The Interaction of Leptin, Zeranol and Gossypol in Breast Cancer

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The interaction of leptin, zeranol and gossypol in breast cancer development Thesis Presented in Partial Fulfillment of the Requirements for the Degree of Master in Science in the Graduate School of The Ohio State University

By Pingping Xu, M.S Graduate Program in Veterinary Bioscience The Ohio State University

2010 Thesis Committee: Dr. Young C. Lin, Advisor Dr. Huey-Jen Lee Lin Dr. Robert J.Lee

Pingping Xu

2010

Abstract

Both breast cancer and obesity are very serious problems in the U.S. Leptin,

mainly secreted by adipocytes, is a product of the obese (ob) gene and plays an important

role in breast cancer development. Leptin expression is up-regulated in obesity and it can

promote breast cancer cell growth. On the other hand, exposure to environmental

estrogens has been found to be directly related to breast cancer. Zeranol (Z), is a non-

steroidal anabolic growth promoter with estrogenic activity and widely used in the U.S

beef industry. Another natural compound extracted from cottonseed and used as an

anticancer chemopreventive agent, gossypol, can inhibit breast cancer growth. It is

suggested gossypol could be used as potential chemopreventive food components.

The current study demonstrated that Z stimulated the proliferation of primary cultured

human normal and cancerous breast epithelial cells and sera collected from Z-implanted beef heifers stimulated the growth of pre-adipocytes isolated from breast cancer patients.

This study is focusing on evaluation of Z and bio-active Z-containing sera collected from

Z-implanted beef and its adverse health risk to human consumption of Z-containing meat produced from Z-implanted beef cattle. We hypothesize that Z might increase the risk of breast cancer in obese women. Cell proliferation assay, ELISA analysis, RT-PCR and

Western blotting were conducted.

The primary cultured human normal breast epithelial cells (HNBECs) and pre-adipocytes

(HNBPADs) were isolated from human normal breast tissues and cultured, the primary

cultured human breast cancer epithelial cells (HBCECs) were isolated from breast cancer ii patients and cultured. The MTT assay to evaluate the proliferation of HNBECs, HBCECs,

and HNBPADs was carried out. The sera used in the MTT assay were: non-zeranol

serum (NZS) dropped from the non-zeranol-implantation beef heifers at day 0 (NZS-D0) and at day 30 post zeranol-implantation (NZS-D30); zeranol-serum (ZS) harvested from zeranol implantation heifers at day 0 (ZS-D0) and day 30 post zeranol-implantation (ZS-

D30). The concentration for both NZS and ZS was 0.2, 1.0 and 5.0% in cultured medium.

Our result shows both Z and lp can increase primary cultured HNBECs, HNCECs, PADs and MCF-7 Adr cell growth. ZS have a greater power to stimulate HNBECs and

HNBPADs growth than NZS and the combination of Z with leptin increase HNBECs and

HNBPADs cyclin D1 mRNA expression and down-regulate P53 or PTPg mRNA expression while (-)-gossypol revert its effect in MCF-7 Adr cells, primary cultured

PADs, HBCECs and HBNECs.

Leptin can increase the breast cancer cells’ sensitivity to Z and Z increase the ObR mRNA expression both in primary cultured HBCECs and MCF-7 Adr cells. Z stimulates primary cultured HNBECs and PADs secrete leptin. These results pointed out that the leptin may be involved in enhancing the HNBPADs sensitivity to ZS and there is interaction between Z and leptin in HNBPADs.

It suggests Z maybe more harmful to obese patients and might play a role in breast cancer

development and (-)-gossypol may serve as a potential chemopreventive agent and play

an important role in breast cancer chemoprevention.

iii

Dedication

Dedicated to my parents, my husband, my son and my daughter

Acknowledgments

I would like to express my appreciation to my advisor Dr. Young C. Lin for his guidance, patience, insight and support throughout the course of this research during the past three years.

I also acknowledge the efforts and contribution of my committee members, Dr. Huey-Jen

Lee Lin and Dr. Robert J. Lee, who provide me advice and direction during the course of my project.

A special note of thanks is extended to all the members in Dr. Lin’s laboratory for their friendly help in this project.

My sincere gratitude to my parents, my husband, my son and my daughter for continued support and enjoy the life with me.

Vita

April 18, 1965…………..born in Guanyun County, Jiangshu Province, P.R.China

1982--1987 …………….Bachelor of Medicine, Zhejiang University, P.R.China

1987—1996 ……. ……..Worked in Quzhou Municipal Sanitary and Anti-epidemic Station, P.R.China.

1996---2010 ……………Worked as an instructor in Jinhua College of Profession and Technology, P. R. China.

2004--2005 …………….Graduate student in the Department of Human Nutrition in OSU

2007--2010 …………….Graduate student in the Department of Veterinary Bioscience in OSU

Publications

Pingping Xu, Weiping Ye, Robert Jen, Shu-Hong Lin, Chieh-Ti Kuo, Young C. Lin

Mitogenic activity of Zeranol in human breast cancer cells is enhanced by leptin and suppressed by gossypol Anticancer Res 29(11): 4621-4628, 2009

Weiping Ye, Pingping Xu, Walter R. Threlfall, Robert Jen, Hong Li, Shu-Hong Lin, Cheik-Ti

Kuo, Young C. Lin Zeranol Enhances the Proliferation of Pre-adipocytes in Beef Heifers

Anticancer Res 29(12): 5045-5052, 2009

Pingping Xu, Weiping Ye, Hong Li, Shu-Hong Lin, Chieh-Ti Kuo, Young C. Lin

Zeranol (Z)-containing beef serum stimulates proliferation of human normal breast

epithelial cells (HNBECs), AACR annual meeting, Denver, CO Apr. 22, 2009

vi Pingping Xu, Weiping Ye, Chieh-Ti Kuo, Young C. Lin (-)-Gossypol inhibits zeranol and leptin-induced MCF-7 adr cell proliferation AACR annual meeting, San Diego, CA.

Apr. 13, 2008

Pingping Xu, Weiping Ye, Qiakuang Yuan Measurement of Particular Size in Aerosol

Atomized by Different Media. China Public Health 17(3):265, 2001

Weiping Ye, Qiakuang Yuan, Pingping Xu Study on A Compound Disinfectant of

Glutaraldehyde Chinese Journal of Disinfection. 16(4):209-12,1999

Pingping Xu. Evaluation of the Albendazole’s Effectiveness to Verminosis Zhejiang

Preventive Medicine and Disease Surveillance 6:74, 1994

Pingping Xu Investigating the Rate of Students’ Tooth Brushing in Quzhou Elementary

School. Zhejiang Preventive Medicine and Disease Surveillance 5:16, 1993

Field of Study

Major Field: Veterinary Biosciences Study in breast cancer and obesity

vii

Table of Contents

Page Abstract ……………………………………………………………………………...…ii

Dedication……………………………………………………………………………...iv

Acknowledgements…………………………………………………………….….…...v

Vita…………………………………………………………………………….………vi

List of Figures………………………………………………………………………….x

Abbreviations..………………………………………………………………………..xiii

CHAPTERS

1. Introduction……………………………………………………….…………….1

2. The Interaction of Leptin, Zeranol and Gossypol in Human Normal and Cancer Breast Epithelial Cells

Abstract………………………………………………………….….……..…….6

Introduction………………………………………………………………….…..8

Materials and Methods…………………………………………………………10

Results…………………………………………………………………….……21

Discussions…………………………………………………...... 24

3. The Interaction of Leptin, Zeranol and Gossypol in Human Normal Breast Pre-

adipocyte Growth

Abstract……………………………………………………………..………….53

Introduction……………………………………………………………….……55 viii Materials and Methods…………………………………………………….….57

Results……………………………………………………………………..….63

Discussions………………………………………………………………..….65

4. Conclusion remarks…………………………………………….…………….79

Literature cited…………………………………………….…..….……..………..82

List of Figures

Figure Page

Figure 2.1 The effect of leptin on MCF-7 Adr cell growth………………………..…..30

Figure 2.2 Effect of leptin on primary cultured HBCECs growth…………………..…31

Figure 2.3 Comparison of effective dose of leptin on primary cultured HBCECs and

HBNECs growth……………………………………………..……….…..…32

Figure 2.4 The effect of Zeranol on MCF-7 Adr cell growth………………………..…33

Figure 2.5 Effect of zeranol on primary cultured HBCECs growth……………………34

Figure 2.6 Comparison of MCF-7 Adr cell growth with pre-lp and post-lp treatment...35

Figure 2.7 Comparison of the primary cultured HBCECs growth with pre-lp and

post-lp treatment……..……….………………………………………...…36

Figure 2.8 Comparison of the proliferative effect of leptin, zeranol, pre-lp and

post-lp treatment in MCF-7 Adr cells…………………………….….……37

Figure 2.9 Comparison of the proliferative effect of leptin, zeranol, pre-lp and

post-lp treatment in MCF-7 Adr cells………………………………..……38

Figure 2.10 Effects of leptin (lp), zeranol (Z) and (-)-gossypol (G) on MCF-7 Adr

cell proliferation……………………………………...…………………...39

Figure 2.11 Effects of leptin and (-)-gossypol (G) on primary cultured HBNECs

growth………………………………………………………………………40

Figure 2.12 Effects of none-zeranol serum on ECs isolated from non-leptin cultured

x tissues………………..…….………………………………………………41

Figure 2.13 Effects of non-zeranol serum on ECs from1.5 nM of leptin cultured

tissues…………………...………………………………………………….42

Figure 2.14 Effects of zeranol serum on ECs from non-leptin cultured tissues……….43

Figure 2.15 Effects of zeranol serum on ECs from 1.5 nM of leptin cultured tissues...44

Figure 2.16 Effects of leptin and zeranol and (-)-gossypol alone or combination on

cyclin D1 expression in MCF-7 Adr cells…….………………………….45

Figure 2.17 Effects of leptin and zeranol on cyclin D1 expression in HBCECs….…..46

Figure 2.18 Effects of leptin, zeranol and (-)-gossypol on cyclin D1 expression in

HBNECs………………………………..…………………………….….47

Figure 2.19 Zeranol up-regulated Cyclin D1 protein expression in HBNECs……..….48

Figure 2.10 Effects of leptin, zeranol and (-)-gossypol alone or combination

on P53 expression in MCF-7/Adr cells…………………………………...49

Figure 2.21. Effects of Zeranol on ObR expression in MCF-7 Adr cells…..……….…50

Figure 2.22 Effects of leptin and zeranol on ObR expression in HBCECs…..……..….51

Figure 2.23 Zeranol increased the HNBECs secrete leptin……….………….……..….52

Figure 3.1 The Effect of leptin in the pre-adipocyte growth……...………….……..….69

Figure 3.2 The comparison of the average effective dose of leptin in the PADs and

HBNECs……………………………….…………………….…………....70

Figure 3.3 Exposure of leptin changed the PADs sensitivity to zeranol……………...71

Figure 3.4 The effect of ZS-D0 and ZS-D30 in the proliferation of pre-adipocytes

isolated from non-leptin cultured tissues…………………….…..……..…72

Figure 3.5 Comparison of effect of ZS-D0 and ZS-D30 in the proliferation of

xi pre-adipocyte isolated from 6 nM leptin cultured tissues………..…………73

Figure 3.6 leptin increase the cyclin D1 mRNA expression in PADs ……..………….74

Figure 3.7 The effect of zeranol, leptin and (-)-gossypol in cyclin D1 mRNA

expression in PADs…..……………….……………….………………..…75

Figure 3.8 Zeranol increase the cyclin D1 protein expression in PADs…… ………...76

Figure 3.9 Leptin up-regulate Cyclin D1 protein expression in PADs …………….…77

Figure 3.10 Zeranol stimulates human normal breast PADs secret leptin……..……....78

xii

Abbreviations

BMI : Body Mass Index

BSA: Bovine Serum Albumin

DCC: Dextran-Coated Charcoal

DMEM: Dulbecco’s Modified Eagle’s Medium

DMSO: Dimethyl Sulfoxide

ELISA: Enzyme-Linked Immunosorbent Assay

ER: Estrogen Receptor

FBS: Fetal Bovine Serum

(-)-G: (-)-Gossypol

HBNECs: Human Breast Normal Epithelial Cells

HBCECs: Human Breast Cancer Epithelial Cells

HBNPADs: Human Normal Breast Pre-adipocytes

Lp: Leptin

MTS: 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H- tetrazolium

NZS: Non-Z-Serum

ΝΖS-D0: NZS Dropped from Heifers Implanted with Z before Z-implantation

ΝΖS-D30: NZS Dropped from Heifers Implanted with Z 30 Days after Z-implantation

xiii ObR: Leptin Receptor

PMS: phenazine methosulfate

PTPγ: Protein Tyrosine Phosphatase γ

PVDF: Polyvinylidine Fluoride

SD: Standard Deviation

Z: Zeranol

ΖS: Z Serum

ΖS-D0: ZS Dropped from Heifers Implanted with Z before Z-implantation

ΖS-D30:ZS Dropped from Heifers Implanted with Z 30 Days after Z-implantation

xiv

Chapter 1

General Introduction

Breast cancer is a worldwide disease, causing over 40,000 women to die of each year in

the U.S. [1]. One of the currently known risk factors of breast cancer is obesity, which

has become a major public health concern [2]. According to the World Health

Organization, Body Mass Index {BMI, MBI=Weight(in kg)/height2(in cm)} can be used

as a standard to evaluate human weight [2], BMI between 18.5 to 24.9 is regarded normal,

25.0 to 29.9 overweight, and more than 30 obesity. According to this standard, the

Centers for Disease Control and Prevention reported about 2/3 adults in the United States

are overweight and 1/3 are obesity [3]. The incidence of breast cancer is increased with obesity, and morbidity is also increased in obese cancer patients as compared to cancer patients with normal or low weight [2]. The relationship between breast cancer and obesity has been studied for more than 40 years [4].

