MiR-422a promotes adipogenesis via MeCP2 downregulation in human bone marrow mesenchymal stem cells.

Methyl-CpG binding protein 2 (MeCP2) is a ubiquitous transcriptional regulator. The study of this protein has been mainly focused on the central nervous system because alterations of its expression are associated with neurological disorders such as Rett syndrome. However, young patients with Rett syndrome also suffer from osteoporosis, suggesting a role of MeCP2 in the differentiation of human bone marrow mesenchymal stromal cells (hBMSCs), the precursors of osteoblasts and adipocytes. Here, we report an in vitro downregulation of MeCP2 in hBMSCs undergoing adipogenic differentiation (AD) and in adipocytes of human and rat bone marrow tissue samples. This modulation does not depend on MeCP2 DNA methylation nor on mRNA levels but on differentially expressed miRNAs during AD. MiRNA profiling revealed that miR-422a and miR-483-5p are upregulated in hBMSC-derived adipocytes compared to their precursors. MiR-483-5p, but not miR-422a, is also up-regulated in hBMSC-derived osteoblasts, suggesting a specific role of the latter in the adipogenic process. Experimental modulation of intracellular levels of miR-422a and miR-483-5p affected MeCP2 expression through direct interaction with its 3' UTR elements, and the adipogenic process. Accordingly, the knockdown of MeCP2 in hBMSCs through MeCP2-targeting shRNA lentiviral vectors increased the levels of adipogenesis-related genes. Finally, since adipocytes released a higher amount of miR-422a in culture medium compared to hBMSCs we analyzed the levels of circulating miR-422a in patients with osteoporosis-a condition characterized by increased marrow adiposity-demonstrating that its levels are negatively correlated with T- and Z-scores. Overall, our findings suggest that miR-422a has a role in hBMSC adipogenesis by downregulating MeCP2 and its circulating levels are associated with bone mass loss in primary osteoporosis.

Decipher non-canonical SPAST splicing mutations with the help of functional assays in patients affected by spastic paraplegia 4 (SPG4).

Flowchart showing the molecular approach used to decipher the non-canonical splicing mutations.

The Current State of Chromatin Immunoprecipitation (ChIP) from FFPE Tissues.

Cancer cells accumulate epigenomic aberrations that contribute to cancer initiation and progression by altering both the genomic stability and the expression of genes. The awareness of such alterations could improve our understanding of cancer dynamics and the identification of new therapeutic strategies and biomarkers to refine tumor classification and treatment. Formalin fixation and paraffin embedding (FFPE) is the gold standard to preserve both tissue integrity and organization, and, in the last decades, a huge number of biological samples have been archived all over the world following this procedure. Recently, new chromatin immunoprecipitation (ChIP) techniques have been developed to allow the analysis of histone post-translational modifications (PTMs) and transcription factor (TF) distribution in FFPE tissues. The application of ChIP to genome-wide chromatin studies using real archival samples represents an unprecedented opportunity to conduct retrospective clinical studies thanks to the possibility of accessing large cohorts of samples and their associated diagnostic records. However, although recent attempts to standardize have been made, fixation and storage conditions of clinical specimens are still extremely variable and can affect the success of chromatin studies. The procedures introduced in the last few years dealt with this problem proponing successful strategies to obtain high-resolution ChIP profiles from FFPE archival samples. In this review, we compare the different FFPE-ChIP techniques, highlighting their strengths, limitations, common features, and peculiarities, as well as pitfalls and caveats related to ChIP studies in FFPE samples, in order to facilitate their application.

A novel POLR3A genotype leads to leukodystrophy type-7 in two siblings with unusually late age of onset.

BACKGROUND: Leukodystrophies are familial heterogeneous disorders primarily affecting the white matter, which are defined as hypomyelinating or demyelinating based on disease severity as assessed at MRI. Recently, a group of clinically overlapping hypomyelinating leukodystrophies (HL) has been associated with mutations in RNA polymerase III enzymes (Pol III) subunits. CASE PRESENTATION: In this manuscript, we describe two Italian siblings carrying a novel POLR3A genotype. MRI imaging, genetic analysis, and clinical data led to diagnosing HL type 7. The female sibling, at the age of 34, is tetra-paretic and suffers from severe cognitive regression. She had a disease onset at the age of 19, characterized by slow and progressive cognitive impairment associated with gait disturbances and amenorrhea. The male sibling was diagnosed during an MRI carried out for cephalalgia at the age of 41. After 5 years, he developed mild cognitive impairment, dystonia with 4-limb hypotonia, and moderate dysmetria with balance and gait impairment. CONCLUSIONS: The present study provides the first evidence of unusually late age of onset in HL, describing two siblings with a novel POLR3A genotype which showed the first symptoms at the age of 41 and 19, respectively. This provides a powerful insight into clinical heterogeneity and genotype-phenotype correlation in POLR3A related HL.

