Potential Mechanisms of Action of Kaempferol in the Prevention

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POTENTIAL MECHANISMS OF ACTION OF KAEMPFEROL IN THE PREVENTION

OF BREAST CANCER

A Dissertation

Presented to the Faculty of the Graduate School

of Cornell University

in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

Jinzhou Li

August 2015

POTENTIAL MECHANISMS OF ACTION OF KAEMPFEROL IN THE PREVENTION

OF BREAST CANCER

Jinzhou Li, Ph.D.

Cornell University 2015

Epidemiological studies have shown that regular consumption of fruits and vegetables could reduce the risk of cancer. Phytochemicals in fruits and vegetables have been suggested to be responsible for their health benefits. Asparagus and one of its major phytochemical, Kaempferol, have been reported to have anti-cancer activities. However, the mechanisms of the anticancer activities are not completely understood.

Seven varieties of asparagus were compared for their antioxidant content, antioxidant activities, and anticancer activities. The results showed that asparagus are rich in phenolic and flavonoids. They also show antioxidant activities in chemical assays and in vitro assays. Asparagus possesses potent anticancer abilities in inhibiting proliferation of HepG2 human liver cancer cells. Among the different varieties, Apollo has the highest phenolic content, flavonoid content, antioxidant activities, and anticancer activities. We further studied kaempferol’s effects in breast cancer cells growth and metastasis.

The anti-proliferative activity and cytotoxicity of kaempferol against MCF-7 human breast cancer cells were measured by the methylene blue assay. All the key

proteins regulating cell proliferation through signaling transduction pathways were determined by Western blot assay. Kaempferol exhibited potent anti-proliferative activity against MCF-7 human breast cancer cells in a dose-dependent manner. No cytotoxicity was observed at the concentrations tested. Kaempferol significantly down-regulated expression of PCNA, CDK-4, Cyclin D1, and estrogen-receptor alpha, and up-regulated expression of p21, and p-p53 in MCF-7 human breast cancer cells in dose-dependent manners when compared to the control. Kaempferol specifically inhibited estradiol-induced cell proliferation in MCF-7 human breast cancer cells.

Kaempferol also exhibited anti-proliferative activity against MDA-MB-231 human breast cancer cells in a dose-dependent manner. No cytotoxicity was observed at concentrations below 100 µM in MDA-MB-231 cells. Kaempferol significantly inhibited MDA-MB-231 human breast cancer cell migration observed by scratch assay. The activity of MMP-2, a critical enzyme for cancer cell metastasis, was assessed by zymography assay, and it was dramatically inhibited in a dose-dependent manner. Cell transformations studied by soft agar assay were also inhibited by kaempferol in a dose-dependent manner.

In conclusion, we demonstrated that asparagus and kaempferol exhibited anticancer activities through effects on antioxidant activity, cancer cell proliferation, migration and metastasis. These data are important in understanding the protective effects of fruits and vegetables in the prevention of breast cancer.

BIOGRAPHIC SKETCH

Jinzhou Li was born in Yongnian, Hebei, China. Currently he is a Ph.D. candidate in Dr. Rui Hai Liu’s lab studying health benefits of phytochemicals in fruits and vegetables. He received his B.E. degree in Food Science and Engineering at

Tianjin University in China in 2005. He received his M.S. degree in Human Nutrition at

Columbia University in 2010.

Prior to join the PhD program, Jinzhou has interned in Nanjing University

Organic Food Research and Development Institute with Professor Wenlong He to

study organic agriculture practice and policy. During his stay at Cornell, Jinzhou

joined the IFT product development team and won the second place in final 2011.

Jinzhou had co-captained several trips for Cornell students doing project in China

organized by smart program. He also worked as a vice president in Cornell China

Association for the Promotion of Agriculture. He has helped organize the

Cornell-China forum in 2012 and 2014. He also helped his family business set up a

branch in the US, and successfully marketed the products to US market.

Jinzhou’s interest in Food Science comes from his experience in his family

food business, which is doing organic vegetables and fruits growing, processing, and

exporting. Involved in the family business these years, Jinzhou feels amazed that

running the business can make profit and take the social responsibility at the same

time. He also realized that only with a wide knowledge about food science and technology, the company can sustainably develop for a long time. Now as Jinzhou is getting his PhD degree, he is excited to make full use of his knowledge gained at

Cornell to develop the business and help more people.

ACKNOWLEDGEMENT

First I would like to thank the unconditional love from my family, without their support I cannot reach this far. I want to thank my mother, my father, my wife, and my babies. They give me courage and motivation. Because of them, I become fearless when I face challenge. I know no matter what happen to me, they will be there with me. Their love brings happiness to my life and work.

I want to thank my lab mates. They have helped me a lot for my research and

PhD life. I want to thank Xue, Ran, Xinbo, Dan, Gu, Hao, Ningbo, Caroline, Hongyu,

Xi and all other people in my lab. They give me helpful advices for my project. We

discuss to solve the problem together. They are all talent people and inspired me. I

really enjoyed the five years when I stayed with them.

I want to thank my friends, who always get my back. My friends Sen, Jie,

Boteng and others are always there when I want to share my happiness and sadness

with. And when I need some help, they just do it without any hesitate.

I want to thank my aunt; she helps us taking care of the babies, so life will not

be too stressful.

I want to thank the people who have helped my experiment. Sungsoo Yoo

and Zeping Zhao showed me some experiment techniques step by step with patience.

I would like to thank Professor Jane Pleasant, Jennifer nelson, and Brian Henehan for

help me edit my dissertation. There are also many other people gave me unreserved

advices for my project even they do not know me.

I want to thank my minor advisor Dr. Miller and Dr. Gomez. They not only

gave me very helpful advices on my minor field, but also very patiently discuss with

me about my career path.

In the end, I want give my appreciation to my advisor Dr. Rui Hai Liu and his

wife Dr. Li. Dr, Liu has given me huge understanding and flexibility. Without his

support I cannot harvest so many things in my study, family, and business. I have learned a lot from him. Dr. Li is like my family member. My heart always feels warmth when I stay in Ithaca.

Thank all the people in my life.

TABLE OF CONTENTS

CHAPTER ONE INTRODUCTION ...... 1

1.1 Fruits and vegetables in cancer prevention ...... 1

1.1.1 Cancer background and statistics ...... 1

1.1.2 Consumption of fruits and vegetables for cancer prevention: epidemiology evidence...... 2

1.2 Phytochemicals for cancer prevention ...... 3

1.2.1 Phytochemicals ...... 3

1.2.2 Intake in the US, bioavailability and absorption ...... 4

1.2.3 Different phytochemicals for cancer prevention ...... 5

1.2.4 Phytochemical synergy and whole foods benefits for cancer prevention ...... 16

1.3 Asparagus and its health benefits ...... 19

1.3.1 General introduction ...... 19

1.3.2 Asparagus nutrition composition and phytochemicals ...... 20

1.3.3 Bioactive compounds of asparagus ...... 22

1.4 Kaempferol ...... 24

1.4.1 Chemistry of kaempferol ...... 24

1.4.1.1 Structure and derivatives ...... 24

1.4.2 Anticancer activities of Kaempferol...... 28

1.5 Potential mechanisms of action ...... 30

1.5.1 Antioxidant effects and Redox regulation ...... 30

1.5.2 Antiproliferation and cell cycle arrest ...... 32

1.5.3 Induce Apoptosis and Autophagy ...... 34

1.5.4 Angiogenesis and metastasis inhibition ...... 39

1.5.5 Estrogen modulating activity ...... 41

1.5.6 Anti-inflammation ...... 43

III

1.5.7 Drug effect improvement ...... 46

1.5.8 Other anti-carcinogenesis mechanism ...... 49

1.6 Overall objectives, significance, and future implications ...... 50

1.6.1 Rationale ...... 50

1.6.2 Hypotheses...... 51

1.6.3 Objectives ...... 52

1.6.4 Implications and future research ...... 52

CHAPTER TWO PHYTOCHEMICAL PROFILES, ANTIOXIDANT ACTIVITY AND ANTI-PROLIFERATIVE ACTIVITY OF SEVEN VARIETIES OF ASPARAGUS...... 54

2.1 Introduction ...... 54

2.2 Materials and Methods ...... 55

2.2.1 Chemicals and materials ...... 55

2.2.2 Asparagus samples and sample Preparation ...... 56

2.2.3 Phytochemical extractions of asparagus ...... 57

2.2.4 Determination of total phenolic contents ...... 57

2.2.5 Determination of total flavonoids ...... 58

2.2.6 Determination of Total Antioxidant Activity ...... 59

2.2.7 Cell Culture ...... 60

2.2.8 Cytotoxicity and Inhibition of Proliferation Assays ...... 61

2.2.9 Cellular Antioxidant Activity (CAA) ...... 62

2.2.10 Statistical Analysis ...... 63

2.3 Results ...... 64

2.3.1 Total Phenolic Content ...... 64

2.3.2 Total Flavonoid Content ...... 64

2.3.3 Total Antioxidant Activity ...... 65

2.3.4 Antiproliferative activity ...... 65

2.3.5 Cellular Antioxidant Activity ...... 65

2.4 Discussion ...... 74

2.4.1 Phenolic content ...... 75

2.4.2 Flavonoids content ...... 75

2.4.3 Total antioxidant activity ...... 76

2.4.4 Antiproliferative activity and cytotoxicity...... 77

2.4.5 Cellular antioxidant activity ...... 78

2.4.6 Summary ...... 79

CHAPTER THREE FLAVONOL KAEMPFEROL INHIBITED PROLIFERATION AND MIGRATION OF MDA-MB-231 HUMAN BREAST CANCER CELLS THROUGH DOWN-REGULATION OF MMP-2 ENZYME ACTIVITY ...... 81

3.1 Introduction ...... 81

3.2 Materials and Methods ...... 82

3.2.1 Chemicals ...... 83

3.2.2 Cell culture ...... 83

3.2.3 Cytotoxicity evaluation ...... 83

3.2.4 Antiproliferative activity evaluation ...... 84

3.2.5 Cell migration examination (Scratch Assay) ...... 85

3.2.6 Matrix metalloproteinase-2 (MMP-2) activity analysis ...... 85

3.2.7 Cell transformation investigation ...... 86

3.2.8 Statistical analysis ...... 88

3.3 Results ...... 88

3.3.1 Antiproliferative activities and cytotoxicity of Kaempferol towards MDA-MB-231 human breast cancer cells ...... 88

3.3.2 Effects of Kaempferol on migration activities of MDA-MB-231 human breast cancer cells . 89

3.3.3 Inhibition of MMP activities of MDA-MB-231 human breast cancer cells by kaempferol. ... 89

3.3.4 Effect of kaempferol on MDA-MB-231 human breast cancer cells transformation ...... 90

3.4 Discussion ...... 100

3.4.1 Antiproliferative activity of kaempferol towards MDA-MB-231 human breast cancer cells ...... 100

3.4.2 Anti-migration effect of kaempferol in MDA-MB-231 human breast cancer cells ...... 101

3.4.3 MMP inhibition effect of kaempferol in MDA-MB-231 human breast cancer cells ...... 102

3.4.4 Kaempferol’s effect on transformation of MDA-MB-231 human breast cancer cells ...... 103

3.4.5 Summary ...... 104

CHAPTER FOUR KAEMPFEROL INHIBITS PROLIFERATION OF MCF-7 HUMAN BREAST CANCER CELLS THROUGH SUPPRESSING EXPRESSION OF ESTROGEN RECEPTOR ALPHA AND REGULATING P38/MAP KINASE SIGNAL TRANSDUCTION PATHWAY ...... 105

4.1 Introduction ...... 105

4.2 Materials and Methods ...... 107

4.2.1 Chemicals...... 107

4.2.2 Cell culture...... 107

4.2.3 Cytotoxicity evaluation ...... 108

4.2.4 Antiproliferative activity evaluation ...... 109

4.2.5 Preparation of protein samples...... 109

4.2.6 Western Blot assay...... 110

4.2.7 Statistical analysis ...... 111

4.3 Results: ...... 112

4.3.1 Antiproliferative activities and cytotoxicity of Kaempferol towards MCF-7 human breast cancer cells ...... 112

4.3.2 Effects of kaempferol on expression of proteins involved in the proliferation and cell cycle in MCF-7 human breast cancer cells...... 112

4.3.3 Regulation of proteins involved in the P38/MAPK pathway ...... 113

4.3.4 Effects of Kaempferol on expression of estrogen receptor alpha...... 113

4.3.5 Effects of kaempferol on estradiol induced cell proliferation in MCF-7 human breast cancer cells ...... 114

4.4 Discussion ...... 121

4.4.1 Antiproliferative activity of kaempferol in MCF-7 human breast cancer cells ...... 121

4.4.2 Kaempferol’s effects on cell signaling pathway in MCF-7 human breast cancer cells ...... 122

4.4.3 Summary ...... 124

REFERENCES: ...... 126

VII

TABLE OF FIGURES

Figure 1 Classification of Dietary Phytochemicals ...... 4

Figure 2 Chemical structures of kaempferol and related compounds ...... 25

Figure 3 Total Phenolics of Various asparagus ...... 67

Figure 4 Total Flavonoid content of various asparagus ...... 68

Figure 5 Total antioxidant activity of various asparagus ...... 68

Figure 6 Inhibition of HepG2 human liver cancer cell proliferation and cytotoxicity of various asparagus ...... 71

Figure 7 CAA values of seven varieties Asparagus ...... 73

Figure 8 Effect of kaempferol on cell proliferation and cytotoxicity in MDA-MB-231 human breast cancer cells ...... 92

Figure 9 Evaluation of kaempferol on HGF-stimulated cell motility ...... 94

Figure 10 Evaluation of kaempferol on MMP-2 enzyme activity ...... 95

Figure 11 Different concentrations of kaempferol inhibit the MDA-MB-231 human breast cancer cells colony formation in soft agar ...... 98

Figure 12 Effect of kaempferol on cell proliferation and cytotoxicity in MCF-7 human breast cancer cells ...... 115

Figure 13 Effects of kaempferol on PCNA, Cyclin D1, and CDK-4 expression in MCF-7 human breast cancer cells ...... 116

Figure 14 Effects of kaempferol on p21, p-p53, and p-p38 expression in MCF-7 human breast cancer cells ...... 117

Figure 15 Effects of kaempferol and p-p38 inhibitor SB 203580 on Estrogen Receptor Alpha expression in MCF-7 human breast cancer cells ...... 118

Figure 16 Effects of kaempferol on estradiol induced proliferation of MCF-7 human breast cancer cells ...... 119

Figure 17 Signaling Transduction Pathway Summary ...... 120

VIII

Chapter one Introduction

1.1 Fruits and vegetables in cancer prevention

1.1.1 Cancer background and statistics

Cancer is a prevailing disease worldwide; about one fourth of all deaths are

due to cancer. According to American Cancer Society’s statistical data, a total of

1,665,540 new cancer cases and 585,720 cancer deaths are estimated to occur in the United States in 2014(1). Globally, around 12.7 million cancer cases and 7.6 million cancer deaths are estimated in 2008(2). Many researchers have worked on

cancer for many decades. The results are promising; modern cancer treatments show

great progress. From 2006 to 2010, cancer death rates in the US declined by 1.8%

per year in men and by 1.4% per year in women. In last two decades, the total cancer

death rate decreased from 215.1 per 10,000 people in 1991 to 171.8 per 10,000

people in 2010. During this period, approximately 1,340,400 cancer deaths have

been avoided due to this 20% decline in the US(1). However, challenges still exist.

First, current cancer treatments damage healthy cells, when they destroy cancer cells;

this will compromise the normal human body system； second, because the cancer

cells easily develop drug resistance, some effective cancer treatments may become

useless over time. Third, the cancer reoccurrence is common; helping patients to

avoid reoccurrence is important (3). Considering all the challenges, an alternative

treatment is urgently needed. This treatment should present the following

characteristics: i) nontoxic, ii) efficient to treat cancer by itself or assist the current

treatment by improving the effects or lowering the side effects, and iii) can be used by

people over long term to avoid cancer reoccurrence.

1.1.2 Consumption of fruits and vegetables for cancer prevention: epidemiology evidence

Vegetables, fruits and whole grains (VFG) have all the above hallmarks.

Researchers have found that VFG consumption is inversely associated with certain cancer risks. Studies of commonly consumed vegetables and fruits showed that their extracts can inhibit HepG2 human liver-cancer cells proliferation (4, 5). A multivariable Cox proportional hazard models study in Europe found that by increasing the fruits and vegetables consumption by 100 g per day, lung cancer risk was significantly reduced(6). A case control study in Uruguay showed that higher

consumption of both fruits and vegetables can decrease the risk of esophagus cancer,

lung cancer, breast cancer and prostate cancer, and at all sites combined(7). In a

cohort study which followed 71058 women for 16 years in the United States, risk for

estrogen receptor (ER) negative breast cancer was significantly decreased in

association with increased consumptions of fruits (RR = 0.88; 95% CI = 0.80–0.97)

and vegetables (RR = 0.94; 95% CI = 0.88–0.99) (8). Another cohort study in Italy studied 8984 women for 9.5 years; the results showed that risk for breast cancer was

significantly decreased in association with increased consumptions of salads and

vegetables (RR = 0.66; 95% CI=0.47–0.94) (9). A case control study in Cyprus also

found that risk for breast cancer was significantly decreased in association with

increased intake of vegetables and salads (OR = 0.74, 95% CI: 0.57-0.96) (10).

Whole grain is also significantly associated with lower invasive colorectal cancer risks in a prospective National Institutes of Health-AARP diet and Health study (11). The phytochemicals in VFG are believed to play a very important role in these beneficial activities.

1.2 Phytochemicals for cancer prevention

1.2.1 Phytochemicals

Phytochemicals are defined as bioactive nonnutrient plant compounds in fruits, vegetables, grains, and other plant foods that have been linked to reducing the risk of major chronic diseases (12). Phytochemicals can be generally categorized as carotenoids, phenolic, alkaloids, nitrogen-containing compounds, and organosulfur compounds. And there are many other phytochemicals under each category (Figure 1)

(12). Since phytochemicals are widely distributed in plants, each day people generally intake fair amounts of the phytochemicals from vegetables and fruits.

FIGURE 1 CLASSIFICATION OF DIETARY PHYTOCHEMICALS

Figure 1. Classification of Dietary Phytochemicals (Adapted from Liu 2004)

1.2.2 Intake in the US, bioavailability and absorption

Some of the results for digestion, absorption, and kinetics of phytochemical bioavailability are briefly introduced here. During digestion, flavonols and proanthocyanidins do not change much. Quercetin is easily degraded; it is not stable at low pH during gastric and pancreatic digestions, while it is stable at pH 2 and pH

5.5. At pH 1-7 trans-resveratrol is stable; there are not much change in caffeic acid, catechin, rutin, chlorogenic acid, and coumaric acids during digestion (13, 14). For

absorption, phenolics are mainly absorbed in the small intestine and only some in large intestine. 95% of caffeic acid was found absorbed in the small intestine and stomach; less than 20% catechin and epicatechin were absorbed (13). Flavonoids normally are absorbed poorly in the small intestine. Because their molecule weight is large, they cannot be transported through passive diffusion pathways. Also, since the efflux transporters recognize the flavonoid better than influx transporters do, they are expelled more (15). After digestion and absorption, phytochemicals will go through different changes and elimination. Based on some phytochemical kinetics studies, quercetin is absorbed very fast after ingestion, and no interactions are found between quercetin and other food (16). After ingestion, lignan is easily absorbed into plasma; at least 40% of ingested lignan can be metabolized and found in plasma (17).

Phytosterol has very low bioavailability after ingestion; only 0.6%-7.5% can be transported through gut epithelia cells (17). Normally the bioavailability of carotenoids is low in human body, and there are many factors affecting it, such as processing and matrix composition. Studies show that heat processing and homogenization has a positive effect on it, while dietary fiber will lower the bioavailability. In addition, at least some fat is needed to help the absorption of the carotenoids (18, 19). Caffeine is transported into the gut primarily by passive diffusion, and it is directly transported without form change (20). Generally, proanthocyanidins, galloylated tea catechins, and anthocyanins are the least absorbed phenolics (13, 14).

1.2.3 Different phytochemicals for cancer prevention

Phytochemicals have been found to possess various anticancer activities in in vitro

and in vivo studies. The following is a summary of research about the effects of some

phytochemicals on cancer, especially breast cancer.

1.2.3.1 Carotenoids

Carotenoids are primarily found in cruciferous, yellow-green vegetables and various fruits. They are natural pigments (21) . The major carotenoids that have been

studied for health benefits include α-carotene, β-carotene, and lycopene.

In Nesaretnam’s study, β-carotene was found to inhibit proliferation of ER

positive MCF-7 human breast cancer cells but not ER negative MDA-MB-231 human breast cancer cells. However, they also found that β-carotene did not affect the level of pS2 mRNA which is an estrogen-regulated gene and serves as a marker for the

Estrogen-positive tumors. Β-carotene did not directly affect the breast cancer cells through estrogen-regulated pathway. One possible explanation is that β-carotene diffuses through the cell membrane and acts on the nuclear receptor, and then activates growth factors (21). Later Nesaretnam’s team did another in vivo study to

test the effect of β-carotene on MCF-7 human breast cancer cells. 48 nude mice were injected with β-carotene at 4 weeks of age, and then they were compared with the control groups which were not injected with β-carotene. The results showed that the carcinogenesis rate in experimental group was significantly lower than the control group, and the cancer metastasis rate was also lower. In addition they found that the level of natural killer cells and B-lymphocytes significantly increased in experiment

groups. , This may be the reason why β-carotene can inhibit the MCF-7 human breast cancer cells growth in nude mice (22).

Many epidemiology studies have also been conducted on the effect of

β-carotene on breast cancer. A recently finished cohort study by Nagel’s team in

Europe showed that β-carotene intake can decrease breast cancer risk in women who are postmenopausal and using exogenous hormones(23). However, most of the studies found no significant correlation between β-carotene intake and breast cancer rate (24, 25).

Lycopene has also drawn much attention from researchers because of its inhibitory ability on breast cancer. In an epidemiology study Zhang et al. found that

Lycopene has potential to decrease breast cancer risk (26). Karas et al. also found that lycopene can inhibit the MCF-7 human breast cancer cell growth by intervening with the IGF1 and G1 cell cycle (27, 28). Voskuil et al. did a cross trial, double blinded study; they found serum IGF1 level in experimental group with lycopene was significantly decreased in healthy women with high breast cancer risk but not in the cancer survivor women, which indicates that lycopene can benefit the high breast cancer risk in a healthy population. Di et al. found that lycopene can lower skin toxicity in breast cancer patients when they get external beam radiotherapy less than 500 ml

PTV (29). However, some epidemiology studies are controversial; in Sesso et al.’s prospective cohort study, they found no correlation between breast cancer risk and plasma lycopene (30).

1.2.3.2 Phenolic acids

Phenolic acids are a class of compounds containing a phenolic ring and an

organic carboxylic acid function. They are found in many plants. Phenolic acids primarily include Hydroxybenzoic acids and Hydroxycinnamic acids (Figure 1).

Hydroxybenzoic acids have been studied most. They include gallic acid and

protocatechuic acid. Gallic acid is a natural 3,4,5-trihydroxybenzoic acid; it is found

in gallnuts, tea leaves, oak bark and other plants. Gallic acid is shown to have

relatively similar absorption in drinking tea as from swallowing tablets(31). In a recent

study, Hsu et al. found gallic acid can inhibit MCF-7 human breast cancer cells growth

as a dose-dependent trend. The mechanism is that gallic acid can induce G2/M

phase cell cycle arrest (32). In addition, Garcia-Rivera et al. proved that gallic acid

possesses the ability to inhibit MDA-MB-231 human breast cancer cells. They found

that gallic acid inhibits the growth of MDA-MB-231 human breast cancer cells by

suppressing the NF-κ B activation (33). Yin et al also found protocatechuic acid has

the ability to inhibit the growth of MCF-7 human breast cancer cells. It can increase

caspas-3 activity and suppress vascular endothelial growth factor (VEGF) production

(34).

Hydroxycinnamic acids are polyphenols with the structure of C6-C3 skeleton.

They can be found in almost all the plants. Caffeic acid and ferulic acid are the major

phytochemicals in this category (Figure 1.). Ferulic acid is the most abundant

phenolic; studies showed that 11-25% of free ferulic acid in food can be absorbed by 8

humans (35). Additionally, the free form of ferulic acid has higher bioavailability than

bound forms (36). Of all hydroxycinnamic acids, caffeic acid and ferulic acid

derivatives have been found to inhibit breast cancer growth. It is very interesting that

caffeic acid and ferulic acid themselves cannot inhibit breast cancer growth, but their

derivatives have antiproliferative effects on MCF-7 human breast cancer cells.

Serafim et al. also found that this new lipophilic derivatives can inhibit cell proliferation and induce cell cycle in MCF-7 human breast cancer cells (37). Watabe et al also found that caffeic acid phenethyl ester (CAPE) can induce apoptosis in MCF-7 human breast cancer cells. The effect is through inhibiting NF-kB and activating fas signaling pathway (38). Wu et al also found CAPE derived from honeybees can inhibit MCF-7 and MDA-MB-231 human breast cancer cell growth in vitro and vivo without affecting normal cells. CAPE can also decrease VEGF formation by MDA-231 cells, which indicates it has the potential to inhibit angiogenesis and metastasis of breast cancer cells(39).

1.2.3.3 Flavonoids

Flavonoids generally exist in photosynthetic plants. Many studies have

shown that flavonoids can lower the risk of chronic diseases. Flavonoids are

characterized by a structure that has two rings linked by 3 carbons that are usually in

oxygenated heterocycle ring. They can be categorized as flavonols, flavones,

catechins, flavanones, anthocyanidins and isoflavonoids. They have been found to

possess the ability to inhibit breast cancer growth (12).

1.2.3.3.1 Flavonols

Quercetin is ubiquitously contained in plants in nature; many studies have

identified quercetin’s anti-breast cancer ability through different mechanisms.

Quercetin from different foods showed different bioavailability in humans. Studies

found that bounding with glucose increases quercetin absorption by humans (40).

Duo et al. found that quercetin can inhibit MCF-7 human breast cancer cells growth

and it induces apoptosis through down-regulating Bcl-2 protein expression and up-regulating Bax expression (16, 41). Chou et al. and Chien et al. obtained these results when MCF-7 human breast cancer cells were exposed to quercetin; there is

apoptosis in a majority of the cells. The possible signaling pathway involved is

through directly activating the caspase cascade through the mitochondrial pathway

(42, 43). Avila et al found that quercetin causes cell cycle to arrest through down-regulating the mutant p53 protein in MDA-MB-468 human breast cancer cells

(44). In addition, Oh et al. found that quercetin can inhibit angiogenesis in tamoxifen-resistant breast cancer cells. Quercetin can inhibit the level of VEGF protein by suppressing the activities of hypoxia inducible factor-1α and AP-1(45).

Scambia et al. also found that quercetin can increase the type-II estrogen-binding site protein level in MCF-7 and MDA-MB-231 human breast cancer cells (46).

Kaempferol can be isolated from tea, broccoli, grapefruit, and other plants. It is one of the most commonly found dietary phytoestrogens. Oh et al found that kaempferol can inhibit MCF-7 human breast cancer cell growth via ER-dependent

pathway; it can either increase or decrease the estrogen activity to maintain a balanced estrogen level (47). Furthermore, Kaempferol can also inhibit the proliferation and induce apoptosis in MCF-7 human breast cancer cells, Diantini et al. and MacPherson et al. found that kaempferol induces apoptosis through ERK activation and caspase cascade pathway activation (48, 49).

Myricetin is naturally found in many grapes, berries, walnuts, and other plants.

Myricetin has been suggested to regulate estrogen-like activity in breast cancer cells.

Thus, it can work as a potential factor in breast tumor growth inhibition. Maggiolini et al. found that myricetin can transactivate endogenous ER α and inhibit proliferation in

MCF-7 human breast cancer cells (50).

1.2.3.3.2 Flavones

Natural flavones are normally found in parsley, celery, and citrus peels. Of all the natural flavones, apigenin and luteolin have been found to possess the potential of inhibiting breast cancer (Figure 1).

Apigenin is generally found in many fruits and vegetables, including oranges, parsley, onions, wheat sprouts, and tea. Luteolin is contained in celery, green pepper, thyme, perilla, chamomile tea, and carrots (51). Their bioavailability is higher in the diet than the pure compound in rats (52). Apigenin has been found to inhibit breast cancer cell growth by inducing apoptosis, causing cell arrest, inhibiting metastasis, and decreasing estrogen receptor(53). Seo et al. found that apigenin can inhibit

MCF-7 human breast cancer cells growth by inducing apoptosis. The effect is through inhibiting the p53 and NF-kappa B expression (54). Choi et al. also found that apigenin can induce SK-BR-3 breast cancer cells apoptosis through activating p53 and BAX protein; it induced MDA-MB-453 human breast cancer cell apoptosis by the mitochondria/caspase-pathway (55, 56). Additionally, apigenin has been shown to inhibit t metastasis by blocking the VEGF mRNA and inhibiting HGF through blocking the PI3K/Akt pathway. Furthermore, by inhibiting both the estrogen-dependent and estrogen-independent signaling pathway, apigenin also can inhibit the growth of

MCF-7 human breast cancer cells (57, 58). Luteolin also has been found to induce apoptosis of breast cancer cells by activating ERK and p38 protein expression (59).

1.2.3.3.3 Catechins

Catechins are found principally in green tea. They can also be found in smaller amounts in grapes, black tea, chocolate and wine. One major catechin, EGCG, draws many people’s attention, because EGCG is absorbed very fast after people drink green tea and black tea. Interestingly, EGCG in hot tea has higher availability than that in iced tea (60, 61). EGCG has been found to possess great potential to lower breast cancer risk in many green tea studies (62, 63). Sen et al. found that EGCG can inhibit breast cancer cells metastasis by down-regulating Gelatinase-A and

Gelatinase-B (MMP-2, MMP-9) (64, 65). Furthermore, Kim et al. found that inhibiting

the Wnt signaling pathway is a potential mechanism of inhibiting breast cancer cells

growth by EGCG(66). Guo et al. also found EGCG can change the gene expression in nuclear and cytoplasmic fractions to inhibit breast cancer cells growth (67).