Leptin, a transcriptional product of the ob gene, plays an important role in breast cancer development and has been studied since its discovery in 1994 [5]. It is a 167- amino acid protein with a molecular mass of 16 kDa. In human, leptin is encoded by a gene located on chromosome 7q31.3 [6]. Besides its involvement in appetite regulation and energy balance by sending signals to the hypothalamus[7], leptin has a number of other regulatory functions such as ensuring normal mammary gland development, bone 1 development, fetal development, sex maturation, angiogenesis, lactation, hematopoiesis, and immune responses [2, 3]. Additionally, leptin is necessary for normal mammary gland development in rodents [2, 6]. Animals and humans with defective leptin or with mutated leptin receptor genes are obese [3, 8]. The serum level of leptin in obese cancer patients was determined to be significantly higher than that in a healthy control group. In clinical studies, the serum leptin level in prostate cancer patients was found to be higher than that in a healthy control group, and was correlated with prostate-specific antigen [9].

In a breast cancer research program, 60% (20/35) of the patients expressed leptin, while none out of four cases with normal breast tissue expressed leptin [10].

Leptin has a direct mitogenic effect on human breast cancer cells [11]; therefore, the inhibition of leptin may contribute to the prevention and treatment of breast cancer

[12]. Leptin expression is upregulated in obesity [3] and it can promote breast cancer cell growth by directly affecting the estrogen receptor (ER) pathway [3]. The leptin receptor

(ObR), localized in the cell membrane of various types of tissues, including breast cancer

[13], has been identified as having one long isoform and five short isoforms [3]. Both short and long isoforms are expressed in normal mammary epithelial cells during pregnancy and lactation [2, 10]. Such expression suggests an autocrine action of leptin in the regulation of mammary gland growth and development. Furthermore, because it is commonly expressed in breast cancer, the gene could serve as a tumor marker, or a prognostic or diagnostic factor [14, 15].

Like other growth factors and cytokines, leptin is present in human serum and plays a role in human cancer development [3]. Because leptin was found to be associated with several types of cancer [3, 16] researchers have attempted to find the relationship

2 and mechanisms of leptin action in several prostate cancer [9], gastric cancer [17], esophageal adenocarcinoma [18], hepatocellular carcinoma [19], gallbladder cancer [20], cholangiocarcinoma [21], and breast cancer [22-27]. Ishikawa et al. found that leptin was overexpressed in breast cancer cells [12] and likewise concluded that high leptin levels in obese breast cancer patients might play a role in the development of antiestrogen resistance [22]. Leptin is not expressed in normal breast tissue but exists near malignant breast lesions [11], while its receptors were detectable in cancer cells but undetectable in normal mammary epithelial cells [12]. In addition to its mitogenic effects, leptin can promote T47-D cell line proliferation [13] and a high level of leptin might contribute to the development of a more aggressive malignant phenotype [28]. ICI 182, 780 is a pure estrogen antagonist approved for treatment of breast cancer patients who failed to respond to tamoxifen therapy. Treatment of these cells with ICI 182,780 led to fast degradation of membrane ER α , which reduced nuclear expression of the receptor and

ER α -dependent transcription, and produced significant growth inhibition. Leptin was able to counteract the cytostatic activity of ICI 182,780, as well as the effect of this compound on the expression of ERα and on ER α -dependent transcription [29]. The power of leptin to stimulate human MCF-7 cell growth and to counteract the effects of

ICI 182, 780 strongly suggests that leptin acts as a paracrine/endocrine growth factor towards mammary epithelial cells [22]. Chen et al. also found that leptin increases ZR-

75-1 breast cell growth by up-regulating cyclin D1 and down-regulating P53 [30].

Because it stimulates estrogen biosynthesis through induction of aromatase activity and modulation of ER α activity, leptin has been characterized as a growth factor for breast

3 cancer [3, 15]. High levels of leptin in obese breast cancer patients might play a

remarkable role in breast cancer cell proliferation, invasion, and metastasis [2]

Estrogen has been regarded as a positive regulator of leptin production [31], and the leptin level in breast cancer patients treated with tamoxifen is significantly higher than levels in the control group [32]. Thorn et al. found that as another risk of breast cancer development, estrogen can modulate ObR expression in some estrogen-responsive tissues [33]. Zearalenone which is a stable natural product with mimics estrogen activity is a carcinogen and thus hazardous to human health [34]. Z, produced from zearalenone, is a non-estrogenic anabolic growth promoter approved by Food and Drug

Administration (FDA) because of the great commercial benefits and is widely used to stimulate cattle growth in the U. S. beef industry [35]. FDA approved the usage of Z in beef cattle industry based on the toxicity information. However, European Union declines to import beef products with residues of hormonal implantation from the US because of potential health concerns. Both zearalenone and Z can bind to the active site of human

ER α and ER β in a similar manner to17 β-estradiol [36]. As food contaminant, the intake of Z is very hard to avoid [34]. Researcher found that beef is a highly consumed meat in the US, averaging 67 pounds per person per year, low income families consume more beef than other income families. Researchers also found that Z did not change the serum leptin level in growing wethers [37]. At low concentration, it can increase ERα-positive cell growth, but a high concentration of Z can reduce growth of both ERα-positive and - negative cell lines [38]. Moreover, our previous data showed that Z was able to transform normal human breast epithelial cell and increase human breast cell growth in a

4 dose-dependent manner [35] and can down-regulate estrogen-regulated human breast

cancer candidate suppressor gene, protein tyrosine phosphatase γ (PTPγ) expression [39].

Another natural polyphenolic compound extracted from cottonseed and used as

an anticancer chemopreventive agent, gossypol, can inhibit various types of cancer cell

growth such as colon [40], prostate [41], and breast cancer [42, 43]. It is suggested

gossypol could be used as potential chemopreventive food components. Our laboratory

also demonstrated that gossypol has anticancer activity against multidrug resistant human

breast cancer cell line, MCF-7 Adr cell [43], with (-)-gossypol having the strongest effect

among the three isoforms (data not shown).

The objective of this research was to investigate the interaction of leptin, Z and

gossypol in breast cancer development and the mechanisms of the suppression of Z- and

leptin-induced proliferation of breast normal and cancer cells by using (-)-gossypol as the

main chemopreventive agent. The hypothesis is (-)-gossypol can inhibit the leptin-or zeranol- induced human normal or cancer cell growth.

Chapter 2

The Interaction of Leptin, Zeranol and Gossypol in Human Normal and Cancer Breast Epithelial Cells

Abstract

Both breast cancer and obesity are very serious problems in the U.S and their

relationship has been studied for many years. Leptin, mainly secreted by adipocytes, is a product of the obese (ob) gene and plays an important role in breast cancer development.

Leptin expression is up-regulated in obesity and it can promote breast cancer cell growth.

On the other hand, exposure to environmental estrogens has been found to be directly related to breast cancer. Zeranol (Z), is a non-steroidal anabolic growth promoter with estrogenic activity and widely used in the U.S beef industry. Another natural compound extracted from cottonseed and used as an anticancer chemopreventive agent, gossypol, can inhibit breast cancer growth. It is suggested gossypol could be used as potential chemopreventive food components.

The current study demonstrated that Z stimulated the proliferation of primary cultured human normal breast epithelial cells (HBNECs) and primary cultured human breast cancer epithelial cells (HBCECs) and sera collected from Z-implanted beef heifers

6 stimulated the growth of HBNECs and HBCECs isolated from human breast tissues. This

study is focusing on evaluation of Z and bio-active Z-containing sera collected from Z-

implanted beef and its adverse health risk to human consumption of Z-containing meat

produced from Z-implanted beef cattle. We hypothesize that Z might increase the risk of

breast cancer in obese women. Cell proliferation assay, ELISA analysis and RT-PCR

were conducted.

The primary cultured HNBECs were isolated from human normal breast tissues and

primary cultured HBCECs were isolated from breast cancer patients and cultured. The

MTT assay to evaluate the proliferation of HNBECs, HBCECs and breast cancer cell line

MCF-7 Adr cells was carried out. The sera used in the MTT assay were: non-zeranol serum (NZS) dropped from the non-zeranol-implantation beef heifers at day 0 (NZS-D0) and at day 30 post zeranol-implantation (NZS-D30); zeranol-serum (ZS) harvested from zeranol implantation heifers at day 0 (ZS-D0) and day 30 post zeranol-implantation (ZS-

D30). The concentration for both NZS and ZS was 0.2, 1.0 and 5.0% in cultured medium.

Our results show both Z and lp can increase primary cultured HNBECs, HNCECs and

MCF-7 Adr cell growth. ZS have a greater power to stimulate HNBECs growth than NZS and the combination of Z with leptin increase HNBECs cyclin D1 mRNA expression and down-regulate P53 or PTPγ mRNA expression while (-)-gossypol revert its effect in

MCF-7 Adr cells, HBCECs and HBNECs. Leptin can increase the breast cancer cells’ sensitivity to Z and Z increase the ObR mRNA expression both in primary cultured

HBCECs and MCF-7 Adr cells. Z stimulates primary cultured HNBECs secrete leptin.

These results suggest there is interaction between leptin and Z in HBCECs.

7 It suggests Z maybe more harmful to obese patients and might play a role in breast cancer

development and (-)-gossypol may serve as a potential chemopreventive agent and play

an important role in breast cancer chemoprevention.

Introduction

Breast cancer remains a serious problem in the U. S. It is estimated that about more than

one-fourth of cancer patients were breast cancer patients in 2009, and it ranks the second leading cause of cancer-related deaths [1]. According to clinical cancer statistics, it is estimated that 182,500 new cases arise each year and 40,500 die from breast cancer in

2009 [1]. Epidemiological studies suggest that there are many risk factors associated with breast cancer such as dietary fat and environmental estrogenic endocrine disruptors. The relationship between dietary fat intake and risk of breast cancer has been studied in ecological, etiologic, and intervention research and animal experimentation. Another of

currently known risk factors of breast cancer is obesity which has become a major public

health concern [44]. The incidence of breast cancer is increased by 30-50% in obese

cancer patients compared to the cancer patients with normal weight, and the morbidity is

also increased in obese cancer patients as compared to cancer patients with normal or low

weight [2]. The molecular links between breast cancer and obesity have been studied for

many years [4]. Obesity significantly increases the incidence rate and chance of

morbidity of breast cancer. Leptin, mainly secreted by adipocytes, plays an important role in breast cancer development. Leptin expression is up-regulated in obesity and it can promote breast cancer cell growth. Z is widely used as an anabolic growth promoter approved by FDA based on its toxicity information to stimulate cattle growth in the U. S. 8 beef industry. However, European Union declines to import beef products with residues of hormonal implantation from the US because of potential health concerns. Recent research found that Z might not be as safe as the FDA claimed before. (–)-Gossypol, a

natural polyphenolic compound extracted from cottonseed, is an anticancer

chemopreventive agent. Breast cancer cell lines play a pivotal role in biomedical research,

the investigation of the mechanisms of the suppression of Z- and leptin-induced

proliferation of MCF-7 Adr cells was conducted by using (–)-gossypol as the main

chemopreventive agent. Because cells from patients have extensive chromosomal

rearrangement, oncogenic mutations, multiple sites of allele loss and gene amplification,

there is a widespread belief that the cell lines are not representative of the tumors from

which they are derived [45]. We also found that 2.5% of Z-containing sera harvested

from 60-day post 72 mg Z pellets implanted beef cattle are capable in transfrorming the

human normal breast epithelial cell line, MCF-10A to neoplastic breast cancer cells in

21-day in culture (paper in preparation). It was reported that leptin can impact

transformed breast cancer cells to induce an alteration to a more aggressive phenotype

and leptin could potentially serve as a tumor marker. It is necessary to understand

whether leptin and Z have effects on cyclin D1 and ObR mRNA expression in breast cancer epithelial cells which were isolated from the normal and cancerous tissues from normal breast or cancer patients. We also evaluated the effect of the Z-S in human normal

breast epithelial cell growth. This study is also focusing on evaluation of bio-active Z-

containing sera collected from Z-implanted beef and its adverse health risk to human

consumption of Z-containing meat produced from Z-implanted beef cattle.

9 Materials and Methods

Animal treatment and blood sampling. Ralgro Magnum® (RM, commercial Z pallet)

was purchased from Schering-Plough Corp, Kenilworth, NJ, USA in the form of

cartridges, each containing 72 mg Z. Twenty cross bred Angus beef heifers (about one

year old) purchased from the Department of Animal Science were randomly divided into

two groups according to their initial body weights. The treatment group consisted of 10

heifers each implanted subcutaneously in the ear with 72 mg of Z (Ralgro Magnum®)

while the other 10 heifers without implanted Z as controls. All samples from treatment

group were studied as Z containing samples while samples from non-Z implanted heifers as control. Both groups were raised at the same environment in a Beef Cattle Barn located at the Ohio State University Livestock Facilities. The body weight of each heifer was measured twice a month. The RM implantation was performed on Sept 14, 2007.

Biopsies were performed by veterinary surgeon occurred at the beginning of each month

from September to December, 2007. Muscle and fat tissue were taken from the dorsal

side of each heifer and divided into three portions. The first portion of the tissue was

fixed in 4 % formalin for histopathologic evaluation. The second portion was placed in a

50 ml centrifuge tube to be used for proteomic analysis, while the last small piece of either muscle or fat tissue was placed in a cyrovial for RNA isolation. The remaining components of fat tissue were used to isolate pre-adipocytes. Blood sample (100 ml) was withdrawn from the jugular vein. Urine and feces were also taken at the same time from the selected heifers. All of the biological samples were then placed into a coolant with ice and transferred to lab for further process. Five heifers in the control group and five

10 heifers in the experiment group were slaughtered in a slaughter house of Meat Research

Laboratory at the Department of Animal Science of the Ohio State University after two months of Z-implantation, and the remaining 10 heifers were slaughtered at the same place after four months of Z-implantation. Blood, adipose tissue, muscle, mammary gland, liver, kidneys, pancreas, ovarian and pituitary were collected during the slaughtering. The small piece of each organ was fixed in 4 % formalin immediately after they were transferred to our lab, and the rest part were stored at -20 ºC for future analysis.

