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Clinical Biochemistry 42 (2009) 1187 – 1189
Hexosaminidase as a new potential marker for larynx cancer Ewa Olszewska a,⁎, Malgorzata Borzym-Kluczyk b , Ireneusz Rzewnicki a , Justyna Rutkowska a , Malgorzata Knas b , Marek Rogowski a , Edyta Waniewska a , Romuald Wielgosz c a
Department of Otolaryngology, Medical University of Bialystok, ul. Sklodowskiej 24 A, 15-274 Bialystok, Poland b Department of Pharmaceutical Biochemistry, Medical University of Bialystok, Poland c Department of Otolaryngology, Krupp Hospital, Essen, Germany Received 22 January 2009; received in revised form 22 February 2009; accepted 1 March 2009 Available online 17 March 2009
Abstract Objectives: Larynx squamous cell carcinoma is one of the most common forms of cancer in the area of the neck. The aim of our study was to investigate the activities of N-acetyl-β-D-hexosaminidase (HEX) in larynx cancer compared with the specimens from the healthy space of the tumor that served as controls. Design and methods: Larynx cancer (n = 15) and normal healthy tissue around the tumor (n = 15) were collected from the patients during total laryngectomy. Specimens were immediately frozen in − 80 °C. To assess hexosaminidase activity, release of p-nitrophenol from p-nitrophenol derivatives was used. Results: We observed a significantly higher activity of the investigated enzyme in all laryngeal cancer specimens compared with that in healthy tissue homogenates. The differences were statistically significant. Conclusions: It could be assumed that HEX may release particular sugars from the ends of oligosaccharide chains of glycocalyx proteins, changing adhesive forces binding together cells, and the communication between cells and elements of extracellular matrix. © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Hexosaminidase; HEX activity; Larynx cancer
Introduction Larynx squamous cell carcinoma is one of the most common forms of cancer in the area of the neck. However the discussion on its etiology and possible markers is still open. We have reported an important role of a group of enzymes called lysosomal exoglycosidases as markers for several types of cancer such as renal cancer and salivary gland cancer. Among them the highest activity demonstrates Nacetylo-β-D-hexosaminidase (HEX) . Hex (EC 22.214.171.124) catalyzes the release of terminal, non-reducing ends of the oligosaccharide chain of N-acetyl-β-D-glucosamine and Nacetyl-β-D-galactosamine in glycoproteins, GM2-gangliosides, and glycosaminoglycans (GAGs), including chondroitin 4sulfate, chondroitin 6-sulfate, hyaluronic acid, keratan sulfate ⁎ Corresponding author. Fax: +48 85 746 8697. E-mail address: [email protected]
and dermatan sulfate. During the course of catalysis, an oxonium ion-like transition state is thought to be generated, which is stabilized by a deprotonated carboxyl group from the enzyme . Human hexosaminidase has two major isoenzymes, A and B. Hexosaminidase A (HEXA) is a heterodimer composed of α and β subunits, whereas hexosaminidase B (HEXB) is a homodimer composed of β only subunits β. Both isoenzymes recognize terminal N-acetylglucosamine and Nacetylgalactosamine, but only isoenzyme A recognizes 6sulfated residues of these sugars . HEX is active in most of the tissues and organs such as kidneys , spleen  and liver . HEX is produced by chondrocytes, neutrophil granulocytes, macrophages, mast cells, leucocytes and cells of synovial membrane . Although hexosaminidase was shown in different diseases, its activity in larynx cancer has never been assessed before. For the first time, we investigated the activities of HEX in laryngeal cancer compared with that in a healthy specimen.
0009-9120/$ - see front matter © 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2009.03.003
E. Olszewska et al. / Clinical Biochemistry 42 (2009) 1187–1189
Materials and methods
Specimens of larynx cancer (n = 15) and normal healthy tissue in the surroundings of a tumor (n = 15) were taken from adult patients during total laryngectomy. Among the 15 cancers of the larynx 9 were localized in the vocal folds, anterior commissure and sinus of Margagni and 6 in the subglottic area. The age of patients ranged between 51 and 72 years old (mean age: 58.53). The history of larynx cancer ranged from 2 months to 10 months. Specimens were immediately frozen in − 80 °C.
