DOI:http://dx.doi.org/10.7314/APJCP.2015.16.12.4949
Sex Hormone Receptor Gene Expression and the Predominance of Hepatocellular Carcinoma in Males
RESEARCH ARTICLE
Implications of Sex Hormone Receptor Gene Expression in the
Predominance of Hepatocellular Carcinoma in Males: Role of
Natural Products
Hanaa H Ahmed1*, Wafaa Gh Shousha2, Aziza B Shalby1, Hatem A El-Mezayen2,
Nora N Ismaiel3, Nadia S Mahmoud1
Abstract
The present study was planned to investigate the role of sex hormone receptor gene expression in the
pathogenesis of hepatocellular carcinoma (HCC). Adult male Wistar rats were divided into seven groups. Group
(1) was negative control. Groups (2), (5), (6), and (7) were orally administered with N-nitrosodiethylamine for
the induction of HCC, then group (2) was left untreated, group (5) was orally treated with curcumin, group
(6) was orally treated with carvacrol, and group (7) was intraperitoneally injected with doxorubicin, whereas
groups (3) and (4) were orally administered only curcumin and carvacrol, respectively. The HCC group showed
significant upregulation in the androgen receptor (AR) and the estrogen receptor-alpha (ERα) gene expression
levels in the liver tissue. On the contrary, HCC groups treated with either curcumin or carvacrol showed
significant downregulation in AR and ERα gene expression levels in the liver tissue. In conclusion, the obtained
data highlight that both AR and ERα but not estrogen receptor-beta (ERβ) gene expression may contribute to
the male prevalence of HCC induced in male rats. Interestingly, both curcumin and carvacrol were found to
have a promising potency in alleviating the male predominating HCC.
Keywords: Hepatocellular carcinoma - male - androgen receptor - estrogen receptor - curcumin - carvacrol
Asian Pac J Cancer Prev, 16 (12), 4949-4954
Introduction
Hepatocellular carcinoma (HCC) incidence and
mortality have globally increased over the last four
decades (Abdelaziz et al., 2014; Zekri et al., 2015). Liver
cancer is the fifth most common cancer in males, and the
seventh most commonly diagnosed cancer in females (Gao
et al., 2012; Zhong et al., 2014).
In fact, HCC has a remarkable increased predominance
in males, with a male to female ratios averaging between
2:1 and 4:1 in most published series (Nordenstedt et al.,
2010; Zekri et al., 2015). Not only males develop HCC
more often than females, but once they develop it, they
also die easier than females. In general, the prognosis for
the disease is more benign in females than in males and
women have a better survival and a reduced recurrence
of the disease after the treatment (El-Serag et al., 2001).
From all these evidences, the interest of some researchers
has been focused since the 80’s on the possible importance
of sex hormones in determining such preference for the
male gender (Maria et al., 2002).
The expression and functional status of the androgen
receptor (AR) and the estrogen receptor (ER) appear to
play a significant role in the carcinogenesis of all hormones
sensitive organs. The expression and activation of these
receptors were also reported to be greatly increased in the
liver tissues of male and female rodents during chemically
induced liver carcinogenesis (Maria et al., 2002).
Curcumin, a natural polyphenol found in the dietary
spice turmeric used traditionally in medicine in India
and other Asian countries, has been demonstrated to
have anticancer activity, including prevention of tumor
initiation, promotion, metastasis, and angiogenesis in
experimental animal models, against a wide range of
tumor cells without promoting the development of side
effects (Yallapu et al., 2013).
Carvacrol is a monoterpenic phenol found in the
essential oils extracted from aromatic plants such as,
thyme and oregano (Esiyok et al., 2004). Carvacrol is
widely used as a food flavoring agent, a food preservative
agent and a cosmetic ingredient. Recently, many studies
have shown that carvacrol possess a variety of biological
and pharmacological properties including antioxidant,
antibacterial, antifungal, anticancer, anti-inflammatory
and hepatoprotective activities (Suntres et al., 2015).
