Pharmacological Research 70 (2013) 90–101 Contents lists available at SciVerse ScienceDirect Pharmacological Research jo u r n al hom epage: www.elsevier.com/locat e/yphrs PIM1 kinase inhibitors induce radiosensitization in non-small cell lung cancer cells Wanyeon Kim a , HyeSook Youn b , TaeWoo Kwon a , JiHoon Kang a , EunGi Kim a , Beomseok Son a , Hee Jung Yang a , Youngmi Jung a , BuHyun Youn a,∗ a Department of Biological Sciences, Pusan National University, Busandaehak-ro 63, Geumjeong-gu, Busan, 609-735, South Korea b Department of Bioscience & Biotechnology/Institute of Bioscience, Sejong University, 98 Gunja-dong, Gwangjin-gu, Seoul, 143-747, South Korea a r t i c l e i n f o Article history: Received 7 November 2012 Received in revised form 7 January 2013 Accepted 14 January 2013 Keywords: Radiosensitizer PIM1 inhibitor SGI-1776 ETP-45299 Tryptanthrin Non-small cell lung cancer a b s t r a c t Radiotherapy plays a critical role in the treatment of non-small cell lung cancer (NSCLC). However, radioresistance is a major barrier against increasing the efficiency of radiotherapy for NSCLC. To understand the mechanisms underlying NSCLC radioresistance, we previously focused on the poten- tial involvement of PIM1, PRAS40, FOXO3a, 14-3-3, and protein phosphatases. Among these proteins, PIM1 functioned as an oncogene and was found to act as a crucial mediator in radioresistant NSCLC cells. Therefore, we investigated the use of PIM1-specific inhibitors as novel therapeutic drugs to regulate radiosensitivity in NSCLC. After structure-based drug selection, SGI-1776, ETP-45299, and tryptanthrin were selected as candidates of PIM1 inhibitors that act as radiosensitizers. With irradiation, these drugs inhibited only PIM1 kinase activity without affecting PIM1 mRNA/protein levels or cellular localization. When PIM1 kinase activity was suppressed by these inhibitors, PRAS40 was not phosphorylated. Conse- quently, unphosphorylated PRAS40 did not form trimeric complexes with 14-3-3 and FOXO3a, leading to increased nuclear localization of FOXO3a. Nuclear FOXO3a promoted the expression of pro-apoptotic pro- teins such as Bim and FasL, resulting in a radiosensitizing effect on radioresistant NSCLC cells. Moreover, an in vivo xenograft mouse model confirmed this radiosensitizing effect induced by PIM1 inhibitors. In these model systems, tumor volume was significantly reduced by a combinational treatment with irradi- ation and PIM1 inhibitors compared to irradiation alone. Taken together, our findings provided evidence that PIM1-specific inhibitors, SGI-1776, ETP-45299, and tryptanthrin, can act as novel radiosensitizers to enhance the efficacy of radiotherapy by inhibiting irradiation-induced signaling pathway associated with radioresistance. © 2013 Elsevier Ltd. All rights reserved. 1. Introduction Lung cancer is the most frequent cause of cancer death through- out the world. Lung cancer is categorized into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) based on cancer cell histology [1]. Between the two types, NSCLC accounts for about 80–85% of total lung malignancies [2]. Only 15–20% of Abbreviations: ASK1, apoptosis signaling kinase 1; ChIP, chromatin immunopre- cipitation; CK2, casein kinase 2; DMSO, dimethyl sulfoxide; FOXO, the mammalian forkhead box transcription factors of the O classification; EGFR, epidermal growth factor receptor; FasL, Fas ligand; FRE, FOXO-response element; HA, Hemagglutinin; HNSCC, head and neck squamous cell carcinoma; IR, ionizing radiation; ITC, isother- mal titration calorimetry; NSCLC, non-small cell lung cancer; PRAS40, proline-rich AKT substrate of 40 kDa; SAR, structure–activity relationship; SCLC, small cell lung cancer; SD, standard deviation. ∗ Corresponding author at: #303 Biology Building, Department of Biological Sciences, Pusan National University, Busandaehak-ro 63, Geumjeong-gu, Busan, 609-735, South Korea. Tel.: +82 51 510 2264; fax: +82 51 581 2962. E-mail address: bhyoun72@pusan.ac.kr (B. Youn). NSCLC patients diagnosed at early stage could be cured success- fully by surgical resection, while over 50% of NSCLC patients were often diagnosed at advanced stages and curative surgical removal could not be performed to their tumors. Therefore, radiotherapy is preferred to an alternative treatment for them [3]. However, recur- rence rates and metastasis still remain high and the therapeutic outcome of most NSCLC patients is not satisfactory. The emer- gence of radioresistance is regarded as one of the major factors for therapy failure. Although enhanced efficacy of radiotherapy could be expected by dose escalation, this protocol might be restricted by considerable cytotoxicity associated with high dose irradia- tion. Thus, there are many efforts to regulate radioresistance and develop potent radiosensitizers for establishing better treatment strategies. Various experimental and clinical studies suggest that radioresistance is positively associated with the overexpression and activation of specific genes, including ones encoding epider- mal growth factor receptor (EGFR), c-Raf, p53 and PIM1 [4–7]. Although such findings have contributed to limited information of the mechanism underlying cellular radioresistance, there are presently no molecular targeted therapies that can be effectively 1043-6618/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phrs.2013.01.005