Hepatoprotective effect of hesperidin in hepatocellular carcinoma: Involvement of Wnt signaling pathways

Accepted Manuscript Hepatoprotective effect of hesperidin in hepatocellular carcinoma: Involvement of Wnt signaling pathways

Randa A. Zaghloul, Nehal M. Elsherbiny, Hany I. Kenawy, Amr El-Karef, Laila A. Eissa, Mamdouh M. El-Shishtawy PII: DOI: Reference:

S0024-3205(17)30356-9 doi: 10.1016/j.lfs.2017.07.026 LFS 15278

To appear in:

Life Sciences

Received date: Revised date: Accepted date:

29 May 2017 18 July 2017 23 July 2017

Please cite this article as: Randa A. Zaghloul, Nehal M. Elsherbiny, Hany I. Kenawy, Amr El-Karef, Laila A. Eissa, Mamdouh M. El-Shishtawy , Hepatoprotective effect of hesperidin in hepatocellular carcinoma: Involvement of Wnt signaling pathways, Life Sciences (2017), doi: 10.1016/j.lfs.2017.07.026

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Hepatoprotective Effect of Hesperidin in Hepatocellular Carcinoma: Involvement of Wnt Signaling Pathways Randa A Zaghloul1*, Nehal M Elsherbiny1#, Hany I Kenawy2, Amr El-Karef3, Laila A Eissa1,

2

IP

Dept. of Biochemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt.

CR

1

T

Mamdouh M El-Shishtawy1*.

Dept. of Microbiology and Immunology, Faculty of Pharmacy, Mansoura University, Mansoura

Dept. of Pathology, Faculty of Medicine, Mansoura University, Mansoura 35516, Egypt.

M

AN

3

US

35516, Egypt.

*Corresponding authors: [email protected] (Mamdouh M El-Shishtawy) and

ED

[email protected] (Randa A Zaghloul), Dept. of Biochemistry, Faculty of Pharmacy, Mansoura

#

PT

University, Mansoura 35516, Egypt.

Present address: Dept. of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Tabuk,

CE

Tabuk 71491, Saudi Arabia.

AC

Abbreviations: AFP, alpha-fetoprotein; ALT, alanine aminotransferase; ANOVA, one-way analysis of variance; AST, aspartate aminotransferase; cDNA, complementary DNA; Ct, threshold Cycle; DAB, 3, 3′-diaminobenzidine; ELISA, Enzyme-Linked Immunosorbent Assay; GAPDH, glyceraldehyde 3phosphate dehydrogenase; H&E, Hematoxylin and Eosin; HCC, hepatocellular carcinoma; HP, Hesperidin; IHC, immunohistochemistry; i.p., intra-peritoneal; MDA, Malondialdhyde; MPO, myeloperoxidase; qPCR, quantitative polymerase chain reaction; S.E.M., standard error of the mean; SOD, superoxide dismutase; TAC, total antioxidant capacity; TMB, tetramethylbenzidine.

-1-

ACCEPTED MANUSCRIPT Abstract Aims: Wnt3a and Wnt5a are ligands orchestrating the canonical and non-canonical pathways, respectively, with involvement in hepatocellular carcinoma (HCC). Hesperidin (HP) is a natural product found in citrus fruits and reputed for its antitumor activity. The present study aims to

IP

focusing on its potential role on Wnt3a and Wnt5a signaling pathways.

T

investigate the potential hepatoprotective effect of HP against thioacetamide (TAA)-induced HCC

CR

Main methods: Forty rats were equally divided into groups; normal control, HP control (receiving HP, 150 mg/Kg/day), HCC (receiving TAA, 200 mg/Kg twice weekly for 14 weeks) and HP-HCC

US

(receiving HP and TAA). Gene expressions of Wnt3a, Wnt5a, β-catenin and Cyclin D1 were assessed

AN

by qPCR, while their protein levels, along with active caspase-3 level, were quantified by ELISA and immunohistochemistry. Liver functions, oxidative stress parameters and myeloperoxidase activity were

ED

absence of HP (100 μM) was performed.

M

measured. MTT assay of hepG2 cells treated with recombinant Wnt3a (10 ng/ml) in presence or

PT

Key findings: HCC group exhibited a significant increase in Wnt3a, β-catenin, Cyclin D1 and Wnt5a gene expressions, as well as, their protein levels. HP significantly prevented TAA-activated Wnt3a/β-

CE

catenin and Wnt5a pathways. Moreover, HP exerted hepatoprotective effect by significantly improving

albumin level.

AC

the oxidative imbalance, inflammation and liver function parameters, serum ALT, AST activities, and

Significance: Our study is the first to report the possible role of Wnt3a/β-catenin and Wnt5a pathways in TAA-induced early HCC model in rats. HP has a prophylactic effect against hepatocarcinogenesis via preventing the induction of both canonical and non-canonical Wnt pathways.

-2-

ACCEPTED MANUSCRIPT Highlights: 

Thioacetamide-induced Hepatocellular carcinoma model in rats was associated with activation of both Wnt3a-canonical and Wnt5a-non-canonical pathways.



Hesperidin has a hepatoprotective effect through its antioxidant and anti-inflammatory

Hesperidin has an antitumor activity through combating Wnt signaling pathways.

IP



T

activities.

AC

CE

PT

ED

M

AN

US

CR

Keywords: Hepatocellular carcinoma, Wnt3a, Wnt5a, β-catenin, Hesperidin, Thioacetamide.

-3-

ACCEPTED MANUSCRIPT 1. Introduction Hepatocellular carcinoma (HCC) is a cancer of liver with high incidence worldwide [1, 2]. Unfortunately, no well-established and effective systemic or adjuvant therapies for HCC have been achieved [3]. Where, Sorafenib, a kinase inhibitor, is the standard therapy for advanced HCC [4].

T

However, its efficacy is hindered by rapid treatment evasion [5].

IP

HCC is characterized by inter- and intra-tumor heterogeneity [6] with tendency to metastasize [7]. A

CR

definite mechanism of hepatic carcinogenesis remains unclear, although several possible pathways

US

have been reported [6]. These pathways included growth factors like insulin-like growth factor-Ⅱ [8], transforming growth factor-α [9, 10] and -β, tumor protein 53 and Wnt signaling pathways, all cross-

AN

passing at certain point [11-13].

M

Wnts are glycoproteins involved in embryogenesis and continuing on to adulthood [14], where the

ED

pathway is almost significantly inactive with exceptions [15]. Wnts have been playing a great, yet not fully-understood, role as regulators of carcinogenesis [16]. Moreover, Wnts have been implicated in

PT

nearly half of the known tumors in human, either by mutations or the up- or down-regulation of

CE

members of their pathways [15] through β-catenin-dependent and -independent pathways [17]. Wnt3a is a ligand activating Wnt canonical pathway [18]. Wnt3a interacts with its membrane-bound

AC

receptor (Frizzled) to initiate a series of events that eventually lead to the disruption of a complex known as the destruction complex. This complex is responsible for β-catenin phosphorylation and its subsequent proteasomal degradation

[19]. However, β-catenin, in its non-phosphorylated form,

accumulates in the cytoplasm, forming a complex with T cell factor/lymphoid enhancer factor [19]. The complex translocate to the nucleus and enhance the expression of Wnt-targeted genes including; the cyclins and others involved in different cellular processes required for survival, differentiation and angiogenesis [20]. Approximately 70% of the reported human HCC cases have shown an accumulation

-4-

ACCEPTED MANUSCRIPT of β-catenin in the nucleus [21], yet limited studies have tackled the involvement of Wnt3a in HCC [18]. Wnt5a is a Wnt ligand involved in the non-canonical pathway with contradictory roles in cancer, depending on its functioning isoform [22]. For instance, Wnt5a-Long chain isoform acts as tumor-

T

suppressor in certain cancer cell lines [22]. In contrast, Wnt5a-Short chain isoform promotes cancer

IP

cell growth [23]. Activation of the non-canonical pathway has been observed during hepatic stellate

CR

cells activation, during liver cirrhosis and in HCC [24], suggesting the possible role of Wnt5a during the early events of hepatocarcinogenesis.

