# Preclinical Assays for Identifying Cancer Chemopreventive Phytochemicals

Authors: Takeru Oyama; Yumiko Yasui; Shigeyuki Sugie; Takuji Tanaka
Journal: Scholarly Research Exchange
 (2009)

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## Abstract

Dietary factors influence carcinogenesis in a variety of tissues. The consumption of fruits and vegetables is associated with a decreased risk of several types of epithelial malignancies. In addition, there are interrelationships between diet, environmental factors, and genetics that can affect cancer risk. Potential chemopreventive agents against cancer development can be found among nutritive and/or nonnutritive compounds in inedible and edible plants. To identify potential cancer chemopreventive agents, scientists are evaluating hundreds of phytochemicals for the prevention of cancer. This short review article describes in vitro and in vivo assays reported to identify potential cancer preventive compounds from plants.


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## Body

## 1. Introduction

Cancer
mortality  rates in the developed countries have increased throughout this century. It is
already the leading cause of death in some Western countries [1, 2].
In Japan, the progressive introduction of Western dietary habits, especially
increased fat intake and reduced carbohydrate and dietary fiber intake, has
increased the
incidence of colon cancer and related deaths [3]. Great advances
have been made in the pharmacologic-based treatment of malignant epithelial neoplasms (cancers). In addition, there is a
marked increase in the
understanding of cell and molecular mechanisms underlying carcinogenic processes. However, therapy for
advanced neoplastic disease remains limited. This may be due to the fact that advanced
neoplasms contain a large number of genetic and molecular alterations that contribute to the maintenance of
their neoplastic progression.The
chemopreventive approach against cancer development is highly attractive.
Although highly attractive from a theoretical point of view, practical
limitations may exist with respect to developing novel and effective
chemopreventive agents. Most importantly, practical clinical endpoints are not
definite. The efficacy of
cancer chemoprevention can
be determined by comparing the incidence of nondeveloped disease in a treated group
to that of a control group. Such a clinical trial is labor intensive, very
costly, and time-consuming. In
addition,
practically such
trials cannot be conducted for
the rapidly increasing number of chemopreventive agents being identified. These
considerations suggest the use of certain intermediate biomarkers as indicators
of clinical efficacy. Validated intermediate biomarkers thus may allow
relatively small chemopreventive trials to be conducted within a short-term
period. However, there are no universal approaches to determine intermediate
biomarkers and their method of validation has still not been proven.Some
herbal and botanical products are likely to possess cancer preventive
activities [4–7]. Many cancer patients use
complementary and alternative medicines, including phytochemicals in addition
to, or following the failure of standard cancer therapy [8]. The term
phytochemical applies to any plant-based substance, but in the field of
nutrition and cancer this
term is usually applied to nutritive and nonnutritive chemicals that occur
naturally in fruits and vegetables. A diet rich in fruits and vegetables has
long been suggested to correlate with reduced risk of certain epithelial
malignancies, including
cancers in the lung, colon, prostate, oral cavity, and breast [4–7, 9–12]. Also, the cancer prevention potential of Mediterranean
diets based mainly on olive tree products is known [13]. The major component of the leaves and unprocessed olive
drupes of Olea europaea is oleuropein and the majority of polyphenols found in
olive oil or table olives are derived from its hydrolysis. Oleuropein is a
novel, naturally occurring antioxidant compound, which may possibly be used to
prevent cancer [14–16] and cardiotoxicity
induced by doxorubicin [17]. Searching
for medicinal benefits from edible or
inedible plants is not a new idea since numerous modern medicines have plant origins.
Given that the ingestion of some plant foods results in reduced risk for
cancer, researchers are delving into the identification of phytochemicals with
cancer preventive ability in studies in vitro (cell culture), in vivo (model animals) and those in
humans [18]. Phytochemicals can be roughly classified into
four groups based on their mechanisms of chemopreventive action, as shown in Table 1.
Preclinical studies focus on the identification of possible cancer preventive
agents, short-term pharmacology, and assessment of toxicity. Agents that are
within tolerable safety limits in humans then moved clinical trials to test
their efficacy.Table 1
Proposed mechanisms of phytochemicals for cancer prevention.
Food sourcesChemicalsModification of carcinogen metabolismAntioxidant and/or anti-inflammatory propertiesModification of cancer cell biologyInduction of differentiationAntiangiogenesisApoptosis inductionRosemaryCarnosol++++Rosmarinic acid+++++Ursolic acid+++++CarrotsCarotenoids++++++TumericCurcumin++++++GarlicDiallyl sulfide++++++GingkoGingkolides+++CrucifersIsothiocyanates++++++Sulforaphane++++++CitrusLimonene++++++MintMenthol+++++CherriesPerillyl alcohol++++++OnionQuercetin++++++Grape seedResveratrol++++++Milk thistleSilymarin++++++SoybeanIsoflavones++++++TeaCatechins++++++OliveOleuropein++++++This review briefly summarizes
the in vitro and in vivo assays
used
to discover possible new chemopreventive agents possessing novel mechanisms of
action. Surely there are many more assay systems
currently used in different laboratories to find candidate cancer
chemopreventive agents and determine their efficacy. Because of limited space, only
limited assays that might be useful for carcinogenesis and chemoprevention studies
are herein introduced.


