Asian J Androl 2006; 8 (): – . 1 . Correspondence to: Dr Horng-Heng Juang, Department of Anatomy, Chang Gung University, 259 Wen-Hua 1st Road, Kwei-Shan, Tao- Yuan 333, Taiwan, China. Tel: +886-3-2118-800 ext. 5071, Fax: +886-3-2118-112 E-mail: hhj143@mail.cgu.edu.tw Received 2005-12-14 Accepted 2006-01-06 Manganese antagonizes iron-blocking mitochondrial aconitase expression in human prostate carcinoma cells Ke-Hung Tsui 1,2 , Phei-Lang Chang 1 , Horng-Heng Juang 2,3 1 Department of Urology, 2 Chang Gung Molecular Image Center, Chang Gung Memorial Hospital, 3 Department of Anatomy, Chang Gung University; Kwei-Shan, Tao-Yuan 333, Taiwan, China Abstract Aim: To investigate the possible role of manganese in the regulation of mitochondrial aconitase (mACON) activity human prostate carcinoma cell line PC-3 cells. Methods: The mACON enzymatic activities of human prostate carcinoma cell line PC-3 cells were determined using a reduced nicotinamide adenine dinucleotide-coupled assay. Immunoblot and transient gene expression assays were used to study gene expression of the mACON. The putative response element for gene expression was identified using reporter assays with site-directed mutagenesis and electro- phoretic mobility-shift assays. Results: In vitro study revealed that manganese chloride (MnCl2) treatment for 16 h inhibited the enzymatic activity of mACON, which induced the inhibition of citrate utility and cell proliferation of PC- 3 cells. Although results from transient gene expression assays showed that MnCl2 treatment upregulated gene translation by approximately 5-fold through the iron response element pathway, immunoblot and reporter assays showed that MnCl2 treatments inhibited protein and gene expression of mACON. This effect was reversed by co- treatment with ferric ammonium citrate. Additional reporter assays with site-directed mutagenesis and electrophoretic mobility-shift assays suggested that a putative metal response element in the promoter of the mACON gene was involved in the regulation of MnCl2 on the gene expression of mACON. Conclusion: These findings suggest that manganese acts as an antagonist of iron, disrupting the enzymatic activity and gene expression of mACON and citrate metabolism in the prostate. (Asian J Androl 2006 May; 8: –) Keywords: citrate; adenosine triphosphate; proliferation; PC-3; metal response element; prostate carcinoma cell lines . Original Article . © 2006, Asian Journal of Andrology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences. All rights reserved. DOI: 10.1111/j.1745-7262.2006.00139.x 1 Introduction Aconitase (aconitase hydratase, EC4.2.1.3) is the enzyme responsible for the interconversion of citrate and isocitrate in the citric acid cycle [1]. Our previous in vitro study using the stable-transfected mitochondrial aconitase (mACON) antisense human prostate carcinoma cell cell line PC-3 illustrated the key role of mACON in citrate util- ity and bioenergy [2]. There are two different aconitases in mammalian cells, cytosolic and mitochondrial, which are encoded by two different genes [3]. Both aconitases contain a (4Fe-4S) cluster that is required for their enzy- matic activities. The conserved iron responsive element (IRE) contains an approximately 30-nucleotide, RNA hairpin stem-loop located in the 5'-untranslated region of ferritins, 5-aminolevulinate synthase and mACON