1.0 INTRODUCTION

Cancer is a broad group of various diseases characterized by

unregulated, uncontrolled abnormal cell proliferation. In a normal

healthy body, cells grow, die, and are replaced in a very controlled

manner. This process of replication is carefully initiated and

terminated to generate a specific amount of cells by a cascade of

signal rendering and silencing mechanisms mediated by growth

factors, inhibitors, and other associated molecules bearing in a

multi-step fashion signals to the nucleus with each step along the

way well monitored and controlled. This ordered signal transduction

sends to the nucleus the need to grow and divide or the need to stop

growing. Changes in the genetic material of cells (mutation) by

environment or internal factors sometimes result in a loss of

restraint on cell proliferation, culminating in cells that do not

die (loss of apoptosis), or that continue to multiply until a mass

of cancer cells or tumor developed. A portion of the mass of

cancerous cells can detach, enters the blood stream or the lymphatic

system, transport and start a new colony elsewhere and thereby

invade and destroy surrounding healthy tissues and organs via a

process called metastasis. Metastasis is a noxious character of

malignant cancers. Benign cancers on the other hand are less

offensive; they do not grow uncontrollably, do not invade

neighboring tissues, and do not spread throughout the body.

NORMAL CELL COMMUNITYWITH A TIGHT LEASH

CANCER CELLS FORM TUMORWHICH CONTINUES TO ANGIOGENESIS SUPPLIES

TUMOR CELLS WITH MORE

Figure 1: PICTORIAL DEPICTION OF HOW CANCER CELLS GROW AND INVADESURROUNDING TISSUES AND ORGANS

1.1 CANCER AS A MENACE

There are over 200 different known cancers that afflict humans. In

2007, cancer caused about 13% of all human deaths worldwide

(7.9 million). Rates are rising as more people age and as mass

lifestyle changes occur in the developing world. In 2009, three

hundred and twenty thousand, four hundred and sixty seven (320,467)

new cases of cancer were diagnosed in the UK (Jemal et al., 2011).

In the US, one in every four people dies of cancer. In the UK, more

than one in three people will develop some form of cancer during

their lifetime (NHS health apps Library).

CANCER CELLS MIGRATEVIA BLOODSTREAM CANCER CELLS INVADE

TISSUES AND ORGANSA HEALTHY BODY BREAKS

DOWN

To put an end to this menace therefore requires exploiting every

process known to be involved with Cancer. Interestingly many if not

all of the peculiar adaptive processes employ by cancer cells from

pre-malignant stage to metastatic and invasive stage sits on one key

metabolic figure; SUGAR. The metabolism of sugar plays a central

role in tumorigenesis. Exploiting sugar in cancer therapy is

therefore a reasonable target in the quest to end the scourge of

cancer.

Also targeted in cancer therapy include signal transduction in

cells, cell division, DNA replication, transcription, and

translation, metastasis, cell death (apoptosis), angiogenesis etc

all of which still have one or more connection to sugar as a major

participant in their metabolic pathway. While exploiting signal

transduction seeks to active and deploy speed breakers (e.g.

tyrosine kinase inhibitors) and brakes on cell proliferation by

finding a way of de-amplifying multiplying signals in the cell,

amplifying contact, some studies try to exploit mutations of proto

Oncogenes (genes liable to become mutated and cause cancer),

oncogenes, tumor suppressor genes etc.

However, the main quest here is find to seek a way of exploiting

sugar-the body primary fuel, in the diagnosis and treatment of

cancer.

CHAPTER TWO: KEY EVENTS WITHIN THE MASS OF CANCER CELLS

Having established the fact that a tumor is a mass of cancer cells,

researchers endeavor to expose the happenings within a tumor

community and seek to exploit these occurrences in cancer diagnosis

and treatment. Some of the usual happenings within a tumor community

include the followings;

2.1 ANGIOGENESIS

Angiogenesis is the physiological process through which new blood

vessels form from pre-existing vessels by dividing (the blood vessel

splitting into two wall to wall) or by growing branches called

sprouts, the latter is used by cancer cells in new blood vessels

formation with the new vessels shooting out from the existing ones

into the tumor. Angiogenesis is different from vasculogenesis in

that with vasculogenesis, entirely new i.e. de novo-formation of blood

vessels takes place especially where there are no pre-existing blood

vessels e.g. in the developing embryo (Flamme et al., 1997).

The development of blood vessels is an essential step in the growth

of a tumor. Key players in this process are growth factors capable

of stimulating blood vessels formation. Some notable growth factors

are fibroblast growth factor (FGF) (Ornitz DM, Itoh N., 2001) and

vascular endothelial growth factor (VEGF) tasked with the job of

inducing capillary growth into tumor, which in turn supply required

nutrients, allowing the tumor to continue expanding. Angiogenesis

also serves as a waste disposal, taking away the biological end

products secreted by rapidly dividing cancer cells. Whether

supplying nutrients or disposing waste, angiogenesis is a necessary

and required step for transition from a small harmless cluster of

cells (often said to be about the size of the metal ball at the end of a ball-point pen) to

a large tumor.

