Genistein Prevents Development of Spontaneous Ovarian Cancer and Inhibits

Tumor Growth in Hen Model

Kazim Sahin1 Engin Yenice

2 Birdal Bilir

3 Cemal Orhan

1 Mehmet Tuzcu

4 Nurhan Sahin

1

Ibrahim H Ozercan5 Nashwa Kabil

6 Bulent Ozpolat

67and Omer Kucuk

89

1Department of Animal Nutrition Faculty of Veterinary Science Firat University Elazig Turkey

2Poultry Research Institute Ankara Turkey

3Department of Pathology and Laboratory Medicine Emory University Atlanta GA USA

4Division of Biology Faculty of Science Firat University Elazig Turkey

5Department of Pathology Faculty of Medicine Firat University Elazig Turkey

6Department of Experimental Therapeutics Unit 422 The University of Texas MD Anderson

Cancer Center Houston Texas USA

7Center for RNA Interference and Non-Coding RNA The University of Texas MD Anderson

Cancer Center Houston Texas USA

8Department of Hematology and Medical Oncology Emory University Atlanta Georgia USA

9Winship Cancer Institute Emory University Atlanta Georgia USA

Corresponding authors Bulent Ozpolat MD PhD Associate Professor Department of

Experimental Therapeutics Unit 422 The University of Texas MD Anderson Cancer Center 1515

Holcombe Boulevard Houston TX 77030 Phone 713-563-0166 Fax 713-792-0362 E mail

Bozpolatmdandersonorg Omer Kucuk MD Professor of Hematology-Oncology and Urology

Winship Cancer Institute of Emory University1365 Clifton Road NE Room C-2110

Atlanta GA 30322 Phone(404) 778-3460 Fax (404) 778-5520

Running title Genistein prevents development of ovarian cancer

Keywords Genistein ovarian cancer spontaneous treatment chicken hen model

Chemoprevention

Conflict of interest The authors have no conflicts of interest to disclose

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ABSTRACT

Genistein the major isoflavone in soybean has been reported to exert anticancer effects

on various types of cancer including ovarian cancer however its chemopreventive effects and

mechanisms of action in ovarian cancer have not been fully elucidated in spontaneously

developing ovarian cancer models In the present study we demonstrated the preventive effects

and mechanisms of genistein in the laying hen model that develops spontaneous ovarian cancer

at high incidence rates Laying hens were randomized to three groups control (301 mghen n =

100) or low (5248 mghen n=100) and high genistein supplementation (10626 mghen per day

per group) At the end of 78 weeks hens were euthanized and ovarian tumors were collected and

analyzed We observed that genistein supplementation significantly reduced the ovarian tumor

incidence (p = 0002) as well as the number and size of the tumors (p= 00001) Molecular

analysis of the ovarian tumors revealed that genistein downregulated serum malondialdehyde

(MDA) a marker for oxidative stress and the expression of NF-κB Bcl-2 and whereas it

upregulated Nrf2 HO-1 and Bax expression at protein level in ovarian tissues Moreover

genistein intake decreased the activity of mTOR pathway as evidenced by reduced

phosphorylation of mTOR p70S6K1 and 4E-BP1 Taken together our findings strongly support

the potential of genistein in the chemoprevention of ovarian cancer and highlight the effects of

the genistein on the molecular pathways involved in ovarian tumorigenesis

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INTRODUCTION

Ovarian cancer is the most lethal gynecologic malignancy and the fifth leading cause of

cancer-related mortality among women in the United States An estimated 22280 new cases of

ovarian cancer and 14240 deaths (accounting for 5 of cancer deaths among women) are

expected in the US in 2016 (1) Ovarian cancer is a molecularly and histopathologically

heterogeneous disease associated with risk factors including family history of breast or ovarian

cancer age at diagnosis race and smoking (2-4) The current standard of care for ovarian cancer

involves cytoreductive surgery followed by adjuvant chemotherapy (5) However the lack of

reliable screening tests for detection of ovarian cancer at early stages high rate of recurrence

after surgery and resistance to available chemotherapeutic drugs lead to poor prognosis and high

mortality with an overall 5-year survival rate of 46 (1 6-8) Therefore there is an urgent need

