Transforming growth factor-beta (TGF-β) is a multifunctional cytokine that regulates many biological events including cell motility and angiogenesis. Here, we investigated the role of elevated TGF-β2 level in triple negative breast cancer (TNBC) cells and the inhibitory effect of silibinin on TGF-β2 action in TNBC cells. Breast cancer patients with high TGF-β2 expression have a poor prognosis. The levels of TGF-β2 expression increased significantly in TNBC cells compared with those in non-TNBC cells. In addition, cell motility-related genes such as fibronectin (FN) and matrix metalloproteinase-2 (MMP-2) expression also increased in TNBC cells. Basal FN, MMP-2, and MMP-9 expression levels decreased in response to LY2109761, a dual TGF-β receptor I/II inhibitor, in TNBC cells. TNBC cell migration also decreased in response to LY2109761. Furthermore, we observed that TGF-β2 augmented the FN, MMP-2, and MMP-9 expression levels in a time- and dose-dependent manner. In contrast, TGF-β2-induced FN, MMP-2, and MMP-9 expression levels decreased significantly in response to LY2109761. Interestingly, we found that silibinin decreased TGF-β2 mRNA expression level but not that of TGF-β1 in TNBC cells. Cell migration as well as basal FN and MMP-2 expression levels decreased in response to silibinin. Furthermore, silibinin significantly decreased TGF-β2-induced FN, MMP-2, and MMP-9 expression levels and suppressed the lung metastasis of TNBC cells. Taken together, these results suggest that silibinin suppresses metastatic potential of TNBC cells by inhibiting TGF-β2 expression in TNBC cells. Thus, silibinin may be a promising therapeutic drug to treat TNBC.
Silibinin TGF-β2 Fibronectin MMP-2 MMP-9 Triple negative breast cancer
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Compliance with ethical standards
Human and animal rights and informed consent
The mice were kept in pathogen-free animal housing in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and were used according to protocols approved by the appropriate Institutional Review Board of the Samsung Medical Center (Seoul, Korea).
Conflicts of interest
This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (HI14C3418), and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1D1A1A01057585).
Supplement 1TGF-β2, FN, MMP-2, and MMP-9 expression levels and cell migration are suppressed by silibinin in TNBC cells. (A) After serum starvation for 24 h, TNBC cells were treated with 50 μM Sil for 24 h. TGF-β2 protein expression level was analyzed by confocal microscopy. (B) After serum starvation for 24 h, MDA-MB231 TNBC cells were treated with or without 25 and 50 μM Sil for 24 h. FN protein expression levels were analyzed by western blotting. MMP-2 and MMP-9 protein expression levels were analyzed by zymography. (C) The migrating ability of TNBC cells was analyzed using the wound healing assay. These results are representative of three independent experiments. Con, control; Sil, silibinin. (TIF 1565 kb)
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