US

According to the WHO, more than half of the world population depends on natural products for their

AN

health care. Hesperidin (HP) is a natural polyphenolic flavanone glycoside of citrus fruits and vegetables [25]. HP is metabolically hydrolyzed into the aglycones active form (Hesperitin) in rats and

M

humans [26]. HP has exhibited multiple bioactivities including; anti-inflammatory [27], radical

ED

trapping ability and anti-oxidant [28, 29], anti-diabetic [30] and anticancer [31] activities.

PT

Therefore, the present study was undertaken to investigate the involvement of Wnt3a and Wnt5a in thioacetamide (TAA)-induced early HCC in rats. In addition, we explored the role of HP in the

AC

CE

protection against HCC through both Wnt3a canonical and Wnt5a non-canonical pathways.

-5-

ACCEPTED MANUSCRIPT 2. Materials and Methods 2.1 Natural product, HP HP, (CAS no. 520-26-3) with purity ≥ 95%, was purchased from Abcam Co. (Cambridge, MA, USA). HP was prepared as a suspension in 2 ml of 0.5% carboxymethyl cellulose (cmc) just before

T

administration, due to its poor solubility. HP was administered orally to rats by a feeding needle in a

IP

dose of 150 mg/kg/day [32].

CR

2.2 Animal treatment outlines

US

Animal protocol was approved by the ethical committee of Faculty of Pharmacy, Mansoura University, Mansoura, Egypt. Healthy adult male Sprague Dawley rats were purchased from the Vaccine and

AN

Immunization Authority (Helwan, Cairo, Egypt) and housed (Animal House, Faculty of Pharmacy,

M

Mansoura University, Mansoura, Egypt) under standard conditions of temperature (25°C ± 2) with a

ED

regular 12 h light/dark cycle and free access to standard animal chew. Forty rats weighing 180-200 g were kept for a week for acclimatization before being randomly divided into four equal groups (n=10)

PT

according to the following scheme:

2 weeks

Normal control

AC

HP control

14 weeks

CE

Groups

HCC HP-HCC

2 ml of 0.5% cmc, orally, once daily. 150 mg/kg/day HP. 200 mg/kg of TAA (Sigma Aldrich Chemicals Co., St Louise, MO, USA) in 0.2 ml phosphate buffered saline (PBS) by Intraperitoneal (i.p.) injection twice weekly [33]. TAA and HP. -6-

ACCEPTED MANUSCRIPT

2.3 Collection of biological samples At the end of the study, TAA-induced HCC development was confirmed by elevation of serum level of alpha-fetoprotein (AFP) as well as histopathological study. Mortality rate was calculated in different

T

groups. Blood samples were collected from the orbital sinus of 12 h fasting animals and allowed to clot

IP

for 30 min before centrifugation at 4000 r.p.m. for 5 min to separate serum. Livers of sacrificed rats

CR

were removed and dissected into four sections; the first section was fixed in 10% buffered formalin for immunohistochemical and histopathological studies. The second section was homogenized in ice cold

US

PBS (10%) using Omni-125 hand held homogenizer (Omni international, GA, USA) to assess

AN

oxidative stress parameters. The third section was homogenized in PBS, containing protease inhibitor cocktail set I (Calbiochem, CA, USA), and was ultrasonicated (10%, 5 x 30 sec) using Soniprep 150

M

Plus (MSE, UK) for ELISA assay. Last section was immediately stored in liquid nitrogen and at -80°C

PT

2.4 Assessment of liver function

ED

for real-time quantitative polymerase chain reaction (qPCR) analysis.

Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) activities and albumin level

CE

were measured using standard colorimetric methodologies by commercially available kits

AC

(Biodiagnostics, Giza, Egypt) according to manufacturer's protocol. 2.5 Assessment of serum AFP Serum AFP level was determined using commercially available Enzyme-Linked Immunosorbant Assay (ELISA) kit (DiaMetra, S.r.I, Italy) according to manufacturer's instructions. 2.6 Assessment of oxidative stress status

-7-

ACCEPTED MANUSCRIPT Hepatic malondialdehyde (MDA) content, total antioxidant capacity (TAC), superoxide dismutase (SOD) activity were measured using commercially available colorimetric kits (Biodiagnostic, Giza, Egypt) according to manufacturer's instructions. 2.7 Assessment of hepatic inflammation pro-inflammatory

peroxidase

enzyme

myeloperoxidase

(MPO)

was

determined

by

T

The

IP

tetramethylbenzidine (TMB) method [34] following the instructions that was previously described by

CR

Makled et al. [35].

US

2.8 Histopathological study

Liver tissues fixed in 10% buffered formalin were embedded in paraffin and cut into 5-µm thick

AN

anonymously coded sections by microtome. Liver sections were stained with Mayer's hematoxylin and

M

eosin (H&E) and viewed under microscope. Histopathological diagnosis of HCC as well as changes in different groups was conducted and photographed using a digital camera-aided computer system

ED

(Nikon digital camera, Tokyo, Japan).

PT

2.9 Determination of hepatic Wnt3a, Wnt5a and β-catenin protein levels

CE

Hepatic Wnt3a, Wnt5a and β-catenin protein levels were measured in the ultrasonicated liver homogenates using commercially available ELISA kits (Cloud-Clone Corp., TX, USA) according to

AC

manufacturer’s protocol.

2.10 Determination of hepatic Wnt3a, Wnt5a and β-catenin, Cyclin D1 gene expression Hepatic Wnt3a, Wnt5a, β-catenin and Cyclin D1 gene expressions were measured by real-time quantitative polymerase chain reaction (qPCR). Total RNA was isolated from rat livers (4-5 rats per group) using TRIzol® Reagent (Life Technologies, Ca, USA), extracted by chloroform/isopropanol. Total RNA yield and quality were determined by Nanodrop (Thermo Fisher Scientific Inc., TX, USA) using 260 and 260/280 nm ratio, respectively. One µg of total RNA was reverse-transcribed into -8-

ACCEPTED MANUSCRIPT complementary DNA (cDNA) by two-step QuantiTect® Reverse Transcription Kit (Qiagen, Ca, USA). Obtained cDNA were diluted 1:2.5 using RNase/DNase free water. Primers sets (table 1) were designed by Primer Express 3.0 (Applied Biosystems, CA, USA) and purchased from Vivantis Co. (Selangor DarulEhsan, Malaysia). A PCR step was subsequently carried out on PikoReal 96 Real-Time PCR System (Thermo Fisher Scientific Inc., USA), using EvaGreen® qPCR Mix Plus (Solis BioDyne,

IP

T

Tartu, Estonia). A reaction of 19 µl of EvaGreen, forward and reverse primers and RNase/DNase free

CR

water were added to 1 µl of cDNA. Thermal cycling conditions included initial activation step at 95°C for 7 min followed by 35-45 cycles of 95°C for 15 seconds, 55-60°C for 30 seconds

US

(according to the primer used) and 72°C for 15-20 seconds. The efficiency of amplifications was

AN

considered acceptable in the range of 90-105 %. Melting curve and agarose gel electrophoresis were uses to assess the specificity of the PCR product. Threshold Cycle (Ct) values of each gene of samples

ED

reference gene using the 2-ΔΔCt method.