Our previous studies have documented the antitumor
1
Hormones Department, Medical Research Division, National Research Centre, Dokki, Giza, Egypt, 2Chemistry Department, Faculty
of Science, Helwan University, Cairo, Egypt; 3Molecular Genetics and Enzymology Department, Human Genetics and Genome
Research, National Research Centre, Dokki, Giza, Egypt *For correspondence: Hanaaomr@yahoo.com
Asian Pacific Journal of Cancer Prevention, Vol 16, 2015
4949
Hanaa H Ahmed et al
efficacy of curcumin and carvacrol against HCC induced
chemically in rats. The effect of these natural products
was achieved via hindering the tumor cells proliferation,
promoting apoptosis and inhibiting the angiogenesis
(Ahmed et al., 2013; Ahmed et al., 2015).
The current study was constructed to evaluate the role
of sex hormone receptors (AR and ER) gene expression
in the pathogenesis of chemically induced hepatocellular
carcinoma in male rats and shed light on the potential
significance of curcumin and carvacrol in mitigating the
male predominant HCC with special concern on their
possible mechanisms of action.
for six weeks, as previously reported by Jayakumar et
al. (2012); and (7) Doxorubicin-treated group, in which
rats were intraperitoneally injected with doxorubicin as
a reference drug with a dose of 0.72 mg/rat, which is
equivalent to the human dose of 40 mg/m2, according
to equation of Barnes and Paget (1965) once a week for
15 weeks, following the administration of NDEA for six
weeks.
After the experimental period, rats were fasted
overnight and sacrificed by cervical displacement under
diethyl ether anesthesia, then the liver was dissected and
snap-frozen immediately in the liquid nitrogen and stored
at -80°C for the gene expression analysis.
Materials and Methods
Chemicals and reagents
N-nitrosodiethylamine (NDEA) (CAS no. 55-18-5),
Curcumin (Cat# C1386), and carvacrol (Cat# 282197)
were purchased from Sigma-Aldrich Chemicals Co. (St
Louis, MO, USA). Doxorubicin was purchased from
Pharmacia Italia S.P.A, Milan, Italy. All other chemicals
used in the experiment were locally purchased (Egypt)
and were of high analytical grade.
Animals and experimental design
This study was conducted on fifty six adult male Wistar
rats weighing 180-200 g purchased from the animal house
colony of the National Research Centre, Cairo, Egypt.
The rats were kept under strict hygienic conditions and
allowed to feed on a standard rodent chow and water. Rats
were adapted to these conditions for two weeks before
beginning the experiment. The animal experimental
protocol was approved by the Ethical Committee for
Medical Research, National Research Centre, Egypt (No.
12110).
After the acclimation period, the animals were
randomly divided into seven groups with eight rats per
group as follows: (1) Healthy rats served as a negative
control group and were orally received 1-ml vehicle
solution (Tween-80 (1%) in 0.9% normal saline) /rat,
five times a week during the whole experimental period
(21 weeks); (2) HCC group, in which rats were orally
administered NDEA with a dose of 20 mg/kg b.wt. , five
times a week for six weeks, according to the modified
method of Darwish and El-Boghdady (2011); (3)
Curcumin control group, in which rats were orally treated
with curcumin only (dissolved in Tween-80 (1%) in 0.9%
normal saline) with a dose of 100 mg/kg b.wt. (five days a
week) for 15 weeks, as previously reported by Sreepriya
and Bali (2005); (4) Carvacrol control group, in which
rats were orally treated with carvacrol only (dissolved in
Tween-80 (1%) in saline) with a dose of 15 mg/kg b.wt.