Angiogenesis is also required for the spread of a tumor, or

metastasis. Single cancer cells can break away from an established

solid tumor, enter the blood vessel, and be carried to a distant

site, where they can implant and begin the growth of a secondary

tumor. Evidence now suggests the blood vessel in a given solid tumor

may, in fact, be mosaic vessels, composed of endothelial cells and

tumor cells. This allows for substantial shedding of tumor cells

into the vasculature, possibly contributing to the appearance of

circulating tumor cells in the peripheral blood of patients with

malignancies. The subsequent growth of such metastases will also

require a supply of nutrients and oxygen and a waste disposal

pathway, therefore the chaotic process continues (W. Jeffrey Allard

et al. 2004).

Adaptation of cancerous cells causes mutations that destroy or

snuff out processes that are against angiogenesis, for instance the

inhibition of the anti-VEGF enzyme PKG. In normal cells PKG

apparently limits beta-catenin-a pro-angiogenetic substance (Bagri,

A; et al 2010).

The blood vessels created in this way are not exactly the same as

normal blood vessels. It is believed tumor blood vessels are not

smooth like normal tissues, and are not ordered sufficiently to give

oxygen effectively to all of the tissues (Risau, W; Flamme, I.,

1995).

2.1.1 EXPLOITING ANGIOGENESIS IN CANCER TREATMENT

Hindering the supply of nutrients e.g. glucose and oxygen to the

growing mass of cancer cells by the use of anti-angiogenic

substances is a naturally reasonable way of limiting cancer growth.

Since glucose and it’s metabolic processes seem to be central to the

very nature of cancer. The first line of defense against cancer may

be cutting off the supply of sugar to the cancer cells. This may be

achieved by inhibiting angiogenesis in cancer cells or by inhibiting

glucose transporters needed to transport glucose into the rebel

cells e.g. GLUT1.

On the prospect of cutting off the supply by targeting angiogenesis,

research has shown that endothelial cells are genetically more

stable than cancer cells-the latter assume the defensive position of

adapting to all assault-doing this requires a high level of

instability. This genomic stability of endothelial cells gives an

advantage of targeting endothelial cells using anti-angiogenic

therapy, which may be more effective in comparison to chemotherapy

directed at cancer cells as cancer cells rapidly mutate and acquire

'drug resistance' to treatment, endothelial cells are more stable

making them an ideal target for therapies directed against them

(Bagri, A.; et al 2010).

Angiogenesis research is therefore a cutting-edge field in cancer

research. Another application of endothelial cells genetic stability

is the use of radiation therapy on endothelial cell compartment,

rather than on tumor cell compartment. This may be effective as new

blood vessel formation is a relatively fragile process, subject to

disruptive interference at several levels. Again, since tumor cells

evolve resistance and adapt to an otherwise lethal environment

rapidly due to their rapid generation time (days) and genomic

instability (variation), endothelial cells are better targets of

radiation therapy as they possess long generation time (months),

genomic stability (low variation), and thus cannot quickly adapt to

hazardous environment to resist the killing agents (Brown JM,

Giaccia AJ, 1998).

Interestingly, these ideas perfectly depict natural selection in

action at the cellular level, using a selection pressure to target

and differentiate between varying populations of cells. The end

result is the extinction of one species or population of cells

(endothelial cells), followed by the collapse of the ecosystem (the

tumor) due either to nutrient deprivation or self-pollution from the

destruction of necessary waste pathways.

Angiogenesis-based tumor therapy presently relies on natural and

synthetic angiogenesis inhibitors like angiostatin, endostatin and

tumstatin. These are proteins that mainly originate as specific

fragments of pre-existing structural proteins like collagen or

plasminogen.

FDA-approved therapy targeted at angiogenesis in cancer is on the

market in the US. This is a monoclonal antibody directed against an

isoform of VEGF. The commercial name of this antibody is Avastin,

and the therapy approved for use in colorectal cancer in combination

with established chemotherapy.

2.2 HYPOXIA-AN IMPORTANT FEATURE OF CANCER

Control of cellular oxygen concentration under normal physiological

condition is very strict as the body tries to keep the oxygen

concentration within a normal physiological range termed normoxia,

and putting mechanisms in place to forestall oxygen concentration

from getting too high; hyperoxia or from getting too low; hypoxia.

Hyperoxia causes damage secondary to reactive oxygen species while

hypoxia leads to activation of a diverse array of downstream

transcriptional pathways including angiogenesis, glucose metabolism

and apoptosis (C Thirlwell, et al, 2011).