M

were normalized against its own rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a

PT

2.11 Immunohistochemistry (IHC) of Cyclin D1 and caspase-3 Five-μm-thick paraffin-embedded liver sections were deparaffinized by heating and rehydrated using

CE

xylene and descending concentrations of ethanol. Antigen retrieval step was carried out using citrate

AC

buffered solution (pH 6.0). Endogenous peroxidase activity was blocked using 3% hydrogen peroxide for 15 min. All non-specific binding sites were blocked using 1% bovine serum albumin for 1 h. Sections were incubated over night with monoclonal Cyclin D1 and active caspase-3 antibodies (Novus Biologicals, CO, USA) at 4° C. Sections were incubated with horseradish peroxidase conjugate anti-mouse antibody. The chromogen used was 2% 3, 3′-diaminobenzidine (DAB) in 50 mM Trisbuffer (pH 7.6). Slides were counterstained with hematoxylin. Tonsil and breast carcinoma were used as a positive control for both Cyclin D1 and active caspase-3 antibodies and staining was detected in the nuclei and cytoplasm for both antibodies, respectively, according to manufacturer instructions. For -9-

ACCEPTED MANUSCRIPT Cyclin D1, positive nuclei were counted in 10 random high power fields and for caspase-3, cells with positive cytoplasm were counted in 10 randomly selected high power fields. The apoptotic index was calculated as the number of cells × 100/number of high power fields [36]. 2.12 In vitro study and cell viability

T

Human hepatoma cell line (HepG2) was purchased from American Type Culture Collection (ATCC,

IP

Manassas, VA, USA) by The Holding Company for Biological Products & Vaccines (Vacsera, Giza,

CR

Egypt). HepG2 cells were examined for identity and absence of bacteria and mycoplasma before splitting. HepG2 cells were grown in Dulbecco's Modified Eagle's Medium (Lonza, Vervies, Belgium)

US

supplemented with 10% fetal bovine serum (Lonza, Vervies, Belgium) and 1% streptomycin and

AN

penicillin (Lonza, Vervies, Belgium) and incubated for 24 h at 37°C in a 5% CO2 incubator. Once reaching 70% confluence, 2 x 104 cells were plated in 96-well plate and allowed to grow for 24 h in

M

tissue culture incubator at 37°C. Medium was replaced with serum-free medium containing different

ED

doses of HP or 10 ng/ml of recombinant Wnt3a (Cloud-Clone Corp., TX, USA), alone or in combination with HP (100 µg/ml). After 24 h, cell viability was determined using MTT method.

PT

Briefly, 20 µl of MTT solution (5 mg/ml) were added to the medium in each well for 4 h before being

CE

discarded. Wells were treated with 100 µl of 0.04 N isopropanol HCl and were measured spectrophotometrically at 540 nm (Bio-Tek EXL 800, BioTek Instruments, Inc., VT, USA). Each

AC

experiment was repeated at least three times independently. 2.13 Statistical analysis

For descriptive statistics, the mean values ± standard error of mean (S.E.M.), were reported. For comparison between groups, student t-test or one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test were applied. Statistical computations were done using Excel 2010 and statistical significance was predefined as (P < 0.05).

- 10 -

ACCEPTED MANUSCRIPT 3. Results 3.1 Effect of HP on gene expression and protein level of hepatic Wnt3a In HCC group, TAA significantly induced an overexpression in hepatic Wnt3a gene, as well as, a significant increase in its protein level when compared to normal control group (P < 0.05). Where,

T

pretreatment with HP significantly decreased the induced overexpression in hepatic Wnt3a gene and

IP

elevation in protein level when compared to HCC group (P <0.05). Moreover, treatment of hepG2 cells

US

recombinant Wnt3a (10 ng/ml) alone (P < 0.05) (Fig. 1).

CR

with HP (100 µM) significantly decreased cells proliferation when compared to cells treated with

3.2 Effect of HP on gene expression and protein level of hepatic Wnt5a

AN

Hepatic Wnt5a gene expression was significantly increased in HCC group, along with its protein level

M

when compared to normal control group (P < 0.05). Rats pretreated with HP exhibited a significant

to HCC group (P <0.05) (Fig. 2).

ED

down-regulation in hepatic Wnt5a gene expression and a decrease in its protein level when compared

PT

3.3 Effect of HP on gene expression and protein level of hepatic β-catenin

CE

In HCC group, TAA significantly induced an increase in hepatic β-catenin gene expression and its protein level when compared to normal control group (P < 0.05). However, pretreatment with HP

AC

significantly prevented TAA-induced elevation in hepatic β-catenin gene expression and protein level when compared to HCC group (Fig. 3). 3.4 Effect of HP on gene expression and protein level of hepatic Cyclin D1 In HCC group, the downstream target of Wnt3a/β-catenin signaling pathway (Cyclin D1) showed a significant overexpression of gene, as well as, an increase in its protein level (evidenced by a strong IHC staining) when compared to normal control group (P < 0.05). Prophylactic treatment with HP

- 11 -

ACCEPTED MANUSCRIPT significantly prohibited the increase in Cyclin D1 gene expression and protein level (evidenced by a weaker brown IHC staining) when compared to HCC group (P < 0.05) (Fig. 4). 3.5 Effect of HP on TAA-induced hepatic oxidative imbalance In HCC group, TAA exerted a pronounced oxidative damage evidenced by a significant increase in

T

hepatic MDA content and a significant decrease in hepatic TAC and SOD activity when compared to

IP

normal control group (P < 0.01). However, pretreatment of rats with HP protected against the shown

CR

oxidative damage by TAA through a significant decrease in hepatic MDA content, while, producing a

US

significant increase in hepatic TAC and SOD activity (P < 0.01) (Fig. 5). 3.6 Effect of HP on hepatic inflammation

AN

The hepatic pro-inflammatory enzyme (MPO) was significantly activated by TAA treatment in HCC

M

group as compared to normal control (P < 0.001). Pretreatment of rats with HP along with TAA

(Fig. 6).

PT

3.7 Effect of HP on apoptosis

ED

significantly restored MPO activity to a near normal level when compared to HCC group (P < 0.001)

CE

In HCC group, IHC staining of liver tissue sections for active caspase-3 (evidenced by a strong brown staining) indicated a clearly active apoptotic pathway when compared to normal control group (P <

AC

0.05). Where, HCC rats pretreated with HP showed a stronger staining for active caspase-3 protein when compared to HCC group (P < 0.05) (Fig. 7). 3.8 Hepatoprotective effect of HP Liver sections stained with (H&E) have exhibited a hepatoprotective effect of HP evidenced by a significant decrease in the necroinflammatory and fibrotic changes of the hepatic parenchyma that were produced by TAA in HCC group. Moreover, In HCC group, dysplastic and neoplastic nodules were significantly evidenced by the increase in their numbers when compared to normal control group. - 12 -

ACCEPTED MANUSCRIPT However, pretreatment of HCC rats with HP exhibited a decrease in hepatic plate thickness, number of dysplastic and neoplastic nodules and mitotic activity with near reversal to the normal control hepatic structure. Consistent with our results, gross examination of livers of different groups revealed rough nodular

T

surface with faint discoloration in HCC group when compared to the shiny, smooth and reddish

IP

surfaces of normal control livers. HP pretreated HCC group showed a decrease in the nodular invasion

CR

across the liver surfaces rendering them more reddish and smoother when compared to HCC group (Fig. 8).

US

In support, HCC group exhibited a significant elevation in serum ALT and AST activities, as well as, a

AN

significant decrease in serum albumin level (P < 0.001). HP pretreated HCC group exhibited a significant improvement in liver function parameters when compared to HCC group evidenced by

M

significant reduction in serum ALT and AST activities compared to their elevated activities in HCC

ED

group (P < 0.001). Serum albumin level, as well, was significantly elevated by HP pretreatment when

PT

compared with HCC group (P < 0.05) (Tab. 2). 3.9 Antitumor activity of HP in vivo and in vitro

CE

The liver tumor marker, serum AFP level, was significantly increased in HCC group when compared

AC

to normal control group (P < 0.001). Pretreatment with HP significantly decreased serum AFP level when compared to HCC group (P < 0.001); however, it did not decrease serum AFP level to its normal control group baseline level. In HepG2 cells, HP in doses (200 and 400 μM) exhibited significant cell cytotoxicity (63 and 60%, respectively) when compared to vehicle treated control cells. Noticeably, pretreatment with HP has led to an increase in the survival percentage in rats, up to 80% when compared to only 60% in the survival percentage in HCC group (Fig. 9).

