross-linking, and chromosomal breakage and rearrangement (Liu, 2003). These mutations

caused on the DNA by free radicals are crucial for the initiation of the carcinogenesis process (Zhang et

al., 2015) (Figure 4.1).

Catabolic and anabolic metabolism has a role in furnishing biochemical energy and building blocks for

macromolecular synthesis and repair, these metabolic pathways are vital to cancer cell growth and

survival in different tumour microenvironments (Hockenbery et al., 2013). In recent studies, metabolic

pathway can also direct chromatin structure and gene expression, differentiation and steaminess, which is

also associated with increased histone methylation and altered gene expression (Hockenbery et al., 2013).

In a recent study, after the incubation of Escherichia coli plasmid PBR322 for 15 days with fructose and

glucose phosphates metabolites, it resulted in variety of DNA modifications and damage (Liu and Heaney

2011). The intensity of the damage done is in the following order: glucose-1-phosphate < glucose <

glucose-6-phosphate < fructose-1-phosphate < fructose < fructose-6-phosphate (Liu and Heaney 2011).

This result gotten supports the hypothesis that fructose intake may cause cancer growth and also help in

proliferation of cancer cells.

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4.2 INSULIN IN RELATION TO PANCREATIC CANCER

Insulin is produced by the β cells of the pancreas in response to elevated glucose levels. In humans with

normal glucose metabolism, 85% to 94% of the variability of postprandial glucose and insulin responses

can be explained by both the source and the amount of carbohydrate intake (Wolever and Bolognesi,

1996). In addition, Insulin can promote tumor development by inhibiting apoptosis and stimulating cell

proliferation (Nöthlings et al., 2007). It has been observed that insulin acts as a promoter for pancreatic

carcinogenesis (Michaud et al., 2005). It should also be noted that growth factor like insulin is used for

mucosal cells in vitro growth by up regulating systematic insulin-like growth factor (IGF)-I activity (Jiao

et al., 2009). High serum levels of IGF-I and IGFBP-3 were associated with an increased risk of death

from pancreatic cancer (Jiao et al., 2009).

4.3 OXIDATIVE STRESS, INSULIN RESISTANCE AND INFLAMMATION IN PANCREATIC

CANCER

Oxidative stress, insulin resistance and inflammation have been linked to the cause of various chronic

diseases, like: cancer and cardiovascular diseases. Fructose leads to an elevated production of uric acid,

this uric acid enters into the cells through the specific organic anion transporter, which induces an

oxidative stress that has been shown in vascular smooth muscle cells, endothelial cells, adipocytes, islet

cells, renal tabular cells and hepatocytes (Johnson et al., 2013). This oxidative stress can cause oxidative

damage to large biomolecules such as proteins, DNA, lipids, resulting in increased risk of cancer and

cardiovascular diseases (Liu, 2013).

In addition, rat treated with fructose led to hyper insulinaemia, insulin resistance, impaired glucose

tolerance, increased thymidine incorporation and decreased endothelium Nitric Oxide Synthase (eNOS)

activity (Miatello et al., 2001). Uric acid is a potent inhibitor of Nitric Oxide synthase, which later affect

21

Figure 4.1 the mechanism showing how refined fructose promotes carcinogenesis and cancer growth (Liu

and Heaney, 2011).

22

the Nitric Oxide activity, and thus leads to oxidative stress within various cells especially Adipocytes (fat

cells). Also, uric acid-induced oxidative stress is mediated by the stimulation of NADPH Oxidase, which

translocate in the liver and generates oxidized lipids and inflammatory mediators such as Monocyte

Chemoattractant Protein-1 (MCP-1). This mitochondrial oxidative stress has a role in driving insulin

resistance (Lanaspa et al., 2012).

Due to the oxidative stress caused by uric acid in the mitochondria, there is a decrease in the activity of

aconitase (ACO2) in the kerb cycle. This leads to the accumulation of aconitase substrate and citrate,

which are released to the cytosol, where they act as substrate for triglyceride (TG) synthesis through the

activation of ATP Citrate Lyase (ACL). Large amount of fructose expressed in the liver leads to

lipogenesis, triglyceride accumulation, reduced insulin sensitivity and hepatic insulin resistance/glucose

intolerance. It supplies substrates like: glycerol-3-phosphate and Acyl-CoA for De novo Lipogenesis that

can later lead to hepatic insulin resistance (Figure 4.2 and 4.3).

