The new low-toxic histone deacetylase inhibitor S-(2) induces apoptosis in various acute myeloid leukaemia cells.

Histone deacetylase inhibitors (HDACi) induce tumour cell cycle arrest and/or apoptosis, and some of them are currently used in cancer therapy. Recently, we described a series of powerful HDACi characterized by a 1,4-benzodiazepine (BDZ) ring hybridized with a linear alkyl chain bearing a hydroxamate function as Zn(++)--chelating group. Here, we explored the anti-leukaemic properties of three novel hybrids, namely the chiral compounds (S)-2 and (R)-2, and their non-chiral analogue 4, which were first comparatively tested in promyelocytic NB4 cells. (S)-2 and partially 4--but not (R)-2--caused G0/G1 cell-cycle arrest by up-regulating cyclin G2 and p21 expression and down-regulating cyclin D2 expression, and also apoptosis as assessed by cell morphology and cytofluorimetric assay, histone H2AX phosphorylation and PARP cleavage. Notably, these events were partly prevented by an anti-oxidant. Moreover, novel HDACi prompted p53 and α-tubulin acetylation and, consistently, inhibited HDAC1 and 6 activity. The rank order of potency was (S)-2 > 4 > (R)-2, reflecting that of other biological assays and addressing (S)-2 as the most effective compound capable of triggering apoptosis in various acute myeloid leukaemia (AML) cell lines and blasts from patients with different AML subtypes. Importantly, (S)-2 was safe in mice (up to 150 mg/kg/week) as determined by liver, spleen, kidney and bone marrow histopathology; and displayed negligible affinity for peripheral/central BDZ-receptors. Overall, the BDZ-hydroxamate (S)-2 showed to be a low-toxic HDACi with powerful anti-proliferative and pro-apototic activities towards different cultured and primary AML cells, and therefore of clinical interest to support conventional anti-leukaemic therapy.

Effects of sample properties and mass spectroscopic parameters on electrospray ionization mass spectra of size-fractions from a soil humic acid.

Size-fractions from a soil humic acid were separated by preparative size-exclusion chromatography (SEC), desalted, and concentrated by ultrafiltration and vacuum centrifugation without being subjected to any freeze-drying process. After having assessed the lack of formation of any multiple-charged ions by high-resolution Fourier transform ion cyclotron resonance electrospray ionization (ESI) mass spectrometry (MS), the size-fractions were used by direct infusion to compare the molecular ion distribution by both atmospheric pressure chemical ionization (APCI)- and ESI-MS in negative mode. The weight- (Mw) and number-averaged (Mn) molecular weight obtained by ESI-MS were invariably larger than by APCI-MS for all size-fractions, thereby indicating that ESI is more efficient than APCI to evaluate the molecular mass distribution of humic samples. No substantial difference was observed when concentration and pH of unfreeze-dried humic size-fractions were varied. The negative mode was applied to assess the effect of cone voltage from -20 to -60 V on ESI of the humic size-fractions further separated through an on-line SEC column. The resulting mass spectra and Mw and Mn values suggested that the variation of cone voltage in ESI-MS affects the ionization potential of associated humic molecules more in solution rather than their fragmentation. These findings agree with previous observations which indicated a limitation of ESI in providing consistent mass detection for a complex mixture of heterogeneous humic molecules, especially when they are aggregated by a freeze-drying process.

Plant tolerance to mercury in a contaminated soil is enhanced by the combined effects of humic matter addition and inoculation with arbuscular mycorrhizal fungi.

In a greenhouse pot experiment, lettuce plants (Lactuca sativa L.) were grown in a Hg-contaminated sandy soil with and without inoculation with arbuscular mycorrhizal fungi (AMF) (a commercial inoculum containing infective propagules of Rhizophagus irregularis and Funneliformis mosseae) amended with different rates of a humic acid (0, 1, and 2 g kg(-1) of soil), with the objective of verifying the synergistic effects of the two soil treatments on the Hg tolerance of lettuce plants. Our results indicated that the plant biomass was significantly increased by the combined effect of AMF and humic acid treatments. Addition of humic matter to soil boosted the AMF effect on improving the nutritional plant status, enhancing the pigment content in plant leaves, and inhibiting both Hg uptake and Hg translocation from the roots to the shoots. This was attributed not only to the Hg immobilization by stable complexes with HA and with extraradical mycorrhizal mycelium in soil and root surfaces but also to an improved mineral nutrition promoted by AMF. This work indicates that the combined use of AMF and humic acids may become a useful practice in Hg-contaminated soils to reduce Hg toxicity to crops.

Genista sessilifolia DC. extracts induce apoptosis across a range of cancer cell lines.

