关键词: 萝卜硫素, 乳腺癌, 4T1细胞, 上皮-间质转化, 细胞迁移

Abstract: Background and purpose: Expression level of histone deacetylase 5 (HDAC5) is abnormally high in breast cancer, and HDAC5 synergically promotes proliferation and migration of breast cancer cells by cross-talking with LSD1. In this study, the effects of sulforaphane (SFN) on invasion, migration and epithelial-mesenchymal transition (EMT) of mouse breast cancer 4T1 cells were evaluated, and the role of HDAC5 in the treatment with SFN was observed. Methods: The effects of SFN on proliferation of 4T1 cells was analyzed by cell counting kit-8 (CCK-8) method. The cytotoxicity of SFN was detected by lactate dehydrogenase release assay. 4T1 cells were transfected with pcDNA3.1(+)-flag-HDAC5 plasmid, and the single cell clone was obtained by G418 screening. Then the monoclonal cell lines with stable high expression of HDAC5 were detected and identified using Western blot. The effects of HDAC5 overexpression and SFN treatment on the migration and invasion of 4T1 cells were analyzed with scratch test and transwell method. The effects of SFN on expressions of EMT markers E-cadherin, N-cadherin, vimentin and matrix metalloproteinase-9 (MMP-9) in 4T1 cells were tested using Western blot. The effect of SFN treatment on proliferation and metastasis in xenograft tumor model of 4T1 cells in vivo and the relationship between SFN and HDAC5 expression were observed in BALB/c mice. The Ki-67 proliferative index in xenograft tumor and lung metastatic tumor tissues was detected using immunohistochemical analysis. The heart, liver and kidney tissues of tumor-bearing mice were taken and detected using H-E staining, therefore to evaluate the toxic effects of SFN on tumor-bearing mice. Results: SFN inhibited the proliferation (P<0.01), migration and invasion of 4T1 cells in vitro, and HDAC5 overexpression partially resisted the migration and invasion of 4T1 cells. Data showed that SFN significantly downregulated the expressions of HDAC5, MMP-9, N-cadherin and vimentin in 4T1 cells, while SFN upregulated the expression of E-cadherin. SFN significantly inhibited proliferation and lung metastasis of 4T1 cells in mice, and downregulated HDAC5 expression level and Ki-67 positive rate in tumor tissues. Importantly, HDAC5 overexpression could partially resist the inhibitory effect of SFN on proliferation and lung metastasis of 4T1 cells in mice. SFN had no significant toxicity in tumor-bearing mice. Conclusion: SFN could inhibit the proliferation, invasion and migration of mouse breast cancer 4T1 cells both in vitro and in vivo, and the molecular mechanism is closely related to SFN-induced HDAC5 downregulation and EMT inhibition.

Key words: Sulforaphane, Breast cancer, 4T1 cells, Epithelial-mesenchymal transition, Cell migration