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ORIGINAL ARTICLE
Year : 2017  |  Volume : 13  |  Issue : 1  |  Page : 62-68

Beneficial influence of ellagic acid on biochemical indexes associated with experimentally induced colon carcinogenesis


Department of Biochemistry, Cell Biology Unit, University of Madras, Chennai, Tamil Nadu, India

Date of Web Publication16-May-2017

Correspondence Address:
Sudhandiran Ganapasam
Department of Biochemistry, University of Madras, Maraimalai Campus (Guindy), Chennai - 600 025, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.172715

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 > Abstract 

Objective: To elucidate the key biochemical indexes associated with 1, 2-dimethylhydrazine (DMH)-induced colon carcinogenesis and the modulatory efficacy of a dietary polyphenol, ellagic acid (EA).
Materials and Methods: Wistar rats were chosen to study objective, and were divided into 4 groups; Group 1-control rats; Group 2-rats received EA (60 mg/kg body weight/day, orally); rats in Group 3-induced with DMH (20 mg/kg body weight) subcutaneously for 15 weeks; DMH-induced Group 4 rats were initiated with EA treatment. We examined key citric acid cycle enzymes such as isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase and the activities of respiratory chain enzymes NADH dehydrogenase and Cytochrome-C-oxidase and membrane-bound enzyme profiles (Na +/K + ATPase, Ca 2+ ATPase and Mg 2+ ATPase), activities of lysosomal proteases such as β-D-glucuronidase, β-galactosidase and N-acety-β-D-glucosaminidase and cellular thiols (oxidized glutathione, protein thiols, and total thiols).
Results: It was found that administration of DMH to rats decreased both mitochondrial and membrane-bound enzymes activities, increased activities of lysosomal enzymes and further modulates cellular thiols levels. Treatment with EA significantly restored the mitochondrial and ATPases levels and further reduced lysosomal enzymes to near normalcy thereby restoring harmful effects induced by DMH.
Conclusion: EA treatment was able to effectively restore the detrimental effects induced by DMH, which proves the chemoprotective function of EA against DMH-induced experimental colon carcinogenesis.

Keywords: Colon cancer, dimethylhydrazine, ellagic acid, lysosomal proteases, mitochondrial enzymes


How to cite this article:
Syed U, Ganapasam S. Beneficial influence of ellagic acid on biochemical indexes associated with experimentally induced colon carcinogenesis. J Can Res Ther 2017;13:62-8

How to cite this URL:
Syed U, Ganapasam S. Beneficial influence of ellagic acid on biochemical indexes associated with experimentally induced colon carcinogenesis. J Can Res Ther [serial online] 2017 [cited 2019 Dec 15];13:62-8. Available from: http://www.cancerjournal.net/text.asp?2017/13/1/62/172715


 > Introduction Top


Colon cancer represents one of the most leading causes of cancer-related morbidity and mortality worldwide. It is a multi-step process involving genetic and epigenetic changes that provide tumor cells with a selective advantage to expand the clones.[1],[2] The epidemiological and molecular basis of colon cancer has been characterized in details, and intense efforts are currently focused to improve risk assessment for primary stages of disease. Lifestyle habits such as physical inactivity, stress, alcohol consumption, and intake of high animal fat/red meat significantly increase the risk of colon cancer. Other studies have shown that diets high in fiber may reduce the risk of colon cancer.[3],[4] Natural or synthetic compounds exist universally and are expected to be pharmacologically safe, that possess potent antioxidant properties should constitute an effective drug regimen for chemoprevention of various cancers.[5]

Free radical and nonradical oxidizing species are frequently produced in animals treated with carcinogens, and mounting evidence suggests that these free radicals and electrophiles mediated oxidative stress plays an important role in all stages of chemical carcinogenesis.[6] 1,2-dimethylhydrazine (DMH) is a potent carcinogen with a capability to induce the enormous amount of free radicals, which are potentially dangerous byproducts of cellular metabolism that have a direct effect on cell development, growth, and survival. DMH is an alkylating agent that metabolizes in the liver to form azoxymethane and methylazoxymethanol later on transported to colon via blood to generate its ultimate carcinogenic metabolite, diazonium ion that elicits an oxidative stress by methylating biomolecules of colonic epithelial cells and leads to significant oxidative damage in the cell structure and macromolecules such as DNA, RNA, protein and lipids.[7] DMH-induced colon carcinogenesis in rats is a widely used experimental model among cancer chemoprevention studies, and this model mimics human colonic epithelial neoplasms in histology, morphology, and anatomy.[8]

