Flaxseed Lignans as Important Dietary Polyphenols for Cancer Prevention and Treatment: Chemistry, Pharmacokinetics, and Molecular Targets

Abstract

Cancer causes considerable morbidity and mortality across the world. Socioeconomic, environmental, and lifestyle factors contribute to the increasing cancer prevalence, bespeaking a need for effective prevention and treatment strategies. Phytochemicals like plant polyphenols are generally considered to have anticancer, anti-inflammatory, antiviral, antimicrobial, and immunomodulatory effects, which explain their promotion for human health. The past several decades have contributed to a growing evidence base in the literature that demonstrate ability of polyphenols to modulate multiple targets of carcinogenesis linking models of cancer characteristics (i.e., hallmarks and nutraceutical-based targeting of cancer) via direct or indirect interaction or modulation of cellular and molecular targets. This evidence is particularly relevant for the lignans, an ubiquitous, important class of dietary polyphenols present in high levels in food sources such as flaxseed. Literature evidence on lignans suggests potential benefit in cancer prevention and treatment. This review summarizes the relevant chemical and pharmacokinetic properties of dietary polyphenols and specifically focuses on the biological targets of flaxseed lignans. The consolidation of the considerable body of data on the diverse targets of the lignans will aid continued research into their potential for use in combination with other cancer chemotherapies, utilizing flaxseed lignan-enriched natural products.

Full text

Pharmaceuticals 2019, 12, 68; doi:10.3390/ph12020068 www.mdpi.com/journal/pharmaceuticals
Review
Flaxseed Lignans as Important Dietary Polyphenols
for Cancer Prevention and Treatment: Chemistry,
Pharmacokinetics, and Molecular Targets
S. Franklyn De Silva * and Jane Alcorn *
Drug Discovery & Development Research Group, College of Pharmacy and Nutrition, 104 Clinic Place,
Health Sciences Building, University of Saskatchewan, Saskatoon, Saskatchewan (SK), S7N 2Z4, Canada
* Correspondence: jane.alcorn@usask.ca (J.A.); f.desilva@usask.ca (S.F.d.S.); Tel.: +1-306-966-6365 (J.A.)
Received: 22 March 2019; Accepted: 30 April 2019; Published: 5 May 2019
Abstract: Cancer causes considerable morbidity and mortality across the world. Socioeconomic,
environmental, and lifestyle factors contribute to the increasing cancer prevalence, bespeaking a
need for effective prevention and treatment strategies. Phytochemicals like plant polyphenols are
generally considered to have anticancer, anti-inflammatory, antiviral, antimicrobial, and
immunomodulatory effects, which explain their promotion for human health. The past several
decades have contributed to a growing evidence base in the literature that demonstrate ability of
polyphenols to modulate multiple targets of carcinogenesis linking models of cancer characteristics
(i.e., hallmarks and nutraceutical-based targeting of cancer) via direct or indirect interaction or
modulation of cellular and molecular targets. This evidence is particularly relevant for the lignans,
an ubiquitous, important class of dietary polyphenols present in high levels in food sources such as
flaxseed. Literature evidence on lignans suggests potential benefit in cancer prevention and
treatment. This review summarizes the relevant chemical and pharmacokinetic properties of
dietary polyphenols and specifically focuses on the biological targets of flaxseed lignans. The
consolidation of the considerable body of data on the diverse targets of the lignans will aid
continued research into their potential for use in combination with other cancer chemotherapies,
utilizing flaxseed lignan-enriched natural products.
Keywords: Flaxseed lignans; Dietary polyphenols; Phytochemicals; Cellular/molecular targets;
Pharmacokinetics; Chemopreventive; Chemotherapeutic; Hallmarks of cancer; Quality of life
1. Introduction
The exploration of alternative strategies for cancer prevention and treatment has become
necessary owing to the high costs of current chemotherapies, prolonged time in regulatory
authorization processes for new cancer treatments, and the considerable expenditure associated
with taking a medicinal agent from bench to bed-side. Repositioning of noncancer therapeutics, such
as plant polyphenols, to treat cancer offers an alternate strategy to address these challenges. Such
therapeutic interventions are usually associated with lower costs and manageable toxicity with
concomitant improvement in quality of life. Plant polyphenols have a long history of proposed
benefits in the prevention and treatment of a chronic disease like cancer [1–4]. Although the
evidence for health benefits of plant polyphenols is available throughout the literature, the flaxseed
polyphenols have gained increasing attention. Flaxseed contains numerous nutrient and
non-nutrient chemical constituents, like α-linolenic acid, fiber, and lignans, which can support our
well-being [5–20]. More recently, polyphenols of flaxseed—the lignans—have sparked increased
interest mostly attributing to their antioxidative, anti-inflammatory, anti-atherosclerogenic, and

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antiestrogenic potential, thus suggesting ability to reduce risk and protect against cancer [16,21–38].
Such attributes have compelled expansion of investigations into lignan mechanisms of action.
Plant lignans [39], different from lignins (racemic polymers that are components of the plant
cell wall [39,40]), are non-nutrient, noncaloric, bioactive phenolic plant compounds [39]. Diverse
lignanoid constituents from plant-based food resources [41], like secoisolariciresinol diglucoside
(SDG) [42], lariciresinol [41], isolariciresinol [43], 7-hydroxymatairesinol [44], matairesinol (MAT)
[45], pinoresinol, arctigenin, syringaresinol [46], and asarinin [47], can be precursors of
enterolignans—the mammalian-derived lignans—following oral consumption of plant lignans [39].
The primary intent of this review is to consolidate the evidence of lignan pharmacokinetics and
modulation of cellular processes and cell signaling pathways within the cancer phenotype so as to
provide opportunity to direct future investigations into the role and benefit of the dietary
polyphenols, specifically flaxseed lignans, in the prevention and treatment of cancer (i.e.,
complementary and integrative medicine [48]).
2. Growing Use of Naturally Derived Products
Unsatisfactory results of Western medicine have given complementary and alternative
treatment options more attention [49]. Many patients rely on phytochemicals and herbal medicines
(collectively referred to as natural health products (NHPs) for the purpose of this review) for
primary health care, especially in the developing world [50]. In developed countries, NHPs are used
to promote healthier living [50]. Although several NHPs have promising effects with wide
utilization, some remain untested with clinical use unmonitored and undocumented [50], while
some NHPs have safety concerns [51]. Existence of a regulatory framework for NHPs provides
greater reassurance to consumers; however, regulations and product quality specification vary
among countries [49]. As an example, the Dietary Supplement Health and Education Act (DSHEA)
of 1994 provided the U.S. FDA the authority to implement Good Manufacturing Practices (GMP) for
dietary supplements and ensure safety of such products, and the framework of the Federal Food,
Drug, and Cosmetic Act, which led to the DSHEA, provides the necessary framework needed by the
Food and Drug Administration (FDA) to regulate dietary supplements [52]. Additionally, in
Canada, NHPs approved by Health Canada (e.g., herbal remedies [53]) are regulated under the
Natural and Non-Prescription Health Products Directorate [54]. This allows for large production
and lower prices (due to competition) by companies, even though NHPs are regulated somewhat
similar to pharmaceutical drugs under the Natural Health Products Regulation (NHPR). These
regulations protect Canadians by ensuring that the products obtained meet their health needs
[55,56]. Regardless of the preclinical evidence of NHPs, translational capabilities into the clinic can
be hampered by similar factors encountered by drugs in development such as the dose size and
dosage forms and the variability in outcomes caused by gender, ethnicity, and comorbidities [55].
3. Cancer, the Unmet Medical Need
Cancer was identified as an important human disease thousands of years ago [57]. In the
subsequent thousands of years cancer patients faced little hope for cure and survival, a situation
unchanged for some cancers and clearly an unmet medical need for these patients. Globally, cancer
contributes to considerable mortality with estimates projected to increase from 14 million new cases
per year in 2012 [2] to an estimated 19.3 million cases yearly by 2025 [58,59]. Colorectal, liver, breast,
gastric, prostate, cervical, and lung cancers remain the principal causes of cancer deaths [2], and the
majority of cancer related mortality occurs in low- and middle-income countries [60,61]. Surgery,
radiotherapy, and systemic therapy, which include general chemotherapy, hormonal therapy,
immunotherapy, and targeted therapies, are the current treatments of cancer [58]. In too many
patients these treatments fail, and cancer remains a major challenge to clinical interventions. A need
exists to discover more effective ways of targeting cancer and new avenues of disease management
might offer some potential.
Today’s improved understanding of the characteristics of cancer offer renewed hope for
treating cancer. The pioneering work of Weinberg and colleagues to categorize the cancer

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characteristics into distinct tumor properties, the so-called “Hallmarks of Cancer”, provide a
framework around which to rally the considerable scientific and technological advances to identify
more effective treatments for different cancer phenotypes [62,63]. Among the different cancer
characteristics, mutations enable malignant cells both continuity and survival. These driver
mutations stem from various mechanisms including carcinogen exposure [57]. Carcinogenesis is a
complex multifactorial and multistep process separated into three closely related stages: initiation,
promotion, and progression [64–67] (Figure 1). The first stage, initiation, follows usually from
carcinogen (or its metabolite) exposure and is traditionally considered an irreversible step with one
or more genetic alterations resulting from DNA mutations, transitions, transversions, or deletions
[65,68]. Promotion is the second stage and is considered a reversible stage where the proliferation of
neoplastic cells takes prominence [65]. This stage does not involve DNA structural changes but
rather changes in genome expressions brought out by promoter–receptor interactions [68]. Tumor
progression is the last stage where neoplastic transformation occurs followed by tumor growth,
invasion, and metastasis [65]. Stages of carcinogenesis are governed by proto-oncogenes, cellular
oncogenes, and tumor suppressor genes. These genes and their protein products may serve as
druggable targets for cancer treatment.
In order to reduce the incidence and mortality of different malignancies, effective preventive
strategies that impede tumorigenesis are needed [69]. Although the heterogeneity of tumors and
tumor development may pose a challenge for successful therapeutic interventions [70], the initial
stages of carcinogenesis is usually associated with a lower burden of molecular and cellular
aberrations such that chemopreventive or early chemotherapy is more likely to achieve therapeutic
efficacy as compared with treatment of more advanced stages of tumorigenesis [70]. Furthermore, it
is well known that cancer development and progression is associated with inflammation. Hence,
early preventive or treatment strategies should include anti-inflammatory therapies. While
recruitment and activation of inflammatory cells due to mutations that initiate cancer may trigger
cancer-intrinsic inflammation, a multitude of factors (e.g., toxin exposure, microbial infections,
autoimmune disease, and obesity) are responsible for cancer-extrinsic inflammation [71].
Epidemiologic studies reveal that ~20% of all cancers emerge as a direct result of long-standing
inflammatory disease [71–73]. For these reasons inflammation is a frequent mechanism of action for
diverse cancer risk factors [71]. Therefore, various anti-inflammatory agents such as selective
cyclooxygenase-2 inhibitors, nonsteroidal anti-inflammatory drugs, and natural health products
with anti-inflammatory properties have been identified as potential chemopreventive agents
[69,71,74–79].
4. Cancer Prevention
The sequence of events in the multistage process of carcinogenesis provides opportunities for
intervention with the goal of preventing, reversing, or delaying tumor development and progression
[80]. Interventions generally fall into three categories of prevention, namely primary (preventing
disease or injury), secondary (reducing impact of disease or injury), and tertiary (reducing impact of
ongoing disease or injury having lasting consequences) [81–88]. These categories are based on the
concept of chemoprevention first proposed in the early 1970s by Sporn [85,89], and extended by
Wattenberg, who suggested the selective inhibition of carcinogenesis in any of the phases of
cancer—initiation, promotion, or progression [90,91]. Primary chemopreventives block the disease
by inhibiting mutagenesis, cancer initiation, and tumor promotion [65]. During early stages of
tumorigenesis, secondary chemopreventive agents inhibit tumor progression by interfering with
signal transduction, hormones, angiogenesis, antioxidant activity, and immune status [65]. The third
class promotes chemoprevention by blocking cancer invasion and metastasis in patients usually
after initial therapy [65] through mechanisms including activation of antimetastatic processors and
modulation of cell adhesion factors or extracellular matrix degradation components [3,65,88] (Figure
1). However, interventions that interfere with all three phases will likely bring about a more
meaningful degree of cancer prevention [81].

