Hexosaminidase as a new potential marker for middle ear cholesteatoma

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Clinical Biochemistry 39 (2006) 1088 – 1090

Hexosaminidase as a new potential marker for middle ear cholesteatoma Ewa Olszewska a,⁎, Malgorzata Borzym-Kluczyk b , Slawomir Olszewski b , Marek Rogowski a , Krzysztof Zwierz b a

Department of Otolaryngology, Medical University, ul. Sklodowskiej 24 A, 15-274 Bialystok, Poland b Department of Pharmaceutical Biochemistry, Medical University of Bialystok, Poland Received 30 March 2006; received in revised form 16 August 2006; accepted 28 August 2006 Available online 14 September 2006

Abstract Objectives: The study aim was to investigate the activities of hexosaminidase (HEX) in cholesteatoma tissue compared with that in normal skin. Design and methods: The enzyme activities were determined using the Chatterjee et al. method in the modification of Zwierz et al. in cholesteatoma and skin. Results: Significantly higher activity of hexosaminidase was observed in cholesteatoma tissue compared with the skin. Conclusions: Hex may be considered as a new pathogenetic factor in that destructive lesion. © 2006 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Hexosaminidase; HEX activity; Cholesteatoma; Normal retroauricular skin

Introduction Cholesteatoma is a destructive middle ear disease characterized by the progressive expansion of keratinizing squamous epithelium in the middle ear and mastoid and a chronic inflammatory reaction of the subepithelial connective tissue. Its exact pathogenesis remains unknown. We have reported the invasive and hyperproliferative behavior of cholesteatoma epithelium as well as altered differentiation, aggressiveness and recidivism of this lesion [1]. The aggressiveness of cholesteatoma is strictly related to the resorption of bone in the area adjacent to cholesteatoma perimatrix. Erosion caused by bone resorption of the ossicular chain and otic capsule may result in hearing loss, vestibular dysfunction, facial paralysis and intracranial complications. Bone resorption is stimulated by a variety of factors, including inflammation, keratin, cytokines, such as interleukins (IL-1α, IL-1β, IL-6), interferon (INFβ) which are known to be released by cholesteatoma. However, the crucial groups of enzymes, which significance in the

⁎ Corresponding author. Fax: +48 85 746 8697. E-mail address: [email protected] (E. Olszewska).

process of bone resorption is not completely known, are lysosomal glycosidases. Among them the highest activity demonstrates N-acetylo-β-D-hexosaminidase (HEX). HEX (EC catalyzes the release of terminal, non-reducing ends of oligosaccharide chain of N-acetyl-β-D-glucosamine and N-acetyl-β-D-galactosamine in glycoproteins, GM2-gangliosides, and glycosaminoglycans (GAGs), including chondroitin 4-sulfate, chondroitin 6-sulfate, hyaluronic acid, keratan sulfate and dermatan sulfate [2]. Although hexosaminidase was shown in different diseases, its activity in cholesteatoma has never been assessed before. For the first time, we investigated the activities of HEX in cholesteatoma tissue compared with that in normal skin. Material and methods Human cholesteatoma (n = 15) and normal retroauricular skin (n = 15) were taken from the same patients during the surgical procedures due to chronic otitis media. The age of patients ranged between 38 and 72 years old (mean age: 45.7). The history of chronic otitis media ranged from 2 months to 7 years. After removal, the specimens were immediately frozen in − 80°C. Homogenates of cholesteatoma and skin specimens

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E. Olszewska et al. / Clinical Biochemistry 39 (2006) 1088–1090

were then centrifuged for 30 min (12,000 × g) at 4°C. Supernatant was stored at − 70°C for further studies. The following reagents were used for the study: p-nitrophenyl-β-DN-acetyl-glucosaminide (Sigma, St. Louis, MO, USA) and other reagents from Polish Chemical Reagents, Gliwice, Poland. Activity of the secretory granule-associated enzyme β-hexosaminidase in cholesteatoma and skin homogenates was determined by Chatterjee et al. [3] method with the modification of Zwierz et al. [4]. Statistical analysis was conducted using STATISTICA StatSoft program. As data have normal distribution, the Student's t test was used to determine the significance of difference; p < 0.05 was regarded as significant. Results In 14 out of 15 specimens, we observed significantly higher activity of HEX in cholesteatoma tissue compared with that in normal retroauricular skin. Release of HEX from the activated cells was approximately 1.5- to 5.5-fold as compared to controls. The descriptive statistics of cholesteatoma is shown in Fig. 1. Discussion In the pathogenesis of cholesteatoma, numerous pathogenetic factors occur. The aggressiveness of cholesteatoma behavior is related to the process of bone destruction which is stimulated by a variety of factors including inflammatory

Fig. 1. Parametrical statistics using Student's t test.


