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HEPATOCELLULAR CARCINOMA IN RAT MODEL .... smoked, salted and dried fish, cured meat, alcoholic beverages as well as in ground water .... tissue sections were collected on clean glass slides and left in the oven at 40ºC for dryness.
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Hanaa et al.

World Journal of Pharmacy and Pharmaceutical Sciences

Volume 2, Issue 5, 2367-2396.

Research Article

ISSN 2278 – 4357

IN VIVO ANTITUMOR POTENTIAL OF CARVACROL AGAINST HEPATOCELLULAR CARCINOMA IN RAT MODEL Hanaa H. Ahmed1* , Wafaa Gh. Shousha 2 , Hatem A. El-Mezayen 2 ,Nora N. Ismaiel 3 , Nadia S. Mahmoud1 1

Hormones Department, Medical Research Division, National Research Centre, Dokki, Giza, Egypt. 2

3

Chemistry Department ,Faculty of Science, Helwan University, Cairo , Egypt.

Molecular Genetics and Enzymology Department, Human Genetics and Genome researches, National Research Centre, Dokki, Giza, Egypt.

Article Received on 02 August 2013, Revised on 23 August 2013, Accepted on 27 September 2013

ABSTRACT This study aimed to investigate the efficacy of carvacrol against hepatocellular carcinoma-induced in rats. Forty male rats were divided into 5 groups. Group (1) was negative control. Groups (2), (4) and (5) were orally administrated diethylnitrosamine for induction of

*Correspondence for Author:

hepatocellular carcinoma then group (2) was left untreated ; group (4) was treated orally with carvacrol, while, group (5) were intraperitoneal

* Prof. Hanaa H. Ahmed

injected with doxorubicin. Group (3) was orally treated with carvacrol

Professor of Biochemistry

only. Serum alpha-fetoprotein (AFP), alpha L-fucosidase (AFU) and

Head of Hormones Department

vascular endothelial growth factor (VEGF) levels were assayed using

Medical Research Division National Research Centre,

ELISA technique. Gamma glutammyl transferase (GGT) gene

12622, Egypt. .

expression was detected by RT-PCR. Immunohistochemical analysis

[email protected]

of proliferating cell nuclear antigen (PCNA) and Ki-67 expression was performed. Apoptosis was detected using DNA fragmentation assay.

Also, histological investigation of liver tissue was achieved. The untreated cancer group showed significant elevation in the studied biochemical markers and appreciable increase in GGT expression. Moreover, cancer group exhibited remarkable increase in PCNA and Ki-67 expression. Furthermore, this group revealed no DNA fragmentation. Histopathological investigation of liver tissue sections in cancer group revealed typical anaplasia. In contrast, the treated groups showed significant depletion in the studied tumor markers and www.wjpps.com

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downregulation in GGT gene expression. Also, these groups displayed marked decrease in PCNA and Ki-67 expression. Carvacrol treated group revealed obvious DNA fragmentation. While, doxorubicin treated group displayed smear DNA patterns. Interestingly, treatment with carvacrol showed marked improvement in the histological feature of liver tissue. While, treatment with doxorubicin revealed no remarkable difference from the cancer group. This study indicated the promising therapeutic potential of carvacrol against hepatocellular carcinoma through its antiangiogenic, antiproliferative and apoptotic effects. Keywords: Hepatocellular carcinoma, carvacrol, proliferation, apoptosis, angiogenesis, in vivo. INTRODUCTION Hepatocellular carcinoma (HCC) is a malignant neoplasm of hepatocytes that is considered as the most common primary cancer of liver, the fifth most common malignancy worldwide and the third leading cause of cancer death, exceeded only by cancers of the lung and stomach. [1] It is a major health problem worldwide, due to its high incidence and high rates of mortality. [2]

This cancer varies widely in incidence throughout the world, higher incidence areas are

East Asian countries such as China and Taiwan and in sub-Saharan Africa, while low incidence areas are western countries such as the USA, Europe and Australia. [3] In Egypt, HCC is the second most frequent cause of cancer incidence and mortality among men.

