Cytotoxic Effects of Peanut Phenolic Compounds Possessing Histone Deacetylase Inhibitory Activity on Human Colon Cancer Cell Lines
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Turkish Journal of Biology Turk J Biol (2016) 40: 1258-1271 http://journals.tubitak.gov.tr/biology/ © TÜBİTAK Research Article doi:10.3906/biy-1601-23 Cytotoxic effects of peanut phenolic compounds possessing histone deacetylase inhibitory activity on human colon cancer cell lines 1 2 2 1,3, Somprasong SAENGLEE , Sanun JOGLOY , Aran PATANOTHAI , Thanaset SENAWONG * 1 Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand 2 Department of Plant Science and Agricultural Resources, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand 3 Natural Product Research Unit, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand Received: 08.01.2016 Accepted/Published Online: 08.04.2016 Final Version: 16.12.2016 Abstract: Phenolic compounds present in our diet play an important role in colon cancer chemoprevention. Previous results demonstrated that peanut testa extract inhibited both histone deacetylase (HDAC) activity and the growth of colon cancer cells. In this study, four identified phenolic compounds in peanut testae (resveratrol, p-coumaric acid, ferulic acid, and sinapinic acid) were investigated for their HDAC inhibitory and anticancer activities against colon cancer cell lines. In vitro study revealed that resveratrol exhibited the greatest HDAC inhibitory activity. Molecular docking studies demonstrated that all four compounds could bind both HDAC1 and HDAC2. Resveratrol exhibited the most effective antiproliferative activity against both human colon adenocarcinoma (HT29) and human colorectal carcinoma (HCT116) cells. Apoptosis induction by ferulic acid and resveratrol appeared to be associated with p53 activation in HCT116 cells. However, resveratrol, p-coumaric acid, ferulic acid, and sinapinic acid induced apoptosis of HT29 cells in a p53-independent manner. Low-concentration treatments of p-coumaric and ferulic acids resulted in cell cycle arrest of HCT116 cells. In contrast, high-concentration treatments of p-coumaric and ferulic acids showed cell death activation as evidenced by increased sub-G1 fractions. The induction of p21 by p-coumaric acid and resveratrol correlated well with the decreased CDK4 levels and cell cycle arrest. Resveratrol, p-coumaric acid, ferulic acid, and sinapinic acid caused activation of pERK1/2 in HCT116 cells, whereas ferulic and sinapinic acids caused downregulation of pERK1/2 in HT29 cells. These results suggest that these peanut phenolics may be potential antineoplastic agents for colon cancer chemoprevention/chemotherapy. Key words: HDAC inhibitor, peanut (Arachis hypogaea L.), p-coumaric acid, ferulic acid, sinapinic acid, resveratrol 1. Introduction (Udenigwe et al., 2008; Khaopha et al., 2012). Peanut testae Colon cancer, one of the most common cancers in have been shown to possess potent antioxidant, anticancer, developed countries, is closely linked with dietary and and antiinflammatory activities (Chang et al., 2006). We lifestyle practices (Jemal et al., 2010). Dietary intake of have previously demonstrated that phenolic-rich extracts plant-derived phenolic-rich ingredients might be a good of peanut testae exhibited histone deacetylase (HDAC) and feasible strategy for preventing and controlling the inhibitory and anticancer activities in several human incidence of colorectal cancer (Center et al., 2009). Phenolic cancer cells (Khaopha et al., 2015). However, the types of compounds are the most abundant antioxidants among the phenolic compounds conferring HDAC inhibitory and bioactive phytochemicals present in our diet (Edwards et anticancer properties to peanut testa extracts remains to al., 2010), serving as natural cancer chemopreventive agents be investigated. Our previous study (Khaopha et al., 2015) (Link et al., 2010). Generally, the underlying mechanisms demonstrated that peanut testa extracts possessing HDAC for their anticancer effects include antioxidation, inhibitory activity could inhibit the growth of human antiproliferation, and the activation and/or inhibition of colon adenocarcinoma (HT29) cells and human colorectal some subcellular signaling pathways, namely apoptosis and carcinoma (HCT116) cells. However, the cytotoxic effects of cell cycle progression (Yang et al., 2001). Among plant- the phenolics found in peanut testa extracts on colon cancer based diets, peanut seeds (Arachis hypogaea L) are popularly cell lines HT29 (p53-mutant) and HCT116 (p53-wild type) consumed worldwide and the peanut testa has been found have not yet been explored. to contain several types of phenolic compounds, including HDAC inhibitors have been shown to cause resveratrol, p-coumaric acid, ferulic acid, and sinapinic acid transcriptional activation of a