The enzymes, volume 37

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CONTRIBUTORS
Ruby John Anto
Cancer Research Program, Division of Cancer Research, Rajiv Gandhi Centre
for Biotechnology, Thiruvananthapuram, Kerala, India
Jayesh Antony
Cancer Research Program, Division of Cancer Research, Rajiv Gandhi Centre
for Biotechnology, Thiruvananthapuram, Kerala, India
Suresh Awale
Frontier Research Core for Life Sciences, University of Toyama, Toyama, Japan
Dominique Bernard-Gallon
Department of Oncogenetics, Centre Jean Perrin—CBRV, and EA 4677 “ERTICA,”
University of Auvergne, Clermont-Ferrand, France
Yves-Jean Bignon
Department of Oncogenetics, Centre Jean Perrin—CBRV, and EA 4677 “ERTICA,”
University of Auvergne, Clermont-Ferrand, France
Elena De Gianni
Interdepartmental Center for Industrial Research, Alma Mater Studiorum-University
of Bologna, Rimini, Italy
Nasim Faridi
Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares
University, Tehran, Iran
Carmela Fimognari
Department for Life Quality Studies, Alma Mater Studiorum-University of Bologna,
Rimini, Italy
Neel M. Fofaria
Department of Biomedical Sciences and Cancer Biology Center, Texas Tech University
Health Sciences Center, Amarillo, Texas, USA
Laurent Guy

EA 4677 “ERTICA,” University of Auvergne, and Department of Urology, CHU Gabriel
Montpied, Clermont-Ferrand, France
Hamid Heidarzadeh
Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares
University, Tehran, Iran
Gae¨lle Judes
Department of Oncogenetics, Centre Jean Perrin—CBRV, and EA 4677 “ERTICA,”
University of Auvergne, Clermont-Ferrand, France

ix


x

Contributors

Seher Karsli-Ceppioglu
Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Marmara University,
Istanbul, Turkey; Department of Oncogenetics, Centre Jean Perrin—CBRV, and EA 4677
“ERTICA,” University of Auvergne, Clermont-Ferrand, France
Sung-Hoon Kim
Cancer Preventive Material Development Research Center, College of Korean Medicine,
Department of Pathology, Kyung Hee University, Seoul, South Korea
G. Mohan Shankar
Cancer Research Program, Division of Cancer Research, Rajiv Gandhi Centre
for Biotechnology, Thiruvananthapuram, Kerala, India
Shuji Nakano
Graduate School of Health and Nutritional Sciences, Nakamura Gakuen University,
Johnan-ku, Fukuoka, Japan
Ahmad Nasimian

Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares
University, Tehran, Iran
Marjolaine Ngollo
Department of Oncogenetics, Centre Jean Perrin—CBRV, and EA 4677 “ERTICA,”
University of Auvergne, Clermont-Ferrand, France
Mai Thanh Thi Nguyen
Faculty of Chemistry, University of Science, Vietnam National University, Hochiminh City,
Viet Nam
Nhan Trung Nguyen
Faculty of Chemistry, University of Science, Vietnam National University, Hochiminh City,
Viet Nam
Misaki Ono
Graduate School of Health and Nutritional Sciences, Nakamura Gakuen University,
Johnan-ku, Fukuoka, Japan
Fre´de´rique Penault-LLorca
Department of Oncogenetics, Centre Jean Perrin—CBRV, and EA 4677 “ERTICA,”
University of Auvergne, Clermont-Ferrand, France
Alok Ranjan
Department of Biomedical Sciences and Cancer Biology Center, Texas Tech University
Health Sciences Center, Amarillo, Texas, USA
Abbas K. Samadi
Sanus Bioscience, San Diego, California, USA
Sanjay K. Srivastava
Department of Biomedical Sciences and Cancer Biology Center, Texas Tech University
Health Sciences Center, Amarillo, Texas, USA, and Cancer Preventive Material
Development Research Center, College of Korean Medicine, Department of Pathology,
Kyung Hee University, Seoul, South Korea


Contributors

xi

Mikako Takeshima
Graduate School of Health and Nutritional Sciences, Nakamura Gakuen University,
Johnan-ku, Fukuoka, Japan
Fuyuhiko Tamanoi
Department of Microbiology, Immunology and Molecular Genetics, University
of California, Los Angeles, California, USA
S. Zahra Bathaie
Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares
University, Tehran, Iran, and Department of Microbiology, Immunology and Molecular
Genetics, University of California, Los Angeles, California, USA


PREFACE
In recent decades, the important role of phytochemicals in dietary and as a
functional food as well as for therapeutic uses has attracted attention of a large
number of scientists in different fields including molecular and cellular
science, medical science, and food science. In this (Volume 37) and previous
(Volume 36) volumes of “The Enzymes,” we attempted to compile studies
on these topics and to discuss the mechanism of action of the phytochemicals
in both cancer prevention and cancer treatment. Molecular mechanism of
the anticancer effect of isoprenoids, polyphenols, and flavonoids was
described in the previous volume. In the current volume (Volume 37),
we continued and expanded the discussion to include some other families
of compounds including quercetin, withanolides, dihydrochalcones,
isothiocyanates, phytoestrogens, and sulfur-containing compounds. In
Chapter 1, we summarized possible molecular mechanisms of anticancer
compounds, especially phytochemicals and natural products. Detailed discussion on the mechanisms involving specific compounds can be found

in other chapters. We hope that these discussions provide helpful guidelines
for new researches on the mechanism of action of natural products. We are
grateful to the authors for providing excellent and informative chapters in a
timely fashion. We also thank Mary Ann Zimmerman and Helene Kabes of
Elsevier for their guidance and encouragement during the preparation of this
volume.
S. ZAHRA BATHAIE
FUYUHIKO TAMANOI
June 2015

xiii


CHAPTER ONE

How Phytochemicals Prevent
Chemical Carcinogens and/or
Suppress Tumor Growth?
S. Zahra Bathaie*,†,1, Nasim Faridi*, Ahmad Nasimian*,
Hamid Heidarzadeh*, Fuyuhiko Tamanoi†
*Department of Clinical Biochemistry, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
†
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles,
California, USA
1
Corresponding author: e-mail addresses: ;

Contents
1. Introduction
2. Phytochemicals Application in Chemoprevention Strategies

