| ObjectiveLung cancer is one of the most common malignant tumors in the world. Metastasis and relapse are main reasons to death. E - cadherin/β - catenin ( E - cad/Cat) complex play an important role in tumor metastasis and invasion, p - catenin plays critical roles both in intercellular adhesion and in the WNT signaling pathway. When localized in the membrane, β - catenin acts as a cadherin - interacting protein by linking cadherin to a - catenin and, hence, mediating anchorage of the cadherin complex to the cortical actin cytoskelelon. In addition, the WNT signaling can stabilize β - catenin and cause it to accumulate in the cytoplasm and nucleus where it can act as a tran-scriptional coactivator by associating with the TCF/LEF family of transcription factors. Because of its important function in tumor metastasis and carcinogenesis, in this study, we used antibodies to β - catenin and to phospho -β - catenin ( specific for phosphorylated serine 33, serine 37, and threonine 41 residues) to immunohistochemically evaluate the expression levels and subcellular localization patterns in NSCLC and analyzed the correlation between their expressions and clinicopathological parameters and with overall survival.Materials and methods1. MaterialsAll the 120 samples used in this study were obtained from the patients hospitalized in the first affiliated hospital of China medical university between 2001 and 2002 and Anshan tumor hospital between 1980 and 2001. Follow - up were available in 67 patients. 53 fresh NSCLC samples and corresponding normal lung tissue used in Western Blot test were obtained from the first affiliated hospital of China medical university.2. Main agentsPrimary antibody were monoclonal anti - β - catenin ( Transduc-tion Laboratories, Lexington, KY) and polyclonal anti - phospho - β - catenin ( specific for phosphorylated serine 33, serine 37, and thre-onine 41 residues, Cell signaling Technologies). The secondary antibody and BCIP/NBT kit were bought from Huamei Biology Company.3. Method3. 1 immunohistochemistryWe detected expression of β - catenin and phospho - β - catenin in NSCLC with anti - β - catenin antibody (1:600 ) and anti - phospho - 3 - catenin antibody ( 1:50 ). Evaluation of immunostaining: normal bronchial epithelium showed membranous staining of β - catenin. In NSCLC, ratio of positive cell number to total cell number ≤ 90% meant decrease of expression, > 90% meant normal expression. Normal bronchial epithelium showed no staining with phospho - β -catenin. In NSCLC, brown granule appearing in cytoplasm or nucleus meant positive expression. Ratio of positive cell number to total cellnumber < 10% meant negative expression and ≥ 10% meant positive expression.3.2 Western BlotRapidly homogenize tissues (1 -2g) in 5 volumes of lysis buffer (50mM Triscl PH7.4, 150mM Nacl, 0. 1% SDS, 1% Triton-100, ImM EDTA PH8.0, Aprotinin 12ul/10ml, PMSF 60ul/10ml) , Centrifuge the homogenate (13000rpm, 4℃ , ) for 30 minutes to pellet insoluble material. Electrophoresis; transfer proteins from gel to PVDF membrane; blocking; incubation with primary antibody; incubation with secondary antibody; substrate incubation; image analysis.4. Statistics analysisAll the data were analyzed with SPSS For Windows 10. 0 software. Survival analysis used Cox model; relationship between expression of β - catenin and phospho - β - catenin and clinical pathology characters was analyzed with chi - squared test and precise probability method. The cut - off for statistical significance was defined as P <0. 05.Results1. immunohistochemistry;abnormal expression rate of β -catenin and phospho - β - catenin were 80% and 53. 3% respectively. Expression of β - catenin was associated with that of phospho - β - catenin. Abnormal expression rate of β - catenin in squamous cell carcinoma and adenocarcinoma were 76. 7% and 83. 3% respectively. There was no apparent difference between the expression level of adenocarcinoma and squamous cell carcinoma.
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