Decreased severity of ovarian cancer and increased survival in hens fed a flaxseed-enriched diet for 1 year.

Abstract

With the exception of the laying hen, no other animal model of spontaneous ovarian surface epithelial cancer replicates the human disease. Flaxseed is the richest vegetable source of omega-3 fatty acids, which are chemopreventive in breast cancer and may be important in other cancers. The objective of this study was to determine if a flaxseed-enriched diet had a chemopreventive effect on ovarian cancer in the laying hen.
White Leghorn hens were fed with 10% flaxseed-enriched or standard diet for 1 year. The incidence and severity of ovarian cancer were determined by gross pathology and histology in the two groups. General health markers were also measured. Eggs were collected and analyzed by gas chromatography to determine omega-3 fatty acid levels.
A significant reduction in late stage ovarian tumors was detected in the flaxseed-fed hens. Incidence rates of ovarian cancer were not significantly different between the two groups. The results indicate that a flaxseed diet increases overall survival in the laying hen. Flaxseed-fed hens' eggs incorporated significantly more omega-3 fatty acids compared to control hens.
These findings show that 10% flaxseed supplementation for 1 year in the laying hen results in a significant reduction in the severity of ovarian cancer, but no change in the incidence of the disease. Hens fed flaxseed had overall better health and reduced mortality. These findings may provide the basis for a clinical trial that evaluates the efficacy of flaxseed as a chemosuppressant of ovarian cancer in women.

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Decreased severity of ovarian cancer and increased survival in hens fed a
flaxseed-enriched diet for 1 year
Kristine Ansenbergera, Cassandra Richardsa, Yan Zhugeb, Animesh Baruab, Janice M. Bahrc,
Judith L. Luborskyb, Dale Buchanan Halesd,⁎
aDepartment of Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL, USA
bDepartment of Pharmacology, Rush University Medical Center, Chicago, IL, USA
cDepartment of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL, USA
dDepartment of Physiology, Southern Illinois University Carbondale, School of Medicine, Life Science II, Room 250 (M/C 6512), 1125 Lincoln Drive, Carbondale, IL 62901, USA
a b s t r a c ta r t i c l ei n f o
Article history:
Received 16 October 2009
Available online 13 February 2010
Keywords:
Ovarian cancer
Flaxseed
Omega-3 fatty acids
Laying hen
Dietary intervention
Objective. With the exception of the laying hen, no other animal model of spontaneous ovarian surface
epithelial cancer replicates the human disease. Flaxseed is the richest vegetable source of omega-3 fatty
acids, which are chemopreventive in breast cancer and may be important in other cancers. The objective of
this study was to determine if a flaxseed-enriched diet had a chemopreventive effect on ovarian cancer in the
laying hen.
Methods. White Leghorn hens were fed with 10% flaxseed-enriched or standard diet for 1 year. The
incidence and severity of ovarian cancer were determined by gross pathology and histology in the two
groups. General health markers were also measured. Eggs were collected and analyzed by gas
chromatography to determine omega-3 fatty acid levels.
Results. A significant reduction in late stage ovarian tumors was detected in the flaxseed-fed hens.
Incidence rates of ovarian cancer were not significantly different between the two groups. The results
indicate that a flaxseed diet increases overall survival in the laying hen. Flaxseed-fed hens' eggs incorporated
significantly more omega-3 fatty acids compared to control hens.
Conclusions. These findings show that 10% flaxseed supplementation for 1 year in the laying hen results in
a significant reduction in the severity of ovarian cancer, but no change in the incidence of the disease. Hens
fed flaxseed had overall better health and reduced mortality. These findings may provide the basis for a
clinical trial that evaluates the efficacy of flaxseed as a chemosuppressant of ovarian cancer in women.
© 2010 Elsevier Inc. All rights reserved.