few silenced tumor * Correspondence: [email protected] 1258 SAENGLEE et al. / Turk J Biol suppressor genes by reversible epigenetic changes (Marks Antibodies against H3, p21, CDK4, β-actin, pERK1/2, et al., 2000; Kim et al., 2013). At present, there remains a and ERK1/2 proteins were purchased from Cell Signaling continuous need for exploring new HDAC inhibitors with Technology. Anti-p53 antibody was purchased from Santa improved pharmacological efficacy and unique modes of Cruz Biotechnology. The Vybrant Apoptosis Assay Kit action. The loss of protein expression involved in growth #2 and MTT were purchased from Molecular Probes, inhibition and induction of apoptosis is one of the most Invitrogen. common reasons for uncontrolled growth of cancer cells 2.2. Cell culture (Roberts et al., 2002). Tumor suppressor protein p53 plays The colon cancer cell lines (HT29 and HCT116) and a key role in preventing tumor cell growth (Banin et al., noncancer cell lines (Vero) used in this study were 1998), whereas cyclins and cyclin-dependent kinases maintained in RPMI-1640 medium supplemented with (CDKs) regulate cell cycle progression. The induction 10% fetal bovine serum, penicillin (100 U/mL), and of p21, a CDK inhibitor, is responsible for arresting cell streptomycin (100 µg/mL) (GIBCO-BRL). The cells were cycle progression via p53-dependent and -independent incubated in a humidified incubator with an atmosphere pathways (Gartel, 2005). Decreased CDK4 expression and of 5% CO2 at 37 °C. WAF1 increased p21 expressionwere associated with cell cycle 2.3. In vitro HDAC inhibitory activity arrest (Cho et al., 2001; Sherr et al., 2002). Moreover, a In vitro HDAC inhibitory activity was determined mitogen-activated protein kinase (MAPK) or extracellular using the Fluor-de-Lys HDAC Fluorometric Activity signal-regulated kinase 1/2 (ERK1/2) signaling cascade Assay Kit (Biomol). Four peanut phenolics, resveratrol, is of great importance in regulating various cellular p-coumaric acid, ferulic acid, and sinapinic acid, at various responses, including proliferation, differentiation, survival, concentrations, were assessed for their HDAC inhibitory and cell death. The cascade is activated through receptor- activity against the mixture of HDAC enzymes from the mediated signaling stimuli (Mordret, 1993). The ERK HeLa nuclear extract provided with the kit. The assay was signaling is synonymous with cell proliferation and found performed according to the manufacturer’s instructions. to be deregulated in about one-third of all human cancers Fluorescence was measured using a Spectra Max Gemini (Dhillon et al., 2007). In the ERK/MAPK model, ERK1/2 XPS microplate spectrofluorometer (Molecular Devices) is activated upon phosphorylation by MEK1/2, which is with excitation at 360 nm and emission at 460 nm. itself activated when phosphorylated by Raf (Johnson 2.4. In silico molecular docking studies et al., 1996). Cell death could be induced through ERK AutoDock tools were used to analyze the binding energy activation by the generation of reactive oxygen species (ΔG) and inhibitory constant (Ki). The Molecular Graphics (ROS) and endoplasmic reticulum stress predominantly Laboratory tools and AutoDock 4.2 were downloaded via the PERK-eIF2α pathway (Arai et al., 2004). However, from www.scripps.edu. Cygwin (for data storage) and the mechanisms used by peanut phenolic compounds to Discovery Studio Visualizer 4.0 were downloaded from inhibit cancer cell growth remain to be explored. www.cygwin.com and www.accelerys.com, respectively. The investigation of HDAC inhibitory activity of The chemical structure of the phenolic compounds was phenolic compounds found in peanut seeds may lead to drawn in the form of a chemical diagram using Spartan the discovery of safe and potentially chemopreventive Student 4.1.1. The human HDAC1 and HDAC2 crystal natural agents. To validate that peanuts are a natural structures were collected from a databank (http://www. source of HDAC inhibitors, the HDAC inhibitory activity rcsb.org/pdb/staticHelp.do?p=help/ligandSearch.html). of peanut phenolic compounds, including resveratrol, For each ligand, the 10 best positions were generated and p-coumaric acid, ferulic acid, and sinapinic acid, was scored using the AutoDock 4.2 scoring functions according demonstrated both in vitro and in cancer cell lines. The to methods previously described (Park et al., 2006). The effects of these phenolic compounds on the proliferation, conformations with the best docking, as determined from apoptosis, and cell cycle progression of colon cancer cells the clustering histogram, were those with low binding were demonstrated. In addition, the levels of p21, CDK4, energy. Binding energy is a measure of the affinity of a p53, and pERK1/2 upon treatment with these phenolic ligand–protein complex, or the difference between