Cristina Cellai

Altered cholesterol ester cycle in skin fibroblasts from patients with Alzheimer's disease.

Intracellular cholesterol metabolism was reported to modulate amyloid-beta (Abeta) generation in Alzheimers disease (AD). Results presented herein demonstrated that, like brain cells, cultured skin fibroblasts from AD patients contained more cholesterol esters than fibroblasts from healthy subjects. Particularly, Oil Red-O, Nile Red, and filipin staining highlighted higher levels of neutral lipids which responded to inhibitors of acyl-coenzyme A:cholesterol acyl-transferase (ACAT-1), associated with an increase in free cholesterol. ACAT-1 mRNA levels increased significantly in AD fibroblasts, whereas those of sterol regulatory element binding protein-2, neutral cholesterol ester hydrolase, and ATP-binding cassette transporter member 1 were markedly down-regulated. Instead, mRNA levels of low-density lipoprotein receptor, hydroxy-methyl-glutaryl-coenzyme A reductase, caveolin-1, and amyloid-beta protein precursor (AbetaPP) were virtually unchanged. Notably, mRNA levels of both beta-site AbetaPP-cleaving enzyme 1 (BACE1) and neprilysin were significantly down-regulated. An increase in Abeta(40) and Abeta(42) immunostaining and a decrease in BACE1 active form were also found in AD versus control fibroblasts. Altogether, these findings support the hypothesis that the derangement of cholesterol homeostasis is a systemic alteration involving central but also peripheral cells of AD patients, and point to cholesterol ester levels in AD fibroblasts as an additional metabolic hallmark useful in the laboratory and clinical practice.

Growth inhibition and differentiation of human breast cancer cells by the PAFR antagonist WEB-2086

WEB-2086 – an antagonist of platelet-activating factor receptor (PAFR) with known anti-inflammatory, antiangiogenic and antileukaemic properties – also proved to inhibit the proliferation in human solid tumour cell lines of different histology, and with much higher efficacy than in normal fibroblasts. A detailed analysis of WEB-2086 anticancer activity was then performed focusing on breast adenocarcinoma MCF-7 and MDA-MB-231 cells. WEB-2086-treated cells, either expressing (MCF-7) or unexpressing (MDA-MB-231) the oestrogen receptor (ER)α, underwent a dose-dependent growth arrest (IC50=0.65±0.09 and 0.41±0.07 mM, respectively) and accumulation in G0–G1 phase. WEB-2086 also induced morphological and functional changes typical of mature mammary phenotype including (i) cell enlargement and massive neutral lipid deposition (best accomplished in MCF-7 cells); (ii) decrease in motility and active cathepsin D levels (mainly observed in highly invasive MDA-MB-231 cells). The expression of ERα was neither increased nor reactivated in treated MCF-7 or MDA-MB-231 cells, respectively. WEB-2086-induced differentiation in breast cancer cells involved the upregulation of PTEN, a key tumour suppressor protein opposing tumorigenesis, and was apparently independent of p53, PAFR, peripheral benzodiazepine receptor and ERα status. Overall, WEB-2086 can be proposed as an effective antiproliferative and differentiative agent with interesting translational opportunities to treat breast cancers in support to conventional chemotherapy.

Specific PAF antagonist WEB-2086 induces terminal differentiation of murine and human leukemia cells

A pharmacological approach to neoplasia by differentiation therapy relies on the availability of cytodifferentiating agents whose antitumor efficacy is usually assayed first on malignant cells in vitro. Using murine erythroleukemia cells (MELCs) as the model, we found that WEB‐2086, a triazolobenzodiazepine‐derived PAF antagonist originally developed as an anti‐inflammatory drug, induces a dose‐dependent inhibition of MELC growth and hemoglobin accumulation as a result of a true commitment to differentiation. MELCs treated for 5 days with 1 mM WEB‐2086 show ≥ 85% benzidine‐positive cells, increased expression of α‐ and β‐globin genes, and down‐regulation of c‐Myb. This differentiation pattern, which does not involve histone H4 acetylation and is abrogated by the action of phorbol 12‐myristate 13‐acetate, recalls the pattern induced by hexamethylene bisacetamide (HMBA). In addition to MELCs, human erythroleukemia K562 and HEL and myeloid HL60 cells are massively committed to maturation by WEB‐2086 and, with some differences, by its analog, WEB‐2170. This suggests that WEB‐2086, structurally distant from other known inducers, might be a member of a new class of cytodifferentiation agents active on a broad range of transformed cells in vitro and useful, prospectively, for anticancer therapy due to their high tolerability in vivo.