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In general, chemoprevention can be achieved through reduction in bioactivation of
procarcinogens, obstruction of expansion of additional malignant cells, or through suppression of
metabolism of specific compounds to reduce toxicity [4,92]. This understanding has led to four
notable categories of chemoprevention and include medications, hormones (i.e., antiestrogens and
antiandrogens), vaccines, and dietary agents [93]. Only a handful of agents have been clinically
approved for cancer chemoprevention (e.g., the anti-inflammatory drugs, aspirin, celecoxib and
diclofenac) [70,71], with several others suggested as possible chemopreventive agents (e.g., the
anti-hypercholesterolemic statins, the antidiabetic drug metformin, and antiosteoporosis
bisphosphonates) [94]. The complexity associated with cancer pathogenesis has otherwise limited
our ability to identify primary, secondary, or tertiary interventions that effectively reduce cancer risk
or progression. The cost of patient survival and quality of life, though, continue to drive research
into effective chemopreventive interventions [70,71,91,95].
Figure 1. Polyphenolic phytochemicals (e.g., lignans) block and suppress carcinogenesis.
Carcinogenesis is a multistage process of initiation, promotion, and progression. Carcinogens may
initiate carcinogenesis by causing the conversion of a normal cell into an “initiated cell”, a process
that is irreversible and involves genetic mutations. Initiated cells further transform into

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pre-neoplastic cells during the stage of promotion, and subsequently progress into neoplastic cells.
Polyphenolic phytochemicals are capable of interfering with cellular and molecular processors in
various stages of carcinogenesis. Phytochemicals may block cancer initiation through inhibition of
procarcinogen activation into electrophilic species and their subsequent interaction with DNA.
Alternatively, phytochemicals can stimulate carcinogen detoxification and their subsequent
elimination from the body. Phytochemicals may suppress cancer by interfering with cancer
promotion (a reversible process that involves nongenetic changes) or by regulating cancer
progression, a complex process that involves both genetic and nongenetic changes as well as cell
survival. Some polyphenols can act as blocking agents; others act as both blocking and suppressing
agents, and some function as suppressing agents to modulate autophagy, cell cycle, and
differentiation, thus affecting cancer cell proliferation. Adapted from references [3,4,67].
An active avenue of research in chemoprevention involves natural chemicals derived from
plants (i.e., phytochemicals). Over 80,000 species of plants are utilized in healthcare management,
while more than 60% of the existing anticancer drugs come from nature [96]. The broad selection of
biologically active, structurally different natural compounds continues to aid the process of cancer
drug discovery with respect to chemoprevention and chemotherapy [97,98]. An abundance of
phytochemical constituents with preventive anticancer properties against cancers such as lung,
breast, ovarian, prostate, thyroid, and colon have been reported throughout the literature [92,99–
109]. These phytochemicals have not seen wide application despite the limitations of current
treatment methods [110] as general Western practices often dismiss their value for patient treatment.
For this reason, researchers covering a wide area of health research have turned their focus on
alternate ways to address the issues related to general western practices and to capitalize on the
protective effects of phytochemicals [111].
5. Alternate Approaches to Malignant Disease
The Halifax Project—an international task force comprising of 180 scientists—has posed a
“broad-spectrum therapeutic approach” as an alternate low-toxicity strategy to mitigate the
problems of cancer chemotherapy [58]. Following from a rigorous examination of the cancer
hallmarks, this interdisciplinary group identified 74 high-priority targets. Many of the suggested
therapeutic approaches for these targets were phytochemicals with evidence of low toxicity [58].
Such phytochemicals are also commonly considered complementary and alternative medicines
(CAMs), and are associated with integrative medicine. For cancer, integrative medicine is based on a
foundation of lifestyle therapies, drawing attention to diet, dietary components, and physical
activity [58,112,113]. It focuses on patient quality of life and demands marshalling of all therapeutic
and lifestyle strategies to ensure the best outcomes and optimal health of the patient [112,114].
Phytochemicals as CAMs should be included as a strategic lifestyle intervention in a broad-spectrum
approach to cancer disease management [58]. Already, the potential psychological and
socioeconomic benefits of CAM use is exemplified by studies that report 32–66% of cancer patients
having used cost-effective CAMs as a means to improve quality of life and therapeutic outcomes
[115]. Furthermore, studies employing a combination of clinically relevant chemotherapeutic drugs
with natural bioactive compounds demonstrate enhancement in antitumor effects and reduction in
side-effects [111,112,116]. Some reports also document the potential of phytochemicals in
overcoming chemoresistance and radioresistance of malignant cells [111,117]. Hence, the
repositioning of traditionally considered “noncancer”, nontoxic phytochemical therapies with
promising antineoplastic characteristics may help achieve better therapeutic outcomes and reduced
toxicity profiles [118]. Convincing practitioners of this broad-spectrum therapeutic approach will be
important to ensure a larger number of patients achieve improved quality of life and cancer
treatment outcomes with phytochemical interventions.
6. Potential of Dietary Phytochemicals for Malignant Disease

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The World Health Organization (WHO) reported that approximately 65% of the world’s
population relied on plant-derived drugs for their primary health care by 1985 [119]. These therapies
demonstrate potential, but their safe and rational use in Western medicine is limited by a lack of
rigorous scientific investigation of their potential therapeutic and adverse effects, mechanisms of
action, and interactions with pharmaceuticals and functional foods [50]. Although the use of dietary
phytochemicals in cancer treatment has had a long history, their efficacy is variable due to their
complexity, their poorly defined targets and modes of action, and lack of knowledge of effective
doses [120]. Nonetheless, a number of phytochemicals have been applied successfully in the clinical
setting such as metformin and nonsteroidal anti-inflammatory drugs (NSAIDs) [121,122]. As well,
the complexity and diversity in structure of phytochemicals make these compounds an often
exploited scaffold to aid the discovery and synthesis of analogs that share similar structures but with
improved and modified efficacy [123–125]. High-throughput screening (HTS), a specialized tool
using automation to screen compound libraries against the drug target within a short period of time
[126], has made the rediscovery of phytochemicals even more feasible [55]. The pleiotropic,
multitarget effects of phytochemicals as well as polypharmacology also resonate within the
emerging paradigm in drug discovery [127–130]. These factors identify dietary phytochemicals as an
invaluable resource for new treatment options in current unmet medical needs, such as cancer. Yet
few randomized clinical trials document the use of dietary phytochemicals in combination with
standard of care treatments against human malignancies. An ability of phytochemicals to enhance
the efficacy of standard treatments against cancer warrants an investigation into the wide range of
biologically active compounds that have been isolated, identified, and tested for their application as
treatments for cancer [131].
6.1. Dietary Polyphenols as Principal Phytochemicals for Malignant Disease
An inverse relationship exists between the high consumption of fruits and vegetables and a
reduced risk of cancer [132,133], with an average 35% of all human cancer mortality directly
attributed to diet [4,134]. Such statistics prompted organizations such as the WHO, the American
Cancer Society (ACS), the American Institute of Cancer Research (AICR), and the U.S. National
Cancer Institute (NCI) to establish dietary guidelines in an attempt to reduce cancer risk [4]. These
guidelines are complemented by ongoing clinical trials that investigate diet and dietary supplements
for the prevention of cancer [4]. Although food is generally perceived as providing nutritional value,
phytochemicals have an additional potential to modulate molecular and cellular targets [135]. Their
influence on biological function suggests that institution of an adequate, economical, and rapid
system for evaluation and testing of phytochemicals with potential anticancer properties may
augment the current dietary guidelines or identify lead compounds for drug discovery in different
cancer phenotypes [96,136].
Phytochemicals (“phyto” in Greek means plant) are bioactive non-nutritive chemical
components of plant-based diets such as fruits, vegetables, nuts, and grains [4,137] produced as
primary and secondary metabolites of the plant [65]. These are generally classified into polyphenols,
alkaloids, carotenoids, and organosulfur compounds [135,138] (Table 1). Primary metabolites are
involved in plant functions such as respiration, development and photosynthesis, while secondary
metabolites play a role in defense against herbivores and pathogens, attracting pollinators, and
protection against ultraviolet radiation [139]. These secondary metabolites can have benefit in
vertebrates as chemopreventive agents, drugs, herbicides, and antibiotics [65,139], and their chronic
exposure is suggested to have health benefits for neurodegenerative disorders, cancer, diabetes and
cardiovascular disease [140–142]. Polyphenols are an important class of beneficial secondary
metabolites found in food and drink sources from vegetables, fruits, nuts, spices, grains, coffee tea,
and wine [65].

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Table 1. The classification of phytochemicals. Adopted from references [135,138,139].
Classification Representative
Members
Examples of Dietary
Sources
Poly-Phenolics
Phenolic Acids
Hydroxycinnamic
acids
p-Coumaric, caffeic,
ferulic, sinapic
Barley, eucalyptus,
coffee, Arabidopsis,
Hibiscus, cereal grains
Hydroxybenzoic
acids
Gallic, vanillic,
syringic, ellagic
Chestnuts (boiled or
roasted), witch hazel, tea
leaves, oak bark,
rhubarb, pomegranate,
grapes, chocolate, wine
Lignans
Plant Lignans
sesamin,
secoisolariciresinol
diglucoside,
lariciresinol,
isolariciresinol,
7-hydroxymatairesinol,
matairesinol,
pinoresinol, arctigenin,
syringaresinol, asarinin
Flaxseed, pumpkin,
sunflower, poppy, rye,
oats, barley, wheat, oat,
rye, berries
Mammalian
Lignans
(enterolignans)
Enterodiol,
enterolactone
Stilbenes Grapes
Other Phenolics Coumarins Tonka bean, vanilla
grass
Tannins Eucalyptus, geranium
Flavonoids
Flavonols Quercetin, kaempferol,
myricetin
Aloe Vera, European
elderberry, soy, St John’s
wort, tomatoes, red
onions
Flavones Apigenin, luteolin
Celery, parsley,
chamomile tea, green
peppers, thyme, oregano
Flavanols
(catechins)
Catechin, epicatechin,
epigallocatechin gallate
White tea, green tea,
persimmon,
pomegranate, cocoa
beans
Flavanones Eriodictyol, hesperetin
Citrus fruits, rose hip,
mountain balm
Anthocyanidins Cyanidin,
pelargonidin, malvidin
Grapes, berries, red
cabbage, red onions,
plums, kidney beans,
geranium
Isoflavonoids Genistein, glycitein
Lupin, fava beans, soy,
coffee
Alkaloids Poppy, tomatoes,
potatoes
Carotenoids
α-carotene,
β-carotene,
lutein,
zeaxanthin,
lycopene
Carrots, broccoli,
spinach, zucchini
Organosulfur
compounds
Isothiocyanates,
indoles, allyl
sulfur
compounds
Cabbage, broccoli,
spinach, garlic, onions
Plant polyphenolic secondary metabolites are synthesized from carbohydrates through the
shikimate pathway [143,144]. Although these metabolites may exist as insoluble or bound forms
[144], they are present generally as glycosylated forms with single or multiple sugar or carbohydrate
residues conjugated to a hydroxyl functional (–OH) group or an aromatic ring involving a
co-translational or post-translational enzymatic process. Over 8000 plant-based polyphenols have

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been identified [65,145], and are divided into a number of classes based on chemical structure,
source, and biological function including the flavonoids (flavonols, flavones, flavanones, catechins,
anthocyanidins, and isoflavones), phenolic acids (benzoic acids and cinnamic acids), stilbenes,
lignans, coumarins, tannins, and other polyphenols (e.g., curcumin, rosmarinic acid, gingerol)
[137,139,141,146]. More broadly, polyphenolics can be classified as either being flavonoid and
nonflavonoids based on their abundance [65,139]. There are over 4000 types of diverse flavonoids
accounting for ~60% of structurally-related dietary polyphenols [80,141], while ~30% of dietary
polyphenols are phenolic acids (i.e., hydroxy-cinnamic and hydroxy-benzoic acids) [80,141].
Flavonoids, phenolic acids, stilbenes and lignans are the most abundantly occurring plant
polyphenols [80].
The literature provides ample evidence for the anticancer properties of polyphenols [2,140,147–
150]. The key anticancer characteristics of polyphenols include anti-inflammatory and antioxidative
effects, immunomodulation, and modulation of molecular/cellular targets within signaling
pathways involved with cell proliferation, survival, differentiation, angiogenesis, migration, and
hormonal activities [2,151,152]. In general, the pleiotropic effects of dietary polyphenols usually
follow from their multitarget effects having the ability to impact an entire process or several
processors of the malignant disease condition or status. Polyphenols typically exhibit low to
moderate affinity for their targets. However, their ability to simultaneously modulate multiple
targets with low affinity is suggested to account for their effects in the cancer phenotype [153,154].
Since bioactivity is not only dependent upon the interaction of the polyphenol with its target sites,
but also on the chronic exposure to the polyphenol, the increasing popularity of polyphenols have
led to the emergence of two new terms, ‘nutridynamics’ and ‘nutrikinetics’ [155,156]. These terms,
similar in meaning to drug pharmacodynamics and pharmacokinetics, are expected to make
significant contributions in our understanding of the relationship between disease phenotypes and
bioactivity, as well as the interplay between chronic exposure and the host’s physiology including
digestion, metabolism, and gastrointestinal microflora [157,158].
6.2. General Properties of Polyphenols and Evidence on Health
As drug discovery efforts continue to move away from single target drugs, the multitarget
characteristics of polyphenols, such as the lignans, warrant further attention to fully grasp their
potential use in the clinic. Diet-derived polyphenols have gained popularity among nutritionists,
food scientists, and consumers during recent years for their health-promoting and chemopreventive
properties [141,159]. The beneficial effects on human health by long-term polyphenol rich diet
consumption is linked to the modulation of cell proliferation, body weight, chronic disease, and
metabolism [160]. The antioxidant and anti-inflammatory potential of polyphenols as indicated in
animal, human, and epidemiologic studies, suggest chemopreventive or therapeutic effects for a
number of noncommunicable diseases such as neurodegenerative disorders, obesity, diabetes,
cardiovascular disease, osteoporosis, gastrointestinal issues, pancreatitis, and cancer [160–162].
Overconsumption of dietary polyphenols, especially when they are not consumed in a form of a
food matrix, though, may result in adverse effects on health [160,163,164]. Our understanding of the
mechanisms underlying the potential health benefits largely arise from in vitro studies and,
therefore, a certain degree of uncertainty exists if these mechanisms hold true in human patients
[160,165–168]. Nonetheless, polyphenol mechanism of action has greater complexity than the long
standing belief that polyphenols form stabilized chemical complexes to negate free radicals and
prevent further reactions [160,169], or result in the production of hydrogen peroxide (H
2O2) for
protection against oxidative stress to aid in the immune response and modulate cell growth
[160,169,170].
6.2.1. General Pharmacodynamic (or Nutridynamic) Effects of Polyphenols
In general, nutridynamic effects of polyphenols can be broadly summarized and grouped based
on the following general molecular mechanisms [92]; (a) modulation of phase I and II drug
metabolizing enzymes (e.g., cytochrome P450s and UDP-glucuronyltransferases) [69,80,141,171–