It should also be noted that inflammation is associated with high level of oxidative stress that can damage

most of the body tissues and the genetic material (DNA), which lead ultimately to cancer formation

(Banzzan et al., 2013).

In a large, nested case control study, the mutual adjustment of peripheral insulin resistance marker,

hyperglycaemia or pancreatic cells dysfunctions were associated with pancreatic adenocarcinoma

(Wolpin et al., 2013). This finding highlights the association between type II diabetes mellitus, obesity

and pancreatic adenocarcinoma risk and might suggest the correction of insulin resistance as a preventive

strategy (Pericleous et al., 2014) (Figure 4.4).

23

Figure 4.2 the mechanism showing how fructose supports the up regulation of fatty acid synthesis and the

triglyceride synthesis (Johnson et al., 2013).

24

Fig 4.3 Fructose causes Hepatic Insulin Resistance from the synthesis of triglyceride (Basciano et al.,

2005).

25

4.4 PANCREATIC CANCER AND DIABETES MELLITUS

Chronic administration of fructose or sucrose to animals did not only cause insulin resistance but also

resulted to the development of type II diabetes. In tissue studies, the pancreatic islets show hyalinosis and

macrophage infiltration, similar to what is observed in humans having type II diabetes. The mechanisms

of how fructose induces this changes is not known, because the GLUT 5 transporter which is the primary

transporter of fructose is not expressed in the islet cells. However, Johnson et al. (2013) reported an up

regulation of the Urate transporter (URAT-1) in islet of fructose fed rats in association with increased

expression of MCP-1. Also, the incubation of cultured insulin secreting with uric acid lead to oxidative

stress and synthesis of MCP-1. This oxidative stress in islet cells is considered to have a major role in

causing the pancreatic islet dysfunction in type II diabetes.

4.5 PANCREATIC CANCER AND OBESITY

Obesity rates have doubled since 1980 and 2014 (World Health Organization, 2016). In 2014, 13% of the

World’s adult population were obese (World Health Organization, 2016). This obesity has been linked to

other health consequences including: cardiovascular diseases (mainly heart and stroke, which were the

leading cause of death in 2012.), diabetes, muscoskeletal disorders (osteoarthritis) and some cancers.

Obesity is caused by an imbalance between calorie intake (increase in high carbohydrate diet), and a

sedentary lifestyle.

The consumption of sugar and fructose as sweeteners with low sodium and fat intake has increased as the

same time as nutrition-linked chronic diseases have reached epidemic proportions in humans. This

increased consumption of sweetened foods and beverages expose the human body to high amounts of

fructose than those that naturally occur in fruits and vegetables. Also, the correlation to chronic diseases

has lead to the implications that excess fructose is involved in developing insulin resistance and also

obesity (Dekker et al., 2010). Fructose tends to behave more like fat than other carbohydrate in respect to

26

insulin resistance, leptin production, and post prandial TG levels. It also does not cross the brain barrier

and could potentially contribute to increase energy intake. Studies in humans have reported weight gain

during prolonged fructose consumption (Teff et al., 2004).

4.6 BODY MASS INDEX AND PHYSICAL ACTIVITY IN RELATION TO PANCREATIC

CANCER

Body Mass Index (BMI) is an approximate measure of whether someone is over- or under weight,

calculated by dividing their weight in kilograms (kg) by the square of their height in metres (m2).

Glycaemic Index (GI) is an empirical measure of blood glucose response after consumption of a specific

food (Nöthlings et al., 2007). While Glycaemic Load (GL) is the GI of a food multiplied by its

carbohydrate content, which reflects both the quantity and quantity of dietary carbohydrates, consumed

(Michaud et al., 2002). The Body Mass Index and physical activity were two factors considered in a

cohort study of US women in relation to the risk of pancreatic cancer. They observed that among women

with high compared with those with low glycaemic load or glycaemic index scores, there was a consistent

although not a significant increase in pancreatic cancer risk when BMI was high (≥25kg/m2) (Michaud et

al., 2002). In contrast, glycaemic load or glycaemic index did not increase the risk of pancreatic cancer in

these women who had BMIs of less than 25kg/m2.