for novel therapeutic agents that target specific molecular defects and have the potential to

prevent ovarian cancer and improve outcomes for ovarian cancer patients

Epidemiological studies have demonstrated that the incidence of ovarian cancer as those

of breast and prostate cancers is much lower in Asian countries where soy foods are consumed

in larger amounts compared to Western countries suggesting the association between high

dietary intake of soy isoflavones and reduced risk of ovarian cancer (9-13) As the major

biologically active isoflavone in the soy diet genistein has been extensively investigated for its

chemopreventive and chemotherapeutic potential in various types of cancer Genistein is a

naturally occurring nonsteroidal plant compound with a structural similarity to the steroid

hormone estradiol (17β-estradiol) that functions as selective estrogen receptor modulators (14-

16) Several studies have shown that genistein inhibits ovarian carcinogenesis through

pleiotropic molecular mechanisms by targeting multiple signaling pathways associated with the

hormonal activity cell cycle apoptosis angiogenesis and metastasis (17-22) In addition

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genistein has been reported to have antioxidant properties and to modulate cytokine synthesis in

ovarian cancer cells (23 17)

Although a number of mouse models for human ovarian cancer have been developed the

non-spontaneous nature of these models and the dissimilarities in the histopathology of ovarian

cancer between mouse and human limit the clinical relevance leading to an inappropriate animal

model to study human ovarian cancer (24) On the other hand the laying hen which is the only

non-human animal that spontaneously develops ovarian cancer with a high prevalence provides

a natural experimental model that recapitulates the pathogenesis of human ovarian cancer (25)

The key similarities between the ovarian cancer in the hen model and the one in human include

epidemiological histological and molecular characteristics supporting the laying hen as a

relevant preclinical model to study the molecular mechanisms underlying the spontaneous onset

and progression of human ovarian cancer and to test the chemopreventive and therapeutic effects

of novel agents on the disease (25-30) In the light of these recent findings we investigated the

effects of genistein on spontaneous ovarian cancer using the laying hen model providing further

mechanistic insights into the preventive effects of genistein on the pathogenesis of ovarian

cancer

MATERIALS AND METHODS

Animals and experimental design

A total of 300 brown laying hens (104 weeks old ATAK-S hybrid Gallus domesticus)

were used for the study in accordance with animal welfare regulations and under the Guide for

the Care and Use of Laboratory Animals of the Institute at the Ankara Poultry Research Station

Turkey (Elazig Turkey) The animal protocol was approved by the Institutional Animal Care

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and Use Committee at the Ankara Poultry Research Station Turkey Hens were fed either a basal

diet containing 1683 crude protein (CP) or 1115 megajoule (MJ) kg of metabolizable energy

(ME) and 2239 and mg of genisteinkg of diet or the basal diet reconstituted with addition of

400 mg or 800 mg of genistein per kilogram of diet at the expense of corn The genistein

contained 98 a glycone and 2 starches as a carrier (Bonistein DSM Nutritional Products

Istanbul Turkey) Daily total diet intake was 1343 1336 and 1332 gday per animals in

control low and high genistein groups respectively Animals received genistein 301 5248 and

10626 mghen per day in control low genistein and high genistein groups respectively The

dosage was chosen based on previously reported dosage in poultry (31 32) The nutrient

composition of the standard diet is listed in Supplementary Table 1 Diets were prepared in

batches and stored in black plastic containers at 4degC to avoid photooxidation The birdhouse was

set to a 16L8D cycle Water and diets were offered for ad libitum consumption throughout the

experiment The animal experiment lasted 78 weeks (from 104 to 182) weeks

Sample collection

Blood samples were collected at the end of the study from the hens via the axillary vein

and centrifuged at 3000 g for 10 minutes for obtaining serum After hens are euthanized and

ovaries and surrounding tissues were removed and the morphological and histological changes

were evaluated and compared Tumor incidence and sizes and were measured Tumor types were

determined by histological examination using hematoxylin and eosin staining of tissue sections

Ovarian tumors were identified as strictly cellular masses confined to the ovary Ovarian tumor

rates were presented in the present study

Tissue and serum samples and tumor tissues were immediately frozen and stored at -80degC

until analysis Tissue samples were fixed in 10 neutral-buffered formalin routinely processed

