The quantification of NF-B activation was performed using Trans-AM NF-B kit from ActiveMotif Effects of PDTC on tumor growth in nude mice PDTC was chosen for further investigation in this study

The quantification of NF-B activation was performed using Trans-AM NF-B kit from ActiveMotif Effects of PDTC on tumor growth in nude mice PDTC was chosen for further investigation in this study. either PDTC or paclitaxel alone. This study suggests that breast cancer stem-like cells could be selectively inhibited by targeting signaling pathways important for breast cancer stem-like cells. = test. 0.05 was considered statistically significant. Results PTL, PDTC and DETC preferentially inhibit MCF7 sphere cell proliferation It has been shown that NF-B pathway specific inhibitors, including MG-132, PTL and PDTC could selectively inhibit leukemia stem cell proliferation [25C27]. To study the sensitivity of breast cancer stem-like cell to NF-B pathway inhibitors, RGS16 11 compounds targeting different actions of the NF-B pathway, including antioxidants Curcumin [32]; PDTC [33]; DETC [34]; Quercetin [35], NF-B phosphorylation inhibitors Sulfasalazine [36]; Sulindac [37]; Ibuprofen [38]; PTL [39] and NF-B degradation inhibitors MG-132 [40]; Cyclosporin A [41]; Genistein [42], were tested in this study. MCF7 sphere cells were used as a model of breast cancer stem-like cells [16, 43]. Among all the inhibitors, antioxidants which inhibit NF-B activation including PDTC and its analog DETC, and NF-B phosphorylation inhibitors PTL were shown to preferentially inhibit sphere cell proliferation. As shown in Darusentan Darusentan Fig. 1, PTL, PDTC and DETC at 1 M inhibited MCF7 sphere cell growth by 33.2, 50.8 and 52.2%, respectively, but did not show obvious growth inhibition effect on MCF7 bulk cells. In contrast, cancer drug paclitaxel gave better growth inhibition effect on bulk MCF7 cells by 44.5% at 2.5 nM but only inhibited sphere cells by 6.1%. These data indicate that, unlike paclitaxel that act primarily on replicating bulk MCF7 cells, PTL, PDTC and DETC selectively inhibited MCF7 sphere cell proliferation over MCF7 bulk cells. Open in a separate window Fig. 1 Growth inhibition of PTL, PDTC, DETC and paclitaxel on MCF7 sphere cells compared with MCF7 bulk cells PTL, PDTC and DETC could preferentially inhibit MCF7 SP cell proliferation and colony formation Apart from sphere Darusentan cells cultured from MCF7 cells, side population (SP) cells isolated from MCF7 cells were also found to enrich breast cancer stem-like cells, which showed higher colony formation ability in vitro and in vivo tumorigenicity than non-SP fraction [18]. MCF7 cells were shown to contain 1.2% SP cells, which could significantly be blocked by ABC transporter inhibitor verapamil (0.12%) (Fig. 2a). It was of interest to test whether the various NF-B inhibitors that are preferentially active against sphere cells as above have similar effect on MCF7 SP cells. For this purpose, both SP and non-SP cells were sorted out by flow cytometry as indicated by trapezoids around the left (R5) and right (R8), respectively (Fig. 2a), and seeded into 96 well plates at 500 cells/well. After overnight incubation, both SP and non-SP cells were treated with PDTC, DETC and PTL for 3 days. Cell proliferation was decided using a fluorescence-based cell proliferation assay as indicated above. Interestingly, unlike cancer drug paclitaxel (2.5 nM), which inhibited MCF7 SP cells by 39.3%, and non-SP cells by 52.2%, all the three compounds showed preferential inhibition for the MCF7 SP cells over the non-SP cells (Fig. 2b). PTL (5 M) inhibited MCF7 SP cell growth by 95.2% and non-SP cells by 66.1% (Fig. 2b). Both PDTC and DETC also showed higher ability to inhibit both SP and non-SP proliferation. As shown in Fig. 2b, 1 M of PDTC and DETC preferentially inhibited MCF7 SP cells by 86.3 and 94.5%, but inhibited non-SP cells by 74.4 and 71.9%, respectively. Open in a separate window Fig. 2 (a) A representative MCF7 SP profile for sorting. MCF7 cells were stained as.