INTRODUCTION:

Colon cancer is the third most common cause of cancer related death in USA according to 2008 American Cancer Society statistics. The carcinogenesis of colon cancer has been associated with both genetic and environmental factors [1]. Many genetic defects in colon cancer have been found and these play important roles in the carcinogenesis of colon cancer [2]. Epidemiologic studies have shown an association between dietary factors, especially consumption of red meat, and increased risk of colon cancer [3]. The main sources of branched-chain fatty acids in humans are from beef, milk and dairy products [4]. The degradation of branched-chain fatty acids is predominantly due to oxidation.

A Well characterized enzyme, methylacyl Co-A racemase (AMACR) plays an important role in oxidation of branched-chain fatty acids because it catalyzes the conversion of several (2R)-methyl-branched-chain fatty acyl-Co, Asto their (S)-stereoisomers in mitochondria and peroxisome [5]. Only stereoisomers with the 2-methyl group in the (S)-configuration can be degraded via oxidation [6]. High expression of AMACR was found only in colon adenomas and in colon carcinomas as compared to normal colon and non-neoplastic polyps [7]. The AOM colon cancer model is extensively used in the study of the underlying mechanisms of human sporadic colon cancer. AOM is a potent carcinogen causing a high incidence of colon cancer in rodents[8]. Development of this cancer closely mirrors the pattern seen in humans. Repetitive intra-peritoneal treatment of rodents with AOM causes tumours specifically in the distal colon. This can be easily and reliably achieved, particularly in susceptible rats and mice, making it a useful model for colon cancer[9]. Additionally, oxygen free radicals generated by a number of processes in vivo are highly reactive and toxic [10]. However, biological systems have evolved an array of enzymic and non-enzymic antioxidant defines mechanisms to combat the deleterious effects of oxygen free radicals. It is a well known fact that oxidative stress arises when there is an imbalance between oxygen free radicals formation and scavenging by antioxidants[11]. Excessive generation of oxygen free radicals can cause oxidative damage to biomolecules resulting in lipid peroxidation (LPO), mutagenesis and carcinogenesis. In this connection, oxygen free radicals induced LPO has been implicated in neoplastic transformation [12]. Free radicals are often generated by various environmental contaminants when exposed to living systems. Polycyclic aromatic hydrocarbons (PAHs) are one of the environmental contaminant and well recognized for its capacity to produce free radicals and the products are formed by incomplete combustion of organic matter [13]. The main sources of PAHs include industrial and domestic oil furnaces, gasoline, and diesel engines. PAHs are widely distributed in our environment and are implicated in various types of cancer.

MATERIALS AND METHODS:

(i) Chemicals:

D-Pinitol and AOM were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). All the other chemicals used in this study were of analytical grade available commercially.

(ii) Animals:

Female Sprague–Dawley rats at the age group of 45-48 days were procured from the Central Animal House Facility, Dr.ALM PGIBMS, University of Madras, Taramani. The animals were housed in well ventilated large spacious polypropylene cages and had 12 h light and dark cycle throughout the experimental period. The animals received a balanced diet of commercially available pellet rat feed and water ad libitum. The Guidelinesfor Breeding and Experiments on Animals, 1998 defined by the Ministry of Social Justice and Empowerment of India were followed and the protocol was approved by the InstitutionalAnimal Ethics Committee (IAEC No. 01/023/2013).

(iii) Experimental design:

The rats were divided into four groups and each group consisting six animals. Group I rats received single dose of 1 ml of distilled water given orally throughout the experimental period, served as vehicle treated control. Rats in Groups II and III were induced colon carcinogenesis by providing single dose of 12 mg/kg body weight of AOM in 1ml distilled water orally. Group II rats received no other treatment. After 60 days the cancer bearing Group III rats received D-Pinitol at the concentration of 300 mg/kg body weight for45 days orally. Group IV rats were treated with D-Pinitol alone at the concentration of 300mg/kg body weight for 45 days orally. At the end of the experimental period all the rats were sacrificed by cervical dislocation. Colon and liver tissues were dissected out and tissue homogenates were prepared in 0.1M Tris-HCl buffer pH 7.4 which was stored at 80° C, until its use for further analysis.