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for histology and embedded in paraffin Tissue blocks were used to prepare sections (6 m)

were cut The slides were stained with hematoxylin and eosin (HampE) and were evaluated based

on the histopathology classification system listed in Table 1 (26 33 34)

Analysis of serum levels of Genistein by high-performance liquid chromatography (HPLC)

At the end of the study blood samples were collected from 12 birds randomly chosen

from each treatment group Blood samples were centrifuged at 3000 g for 10 min and sera were

collected Sera samples were kept on the ice and protected from light until they were processed

to prevent any artifactual oxidation during the experiments Samples were stored at ndash80C until

analysis Serum genistein and daidzein concentrations were measured by high-performance

liquid chromatography (HPLC) (Shimadzu Tokyo Japan) using Shimadzu fluorescence RF-10

AxL detector and C18minus ODS-3 5microm 46 times 250 mm column The serum isoflavone (genistein

and daidzein) levels were measured by the method of our previous study (35) To 200 microl of

serum were added 200 microl of b-glucuronidase type H-5 solution (Sigma Chemical St Louis MO)

in 02 M sodium acetate buffer pH 50 (3500 units of b-glucuronidase and 193 units of

sulfatase) The mixture was incubated at 37C in a shaking water bath for 2 h and then treated

with 3600 microl of methanolacetic acid (955 volvol) The mixture was vortexed for 30 s

sonicated for 30 s vortexed again for 30 s and centrifuged for 15 min at 4C and 800 g The

supernatants were evaporated We then dissolved the sample with 80 methanol at the same

volume of serum Elution was performed at a flow rate of 1 mlmin using the following linear

gradient methanolacetic acid (955 volvol A) wateracetic acid (955 volvol B) and A (by

vol) at 30 for 10 min from 30 to 70 in 35 min and from 70 to 30 in 5 min (35)

Chemical analyses of the diet samples were performed using procedures of Association of

Official Analytical Chemists (36)

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Detection of serum malondialdehyde concentrations using HPLC

Serum levels of malondialdehyde (MDA) a marker for oxidative stress (n =12) were

measured using HPLC with an LC-20AD pump SIL-20A autosampler SPD-20A ultraviolet-

visible spectroscopy detector (at C18-ODS-3V and 5 m with a 46 times 250 mm column) and

CTO-10ASVP column oven (Shimadzu) as described previously (37) Tissue samples (300 L)

were homogenized in a mixture of 200 L of HClO4 (05 M) and 100 L of 500-ppm 2[6]-di-

tert-butyl-p-cresol Next the samples were centrifuged and supernatants were injected (injection

volume 20 L) into an HPLC system The mobile phase was 30 mM KH2PO4-methanol (825 +

175 vv pH 36) the flow rate was 12 mLminute and detection at 250 nm

Western blot analysis

Western blot analysis was performed as described previously (38) Proteins were

extracted from ovarian tumor samples and were homogenized at 110 (w v) in 10 mM Tris-HCl

buffer at pH 74 containing 01 mM NaCl 01 mM phenylmethylsulfonyl fluoride and 5 M

soluble soybean powder (Sigma St Louis MO USA) as a trypsin inhibitor Samples underwent

centrifugation at 15000 g at 4degC for 30 minutes for obtaining a supernatant Supernatants were

mixed with Laemmli sample buffer and boiled for 5 minutes Aliquots containing 20 g of

protein were subjected to 10 sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE)

electrophoresis and subsequently transferred to nitrocellulose membranes (Schleicher amp Schuell

BioScience) Nitrocellulose membranes were washed twice for 5 minutes in phosphate-buffered

saline and blocked with 1 bovine serum albumin in phosphate-buffered saline for 1 hour prior

to application of primary antibodies Antibodies against nuclear factor (NF)-B Bcl-2 Bax p-

mTOR p-P70S6k p-4E-Bp1 nuclear factor erythroid 2 (Nrf2) and heme oxygenase 1 (HO-1)

were diluted at 11000 in the buffer containing 005 Tween-20 and used Membranes were

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striped and used blotted for with other antibodies All antibodies were purchased from Abcam

(Cambridge UK)