Introduction
A woman's risk of developing invasive ovarian cancer during her
lifetime is about 1 in 71 and her lifetime chance of dying from this
disease is about 1 in 95. Research into the discovery of early detection
markers and therapeutics continues, but there are still no effective
treatments for ovarian cancer today. This makes the search for
effective ovarian cancer chemopreventive agents ever more impor-
tant [1]. As researchinto the effect of nutrition on cancer development
continues, it is increasingly evident that nutrition plays a major role
[2]. The American Institute for Cancer Research estimates that 30–40%
of all cancers can be prevented by appropriate diets, physical activity,
and maintenance of appropriate body weight [3]. Obesity, nutrient
sparse foods such as concentrated sugars, refined flour products, low
fiber intake, consumption of red meat, and an imbalance of omega-3
(OM-3FA) and omega-6 (OM-6FA) fatty acids all contribute to excess
cancer risk [4]. OM-3FA and OM-6FA are polyunsaturated fats that are
essential fatty acids because they cannot be synthesized by the body
and must be obtained from the diet [5]. OM-3FA has been shown to
reduce inflammation and help prevent risk factors associated with
chronic diseases such as heart disease, cancer, and arthritis [4]. OM-
3FA also appear to be important for proper brain and behavioral
functions [4]. Epidemiologic studies indicate that populations that
consume high amounts of OM-3FA have lower incidences of breast,
prostate, and colon cancers than do those that consume lower
amounts of OM-3FA [6–8].
Dietary intervention with flaxseed to prevent cancer was first
proposed by Budwig in the 1950s [9]. Flaxseed is the richest plant
source of one kind of OM-3FA, alpha-linolenic acid (ALA). In several
laboratory studies, flaxseed inhibited the formation of colon, breast,
skin, and lung tumors [10]. Flaxseed supplementation reduces
epithelial cell proliferation in female rats, suggestive of a protective
effect against breast (and possibly ovarian) cancer [11]. Flaxseed
supplementation significantly reduces the incidence of breast tumors
in thedimethylbenzanthracene(DMBA)rat model,aswellas protects
against colon carcinogenesis [12,13]. The addition of 10% flaxseed to
the diet decreases metastatic spread in breast cancer xenograft
Gynecologic Oncology 117 (2010) 341–347
⁎ Corresponding author. Fax: +1 618 453 1517.
E-mail address: dhales@siumed.edu (D.B. Hales).
0090-8258/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.ygyno.2010.01.021
Contents lists available at ScienceDirect
Gynecologic Oncology
journal homepage: www.elsevier.com/locate/ygyno

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Author's personal copy
models [14]. These data indicate that flaxseed supplementation may
act to suppress metastatic cancer spread as well as prevent initial
tumor formation.
Animal models that can be used for large-scale dietary inter-
vention studies for human ovarian cancer are lacking. We, and others,
have characterized the laying hen (Gallus domesticus) as an animal
model for the study of ovarian cancer [15–17]. Several studies have
demonstrated the utility of the laying hen for pre-clinical trials and
dietary intervention studies [18–20]. One of the most prevalent
theories about the etiology of ovarian cancer is the “incessant
ovulation hypothesis”, which was proposed by Fathalla in 1971 [21].
He hypothesized that the inflammation associated with continuous
ovulation leaves ovarian surface epithelial (OSE) cells susceptible to
malignant transformation. The observation that egg-laying domestic
hens frequently develop ovarian cancer supports this hypothesis [22].
Hens spontaneously develop ovarian adenocarcinomas that are
similar in histological appearance to human ovarian carcinomas and
share similar symptoms of the disease, such as perfuse ascites fluid
and peritoneal metastatic dissemination [15,23,24].
The laying hen provides a model for conducting large-scale dietary
intervention studies with statistically significant numbers of animals.
The purpose of this study was to determine if flaxseed slowed the
development, or progression, of ovarian cancer in hens fed a diet
enrichedwithomega-3fattyacidscomparedtohensfedacontroldiet.
