expression of metallothionein in pleomorphic adenoma

International Journal of Clinical Dentistry Volume 8, Number 1

ISSN: 1939-5833 © 2015 Nova Science Publishers, Inc.

EXPRESSION OF METALLOTHIONEIN IN PLEOMORPHIC ADENOMA André Luis Ribeiro Ribeiro, DDS, MSc1, Sérgio de Melo Alves-Junior, DDS, MSc, PhD2, Nayara Cristina Monteiro Carneiro, DDS2, Yonara Maria Freire Soares Marques, DDS, MSc, PhD3, João de Jesus Viana Pinheiro, DDS, PhD4, and Décio dos Santos Pinto Junior, DDS, MSc, PhD5 1

Department of Oral and Maxillofacial Surgery, School of Dentistry, University Center of Pará-CESUPA, Belém, Brazil 2 Department of Oral Pathology, School of Dentistry, Federal University of Pará-UFPA, Belém, Brazil 3 Department of Oral Pathology, School of Dentistry, São Paulo State University Júlio de Mesquita Filho - UNESP, Araraquara, Brazil 4 Department of Oral Pathology, School of Dentistry, Federal University of Pará-UFPA, Belém, Brazil 5 Department of Oral Pathology, School of Dentistry, University of São Paulo, São Paulo, Brazil

ABSTRACT Purpose: Pleomorphic adenoma (PA) is a benign salivary gland tumor (SGT) containing different cell types, including myoepithelial cells. Metallothionein (MT) is a protein that has multiple functions in various neoplasm and it is associated with a worse prognosis in cancer. This study aimed to evaluate the immunoexpression of MT in PA. Methods: The expression of MT was assessed by immunohistochemistry in 13 cases of PA. The labeled cells were counted and analyzed. Results: All PA samples showed MT expression with an average rate of positive cells of 12.29%. A higher expression of MT was observed in myoepithelial cells, with both cytoplasmic and nuclear labeling. These results drove us the conclusion that PA expresses MT at low levels. MT was present in the nucleus and cytoplasm of neoplastic 

Corresponding Author: André Luis Ribeiro Ribeiro, Address: Travessa 9 de Janeiro, 827, Faculdade de Odontologia, Departamento de Cirurgia e Traumatologia Buco-Maxilo-Facial, Centro Universitário do ParáCESUPA. Belém-PA, Brazil, Postal code: 66013 090, Phone number:55 9132415180, 55 9181181887, Email: [email protected]

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A. L. Ribeiro Ribeiro, S. de Melo Alves-Junior, N. C. Monteiro Carneiro et al. cells, with more intense staining in the myoepithelial cells. These results suggest a minor role of MT in the biological behavior of PA. However, the nuclear MT may influence the pathogenicity of PA through myoepithelial cells, being responsible for processes involving cell proliferation, protein synthesis and anti-apoptotic activity.

Keywords: Pleomorphic adenoma; salivary gland tumor; myoepithelial cell; metallothionein

Clinical Significance Pleomorphic adenoma (PA) is the most prevalent neoplasm of the salivary gland, accounting for approximately 73.6%2 to 88.5% of benign tumors and about 47.7%2 to 63.7% of all salivary gland tumors. Furthermore, the PA shows architectural pleomorphism with a variety of histologic appearances in different areas of the same tumor. PA presents different cell types, as epithelial, mesenchymal and myoepithelial cells, which each type of cells playing different roles in tumor development. Taking together, these characteristics place the PA as one of the most relevant salivary gland tumors. We have assessed by immunohistochemistry the expression of metallothionein (MT) in PA. MT has several cellular functions, including detoxification of toxic metals, such as cadmium and mercury; intracellular storage of essential metals in perinatal development; protection against free radicals and oxidative injuries; a role in cell proliferation and anti-apoptotic activity; and the most known function is the metabolism and control of essential metals. Our results showed a low MT expression with an average rate of positive cells of 12.29%. The PA expresses low levels of MT, which suggest a minor role of MT in its biological behavior.

