Expression Of 14-3-3zeta And Its Influence On Tumor Development And Apoptosis In Stage T₁ Non-small Cell Lung Cancer

Lung cancer is a malignant tumor and does harm to human health seriously.lt has been one of the leading causes of cancer death for urban residents in China.So it is important to detect the molecular mechanism of lung cancer invation and metastasis. Better understanding of the biological mechanisms in non-small cell lung cancer (NSCLC) is still needed to clarify the patients'progression,prognosis and achieve the most effective treatments. Research on relationship between 14-3-3 protein and lung cancer has become the focus now.14-3-3 proteins are highly conserved small acidic proteins.Study shows 14-3-3 proteins can bind many signal proteins,such as kinase,phosphatase and transmembrane receptor et al. The 14-3-3 proteins play crucial roles in many biological processes including regulation of signaling,cell cycle and apoptosis.There are seven isoforms in mammalian genes:ζ,β,γ,ε,σ,ηandτ. Isoform s andζcan be found in normal lung tissue butβ,γ,σ,ε,ζandτare expressed in lung cancer tissue all.14-3-3 protein isoforms are relevant to tumorgenesis and regarded as potential tumor suppressor gene or oncogene. Thus intensive understanding the function of different 14-3-3 isoforms may offer us a new idea in aiticancer therapies.14-3-3ζis one of the isoform of 14-3-3 proteins and its exact function is unclear now. Research of 14-3-3ζfocus on its regulation function in apoptosis. Incontrollable of apoptosis program is associated with the occurrence, development and prognosis in malignant tumor. Bcl-2 family is one of the most important protein family controlling apoptosis. Bad is one of the promoting apoptosis factor and its expression is of important significance in tumor. Ichinose reported that phosphorylated Bad lost its activity and inactive Bad(p-Bad) could enhance vicious transformation of glioblastoma and prostatic cancer.Research shows that 14-3-3ζ, can inhibit apoptosis through different mechanisms,such as by regulating the Bcl-2 family.14-3-3ζbinds the phosphorylated BAD and insulates it in the cytoplasm, prevents its binding with Bcl-2 and Bcl-XL and so as to inhibiting cell apoptosis. Akt is a serine-threonine kinase located in the downstream of phosphatidylinositol-3 - kinase (PI3-K) and can be activated by many cell survival promoting signal protein.Taso found that activation of Akt involved in early stage of canceration and may be the important event in NSCLC origination. Powell found that Akt phosphorylated Ser-58 on 14-3-3zeta both in vitro and in intact cells.Apoptosis regulated by 14-3-3ζmight control tumor occurrence, progression, invation and matastasis to some extent. So it is important for us to study the expression of apoptosis facters and its association with 14-3-3ζin NSCLC.Malignancy often followed by invation and matastasia. Tumor invation and matastasia is a comolicated process involves many multiple steps and facters and its precondition is the loss of cell adherency. Study showed that E-cadherin had the most close relationship with the abnormal cell-cell adhesion and the proliferation,invation and matastasis of epithelial tumors.Function of cell-cell adhesion mediated by E-cadherin is associated with catenins andβ-catenin plays a key role in the regulation of cell adherency.Research on the relationship between 14-3-3ζand adhension occurs recently and thorough understanding of this relationship may intensify our knowledge and research on the invation and matastasis in NSCLC.To test 14-3-3ζexpression and its relationship with invation, matastasis, prognosis and apoptosi in stage T1NSCLC tissues, western blotting, double labeling immunofluorescence and confocal laser scanning microscopy, immunohistochemical and RT-PCR analysis were carried out. Understanding the relationship between 14-3-3ζ protein and lung cancer and revealing the molecular mechanism will offer us a new idea to beat the disease.MATERIALS AND METHODSThe study population included 110 patients who underwent resection of tumor for pathological stage T1 NSCLC between 2000 and 2006 at Department of Thoracic Surgery, First Affiliated Hospital of Liaoning Medical colledge. All cases were full of follow-up and cut-off date for April 2008, calculation of surial from surgery to cut-off date or to date dead of recurrence and metastasis.50 patients who underwent resection of tumor for pathological stage T1 NSCLC between November,2007 and November, 2008 at Department of Thoracic Surgery, First Affiliated Hospital of China Medical University. None of the patients received chemotherapy or radiotherapy before the operation.To test 14-3-3ζ,β-catenin, E-cadherin, p-Bad and p-Akt expression in stage T1 NSCLC and normal lung tissues, western blotting, immunohistochemical analysis were carried out. To test 14-3-3ζmRNA content in NSCLC and normal lung tissues,RT-PCR analysis were carried out. Double labeling immunofluorescence and confocal laser scanning microscopy analysis were carried out to test the coexistence of 14-3-3ζwithβ-catenin and E-cadherin.RESULTS一,Expression of 14-3-3ζin stage Ti NSCLCImmunohistochemical, western blotting and RT-PCR analysis were carried out to test 14-3-3ζprotein and 14-3-3ζmRNA expression in stage T1 NSCLC and normal lung tissues. Result shows that more strong expression of 14-3-3ζwas observed in stage T1 NSCLC tissues than in normal lung cancer tissue. Overexpression of 14-3-3ζprotein did not show any correlation with age, gender and histological types,but was significantly correlated with histological grades and lymph node metastasis. Companied with lower lung cancer differentiation,14-3-3ζexpression increased. The 14-3-3ζ expression was higher in the lymph node metastasis positive cancer tissues than in the negatie cases. The survival curves analysis showed that 14-3-3ζexpression is closely related to the prognosis of patients.