Mechanism and stereoselectivity of HDAC I inhibition by (R)-9-hydroxystearic acid in colon cancer.

9-Hydroxystearic acid (9-HSA) belongs to the endogenous lipid peroxidation by-products that decrease in tumors, causing as a consequence the loss of one of the control mechanisms on cell division. It acts as a histone deacetylase (HDAC, E.C 3.5.1.98) inhibitor, and the interaction of the two enantiomers of 9-HSA with the catalytic site of the enzyme, investigated by using a molecular modelling approach, has been reported to be different. In this work we tested out this prediction by synthesizing the two enantiomers (R)-9-HSA (R-9) and (S)-9-HSA (S-9) starting from the natural source methyl dimorphecolate obtained from Dimorphotheca sinuata seeds and investigating their biological activity in HT29 cells. Both enantiomers inhibit the enzymatic activity of HDAC1, HDAC2 and HDAC3, R-9 being more active; R-9 and S-9 inhibitory effect induces an increase in histone H4 acetylation. We also demonstrate that the antiproliferative effect brought about by R-9 is more pronounced as well as we observe increase of p21 transcription and protein content, while the expression of cyclin D1 is decreased. Starting from these observations it can be hypothesized that the interaction of R-9 with HDAC1 induce conformational changes in the enzyme causing loss of its interaction with other proteins, like cyclin D1 itself.

Substituted 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinones and analogues: synthesis, cytotoxic activity, and study of the mechanism of action.

The synthesis of substituted 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinones and analogues is reported. Their cytotoxic activity was evaluated according to protocols available at the National Cancer Institute (NCI), Bethesda, MD. The action of selected compounds was examined for potential inhibition of tubulin assembly in comparison with the potent colchicine site agent combretastatin A-4. The most potent compounds also strongly and selectively inhibited the phosphorylation of the oncoprotein kinase Akt in cancer cells. The effect of the most interesting compounds was examined on the growth of HT-29 colon cancer cells. These compounds caused the cells to arrest in the G2/M phase of the cell cycle, as would be expected for inhibitors of tubulin assembly.

Substituted E-3-(3-indolylmethylene)-1,3-dihydroindol-2-ones with antitumor activity. In depth study of the effect on growth of breast cancer cells.

The synthesis of new substituted E-3-(3-indolylmethylene)-1,3-dihydroindol-2-ones is reported. The antitumor activity was evaluated according to protocols available at the National Cancer Institute (NCI), Bethesda, MD. Structure-activity relationships are discussed. The action of selected compounds was investigated in MCF-7 breast cancer cells. The ability of these derivatives to inhibit cellular proliferation was accompanied by increased level of p53 and its transcriptional targets p21 and Bax, interference in the cell cycle progression with cell accumulation in the G2/M phase, and activation of apoptosis.

Histone post-translational modifications by HPLC-ESI-MS after HT29 cell treatment with histone deacetylase inhibitors.

The goal of the present work is to establish a correlation between the degree of histone post-translational modifications and the effects caused by treatment of HT29 colon cancer cells with class I-selective (MS-275 and MC1855), class II-selective (MC1568), and non-selective (suberoylanilide hydroxamic acid (SAHA) histone deacetylase inhibitors (HDACi). This correlation could afford a mean to better understand the mechanism of action of new, more potent, and selective HDACi directly on the cells. To this end, LC coupled to MS was applied in studies of time and concentration-dependent treatment with HDACi in HT29 cells. The results were correlated to their potency of histone deacetylase inhibition and to their effects on the cell cycle. The results indicate that the four tested inhibitors show a different pattern of time- and concentration-dependent modification after treatment of HT29 cells. At the selected concentrations, they cause different histone hyperacetylation and different cell cycle effects. In particular, SAHA (non-selective HDACi) affected hyperacetylation of all histones and caused massive cell death. MC1855 (class I-selective HDACi, hydroxamate) proved to be more potent and less toxic (cell arrest in G2/M phase) than SAHA. MS-275 (class I-selective HDACi, benzamide) exhibited a higher degree of hyperacetylation of H4 and a lower degree of H2A, H2B, and H3 acetylation, causing a cell arrest in G0/G1 phase. On the contrary, MC1568 (class II-selective HDACi) produced only a modest hyperacetylation of H4, was ineffective on the other histones, and showed no effect on cell cycle in HT29 cells.