INTRODUCTION Pleomorphic adenoma (PA) is the most prevalent neoplasm of the salivary gland [1, 2], and is considered a benign lesion whose main characteristic is its high architectural pleomorphism, showing a variety of histologic appearances in different areas of the same tumor [3]. The cell population of PA is dense, consisting primarily of epithelial, mesenchymal and myoepithelial cells [4, 5]. The mechanisms related to the pathogenesis of PA have not been fully elucidated, being important to know the proteins that can influence tumor biology and thus allowing a better understanding of its clinical behavior. PA is the most common salivary gland tumor (SGT), accounting for approximately 73.6% [2] to 88.5%[1] of benign tumors and about 47.7% [2] to 63.7% [1] of all SGTs. PA affects the salivary glands in different ways, Lukšić, et al. [2] found a prevalence of 33.1% in the parotid gland, 10.9% in the minor salivary glands, 3.6% in the submandibular gland, and 0% in the sublingual gland, out of all SGT. Cases of PA in the sublingual gland are very rare [6]. Different types of cells form a PA, including epithelial, mesenchymal and myoepithelial components [4,5,7]. Among the cell types that form the tumor, myoepithelial cells are noteworthy because of their participation in several processes in the pathogenesis of PA, including the formation of myxoid, chondroid and osteoid matrix [8-10]. The elucidation of mechanisms involved in the tumorigenesis of PA remains a challenge. Among the candidates

Metallothionein in Pleomorphic Adenoma

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to influence the biological behavior of PA, especially in myoepithelial cells, are the metallothioneins (MTs). MT is a protein of low molecular weight (about 6–7 kDa) that forms a family with four known isoforms (MT-1, MT-2, MT-3 and MT-4) [11]. The molecular structure of MT contains a considerable amount of residues of the amino acid cysteine, which is directly related to its high affinity for metal ions [12]. MT has several cellular functions, the main and most known of these being related to the metabolism and control of essential metals, such as copper and zinc, although the exact nature of these functions remains undefined [11]. Among the other functions that have been proposed for MT are: detoxification of toxic metals, such as cadmium and mercury [13], intracellular storage of essential metals in perinatal development [14, 15], protection against free radicals and oxidative injuries, and a role in cell proliferation and anti-apoptotic activity [16, 17]. The presence of MT has been reported in many human tumors including those of the breast, colon, kidney, liver, lung, ovary, prostate, and salivary glands [14, 16]. However, it is not expressed in all human tumors and may depend on several factors such as: cellular origin, differentiation, proliferation index of tumors, and other tissue factors and genetic mutations [18]. High levels of MT are observed in tissues that are undergoing rapid growth and development, which may be related to the need to provide copper and zinc for the metabolism of nucleic acids, protein synthesis, and other metabolic processes. It may also indicate that there is a close relationship between MTs and neoplasms [19, 20]. Several studies have shown that there are low levels of metallothionein in normal adult tissues, which can be explained by the decrease in the synthesis of this protein after the postnatal period [13, 15]. On the other hand, an increase in the re-expression of metallothionein has been observed in SGT, which suggests that this protein may serve as an important tumor marker, and the increased expression of which is related to a worse prognosis [21]. Based on the relation between MT and the biological behavior of several malignancies, especially in tumors involving myoepithelial cells, this study aimed to evaluate the expression of MT in PA, given that myoepithelial cells are known to play an important role in the pathogenesis of this neoplasm.

