The Relationship Between TSG101 Protein And Notch3 Receptor In Lung Cancer

IntroductionRecently,TSG101 protein has been found to have association with various human tumors and its aberrant transcripts occurred in many kind of cancer.But its role in tumorigenesis is controversial,and has a defined role in the endocytic pathway.Defects in this machinery will induce dysregulated of the lysosome-dependent degradation of cell-surface receptors,then cell-cell signaling will be disordered.Signaling by Notch receptors has multiple and essential roles in many cell fate decision and in patterning events.Endocytosis regulates the steady-state level of these receptors in differernt time and different space and ensure the normal cell proliferation and differentiation.Some reports indicate that Drosophila cells with impaired Tsg101 function shows accumulation of the Notch receptor in intracellular compartments of cytoplasm.This causes increased Notch-mediated signaling and the overproliferation of Drosophila imaginal discs.In mammals,there are four Notch receptors,Notch3 receptor plays an important role in regulating airway epithelial development and differentiation.So we investigate the expression of TSG101 protein and Notch3 receptor in human lung cancer,and the possiblely relationship between them.Materials and Methods1,Samples65 lung cancer tissues samples(38 lung squamous carcinoma and 27 lung adenocarcinomas),the other 24 fresh lung cancer tissue samples and 24 fresh non-cancerous tissue were obtained from patients who had surgery in the First Affiliated Hospital of China Medical University.6 lung cancer cells(SK is squamous cell carcinoma cells,BE1 is gaint cell carcinoma cells,460 is large cell undifferentiated carcinoma cells,LTE and SPC are adenocarcinoma cells,446 is small cell lung carcinoma cells).2,ReagentThe anti-human TSG101 mouse monoclonal antibody(sc-7964) and anti-human Notch3 receptor polyclonal antibody(sc-5593) were provided by Santa Cruz company (USA).3,Methods(1)Immunohistochmistry:Detect the TSG101 protein(1:200),Notch3 receptor(1:200) expression by Immunohistochemistry S-P method in lung cancer tissue. Detect Notch3 receptor(1:200) expression by Immunohistochemistry S-P method in SPC cells.(2)Western blot:Detect the expression of TSG101 protein and Notch3 receptor in lung cancer tissues/noncancerous tissues and lung cancer cell lines.①Electrophoresis;②transfer proteins from gel to PVDF membrane(Notch3 receptor: 200 mA,500min,4℃);③blocking(TBS/0.3%Tween-20/5%CS RT 2hrs);④incubation with primary antibody(1:400);⑤detection by ECL.β-actin of Notch3 receptor was obtained by cutting the gel and transfering respectively.(3) Antibody Blocking:SPC cells were incubated with serum-free medium 1640 for 8hrs,then devided them into 3 lots.One was incubated without antibody,the other was incubated with normal mouse IgG,the third was incubated with anti- TSG101 protein for 48hrs respectively.Then to detect the expression of Notch3 receptor in SPC cells after blocking the function ofTSG101 protein with WB and Immunohistochemistry.4,Statistical analysisAll the data are analyzed with SPSS for Windows 13.0 software.We use x~2 test to analyze the correlation between clinic pathologic parameters of lung cancer and expression of TSG101 protein and Notch3 receptor,we use the permutation test for the Spearman correlation coefficient to analyze the correlation between expression of TSG101 protein and Notch3 receptor.The result of Western Blot is analyzed by t test. The statiscal significance is defined as P＜0.05.Result1,Immunohistochmistry TSG101 protein localized in cytoplasm and/or nucleus.The staining of TSG101 protein in tumor tissues was significantly lower than that in normal lung tissue.Notch3 receptor localized in cytoplasm and nucleus and menbrane.The staining of Notch 3 receptor was higher in tumor tissues.These changes were correlated with the cellular differentiation and lymph node metastases.And TSG101 protein is positively negative correlation with Notch3 receptor.And the staining of Notch3 receptor in SPC cell cytoplasm was raised when blocked the function ofTSG101 protein.2,Western BlotThe expression of TSG101 protein in tumor tissues and cell lines were significantly lower than that in the neighboring noncancerous tissue and normal cell line,the expressions of Notch 3 receptor was higher in tumor tissues and cell lines.The detection level of the expression of Notch3 receptor in SPC cells was raised when the function ofTSG101 protein was blocked.ConclusionThere's some relationship between the down- regulation of TSG101 protein and the up- regulation of Notch 3 receptor in human lung cancer.