Cytotoxic Effects of Peanut Phenolic Compounds Possessing Histone Deacetylase Inhibitory Activity on Human Colon Cancer Cell Lines

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 the compounds were also investigated. energy of the complex and the sum of the energies of each molecule separately. The binding energy of the individual 2. Materials and methods compound was calculated using the following formula: 2.1. Materials binding energy = A + B + C – D, Propidium iodide, sodium butyrate (purity ≥98.5%), where A denotes the final intermolecular energy + Van der resveratrol (purity ≥99.0%), p-coumaric acid (purity Waals energy + hydrogen bonds + desolvation energy + ≥98.8%), ferulic acid (purity ≥99.0%), and sinapinic acid electrostatic energy (kcal/mol), B denotes the final total (purity ≥98.0%) were purchased from Sigma-Aldrich. internal energy (kcal/mol), C denotes the torsional free

1259 SAENGLEE et al. / Turk J Biol energy (kcal/mol), and D denotes the unbound system 2.8. Western blot analysis energy (kcal/mol). The inhibitory constant (Ki) is the Levels of the proteins of interest were detected by western concentration of the inhibitor required to produce half of blot following the established method. The total cellular the maximum inhibition. proteins were prepared from drug-treated and solvent- 2.5. Cell growth inhibition treated cells using RIPA lysis buffer. Equal amounts of Cell growth inhibition was measured by MTT assay. protein (30 µg) were separated by 10%–12.5% SDS-PAGE Briefly, colon cancer (HT29 and HCT116) and noncancer and transferred to a polyvinylidene fluoride (PVDF) (Vero) cell lines were seeded in 96-well plates at a density membrane. The membrane was then blocked with 5% of 8 × 103 cells per well for 24 h. Cells were treated with skim milk for 1 h and subsequently incubated overnight resveratrol (0–200 µM), p-coumaric acid (0–3 mM), with a primary antibody: anti-p21, antiacetylated H3, ferulic acid (0–3 mM), sinapinic acid (0–3 mM), and NaB anti-CDK4, anti-pERK1/2, anti-ERK1/2, anti-p53, or anti- (0–6 mM) and incubated for 24, 48, and 72 h. Dimethyl β-actin. The proteins were then probed with a horseradish sulfoxide (DMSO) was used at 0.5% as the vehicle control. peroxidase-conjugated secondary antibody at room The culture medium was then replaced with fresh medium temperature. Protein bands were visualized using ECL containing 1.2 mM MTT and incubated for 2 h at 37 °C. prime chemiluminescence (Amersham). After 2 h of incubation, formazan produced in the culture 2.9 Statistical analysis cells was dissolved with DMSO, and the absorbance at 550 Data are expressed as the mean ± standard deviation (SD) nm was measured with a microtiter plate reader (Bio-Rad from three independent experiments. Duncan’s post hoc Laboratories) using 655 nm as a reference wavelength. test was carried out using SPSS 20.0 (IBM Corporation). 2.6. Apoptosis analysis by flow cytometry Significant differences (P < 0.05) among phenolic Induction of apoptosis was assayed using the Vybrant compounds were analyzed using one-way analysis of Apoptosis Assay Kit #2 (Molecular Probes, Invitrogen) variance. according to the manufacturer’s instructions with a BD FACSCanto II flow cytometer (Becton Dickinson). To 3. Results start, cells (~1 × 106) were seeded into a 5.5-cm dish and 3.1. HDAC inhibitory activity of four peanut phenolic incubated for 24 h. The cells were then treated with three compounds different concentrations of resveratrol (50, 100, and 150 The HDAC inhibitory activity of four peanut phenolic µM), p-coumaric acid (1, 2.5, and 5 mM), ferulic acid (1, compounds (Udenigwe et al., 2008; Khaopha et al., 2012) 2.5, and 5 mM), and sinapinic acid (1, 2.5, and 5 mM) or was investigated both in vitro and in silico in comparison solvent (DMSO, 0.37%) for 24 h. After incubation, the with a well-known HDAC inhibitor, sodium butyrate treated cells were harvested, centrifuged, and washed (NaB). As shown in Table 1, all four phenolic compounds twice with ice-cold PBS. Cells were suspended in 100 µL could inhibit HDAC activity in vitro and resveratrol had ice-cold annexin-binding buffer and stained with Alexa the lowest IC50 value (0.4 ± 0.05 mM) against the crude Fluor 488-annexin V and propidium iodide (PI) solutions. HDAC enzymes from the HeLa nuclear extract, followed The samples were then kept in the dark for 15 min at room by ferulic acid (1.9 ± 0.1 mM), sinapinic acid (2.3 ± 0.1 temperature and analyzed by flow cytometry. mM), and p-coumaric acid (2.6 ± 0.1 mM). Compared to

