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High level expression of histone deacetylase 1 (HDAC1) plays a pivotal role in the pathobiology of cancer [1]. The endogenous fatty acid (9R)-9-hydroxystearic acid, 9R, is a natural HDAC1 inhibitor, which exerts its antiproliferative activity by arresting cancer cells growth in G0/G1 phase [2]. However, one of its major limitations is the unfavorable pharmacokinetic that hampers its therapeutic effectiveness. To overcome this constraint, 9R keratin nanoparticles (9R@Ker) were prepared and described here for the first time. Keratin was selected as carrier because possesses excellent biocompatibility and low toxicity to cells, thus resulting very promising material for drug delivery applications [3]. The formation of 200 nm nanoparticles (NPs) was induced by hydrophobic interactions between 9R and the hydrophobic protein domains, affording water-stable NPs with no need of toxic cross-linking agents. NPs were characterized in terms of particles size distribution, zeta potential, morphology, thermogravimetric behavior and drug release profile. Moreover, in vitro uptake and cytotoxicity was evaluated using human colorectal adenocarcinoma cells (HT29). Our data revealed that 9R@Ker are efficiently internalized by tumor cells, altering membrane lipidic composition. In vitro results demonstrate that the activity of 9R as free or loaded onto NPs is similar in terms of anti-proliferative effect. Remarkably, some significant differences were observed in the cell cycle analysis, showing that while free 9R caused a growth arrest in the G0/G1 phase, 9R@Ker induced a S phase arrest, thus promoting apoptosis (Fig.1). Additional studies are ongoing to better elucidate the underpinning biochemical mechanism of action of our newly developed delivery system.

Recent findings identified a new class of endogenous lipids, branched Fatty Acid esters of Hydroxy Fatty Acids (FAHFAs), able to behave as specific signaling molecules that can regulate the cellular metabolism [1]. Among FAHFAs, the Palmitic-Acid-9-Hydroxy-Stearic Acid (9-PAHSA) seems to exert favorable metabolic effects in obesity-related diseases and type 2 diabetes, causing an increase in insulin sensitivity and glucose uptake. It has been recently reported that the FAHFAs content in human serum of breast cancer patients was significantly decreased compared to healthy controls [2], thus it is very interesting to investigate the possible involvement of these lipid molecules also in cancer transformation and progression. Previous studies of our research group have shown that the administration of 9-hydroxy-stearic acid (9-HSA) to colon carcinoma cells (HT29) induces strong antiproliferative and differentiating effects, with a cell cycle arrest in G0/G1 phase [3]. Since 9-HSA can be produced by the hydrolysis of 9-PAHSA, it is conceivable that also this lipid could act as signaling molecule. Consequently, the aim of this research was the characterization of the glucose metabolism of HT29 cells treated with 9-HSA. As a first step, the effect of 9-HSA on the lipid organization of HT29 cells was studied, showing an increase of the fluidity of plasma membrane. The treatment of HT29 with 9-HSA for 1 h provokes a significant increase of the glucose transporter Glut1 (and in a lesser extent of Glut3) on the plasma membrane, as a result of a translocation from intracellular stores, since the level of expression of the two transporters were unchanged, as determined by RT-PCR. Accordingly, the higher amount of Glut1 on the cell surface caused also an increase in glucose uptake into the cells. RT-PCR analysis showed also a significant increase of MCT1, the monocarboxylate transporter, in HT29 cells treated with 9-HSA. MCT1 is commonly overexpressed by cancer cells to maintain lactate and pH homeostasis [4]. Therefore, lactate production was measured in HT29 cells upon 9-HSA treatment for 1 h. Results show that lactate production was significantly increased, indicating a cellular metabolic shift toward glycolysis. The acute metabolic changes observed in HT29 cells are typical cellular responses to a signal molecule, supporting the hypothesis of a signaling role for 9-HSA in cancer cells. 1. M.M. Yore et al. Cell, 159:318-332, 2014. 2. Q.F. Zhu et al., J. Chromatogr. B, 1061-1062:34-40, 2017. 3. N. Calonghi et al., Biochem. Biophys. Res. Commun., 314:138-142, 2004 4. J. Adijanto and N.J. Philp, Curr. Top. Membr., 70:275-311, 2012.