(iv) Biochemical Estimation:

The colon and liver tissue homogenates were used for estimation of enzymic and non-enzymic antioxidants such as Superoxide dismutase (SOD)[14], Catalase (CAT)[15], Glutathione Peroxidase (GPx)[16], Reduced glutathione (GSH)[17], Vitamin-E (α-tocopherol) [18] and Vitamin-C (ascorbic acid)[19]. Lipid peroxidation (LPO) [20] levels in breast tissue homogenate were estimated.

STATISTICAL ANALYSIS:

The values are expressed as Mean± S.D for six rats in each group. Statistically, significance differences between the groups were calculated using One-way Analysis of Variance (ANOVA) followed by the Student’s Turkey’s for multiple comparisons using Statistical Package for Social Sciences (SPSS) computer package. Values of p< 0.05 were considered to be significant.

RESULTS:

Table 1.Effect of D-Pinitol on enzymic and non-enzymic antioxidants in the liver of control and experimental animals.

Parameters Group I Group II Group III Group IV

(control) (AOM) (AOM+ D-Pinitol) (D-Pinitol)

Superoxide

Dismutase 7.49±0.28 3.49±0.27a* 6.37±0.28 a* b* 7.38±0.56 a

Catalase 64.85±4.26 42.62±3.98a* 58.89±4.01a* b* 63.91±3.63 a

Glutathione

Peroxidase 4.91±0.34 2.11±0.19a* 3.33±0.22 a* b* 4.62±0.39 a NS

Reduced

Glutathione 8.65±0.21 4.43±0.10a* 6.33±0.24 a* b* 8.27±0.39 a

VIT -E 6.42±0.56 3.88±0.38a* 4.04±0.27 a*b* 6.52±0.31 a NS

VIT – C 0.91±0.06 0.12±0.05a* 0.59±0.07 a* b* 0.87±0.07 a NS

Unit are expressed as: SOD=units /mg protein: CAT=I moles of H₂O₂ consumed /mg protein/min: GPx=I g of GSH utilized/mg protein/min: GSH= Ig/mg protein/min; VIT-E and VIT-C=mg/g of wet tissue. Each value represents mean ± SD of six animals; a – Group II, III and IV compared with Group I; b – Group III compared with Group II. Statistical significance-*p<0.001; #p<0.01; @p<0.05; NS – No significant.

Table 2.Effect of D-Pinitol on enzymic and non-enzymic antioxidants in the colon of control and experimental animals.

Parameters Group I Group II Group III Group IV

(Control) (AOM) (AOM+ D-Pinitol) (D-Pinitol)

Superoxide

Dismutase 15.44±0.10 6.63±0.61a* 12.21±0.94 a* b* 15.01±0.25 a

Catalase 68.08±2.19 42.78±4.62a* 51.26±3.98a* b* 67.85±2.74 a

Glutathione

Peroxidase 12.44±0.74 6.46±0.41a* 8.47±0.83 a* b* 12.28±0.16 a

Reduced

Glutathione 14.18±0.61 7.11±0.49a* 9.24±0.26 a* b* 14.08±0.92 a

VIT - E 5.11±0.42 3.54±0.73a* 4.29±0.14 a*b* 6.03±0.38 a

VIT -C 3.49±0.79 1.69±0.06a* 2.82±0.47 a* b* 3.41±0.16 a NS

Units are expressed as: SOD=units /mg protein: CAT=I moles of H₂O₂ consumed /mg protein/min: GPx=I g of GSH utilized/mg protein/min: GSH= Ig/mg protein/min; VIT-E and VIT-C=mg/g of wet tissue. Each value represents mean ± SD of six animals; a – Group II,III and IV compared with Group I; b – Group III compared with Group II. Statistical significance-*p<0.001; #p<0.01; @p<0.05; NS – No significant.

