The Effect of Indole-3-Carbinol and 3,3’-Diindolylmethane on Fatty Acid Synthase and Sp1 in Breast Cancer Cells

THE EFFECT OF INDOLE-3-CARBINOL AND 3,3’-DIINDOLYLMETHANE ON FATTY ACID SYNTHASE AND SP1 IN BREAST CANCER CELLS

George Eramiah Saati

A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Nutritional Sciences University of Toronto

THE EFFECT OF INDOLE-3-CARBINOL AND 3,3’-DIINDOLYLMETHANE ON FATTY ACID SYNTHASE AND SP1 IN BREAST CANCER CELLS

Master of Science, 2009 George Eramiah Saati Graduate Department of Nutritional Sciences University of Toronto

ABSTRACT

Fatty acid synthase (FAS), an enzyme that is over-expressed in many cancers, is

necessary for cancer cell proliferation. Previously, we have shown that FAS in cancer cells is

regulated at least in part, by Sp1. Indole-3-carbinol (I3C) and its acid condensation product, 3,3’-

diindolylmethane (DIM) modulate various transcription factors involved in regulating cellular

proliferation and apoptosis. The objective of this study was to determine whether reductions in

breast cancer cell proliferation caused by I3C and/or DIM occur as a result of reductions in FAS.

DIM and, in some cases, I3C reduced FAS expression in three breast cancer cell lines. However,

addition of palmitate or oleate to DIM-treated MCF-7 cells did not restore proliferation. DIM- associated reduction in proliferation of MCF-7 cells also results in a reduction of Sp1 expression, and down-regulation of FAS occurs after inhibition of proliferation. Thus, the anti-proliferative effect of I3C and DIM may be due to their effect on down-regulating Sp1, which in turn could modify several Sp1-associated genes, including FAS.

ACKNOWLEDGEMENTS

There are a number of special people who were instrumental in making my graduate

experience, one of great enlightenment, excitement and accomplishment. I would like to extend

my sincere gratitude to them here.

First, I would like to thank my supervisor Dr. Michael Archer. Over the past two years,

Dr Archer has shared with me a wealth of knowledge, and given me the encouragement I’ve

needed to successfully complete this degree. He is a true gentleman and everyday around him has truly felt like a privilege. The ways he approaches situations in science and life are some of the most rewarding experiences I’ve ever received. Dr. Archer has helped me mature as an academic and a person. I will take what I have learned from him and do my best to apply as

much of it as I can to my life, in hopes that someday I too can lead myself, my family or an

organization to success – like him.

I would also like to thank my committee members, Dr. Ahmed El-Sohemy and Dr.

Richard Bazinet. I feel very privileged to have met these gentlemen and believe they both will

change the way our world understands nutrition. Dr. El-Sohemy, as one of my committee

members, I am sincerely grateful for your help and advice in completing my thesis work and

experiments. I also would like to thank you for introducing me to the world of nutritional

sciences and research, and for granting me the reference letters and referrals that made it into the

hands of Dr. Archer. Without your help, I don’t know if I would have made it this far

academically. Dr. Bazinet, I am extremely honoured to have had the opportunity to have had you

on my committee. Your unparalleled wisdom, expertise and support were paramount in the

progress and completion of this project. I thank you whole-heartedly for your all your help and suggestions.

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I will always be grateful to the people of Archer lab, whose help has been invaluable in all the years that I have worked in their lab. I would like to thank Suying Lu, Kafi Ealey,

Guodong Liu, Dominic Lau and Wanli Xuan for all their helpful advice and technical assistance.

I am grateful for their friendship and for making my laboratory experience an enjoyable one.

I also want to thank my external examiner, Dr. Wendy Ward, and my examination chair,

Dr. Hanley for being very accommodating and for offering helpful feedback and comments.

Next, I’d like to thank the many graduate students and friends who have made my stay here memorable. To Jennifer Truan, Amin Esfahani, Pedro Huot, Sandra Sacco, Alireza

Jahanmihan, Dennis Wagner, Jovanna Kaludjerovic, Sandra Reza-Lopez, Josh Green, Amanda

Carleton and Julie Mason, I will always be grateful for meeting you. The coffee breaks, the pubs, the restaurants downtown and, of course, the troubleshooting of experiments – I’m very thankful for being able to have shared these experiences with you.

Lastly, I would like to express my utmost love and gratitude to my family. My mother and father have given me an enormous amount of help and understanding and have provided me with food and shelter every single day. Also, my brother and my sister have always been there to lighten my day when I get home. Thank you all for putting up with me during my ups and downs. My family’s continued love and support over the years have helped me become who I am today, and for that, I will be forever grateful.

TABLE OF CONTENTS

CHAPTER ONE: INTRODUCTION AND LITERATURE REVIEW…………………...….1

Introduction To Diet and Cancer………………………………………………………………….2 Introduction To Thesis…………………………………………………………………………….4

1.1 Cruciferous Vegetables………………………………………………………………………..4 1.1.1 Glucosinolates……………………………………………………………………………….5 1.1.2 Activation of Glucosinolates by Myrosinase………………………………………………..8 1.1.3 Factors Influencing Myrosinase Activity…………………………………………………....9 1.1.4 Bioactive Glucosinolate Products………………………………………………………….11

1.2 Indole-3-Carbinol (I3C) and 3,3'-Diindolylmethane (DIM)…………………………………11 1.2.1 Tissue Distribution, Intake Levels and Physiologic Concentrations………………………12 1.2.2 Cancer Protective Effects of I3C and DIM in Rodents ……………………………………13 1.2.3 Cancer Protective Effects of I3C and DIM in Humans……………………………………17 1.2.4 Anticancer Effects of I3C and DIM in vitro: Possible Mechanisms of Action ……………17

1.3 Fatty Acid Synthesis…………………………………………………………………………22 1.3.1 Fatty Acid Synthase (FAS) and de novo Generation of Palmitate…………………………23 1.3.2 FAS in Normal Tissues…………………………………………………………………….26 1.3.3 Inhibitors of FAS…………………………………………………………………………..27 1.3.4 Regulation of FAS…………………………………………………………………………30 1.3.4.1 Short-term Regulation of FAS…………………………………………………………...30 1.3.4.2 Long-term Regulation of FAS…………………………………………………………...31 1.3.5 Transcription of FAS………………………………………………………………………32

1.4 FAS and Cancer……………………………………………………………………………...35 1.4.1 FAS and Breast Cancer ……………………………………………………………………36 1.4.2 FAS and Other Cancers……………………………………………………………………38 1.4.3 Regulation of FAS in Cancer………………………………………………………………38 1.4.4 Transcription Factors Regulating FAS…………………………………………………….40 1.4.5 Specificity Protein 1 (Sp1) in Cancer…………...…………………………………………42

1.5 Research Hypothesis and Objectives………………………………………………………...44

CHAPTER TWO: MATERIALS AND METHODS…………………………………………46

2.1 Cell Culture Conditions……………………………………………………………………...47 2.2 Cell Treatment……………………………………………………………………………….47 2.3 Cell Viability Analysis……………………………………………………………………….48 2.4 Preparation of Whole Cell Extracts and Western Blot Analysis……………………………..48 2.5 Preparation of Total RNA……………………………………………………………………49 2.6 Preparation of cDNA and Real-time PCR Analysis………………………………………….50 2.7 Rescue Experiments………………………………………………………………………….51 2.8 Inhibition of FAS with Cerulenin……………………………………………………………52 2.8 Statistical Analysis…………………………………………………………………………...52

CHAPTER THREE: RESULTS……………………………………………………………….53

3.1 The Effect of I3C on MCF-7 Cells…………………………………………………………..54 3.2 The Effect of DIM on MCF-7 Cells………………………………………………………….56 3.3 The Effect of I3C and DIM on MDA-MB-231 Cells………………………………………..59 3.4 The Effect of I3C and DIM on SKBR-3 Cells……………………………………………….61 3.5 MCF-7 Proliferation Restoration Experiments………………………………………………61 3.6 The Effect of DIM on MCF-10A Cells………………………………………………………64 3.7 The Effect of Cerulenin on MCF-10A Proliferation…………………………………………66

CHAPTER FOUR: GENERAL DISCUSSION AND CONCLUSION……………………...68

4.1 Discussion……………………………………………………………………………………69 4.2 Conclusion…………………………………………………………………………………...77 4.3 Implications………………..………………………………………………………………...77 4.4 Limitations and Future Directions..………………………………………………………….78

REFERENCES…………………………………………………………………………………81

LIST OF TABLES

Table 1.1 - Food sources of selected glucosinolates and their hydrolysis products that are currently under investigation for their cancer chemopreventative properties…………………….7

Table 4.1 – The effect of I3C on proliferation, FAS and Sp1 expression in MCF-7, MDA-MB- 231 and SKBr-3 cells……………………………………………………………….……………70 Table 4.2 – The effect of DIM on proliferation, FAS and Sp1 expression in MCF-7, MDA-MB- 231, SKBr-3 and MCF-10A cells………………………………………….…………………….70

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LIST OF FIGURES

Figure 1.1 - General chemical structure of a glucosinolate molecule…………………………….5

Figure 1.2 - Bioactivation of glucosinolates……………………………………………………...9

Figure 1.3 - Molecular structures of I3C and DIM……………...…………………………...….12

Figure 1.4 – Glycolysis and fatty acid synthesis………………………………………………...23

Figure 1.5 - Palmitate synthesis in FAS enzyme complex……………………………………...25

Figure 1.6 - The FAS promoter region and transcription factor binding areas………………….32

Figure 1.7 - Regulation of FAS expression in cancer by growth factors and steroid hormones…….……………………………………………………………………...39

Figure 1.8 - Post-translational regulation of FAS in cancer by USP2a…………………………40

Figure 3.1 - The effect of I3C on MCF-7 proliferation after 48 h and 72 h…………………….55

Figure 3.2 - The effect of I3C on FAS expression in MCF-7 cells……………………………...55

Figure 3.3 - The effect of DIM on MCF-7 proliferation and FAS expression………………….57

Figure 3.4 - 48h time-course evaluation of DIM on MCF-7 cells……………………………...58

Figure 3.5 – The effect of I3C and DIM on MDA-MB-231cells……………………………….60

Figure 3.6 – The effect of I3C and DIM on SKBR-3 cells…………………………………...... 62

Figure 3.7 - Rescue of DIM-treated MCF-7 Cells with palmitate and oleate…………………..63

Figure 3.8 - The effect of DIM on MCF-10A…………………………………………………..65

Figure 3.9 - The effect of cerulenin on MCF-7 and MCF-10A proliferation………..………….67

Figure 4.1 - I3C/DIM modulates nuclear transcription factor Sp1 and several cancer genes...... 76

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LIST OF ABBREVIATIONS

ACC acetyl-CoA carboxylase ACL ATP-citrate lyase APC adenomatous polyposis coli AhR aryl hydrocarbon receptor AOM azoxymethane ATP adenosine triphosphate BRCA1/2 breast cancer susceptibility protein 1 or 2 cAMP cyclic AMP cAPK cAMP-dependent protein kinase CDK cyclin dependent kinase ChRE carbohydrate response element CIN cervical intraepithelial neoplasia CLA conjugated linoleic acid CPT-1 carnitine palmitoyltransferase-1 CRE cAMP response element CYP cytochrome P450 enzyme DDS dextran sodium sulfate DH β –hydroxyacyl-ACP dehydrase DIM 3,3’-diindolylmethane DMBA 7,12-dimethylbenz (a)-anthracene EGCG epigallocatechin-3-gallate EGF epidermal growth factor EGFR epidermal growth factor receptor ER estrogen receptor ERD enoyl-ACP reductase ERK1/2 extracellular signal-regulated kinase 1 or 2 FAS fatty acid synthase FASKOL FAS knock out in liver GF growth factor GFR growth factor receptor GS glucosinolate HsTL hormone sensitive triacylglycerol lipase I3C indole-3-carbinol IRE insulin response element KS β-ketoacyl synthase KR β-ketoacyl-ACP reductase MAPK mitogen-activated protein kinase MAT malonyl-CoA-ACP transferase MNU methylnitrosourea NFκB nuclear factor κB NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

PEITC phenethyl isothiocynate PhIP 2-amino-1methyl-6-phenylimidazo[4,5-b]pyridine PI3 phosphoinositide-3-kinase SFN sulforaphane SH steroid hormone SHR steroid hormone receptor Sp1 specificity protein 1 SRE sterol response element SREBP sterol regulatory binding protein TE palmitoyl thioesterase TRAMP transgenic adenocarcinoma of the mouse prostate USF upstream stimulatory factor USP2a ubiquitin-specific protease 2a VIN vulval intraepithelial neoplasia XRE xenobiotic response element

CHAPTER ONE:

INTRODUCTION AND LITERATURE REVIEW

Introduction to Diet and Breast Cancer

Breast cancer is the most frequent malignancy occurring in women worldwide, and the

second leading cause of cancer death in Canadian women [1]. Globally, incidence and mortality

rates of breast cancer vary dramatically, with industrialized countries, excluding Japan, having

the highest incidence. Amongst industrialized populations, North Americans have the highest

incidence of breast cancer, with rates that are approximately three times higher than the Japanese

[2].

Inheritance of high penetrance susceptibility genes, such as BRCA1 and BRCA2, account for approximately 5% of breast cancer cases, whereas the factors responsible for the remaining

95% remain unclear. However, many epidemiological studies, particularly studies that followed

migrants from lower risk to higher risk countries, have provided strong evidence that diet and

environment play significant roles in the etiology of breast cancer. For example, data from 1962-

1971 on Italians living in Australia showed that while the breast cancer-associated mortality rate in recently arrived Italian migrants was low, it had risen to match that of the Australian-born

population in immigrants that resided there for 17 years or more [3]. Ziegler et al [4] showed that

in females of Chinese, Japanese or Filipino origin who had migrated to the US, breast cancer risk

had increased by as much as 80% in those who resided there for greater than 10 years. Similarly,

Shimizu et al [5] have shown that breast cancer incidence rates in US-born Japanese females is

2.6-fold higher than for Japanese women living in Japan and that, for ‘early-in-life’ Japanese

migrants to the USA, the incidence is 2.4-fold higher. Furthermore, Kolonel et al [6] have shown breast cancer incidence increased nearly three times in first generation Japanese women who had migrated to Hawaii. Additionally, they showed that in the second generation, incidence rates increased further to between four and five times. Overall, the evidence from migration studies

3 indicates that over time, changes in diet and/or environment can affect breast cancer incidence rates.

Although changes in both diet and environment occur following migration from countries with low to countries with high breast cancer incidence rates, the main factor responsible has not been determined. There is no published data, however, indicating that environmental change may be responsible for the increases in breast cancer risk. On the other hand, there is some evidence that implicates changes in diet or in cooking practices. The diets and cooking practices of migrants are known to become progressively more ‘Westernized’ following migration [7-8] and in Japan, for example, the ‘Westernization’ of diet has been found to correlate with increases in breast cancer incidence [8-9].

The Western diet is characterized by high intakes of red meat, fat, refined grains and dairy products [8]. Epidemiological evidence indicates that breast cancer is positively associated with consumption of diets high in meat and animal fat. In Europe and North America, for instance, where meat products contribute 14-15% of the dietary energy supply, while cereal and grain products only contribute 23%, the rate of breast cancer is amongst the highest in the world, while in Africa, where the cereals and grain products contribute up to 65% and meat products only contribute 2-5% of the dietary energy supply, the rate of breast cancer is relatively low [7].