2.1 Blocking Initiation/Reversing Promotion
2.2 Activating Phase II Detoxifying Enzymes
2.3 Prooxidant/Antioxidant Activities
2.4 Protection Against Radiation
2.5 Alteration in Signaling Pathways
2.6 Effect on Cell–Cell Adhesion Machinery
2.7 Induction of Epigenetic Changes
3. Phytochemicals Usage as Chemotherapeutic
3.1 Inhibition of Enzymes
3.2 Direct Binding to Biomacromolecules
3.3 Epigenetic Alteration/Chromatin Modification
3.4 RNA Modulation
3.5 Autophagy and UPR
3.6 Apoptosis Induction
3.7 Cell Cycle Arrest
3.8 Inhibiting Angiogenesis
3.9 Adjuvant/Combinatorial Therapy
4. Summary
References

2
5
7
9
11
12
12
13
13
14

14
17
18
22
24
25
28
30
31
33
33

Abstract
Phytochemicals are a powerful group of chemicals that are derived from natural
resource, especially with plants origin. They have shown to exhibit chemoprevention
and chemotherapeutic effects not only in cell lines and in animal models of cancer

The Enzymes, Volume 37
ISSN 1874-6047
/>
#

2015 Elsevier Inc.
All rights reserved.

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2

S. Zahra Bathaie et al.

but also some of them are in the clinical trial phase I and II. Despite numerous reports of
these phytochemical effects on cancer, an overview of the mechanisms of their action
and their effects on various cellular and molecular functions important in the inhibition
of cancer progression has been lacking. In this review, we attempt to catalogue various
studies to examine the effect of phytochemicals in cancer initiation, promotion, signaling, and epigenetic changes. Because of the numerous studies in these topics, we only
pointed out to some examples in each section.

1. INTRODUCTION
Cancer is a growing health problem around the world; particularly
with the steady increase in life expectancy, rising levels of urbanization
and industrialization, increasing the fast food consumption, and the subsequent changes in environmental conditions, including the lifestyle and production of various pollutions.
On World Cancer Day 2014, a new global cancer report was compiled
by UN Agency, the International Agency for Research on Cancer (IARC),
showing that as a single entity, cancer is the biggest cause of mortality worldwide with an estimated 8.2 million deaths from cancer in 2012. Thus, this
report suggests that cancer is now the world’s biggest killer—with the
number of cases set to explode in coming years. In fact, World Health
Organization (WHO) indicates a 70% increase over next 20 years in worldwide cancer cases. Low- and middle-income countries are most at risk of
cancer overwhelming their health systems and hindering economic growth,
as they have the least resources and infrastructure to cope with the predicted
levels of disease escalation. Restrictions on alcohol and sugar need to be considered, say WHO scientists as there now exists a “real need” to focus on
cancer prevention by tackling smoking, obesity, and drinking. Compiled
by IARC, The World Cancer Report series is recognized as an authoritative
source of global perspective and information on cancer. The first volume
appeared in 2003 and the second in 2008. The third volume in the series
was released in 2014.
The concept of a “magic bullet” was popularized by “Paul Ehrlich”
(March 14, 1854–August 20, 1915) a German physician and scientist,
who worked in the fields of hematology, immunology, and chemotherapy.

He defined a “magic bullet” as an ideal therapeutic agent that would be
created and killed only the organism targeting a disease. He reasoned that
if a compound could be made that selectively targeted a disease-causing
organism, then it could be selectively delivered to that organism; this


Phytochemicals Prevent Chemical Carcinogens

3

compound or “magic bullet” could only kill the target organism. This
concept is now known as “targeted therapy” [1].
Since there appears to be no “magic bullet” to treat a diverse type of cancer, it has been apparent that cancer risks can be reduced by eliminating or at
least minimizing the exposure to known carcinogens [2]. In 1981, Doll and
Peto in a report based on the statistical and epidemiological data have
announced that among all risk factors of cancer—tobacco, alcohol, occupation, and so on—about 35% (10–70%) of human cancer mortality is attributed to diet [3]. Although, it is a wide range variant, but it indicates the
importance of diet as a risk factor of cancer. On the other hand, an inverse
relationship between the risk of specific cancers and consumption of vegetables and fruits have been reported [2]. These indicate the importance of
Phyto products in diet and in the life.
Phytochemicals (“Phyto” is from the Greek word meaning plant) are
nonnutritive components in the plant-based diet that possess substantial
anticarcinogenic and antimutagenic properties [2]. Phytochemicals have different roles in both cancer prevention and treatment. Despite remarkable
progress in understanding the carcinogenic process and devising preventive/therapeutic effects of phytochemicals, the mechanisms of action of most
phytochemicals have not yet been fully understood. Bioavailability, toxicity,
pharmacodynamic, and pharmacokinetics of the plant components(s) should
be investigated. Oral consumption of some phytochemicals results in lower
plasma/serum concentration. The reasons for this include: low intestinal
absorption, degradation by intestinal enzymes, and/or metabolization by
phase I and/or II detoxifying enzymes. For example, crocin intestinal
absorption is low and most of the orally consumed crocin appeared in the

feces of rats [4]. In addition, it is degraded by the intestinal enzymes and after
2 h of oral administration of crocin, crocetin was detected in the serum of
human subject [5]. Thus, oral administration of crocin may have low efficacy
for therapeutic purposes, and it should be better that it is administered via
injection [6].
In addition, adverse (or side) effects of phytochemicals should be considered. For example, there are several hundred published research articles and
many review papers about the beneficial effects of resveratrol in various diseases, in both in vivo and in vitro studies [7–11]. Resveratrol is the most
important stilbene related to cancer, and it is present in the foods like peanuts, pistachios, grapes, red and white wine, blueberries, cranberries, and
even cocoa and dark chocolate. It possesses a natural antiproliferative activity, due to its role as a phytoalexin (plant antibiotic). It also increased the