## 2. Anti-Inflammatory, Antioxidative, Antiangiogenic, and/or
Apoptosis-Inducing Compounds in the  Prevention of  Cancer Development

One
of the most exciting areas of cancer chemoprevention is the effect of
anti-inflammatory compounds against CRC. Almost a dozen case-control and cohort
studies have concluded that the daily or alternate day consumption of aspirin
over extended periods results in halving one's risk for colorectal cancer (CRC) [19]. Several
prospective studies are underway to specifically assess whether the daily
consumption of aspirin affects the recurrence rates of colorectal adenoma in
high-risk subjects for CRC. These studies strongly suggest that eicosanoids,
especially prostaglandins (PGs),
are involved in colorectal oncogenesis [20]. Two genes
encode the two major forms of cyclooxygenase (COX) that is responsible for
the metabolic conversion of dietary arachidonate to PGs, thromboxanes, and leukotrienes.
The constitutive form, COX-1, is present in different types of cells. COX-2, the isoform
inducible by growth factors and tumor promoters, is rarely found in the normal
intestinal epithelial cells but is overexpressed in colorectal neoplasms [21–23]. Since COX-1 is a housekeeping
gene, drugs for COX-1 inhibition cause side effects, such as ulceration.
The kidney function, platelet
aggregation, and the ulceration
in stomach are strongly affected by the overuse or overdose of aspirin and other nonsteroidal
anti-inflammatory drugs (NSAIDs)
[24]. These limit
the wide-scale application of NSAIDs in the general public for the
chemoprevention of CRC. Although NSAIDs are excellent chemopreventive agents in
animal models for CRC, dietary aspirin does not inhibit azoxymethane (AOM)-induced colon carcinogenesis in rats that received
a nonhigh fat diet. Because
of side effects of NSAIDs that affect both COX-1 and COX-2 expressions, specific
COX-2 inhibitors (-coxibs) were introduced to obtain cancer chemopreventive ability without
side effects [25, 26].
They are indeed effective for CRC development in animal studies [27]. However,
certain COX-2 inhibitors have
been reported to increase the risk of ischemic heart diseases
[28].Many
phytochemicals and/or botanicals are routinely tested for anti-inflammatory
properties in the hope of finding new sources of medicines for the treatment of
chronic inflammation and for the cancer chemoprevention. The plant world is
apparently rich in sources of COX and/or inducible nitric oxide (iNOS) inhibitors that are involved in
inflammation-related carcinogenesis
[29]. Several of
these have been found to inhibit cancer development in certain animal models.
Curcumin [30] is able to
inhibit the COX activity [31] in the skin [32] and suppresses
skin cancer development in mice [33], tongue [34] and CRC in rats
[35]. Tea [36, 37],
given to human volunteers in a clinical trial, could reduce PGE2 activity in the rectal mucosa [38]. Resveratrol [39] is a potent
inhibitor of COX [40] and is an
inhibitor in the mouse skin and rat mammary carcinogenesis models [41]. Silymarin [42] a COX and iNOS
inhibitor [43] was also found
to inhibit skin [44, 45], colon [46], tongue [47], and prostatic [45, 48] cancer development. However, the cancer preventive
ability of carnosol and ginger substances, both COX inhibitors [49, 50],
has not fully been investigated [51, 52].
Numerous phytochemicals thus remain to be tested in
animal models for the most common cancers such as colon, mammary, prostate, and
lung. Nobiletin [53–56], zerumbone [57, 58],
and garcinol [59–61] are potent anti-inflammatory
compounds present in plants
that
inhibit carcinogenesis in different tissues. A recent study demonstrated the chemopreventive ability of
zerumbone, which has been shown to possess anti-inflammatory effects in
mouse lung and colon carcinogenesis [62]. These phytochemicals [63], for example, curcumin [31, 64], resveratrol [65, 66], silymarin [67], from plants have multifunctional
effects, including COX-2 inhibition and anti-inflammatory function, on a
variety of events that involved carcinogenesis [68–70].Most phytochemicals with chemopreventive
ability [71] have direct antioxidant activity but many
can also induce oxidative stress within cells when applied at high doses [11, 72]. For example, a simple phenolic acid protocatechuic
acid is a potential cancer chemopreventive agent in a variety of tissues [73], but it enhances tumor promotion and oxidative stress in female ICR mouse skin
via enhancing a promoter-induced inflammatory responses and promotion by
affecting tyrosinase-dependent oxidative metabolism of protocatechuic acid [74]. Therefore, care
should be paid for applying strong antioxidants to clinical use as a
chemopreventive agent. However, recently anti-inflammatory, antioxidative
chemopreventive phytochemicals targeting signal transduction mediated NF-2
related factor-2 (Nrf2), nuclear factor-kappaB (NF-κB), and activator
protein (AP)-1 that are redox-sensitive factors have been
highlighted [72]. We recently
have demonstrated that such a compound and melatonin effectively suppress
inflammation-associated colon tumorigenesis [62, 75].Angiogenesis
the formation of new blood vessels from existing vasculature has been
associated with neoplasms. Angiogenesis is critical to the transition of
premalignant lesions in a hyperproliferative state to the malignant phenotype,
which leads to tumor growth and metastasis. The intensity of angiogenesis as assessed by
counting of microvessels in neoplastic tissue acts as a prognostic factor for
many solid tumors, including CRC [76, 77].
Similarly, expression of angiogenic growth factors is associated with prognosis of a variety of cancers [76, 77].
Angiogenesis enables tumors to grow larger than 1-2 mm in diameter, invade
surrounding tissue, and metastasise. Angiogenesis is already targeted by
chemopreventive agents at various stages of drug development or in clinical
practice [76, 77].
The biomarker measured in chemoprevention must have the potential for
modification by therapeutic interventional agents. In this regard, angiogenesis
is a particularly attractive biomarker. There are in vivo and in vitro assays using human endothelial
cell neoplasms [78], umbilical vein endothelial cells [79] and for screening potential
anti-angiogenic effects of candidate chemicals. Potentially
nontoxic anti-angiogenic dietary compounds include green tea polyphenols, genistein, curcumin,
resveratrol, linoleic acid, hesperidin, naringenin, and allyl disulfide [80].The
defect in apoptosis mechanism is recognized as an important cause of
carcinogenesis [5, 6].
A dysregulation of proliferation alone is not sufficient for cancer development
as suppression of apoptotic signaling is also required. Cancer cells acquire
resistance to apoptosis by overexpression
of anti-apoptotic proteins
and/or by the downregulation
or mutation of pro-apoptotic
proteins. Various studies indicate that dietary constituents, particularly
phytochemicals, can modulate the complex multistage process of carcinogenesis by several mechanism(s), including apoptosis-inducing
effects [81].
They include (−)-epigallocatechin gallate,
curcumin, genistein, indole-3-carbinol, resveratrol, isothiocyanates, luteolin,
lycopene, caffeic acid, apigenin, sylymarin, gingerol, and capsaicin [81].