(five days a week) for 15 weeks, as previously reported
by Jayakumar et al. (2012); (5) Curcumin-treated group,
in which rats were orally treated with curcumin with a
dose of 100 mg/kg b.wt. , five times a week for 15 weeks,
following the administration of NDEA for six weeks, as
previously reported by Sreepriya and Bali (2005); (6)
Carvacrol-treated group, in which rats were orally treated
with carvacrol with a dose of 15 mg/kg b.wt. , five times a
week for 15 weeks, following the administration of NDEA
4950
Asian Pacific Journal of Cancer Prevention, Vol 16, 2015
Gene expression method
RT-PCR analysis: The total RNA was extracted
from the liver tissue of rats in each group using
TRIzol reagent (Bioshop Canada Inc.). Isolated RNA
was reverse transcribed into complementary DNA
(cDNA) using Revert Aid first strand cDNA synthesis
kit (Fermentas Co., USA). The subsequent PCR was
performed using 5 µg of cDNA in a final volume of 20
μl containing 10x PCR buffer, 10mM dNTPs, 5U/μl of
Taq DNA polymerase (Fermentas, USA), and 10µM
of each specific primer. Glyceraldehyde 3 phosphate
dehydrogenase (GAPDH) (Metabion-Germany) was
used as internal control with primer sequences: forward,
5`CAAGGTCATCCATGACAACTTTG3`, and reverse,
5`GTCCACCACCCTGTTGCTGTAG3. Primer sequences
for androgen receptor (AR) (Metabion-Germany)
were forward, 5`GTGTCGTCTCCGGAAATGTT3`,
and reverse, 5`GGAATCAGGCTGGTTGTTGT3`
according to Staub et al. (2005) published sequence,
while primer sequences for ERα (Metabion-Germany)
were forward, 5`AGTGAAGCCTCAATGATGGG
3`and, reverse, 5`CAAAGATCTCCACCATGCCT3`
according to Staub et al. (2005) published sequence
and those for ERβ (Metabion-Germany) were forward,
5`CTACTGAACGCGGTGACAGA3`, and reverse,
5`CGTGTGAGCATTCAGCATCT3` according to Staub
et al. (2005) published sequence. The PCR cycling was
performed using a gradient thermal cycler (BioRad, USA)
as follows: initial denaturation at 94ºC for 5 minutes,
followed by denaturation at 94ºC for 30 seconds was
done. Amplification was carried out using 35 cycles with
an annealing temperature at 58ºC for AR and GAPDH
and at 55ºC for ERα and ERβ for 30 seconds, followed
by extension at 72ºC for 1 minute and a final extension at
72ºC for 8 minutes. The PCR products were separated on
2% agarose gel and visualized by the gel documentation
system. The amplified products’ size was determined by
comparison to a DNA ladder (100 bp) (Fermentas, USA).
All gene expression levels were semiquantified using
LabImage analysis (LabImage2.7.0, Kapelan GmbH)
software and were normalized against GAPDH gene
expression.
Statistical analysis
All results were represented as the mean + SE of the
mean. Data were analyzed by one-way analysis of variance
(ANOVA) using the SPSS software program (version
DOI:http://dx.doi.org/10.7314/APJCP.2015.16.12.4949
Sex Hormone Receptor Gene Expression and the Predominance of Hepatocellular Carcinoma in Males
14.0; SPSS Inc., Chicago, IL, USA) followed by the least
significant difference (LSD) to compare the significance
between the groups. P-values<0.05 were considered
statistically significant.
Results
Gene expression results
AR gene expression: As shown in Figure 1 and
Table 1, the results showed that there was a significant
overexpression of the AR gene (p<0.05) in the liver tissue
of the HCC group (lane 2) compared with that in the
negative control group (lane 1). The AR gene showed low
expression levels in the liver tissue of rats in the curcumin
control group (lane 6) and the carvacrol control group
(lane 7). There was an insignificant change (p>0.05) in
the AR gene expression level in the liver tissue of rats in
curcumin or carvacrol control group compared with that
in the negative control group. The AR gene expression
level was shown to be significantly downregulated
(p<0.05) in the liver tissue obtained from rats in the
curcumin-treated group (lane 4) and in the liver tissue of
rats in the carvacrol-treated group (lane 5) as compared to
that in the untreated HCC group. Similarly, it was found
to be significantly downregulated (p<0.05) in the liver
tissue of rats in the doxorubicin-treated group(lane 3) as
compared to that in the liver tissue of rats in the untreated
HCC group.