The nitrocellulose membrane was incubated at 4degC with antibodies overnight Western

blots were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG

(Abcam Cambridge UK) Specific binding was detected using diaminobenzidine and hydrogen

peroxide as substrates Protein loading was controlled using an anti--actin antibody (Sigma)

Samples were analyzed in quadruplicate under each experimental condition and protein levels

were measured densitometrically using the image analysis software program ImageJ (National

Institutes of Health)

Statistical analysis Tumor incidences in the control and experimental groups were

evaluated statistically using the χ2 test Data were analyzed via analysis of variance using the

general linear model with the SAS program (2002) (SAS Institute Inc) to determine the effects

of genistein supplementation on tumor size protein expressions and serum metabolites When a

significant F statistic (P le 005) in the analysis of variance was noted the least squares mean

procedure was performed to separate means that were significantly different (P lt 005) Linear

and quadratic polynomial contrasts of the responses were used to evaluate the effects of the three

dosages of genistein administered to the animals for serum metabolites

RESULTS

Genistein reduces the incidence and number of spontaneous ovarian tumors in laying hens

To investigate the effects of genistein supplementation on the development of

spontaneous ovarian tumors a total of 300 laying hens at age of 182 weeks was randomized to

three groups (n = 100 per group) (i) control (301 mgday genistein) (ii) low-dose genistein

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(5248 mghen per day) and (iii) high-dose genistein (10626 mghen per day) (Supplementary

Figure 1) At the end of 78 weeks administration of genistein the study was terminated and

necropsy was performed for the examination of gross pathology and microscopy of the tumors

(Figure 1 and 2) In hens revealed a significant effect of genistein on the incidence of

spontaneous ovarian cancer (Table 2) Thirty percent of the hens in the control group developed

ovarian tumors as reported previously (25) The incidence of ovarian cancer significantly

decreased in both LG- and HG-treated hens (19 and 10 respectively) compared with control

group (p = 0002) indicating that genistein acts in a dose-dependent manner Based on the

histopathological assessment of the tumors two subtypes of ovarian cancer including serous and

mucinous carcinomas were observed in these hens representative images are illustrated in Figure

2B-C The histopathology grading system based on mitotic developments and cellular

differentiation is illustrated in Table 1 for the ovary There is no a significantly lower incidence

of adenocarcinoma in birds receiving genistein compared to the control birds (p gt 005) In the

control group 63 of tumor-bearing hens developed serous carcinoma whereas 37 of them

had mucinous carcinoma However there was no significant difference in the incidence of each

subtype of ovarian tumors between control and treatment groups (Table 2)

Additionally genistein treatment significantly reduced both the number and size of

ovarian tumors compared to the control group (Table 2) Hens in the control group had an

average of 37100 tumors (37 tumors in total number of animals 100) whereas low- and high

genistein-treated hens had 19100 and 10100 tumors respectively Average sizes of the tumors

were 281 109 and 047 mm in the control LG and HG groups respectively (p = 00001)

At the end of 78 weeks administration of genistein we also observed that overall

survival rates in genistein groups were higher compared to that of control group although the

differences were not statistically significant (Table 2) In control group 83 of the hens stayed

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alive until the end of the experiment while 88 and 91 of the animals were alive in the LG and

HG groups respectively

Genistein supplementation results in enhanced genistein levels in serum and reduced MDA

in the ovary of laying hens

To demonstrate that the lower ovarian cancer incidence in the treatment groups was

specifically due to the genistein intervention we measured serum levels of genistein and

daidzein in 12 hens per group using HPLC (Figure 3A-B) As expected hens treated with low or

high genistein fed groups had significantly higher serum genistein levels (25183 nmoll and

35800 nmoll respectively) compared with the control animals (14875 nmoll) (p lt 005) and

the increase in the level of serum genistein was dose-dependent However genistein

supplementation had no effect on serum level of daidzein in any of the treatment groups (Figure

3B)

Several studies have shown that oxidative stress is involved in a wide variety of cancers

including ovarian cancer (35) To determine the effects of genistein on the hen ovary in the

context of oxidative stress we analyzed the levels of MDA which is a widely used marker of

oxidative stress in the ovaries of 12 hens per group using HPLC (Figure 3C) Our data showed

that treatment of hens with genistein significantly and dose-dependently decreased the level of