The goal of this study was to provide data on the dose-dependent production of dicentrics and micronuclei in human lymphocytes irradiated with 22.6 MeV protons and to estimate the possible contribution of intracellular superoxide dismutases (SOD) to the relative biological effectiveness (RBE) of protons. For the dose-response study, heparinized whole blood of a healthy volunteer was irradiated with protons and X-rays employing radiation doses of 0.5-4 Gy. Three biological endpoints were analyzed: chromosomal aberrations, micronuclei, and specific activity of cytosolic (CuZnSOD) and mitochondrial (MnSOD) superoxide dismutases in harvested human blood cells. Dicentric dose-response curves fit a linear-quadratic form (alpha = 0.094 +/- 0.006, beta = 0.032 +/- 0.001) induced with X-rays and (alpha = 0.119 +/- 0.057, beta = 0.029 +/- 0.014) for 22.6 MeV protons. Protons were more effective than X-rays in producing exchanges, particularly at 0.5 and 1 Gy. In contrast to X-ray irradiated samples where a significant increase in the specific activity of MnSOD was recorded (up to a radiation dose of 1 Gy), irradiation with protons markedly reduced its activity. As a consequence of the reduced activity of MnSOD, the chromosomal dose-response curve became quadratic. The RBE for dicentrics varies with dose (from 2.2 to 1.01) and reduced activity of MnSOD is an important contributor to the RBE of protons. SODs, particularly MnSOD, play an important role in defending DNA from reactive oxygen species. A reduced activity of SOD, particularly MnSOD, is an important contributor to the RBE of protons.

Introduction. Sulforaphane (SFN), a dietary isothiocyanate isolated from Brassicaceae, has been shown to prevent different cancers in laboratory animals [1]. In particular, SFN protects against colon carcinogenesis in vivo and causes a G0/G1 growth arrest and apoptosis in human colon cancer cells, by inducing p21Cip1/Waf1[2]. SFN also acts as an inhibitor of histone deacetylases (HDACs) in human embryonic kidney 293 cells and HCT116 colon cancer cells [3]. The objective of this study is to evaluate the ability of the HDAC inhibitor SFN to induce cytotoxic and cytostatic effects in HT29 colon cancer cell line. Materials and methods. Cell viability was evaluated by measuring MTT reduction. The cells were plated in 6-well dishes at equal density, grown for 24 hours, and then treated with 5 M SFN. 3H-thymidine (1 μCi/mL) was added for the last 6 hours of the incubation. The cells were washed in ice-cold PBS and 5 % trichloroacetic acid was added for 30 minutes at 4 °C, and then incubated with 0.5 N NaOH for 1 hour at 50 °C. Cell lysates were assayed for protein content by Bio Rad assay kit and measured for incorporated radioactivity. Counts were normalized for total cellular protein. To test SFN effects on HDAC activity, the total histone acetylation level was measured by pulse labelling experiments with 3H-acetate and the acetylation status of the histone classes were assayed by Western Blot and HPLC/MS. Results. SFN negatively affects HT29 growth in a dose- and time-dependent manner with a 24h IC50 equal to 22.3 M  2.4. At concentrations as low as 5 M, a significant inhibition of cell proliferation is observed, accompanied by a 47% decrease of the mitotic index, with respect to the control. Histones extracted from SFN treated HT29 cells show a 63% increase in the acetylation status; in particular SFN markedly prolonges the half- life of the acetyl groups on histone H4.

In the present study, assays were improved for the determination of catecholamines in human plasma. High-performance liquid chromatography with electrochemical detection was employed for quantitative analysis. The influence of various parameters on chromatographic performance, such as the composition and the pH of the mobile phase, and the detection potential, was investigated. An accurate solid-phase extraction procedure, after catecholamine complexation with diphenylborate, was developed. The efficiency yield for all catecholamines was in the range 92-98%. Relative standard deviation values for repeatability and for intermediate precision were less than 2% and 3%, respectively, for all three analytes.

Enantioenriched 7-, 8-, 9-, and 10-hydroxystearic acids (HSA) were obtained, for the first time, by kinetic resolution of their racemates with lipases CALB and PS, in the presence of vinyl acetate. Among them, the best results were obtained for 7-HSA and 9-HSA, whose enantiomeric excess was around 55%. The same resolutions carried out on the hydroxy esters completely failed. For the acid substrates neither the Kazlauskas’ rule nor the 3D-QSAR model could be applied, since both models are focused on the CALB alcohol-pocket evaluation and not on the acyl-pocket one. Therefore, a semiquantitative approach was used, whose results were in accordance with our findings, as far as the absolute configuration of the product is concerned.

A physical hydrogel prepared with the low-molecular-weight hydrogelator (LMWHG) CH2(C3H6CO-L-Phe-D-Oxd-OH)2 and water/ethanol mixture was applied as a potential “Trojan Horse” carrier into cells. By SEM and XRD analysis we could demonstrate that a fibrous structure is present in the xerogel, making a complex network. The gelator is derived from α-amino acids (Thr, Phe) and a fatty acid (azelaic acid) and is biocompatible: it was dosed to IGROV-1 cells, which internalized it, without significantly affecting the cell proliferation. To check the internalization process by confocal microscopy, fluorescent hydrogels were prepared, introducing the fluorescent dansyl moiety into the mixture.

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.

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.