Theresultsindicatethatadietenrichedwithflaxseedincreasesoverall
health in the laying hen. No significant differences in the incidence
rates of ovarian cancer were found between the two groups; however,
a significant decrease in late stage ovarian tumors was observed in
hens fed flaxseed. New insight into the ability of flaxseed and OM-3FA
to decrease ovarian cancer may establish the foundation for clinical
studies to test the efficacy of dietary intervention with flaxseed for the
prevention and suppression of ovarian cancer in women.
Materials and methods
Animal care
Three hundred eighty-seven single-comb 2.5-year-old White
Leghorn hens were used for this study. Hens were maintained three
per cage, provided with measured feed and water ad libitum and
exposed to a photoperiod of 17 h light/7 h dark, with lights on at
05:00 h and lights off at 22:00 h. Animal management and procedures
were reviewed and approved by the Institutional Animal Care and Use
Committees at the University of Illinois at Urbana-Champaign and
University of Illinois at Chicago.
Composition of meals
Two and a half year old hens were randomly divided into control
and flaxseed groups with 194 and 193 hens, respectively. The
composition of the test diets is shown in Table S1 (Supplementary
material). A percentage of both corn and soy were removed from the
experimental diet to keep the diet isocaloric with the addition of the
flaxseed. The hens were fed 110 g of food a day and were provided
water ad libitum. Hens on the flaxseed diet consumed around 11 g of
flaxseed per day, or 6.2 g/kg body weight.
Tissue collection
HenswereeuthanizedbyCO2asphyxiation.Uponnecropsy,ovaries
were removed and small yellow follicles (6–8 mm) and pre-ovulatory
follicles (9–35 mm) were removed from ovaries of the normal hens.
Suspected abnormal ovarian tissues were noted and confirmed as
cancerous or normal ovarian tissue by histology. Tumors were
classified by stage as shown in Table 1. Tumors were characterized
based on the size of the ovarian tumor, oviductal involvement, and if
there was any ascites fluid and peritoneal metastases present by gross
observation. Basic histology was performed on early stage tumors to
determine the extent of tumor growth [25].
Experimental design
The hens were fed either a 10% flaxseed-enriched diet or standard
(control) diet for 1 year. Ovulation frequency was measured by
counting the total number of eggs laid each week by the whole group
(flax or control), then dividing by the total number of hens in each
group. At the 6- and 9-month time points, 20 hens from each group
were randomly selected, euthanized, and necropsied, to ensure that
the OM-3FA from the flaxseed were incorporating into the ovary. Eggs
were collected both at these time points and at the end of the study
from both groups of hens and measured for total omega-3 fatty acid
content by gas chromatography.
Gas chromatography
MeasuringOM-3FAineggyolksisacommonpracticeinthepoultry
industryasawaytomeasuretotalbodyOM-3FAincorporation[26,27].
Theyolkwasseparatedfromtherestoftheegganddiluted1:25witha
phosphate buffered saline solution. A solution containing 30 μg 17:0
standard (Sigma-Aldrich), 30 μg 22:2 standard (Sigma-Aldrich), and
200 μg butylated hydroxy toluene (Sigma-Aldrich) in methanol was
then added to the diluted egg yolks. Lipid extraction was performed
using HPLC grade chloroform, water, and HPLC grade hexane (Sigma-
Aldrich). The lipids were then dried under a stream of nitrogen and
methylated using the Instant Metanolic HCl kit (Alltech). After
methylation,thelipidswereextractedtwicewithhexane.Afterdrying
undernitrogen,thelipidswereredissolvedinhexaneandinjectedinto
the Shimadzu 17A gas chromatograph equipped with a flame
ionizationdetector.Hydrogenwasusedasthecarriergasandnitrogen
as the make up gas with an Omegawax column. Fatty acids were
determined from their retention time and quantified by integration of
area under peak after normalization to 17:0 and 22:2 standards.
Statistical analysis
Statistical analysis was performed with GraphPad InStat by using
one-way ANOVA with Student–Newman–Keuls comparison and also
2-way Contingency Table and Chi-Square Tests. A value of Pb0.05 was
considered significant whereas a value of Pb0.01 was considered
highly significant.
