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ORIGINAL ARTICLE
Year : 2012  |  Volume : 2  |  Issue : 2  |  Page : 151-155

Protective effect of 18β-glycyrrhetinic acid on cell surface glycoconjugates abnormalities in 7,12-dimethylbenz(a)anthracene-induced hamster buccal pouch carcinogenesis


Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar, Tamil Nadu, India

Date of Submission20-Aug-2011
Date of Acceptance30-Sep-2011
Date of Web Publication9-May-2012

Correspondence Address:
Shanmugam Manoharan
Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar - 608 002, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2231-0738.95990

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   Abstract 

Aim: The aim of this study was to evaluate the protective effect of 18β-glycyrrhetinic acid against cell surface glycoconjugates (protein-bound hexose, hexosamine, sialic acid, and fucose) abnormalities in 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch carcinogenesis. Materials and Methods: Topical application of DMBA three times a week for 14 weeks on the buccal pouches of hamsters resulted in well-developed squamous cell carcinoma. Glycoconjugates status in plasma and tumor tissues were estimated using specific and sensitive colorimetric methods. Results: Increases in plasma and tumor tissue glycoconjugates were noticed in hamsters treated with DMBA. Oral administration of glycyrrhetinic acid at a dose of 45 mg/kg body weight restored the status of glycoconjugates in hamsters treated with DMBA. Conclusion: The results of this study suggest that glycyrrhetinic acid might provide protection against cell surface abnormalities during DMBA-induced buccal pouch carcinogenesis in hamsters.

Keywords: Dimethylbenz(a)anthracene, glycoconjugates, hamsters, oral cancer


How to cite this article:
Manoharan S, Kowsalya R, Baskaran N, Silvan S, Sindhu G, Vinothkumar V. Protective effect of 18β-glycyrrhetinic acid on cell surface glycoconjugates abnormalities in 7,12-dimethylbenz(a)anthracene-induced hamster buccal pouch carcinogenesis. Int J Nutr Pharmacol Neurol Dis 2012;2:151-5

How to cite this URL:
Manoharan S, Kowsalya R, Baskaran N, Silvan S, Sindhu G, Vinothkumar V. Protective effect of 18β-glycyrrhetinic acid on cell surface glycoconjugates abnormalities in 7,12-dimethylbenz(a)anthracene-induced hamster buccal pouch carcinogenesis. Int J Nutr Pharmacol Neurol Dis [serial online] 2012 [cited 2019 Sep 16];2:151-5. Available from: http://www.ijnpnd.com/text.asp?2012/2/2/151/95990


   Introduction Top


Oral cancer is one of the most common malignant neoplasms worldwide, accounting for 3%-4% of all cancers in Western countries and 40%-50% of all cancers in developing countries, including India. [1] The organ- and site-specific carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) is commonly used to develop oral carcinogenesis in Syrian golden hamsters. DMBA-induced cell surface abnormalities in the buccal mucosa of hamsters closely resembles that of human oral tumor. [2],[3]

Glycoconjugates play a prominent role in cell differentiation, cellular maturation, cell migration, and cellular adhesion. Malignant transformation is usually associated with abnormalities in the structure and function of cell surface glycoconjugates. [4],[5] As they migrate through the body tumor cells utilize the oligosaccharide moieties of glycoproteins to escape from host immune cells. [6] Sialic acid, a nine-carbon sugar, also known as N-acetylneuraminic acid, has an important role in cell recognition and in defining the antigenic characteristics of cells. Sialic acid, due to its negative charge, inhibits intercellular interaction. Cell surface sialylation determines the invasiveness and metastatic potential of tumor cells. [7] Fucose, a terminal pentose sugar of glycoproteins, is one of the essential sugars as it can retard the growth of cancer cells. [8] Thus, measurement of the status of plasma glycoproteins, sialic acid, and fucose could be used as a biomarker in the diagnosis, staging, and treatment of cancer. [9]

Glycyrrhetinic acid is found in the roots of Glycyrrhiza glabra and licorice. Glycyrrhetinic acid, one of the major active constituents of these herbs, has been shown to possess inhibitory effect on cell differentiation and DNA synthesis in experimental carcinogenesis. [10] Glycyrrhetinic acid has shown significant anti-inflammatory, antiulcer, and antiviral activities, as well as antitumor activity in experimental carcinogenesis. [11] Previous studies from our laboratory have reported the antigenotoxic potential of glycyrrhetinic acid in DMBA-induced genotoxicity. [12] The present study investigated the protective effect of glycyrrhetinic acid on DMBA-induced cell surface abnormalities in the buccal mucosa of golden Syrian hamsters.


   Materials and Methods Top


Chemicals

DMBA and glycyrrhetinic acid were obtained from Sigma-Aldrich Chemicals Pvt. Ltd., Bangalore, India. All other chemicals used were of analytical grade and were purchased from HiMedia Laboratories, Mumbai, India.

