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Biomedicine & Preventive Nutrition 2 (2012) 1–8

Available online at

www.sciencedirect.com

riginal article

poptosis associated inhibition of DEN-induced hepatocellular carcinogenesis byllagic acid in experimental rats

risesharam Srigopalrama, Soundherrajan Ilavenilb, Indira A. Jayraaja,∗

Department of Biochemistry, Kongunadu Arts and Science College (Autonomus), Coimbatore, Tamil Nadu, IndiaCentre for Research and Development, PRIST University, Thanjavur 613 403, Tamil Nadu, India

r t i c l e i n f o

rticle history:eceived 3 December 2011ccepted 14 December 2011

eywords:llagic acidiethylnitrosaminepoptosisepatocarcinogenesis

a b s t r a c t

Ellagic acid (EA), a natural polyphenol, has showed a wide variety of biological activities which makeit a good candidate for the treatment of many oxidative stress-mediated diseases. The present study isaimed to evaluate its therapeutic potential by estimating the levels of lipid peroxidation and assayingactivities of various marker enzymes in DEN-induced liver cancer bearing rats. The daily oral admin-istration of EA (30 mg/kg bwt) to liver cancer bearing rats demonstrated a significant (P < 0.05) declinein lipid peroxidation, key marker enzyme (AST, ALT, ALP, LDH, �-GT and 5′NT) levels and increase inenzymic antioxidants (SOD, CAT, GPx, GR and GST) status. Hematoxylin and eosin (HE) staining andscanning electron microscope (SEM) analysis suggesting that maintenance of cell structure and integrityand also modulation of nucleic acids thereby exhibiting anticancer potential of EA in liver cancer bear-

canning electron microscope

itochondrial permeability ing rats. Further, EA administration attenuated the agryophillic nucleolar organizing regions (AgNORs)and increases cell death execution through the activation of Caspase-3. Thus, the modulatory effects ofEA on attenuating the lipid peroxidation, AgNORs, and downregulation of key marker enzyme activitiesand upregulation of total protein content, enzymic antioxidants, and caspase-3 afford an assurance fortreatment of liver cancer in the future.

. Introduction

Hepatocellular carcinoma (HCC), a highly aggressive form ofolid tumor, has been increasing in South East Asia [1]. Primary hep-tocellular carcinoma (HCC) is one of the most frequently occurringorms of a solid tumor. It exhibits a high prevalence with 620,000ases per year reported worldwide of which more than 80% of casesre reported from China, Africa and South East Asia[2]. It is highlyggressive, as shown by the mortality of 595,000 cases per year thatearly matches the incidence of this tumor type [3]. HCC presentsith limited therapeutic options. Hence, a thorough understand-

ng of the biological bases of this malignancy might suggest newtrategies for effective treatment.

Diethylnitrosamine (DEN), one of the most important environ-ental carcinogens, which is known to cause perturbations in the

uclear enzymes involved in DNA repair/replication and is nor-ally used as a carcinogen to induce liver cancer in animal models

4]. N-nitroso compounds are known hepatocarcinogenic agents

nd have been implicated in the etiology of several human cancers5]. These compounds are considered to be effective health hazardso man.

∗ Corresponding author. Tel.: +91 98 42225776.E-mail address: indirajayraaj@gmail.com (I.A. Jayraaj).

210-5239/$ – see front matter © 2012 Elsevier Masson SAS. All rights reserved.oi:10.1016/j.bionut.2011.12.003

© 2012 Elsevier Masson SAS. All rights reserved.

One approach to control liver cancer is chemoprevention–whendisease is prevented, slowed or reversed substantially by theadministration of one or more non-toxic naturally occurringor synthetic agents. In this regard, recently naturally occurringpolyphenols are receiving increased attention because of theirpromising efficacy in several cancer models [6]. EA is one of suchnaturally occurring compounds present in many plant foods suchas carrot, tomato, strawberry and blueberry [7]. It has been docu-mented that EA possess anti-oxidative activities such as scavengingfree radicals and chelating metal ions [8].

