Marianna Lucafò

New potential therapeutic approach for the treatment of B-Cell malignancies using chlorambucil/hydroxychloroquine-loaded anti-CD20 nanoparticles.

Current B-cell disorder treatments take advantage of dose-intensive chemotherapy regimens and immunotherapy via use of monoclonal antibodies. Unfortunately, they may lead to insufficient tumor distribution of therapeutic agents, and often cause adverse effects on patients. In this contribution, we propose a novel therapeutic approach in which relatively high doses of Hydroxychloroquine and Chlorambucil were loaded into biodegradable nanoparticles coated with an anti-CD20 antibody. We demonstrate their ability to effectively target and internalize in tumor B-cells. Moreover, these nanoparticles were able to kill not only p53 mutated/deleted lymphoma cell lines expressing a low amount of CD20, but also circulating primary cells purified from chronic lymphocitic leukemia patients. Their safety was demonstrated in healthy mice, and their therapeutic effects in a new model of Burkitts lymphoma. The latter serves as a prototype of an aggressive lympho-proliferative disease. In vitro and in vivo data showed the ability of anti-CD20 nanoparticles loaded with Hydroxychloroquine and Chlorambucil to increase tumor cell killing in comparison to free cytotoxic agents or Rituximab. These results shed light on the potential of anti-CD20 nanoparticles carrying Hydroxychloroquine and Chlorambucil for controlling a disseminated model of aggressive lymphoma, and lend credence to the idea of adopting this therapeutic approach for the treatment of B-cell disorders.

MicroRNAs as tools to predict glucocorticoid response in inflammatory bowel diseases

In spite of the introduction in therapy of highly effective biological agents, glucocorticoids (GCs) are still employed to induce remission in moderate to severe inflammatory bowel diseases (IBD), but considerable inter-individual differences in their efficacy and side effects have been reported. The effectiveness of these drugs is indeed very variable and side effects, particularly severe in pediatric patients, are common and often unpredictable: the understanding of the complex gene regulation mediated by GCs could shed light on the causes of this variability. In this context, microRNAs (miRNAs) represent a new and promising field of research. miRNAs are small non-coding RNA molecules that suppress gene expression at post-transcriptional level, and are fine-tuning regulators of diverse biological processes, including the development and function of the immune system, apoptosis, metabolism and inflammation. Emerging data have implicated the deregulated expression of certain miRNA networks in the pathogenesis of autoimmune and inflammatory diseases, such as IBD. There is a great interest in the identification of the role of miRNAs in the modulation of pharmacological response; however, the association between miRNA and GC response in patients with IBD has not yet been evaluated in a prospective clinical study. The identification of miRNAs differently expressed as a consequence of GC treatment in comparison to diagnosis, represents an important innovative approach that could be translated into clinical practice. In this review we highlight the altered regulation of proteins involved in GC molecular mechanism by miRNAs, and their potential role as molecular markers useful for predicting in advance GC response.

Profiling the molecular mechanism of fullerene cytotoxicity on tumor cells by RNA-seq.

The interest on functionalized fullerenes in the field of nanomedicine has seen a significant increase in the past decade. However, the different methods employed to increase C60 solubility profoundly influence the physicochemical properties and the toxicological effects of these compounds, thus complicating the evaluation of their toxicity and potential therapeutic use. Here we report a whole-transcriptome RNA-seq analysis assessing the effect of two fullerenes (1 and 2) on gene expression in the human MCF7 cell line. Although these two compounds had previously been characterized by in vitro studies as having a cytotoxic and null effect respectively, to date the mechanisms at the basis of this different behavior and, more in general, at the basis of the effect of most fullerene derivatives in living cells are still completely unknown. Our data evidence that: (a) fullerene 2 caused a significant, time-dependent alteration of gene expression, whereas 1 only had a negligible effect; (b) the biological processes mostly influenced over the 48h experimental time course were transcription, protein synthesis, cell cycle progression and cell adhesion; (c) the gene expression signature of 2-treated cells was strikingly similar to those induced by selective inhibitors of mTOR signaling, thus suggesting an effect on this pathway for fullerene 2. Our work represents the first approach toward the application of RNA-seq to the study of the molecular mechanisms underlying the interaction of fullerenes with cellular systems and provides an objective view of the feasibility and the safety of these nanomaterials for a medical application.

