Tissue Inhibitor of Matrix Metalloproteinase-2 in Nasopharyngeal Carcinoma

Amr Ahmed El Badry, MD; Amal Abou El-Fadle, MD; Abdel Latif El-Balshy, MD

In This Article

Subjects and Methods

Our study comprised 30 patients with NPC who attended the Kasr El-Aini ENT Clinic during the period 2004-2006 and 20 controls (during endoscopic sinus operation for sinusitis or allergic nasal polypi and well known to have no malignancy). NPC patients were subjected to full history taking, clinical examination with respect to nasopharyngeal region, computed tomography, magnetic resonance imaging, and nasopharyngoscopy to determine the full extent of the local spread of the tumor. Cases were categorized according to TNM classification.[16]

Nasopharyngeal biopsies (from the NPC patients and the controls) were divided into 2 parts. The first specimen was studied and graded pathologically as one of the 3 predominant histologic types according to the World Health Organization (WHO) classification,[17] and the second was transferred into a liquid nitrogen container and washed with ice-cold saline. Fat and necrotic tissues were removed and tissues were stored at -80°C until used.

TIMP-2 protein expression was detected using immunoblotting technique (Western blot) according to the method of Sambrook.[18]

Sample preparation: Tissue samples were transferred into a clean Eppendorf tube and solubilized in 500 microliters ice-cold lysing buffer (HEPES 0.1mol/L, glycerol 10%, K2EDTA 1 mol/L, Triton-X-100 10 mol/L, and NaCl 0.5 mol/L). Protease inhibitors (benzamidine 10 mmol/L, beta-mercaptoethanol 10 mmol/L, aprotinin 5 mg/L, and PMSF 0.39 mmol/L) were freshly added to the lysing buffer before use. Solubilized tissues were sonicated on ice using an ultrasonic homogenizer (Ultra-Turax T25, IKA-Lab, Switzerland) for 3 bursts of 1 minute each, then centrifuged for 30 minutes at 18,000 rpm at 4°C. The supernatant was collected, aliquoted, and stored at -80°C until used.

Protein concentration was measured according to the method of Bradford.[19] SDS-polyacrylamide gel electrophoresis of proteins (SDS/PAGE): 1-dimensional slab gel eletrophoresis was done according to the standard protocol of Laemmli[20] with some modifications.

Electrophoretic separation of protein samples was followed by quantitative electrotransfer as described by Sambrook[18] using a mini transcellulose membrane (Hoefer Scientific Instruments, San Francisco, California).

Version 10 Statistical Package for Social Science software (SPSS Inc., Chicago, Illinois) was used to analyze the data. Univariate analysis was performed using the Chi-square method to find out the relation between various qualitative data. Comparisons of variables among subgroups were made using the Mann-Whitney test. Pearson's x2 test with continuity correction and Mann-Whitney test were used to assess correlations between TIMP-2 expression and clinicopathologic parameters of nasopharyngeal carcinomas.


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