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Biochemical/metabolic changes associated with hepatocellular carcinoma
development in mice
Article in Tumor Biology · February 2014
DOI: 10.1007/s13277-014-1714-6 · Source: PubMed
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Biochemical/metabolic changes associated
with hepatocellular carcinoma development
in mice
Nahla E. El-Ashmawy, Hoda A. ElBahrawy, Maha M. Shamloula & Ola
A. El-Feky
Tumor Biology
Tumor Markers, Tumor Targeting and
Translational Cancer Research
ISSN 1010-4283
Tumor Biol.
DOI 10.1007/s13277-014-1714-6
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Author's personal copy
Tumor Biol.
DOI 10.1007/s13277-014-1714-6
RESEARCH ARTICLE
Biochemical/metabolic changes associated with hepatocellular
carcinoma development in mice
Nahla E. El-Ashmawy & Hoda A. El-Bahrawy &
Maha M. Shamloula & Ola A. El-Feky
Received: 15 December 2013 / Accepted: 29 January 2014
# International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related mortality after lung and stomach
cancers. This work was undertaken to investigate some of the
biochemical mediators/pathways associated with or implicated in the pathogenesis of HCC. Male albino mice were classified into two groups: normal control group and HCC group.
Early stage HCC was induced by injection of
diethylnitrosamine (DEN) i.p. 200 mg/kg as a single dose,
and after 2 weeks, the mice were given i.p. injection of
thioacetamide (TAA) 100 mg/kg twice per week for 4 weeks.
Mice were left for further 2 weeks without any treatment, after
which, mice were sacrificed; blood and liver samples were
collected. Serum was used for determination of activities of
glucose-6-phosphate dehydrogenase (G6PDH) and aldolase
as well as levels of insulin-like growth factor-1 (IGF-1) and
epithelial cadherin (E-cadherin). One portion of the liver was
used for histopathological examination and immunohistochemical staining of the tumor suppressor p53 protein. Another portion of the liver was used for determination of citrate
synthase activity. Induction of HCC in mice resulted in significant increase in G6PDH and aldolase activities, and Ecadherin level, but significant decrease in IGF-1. HCC mice
group showed moderate expression of p53 protein. These
results suggest that the molecular pathogenesis of HCC in
mice involves reduction of serum level of IGF-1 and increased
serum level of E-cadherin accompanied by dysregulation of
N. E. El-Ashmawy : H. A. El-Bahrawy : O. A. El-Feky (*)
Department of Biochemistry, Faculty of Pharmacy, Tanta University,
Tanta, Egypt
e-mail: dr_ola23@yahoo.com
M. M. Shamloula
Department of Pathology, Faculty of Medicine, Tanta University,
Tanta, Egypt
p53 protein expression. HCC was also associated with
reprogrammed metabolic profile shifted toward increased glycolysis and lipogenesis.
Keywords Hepatocellular carcinoma . IGF-1 . E-cadherin .
p53 . G6PDH . Aldolase
Introduction
Hepatocellular carcinoma (HCC) is the most common cause
of primary liver neoplasms and the fourth most frequent type
of cancer worldwide following lung, breast, and bowel cancers with an increasing incidence, causing one million deaths
per year [10].
A study conducted by Cairo Liver Center in 2010 revealed
that HCC has nearly doubled over the last decade, and there is
a growing incidence of HCC in Egypt (10–120 cases/
100,000), which represents the leading cause of death among
all other cancer sites [11].
Several factors contribute to the pathogenesis of HCC;
hepatitis B and C account for more than 70 % of HCC
worldwide. Additional etiological factors include toxins and
drugs (e.g., alcohol, aflatoxins, anabolic steroids, and vinyl
chloride), metabolic liver diseases (e.g., hereditary hemochromatosis, α1-antitrypsin deficiency), steatosis, nonalcoholic
fatty liver disease, and diabetes [7].
Different genes have been implicated in
hepatocarcinogenesis including genes involved in growth inhibition and apoptosis (e.g., tumor suppressor gene; p53), and
genes responsible for cell-cell interaction and signal transduction. Mutations in the p53 tumor suppressor gene are among
the most common alterations which play an important role in
either initiation or progression of HCC [7].
Hepatocellular carcinoma also showed resistance to apoptosis mediated by death receptors such as Fas receptor. The
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majority of the HCCs show one or more alterations in the Fas
pathway molecules, which inhibit Fas-mediated apoptosis.
Loss of response to Fas in HCC cells may be produced either
by downregulation of Fas expression, concomitant with decreased expression of downstream molecules, such as Fasassociated protein with death domain (FADD), or by upregulation or overactivation of molecules that counteract its proapoptotic effect, including nuclear factor kappa B (NF-κB),
Bcl-2–associated X protein (Bcl-2), or B-cell lymphoma-extra
large (Bcl-xl) [33].
