Aciculatin Induces Apoptosis via p53 Pathway Figure 3. Accumulation of p53 is observed following aciculatin treatment in HCT116 and A549 cells via a proteasome degradation pathway. A, HCT116 and A549 cells were treated with aciculatin (10 mM) for the indicated periods and then harvested for detection of p53-related proteins. The levels of p53, phospho-ser15-p53, c-H2AX, and the p53 downstream target MDM2 were then determined in HCT116 cells. The levels of p53, MDM2, c-H2AX, cleaved caspase-9 and PARP were determined in A549 cells. The star marks a non-specific band. B, Nuclear extraction of HCT116 cells was performed after aciculatin treatment at the indicated time points. Aciculatin-induced nuclear accumulation of p53 was shown to be time- dependent. C, HCT116 cells were treated with aciculatin (10 mM) at different time points followed by extraction of total RNA. The p53 mRNA was co- amplified with GAPDH. D, HCT116 cells were pretreated with aciculatin (10 mM) for 3 h, followed by treatment with cycloheximide (20 mg/ml) with or without aciculatin (10 mM) for the indicated periods. E, HCT116 and A549 cells were co-treated with 10 mM MG132 and 10 mM aciculatin for 6 h and then harvested for p53 detection by immunoblotting. doi:10.1371/journal.pone.0042192.g003 Aciculatin induces cell cycle arrest and apoptosis through up-regulation of p21WAF1/CIP1 and induction of caspase activity We next examined the effect of aciculatin on G0/G1 arrest cell cycle regulatory proteins. The cyclin-dependent kinase inhibitors p21 and p27 are crucial regulators of G0/G1 arrest. As shown in Figure 2A, p21 was significantly upregulated and retinoblastoma protein (Rb) was dephosphorylated without any apparent change in p27 after 4 h of aciculatin treatment. Both intrinsic (caspase-9) and extrinsic (caspase-8) apoptotic pathways were detected after 8 h of treatment, and the downstream apoptotic effector caspase-3 was also activated (Figure 2B). Concentration-dependent increase in apoptosis was also identified 24 h after aciculatin treatment (Figure 2C). These results demonstrate that aciculatin treatment can trigger HCT116 cell cycle arrest at the G0/G1 phase and subsequently induce apoptosis. Aciculatin increased the expression of the p53 protein and its downstream effectors via a proteasome-mediated degradation pathway The phenomena our prior data revealed are believed to be associated with the tumor suppressor protein p53. Thus, the expression of p53 was examined at a series of time points. As shown in Figure 3A, aciculatin significantly induced p53 expression in a time-dependent manner. The p53 downstream product MDM2, which is also a major regulator of ubiquitin- mediated p53 degradation, was found to be upregulated following the accumulation of p53. Interestingly, there was a decrease in MDM2 during the first few hours of aciculatin treatment. We then confirmed the correlation between aciculatin and DNA damage. Our data indicate that aciculatin only slightly increased phospho- ser15-p53, which is the downstream effect of DNA damage. The DNA double-strand break marker phosphorylated histone 2AX (c- PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e42192