MDA in the ovary (p lt 005) The average level of ovarian MDA was 304 nmolmg in the

control group while it was 199 nmolmg and 121 nmolmg in low- and high genistein-fed

groups respectively These results confirm that genistein treatment could ameliorate oxidative

stress in the hen ovary

Genistein decreases the expression of NF-κB and Bcl-2 while increasing the expression of

Bax in the ovary of laying hens

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To characterize the molecular mechanisms underlying the genistein-induced changes in

the pathogenesis of ovarian cancer we first examined the effect of genistein on the nuclear

factor-kappa B (NF-κB) signaling pathway which has been shown as one of the key survival

pathways activated by oxidative stress (40) As illustrated in Figure 4A both low and high

genistein treatments significantly reduced the protein expression level of NF-κB indicating that

genistein mediates its anti-tumor effects on ovarian cancer through NF-κB signaling pathway In

addition the expression levels of pro-survival Bcl-2 and pro-apoptotic Bax which are

transcriptionally regulated by NF-κB were also analyzed (Figure 4B-C) Our findings showed

that genistein significantly downregulates Bcl-2 whereas significantly upregulates Bax leading

to a reduced ratio of Bcl-2 to Bax and consequently inducing apoptosis

Genistein suppresses the mTOR pathway in the ovary of laying hens

To further explore the mechanisms involved in the genistein-induced changes in the

ovarian carcinogenesis we studied the effects of genistein on the mammalian target of

rapamycin (mTOR) survival signaling which has also been shown to be associated with

oxidative stress (41) mTOR which is a downstream target of AKT is a SerThr kinase that

phosphorylates p70S6K and 4EBP1 (42) As shown in Figure 5A-C treatment of hens with low

or high genistein significantly decreased the levels of phosphorylated proteins of mTOR

p70S6K and 4EBP1 leading to the inactivation of the mTOR signal transduction These data

demonstrate that mTOR signaling pathway also participates in the genistein-induced responses in

ovarian cancer cells

Genistein upregulates Nrf2 and HO-1 in the ovary of laying hens

It is well established that activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is

one of the major mechanisms in the cellular defense against oxidative stress (43) Similar to

Nrf2 its downstream protein HO-1 has also a protective effect against oxidative stress (44) As

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illustrated in Figure 6A-B significantly increased levels of Nrf2 and HO-1 were observed in the

genistein-treated groups indicating that genistein exerts its chemopreventive effect on the ovary

by activating the Nrf2-induced cellular stress responses

DISCUSSION

Although surgical and chemotherapeutic interventions have improved the overall survival

rates effective treatment of ovarian cancer is limited due to the major challenges such as

clinicopathological and genetic heterogeneity lack of early detection strategies for the disease

tumor recurrence and resistance to conventional chemotherapeutic drugs (6-8 45) Therefore

chemoprevention of ovarian cancer by non-toxic naturally occurring or synthetic agents

provides a rational approach to reduce the incidence and mortality rates of ovarian cancer

Epidemiological studies have shown that the dietary intake of soybean is associated with reduced

risks of various types of cancer including ovarian cancer (46) Genistein the most abundant

isoflavone in soybean has been reported to play a key role in the prevention of ovarian cancer

(17-23) Although numerous epidemiological and in vitro studies have demonstrated that

genistein is an effective anti-tumor agent in the chemoprevention of ovarian cancer there is a

lack of well-characterized studies that address the efficacy and mechanisms of action of genistein

in a biologically relevant in vivo model of spontaneous ovarian carcinogenesis In the present

study therefore we utilized the laying hen model to prospectively test the chemopreventive

effects of genistein on the incidence of spontaneous ovarian cancer and to further investigate the

molecular mechanisms underlying the actions of genistein on the initiation and progression of

ovarian cancer

Laying hens have been shown to develop spontaneous ovarian cancer at a high rate

providing an appropriate natural experimental model of human ovarian cancer (25) The fact that

laying hens and humans share some similarities in reproductive physiology and hens have also

