| Breast cancer is one of the most common malignant tumors in the world. The incidence of breast cancer is quite high in China and is now the most common female malignant tumors. Considerable progress has been made during recent decades in the diagnosis and treatment of primary cancer. As a result, the overall mortality rate from breast cancer has significantly decreased. Despite these advances, patients that present with metastatic disease or who relapse with disseminated tumors usually have poor prognosis. Metastatic tumors are often refractory or only partially sensitive to current therapeutic strategies and are the primary cause of cancer-related mortality. Therefore, it is of important significance to further explore the mechanism and search for new target for the inhibition of metastasis so as to decrease mortality and prolong life span of breast cancer patients.Ezrin was found to be a crucial molecule in the dissemination of some malignant tumors in 2004. Studies showed that Ezrin involved in many biological processes in tumor metastasis, such as the interaction between cancer cells and between cancer and matrix through signal transduction and metastasis-associated cell surface molecules. Ezrin is a key regulatory molecule associated with carcinogenesis and metastasis of many malignant tumors such as rhabdomyosarcoma and osteosarcoma. Up to now, how does Ezrin function as a central organizer for tumor dissemination in breast cancer and other tumors is still not quite clear. To further explore the putative role of Ezrin in the carcinogenesis and metastasis of breast cancer and to set scientific bases for corresponding molecular targeting treatment, we evaluated the expression of Ezrin in the breast cancer tissues and observed the role of anti-sense RNA and cytotoxic drug on the expression of Ezrin as well as the proliferation and invasion of human breast cancer cell lines MDA-MB-231 in vitro. At the same time, the regulatory efffect RhoA, a upstream regulatory signal, on the expression of Ezrin was also studied.The study includes the following four parts:1 Ezrin expression has putative significance of the carcinogenesis and prognosis of infitrating ductal carcinoma of breastObjective: To explore the putative significance of Ezrin expression on the carcinogenesis and prognosis of infiltrating ductal carcinoma of breast.Methods:S-P immunohistochemical method was used to assess the expression of Ezrin in 88 cases of infiltrating ductal carcinoma of breast (with intact clinicpathological and follow-up information) and 54 cases of different intraductal hyperplastic lesions. The relationship between Ezrin expression and the clinicpathological parameters in infiltrating ductal carcinoma of breast was analyzed. The prognostic significance of Ezrin expression in infiltrating ductal carcinoma of breast was statistically studied with Kaplan-Meier and COX models.Results:1.1 Expressions of Ezrin in different pathological changes of breast ducts The strong positive rates of Ezrin expression in atypical hyperplasia and infiltrating ductal carcinoma cases were significantly higher than those in normal ductal epithelium and simple ductal hyperplasia groups (P<0.05), while that in infiltrating ductal carcinoma was significantly higher than that in atypical hyperplasia cases (P<0.05). From normal ductal epithelium to simple ductal hyperplasia, atypical hyperplasia and infiltrating ductal carcinoma, the strong positive rates of Ezrin expression increased gradually.1.2 The relationship between Ezrin expression and clinicpathological parameters of patientsThe strong Ezrin expression in infiltrating ductal carcinoma was positively correlated with axillary lymph node metastasis, histological grading, and TNM stages. The strong positive rates of Ezrin expression in the cases of axillary lymph node metastasis, higher histological grading, and advanced TNM stages were much higher than those of the controls (P<0.05).1.3 The prognosis significance of Ezrin expressionThe patients with strong positive expression of Ezrin had shorter survival time than those of controls during our follow-up period (P<0.05). According to the COX analogue multiplicity, the strong positive expression of Ezrin, state of axillary lymph node, and TNM stage could be the independent index of prognosis for the breast infitrating ductal carcinoma patients.1.4 The relationship between expression of Ezrin, CD44v6 and E-Cadherin According to the analysis of Spearman rank correlation, the strong Ezrin expression in infiltrating ductal carcinoma was positively correlated with the expression of CD44v6, while negatively correlated with the expression of E-Cadherin (P<0.05).2 Anti-sense RNA inhibits the expression of Ezrin and invision of breast cancer cellsObjective: To observe the influence of proliferation and invasion of human breast cancer cells MDA-MB-231 after inhibition of Ezrin expression by anti-sense RNA.Methods:Human breast cancer cell line MDA-MB-231 with highly metastatic ablity was transfected with anti-pCR3.1-ezrin with lipofectamine. Western-blot and RT-PCR were used to determine the expression of Ezrin. The proliferative ability of cells was tested by MTT assay, and the metastatic ability of cells was investigated by Boyden cabin.Results:2.1 Anti-sense RNA impacted the expression of Ezrin protein After 24 h transfection with anti-sense RNA, Ezrin protein expression in MDA-MB-231 cells was significantly reduced by 24.24% and 22.22%, respectively, as compared with that of transfecting with vacuity plasmid group and that of blank