Reagents. Recombinant human leptin was ordered from the R&D Systems (Minneapolis,

MN) and was prepared as stock solution of 1 mg/ml in sterile 20 mM Tris/HCl (pH 8.0) at - 20°C; (–)-gossypol was provided by USDA Southern Regional Research Center

(New Orleans, LA), and was prepared as 50% stock solution in dimethyl sulfoxide

(DMSO); Z was purchased from Sigma Chemical Company (St. Louis, MO).

Tissue culture. Human normal breast tissues were sterilized in 70% ethanol for 30 s, and then washed three times with fresh DMEM/F12. In vitro organ cultured human normal breast tissues were treated with leptin at 0, 1.5, 3.0, and 6.0 nM in DMEM/F12 medium supplemented with 5% dextran-coated charcoal (DCC) stripped fetal bovine serum (FBS)

2 and cultured in a 10 cm cell culture plate in a humidified incubator (5% CO2, 95% air,

37°C) for 96 h. All medium was changed every 48 h. Human breast cancer tissues were not cultured and cells were isolated described as below.

Primary cultured human normal and cancer breast epithelial cells isolation: The cultured human normal breast tissues and cancer tissues were minced and then digested using digestion buffer which is consist of phenol red-free high calcium Dulbecco’s modified Eagle’s medium and Ham’s F12 medium (1:1) (DMEM/F12 ) (1.05 mM CaCl2) 11 with 2% Bovine Serum Albumin (BSA) (Invitrogen, Carlsbad, CA) containing 10ng/ml

Cholera toxin (Sigma, St. Louis, MO) 6300U/ml Collagenase (Invitrogen), and 100U/ml

Hyalurinidase (Calbiochem, Gibbstown, NJ). After the mixture was incubated in a humidified incubator (5% CO2, 95% air, 37°C) overnight, the solution was transferred to a 50 ml tube and centrifuged in 1,200 rpm for 5 min. The upper, middle and lower layers were separated and centrifuged again. The upper containing pre-adipocytes and middle layer containing stromal cells were transferred to another 15 ml tube separately while the lower layer containing epithelial cells remained in the tube. All the pellets were washed by DMEM/F12 medium with antibiotic-antimycotic (100 unit/ml penicillin G sodium,

100 μg/ml streptomycin sulfate and 0.25 μg/ml amphotericin B) (GibcoBRL, Bethesda,

MD) and centrifuged again. This wash procedure was repeated for three times. The final pellet in the tube contains primary cultured human breast epithelial cells and very few stromal cells. The pellet was resuspended in 10 ml low calcium (0.04 mM CaCl2)

DMEM/F12 medium supplemented with 10% of low calcium FBS ( Atlanta Biologicals,

Norcross, GA) and then transferred into a T 75 flask for culturing.

Primary human breast epithelial cell culture. The isolated primary cultured human normal breast and breast cancer epithelial cells (HNBECs and HBCECs) were cultured in

2 a 75 cm culture flask in a humidified incubator (5% CO2, 95% air, 37°C) with 10 ml low calcium (0.04 mM CaCl2) DMEM/F12 mixture (Atlanta Biologicals, Norcross, GA) supplemented with 10% of Chelex-100 (Bio-Rad Laboratories, Richmond, CA) treated

FBS (Invitrogen). The low calcium DMEM/F12 medium was changed every two days.

Only HNBECs and HBCECs can survive in this medium so that the growth of the stromal cells isolated from the same tissue will be stopped and the purity of HNBECs and

12 HBCECs was guaranteed. When the cells grew to 85-90% confluence, cells were washed

with 10 ml of calcium- and magnesium-free Phosphate Buffered Saline (PBS, pH7.3),

and then trypsinized with 3 ml of 0.25% trypsin-5.3 mMEDTA (Invitrogen) for 10

minutes at 37 °C. The trypsinization was stopped by the addition of 10 ml of DMEM/F12

medium with 10% FBS. After centrifugation, the dissociated cells were re-suspended in

low calcium DMEM/F12 medium with 10% low calcium FBS and sub-cultured into 75

cm2 culture flasks at a ratio of 1 flask to 5 flasks. All experiments were conducted on

primary cultured human normal breast epithelial cells and human breast cancer epithelial

cells not generated beyond the fourth passage.

Cell line culture. MCF-7 and MCF-7 Adr cells were purchased from the American Type

Culture Collection (ATCC, Manassas, VA) and cultured in phenol red-free high calcium

DMEM/F12 (1.05 mM CaCl2) containing 5% FBS and antibiotic-antimycotic (100

unit/ml penicillin G sodium, 100 μg/ml streptomycin sulfate and 0.25 μg/ml

amphotericin B) (GibcoBRL, Bethesda, MD) in a 75 cm2 culture flask in a humidified

incubator (5% CO2, 95% air, 37°C). When the cells grew to 85-90% confluence, cells

were subcultured into 75 cm2 culture flasks at a ratio of 1 flask to 5 flasks as described above. Cells were dissociated using 1 ml of 0.5% trypsin-5.3 mM EDTA (GibcoBRL) in

PBS for 3 minutes at 37°C. The trypsinization was stopped by the addition of 10 ml of culture medium with 10% FBS. After centrifugation, the dissociated cells were resuspended in the culture medium with 10% FBS and subcultured into 75 cm2 culture flasks at a ratio of 1 flask to 5 flasks.

Cell proliferation assay (MTT assay). One hundred µl cells (MCF-7 cells, HNBECs and HBCECs) in 3,000/well were placed into 96-well plates in DMEM/F12 high calcium

13 medium for MCF-7 Adr cells and DMEM/F12 low calcium medium for HNBECs and

HBCECs and incubated in 37°C for 24 h. After this time, the medium was changed by

100 µl phenol red-free high calcium DMEM/F12 supplemented with 0.2% BSA for

MCF-7 Adr cells and phenol red-free low calcium DMEM/F12 supplemented with 0.2%

BSA for HNBECs and HBCECs, the plate was incubated at 37°C for another 24 h.

Treatment of 1, 1.5, 3, 4.5, 6, or 12 nM leptin or 5, 10, 20, or 30 nM Z was given, 0.1%

DMSO being given to the control group. Twenty four h later, cell growth was measured.

The cell proliferation was measured by adding 20 µl of a fresh mixture of 3-(4, 5- dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium

(MTS) and phenazine methosulfate (PMS) (20:1) solution (Promega, Madison, WI, USA) to each well. After incubation at 37°C for 1-5 h, OD values were measured by kinetic microplate reader (Molecular Devices Cooperation, Menio Park, CA, USA) at 490 nm wavelength and cell growth was compared.

In the MTT assay in MCF-7 Adr cells of combination of leptin with Z and (–)-

gossypol, the concentration used was 6 nM, 30 nM, and 5.0 µM for leptin, Z, and (–)-

gossypol respectively, alone or combined; 0.1% DMSO was given to the control group.

After 24- h treatment, cell growth was measured as described above.

In the experiment investigating leptin-induced MCF-7 Adr cells’ sensitivity to Z,

treatment was given to MCF-7 Adr cells in five groups: i) control group (CT): cells were only treated with 0.1% DMSO; ii) leptin group (lp): cells were only treated with 6 nM leptin for 48 h; iii) Z only group (Z): cells were only treated with 10, 20, or 30 nM Z; iv) pre-treatment with leptin group (pre-lp): cells were treated with 6 nM leptin for 24 h then

14 treated with 10, 20, or 30 nM Z for 24 h; v). post-treatment with leptin group (post-lp):

cells were treated with 10, 20, or 30 nM Z for 24 h then 6 nM leptin for the following 24

h; All media were changed every 24 h. After 48 h treatment, cell growth was measured as

described above.

In the experiment investigating leptin-induced HBCECs sensitivity to Z, 4,000

HBCECs were planted in each well of 96-well plate within 100 μl low calcium

DMEM/F12 medium and incubated in 37°C for 24 h. The following day, medium was

changed by 100 µl low calcium DMEM/F12 supplemented with 0.2% BSA, and

HBCECs were incubated in 37°C for another 24 h. The cells were treated in five groups: i)

control group (CT): 0.1% DMSO was given to control group. ii) only treatment with

leptin group (lp): only treated with 3 nM leptin for 48 h; iii) The pre-treatment with leptin

group (pre-lp): treated with 3 nM leptin for 24 h and 1.25, 2.5, or 5 nM Z in following 24

h; iv) post-treatment with leptin group (post-lp): firstly treated with 1.25, 2.5, or 5 nM Z

for 24 h and 3 nM leptin in following 24 h; and v) only treatment with Z group (Z): only

treated with 1.25, 2.5, or 5 nM Z for 48 h. All media were changed every 24 h. After 48 h

treatment, cell growth was measured by the methods described above.

A total volume of 100 µl medium containing 4,000 HNBECs/well was seeded in

96-well plates in low calcium DMEM/F12 medium and incubated in 37°C for 24 h. The following day, medium was replaced by 100 µl low calcium DMEM/F12 supplemented

with 0.2% BSA and incubated in 37°C for another 24 h. After the treatment of 1.5, 3.0,

6.0, 12.0 nM of leptin was given to HNBECs isolated from control human normal breast

without leptin cultured tissues for 0, 6, 12, and 24 h, 0.1% DMSO to control group, the

proliferation of HBCECs was measured by the methods described above.

15 The sera used in the MTT assay were: control serum (CS) dropped from the non-Z implantation beef heifers at day 0 (NZS-D0) and at day 30 post Z-implantation (NZS-

D30); Z-serum (ZS) harvested from Z implantation heifers at day 0 (ZS-D0) and day 30 post Z-implantation (ZS-D30). The concentration for both CS and ZS was 0.2, 1.0 and

5.0% in cultured medium and was given to HNBECs isolated from non-leptin and 1.5 nM of leptin cultured human normal breast tissues for 6 h.

In the investigation of the effect of the combination of leptin with (-)-gossypol in

HBNECs growth, 4,000 HBNECs isolated from non-leptin cultured tissues were seeded

in 96-well plates in low calcium DMEM/F12 medium and incubated in 37°C for 24 h.

The following day, medium was replaced by 100 µl low calcium DMEM/F12

supplemented with 0.2% BSA and incubated in 37°C for another 24 h. The treatment of

1.5, 3, 6 nM of leptin combined with or without 10 μM (-)-gossypol, 0.1% DMSO as

control was given, the proliferation of HBCECs was measured by the methods described

above.

Cell treatment for RNA and PCR analyses. A total of 105 viable cells/well MCF-7 Adr

cells were seeded in 6-well plates in 5 ml phenol red-free high calcium DMEM/F12 medium. Twenty four h later, the medium was replaced with phenol red-free high calcium DMEM/F12 supplemented with 5% dextran-coated charcoal (DCC), and the cells were cultured overnight. After 24 h, 6 nM leptin, 10, 20, or 30 nM Z alone or

combined with 6 nM leptin, and the combination of 6 nM leptin with 30 nM Z and 5 μM

(–)-gossypol was given to MCF-7 Adr cells which were then incubated for a further 24 h.

16 A total of 105 viable cells/well HBCECs were seeded in 6-well plates in 5 ml low

calcium DMEM/F12 supplemented with 10% of Chelex-100 (Bio-Rad Laboratories,

Hercules, CA) treated FBS (Invitrogen) medium. Twenty -four h later, medium was

replaced with low calcium DMEM/F12 supplemented with 10% DCC stripped FBS, and

the cells were cultured overnight. After 24 h, medium was changed and 24 h treatment

with leptin and zeranol was given to five groups: i) pre-treatment with leptin group (pre-

lp): cells were treated with 3 nM leptin for 24 h and 5 nM Z in following 24 h; ii) post

treatment with leptin group (post-lp): cells were treated with 5 nM Z for 24 h and 3 nM

leptin in following 24 h; iii) leptin group (lp): cells were only treated with 3 nM leptin for

48 h; iv) only Z group (Z): cells were treated with 5 nM Z for 48 h; and v) control group

(CT): cells were only treated with 0.1% DMSO. All media were changed every 24 h.

Viable HNBECs (10 5 / well) were seeded in 6-well plates in 5 ml low calcium

DMEM/F12 supplemented with 10% FBS medium. 24 h later, medium was replaced with

low calcium DMEM/F12 supplemented with 10% Dextran Coated Charcoal stripped FBS, and those cells were cultured overnight. Twenty four h later, medium was changed and

24 h treatment with leptin and zeranol was given. The concentration for leptin was 1.5, 3, and 6 nM, for Z was 5, 10, and 20 nM. The combination of 1.5 nM leptin and 5 nM Z with or without 3 μM (-)-gossypol was also given, 0.1% DMSO was given to control group.

RNA isolation and cDNA synthesis: After cells were treated for 24 or 48 h, the only cultured medium in HNBECs was collected for leptin measurement, the other media were discarded. Total RNA was isolated in 1 ml TRIZOL Reagent (Invitrogen, Carlsbad, CA,

USA) according to the manufacturer’s instructions. RNA concentration was measured by

17 DU-70 spectrophotometer (Beckman Instruments Inc. Fullerton, CA, USA). RNA (1 μg) from cultured cells was reverse transcribed with 200 U M-MLV Reverse Transcriptase

(Invitrogen) at 37°C for 50 minutes and then 70°C for 15 minutes in the presence of 1 μl

10 mM dNTP (10 mM each dATP, dGTP, dCTP, and dTTP at neutral PH) (Invitrogen,),

1 μl 50 μM Random hexamer (Qiagen, Valencia, CA), RNase Inhibitor (Invitrogen), 10

μl 5X First Strand buffer, 5 μl 0.1M DTT and 1μl RNase Inhibitor (Invitrogen) in a total volume of 50 μl in a gradient mastercycle (Eppendorf®, Westbury, NY).