In addition, they also found out that fructose intake was associated with an elevated risk of pancreatic

cancer among women who were obese but not among lean women. Similarly, there was a statistically

significant increase of 86% of pancreatic cancer risk in inactive women with either high or low fructose

intake. In a multi-ethnic cohort study, the physical activity was not a factor in determining the risk of

pancreatic cancer, but they found an elevated risk in pancreatic cancer with participants that consumed

fructose who were obese/overweight (Nöthlings et al., 2007).

27

Figure 4.4 Potential mechanisms for fructose-induced insulin resistance (Tappy and Lê, 2010).

28

4.7 RECENT ADVANCES IN FRUCTOSE METABOLISM AND MECHANISM IN

PANCREATIC CANCER CELLS

In the 1920s, Otto Warburg observed that some proliferating tissues, especially tumour cells, display

glucose uptake, and even in the presence of oxygen preferentially metabolise glucose-derived pyruvate to

lactate. This phenomenon of aerobic glycolysis is known as the ‘Warburg Effect’ (Cohen et al., 2015).

However, some biotechnologist recognise that as glucose utilization increases, so does lactate production

from anaerobic glycolysis, which limits growth as pH becomes increasingly acidic (Port et al., 2012).

This is the same challenge cancer cells face. Substituting fructose into culture media limits growth, but

simultaneously decreases lactate levels and increases protein yields, suggesting that once inside cells,

fructose-derived carbons are directed away from glycolysis and into pathways that generate metabolites

used for protein synthesis (Port et al., 2012).

In addition, the knockdown of TKT in the non-oxidative PPP drastically reduced tumour growth

regardless tumor growth regardless of the accumulation of ribose intermediates suggesting that ribose was

not a limiting factor in cancer growth (Xu et al., 2016).

On the other hand, TKT knockdown resulted in a decrease of NADPH and an increase of ROS, further

suggesting that oxidative stress homeostasis is an important determining factor in cancer growth (Xu et

al., 2016).

Also, current observation was made that the mean circulating fasting fructose levels in pancreatic cancer

cells were 2.5 fold higher in comparison with fasting fructose level in healthy patient. The role of fructose

to generate nucleic acids is supported by the demonstration of elevated production of uric acid, as a by-

product of purine metabolism and this provides important mechanistic insight into a recent report of

increased serum uric acid levels in patients with high fructose consumption (Charrez et al., 2015).

29

Furthermore, hyperinsulinaemia; metabolic syndrome and type II diabetes mellitus have all been

associated with pancreatic cancer (Pericleous et al., 2014). Interestingly, the drug metformin that is used

to treat diabetes appears to reduce pancreatic cancer (Pericleous et al., 2014).

Finally, fructose is the only sugar that raises uric acid concentration in the blood. Serum uric acid

concentration has been shown to have a strong positive association with the risk of pancreatic cancer

mortality in men (Jiao et al., 2009).

30

CHAPTER FIVE

DIETARY FACTORS IN RELATION TO PANCREATIC CANCER

Dietary factors have been thought to account for about 30% of cancers in Western countries, thus making

diet second only to tobacco smoking as a preventable cause of cancer. Diet contribution to cancer risk in

developing countries has been considered to be lower, perhaps around 20%. Therefore, unravelling the

effects of diet is of great public importance (Key et al., 2004).

5.1 HIGH FRUCTOSE CORN SYRUP (HFCS) IN RELATION TO PANCREATIC CANCER

High Fructose Corn Syrup (HFCS) is the most preferred used sweetener, which is found in processed

foods, baked goods, condiments, soft drinks, candy, dairy products and concentrated fruit juices (Bray,

2013). The consumption of this principal form of fructose has increased as the same time with most

common human disease (including: obesity, diabetes, cardiovascular diseases and cancer).

In a prospective study conducted, a significantly greater risk of pancreatic cancer associated in both men

and women with high consumption of added sugar, soft drinks, and sweetened fruit soups or stewed fruit

was observed than low consumption of those items (Larsson et al., 2006). Also, no association was found

between the consumption of jam or marmalade or sweets and pancreatic cancer risk. They reported that

in a case control study, women with high intake of added sugars had a 3.7 times greater risk of pancreatic

cancer than those who had low intakes, but this association with high intake was not significant in men. In

a cohort study conducted by Michaud et al. (2002), they observed that the strongest risk of pancreatic

cancer was associated with fructose intake. Foods that contribute to this dietary fructose (as a

monosaccharide) include: soda, punch, and fruit juices, which accounts for a higher percentage of dietary

glycaemic load.

