New histone deacetylase inhibitors as potential therapeutic tools for advanced prostate carcinoma

The anti-epileptic drug valproic acid is also under trial as an anti-cancer agent due to its histone deacetylase (HDAC) inhibitory properties. However, the effects of valproic acid (VPA) are limited and concentrations required for exerting anti-neoplastic effects in vitro may not be reached in tumour patients. In this study, we tested in vitro and in vivo effects of two VPA-derivatives (ACS2, ACS33) on pre-clinical prostate cancer models. PC3 and DU-145 prostate tumour cell lines were treated with various concentrations of ACS2 or ACS33 to perform in vitro cell proliferation 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and to evaluate tumour cell adhesion to endothelial cell monolayers. Analysis of acetylated histones H3 and H4 protein expression was performed by western blotting. In vivo tumour growth was conducted in subcutaneous xenograft mouse models. Tumour sections were assessed by immunohistochemistry for histone H3 acetylation and proliferation. ACS2 and ACS33 significantly up-regulated histone H3 and H4 acetylation in prostate cancer cell lines. In micromolar concentrations both compounds exerted growth arrest in PC3 and DU-145 cells and prevented tumour cell attachment to endothelium. In vivo, ACS33 inhibited the growth of PC3 in subcutaneous xenografts. Immunohistochemistry and western blotting confirmed increased histone H3 acetylation and reduced proliferation. ACS2 and ACS33 represent novel VPA derivatives with superior anti-tumoural activities, compared to the mother compound. This investigation lends support to the clinical testing of ACS2 or ACS33 for the treatment of prostate cancer

Extracellular release of the ‘differentiation enhancing factor’, a HMG1 protein type, is an early step in murine erythroleukemia cell differentiation

AbstractDifferentiation enhancing factor (DEF) is a 29 kDa protein expressed in murine erythroleukemia (MEL) cells and active in promoting a significant increase in the rate of hexamethylenebisacetamide induced differentiation of these cells. The factor was recently shown to possess an amino acid sequence identical to that reported for one of the HMG1 proteins, designated as ‘amphoterin’ on the basis of its highly dipolar sequence. In the present study, we have expressed DEF cDNA in an E. coli strain and found that the recombinant protein has functional properties identical to those observed with native DEF. Furthermore, we demonstrate that, following MEL cell stimulation with the chemical inducer, DEF is secreted in large amounts in the extracellular medium. In fact, the N-terminal sequence and the partial amino acid sequence of tryptic peptides from the secreted protein correspond to those of DEF isolated from the soluble fraction of resting MEL cells. These results are indicative for an extracellular localization as the site of action of DEF and suggest a novel function for proteins belonging to the HMG1 family. Finally, the early decay of DEF mRNA, in chemical induced MEL cells, support the hypothesis that the involvement of the enhancing factor occurs and is completed in the early phases of cell differentiation

MZe786 Rescues Cardiac Mitochondrial Activity in High sFlt-1 and Low HO-1 Environment

Hypertensive disorder in pregnancy is a major cause of maternal and perinatal mortality worldwide. Women who have had preeclampsia are at three to four times higher risk in later life of developing high blood pressure and heart disease. Soluble Flt-1 (sFlt-1) is elevated in preeclampsia and may remain high postpartum in women with a history of preeclampsia. Heme oxygenase-1 (Hmox1/HO-1) exerts protective effects against oxidative stimuli and is compromised in the placenta of pregnant women with preeclampsia. We hypothesized that sFlt-1 inhibits cardiac mitochondrial activity in HO-1 deficient mice. HO-1 haplo-insufficient mice (Hmox1+/−) were injected with adenovirus encoding sFlt-1 (Ad-sFlt-1) or control virus (Ad-CMV). Subsequently, they were treated daily with either placebo or MZe786 for six days, when the heart tissue was harvested to assess cardiac mitochondrial activity. Here, we show that the loss of HO-1 disturbed cardiac mitochondrial respiration and reduced mitochondrial biogenesis. The overexpression of sFlt-1 resulted in the inhibition of the cardiac mitochondrial activity in Hmox1+/− mice. The present study demonstrates that the hydrogen sulfide (H2S) releasing molecule, MZe786, rescues mitochondrial activity by stimulating cardiac mitochondrial biogenesis and antioxidant defense in Hmox1−/− mice and in Hmox1+/− mice exposed to a high sFlt-1 environment

