doi: 10.1111/jop.

12171

J Oral Pathol Med (2015) 44: 167177

2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
wileyonlinelibrary.com/journal/jop

Molecular and cellular cues of diet-associated oral

carcinogenesiswith an emphasis on areca-nut-induced
oral cancer development
Wan-Chun Li1,2,3, Pei-Lun Lee2, I-Chiang Chou1,4, Wan-Jung Chang2, Shu-Chun Lin1,2,5, Kuo-Wei
Chang1,2,5
1

Department of Dentistry, School of Dentistry, National Yang-Ming University, Taipei, Taiwan; 2Institute of Oral Biology, School of
Dentistry, National Yang-Ming University, Taipei, Taiwan; 3Department of Education and Research, Taipei City Hospital, Taipei, Taiwan;
4
Department of Dentistry, Zhong-Xiao Branch, Taipei City Hospital, Taipei, Taiwan; 5Department of Stomatology, Taipei Veterans
General Hospital, Taipei, Taiwan

In modern times, potent dietary carcinogens are key

contributors for neoplastic development. For oral squamous cell carcinoma (OSCC), one of the leading cancer
types in developing countries, main oncogenic inducers/
enhancers, including areca nut chewing, tobacco smoking, and alcohol consumption, were shown to promote
cancer initiation/progression. Over decades, studies from
different laboratories have identified underlying cellular
and molecular mechanisms for carcinogen-induced
OSCC. In this review, we will give an overview of where
we are in understanding potential oral carcinogenic
factors stimulated OSCC tumorigenesis, especially those
associated with areca nut chewing in Asians, aiming to
provide future scope of possible interception.
J Oral Pathol Med (2015) 44: 167177
Keywords: areca nut; curcumin; oral cancer; tea polyphenol

Introduction
The formation of oral squamous cell carcinoma (OSCC), a
subcategory of head and neck cancers, is the outcome of
multiple hit-and-go events acquired mostly from oral intake,
which affects oral epithelial/mucosal tissues. Overall intraand extracellular carcinogenic input triggers aberrant genetic
accumulation, defective epigenetic regulation, and pathological microenvironment resulting in sequential pathological
transformation (1). The neoplastic process could be evident
by several abnormal pathological features (hyperkeratosis,
epithelial hyperplasia, and dysplasia), phenotypic alterations
Correspondence: Kuo-Wei Chang, DDS, PhD, Department of Dentistry,
School of Dentistry, National Yang-Ming University, No. 155, Li-Nong St.,
Sec.2, Taipei, Taiwan 112. Tel: +8862-28267223, Fax: +8862-28264053,
E-mail: ckcw@ym.edu.tw
Accepted for publication January 23, 2014

(invasion, enhanced chemoattraction of lymphocytes, and

angiogenesis) as well as leukoplakia, erythroleukoplakia,
lichenoid changes, and other pre-cancerous lesions clinically
identied (2). Several dened oral carcinogens or tumorpromoting materials, including alcohol consumption, tobacco
smoking, areca nut chewing, and viral infection, are risk
factors for oral malignancy (36). At the molecular level,
earlier studies were initiated by examination of differential
gene mutations or aberrant expression in cancer cells/tissues
compared with normal oral tissues. Many studies from our
laboratory showed that deregulated expression of genes for
proliferation, cell mobility, survival, angiogenesis, and
immune modulation could be implicated for areca-associated
OSCC pathogenesis (714). The importance of environmental challenges including infection of oncogenic human
papillomavirus (e.g., HPV16, 18, 31) (1517) and imbalanced metabolism such as diabetes mellitus (1822) linking
to oral carcinogenesis has also been discussed elsewhere. In
contrast, various anti-oncogenic natural compounds such as
tea polyphenols and curcumin were also found to counteract
oral carcinogenesis by means of various regulations (23, 24).
To further explore mechanisms underlying different
stages of oral tumorigenesis, tissues from normal, pre-cancerous, early, and late stages of tumors are required to
elucidate dynamic pathological process of oral carcinogenesis. However, the use of clinical samples only represented
snapshots but not systemic conditions of disease
progression, while the limited access to clinical samples
from the same individuals restricted comprehensive
interpretation of cellular and molecular cues important for
oral cancer formation. Most mechanistic analysis was
therefore performed in in vitro cultured oral cells. Despite
the treatment in vitro may not truly reect in vivo
metabolism of candidate oncogenic substances, both acute
and prolonged cytotoxic response in cultured cells, either
from isolated primary oral epithelial/mesenchymal cells or
established OSCC cell lines, are indeed of great help to
identify essential mechanisms of OSCC formation. Rodent

Overview of diet-mediated oral cancer

Li et al.