Dissecting Molecular Features of Gliomas: Genetic Loci and Validated Biomarkers.

Recently, several studies focused on the genetics of gliomas. This allowed identifying several germline loci that contribute to individual risk for tumor development, as well as various somatic mutations that are key for disease classification. Unfortunately, none of the germline loci clearly confers increased risk per se. Contrariwise, somatic mutations identified within the glioma tissue define tumor genotype, thus representing valid diagnostic and prognostic markers. Thus, genetic features can be used in glioma classification and guided therapy. Such copious genomic variabilities are screened routinely in glioma diagnosis. In detail, Sanger sequencing or pyrosequencing, fluorescence in-situ hybridization, and microsatellite analyses were added to immunohistochemistry as diagnostic markers. Recently, Next Generation Sequencing was set-up as an all-in-one diagnostic tool aimed at detecting both DNA copy number variations and mutations in gliomas. This approach is widely used also to detect circulating tumor DNA within cerebrospinal fluid from patients affected by primary brain tumors. Such an approach is providing an alternative cost-effective strategy to genotype all gliomas, which allows avoiding surgical tissue collection and repeated tumor biopsies. This review summarizes available molecular features that represent solid tools for the genetic diagnosis of gliomas at present or in the next future.

Playing with Structural Parameters: Synthesis and Characterization of Two New Maltol-Based Ligands with Binding and Antineoplastic Properties.

Two maltol-based ligands, N,N'-bis((3-hydroxy-4-pyron-2-yl)methyl)-1,4-piperazine (L1) and N,N',N'-tris((3-hydroxy-4-pyron-2-yl)methyl)-N-methylethylendiamine (L2), were synthesized and characterized. L1 and L2, containing, respectively, two and three maltol units spaced by a diamine fragment, were designed to evaluate how biological and binding features are affected by structural modifications of the parent compound malten. The acid-base behavior and the binding properties towards transition, alkaline-earth (AE) and rare-earth (RE) cations in aqueous solution, studied by potentiometric, UV-Vis and NMR analysis, are reported along with biological studies on DNA and leukemia cells. Both ligands form stable complexes with Cu(II), Zn(II) and Co(II) that were studied as metallo-receptors for AE and RE at neutral pH. L1 complexes are more affected than L2 ones by hard cations, the L1-Cu(II) system being deeply affected by RE. The structural modifications altered the mechanism of action: L1 partially maintains the ability to induce structural alterations of DNA, while L2 provokes single strand (nicks) and to a lesser extent double strand breaks of DNA.

Effects of Fifty-Hertz Electromagnetic Fields on Granulocytic Differentiation of ATRA-Treated Acute Promyelocytic Leukemia NB4 Cells.

BACKGROUND/AIMS: Life on Earth is constantly exposed to electromagnetic fields (EMFs) and the effects induced by EMFs on biological systems have been extensively studied producing different and sometimes contradictory results. Extremely low-frequency electromagnetic fields (ELF-EMFs) have shown to play a role in regulating cell proliferation and differentiation, although how EMFs influence these processes remains unclear. Human acute promyelocytic leukemia (APL) cells are characterized by the arrest of differentiation at the promyelocytic stage due to epigenetic perturbations induced by PML/RARα fusion protein (Promyelocytic Leukemia protein - PML/Retinoic Acid Receptor alpha - RARα). Therapeutic administration of all-trans retinoic acid (ATRA) re-establishes the leukemogenic mechanism re-inducing the normal differentiation processes. METHODS: We studied the effects of ELF-EMFs (50 Hz, 2 mT) on the ATRA-mediated granulocytic differentiation process of APL NB4 cells (a cell line established from the bone marrow of a patient affected by the acute promyelocytic leukemia) by monitoring cellular proliferation and morphology, nitrob lue tetrazolium (NBT) reduction and the expression of differentiation surface markers. Finally, we investigated mechanisms focusing on reactive oxygen species (ROS) generation and related molecular pathways. RESULTS: ELF-EMF exposure decreases cellular proliferation potential and helps ATRA-treated NB4 cells to mature. Furthermore, the analysis of ROS production and the consequent extracellular signal regulated kinases (ERK1/2) phosphorylation suggest that a changed intracellular oxidative balance may influence the biological effects of ELF-EMFs. CONCLUSIONS: These results indicate that the exposure to ELF-EMF promotes ATRA-induced granulocytic differentiation of APL cells.

