Biologia 67/3: 616—621, 2012 Section Cellular and Molecular Biology DOI: 10.2478/s11756-012-0037-6 Hypoxia inhibition of camptothecin-induced apoptosis by Bax loss Kyoungsook Park 1,2,3 , Abdela Salah Woubit 1 , Cesar D. Fermin 1 , Gopal Reddy 1 , Tsegaye Habtemariam 1 , Jin Woong Chung 4 , Minseo Park 5 , Dai-Wu Seol 6,7 * & Moonil Kim 1,2,3 * 1 Department of Pathobiology, College of Veterinary Medicine Nursing & Allied Health (CVMNAH), Tuskegee University, Tuskegee, AL 36088, USA; e-mail: kimm@mytu.tuskegee.edu 2 BioNanotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, 52 Eoeun-Dong, Yuseong- Gu, Daejeon 305–333, Korea 3 School of Engineering, University of Science and Technology (UST), Daejeon 305–333, Korea 4 Department of Biology and Biomedical Science, Dong-A University, Busan 604–714, Korea 5 Department of Physics, Auburn University, Auburn, AL 36849, USA 6 Faculty of Pharmacy, College of Pharmacy, Chung-Ang University, Seoul 156–756, Korea; e-mail: seold@cau.ac.kr 7 Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA Abstract: Tumor cell hypoxia is linked to the resistance of human solid tumors to the various anti-cancer therapies: thus, its exploitation has been considered to be a potential target for cancer treatment. Previously, we demonstrated for the first time that hypoxia inhibits apoptosis induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) through blocking translocation of Bax, a pro-apoptotic protein, from the cytosol to the mitochondria. Nevertheless, the molecular mechanism coupling hypoxia to resistance for drugs, especially for anti-cancer chemotherapeutics, still remains to be elucidated. Here, we demonstrate that hypoxia attenuates camptothecin (CPT)-induced apoptosis by decreasing the protein levels of Bax, thereby leading to resistance to the drug. DNA damage after exposure to CPT resulted in an increase of p53, and a concomitant up-regulation of p21, regardless of oxygen content. Under normoxic condition, CPT induced expression of p53 and its down-stream target molecule Bax as well, in the presence of increased p21. In contrast, when pre- exposed to hypoxia, Bax-inducing activity of CPT was completely lost and the Bax level was even decreased, although CPT increased both p53 and p21 as observed under normoxic condition. Our data indicate that hypoxia attenuates apoptosis via Bax. Our data also suggest that hypoxia regulates tumor cell apoptosis differentially, through regulating Bax translocation or through down-regulating Bax levels, depending on death-inducing signals as shown by TRAIL- or CPT-induced apoptosis. Key words: Bax; camptothecin; apoptosis; hypoxia; colon cancer. Abbreviations: CPT, camptothecin; 5-FU, 5-fluorouracil; IPTG, isopropyl β-D-thiogalactopyranoside; ROS, reactive oxy- gen species; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand. Introduction Hypoxic cells are present in many human solid tumors. Hypoxia is known to lead to a negative effect on therapy such as radiation therapy and chemotherapy (Graeber et al. 1996; Dong et al. 2001; Harris 2002). In addition, tumor hypoxia is strongly related to cancer propaga- tion, increased metastatic potential, and poor prognosis (Teicher 1994; Vaupel et al. 2001; Shannon et al. 2003). For these effects, hypoxia has been thought to be critical setback in cancer treatments, because it renders tumor cells more aggressive and resistant to apoptosis. Thus, it is obvious that tumor cell responses to hypoxia are critical for tumor progression as well as tumor therapy. Most chemotherapy agents including camptothecin (CPT) destroy tumors by affecting DNA replication in cells (Rustum & Cao 1999; Trimmer & Essigmann 1999; Pommier 2006). During DNA synthesis, CPT works as a topoisomerase I inhibitor, thereby causing double- strand DNA damage (Liu et al. 1996; Pommier 2009). Studies have demonstrated that once activated by DNA damage or agent that stimulates DNA damage, cells stimulate expression of a tumor suppressor protein p53, which in turn up-regulates p21 expression to induce cell growth arrest in G1 or G2 phase until DNA repair pro- cess has been completed (Bunz et al. 1998; Hamada et al. 2009). If DNA damage is too severe, this po- tent tumor suppressor stimulates pro-apoptotic Bax to eliminate injured cells, thereby leading to apoptotic cell death (Chipuk et al. 2004). The mitochondrial apoptotic events are largely controlled by the Bcl-2 family of proteins, which con- * Corresponding author c 2012 Institute of Molecular Biology, Slovak Academy of Sciences