Reverse transcription polymerase chain reaction (RT-PCR). RT-PCR conditions were

optimized for every primer and performed with a thermocycler Gene Amp PCR

(Eppendorf®). A volume of 2 μl of the newly synthesized cDNA was used as templates for RT-PCR. PCR conditions were optimized for MgCl2 concentration, annealing

temperature, and cycle number for the amplification of each of the PCR products (cyclin

D1, P53, or ObR). Under optimal conditions, 1 U platinum Taq DNA polymerase

(Invitrogen) was added in a total volume of 25 μl.

Primers for cyclin D1: Upper primer: 5’-GCT CCT GTG CTG CGA AGT GG-3’,

lower primer: 5’-TGG AGG CGT CGG TGT AGA TG-3’ (product size 372 bp), at 95°C

for 5 min, 27 cycles of 94°C for 45 s, 54°C for 45 s, 72°C for 60 s, then extension at

72°C for 10 min. Primers for P53: Upper primer: 5’-CAT GAC GGA GGT TGT GAG

GC-3’, lower primer: 5’-CGC AAA TTT CCT TCC ACT CG-3’ (product size 336 bp),

at 95°C for 5 min, 31 cycles of 95°C for 45 s, 57°C for 45 s, and 72°C for 45 s, then

extension at 72°C for 10 min. Primers for ObR common domain: Upper primer: 5’-CAT

TTT ATC CCC ATT GAG AAG TA-3’, lower primer: 5’-CTG AAA ATT AAG TCC

TTG TGC CCA G-3’ (product size 273 bp), at 95°C for 5 min, 30 cycles of 95°C for 40 s, 18 60°C for 50 s, 72°C for 50 s, then extension at 72°C for 10 min. Primers for 36B4: Upper

5’- AAA CTG CTG CCT CAT ATC CG-3’, lower 5’- TTT CAG CAA GTG GGA AGG

TG-3’ (product size 563 bp), at 95°C for 5 min, 24 cycles of 95°C for 60 s, 63°C for 60 s,

72°C for 60 s, then extension at 72°C for 10 min. Pure H2O was used as negative control

in order to detect genomic DNA contamination and 36 B4 as internal control whose RNA

is unmodified by treatment.

The final RT-PCR products (10 µl) mixed with 1 µl 10 X loading buffer were

separated on 1.5% agarose gel and visualized by staining with ethidium bromide.

Electronic images were taken by a FUJIFILM LAS-3000 image system (FUJIFILM

Medical Systems USA, Inc. Stanford, CT, USA). The densities of specific bands were

quantified by ImageQuant software (Molecular Dynamics, Sunnyvale, CA, USA). The

results were presented as the ratio of cyclin D1 to 36B4, p53 to 36 B4, and ObR to 36 B4.

Western blotting assay. Primary cultured HNBECs isolated from non-leptin and 1.5 nM leptin cultured tissue were placed in a 10 cm culture dish with a density of 1 × 10 6 viable cells/well within 10 ml low calcium DMEM/F12 supplement with 10% FBS and cultured overnight. The media was replaced with low calcium DMEM/F12 supplemented with 5%

DCC treated FBS and remained cultured for another 24 hr. Primary cultured HNBECs were then treated with 1.5, 3 nM leptin, 5, 10, 20 nM Z, 0.1% DMSO as a vehicle control.

After 24 h treatment, culture media were collected for leptin measurement and proteins were isolated from the control group and each treatment group using M-PER® mammalian protein extraction reagent (Pierce, Rockford, IL) according to the manufacturer’s instructions. Culture media were collected for further study, and 200 µl of extraction reagent was added in each dish. Afterwards, each dish was placed on an orbital

19 shaker to be digested for 5 min. Digested products were collected and transferred to a 1.5 ml centrifuge tube. Mixture was centrifuged at 19,000 x rpm for 15 minute, and the supernatant was then transferred to a new 0.5 ml centrifuge tube. Protein concentrations were measured using a Mciro BCATM protein assay reagent kit (Pierce) followed by the manufacturer’s protocol. A total of 50 µg of proteins from each treatment group were separated by SDS-PAGE and then transferred to a Polyvinylidine Fluoride (PVDF) membrane (Bio-Rad Laboratory, Hercules, CA). The membrane was first blotted in

Phosphate Buffered Saline - Tween 20 (PBST) containing 10% fat free dry milk for 1 h and then incubated with primary antibody (Cyclin D1 1: 1000 dilution and p53 1: 1000 dilution, Cell Signaling Technology® Danvers, MA; β actin, 1:2000 dilution) ( Santa

Cruz Biotechnology, Inc. Santa Cruz, CA) for 1 h. The membrane was rinsed in PBST three times, each time last for 5 minutes. In the following step, the membrane was incubated within the second antibody for 1 h. After washing the membrane in PBST three times, the membrane was detected by using Fuji Imagine System. Pictures were taken by

FujiFilm LAS-300 imagine system (FUJIFILM Medical Systems USA, Inc.). The protein ratios of cyclin D1 to β actin were calculated by measuring the density of the specific band using Multi-Gauge (v3.0).

Leptin Measurement. Culture media were collected before protein extraction as previous statement. The human leptin immunoassay kit for leptin measurement was purchased from R&D Systems (Minneapolis, MN). The particulates in cell culture supernates were removed by centrifugation in 1,200 rpm for 5 min and assay immediately according to the manufacturer’s instructions. Assay was stopped by adding 50 μl stop solution to each well and OD values were measured within 30 min by kinetic microplate reader

20 (Molecular Devices Cooperation, Menio Park, CA) at 450 nm wavelength and leptin

concentration was compared.

Statistical analysis. The results for the cell proliferation assay are presented as

mean ± standard deviation (SD) for 4 replicate culture wells. Analysis was performed by

using Minitab 15 (Minitab Inc. PA). Statistical difference was determined by using two-

sample t-test analyses for independent samples. P-values of less than 0.05 were considered statistically significant.

Results

Leptin increased cell proliferation of MCF-7Adr cell, HBCECs and HBNECs. As shown in Figure 2.1, 6 and 12 nM leptin significantly increased MCF-7Adr cell growth by at least 20% and in a dose-dependent manner. Leptin increased all the epithelial cell growth from 7 breast cancer patient tissues and 7 normal breast tissues disregarding match or not (data not shown). Figure 2.2 shows leptin significantly increased HBCECs growth in a dose-dependent manner and HBCECs were more sensitive to leptin than

MCF-7 Adr cells. Figure 2.3 shows that the average effective dose of all three matched

HBCECs were more sensitive to leptin than HBNECs which were isolated from the same patients.

Z increased breast cancer cell growth. Our data showed that 20 and 30 nM Z treated for

24 h can significantly increase MCF-7 Adr cell growth by 26% and 35% respectively

21 (Figure 2.4) and HBCECs were more sensitive to Z than MCF-7 Adr cells. Z at 5 nM for

24 h increased the HBCECs growth in a dose-dependent manner (Figure 2.5).

Leptin induced breast cancer cell sensitivity to Z. Figure 2.6 shows the MCF-7 Adr

cells treated with 6 nM leptin and 30 nM Z exhibited the most cell proliferation compared

to the 10 nM and 20 nM Z groups and significant difference between pre-lp and post-lp

was found in this group. This significant difference was also found in the primary cultured HBCECs between pre-lp and post-lp treatment (figure 2.7). As shown in Figure

2.8, MCF-7 Adr cells pre-treated with leptin grew significantly faster than post-lp, Z and leptin groups. As shown in Figure 2.9, 3 nM leptin and 5 nM Z significantly increased the growth of HBCECs after 48 h treatment and 5 nM Z stimulates cell growth more potent than that of 3 nM leptin. However, the pre-lp group had the statistic significance in stimulating cell growth, compared to the post-lp group. The pre-lp group is the best to stimulate HBCECs growth compared to the groups treated with 5 nM Z.As shown in

Figure 2.7, compared to the combination of 3 nM leptin with 1.25, 2.5 nM Z, the combination of 5 nM Z with 3 nM leptin is the best for increasing the sensitivity of primary cultured HBCECs to Z for proliferation.

Besides, the combination of 6 nM leptin with 30 nM Z significantly promoted cell growth compared to 6 nM leptin and 30 nM Z alone while (–)-gossypol at 5 µM significantly

suppressed the stimulation induced by Z combined with leptin (Figure 2.10). Figure 2.11

shows the effects of leptin and (-)-gossypol {(-)-G} on primary cultured HBNECs growth.

Compared to control group, 3 and 6 nM leptin can significantly increase HBNECs growth.

Significant difference was found between 3 and 6 nM leptin combined with and without

10 μM (-)-gossypol (*, p<0.05) 22 Z-containing serum increases primary cultured HNBECs growth. Figure 2.12 shows that NSZ-D0 and NSZ-D30 has at all doses no effect on primary cultured HNBECs isolated from non-leptin cultured tissue but it can stimulate the primary cultured

HNBECs growth isolated from leptin cultured tissue at the same dose (Figure 2.13).

Figure 2.14 indicates the ZS-D0 and ZS-D30 at 0.2, 1, and 5% concentration can increase the primary cultured HNBECs isolated non-leptin leptin cultured tissue compared to control group, respectively. Figure 2.15 indicates ZS-D0 and ZS-D30 at 0.2,

1, and 5% concentration can increase the primary cultured HNBECs isolated 1.5 nM of leptin cultured tissue with the significant difference between the ZS-D0 and ZS-D30 at all dose.

The effect of leptin, Z and (-)-gossypol in cyclin D1 expression in breast cells.

Figure 2.16 shows that 6 nM leptin and 30 nM Z alone increased cyclin D1 mRNA expression without significant difference. When they were combined, cyclin D1 mRNA expression was significantly increased while (–)-gossypol at 5 µM counteracted the stimulatory effect. In HBCECs, the pre-treated with leptin group can significantly increase the cyclin D1 expression (Figure 2.17). This result shows that the lp group and the pre-lp group significantly increased cyclin D1 expression in mRNA level in HBCECs when compared to control group. Besides, HBNECs were treated with 1.5, 3, 6 nM leptin, 5, 10, 20 nM Z, the combination of 5 nM Z and 1.5 nM leptin with or without 3

μM (-)-gossypol for 24 h. Significant differences in cyclin D1 expression compared to the control group at * p<0.05 was found in the group of treated with 3 and 6 nM leptin and 10 and 20 nM Z. But, when 1.5 nM leptin combined with 5 nM Z, significant difference was found, and the effective combination can be suppressed by adding 3 μM 23 (-)-gossypol (Figure 2.18). Consistent with the mRNA expression, the Cyclin D1 protein

expression was also increased with the 24 h treatment of Z in HBNECs. (Figure 2.19)

Effect of leptin and Z on P53 expression in MCF-7 Adr cells. 6 nM leptin and 30 nM Z

reduced P53 mRNA expression without significant difference as compared to the control

group. When they were combined at the dose, the P53 mRNA expression was

significantly reduced compared to the control group, while (-)-gossypol at 5 µM reversed

their combination effect (Figure 2.20). There was a significant difference between the

combination of leptin and Z with and without (–)-gossypol.

Z significantly increased ObR expression in breast cancer cells. As shown in Figure

2.21, compared to the control group, 30 nM Z can significantly increased ObR expression in MCF-7 Adr cells by about 40%, while 10 and 20 nM Z increased the ObR expression but not significantly. Z (5 nM) also can significantly increase the ObR expression in

HBCECs (Figure 2.22). As shown in Figure 2.22, compared to the control, the 5 nM Z group and the post-lp group (treated with 5 nM Z for 24 h and then treated with 3 nM leptin for following 24 h) significantly improved the ObR gene expression.

Z increased HBNECs secrete leptin. As shown in Figure 2.23, 20 nM Z significantly increased the HNBECs secret leptin, compared to the control group at * p<0.05.

Discussions:

Cyclin D1 is a cell cycle regulator and plays an important role in cell growth. It is the

product of the CCND1 gene which is located on chromosome 11q13 [46]. The cyclin-

24 dependent kinases (CDKs) can not regulate cell growth without the cyclin subunit. By

binding to cyclin D, cyclinD-CDK 4/6 constitutes the engine of the cell cycle machinery

and affects the G1 phase in cell growth. The cyclin D1 level can be modulated by

changing growth factors in the medium used to culture cells. Cyclin D1 was found to be

overexpressed in over 50% of the breast cancer patients and known as one of the most

overexpressed proteins in breast cancer [46]. Leptin stimulates breast cancer cell growth

by up-regulating cyclin D1 expression. Moreover, Garofalo et al. found leptin could

modulate both estrogen synthesis and estrogen receptor (ER) α activity [3]. Besides

controlling the cell cycle, cyclin D1 was found to be associated with ER [46]. Cyclin D1

can bind to the ER and stimulate its transcriptional activities. The cyclin D1 and ER complex may play a role in stimulating the tumor cell proliferation. On the other hand,

P53 is a tumor suppressor gene. It is reported that cyclin D1 expression can be regulated by P53 [46].

Our data show that 6 nM leptin or 30 nM Z alone had no affect on the cyclin D1

expression but their combination significantly increased the expression of cyclin D1

comparing to the control group. In our cell proliferation assay, both MCF-7 Adr cells and

HBCECs pre-treated with leptin increased their sensitivity to Z. This can partly be

explained by the fact that the combination resulted in high expression of cyclin D1. It is

possible that if obese healthy women or breast cancer patients have higher leptin in their

serum, the sensitivity of normal or cancerous breast cells may be increased to Z contained

in beef product. Under such circumstances, the risk of breast cancer may be increased by

Z-contained in beef because the breast cancer patients expressed more leptin than control

group and leptin can induce human breast cancer epithelial cell more sensitive to Z [10].