[4]

Its incidence is expected to increase significantly in the next decade because of high

prevalence rate of HCV in the general population which accounts for most of the cirrhosis and HCC cases. [5] Hospital-based studies from Egypt have reported an increase in the relative frequency of all liver-related cancers in Egypt (>95% as HCC), from 4.0% in 1993 to 7.3% in 2003. [6] Major risk factors for HCC include chronic infection with HBV or HCV, alcoholic liver disease, and most probably nonalcoholic fatty liver disease. Less common causes include hereditary hemochromatosis, alpha1-antitrypsin deficiency, autoimmune hepatitis, some porphyrias, and Wilson’s disease. The distribution of these risk factors among patients with hepatocellular carcinoma is highly variable, depending on geographic region and race or ethnic group.[7] Most of these risk factors lead to the formation and progression of cirrhosis, which is present in 80 to 90% of patients with hepatocellular carcinoma.[8]

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Hepatic injury induced by different risk factors causes necrosis followed by hepatocyte proliferation. Repeated cycles of necrosis-liver regeneration foster a chronic liver disease leading to cirrhosis. Cirrhosis is characterized by the formation of hyperplastic liver nodules, surrounded by collagen deposition and scarring of liver as well as regenerative nodules. During this process, different subtypes of foci of altered hepatocytes (FAH) develop in the liver, followed by low-grade dysplastic nodules (DNs), and then by high-grade DNs that are considered as premalignant lesions .Subsequently, HCC develop which can be further classified into well differentiated, moderately differentiated and poorly differentiated tumors. [9]

Diethylnitrosamine (NDEA) is found in a wide variety of foods such as cheese, soybeans, smoked, salted and dried fish, cured meat, alcoholic beverages as well as in ground water having a high level of nitrates.[10] In rats, NDEA is a potent hepatocarcinogen influencing the initiation stage of carcinogenesis during a period of enhanced cell proliferation accompanied by hepatocellular necrosis and induces DNA carcinogen adducts, DNA-strand breaks and in turn hepatocellular carcinomas without cirrhosis through the development of putative preneoplastic focal lesions.[11] The treatment of hepatocellular carcinoma (HCC) remains a dismal, with 1- and 3-year survival rates of 20% and 5%, respectively and a median survival of 8 months. The current therapies for HCC include hepatic artery embolization and chemotherapy, radiofrequency ablation, and cryoablation [12] as well as liver transplantation which can provide survival rate of 28% at 3 years. [13] Although these therapies play an important role in HCC treatment, but its therapeutic outcome remains very poor due to its toxic side effects. Moreover, there is a significant resistance to available chemotherapeutic agents and liver cannot tolerate frequent doses of radiation.[14]Therefore; there is an urgent need for the naturally occurring agents and plants extracts that have anticancer potential with low toxicity and side effects.[15] Natural dietary agents including fruits, vegetables, and spices have attracted a great attention from both scientific community and the general public due to their demonstrated ability to suppress cancers.[16] Carvacrol is a predominant mono-terpenicphenol which occurs in many essential oil of the family Labiatae including Origanum, Ocimum and Thymus species. [17] It was reported to have a broad range of pharmacological properties including antiinflammatory,

[18]

antioxidant,

[19]

antitumor activities,

[20]

anti-proliferative and anti-

carcinogenic effects which are strongly supported by in vitro studies. [21] www.wjpps.com

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The current study was undertaken to elucidate the possible therapeutic potential of carvacrol against diethylnitrosamine-induced hepatocellular carcinoma in male rats with special concern on its mechanism of action. MATERIALS AND METHODS 1. Materials Chemicals and reagents N-nitrosodiethylamine (NDEA) (CAS no. 55-18-5) and Carvacrol (Cat # 282197) were purchased from Sigma-Aldrich Chemicals Co. (St Louis, MO, USA). Doxorubicin was supplied from Pharmacia Italia S.P.A, Milan, Italy. All other chemicals used were of high analytical grade and were locally purchased (Egypt). Animals and experimental design Forty adult male Wistar rats weighing 170-200 g were obtained from the Animal House Colony of the National Research Centre, Cairo, Egypt. The animals were housed in polypropylene cages in an environmentally controlled clean air room with a temperature of 25±1˚C, an alternating 12h light/12h dark cycle, a relative humidity of 60 ± 5% and free access to tap water and a standard rodent chow (Wadi El Kabda Co., Cairo, Egypt). Rats were allowed to adapt to these conditions for 2 weeks before beginning the experimental protocol. The animal experimental protocol was approved by the Ethical Committee for Medical Research, National Research Centre, Egypt. After the acclimatization period, the animals were divided into five groups; each group was comprised of eight rats: (1): Normal healthy animals served as negative control group. (2): Hepatocellular carcinoma (HCC) group in which rats were orally administered NDEA (dissolved in 0.9% normal saline), in a dose of 20 mg/kg b.wt. five times a week for six weeks according to the modified method of Darwish and El-Boghdady.[22] (3): Carvacrol group in which rats were treated orally only with carvacrol (dissolved in Tween-80 (1%) in saline) in a dose of 15 mg/kg b.wt. (five days a week) for 15 weeks. (4): Carvacrol-treated group in which rats were treated orally with carvacrol (dissolved in Tween-80 (1%) in saline) in a dose of 15 mg/kg b.wt. five times a week for 15 weeks, following the administration of NDEA for six weeks, as previously reported by Jayakumar et al. [23] (5): Doxorubicin-treated group in which rats were intraperitoneally injected with doxorubicin as a reference drug (dissolved in 0.9% saline) in a dose of 0.72 mg/rat which is equivalent to the human dose 20