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high ovulatory rates led to the possibility of a similar pathogenesis associated with ovulation-

induced DNA damage to ovarian cells in both hen and human ovarian cancers (47) Histological

classification of ovarian tumors based on the tumor stage and grade have indicated that similar to

humans four subtypes including serous endometrioid mucinous and clear cell carcinomas are

observed in hens (26) Recent studies have reported that several biomarkers of human ovarian

cancer are also expressed in ovarian tumors of hens including cytokeratin epidermal growth

factor receptor (EGFR) cytochrome P450 family member CYP1B1 proliferating cell nuclear

antigen (PCNA) vascular endothelial growth factor (VEGF) CA125 and HER2 (28 30 48 49)

Moreover as in human ovarian tumors molecular alterations in p53 and Ras genes have been

identified in ovarian tumors of hens (29) Based on the findings validating that ovarian cancer in

the laying hen model recapitulates the etiology and disease progression in human hens have

been previously used in studies testing the effects of chemopreventive agents such as oral

contraceptives aspirin and flaxseed in the prevention of ovarian cancer (50 51)

To the best of our knowledge the present study is the first study to investigate the

chemopreventive effects of genistein in the laying hen model of ovarian cancer In this pilot

study we conducted a three-armed randomized controlled trial to assess the effects of genistein

intervention on the incidence of spontaneous ovarian cancer in laying hens Our data

demonstrated that genistein significantly and dose-dependently reduced the incidence rate of

ovarian cancer consistent with previously published epidemiological and in vitro findings In

addition we observed an increased survival rate in genistein-treated animals although the effect

was not significant Histological analysis of the ovarian tumors revealed that two subtypes of

ovarian cancer serous and mucinous carcinomas were observed in these hens However the

prevalence of different subtypes did not significantly vary between control and genistein-treated

hens Genistein intake also significantly decreased the number and size of ovarian tumors in

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hens indicating the inhibitory effect of genistein on the growth of ovarian cancer cells Analysis

of genistein levels in the serum showed a dose-dependent increase in hens fed the diet containing

genistein whereas there was no change in the serum daidzein levels of these animals confirming

that the tumor inhibitory effect has been linked specifically to genistein

It is well documented that tumor initiation and progression in the ovary has been

associated with chronic inflammation which is activated by oxidative stress (52) Therefore we

first assessed the level of MDA which is a biomarker for oxidative stress in the ovaries of the

control and genistein-fed animals Our results showed that genistein supplementation resulted in

a significant dose-dependent reduction of MDA levels in the ovary suggesting that genistein

exerts its chemopreventive effects on the ovary via oxidative stress-induced signaling pathways

Based on this finding we hypothesized that genistein could ameliorate oxidative stress and

inflammatory responses in the ovary through regulation of NF-κB mTOR and Nrf2 pathways

which are involved in the pathogenesis of ovarian cancer

NF-κB signaling which is a critical molecular link between inflammation and cancer is

known to regulate key processes in several malignancies including ovarian cancer (53-55)

Activation of NF-κB cascade has been shown to correlate with clinical outcome in ovarian

cancer patients and is associated with growth and progression of ovarian tumors (56-58)

Analysis of gene expression microarrays in ovarian cancer cells treated with highly specific NF-

κB inhibitors revealed that NF-κB pathway regulates genes associated with cell proliferation

adhesion invasion angiogenesis and the creation of a pro-inflammatory microenvironment

including TNF cytokine network (54) Targeting NF-κB pathway is therefore of interest in the

suppression of inflammatory processes Our data showed that genistein supplementation

significantly reduced the expression of NF-κB as well as its downstream targets Bcl-2 and Bax at

the protein level resulting in the induction of apoptosis

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It has been reported that PI3KAKTmTOR signaling pathway is frequently activated in

ovarian cancer (59) It is a complex signaling network transducing signals from various growth

factors and cytokines (eg EGF heregulin and TGF) through receptor tyrosine kinases and G

protein-coupled receptors into intracellular messages by generating phospholipids which

activate downstream effectors including AKT and mTOR via phosphorylation (42) Once

activated mTOR phosphorylates two key translation-regulating factors ribosomal protein S6

kinase (p70S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1)

resulting in increased translation of target genes involved in cell cycle cell survival metabolism

motility angiogenesis chemoresistance and genomic instability (60) In our study we observed

that genistein significantly reduced the activation of mTOR and its downstream targets p70S6K

and 4EBP1 by inhibiting the phosphorylation of these proteins in ovarian cancer cells suggesting

that genistein may have the potential to enhance the efficacy of therapeutic agents that target

