Green tea phenolics inhibit butyrate-induced differentiation of colon cancer cells by interacting with monocarboxylate transporter 1

Abstract

Diet has a significant impact on colorectal cancer and both dietary fiber and plant-derived compounds have been independently shown to be inversely related to colon cancer risk. Butyrate (NaB), one of the principal products of dietary fiber fermentation, induces differentiation of colon cancer cell lines by inhibiting histone deacetylases (HDACs). On the other hand, (-)-epicatechin (EC) and (-)-epigallocatechin gallate (EGCG), two abundant phenolic compounds of green tea, have been shown to exhibit antitumoral properties. In this study we used colon cancer cell lines to study the cellular and molecular events that take place during co-treatment with NaB, EC and EGCG. We found that (i) polyphenols EC and EGCG fail to induce differentiation of colon adenocarcinoma cell lines; (ii) polyphenols EC and EGCG reduce NaB-induced differentiation; (iii) the effect of the polyphenols is specific for NaB, since differentiation induced by other agents, such as trichostatin A (TSA), was unaltered upon EC and EGCG treatment, and (iv) is independent of the HDAC inhibitory activity of NaB. Also, (v) polyphenols partially reduce cellular NaB; and (vi) on a molecular level, reduction of cellular NaB uptake by polyphenols is achieved by impairing the capacity of NaB to relocalize its own transporter (monocarboxylate transporter 1, MCT1) in the plasma membrane. Our findings suggest that beneficial effects of NaB on colorectal cancer may be reduced by green tea phenolic supplementation. This valuable information should be of assistance in choosing a rational design for more effective diet-driven therapeutic interventions in the prevention or treatment of colorectal cancer.

Keywords: (−)-epicatechin; (−)-epigallocatechin gallate; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; AP; Butyrate; Colon cancer; Differentiation; EC; EGCG; Epicatechin; Epigallocatechin gallate; HDAC; MCT1; MTT; NaB; TSA; alkaline phosphatase; butyrate; histone deacetylase; monocarboxylate transporter 1; trichostatin A.

Fig. 1

Dose–effect curves of (−)-epicatechin and (−)-epigallocatechin gallate on cell viability. HT29 cell cultures were treated with increasing doses of (−)-epicatechin (EC) (A) or (−)-epigallocatechin gallate (EGCG) (B) as indicated on the x axis for 72 h. Cell viability was expressed as a percentage with respect to untreated control. IC20 (EC) = 100 μM and IC20 (EGCG) = 20 μM.

Fig. 2

Phenolics reduce butyrate-induced differentiation. (A) HT29 cells were treated with butyrate (NaB) 2 mM or with NaB and phenolics (EC 100 μM and EGCG 20 μM) for 24, 48 and 72 h and alkaline phosphatase (AP) activity was measured and normalized by protein level. The data are normalized and statistically tested with respect to NaB-treated cells. Mean ± SD; n ≥ 3;**Different from NaB, p < 0.01. (B) HT29 cells were treated with NaB 2 mM or with phenolics alone (EC 100 μM and EGCG 20 μM) for 24, 48 and 72 h and AP activity was measured. The data are normalized and statistically tested with respect to Ctrl cells. Values are mean ± SD; n ≥ 3; */**Different from Ctrl, p < 0.05/p < 0.01.

Fig. 3

Phenolics effect on differentiation is not histone deacetylase-related. (A) HT29 cells were treated with TSA at 180 nM or with TSA in the presence of phenolics (EC 100 μM and EGCG 20 μM) for 48 h and AP activity was measured. Data are normalized and statistically tested with respect to TSA-treated cells. Mean ± SD; n = 3; **Different from TSA, p < 0.01. (B) Histone deacetylase (HDAC) activity determined in nuclear extracts from HT29 cells after treatment with NaB or NaB with phenolics for 30 min. The data are normalized and statistically tested with respect to Ctrl cells. Values are mean ± SD; n ≥ 2; **Different from Ctrl, p < 0.01. (C) HDAC activity determined in nuclear extracts from HT29 cells after treatment with NaB or phenolics for 30 min. The data are normalized and statistically tested with respect to NaB cells. Mean ± SD; n ≥ 2; **Different from NaB, p < 0.01.

Fig. 4

Phenolics impair NaB influx into the cells. HT29 cells were treated with NaB 2 mM or with NaB and phenolics (EC 100 μM or EGCG 20 μM) for 48 h. Acute [¹⁴C]-NaB uptake was subsequently measured as described in Materials and methods. Results are normalized and statistically tested with respect to NaB cells. Values are mean ± SD; n ≥ 2; **Different from NaB, p < 0.01.

Fig. 5

Effect of NaB and phenolics on monocarboxylate transporter 1 (MCT1). (A) After 48 h of incubation with NaB, phenolics or both, HT29 lysates were probed against MCT1 in a Western blot, using β-actin as a loading control. A representative blot is shown. (B) EC and EGCG antagonize plasma membrane redistribution of MCT1 caused by NaB. HT29 cells were incubated with NaB or NaB/phenolics for 48 h and then lysed and laid at the bottom of an OptiPrep density gradient. After ultracentrifugation, fractions were collected from the top (lipid rafts low-density fractions) to the bottom of the gradient (high-density non-raft fractions). Proteins in the fractions were separated on 10% SDS–PAGE and blots were probed with anti-MCT1 or anti-flotillin antibodies.