The development of HCC is a multistage process. In the
molecular aspect, dysregulation of pleiotropic growth factors
(e.g., IGF-1, transforming growth factor-alpha, hepatocyte
growth factor, and EGF), adhesive molecules (e.g. Ecadherin), and metabolic pathways represents a central protumorigenic principle in human hepatocarcinogenesis [9, 45].
Some autocrine signal activators, such as epidermal growth
factor receptor (EGFR) ligands, might protect liver tumor cells
from apoptosis induced by stress, physiological factors, or
pro-apoptotic drugs. Dysregulation of growth factor signaling,
including EGF and IGF-1 pathways, has been well established
in human HCCs [2].
The transforming growth factor-beta (TGF-β) family of
cytokines plays a physiological role during embryonic development, and its misregulation can result in tumorigenesis.
TGF-β1 is an important regulatory suppressor factor in hepatocytes, inhibiting proliferation and inducing cell death. Paradoxically, TGF-β may also modulate other pro-tumorigenic
processes, such as cell invasion, immune regulation, or microenvironment modification. The escape from the antiproliferative and pro-apoptotic actions of TGF-β might be a prerequisite for hepatocarcinoma progression [25].
This study was conducted to identify some of the pathogenetic mediators or molecular mechanisms implicated in HCC
chemically induced in mice.
Materials and methods
Experimental design
Sixty male albino mice were utilized in this study, 15–30 g
each. Mice were purchased from the animal house of Giza
Institute of Ophthalmology, Cairo, Egypt. Mice were weighed
and housed in wire cages for 2 weeks under identical environmental conditions for adaptation and allowed free access to
balanced laboratory diet and water ad libitum. The diet contains about 54 % carbohydrates, 37.2 % protein, and 6 % fat
[15]. After acclimatization period, mice were weighed and
randomly divided into two groups: group 1: normal control
group, 10 mice received the vehicle; group 2: HCC group, 50
mice. For induction of HCC, 200 mg/kg of diethylnitrosamine
(DEN) (Sigma-Aldrich Inc. USA) was injected i.p. as a single
dose [22]. After 14 days, the mice were subjected to i.p.
injection of thioacetamide (TAA) (Sigma-Aldrich Inc. USA)
100 mg/kg twice per week for 4 weeks [32]. Then the mice
were left for further 2 weeks without any treatment.
At the end of the experiment (8 weeks), mice were weighed
then anesthetized by ether and blood was collected by cardiac
puncture. The survival rate within each group was calculated
as number of live animals after 8 weeks/number of animals at
the start of the experiment×100 [8]. Blood samples were
centrifuged for 12 min at 3,000 rpm, at 4 °C using cooling
centrifuge (Sigma 3K15, Germany). The obtained serum was
subdivided into four portions; one portion (250 μL) was used
for immediate determination of activity of glucose-6phosphate dehydrogenase enzyme (G6PDH), and other portions were stored at −20 °C until used for biochemical analysis
of aldolase enzyme activity (250 μL serum), IGF-1 (10 μL
serum), and E-cadherin (50 μL serum).
Mice were sacrificed and their livers were dissected. Fresh
liver was washed twice with ice-cold saline, dried on clean
paper towels, and weighed. Relative liver weight was calculated as liver weight (g)/final body weight (g)×100. The liver
was minced quickly and divided into two portions. One portion of the liver was kept in 10 % formalin for histopathological examination and immunohistochemical staining of the
tumor suppressor gene p53 product. The second portion was
kept frozen in liquid nitrogen at −80 °C till determination of
the activity of citrate synthase enzyme.
Serum analysis
IGF-1 and E-cadherin were determined by mouse IGF-1 and
mouse E-cadherin ELISA kits, respectively, according to
manufacturer instructions. The ELISA kits were purchased
from Boster Biological Technology, Ltd. (China). The concentrations of IGF-1 and E-cadherin were determined by
ELISA kit according to the manufacturer procedures and
expressed as nanogram per milliliter. The enzyme activities
of G6PDH [43] and aldolase [35] were determined by measurement of the rate of absorbance change at 340 nm using
kits obtained from Randox Laboratories Ltd. Company
(England).
Determination of liver citrate synthase activity
Liver extract was prepared according to Morgunov and
Srere [30]. For 50-mg liver tissue samples, Cellytic MT
reagent (Sigma-Aldrich Inc. USA) was added in the ratio
of 1:20 w/v and protease inhibitor cocktail (Sigma-Aldrich
Inc. USA) was added in the ratio of 20:1w/v. The mixture
was homogenized under cooling, centrifuged at 15,000×g
for 10 min at 4 °C, and the protein-containing supernatant
was separated and used for the determination of citrate
synthase activity. The reaction mixture contains the
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Tumor Biol.
following: 100 μL of 1.01 mM dithionitro benzoic acid
(DTNB), 25 μL of 10 % triton X-100, 50 μL of 10 mM
oxalacetate, 25 μL of 12.2 mM acetyl-CoA, and 790 μL
of redistilled water. Supernatant (20 μL) was added,
mixed carefully, and incubated for 10 min at 30 °C. The
yellow product 5-thio-2-nitrobenzoic acid (TNB) was
measured spectrophotometrically at 412 nm [44]. The
protein content was determined according to Fleury and
Eberhard [12] using kits obtained from Biodiagnostics Co.