OBJECTIVES: Restorative properties of medicinal plants such as Genista sessilifolia DC. have often been suggested to occur, in epidemiological studies. However, full characterization of effective principles responsible for this action has never previously been performed. Here, we have characterized G. sessilifolia's anti-cancer effects and identified the chemical components involved in this anti-tumour action. MATERIALS AND METHODS: Cell cycle, apoptosis, necrosis, differentiation analyses, high-performance liquid chromatography, western blotting, RNA extraction, real-time PCR and primers have all been observed/used in the study. RESULTS: We report that G. sessilifolia methanol extract has anti-cancer activity on solid and haematological cancer cells. G. sessilifolia extract's anti-proliferative action is closely bound to induction of apoptosis, whereas differentiation is only weakly modulated. Analysis of G. sessilifolia extract, by high-performance liquid chromatography, identifies fraction 18-22 as the pertinent component for induction of apoptosis, whereas fractions 11-13 and 27-30 both seem to contribute to differentiation. G. sessilifolia extract induces apoptosis mediated by caspase activation and p21, Rb, p53, Bcl2-associated agonist of cell death (BAD), tumour necrosis factor receptor super-family, member 10 (TRAIL) overexpression and death receptor 5 (DR5). Accordingly, fraction 18-22 inducing apoptosis was able to induce TRAIL. CONCLUSIONS: Our results indicate that G. sessilifolia extract and its fraction 18-22 containing genistin and isoprunetin, were able to induce anti-cancer effects supporting the hypothesis of a pro-apoptotic intrinsic content of this natural medicinal plant.

Design, synthesis and preliminary evaluation of a series of histone deacetylase inhibitors carrying a benzodiazepine ring.

A series of new histone deacetylase inhibitors were designed and synthesized based on hybridization between SAHA or oxamflatin and 5-phenyl-1,4-benzodiazepines. The compounds were tested for their enzyme inhibitory activity on HeLa nuclear extracts, and on human recombinant HDAC1 and HDAC6. Antiproliferative activity was tested on different cancer cells types, while proapoptotic activity was primarily tested on NB4 cells. The compounds showed IC50 values similar to those of SAHA. Compound (S)-8 displayed interesting activity against hematological and solid malignancies.

Anti-leukemia activity of chaetocin via death receptor-dependent apoptosis and dual modulation of the histone methyl-transferase SUV39H1.

Epigenetic deregulation is involved in acute myeloid leukemia (AML) pathogenesis and epigenetic targeting drugs are in clinical trial. Since the first results with histone-deacetylase inhibitors in AML are controversial, novel single and combined treatments need to be explored. It is tempting to combine chromatin-targeting drugs. SUV39H1, the main methyl-transferase for lysine 9 tri-methylation on histone H3, interacts with oncogenes involved in AML and acts as a transcriptional repressor for hematopoietic differentiation and immortalization. We report here that pharmacological inhibition of SUV39H1 by chaetocin induces apoptosis in leukemia cell lines in vitro and primary AML cells ex vivo, and that it interferes with leukemia growth in vivo. Chaetocin treatment upregulates reactive oxygen species (ROS) production as well as the transcription of death-receptor-related genes, in a ROS-dependent manner, leading to death receptor-dependent apoptosis. In addition to its direct inhibition by chaetocin, SUV39H1 is indirectly modulated by chaetocin-induced ROS. Accordingly, chaetocin potentiates other anti-AML drugs, in a ROS-dependent manner. The decryption of a dual mechanism of action against AML involving both direct and indirect SUV39H1 modulation represents an innovative read-out for the anticancer activity of chaetocin and for its synergy with other anti-AML drugs, suggesting new therapeutic combination strategies in AML.

UHRF1 coordinates peroxisome proliferator activated receptor gamma (PPARG) epigenetic silencing and mediates colorectal cancer progression.

Peroxisome proliferator-activated receptor gamma (PPARG) inactivation has been identified as an important step in colorectal cancer (CRC) progression, although the events involved have been partially clarified. UHRF1 is emerging as a cofactor that coordinates the epigenetic silencing of tumor suppressor genes, but its role in CRC remains elusive. Here, we report that UHRF1 negatively regulates PPARG and is associated with a higher proliferative, clonogenic and migration potential. Consistently, UHRF1 ectopic expression induces PPARG repression through its recruitment on the PPARG promoter fostering DNA methylation and histone repressive modifications. In agreement, UHRF1 knockdown elicits PPARG re-activation, accompanied by positive histone marks and DNA demethylation, corroborating its role in PPARG silencing. UHRF1 overexpression, as well as PPARG-silencing, imparts higher growth rate and phenotypic features resembling those occurring in the epithelial-mesenchymal transition. In our series of 110 sporadic CRCs, high UHRF1-expressing tumors are characterized by an undifferentiated phenotype, higher proliferation rate and poor clinical outcome only in advanced stages III-IV. In addition, the inverse relationship with PPARG found in vitro is detected in vivo and UHRF1 prognostic significance appears closely related to PPARG low expression, as remarkably validated in an independent dataset. The results demonstrate that UHRF1 regulates PPARG silencing and both genes appear to be part of a complex regulatory network. These findings suggest that the relationship between UHRF1 and PPARG may have a relevant role in CRC progression.