2.7. Cell cycle analysis by flow cytometry the IC50 value of NaB (1.02 ± 0.1 mM), only resveratrol Cell cycle phase distribution was evaluated using a method possessed more potent HDAC inhibitory activity than NaB as previously described by Poolman and Brooks (1998). in vitro. To support the in vitro results, an in silico study or Cells were seeded into a 5.5-cm dish (~1 × 106 cells/ molecular docking was conducted using these compounds dish), incubated for 24 h, and treated with three different as ligand molecules docking with crystal structures of concentrations of resveratrol (50, 100, and 150 µM), both human HDAC1 and HDAC2, the two major HDAC p-coumaric acid (1, 2.5, and 5 mM), ferulic acid (1, 2.5, and family members found in most transcriptional repression 5 mM), and sinapinic acid (1, 2.5, and 5 mM) or solvent complexes (Hassig et al., 1998). The results revealed that (DMSO, 0.37%) for 24 h. The cells were then harvested, all of the inhibitor compounds bind with one or more centrifuged, and fixed in ice-cold 70% ethanol. The fixed amino acids in the active pockets of the enzymes (data cells were again centrifuged and resuspended in 300 µL not shown). The calculated binding energy (ΔG) and of PBS. The cells were incubated with RNase A (final inhibitory constant (Ki) of the individual compounds concentration of 0.2 mg/mL) at 37 °C for 1 h and stained are shown in Table 1. Theoretically, all four phenolic with a propidium iodide solution (final concentration of 50 compounds showed very good binding energy compared µg/mL) at room temperature for 45 min. The labeled cells to that of the well-known HDAC inhibitor NaB. According were analyzed using a BD FACSCanto II flow cytometer to the binding energy and inhibitory constant, all four (Becton Dickinson). phenolic compounds (ΔG = –3.4 to –4.9 kcal/mol, Ki =

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Table 1. Half maximal inhibitory concentrations (IC50) of phenolic compounds after exposure to HDAC enzymes in vitro and their binding energy and inhibitory constants against HDAC1 and HDAC2 in silico.

In vitro In silico HDACsa HDAC1 HDAC2 Compounds Chemical structure Binding Inhibitory Binding Inhibitory IC valuesb 50 energy (ΔG) constant energy (ΔG) constant (mM) (kcal/mol) (Ki) (kcal/mol) (Ki)

p-Coumaric acid 2.6 ± 0.1 –3.5 2.9 mM –5.1 170.8 µM

Ferulic acid 1.9 ± 0.1 –4.3 673.3 µM –4.6 451.8 µM

Sinapinic acid 2.3 ± 0.1 –3.4 3.3 mM –3.9 1.5 mM

Resveratrol 0.4 ± 0.1 –4.9 271.6 µM –4.8 328.7 µM

Sodium butyratec 1.0 ± 0.1 –2.9 8.0 mM –4.7 375.3 µM a The mixture of HDAC enzymes in the HeLa nuclear extract. b IC50 values are shown as mean ± SD from three independent experiments, representing the concentrations of the indicated compounds that inhibit 50% of HDAC activity. cA well-known HDAC inhibitor.

0.3 to 3.3 mM) appeared to interact with HDAC1 more acids exhibited similar IC50 values against HT29, HCT116, efficiently than NaB (ΔG = –2.9 kcal/mol, Ki = 8.0 mM). In and Vero cells at 72 h of exposure. In line with our findings, contrast, only p-coumaric acid (ΔG = –5.1 kcal/mol, Ki = previous reports documented the growth inhibition effects 170.8 mM) showed a better interaction with HDAC2 than of plant phenolics on colon cancer cells (Yi et al., 2005). NaB (ΔG = –4.7 kcal/mol, Ki = 375.3 µM). These results In addition, p-coumaric, ferulic, and sinapinic acids were indicate that all four phenolic compounds found in peanut more cytotoxic than NaB. However, it has been reported testa extracts (Khaopha et al., 2015) function as HDAC that sinapinic acid inhibited the growth of HeLa and Jurkat inhibitors capable of inhibiting HDAC enzymes. cells less effectively than sodium butyrate (Senawong et al., 3.2. Antiproliferative activity of four peanut phenolic 2013). compounds 3.3. Hyperacetylation of histone H3 by peanut phenolic To determine the antiproliferative activity of these compounds in colon cancer cells compounds, the MTT assay was used to evaluate their As presented above, we observed that all four phenolic growth inhibition activity against human colon cancer compounds possessed in vitro HDAC inhibitory activity cell lines HT29 and HCT116 and noncancer (Vero) cells. and inhibited the growth of both colon cancer cell lines. A dose-response curve showed that all four phenolic Therefore, we aimed to explore the HDAC inhibitory compounds inhibited the proliferation of colon cancer cell activity of these compounds in both cancer cell lines by lines in a time-dependent and dose-dependent manner determining the acetylation status of histone H3 using (data not shown). Cellular sensitivity was determined western blot analysis. As demonstrated in Figure 1, all by calculating the IC50 against each compound (Table 2). four phenolic compounds, p-coumaric acid (Figures 1A Resveratrol produced a more profound cytotoxic effect and 1B), ferulic acid (Figures 1C and 1D), sinapinic acid in colon cancer cells. Consistent with our previous report (Figures 1E and 1F), and resveratrol (Figures 1G and 1H), (Senawong et al., 2014), p-coumaric, ferulic, and sinapinic caused hyperacetylation of histone H3 in both HT29 and

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Table 2. Antiproliferative activity of the four phenolic compounds represented by IC50 values after exposures to colon cancer cell lines for 24, 48, and 72 h.

