Early growth response protein 1 upregulation and nuclear translocation by 2 0 -benzoyloxycinnamaldehyde induces prostate cancer cell death Hye-Sook Kang a , Jiyeon Ock a , Heon-Jin Lee a,c , Yu-Jin Lee b , Byoung-Mog Kwon b,⇑ , Su-Hyung Hong a,⇑ a Department of Oral Microbiology, School of Dentistry, Kyungpook National University, Daegu 700-412, South Korea b Laboratory of Chemical Biology and Genomics, Korea Research Institute of Bioscience and Biotechnology, Daejon 305-806, South Korea c Craniofacial Dysfunction Research Center, School of Dentistry, Kyungpook National University, Daegu 700-412, South Korea article info Article history: Received 18 September 2012 Received in revised form 31 October 2012 Accepted 2 November 2012 Keywords: 2 0 -Benzoyloxycinnamaldehyde (BCA) Prostate cancer cells Early growth response protein 1 (EGR1) Activating transcription factor 3 (ATF3) NSAID-activated gene 1 protein (NAG-1) Growth arrest and DNA-damage-inducible protein alpha (GADD45A) Importin-7 (IPO7) abstract 2 0 -Benzoyloxycinnamaldehyde (BCA) induces apoptosis in human cancer cells through ROS generation. BCA upregulates proapoptotic genes such as activating transcription factor 3 (ATF3), NSAID-activated gene 1 protein (NAG-1), and growth arrest and DNA-damage-inducible protein alpha (GADD45A) in pros- tate cancer cells. These genes are known to be induced by transcription factor early growth response pro- tein 1 (EGR1). BCA induces significant EGR1 upregulation, while EGR1 knockdown decreases the induction of these genes with concurrent alleviation of cell death by BCA. Antioxidant glutathione pre- treatment with BCA removes EGR1 expression increase, suggesting that EGR1 upregulation is dependent on oxidative stress generated by BCA. In prostate cancer cells, EGR1 localizes in the cytoplasm; however, BCA remarkably upregulates EGR1 nuclear translocalization, suggesting its possible effect as a transcrip- tional activator. BCA induces transient upregulation of importin-7 (IPO7) which is critical for EGR1 nuclear translocation, and IPO7 knockdown led to a significant decrease in chemosensitivity to BCA. Taken together, our findings suggest that BCA induces prostate cancer cell death via EGR1 upregulation and nuclear translocalization, followed by activation of proapoptotic target genes. Ó 2012 Elsevier Ireland Ltd. All rights reserved. 1. Introduction 2 0 -Benzoyloxycinnamaldehyde was originally synthesized as a derivative of 2 0 -hydroxycinnamaldehyde (HCA) [1], which is a nat- ural compound isolated from the bark of Cinnamomum cassia Blume [2]. These compounds are characterized as antitumor agents in diverse cancer cell lines, and they may induce apoptosis via ROS generation and caspase-3 activation [3]. BCA/HCA-induced apopto- sis has also been found to be associated with the inhibition of proteasome activity [4]. BCA shows therapeutic selectivity in K-ras-transformed cells through downregulation of thiol antioxi- dants [5], which is a promising result for antitumor drug develop- ment. BCA was also approved for clinical tests by the Korean Food and Drug Administration on January, 2011 and it is now under clin- ical test. There has been little investigation of the effect of BCA on prostate cancer which is the second leading cause of deaths from cancer [6]. Therefore, we investigated the effect of BCA and its molecular mechanisms in prostate cancer cell lines such as andro- gen-dependent (LNCap) and androgen-independent (PC-3, DU145) cells. In our previous study, pretreatment of antioxidant, glutathione (GSH) with BCA in cancer cells eliminated the growth inhibition ef- fect [3], suggesting that ROS may play a key role in BCA-induced cell death. A previous study demonstrated that oxidative stress in- duces early growth response gene-1 (EGR1) expression in renal epithelial cells [7]. Furthermore, EGR1 downstream genes such as ATF3, NAG-1, and GADD45A were upregulated by HCA or BCA in our DNA microarray analysis (data unpublished). Therefore, we at- tempt to investigate the mechanism behind BCA-induced prostate cancer cell death through the EGR1. EGR1, a member of the immediate-early gene family, is involved in the regulation of cell growth and differentiation in re- sponse to signals such as mitogens, growth factors, and stress [8] and [9]. Previous reports indicate that EGR1 is a prominent tumor suppressor and that many human tumor cell lines express little or 0304-3835/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2012.11.006 Abbreviations: BCA, 2 0 -benzoyloxycinnamaldehyde; HCA, 2 0 -hydroxycinnam- aldehyde; EGR1, early growth response protein 1; ATF3, activating transcription factor 3; GSH, glutathione; NAG-1, NSAID-activated gene 1 protein; GADD45A, growth arrest and DNA-damage-inducible protein alpha; IPO7, Importin-7; MTT, 3- [4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide; ROS, reactive oxygen species. ⇑ Corresponding authors. Addresses: 2177 Dalgubeol-daero, Jung-gu, Daegu 700- 412, South Korea. Tel.: +82 53 660 6831; fax: +82 53 425 6025 (S.-H. Hong), 111 Gwahak-ro Yoosung-gu Daejon 305-806, Korea. Tel.: +82 42- 860 4557 (B.-M. Kwon). E-mail addresses: kwonbm@kribb.re.kr (B.-M. Kwon), hongsu@knu.ac.kr (S.-H. Hong). Cancer Letters 329 (2013) 217–227 Contents lists available at SciVerse ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet