Novel Insights into the Biological Effects of the Isoprenoid Derivative N6-Isopentenyladenosine: Involvement of the Metabolic Sensor Ampk in Angiogenesis Inhibition

2012 - 2013N6-isopentenyladenosine (iPA) is a modified adenosine characterized by an isopentenyl chain derived by dimethylallyl pyrophosphate (DMAPP), an intermediate of the metabolic pathway of mevalonate, that is known to be deregulated in cancer. iPA is an endogenous isoprenoid-derived product present in mammalian cells as a free nucleoside in the cytoplasm, or in a tRNA-bound form, displaying well established pleiotropic biological effects, including a direct anti-tumor activity against several cancers. However, the precise mechanism of action of iPA in inhibiting cancer cell proliferation remains to be clarified. In this work, we investigated whether iPA could directly interfere with the angiogenic process, fundamental to cancer growth and progression, and if the growth and proliferation of human melanoma cells, known for their highly angiogenic phenotype, could be affected by the treatment with iPA. Finally, we investigated if iPA could have an immunomodulatory role targeting directly human natural killer (NK) cells, components of innate immunity that participate in immunity against neoplastic cells, in order to provide a cooperative and multifactorial mode of action of iPA to arrest cancer growth. To evaluate the potential involvement of iPA in angiogenesis, we employed human umbilical vein endothelial cells (HUVECs) as a suitable in vitro model of angiogenesis, by evaluating the viability, proliferation, migration, invasion, tube formation, and molecular mechanisms involved. Data were corroborated in mice by using a gel plug assay. iPA dose- and time-dependently inhibited all the neoangiogenesis stages, with an IC50 of 0.98 μM. We demonstrated for the first time that iPA was monophosphorylated into iPA 5'-monophosphate (iPAMP) by adenosine kinase (ADK) inside the cells. iPAMP is the active form that inhibits angiogenesis through the direct activation of AMP-kinase (AMPK). Indeed, all effects were completely reversed by pre-treatment with 5-iodotubercidin (5-Itu), an ADK inhibitor. The isoprenoid intermediate isopentenyl pyrophosphate (IPP), which shares the isopentenyl moiety with iPA, was ineffective in the inhibition of angiogenesis, thus showing that the iPA structure is specific for the observed effects. Thus, iPA is a novel AMPK activator and could represent a useful tool for the treatment of diseases where excessive neoangiogenesis is the underlying pathology... [edited by author]XII n.s

Role of intracellular and extracellular annexin A1 in migration and invasion of human pancreatic carcinoma cells

Background: Annexin A1 (ANXA1), a 37 kDa multifunctional protein, is over-expressed in tissues from patients of pancreatic carcinoma (PC) where the protein seems to be associated with malignant transformation and poor prognosis. Methods: The expression and localization of ANXA1 in MIA PaCa-2, PANC-1, BxPC-3 and CAPAN-2 cells were detected by Western Blotting and Immunofluorescence assay. Expression and activation of Formyl Peptide Receptors (FPRs) were shown through flow cytometry/PCR and FURA assay, respectively. To investigate the role of ANXA1 in PC cell migration and invasion, we performed in vitro wound-healing and matrigel invasion assays. Results: In all the analyzed PC cell lines, a huge expression and a variable localization of ANXA1 in sub-cellular compartments were observed. We confirmed the less aggressive phenotype of BxPC-3 and CAPAN-2 compared with PANC-1 and MIA PaCa-2 cells, through the evaluation of Epithelial-Mesenchymal Transition (EMT) markers. Then, we tested MIA PaCa-2 and PANC-1 cell migration and invasiveness rate which was inhibited by specific ANXA1 siRNAs. Both the cell lines expressed FPR-1 and -2. Ac2-26, an ANXA1 mimetic peptide, induced intracellular calcium release, consistent with FPR activation, and significantly increased cell migration/invasion rate. Interestingly, in MIA PaCa-2 cells we found a cleaved form of ANXA1 (33 kDa) that localizes at cellular membranes and is secreted outside the cells, as confirmed by MS analysis. The importance of the secreted form of ANXA1 in cellular motility was confirmed by the administration of ANXA1 blocking antibody that inhibited migration and invasion rate in MIA PaCa-2 but not in PANC-1 cells that lack the 33 kDa ANXA1 form and show a lower degree of invasiveness. Finally, the treatment of PANC-1 cells with MIA PaCa-2 supernatants significantly increased the migration rate of these cells. Conclusion: This study provides new insights on the role of ANXA1 protein in PC progression. Our findings suggest that ANXA1 protein could regulate metastasis by favouring cell migration/invasion intracellularly, as cytoskeleton remodelling factor, and extracellularly like FPR ligand

