INTRODUCTION:

Oral squamous cell carcinoma (OSCC) is the fifth most common malignancy of the head and neck and growing problem worldwide. It has been estimated that 300,000 cases annually, generally most common in developing countries.¹ The morbidity and mortality rate of oral cancer is high in India and accounts for approximately 40-50% of all cancers.² Tobacco smoking, betel quid chewing, with or without tobacco, and alcohol consumption are identified as the major risk factors for the development of oral cancer.³ 7,12- dimethyl benz(a)anthracene (DMBA) is commonly used potent organ and site specific carcinogen, causes metabolic activation produces the ultimate carcinogen, dihydrodiol epoxide, to induce squamous cell carcinomas in the hamster buccal pouches by over production of ROS.⁴ Oral squamosa cell carcinoma induced by this carcinogen is morphologically and histologically similar to that of human oral tumor, as well as it express many biochemical and molecular markers that expressed in human ⁵.

Glycoproteins are a family of complex proteins that have oligosaccharide chain covalently linked to their polypeptide back bones. Carbohydrate moieties such as glucose, galactose, fucose, mannose, and derivatives of sialic acid as well as acetylated derivatives of hexosamine⁶.

Screening of glycoproteins along with other clinical and biochemical parameters in pre-oral and cancerous lesions may be useful in establishing diagnosis, staging disease, detecting metastasis and identifying the risk for recurrence as well as for evaluating therapeutive response^7.Glycoproteins have also been implicated in the transport of metabolites across cell membranes. It performs basic biological functions including in protein sorting, immune defense, receptor function, cell recognition, inflammation, pathogenicity, fertilization, and degradation of blood clots, metastasis and other cellular process ⁸. Glycoconjugates are essential for the assembly of biomembranes or genesis of biomembrane and their levels have been found to be elevated in neoplastic transformation, it is often associated with profound alterations in cell membrane protein by aberrant glycosylation⁹.

The sialic acid is the prevalent monosaccharide of cell surface glycoconjugates in animal cells and tissues. It plays an important biological process involving cell-cell recognition and interaction, masking effects of cell surface antigen, differentiation of cells and neoplastic transformation¹⁰.Increased activities of sailyl transferase on cell surface glycoconjugates are among the key molecular changes associated with malignant transformation and cancer progression. Numerous investigators have reported that possible relation between increased sialic acid levels and various malignancies¹¹. Fucose is one of the essential sugars for the optimal function of cell-cell communication in mammalian cells. It plays a significant role in many diseases including cancer and its metastasis. Lipid bound sialic acid regarded as a tumour marker of several cancers as well as to follow up the effects of anticancerous treatment¹². Previous studies from our laboratory have examined the significant correlation between glycoconjugates levels (Hexose, hexosamine, sialic acid, lipid bound sialic acid and fucose) and squamosa cell carcinoma¹³.

Chemoprevention is a novel approach to control, suppress, reverse, and prevent the tumor formation by using natural or synthetic entities¹⁴. Several studies reported that medicinal plants and their products have shown to chemopreventive and chemotherapeutic effects in animal models¹⁵. It has been suggested that medicinal plants to posses anti-carcinogenic, anti-mutagenic and anticell proliferating properties are considered as novel chemo preventive agents¹⁶. Annona squamosa (annonaceae) is commonly known as custard apple, a native of west Indies and is now cultivated throughout India¹⁷. Chavan et. al.¹⁸ reported that Annona squamosa posses several medicinal properties and used in folkoric medicine for the treatment of several diseases including cancer. Annonaceous acetogenins constitute a series of natural products isolated exclusively from Annonaceae species that are widely distributed in tropical and sub-tropical¹⁹. Londerhausen et al.²⁰ reported that acetogenins inhibit complex I mitochondrial oxidative phosphorylation with an activity several times that of rotenone. Mosinone-A is one of the novel mono-tetrahydrofuran ring acetogenins, from the bark of Annona squamosa, showing cytotoxic selectivities for the human pancreatic carcinoma cell line²¹. However, no scientific report is available in literature about its modifying effects on glycoconjugate levels in DMBA induced hamster buccal pouch carcinogenesis. The present study is therefore, designed to examine the protective effects of Mosinone-A on cell surface glycocojugates abnormalities during DMBA induced oral carcinogenesis.

