Since the multifunctionality of transglutaminase 2 (TG2) includes extra- and intracellular functi... more Since the multifunctionality of transglutaminase 2 (TG2) includes extra- and intracellular functions, we investigated the effects of intracellular administration of TG2 inhibitors in three breast cancer cell lines, MDA-MB-231, MDA-MB-436 and MDA-MB-468, which are representative of different triple-negative phenotypes, using a patch-clamp technique. The first cell line has a highly voltage-dependent a membrane current, which is low in the second and almost absent in the third one. While applying a voltage protocol to responsive single cells, injection of TG2 inhibitors triggered a significant decrease of the current in MDA-MB-231 that we attributed to voltage-dependent K+ channels using the specific inhibitors 4-aminopyridine and astemizole. Since the Kv10.1 channel plays a dominant role as a marker of cell migration and survival in breast cancer, we investigated its relationship with TG2 by immunoprecipitation. Our data reveal their physical interaction affects membrane currents in ...
We have investigated motility in breast cancer cell lines in association with the expression of T... more We have investigated motility in breast cancer cell lines in association with the expression of Transglutaminase type 2 (TG2) as well as upon the administration of Doxorubicin (Dox), an active cytotoxic agent that is employed in chemotherapy. The exposure of MCF-7 cells to the drug increased TG2 levels, triggering epithelial–mesenchymal transition (EMT), thereby supporting cell motility. The effects of Dox on the movement of MCF-7 cells were counteracted by treatment with NC9, a TG2 inhibitor, which induced morphological changes and also reduced the migration of MDA-MB-231 cells exhibiting high levels of TG2. The physical association of TG2 with the cytoskeletal component vimentin appeared pivotal both in drug-treated MCF-7 and in MDA-MB-231 cells and seemed to be independent of the catalytic activity of TG2. NC9 altered the subcellular distribution of TG2 and, consequently, the co-localization of TG2 with vimentin. Furthermore, NC9 induced a nuclear accumulation of TG2 as a prelude...
Long non-coding RNAs are nucleotide molecules that regulate transcription in numerous cellular pr... more Long non-coding RNAs are nucleotide molecules that regulate transcription in numerous cellular processes and are related to the occurrence of many diseases, including cancer. In this regard, we recently discovered a polyadenylated long non-coding RNA (named TG2-lncRNA) encoded within the first intron of the Transglutaminase type 2 gene (TGM2), which is related to tumour proliferation in human cancer cell lines. To better characterize this new biological player, we investigated the effects of its suppression in MCF-7 breast cancer cells, using siRNA treatment and RNA-sequencing. In this way, we found modifications in several networks associated to biological functions relevant for tumorigenesis (apoptosis, chronic inflammation, angiogenesis, immunomodulation, cell mobility, and epithelial–mesenchymal transition) that were originally attributed only to Transglutaminase type 2 protein but that could be regulated also by TG2-lncRNA. Moreover, our experiments strongly suggest the ability...
Despite convincing experimental evidence, epidemiological studies on the effects of serum uric ac... more Despite convincing experimental evidence, epidemiological studies on the effects of serum uric acid (SUA) on bone health are still conflicting since factors influencing SUA bioavailability have not been adequately considered. To shed some light on this issue, we investigated the impact of adiposity and menopause status on the relationship between SUA and bone health. We examined SUA in relation to bone mineral density (BMD) at different skeletal sites and with markers of bone metabolism in 124 pre-menopausal and 234 post-menopausal women and assessed whether adiposity, evaluated by anthropometry and dual x-ray absorptiometry (DXA), might have a discriminant role. After conservative adjustment (covariates: age, hormones treatment, smoking and time since menopause), SUA showed a significant and positive association with total hip BMD (β = 0.220, p < 0.01) among postmenopausal women, maintained also after adjustment for legs adiposity. Notably, stratification for waist circumference...