- 13 -

ACCEPTED MANUSCRIPT

4. Discussion HCC is a major health issue worldwide [37]. Over the last decade, Egypt has suffered from alarming increase in the incidence of HCC [38], occupying the first rank in Hepatitis C virus-related HCC [39].

T

Where, neither standard systemic therapy for HCC nor surgical intervention has been shown to be

IP

effective without limitations [40-42]. Alternative therapeutic options for HCC have been considered

CR

with increasing interests in safer, more effective and cheaper compounds including; natural products [43], repositioned drugs [44] or immunotherapies [45]. HP is a natural flavanone glycoside abundant in

US

the peel of citrus fruits with anti-inflammatory [27], anti-oxidant and anticancer [31] activities.

AN

TAA is a well-known hepatotoxin that induces hepatic lesions mimicking the majority of cases of human liver diseases [46] and eventually leads to HCC [33]. In our study, livers of HCC group

M

revealed a nodular surface with uneven pale yellowish and white discoloration confirming the early

ED

incidence of HCC. In support, their stained liver sections showed evidence of dysplastic changes and

PT

mitotic activity. Moreover, TAA has induced extensive hepatocyte damage and reduced synthetic function in the HCC group indicated by elevated serum ALT and AST activities, and decreased serum

CE

albumin level, respectively, when compared to normal control group.

AC

Our study has shown the pronounced hepatoprotective effect of HP on TAA-induced HCC model in rats. This was evidenced by the more reddish and smoother surface of livers, as well as, the restoration of liver architecture in liver sections of HP-HCC group. Interestingly, HP minimized TAA-induced liver damage and dysfunction improved serum liver function markers. This hepatoprotective effect might be attributed to the anti-oxidant [47, 48] and anti-inflammatory [49] activities of HP. TAA exerts its hepatotoxic activity through its active metabolites TAA-sulfoxide and -sulfdioxide [50]. Both intermediates induce hepatic necrosis in liver perivenous areas via compromising the oxidative

- 14 -

ACCEPTED MANUSCRIPT balance in the normal state leading to hepatocarcinogenicity [51]. This oxidative imbalance has been explained as a defeat in the anti-oxidant defense system including; the anti-oxidant enzymes such as SOD, as well as, the anti-oxidant molecules such as α-tocopherol and carotenoids [52]. As a result, the induced oxidative imbalance provokes the pro-inflammatory mediators that activate reactive oxygen species-producing inflammatory cells, thus exaggerating the oxidative stress at the inflammatory

IP

T

lesions [53].

CR

HCC group showed a great oxidative imbalance leading to an increase in hepatic MDA content and a decrease in the hepatic TAC. Treatment with TAA alone compromised the antioxidant defense system

US

by reducing hepatic SOD activity compared to normal control group. TAA induced oxidative damage

liver pro-inflammatory enzyme MPO activity.

AN

is thought to cause the observed liver cell injury and inflammation indicated by elevated activity of the

M

From our observations, HP might exert its antioxidant activity by elevating TAC level, while reducing

ED

TAA-induced lipid peroxidation as shown by the reduced hepatic MDA level. HP, as a natural

scavenging property [54].

PT

polyphenolic compound, has been reported to exert its antioxidant activity through free radical

CE

Moreover, we suggest that the antioxidant activity of HP could be attributed to the observed restoration

AC

of the antioxidant enzyme activity of SOD compared to TAA treatment alone, which has been reported before, among other antioxidant molecules and enzymes [53]. The antioxidant activity of HP was accompanied by a reduction in MPO activity indicating a reduced inflammatory response in liver tissue. HP protected against both inflammation and oxidative damage and broke the vicious cycle contributing to the pathogenesis of liver injury at later stages [53]. Beside the hepatoprotective effect of HP, it also has an antitumor activity. Whereas, neoplastic and dysplastic nodules were evidenced in liver sections of HCC group. However, HP was able to decrease their numbers in HCC pretreated group. Serum AFP, the golden marker for diagnosis of HCC, was - 15 -

ACCEPTED MANUSCRIPT significantly decreased in HP-HCC group when compared to HCC group. Interestingly, the survival rate in HP-HCC group was markedly elevated when compared to HCC group. In agreement with the observed results, HP has been reported to exert an antitumor activity in vitro [53]. Finally, HP induced a decrease in cell viability of HepG2 cells in doses (200 and 400 µM) when compared to untreated cells. A proposed mechanism of HP hepatoprotective and antitumor activities could be linked to its

IP

T

action on the active Wnt3a-canonical pathway by TAA.

CR

Wnt3a is an oncogenic gene previously implicated in HCC [18] and other human malignancies [55]. Studies has reported Wnt3a as an important regulator of HCC cell growth in vitro [56] and in vitro

US

[57]. However, limited data are available on neither the relationship between Wnt3a and HCC nor its

AN

therapeutic targeting value [18].

The gene expression and protein level of hepatic Wnt3a were elevated in HCC group when compared

M

to normal control group. This elevation by TAA could indicate a major role of Wnt3a canonical

ED

pathway activation in the early events of HCC [58]. In accordance, elevation in Wnt3a level has been associated with induced HCC cells proliferation using high levels of glucose [59]. The proposed action

PT

of Wnt3a is thought to be by binding to its receptor Frizzled, initiating a chain of events, including the

CE

dissociation of the destruction complex, to finally promoting β-catenin accumulation in the cytoplasm and its translocation to the nucleus [20].

AC

To this end, our study has reported a TAA-induced overexpression of β-catenin encoding gene in HCC rats when compared to normal control group. In agreement with the gene expression, liver total βcatenin protein level was significantly elevated in HCC group when compared to normal control group. The elevation of β-catenin level could be directly attributed to Wnt3a elevation that prevented the destruction complex from inducing proteasomal degradation of β-catenin and its subsequent accumulation in the cytoplasm and nucleus. Previously, glucose-induced Wnt3a activation was

- 16 -

ACCEPTED MANUSCRIPT associated with elevation of β-catenin level [59]. In many types of cancer, β-catenin accumulation has been reported to correlate with tumor progression and poor prognosis [60, 61]. Our in vitro experiments could show that recombinant Wnt3a-induced proliferation of hepG2 cells was abrogated by HP co-treatment with recombinant Wnt3a, suggesting a significant role of HP in

T

countering the effect of Wnt3a. In addition, treatment of rats with HP was able to decrease TAA-

IP

induced activation of Wnt3a gene expression and protein level.

CR

This effect was accompanied by a decrease in total β-catenin protein level compared to TAA-induced HCC group. This may indicate that in the absence of sufficient Wnt3a activation, β-catenin could no

US

longer escape its fate by the destruction complex. Moreover, the observed effect of HP on down-

AN

regulating TAA-induced β-catenin gene activation could indicate an additional direct action on βcatenin gene expression. Similarly, a study has indicated the potential role of HP in reducing β-catenin

M

gene expression to prevent hepatic metastasis in colorectal cancer [62].