a IC50 values Phenolic compounds HT29 cells HCT116 cells Vero cellsb

24 h 48 h 72 h 24 h 48 h 72 h 24 h 48 h 72 h

p-Coumaric acid (mM) >3 1.3 ± 0.2 1.0 ± 0.2 >3 2.4 ± 0.3 1.3 ± 0.8 >3 >3 2.9 ± 0.7

Ferulic acid (mM) >3 2.4 ± 0.1 2.2 ± 0.2 >3 2.5 ± 0.9 2.1 ± 0.4 >3 >3 2.8 ± 0.6

Sinapinic acid (mM) >3 2.7 ± 0.3 2.6 ± 0.4 >3 3.0 ± 0.5 2.7 ± 0.1 >3 >3 >3

Resveratrol (µM) >200 131.2 ± 9.8 99.8 ± 8.7 190.3 ± 1.2 129.6 ± 6.3 99.8 ± 12.2 >200 193.4 ± 8.8 166.3 ± 0.8

Sodium butyratec (mM) >6 5.4 ± 0.7 3.7 ± 0.2 >6 4.5 ± 0.7 3.8 ± 0.3 >6 5.0 ± 0.5 4.1 ± 0.5

a IC50 values or half maximal inhibitory concentrations are shown as means ± SD from three independent experiments. bNoncancer cell line. cA well-known HDAC inhibitor.

HCT116 cells, indicating that all four compounds could decrease of the G0/G1 population when treated with inhibit HDAC activity in a cellular context compared with resveratrol, p-coumaric acid, ferulic acid, and sinapinic the HDAC inhibitor NaB (Figures 1I and 1J). Among the acid. NaB caused an increase of cells in the S phase but four compounds, resveratrol was shown to possess the most a decrease in the G2/M phase. In contrast, the HCT116 potent HDAC inhibitory activity in both HT29 (Figure cells were arrested in the G0/G1 phase after treatment with 1G) and HCT116 (Figure 1H) cells, which correlates well p-coumaric acid, ferulic acid, and sinapinic acid, whereas with its activity in vitro (Table 1). an S phase arrest was observed with the resveratrol 3.4 Peanut phenolic compounds induce apoptosis in treatment. Of note, the NaB-treated HCT116 cells showed human colon cancer cell lines a prominent sub-G1 fraction. To elucidate the mechanism underlying cell growth 3.6. Peanut phenolic compounds induced inhibition of inhibition by the peanut phenolic compounds, induction ERK1/2 pathway of apoptosis in colon cancer cell lines was examined. As The effects of the phenolic compounds on the MAPK (ERK) shown in Figure 2, all four phenolic compounds exhibited signaling pathway of colon cancer cells were investigated a significant effect on induction of apoptosis in both HT29 by western blot analysis. p-Coumaric acid caused an and HCT116 cells. In the HT29 cells, treatments with increase in the level of pERK1/2 in both the HT29 (Figure increasing concentrations of p-coumaric acid (Figure 3A) and the HCT116 (Figure 3B) cells. In contrast, ferulic 2A), ferulic acid (Figure 2B), sinapinic acid (Figure 2C), acid caused an inhibition of the MAPK signaling pathway resveratrol (Figure 2D), and NaB (Figure 2E) increased in the HT29 cells (Figure 3C), but activated the MAPK the percentage of apoptotic cells. Similarly, the percentage signaling pathway in the HCT116 cells (Figure 3D). of HCT116 apoptotic cells also increased after treatment Similarly, sinapinic acid decreased the pERK1/2 level in the with these phenolic compounds (Figures 2F–2I) and HT29 cells (Figure 3E), but increased the pERK1/2 level in NaB (Figure 2J). In addition, the percentage of apoptotic the HCT116 cells (Figure 3F). Resveratrol increased the noncancer cells was lower than that of apoptotic cancerous level of pERK1/2 at all concentrations tested in the HT29 cells, indicating that the peanut phenolic compounds cells (Figure 3G), and at the highest concentration (200 (Figures 2K–2N) were less toxic to a noncancer cell line µM) in the HCT116 cells (Figure 3H). Furthermore, the than to cancerous cell lines. In contrast, NaB exhibited NaB treatments caused a reduction of pERK1/2 in both more toxicity to a noncancer cell line (Figure 2O) than to the HT29 (Figure 3I) and the HCT116 (Figure 3J) cells, cancerous cell lines (Figures 2E and 2J). especially at high concentration treatments. 3.5. Peanut phenolic compounds induce cell cycle arrest 3.7. Western blot analysis of cell cycle and apoptosis in human colon cancer cell lines regulatory proteins Cell cycle arrest was further examined by flow cytometry To explore the molecular mechanisms underlying growth after treatment with the phenolic compounds. As presented inhibition of colon cancer cells, HT29 (p53-mutant) and in Table 3, the HT29 cells showed a significant increase HCT116 (p53-wild type), by peanut phenolic compounds, in the number of sub-G1 cells along with concomitant the expression of some cell cycle and apoptosis regulatory

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HT29 HCT116 A B Ac-H3 Ac-H3 -actin -actin 3 3 ease) 2 ease) 2 1 1 Ac-H3 0 Ac-H3 0 C 1.0 2.5 5.0 C 1.0 2.5 5.0 (fold inc r (fold inc r p-Coumaric acid (mM) p-Coumaric acid (mM) C D Ac-H3 Ac-H3 -actin -actin 3 2 ease) 2 ease) 1 1 Ac-H3 0 Ac-H3 0 (fold inc r