4

S. Zahra Bathaie et al.

antitumor activity of several other drugs, such as rapamycin in breast cancer
and gemcitabine in pancreatic cancer, both in vitro and in vivo [12]. Resveratrol affects all three stages of carcinogenesis, including: tumor initiation,
promotion, and progression. It was found that it acts as an antioxidant
and antimutagen, and induces phase II drug-metabolizing enzymes (antiinitiation activity). It also mediated anti-inflammatory effects and inhibited
COX1 and hydroperoxidase functions, as well as both COX-2 and MM-92
expression. It is a potent inhibitor of nuclear factor NF-κB activation in
DMBA3-induced breast cancer in female Sprague-Dawley rats and other
tumor types. Treatment of human breast cancer MCF-7 cells with resveratrol, in addition to the suppression of NF-κB activation, inhibited proliferation at S/G2/M phase (antipromotion activity). Extensive in vitro
studies also revealed multiple intracellular targets of resveratrol, which in
addition to inflammation, cell growth, and proliferation affect other targets
like apoptosis, angiogenesis, invasion, and metastasis. Resveratrol induces
human promyelocytic leukemia cell differentiation (antiprogression activity). It inhibited the development of preneoplastic lesions in carcinogentreated mouse mammary glands in culture and inhibited tumorigenesis in
a mouse skin cancer model. Several other known targets of resveratrol are
including: tumor suppressor p53 and Rb4; cell cycle regulators, cyclins,
CDKs, p21WAF1, p27KIP and INK, and the checkpoint kinases ATM/

ATR; transcription factors NF-κB, AP-1, c-Jun, and c-Fos; angiogenic
and metastatic factors, VEGF, and matrix metalloprotease 2/9; and apoptosis
and survival regulators, Bax, Bak, PUMA, Noxa, TRAIL, APAF, surviving,
Akt, Bcl-2, and Bcl-xL. In some conditions, it also exerts the prooxidant
activity and cause oxidative DNA damage that may lead to cell cycle arrest
or apoptosis [13–15]. In contrast to the above-mentioned data, some papers
also reported its adverse effects and show some hints about its application for
chemoprevention or, even, its therapeutic effects in human subjects [16–21].
The renal toxicity of resveratrol in rat has been observed at the dose
of 3000 mg/kg BW5 per day, but the dose of 300 was not toxic [19].
However, it has been reported that low concentration (5 mg/kg BW) of
resveratrol promotes breast cancer in mice and has a role in metastasis. Resveratrol (50 mg/kg BW) induced tumor growth in both MDA-MB-2316
1
2
3
4
5
6

Cyclooxygenase.
Matrix metalloprotease-9.
7,12-dimethylbenz(α)anthracene.
Retinoblastoma.
Body weight.
Mammary carcinoma-derived cell.


Phytochemicals Prevent Chemical Carcinogens

5

(ERαÀ, ERβ+) and MDA-MB-435 (ERÀ and highly aggressive) breast cancer cells. Investigation of the role of resveratrol in breast cancer metastasis
indicated the lung metastasis in mice bearing MDA-MB-231 tumor, while
metastasis of lung, liver, kidney, and bone from mice bearing MDA-MB435 mammary tumors have been observed [18]. Resveratrol also affects
the endocrine function and accelerates development of MNU7-induced
mammary carcinomas of female rat [20]. Thus, resveratrol effect is dependent to both concentration and tumor type. Since impressive numbers of
positive results were published, more attention on its safety should be considered for clinical usage of resveratrol.
In the present chapter, regardless of the phytochemical type, we focus on
molecular mechanisms involved in the prevention or therapeutic activities
of phytochemicals. Figure 1 summarized the most important aspect of
molecular mechanism of phytochemicals action. Because of the considerable
studies on the molecular mechanisms of many phytochemicals functions,
and the extensive reviews presented by the experts in the volumes 36 and
37 of The Enzymes, we only presented here a few examples for each mechanism with the goal to provide a guidance to check for each phytochemical
by researchers in the future.

2. PHYTOCHEMICALS APPLICATION IN
CHEMOPREVENTION STRATEGIES
While there is no “magic bullet” that can completely cure cancer, like
many types of diseases, cancer might be prevented. To achieve this purpose,
all the risk factors should be recognized completely and avoided. Without
complete identification of risk factors, this type of prevention is difficult
to implement. For primary prevention, there is a need for large lifestyle
changes, but this is not easy to implement.
The population-based studies indicated the potential of some macronutrients (like fibers) and micronutrients (for example, vitamins and some trace
elements) in vegetables and fruits to reduce the risk of cancer. While, some
macronutrients like carbohydrates and lipids increase the risk of some diseases including cancer. The most exciting results have been obtained with
antioxidant vitamins and their precursors, as well as the components which

7

N-methyl-N-nitrosourea.


6

S. Zahra Bathaie et al.

Effect on telomerase
Inhibition of
topoisomerase I or II
Activation or inhibition of
specific rate-limiting/key
enzyme in a pathway

Inhibiting phase I
enzymes (Cyt P450)
Induction of phase II
enzymes
Induction/inhibition the
antioxidant defense
system

Induction of mutation
Induction of DNA break
Binding to DNA/DNA
adduct formation

Stabilization of specific
DNA structure, such as

telomeric DNA
Inhibition of DNA
replication or transcription

Inside the cell
Binding to RNA
Inhibition of HDAC

Epigenetic
alterations/chromatin
modification

Activation of histone
acetyltransferase
Activation/ inhibition of
histone methyltransferase
DNA methyltransferase
inhibition/ DNA
demethylase activation

Bind microtubules disrupt
or stabilize microtubules

Phytochemicals
as
chemopreventive
or
chemotherapeutic
agents

Induction of apoptosis
Induction of cell cycle
arrest/mitotic disruption

Binding to a
receptor and
begining/affecting
a signaling pathway
As a vitamin
activates specific
enzyme

Induction of cell
differentiation
Inhibition of cell proliferation
Inhibition of oncogene
expression
Activation/induction of tumor
suppressor gene expression
Inhibiting angiogenesis

Application in
adjuvant/ combinatorial
therapy

Figure 1 Important aspects of the mechanisms of action of various phytochemicals.

are found in dark, leafy green vegetables, and yellow/orange/red fruits and
vegetables. NCI8 has produced a series of guidelines featuring each color of
the “rainbow” of fruits and vegetables.

8

National Cancer Institute of USA.


Phytochemicals Prevent Chemical Carcinogens

7

Different mechanisms involved in prevention with consideration to the
more known phytochemicals that act through these mechanisms will be
reviewed in the following sections.

