The cells were incubated for 16 hours. BIIB057 ic50 Afterwards, the culture medium was exchanged with 1.8mL D-MEM containing 10% FBS and 0.2mL PBS, CV containing

encapsulated FITC, EV containing encapsulated FITC, or EPV containing encapsulated FITC. The cells were then kept for 15min in a CO2 incubator at 37°C. After incubation, the OST cells were washed with cold PBS twice, followed by flow cytometric analysis. 3. Results 3.1. Effect of ESA on the Viabilities of OST Cells and LM8 Cells The viabilities of OST cells and LM8 cells were measured in the concentration range from 10μg/mL to 50μg/mL to evaluate the possible anticancer activity of ESA. As shown in Figure 1(a), the Inhibitors,research,lifescience,medical proliferations of both osteosarcoma cell types were inhibited by ESA. The inhibitory effect against the cell viability increased with increasing amounts of added ESA. Addition of 50μg/mL ESA, for example, decreased the cell viabilities of OST cells and LM8 cells to 54.7 ± 11.4% and 41.7 ± 12.3%, respectively. Furthermore, Figure 1(b) shows that the cell viabilities decreased with increasing elapsing time. The Inhibitors,research,lifescience,medical cell proliferation was inhibited completely by the addition of 50μg/mL ESA after incubation for 48 hours. These experiments clearly demonstrate the anticancer activity of ESA in the case of these osteosarcoma cells. Figure 1 Cytotoxic effect of ESA on either OST cells or LM8 cells, as evaluated by means of

propidium iodide staining. (a) Variation Inhibitors,research,lifescience,medical of the cell viability with increasing ESA concentration during incubation for 24 hours. (b) Time courses of the cell viabilities … 3.2. Apoptosis Induction by ESA in Both OST Cells and LM8 Cells as Determined by Means of a Double Staining Test Previously, we have already demonstrated that ESA induces apoptosis in carcinoma cells [4]. The findings presented above about the inhibition of sarcoma Inhibitors,research,lifescience,medical cell proliferation (see Section 3.1.) suggested that ESA may also induce apoptosis in sarcoma cells.

Therefore, apoptosis induction in either OST Inhibitors,research,lifescience,medical cells or LM8 cells by ESA was examined by means of the double staining test for Annexin V-PE and 7-ADD. The numerical values obtained from this analysis are displayed Canertinib nmr in Figure 2 and summarized in Table 1. As shown in Figure 2(a) and Table 1, the relative amount of cells in the lower right part of the diagram (indicating early stages of apoptosis) was 74.8% at an elapsing time of 3 hours after adding ESA, while in the case of the control cells (PBS-treated only, no ESA), the amount of the cells was 14.2% in the same part. Moreover, the amount of cells in the upper right part of the diagram (indicating dead cells) increased from 22.5% (at 3 hours after ESA addition) to 71.0% (at 24 hours). These results clearly show that ESA induced apoptosis in OST cells. Figure 2 Apoptotic induction in either (a) OST cells or in (b) LM8 cells after adding ESA. The cells were cultured in 10% FBS D-MEM with 50μg/mL ESA (bottom panel). As control, only PBS (no ESA) was added (top panel). The cells were incubated …