Histone deacetylase 1: a target of 9-hydroxystearic acid in the inhibition of cell growth in human colon cancer

Recent studies have shown that an endogenous lipoperoxidation product, 9-hydroxystearic acid (9-HSA), acts in colon carcinoma cells (HT29) as a growth inhibitor by inducing p21(WAF1) in an immediate-early, p53-independent manner and that p21(WAF1) is required for 9-HSA-mediated growth arrest in HT29 cells. It is conceivable, therefore, to hypothesize that the cytostatic effect induced by this agent is at least partially associated with a molecular mechanism that involves histone deacetylase 1 (HDAC1) inhibition, as demonstrated for sodium butyrate and other specific inhibitors, such as trichostatin A and hydroxamic acids. Here, we show that, after administration, 9-HSA causes an accumulation of hyperacetylated histones and strongly inhibits the activity of HDAC1. The interaction of 9-HSA with the catalytic site of the enzyme has been highlighted by computational modeling of the human HDAC1, using its homolog from the hyperthermophilic Aquifex aeolicus as a template. Consistent with the experimental data, we find that 9-HSA can bind to the active site of the protein, showing that the inhibition of the enzyme can be explained at the molecular level by the ligand-protein interaction.     

Acetylation of the Entamoeba histone H4 N-terminal domain is influenced by short-chain fatty acids that enter trophozoites in a pH-dependent manner

Treatment of higher eukaryotic cells with short-chain fatty acids (SCFA) such as butyrate causes decreased levels of histone deacetylase (HDAC) activity and hyperacetylation of histones, and thereby affects gene expression, cell growth and differentiation. Entamoeba parasites encounter high levels of SCFA in the host colon, and in vitro these compounds allow trophozoite stage parasites to multiply but prevent their differentiation into infectious cysts. The Entamoeba invadens IP-1 histone H4 protein has an unusual number of lysines in its N-terminus, and these become hyperacetylated in trophozoites exposed to the HDAC inhibitors trichostatin A (TSA) or HC-toxin, but not in trophozoites exposed to butyrate. We have now found that several other commonly studied isolates of Entamoeba parasites also have an extended set of histone H4 acetylation sites that become hyperacetylated in response to TSA, but hypoacetylated in response to butyrate, suggesting an unusual sensitivity of this parasite's histone modifying enzymes to SCFA. Butyrate was found to enter trophozoites in a pH-dependent manner consistent with diffusive entry of the un-ionised form of the fatty acid into the amoebae. Transit of the Entamoeba organism through areas of the host intestine with distinct pH and SCFA concentrations would therefore result in very different levels of SCFA within the parasite. Entamoeba appears to have acquired unique alterations of its histone acetylation mechanism that may allow for its growth in the presence of varying amounts of the bacterial fermentation products.     

Transient vs. prolonged histone hyperacetylation: effects on colon cancer cell growth, differentiation, and apoptosis

The role of histone hyperacetylation in regard to growth, differentiation, and apoptosis in colon cancer cells was assessed in an in vitro model system. HT-29 cells were grown in +/-10% fetal bovine serum with either 5 mM sodium butyrate or 0.3 microM trichostatin A [single dose (T) or 3 doses 8 h apart (TR)] for 24 h. Serum-starved HT-29 cells were further treated with epidermal growth factor or insulin-like growth factor I for an additional 24 h. Apoptosis was quantified with propidium iodide and characterized by electron microscopy. Northern blot analyses were performed with cDNA probes specific for intestinal alkaline phosphatase, Na-K-2Cl cotransporter, the cell cycle inhibitor p21, and the actin control. Flow cytometric analysis revealed a time-dependent growth suppression along with early induction of p21 mRNA in the butyrate, T, and TR groups. Histone hyperacetylation, assessed by acid-urea-triton gel electrophoresis, was transient in the T group but persisted for up to 24 h in the butyrate and TR groups. Induction of apoptosis, growth factor unresponsiveness, and differentiation occurred in the butyrate- and TR-treated cells but not those treated with a single dose of trichostatin A. Thus transient hyperacetylation of histones is sufficient to induce p21 expression and produce cellular growth arrest, but prolonged histone hyperacetylation is required for induction of the programs of differentiation, apoptosis, and growth factor unresponsiveness.     