Ltd. (Egypt), and citrate synthase activity was expressed
as micromole per minute per milligram of protein.
Histopathology
The liver sections were prepared (3–5 μm thick) and stained
with hematoxylin and eosin (H and E). Then, sections were
investigated under light microscope (Leica, Switzerland)
using image analysis system under magnification ×400.
The liver sections were investigated by a pathologist.
Hepatocytes were seen with blue nuclei and pink to red
cytoplasm.
Immunohistochemical detection of p53 protein
p53 protein was detected by immunostaining of the liver
sections prepared from formalin-fixed, paraffin-embedded
liver, using an Invitrogen kit (HistostainTM-SP kit). The kit
utilizes the labeled streptavidin-biotin (LAB-SA) staining
methodology. The primary antibody was mouse monoclonal
antibody specific for p53 (PAb 240, Invitrogen Corporation,
Camarillo, CA, USA). The slides were investigated with light
microscopy (Leica, Switzerland) by a pathologist for number
of positive cells and color intensity. Strongly p53-stained cells
(+++) are those showing nuclei/cytoplasm with dark brown
color and highest number of apoptotic bodies or figures.
Moderately p53-stained cells (++) are those showing intermediate golden brown color and modest number of apoptotic
bodies or figures. Weakly p53-stained cells (+) are those
showing light brown color and least number of apoptotic
bodies or figures [19].
Results
Survival rate, body weight, and relative liver weight
The survival rate was 20 % in the HCC group compared to
80 % in the normal control group (Fig. 1). HCC group showed
a significant body weight loss (P<0.05) and a significant
increase in relative liver weight (P<0.05) compared with
normal control group (Table 1).
Serum concentrations of IGF-1 and E-cadherin
The results indicated that HCC group showed a significant
decrease in serum IGF-1 concentration (P>0.001, ↓12.19 %),
but a significant increase in serum E-cadherin concentration
(P>0.01, ↑24.69 %) compared with normal control group
(Table 2).
G6PDH, aldolase, and citrate synthase activities
HCC group showed a significant increase in serum G6PDH
activity (P<0.01, ↑39.96 %) and serum aldolase activity
(P<0.001, ↑81.13 %), whereas the liver citrate synthase activity showed nonsignificant increase compared with normal
control group (Table 3).
Correlation studies
Serum IGF-1 showed a significant negative correlation with
aldolase activity (r=−0.62, P<0.01) (Fig. 2), whereas serum
E-cadherin exhibited a significant positive correlation with
aldolase activity (r=0.50, P<0.05) (Fig. 3). Enzyme activity
of G6PDH exhibited significant positive correlation with each
of aldolase activity (r=0.59, P<0.05) (Fig. 4) and the liver
citrate synthase activity (r=0.64, P<0.01) (Fig. 5).
Liver histopathology
The liver sections from normal control group showed the
normal liver architecture (Fig. 6) whereas the liver sections
from HCC mice group showed malignant cells characterized
by a large nucleus, having an irregular size and shape
Analysis of data was performed with Statistical Package
for Social Science (SPSS) version 17. Data are presented as mean ± SEM. Comparison between the studied
groups was performed with one-way ANOVA (F-testing). Correlation between variables was evaluated using
Pearson’s correlation coefficient. P<0.05 was considered
statistically significant.
Survival rate (%)
100
Statistical analysis
80
80
60
40
20
20
0
Normal control
HCC
Fig. 1 Survival rate as percentage of number of animals in each group
which survived for 8 weeks in the experiment. HCC, hepatocellular
carcinoma
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Tumor Biol.
Table 1 Effect of hepatocellular carcinoma on body weight and relative
liver weight of mice
Parameter
Normal control
group (n=8)
HCC group
(n=10)
Body weight difference (g)
Relative liver weight (g)
↑1.75±0.25
3.49±0.23
↓3.50±0.43a
4.11±0.15a
Body weight difference=final body weight−initial body weight
Enzyme
Normal control
group (n=8)
HCC group
(n=10)
Serum G6PDH activity (mU/mL)
Serum aldolase activity (U/L)
Liver citrate synthase activity
(μmole/min/mg protein)
5.18±0.48
0.53±0.06
2.88±0.29
7.25±0.51a
0.96±0.04a
3.26±0.26
HCC hepatocellular carcinoma, G6PDH glucose-6-phosphate
dehydrogenase
HCC hepatocellular carcinoma
Values are mean±SEM, P>0.05 was significant
a
Table 3 Effect of hepatocellular carcinoma on some metabolic enzymes
Values are mean±SEM, P>0.05 was significant
Significant versus normal control group
a
Immunohistochemical staining of p53 protein
Immunohistochemical staining of the liver sections from normal control group showed very weak expression (+) of p53
(Fig. 8), whereas the liver sections from HCC group showed
moderate expression (++) of p53 (Fig. 9).