168

models were also applied to induce oral neoplastic lesions in

vivo by treatment for various dietary carcinogens using
different methods including painting onto buccal pouch or
administration in drinking water (2527). More recently,
identication of epigenetic modulators such as non-coding
small RNAs for OSCC pathology was also carried out.
For example, disruption was frequently identied in OSCC
(2831). Taken together, this review aims to provide an
overview of carcinogenic effects of dietary uptakes, with
main emphasis on areca nut constitutes.

Areca (betel) nut chewing vs. oral

carcinogenesis
Areca nut is a nut of tropic palm. Areca quid (or called betel
quid in some regions) contains areca nut, inorescence piper
betel (also called betel leaf), slaked lime, tobacco, liqueed
catechu, and other spicy ingredients (32). Areca chewing is a
popular oral habit in South Asia; it is the fourth most common
addictive substance. Despite the different ways in which
areca nut is consumed in various regions, areca chewing
manifests general pharmacological effects, including euphoria, central nervous system stimulation, vertigo, salivation,
miosis, tremor, and bradycardia. Physiologically, areca
chewing could produce a sense of well-being, euphoria,
heightened alertness, sweating, salivation, and increased
body temperature leading to habituation and addiction,
mainly via the modulation on the central and autonomic
nervous system (33). Epidemiological evidences showed that
areca use is closely associated with various oral pathogenesis
such as oral submucous brosis (OSF), OSCC, periodontal
disorders, and others (34). Areca-associated OSCC is the
third most common neoplasia in developing countries,
comprising around 50% of all malignancies in some nations
of South Asia (3537). An early study demonstrated
epidermal thickening of the tongue, esophagus, and forestomach of rats in groups fed with areca nut diet for 480 days,
implying its carcinogenic activity (38). Although malignant
tumors were not activated in this experiment, the observation
of pre-cancerous-like epithelial lesions suggested other
milder carcinogens or cocarcinogens in areca nut. It was
reported that areca chewers often swallow the juice in direct
contact with oropharynx and esophagus, thereby leading to a
higher risk of cancer in these anatomic sites, indicating that
the quid also contains other milder carcinogens or cocarcinogens, possibly mediated by the formation of areca-nutspecic or tobacco-specic N-nitrosamines (39, 40). In
addition, Atkinson et al. (41) reported that a high incidence
of OSCC in the New Guinea population might be resulted
from the habit of chewing areca quid, even though the areca
quid which is never mixed with tobacco, implying that areca
nut could also have a carcinogenic effect when chewed
without tobacco (42).
Areca nut itself comprises a number of compounds
identied as potential tumor inducers or enhancers. For
example, alkaloid is one of the main constituents in areca
nut. Around 8595% of total alkaloid content in areca nut is
arecoline, while others are arecaidine, guvacoline, and
guvacine (43). In addition to areca nut alkaloids, studies
have been carried out to test the mutagenic and carcinogenic
effects of other alternative constituents in areca quid using
J Oral Pathol Med