Results
Incorporation of OM-3FA into egg yolk
The incorporation of OM-3FA into tissue was measured by gas
chromatography as shown in Fig. 1. Total OM-3FA content was
determined in egg yolks collected from control and flaxseed-fed hens
at the 12-month time point. Six egg yolks from 6 hens in each group
were used to measure total OM-3FA. Flax eggs incorporated 78.20 μg
OM-3FA/mg yolk while control eggs only incorporated 32.05 μg OM-
3FA/mg yolk. There was significantly more OM-3FA incorporated into
the eggs from flaxseed-fed hens when compared to the control group
(Pb0.007).
Tumor incidence and severity
After feeding the hens flaxseed for 1 year, we found no significant
difference in the incidence of total ovarian cancers between the
flaxseed-fed hens (24%) and control-fed hens (22%; Pb0.1; Fig. 2A).
Tumors from both groups were analyzed and characterized according
to Table 1. Hens fed flaxseed had more early stage tumors that were
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confined to the ovary and oviduct compared to the control diet hens,
indicative of the chemosuppressive actions of flaxseed (Fig. 2B). In
contrast, hens on the control diet had significantly more late stage
tumors (T3/T4) that presented with ascites fluid and metastases
compared to the flaxseed group (61% vs. 47%; Pb0.05).
Effect of flaxseed on egg laying frequency
No significant differences in egg laying frequency were seen
between the two groups after feeding for 1 year (Fig. 3), indicating
that diet had no effect on ovulation. There appears to be a decrease in
Table 1
Histological staging of ovarian cancer in the laying hen.
20×40×
T0—Normal ovary, follicles
present by histology
T1—Tumor confined to the
ovary, some areas may
be normal
T2—Most of the ovary is
abnormal, may have
oviductal involvement
(continued on next page)
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egg laying frequency in the last quarter of the study in both diet
groups, but these data were not statistically significant.
Effect of flaxseed on body weight
The graph in Fig. 4 represents the average weights of hens on the
control diet and flaxseed-enriched diet necropsied at the 6- and 12-
month time points. At 6 months, the control-fed hens' average weight
was 1.83 kg while the flaxseed-fed hens weighed significantly less at
1.67 kg (Pb0.05). By 12 months, the control diet hens' weight had
significantly decreased to 1.58 kg, while the flaxseed-fed hens' weight
remained stable at 1.71 kg (Pb0.001). There was no difference in food
intake in hens fed flaxseed compared to the control diet.
Effect of flaxseed on mortality
Flaxseed increased overall survival of the hens over the course of
the 1-year study (Fig. 5). Initially, 193 hens were in the control group
and 194 hens in the flax group. Twenty hens from each group were
euthanized at the 6- and 9-month time points. Of the remaining 153
hens in the control diet group, only 78 hens survived to the 12-month
time point (51.6%), compared to 111 flaxseed hens out of the
remaining 154 (72.1%; Pb0.0001). Mortality rates include all natural
causes of death over the total study.
Discussion
The goal of this study was to determine if a diet enriched with
flaxseed for 1 year prevented ovarian cancer development and
progression in the laying hen. Flaxseed-fed hens had fewer late
stage tumors, but no statistically significant reduction of total ovarian
cancers was found between the two diet groups. Hens fed a flaxseed-
rich diet over 1 year had better overall health and reduced mortality
compared to hens fed the control diet.
A significant reduction in severity of ovarian tumors was observed
in hens fed flaxseed, with more tumors confined to the ovary and
decreased metastatic spread. This is an important finding as the
metastases that accompany late stage ovarian cancer are the main
Table 1 (continued)
20×40×
T3—Tumor has spread to the
whole ovary, other organs
may be involved, ascites
may be present
T4—Extensive peritoneal
metastases and
ascites fluid
T0—Stage 0. The ovary is normal and presents with large pre-ovulatory follicles and small peri-ovulatory follicles. The tissue is organized into follicular structures around the edge of
the ovary and the internal stromal tissue in the medullary region of the ovary. T1—Stage 1. The ovary is mostly normal, except for a small area of early stage cancerous tissue, seen
here surrounding an otherwise normal pre-ovulatory follicle. The earliest lesions seen in the hen are acini created by a single layer of cells surrounding a lumen (black arrows) [22].