Activities of enzymic and non-enzymic antioxidants in liver and colon of control and experimental animals are presented in Table 1and2. Group II cancer-bearing animals showed a significant reduction in both enzymic and non-enzymic antioxidant levels when compared to control animals. Administration of D-Pinitol in group III animals significantly increased the antioxidant levels when compared to Group II animals. No significant changes were observed in Group IV drug control animals when compared to Group I animals.

(ii)Lipid peroxidation:

Graph:1. The levels of lipid peroxidation in basal and in the presence of inducers in colon of control

Each value represents mean ± SD of six animals a – Group II, III and IV compared with Group Ib – Group III compared with Group II*p<0.001; #p<0.01; @p<0.05; NS – No significant Graph 1 presents the activity of LPO in colon of control and experimental animals. Colon of Group II cancer-bearing animals showed a significant increase in LPO levels when compared to Group I control animals

Graph 1 presents the activity of LPO in colon of control and experimental animals. Colon of Group II cancer-bearing animals showed a significant increase in LPO levels when compared to Group I control animals. However, the levels of LPO were decreased significantly in Group III animals when compared with Group II animals. No significant changes were found in drug control animals when compared to control animals.

DISCUSSION:

Colon carcinoma is the third most common form of cancer in men and women, and the third leading cause of death of cancer, accounting for about 55,000 deaths per year in the United States [21]. However, current treatments used for colon cancer such as radiation, chemotherapy produce various side effects. 75% of patients with colorectal cancer present with localised disease, however, despite curative surgery, around 40% of patients still experience disease relapse leading to morbidity and eventual mortality [22]. The use of adjuvant chemotherapy eliminates microscopic disease, with the hope of preventing recurrent disease [23].The most active drug in colorectal cancer, the anti-metabolite 5-ftuorouracil (5-FU), was developed more than 40 years ago[24]. 5-FU exerts its anticancer effects through inhibition of thymidylatesynthase (TS) and incorporation of its metabolites into RNA and DNA [25]. Considering these effects, it is of interest that innovative new strategies will be required to treat colon cancer. Therefore searching for effective chemotherapeutic agents is important to improve the survival rate of patients with advanced or recurrent colon cancer [26]. Hence, the development of novel drugs of natural origin or plant derived compounds is desired. The natural chemotherapeutic agents have low side effects, toxicity and are involved in the carcinogenic potential by modulating carcinogen detoxification, inhibiting lipidperoxidation, or by improving in vivo antioxidants defence mechanism [27].In the present study, D-Pinitol was selected as a compound from natural origin to evaluate anticancer potency in AOM induced colon cancer animals [28]. Lipid peroxidation (LPO) has been implicated in several pathologic conditions including aging, hepatotoxicity, hemolysis and cancer[29]. It is regarded as one of the basic mechanism of cellular damage caused by free radicals. Increased lipid peroxidation alters membrane fluidity and membrane potential and there by leading to loss of cellular function and cell death [30]. Malondialdehyde is the major end product of LPO and readily reacts with DNA to form DNA-MDA adduct. The increased levels of LPO in Group-II cancer bearing animals of the present investigation may be due to the free radicals induced by AOM[31]. However the administration of D-Pinitol decreased the LPO levels in drug treated animals indicating that it is a good free radical scavenger[32]. Naturally there is a dynamic balance between the amount of free radicals generated in the body and antioxidant defence system that scavenge them and protect the body against their deleterious effects [33]. In our study, the antioxidants such as SOD, CAT, GPx, VIT-E and VIT-C activities were significantly lowered in Group-II cancer bearing animals which could be due to altered antioxidant status caused under oxidative stress condition and increased concentration of Reactive oxygen species (ROS)by AOM [34]. On contrary, upon administration of D-Pinitol the levels were reverted to near normal when compared to the control animals, Thus signifying its role in scavenging the free radicals, which in turn may readily account for its antioxidant nature [35]. Xenobiotics may exert their pathological effects through generation of ROS which is related to the aetiology of cancer.