Furthermore, it has recently been shown that cancer rates in cities within economically developing countries are now approximating the cancer rates of western cities. In Singapore, for example, the incidence of breast cancer approximately doubled between the 1970s and 1990s

[11]. The shift in breast cancer pattern here is thought to be a result of rapid economic development in this area, where energy dense foods such as meat and dairy products are becoming more affordable, and are replacing traditional phytochemical-rich fruits, vegetables

and legumes. This idea is supported by data from India, where cancers associated with Western

diets are also becoming more common in the more urbanized areas of the country [12].

Numerous dietary components have been hypothesized to reduce the risk of breast cancer

[13-23]. Epidemiological evidence indicates that high levels of cruciferous vegetable intake are

associated with reduced risk of acquiring several types of cancers, including breast cancer. For

example, in a cohort study of 628 men, Cohen et al. showed that consumption of 3 or more

servings of cruciferous vegetables per week was associated with a 41% decreased risk of prostate

cancer, compared with those eating less than one serving per week [24]. Data from the Health

Professionals Follow-up Study indicates that high cruciferous vegetable consumption may

reduce bladder cancer risk, while other vegetables and fruits may not confer appreciable benefits

against this cancer [25]. Furthermore, in an analysis of 87 case-control studies, Verhoven et al showed that 67% of the studies showed an inverse relationship between consumption of cruciferous vegetables and risk of cancer at various sites (including lung, breast, and colon) [26].

Introduction to Thesis

This thesis will focus on the protective effects of cruciferous vegetable consumption.

Specifically, it will describe our investigation of the effect of the bioactive crucifer component,

indole-3-carbinol (I3C), and its derivative product, 3,3’-diindolylmethane (DIM), on the

expression of fatty acid synthase (FAS), an enzyme involved in cancer cell proliferation, in

cultured breast cancer cells.

1.1 - Cruciferous Vegetables

Cruciferous vegetables come from plants in the family known to botanists as Cruciferae

or Brassicaceae. Plants in this family have flowers with four equal-sized petals in the shape of a

‘crucifer’ or cross. “Brassica” is the Latin term for cabbage. Many commonly consumed cruciferous vegetables come from the Brassica genus, including broccoli, Brussel sprouts, cabbage, cauliflower, bok choy, turnips, collard greens and kale. Although not in the Brassica genus, watercress, radish and arugala are also cruciferous vegetables [27].

Like other vegetables, cruciferous vegetables contain a number of components with cancer chemopreventative properties, including folate, fiber, carotenoids and minerals [27].

However, cruciferous vegetables are unique in that they are rich sources of glucosinolates. As will be discussed in the following sections, glucosinolates may play a significant role in the association between cruciferous vegetable consumption reduced cancer rates.

1.1.1 - Glucosinolates

Glucosinolates are a class of organic compounds that give cruciferous vegetables and some condiments, such as wasabi and mustard, their pungent aromas and spicy (some say bitter) taste. The main function of glucosinolates in plants is as natural pesticides and as a defense against herbivores [28].

Figure 1.1: General chemical structure of a glucosinolate molecule

Over 120 different glucosinolates have been identified [27]. Figure 1.1 shows the general chemical structure of glucosinolate molecules. The central carbon of all glucosinolates is bound

6 to a thioglucose group to a sulfate group via the nitrogen atom. The central carbon of each glucosinolate is also bound to a side group (designated R in Figure 1.1), and this is what makes each glucosinolate unique. The R-group is derived from protein amino acids and the diversity of glucosinolates is obtained by secondary modifications of the side chain and/or glucose moiety.

Glucosinolates are classified as aliphatic (e.g. alkyl, alkenyl, hydroxyalkenyl or w- methylthioalkyl), aromatic (e.g. benzyl, substituted benzyl) or heterocyclic (e.g. indolyl) [29], with the R-group of the aliphatic glucosinolates being derived from methionine, alanine, leucine or valine; and those of aromatic and heterocyclic glucosinolates being derived from tryptophan, phenylalanine or tyrosine.

Worldwide, cruciferous vegetable intake varies. It has been estimated that on average,

North Americans consume 14.7mg of glucosinolates from cruciferous vegetables daily [30]. On the other hand, in Japan, a country associated with lower incidence of several cancers (including breast cancer), it has been estimated that glucosinolate consumption from cruciferous vegetables averages 112mg per day [31]. However, it should be noted that in Japan, radishes are the primary source of glucosinolates, as they make up more than 1% of all food sold.

Table 1.1: Food sources of selected glucosinolates and their hydrolysis products that are currently under investigation for their cancer chemopreventative properties.

Each cruciferous vegetable contains a mixture of glucosinolates. Table 1.1 lists a number of cruciferous vegetables and the glucosinolates they contain. A number of comparative studies of glucosinolate distribution and variability between specific cruciferous vegetables have been performed. These studies have compared the glucosinolate levels in the edible portions of some of the most widely consumed cruciferous vegetables and suggest there are considerable differences in the glucosinolate levels between each type of crucifer. For example, Carlson et al

[32] reported 13 different glucosinolates in broccoli, Brussel sprouts, cauliflower and kale. The authors also showed that certain cultivars were highly correlated in their glucosinolate pattern and could be grouped together. Broccoli, for instance, formed one group with higher levels of glucoraphinin; Brussel sprouts and cauliflower with higher levels of glucobrassicin formed a second group; collard greens with higher levels of progoitrin formed a third group, and mustard greens with higher levels of sinigrin formed a fourth group. Radishes are a rich source of glucoraphasatin. Similar studies have since been performed comparing a wider array of crucifers and have demonstrated that the glucosinolate content of each cruciferous vegetables varies -

where each type of cruciferous vegetable contains higher levels of certain glucosinolates and

lower levels of other glucosinolates [33-34].

Additionally, it has been determined that glucosinolate levels in crucifers are also affected by the growing location. Shelp et al. [35] evaluated glucosinolate distribution in two broccoli cultivars grown at three different locations. They reported that differences in glucosinolate content among growing sites were greater than differences between cultivars.

Factors that may have contributed to these differences included soil type, sulphate and nitrate fertilizer application, plant spacing and date of harvest. Higher levels of glucosinolates were found in plants grown on clay soil than on sandy soil and in plants treated with sulphate than in

plants treated with nitrate. Hence, environment and factors associated with culture conditions

significantly influence cruciferous vegetable glucosinolate distribution.

1.1.2 - Activation of Glucosinolates by Myrosinase

Although glucosinolates are associated with inhibition of carcinogenesis, it is actually

their hydrolysis products, not the glucosinolates themselves that are biologically active.

Hydrolysis of glucosinolates is catalyzed by the enzyme myrosinase, also known as β-

thioglucoside glucohydrolase [28]. Glucosinolates co-exist with myrosinase in cruciferous

vegetables; however, the two are kept apart from each other as myrosinase is stored in

specialized cells known as myrosin cells. Upon wounding of the vegetable, for example, after

chopping, crushing, chewing or freeze-thawing, myrosinase may be released from the myrosin

cells. Myrosinase then acts by cleaving glucsinolates at their thioglycoside linkage to produce

glucose and an unstable aglycone thiohydroximate-O-sulfonate, which, in turn, spontaneously

rearranges to yield an isothiocyanate, nitrile or thiocynate product (Figure 1.2) [29].

Figure 1.2: Bioactivation of glucosinolates. Hydrolysis of glucosinolates by myrosinase to form nitriles, thiocyanates and isothiocyanates [image taken from ref 8].

The extent of hydrolysis of glucosinolates by plant myrosinase and the structure and

concentration of metabolites formed is influenced by various features of the hydrolysis

environment (such as the presence of ascorbic acid or Fe2+, and extrinsic factors such as pH and

temperature) [36, 37].

1.1.3 - Factors Influencing Myrosinase Activity

Several factors influence the glucosinolate-myrosinase hydrolysis reaction. Indeed, the activity of myrosinase can vary extensively between and within brassica species and in different parts of the plant [37]. However, cruciferous vegetables undergo a variety of post-harvest

processes, including storage, preparation and cooking, before they are consumed. During these

stages myrosin cells may deteriorate and the concentration of glucosinolates becomes altered, as

10 the bioactive glucosinolate products themselves deteriorate after a few days [38]. This, in turn, leads to considerable uncertainty in assessing rates of exposure to glucosinolates and their metabolites at target tissues.

Upon cooking cruciferous vegetables, the glucosinolate-myrosinase system can further become altered as a result of partial or total inactivation of myrosinase, thermal or plant myrosinase mediated breakdown of glucosinolates, loss of enzymatic cofactors, leaching of glucosinolates and their metabolites into the cooking medium or thermal degradation of the metabolites. These changes are mostly influenced by the duration and method of cooking, the type of vegetable matrix and the extent of its cellular disruption, and the chemical structure of the glucosinolate precursors [38]. Furthermore, the thermostability of myrosinase varies depending on the crucifer source. For example, myrosinase extracted from broccoli loses approximately 90% of its activity after heating at 60oC for 3 minutes at pH 6.5, whereas 30 minutes of heating homogenized red or white cabbage at pH 7 and 70oC is required to reduce its myrosinase activity by 90% [37, 39-40].

Following ingestion, a second phase of hydrolysis of glucosinolates may occur in the gastrointestinal tract under the action of the colonic microflora. Intestinal microflora that come in contact with intact glucosinolates activate them by digesting the glucosinolates themselves, cleaving the glucosinolate at its thioglycoside linkage, to produce the bioactive compound. The digestive and post-absorptive fate of glucosinolates in vivo may be influenced by factors such as the activity of plant myrosinase ingested, the extent of chewing, the nature of the meal containing the crucifers, the composition of the gut microflora and genotypic variation in post- absorptive metabolism [41-42].

1.1.4 - Bioactive Glucosinolate Products

As mentioned above, myrosinase-mediated glucosinolate hydrolysis reactions yield

products shown to have cancer chemopreventative properties. For example, gluconasturiin, the

major glucosinolate of watercress, is the precursor of phenethyl isothiocynate (PEITC) [56]. In

vitro, PEITC induces apoptosis of MCF-7 human breast cancer cells, and has been shown to inhibit tumorigenesis in rodents [43-46]. Glucoraphinin, a major glucosinolate in broccoli, is the precursor of sulforaphane (SFN), that like PEITC, also exerts cancer preventative properties such as the induction of apoptosis and the reduction of cellular proliferation of various cancer cell lines [43, 47-49]. Table 1.1 lists other isothiocyanates and indoles that are currently under investigation for their cancer chemopreventative properties, along with their glucosinolate precursors.

1.2 – Indole-3-Carbinol (I3C) and 3,3’–Diindolylmethane (DIM):

One of the most extensively studied glucosinolate hydrolysis products known to have

anticancer effects is indole-3-carbinol (I3C), also called 3-(hydroxymethyl)-indole (Figure). I3C

can be derived from several cruciferous vegetables (including broccoli, Brussel sprouts and

cabbage) and is produced by the hydrolysis of glucobrassicin [27].

In a low pH environment, such as that in the stomach, I3C is converted to a number of

oligomeric products (Figure 1.3a). It has been suggested that the observed biological activity of

I3C may be attributable mainly to the acid condensation products, in particular, 3,3’–diindolylmethane (DIM) (Figure 1.3b). DIM is the major in vivo I3C derivative making up 5.9-11% of the total amount of I3C condensation products in the stomach [50].

Figure 1.3: Molecular structures of (a) I3C and (b) DIM

Both I3C and DIM are known to have chemopreventative properties. The following section will discuss I3C intake levels, I3C and DIM tissue distribution levels, physiologically relevant in vitro doses of I3C and DIM, and will review much of the existing literature on the effect of I3C and/or DIM on cancer.

1.2.1 – Tissue Distribution, Intake Levels and Physiologic Concentrations

The tissue distribution of I3C has been determined in mice using radio-labelled I3C. Both

I3C and DIM have been detected in liver, kidney, lungs, heart, plasma and brain samples of mice treated with 250mg/kg of I3C as early as 15 minutes after administration [69]. These results suggest that I3C is rapidly absorbed and distributed to a number of well-perfused tissues, where it is converted to DIM to perform its anticancer actions.

Plasma and tissue levels of I3C and DIM have been extrapolated from animal studies, since levels for humans are not available. Mice fed 250mg/kg of I3C produced maximum concentrations of 29 and 170 μM I3C in their plasma and liver, respectively (15 min). I3C produced maximum concentrations of 4 and16 μM DIM in their plasma and liver, respectively

(after 2 h) [52]. By allometric scaling, Howells et al. have determined the I3C dose given to mice would equate to a 20mg/kg dose in humans (or 1.2g of I3C/d). To achieve intake, I3C must be taken as a supplement, since dietary intake of glucobrassicin, the precursor of I3C in cruciferous vegetables is much lower. In the United States and the United Kingdom, estimated per capita intakes of glucobrassicin are 8.1 and 19.4mg/d, respectively [53,54], whereas in some populations in Asia, the level reaches 46mg/d [55]. As will be further explained below, I3C, taken as a supplement, has been shown to have chemopreventative effects at doses ranging between 200-800mg/d.

In vitro studies, many of which are mentioned below, that have investigated the mechanisms involved in the anticancer effect of I3C have treated cells with doses of I3C ranging from 10 nM to 250 μM. Howells et al. have reviewed a number of in vivo and in vitro studies to determine physiologically relevant in vitro doses of I3C and DIM. They suggest that serum concentrations of up to 150 µM for I3C and 50 µM for DIM are physiologically relevant to humans [52]. These concentrations have been considered in the design of our in vitro studies.

1.2.2 – Cancer Protective Effects of I3C and DIM in Rodents

I3C in Rodents

It has been demonstrated that I3C is active against a number of carcinogen-induced and spontaneous tumors in multiple tissues in rodents.

Breast Cancer - Several studies have evaluated the efficacy of I3C in the prevention of chemically-induced mammary tumors. Using the 7,12-dimethylbenz[α]anthracene (DMBA)- model, Grubbs et al [56] have shown that mammary tumor multiplicity may be reduced by 91-

96% in Sprague-Dawley rats after administration of 50mg I3C/kg of diet per day five times a week during the initiation phase. Similarly, 50mg I3C/kg of diet given both prior to and after

treatment with MNU (a direct acting carcinogen) caused a 65% decrease in mammary tumor

multiplicity in Sprague Dawley rats [56]. Bradlow et al [57] have shown that C3H/OuJ mice

given diets containing 500-2000 ppm I3C for 8 months have significantly lower spontaneous

mammary tumor incidence and multiplicity compared to those given the a diet without I3C.

Furthermore, the mice given the high I3C dose also had a prolonged tumor latency.

Liver Cancer – I3C supplementation has effectively reduced tumor formation in liver.

Manson et al [58] have shown that I3C (making up 0.5% of the diet) fed to male Fischer rats 2

weeks prior to administration of aflatoxin B1 reduced the development of preneoplastic hepatic

lesions by 53%.

Lung Cancer - Morse et al [59] showed a 40% inhibition of NNK- induced pulmonary

adenoma multiplicity (NNK is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, an important lung carcinogen present in tobacco smoke) in A/J mice pre-treated with I3C. In a similar study,

Kassie et al [60] tested the chemopreventative efficacy of I3C against lung tumorigenesis induced by a mixture of NNK and benzo[a]pyrene (BaP) in A/J mice. They showed that of 112

μg I3C/g of diet for 23 weeks resulted in a reduction of lung tumor multiplicity by 86%.