The multifunctional protein Transglutaminase type 2, is associated with cancer epithelial mesench... more The multifunctional protein Transglutaminase type 2, is associated with cancer epithelial mesenchymal transition, invasiveness, stemness and drugs resistance. Several variant isoforms and non-coding RNAs are present in cancer and this report explored the expression of these transcripts of the TGM2 gene in cancer cell lines after induction with all-trans retinoic acid. The expression of truncated variants along with two long non-coding RNAs, was demonstrated. One of these is coded from the first intron and the Last Exon Variant is constituted by a sequence corresponding to the last three exons and the 3′UTR. Analysis of ChIP-seq data, from ENCODE project, highlighted factors interacting with intronic sequences, which could interfere with the progression of RNApol II at checkpoints, during the elongation process. Some relevant transcription factors, bound in an ATRA-dependent way, were found by RNA immunoprecipitation, notably GATA3 mainly enriched to Last Exon Variant non-coding RNA. The involvement of NMD in the regulation of the ratio among these transcripts was observed, as the prevalent recovering of Last Exon Variant to phUPF1-complexes, with decrease of the binding towards other selective targets. This study contributes to identify molecular mechanisms regulating the ratio among the variants and improves the knowledge about regulatory roles of the non-coding RNAs of the TGM2 gene.
The changes in hemoglobin (Hb) profile following autologous blood transfusion (ABT) for the first... more The changes in hemoglobin (Hb) profile following autologous blood transfusion (ABT) for the first time were studied for anti-doping purposes. Twenty-four healthy, trained male subjects (aged 18‒40) were enrolled and randomized into either the transfusion (T) or control (C) groups. Blood samples were taken from the T subjects at baseline, after withdrawal and reinfusion of 450 ml of refrigerated or cryopreserved blood, and from C subjects at the same time points. Hematological variables (Complete blood count, Reticulocytes, Immature Reticulocytes Fraction, Red-cell Distribution Width, OFF-hr score) were measured. The Hb types were analyzed by high-performance liquid chromatography and the Hemoglobin Profile Index (HbPI) arbitrarily calculated. Between-group differences were observed for red blood cells and reticulocytes. Unlike C, the T group, after withdrawal and reinfusion, showed a significant trend analysis for both hematological variables (Hemoglobin concentration, reticulocytes...
Cytokine-induced killer (CIK) cells, a heterogeneous T cell population obtained by differentiatio... more Cytokine-induced killer (CIK) cells, a heterogeneous T cell population obtained by differentiation of peripheral blood mononuclear cells (PBMC), represent a promising immunological approach in cancer. Numerous studies have explored the role of CD38, CD39, CD203a/PC-1, and CD73 in generating extracellular adenosine (ADO) and thus in shaping the tumor niche in favor of proliferation. The findings shown here reveal that CIK cells are able to produce extracellular ADO via traditional (CD39/CD73) and/or alternative (CD38/CD203a/CD73 or CD203a/CD73) pathways. Transcriptome analysis showed the mRNA expression of these molecules and their modulation during PBMC to CIK differentiation. When PBMC from normal subjects or cancer bearing patients were differentiated into CIK cells under normoxic conditions, CD38 and CD39 were greatly up-regulated while the number of CD203a, and CD73 positive cells underwent minor changes. Since hypoxic conditions are often found in tumors, we asked whether CD39,...
The type 2 isoenzyme is the most widely expressed transglutaminase in mammals displaying several ... more The type 2 isoenzyme is the most widely expressed transglutaminase in mammals displaying several intra- and extracellular activities depending on its location (protein modification, modulation of gene expression, membrane signalling and stabilization of cellular interactions with the extracellular matrix) in relation to cell death, survival and differentiation. In contrast with the appreciable knowledge about the regulation of the enzymatic activities, much less is known concerning its inducible expression, which is altered in inflammatory and neoplastic diseases. In this context, we first summarize the gene's basic features including single-nucleotide polymorphism characterization, epigenetic DNA methylation and identification of regulatory regions and of transcription factor-binding sites at the gene promoter, which could concur to direct gene expression. Further aspects related to alternative splicing events and to ncRNAs (microRNAs and lncRNAs) are involved in the modulation...
