EXCLI Journal 2020;19:1038-1051 – ISSN 1611-2156 Received: January 30, 2020, accepted: July 09, 2020, published: July 23, 2020 Original article: EFFECT OF SODIUM BUTYRATE ON HDAC8 mRNA EXPRESSION IN COLORECTAL CANCER CELL LINES AND MOLECULAR DOCKING STUDY OF LHX1 - SODIUM BUTYRATE INTERACTION Flora Forouzesh (PhD)a*, Mahsa Ghiaghi (MSc)b, Hamzeh Rahimi (PhD)c a Assistant professor, Department of Genetics, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran. E-mail: [email protected] b Student of Master Science of Genetics, Department of Genetics, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran. E-mail: [email protected] c Assistant professor, Department of Molecular Medicine, Pasteur Institute of Iran, Tehran, Iran. E-mail: [email protected] * Corresponding author: Dr. Flora Forouzesh, Faculty of Advanced Science and Technology, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran. Dr Sharyati street, Shahid Khaghani street. P.O. Box: 193951495, Tehran, Iran. Tel: +98(21) 22006660-7, Fax: +98(21) 22600714, E-mail: [email protected], [email protected] http://dx.doi.org/10.17179/excli2020-2010 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/). ABSTRACT Colorectal cancer (CRC) is the third most common type of cancer and the fourth leading cause of cancer related deaths worldwide. The Histone Deacetylase 8 (HDAC8) gene is a gene with unique features which can be used as a potential target for drug design. The LHX1 transcription factor is an important transcription factor for this gene. The aim of this study was to investigate the effect of sodium butyrate (NaB) as a histone deacetylase inhib- itor (HDACi) on the expression of the HDAC8 gene in the colorectal cancer cell line, and the molecular docking of the LHX1 transcription factor with NaB. For this purpose, HCT-116 and HT-29 cell lines were treated with different concentrations of NaB (6.25 mM to 150 mM) at 24, 48 and 72 hours. Subsequently, RNA was extract- ed from the treated and untreated cells and cDNA was synthesized. Quantitative Real-Time-PCR was done to investigate the mRNA expression of HDAC8. Molecular docking was also performed to investigate the interac- tion between NaB and LHX1. Based on Real-time-PCR results, the concentration of 150 mM of NaB after 24 hours in HT-29 and HCT-116 cell lines caused a significant reduction in mRNA expression of HDAC8 (P<0.05). After 48 hours of treatment, there was a significant decrease in the mRNA expression of HDAC8 at all concentrations (P<0.05). The docking results showed that LHX1 and NaB interacted best at the lowest energy levels. Our results also showed that NaB bonded strongly to LHX1. In addition, our results demonstrated that NaB bound to the LHX1 transcription factor and inhibited the function of this factor and consequently decreased the transcription from the HDAC8 gene which resulted in cell death. Future studies are needed to assess the like- ly molecular mechanisms of NaB action on gene expression. Keywords: Colorectal cancer, sodium butyrate, HDAC8 gene, LHX1 transcription factor, docking, HT-29, HCT-116 cell lines 1038 EXCLI Journal 2020;19:1038-1051 – ISSN 1611-2156 Received: January 30, 2020, accepted: July 09, 2020, published: July 23, 2020 INTRODUCTION pressed in several types of tumors. The Colorectal cancer (CRC) is the fourth pharmacological inhibition of HDACs also most common cause of death from cancer in known as HDACi may be considered as new the world (Ansari et al., 2006; Mundade et method for cancer therapy (Bolden et al., al., 2014; Stigliano et al., 2014). As it has 2006; Minucci and Pelicci, 2006; Hildmann been found, cancer is the result of a wide et al., 2007; Riester et al., 2007; range of genetic and epigenetic changes. Shuttleworth et al., 2010). It has been ob- Histone acetylation is one of such epigenetic served that, HDACi can change the balance changes (Esteller, 2002; Johnstone, 2002; between HATs and HDACs and therefore Iizuka and Smith, 2003). Acetylation levels lead to a hyperacetylated state of histones are the result of the balance of the activities that causes cell cycle arrest, inducing tran- between histone deacetylase (HDAC) en- scriptional inhibition and apoptosis which are valid strategies for cancer treatment zymes and histone acetyltransferase (HAT) enzymes (Bannister and Kouzarides, 2011). (Bolden et al., 2006). The HDAC8 gene is a class I HDAC gene One of these HDACis is sodium butyrate (Hu et al., 2000; Van den Wyngaert et al., (NaB). Butyrate is produced by anaerobic 2000; Wawruszak et al., 2019), which is pre- bacterial fermentation of dietary fibers and is sent in the nucleus and cytoplasm of cells a short-chain fatty acid (Emenaker et al., distinctively (Waltregny et al., 2004; Li et 2001; Hinnebusch et al., 2002). One of the al., 2014). HDAC8 is expressed in normal