## 3. Mechanistic Screening Assays for Detecting Potential
Chemopreventive Compounds

Potential
chemopreventive agents are systematically screened in a battery of short-term
assays which determine the inhibition or induction of biochemical and molecular
processes involved in carcinogenic processes. As summarized in Table2, these mechanistic-based assays are
roughly divided into three major categories: (i) antimutagenesis assays
which evaluate carcinogenesis blocking activities, (ii) antiproliferative and antiprogression screening
assays, and (iii) assays assessing antioxidant and anti-inflammatory mechanisms.Table 2
Various assays for chemopreventive mechanisms.
CategoriesAssaysCulture
cells or enzymesMeasurements
(effects)AntimutagenesisB(a)P-DNA adduct formationBronchial cells (human)DNA
damage (inhibition)NAD(P)H:quinone reductaseLiver
cells (human)Detoxification
(induction)GSHS-transferaseLiver
cells (human)Detoxification
(induction)GSH
synthesis & GSSG reductionLiver
cells (rat)Detoxification
(induction)AntiproliferationTPA-induced
ODCTracheal
epithelial cells (rat)Proliferative
activity (inhibition)Normal
epithelial cell proliferationPrimary
keratinocytes (human)Proliferative
activity (inhibition)Poly(ADP-ribose)polymerasePrimary
fibroblasts (human)DNA
damage (inhibition)Calmodulin
regulated phosphodieaateraseLeukemia
cells (HL60)Signal
transduction regulation (inhibition)TPA-induced
tyrosine kinaseLeukemia
cells (HL60)Signal
transduction regulation (inhibition)EGFRA431
(human) and 3T3 (mouse) cellsSignal
transduction regulation (inhibition)ras farnesylationBrain
farnesyl transferase (rat)Signal
transduction regulation (inhibition)HMG-CoA
reductaseLiver
HMG-CoA reductase (rat)Signal
transduction regulation (inhibition)Steroid
aromatasePMSG-stimulated
ovarian aromatase (rat)Estrogenic
activity (inhibition)Estrogen
receptorBreast
cancer cells (MCF-7)Estrogenic
activity (inhibition)5α-reductaseProstate
5α-reductase
(rat)Androgenic
activity (inhibition)Cell
differentiationLeukemia
cells (HL60)Differentiation
(induction)DNA
fragmentationLeukemia
cells (HL60) or histiocytic lymphoma cells (U937 cells)Apoptosis
(induction)AA metabolismMacrophages/keratinocytes
(human)Anti-inflammatory
activity
(AA
metabolism inhibition)TPA-induced
active oxygenLeukemia
cells (HL60)Active
oxygen (inhibition)Antioxidant/Anti-inflammationCOX-2Placental
COX-2 (sheep)Anti-inflammatory
activity
(COX-2
inhibition)5-LOXRBL-1
cells (rat)Anti-inflammatory
activity
(5-LOX
inihibition)AA,
arachidonic acid; B(a)P, benzo[a]pyrene; COX, cyclooxygenase; EGFR,
epidermal growth factor receptor; GSH, glutathione; GSSG, oxidized glutathione;
HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; LOX, lipoxygenase; PMSG,
pregnant mares' serum gonadotropin; TPA, 12-O-tetradecanoylphorbol-13-acetate.In
addition to these assays, studies
are in progress to establish assays utilizing DNA
microarray and proteomics to facilitate the discovery of new chemopreventive
agents and novel molecular mechanisms of action [82, 83].
Such new emerging technologies
allow the screening and monitoring of the expression levels of thousands of
genes simultaneously [84]. More
importantly, the technology to make customized gene chips with specific genes
is also possible. In addition
to monitoring the alterations
in gene expression patterns in tissues undergoing carcinogenesis, these chips
can be utilized to evaluate subjects at risk, such as those carrying specific
germline mutations and genetic polymorphisms [85].


## 4. In Vitro Efficacy
Model Systems

Besides
the mechanistic assays mentioned above, the systematic evaluation of cancer
preventive agents includes screening compounds in short-term in vitro screens which aim to select
agents for subsequent whole animal testing with insight into potential
mechanisms of action. Use of primary cultured cells without aneuploidy is ideal
because they possess relatively intact drug metabolizing systems and normal
gene numbers. Epithelial cells used are rat tracheal epithelial cells, human
lung cells, hyperplastic alveolar nodules in mouse mammary gland organ cultures,
JB6 epidermal cells, and human foreskin epithelial cells. In each assay, the substances are tested
over a wide range of concentrations to determine EC50 values. The assays include (i) a rat tracheal epithelial
cell (RTE) assay which measures the ability of candidate
chemopreventive agents to block the benzo[a] pyrene (B[a]P)-induced
transformation of primary RTE cells [86]; (ii) an anchorage independence
assay which is an effective method for detecting compounds that block
carcinogenesis in the postinitiation stages and evaluates inhibition of
anchorage independence in human lung tumor (A427) cells [87]; (iii) a mouse mammary gland
organ cultures (MMOCs)
assay which assesses
the inhibitory activity of test chemicals on the development of carcinogen-induced
hyperplastic alveolar nodules (HANs)
in
MMOC [88]. This assay is similar in
appearance to the alveolar nodules produced in mouse mammary glands in vivo [89]; (iv) an in vitro assay for antipromoters or antiprogressors which is
designed to identify chemopreventive agents effective in the promotion or
progressive stages of carcinogenesis in JB6 epidermal cells [90]; and (v) a human foreskin
epithelial assay which determines the inhibitory potential of chemopreventive
agents in blocking cell growth stimulation induced by the carcinogen propane
sulfone [91].


## 5. In Vivo Short-Term Screening Assays

This
type of short-term assays identifies agents that might block or arrest
carcinogenesis in the early stages. Two experimental models, which reflect
major cancers in humans, are being used. They include the rat and mouse
colorectal aberrant crypt
foci (ACF)
assay [11, 12, 92, 93] and a rat model of breast ductal carcinoma in situ model (DCIS) [94].


