2016;7:79417C79427. results were obtained. Since activation of mTOR is important for cancer cell proliferation , the activity of CZ415 on OS cell proliferation was tested next. BrdU incorporation ELISA assay was performed. Results in Figure ?Figure1D1D showed that treatment of CZ415 (at 25-1000 nM) in U2OS cells significantly decreased BrdU ELISA OD, suggesting its anti-proliferative activity. Similarly, in the primary human OS cells (primary OS1 and primary OS2), CZ415 (25/100 nM) largely inhibited BrdU incorporation (Figure ?(Figure1E).1E). Notably, for the BrdU assay, OS cells were treated with CZ415 for only 24 hours, when no significant survival reduction/cell death was noticed. Collectively, these results suggest that CZ415 is anti-survival and anti-proliferative to human OS cells. CZ415 provokes apoptosis in OS cells Next, we tested the potential activity of CZ415 on OS cell apoptosis. Caspase-9 activity assay results in Figure ?Figure2A2A demonstrated that CZ415 concentration-dependently activated caspase-9 in U2OS cells. Meanwhile, Histone DNA apoptosis ELISA OD was increased following CZ415 (at 25-1000 nM) treatment in U2OS cells (Figure ?(Figure2B).2B). Further, the percentage of U2OS cells with TUNEL Deoxycholic acid positive Deoxycholic acid nuclei was also significantly elevated with CZ415 (at 25-1000 nM) treatment (Figure ?(Figure2C).2C). These results confirm that CZ415 induced apoptosis in U2OS cells (Figure 2A-2C). On the other hand, same CZ415 treatment failed to induce significant apoptosis in primary osteoblasts (Figure ?(Figure2C),2C), confirming selective activity of CZ415 against cancerous cells. The pro-apoptosis activity of CZ415 was also observed when added to primary OS cells (primary OS1 and primary OS2), where CZ415 (at 25-1000 nM) treatment significantly increased Histone DNA apoptosis ELISA OD (Figure ?(Figure2D).2D). Collectively, these results confirm that CZ415 provokes apoptosis in OS cells. Open in a separate window Figure 2 CZ415 provokes apoptosis in OS cellsU2OS cells (A-C), primary murine osteoblasts (Osteoblasts, C), or the primary human OS cells (primary Deoxycholic acid OS1 and primary OS2) (D) were treated with designated concentration of CZ415, cells were further cultured for indicated time. Cell apoptosis was tested by listed assays. For each assay, n=5. *group C. Experiments in this figure were repeated five times, with similar results were obtained. CZ415 disrupts OS cell cycle progression, causing G1-S arrest Activation of mTOR is vital for cancer cell cycle progression . Several cell cycle proteins, including Cyclin D1 and Cyclin E, were mTOR-dependent . Thus, the potential activity of CZ415 on cell cycle progression was tested. Quantified results in Figure ?Figure3A3A showed that treatment with CZ415 Deoxycholic acid (100 nM for 24 hours) in U2OS cells led to increase of G1 phase, but significant reduction of S and G2M phases. These results imply that CZ415 possibly induced G1-S arrest in U2OS cells (Figure ?(Figure3A).3A). Similarly in the primary OS cells, G1 phase increase and S/G2M phase decrease were observed after CZ415 (100 nM for 24 hours) treatment (Figure ?(Figure3B).3B). Therefore, CZ415 disrupts OS cell cycle progression, causing G1-S arrest to favor proliferation inhibition. Open in a separate window Figure 3 Rabbit Polyclonal to PRKAG1/2/3 CZ415 disrupts OS cell cycle progression, causing G1-S arrestU2OS cells (A) or the primary human OS cells (primary OS1) (B) were treated with CZ415 (100 nM) for 24 hours, cell cycle was analyzed by PI-FACS assay, and results were quantified. For each assay, n=3. *group Deoxycholic acid C. Experiments in this figure were repeated three times, with similar results were obtained. CZ415 blocks mTORC1 and mTORC2 activation in OS cells Since CZ415 is a newly-developed mTOR kinase inhibitor [17, 18], it presumably should block mTORC1 and mTORC2 activation. Indeed, in the U2OS cells, treatment of CZ415 (100 nM, 3 hours) blocked p-S6K1 (Thr-389, the indicator of mTORC1 activation) and p-AKT (Ser-473, the indicator of mTORC2 activation)  (Four sets of blot data were quantified in Figure ?Figure4A).4A). ERK-MAPK activation, tested by p-ERK1/2, was not affected by the same CZ415 treatment (Figure ?(Figure4A).4A). Similar results were also achieved in the primary human OS cells (Primary OS1), where CZ415 (100 nM, 3 hours) almost blocked activation of mTORC1 (p-S6K1) and mTORC2 (p-AKT, Ser-473), but not ERK (Four sets of blot data were quantified in Figure ?Figure4B).4B). On the other hand, in the primary osteoblasts, basal activation and expression of AKT-S6K1 were much lower than those in the OS cells (Four sets of blot data were quantified in Figure ?Figure4C),4C), which could be the primary reason of ineffectiveness of CZ415 in these non-cancerous cells (Figures ?(Figures11 and ?and2).2). Collectively, these results demonstrate that CZ415 blocks mTORC1 and mTORC2 activation in OS cells. Open in a separate window.