Identification of pharmacologically safe chemopreventive agents to prevent cancer has gained importance in recent years. Ellagic acid (EA) (4,4', 5,5', 6,6' - hexahydroxy diphenic acid 2, 6, 2',6'-dilactone) [Figure 1] is a naturally occurring polyphenolic compound which is found in many fruits (raspberries, strawberries, grapes, pomegranate, black currants), nuts (walnuts, almonds) and plant extracts (green tea, longan seeds). EA has been established to have a wide spectrum of pharmacological effects such as anti-inflammatory, anti-carcinogenic and antioxidant properties both in vivo and in vitro.[9],[47] Our recent studies have also proved the influence of EA on modulating lipid peroxidation (LPO), antioxidant potential, phase I, II enzymes and glycoprotein profile during colon carcinogenesis.[8],[9] EA has been reported to inhibit potentially various LPO's, directly scavenges different toxic radicals and hampers the accumulation of reactive oxygen species and the radical chain reaction in a concentration-dependent manner.[10] This study was aimed to determine if EA treatment can alleviate the toxic effects produced during DMH-induced colon cancer, using mitochondrial enzymes, membrane-bound enzymes, lysosomal proteases, and cellular thiols as biochemical endpoints of chemoprevention.
Figure 1: (a) Structure of ellagic acid (b) the activities of the respiratory marker enzymes such as NADH dehydrogenase and Cytochrome-C-oxidase in the colon of control and experimental groups of animals. Results were expressed as mean ± standard deviation (n = 6). Values are statistically significant at P< 0.05; aDMH versus control; bDMH + ellagic acid versus DMH. DMH: dimethylhydrazine

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 > Materials and Methods Top


Source of chemicals

EA and 1, 2-DMH was purchased from Sigma Chemicals Company (St. Louis, MO, USA). All other chemicals and reagents used in this study were of analytical grade.

Experimental animals

Male, Wistar strain albino rats weighing approximately 150–180 g were obtained from the Laboratory Animal Maintenance Unit, Tamil Nadu Animal Science and Veterinary University, Chennai, India. The animals were acclimatized to the laboratory conditions for a period of 2 weeks. They were maintained at an ambient temperature of 25 ± 2°C and 12/12 h of the light-dark cycle and given a standard rat feed (Hindustan Lever Ltd., Bangalore, India) and water ad libitum. All the experiments involved with animals were conducted according to the ethical norms approved by Ministry of Social Justices and Empowerment, Government of India and Institutional Animal Ethics Committee Guidelines (Approval No. 01/019/08). Care was taken to minimize animal suffering.

Tumor induction

For induction of colon cancer, rats (n = 6) received a dosage of 20 mg/kg body weight DMH (dissolved freshly in 0.9% NaCl solution) subcutaneously (SC) once in a week for 15 weeks.[8] Control rats were given SC dose of saline alone.

Experimental design

The animals were divided into four groups (n = 6). Group 1-control rats received physiological saline. Group 2 rats received EA alone (dissolved in water, 60 mg/kg body weight, p.o.) throughout the experimental period. Group 3 rats received SC injection of DMH (20 mg/kg body weight) once a week for 15 weeks and Group 4 rats were administered DMH for the first 15 weeks followed by treatment with EA 60 mg/kg body weight, p.o. every day as reported earlier [9] for the remaining 15 weeks. After the experimental period, the rats were anesthetized by diethyl ether followed by cervical decapitation after an overnight fast. The colon was excised and rinsed with ice-cold saline, blotted dry on filter paper and weighed. 10% (w/v) homogenates of colon tissue were prepared in 0.1 M Tris-HCl buffer (pH 7.4) using a tissue homogenizer with Teflon pestle at 4°C and the resultant tissue homogenate was used for the biochemical measurements.