International journal of molecular sciences, Jan 26, 2017
The involvement of microRNAs in the control of repressors of human γ-globin gene transcription ha... more The involvement of microRNAs in the control of repressors of human γ-globin gene transcription has been firmly demonstrated, as described for the miR-486-3p mediated down-regulation of BCL11A. On the other hand, we have reported that miR-210 is involved in erythroid differentiation and, possibly, in γ-globin gene up-regulation. In the present study, we have identified the coding sequence of BCL11A as a possible target of miR-210. The following results sustain this hypothesis: (a) interactions between miR-210 and the miR-210 BCL11A site were demonstrated by SPR-based biomolecular interaction analysis (BIA); (b) the miR-210 site of BCL11A is conserved through molecular evolution; (c) forced expression of miR-210 leads to decrease of BCL11A-XL and increase of γ-globin mRNA content in erythroid cells, including erythroid precursors isolated from β-thalassemia patients. Our study suggests that the coding mRNA sequence of BCL11A can be targeted by miR-210. In addition to the theoretical p...
The long non-coding RNAs (lncRNAs) are matter of intense investigation as potential regulators of... more The long non-coding RNAs (lncRNAs) are matter of intense investigation as potential regulators of gene expression. In the case of the transglutaminase 2 gene (TGM2) the databases of genome sequence indicate location of a lncRNA (LOC107987281) within the first intron. This lncRNA is 1000 bp long, arises from 2 exons and starts few nucleotides 3' of the first splicing site of translated TGM2. We have analysed correlations between expression of LOC107987281 lncRNA and TGM2 mRNA by real-time PCR in K562 cell line untreated or treated with the anticancer drugs TPA (12-O-tetradecanoylphorbol-13-acetate), Docetaxel and Doxorubicin. In the treated cells the lncRNA increase follows the trend of TGM2 transcript. To validate this finding we used HumanExon1_0ST Affymetrix; chip data were background-adjusted, quantile-normalized and summarized using robust multi-array average analysis implemented in the R package. The probesets recognize sequences inside each exon, near intronic splicing sit...
Molecular medicine (Cambridge, Mass.), Oct 9, 2017
Adoptive immunotherapy with Cytokine Induced Killer (CIK) cells has shown antitumor activity agai... more Adoptive immunotherapy with Cytokine Induced Killer (CIK) cells has shown antitumor activity against several kinds of cancers in preclinical models and clinical trials. CIK cells are a subset of ex vivo expanded T lymphocytes with T-NK phenotype and MHC-unrestricted antitumor activity. Literature provides scanty information on cytokines, chemokines and growth factors secreted by CIK cells. Therefore, we investigated the secretory profile of CIK cells generated from tumor patients. The secretome analysis was performed at specific time points (day 1, day 14 and day 21) of CIK cells expansion. Mature CIK cells (day 21) produce a great variety of interleukins and secreted proteins that can be divided into 3 groups based on their secretion quantity: high (IL-13, RANTES, MIP-1α and 1β), medium (IL-1Ra, IL-5, IL-8, IL-10, IL-17, IP-10, INF-γ, VEGF and GMCSF) and low (IL-1β, IL-4, IL-6, IL-7, IL-9, IL-12, IL-15, Eotaxin, PDGF-bb, FGF basic, G-CSF and MCP-1) secreted. Moreover, comparing PBM...
MicroRNAs (miRNAs) are small non-coding RNA molecules capable of silencing mRNA targets. miRNA dy... more MicroRNAs (miRNAs) are small non-coding RNA molecules capable of silencing mRNA targets. miRNA dysregulation has been linked to cancer development, cardiovascular and neurological diseases, lipid metabolism, and impaired immunity. Therefore, miRNAs are gaining interest as putative novel disease biomarkers and therapeutic targets. Recent studies have shown that purinergic surface receptors activated by extracellular nucleotides (ATP, ADP, UTP, UDP), and by nucleosides such as adenosine (ADO), are subject to miRNA regulation. This opens a new and previously unrecognized opportunity to modulate the purinergic network with the aim of avoiding abnormal activation of specific receptor subtypes. miRNA technology will hopefully contribute strategies to prevent purinergic-mediated tissue damage in conditions of neurodegeneration, atherosclerosis, transplantation, and even neoplasia.