Mineralized tissues grow through biologically controlled processes in which specific macromolecules are involved. Some of these molecules, which are present in very low concentrations and are difficult to localize and characterize, become entrapped inside the mineralized tissue. Herein, a protein fragment, GP, which was obtained by the alkaline digestion of the green sheet of the abalone shell, is used as a probe to study the changes in molecular structure that occur during the precipitation of calcium carbonate. This important goal was achieved by exploiting a fluorescent tag in GP. The experimental results that were obtained by using spectroscopic-, chromatographic-, and microscopic techniques indicate that GP controls the precipitation kinetics and morphology of calcium carbonate crystals, and that it only undergoes structural reorganization when entrapped inside calcium carbonate crystals. To the best of our knowledge, this report represents one of the first studies on the conformational changes of a protein fragment that is involved in biomineralization processes on moving from the solution phase into the mineral phase.

The abilities of an ideal wound dressings can be summarised as follows: increase healing, decrease pain, safety, cost-effectiveness, flexibility, moisture-permeability and long term activity. However, controlling the release of water soluble drugs from bioactive dressings is challenging because of their hydrophilic nature. To that aim, several approaches have been undertaken, involving the formation of inter- and intra-molecular covalent bonds, control of morphology and surface modification options or synthesis of new composite polymers. This study concerns on an original formulative/manufacturing approach for the development of a multi-composite wound dressing able to control the release of a water soluble API (lidocaine HCl) for several days. The prepared multi-composite wound dressing is a microstructured spongy matrix, which embeds solid lipid microparticles (SLMs). The release control is due to a particular ratio between two biopolymers (chitosan and alginate) and to the presence of SLMs within the polycomplex. SLMs have been prepared by spray congealing, a low cost, easy and solvent free technology; then they have been incorporated into different polyelectrolyte complexes, which have been freeze dried to obtain the spongy matrices. Moreover, the long-term delivery system, with the addition of extra cellular matrix components as hyaluronan and cysteine, is able to improve the proliferation of fibroblasts as observed by confocal laser scanning microscope, suggesting the potential usefulness of this multifunctional platform in the wound healing process. It can be concluded that the combination of two simple technologies and of different ready available biomaterials produced interesting results in the wound healing process.

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 lastly activation of apoptosis.

It has been reported that the interaction of the two enantiomers of 9-HSA with the catalytic site of hystone deacetylase, using a molecular modelling approach, is different. The interaction energy of the (R) enantiomer was lower as compared with the (S) indicating a more stable complex formation (N. Calonghi et al 2005). In this work (R) and (S)-9HSA were isolated and their biological activity tested in HT29 cell line. The conclusions that can be drawn from this work can be summarized as follows: (i) Both enantiomers inhibit the enzymatic activity of HDAC1, HDAC2 and HDAC3, being (R) more active; (ii) (R) and (S) inhibitory effect induces an increase in histone H4 acetylation; (iii) the antiproliferative effect induced by (R) is more pronounced as much as the increase of p21 transcription and protein level, while the expression of Cyclin D1 is decreased. It can be hypothesized that the interaction of (R)-9HSA with HDAC1 may induce conformational changes in the enzyme causing loss of its interaction with other proteins, like cyclin D1 that, being released from the complex, can be ubiquitinated, thus losing the interaction with CDK and thereby stopping the cell proliferation.

Histone proteins are subject to a range of post-translational modifications in living cells. The combinatorial nature of these modifications reflects in the so called "histone code" that modulates chromatin structure and function during development, growth, differentiation, and homeostasis of cells. Histone acetylation and deacetylation are controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs) enzymes. HDAC inhibitors (HDACi) increase acetylation levels, induce open chromatin structure and increased gene transcription, being found to function as potential chemotherapeutic in tumour cell types (1). Deciphering of the histone code is hampered by the lack of analytical methods for monitoring the combinatorial complexity of reversible multisite modifications of histones, including acetylation and methylation. To address this problem, it is here reported a LC-MS approach for qualitative and quantitative proteomic analysis of histones extracted from human colon cancer cells after treatment with HDACi. LC-MS-based methods were applied in a quantitative proteomic study of the dose and time response effect of HDACi on histone acetylation in HT29 colon cancer cell cultures. A correlation was established between the degree of histone post-translational modifications and the cell cycle effects caused by treatment of HT29 colon cancer cells with selective and non selective HDACi. This correlation affords a mean to better understand the mechanism of action of new, more potent and selective HDACi directly on the cells. On the basis of these studies, LC-MS-based quantitative proteomic analysis of histone post-translational modifications results as a viable approach for functional analysis of colon cancer candidate drugs, such as HDACi.

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.

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.