Biochemical analysis

Mitochondrial enzymes

Mitochondria from colon tissue were isolated by the method described by Johnson and Lardy [11] and the following parameters were analyzed. Protein was estimated by the method of Lowry et al.[12] Citric acid cycle enzymes were analyzed by described in their methods isocitrate dehydrogenase (ICDH),[13] alpha-ketoglutarate dehydrogenase (alpha-KDH),[14] succinate dehydrogenase (SDH),[15] malate dehydrogenase (MDH).[16] The activity of respiratory chain enzyme NADH dehydrogenase was determined by Minakami et al.[17] and Cytochrome-C-oxidase by Pearl et al.[18]

Membrane-bound phosphatases

Na + K +-ATPase activity was assayed as described by Bonting.[19] The activity of Mg 2+ ATPase was assayed by Ohnishi et al.,[20] Ca 2+-ATPase by Hjertén and Pan.[21] The enzyme activity is expressed as μmoles of inorganic phosphorus liberated/min/mg protein. The phosphorus content of the supernatant was estimated by Fiske and Subbarow.[22]

Lysosomal hydrolases

Lysosomal fraction was isolated as described by Wattiaux et al.[23] The colon tissue homogenate was subjected to differential centrifugation and the different fractions were separated as follows: Structural proteins, nucleus, and cell debris at 600 g for 10 min, lysosomes at 11,000 rpm for 10 min. The activities of lysosomal enzymes, i.e., β-D-glucuronidase [24] β-galactosidase [25] and N-Acetyl-β-D-glucosaminidase [26] were assayed.

Cellular thiols

Oxidized glutathione (GSSG) was estimated by the method of Griffth.[27] Virtually all the nonprotein sulfhydryl content of the cell is in the form of reduced glutathione. Total thiols content was estimated by Sedlak and Lindsay [28] using Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid) or DTNB), which was reduced by thiol groups to from one mole of 2-nitro 5-merc aptobenzoic acid per mole of thiol, which has an absorption maximal at 412 nm. Protein thiols (PSH) were calculated by subtracting the non-PSH from total thiols.

Statistical analysis

All the grouped data were significantly evaluated with SPSS/10 software (Chicago, IL, USA). Hypothesis testing method included one-way analysis of variance followed by least significant difference test P values of <0.05 were considered to indicate statistical significance. All these results were expressed as Mean ± standard deviation for six rats in each group.


 > Results Top


Effect of ellagic acid on body weight changes

The effect of DMH and EA on the change in body weight and percentage of body weight gain is shown in [Table 1]. Induction of DMH to rats (Group 3) resulted in the loss of body weight, which also exhibited decreased the percentage of body weight gain when compared with the control rats. Administration of EA to DMH-induced rats (Groups 4) showed a significant increase in body weight when compared with DMH-induced rats. Body weight changes in EA (Group 2) treated rats were monitored and compared with the control to determine the toxic effects if any, of EA during the treatment period. No noticeable changes were observed between the groups that prove the nontoxic nature of EA.
Table 1: Effect of EA on body weights of rats subjected to DMH

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Ellagic acid effect on incidence of colonic neoplasms

Incidence of colonic neoplasms and effect of EA on incidence of colonic tumors in DMH-induced rats are summarized in [Table 2]. There were no tumors in control and EA treated rats. In animals given DMH, the tumor incidence was 100%. EA administration to DMH-induced group significantly suppressed tumor formation.
Table 2: Incidence of colonic neoplasms

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Ellagic acid stabilizes mitochondrial enzyme system

Mitochondrion is now gaining importance in cancer research because of its principal role as a regulator of energy balance and it appears to be the primary target for oxidative stress-induced damage during cancers as it has been suggested to be the main source of free radical production.[29] The influence of EA on the activities of key citric acid cycle enzymes is shown in [Table 3]. The activities of citric acid cycle enzymes ICDH, alpha-KDH, MDH and SDH were found to be significantly (P < 0.05) reduced in the colon mitochondria of the tumor-bearing DMH-induced rats (Group 3). On EA treatment, the activities of these enzymes were sustained to near normal status in Group 4 animals compare to colon cancer induced animals.
Table 3: Effect of 1,2-dimethylhydrazine and ellagic acid on the activities of major citric acid cycle enzymes in colon mitochondria

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[Figure 1]b depicts the activities of the respiratory marker enzymes such as NADH dehydrogenase and Cytochrome-C-oxidase in the colon of control and experimental groups of animals. A notable descend in the activities of these enzymes were prominent in DMH-induced rats. EA treatment to DMH-induced animals reversed this alteration by inducing the activities of these enzymes and thereby detoxified the carcinogen effectively.