The regulatory elements of araBAD operon, contrary to lac-based expression systems, afford hypersynthesis of murine, and human interferons in Escherichia coli.

The overexpression of four different interferons, i.e., murine interferon alpha1 and human interferons alpha1, alpha 8, and alpha 21 was challenged in Escherichia coli. Synthetic genes coding for these interferons were designed, assembled, and cloned into the vector pET9a (using the NdeI and BamHI sites), placing interferon expression under the control of phage T7 promoter. Despite an intensive screening for optimal culture conditions, no interferon synthesis was observed using overexpression systems based on the regulatory elements of lac operon (e.g., in E. coli BL21DE3). On the contrary, high levels of interferon expression were detected in E. coli BL21AI, which chromosome contains the gene coding for phage T7 RNA polymerase under the control of the araBAD promoter. To analyze the reasons of this striking difference, the molecular events associated with the lack of interferon expression in E. coli BL21DE3 were studied, and murine interferon alpha1 was chosen as a model system. Surprisingly, it was observed that this interferon represses the synthesis of T7 RNA polymerase in E. coli BL21DE3 and, in particular, the expression of lac operon. In fact, by determining beta-galactosidase activity in E. coli BL21AI, a significantly lower LacZ activity was observed in cells induced to interferon synthesis.

Antitumor activity of new substituted 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinones and 3-(5-imidazo[2,1-b]thiadiazolylmethylene)-2-indolinones: selectivity against colon tumor cells and effect on cell cycle-related events.

The synthesis of new 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinones and 3-(5-imidazo[2,1-b]thiadiazolylmethylene)-2-indolinones is reported. The antitumor activity was evaluated according to the protocols available at the National Cancer Institute (NCI), Bethesda, MD. To investigate the mechanism of action of the most potent antitumor agent of this series, its effect on growth of HT-29 colon carcinoma cells was studied. Its ability to inhibit cellular proliferation was mediated by cell cycle arrest at the G2/M phase, accompanied by inhibition of ornithine decarboxylase (ODC), the limiting enzyme of polyamine synthesis, and followed by induction of apoptosis.

Parallel synthesis and cytotoxicity evaluation of a polyamine-quinone conjugates library.

A library of 24 derivatives designed by combining two natural products-derived fragments was prepared and tested to determine their anticancer potential in HT29 colon cancer cells. All library members inhibit cell proliferation as measured by MTT mitochondrial functional assay, with IC50 values in the 1-100 microM range. Entry 1b caused apoptotic EGFR-mediated intracellular signaling. Thus, polyamino-quinones emerged as readily accessible and easily diversified scaffolds for anticancer lead discovery.

Analysis of human histone H4 by capillary electrophoresis in a pullulan-coated capillary, LC-ESI-MS and MALDI-TOF-MS.

The object of the present study was the analysis of the human histone H4 (a core histone) in order to evaluate the state of its acetylation. Capillary electrophoresis (CE) using a pullulan-coated capillary provides a rapid and efficient approach to the separation of monoacetylated, diacetylated and triacetylated H4 isoforms from human cells. By using a simple running buffer of 100 mM triethanolamine-phosphate solution at pH 2.5 and exploiting the effectiveness of pullulan-based coverage in preventing adsorptive phenomena, the separation of the differently acetylated isoforms was achieved in less than 15 min with high efficiency and reproducibility. The proposed method was for the first time applied in the analysis of histone H4 fractions obtained from cell lines treated with different histone deacetylase (HDAC) inhibitors, used as potential anticancer drugs. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) analysis demonstrated that the acetylation occurred in the histone H4 tail, whereas the CE separation allowed for a fast determination of the percentages of H4 acetylated isoforms in real samples; the results were in agreement with those obtained from liquid chromatography electrospray ionisation mass spectrometry (LC-ESI-MS) analysis. Therefore, the proposed CE method is a useful complementary support to the hyphenated techniques for the rapid monitoring of the activity of HDAC inhibitors.

