The Expression And Significance Of Annexin â…¡ In Lung Squamous Cell Cancer And Adenocarcinoma

IntroductionAnnexinⅡis a member of the Annexins family,it also be knomn as Annexin A2,CalpactinⅠheavy chain,LipocortinⅡ,P36.It is a calcium-dependent phospholipid-binding protein that plays a role in many cellular functions,including apoptosis,signal transduction,and cellular motility.AnnexinⅡexists in endothelial cell,monocyte,macrophage,nerve cell and some tumour cells,it lies cellular membranes or the cytoplasmic face of cellular membranes.It high to express in hyperplasia transform cell,low to express in split up ending cell.In recent years, We discover annexinⅡunusually express in tumours,such as gastric carcinoma, cancer of colon,prostate cancer,hepatocyte hepatocarcinoma,pancreas conduit gland cancer,etc.and correlate with invading and transformation moistening or prognosis of tumour.At present,it is also not clear about the relationship of the expression of annexinⅡin lung sguamous cell cancer and adenocarcinoma and malignant intensity of tumour attacking ability.This research through measure the expression of annexinⅡin lung squamous cell cancer and adenocarcinoma,analyze the relationship its expression with clinical pathologic factor,understand the role of annexinⅡin the development in lung cancer.And through measure the expression of annexinⅡin different attack ability of lung cancer cell,research the relation of between annexinⅡand lung outside the body of lung cancer cell attack ability furtherly.Meteral And Methods1.Tissue samplesForty fresh lung cancer samples with corresponding noncancerous tissue were obtained from the First Affiliated Hospital of China Medical University between May, 2006 and July,2007.One hundred and twenty lung cancer tissue samples were obtained from patients underwent surgical resection in the First Affiliated Hospital of China Medical University between 2000 and 2006.All were fixed in formaldehyde,embedded in paraffin.Human lung cancer cell lines BE1,A549,LH7,A2 and Homo sapien bronchusepithelial were come from China medicine university's laboratry.2.ReagentsThe primary antibody anti-human annexinⅡmouse monoclonal antibody produced in Santa Cruz Co.S-P kit was purchased from Fuzhou Maixin Biocompany.DAB agent kit and anti-humanβ-actin rabbit polyclonal antibody were bought from Zhongshan Biotechnology and Beijing Zhongshan Golden Bridge Biotechnology Co,Ltd.RPMI 1640 were purchased from Gibco.3.Method(1)Western BlotFresh tissue(1-2g)or collection cells in 5 volumes of lysis buffer was homogenized,treated with ultrasonic wave and centrifuged for 30 minutes.Then it followed by the whole procedure,including electrophoresis,transfer,blocking, incubation with primary antibody and enzyme conjugated compound,and dipping with DAB.It was determined as positive case that the special bands appeared in PVDF membrane,and then examine the gray value of the bands.(2)ImmunohistochemistryImmunohistochemical staining was performed using annexinⅡ(1:200) according to S-P method.The sections were deparaffinized,water processed,treated by 0.1%trytsin to expose antigen and incubated with primary antibody at 4℃overnight. The color was developed with diaminobenzidine and the section were couterstained with harmatoxylin. The expression of annexinⅡby IHC staining was graded for both the extent and intensity of label.The extent of staining was evaluated on a scale of 0-2:＜50%,0; 50-75%,1;＞75%,2 and the intensity of staining was evaluated on a scale of 0-2:no yellow was(-)0,buff was(+)1,yellow was(++)2.The case which the product of the two score were 2 or over 2 were positive ones.4.Statistical analysisThe statistical software SPSS 13.0 was applied for data analysis.Chi-square and t-test were used to study the relationship between annexinⅡexpression and clinicopathological characteristics,respectively.P＜0.05 was significative in statistic.Results1.Expression of annexinⅡin Lung Squamous Cell Carcinoma and Adennocarcinoma and it's neighboring noncancerous tissueAs a result of the Western Blot analysis on 40 cases of fresh Lung Squamous Cell Carcinoma and Adennocarcinoma and their neighboring noncancerous tissue,a 36kDa band corresponding annexinⅡprotein was detected in both cancer and para-carcinoma tissue.But the level of annexinⅡexpression was higher in cancer tissue than in para-carcinoma(p=0.02).There are 79 cases of Lung Squamous Cell Carcinoma and Adennocarcinoma tissue that appeared yellow or even brown particles in cytoplasm of tumor cell among 120 cases,and no signal was observed in stroma.2.Expression of AnnexinⅡin Lung cancer cell linesAs a result of the Western Blot analysis on four Lung cancer cell lines BE1,A549,LH7,A2 and Homo sapien bronchusepithelial,a 36kDa band corresponding annexinⅡprotein was detected in all cells,But the level of AnnexinⅡexpression was higher in BE1,A549,LH7,A2 than in Homo sapien bronchusepithelial,and the level of annexinⅡexpression was significantly higher in BE1(the cell with high metastasis) than in LH7(the cell with low metastasis)(p＜0.05). 3.The relationship between the expression of AnnexinⅡand pathological features,respectivelyThe expression of annexinⅡwas correlated closely with clinic stage(p=0.021),lymph node metastasis(p=0.030)and differentiation deagree(p=0.000),but had no relationship with gender(p=0.441),age(p=0.703)and historical type(p=0.844).Conclusion1.AnnexinⅡprotein are overexpress in Lung Squamous Cell Carcinoma and Adennocarcinoma,and the level were higher in cancer tissues than in para-carcinoma.2.The overexpression of annexinⅡis correlated with differentiation deagree,lymph node metastasis and clinic stage.3.The expression level of annexinⅡin lung carcinoma cell was higher than in HBE.4.The expression level of annexinⅡin the cell with high metastasis was higher than the cell with low metastasis.