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173]; (b) inhibition of reactive oxygen species and modulation of antioxidant activity [4,141,171,174–
176]; (c) inhibition of multidrug resistance (e.g., c-Myc and HDACs) [4,80,141,176,177]; (d)
modulation of inflammation [69,141,172,175,177]; (e) modulation of androgen and estrogenic activity
[141,176,178–181]; (f) inhibition of tyrosine kinases [80,141,176,177,182]; (g) modulation of matrix
metalloproteinases, epithelial-to-mesenchymal transition [183], and metastases [80,91,141,172,177];
(h) modulation of angiogenesis [91,141,171,177,184]; (i) inhibition of cell cycle regulators and
induction of cell cycle arrest [80,141,171,177,185]; (j) induction of apoptosis [80,91,141,171,175]; (k)
inhibition of cell growth and proliferation [91,141,174,175,177]; (l) modulation of endoplasmic
reticulum-stress and type II programmed cell death or autophagy [141,175,176,185–187]; (m)
modulation of mitogen-activated protein kinases [69,141,171,176,177]; (n) modulation of PI3K-AKT
signaling [4,69,141,177,185]; (o) modulation of JNK pathway [80,141,176,177,185]; (p) modulation of
glucose and lipid [69,171,174,185,188,189]; and (q) hepatoprotective effects [190–194]. However, only
a few polyphenols (e.g., flavonoids) have gained approval as NHPs, some with defined health
claims, and none have been widely approved for clinical use [92].
6.2.2. General Pharmacokinetic (Or Nutrikinetic) Characteristics of Polyphenols
Absorption and disposition (i.e., nutrikinetics) characteristics play an important role in
exposure to dietary polyphenols and their eventual therapeutic effects. With oral consumption,
nutrikinetic processes ultimately determine the concentration and persistence of polyphenolic
compounds at their target sites. Since both genetic and epigenetic factors influence the nutrikinetics
of polyphenols, these factors often result in considerable interindividual variation in blood and
tissue exposure levels [137,195–200]. Despite the importance of nutrikinetics as a determinant of
polyphenolic action, only a handful of in vivo studies have systematically addressed the factors that
contribute to the differences in their absorption and disposition characteristics [137].
Dietary polyphenols must become systemically available to influence cancer treatment. Many
plant polyphenols first undergo modification by gastrointestinal enzymes and/or bacteria to
produce metabolites that are more or less systemically biologically active. The initial metabolic
transformations typically involve deglycosylation to release aglycones into the gastrointestinal tract
lumen following enzymatic breakdown of polymeric forms with subsequent deconjugation of
monomeric forms by β-glucosidases on the brush border membrane or by the resident (small
intestine and colon) gut bacteria [137,143,144]. These aglycones may undergo absorption or be
further subjected to microbial enzymatic transformations including ring fission, α/β-oxidation,
dihydroxylation, dehydrogenation, and demethylation reactions [137,144,201–203], with their
subsequent absorption from the gastrointestinal lumen. Given their interactions with intestinal
bacteria, polyphenols also can induce intestinal microbial changes [144], with reports that identify a
polyphenol–gut microbiota interaction that either contributes to or prevents the development of
disease [144,204,205].
During their permeation across the intestinal epithelium or with passage through the liver,
aglycones or their metabolites may undergo extensive first-pass metabolism. These metabolic
transformations typically involve conjugation reactions, with glucuronic acid or, to a lesser extent,
with glutathione or sulfate [137]. UDP-glucuronosyltransferases (UGT), sulfotransferases (SULTs),
and glutathione-S-transferases (GST) carry out conjugation reactions in both enterocytes and
hepatocytes to produce conjugates that are excreted into the bile or become systemically available
with subsequent excretion by the kidney into the urine [137]. Conjugates excreted into bile may
undergo enterohepatic recycling making available the nonconjugated form for absorption following
deconjugation by intestinal and/or microbial β-glucuronidase [137]. Typically, the aglycones are
more biologically active, but the glycosidic forms, and rarely the glucuronide conjugates, have
biological activity [137,206–213].
An important consideration in the oral bioavailability of phytochemicals is the role of intestinal
epithelial transporters. Plasma membrane ATP-binding cassette (ABC) transporters play a vital role
in the systemic availability of a number of dietary polyphenols or their metabolites. These
ATP-dependent transmembrane efflux transporters are expressed on the apical or basolateral

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epithelial membrane, depending on the isoform. On the basolateral membrane, ABC transporters
actively efflux phytochemical conjugates from intestinal cells (where conjugation occurred) into the
portal blood supply. When expressed on the apical side of the epithelium, ABC transporters efflux
phytochemicals back into the intestinal lumen to cause reductions in oral bioavailability [137].
P-Glycoprotein (Pgp/ABCB1/MDR1), multidrug resistance proteins (MRPs/ABCCs), and the breast
cancer resistance protein (BCRP/ABCG2) are the key ABC efflux transporters [137,214,215] known to
influence systemic availability of a number of dietary polyphenols [216,217]. For example,
enterolactone is a substrate and competitive inhibitor of ABCG2 [218]. These ABC transporters
exhibit several genetic polymorphisms that may influence the systemic availability of these
compounds, which can contribute to considerable interindividual variation in their oral
bioavailability [219–221].
Members of the solute carrier (SLC) family of transporters also contribute to the intestinal
epithelial uptake of certain dietary polyphenolic compounds. The polar glycosidic forms of the
dietary polyphenols typically exploit SLC transporters to ensure their systemic availability [142,222–
225]. Glucosides could be transported into enterocytes by sodium-dependent glucose transporters
(SGLT) such as SGLT-1 [142]. Once inside they can be hydrolyzed by cytosolic β-glucosidase to their
aglycone forms [142]. Additionally, in the small intestine brush border membrane, extracellular
hydrolysis of several glucosides can be carried out by lactase phloridzine hydrolase [142]. Although
it is speculated that both enzymes are involved this process, their relative contribution towards
different glucosides is unknown [142]. The aglycone metabolites have sufficient lipophilicity for
passive diffusion to be the principal transport process during absorption. However, some
polyphenols show ability to inhibit SLC transporters to influence the uptake of substrates of such
transporters [215].
The relatively limited information on the tissue distribution of dietary polyphenols largely
comes from preclinical evaluations with rodent models. Polyphenol compounds generally
accumulate in highly perfused tissues such as the liver, kidney, heart, lung, and intestine, and many
are present predominantly in their conjugated forms [142,145,226–231]. Tissue specific accumulation
is observed as well, as is the case for flaxseed lignans which accumulate in prostate and breast tissue
[232,233]. Extent of plasma protein binding, which can function to limit availability of polyphenols
to tissue sites depending upon relative affinity between plasma protein and tissue binding sites,
tends to increase with increasing lipophilicity of the compounds [213,230,234–237]. However, the
polar conjugates of dietary polyphenols exhibit very limited plasma protein binding characteristics.
Finally, expression of ABC transporters at tissue–blood barriers might limit access of certain
polyphenols to such tissues preventing their accumulation and possible activity at such sites. For
example, tumor cells typically overexpress ABC transporters to restrict access of a broad range of
chemically unrelated pharmacological therapeutics to the cancer cell (aka multidrug resistance or
MDR) [215]. ABC transporters also function to reduce intracellular concentrations of polyphenols,
but as competitive inhibitors [215] polyphenols may enhance the cellular concentration and
pharmacological response of chemotherapeutic drugs [238–240].
Most polyphenols are eliminated by intestinal and hepatic metabolism [241,242]. Aglycones of
plant polyphenols that bypass first-pass metabolism are typically eliminated via hepatic phase II
metabolism with the subsequent excretion of these metabolites by the biliary or renal system [241–
243]. First-pass and systemic phase II metabolism are typically considered inactivation processes
that result in loss of biological activity [241]. Very limited phase I metabolism occurs yielding
primarily aromatic hydroxylated metabolites largely mediated by the cytochrome P450 enzyme
superfamily [241,244,245]. These hydroxylated metabolites undergo further phase II conjugation and
subsequent excretion by the kidney [241,242]. Given the extensive first-pass metabolism and ability
to undergo enterohepatic recirculation, fecal excretion represents a major route of elimination of
many dietary polyphenols, while fecal and urinary excretion is the principal route of elimination for
the metabolites of polyphenols [231,241,246].

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7. Challenges Associated with Cancer Prevention and Dietary Polyphenols
Screening, early detection, and chemoprevention are widely accepted as the major strategies to
address the burden of cancer [85]. Chemoprevention as a major strategy is viewed less
optimistically, as there exists a lack of clear understanding of the benefits of chemoprevention. The
clustering of various approaches including dietary manipulation, NHPs such as polyphenols, and
repurposed “benign drugs” into the idea of chemoprevention has done little to mitigate the
uncertainty associated with the value of chemoprevention in reducing cancer risks [85]. Nonetheless,
preventive strategies support the rationale for the early disruption of the carcinogenic process,
which can avoid the treatment difficulties arising with the complexity and heterogeneity of more
advanced stage cancers [85]. Chemoprevention has potential to address the many intrinsic genetic,
epigenetic, and environmental factors that influence individual risk for cancer [85,247–252]. Hence, a
basic rethinking of the nature of carcinogenesis as proceeding in a nonlinear fashion with irregular
interruptions owing to changes in genetics and epigenetics [85] may benefit and expand the
understanding and application of chemoprevention [85].
A variety of obstacles have hindered the development of dietary polyphenols as clinically
approved chemopreventives. There has been a general inability to confirm their effect in reducing
cancer risk due to a number of factors that include the lack of appropriate experimental models, the
costs and time associated with epidemiological studies, as well as variations in length of exposure
and adherence data in clinical populations, difficulties in evaluating the dietary intake of
polyphenols, the variation in composition of polyphenols among different dietary sources,
degradation or alteration of polyphenol chemical structures that may result in loss of bioactivity,
variability and unpredictability in pharmacokinetic profiles, the impact of the microbiome on
polyphenol bioactivity, and drug–polyphenol interactions [92,160,230,253–260]. To address these
obstacles, research is focusing on improved extraction and purification methodology [92,261–264],
development of microbial production systems for plant polyphenols [265–270], formulation of
polyphenols into micro- or nanodelivery systems [271–274], development of antibody directed
enzyme prodrug therapy approaches [275–278], administration of glycosidic derivatives
[92,279,280], use of bioenhancers [281–283] or specific ATP binding cassette transporter inhibitors
[92,284], use of antibiotics or other natural products to modulate intestinal microflora [260,285],
development of novel polyphenol derivatives by modification of chemical structures [286–290], and
polyphenol complexing with protein or phospholipids [92,291–295]. To date though, much of the
investigation into polyphenols have involved in vitro evaluations and a sparsity of clinical trials
using well-defined amounts of polyphenolic compounds [296] suggests a need for well-conducted
clinical investigations to resolve their safety and efficacy as chemopreventives. The substantive
evidence that exist beyond their publicized antioxidant properties [259,297], for their selective
actions on a plethora of cellular and molecular signaling pathways, warrant their investigation in
human clinical populations so that we may realize the health benefits of polyphenols in chronic
diseases such as cancer [298–301].
8. Polyphenols of Flaxseed as Important Phytochemicals in Malignant Disease
Flax is well known for its usefulness as a source for industrial oil and fiber [109,302]. Canada
and the United States are among the top producers of flax [15]. Flaxseed is considered to be a
multicomponent system consisting of plant-based dietary fiber (insoluble 20–30% and soluble fiber
9–10%), oil (triacylglyceride fatty acid typically include linolenic 52%, linoleic 17%, oleic 20%,
palmitic 6%, and stearic 4% acids), minor lipids and lipid-soluble components (tocopherols,
monoacylglycerides, diacylglycerides, sterols, sterol-esters, phospholipids, waxes, free fatty acids,
and carotenoids), protein, soluble polysaccharides, vitamins, minerals, lignans, and other phenolic
compounds [15,303,304]. Various flaxseed products, such as whole and ground flaxseed, defatted
flaxseed meal, and flaxseed oil, are available with suggested health benefits [305–314] and even
health claims [308,315–318]. Although these products contain a number of bioactive substances
including α-linolenic acid and the linusorbs (cyclolinopeptides) [5,319–321], lignans receive
increasing attention for their health effects [322].