Promoter-specific relevance of histone modifications induced by dexamethasone during the regulation of pro-inflammatory mediators.

Glucocorticosteroids (GCs) are widely used to treat different kinds of chronic inflammatory and immune diseases through transcriptional regulation of inflammatory genes. Modulation of gene expression by GCs is known to occur through diverse mechanisms of varying relevance to specific classes of genes. Epigenetic modifications are indeed a pivotal regulatory feature of glucocorticoid receptor and other transcription factors. In this study, histone post-translational modifications were investigated for their involvement in the regulation of selected pro-inflammatory genes - expressed in human monocyte-derived macrophages - in response to treatment with synthetic GC dexamethasone (DEX). We show that histone tail acetylation status is modified following DEX administration, through distinct and alternative mechanisms at the promoters of interleukin-8 and interleukin-23. In addition to histone H3 acetylation, our results demonstrate that H3 lysine 4 trimethylation is affected following drug treatment.

A preorganized metalloreceptor for alkaline earth ions showing calcium versus magnesium selectivity in water: biological activity of selected metal complexes.

The N,N'-bis[(3-hydroxy-4-pyron-2-yl)methyl]-N,N'-dimethylethylendiamine (Malten = L) forms the highly stable [CuH(-2)L] species in water, in which the converging maltol oxygen atoms form an electron-rich area able to host hard metal ions. When considering the alkaline earth series (AE), the [Cu(H(-2)L)] species binds all metal ions, with the exception of Mg(2+), exhibiting the relevant property to discriminate Ca(2+) versus Mg(2+) at physiological pH 7.4; the binding of the AE metal is visible to the naked eye. The stability constant values of the trinuclear [AE{Cu(H(-2)L)}2](2+) species formed reach the maximum for Ca(2+) (log K=7.7). Ca(2+) also forms a tetranuclear [Ca{Cu(H(-2)L)}]2(4+) species at a high Ca(2+) concentration. Tri- and tetranuclear calcium complexes show blue- and pink-colored crystals, respectively. [Cu(H(-2)L)] is the most active species in inducing DNA alterations. The DNA damages are compatible with its hydrolytic cleavages.

An aza-macrocycle containing maltolic side-arms (maltonis) as potential drug against human pediatric sarcomas.

BACKGROUND: Identification of new drugs against paediatric sarcomas represents an urgent clinical need that mainly relies on public investments due to the rarity of these diseases. In this paper we evaluated the in vitro and in vivo efficacy of a new maltol derived molecule (maltonis), belonging to the family of molecules named hydroxypyrones. METHODS: Maltonis was screened for its ability to induce structural alteration of DNA molecules in comparison to another maltolic molecule (malten). In vitro antitumour efficacy was tested using a panel of sarcoma cell lines, representative of Ewing sarcoma, osteosarcoma and rhabdomyosarcoma, the three most common paediatric sarcomas, and in normal human mesenchymal primary cell cultures. In vivo efficacy was tested against TC-71 Ewing sarcoma xenografts. RESULTS: Maltonis, a soluble maltol-derived synthetic molecule, was able to alter the DNA structure, inhibit proliferation and induce apoptotic cell death in paediatric sarcoma cells, either sensitive or resistant to some conventional chemotherapeutic drugs, such as doxorubicin and cisplatin. In addition, maltonis was able to induce: i) p21, p15 and Gadd45a mRNA upregulation; ii) Bcl-2, survivin, CDK6 and CDK8 down-regulation; iii) formation of γ-H2AX nuclear foci; iv) cleavage of PARP and Caspase 3. Two independent in vivo experiments demonstrated the tolerability and efficacy of maltonis in the inhibition of tumour growth. Finally maltonis was not extruded by ABCB1, one of the major determinants of chemotherapy failure, nor appeared to be a substrate of the glutathione-related detoxification system. CONCLUSIONS: Considering that treatment of poorly responsive patients still suffers for the paucity of agents able to revert chemoresistance, maltonis may be considered for the future development of new therapeutic approaches for refractory metastatic patients.

Modulating the sensor response to halide using NBD-based azamacrocycles.