C 1.0 2.5 5.0 (fold inc r C 1.0 2.5 5.0 Ferulic acid (mM) Ferulic acid (mM) E F Ac-H3 Ac-H3 -actin -actin 2 3 ease) ease) 1 2 1 Ac-H3 0 Ac-H3 0 C 1.0 2.5 5.0 C 1.0 2.5 5.0 (fold inc r (fold inc r Sinapinic acid (mM) Sinapinic acid (mM) G H Ac-H3 Ac-H3 -actin -actin 2 3 ease) ease) 2 1 1 Ac-H3 Ac-H3 0 0 (fold inc r (fold inc r C 50 100 150 C 50 100 150 Resveratrol (μM) Resveratrol (μM) I J Ac-H3 Ac-H3 -actin -actin 4 6 ease) ease) 3 4 2 2

1 Ac-H3 Ac-H3 0 0

C 1.0 2.5 5.0 (fold inc r C 1.0 2.5 5.0 (fold inc r NaB (mM) NaB (mM) Figure 1. Hyperacetylation of histone H3 by peanut phenolic compounds and NaB. Acetylated histone-H3 levels increased in HT29 (A, C, E, G, and I) and HCT116 (B, D, F, H, and J) cells after exposure to varying concentrations of phenolic compounds for 24 h. The control (C) represents the level of acetylated (Ac)-H3 in the solvent control treatment. β-actin was used as a loading control. Quantification of the relative expression of Ac-H3 in HT29 and HCT116 cells was determined using Genetool software (Genesys).

1263 SAENGLEE et al. / Turk J Biol * * * 5.0 5.0 5.0 * * * 2.5 2.5 2.5 NaB (mM) NaB (mM) NaB (mM) * 1.0 1.0 1.0 Early apoptosis Late apoptosis Late apoptosis Early apoptosis Early apoptosis Late apoptosis

P P P S J S S E O * μ M ) μ M ) * * μ M ) 200 200 200 ol ( ol ( ol ( * * * 150 150 150 * * 100 100 100 Resverat r Resverat r Resverat r Late apoptosis Early apoptosis Late apoptosis Early apoptosis Early apoptosis Late apoptosis

P P P S S I D S N * * 5.0 5.0 5.0 * * 2.5 2.5 2.5 * 1.0 1.0 1.0 Sinapinic acid (mM) Early apoptosis Late apoptosis Early apoptosis Sinapinic acid (mM) Late apoptosis Early apoptosis Late apoptosis Sinapinic acid (mM)

P P P S C S S M H * * * 5.0 5.0 5.0 -coumaric acid (A, F, and and acid (A, F, p -coumaric 24 h with for incubated were cells (Vero) noncancer and HT29, HCT116, * * * 2.5 2.5 2.5 * * * 1.0 1.0 1.0 Ferulic acid (mM) Ferulic acid (mM) Ferulic acid (mM) Late apoptosis Early apoptosis Late apoptosis Early apoptosis

Early apoptosis Late apoptosis P P P

S S S L B G * * * 5.0 5.0 5.0 * * 2.5 2.5 2.5 * * * * 1.0 1.0 1.0 Early apoptosis Early apoptosis p - Coumaric acid (mM)

Late apoptosis Late apoptosis

Early apoptosis Late apoptosis p - Coumaric acid (mM) p - Coumaric acid (mM) P P P

S S S F K A 0

60 50 40 30 20 10

60 50 40 30 20 10 60 50 40 30 20 10 Number of apoptotic cells (%) (%) cells apoptotic of Number Number of apoptotic cells (%) (%) cells apoptotic of Number Number of apoptotic cells (%) (%) cells apoptotic of Number 16 ero V HT29 HCT 1 K), ferulic acid (B, G, and L), sinapinic acid (C, H, and M), resveratrol (D, I, and N), and the well-known HDAC inhibitor NaB (E, J, and O). S and P represent solvent (DMSO) and and (DMSO) solvent P represent O). S and and (E, J, NaB inhibitor HDAC the well-known N), and I, and (D, M), resveratrol acid (C, H, and L), sinapinic G, and K), ferulic acid (B, (P < 0.05). treatment and control betweensolvent difference *: Significant respectively. treatments, camptothecin induction. apoptosis on compounds phenolic of effect 2. The peanut Figure