### 5.1. ACF Assay

The ACF assay is a short-term
model which can identify agents that may be effective in preventing CRC [11, 12, 92, 93]. ACF, which were first described by
Bird [95], are putative
preneoplastic lesions consisting of aggregates of single and multiple crypt
cells that exhibit hyperplasia and/or dysplasia and are thought to be the
earliest detectable lesions of CRC [96–99]. Two different protocols have been
developed: one which identifies compounds that inhibit initiation and a second
treatment schedule which evaluates potential chemopreventive agents during the
postinitiation phase of colorectal carcinogenesis. Details of these regimens
have been described previously [11, 12, 100]. In the former (the initiation
protocol), rats are given a test agent in the diet one week prior to the administration of a
colonic carcinogen, such as AOM and continuing throughout the five-week study
period. In the latter regimen (the postinitiation protocol), rats are first
treated with AOM, followed four weeks later by a test agent, which is given for
additional weeks. Animals are sacrificed and the ACF frequency is determined by microscopic
evaluation.


### 5.2. DCIS Assay

The DCIS assay provides
both toxicity and efficacy data for identifying candidate chemopreventive
agents prior to testing in common mammary carcinogenesis models. Therefore, the induction of mammary
tumorigenesis is initiated in weanling female SD rats by the intraperitoneal (i.p.)
injection of the carcinogen N-methy1-N-nitrosourea (MNU) [94]. In general,
test agents are administered in the diet, starting one week after the carcinogen
administration,
and thereafter are continued
until the termination of
the study (45–50 days later). Mammary tissue specimens are excised and
processed for histopathological analysis. The efficacy is estimated as the percent reduction in
the number of DCIS lesions in
comparison to controls that
receive a carcinogen alone.


## 5.1. ACF Assay

The ACF assay is a short-term
model which can identify agents that may be effective in preventing CRC [11, 12, 92, 93]. ACF, which were first described by
Bird [95], are putative
preneoplastic lesions consisting of aggregates of single and multiple crypt
cells that exhibit hyperplasia and/or dysplasia and are thought to be the
earliest detectable lesions of CRC [96–99]. Two different protocols have been
developed: one which identifies compounds that inhibit initiation and a second
treatment schedule which evaluates potential chemopreventive agents during the
postinitiation phase of colorectal carcinogenesis. Details of these regimens
have been described previously [11, 12, 100]. In the former (the initiation
protocol), rats are given a test agent in the diet one week prior to the administration of a
colonic carcinogen, such as AOM and continuing throughout the five-week study
period. In the latter regimen (the postinitiation protocol), rats are first
treated with AOM, followed four weeks later by a test agent, which is given for
additional weeks. Animals are sacrificed and the ACF frequency is determined by microscopic
evaluation.


## 5.2. DCIS Assay

The DCIS assay provides
both toxicity and efficacy data for identifying candidate chemopreventive
agents prior to testing in common mammary carcinogenesis models. Therefore, the induction of mammary
tumorigenesis is initiated in weanling female SD rats by the intraperitoneal (i.p.)
injection of the carcinogen N-methy1-N-nitrosourea (MNU) [94]. In general,
test agents are administered in the diet, starting one week after the carcinogen
administration,
and thereafter are continued
until the termination of
the study (45–50 days later). Mammary tissue specimens are excised and
processed for histopathological analysis. The efficacy is estimated as the percent reduction in
the number of DCIS lesions in
comparison to controls that
receive a carcinogen alone.


## 6. Animal Efficacy Assays

The
use of animal efficacy models to establish organ specificity and to generate dose-response, toxicity, and other pharmacological data is a crucial
component of the determinant
process for chemoprevention agents. These assays with thetoxicity tests
are used for decisions regarding recommendations for clinical use. Numerous
animal models are used to study inhibition of chemical carcinogenesis in
rodents. Important criteria considered in selecting an in vivo model for screening cancer chemoprevention agents include
(i) short study duration and induction of carcinogenesis; (ii) target-specific
experimental model evidenced by the induction of cancer in the target tissues
comparable in such factors as histological type and hormone dependence to those
found in humans; (iii) evaluation of in vitro mechanistic activities, efficacy profiles, and relevant published data
prior to the selection of models for a given possible chemopreventive agent.
Typically, test agents are administered in the diet unless problems with
stability are encountered. During the course of chemoprevention studies a
maximum tolerated dose (MTD),
defined as the highest dose level that does not cause ≥10% reduction or gain in body weight over a
six-week period, is determined. The treatment schedules include the administration of test
agents either before, concurrently, or following exposure to the carcinogen. Efficacy is based upon the percent inhibition of
tumor incidence and/or multiplicity, or increased tumor latency in comparison to carcinogen-treated
controls. Representative carcinogenesis models are listed in Table3.Table 3
Preclinical
animal models for identifying chemoprevention efficacy.
Target tissuesSpecies and carcinogensInduced tumorsOral cavity:
tongue or buccal pouchHamster (buccal pouch): DMBA*SCC, PAPMouse (tongue): 4-NQORat (tongue):
4-NQOColonMouse: AOM, DMH, MAM
acetateADC, AD, ACFRat: AOM, DMH, MAM
acetate, MNUEsophagusRat: Nitrosaminea
(MNAM, NMBA), 4-NQOSCC, PAPMouse: 4-NQOForestomachMouse: B(a)PSCC, PAPLiverMouse: variousHCC, ADRat: 2-AAF, DEN, DMN,3′-Me-DABLungMouse: B(a)P, DMBA, NNK, Urethane, 4-NQOSCC, ADC, ADHamster: DEN, MNU
(trachea)BreastMouse: DMBAADC, AD, fibroadenomaRat: DMBA, MNUPancreaseHamster (duct cell)): BOPADC, AD, acinar cell carcinomaRat (acinar cell): azaserineSkinMouse: UV radiation,
B(a)P/TPA, DMBA, DMBA/TPA, MCSCC, PAPGlandular stomachRat: MNNG, MNUADCUrinary bladderMouse: OH-BBNTCCRat: MNU, OH-BBN2-AAF,
2-acetylaminofluorene; ACF, aberrant crypt foci; AD, adenoma; ADC,
adenocarcinoma; AOM, azoxymethane; B(a)P,
benzo[a]pyrene; BOP, N-bis(2-oxopropyl)nitrosamine; DEN,
diethylnitrosamine; DMBA, 7,12-dimethylbenz[a]anthracene;
DMH, 1,2-dimethylhydrazine; DMN, dimethylnitrosamine; HCC, hepatocellular
carcinoma; MAM acetate, methylazoxymethanol acetate; MC, 3-methylcholanthrenel;
3′-Me-DAB, 3′-methyl-4-dimethylaminoazobenzene; MNAN, N-methyl-N-amylnitrosamine;
MNNG, N-methyl-N’-nitro-N-nitrosoguanidine;
MNU, N-methyl-N-nitrosourea; NMBA, N-nitrosomethylbenzylamine;
NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; 4-NQO, 4-nitroquinoline
1-oxide; OH-BBN, N-butyl-N-(4-hydroxybutyl)nitrosamine; PAP,
squamous cell papilloma; SCC, squamous cell carcinoma; TCC, transitional cell
carcinoma; TPA, 12-O-tetradecanoylphorbol-13-acetate.


