Histological Evaluation of the Effect of Bee Venom on Squamous Cell Carcinoma of the Tongue

Background: Bee venom (apitoxin) composition contains numerous biologically active peptides, including melittin (main component), apamin, peptide mast cell degranulation and enzymes and non-peptide components such as histamine. While the effect of bee venom, according to the literature, would be beneficial in rheumatic diseases, evidence of its action on tumor pathology is inconclusive. In this paper, the potential chemopreventive effect of apitoxin on tumor lesions of the tongue was studied. Materials and Methods: Two groups of rats were used. Both groups were subjected to the carcinogenic action of 4-NQO. One group served as a control, and the other group (experimental) received subcutaneous bee venom that was applied weekly. The incidence of dysplastic lesions and tongue squamous cell carcinoma (TSCC) in both groups was determined microscopically. In addition, peritumoral mast cell density was determined at 30 weeks of exposure to a carcinogen. Results: The results showed no significant reduction in the incidence of TSCC. The peritumoral mast cell density found in dysplastic lesions and TSCC was lower than in the normal tongue tissue. Conclusions: The findings of this study indicate poor action of bee venom as a preventive drug in the emergence of TSCC. The lack of recruitment of mast cells indicates that every tumor is influenced by its particular microenvironment. Zavala Walther 1 , Foscolo Mabel 2 1. Ph.D; Faculty of Dentistry, Cuyo National University. Mendoza, Argentina. 2. National Council of Scientific and Technical Research of Argentina (CONICET) staff, Argentina. Submission: 29 June 2016 Accepted: 7 July 2016 Published: 25 July 2016 www.ijsrm.humanjournals.com Citation: Zavala Walther et al. Ijsrm.Human, 2016; Vol. 4 (1): 14-23. 15 INTRODUCTION Globally, oral cavity cancer ranks fifth among the different known malignancies. In the United States, Siegel et.al. (2015) reported that there are 14,000 new cases per year of lingual cancer, and it is estimated that approximately 50% will die as a result thereof [1, 2]. In addition to smoking and drinking alcohol, which remain the two major carcinogenic factors leading to lingual cancer, human papillomavirus infections (HPV) and low socioeconomic status are implicated as risk factors leading to the development of this cancer [3, 4]. The current treatments of surgery, radiotherapy and chemotherapy, alone or in combination, have not shown progress in increasing survival. The primary treatment is associated with high mortality and loss of organ function, while 50% die of recurrences [5]. On the other hand, there is growing evidence on the effects of new substances in certain cancers, including synthetic compounds and products of animal, vegetable or mineral origin. Among the animal substances being studied are components of some poisons such as those from scorpion [6] or bee [7]. Bee venom (apitoxin) contains in its composition biologically active peptides, including melittin (main component), apamin, peptide mast cell degranulation, enzymes (phospholipases, hyaluronidase) and histamine as a non-peptide component [8]. While the effect of apitoxin, according to the literature, would be beneficial in diseases of rheumatic origin, evidence of its action on tumor pathology is inconclusive, although some studies have reported a strong apoptotic action of bee venom in lung cancer [9] and liver [10] tumors. Based on this previous evidence, the present work was designed to study the possible chemopreventive effect of apitoxin in tongue tumor lesions, and a secondary objective was to examine migration of mast cells in the peritumoral area. MATERIALS AND METHODS Twenty Wistar male rats were used for this study. The experimental procedure was carried out in compliance with ethical principles for animal research, according to the revised protocol www.ijsrm.humanjournals.com Citation: Zavala Walther et al. Ijsrm.Human, 2016; Vol. 4 (1): 14-23. 16 approved by the Institutional Committee on Care and Use of Experimental Animals (CICUAL) of the Faculty of Medicine of the Cuyo National University. The rats were separated randomly into two groups: 10 animals were assigned to the control group (group 1), and 10 were assigned to the experimental group (group 2). To achieve the development of lingual squamous cell carcinoma, nitroquinoline (4-NQO) was used. The substance 4-NQO was obtained as a powder (Sigma, St. Louis, MO, USA, cat. # N8141) and was dissolved in drinking water at a final concentration of 0.02 g / l (20 ppm). Both groups were subjected to the action of 4-NQO dissolved in drinking water. The water was changed once a week, and food was provided ad libitum. Common clinical controls were measured, including weight, diet and change in hair color. On the other hand, rats in the experimental group (group 2) were not only given water with 4NQO but were also provided with a dose of subcutaneous apitoxin every week. Bee venom was diluted (Apismillifera 3X, equivalent to 1 mg / ml) at 0.5 mg / kg. Getting tongue tissue samples: The animals in both groups were sacrificed 30 weeks after the start of the administration of specific treatments. The tongue was removed and photographed for macroscopic analysis. The whole tissue was separated into two sections: the anterior segment (in front of the V lingual) and posterior segment (behind the lingual V). Each tissue sample was set in paraformaldehyde 10% solution. Sections of tongue samples were embedded in paraffin and then cut and stained with hematoxylin-eosin (H&E) in accordance with the standard protocol. In addition, Toluidine blue staining was also performed to identify mast cells in the tissue sections. Macroscopic evaluation: The samples were classified as clinically normal tissue (TN), white lesion (WL), exophytic lesion (EL) or ulcerated lesion (UL). www.ijsrm.humanjournals.com Citation: Zavala Walther et al. Ijsrm.Human, 2016; Vol. 4 (1): 14-23. 17 Microscopic or histopathologic evaluation: The histological analysis was performed using light microscopy (Zeiss Axiostar). Tongue tissue sections were grouped according to pathological diagnosis: a) normal (TLN), b) epithelial dysplasia (D) and c) squamous cell carcinoma (SCC). Quantification of mast cells: In samples stained with toluidine blue, mast cells showed purple granules in the cytoplasm. Mast cells in the peritumoral zone were quantified by counting the positively stained cells on each tissue section and were expressed as cells/mm2 tissue area. Statistical analysis: Data are expressed as the means ± SD. Statistical significance comparing different sets of mice was determined by analysis of variance (ANOVA) and Tukey post-hoc test using InStat GraphPad software (InStat Software, San Diego, CA). A P value < 0.05 was considered statistically significant.