Figure 1.1 Structure of Mosinone-A

MATERIALS AND METHODS:

Chemicals:

The carcinogen, 7, 12-dimethylbenz(a)anthracene was obtained from Sigma-Aldrich Chemical Pvt. Ltd. Bangalore, India. All other chemicals used were of analytical grade, marketed by Himedia laboratories Bangalore and Sisco Research Laboratories Pvt, Ltd, Mumbai, India.

Animals:

Male golden Syrian hamsters, 8-10 weeks old, weighing 80-120g were purchased from National Institute of Nutrition, Hyderabad, India and maintained in Central Animal House, Rajah Muthaiah Medical College and Hospital, Annamalai University. The animals were housed in polypropylene cages and provided standard pellet diet and water ad libitum. The animals were maintained under controlled conditions of temperature and humidity with a 12h light /dark cycle

Isolation of Mosinone-A:

Mosinone-A was isolated from Annona squamosa bark by the method of Maclaughlin²². The dried and pulverized bark of Annona squamosa was extracted with ethanol. The residues were portioned between chloroform and water. Further portioned between 90% methanol and hexane to got hexane soluble residues, then the hexane soluble residue was subjected into column chromatography over slica gel using hexane and chloroform, then chloroform and methanol as a solvent system. The fractions were combined on the basis of HPTLC. Then the combined fractions were run into column chromatography to get the final product of Mosinone-A. The identification of isolated Mosinone-A was done by LC-MS and NMR. The isolated Mosinone-A was compared with the reference Mosinone-A purchased from Lock chemicals, china. The yield and purity of the isolated Mosinone-A was found to be 0.21% and >90% respectively. For experimental Mosinone-A was first dissolved in 0.5% DMSO.

Experimental protocol:

The local institutional animal ethics committee, Annamalai University, Annamalai Nagar, India, has approved the experimental design. A total number of 40 golden Syrian hamsters were randomized into 4 group of 10 animals in each. Group I animals were served as untreated control. Groups II and III animals were painted with 0.5% DMBA in liquid paraffin three times per week for 14 weeks on the. left buccal pouches(No:4 brush). Group II animals received no other treatment. Group III animals were orally administered with Mosinone-A (2 mg kg^-1 bw) starting one week before the exposure to the carcinogen and continued on days alternate to DMBA painting, until the sacrification of the animals. Groups IV animals were received Mosinone-A alone throughout the experimental period. The experiment was terminated at the end of 14^th week and all animals were sacrificed by cervical dislocation. Biochemical studies were conducted on plasma, erythrocytes and buccal mucosa of control and experimental animals in each group.

Biochemical analysis:

After plasma separation, the erythrocyte membrane was prepared by the method of Dodge et. al.²³. Modified by Quist²⁴.The protein bound hexose, hexosamine, total sialic acid and fucose in plasma, erythrocyte membrane and buccal mucosa tissues were estimated by the methods of Niebes et. al²⁵, Wanger²⁶, Warren²⁷ and Dische and Shettles²⁸ respectively. Plasma lipid bound sialic acid level was determined by the method of katopodis and Stock. ^29.

Statistical analysis:

Values are expressed as mean ± SD. Statistical comparisons were performed by One-way analysis of variance (ANOVA) followed by Duncan’s Multiple Range Test (DMRT). The values were considered statistically significant if the p-value were less than 0.05.

Figure 1: Histopathological changes observed in hamster buccal pouch carcinogenesis

Control

DMBA

DMBA + Mosinone- A

Mosinone -A alone

RESULTS:

Table 1 shows the effect of tumor volume; tumor burden and histopathological changes in DMBA treated hamsters. We have noticed 100% tumor formation with mean tumor volume (280.1536.0 mm³) and tumor burden (840.5 ±97.2mm³) in DMBA alone painted hamsters (Group II). Oral administration of Mosinone-A at a dose of 2mg kg^-1 bw significantly prevented the tumor incidence, tumor volume and tumor burden in DMBA painted hamsters (Group III). No tumors were observed in control animals (Group I) and Mosinone-A alone administered animals (Group IV). We have observed severe keratosis, hyperplasia, dysplasia and squamous cell carcinoma in the buccal mucosal tissues of hamsters painted with DMBA alone (Group II). A mild to moderate preneoplastic lesions (hyperplasia, Keratosis and dysplasia) were noticed in groups III animals.