1.2.3.3.4 Isoflavonoids

Isoflavonoids are contained in plants of the legume family; they are famous for their role as phytoestrogens. Studies have found that isoflavonoids can lower breast cancer risk(68). Especially genistein and daidzein have been proven to be very efficient. Because of their high efficiency, there has been more research on isoflavonoids than other phytochemicals in related breast cancer research.

Genistein has more bioavailability in food than in solid form, since genistein has low water solubility. Its bioavailability can be increased by adding a starch component (69). It has gotten many researchers' attention because its inhibition functions affect many different kinds of breast cancer. As a phytoestrogen, genistein can work similarly to estrogen or anti-estrogen to maintain the appropriate estrogen level in breast cancer. Thus it inhibits breast cancer growth. Marik et al. found genistein can inhibit MCF-7, 21PT and T47D breast cancer cell growth through this mechanism (70). In addition, genistein can induce cell cycle arrest and apoptosis in breast cancer cells. Park et al. found that genistein can induce G2/M arrest in T47D and MDA-MB231 cells (71). Li et al. also found the similar results; genistein induced cell apoptosis in MDA-MB-231 human breast cancer cells through inhibiting the mEK5/ERK5/NF- κB pathway (72, 73). Montales et al. found that genistein can suppress the stem-like/progenitor cells in MMTV-wnt-1 transgenic mice (74). In the 13

study by Ra et al., genistein suppressed breast cancer growth by inhibiting the

expression of the fatty acid synthase (75). Farina et al. found that genistein inhibited

the migration of breast cancer cells in F3ll mammary carcinoma mouse models (76).

Furthermore, Privat et al. found that genistein can also inhibit the breast cancer

SUM1315MO2 cells which carry the BRCA1 mutation (77).

Daidzein is another important isoflavnoid, and it is mainly found in soy products. Daidzein is poorly water soluble; thus it is better absorbed in diet form.

Researchers found the self-microemulsifying drug delivery system, composed of oil,

surfactant and cosurfactant, can enhance its bioavailability (78). Its breast cancer

inhibitory effect also accounts for part of the reason why soy diets can lower breast

cancer risk. Jin et al. found that daidzein can induce MCF-7 human breast cancer apoptosis through the mitochondrial pathway (79). Choi et al. also found that daidzein can cause MCF-7 cell cycle arrest at G1 and G2/M phase (80).

1.2.3.4 Tannins

Tannins are a bitter plant polyphenolic compound; they are contained in many species of plants. Reported as anti-nutrients, tannins have been found to inhibit breast cancer growth. Bawadi et al. found that black bean concentrated tannins can inhibit MCF-7 human breast cancer growth; they can also inhibit the cancer migration

by decreasing VEGF protein and MMP2/MMP9 levels (81).

1.2.3.5 Nitrogen-containing compounds

Nitrogen-containing compounds are a category of phytochemicals that contain nitrogen. The bisphosphonates in nitrogen-containing compounds are much studied for their anti-cancer activities. In a retrospective study, Park et al. found that nitrogen-containing bisphosphonate can increase the survival time of hormone receptor negative breast cancer patients with bone metastases (82). Merrell et al. supported these conclusions in their study. The results showed that the mechanism for this process is through inhibiting the mevalonate pathway and activating p38 MAP kinase (83).

1.2.3.6 Organosulfur compounds

Organosulfur compounds are organic compounds that contain sulfur. Some dietary organosulfur compounds that possess anti breast cancer ability are isothiocyanates and indoels (Figure 1).

Organic isothiocyanates are dietary components often found in cruciferous vegetables. Tseng et al. found that benzyl isothiocyanate (BITC) and beta-phenethyl isothiocyanate (PETTC) were able to inhibit the growth of MCF-7 human breast cancer cells as chemotherapeutic agent daunomycin(84). Kang et al. studied the mechanism by which isothiocyanates prevent breast cancer. They found that BITC and PETTC can inhibit the mitogenic estrogen level in ER-positive breast cancer cells.

In addition, indoles had also been found to inhibit the growth of breast cancer cells by suppressing the PI3 kinase-alpha pathway and mTOR pathway in MDA-MB-361 human breast cancer cells (85). 15

1.2.4 Phytochemical synergy and whole foods benefits for cancer prevention

In addition to the individual phytochemical, many studies on phytochemical-phytochemical synergy, phytochemical-drug synergy, and whole foods synergy have been done. Most of them showed a better result than individual use, and it is more applicable to real life.

Many phytochemicals are effective individually, working in vitro but not in vivo.

In the human body phytochemicals normally don’t appear in pure form; thus research on combinations of different phytochemicals is necessary to simulate the effects of natural foods. . Hsieh and Wu conducted research on the synergy effect of EGCG, resveratrol and gamma-tocotrienol. They found that when one phytochemical was added to another, there was a significant improved effect. However when three were combined there was no improvement over two together (86). Sahin et al. also found that when treating DMBA induced female mice, the lycopene and genistein combination was more efficient than the individual one. Rats treated with DMBA for

20-week periods developed mammary tumors with 100% tumor incidence. Lycopene inhibited breast cancer incidence to 70%, genistein inhibited to 60% and a combination of both inhibited to 40%. The study proved the synergistic protection of lycopene and genistein against the DMBA induced breast cancer in rat (87).

Regular chemoprevention treatment normally brings side effects, such as discomfort to the body or drug resistance. Treating the tumor with medicine and phytochemicals together has been found to improve the efficacy of the drug and 16

reduce the toxicity and damage to body. Scambia et al. conducted research on

ADR-resistant MCF-7 human breast cancer cells; quercetin together with ADR

decreased the level of p-glycoprotein, an indicator of malignancy of the ADR resistant

MCF-cancer cells, compared to ADR alone (88). Akbas et al. found that quercetin can

increase the antiproliferation effect of topotecan in MCF-7 human breast cancer cells

by 1.4 fold and in MDA-MB-231 human breast cancer cells by 1.3 fold. Quercetin

combined with topotecan also increased the ROS and nitrite levels in both cells

compared to those treated with only topotecan (89). Ferenc et al. found that using

genistein can significantly improve the result of photodynamic therapy with hypericin

in MCF-7 and MDA-MB-231 human breast cancer cells. The apoptosis they induced

is better targeted to the right cells; moreover, caspase-7, and PARP cleavage are

both activated when genistein is added. All of these indicate adding genistein to

photodynamic therapy with hypericin can improve the effects in reducing the proliferation and inducing the apoptosis in MCF-7 and MDA-MB-231 human breast cancer cells compared to hypericin alone (90). Wong and Chiu combined quercetin with vincristine to treat ER negative breast cancer. They found the molar ratio of

quercetin to vincristine was best at 1:2 (91). Lattrich et al. also found that high-dose

genistein (10 µmol/l) enhanced the inhibitory effect of trastuzumab in

HER2-overexpressing, ER positive BT-474 breast cancer cells (92). Staedler et al studied the combination effect of quercetin and doxorubicin; they found that when highly invasive breast cancer cells were treated with quercentin and doxorubicin,

quercentin improved the inhibitory effect of doxorubicin in breast cancer cells, while it reduced the cytotoxic side effects in non-tumoral cells. The study showed that a quercetin and doxorubicin combination can be a good treatment for breast cancer

(93).

Many recent studies concentrate on whole food extracts, which contain complex phytochemicals. Many in vitro studies found that the whole food extracts present strong inhibitory effects in breast cancer cells. Phadhan et al. reported that limonia acidissima linn extracts can inhibit SKBR3 and MDA-MB-435 breast cancer cell lines. It also induced the cell arrest in G2/M phase in MDA-MB-435 cells but not in the SKBR3 cells (94). Plastina et al. found that Ziziphus jujuba fruit extracts suppressed the proliferation of MCF-7 and SKBP3 breast cancer cells, but there was no effect for apoptosis (95). Dikmen et al. reported that Punica granatum L. Fruit Peel extract inhibited the MCF-7 human breast cancer cell proliferation as a dose-dependent manner; it also induced apoptosis as the concentration increased

(96). In vivo studies it has also been done on the prevention effects of whole foods in breast cancer. Dai et al. found that graviola fruit extract significantly reduced the key protein markers EGFR, p-EGFR, and p-ERK expression in MDA 468 tumors in a

5-week mice feeding study (97). Wang et al. also found litchi fruit pericarp extract exhibited anti-breast cancer potential in breast cancer nude mice following 10 weeks feed. Litchi fruit pericarp showed a dose and time dependent suppression effect on

the cell growth, and it significantly reduced colony formation and bromodeoxyuridine

incorporation of breast cancer cells in mice (98).

Furthermore, there are many epidemiology studies on the whole foods’ anti

breast cancer effect; however, the results are contradictory. An Italian retrospective

study found that consuming vegetables, especially leafy and fruiting vegetables, can

significantly lower the risk of breast cancer, while overall fruit consumption was not

associated with breast cancer prevention (99). In a 12 year study with 51,928 black

women, the author found no correlation between total fruits and vegetables

consumption and breast cancer risk. However, consuming vegetables was

associated with lowering risk of ER negative breast cancer. In addition, eating

cruciferous vegetables and carrots can also significantly decrease breast cancer risk

(100). In a whole grain large prospective cohort study (101), no association between a whole grain diet and breast cancer risk was found. Similar results were also found in several other studies (102-104).

1.3 Asparagus and its health benefits

1.3.1 General introduction

Asparagus, scientific name Asparagus officinalis, is a perennial vegetable that flowers in the spring. People have normally cultivate it as a vegetable crop. It is a very

important economic crop grown in many temperate places all over the world (105).

The earliest cultivated asparagus was found in Egypt as early as 3000 B.C., and later

it was also found in European cultures, including early Greek and Roman cultures.

Today China is the largest commercial producer with 587,500 tons per year; Peru is second with 186,000 tons produced per year. They are currently the world's largest producers and exporters of asparagus. Next in line commercial production are the

United States (102,780 tons) and Mexico (67,247 tons). (106)

1.3.2 Asparagus nutrition composition and phytochemicals

Asparagus is one of the most nutritious vegetables (107). It is low in calories and sodium. 100 g of fresh asparagus spears provide just 20 calories and 5 mg sodium. Besides, asparagus contains high amounts of folic acid. 100 g of spears provide about 54 µg or 14% of RDA of folic acid. Folic acid is necessary for blood cell formation, growth, and prevention of neural tube defects (108). Asparagus also contains fair amounts of fiber; 100 g of fresh spears provide 2.1 g of fiber, which has been shown to lower the colon cancer risk (109). Asparagus also has a rich

B-complex group of vitamins such as thiamin, riboflavin, niacin, vitamin B-6

(pyridoxine), and pantothenic acid. These vitamins are essential for optimum cellular enzymatic and metabolic functions. Asparagus is also a good source of vitamin K; it provides about 35% of DRI. Vitamin K has a potential role in bone health, promoting osteotrophic (bone formation) activity. Adequate vitamin-K levels in the diet help to limit neuronal damage in the brain; thus, asparagus has an established role in the treatment of patients suffering from Alzheimer’s disease (110). Asparagus has moderate amounts of copper, iron, and small amounts of other essential minerals and

electrolytes such as calcium, potassium, manganese, and phosphorus. Potassium is an important component of cell and body fluids that helps control heart rate and blood pressure by countering effects of sodium. Manganese is used as a co-factor for the antioxidant enzyme, superoxide dismutase. Copper is required in the production of red blood cells. Iron is required for cellular respiration and red blood cell formation

(111). One more benefit of asparagus: it contains high levels of the amino acid asparagine, which serves as a natural diuretic, and increased urination not only releases fluid but helps rid the body of excess salts. This is especially beneficial for people who suffer from edema (an accumulation of fluids in the body's tissues) and those who have high blood pressure or other heart-related diseases (112).

Asparagus has been found to possess various health benefits, such as neuroprotective, antioxidant, hypolipidaemic, hypoglycaemic, hepatoprotective, and anti-tumorigenic activities in vitro or vivo in one study, an asparagus-containing diet was used to feed spontaneously hypertensive rats to test whether it can prevent hypertension. The results showed that the 5% fresh green asparagus diet significantly lowered the systolic blood pressure level compared with the control (159 ± 4.8 mmHg vs 192 ± 14.7 mmHg); asparagus inhibited angiotensin-converting enzyme (ACE) activity (113). In another study, asparagus was found to show neuroprotective effects in senescence-accelerated mice. Enzyme-treated asparagus extract (ETAS) can up-regulate the mRNA level of heat-shock protein 70 and heme oxygenase-1 in

NG108-15 neuronal cells. It can also attenuate the cognitive impairment caused by

contextual fear memory in SAMP8 mice (114). Asparagus extracts have also been found to have hypocholesterolemic and hepatoprotective effects on rats. When rats

with high cholesterol were fed asparagus (500 mg/kg body weight (bw)/day) for 5

weeks, the total cholesterol, and low-density lipoprotein cholesterol levels were

decreased, while the high-density lipoprotein cholesterol level was increased,

compared with the high cholesterol group. In addition a significant increase in enzyme

activity from multiple hepatic antioxidant systems, such as superoxide dismutase,

catalase, and glutathione reductase/peroxidase as well as a decrease in

malondialdehyde concentrations compared to high cholesterol group were observed

(115). Asparagus also showed anticancer activities in different tumors. Methanolic

extract of white asparagus shoots can inhibit colon carcinogenesis by regulating the

TRAIL death-receptor signaling pathway in human colon carcinoma cells (SW480)

and their derived metastatic cells (SW620) (116).

1.3.3 Bioactive compounds of asparagus

In addition to the basic nutrients, asparagus also contains varieties of

phytochemicals that possess different health benefits.

Asparagus saponins, which are a group of steroids found in the asparagus

spear and root, have shown strong anticancer activities in different cancer cells. The

content of saponins in asparagus is 0.03% in dry asparagus and 0.002% in fresh

asparagus. In 1996 Shao tested the effects of crude saponins extracts from

asparagus shoots (ACS) in human leukemia HL-60 cells. He found that the ACS can

inhibit HL-60 cells growth in a dose-dependent trend. When ACS were added to

HL-60 cells and incubated at 37°C for 24 h at the dose of 50, 200, or 400pg/ml, the

growth of HL-60 cells were inhibited by 53, 88 and 94%. Meanwhile, when HL-60 cells were treated with ACS at 6.25, 50 or 200 pg/ml at 37°C for 2 hours, the DNA synthesis was inhibited by 40.19, 82.98, and 90.89%, respectively (117). Later in

1997, Shao identified two steroid saponins from asparagus seeds. These two

saponins are methyl protodioscin and protodioscin. It was also found that both

saponins can inhibit DNA synthesis in a dose-dependent manner. The IC50 value

(the half maximal inhibitory concentration) for protodioscin is 9 uM and 19 uM for

methyl protodioscin. Further, protodioscin was chosen to test its effects on the

HL-60 cells growth. The IC50 was found to be 15 uM (118). The effects of

asparagus saponins on other cancer cell lines were also evaluated by the National

Cancer Institute Development Therapeutics Program (USA). It was found that

asparagus can inhibit growth of various cancer cells, with the most effect on colon

cancer cells (119). Saponins extract from asparagus has also been found to inhibit

tumor migration and invasion. Asparagus saponins can significantly inhibit migration

and invasion of MDA-MB-231 human breast cancer cells at a concentration of 400 μg

/mL. This mechanism is shown through targeting the Rho GTPase signaling

pathway(120). Besides, saponins were also found to lower the cholesterol level in

plasma by inhibiting cholesterol absorption in humans (121). There are now more

than 10 different saponins found in asparagus (122).

Polysaccharide is another important class of bioactive compounds found in

asparagus; it has been shown to have immunity improving, anti-aging and antitumor

activities. Polysaccharide extraction from Asparagus officinalis has been tested on

HeLa and BEL-7404 cells. It can inhibit cells growth in a dose-dependent manner. At

a concentration of 10 mg/mL, the polysaccharide can inhibit HeLa cells growth by

83.96% (105). When investigators injected a sarcoma mouse model (S180) mice with asparagus extracts for 7 days, the expression of CD25 in lymphocytes was much

higher than the control group and normal group. This showed that asparagus can

improve immunity of S180mice by improving its erythrocyte function (123).

According to several studies, asparagus also contains other phytochemicals,

such as hydroxycinnamic acids (HCA), sulfur containing acids, sterols, fructans, rutin,

diosgenin, and kaempferol. Kaempferol, an important bioactive compound, is a major

flavonoid in asparagus. According to the USDA database, kaempferol content in

asparagus is around 1.39 mg per 100 g(124). Kaempferol has been found to possess

different functions. It may account for some of asparagus’ health benefits. However,

more studies need to be done to clarify it.

1.4 Kaempferol

1.4.1 Chemistry of kaempferol

1.4.1.1 Structure and derivatives

Kaempferol is widely distributed in plants. Its structure is

3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one (Figure 2). It has many derivatives; kaempferol-3-O-glucoside (astragalin), kaempferol-3-O-beta-isorhamninoside, and kaempferol-7-O-beta-D-glucoside have shown anticancer potential are (Table 1.1) (125).

FIGURE 2 CHEMICAL STRUCTURES OF COMPOUNDS

Compound name R1 R2

Kaempferol H H

Kaempferol-3-O-glucoside Glucoside H kaempferol-3-O-beta-isorhamninoside Gal(6-1)Rha(3-1)Rha Isorhamninoside

kaempferol-7-O-beta-D-glucoside H Glucoside

Figure 2 Chemical structures of kaempferol and related compounds

1.4.1.2 Food sources and content

Kaempferol has been found distributed widely in Pteridophyta, Pinophyta and

Magnoliophyta in nature. It also exists in many vegetables and fruits, such as asparagus, broccoli, leek, onion, chives, Chinese cabbage, cucumber, tea, apple, peach, blackberries, raspberries, and grapefruit. It is also found in traditional Chinese medicines (125).

1.4.1.3 Absorption, distribution and metabolism

No matter how efficient a compound treatment can be, it only works when it reaches the target after absorption and metabolism. Kaempferol in different forms is taken orally most of the time. Studying its bioavailability and metabolism will help its application in practice.

An animal study on metabolism found that oral bioavailability of kaempferol in rats was around 2%. Quercetin and kaempferol-glucuronides are major conjugated form found in plasma. , There are phase I oxidative metabolism and phase II glucuronidation in both the intestine and liver (126). In a pharmacokinetic study, the bioavailability of kaempferol from endive was evaluated. Eight subjects were recruited and fed endive containing approximately 9 mg kaempferol. Throughout 24 hours, their plasma and urine samples were tested for kaempferol level and quercetin level.

The results showed that after 5.8 hours, the plasma kaempferol level reached a maximum of 0.1 µM; an average of 1.9% of kaempferol intake had been excreted in

24 h. Meanwhile there was no quercetin detected in either plasma or urine samples.

This result indicated that kaempferol has relatively higher bioavailability than 26

quercetin, even in low dose. Since quercetin was detected, the kaempferol was not converted to quercetin, which indicated that there was no phase I metabolism. In plasma a conjugated kaempferol metabolite- kaempferol-3-glucuronide accounted for

55%-80% of total kaempferol. This suggested there was phase II metabolism involved. Furthermore, the study also indicated that different kaempferol derivatives were absorbed in different ways. Kaempferol-3-glucoside accounts for the early peak in plasma at 0.5 h, so it is absorbed faster in the small intestine, while kaempferol-3-glucuronide is absorbed in the distal intestine and colon (127). In another similar study, 7 healthy subjects received kaempferol and were tested for their urea excretion after 2-8 h. They found 6.10 +/- 5.50% and 5.40 +/- 5.40% of the kaempferol dose for male and female subjects in urea samples (128). Some modern technology can now increase the bioavailability of kaempferol. By using a

PEO-PPO-PEO nanoparticle incorporate, kaempferol's inhibition effect was improved on ovarian cancer (129).

While kaempferol is mostly present in the food form, it is also important to know how kaempferol is affected by processing and storage. A study about the effects of kaempferol in processed onions found that most of the kaempferol was lost during the pre-blanch process, which included peeling, trimming and chopping. The content of kaempferol dropped from 0.96 mg/100 g to 0.35 mg/100g. After blanching, the content was pretty stable during further cooking, frying, or warm-holding (130).

Another study examined the effects of transport and marketing on kaempferol in

broccoli heads. The heads were first placed in 1 or 4◦C at 99% relative humidity for

different days to simulate the transport conditions. Then they were put in an

environment with temperature and relative humidity similar to markets. Kaempferol

contents were not significantly changed, suggesting current transport and marketing

practices are not likely to affect the kaempferol content(131). Research on strawberry,

acerola, pitanga and cashew-apple found that processed juices made from these

fruits lost almost half the kaempferol, compared with raw fruits (132).

1.4.2 Anticancer activities of Kaempferol

1.4.2.1 Epidemiology evidences

Kaempferol has been inversely associated with the risk of different cancers in

human trial studies. Kaempferol intake is inversely associated with cancer and

inflammation markers, such as the serum interleukin-6 (IL-6), which indicates inflammation and carcinogenesis. In an intervention arm of the Polyp Prevention Trial, subjects with high intake of kaempferol had low serum IL-6 concentration (highest

versus lowest kaempferol intake quartile, 1.80 versus 2.09 pg/mL). The decrease in

IL-6 level was also inversely associated with high-risk (OR, 0.44; 95% CI, 0.23-0.84)

and advanced adenoma recurrence (OR, 0.47; 95% CI, 0.19-1.18) (133). In another

population-based case-control study among tobacco smokers daily intake of 2 mg

kaempferol significantly decreased the lung cancer rate (OR, 0.68; 95% CL,

0.51-0.90) (134). A cohort study among the male smokers in Finland examined the

effects of alpha-Tocopherol, beta-Carotene on cancer prevention. The results 28

showed that when subjects took more than 1.29 mg per day kaempferol without

alpha-tocopherol and/or beta-carotene, the pancreatic cancer rate decreased (OR,

0.37; 95% CI, 0.17-0.79) (135). The same group examined the U.S. Department of

Agriculture flavonoid database and found that high intake of flavones significantly

reduced the risk of advanced adenoma recurrence (OR, 0.24; 95% CI, 0.11-0.53)

when the 4th quartile intake compared with the first quartile intake. Kaempferol

exhibited a similar pattern (OR, 0.44; 95%CI, 0.22-0.89). In a Nurses’ Health Study of

66,940 women to determine the correlation between 5 common dietary flavonoids intake and epithelial ovarian cancer rate, it found no firm association between total intake of the 5 flavonoids and ovarian cancer incidence. However, kaempferol alone can significantly decreased the rate by highest quintile verses lowest quintile intake

(OR, 0.6, 95%CI, 0.42-0.87)(136).

1.4.2.2 In vivo and in vitro studies

Kaempferol has also been found to possess cancer prevention characteristics in animal models. In one study, kaempferol’s cancer prevention effects were evaluated by feeding it to male wistar rats treated with 1,2-dimethyl hydrazine, a colorectal cancer inducer. After 16 weeks, the 1,2-dimethyl hydrazine increased the

erythrocyte lysate and liver thiobarbituric acid reactive substances level; it also

induced rejuvenated antioxidant enzymes catalase, super oxide dismutase and

glutathione peroxidase. Kaempferol can inhibit these inductions in a dose response

manner compared with control. And the effects were comparable to Irinotecan, a

well-known anticancer agent for colon cancer (137). Kaempferol can also inhibit

cancer cell growth in different ways. Ginkgo biloba extract kaempferol has been found to inhibit the proliferation of pancreatic cancer cell lines MIA PaCa-2 and Panc-1.

Treated with 70 μM kaempferol for 4 days, MIA PaCa-2 and Panc-1 cells proliferation were significantly suppressed (138). Kaempferol can also induce apoptosis in human HCT116 colon cancer cells. When the cells were incubated with 60 μM kaempferol, the number of the cells in sub-G1 phase was significantly increased over time (139). In another study, kaempferol inhibited angiogenesis factor of two ovarian cancer cell lines, OVCAR-3 and A2780/CP70. Kaempferol can directly inhibit

VCAR-3-induced angiogenesis and tumor growth significantly in chorioallantoic membranes of chicken embryos(140).

1.5 Potential mechanisms of action

Kaempferol has drawn much attention of researchers to study its function on

cancer, and it is found that kaempferol showed anticancer activities through different

mechanisms. Some of these are described below.

1.5.1 Antioxidant effects and Redox regulation

Many phytochemicals possess both antioxidant ability in normal cells and

pro-oxidant ability in cancer cells. Through these functions they can prevent the

carcinogen damage on normal cells and increase the repair process such as

apoptosis in cancer cells (141). One good explanation for the function is that

antioxidants can interfere with the reactive oxygen species (ROS) level. ROS in normal cells is balanced with a cell’s self-antioxidant defense in redox system.

However, under certain conditions, such as oxygen free radicals, UV exposure, gamma-radiation and other carcinogens, ROS will be produced excessively and induce oxidative stress. Then the stress will promote damage to cell structure, including proteins, lipids, membranes and DNA, which leads to carcinogenesis (142).

In cancer cells ROS level is suppressed, because increased ROS level is associated with induction of apoptosis program. Antioxidants can improve the redox balance by regulating ROS, and it can improve the defense systems which include several phase

II enzymes such as Glutathione peroxidases (GPx), catalase, superoxide dismutase

(SODs) (141).

Studies found that vegetables and fruits’ cancer inhibition effects are associated with their antioxidant ability (4, 5, 143, 144). Kaempferol, as a natural antioxidant, also has such ability; it can efficiently scavenge the free radicals (145,

146). A study about the antioxidant effects of kaempferol on colorectal carcinoma in rats found that kaempferol significantly improved the antioxidant status. The study used 1.2-dimethyl hydrazine (DH) to induce colorectal cancer in rats. Rats were fed

50, 100, 200 mg/Kg body weight of kaempferol or they received irinotecan intravenously; after 16 weeks, lipid peroxidation products, catalase, SODs and GPx level in blood were tested. The results showed that kaempferol decreased

DH-induced thiobarbituric acid level and increased catalase, SODs and GPx levels,

compared with the control group, in a dose response manner; 200 mg/Kg body weight

per day of kaempferol was comparable to Irinotecan, a common anticancer drug for colon cancer (137). Another study examined the protective ability of kaempferol in rat

H4IIe cells against oxidative stress apoptosis by H2O2. Up to 25 µM kaempferol

decreased H2O2 induced ROS level, dependent on time and concentration. It also decreases the H2O2 induced caspase-3 activity as doses increased (125). Another study found the cellular ROS level is up-regulated by kaempferol in MCF-7 human

breast cancer cells, and it can enhance the apoptosis effect. Using antioxidant

N-acetyl cysteine negatively affected the apoptosis caused by kaempferol; this

suggested that the increased ROS stress by kaempferol may also contribute to its

apoptosis induction (48). Another study found that kaempferol increased ROS

generation and induced apoptosis in Glioblastoma cells. When the antioxidants

N-acetylcysteine (NAC) is used, the sensitivity of kaempferol induced apoptosis is

reduced. Phase II enzyme superoxide dismutase and thioredoxin was

down-regulated by kaempferol. Knockdown of these two enzymes significantly

increased the apoptosis effect of kaempferol. All of these suggest that kaempferol

induced apoptosis through increasing ROS stress in Glioblastomacells (147). Similar

results in HeLa cells showed that kaempferol can elevate the ROS level, and it is

related to the MAPK-mediated apoptosis induced by kaempferol (148).

1.5.2 Antiproliferation and cell cycle arrest

Cells in the human body are proliferating all the time. One reason is to help the body grow in a needed situation; the other reason is to make up for the dying cells

every moment to maintain the balance. The proliferating cells will grow larger increasing the DNA, and doubling into two daughter cells. These steps happen in a

cycle, called the G1/G0, S, G2, M phase cell cycle (149). In normal cells, cell proliferation is kept in balance by Cyclins and Cyclin-dependent kinase. When cells

need to proliferate, they receive the mitogenic growth factors signal, and then grow

and divide normally. When the environment is abnormal, such as the DNA is

damaged or the cells decide not to grow, the cells will halt at check points in the

different cell cycle phases or become quiescence at G1/G0 phase or arrest at G2/M

phase. However, in cancer cells, these signals have been interrupted, and the

oncogenic factors override these signals, so the cells surpass the checkpoints and

proliferate even in the abnormal environment (150). Kaempferol is found to inhibit the

cancer cell proliferation and induce the cell cycle arrest. In one study researchers

used ginkgo extract kaempferol to treat pancreatic cancer cell lines MIA PaCa-2 and

Panc-1. Tested by MTS assay, the results showed that when both cell lines were

treated with 70 µM kaempferol for 4 days, proliferation was significantly suppressed

(138, 151). Research on kaempferol derivative kaempferol-7-O-β-D-glucoside (KG)

also showed that KG has strong antiproliferative effect against HeLa carcinoma cells

while it did not affect the normal HEK293 human embryonic kidney cell. The flow

cytometry experiment also showed that KG increased G2/M cell cycle arrest.

Furthermore KG was shown to down-regulate the Cyclin B1 and Cdk1 proteins while it had no effects on the p53/p21 protein. This indicated that KG induces HeLa cell cycle arrest in a p53-independent manner (152). Another study about KG also found similar results on inhibiting cancer proliferation; besides HeLa cancer cells, KG also significantly inhibited 6 other cancer cell lines and had less cytotoxicity on normal human cells. The KG treated A375melanoma cell and leukemia HL60 cells increased their G1 phase cells arrest and decreased G2/M phase cell cycle arrest(153). Another group used Kaempferol to treat HL60 cells; they found that when the treatment was more than 10 µM, there was a significant increase of S phase and G2/M cell cycle arrest but there was not much apoptosis inducing effect. These results indicate that kaempferol inhibited HL60 cells growth primarily by cell cycle alteration (154, 155).

