Evaluation of Antitumoral activity of bone targeted drugs/conventional chemotherapies and identification of biomarkers for the selection of patients with breast cancer for the bone targeted therapy in adjuvant setting

Bone metastases are responsible for different clinical complications defined as skeletal-related events (SREs) such as pathologic fractures, spinal cord compression, hypercalcaemia, bone marrow infiltration and severe bone pain requiring palliative radiotherapy. The general aim of these three years research period was to improve the management of patients with bone metastases through two different approaches of translational research. Firstly in vitro preclinical tests were conducted on breast cancer cells and on indirect co-colture of cancer cells and osteoclasts to evaluate bone targeted therapy singly and in combination with conventional chemotherapy. The study suggests that zoledronic acid has an antitumor activity in breast cancer cell lines. Its mechanism of action involves the decrease of RAS and RHO, as in osteoclasts. Repeated treatment enhances antitumor activity compared to non-repeated treatment. Furthermore the combination Zoledronic Acid + Cisplatin induced a high antitumoral activity in the two triple-negative lines MDA-MB-231 and BRC-230. The p21, pMAPK and m-TOR pathways were regulated by this combined treatment, particularly at lower Cisplatin doses. A co-colture system to test the activity of bone-targeted molecules on monocytes-breast conditioned by breast cancer cells was also developed. Another important criticism of the treatment of breast cancer patients, is the selection of patients who will benefit of bone targeted therapy in the adjuvant setting. A retrospective case-control study on breast cancer patients to find new predictive markers of bone metastases in the primary tumors was performed. Eight markers were evaluated and TFF1 and CXCR4 were found to discriminate between patients with relapse to bone respect to patients with no evidence of disease. In particular TFF1 was the most accurate marker reaching a sensitivity of 63% and a specificity of 79%. This marker could be a useful tool for clinicians to select patients who could benefit for bone targeted therapy in adjuvant setting.

CYP17A1 polymorphisms and clinical outcome of patients with metastatic castration-resistant prostate cancer treated with abiraterone

Background. Abiraterone acetate is a potent inhibitor of cytochrome P450 17 α-hydrolase (CYP17A1) that causes a reduction in the synthesis of testosterone in the adrenal glands, testes and tumor microenvironment. Blocking androgen production, abiraterone has been shown to prolong progression-free survival (PFS) and overall survival (OS) in patients with metastatic castration-resistant prostate cancer (CRPC) previously submitted to chemotherapy. The aim of our study was to verify the role of single nucleotide polymorphisms (SNPs) in predicting clinical outcome in CRPC patients treated with abiraterone after chemotherapy. Methods. We analyzed 48 CRPC consecutive patients treated with abiraterone after at least one chemotherapeutic regimen with docetaxel. DNA was extracted from peripheral blood and genotyped for four polymorphisms in the CYP17A1 gene (rs743572, rs10883783, rs17115100, rs284849). PFS and OS survival curves were used to identify statistical associations between haplotypes and clinical outcome. Results. Forty-eight Caucasian patients with metastatic CRPC treated with abiraterone were genotyped for polymorphisms in the CYP17A1 gene. All samples were evaluable for both sequencing and TaqMan Genotyping assay. The CRPC patients treated with abiraterone had a median PFS and OS of 7.6 months (95% CI: 4.3-10.5) and 17.6 months (95% CI: 10.5-19.0), respectively Statistical analyses highlighted a difference approaching statistical significance (log-rank test p = 0.0534) between rs10883783 and PFS. Other polymorphisms were not associated with a benefit from treatment with abiraterone. Conclusions. In our case series of 48 treated patients, rs10883783 only was identified as a possible predictive marker, results showing a trend toward statistical significance. Further analysis of this polymorphism is needed in larger series of patients to confirm our findings.

Involvement of the opioid system and BDNF in neuroplastic alterations occurring in neuropathic pain conditions