A marker is synthesized by the tumor and released into the cir-culation, but it may be produced by normal tissues in responseto invasion by cancer cells [9]. A variety of substances, includ-ing enzymes, hormones, and proteins can be considered as tumormarkers. Analysis of tumor markers can be used as an indicatorof tumor response to therapy. Sensitive and specific liver cancermarker enzymes are used as indicators of liver injury. Analysisof these marker enzymes reflects mechanisms of cellular dam-age and subsequent release of proteins and extracellular turnover[10]. Lipid peroxidation generates a complex variety of products,many of which are reactive electrophiles; some of these react with

protein and DNA and as a result are toxic and mutagenic [11].Taking the above into account, our present study was carried outto assess the efficacy of EA on DEN-induced HCC in experimentalrats.

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. Materials and method

.1. Animals

Male wistar albino rats, weighing 150–180 g, procured from themall Animal Breeding Centre, Agricultural University, Mannuthy,erala were used. Animals were acclimatized under standard labo-atory conditions at 25◦ ± 2 ◦C and normal photoperiod (12 h light:ark cycle). The animals were fed with standard rat chow and waterd libitum. The food was withdrawn 18–24 h before the experiment.he care and use of laboratory animals were done according to theuidelines of the Council Directive CPCSEA, India (No: 659/02/a)bout Good Laboratory Practice (GLP) on animal experimentation.ll animal experiments were performed in the laboratory accord-

ng to the ethical guidelines suggested by the Institutional Animalthics Committee (IAEC).

.2. Hepatocarcinogenesis initiation using diethylnitrosamineDEN)

The experimental hepatocarcinogenesis was induced by usingEN (Sigma, USA). DEN is the most important environmental car-inogen among nitrosamines and primarily induces tumors of liver12]. The presence of nitrosamines and their precursors in humannvironment, together with the possibility of their endogenousormation in human body from ingested secondary amines anditrites, have led to the suggestions of their potential involvement

n HCC [13]. It is now widely used as a standard experimental modelor HCC [14].

.3. Experimental design

The experimental animals were divided into four groups, eachroup containing six animals, analyzed for a total experimentaleriod of 16 weeks as follows:

group 1: normal control rats fed with standard rat chow and puredrinking water;group 2 (EA alone): rats were orally given EA (30 mg/kg bodyweight) in the form of aqueous suspension daily once a day for16 weeks. This dose of EA is set based on the effective dosagefixation studies;group 3: rats were induced with DEN (0.01%) alone in drinkingwater for 16 weeks;group 4: rats were administered DEN (0.01%) in drinking waterfor the first 10 weeks followed by post-treatment with EA as ingroup 2 for the remaining 6 weeks.

At the end of 16 weeks, experimental rats (n = 6 per group) wereacrificed. The liver tissues were collected and homogenized by

Teflon® homogenizer in phosphate buffered saline, pH 7.4 andamples were stored at −80 ◦C for further assays.

.4. Biochemical studies

The protein content was estimated by the method of Lowry et al.15] using bovine serum albumin (BSA) as standard. The DNA wasstimated by the method of Burton [16] and RNA was estimatedy the method of Rawal et al. [17]. The macromolecular damageuch as LPO was estimated by the method of Ohkawa et al. [18].he activities of aspartate transaminase (AST), alanine transam-nase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase

LDH) were estimated by the method of King [19–21]. Gamma-lutamyl transpeptidase (�-GT) was estimated by the method ofosali and Rau [22]. 5′-nucleotidase (5′NT) was estimated by theethod of Luly et al. [23], and the activities of the antioxidant

eventive Nutrition 2 (2012) 1–8

enzymes superoxide dismutase (SOD) Misra and Fridovich [24],catalase (CAT) Sinha [25], glutathione peroxidase (GPx) Rotrucket al. [26], glutathione transferase (GST) Habig et al. [27], and glu-tathionereductase (GR) Maron et al. [28] were estimated in liver.