Discussion
Hepatocellular carcinoma was induced in mice in the present
work by DEN/TAA and was associated with loss of body
weight and increase of relative liver weight, which are common features of HCC [3]. Early stage hepatocellular carcinoma was evidenced by the histopathological results, which
indicated that the liver of HCC mice group showed malignant
cells characterized by a large nucleus, having an irregular size
and shape (pleomorphism) and irregular border [37]. Malignant cells showed a small cytoplasmic amount, frequently
with vacuoles and consequently have increased nuclearcytoplasmic (N/C) ratio. These results were in line with those
recorded by Roncalli et al. [37].
Table 2 Effect of hepatocellular carcinoma on IGF-1, E-cadherin serum
concentrations
Parameter
Normal control group (n=8) HCC group (n=10)
IGF-1 (ng/mL)
34.61±0.76
E-cadherin (ng/mL) 5.67±0.34
30.39±0.74a
7.07±0.48a
HCC hepatocellular carcinoma, IGF-1 insulin-like growth factor type-1
Values are mean±SEM, P>0.05 was significant
a
Significant versus normal control group
The pathological manifestations of HCC in the liver were
associated with a moderate expression of p53 in the present
study. Similar observations were also reported in other carcinoma like ovarian cancer [29]. Activated p53 functions as a
transcription factor to regulate the expression of many different downstream genes, whose products are implicated in cell
cycle arrest, DNA repair, or apoptosis. To achieve proper
function, p53 is tightly regulated by means of posttranslational
modifications, cofactor binding, and subcellular localization.
In normal cells, the function of p53 is tightly controlled by
mouse double minute 2 homolog (MdM2), which is E3 ubiquitin ligase enzyme implicated in the inactivation of the tumor
suppressor by accelerating its nuclear export to cytoplasm and
degradation by the 26S proteasome keeping p53 in low cellular level [28].
The tumor suppressor gene p53 is frequently mutated in
human cancers. Its product, the p53 protein, is induced and
phosphorylated by various forms of cellular stress, including
γ-irradiation, UV radiation, DNA cross-linking, oxidative
stress, hypoxia, and chemical agents. Phosphorylation of
p53 within its amino-terminal domain facilitates p53 stabilization by disrupting p53-MdM2 interaction leading to p53
protein accumulation which is regarded as a hallmark of
cancer cells [28].
Our study reported moderate (++) expression of p53 in
HCC group compared to weak (+) expression in normal
control group which is in line with the results of Qu et al.
[36]. This could be explained on the basis that the mutant p53
protein expressed in HCC often do not induce MDM2 and are
Serum aldolase activity
U/L
(pleomorphism) and irregular border. Malignant cells also
have a small cytoplasmic amount, frequently with vacuoles
and consequently have increased nuclear-cytoplasmic (N/C)
ratio. Normal structures may disappear from the cytoplasm
(cytoplasmic dissociation) (Fig. 7).
Significant versus normal control group
1.4
1.2
1
0.8
0.6
0.4
0.2
0
r=0.62
P<0.01
20
25
30
35
Serum IGF-1 concentration ng/mL
40
Fig. 2 Correlation between serum insulin-like growth factor type-I (IGF1) concentration and serum aldolase activity; n=18
Author's personal copy
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Liver citrate synthase
activity (µmole/min/mg
protein)
Serum aldolase activity
U/L
Tumor Biol.
r=0.50
P<0.05
4
6
8
10
5
4
3
2
r=0.64
P<0.01
1
0
2
4
6
8
10
Serum G6PDH activity mU/mL
Serum E-cadherin concentartion ng/mL
Fig. 5 Correlation between serum glucose-6-phosphate dehydrogenase
(G6PDH) activity and liver citrate synthase activity; n=18
thus able to accumulate at very high concentrations. This leads
to the accumulation of unfolded proteins, which initiates
transcriptional and translational-signaling pathways known
as the unfolded protein response (UPR). UPR is an adaptive
response that involves the upregulation of the expression of
p53 [20].
Increased proliferation is a prominent feature of HCC and
is achieved by increased expression of growth factors and
their receptors such as transforming growth factor-α
(TGF-α), insulin-like growth factor-II (IGF-II), and hepatocyte growth factor (HGF) [34].
In the current study, serum level of IGF-1 was significantly
reduced in HCC mice group compared to normal control
group. Our results were in agreement with other reports demonstrating low IGF-1 levels in HCC patients [16] and were
opposite to those in other malignancies as prostate carcinoma
[31], breast carcinoma [1], and colorectal carcinoma [24],
reflecting the possible specificity of IGF-1 for HCC.