ERα gene expression
As indicated in Figure 2 and Table 1, the results
Figure 1. Agarose Gel Electrophoresis Shows AR
mRNA Expression in the Liver Tissue by RT-PCR
Analysis. GAPDH expression with 496 bp, AR expression
with 266 bp. Negative control group (Lane 1), HCC group (Lane
2), doxorubicin-treated group (Lane 3), curcumin-treated group
(Lane 4), carvacrol-treated group (Lane 5), curcumin control
group (Lane 6), carvacrol control group (Lane 7). DNA ladder
(100 bp) (Lane M)
Table 1. The Gene Expression Level of AR and ERα in
the Liver Tissue of Rats in the Different Groups
Groups
Negative control group
HCC group
Doxorubicin-treated group
Curcumin-treated group
Carvacrol-treated group
Curcumin control group
Carvacrol control group
AR expression
ERα expression
0.4830±0.003
0.1308±0.09506
0.9745±0.03907a 0.5320±0.01504a
0.6498±0.02064b No expression
0.2303±0.00862b No expression
0.4110±0.00881b No expression
0.2325±0.02346 No expression
0.3473±0.02315 No expression
Values are expressed as the mean ± standard error (SE) of mean for four
samples/ group; a: Significant change at p<0.05 in comparison with the
negative control group; b: Significant change at p<0.05 in comparison
with the HCC group; HCC hepatocellular carcinoma
Figure 2. Agarose Gel Electrophoresis Shows ERα
mRNA Expression in the Liver Tissue by RT-PCR
Analysis. GAPDH expression with 496 bp, ERα expression
with 281 bp. Negative control group (Lane 1), HCC group (Lane
2), doxorubicin-treated group (Lane 3), curcumin-treated group
(Lane 4), carvacrol-treated group (Lane 5), curcumin control
group (Lane 6), carvacrol control group (Lane 7).DNA ladder
(100 bp) (Lane M)
Figure 3. Agarose Gel Electrophoresis Shows ERβ
mRNA Expression in the Liver Tissue by RT-PCR
Analysis. GAPDH expression with 496 bp, ERβ expression
with 255 bp. Negative control group (Lane 1), HCC group (Lane
2), doxorubicin-treated group (Lane 3), curcumin-treated group
(Lane 4), carvacrol-treated group (Lane 5), curcumin control
group (Lane 6), carvacrol control group (Lane 7). DNA ladder
(100 bp) (Lane M)
revealed that there was a significant upregulation in the
ERα gene expression (p<0.05) in the liver tissue of the
HCC group (lane 2) compared with that in the liver tissue
of the negative control group (lane 1). The ERα gene was
not expressed in the liver tissue of rats in the curcumin
control group (lane 6) and the carvacrol control group
(lane 7). The ERα gene expression level was shown to
be decreased to an undetectable level in the liver tissue
obtained from rats in the curcumin-treated group (lane 4)
and in the liver tissue of rats in the carvacrol-treated group
(lane 5). Likewise, ERα gene expression was found to be
decreased to undetectable level in the liver tissue of rats
in the doxorubicin-treated group (lane 3).
ERβ gene expression
As illustrated in Figure 3, the results showed that
ERβ gene was not expressed in the liver tissue of rats
in the negative control group (lane 1) as well as in that
of the HCC group (lane 2). Similarly, there was no ERβ
expression in the liver tissue of rats in the curcumin control
group (lane 6) and the carvacrol control group (lane 7).
ERβ expression was shown to be absent in the liver tissue
obtained from rats in the curcumin-treated group (lane 4)
and in the liver tissue of rats in the carvacrol-treated group
(lane 5). Similarly, ERβ gene was not expressed in the liver
tissue of rats in the doxorubicin-treated group (lane 3).
Discussion
Hepatocellular carcinoma was found to show
Asian Pacific Journal of Cancer Prevention, Vol 16, 2015
4951
Hanaa H Ahmed et al
significantly higher prevalence in males, an effect related
to sex hormones (Li et al., 2012). Liver is a hormonesensitive organ that was found to express estrogen
receptors (ER) as well as androgen receptors (AR) in both
male and female patients (Villa et al., 2002).
Androgen receptor (AR) is critical for the development
and maintenance of the male sexual phenotype. It is a type
of nuclear receptor that regulates the gene transcription
when activated by the androgens (Heinlein and Chang,
2002).
The current study reveals a significant AR
overexpression in the liver tissue of the HCC group.
Indeed, these results are in line with those published by
Eagon et al. (1996) who reported that the expression and
activation of AR are greatly increased in the liver tissue
of male and female rodents during the chemical induction
of the liver carcinogenesis. Ma et al. (2008) reported that
the AR expression is elevated in the liver of the male
patients with HCC as compared to the healthy subjects.
Also, this study shows that the loss of hepatic AR in mice
bearing HCC resulted in the reduction of HCC incidence.
Moreover, this study suggested that the AR signaling
promotes hepatocarcinogenesis via the enhancement of
the cellular oxidative stress and DNA damage as well
as suppression of p53-mediated DNA damage/repairing
system and cell apoptosis.