The stable isotope-based dynamic metabolic profile of butyrate-induced HT29 cell differentiation

Stable isotope-based dynamic metabolic profiling is applied in this paper to elucidate the mechanism by which butyrate induces cell differentiation in HT29 cells. We utilized butyrate-sensitive (HT29) cells incubated with [1,2-13C2]glucose or [1,2-13C2]butyrate as single tracers to observe the changes in metabolic fluxes in these cells. In HT29 cells, increasing concentrations of butyrate inhibited glucose uptake, glucose oxidation, and nucleic acid ribose synthesis in a dose-dependent fashion. Glucose carbon utilization for de novo fatty acid synthesis and tricarboxylic acid cycle flux was replaced by butyrate. We also demonstrated that these changes are not present in butyrate-resistant pancreatic adenocarcinoma MIA cells. The results suggest that the mechanism by which colon carcinoma cells acquire a differentiated phenotype is through a replacement of glucose for butyrate as the main carbon source for macromolecule biosynthesis and energy production. This provides a better understanding of cell differentiation through metabolic adaptive changes in response to butyrate in HT29 cells, demonstrating that variations in metabolic pathway substrate flow are powerful regulators of tumor cell proliferation and differentiation.     

Validation of NCM460 cell model as control in antitumor strategies targeting colon adenocarcinoma metabolic reprogramming: Trichostatin A as a case study

Background

Cancer cells have extremely active metabolism, which supports high proliferation rates. Metabolic profiles of human colon cancer cells have been extensively studied, but comparison with non-tumour counterparts has been neglected.

Methods

Here we compared the metabolic flux redistribution in human colon adenocarcinoma cells (HT29) and the human colon healthy cell line NCM460 in order to identify the main pathways involved in metabolic reprogramming. Moreover, we explore if induction of differentiation in HT29 by trichostatin A (TSA) reverts the metabolic reprogramming to that of NCM460. Cells were incubated with [1,2-¹³C₂]-d-glucose as a tracer, and Mass Isotopomer Distribution Analysis was applied to characterize the changes in the metabolic flux distribution profile of the central carbon metabolism.

Results

We demonstrate that glycolytic rate and pentose phosphate synthesis are 25% lower in NCM460 with respect to HT29 cells. In contrast, Krebs cycle activity in the former was twice that recorded in the latter. Moreover, we show that TSA-induced HT29 cell differentiation reverts the metabolic phenotype to that of healthy NCM460 cells whereas TSA does not affect the metabolism of NCM460 cells.

Conclusions

We conclude that pentose phosphate pathway, glycolysis, and Krebs cycle are key players of colon adenocarcinoma cellular metabolic remodeling and that NCM460 is an appropriate model to evaluate the results of new therapeutic strategies aiming to selectively target metabolic reprogramming.

General significance

Our findings suggest that strategies to counteract robust metabolic adaptation in cancer cells might open up new avenues to design multiple hit and targeted therapies.     

Complementary roles for histone deacetylases 1, 2, and 3 in differentiation of pluripotent stem cells

Abstract In eukaryotic cells, covalent modifications to core histones contribute to the establishment and maintenance of cellular phenotype via regulation of gene expression. Histone acetyltransferases (HATs) cooperate with histone deacetylases (HDACs) to establish and maintain specific patterns of histone acetylation. HDAC inhibitors can cause pluripotent stem cells to cease proliferating and enter terminal differentiation pathways in culture. To better define the roles of individual HDACs in stem cell differentiation, we have constructed "dominant-negative" stem cell lines expressing mutant, Flag-tagged HDACs with reduced enzymatic activity. Replacement of a single residue (His→Ala) in the catalytic center reduced the activity of HDACs 1 and 2 by 80%, and abolished HDAC3 activity; the mutant HDACs were expressed at similar levels and in the same multiprotein complexes as wild-type HDACs. Hexamethylene bisacetamide-induced MEL cell differentiation was potentiated by the individual mutant HDACs, but only to 2%, versus 60% for an HDAC inhibitor, sodium butyrate, suggesting that inhibition of multiple HDACs is required for full potentiation. Cultured E14.5 cortical stem cells differentiate to neurons, astrocytes, and oligodendrocytes upon withdrawal of basic fibroblast growth factor. Transduction of stem cells with mutant HDACs 1, 2, or 3 shifted cell fate choice toward oligodendrocytes. Mutant HDAC2 also increased differentiation to astrocytes, while mutant HDAC1 reduced differentiation to neurons by 50%. These results indicate that HDAC activity inhibits differentiation to oligodendrocytes, and that HDAC2 activity specifically inhibits differentiation to astrocytes, while HDAC1 activity is required for differentiation to neurons.