Aciculatin Induces Apoptosis via p53 Pathway aciculatin (10 mM) for different periods. The DNA proportion was subsequently analyzed by PI staining. The cell cycles of p53-WT and p53-KO HCT116 cells after treatment of 18 h are shown. The G1 proportions of both cell lines were compared. Mean 6 SE values from 3 independent experiments. *P,0.05 and ***P,0.001. C, p53-WT and p53-KO HCT116 cells were incubated with aciculatin (10 mM) for 24 h and 48 h and then harvested for detection of p53, caspase activation, and cleavage of PARP (upper). p53-WT and p53-KO cells were treated with vehicle (0.1% DMSO) or aciculatin (10 mM) for 24 h and were subsequently analyzed by PI staining. The induction of sub-G1 phase of each group was determined (lower bar chart). **P,0.005. D, HCT116 cells were treated with various concentrations of aciculatin (5, 7.5, and 10 mM). After 24 h and 48 h aciculatin treatment, The levels of p53, p21, pRb and PUMA were then determined (upper). p53 siRNA was used to knock down the p53 level of HCT116 and A549 cells, which were then treated with aciculatin for 24 h. The cells were harvested for detection of p53 and apoptotic related proteins (lower). E, p53 knock-down HCT116 and A549 cells were treated with aciculatin (10 mM) for 40 h and then double stained with annexin V-FITC and PI. The percentages of fluorescently labeled cells were determined by flow cytometry. doi:10.1371/journal.pone.0042192.g005 upregulated after 24 h and 48 h of aciculatin treatment in a concentration-dependent manner. (Figure 5D). Figure 5D also shows knockdown of the p53 protein level by siRNA in p53 wild- type cells HCT116 and A549; this resulted in significant inhibition of p53 expression after aciculatin treatment, an effect that lasted for up to 24 h. This p53 depletion led to abrogation of aciculatin- induced p21 and dephosphorylation of Rb; also the apoptotic proteins PUMA, caspase-9, caspase-3 and PARP. After cells were double stained with annexin V-FITC and PI for apoptosis and necrosis, the results revealed that p53 knock-down HCT116 and A549 cells were both more resistant to aciculatin induced apoptosis than wild-type cells (Figure 5E). Based on these data, we suggest that p53 is a pivotal mediator of aciculatin-induced apoptosis. Aciculatin inhibited HCT116 tumor cell growth in the mouse xenograft models To examine the in vivo anti-cancer activity of aciculatin, we established HCT116-derived xenograft models in severe combined immunodeficient (SCID) mice. Both control and treated mice were sacrificed, and the HCT116-xenografted tumor tissues were examined. As shown in Figure 6A, our results demonstrated that intraperitoneal administration of aciculatin (30 mg/kg) once daily significantly inhibited tumor growth from day 8, without any loss of body weight; this suggests that there was no obvious cytotoxicity in vivo. We also stained the HCT116-xenografted tumor tissues with Hematoxylin and Eosin (Figure 6B–a, b), p53 antibody (Figure 6B–c, d) and Ki-67 (Figure 6B–e, f). The results revealed that treatment with aciculatin significantly increased p53 expres- sion and the cell proliferation marker Ki67. These observation correlates with the in vitro studies. The tumor tissue slices were also stained with TUNEL reagent and the results indicates that aciculatin-treated tumor did undergo apoptosis (Figure 6C). Discussion The tumor suppressor protein p53 has been a promising target of anti-cancer therapy for decades. It plays an essential role in cell cycle regulation and apoptosis. Here, we demonstrated that aciculatin is a compelling p53 inducer and also activates the downstream effectors of p53. p21, a well-known transcriptional target of p53, is the major regulator of the G0/G1 cell cycle phase. As shown in Figure 2A and 5D, p21 expression was increased and pRb was hypophosphorylated after aciculatin treatment. p21 has previously been shown to bind to Cdk/cyclin complexes, inducing hypophosphorylated pRb to sequestrate E2 gene promoter region binding factor 1 (E2F-1), thereby inhibiting the ability of E2F-1 to carry out the cell cycle [19]. Some studies have indicated that HCT116 cells are prone to remain in G0/G1 phase rather than undergo apoptosis with p53 induction [20,21]. In this situation, p21 induction is regarded as protection and mediates cell cycle arrest to suppress apoptosis [5]. However, a high percentage of HCT116 cells do undergo apoptosis after G0/G1 arrest, as was shown here with the aciculatin-treated cells. Furthermore, a recent study suggested that p21 induced by nongenotoxic p53 activation, unlike stress-induced p53 activation, does not affect the apoptotic response [22], which could explain the role of p21 induced by nongenotoxic aciculatin. Another p53 downstream target, PUMA, was also upregulated after aciculatin treatment (Figure 5D). PUMA is a potent pro-apoptotic factor and has been reported to mediate p53-dependent apoptosis [20,23]. These results indicate that the aciculatin-induced p53 pathway is growth inhibitory and cytotoxic in HCT116 cells. Aciculatin is a C-glycosidic flavonoid with more indestructible structure than O-glycosidic flavonoids. Several flavonoids such as wogonin, apigenin, luteolin, and quercetin have been documented to induce p53 activity [23,24]. Here, aciculatin induced accumu- lation of p53 by inhibiting proteasome-dependent degradation at lower concentrations compared to other flavonoids (Figure 3C–E). It has been reported that p53 degradation is decreased in response to cellular stresses, especially DNA damage [8]. Recently, flavonoid interactions with DNA and RNA have been studied [25], and aciculatin has been documented to have DNA-binding ability [2]. Therefore, we checked for DNA damage after aciculatin treatment of HCT116 cells. However, no significant DNA damage was observed in the aciculatin-treated HCT116 cells. The histone H2AX is immediately phosphorylated to c- H2AX when there is a DNA double-strand break in chromatin [26]. In this study, c-H2AX did not appear until 24 h after aciculatin treatment, and this delayed effect was more likely a consequence of apoptosis. The COMET assay also proved no significant DNA breaks in early stage of aciculatin treatment. But our western blotting results still revealed faint upregulation of phospho-ser15-p53, which correlates with DNA damage and may contribute to the transcriptional activity of p53. Collectively, these results suggest that aciculatin is a DNA-binding agent, but one that does not induce significant DNA damage. Mechanisms other than genomic damage could be responsible for the p53 upregulation and anti-cancer activity of aciculatin. Therapeutic agents that can activate the p53 pathway without genotoxicity are potentially safer agents to use in cancer therapy [7,27]. This concept has already been considered in the development of drugs targeting the p53-MDM2 interaction. In this study, the ability of aciculatin to ablate MDM2 mRNA was noted (Figure 4B). Although the abrogation of MDM2 mRNA by some natural products, including genistein and gambogic acid, has been reported [28,29], to our knowledge this is the first time to prove that overexpressed MDM2 could partially reverse the flavonoid-induced p53 accumulation in HCT116 cells. Phosphor- ylation of p53 (Figure 3A) could interrupt the p53-MDM2 interaction; this may resulted in the residual p53 level when MDM2 over-expression (Figure 4C). The contradictory observa- tion that p53 stability is not responsive to changes in endogenous MDM2 level has also been reported [30]. However, we demonstrated that the depletion of MDM2 dose plays an PLOS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e42192