Our study suggests that the measurement of serum IGF-1
may be an important early marker for the diagnosis of early
stage HCC. Reduced IGF-1 could be explained by the increased oxidative damage in cirrhosis and HCC, leading to
increased damage of parenchymal liver cell and decrease in
IGF-1 synthesis [14].
Another explanation was provided by Mazziotti et al. [26],
who proposed that IGF-1 was low in HCC patients because of
reduced ability of growth hormone to stimulate IGF-1 synthesis due to either a reduction of growth hormone receptors
number in the diseased liver or a post receptor defect. Low
circulating IGF-1 levels in HCC may also be derived from an
inhibitory effect by some tumor cytokines, like transforming
growth factor-beta and platelet-derived growth factor [5].
Cancer progression is a multistep process in which some
adhesion molecules play a pivotal role in the development of
recurrent, invasive, and distant metastasis. E-cadherin is an
epithelial cell adhesion molecule that helps establish and
maintain intercellular connections. Loss of E-cadherin function is a critical factor in the initial stages of cancer invasion
and is associated with poor prognosis in a variety of epithelial
carcinomas including HCC [6].
Furthermore, E-cadherin is well known to be an invasive
suppressor in tumor progression because expression levels for
E-cadherins were reduced in almost all the malignant tumors.
Additionally, some reports suggested the possibility that some
carcinoma cells lose E-cadherin expression during the process
of detaching from the primary sites and infiltrating other sites
[6].
Our results showed a significant increase in serum concentration of E-cadherin in HCC group compared to normal
control group, which was in agreement with Soyama et al.
[41], who demonstrated that a significant increase in Ecadherin level was observed in HCC patients. It could be
suggested that E-cadherin dysfunction in tumor cells was
partly mediated by the degradative action of proteases secreted from these cells because soluble E-cadherin with a molecular weight of about 80 kDa remarkably increased in the
circulation, and it can reasonably be derived from proteolytic
digests of the cell-surface of E-cadherin [40].
Serum aldolase activity
U/L
Fig. 3 Correlation between serum E-cadherin concentration and serum
aldolase activity; n=18
1.4
1.2
1
0.8
0.6
0.4
0.2
0
r=0.59
P<0.05
2
4
6
8
10
Serum G6PDH activity mU/mL
Fig. 4 Correlation between serum glucose-6-phosphate dehydrogenase
(G6PDH) activity and serum aldolase activity; n=18
Fig. 6 Histopathology of liver section from normal control group showing normal cells (arrow) (H&E, ×400)
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Tumor Biol.
Fig. 7 Histopathology of liver section from HCC group showing tumor
cells with cytoplasmic dissociation (CD), increased nuclear-cytoplasmic
ratio (arrow), and pleomorphism (H&E,×400)
Cancer cells have nutritional requirements that are different
from normal cells. They need to take up, generate, and use
nutrients differently in order to divide and grow rapidly. They
accomplish this change by accumulating stable mutations in
genes that are key regulators of metabolism [4]. Altered
metabolism in cancer cell was discovered by the Nobel Prize
winner Otto Warburg in the 1920s. The “Warburg phenomenon” consists of an increase in glycolysis that is maintained in
conditions of high oxygen tension (“aerobic glycolysis”) and
gives rise to enhanced lactate production [4].
The metabolic profile of the liver tumors in the present
work indicated significant increase in G6PDH activity, and
aldolase activity, but insignificant increase in citrate synthase
activity. Our data were supported by other previous findings;
Frederiks et al. [13] demonstrated that chemically induced
HCC in rats was characterized by increased G6PDH activity.
Sharma et al. [39] reported an increased aldolase activity in
chemically induced HCC in rats. Opposite to our findings,
Schlichtholz et al. [38] reported a significant increase in citrate
synthase activity in human pancreatic cancer.
Since glycolysis is the most favorable pathway that promotes the invasion and metastasis of tumor cells. Aldolase
activity in the present study was increased in HCC mice group
indicating enhanced rate of glycolysis. Cancer cells synthesize
Fig. 8 Immunostaining of p53 in liver section from normal control group
showing very weak (+) cytoplasmic p53 expressing cells (arrow) (LABSA, ×400)
Fig. 9 Immunostaining of p53 in liver section from HCC group showing
moderate (++) cytoplasmic/nuclear expression of p53 (arrow) with central area of necrosis (N) (LAB-SA, ×400)
great quantities of macromolecules and lipids to proliferate
and build new cells, while requiring to continuously producing ATP and cofactors (NAD+, NADPH) in order to sustain
synthetic pathways. Therefore; these cells consume glucose in
excessive manner [17].