1.5.3 Induce Apoptosis and Autophagy

Under certain stress or in abnormal situations, organisms will try to get rid of the defective or mal-functioning cells from the tissue. One of the important functions to precede this is apoptosis. The cancer occurs when there are irregular cell cycles which are induced by the DNA intervention or by passing the wrong DNA to sister cells. The body will first try to fix these abnormal factors through DNA repair or cell cycle arrest. However, when the damage or abnormal situation cannot be resolved, the cell's suicide program, which is called apoptosis is activated (156). Apoptosis is a vital process whereby the organisms use to stop the cancer growth, and kaempferol has shown strong pro-apoptosis effects in different cancer cells.

There are two major signaling pathways which can trigger apoptosis. One is called intrinsic apoptotic program, which is through mitochondria. The other is called extrinsic apoptotic program which uses cell surface receptors. Both signaling pathways will induce the cytoplasmic caspase cascade and induce the cell apoptotic death in the end. In intrinsic apoptotic program, caspase cascade will be activated by cytochrome c; cytochrome c release is controlled by opening and closing the mitochondrial outer membrane channel which is affected by Bcl-2 (negative) and other relatives of Bcl-2 (negative) such as Bax, Bad, and Bid. The upstream regulator for these proteins is p53 and its regulation factors (151, 157, 158). Several studies found that kaempferol can induce apoptosis through this pathway. One study found that when treated with 50 µM kaempferol, the MCF-7 human breast cancer cells showed an increasing number of the apoptosis cells and cleaved PAPR level over time. Western Blot analysis showed that Bax, cleaved Caspase-9, Caspase-3, and

Caspases-7 level all increased. The PLK-1, which they found participated in the process, had decreased (159). Another study found that kaempferol can induce apoptosis in human HCT116 colon cancer cells. The results showed that increased concentrations of kaempferol up-regulated the levels of cleaved PARP, Caspase-3, cytochrome-c, p-p53, bax, PUMA, while they down-regulated Bcl-2 protein level.

There was no obvious effect on Caspase-8 protein, indicating the extrinsic apoptotic program was not involved in the pathway. They identified Ataxia-Teleangiectasia

Mutated protein as the upstream activator in this pathway (139). Another group of

researchers treated the K562 leukemia cell line and the U937 promyelocitic human leukemia cell line with 50 µM kaempferol. They found that the apoptosis cells increased. For both cells kaempferol increased Bax, cytosol cytochrome c, and cleaved PARP levels. It increased the activation of Caspase-3 and Caspase-9. It also decreased the Bcl-2, mitochondrial cytochrome c levels. In addition, they found

SIRT3 up-regulation and PI3K inhibition are involved in the upstream regulation (160).

Kaempferol also induced apoptosis in ovarian cancer cells, which include

A2780/CP70, A2780/wt, and OVCAR-3 cell lines. 80 µM kaempferol treatment increased Caspase 3/7 level in first two hours, and the level was decreased; using

Caspase-9 inhibitor significantly offset this effect which indicated that it is through the intrinsic pathway. When the dose of kaempferol treatment was increased , p53, Bad,

Bax protein expression were up-regulated, while Bcl-xl protein expression was down-regulated (161). Kaempferol can also induce apoptosis in oral cavity cancer cells through Caspase-3 pathway (162).

For extrinsic apoptotic program, there are fewer studies; one study on kaempferol 3-O-beta-isorhamnioside (K3O-ir) from Rhamnusalaternus L. found

K3O-ir can induce apoptosis in human lymphoblastoid cells through the extrinsic pathway. Kaempferol treatment increased level of cleaved PARP and cleaved

Caspase-3; and it increased Caspase-8 protein, which indicated it was through the extrinsic program (163).

Additionally, Endoplasmic Reticulum (ER) stress was also involved in the

apoptotic process; some researchers used kaempferol to treat U-2 OS osteosarcoma

cells and induce apoptosis. Their results showed that besides the activation of the

intrinsic pathway, the ER stress was also up-regulated by kaempferol. They found

cytoplasmic Ca2+, GADD153, GRP78, GRP94, ATF-6α, ATF-6β, Caspase-4,

Caspase-12, Calpain 1 and Calpain 2 protein levels were increased; when Calpain

inhibitor BAPTA was used, the apoptosis effect was weaker. These results indicate

ER stress involved in this kaempferol induced apoptosis effect (164). Kaempferol was

found to induce MCF-7 human breast cancer cell apoptosis through MAPK pathway;

ERK activation played an important role in apoptosis. By using the TUNEL assay, 30

µM kaempferol treatment clearly induced MCF-7 human breast cancer cell apoptosis;

the cleaved PARP, Phospho MEK1, Phospho ELK1, and Phospho ERK levels also

were increased with time from 0 to 24 h. When the researchers treated the cells with

MEK1 inhibitor PD98059 or transfected the MCF-7 cells with Kinase-inactive ERK

mutant, the cell viability number increased and the cleaved PARP decreased,

confirming the ERK pathway was involved in the apoptosis induce effect of

kaempferol in MCF-7 human breast cancer cells (48). Another study achieved similar

results in A549 lung cancer cells. Kaempferol can induce apoptosis by up-regulating

the Bax and Caspase-7, cleaved PARP protein expression and down-regulating the

Bcl-2, Bcl-Xl protein expression. For the upstream regulator, total Akt and

phosphorylated Akt levels were decreased, and MEK1/2 level was increased.

However, using Akt inhibitor did not affect apoptosis results while MEK inhibitor did, suggesting that the upstream activator was through MAPK pathway (165).

When cells are under energetic stress, autophagy will also be induced. The autophagy cells are still alive and the process is reversible. It is considered an early survival mechanism against apoptosis. Recently a study also found that before kaempferol induces apoptosis, it could also induce autophagy in the beginning period.

The researchers tested 100 and 200 µM kaempferol’s effects on HeLa cells and gastric adenocarcinoma AGS. They found in the first 6 h the extracellular lactate was significantly decreased, especially in a low-glucose environment; after 12 h treatment the total oxygen consumption was significantly reduced. The respiration site was in complex I. Total ATP was also decreased by kaempferol after 12h in low-glucose medium but not in high-glucose one. They found this energetic failure was associated with autophagy; 12 h kaempferol treatment changed the cell phenotype and increased the autolysosomes, and colocalization of microtubule-associated protein 1 light chain 3 (LC3), lysosomes, and LC3 protein in the low glucose condition. When autophagy inhibitor wortmannin, 3-MA, was added to the cells or transfected the cells with siRNA against autophagy, there were 40% more apoptosis cells than with kaempferol treatment alone; when pretreated with kaempferol for 12-24 h, and the medium replaced with fresh medium for 48 h, kaempferol protected the cells from apoptosis. The researchers also proved that inhibition of glucose uptake is required for this process; the autophagy is through AMPK/mTOR pathway (148).

1.5.4 Angiogenesis and metastasis inhibition

Proposed by Dr. Folkman, angiogenesis has been acknowledged for its important role in cancer improvement and has attracted many researchers in recent years (166). Although malignant cancer cells have acquired the ability to keep proliferating and resist to apoptosis, like normal cells, they still need to absorb nutrients and oxygen in order to grow, or they will die. This explained why initial primary tumor cells only grew around vascular vessels and the tumor size normally was no more than 0.2 mm. To further develop, cancers cells need to get close to the blood vessel. Thus many cancer cells later develop the ability to stimulate new blood vessels to grow from existing ones; this process is called angiogenesis (167). This process involves many interactions between cancer cells, stroma cells, epithelial cells, extra cellular matrix, and a series of signaling transductions. Basically, when cancer cells are far from capillaries, they will suffer hypoxia, which will stimulate the cancer cells to release platelet-derived growth factor (PDGF) which will recruit the stroma cells (most are fibroblasts, macrophages and neutral cells). Then the stroma cells will release the angiogenic factors such as VEGF. These angiogenic proteins bind to their according receptor on endothelial cells on the blood vessel and stimulate capillary growth (151, 168). One thing to mention is even after the angiogenic factors secrete, they do not directly contact with their receptors since an extra cellular matrix (ECM) separates them. In this situation Matrix metalloproteinase (MMP) will be secreted.

After the ECM are degraded by MMP, the angiogenic switch will be turned on, and

angiogenesis can happen successfully(169). Considering that the cancer cells can

always mutate to resist the new drugs which target the cancer cells directly,

angiogenesis avoids this problem. The new treatment can work on the stroma cells instead of cancer cells to interrupt the angiogenesis. Besides, since it is a chronic way for the angiogenesis to happen, the daily consumption of phytochemicals which naturally exist in fruits and vegetables can have a great healthy benefit towards this.

Although there are not many studies about the antiangiogenesis ability of

kaempferol, several studies indicate kaempferol has such potential. One study found

10 µM kaempferol can significantly reduce the VEGF induced angiogenesis in the

Human Umbilical Vein Endothelial Cells (HUVEC) by 15% (170). Another study

used kaempferol to treat OVCAR-3 and A2780/CP70 human ovarian cancer cells. It

was found that in both cells kaempferol significantly reduced the mRNA and protein

levels of VEGF; kaempferol also reduced blood vessel formation in the Chicken

Embryo assay; in additional tests, HIF-α, p-AKT protein levels and ESRRA mRNA

levels were decreased, while HIF-β, ERK protein level and PRARGC1A mRNA levels

did not change. The results suggest that kaempferol inhibits angiogenesis and VEGF

expression through HIF and ESRRA pathway (140). The same group did another

study later to identify an exclusive pathway for kaempferol’s angiogenesis inhibition

effect on ovarian cancer. In this study they found kaempferol can inhibit the OVCAR-3

and A2780/CP70 cells VEGF secretion time dependently. It can also inhibit the

culture medium induction of tubes formation by HUVEC cells. They also found that

kaempferol suppressed the VEGF secretion and angiogenesis by down-regulating phos-ERK, NF kappa B, cMyc level and up-regulating p21 level. The pathway was confirmed by plasmid transfect; they found the signal was correlated and could not be

reversed (140). Kaempferol was also found to suppress the HGF induced

medulloblastoma cell migration. 20µM kaempferol clearly decreased expression of

p-Met and p-Akt induced by HGF; pretreatment by 20 µM kaempferol also inhibited

the medulloblastoma cell migration and morphological changes induced by HGF

(171). Besides, kaempferol inhibited the MMP-3 secretion in MDA-MB-231 human

breast cancer cells in a dose- dependent manner; the IC50 is 45 µM. It also inhibited

the in vitro invasion of MDA-MB-231 cells in a dose-dependent manner; the IC50 is 30

µM (172).

1.5.5 Estrogen modulating activity

Estrogen has been considered to play an important role in cancer growth,

especially for these estrogen dependent cancers like breast cancer and ovarian

cancer. Many years ago researchers found that large doses of estrogen can induce different cancers in animal and human (173-175). They also found that estrogen can enhance the effects of other carcinogens (176, 177). Later the mechanism of estrogen involvement in the carcinogenesis process has been made clearer.

Estrogen can bind to ER and form complexes, and then it bind to a specific sequence

called an estrogen response element (ERE). ERE will induce gene expression and

stimulate the synthesis of effector proteins which promote mitotic cascade to increase

cell division and cancer risk(178). Currently two major ERs are ERα and ERβ.

Different cells may contain different ERs, which provide a target for modulating the estrogen-estrogen receptor pathway by some compounds called selective ER

modulators (SERMs). Commonly used SERMs today are Aromatase Inhibitors, the

antiestrogen such as tamoxifen and raloxifene, ER agonists (179). Although the

SERMs have efficiently reduced some cancer risks, there cause effects, including

discomfort or drug resistance. , Thus a better SERM is needed. Kaempferol is one of

the most widely distributed natural phytoestrogens found to possess such potential in

many studies.

Aromatase is an important enzyme in the process that converts androgens

into estrogens; inhibition of aromatase is considered an efficient way to treat estrogen

related cancers. In a study kaempferol has been found to decrease aromatase

enzyme activity in MCF-7 human breast cancer cells; the IC50 is 61 µM and Ki is

27.2 µM (180). Researchers used different concentrations of kaempferol to treat

MCF-7 human breast cancer cells; kaempferol inhibited the ER-α mRNA and protein

levels, and increased the ER-α protein aggregation in nuclei. It also decreased the

progesterone receptor, cyclin D1, and insulin receptor substrate level. Proteasome

and lysosome inhibitor did not completely block the inhibition effect of kaempferol,

suggesting another pathway is involved. Kaempferol also inhibited the MCF-7 human

breast cancer cell proliferation induced by estradiol, suggesting that kaempferol's

antiproliferation effects in vitro is partly through modulation of ER-α (181). Kaempferol

also showed biphasic effects on estrogenicity in MCF-7 human breast cancer cells.

When kaempferol was lower than 10 µM, it alone increases the ERE expression in a

dose-response manner; when kaempferol concentration was more than 10 µM it

-8 began to decrease the ERE expression. With 10 µM E2 and kaempferol combination,

kaempferol significantly inhibited the expression of E2 induced ERE and downstream

protein pS2 in a dose-response manner. The inhibition effect is similar to tamoxifen.

-8 When the cells were treated with both 10 µM E2 and kaempferol, E2 induced cell

proliferation was completely inhibited. When kaempferol concentration was less than

10 µM, this effect was offset in a dose response manner as E2 concentration

increased to 10-4µM. More than 10 µM kaempferol did not have an effect. This

indicates the antiproliferation effect of kaempferol is estrogen dependent when its

concentration is lower than 10 µM; additionally, 10 µM kaempferol also inhibited

malignant transformation in MCF-7 human breast cancer cells caused by estrogen

response by focus assay (47). Some researchers studied the correlation between

kaempferol's estrogen activities and apoptosis. They found that kaempferol

possesses much weaker estrogen binding affinity and could not inhibit the estradiol

induced ER activation in HepG2 cells. But kaempferol can induce ERE expression in

MCF-7 cells. Although kaempferol can up-regulate the Bcl-2 protein level and induce

apoptosis, there was no clear relationship between kaempferol estrogencity and

apoptosis (182).

1.5.6 Anti-inflammation

Inflammation has been found to play several roles in different carcinogenesis

processes. Initially, inflammation can trigger ROS stress which will damage DNA;

then inflammation also induces the secretion of growth factors including EGF and

FGF. These growth factors can promote cancer cell proliferation and help cancer

cells resist apoptosis. Furthermore, the pro-inflammatory cytokines, such as TNF-α

and Interleukins, produced in inflammation can stimulate the survival pathways for

cancer cells, and help cancer cells avoid the apoptosis(183). Thus targeting inflammation will be a good way to inhibit carcinogenesis and inhibit cancer growth.

Kaempferol has been found to exhibit different anti-inflammation effects in different studies.

Cyclooxygenase-2 (COX-2) is found highly expressed in many cancers.

COX-2 can convert arachidonic acid (AA) to Prostaglandin E2 (PGE2), which has been shown to promote carcinogenesis in different cancers(183). In a study, treating

LNCaP human prostate cancer cells with 1 µM kaempferol significantly reduced the

PGE2 level; after the cells were treated with 0.1µM or 1 µM kaempferol for 24 h, its mRNA level for COX-2 and its down regulator PPARγ were decreased, while the protein level was not significantly changed (184). Kaempferol was also found to inhibit the COX-2 protein and mRNA level in dose-dependent manners in Chang Liver cells; moreover, kaempferol also inhibited the NF-κB activation and expression of IKKα and

IκBα proteins, suggesting that the anti-inflammation effect is through NF-κB pathway

(185). In another study, researchers treated JB6 P+ mouse epidermal cells with 40

µM kaempferol for 30 minutes. Kaempferol significantly reduced the UVB-induced

COX-2 protein ERKS, p38, and JUNKs levels; it inhibited COX-2 and AP-1 activation.

Src is a upstream activator for MAPKs. Kaempferol inhibited expression of Src in mouse skin while not in vitro; they also found that kaempferol worked by competing with ATP for Src binding (186). Kaempferol was also found to have the best activity of inhibiting the fatty acid amide hydrolase among 16 common flavonoids; fatty acid amide hydrolase inhibition is associated with suppression of PPARγ expression

(187).

Kaempferol also exhibited different inhibitory effects on pro-inflammatory cytokines. 30 µM kaempferol successfully decreased the IL-1 β and INF-α mRNA levels which were induced by LPS in J774.2 macrophage cells (188). 40 µM kaempferol inhibited IL-4 induced STAT 6 phosphorylation and CD23 expression significantly; it also increased apoptosis which is suppressed by IL-4 first. Kaempferol inhibited the JAK3 level, but not IL-4 induced Src or JAK1 activity. Furthermore, the researchers confirmed that kaempferol only showed this inhibition effect in JAK3 expressing cells and JAK3 dependent cytokines (189). In another study, kaempferol inhibited the IL-8 promoter activation and gene expression induced by TNF-α in HEK

293 cells in a dose-response manner; it also decreased the TNF-α induced IκBα, phospho-IκBα protein levels, p65 nuclear localization, and ROS production without affecting the viability of the cells(190). Additionally, kaempferol was found to inhibit

TNF-α induced nuclear hormone receptor liver-X-receptor alpha mRNA expression

(191). Other researchers found that kaempferol increased cytokine

Granulocyte-macrophage colony-stimulating factor (GM-CSF) level. The process was

through ER/Golgi, PLC, PKC, and MEK/ERK pathways (192).

Kaempferol also showed anti-inflammation effects in vivo. Female C58BL/6J

mice were fed with 0.1 and 0.3% kaempferol for three weeks, and 2% dextran sulfate

sodium (DSS) was used as an inducing agent. Kaempferol significantly decreased

the DSS induced plasma NO and PGE2 concentrations, COX-2 and iNOS mRNA

level, and TNF-α, IL-1β, IL-6 mRNA (193).

1.5.7 Drug effect improvement

In the middle of last century, scientists found that certain chemicals can kill

tumors while having limited damage to normal cells. Since then, many chemotherapy

drugs have been developed, some of which have been effective in treating cancer.

However, one large challenge for these drugs is that cancer cells easily develop

resistance. Multidrug resistance (MDR) has greatly reduced drug effectiveness.

Finding a safe compound to reduce this resistance is critical. Kaempferol in many

studies has been found to decrease this resistance in different cancer cells. Adding

kaempferol with regular drugs can also increase the sensitivity of cancer to drugs.

P-glycoprotein (Pgp) is an ATP-dependent drug efflux pump that will pump

many foreign substances out of cells. High levels of Pgp are responsible for the low

efficacy of chemotherapy drugs. Kaempferol has been found to decrease Pgp level in

several cancer cell lines. In one study researchers treated MDR human cervical carcinoma KB-V1 cells (high Pgp expression) with several flavonoids. Kaempferol showed the best effect. 10 and 30 µM kaempferol significantly increased cell sensitivity and decreased relative resistance of KB-V1 cells to vinblastine and paclitaxel, two common anti-cancer drugs. Meanwhile it did not show such effect in drug-sensitive KB-3-1 cells. Furthermore, kaempferol increased accumulation and decreased efflux of Rh123 and 3[H]vinblastine in KB-V1 cells but not in KB-3-1 cells;

48 h treatment with kaempferol significantly decreased Pgp protein expression in

KB-V1 cells (194). Two kaempferol derivatives, kaempferol-3-O-methyl ether (K3O) and kaempferol-3,4'-O-dimethyl ether (K34O) from Zingiberzerumber showed similar effects. They increased the accumulation and decreased the efflux of

[3H}-daunomycin in Pgp overexpressed MCF-7 cells. The effect is as good as the effect of common Pgp inhibitor verapamil (195). In vivo kaempferol also improved the drug efficacy; the researchers gave mice tamoxifen both intravenously and orally. At the same time they fed them with or without kaempferol; the absolute oral bioavailability and the maximum plasma concentration of tamoxifen were significantly higher in the kaempferol fed group. There were no other changes or differences among different concentrations (196).

Kaempferol can also improve the sensitivity of cancer cells to treatment and improve the drug effect. Tumor necrosis factor-related apoptosis inducing ligand

(TRAIL/Apo2L) is an efficient drug for cancer treatment; however resistance often will

develop. Kaempferol significantly increased the inhibitory effect of TRAIL on cell viability in U252 and U87 glioma cells but not in U373 cells. Kaempferol also improved the sensitivity of NCH89 and NCH149 glioblastoma cultures. Combination of TRAIL and kaempferol significantly increased the level of caspase-9, caspase-7, and PARP levels in U87 and U251 cells. Kaempferol can inhibit the surviving and

XIAP expression in U251 and U87 cells. Because overexpression of surviving attenuated the effect of TRIAL and kaempferol combination treatment on cell

cytotoxicity in U251 cells, it indicated that kaempferol enhanced the TRIAL efficacy by

suppressing the surviving. Furthermore, the researchers also confirmed the process

is through proteasome degradation and Akt activity inhibition (197). Another study

also found that combination of kaempferol and TRAIL significantly increased the

apoptosis induction and cleaved PARP compared to treating colon cancer SW480

cells with TRAIL or kaempferol alone. The apoptosis was through caspase and TRAIL

receptor. Kaempferol also increased the level of TRAIL receptors DR5 and DR4.

Transfecting the cells with DR5 siRNA not DR4 siRNA blocked the sensitizing effect,

indicating that the combination effect is through DR5. Furthermore, the combination

also showed similar effect in colon cancer DLD-1 and prostate cancer PC3 cells,

while it has low toxicity in peripheral blood mononuclear normal cells (198). Among 10

chemicals, kaempferol showed the best effect to increase OVCAR-3 ovarian cancer

cells sensitive to cisplatin. The combination of cisplatin and kaempferol significantly

reduced cell viability, ABCC6, cMyc mRNA levels, and increased CDKN1A mRNA

level, caspase-3 protein levels compared to cisplatin alone(199). In addition,

kaempferol increased the antiproliferation effect of 5-fluorouracil in pancreatic cancer

cells and the quercetin bioavailability by inhibiting Abcg2 protein (200).

1.5.8 Other anti-carcinogenesis mechanism

There are also other anti-carcinogenesis effects by kaempferol. The aryl

hydrocarbon receptor (AHR) is a ligand-activated transcription factor. It regulates the

Cytochrome P450, family 1, member A1 (CYP1A1) enzyme which is involved in

metabolically activating and detoxifying. Activation of the AHR has been associated

with cell proliferation and tumor formation(201, 202). Kaempferol was found to inhibit

AHR and CYP1A1 expression in different studies. Among 15 flavonoids, kaempferol

showed the strongest inhibitory effect on AHR activated reporter activity in MCF-7

cells; it also inhibited the formation of AHR/ARNT DNA-binding complex and

increased the CYP1A1 expression (203). Kaempferol inhibited dioxin-induced

CYP1A1 and CYP1B1 expression, and this process is independent of ER pathway

(204). However, the kaempferol inhibition on AHR is controversial; in another study,

although kaempferol inhibited dioxin induced CYP1A1 level in Rl95-2 endometrial

cancer cells, the AHR was not significantly affected (205). Kaempferol was found to reduce the viability of human hepatoma cancer cells. The effect is through inhibiting the HIF-1α activity by decreasing the accumulation of HIF-1α in the nucleus. It also

inhibited the p44/p42 MAPK activations (206). Kaempferol can inhibit the

β-catenin/Tcf binding which is found in human carcinogenesis; kaempferol inhibited

the Tcf complex binding to DNA and decreased the c-Myc, AXIN 2, and Cyclin D1 protein levels in β-catenin activated HEK293 cells(207) . A kaempherolglycoside with an acetyl group was found to inhibit the Topoisomerases, of which the inhibitor was considered to be an anti-cancer agent (208).

1.6 Overall objectives, significance, and future implications

1.6.1 Rationale

Asparagus is a nutritious vegetable; it is rich in many nutrients, such as folic

acids, B vitamins, vitamin K and minerals. It also contains many phytochemicals,

including flavonoids, saponins and polysaccharides. Studies have found that

asparagus showed strong antioxidant activity and anticancer properties (119, 209).

However, since there are many different varieties of asparagus, health benefits may

differ according to variety. Thus, comparing different varieties of asparagus for their

antioxidant and anticancer activities will provide useful information for consumers.

MDA-MB-231 human breast cancer cells are triple-negative cancer cells; this

means that they lack receptors for estrogen, progesterone, and HER-2 epidermal

growth factor. They account for 15% of all types of breast cancer (210).

MDA-MB-231 human breast cancer cells do not have the specific hormone target;

hence they are less sensitive to the traditional hormone chemotherapy. They are

characterized by an aggressive clinical history with poor disease-free and overall

survival (211). Currently there is very little data available about prevention strategies

for the occurrence and reoccurrence of this disease. It is found that MDA-MB-231 50

human breast cancer cells more easily metastasize to the cerebrum and visceral

sites, compared with other types of breast cancers in patients (212). Thus, targeting

their migration and metastasis may be a potential way to improve treatment.

Kaempferol, a widely consumed flavonoid, has been shown to possess such

potential; so studying kaempferol’s effects on MDA-MB-231 human breast cancer

cells many help improve patients’ survival rate and quality of life.

Most of the human breast cancers are still hormone sensitive, and MCF-7

human breast cancer cells belong to this group. Because it is hormone sensitive,

estrogen will promote MCF-7 proliferation. Estrogen will bind to estrogen receptor

and activate expression of a series of proliferation signaling proteins(178).

Kaempferol is one of the most common phytoestrogens found in fruits and

vegetables; it can compete with estrogen to bind with estrogen receptors to

suppress the proliferation activities of MCF-7 human breast cancer cells (181). It is

important to study the effects of kaempferol on MCF-7 proliferation and the related

signaling proteins. The findings will help us better understand the phytochemicals’

effects in prevention of breast cancer and the mechanisms through which they are

working.

1.6.2 Hypotheses.

Hypothesis 1: there are differences in the phenolic content, flavonoids content, antioxidant activities, and anticancer activities among the seven varieties of asparagus. 51

Hypothesis 2: Kaempferol inhibits proliferation, migration and metastasis of

MDA-MB-231 human breast cancer cells at proposed concentrations without cytotoxicity.

Hypothesis 3: Kaempferol inhibits cell proliferation of MCF-7 Human Breast

Cancer Cells through suppressing the expression of estrogen receptor alpha.

1.6.3 Objectives

I. To compare antioxidant and anticancer activities of seven varieties of

asparagus

II. To determine anti-proliferative activity and cytotoxicity of kaempferol toward

MDA-MB-231 human breast cancer cells, and to determine the effect of kaempferol

on migration and metastasis in MDA-MB-231 human breast cancer cells.

III. To determine anti-proliferative activity and cytotoxicity of kaempferol toward

MCF-7 human breast cancer cells, and to determine the mechanisms of action of

kaempferol in inhibiting cell proliferation in MCF-7 human breast cancer cells.

1.6.4 Implications and future research

In this study, we demonstrated that different varieties of asparagus have

different antioxidant and anticancer activities, and the Apollo and Purple Passion

asparagus showed the strongest effects. We also show that kaempferol has potential

to inhibit angiogenesis and metastasis in MDA-MB-231 human breast cancer cells. In addition, it also exhibited anti-cancer activity through inhibiting cell proliferation of

MCF-7 breast cancer cells by suppressing the expression of estrogen-receptor alpha.

These data are important in understanding the protective effects of fruits and vegetables in the prevention of breast cancer. To better evaluate its health benefits in human, an animal study should be pursued as the next step.

Chapter Two Phytochemical Profiles, antioxidant activity and

anti-proliferative activity of seven varieties of asparagus

2.1 Introduction

Cardiovascular disease (CVD) and cancer are the top two major causes of death in the United States. In 2010 they accounted for 47 % of all deaths (1).

Among many prevention strategies, vegetables and fruits consumption is one of the most effective. Epidemiology studies have found that vegetable and fruit consumption is inversely correlated with the reduced risks of both diseases (10, 213).

Phytochemicals are defined as a group of bioactive, non-nutrient compounds found in vegetables, fruits, grains and other plants (12). Phytochemicals in fruits and vegetables may be responsible for their chronic disease reducing effect (12).

Vegetables and fruits contain many phytochemicals such as phenolics and flavonoids.

Some of them can work as antioxidants to prevent CVD by scavenging free radicals.

Furthermore, they can also prevent CVD by inhibiting LDL oxidation and regulating lipid profiles (214). Phytochemicals in vegetables and fruits also showed anticancer activities by different mechanisms. The phenolics and flavonoids in fruits and vegetables have been found to inhibit cancer growth by inducing cell cycle arrest, inducing apoptosis, inhibiting angiogenesis, regulating hormone interactions, inhibiting inflammation, and regulating different cell signaling pathways in cancer (117,

215, 216). The U.S. Department of Agriculture recommends that people consume at

least two and half cups of vegetables and fruits each day to reduce the risk of developing chronic diseases (217).

Asparagus is a popular vegetable consumed in the US. It is one of the most nutritious vegetables, and contains many B vitamins, vitamin K, and iron. It is also rich in phytochemicals, including saponins, rutin, kaempferol and other flavonoids.

Studies have found that asparagus has various health benefits, including neuroprotective, antioxidant, hypolipidaemic, hypoglycaemic, hepatoprotective, and anti-tumorigenic activities. Different studies have found that it is one of the vegetables with highest antioxidant activities in US (4, 105, 218). Besides, asparagus extract can also inhibit different cancer cell growth(118). However, there are many different varieties of asparagus. Just by color, there are green asparagus, white asparagus, and purple asparagus. Moreover, there are many subtypes under each color of asparagus. Consumers may be confused about which variety of asparagus has more health benefits. Currently there are few studies about the phytochemical profiles and health benefits of different varieties of asparagus. Hence the objective of this study was to compare phytochemicals profiles, antioxidant activities, and anticancer activities among different asparagus varieties.