PI3KAKTmTOR signaling cascade in ovarian cancer cells

Nrf2 signaling pathway has been shown as one of the major defense mechanisms to

protect cells against oxidative stress (61) Under basal conditions Nrf2 is present in the

cytoplasm and kept transcriptionally inactive through binding to its inhibitor Kelch like-ECH-

associated protein 1 (Keap1) which targets Nrf2 to ubiquitination and the subsequent

proteasomal degradation (62 63) In the presence of oxidative stress the cysteine residues of

Keap1 become oxidized resulting in disruption of the Nrf2-Keap1 complex This dissociation

allows the translocation of Nrf2 to the nucleus where it binds to antioxidant response elements

(AREs) resulting in the transcription of its downstream target genes (64) Interestingly aberrant

activation of Nrf2 is also observed in ovarian cancer and high levels of Nrf2 expression are

associated with poor prognosis in patients with ovarian cancer (65) Recent studies have

demonstrated that not only healthy cells but also various cancer cells including ovarian tumors

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can protect themselves against oxidative stress by activating the transcription factor Nrf2 the

master regulator of antioxidant genes suggesting a dual role of Nrf2 in carcinogenesis (66 67)

Therefore these findings may suggest that activation of Nrf2 could be utilized as a cancer

prevention strategy whereas inhibition of Nrf2 could be effective in cancer treatment (68 69)

Our results showed that genistein intervention significantly and dose-dependently increased the

expression levels of Nrf2 and its downstream target HO-1 in hen ovarian tumors indicating the

involvement of antioxidant activity of genistein in chemoprevention of ovarian cancer

In conclusion our findings in laying hen model of spontaneous ovarian cancer indicate

that genistein is a potent agent in chemoprevention of ovarian cancer through acting its effects by

modulating NF-κB mTOR and Nrf2 signaling pathways These results provide further support

and mechanistic insights into the chemopreventive effects of genistein on ovarian cancer in a

biologically relevant in vivo model providing a strong rationale for clinical studies to assess the

protective effects of genistein which may ultimately lead to better clinical outcomes and

improved overall survival rates for patients diagnosed with ovarian cancer

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Butzow R Godwin AK Testa JR Nathanson KL Gimotty PA Coukos G Weber BL

Degenhardt Y Frequent genetic abnormalities of the PI3KAKT pathway in primary ovarian

cancer predict patient outcome Genes Chromosomes Cancer 2011 50606-618

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149274-293

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in stress response and cancer evolution Genes Cells 2011 16123-140

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6636-44

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Nrf2 identifies novel targets in cell survival response through ChIP-Seq profiling and network

analysis Nucleic Acids Res 2010 385718-5734

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2012 271918-1924

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AP Hecht JL Cannistra SA Keap1 mutations and Nrf2 pathway activation in epithelial ovarian

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with Nrf2-ARE pathway Food Sci Hum Wellness 2013 222-28

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Pharmacol 2012 69485-494

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Table 1 Histopathology classifications of the reproductive tumors of hens

Adenocarcinoma Characteristics

Grade 1 Well-differentiatedndashmitosis rare to absent defined pattern most

common classification

Grade 2 Intermediate differentiationndashmitosis rare to occasional tubular

pattern present but not distinct

Grade 3 Poorly differentiatedndashmitosis common cells highly anaplastic least

common classification

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Table 2 Effect of genistein rich diet on the development of spontaneous ovarian cancer in hens

Item Dietary Genistein levels mghen per day --P--

X2 301 5248 10626

Tumor ()

Incidence

Reduction

30100

30

-

19100

19

-367

10100

10

-667

0002

X2=12701

Serous carcinoma ()

Incidence

Reduction

19100

19

-

11100

11

-421

6100

6

-684

0017

X2=8144

Mucinous carcinoma ()

Incidence

Reduction

11100

11

-

8100

8

-273

4100

4

-636

0175

X2=3485

Adenocarcinoma incidence ()