Ligand L (2,6-bis{[7-(7-nitrobenzo[1,2,5]oxadiazole-4-yl)-3,10-dimethyl-1,4,7,10-tetraazacyclododeca-1-yl]methyl}phenol) is a fluorescent sensor that is useful for detecting Cu(II), Zn(II), and Cd(II). Some of the complexes formed are able to sense the presence of halides in solution. L passes through the cellular membrane, becoming fluorescent inside cells. The H(-1)L- species is able to form dinuclear complexes with [M(2)H(-1)L]3+ stoichiometry with Cu(II), Zn(II), and Cd(II) ions, experiencing a CHEF effect upon metal coordination in an acetonitrile/water 95:5 (v/v) solution. In all three of the complexes investigated, the metal cations are coordinatively unsaturated and can therefore bind secondary ligands as anionic species. The crystal structure of [Cd(2)(H(-1)L)Cl(2)](ClO(4))·4H(2)O is discussed. The Zn(II) complex behaves as an OFF-ON sensor for fluoride and chloride anions.

Pathology tissue-chromatin immunoprecipitation, coupled with high-throughput sequencing, allows the epigenetic profiling of patient samples.

Epigenetic alterations in the pattern of DNA and histone modifications play a crucial role in cancer development. Analysis of patient samples, however, is hampered by technical limitations in the study of chromatin structure from pathology archives that usually consist of heavily fixed, paraffin-embedded material. Here, we present a methodology [pathology tissue-ChIP (PAT-ChIP)] to extract and immunoprecipitate chromatin from paraffin-embedded patient samples up to several years old. In a pairwise comparison with canonical ChIP, PAT-ChIP showed a high reproducibility of results for several histone marks and an identical ability to detect dynamic changes in chromatin structure upon pharmacological treatment. Finally, we showed that PAT-ChIP can be coupled with high-throughput sequencing (PAT-ChIP-Seq) for the genome-wide analysis of distinct chromatin modifications. PAT-ChIP therefore represents a versatile procedure and diagnostic tool for the analysis of epigenetic alterations in cancer and potentially other diseases.

Small molecule-induced epigenomic reprogramming of APL blasts leading to antiviral-like response and c-MYC downregulation.

Acute promyelocytic leukemia (APL) is an aggressive subtype of acute myeloid leukemia (AML) in which the PML/RARα fusion protein exerts oncogenic activities by recruiting repressive complexes to the promoter of specific target genes. Other epigenetic perturbations, as alterations of histone H3 lysine 9 trimethylation (H3K9me3), have been frequently found in AMLs and are associated with leukemogenesis and leukemia progression. Here, we characterized the epigenomic effects of maltonis, a novel maltol-derived molecule, in APL cells. We demonstrate that maltonis treatments induce a profound remodulation of the histone code, reducing global H3K9me3 signal and modulating other histone post-translational modifications. Transcriptomic and epigenomic analyses revealed that maltonis exposure induces changes of genes expression associated with a genomic redistribution of histone H3 lysine 4 trimethylation (H3K4me3) and lysine 27 acetylation (H3K27ac). Upregulation of interferon alpha and gamma response and downregulation of c-MYC target genes, in function of c-MYC reduced expression (monitored in all the hematopoietic neoplasms tested), represent the most significant modulated pathways. These data demonstrate the ability of maltonis to epigenetically reprogram the gene expression profile of APL cells, inducing an intriguing antiviral-like response, concomitantly with the downregulation of c-MYC-related pathways, thus making it an attractive candidate for antileukemic therapy.

SIRT6 promotes metastasis and relapse in HER2-positive breast cancer.

The histone deacetylase sirtuin 6 (SIRT6) has been endowed with anti-cancer capabilities in many tumor types. Here, we investigate the impact of SIRT6-overexpression (SIRT6-OE) in Delta16HER2 mice, which are a bona fide model of HER2-positive breast cancer. After an initial delay in the tumor onset, SIRT6-OE induces a more aggressive phenotype of Delta16HER2 tumors promoting the formation of higher number of tumor foci and metastases than controls. This phenotype of SIRT6-OE tumors is associated with cancer stem cell (CSC)-like features and tumor dormancy, and low senescence and oxidative DNA damage. Accordingly, a sub-set of HER2-positive breast cancer patients with concurrent SIRT6-OE has a significant poorer relapse-free survival (RFS) probability than patients with low expression of SIRT6. ChIP-seq, RNA-seq and RT-PCR experiments indicate that SIRT6-OE represses the expression of the T-box transcription factor 3 (Tbx3) by deacetylation of H3K9ac. Accordingly, loss-of-function mutations of TBX3 or low TBX3 expression levels are predictive of poor prognosis in HER2-positive breast cancer patients. Our work indicates that high levels of SIRT6 are indicative of poor prognosis and high risk of metastasis in HER2-positive breast cancer and suggests further investigation of TBX3 as a downstream target of SIRT6 and co-marker of poor-prognosis. Our results point to a breast cancer subtype-specific effect of SIRT6 and warrant future studies dissecting the mechanisms of SIRT6 regulation in different breast cancer subtypes.