The development of drugs able to inhibit the expression of pro-inflammatory genes is of great int... more The development of drugs able to inhibit the expression of pro-inflammatory genes is of great interest in the treatment of cystic fibrosis (CF). Chronic pulmonary inflammation in the lungs of patients affected by CF is characterized by massive intra-bronchial infiltrates of neutrophils. This process is initiated upon interaction of pathogens (including Pseudomonas aeruginosa) with surface bronchial cells. Consequently, they release cytokines, the most represented being the potent neutrophilic chemokine Interleukin (IL)-8 and the pro-inflammatory cytokine IL-6. The chronic inflammatory process is crucial, since it leads to progressive tissue damage and severe respiratory insufficiency. In order to reduce the adverse effects of the excessive inflammatory response, one of the approaches leading to inhibition of IL-8 and IL-6 gene expression is the transcription factor (TF) decoy approach, based on intracellular delivery of double stranded oligodeoxynucleotides (ODNs) mimicking the binding sites of TFs and causing inhibition of binding of TF-related proteins to regulatory sequences identified in the promoters of specific genes. Since the promoters of IL-8 and IL-6 contain consensus sequences for NF-κ B and Sp1, double stranded TF &quot;decoy&quot; ODNs targeting NF-κB and Sp1 can be used. Alternatively, screening of drugs targeting relevant TFs can be performed using drug cocktails constituted by extracts from medicinal plants inhibiting TF/DNA interactions. Finally, virtual screening might lead to identification of putative bioactive molecules to be validated using molecular and cellular approaches. By these means, low-molecular drugs targeting NF-κB and inhibiting IL-8 gene expression are available for pre-clinical testing using experimental systems recapitulating chronic pulmonary inflammation of patients affected by CF.
The design, synthesis and in vitro activities of novel alpha-bromoacryloyl pyrazole, imidazole an... more The design, synthesis and in vitro activities of novel alpha-bromoacryloyl pyrazole, imidazole and benzoheterocyclic derivatives of distamycin A, in which the amidino moiety has been replaced by moieties of different physico-chemical features are described, and the structure-activity relationships are discussed. In spite of the relevance of these modifications on the distamycin frame, these derivatives showed significant growth inhibitory activity against mouse leukemia L1210 cells. Therefore, the presence of the amidino moiety, and in general of a basic moiety, is not an absolute requirement for biological activity of alpha-bromoacrylic derivatives of distamycin.
Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics, 2003
Double-stranded molecules based on peptide nucleic acids (PNAs)-DNA chimeras carrying binding sit... more Double-stranded molecules based on peptide nucleic acids (PNAs)-DNA chimeras carrying binding sites for known transcription factors could be of great interest in decoy pharmacotherapy of neoplastic diseases. For instance, decoy molecules recognizing Sp1 and NF-kappaB transcription factors were found to inhibit tumor cell growth and invasion activity. In this respect, we have recently found that double-stranded PNA-DNA chimeras carrying NF-kappaB binding sites inhibit the binding of NF-kappaB p52 and p50 transcription factors to target DNA molecules. In this article we determined the resistance of double-stranded decoy molecules based on PNA-DNA chimeras to exonucleases (both 3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;--&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; and 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;--&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; exonucleases), endonucleases, and 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-phosphatases. In addition, we performed experiments aimed at determining the resistance of these molecules in cellular extracts and serum. Finally, we used liposomes as protective agents in experimental conditions in which the decoy molecules employed were found to be unstable (high concentrations of enzymes, cellular extracts, or serum). The results obtained demonstrated that decoy molecules based on PNA-DNA chimeras are more resistant than DNA-based decoys to exo- and endonucleases, serum, and cellular extracts. In addition, the resistance of DNA/PNA hybrids in the presence of high concentrations of serum and cellular extracts was increased after complexation to cationic liposomes, due to the fact that double-stranded PNA-DNA-PNA chimeras bind to these delivery systems. The results obtained in the present study support the proposal of molecules based on PNA-DNA chimeras for an efficient decoy treatment of tumor cells both in vitro and in vivo.