A Cationic [60] Fullerene Derivative Reduces Invasion and Migration of HT-29 CRC Cells in Vitro at Dose Free of Significant Effects on Cell Survival

Nanomaterials with unique characteristics exhibit favorable therapeutic and diagnostic properties, implying their enormous potential as biomedical candidates. C60 has been used in gene- and drug-delivery, as imaging agents, and as photosensitizers in cancer therapy. In this study, the influences of a cationic functionalized fullerene on cellular behavior of human colorectal cancer cell line (HT-29) were investigated. Results indicated that HT-29 treated with the studied compound showed a lower sensitivity but a significant impairment in migration and invasion by interfering with the activities of matrix metalloproteinases (MMP-2 and 9). The presence of fullerene also altered the capacity of adhesion-related proteins to perform their activity, thereby inducing dramatically adverse effects on the cell physiological functions such as cell adhesion. Thus, our study suggests that this compound is a new potential anti-metastatic effector and a therapeutic component for malignant colorectal cancer.

Identification and Characterization of a Novel Family of Cysteine-Rich Peptides (MgCRP-I) from Mytilus galloprovincialis

We report the identification of a novel gene family (named MgCRP-I) encoding short secreted cysteine-rich peptides in the Mediterranean mussel Mytilus galloprovincialis. These peptides display a highly conserved pre-pro region and a hypervariable mature peptide comprising six invariant cysteine residues arranged in three intramolecular disulfide bridges. Although their cysteine pattern is similar to cysteines-rich neurotoxic peptides of distantly related protostomes such as cone snails and arachnids, the different organization of the disulfide bridges observed in synthetic peptides and phylogenetic analyses revealed MgCRP-I as a novel protein family. Genome- and transcriptome-wide searches for orthologous sequences in other bivalve species indicated the unique presence of this gene family in Mytilus spp. Like many antimicrobial peptides and neurotoxins, MgCRP-I peptides are produced as pre-propeptides, usually have a net positive charge and likely derive from similar evolutionary mechanisms, that is, gene duplication and positive selection within the mature peptide region; however, synthetic MgCRP-I peptides did not display significant toxicity in cultured mammalian cells, insecticidal, antimicrobial, or antifungal activities. The functional role of MgCRP-I peptides in mussel physiology still remains puzzling.

RNA-seq analysis of the whole transcriptome of MDA-MB-231 mammary carcinoma cells exposed to the antimetastatic drug NAMI-A

Gene expression profiling has been introduced into drug development to understand the activity of chemical entities in pre-clinical settings. The present study investigates the changes induced in gene expression by the ruthenium-based compound NAMI-A. The genes differentially expressed by NAMI-A are evaluated through whole-transcriptome analysis and RNA-sequencing in the metastatic MDA-MB-231 mammary carcinoma cells, in comparison to the non-tumorigenic HBL-100 mammary gland cells. NAMI-A treatment rapidly induces a relevant gene up-regulation that quickly returns to normal values. These changes differ between MDA-MB-231 and HBL-100 cells, highlighting the selectivity of the NAMI-A induced transcriptional perturbation in the invasive rather than in the non-tumorigenic cells. The transcriptional response, in the invasive MDA-MB-231 cells, comprises a set of early-response transcription factors and reveals a pharmacological signature in good agreement with the most peculiar NAMI-A behavior as a metastasis inhibitor such as cell cycle regulation and ECM remodeling. Globally, the results of this study indicate some transcription factors influencing the expression and activity of many downstream genes and proteins fundamentally involved in the functional effects of NAMI-A in vitro and in vivo.

Glucocorticoid pharmacogenetics in pediatric idiopathic nephrotic syndrome

Idiopathic nephrotic syndrome represents the most common type of primary glomerular disease in children: glucocorticoids (GCs) are the first-line therapy, even if considerable interindividual differences in their efficacy and side effects have been reported. Immunosuppressive and anti-inflammatory effects of these drugs are mainly due to the GC-mediated transcription regulation of pro- and anti-inflammatory genes. This mechanism of action is the result of a complex multistep pathway that involves the glucocorticoid receptor and several other proteins, encoded by polymorphic genes. Aim of this review is to highlight the current knowledge on genetic variants that could affect GC response, particularly focusing on children with idiopathic nephrotic syndrome.