The new H2S-releasing compound ACS94 exerts protective effects through the modulation of thiol homoeostasis

The synthesis of a new dithiolethione-cysteine ethyl ester hybrid, ACS94, its metabolites, and its effect on GSH levels in rat tissues and on the concentration of circulating H2S is described. ACS94 rapidly enters the cells, where it is metabolised to cysteine and the dithiolethione moiety ACS48. Experiments performed through the oral administration of ACS94 to healthy rats showed that it is capable of increasing the GSH levels in most of the analysed organs and the concentration of circulating H2S. Although the increase in GSH concentration was similar to that obtained by ACS48 and N-acetylcysteine ethyl ester, the H2S increase was long-lasting and more evident with respect to the parent molecules. Moreover, a decrease of homocysteine in several rat organs and in plasma was noted. This effect may represent a potential therapeutic use of ACS94, as hyperhomocysteinaemia is considered a risk factor for cardiovascular diseases. Lastly, ACS94 was more efficient than N-acetylcysteine in protecting the liver and kidneys against acute acetaminophen toxicity

MZe786, a hydrogen sulfide-releasing aspirin prevents preeclampsia in heme oxygenase-1 haplodeficient pregnancy under high soluble flt-1 environment

Preeclampsia affects one in twelve of the 130 million pregnancies a year. The lack of an effective therapeutic to prevent or treat it is responsible for an annual global cost burden of 100 billion US dollars. Preeclampsia also affects these women later in life as it is a recognised risk factor for cardiovascular disease, stroke and vascular dementia. Our laboratory demonstrated that preeclampsia is associated with high soluble fms-like tyrosine kinase 1 (sFlt-1) and low heme oxygenase-1 (HO1/Hmox1) expression. Here we sought to determine the therapeutic value of a novel H 2S-releasing aspirin (MZe786) in HO-1 haploid deficient (Hmox1 +/−) pregnant mice in a high sFlt-1 environment. Pregnant Hmox1 +/− mice were injected with adenovirus encoding sFlt-1 or control virus at gestation day E11.5. Subsequently, Hmox1 +/− dams were treated daily with a number of treatment regimens until E17.5, when maternal and fetal outcomes were assessed. Here we show that HO-1 compromised mice in a high sFlt-1 environment during pregnancy exhibit severe preeclampsia signs and a reduction in antioxidant genes. MZe786 ameliorates preeclampsia by reducing hypertension and renal damage possibly by stimulating antioxidant genes. MZe786 also improved fetal outcome in comparison with aspirin alone and appears to be a better therapeutic agent at preventing preeclampsia than aspirin alone