different experimental models. These components include

polyphenols, tannin, metal ions, and safrole. Studies have
identied areca ingredients as the synergistic or promoting
agents for multistep chemical carcinogenesis in different
animal models (8, 44). Nonetheless, the extracted areca nut
compounds described in previous sections to monitor the
physiological impact on oral tumorigenesis are pure chemicals, raising an argument whether the effect of the treatment
could truly recapitulate physiological conditions of oral cells
exposed to areca nut consumption environment. To address
this issue, the crude extract from areca nut (ANE) was
chosen to use in many studies. ANE could be prepared in
two ways, based on the involvement of areca nut husk,
thereby resulting in ripe ANE (rANE, used in most studies)
and tender ANE (tANE) for research uses (45, 46). The
extract of areca nut is tumorigenic on mouse skin and elicits
sarcoma in the connective tissue of mice and rats. It also
induces hepatocellular carcinoma (HCC) in mice upon
gastric intubation, suggesting that ANE is capable of
stimulating cancer formation (47). As ANE-induced oral
cancer progression was emphasized, the established OSCC
cell lines were often used. Interestingly, the molecular clues
underlying ANE-mediated tumor development are similar to
the factors involved in cancer initiation, although the
administrative concentration of ANE (approximately 5 lg/
ml) in normal human oral keratinocyte (NHOK)/normal
human oral broblast (NHOF) is much lower than the dose
treated in OSCC cell lines (approximately 20 lg/ml).
Areca quid-induced cytotoxicity and genotoxicity
Early studies indicated that arecoline treatment led to
increased sister chromatid exchange in mouse bone marrow
cells as well as higher frequency of micronuclei, an
unscheduled mitotic debris during cell division, in Chinese
hamster ovary cells (48, 49). Further investigation suggested
that arecoline could induce cell cycle arrest at prometaphase
by deregulating mitotic spindle assembly supporting the role
of arecoline in producing progeny that carried various
chromosomal abnormalities and genomic instability (50). It
was shown that the expression of p53-regulated p21 and
p53-activated DNA repair was repressed by arecoline
administration in human epithelial cells, suggesting that
arecoline-mediated p53 inhibition plays an important role in
areca-nut-associated tumorigenesis (51). As for the effect on
primary oral cells, arecoline was cytotoxic but not genotoxic
to cultured human buccal broblasts, and this arecolinemediated cytotoxicity could be overcome after the treatment
of antioxidants glutathione and glycyrrhizin (52). The
synergistic effect with the cigarette smoking product
nicotine was found to enhance arecoline-associated cytotoxicity (53). To explore susceptible genes accounting for
arecoline-induced cellular damage, a high-throughput
genetic screening for human gingival broblasts treated
with or without arecoline was carried out. Twelve candidate
genes associated with xenobiotic metabolism, maintenance
of genomic stability, DNA damage or repair, stress
response, and the TGFb signaling pathway were uncovered,
presenting potential targets for how arecoline induced
cytotoxicity and initiated cellular impairments (54).
Using in vivo models, the tumorigenic effect of areca nut
alkaloids was also investigated. Lin et al. (55) demonstrated

Overview of diet-mediated oral cancer

Li et al.

172

Areca nut
extract

Tobacco
ingredients
Tumor progression:
- ERK
Cox2
PGE2
Vimen n
- Akt
Vimen n
Cell mo lity
- NF-B
[ROS]
p38
HIF1
Autophagy
Curcumin
Tea polyphenols

Overdosed
Alcohol

Tumor ini a on:

- Genotoxicity
- NF-B
ROS
Cytotoxicity

MMP2

Areca nut
extract

Niche eect:
- Invasion
- Akt
Focal adhesion

Normal oral epithelium

Neoplas c oral
epithelium

ANE s mulated oral

fibroblasts

Premalignant oral
epithelium

Normal oral fibroblasts

Cancer associated
fibroblasts

Figure 1 Diagraphic illustration of dietary-associated oncogenic effects. The tumorigenic promoters including areca nut, tobacco, and alcohol could trigger
various cellular and molecular events key for oral cancer formation, while curcumin and tea polyphenols suppress cancerous phenotypes by antagonistic
mechanisms.

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N-nitrosonornicotine, polycyclic aromatic hydrocarbons (e.g.,

benzo[a]pyrene), and aromatic amines have the most potent
links to OSCC, mainly through induction of DNA adducts
(117). Ethanol itself is not carcinogenic in animal study, as its
metabolites such as acetaldehyde could cause DNA damage
leading to higher risk of OSCC. It is interesting to note that
the anatomic sites in close contact with alcohol are at higher
risk to develop tumors, indicating the facilitating role of
alcohol for oral tumor formation (118). Epidemiologically, a
recent large cohort analysis conducted by the International
Head and Neck Cancer Epidemiology (INHANCE) consortium showed the attributable risk of smoking and drinking
combined for cancer formation to be 64%, implying the joint
effect of these two components in major proportion of
patients with head and neck cancer (119). Bruguere et al.
(120) found relative risk value of 13.5 for OSCC when 100
159 g of alcohol was consumed daily, while men who both
smoke and drink are nearly 38 times more likely to develop
head and neck cancers than men who do neither (121). A
casecontrol study showed that the odds ratio was 123 times
for Taiwanese patients with OSCC who smoked, drank
alcohol, and chewed areca quid than non-users, suggesting
synergetic effect of alcohol and tobacco consumption in
areca nut chewers (122). The underlying molecular cues for
smoking and drinking are still poorly understood. Early
studies using PCR-single-strand conformation polymorphism and DNA sequencing to analyze the conserved exon
regions (exon 59) of p53 gene in OSCC specimens
identied that tobacco smoking drives an important contribution to p53 mutation, while alcohol might enhance these
mutagenic effects (109). Overall, alcohol consumption and
smoking could be dened as enhancers for areca-associated
oral carcinogenesis, although further analysis for molecular
players for this synergetic impact would be required.
J Oral Pathol Med