1264 SAENGLEE et al. / Turk J Biol 10.4 ± 0.8 16.8 ± 0.8 28.3 ± 0.4 4.7 ± 0.1 48.5 ± 0.3 31.0 ± 0.7 12.3 ± 0.1 47.0 ± 1.8 5.0 5.0 10.9 ± 0.9 17.6 ± 1.0 27.8 ± 0.6 5.4 ± 0.1 48.2 ± 0.3 40.4 ± 0.6 12.6 ± 0.0 35.9 ± 1.9 2.5 2.5 21.9 ± 0.9 13.8 ± 0.8 17.0 ± 0.6 5.2 ± 0.2 51.0 ± 0.2 64.6 ± 0.6 9.5 ± 0.1 16.0 ± 1.6 1.0 1.0 NaB (mM) NaB (mM) NaB 18.4 ± 1.3 10.0 ± 3.1 22.5 ± 5.5 14.4 ± 2.1 18.4 ± 1.3 24.5 ± 5.1 26.6 ± 1.0 49.1 ± 7.6 22.5 ± 5.5 26.6 ± 7.4 26.6 ± 1.0 29.8 ± 7.2 31.7 ± 2.8 14.0 ± 1.5 20.3 ± 5.7 15.8 ± 6.4 5.0 5.0 150 150 23.2 ± 3.9 10.9 ± 1.4 19.7 ± 5.1 16.5 ± 2.5 23.2 ± 4.0 26.3 ± 9.3 39.8 ± 7.1 68.9 ± 1.7 19.7 ± 5.1 11.8 ± 5.7 39.8 ± 7.0 36.8 ± 3.8 15.9 ± 1.8 2.8 ± 0.1 34.6 ± 4.3 23.2 ± 6.1 2.5 2.5 100 100 32.1 ± 1.9 15.7 ± 0.6 21.6 ± 1.3 14.2 ± 0.8 32.1 ± 1.9 25.5 ± 0.4 28.1 ± 8.8 65.5 ± 2.8 21.6 ± 1.1 19.9 ± 8.0 28.1 ± 8.7 36.3 ± 5.5 15.8 ± 0.3 2.1 ± 0.3 19.9 ± 6.1 16.6 ± 4.7 1.0 1 50 50 Ferulic acid (mM) Ferulic acid (mM) Ferulic Resveratrol (µM) Resveratrol (µM) Resveratrol 25.5 ± 4.7 17.1 ± 0.3 10.0 ± 2.7 15.2 ± 5.6 31.6 ± 7.1 17.4 ± 6.1 35.3 ± 10.1 53.5 ± 0.7 21.9 ± 5.7 10.0 ± 2.7 35.3 ± 9.9 80.6 ± 4.2 28.1 ± 2.1 1.7 ± 0.2 11.5 ± 1.3 6.9 ± 6.5 5.0 5 5.0 5.0 30.0 ± 9.3 18.2 ± 0.3 19.6 ± 1.3 17.1 ± 2.2 25.1 ± 6.3 16.4 ± 1.8 28.4 ± 1.3 67.7 ± 3.3 19.9 ± 4.9 19.6 ± 1.3 28.4 ± 1.3 78.2 ± 2.9 20.6 ± 6.0 1.8 ± 0.2 5.5 ± 0.5 3.5 ± 2.8 2.5 2.5 2.5 2.5 a a a a HT-29 cells HT-29 cells HCT-116 18.4 ± 5.1 16.2 ± 1.3 18.5 ± 1.0 16.7 ± 3.6 19.7 ± 2.4 16.5 ± 7.8 45.4 ± 1.6 67.3 ± 0.1 15.1 ± 6.3 18.5 ± 1.0 45.4 ± 1.6 65.5 ± 4.7 15.3 ± 4.3 1.9 ± 0.2 4.4 ± 0.3 3.2 ± 1.7 HT-29 cells HT-29 acid (mM) p -Coumaric cells HCT-116 acid (mM) p -Coumaric 1.0 1.0 Sinapinic acid (mM) Sinapinic acid (mM) Sinapinic 1.0 1.0 b b 9.1 ± 2.1 17.3 ± 0.1 Control 16.1 ± 1.7 17.8 ± 4.1 66.0 ± 2.5 57.7 ± 0.2 7.6 ± 2.4 1.9 ± 0.2 Control . Analysis of the cell cycle distribution of the colon cancer cell lines treated with different phenolic compounds for 24 h. for compounds phenolic different with treated cell lines cancer the colon of the cell cycle distribution of . Analysis Cell cycle phase Cell cycle phase S S Cell cycle phase G2/M G2/M S S G0/G1 G0/G1 G2/M G2/M G0/G1 G0/G1 Sub-G1 Sub-G1 Sub-G1 Sub-G1 Cell cycle phase Cells were treated with a solvent. with treated Cells were Cell numbers are presented as a percentage of the total analyzed cells and the values are shown as mean ± SD from three independent experiments. experiments. independent three from ± SD mean as shown are the values and cells analyzed the total of a percentage as presented are Cell numbers Table 3 a b

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HT29 HCT116 A B pERK1/2 pERK1/2 Total ERK1/2 Total ERK1/2 3 3 ease) 2 ease) 2 1 1 pERK1/2 pERK1/2 (fold inc r 0 (fold inc r 0 p-Coumaric acid - - 2 3 4 5 (mM) p-Coumaric acid - - 2 3 4 5 (mM) EGF - + + + + + EGF - + + + + + C D

pERK1/2 pERK1/2 Total ERK1/2 Total ERK1/2 2 3 ease) ease) 2 1 1 pERK1/2 pERK1/2 (fold inc r

0 (fold inc r 0 Ferulic acid - - 2 3 4 5 (mM) Ferulic acid - - 2 3 4 5 (mM) EGF - + + + + + EGF - + + + + + E F

pERK1/2 pERK1/2 Total ERK1/2 Total ERK1/2 2 3 ease) ease) 2 1 1 pERK1/2 pERK1/2 (fold inc r (fold inc r 0 0 Sinapinic acid - - 2 3 4 5 (mM) Sinapinic acid - - 2 3 4 5 (mM) EGF - + + + + + EGF - + + + + +