CONCLUSION:

The data of this experiment suggest that, D-Pinitol positively modulated the antioxidant activity and reduce the lipid peroxidation by quenching and detoxifying the free radicals. Therefore, understanding the potential beneficial or adverse effects of natural products that are extensively used by the human population is very important to implement public health safety measures. Hence, it is concluded from the present investigation that the bioflavonoid hesperidin attenuates the LPO, normalizes the status of marker enzymes, protein content and DNA and RNA levels via the free radical scavenging activity and thereby exhibiting anticancer potency during by AOM against colorectal carcinoma.

ACKNOWLEDGEMENT:

The authors extremely grateful to Dr. R. Venkatakrishna Murali, M.D., Ph.D., Professor and Head, Department of Pharmacology and Environmental Toxicology, Dr. A.L. Mudhaliar Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai–600113 for providing the laboratory facility.

REFERENCES:

[1] Giovannucci E, Willett WC. Dietary factors and risk of colon cancer. Ann Med 1994; 26:443–52.

[2] Truswell AS. Meat consumption and cancer of the large bowel. EurJ Clin Nutr 2002; 56 (Suppl 1):S19–24.

[3] Willett WC. Diet and cancer. Oncologist 2000; 5:393–404

[4] Flanagan VP, Ferretti A, Schwartz DP, et al. Characterization of twosteroidal ketones and two isoprenoid alcohols in dairy products. J. Lipid Res 1975; 16:97–101.

[5] Ferdinandusse S, Denis S, Ijlst L, et al. Subcellular localization and physiological role of a methylacyl-CoA racemase. J Lipid Res2000; 41:1890–6.

[6] Wanders RK, Breken P, Ferdinandusse S, et al. Peroxisomal fattyacid and oxidation in humans: enzymology, peroxisomal metabolite transporters and peroxisomal diseases. Biochem Soc Trans 2001; 29:250–67.

[7] Xu J, Stolk JA, Zhang X, et al. Identification of differentially expressed genes in human prostate cancer using subtraction and microarray. Cancer Res 2000; 60:1677–82.

[8] Marnett LJ. Oxyradicals and DNA damage. Carcinogenesis 2000; 21:361–70.

[9] Hristozov D., Gadjeva V., Vlaykova T., Dimitrov G. Evaluation of oxidative stress in patients with cancer. Arch Physiol Biochem 2001; 109:331–6.

[10] Rubin MA, Zhou M, Dhanasekaran SM, et al. Methylacylcoenzyme a racemase as a tissue biomarker for prostate cancer. JAMA 2002;287:1662–70.

[11] Lou J, Zha S, Gage WR, et al. Methylacyl-CoA racemase: a newmolecular marker for prostate cancer. Cancer Res 2002; 62:2220–6.

[12] Jiang Z, Woda BA, Rock KL, et al. P504S: a new molecular marker for the detection of prostate carcinoma. Am J SurgPathol2001; 25:1397–404.

[13] Jiang Z, Wu CL, Woda BA, et al. P504S/methylacyl-CoA racemase: a useful marker for diagnosis of small foci of prostatic carcinoma on needle biopsy. Am J Surg Pathol 2002; 26:1169–74.

[14] Habig W.H, Pabst M.J, Jakoby W.B, Glutathione S-transferases. The first enzymatic step in mercapturic acid formation, J. Biol. Chem, 249: 7130–7139,(1974).

[15] Kolanjiappan K, Manoharan S, Kayalvizhi M, Measurement of erythrocyte lipids, lipid peroxidation antioxidants and osmotic fragility in cervical cancer patients, Clinical Chimica ACTA, 326: 143– 149, (2002).

[16] Giftson Senapathy J, Jayanthi S, Viswanathan P, Umadevi P, Nalini N, Effect of gallic acid on xenobiotic metabolizing enzymes in 1,2-dimethylhydrazine induced colon carcinogenesis in Wistar rats – A chemopreventive approach, Food and Chemical Toxicology, 49:887-892(2011).

[17] Ruvolo PP, Deng X, May WS, Phosphorylation of Bcl-2 and regulation of apoptosis, leukemia,15(4):515-522,(2001).