Anti-oncogenic dietary moleculestea

polyphenols and curcumin
From the aspect of chemoprevention, the use of natural
substances derived from diet could provide a safe strategy to
inhibit cancer with limited toxicity. Two such agents, green
tea and the spice curcumin, have been extensively investigated (123126). In general, tea polyphenols and curcumin
could inhibit oral malignancy via a number of cellular and
molecular mechanismsmost of those targets shared signals involved in areca-associated oral oncology (127129).
Recent studies determined that curcumin hinders arecoline
or ANE-associated oral cancer development, probably via
the regulation of heat-shock protein, HIF, or placenta
growth factor (130132).
Several studies identied the benets of using tea
polyphenols for suppression of cancer development, mainly
through its major constituent ()-epigallocatechin-3-gallate
(EGCG) (133). Using cultured OSCC cells, in vitro effects
of EGCG contained several key steps: (1) to inhibit cell
proliferation via ROS-mediated apoptosis induction and cell
cycle arrest as well as telomerase activity; (2) to modulate
transcription factors such as NF-jB and activator protein-1
(AP1); and (3) to reduce cell migration and invasion by
decreased expression of MMPs and urokinase plasminogen
activator serine protease (u-PA) (134136). As for animal
models of oral carcinogenesis, treatment with 0.6% green
tea reduced the number of visible tumors by 35% and
reduced tumor volume by 57% in the DMBA-induced
hamster buccal pouch cancers. Immunohistochemical analyses showed that tea increased the apoptotic index of the
tumors as well as decreased the proliferation index and
microvessel density (137). Recent examination giving green
tea polyphenols extracts to 4-nitroquinoline 1-oxide (4NQO) induced oral tongue carcinogenesis in rats was

Overview of diet-mediated oral cancer

Li et al.

170

Table 2 Oncogenic effects of areca nut extract treatment

Cell type
Mouse skin/liver
Human normal
oral epithelium

Human OSCC
cells

Human normal oral

broblasts

Effects
Sarcoma/hepatocellular
carcinoma
Increased senescence
Hyperdiploid chromosomal
changes
Cell cycle arrest
Increased PI3K/Akt ?
Increased dedifferentiation
Increased Rac/Rho/
Phospho-JNK
Increased broblastoids
Increased stress ber/
lamellipodin
Increased NF-kB ? Increased
Cox2/ProstaglandinE2/IL6/TNFa
Increased JNK/ERK/p38 ?
Increased inammation
Increased MMP9 ? Increased
NFkB ? Decreased cell
viability/Decreased re-epithelization
Increased lysyl oxidase activity
Muscarinic M4 receptor-mediated
signaling ? Increased cell motility
Increased oxidative stress
? Increased
HIF1a ? Increased autophagy
Increased NF-kB ? Increased
miR146a
Increased AuroA ? Increased
autophagy
Increased senescence
Cell cycle arrest
Increased MMP2 secretion ?
Increased PBMC invasiveness
Decreased FANCG ? Decreased
DNA repair ? Increased miR23a
Increased Proto-oncogene c-fos/c-jun

References
47
6668

74
80, 82

110,111
112
113
12
81
102
106
107
73
73, 84
105
108

(76). Further study to examine the effect of arecoline on the

expression of Cyr61, an important matricellular protein in
promoting many types of tumors, revealed that arecoline
stimulated Cyr61 synthesis in human gingival epithelial SG
cells leading to putative carcinogenesis. The Cyr61-mediated tumorigenic effect could be attenuated with the
treatment of various inhibitors to abrogate ERK, reactive
oxidative species (ROS), ROCK, and HMG-CoA reductase,
demonstrating distinctive molecular regulations may underlie arecoline-related oral pathogenesis (77). The regulation
of environmental-sensing integrins upon areca nut ingredient treatment was also reported. The up-regulation of
integrin avb6 was detected in approximately 50% of human
OSF suggesting integrin may be a potential component
involving in arecoline-induced oral aberrations. A recent
study using oral keratinocyte-derived cells genetically
modied to express high avb6 indeed showed that arecoline-dependent avb6 up-regulation promoted keratinocyte
migration and induced invasion, providing a possibility that
this mechanism may support malignant transformation (78).
In addition to areca nut alkaloids, safrole treatment could
also regulate tissue inhibitor of metalloproteinase-1 (TIMP1), a key matrix remodeling protein in isolated buccal
mucosal broblasts from patients with OSF, providing a
possible pathologic mechanism of OSF (79).
J Oral Pathol Med