Table 2-4 show the status of glycoconjugates in plasma (protein bound hexose, protein bound hexosamine, total sialic acid, lipid bound sialic acid and fucose), erythrocyte membrane (Protein bound hexose, Protein bound hexosamine and total sialic acid) and buccal mucosa (protein bound hexose, total sialic acid, and fucose) in control and experimental animals in each group. The levels of glycoconjugates were significantly increased in plasma and buccal mucosa whereas decreased in erythrocyte membranes of tumour bearing hamsters as compared to control hamsters. Oral administration of Mosinone - A at a dose of 2mg/kg b.w. to DMBA painted hamsters brought back the status of glycoconjugates to near normal range in plasma, erythrocyte membrane and buccal mucosa. Hamsters treated with Mosinone-A alone showed no significant differences in glycoconjugates status as compared to control hamsters.

DISCUSSION:

Oral administration of Mosinone-A significantly prevented the tumor formation, tumor volume and burden in DMBA painted hamsters. It indicates that Mosinone-A has potent chemopreventive efficacy in experimental buccal pouch carcinogenesis. Neoplastic transformation is associated with altered cell surface carbohydrate composition of the cell membrane. Extracellular matrix plays an important role of biological process in the development of tissue repair and metastasis by regulating cell proliferation, differentiation, adhesion and migration³⁰.

Malignant tumor in the body stimulates the synthesis of more glycoprotein’s in the liver, which subsequently enter into the circulation³¹. Reduction in erythrocyte membrane glycoprotein in cancer bearing hamsters is probably due to increased membrane degradation as well as increased shedding into the circulation. Increased the levels of plasma glycoconjugates observed in the present study are probably due to shedding from buccal mucosa tumor tissues or due to release of glycoconjugates from the cell membrane which has been shown to undergo increased turnover in cancerous condition.

Table 1. Histopathological changes in the buccal pouches of DMBA painted golden Syrian hamsters (n=10)

Parameters	Control (Group- I)	DMBA (Group-II)	DMBA + Mosinone- A (2mg/kg b.wt) (Group-III)	Mosinone -A alone (2mg/kg b.wt) (Group-IV)
Tumor incidence (oral squamous cell carcinoma)	0	100%	18%	0
Total number of tumors/animals	0	28(10)	4(2)	0
Tumour volume (mm³)	0^a	280.5±36.0^b	99.96±6.84^c	0^a
Tumour burden(mm³)	0^a	840.0± 97.2^b	297.5±22.5^c	0^a
Keratosis	No change	Severe	Moderate	No change
Hyperplasia	No change	Severe	Moderate	No change
Dysplasia	No change	Severe	Mild	No change
Squamous cell carcinoma	-------	Moderately Differentiated	Well Differentiated	-------

Values are expressed as ± SD for 10 animals in each group. Tumor volume was measured using the formula

Table 2: Protein bound hexose, hexosamine, sialic acid and fucose levels in plasma of control and experimental animal in each group (n=10).

Groups

Treatment

Protein bound hexose (mg/ dl )

Protein bound hexosamine(mg/ dl)

Total sialic acid (mg/ dl )

Lipid bound sialic acid (mg/ dl)

Fucose

(mg/ dl)

Control

83.96 ±10.2^a

70.19 ±8.54^a

53.18± 6.47^a

15.15± 1.84^a

8.55 ±1.04^a

DMBA

126.49±16.8^b

105.19 ±7.20^b

83.15 ±11.0^b

31.16 ±3.52^b

25.54±3.11^b

DMBA + Mosinone-A (2mg/kg b.wt)

99.59 ±10.5^c

86.79 ±8.52^c

64.43 ±8.68^c

23.66± 2.99^c

13.01±4.95^c

Mosinone-A alone

(2mg/kg b.wt)

