10(9)-hydroxystearic-acid and Colonic-Neoplasms

Histone deacetylases, HDACs, have been demonstrated to play a critical role in epigenetic signaling and were found to be overexpressed in several type of cancers; therefore, they represent valuable targets for anticancer therapy. 9-Hydroxystearic acid has been shown to bind the catalytic site of HDAC1, inducing G0/G1 phase cell cycle arrest and activation of the p21

Topics: Adenocarcinoma; Albumins; Cell Membrane; Cell Proliferation; Cell Survival; Chemistry Techniques, Synthetic; Colonic Neoplasms; Drug Discovery; HCT116 Cells; Histone Deacetylase 1; HT29 Cells; Humans; Keratins; Nanoparticles; Reactive Oxygen Species; S Phase Cell Cycle Checkpoints; Signal Transduction; Solubility; Stearic Acids

The epidermal growth factor has long been known to be strictly correlated with the highly proliferating activities of cancer cells and primary tumors. Moreover, in the nucleus, the epidermal growth factor/epidermal growth factor receptor complex (EGF/EGFR) functions as a transcriptional regulator that activates the cyclin D1 gene. 9-hydroxystearic acid (9-HSA) induces cell proliferation arrest and differentiation in HT29 colon cancer cells by inhibiting histone deacetylase 1 (HDAC1). 9-HSA-treated HT29, when stimulated with EGF, are not responsive and surprisingly undergo a further arrest. In order to understand the mechanisms of this effect, we analyzed the degree of internalization of the EGF/EGFR complex and its interactions with HDAC1. It appears that HDAC1, as modified by 9-HSA, is unable to associate with cyclin D1, interfering with the cell proliferation program, and sequesters the EGF/EGFR complex interrupting the transduction of the mitogenic signal.

Topics: Adenocarcinoma; Cell Proliferation; Colonic Neoplasms; Cyclin D1; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Histone Deacetylase 1; Histone Deacetylases; HT29 Cells; Humans; Signal Transduction; Stearic Acids

N-methylformamide (NMF) is an anti-proliferative, differentiating agent studied in several cell lines as well as in preclinical and clinical trials, whose mechanisms of action are still unclear. 9-Hydroxystearic acid (9-HSA) is an endogenous product of lipid peroxidation recently identified as a new histone deacetylase 1 inhibitor. Both agents show the same anti-proliferative effects by arresting colon cancer cell growth in G0/G1. We addressed two questions. (i) Do they act by regulating G0/G1 checkpoint proteins? (ii) Does 9-HSA have differentiating effects comparable to those of NMF? The effects of NMF and 9-HSA on growth, differentiation and invasiveness of HT29, a colon cancer cell line, have been compared by using immunoprecipitation analysis, confocal microscopy, enzyme assays and invasiveness tests. The results show that the G1 arrest caused by NMF is a cell cycle exit due to p27 induction, whereas 9-HSA has no effect on the induction of this inhibitor. Evidence is presented that the arrest in early G0/G1 induced by 9-HSA is associated with the conversion of HT29 characteristics to those of a more benign phenotype, whereas the arrest in the late G1 in response to NMF is not followed by a decrease in tumorigenicity. The failure of NMF in cancer therapy indicates that both anti-proliferative and differentiating characteristics are required for an anti-tumoral agent to be effective.

Topics: Cell Cycle; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cytoskeleton; Formamides; Humans; Microscopy, Confocal; Stearic Acids

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.

Topics: Acetylation; Catalytic Domain; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Histones; Humans; Ligands; Models, Molecular; Protein Binding; Stearic Acids

Growing evidence supports the critical role of lipid peroxidation products in the control of cell proliferation. In previous studies we demonstrated the efficient restriction of the proliferation rate in several cell lines resulting from the in vitro treatment with endogenous lipid polar components of cell membranes. Among these, 9-hydroxystearic acid (9-HSA), a primary intermediate of lipid peroxidation, induced a significant arrest in G0/G1 in HT29 colon cancer cells. In response to 9-HSA treatment of HT29 we observed cell growth arrest and increase in p21(WAF1) expression both at the transcriptional and the translational levels. Growth of p21(WAF1)-deleted HCT116 human colon carcinoma cells was not inhibited by 9-HSA. We present evidence that p21(WAF1) is required for 9-HSA mediated growth arrest in human colon carcinoma cells.

Topics: Antineoplastic Agents; Cell Division; Cell Line, Tumor; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Humans; Reverse Transcriptase Polymerase Chain Reaction; Stearic Acids; Up-Regulation

A sensitive, specific, accurate and reproducible gas chromatography/mass spectrometry method was developed for the assay of 9- and 10-hydroxystearic acids in samples obtained as cell extracts. The preparation of the samples required specific procedures to allow the analysis of both the free and the conjugated hydroxy acids as the corresponding methyl esters. The quantification used propyl-paraben as the internal standard and monitoring of a specific fragment of each isomeric hydroxy acid methyl ester, and allowed quantification of the conjugate and the free fractions of both 9- and 10-hydroxystearic acids. This method is suitable for identification and quantification (LOQ 1.8 and 4.4 ng, respectively) of these important metabolites of lipid peroxidation. In particular the development of an assay for the free 9-hydroxystearic acid methyl ester makes the method a reliable analytical tool for investigations of the role of this metabolite in the mechanisms of tumour cell proliferation.

Topics: Calibration; Carcinoma; Colonic Neoplasms; Gas Chromatography-Mass Spectrometry; Humans; Lipid Peroxidation; Reproducibility of Results; Sensitivity and Specificity; Stearic Acids; Tumor Cells, Cultured

The in vitro effects of hydroxystearic acid on the proliferation of human colon carcinoma cells (HT29) and human embryonic intestine cells (I407) were examined and compared to previous results obtained in murine C108 lung carcinoma cells. The cells were cultured in the presence, or in the absence, of hydroxystearic acid and tested for cell proliferation and viability; the distribution of cells in the cell cycle was evaluated by flow cytometry. Results show that hydroxystearic acid is also an inhibitor of human cell proliferation, and not only of murine C108 cells. Differently from C108 cells, which upon treatment with hydroxystearic acid accumulate in G2-M phases, hydroxystearic acid-treated HT29 cells increase significantly in numbers in G0-G1; I407, embryonic cells used as a control, when treated show only a slight increase in G0-G1.

Topics: Adenocarcinoma; Animals; Cell Cycle; Cell Division; Cells, Cultured; Colonic Neoplasms; Depression, Chemical; Humans; Intestines; Lung Neoplasms; Mice; Stearic Acids; Tumor Cells, Cultured