Increased G6PDH activity in HCC mice group suggests an
important role of G6PDH in maintaining the antioxidant capacity of cancer cells, but there are also indications that
NADPH induces tumor growth by affecting redox state of
transcription factors [21]. Increased G6PDH activity in HCC
provides reducing power for regeneration of reduced glutathione (GSH) and other detoxification processes, thus, increasing
intracellular GSH levels and the activation of the redoxsensitive transcription factor. Nuclear factor-κB (NF-κB)
could play a major role in inducible chemoresistance. This
cell survival transcription factor, which is subject to regulation
by GSH, has been shown to be constitutively activated in
many cancer cells. NF-κB has been shown to be associated
with the proliferation of tumor cells, with invasion, angiogenesis, and the production of metastasis [23].
Malignant cells have increased ability to synthesize lipid
molecules (lipogenesis) to promote proliferation, invasion,
and metastasis [17]. G6PDH is a lipogenic enzyme that provides NADPH for fatty acid biosynthesis [42]. This could be
another explanation of increased G6PDH activity in HCC
group in the present work.
In tumor cell, citrate synthase activity is elevated, condensing acetyl-CoA and oxaloacetate (OAA); thus, citrate increases and ketone bodies decrease. Consequently, decreased
ketone bodies formation will stop stimulating pyruvate carboxylase. In tumors, the OAA needed for citrate synthase will
presumably come from phosphoenolpyruvate (PEP), via reversible PEP carboxykinase or other sources. The quiescent
pyruvate carboxylase will not process the pyruvate produced
by alanine transamination after proteolysis, leaving even more
pyruvate to lactate dehydrogenase, increasing the lactate released by the tumor cell and nicotinamide adenine dinucleotide (NAD+) required for glycolysis [18].
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Tumor Biol.
Citrate, which moves outside the mitochondria, in exchange with malate is restored in cytosol into OAA and
acetyl-CoA by ATP citrate lyase. While acetyl-CoA feeds
fatty acid synthesis, OAA is converted into pyruvate via two
successive reactions: the first converts OAA into malate by
malate dehydrogenase, a reaction producing NAD+, whereas
the second reaction converts malate into pyruvate by the malic
enzyme, hence generating NADPH which is required for lipid
biosynthesis [17].
Chronic liver inflammation due to viral infection,
metabolic injury, and hepatotoxic drugs is another
mechanism underlying HCC development. It has been
showed that inflammation has an important role in initiation, promotion, and progression of tumors through
increased production of extracellular matrix proteins,
cytokines, growth factors, and products of oxidative
stress [27].
NF-κB is a protein complex that controls the transcription
of DNA and involved in cellular responses to stimuli such as
stress, cytokines, free radicals, and ultraviolet irradiation.
NF-κB might be activated by cytokines or interleukins, such
as tumor necrosis factor-alpha (TNF-α), chemokines, and
viral proteins, which likely will promote cell survival of
precancerous hepatocytes. Furthermore, cellular pathways
such as epidermal growth factor receptor (EFGR)-mediated
cascade can activate NF-κB signaling leading to inhibition of
c-Myc-induced apoptosis Myc [c-Myc is a regulator gene that
codes for a transcription factor].
NF-κB signaling can also activate pro-survival factors such
as Bcl-xl and the X-linked inhibitor of apoptosis protein
(XIAP). The generation of pro-inflammatory cytokines and
growth factors produced by tumor-infiltrating macrophages,
lymphocytes, and other cell types in the tumor microenvironment provokes activation of NF-κB, protects against proapoptotic host immune defense mechanisms, influences cell
differentiation, and exerts pro-angiogenic effects which stimulate the growth of cancer cells, tumor invasiveness, and
metastasis [27]. This mechanism, in agreement with results
of the present work regarding increased expression of mutant
p53, increased degradation of E-cadherin leading to increased
ability of cells toward metastasis and increased ability of cells
for growth through shifted metabolism toward glycolysis and
lipogenesis.
In summary, development of HCC displays changes that
aid them to escape from cell death as indicated by increased
expression of mutant p53 protein, increased ability of cells for
metastasis, and invasiveness indicated by increased soluble Ecadherin in serum and promoted cells’ ability of proliferation
through shifted metabolism toward glycolytic energy production and de novo lipogenesis. Interference with the molecular
pathways of HCC and detection of early diagnostic markers
may help intervention and effective management of the
disease.
Conflicts of interest None
References
1. Baglietto L, English DR, Hopper JL, Morris HA, Tilley WD, Giles
GG. Circulating insulin-like growth factor-I and binding protein-3
and the risk of breast cancer. Cancer Epidemiol Biomarkers Prev.
2007;16(4):763–8.
2. Breuhahn K, Longerich T, Schirmacher P. Dysregulation of growth
factor signaling in human hepatocellular carcinoma. Oncogene.
2006;25(27):3787–800.
3. Bialecki ES, Di Bisceglie AM. Diagnosis of hepatocellular carcinoma. HPB. 2005;7(1):26–34.
4. Brahimi-Horn MC, Chiche J, Pouyssegur J. Hypoxia signaling controls metabolic demand. Curr Opin Cell Biol. 2007;19(2):223–9.
