TheProstate70:1074^1086(2010) AWater-SolubleParthenolideAnalogueSuppresses InVivo Prostate Cancer GrowthbyTargetingNFk B andGeneratingReactiveOxygenSpecies Rajasubramaniam Shanmugam, 1 Praveen Kusumanchi, 1 Liang Cheng, 2 Peter Crooks, 3 Sundar Neelakantan, 3 William Matthews, 4 Harikrishna Nakshatri, 5,6,7 and Christopher J. Sweeney 1,5,8 * 1 Departmentof Medicine,Indiana University,Indianapolis,Indiana 2 Departmentof Pathology,Indiana University,Indianapolis,Indiana 3 College of Pharmacy,Universityof Kentucky, Lexington,Kentucky,United States 4 Leuchemix Inc.,Woodside,California 5 Departmentof Surgery,Indiana University,Indianapolis,Indiana 6 Walther Cancer Institute,Indianapolis,Indiana 7 Departmentof Biochemistryand Molecular Biology,Indiana University,Indianapolis,Indiana 8 Departmentof Medicine,Dana-Farber Cancer Institute, Boston, Massachusetts BACKGROUND. To characterize the molecular changes associated with DMAPT-induced prostate cancer cell death and its in vivo activity. METHODS. CWR22Rv1 and PC-3 were subjected to flow cytometry, electrophoretic mobility shift assays, and Western blot studies to measure DMAPT’s ability to generate reactive oxygen species (ROS), inhibit NFkB DNA binding, and cause changes in anti-apoptotic proteins. N-acetyl cysteine (NAC) and short hairpin RNA (shRNA) were used to determine the contribution of ROS and JNK2 activation, respectively. The BrdU incorporation assay was used to measure proliferation and trypan blue studies assessed cell viability after DMAPT treatment. The in vivo activity of DMAPT as a single agent and in combination with bicalutamide or docetaxel was assessed in a subcutaneous xenograft model with athymic nude female mice. RESULTS. DMAPT generated ROS with subsequent JNK activation and inhibited NFkB DNA binding and expression of NFkB-regulated anti-apoptotic proteins. DMAPT increased necrotic and apoptotic cell death in a cell-type-dependent manner and both types of cell death were blocked by NAC. Additionally, shRNA JNK2 partially blocked the anti-proliferative activity of DMAPT. DMAPT inhibited CWR22Rv1 and PC-3 cellular proliferation by 100% with 10 and 20 mM respectively and in vivo, DMAPT was more effective at inhibiting growth than biclutamide (CWR22v1) and docetaxel (PC-3). CONCLUSIONS. DMAPT promotes cell death by both generating ROS and inhibition of NFkB. Its in vivo activity supports the conduct of clinical trials in patients with castrate-resistant disease. Prostate 70: 1074 – 1086, 2010. # 2010 Wiley-Liss, Inc. KEY WORDS: parthenolide analogue; apoptosis; androgen independence Rajasubramaniam Shanmugam and Praveen Kusumanchi contrib- uted equally to this work. Grant sponsor: Department of Defense; Grant number: DAMD W81XWH-06-1-0225; Grant sponsor: Walther Cancer Institute. *Correspondence to: Christopher J. Sweeney, MBBS, Lank Center for Genitourinary Oncology, Dana-Farber Cancer Institute, 44 Binney Street, DA-1230, Boston, MA 02115. E-mail: christopher_sweeney@dfci.harvard.edu Received 29 December 2009; Accepted 13 January 2010 DOI 10.1002/pros.21141 Published online 5 March 2010 in Wiley InterScience (www.interscience.wiley.com). ß2010Wiley-Liss,Inc.