ED

Furthermore, we investigated the effect of HP on the oncogenic marker, Cyclin D1, an important

PT

downstream target of β-catenin and a key regulator of cell cycle progression [63]. Cyclin D1 gene and protein were significantly elevated in HCC group confirming the development of HCC, as Cyclin D 1

CE

has been associated with advanced stages of human HCC [64]. HP-HCC group showed a significant

AC

decrease in Cyclin D, gene expression and protein, when compared to HCC group. Thus, HP could potentially be playing an important role in opposing the activation of Wnt3a-initiated canonical pathway during hepatocarcinogenicity provoked by TAA treatment of rats. On the other hand, Wnt5a has been considered as a non-canonical Wnt ligand with involvement in cell migration, invasiveness and metastasis [24]. Moreover, Wnt5a was reported to be elevated in activated hepatic stellate cell culture, pre-neoplastic cirrhotic liver tissues and HCC, suggesting a role in the early events of hepatocarcinogenesis [65]. In this study, overexpression in hepatic Wnt5a gene, as well as an elevation in its protein level was observed in HCC group. - 17 -

ACCEPTED MANUSCRIPT Although The non-canonical pathway has been suggested to antagonize the canonical pathway [24]. However, in our study, both pathways were active in early events of TAA-induced HCC in rats. Nevertheless, comparing between a pre-cancerous state induced by 10 weeks administration of TAA (unpublished data) and a cancerous state in HCC group, hepatic Wnt5a was more, elevated as gene and protein, in the pre-cancerous state. Moreover, Wnt5a started to decline significantly on both the gene

IP

T

expression and protein levels in HCC group when compared to the pre-cancerous state (unpublished

CR

data).

Unlike Wnt5a, hepatic Wnt3a was significantly overexpressed on the gene level in HCC state but

US

indifferently elevated on the protein level when compared to the pre-cancerous state (unpublished

AN

data). These findings may suggest the role of Wnt5a as a tumor suppressor gene in early tumor stage of liver, but a loss of function in the advanced stage of HCC tumorigenesis [24].

M

HP was able to significantly reverse the TAA-induced elevation of gene expression of Wnt5a and

ED

subsequently the protein level in HCC group. One possible explanation to these findings could be attributed to the pro-apoptotic action of HP against active Wnt5a secreting-stellate cell activation that

PT

stimulates the death process [66]. In our study, HP possessed pro-apoptotic activity by stimulating the

CE

intrinsic apoptotic pathway, caspase-3, in HP-HCC group when compared to both HCC and normal control groups. The pro-apoptotic activity of HP may also be related to its ability to inhibit the

AC

activated Wnt3a/β-catenin pathway. This finding is in agreement with a previous study where inhibition of Wnt3a/β-catenin pathway was associated with the activation of the apoptotic marker caspase-3 [67]. 5. Conclusions In summary, the current study shaded some light on the important role of two Wnts ligands, Wnt3a and Wnt5a, in experimentally induced hepatocarcinogenicity by TAA. Moreover, the data presented here suggest that HP exhibits an antitumor and hepatoprotective effects against the development of HCC. - 18 -

ACCEPTED MANUSCRIPT These actions could be attributed to the antioxidant, anti-inflammatory and pro-apoptotic activities of HP, beside the interference with both the canonical, Wnt3a/β-catenin pathway, and the non-canonical, Wnt5a, pathway. Disclosure:

T

Financial support: This research did not receive any specific grant from funding agencies in the public,

IP

commercial, or not-for-profit sectors.

CR

Conflict of interest: none.

US

Acknowledgement

We would like to thank M. El-Mesery, PhD, Dept. of Biochemistry, Faculty of Pharmacy, Mansoura

AC

CE

PT

ED

M

AN

University, Mansoura 35516, Egypt, for his assistance in the in vitro study.

- 19 -

ACCEPTED MANUSCRIPT 6. Figure legend: Fig. 1 Effect of HP on hepatic Wnt3a; a. gene expression of hepatic Wnt3a was up-regulated in HCC group when compared to normal control group. Pretreatment with HP decreased the overexpressed hepatic Wnt3a gene. b. protein level of hepatic Wnt3a was significantly elevated in HCC group when

T

compared to normal control group. HP significantly decreased hepatic Wnt3a protein level elevated by

IP

TAA in HCC group. c. HepG2 cell treated with recombinant Wnt3a (10 ng/ml) showed a significant

CR

increase in cell proliferation when compared to untreated hepG2 cells. HP significantly reduced Wnt3a-induced proliferation in hepG2 cells. HCC= Hepatocellular carcinoma; HP= Hesperidin; TAA=

US

Thioacetamide; Values are expressed as mean ± S.E.M.; a = Significant at P < 0.05 against normal

AN

control group, b = Significant at P < 0.05 against HCC group. c = Significant at P < 0.05 against vehicle untreated hepG2 cells; d = Significant at P < 0.05 against Wnt3a (10 ng/ml)-treated hepG2

M

cells.

ED

Fig. 2 Effect of HP on hepatic Wnt5a; a. gene expression of hepatic Wnt5a was up-regulated in HCC group when compared to normal control group. Pretreatment of HCC rats with HP induced a down-

PT

regulation of the overexpressed hepatic Wnt5a gene. b. protein level of hepatic Wnt5a was

CE

significantly elevated in HCC group when compared to normal control group. HP significantly reduced hepatic Wnt5a protein level elevated by TAA in HCC group. HCC= Hepatocellular carcinoma; HP=

AC

Hesperidin; TAA= Thioacetamide; Values are expressed as mean ± S.E.M.; a = Significant at P < 0.05 against normal control group, b = Significant at P < 0.05 against HCC group. Fig. 3 Effect of HP on hepatic β-catenin; a. gene expression of hepatic β-catenin was up regulated in HCC group when compared to normal control group. Pretreatment of HCC rats with HP induced a down-regulation of overexpressed hepatic β-catenin gene. b. protein level of hepatic β-catenin was significantly elevated in HCC group when compared to normal control group. HP significantly reduced hepatic β-catenin protein level elevated by TAA in HCC group. HCC= Hepatocellular carcinoma; HP= - 20 -

ACCEPTED MANUSCRIPT Hesperidin; TAA= Thioacetamide; Values are expressed as mean ± S.E.M.; a = Significant at P < 0.05 against normal control group, b = Significant at P < 0.05 against HCC group. Fig. 4 Effect of HP on hepatic Cyclin D1; a. gene expression of Cyclin D1 was significantly upregulated in HCC group as compared to normal control group. Pretreatment of HCC rats with HP

T

significantly down-regulated the gene expression of Cyclin D1 when compared to HCC group. b.

IP

immunohistochemical staining of liver sections for Cyclin D1 revealed a strong staining of the protein

CR

in the hepatocytes nuclei of HCC group compared to normal control group. Pretreatment of HCC rats with HP exhibited a weaker staining for Cyclin D1 when compared to HCC group.

HCC=

US

Hepatocellular carcinoma; HP= Hesperidin; TAA= Thioacetamide; Values are expressed as mean ±

AN

S.E.M.; a = Significant at P < 0.05 against normal control group, b = Significant at P < 0.05 against HCC group.

M

Fig. 5 Effect of HP on hepatic oxidative stress parameters; a. elevated hepatic MDA content and

ED

decreased TAC were significant in HCC group compared to normal control group. Pretreatment of HCC rats with HP significantly reduced hepatic MDA content and increased hepatic TAC when

PT

compared to HCC group. b. decreased SOD activity was significant in HCC group as compared to

CE

normal control group. HP pretreatment significantly reduced hepatic MDA content and increased hepatic TAC when compared to HCC group. HCC= Hepatocellular carcinoma; HP= Hesperidin;

AC

MDA= Malondialdhyde; TAC= Total antioxidant capacity; SOD= Superoxide dismutase; Values are expressed as mean ± S.E.M.; a = Significant at P < 0.01 against normal control group, b = Significant at P < 0.01 against HCC group. Fig. 6 Effect of HP protection on hepatic MPO activity. Hepatic MPO was significantly active in HCC group when compared to normal control group. A significant decrease in hepatic MPO activity was found in HCC group pretreated with HP when compared to HCC group. HCC= Hepatocellular carcinoma; HP= Hesperidin; MPO= Myeloperoxidase activity; Values are expressed as relative mean ± - 21 -

ACCEPTED MANUSCRIPT S.E.M.; a = Significant at P < 0.001 against normal control group, b = Significant at P < 0.001 against HCC group. Fig. 7 Effect of HP protection on apoptosis; immunohistochemical staining of liver sections for active caspase-3 revealed a strong staining of the protein in HCC group as compared to normal control

T

group. HP pretreatment of HCC rats exhibited a very strong staining for hepatic active caspase-3 when

IP

compared to HCC group. HCC= Hepatocellular carcinoma; HP= Hesperidin; TAA= Thioacetamide;

CR

Values are expressed as mean ± S.E.M.; a = Significant at P < 0.05 against normal control group, b = at P < 0.05 against HCC group.