5. Clemmons DR. Insulin-like growth factor-1 and its binding proteins.
In: De Groot LJ, Jenmeson JL, Burger HG, editors. Endocrinology.
4th ed. Philadelphia: WB Saunders Company; 2001. p. 439–60.
6. Conacci-Sorrell M, Simcha I, Ben-Yedidia T, Blechman J, Savagner
P, Ben-Ze’ev A. Autoregulation of E-cadherin expression by
cadherin-cadherin interactions: the roles of beta-catenin signaling,
Slug, and MAPK. J Cell Biol. 2003;163(4):847–57.
7. Cornellà H, Alsinet C, Villanueva A. Molecular pathogenesis of
hepatocellular carcinoma. Alcohol Clin Exp Res. 2011;35(5):821–5.
8. Cosetti M, Yu GP, Schantz SP. Five-year survival rates and time
trends of laryngeal cancer in the US population. Arch Otolaryngol
Head Neck Surg. 2008;134(4):370–9.
9. DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB. The
biology of cancer: metabolic reprogramming fuels cell growth and
proliferation. Cell Metab. 2008;7(1):11–20.
10. El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology
and molecular carcinogenesis. Gastroenterology. 2007;132(7):2557–76.
11. El-Zayadi AR, Badran HM, Shawky S, Emara S, El-Bareedy A,
Sobhi M. Effect of surveillance for hepatocellular carcinoma on
tumor staging and treatment decisions in Egyptian patients. Hepatol
Int. 2010;4(2):500–6.
12. Fleury P, Eberhard R. Determination of proteins by photometric,
biuret method according to the technique of Gornall. Ann Biol
Clin. 1951;9(10–12):453–66.
13. Frederiks WM, Vizan P, Bosch KS, Vreeling-Sindelárová H, Boren J,
Cascante M. Elevated activity of the oxidative and non-oxidative
pentose phosphate pathway in (pre)neoplastic lesions in rat liver. Int J
Exp Pathol. 2008;89(4):232–40.
14. García-Fernández M, Castilla-Cortázar I, Díaz-Sanchez M, Navarro
I, Puche JE, Castilla A, et al. Antioxidant effects of insulin-like
growth factor-1 (IGF-1) in rats with advanced liver cirrhosis. BMC
Gastroenterol. 2005;5(7):1–8.
15. Gebhardt SE, Thomas RG. Nutritive value of foods. U.S. Department
of Agriculture, Agricultural Research Service, Nutrient Data
Laboratory, Beltsville, Maryland. Home Gard Bull. 2002;72:45–62.
16. Ibrahim AS, Attia HA, Rabea AM, El-Gayar AM. Serum levels of
glycosaminoglycans (GAGs) and insulin like growth factor-1 (IGF-1)
as diagnostic markers for early hepatocellular carcinoma in cirrhotic
patients with or without diabetes. J Med Lab Diagn. 2013;4(1):8–20.
17. Icard P, Poulain L, Lincet H. Understanding the central role of citrate
in the metabolism of cancer cells. Biochim Biophys Acta.
2012;1825(1):111–6.
18. Israël M and Schwartz L. The metabolic advantage of tumor cells.
Mol Cancer. 2011;10(70): doi: 10.1186/1476-4598-10-70
19. Jadali F, Sayadpour D, Rakhshan M, Karimi A, Rouzrokh M,
Shamsian BS, et al. Immunohistochemical detection of p53 protein
expression as a prognostic factor in Wilms tumor. Iran J Kidney Dis.
2011;5(3):149–53.
Author's personal copy
Tumor Biol.
20. Kaufman RJ, Scheuner D, Schroder M, Shen X, Lee K, Liu CY, et al.
The unfolded protein response in nutrient sensing and differentiation.
Nat Rev Mol Cell Biol. 2002;3(6):411–21.
21. Kuo WY, Lin JY, Tang TK. Human glucose-6-phosphate dehydrogenase (G6PD) gene transforms NIH 3T3 cells and induces tumors in
nude mice. Int J Cancer. 2000;85(6):857–64.
22. Kushida M, Kamendulis LM, Peat TJ, Klaunig JE. Dose-related
induction of hepatic preneoplastic lesions by diethylnitrosamine in
C57BL/6 mice. Toxicol Pathol. 2011;39(5):776–86.
23. Lou H, Kaplowitz N. Glutathione depletion down-regulates tumor
necrosis factor alpha-induced NF-kappaB activity via IkappaB
kinase-dependent and -independent mechanisms. J Biol Chem.
2007;282(40):29470–81.
24. Ma J, Giovannucci E, Pollak M, Chan JM, Gaziano JM, Willett W,
et al. Milk intake, circulating levels of insulin-like growth factor-I,
and risk of colorectal cancer in men. J Natl Cancer Inst. 2001;93(17):
1330–6.