AR was shown to regulate the expression of
transforming growth factor-beta 1 (TGF-β1) through
binding to the androgen response element on the TGF-β1
gene promoter, suggesting that such activation might
regulate the progression of HCC in both human and animal
models (Villa ,2009).
Recently, Awuah and Monga (2012) have elegantly
unveiled one of the mechanisms of sex-related disparity
of HCC depending on the existing finding that AR and
testosterone contribute to the HCC predominance in
males. It was found that the presence of androgens in
males engages AR to stimulate the cell cycle-related kinase
(CCRK) expression. Then CCRK activates β-catenin
signaling which in turn stimulates the expression of
the epidermal growth factor receptor and cyclin-D1
thus promoting the cell proliferation. These authors
also suggested a mechanism by which Wnt/β-catenin
signaling may cause tumor progression in males. β-catenin
activation in males with HCC leads to high expression
and activation of AR which in turn results in high CCRK
expression that stimulates a vicious cycle of cell growth
and proliferation.
The suggested mechanism for overexpression of AR
in the HCC group in the present study is related to the
inflammatory signaling associated with the chemical
induction of HCC by NDEA. Naugler et al. (2007) stated
that NDEA administration causes a greater increase in the
serum interleukin-6 (IL-6) concentration in male mice.
IL-6 has been found to induce the AR gene expression
level and activity via mitogen activated protein kinase
(MAPK) pathway in prostate cancer cells (Lin et al.,
2001).
The present results show that curcumin treatment
in HCC group significantly downregulated the AR
gene expression level in the liver tissue. This result is
4952
Asian Pacific Journal of Cancer Prevention, Vol 16, 2015
in accordance with that of Nakamura et al. (2002) who
reported that curcumin downregulates AR gene expression
and activation in the prostate cancer cell lines. Curcumin
was shown to inhibit the IL-6 production in human
multiple myeloma cells (Bharti et al., 2003). Hence, the
current study suggests that the downregulation of the AR
gene expression level due to the treatment of the HCC
group with curcumin may be attributed to the ability of
curcumin to reduce IL-6 production.
The results of the current study have shown that
carvacrol administration in the HCC group significantly
downregulates the AR gene expression level in the liver
tissue. This finding is in accordance with that of Alexander
and Ekunwe (2012) who demonstrated that the treatment
of the human prostate cancer cell (PC-3) with the aqueous
extract of Ocimum gratissimum leaf decreases the AR
gene expression level.
The suggested mechanism underlying the reduction of
the AR gene expression level by carvacrol could be due
to the significant reduction of IL-6 levels by carvacrol.
Such effect might be occurred through the attenuation of
the NF-κB pathway by carvacrol (Aristatile et al., 2013)
which in turn results in a reduction of IL-6.
The current results show that doxorubicin significantly
downregulated the AR expression in the liver tissue of rats
in the HCC group. This finding is in an agreement with
that of Rokhlin et al. (2006) who stated that doxorubicin
suppresses the AR gene expression in the prostate cancer
cell line. The underlying mechanism for this action of
doxorubicin is not known.
Two subtypes of estrogen receptor (ER), ERα and
ERβ, have been identified in the human liver tissue
(Moggs and Orphanides, 2001).
The present study demonstrates that the ERα was
significantly overexpressed in the liver tissue of rats in
the HCC group, and expressed at a low level in that of the
negative control group, whereas no expression of ERβ has
been found in the liver tissue from the normal or the HCC
group at all. These results are in an agreement with the
previous results reported by Alvaro et al. (2000).
The roles of the ERs have been widely investigated
in other cancers than hepatocellular carcinoma. For
example, the mutated ERα expression is involved in
the breast carcinogenesis (Hayashi and Yoshida, 2000),
while the ERβ expression suppresses the breast cancer
cell proliferation and tumor formation (Paruthiyil et
al., 2004). Although the precise role of ERα in HCC is
unknown (Huang et al., 2006), previous study of Waalkes
et al. (2004) suggested that the underlying basis of ERα
overexpression in the liver tissue of HCC-induced mice
may be related to the hypomethylation of the ERα
promoter which have been associated with the ERα
activation.
