Ruyong Formula (RYF) Attenuates the Abnormal Phenotypic Changes of Thymic Epithelial Cells in 4T1 Breast Cancer Mice

Abstract BackgroundEpithelialization of the breast epithelial cell is the critical step in breast cancer, but the phenotypic changes of thymus epithelial cells (TEC) and the following immune abnormalities during the development of breast cancer are rarely examined. Ruyong Formula (RYF) has been used for thousands of years, our previous researches have shown it could attenuates atrophied thymic epithelial tissue in breast cancer mice, but the mechanism is still unknown.MethodsHPLC was used to analyze the chemical components of RYF. The 4T1 breast cancer mice model was established to study the anticancer effects of RYF. The efficacy of RYF on tumor volume, anti-tumor rate and organ index were observed. The thymus tissue were stained with Hematoxylin-Eosin (H&E) to observe the morphological changes. Cell phenotype marker, such as E-cadherin, α-tubulin and Vimentin were observed by immunofluorescence staining in TGF-β1-induced iTECs after RYF treatment. The mRNA levels of phenotypic markers and phenotype-related transcription factors, including E-cadherin, Vimentin, Zeb-1, Snail 1 and Smad 2 were detected by qPCR. The effect of RYF on the activation of Smad pathway in TGF-β1 induced iTECs was detected by luciferase reporter assay.ResultsRYF could reduce the metastatic rate and the number of pulmonary metastases in breast cancer mice and increased anti-tumor rates. Compared with the thymus in normal group, RYF increased the number of thymocytes in the cortex regions. In vitro study indicated the EMT promotion effect of TGF-β1, shown as the decreasing of E-cadherin and up-regulation of the Vimentin’s expression. The level of Snail 1 and Zeb 1 increased significantly, and the mRNA levels of Smad 2 was up-regulated. Compared with TGF-β1 group, RYF-treated TECs reversed the proteins expression of E-cadherin and Vimentin and the mRNA levels of Snail 1 and Zeb 1. RYF also promoted the proliferation of iTECs, and confronted the TGF-β1 induced phenotypic transition in iTECs.ConclusionThe abnormal thymic function of breast cancer mice was mainly due to the disorder of cortex and medulla regions cells and the atrophy of cortex. Interestingly, RYF could reverse the phenotypic changes of TECs in breast cancer by inhibiting the TGF-β1/Smad pathway.