### 6.1. Head and Neck Carcinogenesis Models

Several
well-established models of oral and respiratory tract cancer have been developed.
In the hamster buccal pouch model [101], the carcinogen
7,12-dimethylbenz[a]anthracene (DMBA) is topically applied over a 12-week
period, thus resulting in buccal
pouch squamous cell carcinomas [102]. Rats [4] and mice [103] develop tongue
cancers when exposed to
4-nitroquinoline 1-oxide (4-NQO)
[104]. In rats,
tongue squamous cell carcinomas and dysplasia can be induced by 4-NQO in
drinking water (20 ppm) for 8 weeks [4]. Tongue
dysplasia occurs during 4-NQO treatment and the incidence of tongue squamous
cell carcinoma is over 50% at 32 weeks after the exposure. In this model, test chemicals can be
orally administered either before, during, or following 4-NQO exposure [4]. For the induction of lung
adenosquamous cell carcinoma or squamous cell carcinoma of hamsters, MNU is
administered over a 15-week period, thus resulting in approximately 40–50% of the treated animals after 6
months [105]. Starting one
week prior to MNU exposure, a test compound is administered over 180 days. In
the second model, hamsters are given the carcinogen diethylnitrosamine (DEN), subcutaneously, twice per week over a 20-week
period, resulting in the formation of lung adenocarcinomas in about 50% and
tracheal tumors in over 90%, of treated animals [106]. As in the MNU
model, test agents are given prior to carcinogen exposure. The tobacco-specific
nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) can induce lung tumors in A/J mice and a large number of compounds are now
available that inhibit the mouse
lung tumorigenesis induced by NNK [107]. Another lung
cancer bioassay is the strain A mouse model [108]. Strain A mice
spontaneously develop lung tumors as early as 3-4 weeks, with lung tumor
incidences approaching 100% at 24 months of age. A/J mice are also utilized for
studies on colon tumorigenesis because of their susceptibility to colonic
carcinogens [109].


### 6.2. Colorectal Carcinogenesis Models

Potential
inhibitors of colorectal carcinogenesis can be assessed utilizing models in
both rats and mice [11, 12, 110, 111]. According to established
protocols, 1,2-dimethylhydrazine (DMH),
AOM, or methylazoxymethanol (MAM)
acetate is administered intraperitoneally or subcutaneously, thus resulting in colorectal
adenocarcinoma development within 32–40 weeks in either species. DMH is first
activated to form AOM and then is metabolized by the liver to form MAM, the
ultimate carcinogen, which is excreted via glucuronide conjugation. In the AOM
induction model, a single or multiple (up to 3 times) subcutaneous dose of AOM
in male F344 rats results in the occurrence of colorectal adenocarcinoma and
adenoma in approximately 70% of treated animals by 40 weeks. Again, the test agents can be
orally administered either before, during, or following carcinogen treatment [112]. In comparison to rats, mice should
receive multiple exposures of a colonic carcinogen to induce colonic tumors and
tumor development needs long-term period. A mouse model recently established for
colorectal carcinogenesis [113], in which
different colonic carcinogens are
followed by a colitis-inducing
agent, dextran sodium sulfate (DSS),
is quite useful  to identify potential chemopreventive agents within a
short-term period [68, 114].


### 6.3. Mammary Carcinogenesis Models

Chemopreventive efficacy against mammary gland carcinogenesis
is routinely assessed by either the MNU- or DMBA-induced models [115].
Both protocols utilize female SD rats and require that the carcinogen is given as a single dose at 50 days of age. In some instances,
the carcinogen is administered to older animals (180 days) which is more
representative of the human target population. Tumor incidences at 120 days
after carcinogen treatment are similar, ranging from 80–100% in the DMBA
protocol and 75–95% in the MNU model. However, the histological types of tumors induced by the two carcinogens are different.
DMBA-induced mammary tumors are predominantly adenoma and fibroadenoma, with
some adenocarcinoma, whereas MNU-induced mammary tumors are invasive
adenocarcinomas. The chemopreventive activity
of the test agents is determined by the percent reduction in tumor
incidence or percent increase in tumor latency relative to controls treated
with the carcinogen alone. These models produce hormonally responsive
tumors. In addition to these
chemically-induced mammary carcinogenesis models, several genetically engineered
animals have been introduced to investigate breast carcinogenesis and evaluate the
efficacy of candidate chemopreventive agents. They include a COX-2 overexpressing
mouse model [116], a Ras-driven mouse mammary
tumorigenesis model [117], and a HER-2/neu transgenic mouse model [118]. In addition, mammary stem
cell models can be used for studying how the hormonally regulated paracrine interactions influence stem
cells and the stem cell niche during mammary carcinogenesis [119]. Another interesting model system for
understanding normal human breast development or tumorigenesis is the orthotopic
xenograft model that  has the potential to improve the understanding
of crosstalk between tissue stroma and the epithelium as well as factors
involved in breast stem cell biology tumor initiation and progression [120].