Grade 1 530

167

719

368

510

500

0084

X2=4943

Grade 2 1830

600

819

421

310

300

0196

X2=3258

Grade 3 730

233

419

211

210

200

0968

X2=0064

Number of tumors1 37100

19100 10100 00001

Sizehen mm1

281plusmn050a

109plusmn027b 047plusmn016

b 00001

Sizehen with tumors only

mm1

686plusmn085 526plusmn081 470plusmn067 0249

Size range of tumors mm 1ndash22 1ndash12 2ndash8 -

Survival () 83100

83

88100

88

91100

91

0228

X2=2953

1Data are presented as the means and standard errors a-b Means in the same line without a common

superscript differ significantly (P lt 005)

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FIGURE LEGENDS

Figure 1 Gross pathology of ovaries in laying hens with tumor (A-D) Tumors restricted to the

ovary Primary malignant ovarian tumors in hens Multiple solid tumor masses are observed

Figure 2 Histopathology of normal ovary (A) and ovarian carcinoma (B-C) in laying hens

Arrows indicate serous (B) and mucinous (C) ovarian tumors Hematoxylin and eosin staining

original magnification x40

Figure 3 The effects of genistein supplementation on serum concentration of genistein (A) and

daidzein (B) and ovarian tissues of MDA (C) in laying hens a-c Means in the same line without

a common superscript differ significantly (P lt 005)

Figure 4 Effects of genistein on NF-B (Panel A) Bcl-2 (Panel B) and Bax (Panel C)

expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a representative

blot for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the

same line without a common superscript differ significantly (P lt 005)

Figure 5 Effects of genistein on p-mTOR (Panel A) p-p70S6K1 (Panel B) and p-4E-BP1 (Panel

C) expressions in hen ovarian tissue Blots were repeated at least 3 times (n=3) and a

representative blot for each is shown (Supplementary Fig 2) Values are means plusmn SD Data are

percent of the control a-c Means in the same line without a common superscript differ

significantly ( P lt 005)

Figure 6 Effects of genistein on Nrf2 (Panel A) and HO-1(Panel B) expressions in hen ovarian

tissue Blots were repeated at least 3 times (n=3) (Supplementary Fig 2) and a representative blot

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for each is shown Values are means plusmn SD Data are percent of the control a-c Means in the

same line without a common superscript differ significantly (P lt 005)

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Figure 1 Cancer Research

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Figure 2 Cancer Research

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301 5248 10626

0

100

200

300

400

500

c

b

a

A

Genistein levels mghen per day

Gen

iste

in n

mo

ll

301 5248 10626

0

20

40

60

80

B

Genistein levels mghen per day

Dai

dze

in n

mo

ll

301 5248 10626

0

1

2

3

4a

b

c

C

Genistein levels mghen per day

Ova

ry M

DA

n

mo

lm

g p

rote

in

Figure 3 Cancer Research

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Figure 4 Cancer Research

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301 5248 10626

0

50

100

150

a

b

c

A

-actin

p-mTOR

Genistein levels mghen per day

p-m

TO

R

pe

rce

nt

of

co

ntr

ol

301 5248 10626

0

50

100

150

a

b

c

B

-actin

p-p70S6K1

Genistein levels mghen per day

p-p

70

S6

K1

p

erc

en

t o

f co

ntr

ol

301 5248 10626

0

50

100

150

a

b

c

C

-actin

p-4E-BP1

Genistein levels mghen per day

p-4

E-B

P1

p

erc

en

t o

f co

ntr

ol

Figure 5 Cancer Research

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Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited Author Manuscript Published OnlineFirst on January 16 2019 DOI 1011581940-6207CAPR-17-0289

301 5248 10626

0

50

100

150

200

250

c

b

a

A

-actin

Nrf2

Genistein levels mghen per day

Nrf

2

pe

rce

nt

of

co

ntr

ol

301 5248 10626

0

50

100

150

200

250

c

b

a

B

-actin

HO-1

Genistein levels mghen per day

HO

-1 p

erc

en

t o

f co

ntr

ol

Figure 6 Cancer Research

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Published OnlineFirst January 16 2019Cancer Prev Res Kazim Sahin Engin Yenice Birdal Bilir et al Cancer and inhibits Tumor Growth in Hen ModelGenistein Prevents Development of Spontaneous Ovarian

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