Chronic pain affects one in five adults, reducing quality of life and increasing risk of developing co-morbidities such as depression. Neuropathic pain results by lesions to the nervous system that alter its structure and function leading to spontaneous pain and amplified responses to noxious and innocuous stimuli. The Opioid System is probably the most important system involved in control of nociceptive transmission. Dynorphin and nociceptin systems have been suggested key mediators of some neuropathic pain aspects. An important role also for BDNF has been recently suggested since its involvement in the peripheral and central sensitization phenomena is known. We studied neuroplastic alterations occurring in chronic pain in mice subjected to the chronic constriction injury (CCI). We investigated gene expression alterations of both BDNF and Opioid System at spinal level at different intervals of time. A transient upregulation of pBDNF and pDYN was observed in spinal cord, while increasing upregulation of ppN/OFQ was found in the DRGs of injured mice. Development of neuropathic behavioral signs has been observed in ICR/CD-1 and BDNF+/+ mice, subjected to CCI. A different development of these signs was observed in BDNF+/-. We also studied gene expression changes of investigated systems in different brain areas fourteen days after surgery. We found pBDNF, pDYN, pKOP, ppN/OFQ and pNOP gene expression alterations in several areas of CCI mice. In the same brain regions we also determined bioactive nociceptin peptide levels, and elevated N/OFQ levels were observed in the amygdala area. Histone modifications studies have been performed in BDNF and DYN gene promoters of CCI animal spinal cord showing selected alterations in pDYN gene promoter. In addition, a preliminary characterization of the innovative NOP-EGFP mice was performed. Overall, our results could be useful to understand which and how neuropeptidergic systems are involved in neuroplastic mechanism occurring in neuropathic pain.

Study of novel natural compounds as potential drugs in the treatment of acute leukemias

Introduction: Among all cancer types leukemia represents the leading cause of cancer death in man younger than 40 years. Single-target drug therapy has generally been highly ineffective in treating complex diseases such as cancer. A growing interest has been directed toward multi-target drugs able to hit multiple targets. In this context, plant products, based on their intrinsic complexity, could represent an interesting and promising approach. Aim of the research followed during my PhD was to indentify and study novel natural compounds for the treatment of acute leukemias. Two potential multi-target drugs were identified in Hemidesmus indicus and piperlongumine. Methodology/Principal Findings: A variety of cellular assays and flow cytometry were performed on different cell lines. We demonstrated that Hemidesmus modulates many components of intracellular signaling pathways involved in cell viability and proliferation and alters gene and protein expression, eventually leading to tumor cell death, mediated by a loss of mitochondrial transmembrane potential, raise of [Ca2+]i, inhibition of Mcl-1, increasing Bax/Bcl-2 ratio, and ROS formation. Moreover, we proved that the decoction causes differentiation of HL-60 and regulates angiogenesis of HUVECs in hypoxia and normoxia, by the inhibition of new vessel formation and the processes of migration/invasion. Clinically relevant observations are that its cytotoxic activity was also recorded in primary cells from acute myeloid leukemia (AML) patients. Moreover, both Hemidesmus and piperlongumine showed a selective action toward leukemic stem cell (LSC). Conclusions: Our results indicate the molecular basis of the anti-leukemic effects of Hemidesmus indicus and indentify the mitochondrial pathways, [Ca2+]i, cytodifferentiation and angiogenesis inhibition as crucial actors in its anticancer activity. The ability to selectively hit LSC showed by Hemidesmus and piperlongumine enriched the knowledge of their anti-leukemic activity. On these bases, we conclude that Hemidesmus and piperlongumine can represent a valuable strategy in the anticancer pharmacology.

Hypertension, hypercholesterolemia, hyperaldosteronism: a genetic perspective for personalized therapy.

Essential, primary, or idiopathic hypertension is defined as high BP in which secondary causes such as renovascular disease, renal failure, pheochromocytoma, hyperaldosteronism, or other causes of secondary hypertension are not present. Essential hypertension accounts for 80-90% of all cases of hypertension; it is a heterogeneous disorder, with different patients having different causal factors that may lead to high BP. Life-style, diet, race, physical activity, smoke, cultural level, environmental factors, age, sex and genetic characteristics play a key role in the increasing risk. Conversely to the essential hypertension, secondary hypertension is often associated with the presence of other pathological conditions such as dyslipidaemia, hypercholesterolemia, diabetes mellitus, obesity and primary aldosteronism. Amongst them, primary aldosteronism represents one of the most common cause of secondary hypertension, with a prevalence of 5-15% depending on the severity of blood pressure. Besides high blood pressure values, a principal feature of primary aldosteronism is the hypersecretion of mineralcorticoid hormone, aldosterone, in a manner that is fairly autonomous of the renin-angiotensin system. Primary aldosteronism is a heterogeneous pathology that may be divided essentially in two groups, idiopathic and familial form. Despite all this knowledge, there are so many hypertensive cases that cannot be explained. These individuals apparently seem to be healthy, but they have a great risk to develop CVD. The lack of known risk factors makes difficult their classification in a scale of risk. Over the last three decades a good help has been given by the pharmacogenetics/pharmacogenomics, a new area of the traditional pharmacology that try to explain and find correlations between genetic variation, (rare variations, SNPs, mutations), and the risk to develop a particular disease.

