Expression And Significance Of A Group Of Apoptosis-Associated Proteins Including P53, Caspase 3, Cathepsin D, Survivin And Fas In Non-Small-Cell Lung Cancer

IntroductionApoptosis is an important and necessary physiological process of humanbeing.In the past years the abnormity of apoptosis has been recognized as an important mechanism involed in oncogene and tumor process.The mechanism by which p53 repress oncogene is widely thought to be associated with it's function to supervise and repaire of the change of genes.If ia can't repaire them then it will lead the cell to a process of apoptosis.The function is partly contributed to the bcl-2 family,caspase 3 is a effective proteinase in the process of apoptosis.Cathepsin D(Cat D)is widely found in normal tissuse and mainly disttributedinthe cellular endosomes.The report about its expression in tumor cells are not so consistent.Recently the research about this proteinase was mainly consentrated in the research about its possible function in the process of apoptosis.Survivin is a member of the IAPs(inhibitor of apoptosis protein, IAP)and was widly researched.Its high expression was found in many tumors and was reproted to be associated with the tumor progression.Fas belongs to the death receptor proteins.It bands to its ligan Fas-L and then transducts the process of apoptosis.It also is involed in the interaction between the cancer cells and the immunal cells in the tumor tissues.In this study we investigate the expression of a group of apoptosis associated proteins including p53,caspase 3,cat D,survivin and fas in the NSCLC tissues to access the meanings of this expression and the possible apoptosis signal conduction work by these protein in lung cancer cells.Materials and Methods1.Materials80 primary lung cancer tissue samples and 15 neighboring noncancerous tissue samples were obtained from patients who had surgery in the First Affiliated Hospital, China Medical University between 2001 and 2005.2.ReagentsAntibodies were as follow:anti-P53 IgG(mouse monoclonal antibody); anti-caspase 3 IgG(mouse monoclonal antibody);anti-cathepsin D IgG(rabbit polyclonal antibody);anti-survivin IgG(rabbit polyclonal antibody);anti-Fas IgG (rabbit polyclonal antibody).3.ImmunohistochemistryTissue sections(4μm thick)of formalin-fixed,paraffin- embedded specimens were deparaffinized in xylene,rehydrated in graded alcohol,and transferred to PBS.The slides were rinsed 3 times with PBS,and antigen retrieval was performed with pressure-cooker for 75 second.Endogenous peroxidase was blocked by the use of 3% hydrogen peroxide in PBS for 20 min.The samples were washed three times with PBS and incubated for 30 min at room temperature with a protein blocking solution.Excess blocking solution was drained,and the samples were incubated overnight at 4℃with anti-caveolin-1 antibody and anti-CD34 antibody.Immunodetection was performed with diaminobenzidine as the chromogen and counterstained with hematoxylin and dehydrated in alcohol before mounting.4.Immunohistochemical evaluationP53 was a nuclear staining protein,and caspase 3 a nuclear and partly cytoplasmic staining protein,survivin a cytoplasmic staining protein,cathepsin D a cytoplasmic staining protein,and Fas was a cytoplasmic and membrane staining protein.Both staining intensity(Ⅰ)and scope(S)were counted.It was considered negative when less than 10%of all caner cells were stained.5.Statistic analysisAll the data were analyzed with SPSS for Windows 13.0 software.The Chisquare test and precise probability method were adopted to compare the clinicopathological characteristics with the expressions of p53,caspase 3,cat D, survivin and fas.Spearman correlation test was used to analyse the the correlation between the expression of these proteins.Statistical significance was defined as P＜0.05.Results(1)The positive percentage of p53 in lung cancer tissues was 53.75%,much higher than that in normal lung tissues(P＜0.01).In NSCLC,there was no correlation between the expression of p53 and the pathogy and clinical data such as sex,years, pathology type,and pathology and clinical stages(p＞0.05).(2)The positive percentage of p53 in lung tissues was 46.67%.The positive percentage of p53 in lung cancer tissues was 53.75%.There was no significant difference between the expression of caspase 3 in theses two parts(p＞0.05).In NSCLC, there was no correlation between the expression of caspase 3 and the pathogy and clinical data such as sex,years,pathology type,and pathology and clinical stages(p＞0.05).(3)The positive percentage of p53 in lung tissues was 53.33%.The positive percentage of p53 in lung cancer tissues was 63.75%.There was no significant difference between the expression of cat D in theses two parts(p＞0.05).In NSCLC, there was no correlation between the expression of cat D and the pathogy and clinical data such as sex,years,pathology type,and pathology and clinical stages(p＞0.05).(4)The positive percentage of p53 in lung cancer tissues was 47.50%,much higher than that in normal lung tissues(P＜0.01).In NSCLC,there was no correlation between the expression of survivin and the pathogy and clinical data such as sex,years, pathology type,and pathology and clinical stages(p＞0.05). (5)Fas was localized in cell membrane and cytoplasm.It showed expression in both the normal surrounding lung tissues and NSCLC cancer cells.The positive percentage of p53 in lung tissues was 60%.The positive percentage of p53 in lung cancer tissues was 50%.There was no significant difference between the expression of cat D in theses two parts(p＞0.05).In NSCLC,there was no correlation between the expression of Fas and the pathogy and clinical data such as sex,years,pathology type,and pathology and clinical stages(p＞0.05).6.The correlation between the expression of these proteins:there was a postive correlation between the expression of p53 and survivin(R=0.262,P=0.020).And there was a postive correlation between the expression of cat D and fas(R=0.375,P=0.001).Conclusions(1)The expression of p53 and survivin in NSCLC cancer cells was much higher than that in normal lung tissues.(2)There was no significant difference between the expression of caspase 3,fas, cat D in NSCLC cancer cells and the surrounding lung tissues.(3)In NSCLC,there was no correlation between the expression of p53,caspase 3,cat D,survivin and Fas and the pathogy and clinical data such as sex,years, pathology type,and pathology and clinical stages(4)There was a postive correlation between the expression of p53 and survivin in NSCLC cancer cells.(5)There was a postive correlation between the expression of cat D and Fas in NSCLC cancer cells.