Despite the strict regulation of cellular oxygen concentration, the

unrestrained cell multiplication of cancer cells generates more

cells to be “fed” oxygen and glucose; this necessitates an urgent

angiogenesis which generates impaired microvasculature for

micronutrient supply and oxygenation. As this vasculature develops

in a chaotic way with structural malformations, regions of hypoxia

are present within all solid tumors. Normal oxygen tension in

healthy tissue is 7% (53 mmHg), levels of oxygenation in tumors may

vary from physiological levels (7%) to severe hypoxia (< 1%) which

is usually found in areas adjacent to necrotic tissue (Heddleston

JM, et al, 2010).

Within the same region of a given tumor, levels of oxygenation may

cycle due to poor vasculature and limited oxygen diffusion,

resulting in intermittent periods of hypoxia (Heddleston JM, et al,

2010). These cyclical episodes of hypoxia lead to increased

metastatic potential of cancer cells (Cairns RA, et al, 2001).

Hypoxia is therefore a promoter of cancer progression, invasion and

metastasis (Pennacchietti S; et al 2003).

Hypoxia may also be responsible for the voracious appetite of cancer

cells for glucose because intra-tumoral hypoxia causes the hypoxia-

inducible factor (HIF)1α pathway to be activated in many tumors

cells, resulting in the direct up-regulation of lactate

dehydrogenase (LDH) and increased glucose consumption (Cairns RA, et

al 2011.).

Another important effect of hypoxia is autophagy or self-eating-

process by which cells degrade own cellular components to survive

during starvation or to eliminate damaged organelles after oxidative

stress. Mitophagy, or mitochondrial-autophagy, is particularly

important to remove damaged or reactive oxygen species (ROS)-

generating mitochondria or a result of cancer genes shutting down

the mitochondria because they are involved in the cell's apoptosis

program which would otherwise kill cancerous cells. An autophagy or

mitophagy program can be triggered by hypoxia (Azad MB, Gibson SB;

2010).

Degrading the mitochondria in this way may explain the lack of

apoptosis in cancer cells. The hypoxia inducible factor can be said

to be the mediator and key promoter of many characteristics of

cancer cells, being involved in angiogenesis, up regulation of

glycolytic enzymes, cancer’s genetic instability making cancer cells

evolve and remain adamant to therapy and the loss of apoptosis.

DIFFERENTIATED TISSUE

GLUCOSE

PYRUVATE PYRUVATE PYRUVATE

LACTATELACTATE

OXIDATIVE PHOSPHORYLATION: 36 MOL ATP/MOL GLUCOSE

ANAEROBIC GLYCOLYSIS: 2 MOL ATP/MOL GLUCOSEAEROBIC GLYCOLYSIS (WARBURG EFFECT): 4 MOL ATP/MOL GLUCOSE

-O2+O2

GLUCOSE

TUMOR

+/-O2

CO2

O2 O2

2.3 WARBURG’S HYPOTHESIS AND EFFECT

Warburg's hypothesis was postulated by the Nobel laureate Otto

Heinrich Warburg in 1924 (O. Warburg, K. Posener, E. Negelein: Ueber

den Stoffwechsel der Tumoren, 1924). Also called The Warburg Theory

of Cancer, it can be summarized thus; that the driver of

tumorigenesis is an insufficient cellular respiration caused by

insult to mitochondria (Warburg O., 1956).

PROLIFERATIVE

Figure 2: Comparison of ATP generation of aerobic glycolysis to

Oxidative phosphorylation36mol ATP/mol glucose

Anaerobic glycolysis 2 mol ATP/mol glucose

Aerobic glycolysis (Warburg effect)4mol ATP/mol glucose

Warburg hypothesized that cancer, malignant growth, and tumor growth

are caused by the fact that tumor cells mainly generate energy (as

e.g. adenosine triphosphate ATP) by non-oxidative breakdown of

glucose (glycolysis). This is in contrast to "healthy" cells which

mainly generate energy from oxidative breakdown of pyruvate.

Pyruvate is an end-product of glycolysis, and is oxidized within the

mitochondria. Hence, according to Warburg, the driver of cancer

cells should be interpreted as stemming from a lowering of

mitochondrial respiration. Warburg reported a fundamental difference

between normal and cancerous cells to be the ratio of glycolysis to

respiration; this observation is also known as the Warburg effect.

Figure 3: Transport of glucose and its metabolites in mammalian cells. Under normoxic conditions glucose is metabolized in mitochondria to H2O and CO2 producingabout 36 mol of ATP per mole of glucose. Under anaerobic conditions or in the presence of defects in mitochondria glucose is metabolized to lactic acid

The Warburg effect or aerobic glycolysis can be defined as the

propensity of cancer cells to take up high levels of glucose and to

secrete lactate in the presence of oxygen (Warburg O., 1956; Dang

CV., 2009).