A promising strategy for protecting cardiac cells against oxidative damage may be through the induction of endogenous phase 2 enzymes. Sulforaphane (SF), a naturally occurring isothiocyanate from Brassicaceae, demonstrated a strong chemopreventive effect mediated by Nuclear factor E2-related factor-2 (Nrf2), a transcription factor that binds to the Antioxidatn responsive Element orchestrating antioxidant and cytoprotective responses. No data are available to support a similar role of SF in cardioprotection. Using cultured rat cardiomyocytes we have characterized the time-dependent gene induction of phase 2 enzymes and Nrf2 elicited by SF, the corresponding protein expression and Nrf2 translocation to the nucleus. Methods: Cultured rat cardiomyocytes were supplemented with 5 mM SF for different times (6-48 h). The gene induction of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST), NAD(P)H-quinone reductase 1 (NQO1), thioredoxine reductase (TR) and Nrf2 were determined by RT-PCR. Western blot analyses of GR, GST, GPx, TR, NQO1 and Nrf2 were performed using specific antibodies. Nrf2 translocation to the nucleus was evaluated by laser confocal microscopy. Results: Both the mRNA and protein levels of GR, GST, NQO1 and TR significantly increased after SF supplementation in a time-dependent manner. GR, GST, NQO1 mRNA increased after 3h treatment, while all protein expressions appear significantly different from unsupplemented cells only at longer exposure times (24, 48h) apart of NQO1 and TR which expressions increased after 6h and 12h respectively. This is probably due to the delay between gene induction and active protein synthesis and confirm our previous data on the time course of phase 2 enzymes activities (1). GPx was not affected by SF at any time point. SF caused an increase of Nrf2 mRNA and laser confocal microscopy evidenced its nuclear translocation. In this study we have demonstrated that SF modulates phase 2 enzymes and Nrf2 induction in cardiomyocytes, eliciting an indirect antioxidant activity to counteract oxidative stress that characterizes many cardiovascular diseases. Supported by MIUR (PRIN 2005) and Fondazione del Monte di Bologna e Ravenna 1. E. Leoncini et al., Nutrition, Oxygen Biology and Medicine (P.Cillard, L.Packer et al eds)2007,55

Among active compounds introduced with diet or originating from cellular metabolism, 9-hydroxystearic acid 89-HSA) and sulforaphane (SFN) are two promising modulators of cell proliferation and differentiation. 9-HSA is a dietary fatty acid, contained in apple skin, in the cutin of Finnish berries and in the seed oil of sea bucktorn, as well as an endogenous lipoperoxidation product, identifiee in several normal and neoplastic human cell lines. SFN is the major isothiocyanate derived from Brassicaceae, largely studied for its chemiopreventive activity. They both exhibit antiproliferative effects, inducing growth arrest and apoptosis in various cancer cell lines. In particular, their administration to HT29, a human colon adenocarcinoma cell line, causes a proliferative arrest mediated by a direct activation of p21WAF1 gene, bypassing p53. These effects have been found to be related to the inhibition of histone deacetylases (HDACs): despite their different chemical structures, 9-HSA and SFN are both able to directly interact with the catalitic site of HDAC1, leading to a reduced enzymatic activity. In this study we have investigated 9-HSA and SFN effects on core histones acetylation status, and its correlation with HT29 cell growth and differentiation.

The nucleosome is the basic structural unit of eukariotic chromosomes and consists of a DNA molecule associated with a histone octamer comprised of pairs of the core histones H2A, H2B, H3 and H4. Linker DNA and histone1 to form chromatin join the nucleosomes. Histones play an important role in transcription, DNA replication, DNA repair and recombination. Post-translational modifications of specific residues in the core histones (acetylation, methylation, phosphorylation, etc.) have been demonstrated to be critical to their regulatory function. In particular acetylation is a very specific phenomenon with various isoforms playing distinct roles. Increased acetylation is generally correlated with transcriptionally active or poised genes. Histone deacetylases’ inhibitors (short chain fatty acids, such as sodium butyrate, hydroxamic acids such as thrichostatin A) have been described as potential cancer therapeutics in a variety of preclinical studies1. Inhibitors’ treatment of cells resulted in the increase of highly acetylated isoforms of the histones, which affect gene expression, cell differentiation and apoptosis. It is here described the application of reversed-phase high-pressure liquid chromatography under gradient conditions and mass spectrometry (LC-ESI-MS) to analyse global modification levels of core histones. The LC-MS method was optimised using histones extracted from HT 29 colon cancer cell line2. Histones eluted from the column were detected with Q-Tof and ion trap mass spectrometers with an electrospray source put in parallel and UV detection at 214 nm. These methods were then applied to the characterisation of changes in histone modification in HT29 treated with histone deacetylase inhibitors such as valproic acid, sodium butyrate and 9-hydroxystearic acid. Acetylation were found located in the H4 histone tail by MALDI-Tof analysis of tryptic and arginase peptide digests. 1Rasheed WK, Johnstone RW, Prince H,Histone deacetylase inhibitors in cancer therapy Expert Opin Investig Drugs. 2007 May;16(5):659-78. 2Naldi M, Andrisano V, Fiori J, Calonghi N, Pagnotta E, Parolin C, Pieraccini G,Masotti L. Histone proteins determined in a human colon cancer by high-performance liquid chromatography and mass spectrometry. J Chromatogr A. 2006 Sep 29;1129(1):73-81.

