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Aim To study the effect of estrogen on the migration of breast cancer cells and the possible underlying mechanisms.Methods Human breast cancer cell lines MCF-7(estrogen receptor, ER+) and MDA-MB-468(ER-) were employed as a model system.Cells were treated with E2 and pretreated with CANP inhibitor(calpeptin)where needed.Wound-healing assay was applied to evaluate cell migration, Western blot assay was performed to observe protein level, and fibronectin expression was silenced by specific siRNA transfection.Results ① Treatmentof MCF-7 and MDA-MB-468 cellswith E2(50 nmol·L-1) increased cell migration by(51.55±5.50) ％(P<0.01)and (40.78±4.78)％(P<0.05), respectively;② E2 significantly up-regulated the expression of FN protein in MCF-7 and MDA-MB-468 breast cancer cells, which was 2.11 times(P<0.05)and 1.86 times(P<0.01), respectively;③ Pretreatment with calpeptin(10 μmol·L-1) decreased E2-induced cell migration by (49.55±6.44)％(P<0.05) in MCF-7 and(36.85±4.40)％ (P<0.01)in MDA-MB-468 cells;④ Calpeptin pretreatment inhibited E2-induced fibronectin up-regulation by(80.12±4.55)％ and(78.84±5.70) ％(P<0.01), respectively;⑤ Knockdown of FN with siRNA suppressed cell E2-induced migration by(40.65±5.80)％(P<0.01)in MCF-7 and(40.88±6.02)％(P<0.05)in MDA-MB-468 cells.Conclusion E2 stimulates the migration of breast cancer cells with or without ER expression and a calpain-FN signaling pathway may be involved in the E2 action.
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Background and purpose:Ethanol has been reported to stimulate progression of breast cancer, yet the underlying mechanism is not fully understood. This study aimed to investigate effects of ethyl alcohol (EtOH) on the calcium-activated neutral protease (CANP)-cyclin E/focal adhesion kinase (FAK) signaling and cell migration in breast cancer cells, as well as the role of epidermal growth factor receptor (EGFR) in the EtOH-stimulated effects, in order to assess the signaling mechanism(s) underlying how EtOH enhances cancer progression.Methods:Human breast cancer cell line MCF-7 was employed as a model system, with MCF-10A mammary epithelial cells as control. In vitro wound healing assay was carried out to evaluate EtOH-induced cell migration. The effects of EtOH or epidermal growth factor on the proteolysis of cyclin E/FAK were detected by Western blot. EGFR inhibitor (EGFR-I) and a speciifc inhibitor for CANP, Calpeptin, were applied to pretreat cultured cells to explore their inlfuences on the cell migration and cyclin E/FAK proteolysis triggered by EtOH.Results:Treatment of model cells with EtOH (0.3%) stimulated significant proteolysis of cyclin E/FAK in a dose-/time-dependent manner and increased migration (+47.30%,P<0.05) in MCF-7 breast cancer cells, but had no signiifcant effect on migration in MCF-10A cells. Pretreatment with Calpeptin (10 μmol/L) signiifcantly reduced EtOH (0.3%)- or EGFR (10 ng/mL)-induced cyclin E/FAK truncation. EGFR-I (3 μmol/L) pro-foundly reduced EtOH-indcued CANP dependent proteolysis of CANP1 and cyclin E/FAK as well as cell migration (-53.00%,P<0.01).Conclusion:EtOH signiifcantly stimulates activation of CANP via EGFR pathway, resulting in proteolysis of cyclin E/FAK and migration in MCF-7 breast cancer cells, suggesting EGFR-CANP signaling to be a potential target for suppression of metastasis in breast cancer.