2.5. Histopathological studies

A portion of the liver was cut into two to three pieces ofapproximately 6 mm3 sizes and fixed in phosphate buffered 10%formaldehyde solution. After embedding in paraffin wax, thin sec-tions of 5 �m thickness of liver tissues were cut and stainedwith haematoxylin–eosin. The thin sections of liver were madeinto permanent slides and examined [29] under high-resolutionmicroscope with photographic facility and photomicrographs weretaken. For scanning electron microscope (SEM), the intact liversamples were prepared according to methods described previouslyreported [30].

2.6. Argyophillic nucleolar organizing regions (AgNORstaining)

AgNOR staining was performed according to the method of Plo-ton et al. [31]. The liver sections were dewaxed in xylene andrehydrated through decreasing concentrations of ethanol to dis-tilled deionised water. The AgNOR solution was freshly preparedby dissolving gelatin at a concentration of 2 g/dl in 1 g/dl aque-ous formic acid. This solution was added to 50 g/dl aqueous silvernitrate solution (1:2, v/v). This final solution was then immediatelypoured on to the slides, which were left in the dark at room tem-perature for 45 min. The silver colloid was washed from the sectionwith deionised water and the sections were dehydrated through agraded series of ethanol to xylene. For quantification, a mean of 10different areas of sections were chosen to determine the homoge-nous AgNOR quantification throughout all groups and at least 500cells were counted, the AgNOR dots were easily identified as blackpoints within the nuclei.

2.7. Immunohistochemistry

Immunohistochemical staining was carried out following themethod of Ramakrishnan et al. [32]. The tissue sections were depar-raffinized in two changes of xylene at 60 ◦C for 20 min each andhydrated through a graded series of alcohol, the slides were incu-bated in a citrate buffer (pH 6.0) for three cycles of 5 min each ina microwave oven for antigen retrieval. The sections were thenallowed to cool to room temperature and then rinsed with 1 × Tris-buffered saline (TBS), and treated with 0.3% H2O2 in methanol for10 min to block endogenous peroxidase activity. Non-specific bind-ing was blocked with 3% BSA at room temperature for 1 h. Thesections were then incubated with caspase-3 (Santa Cruz Biotech-nology, USA) rabbit polyclonal antibody at a dilution of 1:500 at 4 ◦Covernight. The slides were washed with TBS and then incubatedwith anti-rabbit HRP-labeled secondary antibody (Genei, Banga-lore, India) at a dilution 1:5000 for 1 h in room temperature. Theperoxidase activity was visualized by treating the slides with 3,3′-diamino benzidine tetrahydrochloride (SRL, Mumbai, India), theslides were counterstained with Meyer’s hematoxylin. Negativecontrols were incubated with TBS instead of primary antibod-ies. Quantitative analysis was made in a blinded manner under alight microscope. Each section was examined at high magnification(100 ×).

3. Results

Abnormalities in DNA content are associated with malignancy.It is an indicator of proliferating activity in tumor conditions. Hence,it is important to determine DNA content in cancer state. The effect

S. Srigopalram et al. / Biomedicine & Preventive Nutrition 2 (2012) 1–8 3

Table 1Effect of ellagic acid (EA) on the levels of nucleic acid in liver of control and experimental animals.

Parameters (mg/g wet tissue) Control EA alone DEN DEN + EA

DNA 5.78 ± 0.20 5.62 ± 0.20 9.83 ± 0.92a 6.76 ± 0.32b

RNA 3.36 ± 0.13 3.48 ± 0.14 6.12 ± 0.66a 4.31 ± 0.28b

Values are expressed as mean ± SD (n = 6). Statistical significance P < 0.05.a Comparisons are made with group 1 (control).b Comparisons are made with group 3 (DEN-induced).

Table 2Effects of ellagic acid (EA) on serum enzymic antioxidant status in the liver of control and experimental group of rats.