The tumor has not broken through the basement membrane to invade the rest of the ovary. There is no tumor on the surface. T2—Stage 2. The majority of the ovary is abnormal and
there may be oviductal involvement. The tumor has broken through the basement membrane (black arrows) and transformed the normal ovary. Tumor may be on the surface of the
ovary. T3—Stage 3. The tumor has taken over the whole ovary and may have metastasized to other organs within the peritoneum. Ascites may be present. T4—Stage 4. The ovary is
completely cancerous. Extensive peritoneal metastases and ascites fluid are seen by gross observation (20× bar=50 μm, 40× bar=100 μm).
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cause of death from this disease [28]. Over 60% of all women
diagnosed with ovarian cancer present at stage 3 or 4 when
metastases has already occurred [29]. If the cancer is found at an
early stage, when the tumor is still confined to the ovary, women have
a much better prognosis and much better treatment options
compared to a late stage diagnosis when it has already metastasized.
Flaxseed supplementation for 1 year is able to reduce the severity of
ovarian cancer in hens. This finding may have therapeutic potential as
women at high risk for developing ovarian cancer could supplement
their diet with flaxseed to prevent tumors from forming metastases.
Feeding hens a flaxseed-supplemented diet for 1 year did not
prevent ovarian cancer. No statistical differencein incidence rates was
detected between the control group and flax group. This may be
because the diet was not administered until the hens were 2.5 years of
age. By this age, a hen has ovulated approximately 500 times, similar
to the number of ovulations of a woman entering menopause [22]. At
2.5–3.5 years of age, the incidence rate of ovarian cancer in hens is
between 4% and 20%. A chemoprevention with flaxseed may not have
been seen because the damage caused by ovulation had already
occurred. A 1-year feeding study is not adequate to test if flaxseed
supplementation prevents ovarian cancer. A new 4-year study is
currently being conducted in order to determine if long-term dietary
intervention withflaxseedmay reducethe incidence ofovariancancer
development. In this new study, a 10% flaxseed-supplemented diet
has been provided to the hens as soon as they commenced egg laying
Fig. 1. Flaxseed-fed hens lay eggs with higher OM-3FA content. Eggs were collected
from control and flaxseed-fed hens at time of necropsy after 12 months on the diet.
There was no difference in the number of eggs laid between the groups. Six egg yolks
from each group were measured for total OM-3FA content by gas chromatography. Yolk
was separated from the rest of the egg and diluted with a phosphate buffered saline
solution. Lipids were extracted with hexane and measured by a gas chromatograph.
Fatty acids were determined from their retention time and quantified by integration of
area under peak after normalization to 17:0 and 22:2 standards. Eggs from flaxseed-fed
hens contained significantly more OM-3FA compared to control-fed hens (Pb0.007).
Fig. 2. Flaxseed suppresses the severity of ovarian cancer in the laying hen. Hens were
fed a diet enriched with 10% flaxseed or a control diet for 1 year. After 1 year, the hens
were euthanized, necropsied, and examined for the presence of ovarian cancer. (A)
There was no significant difference in incidence rates between the two groups. (B)
There was a reduction in the severity of the disease in hens fed flaxseed. Hens fed
flaxseed had more ovarian tumors in stage T1/T2, when the tumor was still confined to
the ovary (Pb0.05). Hens on the control diet had more late stage tumors (T3/T4),
where the cancer had spread to other organs (Pb0.05).
Fig. 3. Egg laying frequency is unaffected by the addition of flaxseed to the diet. Eggs
were collected and counted weekly over the 1-year study from the two groups. There
were no significant differences in the numbers of eggs laid in the flaxseed-fed group
compared to the control group. Egg laying frequency in both groups appeared to slow in
the last quarter of the study, but this change was not significant.