The Warburg Effect describes the observation that cancer cells, and

many cells grown in-vitro, exhibit glucose fermentation even when

enough oxygen is present to properly respire. In other words,

instead of fully respiring in the presence of adequate oxygen,

cancer cells ferment. The current popular opinion is that cancer

cells ferment glucose while keeping up the same level of respiration

that was present before the process of carcinogenesis (Vazquez, A.,

et al, 2010).

That cancer cells consistently use more glucose and produce more

lactic acid than normal tissue may represents adaptation to intra-

tumoral hypoxia due to disordered angiogenesis and blood flow

(explained above), with recent researches it is revealed that much

seems to be due to the curious phenomenon of “aerobic glycolysis”—

i.e. continued use of the glycolytic metabolic pathway even in the

presence of adequate oxygen. Warburg explained his observation with

his hypothesis that proposed aerobic glycolysis was a manifestation

of a deficiency in respiration. However, extensive research clearly

demonstrated this to be untrue.

From a casual and hasty perception, aerobic glycolysis should not

contribute positively to cell’s fitness and survival, in fact it

should do more damage than good to the survival of cancer cells

because; it is much less efficient in energy production than is

aerobic metabolism—specifically glycolysis produces only 2 mol

ATP/mole of glucose while oxidative metabolism of glucose results in

about 36 molATP/mole of glucose; also glycolysis increases acid

production resulting in a highly acidic extra-cellular environment.

This should result in local toxicity including cell death and extra-

cellular matrix degradation due to release of proteolytic enzymes

(Williams, A. C., Collard, T. J., & Paraskeva, C., 1999; Park, H.

J., et al, 1990; Rohzin, J., et al, 1994; Cuvier, C., Jang,

A.,&Hill, R. P., 1997).

How then do cancer cells survive if increase glycolysis is a shot in

the leg for them? A deeper study of cancer cells sugar metabolism

reveal how cancer cells beat the odds through sugar metabolism;

2.4 SUGAR METABOLISM AND CANCER CELLS SURVIVAL

Cancer cells strategy to overcoming consistent environmental

pressures is the up-regulated glycolytic metabolic pathways (Gatenby

& Gillies, 2004).

Under hazardous conditions (hypoxia and acidosis), cellular traits

that promote constitutive up-regulation of glycolysis and resistance

to acid-induced toxicity occur in cancer cells. Mathematical models

and empirical observation suggest the growth advantage conferred by

this combination of phenotypic traits is substantial because the

cells create an acidic local environment (due to up-regulated

glycolysis) that is toxic to other cells (Gatenby & Gawlinski,

1996).

There is some evidence that this advantage along with acid-induced

degradation of extracellular matrix is critical for tumor invasion

into normal host tissue (Gatenby et al., 2006).

Studying extensively the multi-faceted manipulative use of sugar

metabolism by cancer cells opens our eyes to the elusive nature of

this menace greatly. A very interesting idea suggested that

increased sugar metabolism including glycolysis (which has cropped

up as a chief culprit in cancer survival plot) and also pentose

phosphate metabolism may have a way of conferring other measures of

adaptive stone walling power to cancer cells, if it allows excess

pyruvate to be available for lipid synthesis or providing essential

anabolic substrates, such as ribose for nucleic acid synthesis

(Homem de Bittencourt, Peres, Yano, Hiratea, & Curi, 1993).

Glucose consumption through the pentose pathway may also provide

essential reducing equivalents (NADPH) to reduce the toxicity of

reactive oxygen species conferring resistance (Kondoh, Lieonart,

Bernard, & Gil, 2007; Kondoh, Lieonart, Gil, Beach, & Peters, 2005).

These evolutionary advantages can explain the remarkable prevalence

of the glycolytic phenotype in human cancers and the otherwise

puzzling observation that malignant cells remain glycolytic even in

the presence of normoxia. This conceptual model is supported by

empirical studies that demonstrate constitutive upregulation of

glycolysis is consistently observed during the transition from

premalignant lesions and invasive cancer (Gambhir, 2002; Abbey et

al, 2006; Yasuda et al., 2001; Abbey et al., 2004).

The molecular basis for evolution of the glycolytic phenotype has

been clarified by recent advances in understanding the hypoxia-

inducible factor, HIF-1 system (Ryan, Lo, & Johnson, 1998).

It is clear that up-regulation of the HIF system elicits a multi-

phasic response that includes increased expression of components of

the glycolytic pathways including membrane glucose transporters

(Greijer et al., 2005). At present over 70 genes directly regulated

by HIF have been identified and several hundred are directly or

indirectly influenced by HIF. HIF stabilization occurs in response

to diminished oxygen concentrations.

Interestingly, increased levels of pyruvate (a glycolytic product)

also stabilize HIF providing a potential feed forward loop which may

be critical to aerobic glycolysis (Vangellur, Phillips, Bogenesch, &

LaPres, 2005; Kim, Tchernyshyov, Semenza, & Dang, 2006). HIF

activity can also be stabilized in the presence of oxygen by growth

Figure 4: A summary of the HIF system demonstrating several factors whichgovern the level of HIF-1 and some of its protean effects on cell

metabolism, survival, proliferation and their microenvironment which

factors that also participate in carcinogenesis, including ras, HSP

90, Cox 2, HER, and the AKT/mTOR pathway (Sang, Stiehl, et al,

2003).

Cells with loss of HIF activity demonstrate reduced (but not

completely absent) expression of glucose transporter and glycolytic

enzymes in response to hypoxia when compared to normal (Vengellur,

Woods, Ryan, Johnson, & LaPres, 2003).

Interestingly, HIF1 activation can also lead to expression of cell

death factors so that full activation by severe hypoxia can also

lead to apoptosis and necrosis (Burke et al., 2003; Cramer et al.,

2003). Clearly, much additional work will be required to fully

understand the complex mechanisms involved in hypoxic response,

metabolic controls, and adaptation to acidosis in cancer

progression. Despite gaps in understanding, the complex pathways

that result in increased glycolytic metabolism in the vast majority

of human cancers provide multiple potential targets for treatment

strategies designed to alter tumor glucose metabolism (Semenza,

2003).

Since this phenotype emerges during carcinogenesis, it may represent

a possible target in cancer prevention. In advanced metastatic

cancers, understanding of the molecular and physiological causes and

consequences of upregulated glycolysis may lead to targeted

therapies.

CHAPTER THREE: EXPLOITING SUGAR IN CANCER DIAGNOSIS AND THERAPY

3.1 EXPLOITING SUGAR IN CANCER DIAGNOSIS

The Warburg effect has important medical applications, as high

aerobic glycolysis by malignant tumors is exploited clinically to

diagnose and monitor treatment responses of cancers by imaging

uptake of 2-18F-2-deoxyglucose (FDG) (a radioactive modified

hexokinase substrate) with positron emission tomography (PET).

Positron Emission Tomography (PET) is a nuclear imaging technique

that takes the advantage of the fact that cancer cells absorb

glucose more than normal cells because of their dependence on low

energy yielding aerobic glycolysis, which extract only about 5% of

the available energy in the glucose, calling for more glucose

absorption. This voracious appetite for glucose is taken advantage

of in the use of radioactive tracers that allows delving deeper into

the physiologic processes occurring intracellularly. PET primarily

looks at one specific tracer the 18F-labeled glucose analog 2-

deoxyglucose (2DG) (Zhang et al., 2006), which can be administered

through a vein and observed with the PET scanner after a while. The

PET scanner is used to take pictures that show the use of the

radioactively label glucose by different organs and tissues in the

body. Cancer cells use more glucose than normal cells, and the

ability to detect that on an image is a way of diagnosing the

presence of cancer-and evaluating tumor size and a very effective

way of doing that.

One of the hurdles FDG-PET has helped to scale is the pre-operative

staging of the carcinoma of the stomach which has a poor prognosis

because many patients have advanced disease at the time of diagnosis

(Siewert et al, 1998). Therefore, pretreatment assessment and

staging of disease is essential for managing gastric carcinoma. The

tumor stage provides the basis for selecting the most appropriate

therapeutic strategy (Fleming et al., 1997). Preoperative staging

currently relies on a standard noninvasive imaging modality of

spiral-computed tomography (CT) of the abdomen and pelvis. However,

CT is an anatomy-based diagnostic technique with certain drawbacks,

including limited sensitivity from falsenegative findings due to

non-enlarged invaded lymph nodes and limited specificity from false-

positive findings due to enlarged inflammatory lymph nodes.

Therefore, a better preoperative evaluation strategy would greatly

aid the preparation of treatment plans for patients with gastric

carcinoma. Positron emission tomography (PET) using the radiolabeled

glucose analogue, 18 F-fluorodeoxyglucose (FDG), as a tracer is a well

established imaging technique that offers new perspectives in

staging malignant diseases. FDG-PET scanning enables observation of

altered glucose metabolism in neoplastic cells (Tschmelitsch et al,

2000). Images from PET scanners are complementary to traditional

morphologic images, such as those produced by CT, and may be more

sensitive because functional changes often precede anatomic changes.

Jian Chen et al in a research report on the application of FDG-PET

in the pre-operative staging of gastric adenocarcinoma that proved

that FDG-PET improves the preoperative TNM staging of gastric

adenocarcinoma. Based on its superior specificity, FDG-PET can

facilitate the selection of patients for a curative resection by

confirming a nodal status identified by CT (Jian Chen et al, 2005).