US

Fig. 8 Hepatoprotective effect of HP; a. Histopathological examination of liver sections stained with

AN

(H&E) stain of different rat groups. b. Images of rat livers [all images were taken at the same zooming, magnification power and distance (10 cm) from camera's eye lens]. c. Liver sections examination

M

revealed an increase in the number of dysplastic and neoplastic nodules in HCC rats, (n= 4).

ED

Pretreatment of HCC rats with HP significantly reduced their number in liver sections; HCC= Hepatocellular carcinoma; HP= Hesperidin. Values are expressed as mean ± S.E.M.; a = Significant at

PT

P < 0.05 against normal control group, b = Significant at P < 0.05 against HCC group.

CE

Fig. 9 Antitumor effect of HP; a. Serum AFP level (ng/ml) was significantly increased in HCC group

AC

when compared to normal control group, which was significantly decreased in HP-HCC group. b. Kaplan’ Meier representation of survival rate; indicating a marked increase in survival in HP-HCC group when compared to HCC group. c. Cell viability (%) of hepG2 cells treated with (0,50,100, 200, 400 µM) of HP. HCC= Hepatocellular carcinoma; HP= Hesperidin; AFP= Alpha-fetoprotein; Values are expressed as mean ± S.E.M.; a = Significant at P < 0.001 against normal control group, b = Significant at P < 0.001 against HCC group, c = significant against HepG2 cells treated with (0, 50 and 100 µM) of HP, d = Significant against HepG2 cells treated with (0, 50, 100 and 200 µM) of HP.

- 22 -

ACCEPTED MANUSCRIPT

7. Tables legends Tab. 1 Primers sets

CR

IP

T

GAPDH= Glyceraldehyde 3-phosphate dehydrogenase; F= forward; R= reverse.

AN

US

Tab. 2 Effect of HP protection on liver function parameters

M

n= Number; ALT= Alanine aminotransferase; AST= Aspartate aminotransferase; HCC= Hepatocellular carcinoma; HP= Hesperidin, Values are expressed

AC

CE

PT

ED

as mean ± S.E.M.; a = Significant at P < 0.01 against normal control group, b = at P < 0.01 against HCC group; c= Significant at P < 0.05 against HCC

- 23 -

ACCEPTED MANUSCRIPT 8. Figures Fig. 1 a

b

ED

M

AN

US

CR

IP

T

a

PT

b

AC

CE

a

b

- 24 -

ACCEPTED MANUSCRIPT c

c

AC

CE

PT

ED

M

AN

US

CR

IP

T

d

- 25 -

ACCEPTED MANUSCRIPT Fig. 2

a

AC

CE

PT

ED

M

AN

US

CR

IP

T

a

- 26 -

b

ACCEPTED MANUSCRIPT b

AC

CE

PT

ED

M

AN

US

CR

IP

T

a

- 27 -

b

ACCEPTED MANUSCRIPT Fig. 3 a

b

ED

M

AN

US

CR

IP

T

a

PT

b

AC

CE

a

b

- 28 -

ACCEPTED MANUSCRIPT Fig. 4 a

AC

CE

PT

ED

M

AN

US

CR

IP

T

a

- 29 -

b

ACCEPTED MANUSCRIPT b No treatment

HP

US

CR

IP

T

Control

ED

M

AN

HCC

PT

a

AC

CE

b

- 30 -

ACCEPTED MANUSCRIPT Fig. 5 a

a

CR

IP

T

b

ED

M

AN

US

a

CE

PT

b

AC

b

a

- 31 -

b

ACCEPTED MANUSCRIPT Fig. 6

a

AC

CE

PT

ED

M

AN

US

CR

IP

T

b

- 32 -

ACCEPTED MANUSCRIPT Fig. 7 No treatment

HP

b

T

Control

US

CR

IP

a

CE

PT

ED

M

AN

HCC

b

AC

a

- 33 -

ACCEPTED MANUSCRIPT Fig. 8 a

HP

No treatment

US

CR

IP

T

Control

AC

CE

PT

ED

M

AN

HCC

- 34 -

ACCEPTED MANUSCRIPT b HP

No treatment

US

CR

IP

T

Control

AC

CE

PT

ED

M

AN

HCC

- 35 -

ACCEPTED MANUSCRIPT

c

a

AC

CE

PT

ED

M

AN

US

CR

IP

T

b

- 36 -

ACCEPTED MANUSCRIPT Fig. 9 a

AN

US

CR

IP

T

a

AC

CE

PT

ED

M

b

- 37 -

b

ACCEPTED MANUSCRIPT c

c

AC

CE

PT

ED

M

AN

US

CR

IP

T

d

- 38 -

ACCEPTED MANUSCRIPT 9. Tables Tab. 1

Name*

Amplicon

Melting

Sequence

size

Temp.

193

58°C

NM_001107005.2

155

62°C

NM_022631.2

135

62 °C

NM_053357.2

147

62°C

NM_171992.4

130

61.9°C

Sequence

5`- CAACGGGAAACCCATCACCATC-3`

Wnt3a F

5`- AGGGCACTAACAAGTCGGGTTC-3`

5`- CACTGGGCATGATCTCCACGTA-3`

Wnt5a F

5`- GACGCACGAGAAAGGGAACG-3`

M

AN

Wnt3a R

5`- TGGCACTTACAGGCTACATCTGC-3`

β-catenin F

5`- CCACAGGACTACAAGAAACGGC-3`

ED

Wnt5a R

5`- TGGCGATATCCAAGGGCTTCTC-3`

Cyclin D1 F

5`- AACTTCCTCTCCTGCTACCGC-3`

CE

PT

β-catenin R

5`- GCCTTGGGATCGATGTTCTGC-3`

AC

Cyclin D1 R

CR

5`- ACGCCAGTAGACTCCACGACAT-3`

US

GAPDH R

IP

NM_017008.4

T

GAPDH F

Reference

- 39 -

ACCEPTED MANUSCRIPT Tab. 2 ALT

AST

(g/dl)

(U/L)

(U/L)

Normal control group, (n=9)

3.8±0.11

42.4±1.93

63.92±0.3

HP control group, (n=10)

3.6±0.12

32.2±1.816

IP

CR

a

2.6±0.12

HP- HCC group, (n=8)

3.37±0.08

c

93.72±4.61

AC

CE

PT

ED

M

AN

HCC group, (n=6)

T

Albumin

US

Group

- 40 -

49.22±2.005

a

b

71.85±1.7

130±8.3

a

63.51±1.86

b

ACCEPTED MANUSCRIPT 8. References [1]

Lamarca A, Mendiola M, Barriuso J. Hepatocellular carcinoma: Exploring the impact of ethnicity on

molecular biology. Crit Rev Oncol Hematol 2016. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55:74-108.

[3]

Maluccio M, Covey A. Recent progress in understanding, diagnosing, and treating hepatocellular

T

[2]

Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. Sorafenib in advanced hepatocellular

CR

[4]

IP

carcinoma. CA Cancer J Clin 2012;62:394-399.