Furthermore, compared to the lung tissues of untreated mice, I3C inhibited expression of SP-C,

L-plastin, annexin A1, and haptoglobin – several proteins associated with lung cancer. In addition, I3C was shown to suppress breast cancer cell metastases, as well as inhibit the formation of lung surface metastatic nodules when poorly invasive MCF-7 or highly invasive

MCF-468 cells were injected intravenously into mice.

Tongue Cancer - Tanaka et al [61] have shown that a diet containing I3C at a dose of

1000 ppm inhibits 4-nitroquinoline 1-oxide (4NQO-) induced neoplasm formation in ACI/N rat tongues, when given during both the initiation and post-initiation phases of carcinogenesis.

Endometrial Cancer - Kojima et al [62] showed that a diet containing 1000 ppm I3C resulted in a 44% reduction in the occurrence of spontaneous endometrial adenocarcinomas in

Donryu rats.

Cervical Cancer – Transgenic mice expressing the human papillomavirus type 16 under a keratin 14 promoter (K14-HPV16 mice), developed cervical cancer when they were given 17β- estradiol chronically. Jin et al [63] showed that mice fed I3C at a dose of 2000ppm in the diet had a 68% lower in cervical-vaginal cancer incidence after 6 months compared to the unsupplemented group. Furthermore, they showed that I3C prevented morbidity associated with retention of fluid in the bladder that frequently occurred with the higher estradiol dose.

Additionally, I3C appeared to reduce skin cancer in the transgenic mice.

DIM in Rodents

Several studies have been published on the effect of DIM on carcinogen-induced tumors in rodents. As with I3C, the results from the existing data appear promising as they provide evidence of a strong protective effect for DIM against the formation of certain cancers.

Breast Cancer – Chen et al [64] showed that administration of 5 mg/kg DIM every other day for a total of 20 days significantly inhibited DMBA-induced mammary tumorigenesis in female Sprague–Dawley rats. Using a complementary in vivo Matrigel plug angiogenesis assay,

Chang et al [65] have demonstrated that, compared with vehicle control, neovascularisation of mammary tumors in female C57BL/6 mice may be inhibited by up to 76% following the administration of 5mg/kg DIM. Furthermore, they have shown that this dose of DIM also inhibits the growth of human MCF-7 cell tumor xenografts by up to 64% in female athymic (nu/nu) mice compared to vehicle control [66]. Finally, McDougal et al [67] have shown that DIM significantly inhibits mammary tumor growth in female B6C3F1 mice implanted with a T47D

(estrogen sensitive human breast cancer cell line) xenograft, at a low DIM dose of 1mg/kg of

body weight.

Colon cancer – In an azoxymethane (AOM)-initiated colon carcinogenesis model, Kim et

al [68] have showed that DIM administration dramatically decreased the number of colon tumors

in BALB/c mice treated with 20mg DIM/kg. Furthermore, the antiinflammatory effects of DIM

in dextran sodium sulfate (DSS)-induced colitis was also inestigated in this study, and it was

shown that DIM significantly attenuated the loss of body weight and shortening of the colon -

two severe symptoms in this colitis model. Furthermore, they showed an associated reduction in

the disruption of the colonic architecture, a significant reduction in colonic myeloperoxidase

activity and production of prostaglandin E(2), nitric oxide, and proinflammatory cytokines – all

of which contribute to colitis.

Lung Cancer - In a follow-up to the study described for I3C in the previous section,

Kassie et al [69] tested the efficacy of DIM against lung tumorigenesis induced by a mixture of

NNK and benzo[a]pyrene (BaP) in A/J mice. They showed that DIM at 30 μg/g of diet for 23

weeks resulted in a reduction of NNK plus BaP-induced lung tumor multiplicity by 66%. They showed differences in the relative abundance of proteins associated with lung tumors in the pulmonary tissues of carcinogen-treated versus untreated mice. Specifically they found that fatty acid synthase, transketolase, pulmonary surfactant-associated protein C (SP-C), L-plastin,

annexin A1, and haptoglobin increased, whereas transferrin, alpha-1-antitrypsin, and apolipoprotein A-1 decreased.

Prostate Cancer – Nachshon-Kedmi et al [70] have demonstrated that DIM given to male

C57BL/6 mice 3 times a week for 3 weeks at doses of 2.5-10mg/kg body weight inhibits tumor growth of TRAMP-C2 (a mouse prostate cancer cell line) xenografts by up to 46%, compared to

vehicle control. Histological examination of the tumors from treated groups revealed increased

apoptotic activity and decreased cell proliferation compared with the control tumors.

1.2.3 – Cancer Protective Effects of I3C and DIM in Humans

In humans, the bioavailabilities of I3C and DIM have not been determined. As well, no

studies have been published on the in vivo effect of DIM in humans [52]. However, phase I and

phase II clinical trials have shown that orally administered I3C and/or its acid-condensation products have dose-dependent anticancer effects [71-73]. For example, female subjects with

cervical intraepithelial neoplasia (CIN) or vulval intraepithelial neoplasia (VIN) treated with

either 200 or 400 mg/d of I3C showed complete regression of CIN after 12 weeks of treatment

compared to placebo groups [74-76].

In vivo, estrogen is metabolized to several hydroxylated forms. The 2-OH derivative is

the protective, less potent estrogen; while, 16-OH estrogen, the potent form of estrogen,

increases estrogenic effects (e.g. breast cell mitosis) and accumulation of it is associated with

high risk of breast cancer. I3C treatments of 200-800 mg/d have been shown to significantly

elevate the 2-hydroxyestrone to 16α-hydroxyestrone ratio in these women compared to placebo

group, the degree of elevation depending on the administered dose [72,74].

1.2.4 – Anticancer Effects of I3C and DIM in vitro: Possible Mechanisms of Action

I3C and DIM have been studied in a number of cell lines. Both I3C and DIM have been

shown to modify nuclear transcription factors (e.g. ER, AhR, NFκB and Sp1) involved in cancer-

related cell processes including xenobiotic metabolism, cell cycle progression, proliferation

apoptosis and metastasis. This section will review the mechanisms through which I3C and DIM

have been shown to exert their anticancer effects.

Estrogen Receptor (ER)

Estrogens exert their effects by binding to estrogen receptors (ERs). Within the nucleus, the estrogen-ER complex can bind to DNA sequences in genes known as estrogen response

elements (EREs), and enhance the transcription of estrogen responsive genes.

When added to breast cancer cells in culture, I3C has been found to inhibit cell

proliferation. The decreased proliferation occurs because I3C modifies the ER phospholylation

and DNA binding domains, which in turn, inhibits the transcription of estrogen-responsive genes

[77]. Meng et al have shown a dose-dependent repression in estrogen activated ER signaling and transcriptional activity when I3C is added to MCF-7 cells at 10-125µM [78]. Similarly, DIM has been shown to interfere with ER-DNA binding, signaling and ER expression [79,80].

Estrogen dependent genes essential for tumor cell viability, such as epidermal growth factor receptor (EGFR) in breast cancer, have been shown to decrease with I3C and DIM treatment (75 µM and 25 µM, respectively) [81, 82]. Similarly, I3C (50 µM) has been shown to inhibit phosphoinositide-3-kinase (PI3K), resulting in the inhibition of protein kinase B (Akt) phosphorylation and decreased survival of cancer cells dependent on this pathway [83]. As well, breast cancer susceptibility proteins (BRCA1/2) have been shown to be upregulated after

I3C/DIM treatment, and a mechanism involving ER modification by I3C/DIM has been proposed [77]. Hence, the anticancer effect of I3C/DIM can partly be explained by its ability modify the ER, which leads to changes in the expression of genes that inhibit cellular proliferation and promote apoptosis.

The Aryl Hydrocarbon Receptor (AhR)

The AhR is a nuclear transcription factor that can be activated in the cytoplasm by binding to several types of aromatic compounds (e.g. dioxins, flavonoids) including I3C and

DIM [77]. Binding allows the AhR to enter the nucleus where it forms a complex with the AhR

nuclear translocator (Arnt) protein. This Ahr/Arnt complex binds to specific DNA sequences in

genes known as xenobiotic response elements (XRE) and enhances their transcription. Genes for

a number of CYP (e.g. CYP1A1, CYP1B1) enzymes and several phase II enzymes (e.g.

glutatione-S-transferase) are known to contain XREs. By increasing the activity of

biotransformation enzymes, I3C and DIM are thought to exert part of their anticancer effect by

enhacing the metabolism or elimination of potential carcinogens or toxins [52].

As mentioned above increased levels of circulating 16-OH estrogen is known to enhance

breast cancer cell proliferation and tumorigenesis [77]. Steroid hydroxylases related to CYP1A1

catalyze the formation of 2-OH estrogen, and can increase the ratio of circulating 2-OH:16-OH

estrogen [52]. Ociepa-Zawal et al. have shown that MCF-7 cells increase transcription and

activity of CYP1A1 following 48 hour treatment with 100µM I3C [84], while Chen et al [64]

have similarly shown that 24 hour treatment with DIM at 50 µM induces CYP1A1 gene

expression in these cells. As well, rats given I3C or DIM have been shown to increase the

capacity of hepatic microsomes to convert estrogen to 2-OH estrogen and increased expression

of CYP1A1 [85]. Hence, part of the protective effect of I3C and DIM includes its influence on

the AhR to induce hormone and xenobiotic metabolism.

Nuclear Factor κB (NFκB)

The anticancer effect of I3C and DIM has also been suggested to be partly due to an increase in cell apoptosis (programmed cell death). NFκB is a transcription factor that has an important role in regulating the genes involved with the apoptotic process. NFκB binds to the promoter region of the Bcl-2 gene (antiapoptotic protein) and allows its expression. When NFκB expression is inhibited, Bax (a proapoptotic protein) is released from the Bax/Bcl-2 dimer and

can induce apoptosis [77]. Studies with several breast cancer lines indicate that the relative

amounts of Bax and Bcl-2 proteins are highly predictive of sensitivity to apoptosis in mammary

tumor cells.

Translocation of Bax protein from the cytosol to the mitochondria results in

mitochondrial depolarization and the release of apoptosis associated factors in the mitochondria

(e.g. apoptosis-protease-activated factor 1, apoptosis-inducing factor 1 and cytochrome c) [77].

Rahman et al. [86] showed that apoptosis was induced in MCF-7 cells treated with 60 µM I3C for 48 h, with an associated decreased level of Bcl-2. Similarly DIM has been shown to increase the Bax/Bcl-2 ratio in MCF-7 cells [87]. Hence, I3C/DIM modulates apoptosis by inhibiting the

activation of NFκB, which allows for the release of apoptosis associated factors resulting from

increased levels of free mitochondrial Bax.

Specificity Protein 1 (Sp1)

I3C and DIM have been shown in vitro to modulate nuclear transcription factors that are involved in regulating downstream cellular events such as cell cycle progression. The nuclear transcription factor specificity protein 1 (Sp1) is a sequence-specific DNA-binding protein that activates a broad and diverse spectrum of mammalian and viral genes. It acts by recognizing GC- rich promoter elements and interacting with DNA through three zinc fingers located at the C- terminal domain, leading to expression of those genes [88]. In cancer, Sp1 protein plays a critical role in the growth and metastasis of many tumor types by regulating expression of cell cycle genes [77].

I3C and DIM have been shown to regulate expression of cyclin-dependent kinase 6

(CDK6), an enzyme involved in cell cycle control, in MCF-7, MDA-MB231 and LNCaP cells.

I3C/DIM disrupts the interactions of Sp1 with its DNA-binding site within the CDK6 promoter,

leading to suppression of CDK6 expression and cell cycle arrest at the G1 phase [89-92].

Similarly, MCF-7 and MDA-MB231 cells treated 50uM DIM for 48 hours have decreased expression of CDK6 and G1 cell cycle arrest [122, 124-125]. Futhermore, it has also been shown

that the G1 cell cycle arrest is due to elevated p21 levels (a CDK inhibitor). DIM is thought to

induce the binding of Sp1 to the Sp1-responsive elements of p21 in its promoter region, thereby

increasing p21 expression in MCF-7 cells [89]. Hence, by modifying Sp1 binding, I3C/DIM acts

on cancer cells to arrest cell cycle progression and decrease the proliferation rate of cancer cells.

The relationship between I3C/DIM, Sp1 and proliferation will be further explored in

relation to my thesis experiments in the next section.

Metastasis

I3C has also been shown to affect breast cancer cell motility. Brew et al [93]

demonstrated that 100uM I3C significantly decreases in vitro migration of MDA-MB231 cells -

an indole-sensitive, highly invasive breast cancer cell line. I3C treatment activates Rho kinase

activity in these cells, resulting in the formation of localized stress fibers and peripheral focal

adhesions, which decrease overall cell motility.

Furthermore, elastase (an intracellular protease) activity has been shown to be inhibited

in MDA-MB231 cells treated with 100uM I3C [94]. High elastase activity is a marker for breast

cancer survival and metastasis progression. Inhibition of elastase causes the accumulation of 50- kDA cyclin E and loss of 35kDA cyclin E (the larger form is typically expressed in normal mammary tissue, while the smaller form is associated with poor clinical outcomes). Disruption of the proteolytic processing of the 50-kDA cyclin E into the smaller form by I3C inhibits cell cycle progression and cell motility. This inhibition effect of I3C on elastase, however, is specific to I3C since 50 μM DIM did not produce similar results [94].

1.3 - Fatty Acid Synthesis

Fatty acid synthesis is a biochemical process common to all animals that converts excess

dietary glucose to the non-essential fatty acid palmitate. The process begins with glycolysis

(Figure 1.4). Glucose is converted to glucose-6-phosphate by hexokinase, which then enters the glycolytic pathway to generate pyruvate, the major glycolytic end-product, and ATP. Cancer cells produce excess pyruvate as they have an elevated rate of glucose catabolism compared to non-cancer cells [95]. Most pyruvate is converted to lactate in the cytoplasm by lactate-

dehydrogenase and is secreted from the cell; however, some pyruvate is taken up by the

mitochondria and enters the citric acid cycle, producing acetyl coenzyme A (CoA) [96].

In the mitochondria, acetyl CoA is converted to citrate, that can be transported to the

cytosol by tricarboxylate translocase. Citrate, in the cytoplasm, is then converted back to acetyl-

CoA by ATP citrate lyase (ACL), where a portion of it is carboxylated to malonyl-CoA by

acetyl-CoA carboxylase (ACC). Fatty acid synthase (FAS) then performs the condensation of

acetyl-CoA and malonyl-CoA (and NADPH as a reducing agent) to produce palmitate, a 16-

carbon saturated fatty acid (further described below), and other saturated long-chain fatty acids.

Saturated long-chain fatty acids may further be modified by elongases (in the

mitochondria and endoplasmi reticulum) or desaturases to form more complex fatty acids, that

may be used for the synthesis of various cellular lipids such as phospholipids, triglycerides and

cholesterol esters [96].

Figure 1.4: Glycolysis and fatty acid synthesis .[image taken from ref 95]

1.3.1 - Fatty Acid Synthase (FAS) and de novo Generation of Palmitate

Of the various enzymes mentioned in the previous section, FAS is the major biosynthetic

enzyme involved in de novo palmitate synthesis. FAS may also produce smaller amounts of myristate, laureate, and even shorter-chain fatty acids. Two kinds of FAS proteins are classically

recognized: cytosolic FAS (FAS I) and mitochondrial FAS (FAS II). Cytosolic FAS is primarily

responsible for de novo fatty acid synthesis, whereas mitochondrial FAS provides octanoyl

precursors required for the essential lipoylation pathway [97,98].