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mg/m2 according to Barnes and Paget equation [24] once a week for 15 weeks, following the administration of NDEA for six weeks. Blood sampling At the end of the experimental period, the animals were fasted overnight (12-14 h) and the blood samples of each animal were collected, under diethyl ether anesthesia, from the retroorbital venous plexus in a clean dry centrifuge tubes without any anticoagulant agent and allowed to coagulate for 45 minutes at room temperature to obtain the sera to be used for biochemical analysis. Serum samples were separated by centrifugation at 1800 xg for 15 minutes at 4˚C using cooling centrifuge. Aliquots of serum were frozen and stored at -20˚C for further biochemical analysis. Liver tissue sampling After blood collection, the animals were sacrificed by cervical decapitation, dissected and the whole liver of each rat was rapidly excised and thoroughly washed with isotonic saline. The whole liver of animals in each group was divided into three portions; the first and second portions were fixed in formal saline (10%) for 24 h for immunohistochemical analysis and histological examination respectively. Whereas, the third portion was snap-frozen directly in liquid nitrogen and stored at -80°C prior to RNA isolation for gene expression analysis. 2. METHODS Biochemical analysis Serum alpha-fetoprotein was estimated by enzyme linked immunosorbent assay (ELISA) technique using a rat alpha-fetoprotein (AFP) ELISA kit purchased from Glory Science Co., Ltd (USA), according to the manufacturer’s instructions provided with AFP assay kit. Serum vascular endothelial growth factor was estimated by rat enzyme linked immunosorbent assay (ELISA) technique using a kit purchased from Glory Science Co., Ltd (USA), according to the manufacturer’s instructions provided with VEGF assay kit. Serum alpha-L-fucosidase activity was estimated by colorimetric method using Biodiagnostic AFU assay kit purchased locally (Egypt), according to the method described by Zielke et al. [25] Immunohistochemical method Liver portion of rats in the different groups, fixed in 10% formal saline for 24 hours, was washed in tap water and then subjected to serial dilutions of alcohol (methyl, ethyl and absolute ethyl) for dehydration. Specimens were cleared in xylene and embedded in paraffin

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at 56 degree in hot air oven for 24 hours. Sections were cut into 4µ by slidge microtome then fixed on positive slides in a 65ºC oven for 1 hr. Slides were placed in a coplin jar filled with 200 mL of triology working solution (Cell Marque, CA-USA. Cat# 920p-06) which is a product that combines the three pretreatment steps: deparaffinization, rehydration and antigen unmasking. Then, the jar is securely positioned in the autoclave which was adjusted so that temperature reached 120oC and maintained stable for 15 minutes after which pressure is released. Thereafter, the coplin jar is removed to allow slides to cool for 30 minutes. Sections were then washed and immersed in Tris-buffer saline (TBS) to adjust the pH; this is repeated between each step of the IHC procedure. Quenching endogenous peroxidase activity was performed by immersing slides in 3% hydrogen peroxide for 10 minutes. Broad spectrum LAB-SA detection system (Invitrogen, USA) was used to visualize any antigen-antibody reaction in the tissues. Background staining was blocked by putting 2-3 drops of 10% goat non immune serum blocker on each slide and incubating them in a humidity chamber for 10 minutes. Without washing, excess serum was drained and the working dilution (1:100) of the two primary antibodies; proliferating cell nuclear antigen (PCNA) monoclonal antibody PC10 (Lab Vision Co., Fremont, California, USA. Cat # MS-106-R7) and Ki-67 polyclonal antibody (Lab Vision Co., Fremont, California, USA. Cat# RB-9043-R7) were prepared. Two-three drops of the working dilution were applied. Then, slides were incubated in the humidity chamber overnight at 4ºC. Henceforward, biotinylated secondary antibody was applied on each slide for 20 minutes followed by 20 minutes incubation with the streptavidin horse reddish peroxidase (HRP) enzyme conjugate. 3,3´-diaminobenzidine (DAB) chromogen was prepared and 2-3 drops were applied on each slide for 2 minutes. DAB was rinsed, after which counterstaining with Mayer hematoxylin and cover slipping were performed as the final steps before slides were examined under the light microscope.

