Mechanism and stereoselectivity of HDAC I inhibition by (R)-9-hydroxystearic acid in colon cancer Carola Parolin a , Natalia Calonghi a, ⁎, Enrica Presta a , Carla Boga b , Paolo Caruana b , Marina Naldi c , Vincenza Andrisano c , Lanfranco Masotti a , Giorgio Sartor a a Department of Biochemistry “G. Moruzzi”, Alma Mater Studiorum, University of Bologna, Via Irnerio 48, 40126, Bologna, Italy b Department of Organic Chemistry “A. Mangini”, Alma Mater Studiorum, University of Bologna, Viale Risorgimento 4, 40136, Bologna, Italy c Department of Pharmaceutical Sciences, Alma Mater Studiorum, University of Bologna, Via Belmeloro 6, 40126, Bologna, Italy abstract article info Article history: Received 1 March 2012 Received in revised form 29 June 2012 Accepted 9 July 2012 Available online 17 July 2012 Keywords: 9-hydroxystearic acid Colon cancer Histone deacetylase Cyclin D1 Protein interaction Stereoselectivity 9-Hydroxystearic acid (9-HSA) belongs to the endogenous lipid peroxidation by-products that decrease in tumors, causing as a consequence the loss of one of the control mechanisms on cell division. It acts as a histone deacetylase (HDAC, E.C 3.5.1.98) inhibitor, and the interaction of the two enantiomers of 9-HSA with the catalytic site of the enzyme, investigated by using a molecular modelling approach, has been reported to be different. In this work we tested out this prediction by synthesizing the two enantiomers (R)-9-HSA (R-9) and (S)-9-HSA (S-9) starting from the natural source methyl dimorphecolate obtained from Dimorphotheca sinuata seeds and investigating their biological activity in HT29 cells. Both enantiomers inhibit the enzymatic activity of HDAC1, HDAC2 and HDAC3, R-9 being more active; R-9 and S-9 inhibitory effect induces an increase in histone H4 acetylation. We also demonstrate that the antiproliferative effect brought about by R-9 is more pronounced as well as we observe increase of p21 transcription and protein content, while the expression of cyclin D1 is decreased. Starting from these observations it can be hypothesized that the interaction of R-9 with HDAC1 induce conformational changes in the enzyme causing loss of its interac- tion with other proteins, like cyclin D1 itself. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Chromatin architecture is now generally recognized as an important factor in the regulation of gene expression. The fundamental subunit of chromatin, the nucleosome, is composed of an octamer of four core histones, i.e. an H3/H4 tetramer and two H2A/H2B dimers, surrounded by 146 bp of DNA [1]. Chemical modification of the nucleosome can allow interactions of these inaccessible DNA sequences with the molec- ular machinery involved in gene expression [2]. Transcriptional regulation in eukaryotes occurs within a chromatin setting, and it is strongly influenced by the post-translational modifica- tion of histones, such as methylation, phosphorylation and acetylation. Histone acetylation is carried out by a group of proteins called histone acetyl transferases (HATs E.C 2.3.1.48), and the acetyl groups can be removed by histone deacetylases (HDACs E.C 3.5.1.98), thereby regulating the expression of many genes, some of which are involved in apoptosis and cell proliferation. Eighteen different human HDAC isoforms have been described [2,3] and these can be divided into four classes based on structural homolo- gies between human and yeast HDACs. Class I HDACs (HDAC1, 2, 3, and 8) are related to the yeast RPD3 deacetylase, and are primarily found in the nucleus with the exception of HDAC3 [4], which is found both in the nucleus and in the cytoplasm, and in association with the plasma membrane [4]. Class II HDACs are divided into two subclasses: class IIa (HDAC4, 5, 7, and 9) and class IIb (HDAC6 and 10), and are ho- mologous to the yeast Hda1 deacetylase. This class of HDACs is able to shuttle in and out of the nucleus depending on different signals. Class III HDACs consist of seven HDACs (SIRT1 to SIRT7) and share homolo- gies with the yeast silent information regulator 2 (Sir2) family [5,6]. This class of HDACs has a unique catalytic mechanism that requires the co-factor NAD + for activity. The last class of HDACs, class IV, has only one member (HDAC11) which is structurally related to both class I and class II [7,8]. Classes I, II, and IV require Zn 2+ for activity. Emerging data now implicate histone modification in the pathobiology of cancer and other diseases, and a common finding in cancer cells is a high level expression of HDAC isoenzymes and a corresponding hypoacetylation of histones [9,10]. A study of normal and malignant tissues has revealed a consistent pattern: higher levels of histone acetylation in normal lymphoid tissue as compared to lymphomas, and in normal epithelium as compared to colon adenocarcinomas [11], as well as higher HDAC1 expression in Biochimica et Biophysica Acta 1821 (2012) 1334–1340 Abbreviations: 9-HSA, 9-hydroxystearic acid; R-9, (R)-9-hydroxystearic acid; S-9, (S)-9-hydroxystearic acid; RT-PCR, Real time PCR; HDAC, histone deacetylase; HAT, histone acetyltransferase; PI, propidium iodide; CBP, chromatin bound proteins ⁎ Corresponding author. Tel.: +39 051 2091231; fax: +39 051 2091234. E-mail address: natalia.calonghi@unibo.it (N. Calonghi). 1388-1981/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2012.07.007 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbalip