Expression Of DLK/ZIPK And Integrinβ1 And Clinical Significance In Cervical Intraepithelial Neoplasia And Cervical Squamous Cell Carcinoma

ObjectivesApoptosis or programmed cell death (PCD)is a universal physiological cell death form. It can be initiated either by extrinsic signals or by intrinsic signals,mechanically depending on the dynamic balance between pro-apoptotic and anti-apoptotic pathways. Defective apoptotic processes have been implicated in an extensive variety of human diseases including cancers.Blocked apoptosis thus enhancing the viability of tumor cells is conducive to tumor cell escape immune surveillance as well as chemotherapy or radiotherapy induced cell death.Normal cervical epithelium becomes dysplastic before potentially developing into carcinoma.It is reported that apoptosis played an important role in CIN (cervical intraepithelial neoplasia) and cervical squamous cell carcinoma (SCC).Death-associated protein kinase (DAPk) family has emerged as a novel subfamily of pro-apoptotic serine/threonine kinase in the last 10 years. Although the functions of DAPk have been well documented, thoseof other family members remain uncertain. Dlk/ZIP kinase (DAPk like kinase/Zipper interacting protein kinase) is a member of DAPk family,which shared 81% kinase domain similarity to DAPK. More evidences were then accumulated to reveal that cytoplasmic Dlk/ZIPK could sensitize cells to various apoptotic stimuli, such as TNF-a, Fas ligands and anoikis.It involved in the regulation of cell apoptosis by inhibiting adhesion between cell-cell or cell- extracellular matrix and by degradating adhesion dependent molecules integrin and integrin-mediated survival signals. It is reported that Dlk was significantly down-regulated in cervical carcinoma specimens and gastric carcinoma, compared with normal adjacent tissues. It is expected to act as a tumor suppressor in tumorigenesis,and may become new tumor markers.However, the role of Dlk/ZIPK and integrinβ1 on the diagnosis and treatment in Early lesions in cervical need to be further studied.This study was conducted to delineate the role of apoptosis-related proteins in various stages of development in cervical neoplasia. To investigate the expression of DLK /ZIPK and Integrinβ1 and clinical significance in cervical intraepithelial neoplasia (CIN) and squamous cell carcinoma(SCC).Methods1.A total of 130 paraffin-embedded cervical tissue blocks were obtained from the Department of Gynecologic Oncology at the Cancer Affiliated Hospital of Shantou University Medical College in the period between March 2007 and April 2010, and clinical data of these patients was collected. These included 15 normal cervical epithelium , 30 CIN and 85 SCC. No one patient has undergone preoperative radiotherapy,nor chemotherapy. Their age bracket is from 27 to 74 years old, with an average age of 44.6 years.2.The immunohistochemical detection system S-ABC was used to detect the expression of DLK /ZIPK and Intβ1 in normal cervical epithelium, cervical intraepithelial neoplasia and squamous cell carcinoma3. Serial sections of thick 4μm, endogenous peroxidase was first blocked with H2O2. After normal goat serum blocking,slides wereincubated with anti-Dlk antibody and anti-integrinβ1 antibody, working concentration 1:100 and 1:50. Cytoplasmic staining appears brown as positive cells.The staining intensity of protein Dlk/ZIPK and integrinβ1 was quantified by semi-quantitative used in reference.4. Statistical analysis was done using SPSS software version 13.0. The chi-square test was used to analyze the correlation between DLK /ZIPK and Intβ1 expression and clinico-pathological variables,such as patient age ,tumor size, depth of myometrial invasion, histopathologic grade, lymph vascular invasion and lymph node metastasis.The p values of less than 0.05 were considered statistically significant. Results1.The positive rate of DLK /ZIPK in NE was 93.3%, much higher than those 63.3% in CIN and 32.9% in SCC(P<0.05).In CIN, but DLK / ZIPK protein expression did not showed a downward trend , 66.7% in CINII vs 60.0% in CINIII.2.The relationship between DLK /ZIPK protein expression and the clinicopathological significance of SCC: the positive rate of DLK / ZIPK in squamous cell carcinoma of stage IA,stage Ib1, stageIb2, stage IIA decreased gradually.they were 40.0%,32.5%,33.3%,26.7%. difference between every two of them were not statistically significant. neither between clinical Phase I and II. DLK /ZIPK low expression was closely correlated with tumor size, depth of myometrial invasion, histopathologic grade, and lymph node metastasis(LNM) (P<0.05), but not with age and vessel invasion(p>0.05).3. Weak expression (13.3% )of Integrinβ1 was found in NE,while the positive rates of Integrinβ1 are 63.3 % in CIN and 85.9% in SCC. Differences among them were statistically significant.(P < 0.05),however, Difference between CINII and CINIIIwas not statistically significant, due to positive rate 60.0% in CINIIvs66.7% in CINIII.4. The relationship between Integrinβ1 protein expression and the clinico - pathological significance of SCC: the positive rate of Integrinβ1in squamous cell carcinoma of stage IA,stage Ib1, stageIb2, stage IIA increased gradua lly.they were 73.3%,87.5%,86.7%,93.3%. differences between every two of them were not statistically significant. neither between clinical Phase I and II. Intβ1 high expression was closely correlated with LNM and histopathologic grade(p<0.05),but not with age ,tumor size,depth of myometrial invasion and vessel invasion(p>0.05).5. In the 30 cases of CIN tissues, there were 18 cases (60.0%), which expression of DLK / ZIPK was consistent with expression of Intβ1,that is, 13 cases(43.3%) both positive expressioned and both negative in 5 cases(38.5%). by Spearman correlation analysis, DLK / ZIPK expression in CIN tissue had no correlation with the expression of Intβ1 (r = 0.139, P = 0.465). In the SCC, the cases number of the DLK / ZIPK positive together with Intβ1 positive was 19, accounting for 22.4%,while 5 caseswere both negative, accounting for 5.9%, DLK / ZIPK expression in CIN tissue was negatively correlated with the expression of Intβ1 (r =- 0.244, P = 0.024) .Conclusions1.DLK/ZIPK and Intβ1 is closely related to the occurrence and development of cervical cancer. The expression rate decreased with the progress of disease, DLK /ZIPK may be an inhibitor of cervical carcinoma by promoting cell apoptosis and it might inhibit cancer metastasis by neoplastic differentiation. Thereby DLK/ZIPK is expected to become a new tumor suppressor gene.The expression of integrinβ1 might be related to the differentiation metastasis and invasion of cervical cancer and might reflect the prognosis of cervical cancer to some extent. It could be used as indicators of prognosis for scc, and provide a new way for targeted therapy.2. In the process CINⅡ→CINⅡ, the expression DLK / ZIPK had no correlation with resistanced by the Intβ1-mediated cell survival signals. In SCC,DLK / ZIPK expression decreased, while the expression of Intβ1increased,there was a negative correlation between the two. Both synergisticly promoted the development of cervical cancer ,leading to an imbalance between cell proliferation and abnormal apoptosis .it hinted that they may be an important part in the cell apoptosis signal transduction network.