There is substantial evidence to support the theory that oxidative stress plays an important role in the pathophysiology of CVD (1). Accordingly, several naturally-occurring antioxidant compounds have been utilized to counteract oxidative cardiac injury (2,3). Another promising strategy for protecting cardiac cells against oxidative stress may be through the induction of endogenous antioxidants and phase II enzymes. Sulforaphane (SF) is a naturally-occurring isothiocyanate that is highly concentrated in Cruciferous vegetables. Many studies have shown a strong chemopreventive effect of SF through its ability to induce phase II detoxifying enzymes by activating antioxidant-response element (ARE) through the induction of Nrf2 (4), but no data are available to support a similar role for SF in cardioprotection. Using cultured rat cardiomyocytes, the time-dependent induction of cellular antioxidants and phase II enzymes by SF and its ability to protect cardiac cells against oxidative stress have been characterised, and the translocation of nrf2 to the nucleus after SF supplementation has been investigated. Cultured rat cardiomyocytes were prepared and grown as described previously (5). Cells were supplemented with 5 mM-SF for different time periods (6, 12, 24 and 48 h). The activities of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST) and NAD(P)H-quinone reductase 1 (NQO1) were determined spectrophotometrically, and the content of intracellular reduced glutathione (GSH) was estimated using the fluorescent indicator monochlorobimane. Western-blot analyses of GR, GST, GPx and NQO1 were performed using specific antibodies and following the manufacturer’s recommended protocols. ROS formation was determined by a spectrofluorimetric method using 2',7'-dichlorofluorescein diacetate. Cell viability was evaluated by MTT assay and Nrf2 translocation to the nucleus by laser confocal microscopy using specific antibodies. Incubation of cardiomyocytes with SF resulted in a significant elevation of cellular GSH content for all exposure times. SF supplementation also led to a time-dependent increase in GR, GST and NQO1 activities. Accordingly, a significant increase in GR, GST and NQO1 expression was observed. In contrast, incubation of cardiomyocytes with SF for 6–48 h did not result in any significant increase in cellular GPx activity and expression, in agreement with published data (6). Laser confocal microscopy revealed the translocation to the nucleus of Nrf2 after SF supplementation. SF pretreatment led to a decreased intracellular accumulation of ROS and marked cytoprotection after exposure to 100 μM-H2O2. The results demonstrate, for the first time, that a number of endogenous antioxidants and phase II enzymes can be induced in cultured cardiomyocytes by low micromolar concentrations of SF, and that this nutritionally-mediated up-regulation of cellular defences is accompanied by a markedly increased resistance to cardiac cell injury elicited by peroxide. 1. Ceconi C, Boraso A, Cargnoni A & Ferrari R (2003) Arch Biochem Biophys 420, 217 -21. 2. Angeloni C, Spencer JP, Leoncini E, Biagi PL & Hrelia S (2007) Biochimie 89, 73-82. 3. Bandyopadhyay D, Chattopadhyay A, Ghosh G & Datta AG (2004) Curr Med Chem 11, 369-387. 4. Fimognari C & Hrelia P (2007) Mutat Res 635, 90-104. 5. Hrelia S, Fiorentini D, Maraldi T, Angeloni C, Bordoni A, Biagi PL & Hakim G (2002) Biochim Biophys Acta 1567, 150–156. 6. Cao Z & Li Y (2004) Eur J Pharmacol 489, 39–48.

The application of reversed-phase high-pressure liquid chromatography under gradient conditions and electrospray ion trap mass spectrometry (LC–ESI–MS) to the analysis of global modification levels of core histones is described. The optimised LC–ESI–MS method was applied for the first time to the characterisation of histones extracted from HT29, a human colon cancer cell line. Eight histones (H1-1, H1-2, H2A-1, H2A-2, H2B, H3-1, H3-2, H4) were separated on a C4 stationary phase with complete resolution, never reached in previous HPLC–MS methods, by using a gradient elution with the combined presence of heptafluorobutyric acid and formic acid as acidic modifiers in the mobile phase. Heptafluorobutyric acid was found to improve selectivity, whereas the presence of formic acid decreased ion suppression. Histones eluted from the column were detected with an ion trap mass spectrometer with an electrospray source. The peak averaged mass spectra were reconstructed by Mag Tran 1.0 software and the mass of the various isoforms of histones were derived. Method validation was conducted by performing the same sample analysis by coupling LC–ESI to a quadrupole-time-of-flight mass spectrometer (Q-TOF). The number of histone forms and their mass were found to differ not significantly from those obtained by ion trap mass spectrometer. Also the relative modifications abundance within the same histone type was found following the same trend as the two mass analysers. This method was then applied to the characterisation of changes in histone modification in HT29, never analysed by LC–MS before, treated with histone deacetylase inhibitors such as valproate and sodium butyrate, also used in preclinical trials as anticancer drugs. In particular, both the inhibitors produced a significant increase in H4 histone acetylated forms: 89% increase of the diacetyl dimethyl H4 form was observed with 1 mM valproate supplementation, whereas 5 mM butyrate led to a 68% increase of the same form. Triacetyl monomethyl H4 (11 377 Da) and triacetyl dimethyl H4 (11 390 Da) were found only in cells treated with butyrate. Selective changes of H3 histone were detected with butyrate, in agreement with recently reported western blotting studies. Modifications in the H2A histone degree of acetylation were revealed by treatment of the cells with butyrate (H2A-1, H2A-2) and valproate (H2A-2). The results of the proposed methodology confirmed that inhibition of histone deacetylases caused histone hyperacetylation, responsible for decondensation and reorganization of interphase dynamic chromatin. This method resulted in selective and sensitive method to monitor variations in the acetylation and methylation state of histones after treatment of HT29 with inhibitors, and is therefore suitable for further application in new drug discovery for tumour therapy.