Animals

Forty male golden Syrian hamsters, 8 weeks old and weighing 80-120 g, were obtained from the National Institute of Nutrition, Hyderabad, India, and maintained in the Central Animal House, Rajah Muthiah Medical College and Hospital, Annamalai University. The animals were housed in polypropylene cages and provided standard pellet diet and water ad libitum. They were maintained under controlled conditions of temperature and humidity, with a 12-hour light/dark cycle.

Experimental design

The institutional Animal Ethics Committee (Reg. No. 160/1999/CPCSEA). Annamalai University, Annamalainagar, India, approved the experimental design (Proposal No. 550: dated 20.03.2008). Animals were maintained in accordance with the guidelines of the Ethical Committee for Animal Care of Annamalai University and complied with the Indian national laws on animal care and use.

A total of 40 hamsters were randomized into four groups of ten animals each. Group I animals served as control and were painted with liquid paraffin on their left buccal pouches three times a week for 14 weeks. Groups II and III animals were painted on their left buccal pouches with 0.5% DMBA in liquid paraffin three times a week for 14 weeks; while group II animals received no further treatment, group III animals received oral glycyrrhetinic acid at a dose of 45 mg/kg body weight/day, starting 1 week before exposure to the carcinogen and continued on the days alternate to DMBA painting, until the sacrifice of the animals. Group IV animals received oral glycyrrhetinic acid alone throughout the experimental period. The experiment was terminated at the end of the 16 th week and all animals were sacrificed by cervical dislocation. Histological studies

For histopathological examination, buccal mucosa was fixed in 10% formalin and routinely processed and embedded in paraffin, after which 2-3 μm sections were made for histological studies. For detection of glycoconjugates, the tissue sections of buccal mucosa were immersed in a solution of 0.1% periodic acid for 15 minutes at 50°C. The slides were washed in running tap water and immersed in Schiff's reagent for 40 minutes. Subsequently, the sections were washed in running tap water for 10 minutes, counterstained with hematoxylin, dehydrated in graded ethanol, cleared in xylene, and mounted in resinous medium.

Biochemical analysis

The precipitate obtained after treating the plasma with 95% ethanol was used for the estimation of protein-bound hexose and hexosamine. The defatted tissues obtained after treating buccal mucosa with methanol and chloroform was used for the estimation of glycoprotein. To the dry defatted tissues remaining after lipid extraction, 0.1N H 2 SO 4 was added and it was hydrolyzed at 80°C for 1 hour. It was then cooled and the aliquot was used for sialic acid estimation. To the remaining solution 0.1N sodium hydroxide was added and it was kept in an ice bath for 1 hour. Protein-bound hexose and fucose were estimated from these aliquots. The protein-bound hexose, hexosamine, total sialic acid, and fucose were estimated by the methods of Niebes, [13] Wagner et al., [14] Warren et al., [15] and Dische and Shettles, [16] respectively. Plasma lipid-bound sialic acid level was determined by the method of Katopodis and Stock. [17]

Statistical analysis

The data are expressed as mean±SD. Statistical comparisons were performed using one-way analysis of variance (ANOVA), followed by Duncan's multiple range test (DMRT). The results were considered statistically significant at P ≤ 0.05.


   Results Top


The levels of glycoconjugates in plasma (protein-bound hexose, hexosamine, total sialic acid, lipid-bound sialic acid, and fucose) and buccal mucosa (protein-bound hexose, total sialic acid, and fucose) of control and experimental hamsters in each group are shown in [Figure 1] and [Figure 2], respectively. The levels of glycoconjugates in plasma and buccal mucosa were significantly increased in hamsters painted with DMBA as compared to control hamsters. Oral administration of glycyrrhetinic acid to DMBA-painted hamsters brought back the levels of the above-mentioned glycoconjugates to near normal ranges. No significant difference was noticed in the levels of plasma and buccal mucosa glycoconjugates between glycyrrhetinic acid-alone treated hamsters and control hamsters.
Figure 1: Status of plasma glycoconjugates (protein-bound hexose, hexosamine, total sialic acid, lipid-bound sialic acid, and fucose) in control and experimental hamsters in each group. Values are expressed as mean±SD (n=10). Values that do not share a common superscript between groups differ significantly at P<0.05

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Figure 2: Status of buccal mucosa glycoconjugates (protein-bound hexose, total sialic acid, and fucose) in control and experimental hamsters in each group. Values are expressed as mean±SD (n=10). Values that do not share a common superscript between groups differ significantly at P<0.05