25 Moreover, we found that primary cultured human normal breast epithelial cells were more sensitive to leptin and Z than are MCF-7Adr cells and leptin increased the HBCECs growth at a much lower dose than HNBECs.

According to other researchers, the serum level of leptin in breast cancer patients is higher than that in controls [32]. Figure 2.8 shows that 30 nM Z stimulated cell growth more than 6 nM leptin in MCF-7 Adr cells. However, cells pre-treated with 6 nM leptin grew more than those only treated with 30 nM Z for 48 h (Figure 2.6). The pre-treatment of 6 nM leptin significantly stimulated cell growth, comparing to the other groups. This result supports our hypothesis that pre-treatment of 6 nM leptin for 24 h can increase the sensitivity of MCF-7Adr cell to Z and thus enhance the proliferation of MCF-7 Adr cells.

The same patent was found in primary cultured HBCECs. Figure 2.9 shows that 5 nM Z stimulated cell growth more than 3 nM leptin in HBCECs. However, cells pre-treated with 3 nM leptin grew more than those only treated with 5 nM Z for 48 h (Figure 2.9).

The pre-treatment of 3 nM leptin significantly stimulated cell growth, comparing to the other groups. This result supports our hypothesis that pre-treatment of 3 nM leptin for 24 h can increase the sensitivity of HBCECs to Z and thus enhance the proliferation of

HBCECs. All these results suggest a possible relationship between obesity and breast cancer and points to the potential risk for breast cancer in obese people, especially in those consuming animal products implanted with Z because leptin can increase the cell sensitivity to Z.

On the other hand, like estrogen modulating the ObR expression in some estrogen-responsive tissues [31], 30 nM Z can increase ObR expression in MCF-7 Adr cells and 5 nM Z increases ObR expression in HBCECs so as to strengthen the action of

26 leptin. The molecular action of leptin was initiated by leptin binding to leptin receptor

ObR. When treated with Z, the MCF-7 Adr cells and HBCECs increased their expression

of ObR and thus the action of leptin was amplified. This result indicates that Z might be

more harmful to obese people than normal weight people in increasing breast cancer risk

[38] because their consumption of Z-containing products may amplify their chances of

having breast cancer. However, (–)-gossypol can reverse the effect of the combination of leptin with Z on cell growth; it could be used in the treatment of breast cancer, especially in obese multidrug resistant patients.

Our data shows in this study there is no observable proliferative stimulation by exposing the NZS for 6 h in primary cultured HNBECs isolated from the non-leptin cultured tossues (Figure 2.12). However, the proliferation of primary cultured HNBECs isolated from 1.5 nM leptin cultured tissues was significantly increased by treatment of

ZS at all doses for 6 h (Figure 2.15). As can be seen in Figure 2.14 and Figure 2.15, ZS increases primary cultured HNBECs isolated from leptin cultured tissues more than that isolated from non-leptin cultured tissues. Comparing Figure 2.12 to Figure 2.13, Figure

2.14 to Figure 2.15, the result showed that the HNBECs isolated from 1.5 nM of leptin cultured tissues grow faster than those isolated from non-leptin cultured tissues with the same treatment of ZS or NZS. It demonstrates that leptin can stimulate HNBECs growth.

Meanwhile, comparing Figure 2.12 to Figure 2.14, Figure 2.13 to Figure 2.15, ZS increases primary cultured HNBECs isolated from leptin cultured tissues more than those

isolated from non-leptin cultured tissues. The stimulatory effect of ZS is greater than that

of NZS in primary cultured HNBECs isolated from both with or without leptin cultured

tissues. The significant difference between the ZS-D0 and ZS-D30 at 0.2, 1, and 5% 27 concentration was only found in the primary cultured HNBECs isolated from 1.5 nM leptin cultured tissues. The only difference between NZS and ZS is the implantation of Z pallet, ralgro and its metabolic material in the beef blood. This result imply that some, not-yet-defined, growth factors which are responsible for stimulatory action in the primary cultured HNBECs proliferation may be secreted by the Z-implanted heifers into blood circulation. We might attribute the stimulatory effect of ZS on the primary cultured

HNBECs to the implantation of Z.

It seems that leptin can stimulate primary cultured HNBECs growth and ZS-D30

can improve the leptin induced growth. This result is consistent with the proliferative

effect of leptin in HBCECs and increased HBCECs sensitivity to Z because the cells in

pre-lp group grew faster than those treated with Z alone.

Considering the leptin level is higher in obese women than in normal or lower

weight women, this result suggests the obese women maybe more sensitive to Z. This

also proved our hypothesis that obese women may have higher risk of breast cancer when

they are consuming beef products containing Z might be right. The current study showed

leptin can increase cell sensitivity to Z both in primary cultured human breast cancer

epithelial cells and breast cancer cell line, MCF-7 Adr cells [47].

In summary, we found mitogenic activity of Z in human breast cancer cells is

enhanced by leptin and suppressed by gossypol. Leptin appears to increase MCF-7 Adr

cell growth via increasing cyclin D1 mRNA expression. Leptin improves the MCF-7 Adr

cell sensitivity to Z and Z can strengthen the leptin by increasing ObR expression in the

MCF-7 Adr cell and in HBNECs and HBCECs and by resulting in HBNECs secreting

leptin. Both leptin and Z up-regulated cyclin D1 expression in HBNECs and HBCECs,

28 however, (–)-Gossypol can counteract the growth of breast cancer cells induced by leptin

alone or combined with Z by down-regulating cyclin D1, and up-regulating P53 mRNA

expression. More mechanisms will be further studied in the future. This study is the first

to reveal that (–)-gossypol as a food component in cottonseed products may serve as a

potential chemopreventive agent to suppress the stimulatory effect of Z and leptin on human breast cancer cells.

0.7

* 0.6 * 0.5

0.4

0.3

Growth Cell 0.2

0.1

0 03612 leptin (nM)

Figure 2.1 The effect of Leptin on MCF-7 Adr cell growth.

MCF-7 Adr cells were treated with 3,6,12 nΜ of leptin, 0.1% DMSO as control. The symbols “*” indicate significant differences in MCF-7 Adr cell growth compared to the control group (p<0.05).

0.8 * * * 0.6

0.4 Cell growth 0.2

0 CT 3 6 12 leptin (nM)

Figure 2.2. Effect of leptin on primary cultured HBCECs growth. Primary cultured

HBCECs were treated with 0, 1.5, 3 and nM leptin for 24 hrs. Significant differences in

HBCECs growth compared to the control group at * p<0.05.

7 ** 6 N=3 5

Leptin (nM) 2

0 HBCECs HBNECs

Figure 2.3. Comparison of effective dose of leptin on primary cultured HBCECs and

HBNECs growth.

Primary cultured HBCECs and HBNECs from the same patients were treated with 0, 0.75,

1.5, 3, 6 and 12 nM leptin. Average sensitive dose of leptin were calculated and t-test was conducted. The average dose of leptin in HBCECs was significant lower that that in

HBNECs growth. p<0.05.

0.8

** 0.6 *

0.4

Growth Cell 0.2

0 0102030 Zeranol (nM)

Figure 2.4. The effect of Zeranol on MCF-7 Adr cell growth.

MCF-7/ Adr cells were treated with 10, 20 30 nM Zeranol for 24 hrs, 0.1 % DMSO as control. The symbols “*”and “**”indicate significant differences in MCF-7 Adr cell growth compared to the control group (*, p<0.05; **, p<0.01).

1.0 * * * 0.8

h 0.6

growt

ll 0.4 e

C 0.2

0.0 CT 5 10 20

Zeranol (nM)

Figure 2.5. Effect of zeranol on primary cultured HBCECs growth.

Primary cultured HBCECs were treated with 0, 5, 10 and 20 nM zeranol fro 24 hrs, 1%

DMSO as control. Cell was measured and compared. Significant differences in HBCECs growth compared to the control group at * p<0.05, ** p<0.01.

* 1.2 post-lp pre-lp ** 1.0

th 0.8 ** ** 0.6 * grow ll e

C 0.4

0.2

0.0 0102030 Zeranol (nM)

Figure 2.6 Comparison of MCF-7 Adr cell growth with pre-lp and post-lp treatment.

MCF-7 Adr cells were treated with 10, 20, and 30 nM zeranol or 6 nM leptin in 1st and/or 2nd 24 hours. 0.1% DMSO as control. All medium was changed every 24 hrs, 48 h later, cell growth was measured. The symbols “*” and “**” indicate significant differences in MCF-7 Adr cell growth compared to the control group and significant difference between the groups of pre-lp and post-lp (*, p<0.05; **, p<0.01).

1.4 * post-lp pre-lp 1.2 *

1 * * 0.8

0.6

growth Cell 0.4

0.2

CT 1.25 2.5 5

Zeranol (nM)

Figure 2.7 Comparison of the primary cultured HBCECs growth with pre-lp and post-lp treatment.

HBCECs were treated with 1.25, 2.5, and 5 nM zeranol or 3 nM leptin in 1st and/or 2nd

24 hours, 0.1% DMSO as control. Forty eight h later, cell growth was measured.

Significant differences in HBCECs growth compared to the control group at * p<0.05.

1.2 * *

* ** 1.0 * 0.8 *

0.6

* Cell Growth 0.4

0.2

0.0 CT lp Z pre-lp post-lp

Figure 2.8 Comparison of the proliferative effect of leptin, zeranol, pre-lp and post- lp treatment in MCF-7 Adr cells.

MCF-7/Adr cells were treated with 6 nΜ leptin (lp) or 30 nM Zeranol (Z) in the 1st and/

or 2nd 24 hours, 0.1% DMSO as control. Forty eight h later, cell growth was measured.

The symbols “*” and “**” indicate significant differences in MCF-7 Adr cell growth

compared to the control group and between any two groups (*, p<0.05; **, p<0.01).

1.5 * *

* 1.2 * * 0.9 *

0.6

growth Cell

0.3

0 CT lp lp+Z Z+lp Z

Figure 2.9 Comparison of the proliferative effect of leptin, zeranol, pre-lp and post- lp treatment in HBCECs.

HBCECs were treated with 3 nΜ leptin (lp) or 5 nM Zeranol (Z) in the 1st and/ or 2nd 24

hours. Lp+Z means the cells were treated with leptin in 1st 24 hrs and then Z in 2nd 24 hrs;

Z+lp means cells were treated with Z in 1st 24 hrs and then leptin in 2nd 24 hrs. The symbols “*” and “**” indicate significant differences in MCF-7 Adr cell growth compared to the control group and between any two groups (*, p<0.05; **, p<0.01).

* ** 0.6 * ** 0.5 ** ** 0.4 *

0.3

0.2 Growth Cell 0.1

0 CT lp Z lp+Z lp+Z+(-)-G

Figure 2.10 Effects of leptin (lp), Zeranol (Z) and (-)-Gossypol (G) on MCF-7/Adr cell proliferation.

MCF-7/Adr cells were treated with 6 nM leptin (lp), 10 nM Zeranol (Z) and 5 μM (-)-

Gossypol (G) alone or combination for 24 hrs and cell growth was measured. The symbols “*”and “**”indicate significant differences in MCF-7 Adr cell growth compared to the control group and between any two groups (*, p<0.05; **, p<0.01).

* * lp 0.6 * lp+(-)-G *

0.4

Cell growth

0.2

0 01.536 Lep tin (n M)

Figure 2.11 Effects of leptin and (-)-gossypol (G) on primary cultured HBNECs growth

Primary human normal breast epithelial cells were isolated from non-leptin cultured tissues and treated with 1.5, 3, 6 nM leptin alone or combined with 10 μM (-)-gossypol for 24 hrs. Cell growth was measured. Significant difference in HBNECs between with or without (-)-gossypol treatment at 3 and 6 nM leptin group (*, p<0.05).

0.3 NZS-D0 NZS-D30

0.2

Cell growth 0.1

0 0.0 0.2 1.0 5.0 N on-Z-implanted serum (%)

Figure 2.12 Effects of None-Zeranol serum on ECs isolated from non-leptin

cultured tissues.

Primary human normal breast epithelial cells were isolated from non-leptin cultured

tissues and treated with 0.2%, 1%, and 5% NZS-D0 and NZS-D30 at cultured medium for 24 hrs. Cell growth was measured. No significant difference was found at any group compared to control group (0.0% group)

NZS-D0 NZS-D30 0.8 * * * * * *

0.6

0.4

growth Cell

0.2

0.0 0.00.21.05.0

Non-Z-implanted serum (%)

Figure 2.13 Effects of non-Zeranol serum on ECs from1.5 nM of leptin cultured

HNBTs.

Primary human normal breast epithelial cells were isolated from non-leptin cultured

tissues and treated with 0.2%, 1%, and 5% NZS-D0 and NZS-D30 at cultured medium for 24 hrs. Cell growth was measured. No significant difference was found between NZS-

D0 and NZS-D30 at any dose.

0.4 ZS -D0 ZS -D30 * **

0.3 ** *

0.2

Cell growth Cell 0.1

0 0.0 0.2 1.0 5.0

Z-implanted serum (%)

Figure 2.14. Effects of Zeranol serum on ECs from non-leptin cultured tissues.

Primary human normal breast epithelial cells were isolated from non-leptin cultured tissues and treated with 0.2%, 1%, and 5% ZS-D0 and ZS-D30 at cultured medium for 24 hrs. Cell growth was measured. No significant difference was found between ZS-D0 and

ZS-D30 at any dose.

ZS-D0 ZS-D30

* * * 1.2

** * 1 * * * 0.8

0.6 0.4 Cell Growth Cell 0.2 0

00.21 5 ZS (%)

Figure 2.15 Effects of Zeranol serum on ECs from 1.5 nM of leptin cultured

HNBTs.