[5]

US

carcinoma. N Engl J Med 2008;359:378-390.

Duncan JS, Whittle MC, Nakamura K, Abell AN, Midland AA, Zawistowski JS, et al. Dynamic

AN

reprogramming of the kinome in response to targeted MEK inhibition in triple-negative breast cancer. Cell

[6]

M

2012;149:307-321.

Lu LC, Hsu CH, Hsu C, Cheng AL. Tumor Heterogeneity in Hepatocellular Carcinoma: Facing the

Qu W, Zhu ZJ, Sun LY, Wei L, Liu Y, Zeng ZG. Salvage liver transplantation for hepatocellular carcinoma

PT

[7]

ED

Challenges. Liver Cancer 2016;5:128-138.

[8]

CE

recurrence after primary liver resection. Clin Res Hepatol Gastroenterol 2015;39:93-97. Zaghloul RA, El-Shishtawy MM, El Galil KH, Ebrahim MA, Metwaly AA, Al-Gayyar MM. Evaluation of

AC

antiglypican-3 therapy as a promising target for amelioration of hepatic tissue damage in hepatocellular carcinoma. Eur J Pharmacol 2015;746:353-362. [9]

Breuhahn K, Vreden S, Haddad R, Beckebaum S, Stippel D, Flemming P, et al. Molecular profiling of

human hepatocellular carcinoma defines mutually exclusive interferon regulation and insulin-like growth factor II overexpression. Cancer Res 2004;64:6058-6064.

- 41 -

ACCEPTED MANUSCRIPT [10]

Zhang J, Wang WL, Li Q, Qiao Q. Expression of transforming growth factor-alpha and hepatitis B

surface antigen in human hepatocellular carcinoma tissues and its significance. World J Gastroenterol 2004;10:830-833. [11]

Bosch FX, Ribes J, Cleries R, Diaz M. Epidemiology of hepatocellular carcinoma. Clin Liver Dis

Breuhahn K, Longerich T, Schirmacher P. Dysregulation of growth factor signaling in human

IP

[12]

T

2005;9:191-211, v.

[13]

CR

hepatocellular carcinoma. Oncogene 2006;25:3787-3800.

Idobe Y, Murawaki Y, Kitamura Y, Kawasaki H. Expression of transforming growth factor-beta 1 in

US

hepatocellular carcinoma in comparison with the non-tumor tissue. Hepatogastroenterology 2003;50:54-59. Willert K, Nusse R. Wnt proteins. Cold Spring Harb Perspect Biol 2012;4:a007864.

[15]

Ahmed K, Shaw HV, Koval A, Katanaev VL. A Second WNT for Old Drugs: Drug Repositioning against

M

AN

[14]

WNT-Dependent Cancers. Cancers (Basel) 2016;8.

Giles RH, van Es JH, Clevers H. Caught up in a Wnt storm: Wnt signaling in cancer. Biochim Biophys

ED

[16]

Neth P, Ciccarella M, Egea V, Hoelters J, Jochum M, Ries C. Wnt signaling regulates the invasion

CE

[17]

PT

Acta 2003;1653:1-24.

capacity of human mesenchymal stem cells. Stem Cells 2006;24:1892-1903. Pan LH, Yao M, Cai Y, Gu JJ, Yang XL, Wang L, et al. Oncogenic Wnt3a expression as an estimable

AC

[18]

prognostic marker for hepatocellular carcinoma. World J Gastroenterol 2016;22:3829-3836. [19]

Gordon MD, Nusse R. Wnt signaling: multiple pathways, multiple receptors, and multiple transcription

factors. J Biol Chem 2006;281:22429-22433. [20]

Clevers H, Nusse R. Wnt/beta-catenin signaling and disease. Cell 2012;149:1192-1205.

[21]

Wong N, Lai P, Pang E, Fung LF, Sheng Z, Wong V, et al. Genomic aberrations in human hepatocellular

carcinomas of differing etiologies. Clin Cancer Res 2000;6:4000-4009. - 42 -

ACCEPTED MANUSCRIPT [22]

Bi L, Liu X, Wang C, Cao Y, Mao R, Li P, et al. Wnt5a involved in regulation of the biological behavior of

hepatocellular carcinoma. Int J Clin Exp Pathol 2014;7:987-995. [23]

Bauer M, Benard J, Gaasterland T, Willert K, Cappellen D. WNT5A encodes two isoforms with distinct

functions in cancers. PLoS One 2013;8:e80526. Li P, Cao Y, Li Y, Zhou L, Liu X, Geng M. Expression of Wnt-5a and beta-catenin in primary

T

[24]

Sacco SM, Saint C, LeBlanc PJ, Ward WE. Maternal Consumption of Hesperidin and Naringin Flavanones

CR

[25]

IP

hepatocellular carcinoma. Int J Clin Exp Pathol 2014;7:3190-3195.

[26]

US

Exerts Transient Effects to Tibia Bone Structure in Female CD-1 Offspring. Nutrients 2017;9. Habauzit V, Sacco SM, Gil-Izquierdo A, Trzeciakiewicz A, Morand C, Barron D, et al. Differential effects

AN

of two citrus flavanones on bone quality in senescent male rats in relation to their bioavailability and

[27]

M

metabolism. Bone 2011;49:1108-1116.

Wei D, Ci X, Chu X, Wei M, Hua S, Deng X. Hesperidin suppresses ovalbumin-induced airway

Hirata A, Murakami Y, Shoji M, Kadoma Y, Fujisawa S. Kinetics of radical-scavenging activity of

PT

[28]

ED

inflammation in a mouse allergic asthma model. Inflammation 2012;35:114-121.

hesperetin and hesperidin and their inhibitory activity on COX-2 expression. Anticancer Res 2005;25:3367-

Selvaraj P, Pugalendi KV. Hesperidin, a flavanone glycoside, on lipid peroxidation and antioxidant

AC

[29]

CE

3374.

status in experimental myocardial ischemic rats. Redox Rep 2010;15:217-223. [30]

Umeno A, Horie M, Murotomi K, Nakajima Y, Yoshida Y. Antioxidative and Antidiabetic Effects of

Natural Polyphenols and Isoflavones. Molecules 2016;21. [31]

Yumnam S, Hong GE, Raha S, Saralamma VV, Lee HJ, Lee WS, et al. Mitochondrial Dysfunction and

Ca(2+) Overload Contributes to Hesperidin Induced Paraptosis in Hepatoblastoma Cells, HepG2. J Cell Physiol 2016;231:1261-1268.

- 43 -

ACCEPTED MANUSCRIPT [32]

Balakrishnan A, Menon VP. Protective effect of hesperidin on nicotine induced toxicity in rats. Indian J

Exp Biol 2007;45:194-202. [33]

Dasgupta A, Chatterjee R, Chowdhury JR. Thioacetamide-induced hepatocarcinoma in rat. Oncology

1981;38:249-253. Suzuki K, Ota H, Sasagawa S, Sakatani T, Fujikura T. Assay method for myeloperoxidase in human

T

[34]

Makled MN, El-Awady MS, Abdelaziz RR, Atwan N, Guns ET, Gameil NM, et al. Pomegranate protects

CR

[35]

IP

polymorphonuclear leukocytes. Anal Biochem 1983;132:345-352.

liver against cecal ligation and puncture-induced oxidative stress and inflammation in rats through TLR4/NF-

McPartland JL, Guzail MA, Kendall CH, Pringle JH. Apoptosis in chronic viral hepatitis parallels

AN

[36]

US

kappaB pathway inhibition. Environ Toxicol Pharmacol 2016;43:182-192.

histological activity: An immunohistochemical investigation using anti-activated caspase-3 and M30 Cytodeath

M

antibody. International Journal of Experimental Pathology 2005;86:19-24. Njei B, Rotman Y, Ditah I, Lim JK. Emerging trends in hepatocellular carcinoma incidence and mortality.