The treatment with ANE induced the transition of

polygonal morphology into broblastoid, formation of
lamellipodia, and stress bers in NHOK and OECM-1 cells,
which indicated that ANE administration impaired cytoskeletal network of oral keratinocytes. Further mechanistic
analysis showed that active Rac and Rho GTPase and
phosphorylation of JNK could be important in regulating
morphological changes under ANE treatment (80). A recent
study deciphered that the stimulation of a muscarinic M4
receptor-mediated signaling cascade by ANE could also
promote the migration of OSCC cells. The increased cell
motility was regulated via the activation of Src and ERK
kinases (81). In both clinical and cellular model, investigations showed the association of areca-stimulated vimentin
expression, cell mobility, and progression of OSCC,
implying tumorigenic effect of ANE could be through
regulation of cytoskeletal proteins (82). Moreover, elevated
content of metal ion copper in the areca nut was detected in
OSF tissues, supporting the idea that copper is an initiating
factor for OSF while this pathological brogenic stimulation
may be mediated by the up-regulation of lysyl oxidase
(LOX) activity (83). The contribution of this metastaticrelated enzyme LOX using both in vitro and in vivo oral
cancer pathogenesis was further proved in a later study.
LOX was enhanced upon ANE treatment through AKT
activation. As the LOX expression was associated with cell
migration and invasion, and tumorigenesis, this indicated
that LOX facilitated oncogenic transformation in OSCC
cells (12). Taking the niche effect into account, the ANEactivated conditional medium of NHOF promoted various
malignant properties of OSCC cells, probably via the
paracrine activity of MMP-2 produced from NHOF (73). In
addition, the MMP-2 secreted by ANE-treated NHOF also
enhanced the invasiveness of polymorphonuclear leukocytes, indicating the relevance between inammatory processes with oral carcinogenesis in areca chewers (84).
Epigenetic manipulation and microRNA changes in arecanut-associated oral carcinogenesis
The molecular cues underlying the change in cellular
phenotype or gene expression other than the change in
gene sequences could be dened as epigenetic regulations.
A recent report implied that another class of regulatory
player of areca-nut-associated pathogenesis is via
epigenetic modulation. Using histological analysis, the
frequency of hypermethylation on Ras association domaincontaining protein 1A (RASSF1A) and p16 promoter in
OSCC and verrucous carcinoma (VC) tissues (93% and
63% in OSCC and 100% and 67% in VC for RASSF1A
and p16, respectively) was reported recently (85). Upon the
exposure of arecoline, human leukemia K-562 cells
exhibited epigenetic alterations based on the detection of
attenuation of a panel of genes catalyzing histone methylation, acetylation, and demethylation (86). In addition,
OSCC-related microRNAs (miRNAs) were also identied.
Once discovered, miRNAs have drawn lots of attention in
medical research not only because of their role in
regulating various physiological conditions but also due
to great potential to serve as biomarkers for early diagnosis
and prognosis of diseases (87, 88). miRNA is a class
of small single-stranded RNAs comprised of 22- to

Overview of diet-mediated oral cancer

Li et al.

