An NQO1-Initiated and p53-Independent Apoptotic Pathway Determines the Anti-Tumor Effect of Tanshinone IIA against Non-Small Cell Lung Cancer Fang Liu., Guo Yu . , Guangji Wang*, Huiying Liu, Xiaolan Wu, Qiong Wang, Miao Liu, Ke Liao, Mengqiu Wu, Xuefang Cheng, Haiping Hao* State Key Laboratory of Natural Medicines, Key Lab of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, China Abstract NQO1 is an emerging and promising therapeutic target in cancer therapy. This study was to determine whether the anti- tumor effect of tanshinone IIA (TSA) is NQO1 dependent and to elucidate the underlying apoptotic cell death pathways. NQO1+ A549 cells and isogenically matched NQO1 transfected and negative H596 cells were used to test the properties and mechanisms of TSA induced cell death. The in vivo anti-tumor efficacy and the tissue distribution properties of TSA were tested in tumor xenografted nude mice. We observed that TSA induced an excessive generation of ROS, DNA damage, and dramatic apoptotic cell death in NQO1+ A549 cells and H596-NQO1 cells, but not in NQO12 H596 cells. Inhibition or silence of NQO1 as well as the antioxidant NAC markedly reversed TSA induced apoptotic effects. TSA treatment significantly retarded the tumor growth of A549 tumor xenografts, which was significantly antagonized by dicoumarol co-treatment in spite of the increased and prolonged TSA accumulations in tumor tissues. TSA activated a ROS triggered, p53 independent and caspase dependent mitochondria apoptotic cell death pathway that is characterized with increased ratio of Bax to Bcl- xl, mitochondrial membrane potential disruption, cytochrome c release, and subsequent caspase activation and PARP-1 cleavage. The results of these findings suggest that TSA is a highly specific NQO1 target agent and is promising in developing as an effective drug in the therapy of NQO1 positive NSCLC. Citation: Liu F, Yu G, Wang G, Liu H, Wu X, et al. (2012) An NQO1-Initiated and p53-Independent Apoptotic Pathway Determines the Anti-Tumor Effect of Tanshinone IIA against Non-Small Cell Lung Cancer. PLoS ONE 7(7): e42138. doi:10.1371/journal.pone.0042138 Editor: Siyaram Pandey, University of Windsor, Canada Received March 16, 2012; Accepted July 2, 2012; Published July 27, 2012 Copyright: 2012 Liu 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 study was financially supported by a Foundation for the Author of National Excellent Doctoral Dissertation of China (200979), Natural Science Foundation of Jiangsu province (BK2011065), Science Foundation for Youth Scholars of Ministry of Education of China (200803161012), the Program for New Century Excellent Talents in University (NCET-09-0770), and National Natural Science Foundation China (Nos. 91029746, 30801422). 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: hhp_770505@yahoo.com.cn (HH); guangjiwang@hotmail.com (GW) . These authors contributed equally to this work. Introduction Non-small cell lung cancer (NSCLC) accounts for approxi- mately 80,85% of all cases of lung cancer, and is the most common cause of death in men and second to breast cancer in women [1]. Combination chemotherapy, usually platinum-based, is currently the first-line therapy of choice for NSCLCs. However, the prognosis for patients with advanced NSCLC remains poor with a median survival time of 8 to11 months and a 1-year survival rate of 30% [2,3]. The long term survival (5-year) rate was even poor at around 15% [4]. The recent development of various molecular target drugs and their combination with chemotherapy drugs improves the outcome of NSCLC therapy; however, it remains disappointing in the therapy of advanced NSCLC. Obviously, there is an urgent need to identify new therapeutic targets and to develop tumor-selective chemotherapeutic drugs specific for NSCLCs. NAD(P)H:quinone oxidoreductase (NQO1, EC 1.6.99.2) is a cytosolic flavoenzyme that catalyzes the obligatory two-electron reduction of a variety of quinone substrates, using both NADH and NADPH as electron donors [5]. Originally, NQO1 was widely believed to be a detoxification enzyme in view of its two- electron reduction property, bypassing the one-electron reduction producing unstable and highly reactive semiquinone [6,7]. Lately, it was found that some quinones such as mitomycin C, streptonigrin, E09, and RH1 [8,9,10,11,12] were bioactivated by NQO1. The bioactivation property of NQO1 promises it an ideal target for developing anti-tumor drugs, because various human tumors [13] have elevated NQO1 activities. In the case of lung tumors, NQO1 activity is increased up to 80-fold in NSCLC tumors relative to normal lung, and 20,35-fold relative to SCLC cell lines [14]. Such a differentiated expression mode of NQO1 between tumors and normal tissues suggests that NQO1 target drugs would be highly selective in killing tumor cells while saving normal tissues. RH1 is a drug candidate bioactivated by NQO1 to produce hydroquinone in the activation of the aziridine rings and subsequent DNA alkylation and interstrand cross-linking. In this case, NQO1 is utilized as a tumor selective enzyme to bioactivate the prodrug and thus to realize a tumor specific toxicity. In addition to its property as an oxidoreductase, NQO1 has been also found directly involved in stabilizing the vital tumor suppressors p53/p73/p33 [15,16]. Moreover, NQO1 polymorphism that leads to the enzyme inactivity has been found to be a strong PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e42138