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.

Introduction. Sulforaphane (SFN), a dietary isothiocyanate isolated from Brassicaceae, has been shown to prevent different cancers in laboratory animals [1]. In particular, SFN protects against colon carcinogenesis in vivo and causes a G0/G1 growth arrest and apoptosis in human colon cancer cells, by inducing p21Waf1[2]. SFN also acts as an inhibitor of histone deacetylases (HDACs) in human embryonic kidney 293 cells and HCT116 colon cancer cells [3]. The objective of this study is to evaluate the ability of the HDAC inhibitor SFN to induce antiproliferative and differentiating effects in HT29 colon cancer cell line. Materials and methods. Cell viability was evaluated by measuring MTT reduction. Cell proliferation was assayed by measuring 3H-thymidine incorporation and by normalizing counts to mg protein. To test SFN effects on HDAC activity, the total histone acetylation level was measured by pulse labelling experiments with 3H-acetate and by Western Blot by labelling a nitrocellulose membrane with an anti-acetylated lysine antibody. Histone classes were separated and identified by HPLC/MS, and the post-translational modifications were determined. The effects of SFN on cell differentiation and invasivity were detected after 120h administration by immunofluorescence microscopy, by the kinetic determination of alkaline phosphatase activity, and by means of a cell invasion kit. Results. SFN negatively affects HT29 growth in a dose- and time-dependent manner with a 24h IC50 equal to 22.3 M  2.4. At concentrations as low as 5 M, a significant inhibition of cell proliferation is observed, accompanied by a 47% decrease of the mitotic index, with respect to the control. Histones from SFN treated cells show a 63% increase in the acetylation status; in particular SFN selectively prolongs the half- life of the acetyl groups on histone H4. These data are associated to a diminished cell invasivity, that decreases of 25% in SFN treated HT29. Antiproliferative and differentiating effects of SFN can therefore be attributed to the inhibition of HDAC. References. [1] Chung F.L., et al. (2000) Carcinogenesis 21, 2287–2291. [2] Shen G., et al. (2006) Cancer Chemother Pharmacol 57, 317–327. [3] Myzak M.C. et al. (2004) Cancer Research 64, 5767–5774

INTRODUCTION The endogenous lipoperoxidation product 9-hydroxystearic acid (9HSA) is an inhibitor of HDAC1 and its administration to HT29, a colon adenocarcinoma cell line, induces a proliferative arrest mediated by a direct activation of the p21 WAF 1 gene, bypassing p53 [1]. In a recent work the interaction of 9-HSA with the catalytic site of the 3D model of HDAC1 has been tested with a docking procedure. Noticeably, when interacting with the site, the (R)-9-enantiomer is more stable than the (S) one: in fact the energies of interaction are -8.45 kcal/mol and -1.97 kcal/mol for the (R) and (S) isomer, respectively, and the estimated free energies of binding are -6.31 kcal/mol and +4.98 kcal/mol for (R) and (S), respectively [2]. In this work (R) and (S)-9HSA were isolated and their biological activity tested in HT29. MATERIALS AND METHODS (R)-9HSA and (S)-9HSA were obtained in enantiomerically pure forms, through a multistep procedure starting from Dimorphotheca sinuata seeds. Cell viability was evaluated by MTT assay. HT29 cells were treated with 50 μM of (R)-9HSA or (S)-9HSA after 24 hours of seeding and their effects on cell growth were evaluated by flow citometry. The effect of the two enantiomers on the HDAC1 activity was tested as histone acetylation level by pulse labelling experiments with 3H-acetate. Histone isoforms were separated and identified by HPLC/MS to determine post-translational modifications. RESULTS At the concentration of 50 μM R 9HSA shows a stronger antiproliferative effect in HT29 cells in comparison with the S isomer, as indicated by a more remarkable arrest in G0/G1 associated to the higher hyperacetylation of nucleosamal histones. These results show for the first time that (R)-9HSA induces antiproliferative effects at concentrations lower than those of the racemic mixture and they suggest that the interaction with HDAC1 is strictly stereospecific. [1] Calonghi N. et al. (2004) BBRC 314:138-142.