Groups SOD CAT GPx GR GST

Control 2.76 ± 0.14 6.78 ± 0.52 14.12 ± 1.08 11.32 ± 0.33 4.07 ± 0.17EA alone 2.72 ± 0.13 6.70 ± 0.50 14.06 ± 0.99 11.26 ± 0.29 4.12 ± 0.16NDEA 0.31 ± 0.37a 1.08 ± 0.85a 8.31 ± 0.72a 3.39 ± 0.78a 1.96 ± 0.43a

EA + NDEA 2.68 ± 0.15b 6.32 ± 0.47b 13.78 ± 1.12b 10.76 ± 0.37b 3.89 ± 0.37b

Values are expressed as mean ± S.D. (n = 6). Statistical significance P < 0.05. Activity is expressed as �mol of GSH Oxidized per min per mg of protein for GPx; units per minper mg of protein for GST; 50% inhibition of epinephrine auto oxidation for SOD; �mole Of hydrogen peroxide decomposed per min per mg of protein for CAT and �mole ofNADPH oxidized/(min mg protein) for GR.

a Comparisons are made with group 1 (control).b Comparisons are made with group 3 (NDEA-induced).

Table 3Effects of ellagic acid (EA) on the activities of marker enzymes in the liver of control and experimental group of rats.

Groups AST ALT ALP LDH � GT 5′NT

Control 2.76 ± 0.12 12.83 ± 0.46 26.03 ± 0.86 2.01 ± 0.16 4.92 ± 0.15 2.78 ± 0.12EA alone 2.72 ± 0.13 12.89 ± 0.47 26.07 ± 0.84 2.03 ± 0.15 4.90 ± 0.12 2.76 ± 0.13DEN 7.83 ± 0.34a 40.36 ± 0.79a 41.36 ± 1.06a 7.72 ± 0.45a 8.36 ± 0.72a 8.73 ± 0.52a

EA + DEN 3.06 ± 0.18b 16.16 ± 0.56b 27.12 ± 0.79b 2.97 ± 0.19b 5.12 ± 0.19b 2.82 ± 0.12b

Values are expressed as mean ± SD (n = 6). Statistical significance P < 0.05. Activity is expressed as IU/L for AST, ALT, ALP, LDH, �GT and 5′NT.

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fewer neoplastically-transformed cells.SEM of liver of control and EA alone (Fig. 4A and B) maintained

a similar kind of architecture. Hepatocyte damage and membranedeformation were found in DEN-induced group 3 animals (Fig. 4C).

a Comparisons are made with group 1 (control).b Comparisons are made with group 3 (DEN-induced).

f EA on the levels of nucleic acids (DNA and RNA) in control andxperimental group of rats are shown in Table 1. In DEN-inducedroup 3 animals, the levels of nucleic acids were found to be sig-ificantly (P < 0.05) increased when compared to control group ofats. Conversely, these elevated levels were significantly (P < 0.05)ecreased in EA treated group 4 animals when compared to group

animals.Proteins play a significant role in the maintenance of cell struc-

ure and integrity. Abnormalities in protein content results inevere deformities in cancer conditions. The total protein content iniver of control and experimental animals are shown in Fig. 1. There

as a significant decrease in total protein levels were observed inroup 3 cancer bearing animals when compared to group 1 controlroup. On the other hand, the total protein content significantlyncreased (P < 0.05) in liver of EA treated group 4 animals whenompared to cancer bearing group 2 animals.

LPO is an important consequence of oxidative stress. The effectf EA on LPO in liver of control and experimental rats are given inig. 2. The levels of LPO were increased significantly (P < 0.05) inEN induced group 3 animals when compared to control group ofnimals. Whereas, they appeared to be neutralized to near normaln animals treated with EA.

Effect of EA on the enzymic antioxidant levels of control andxperimental group of animals are shown in Table 2. Antioxi-ant levels decreased significantly (P < 0.05) in DEN-induced group

animals, whereas they remained near normal level after EAdministration. Thus, EA restored the changes by its antioxidantfficacy.

Effect of EA on the activities of pathophysiological key markernzymes in the liver of control and experimental group of rats areiven in Table 3. A significant (P < 0.05) increase in the activitiesf these enzymes were noted in DEN-induced group 3 rats when

compared with control group of rats. On the other hand, EA admin-istration showed significant (P < 0.05) reduction in the activities ofthese enzymes when compared with cancer bearing group 3 rats.