MATERIALS AND METHODS Immunohistochemistry Thirteen cases of PA were retrieved from the files of the Department of Oral Pathology of the University of Sao Paulo – USP for histological and immunohistochemical investigation. This study was approved by the ethics committee of the Institute of Health Sciences at the Federal University of Pará (number: 0047.0.073.000-11). Formalin-fixed, paraffin-embedded tissues were studied by immunohistochemistry. Three-micron thick sections were obtained and mounted on poly-D-lysine-coated slides (Sigma Chemical Corp., St Louis MO, USA). The sections were de-waxed in xylene and rehydrated in graded ethanol. Antigen retrieval was performed in Pascal chambers (Dako, Carpinteria, CA, USA) for 30 seconds. Sections were immersed in 3% H2O2 in methanol for 20 minutes for the

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A. L. Ribeiro Ribeiro, S. de Melo Alves-Junior, N. C. Monteiro Carneiro et al.

inhibition of endogenous peroxidase activity and then blocked with 1% bovine serum albumin (BSA, Sigma) in phosphate-buffered saline (PBS) for 1 hour. The slides were incubated with primary antibody against metallothionein I and II (clone E9, Dako, Carpinteria, CA, USA). The primary antibodies were diluted in PBS 1:100 and incubated for 1 hour at room temperature. Subsequently, the sections were incubated for 30 minutes with the biotin-free horseradish peroxidase (HRP)-labeled polymer of the EnVision Plus Detection System (Dako, Carpinteria, CA, USA). Diaminobenzidine (Sigma) was used as a chromogen, and the sections were counterstained with Mayer's hematoxylin (Sigma). The replacement of specific primary antibodies with non-immune sera generated negative controls.

Immunostaining Evaluation The cellular and histological patterns of MT immunolocalization were evaluated. This received a supplementary quantification via indexes of cells (IMT) among at least 4000 tumoral cells to be counted. This latter number was large enough to achieve a variation coefficient of the mean of less than 20% in multiple resamplings of every given case (not shown).

Statistical Analysis Data were analyzed using the Graph Pad Prism 5 software (Graph Pad Software, Inc., San Diego, CA, USA). Differences between groups were assessed using the Student’s t test.

RESULTS All PA samples showed MT expression. The percentage of cells that stained positive ranged from 6% to 25%, with an average of 12.29% of cells. MT was expressed in the nucleus and in the cytoplasm of the neoplastic cells, being distributed evenly between the nucleus and cytoplasm in five samples and with predominantly nuclear staining in eight samples. The expression of MT in the distinct cell types that compose the tumor was differentiated (Figure 1A). The duct-like structures of the tumor consist of two layers of cells, the inner layer being formed by epithelial cells and the outer layer formed by myoepithelial cells. The staining on the myoepithelial cells was more intense than in the other cell types (Figure 1B and 1C). The epithelial cells of the inner layer of the duct-like structures showed rare and occasional labeling of MT (Figure 1B). Myoepithelial cells expressed MT at a higher intensity in the nucleus compared to the cytoplasm of the same cells (Figure 1D). Statistical analysis provided strong evidence that the majority of cells in PA do not express MT (Figure 2A), but when MT was present, nuclear staining was more prevalent in the majority of cases (Figure 2B).


Figure 1. MT are expressed in PA neoplastic cells. MT was expressed differentially between neoplastic cells of the PA (A and B – X400). The staining of the outer layer corresponding to myoepithelial cells was more intense than in the other cells types (C – X630). A strong nuclear staining in myoepithelial cells could be observed. (D – X630). Scale bars: 20 µm.

Figure 2. Comparison of the percentage of labeled and unlabeled cells and location of metallothionein in pleomorphic adenoma. There was a prevalence of unlabeled cells (A) as well as nuclear localization of MT (B). Significance: ***p < 0.05; ****p < 0.0001.