Mammalian FAS consists of two identical multifunctional 250 kDa polypeptides. The monomers are arranged in a head-to-tail configuration and are held together by two thio-disulfide

bonds. Each monomer includes seven catalytic domains, and each of the domains has a distinct

activity required for fatty acid biosynthesis [95]. The enzymes in the FAS complex include:

malonyl/acetyl-CoA-ACP transferase (MAT), β-ketoacyl synthase (KS), β-ketoacyl-ACP

reductase (KR), β –hydroxyacyl-ACP dehydrase (DH), enoyl-ACP reductase (ER) and palmitoyl

thioesterase (TE). The clustering of the domains in FAS offers great efficiency and coordination

during palmitate synthesis [96].

The reactions catalyzed by each domain in mammalian FAS are illustrated in Figure 5.

Synthesis of palmitate begins with the “loading” of FAS with malonyl-CoA. The malonyl group

originally linked as a thioester in malonyl-CoA, is first transferred first to the anchoring protein

acyl-carrier protein (ACP) (1) and then to the Cys residue KS enzyme to form malonyl-ACP, the

growing fatty acid (2a). Similarly, an acetyl group is transferred from acetyl-CoA to ACP to

form acetyl-ACP (2b).

In the next step (3), known as the condensation reaction, malonyl-ACP is decarboxylated, driving a resulting carbanion to attack the acetyl-thioester. This reaction, in turn, results in the formation of a four-carbon β-keto-ACP.

Two reductions and a dehydration (4-6) then convert the β-keto group to an alkyl group, where the coenzyme in both reductive steps is NADPH. Here, NADPH is provided in a reaction catalyzed by malic enzyme or can be acquired through the pentose phosphate pathway.

At this point, the acyl group, originally an acetyl group, has been elongated by a C2 unit.

The butyryl group is then transferred from ACP to the Cys-SH in the KS domain (a repeat of

2b). In each reaction cycle, the ACP is “reloaded” two carbon atoms. Seven such cycles are required to form palmitoyl-ACP. The thioester bond is then hydrolyzed by palmitoyl thioesterase

(7), yielding palmitate, and regenerating the enzyme for a new round of synthesis [98].

2a 2b

thioesterase

Palmitate

Figure 1.5: Palmitate synthesis in FAS enzyme complex [image taken from ref 98]

1.3.2 - FAS in Normal Tissues

FAS expression varies in a tissue specific manner. Most normal tissues, such as the skin

or skeletal muscle, express FAS at low levels and synthesize low levels of palmitate [99]. Cells

from these tissues acquire fatty acids from circulating levels made available by the diet rather

than via de novo synthesis.

Liver and adipose tissues, on the other hand, are highly lipogenic tissues and express FAS

at relatively higher levels. Fatty acid synthesis in adipose tissue, for example, occurs 10-1000

times more rapidly than it does in skeletal muscle cells [101]. In adipose, FAS functions to

maintain energy balance for the organism, converting excess calories from the diet into storage

lipid. In the liver, FAS functions to synthesise fat from protein and carbohydrate when dietary

sources of fat are lacking.

In mammals, expression of FAS in certain tissues also depends on stage in development.

For example, in the maturing fetal lung, FAS is elevated to provide the fetus with palmitic acid

for the synthesis of lecithin. After birth, FAS levels in the lungs of newborns lower and steadily

plateau to adult lung levels. Similarly, during lactation, FAS is elevated in the mammary gland to

supply medium chain fatty acids, such as myristate, for breast milk [101]. As the frequency

breast-feeding decreases, FAS levels in the mammary gland also decrease.

Few diseases directly involve the fatty acid synthesis pathway, although variations of

FAS activity may have an influence on diseases such as obesity and diabetes. Nonetheless, fatty acid synthesis has been shown to be crucial for development. In mice, FAS homozygous knockouts are non-viable, with most of the embryos dying before implantation [101]. As well, most heterozygous FAS knockout mice die at various stages of embryonic development [102].

Surviving heterozygous mice possess 50% and 35% lower FAS mRNA and activity compared to wild types, suggesting that the lack of FAS gene has significant teratogenic consequences.

Additionally, conditional knockout FASKOL (FAS knockout in liver) mice develop fatty livers

and liver disease when consuming fat free diets [103]. In these mice, fatty acids are mobilized

from the adipose tissue to the liver. However, due to the liver’s inability to synthesize new fatty

acids, the incoming fatty acids are not oxidized and build up. This suggests that FAS, is

necessary to maintain normal health and that a basal level of de novo fatty acid synthesis is

required.

1.3.3 - Inhibitors of FAS

A number of molecules that target FAS and inhibit its activity exist in nature or have

been developed.

Cerulenin

Cerulenin ([2S,3R] 2,3-epoxy-4-oxo-7E,10E-dodecadienamide) is an antifungal antibiotic found naturally in the industrial strain of the fungus cephalosporium ceruleans [104].

Cerulenin covalently binds to the FAS β-ketoacyl synthase moiety, thus preventing the condensation reaction between the elongating fatty acid chain and successive acetyl or malonyl residues [105]. In vitro, cerulenin causes dose-dependent decreases in cell proliferation of breast, ovarian, prostate and colon cancer cells [106-108]. Pro-apoptotic effects have been shown in colon cancer cells as well [109]. The clinical relevance of cerulenin, however, is limited because of the chemical instability caused by its very reactive epoxy group. Furthermore, its been demonstrated that a dose of 30 milligrams per kilogram of cerulenin inhibits feeding and induces dramatic weight loss of mice by a mechanism similar to, but independent of, leptin signaling

[110].

C75

C75 (4-methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid) is a cerulenin-derived

semi-synthetic FAS inhibitor. Like cerulenin, C75 covalently binds to the FAS β-ketoacyl

synthase moiety and prevents the condensation reaction. Unlike cerulenin, C75 lacking the

reactive epoxy group making it a more stable and effective treatment [111]. C75 also increases

activity of carnitine palmitoyltransferase-1 (CPT-1), an enzyme involved in fatty acid oxidation; hence, making it a more potent weight-loss-promoting drug than cerulenin. Both lean and obese

mice treated with C75 undergo rapid and profound weight loss and loss of adipose mass [112].

Furthermore, cells and mice treated with doses of C75 as low as 10uM have shown cytotoxic

effects [113]; hence, precluding it from being developed as a safe drug.

Orlistat

The β-lactone Orlistat (1-(3-hexyl-4-oxo-oxetan-2-yl)tridecan-2-yl 2-formylamino-4- methyl-pentanoate) is a US FDA-approved anti-obesity drug [114]. Orlistat elicits its effect by blocking the palmitoyl thioesterase domain in the FAS complex, which is responsible for releasing palmitate from FAS [115]. Orlistat has been shown to arrest proliferation and induce apoptosis of LNCaP, DU-145, and PC-3 prostate cancer cell lines [116]. Furthermore, it has been shown to inhibit growth of PC-3 tumors in male athymic nude mice [115]. Clinically, however,

Orlistat has poor solubility and low oral bioavailability [117]; hence, it may not necessarily be able to act on tumors other than those confined to the gastrointestinal tract.

Triclosan

Triclosan (2,4,4’-trichloro-2’-hydroxydiphenyl ether) is a widely-used antibiotic in soaps, mouthwashes and other oral health care products, and blocks FAS activity by inhibiting the

29 enoyl-ACP reductase domain [118]. Triclosan has been shown to inhibit MCF-7 and SKBr-3 proliferation by as much as 50% when given at a concentration of 50 uM [119]. A diet containing 1000 ppm triclosan has been shown to suppress MNU-induced-mammary carcinogenesis of Sprague-Dawley rats [120]. Furthermore, acute, subacute/subchronic, and chronic toxicity tests have determined that triclosan is neither an acute oral toxicant nor that it acts as a carcinogen, mutagen, or teratogen [121]. However, triclosan has been associated with side effects, the major one being that it disrupts the colonic bacteria of rats, dogs and rabbits.

Dietary compounds

Several dietary compounds have been shown to reduce FAS activity. For example, epigallocatechin-3gallate (EGCG), a component of green tea, acts similarly to cerulenin and C75 by blocking the FAS KS domain [122]. Other EGCG-related naturally occurring polyphenols

(the flavonoids luteolin, quercetin, and kaempferol) also block FAS KS activity [123]. Water- soluble organosulfur compounds of garlic have been shown to inhibit FAS activity by inhibiting the enoyl-ACP reductase domain, similarly to triclosan [124]. Furthermore, a diet given to male

Wistar containing 0.2% dietary sesamin, the most abundant lignin in sesame seeds, results in the reduction of both FAS activity and mRNA levels [125]. As well, rats consuming a diet containing 0.2% curcumin, a bioactive component in turmeric, have reductions in their hepatic

FAS activity [126]. And finally, extracts from traditional Chinese medicinal herbs such as ginko leaf and fleeceflower root have also been shown to directly inhibit FAS activity in MDA-MB435 breast cancer and PC-3 prostate cancer cell lines [127].

1.3.4 - Regulation of FAS

Regulation of FAS may be classified as either short-term (minutes to hours) or long-term

(hours to days). Short-term regulation usually involves modification of FAS enzyme activity,

while long-term regulation refers to the regulation of FAS at its mRNA expression or enzyme

synthesis/breakdown level.

1.3.4.1 - Short-term regulation

In normal tissues, FAS activity in the short-term is regulated by circulating fatty acids and by hormones affected by the diet (i.e insulin and glucagon). During fasting, for example, as insulin and free fatty acid levels decrease in the blood, while glucagon, epinephrine and norepinephrine levels rise. The increasing concentration of these hormones causes an elevation of cAMP levels in the lipogenic tissues (i.e. the liver and adipose). cAMP is an allosteric activator of cAMP-dependent protein kinase (cAPK), which, in turn, phosphorylates and

activates several enzymes in the β-oxidation pathway including hormone-sensitive

triacylglycerol lipase (HsTL) in the liver and adipose tissue. (HsTL frees fatty acids for

metabolism by the liver and muscle). cAPK also inactivates acetyl-CoA carboxylase – the

enzyme that converts acetyl-CoA to malonyl CoA - reducing the level of substrate available for

palmitate synthesis by FAS [128]. Therefore, during fasting, glucagons, epinephrine and

norepinephrine signals cause a reduction in FAS activity by limiting the amount of available

malonyl-CoA levels.

On the other hand, during the fed state, as free fatty acid and insulin levels in the blood

rise, FAS activity in the liver and adipose increase compared to the fasting state. Insulin has the

opposite effect of glucagon, epinephrine and norepinephrine: it decreases cAMP levels, leading

to a reduction in cAPK production. A reduction in cAPK leads to the dephosphorylation and

inactivation of of HsTL, decreasing the amount of fatty acids available for oxidation. More

importantly, less cAPK also results in the activation of acetyl-CoA carboxylase. This leads to

increased malonyl-CoA synthesis, which in turn, increases the amount of substrates availability

for palmitate synthesis by FAS [128]. Therefore, during the fed state, insulin signals increase

FAS activity by elevating the amount of available malonyl-CoA levels.

1.3.4.2 - Long-term Regulation

Long-term regulation of FAS activity involves modifications in the expression of FAS,

and is highly dependent on the nutritional and hormonal conditions in lipogenic tissues.

Nutritional Regulation of FAS

Nutritional and hormonal stimuli have been shown to regulate FAS transcription.

Carbohydrate fasting and re-feeding, for example, up-regulates FAS expression, and insulin has

been implicated as a possible regulator of this elevation in FAS. Lakhshmanan et al have shown

that feeding a high carbohydrate, fat-free diet to rats fasting for 48-h causes a supraphysiologic increase of hepatic FAS expression and activity compared to normally fed rats [129]. In a follow- up study, they showed that male Holtzman rats – a strain of diabetic rats – do not exhibit the same increase in hepatic FAS activity upon refeeding after a 48-h fast [130]. However, after treatment with insulin, FAS expression in these rats becomes elevated to the supraphysiologic levels observed in the normal “fasting and re-fed” rats. Similarly, insulin has been shown to stimulate an approximately five- and three-fold increase in FAS mRNA and transcription rate in adipocytes and hepatoma cells, respectively; glucagon, on the other hand, suppressed FAS expression in these cells [131]. These results therefore indicate that FAS expression is highly responsive to the dietary insulin response.

Regulation of FAS in Hormone-Sensitive Tissues

As mentioned above, FAS is also highly expressed in hormone-sensitive cells such as in

the mammary glands. During lactation, stimulation of FASN expression and activity in the

mammary glands are considered to be due to the increases in cortisol, prolactin, and insulin and

the decrease in progestins [132]. Furthermore, during the menstrual cycle, the expression of FAS

in the human endometrium is closely linked to the expression of the proliferation antigen Ki-67,

estrogen receptor, and progesterone receptor, which suggest a functional connection between

FAS and the estradiol (E2)/estrogen receptor–dependent signaling in the normal control of endometrial cell proliferation [133].

1.3.5 - Transcription of FAS

Several transcription factor binding regions have been identified in the FAS promoter

such as the sterol response element (SRE), insulin response element (IRE), carbohydrate

response element (ChRE) and the cAMP response element (CRE). Transcription factors that

have been identified to bind these DNA elements in the FAS promoter and determine the degree

of cellular FAS transcript expression including: sterol response element binding proteins

(SREBP), upstream stimulatory factors (USF), and specificity protein (Sp1) [95,130]. Figure 1.6

illustrates showing the binding areas for these transcription factors in the FAS promoter.

SRE IRE

E-Box (USF) (SREBP) __(Sp1)__ (SREBP) E-Box (USF)

Figure 1.6: The FAS promoter region showing the binding areas for transcription factors involved in the regulation of FAS expression.

This section discusses the relationships between these transcription factors and FAS in

normal tissues.

SREBP-1

SREBP-1 belongs to the basic-helix-loop-helix leucine zipper class of transcription

factors. There are two isoforms of SREBP-1, 1a and 1c, both of which are derived from the same gene but through alternative splicing. The major isoform expressed in most cells is SREBP-1a, whereas SREBP-1c is the major isoform in animal liver and adipose tissue [134,135]. SREBP-1

is necessary for lipogenesis stimulation, as SREBP-1 knockout mice have an impaired FAS

response to carbohydrate feeding [136]. In contrast, transgenic mice that overexpress SREBP-1c

in the liver, have an increased level of both FAS mRNA and activity [137]. SREBP-1 has two

potential binding sites on the FAS promoter. One is the sterol response element (SRE) located at

position -150 of the proximal promoter, the other is the E-box motif at position -65, that is the

binding site of USF [138]. However, SREBP-1 binding to the E-box occurs in vitro only, and

studies show that the physiologically relevant site of SREBP-1 binding is the SRE at -150 [139-

141].

USFs

USFs are members of the basic-helix-loop-helix (bHLH) family of transcription factors

that bind to the E-box motif on the FAS promoter [142]. Two forms of USF, USF-1 and USF-2

are encoded by separate genes, and studies have identified that USFs are major components of

the protein complex binding to the insulin response sequence FAS-IRS-A, located at position -65

and at position -332 in the proximal FAS promoter region [143, 144]. USF binding to the E-

boxes is required for insulin regulation of FAS, as mutations within the E-box completely

remove the insulin response [143], and USF-1 and USF-2 -/- mice show reduction in FAS transcription after fasting/refeeding [145].