Differential expression of GAS5 in rapamycin-induced reversion of glucocorticoid resistance

This study evaluates the association between the long noncoding RNA GAS5 levels and the anti‐proliferative effect of the glucocorticoid (GC) methylprednisolone (MP) alone and in combination with rapamycin in peripheral blood mononuclear cells (PBMCs) obtained from healthy donors. The effect of MP, rapamycin, and MP plus rapamycin was determined in 17 healthy donors by labelling metabolically active cells with [methyl‐3H] thymidine and the expression levels of GAS5 gene were evaluated by real‐time RT‐PCR TaqMan analysis. We confirmed a role for GAS5 in modulating GC response: poor responders presented higher levels of GAS5 in comparison with good responders. Interestingly, when PBMCs were treated with the combination of rapamycin plus MP, the high levels of GAS5 observed for each drug in the MP poor responders group decreased in comparison with rapamycin (P value = 0.0134) or MP alone (P value = 0.0193). GAS5 is involved in GC resistance and co‐treatment of rapamycin with GCs restores GC effectiveness in poor responders through the downregulation of the long noncoding RNA. GAS5 could be considered a biomarker to personalize therapy and a novel therapeutic target useful for the development of new pharmacological approaches to restore GC sensitivity.

Role of the Long Non-Coding RNA Growth Arrest-Specific 5 in Glucocorticoid Response in Children with Inflammatory Bowel Disease

Glucocorticoids (GCs) are widely employed in inflammatory, autoimmune and neoplastic diseases, and, despite the introduction of novel therapies, remain the first‐line treatment for inducing remission in inflammatory bowel disease (IBD). Given the high incidence of suboptimal response, associated with a significant number of side‐effects, that are particularly severe in paediatric patients, the identification of subjects that are most likely to respond poorly to GCs is extremely important. Recent evidence suggests that the long non‐coding RNA (lncRNA) GAS5 could be a potential marker of GC resistance. To address this issue, we evaluated the association between the lncRNA GAS5 and the efficacy of steroids, in terms of inhibition of proliferation, in two cell lines derived from colon and ovarian cancers, to confirm the sensitivity and specificity of these lncRNAs. These cells showed a different sensitivity to GCs and revealed differential expression of GAS5 after treatment. GAS5 was up‐regulated in GC‐resistant cells and accumulated more in the cytoplasm compared to the nucleus in response to the drug. The functions of GAS5 were assessed by silencing, and we found that GAS5 knock‐down reduced the proliferation during GC treatment. Furthermore, for the first time, we measured GAS5 levels in 19 paediatric IBD patients at diagnosis and after the first cycle of GCs, and we demonstrated an up‐regulation of the lncRNA in patients with unfavourable steroid response. Our preliminary results indicate that GAS5 could be considered a novel pharmacogenomic marker useful for the personalization of GC therapy.

Effects of Two Fullerene Derivatives on Monocytes and Macrophages

Two fullerene derivatives (fullerenes 1 and 2), bearing a hydrophilic chain on the pyrrolidinic nitrogen, were developed with the aim to deliver anticancer agents to solid tumors. These two compounds showed a significantly different behaviour on human neoplastic cell lines in vitro in respect to healthy leukocytes. In particular, the pyrrolidinium ring on the fullerene carbon cage brings to a more active compound. In the present work, we describe the effects of these fullerenes on primary cultures of human monocytes and macrophages, two kinds of immune cells representing the first line of defence in the immune response to foreign materials. These compounds are not recognized by circulating monocytes while they get into macrophages. The evaluation of the pronecrotic or proapoptotic effects, analysed by means of analysis of the purinergic receptor P2X7 activation and of ROS scavenging activity, has allowed us to show that fullerene 2, but not its analogue fullerene 1, displays toxicity, even though at concentrations higher than those shown to be active on neoplastic cells.