control group (P<0.05).2.2 Anti-sense RNA impacted the expression of Ezrin mRNA After 24 h transfection with anti-sense RNA, Ezrin mRNA expression in MDA-MB-231 cells was significantly reduced as compared with those of vacuity plasmid group and blank control group (P<0.05). Ezrin mRNA expression in both vacuity plasmid group and blank control group had not significant difference (P>0.05).2.3 Anti-sense RNA impacted the proliferation of MDA-MB-231 cells The MTT assay showed that OD value in anti-pCR3.1- ezrin group,vacuity plasmid group and blank control group were 0.41±0.018, 0.765±0.058, and 0.795±0.061, respectively. The results suggested that the proliferation of MDA-MB-231 cells was inhibited significantly in anti-pCR3.1-ezrin group (P<0.05).2.4 Anti-sense RNA impacted the invasive ability of MDA-MB-231 cells After 24 h transfection with anti-sense RNA, the invasive ability of MDA-MB-231 cells was 50.5%±3.2% as compared with that of blank control group (P<0.05). The invasive ability of MDA-MB-231 cells in vacuity plasmid group was almost the same of that of blank control group3 RhoA regulates the expression of Ezrin as a upstream regulatory signal Objective: To investigate whether RhoA regulates the expression of Ezrin as a upstream regulatory signal.Methods:Western-blot was used to determine the effect of fasudil, a special RhoA kinase inhibitor, on EGF induced the expression of RhoA, p-RhoA, and Ezrin.Results:3.1 EGF impacted the phosphorylation of RhoA Western-blot results showed that the level of RhoA phosphorylation ascended gradually after induction with EGF for 5 min, and increased up to peak for 30 min. The protein expression of RhoA had no marked changes.3.2 EGF impacted the expression of Ezrin Western-blot results showed that the expression of Ezrin ascended gradually after different time treatments with EGF, and increased up to peak time for 24 h.3.3 Fasudil impacted both the phosphorylation of RhoA and the expression of Ezrin induced by EGFWestern-blot results showed that phosphorylation of RhoA had been significantly reduced by 72.73% and the expression of Ezrin had been significantly reduced by 51.28% at their apex time after the pre-treatment with fasudil.4 Low dose epirubicin inhibit the migration of breast cancer cells is associated with the expression of Ezrin Objective: To investigate the mechanisms on low dose epirubicin (EPI) inhibit the migration of breast cancer cellsMethods:Influence on expression of Ezrin and migration of breast cancer cells after treated by low dose epirubicin was observed. After 4 h treatment with defferent low dose epirubicin, western-blot, immunocytochemistry, and RT-PCR were used to detect the effect of epirubicin on the expression of Ezrin, CD44, and E-Cadherin. Cell motility was measured by using monolayer wounding protocols, and cell vigor was measured with MTT assay.Results:4.1 EPI impacted the protein expression of Ezrin, CD44 and E-Cadherin Western-blot results showed that when the cells were pretreated for 4 hours with EPI (2.5, 5 and 10μg/ ml), both Ezrin and CD44 protein expressions were decreased significantly as compared with those of control (P<0.05). However, E-Cadherin protein expression was ascended significantly as compared with that of control (P<0.05). These changes could also be seen in the results of immunocytochemistry. The functions of EPI were in concentration-dependent manner.4.2 EPI impacted the mRNA expression of Ezrin, CD44 and E-Cadherin RT-PCR results showed that when the cells were pretreated for 4 hours with EPI (2.5, 5, and 10μg/ ml), both Ezrin and CD44 mRNA expressions were decreased significantly as compared with those of control (P<0.05). However, E-Cadherin mRNA expression was ascended significantly as compared with that of control (P<0.05). The functions of EPI were in concentration-dependent manner.4.3 EPI impacted the migration ability of MDA-MB-231The groups incubated with EPI were unable to close the wound and exhibited an obviously reduction in migration rate as compared with the control. The reduction rates were 21.4%, 50.0%, 64.3% and 85.7% at defferent dose of EPI (1, 2.5, 5 and 10μg/ ml) respectively. The control almost closed the wound.4.4 Low dose EPI did not show significant cytotoxic effects MTT results showed that the absorption values at 570 nm of experimental groups (1, 2.5, 5 and 10μg/ ml) were 0.92±0.26, 0.91±0.35, 0.97±0.06, 0.89±0.21 respectively. There were no significant difference between experimental groups and control group (0.97±0.40).Conclusions1 Ezrin may play an important role in the carcinogenesis of infiltrating ductal carcinoma of breast.2 Strong expression of Ezrin could be used as an indicator of poor prognosis for the patients with infiltrating ductal carcinoma of breast.3 The strong Ezrin expression in infiltrating ductal carcinoma was positively correlated with the expression of CD44v6, while negatively correlated with the expression of E-Cadherin, and this may be its functional mechanisms.4 Ezrin was related with the invasion of breast cancer cells in vitro. The inhibition of Ezrin could suppress proliferation and invasion of breast cancer cells. 5 RhoA may regulatd the expression of Ezrin as a upstream regulatory signal.6 Low dose EPI inhibited the migration ability of breast cancer cells in concentration-dependent manner, and dose not express cytotoxic effects.7 Low dose EPI decreased the expression of Ezrin and CD44 in concentration-dependent manner in both protein and gene levels, and increased the expression of E-Cadherin in the same way.
|
PDF Full Text Request