2.2 Materials and Methods

2.2.1 Chemicals and materials

Ethanol (EtOH, anhydrous, 100%), potassium hydroxide (KOH), sodium

borohydride (NaBH4), aluminum chloride, chloranil, tetrahydrofuran (THF), quercetin

dehydrate, catechin hydrate, vanillin, Folin-Ciocalteu reagent, and dichlorofluorescin

diacetate (DCFH-DA) were obtained from Sigma Chemical Co. (St. Louis, MO).

Sodium hydroxide (NaOH), potassium dihydrogen phosphate (KH2PO4), potassium

phosphate, methanol, alcohol, acetone, acetic acid, hydrochloric acid (HCl) and

dipotassium hydrogen phosphate (K2HPO4) were purchased from J.T. Baker

(Phillipsburg, NJ). Dimethyl sulfoxide and sodium bicarbonate (NaHCO3) were

purchased from Fisher Scientific (Pittsburgh, PA). Gallic acid was purchased from

ICN Biomedical Inc. (Costa Mesa, CA). 2, 2'-azobis-amidinopropane (ABAP) was purchased from Wako chemicals (Richmond, VA). All reagents used were of analytical grade. HepG2 human liver cancer cells were obtained from the American

Type Culture Collection (ATCC) (Rockville, MD). Williams’ Medium E (WME) and

Hanks’ Balanced Salt Solution (HBSS) were purchased from Gibco Life Technologies

(Grand Island, NY). Fetal bovine serum (FBS) was obtained from Atlanta Biologicals

(Lawrenceville, GA).

2.2.2 Asparagus samples and sample Preparation

Seven asparagus varieties were provided by Hebei Qimei Agriculture Science

Technology Co, Ltd. All of the asparagus varieties were available on the market. The

7 varieties are Apollo, Purple Passion, UC800, Crown, Grande, Green, Harvest, and

Atlas. All the samples were harvested in 2009. Each sample was weighed 100 g fresh

weight in triplicate; then they were put in individual bags and processed by freeze drying. Samples were stored at -80 ºC until use.

2.2.3 Phytochemical extractions of asparagus

Phytochemicals of asparagus were extracted using the method as reported previously by our lab (219). Briefly, 100 g fresh asparagus equivalent freeze dry samples were blended for 5 min in a Waring blender using 200 mL chilled 80% acetone at medium speed. The mixture was then homogenized in a Polytron homogenizer for 3 min and filtered with vacuum under an ice bath. Supernatants were collected and then another 20 mL chilled 80% acetone was added to the residues and the extraction was repeated three times. The acetone in the supernatants was evaporated using a rotary evaporator under vacuum at 45°C until the weight of the supernatants was less than 10% of the original weight. The free phytochemical extracts were brought to 100 mL in water and were kept at -40°C until analysis. All extractions were performed in triplicate.

2.2.4 Determination of total phenolic contents

The total phenolic contents of asparagus were determined by the

Folin-Ciocalteu colorimetric method (220), and modified in our laboratory (221). All extracts were diluted with Milli-Q water to obtain readings that fall within the standard curve range of 0.0 to 600.0 µg gallic acid/mL. Briefly, for each analysis,

Folin-Ciocalteu reagent (0.1 mL) was added to the diluted extract solution and

allowed to react for 6 minutes to ensure that the Folin-Ciocalteu reagent reacted completely with the oxidizable phenolates in the sample. Then, 1 mL of 7% sodium carbonate solution was added to neutralize the samples. The samples were mixed and allowed to stand for 90 minutes at room temperature. After the color was developed, the absorbance of the results was read at 760 nm using a MRX II Dynex spectrophotometer (Dynex Technologies, Inc., Chantilly, VA). Results were compared with the standard curve of gallic acid concentrations and expressed as milligram per 100 gram of fresh weight for triplicate. Data were reported as mean ±

SD.

2.2.5 Determination of total flavonoids

The total flavonoid contents of asparagus were determined using the sodium borohydride/chloranil protocol (SBC) developed in our laboratory (72, 222). Briefly, 1 mL phytochemical extracts of tested samples were added into test tubes (15 x 150 mm), solvent evaporated to dryness under nitrogen gas, and reconstituted in 1 mL of terahydrofuran/ethanol (THF/EtOH, 1:1, v/v). Catechin standard (0.3-10.0 mM) was prepared fresh in 1 mL of THF/EtOH (1:1, v/v). Then, 0.5 mL of 50 mM NaBH4 solution and 0.5 mL of 74.6 mM AlCl3 solution were added into each test tube with sample or standard. Then the test tubes were shaken in an orbital shaker at room temperature for 30 minutes. An additional 0.5 mL of 50.0 mM NaBH4 solution was added into each test tube with continued shaking for another 30 minutes at room temperature. Then, 2.0 mL of 0.8 M Chilled acetic acid solution was added into each

test tube and kept in the dark for 15 min after a thorough mixing. Then, 1 mL 20.0 mM chloranil was added in each tube and heated at 95 ˚C with shaking for 60 min. The reaction solutions were cooled using tap water, and the final volume was brought to 4

mL using methanol. Then, 1 mL 16% (w/v) vanillin was added into each tube and

mixed. Then 2 mL 12 M HCl was added into each tube and kept in the dark for 15 min

after mixing thoroughly. The reaction solutions were centrifuged at 1400×g for 5

minutes, and 200 μL of each solution were added into a 96-well plate in duplicate and

the absorbance was measured at 490 nm using a MRX Microplate Reader with

Revelation workstation (Dybex Technologies, Inc., Chantilly, VA). Results were

calculated by using the standard curve of catechin hydrate concentration. Total

flavonoid content was expressed as milligram per 100 gram of fresh weight of sample.

Data were reported as mean ± SD for at least triplicates.

2.2.6 Determination of Total Antioxidant Activity

Total antioxidant activity was measured using the hydrophilic peroxyl radical

scavenging capacity (Hydro-PSC) assay as reported previously (223). 75 mM

phosphate buffer (pH 7.4) was used to dilute Ascorbic acid and phytochemical

extracts in appropriate concentration. Ascorbic acid was made fresh and diluted to

6.3, 4.8, 3.2, 2.4 and 1.0 μg/mL. Gallic acid was made fresh and diluted to 5.5, 3.5,

2.7, 1.4, 0.9 μg/mL. The reaction mix contained 75 mM phosphate buffer (pH 7.4), 40

mM ABAP, 13.26 μM DCFH dye, and the appropriate concentrations of the pure

antioxidant compound or sample extracts. 1 mM KOH was used to prehydrolyze the

dye to remove the diacetate moiety just prior to use in the reaction, which was carried out at 37°C, in a total volume of 250 µL using a 96-well plate. Fluorescence generation was monitored (excitation at 485 nm and emission at 538 nm) with a

Fluoroskan Ascent fluorescent spectrophotometer (Thermo Lab systems, Franklin,

MA). Data were acquired with the Ascent Software, version 2.6 (Thermo Lab systems,

Franklin, MA) running on a PC. The areas under the fluorescence reaction time kinetic curve (AUC) for both control and samples were integrated and used as the basis for calculating peroxyl radical scavenging capacity (PSC) using the equation

PSC (value) = 1 - (SA/CA), where SA is AUC for the sample or standard dilution and

CA is AUC for the control reaction. Compounds or extracts inhibiting the oxidation of

DCFH produced smaller SA and higher PSC values. The parameter EC50 was defined as the dose required to cause a 50% inhibition (PSC unit = 0.5) for each pure compound or sample extract, and was used as the basis for comparing the antioxidant activities of different compounds or samples. Results obtained for antioxidant activities of sample extracts were expressed as μmol of vitamin C equiv./100g of fresh sample ± SD for triplicate analyses.

2.2.7 Cell Culture

HepG2 cells were grown in Complete Medium (WME supplemented with 5%

FBS, 10 mM Hepes, 2 mM Lglutamine, 5 μg/mL insulin, 0.05 μg/mL hydrocortisone,

50 units/mL penicillin, 50 μg/mL streptomycin, and 100 μg/mL gentamycin) and were

maintained at 37 °C and 5% CO2 as described previously (215). Cells used in this

study were between passages 10 and 38.

2.2.8 Cytotoxicity and Inhibition of Proliferation Assays

The cytotoxicity against HepG2 human liver cancer cells was determined by

the method developed in our laboratory (215). HepG2 cells in 100μL growth media were placed in each well of a 96-well flat-bottom plate at a density of 4.0 × 104

cells/well. After 24 h of incubation with 37 °C and 5% CO2, the growth medium was

removed, each well washed with 100 μL of PBS, and replaced by media containing

different concentrations of sample tested. Control cultures received the extraction

solution minus the extracts, and blank wells contained 100 μL of growth medium with

no cells. After another 24 h of incubation, cytotoxicity was determined by the

methylene blue assay (224). Cytotoxicity was measured as percentage compared to

control. The absorbance at 570 nm was read by using a MRX Microplate Reader.

More than 10% cells number reduction was considered to be cytotoxic. A minimum of

three replications for each sample was used to determine the cytotoxicity.

Antiproliferative activity toward HepG2 human liver cancer cells was

determined using the method reported previously (225). Briefly, HepG2 cells in 100 μL

growth media were placed in 96-well plate at a density of 2.5 × 104 cells/well. After 4 h

of incubation at 37 °C with 5% CO2, the growth medium was replaced by media

containing different concentrations of extract. Control cultures received the extraction solution minus the sample extracts, and blank wells contained 100 μL of growth 61

medium with no cells. After 72 h of incubation, cell proliferation was determined by

the methylene blue assay at absorbance of 570 nm (224). Cell proliferation (percent)

was measured as percentage compared to control. All measurements were

conducted in triplicate.

2.2.9 Cellular Antioxidant Activity (CAA)

The CAA assay was performed using the protocol described previously by

our laboratory (143, 144, 226). Briefly, HepG2 human liver cancer cells were seeded

at a density of 6 × 104/well in a 96-well microplate in 100 μL of complete medium per

well in a humidified 5% CO2 incubator at 37 °C. Twenty-four hours after seeding, the

growth medium was removed, and the wells were washed with 100 μL of PBS. Wells

were then treated in triplicate with 100 μL of treatment medium containing solvent

control, control extracts, or tested extracts plus 25 μM DCFH-DA for 1 h. Then certain wells were washed with 100 μL of PBS (i.e., PBS wash protocol) and certain wells were not washed (i.e., no PBS wash protocol). PBS wash protocol means that before the ABAP is added cells are pretreated with asparagus extract; while no PBS wash means that cells are treated with asparagus extracts and ABAP together. After 600

μM ABAP was applied to the cells in 100 μL of oxidant treatment medium (HBSS with

10 mM Hepes), the 96-well microplate was placed into a Fluoroskan Ascent FL plate reader at 37 °C. Emission at 538 nm was measured after excitation at 485 nm every 5 min for 1 h.

After blank subtraction and subtraction of the initial fluorescence values, the area

under the curve for fluorescence versus time was integrated to calculate the CAA value at each concentration of extract as follows:

CAA unit = 100 − (∫ SA/∫ CA) × 100

where ∫ SA is the integrated area under the sample fluorescence versus time

curve, and ∫ CA is the integrated area from the control curve. The median effective

dose (EC50) was determined for the sample extracts from the median effect plot of log

( fa/fu) versus log(dose), where fa is the fraction affected (CAA unit), and fu is the

fraction unaffected (1-CAA unit) by the treatment. The EC50 values were stated as the mean ± SD for triplicate sets of data obtained from the same experiment. EC50 values

were converted to CAA values, which are expressed as micromoles of quercetin

equivalents (QE) per 100 g of FW, using the mean EC50 value for quercetin from at

least four separate experiments.

2.2.10 Statistical Analysis

Statistical analyses were conducted using SigmaPlot Version 11.0 (Systat

Software, Inc., Chicago, IL) and dose-effect analysis was performed using Calcusyn

software version 2.0 (Biosfot, Cambridge, U.K.). Results were subjected to ANOVA

and differences between means were located using Tukey’s multiple comparison test.

Significance was determined at p < 0.05. All data were reported as the mean ± SD for

the three replications.

2.3 Results

2.3.1 Total Phenolic Content

Total phenolic contents of different varieties of asparagus are shown in figure

3, expressed as milligrams of gallic acid equivalent per 100 g fresh weight. Apollo asparagus has the highest content, which is 113.1 ± 4.5 mg GA equiv/100 g FW, while Atlas asparagus has the lowest content which is 50. 3 ± 2.7 mg GA equiv/100 g

FW. The content of other varieties are: Purple passion asparagus (103.6 ± 1.5);

UC800 asparagus (72.1± 2.8); Crown asparagus (65.5± 3.2); Grande asparagus

(63.8± 3.9); Green Harvest asparagus (59.3± 3.1); There are significant differences among different varieties of asparagus.

2.3.2 Total Flavonoid Content

Flavonoid content in samples was determined using the SBC assay is shown in figure 4. The results are expressed as milligrams of catechin equivalents per 100 g of sample on a fresh weight basis. Apollo asparagus has the highest total flavonoid content which is 75.7±5.2 mg catechin equiv/100 g FW; Atlas asparagus has the lowest flavonoid content which is 19.0±2 mg catechin equiv/100 g FW. Other asparagus flavonoids content are as following: Purple passion asparagus (63.±2.3);

UC800 asparagus (27.3± 2.1); Crown asparagus (25.4± 2); Grande asparagus (32.6±

2.8); Green Harvest asparagus (22.3± 1.6). There are significant differences among the different varieties of asparagus.

2.3.3 Total Antioxidant Activity

The total antioxidant activities, measured by PSC assay for asparagus are

presented in figure 5, expressed as μmol of vitamin C equiv/ 100 g fresh weight basis.

Apollo asparagus has the highest total antioxidant activity which is 507 ± 30.81 μmol

of vitamin C equiv/ 100 g FW. Atlas showed the lowest antioxidant activity which is

300.1 ± 7.67 μmol of vitamin C equiv/ 100 g FW. The PSC value for other asparagus are Purple passion asparagus (481.6±13.8); UC800 asparagus (352.± 7.5); Crown

asparagus (382.7± 13.5); Grande asparagus (440.7± 21.3); Green Harvest

asparagus (336.4± 5.8). There are significant differences among the different

varieties of asparagus.

2.3.4 Antiproliferative activity

The antiproliferative activities and cytotoxic activities in HepG2 human liver cancer cells by different varieties of asparagus are shown in figure 6. All varieties of asparagus significantly inhibited the proliferation of HepG2 human liver cancer cells in

a dose-dependent manner under 100 mg/mL when compared to control. Apollo

asparagus has the strongest antiproliferative activities; its EC50 is 32.5±2.4 mg/mL.

For other varieties, the EC50 values range from 33.2 ±1.5 mg/mL (Purple passion asparagus) to 53.5± 1.5 mg/mL (Atlas asparagus). The lower the EC50, the stronger is

the antiproliferative activity.

2.3.5 Cellular Antioxidant Activity

The cellular antioxidant activities of asparagus were measured by the CAA

assay (226). The EC 50 and CAA Values are listed in figure 7. The CAA values are

expressed as μmol QE/ 100 g asparagus. In the no PBS wash groups, Apollo

asparagus has the highest CAA value which is 32.1 ± 1.8 μmol of QE/100g

asparagus, and the EC50 is 84.2 ± 3.8 mg/mL; the Grande asparagus has the lowest

CAA value which is 21.7 ± 2.4 μmol of QE/100 g, the EC50 is 107.9 ± 6.1 mg/mL. In

the PBS wash group, the results showed a similar pattern; Apollo asparagus has the

highest CAA value which is 8.4 ± 0.2 μmol of QE/100g, while Grande asparagus has

the lowest CAA value which is 4.7± 0.5 mol of QE/100g.

FIGURE 3 TOTAL PHENOLIC OF VARIOUS ASPARAGUS

140

120 a b 100

80 c d d,e e 60 f

Total phenolic content (mg GA equiv./100 g, FW) g, GA equiv./100 (mg content Totalphenolic 0

Atlas Appollo UC800 Grande Champion Purple Passion Green Harvest

Figure 3. Total Phenolics of Various asparagus varieties (mean ± SD, n=3). Bars with no letters in common are significantly different (p < 0.05).

FIGURE 4 TOTAL FLAVONOID CONTENT OF VARIOUS ASPARAGUS

100

a 80

40 c d de ef f 20

Total flavonoid content (mg catechin equiv./100 g, FW) g, equiv./100 catechin (mg Totalcontent flavonoid Atlas Appollo UC800 Grande Champion Purple Passion Green Harvest

Figure 4. Total Flavonoid content of various asparagus varieties (mean ± SD, n=3).

Bars with no letters in common are significantly different (p < 0.05).

FIGURE 5 TOTAL ANTIOXIDANT ACTIVITY OF VARIOUS ASPARAGUS

600

a a 500 b

400 c d d e 300

mol vitamin C equiv./ 100 g, FW) g, 100 equiv./ C molvitamin 200 µ

100

PSC values ( PSCvalues 0

Atlas Apollo UC800 Grande Champion Purple Passion Green Harvest

Figure 5 Total antioxidant activity of various asparagus varieties (mean ± SD, n=3).

Bars with no letters in common are significantly different (p < 0.05).

120

100

Control 80 Atlas UC800 Champion Green Harvest 60 Grande Purple Passion Apollo 40 Cell Proliferation (%) Proliferation Cell

0 0 20 40 60 80 100 120 Asparagus extracts (mg/mL)

FIGURE 6 PERCENT INHIBITION OF HEPG2 CELL PROLIFERATION AND CYTOTOXICITY

140

120

100

Control 80 Atlas UC800 60 Champion Green Harvest Grande 40 Purple Passion

Cell Cytotoxicity (%) CellCytotoxicity Apollo 20

-20 0 20 40 60 80 100 120 Asparagus extracts (mg/mL)

Figure 6. Inhibition of cell proliferation (A) and cytotoxicity (B) of HepG2 human liver cancer cells by asparagus extracts of the seven varieties (mean ± SD, n =3)

40 A a

30 b mol b µ b b b b

10 Cellular antioxidant activity ( activity antioxidant Cellular quercetin equivalents/100 g asparagus) g equivalents/100 quercetin

Atlas Apollo UC800 Grande Champion Purple Passion Green Harvest

FIGURE 7 CAA VALUES OF SEVEN VARIETIES ASPARAGUS

a B

8 b mol/ µ bc d 6 d bcd d

2 Cellular antioxidant activity ( activity antioxidant Cellular asparagus) g equivalents/100 quercetin

Atlas Apollo UC800 Grande Champion Purple Passion Green Harvest

Figure 7. CAA values of seven varieties Asparagus without PBS wash protocol (A) and with PBS wash protocol (B) (mean±SD, n=3). Bars with no letters in common are significantly different (p < 0.05).

2.4 Discussion

Epidemiology studies have found that diet is closely correlated with chronic diseases risks(227). Specifically, researchers reported that vegetables and fruits consumption is inversely associated with the reduced risk of different chronic diseases such as CVD, cancer, diabetes, obesity, and Alzheimer disease(228, 229).

The 2010 Dietary Guidelines for Americans recommend that people should eat two and half cups of fruits and vegetables each day to prevent the chronic diseases (230).

Different reasons explain the health benefits of fruits and vegetables. One is that fruits and vegetables contain phytochemicals. Phytochemicals are defined as bioactive nonnutrient compounds found in fruits and vegetables associated with reduction of chronic diseases(12). Many phytochemicals are antioxidants. They can reduce free radicals, which damage cell membranes and DNA, and thus prevent cancer. The antioxidants in fruits and vegetables will prevent cancer and chronic disease by reducing free radicals.

Asparagus has been found to contain many nutrients and phytochemicals, such as Vitamin C, Vitamin A, Vitamin E, ferulic acid, protodioscin, and kaempferol(119). Different studies have found that asparagus can prevent cancer, protect neuro function, and reduce CVD(105, 114); however, for different varieties of asparagus, which variety has the best health benefits is unknown, hence in this study we tried to determine the differences of phytochemicals content, antioxidant activities, and anticancer activities among 7 varieties of asparagus.

2.4.1 Phenolic content

Phenolics are produced in the secondary metabolism process in plants; they are important for normal human development and when responding to stress conditions. Different studies have found that phenolic consumption can reduce risks of chronic diseases, including CVD, cancer, and neurodegeneration (231). Phenolics have played an important role in the health benefits of fruits and vegetables (222,

232). Asparagus has been reported to contain high amounts of phenolics, around 80 mg GA equiv/ 100 g FW (143). In this study, the total phenolic ranges from 50.3 mg

GA equiv/ 100g FW to 113.1 mg GA equiv/100 g FW, so the results are consistent with the previous finding. The study found that green asparagus Apollo and purple asparagus Purple Passion have the highest phenolic content, while white asparagus

Atlas has the lowest phenolic content. These results are similar with the previous finding. Japanese researcher Maeda studied polyphenol concentration of the extracts of green, purple, and white asparagus, and found a similar pattern (233).

Purple asparagus contains many phenolic perhaps because it is rich in anthocyanin.

White asparagus grows under the soil. Without exposure to light, the phenolics are apparently lower than other varieties.

2.4.2 Flavonoids content

Flavonoids are the most widely distributed group of phenolics. Flavonoids have played an important role in the health benefits of phenolics. In different biological and epidemiology studies, flavonoids have been found to possess properties of anticancer, antiinflammation, antiallergy, CVD reducing, and antioxidant activities (234, 235). Asparagus contains many flavonoids, such as rutin and kaempferol. In this study, a new method called SBC developed in our lab was used to evaluate the total flavonoid content in asparagus. Previously, the flavonoid content was tested by aluminum chloride (AlCl3) colorimetric assay or high performance liquid chromatography (HPLC)(216). However, both of these methods have limitations. AlCl3 assay can only measure certain types of flavonoids, so it cannot reflect the total flavonoids level; HPLC can accurately measure the flavonoids, but it can only test individual flavonoid by adding different standard, so it is time consuming and costly. And sometimes there are unknown flavonoids in the samples. The SBC assay can overcome these limitations. It can detect all kinds of flavonoids, including flavones, flavonols, flavanones, flavanols, isoflavonoids, and anthocyanin’s (72).

According to our study, the total flavonoid content is highest in Apollo asparagus

(75.79 mg catechin equiv/ 100g FW) and Purple Passion asparagus (63.05 mg catechin equiv/100 g FW), while lowest in Atlas asparagus (19.01 mg catechin equiv/100 g FW). The pattern is similar with the phenolic content.

2.4.3 Total antioxidant activity

One of the major health benefits from phytochemicals is that they have antioxidant activity. In the human body, free radicals oxidation can cause a series of chronic diseases, such as CVD and cancer. Phytochemicals can remove free radicals to prevent these diseases. Among the seven different varieties of asparagus, green asparagus Apollo has the strongest total antioxidant activity; with a PSC value is

507.5 μmol of vitamin C equiv/100 g FW; Purple Passion has next strongest activity with PSC value of 481.6 μmol of vitamin C equiv/100 g FW. The white asparagus

Atlas has the lowest total antioxidant activity; its PSC value is 300 μmol of vitamin C equiv/100 g FW. The antioxidant activity of phytochemicals mainly depends on its ability to remove free radicals. Flavonoids are very effective for this; total antioxidant activities can be reflected by total flavonoids content to some extent (236). And the finding for total antioxidant activities for different variety asparagus are also consistent with the previous study (233).

2.4.4 Antiproliferative activity and cytotoxicity.

One of the hallmarks of cancer is that cells proliferate without control.

Therefore antiproliferation is an important factor in studying anticancer activity (237).

Besides, to make sure the anti-proliferative effect was not caused by cytotoxicity, it is necessary to perform cytotoxicity assay. In this study, the HepG2 human liver cancer cells are used as the model. As showed in figure 6, the green asparagus Apollo showed the strongest antiproliferative effect with EC50 value of 32.51 ± 2.46 mg/mL; the second strongest antiproliferative asparagus variety is Purple Passion, and its

EC50 value is 33.26 ± 1.5 mg/mL. The white asparagus Atlas has the lowest antiproliferative effect, with EC50 value of 53.52 ± 1.58 mg/mL. No cytotoxicity was observed for all the concentrations tested for the 7 different varieties of asparagus.

The antiproliferative effects are consistent with the phenolics content and flavonoids content. It may indicate that phenolics and flavonoids played an important role in the anticancer effects of asparagus. Currently few studies have compared the antiproliferation effects of different varieties of asparagus, so this study enhances our understanding of the direct health benefits of different varieties of asparagus.

2.4.5 Cellular antioxidant activity

Even though a phytochemical has antioxidant activity in chemical assay, this doesn’t mean it is equally active in cells, because many phytochemicals cannot be up taken (238). Many chemistry assays do not have similar environment as the biological system. To test the real antioxidant benefit of phytochemicals we need to have an assay which can take bioavailability, metabolism, and cellular uptake into account. To consider these characteristics, the human model or animal model will be most accurate; however, they are time consuming and costly and are thus inefficient for initially screening for phytochemical antioxidants. Cell models better for initial screening as cells are normally easier to grow. In this study, we used CAA assay, an assay developed in our lab to test the cellular antioxidant activity (226). This assay can mimic the cellular process in the human body to some extent. In this study, the

CAA value was expressed as μmol QE /100 g FW. For the No PBS wash group, the

results are shown in figure 7, and Apollo green asparagus has the highest CAA value, which is 32.1 ± 1.3 μmol QE /100 g FW. There are no significant differences among the other varieties. The CAA values of these varieties range from 22 ± 1.2 to 24.6 ±

2.3 μmol QE /100 g FW. The EC50 for Apollo green asparagus without PBS wash is

84.3 ± 3.8 mg/mL. For the PBS wash group, there was a similar pattern. Apollo green asparagus had significantly higher CAA value than the other varieties. Its CAA value is 8.4 ± 0.2 μmol QE /100 g FW. The CAA values for other varieties range from 4.8 ±

0.9 to 6.3 ± 1.2 μmol QE /100g FW, with no significant differences among them..

The EC50 for Apollo green asparagus is 240.69 ± 19.39 mg/mL. The result is consistent with the previous finding that the CAA value of asparagus is 4.35 μmol QE

/100g FW with PBS wash (143). PBS wash group has lower CAA value because the final products of antioxidative reaction left outside the cell have been washed away.

The CAA value is not correlated with the PSC value; it means that not all antioxidant reaction happened in the cells. Especially for white asparagus Atlas, although its total phenolic and flavonoids content are both the lowest, its CAA value is not. It indicates that white asparagus Atlas contains some antioxidants, which are more readily taken up by cells than that of other varieties of asparagus.

2.4.6 Summary

In summary, asparagus consumption has been associated with reduction of different chronic diseases. In this study, the total phenolic content, total flavonoid content, total antioxidant activities, antiproliferative activity and cellular antioxidant

activities were compared among seven asparagus varieties. All asparagus varieties contain high phenolic content (50.3±2.7 to 113.1±4.5 mg GA equiv/100 g FW), high total flavonoid content (19 ±2 to 75.7 ± 5.2 mg catechin equiv. 100 g FW); they have strong antiproliferative activities, and antioxidant activities in chemical assay and in vitro assay. Our findings also indicate that phytochemicals may play an important role in the health benefits of asparagus consumption. Of all the varieties we tested, Apollo green asparagus and Purple Passion purple asparagus showed the strongest activity.

Further studies need to be done to find the related reasons and more direct health benefits of asparagus for humans.

Chapter three Flavonol Kaempferol Inhibited Proliferation and

Migration of MDA-MB-231 Human Breast Cancer Cells Through

Down-regulation of MMP-2 Enzyme Activity

3.1 Introduction

Breast cancer is a prevalent disease. According to American Cancer Society there were 229,060 new breast cancer cases and 39,920 deaths from breast cancer in the US in 2012(239). Globally, around 1,383,500 breast cancer cases and 458,400 breast cancer deaths are estimated to have occurred in 2008(2). It is reported that one eighth of women in the US will develop breast cancer in their life time; breast cancer is the second leading cause of death in women (240). Although chemotherapy, radiation therapy, and surgery have improved the patients’ survival time (241), side effects and reoccurrence often come along with them. Thus, new preventive strategies and alternative treatments for breast cancer are needed.

Vegetable and fruit consumption have been found inversely associated with breast cancer risk (99, 216, 242). Phytochemicals are suggested to play the key role in the cancer preventive effects of fruits and vegetables (12, 243). As natural ingredients, phytochemicals are a safe and effective alternative agent for breast cancer treatment.

The flavonoid kaempferol is a widely distributed phytochemical in fruits and vegetables. It has been found to possess a variety of anti-cancer properties. Studies

reported that kaempferol can suppress breast cancer cells by inducing apoptosis,

regulating ROS level, reducing estrogen activities and other mechanisms (48, 159,

181). However, there is little evidence for anti-migration and anti-metastasis effects of

kaempferol on breast cancer. Recently more and more scientific evidence suggests

that anti-migration and anti-metastasis can be an effective way to inhibit breast

cancer (48, 244). Because human breast cancer migration takes years to happen,

anti-migration can be a good target for cancer prevention using phytochemicals,

which mostly are consumed from daily diet(245). Thus, the study on kaempferol’s

effect on breast cancer migration and its related mechanism is greatly needed.

In this study, to test the anti-proliferative activity and cytotoxicity of kaempferol

on MDA-MB-231 human breast cancer cells, we used a modified methylene blue

assay developed in our lab (224). We also studied the anti-migration effect of

kaempferol on MDA-MB-231 human breast cancer cells by scratch assay. Then we

used a zymography assay to investigate the effect of kaempferol on matrix

metalloproteinase-2 (MMP-2) enzyme of MDA-MB-231 cells. Furthermore, we

determined the effect of kaempferol on transformation of MBA-MD-231 human breast cancer cells by soft agar assay. We accomplished our objective to determine the mechanism of action of kaempferol on migration and metastasis in MDA-MB-231 human breast cancer cells.

3.2 Materials and Methods

3.2.1 Chemicals

α-Minimum Essential Medium Alpha Medium (MEM), Hepes, insulin, fetal

bovine serum (FBS), gentamicin, penicillin and streptomycin were purchased from

GIBCO (Life Technologies, Grand Island, NY). Hepatocyte growth factor (HGF),

kaempferol, gelatin, and agarose were purchased from Sigma-Aldrich (St Louis, MO,

USA). All other reagents used in the study were of analytical grade.

