Figure 4. Aciculatin treatment attenuates MDM2 mRNA, contributing to p53 accumulation. A, HCT116 and A549 cells were co-treated with 10 mM MG132 and 10 mM aciculatin for 6 h (HCT116) and 3 h (A549) then harvested for detection of MDM2 by immuno- blotting. B, HCT116 and p53-KO HCT116 cells were treated with aciculatin (10 mM) at different time points followed by extraction of total RNA. The MDM2 mRNA was co-amplified with GAPDH. C, The MDM2 plasmid was introduced into the HCT116 cells to induce over- expression of the MDM2 protein; the cells were then treated with aciculatin for 3 h. Protein levels of p53 and MDM2 were detected by western blotting. doi:10.1371/journal.pone.0042192.g004 H2AX) appeared until the later stages of aciculatin treatment. In addition, to check DNA single and double strand breaks, single- cell gel electrophoresis assay (COMET assay) was performed. The results showed that there is no obvious DNA damage after 6 h treatment of 10 mM aciculatin with a novel DNA damage agent QS-ZYX-1-61 (published by our group previously) as the positive control [17]. (Figure S1). These results indicate that DNA damage is not an obvious early effect of aciculatin treatment. We than verified this p53-dependent effect in another p53 wild type cancer cells A549. Aciculatin also triggered p53 accumulation, early MDM2 depletion (3 h) and the subsequent apoptosis pathway including cleaved caspase-9 and cleaved PARP in A549. To investigate the protein level of transcription factor p53 accumu- lated in nucleus by aciculatin, the nuclear fractions were extracted and analyzed. Figure 3B shows that p53 was increased in nucleus in a time-dependent manner after aciculatin treatment. To determine the mechanism of aciculatin-induced p53 accumula- Aciculatin Induces Apoptosis via p53 Pathway tion, we first determined the mRNA level of p53. Figure 3C shows that there was no prominent change in p53 mRNA level. In normal circumstances, p53 protein has a very short half-life (approximately 15–30 min) due to rapid proteasome degradation. Therefore, we determined whether aciculatin increases half-life of p53. When cycloheximide was used to inhibit protein biosynthesis, we observed that aciculatin-induced p53 was stabilized and had a longer half-life (Figure 3D). Furthermore, we introduced the proteasome inhibitor MG132 to block the degradation pathway. Figure 3E shows that the protein level of p53 was not increased by aciculatin treatment after MG132 pre-treatment in HCT116 and A549 cells. These results demonstrated that the proteasome- mediated degradation pathway is important for aciculatin-induced p53 accumulation. Aciculatin decreases MDM2 at a transcriptional level, which is critical for p53 accumulation In our previous study, no obvious DNA damage signals have been found. This may imply that this pathway is not the only mechanism contributing to p53 accumulation. MDM2 mediates p53 ubiquitination, leading to proteasomal degradation of p53. MDM2 also controls its own degradation by auto-ubiquitylation [18]. In this study, MDM2 depletion was found in the early stages of aciculatin treatment (Figure 3A). Thus, we next evaluated the mechanism of MDM2 ablation to determine whether this decrement was involved in aciculatin-induced p53 accumulation. After co-treatment with aciculatin and MG132, a proteasome inhibitor, the MDM2 protein level still declined remarkably in HCT116 and A549 cells (Figure 4A). These results indicate that other proteasome-independent regulatory pathways are active. We then examined the mRNA level and found that MDM2 mRNA started to decrease after 1 h of aciculatin treatment. Same result can be achieved in p53-KO HCT116, suggesting that aciculatin induced MDM2 depletion is not a p53-related effect (Figure 4B). To identify the importance of MDM2 in p53 accumulation, we investigated aciculatin-induced p53 accumulation after over- expressing MDM2 in HCT116 cells. Following aciculatin treat- ment, over-expression of MDM2 reversed the p53 level (Figure 4C). Collectively, these results indicate that aciculatin can induce accumulation of p53 through MDM2 mRNA depletion. Role of p53 in aciculatin-mediated cell cycle arrest and apoptosis Next, we aimed to identify the role of p53 in aciculatin-induced HCT116 cell cycle arrest and apoptosis. First, we compared the cytotoxicity of aciculatin in a HCT116 p53-WT (wild-type) and p53-KO (knockout) system. As shown in Figure 5A, we found a significant difference in the IC50 between these 2 cell lines; the WT cells (IC50 value, 5.88 mM) were more sensitive to aciculatin than the p53-KO cells (IC50 value, 9.13 mM). Furthermore, the proportion of aciculatin-induced G0/G1 arrest in the WT cells was about 3-fold higher than that in the p53-KO cells after 18 h of aciculatin treatment (Figure 5B). We then used western blotting to examine p53 and apoptotic proteins in p53-WT and p53-KO HCT116 cells during aciculatin treatment. Figure 5C shows that the p53-KO cells expressed lower levels of caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). The induction of sub-G1 was also examined after aciculatin treatment and results indicate that p53-KO cells were more resistant to aciculatin-induced sub- G1 increment (lower bar chart). The p53 direct transcriptional targets, p21 and PUMA (p53 upregulated modulator of apoptosis) are related to growth arrest and apoptosis. Both of them were PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e42192