Sp1

Sp1 is a member of family of the Specificity Protein/Kruppel-like transcription factors.

Sp1 is a ubiquitously expressed, sequence-specific DNA-binding transcription factor that activates a broad and diverse spectrum of mammalian and viral genes [88]. Sp1 recognizes

GC/GT boxes in gene promoters and interacts with DNA through three C2H2-type zinc fingers located at the C-terminal domain. Based on results of crystal structure and NMR studies, each of the three zinc fingers in Sp1 recognizes three bases in one strand, and a single base in the complementary strand of the GC-rich elements where the consensus Sp1 binding site located 5’-

(G/T)GGGCGG(G/A)(G/A)(C/T)-3’[146]. Wolf et al have shown that the 5' region of the rat fatty acid synthase (FAS) gene has a high GC content between -900 and +500, implying several binding sites for members of the Sp1 family of transcription factors [147]. Using SL2 and H4IIE cells in conjunction with FAS promoter/luciferase constructs either successively deleted or containing defined deletions, Wolf et el characterized seven GC boxes--GC-I to GC-VII--located between -557 and -83 in the FAS promoter. In vitro DNAse I-footprinting, electrophoretic mobility shift assays, and the yeast one-hybrid system indicated that Sp1 interacts with GC-I to

GC-VII and that each of the GC boxes conferred Sp1-dependent transcription.

Sp1 is required in the expression of several genes; however, the specific physiological function of Sp1 has not been determined [148]. Results of gene knockout studies in mice have provided valuable insight on some of the critical functions of this gene. For example, Marin et al showed that Sp1 -/- embryos exhibit multiple abnormalities and retarded development and embryolthality on day 11 of gestation [149]. Furthermore, Sp1 -/- mice also exhibit defects in

late tooth formation and death at birth [150,151]. Therefore, Sp1 is a critical factor required for

normal physiolocial development.

1.4 - FAS and Cancer

As mentioned above, FAS is closely regulated by nutritional and hormonal factors and

normally remains at very low levels in normal cells. These tissues sufficiently obtain any

necessary fatty acids from the diet. On the other hand, in 1989, Kuhajda et al identified FAS as a

molecule that was highly expressed in the tumor cells of breast cancer patients [152]. Indeed,

immunohistochemical results of surgical samples have since revealed that FAS is highly

expressed in a number of cancers, including the breast, endometrium, prostate, colon, thyroid

gland, ovaries, stomach and lungs; and that FAS is a prognostic marker of these types of cancer

[153-161]. In all these cases, FAS expression has been found to be significantly higher in the tumor sections of these tissues compared to their surrounding normal tissues. In addition, several rodent studies have shown that chemically or virally induced mammary tumors also overexpress

FAS [162-164]. Similar studies have also shown positive associations between FAS overexpression and cancer aggression [153, 154, 156, 157].

The function of FAS overexpression in cancer, and how it contributes to cancer growth and development remains unclear. However, it is hypothesized that FAS over-expression

contributes to cellular proliferation through adequately supplying membrane lipids, as well as

supplemental energy, via β-oxidation of fatty acids in cancer cells [165]. It has also been

suggested that palmitate and myristate, the major products of FAS, may be required at high

levels in cancer cells for the lipidation of signalling proteins such as G-protein subunits, nonreceptor tyrosine kinases, and Ras proteins [166,167]– all of which are required in cancer- associated pathways.

For our experiments, we have focussed on FAS expression in breast cancer cells. The

following section will briefly review the current literature on FAS overexpression in breast

cancer.

1.4.1 - FAS and Breast Cancer

In human breast cancer patients, FAS overexpression is associated with poor prognosis

[157, 168, 169]. For example, Alo et al. have shown that FAS overexpression in malignant breast

tumors is associated with a four-fold increase in risk of death [157]. Similarly, Jensen et al. observed a nine-fold increase in risk when FAS expression occurred along with a high proliferative index (>17%) [168]. In addition to the expression of FAS in fully malignant breast

tumors, human breast cancer patients also have elevated serum FAS levels compared to normal

control subjects using a polyclonal-monoclonal sandwich enzyme immunosorbent assay [170].

Furthermore, high levels of FAS have been found in both in situ duct and lobular carcinomas –

both of which are direct precursor lesions to invasive breast cancer [158, 170]. Hence, elevated

FAS expression in the blood and certain breast regions are indicators of breast cancer (and/or

possibly cancers in other regions).

Cell culture and rodent studies involving FAS inhibitors or siRNA indicate that its over- expression may indeed be required for enhancing certain cancer processes such as cellular proliferation and cell cycle progression, while reducing other processes such as apoptosis.

FAS and cell proliferation, cell cycle and tumour growth

Liu et al have shown that inhibition of FAS by triclosan reduces MCF-7 and SKBr3 breast cancer cell viability and growth [171]. Similarly, Pizer et al showed that inhibition of FAS with C75 reduces cell proliferation and causes a profound inhibition of DNA replication and S

phase progression of MCF-7 cells [172]. Menendez et al showed that the FAS inhibitor orlistat

reduces cell proliferation of SKBr3 cells, by leading to S-phase cell cycle arrest and by down-

regulating HER2/neu oncogene expression [173] (HER2/neu is a cell membrane-bound receptor

tyrosine kinase, commonly involved in the pathogenesis of breast cancer). Knowles et al showed

that in MDA-MB435 breast cancer cells, ablation of FAS activity with orlistat or siRNA caused a dramatic downregulation of Skp2, a component of an E3 ubiquitin ligase that tags p27Kip1 for

degradation by the proteasome [174] (p27Kip1 is an inhibitor protein that prevents the activation of cyclin E-CDK2 or cyclin D-CDK4 complexes, and thus controls the cell cycle progression at

G1). The reduction in Skp2, in turn, resulted in an upregulation of p27Kip1 and a cell-cycle arrest

at the G1/S boundary, ultimately reducing the proliferation rate of these cells. Furthermore, Lu et

al showed that MNU-induced rats fed diets containing 1000 ppm triclosan have a significant

reduction in the size and incidence of mammary tumors compared to control rats [121].

FAS and apoptosis

Pizer et al report that within 6 h after exposure to cerulenin, ZR-75, SKBr3 and MCF-7

breast cancer cells express lower levels of FAS [175]. Within the same interval, loss of

clonogenic capacity occurs, followed by DNA fragmentation and morphological changes

characteristic of apoptosis. Similar outcomes have also been observed following triclosan

treatment [171].

Moreover, FAS inhibition also sensitizes breast cancer cells to other apoptotic agents.

Menedez et al showed that pharmalogical and siRNA mediated inhibition of FAS synergistically

enhances Taxo (Paclitaxel)-induced apoptosis in MDA-MB-231 and MCF-7 cells [176]. In

MDA-MB-231 cells, FAS inhibition suppresses HER2/neu oncogene overexpression, and synergistically enhances apoptosis by anti-HER2 antibody trastuzumab [177]. Furthermore,

Kumar-Sinha et al have shown that inhibition of FAS preferentially induces apoptosis in HER2- transfected breast epithelial cells compared to matched vector-transfected control cells [178].

1.4.2 - FAS and Other Cancers

Elevated FAS expression is also a characteristic of other types of cancer. Several studies have evaluated FAS overexpression in colon cancer. Rashid et al used immunohistochemistry to show that FAS was high in essentially all colon cancer samples evaluated, in comparison to their normal surrounding mucosa [159]. FAS has also been shown to be overexpressed in most human colon adenomas [179]. In addition, inhibition of FAS in HCT116 colon cancer cells results in a reduction of their proliferation [180].

A number have studies show that FAS expression is associated with prostate cancer progression [153,154]. Both LNCaP and PC-3 prostate cancer cells have been shown to overexpress FAS. Orlistat inhibits proliferation and induces apoptosis in both of these cell lines

[115]. Furthermore, FAS has also been shown to be elevated in the endometrium, ovaries, stomach, lungs and tongue [156, 160, 161, 181].

1.4.3 - Regulation of FAS in Cancer

The ultimate mechanism responsible for tumor associated FAS expression is not completely understood; however, several hormones and signalling pathways have been shown to be involved in FAS over-expression (all of which are illustrated in Figure 1.7).

At the transcriptional level, growth factors (GFs) and GF receptors (GFRs) have emerged as major contributors to FAS over-expression in tumour cells. For example, FAS expression in breast, prostate and ovarian cancer cell lines has been shown to be stimulated by epidermal growth factor (EGF), and by the EGF receptor (EGFR) [182], which in turn, results in over

expression of FAS by activating the phosphatidylinositol-3 kinase (PI3K)–Akt signalling pathway.

EGFs and EGFRs have also been shown to affect FAS expression through the mitogen- activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK1/2) signalling

cascade in tumour cells [182].

Steroid hormones (SHs) including estrogen, progestins and androgens also have an

important role in regulating FAS gene expression and the FAS biosynthetic pathway as part of

the SH-driven cellular response [182]. The regulatory effects of SH on FAS gene expression,

downstream of the SH receptors (SHRs) ER, PR and androgen receptor (AR) also involve

aberrant activation of the PI3K–Akt and ERK1/2 signal transduction pathways [182].

Figure 1.7: Regulation of FAS expression in cancer by growth factors and steroid hormones [image taken from ref 183].

Post-translational regulation may also contribute to the upregulation of FAS in cancer

cells. In LNCaP prostate cancer cells, FAS has been found to interact with the pre-proteosomal ubiquitin-specific protease USP2a, which removes ubiquitin and strongly stabilizes FAS (Figure

1.8) [182]. Knockdown of USP2a reduces FAS expression, decreases cell proliferation and induces cell death, whereas apoptosis is rescued by FAS overexpression [183]. The relevance of this regulatory mechanism on FAS expression in other tumours, however, is largely unknown.

Figure 1.8: Post-translational regulation of FAS in cancer. Interaction between FAS and USP2a prevents proteasomal degradation of FAS [image taken from ref 183].

1.4.4 - Transcription Factors Regulating FAS in Cancer

Although several transcription factors contribute to the regulation of expression of FAS in

cance, the existing literature has primarily focused on two in particular: SREBP-1c and Sp1.

SREBP-1c

FAS over-expression in both nutritionally controlled endogenous fatty acid synthesis in

liver and adipose tissue and in cancer occurs through the modulation of the expression of the

transcription factor SREBP-1c. In both cases, hormones stimulate FAS transcription when

SREBP-1c interacts with the SREBP-binding site at the endogenous FAS promoter. Although intracellular signalling cascades that regulate FAS expression in normal and tumour cells seem to share identical downstream elements, including PI3K, MAPK and SREBP1c, the upstream mechanisms controlling FAS expression in cancer cells may be different, as tumour-associated

FAS seems to be insensitive to nutritional signals [182].

In lipogenic cells, the effects of carbohydrates and dietary fatty acids on FAS expression are mainly mediated by hormones, which stimulate (insulin, tri-iodothyronine and glucocorticoids) or inhibit (leptin, glucagon and cyclic AMP) FAS-dependent lipogenesis. In tumour cells, SREBP-1c expression and activation will be driven by aberrant GF and GFR and/or SH and SHR-driven signalling. Supporting this notion, inhibitors of PI3K and MAPK downregulate SREBP1c and decrease FAS transcription, ultimately reducing neoplastic lipogenesis in cultured cancer cells. Furthermore, FAS overexpression by oncogenic stimuli can also be abrogated by deletion of the major SREBP binding site from the FAS promoter. As well,

Akt stimulates the synthesis and nuclear accumulation of activated SREBP1c [182].

Sp1

Sp1 is over-expressed in a variety of cancers including breast cancer, and plays an important role in cell growth regulation by modulating the expression of several cell cycle regulatory proteins [148]. As mentioned above, Sp1 regulates gene expression by binding to

‘Sp1 sites’ that include GC-boxes. The promoters of the genes encoding two enzymes of fatty acid synthesis, ACL and ACC, are known to contain Sp1 sites [182], and recently, we have shown that FAS over-expression in cancer cells is also regulated by Sp1. Using the estrogen- responsive MCF-7 human breast cancer cell line, Lu et al [184] demonstrated by RNA interference and ChIP analysis that Sp1 plays a role in regulating FAS expression at the FAS promoter level. Interferce of Sp1 binding at the FAS promoter resulted in a reduced expression on FAS and reduced cell proliferation. Similarly, blocking Sp1 binding sites with the drug mithramycin, also caused a decrease in FAS expression and proliferation. Conversely, 17β- estradiol, a stimulator of MCF-7 proliferation, increased binding of Sp1 to the FAS promoter, increased FAS expression and increased cell proliferation; suggesting that the transcription factor

Sp1 is a molecular link between de novo lipogenesis and proliferation in breast cancer cells

[184]. The next and final section of this chapter briefly reviews the existing studies of Sp1 in cancer.

1.4.5 -Sp1 and Cancer

Sp1 has been shown to be elevated in a number of cancers. Zanetti et al [185] showed that Sp1 was elevated in the breast carcinomas of 11 out of 14 patients, whereas only 1 of 5 benign breast lesions expressed detectable levels of Sp1. Chiefari et al [186] showed that Sp1 was over-expressed in thyroid tumours compared to normal tissues, whereas, Shi et al [187] showed that Sp1 is over-expressed in pancreatic tumours compared to normal tissues. Hosoi et al

[188] have shown that DNA-dependent protein kinases Ku70 and Ku80 are upregulated in colon

tumours compared to normal and adjacent tissues. Sp1 is known to regulate these genes and was

also shown to be elevated in these tumours. A promoter analysis confirmed that constitutive

expression of these kinases was due to the increased interaction of Sp1 with the GC-rich regions

in the promoters of these genes [188].

There is a significant amount of evidence indicating that Sp1 over-expression is a critical

factor in the regulation of several cancer cell processes, specifically the expression of genes

involved in cell cycle progression, angiogenesis, apoptosis and metastasis.

Cell Cycle Progression

In various cancer cell lines, Sp1 has been shown to play an important role in sustaining

cell cycle progression. For example, inhibition of Sp1 suppressed cell growth and caused G1

phase arrest in human glioblastoma, lung and pancreatic cancer cells [189, 190]. A dominant- negative Sp1 mutation in Hela cells decreased cell growth, led to G1 phase arrest, reduced expression of cyclin D1 and increased expression of p27 [191]. Another study in Hela cells showed that ectopic expression of dominant negative Sp1 reduced growth rate by prolonging the

S phase [192]. As well, in serum-starved MCF-7 cells, Abdelrahim et al showed that Sp1 siRNA transfection blocked estrogen-stimulated cell cycle progression by causing G1 phase arrest [193].

Angiogenesis

Vascular endothelial growth factor (VEGF), a growth factor involved in angiogenesis, has been shown to be regulated by Sp1 in various cancers [189,190]. Wang et al [194] showed that Sp1 was highly expressed in nuclei of gastric tumour cells, whereas minimal to non- detectable levels were detected in stromal or normal glandular cells within or surrounding the

tumours. They also showed that the survival of patients with high Sp1 expression was

significantly decreased compared to patients with weak to non-detectable Sp1 expression.

Similarly, Shi et al showed that Sp1 as over-expression in pancreatic tumour cell lines correlates with elevated VEGF levels [187].

Apoptosis and Metastsis

Resistance to apoptosis has been associated with over-expression of Sp1 in cancer cells.