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Glycoconjugates expression patterns in the buccal mucosa of control and experimental animals in each group are shown in [Figure 3]a-d. Increased glycoconjugates expression was noticed in the buccal mucosa of tumor-bearing hamsters. Oral administration of glycyrrhetinic acid to DMBA-painted hamsters significantly reduced the expression of glycoconjugates in the buccal mucosa. Glycoconjugates expression patterns in glycyrrhetinic acid-alone treated and control hamsters were similar
Figure 3: Glycoconjugates expression pattern in the buccal mucosa of control and experimental animals in each group. (a) Normal glycoconjugates expression in the control hamsters (40×); (b) overexpression of glycoconjugates in hamsters treated with DMBA alone (40×); (c) lowered expression of glycoconjugates in DMBA + glycyrrhetinic acid-treated hamsters (40×); (d) normal glycoconjugates expression in hamsters treated with glycyrrhetinic alone (40×)

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   Discussion Top


Glycoproteins are considered as biomarkers that can be used for the diagnosis and assessment of therapeutic response of several cancers, including oral cancer. [18] Abnormal glycosylation of glycoproteins and glycolipids are involved in the process of neoplastic transformation. [19] Aberrant expression of glycoproteins could contribute to defects in cell-cell communication, invasiveness, and metastatic characteristics of cancer cells. Overexpression of glycoproteins, sialic acid, lipid-bound sialic acid, and fucose has been well documented in DMBA-treated animals and human cancers. [20],[21] Abnormal secretion and turnover of glycoproteins occurring in highly proliferated tumor cells could account for increased plasma glycoproteins. [22],[23] The higher content of glycoproteins in tumor cells was also confirmed by histopathological studies using periodic acid/Schiff base staining.

Earlier studies have demonstrated that the concentrations of fucose and sialic acid are overexpressed in the cell surface during malignant transformation. [3],[20],[21],[22] It has been well documented that tumor cells have increased sialic acid and fucose concentrations as compared to their adjacent normal counterparts. [24] Increased synthesis or secretion of sialoglycoproteins from tumor cells could account for increased concentrations of plasma sialic acids. Earlier studies have demonstrated higher concentration of fucose in plasma and tumor tissues of cancer patients and tumor-bearing animals. [25],[26] In our study, oral administration of glycyrrhetinic acid at a dose of 45 mg/kg body weight restored the status of glycoconjugates in the plasma and buccal mucosa of hamsters treated with DMBA. The present results suggest that glycyrrhetinic acid significantly protected against cell surface abnormalities during DMBA-induced oral carcinogenesis.


   Conclusion Top


The present study demonstrates the protective effect of glycyrrhetinic acid on cell surface abnormalities during DMBA-induced hamster buccal pouch carcinogenesis. The protective effect of glycyrrhetinic acid may be due to its inhibitory effect on the activities of the enzymes involved in the process of glycosylation, sialylation, and fucosylation. However, further studies are needed to study the modulating effect of glycyrrhetinic acid on the activities of enzymes involved in the glycosylation processes during DMBA-induced hamster buccal pouch carcinogenesis.

 
   References Top

1.Warnakulasuriya S. Living with oral cancer: Epidemiology with particular reference to prevalence and life-style changes that influence survival. Oral Oncol 2010;46:407-10.   Back to cited text no. 1
    
2.Manoharan S, Vasanthaselvan M, Silvan S, Baskaran N, Singh AK, Vinoth Kumar V. Carnosic acid: A potent chemopreventive agent against oral carcinogenesis. Chem Biol Interact 2010;188:616-22.   Back to cited text no. 2
    
3.Manoharan S, Kavitha K, Balakrishnan S, Rajalingam K. Clerodendron inerme protects cellular integrity during 7,12-dimethylbenz[a]-anthracene-induced hamster buccal pouch carcinogenesis. Afr J Tradit Complement Altern Med 2008;5:213-22.   Back to cited text no. 3
    
4.Murray RK, Glycoproteins. In: Murray RK, Granner DK, Mayes PA, Rodwell VW, editors. Harper's biochemistry. 24th ed. USA: Appleton and Lange; Prentice-Hall; 1996. p. 648-66.   Back to cited text no. 4
    
5.Sankaranarayanan C, Pari L. Influence of thymoquinone on glycoprotein changes in experimental hyperglycemic rats. Int J Nutr Pharmacol Neurol Dis 2011;1:51-5.  Back to cited text no. 5
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6.Dabelsteen E. Cell surface carbohydrates as prognostic markers in human carcinomas. J Pathol 1996; 179:358-69.  Back to cited text no. 6
    
7.Bryne M, Kjaerheim A, Schreurs O, Dabelsteen E. Cell surface carbohydrates are involved in various biological process. Tidsskr Nor Laegeforen 1992;112:2859-62.  Back to cited text no. 7
    