Primary human normal breast epithelial cells were isolated from 6 nM leptin cultured tissues and treated with 0.2%, 1%, and 5% ZS-D0 and ZS-D30 at cultured medium for 24 hrs. Cell growth was measured. Significant difference was found between ZS-D0 and ZS-

D30 at all dose.

Cyclin D1

36 B4

0.8 * *

0.6

0.4

0.2

B4) (Cyclin D1/36

relative mRNAexpression 0 CT lp Z lp+Z lp+Z+(-)-G

Figure 2.16 Effects of leptin and Zeranol and (-)-Gossypol alone or combination on cyclin D1 expression in MCF-7/Adr cells.

The 24 hr treatment of 6 nM lp, 30 nM zeranol and 5mM (-)-gossypol was given and cyclinD1 mRNA was amplified. The symbol “*” indicates significant differences in cyclin D1 expression compared to the control group, and between the combination of leptin and Zeranol with or without Gossypol groups (p<0.05).

CyclinD1 36B4

4 *

* 3.6

3.2

2.8 (cyclin D1/36B4) 2.4 Relative mRNA Expression Expression mRNA Relative 2 CT lp Z post-lp pre-lp

Figure 2.17 Effects of leptin and zeranol on cyclinD1 expression in HBCECs.

HBCECs were treated with 3 nM leptin, 5 nM zeranol in 1st or 2nd 24 hours. All media were changed every 24 hrs. Then RNA was extracted, cDNA was synthesized and cyclinD1 was amplified. Significant differences in cyclinD1 expression compared to the control group at * p<0.05.

Cyclin D1

36 B4

* ** 7 * * * 6 * 5

4 3

(Cyclin D1/36B4) 2 1 Relative mRNAExpression 0 1 2 3 4 5 6 7 8 9

Figure 2.18 Effects of leptin, zeranol and (-)-gossypol on cyclin D1 expression in

HBNECs.

HBNECs were treated with 1.5, 3, 6 nM leptin, 5, 10, 20 nM zeranol, the combination of

5 nM zeranol and 1.5 nM leptin with or without 3 μM (-)-gossypol for 24 hrs. 1:CT; 2:

1.5 nM lp; 3: 3 nM lp;4: 6 nM lp; 5:5 nM Z; 6: 10 nM Z; 7: 20 nM Z; 8:5 nM Z+1.5 nM lp; 9: 5 nM Z+1.5 nM lp+3mM (-)-G. Then RNA was extracted, cDNA was synthesized

and cyclin D1 was amplified. Significant differences in cyclinD1 expression compared to

the control group at * p<0.05.

Cyclin D1

β actin

0.7

0.6

0.5

actin)

β 0.4

0.3

0.2 (Cyclin D1/ D1/ (Cyclin 0.1

Relative Protein Expression Relative Protein Expression 0 CT 5.0 10 20

Figure 2.19 Zeranol up-regulated Cyclin D1 protein expression in HBNECs

Primary cultured human normal breast epithelial cells were treated with 5, 10, and 20 nM zeranol, 0.1% DMSO as control. 24 h later, protein was extracted and western blot was conducted. Cyclin D1 expression was compared.

P53

36B4

2.2 * 2.1

1.9 * 1.8

mRNA Relative 1.7 Expression (P53/36B4) Expression 1.6 CT lp Z lp+Z lp+Z+(-)-G

Figure 2.20 Effects of leptin, Zeranol and (-)-Gossypol alone or combination on

P53 expression in MCF-7/Adr cells.

The 24 h treatment of 6 nM lp, 30 nM zeranol and 5μM (-)-gossypol was given and RNA was extracted. Then cDNA was synthesized and P53 was amplified. The symbol “*” indicates significant differences in P53 expression compared to the control group and between the groups of the combination of leptin and Zeranol with or without (-)-

Gossypol (p<0.05).

ObR 36B4

0.8 * 0.6

0.4

(ObR/36B4) 0.2

0 Relative mRNA Expression Expression mRNA Relative CT 10 20 30 Zeranol (nM)

Figure 2.21 Effects of Zeranol on ObR expression in MCF-7 Adr cells. The 24 h treatment of 10, 20, and 30 nM zeranol was given and RNA was extracted. Then cDNA was synthesized and the ObR gene was amplified. The symbol “*” indicates significant difference in ObR mRNA expression compared to the control group (p<0.05).

ObR

36B4

3.0

2.5 * * 2.0

1.5

(ObR/36B4) 1.0

0.5 Relative mRNA Expression mRNA Relative 0.0 CT lp Z post-lp pre-lp

Figure 2.22 Effects of leptin and zeranol on ObR expression in HBCECs.

HBCECs were treated with 3 nM leptin and 5 nM zeranol in 1st or 2nd 24 hours. Then

RNA was extracted, cDNA was synthesized and ObR gene was amplified. Significant differences in ObR gene expression compared to the control group at * p<0.05.

0.2

0.16 * 0.12

0.08

leptin in medium (nM) medium in leptin 0.04

0 CT 5 10 20 zeranol (nM)

Figure 2.23 Zeranol increased the HNBECs secret leptin

HNBECs were treated with 5, 10, and 20 nM zeranol for 24 hrs and culture medium were collected and the human leptin immunoassay was conducted. Zeranol at 20 nM can significantly increase the HNBECs secret leptin, compared to the control group at * p<0.05.

Chapter 3

The Interaction of Leptin, Zeranol and Gossypol in Human Normal Breast Pre-adipocyte Growth

Abstract

Adipocytes account for more than 90% of human breast volume and secrete some

adipocytokines which play a role in breast cancer development. Leptin, one of the adipocytokines, is mainly secreted by adipocytes and plays an important role in breast cancer development. Leptin expression is up-regulated both in obesity and breast cancer patients. It can promote breast cancer cell growth. On the other hand, exposure to environmental estrogens has been found to be directly related to breast cancer. Zeranol

(Z), is a non-steroidal anabolic growth promoter with estrogenic activity and widely used in the U.S beef industry because of the commercial benefit. Gossypol, another natural compound extracted from cottonseed, can inhibit breast cancer growth. It is suggested gossypol could be used as potential chemopreventive food components.

This study demonstrated that Z and ZS stimulated the growth of pre-adipocytes isolated from human normal breast tissues. This study is focusing on evaluation of Z and bio- active Z-containing sera collected from Z-implanted beef and its adverse health risk to human consumption of Z-containing meat produced from Z-implanted beef cattle. We

53 hypothesize that Z might increase the risk of breast cancer in obese women. Cell

proliferation assay, ELISA analysis and RT-PCR were conducted.

The primary cultured human normal breast pre-adipocytes (HNBPADs) were isolated

from human normal breast tissue and cultured. The MTT assay to evaluate the

proliferation of HNBPADs was carried out. The sera used in the MTT assay were: non-

zeranol serum (NZS) dropped from the non-zeranol-implantation beef heifers at day 0

(NZS-D0) and at day 30 post zeranol-implantation (NZS-D30); zeranol-serum (ZS)

harvested from zeranol implantation heifers at day 0 (ZS-D0) and day 30 post zeranol-

implantation (ZS-D30). The concentration for both NZS and ZS was 0.2, 1.0 and 5.0% in

cultured medium.

Our result shows both Z and lp can increase primary cultured HNBECs, HBCECs,

HNBPADs and MCF-7 Adr cell growth. ZS have a greater power to stimulate HNBPADs

growth than NZS and the combination of Z with leptin increase HNBPADs cyclin D1

mRNA expression both in mRNA and protein levels and down-regulate PTPγ mRNA expression while (-)-gossypol revert its effect in MCF-7 Adr cells, primary cultured

HNBPADs.

Leptin can increase the breast HNBPADs’ sensitivity to Z and ZS. On the other hand, and Z stimulates primary cultured HNBPADs secrete leptin. These results pointed out that the leptin may be involved in enhancing the HNBPADs sensitivity to ZS and there is interaction between Z and leptin in HNBPADs.

It suggests Z maybe more harmful to obese patients and might play a role in breast cancer

development and (-)-gossypol may serve as a potential chemopreventive agent and play

an important role in breast cancer chemoprevention.

54 Introduction

Breast cancer is the leading cancer in women in the United States. Exposure to environmental estrogens has been found to be directly related to the increase in breast cancer incidence. Z is a non-steroidal anabolic growth promoter with estrogenic activity and widely used in the U.S beef industry for improvement of weight gain, feed efficiency and marbling. Our previous study demonstrated that Z stimulated the proliferation of primary cultured human normal breast cells and MCF-7 cells. One of known risk factors of breast cancer is obesity which has become a major public health concern in the U.S.

The condition of obesity is characterized by increases in fat cell number, fat cell size, or a combination of the two [6]. It is assumed that a gradual accumulation of body fat after birth is mainly due to increasing fat cell size, not fat number, and total fat cell number increases only in severe obesity [6]. Adipocytes were thought to be extremely stable and food restriction alone does not lead to a decrease in adipocyte number, but in adipocyte size [48]. Leptin, mainly secreted by adipocytes, is a product of the obese (ob) gene and plays an important role during pregnancy and fetal development [6]. One of the major functions of leptin is to regulate the energy balance and appetite regulation (food intake)

[6]. Leptin targets the brain to increase loss of adipose tissue by increasing adipocyte apoptosis.

In human breast, adipocytes account for about 90% of the bulk while epithelial cells only make up the 10% [14]. Fat tissue contains small and large adipocytes as well as stromal and endothelial cells [6]. Various hormones such as insulin and sex steroid have been associated with dynamic changes of the adipose tissue and are believed to contribute to the signals that cause adipocyte differentiation [6]. An increase in breast cancer risk is 55 associated with higher BMI levels and this association is restricted to women with ER+ tumors [2]. In fact, obesity is not associated with the deficiency of leptin but resistance to leptin [29]. Leptin was found to increase the estradiol-induced activation of ERα,

suggesting that leptin and estrogen might cooperate in the growth of estrogen-dependent

breast cancer cells [29]. As previous stated, leptin might be involved in the resistance of

breast cancer cells to anti-estrogen therapies [22]. In general, adipose tissue mass is

determined by a balance of lipolysis, lipogenesis, and adipocyte proliferation [49]. The

adipogenesis is a complex process of differentiation of preadipocytes [49]. The

mechanism is still not fully understood. Researchers found that leptin does not act

directly to induce adipocyte apoptosis, leptin acts only centrally to initiate a signal to the

adipose tissue to cause apoptosis [49]. Leptin had no effects on apoptosis of either

preadipocytes or mature adipocytes within 24/48 hr incubation in 3T3-L1 adipocytes, but

can act directly to inhibit maturation of preadipocytes and suppressed lipid accumulation

during 3T3-L1 differentiation [49].

This study is focusing on evaluation of bio-active Z-containing sera collected

from Z-implanted beef and its adverse health risk to human consumption of Z-containing

meat produced from Z-implanted beef cattle. The objective of this study was to

investigate the intervention of leptin, Z or ZS, and (-)-gossypol on the primary cultured

human breast preadipocytes growth. We hypothesize that obese women may have higher

risk of breast cancer when they are consuming beef products containing

Z. .

56 Materials and methods

Biological sample resource, reagents, human breast tissues, tissue culture. All process

was the same described in chapter 2.

Primary cultured human normal breast pre-adipocytes isolation. Primary cultured

human normal pre-adipocytes were isolated described in the chapter 2 and cultured. After

96 h cultured, the human normal breast fat tissues were minced and then digested in

digestion buffer which is consist of phenol red-free high calcium DMEM/F12 (1.05 mM

CaCl2) with 2% Bovine Serum Albumin (Invitrogen) containing 10ng/ml Cholera toxin

(Sigma) 6300U/ml Collagenase (Invitrogen), and 100U/ml Hyalurinidase (Calbiochem).

After the mixture was incubated in 37°C overnight, the solution was transferred to a 45

ml tube and centrifuged in 1,200 rpm for 5 min. The upper, middle and lower layers were

separated and centrifuged again. The upper layer containing pre-adipocytes was

transferred to another 5 ml tube and was washed by DMEM/F12 medium with antibiotic-

antimycotic (100 unit/ml penicillin G sodium, 100 μg/ml streptomycin sulfate and 0.25

μg/ml amphotericin B) (Invitrogen) and centrifuged again. This wash procedure was repeated for three times. The final pellet in the tube contains primary cultured human normal breast pre-adipocytes (HNBPADs). The pellet was resuspended in 10 ml high calcium (1.05 mM CaCl2) DMEM/F12 medium supplemented with 10% FBS ( Atlanta

Biologicals, Norcross, GA) and then transferred into a T75 flask for culturing.

Cell culture. The isolated primary cultured HNBPADs were allowed to attach and

2 cultured in a 75 cm culture flask in a humidified incubator (5% CO2, 95% air, 37°C)

with 10 ml high calcium (1.05 mM CaCl2) DMEM/F12 mixture (Atlanta Biologicals)

supplemented with 10% FBS (Invitrogen). The DMEM/F12 medium was changed every 57 two days. When the cells grew to 85-90% confluence, cells were washed with 10 ml of

calcium- and magnesium-free Phosphate Buffered Saline (PBS, pH7.3), and then trypsinized with 3 ml of 0.25% trypsin-5.3 mM EDTA (Invitrogen) for 10 minutes at 37

°C. The trypsinization was stopped by addition of 10 ml of DMEM/F12 medium with

10% FBS. After centrifugation, the dissociated cells were re-suspended in high calcium

DMEM/F12 medium with 10% FBS and sub-cultured into 75 cm2 culture flasks at a ratio

of 1 flasks to 5 flasks. All experiments were conducted on primary cultured normal

HNBPADs not generated beyond the fourth passage.