2.1 Blocking Initiation/Reversing Promotion
The study of experimentally induced carcinogenesis in model animals indicated that tumor development consists of sequential separate steps: initiation,
promotion, and progression. After the initial uptake or exposure to a
carcinogen, the initiation step which is a rapid (may be 1–2 days) and irreversible process is beginning. This step involves a chain of extracellular and intracellular events. At first, the carcinogen should be distributed and transported to
organs and tissues. Then, metabolic activation and detoxification can occur. It
is also possible that the covalent interaction of reactive species with DNA
results in genotoxic damages. In contrast, tumor promotion is relatively slow
(>10 years) and is known as a reversible process. In this step, actively proliferating preneoplastic cells accumulate. The last step, progression, is the neoplastic
transformation and in some types of cancer, its duration is less than a year. It
involves the growth of a tumor with the potential of invasion and metastasis [2].
A preventive strategy may block the initiation through different mechanisms. It may prevent the carcinogen from reaching the target sites, from
undergoing metabolic activation or from interacting with the target cellular
macromolecule (DNA, RNA, and proteins); i.e., preventing the DNA
damage. It may also be accomplished through detoxification of carcinogen
by phase II enzymes, scavenging free radicals by antioxidants, or through
binding to DNA and DNA-adduct formation may prevent the attack of free
radical to DNA. In addition to the mitochondrial source of reactive oxygen

species,9 other important sources, both enzymatic and nonenzymatic, of the
ROS production are shown in Fig. 2.
However, after initiation, a suppressing agent inhibits the malignant cell
transformation in either the promotion or the progression steps. Chemopreventive phytochemicals can block initiation or reverse the promotion stage
of multistep carcinogenesis or suppress proliferation of early preneoplastic
lesions. They can also delay, interrupt, or terminate the progression of precancerous cells into malignant ones [2]. These mechanisms will be discussed
later in this chapter.
I3C10 is a glucosinolate obtained from cruciferous vegetables. The preventive effect of I3C and its mechanism of action have been investigated
9
10

ROS.
Indole-3-carbinol.


8

S. Zahra Bathaie et al.

Fenton's reaction

Nonenzymatic
sources

Haber–weiss reaction

Monoamine
oxidase
(MAO)

NADPH
oxidase/respiratory
burst oxidase

Reactive
oxygen
species
are
generated
from:

NADPH-like
oxidase

Enzymatic
sources

Xanthine
oxidoreductase
(XOR)

ROS generation
by
arachidonic acid

Cytochrome
P450 oxidase

Myeloperoxidase

The heme-containing enzyme present in outer
mitochondrial membrane catalyzes oxidative
deamination of amines, and thus produces H2O2
in matrix and cytosol

The multicomplex enzyme located in plasma membrane
of neutrophils. It contains several components. Upon
stimulation, cytoplasmic subunits activate gp91 and cause
respiratory bursts that activates superoxides, and releases
them into the phagosomes

Present in endothelia, fibroblast, mesangial,
osteoclast, chondrocytes, and smooth muscles
activated by hormones and cytokines
generate superoxides

Present in the form of Xanthine Dehydrogenase (XD).
XOR catalyzes the conversion of hypoxanthine into xanthine,
and then into uric acid.
XD and XOR are transformed.
XD is transformed into XO irreversibly by proteolysis
and reversibly by oxidation of sulfhydryls
and produce large amount of H2O2 and O2

During the metabolism of arachidonic acid, ROS is
generated intracellularly in which cyclooxygenase,
lipooxygenase, cytochrome P450 oxidase enzyme
system are involved

The heme-containing enzyme, present in mitochondria.

Participates in metabolism of cholesterol, steroids,
hormones, catabolism of bile acids, arachidonic acid,
and eicosanoids, hydroxylation of vitamin D3 and retinoic
acid, produces highly reactive hypochloric acids

The heme-containing enzyme,
present in neutrophils and eosinophils
catalyzes the H2O2 with various
substrates to form
highly reactive hypochloric acids

Figure 2 Various sources of reactive oxygen species in the body.

using the mammoplasty-derived 184-B5 cells. Initiation of carcinogenesis
was induced by chemical carcinogen BP11 or with oncogene (HER) induction and the resulted cells were named as 184-B5/BP and 184-B5/HER,
respectively. The results showed that treatment of 184-B5/BP, 184-B5/
HER, and MDA-MB-231 cells with I3C resulted in a decrease in
11

Benzopyrene.


Phytochemicals Prevent Chemical Carcinogens

9

proliferation, a significant increase in the estradiol (E2) metabolite ratio and
in cellular apoptosis, and inhibition of cell growth. It was concluded that
the preventive effect of I3C on human mammary carcinogenesis possible
is through regulation of cell cycle progression, increase the formation of

antiproliferative E2 metabolites and induction of cellular apoptosis [22].

2.2 Activating Phase II Detoxifying Enzymes
Elimination of potential carcinogen from the body has been known
as a highly effective strategy for reducing susceptibility to carcinogens.
These mechanisms include: conjugation with endogenous ligands, chemical
modification of reactive features of molecules that can damage DNA and
other macromolecules, and the generation or increase of cellular antioxidants. This may happen through the conjugating enzymes and phase II
drug-metabolizing (or detoxifying) enzymes/proteins.
The phase II enzyme induction system is an important component of
the cellular stress response in which a diverse array of electrophilic and oxidative agents can be removed from the cell before they are able to damage
biomacromolecules. The 50 -flanking regions of these genes contain a common cis-element, known as the antioxidant-responsive element.12 Basic
leucine zipper,13 and helix-loop-helix14 transcription factors (such as
NRF2, JUN, FOS, FRA, MAF, and AH receptor) bind of these ARE
sequences and regulate expression of some of the stress-response genes
and induce phase II enzymes. The final result of these processes is the detoxification of carcinogens and protection against oxidative stress [23–25].
Antioxidants also exert their protective effects not only by scavenging
ROS15 but also by inducing de novo expression of the aforementioned
genes including phase II enzymes. Many xenobiotics can also activate
stress-response genes in a manner similar to that achieved by antioxidants.
These genes encode proteins/enzymes such as glutathione,16 catalase,17
superoxide dismutase,18 glutathione reductase,19 glutathione peroxidase,20

12
13
14
15
16
17
18

19
20

ARE.
bZIP
HLH.
Reactive oxygen species.
GSH.
CAT.
SOD.
GR.
GPx.


