| INTRODUCTIONTumor cell invasion and subsequent spread are the leading causes of cancer-related mortality. There are two critical events required for metastasis--degradation of tissue barrier and neoangiogenesis. HSPGs ( heparan sulfate pro-toglycans) , which are the main components of basement membrane ( BM) and extracellular matrix (ECM) , play an important role in many cellular processes such as cell invasiveness. It has been found that heparanase (hpa) can degrade HSPG by cleavage of heparan sulfate side chains and coordinates with a series of enzymes and growth factors, such as bFGF, to promote tumor invasion , metastasis and angiogenesis.In the present study, we assessed the presence of heparanase and bFGF in non small cell lung cancer ( NSCLC) and their roles in the invasion, metastasis and prognosis of NSCLC.MATERIALS AND METHODS1. Tissue samplesA total of one hundred and fifteen NSCLC with neighboring forty-five cases of noncancerous tissue samples were obtained from patients underwent surgical resection in the Anshan Tumor Hospital between 1980 and 2001, fixed in 4% paraformaldehyde, embedded in paraffin. Forty freshly taken NSCLC tissue samples with corresponding non-cancerous tissues were obtained from the First Affiliated hospital of China Medical University between 2001 and 2002.2. ReagentsThe primary antibody anti-human heparanase mouse monoclonal antibodyH-130 was provided by Insight Biophannaceuticals Ltd.. The anti-human bFGF rabbit polyclonal antibody, the Ultro-sensitive S-P kit and DAB agent kit were bought from Fujian Maxin Biological Company. The goat-anti-mouse second antibody was bought from Huamei Biological Company.3. Methods3. 1 Imrnunohistochemistry for heparanase and bFGFImmunohistochemical staining was performed using an Ultro-sensitive S-P kit according to the instructions provided by the manufacturer. Color was developed using DAB/H20 solution. After the areas of tumor tissue had been chosen by random under low power, one hundred cells within tumors were counted in each of 10 fields at 200 x magnification. Cases defined as positive were regarded by both staining intensity and the percentage of immunoreactive tumor cells.3. 2 Western blot analysis of heparanaseRapidly homogenize tissues (l-2g) in 5 volumes of lysis buffer (50mM Tris-HCL PH7.4, 0. 1% SDS, 1% Triton-100, 1 mM EDTA PH8.0, Aprotin-in 12ul/10ml, PMSF 60ul/10ml) centrifuge the homogenate( 10,000rpm, 4C ) for 30 minutes to pellet insoluble material. (1) Electrophoresis (2) transfer proteins from gel to membrane (3) blocking (4) incubation with primary antibody (5) enzyme conjugate incubation (6) substrate incubation.3.3 In situ hybridization of heparanaseParaffin-embedded specimens (4 m) were deparaffinized and rehydrated. The steps of hybridization were performed using a kit according to the recommendations of the manufacturer.4. Statistical analysisStatistical Product and Services Solutions (SPSS) statistical software ( version 10.00) was applied for data analysis. Chi-square and Student' s t test were used to compare the clinicopathological characteristics with the enzyme expression. Postoperative survival periods were computed by the Kaplan-Meier method and compared by the Log rank test. A P-value less than 0. 05 was considered significant. The Cox stepwise regression analysis of which the level of significance was set at P < 0. 1 was used to evaluate the statistical strength of independent association between selected covariates and patient survival.RESULTS1. Expression of heparanase protein and mRNA in NSCLCStaining in immunoreactive tumor cells were observed for both cytoplasmic and membrane associated with many cells showing more intense signals on the cell surface. Heparanase was particularly prominently expressed at the edge of tumor cell groups. There were also some levels of expression in BM adjacent to the strongly stained tumor cells. None of the normal epithelia of bronchi and alveoli had a detectable level of heparanase. Ca...
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