Statistical analysis was conducted using STATISTICA StatSoft program. As data has a normal distribution the Student's t test was used to determine the significance of difference; p b 0.05 was regarded as significant. When significant differences were detected, test groups were compared to controls using multiple comparison test. T test was used to perform pairwise comparisons where indicated.
Preparation of homogenate
In 15 specimens we observed significantly higher activity of HEX in larynx cancer homogenates compared with healthy tissue homogenates. Mean release of HEX from the activated cells was 81.76027 nkat/g wet tissue compared with 24.45867 nkat/g wet tissue in healthy tissue homogenates. The descriptive statistics of larynx cancer is shown on Fig. 1. Parametrical statistics using Student's t test is: P = 0.000004. Pearson's coefficient of variation is shown on Fig. 2.
Larynx cancer and healthy tissue specimens were thawed out and weighed. Specimens were suspended in 0.05 M citric acid at a 1:9 ratio (w/v) and homogenized for 2 min using a homogenizer. Homogenates were then centrifugalized for 30 min (12,000 ×g) at 4 °C. Supernatant was stored at − 70 °C for further studies.
Reagents ⁎ p-nitrophenyl-β- D -N-acetyl-glucosaminide was from Sigma, St. Louis, MO, USA and other reagents were from Polish Chemical Reagents, Gliwice, Poland. N-acetylo-β-D-hexosaminidase release and assay Activity of the secretory granule-associated enzyme βhexosaminidase in larynx cancer and skin homogenates was determined by the mean of p-nitrophenol release from pnitrophenol derivatives used. The wavelength used to measure the absorbance was 410 nm.
Discussion We demonstrated, for the fist time, that HEX is present in larynx cancer tissue. The revealed level of HEX was also found to be substantially increased compared to that in healthy tissue. However we have not observed the correlation between the level of HEX activity and the site of cancer origin and the length of disease history. The activity of N-acetylo-β-D-hexosaminidase in larynx cancer is hardly introduced. Owing to the essentially important role of HEX in variety of tumors it may be assumed, HEX takes part significantly in the precise mechanisms of larynx
Fig. 1. Parametrical statistics using Student's t test.
E. Olszewska et al. / Clinical Biochemistry 42 (2009) 1187–1189
Fig. 2. Scatter diagram of larynx cancer versus skin. Pearson's coefficient of variation.
cancer development. The process of cancer development in the larynx induces molecular and cellular defects. Those defects are manifested in the form of invasion, migration, hyperproliferation, aggressiveness and recurrence. The correlation between the intracellular activity of lysosomal exoglycosidases and metastatic capacity seems not to be excluded. Bosmann et al. observed two melanoma cell lines and noticed the high metastatic activity levels of certain hydrolytic enzymes than those which were present in a line of low metastatic activity . Activity of lysosomal exoglycosidases are proposed to be considered as biochemical markers for renal, colon, carcinomas, glial tumors and pleomorfic adenomas [8,9]. Oktem et al. emphasize the clinical value of measuring urinary N-acetylbeta-D-glucosaminidase (U-NAG) levels in patients with laryngeal squamous cell carcinoma. They obtained results, which suggest that the level of U-NAG in urine might be used in the diagnosis of laryngeal carcinoma and the early detection of recurrences during follow-up evaluation. In the postoperative period, they observed a trend to a significant decrease in urinary U-NAG levels compared to preoperative results of measurements in 38 patients with laryngeal squamous cell carcinoma . Our study has demonstrated the increased activity of HEX in larynx cancer tissue. We will conduct further studies with the enlarged study group. We will also search for the distribution of HEX isoenzymes and the correlation of the enzyme activity with the site origin and predisposition to metastasis.
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