INTRODUCTION: 9-hydroxystearic acid (9HSA) induces cell proliferation arrest in G0/G1 by inhibiting HDAC1 and inducing p21 expression. Serum-starved cells are known to exit G1 and enter the G0 phase, but treatment with either serum or specific growth factors induces them to reenter the cycle. On the contrary, 9HSA treated cells, if stimulated with EGF, are not responsive and undergo a further arrest. In the effort of understanding the possible mechanisms of this effect we analysed the degree of internalisation of the EGF/EGFR complex and the interactions between the complex and other proteins, such as HDAC1 and pRb. The phosphorilation level of EGFR was indicative of its activity in the cells. Cyclin D1 promotor being one of the most important EGF/EGFR target involved in cell proliferation, we also analysed its expression, complexation, and localization with HDAC1. MATERIALS AND METHODS: HT29 colon adenocarcinoma cells, serum starved (SS) were treated with 9HSA, EGF, or both. By citofluorimetry we analysed the levels of EGF/EGFR internalisation and cyclin D1 expression. Cyclin D1 expression, HDAC1/cyclin D1 and HDAC1/EGFR complexes were analysed by confocal microscopy. Furthermore EGFR activity and interactions with HDAC1 and pRb were evaluated by co-immunoprecipitation and WB. RESULTS: In response to EGF treatment, SS cells exhibited an increase in 3H-thymidine incorporation, whereas 9HSA treated cells were uresponsive to EGF. There was no quantitative difference in EGF/EGFR internalisation as seen in citofluorimetry. There was no quantitative difference in cyclin D1 expression in 9HSA treated- and EGF stimulated-HT29. The interaction between cyclin D1 and HDAC1 was visible in cells that respond to EGF after serum starvation but not after 9HSA treatment. EGFR/HDAC1 complex quantitatively increased in 9HSA treated but not in SS cells after EGF stimulation and localised outside the nucleus: this does not allow HDAC1 to interacts with cyclin D1. PRb was increased in its low phosphorilation condition by EGF stimulation in 9HSA treated cells, justifing the further arrest of the cell proliferation. EGFR was phosphorilated in cells treated with 9HSA and stimulated with EGF, as if the mitogen signal was blocked. This work was supported by grants from COFIN 2004

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.

Introduction. 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 (1). 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 -tocopherol (-TC). 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 cytoskeleton and nuclear structural integrity. GTE supplementation shows protective effects on cardiomyocyte cytoarchitecture and viability, due to GTE ability to increase cytosolic antioxidant activity. Materials and Methods. Primary heart cell cultures were obtained by isolation of cardiomyocytes from the ventricles of 2–4 day old Wistar rats. In some dishes, 50 μg/mL GTE or 20 μM -TC were added to the culture medium 24 hours before the induction of hypoxia. Cell viability and cellular damage elicited by H/R treatment was evaluated by measuring MTT reduction. In both normoxic condition and H/R, cytosolic antioxidant activity was determined in cardiomyocytes using the method based on the ability of the antioxidant molecules in the sample to reduce the radical cation of ABTS, and measured as quenching of the absorbance at 740 nm. The effects of hypoxia on the microtubule network (MT) and on the nuclear morphology were investigated by fluorescence and immunofluorescence methods. MT was stained and integrity, in the absence or presence of GTE or -TC, was observed by confocal microscopy. Results. The ability of cardiomyocytes to reduce MTT was drastically reduced after H/R (67.32 ± 4.52%, p

Constitutive tyrosine kinase (TK) activity of p210 bcr-abl fusion protein is the causative event of Chronic Myeloid Leukemia (CML). Accordingly, its inhibition abrogates the proliferative advantage and apoptosis resistance of clonal leukemic progenitors and induces the disease remission and the emergence of normal hematopoiesis. Imatinib mesylate (IM), the first TK inhibitor approved for clinical use in CML, let to hope to have a cure for Ph1-(bcr-abl)-positive leukemias. Unfortunaley, the IM resistance apparent from longer clinical follow-up emerged as the drug major limitation. It imposes the development of combined therapies capable of targeting genes located downstream of bcr-abl and endowed with a bcr-abl-independent role in CML progression towards the fully transformed phenotype of blast crisis. Src kinase inhibitors are now regarded as the most promising drugs as they target p210 constitutive phosphorylation at Tyr177. We investigated the in vitro effects of a new compound, a 4-anilino-3-quinolinecarbonitrile (SKI-606) alone or in combination with IM on proliferation and survival of bcr-abl-transducing 32D cell clones. Here we demonstrate that SKI606 and IM combined treatment is more effective than IM alone in inhibiting the enzymatic activity of Cdk2, the cyclin-dependent kinase mostly responsible for p210 TK-induced acceleration of cell cycle progression. Cdk2 activity was prevented by the enhanced effects of drug combination on Chk2-Cdc25A axis and PI3K/Akt pathway (leading to increased nuclear import of Cdk inhibitors p21 and p27 and to cyclin D downmodulation). Drug combination effects on Chk2-Cdc25A and PI3K/Akt were greater in IM-sensitive bcr-abl-expressing cells compared to IM-resistant cells and were abrogated by IL3. In conclusion, the combined use of TK and Src kinase inhibitors may prevent IM resistance and, therefore, improve the prognosis of CML at clinical diagnosis, but its advantage in the treatment of IM-resistant CML is likely marginal and conditional upon the mechanisms responsible for IM resistance