Methanethiosulfonate derivatives as ligands of STAT3-SH2 domain

It is well known that inflammatory conditions in selected organs increase the risk of cancer. Compounds of the inflammatory tumor microenvironment include leukocytes, cytokines, complement components, are orchestrated by transcription factors, such as STAT-3 (Signal Transducer and Activator of Transcription 3) and NF-kB. Therefore drugs able to inhibit one or both transcription factors could be useful tools to treat cancer disease. Two main approaches have been explored to inhibit STAT-3 signalling: \u2022 indirect, inhibiting the upstream tyrosine kinases that are responsible for STAT-3 activation or blocking factors such as JAK, Src, Bcr-Abl, FLT3 and EGFR that are involved in the activation of STAT-3 signalling. This kind of inhibition induces tumour-cell apoptosis but is poor selective. \u2022 direct, by interaction of small molecules with the protein. In this selective approach the starting point is the crystallographic structure of STAT-3 SH2 domain. S-methyl methanethiosulfonate, isolated from cauliflower has been shown to inhibit colon tumor incidence when administered to rats during the post-initiation phase of carcinogenesis [1]. Recently, a new methanethiosulfonate derivative of valproic acid (ACS33) was reported by some of us to show good in vitro antiproliferative activity and to inhibit in vivo the growth of PC3 in subcutaneous xenograft mice models [2]. Fig.1: Structures of the studied thiosulfonate hybrids. Since the influence of methanethiosulfonates on STAT-3 activity has not been yet studied, we decided to synthesize a set of thiosulfonate-drug hybrids (Fig.1) and to submit them and their parent compounds to the AlphaScreen-based assay, to investigate their ability to bind STAT-3 SH2 domain. Moreover, in order to check the selectivity of our molecules on STAT-3, other SH2-containing proteins, such as STAT-1, exhibiting a high degree of sequence homology to STAT-3, have also been tested. Results showed that most of the synthesized thiosulfonate-hybrids are able to strongly and selectively bind STAT-3 SH2 domain, whereas the parent drugs were completely devoid of this ability. Studies are ongoing to better define the profile of our new methanethiosulfonate derivatives as potential dual STAT-3/NFkB inhibitors. References 1. Reddy, B. S.; Kawamori, T.; Lubet, R.; Steele, V.; Kelloff, G.; Rao, C. V. Chemopreventive effect of S-methylmethane thiosulfonate and sulindac administered together during the promotion/progression stages of colon carcinogenesis Carcinogenesis 1999, 20, 1645-8. 2. Wedel S. A.; Sparatore A.; Del Soldato P.; Al-Batran S. E.; Atmaca A.; Juengel E.; Hudak L.; Jonas D.; Blaheta R. A. New histone deacetylase inhibitors as potential therapeutics tools for advanced prostate carcinoma. J. Cell. Mol Med 2008, 12, 2457-66

H(2)S-Releasing Aspirin Protects against Aspirin-Induced Gastric Injury via Reducing Oxidative Stress

The aim of this study was to examine the effect of ACS14, a hydrogen sulfide (H(2)S)-releasing derivative of aspirin (Asp), on Asp-induced gastric injury. Gastric hemorrhagic lesions were induced by intragastric administration of Asp (200 mg/kg, suspended in 0.5% carboxymethyl cellulose solutions) in a volume of 1 ml/100 g body weight. ACS14 (1, 5 or 10 mg/kg) was given 30 min before the Asp administration. The total area of gastric erosions, H(2)S concentration and oxidative stress in gastric tissues were measured three hours after administration of Asp. Treatment with Asp (200 mg/kg), but not ACS14 (430 mg/kg, at equimolar doses to 200 mg/kg Asp), for 3 h significantly increased gastric mucosal injury. The damage caused by Asp was reversed by ACS14 at 1-10 mg/kg in a concentration-dependent manner. ACS14 abrogated Asp-induced upregulation of COX-2 expression, but had no effect on the reduced PGE(2) level. ACS14 reversed the decreased H(2)S concentrations and blood flow in the gastric tissue in Asp-treated rats. Moreover, ACS14 attenuated Asp-suppressed superoxide dismutase-1 (SOD-1) expression and GSH activity, suggesting that ACS14 may stimulate antioxidants in the gastric tissue. ACS14 also obviously inhibited Asp-induced upregulation of protein expression of oxidases including XOD, p47(phox) and p67(phox). In conclusion, ACS14 protects Asp induced gastric mucosal injury by inhibiting oxidative stress in the gastric tissue

Hydrogen sulfide releasing molecule MZe786 inhibits soluble Flt-1 and prevents preeclampsia in a refined RUPP mouse model