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The lignans of flaxseed were once marketed in a highly concentrated standardized formulation
as BeneFlax®, a ∼38% secoisolariciresinol diglucoside (SDG)-enriched product (Archer Daniel’s
Midland) approved by both the U.S. Food and Drug Administration Agency and Health Canada
that ensured a significant source of lignan with oral consumption [323]. BeneFlax® demonstrated
good tolerability and safety with long-term supplementation [323,324]. Additionally, Goyal et al.
exhaustively listed the traditional and medicinal uses of different flax forms such as flaxseed tea,
flaxseed flour and flaxseed drink for various health conditions as well as of various medicinal
preparations using flaxseed oil [15]. Flaxseed oil, whole seed, ground whole seed, fully defatted
flaxseed meal, partially defatted flaxseed meal, flaxseed hulls, flaxseed mucilage extracts, flaxseed
oleosomes, and flaxseed alcohol extracts are among the many different types of available flaxseed
products for consumption [304]. However, most of these commercially available products contain
insufficient amounts of lignan, with levels of consumption unlikely to achieve therapeutic
concentrations. The use of such products containing relatively modest to low lignan levels in studies
investigating the health effects of lignans have contributed to inconclusive and unsatisfactory results
of flaxseed lignan interventions [325–329]. The mounting preclinical and clinical evidence, though,
suggests a need to revisit the requirement for lignan enriched products, particularly as the role of
flaxseed lignans against chronic disease such as cancer continues to attract increased attention
[22,33,303,330–347]. These include detailed investigations into the molecular mechanisms in order to
relate to lignan safety and efficacy in malignant disease.
8.1. Lignans of Flaxseed
Naturally occurring plant lignans are present in vegetables, fruits, and whole grains [22],
although the major source is flaxseed (the richest known source with 9–30 mg per gram, with lignan
production at 75–800 times over other sources [28,348]) followed by sesame and rye bran [349]. The
seed of Linum usitatissimum (Linum, a Celtic word for lin or “thread,” and usitatissimum, a Latin
word for “most useful” [302]) contains a rich source of the plant lignan, secoisolariciresinol
diglucoside (SDG) [350], and contains minor amounts of other lignans [351,352] and
cyanide-containing substances [213,303,353]. Biosynthesis of lignans in flaxseed is reported to occur
through the following pathways that include phenylpropanoid pathway, stereospecific coupling by
dirigent proteins, biosynthesis of dibenzylbutane lignans, and glycosylation of lignans into SDG
[6,354–356]. SDG primarily exists in the seed hulls [350] with an average of 32 nmol/mg hull
compared with 9.2 nmol/mg in the other parts of the seed [357]. Flax is an old agronomic crop with
over 300 species [318]. Newer cultivars, though, can contain higher concentrations of lignans in the
hull [358,359]. Differences in climatic conditions and methods of cultivation can influence the
percentage composition of the various bioactive compounds [213,360,361]. In 1956, Bakke and
Klosterman were first to isolate SDG from flaxseed [213]. However, scientific interest grew in the
early 1970s with the discovery of enterodiol and enterolactone, later referred to as the mammalian
lignans [362,363] when Axelson et al. identified SDG as the precursor for mammalian lignans
[213,364]. Traditionally, plant lignans are considered phytoestrogens along with stilbenes and
flavonoids containing the dibenzyl butane scaffold [213,365,366]. Today, lignan interest as potential
bioactive compounds goes beyond the long-held belief of estrogenic effects as their health benefits
undergo further scrutiny at the molecular level.
8.2. Chemistry and Pharmacokinetics (or Nutrikinetics) of Lignans
Lignans are a complex class of polyphenolic bioactive phytochemicals. Lignans can be
described as stereospecific dimers of monolignols (aka cinnamic alcohols) bonded at carbon 8, which
can exist free or bound to sugars in plants [39]. Secoisolariciresinol, syringaresinol, and pinoresinol
are commonly found lignan diglucosides [39]. The major lignan, SDG, exists as two
enantiomers—(+) and (-)—with varying distribution in different Linum species where some species
predominatly contain one of the two enantiomers, while others contain both (e.g., L. elegans and L.
flavum) [6,302]. Additionally, hydroxycinnamic acid derived monolignols can be dimerized into
lignans (monolignol dimers) or polymerized into lignins (insoluble dietary fibers composed of

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p-coumaryl, coniferyl, and sinapyl hydroxycinnamic alcohol large polymers [367,368]) [39].
Although lignans are not categorized as dietary fibers, lignans and lignins share some chemical
characteristics [39,369]. Mataresinol (MAT), SDG, lariciresinol, and pinoresinol are the most
common plant lignans, but the lignans arctigenin, syringaresinol, cyclolariciresinol (isolariciresinol),
medioresinol, 7′-hydroxymatairesinol, and 7-hydroxysecoisolariciresinol, sesamin, and the lignan
precursor sesamolin, can also exist in plant-based food [39,138,370].
Figure 2. Chemical composition of flaxseed. (a) Schematic representation of the lignan
macromolecule. The principal flaxseed lignan, secoisolariciresinol diglucoside (SDG), exists as a
macromolecule in the flaxseed hull. This polymer complex is composed of five SDG structures held
together by four hydroxy-methylglutaric acid (HMGA) residues with the hydroxycinnamic acids,
p-coumaric glucoside (4-O-β-d-glucopyranosyl-p-coumaric acid or linocinnamarin) (CouAG), and
ferulic acid glucoside (4-O-β-d-glucopyranosyl ferulic acid) (FeAG) as end units linked to the
glucosyl moiety of SDG. The backbone moieties of this macromolecule are represented by the circles.
The overlapping circles represent the linker molecule HMGA and the squares represent the terminal
units. The terminal unit can be CouAG/FeAG or HMGA. (b) Postulated structure of the lignan
oligomer. The SDG–HMGA polymer complex is converted into its monomer units—3-HMGA and
SDG—by hydrolysis (average size, n = 3). Flaxseed contains high levels of the lignan oligomer (with
ester linkages to HMGA, cinnamic acid, and other phenolic glucosides), which undergoes conversion
to its aglycone, secoisolariciresinol (SECO), with further biotransformation into mammalian lignans
by the action of the colonic bacteria in mammalian systems. Adopted from references
[234,302,348,371,372]
.
Flaxseed lignans are formed by the coupling of two coniferyl alcohol residues that become
integrated into an oligomeric polymeric structure, termed the lignan macromolecule [371,373,374]
(Figure S1). This polymer complex is composed of five SDG structures held together by four
hydroxy-methylglutaric acid (HMGA) residues (3-hydroxy-3-methyl glutaric acid [371]) with the
hydroxycinnamic acids, p-coumaric glucoside (4-O-β-d-glucopyranosyl-p-coumaric acid or

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linocinnamarin [371]), and ferulic acid glucoside (4-O-β-d-glucopyranosyl ferulic acid [371]) as end
units linked to the glucosyl moiety of SDG [354,372,374–376]. HMGA is considered the backbone of
the lignan macromolecule [374] (Figure 2). The flavonoid herbacetin diglucoside (HDG) is also part
of the lignan macromolecule attached via ester linkages with HMGA, similar to SDG [374,377]. This
complex is a straight chain oligomeric complex with an average molecular weight of 4000 Da [371]
and the average chain length of the complex is three SDG moieties with a hydroxycinnamic acid at
each of the terminal positions [372]. Additionally, caffeic acid glucoside (CaAG) has also been
isolated from the flaxseed lignan macromolecule [378]. The suggestion that the different phenolic
compounds of flaxseed exist in acylated forms adds further complexity to the lignan polymer
composition of flaxseed [371].
The bioactivity of the lignans requires their removal from the oligomeric macromolecule
structure upon oral consumption of the seed hull. The mechanism of release of SDG from the
complex is uncertain, but the cleavage of the glucose groups of SDG is thought to be mediated by
β-glucosidase and bacterial fermentation in the gastrointestinal tract to yield its aglycone,
secoisolarisiresinol (SECO) [379]. SDG-deglycosolating bacteria strains Clostridium sp.
SDG-Mt85-3Db (DQ100445), Bacteroides ovatus SDG-Mt85-3Cy (DQ100446), Bacteroides fragilis
SDG-Mt85-4C (DQ100447), and B. fragilis SDG-Mt85-5B (DQ100448) are mainly responsible for
conversion in the human gastrointestinal tract [55,380]. Alternative bacterial species (e.g.,
Butyrivibrio fibrosolvens, Peptostreptococcus anaerobius, and Fibrobacter succinogens in cow [381] and
Klebsiella [382], Bacteroides distasonis, Clostridium cocleatum, Butyribacterium methylotrophicum,
Eubacterium callendari, Eubacterium limosum, Ruminococcus productus, Peptostreptococcus productus,
Clostridium scindens, Eggerthella lenta and ED-Mt61/PYG-s6 in human [179,380,383,384] and
Ruminococcus gnavus in goat [385], Prevotella spp., and B. proteoclasticus [381]) are also involved in
various reactions with lignan conversion [380,381]. SECO may undergo further bacterial
demethylation and dihydroxylation reactions to produce the mammalian lignan, enterodiol (ED),
which undergoes further oxidation to enterolactone (ENL) by microbes. Streptomyces avermitilis
MA-4680 and Nocardia farcinica IFM10152 bacteria have the highest hydroxylation activity for ED
[386]. The microorganism P450 enzyme, Nfa45180, is reported to show the highest hydroxylation
activity towards ED, especially for ortho-hydroxylation of the aromatic ring in vivo [386]. Other plant
lignans in flaxseed, such as matairesinol (MAT), pinoresinol (pinoresinol diglucoside [387]), and
lariciresinol (isolariciresinol [351]) that are found in minor amounts, are also converted into the
mammalian lignans ENL and ED [46,388] (Figure 3). Hence, following consumption, SDG is released
from the macromolecule (the exact location within the gastrointestinal tract is unknown) and is
principally converted to the mammalian lignans in the lower intestine, either by the brush border
enzymatic activity of the gut mucosa or by bacterial enzymatic activity [379,389]. Oral antimicrobial
drugs are known to decrease serum concentrations of mammalian lignans highlighting the
importance of gastrointestinal flora in the production of mammalian lignan [390]. A detailed
composition of the flax lignan macromolecule, history of lignans and the analytical methods used to
identify lignans as well as extraction, isolation and purification techniques are described in previous
reviews [28,391].

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Figure 3. Lignan chemical structure and metabolites. Plant lignans are converted to various
metabolites including the mammalian lignans (enterolactone (ENL) and enteroldiol (ED)) and their
phase II metabolites such as glucuronide conjugates. The conversion of plant lignan
secoisolariciresinol diglucoside (SDG) into the mammalian lignans can by separated into four
catalytic reactions in order of O-deglycosylation (SDG to the its aglycone, SECO), O-demethylation
(SECO to 2,3-bis (3’-hydroxybenzyl)butyrolactone/2,3-bis(3,4-dihydroxybenzyl)butane-1,4-diol),
dehydrogenation (2,3-bis(3,4-dihydroxybenzyl)butane-1,4-diol to ED), and dihydroxylation (ED to
ENL). Adopted from references [42,380,381,384].
Bioactivity of the lignans also requires their adequate systemic exposure following oral
consumption. Systemic exposure of the lignans is generally quite low due to their limited oral
bioavailability. As a polar molecule, SDG does not undergo oral absorption due to its poor
permeation characteristics across the gastrointestinal mucosa [199,213,234,323,392,393]. The
lipophilicity of the aglycone SECO and the mammalian lignans encourages passive diffusion across
the gastrointestinal mucosa [392]. However, these lignan metabolites (Table S1) are subject to
extensive first-pass metabolism, primarily through phase II conjugation reactions, before they enter
the systemic circulation resulting in their rather limited bioavailability [55,323,379,392–399].

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Glucuronidation by UDG-glucuronosyltransferases (UGTs) is the principal conjugative reaction,
although sulfation by sulfotransferases (STs) and, to a minor extent, methylation by
catechol-O-methyltransferase also contribute to lignan metabolite metabolism [246]. Although the
ST isoforms involved in lignan metabolism are unknown, animal studies suggest the UGT2B
subfamily is principally responsible for the glucuronidation of lignans to mono- and diglucuronic
acid conjugates [55,400]. The extent of conjugation relates to the order of lipophilicity (SDG < SECO <
ED < ENL) [392], and therefore indicates that metabolism, excretion, and the ratios of each conjugate
and aglycone may vary depending upon the cell and tissue type. Polar conjugates of the lignans
produced in enterocytes are transported out of the cell to the portal blood supply through the
activity of the multidrug resistance-like protein, MRP3 [242]. Such polar metabolites bypass the liver
and are ultimately excreted by the kidney, but lignans escaping intestinal first-pass metabolism
undergo additional hepatic phase II metabolism and to a limited extent, cytochrome P450 enzyme
mediated metabolism [46,55,213]. Relevant lignan–drug interactions have not been identified, but
lignans may reversibly inhibit several cytochrome P450 enzymes at high concentrations [213,401].
Additionally, SECO and ENL can activate the nuclear receptor pregnane X receptor (PXR), which
may modulate the induction of phase I and II enzymes and, in turn, alter systemic and tissue
concentrations of other substrates of these enzymes [213,402].
Given the propensity for hepatic phase II metabolism, SECO, ED, and ENL undergo
enterohepatic recirculation [403] (Figure 4). Reabsorption of nonconjugated lignans result in
fluctuations in plasma concentrations, as evidenced by secondary peaks in the oral plasma
concentration-time profile [213], and prolongation of their mean residence times in the body
[199,213]. Approximately 20–50% of glucuronide and sulphate conjugates are excreted into the bile
[213,404], and of this amount 80% is deconjugated by β-glucuronidase of intestinal microflora in the
intestinal lumen [213,405]. β-glucuronidase activity has been detected in various bacterial genera
such as Bacteroides, Bifidobacterium, Eubacterium, and Ruminococcus, belonging to the prominent
human intestinal microbiota [406], and specifically genes encoding β-glucuronidase have been
described in Escherichia coli, Lactobacillus gasseri, Clostridium perfringens, Staphylococcus aureus, and
Thermotoga maritina [406]. Due to the high β-glucuronidase activity, such glucuronides are more
likely to be hydrolyzed back to their aglycone forms for reabsorption or their fecal excretion. As a
result, physiologically relevant lignan concentrations in the range of 10 to 1000 μM are likely
achievable in the colon lumen [407,408]. Enterohepatic recirculation also results in 10–35% of
conjugated and unconjugated lignan excretion by the fecal route [213,409–412]. However, a
considerable proportion is excreted by the kidney as glucuronide conjugates with permanent
removal from the body [213,413]. Generally, a good correlation exists between plasma
concentrations and urinary excretion of various lignan metabolites [213,411,414], but the relative
ratio and extent of urinary excretion can vary depending upon population characteristics, e.g.,
postmenopausal women with or without breast cancer [199,213,415–417]. Additionally, small
portions of enterolignans have been reported to be found in certain animal based food such as milk
[39,418–420] and therefore can be considered as another route of excretion.
Blood and tissue levels of the lignans and their conjugative metabolites show a high degree of
interindividual variability due to variation in their absorption and disposition (distribution and
elimination) characteristics as well as differences in diet, microflora, gender, and age [213,421].
Extensive first-pass metabolism results in concentrations of unconjugated SECO, ED, and ENL in the
lower nanomolar range [323,421–424], with concentration of the conjugated forms 250 times or more
higher than the unconjugated lignans [55,213,323,379,393]. Low plasma concentrations are also due
to their wide distribution throughout the body with detection in tissues such as the intestine, liver,
lung, kidney, breast, heart, and brain with higher levels in liver and kidney
[213,232,392,404,425,426]. In humans, plasma protein binding of flaxseed lignans is unknown but in
rat plasma the unbound fraction for SECO, ED, and ENL was 33%, 7%, and 2%, respectively [234].
Plasma protein binding of the conjugated metabolites of SECO, ED, and ENL is likely very low given
the polar nature of these metabolites. Additionally, an erythrocyte partitioning study indicated no
accumulation of ENL in erythrocytes [234]. Despite low blood levels of ENL and the polar nature of

