Significance Of Claudin-3 And Claudin-4 In Endometrial Malignant Transformation

PART IExpression of claudin-3 and claudin-4 in normal, hyperplastic and malignant endometrial tissueObjective: To investigate the expression of claudin-3 and claudin-4 in normal, hyperplastic and malignant endometrial tissue, and to clarify the roles of claudins in endometrial tumorigenesis.Methods: Levels of protein and mRNA expression of claudin-3 and claudin-4 in human endometrial tissue were determined (proliferative phase (PE, n=25); secretory phase (SE, n=25); simple hyperplasia (SH, n=20); complex hyperplasia (CH, n=12); atypical hyperplasia (AH, n=15); endometrioid adenocarcinoma (EEC, n=30)) using immunohistochemistry, western blot and real-time PCR, respectively. Morphologic changes of tight junctions (TJs) were also observed in normal, hyperplastic and malignant endometrial tissue.Results: Absence or weak staining for claudin-3 and claudin-4 was observed in PE, SE, SH and CH, while medium to intense staining was detected in AH and EEC. Staining of claudin-3 and claudin-4 was predominantly localized to the glandular epithelial cell membrane. Expression of claudin-3 and claudin-4 was significantly increased in the groups of AH and EEC in comparison with the groups of CH, SH and normal cyclic endometrium at both protein and mRNA levels. The highest expression was observed in EEC. Although no relevance was found with regard to FIGO stage and histologic grade, overexpression of claudin-3 and -4, especially claudin-4, significantly correlated with myometrial invasion.Conclusion: Transmission electron microscopy analysis indicated morphological disruptions of TJs may lag behind the increase of claudins expression. Claudin-3 and claudin-4 are strongly expressed in AH and EEC, but less frequently in normal endometrium. The up-regulation of claudins expression during endometrial carcinogenesis suggests their potential utility as diagnostic and prognostic biomarkers. PART IIRegulation of claudin-3 and claudin-4 expression inendometrial adenocarcinoma cell line by megestrol acetateObjective: To determine the expression change of claudin-3 and claudin-4 in Ishikawa endometrial adenocarcinoma cell line in response to progesterone, and to evaluate the role of claudin-3 and claudin-4 in the anti-cancer effects of progesterone.Methods: MTT assay was used to determine the inhibitory concentration 50% (IC50) of megestrol acetate (MA) in treating Ishikawa. After the Ishikawa cell was treated with MA at IC50, cell apoptosis was examined by flow cytometry and transmission electron microscopy. The mRNA and protein expression levels of claudin-3 and claudin-4 were further quantified by real-time PCR and western blot. The localization of claudins was examined by immunofluorescent staining.Results: The inhibitory effects of megestrol acetate on the growth of Ishikawa cell were dose and time dependent. The IC50 of MA on Ishikawa was 15mg/l after incubated for 72 hours. Apoptosis percentage was elevated from (0.06±0.02)% to (3.74+0.69)% after MA treated. Ishikawa cell expresses claudin-4, not claudin-3. No significant difference for expression of claudin-4 at both protein and mRNA levels was found before and after MA use. However, The localization of claudin-4 transferred from cytomembrane to cytoplasm and nucleus.Conclusion: Megestrol acetate can inhibit the growth of Ishikawa cell, and apoptosis can be easily detected after MA use. Ishikawa cell expresses claudin-4, not claudin-3. The localization change of claudin-4 may involved in the anti-cancer effects of progesterone. PART IIIAltered expression of claudin-3 and claudin-4 in ectopicendometrium of women with endometriosisObjective: To investigate the expression of claudin-3 and claudin-4 in the eutopic and ectopic endometrium of women with endometriosis and to evaluate the role of claudin-3 and claudin-4 on the pathogenesis of endometriosis.Methods: Thirty-five women with endometriosis and 35 controls were studied. The expression of claudin-3 and claudin-4 were investigated using immunohistochemistry, western blot and real-time PCR, respectively. Morphologic changes of tight junctions were also observed in different kinds of endometria.Results: Expression of claudin-3 and claudin-4 were significantly lower in the ectopic endometriotic tissue than in the eutopic endometrium from women with endometriosis and normal control at both mRNA and protein levels. No significant difference was found between eutopic endometrium from women with endometriosis and normal endometrium from women without endometriosis. Expression of claudin-3 and claudin-4 was not influenced by the menstrual cycle in endometrial samples deriving from women with endometriosis and healthy controls.Conclusion: Down-regulated expression of claudin-3 and claudin-4 in ectopic endometrium suggests that claudin-3 and claudin-4 might play a pathogenic role in the formation of endometriosis.