25. Massague J. TGFbeta in Cancer. Cell. 2008;134(2):215–30.
26. Mazziotti G, Sorvillo F, Morisco F, Carbone A, Rotondi M,
Stornaiuolo G, et al. Serum insulin-like growth factor I evaluation
as a useful tool for predicting the risk of developing hepatocellular
carcinoma in patients with hepatitis C virus-related cirrhosis: a prospective study. Cancer. 2002;95(12):2539–45.
27. Merle P, Trepo C. Molecular mechanisms underlying hepatocellular
carcinoma. Viruses. 2009;1(3):852–72.
28. Michael D, Oren M. The p53 and Mdm2 families in cancer. Curr
Opin Genet Dev. 2002;12(1):53–9.
29. Milde-Langosch K, Hagen M, Bamberger AM, Löning T. Expression
and prognostic value of the cell-cycle regulatory proteins, Rb,
p16MTS1, p21WAF1, p27KIP1, cyclin E, and cyclin D2, in ovarian
cancer. Int J Gynecol Pathol. 2003;22(2):168–74.
30. Morgunov I, Srere PA. Interaction between citrate synthase and
malate dehydrogenase: substrate channeling of oxaloacetate. J Biol
Chem. 1998;273(45):29540–4.
31. Mucci LA, Stark JR, Pollak MN, Li H, Kurth T, Stampfer MJ, et al.
Plasma levels of acid-labile subunit, free insulin-like growth factor-I,
and prostate cancer risk: a prospective study. Cancer Epidemiol
Biomarkers Prev. 2010;19(2):484–91.
32. Novosyadlyy R, Dargel R, Scharf JG. Expression of insulin-like
growth factor-I and insulin-like growth factor binding proteins during
thioacetamide-induced liver cirrhosis in rats. Growth Horm IGF Res.
2005;15:313–23.
33. Otsuka M, Kato N, Taniguchi H, Yoshida H, Goto T, Shiratori Y,
et al. Hepatitis C virus core protein inhibits apoptosis via enhanced
Bcl-xL expression. Virology. 2002;296(1):84–93.
View publication stats
34. Paiva C, Oshima CT, Lanzoni VP, Forones NM. Apoptosis, PCNA
and p53 in hepatocellular carcinoma. Hepatogastroenterology.
2002;49(46):1058–61.
35. Pinto PV, Kaplan A, Van Dreal PA. Aldolase. II. Spectrophotometric
determination using an ultraviolet procedure. Clin Chem. 1969;15(5):
349–60.
36. Qu L, Huang S, Baltzis D, Rivas-Estilla AM, Pluquet O, Hatzoglou
M, et al. Endoplasmic reticulum stress induces p53 cytoplasmic
localization and prevents p53-dependent apoptosis by a pathway
involving glycogen synthase kinase-3β. Genes Dev. 2004;18(3):
261–77.
37. Roncalli M, Terracciano L, Di Tommaso L, David E, Colombo M.
Liver precancerous lesions and hepatocellular carcinoma: the histology report. Dig Liver Dis. 2011;43(4):S361–72.
38. Schlichtholz B, Turyn J, Goyke E, Biernacki M, Jaskiewicz K,
Sledzinski Z, et al. Enhanced citrate synthase activity in human
pancreatic cancer. Pancreas. 2005;30(2):99–l04.
39. Sharma S, Lakshmi KS, Chitra V, Lakshmi AI, Pharm M.
Anticarcinogenic activity of allylmercaptocaptopril against
aflatoxin-B1 induced liver carcinoma in rats. Eur J Gen Med.
2011;8(1):46–52.
40. Shen Y, Hirsch DS, Sasiela CA, Wu WJ. Cdc42 regulates E-cadherin
ubiquitination and degradation through an epidermal growth factor
receptor to Src-mediated pathway. J Biol Chem. 2008;283(8):5127–
37.
41. Soyama A, Eguchi S, Takatsuki M, Kawashita Y, Hidaka M, Tokai H,
et al. Significance of the serum level of soluble E-cadherin in patients
with HCC. Hepatogastroenterology. 2008;55(85):1390–3.
42. Takeuchi H, Nakamoto T, Mori Y, Kawakami M, Mabuchi H,
Ohishi Y, et al. Comparative effects of dietary fat types on
hepatic enzyme activities related to the synthesis and oxidation of fatty acid and to lipogenesis in rats. Biosci Biotechnol
Biochem. 2001;65(8):1748–54.
43. Tian WN, Pignatare JN, Stanton RC. Signal transduction proteins
that associate with the platelet-derived growth factor (PDGF) receptor mediate the PDGF-induced release of glucose-6-phosphate dehydrogenase from permeabilized cells. J Biol Chem. 1994;269(20):
14798–805.
44. Trounce IA, Kim YL, Jun AS, Wallace DC. Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines. Methods Enzymol.
1996;264:484–509.
45. Wu J and Zhu AX. Targeting insulin-like growth factor axis in
hepatocellular carcinoma. J Hematol Oncol. 2011;4(30): doi: 10.
1186/1756-8722-4-30