Sp1 sites have been located in the promoters of the anti-apoptotic genes Bcl-2, Bcl-3, Bcl-x, survivin and Bak [195]. Sp1 sites have also been located in the promoter regions of the matrix metalloproteinase-2 (MMP-2), a gene associated with metastasis in cancer. Bae et al [196] investigated invasiveness of SNU-484 and SNU-638 gastric adenocarcinoma cell lines. They showed that Bcl-w and Bcl-2 were overexpressed in both cell lines, and this was associated with their migratory and invasive potentials. Blocking Sp1 using pharmacologic inhibitors, dominant- negative mutants, or small interfering RNA reduced Bcl-w and Bcl-2 expression and increased cell apoptosis. Furthermore, blocking Sp1 also reduced MMP-2 expression and reduced cell invasion. Overall, their results suggest that Sp1 overexpression in cancer may be necessary for cell survivability and invasiveness.

1.5 Research Hypothesis and Objectives

As described above, in contrast to normal cells, most cancer cells over-express the

enzyme FAS to provide lipids for membrane production to support a high rate of proliferation

[152]. This differential degree of FAS expression between normal and cancer cells make FAS an

ideal target for cancer prevention.

Sp1 binding sites exist in regions of the FAS gene promoter [148] and previous findings in our lab suggest that Sp1 regulates the expression of FAS in MCF-7 breast cancer cells [182].

More specifically, we showed that binding of Sp1 at the FAS promoter coordinately regulates

FAS over-expression and proliferation in MCF-7 cells.

While drugs that inhibit FAS (e.g. cerulenin and orlistat) effectively inhibit cancer cell proliferation, they produce undesirable side effects [110, 113, 117, 121]. Dietary compounds, on the other hand, that are capable of inhibiting FAS are being sought as an alternative to these drugs. Two possible compounds include the dietary indoles I3C and DIM, which have been shown to reduce cancer cell proliferation and modify the expression of Sp1-regulated enzymes in cancer cells.

Therefore, the objective of our study was to determine if the I3C or DIM-induced reduction of MCF-7 growth was caused by a reduction in FAS expression. Since both compounds have been shown to reduce expression of Sp1-associated cancer genes, we hypothesized that I3C and DIM will reduce MCF-7 proliferation and will also reduce FAS expression.

Furthermore, we also chose to analyze the effect these indole compounds have on breast cancer cells that express FAS at levels that differ from that of MCF-7 cells. Menendez et al [165] have shown that MCF-7 cells have a moderate level of FAS over-expression, whereas comparatively, MDA-MB-231 cells have a low FAS expression level, while SKBR-3 cells have a very high FAS over-expression level (indeed, 28% of total soluble proteins expressed in

SKBR-3 cells is FAS [165]). Therefore, the specific objectives of our study were to determine 1) if I3C or DIM affect the proliferation and FAS expression of three different breast cancer cells lines and one normal mammary tissue line 2) if the I3C or DIM-induced reduction of cell growth can be reversed by fatty acid supplementation and 3) if Sp1 is affected by I3C or DIM.

CHAPTER TWO:

MATERIALS AND METHODS

2.1 – Cell Culture Conditions

MCF-7, MDA-MB-231, SKBr3 and MCF-10A cell lines were purchased from the

American Type Cell Culture collection and routinely maintained in T-75 flasks (75 cm2 area) and

subcultured every 5-7 days (~80-90% confluency) with medium renewal every 1-2 days. Cells of

4-7 passage generations were used for all experiments. All cell culture reagents were obtained

from Gibco-Invitrogen (Burlington, ON, Canada) and cell culture materials were obtained from

BD Biosciences (Missisauga, ON, Canada) except where noted. MCF-7 and MCF-10A cells were cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM) F12, SKBr-3 in

McCoy’s 5A medium, and MDA-MB-231 in Iscove’s Modified Dulbecco’s Medium (IMDM).

All media contained 1% penicillin/streptomycin and were supplemented with 10% fetal bovine

serum (FBS, Sigma-Aldrich Canada Ltd., Oakville, ON, Canada).

o Cells were maintained at 37 C in a humidified atmosphere of 95% air and 5% CO2. As

well, cells were screened periodically for contamination.

2.2 - Cell Treatment

Indole-3-carbinol (I3C) was obtained from Sigma-Aldrich Canada Ltd and 3,3’-diindolylmethane (DIM) from Designed Nutritional Products (Orem, Utah, USA). Stock solutions of 200 mM I3C and 100 mM DIM were prepared by dissolving each indole compound in dimethyl

sulphoxide (DMSO) (Hybri-max®, Sigma-Aldrich Canada Ltd.). Stock solutions were stored at

−20 °C in the dark prior to use. Final concentrations were made such that diluting the I3C or

DIM stock into media would result in an initial concentration of 100 μM I3C or 50 μM DIM (or

none in control), 10% FBS (v/v) and 0.05% DMSO (v/v).

The half-life of I3C in medium is approximately 40 hours, while that of DIM is

approximately 35 h; hence, the medium was changed once a day for a total of 72 h for I3C

treated cells and 48 h for DIM treated cells. These incubation durations are commonly used in studies testing the effects of I3C or DIM in culture.

2.3 - Cell Proliferation Analysis

Cell proliferation was determined using a standard colorimetric MTT (3-4, 5-

dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide) assay. MTT is converted from a yellow-

coloured tetrazolium salt to a purple-coloured formazan in the cells by cleavage of the

tetrazolium ring by mitrochondrial dehydrogenases, the activity of which is linear to the cell

number.

Briefly, cells were plated at a density of 5x104/100μl/well in 96-well microtitre plates and allowed an overnight period for attachment. Then the medium was removed and fresh medium alone (control) or fresh medium final concentrations of 100 μM I3C or 50 μM for DIM were

added to cultures in parallel.

Following this treatment, cells were treated with phenol-red-free DMEM medium

containing 5% MTT (that is, the medium did not contain FBS, I3C or DIM). The cells were

incubated with the MTT dye for 4 hours at 37oC. After removing the supernatants, the MTT-

formazan crystals were dissolved with a solution of 0.4N HCl in isopropanol (100μl/well) and

the absorbance was measured at 570 nm in a multi-well plate reader (Bio-Rad Microplate

Reader, Model 550).

2.4 - Preparation of Whole Cell Extracts and Western Blot Analysis

Cells were washed with ice-cold phosphate-buffered saline then incubated with cold lysis buffer (50mM Tris-HCl, pH 7.5, 1 mM EDTA, 1mM EGTA, 0.5 mM Na3VO4, 10 mM

glycerophosphate, 5 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 40 µg/ml each of

pepstatin A, aprotinin, and leupeptin) for 30 min, and centrifuged at 16,000 x g for 15 min.

Supernatants were stored at -80ºC for Western blot analysis. Due to their varying level of FAS protein, for MCF-7, MDA-MB-231, SKBr-3 and MCf-10A cells, 5, 5, 1 and 12ug of protein was injected into each western gel well, respectively.

The proteins from whole cell extracts were separated by 10% SDS-PAGE, and were transferred onto PVDF membranes (Bio-Rad Laboratories, Inc., Hercules, CA) using a semi-dry blotter (C.B.S. Scientific Co., Del Mar, CA). Membranes were blocked with 5% non-fat dried milk in TTBS (10 mM Tris-HCl, pH 8.0, 100 mM Nacl, and 0.05% Tween 20) for 1 h at room temperature, then incubated overnight at 4ºC with either mouse monoclonal anti-human FAS antibody at 1:1000 dilution (BD Transduction Laboratories, Missisauga, Ontario, Canada) or mouse monoclonal anti-human Sp1 antibody (Santa Cruz Biotechnology, Inc.) at 1:5000 dilution for 45 min at room temperature. Bands were visualized on Kodak X-ray imaging film, which was developed using a Kodak M35A X-OMAT Processor (Toronto, ONT).

Membranes were stripped in 62.5 mM Tris pH 6.7, 100 mM β-mercaptoethanol and 2%

SDS, for 30 min at 50ºC.β -actin was used as a loading control. Bands were quantified using a

FluorChem digital imager (Alpha Innotech Corp, San Leandro, CA, USA).

2.5 - Preparation of Total RNA

Total RNA was isolated from MCF-7 cells using TRI REAGENTTM (Sigma-Aldrich

Canada Ltd.) according to the manufacturer’s protocol for cell culture RNA preparation. Briefly,

3 ml of medium containing cells were plated in 100x20mm culture dishes at a cell density of

5x105/ml. Cells were allowed one day to attach and grow to approximately 30% confluency.

Next, cells were treated with FBS containing medium only or with medium containing DIM (and

FBS) for 2 days. The medium was then removed and 1 ml of TRI REAGENTTM was applied to the cell monolayer. The lysate was then collected, mixed with isopropanol (0.5ml), allowed to stand for 10 minutes, and centrifuged at 12,000g for an additional 10 minutes at 4ºC. The

supernatant was removed and the RNA pellet was washed by adding 1ml of 75% ethanol,

vortexing and centrifuging for 5 minutes at 4ºC. The RNA pellet was then air-dried for 5-10

minutes, dissolved in RNase-free dH2O and stored at -80ºC. RNA concentrations were measured

at 260 and 280 nm to ensure a ratio of >1.7, indicating the RNA is free of proteins and DNA.

2.6 - Preparation of cDNA and Real-time PCR Analysis

First Strand cDNA Synthesis KitTM by Fermentas (Burlington, ONT) was used to prepare

cDNA for real-time PCR according to the manufacturers instructions. Two µg of total RNA, 2µl

of primer, 12 µl of DEPC-treated water, 8 µl of 5x reaction buffer, 2 µl of RiboLock RNase

inhibitor 4 µl of 10mM dNTP and 4 µl of reverse transcriptase were mixed and incubated for 60

minutes at 37ºC. Heating the tubes to 70ºC for 5 minutes terminated the reaction.

All real-time PCR analysis was done using an ABI Prism 7000 Sequence Detection

System (Applied Biosystems, USA). Sp1 and β-actin primers and probes were purchased from

Applied Biosystems (Foster City, CA, USA). Each PCR-reaction was performed in triplicate on

a 96-well PCR plate, and each reaction well contained 1 µl of cDNA products, 1 µl of either Sp1

or β-actin primers and probes, 8 µl of distilled water, and 10 µl of Taqman® Universal PCR

Master Mix (Applied Biosystems, USA). Two amplification efficiency tests were performed,

using cDNA samples prepared from two different experiments, to calibrate the system and

confirm that β-actin was indeed a suitable control gene for this experiment.

Quantification of Sp1 expression was accomplished by measuring the fractional cycle

number at which the amount of expression reached a fixed threshold (CT). The relative

quantification was given by the average CT values (from the triplicates) for Sp1 and β-actin. CT

values were averaged and the Sp1 CT value was subtracted from the β-actin CT to obtain ΔCT. The

-ΔΔCT relative expression level of Sp1 was determined as 2 where ΔΔCT represents ΔCT from DIM

treated cells minus ΔCT from untreated cells.

2.7 - Rescue Experiments

Palmitic acid and oleic acid were obtained from Sigma-Aldrich Canada Ltd. Stock

solutions of 60 mM of each fatty acid were prepared by dissolving palmitic acid in DMSO and

oleic acid in ethanol. Stock solutions were protected from light with aluminum foil and stored at

−20 °C in the dark prior to use.

MCF-7 cells were plated at a density of 5x104/100μl/well in 96-well microtitre plates in

DMEM-F12 medium containing 10% FBS and 1% BSA (BSA, fraction V, Sigma-Aldrich

Canada Ltd.) and allowed an overnight period for attachment. For these experiments, medium

containing BSA was always prepared fresh before fatty acid stock solutions were diluted in it.

Rescue with palmitate

Cells were treated with medium alone, medium containing 20 µM palmitic acid, medium

containing 50 μM for DIM, or medium containing 20 µM palmitic acid and 50 µM DIM, for

48h. Testing was also performed with 30 µM palmitic acid and 25 µM DIM. The total

concentration of DMSO in the medium was kept contant between treatments.

Rescue with oleate

Cells were treated with medium alone, medium containing 7.5-30 µM oleic acid alone,

medium containing 50 µM DIM alone or with medium containing 7.5-30 µM oleic acid and 50

µM DIM, for 48h. The total concentration of ethanol and DMSO in the medium was kept contant

between treatments.

2.8 - Inhibition of FAS with Cerulenin

Cerulenin was purchased from Sigma-Aldrich Canada Ltd. A stock solution of 10mg/ml was prepared by dissolving the compound in DMSO. The stock solution was protected from light with aluminum foil and stored at −20 °C in the dark prior to use.

MCF-7 cells were treated with a final concentration 5-10 μg/ml of cerulenin as positive controls during the palmitate and oleate rescue experiments (described above). The effect of 10

μg/ml cerulenin on MCF-10A was also tested. In all cases, the total concentration of DMSO in the medium was kept contant between treatments.

2.9 - Statistical Analysis

All statistical analyses were performed using GraphPad Prism 4.0 software (GraphPad,

San Diego, CA, USA). Student’s t-tests were used to compare two means, while comparisons of multiple means were analyzed with a one-way ANOVA (with Tukey’s post-hoc tests). All values were expressed as means +/- SEM. For proliferation figures, means were derived from 24 similar repetitions. For western blot densitometry figures, means were derived from 3 separate repetitions.

CHAPTER THREE:

RESULTS

3.1 - The Effect of I3C on MCF-7 Cells

I3C inhibits proliferation

Several studies have measured the effect of I3C on cell proliferation, with incubation times ranging between 2 and 96 h [78-92]. We chose to use an incubation time of 72 h, which is based the I3C exposure durations used in MCF-7 cell studies by Firestone et al [89-92]. In addition, we tested the effects of I3C on MCF-7 cell proliferation at 48 h, since several cancer- associated proteins in MCF-7 cells have also been shown to be affected by I3C after this exposure duration.

There were significantly fewer cells in wells treated with 100 μM I3C. As shown in

Figure 3.1, I3C added to the culture medium significantly inhibited MCF-7 proliferation after 48 h of incubation by 32% compared to untreated cells. After 72 h, I3C inhibited cell proliferation by 47% compared to untreated cells.

I3C does not alter FAS expression

To determine if the effect of I3C on cell proliferation was associated with changes in

FAS protein expression, we performed Western Blot analysis on extracts of MCF-7 cells grown in the presence of 100 uM I3C. As shown in Figure 3.2, FAS was not significantly affected by

I3C after 48 or 72 h.

100 100 * * 80 80 60 60

40 40 PercentControl (%) of PercentControl (%) of PercentControl (%) of PercentControl (%) of 20 20

0 0 Control I3C Control I3C

Figure 3.1: The effect of I3C on MCF-7 proliferation after 48 h (left panel) and 72 h (right panel). All cells were treated with 100 μM I3C. Values are means ± SEM. (*) indicates a significant difference in mean value compared to untreated cells (p<0.01).

100 100

80 80

60 60

40 40 PercentControl (%) of PercentControl (%) of PercentControl (%) of PercentControl (%) of 20 20

0 0 Control I3C Control I3C

FAS FAS

β-actin β-actin

Figure 3.2: The effect of I3C on FAS expression in MCF-7 cells after 48 h (left panel) and 72 h (right panel). Cells were treated with 100 μM I3C. Values are means ± SEM. The actual blots used to generate the above quantivative representations are shown below. In all cases, representative blots of samples from three independent experiments are shown, with β-actin as a loading control.