An imbalance in angiogenic growth factors and poor utero-placental perfusion are strongly associated with preeclampsia. The reduced utero-placental perfusion (RUPP) model that mimics insufficient placental perfusion is used to study preeclampsia. The aim of this study was to develop a refined RUPP model in C57Bl/6 J mice to test the efficacy of MZe786 as a potential inhibitor of soluble Flt-1 for preeclampsia therapy. Murine RUPP (mRUPP) was induced through bilateral ligation of the ovarian arteries at E11.5 that resulted in typical preeclampsia symptoms including increase in mean arterial pressure (MAP), kidney injury and elevated soluble Flt-1 (sFlt-1) levels in the maternal plasma and amniotic fluid. The murine RUPP kidneys showed tubular and glomerular damage along with increased oxidative stress characterised by increased nitrotyrosine staining. The mRUPP displayed abnormal placental vascular histology, reduced expression of placental cystathionine γ-lyase (CSE), the hydrogen sulfide (H 2S) producing enzyme, and resulted in adverse fetal outcomes (FGR). Importantly, oral administration of hydrogen sulfide (H 2S)-releasing compound MZe786 from E11.5 to E17.5 successfully prevented the development of preeclampsia. Specifically, MZe786 treatment reduced maternal MAP and kidney nitrotyrosine staining and improved fetal outcome. The circulation levels of sFlt-1 were dramatically decreased in MZe786 treated animals implying that H 2S released from MZe786 offered protection by inhibiting sFlt-1 levels. MZe786 prevent preeclampsia and warrant a rapid move to randomised control clinical trial

Therapeutic effect of hydrogen sulfide-releasing L-dopa derivative ACS84 on 6-OHDA-induced Parkinson's disease rat model

Parkinson's disease (PD), characterized by loss of dopaminergic neurons in the substantia nigra, is a neurodegenerative disorder of central nervous system. The present study was designed to investigate the therapeutic effect of ACS84, a hydrogen sulfide-releasing-L-Dopa derivative compound, in a 6-hydroxydopamine (6-OHDA)-induced PD model. ACS84 protected the SH-SY5Y cells against 6-OHDA-induced cell injury and oxidative stress. The protective effect resulted from stimulation of Nrf-2 nuclear translocation and promotion of anti-oxidant enzymes expression. In the 6-OHDA-induced PD rat model, intragastric administration of ACS84 relieved the movement dysfunction of the model animals. Immunofluorescence staining and High-performance liquid chromatography analysis showed that ACS84 alleviated the loss of tyrosine-hydroxylase positive neurons in the substantia nigra and the declined dopamine concentration in the injured striatums of the 6-OHDA-induced PD model. Moreover, ACS84 reversed the elevated malondialdehyde level and the decreased glutathione level in vivo. In conclusion, ACS84 may prevent neurodegeneration via the anti-oxidative mechanism and has potential therapeutic values for Parkinson's disease

Benzimidazole derivatives endowed with potent antileishmanial activity

Two sets of benzimidazole derivatives were synthesised and tested in vitro for activity against promastigotes of Leishmania tropica and L. infantum. Most of the tested compounds resulted active against both Leishmania species, with IC50values in the low micromolar/sub-micromolar range. Among the set of 2-(long chain)alkyl benzimidazoles, whose heterocyclic head was quaternised, compound 8 resulted about 100-/200-fold more potent than miltefosine, even if the selectivity index (SI) versus HMEC-1 cells was only moderately improved. In the set of 2-benzyl and 2-phenyl benzimidazoles, bearing a basic side chain in position 1, compound 28 (2-(4-chlorobenzyl)-1-lupinyl-5-trifluoromethylbenzimidazole) was 12-/7-fold more potent than miltefosine, but exhibited a further improved SI. Therefore, compounds 8 and 28 represent interesting hit compounds, susceptible of structural modification to improve their safety profiles