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DISCUSSION Our results demonstrated the presence of MT in all samples of PA. MT was observed in all cell types that form the tumor, but with a greater intensity in myoepithelial cells. MT was present in both the nucleus and cytoplasm of neoplastic cells, but in most cases nuclear staining was predominant. These results suggest that MT is expressed in small quantities in PA, especially when compared to malignant neoplasms of the salivary gland [21]. In turn suggesting that MT plays a secondary role in the behavior of PA. However, when MT was expressed, it was located mostly in the nuclei, where MT may play a role in cell biology, especially in myoepithelial cells. These findings led us to investigate the possible influence of MT in PA. The MTs are a family of low molecular weight proteins that are rich in cysteine and are characterized by their binding of divalent metals [16]. MTs are responsible for performing various functions in the human body, some of them directly related to various pathophysiological processes such as the homeostasis of metal ions, redox control of substances, protection against damage from oxidative agents, cell proliferation and drug resistance, and radiotherapy. There is also strong evidence of their involvement in the process of carcinogenesis and neoplastic invasion [12, 22]. The role of MT in neoplasm biology has not yet been fully clarified, however, some studies have suggested that it may serve as an important intracellular protein for storage of essential metals, providing copper and zinc for tissues that are undergoing rapid growth and development. These metals are used in nucleic acid metabolism, protein synthesis, and other metabolic processes [19, 20]. This also takes place during perinatal development, during which high levels of endogenous hepatic copper and zinc bound to metallothionein are observed [14 16]. Thus, the high expression of MT in tissues may be related to high proliferative activity and protein synthesis. Alves, et al. [21] studied the expression of MT in adenoid cystic carcinoma (ACC), which has three distinct histological subtypes: tubular, which has a more indolent clinical behavior; cribriform, with a median clinical behavior; and solid, which presents the most aggressive clinical behavior. PA has a much lower percentage of cells expressing MT than the three variants of ACC. While the percentage of MT-positive cells in PA was 12.29%, the rate in ACC samples was 64.34% for tubular, 88.18% for cribriform, and 91.58% for solid subtypes. Therefore, the expression of MT in PA is five-fold lower than the most indolent subtype of ACC (tubular), which suggests that the reduced expression of MT in PA may be related to the less aggressive clinical behavior of this neoplasm. MT is a cytoplasmic protein that is transferred to the nucleus when the cell is undergoing rapid growth or differentiation, requiring high concentrations of zinc for the synthesis of its DNA and transcription factors. The nuclear localization of MT may also help protect DNA against oxidative damage and apoptosis [23]. The temporary presence of MT and zinc in the cell nucleus is also seen during the progression from the G1 to S phase of the cell cycle [24, 25]. MT shows strong nuclear and cytoplasmic expression in tumor cells of breast tumors, especially in myoepithelial cells. In these tumors, MT is able to increase the cell proliferation rate by two- to three-fold [25]. The real role of the higher expression of MT in neoplastic myoepithelial cells compared with other cell types that comprise the tumor has not yet been fully clarified. In their study of


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ACC, Alves, et al. [21] reported that MT can serve as a powerful tumor marker of these cells and that its expression is related to a poor prognosis in various neoplasms. A probable reason for the higher expression of MT in neoplastic myoepithelial cells is that myoepithelial cells need a lot of energy to perform their contractile functions, which in turn involve a large amount of contractile proteins that suffer high ATP hydrolysis, such as actin, myosin, calponin, and caldesmon [26]. These proteins are organized into microfilaments or myofilaments that form a large part of the cytoskeleton of myoepithelial cells. This energy consumption is Zn-dependent and MT could work as a reservoir of zinc in these cells. The transcription gene of MT I and II is activated by a range of external stimuli that are generally related to glucocorticoids, catecholamines, reactive oxygen and nitrogen species, toxic metals and proinflammatory cytokines [27]. Little is known about the actual molecular process that results in elevated expression of MT in some tumors, especially those that are malignant. In this study, although PA demonstrated positivity for MT in all samples, MT expression was relatively low when compared to its expression in ACC [21]. The high expression of MT in malignant tumors is directly related to the worse prognosis of these lesions, which helps explain the lower expression of MT in benign tumors, as seen in this study. The immunoexpression of MT was compared in benign and malignant tumors, with higher MT expression being found in malignant tumors in association with increased mortality [28]. This difference can be attributed to the fact that neoplastic cells of malignant tumors grow faster and require larger amounts of zinc to proliferate than those of benign tumors. Another theory regarding the function(s) of MT in cancer cells concerns its relationship with tumor invasion through proteolytic enzymes such as matrix metalloproteinases (MMPs). MMPs are zinc-dependent enzymes that degrade collagen and extracellular components that are directly related to increased cellular proliferation and the local invasiveness of neoplasms [17, 29, 30]. MT is the major intracellular zinc-binding protein and also plays an important role in the intake, distribution, storage and release of zinc for cell metabolism [12]. Hence it is assumed that MT can serve as a reservoir of zinc and that the newly synthesized apometalloproteins may use this source of zinc to perform their function [12, 17]. An understanding of the biological processes that may influence the clinical behavior of tumors is crucial to the establishment of targets that could lead to specific therapies, providing better results and a more favorable prognosis in the treatment of such lesions. Thus, our results confirm the expression of MT in PA, as well as its nuclear and cytoplasmic localization, with higher staining in myoepithelial cells. Despite constant expression of MT, the percentage of positive cells was relatively low. These results suggest a minor role of MT in PA biology. However, when MT was present, it was located in the nucleus of myoepithelial cells. These cells play a key role in the pathogenicity of SGT and the MT in these cells may be responsible for processes involving proliferation, protein synthesis and anti-apoptotic activity in PA.