New antitumor imidazo[2,1-b]thiazole guanylhydrazones and analogues.

The synthesis of new antitumor 6-substituted imidazothiazole guanylhydrazones is described. Moreover, a series of compounds with a different basic chain at the 5 position were prepared. Finally, the replacement of the thiazole ring in the imidazothiazole system was also considered. All the new compounds prepared were submitted to the NCI cell line screen for evaluation of their antitumor activity. A few selected compounds were submitted to additional biological studies concerning effects on the cell cycle, apoptosis, and mitochondria.

Substituted E-3-(2-chloro-3-indolylmethylene)1,3-dihydroindol-2-ones with antitumor activity. Effect on the cell cycle and apoptosis.

The synthesis and antitumor activity of new E-3-(2-chloro-3-indolylmethylene)1,3-dihydroindol-2-ones is described. They were studied at the National Cancer Institute, taking into consideration the 50% growth inhibitory power (pGI50), the cytostatic effect (pTGI = total growth inhibition), and the cytotoxic effect (pLC50). All the compounds were potent growth inhibitors, with mean pGI50 ranging from 5.26 to 7.72. They were also analyzed with NCI COMPARE algorithm. Further studies were dedicated to the effects on the cell cycle and apoptosis.

Green tea protects cytoskeleton from oxidative injury in cardiomyocytes.

Cardiac ischemia/reperfusion injury results in oxidative stress and poor physiological recovery. Episodes of hypoxia/reoxygenation (H/R) cause some subtle functional and structural alterations in sarcolemma, mithocondria, sarcoplasmic reticulum, nucleus, as well as cytoskeleton. In this report, by using cultured rat cardiomyocytes and laser confocal microscopy we have verified the possibility to counteract cytoskeleton alterations induced by H/R with the supplementation of an antioxidant agent, a green tea extract (GTE), and compared its effects to those of alpha-tocopherol. Moreover the effects of GTE on cell viability and cytosolic antioxidant activity have been evaluated. H/R induced myocardial damage occurs as histological alterations such as degeneration and disorganization of the cytoskeleton and loss of structural integrity of the nucleus. GTE supplementation increases cytosolic antioxidant activity and shows protective effects on cardiomyocyte cytoarchitecture and viability.

Synthesis and antitumor activity of guanylhydrazones from 6-(2,4-dichloro-5-nitrophenyl)imidazo[2,1-b]thiazoles and 6-pyridylimidazo[2,1-b]thiazoles(1).

The design and synthesis of antitumor imidazothiazole guanylhydrazones are reported. The compounds were submitted to NCI for testing. All but one were more active than methyl-GAG. A few compounds were selected for further studies in search of a possible mechanism of action. The results from these studies and a final search with the NCI COMPARE algorithm suggest that the guanylhydrazones described in this paper are acting through a novel mechanism of action.

N-methylformamide and 9-hydroxystearic acid: two anti-proliferative and differentiating agents with different modes of action in colon cancer cells.

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.

9-Hydroxystearic acid interferes with EGF signalling in a human colon adenocarcinoma.

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.

Antitumor activity of new substituted 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinones and study of their effect on the cell cycle.

This paper reports the synthesis of a new series of 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinones which were tested as potential antitumor agents at the National Cancer Institute. Two derivatives are now under review by BEC (Biological Evaluation Committee of NCI). To investigate the mechanism of action, the effect on cell cycle progression was studied by monitoring them in colon adenocarcinoma HT-29: both were able to block HT-29 in mitosis. 3-[(2,6-Dimethylimidazo[2,1-b]thiazol-5-yl)methylene]-5-chloro-2-indolinone was the most active compound.

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.