### 6.4. Urinary Bladder Carcinogenesis Models

Urinary
bladder neoplasms are typically induced by the carcinogenN-butyl-N-(4-hydroxybutyI)nitrosamine
(OH-BBN) which can induce invasive transitional
cell carcinomas morphologically similar to those found in humans [121, 122].
This carcinogen is given either intragastrically or in
drinking water
over an 8-week
period to 50-day old BDF mice (C57BL/6 × DBS/2-Fl) or F344 rats, thus resulting in a 40–50% incidence
of bladder tumor incidence at 180 days after OH-BBN treatment. Treatment schedules for a
test agent administration are as described above [123].


### 6.5. Skin Carcinogenesis Models

Agents effective in inhibiting skin carcinogenesis are identified in a two-stage skin
carcinogenesis model utilizing DMBA and TPA,
which are applied topically to the back skin of SENCAR or CD-1 mice [124, 125].
Both strains of mice are highly susceptible to skin tumor induction. Skin
papillomas appear as early as 6 weeks postcarcinogen treatment, eventually
progressing to squamous cell carcinomas by 18 weeks [126]. Test agents
are generally administered in the diet or in some experiments are topically
applied according to several predefined treatment regimens. As to melanoma chmoprevention study, we
should read an elegant review for new perspectives of this research area [127]. In addition to in vitro screening assay [128], several genetically altered animal (mouse)
models of melanoma [129–132], including hepatocyte growth
factor/scatter factor (HGF/SF) transgenic mice [133–136], have been introduced for prevention [137] and biology [138] of this neoplasm. Also, a
three-dimensional skin reconstruction model [139] is useful for determining the therapeutic
efficacy of selected chemicals or drugs in cultured melanoma cells [140]. While epidemiological studies suggest
sunlight as an etiologic agent for the pathogenesis of melanoma, recent
experimental investigations by the group Meyskens, Jr. indicated that elevation
of reactive oxygen species follows from melanin serving as a redox generator [127] and this may be involved in the etiology
and pathogenesis of cutaneous melanoma. Such findings will help to establish
novel preventive and therapeutic approaches to this malignancy.


## 6.1. Head and Neck Carcinogenesis Models

Several
well-established models of oral and respiratory tract cancer have been developed.
In the hamster buccal pouch model [101], the carcinogen
7,12-dimethylbenz[a]anthracene (DMBA) is topically applied over a 12-week
period, thus resulting in buccal
pouch squamous cell carcinomas [102]. Rats [4] and mice [103] develop tongue
cancers when exposed to
4-nitroquinoline 1-oxide (4-NQO)
[104]. In rats,
tongue squamous cell carcinomas and dysplasia can be induced by 4-NQO in
drinking water (20 ppm) for 8 weeks [4]. Tongue
dysplasia occurs during 4-NQO treatment and the incidence of tongue squamous
cell carcinoma is over 50% at 32 weeks after the exposure. In this model, test chemicals can be
orally administered either before, during, or following 4-NQO exposure [4]. For the induction of lung
adenosquamous cell carcinoma or squamous cell carcinoma of hamsters, MNU is
administered over a 15-week period, thus resulting in approximately 40–50% of the treated animals after 6
months [105]. Starting one
week prior to MNU exposure, a test compound is administered over 180 days. In
the second model, hamsters are given the carcinogen diethylnitrosamine (DEN), subcutaneously, twice per week over a 20-week
period, resulting in the formation of lung adenocarcinomas in about 50% and
tracheal tumors in over 90%, of treated animals [106]. As in the MNU
model, test agents are given prior to carcinogen exposure. The tobacco-specific
nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) can induce lung tumors in A/J mice and a large number of compounds are now
available that inhibit the mouse
lung tumorigenesis induced by NNK [107]. Another lung
cancer bioassay is the strain A mouse model [108]. Strain A mice
spontaneously develop lung tumors as early as 3-4 weeks, with lung tumor
incidences approaching 100% at 24 months of age. A/J mice are also utilized for
studies on colon tumorigenesis because of their susceptibility to colonic
carcinogens [109].


## 6.2. Colorectal Carcinogenesis Models

Potential
inhibitors of colorectal carcinogenesis can be assessed utilizing models in
both rats and mice [11, 12, 110, 111]. According to established
protocols, 1,2-dimethylhydrazine (DMH),
AOM, or methylazoxymethanol (MAM)
acetate is administered intraperitoneally or subcutaneously, thus resulting in colorectal
adenocarcinoma development within 32–40 weeks in either species. DMH is first
activated to form AOM and then is metabolized by the liver to form MAM, the
ultimate carcinogen, which is excreted via glucuronide conjugation. In the AOM
induction model, a single or multiple (up to 3 times) subcutaneous dose of AOM
in male F344 rats results in the occurrence of colorectal adenocarcinoma and
adenoma in approximately 70% of treated animals by 40 weeks. Again, the test agents can be
orally administered either before, during, or following carcinogen treatment [112]. In comparison to rats, mice should
receive multiple exposures of a colonic carcinogen to induce colonic tumors and
tumor development needs long-term period. A mouse model recently established for
colorectal carcinogenesis [113], in which
different colonic carcinogens are
followed by a colitis-inducing
agent, dextran sodium sulfate (DSS),
is quite useful  to identify potential chemopreventive agents within a
short-term period [68, 114].