Characterization of the Caspr2 and NLGN2 ligands: a proteomic and biochemical approach

Autism Spectrum Disorder (ASD) is a range of early-onset conditions classified as neurodevelopmental disorders, characterized by deficits in social interactions and communication, as well as by restricted interest and repetitive behaviors. Among the proteins associated with this spectrum of disease there are Caspr2, α-NRXN1, NLGN1-4. Caspr2 is involved in the clustering of K+ channels at the juxtaparanodes, where it is proposed to bind TAG-1. Recent works reported a synaptic localization of Caspr2, but little is know on its role in this compartment. NRXNs and their ligand NLGNs, instead, have a well-defined role in the formation and maintenance of synapses. Among the neuroligins, NLGN2 binds NRXNs with the lowest affinity, suggesting that it could have other not yet characterized ligands. The aim of this work was to better characterize the binding of Caspr2 to TAG-1 and to identify new potential binding partner for Caspr2 and NLGN2. Unexpectedly, using Isothermal Titration Calorimetry and co-immunoprecipitation experiments the direct association of the first two proteins could not be verified and the results indicate that the first evidences reporting it were biased by false-positive artifacts. These findings, together with the uncharacterized synaptic localization of Caspr2, made the identification of new potential binding partners for this protein necessary. To find new proteins that associate with Caspr2 and NLGN2, affinity chromatography in tandem with mass spectrometry experiments were performed. Interestingly, about 25 new potential partners were found for these two proteins and NLGN1, that was originally included as a control: 5 of those, namely SFRP1, CLU, APOE, CNTN1 and TNR, were selected for further investigations. Only the association of CLU to NLGN2 was confirmed. In the future, screenings of the remaining candidates have to be carried out and the functional role for the proposed NLGN2-CLU complex has to be studied.

Drugs down-regulating E2F-1 expression hinders cell proliferation through a p53-indipendent mechanism

E2F-1 is a transcription factor that plays a key role in cell-cycle control at G1/S check-point level by regulating the timely expression of many target genes whose products are required for S phase entry and progression. In mammalian cells, E2F-1 is negatively regulated by hypo-phosphorylated Retinoblastoma protein (pRb) whereas it is protected against degradation by its binding to Mouse Double Minute 2 protein (MDM2). In this study we experimented a drug combination in order to obtain a strong down-regulation of E2F-1 by acting on two different mechanisms of E2F-1 regulation mentioned above. This was achieved by combining drugs inhibiting the phosphorylation of pRb with drugs inactivating the MDM2 binding capability. The mechanism of action of these drugs in down-regulating E2F-1 level and activity is p53 independent. As expected, when combined, these drugs strongly inhibits E2F-1 and hinder cell proliferation in p53-/- and p53-mutated cells by blocking them in G1 phase of cell cycle, suggesting that E2F-1 down-regulation may represent a valid chemotherapeutic approach to inhibit proliferation in tumors independently of p53 status.

Discovery and Pharmacological Characterization of a Novel Small Molecule Antagonist of Integrin α4β1: Focus on Antiallergic Activity

Allergy is a common hypersensitivity disorder that affects 15% to 20% of the population and its prevalence is increasing worldwide. Its severity correlates with the degree of eosinophil infiltration into the conjunctiva, which is mediated by chemokines that stimulate the production of adhesion molecules like intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on the endothelial cell surface. The α4β1 and α4β7 integrins are expressed in eosinophils and contribute to their activation and infiltration in AC through the binding to VCAM-1 or fibronectin, expressed on vascular endothelial cells. Blockade of α4 integrins might be a therapeutical achievement in allergic eye diseases. DS 70, that show an IC50 in the nanomolar range against α4β1 integrin in Jurkat cells and in the eosinophilic cell line EOL-1. This compound was able to prevent cell adhesion to VCAM-1 and FN in vitro. In a scintillation proximity assay DS70 displaced 125I-FN binding to human α4β1 integrin and, in flow cytometry analysis, it antagonized the binding of a primary antibody to α4β1 integrin expressed on the Jurkat cells surface as well. Furthermore, we analysed also its effects on integrin α4β1 signalling. In an vivo model of allergic conjunctivitis, topical DS70 reduced the clinical aspects of EPR (early phase reaction) and LPR (late phase reaction), by reducing clinical score, eosinophil accumulation, mRNA levels of cytochines and chemochines pro-inflammatory and the conjunctival expression of α4 integrin. In conclusion, DS70 seems a novel antiallergic ocular agent that has significant effects on both early and late phases of ocular allergy.