31

Furthermore, in two prospective cohort work done by Schernhammer et al. (2005), they failed to find a

significant overall association between sweetened soft drinks and pancreatic cancer. Although, the data

they got may suggest a modesty higher pancreatic cancer risk in both sedentary and overweight women

who had high consumption of sweetened sugar and soft drinks.

5.2 FRUIT AND FRUIT JUICES IN RELATION TO PANCREATIC CANCER

Fructose which is fruit sugar, is a simple ketogenic monosaccharide found in many plants, where it is

often bonded to glucose to form the disaccharide sucrose. For the past years fruits have been known to

stop or fight against different human disease. From this two study carried out, their findings may question

the benefits of fruit we are meant to believe in. In a multi-ethnic study conducted by Nöthlings et al.

(2007), they found an association of pancreatic cancer risk with obese men not in obese woman who had

a high consumption of fruit juices and not with high intake of soda. A prospective cohort study conducted

by Jiao et al. (2009), reported a greater risk of pancreatic cancer from free fructose in fruits and fruit

juices than from other sources (including: soda and soft drinks). The analysis showed that fruits but not

fruit juices were associated with a greater risk of pancreatic cancer.

5.3 PHYTOCHEMICALS AND ANTIOXIDANTS IN FRUITS IN RELATION TO PANCREATIC

CANCER

Phytochemicals are simply the bioactive non-nutrient plant components in fruit, vegetables, grains and

other plant foods. These phytochemicals and antioxidants could inhibit cell proliferation and induce

cancer cell death. Johnson et al. (2013), reported that natural fruit contain numerous substance that can

block the effect of fructose including: potassium, vitamin C and antioxidant such as: resveratrol, quercetin

and other flavonols. Where as fructose from added sugars is linked with hypertension, fructose from

natural fruit is not.

32

In a recent study, reported by Pericleous et al. (2014), citrus fruits (orange, tangerine, lime, lemon and

grape fruit) are rich in flavonoids such as hesperidin, rutin and diosmin, which have been shown to have

antitumor, anti-proliferative and pro-apoptotic properties. They are also rich in carotenoids such as β-

carotene and lutein that may also decrease the risk of cancer (Martí et al., 2009). Citrus fruits also contain

citrus limonoids compounds such as limonin and nomilin that were found to possess antioxidant and

anticancer properties (Patil et al., 2010). Bennett et al. (2012) reported that several clinical trials using

antioxidants and natural compounds have been conducted to observe if this compounds have anticancer

and chemo preventive properties. Compounds such as selenium, Vitamin E, and carotene have been

shown to exhibit pharmacologic and biologic effects as anticancer agents.

Although, dietary supplement does not have the same effect as natural fruit. He explained that the natural

combination of phytochemicals in fruits and vegetables is responsible for the potent antioxidant (Liu,

2003). This was achieved by using the effect of apple extract with skin that reduced tumour growth

compared with apple extract without skin.

Nutrients commonly found in fruits and vegetables have been inversely associated with several other

cancers including pancreatic cancer (Chan et al., 2005). Potential mechanisms of action include

antioxidant protection against free-radical damage to DNA and polyunsaturated fats (e.g., vitamin C,

carotenoids, tocopherols, and selenium), apoptosis ((e.g., indole-3 carbinol in cruciferous vegetables),

enhancing immune function (e.g., carotenoids, vitamin C, and vitamin E), modulating hormonal pathways

linked to cancer, such as sex hormones (e.g., soy and lignans) or insulin-like growth factor (e.g.,

lycopene), inhibiting cellular proliferation (e.g., carotenoids), and ensuring proper DNA methylation and

gene expression (e.g., folate; Chan et al., 2005).

33

5.4 GLUCOSE IN RELATION TO PANCREATIC CANCER

Pancreatic ductal adenocarcinoma (PDA) exhibits an elevated capacity for glucose uptake (Sousa et al.,

2014). This metabolic changes involving glucose are driven by the oncogenic Kras (Sousa et al., 2014).