8.Glick MC. Fucosylation-A role in cell function. In: Walborg EF, editor. Glycoproteins and glycolipids in disease processes. Washington DC: Amer Clin Soc 1978;22:404-11.   Back to cited text no. 8
    
9.Goodarzi MT, Shafiel M, Nomani H, Shahriarahmadi A. Relationship between total and lipid bound serum sialic acid and some tumor markers. Iran J Med Sci 2005;30:124-7.   Back to cited text no. 9
    
10.Nishino H, Yoshioka K, Lwashima A, Takizawa H, Konishi S, Okamoto H, et al. Glycyrrhetic acid inhibits tumour promoting activity of teleocidin and 12-O-tetradecanoylphorbol-13-acetate in two stage mouse skin carcinogenesis. Jpn J Can Res 1986;77:33-8.  Back to cited text no. 10
    
11.Finney RS, Somers GF. The anti-inflammatory activity of glycyrrhetinic acid and derivatives. J Pharm Pharmacol 1959;10:613-20.  Back to cited text no. 11
    
12.Kowsalya R, Manoharan S, Silvan S, Baskaran N, Palanimuthu D. Antigenotoxic effect of 18ß-glycyrrhetinic acid in 7,12-dimethylbenz(a)anthracene induced genotoxicity in Golden Syrian hamsters. J Cell Tissue Res 2011;11:2839-46.   Back to cited text no. 12
    
13.Niebes P. Determination of enzymes and degradation product of glycosaminoglycan metabolism in the serum of health and various subjects. Clin Chim Acta 1972;42:399-408.   Back to cited text no. 13
    
14.Wagner WD. A more sensitive assay discriminating galactosamine and glucosamine in mixture. Anal Biochem 1979;94:369-94.   Back to cited text no. 14
    
15.Warren L. Thiobarbituric acid and assay of sialic acid. J Biol Chem 1959;30:171-80.  Back to cited text no. 15
    
16.Dische L, Shettles LB. Specific color reactions of methyl pentoses and spectrophotometric micromethod for their determination. J Biol Chem 1948;175:595-604.  Back to cited text no. 16
    
17.Katopodis NN, Stock CC. Improved method to determine lipid bound sialic acid in plasma. Res Commun Chem Pathol Pharmacol 1980;30:171-80.  Back to cited text no. 17
    
18.Dennis JW, Granovsky M, Warren CE. Glycoprotein glycosylation and cancer progression. Biochem Biophys Acta 1999;1473:21-34.  Back to cited text no. 18
    
19.Dabelsteen E, Clausen H, Mandel MU. Aberrant glycosylation in oral malignant and premalignant lesions. J Oral Pathol Med 1991;20:361-8.   Back to cited text no. 19
    
20.Manoharan S, Padmanabhan M, Kolanjiappan K, Ramachandran CR, Suresh K. Analysis of glycoconjugates in patients with oral squamous cell carcinoma. Clin Chim Acta 2004;339:91-6.  Back to cited text no. 20
    
21.Senthil N, Manoharan S, Balakrishnan S, Ramachandran CR, Muralinaidu R, Rajalingam K. Modifying effects of Piper longum on cell surface abnormalities in 7,12-dimethylbenz[a]anthracene induced hamster buccal pouch carcinogenesis. Intl J Pharmacol 2007;3:290-4.  Back to cited text no. 21
    
22.Pugalendhi P, Manoharan S, Suresh K, Baskaran N. Genistein and daidzein in combination protect cellular integrity during 7, 1 2-dimethylbenz(a)anthracene (DMBA) induced mammary carcinogenesis in Sprague Dawley rats. Afr J Tradit Complement Altern Med 2009;6:94-102.  Back to cited text no. 22
    
23.Baskaran N, Manoharan S, Silvan S, Palanimuthu D, Rajasekaran D, Karthikeyan S, et al. Protective effect of coumarin on cell surface glycoconjugates abnormalities during 7,12- dimethylbenz(a)anthracene (DMBA) induced oral carcinogenesis. Int J Biol Med Res 2011;2:643-7.  Back to cited text no. 23
    
24.Narayanan S. Sialic acid as a tumour marker. Anal Clin Lab Sci 1994;24:376-84.   Back to cited text no. 24
    
25.Delphine W, Silvia CR, Vasudevan DM, Sudhakar Praphu K. Evaluation of serum glycoproteins in oral carcinoma. Indian J Clin Biochem 2001;16:113-5.  Back to cited text no. 25
    
26.Silvan S, Manoharan S, Baskaran N, Karthikeyan S, Prabhakar M. Protective effect of apigenin on 7,12-dimethylbenz(a)anthracene induced glycoconjugates in the plasma and buccal mucosa of golden Syrian hamsters. Int J Pharm Sci Res 2011;2:1753-8.  Back to cited text no. 26
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]



 

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