ACKNOWLEDGMENTS The author Andre Luis Ribeiro Ribeiro is grateful to CAPES foundation, Ministry of Education of Brazil, for funding his scholarship (grant no. 0698130).

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REFERENCES [1]

[2]

[3] [4] [5] [6]

[7]

[8]

[9]

[10]

[11] [12] [13]

[14] [15]

[16]

Fonseca FP, Carvalho Mde V, de Almeida OP, Rangel AL, Takizawa MC, Bueno AG, Vargas PA. Clinicopathologic analysis of 493 cases of salivary gland tumors in a Southern Brazilian population. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. 2012; 114:230-9. Lukšić I, Virag M, Manojlović S, Macan D. Salivary gland tumours: 25 years of experience from a single institution in Croatia. J. Craniomaxillofac. Surg. 2012; 40:e75-81. Neville BW, Damm DD, Allen CM, Bouqout J. Oral and maxillofacial pathology. 3rd ed., St. Louis, Saunders Elsevier, 2009. Witt RL. The significance of the margin in parotid surgery for pleomorphic adenoma. Laryngoscope 2002; 112:2141-54. Pires FR, Pringle GA, de Almeida OP, Chen SY. Intra-oral minor salivary gland tumors: a clinicopathological study of 546 cases. Oral Oncol. 2007; 43:463-70. Dalampiras S, Andreadis D, Epivatianos A, Sakas L, Stanko P. Pleomorphic adenoma of the sublingual salivary gland: an unusual diagnostic challenge. Med. Princ Pract. 2012; 21:282-4. Lingam RK, Daghir AA, Nigar E, Abbas SA, Kumar M. Pleomorphic adenoma (benign mixed tumour) of the salivary glands: its diverse clinical, radiological, and histopathological presentation. Br. J. Oral Maxillofac. Surg. 2011; 49:14-20. Nagao T, Sato E, Inoue R, Oshiro H, Takahashi R, Nagai T, Yoshida M, Suzuki F, Obikane H, Yamashina M, Matsubayashi J. Immunohistochemical analysis of salivary gland tumors: application for surgical pathology practice. Acta Histochem Cytochem 2012; 45:269-82. Batsakis JG, Kraemer B, Sciubba JJ. The pathology of head and neck tumors: the myoepithelial cell and its participation in salivary gland neoplasia, Part 17. Head Neck Surg 1983; 5:222-33. Dardick I, van Nostrand AW, Phillips MJ. Histogenesis of salivary gland pleomorphic adenoma (mixed tumor) with an evaluation of the role of the myoepithelial cell. Hum Pathol 1982; 13:62-75. Vasa´k M. Advances in metallothionein structure and functions. J. Trace Elem. Med. Biol. 2005; 19:13-7. Vasák M, Hasler DW. Metallothioneins: new functional and structural insights. Curr. Opin. Chem. Biol. 2000; 4:177-83. Cherian MG. The significance of the nuclear and cytoplasmic localization of metallothionein in human liver and tumor cells. Environ. Health Perspect 1994; 102 Suppl 3:131-5. Nartey N, Cherian MG, Banerjee D. Immunohistochemical localization of metallothionein in human thyroid tumors. Am. J. Pathol. 1987; 129:177-82. Cherian MG, Huang PC, Klaassen CD, Liu YP, Longfellow DG, Waalkes MP. National Cancer Institute workshop on the possible roles of metallothionein in carcinogenesis. Cancer Res. 1993; 53:922-5. Cherian MG, Jayasurya A, Bay BH. Metallothioneins in human tumors and potential roles in carcinogenesis. Mutat. Res. 2003; 533:201-9.