ED

[37]

[38]

PT

Hepatology 2015;61:191-199.

Anwar WA, Khaled HM, Amra HA, El-Nezami H, Loffredo CA. Changing pattern of hepatocellular

Alavian SM, Haghbin H. Relative Importance of Hepatitis B and C Viruses in Hepatocellular Carcinoma

AC

[39]

CE

carcinoma (HCC) and its risk factors in Egypt: possibilities for prevention. Mutat Res 2008;659:176-184.

in EMRO Countries and the Middle East: A Systematic Review. Hepat Mon 2016;16:e35106. [40]

Lin S, Hoffmann K, Schemmer P. Treatment of hepatocellular carcinoma: a systematic review. Liver

Cancer 2012;1:144-158. [41]

Waly Raphael S, Yangde Z, Yuxiang C. Hepatocellular carcinoma: focus on different aspects of

management. ISRN Oncol 2012;2012:421673.

- 44 -

ACCEPTED MANUSCRIPT [42]

Llovet JM, Bruix J. Molecular targeted therapies in hepatocellular carcinoma. Hepatology

2008;48:1312-1327. [43]

Fathy SA, Emam MA, Abo Agwa SH, Abu Zahra FA, Youssef FS, Sami RM. The antiproliferative effect of

Origanum majorana on human hepatocarcinoma cell line: suppression of NF-kappaB. Cell Mol Biol (Noisy-le-

Li W, Yu J, Lian B, Sun H, Li J, Zhang M, et al. Identifying prognostic features by bottom-up approach

[45]

CR

and correlating to drug repositioning. PLoS One 2015;10:e0118672.

Ma W, Wu L, Zhou F, Hong Z, Yuan Y, Liu Z. T Cell-Associated Immunotherapy for Hepatocellular

US

Carcinoma. Cell Physiol Biochem 2017;41:609-622.

Wang ZW, Lu XF, Tuo HY, Cheng XL, Guo M. [The protective effect of Yuyin Ruangan Decoction on

AN

[46]

IP

[44]

T

grand) 2016;62:80-84.

[47]

M

experimental hepatic injury]. Zhongguo Ying Yong Sheng Li Xue Za Zhi 2015;31:76-79. Oguzturk H, Ciftci O, Cetin A, Kaya K, Disli OM, Turtay MG, et al. Beneficial effects of hesperidin

Li X, Hu X, Wang J, Xu W, Yi C, Ma R, et al. Short-Term Hesperidin Pretreatment Attenuates Rat

PT

[48]

ED

following cis-diamminedichloroplatinum-induced damage in heart of rats. Niger J Clin Pract 2016;19:99-103.

Myocardial Ischemia/Reperfusion Injury by Inhibiting High Mobility Group Box 1 Protein Expression via the

Xiong YJ, Chu HW, Lin Y, Han F, Li YC, Wang AG, et al. Hesperidin alleviates rat postoperative ileus

AC

[49]

CE

PI3K/Akt Pathway. Cell Physiol Biochem 2016;39:1850-1862.

through anti-inflammation and stimulation of Ca(2+)-dependent myosin phosphorylation. Acta Pharmacol Sin 2016;37:1091-1100. [50]

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;15:313323.

- 45 -

ACCEPTED MANUSCRIPT [51]

Rekha RD, Amali AA, Her GM, Yeh YH, Gong HY, Hu SY, et al. Thioacetamide accelerates

steatohepatitis, cirrhosis and HCC by expressing HCV core protein in transgenic zebrafish Danio rerio. Toxicology 2008;243:11-22. [52]

Chen T, Zamora R, Zuckerbraun B, Billiar TR. Role of nitric oxide in liver injury. Curr Mol Med

Shaban NZ, Ahmed Zahran AM, El-Rashidy FH, Abdo Kodous AS. Protective role of hesperidin against

IP

[53]

T

2003;3:519-526.

[54]

CR

gamma-radiation-induced oxidative stress and apoptosis in rat testis. J Biol Res (Thessalon) 2017;24:5. Cetin A, Ciftci O, Otlu A. Protective effect of hesperidin on oxidative and histological liver damage

Lamb R, Ablett MP, Spence K, Landberg G, Sims AH, Clarke RB. Wnt pathway activity in breast cancer

AN

[55]

US

following carbon tetrachloride administration in Wistar rats. Arch Med Sci 2016;12:486-493.

[56]

M

sub-types and stem-like cells. PLoS One 2013;8:e67811.

Lai JP, Oseini AM, Moser CD, Yu C, Elsawa SF, Hu C, et al. The oncogenic effect of sulfatase 2 in human

ED

hepatocellular carcinoma is mediated in part by glypican 3-dependent Wnt activation. Hepatology

[57]

PT

2010;52:1680-1689.

Yang T, Cai SY, Zhang J, Lu JH, Lin C, Zhai J, et al. Kruppel-like factor 8 is a new Wnt/beta-catenin

Suarez MI, Uribe D, Jaramillo CM, Osorio G, Perez JC, Lopez R, et al. Wnt/beta-catenin signaling

AC

[58]

CE

signaling target gene and regulator in hepatocellular carcinoma. PLoS One 2012;7:e39668.

pathway in hepatocellular carcinomas cases from Colombia. Ann Hepatol 2015;14:64-74. [59]

Chouhan S, Singh S, Athavale D, Ramteke P, Pandey V, Joseph J, et al. Glucose induced activation of

canonical Wnt signaling pathway in hepatocellular carcinoma is regulated by DKK4. Sci Rep 2016;6:27558. [60]

Lachenmayer A, Alsinet C, Savic R, Cabellos L, Toffanin S, Hoshida Y, et al. Wnt-pathway activation in

two molecular classes of hepatocellular carcinoma and experimental modulation by sorafenib. Clin Cancer Res 2012;18:4997-5007.

- 46 -

ACCEPTED MANUSCRIPT [61]

Inagawa S, Itabashi M, Adachi S, Kawamoto T, Hori M, Shimazaki J, et al. Expression and prognostic

roles of beta-catenin in hepatocellular carcinoma: correlation with tumor progression and postoperative survival. Clin Cancer Res 2002;8:450-456. [62]

Tao L, Yang JK, Gu Y, Zhou X, Zhao AG, Zheng J, et al. Weichang'an and 5-fluorouracil suppresses

Ge YX, Wang CH, Hu FY, Pan LX, Min J, Niu KY, et al. New advances of TMEM88 in cancer initiation and

IP

[63]

T

colorectal cancer in a mouse model. World J Gastroenterol 2015;21:1125-1139.

[64]

CR

progression, with special emphasis on Wnt signaling pathway. J Cell Physiol 2017.

Kannangai R, Wang J, Liu QZ, Sahin F, Torbenson M. Survivin overexpression in hepatocellular

Bengochea A, de Souza MM, Lefrancois L, Le Roux E, Galy O, Chemin I, et al. Common dysregulation of

AN

[65]

US

carcinoma is associated with p53 dysregulation. Int J Gastrointest Cancer 2005;35:53-60.

[66]

M

Wnt/Frizzled receptor elements in human hepatocellular carcinoma. Br J Cancer 2008;99:143-150. Kordes C, Sawitza I, Haussinger D. Canonical Wnt signaling maintains the quiescent stage of hepatic

Kavitha K, Vidya Priyadarsini R, Anitha P, Ramalingam K, Sakthivel R, Purushothaman G, et al.

PT

[67]

ED

stellate cells. Biochem Biophys Res Commun 2008;367:116-123.

Nimbolide, a neem limonoid abrogates canonical NF-kappaB and Wnt signaling to induce caspase-dependent

AC

CE

apoptosis in human hepatocarcinoma (HepG2) cells. Eur J Pharmacol 2012;681:6-14.

- 47 -