## 6.3. Mammary Carcinogenesis Models

Chemopreventive efficacy against mammary gland carcinogenesis
is routinely assessed by either the MNU- or DMBA-induced models [115].
Both protocols utilize female SD rats and require that the carcinogen is given as a single dose at 50 days of age. In some instances,
the carcinogen is administered to older animals (180 days) which is more
representative of the human target population. Tumor incidences at 120 days
after carcinogen treatment are similar, ranging from 80–100% in the DMBA
protocol and 75–95% in the MNU model. However, the histological types of tumors induced by the two carcinogens are different.
DMBA-induced mammary tumors are predominantly adenoma and fibroadenoma, with
some adenocarcinoma, whereas MNU-induced mammary tumors are invasive
adenocarcinomas. The chemopreventive activity
of the test agents is determined by the percent reduction in tumor
incidence or percent increase in tumor latency relative to controls treated
with the carcinogen alone. These models produce hormonally responsive
tumors. In addition to these
chemically-induced mammary carcinogenesis models, several genetically engineered
animals have been introduced to investigate breast carcinogenesis and evaluate the
efficacy of candidate chemopreventive agents. They include a COX-2 overexpressing
mouse model [116], a Ras-driven mouse mammary
tumorigenesis model [117], and a HER-2/neu transgenic mouse model [118]. In addition, mammary stem
cell models can be used for studying how the hormonally regulated paracrine interactions influence stem
cells and the stem cell niche during mammary carcinogenesis [119]. Another interesting model system for
understanding normal human breast development or tumorigenesis is the orthotopic
xenograft model that  has the potential to improve the understanding
of crosstalk between tissue stroma and the epithelium as well as factors
involved in breast stem cell biology tumor initiation and progression [120].


## 6.4. Urinary Bladder Carcinogenesis Models

Urinary
bladder neoplasms are typically induced by the carcinogenN-butyl-N-(4-hydroxybutyI)nitrosamine
(OH-BBN) which can induce invasive transitional
cell carcinomas morphologically similar to those found in humans [121, 122].
This carcinogen is given either intragastrically or in
drinking water
over an 8-week
period to 50-day old BDF mice (C57BL/6 × DBS/2-Fl) or F344 rats, thus resulting in a 40–50% incidence
of bladder tumor incidence at 180 days after OH-BBN treatment. Treatment schedules for a
test agent administration are as described above [123].


## 6.5. Skin Carcinogenesis Models

Agents effective in inhibiting skin carcinogenesis are identified in a two-stage skin
carcinogenesis model utilizing DMBA and TPA,
which are applied topically to the back skin of SENCAR or CD-1 mice [124, 125].
Both strains of mice are highly susceptible to skin tumor induction. Skin
papillomas appear as early as 6 weeks postcarcinogen treatment, eventually
progressing to squamous cell carcinomas by 18 weeks [126]. Test agents
are generally administered in the diet or in some experiments are topically
applied according to several predefined treatment regimens. As to melanoma chmoprevention study, we
should read an elegant review for new perspectives of this research area [127]. In addition to in vitro screening assay [128], several genetically altered animal (mouse)
models of melanoma [129–132], including hepatocyte growth
factor/scatter factor (HGF/SF) transgenic mice [133–136], have been introduced for prevention [137] and biology [138] of this neoplasm. Also, a
three-dimensional skin reconstruction model [139] is useful for determining the therapeutic
efficacy of selected chemicals or drugs in cultured melanoma cells [140]. While epidemiological studies suggest
sunlight as an etiologic agent for the pathogenesis of melanoma, recent
experimental investigations by the group Meyskens, Jr. indicated that elevation
of reactive oxygen species follows from melanin serving as a redox generator [127] and this may be involved in the etiology
and pathogenesis of cutaneous melanoma. Such findings will help to establish
novel preventive and therapeutic approaches to this malignancy.


## 7. Transgenic and Gene-Knockout Animal Models

Animal
models that mimic the specific
characteristics of human carcinogenesis may prove to be a valuable resource in both evaluating chemopreventive
efficacy and identifying appropriate biomarkers for measuring the chemopreventive
activity. Transgenic and gene knockout mice that carry well-characterized
genetic lesions predisposing them
to
carcinogenesis are appropriate models for chemoprevention testing (Table4). Some of the best developed models
include the multiple intestinal neoplasia (Min) mouse [141] and other
strains possessing lesions in the Apc gene [142]. The Min mouse carries an Apc mutation similar to that found in
human familial adenomatous polyposis (FAP)
patients. These
mice are predisposed to develop predominantly small intestinal adenomas, but a
few in the large intestine. By
manipulating two or more carcinogenesis-associated genes, such as modifier
genes, in a single animal, closer approximations of human carcinogenesis may be
possible. Numerous
colonic tumors develop in the large bowel in Min mice at 3 weeks after
one-week-exposure of DSS [143], thus suggesting the importance of
gene-environmental interaction in cancer development [68].
It might be feasible to knock out p53 in an animal that already carries
another tumor suppressor defect such as Apc or p16. Recently, new transgenic
animal models for mammary [144], tongue [145, 146],
pancreas [147], and gall
bladder [148] cancers have
been reported. These models might be useful for discovering possible novel
cancer chemopreventive agents.Table 4
Transgenic/gene KO mice or rats for identifying chemoprevention efficacy.
GenesTarget
tissuesGenetic
lesionsInduced
tumorsMinIntestineHeterozygousApc 2549ADApcIntestineHeterozygousApc 1638ADMLH1/Apc 1638IntestineHeterozygous
MLH1 andApc 1638ADMsh2/MinIntestineHeterozygous
MLH2 andApc 2549ADpimLymphatic
systemAmplifiedpim-1T-cell
lymphomaTG:ACSkinHa-ras mutationPAP,
possibly carcinomaTSG-p53SkinHeterozygous
p53 deficientPAP,
possibly carcinomaA/JxTSG-p53LungHeterozygous
p53 deficientADA/JxUL53LungHeterozygous
p53 mutantADTGFβ1Liver,
lungHeterozygous
TGFβ1
mutantAD,
carcinomav-Ha-rasSkinHa-ras + Human keratin K-1HP,
PAPK14-HPV16SkinHPV-infected
(K14-HPV16 heterozygote), estradiol-treated + SV40 T-antigenPAP,
condylomaCervixHPV-infected
(K14-HPV16 heterozygote), estradiol-treated + SV40 T-antigenDysplasiarPB-SV40 Tag transgeneProstateSV40 large tumor T antigen (Tag)Dysplasia,
AD, ADC
(TRAMP
model)C3(1)-SV40ProstateHeterozygous rat prostate steroid binding gene [C3(1)]
+ SV40 T-antigenDysplasia,
AD, ADCBreastHeterozygous rat prostate steroid binding gene [C3(1)]
+ SV40 T-antigenADCErbB-2Gall
bladderBK.ErbB-2AD,
ADCHuman
c-Ha-rasBreastJcl/SD-TgN(Hras Gen)128NccADCUrinary
bladderJcl/SD-TgN(Hras Gen)128NccTCCProstateJcl/SD-TgN(Hras Gen)128NccADC,
PINTongueHuman
prototype c-Ha-ras gene with its own promoter regionPAP,
SCCAD,
adenoma; ADC, adenocarcinoma; HP, hyperplasia; HPV, human papilloma virus; PAP,
squamous cell papilloma; PIN, prostate intraepithelial neoplasia; TCC,
transitional cell carcinoma; TGF, transforming growth factor.