Beneficial influence of ellagic acid on membrane-bound-phosphatases

Membrane-bound enzymes are sensitive indices of an altered cellular environment and could form one approach toward the understanding of the biochemical basis of the pathogenesis of diseases.[30] [Table 4] represents the activities of Na +/K +, Mg 2+, Ca 2+ and total ATPases in the colon of control and experimental groups of rats. Activities of ATPases showed significant (P < 0.01) decrease in Group 3 when compared to control (Group 1) animals. Group 4 (DMH + EA) had a significant increase in the activities of ATPases when compared to Group 3. No significant changes were found between Group 1 (control) and Group 2 (EA).
Table 4: Effect of ellagic acid on the activities of membrane-bound phosphatases in the colon of experimental group of rats

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Ellagic acid administration reduces the levels of lysosomal hydrolases

Membrane integrity is important for cell viability and the lysosomal membrane is a potential site for free radical attack subsequently causing loss of membrane stability. Lysosomal enzymes such as β-D-galactosidase, β-D-glucuronidase, and β-D-N-acetylglucosaminidase in the colon tissue of control and experimental animals are shown in [Figure 2]. The activities of these enzymes found to be significantly (P < 0.05) increased in the tumor-bearing animals (Group 3), whereas EA supplementation decreases the activities of the above enzymes.
Figure 2: Graph represents the effect of ellagic acid on the levels of lysosomal hydrolases in the colon of control and experimental groups of rats. Activity is expressed as μmol of p-nitrophenol liberated/min/mg of protein for β-D-glucuronidase, β-D-galactosidase and β-D-N-acetyl glucosaminidase. Results were expressed as mean ± standard deviation (n = 6). Values are statistically significant at P< 0.05; aDMH versus control; bDMH + ellagic acid versus DMH. DMH: dimethylhydrazine

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Ellagic acid modulates the levels of cellular thiols

[Figure 3] shows the levels of cellular thiols in the colon of control and experimental group of animals. The levels of PSH and total thiols were markedly decreased, whereas the level of GSSG found to be increased in DMH-induced colon cancer group of animals. Supplementation of EA to rats with colon cancer increased the levels of PSH and total thiols and also decreased the level of GSSG. No significant changes were found in control and EA alone treated group of rats.
Figure 3: Graph represents the effect of ellagic acid on the levels of cellular thiols in the colon of control and experimental groups of rats. Results are expressed as mean ± standard deviation (n = 6). Values are statistically significant at P< 0.05; aDMH versus control; bDMH + ellagic acid versus DMH. DMH: dimethylhydrazine

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 > Discussion Top


Colon cancer is one of the most common malignancies in many regions of the world. The strong evidence that the majority of large bowel cancers are attributable to environmental factors means that it is a potentially preventable disease.[2] Therefore, a strategy to arrest or reverse preneoplastic changes in the colonic epithelium by natural or a synthetic agent before invasive cancer develops is a rational approach for reducing the burden of colon cancer.[5] The quest of identifying new chemopreventive agents, which can either abolish or delay the development of carcinogenesis, has become an ideal strategic paradigm to struggle against cancer. EA is one such polyphenol compound has attracted considerable attention due to its potential antioxidant and chemopreventive properties against certain carcinogens and mutagens.

The NAD +/NADP + linked citric acid cycle enzymes regulate the NADH levels through the allosteric stimulation by ADP, the content of which increases with the breakdown of ATP. These NAD +/NADP + dependent major enzymes of citric acid cycle ICDH, alpha-KDH, MDH are involved in the maintenance of the reduced redox state in mitochondria in order to provide the reducing power to generate ATP via oxidative phosphorylation. Unlike the other citric acid cycle enzymes, which are soluble in the mitochondrial matrix, the FAD-dependent SDH is an integral protein of the inner mitochondrial membrane, which is a strategic location for the regeneration of its prosthetic group.[31] In this study, significant decrease in the activity of these enzymes was observed in DMH-induced rats, might be due to the alterations in cancer cell morphology, ultrastructural changes and the ability of mitochondria to undergo metabolic changes. EA treatment increased the activities of these enzymes close to near normalcy suggesting its chemoprotective nature.[9],[32] NADH dehydrogenase and Cytochrome-C-oxidase are the major mitochondrial respiratory chain enzymes that play a critical role in producing energy-rich compounds like ATP.[33] An increased LPO has been reported to alter the lipid environment of the membrane thus affecting the activity of these respiratory chain enzymes in DMH-induced rats.[8] Treatment with EA attenuated LPO and restored membrane integrity leading to a marked increase in the above-mentioned respiratory chain enzymes.