Cell proliferation assay (MTT assay): A total volume of 100 µl medium containing

4,000 HNBPADs isolated from non-leptin, 1.5 and 6 nM leptin cultured tissues were

seeded in each well of 96-well plate in DMEM/F12 medium and incubated in 37°C for 24

h. The following day, medium was replaced by 100 µl DMEM/F12 supplemented with

0.2% BSA and incubated in 37°C for another 24 h. After the treatment of 0.75, 1.5, 3.0,

6.0 nM of leptin and 0, 2.5, 5, 10, 20 nM of Z, non-Z-serum(NZS) and Z-serum(ZS)

before Z implantation (D0) and 30 days post Z implantation (D30) at 0.2, 1, 5% at

cultured medium were given to HNBPADs for 24 h, 0.1% DMSO to control group, the

proliferation of HNBPADs was measured by adding 20 µl fresh mixture of 3-(4,5-

dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium

(MTS) and phenazine methosulfate (PMS) (20: 1) solution (Promega, Madison, WI) to

each well. After incubated in 37°C for 1 to 3 h, O.D. values were measured by kinetic

microplate reader (Molecular Devices Cooperation, Menio Park, CA) at 490 nm

wavelength and cell growth was compared.

58 Cell treatment. Viable HNBPADs (10 5 / well) isolated from non-leptin cultured tissues were seeded in 6-well plates in 5 ml DMEM/F12 supplemented with 10% FBS medium.

Twenty four h later, medium was replaced with DMEM/F12 supplemented with 10%

Dextran Coated Charcoal stripped FBS, and those cells were cultured for 24 h, then the

treatment of serum harvested from heifers was given. The control serum was harvested

from heifers without Z-implantation at day 0 (NZS-D0) and 30 day (NZS-D30) and

experimental serum was harvested from heifers with Z-implantation at day 0 (ZS-D0) and

30 day (ZS-D30). The concentration of all control and experimental serum was 0, 0.2,

1.0 and 5.0% in culture medium. The 24 h treatment is 0, 0.5, 1, 2 nM leptin alone, 2.5, 5,

10 nM Z alone for HNBPADs isolated from non-leptin cultured tissue. The cells were

also treated with 3 nM leptin, 3 nM Z and 3 μM (-)-gossypol, alone or combination for 24 h, and cyclin D1 expression was measured. 0.1% DMSO was treated as control.

Preadipocyte mRNA isolation and cDNA synthesis. After HNBPADs were treated for

24 h, total RNA was isolated in 1 ml TRIZOL Reagent (Invitrogen) according to

manufacturer’s instructions. RNA concentration was measured by DU-70

spectrophotometer (Beckman Instruments Inc.). RNA (1μg) from cultured cells were

reverse transcribed with 200 U M-MLV Reverse Transcriptase (Invitrogen) at 37°C for

50 minutes then 70°C for 15 min in the presence of 1 μl 10 mMdNTP (10 mM each

dATP, dGTP, dCTP, and dTTP at neutral PH) (Invitrogen), 1 μl 50 μM Random hexamer

(Qiagen), RNase Inhibitor (Invitrogen, Carlsbad, CA), 10 μl 5 X First Strand buffer, 5 μl

0.1M DTT and 1 μl RNase Inhibitor (Invitrogen) in a total volume of 50 μl in a gradient

mastercycle (Eppendorf®).

59 Reverse transcription polymerase chain reaction. RT-PCR conditions were optimized

for every primer pairs and performed with a thermocycler Gene Amp PCR (Eppendorf).

The newly synthesized cDNA (2 μl) was used as templates for RT-PCR and 1 U platinum

Taq DNA polymerase (Invitrogen) was added in a total volume of 25 μl. MgCl2 concentration, annealing temperature, and cycle number for the amplification of the PCR product, cyclin D1, were optimized. The primer sequences were 5’-GCT CCT GTG CTG

CGA AGT GG-3’ (sense) and 5’-TGG AGG CGT CGG TGT AGA TG-3’ (antisense, product size 372 bp) PCR condition was denatured at 95°C for 5 min, 27 cycles of 94°C for 45 s, 54°C for 45 s, 72°C for 60 s, then extension 72°C for 10 min. The primer sequences for 36B4, an internal control, was 5’- AAA CTG CTG CCT CAT ATC CG-3’

(sense) and 5’- TTT CAG CAA GTG GGA AGG TG-3’ (antisense, product size 563 bp).

PCR condition 95°C for 5 min, 24 cycles of 95°C for 60 s, 63°C for 60 s, 72°C for 60 s, then extension 72°C for 10 min. The 36 B4 is internal control whose RNA is unmodified by treatment.

The final RT-PCR products (10 µl) mixed with 1 µl 10 X loading buffer were separated on 1.5% agarose gel and visualized by staining with ethidium bromide. Electronic pictures were taken under FUJIFILM LAS-3000 image system (FUJIFILM Medical

Systems USA, Inc.). The densities of specific band were quantified by ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The results were presented as the ratio of cyclin D1 to 36B4.

Western blotting assay Primary cultured human normal PADs were separately seeded in a 100 mm dish (3 X 105 cells/dish) in DMEM/F12 medium supplemented with 10% FBS

and cultured overnight. Then the medium was replaced with DMEM/F12 supplemented

60 with dextran charcoal coated (DCC) -stripped 5% FBS. After cultured for 24 hrs, cells

were treated with 0.1% DMSO as vehicle control, 0.5 and 1 nM leptin, and 2.5, 5, 10 nM

zeranol in DMEM/F12 supplemented with 10% FBS media for 24 hrs. Cells in each dish

were lysed by adding 0.5 ml of M-PERTM Mammalian Protein Extraction Reagent

(Thermo, Rockford, IL) and 5 μl 100X protease inhibitor (Thermo Scientific, Waltham,

MA). Dishes were gently shaked on an orbital shaker at 4 ºC for 5 minutes. Then, the cell

lysate was collected into a microcentrifuge tube and samples were centrifuged at 27,000

X g at 4 ºC for 10 minutes to pellet the cell debris. Finally, the supernatant layer

containing the proteins was transferred to a clean tube for analysis. The concentration of

total protein was determined by using Micro BCATM Protein Assay (Pierce

Biotechnology Inc., Rockford, IL) according to the manufacturer’s instructions. Protein

(50 μg) from each sample was mixed with appropriate volume of 3 X Laemmli sample

buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 25% glycerol, 0.01% Bromophenol blue)

(Bio-Rad Laboratories) and was then denatured at 100 ºC for 5 min. Mixtures were

loaded onto ready gel 10% Tris-HCl Gel for SDS-PAGE (Bio-Rad Laboratories) and

electrophorsed at 100 volts for about 1 hour in 1X Tris/Glycine/SDS buffer (25 mM Tris-

HCl, 250mM glycine, 0.1% SDS, pH 8.3). The separated proteins were than transferred

to a piece of polyvinylidene fluoride (PVDF) membrane (Bio-Rad Laboratories) under

current density at 0.8 mA/cm2 for approximate time according to the manufacturer’s

instruction. After the protein was transferred, the membrane was immediately soaked in

100% methanol for 10 seconds and then placed on a piece of filter paper until dry. Then,

the membrane was blocked in PBST (PBS + 0.1% Tween-20) solution containing 10%

non-fat milk and incubated at 4 ºC for overnight. The membrane was then rinsed with

61 PBST and blotted with primary antibody as followings: cyclin D1 antibody (1:500 dilution, sc-246, mouse monoclonal antibody), p53 antibody (1:500 dilution, raised in mouse Ab-2) ( Calbichem), p21 antibody (1: 2000 dilution, mouse IgG2a, Cell Signaling) or β-actin antibody (1:1000 dilution, sc-1615, affinity-purified goat polyclonal antibody, )

(Santa Cruz Biotechnology, Inc.) and incubated at room temperature for 1 hour. After that, the membrane was rinsed with PBST and incubated with secondary antibody as followings: ECL anti-mouse IgG linked to horseradish peroxidase (HRP) (1:1000 dilution, sc2020) (Santa Cruz Biotechnology, Inc.) or a donkey anti-goat IgG HRP

(1:1000 dilution, NA931V) ( Amersham Biosciences) for 1 h. The membrane was rinsed again with PBST, incubated for 5 minutes with ECL-Plus Western blotting detection reagents (Amersham pharmacia biotech, Piscataway, NJ) and then photos were taken using FUJIFILM LAS-3000 image system (FUJIFILM Medical Systems USA, Inc.). The densities of specific band were quantified by ImageQuant software (Molecular

Dynamics).

Leptin measurement. Primary culture human normal HNBPADs (3 X 105 cells/dish)in

DMEM/F12 medium supplemented with 10% FBS were seeded in 100mm dish and

cultured overnight. Then the medium was replaced with DMEM/F12 supplemented with

dextran charcoal coated (DCC) -stripped 5% FBS. After cultured for 24 hrs, cells were treated with 0.1% DMSO as vehicle control and 2.5 nM zeranol in DMEM/F12 supplemented with 10% FBS media for 24 hrs. Culture media were collected and leptin measurement was conducted by human leptin immunoassay kits (Minneapolis). The particulates in cell culture supernates were removed by centrifugation in 1,200 rpm for 5 min and assay immediately according to the manufacturer’s instructions. Assay was

62 stopped by adding 50 μl stop solution to each well and OD values were measured within

30 min by kinetic microplate reader (Molecular Devices Cooperation) at 450 nm

wavelength and leptin concentration was compared.

Statistical Analysis

The results for cell proliferation assay are presented as the relative cell growth comparing to control group ± Standard deviation (SD) for 4 replicate culture wells. The results for mRNA expression are presented as the ratio of cyclin D1 to 36B4 comparing to control group ± Standard deviation (SD) for 3 replicate culture wells. Analysis was

performed by using Minitab 15 software (Minitab Inc.). Statistical difference was

determined by using two sample t-test analysis for cell proliferation and One-way

ANOVA analysis for mRNA expression. p-values of less than 0.05 were considered

statistically significant difference

Results

Leptin can stimulate primary cultured HNBPADs growth in a dose-dependent manner

As figure 3.1 showed, primary cultured HNBPADs are sensitive to 0.4 nM of leptin in a dose-dependent manner. Compared to HBNECs, the average effective dose of leptin in

HNBPADs was significantly lower than that in HBNECs (Figure 3.2)

Z can stimulate primary cultured HNBPADs growth

63 Figure 3.3 showed that Z can stimulate the proliferation of the primary cultured

HNBPADs isolated from non-leptin or 6 nM leptin cultured human normal breast tissues when compared to the control group. Compared to the control group, Z at 2.5 nM and above can significantly increase the cell isolated from non-leptin cultured tissue growth, but cells isolated from 6 nM leptin cultured tissue were responsive to 1.25 nM and above concentration of Z.

ZS stimulates primary cultured HNBPADs growth

Figure 3.4 shows comparing to the control group, all the treatment can increase

HNBPADs growth, but there was not significant difference between groups treated by

NZS-D0 and NZS-D30 at all doses in the proliferation of HNBPADs isolated from non- leptin cultured tissue. Figure 3.5 shows compared to the control group, all the dose of treatment of ZS-D0 and ZS-D30 can significantly increase the proliferation of preadipocytes isolated from 6 nM leptin cultured tissue. The significant difference in the cell growth between groups treated with ZS-D0 and ZS-D30 was found in the concentration of 1% and 5% in cultured medium in the preadipocytes isolated from 6 nM leptin cultured tissue.

Both Z and the combination of Z with leptin can up-regulate HNBPADs cyclin D1 mRNA expression but (-)-gossypol can counteract this effect. Figure 3.6 shows the cyclin D1 expression was up-regulated by leptin treatment in HNBPADs. Leptin at 1 and

2 nM can significantly increase the HNBPADs express cyclin D1. Moreover, 3 nM Z and the combination of 3 nM Z and 1.5 nM leptin can also significantly increase the

HNBPADs expression cyclin D1. Significant difference was found between the combination groups treated with or without 3 μM(-)-gossypol (Figure 3.7).

64 Both Z and leptin can up-regulate HNBPADs cyclin D1 protein expression. As

shown in Figure 3.8 and Figure 3.9, 2.5, 5, 10 nM zeranol and 0.5, 1 nM leptin can

increase Cyclin D1 protein expression in PADs isolated from non-leptin cultured human normal breast tissue.

Z increased HBNECs secrete leptin. As shown in Figure 3.10, 2.5 nM Z can

significantly increase the HNBECs secrete leptin by about 40%, compared to the control

group at * p<0.05.

Discussions:

Cyclin D1 is a cell cycle regulator and plays an important role in cell growth. The cyclin-

dependent kinases (CDKs) can not regulate cell growth without the cyclin subunit. By

binding to cyclin D, cyclin D-CDK 4/6 constitutes the engine of the cell cycle machinery

and affects the G1 phase in cell growth. Among several cyclins, cyclin D1 protein plays

an important role in regulating the progress of the cell during the G1 phase of the cell

cycle [46]. Cyclin D1-cdk4 complexes are known to phosphorylate Rb and Rb-like proteins, which release a transcription factor (E2F) with resultant effects on gene transcription. Researchers found that cyclin D1 expression is strongly associated with

estrogen receptor positivity. Cyclin D1 was found to be overxpressed in up to 50% of

human breast cancer [46]. The overexpression of cyclin D1 was found to be statistically

correlated with ER positivity and well differentiated tumors, and it was seen in 79% of

ER positive tumors and 45% of ER negative tumor [46]. Cyclin D1 expression was

detected at levels significantly greater than in normal breast epithelium in the earliest 65 proliferative epithelial lesions of the breast with a further significant increase accompanying the progression to any form of cancer [50, 51]. Cyclin D1 protein is overexpressed in hyperplasia and intraductal carcinoma of the breast [50]. Because of its established role as a major human oncogene, the therapeutic exploition of cyclin D1 holds tremendous promise and should be vigorously pursued.