82.80± 8.34^a

72.29 ±9.50^a

52.00 ±6.33^a

15.00 ±4.50^a

7.98 ±2.05^a

Values are expressed as mean ± SD for ten animals in each group. Values not sharing a common superscript letter differ significantly at p<0.05(DMRT)

Table 3: Protein bound hexose, hexosamine and total sialic acid levels in erythrocyte membrane of control and experimental animals in each group (n=10)

Groups

Treatment

Protein bound hexose (mg / dl )

Protein bound hexosamine (mg / dl )

Total sialic acid (mg / dl)

Control

120.01± 14.6^a

95.44± 9.79^a

44.57± 5.42^a

DMBA

83.01± 9.96^b

61.17 ± 5.84^b

25.59± 3.39^b

DMBA+Mosinone-A (2mg/kg b.wt)

99.02± 12.0^c

77.88± 7.58^c

34.49± 3.83^c

Mosinone-A alone

(2mg/kg b.wt)

122.60 ± 14.8^a

97.09 ± 9.50^a

43.50± 5.69^a

Values are expressed as mean± SD for ten animals in each group. Values not sharing a common superscript letter differ significantly at p<0.05 (DMRT).

Table 4 : Protein bound hexose sialic acid and fucose in buccal mucosa of control and experimental animals in each group (n=10)

Groups	Treatment	Protein bound hexose (mg / g protein)	Total sialic acid (mg / g protein )	Fucose (mg / g protein)
1	Control	106.34 ±13.2^a	16.29± 2.16^a	12.68 ±1.68^a
2	DMBA	150.50± 13.5^b	31.31± 2.15^b	31.55± 4.52^b
3	DMBA + Mosinone-A (2mg/kg b.wt)	126.09 ±16.8^c	24.97 ±3.85^c	21.51± 3.15^c
4	Mosinone-A alone (2mg/kg b.wt)	104.24 ± 7.52^a	15.98± 4.52^a	14.15 ±4.32^a

Values are expressed as mean± SD for ten hamsters in each group. Values not sharing a common superscript letter differ

Sialic acid residue on the surface of the erythrocyte membranes are responsible for a net negative charge on the cell surface and confer rigidity to the cell membrane³². Several studies have documented that malignant cells have more sialic acid in their cell membrane than in normal cells. Neoplasm often have increased concentration of sialic acid on the tumor cell surface and sialoglycoproteins are shed or secreted by these cells increasing their concentration in blood and increased excretion of urine in cancer patients ³³. Suresh et. al.³⁴ reported that increased glucosyl transferase activity could be responsible for over expression of cell surface glycoconjugates in malignant tumors. Our results corroborate these observations. Fucose has profound role in cell-cell communication and its altered pattern in the cell surface may lead to neoplastic transformation and metastasis. Serum fucose level is better biochemical tumor marker than sialic acid levels in oral squamous cell carcinoma ³⁵. Elevated levels of fucose in DMBA bearing hamsters may have resulted from increased activity of serum fucosyl transferase.

Oral administration of Mosinone-A not only prevented the tumor formation but also significantly restored the status of glycoconjugates in plasma, erythrocyte membrane and tumor tissues of DMBA painted hamsters. Mosinone-A may alter cell membrane glycoprotein synthesis and structure indicating its potent antioxidant property. Present study thus demonstrate that Mosinone-A have maintained the structural integrity of the cell membrane and activate the enzymes involved glycosylation process during DMBA induced hamster buccal pouch carcinogenesis. The protective effect of Mosinone-A on cell surface glycoconjugates is probably due to their inhibitory role on glycoprotein synthesis or on glycosyl transferase and fucosyl transeferase activity. Our results thus demonstrate the chemopreventive efficacy of Mosinone-A and their modifying effect on cell surface glycoconjugates in DMBA induced hamster buccal pouch carcinogenesis.

ACKNOWLEDGEMENT:

Financial assistance from the Department of Science and Technology (DST) New Delhi, India is gratefully acknowledged.

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Received on 25.12.2009 Modified on 09.02.2010

Asian J. Research Chem. 3(2): April- June 2010; Page 459-463

INTRODUCTION:

Parameters