functions of butyrate is the anti- tissues, but its expression increases in can- inflammatory effect that plays a role in in- cerous tissues (Song et al., 2015). For exam- hibiting histone deacetylase. Butyrate specif- ple, HDAC8 is correlated with T-cell lym- ically inhibits the class I HDAC. And it has phoma, childhood neuroblastoma and gas- the highest level of inhibition for HDAC8 tric, colon, lung, breast cancers. In addition, (Cummings and Macfarlane, 1991). Hence, HDAC8 shows functional abundance with the production of butyrate in the colon may HDAC6 when overexpressed in Hela and protect against colon cancer and inhibit the cervical cancer cells (Vanaja et al., 2018). As development of colon cancer (Emenaker et a result, HDAC8 can potentially be an attrac- al., 2001; He et al., 2001). As a result of such tive target for structure-based drug design findings, identifying the chemical properties because it exhibits a number of unique char- that govern the action of NaB on HDAC8 acteristics allowing the development of se- could be significant for the development of lective inhibitors. One particularly interest- new selective drugs with low side effects. It ing feature in this regard is the formation of is also known that logical drug design in- the foot pocket also called the acetate release volves investigating new molecules that complement the molecular target in struc- channel, which is a sub-pocket observed in class I HDACs, such as HDAC1, 2, 3 and 8, ture, aiming to reach an allosteric site or the but not in class II isotypes such as, HDAC4 active site (Bieliauskas and Pflum, 2008; Estiu et al., 2008, 2010; Nussinov and Tsai, and 7 X-ray structures (Hildmann et al., 2007). HDACs are regulators of gene ex- 2012). Moreover, molecular docking can be pression, that affect cell cycle arrest, apopto- defined as a simulation process where a lig- sis, angiogenesis, and the differentiation of and position is estimated at a predicted or different cell types (Hildmann et al., 2007; pre-defined binding site. This process can be Riester et al., 2007). Moreover, abnormal used widely; with minimal effort and ex- histone modification patterns have been pense, and it may increase the effectiveness found to be closely associated with several of obtaining potential drugs. Through mo- diseases such as cancers (Ma et al., 2019). lecular docking, we aim to clarify the benefi- According to the Human Protein Atlas cial HDAC8 structural features which may (2019), these types of class I HDACs are ex- help to obtain knowledge for drug develop- 1039 EXCLI Journal 2020;19:1038-1051 – ISSN 1611-2156 Received: January 30, 2020, accepted: July 09, 2020, published: July 23, 2020 ment in the future using some known selec- study between NaB and LHX1 as a tran- tive HDACis (Knegtel et al., 1997; Alonso et scription factor of HDAC8 was done in this al., 2006). study. The LHX1 protein is one of the transcrip- tion factors involved in the expression of the MATERIALS AND METHODS HDAC8 gene. The LIM homeobox 1 protein Cell culture of the human cell is encoded by the LHX1 HCT-116 (the human colorectal carci- gene. This protein is a transcription factor, noma) and HT-29 (the colorectal adenocar- important for control of the development and cinoma) cell lines were obtained from the differentiation of neural, lymphoid, renal and Iranian Biological Research Center (Pastor, urogenital cells. The LHX protein plays a Tehran, Iran). HCT-116 and HT-29 cell lines role in determining the origin and identity of were cultured in Dulbecco's modified Eagle's the cell. In humans, the encoding genome is medium (DMEM) and RPMI 1640, respec- located on chromosome 17q11.2-q12. Its tively, ( Gibco by life technologies, Germa- molecular weight is 44.808 Dalton and it ny) including 10 % fetal bovine serum (FBS; contains 406 amino acids. LHX1 binds to Gibco by life technologies, Germany) and DNA through its homeodomains and is an 1 % penicillin/streptomycin (Dacell, Iran) at important evolutionary regulator. The LHX1 37 °C and 5 % CO2. The cells were grown to protein contains 3 domains, which include: approximately 80 % confluence. A homeobox domain for DNA binding, and two LIM domains, which are involved in the Cell treatment and cytotoxicity assay ion-zinc connection. The LIM domains in- CRC cell lines were plated at 50×103 volved in zinc ion interconnection, play a cells per well in a 200 μL volume in 96-well role in cytoskeleton organization, cell adhe- plates. CRC cell lines were treated with the sion, epithelial development, gene transcrip- medium containing 6.25 to 200 mM of NaB, tion, mRNA trafficking, translation, protein (Biobasic, Canada Inc.) according to our folding, and chromatin remodeling. In addi- previous study, (Ghiaghi et al., 2019) for 24 tion, LIM domains have seven conserved and 48 hours.