3.2 - The Effect of DIM on MCF-7 Cells

DIM inhibits proliferation and FAS expression

Firestone et al [89-92] have shown that DIM has inhibitory effects on MCF-7 cell

proliferation similar to I3C. We tested the effect 50 uM DIM on MCF-7 proliferation for a 48 h incubation duration. In confirmation of the results from the literature, 50 uM DIM reduced the

MCF-7 proliferation by 49% (Figure 3.3). However, unlike with I3C, DIM treatment resulted in a 78% reduction in FAS expression.

48-h time-course of effect of DIM on MCF-7 proliferation and FAS expression

Since DIM reduced both cell proliferation and FAS expression, we further explored this

relationship by performing a time course experiment over the 48 h period. Furthermore, we also

determined if and when Sp1 protein expression was affected in MCF-7 cells treated with DIM.

As shown in Figure 3.4a, DIM significantly inhibited MCF-7 cell growth as early as 12 h

after the treatment application. However, after 12 h, FAS expression in DIM-treated cells was not significantly reduced (Figure 3.4b). There was inhibition of both proliferation and FAS expression at 24 h- 48 h.

Re-probing the same membranes with an anti-body for Sp1, showed a 84% reduction in

Sp1 expression due to DIM treatment at 24 h. At 36 and 48 h, there appeared to be little to no expression of Sp1.

100

80 *

PercentControl (%) of 20

0 Control DIM

100

60 *

PercentPercentControlControl (%) (%) of of 20

0 Control DIM

FAS

β-actin

Figure 3.3: The effect of DIM on MCF-7 proliferation (upper panel) and on FAS expression (lower panel). Cells were treated with 50μM DIM. Values are means ± SEM. For FAS expression, quantitative representations are shown above, and the actual blots are shown below, with β-actin as the loading control. (*) indicates a significant difference in mean value compared to untreated cells (p<0.01).

12h 24h 36h 48h

(a) 100 * * * *

40 Percent of Control (%) Control of Percent Percent of Control (%) Control of Percent 20

0 Ctrl DIM Ctrl DIM Ctrl DIM Ctrl DIM

(b) 100 * * *

20 Percent of Control (%) Control of Percent (%) Control of Percent 0 Ctrl DIM Ctrl DIM Ctrl DIM Ctrl DIM

20 Percent of Control (%) Control of Percent Percent of Control (%) Control of Percent

0 Ctrl DIM Ctrl DIM Ctrl DIM Ctrl DIM FAS

Sp1

β-actin

Figure 3.4 – 48 h Time-course evaluation of DIM on MCF-7 (a) proliferation, (b) FAS and (c) Sp1 expression. Cells were treated with 50μM DIM. Values are means ± SEM. (*) indicates a significant difference in mean value compared to untreated cells (P<0.01). For FAS and Sp1 expression, the actual western blots are shown below, with β-actin as the loading control.

DIM does not affect Sp1 transcript level

As mentioned in Chapter 1, Sp1 is known to regulate FAS expression in MCF-7 cells

[184]. Since we observed that Sp1 protein was reduced in MCF-7 cells after DIM treatment, we

next determined whether this observed reduction was due to a reduction of Sp1 transcript. Real- time PCR analysis indicated, however, that Sp1 transcript levels were unchanged after 48-hours in DIM treated cells in comparison to untreated cells (data not shown).

3.3 - The Effect of I3C and DIM on MDA-MB-231 Cells

Both compounds inhibit proliferation and FAS and Sp1

Although nearly all cancer cell-lines over-express FAS in comparison to their

untransformed normal counterparts, the degree of FAS over-expression varies between cell lines

[165]. For example, MDA-MB-231 cells have been shown to have a lower FAS expression than

MCF-7 cells. Hence, we determined whether I3C or DIM affect proliferation and FAS expression of MDA-MB-231 cells.

As shown in Figure 3.5a, 100 µM I3C or 50 µM DIM resulted in a 24% and 26%

inhibition of MDA-MB-231 proliferation, respectively. In addition, cells treated with I3C had a

38% and 57% reduction in FAS and Sp1 expression, respectively (Figure 3.5b), while DIM

treatment caused 56% and 73% reductions in FAS and Sp1 (Figure 3.5c).

(a) (a)

100 100 * * 80 80 60 60

40 40

Percent of (%) Control Percent Percent of (%) Control Percent Percent of (%) Control Percent Percent of of (%) (%) Control Control Percent Percent 20 20 0 0 Control I3C Control DIM

(b) (b) FAS Sp1 FAS Sp1

100 100 * * * * 80 80

60 60

40 40

Percent of (%) Control Percent Percent of (%) Control Percent Percent of (%) Control Percent Percent of (%) Control Percent 20 20

0 0 Control I3C Control I3C Control DIM Control DIM

FAS FAS

Sp1 Sp1

β-actin β-actin

Figure 3.5 - The effect of I3C (left panel) and DIM (right panel) on MDA-MB-231 (a) proliferation and (b) FAS and Sp1 expression. Cells were treated with 100μM I3C for 72h or 50μM DIM for 48h. Values are means ± SEM. (*) indicates a significant difference in mean value compared to untreated cells (P<0.01). For FAS and Sp1 expression, the actual western blots are shown below (c), with β-actin as the loading control.

3.4 -The Effect of I3C and DIM on SKBR-3 Cells

Both compounds inhibit proliferation and FAS, only DIM inhibits Sp1

Approximately a third of the total soluble protein in SKBr-3 cells is FAS [165]. As shown

in Figure 3.6, treatment of these cells with 100 µM I3C or 50 μM DIM resulted in an inhibition of cell proliferation by 26% and 45%, respectively. Furthermore, I3C and DIM treatment resulted in FAS expression reductions of 66% and 73% compared to untreated cells. In addition, DIM treatment resulted in a 13% reduction of Sp1 expression. However, I3C did not affect Sp1 expression in this cell line.

3.5 - MCF-7 Proliferation Restoration Experiments

Palmitate does not restore MCF-7 growth

To test whether the downregulation of FAS by DIM in MCF-7 cells caused the inhibition

of proliferation, we next attempted to restore proliferation with palmitate.

Figure 3.7a shows that 20 μM palmitate added MCF-7 cells grown in the presence of

50uM DIM for 48 hours did not restore cell growth. Furthermore, increasing the concentration of

palmitate to 30uM and reducing the concentration of DIM to 25uM did not restore the amount of

cell growth compared to cells treated with DIM alone (Figure 3.7b). Addition of 20 or 30uM of

palmitate to cells in the absence of DIM had no effect on proliferation.

(a) (a)

100 100 * *

80 80

60 60

40 40 Percent of Control (%) Control of Percent Percent of Control (%) Control of Percent 20 20 0 0 Control I3C Control DIM

(b) (b)

FAS Sp1 FAS Sp1

100 100 * * *

80 80

60 60

40 40

(%) Control of Percent Percent of Control (%) Control of Percent 20 20

0 0 Control I3C Control I3C Control DIM Control DIM

FAS FAS Sp1 Sp1 β-actin β-actin

Figure 3.6 - The effect of I3C (left panel) and DIM (right panel) on SKBr-3 (a) proliferation and (b) FAS and Sp1 expression. Cells were treated with 100μM I3C for 72h or 50μM DIM for 48h. Values are means ± SEM. (*) indicates a significant difference in mean value compared to untreated cells (P<0.01). For FAS and Sp1 expression, the actual western blots are shown below (c), with β-actin as the loading control.

(a) (b)

125 125 a a 100 100

a a 75 75

50 50

25 25 Percent of Control of Percent (%) Percent of Control of Percent (%) 0 0

DIM DIM Control Control Palmitate Palmitate + Palmitate + Palmitate

(c)

125

100

a b c d 75

Control of Percent (%)

OA DIM OA uM Control 30uM OA 15uM OA 7.5uM OA + 30uM OA + 15 uM 7.5

Figure 3.7 – Attempted rescue of DIM-treated MCF-7 cells with palmitate (a) and (b) and with oleic acid (c). Treated cells in (a) were incubated with 20μM palmitate, 50 μM DIM or both. Treated cells in (b) were incubated with 30μM palmitate, 25 μM DIM or both. Treated cells in (c) were incubated with 7.5- 30μM oleic acid (OA), 25 μM DIM or combinations of 25 μM DIM and 7.5-30μM OA. Values are means ± SEM. One-way ANOVA determined differences between means. Different letters indicate means that are significantly different from one another (P<0.01).

Oleate does not restore MCF-7 growth

Menendez et al [165] have shown that the addition of oleate to the medium of FAS-

inhibited cells is an effective means of restoring cell growth.

Addition of 7.5-30uM oleic acid MCF-7 cells grown in the absence of DIM had no effect

on proliferation (Figure 3.7c). However, in the presence of 50uM DIM, increasing the final

concentration of oleic acid from 7.5 to 30uM progressively decreased cell growth. Furthermore,

7.5-30 μM oleic acid did not restore cells treated with the FAS inhibitor cerulenin after 48 hours;

in contrast, it progressively reduced cell growth compared to cells cultured in the absence of

cerulenin.

3.6 – The Effect of DIM on MCF-10A cells

DIM does not affect MCF-10A proliferation, FAS or Sp1 expression

Unlike the breast cancer cell lines, MCF-10A cells have the characteristics of normal breast epithelial cells in that they are non-tumorigenic and grow in a culture controlled by

hormones and growth factors [197]. Although the effects of I3C and DIM on various cancer cell

lines are well established, the effect of these indoles on the proliferation of this cell lines have

not been reported.

Since it appeared as though there was a stronger anti-proliferative effect with DIM on the three breast cancer cell lines than there was with I3C, we next only tested the effect of DIM

MCF-10A cells. As shown in Figure 3.8 50 µM DIM did not affect MCF-10A cell proliferation after 48 and 72 h incubation periods. Furthermore, DIM treatment did not alter FAS or Sp1 expression of MCF-10A cells after 48 h compared to untreated cells.

100

40 Percent of Control (%) Control of Percent

0 Control DIM

b) FAS Sp1

100

Percent of Control (%) Control of Percent 20

0 Control DIM Control DIM

FAS Sp1 β-actin

Figure 3.8 - The effect of DIM on MCF-10A (a) proliferation and (b) FAS and Sp1 expression. Cells were treated with 50μM DIM for 48 h. Values are means ± SEM. For FAS and Sp1 expression, the actual western blots are shown below (c), with β-actin as the loading control.

3.7 – The Effect of Cerulenin on MCF-7 and MCF-10A Proliferation

Cerulenin inhibits MCF-7 cell proliferation but does not affect MCF-10A cells.

Cerulenin is a known synthetic inhibitor of FAS activity [104]. Several studies have

shown that cerulenin reduces breast cancer cell proliferation and increases apoptosis [106-109].

In confirmation of the results in the literature, 10 μg/ml cerulenin reduced the MCF-7

proliferation by 69% after 48 h (Figure 3.9a). However, Figure 3.9b shows that the same

concentration of cerulenin does not affect MCF-10A growth, even after 72 h.

(a)

100

80 *

40 Percent of Control (%) Control of Percent

0 Control Cerulenin

(b) 100

40 (%) Control of Percent 20

0 Control Cerulenin

Figure 3.9 - The effect of cerulenin on (a) MCF-7 and (b) MCF-10A proliferation. MCF-7 and 10A cells were treated with 10μg/ml cerulenin for 48 and 72 h, respectively. Values are means ± SEM. (*) indicates a significant difference in mean value compared to untreated cells (P<0.01).

CHAPTER FOUR:

GENERAL DISCUSSION AND CONCLUSION

4.1 - Discussion

Both I3C and DIM have been shown by others to suppress the proliferation of various

cancer cell lines including those of breast [77-94], colon [198-200], prostate [201-204], lung

[205] and endometrium [206, 207]. Our first task was to attempt to reproduce the antiproliferative effect of these compounds on MCF-7 cells in our hands. Concentrations of 10-

150 µM I3C and 1-100 µM DIM have been shown to reversibly inhibit MCF-7 proliferation. As discussed in Chapter 1, concentrations of up to 150 µM for I3C and 50 µM for DIM are relevant to humans. I3C and DIM must be taken as a supplement, however, to achieve a comparable serum concentration, since dietary intake of glucobrassicin, the precursor of I3C in cruciferous vegetables, is much lower [52-55]. The doses and exposure durations for our experiments were based on those established in studies on MCF-7 cells by the Bjeldanes and Firstone group [89-

92], who have demonstrated strong antiproliferative effects with concentrations of 100 µM for

I3C and 50µM for DIM, and exposure times of 72 and 48h, respectively. For example, Cram et al [89] and Cover et al [91] showed an 80% reduction in proliferation after 72 h treatment with

100 µM I3C, while Chang et al [90] and Hong et al [66] showed that 48 h treatment with 50 µM

DIM caused an approximately 55% reduction in proliferation. Under similar, though not identical conditions, we showed that I3C and DIM suppressed growth of MCF-7 cells by 47 and

49%, respectively (Figures 3.1 and 3.3).

Our next objective was to determine whether I3C and DIM have a similar anti- proliferative effect on breast cancer cell lines that have lower and higher amounts of FAS expression compared to MCF-7 cells. For MDA-MB-231 cells, Cover et al [91] showed that 100

µM I3C caused a 31% reduction of proliferation after 72 h, while Moiseeva et al [208] showed that 50 µM DIM caused a reduction of 19% after 48 h. Consistent these findings, we showed that

100 µM I3C suppressed MDA-MB-231 growth by 24 % after 72 h (Figure 3.5a), while 50 µM

DIM suppressed MDA-MB-231 growth by 26% after 48 h. Furthermore, we showed that 100

µM I3C reduced proliferation of a third breast cancer line, SKBr-3 by 26% after 72 h, while 50

µM DIM reduced SKBr-3 proliferation by 45% after 48 h (Figure 3.6a). To our knowledge, no previous studies have shown these indole compounds to reduce SKBr-3 proliferation. Hence, I3C and DIM suppress the growth of breast cancer cell growth in culture to similar extents, regardless of their degree of FAS over-expression. Tables 4.1 and 4.2 summarize our results for the effect that I3C and DIM had on proliferation, FAS and Sp1 expression on all cell lines tested.

MCF-7 MDA-MB-231 SKBr-3

Proliferation ↓ ↓ ↓

FAS No change ↓ ↓

Sp1 Not measured ↓ No Change

Table 4.1 – The effect of I3C on proliferation, FAS and Sp1 expression in MCF-7, MDA-MB- 231 and SKBr-3 cells.

MCF-7 MDA-MB-231 SKBr-3 MCF-10A

Proliferation ↓ ↓ ↓ No Change

FAS ↓ ↓ ↓ No Change

Sp1 ↓ ↓ ↓ No Change

Table 4.2 – The effect of DIM on proliferation, FAS and Sp1 expression in MCF-7, MDA-MB- 231, SKBr-3 and MCF-10A cells.

Using the same number of doubling times we had for the cancer cell lines, we next tested whether DIM would affect the growth of the non-tumorigenic mammary epithelial cell line,

MCF-10A. For these experiments we selected to test only the effect DIM had on proliferation, since we had already shown that DIM causes a greater reduction of MCF-7 proliferation than

I3C. Interestingly, we found that DIM did not affect MCF-10A cell growth (Figure 3.8a). This novel observation suggests that the inhibitory effect of DIM may be specific to cancer versus non-cancer cells. Rahman’s labratory have shown that this effect may also be true for I3C [209].

