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Histone deacetylase inhibition results in a common metabolic profile associated with HT29 differentiation - PubMed

Histone deacetylase inhibition results in a common metabolic profile associated with HT29 differentiation

Gema Alcarraz-Vizán et al. Metabolomics. 2010 Jun.

Abstract

Cell differentiation is an orderly process that begins with modifications in gene expression. This process is regulated by the acetylation state of histones. Removal of the acetyl groups of histones by specific enzymes (histone deacetylases, HDAC) usually downregulates expression of genes that can cause cells to differentiate, and pharmacological inhibitors of these enzymes have been shown to induce differentiation in several colon cancer cell lines. Butyrate at high (mM) concentration is both a precursor for acetyl-CoA and a known HDAC inhibitor that induces cell differentiation in colon cells. The dual role of butyrate raises the question whether its effects on HT29 cell differentiation are due to butyrate metabolism or to its HDAC inhibitor activity. To distinguish between these two possibilities, we used a tracer-based metabolomics approach to compare the metabolic changes induced by two different types of HDAC inhibitors (butyrate and the non-metabolic agent trichostatin A) and those induced by other acetyl-CoA precursors that do not inhibit HDAC (caprylic and capric acids). [1,2-(13)C(2)]-d-glucose was used as a tracer and its redistribution among metabolic intermediates was measured to estimate the contribution of glycolysis, the pentose phosphate pathway and the Krebs cycle to the metabolic profile of HT29 cells under the different treatments. The results demonstrate that both HDAC inhibitors (trichostatin A and butyrate) induce a common metabolic profile that is associated with histone deacetylase inhibition and differentiation of HT29 cells whereas the metabolic effects of acetyl-CoA precursors are different from those of butyrate. The experimental findings support the concept of crosstalk between metabolic and cell signalling events, and provide an experimental approach for the rational design of new combined therapies that exploit the potential synergism between metabolic adaptation and cell differentiation processes through modification of HDAC activity.

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Figures

Fig. 1
Fig. 1

Antiproliferative effect of NaB (a), caprylic (b) and capric acids (c). In combined treatments, a constant dose of 3 mM NaB was used while increasing doses of caprylic and capric were added. Results are normalized by the untreated control

Fig. 2
Fig. 2

Percentage of cells in a each cell cycle phase and b early/late apoptosis or necrotic state under the different fatty acid treatments. The IC50 of each fatty acid was used. Ct: Control; NaB: Butyrate; 1: Caprylic; 2: Capric. P < 0.01 (*) was considered to indicate significant differences in percentages compared to the control

Fig. 3
Fig. 3

Alkaline phosphatase activity induced by the different treatments at their IC20 and IC50 doses. An arbitrary value of 1 was assigned to the controls. Treatments are shown as the value per one with respect to the control. Ct: Control; NaB: Butyrate; 1: Caprylic; 2: Capric. P < 0.01 (*) was considered to indicate significant differences of the treatments compared to the corresponding control

Fig. 4
Fig. 4

a Determination of glucose consumption and lactate production under the different treatments at their respective IC20. b Total 13C lactate enrichment from labeled glucose of the different treatments was calculated as Σmn = m1 + 2 × m2 + 3 × m3. c Glycolytic rate expressed as m2lactate/(m2glucose/2). m2glucose represented 48.08% of total glucose.Ct: Control; NaB: Butyrate; TSA: Trichostatin A; 1: Caprylic; 1b: Caprylic 0.4 mM; 2: Capric. P < 0.01 (*) was considered to indicate significant differences

Fig. 5
Fig. 5

RNA ribose isotopomer distribution of 13C enrichment under the different treatments, expressed as % of total ribose (a). In b, the contribution of oxidative vs. nonoxidative branches of PPP was calculated from the isotopomeric distribution as ox/nonox = (m1 + m3)/(m2 + m3 + 2 × m4). c Pyruvate dehydrogenase contribution under the different treatments expressed as m2 of C4–C5 carbons of glutamate obtained from labeled glucose (% of total glutamate). Pyruvate dehydrogenase activity relative to β-oxidation (m2 C4–C5) was calculated by subtracting the m2 of m/z 152 (glutamate fragment C2–C4) from the m2 of m/z 198 (glutamate fragment C2–C5). Ct: Control; NaB: Butyrate; TSA: Trichostatin A; 1: Caprylic; 1b: Caprylic 0.4 mM; 2: Capric. P < 0.01 (*) was considered to indicate significant differences

Fig. 6
Fig. 6

Measure of β-oxidation of butyrate: glutamate isotopomer distribution under the conditions containing U-13C4-butyrate as a tracer (% of total glutamate). Ct: Control; NaB: Butyrate; 1: Caprylic; 1b: Caprylic 0.4 mM; 2: Capric. P < 0.01 (*) was considered to indicate significant differences

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