3.2.2 Cell culture

MDA-MB-231 human breast cancer cells were obtained from American Type

Culture Collection (ATCC, Rockville, MD). MDA-MB-231 human breast cancer cells

were cultured in α-MEM containing 10 mM Hepes, 10 g/mL insulin, 50 units/mL

penicillin, 50 g/mL streptomycin, 100 µg/mL gentamicin, and10% FBS as described

previously(246). Cells were incubated at 37°C with 5% CO2, and were seeded and/or

subcultured when they were in exponential growth phase (224).

3.2.3 Cytotoxicity evaluation

The cytotoxicity of kaempferol against MDA-MB-231 human breast cancer cells was determined by methylene blue assay as previously described (224, 238).

Cells in 100 µL fresh medium were plated in 96 wells plate with the concentration of 4

4 x 10 cells/ well. After 24 hours incubation with37 °C and 5% CO2, growth medium

was removed and cells were treated with 100 µL fresh medium with different

concentrations of kaempferol as samples or only dimethyl sulfoxide (DMSO) as

control. After another 24 hours incubation, cells were washed with 100 µl

phosphate-buffered saline (PBS), and 50 µl methylene blue staining buffer (98%

Hanks Balanced Salt Solution (HBSS), 0.67% glutaraldehyde, 0.6% methylene blue)

was added to each well, and incubated for 1 hour. Then the methylene blue staining

solution was removed, and the plate was washed with deionized water three times.

After the wells were dry, a volume of 100 µl elution buffer (1% (v/v) acetic acid, 49%

(v/v) PBS, and50% (v/v) ethanol) was added per well. The plate was then put on a

bench shaker for 20 minutes until the buffer was uniform. At the end, the absorbance

was read at 570 nm using a MRX Microplate Reader (Dynex Technologies, Inc.,

Chantilly, VA).Cytotoxicity was measured as percentage compared to control. More

than 10% cells number reduction was considered to be cytotoxic. All measurements

were conducted in triplicate.

3.2.4 Antiproliferative activity evaluation

The antiproliferative activity toward MDA-MB-231 human breast cancer cells was assessed by methylene blue assay reported previously (224, 232). 100 µl fresh medium with cells were seeded in 96 well at the concentration of 2.5 x 104cells/well.

After 6 hours incubation with 37 °C and 5% CO2, the growth medium was removed

and the cells were treated with 100 µl fresh medium with different concentrations of

kaempferol as samples or only dimethyl sulfoxide (DMSO) as control. After 72 hours

incubation, cell proliferation was determined by methylene blue assay at absorbance

of 570 nm (224). Antiproliferative activity was measured as percentage compared to

control. All measurements were conducted in triplicate.

3.2.5 Cell migration examination (Scratch Assay)

Cell migration of MDA-MB-231 human breast cancer cells was determined by

scratch assay (244). Briefly, 1.5 mL fresh medium with MDA-MB-231 human breast

cancer cells were plated in a six-well plate at a concentration of 5 x 105 cells/well. The plate was incubated for 24 hours at 37 °C with 5% CO2 to allow cells to grow

confluent, and the medium was then replaced with 1.5 mL Serum free medium (SFM)

to starve cells for 24 hours. Then the conditioned medium was removed. The mono

cells layer was scratched using a 200 µl pipette tip. Cells debris were washed out by

warm PBS twice, then different concentrations of kaempferol in 1.5 mL warm SFM

with or without HGF (40 ng/mL) were added to each well. Cells pictures were taken at

three marked sites per well at 0 hour, 12 hours and 24 hours by digital camera under

an inverted microscope. Cell migrations rate were calculated by the following formula:

rate (%) = B/A x 100, where A is the area of beginning scratch, and B is the

uncovered area at 12 or 24 hours. The area was analyzed by Adobe Photoshop CS 5

(Adobe Systems Inc. California, USA)

3.2.6 Matrix metalloproteinase-2 (MMP-2) activity analysis

Zymography assay was conducted using the method described previously

(247) with a modification. Briefly, MDA-MB-231 cells were seeded in a 6 well plate

with SFM at a density of 1 x 106 cells/well. After 24 hours starvation in incubator with

37 °C and 5% CO2, cells were treated with different concentrations of Kampferol with

HGF (40 ng/mL) and incubated for 24 hours. The medium was then harvested and centrifuged for 3 minutes at 1000~1100 RPM, and the supernatants were collected and mixed with loading buffer ( 2% sodium dodecyl sulfate (SDS), 0.1% bromophenol blue, 40% glycerol, 50% ammediol solution ). 20 µl samples were loaded on 10% acrylamide gel with gelatin (0.8 mg/mL) and without SDS. After gel was run for 1 hour at 150 V, it was washed four times with 20 mL enzyme renaturing buffer (200 mM

NaCl, 5 mM CaCl2, 0.7 µM ZnCl2, 2.5% (v/v) Triton X-100,0.02% (w/v) NaN3, and 50 mM Tris, pH 7.5) for 15 minutes each time with gentle agitation at room temperature.

Then it was transferred to developing buffer (Renaturing buffer without Triton X-100) and agitated on shaker for 1 hour. After the gel was incubated in incubator for 20 hours at 37 °C, the developing buffer was decanted. The gel was stained by staining solution (0.125% Coomassie brilliant blue, 1.25 L methanol, 0.5 L acetic acid, and

0.75 L water) for 20 minutes and then destained by destaining solution (1.5L methanol, 3.5 L water, and 50 mL formic acid) until clear bands were visible. Bands were visualized on scanner and quantified by ImageJ software version 1.46r (Wayne

Rasband, National Institutes of Health, Maryland, USA). MMP-2 activity was expressed as percentage compared to control. All measurements were conducted in triplicate.

3.2.7 Cell transformation investigation

The effect of kaempferol on MDA-MB-231 human breast cancer cell transformation was investigated by soft agar assay(248). Briefly, 3% sterilized agrose solution was microwaved for 30-45 seconds to dissolve well and kept in 55°C water bath. Then it was mixed with SFM and FBS to form 10% FBS, 0.6% agrose solution.

2 mL such solution was applied to each well of 6 well plates. The base layers were formed 1 hour after the solution solidified in 37°C hood. For the cell layer, MDA human breast cancer cells were harvested and diluted in SFM to concentration of 12 x 103 cells/mL; 3% agrose solution was taken out from 55°C into 42°C water bath to

lower the temperature for a while, then it was mixed with FBS, SFM and kaempferol

to form 20% FBS, 0.6 % agarose, and different concentrations of kaempferol solution

which include 0, 40, 80, and 120 µM. Then 2 mL cell solution and 2 mL kaempferol

agarose solution was mixed, and then it was distributed 1 mL per well on the top of

the base layer in triplicate. The final cell layers contain 6 x 103 cells in medium with

0.3% agarose and 0, 20, 40, and 60 µM kaempferol. The plate was kept horizontal for 15 minutes in 37°C hood and 15 minutes in 4°C refrigerator. After layers were solidified, the plates were placed into an incubator with 37 °C and 5% CO2.The

cultures were fed once a week with 0.3% agarose medium containing different

concentrations of kaempferol. The colonies were then observed under 40X final

magnification inverted microscope, and the number of colonies with diameter larger

than 50 µm was counted at 4th week. The colonies were also microscope

photographed for random 9 fields 2 weeks, 3 weeks, 4 weeks and 5 weeks after

plating to analyze the colonies grade. Colonies with diameter of n x 50 µm were given

a grade n. The colony grade under each concentration of kaempferol was then

quantified as colony grade per 1000 cells in triplicate.

3.2.8 Statistical analysis

Statistical analyses were performed using Sigmaplot software version 11.0

(Systat Software, Inc. Chicago, IL) and dose-effect analysis was performed using

Calcusyn software version 2.0 (Biosoft, Cambridge, UK). Data were statistically

analyzed by JMP software version 9.0.2 (SAS Institute Inc. North Carolina, USA) and

presented as mean ±standard deviation (SD) for at least three replicates. Significance

was determined at p value of <0.05 by analysis of variance (ANOVA) followed by

student’s t test.

3.3 Results

3.3.1 Antiproliferative activities and cytotoxicity of Kaempferol towards MDA-MB-231

human breast cancer cells

The antiproliferative activities and cytotoxicity of kaempferol was determined

by treating MDA-MB-231 human breast cancer cells with various concentrations

(0-100 µM). Compared to the control, which was DMSO treated alone, there was no

cytotoxicity observed for kaempferol for MDA-MB-231 cells at concentrations lower than 100 µM (Figure 8). Kaempferol exhibited significant antiproliferative activity in a 88

dose-dependent manner; it inhibited cell proliferation by 38.6± 2.9% at the concentration of 100 µM (Figure 8).

3.3.2 Effects of Kaempferol on migration activities of MDA-MB-231 human breast cancer cells

Because MDA-MB-231 human breast cancer cells are highly invasive, we investigated the effects of kaempferol on the HGF-induced migration activities of

MDA-MB-231 cells by scratch assay. 12 h or 24 h after the monolayers were scratched, the HGF+DMSO treated group showed a much higher migration rate than no HGF treated and HGF+Kaempferol treated groups (Figure 9A). The migration rate is calculated as stated in method. After wounding for 12 and 24 h, three pre-marked spots in each well were chosen, and the area in each spot was analyzed by

Photoshop CS 5. The results showed that under 20 µM and 40 µM kaempferol treatments, cell migration was inhibited by 60.1 ± 11.6% and 70.8 ± 6.3%, while in control group it was inhibited by 9.13 ± 1.16% at 24 hours (Figure 9B).

3.3.3 Inhibition of MMP activities of MDA-MB-231 human breast cancer cells by kaempferol.

MMP played an important role in cancer metastasis by digesting the extracellular matrix, which is required for constant remodeling when the cancer cells invade and migrate (247). The effects of kaempferol on MMP activities of

MDA-MB-231 human breast cancer cells were evaluated by zymography assay. After

24 h treatment with kaempferol and 24 h enzyme activity, the gelatin gel which is the substrate of MMP-2 was stained, and then it was destained a couple of times until there were clear bands visible on the gel. The results showed that as the kaempferol concentrations increased; the bands became smaller. MMP-2 enzyme expressions were inhibited by kaempferol significantly in a dose-response manner (Figure 10A).

The results were also quantified by analyzing the band area by ImageJ software.

Enzyme inhibition activities were expressed as peak of band at different concentrations compared with that of control. At concentrations of 20, 40, 60 µM, the

MMP-2 enzyme activities were inhibited by 48.4 ±9.9, 66.2 ± 7.6, 89.6 ± 4.6% when compared to the control (Figure 10B).

3.3.4 Effect of kaempferol on MDA-MB-231 human breast cancer cells transformation

For normal cells the interactions between them will suppress the cells overgrowth, while transformed cells have lost these contact inhibitions. Normal cells need to attach to a solid surface to grow; however the transformed cells become anchorage independent(248). Thus soft agar assay is a good model to study transformation of cells. 4 weeks after plating the cells, colonies were formed in the top soft agar layer. The numbers of colonies with diameter larger than 50 µm were significantly decreased by kaempferol in a dose-response manner (Figure 11A).

Treated with 0, 20, 40, and 60µM kaempferol, the colonies numbers were 394 ± 13,

306 ± 34, 249 ± 7, and 193 ± 12 (Figure 11B). Moreover, after taking the size of the

colonies into consideration, the suppression effect was even more obvious. At 4th week, the colony grade was inhibited to 143.9 ± 4.6 compared with the control which is 410.6 ± 15.1 (Figure 11C).

Figures

FIGURE 8 EFFECT OF KAEMPFEROL ON CELL PROLIFERATION AND CYTOTOXICITY IN MDA-MB-231 CELLS

120

100

80 * * * * 60 Proliferation Control Cell Proliferation * * Cytotoxicity Control 40 Cytotoxicity Cytotoxicity(%)

Cell Proliferation (%) Proliferation Cell 20

-20 0 10 20 30 40 50 60 70 80 90 100 Concentration of Kaempferol (µΜ)

Figure 8. Effect of kaempferol on cell proliferation and cytotoxicity in MDA-MB-231 human breast cancer cells (mean ±SD, n =3). Values marked with * are significantly different compared to the control (p < 0.05).

24 No HGF HGF HGF + 20µM HGF + 40 µM HGFC

BFigure 9 Evaluation of kaempferol on HGF-stimulated cell motility

120 12 h 24 h 100 a

b 60

c cd cd 40 de

f ef 20 f Closused area to Original area ratio (%) ratio area to Original area Closused

_ HGF (40 ng/mL) + + + Kaempferol (µM) _ _ 20 40

Figure 9. Evaluation of kaempferol on HGF-stimulated cell motility in MDA-MB-231 human breast cancer cells. (A) Confluent monolayers were scratched with a 200 µl

plastic pipette tip and incubated in SFM in presence of either with or without HGF (40

ng/mL), and HGF plus Kaempferol ( 20 µM and 40 µM ) for 0 h, 12 h, 24 h. The

results were observed under phase contrast microscope and photographed. (B) The

uncovered scratch area is determined by Photoshop CS5, and wound closure rate is

calculated by closed scratched area at different time divided by original scratched

area. Bar graphs indicate quantification of at least three scratches. Bars with no

letters in common are significantly different (p < 0.05).

A Concentrations of Kaempferol (µM)

0 20 40 60

FIGURE 10 EVALUATION OF KAEMPFEROL ON MMP-2 ENZYME ACTIVITY

1.2

a 1.0

b .6

c .4

Relavant MMP-2 Enzyme activity Enzyme MMP-2 Relavant .2 d

0.0 0 20 40 60

Concentrations of Kaempferol (µΜ)

Figure 10. Evaluation of kaempferol on MMP-2 enzyme activity. (A) Inhibition of

MMP-2 activity in SFM from MDA-MB-231 human breast cancer cells treated with

HGF (40 ng/mL) and Kaempferol (0, 20, 40, 60 µmol/L) for 24 h was evaluated via gelatin zymography. Equal amounts of sample were run on 10% acrylamide gels and stained with Coomassie blue. Bands were visualized by camera. (B) Results are

analyzed by Image J software. Data is presented as the treatment blank area divided by 0 µM concentration treatment blank area. Bar graphs indicate quantification of at least three separate zymography. Bars with no letters in common are significantly different (p < 0.05).

Control 20 µM

40 µM 60 µM

500

a 400

300 c

d 200

Number of Colonies formed of Number Colonies 100

0 0 20 40 60 Concentrations of Kaempferol (µΜ)

FIGURE 11 DIFFERENT CONCENTRATIONS OF KAEMPFEROL INHIBIT THE MDA-MB-231 BREAST CANCER CELLS COLONY FORMATION IN

SOFT AGAR

500

Control 20 µΜ µΜ 400 40 60 µΜ

300

200

Colony grade per 1000 cells 1000 per grade Colony 100

0 0 weeks 2 weeks 3 weeks 4 weeks

Figure 11. Different concentrations of kaempferol inhibit the MDA-MB-231 human breast cancer cell colony formation in soft agar. Single cell suspensions of 6x103

MDA-MB-213 cells were seeded in soft agar as described in materials and methods.

Colonies were microscopic fields photographed (40 X final magnification) 4 weeks after plating. (A) Photographs of representative microscopic fields containing colonies that developed in soft agar. (B)The numbers of colonies with diameter more than 50 µm were counted. Values indicate quantification of at least three separate wells. Bars with no letters in common are significantly different (p < 0.05) (C) Colonies were also microscopic fields photographed for 9 random fields 2 weeks, 3 weeks, and

4 weeks after plating. Colonies with diameter of n x 50 µm was given a burden grade

n. The values were quantified as colony grade per 1000 cells in triplicate.

3.4 Discussion

It is reported that the incidence and mortality of breast cancer in Asia is lower

than that in Western countries (2). One possible explanation is that Asians consume

more plant food that is rich in flavonoids. Indeed, recent studies have found

vegetables and fruits consumption are linked to a reduced risk of developing breast cancer (10, 216, 249). And phytochemicals are considered the major components to cause this effect (215). Flavonoid Kaempferol is commonly found in vegetables, fruits, and other edible plants. Epidemiology studies have reported that daily intake of kaempferol significantly decreased the risk of lung cancer, pancreatic cancer , and advanced adenoma recurrence (133-135). It had also been found to possess various anti-cancer properties in animal studies and in vitro studies. Kaempferol can prevent colorectal carcinoma in rats by its antioxidative ability, inducing apoptosis and modulating the estrogen activities in MCF-7 human breast cancer cells (137, 159,

181). However, there are few studies about kaempferol’s effects on breast cancer migration. Hence here we investigated anti-migration ability of kaempferol on

MDA-MB-231 human breast cancer cells.

3.4.1 Antiproliferative activity of kaempferol towards MDA-MB-231 human breast cancer

cells

Unlimited proliferation is a one of the hall marks of cancer cells(250); therefore, halting the proliferation is an efficient way to prevent cancer. In many

studies phytochemicals have been found to slow the proliferation of breast cancer 100

cells, and showed no or very mild cytotoxicity. In this study, cytotoxicity of kaempferol was not observed in MDA-MB-231 human breast cancer cells when the concentration of kaempferol was lower than 100 µM. This is consistent with a previous study (48).

When kaempferol concentration is increased to 100 µM, the inhibition rate is 38.6 ±

2.9%.

3.4.2 Anti-migration effect of kaempferol in MDA-MB-231 human breast cancer cells

It is well documented that cancer cells may be nongrowing in human body for a long time. They will locate within 0.2 mm from blood vessels. Since the oxygen supplied by the blood vessels can only diffuse to this distance, the cells situated further than this radius will suffer hypoxia and become necrotic(251, 252). In addition, cancer cells also need effective interactions with blood vessels to acquire nutrients and excrete waste. To overcome this hindrance and continue growing, cancer cells need to stimulate angiogenesis, and then migrate to a better environment. This angiogenesis and migration process has been found very similar to the wound healing process of normal tissue(253). Hence scratch assay (wound healing assay) is a very good model to investigate the migration characteristics of cancer cells. In our study, kaempferol showed strong inhibitory effect on movement of cancer cells in scratch assay in a dose-response manner. After the cells were scratched by 200 ul pipette, cells tried to close this area at different speeds; at 24 h, the HGF induced control group without kaempferol treatment almost covered the scratch area, while the treatment group left most of the area blank. Compared to control which inhibited

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migration by 9.13 ± 1.16%, 20 and 40 µM kaempferol inhibited cell migration by

60.1 ± 11.6 and 70.8 ± 6.3% at 24 h after the scratch. Although no previous data that

showed kaempferol’s effect on scratch motility of MDA-MB-231 human breast cancer cells, kaempferol showed its anti-migration ability in another assay (48, 172), and our

results showed kaempferol’s anti-migration effect in a further step.

3.4.3 MMP inhibition effect of kaempferol in MDA-MB-231 human breast cancer cells

To induce new blood vessel formation, at some point in time cancer cells

acquire the ability to release the angiogenic factors such as vascular endothelial

growth factor (VEGF) and fibroblast growth factor (FGF). These angiogenic factors

will work on endothelial cells, and then angiogenesis can begin. However, angiogenic

factors alone are not enough to begin the process, since ECM will block these factors

and prevent them contacting with endothelial cells. The 'angiogenic switch' will be

turned on until the ECM is broken down by MMP (254). Besides, MMP can also

degrade and remodel the ECM. This is very important in the process of

epithelial-mesenchymal transition which enables cancer cells to become invasive and

acquire motility (255). Thus inhibition of MMP is an effective way to inhibit tumor

migration and metastasis. MMP-2 is a member of MMP family. It is found largely

expressed in different tumors and can degrade laminin which is an important

component of ECM. Inhibition of MMP-2 activity will slow the cancer cell growth (256).

There are already some angiogenic drugs which are matrix metalloproteinases

(MMPs) inhibitors in clinical trials(257). Recently many studies also found food or its

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active compound to possess such potential. Grape extracts of 4 varieties showed

they can inhibit the expression of MMP-2 in HUVE cells (247).

Epigallocatechin-3-gallate (EGCG) can down-regulate MMP-2 level in MCF-7 human breast cancer cells (64). Soy isoflavone genistein inhibited the MMP-2 expression in

HCC 1395 breast cancer cells (258). In our study, kaempferol significantly suppressed the MMP-2 activity in a dose-dependent manner. 60 µM kaempferol inhibited the MMP-2 enzyme activity by 89.6 ± 4.6% compared to the control.

3.4.4 Kaempferol’s effect on transformation of MDA-MB-231 human breast cancer cells

The process of cell migration involves many heterotypic interactions between

different cells. To stimulate angiogenesis, communications among platelets,

neutrophils, mast cells, lymphocytes and other cells are needed. In normal tissues,

these interactions create a balanced environment where cells will not overgrow.

However, cancer cells have overcome this barrier and they become independent from

these interactions (250). In addition, transformation of cancer cells is a prerequisite

for cancer migration. Thus it can help explain kaempferol’s effect on MDA-MB-231

human breast cancer cell transformation and further migration by using soft agar

assay. Previously, kaempferol has been found to inhibit EGF-induced colony

formation of JB6 C141 cells and immortalized lung epithelial (BEAS-2B) cells in soft

agar (203, 259). But there has been no research studying its effect on breast cancer

cells. In our study, kaempferol decreased colony numbers and size in a

dose-dependent manner; 4 weeks after plating, 60 µM kaempferol inhibited the

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colony number by 49.1± 4.8%. We also found that kaempferol inhibited colony grade, which considers both the number and size factors, by 35.1± 2.5%.

3.4.5 Summary

In summary, kaempferol can be used as a potential chemopreventive compound for breast cancer. It can slow proliferation, inhibit migration, and suppress transformation of MDA-MB-231 human breast cancer cells.

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Chapter four Kaempferol Inhibits Proliferation of MCF-7 Human Breast Cancer

Cells through Suppressing Expression of Estrogen Receptor alpha and Regulating

P38/MAP Kinase Signal Transduction Pathway

4.1 Introduction

Breast cancer is the second leading cause of cancer deaths in women in the

US. According to American Cancer Society 1,665,540 new cancer cases and 585,720 cancer deaths are estimated to occur in the United States in 2014(1). Globally, around 12.7 million cancer cases and 7.6 million cancer deaths are estimated to have occurred in 2008. One in eight women will develop breast cancer in her lifetime(2).

Currently there are several kinds of breast cancer treatments such as radiotherapy, surgery, adjuvant chemotherapy, monoclonal antibody immunotherapy, and hormone therapy. Although these treatments are effective to some extent, there are always side effects, including drug resistance, depression of immune system, fatigue, tendency to bleed easily, gastrointestinal distress, and hair loss (260). Hence, an

alternative treatment is urgently needed. Epidemiological studies have shown that

regular consumption of fruits and vegetables could reduce the risk of breast cancer. A

EPIC study in Italy demonstrated that consumption of vegetables and fruits

significantly decreased breast cancer risk among 31,000 women after a median

follow-up of 11.25 years (261). Phytochemicals are bioactive nonnutrient plant

compounds in fruits, vegetables, grains, and other plant foods that have been linked

to reducing the risk of major chronic diseases (12). They have been suggested to be

105

responsible for the anticancer effects of fruits and vegetables(12). Flavonoids are a large group of phytochemicals found in vegetables and fruits; they are secondary metabolites in plants. Regular consumption of flavonoids has been found to reduce

cancer risk. In a longitudinal study, 9959 subjects were followed from 1967 to 1991.

Total flavonoid consumption was found to significantly reduce the incidence of all

types of cancers (highest vs. lowest quartile RR 0.8; 95% CI 0.67-0.96) (262).

About 75% of breast cancers grow in response to the growth hormone; these

are called endocrine receptor positive (ER positive) cancer. Because they have this

characteristic, the common hormone therapy is effective to treat these cancers.

However, because there are side effects, we need to find some alternative, natural,

and safe sources for breast cancer prevention. Phytoestrogens is a group of

phytochemicals in plants. They have a similar structure as estrogen, but their

activities are much weaker. So by routinely consuming the phytoestrogen, breast

cancer risk can be reduced. In a case control study, the researchers studied soyfood

consumption during adolescent period in Chinese women and the breast cancer risk

later in their life. They found that adolescent soyfood intake was inversely associated

with breast cancer risk (P for trend < 0.001) (263). Kaempferol is one of the most

widely distributed flavonoids and phytoestrogens in plants. It is normally found in

asparagus, broccoli, leek, onion, apples, and other plant foods. It has been reported

to have different anti-cancer properties, such as antioxidants, cell cycle arrest,

106

apoptosis induction, anti-inflammation, and phytoestrogen (48, 159, 181). However, the mechanism for its phytoestrogen health effect is not completely understood.

In this paper we studied the antiproliferative activity and cytotoxicity of kaempferol against MCF-7 human breast cancer cells using the methylene blue assay developed in our laboratory. We also investigated the effects of kaempferol on cell signaling transductions in MCF-7 human breast cancer cells by Western Blot assay. Our objective was to determine the mechanisms of action of kaempferol in inhibiting cell proliferation in MCF-7 human breast cancer cells.

4.2 Materials and Methods

4.2.1 Chemicals.

α-Minimum Essential Medium (MEM), Hepes, insulin, fetal bovine serum

(FBS), gentamicin, penicillin and streptomycin were purchased from GIBCO (Life

Technologies, Grand Island, NY). Methanol, phosphate-buffered saline (PBS), sodium hydroxide and xylene were purchased from Fisher Scientific (Pittsburgh, PA).

Kaempferol, Estradiol alpha, folin-Ciocalteu reagent, hyrdrochloric acid, Igepal, and

SB203580 were purchased from Sigma Chemical Co. (St. Louis, MO). Ultrapure Tris

(base) and Tris (acid) were purchased from J. T. Baker (Phillipsburg, NJ). All other reagents used in the study were of analytical grade.

4.2.2 Cell culture.

107

MCF-7 human breast cancer cells were obtained from American Type Culture

Collection (ATCC, Rockville, MD), and cultured in α-MEM containing 10 mMHepes,

10 g/mL insulin, 50 units/mL penicillin, 50 g/mL streptomycin, 100 µg/mL gentamicin, and10% FBS as described previously(246). Cells were incubated at 37°C with 5%

CO2, and were seeded and/or subcultured when they were in exponential growth phase (224).

4.2.3 Cytotoxicity evaluation

The cytotoxicity of kaempferol against MCF-7 human breast cancer cells was determined by methylene blue assay as previously described (224, 238). Cells in 100

µl fresh medium were plated in 96 wells plate with the concentration of 4 x 104 cells/ well. After 24 hours incubation with 37 °C and 5% CO2, the growth medium was removed and cells were treated with 100 µl fresh medium with different concentrations of kaempferol as samples or only dimethyl sulfoxide (DMSO) as control. After another 24 hours incubation, cells were washed with 100 µl phosphate-buffered saline (PBS). 50 µl methylene blue staining buffer (98% Hanks

Balanced Salt Solution (HBSS), 0.67% glutaraldehyde, 0.6% methylene blue) was added to each well, and they incubated for 1 hour. Then the methylene blue staining solution was removed, and the plate was washed with deionized water three times.

After the wells were dry, a volume of 100 µl elution buffer (1% (v/v) acetic acid, 49%

(v/v) PBS, and50% (v/v) ethanol) was added per well. The plate was then put on a bench shaker for 20 minutes until the buffer was uniform. At the end, the absorbance

108

was read at 570 nm using a MRX Microplate Reader (Dynex Technologies, Inc.,

Chantilly, VA).Cytotoxicity was measured as percentage compared to control. More

than 10% cells number reduction was considered to be cytotoxic. All measurements

were conducted in triplicate.

4.2.4 Antiproliferative activity evaluation

The antiproliferative activity toward MCF-7 human breast cancer cells was

assessed by methylene blue assay as reported previously (224, 232). 100 µl fresh

medium with cells were seeded in 96 wells at the concentration of 2.5 x 104 cells/well.

After 6 hours incubation with 37 °C and 5% CO2, the growth medium was removed

and cells were treated with 100 µl fresh medium with different concentrations of

kaempferol as samples or only dimethyl sulfoxide (DMSO) as control. After 72 hours

incubation, cell proliferation was determined by methylene blue assay at absorbance

of 570 nm (224). Antiproliferation activity was measured as percentage compared to

control. All measurements were conducted in triplicate.

4.2.5 Preparation of protein samples.

Protein samples of cells were obtained using the method as reported

previously in our laboratory (264). Briefly, MCF-7 human breast cancer cells were

seeded at a density of 0.5 × 106 cells/well in 6-well-plate and treated with different concentrations of kaempferol (0, 25, 50 and 75 μM) after 8 h attachment. After a 24 h of incubation, growth media were removed, and cells were washed twice with ice-cold

109

PBS, and then cells were scraped off and collected at a centrifuge of 12000 g for 5 min at 4 °C. Each treatment was triplicated in six wells, and the cells receiving the same level of treatment were combined together for protein extraction and further

Western Blot analysis.

Harvested cells were lysed using lysis buffer (50 mM Tris, pH 7.4; 1% Igepal;

150 mM sodium chloride; 1 mM EDTA) with protease inhibitors (1 μg/mL aprotinin; 1

μg/mL leupeptin; 1 μg/mL pepstain; 1 mM PMSF; 1 mM sodium orthovanadate; 1 mM sodium fluoride). Cell lysates were placed on ice for 30 min with vortex every 5 minutes to facilitate protein extraction. The lysates were then centrifuged at 12000 g for 15 min at 4 °C. The supernatants were collected for protein concentrations by

Lowry protein assay using a Sigma Diagnostics Micro Protein Determination Kit and a

Dynex Microplate Reader (Dynex Technologies, Inc., Chantilly, VA) as described previously (265).