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the conjugates, ENL and its conjugates seem to concentrate in body fluids such as breast milk,
intestinal fluid, prostatic and breast cyst fluid [381,420,424,427,428]. Accumulation is also most
prominent with chronic administration of flaxseed lignans compared to acute administrations
[213,233,429].
Although lignan accumulation into solid tumors is unknown, tumors commonly possess poorly
formed, highly permeable vasculature that results in the accumulation of various macromolecules
(e.g., plasma protein albumin) within the tumor microenvironment [430–432]. Several studies have
suggested tumors as sites of albumin catabolism, and the presence of putative albumin-binding
proteins on tumor cell surfaces [433]. Therefore, taking this into consideration, it is possible that
albumin bound lignans may accumulate in the tumor environment independently and/or released
upon albumin catabolism, e.g., similar to albumin conjugated drugs used for increasing intratumoral
accumulation of drugs for antitumor effects [434]. Similarly, the conjugated metabolites of the
aglycone and mammalian lignans may gain easy access to the tumor microenvironment due to the
leakiness of the tumor vasculature.
The biological interactions of SECO, ED, ENL, and their phase II conjugates within the
molecular and cellular environment remains unclear. Phase II enzyme reactions are typically
considered as deactivation pathways. Hence, extensive first-pass metabolism, which results in high
levels of circulating phase II conjugates traditionally considered to be inactive metabolites [400],
raises questions on how lignans exhibit health benefit following oral consumption. Recent evidence,
though, may suggest the conjugative metabolites of the mammalian lignans exert pharmacological
activity in certain cellular contexts [435]. Furthermore, evidence exists of the ability of polyphenolic
glucuronide conjugates to undergo deconjugation reactions in specific tissues such as the
inflammatory sites of the tumor microenvironment due to extracellular availability of
β-glucuronidase, which expresses optimal enzyme activity at low pH [142,436–440], as well as
evidence of the ability of the vascular endothelium to deconjugate certain glucuronide conjugates
[436,441]. This suggests that high circulating glucuronide conjugates might act as aglycone carriers
with release of the aglycones at target sites upon deconjugation [436,442].

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Figure 4. Flaxseed lignan absorption, first-pass metabolism, and enterohepatic recycling. The
flaxseed lignan, secoisolariciresinol diglucoside (SDG), is biotransformed by bacteria in the
gastrointestinal tract upon oral intake. Due to their lipophilicity, the aglycones and mammalian
lignans may cross biological membranes via passive diffusion. With permeation into the enterocyte a
portion of the aglycone and mammalian lignans undergo first-pass metabolism by phase II enzymes
(e.g., UDP-glucuronosyltransferases (UGT), sulfotransferases (ST)). The polar, water-soluble
glucuronide and sulfate conjugates require transport across the basolateral membrane of the
intestinal epithelium by active transporters to gain access to the portal blood supply. Unmetabolized
aglycone and mammalian lignans enter the hepatocyte by passive diffusion and undergo phase II
metabolism by UGTs and STs. The conjugated metabolites are actively transported into the bile and
can be reintroduced into the gastrointestinal tract. Here, they can be deconjugated and undergo
reabsorption, a process called enterohepatic recirculation (EHR). The various lignans and their
corresponding metabolites may elicit biological responses upon entering the systemic circulation by
interacting with various enzymes, transporters, and other cell signaling macromolecules. Elimination
of the conjugated metabolites can occur through either fecal or renal excretion. Adopted from
reference [403].
8.3. Lignans as Therapeutic Agents for Cancer
Cancer involves complex mechanistic changes in multiple interdependent and redundant
cellular signaling networks that ensure initiation, survival, and promotion of carcinogenesis. This
complexity results in many failures of single target therapies in clinical drug development despite
the enormous investments made to advance such products to the market [443]. The effects of
conventional chemotherapy, though, might be enhanced by compounds that have ability to inhibit
and antagonize multiple targets within the complex array of cell signaling processes [444]. This is
supported by an increased focus on multitarget agents in drug discovery and development, which
has gained much needed attention in recent years [445]. The historical presence in the human diet of
phytochemicals such as lignans, though, may have an advantage over synthetic compounds due to
their coevolutionary exposure. Given their possible multiple therapeutic effects, we have witnessed
an increased investment into the investigation of their mechanisms of actions in order to more fully
understand their antitumor effects [443].
Flaxseed lignans have a long history of purported health benefits [25,28,38,304] (Figure 5). For
cancer, flaxseed is consumed for both chemopreventive and treatment purposes [15,318,446,447].
Studies with preclinical models of cancer clearly have demonstrated therapeutic benefits of lignan
rich diets with evidence of reductions in early tumorigenesis [448,449], as well as inhibition of tumor
growth, angiogenesis, and progression of the disease [450,451]. Such evidence supports the putative
relevance of lignans in carcinogenesis [1,15,55,174]. However, clear evidence of benefit in human
clinical populations is confounded by the numerous epidemiological and population-based studies
that report an unclear accounting of the daily lignan dose and, hence, uncertain lignan exposure
levels [325,452–457]. The availability of standardized lignan-enriched products now provides
opportunity to clearly understand daily dose exposures and ensure adequate therapeutic levels for
clinical benefit. Such lignan-enriched products have demonstrated good safety and tolerability in
vulnerable populations, such as frail elderly adults [323,458], as well as in other preclinical and
human clinical trials [32,55,459–461], except during pregnancy [462–465] and lactation [420], or with
products that produce high ED levels [466,467]. These lignan-enriched products can guarantee
pharmacological lignan doses, which will allow us to address past inconclusive epidemiological
studies of the effect of lignans (and other polyphenols) on human cancer risk and therapy
[55,323,326,333,417,452,468–481].

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Figure 5. Protective health benefits of lignans. Lignans are polyphenolic phytochemicals that have
varying biological activities under several contexts. Lignan containing diets or supplements can
support general health as well as target many diseases.
8.4. Linking Benefits of Flaxseed with Cancer Associated Chronic Diseases
Cancer shares a number of risk factors common to other chronic disease states [482]. This is
emphasized by statistics that indicate cancer, diabetes, and cardiovascular disease (CVD) were
responsible for 71% of deaths globally in 2015 [482]. Chronic diseases, including type 2 diabetes
[483–485], and CVD risk factors, such as cholesterol level [486–491], heart rate [492,493], blood
pressure [494–498], uric acid [499–503], and chronic kidney disease [504–509], as well as pulmonary
disease [510], are associated consistently with the risk of cancer [482]. The abundant evidence
confirming the health promoting beneficial effects of flaxseed in chronic disease can be grouped
according to health benefits in (1) the cardiovascular system (e.g., platelet aggregation,
atherosclerosis, hyperlipidemia, and dyslipidemia [7,9,10,17,19,24,26,27,35,485,511–518]); (2) insulin
resistance, glycemic control, and obesity [9,25,27,35,519–526]; (3) inflammation
[5,7,8,19,25,27,514,527,528] and oxidative stress [5,12,21,23,35,407,513,514,529–534]; (4) hepatic
[28,535] and renal systems (e.g., lupus nephritis) [15,28,435,536]; (5) the immune and nervous system
[7,15,28,537–543]; (6) the reproductive system [8,11,14,25,28,33,311,352,514,542,544–546]; and (7) the
gut microbiome [547–549]. Detailed discussions on the relationship between chronic disease and
flaxseed can be found in our previous review [25] and others [5,6,8,15,16,21,34,304,513]. This
collective epidemiological, observational, and preclinical evidence support the idea of flaxseed
lignans as qualified candidates for risk reduction and treatment of chronic disease (Table S2)
warranting additional clinical trials with known pharmacological doses to provide the evidence base
to support their use clinically [21,513].
8.5. Purposing Lignans into Established Models of Cancer Characteristics

Pharmaceuticals 2019, 12, 68 20 of 67
Several models have been elaborated to describe the wide range of properties and
characteristics of cancer [62,550]. These models aid in understanding both the complexity of cancer
pathogenesis and the various processes contributing to cancer, as well as to focus research efforts on
identifying possible chemopreventive agents or therapeutics [59]. These models provide an
organizing framework to explain responses to a targeted therapy, where cancers may modify their
dependence on a particular hallmark, while enhancing the activity of another [551]. The “hallmarks
of cancer” model established in 2000 by Hanahan and Weinberg identified the six cancer hallmarks
of evading growth suppressors, resisting cell death, activating invasion and metastasis, enabling
replicative immortality, sustained proliferative signaling, and inducing angiogenesis [63]. This
model was subsequently updated in 2011 with further inclusion of two enabling characteristics
(genomic instability and tumor-promoting inflammation that support cancer cells to acquire the
hallmarks) and two emerging hallmarks (deregulation of cellular energetics and avoidance of
immune destruction) [59,62]. In a model (signaling pathways and cellular processes) articulated by
Vogelstein et al. in 2013 [550], tumors contain two to eight “driver gene” mutations that drive cancer
growth, while the remaining “passenger” mutations do not add to the selective growth advantage
[59]. Genes either contain intragenic mutations (Mut-driver genes) or epigenetic alterations
(Epi-driver genes), both of which are responsible for carcinogenesis as well as a selective growth
advantage. According to this model, twelve major signaling pathways drive cancer growth and
include (a) cell survival: PI3K (phosphatidylinositide 3-kinase), MAPK (mitogen-activated protein
kinase), RAS (rat sarcoma), STAT (signal transducers and activators of transcription), cell
cycle/apoptosis, and TGFβ (transforming growth factor β); (b) cell fate: NOTCH, HH (Hedgehog),
APC (Adenomatous polyposis coli), chromatin modification, and transcriptional regulation; and (c)
genome maintenance: DNA damage control related pathways [59]. Finally, K.I. Block’s model
(pathways of progression and contributing metabolic factors) of nutraceutical-based targeting of
cancer lists nine “pathways of progression” (proliferation, apoptosis, treatment resistance, immune
evasion, angiogenesis, metastasis, cell-to-cell communication, differentiation, and immortality) and
six “metabolic terrain factors” (oxidation, inflammation, glycemia, blood coagulation, immunity,
and stress chemistry) that influence the quality of life of all cancer patients [59,552]. Together, these
models clearly demonstrate the interrelationships of different signaling network pathways and the
enormous number of targets that require interrogation for cancer prevention and therapeutic
management.
Many phytochemicals are known to modulate multiple targets within these complex cancer
processes [444,553–556]. In particular, flaxseed lignans may concurrently target various complex
interdependent pathways involved in cancer progression and survival raising the possibility that
lignans could be incorporated into a design of a broad-spectrum combination chemotherapy [557]
(Figure 6). Drug discovery programs today have moved away from the single-target approach and
currently consider systems biology approaches to improve pharmacological network understanding
[59]. The complexities in tumor heterogeneity and in the interconnections amongst the various
growth factors, cytokines, chemokines, transcription factors, and the proteome makes systems
biology approaches exceedingly more relevant [558,559]. It also makes the broad-spectrum
multitargeted approach to cancer highly significant [59]. In recognition of this changing paradigm to
cancer discovery, we compiled the known lignan targets alongside their potential identification
within the different cancer characteristics models listed above (Table 2).

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Figure 6. The cellular and molecular targets of lignans. Flaxseed lignans have the ability to target
multiple pathways in cancer given the evidence from both in vitro and in vivo evaluations (Table 2).
Cancer metastases can be inhibited by the modulation of the cytoskeleton and cell motility
processors. Modulation of cell growth and differentiation as well as cell cycle arrest interfere with
tumor proliferation and survival. Starving tumors by targeting angiogenesis as well as triggering
apoptosis leads to inhibition of progression and survival. Interfering with different cell signaling
pathways linking AKT and ERK modulates cell metabolism and disfavors progression and survival.
8.6. The Multitarget Effects of Lignans in Cancer (Nutridynamics)
In the following section, the ability of the lignans to influence the cancer phenotype is broadly
organized according to the cancer hallmarks [62]. Lignan modulation of a specific target is
highlighted under specific areas linked to hallmarks, although any one target might have
overlapping function in the different hallmarks. Examples are provided, but due to the complexity
and interconnections among molecular signaling networks, flaxseed lignans are able to impact an
array of targets leading to the modulation of various signaling cascades in the different stages of the
malignant disease to disfavor progression. The reader is referred to the review by Teponno et al.
[560] for detailed information on other lignans and neolignans.
8.6.1. Antioxidant and Anti-Inflammatory Properties
Lignans are well recognized for their antioxidant and anti-inflammatory activities, key
properties that contribute to their multitarget effects [55,561]. As polyphenolic compounds, lignans
can act as direct antioxidants (e.g., direct scavenging of hydroxyl radical) [562] or through indirect
mechanisms such as modulation of the expression of antioxidant enzymes [563,564]. A number of in
vitro studies have shown the lignans to be effective direct antioxidants [565]. For example, the direct
antioxidant activity of SECO, ED, and EL exceeded vitamin E, a typical comparator, by
approximately 4.5 to 5 times, with SDG showing similar activity to vitamin E [38,530]. ED and EL are
reported to be effective inhibitors of lipid peroxidation in vitro [407], and in a model of lipid
autoxidation, SECO showed much better antioxidant activity than SDG [566]. There is no significant
difference between SECO/SDG and BHT—a food preservative known to cause liver toxicity—to