The nucleosome is the basic structural unit of eukariotic chromosomes and consists of a DNA molecule associated with a histone octamer comprised of pairs of the core histones H2A, H2B, H3 and H4. The nucleosomes are joined by linker DNA and histone1 to form chromatin. Histones play an important role in transcription, DNA replication, DNA repair and recombination. Post-translational modification of specific residues in the core histones (acetylation, methylation, phosphorylation, etc.) has been demonstrated to be critical to their regulatory function. In particular acetylation is a very specific phenomenon with various isoforms playing distinct roles. Increased acetylation is generally correlated with transcriptionally active or poised genes. Histone deacetylases’ inhibitors (short chain fatty acids, such as sodium butyrate, hydroxamic acids such as thrichostatin A) have been described as potential cancer therapeutics in a variety of preclinical studies. Inhibitors’ treatment of cells resulted in the increase of highly acetylated isoforms of the histones, which affect gene expression, cell differentiation and apoptosis. It is here described the application of reversed-phase high-pressure liquid chromatography under gradient conditions and mass spectrometry (LC-MS) to analyse global modification levels of core histones.

9-hydroxystearic acid (9-HSA) belongs to the class of lipid peroxidation by-products, that greatly diminish in tumours, becoming unable to exert their normal controlling functions on cell division; its binding with the catalityc site of HDAC1 inhibits the activity of the enzyme and causes histone hyperacetylation as well as growth inhibition p21WAF1 mediated as well as differentiation. Recent work demontrates that cyclin D1 enhances recruitment of HDAC1 to target genes of differentiation. In particular cyclin D1 has been demonstrated to inhibit basal and ligand-induced PPARgamma activity through a direct interaction with HDAC1. In this work we investigated the effects of 9-HSA on cyclin D1 expression and localization and on its interaction with CDK4 and HDAC1, by using as a cellular model HT29 adenocarcinoma cell line. A flow cytometric cyclin D1 expression /DNA content analysis revealed that the percentage of cyclin D1 positive cells decreased of 25% in 9-HSA treated cells and that 9-HSA induced different patterns of cyclin D1 through the cell cycle phases. Modification of cyclin D1 expression as well as of its localization mediated by 9-HSA in tumour cells can have two effects: i) to substain an early inhibition of proliferation; ii) to promote a precise differentiation program by acting as a transcriptional factor or transcriptional co-factor. Immunofluorescence analysis in confocal microscopy has been used to monitor cyclin D1/CDK4 and cyclin D1/HDAC1 complexe formation when 9-HSA occupies the HDAC1 active site. The diminished expression of cyclin D1 is associated to a dimished presence of both CDK4/cyclin D1 and cyclin D1/HDAC1 complexe in treated cells. Moreover 9-HSA inhibition of HDAC1 removes the enzyme from chromatin binding proteins and results in a new clustering of PPARin nuclear membrane. This work presents preliminary data indicating that 9-HSA modulates cyclin D1 activity both as cell cycle and transcriptional regulator.

Extended survival of clonal hematopoietic progenitors associated with the bcr-abl expression has a key role in the pathogenesis of Chronic Myeloid Leukemia (CML) and may concur to promote the disease progression towards the fully transformed, drug-resistant phenotype of blast crisis. Here we demonstrated that p210 bcr-abl protein tyrosine kinase (TK) activity protects bcr-abl-transducing 32D cell clones from apoptotic death through multiple pathways preserving mitochondrial membrane integrity. P210 TK prevents Trail/DR4, Bim and Bax induction and caspase 9 and Bid activation and upregulates the expression of anti-apoptotic BclxL in the cytoplasm. Moreover, it precludes the integration and aggregation of pro-apoptotic signals Bid, Bax and Bak at mitochondrial membranes letting, in turn, pore opening and apoptogenic molecule leakage from intermembrane spaces. Our results underscore the central role of Akt serine/threonine kinase, activated by p210 TK downstream of phosphatidyl-inositol 3 kinase (PI3K), in leukemic progenitor failure to undergo apoptotic death and support that pharmacological targeting of its enzymatic activity may implement the effects of TK inhibitor Imatinib mesylate (IM).