G H pERK1/2 pERK1/2 Total ERK1/2 Total ERK1/2 3 3 ease) ease) 2 2

1 1 pERK1/2 pERK1/2 (fold inc r (fold inc r 0 0 Resveratrol - - 50 100 150 200 (μM) Resveratrol - - 50 100 150 200 (μM) - EGF + + + + + EGF - + + + + + I J pERK1/2 pERK1/2 Total ERK1/2 Total ERK1/2 2 2 pERK1/2 ease) ease) 1 1 pERK1/2 pERK1/2 (fold inc r

(fold inc r 0 0 NaB - - 2 3 4 5 (mM) NaB - - 2 3 4 5 (mM) EGF - + + + + + EGF - + + + + +

Figure 3. The effect of peanut phenolic compounds on ERK1/2 activation. HT29 and HCT116 cells were exposed to 0.37% DMSO (solvent control) and varied concentrations of p-coumaric acid (A and B), ferulic acid (C and D), sinapinic acid (E and F), resveratrol (G and H), and NaB (I and J) for 4 h. Serum-starved HT29 and HCT116 cells were stimulated with 750 ng/mL EGF for 1 h after 3 h of preincubation with or without phenolic compounds. Total ERK1/2 was used as a loading control. Quantification of the relative expression of pERK1/2 in HT29 and HCT116 cells was determined using Genetool software (Genesys).

1266 SAENGLEE et al. / Turk J Biol proteins were evaluated by western blotting. In this study, 2005). In addition, HDAC inhibitor-induced accumulation three proteins related to cell growth inhibition (p21, of acetylated histones may affect cell cycle progression CDK4, and p53) were investigated. p21 is one of the p53- by altering the ability of tumor cells to undergo mitosis regulated protein targets of HDAC inhibitor-mediated (Warrener et al., 2003). However, the relationship between suppression to control cell cycle progression. In the HT29 hyperacetylation of histone proteins and cell growth cells, p-coumaric acid (Figure 4A) induced p21 expression, inhibition is still unclear. Previous studies demonstrated whereas ferulic (Figure 4B) and sinapinic (Figure 4C) that combination treatments of HDAC inhibitors and the acids decreased p21 level. Resveratrol (Figure 4D) and current anticancer drug cisplatin may lead to enhanced NaB (Figure 4E) also increased the p21 level compared to anticancer efficacy and reduced cytotoxicity to normal the solvent control treatment (Figure 4F). CDK4, a protein cells (Shen et al., 2007; Ong et al., 2012; Asgar et al., 2016). associated with cell cycle regulation, was also detected in Drug combination studies between the peanut phenolic- the same condition. In the HT29 cells, all of the phenolic based HDAC inhibitors and current anticancer drugs such compounds and NaB caused a downregulation of CDK4 as cisplatin and 5-fluorouracil are of future interest with (Figure 4G). Mutant p53 protein expression was constant the aim to reduce toxicity and resistance based on their for the phenolic compounds and NaB treatments (Figure differences in drug targets. Consistent with our results, 4H) in the HT29 cells. resveratrol has been found to inhibit proliferation in a In HCT116 cells, p21 expression was increased when number of cancer cell lines (Agrawal et al., 2002). Moreover, treated with p-coumaric acid (Figure 4I), but it decreased resveratrol as well as some other phenolic compounds when cells were treated with ferulic (Figure 4J) and sinapinic of natural origin, including curcumin and capsaicin, (Figure 4K) acids. Furthermore, resveratrol (Figure 4L) at exhibited growth inhibition against HL-60, K-562, MCF- a concentration of 150 µM caused a decrease of p21 in the 7, and HeLa cells (Roy et al., 2002). It is noteworthy that HCT116 cells. The increase of p21 upon treatments with all four phenolic compounds in this study were less toxic p-coumaric acid and NaB (Figure 4M) appeared to be on noncancer cells than cancer cells, consistent with independent of p53 in the HCT116 cells (Figure 4N). All many other phenolic compounds (Nakajima et al., 2009). four phenolic compounds and NaB caused a reduction in Similarly, the noncancer cell line appeared to be resistant CDK4 expression in the HCT116 cells (Figure 4O). In the to both sodium butyrate and phenolic-rich extracts of HCT116 cells, the expression of p53 remained unaltered Hydnophytum formicarum Jack rhizome (Senawong et al., after the p-coumaric acid and sinapinic acid treatments, 2013). but it was upregulated when treated with ferulic acid and According to the apoptosis results, it is evident that resveratrol (Figure 4P). However, the p53 level seemed to apoptosis induction is the underlying mechanism for decrease after NaB treatment (Figure 4P). Interestingly, the cancer cell growth inhibition by the four peanut phenolic resveratrol-treated HCT116 cells exhibited an increased compounds (Figure 2). Regarding phenolic compound- p53 level (Figure 4P) but unaltered or decreased p21 levels induced apoptosis, phenolic-rich extracts and sinapinic (Figure 4N), suggesting that the increase of p53 level in acid from H. formicarum also inhibited the growth of HeLa this cell line may not cause G0/G1 cell cycle arrest. In this cells by induction of apoptosis (Senawong et al., 2013). context, the role of p21 is particularly complicated, as this Similarly, the natural phenolic compounds curcumin, protein can be activated by p53-independent mechanisms resveratrol, and capsaicin act as chemopreventive agents upon exposure to p-coumaric acid and NaB (Figure 4N). by inducing apoptosis in tumor cells (Roy et al., 2002). Phenolic compounds from blueberries also showed 4. Discussion growth inhibition and apoptosis induction of colon cancer The principal aim of cancer chemoprevention is the use of cell lines (Yi et al., 2005). Resveratrol induced a decrease in pharmacological or natural agents to prevent or stop the proliferation rates and an increase in apoptosis in prostate process of tumorigenesis. Here we demonstrated that all cancer cell lines in a dose-dependent manner (Benitez et four phenolic compounds found in peanut testa extracts al., 2007). In addition, p-coumaric and ferulic acids have (Khaopha et al., 2015) possess HDAC inhibitory activity previously been reported to induce cancer cell apoptosis and may inhibit the growth of colon cancer cells. Among (Jaganathan et al., 2013; Peng et al., 2013). the four phenolic compounds tested, resveratrol exhibited The cell cycle profiles indicate that all four phenolic the most effective HDAC inhibition (Table 1; Figure 1) and compounds inhibited cancer cell growth via induction cell growth inhibition (Table 2). An imbalance between of not only apoptosis but also cell cycle arrest (Table 3). acetylation and deacetylation changes the structure and Moreover, a noncancer cell line (Vero cells) was not the activity of histone proteins, influencing cancer cell arrested at any particular phase of the cell cycle (data not functions such as gene expression, cell cycle progression, shown). All four phenolic compounds and NaB induced and cell death pathways (Drummond et al., 2005; Peart et al., a small increase in sub-G1 in the Vero cells. Our findings