Serum Metabolic Profile in Multiple Sclerosis Patients

Multiple sclerosis (MS) is a progressive demyelinating process considered as an autoimmune disease, although the causes of this pathology have not been yet fully established. Similarly to other neurodegenerations, MS is characterized by a series of biochemical changes affecting to different extent neuronal functions; great attention has been given to oxidative/nitrosative stress and to alterations in mitochondrial functions. According to previous data, MS patients show significant changes in the circulating concentrations of different metabolites, although it is still unclear whether uric acid undergoes to decrease, increase, or no change under this pathological condition. In this study, we report the serum metabolic profile in terms of purines, pyrimidines, creatinine, malondialdehyde, ascorbic acid, nitrite, and nitrate in a group of 170 MS patients. The results show increase in circulating uric acid and other oxypurines (hypoxanthine and xanthine), as well as in uridine and β-pseudouridine. The concomitant increase in circulating creatinine, malondialdehyde, nitrite, and nitrate, and decrease in ascorbic acid, demonstrates that MS induces alteration in energy metabolism and in oxidants/antioxidants balance that can be monitored in serum of MS patients

Novel potent liposome agonists of triggering receptor expressed on myeloid cells 2 phenocopy antibody treatment in cells

The receptor Triggering Receptor Expressed on Myeloid cells 2 (TREM2) is associated with several neurodegenerative diseases including Alzheimer's Disease and TREM2 stimulation represents a novel therapeutic opportunity. TREM2 can be activated by antibodies targeting the stalk region, most likely through receptor dimerization. Endogenous ligands of TREM2 are suggested to be negatively charged apoptotic bodies, mimicked by phosphatidylserine incorporated in liposomes and other polyanionic molecules likely binding to TREM2 IgV fold. However, there has been much discrepancy in the literature on the nature of phospholipids (PLs) that can activate TREM2 and on the stability of the corresponding liposomes over time. We describe optimized liposomes as robust agonists selective for TREM2 over TREM1 in cellular system. The detailed structure/activity relationship studies of lipid polar heads indicate that negatively charged lipid heads are required for activity and we identified the shortest maximally active PL sidechain. Optimized liposomes are active on both TREM2 common variant and TREM2 R47H mutant. Activity and selectivity were further confirmed in different native TREM2 expressing cell types including on integrated cellular responses such as stimulation of phagocytic activity. Such tool agonists will be useful in further studies of TREM2 biology in cellular systems alongside antibodies, and in the design of small molecule synthetic TREM2 agonists

Multicenter Observational Retrospective Study on Febrile Events in Patients with Acute Myeloid Leukemia Treated with Cpx-351 in "Real-Life": The SEIFEM Experience

: In the present study, we aimed to evaluate the absolute risk of infection in the real-life setting of AML patients treated with CPX-351. The study included all patients with AML from 30 Italian hematology centers of the SEIFEM group who received CPX-351 from July 2018 to June 2021. There were 200 patients included. Overall, 336 CPX-351 courses were counted: all 200 patients received the first induction cycle, 18 patients (5%) received a second CPX-351 induction, while 86 patients (26%) proceeded with the first CPX-351 consolidation cycle, and 32 patients (10%) received a second CPX-351 consolidation. A total of 249 febrile events were recorded: 193 during the first or second induction, and 56 after the first or second consolidation. After the diagnostic work-up, 92 events (37%) were classified as febrile neutropenia of unknown origin (FUO), 118 (47%) were classifiable as microbiologically documented infections, and 39 (17%) were classifiable as clinically documented infections. The overall 30-day mortality rate was 14% (28/200). The attributable mortality-infection rate was 6% (15/249). A lack of response to the CPX-351 treatment was the only factor significantly associated with mortality in the multivariate analysis [p-value: 0.004, OR 0.05, 95% CI 0.01-0.39]. Our study confirms the good safety profile of CPX-351 in a real-life setting, with an incidence of infectious complications comparable to that of the pivotal studies; despite prolonged neutropenia, the incidence of fungal infections was low, as was infection-related mortality