The cyclin D1 level can be modulated by changing growth factors in the culture medium. Leptin stimulates breast cancer cell growth by up-regulating cyclin D1 expression [30]. Moreover, Garofalo et al. found that leptin could modulate both estrogen synthesis and ER α activity [3, 15]. Besides controlling the cell cycle, cyclin D1 was found to be associated with ER [46]. Cyclin D1 can bind to the ER and stimulate the

ER’s transcriptional activities. The cyclin D1 and ER complex may play a role in stimulating the tumor cell proliferation. Because leptin stimulates estrogen biosynthesis through induction of aromatase activity and modulation of ER α activity, it has been characterized as a growth factor for breast cancer [15]. High levels of leptin in obese breast cancer patients may play a remarkable role in breast cancer cell proliferation, invasion, and metastasis [15, 52]. Leptin stimulates breast cancer cell growth by up- regulating cyclin D1 expression. It was also reported that leptin can directly inhibit the maturation of 3T3-L1 preadipocyte but can not directly induce adipocyte apoptosis.

In our cell proliferation assay, as Figure 3.4 and Figure 3.5 showed, ZS also increases

HNBPADs isolated from both non-leptin and 6 nM leptin cultured tissue. Comparing

Figure 3.4 and Figure 3.5, it is obvious that the preadipocytes isolated from non-leptin cultured tissue were more sensitive to ZS-D30 than to NZS-D0 and NZS-D30 and, there is significant difference between ZS-D0 and ZS-D30 in the HNBPADs isolated from

66 6nM leptin cultured tissue. This result implied that leptin also increase the HNBPADs

sensitivity to ZS-D30 because HNBPADs isolated from leptin cultured tissue grew faster than those isolated from non-leptin cultured tissue. ZS increased HNBPADs isolated from both leptin cultured tissue and non-leptin cultured tissue more than NZS did. It is suggested that leptin can stimulate primary cultured HNBPADs growth and ZS can

improve the leptin induced growth.

According to Figure 3.3, we reasoned that HNBPADs isolated from 6 nM leptin cultured

tissue increased their sensitivity to Z than those isolated from non-leptin cultured tissue.

This can partly be explained by the fact that the combination resulted in high expression

of cyclin D1. It is possible that if obese healthy women or breast cancer patients have

higher leptin in their serum, the sensitivity of HNBPADs may be increased to Z.

Moreover, we found that primary cultured human normal breast epithelial cells and

MCF-7Adr cells can induce the cell sensitivity to Z [53]. Under such circumstances, the

risk of breast cancer may be increased because the breast cancer patients expressed more

leptin than control group [12].

According to other researchers, the serum level of leptin in breast cancer patients is

higher than that in controls (32). Our data showed (Figure 3.7) that 6 nM leptin alone had

no affect on the cyclin D1 expression but their combination significantly increased the

expression of cyclin D1 comparing to the control group. This result supports our hypothesis that 6 nM leptin can increase the preadipoicyte sensitivity to Z containing serum and thus enhance the proliferation of preadipocytes. This suggests a possible relationship between obesity and breast cancer and points to the potential risk for breast cancer in obese people because leptin can increase the cell sensitivity to Z.

67 On the other hand, like estrogen modulating the leptin expression in some estrogen-responsive tissues [31], we found 20 nM Z can increase HNBPADs secrete and thus the action of leptin was amplified. This result indicates that Z might be more harmful to obese people than normal weight people in increasing breast cancer risk [38] because their consumption of Z-containing products may amplify their chances of having breast cancer. However, (–)-gossypol can reverse the effect of the combination of leptin with Z on cell growth; it could be used in the treatment of breast cancer, especially in obese multidrug resistant patients.

In summary, leptin appears to increase HNBPADs growth via increasing cyclin

D1 expression in both mRNA and protein levels. Leptin improves the HNBPADs sensitivity to Z and Z can strengthen the leptin by inducing HNBPADs secret leptin. (–)-

Gossypol can counteract the growth of breast preadipocyte induced by leptin alone or combined with Z by down-regulating cyclin D1 mRNA expression. More mechanisms will be further studied in the future. Our report is the first to reveal that (–)-gossypol as a food component in cottonseed products may serve as a potential chemopreventive agent to suppress the stimulatory effect of Z and leptin on human breast preadipocytes. This result proved that our hypothesis that obese women may have higher risk of breast cancer when they are consuming beef products containing Z might be right. Moreover, our previous investigation showed that leptin can increase cell sensitivity to Z both in primary cultured human breast cancer epithelial cells and MCF-7 Adr cells [47]

0.5 * * * 0.4

0.3

0.2

growth PAD 0.1

CT 0.4 0.8 1.6 Leptin (nM)

Figure 3.1 The Effect of leptin in the pre-adipocyte growth.

Primary cultured human normal breast pre-adipocytes were isolated from non-leptin cultured tissues. The treatment of 0.4, 0.8, 1.6 nM leptin was given for 24 hrs, cell growth was measured. The symbols “*” indicate significant differences in MCF-7 Adr cell growth compared to the control group (p<0.05).

6 N= 7

3 p=0.019 2 Sensitive Sensitive ofdose lp N=2 1

0 PAD HBNECs

Figure 3.2 The comparison of the average effective dose of leptin in the PAD and

HBNECs. PADs isolated from two patients and HBNECs from 7 patients were tested their minimum sensitive dose to leptin and the average sensitive dose to leptin was calculated and compared. The PADs were more sensitive to leptin than HBNECs at the level of P value<0.05 (P=0.019).

1.0 ** CT 6 nM leptin ** ** ** ** 0.8 *

th 0.6 ** grow

e 0.4

0.2

0.0 0 1.25 2.5 5 10

zeranol (nM) Figure 3.3 Exposure of lp changed the PADs sensitivity to zeranol. Primary cultured human normal breast pre-adipocytes were isolated from non-leptin and 6 nM leptin cultured tissues. The treatment of 1.25, 2.5, 5, 10 nM zeranol was given for 24 hrs, cell

growth was measured. The symbols “*” indicate significant differences in PADs growth

compared to the control group (p<0.05). 1.25 nM zeranol already significantly increase the proliferation of PAD exposure to 6 nM leptin but 2.5 nM zeranol began significantly increase the proliferation of PAD isolated from non-leptin cultured tissues.

0.5

ZS-D0 ZS-D30 * * * 0.4 * 0.3 * *

0.2 GrowthCell

0.1

0 0.0 0.2 1.0 5.0 Z-im planted Serum (%)

Figure 3.4 The effect of ZS-D0 and ZS-D30 in the proliferation of pre-adipocyte isolated from non-leptin cultured tissues.

Primary cultured human normal breast pre-adipocyte isolated from non-leptin cultured tissues were treated with ZS-D0 and ZS-D30 at 0.2%, 1% and 5% at cultured medium for

24 hrs, cell growth was measured. All the treatment can significantly increase cell growth,

The symbols “*” indicate significant differences in PAD growth compared to the control group(p<0.05). However, there was no significant difference between groups treated with

ZS-D0 and ZS-D30 at all doses.

* * 0.9 ZS-D0 ZS-D30 ** **

* 0.6 * * *

0.3 Cell Growth

0 CT 0.2 1 5

Z-implanted Serum (%)

Figure 3.5 Comparison of effect of ZS-D0 and ZS-D30 in the proliferation of pre- adipocyte isolated from 6 nM leptin cultured tissues.

Primary cultured human normal breast pre-adipocyte isolated from 6 nM leptin cultured tissues were treated with ZS-D0 and ZS-D30 at 0.2%, 1% and 5% at cultured medium for

24 hrs, cell growth was measured. The symbols “*” indicate significant differences in

PAD growth compared to the control group (p<0.05). There were significant difference between cells treated with ZS-D0 and ZS-D30 at 1% and 5% groups. Marked as *.

CyclinD1

36 B4

1 * 0.8 * 0.6

0.4

(cyclin D1/36B4) 0.2

Relative mRNA Expression 0 CT 0.5 1 2 leptin (nM)

Figure 3.6 A Leptin increases cyclin D1 mRNA expression in PADs

PAD treated with 0.5, 1, 2 nM leptin for 24 hrs, cyclinD1 mRNA expression was tested and significant difference was found. Leptin at 1 and 2 nM can significantly increase the

PADs express cyclin D1 , compared to the control group at * p<0.05.

CyclinD1

36 B4

1.6 * ** 1.2 *

0.8

D1/36B4) (cyclin 0.4

Relative mRNA Expression 0.0 CT Z Z+lp Z+lp+(-)G

Figure 3.7 Cyclin D1 mRNA expression in HNPADs.

HNPADs were treated with 1.5 nM lp, 3 nM Z, and 3 μΜ (-)-G for 24 hrs and mRNA

was extracted. Cyclin D1 was amplified. Compared to the control group, 3nM zeranol

and the combination of 3 nM zeranol with 1.5 nM leptin can significantly increase cyclin

D1 expression, 3 μM (-)-gossypol can counteract the effect. Significant between the

combination groups with or without (-)-gossypol (*, p<0.05).

Cyclin D1

β actin

0.4 n

0.3

actin) β 0.2

0.1 (Cyclin D1/ (Cyclin

Relative Protein Expressio Protein Relative 0 CT 2.5 5 10 Zeranol (nM)

Figure 3.8 Zeranol increases Cyclin D1 expression in PADs.

Primary cultured human normal PADs isolated from non-leptin cultured tissue were treated with 2.5, 5, 10 nM zeranol for 24 hrs, protein concentration was measured and

Western Blotting was conducted. Cyclin D1 protein expression was compared.

Cyclin D1

β actin

0.6

0.5

actin) 0.4 β 0.3

0.2

(Cyclin D1/ D1/ (Cyclin 0.1

0 Relative Expression Protein CT 0.5 1 Leptin (nM)

Figure 3.9 Leptin up-regulate Cyclin D1 protein expression in PADs.

Primary cultured human normal PADs isolated from non-leptin cultured tissue were treated with 0, 0.5, 1 nM leptin for 24 hrs, protein concentration was measured and

Western Blotting was conducted. cyclin D1 protein expression was compared.

0.25

* 0.20

0.15

0.10 leptin (nM) leptin

0.05

0.00 CT 2.5nM Z

Figure 3.10 Zeranol stimulates human normal breast PADs secret leptin.

Primary cultured human normal breast PADs were treated with 2.5 nM zeranol for 24 hrs

and culture medium were collected. The human leptin immunoassay was conducted.

Zeranol at 2.5 nM can significantly increase the HNBECs secret leptin, compared to the control group at * p<0.05.

Chapter 4 Conclusion Remarks

Breast cancer cell lines play a pivotal role in biomedical research. Because cells from

patients have extensive chromosomal rearrangement, oncogenic mutations, multiple sites

of allele loss and gene amplification, there is a widespread belief that the cell lines are not

representative of the tumors from which they are derived [45]. It is very necessary to

investigate whether leptin and Z have effects on cyclin D1 and ObR mRNA expression in breast cancer epithelial cells which were isolated from the cancerous tissues from breast cancer patients. Our current data showed that the combination of leptin and Z can increase the cyclin D1 and ObR mRNA expression in both MCF-7 Adr cell and primary cultured human cancer epithelial cell.

All data observed both Z and lp can increase primary cultured HBNECs, HBCECs,

HNBPADs and MCF-7 Adr cell growth and Primary cultured human normal breast

HNBPADs are more sensitive to lp than HBNECs, HBCECs, MCF-7 Adr cells. It might also implicate that the obese individuals with higher leptin expression than that of normal weight ones may be at greater risk of breast cancer. Z enhances leptin induced proliferation in primary cultured human breast cancer epithelial cells. Leptin and Z can enhance HBCECs growth via increasing cyclin D1 mRNA expression and down-regulate

P53 or PTPγ mRNA expression. Our results also demonstrated exposure to leptin can increase the HBCECs, HBNECs and HNBPADs sensitivity to Z or ZS which greatly enhanced the mitogenic action of leptin in these cells. The experimental data observed

79 suggests that the prognosis of obese cancer patients with higher leptin expression would be worse if they consumed beef products from growth promoter, Z-implanted beef cattle.

This result is the first to reveal leptin can improve HBCECs’ sensitivity to Z.

Furthermore, Z can strengthen the potency of leptin by increasing ObR expression in

HBCECs and induce HBNECs and HNBPADs secrete leptin. Z maybe more harmful to obese patients and might play a role in breast cancer development.

Meanwhile, (-)-gossypol revert the effect of the combination of leptin and Z in MCF-7

Adr cells, or primary cultured HNBPADs, HBCECs and HBNECs. It can serve as a potential chemopreventive agent and play an important role in breast cancer chemoprevention.

Our results suggest that the leptin may be involved in enhancing the breast cancer epithelial cells and breast cancer cell line MCF-7 Adr cells sensitivity to Z and the primary cultured HBNECs sensitivity to ZS. Leptin and Z and their combination can increase normal or cancer epithelial cells up-regulate cyclin D1 while (-)-gossypol can counteract this effect.

In summary, all the results imply that leptin, Z and ZS stimulate primary cultured

HBNECs and HNBPADs growth. Leptin may be involved in enhancing the HBCECs and HNBPADs sensitivity to Z and ZS by up-regulate cyclin D1 expression in

HNBPADs. Z can strengthen this effect while (-)-gossypol can counteract the effect.

Further investigation of the interactions involving leptin and Z in human breast normal cancer epithelial cells and pre-adipocytes is in progress.

It has been reported that breast cancer cells express high levels of aromatase, which can convert androgen into estrogen, and results in high concentrations of estrone (E1) and

80 estradiol (E2) in breast tissue. This might be the major reason of high risk of breast cancer in postmenopausal women and obese women. On the other hand, leptin was reported to increase both normal and cancer cells by activating signal transducer and activator of transcription 3 (STAT 3) and extracellular regulated kinase (ERK)1/2 pathways [23, 26, 27, 52], it can be tested if there is interaction between leptin and Z in the aromatase and STAT 3 activity in PAD or mature adipocytes in the future. The future attention also could be focus on the interaction of leptin, Z and gossypol in the primary cultured human normal pre-adipocyte differeciation and mature adipocytes.

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