29

[17] Ribeiro AL, Nobre RM, Rocha GC, Lobato IHS, Alves-Junior SM, Jaeger RG, Pinheiro JJV. Expression of metallothionein in ameloblastoma. A regulatory molecule? J. Oral. Pathol. Med. 2011; 40:516-9. [18] Weinlich G, Zelger B. Metallothionein overexpression, a highly significant prognostic factor in thin melanoma. Histopathology 2007; 51:280-3. [19] Moffatt P, Denizeau F. Metallothionein in physiological and physiopathological processes. Drug Metab. Rev. 1997; 29:261-307. [20] Tapiero H, Tew KD. Trace elements in human physiology and pathology: zinc and metallothioneins. Biomed Pharmacother 2003; 57:399-411. [21] Alves SM, Cardoso SV, Bernardes VF, Machado VC, Mesquita RA, Carmo MAV, Aguiar MCF. Metallothionein immunostaining in adenoid cystic carcinomas of the salivary glands. Oral Oncol. 2007; 43:252-6. [22] Aguiar MCF, Reis DG, Fonseca LMS, Cardoso SV, Leite JA, Carmo MAV. Immunohistochemical evaluation of metallothionein in palatal mucosal cells of mice treated with 4NQO. Med. Oral Patol. Oral Cir. Bucal. 2006; 11:315-8. [23] Cherian MG, Apostolova MD. Nuclear localization of metallothionein during cell proliferation and differentiation. Cell. Mol. Biol. 2000; 46:347-56. [24] Nagel WW, Vallee BL. Cell cycle regulation of metallothionein in human colonic cancer cells. Proc. Natl. Acad. Sci. USA 1995; 92:579-83. [25] Huang J, Tan PH, Jayabaskar T, Bay BH. Prognostic significance of glutathione-Stransferase-pi in invasive breast cancer. Mod. Pathol. 2003; 16:558-65. [26] Man YG, Sang QX. The significance of focal myoepithelial cell layer disruptions in human breast tumor invasion: a paradigm shift from the "protease-centered" hypothesis. Exp. Cell. Res. 2004; 301:103-18. [27] Haq F, Mahoney M, Koropatnick J. Signaling events for metallothionein induction. Mutat. Res. 2003; 533:211-26. [28] Pedersen MØ, Larsen A, Stoltenberg M, Penkowa M. The role of metallothionein in oncogenesis and cancer prognosis. Prog. Histochem Cytochem 2009; 44:29-64. [29] Pinheiro JJ, Freitas VM, Moretti AI, Jorge AG, Jaeger RG. Local invasiveness of ameloblastoma. Role played by matrix metalloproteinases and proliferative activity. Histopathology 2004; 45:65-72. [30] Ribeiro AL, Nobre RM, Alves-Junior SM, Kataoka MS, Barroso RF, Jaeger RG, Pinheiro JJVl. Matrix metalloproteinases, tissue inhibitors of metalloproteinases, and growth factors regulate the aggressiveness and proliferative activity of keratocystic odontogenic tumors. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. 2012; 114:48796.

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