Constitutive and inducible expression of megakaryocyte-specific genes in Friend erythroleukaemia cells

Friend murine erythroleukaemia cells (MELC) were analysed by semiquantitative RT‐PCR for the constitutive and inducible expression of megakaryocyte‐specific genes. Uninduced MELC expressed detectable levels of mRNAs for acethylcholinesterase (AChE), platelet factor‐4 (PF4), glycoprotein IIb (GPIIb) and von Willebrand factor (VWF), whereas the erythroid α‐ and β‐globin genes were not transcribed appreciably. However, MELC exposed to 5 mm hexamethylene bisacetamide (HMBA) or 1.5% dimethyl sulphoxide (DMSO) seemed to be channelled towards a mixed erythroid/megakaryocytic phenotype characterized by unaltered levels of VWF mRNA, increased levels of AChE, GPIIb and PF4 mRNA, and simultaneous induction of the globin genes. Megakaryocyte‐related genes were expressed, in the absence of globin gene transcription, by MELC treated with either phorbol‐12‐myristate acetate (PMA; 100 ng/ml) or colcemid (40 nm), an antimicrotubule agent capable of promoting polyploidization in this model. Moreover, PMA and colcemid induced also de novo expression of the thrombopoietin receptor c‐mpl. PMA and colcemid did not affect high basal c‐myb mRNA levels which, in turn, were down‐regulated upon HMBA or DMSO induction. Additionally, both uninduced and induced MELC exhibited significant levels of Epo‐R and IL‐3R mRNAs, whereas no expression of granulocyte/macrophage‐related genes was detected. Megakaryocyte gene expression of MELC was also compared to that of other haemopoietic cell populations from normal mice and mice infected with the anaemic strain of the Friend virus. According to our results, MELC should be seen as an unique erythro‐megakaryocytic model of differentiation, potentially useful for studying molecular events governing lineage commitment as well as some steps of megakaryocytopoiesis.

Design, synthesis and preliminary biological evaluation of new hydroxamate histone deacetylase inhibitors as potential antileukemic agents

This study concerns the synthesis of new histone deacetylase inhibitors (HDACi) characterized by a 1,4-benzodiazepine ring used as the cap, joined through an amide function or a triple bond as connection units, to a linear alkyl chain bearing the hydroxamate function as Zn2+-chelating group. Biological tests performed in human acute promyelocytic leukemia NB4 cells showed that new hybrids can induce histone H3/H4 acetylation, growth arrest, and also apoptosis. Notably, chiral compounds exhibit stereoselective activity.

Effectiveness of the Histone Deacetylase Inhibitor (S)-2 against LNCaP and PC3 Human Prostate Cancer Cells

Histone deacetylase inhibitors (HDACi) represent a promising class of epigenetic agents with anticancer properties. Here, we report that (S)-2, a novel hydroxamate-based HDACi, shown previously to be effective against acute myeloid leukemia cells, was also a potent inducer of apoptosis/differentiation in human prostate LNCaP and PC3 cancer cells. In LNCaP cells (S)-2 was capable of triggering H3/H4 histone acetylation, H2AX phosphorylation as a marker of DNA damage and producing G0/G1 cell cycle arrest. Consistently, (S)-2 led to enhanced expression of both the protein and mRNA p21 levels in LNCaP cells but, contrary to SAHA, not in normal non-tumorigenic prostate PNT1A cells. Mechanistic studies demonstrated that (S)-2-induced apoptosis in LNCaP cells developed through the cleavage of pro-caspase 9 and 3 and of poly(ADP-ribose)-polymerase accompanied by the dose-dependent loss of mitochondrial membrane potential. Indeed, the addition of the pan-caspase inhibitor Z-VAD-fmk greatly reduced drug-mediated apoptosis while the antioxidant N-acetyl-cysteine was virtually ineffective. Importantly, preliminary data with nude mice xenografted with LNCaP cells showed that (S)-2 prompted a decrease in the tumor volume and an increase in H2AX phosphorylation within the cancer cells. Moreover, the highly metastatic prostate cancer PC3 cells were also sensitive to (S)-2 that: i) induced growth arrest and moderate apoptosis; ii) steered cells towards differentiation and neutral lipid accumulation; iii) reduced cell invasiveness potential by decreasing the amount of MMP-9 activity and up-regulating TIMP-1 expression; and iv) inhibited cell motility and migration through the Matrigel. Overall, (S)-2 has proven to be a powerful HDACi capable of inducing growth arrest, cell death and/or differentiation of LNCaP and PC3 prostate cancer cells and, due to its low toxicity and efficacy in vivo, might also be of clinical interest to support conventional prostate cancer therapy.

