Evidence has been provided that diet and environmental factors directly influence epigenetic mechanisms associated with cancer development in humans. The inhibition of histone deacetylase (HDAC) activity and the disruption of the HDAC complex have been recognized as a potent strategy for cancer therapy and chemoprevention. In the present study, we investigated whether selected plant constituents affect HDAC activity or HDAC1 protein status in the human colon carcinoma cell line HT29. The polyphenols (−)-epigallocatechin-3-gallate (EGCG) and genistein (GEN) as well as two oxidative methyleugenol (ME) metabolites were shown to inhibit HDAC activity in intact HT29 cells. Concomitantly, a significant decrease of the HDAC1 protein level was observed after incubation with EGCG and GEN, whereas the investigated ME metabolites did not affect HDAC1 protein status. In conclusion, dietary compounds were found to possess promising HDAC-inhibitory properties, contributing to epigenetic alterations in colon tumor cells, which should be taken into account in further risk/benefit assessments of polyphenols and alkenylbenzenes.
Cancer is one of the most causes of death in industrial countries. Especially the genesis of tumors of the gastrointestinal tract seems to depend on genetic predisposition, environmental factors, and diet [
Epidemiological studies indicate that cancer incidence might be significantly modulated by an enhanced dietary intake of polyphenols with fruits and vegetables [
In this study we addressed the question whether the polyphenols chlorogenic acid (CGA), genistein (GEN), and EGCG (Figure
Food-derived polyphenols of different classes and structure of methyleugenol and selected oxidative metabolites.
Dimethyl sulfoxide (DMSO), ME, CGA, and catalase were purchased from Sigma-Aldrich (Steinheim, Germany). GEN and EGCG were purchased from Extrasynthese (Genay Cedex, France). The methyleugenol metabolites 1′-OH-ME, 3′-OXO-MIE, and MEE were synthesized and purified according to the procedures previously described by our lab [
HT29 cells, a human colon adenocarcinoma cell line (Accession number ACC 299, Deutsche Sammlung von Mikroorganismen und Zellkultur (DSMZ), Braunschweig, Germany), were grown in Dulbecco’s modified Eagle’s medium (DMEM) high glucose (4.5 g/L, Invitrogen Life Technologies, Karlsruhe, Germany). Cell culture medium was supplemented with 10% fetal calf serum (FCS; PAA, Coelbe, Austria) and 1% penicillin/streptomycin (Invitrogen Life Technologies, Karlsruhe, Germany). Cells were cultured at 37°C in a water-saturated atmosphere containing 5% CO2. Compounds were dissolved in DMSO and added to the medium to yield a final DMSO concentration of 0.5% (v/v). Control experiments were carried out with medium containing 0.5% of DMSO without test compounds.
30,000 (24 h), 20,000 (48 h), or 8,000 (72 h) HT29 cells per well were seeded into 24-well plates and allowed to grow for 24 h before treatment. Thereafter, cells were incubated with the respective test compound for 24 h, 48 h, or 72 h in culture medium. Effects on cell growth were determined according to the general method of Skehan et al. [
The WST-1 (water soluble tetrazolium) cell proliferation assay was performed according to the manufactures protocol (Roche Diagnostics GmbH, Mannheim, Germany). 5,000 or 3,500 HT29 cells per well, respectively, were seeded into 96-well plates and allowed to grow for 24 h. V79 cells were treated with CGA, EGCG, or GEN for 24 h or 48 h under FCS-containing conditions in the presence of catalase (100 U/mL). In each experiment a solvent control (DMSO 0.5%) and a positive control IGPAL-CA630 (0.5%) were included. After treatment, medium was removed, cells were rinsed with 100
HDAC enzyme activity was determined using HDAC Assay Kit obtained from Cayman Chemical Company (Ann Arbor, MI, USA) according to the manufactures protocol. 5,000 HT29 cells per well were seeded in 100
4.5 × 106 HT29 cells per Petri dish were seeded in culture medium and allowed to grow for 48 h. Cells were incubated with CGA, GEN, and EGCG in the presence of catalase (see above), or with ME and its oxidative metabolites at respective concentrations. After 24 h of incubation the culture medium was removed and the cells were washed with ice-cold phosphate buffered saline (PBS) for two times and abraded on ice with 200
Growth inhibitory properties of methyleugenol and selected methyleugenol metabolites in HT29 cells were determined using the sulforhodamine B (SRB) assay over 72 h. No growth inhibitory properties were observed for ME and 1′-OH-ME using concentrations up to 100
Cytotoxic properties of the test compounds in HT29 cells.
