The Relativity And Significance Of The Expressions Of P120～(ctn) And RhoA In Lung Squamous Cell Carcinoma And Adenocarcinoma

IntroductionLung cancer is one of the most lethal malignant tumors, and the primary causes of this lethality include tumor recurrence and metastasis. Dysfunction of adhesion molecule and destruction of cell adhesion are the initial steps of tumor metastasis. pl20 - Catenin (pl20^ctn) , a newly found adhesion molecule, may play an important role in tumor progression and metastasis. RhoA, the core member of Rho GTPase, existing as GDP - binding and GXP - binding status, works as a molecular switch, which dynamically regulate cell adhesion. p120^ctn is main factor to switch these two statuses. Abnormal expression of p120^ctn or RhoA can directly or indirectly affect E - cadherin/catenin complex, cell adhesion and tumor metastasis. We applied Immunohistochemistry S - P method and Western blot to investigate co - expression of p120^ctn and RhoA, as well as their, relationship with clinical pathologic factors.Materials and methods1. MaterialsAll the 124 samples used in this study were obtained from the patients hospitalized in the first affiliated hospital of China Medical University between 2001 and 2002. 36 fresh lung cancer samples and corresponding normal lung tissue used in Western blot test were obtained from the first affiliated hospital of China medical university.Primary antibodies were monoclonal anti - p120 - catenin ( TransductionLaboratories, Lexington, KY) and monoclonal anti - RhoA - catenin (Santa Cruz, NY). The S -P Kit and DAB were bought from Maxin Company. The secondary antibody and BCIP/NBT kit were bought from Huamei Biology Company.2. methods2.1 immunohistochemistryWe detected expression of pl20ctn and RhoA in lung cancer with anti -pl20ctn antibody (1:200) and anti - RhoA antibody (1:100). Five views were examined per slide, and 100 cells were observed per view, at 400 magnification. Labeling scores were determined by the percentage of positive cells per slide (0% -100% ) , for membranous and cytoplasmic staining separately, normal bronchial epithelium showed membranous staining of 3 - catenin. In lung cancer, the staining reactions localized on the cell membranes were graded into three groups, according to the percentage of the positive cells, as followed: + + + ( 76% ) , + + ( S75% ) , + ( 25% ) and - (0% ) of the tumor cells. When 10% of the tumor cells stained for pl20ctn in cytoplasm, the result was defined as ectopic cytoplasmic expression. Either reduced membranous expression or ectopic cytoplasmic expression or both were defined as abnormal expression of pl20ctn. According the result of experiments on RhoA by Horiuchi in ovary tumors, we defined-----+ for normal, and + + + + + for over expression.2. 2 Western blotRapidly homogenize tissues (1 -2g) in 5 volumes of lysis buffer (50mM Triscl PH 7. 4, 150mM NaCl, 0. 1% SDS, 1% Triton - 100, lmM EDTA PH8.0, Aprotinin 12ul/10ml, PMSF 60ul/10ml) , Centrifuge the homogenate (13000rpm, 4V , ) for 30 minutes to pellet insoluble material. Electrophore-sis; transfer proteins from gel to PVDF; blocking; incubation with primary antibody; incubation with secondary antibody; substrate incubation; image analysis.3. Statistics analysisAll the data were analyzed with SPSS for Windows 11.5 software. Survival analysis used Cox model; Relationship between expression of pl20cln, RhoA andclinical pathology characters was analyzed with Person chi - squared test and Fisher precise probability method. The cut - off for statistical significance was defined as P< 0.05.ResultImmunohistochemistryExpression of pl20ctn occurs mainly in the cell membrane of bronchial epithelium. Of 124 lung cancer specimens, 25 (20. 2%) showed normal or preserved membranous expression only, and 99 (79. 8%) showed abnormal expression , including reduced membranous expression, preserved membranous expression accompanied with ectopic cytoplasmic expression, and reduced or absent membranous expression together with ectopic cytoplasmic expression. Over expression rate of RhoA is 62.9 % ( 78/124).We found that abnormal pl20ctn expression occurred more frequently in samples with poor differentiation, advanced tumor stage, and lymph node metastasis (P = 0.044, P = 0.008, and P = 0.0001, respectively) , while no significant associations were found with regard to age and sex. RhoA over expression has no significance with any pathologic factors. pl20CUI abnormal expression and RhoA over expression have obvious agreement and correlation ( Kappa = 0. 523,P<0.001).Western blotThe average level of pl20cm expression in normal lung tissue was significantly higher than that in the lung cancer specimens (60 472. 85 ± 352. 51 and 32 181.06 ±245.98, respectively; P =0.003). pl20CU1 has4 isoforms (iso-forms 14). In normal lung tissues, several specific bands that were 120 kDa (corresponding to isoform 1) and 100 kDa (corresponding to isoform 3 ) appeared, while the lung cancer samples showed faintly stained bands or the absence of isoform 1. RhoA shows bands in 21 KDa, and the average level of protein in tumors is higher than that in normal tissues (P <0.001)DiscussionAltered expression of pl20ctn has been implicated in tumor progression in several malignant carcinomas. The common change in pl20ctn immunostaining in cancer tissues has shown reduced membranous expression and/or increased cyto-plasmic expression. In this study, we also found similar abnormal expression of pl20ctn in Chinese patients with lung cancer. Reduced membranous expression of pl20ctn was observed in 99 of 124 tumors (79. 7% ) , which was similar to the proportion reported by Sarrio et al. for pl20ctn expression in 262 breast cancer (74.8% ). Our results showed that abnormal pl20cln expression was associated with poor differentiation, advanced tumor stage, and lymph node metastasis. Abnormal expression of pl20ctn may damage the function of the E - cad/catenin complex and affects related cell adhesion. As a result, this triggers tumor cells to detach from the primary site, invade surrounding tissues, and metastasize to lymph nodes and distant organs. Acquisition of cell dissociation and motility, induced by aberrations in the E - cad/catenin complex, could thus enhance the release of cancer cells from the primary site and affect the initial steps in the metastasis process.Fritz et al. reported that Rho GTPase expression may up - regulate in human cancers. This hypothesis was proved to be true by several studies. We examined the expression of RhoA in human lung cancer for the first time, and find that it expressed faintly in normal lung tissue while over expressed in lung cancers. This result consisted with the report by Horiuchi and Fritz in other tumors. Since pl20ctnmay inactive RhoA and make it change to GDP - binding status. We may concluded that what we found in cytoplasm might be phosphorylate -pl20ctn and GDP - binding RhoA; what we found in cell membrane might be un-phosphorylate - pl20ctn and GTP - binding RhoA. We also found that abnormal expression of pl20ctnhas obvious agreement and correlation with over expression of RhoA, which implied that abnormal expression of pl20ctn may act as an important factor to induce over expression of RhoA.Reduced membranous pl20ctn expression was also identified in our samples...