Fluorescein conjugates of 9- and 10-hydroxystearic acids: synthetic strategies, photophysical characterization, and confocal microscopy applications.

Different strategies are presented to conjugate a fluorescein moiety to 9- and 10-hydroxystearic acids (HSAs). 5-Amino-fluorescein (5-AF) was used as a starting reagent. When reacted with acyl-chloride-modified HSAs, 5-AF gave rise to stable amide derivatives with a 75% reaction yield. These products exhibited the typical steady-state and time-resolved fluorescence properties of the fluorescein chromophore with absorption at 494 nm and emission at 519 nm. Flow cytometry studies confirmed the distinct proapoptotic effect of underivatized 9-HSA on Jurkat cells and revealed a comparable ability of its amide derivative. Confocal microscopy imaging studies showed that green fluorescence could stain intracellular membranous structures. Moreover, dual-dye labeling with Mito Tracker Red, followed by colocalization analysis, revealed that HSA can move to the mitochondria. Thus, fluorescent derivatives of HSA can be used to monitor the localization of these biologically active molecules in living cells and can provide a useful tool for linking biochemical investigation with optical visualization methods. In contrast, when unmodified HSAs were used, the reaction gave monoesterified and diesterified fluorescein derivatives. These products exhibited unusual steady-state and time-resolved fluorescence properties with the excitation wavelength at 342 nm and the emission wavelength at 432 nm. It is shown that the synthesized HSA amides of fluorescein provide all of the typical photophysical and instrumental advantages of this popular dye, whereas the unusual luminescence and excitation properties of the monoester and diester of the 5-aminofluorescein would make these dyes interesting to explore as potential candidates for two photon excitation applications.

4-hydroxy-2,3 trans-nonenal nuclear concentration inversely correlates with the growth rate of T leukemia cells.

4-hydroxy-2,3 trans-nonenal is the major diffusible product generated by linoleic and arachidonic acids peroxidation. Its endogenous content is inversely related to the rate of cell proliferation and directly related to the level of cell differentiation. As previously reported, the nuclear localization of the aldehyde has been observed by means of a fluorescent antibody and confocal microscopy, and its concentration measured by electrospray/mass spectrometry, a sensitive and selective method for 4-hydroxy-2,3 trans-nonenal determination, on nuclear extracts of leukemic cells. With the aim to establish a possible correlation between the peroxidation product nuclear concentration and cell growth rate, Jurkat 6 leukemic cells have been used and the aldehyde measured by electrospray/mass spectrometry. The cells arrested in G1 show a content of 4-hydroxy-2,3 trans-nonenal significantly increased with respect to control ones.

Substituted E-3-(2-Chloro-3-indolylmethylene)1,3-dihydroindol-2-ones with antitumor activity.

The synthesis and antitumor activity of a new series of E-3-(2-chloro-3-indolylmethylene)1,3-dihydroindol-2-ones is described. Several compounds were active on the primary test (three human cell lines) and entered the second level (60 human cell lines). All of them were potent growth inhibitors with GI(50) ranging from -5.32 to -7.27. Four are now under review by BEC (Biological Evaluation Committee of the NCI). The most potent antitumor derivatives were also evaluated as cardiotonic agents (in view of a possible coanthracyclinic activity). In order to find a possible mechanism of action their effects on cell cycle progression in an adenocarcinoma cell line (HT29) were tested, evidencing that these molecules are able to block HT29 in mitosis. The introduction of new substituents in the indolinone moiety while maintaining the same chloroindole portion generated interesting derivatives. 3-(2-Chloro-5-methoxy-6-methyl-3-indolylmethylene)5-hydroxy-1,3-dihydroindol-2-one was the most active of the whole series. It was more potent than vincristine against seven of the nine tumors considered. Moreover it was selective towards some cell lines such as MDA-MB-435 (breast), OVCAR-3 (ovarian) and SK-MEL-28 (melanoma). Even the introduction of a benzyl ring at the nitrogen of the chloroindole portion, gave rise to potent compounds.