L'uso dei farmaci antibatterici negli ospedali dell'Emilia-Romagna

Obiettivo Analisi di consumi e costi degli antibiotici sistemici negli ospedali dell’Emilia-Romagna dal 2004 al 2011, con attenzione alla variabilità interaziendale e al significato, in termini di resistenza batterica, dell’aumento di alcuni gruppi terapeutici; Sottoanalisi nei reparti pediatrici, individuando i gruppi terapeutici critici, e valutazione delle reazioni avverse pediatriche da antibiotici segnalate, per il periodo in esame. Metodi I dati di consumo e spesa degli antibiotici sistemici per il periodo 2004-2011 sono stati ottenuti dal database regionale AFO e le giornate di degenza per ogni reparto dal database regionale di dimissione ospedaliera SDO. Le segnalazioni di sospette reazioni avverse da antibiotici tra gennaio 2004 e dicembre 2011 sono state estratte dal database nazionale VigiSegn. Risultati Negli otto anni, il consumo di antibiotici negli ospedali dell’Emilia-Romagna è aumentato del 27% e la spesa del 3%. Il consumo è apparso nettamente superiore nei reparti chirurgici che medici. La prima classe per consumo e spesa sono le penicilline/inibitori delle beta lattamasi. Nei reparti pediatrici, sono stati utilizzati 65 principi attivi diversi e amoxicillina/acido clavulanico è stato il più usato (26% del totale del 2011). Tra gli antibiotici critici, le cefalosporine di terza generazione sono state le più consumate in tutti i reparti pediatrici nel 2011. Tra le molecole il cui uso ospedaliero è vincolato, spiccano il linezolid e la teicoplanina che, comunque, hanno inciso più di tutte nella spesa del 2011 (18% e 15%, rispettivamente). Per la farmacovigilanza, i bambini (3-13 anni) sono stati coinvolti in 23 casi, mentre gli infanti (≤2 anni) solo in 4. L’associazione amoxicillina/acido clavulanico è stata più frequentemente segnalata (n=7), e soltanto 2 casi erano gravi. Conclusioni I risultati mostrano un quadro critico sul massiccio uso delle cefalosporine di terza generazione e sull’incremento del linezolid, da approfondire se per inappropriatezza d’uso oppure per aumento delle resistenze batteriche.

Role of Non-coding RNAs in chemotherapeutic treatments

The transcribed ultraconserved regions (T-UCRs) are a group of long non-coding RNAs involved in human carcinogenesis. The factors regulating the expression of T-UCRs and their mechanism of action in human cancers are unknown. In this work it was shown that high expression of uc.339 associates with lower survival in 204 non-small cell lung cancer (NSCLC) patients. Moreover, it was shown that uc.339 found up-regulated in archival NSCLC samples, acts as a decoy RNA for miR-339-3p, -663-3p and -95-5p. So, Cyclin E2, a direct target of three microRNAs is up-regulated, inducing cancer growth and migration. Evidence of this mechanism was provided from cell lines and primary samples confirming that TP53 directly regulates uc.339. These results support a key role for uc.339 in lung cancer.

Neurodegenerative diseases: molecular mechanisms and new strategies for neuroprotection

The term neurodegeneration defines numerous conditions that modify neuron’s normal functions in the human brain where is possible to observe a progressive and consistent neuronal loss. The mechanisms involved in neurodegenerative chronic and acute diseases evolution are not completely understood yet, however they share common characteristics such as misfolded proteins, oxidative stress, inflammation, excitotoxicity, and neuronal loss. Many studies have shown the frequency to develop neurodegenerative chronic diseases several years after an acute brain injury. In addition, many patients show, after a traumatic brain injury, motor and cognitive manifestations that are close to which are observed in neurodegenerative chronic patients. For this reason it is evident how is fundamental the concept of neuroprotection as a way to modulate the neurodegenerative processes evolution. Neuroinflammation, oxidative stress and the apoptotic process may be functional targets where operate to this end. Taking into account these considerations, the aim of the present study is to identify potential common pathogenetic pathways in neurodegenerative diseases using an integrated approach of preclinical studies. The goal is to delineate therapeutic strategies for the prevention of neuroinflammation, neurodegeneration and dysfunctions associated to Parkinson’s disease (PD) and cerebral ischemia. In the present study we used a murine model of PD treated with an isothiocyanate, 6-MSITC, able to quench ROS formation, restore the antioxidant GSH system, slow down the apoptotic neuronal death and counteract motor dysfunction induced by 6-OHDA. In the second study we utilized a transgenic mouse model knockout for CD36 receptor to investigate the inflammation involvement in a long term study of MCAo, which shows a better outcome after the damage induced. In conclusion, results in this study allow underlying the connection among these pathologies, and the importance of a neuroprotective strategy able to restore neurons activity where current drugs therapies have shown palliative but not healing abilities.