Based on the work of Jian Chen et al, PET may play a complementary

role in pretreatment evaluation, as results of this research showed

FDG-PET upstaged 6% patients from the false-negative CT findings,

and downstaged 9% patients from CT false-positive findings.Also, PET

provided important information for making decisions regarding

treatment; splenectomy was performed in patient(s) confirmed to have

a spleen metastasis, and lymphadenectomy was performed in another

patient. Therefore, those patients who benefited from the FDG-PET

detection method were treated with a timely curative resection,

without the need for any extra neoadjuvant chemotherapy.

3.2 EXPLOITING SUGAR IN CANCER TREATMENT

Cancer cells with increased aerobic glycolysis due to activated

oncogenes (including Ras, Her-2, and Akt) or loss of tumor

suppressor function (including TCS1/2, p53, LKB1) has been shown to

undergo rapid apoptosis when placed in culture conditions with low

glucose concentrations (Inoki, Zhu, & Guan, 2003; Xu et al., 2005;

Jones et al., 2005). If such low glucose concentration environment

can therefore be created through glucose deprivation, it is expected

that that will lead to cancer cells apoptosis. In human patients,

these culture conditions could be mimicked in vivo by transiently

reducing blood and interstitial glucose concentrations through

administration of insulin, diet alteration and lifestyle adjustment.

3.2.1 ANTI-GLYCOLYTIC MEASURES

Since cancer cells rely on anaerobic metabolism to produce a

variable but generally significant portion of their energy

requirements, inhibition of the glycolytic pathway is an obvious

approach that may exploit the high glucose consumption by cancer

cells. There are evidences that support this approach; that

inhibition of glycolysis can result in cancer cell’s death

particularly in hypoxic environment due to ATP depletion (Jones et

al., 2005).

3.2.1.1 BROMOPYRUVATE: HEXOKINASE AND G-3-P DEHYDROGENASE INHIBITOR

An obvious therapeutic target in the glycolytic pathways is

hexokinase which catalyzes phosphorylation of glucose to glucose-6-

phosphate, the first and rate-limiting step in glucose metabolism.

Bromopyruvic acid is a synthetic brominated derivative of pyruvic acid. It

is being studied as a potential treatment for certain types of cancer.

Initial studies in laboratory animals’ researchers at Johns Hopkins showed

that bromopyruvic acid is effective at eliminating aggressive liver tumors

(Ko et al, 2001).

A study stated that 3-Bromopyruvate markedly reduce ATP

concentrations leading to cytotoxicicity in tumor cells that are

hypoxic or possess mitochondrial defects by inhibiting hexokinase

(Gerschwind et al, 2002).

Another study observed that the mechanism of action of bromopyruvic

acid involves interruption of the glycolytic process by the

inhibition of the enzyme Glyceraldehyde 3-phosphate-dehydrogenase

(Ganapathy-Kanniappan, S; Geschwind, JF 2012).

The research group of J.F. Geschwind reported that intra-arterial

delivery of bromopyruvic acid directly to the site of a tumor

represents a new strategy for stopping the growth of liver cancer

while minimizing toxic side-effects (Geschwind JF, et al. 2002). The

first clinical use of 3-bromopyruvate to treat patients with late

stage liver cancer was reported to have taken place in 2010 under a

compassionate use protocol, where a total of two patients received

3-bromopyruvate intra-arterially. The patients were treated using a

special patented formulation of 3-bromopyruvate invented by Ko,

Pederson and Geschwind.

3.2.1.2 LONIDAMINE

Lonidamine, a derivative of indazole-3-carboxylinc acid decreases

glycolysis in vitro and in vivo probably through inhibition of

mitochondrial bound hexokinase (Floridi et al., 1981). Lonidamine

has been shown to decrease intra-cellular ATP and lactate production

in cancer cells but, interestingly, appears to enhance aerobic

glycolysis in non-transformed cells. Although lonidamine is

Figure 5: The structure of 3-bromopyruvic acid: a potent inhibitor ofG-3-P dehydrogenase and Hexokinase

IUPAC name: 3-Bromo-2-oxopropanoic acid

currently under investigation as a primary therapy for benign

prostatic hypertrophy, its main role in malignant therapy is in

combination with cytotoxic drugs.

3.2.1.3 MANNOHEPTULOSE

Mannoheptulose- a heptose, a monosaccharide with seven carbon atoms

is a hexokinase inhibitor. By occupying the active site of

glucokinase, it prevents glucose phosphorylation thus inhibiting

glycolysis. It is naturally found as D-mannoheptulose in avocado

plants (Persea americana) (Dai et al., 1999). A purified avocado extract

of D-mannoheptulose was added to a number of tumor cell lines tested in

vitro by researchers in the Department of Biochemistry at Oxford University

in Britain, their findings showed that it inhibited tumor cell glucose

uptake by 25 to 75 percent, their findings also show it has a inhibitory

effect on the enzyme glucokinase and on the growth rate of the cultured

tumor cell lines. Lab animals were administered 1.7 mg/g body weight dose

of mannoheptulose for five days and it reduced tumors by 65 to 79 percent

(Board et al., 1995) Based on these studies, there is good reason to

believe that avocado extract could help cancer patients by limiting glucose

use of the cancer cells.

