Dulxanthone A induces cell cycle arrest and apoptosis via up-regulation of p53 through mitochondrial pathway in HepG2 cells Ze Tian 1,2 , Jie Shen 2 , Annie P. Moseman 1 , Quanli Yang 3 , Junshan Yang 2 , Peigen Xiao 2 , Erxi Wu 1 * and Isaac S. Kohane 1 1 Children’s Hospital Informatics Program at Harvard- MIT Division of Health Sciences and Technology, Children’s Hospital Boston, Harvard Medical School, Boston, MA 2 Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China 3 Medical Polymer Center/Institute of Science and Technology, National Family Planning Research Institute, Beijing, China Natural products derived from plants provide a rich source for development of new anticancer drugs. Dulxanthone A was found to be an active cytotoxic component in Garcinia cowa by bioactiv- ity-directed isolation. Studies to elucidate the cytotoxic mecha- nisms of dulxanthone A showed that dulxanthone A consistently induced S phase arrest and apoptosis in the most sensitive cell line HepG2. Furthermore, p53 was dramatically up-regulated, leading to altered expression of downstream proteins upon dulxanthone A treatment. Cell cycle related proteins, such as cyclin A, cyclin B, cyclin E, cdc-2, p21 and p27 were down-regulated. Some apoptosis correlated proteins were also altered following the drug treat- ment. Bcl-2 family members PUMA was up-regulated while Bcl-2 and Bax were down-regulated. However, the expression ratio of Bax/Bcl-2 was increased. This resulted in the release of cyto- chrome C from the mitochondria to the cytosol. Concurrently, Apaf-1 was stimulated with p53 by dulxanthone A. In result, cyto- chrome C, Apaf-1 and procaspase-9 form an apoptosome, which in turn triggered the activation of caspase-9, caspase-3 and down- stream caspase substrates. Lamin A/C and PARP were down- regulated or cleaved, respectively. Moreover, cell cycle arrest and apoptosis in HepG2 cells induced by dulxanthone A were mark- edly inhibited by siRNA knockdown of p53. In summary, dulxan- thone A is an active cytotoxic component of G. cowa. It induces cell cycle arrest at lower concentrations and triggers apoptosis at higher concentrations via up-regulation of p53 through the intrin- sic mitochondrial pathway in HepG2 cells. Dulxanthone A is therefore likely a promising preventive and/or therapeutic agent against Hepatoma. ' 2007 Wiley-Liss, Inc. Key words: Garcinia cowa; dulxanthone A; cytotoxicity; cell cycle; apoptosis; p53; mitochondria Although genetic alterations such as PTEN, BRCA1, BRCA2, RAS, PIK3CA are often found in human cancers, the most com- mon cancer-related genetic change is p53 mutation. 1–3 Inactiva- tion of the tumor suppressor gene p53 has been found in more than half of human malignancies and is frequently encountered in human hepatocellular carcinoma. 4,5 Being a key role in cellular response to various endogenous and exogenous stimulations, p53 serves as the major obstruction for carcinogenesis through the induction of cell cycle arrest and apoptosis. It has been shown that p53-mediated cell cycle arrest is partly achieved through the tran- scriptional activation of a CDK inhibitor p21. However, Myc represses p21 induction through recruitment by Miz-1 to the proxi- mal promoter region of p21. This block in p21 induction shifts the balance away from growth arrest towards apoptosis. 6 p53 is implicated in the two distinct extrinsic and intrinsic apo- ptosis pathways that lead to the activation of caspases, which in turn induce apoptosis. The extrinsic pathway involves the engage- ment of specific death receptors from TNF-R family and the for- mation of this death-inducing-signaling-complex, which promotes a cascade of activation of caspase-8, caspase-3, caspase-6 and cas- pase-7. 7 The intrinsic pathway is associated with an alteration in the ratio of pro-apoptotic to antiapoptotic proteins from the Bcl-2 family. An increase in the ratio triggers the release of mitochon- drial cytochrome C, which forms an apoptosome complex with Apaf-1 and procaspase-9. Caspase-9 is then activated by the apop- tosome and this leads to the activation of caspase-3, which subse- quently mediates apoptosis. 8 In addition, extrinsic and intrinsic ap- optotic pathways are linked by the actions of Bid. Following TNF- R1/Fas receptor activation, cytosolic p22 full-length Bid is cleaved by caspase-8. The truncated p15 tBid translocates to the mitochondria and activates Bax and Bak to initiate mitochondrial disruption processes associated with the intrinsic pathway. 9 Herein, activation or restoration of the function of p53 is an im- portant strategy for anticancer agents. Not only is the use of herbal intervention widespread all over the world, 10 but also natural products are lead compounds for many of the drugs that are currently in use. 11 Hence, it is of inter- est to investigate natural materials as a source of potential, new chemotherapeutic agents for cancer. Garcinia cowa Roxb (Gutti- ferae) is a tropical subcanopy climax tree indigenous to monsoon rain forests in southwest Asia with cooling and detoxification functions. 12 Phytochemical studies showed that xanthone and ben- zophenone are the two main classes of components in Garcinia species, 13–17 some of which have demonstrated cytotoxic activ- ity. 18,19 Gaudichaudione A exhibits strong growth inhibitory effects against Jurkat human leukemic cells, parental murine leu- kemic P388 and P388/DOX-resistant cell lines by the induction of apoptosis through mitochondrial destabilization and caspase-3 activation. 20 Gambogic acid possesses antitumor activity both in vivo and in vitro. 21 It induces irreversible G 2 /M cell cycle arrest in human gastric carcinoma BGC-823 cells by significant decreas- ing cdc2/p34 synthesis, which is mediated through the inhibition of CDK-activating kinase (CDK7/cyclin H) activity. 22 However, the antitumor activities of G. cowa and its xanthone constituents have not been reported yet. The present study investigated the antitumor action of G. cowa extract and dulxanthone A, and gained insights into the mecha- nisms through which dulxanthone A induces cell cycle arrest and apoptosis. Material and methods Extraction and isolation The plant material was collected in Jinghong, Yunnan Province, China, and identified by Professor Shaorong Guo of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College. A voucher specimen (YA-04-0418) has been deposited in the Herbarium of the same institute. The dried and pulverized stems of G. cowa (20 kg) were extracted three times with 95% EtOH for 2 h under reflux, and then extracted twice with 70% EtOH for another 2 h. After Grant sponsor: National Natural Science Foundation of China; Grant number: 30470195; Grant sponsor: National Institute of Health Training Grant; Grant number: 2Ta5 LM 07092-11. *Correspondence to: Children’s Hospital Informatics Program at Har- vard- MIT Division of Health Sciences and Technology, Children’s Hospi- tal Boston, Harvard Medical School, Boston, MA 02115, USA. Fax: 1617-730-0921. E-mail: erxi.wu@childrens.harvard.edu Received 18 February 2007; Accepted after revision 9 July 2007 DOI 10.1002/ijc.23048 Published online 10 September 2007 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 122, 31–38 (2008) ' 2007 Wiley-Liss, Inc. Publication of the International Union Against Cancer