Psidium guajava L. anti-neoplastic effects: induction of apoptosis and cell differentiation.

OBJECTIVES: Curative properties of medicinal plants such as Psidium guajava L. (Myrtaceae) have often been indicated by epidemiological studies on populations in which these fruits are consumed daily. However, complete characterization of the active principles responsible for this ability has never been performed. Here, we have characterized P. guajava's anti-cancer potential and identified the parts of the fruit involved in its anti-neoplastic action. MATERIALS AND METHODS: We studied morphology of our cells, cell cycle characteristics and apoptosis and performed immunostaining, differentiation and western blot analyses. RESULTS: We report that the P. guajava extract exerted anti-cancer control on both haematological and solid neoplasias. P. guajava extract's anti-tumour properties were found to be tightly bound to induction of apoptosis and differentiation. Use of ex vivo myeloid leukaemia blasts corroborated that P. guajava was able to induce cell death but did not exhibit anti-cancer effects on all malignant cells investigated, indicating selective activity against certain types of tumour. Analyses of P. guajava pulp, peel and seeds identified the pulp as being the most relevant component for causing cell cycle arrest and apoptosis, whereas peel was responsible for causing cell differentiation. P. guajava itself and its pulp-derived extract were found to induce apoptosis accompanied by caspase activation and p16, p21, Fas ligand (FASL TNF super-family, member 6), Bcl-2-associated agonist of cell death (BAD) and tumour necrosis factor receptor super-family, member 10b (DR5), overexpression. CONCLUSIONS: Our findings showed that P. guajava L. extract was able to exert anti-cancer activity on cultures in vitro and ex vivo, supporting the hypothesis of its anti malignant pro-apoptotic modulation.

Histone deacetylase inhibitors induce thyroid cancer-specific apoptosis through proteasome-dependent inhibition of TRAIL degradation.

Anaplastic thyroid carcinoma (ATC) is considered one of the most aggressive malignancies, having a poor prognosis and being refractory to conventional chemotherapy and radiotherapy. Alteration in histone deacetylase (HDAC) activity has been reported in cancer, thus encouraging the development of HDAC inhibitors, whose antitumor action has been shown in both solid and hematological malignancies. However, the molecular basis for their tumor selectivity is unknown. To find an innovative therapy for the treatment of ATCs, we studied the effects of deacetylase inhibitors on thyroid tumorigenesis models. We show that HDACs 1 and 2 are overexpressed in ATCs compared with normal cells or benign tumors and that HDAC inhibitors induce apoptosis selectively in the fully transformed thyroid cells. Our results indicate that these phenomena are mediated by a novel action of HDAC inhibitors that reduces tumor necrosis factor-related apoptosis-inducing ligand protein degradation by affecting the ubiquitin-dependent pathway. Indeed, the combined treatment with HDAC and proteasome inhibitors results in synergistic apoptosis. These results strongly encourage the preclinical application of the combination deacetylase-proteasome inhibitors for the treatment of ATC.

Salermide, a Sirtuin inhibitor with a strong cancer-specific proapoptotic effect.

Sirtuin 1 (Sirt1) and Sirtuin 2 (Sirt2) belong to the family of NAD+ (nicotinamide adenine dinucleotide-positive)-dependent class III histone deacetylases and are involved in regulating lifespan. As cancer is a disease of ageing, targeting Sirtuins is emerging as a promising antitumour strategy. Here we present Salermide (N-{3-[(2-hydroxy-naphthalen-1-ylmethylene)-amino]-phenyl}-2-phenyl-propionamide), a reverse amide with a strong in vitro inhibitory effect on Sirt1 and Sirt2. Salermide was well tolerated by mice at concentrations up to 100 muM and prompted tumour-specific cell death in a wide range of human cancer cell lines. The antitumour activity of Salermide was primarily because of a massive induction of apoptosis. This was independent of global tubulin and K16H4 acetylation, which ruled out a putative Sirt2-mediated apoptotic pathway and suggested an in vivo mechanism of action through Sirt1. Consistently with this, RNA interference-mediated knockdown of Sirt1, but not Sirt2, induced apoptosis in cancer cells. Although p53 has been reported to be a target of Sirt1, genetic p53 knockdowns showed that the Sirt1-dependent proapoptotic effect of Salermide is p53-independent. We were finally able to ascribe the apoptotic effect of Salermide to the reactivation of proapoptotic genes epigenetically repressed exclusively in cancer cells by Sirt1. Taken together, our results underline Salermide's promise as an anticancer drug and provide evidence for the molecular mechanism through which Sirt1 is involved in human tumorigenesis.