The role of ERα in the regulation of MAPK cascade
has been further studied in cell lines expressing ERα. It
was found that estrogen-bound ERα can rapidly activate
the ERK and AKT signal transduction pathways leading
to the cell cycle progression and inhibition of apoptosis
(Acconcia et al., 2005).
P53 gene mutations occur in the late stages of
hepatocarcinogenesis and have been found to be
DOI:http://dx.doi.org/10.7314/APJCP.2015.16.12.4949
Sex Hormone Receptor Gene Expression and the Predominance of Hepatocellular Carcinoma in Males
associated with the prognosis and survival of HCC
patients (Malaguarnera et al., 2010). Several lines of
evidence have suggested that p53 regulates the ERα
expression (Angeloni et al., 2004) and downregulates
ERα-responsive genes by interfering with the binding
of ERα to its response element (Jin et al., 2008). So, the
mutation of p53 gene could result in overexpression of
the ERα gene in HCC.
The current study shows that ERα gene expression
was decreased to undetectable level in the liver tissue
of the HCC group treated with curcumin. This result
is supported by a previous study conducted by Shao et
al. (2002) which revealed that curcumin decreases the
expression of the ER transcript in ER-positive (MCF-7)
human breast cancer cell line. Such effect may be as the
result of the upregulation of wild-type of the p53 gene
by curcumin, which is thought to downregulate ERα
expression. Also, the study of Liontas and Yeger (2004)
demonstrated that curcumin is found to upregulate the p53
gene expression level and induce its nuclear translocation
in neuroblastoma.
The present study shows that carvacrol administration
decreased the ERα gene expression to undetectable level
in the liver tissue of HCC group. This inhibitory effect
could be attributed to the induction of the p53 gene that
is responsible for the reduction in the gene expression
level of ERα. Al-Dhaheri et al. (2013) found that the
treatment of the breast cancer cell line (MDA-MB-231)
with the Origanum majorana extract, known to contain
carvacrol, results in downregulation of mutant p53 gene
with consequent upregulation of the wild-type p53.
The present work demonstrates that the ERα gene
expression was absent in the liver tissue of rats in the
HCC group administered doxorubicin. This result is
in accordance with that of Rusetskaya et al. (2009)
which showed that upon culturing of MCF-7 cells with
doxorubicin, a significant downregulation in the ER gene
expression level is observed.
The observed inhibition of the ERα gene expression
by doxorubicin may be due to activation of the p53 gene,
which is known to downregulate ERα. Liu et al. (2008)
reported that doxorubicin produces a cytotoxic effect in
H9c2 cells and neonatal rat cultured cardiomyocytes,
which is associated with the increased activation and
nuclear translocation of p53.
However, further studies are needed to address the exact
molecular mechanism underlying the downregulation of
sex hormone receptors expression level by these natural
products.
In conclusion, the current study emphasizes the
principal role of both AR and ERα but not estrogen
receptor-beta (ERβ) gene expression in the male
predominance of HCC in male rats. Interestingly, both
curcumin and carvacrol showed a great efficacy against
HCC.
References
Abdelaziz AO, Elbaz TM, Shousha HI, et al (2014). Survival
and prognostic factors for hepatocellular carcinoma: an
egyptian multidisciplinary clinic experience. Asian Pac J
Cancer Prev, 15, 3915-20.
Acconcia F, Totta P, Ogawa S, et al (2005). Survival versus
apoptotic 17beta-estradiol effect: role of ER alpha and ER
beta activated non-genomic signaling. J Cell Physiol, 203,
193-201.
Ahmed HH, Shousha WG, Shalby AB, et al (2013). In vivo
antitumor potential of carvacrol against hepatocellular
carcinoma in rat model. World journal of pharmacy and
pharmaceutical sciences, 2, 2367-96.
Ahmed HH, Shousha WG, Shalby AB, et al (2015). Curcumin:
a unique antioxidant offers a multimechanistic approach
for management of hepatocellular carcinoma in rat model.
Tumor Biol, 36, 1667-8.
Al-Dhaheri Y, Eid A, Abu Qamar S, Attoub S, et al (2013).
Mitotic arrest and apoptosis in breast cancer cells induced
by Origanum majorana extract: upregulation of TNF-α and
downregulation of survivin and mutant p53. Plos one, 8,
56649.