25-nucleotide-long non-coding RNA that normally bind to

the 30 untranslated region (30 UTR) of their target mRNA,
leading to translational inhibition and/or mRNA degradation (89). The miRNAs are initially transcribed as long
primary miRNAs, which are processed to a hairpin
structure in the nucleus by RNase Drosha, and further
cleaved to miRNA duplexes by RNase Dicer in cytosol.
The miRNA duplexes are then unwound, and one strand,
termed mature miRNA or guide strand, which has
complimentarity to target mRNAs, is loaded into the
RNA-induced silencing complex. The complimentarity of
miRNAs to target RNAs renders the cleavage or the
translational repression of targeted mRNA (9092). Over
500 miRNAs have been discovered in human genome, and
it has been estimated that they could regulate 7492% of
all protein encoding mRNAs to a certain extent (93).
Considering the complex level of gene expression regulation conferred by miRNAs, miRNAs could regulate a wide
range of biological events including cell growth and
proliferation, differentiation, organogenesis, metabolism,
stress response, stemness, tissue remodeling, and onset/
progression of different diseases such as cancer, cardiovascular diseases, and diabetes through epigenetic regulation (9496).
In general, oncogenic miRNAs are usually overexpressed, driving tumor initiation and progression; tumor
suppressive miRNAs are down-regulated or deleted facilitating cancer development (97). The miRNA expression
proles appear to be tumor- and tissue specic (98). As for
OSCC development, many miRNAs play important roles in
regulating cell malignancy (99, 100). The dysregulation of
these cancerous miRNAs in OSCC induces cell proliferation and anti-apoptosis, promotes cancer metastasis, and
mediates resistance to chemotherapy (94). Among them,
OSCC-related oncogenic miRNAs included miR-21, miR31, miR-184, and miR-221, while let-7, miR-1, miR-133a,
miR-138, miR-145, and miR-375 have shown to be tumor
suppressive (101). The activation of the hypoxia pathway is
important for the pathogenesis of OSCC. ANE up-regulates
hypoxia-inducible factor 1a (HIF1a) in OSCC cells (102).
MiR-31 was identied to be important for OSCC progression by impeding factor-inhibiting hypoxia-inducible factor
to activate HIF based on different in vitro and in vivo assays
(103). Further study showed that miR-210 also targeted
hypoxic markers, including HIF-1a, TWIST, and carbonic
anhydrase IX (CA IX), to correlate tumor recurrence and
reduced survival rate (104). Very recently, studies also
revealed that that ANE could trigger aberrant miRNA
expression. For instance, ANE-induced miR-23a expression
correlated with increased DNA damage proteins and
down-regulated DNA double-strand breaks repair machinery. This ANE-stimulated miR-23a was mediated with a
reduced expression of Fanconis anemia susceptibility genes
(FANCG) that participate in a double-strand break repair
pathway to prevent chromosomal aberrations (105).
Furthermore, ANE treatment up-regulates miR-146a
expression, likely through the activation of NF-jB (106).
Overall, further examination would be required to elucidate
the ANE-induced miRNA expression prole for better
identication of the role of diet-induced miRNA alteration
in oral carcinogenesis.

Areca-nut-induced autophagy
Autophagy is a catabolic mechanism in which cells degrade
unnecessary organelles to ensure survival in adverse
environments. In the regards to long-term incubation of
areca consumption in vivo, a more recent study further
demonstrated that low-dose ANE treatment in OSCC cells
induced oxidative stress and up-regulated HIF1a, and this
therefore led to autophagy (102). The results showed that
10 lg/ml ANE administration on SAS OSCC cells resulted
in acidic vesicles evidenced by acridine orange staining
analysis, LC3-II accumulation, and autophagosomes
genesis. While the blockage of ROS, HIF1a induction,
p38-mediated signal activity, and other events decreased
ANE-induced autophagy. Taken together, the incubation of
ANE in OSCC cells exhibited a protective effect to decrease
cell apoptosis, while ANE treatment induced various
mitogen-mediated signaling pathways, leading to the promotion of cell malignancy. Interestingly, knockdown of
Aurora A oncogene in OSCC cells also resulted in the
autophagy, implicating that this inhibition might be
important for oral cancer therapy (107).

171

Other molecular cues underlying areca-mediated

oncogenesis
Numbers of studies have sought to explore detailed cellular
and molecular events after treatment of areca-nut-associated
constitutes. The effect of ANE treatment on primary
cultured oral cells was initially observed by the detection
of increased proto-oncogenes c-fos and c-jun in NHOF
(108). The correlation between p53 mutation and the
presence of safrole-DNA adducts in oral cancer patients
with areca-nut-chewing history supporting that safrole could
be one of the key players causing p53 mutation (109). Other
studies revealed that ANE could induce an inammatory
response based on the detection of increased production of
COX-2, prostaglandin E2, interleukin 6, and tumor necrosis
factor a (110, 111), while a later study showed that ANEmediated COX-2 overexpression was probably evoked
through induction of mitogen-activated protein kinases
(e.g., JNK1, ERK, and p38) and NF-jB activation (112).
NF-jB activation was shown upon ANE treatment in arecaassociated OC3 cells (67), while similar results were
reported in non-areca-associated OSCC cell lines (112). A
more recent study linking the increased inammation,
reduced differentiation, and altered cell structural protein
organization to ANE. It showed that ANE could activate
NF-jB activation for MMP-9 up-regulation to decrease cell
viability and impede re-epithelization of normal oral
keratinocyte (113). The schematic diagram in Figure 1
summarizes the molecular elements that are known to be
involved in the neoplastic process of oral epithelium, which
are modulated by areca ingredients.

Non-areca-nut-associated oral carcinogenesis

In addition to areca nut, smoking and alcohol consumption
are two other main oncogenic risk factors for oral
tumor development (114, 115). The IARC identied more
than 60 carcinogens in cigarette smoke, and at least 16
carcinogens in unburned tobacco have also been
reported (116). The tobacco-specic nitrosamines including
J Oral Pathol Med

Overview of diet-mediated oral cancer

Li et al.