4.2.6 Western Blot assay.

Western Blot analysis were conducted using the method as described previously (266) with a modification (246). All of the cellular proteins were subjected to electrophoresis on 10% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Lysate proteins after separation were transferred onto an Polyvinylidene fluoride (PVDF) membrane, and the membrane was blocked in 5% nonfat dry milk in TBST (Tris-base buffer solution containing of 0.1% Tween 20) at room temperature for two hours and incubated with primary antibodies (diluted in 1% 110

nonfat dry milk and TBST) overnight at 4 °C. Membranes were then rinsed three

times with TBST and incubated for 2h with a corresponding secondary antibody

diluted in the antibody buffer. After subsequent washing with TBST and deionized

water, membrane-bound antibodies were visualized by the Enhanced

Chemiluminescence kit (Cell Signaling Technology, Inc., Beverly, MA) according to

the manufacturer’s instruction. Protein band intensity was determined with integrated

optical densitometry using Labworks gel imaging and analysis software as described

previously (265).

In this study, mouse monoclonal primary antibodies against human

proliferating cell nuclear antigen (PCNA), CDK-4, Cyclin D1, p21, estrogen receptor-alpha(ER α ), p-p53 and their corresponding secondary antibody, anti-mouse IgG-HRP conjugate, and rabbit polyclonal antibodies against p-p38,were purchased from Santa Cruz Biotechnology Co. (Santa Cruz, CA). Anti-rabbit

IgG-HRP conjugate was purchased from Sigma Chemical Co. (St. Louis, MO). The expression of beta-actin was used as an internal standard control.

4.2.7 Statistical analysis

Statistical analyses were performed using Sigmaplot software version 11.0

(Systat Software, Inc. Chicago, IL) and dose-effect analysis was performed using

Calcusyn software version 2.0 (Biosoft, Cambridge, UK). Data were statistically analyzed by JMP software version 9.0.2 (SAS Institute Inc. North Carolina, USA) and presented as mean ±standard deviation (SD) for at least three replicates. Significance 111

was determined at p value of <0.05 by analysis of variance (ANOVA) followed by

student’s t test.

4.3 Results:

4.3.1 Antiproliferative activities and cytotoxicity of Kaempferol towards MCF-7 human

breast cancer cells

The antiproliferative activities and cytotoxicity of kaempferol was determined

by treating MCF-7 human breast cancer cells with various concentrations (0-100 µM;

Figure 12). Compared to the control, which is DMSO treated alone, there was no

cytotoxicity observed for kaempferol for MCF-7 human breast cancer cells at

concentration lower than 100 µM (Figure 12). Kaempferol exhibited significant

antiproliferative activity in a dose-dependent manner; it inhibited cell proliferation by

77.1% ± 0.58% at the concentration of 100 µM. The EC50 value of kaempferol’s

antiproliferative activity toward MCF-7 human breast cancer cells was 56.3 µM.

4.3.2 Effects of kaempferol on expression of proteins involved in the proliferation and

cell cycle in MCF-7 human breast cancer cells.

To confirm the antiproliferative effect of kaempferol on MCF-7 human breast

cancer cells, we tested the expression of proliferating cell nuclear antigen (PCNA),

the marker of cell proliferation. As shown in Figure 13A, kaempferol inhibited PCNA level in MCF-7 cells in a dose-dependent manner. The PCNA expression in the

MCF-7 cells treated with kaempferol was inhibited by 63% at a concentration of 75

112

µM kaempferol, indicating a direct antiproliferative effect of kaempferol during the

DNA synthesis phase of the cell cycle in MCF-7 human breast cancer cells. To further investigate the modulation of other cell cycle proteins, we also examined the expression of Cyclin D1 and CDK-4 in the MCF-7 human breast cancer cells by kaempferol treatment. The expression of Cyclin D1 and CDK-4 were significantly down-regulated in a dose-dependent manner with the treatment of kaempferol

(Figure 13B and Figure 13C). At a concentration of 75µM kaempferol, the Cyclin D1 level was inhibited by 72% and CDK-4 level was inhibited by 57%.

4.3.3 Regulation of proteins involved in the P38/MAPK pathway

We first investigated whether p21 and phosphorylated-p53 (p-p53) was

involved in cell cycle control in kaempferol-treated MCF-7 human breast cancer cells.

Kaempferol significantly increased the expression of p21 and p-p53 in MCF-7 human

breast cancer cells when compared to the control (Figure 14A and Figure 14B). 75

µM kaempferol significantly increased the p21 protein level by 37% and p-p53 protein

level by 96%. Phosphor-p38 (p-p38) is the upstream protein for p-p53, and our study

also found that kaempferol can up-regulate the expression of p-p38 (Figure 14C). It

indicated that p-p38 was involved in kaempferol’s antiproliferative activity in MCF-7

cells.

4.3.4 Effects of Kaempferol on expression of estrogen receptor alpha.

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Because kaempferol is a phytoestrogen, we also investigated the effects of

kaempferol on the expression of ER α in human breast cancer estrogen-dependent

MCF-7 human breast cancer cells. As shown in Figure 15A, treatment of kaempferol

to MCF-7 human breast cancer cells resulted in a significant decrease in expression

of ER α in a dose-dependent manner. The ER α expression was inhibited by 50.6%

by 75 µM kaempferol. Furthermore, to confirm the p-p38 is involved in this effect,

p-p38 inhibitor SB203580 was used to test its effects on the regulation of ER α by

kaempferol. After adding 10 µM SB203580, we found that kaempferol’s inhibition

effect to ER α had been abrogated (Figure 15B). This confirmed the relevance of

p-p38 to the ER α regulation effect by kaempferol.

4.3.5 Effects of kaempferol on estradiol induced cell proliferation in MCF-7 human breast cancer cells

If kaempferol can inhibit MCF-7 cells proliferation as phytoestrogen, it should

also inhibit proliferation by competing with estrogen. To confirm this, Estradiol alpha

was used to study its effects on kaempferol’s antiproliferative activity in MCF-7 cells.

Estradiol alpha alone had been found to increase the cell numbers in a

dose-dependent manner. When there was only 50 µM kaempferol, the cell number

was inhibited by 40% as in the previous result. After adding different doses of

Estradiol α, this inhibition effect was offset in a dose-dependent manner (Figure 16).

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FIGURE 12 EFFECT OF KAEMPFEROL ON CELL PROLIFERATION AND CYTOTOXICITY IN MCF-7 HUMAN BREAST CANCER CELLS

120 120

100 100

* 80 * Proliferation control 80 * Cell proliferation * Cytotoxicity control 60 * Cytotoxicity 60 * * 40 40 * * Cytotoxicity(%) Cell Proliferation (%) Proliferation Cell 20 20

0 0

-20 -20 0 20 40 60 80 100 120 µ Kaempferol Concentration M

Figure 12. Effects of kaempferol on cell proliferation and cytotoxicity in MCF-7 human breast cancer cells (mean ± SD, n =3). Values marked with * are significantly different compared to the control (p < 0.05).

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FIGURE 13 EFFECTS OF KAEMPFEROL ON PCNA, CYCLIN D1, AND CDK-4 EXPRESSION IN MCF-7 HUMAN BREAST CANCER CELLS

Figure 13. Effects of kaempferol on PCNA, Cyclin D1, and CDK-4 expression in

MCF-7 human breast cancer cells (mean ± SD, n =3). Bars with no letters in common are significantly different (p < 0.05).

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FIGURE 14 EFFECTS OF KAEMPFEROL ON P21, P-P53, AND P-P38 EXPRESSION IN MCF-7 HUMAN BREAST CANCER CELLS

Figure 14. Effects of kaempferol on p21, p-p53, and p-p38 expression in MCF-7 human breast cancer cells (mean ± SD, n =3). Bars with no letters in common are

significantly different (p < 0.05).

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FIGURE 15 EFFECTS OF KAEMPFEROL AND P-P38 INHIBITOR SB 203580 ON ESTROGEN RECEPTOR ALPHA EXPRESSION IN MCF-7 HUMAN

BREAST CANCER CELLS

Figure 15. Effects of kaempferol and p-p38 inhibitor SB 203580 on Estrogen

Receptor Alpha expression in MCF-7 human breast cancer cells (mean ± SD, n =3).

Bars with no letters in common are significantly different (p < 0.05).

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FIGURE 16 EFFECTS OF KAEMPFEROL ON ESTRADIOL INDUCED PROLIFERATION OF MCF-7 HUMAN BREAST CANCER CELLS

180

160 a a a 140

120 b b b 100 c 80 d 60 Cell Proliferation (%) Proliferation Cell

0 control ------50 uM kaempferol - - - - + + + + -10 Estradiol 10 M - + - - - + - -

Estradiol 10 -9 M - - + - - - + - -8 + Estradiol 10 M - - - + - - -

Figure 16. Effects of kaempferol on estradiol induced proliferation of MCF-7 human breast cancer cells (mean ± SD, n =3).

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FIGURE 17 SIGNALING TRANSDUCTION PATHWAY SUMMARY

Figure 17. Potential mechanisms of action of kaempferol in regulating cell proliferation in MCF-7 human breast cancer cells through inhibiting estrogen receptor alpha via direct competitive binding with estradiol and suppressing P38/MAP Kinase signal transduction pathway

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4.4 Discussion

Breast cancer is one of the most often diagnosed cancers and is a major cause of deaths in women. In the last several decades, breast cancer morbidity has steadily decreased (2). Interestingly, Asian women have lower breast cancer incidence rate and morbidity rate. Researchers think that the consumption of more fruits and vegetables in Asian diets compared to western diets is one of the major reasons for this phenomenon (263). The 2010 Dietary Guidelines for Americans recommend that people should eat two and half cups of fruits and vegetables each day to prevent chronic diseases(230). Fruits and vegetables contain many phytochemicals. These phytochemicals can prevent cancer and inhibit cancer growth through different mechanisms, including regulating the key proteins in cancer formation and working as phytoestrogen to modulate estrogen activity(12).

Kaempferol, as one of the most widely distributed phytochemicals and phytoestrogen, has been found to possess different anticancer properties including antioxidant activity, inducing apoptosis and modulating the estrogen activities (137, 159, 181).

However, the mechanisms are not clearly understood. Hence in this paper we investigated kaempferol’s effects on estrogen activity and the expression of related key proteins in MCF-7 human breast cancer cells.

4.4.1 Antiproliferative activity of kaempferol in MCF-7 human breast cancer cells

Uncontrolled proliferation is one of the hallmarks of cancer cells; we first confirmed the antiproliferative effect of kaempferol on MCF-7 human breast cancer 121

cells. In this study kaempferol inhibited the proliferation of MCF-7 human breast cancer cells in a dose-dependent manner. When kaempferol concentration was increased to 100 µM, MCF-7 proliferation was inhibited by 77.1 ± 0.58%, compared to control. The EC50 value was 56.3 µM. In a previous study, we tested the

antiproliferative effect of kaempferol on MDA-MB-231 human breast cancer cells. The

results showed that at 100 µM kaempferol can only inhibit the proliferation by 38.6 ±

2.9%. So kaempferol’s antiproliferation activity in MCF-7 human breast cancer cells was almost twice strong as in MDA-MB-231 human breast cancer cells. One of the major differences between MDA-MB-231 cells and MCF-7 cells is that MCF-7 cells have estrogen receptors while MDA-MB-231 cells don’t. Thus, the antiproliferative effect of kaempferol in MCF-7 human breast cancer cells is very possibly caused by regulation of the estrogen receptor.

4.4.2 Kaempferol’s effects on cell signaling pathway in MCF-7 human breast cancer cells

When an extracellular compound works on cells, it normally delivers the

message by cell signaling transduction. Thus understanding the key cell signaling

proteins level change will help understand the mechanism. Several key proteins

directly involved in cell proliferation are PCNA, Cyclin D1, and CDK-4. PCNA is a

DNA clamp that acts as an important factor for DNA polymerase in eukaryotic cells and is essential for replication. Inhibiting proliferation normally will be reflected by a

decrease of this protein expression; kaempferol in the study had been found to inhibit

the PCNA protein expression in a dose-dependent manner. Cyclins are a family of

122

proteins that control the progression of cells through the cell cycle by activating CDK enzymes (267), and in this study we found that kaempferol can significantly inhibit the

Cyclin D1 and CDK-4 protein expressions in dose-dependent manners. We also investigated the p21 protein which is an upstream protein for PCNA, Cyclin D1, and

CDK-4. P21 regulates cell cycle progression through suppressing the Cyclin D1,

CDK-4, and PCNA expression. It plays a regulatory role in the S phase DNA synthesis and DNA damage repair (268). We found that kaempferol can increase the p21 protein level significantly. P53 is a cellular tumor suppress antigen; it has been described as "the guardian of the genome" because of its role in conserving stability by preventing genome mutation(269). P-p53 is the active form of p53; it has been found to up-regulate the p21 protein level. In our study, p-p53 level was significantly increased by kaempferol. For this effect, one possible explanation is that p38/MAPK pathway was activated. So we tested the effect of kaempferol on p-p38 expression in

MCF-7 human breast cancer cells and found that kaempferol can significantly increase the p-p38 level.

Many breast cancers contain ER α. Estrogen will interact with ER α and promote the tumor growth. Kaempferol is a phytoestrogen, and it has similar structure as estrogen but with much weaker activity. Kaempferol may regulate the ER α activity to suppress the cell proliferation. In this study, kaempferol indeed significantly inhibited the ER α level in a dose-dependent manner. To confirm whether kaempferol has the antiproliferation effect through regulating the hormone receptor, we also used

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Estradiol α to study its effect on kaempferol’s antiproliferative activity in MCF-7 human breast cancer cells. Since MCF-7 breast cancer cells are hormone receptor positive, the results showed that Estradiol α can increase the cell proliferation

compared to the control. More importantly, estradiol α reversed the inhibition effect of

kaempferol on MCF-7 human breast cancer cells. It proves that kaempferol inhibited

MCF-7 human breast cancer cell proliferation through the regulation of ER α.

In addition to the compete binding with estradiol, previous studies also

showed that estrogen receptor regulation may also be controlled by MAPK pathway.

It is reported that p53 can be recruited to ER α minimal promotor to inhibit ER α

protein expression (270). To study whether kaempferol can regulate ER α by affecting

the MAPK pathway, we used p-p38 specific inhibitor SB203580 to study whether it

can abrogate kaempferol’s antiproliferative effect. After we added 10 µM SB203580,

the suppression of kaempferol on estrogen receptor alpha disappeared. Because

p-p38 is the upstream for p-p53, the p-p38 and p-p53 up-regulation may contribute to

decrease the expression of ER α. In another study, ER α has also been found to

inhibit p21 protein level by promoting miR–17 activity(271). The p21 in turn will

down-regulate the Cyclin D1, CDK-4, and PCNA levels.

4.4.3 Summary

In summary, based on the results of this study, we proposed a model as in

figure 17. Kaempferol can inhibit MCF-7 human breast cancer cell proliferation. It

competes with estradiol to bind to ER α. Then it will cause estrogen receptor alpha 124

level to decrease and lead to anti-proliferation. Furthermore, kaempferol can up-regulate p-p38 expression. It consequently increased the p-p53 and p21 protein level, and then decreased the expression of Cyclin D1, CDK-4, and PCNA protein.

The protein changes are closely correlated with the antiproliferative effects. These findings will help us understand the mechanisms of action of kaempferol’s antiproliferative effect on MCF-7 human breast cancer cells. These data are important in understanding the protective effects of fruits and vegetables in the prevention of breast cancer.

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References:

1. Siegel, R.; Ma, J. M.; Zou, Z. H.; Jemal, A., Cancer Statistics, 2014. Ca-a Cancer Journal for Clinicians 2014, 64, 9-29.

2. Jemal, A.; Bray, F.; Center, M. M.; Ferlay, J.; Ward, E.; Forman, D., Global Cancer Statistics. Ca-a Cancer Journal for Clinicians 2011, 61, 69-90.

3. Siegel, R.; DeSantis, C.; Virgo, K.; Stein, K.; Mariotto, A.; Smith, T.; Cooper, D.; Gansler, T.; Lerro, C.; Fedewa, S.; Lin, C. C.; Leach, C.; Cannady, R. S.; Cho, H. S.; Scoppa, S.; Hachey, M.; Kirch, R.; Jemal, A.; Ward, E., Cancer treatment and survivorship statistics, 2012. Ca-Cancer J Clin 2012, 62, 220-241.

4. Chu, Y. F.; Sun, J.; Wu, X. Z.; Liu, R. H., Antioxidant and anti proliferative activities of common vegetables. J Agr Food Chem 2002, 50, 6910-6916.

5. Sun, J.; Chu, Y. F.; Wu, X. Z.; Liu, R. H., Antioxidant and anti proliferative activities of common fruits. J Agr Food Chem 2002, 50, 7449-7454.

6. Buchner, F. L.; Bueno-de-Mesquita, H. B.; Linseisen, J.; Boshuizen, H. C.; Kiemeney, L. A. L. M.; Ros, M. M.; Overvad, K.; Hansen, L.; Tjonneland, A.; Raaschou-Nielsen, O.; Clavel-Chapelon, F.; Boutron-Ruault, M. C.; Touillaud, M.; Kaaks, R.; Rohrmann, S.; Boeing, H.; Nothlings, U.; Trichopoulou, A.; Zylis, D.; Dilis, V.; Palli, D.; Sieri, S.; Vineis, P.; Tumino, R.; Panico, S.; Peeters, P. H. M.; van Gils, C. H.; Lund, E.; Gram, I. T.; Braaten, T.; Martinez, C.; Agudo, A.; Arriola, L.; Ardanaz, E.; Navarro, C.; Rodriguez, L.; Manjer, J.; Wirfalt, E.; Hallmans, G.; Rasmuson, T.; Key, T. J.; Roddam, A. W.; Bingham, S.; Khaw, K. T.; Slimani, N.; Bofetta, P.; Byrnes, G.; Norat, T.; Michaud, D.; Riboli, E., Fruits and vegetables consumption and the risk of histological subtypes of lung cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC). Cancer Cause Control 2010, 21, 357-371.

7. Aune, D.; De Stefani, E.; Ronco, A.; Boffetta, P.; Deneo-Pellegrini, H.; Acosta, G.; Mendilaharsu, M., Fruits, Vegetables and the Risk of Cancer: a Multisite Case-Control Study in Uruguay. Asian Pac J Cancer P 2009, 10, 419-427.

8. Fung, T. T.; Hu, F. B.; Holmes, M. D.; Rosner, B. A.; Hunter, D. J.; Colditz, G. A.; Willett, W. C., Dietary patterns and the risk of postmenopausal breast cancer. Int J Cancer 2005, 116, 116-121.

9. Sieri, S.; Krogh, V.; Pala, V.; Muti, P.; Micheli, A.; Evangelista, A.; Tagliabue, G.; Berrino, F., Dietary patterns and risk of breast cancer in the ORDET cohort. Cancer Epidem Biomar 2004, 13, 567-572.

10. Demetriou, C. A.; Hadjisavvas, A.; Loizidou, M. A.; Loucaides, G.; Neophytou, I.; Sieri, S.; Kakouri, E.; Middleton, N.; Vineis, P.; Kyriacou, K., The mediterranean dietary pattern and breast cancer risk in Greek-Cypriot women: a case-control study. Bmc Cancer 2012, 12.

11. Schatzkin, A.; Mouw, T.; Park, Y.; Subar, A. F.; Kipnis, V.; Hollenbeck, A.; Leitzmann, M. F.; Thompson, F. E., Dietary fiber and whole-grain consumption in relation to colorectal cancer in the NIH-AARP Diet and Health Study. Am J Clin Nutr 2007, 85, 1353-1360.

126

12. Liu, R. H., Potential synergy of phytochemicals in cancer prevention: Mechanism of action. J Nutr 2004, 134, 3479s-3485s.

13. Ginjom, I., Health aspects of wine antioxidants: composition and in vitro bioavailability. 2009.

14. Epriliati, M. I., Nutriomic analysis of fresh and processed fruits through the development of an in-vitro model of human digestive system. 2008.

15. Johnson, L. R., Gastrointestinal Physiology. 6th ed.; Mosby: St. Louis, 2001.

16. Erlund, I.; Freese, R.; Marnietni, J.; Hakala, P.; Alfthan, G., Bioavailability of quercetin from berries and the diet. Nutrition and Cancer-an International Journal 2006, 54, 13-17.

17. Kang, J.; Badger, T. M.; Ronis, M. J. J.; Wu, X. L., Non-isoflavone Phytochemicals in Soy and Their Health Effects. J Agr Food Chem 2010, 58, 8119-8133.

18. Fernandez-Garcia, E.; Carvajal-Lerida, I.; Jaren-Galan, M.; Garrido-Fernandez, J.; Perez-Galvez, A.; Hornero-Mendez, D., Carotenoids bioavailability from foods: From plant pigments to efficient biological activities. Food Res Int 2012, 46, 438-450.

19. Schwartz, S. J., Food processing and matrix affects bioavailability of carotenoids and folates. Abstr Pap Am Chem S 2011, 241.

20. Mao, X.; Chai, Y. P.; Lin, Y. F., Dual regulation of the ATP-sensitive potassium channel by caffeine. American Journal of Physiology-Cell Physiology 2007, 292, C2239-C2258.

21. Nesaretnam, K.; Lim, E. J.; Reimann, K.; Lai, L. C., Effect of a carotene concentrate on the growth of human breast cancer cells and pS2 gene expression. Toxicology 2000, 151, 117-126.

22. Nesaretnam, K.; Radhakrishnan, A.; Selvaduray, K. R.; Reimann, K.; Pailoor, J.; Razak, G.; Mahmood, M. M.; Dahliwal, J. S., Effect of palm oil carotene on breast cancer tumorigenicity in nude mice. Lipids 2002, 37, 557-560.

23. Nagel, G.; Linseisen, J.; van Gils, C. H.; Peeters, P. H.; Boutron-Ruault, M. C.; Clavel-Chapelon, F.; Romieu, I.; Tjonneland, A.; Olsen, A.; Roswall, N.; Witt, P. M.; Overvad, K.; Rohrmann, S.; Kaaks, R.; Drogan, D.; Boeing, H.; Trichopoulou, A.; Stratigakou, V.; Zylis, D.; Engeset, D.; Lund, E.; Skeie, G.; Berrino, F.; Grioni, S.; Mattiello, A.; Masala, G.; Tumino, R.; Zanetti, R.; Ros, M. M.; Bueno-de-Mesquita, H. B.; Ardanaz, E.; Sanchez, M. J.; Huerta, J. M.; Amiano, P.; Rodriguez, L.; Manjer, J.; Wirfalt, E.; Lenner, P.; Hallmans, G.; Spencer, E. A.; Key, T. J.; Bingham, S.; Khaw, K. T.; Rinaldi, S.; Slimani, N.; Boffetta, P.; Gallo, V.; Norat, T.; Riboli, E., Dietary beta-carotene, vitamin C and E intake and breast cancer risk in the European Prospective Investigation into Cancer and Nutrition (EPIC). Breast Cancer Res Tr 2010, 119, 753-765.

24. Verhoeven, D. T. H.; Assen, N.; Goldbohm, R. A.; Dorant, E.; vantVeer, P.; Hermus, R. J. J.; vandenBrandt, P. A., Vitamins C and E, retinol, beta carotene and dietary fibre in relation to breast cancer risk: A prospective cohort study. Brit J Cancer 1997, 75, 149-155.

127

25. Michels, K. B.; Holmberg, L.; Bergkvist, L.; Ljung, H.; Bruce, A.; Wolk, A., Dietary antioxidant vitamins, retinol, and breast cancer incidence in a cohort of Swedish women. Int J Cancer 2001, 91, 563-567.

26. Zhang, S. M.; Tang, G. W.; Russell, R. M.; Mayzel, K. A.; Stampfer, M. J.; Willett, W. C.; Hunter, D. J., Measurement of retinoids and carotenoids in breast adipose tissue and a comparison of concentrations in breast cancer cases and control subjects. Am J Clin Nutr 1997, 66, 626-632.

27. Karas, M.; Amir, H.; Fishman, D.; Danilenko, M.; Segal, S.; Nahum, A.; Koifmann, A.; Giat, Y.; Levy, J.; Sharoni, Y., Lycopene interferes with cell cycle progression and insulin-like growth factor I signaling in mammary cancer cells. Nutr Cancer 2000, 36, 101-111.

28. Nahum, A.; Hirsch, K.; Danilenko, M.; Watts, C. K. W.; Prall, O. W. J.; Levy, J.; Sharoni, Y., Lycopene inhibition of cell cycle progression in breast and endometrial cancer cells is associated with reduction in cyclin D levels and retention of p27(Kip1) in the cyclin E-cdk2 complexes. Oncogene 2001, 20, 3428-3436.

29. Di Franco, R.; Calvanese, M.; Murino, P.; Manzo, R.; Guida, C.; Di Gennaro, D.; Anania, C.; Ravo, V., Skin toxicity from external beam radiation therapy in breast cancer patients: protective effects of Resveratrol, Lycopene, Vitamin C and anthocianin (Ixor (R)). Radiat Oncol 2012, 7.

30. Sesso, H. D.; Buring, J. E.; Zhang, S. M. M.; Norkus, E. P.; Gaziano, J. M., Dietary and plasma lycopene and the risk of breast cancer. Cancer Epidem Biomar 2005, 14, 1074-1081.

31. Shahrzad, S.; Aoyagi, K.; Winter, A.; Koyama, A.; Bitsch, I., Pharmacokinetics of gallic acid and its relative bioavailability from tea in healthy humans. J Nutr 2001, 131, 1207-1210.

32. Hsu, J. D.; Kao, S. H.; Ou, T. T.; Chen, Y. J.; Li, Y. J.; Wang, C. J., Gallic Acid Induces G2/M Phase Arrest of Breast Cancer Cell MCF-7 through Stabilization of p27(Kip1) Attributed to Disruption of p27(Kip1)/Skp2 Complex. J Agr Food Chem 2011, 59, 1996-2003.

33. Garcia-Rivera, D.; Delgado, R.; Bougarne, N.; Haegeman, G.; Berghe, W., Gallic acid indanone and mangiferin xanthone are strong determinants of immunosuppressive anti-tumour effects of Mangifera indica L. bark in MDA-MB231 breast cancer cells. Cancer Lett 2011, 305, 21-31.

34. Yin, M. C.; Lin, C. C.; Wu, H. C.; Tsao, S. M.; Hsu, C. K., Apoptotic Effects of Protocatechuic Acid in Human Breast, Lung, Liver, Cervix, and Prostate Cancer Cells: Potential Mechanisms of Action. J Agr Food Chem 2009, 57, 6468-6473.

35. Bourne, L. C.; Rice-Evans, C., Bioavailability of ferulic acid. Biochem Bioph Res Co 1998, 253, 222-227.

36. Anson, N. M.; van den Berg, R.; Havenaar, R.; Bast, A.; Haenen, G. R. M. M., Bioavailability of ferulic acid is determined by its bioaccessibility. J Cereal Sci 2009, 49, 296-300.

37. Serafim, T. L.; Carvalho, F. S.; Marques, M. P. M.; Calheiros, R.; Silva, T.; Garrido, J.; Milhazes, N.; Borges, F.; Roleira, F.; Silva, E. T.; Holy, J.; Oliveira, P. J., Lipophilic Caffeic and Ferulic Acid Derivatives Presenting Cytotoxicity against Human Breast Cancer Cells. Chem Res Toxicol 2011, 24, 763-774.

128

38. Watabe, M.; Hishikawa, K.; Takayanagi, A.; Shimizu, N.; Nakaki, T., Caffeic acid phenethyl ester induces apoptosis by inhibition of NF kappa B and activation of Fas in human breast cancer MCF-7 cells. J Biol Chem 2004, 279, 6017-6026.

39. Wang, B. S.; Chang, L. W.; Wu, H. C.; Huang, S. L.; Chu, H. L.; Huang, M. H., Antioxidant and antityrosinase activity of aqueous extracts of green asparagus. Food Chemistry 2011, 127, 141-146.

40. Lesser, S.; Wolffram, S., Oral bioavailability of the flavonol quercetin - A review. Current Topics in Nutraceutical Research 2006, 4, 239-256.

41. Hollman, P. C. H.; vanTrijp, J. M. P.; Buysman, M. N. C. P.; VanderGaag, M. S.; Mengelers, M. J. B.; deVries, J. H. M.; Katan, M. B., Relative bioavailability of the antioxidant flavonoid quercetin from various foods in man. Febs Lett 1997, 418, 152-156.

42. Chien, S. Y.; Wu, Y. C.; Chung, J. G.; Yang, J. S.; Lu, H. F.; Tsou, M. F.; Wood, W. G.; Kuo, S. J.; Chen, D. R., Quercetin-induced apoptosis acts through mitochondrial- and caspase-3-dependent pathways in human breast cancer MDA-MB-231 cells. Hum Exp Toxicol 2009, 28, 493-503.

43. Chou, C. C.; Yang, J. S.; Lu, H. F.; Ip, S. W.; Lo, C.; Wu, C. C.; Lin, J. P.; Tang, N. Y.; Chung, J. G.; Chou, M. J.; Teng, Y. H.; Chen, D. R., Quercetin-mediated Cell Cycle Arrest and Apoptosis Involving Activation of a Caspase Cascade through the Mitochondria! Pathway in Human Breast Cancer MCF-7 Cells. Arch Pharm Res 2010, 33, 1181-1191.

44. Avila, M. A.; Velasco, J. A.; Cansado, J.; Notario, V., Quercetin Mediates the down-Regulation of Mutant P53 in the Human Breast-Cancer Cell-Line Mda-Mb468. Cancer Res 1994, 54, 2424-2428.

45. Oh, S. J.; Kim, O.; Lee, J. S.; Kim, J. A.; Kim, M. R.; Choi, H. S.; Shim, J. H.; Kang, K. W.; Kim, Y. C., Inhibition of angiogenesis by quercetin in tamoxifen-resistant breast cancer cells. Food Chem Toxicol 2010, 48, 3227-3234.