Alexander TC, Ekunwe SIN (2012). Investigating the molecular
mechanism of inhibition of proliferation of human prostate
cancer (PC-3) cells by fractionated Ocimum gratissimum
(og) leaf extracts. 9thInternational symposium on recent
advances in environmental health research (Poster). Jackson,
USA, p.42.
Alvaro D, Alpini G, Onori P, et al (2000). Estrogens stimulate
proliferation of intra-hepaticbiliary epithelium in rats.
Gastroenterology, 119, 1681-91.
Angeloni SV, Martin MB, Garcia-Morales P, et al (2004).
Regulation of estrogen receptor-alpha expression by the
tumor suppressor gene p53 in MCF-7 cells. J Endocrinol,
180, 497-504.
Aristatile A, Al-Assaf AH, Pugalendi KV (2013). Carvacrol
suppresses the expression of inflammatory marker genes in
D-galactosamine-hepatotoxic rats. Asian Pac J Trop Med,
6, 205-11.
Awuah PK, Monga SP (2012). Cell cycle-related kinase links
androgen receptor and β-catenin signaling in hepatocellular
carcinoma: Why are men at a loss? Hepatology, 55, 970-4.
Barnes JM, Paget GE (1965). Mechanisms of toxic action. Prog
Med Chem, 4, 18-38
Bharti AC, Donato N, Aggarwal BB (2003). Curcumin
(diferuloylmethane) inhibits constitutive and IL-6-inducible
STAT3 phosphorylation in human multiple myeloma cells.
J Immunol, 171, 3863-71.
Darwish HA, El-Boghdady NA (2011). Possible involvement
of oxidative stress in diethylnitrosamine induced
hepatocarcinogenesis: chemopreventive effect of curcumin.
J Food Biochem, 37, 353-61.
Eagon PK, Elm MS, Epley MJ, et al (1996). Sex steroid
metabolism and receptor status in hepatic hyperplasia and
cancer in rats. Gastroenterology, 110, 1199-1207.
El-Serag HB, Mason AC, Key C (2001). Trends in survival of
patients with hepatocellular carcinoma between 1977 and
1996 in the United States. Hepatology, 33, 62-5.
Esiyok D,Semih Ötles S, Akcicek E (2004). Herbs as a Food
Source in Turkey. Asian Pac J Cancer Prev, 5, 334-9.
Gao J, Xie L, Yang W, et al (2012). Risk factors of hepatocellular
carcinoma - current status and perspectives. Asian Pac J
Cancer Prev, 13, 743-52.
Hayashi S, Yoshida T (2000). Regulation of ER (estrogen
receptor) gene expression in the genesis and development
of breast cancer. Nippon Rinsho, 58, 495-503.
Heinlein CA, Chang C (2002). Androgen receptor (AR)
coregulators: an overview. Endocr Rev, 23, 175-200.
Huang E, Wu C, Lee S, et al (2006). Opposing action of estrogen
receptors α and β on tumor necrosis factor-α gene expression
and Caspase-8-mediated apoptotic effects in HA22Tcells.
Asian Pacific Journal of Cancer Prevention, Vol 16, 2015
4953
Hanaa H Ahmed et al
Mol Cell Biochem, 287, 137-45.
Jayakumar S, Madankumar A, Asokkumar S, et al (2012). Potential
preventive effect of carvacrol against diethylnitrosamineinduced hepatocellular carcinoma in rats. Mol Cell Biochem,
360, 51-60.
Jin W, Chen Y, Di GH, et al (2008). Estrogen receptor
(ER) {beta} or p53 attenuates ER {alpha}-mediated
transcriptional activation on the BRCA2 promoter. J Biol
Chem, 283, 29671-80.
Li Z, Tuteja G, Schug J, Kaestner KH (2012). Foxa1 and Foxa2
are essential for sexual dimorphism in liver cancer. Cell,
148, 72-83.
Lin D, Whitney MC, Yao Z, et al (2001). Interleukin-6 induces
androgen responsiveness in prostate cancer cells through
up-regulation of androgen receptor expression. Clin Cancer
Res, 7, 1773-81.
Liontas A, Yeger H (2004). Curcumin and resveratrol induce
apoptosis and nuclear translocation and activation of p53 in
human neuroblastoma. Anticancer Res, 24, 987-98.