10

S. Zahra Bathaie et al.

gamma-glutamylcysteine synthetase,21 glutathione S-transferase,22 NAD
(P)H:quinone oxidoreductase,23 heme oxygenase-1,24 and UDPglucuronosyltransferase.25 Other enzymes/proteins in this group include:
epoxide hydrolase, dihydrodiol dehydrogenase, leukotriene B4 dehydrogenase, aflatoxin B1 dehydrogenase, and ferritin [23–27].
Several studies indicated the involvement of oxidative DNA damage and
impaired antioxidant defense system in patients with various types of cancer
[28–30]. For example, changes in the oxidant/antioxidant balance and DNA
damage (8-hydroxy-deoxyguanosine26 formation) in gastrointestinal cancer
patients has been reported. In addition, significant increases in glutathione
and decreases in both nitrite and nitrate, SOD, CAT activities, and antioxidant molecules in these patients lead to the suggestion of a mechanism
involved in oxidative stress in gastrointestinal cancer [28]. Another study
indicated the important roles of the antioxidant defense capacity and
DNA repair system against oxidative damage as a known risk factor for pancreatic cancer [29].

The importance of dietary phytochemicals has been shown in various
studies against oxidative stress. A number of phytochemicals have also been
shown to induce expression of phase II enzymes via NRF2 [2]. Among
them, the chemopreventive activity of four common phytochemicals present in cruciferous vegetables, the indoles: I3C, 3,30 -diindolylmethane27;
the isothiocyanates (ITCs): phenethyl isothiocyanate28; and sulforaphane29
has been investigated in HepG2-C8.30 The cytotoxicity of the compounds
and their mechanism of action through the potential activation of Nrf2ARE-mediated transcriptional activation of phase II enzymes has been determined. The results indicated that the indoles like I3C or DIM alone could
induce the expression of Nrf2-related genes. In addition, they can do the
same in combination with the ITCs, SFN, or PEITC, which enhances their
protective role against cancer [31]. The role of various phytochemicals in the
regulation of UGT transcription has also been reviewed [32].
21
22
23
24
25
26
27
28
29
30

γ-GCS.
GST.
NQO.
HO-1.
UGT.
8-Oh-dG.
DIM.
PEITC.

SFN.
Human liver hepatoma cell line.


Phytochemicals Prevent Chemical Carcinogens

11

2.3 Prooxidant/Antioxidant Activities
The protective effect of antioxidants against reactive oxygen/nitrogen
species (ROS/RNS) could be exerted through different ways. The
endogenous antioxidants are classified into two essential groups, small
molecules and enzymes. The antioxidant enzymes were explained in
the last section.
The small molecule family of antioxidants is categorized into watersoluble (ascorbate, uric acid, glutathione, etc.) and lipid-soluble compounds
(tocopherol, ubiquinol, carotenoids, etc.). According to the chemical structure, phytochemicals should belong to each of these classes.
Some phytochemicals have both antioxidant and prooxidant activities
which may differ according to the concentration and/or other conditions.
For example, exposure of cells to low or high concentrations of curcumin
diminishes or enhances the ROS generation, respectively [33].
Another example is ascorbic acid, which has been known for the past
several decades as an antioxidant and anticancer agent. Although it shows
the toxic effect against cancerous cells, normal cells are relatively resistant
to such cytotoxicity. It has been shown that ascorbic acid as a prooxidant
leads to oxidative DNA breakage in lymphocytes and lymphocyte nuclei.
The copper-dependent cellular redox status has been also suggested, which
is an important element in the cytotoxic action of ascorbic acid against cancer cells [34].
Myricetin with both prooxidant and antioxidant activities in different
conditions is the third example. Its antioxidant activity depends on both
the ROS scavenging and iron ion chelation properties. In the presence of

ascorbic acid, myricetin showed antioxidant properties, especially in complex with iron [35]. The dual property is very useful for medical application
of the mentioned phytochemicals.
In a comparative study, the antioxidant and prooxidant activities
of a series of phenolic compounds have been investigated. The results
indicated most of the phenolic compounds have prooxidant activity
at low concentrations. The antioxidant activity usually increases with
an increase in the number of hydroxyl groups and a decrease in
glycosylation [36].
Baicalin, a flavonoid obtained from Sho-saiko-to as a prooxidant showed
the apoptotic effect on Jurkat cells31 [37].
31

Leukemia-derived T-cell line.


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2.4 Protection Against Radiation
In some studies, natural products derived from plant have been studied for
their protective effect against various radiations [38,39]. Recently, a combination of caffeic acid, rosmarinic acid, and trans-cinnamic acid has been used
for their protective effect against γ-radiation in human HaCaT32 cells by
immunocytochemistry. The named compounds protect cells, with various
degrees, against ROS production as a result of irradiation [40].

2.5 Alteration in Signaling Pathways
Many molecular alterations associated with carcinogenesis, in both promotion and progression steps, occur in the cell-signaling pathways, including
the induction of cell cycle arrest and apoptosis or inhibition of signaltransduction pathways that regulate cell proliferation and differentiation.
One of the central components of the intracellular-signaling network that

maintains homeostasis is the family of MAPKs.33 Numerous intracellular
signal-transduction pathways converge with alteration in PKC,34 PI3K,35
and GSK,36 which lead to abnormal COX-2,37 AP-1,38 NF-κB,39 and
c-myc expression. As these factors mediate pleiotropic effects of both external and internal stimuli in the cellular-signaling cascades, they are prime
targets of diverse classes of chemopreventive phytochemicals [2,41,42].
The chemopreventive effect and mechanisms of curcumin have been
well studied. Curcumin has been known as the inhibitor of NF-κB, which
subsequently inhibits the proinflammatory pathways [43]. It also inhibits
AP-1, some enzymes like COX-2 and MMPs, induces cell cycle arrest
(cyclin D1), affects proliferation (EGFR and Akt), survival pathways
(β-catenin and adhesion molecules), and TNF [44]. Although absorption
of curcumin is limited through the intestinal tract, and its low systemic bioavailability decreased its adequate access in certain tissues, but active levels
have been found in the gastrointestinal tract of animal and human [44].

