Doxorubicin and TGFb Inhibitor Figure 2. Enhanced phosphorylation of Smad2 and Smad3 in murine and human breast cancer cells by doxorubicin and TGFb treatment. The murine 4T1 and human MDA-MB-231 breast cancer cells were treated with doxorubicin (25 nM) or TGFb1(2 ng/ml) in the presence or absence of TGFb antagonist TbRI-KI (500 nM) for 7 days. The medium was changed every alternate day. The cell extracts were used for Western blotting analysis to measure the levels of phosphorylated Smad2 (P-Smad2) and phosphorylated Smad3 (P-Smad3) with their respective antibodies. doi:10.1371/journal.pone.0010365.g002 poor clinical outcome in breast cancer patients [33]. TGFb is a potent inducer of EMT. It induces EMT not only during embryonic development but also during tumor progression in vivo [34]. An essential role for TGFb mediated Smad signaling has been demonstrated in EMT associated with tumor metastasis [35]. Therefore, we investigated whether the TGFb- or doxorubicin- induced EMT phenotype can be reversed by the treatment with the TbRI-KI. Due to the spindle shaped morphology of the human breast cancer MBA-MB-231 cells, the detection of additional EMT was difficult. F-actin staining of the cytoskeleton could detect a small number of MDA-MB-231 cells with an enhanced mesenchymal phenotype which was abrogated by the addition of TbRI-KI (Figure S3A and S3B). To study the effect of TbRI-KI on the EMT generated by doxorubicin, therefore we have focused on murine 4T1 cells with an epithelial morphology. The treatment of murine 4T1 cells with TGFb1 (2 ng/ml) and doxorubicin (25 nM) for 7 days generated majority of the cells with mesenchymal spindle-shaped morphology (Figure 3A). Vimentin, which is a marker associated with mesenchymal morphology, showed increased expression after TGFb1 treatment and was significantly reduced by the addition of TbRI-KI (Figure 3B). However, the level of vimentin expression was not increased after doxorubicin treatment (data not shown) suggesting that doxorubicin may activate other pathways in addition to the TGFb pathway to regulate vimentin expression. EMT was evident after treatment with TGFb1 or doxorubicin as detected by polarized organization of the actin cytoskeleton (Figure 3C). We also observed an increased nuclear translocation of the transcrip- tion factor Snail, which is known to promote EMT [36] after the treatment of the 4T1 cells with TGFb1 or doxorubicin (Figure 3C). The cells regained their epithelial phenotype when TbRI-KI was included in the treatment, suggesting the inhibition of EMT as indicated by the re-expression of F-actin along cell membrane and a more even distribution of Snail between cytoplasm and nucleus (Figure 3C). Our results indicated that TbRI-KI has the potential to reverse the EMT process by counteracting the action of TGFb and doxorubicin in inducing EMT in murine breast cancer cells in vitro. Doxorubicin or TGFb treatment generated stem cell phenotype with drug resistance Recently, EMT has been implicated in the generation of stem like cells in normal and transformed human mammary epithelial cells [26,27]. The authors in both studies showed that prolonged treatment of cells with TGFb1 enhanced expression of markers associated with EMT and many properties of stem cells which include expression of cell surface stem cell markers and ability to form mammospheres. We examined whether murine breast cancer 4T1 cells, which showed EMT upon treatment with TGFb1 or doxorubicin, could exhibit stem cell characteristics in vitro. After 7 days of treatment with either doxorubicin or TGFb1, the cells were analyzed by flow cytometry for the murine stem/ progenitor cell marker Sca-1 and the multidrug resistance protein MDR1. A Sca-1 positive population in murine mammary gland has been shown to be enriched for functional stem/progenitor cells as demonstrated by its increased regenerative potential in comparison to the Sca-1 negative cells when transplanted into the cleared mammary fat pads of host mice [37]. Multi drug resistance protein has been implicated in cancer stem cell resistance to chemotherapy [38]. In comparison to the non- treated cells, the number of cells expressing Sca-1 and MDR1 were enhanced by about 2-fold and 5-fold respectively after TGFb1 treatment and by about 1.7-fold and 2-fold respectively after doxorubicin treatment (Figure 4A). The addition of TbR1- KI during doxorubicin treatment reduced the number of MDR1 expressing cells to the basal level (Figure 4B). Doxorubicin treatment showed enhanced MDR1 expression in a dose dependent manner as detected by Western blotting (Figure 4C). Since TGFb1 treatment increased MDR1 expression in 4T1 cells which was detected by both flow cytometry (Figure 4A) and western blotting (Figure 4C), we also tested whether TGFb1 treatment can induce doxorubicin resistance with a cell prolifer- ation assay. We found that treatment with TGFb1 attenuated growth inhibitory activity of doxorubicin (Figure 4D), which indicates that the stem cell phenotype induced by TGFb appears to enable breast cancer cells to acquire resistance to anti-cancer agents like doxorubicin. Thus, our results indicated that doxorubicin or TGFb1 treatment has the potential to generate drug resistant cancer cells with stem/progenitor cell features. TbRI-KI reduced doxorubicin-induced mammosphere formation One defining characteristic of the stemness of a cell is its ability to self-renew and to generate differentiated progeny. Recently, a non-adherent and non-differentiating culture system has been developed [39] in which a small population of stem like mammary epithelial cells, capable of surviving anoikis and proliferating in such conditions, formed discrete clusters of cells termed ‘mammo- PLoS ONE | www.plosone.org 3 April 2010 | Volume 5 | Issue 4 | e10365