Since the multifunctionality of transglutaminase 2 (TG2) includes extra- and intracellular functi... more Since the multifunctionality of transglutaminase 2 (TG2) includes extra- and intracellular functions, we investigated the effects of intracellular administration of TG2 inhibitors in three breast cancer cell lines, MDA-MB-231, MDA-MB-436 and MDA-MB-468, which are representative of different triple-negative phenotypes, using a patch-clamp technique. The first cell line has a highly voltage-dependent a membrane current, which is low in the second and almost absent in the third one. While applying a voltage protocol to responsive single cells, injection of TG2 inhibitors triggered a significant decrease of the current in MDA-MB-231 that we attributed to voltage-dependent K+ channels using the specific inhibitors 4-aminopyridine and astemizole. Since the Kv10.1 channel plays a dominant role as a marker of cell migration and survival in breast cancer, we investigated its relationship with TG2 by immunoprecipitation. Our data reveal their physical interaction affects membrane currents in ...
We have investigated motility in breast cancer cell lines in association with the expression of T... more We have investigated motility in breast cancer cell lines in association with the expression of Transglutaminase type 2 (TG2) as well as upon the administration of Doxorubicin (Dox), an active cytotoxic agent that is employed in chemotherapy. The exposure of MCF-7 cells to the drug increased TG2 levels, triggering epithelial–mesenchymal transition (EMT), thereby supporting cell motility. The effects of Dox on the movement of MCF-7 cells were counteracted by treatment with NC9, a TG2 inhibitor, which induced morphological changes and also reduced the migration of MDA-MB-231 cells exhibiting high levels of TG2. The physical association of TG2 with the cytoskeletal component vimentin appeared pivotal both in drug-treated MCF-7 and in MDA-MB-231 cells and seemed to be independent of the catalytic activity of TG2. NC9 altered the subcellular distribution of TG2 and, consequently, the co-localization of TG2 with vimentin. Furthermore, NC9 induced a nuclear accumulation of TG2 as a prelude...
Long non-coding RNAs are nucleotide molecules that regulate transcription in numerous cellular pr... more Long non-coding RNAs are nucleotide molecules that regulate transcription in numerous cellular processes and are related to the occurrence of many diseases, including cancer. In this regard, we recently discovered a polyadenylated long non-coding RNA (named TG2-lncRNA) encoded within the first intron of the Transglutaminase type 2 gene (TGM2), which is related to tumour proliferation in human cancer cell lines. To better characterize this new biological player, we investigated the effects of its suppression in MCF-7 breast cancer cells, using siRNA treatment and RNA-sequencing. In this way, we found modifications in several networks associated to biological functions relevant for tumorigenesis (apoptosis, chronic inflammation, angiogenesis, immunomodulation, cell mobility, and epithelial–mesenchymal transition) that were originally attributed only to Transglutaminase type 2 protein but that could be regulated also by TG2-lncRNA. Moreover, our experiments strongly suggest the ability...
Despite convincing experimental evidence, epidemiological studies on the effects of serum uric ac... more Despite convincing experimental evidence, epidemiological studies on the effects of serum uric acid (SUA) on bone health are still conflicting since factors influencing SUA bioavailability have not been adequately considered. To shed some light on this issue, we investigated the impact of adiposity and menopause status on the relationship between SUA and bone health. We examined SUA in relation to bone mineral density (BMD) at different skeletal sites and with markers of bone metabolism in 124 pre-menopausal and 234 post-menopausal women and assessed whether adiposity, evaluated by anthropometry and dual x-ray absorptiometry (DXA), might have a discriminant role. After conservative adjustment (covariates: age, hormones treatment, smoking and time since menopause), SUA showed a significant and positive association with total hip BMD (β = 0.220, p < 0.01) among postmenopausal women, maintained also after adjustment for legs adiposity. Notably, stratification for waist circumference...