32
33
34
35
36
37
38
39

Keratinocytes.
Mitogen-activated protein kinases.
Protein kinases C.
Phosphoinositide 3-kinase.
Glycogen synthase kinase.
Cyclooxygenase-2.
Activator protein-1.

Nuclear factor-kappaB.


Phytochemicals Prevent Chemical Carcinogens

13

The chemopreventive effect of quercetin and other flavonoids was also
reported against some types of cancer, in both in vitro and in vivo studies
[45–50].

2.6 Effect on Cell–Cell Adhesion Machinery
β-Catenin, a multifunctional protein, was originally identified as a component of cell–cell adhesion machinery. β-Catenin-mediated signaling, which
regulates developmental processes, may act as a potential link between
inflammation and cancer. The protein β-catenin is an essential component
of intercellular junctions and the Wnt growth factor signaling pathway.
In many cancers, mutation of Wnt pathway components leads to activation
of oncogenes by β-catenin–TCF transcription factor complex [51,52].
Disruption of β-catenin-mediated TCF signaling is a promising strategy
for early chemopreventive intervention [53]. The mechanism by which
agents disrupt β-catenin-mediated TCF signaling is not completely known;
however, some mechanisms have been suggested. They include: (1) physical
inhibition of the β-catenin/TCF complex formation, (2) upregulation of the
ubiquitin-mediated proteosomal degradation of β-catenin, (3) accelerated
the nuclear export of β-catenin, and (4) enhanced sequestration of β-catenin
by E-cadherin [53].
Several dietary phytochemicals, especially those with anti-inflammatory
effect, have been shown to target this molecular pathway [2,51]. In a
review article, the effect of some phytochemicals on modulation of
β-catenin-mediated signaling in various cell lines has been tabulated. For

example, EGCG, resveratrol, and curcumin decrease the β-catenin, while
β-lapachone increases the β-catenin cleavage and genistein decreases
H2O2-induced tyrosine phosphorylation of β-catenin [51].

2.7 Induction of Epigenetic Changes
Epigenetics is defined as heritable and reversible changes in somatic cells that
control gene expression and have an important role in the survival, but these
changes are not encoded in the DNA sequence. Epigenetic mechanisms in
mammals include changes in DNA methylation, histone modifications,
and noncoding RNAs. The reversibility of epigenetic changes makes them
attractive and promising avenues for tailoring both cancer preventive and
therapeutic strategies. We will discuss more about these mechanisms
in Section 3; however, since diet and environmental factors directly influence


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epigenetic mechanisms in human, it can be considered as new strategies to
prevent cancer, cardiovascular diseases, and so on.
Various phytochemicals including dietary polyphenols (soy, genistein,
resveratrol, catechin, and curcumin) may exert their chemopreventive
effects in part by modulating various components of the epigenetic machinery in humans [54,55]. In a study, the inhibition of DNMTs40 activity by
catechol-containing polyphenols in human breast cancer MCF-7 cell line
has been shown. However, the methylation pattern or the expression of
RASSF1A, GSTP1, or HIN1 and the global methylation of histone H3
was not affected [54]. The results of these papers suggested that a long-term
exposure to these phytochemicals in the diet might potentially lead cancer
chemoprevention through epigenetic mechanisms [54,55].

3. PHYTOCHEMICALS USAGE AS CHEMOTHERAPEUTIC
Various phytochemicals have been used to treat cancer. Some of them
are in the clinical trial studies. However, their mechanism of function is very
different. Here, we review these mechanisms in different categories such as
enzyme inhibition, biomacromolecule binding, chromatin modification,
RNA modulation, autophagy, apoptosis, cell cycle, and angiogenesis. We
also discuss combination therapy.

3.1 Inhibition of Enzymes
3.1.1 Inhibition of Topoisomerases I or II
Gossypol is a polyphenolic dinaphthalene isolated from the cotton plant. It
has been firstly known as a potent male contraceptive; but later studies, as
early as 1989, showed that gossypol also possesses effective antitumor activity
against several cancer cells, including ulcerated melanoma, Ehrlich’s ascites
carcinoma,41 and mammary adenocarcinoma 75542 [56]. The result of a
study of the mechanism of gossypol action showed a unique mechanism,
i.e., direct interaction with topoisomerase II, resulting in a decreased topoisomerase II-mediated DNA cleavage [56].
The mechanism of gossypol interaction with topoisomerases has further
been studied recently. Two classes of ligand-topoisomerases complexes have
been defined. Class I ligands stabilize topoisomerase-DNA complexes by
40
41
42

DNA methyltransferases.
EAC.
MAC755.

Phytochemicals Prevent Chemical Carcinogens

15

covalent interaction and induce DNA strand breaks, while class II ligands
without DNA strand breaks interfere with the catalytic function of
topoisomerases. The results of the mentioned study indicated that gossypol
is a potential class II inhibitor that blocks DNA-topoisomerase interaction
with no DNA strand breaks [57].
The effect of topical use of camptothecin in treating psoriasis has been
reported. Inhibition of cell proliferation and promotion of cell differentiation by camptothecin was shown in both mouse model and human cultured
keratinocytes. The therapeutic effects of camptothecin on psoriasis were
explained as a topoisomerase inhibitor and by its multiple effects on
DNA [58].
The (À)-epigallocatechin-3-O-gallate43 deferentially inhibited the
topoisomerases I from different sources. It strongly inhibited topoisomerases
I from wheat germ, calf thymus gland, and Vero cells, but showed no or
weak inhibition against topoisomerases I from carcinoma cells such as
A549,44 HeLa, and COLO 201 cells.45 The substitution of gallic acid at
the 3 position of EGCG increased its inhibitory effect on calf thymus topoisomerase I and human placenta topoisomerase II, but the substitution of
a hydroxyl group at the 39 position increased its inhibitory effect on topoisomerase I. These results suggested that the mentioned positions of the
EGCG play important roles in the process of topoisomerases inhibition [59].
GAX46 isolated from Ganoderma amboinense inhibited topoisomerases
I and IIα in HuH-7 cells47 and sensitized these cancer cells to apoptosis [60].
3.1.2 Effect on Telomerase
Telomerase activity was discovered first in Tetrahymena cell-free system (cell
extract) that adds tandem repeats (TTGGGG) to synthetic telomere
primers [61]. Then, it was shown that the enzyme telomere terminal transferase (telomerase) from Tetrahymena is a ribonucleoprotein (RNP) complex
containing RNA and protein components. Both of these components, in
addition to a G-rich DNA sequence with specific structure are essential

for telomerase activity [62]. Further researches showed that telomerase is
a eukaryotic enzyme with reverse transcriptase activity that formed from different components, including: reverse transcriptase motifs, p133; telomerase
43
44
45
46
47

EGCG.
Adenocarcinomic human alveolar basal epithelial cells.
Colorectal adenocarcinoma.
Ganoderic acid X.
Human hepatoma cells.