Histological examination of liver sections from control (Fig. 3A)and EA alone (Fig. 3B) animals revealed normal architecture andcells with granulated cytoplasm and uniform nuclei. DEN-induced(Fig. 3C) animals showed loss of architecture with granular cyto-plasm and larger hypochromatic nuclei, whereas group 4 (Fig. 3D)animals treated with EA maintained near normal architecture with

Fig. 1. Effect of ellagic acid on total protein content in liver tissues of control andexperimental animals. Each value represents mean ± SD of six animals; Statisticalsignificance: P < 0.05. Comparisons are made with a group 1 (control) and b group 3(DEN-induced). Protein levels are expressed as mg/g wet tissue.

4 S. Srigopalram et al. / Biomedicine & Pr

Fig. 2. Effect of ellagic acid on lipid peroxidation in liver tissues of control andexperimental animals. Each value represents mean ± SD of six animals; Statisticals(

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ignificance: P < 0.05. Comparisons are made with a group 1 (control) and b group 3DEN-induced). Lipid peroxidation is expressed as U/mg of protein.

onversely, ultimate change such as hepatocyte regeneration wasoted in EA administered group 4 (Fig. 4D) animals.

Fig. 5 shows the levels of AgNORs in the liver of control andxperimental group of animals. Tumor-induced group 3 animalshowed a significant increase (P < 0.05) in the numbers of AgNOR

ig. 3. H and E stained section of liver from a normal (group 1) and EA alone (group 2) rand nucleolus. H and E stained section of liver from DEN treated (group 3) rat showing

ection from EA treated (group 4) rat showing improved architecture with few neoplastic

eventive Nutrition 2 (2012) 1–8

nuclei when compared to control group of animals. EA treatedgroup 4 animals showed significant decrease in the numbers ofAgNOR nuclei when compared to cancer bearing animals of group3.

Fig. 6 shows the effect of EA on the activation of caspase-3 in the liver of control and experimental group of animals byimmuno histochemical staining. DEN-induced group 3 animalsshowed downregulation in the expression of caspase-3 when com-pared to control group of animals. However, EA treated group 4animals showed upregulation in the expression of caspase-3 whencompared to the tumor bearing animals of group 3.

4. Discussion

Nucleic acids play an important role during neoplastic trans-formation and the determination of DNA content was moremeaningful with regard to biological and functional aspects of thetumor, because it is an index of proliferative activity in tumor con-ditions. Additionally DNA content is found to be an independentindicator of prognosis, since the size of the tumor often correlateswell with the DNA content of tumor [34]. In the present study,

be due to the increased expression of the enzymes necessary fordifferentiated cell function. Increased RNA level in cancer bear-ing animals may be due to the increased DNA content, this lead

ts showing normal hepatic cells with well preserved cytoplasm Prominent nucleusloss of architecture, granular cytoplasm and neoplastic cells. H and E stained liverally-transformed cells.

S. Srigopalram et al. / Biomedicine & Preventive Nutrition 2 (2012) 1–8 5

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ig. 4. Representative photomicrograph of rat liver section under the scanning elenduced–arrow indicates that morphological alteration in liver, (D) EA + DEN (post-

o an increased transcription and thereby elevated RNA content inancerous condition. Contrarily, EA administration controlled theucleic acid biosynthesis and exerts anticancer effect.

The chemopreventive nature of EA against DEN-induced hep-tocellular carcinogenesis was investigated in the current study.roteins and its synthesis is an important phenomenon in normals well as in cancer conditions. The highest rate of synthesis of tis-ue proteins and major protein mass of the organism is severelyffected in cancer [33]. In the present study, a decrease in proteinontent was observed in cancer bearing animals; this may be dueo the use of host protein for tumor growth. Conversely, EA admin-stration increased the protein content which indicates that EA isnvolved in the maintenance of macromolecular structure.