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prevent/delay the autoxidation process [567]. SDG and SECO are effective antioxidants (attributed to
the 3-methoxy-4-hydroxyl substituents) against 1,1-diphenyl-2-picrylhydrazyl (DPPH))-initiated
peroxyl radical plasmid DNA damage and phosphatidylcholine liposome lipid peroxidation [532].
In an aqueous environment, benzylic hydrogen abstraction and potential resonance stabilization of
phenoxyl radicals are likely to aid in the antioxidant activity of the mammalian lignans [532].
Further details on the antioxidant properties of flaxseed lignans can be found in several other
reviews [38,568,569]. Despite these direct antioxidant effects in vitro, it is debatable whether lignans
attain adequate systemic concentrations with dietary consumption to mediate similar effects in vivo
as lignans largely exist as conjugated metabolites.
The indirect antioxidant activity of lignans is mediated through upregulation of a number of
antioxidant enzymes and phase II detoxifying enzymes. Upregulation of these enzymes is associated
with the nuclear factor erythroid 2 (Nrf2)-linked pathway—a key transcriptional regulator of many
antioxidative and anti-inflammatory pathways [570]. Nuclear factor-κB (NF-κB) is a transcription
factor that is of importance in inflammation and plays a role in development, cell growth, cell
survival, and proliferation [571]. Certain NF-κB-regulated genes play a pivotal role in controlling
reactive oxygen species (ROS), but ROS also has various inhibitory/stimulatory effects in NF-κB
mediated signaling [571]. Transcriptional regulation by Nrf2 is clearly associated with lignan
induction of heme oxygenase-1 (HO-1) expression [562] with subsequent modulation of NF-κB
mediated inflammatory and oxidative pathways [572,573]. Lignans also increase the abundance of
antioxidant genes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase
(GPX) [564,574], and induce the expression of glutathione S-transferases (e.g., GSTM1) and
NAD(P)H dehydrogenase [quinone] 1 (NQO1) [575,576]. SOD2, SOD1, NQO1, CAT, GST, and GPX
are all regulated by NF-κB [571]. SDG in physiological solutions provide DNA radioprotection by
scavenging active chlorine species and reducing chlorinated nucleobases [577], suggesting SDG as a
promising candidate for radioprotection of normal tissue during cancer radiation therapy [578]. The
molecular pathways connected to these various antioxidant activities contribute to the control of
multiple cancer hallmarks such as “resisting cell death”, “genome instability and mutation”,
“deregulating cellular energetics”, and others depending on the context.
The anti-inflammatory properties of lignans are well-documented and are suggested to benefit
chronic inflammatory diseases such as cancer [55,576,579–581]. Lignans can modulate inflammation
through several mechanisms including modulation of immune cell activation through interference
with NF-κB pathway signaling [408]; reductions in proinflammatory cytokines, such as IL-1ß, IL-6,
TNFα, HMGB1, and TGFß1, and cytokine receptors, TNFαR1 and TGFßR1[582]; and
downregulation of cyclooxygenase enzyme activity and levels [583]. Flaxseed also downregulates
microRNA (miRNA) miR-150, which is integrated into immune response-mediated networks [584].
Lignan influence on the inflammatory process clearly impacts the “tumor promoting inflammation”
hallmark of cancer.
8.6.2. Anticarcinogenic and Antimutagenic Properties
Carcinogenesis occurs in several stages and mutagenesis supports the progression of the
malignant disease. As effective antioxidants against DNA damage and lipid peroxidation [532],
lignans are suggested to have chemopreventive properties in cancer, and SDG is emerging as a
potential anticarcinogenic agent [344,582]. Preclinical in vivo studies that have demonstrated
decreased incidence of tumor formation in tumor induction models and reductions in the
procarcinogenic microenvironment following flaxseed lignan supplementation, offer support for
lignans as chemopreventive agents [583,585–588]. Activation of p53 can induce cell cycle arrest as
well as apoptosis in response to DNA damage [589]. The transcriptional activation of target genes of
p53 is critical in cell fate determination after genotoxic stress [589,590]. Oxygen radical-based
alterations at specific nucleotides can lead to mutations that occur when altered bases are copied by
DNA polymerases (replicate the genome) [591]. ROS has been attributed to the pathogenesis of liver,
lung, and prostate cancers [591]. The use of antioxidant therapy (preventive), such as with the
lignans [576,581,582,592], has been suggested to slow tumorigenesis to prevent clinical presentation

Pharmaceuticals 2019, 12, 68 23 of 67
of cancers [591]. In cancer cell model systems, lignans can modulate the percentage of cells in the
different stages of the cell cycle [593], downregulate viral oncogenes E6 and E7, upregulate tumor
suppressor p53, and fail to exhibit genotoxicity in cancer cells [588]. However, depending upon the
cancer type, p53 status, and lignan concentration, flaxseed lignans may have different effects on
cancer prevention and treatment.
Interestingly, the various signaling pathways involved in anticarcinogenic and antimutagenic
effects of lignans could be connected to lignan ability to favorably modulate lipid and glucose
homeostasis [9,519,537,594–596]. High cholesterol, fat, and glucose levels are known to increase the
risk of cancer [491,597–602]. Several studies have shown altered cholesterol metabolism and
accumulation within mitochondria of malignant cells seems to favor continuous cell growth,
survival, and progression [603–606]. The lignans variably influence targets within cellular energy
and lipid homeostasis pathways, including the ability to reduce expression and activity of CPT 1
(carnitine palmitoyltransferase 1), as well as modulate pAMPK (5’ adenosine
monophosphate-activated protein kinase), PPARα (peroxisome proliferator-activated receptor
alpha), FASN (fatty acid synthase), expression and activity of SREBP1c (sterol regulatory
element-binding proteins) and adipogenesis-related genes, such as leptin, adiponectin, glucose
transporter 4 (GLUT-4), and PPARγ (peroxisome proliferator-activated receptor gamma) [213,607–
610]. Additionally, a recent study reports upregulation of INSIG-1 (insulin-induced gene 1) and
alteration in intracellular cholesterol trafficking in Caco2 colorectal adenocarcinoma cells [435].
Collectively, lignan effects on cellular energy metabolism and lipid homeostasis favorably modulate
the cancer hallmarks of “deregulated cellular energetics” and “resisting cell death”.
8.6.3. Anti-proliferative properties
Lignans are known to reduce chemically-induced mammary and colon tumorigenesis
[31,345,582]. In addition to their well-known antioxidative and anti-inflammatory effects, lignans are
purported phytoestrogens with ability to modulate estrogen receptors and other hormonal functions
[611]. Their putative role as phytoestrogens prompted extensive investigation into
hormone-dependent cancers, since hormones play a vital role in their etiology influencing rate of
cancer cell division, differentiation, survival, and metastasis [55,612,613]. Interestingly, lignans
demonstrate weak binding properties to estrogen receptor α (ERα) and ERβ suggesting a limited
potential for estrogenic and antiestrogenic activity [614]. Yet, studies suggest lignans’ ability to
inhibit hormone-dependent cancer cell proliferation, cancer growth, and progression [55,467,615–
617]. This may result from such mechanisms as lignan-mediated reduction in the expression of
hormonal and growth factor receptor expression or binding affinity (e.g., ER, progesterone receptor
(PR), EGFR (epidermal growth factor receptor), and IGF-1R (insulin-like growth factor 1 receptor))
[341,453,618], regulation of plasma sex hormone binding globulin (SHBG) levels [55,619] or binding
affinity with endogenous hormones [55,620], competition with estradiol for the type II estradiol
binding sites (EBS) [621–623], inhibition of aromatase and 17β-hydroxysteroid dehydrogenase and
thereby reducing sex hormone synthesis [55,624–626], modulation of secreted matrix
metalloproteinase (MMP) activities [531], and/or alteration in the expression and activity of cell cycle
regulators and signal transduction networks regulating cell proliferation, survival, and migration
[335,593,613,618,627–629]. The multitarget effects of lignans on hormonal signaling pathways
identify their key role in modulating the important cancer hallmark of “sustaining proliferative
signaling”.
8.6.4. Dysregulated cellular metabolism
A common feature of cancer cell metabolism is the ability to obtain nutrients from the
nutrient-poor tumor environment to maintain viability and make new biomass [630]. Given the
linkage between cell proliferation and cell metabolism [631], the core fluxes such as aerobic
glycolysis, de novo lipid biosynthesis, and glutamine-dependent anaplerosis, have been suggested to
form a stereotyped platform in order to carry out proliferation [631]. Additionally, regulation of
these cellular fluxes are predominantly linked to phosphatidylinositol 3-kinase (PI3K)/protein

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kinase B (Akt)/mechanistic target of rapamycin (mTOR), hypoxia-inducible factor 1 (HIF-1), and
Myc (myelocytomatosis oncogene) mediated signal transduction and gene expression [631].
Interestingly, upregulation of HO-1 is suspected to act through PI3K/Akt and Nrf-2 signaling
pathways [632,633]. PI3K/Akt signaling (master regulator of glucose uptake) stimulates mRNA
expression of GLUT1 glucose transporter and the translocation of its protein to the cell surface [630].
Akt amplifies the activity of the glycolytic enzymes hexokinase, the first enzyme of the glycolytic
pathway (phosphorylates glucose molecules, and prevents their efflux out of the cell), and
phosphofructokinase (catalyzes the main irreversible step) [630]. Akt alone also is capable of
stimulating glycolysis to restore cell size, viability, mitochondrial potential, and ATP levels [630].
Additionally, constitutively active Akt can prevent reductions in ATP levels, which is usually
triggered by the loss of cellular attachment [630]. Lignans have been reported to reduce Akt
signaling [618,629,634], as well as HO-1 and Nrf-2 signaling [562]. Therefore, not only the hallmark
of “sustaining proliferative signaling” but other hallmarks such as “deregulating cellular
energetics”, “enabling replicative immortality”, and “evading growth suppressors” can be targeted
by lignans.
8.6.5. Antiangiogenic Properties
Angiogenesis is a complicated process that depends on the type of tumor [635,636]. Solid
tumors with high vascularization (e.g., ovarian cancer, non-small cell lung cancer, renal cell
carcinoma, hepatocellular carcinoma, and colorectal cancer) have been the main focus of the
development for antiangiogenic drugs [636,637]. The series of events in this complex process include
an initial activation of endothelial cells (EC), which often results in the release of proteases that
causes the degradation of the basement membranes in the surrounding area of existing vessels, and
the migration of ECs to the growing lesion, followed by extensive cell proliferation forming tubes for
new blood vessels [635]. However, unlike normal tissue angiogenesis, tumor blood vessel network is
disorganized and leaky [638]. Lignans may have a role as effective agents in targeting the hallmark
“inducing angiogenesis”. Lignans were shown to inhibit estradiol-induced tumor growth and
angiogenesis in vivo [451]. The antiangiogenesis activity may relate to ability of lignans to reduce
extracellular cancer stroma-derived vascular endothelial growth factor (VEGF) and increase in
placenta growth factor (PIGF), a VEGF family member [331,451]. The platelet-derived growth factor
(PDGF), its receptor, PDGFR, fibroblast growth factor (FGF) and its receptor, FGFR pathways, can
aid in compensatory escape mechanisms facilitating tumor growth from anti-VEGF/VEGFR therapy
drugs, which has been the gold standard pharmaceutical target [636]. However, current
antiangiogenic strategy is investigating novel and emerging agents that target multiple pathways for
treatment [636]. Interestingly, lignans also modulate PDGF signaling pathways making it a
multitargeted agent to suppress tumor growth [628].
8.6.6. Anti-invasive and Antimigratory Properties
The lignans can modulate a number of key targets to reduce cancer cell propensity for invasion
and migration [13,32,55,330,331,467,618,639]. Lignans were shown to reduce metastasis in an
experimental model of melanoma [108,582]. They demonstrate ability to inhibit matrix
metalloproteinases (MMPs), the enzymes responsible for degradation of the extracellular matrix
(ECM) [55,640–642], modulate the phosphorylation of FAK (focal adhesion kinase), Src
(proto-oncogene nonreceptor tyrosine protein kinase Src), and Paxillin, with subsequent modulation
of their key targets (e.g., uPA (urokinase-type plasminogen activator), PAI-1 (plasminogen activator
inhibitor-1), TIMP-1 (TIMP metallopeptidase inhibitor 1) and TIMP-2, RhoA (Ras homolog gene
family, member A), Rac1 (Ras-related C3 botulinum toxin substrate 1), Cdc42 (cell division control
protein 42 homolog), and ITGA2 (Integrin subunit alpha 2)) [627,628], and inhibit organization of the
actin cytoskeleton to influence cell motility and clonogenicity [627,628,642,643]. Given that cancer
relapse and metastasis continue to challenge effective chemotherapy [644,645], such properties
suggest a potential for the use of lignans to target this cancer hallmark.