[26]

Image J software (NIH, version v1.45e, USA) was calibrated and the image is opened on the computer screen for image analysis. Histopathological examination After fixation of the second liver portion of rats in the studied groups in formal saline (10%) for 24 hours, the tissues were then washed in running tap water, dehydrated in series of alcohol (methyl, ethyl and absolute alcohol). The specimens were cleared in xylene and embedded in paraffin at 56 degree in hot air oven for twenty four hours. The paraffin bees wax tissue blocks were sectioned by slidge microtome at thickness of 4 µm. The obtained tissue sections were collected on clean glass slides and left in the oven at 40ºC for dryness.

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The slides were deparaffinized in xylol and then immersed in descending series of alcohol, stained by hematoxylin and eosin stain then examined through the light electric microscope. [27]

Gene expression method RT-PCR analysis Total RNA was isolated from liver tissues of rats in each group using the standard Trizol extraction method (Bioshop Canada Inc.) described by Chomczynski.[28] Two microgram of the isolated RNA was reverse transcribed into cDNA in a total volume of 20 µl using RevertAid first strand cDNA synthesis kit (Fermentas Co.,USA). The PCR was performed using 5 µg of cDNA in a final volume of 20 µl containing 10x PCR buffer, 10mM dNTPs, 2U/µl of Taq DNA polymerase (Fermentas, USA) and 10µM of each appropriate sequencespecific

primers.

dehydrogenase)

The was

housekeeping used

as

gene

internal

GAPDH control

(glyceraldhyde with

a

3

phosphate

primer

sequence;

forward:5`CAAGGTCATCCATGACAACTTTG3`,reverse:5`GTCCACCACCCTGTTGCT GTAG-3`. The primer sequence of gamma glutamyl transferase (GGT) used was; forward: 5'CTCTGCATCTGGCTACCCAC-3',

reverse:

5’-GGATGCTGGGTTGGAAGAGG-3'.

GAPDH primer sequence was designed to yield a PCR product of 496 bp (MetabionGermany). GGT primer sequence (Metabion-Germany) was designed according to CarrascoLegleu et al.

[29]

The PCR cycling was performed using gradient thermal cycler (BioRad,

USA) as follow; The samples were initially denatured at 94ºC for 5 minutes then denatured at 94ºC for 30 seconds. Amplification was carried out using 35 standard PCR cycles with annealing temperature at 57ºC for 30 seconds, extension at 72ºC for 1 minute and final extension at 72ºC for 8 minutes. Aliquots of 15 µl of each PCR sample were loaded into 2 % agarose gel stained with ethidium bromide (EtBr) and separated by electrophoresis at 130 V cm−1 for 30 minutes. The gel was visualized and photographed under the ultraviolet (UV) trans-illuminator to detect successful amplification. The amplified product size was determined by comparison to markers (100 bp) DNA ladder (Fermentas, USA). The amplified product for GGT was 418 bp. Qualitative DNA fragmentation assay Isolation of DNA was performed according to the method of Sambrook et al [30]. Briefly, 0.02 g of liver tissue from different groups of rats was homogenized in lysis buffer and proteinase K. After incubation at 56 °C for 2 h, samples were extracted twice with phenol–chloroform– isoamyl alcohol (25:24:1, by volume) and centrifuged at 12,000 xg for 5 min at 4 °C. DNA

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was precipitated with isopropanol. After centrifugation at 12,000×g for 10 minutes at 4 °C, DNA pellets were then dissolved in deionized water and mixed with DNase–free RNase A. Analysis of DNA fragments was carried out on 2% agarose gel containing EtBr and done at 130 V cm−1 for~1 h. The gel was then examined and photographed under UV light to visualize intra-nucleosomal DNA fragmentation, characteristic of apoptosis. Statistical analysis In the present study, the results were expressed as Mean + S.E of the mean. Data were analyzed by one way analysis of variance (ANOVA) using the Statistical Package for the Social Sciences (SPSS) program, version 14 followed by least significant difference (LSD) to compare significance between groups.[31] Difference was considered significant when P value was < 0.05. Percentage difference representing the percent of variation with respect to the corresponding control group was also calculated using the following formula:

RESULTS Biochemical analyses The data in Table (1) illustrated the effect of carvacrol and doxorubicin treatment on serum levels of AFP, VEGF and AFU in rats bearing hepatocellular carcinoma (HCC). Our results demonstrated that there is significant increase (p