The multifunctional protein Transglutaminase type 2, is associated with cancer epithelial mesench... more The multifunctional protein Transglutaminase type 2, is associated with cancer epithelial mesenchymal transition, invasiveness, stemness and drugs resistance. Several variant isoforms and non-coding RNAs are present in cancer and this report explored the expression of these transcripts of the TGM2 gene in cancer cell lines after induction with all-trans retinoic acid. The expression of truncated variants along with two long non-coding RNAs, was demonstrated. One of these is coded from the first intron and the Last Exon Variant is constituted by a sequence corresponding to the last three exons and the 3′UTR. Analysis of ChIP-seq data, from ENCODE project, highlighted factors interacting with intronic sequences, which could interfere with the progression of RNApol II at checkpoints, during the elongation process. Some relevant transcription factors, bound in an ATRA-dependent way, were found by RNA immunoprecipitation, notably GATA3 mainly enriched to Last Exon Variant non-coding RNA. The involvement of NMD in the regulation of the ratio among these transcripts was observed, as the prevalent recovering of Last Exon Variant to phUPF1-complexes, with decrease of the binding towards other selective targets. This study contributes to identify molecular mechanisms regulating the ratio among the variants and improves the knowledge about regulatory roles of the non-coding RNAs of the TGM2 gene.
The changes in hemoglobin (Hb) profile following autologous blood transfusion (ABT) for the first... more The changes in hemoglobin (Hb) profile following autologous blood transfusion (ABT) for the first time were studied for anti-doping purposes. Twenty-four healthy, trained male subjects (aged 18‒40) were enrolled and randomized into either the transfusion (T) or control (C) groups. Blood samples were taken from the T subjects at baseline, after withdrawal and reinfusion of 450 ml of refrigerated or cryopreserved blood, and from C subjects at the same time points. Hematological variables (Complete blood count, Reticulocytes, Immature Reticulocytes Fraction, Red-cell Distribution Width, OFF-hr score) were measured. The Hb types were analyzed by high-performance liquid chromatography and the Hemoglobin Profile Index (HbPI) arbitrarily calculated. Between-group differences were observed for red blood cells and reticulocytes. Unlike C, the T group, after withdrawal and reinfusion, showed a significant trend analysis for both hematological variables (Hemoglobin concentration, reticulocytes...
Cytokine-induced killer (CIK) cells, a heterogeneous T cell population obtained by differentiatio... more Cytokine-induced killer (CIK) cells, a heterogeneous T cell population obtained by differentiation of peripheral blood mononuclear cells (PBMC), represent a promising immunological approach in cancer. Numerous studies have explored the role of CD38, CD39, CD203a/PC-1, and CD73 in generating extracellular adenosine (ADO) and thus in shaping the tumor niche in favor of proliferation. The findings shown here reveal that CIK cells are able to produce extracellular ADO via traditional (CD39/CD73) and/or alternative (CD38/CD203a/CD73 or CD203a/CD73) pathways. Transcriptome analysis showed the mRNA expression of these molecules and their modulation during PBMC to CIK differentiation. When PBMC from normal subjects or cancer bearing patients were differentiated into CIK cells under normoxic conditions, CD38 and CD39 were greatly up-regulated while the number of CD203a, and CD73 positive cells underwent minor changes. Since hypoxic conditions are often found in tumors, we asked whether CD39,...
The type 2 isoenzyme is the most widely expressed transglutaminase in mammals displaying several ... more The type 2 isoenzyme is the most widely expressed transglutaminase in mammals displaying several intra- and extracellular activities depending on its location (protein modification, modulation of gene expression, membrane signalling and stabilization of cellular interactions with the extracellular matrix) in relation to cell death, survival and differentiation. In contrast with the appreciable knowledge about the regulation of the enzymatic activities, much less is known concerning its inducible expression, which is altered in inflammatory and neoplastic diseases. In this context, we first summarize the gene's basic features including single-nucleotide polymorphism characterization, epigenetic DNA methylation and identification of regulatory regions and of transcription factor-binding sites at the gene promoter, which could concur to direct gene expression. Further aspects related to alternative splicing events and to ncRNAs (microRNAs and lncRNAs) are involved in the modulation...