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S. Zahra Bathaie et al.

proteins p80 and p95; and RNA. Therefore, all four components of
Tetrahymena enzyme are present in a single complex in eukaryotes [63].
It has been shown that epigallocatechin gallate, a major tea catechin,
strongly and directly inhibits telomerase, an enzyme essential for unlocking
the proliferative capacity of U937 monoblastoid leukemia cells and HT29
colon adenocarcinoma cells by maintaining the tips of their chromosomes.
Telomerase inhibition was detected in both cell-free system and in living
cells. In addition, in the presence of nontoxic concentrations of EGCG, life
span of these cells were limited, the telomeres lengths were shortened and
abnormalities in the chromosomes were appeared [64]. Another report indicated that EGCG prevents the carcinogenesis of cervical cancer by induction
of apoptosis and inhibition of telomerase activity. These effects of EGCG are

possibly happening in early cervical lesions [65]. Exposure to EGCG also
reduced cellular proliferation and induced apoptosis in MCF-7 breast cancer
cells. It was shown that the human telomerase reverse transcriptase (hTERT)
mRNA expression was decreased in these cells due to the treatment with
EGCG [66].
The induction of apoptosis due to camptothecin administration was
shown in HL-60 cells. The phenomenon was accompanied by a timedependent decrease in telomerase activity. Determination of the levels of
different components of human telomerase (hTR, human telomerase
RNA), hEST2/hTERT, and TLP1/TP1 (telomerase association protein
1) by RT-PCR, before and after camptothecin treatment showed no difference in the expression of each component. However, the expression of
Bcl-2 was progressively downregulated. These results indicated that the
decreased activity of telomerase in HL-60 cells due to the camptothecin
treatment was closely related to apoptosis induction, with no effect on
the transcription of the genes involved in RNP complex. In addition,
Bcl-2 had no direct effect on the regulation of the expression of telomerase
subunit mRNA [67].
Camptothecin-mediated apoptosis and its antiproliferative effect on psoriasis through inhibition of topoisomerase [58], was also reported in human
keratinocytes HaCaT cells. This effect is accompanied by downregulation of
telomerase activity [68]. Isocamptothecin, another analogue of camptothecin, also showed similar effects on proliferation, apoptosis and telomerase activity of HaCaT cells [69].
Higher concentrations of crocin (pharmacological dose, >3.5 mg/ml)
inhibited growth of MCF-7 [70] and HepG2 [71] cells and induced apoptosis on these cancerous cell lines. The results of the mentioned studies


Phytochemicals Prevent Chemical Carcinogens

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indicated the telomerase (hTERT) inhibitory activity of crocin [70,71].
Furthermore, the in “test tube” experiments indicated direct interaction
of crocin and other saffron components with telomeric DNA structures,

G-quadruplex, and i-motif [72].
3.1.3 Other Enzymes
Curcumin inhibits the migration and invasion of human A549 lung cancer
cells through the inhibition of MMP48-2 and -9 and VEGF49 [73].

3.2 Direct Binding to Biomacromolecules
The results of various researches show direct binding of some phytochemicals to microtubules, DNA, and some other proteins that result in alterations
in cellular processes.
Paclitaxel (taxol) has played a major role in cancer chemotherapy for several decades. In 1967, it was isolated from the bark of Taxus brevifolia
(Northwest Pacific Yew Tree) by Monroe E. Wall and Mansukh C. Wani.
Paclitaxel is a complex diterpene having a taxane ring with a four-membered
oxetane ring and an ester side chain at position C-13. Paclitaxel has a specific
binding site on the microtubule, thus interacts directly with microtubules,
enhances the polymerization of tubulin, and stabilizes microtubules against
depolymerization. This is a unique character of paclitaxel among other chemotherapeutic agents, and its ability to polymerize tubulin in the absence of
GTP50 and microtubule-associated proteins are unusual. It is preferentially
bound covalently to the β-subunit of tubulin and after polymerization produces extensive parallel arrays or stable bundles of microtubules in cells,
in vitro. The mechanism of paclitaxel differs from colchicine in the manner
that colchicine inhibits the microtubule assembly, whereas paclitaxel stabilizes and protects microtubule against disassembly. At a higher dose, paclitaxel suppresses microtubule minus ends detachment from centrosomes.
Paclitaxel stops cells in the G2/M phase, blocks the cell cycle, and thus
the cells are unable to form a normal mitotic apparatus [74,75].
Direct interaction of some phytochemicals with DNA in the in vitro
studies has also been reported. For example, resveratrol [76,77], genistein [77], crocin [78,79], crocetin [79], DNA quercetin, kaempferol, and
delphinidin [80] interaction with nucleic acids has been studied. Most of
48
49
50

Matrix metalloproteinase.
Vascular endothelial growth factor.

Guanosine triphosphate.


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S. Zahra Bathaie et al.

them can protect DNA/RNA from oxidative stress and DNA damage, but
the exact role of this interaction in the in vivo condition needs to be studied
further.
Among these phytochemicals, resveratrol can induce DNA strand break
in the presence of Cu2+ and inhibit DNA polymerases α and δ, which produces some controversy regarding its role as a caretaker compound [76].
Although resveratrol prevents DNA oxidative damage [81], but its carcinogenic effect, especially at low concentration has been reported in mice [18].