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HT29 A B C D E p-Coumaric acid (mM) Ferulic acid (mM) Sinapinic acid (mM) Resveratrol (μM) NaB (mM) C 1 2.5 5 C 1 2.5 5 C 1 2.5 5 C 50 100 150 C 1 2.5 5 p21 CDK4 p53 -actin

HT29 6 F 1.2 G 2 H p21 CDK4 p53 5 1 1.5 4 0.8

3 0.6 1

2 0.4 0.5 1 0.2

Relative band intensity 0 0 0 5.0 50 5.0 50 50 5.0 5.0 2.5 2.5 5.0 5.0 5.0 2.5 2.5 2.5 2.5 1.0 1.0 5.0 2.5 1.0 1.0 1.0 5.0 2.5 1.0 5.0 5.0 2.5 2.5 2.5 1.0 1.0 1.0 1.0

C C 5.0 2.5 C C C C 1.0 C C C C C C C C 1.0 150 100 150

100 C 150 100 Sinapinic p-Coumaric Ferulic Resveratrol NaB p-Coumaric Ferulic Sinapinic Resveratrol NaB p-Coumaric Ferulic Sinapinic Resveratrol NaB acid (mM) acid (mM) acid (mM) (μM) (mM) acid (mM) acid (mM) acid (mM) (μM) (mM) acid (mM) acid (mM) acid (mM) (μM) (mM) HCT116 I J K L M p-Coumaric acid (mM) Ferulic acid (mM) Sinapinic acid (mM) Resveratrol (μM) NaB (mM) C 1 2.5 5 C 1 2.5 5 C 1 2.5 5 C 50 100 150 C 1 2.5 5 p21 CDK4 p53 -actin HCT116 4 N 1.6 O 9 P 1.4 8 3 p21 1.2 CDK4 7 p53 6 1 5 2 0.8 4 0.6 3 1 0.4 2 0.2 1 Relative band intensity 0 0 0 5.0 50 5.0 50 5.0 5.0 5.0 50 2.5 2.5 5.0 5.0 2.5 2.5 2.5 2.5 1.0 1.0 5.0 2.5 1.0 1.0 1.0 5.0 5.0 5.0 2.5 2.5 2.5 1.0 2.5 1.0 1.0 1.0 1.0 5.0 2.5 C C C C C C 1.0 C C C C C C C 1.0 150

100 C 150

100 C 150 100 p-Coumaric Ferulic Sinapinic Resveratrol NaB p-Coumaric Ferulic Sinapinic Resveratrol NaB p-Coumaric Ferulic Sinapinic Resveratrol NaB acid (mM) acid (mM) acid (mM) (μM) (mM) acid (mM) acid (mM) acid (mM) (μM) (mM) acid (mM) acid (mM) acid (mM) (μM) (mM) Figure 4. The effect of peanut phenolic compounds on the expression levels of cell cycle and apoptosis regulatory proteins. The protein expression levels of p21, CDK4, and p53 in HT29 (A–H) and HCT116 (I–P) cells were determined after treatment with phenolic compounds and NaB where β-actin was used as a loading control. Proteins were extracted from cell lysates, separated by SDS-PAGE, and probed with the respective antibodies. C represents the protein level in the solvent control treatment. Quantification of the relative expression of these proteins in HT29 (F–H) and HCT116 (N–P) cells was determined using Genetool software (Genesys). on cell cycle arrest are in agreement with several findings induce growth inhibition along with arrest in the S phase on induction of cancer cell cycle arrest by some phenolic of several human cancer cell lines (Joe et al., 2002). compounds. For example, the growth inhibitory activity Treatment of the HCT116 cells with all four phenolic of ellagic acid was accompanied by induction of arrest of compounds resulted in ERK1/2 activations (Figures 3B, the cell cycle in the G0/G1 phase of human bladder cancer 3D, 3F, and 3H). The observed ERK1/2 activations may T24 cells (Li et al., 2005). Resveratrol was also reported to be associated with oxidative stress upon drug treatment.

