2B, D), all of which were characteristics of cells undergoing apoptosis. On the contrary, control cells were morphologically normal and exhibited no learn more signals of apoptosis (Fig. 2A, C). Figure 2 Transmission electron microscopy observation. After ChA21 (5.4 μg/ml) treatment for 72 h or the tumor tissues removed from nude mice treated ChA21 (40 mg/kg) for 5 weeks, a large number of cells presented a series of ultrastructural changes of apoptosis (B, D). On the contrary, control cells were morphologically normal and exhibited no signals of apoptosis (A, C). (magnification: A, C × 3000; IAP inhibitor B, D × 8000).

Cells cultured on coverslips and tissue sections from the above experiments were stained with the TUNEL agent, and examined by microscopy. Less apoptotic cells were detected in the control group, whereas more apoptotic cells were detected in ChA21 treatment group (Fig. 3). The apoptotic cells on coverslips and tissue sections were counted to calculate the apoptotic index. In vitro, the AI value in ChA21 (5.4 μg/ml) treatment group reached 16.22 ± 1.05, which was higher than that in the controls (6.22 ± 1.09, P < 0.05). In vivo, the AI value in ChA21 (40 mg/kg) treatment group reached 9.16 ± 2.44, which Nutlin3a was also higher

than that in the controls (3.45 ± 0.98, P < 0.05). Figure 3 ChA21 induces apoptosis of SK-OV-3 cells in vitro and in vivo by TUNEL staining. (A): Control group in vitro (B): ChA21 (5.4 μg/ml) group in vitro (C): Control group in vivo (D): ChA21 (40 mg/kg) group in vivo. Cells cultured with coverslips and tissue sections were stained with the Thiamet G TUNEL agent and examined by light microscopy. Less apoptotic cells were detected in control group, whereas more apoptotic cells were detected in ChA21 treatment group. (magnification: × 200) SK-OV-3 cells were incubated with ChA21 (0.2 or 5.4 μg/ml) for 72 h, and flow cytometric analysis was used to measure the death rate. As shown in Fig. 4, there was a significant difference between ChA21 group

and control group in the death rate (%) (P < 0.05). After the treatment of SK-OV-3 cells with ChA21 (0.2 or 5.4 μg/ml) for 72 h, the death rate (%) reached 8.75 ± 0.97, and 19.73 ± 1.99, respectively. Figure 4 ChA21 induces death of SK-OV-3 cells in vitro with PI staining. SK-OV-3 cells were incubated with ChA21 (0.2 or 5.4 μg/ml) for 72 h, and flow cytometric analysis was used to measure the death rate. Significant differences in death rates are represented by asterisk (P < 0.05) and double asterisk (P < 0.01). Expression of Bcl-2 and Bax Detection of the expression of apoptosis-related proteins of Bcl-2 and Bax by immunohistochemistry showed that ChA21 therapy could up-regulate the expression of Bax, and down-regulate the expression of Bcl-2 (Fig. 5), thereby reducing the ratio of Bcl-2/Bax in vitro and in vivo. As shown in Fig. 6, MOD values of Bax in ChA21 group were higher than those in control group (P < 0.