3.3 Epigenetic Alteration/Chromatin Modification
Cancer can be initiated by alterations in genes, such as oncogenes and tumor
suppressor genes. Those regulate cell proliferation, survival, and other
homeostatic functions. In cancer cells, genes are either modified by mutations, which alter the function of the proteins they encode, or through epigenetics, which provide a distinct layer of control for genes transcription.
Some examples of epigenetic control include: chromatin remodeling,
RNA-associated gene silencing, and chromosome inactivation.
In the nucleosome, 146–147 bp of DNA in its native form wraps around
the core histones that is formed from a couple of each of histone proteins
(H2A, H2B, H3, and H4). In this structure, DNA wrapped in 1.6–1.7
superhelical turns around the histone octamer, in the manner which is called
“beads on a string” and produces the fiber with a diameter of 10 mm. The
histone H1 (linker histone) locks the DNA coming in and out of the nucleosome and stabilizes the chromatin fiber in the form of 30 nm fiber in which
DNA wrapped two full turns around the core histone (168–200 bp). The
higher order chromatin structure is formed by self-association of chromatin
fibers and attachment to nuclear matrix that form the 300-nm fibers. More
folding of the fiber produces more condensed structures, 700 nm fiber and

then chromosome [82]. Chromatin has regions of transcriptionally active
and inactive that has been named euchromatin and heterochromatin,
respectively. The interconversion of these two regions for DNA accessibility
to transcription factors is determined by epigenetic through the epigenome
components.
Epigenetic changes can occur through alterations in DNA, histones, and
especially proteins involved in chromatin structure. They include: alteration
in DNA methylation, histones modifications (methylation, acetylation,
phosphorylation, poly-ADP-ribosylation, or ubiquitination), chromatinremodeling complexes, histone chaperones, histone variants and their


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19

enzymatic modifications, and dysregulation of DNA-binding proteins and
noncoding RNAs like the microribonucleic acids (miRNAs) [83,84]. Thus,
the term “epigenetics” refers to the heritable, stable, and perpetual, but
reversible alteration in the gene expression without any changes in DNA
sequence [85].
The role of the epigenome and epigenetic regulation of gene expression
has been studied in various diseases [85–88]. Understanding the mechanisms
of epigenetic regulation of gene transcription will lead to the development
of novel therapies for treatment of diseases and identification of strategies
for preventive intervention. In the present subsections, we try to explain
the effect of some phytochemicals on different aspects of epigenetic regulation. The components could be used for chemoprevention and/or
chemotherapy.
The regulatory effect of some phytochemicals on the epigenome of different cancer cells has been reviewed by Malireddy et al. [84]. Some phytochemicals like curcumin affect all three epigenetic mechanisms (histone
modifications, DNA methylation, and miRNAs) [89]. Here, we explain
some phytochemicals that affect multiple pathways, then the effect of each

phytochemical on each epigenetic pathway will be discussed separately.
Curcumin treatment of HT29 cells51 caused a decrease in the protein
expression of DNA methyl transferases (DNMTs) and subtypes of histone
deacetylases (HDACs 4, 5, 6, and 8). The results suggest that the inhibitory
effect of curcumin on anchorage-independent growth of HT29 cells could,
at least in part, involve the epigenetic demethylation and upregulation
of deleted in lung and esophageal cancer 1 (DLEC1). DLEC1 is a tumor
suppressor gene with reduced transcriptional activity and promoter
hypermethylation in various cancers [90].
EGCG also acts through different epigenetic mechanisms in cancer cells.
For example, the anticancer effect of EGCG (tea catechin) on MCF-7
(ER+)52 and MDA-MB-231 (ERÀ)53 breast cancer cells, through epigenetic mechanism have been shown [91,92]. ERÀ breast cancer is clinically
aggressive and has a poor prognosis. It has been shown that EGCG
reactivates ER expression in MDA-MB-231 cells by a mechanism involving
chromatin remodeling of the ERα promoter and ERα reactivation, through
altering histone acetylation and methylation status. Combination therapy of
51
52
53

human colorectal adenocarcinoma.
Estrogen receptor positive.
Estrogen receptor negative.


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S. Zahra Bathaie et al.

these cells with both EGCG and trichostatin A (HDAC inhibitor) decreased

binding of the transcription repressor complex, Rb/p130-E2F4/5HDAC1-SUV39H1-DNMT1, in the regulatory region of the ERα
promoter, which results in the ERα transcriptional activation [91].
3.3.1 Histone Modifications: Acetylation/Deacetylation and
Methylation/Demethylation
Posttranslational modification of histone proteins plays an important role in
the epigenetic regulation of gene transcription. Acetylation of histones causes their release from chromatin and the appearance of the accessible sites on
DNA for the attack of transcription factors. However, histone deacetylation
prepares the protein to bind DNA and reformation of chromatin structure.
These reactions are catalyzed by histone acetylases (HATs) and HDACs.
HDACs are composed of 18 different enzymes in human that 11 members
of which are zinc dependent. These enzymes are not redundant in function.
These enzymes are classified on the basis of homology to yeast HDACs:
Class I include HDACs 1, 2, 3, and 8, and have high homology in their catalytic sites; Class IIA include HDACs 4, 5, 7, and 9; Class IIB, HDACs 6 and
10, which contains two catalytic sites. While all members of HDACs class
I and II are zinc dependent, class III HDACs, sirtuins 1–7, have an absolute
requirement for NAD +, are not zinc dependent and generally not inhibited
by compounds that inhibit zinc-dependent deacetylases. Class IV or
HDAC11 has conserved residues in the catalytic core region shared by both
class I and II enzymes [93,94].
It has been reported that EGCG treatment significantly inhibited HAT
activity in an hTERT gene of human breast cancer cells MCF-7 and MDAMB-231 [92]. This study showed that EGCG and its more active form
pro-EGCG can remodel chromatin structures of the hTERT promoter
by decreasing the level of acetyl-H3, acetyl-H3K9, and acetyl-H4 to the
hTERT promoter [92].
EGCG, in a dose-dependent manner, decreased HDAC activity and
increased levels of acetylated lysines of histone H3 and H4 (H3-Lys 9 and
14 and H4-Lys 5, 12, and 16), but decreased levels of methylated H3-Lys
9 in A43154 (skin cancer) cells. Therefore, EGCG treatment resulted in
the reexpression of silent tumor suppressor genes, p16INK4a and Cip1/
p21 in both mRNA and protein levels [95].

54

human epidermoid carcinoma.