In malignancy, the cell membrane plays a crucial role in the stimulation and control of cell adhesiveness, mortality, and proliferation in a damaged condition and hence, protection of membranes is of potential importance in the treatment of disease processes.[34] Oxidative stress is a predominant factor in colon cancer, causing membrane lipid oxidation, initiating loss of membrane-bound enzyme activity, cell lysis, altering membrane permeability, and cell function.[35] The protein moiety of Na +/K + ATPases is modified by free radicals and it is well known that as a result of oxidative stress, the membrane loses its component, resulting in increased deformability and cell death. Ca 2+ ATPase is a reflection of energy-dependent calcium transport across the cell membrane and extremely sensitive to hydroperoxides. Mg 2+ ATPase, along with the other ATPases, is also involved in energy-requiring processes in the cell.[36],[37] The metabolic products of DMH generate free radicals and LPO in biological systems, which damage the membrane-bound enzymes and affect the normal functioning of the cell.[38] In the present study, we observed reduced activity of all three membrane-bound ATPases, Na +/K + ATPase, Ca 2+ ATPase and Mg 2+ ATPase in DMH-induced rats. EA treatment brought back the activities of these enzymes to near normal levels. The protective effects of EA in this system could be either due to scavenging peroxides before attacking membrane and/or due to blocking the oxidation of membrane lipids.[8],[9]

Lysosomal hydrolases comprise a variety of proteases, nucleases, glycosidases, sulfatases and lipases. A correlation between expression of lysosomal hydrolases and aggressiveness of breast, lung, brain and gastric cancers has been proposed. The diagnostic and prognostic potential of lysosomal proteases as cancer markers has previously been evaluated.[39],[40] It has been shown that in some types of tumors, the high content of lysosomal proteases is correlated with a higher risk of recurrence and with a poor prognosis. Besides their usefulness as markers, lysosomal enzymes can act as genuine players in cancer development. Free radical-mediated oxidative stress has been reported to induce lysosomal disruption in DMH-induced rat colon adenomas and adenocarcinomas.[41] Our results are in agreement with these findings as we have observed increased activities of lysosomal hydrolases β-D-galactosidase, β-D-glucuronidase, and β-D-N-acetylglucosaminidase in the colon of animals having cancer. EA has been reported to possess antioxidant/free radical-quenching property [42] and its treatment preserved the lysosomal membrane integrity and thereby prevented the leakage of lysosomal proteases.

Free sulfhydryl groups in PSH play the role of highly reactive functional groups in biological systems and participate in several different reactions such as alkylation, arylation, oxidation, thiol-disulfide exchange, etc. Therefore, the modification of PSH groups can result in severe functional damage including loss of enzyme activity.[43] In our study, the levels of PSH and total thiols were noticeably decreased whereas the levels of GSSG found to be significantly increased in DMH-induced animals. LPO is known to deplete the PSH according to the previous study, where they have shown that a significant decrease in PSH was observed only when LPO is developed.[44] Free radicals are able to react directly with protein sulfhydryl groups. In our earlier studies, we have shown that induction with DMH leads to increased LPO and oxidative stress, which supports our present findings.[8],[45] Polyphenols and other natural compounds are known to quench the radicals generated by chemical substances.[45],[46] Similarly, EA is a strong antioxidant [47] that controls LPO and hence it could modulate the levels of thiols.


 > Conclusion Top


The results of this study indicate that EA is a potent free radical quencher, and it alleviated mitochondrial metabolic disturbance and thereby maintains membrane stability. EA supplementation was effective in preserving lysosomal stability. Further EA treatment alleviated the disruptions in membrane-bound enzymes activity and cellular thiols during DMH-induced colon cancer, thus validating its chemopreventive effect. Thus, EA scavenges radical at/near the membrane surface and in the interior of the membranes and these dual effects of EA demonstrating its potent antiperoxidation and chemopreventive effect.

Acknowledgment

The author S. Umesalma gratefully acknowledges the Council of Scientific and Industrial Research, New Delhi, India for the financial assistance in the form of Senior Research Fellowship.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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