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8.6.7. Induction of Apoptosis and Cell Death
Apoptosis plays a pivotal role in the pathogenesis of cancer where limited apoptosis results in
survival of malignant cells. The complex mechanism of apoptosis is linked to many cell signaling
pathways where deregulation can cause malignant transformation, metastasis, and resistance to
anticancer drugs [646]. Consequently, lignan-mediated enhancement of apoptosis can occur through
many mechanisms that are generally categorized into disruption of mitochondrial membrane
potential (mitochondrial mediated cell death) [55,629,634], and activation of the intrinsic or extrinsic
apoptotic pathways through mechanisms such as TRAIL (tumor necrosis factor (TNF)-related
apoptosis-inducing ligand)-induced BID (BH3 interacting-domain death agonist) cleavage [629],
reduction in antiapoptosis proteins, Bcl-2 (B-cell lymphoma 2) and survivin [22,588], caspase
dependent cell death [634], and death receptor-sensitization through decreased expression of death
receptor DR4 expression and TRAIL-DISC (death-inducing signaling complex) proteins, c-FLIPL/S
(cellular FLICE-inhibitory protein: short form; FLICE: (Fas-associated death domain-like interleukin
1β-converting enzyme) and caspase-8, and pGSK-3β (glycogen synthase kinase 3 beta) [629,634].
Flaxseed along with radiation therapy have reported to significantly decrease the p53-responsive
miRNA, miR-34a, which is responsible for regulating cellular senescence and apoptosis related
factors [584]. Dietary flaxseed lignan complex, mainly consisting of SDG, induced radiosensitizing
effects in a model of metastatic lung cancer. SDG is protective against radiation pneumonopathy,
decreasing lung injury and eventual fibrosis, while improving survival indicating its ability to
selectively target malignant cells but spare normal cells [576,581]. Although specific targeting of
apoptosis can be associated with safety issues [646], as one of multiple hallmarks influenced by
lignans, the ability to enhance cell death is an important attribute of the role of lignans in the
therapeutic management of cancer.
Table 2. Cellular targets modulated by flaxseed lignan and lignan metabolites in cancer 1.
Experimental
System and Lignan*
Targets: Molecules
(Protein/Gene) Block’s Model
Hanahan and
Weinberg’s
Model
Vogelstein et
al., Model
MDA-MB231 (BC)
ENL*
↓Ki67, ↓PCNA, ↓FoxM1,
↓Cyclin E1, ↓Cyclin A2,
↓Cyclin B1 ↓Cyclin B2
[627]
Proliferation,
Immortality,
Treatment
resistance
Sustaining
proliferative
signaling,
Evading growth
suppressors
Cell survival
↓pFAK, ↓pPaxillin [627]
↓ERK-1/2, ↓NF-κB,
↓MAPK-p38, ↓CD44
[647]
Proliferation,
Metastasis,
Cell-to-cell
communication
and Immortality
Activating
invasion &
metastasis,
Sustaining
proliferative
signaling
Cell survival,
Cell fate
↓uPA, ↓MMP-2, ↓MMP-9,
↑PAI-1, ↑TIMP-1, ↑TIMP-2
[643]
↓N-cadherin, ↓vimentin,
↑E-cadherin, ↑occludin,
↓Snail [647]
Differentiation,
Metastasis
Activating
invasion &
metastasis
Cell fate
XM (MDA-MB231)
SDG*
↑LIV-1, ↑↓ ZIP2, ZnT-1
[648] Proliferation
Sustaining
proliferative
signaling
Cell survival
MO (basal-like BC)
SDG*
↓Proinflammatory
markers (F4/80, CRP),
↓p-p65 [649]
Inflammation Tumor promoting
inflammation Cell survival
MO (MCF7) (BC)
ENL* ↓VEGF, ↑PIGF [331]
Proliferation,
Treatment
resistance,
Angiogenesis
Inducing
angiogenesis Cell survival
OVX MO (MCF-7)
SDG*
↓ERα, ↓ERβ, ↓EGFR,
↓pS2, ↓IGF-1R, ↓BCL2
Apoptosis,
Proliferation,
Sustaining
proliferative Cell survival

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[341] Glycemia signaling,
Resisting cell
death
↓pMAPK [341] Proliferation
Sustaining
proliferative
signaling
Cell survival
MCF7, MDA-MB231
ENL*
↓MMP2, ↓MMP9
↓MMP14, ± MMP11 [642]
Differentiation,
Metastasis
Activating
invasion &
metastasis
Cell fate
A549, H60 (Lung
cancer)
ENL*
↓pFAK, ↓pSrc, ↓pPaxillin
[628]
Proliferation,
Metastasis,
Cell-to-cell
communication
Activating
invasion &
metastasis,
Sustaining
proliferative
signaling
Cell survival,
Cell fate
↓RhoA, ↓Rac1, ↓Cdc42
[628]
Metastasis,
Cell-to-cell
communication
Activating
invasion &
metastasis
Cell fate
↑↓FAK, PDGF signaling
(AKT1, CCND3). ↓RhoA,
Rac1, Cdc42, ↑ITGA2
[628]
Metastasis,
Differentiation,
Proliferation,
Cell-to-cell
communication
Activating
invasion &
metastasis,
Sustaining
proliferative
signaling
Cell survival,
Cell fate
MG-63
(Osteosarcoma)
ENL and ED*
Biphasic (↑↓) – osteonectin,
collagen I [650]
Proliferation,
Differentiation,
Cell-to-cell
communication
Activating
invasion &
metastasis
Cell fate
↑ALP, ↑osteopontin,
↑osteocalcin [650]
Proliferation,
Differentiation,
Metastasis,
Cell-to-cell
communication
Activating
invasion &
metastasis
Cell fate
WPMY-1 (PS)
ENL*
↑GPER, ↑p-ERK, ↑P53,
↑P21, ↓Cyclin D1[651]
Proliferation,
Immortality
Sustaining
proliferative
signaling
Cell survival
Rat prostate
SDG* ↑GPER [651] Proliferation,
Immortality
Sustaining
proliferative
signaling
Cell survival
WPE1-NA22,
WPE1-NB14,
WPE1-NB11,
WPE1-NB26 and
LNCaP (PC)
ENL*
↑↓DNA licensing genes
(GMNN, CDT1, MCM2,
MCM7) [593]
Proliferation,
Immortality,
Treatment
resistance
Sustaining
proliferative
signaling
Cell survival,
Genome
maintenance
↓miR-106b cluster
(miR-106b, miR-93,
miR-25),↑PTEN [593]
Proliferation,
Angiogenesis
Sustaining
proliferative
signaling
Cell survival
LNCaP
ENL*
↓BRCA1, ↓CDK2,
↓CDKN3, ↓E2F1, ↓KLK3,
↓KLK4, ↓PCNA, ↓PIAS1,
↓PRKCD, ↓PRKCH,
↓RASSF1, ↓TPM1,
↓SLC43A1 [335]
Proliferation,
Immortality,
Differentiation,
Treatment
resistance
Sustaining
proliferative
signaling,
Replicative
immortality,
Evading growth
suppressors
Cell survival,
Genome
maintenance
Cell fate
↓BIRC5, ↓BRCA1,
↓BRCA2, ↓CCNB1,
↓CCNB2, ↓CCNF,
↓CCNG1, ↓CCNH,
↓CDC2, ↓CDC20, ↓CDK2,
↓CDK4, ↓CDK5R1,
↓CDKN1B, ↓CDKN3,
↓CHEK1, CKS1B, ↓CKS2,
↓DDX11, ↓GADD45A,
↓KNTC1, ↓KPNA2,
↓MAD2L1, ↓MCM2,
Proliferation,
Immortality,
Treatment
resistance, Stress
chemistry
Sustaining
proliferative
signaling,
Evading growth
suppressors
Genome
maintenance,
Cell survival

Pharmaceuticals 2019, 12, 68 27 of 67
↓MCM3, ↓MCM4,
↓MCM5, ↓MKI67,
↓MRE11A, ↓PCNA,
↓RBL1, ↓RPA3, ↓SKP2,
↑CCND2 [335]
LNCaP
MAT*
↓pAKT [629]
Treatment
resistance,
Apoptosis,
Proliferation,
Glycemia
Sustaining
proliferative
signaling
Cell survival
↓DR4 [629]
Apoptosis,
Proliferation,
Immortality
Resisting cell
death
Cell survival,
Cell fate
↓TRAIL-DISC proteins
(c-FLIPL/S, caspase-8)
[629]
Apoptosis,
Proliferation
Sustaining
proliferative
signaling,
Resisting cell
death
Cell survival,
Cell fate
↑TRAIL-induced BID
cleavage [629]
Apoptosis,
Proliferation
Resisting cell
death Cell survival
LNCaP
ENL*
↑Cytochrome c release,
↑cleaved caspase-3,
↑PARP [634]
Apoptosis,
Proliferation,
Glycemia,
Immortality,
Oxidation
Deregulated
cellular
energetics, and
Genome
instability and
mutation
Cell survival
↓pAKT, ↓pGSK-3β,
↓pMDM2, ↑P53 [634]
Apoptosis,
Immortality,
Proliferation
Sustaining
proliferative
signaling,
Evading growth
suppressors,
Enabling
replicative
immortality
Cell survival
↑Caspase cell death [634] Apoptosis Resisting cell
death Cell survival
PC3 (PC)
ENL*
↓pIGF-R(IGF-1), ↓pAKT,
↓p-p70S6K1, ↓pGSK3β,
↓pCyclinD1, ↓pERK ½
[618]
Proliferation,
Glycemia,
Immortality
Sustaining
proliferative
signaling,
Activating
invasion &
metastasis
Cell survival,
Cell fate
↓IGF-1 signaling [618] Proliferation,
Glycemia
Sustaining
proliferative
signaling
Cell survival,
Cell fate
↓FASN [213]
Proliferation,
Treatment
resistance
Sustaining
proliferative
signaling,
Deregulated
cellular energetics
Cell survival
HUVEC
(endothelial)
ENL*
↓VEGFR-2 [331] Proliferation,
Angiogenesis
Inducing
angiogenesis Cell survival
Adipocytes
ENL*
↓ROS - oxidative damage,
↓DNMTs, ↓HDACs,
↓MBD2 [334]
Proliferation,
Oxidation,
Inflammation,
Stress chemistry,
Immortality
Cell fate,
Genome
maintenance
Colonocytes-YAMC
ENL and ED* ↓Cyclin D1, ↓Bcl-2 [586]
Proliferation,
Immortality,
Apoptosis
Sustaining
proliferative
signaling,
Resisting cell
death
Cell survival
Colo201 (COC) ↓Bcl-2, ↓PCNA, ↑cleaved Apoptosis, Resisting cell Cell survival

Pharmaceuticals 2019, 12, 68 28 of 67
ENL* caspase-3 [22] Proliferation death
Apc-Min (intestinal)
Diet (flaxseed)* ↓COX-1, COX-2 [652]
Proliferation,
Immortality,
Inflammation
Sustaining
proliferative
signaling, Tumor
promoting
inflammation
Cell survival
Hens
Flaxseed
supplement*
↓COX-2 [583]
Proliferation,
Immortality,
Inflammation
Tumor promoting
inflammation Cell survival
↓Prostaglandin E2, ↓ERα,
↓CYP3A4, ↓CYP1B1,
↓16-OHE1, ↑CYP1A1,
↑2-OHE1 [583]
Proliferation,
Inflammation,
Treatment
resistance, Stress
chemistry
Tumor promoting
inflammation Cell survival
Hela (CC)
ENL*
↓Viral oncogene E6 [588] Proliferation Evading growth
suppressors Cell survival
↓Survivin [588] Apoptosis,
Proliferation
Resisting cell
death, Sustaining
proliferative
signaling
Cell survival
↑pHistone H2AX [588]
Apoptosis,
Immortality,
Proliferation
Resisting cell
death
Cell survival,
Cell fate
Hela
ED* ↑Caspase 3 [588] Apoptosis Resisting cell
death Cell survival
CaSki (CC)
ENL*
↓Viral oncogene E7 [588] Proliferation Evading growth
suppressors Cell survival
↓Bcl-2 [588]
Apoptosis,
Treatment
resistance
Resisting cell
death Cell survival
Hela and CaSki
ENL*
↑P53 [588] Proliferation,
Apoptosis
Evading growth
suppressors
Cell survival,
Genome
maintenance
↑Bax [588]
Apoptosis,
Treatment
resistance
Resisting cell
death Cell survival
Targets: Cellular
Processors
SKBR3 and
MDA-MB231 (BC)
ENL*
↓Cell viability with
anticancer agents
[55,608]
Proliferation,
Treatment
resistance, Stress
chemistry,
Apoptosis
Resisting cell
death, Sustaining
proliferative
signaling,
Evading growth
suppressors
Cell survival
MDA-MB231
ENL*
↑Cell cycle S phase, ↓cell
viability [627]
Apoptosis,
Immortality,
Proliferation
Sustaining
proliferative
signaling,
Evading growth
suppressors
Cell survival,
Genome
maintenance,
Cell fate
↓Actin cytoskeleton
organization [627,647]
↓Epithelial–mesenchymal
transition [647]
Proliferation,
Metastasis
Sustaining
proliferative
signaling,
Activating
invasion &
metastasis
Cell survival,
Cell fate
↓Migration, invasion
[627,642] Metastasis
Activating
invasion &
metastasis
Cell fate
↓Actin, filopodia,
lamellipodia [642]
Proliferation,
Metastasis
Sustaining
proliferative
signaling,
Activating
invasion &
metastasis
Cell survival,
Cell fate

Pharmaceuticals 2019, 12, 68 29 of 67
Anticancer/metastatic/
proliferative/migratory/clo
nogenic [643]
Metastasis
Activating
invasion &
metastasis
Cell fate
MCF7 and
MDA-MB231
SDG and ASECO*
↓Growth [653] Proliferation
Sustaining
proliferative
signaling
Cell survival
ER+ BC (XM)
ENL and ED* ↓Angiogenesis [451] Angiogenesis Inducing
angiogenesis Cell survival
WPMY-1
ENL*
↓proliferation, arrested cell
cycle (G0/G1) [651] Proliferation
Sustaining
proliferative
signaling
Cell survival
Rat model (PH)
SDG*
↓Prostate enlargement, #
papillary projections,
thickness of cell layers
[651]
Proliferation
Sustaining
proliferative
signaling
Cell survival
WPE1-NA22,
WPE1-NB14,
WPE1-NB11,
WPE1-NB26 and
LNCaP
ENL*
↓Metabolic
activity,↑doubling time
[593]
Proliferation,
Stress chemistry,
Oxidation
Sustaining
proliferative
signaling,
Deregulated
cellular
energetics,
Evading growth
suppressors
Cell survival,
Cell fate
Modulated cell cycle [593] Proliferation,
Immortality
Evading growth
suppressors,
Sustaining
proliferative
signaling
Cell survival,
Genome
maintenance
↑Apoptosis [593] Immortality,
Apoptosis
Sustaining
proliferative
signaling,
Resisting cell
death
Cell survival
LNCaP
ENL*
↑Sub-G0 and S, ↓G0/G1,
↓G2/M cell cycle [335]
Proliferation,
Immortality
Sustaining
proliferative
signaling,
Evading growth
suppressors
Cell survival,
Cell fate
↓Cell density, ↓metabolic
activity, ↓PSA, ↑apoptosis
[335]
Proliferation,
Apoptosis
Sustaining
proliferative
signaling,
Resisting cell
death,
Deregulated
cellular energetics
Cell survival,
Cell fate
↑Apoptosis with
anticancer agents [335] Apoptosis Resisting cell
death Cell survival
↓Mitochondrial membrane
potential [634]
Treatment
resistance, Stress
chemistry,
Glycemia,
Oxidation,
Proliferation,
Apoptosis
Deregulated
cellular energetics Cell survival
LNCaP
MAT*
Death receptor sensitizer
(sensitizes TRAIL-induced
apoptosis) [629]
Proliferation,
Apoptosis
Sustaining
proliferative
signaling,
Evading growth
suppressors,
Resisting cell
death
Cell survival,
Cell fate
↑TRAIL-induced
mitochondrial
Proliferation,
Apoptosis
Resisting cell
death, Cell survival