172

Areca nut
extract

Tobacco
ingredients
Tumor progression:
- ERK
Cox2
PGE2
Vimen n
- Akt
Vimen n
Cell mo lity
- NF-B
[ROS]
p38
HIF1
Autophagy
Curcumin
Tea polyphenols

Overdosed
Alcohol

Tumor ini a on:

- Genotoxicity
- NF-B
ROS
Cytotoxicity

MMP2

Areca nut
extract

Niche eect:
- Invasion
- Akt
Focal adhesion

Normal oral epithelium

Neoplas c oral
epithelium

ANE s mulated oral

fibroblasts

Premalignant oral
epithelium

Normal oral fibroblasts

Cancer associated
fibroblasts

Figure 1 Diagraphic illustration of dietary-associated oncogenic effects. The tumorigenic promoters including areca nut, tobacco, and alcohol could trigger
various cellular and molecular events key for oral cancer formation, while curcumin and tea polyphenols suppress cancerous phenotypes by antagonistic
mechanisms.

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N-nitrosonornicotine, polycyclic aromatic hydrocarbons (e.g.,

benzo[a]pyrene), and aromatic amines have the most potent
links to OSCC, mainly through induction of DNA adducts
(117). Ethanol itself is not carcinogenic in animal study, as its
metabolites such as acetaldehyde could cause DNA damage
leading to higher risk of OSCC. It is interesting to note that
the anatomic sites in close contact with alcohol are at higher
risk to develop tumors, indicating the facilitating role of
alcohol for oral tumor formation (118). Epidemiologically, a
recent large cohort analysis conducted by the International
Head and Neck Cancer Epidemiology (INHANCE) consortium showed the attributable risk of smoking and drinking
combined for cancer formation to be 64%, implying the joint
effect of these two components in major proportion of
patients with head and neck cancer (119). Bruguere et al.
(120) found relative risk value of 13.5 for OSCC when 100
159 g of alcohol was consumed daily, while men who both
smoke and drink are nearly 38 times more likely to develop
head and neck cancers than men who do neither (121). A
casecontrol study showed that the odds ratio was 123 times
for Taiwanese patients with OSCC who smoked, drank
alcohol, and chewed areca quid than non-users, suggesting
synergetic effect of alcohol and tobacco consumption in
areca nut chewers (122). The underlying molecular cues for
smoking and drinking are still poorly understood. Early
studies using PCR-single-strand conformation polymorphism and DNA sequencing to analyze the conserved exon
regions (exon 59) of p53 gene in OSCC specimens
identied that tobacco smoking drives an important contribution to p53 mutation, while alcohol might enhance these
mutagenic effects (109). Overall, alcohol consumption and
smoking could be dened as enhancers for areca-associated
oral carcinogenesis, although further analysis for molecular
players for this synergetic impact would be required.
J Oral Pathol Med

Anti-oncogenic dietary moleculestea

polyphenols and curcumin
From the aspect of chemoprevention, the use of natural
substances derived from diet could provide a safe strategy to
inhibit cancer with limited toxicity. Two such agents, green
tea and the spice curcumin, have been extensively investigated (123126). In general, tea polyphenols and curcumin
could inhibit oral malignancy via a number of cellular and
molecular mechanismsmost of those targets shared signals involved in areca-associated oral oncology (127129).
Recent studies determined that curcumin hinders arecoline
or ANE-associated oral cancer development, probably via
the regulation of heat-shock protein, HIF, or placenta
growth factor (130132).
Several studies identied the benets of using tea
polyphenols for suppression of cancer development, mainly
through its major constituent ()-epigallocatechin-3-gallate
(EGCG) (133). Using cultured OSCC cells, in vitro effects
of EGCG contained several key steps: (1) to inhibit cell
proliferation via ROS-mediated apoptosis induction and cell
cycle arrest as well as telomerase activity; (2) to modulate
transcription factors such as NF-jB and activator protein-1
(AP1); and (3) to reduce cell migration and invasion by
decreased expression of MMPs and urokinase plasminogen
activator serine protease (u-PA) (134136). As for animal
models of oral carcinogenesis, treatment with 0.6% green
tea reduced the number of visible tumors by 35% and
reduced tumor volume by 57% in the DMBA-induced
hamster buccal pouch cancers. Immunohistochemical analyses showed that tea increased the apoptotic index of the
tumors as well as decreased the proliferation index and
microvessel density (137). Recent examination giving green
tea polyphenols extracts to 4-nitroquinoline 1-oxide (4NQO) induced oral tongue carcinogenesis in rats was