[18] Ohkawa H, Ohishi N, Yagi K. Assay of lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979; 95: 351–8.

[19] Bergmeyer HU, Scheibe P, Wahlefeld AW. Optimization of methods for aspartate aminotransferase and alanine aminotransferase. Clin Chem1978; 24:58–61.

[20] King J. The transferases-alanine and aspartate transaminases. In: Practical and clinical enzymology. London: Van Nostrand Co. Ltd; 1965. p. 121–38.

[21] Balasubramanian MP, Dhandayuthapani S, Nellaiappan K, Ramalingam K. Comparative studies on phosphomono esterase in helminthes. Helminthologia.1983; 20:111–20.

[22] Luly P, Barnabei O, Tria E. Hormonal control in vitro of plasma membrane bound Na+, K+-ATPase of rat liver. Biochem Biophy Acta 1972;282:447–52.

[23] Rosalki SB, Rau D. Serum-glutamyl transpeptidase activity in alcoholism. ClinChimActa 1972;39:41–7

[24] Yao D, Jiang D, Huang Z, Lu J, Tao JO, Yu Z, Meng X. Abnormal expression of hepatoma specific -glutamyl transferase and alteration-glutamy1 transferasegene methylation status in patients with hepatocellular carcinoma. Cancer2000; 88:761–9.

[25] Meister A. Glutathione. In: Arias IM, Popper H, Schachter D, Schafritz DA,editors. The liver biology and pathology. New York: Reven press; 1982.p. 297-305.

[26] Buckpitt AR, Rollins DE, Mitchell JR. Varying effective sulphydryl nucleophiles on acetaminophen oxidation and sulphydryl adduct formation. Biochem Pharmacol 1979; 28(19): 2941–6.

[27] Makpol S, Shamann NA, Jarien Z, Top AH, Khalid BAK, Wannagan WZ. tocotrienol supplementation and tumor marker enzyme activities in the ratchemically induced cancer. Gen Pharmacol 1997; 28:589–92.

[28] Pradeep K, Park SH, Ko KC. Hesperidin a flavonoglycone protects against irradiation induced hepatocellular damage and oxidative stress in Sprague-Dawley rats. Eur J Pharmacol 2008; 587:273–80.

[29] Thampi BS, Manoj G, Leelamma S, Menon VP. Dietary fiber and lipid peroxidation: effect of dietary fiber on levels of lipids and lipid peroxides in high fat diet. Ind J ExpBiol 1991; 29:563–7.

[30] Stein TP. Cachexia, gluconeogenesis and progressive weight loss in cancer patients. J TheorBiol 1978; 73:51–9.

[31] Waitzberg DL, Goncalves EL, Faintuch J, Bevilah A, Rocha CL, Cologni AM. Effectsof diets on different protein levels on growth of Walker 156 carcinoma in rats. Braz J Med Biol Res 1989; 22: 447–55.

[32] Singletary K, Mac Donald C, Wallig M. Inhibition of rosemary and carnosol of 7,12-dimethylbenz(a)anthracene-induced rat mammary tumorigenesis and in vivo DMBA-DNA adduct formation. Cancer Lett 1996; 104:43–8.

[33] Moselhy SS, Mohamed A, Al mslmani B. Chemopreventive effect of lycopenealone or with melatonin against the genesis of oxidative stress and mammarytumors induced by 7,12 dimethylbenz(a)anthracene in Sprague dawley femalerats. Mol Cell Biochem 2008; 319:175–80.

[34] Fraga, C. G., Leibovitz, R. M., and Tappel, A. L., Lipid peroxidation measured as thiobarbituric-reactive substances in tissue slices: characterization and comparison with homogenates and microsomes. Free Radic. Biol. Med., 4, 155–161 (1988).

[35] Shin, Y. C., and Jeon, J. Y., The physiological activities of pinitol isolated from soybean. Food Ind. Nutr., 30,2680–2688 (2004).

Received on 16.06.2014 Modified on 21.06.2014

Research J. Pharm. and Tech. 7(8): August 2014 Page 845-849