Cohort analysis of novel SPAST variants in SPG4 patients and implementation of in vitro and in vivo studies to identify the pathogenic mechanism caused by splicing mutations.

Introduction: Pure hereditary spastic paraplegia (SPG) type 4 (SPG4) is caused by mutations of SPAST gene. This study aimed to analyze SPAST variants in SPG4 patients to highlight the occurrence of splicing mutations and combine functional studies to assess the relevance of these variants in the molecular mechanisms of the disease. Methods: We performed an NGS panel in 105 patients, in silico analysis for splicing mutations, and in vitro minigene assay. Results and discussion: The NGS panel was applied to screen 105 patients carrying a clinical phenotype corresponding to upper motor neuron syndrome (UMNS), selectively affecting motor control of lower limbs. Pathogenic mutations in SPAST were identified in 12 patients (11.42%), 5 missense, 3 frameshift, and 4 splicing variants. Then, we focused on the patients carrying splicing variants using a combined approach of in silico and in vitro analysis through minigene assay and RNA, if available. For two splicing variants (i.e., c.1245+1G>A and c.1414-2A>T), functional assays confirm the types of molecular alterations suggested by the in silico analysis (loss of exon 9 and exon 12). In contrast, the splicing variant c.1005-1delG differed from what was predicted (skipping exon 7), and the functional study indicates the loss of frame and formation of a premature stop codon. The present study evidenced the high splice variants in SPG4 patients and indicated the relevance of functional assays added to in silico analysis to decipher the pathogenic mechanism.

Multi-use NBD-based tetra-amino macrocycle: fluorescent probe for metals and anions and live cell marker.

Ligand L (4-(7-nitrobenzo[1,2,5]oxadiazole-4-yl)-1,7-dimethyl-1,4,7,10-tetra-azacyclododecane) is a versatile fluorescent sensor useful for Cu(II), Zn(II) and Cd(II) metal detection, as a building block of fluorescent metallo-receptor for halide detection, and as an organelle marker inside live cells. Ligand L undergoes a chelation-enhanced fluorescence (CHEF) effect upon metal coordination in acetonitrile solution. In all three complexes investigated the metal cation is coordinatively unsaturated; thus, it can bind secondary ligands as anionic species. The crystal structure of [ZnLCl](ClO(4)) is discussed. Cu(II) and Zn(II) complexes are quenched upon halide interaction, whereas the [CdL](2+) species behaves as an OFF-ON sensor for halide anions in acetonitrile solution. The mechanism of the fluorescence response in the presence of the anion depends on the nature of the metal ion employed and has been studied by spectroscopic methods, such as NMR spectroscopy, UV/Vis and fluorescence techniques and by computational methods. Subcellular localization experiments performed on HeLa cells show that L mainly localizes in spot-like structures in a polarized portion of the cytosol that is occupied by the Golgi apparatus to give a green fluorescence signal.

Synthesis, basicity, structural characterization, and biochemical properties of two [(3-hydroxy-4-pyron-2-yl)methyl]amine derivatives showing antineoplastic features.

The N,N'-bis[(3-hydroxy-4-pyron-2-yl)methyl]-N,N'-dimethylethylendiamine (malten) and 4,10-bis[(3-hydroxy-4-pyron-2-yl)methyl]-1,7-dimethyl-1,4,7,10-tetraazacyclododecane (maltonis) were synthesized and characterized. The acid-base behavior, structural characterizations, and biochemical studies in aqueous solution were reported. Each compound contains two 3-hydroxy-2-methyl-4-pyrone units (maltol) symmetrically spaced by a polyamine fragment, the 1,4-dimethylethylendiamine (malten), or the 1,7-dimethyl-1,4,7,10-tetraazacyclododecane (maltonis). They are present at physiological pH 7.4 in the form of differently charged species: neutral but in a zwitterion form for malten and monopositive with an internal separation of charges for maltonis. Malten and maltonis are both able to alter the chromatin structure inducing the covalent binding of genomic DNA with proteins, a feature consistent with the known antiproliferative activity exerted by this class of molecules. Solid-state results and MD simulations in water show that malten, because of its molecular topology, should be more prone than maltonis to act as a donor of H-bonds in intermolecular contacts, thus it should give a better noncovalent approach with the negatively charged DNA. Crystal structures of [H(2)malten](2+) and [H(2)maltonis](2+) cations were also reported.