Figure 6: D-Mannoheptulose-a heptosewith glucokinase inhibitory activity.

IUPAC Name: D-Manno-hept-2-ulose

3.2.2 INHIBITING THE ALTERNATIVE SUGAR SOURCE: GLUCONEOGENESIS

Glucose can be made by cells via gluconeogenesis. Joseph Gold, M.D.,

director of the Syracuse (N.Y.) Cancer Research Institute and former

U.S. Air Force research physician, surmised that a chemical called

hydrazine sulfate, used in rocket fuel, could inhibit the excessive

gluconeogenesis (making sugar from amino acids) that occurs in

cachectic cancer patients. Gold's work demonstrated hydrazine

sulfate's ability to slow and reverse cachexia in advanced cancer

patients. A placebo-controlled trial followed 101 cancer patients

taking either 6 mg hydrazine sulfate three times/day or placebo.

After one month, 83 percent of hydrazine sulfate patients increased

their weight, compared to 53 percent on placebo (Chlebowski et al.,

1987). A similar study by the same principal researchers backed by

the National Cancer Institute, Bethesda, followed 65 patients;

hydrazine sulfate was shown to potentiate the lives of those it was

administered to by 17 weeks (Chlebowski et al., 1990).

3.2.2 USING GLUCOSE ANALOGS

2-deoxy glucose has been shown to compete with glucose for trans-

membrane transport, making it useful not just in imaging techniques

but also in limiting glucose absorption. 2DG will not inhibit

glucokinase, infact it is bound by the enzyme and phosphorylated

just like normal glucose-to 2-deoxy glucose phosphate which cannot

be further metabolized. This is employed in the imaging technique

using the 18F labeled 2DG, as the accumulation of this metabolite in

the tumor cells is an obvious signal of the massive appetite that

cancer cells display for glucose or its derivatives. Several studies

have confirmed that 2DG depletes intra-cellular ATP leading to cell

death in vitro (Zhang et al., 2006). it is currently being explored

as an agent to enhance the therapeutic effects of radiation therapy

or cytotoxic drugs such as adriamycin and paclitaxel (Maschek et

al., 2004).

3.2.2 EXPLOITING HIF1 AND OTHER GENES

HIF1 is an essential regulator of adaptation to low oxygen levels.

It is a heterodimer composed of an oxygen regulated α-subunit and a

constitutively expressed β-subunit. The abundance of α subunit is

primarily regulated by a family of prolyl hydroxylases. In normoxia,

prolyl hydroxylase is activated and it directs the degradation of

the α subunit by ubiquiting proteasome pathway (McNeil et al.,

2002). Under hypoxia, porlyl hydroxylase activity decreases which

leads to the stabilization and translocation to the nucleus α

subunit which then heterodimerize with the β-subunit. HIF1 acitivity

is primarily regulated by the abundance of the HIF1- α subunit. HIF1

has also been shown to regulated many pro inflammatory genes such as

tumor necrosis factor- α (TNF- α), interleukin-8 (IL-8) and

vascular endothelial growth factor A (VEGF-A)-which is important to

angiogenesis. HIF1 promotes he expression of phosphofructokinase,

lactose dehydrogenase etc (Semenza GL, 2000).

Having establish the critical role of the HIF system in upregulating

glycolysis as an adaptation to hypoxia and non-adaptively in cancer

(producing aerobic glycolysis) has resulted in considerable interest

in developing inhibiting strategies. Exploiting HIF1 can be achieved

by direct influence; altering transcription or degradation, or by

indirect alterations of HIF by targeting control proteins and also

by alteration of effector proteins (Lee et al., 2006).

Probably the most direct of these is simply inhibiting the

transcription of HIF. PX-478, a recently developed drug that

specifically targets HIF expression, and has demonstrated anti-tumor

effects in animal models (Macpherson & Figg, 2004).

A number of treatment candidates have been developed to indirectly

influence HIF levels by targeting non-hypoxic controlling pathways.

This includes drugs such as CCI-779, which inhibits expression of

Mtor and consequently reduces HIF expression (Georger et al., 2001).

Similarly, drugs that alter expression of heat shock protein (Hsp90)

such as geldanamycin and apigenin also influence HIF1 expression.