46. Scambia, G.; Ranelletti, F. O.; Panici, P. B.; Piantelli, M.; Devincenzo, R.; Ferrandina, G.; Bonanno, G.; Capelli, A.; Mancuso, S., Quercetin Induces Type-Ii Estrogen-Binding Sites in Estrogen-Receptor-Negative (Mda-Mb231) and Estrogen-Receptor-Positive (Mcf-7) Human Breast-Cancer Cell-Lines. Int J Cancer 1993, 54, 462-466.

47. Oh, S. M.; Kim, Y. P.; Chung, K. H., Biphasic effects of kaempferol on the estrogenicity in human breast cancer cells. Arch Pharm Res 2006, 29, 354-362.

48. Kim, B. W.; Lee, E. R.; Min, H. M.; Jeong, H. S.; Ahn, J. Y.; Kim, J. H.; Choi, H. Y.; Choi, H.; Kim, E. Y.; Park, S. P.; Cho, S. G., Sustained ERK activation is involved in the kaempferol-induced apoptosis of breast cancer cells and is more evident under 3-D culture condition. Cancer Biol Ther 2008, 7, 1080-1089.

49. Diantini, A.; Subarnas, A.; Lestari, K.; Halimah, E.; Susilawati, Y.; Supriyatna; Julaeha, E.; Achmad, T. H.; Suradji, E. W.; Yamazaki, C.; Kobayashi, K.; Koyama, H.; Abdulah, R., Kaempferol-3-O-rhamnoside isolated from

129

the leaves of Schima wallichii Korth. inhibits MCF-7 breast cancer cell proliferation through activation of the caspase cascade pathway. Oncol Lett 2012, 3, 1069-1072.

50. Maggiolini, M.; Recchia, A. G.; Bonofiglio, D.; Catalano, S.; Vivacqua, A.; Carpino, A.; Rago, V.; Rossi, R.; Ando, S., The red wine phenolics piceatannol and myricetin act as agonists for estrogen receptor alpha in human breast cancer cells. J Mol Endocrinol 2005, 35, 269-281.

51. Lopez-Lazaro, M., Distribution and Biological Activities of the Flavonoid Luteolin. Mini-Rev Med Chem 2009, 9, 31-59.

52. Chen, Z. J.; Pan, H.; Li, L. P.; Ting, C.; Wang, R. W.; Zeng, S.; Jiang, H. D., The Oral Bioavailability of Luteolin and Apigenin in Chrysanthemum Morifolium Extract Is Much Higher Than When They Are Given as Pure Individual Compounds in Rats. Drug Metab Rev 2008, 40, 54-55.

53. Patel, D.; Shukla, S.; Gupta, S., Apigenin and cancer chemoprevention: progress, potential and promise (review). Int J Oncol 2007, 30, 233-45.

54. Seo, H. S.; Choi, H. S.; Kim, S. R.; Choi, Y. K.; Woo, S. M.; Shin, I.; Woo, J. K.; Park, S. Y.; Shin, Y. C.; Ko, S. K., Apigenin induces apoptosis via extrinsic pathway, inducing p53 and inhibiting STAT3 and NF kappa B signaling in HER2-overexpressing breast cancer cells. Mol Cell Biochem 2012, 366, 319-334.

55. Choi, E. J.; Kim, G. H., Apigenin Induces Apoptosis through a Mitochondria/Caspase-Pathway in Human Breast Cancer MDA-MB-453 Cells. J Clin Biochem Nutr 2009, 44, 260-265.

56. Choi, E. J.; Kim, G. H., Apigenin causes G(2)/M arrest associated with the modulation of p21(Cip1) and Cdc2 and activates p53-dependent apoptosis pathway in human breast cancer SK-BR-3 cells. J Nutr Biochem 2009, 20, 285-290.

57. Mafuvadze, B.; Benakanakere, I.; Hyder, S. M., Apigenin Inhibits Progestin-Induced VEGF at Both mRNA and Protein Secretion Level in T47-D Human Breast Cancer Cells. Endocr Rev 2010, 31.

58. Lee, W. J.; Chen, W. K.; Wang, C. J.; Lin, W. L.; Tseng, T. H., Apigenin inhibits HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and beta 4 integrin function in MDA-MB-231 breast cancer cells. Toxicol Appl Pharm 2008, 226, 178-191.

59. Kim, M. J.; Woo, J. S.; Kwon, C. H.; Kim, J. H.; Kim, Y. K.; Kim, K. H., Luteolin induces apoptotic cell death through AIF nuclear translocation mediated by activation of ERK and p38 in human breast cancer cell lines. Cell Biol Int 2012, 36, 339-344.

60. van het Hof, K. H.; Kivits, G. A. A.; Weststrate, J. A.; Tijburg, L. B. M., Bioavailability of catechins from tea: the effect of milk. Eur J Clin Nutr 1998, 52, 356-359.

61. Del Rio, D.; Calani, L.; Scazzina, F.; Jechiu, L.; Cordero, C.; Brighenti, F., Bioavailability of catechins from ready-to-drink tea. Nutrition 2010, 26, 528-533.

62. Katiyar, S. K.; Mukhtar, H., Tea antioxidants in cancer chemoprevention. J Cell Biochem 1997, 59-67. 130

63. Ahmad, N.; Mukhtar, H., Green tea polyphenols and cancer: Biologic mechanisms and practical implications. Nutr Rev 1999, 57, 78-83.

64. Sen, T.; Moulik, S.; Dutta, A.; Choudhury, P. R.; Banerji, A.; Das, S.; Roy, M.; Chatterjee, A., Multifunctional effect of epigallocatechin-3-gallate (EGCG) in downregulation of gelatinase-A (MMP-2) in human breast cancer cell line MCF-7. Life Sci 2009, 84, 194-204.

65. Sen, T.; Dutta, A.; Chatterjee, A., Epigallocatechin-3-gallate (EGCG) downregulates gelatinase-B (MMP-9) by involvement of FAK/ERK/NF kappa B and AP-1 in the human breast cancer cell line MDA-MB-231. Anti-Cancer Drug 2010, 21, 632-644.

66. Kim, J. Y.; Zhang, X. W.; Rieger-Christ, K. M.; Summerhayes, I. C.; Wazer, D. E.; Paulson, K. E.; Yee, A. S., Suppression of Wnt signaling by the green tea compound (-)-epigallocatechin 3-gallate (EGCG) in invasive breast cancer cells - Requirement of the transcriptional repressor HBP1. J Biol Chem 2006, 281, 10865-10875.

67. Guo, S. Q.; Yang, S. W.; Taylor, C.; Sonenshein, G. E., Green tea polyphenol epigallocatechin-3 gallate (EGCG) affects gene expression of breast cancer cells transformed by the carcinogen 7,12-dimethylbenz[a]anthracene. J Nutr 2005, 135, 2978s-2986s.

68. Yamamoto, S.; Sobue, T.; Kobayashi, M.; Sasaki, S.; Tsugane, S., Soy, isoflavones, and breast cancer risk in Japan. J Natl Cancer Inst 2003, 95, 906-13.

69. Cohen, R.; Schwartz, B.; Peri, I.; Shimoni, E., Improving Bioavailability and Stability of Genistein by Complexation with High-Amylose Corn Starch. J Agr Food Chem 2011, 59, 7932-7938.

70. Marik, R.; Allu, M.; Anchoori, R.; Stearns, V.; Umbricht, C. B.; Khan, S., Potent genistein derivatives as inhibitors of estrogen receptor alpha-positive breast cancer. Cancer Biol Ther 2011, 11, 883-892.

71. Park, J. Y.; Kang, K. S.; Sohn, Y. K., Molecular Mechanism of the G2/M Arrest in Breast Cancer Cell Lines (T47D and MDA-MB231) Induced by Genistein. J Breast Cancer 2010, 13, 382-391.

72. He, X. J.; Liu, D.; Liu, R. H., Sodium Borohydride/Chloranil-Based Assay for Quantifying Total Flavonoids. J Agr Food Chem 2008, 56, 9337-9344.

73. He, X.; Liu, R. H., Phytochemicals of Apple Peels: Isolation, Structure Elucidation, and Their Antiproliferative and Antioxidant Activities. J Agr Food Chem 2008, 56, 9905-9910.

74. Montales, M. T. E.; Rahal, O. M.; Kang, J.; Rogers, T. J.; Prior, R. L.; Wu, X. L.; Simmen, R. C., Repression of mammosphere formation of human breast cancer cells by soy isoflavone genistein and blueberry polyphenolic acids suggests diet-mediated targeting of cancer stem-like/progenitor cells. Carcinogenesis 2012, 33, 652-660.

75. Ra, Y. M.; Yim, J. S.; Song, K. H.; Ko, D. G.; Choi, I. S.; Choi, W. J.; Yoon, D. S., The effects of genistein to expression of fatty acid synthase in breast cancer cells. J Breast Cancer 2007, 10, 127-133.

76. Farina, H. G.; Pomies, M.; Alonso, D. F.; Gomez, D. E., Antitumor and antiangiogenic activity of soy isoflavone genistein in mouse models of melanoma and breast cancer. Oncol Rep 2006, 16, 885-891. 131

77. Privat, M.; Aubel, C.; Arnould, S.; Communal, Y.; Ferrara, M.; Bignon, Y. J., Breast cancer cell response to genistein is conditioned by BRCA1 mutations. Biochem Bioph Res Co 2009, 379, 785-789.

78. Shen, Q.; Li, X.; Yuan, D.; Jia, W., Enhanced Oral Bioavailability of Daidzein by Self-Microemulsifying Drug Delivery System. Chem Pharm Bull 2010, 58, 639-643.

79. Jin, S.; Zhang, Q. Y.; Kang, X. M.; Wang, J. X.; Zhao, W. H., Daidzein induces MCF-7 breast cancer cell apoptosis via the mitochondrial pathway. Ann Oncol 2010, 21, 263-268.

80. Choi, E. J.; Kim, G. H., Daidzein causes cell cycle arrest at the G1 and G2/M phases in human breast cancer MCF-7 and MDA-MB-453 cells. Phytomedicine 2008, 15, 683-690.

81. Bawadi, H. A.; Bansode, R. R.; Trappey, A.; Truax, R. E.; Losso, J. N., Inhibition of Caco-2 colon, MCF-7 and Hs578T breast, and DU 145 prostatic cancer cell proliferation by water-soluble black bean condensed tannins. Cancer Lett 2005, 218, 153-162.

82. Park, I. H.; Ro, J.; Nam, B. H.; Kwon, Y.; Lee, K. S., Potential antitumor effects of nitrogen-containing bisphosphonate in hormone receptor negative breast cancer patients with bone metastases. Bmc Cancer 2009, 9.

83. Merrell, M. A.; Wakchoure, S.; Lehenkari, P. P.; Harris, K. W.; Selander, K. S., Inhibition of the mevalonate pathway and activation of p38 MAP kinase are independently regulated by nitrogen-containing bisphosphonates in breast cancer cells. Eur J Pharmacol 2007, 570, 27-37.

84. Tseng, E.; Scott-Ramsay, E. A.; Morris, M. E., Dietary organic isothiocyanates are cytotoxic in human breast cancer MCF-7 and mammary epithelial MCF-12A cell lines. Exp Biol Med 2004, 229, 835-842.

85. Kang, L. G.; Ding, L.; Wang, Z. Y., Isothiocyanates repress estrogen receptor a expression in breast cancer cells. Oncol Rep 2009, 21, 185-192.

86. Hsieh, T. C.; Wu, J. M., Suppression of cell proliferation and gene expression by combinatorial synergy of EGCG, resveratrol and gamma-tocotrienol in estrogen receptor-positive MCF-7 breast cancer cells. Int J Oncol 2008, 33, 851-859.

87. Sahin, K.; Tuzcu, M.; Sahin, N.; Akdemir, F.; Ozercan, I.; Bayraktar, S.; Kucuk, O., Inhibitory Effects of Combination of Lycopene and Genistein on 7,12-Dimethyl Benz(a)anthracene-Induced Breast Cancer in Rats. Nutr Cancer 2011, 63, 1279-1286.

88. Scambia, G.; Ranelletti, F. O.; Panici, P. B.; Devincenzo, R.; Bonanno, G.; Ferrandina, G.; Piantelli, M.; Bussa, S.; Rumi, C.; Cianfriglia, M.; Mancuso, S., Quercetin Potentiates the Effect of Adriamycin in a Multidrug-Resistant Mcf-7 Human Breast-Cancer Cell-Line - P-Glycoprotein as a Possible Target. Cancer Chemoth Pharm 1994, 34, 459-464.

89. Akbas, S. H.; Timur, M.; Ozben, T., The effect of quercetin on topotecan cytotoxicity in MCF-7 and MDA-MB 231 human breast cancer cells. J Surg Res 2005, 125, 49-55.

132

90. Ferenc, P.; Solar, P.; Kleban, J.; Mikes, J.; Fedorcko, P., Down-regulation of Bcl-2 and Akt induced by combination of photoactivated hypericin and genistein in human breast cancer cells. J Photoch Photobio B 2010, 98, 25-34.

91. Wong, M. Y.; Chiu, G. N. C., Simultaneous liposomal delivery of quercetin and vincristine for enhanced estrogen-receptor-negative breast cancer treatment. Anti-Cancer Drug 2010, 21, 401-410.

92. Lattrich, C.; Lubig, J.; Springwald, A.; Goerse, R.; Ortmann, O.; Treeck, O., Additive effects of trastuzumab and genistein on human breast cancer cells. Anti-Cancer Drug 2011, 22, 253-261.

93. Staedler, D.; Idrizi, E.; Kenzaoui, B. H.; Juillerat-Jeanneret, L., Drug combinations with quercetin: doxorubicin plus quercetin in human breast cancer cells. Cancer Chemoth Pharm 2011, 68, 1161-1172.

94. Pradhan, D.; GitanjaliTripathy; Patanaik, S., Anticancer Activity of Limonia acidissima Linn (Rutaceae) Fruit Extracts on Human Breast Cancer Cell Lines. Trop J Pharm Res 2012, 11, 413-419.

95. Plastina, P.; Bonofiglio, D.; Vizza, D.; Fazio, A.; Rovito, D.; Giordano, C.; Barone, I.; Catalano, S.; Gabriele, B., Identification of bioactive constituents of Ziziphus jujube fruit extracts exerting antiproliferative and apoptotic effects in human breast cancer cells. J Ethnopharmacol 2012, 140, 325-332.

96. Dikmen, M.; Ozturk, N.; Ozturk, Y., The Antioxidant Potency of Punica granatum L. Fruit Peel Reduces Cell Proliferation and Induces Apoptosis on Breast Cancer. J Med Food 2011, 14, 1638-1646.

97. Dai, Y. M.; Hogan, S.; Schmelz, E. M.; Ju, Y. H.; Canning, C.; Zhou, K. Q., Selective Growth Inhibition of Human Breast Cancer Cells by Graviola Fruit Extract In Vitro and In Vivo Involving Downregulation of EGFR Expression. Nutr Cancer 2011, 63, 795-801.

98. Wang, X. J.; Yuan, S. L.; Wang, J.; Lin, P.; Liu, G. J.; Lu, Y. R.; Zhang, J.; Wang, W. D.; Wei, Y. Q., Anticancer activity of litchi fruit pericarp extract against human breast cancer in vitro and in vivo. Toxicol Appl Pharm 2006, 215, 168-178.

99. Masala, G.; Assedi, M.; Bendinelli, B.; Ermini, I.; Sieri, S.; Grioni, S.; Sacerdote, C.; Ricceri, F.; Panico, S.; Mattiello, A.; Tumino, R.; Giurdanella, M. C.; Berrino, F.; Saieva, C.; Palli, D., Fruit and vegetables consumption and breast cancer risk: the EPIC Italy study. Breast Cancer Res Tr 2012, 132, 1127-1136.

100. Boggs, D. A.; Palmer, J. R.; Wise, L. A.; Spiegelman, D.; Stampfer, M. J.; Adams-Campbell, L. L.; Rosenberg, L., Fruit and Vegetable Intake in Relation to Risk of Breast Cancer in the Black Women's Health Study. Am J Epidemiol 2010, 172, 1268-1279.

101. Egeberg, R.; Olsen, A.; Loft, S.; Christensen, L.; Johnsen, N. F.; Overvad, K.; Tjonneland, A., Intake of whole grain products and risk of breast cancer by hormone receptor status and histology among postmenopausal women. Int J Cancer 2009, 124, 745-750.

102. Zhang, C. X.; Ho, S. C.; Chen, Y. M.; Fu, J. H.; Cheng, S. Z.; Lin, F. Y., Greater vegetable and fruit intake is associated with a lower risk of breast cancer among Chinese women. Int J Cancer 2009, 125, 181-188.

133

103. van Gils, C. H.; Peeters, P. H. T.; Bueno-De-Mesquita, H. B.; Boshuizen, H. C.; Lahmann, P. H.; Clavel-Chapelon, F.; Thiebaut, A.; Kesse, E.; Sieri, S.; Palli, D.; Tumino, R.; Panico, S.; Vineis, P.; Gonzalez, C. A.; Ardanaz, E.; Sanchez, M. J.; Amiano, P.; Navarro, C.; Quiros, J. R.; Key, T. J.; Allen, N.; Khaw, K. T.; Bingham, S. A.; Psaltopoulou, T.; Koliva, M.; Trichopoulou, A.; Nagel, G.; Linseisen, J.; Boeing, H.; Berglund, G.; Wirfalt, E.; Hallmans, G.; Lenner, P.; Overvad, K.; Tjonneland, A.; Olsen, A.; Lund, E.; Engeset, D.; Alsaker, E.; Norat, T. A.; Kaaks, R.; Slimani, N.; Riboli, E., Consumption of vegetables and fruits and risk of breast cancer. Jama-J Am Med Assoc 2005, 293, 183-193.

104. Do, M. H.; Lee, S. S.; Kim, J. Y.; Jung, P. J.; Lee, M. H., Fruits, vegetables, soy foods and breast cancer in pre- and postmenopausal Korean women: A case-control study. Int J Vitam Nutr Res 2007, 77, 130-141.

105. Zhao, Q. S.; Xie, B. X.; Yan, J.; Zhao, F. C.; Xiao, J.; Yao, L. Y.; Zhao, B.; Huang, Y. X., In vitro antioxidant and antitumor activities of polysaccharides extracted from Asparagus officinalis. Carbohydrate Polymers 2012, 87, 392-396.

106. Vaughan, J. G. G., Catherine Alison; Nicholson, Barbara, The New Oxford Book of Food Plants. Oxford University Press: 1997.

107. Gebhardt, S.; Lemar, L.; Haytowitz, D.; Pehrsson, P.; Nickle, M.; Showell, B.; Thomas, R.; Exler, J.; Holden, J., USDA national nutrient database for standard reference, release 21. 2008.

108. Houk, V.; Oakley Jr, G.; Erickson, J. D.; Mulinare, J.; James, L. M., Recommendations for the use of folic acid to reduce the number of cases of spina bifida and other neural tube defects. Morbidity and Mortality Weekly Report 1992, 41, 1-7.

109. Giovannucci, E.; Rimm, E. B.; Stampfer, M. J.; Colditz, G. A.; Ascherio, A.; Willett, W. C., Intake of fat, meat, and fiber in relation to risk of colon cancer in men. Cancer Res 1994, 54, 2390-2397.

110. Sato, Y.; Honda, Y.; Hayashida, N.; Iwamoto, J.; Kanoko, T.; Satoh, K., Vitamin K deficiency and osteopenia in elderly women with Alzheimer’s disease. Archives of physical medicine and rehabilitation 2005, 86, 576-581.

111. McDowell, L. R., Minerals in animal and human nutrition. Elsevier Science BV: 2003.

112. Kieslich, M.; Porto, L.; Lanfermann, H.; Jacobi, G.; Schwabe, D.; Böhles, H., Cerebrovascular complications of L-asparaginase in the therapy of acute lymphoblastic leukemia. Journal of pediatric hematology/oncology 2003, 25, 484-487.

113. Sanae, M.; Yasuo, A., Green Asparagus (Asparagus officinalis) Prevented Hypertension by an Inhibitory Effect on Angiotensin-Converting Enzyme Activity in the Kidney of Spontaneously Hypertensive Rats. J Agr Food Chem 2013, 61, 5520-5525.

114. Sakurai, T.; Ito, T.; Wakame, K.; Kitadate, K.; Arai, T.; Ogasawara, J.; Kizaki, T.; Sato, S.; Ishibashi, Y.; Fujiwara, T.; Akagawa, K.; Ishida, H.; Ohno, H., Enzyme-treated Asparagus officinalis Extract Shows Neuroprotective Effects and Attenuates Cognitive Impairment in Senescence-accelerated Mice. Natural Product Communications 2014, 9, 101-106.

134

115. Vazquez-Castilla, S.; De la Puerta, R.; Gimenez, M. D. G.; Fernandez-Arche, M. A.; Guillen-Bejarano, R., Bioactive Constituents from "Triguero" Asparagus Improve the Plasma Lipid Profile and Liver Antioxidant Status in Hypercholesterolemic Rats. International Journal of Molecular Sciences 2013, 14, 21227-21239.

116. Bousserouel, S.; Le Grandois, J.; Gosse, F.; Werner, D.; Barth, S. W.; Marchioni, E.; Marescaux, J.; Raul, F., Methanolic extract of white asparagus shoots activates TRAIL apoptotic death pathway in human cancer cells and inhibits colon carcinogenesis in a preclinical model. Int J Oncol 2013, 43, 394-404.

117. Shao, Y.; Chin, C. K.; Ho, C. T.; Ma, W.; Garrison, S. A.; Huang, M. T., Anti-tumor activity of the crude saponins obtained from asparagus. Cancer Lett 1996, 104, 31-36.

118. Shao, Y.; Poobrasert, O.; Kennelly, E. J.; Chin, C. K.; Ho, C. T.; Huang, M. T.; Garrison, S. A.; Cordell, G. A., Cytotoxic activity of steroidal saponins from Asparagus officinalis. Ixth International Asparagus Symposium 1999, 277-282.

119. Chin, C. K.; Garrison, S. A., Functional elements from asparagus for human health. Proceedings of the Xith International Asparagus Symposium 2008, 219-225.

120. Wang, J.; Liu, Y.; Zhao, J.; Zhang, W.; Pang, X., Saponins extracted from by‐product of Asparagus officinalis L. suppress tumour cell migration and invasion through targeting Rho GTPase signalling pathway. Journal of the Science of Food and Agriculture 2013, 93, 1492-1498.

121. Oakenfull, D.; Sidhu, G. S., Could Saponins Be a Useful Treatment for Hypercholesterolemia. European Journal of Clinical Nutrition 1990, 44, 79-88.

122. Huang, X. F.; Lin, Y. Y.; Kong, L. Y., Steroids from the roots of Asparagus officinalis and their cytotoxic activity. Journal of Integrative Plant Biology 2008, 50, 717-722.

123. Ji, C. F.; Ji, Y. B.; Min, Y. C., The effect of Asparagus Officinalis Polysaccharide on Erythrocyte of S180 Mice Played in Immunological Reaction of lymphocytes. Smart Materials and Nanotechnology in Engineering 2012, 345, 99-103.

124. Bhagwat, S. A.; Haytowitz, D. B.; Harnly, J.; Holden, J. M., Update of the USDA database for the flavonoid content of selected foods. J Nutr 2005, 135, 3050s-3050s.

125. Calderon-Montano, J. M.; Burgos-Moron, E.; Perez-Guerrero, C.; Lopez-Lazaro, M., A Review on the Dietary Flavonoid Kaempferol. Mini-Rev Med Chem 2011, 11, 298-344.

126. Barve, A.; Chen, C.; Hebbar, V.; Desiderio, J.; Saw, C. L. L.; Kong, A. N., Metabolism, Oral Bioavailability and Pharmacokinetics of Chemopreventive Kaempferol in Rats. Biopharm Drug Dispos 2009, 30, 356-365.

127. DuPont, M. S.; Day, A. J.; Bennett, R. N.; Mellon, F. A.; Kroon, P. A., Absorption of kaempferol from endive, a source of kaempferol-3-glucuronide, in humans. Eur J Clin Nutr 2004, 58, 947-954.

128. Bonetti, A.; Marotti, I.; Dinelli, G., Urinary excretion of kaempferol from common beans (Phaseolus vulgaris L.) in humans. Int J Food Sci Nutr 2007, 58, 261-269. 135

129. Luo, H. T.; Jiang, B. B.; Li, B. Y.; Li, Z. L.; Jiang, B. H.; Chen, Y. C., Kaempferol nanoparticles achieve strong and selective inhibition of ovarian cancer cell viability. Int J Nanomed 2012, 7, 3951-3959.

130. Ewald, C.; Fjelkner-Modig, S.; Johansson, K.; Sjoholm, I.; Akesson, B., Effect of processing on major flavonoids in processed onions, green beans, and peas. Food Chem 1999, 64, 231-235.

131. Winkler, S.; Faragher, J.; Franz, P.; Imsic, M.; Jones, R., Glucoraphanin and flavonoid levels remain stable during simulated transport and marketing of broccoli (Brassica oleracea var. italica) heads. Postharvest Biol Tec 2007, 43, 89-94.

132. Hoffmann-Ribani, R.; Huber, L. S.; Rodriguez-Amaya, D. B., Flavonols in fresh and processed Brazilian fruits. J Food Compos Anal 2009, 22, 263-268.

133. Bobe, G.; Albert, P. S.; Sansbury, L. B.; Lanza, E.; Schatzkin, A.; Colburn, N. H.; Cross, A. J., Interleukin-6 as a Potential Indicator for Prevention of High-Risk Adenoma Recurrence by Dietary Flavonols in the Polyp Prevention Trial. Cancer Prev Res 2010, 3, 764-775.

134. Cui, Y.; Morgenstern, H.; Greenland, S.; Tashkin, D. P.; Mao, J. T.; Cai, L.; Cozen, W.; Mack, T. M.; Lu, Q. Y.; Zhang, Z. F., Dietary flavonoid intake and lung cancer - A population-based case-control study. Cancer 2008, 112, 2241-2248.

135. Bobe, G.; Weinstein, S. J.; Albanes, D.; Hirvonen, T.; Ashby, J.; Taylor, P. R.; Virtamo, J.; Stolzenberg-Solomoni, R. Z., Flavonoid intake and risk of pancreatic cancer in male smokers (Finland). Cancer Epidem Biomar 2008, 17, 553-562.

136. Gates, M. A.; Tworoger, S. S.; Hecht, J. L.; De Vivo, I.; Rosner, B.; Hankinson, S. E., A prospective study of dietary flavonoid intake and incidence of epithelial ovarian cancer. Int J Cancer 2007, 121, 2225-2232.

137. Nirmala, P.; Ramanathan, M., Effect of kaempferol on lipid peroxidation and antioxidant status in 1,2-dimethyl hydrazine induced colorectal carcinoma in rats. European Journal of Pharmacology 2011, 654, 75-79.

138. Zhang, Y. Q.; Chen, A. R. Y.; Li, M.; Chen, C. Y.; Yao, Q. Z., Ginkgo biloba extract kaempferol inhibits cell proliferation and induces apoptosis in pancreatic cancer cells. Journal of Surgical Research 2008, 148, 17-23.

139. Li, W.; Du, B. N.; Wang, T. Y.; Wang, S. L.; Zhang, J. H., Kaempferol induces apoptosis in human HCT116 colon cancer cells via the Ataxia-Telangiectasia Mutated-p53 pathway with the involvement of p53 Upregulated Modulator of Apoptosis. Chemico-Biological Interactions 2009, 177, 121-127.

140. Luo, H. T.; Rankin, G. O.; Liu, L. Z.; Daddysman, M. K.; Jiang, B. H.; Chen, Y. C., Kaempferol Inhibits Angiogenesis and VEGF Expression Through Both HIF Dependent and Independent Pathways in Human Ovarian Cancer Cells. Nutrition and Cancer-an International Journal 2009, 61, 554-563.

141. Nair, S.; Li, W. G.; Kong, A. N. T., Natural dietary anti-cancer chemopreventive compounds: redox-mediated differential signaling mechanisms in cytoprotection of normal cells versus cytotoxicity in tumor cells. Acta Pharmacol Sin 2007, 28, 459-472. 136

142. Acharya, A.; Das, I.; Chandhok, D.; Saha, T., Redox regulation in cancer A double-edged sword with therapeutic potential. Oxid Med Cell Longev 2010, 3, 23-34.

143. Song, W.; Derito, C. M.; Liu, M. K.; He, X. J.; Dong, M.; Liu, R. H., Cellular Antioxidant Activity of Common Vegetables. J Agr Food Chem 2010, 58, 6621-6629.

144. Wolfe, K. L.; Kang, X. M.; He, X. J.; Dong, M.; Zhang, Q. Y.; Liu, R. H., Cellular antioxidant activity of common fruits. J Agr Food Chem 2008, 56, 8418-8426.

145. Bhouri, W.; Ben Sghaier, M.; Kilani, S.; Bouhlel, I.; Dijoux-Franca, M. G.; Ghedira, K.; Ghedira, L. C., Evaluation of antioxidant and antigenotoxic activity of two flavonoids from Rhamnus alaternus L. (Rhamnaceae): Kaempferol 3-O-beta-isorhamninoside and rhamnocitrin 3-O-beta-isorhamninoside. Food Chem Toxicol 2011, 49, 1167-1173.

146. Park, J. S.; Rho, H. S.; Kim, D. H.; Chang, I. S., Enzymatic preparation of kaempferol from green tea seed and its antioxidant activity. J Agr Food Chem 2006, 54, 2951-2956.

147. Sharma, V.; Joseph, C.; Ghosh, S.; Agarwal, A.; Mishra, M. K.; Sen, E., Kaempferol induces apoptosis in glioblastoma cells through oxidative stress. Mol Cancer Ther 2007, 6, 2544-2553.

148. Filomeni, G.; Desideri, E.; Cardaci, S.; Graziani, I.; Piccirillo, S.; Rotilio, G.; Ciriolo, M. R., Carcinoma cells activate AMP-activated protein kinase-dependent autophagy as survival response to kaempferol-mediated energetic impairment. Autophagy 2010, 6, 202-216.