Figure 5.2 shows enzymes whose expression is increased by Kras are indicated with an asterisk. Increased

glucose uptake fuels glycolysis (leading to increased lactate production), anabolic pathways such as the

non-oxidative arm of the PPP (producing ribose for nucleotide biosynthesis) and the HBP (producing

precursors for glycosylation) (Sousa et al., 2014). Glutamine is a key metabolite that is utilized to fuel the

TCA cycle and maintains redox homeostasis in PDA through a novel pathway (shown in orange) that

leads to NADPH production (Sousa et al., 2014).

Also, the consumption of high-sugar foods may increase the risk of pancreatic cancer by the direct effects

of increased glucose concentrations. This chronic excess of glucose had toxic effects on the pancreatic

islet and may result in beta cell dysfunction and eventually cell death, a phenomenon regarded as ‘glucose

toxicity’. One of the potential central mechanisms for glucose toxicity is the increased formation of

reactive oxygen species (ROS), when in prolonged excess can cause chronic oxidative stress (Robertson,

2004).

5.5 OTHER DIETARY FACTORS AND PANCREATIC CANCER

5.5.1 Vegetables

People who consumed at least five servings of vegetables per day were inversely associated with the risk

of developing pancreatic cancer than those who did not. Specifically, dark leafy vegetables, cruciferous

vegetables, yellow vegetables, beans, onions, garlic and carrots were found to be associated with reduced

risk of pancreatic cancer (Chan et al., 2005).

34

Antioxidants in Pancreatic

Cancer

DNA damage (vit. C, carotenoids, tocopherols &

selenium)

Apoptosis (indole-3 carbinol)

Enhance immune function

(carotenoids, vit. C & vit. E)

Modulate hormonal

pathway (soy & lignans)

Modulate insulin-like growth facor

(lycopene)

Inhibit cellular proliferation (carotenoids)

Ensure proper DNA methylation

& gene expression

(folate)

Figure 5.1 Effect of natural antioxidants on pancreatic cancer (the purple circle). The red circles represent

what happens to the cancer cells while the blue circles represent the mechanisms taken to fight the cancer

cells.

35

Figure 5.2 Metabolic pathways that are altered by the oncogene Kras (Sousa et al., 2014).

36

Raw vegetables may impart more benefit than cooked vegetables due to their potential increased nutrient

content, lower glycemic index, higher level of enzymes important for phytochemical production, and

increased insoluble fiber content (Chan et al., 2005). In addition, a study done on anti proliferative and

antioxidant activities of common vegetables identified a number of cruciferous and allium vegetables as

foods with exceptional inhibitory activity against tested cancer cell lines, including pancreas and kidney.

The vegetables found to possess very potent inhibitory activities include: garlic, leak, immature green

onion, Brussels sprouts, kale, broccoli and various cabbages (Boivin et al., 2009).

5.5.2 Dietary Meat and Fat

In a large cohort of women, there was no association found between the intake of meat, dairy products,

fat, cholesterol and the risk of pancreatic cancer (Michaud et al., 2003). Rodents fed high-fat

diets experienced a greater incidence of pancreatic tumors than did rodents fed low-fat diets with a similar

caloric content (Michaud et al., 2003). In one study, rodents fed diets rich in saturated fat and also linoleic

acid had the greatest increase in pancreatic tumorigenesis (Michaud et al., 2003). It has been suggested

that the different practices of cooking or processing meat may be related to the risk of pancreatic cancer

(Michaud et al., 2003). Cooking meat at high temperatures can result in the formation of heterocyclic

amines, and processing meats (e.g., curing or smoking) increases N-nitroso compounds (Michaud et al.,

2003). In a case-control study in China, intake of deep-fried foods was not associated with pancreatic

cancer risk, but smoked and cured foods increased the risk of pancreatic cancer (Michaud et al., 2003).

5.6 CONCLUSION

Taken together, fructose consumption has increased over the past years especially in its principal form;

High-Fructose Corn Syrup (HFCS). This has been linked to the epidemiology of different human diseases

such as obesity, diabetes and cancer. This simple sugar with the same chemical formula as glucose has a

different metabolism from that of glucose. It is believed that the antioxidants and phytochemicals in fruits

37

inhibit the effect of fructose to promote carcinogenesis and cancer growth. Therefore, more research is

required in this area of study to ascertain the role played by fructose in carcinogenesis and cancer growth

in HFCS or in its natural form.

38

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