Lipid peroxidation is one of the major mechanisms of cellularnjury caused by free radicals [35]. Administration of DEN has beeneported to generate lipid peroxidation products like MDA thatay interact with various molecules leading to oxidative stress and

arcinogenesis [36]. The level of LPO increases with the administra-ion of DEN during hepatocarcinogenesis. This dynamic action mayurther lead to uncompromised production of free radicals over-helming the cellular antioxidant defense [37]. From the present

tudy, it is evident that increased level of LPO was found in cancerearing animals. However, the administration of EA decreased the

PO level which may be due to the free radical scavenging activityf EA.

Antioxidant enzymes are a major strategy for protecting cellsgainst a variety of endogenous and exogenous toxic compounds

microscope exhibited morphological feature of (A) control, (B) EA alone, (C) DEN-ent).

such as reactive oxygen species (ROS) and chemical carcinogens[41]. SOD, CAT and GPx are involved in the direct elimination ofROS [42]. SOD plays an important role in scavenging superoxideanion free radical and CAT catalyses conversion of hydrogen perox-ide, powerful and potentially harmful oxidizing agent, to water andoxygen [43]. SOD acts as the first line of defense against superoxideradicals. The reduction in activity of these enzymes may be causedby the increase in radical production during NDEA metabolism [44].In the present study, decreased activities of antioxidant enzymeswere observed in cancer bearing animals; this may be due to theincreased production of ROS by DEN metabolism. On the otherhand, EA administration increased the activities of these enzymes;this may be due to the free radical scavenging potential of EA.

Liver damage caused by DEN generally reflects instability of livercell metabolism which leads to distinctive changes in the serumenzyme activities. Serum transaminases, ALP, LDH and �-GT arerepresentative of liver function; their increased levels are indica-tors of liver damage. The elevation of ALT activity is repeatedlycredited to hepatocellular damage and is usually accompanied bya rise in AST [38]. 5′NT is present at the bile canalicular and sinu-soidal surface of plasma membrane of hepatocytes [39] and it isused as a diagnostic tool for liver injury [40]. In the present study,the elevation of these marker enzymes in group 3 animals may be

correlated with the malignancy. Post-treatment with EA attenu-ated the elevated levels of these enzymes. Histopathological andSEM analysis confirm the membrane regeneration and it is sug-gested that EA involves in paraenchymal cell regeneration in liver.

6 S. Srigopalram et al. / Biomedicine & Preventive Nutrition 2 (2012) 1–8

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ig. 5. Histochemical analysis of AgNORs in the liver of control and experimental rhe bar graph representing the number of AgNORs/nuclei in 10 different fields in co

hus, protecting membrane integrity and thereby decreasing thenzyme leakage.

Cell proliferation is thought to play an important role in severalteps of the carcinogenic process. AgNORs are a set of nucleolar

roteins that are necessary for ribosomal biogenesis. They can beemonstrated in formalin-fixed paraffin-embedded tissues by onetep silverstaining, resulting black dots being termed as AgNORs.he amount of AgNOR proteins can be used as a marker of cell

) Control. (B) EA alone. (C) DEN-induced. (D) DEN + EA (post-treatment). (E) showsand experimental groups.

proliferation [45]. In the present study, EA administration signif-icantly reduced the amount of AgNORs in cancer bearing groupwhen compared to control group, this confirms the antiprolifer-ative potential of EA.

The caspase family plays an important role in the reg-ulation of apoptosis. Activation of caspase-3 requires theactivation of initiator caspases, such as caspase-8 or caspase-9, inresponse to pro-apoptotic signals [46]. In the present study, EA

S. Srigopalram et al. / Biomedicine & Preventive Nutrition 2 (2012) 1–8 7

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dministration upregulated the expression of caspase-3; this maye due to the mitochondrial permeability and activation of procas-ases and thereby execute cell death.

. Conclusion

The results of the present study demonstrate that EA attenuatesPO, normalizes pathophysiological marker enzymes and nucleiccid levels, increases antioxidant status and protein content. Fur-her, EA inhibits cell proliferation and induce apoptosis. Thus, theesults of the present investigation have confirmed the efficacy ofA as an effective chemotherapeutic agent.

isclosure of interest

The authors declare that they have no conflicts of interest con-erning this article.

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