The levels of cleaved caspase 3 and caspase 9 showed mild increases up to 24 h, suggesting that the apoptosome pathway was activated by this VPA treatment. Conversely, the levels of bcl-2 and survivin gradually decreased. VPA reduced bcl-2 level by 30% and survivin level by 70%, suggesting that the antiapoptotic activity

was suppressed by this HDAC inhibitor. Figure 6 Time courses of changes in apoptosis-related proteins. Cleaved caspase 3, caspase 9, survivin, bcl-2 and p53 were examined by western blotting with a series of primary antibodies. Lysates were obtained from OCUM-2MD3 cells with exposure to 1 mM VPA up within 48 h incubation. YH25448 Acetylation of tubulin after exposure to VPA Figure 7 shows the status of tubulin acetylation determined by western blotting. Increased acetyl-α-tubulin was detected

by 6 h and the maximal induction was evident by 12 h. Such rapid tubulin acetylation occurred in parallel with increases in acetyl-histone PX-478 mouse H3 and p21WAF1. Figure 7 Acetylation status of α-tubulin selleck kinase inhibitor assessed by western blotting. Acetyl-α-tubulin level was increased after exposure to 1 mM VPA. 50 kDa: monomer; 100 kDa: dimer. Effects of VPA on xenograft model in vivo The time courses of changes in xenografted tumor volume are shown in Figure 8. The mean tumor volume of the VPA-treated group (246.3 ± 56.0 mm3) was significantly reduced by 36.4%, compared with that of the control group (387.5 ± 99.6 mm3) at 4 weeks after treatment (P < 0.01). As shown in Figure

9, immunohistochemical examination of the xenografted tumor revealed upregulation of p21WAF1 in the VPA-treated group. Moreover, degenerated cells with VPA treatment showed reactivity for cleaved caspase 3, indicating caspase 3 activation. TUNEL assay showed that the apoptotic index was significantly higher in the VPA-treated group (42.3% ± 3.5%) than in the control group (7.7% ± 2.5%) as shown in Figure 10 (P < 0.001). Figure 8 In vivo effects of VPA on the growth of tumor xenografts. The results are means ± SD of three different experiments. Figure 9 Effects of VPA on the expression of p21WAF1 and cleaved caspase 3 in xenograft model. Oxymatrine Immunohistochemical examination showed that p21WAF1-positive cells (nuclear staining) were increased compared with the control group. Cleaved-caspase 3-positive cells were observed as apoptotic cells characterized by cell shrinkage and nuclear fragmentation in the VPA-treated group. Original magnification ×400. Figure 10 Effects of VPA on apoptosis in the xenograft model. Shrunken tumor cells showed positive reactivity in TUNEL assay. Apoptotic index of the VPA-treated group was significantly higher than that of the control group. Original magnification ×400. Discussion The results of the present study showed that VPA alone has an antiproliferative effect on a scirrhous gastric cancer cell line (OCUM-2MD3) in vitro and in vivo.