Time makes histone H3 modifications drift in mouse liver.

To detect the epigenetic drift of time passing, we determined the genome-wide distributions of mono- and tri-methylated lysine 4 and acetylated and tri-methylated lysine 27 of histone H3 in the livers of healthy 3, 6 and 12 months old C57BL/6 mice. The comparison of different age profiles of histone H3 marks revealed global redistribution of histone H3 modifications with time, in particular in intergenic regions and near transcription start sites, as well as altered correlation between the profiles of different histone modifications. Moreover, feeding mice with caloric restriction diet, a treatment known to retard aging, reduced the extent of changes occurring during the first year of life in these genomic regions.

Histone H3 Lysine 4 and 27 Trimethylation Landscape of Human Alzheimer's Disease.

BACKGROUND: Epigenetic remodeling is emerging as a critical process for both the onset and progression of Alzheimer's disease (AD), the most common form of neurodegenerative dementia. However, it is not clear to what extent the distribution of histone modifications is involved in AD. METHODS: To investigate histone H3 modifications in AD, we compared the genome-wide distributions of H3K4me3 and H3K27me3 in entorhinal cortices from severe sporadic AD patients and from age-matched healthy individuals of both sexes. RESULTS: AD samples were characterized by typical average levels and distributions of the H3K4me3 and H3K27me3 signals. However, AD patients showed a lower H3K4me3 and higher H3K27me3 signal, particularly in males. Interestingly, the genomic sites found differentially trimethylated at the H3K4 between healthy and AD samples involve promoter regions of genes belonging to AD-related pathways such as glutamate receptor signaling. CONCLUSIONS: The signatures of H3K4me3 and H3K27me3 identified in AD patients validate the role of epigenetic chromatin remodeling in neurodegenerative disease and shed light on the genomic adaptive mechanisms involved in AD.

The indole-3-carbinol cyclic tetrameric derivative CTet inhibits cell proliferation via overexpression of p21/CDKN1A in both estrogen receptor-positive and triple-negative breast cancer cell lines.

INTRODUCTION: Indole-3-carbinol (I3C), an autolysis product of glucosinolates present in cruciferous vegetables, and its dimeric derivative (3,3'-DIM) have been indicated as promising agents in preventing the development and progression of breast cancer. We have recently shown that I3C cyclic tetrameric derivative CTet formulated in γ-cyclodextrin (γ-CD) efficiently inhibited cellular proliferation in breast cancer cell lines. This study aims to analyze the mechanisms involved in the in vitro inhibition of cell proliferation and to evaluate the in vivo antitumor activity of CTet in a xenograft study. METHODS: Estrogen receptor-positive MCF-7 and triple-negative MDA-MB-231 breast cancer cell lines were exposed to CTet to evaluate cell cycle perturbation (propidium iodide staining and cytofluorimetric acquisition), induction of autophagic morphological features (co-localization of LC3b autophagosome marker and LAMP2a lysosome marker by immunofluorescence) and changes in protein expression (immunoblot and microarray-based gene expression analyses). To test the in vivo efficacy of CTet, female athymic nude mice inoculated with MCF-7 cells were i.p. treated with 5 mg/kg/day of CTet for five days/week for two weeks and the tumor mass was externally monitored. RESULTS: CTet induced accumulation in G2/M phase without evidence of apoptotic response induction in both cell lines tested. In triple-negative MDA-MB-231 the autophagic lysosomal activity was significantly up-regulated after exposure to 4 µM of CTet for 8 hours, while the highest CTet concentration was necessary to observe autophagic features in MCF-7 cells. The inhibition of Akt activity and p53-independent p21/CDKN1A and GADD45A overexpression were identified as the main molecular events responsible for CTet activity in MCF-7 and p53-mutant MDA-MB-231 cells. In vivo, CTet administration was able to significantly inhibit the growth of MCF-7 xenotransplanted into nude mice, without adverse effect on body weight or on haematological parameters. CONCLUSIONS: Our data support CTet formulated with γ-CD as a promising and injectable anticancer agent for both hormone-responsive and triple-negative breast tumors.