International journal of molecular sciences, Jan 26, 2017
The involvement of microRNAs in the control of repressors of human γ-globin gene transcription ha... more The involvement of microRNAs in the control of repressors of human γ-globin gene transcription has been firmly demonstrated, as described for the miR-486-3p mediated down-regulation of BCL11A. On the other hand, we have reported that miR-210 is involved in erythroid differentiation and, possibly, in γ-globin gene up-regulation. In the present study, we have identified the coding sequence of BCL11A as a possible target of miR-210. The following results sustain this hypothesis: (a) interactions between miR-210 and the miR-210 BCL11A site were demonstrated by SPR-based biomolecular interaction analysis (BIA); (b) the miR-210 site of BCL11A is conserved through molecular evolution; (c) forced expression of miR-210 leads to decrease of BCL11A-XL and increase of γ-globin mRNA content in erythroid cells, including erythroid precursors isolated from β-thalassemia patients. Our study suggests that the coding mRNA sequence of BCL11A can be targeted by miR-210. In addition to the theoretical p...
The long non-coding RNAs (lncRNAs) are matter of intense investigation as potential regulators of... more The long non-coding RNAs (lncRNAs) are matter of intense investigation as potential regulators of gene expression. In the case of the transglutaminase 2 gene (TGM2) the databases of genome sequence indicate location of a lncRNA (LOC107987281) within the first intron. This lncRNA is 1000 bp long, arises from 2 exons and starts few nucleotides 3' of the first splicing site of translated TGM2. We have analysed correlations between expression of LOC107987281 lncRNA and TGM2 mRNA by real-time PCR in K562 cell line untreated or treated with the anticancer drugs TPA (12-O-tetradecanoylphorbol-13-acetate), Docetaxel and Doxorubicin. In the treated cells the lncRNA increase follows the trend of TGM2 transcript. To validate this finding we used HumanExon1_0ST Affymetrix; chip data were background-adjusted, quantile-normalized and summarized using robust multi-array average analysis implemented in the R package. The probesets recognize sequences inside each exon, near intronic splicing sit...
Molecular medicine (Cambridge, Mass.), Oct 9, 2017
Adoptive immunotherapy with Cytokine Induced Killer (CIK) cells has shown antitumor activity agai... more Adoptive immunotherapy with Cytokine Induced Killer (CIK) cells has shown antitumor activity against several kinds of cancers in preclinical models and clinical trials. CIK cells are a subset of ex vivo expanded T lymphocytes with T-NK phenotype and MHC-unrestricted antitumor activity. Literature provides scanty information on cytokines, chemokines and growth factors secreted by CIK cells. Therefore, we investigated the secretory profile of CIK cells generated from tumor patients. The secretome analysis was performed at specific time points (day 1, day 14 and day 21) of CIK cells expansion. Mature CIK cells (day 21) produce a great variety of interleukins and secreted proteins that can be divided into 3 groups based on their secretion quantity: high (IL-13, RANTES, MIP-1α and 1β), medium (IL-1Ra, IL-5, IL-8, IL-10, IL-17, IP-10, INF-γ, VEGF and GMCSF) and low (IL-1β, IL-4, IL-6, IL-7, IL-9, IL-12, IL-15, Eotaxin, PDGF-bb, FGF basic, G-CSF and MCP-1) secreted. Moreover, comparing PBM...
MicroRNAs (miRNAs) are small non-coding RNA molecules capable of silencing mRNA targets. miRNA dy... more MicroRNAs (miRNAs) are small non-coding RNA molecules capable of silencing mRNA targets. miRNA dysregulation has been linked to cancer development, cardiovascular and neurological diseases, lipid metabolism, and impaired immunity. Therefore, miRNAs are gaining interest as putative novel disease biomarkers and therapeutic targets. Recent studies have shown that purinergic surface receptors activated by extracellular nucleotides (ATP, ADP, UTP, UDP), and by nucleosides such as adenosine (ADO), are subject to miRNA regulation. This opens a new and previously unrecognized opportunity to modulate the purinergic network with the aim of avoiding abnormal activation of specific receptor subtypes. miRNA technology will hopefully contribute strategies to prevent purinergic-mediated tissue damage in conditions of neurodegeneration, atherosclerosis, transplantation, and even neoplasia.