They investigated the effect I3C has on MCF-10A cells in comparison to the tumorigenic breast epithelial cell line MCF-10CA1a (CA1a), and showed that CA1a cells are more sensitive to growth inhibition by 50 µM than MCF-10A cells. These results, along with ours for DIM, indicate that both I3C and DIM do not affect on normal breast cell growth. Since we have shown that both I3C and DIM reduce proliferation of three breast cancer cell lines, we suggest that these indole compounds may be targeting genes important to cancer cell proliferation, but not to normal cell growth.

Kuhajda et al were the first to show that FAS is highly expressed in tumor cells of breast cancer patients [152]. Since then, FAS has been shown to be highly expressed in a number of cancers, including the breast, endometrium, prostate, colon, thyroid gland, ovaries, stomach and lungs [153-164]. It is thought that FAS is over-expressed in cancer tissues for membrane synthesis to sustain an elevated rate of cell division. Hence, we were interested in understanding if the anti-proliferative effect of I3C and DIM involves a modification of FAS expression. The rational behind this idea was that both I3C and DIM have been shown to modify the expression of certain genes in MCF-7 cells requiring the transcription factor Sp1[89, 92] and the FAS gene promoter includes binding sites for Sp1 [184].

Since all three breast cancer cell lines over-express FAS, we expected I3C would reduce the expression of this enzyme in all of them. Our results, however, indicate that I3C and DIM have varying effects on FAS expression, and suggest that the two compounds may be acting to reduce proliferation possibly through different mechanisms. Although I3C caused a reduction in the proliferation of all three breast cancer cell lines, it caused a downregulation of FAS in MDA-

MB-231 cells and SKBr-3 cells (Figures 3.5b and 3.6b) but did not significantly reduce expression of FAS in MCF-7 cells (Figure 3.2). As mentioned above, compared to MCF-7 cells,

SKBr-3 and MDA-MB-231 cells have a relatively higher and lower amounts of FAS, respectively. Thus, these results suggest that the inhibitory effect of I3C on cell proliferation is not caused by a downregulation of FAS.

On the other hand, the reduction in breast cancer cell proliferation we observed after treatment with DIM was associated with a reduction in FAS expression in all three cancer cell lines (Figures 3.3, 3.5b and 3.6b). To better understand whether there is a cause-effect relationship, we performed a time-course experiment on MCF-7 cells treated with DIM because unlike with I3C, DIM caused a reduction in both proliferation and FAS expression. The 48-h time course, however, indicates that a reduction in FAS expression may not necessarily be the cause of the inhibition of proliferation (Figure 3.4 a and b). Thus, at 12 h, MCF-7 cells have a significant reduction in growth compared to untreated cells. However, a reduction in FAS expression was significant only after 24 h of treatment with DIM. Hence, it appears that although

DIM causes a reduction in proliferation and FAS expression, the reduction in FAS expression may be a secondary effect that follows the reduction in proliferation.

We gained further evidence to support this notion from our rescue experiments.

Inhibition of FAS by I3C or DIM would reduce the over-production of palmitate by the cells.

Indeed, inhibition of FAS by cerulenin has been shown to reduce the amount of palmitate in

MCF-7 cells, resulting in supra-physiological accumulations of malonyl-CoA [105, 109].

However, studies showing a rescue effect of FAS-inhibited MCF-7 cells by palmitate have not been performed. Previously, it was shown in our lab that proliferation of MCF-7 cells treated with cerulenin can not be restored with 10-40 µM palmitate (unpublished data), and that greater than 40µM palmitate in the presence of cerulenin is toxic to the cells. Consistent with this finding, Figures 3.7 a and b show that supplementation of DIM-treated MCF-7 cells with 20 or

30 µM palmitate does not restore MCF-7 growth. (For the rescue experiments, we only attempted to restore proliferation of cells treated with DIM, not I3C, because of the greater suppression of MCF-7 proliferation observed with DIM). Since the addition of palmitate to the culture medium does not restore proliferation, this indicates that inhibition of proliferation may involve pathways other than FAS.

We also showed that DIM-treated MCF-7 cell proliferation is not restored when these cells are supplemented with oleate (Figure 3.7c). Menendez et al [165] have shown that oleate, a fatty acid generated de novo from palmitate, can rescue cerulenin-inhibited MCF-7, MDA-MB-

231 and SKBr-3 cell growth. However, several issues arise upon inspection of their analysis, mainly regarding the form and concentration of oleate used to restore cell proliferation. First,

Menendez et al treated the cells with a methyl ester of oleate, a form that would not likely be physiologically relevant. Second, they treated cells with a supraphysiologic concentration of oleate (500ug/ml), and we observed that this concentration was toxic to MCF-7 cells (data not shown). Third, oleate and palmitate are physiologically associated with the serum protein albumin, and Menendez et al did not include albumin in their culture medium. Hence, neither our results nor those in the literature support the notion that oleate restores MCF-7 cell proliferation after inhibition of FAS. Thus, although we showed that inhibition of cell growth by DIM is

associated with a reduction of FAS, DIM is likely acting upon other pathways to suppress cell

growth.

As mentioned in Chapter 1, the regulation and function of FAS differs between cancer

and non-cancer tissues. Most normal cells acquire fatty acids from the circulation and indeed,

MCF-10A cells express low levels of FAS. We showed that DIM had no effect on the

proliferation of these cells or on FAS expression (Figure 3.8). This further suggests that DIM

acts differentially on normal cells and cancer cells, since we showed a DIM-associated reduction

of FAS expression in all three cancer cell lines. As FAS over-expression is important to cancer

proliferation, but not to normal breast cell growth, we suggest that DIM may acting in cancer

cells to target FAS itself or genes involved in regulating FAS over-expression. However, in

Figure 3.9 we show that growth of MCF-7 cells is inhibited in the presence of the specific FAS inhibitor cerulenin, whereas growth of MCF-10A cells is unaffected by this compound. Because the inhibition of FAS activity differs between cancer and non-cancer cells, and since we showed that DIM reduces proliferation before significantly reducing FAS expression, we propose that inhibition of FAS expression may be a secondary effect of I3C and DIM treatment. In turn, we suggest that I3C and DIM may be acting on factor(s) upstream of FAS expression. Since we have previously shown FAS expression is regulated by the transcription factor Sp1, we next tested the effects I3C and DIM have on its expression.

As mentioned in Chapter 1, Sp1 has been shown to be over-expressed in several cancer cell lines [185-188]. Sp1 is necessary for the expression of several genes involved in cancer growth. For example, Sp1 sites have been found in the promoter regions of the CDK2, CDC25A,

CDK6 and p27 genes, all of which are necessary for G1 to S phase cell cycle progression [89,

92, 184]. As well, Sp1 sites have been found in genes important in cell survival such as Bcl-2,

Bcl-w and survivin [77]. Sp1 sites have also been located in the promoter region of the VEGF

and MMP-2 genes, which are necessary for angiogenesis and metastasis, respectively [189, 190,

195, 196] . Our lab has shown that binding of Sp1 to the promoter region of FAS is necessary for

its expression and for proliferation [184]. Hence, modification of the expression of Sp1 could

affect several genes important to cancer cell survival.

Our time-course experiment with MCF-7 cells indicates that at 24 h following DIM

treatment Sp1 levels are reduced by 84% (Figure 3.4c). We speculate that this large reduction in

Sp1 level may be responsible for the reduction in cell growth that we observed. Hence, we

propose that down-regulation of Sp1 by DIM causes a reduction of proliferation by modifying expression of several genes, including FAS. Real-time PCR, however, revealed that the DIM-

associated reduction of Sp1 protein level in MCF-7 cells is not due to a change at the transcript

level. Hence, DIM probably reduces Sp1 protein levels post-transcriptionally.

Furthermore, we showed that DIM caused a reduction in Sp1 expression in all three

cancer cell lines (Figures 3.3, 3.5c and 3.6c). Our early experiments tested the effect of I3C on

MCF-7 cells, but we did not observe a change FAS between treated and untreated cells. Hence,

we did not analyze Sp1 expression in MCF-7 cells after treatement with I3C. We have, on the

other hand, shown that I3C reduces Sp1 levels in MDA-MB-231 cells, but not in SKBr-3 cells

(Figures 3.5c and 3.6 c). Based on our results, it appears that I3C and DIM may be acting differentially on Sp1, since DIM, but not I3C, reduced Sp1 in all cancer cells. Thus, it seems likely that DIM, and not I3C, modifies Sp1-associated gene expression. Indeed DIM is known to accumulate in the nuclei of cells [77] and it has been suggested that I3C is converted to DIM before it enters into the nuclei of cells. Therefore, the effects that DIM may have on transcription factors could appear sooner than they would upon treatment with I3C. In summary, based on our data, DIM reduces Sp1 expression in breast cancer cells, but it is unclear whether I3C acts

similarly. Finally, our results indicate that DIM does not affect Sp1 expression in MCF-10A cells and also does not affect their proliferation (Figure 3.8).

Since we showed that Sp1 in normal cells is unaffected by DIM, but that DIM causes a downregulation of Sp1 protein in breast cancer cells, we suggest that the anticancer effect of

DIM may be due its action on down-regulating Sp1 protein. Hence, genes that are typically over- expressed in cancer and regulated by Sp1, such as FAS, CDC25A, CDK2 and 6, Bcl-2, and

VEGF are likely to be down-regulated upon treatment with DIM as a secondary effect of a reduction of Sp1 protein. In turn, several cellular processes imporortant to cancer cell growth, such as proliferation, apoptosis and angiogenesis would be affected (Figure 4.1).

Figure 4.1 - I3C/DIM modulates nuclear transcription factor Sp1, which is important in the regulation of several cancer genes and processes.

Conclusions

Our investigation was initially directed at understanding whether the anti-proliferative effect I3C and its condensation product DIM on cancer cells involves a change in the expression of FAS. Our results, however, have shown that the inhibitory effect of these indole compounds is more complex than we originally anticipated. Although DIM, and in some cases I3C, was associated with a reduction of FAS expression in breast cancer cells, the reduction in proliferation was likely not caused by changes in FAS, since reductions in FAS occur after a decrease in proliferation. DIM, and in some cases I3C, resulted in a down-regulation of Sp1, a transcription factor required for FAS expression. It seems likely that a reduction in Sp1 level would have caused a downregulation of several genes involved in cell cycle progression as well as in FAS. Interestingly, the effect that I3C and DIM have on cell growth appears to be specific for cancer cells, since these compounds did not appear to affect the growth, FAS or Sp1 levels of non-tumorigenic mammary epithelial cells.

Overall, our results suggest that I3C and DIM regulate Sp1 over-expression post- transcriptionally. Hence, the anticancer effect of these compounds may be due to their affect on several genes regulated by Sp1. Sp1-dependent genes such as FAS, CDK2 and 6, Bcl-2, and

VEGF have all been shown to be down-regulated upon treatment with I3C and DIM [77].

4.3 - Implications

Our findings may have implications for breast cancer therapy and prevention. Although

mortality from breast cancer has declined in the past decade due to advances in diagnosis and

treatment [210], clinical challenges still exist because of the development of drug resistance and

serious side effects [211]. Better approaches need to be developed. Sp1 is over-expressed in 80%

of breast carcinomas, and its expression is associated with poor prognosis [212]. As addressed in

Chapter 1, in breast cancer cells, Sp1 has been shown to play an important role in proliferation

[90, 184, 213], angiogenesis [189, 190], apoptosis [195], invasion and metastasis [196, 214]. Our

research has shown that dietary indole compounds significantly decrease the amount of Sp1

protein. Since Sp1 is crucial for maintaining multiple biological processes that are essential for

the survival and growth of cancer cells, our results suggest that this transcription factor is a

particularly promising target for breast cancer therapy.

4.4 – Limitations and Future Directions

Though our results are consistent with previous findings for the anti-proliferative effect

of I3C/DIM on cultured breast cancer cells, and though our results provide insight into the

mechanistic relationship of this effect, several limitations to our study should be considred. First,

although we have linked our proliferation assays to our western blots, the diameter of the culture

dishes used, and hence the number of cells tested for each assay, differed. We used

materials/techniques that were readily available in our lab, however, dish diameters of equal

lengths would allow us to assess the associations we observed with greater certainty. Secondly,

though literature values have tested these compounds with cell passage generations of less than

20, we specifically used between 4-7, for the most part, and up to 12 for our cerulenin experiments on MCF-10A cells. Although we are unaware of the effect on cell physiology between 4-12 and 20 passages for each of the four cell lines, our results could possibly have been more comparable to literature values had these parameters been the same.

Furthermore, several possible limitations could be considered for our time-course experiment. First, it would have been interesting to know how DIM affects proliferation and Sp1

in MDA-MB-231 and SKBr-3 cells over the 48-h period, along with our MCF-7 cell results. We

could better understand the relationship between DIM, proliferation and Sp1 by looking for

similar trends (to our MCF-7 results) across a larger number of breast cancer cell types. As well,

there may also have been issues with the number of time points we had for our time-course experiment and/or the duration of each interval. Had we measured more intervals over the first

24 h, we could better understand the relationship between the reduction in proliferation and the reduction in Sp1 soon after treatment. As well, we could also be more confident in our time-

course results had we repeated this experiment at least one more time. Variability in the pipetting

of MCF-7 protein, as well as in the densitometry technique we used likely exist, hence,

replication of our results could give us greater confidence in our observations.

Finally, another possible limitation to our study could be that we tested the effect DIM

has on one non-cancer cell line, MCF-10A. In line with out results, it has been previously shown

that DIM does not affect growth of this cell line, but the absorbtion of DIM into this cell line is

not known. It is possible that this cell line may be more resistant to the absorption of DIM.

Performing a similar experiment across more non-cancer mammary cell lines could provide more

evidence to our hypothesis, that DIM acts specifically to reduce the proliferation of breast cancer

cells by a reduction in Sp1, and does not do the same to non-cancer mammary cells.

Our study has provided a strong case in favor of further exploring the effect that I3C and

DIM have on Sp1 and its associated proteins in cancer. While we have been able to show that

DIM and in some cases I3C reduce the total amount of Sp1 protein in treated cancer cells, we

showed that this effect is not at the Sp1 transcript level. We speculate that I3C and DIM could be

activating mechanisms that lead to the degradation of Sp1 protein. This degradation mechanism

should be further investigated. Since Sp1 is known to undergo ubiquitination [215] (which refers

to its post-translational proteosomal degradation by the attachment of one or more ubiquitin

80 monomers), one possible experiment could involve the inhibition of this process with the cell- permeable proteasome inhibitor MG132. It would be particularly interesting to see if inhibition of Sp1 degradation would reduce I3C and DIM’s anti-proliferative effect. Furthermore, it would be interesting to know how genes that are down-regulated by treatment of these indoles (e.g.

FAS), would be affected by inhibition of Sp1 degradation.

As well as reducing MCF-7 and MDA-MB-231 proliferation, both these compounds have been shown to increase the level of apoptosis in these cell lines [77]. Since Bcl-2, a major enzyme controlling apoptosis, also requires Sp1, it would be interesting to test whether its downregulation of Bcl-2 was also caused by these indoles. For this, a time-course experiment similar to the one we prepared for DIM on MCF-7 cells could be performed. Results from this experiment could provide more substance to our notion that a reduction in Sp1 affects several important genes in cancer cells.

Furthermore our study focused on breast cancer cells, but future in vitro experiments should also include cell lines that are derived from other cancers such as prostate, colon, endometrial and lung cancer.

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