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Previous reports have revealed that ERK1/2 activation Similar to our observation, it has been shown that induced ROS generation and endoplasmic reticulum stress resveratrol induced the expression of p21/WAF1 and mainly via the PERK-eIF2α pathway, causing apoptosis decreased CDK4 expression in human vascular smooth (Tan and Chiu, 2013). The activation of MAPKs through muscle cells in which p21 expression was p53-independent intracellular ROS has been documented (Martindale (Lee and Moon, 2005). However, resveratrol induced-cell and Holbrook, 2002). Similarly, intracellular ROS lead to cycle arrest was mediated by increased expression of the activation of ERK1/2 and induce apoptosis in cancer cells modulator proteins p53 and p21 and decreased CDK4 (Shin et al., 2009). The precise role of ROS in the signaling expression in LNCaP cells (Benitez et al., 2007). Some of apoptosis is very complicated, as both the intrinsic research findings with regard to p21 and CDK4 coincide and extrinsic pathways are known to be associated with with our observations only for p-coumaric acid and ROS (Mates et al., 2008). Previous reports demonstrated NaB treatments. Ellagic acid increased p53 and p21 but that oxidative stress induced by chemopreventive drugs decreased CDK2 gene expression, which can lead to the reduced mitochondrial membrane potential and caused G0/G1 arrest of T24 cells (Li et al., 2005). It was reported cytochrome c to be released from the mitochondria, that activation of p53/p21(WAF1/CIP1) expression played a resulting in the activation of caspase-9 (Bagriacik et al., role in G1 arrest and apoptosis in cancer cells treated with 2007). However, our findings suggest a more complicated ellagic acid (Narayanan et al., 1999). role of ERK1/2 inhibition in promoting cell cycle arrest The four peanut phenolic compounds exhibited and apoptosis upon phenolic compound treatment. antiproliferative activity against colon cancer cell lines, It is widely accepted that p53 expression regulates which was accompanied by apoptosis with p53 either apoptosis and/or cell cycle arrest in response to DNA dependently or independently. As these compounds also damage by anticancer agents. In accordance with previous exhibited HDAC inhibitory activity, one may envision reports that HDAC inhibitors caused cell death in a that inhibition of HDAC activity may lead to a decreased p53-dependent manner (Zhao et al., 2006), ferulic acid expression of CDK4 but increased expression of p21, and resveratrol could induce cell death and activate p53 which eventually could block the cell cycle progression. expression in HCT116 cells (Figure 4P). However, both An oncogenic cell signaling process involving pERK1/2 upregulation and downregulation of p21 were observed activation was also responsible for growth inhibition of upon treatment of the colon cancer cells with the four HCT116 cells. This study has shown that peanut phenolic phenolic compounds (Figures 4F and 4N). This suggests compounds possessing HDAC inhibitory activity could that modes of cell death other than apoptosis may operate induce apoptosis and cell cycle arrest of tumor cells, in tumor cells following exposure to sinapinic acid, or, more which could have an important implication in the further generally, to DNA-damaging agents (te Poele et al., 2002). development of cancer chemopreventive agents. Expression of p21 coincides with the hyperacetylation of In summary, a possible mechanism for the protective histones H3 and H4 in its promoter region (Richon et al., effect of peanut phenolic compounds against colon cancer 2002), indicating that HDAC enzymes negatively regulate relates to their antiproliferative and proapoptotic effects, p21 expression in many cell types. However, in HT29 cells, leading to downregulation of cell cycle proteins and an p53-independent induction of p21 expression seems to be increase in the apoptosis of colon cancer cells. a prerequisite for apoptosis (Agrawal et al., 2002) and also seems to sensitize tumor cells to the actions of different Acknowledgments agents. Interestingly, our observations do not comply with This work was supported by the Higher Education Research either view. In fact, while the induction of p21 expression Promotion and National Research University Project of in p53-proficient and -deficient cell lines was responsive Thailand, Office of the Higher Education Commission, to drug treatment, this did not parallel the difference in through the Food and Functional Food Research Cluster apoptotic cells between the HCT116 and HT29 cells. of Khon Kaen University. Grateful acknowledgment is also Regarding CDK4, all phenolic compounds tested in this made to the Thailand Research Fund, the Commission of study caused downregulation of CDK4 in the HCT116 Higher Education, and Khon Kaen University for providing cells (Figure 4O). As with NaB treatment (Sherr, 1994), financial support to this research through a Distinguished the decreased CDK4 level resulting from the phenolic Research Professor Grant to Dr Aran Patanothai. compound treatments may facilitate controlling the cell cycle progression at the G1 phase in HCT116 cells.

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