Aciculatin Induces p53-Dependent Apoptosis via MDM2 Depletion in Human Cancer Cells In Vitro and In Vivo Chin-Yu Lai1, An-Chi Tsai2, Mei-Chuan Chen1, Li-Hsun Chang 1 , Hui-Lung Sun 1 , Ya-Ling Chang 1 , Chien- Chih Chen3, Che-Ming Teng 1 * . , Shiow-Lin Pan2*. 1 Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, 2 Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan Town, Taiwan, 3 Department of Biotechnology, Hungkuang University, Taichung, Taiwan Abstract Aciculatin, a natural compound extracted from the medicinal herb Chrysopogon aciculatus, shows potent anti-cancer potency. This study is the first to prove that aciculatin induces cell death in human cancer cells and HCT116 mouse xenografts due to G1 arrest and subsequent apoptosis. The primary reason for cell cycle arrest and cell death was p53 accumulation followed by increased p21 level, dephosphorylation of Rb protein, PUMA expression, and induction of apoptotic signals such as cleavage of caspase-9, caspase-3, and PARP. We demonstrated that p53 allele-null (2/2) (p53-KO) HCT116 cells were more resistant to aciculatin than cells with wild-type p53 (+/+). The same result was achieved by knocking down p53 with siRNA in p53 wild-type cells, indicating that p53 plays a crucial role in aciculatin-induced apoptosis. Although DNA damage is the most common event leading to p53 activation, we found only weak evidence of DNA damage after aciculatin treatment. Interestingly, the aciculatin-induced downregulation of MDM2, an important negative regulator of p53, contributed to p53 accumulation. The anti-cancer activity and importance of p53 after aciculatin treatment were also confirmed in the HCT116 xenograft models. Collectively, these results indicate that aciculatin treatment induces cell cycle arrest and apoptosis via inhibition of MDM2 expression, thereby inducing p53 accumulation without significant DNA damage and genome toxicity. Citation: Lai C-Y, Tsai A-C, Chen M-C, Chang L-H, Sun H-L, et al. (2012) Aciculatin Induces p53-Dependent Apoptosis via MDM2 Depletion in Human Cancer Cells In Vitro and In Vivo. PLoS ONE 7(8): e42192. doi:10.1371/journal.pone.0042192 Editor: Marie-Jose´e Boucher, University of Sherbrooke, Canada Received January 26, 2012; Accepted July 4, 2012; Published August 13, 2012 Copyright: 2012 Lai et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Science Council of the Republic of China (NSC 99-2628-B-002-024-MY3 and NSC 99-2320-B-400-008- MY3)(http://web1.nsc.gov.tw/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: cmteng@ntu.edu.tw (CMT); apan0826@nhri.org.tw (SLP) . These authors contributed equally to this work. Introduction Discovery and development of anti-cancer drugs is an endless challenge for biochemists and pharmacologists worldwide. Natural products are abundant sources of novel and cytotoxic structures that have the potential to be promising lead compounds for antineoplastic drugs. Aciculatin is a natural compound isolated from the medicinal herb, Chrysopogon aciculatus, which is widely used for the treatment of swellings, common cold, fever, and diarrhea. Aciculatin was first reported in 1991 showing DNA-binding activity and cytotoxicity in vitro [1,2]. A recent study demonstrated that aciculatin exerts dual inhibitory effects on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) due to regulation of the NF-kappaB and c-Jun N-terminal kinase (JNK)/p38 pathways [3]. However, the exact mechanism by which aciculatin exerts its tumor inhibitory effect remains unclear. Tumor suppressor p53 plays a crucial role in arousing response to cellular stresses such as hypoxia, DNA damage, and oncogene activation. In normal circumstances, p53 is rapidly degraded by the E3-ubiquitin ligase murine double minute 2 (MDM2); the half- life of p53 is thus short and its protein level is difficult to detect [4]. Once cells are exposed to the aforementioned stresses, p53 degradation is turned off. Accumulated p53 then forms tetramers and binds to specific DNA domains, leading to transcriptional regulation of genes. The most common effects are DNA repair, cell cycle arrest, senescence, and apoptosis. The downstream cellular responses of p53 could suppress uncontrolled growth, and if necessary, eliminate damaged cells [5]. This strategy has been used in current cancer therapies to induce p53-dependent apoptosis using, for example, DNA damage agents. However, the side effects of this treatment are severe due to genotoxicity. Moreover, even though nearly 50% of human cancers have wild- type p53, some up-regulated pathways block p53 function such as p14ARF deficiency and MDM2 amplification [6]. Therefore, agents that can induce or restore wild-type p53 function and correct the abnormal pathway with lower side effects are ideal candidates for novel anti-cancer drugs [7,8]. MDM2 oncoprotein is an E3 ubiquitin ligase that controls the p53 level in cells. MDM2 is a negative regulator mediating the ubiquitin degradation of p53; MDM2 also inhibits the transcrip- tional activity of p53 and facilitates its nuclear export [9]. Overexpression of MDM2 which impairs p53 function has been found in many human tumors. Moreover, MDM2 interacts with various tumor suppressor proteins including Rb, p21, p19/14ARF, E2F1, p73 and MTBP [10,11,12,13,14]. In this study, we aimed to identify the anti-tumor mechanism of aciculatin with in vitro and in vivo experiments using HCT116 PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e42192