149. Blagosklonny, M. V.; Pardee, A. B., The restriction point of the cell cycle. Cell Cycle 2002, 1, 102-109.

150. Sherr, C. J., Cancer cell cycles. Science 1996, 274, 1672-1677.

151. Robert, A., The Biology of Cancer. 3rd edition ed.; Garland Science, Taylor & Francis Group, LLC: 2007.

152. Xu, W.; Liu, J. W.; Li, C. L.; Wu, H. Z.; Liu, Y. W., Kaempferol-7-O-beta-D-glucoside (KG) isolated from Smilax china L. rhizome induces G(2)/M phase arrest and apoptosis on HeLa cells in a p53-independent manner. Cancer Lett 2008, 264, 229-240.

153. Li, Y. L.; Gan, G. P.; Zhang, H. Z.; Wu, H. Z.; Li, C. L.; Huang, Y. P.; Liu, Y. W.; Liu, J. W., A flavonoid glycoside isolated from Smilax china L. rhizome in vitro anticancer effects on human cancer cell lines. J Ethnopharmacol 2007, 113, 115-124.

154. Bestwick, C. S.; Milne, L.; Pirie, L.; Duthie, S. J., The effect of short-term kaempferol exposure on reactive oxygen levels and integrity of human (HL-60) leukaemic cells. Bba-Mol Basis Dis 2005, 1740, 340-349.

155. Bestwick, C. S.; Milne, L.; Duthie, S. J., Kaempferol induced inhibition of HL-60 cell growth results from a heterogeneous response, dominated by cell cycle alterations. Chem-Biol Interact 2007, 170, 76-85.

156. Danial, N. N.; Korsmeyer, S. J., Cell death: Critical control points. Cell 2004, 116, 205-219.

137

157. Reed, J. C., Mechanisms of apoptosis. Am J Pathol 2000, 157, 1415-1430.

158. Vousden, K. H.; Lu, X., Live or let die: The cell's response to p53. Nat Rev Cancer 2002, 2, 594-604.

159. Kang, G. Y.; Lee, E. R.; Kim, J. H.; Jung, J. W.; Lim, J.; Kim, S. K.; Cho, S. G.; Kim, K. P., Downregulation of PLK-1 expression in kaempferol-induced apoptosis of MCF-7 cells. Eur J Pharmacol 2009, 611, 17-21.

160. Marfe, G.; Tafani, M.; Indelicato, M.; Sinibaldi-Salimei, P.; Reali, V.; Pucci, B.; Fini, M.; Russo, M. A., Kaempferol Induces Apoptosis in Two Different Cell Lines Via Akt Inactivation, Bax and SIRT3 Activation, and Mitochondrial Dysfunction. J Cell Biochem 2009, 106, 643-650.

161. Luo, H. T.; Rankin, G. O.; Li, Z. L.; DePriest, L.; Chen, Y. C., Kaempferol induces apoptosis in ovarian cancer cells through activating p53 in the intrinsic pathway. Food Chem 2011, 128, 513-519.

162. Kang, J. W.; Kim, J. H.; Song, K.; Kim, S. H.; Yoon, J. H.; Kim, K. S., Kaempferol and Quercetin, Components of Ginkgo biloba Extract (EGb 761), Induce Caspase-3-Dependent Apoptosis in Oral Cavity Cancer Cells. Phytother Res 2010, 24, S77-S82.

163. Bhouri, W.; Bouhlel, I.; Boubaker, J.; Kilani, S.; Ghedira, K.; Ghedira, L. C., Induction of apoptosis in human lymphoblastoid cells by kaempferol 3-O-beta-isorhamninoside and rhamnocitrin 3-O-beta-isorhamninoside from Rhamnus alaternus L. (Rhamnaceae). Cell Proliferat 2011, 44, 283-290.

164. Huang, W. W.; Chiu, Y. J.; Fan, M. J.; Lu, H. F.; Yeh, H. F.; Li, K. H.; Chen, P. Y.; Chung, J. G.; Yang, J. S., Kaempferol induced apoptosis via endoplasmic reticulum stress and mitochondria-dependent pathway in human osteosarcoma U-2 OS cells. Mol Nutr Food Res 2010, 54, 1585-1595.

165. Nguyen, T. T. T.; Tran, E.; Ong, C. K.; Lee, S. K.; Do, P. T.; Huynh, T. T.; Nguyen, T. H.; Lee, J. J.; Tan, Y.; Ong, C. S.; Huynh, H., Kaempferol-induced growth inhibition and apoptosis in A549 lung cancer cells is mediated by activation of MEK-MAPK. J Cell Physiol 2003, 197, 110-121.

166. Folkman, J., Tumor Angiogenesis. Adv Cancer Res 1985, 43, 175-203.

167. Folkman, J., Anti-Angiogenesis - New Concept for Therapy of Solid Tumors. Ann Surg 1972, 175, 409-&.

168. Littlepage, L. E.; Egeblad, M.; Werb, Z., Coevolution of cancer and stromal cellular responses. Cancer Cell 2005, 7, 499-500.

169. McCawley, L. J.; Matrisian, L. M., Matrix metalloproteinases: they're not just for matrix anymore! Curr Opin Cell Biol 2001, 13, 534-540.

170. Kim, J. D.; Liu, L. P.; Guo, W. M.; Meydani, M., Chemical structure of flavonols in relation to modulation of angiogenesis and immune-endothelial cell adhesion. Journal of Nutritional Biochemistry 2006, 17, 165-176.

171. Labbe, D.; Provencal, M.; Lamy, S.; Boivin, D.; Gingras, D.; Beliveau, R., The Flavonols Quercetin, Kaempferol, and Myricetin Inhibit Hepatocyte Growth Factor-Induced Medulloblastoma Cell Migration. J Nutr 2009, 139, 646-652.

138

172. Phromnoi, K.; Yodkeeree, S.; Anuchapreeda, S.; Limtrakul, P., Inhibition of MMP-3 activity and invasion of the MDA-MB-231 human invasive breast carcinoma cell line by bioflavonoids. Acta Pharmacol Sin 2009, 30, 1169-1176.

173. Gardner, W. U., Hormonal Aspects of Experimental Tumorigenesis. Adv Cancer Res 1953, 1, 173-232.

174. Kupperman, H. S.; Greenblatt, R. B., The Relationship between the Steroid Hormones and Experimentally-Induced Tumors in Rats. Anat Rec 1946, 96, 529-530.

175. Waldum, H. L.; Brenna, E.; Sandvik, A. K.; Syversen, U.; Falkmer, S., Hormones and carcinogenesis. Endocr-Relat Cancer 1998, 5, 45-48.

176. Weisburg.Jh; Yamamoto, R. S.; Korzis, J.; Weisburg.Ek, Liver Cancer - Neonatal Estrogen Enhances Induction by a Carcinogen. Science 1966, 154, 673-&.

177. Aylsworth, C. F.; Vanvugt, D. A.; Sylvester, P. W.; Meites, J., Role of Estrogen and Prolactin in Stimulation of Carcinogen-Induced Mammary-Tumor Development by a High-Fat Diet. Cancer Res 1984, 44, 2835-2840.

178. Lupulescu, A., Estrogen Use and Cancer Incidence - a Review. Cancer Invest 1995, 13, 287-295.

179. Pasqualini, J. R., The selective estrogen enzyme modulators in breast cancer: a review. Bba-Rev Cancer 2004, 1654, 123-143.

180. Wang, C. F.; Makela, T.; Hase, T.; Adlercreutz, H.; Kurzer, M. S., Lignans and Flavonoids Inhibit Aromatase Enzyme in Human Preadipocytes. J Steroid Biochem 1994, 50, 205-212.

181. Hung, H., Inhibition of estrogen receptor alpha expression and function in MCF-7 cells by kaempferol. Journal of Cellular Physiology 2004, 198, 197-208.

182. Leung, L. K.; Po, L. S.; Lau, T. Y.; Yuen, Y. M., Effect of dietary flavonols on oestrogen receptor transactivation and cell death induction. Brit J Nutr 2004, 91, 831-839.

183. Sobolewski, C.; Cerella, C.; Dicato, M.; Ghibelli, L.; Diederich, M., The Role of Cyclooxygenase-2 in Cell Proliferation and Cell Death in Human Malignancies. International Journal of Cell Biology 2010, 2010.

184. Munoz-Espada, A. C.; Watkins, B. A., Cyanidin attenuates PGE(2) production and cyclooxygenase-2 expression in LNCaP human prostate cancer cells. J Nutr Biochem 2006, 17, 589-596.

185. Garcia-Mediavilla, V.; Crespo, I.; Collado, P. S.; Esteller, A.; Sanchez-Campos, S.; Tunon, M. J.; Gonzalez-Gallego, J., The anti-inflammatory flavones quercetin and kaempferol cause inhibition of inducible nitric oxide synthase, cyclooxygenase-2 and reactive C-protein, and down-regulation of the nuclear factor kappaB pathway in Chang Liver cells. Eur J Pharmacol 2007, 557, 221-229.

186. Lee, K. M.; Lee, K. W.; Jung, S. K.; Lee, E. J.; Heo, Y. S.; Bode, A. M.; Lubet, R. A.; Lee, H. J.; Dong, Z. G., Kaempferol inhibits UVB-induced COX-2 expression by suppressing Src kinase activity. Biochemical Pharmacology 2010, 80, 2042-2049.

139

187. Thors, L.; Belghiti, M.; Fowler, C. J., Inhibition of fatty acid amide hydrolase by kaempferol and related naturally occurring flavonoids. Brit J Pharmacol 2008, 155, 244-252.

188. Kowalski, J.; Samojedny, A.; Paul, M.; Pietsz, G.; Wilczok, T., Effect of apigenin, kaempferol and resveratrol on the expression of interleukin-1 beta and tumor necrosis factor-alpha genes in J774.2 macrophages. Pharmacol Rep 2005, 57, 390-394.

189. Cortes, J. R.; Perez-G, M.; Rivas, M. D.; Zamorano, J., Kaempferol inhibits IL-4-Induced STAT6 activation by specifically targeting JAK3. J Immunol 2007, 179, 3881-3887.

190. Lee, S.; Kim, Y. J.; Kwon, S.; Lee, Y.; Choi, S. Y.; Park, J.; Kwon, H. J., Inhibitory effects of flavonoids on TNF-alpha-induced IL-8 gene expression in HEK 293 cells. Bmb Rep 2009, 42, 265-270.

191. Ee, Y. Y.; Hoong, C. C., Downregulation in the mRNA expression of nuclear hormone receptor liver-X-receptor alpha (LXR-alpha) by TNF-alpha is abolished by the antioxidant kaempferol, but not ascorbic acid, in human hepatocarcinoma HepG2 cells. Asian Biomed 2012, 6, 585-589.

192. Bandyopadhyay, S.; Romero, J. R.; Chattopadhyay, N., Kaempferol and quercetin stimulate granulocyte-macrophage colony-stimulating factor secretion in human prostate cancer cells. Mol Cell Endocrinol 2008, 287, 57-64.

193. Park, M. Y.; Ji, G. E.; Sung, M. K., Dietary Kaempferol Suppresses Inflammation of Dextran Sulfate Sodium-Induced Colitis in Mice. Digest Dis Sci 2012, 57, 355-363.

194. Ackland, M. L.; Van de Waarsenburg, S.; Jones, R., Synergistic antiproliferative action of the flavonols quercetin and kaempferol in cultured human cancer cell lines. In Vivo 2005, 19, 69-76.

195. Chung, S. Y.; Jang, D. S.; Han, A. R.; Jang, J. O.; Kwon, Y.; Seo, E. K.; Lee, H. J., Modulation of P-glycoprotein-mediated resistance by kaempferol derivatives isolated from Zingiber zerumbet. Phytother Res 2007, 21, 565-569.

196. Piao, Y.; Shin, S. C.; Choi, J. S., Effects of oral kaempferol on the pharmacokinetics of tamoxifen and one of its metabolites, 4-hydroxytamoxifen, after oral administration of tamoxifen to rats. Biopharm Drug Dispos 2008, 29, 245-249.

197. Siegelin, M. D.; Reuss, D. E.; Habel, A.; Herold-Mende, C.; von Deimling, A., The flavonoid kaempferol sensitizes human glioma cells to TRAIL-mediated apoptosis by proteasomal degradation of survivin. Mol Cancer Ther 2008, 7, 3566-3574.

198. Yoshida, T.; Konishi, M.; Horinaka, M.; Yasuda, T.; Goda, A. E.; Taniguchi, H.; Yano, K.; Wakada, M.; Sakai, T., Kaempferol sensitizes colon cancer cells to TRAIL-induced apoptosis. Biochem Bioph Res Co 2008, 375, 129-133.

199. Luo, H. T.; Daddysman, M. K.; Rankin, G. O.; Jiang, B. H.; Chen, Y. C., Kaempferol enhances cisplatin's effect on ovarian cancer cells through promoting apoptosis caused by down regulation of cMyc. Cancer Cell Int 2010, 10. 140

200. An, G. H.; Gallegos, J.; Morris, M. E., The Bioflavonoid Kaempferol Is an Abcg2 Substrate and Inhibits Abcg2-Mediated Quercetin Efflux. Drug Metab Dispos 2011, 39, 426-432.

201. Nebert, D. W.; Dalton, T. P.; Okey, A. B.; Gonzalez, F. J., Role of aryl hydrocarbon receptor-mediated induction of the CYP1 enzymes in environmental toxicity and cancer. J Biol Chem 2004, 279, 23847-23850.

202. Fan, Y.; Boivin, G. P.; Knudsen, E. S.; Nebert, D. W.; Xia, Y.; Puga, A., The aryl hydrocarbon receptor functions as a tumor suppressor of liver carcinogenesis. Cancer Res 2010, 70, 212-220.

203. Puppala, D.; Gairola, C. G.; Swanson, H. I., Identification of kaempferol as an inhibitor of cigarette smoke-induced activation of the aryl hydrocarbon receptor and cell transformation. Carcinogenesis 2007, 28, 639-647.

204. MacPherson, L.; Matthews, J., Inhibition of aryl hydrocarbon receptor-dependent transcription by resveratrol or kaempferol is independent of estrogen receptor alpha expression in human breast cancer cells. Cancer Lett 2010, 299, 119-129.

205. Master, Z.; Chaudhary, A.; Sutter, T. R.; Willett, K. L., Effects of flavonoids on CYP1 expression in RL95-2 endometrial carcinoma cells. Food Chem 2012, 133, 912-922.

206. Mylonis, I.; Lakka, A.; Tsakalof, A.; Simos, G., The dietary flavonoid kaempferol effectively inhibits HIF-1 activity and hepatoma cancer cell viability under hypoxic conditions. Biochem Bioph Res Co 2010, 398, 74-78.

207. Park, S.; Choi, J., Inhibition of beta-Catenin/Tcf Signaling by Flavonoids. J Cell Biochem 2010, 110, 1376-1385.

208. Tselepi, M.; Papachristou, E.; Emmanouilidi, A.; Angelis, A.; Aligiannis, N.; Skaltsounis, A. L.; Kouretas, D.; Liadaki, K., Catalytic Inhibition of Eukaryotic Topoisomerases I and II by Flavonol Glycosides Extracted from Vicia faba and Lotus edulis. J Nat Prod 2011, 74, 2362-2370.

209. Fuentes-Alventosa, J. M.; Jaramillo-Carmona, S.; Rodriguez-Gutierrez, G.; Rodriguez-Arcos, R.; Fernandez-Bolanos, J.; Guillen-Bejarano, R.; Espejo-Calvo, J. A.; Jimenez-Araujo, A., Effect of the extraction method on phytochemical composition and antioxidant activity of high dietary fibre powders obtained from asparagus by-products. Food Chemistry 2009, 116, 484-490.

210. Reis-Filho, J. S.; Tutt, A. N. J., Triple negative tumours: a critical review. Histopathology 2008, 52, 108-118.

211. Cleator, S.; Heller, W.; Coombes, R. C., Triple-negative breast cancer: therapeutic options. Lancet Oncol 2007, 8, 235-244.

212. van de Rijn, M.; Perou, C. M.; Tibshirani, R.; Haas, P.; Kallioniemi, C.; Kononen, J.; Torhorst, J.; Sauter, G.; Zuber, M.; Kochli, O. R.; Mross, F.; Dieterich, H.; Seitz, R.; Ross, D.; Botstein, D.; Brown, P., Expression of cytokeratins 17 and 5 identifies a group of breast carcinomas with poor clinical outcome. Am J Pathol 2002, 161, 1991-1996.

141

213. Joshipura, K. J.; Hung, H. C.; Li, T. Y.; Hu, F. B.; Rimm, E. B.; Stampfer, M. J.; Colditz, G.; Willett, W. C., Intakes of fruits, vegetables and carbohydrate and the risk of CVD. Public Health Nutr 2009, 12, 115-121.

214. Chu, Y. F.; Liu, R. H., Apple phytochemicals inhibit human LDL oxidation and induce LDL receptor expression in hepatocytes. Abstr Pap Am Chem S 2005, 229, U31-U31.

215. Liu, R. H.; Sun, J., Antiproliferative activity of apples is not due to phenolic-induced hydrogen peroxide formation. J Agr Food Chem 2003, 51, 1718-1723.

216. Sun, J.; Liu, R. H., Cranberry phytochemical extracts induce cell cycle arrest and apoptosis in human MCF-7 breast cancer cells. Cancer Lett 2006, 241, 124-134.

217. Committee, D. G. A., Report of the dietary guidelines advisory committee on the dietary guidelines for Americans, 2010, to the Secretary of Agriculture and the Secretary of Health and Human Services. Agricultural Research Service 2010.

218. Vinson, J. A.; Hao, Y.; Su, X. H.; Zubik, L., Phenol antioxidant quantity and quality in foods: Vegetables. J Agr Food Chem 1998, 46, 3630-3634.

219. Guo, X. B.; Li, T.; Tang, K. X.; Liu, R. H., Effect of Germination on Phytochemical Profiles and Antioxidant Activity of Mung Bean Sprouts (Vigna radiata). J Agr Food Chem 2012, 60, 11050-11055.

220. Singleton, V. L.; Orthofer, R.; Lamuela-Raventos, R. M., Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Oxidants and Antioxidants, Pt A 1999, 299, 152-178.

221. Liu, R. H.; Sun, J., Antiproliferative activity of apples is not due to phenolic-induced hydrogen peroxide formation. J Agric Food Chem 2003, 51, 1718-23.

222. Zhang, M. W.; Zhang, R. F.; Zhang, F. X.; Liu, R. H., Phenolic Profiles and Antioxidant Activity of Black Rice Bran of Different Commercially Available Varieties. J Agr Food Chem 2010, 58, 7580-7587.

223. Adom, K. K.; Liu, R. H., Rapid peroxyl radical scavenging capacity (PSC) assay for assessing both hydrophilic and lipophilic antioxidants. J Agr Food Chem 2005, 53, 6572-6580.

224. Felice, D. L.; Sun, J.; Liu, R. H., A modified methylene blue assay for accurate cell counting. Journal of Functional Foods 2009, 1, 109-118.

225. Dewanto, V.; Wu, X. Z.; Liu, R. H., Processed sweet corn has higher antioxidant activity. J Agr Food Chem 2002, 50, 4959-4964.

226. Wolfe, K. L.; Liu, R. H., Cellular antioxidant activity (CAA) assay for assessing antioxidants, foods, and dietary supplements. J Agr Food Chem 2007, 55, 8896-8907.

227. Hu, F. B., Dietary pattern analysis: a new direction in nutritional epidemiology. Curr Opin Lipidol 2002, 13, 3-9.

142

228. Hu, F. B., Plant-based foods and prevention of cardiovascular disease: an overview. Am J Clin Nutr 2003, 78, 544s-551s.

229. Liu, R. H., Health benefits of fruit and vegetables are from additive and synergistic combinations of phytochemicals. Am J Clin Nutr 2003, 78, 517s-520s.

230. USDA, U., Dietary guidelines for Americans. Department of Agriculture, Department of Health and Human ServicesU. S. Government Printing Office, Washington 2010.

231. Del Rio, D.; Rodriguez-Mateos, A.; Spencer, J. P. E.; Tognolini, M.; Borges, G.; Crozier, A., Dietary (Poly)phenolics in Human Health: Structures, Bioavailability, and Evidence of Protective Effects Against Chronic Diseases. Antioxid Redox Sign 2013, 18, 1818-1892.

232. You, L. J.; Zhao, M. M.; Liu, R. H.; Regenstein, J. M., Antioxidant and Antiproliferative Activities of Loach (Misgurnus anguillicaudatus) Peptides Prepared by Papain Digestion. J Agr Food Chem 2011, 59, 7948-7953.

233. Maeda, T.; Kakuta, H.; Sonoda, T.; Motoki, S.; Ueno, R.; Suzuki, T.; Oosawa, K., Antioxidation capacities of extracts from green, purple, and white asparagus spears related to polyphenol concentration. Hortscience 2005, 40, 1221-1224.

234. Wolfe, K. L.; Liu, R. H., Structure-activity relationships of flavonoids in the cellular antioxidant activity assay. J Agr Food Chem 2008, 56, 8404-8411.

235. Cazarolli, L. H.; Zanatta, L.; Alberton, E. H.; Figueiredo, M. S. R. B.; Folador, P.; Damazio, R. G.; Pizzolatti, M. G.; Silva, F. R. M. B., Flavonoids: Prospective Drug Candidates. Mini-Rev Med Chem 2008, 8, 1429-1440.

236. Bravo, L., Polyphenols: chemistry, dietary sources, metabolism, and nutritional significance. Nutrition reviews 1998, 56, 317-333.

237. Liu, R. H.; Liu, J. R.; Chen, B. Q., Apples prevent mammary tumors in rats. J Agr Food Chem 2005, 53, 2341-2343.

238. Faller, A. L. K.; Fialho, E.; Liu, R. H., Cellular Antioxidant Activity of Feijoada Whole Meal Coupled with an in Vitro Digestion. J Agr Food Chem 2012, 60, 4826-4832.

239. Siegel, R.; Naishadham, D.; Jemal, A., Cancer Statistics, 2012. Ca-Cancer J Clin 2012, 62, 10-29.

240. DeSantis, C.; Siegel, R.; Bandi, P.; Jemal, A., Breast cancer statistics, 2011. Ca-Cancer J Clin 2011, 61, 409-418.

241. Ravert, P. K.; Huffaker, C., Breast cancer screening in women: An integrative literature review. J Am Acad Nurse Prac 2010, 22, 668-673.

242. Sun, J.; Liu, R. H., Apple Phytochemical Extracts Inhibit Proliferation of Estrogen-Dependent and Estrogen-Independent Human Breast Cancer Cells through Cell Cycle Modulation. J Agr Food Chem 2008, 56, 11661-11667.

143

243. Eberhardt, M. V.; Lee, C. Y.; Liu, R. H., Nutrition - Antioxidant activity of fresh apples. Nature 2000, 405, 903-904.

244. Adams, L. S.; Phung, S.; Yee, N.; Seeram, N. P.; Li, L. Y.; Chen, S. A., Blueberry Phytochemicals Inhibit Growth and Metastatic Potential of MDA-MB-231 Breast Cancer Cells through Modulation of the Phosphatidylinositol 3-Kinase Pathway. Cancer Res 2010, 70, 3594-3605.

245. Singh, R. P.; Agarwal, R., Tumor angiogenesis: a potential target in cancer control by phytochemicals. Curr Cancer Drug Targets 2003, 3, 205-17.

246. Yoon, H.; Liu, R. H., Effect of 2alpha-hydroxyursolic acid on NF-kappaB activation induced by TNF-alpha in human breast cancer MCF-7 cells. J Agric Food Chem 2008, 56, 8412-7.

247. Liu, M.; Liu, R. H.; Song, B. B.; Li, C. F.; Lin, L. Q.; Zhang, C. P.; Zhao, J. L.; Liu, J. R., Antiangiogenetic Effects of 4 Varieties of Grapes In Vitro. Journal of Food Science 2010, 75, T99-T104.

248. Yoo, S. M.; Antonyak, M. A.; Cerione, R. A., The Adaptor Protein and Arf GTPase-activating Protein Cat-1/Git-1 Is Required for Cellular Transformation. J Biol Chem 2012, 287, 31462-31470.

249. Yang, J.; Liu, R. H., Synergistic Effect of Apple Extracts and Quercetin 3-beta-D-Glucoside Combination on Antiproliferative Activity in MCF-7 Human Breast Cancer Cells in Vitro. J Agr Food Chem 2009, 57, 8581-8586.

250. Hanahan, D.; Weinberg, R. A., The hallmarks of cancer. Cell 2000, 100, 57-70.

251. Panigrahy, D.; Singer, S.; Shen, L. Q.; Butterfield, C. E.; Freedman, D. A.; Chen, E. J.; Moses, M. A.; Kilroy, S.; Duensing, S.; Fletcher, C., PPARgamma ligands inhibit primary tumor growth and metastasis by inhibiting angiogenesis. Journal of Clinical Investigation 2002, 110, 923-932.

252. Helmlinger, G.; Yuan, F.; Dellian, M.; Jain, R. K., Interstitial pH and pO2 gradients in solid tumors in vivo: high-resolution measurements reveal a lack of correlation. Nature medicine 1997, 3, 177-182.

253. Flier, J. S.; Underhill, L. H.; Dvorak, H. F., Tumors: wounds that do not heal. New England Journal of Medicine 1986, 315, 1650-1659.

254. Hanahan, D.; Folkman, J., Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996, 86, 353-64.

255. Mizutani, K.; Miki, H.; He, H.; Maruta, H.; Takenawa, T., Essential role of neural Wiskott-Aldrich syndrome protein in podosome formation and degradation of extracellular matrix in src-transformed fibroblasts. Cancer Res 2002, 62, 669-674.

256. Zhi, Y. H.; Song, M. M.; Wang, P. L.; Zhang, T.; Yin, Z. Y., Suppression of matrix metalloproteinase-2 via RNA interference inhibits pancreatic carcinoma cell invasiveness and adhesion. World J Gastroentero 2009, 15, 1072-1078.

144

257. Roy, R.; Yang, J.; Moses, M. A., Matrix metalloproteinases as novel biomarkers and potential therapeutic targets in human cancer. J Clin Oncol 2009, 27, 5287-97.

258. Do, M. H.; Lee, S. S.; Jung, P. J.; Lee, M. H., Intake of fruits, vegetables, and soy foods in relation to breast cancer risk in Korean women: A case-control study. Nutr Cancer 2007, 57, 20-27.

259. Cho, Y. Y.; Yao, K.; Kim, H. G.; Kang, B. S.; Zheng, D.; Bode, A. M.; Dong, Z., Ribosomal s6 kinase 2 is a key regulator in tumor promoter-induced cell transformation. Cancer Res 2007, 67, 8104-8112.

260. Fink, M., Effects of chemotherapy and hormonal therapy for early breast cancer recurrence and 15-year survival. Strahlenther Onkol 2006, 182, 53-55.

261. Masala, G.; Assedi, M.; Bendinelli, B.; Ermini, I.; Sieri, S.; Grioni, S.; Sacerdote, C.; Ricceri, F.; Panico, S.; Mattiello, A., Fruit and vegetables consumption and breast cancer risk: the EPIC Italy study. Breast Cancer Res Tr 2012, 132, 1127-1136.

262. Knekt, P.; Jarvinen, R.; Seppanen, R.; Heliovaara, M.; Teppo, L.; Pukkala, E.; Aromaa, A., Dietary flavonoids and the risk of lung cancer and other malignant neoplasms. Am J Epidemiol 1997, 146, 223-230.

263. Shu, X. O.; Jin, F.; Dai, Q.; Wen, W. Q.; Potter, J. D.; Kushi, L. H.; Ruan, Z. X.; Gao, Y. T.; Zheng, W., Soyfood intake during adolescence and subsequent risk of breast cancer among Chinese women. Cancer Epidem Biomar 2001, 10, 483-488.

264. Yoon, H.; Liu, R. H., Effect of selected phytochemicals and apple extracts on NF-kappaB activation in human breast cancer MCF-7 cells. J Agric Food Chem 2007, 55, 3167-73.

265. Yoon, H.; Liu, R. H., Effect of selected phytochemicals and apple extracts on NF-kappa B activation in human breast cancer MCF-7 cells. J Agr Food Chem 2007, 55, 3167-3173.

266. Liu, R. H.; Jacob, J.; Tennant, B., Chemiluminescent detection of protein molecular weight markers in Western blot techniques. Biotechniques 1997, 22, 594-595.

267. Morris, M. C.; Divita, G., Characterization of the interactions between human cdc25C, cdks, cyclins and cdk-cyclin complexes. J Mol Biol 1999, 286, 475-487.

268. Hiyama, H.; Iavarone, A.; Reeves, S. A., Regulation of the cdk inhibitor p21 gene during cell cycle progression is under the control of the transcription factor E2F. Oncogene 1998, 16, 1513-1523.

269. Levine, A. J.; Momand, J.; Finlay, C. A., The p53 tumour suppressor gene. Nature 1991, 351, 453-456.

270. De Amicis, F.; Giordano, F.; Vivacqua, A.; Pellegrino, M.; Panno, M. L.; Tramontano, D.; Fuqua, S. A. W.; Ando, S., Resveratrol, through NF-Y/p53/Sin3/HDAC1 complex phosphorylation, inhibits estrogen receptor alpha gene expression via p38(MAPK)/CK2 signaling in human breast cancer cells. Faseb J 2011, 25, 3695-3707.

145

271. Liao, X. H.; Lu, D. L.; Wang, N.; Liu, L. Y.; Wang, Y.; Li, Y. Q.; Yan, T. B.; Sun, X. G.; Hu, P.; Zhang, T. C., Estrogen receptor α mediates proliferation of breast cancer MCF–7 cells via a p21/PCNA/E2F1‐dependent pathway. Febs J 2014, 281, 927-942.

146