二,Expression ofβ-catenin in stage T1 NSCLC and Relationship between 14-3-3ζand P-cateninImmunohistochemical, western blotting, double labeling immunofluorescence and confocal laser scanning microscopy analysis were carried out to testβ-catenin protein expression in stage T1 NSCLC and normal lung tissues. Result shows most NSCLC samples showed reduced membranous staining or increased nuclear or cytoplasmic staining. Abnormal expression ofβ-catenin protein did not show any correlation with age, gender and histological types but showed significantly correlation with poor differentiation and lymph node metastasis. Abnormalβ-catenin expression occured more frequently in poor differentiated tumor tissues than in well differentiated cases and more frequently in the lymph node metastasis positive cancer tissues than in the negatie cases. Statistical analysis showed significantly negative correlation between P-catenin protein and 14-3-3ζexpression.三,Expression of E-cad in stage T1 NSCLC and Relationship between 14-3-3ζand E-cadImmunohistochemical,western blotting, double labeling immunofluorescence and confocal laser scanning microscopy analysis were carried out to test E-cad protein expression in stage T1 NSCLC and normal lung tissues. Result indicated most NSCLC samples showed reduced or deletion membranous staining. Abnormal expression of E-cad protein did not show any correlation with age, gender and histological types,but showed significantly correlation with poor differentiation and lymph node metastasis. Abnormal E-cad expression occured more frequently in poor differentiated tumor tissues than in well differentiated cases and more frequently in the lymph node metastasis positive cancer tissues than in the negatie cases. Statistical analysis showed significantly negative correlation betweenβ-catenin protein and 14-3-3ζexpression in stage T, NSCLC.四,Expression of p-Bad in stage T1 NSCLC and Relationship between 14-3-3ζand p-BadImmunohistochemical,western blotting analysis were carried out to test p-Bad protein expression in stage T1 NSCLC and normal lung tissues. Result showed increased expression of p-Bad in stage T1 NSCLC tissues. Expression of p-Bad protein did not show any correlation with age, gender and histological types but showed significantly correlation with poor differentiation and lymph node metastasis. Increased p-Bad expression occured more frequently in poor differentiated tumor tissues than in well differentiated cases and more frequently in the lymph node metastasis positive cancer tissues than in the negative cases. Statistical analysis showed significantly positive correlation between p-Bad protein and 14-3-3ζexpression in stage T1 NSCLC.五,Expression of p-Akt in stage T1 NSCLC and Relationship between 14-3-3ζand p-AktImmunohistochemical,western blotting analysis were carried out to test p-Akt protein expression in stage T1 NSCLC and normal lung tissues. Result showed no expression of p-Akt was observed in normal lung tissue but increased expression of p-Akt was observed in stage T1 NSCLC tissues. Expression of p-Akt protein did not show any correlation with age, gender and histological types,but showed significantly correlated with poor differentiation and lymph node metastasis. Increased p-Bad expression occured more frequently in poor differentiated tumor tissues than in well differentiated cases and more frequently in the lymph node metastasis positive cancer tissues than in the negative cases. Statistical analysis showed significantly positive correlation between p-Akt protein and 14-3-3ζexpression in stage T1 NSCLC. CONCLUSION1. The upregulated expression of 14-3-3∫in stage T1 NSCLC indicate its possibility of promoting NSCLC dovelopment. Overexpression of 14-3-3ζcorrelated with histological grades, lymph node metastasis and poor clinical outcome but not with age, gender and histological types in stage T1NSCLC.lt seems that 14-3-3ζmight be used as develpomental and prognostic biomarkers for NSCLC.2. Expression ofβ-catenin and E-cadherin is increased obviously in stage T1 NSCLC than in normel lung tissue.Abnormal expression ofβ-catenin and E-cadherin correlated with histological grades, lymph node metastasis but not with age, gender and histological types. It seems thatβ-catenin and E-cadherin might be involoved in development and metastasis of NSCLC.3. Abnormalβ-catenin and E-cadherin expression was associated signigicantly with 14-3-3ζexpression in stage T1 NSCLC. It seems that overexpression of 14-3-3ζmight induce abnormal expression ofβ-catenin and E-cadherin so as to affect invation and metastasis of NSCLC.4. Expression of p-Bad and p-Akt is increased obviously in stage T1 NSCLC and is correlated with histological grades, lymph node metastasis signigicantly. It seems that p-Bad and p-Akt might be involoved in development and metastasis of NSCLC.5. Upregulated p-Bad and p-Akt expression was associated signigicantly with positive 14-3-3ζexpression in stage T1 NSCLC. It seems that overexpression of 14-3-3ζmight block apoptosis of NSCLC cells through some molecular mechanism.