Mechanistic insight into WEB-2170-induced apoptosis in human acute myelogenous leukemia cells: The crucial role of PTEN

OBJECTIVE This study aimed to investigate the mechanisms of action of WEB-2170, an inverse agonist of platelet-activating factor receptor, capable of inducing apoptosis in human acute myelogenous leukemia (AML) cells. MATERIAL AND METHODS Gene expression profiling followed by cytofluorimetric, morphologic, and biologic analyses were used to monitor WEB-2170 effects in AML cell lines (ie, NB4, KG1, NB4-MR4, THP1, and U937) and blasts from patients with different AML (M0-M5) subtypes. PTEN silencing with small interfering RNA was also performed. RESULTS We have demonstrated that drug-mediated cytostasis/apoptosis in NB4 cells is characterized by upregulation of cyclin G2, p21/WAF1, NIX, TNF-alpha, and PTEN expression, and downregulation of cyclin D2 and BCL2 expression. We observed an increase in PTEN protein accompanied by a decrease in phospho-extracellular signal-regulated kinase 2 (ERK2) and phospho-AKT, and by forkhead box O3a (FOXO3a) cytoplasmic-nuclear translocation; the mitochondrial cytochrome C release and PARP cleavage marked the late apoptotic steps. We have found that WEB-2170 triggered apoptosis in NB4, KG1, and NB4-MR4 cells where PTEN was expressed, but not in THP1 and U937 cells where PTEN was absent. Finally, we show that PTEN silencing in NB4 cells by PTEN-specific small interfering RNA resulted in a significant reduction of drug-induced apoptosis. CONCLUSION We demonstrated that WEB-2170 is a powerful antileukemic agent with interesting translational opportunities to treat AML and described mechanisms of drug-induced intrinsic and extrinsic apoptosis both in AML cell lines and blasts from AML patients by addressing PTEN as the master regulator of the whole process.

WEB-2086 and WEB-2170 trigger apoptosis in both ATRA-sensitive and -resistant promyelocytic leukemia cells and greatly enhance ATRA differentiation potential.

PAF-receptor antagonists WEB-2086 and WEB-2170 (WEBs) have been previously shown to induce differentiation in murine and human leukemia cells. The present study describes the apoptotic-differentiative effect of WEBs in all-trans-retinoic acid (ATRA)-sensitive (NB4) and -resistant (NB4-007-6 and NB4-MR4) acute promyelocytic leukemia (APL) cell lines as well as blasts from patients with t(15;17) APL. NB4 cells exposed to 0.5–1 mM WEBs underwent striking growth arrest and massive apoptosis without appreciable differentiation; IC50 values after 3-day treatment of NB4 were 0.4 and 0.25 mM for WEB-2086 and WEB-2170, respectively. WEBs induced apoptosis also in the two ATRA-resistant NB4-007-6 and NB4-MR4 cell lines and in blasts from patients with t(15;17) APL. Moreover, subapoptotic WEBs acted synergistically with low-dose (0.025–0.05 μM) ATRA; this allowed to increase ATRA differentiation potential up to 40-fold and to improve both number and intensity of NBT-positive NB4 cells at definitely higher levels than with 1 μM ATRA alone. The powerful antiproliferative-apoptotic activities of WEBs in vitro on ATRA-sensitive, ATRA-resistant APL cells and blasts from patients with APL as well as drug capabilities to enhance ATRA differentiation potential suggested that these agents also due to their recognized tolerability in vivo might improve, alone or in combination, clinical treatment of APL.

Design, synthesis and nootropic activity of new analogues of sunifiram and sapunifiram, two potent cognition-enhancers

A series of amides and sulfonamides, structurally related to DM235 (sunifiram) and MN19 (sapunifiram), derived by ring expansion or contraction, or by inversion of the exocyclic amide function, have been synthesized and tested for cognition-enhancing activity in the mouse passive-avoidance test. Some of the compounds display good antiamnesic and procognitive activity, with higher potency than piracetam, and with a potency similar to the parent compounds.

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.