The development of drugs able to inhibit the expression of pro-inflammatory genes is of great int... more The development of drugs able to inhibit the expression of pro-inflammatory genes is of great interest in the treatment of cystic fibrosis (CF). Chronic pulmonary inflammation in the lungs of patients affected by CF is characterized by massive intra-bronchial infiltrates of neutrophils. This process is initiated upon interaction of pathogens (including Pseudomonas aeruginosa) with surface bronchial cells. Consequently, they release cytokines, the most represented being the potent neutrophilic chemokine Interleukin (IL)-8 and the pro-inflammatory cytokine IL-6. The chronic inflammatory process is crucial, since it leads to progressive tissue damage and severe respiratory insufficiency. In order to reduce the adverse effects of the excessive inflammatory response, one of the approaches leading to inhibition of IL-8 and IL-6 gene expression is the transcription factor (TF) decoy approach, based on intracellular delivery of double stranded oligodeoxynucleotides (ODNs) mimicking the binding sites of TFs and causing inhibition of binding of TF-related proteins to regulatory sequences identified in the promoters of specific genes. Since the promoters of IL-8 and IL-6 contain consensus sequences for NF-κ B and Sp1, double stranded TF &quot;decoy&quot; ODNs targeting NF-κB and Sp1 can be used. Alternatively, screening of drugs targeting relevant TFs can be performed using drug cocktails constituted by extracts from medicinal plants inhibiting TF/DNA interactions. Finally, virtual screening might lead to identification of putative bioactive molecules to be validated using molecular and cellular approaches. By these means, low-molecular drugs targeting NF-κB and inhibiting IL-8 gene expression are available for pre-clinical testing using experimental systems recapitulating chronic pulmonary inflammation of patients affected by CF.
The design, synthesis and in vitro activities of novel alpha-bromoacryloyl pyrazole, imidazole an... more The design, synthesis and in vitro activities of novel alpha-bromoacryloyl pyrazole, imidazole and benzoheterocyclic derivatives of distamycin A, in which the amidino moiety has been replaced by moieties of different physico-chemical features are described, and the structure-activity relationships are discussed. In spite of the relevance of these modifications on the distamycin frame, these derivatives showed significant growth inhibitory activity against mouse leukemia L1210 cells. Therefore, the presence of the amidino moiety, and in general of a basic moiety, is not an absolute requirement for biological activity of alpha-bromoacrylic derivatives of distamycin.
Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics, 2003
Double-stranded molecules based on peptide nucleic acids (PNAs)-DNA chimeras carrying binding sit... more Double-stranded molecules based on peptide nucleic acids (PNAs)-DNA chimeras carrying binding sites for known transcription factors could be of great interest in decoy pharmacotherapy of neoplastic diseases. For instance, decoy molecules recognizing Sp1 and NF-kappaB transcription factors were found to inhibit tumor cell growth and invasion activity. In this respect, we have recently found that double-stranded PNA-DNA chimeras carrying NF-kappaB binding sites inhibit the binding of NF-kappaB p52 and p50 transcription factors to target DNA molecules. In this article we determined the resistance of double-stranded decoy molecules based on PNA-DNA chimeras to exonucleases (both 3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;--&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; and 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;--&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; exonucleases), endonucleases, and 5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-phosphatases. In addition, we performed experiments aimed at determining the resistance of these molecules in cellular extracts and serum. Finally, we used liposomes as protective agents in experimental conditions in which the decoy molecules employed were found to be unstable (high concentrations of enzymes, cellular extracts, or serum). The results obtained demonstrated that decoy molecules based on PNA-DNA chimeras are more resistant than DNA-based decoys to exo- and endonucleases, serum, and cellular extracts. In addition, the resistance of DNA/PNA hybrids in the presence of high concentrations of serum and cellular extracts was increased after complexation to cationic liposomes, due to the fact that double-stranded PNA-DNA-PNA chimeras bind to these delivery systems. The results obtained in the present study support the proposal of molecules based on PNA-DNA chimeras for an efficient decoy treatment of tumor cells both in vitro and in vivo.
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Papers by Nicoletta Bianchi