Fig. 4. Flaxseed-fed hens maintain a steady weight compared to control hens over a 12-
month study. At 6 months, 20 hens from each group were randomly selected to be
euthanized and necropsied. All hens were weighed before necropsy. At the 12-month
time point, the remaining hens were weighed and necropsied. At the 6-month time
point, the flaxseed-fed hens weighed significantly less than the control diet hens
(Pb0.05). By 12 months, the control-fed hens weight had significantly decreased
(Pb0.001), while the weight of the flaxseed-fed hens remained stable.
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(approximately 22 weeks of age), before the damage from ovulation
accumulates. The findings from this study will providemore definitive
results indicating the value of flaxseed as a chemopreventive agent.
The importance of diet in the development of cancer has only been
studied for 30 years, while the chemotherapeutic efficacy of certain
foods in the treatment andprevention of cancer is an age old idea [30].
The increasing awareness that certain compounds, such as lycopene
or selenium, have anti-cancer activities has lead to an increase in
dietary intervention studies [31,32]. However, many of these
compounds that showed promise in vitro show little or no positive
effects once tested in vivo [33,34]. Dietary intervention with
individual compounds can be difficult to study since many individual
compounds can work together synergistically. Thus, given alone as
treatment, effects may be minimal or not seen at all.
The hens were fed a diet enriched in flaxseed as it contains many
nutrients that have been shown to have anti-cancer effects, including
OM-3FA and phytoestrogen lignans. Flaxseed contains the highest
level of plant lignans that are metabolized in the digestive tract to
enterodiol (END) and enterolactone (ENL), which have antioxidant
activities [35]. Lignans have also been shown to alter estrogen
metabolism, which may decrease ovarian cancer risk and improve
health [36,37]. During the course of the 1-year trial study, the overall
health of the hens fed flaxseed was improved based on survival
compared to the hens fed the control diet. Hens were able to
incorporatehigh amounts of OM-3FA into their eggs from the flaxseed
as shown by gas chromatography, yet had no differences in egg laying
frequencies.
Hensfed flaxseedmaintaineda lowsteady weightcomparedtothe
control-fed hens whose weight precipitously declined over the course
of the study, indicative of their failing health. The overall reduced
weightintheflaxseed-fedhensmay haveplayeda roleinthedecrease
in late stage ovarian cancers as obesity and increased body mass index
have been linked to an increased risk for developing ovarian cancer in
women [38]. Hens fed a low-calorie diet also have a reduced risk of
developing the disease [38,39]. Overall mortality rates were also
significantly decreased in hens fed flaxseed compared to control. A
diet high in flaxseed has anti-cancer activity as well as important
overall health benefits. As OM-3FA reduce inflammation and
phytoestrogens can act as antioxidants, the combination of OM-3FA
and phytoestrogens may create a synergistic effect that results in an
overall health benefit. In vitro studies are currently underway to
elucidate the mechanisms by which these compounds act to protect
against tumor progression and improve survival.
The laying hen is an excellent model to study the effects of dietary
intervention on ovarian cancer. The findings here show that dietary
intervention with flaxseed in the laying hen increases overall health
and reduces mortality. A chemosuppressive effect of flaxseed on
ovarian cancer progression was reported after feeding for 1 year.
These findings have important clinical relevance and may provide the
basis for a large-scale clinical trial testing the efficacy of flaxseed as a
chemopreventive agent of ovarian cancer in humans.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was funded by NIH/National Center for Complementary
and Alternative Medicine Grant AT004085 (DBH), American Institute
for Cancer Research Grant 06-A043 (DBH), and NIH Training Grant
T32 HL007692 (KA). We are grateful for the expert histological
support from Patty Mavrogianis and poultry management by Chet
Utterback, Douglas Hilgendorf, and Pam Utterback. We also thank Drs.
Joanna Burdette, Jonna Frasor, and Randy Jaffe for their suggestions
and review of this manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ygyno.2010.01.021.
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