cells, e.g. macrophages, lymphocytes, epithelial cells and fibroblasts which released cytokines e.g. IL-1α, IL-1β, IL-6, TNF-α, TNF-β. Interleukins IL-1α and IL-1β stimulate the resorption of bone and induce synthesis of prostaglandins that play an important role in the process of bone destruction [5]. Lerner has examined the relationship between bone resorption and cell proliferation after stimulation with TGF-β in murine model. The author has proved that TGF-β enhanced the release of HEX [6], however the most important for HEX activation is IL-1β [7]. The increase in HEX activity was observed in several inflammatory diseases such as: rheumatoid arthritis, idiopathic juvenile arthritis, osteoarthritis and chronic glomerulonephritis. Inflammation is always observed in the microenvironment of cholesteatoma. Owing to the essential role of HEX in inflammatory diseases, it may be assumed that significance of HEX is also crucial in the pathogenesis of cholesteatoma. We demonstrated, for the fist time, that HEX is present in the cholesteatoma specimens. The revealed level of HEX was also found to be substantially increased compared to that in normal skin. HEX is produced and released by leucocytes, neutral granulocytes, mast cells, synovial cells and chondrocytes [7]. Most of these cells were easily found in cholesteatoma subepithelial connective tissue. Inflammation is always observed in cholesteatoma. Bacteria exist in complex, surface-attached organization known as biofilms which have been observed in cholesteatoma [8]. The matrix of cholesteatoma creates an environment for the support of biofilm formation. Chole et al. observed Gram-positive and Gramnegative bacteria within acellular deposits among the keratin accumulations in gerbil and human cholesteatoma [8]. Actinomyces actinomycetemcomitans biofilm cells exhibit increased resistance to antimicrobial agents. It may explain the clinical characteristics of infected cholesteatomas and highly resistance to eradication by antimicrobial agents. Kaplan and Fine showed that biofilm colonies of A. actinomycetemcomitans release cells into liquid medium and that these cells can attach to the surface of the culture vessel and form new colonies, enabling the biofilm to spread. The authors described a novel gene (dspB) which encodes a soluble N-acetylglucosaminidase (HEX) that causes the detachment of cells from A. actinomycetemcomitans biofilm colonies [9]. DspB specifically hydrolyzes the glycosidic linkages of poly-β-1,6-N-acetylD-glucosamine [10]. These data are important for establishing a new model of cholesteatoma development and its genesis. We will search for further study concerning the role of HEX in the context of accompanying biofilms. The activity of N-acetylo-β-D-hexosaminidase in middle ear cholesteatoma is hardly introduced. Owing to the essentially important role of HEX in variety of inflammatory diseases, it may be assumed that HEX takes part significantly in the precise mechanisms of cholesteatoma development. The relationship between inflammatory cells and cholesteatoma tissue may induce molecular and cellular defects. Those defects are manifested in the form of invasion, migration, hyperproliferation, aggressiveness and recurrence. Our study has demonstrated the increased activity of HEX in cholesteatoma tissue. We will conduct further studies with the enlarged


E. Olszewska et al. / Clinical Biochemistry 39 (2006) 1088–1090

study group. We will also search for the distribution of HEX isoenzymes and the correlation of the enzyme activity with the severity of inflammation. In our point of view, HEX may be considered as a new pathogenetic factor in that destructive lesion. References [1] Olszewska E, Lautermann J, Koc C, et al. Cytokeratin expression pattern in congenital and acquired pediatric cholesteatoma. Eur Arch Otorhinolaryngol 2005;262(9):751–6. [2] Winchester BG. Lysosomal metabolism of glycoconjugates. Subcell Biochem 1996;27:191–238. [3] Chatterjee S, Velicer LF, Sweeley CC. Glycosphingolipid glycosyl hydrolases and glycosidases of synchronized human KB cells. J Biol Chem 1975;250(13):4972–9.

[4] Zwierz K, Zalewska A, Zoch-Zwierz W. Isoenzymes of N-acetyl-βhexosaminidase. Acta Biochem Polon 1999;46:739–51. [5] Fox SW, Kuller K, Chambers TJ. Activation of osteoclasts by interleukin-1: divergent responsiveness in osteoclasts formed in vivo and in vitro. J Cell Physiol 2000;184:334–40. [6] Lerner UH. Transforming growth factor-beta stimulates bone resorption in neonatal mouse calvariae by a prostaglandin-unrelated but cell proliferation-dependent pathway. J Bone Miner Res 1996;11(11):1628–39. [7] Berenbaum F, Le Gars L, Toussirot E, et al. Marked elevation of serum N-acetyl-β-D-hexosaminidase activity in rheumatoid arthritis. Clin Exp Rheumatol 2000;18:63–6. [8] Chole RA, Faddis BT. Evidence for microbial biofilms in cholesteatomas. Arch Otolaryngol Head Neck Surg 2002;128(10):1129–33. [9] Kaplan JB, Meyenhofer MF, Fine DH. Biofilm growth and detachment of Actinobacillus actinomycetemcomitans. J Bacteriol 2003;185:1399–404. [10] Itoh Y, Wang X, Hinnebusch JB, Preston III JF, Romeo T. Depolymerization of β-1,6-N-Acetyl-D-glucosamine disrupts the integrity of diverse bacterial biofilms. J Bacterial 2005;187(1):382–7.

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