Tumor suppressor genes are normal genes that slow down cell

division, repair DNA mistakes, or signal to the cells when to die (a

process known as apoptosis or programmed cell death). When tumor

suppressor genes don't work properly, cells can grow out of control,

which can lead to cancer. Treating problems in tumor suppressor

genes is more difficult. It would mean restoring normal

tumor suppressor gene functions, which researchers have not yet

figured out how to do effectively.

A major stumbling block lies in how to get new DNA into the cancer

cells. Another problem is that

most cancers have several mutations, so replacing one gene may not

be enough to stop the cancer

cells from growing and spreading.

One major breakthrough was achieved by inserting normal TP53 genes

into viruses and then trying to infect tumor cells with these

viruses. This worked well in the lab, but not in human studies. A

newer approach targets the weakness in the cell caused by the

abnormal tumor suppressor genes, rather than trying to restore

normal gene function. For example, some people inherit a mutation in

one of the BRCA genes (BRCA1 or BRCA2). If the second copy of this

gene is damaged, the gene no longer works and they may develop a

cancer. In cells where a BRCA gene no longer works (like cancer

cells), drugs called PARP inhibitors cause DNA damage that can lead

to cell death. Cells that have normally functioning BRCA genes can

repair this damage. This allows the PARP inhibitor to target the

cancer cells while doing little damage to the normal cells.

3.2.3 EXPLOITING ACIDOSIS

One of the consequences of increased glycolysis is acidification of

the extra-cellular space (Gillies et al., 2002). Acidosis in the

extracellular space brings about resistance to acid-mediated

toxicity, and that these enhance tumor growth and invasion. It also

represents, however, an opportunity for therapy because the tumor

microenvironment is typically extremely acidic which, in combination

with hypoxia, may produce significant cellular stress leading to

intratumoral regions of necrosis. Strategies to enhance this

toxicity and extend the regions of necrosis stem from recognition

that acid escapes from tumors by only two mechanisms: venous efflux

and local diffusion. Using mathematical models, it was demonstrated

that venous outflow can be significantly inhibited using even mild

systemic acidosis (Gatenby et al., 2002). This is due to the fact

that acid flow into intratumoral blood vessels is in part dependent

on the concentration gradient between the interstitial space and the

blood. If the acid concentration in the blood is increased, vascular

outflow of acid from the tumor dramatically decreases and the

intratumoral pH declines precipitously. As a result local acid

concentrations exceed the tolerance of even the tumor cells

triggering extensive tumor necrosis. Theoretical results have not

been explicitly tested but some indirect results are supportive.

Harguindey, Henderson, and Naeher (1979) demonstrated tumor-bearing

rats fed acidic chow had a substantial survival advantage when

compared to control (Harguindey et al., 1979).

Gatenby et al. (2002) found that patients who develop renal failure

(which is typically associated with metabolic acidosis) after

cytoreductive nephrectomy for metastatic renal cancer have a

substantial survival advantage compared to those who maintain normal

post operative renal function (survival of 17 months versus 4

months) (Gatenby et al., 2002).

Kelley, Manon, Buerk, Bauer, and Fraker, 2002) used isolate limb

perfusion in a rat model to examine simultaneous perfusion of acid

and melphalan for treating melanoma. They found the combination

resulted in consistent cure of the animals. However, in addition,

they showed that acid perfusion alone (pH of 6.8 for 10 min) induced

extensive apoptosis in the tumors and significant survival benefit

(Kelley et al., 2002).

CHAPTER FOUR: CONCLUSION

Tumorigenesis though beginning like a normal process in the body has

been shown to fascinatingly evolve abnormal but highly defensively

and offensively equipped cells. Finding a loophole to exploit in

cancer treatment must have been daunting over the years, as cancer

cells learn to adapt to different hazardous environment. However,

the evasiveness of cancer cells seems to solely rely on glucose and

glucose metabolism. The Warburg effect may have wide application

many of which still have gaps that need to be filled, but successful

inhibition of glycolytic enzymes especially hexokinase is given a

thumps up in the treatment of cancer. Tumor cells have been shown to

undergo apoptosis in the absence of the desired sugar. Also since a

lot of adaptive tendencies of cancer cells depend on HIF1

expression, which though emerges in response to hypoxia seems confer

on cancer cells the ability to survive. More research works need to

go into exploiting HIF1 then as it is central to tumorigenesis and

cancer development. Possible areas to exploit may be the up

regulation of the expression of von hippel lindau (VHL) protein-a

tumor suppressor capable of degrading HIF1. With HIF1 llimited,

tumorigenesis is highly restricted in terms of angiogenesis-HIF1 is

implicated in the expression of VEGF, expression of glycolytic

enzymes, and the downregulation of the tumor suppressor genes. Also

much of these works may be use in dietary adjustments for cancer

patients. Research works need to be devoted to exploiting sugar in

cancer metabolism as much prospects exist in future cancer therapy

and diagnosis from this perspective.