Liu J, Mao W, Ding B, Liang C (2008). ERKs/p53 signal
transduction pathway is involved in doxorubicin-induced
apoptosis in H9c2 cells and cardiomyocytes. Am J Physiol
Heart Circ Physiol, 295, 1956-65.
Ma W, Hsu C, Wu M, et al (2008). Androgen receptor is a new
potential therapeutic target for the treatment of hepatocellular
carcinoma. Gastroenterology, 135, 947-55.
Malaguarnera G, Giordano M, Paladina I, et al (2010). Serum
markers of hepatocellular carcinoma. Dig Dis Sci, 55,
2744-55.
Maria ND, Manno M, Villa E (2002). Sex hormones and liver
cancer. Mol Cell Endocrinol, 193, 59-63.
Moggs JG, Orphanides G (2001). Estrogen receptors:
orchestrators of pleiotropic cellular responses. EMBO Rep,
2, 775-81.
Nakamura K, Yasunaga Y, Segawa T, et al (2002). Curcumin
downregulates AR gene expression and activation in prostate
cancer cell lines. Int J Oncol, 21, 825-30.
Naugler WE, Sakurai T, Kim S, et al (2007). Gender disparity
in liver cancer due to sex differences in MyD88-dependent
IL-6 production. Science, 317, 121-4.
Nordenstedt H, White DL, El-Serag HB (2010). The changing
pattern of epidemiology in hepatocellular carcinoma. Dig
Liver Dis, 42, 206-14.
Paruthiyil S, Parmar H, Kerekatte V, et al (2004). Estrogen
receptor beta inhibits human breast cancer cell proliferation
and tumor formation by causing a G2 cell cycle arrest.
Cancer Res, 64, 423-8.
Rokhlin OW, Glover RB, Guseva NV, et al (2006). Mechanisms
of cell death induced by histone deacetylase inhibitors
in androgen receptor–positive prostate cancer cells. Mol
Cancer Res, 4, 113-23.
Rusetskaya NV, Lukyanova NY, Chekhun VF (2009). Molecular
profile and cell cycle in MCF-7 and mcf-7/Dox cells exposed
to conventional and liposomal forms of doxorubicin. Exp
Oncol, 31, 140-3.
Shao Z, Shen Z, Liu C, et al (2002). Curcumin exerts multiple
suppressive effects on human breast carcinoma cells. Int J
Cancer, 98, 234-40.
Sreepriya M, Bali G (2005). Chemopreventive effects of embelin
and curcumin against N- nitrosodiethylamine/ phenobarbitalinduced hepatocarcinogenesis in Wistar rats. Fitoterapia,
76, 549-55.
Staub C, Rauch M, Ferrière F, et al (2005). Expression of
estrogen receptor ESR1 and its 46-kDa variant in the
gubernaculums testis. Biol Reprod, 73, 703-12.
Suntres ZE, Coccimiglio J, Alipour M (2015). The bioactivity
and toxicological actions of carvacrol. Crit Rev Food Sci
4954
Asian Pacific Journal of Cancer Prevention, Vol 16, 2015
Nutr, 55, 304-18.
Villa E (2009). A ndroge n re c e ptor a lte ra tions i n
hepatocarcinogenesis. Digest Liver Dis, 41, 632-3.
Villa E, Grottola A, Colantoni A, et al (2002). Hepatocellular
carcinoma: role of estrogen receptors in the liver. Ann NY
Acad Sci, 963, 37-45.
Waalkes MP, Liu J, Chen H, et al (2004). Estrogen signaling in
livers of male mice with hepatocellular carcinoma induced
by exposure to arsenic in utero. J Natl Cancer Inst, 96,
466-74.
Yallapu MM, Jaggi M, Chauhan SC (2013) Curcumin
nanomedicine: a road to cancer therapeutics. Curr Pharm
Des. 19, 1994-2010.
Zekri A , Youssef AS, Bakr YM, et al (2015). Serum biomarkers
for early detection of hepatocellular. carcinoma associated
with HCV infection in egyptian patients. Asian Pac J Cancer
Prev, 16, 1281-7
Zhong C, Li H, Liu D, et al (2014). Clinical study of hepatectomy
combined with jianpi huayu therapy for hepatocellular
carcinoma. Asian Pac J Cancer Prev, 15, 5951-7