The characteristics and activities of child and adolescent mental health services in Italy: a regional survey

<p>Abstract</p> <p>Background</p> <p>To date, no studies have assessed in detail the characteristics, organisation, and functioning of Child and Adolescent Mental Health Services (CAMHS). This information gap represents a major limitation for researchers and clinicians because most mental disorders have their onset in childhood or adolescence, and effective interventions can therefore represent a major factor in avoiding chronicity. Interventions and mental health care are delivered by and through services, and not by individual, private clinicians, and drawbacks or limitations of services generally translate in inappropriateness and ineffectiveness of treatments and interventions: therefore information about services is essential to improve the quality of care and ultimately the course and outcome of mental disorders in childhood and adolescence.</p> <p>The present paper reports the results of the first study aimed at providing detailed, updated and comprehensive data on CAMHS of a densely populated Italian region (over 4 million inhabitants) with a target population of 633,725 subjects aged 0-17 years.</p> <p>Methods</p> <p>Unit Chiefs of all the CAMHS filled in a structured 'Facility Form', with activity data referring to 2008 (data for inpatient facilities referred to 2009), which were then analysed in detail.</p> <p>Results</p> <p>Eleven CAMHS were operative, including 110 outpatient units, with a ratio of approximately 20 child psychiatrists and 23 psychologists per 100,000 inhabitants aged 0-17 years. All outpatient units were well equipped and organized and all granted free service access. In 2008, approximately 6% of the target population was in contact with outpatient CAMHS, showing substantial homogeneity across the eleven areas thereby. Most patients in contact in 2008 received a language disorder- or learning disability diagnosis (41%). First-ever contacts accounted for 30% of annual visits across all units. Hospital bed availability was 5 per 100,000 inhabitants aged 0-17 years.</p> <p>Conclusion</p> <p>The percentage of young people in contact with CAMHS for mental disorders is in line with those observed in previous epidemiological studies. The overall number of child psychiatrists per 100,000 inhabitants is one of the highest in Europe and it is comparable with the most well equipped areas in the US. This comparison should be interpreted with caution, however, because in Italy, child psychiatrists also treat neurological disorders. Critical areas requiring improvement are: the uneven utilisation of standardised assessment procedures and the limited availability of dedicated emergency services during non-office hours (e.g., nights and holidays).</p

Impairment of circulating endothelial progenitors in Down syndrome

<p>Abstract</p> <p>Background</p> <p>Pathological angiogenesis represents a critical issue in the progression of many diseases. Down syndrome is postulated to be a systemic anti-angiogenesis disease model, possibly due to increased expression of anti-angiogenic regulators on chromosome 21. The aim of our study was to elucidate some features of circulating endothelial progenitor cells in the context of this syndrome.</p> <p>Methods</p> <p>Circulating endothelial progenitors of Down syndrome affected individuals were isolated, <it>in vitro </it>cultured and analyzed by confocal and transmission electron microscopy. ELISA was performed to measure SDF-1α plasma levels in Down syndrome and euploid individuals. Moreover, qRT-PCR was used to quantify expression levels of <it>CXCL12 </it>gene and of its receptor in progenitor cells. The functional impairment of Down progenitors was evaluated through their susceptibility to hydroperoxide-induced oxidative stress with BODIPY assay and the major vulnerability to the infection with human pathogens. The differential expression of crucial genes in Down progenitor cells was evaluated by microarray analysis.</p> <p>Results</p> <p>We detected a marked decrease of progenitors' number in young Down individuals compared to euploid, cell size increase and some major detrimental morphological changes. Moreover, Down syndrome patients also exhibited decreased SDF-1α plasma levels and their progenitors had a reduced expression of SDF-1α encoding gene and of its membrane receptor. We further demonstrated that their progenitor cells are more susceptible to hydroperoxide-induced oxidative stress and infection with Bartonella henselae. Further, we observed that most of the differentially expressed genes belong to angiogenesis, immune response and inflammation pathways, and that infected progenitors with trisomy 21 have a more pronounced perturbation of immune response genes than infected euploid cells.</p> <p>Conclusions</p> <p>Our data provide evidences for a reduced number and altered morphology of endothelial progenitor cells in Down syndrome, also showing the higher susceptibility to oxidative stress and to pathogen infection compared to euploid cells, thereby confirming the angiogenesis and immune response deficit observed in Down syndrome individuals.</p