## 8. Combination Treatment

One
strategy for improving the efficacy
and lessening toxicity is using combinations of agents [149, 150].
Synergistic or additive effects may be observed when two agents with different
mechanisms of action are combined [149]. Such improved
activity of inhibition may allow either or both of the agents to be given at
lower doses, thereby reducing
the
toxic potential. Examples of chemical combinations producing positive
results in experimental animal models include all-trans-N-(4-hydroxyphenyl)retinamide
(4-HPR) and tamoxifen in the rat mammary gland [151] and 2-dinuoromethylorinithine (DFMO) and piroxicam in the rat colon [152, 153]. The identification and evaluation
of other effective agent combinations is an interesting and important research
effort for chemopreventive agent development [154, 155]. Since different treatment schedules of the two drugs can be compared and
evaluated for synergism, additivity, or antagonism using a quantitative method
based on the median-effect
principle of Chou and Talalay [156–159] can identify the ideal
combination treatment for obtaining an improved effect in comparison to a single
exposure of the potential chemopreventive agent [160–162].


## 9. Intermediate Biomarkers

Carcinogenesis
proceeds through a very long preclinical period. The collective hope is that multiple
opportunities exist for chemoprevention to arrest or reverse progression
towards malignancy. Intermediate biomarkers of malignant neoplasms are the
phenotypic, genotypic, and molecular changes that occur during carcinogenesis [69].
An important component of chemopreventive agent development research is the
identification and characterization of intermediate biomarkers that may serve
as surrogate end points for cancer incidence reduction in chemoprevention
clinical trials [163]. Such efforts are critical to the
progress of chemoprevention and have the potential for cost-effective development
of chemopreventive research
[5, 6, 69].
Biomarkers include proliferation,
apoptosis, growth factors and their receptors, genetic alterations, and so forth in the
target tissues [162, 164, 165]. In the hope of faster
progress with fewer subjects and lower total cost, much effort is being
expended on the search for reliable biomarkers to predict the likelihood of
developing cancer and/or to signal the effectiveness of chemopreventive therapy [166].
Considerable attention has
been
paid to identifying those markers that can act as surrogate markers for cancer
development since favorable modulation of the surrogate end-point biomarker
may demonstrate the effectiveness
of a putative preventive treatment. However, the complexity of the biology
challenges the
ability to measure the effectiveness of attempts to arrest or reverse
carcinogenesis, other than through costly and time-consuming prospective trials
with the disease state as the
endpoint. Despite much work, to date no prehistological biological or
molecular intermediate marker has yet been validated for sporadic cancers.For chemoprevention of prostate cancer [167] and other types of cancer,
natural and synthetic agents that may suppress, reverse, or regress precancer and
delay progression to invasive cancer can be used [77]. Epidemiological evidence suggests that environmental factors, such
as diet, play a role in the development and progression of prostate cancer. The
number of potential protective dietary compounds or whole dietary products that
are indicated to have preventive effects is piling up and demands further
evaluation. To face this scientific field, a strategy that combines
prostate-specific antigen (PSA)-based clinical
trials with experimental human xenograft studies to evaluate potential
chemopreventive agents against prostatic cancer is proposed [168, 169]. PSA for prostate adenocarcinoma, even
though has relatively poor specificity, is cheap and easily followed with
minimally invasive procedures.


## 10. Future Direction

Epidemiologic evidence suggests that green tea polyphenols reduce the risk of some forms of cancer, while data for other commonly used
phytochemicals is less convincing. Part of the problem lies in precise
quantitation of foodborn phytochemicals where multiple dietary sources of a
particular phytochemical are involved, as in recent studies of dietary quercetin on the
prevention of heart disease. Future investigations may focus on specific
foodborn phytochemicals and/or botanicals capable of intervening in critical pathways
in carcinogenesis as indicated by the possible use of “natural” NSAIDs in the
prevention of CRC and the soy-based phytoestrogens in the prevention of breast
cancer. Much is to be learned by collaboration between cancer researchers and
ethnopharmacologists for probing dietary prevention of cancer. In addition, the development and validation of new in vitro and in vivo assays of high predictive value for discovering agents with human chemopreventive
potential is needed. Care, however, is needed when extrapolating in vitro data to in vivo models because it cannot be assumed that the effects seen
when cells are exposed directly to active compounds that would be candidate
chemopreventive agents will be seen when they are consumed in the diet. We
should investigate whether they are capable of distribution throughout the body
when they are absorbed after ingestion. The
understanding of the molecular and cellular processes, such as gene and protein
expression, apoptosis, angiogenesis, signal transduction, involved in
carcinogenesis is rapidly increasing. Currently several translational studies using
phytochemicals and bioactive compounds, such as indole-3-carbinol [170], ellagitannins [171], sulforaphane [172], lycopene [173], diallyl trisulfide [174], omega-3 fatty acids [175], proanthocyanidins [176],
green tea polyphenols [177],
genistein [178],
silymarin [179], and curcumin [180, 181], from plants are underway.



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*Source: 475963-2009-04-28.xml*