Pharmaceuticals 2019, 12, 68 30 of 67
depolarization [629] Deregulated
cellular energetics
PC3
ENL*
↓IGF-1 induced
proliferation, ↓cell cycle
arrest (G0/G1) [618]
Proliferation
Sustaining
proliferative
signaling,
Evading growth
suppressors
Cell survival
↓IGF-1 induced migration
[618] Metastasis
Activating
invasion &
metastasis
Cell survival,
Cell fate
A549 and H60
ENL*
↓Migration, invasion
[628] Metastasis
Activating
invasion &
metastasis
Cell fate
↓Density F-actin fibers
[628]
Metastasis,
Proliferation
Activating
invasion &
metastasis,
Sustaining
proliferative
signaling
Cell survival,
Cell fate
YAMC
ENL and ED*
↓Cell growth, ↑ apoptosis
[586]
Proliferation,
Apoptosis
Resisting cell
death, Sustaining
proliferative
signaling,
Evading growth
suppressors
Cell survival
MG-63
ENL and ED*
Biphasic (↓↑)- cell viability,
ALP activity [650] Proliferation
Sustaining
proliferative
signaling
Cell survival,
Cell fate
Mouse model
ENL*
↓Estradiol-induced
endothelial cell infiltration
[331]
Metastasis
Activating
invasion &
metastasis
Cell survival,
Cell fate
Colo201
ENL*
↑Apoptosis (sub-G1
cells),↑cell viability [22]
Proliferation,
Apoptosis
Sustaining
proliferative
signaling,
Evading growth
suppressors,
Resisting cell
death
Cell survival
CC cells
ENL*
↑Cell death, ↓ metabolic
activity in p53+ [588]
Immortality,
Proliferation,
Treatment
resistance,
Glycemia,
Apoptosis
Evading growth
suppressors,
Resisting cell
death,
Deregulated
cellular energetics
Cell survival,
Genome
maintenance
↑Apoptosis (Hela) [588] Apoptosis Resisting cell
death Cell survival
CC cells
ENL and ED* ↓Cell survival [588]
Immortality,
Proliferation,
Apoptosis
Sustaining
proliferative
signaling,
Evading growth
suppressors
Cell survival,
Genome
maintenance
TR C33-A (CC)
ENL and ED*
↓Promoter activity
(Episomal, HPV
oncoproteins) [588]
Proliferation
Sustaining
proliferative
signaling,
Evading growth
suppressors
Cell survival
Hela
ENL*
↑p53 activity [588] Immortality,
Proliferation
Evading growth
suppressors
Cell survival,
Genome
maintenance
No DNA-breaks
(genotoxicity) [588]
Proliferation,
Apoptosis
Resisting cell
death, Evading
growth
suppressors
Cell survival
Hela/CaSki ↑Apoptosis (Caspase 9, Proliferation, Resisting cell Cell survival

Pharmaceuticals 2019, 12, 68 31 of 67
ENL* Caspase 3) [588] Apoptosis death
CaSki
ED* ↑Caspase 3 activity [588] Proliferation,
Apoptosis
Resisting cell
death Cell survival
1 Note: Processors may include anything other than an individual protein/gene target expression
such as cell cycle, invasion, motility, metastases, cell viability, apoptosis, cytoskeletal dynamics, ATP
levels, metabolic rates, oxygen consumption, target activity, etc. Each molecule or processor can be
related to multiple pathways and hallmarks indicated in the models, and therefore what is listed are
some selected examples. The different types of lignans are indicated with an asteric (*); e.g. Lignan*.
Lower case (simple) “p” in certain instances denotes “phosphorylated” protein. Refer to Appendix A
for table abbreviations.
9. Final Remarks
Cancer remains a significant unmet medical need despite the extensive research into possible
pharmaceutical solutions to tackle the various cancer phenotypes. Unfortunately, cancer will
continue to be an important cause of morbidity and mortality in the near future as we witness
increasing urbanization, increasing life expectancy, changing lifestyle, globalization, and changing
environmental factors [4,654–658]. To address this global health dilemma, we may need to adopt a
“broad-spectrum therapeutic approach” into our chemopreventive and therapeutic plans of cancer
mitigation. Such a trend is already being observed as the 2012 U.S. National Health Interview Survey
(NHIS) reported over 30% of adults and 12% of children used atypical approaches to health care [48].
The application of plant-derived bioactives or phytochemicals for disease prevention and treatment
continues to gain attention as a desired approach for preventing or delaying disease [91]. Both
human and preclinical studies suggest synergism of polyphenols such as lignans with existing
therapeutics and, therefore, represent possible candidates for chemoprevention or as combination
treatments with standard therapies such as chemotherapy, radiotherapy, immunotherapy, and gene
therapy [259]. The overall results seem promising, yet the clinical evidence remains inconclusive
[326,477,659–662]. Adoption of dietary polyphenols, like flaxseed lignans, into a “broad-spectrum
therapeutic approach” will require an interdisciplinary approach combining prospective cohort
studies investigating lignan exposure [326,477,481] with mechanistic studies to confirm the health
benefits of flaxseed lignan interventions [4,654–658].
10. Conclusions
Dietary polyphenols represent a diverse array of chemical subgroups with evidence of variable
efficacy in mitigating cancer risk and progression. Despite epidemiological support of possible
benefit, these compounds lack general acceptance as therapeutic modalities in cancer treatment. This
likely relates to an incomplete understanding of their mechanisms of action as well as a general lack
of understanding of their absorption and pharmacokinetic characteristics resulting too often in
exposure levels inadequate to address the disease process. Hence, an important purpose of this
article was to review the scientific evidence of the role of flaxseed lignans in chemoprevention and
on the growth, survival, and progression of malignant cells. This review consolidates years of
unsystematic research with the flaxseed lignans and identifies lignans as having multiple targets and
modes of action within the cancer phenotype. These multitargeted effects are broadly grouped as
modulation of cell signaling and metabolism, cell growth and differentiation, cell motility and
cytoskeletal dynamics, cell cycle, angiogenesis, and apoptosis. Such effects might explain the limited
epidemiological evidence of lignan benefit in cancer, but a systematic approach, which includes
lignan preclinical studies with translational relevance as well as clinical trials utilizing
therapeutically relevant doses, will be needed to clarify their role in cancer. As other
pharmaceuticals (e.g., the statin drugs) undergo repurposing to cancer treatment, a systematic
investigation of polyphenolics such as the lignans might also harness their potential benefits
towards chemoprevention and enhancement of patient longevity and quality of life.

Pharmaceuticals 2019, 12, 68 32 of 67
Supplementary Materials: The following are available online at www.mdpi.com/1424-8247/12/2/68/s1, Figure
S1: Flaxseed Lignan Secoisolariciresinol Oligomer Chemical Structure; Table S1: Various Metabolites of
Enterodiol (ED) and Enterolactone (ENL) Detected in Different Species; Table S2: Clinical Studies on
Flaxseed/Flaxseed Lignans Administration.
Author Contributions: Conceptualization, S.F.d.S.; Methodology and Graphics, S.F.d.S.; Validation, S.F.d.S.
and J.A.; Formal Analysis, S.F.d.S.; Investigation, S.F.d.S.; Writing—Original Draft Preparation, S.F.d.S.;
Writing—Review, Subsequent Drafts, and Editing, S.F.d.S. and J.A; Visualization, S.F.d.S. and J.A; Supervision,
J.A.; Project Administration, J.A.; Funding Acquisition, J.A.
Funding: This review article received no external funding. The scholarships for Shanal Franklyn De Silva were
provided by the University of Saskatchewan College of Pharmacy and Nutrition and College of Graduate and
Postdoctoral Studies (Saskatoon, Saskatchewan).
Acknowledgments: University of Saskatchewan College of Pharmacy and Nutrition Graduate Program’s
Office, Past-Associate Dean Dr. Alfred Remillard and the Dean Dr. Kishor Wasan for their support in numerous
ways including conference travel support for S.F.d.S.
Conflicts of Interest: The authors declare no conflicts of interest. The funders had no role in the design of the
study, in the collection, analyses or interpretation, in the writing of the manuscript, or in the decision to publish
this review.
Abbreviations.
Apc-Min, Mouse tumor model (intestinal and mammary);
AKT1, AKT (RAC-alpha) serine/threonine kinase 1;
ASECO, Anhydro-secoisolariciresinol;
Bax, Bcl-2-associated X protein;
BC, Breast cancer;
Biphasic, ↑lower/↓higher concentration;
CC, Cervical Cancer;
TR, Transfected;
COC, Colon cancer;
ALP, alkaline phosphatase;
miR, micro RNA;
ERK, Extracellular signal–regulated kinases;
subG1, DNA profile representing cells in the G1 stage of the cell cycle;
F-actin, Filamentous actin;
BIRC5, Survivin;
BRCA1, breast cancer type 1 (BCT1) susceptibility protein;
BRCA2, BCT2 susceptibility protein;
CCNB1, Cyclin B1;
CCNB2, Cyclin B2;
CCND2, Cyclin D2;
CCNF, Cyclin F;
CCNG1, Cyclin G1;
CCNH, Cyclin H;
CDC2, Cell division cycle 2;
CDC20, Cell division cycle 20;
CDK2, Cyclin dependent kinase 2;
CDK4, Cyclin dependent kinase 4;
CDK5R1, CDK5 regulatory subunit 1;
CDKN1B, Cyclin dependent kinase inhibitor 1B/p27/Kip1;
CDKN3, Cyclin dependent inhibitor 3/Cip2;
CDT1, DNA replication factor Cdt1;
CHEK1, CHK1 checkpoint homolog;
CKS1B, CDC28 protein kinase regulatory subunit 1B;

Pharmaceuticals 2019, 12, 68 33 of 67
CKS2, CDC28 protein kinase regulatory subunit 2;
COX1/2, Cyclooxygenase 1 & 2;
CRP, C-reactive protein;
CYP, Cytochrome P450;
DDX11, DEAD/H box polypeptide 11;
DNMT, DNA methyl transferases E2F1, Retinoblastoma protein transcription factor;
F4/80 (EMR1), EGF-like module-containing mucin-like hormone receptor-like 1;
GADD45A, Growth arrest and DNA-damage-inducible, alpha;
GMNN, Geminin; GPER, G protein-coupled estrogen receptor 1;
HDACs, Histone deacetylases;
HPV, Human papillomavirus;
ITGA2, Integrin subunit alpha 2;
KLK3, Prostate-specific antigen (PSA)/kallikrein-3;
KLK4, Kallikrein 4; KNTC1, Kinetochore associated 1;
KPNA2, Karyopherin alpha 2;
LIV-1, Zinc transporter SLC39A6;
MAD2L1, MAD2 mitotic arrest deficient-like 1;
MBD2, Methyl-CpG-Binding domain protein;
MCM2, Mini-chromosome maintenance 2/mitotin; MCM2/7, Mini-chromosome maintenance
complex component 2/7;
MCM3, Mini-chromosome maintenance 3;
MCM4, Mini-chromosome maintenance 4;
MCM5, Mini-chromosome maintenance 5;
MCM2/7, Mini-chromosome maintenance complex component 2/7;
MKI67, Antigen identified by mAb Ki-67;
MBD2, Methyl-CpG-Binding domain protein;
MO, Mouse orthotopic;
MRE11A, Meiotic recombination 11 homolog A;
16/2-OHE1, 16/2-hydroxyestrone;
OVX, Ovariectomized rat;
p21 (p21WAF1/Cip1), Cyclin-dependent kinase inhibitor 1 (CDK-interacting protein 1);
P53, Tumor protein p53 (aka “guardian of the genome”);
p65, Transcription factor p65 (nuclear factor NF-kappa-B p65 subunit);
p70S6K1, Ribosomal protein S6 kinase beta-1 (S6K1)/p70S6 kinase 1;
PARP, poly-ADP ribose polymerase;
PC, Prostate cancer; PCNA, Proliferating cell nuclear antigen;
PDGF, Platelet-derived growth factor;
PH, Prostatic hyperplasia;
PIAS1, E3 SUMO-protein ligase PIAS1;
PRKCD, Protein kinase C delta type;
PRKCH, Protein kinase C eta type;
Prostaglandin E2, Dinoprostone;
PS, Prostate Stromal;
pS2 (TFF1), Trefoil factor family 1;
RASSF1, Ras association domain-containing protein 1;
RBL1, Retinoblastoma-like 1/p107;
RPA3, Replication protein A3;
SKP2, S-phase kinase-associated protein;
SLC43A1, Large neutral amino acid transporter small subunit 3;
TPM1, Tropomyosin alpha-1;
VEGFR, Vascular endothelial growth factor receptor; XM, Xenograft model;
YAMC, Young adult mouse colon;

Pharmaceuticals 2019, 12, 68 34 of 67
ZIP2, Zinc transporter SLC39A2;
ZnT-1, Zinc transporter protein 1.
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