Overview of diet-mediated oral cancer

Li et al.

carried out. The decreased average number and volume of

tumors were detected in test groups compared with control
rats (138). Despite all experimental results supporting the
anticancer effect of using tea polyphenols for oral carcinogenesis, different clinical trials showed inconsistent outcomes, indicating the potential discrepancies between
human and rodents, or the needs to select more suitable
subset of subjects for clinical trial (139, 140).
Curcumin is one of the major phenolic antioxidant and
anti-inammatory agents in the spice turmeric (Curcuma
longa). There are three major curcuminoids that constitute
curcumin: curcumin (curcumin I, 75%), demethoxycurcumin (curcumin II, 20%), and bisdemethoxycurcumin
(curcumin III, 5%) (141). Early studies have demonstrated
the activity of curcumin to repress different cancer growth,
including skin and gastrointestinal tract using animal
models (142, 143). The mechanistic analysis indicated
that curcumin had versatile potential to suppress cancer
growth by down-regulation of proto-oncogene expression
and protein kinase activity, suppressed inammation,
inhibition of angiogenesis, and DNA-mutation-mediated
genetic instability (144). Various cell culture studies have
shown that curcumin induced apoptosis in malignant cells
by inhibition of different transcription factors and secondary messengers such as NF-jB, AP1, c-Jun, and JakSTAT signal (145, 146). In addition, curcumin targeted
elements in the mTOR pathway for cancer abrogation
(147). Curcumin also increased the levels of several
endogenous antioxidants via the Nrf2 pathway to
strengthen self-defense mechanism against ROS (148).
More recent investigations demonstrated that curcumin
regulated ANE-associated tumorigenic phenotypes, including autophagy, cell mobility, IGFBP-5-mediated mitogenic
signaling activity, and broblast-tumor cell interaction,
leading to decreased OSCC development (23, 113, 149,
150). Curcumin is also proven for its potential to attenuate
carcinogenesis induced by chemical carcinogens, at both
initiation and progression stages under various preclinical
studies (151). In addition, a recent study identied that
curcumin increased miR-145 expression and decreased
Sox9-mediated regulation cascade, which underlie the
elimination of stemness in OSCC (152). However, there
is an urgent need to validate the efcacy of curcumin in
adjuvant therapy or in prevention of oral carcinogenesis.

Conclusions and future perspective

Oral neoplastic formation is the cellular process in response
to numbers of aberrant genetic and epigenetic regulations
resulting from environmental challenges. Among all oncogenic stimuli, areca nut ingredients play an essential role in
regulating oncogenic phenotypes. It could be concluded that
areca nut induced cytotoxicity and genotoxicity in normal
oral epithelium leading to greater susceptibility for tumor
formation. Prolonged incubation of areca nut ingredients
provides substantial oncogenic inputs resulting in cellular
transformation of normal oral epithelium into malignant
cells, mainly by the activation of various intrinsic tumorigenic signals, including ERK, AKT, and NF-jB pathways.
Interestingly, the external oncogenic contributions from
surrounding stroma for cancer progression have been also

recognized. In contrast, the studies showed that curcumin

and tea polyphenols play antagonistic roles to abrogate oral
carcinogenesis by targeting areca-nut-related molecular cues
(Figure 1).
Although much effort has been made to uncover detailed
molecular cues of diet-mediated carcinogenesis, how to
dene better experimental models in order to truly recapitulate physiological pharmacokinetics of these diet compounds on either normal or malignant oral cells remains a
big challenge. Moreover, the limited success of chemopreventive natural compounds in the clinical setting due
probably to their poor bioavailability, rapid rate of metabolism, or both, restrict the application in medical practice
would also need to be addressed. A new strategy for
development of optimal delivery systems to bypass poor
oral bioavailability as well as to reduce hepatic metabolism
to reach an effective therapeutic concentration in the blood
stream would be a key in the future to improve the success
of OSCC interception.

173

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Acknowledgements
This work is supported by Grants NSC102-2314-B-010-010 and NSC102-2628B-010-011-MY3 from National Science Council, Taiwan, as well as Grants
99TPECH06 and 100TPECH06 from Taipei City Hospital/Department of
Health, Taipei City Government. We also would like to thank Ms Courtney
Anne Curtis for critical review and English edition for the manuscript.

J Oral Pathol Med