Compound | IC50-value |
||
---|---|---|---|
24 h | 48 h | 72 h | |
EGCG | n.d.a, b | n.d.a, b | 40 ± 6c |
GEN | n.d.a, b | >100b | ~50d |
CGA | n.d.a, b | n.d.a, b | 205 ± 53c |
ME | n.d.a | n.d.a | n.d.a |
1′-OH-ME | n.d.a | n.d.a | n.d.a |
3′-OXO-MIE | n.d.a | >100 | 100 ± 14 |
MEE | n.d.a | >100 | >100 |
aNo cytotoxicity up to the highest concentration implemented in the testing; bdata was performed in the presence of catalase (100 U/mL) to avoid hydrogen peroxide formation; cwithout catalase; previously reported by Kern et al. [
Inhibition of tumor cell growth
Cytotoxicity of CGA, GEN, and EGCG was determined in the presence of catalase (100 U/mL) to avoid hydrogen peroxide formation after an incubation period of 24 h or 48 h using the WST 1 (water soluble tetrazolium salt) assay. CGA, GEN, and EGCG did not exhibit cytotoxic effects up to the highest concentration implemented in our further investigations on HDAC expression and activity (data not shown).
To address the question whether polyphenols or ME and its respective metabolites modulate HDAC activity in intact cells, HT29 cells were incubated with the test compounds for 24 h and enzyme activity was determined in an HDAC cell-based activity assay kit. The specific HDAC inhibitor trichostatin A (Figure
Inhibition of HDAC1 activity in HT29 cells after 24 h of incubation with (a) polyphenols in the presence of catalase (100 U/mL) or (b) methyleugenol and methyleugenol metabolites. HDAC activity was determined by the metabolic rate of a specific HDAC1 substrate. Recombinant HDAC1 was included as positive control (PC) and trichostatin A (TA) as a specific HDAC inhibitor. The data presented are mean ± SD of at least three independent experiments, each performed in a duplicate. The significances indicated were calculated in relation to the solvent control DMSO 0.5% v/v (Student’s
Within the group of alkenylbenzenes ME and 1′-OH-ME did not affect HDAC activity up to 100
We further addressed the question whether the modulation of HDAC activity by secondary plant constituents in HT29 cells is associated with changes in the amount of HDAC protein. The impact of the test compounds on HDAC1 protein status in HT29 cells after 24 h of incubation was detected by western blot analysis using a goat polyclonal antibody against human HDAC1. Incubation of HT29 cells with CGA increased the HDAC1 protein level up to 250
Western blot analysis and representative western blots of HDAC1 expression in HT29 cells after 24 h treatment with polyphenols in the presence of catalase (100 U/mL) ((a) and (b)) or methyleugenol and respective metabolites ((c) and (d)). DMSO: solvent control (0.5% v/v),
After treatment of HT29 cells with methyleugenol or the selected metabolites, no changes in the amount of HDAC1 protein were detected by western blot analysis (Figures
Dietary polyphenols have been demonstrated to exhibit cancer preventive and cancer therapeutic activity [
Furthermore, it has to be mentioned that cytotoxic effects of the polyphenols EGCG, GEN, and CGA in the implemented concentration range has been intensively discussed [
Taken together, a hydrogen peroxide-scavenging system to
Within the class of alkenylbenzenes only the two oxidative metabolites 3′-OXO-MIE and MEE observed significant growth inhibitory properties on HT29 cells (Figure
The levels of HDAC activity within cells can be altered via direct inhibition of the HDAC enzyme and changes in HDAC protein levels. In the study presented here, the metabolites 3′-OXO-MIE and MEE were firstly reported to potently diminish HDAC enzyme activity (Figure
HDAC inhibition leads to genomic instability by a variety of mechanisms. This effect may contribute to the cytotoxicity of these drugs. Furthermore, HDAC inhibitors sensitize DNA to exogenous genotoxic damage and induce the generation of reactive oxygen species. At least, HDAC inhibitors could induce chromosome missegregation [
The present study shows that the polyphenols EGCG and GEN and the two ME metabolites 3′-OXO-MIE and MEE potently diminished the activity of HDAC in intact colon carcinoma cells. We further demonstrated that modulation of HDAC activity is associated with the suppression of HDAC1 protein status by polyphenols, whereas the ME metabolites did not affect the protein level of HDAC1. These results illustrate an interference of EGCG and GEN with epigenetic pathways which may contribute to the idea that dietary polyphenols have potentially chemopreventive effects. Furthermore, we show that via bioactivation of the prominent food carcinogen ME, metabolites not only with potential genotoxic, but also with HDAC inhibitory properties will be generated which may contribute to their DNA-damaging properties. In summary, the results presented reveal that more investigations on the mechanism of action for future risk/benefit assessment of polyphenols and alkenylbenzenes will be necessary.
The study was supported by the “Institut Danone Ernährung für Gesundheit e.V.”