http://elea.unisa.it/xmlui/handle/10556/1452 2026-04-20T11:30:18Z 2026-04-20T11:30:18Z Picardi, Paola http://elea.unisa.it/xmlui/handle/10556/1477 2025-04-30T14:05:11Z 2014-03-19T00:00:00Z

Novel Insights into the Biological Effects of the Isoprenoid Derivative N6-Isopentenyladenosine: Involvement of the Metabolic Sensor Ampk in Angiogenesis Inhibition Picardi, Paola N6-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] 2012 - 2013

2014-03-19T00:00:00Z Passaretti, Federica http://elea.unisa.it/xmlui/handle/10556/1468 2025-04-30T14:13:42Z 2014-03-21T00:00:00Z

Molecular pathways involved in metabolic control of CCL5 in adipocytes Passaretti, Federica Obesity is a chronic disorder characterized by a tonic low-grade activation of the innate immune system that affects steady-state measures of metabolic homeostasis over time. In addition, obesity is often accompanied by elevations in tissue and circulating FFA concentrations. Systemic levels of FFAs can induce inflammatory cascades in adipocytes and macrophages through TLR4-dependent effect. Signaling through TLR4 activates a broad range of intracellular cascades that include stimulation of IKK-β, NF-kB, JNK and AP1. Indeed, in addition to store excess calories in the form of lipid, adipose tissue produces classical cytokines and chemokines such as MCP-1, IL-8 and CCL5. CCL5, as other chemokines, participates in mediating leukocyte infiltration of adipose tissue. Moreover circulating CCL5 concentrations are elevated in obesity, impaired glucose tolerance (IGT) and type 2 diabetes. In this study I have investigated the molecular mechanisms involved in the metabolic control of CCL5 expression in adipocytes. Cytokine/growth factor screening of conditioned media from 3T3-L1 preadipocytes and adipocytes revealed that adipocytes secreted higher amount of CCL5 compared to their undifferentiated precursors. Higher concentrations of glucose and fatty acids (oleate and palmitate) increased CCL5 secretion by 3T3-L1 adipocytes. Moreover, both oleate and palmitate enhanced CCL5 mRNA levels and induced an activation of JNK, NF-kB, MAPK and PI3K/AKT pathways. In cells treated with JSH23, a NF-kB inhibitor, the effect of FFAs on CCL5 mRNA levels was reduced thus indicating a direct involvement of NF-kB. Treatment of the cells with SP600125, a JNK inhibitor, also significantly reduced the stimulatory effect of oleate and palmitate on CCL5 mRNA and interestingly prevented FFA-induced NF-kB binding to CCL5 promoter. I have also obtained evidence that insulin exerted an inhibitory effect on CCL5 mRNA and counteracted fatty acid-induced stimulation. Both PD98059 and LY294002, inhibitors of MAPK and PI3K, respectively, increased CCL5 expression levels reverted anti-inflammatory effect of insulin in presence of fatty acids. Consistently, insulin exposure reduced NF-kB recruitment onto CCL5 promoter, and almost completely prevented fatty acid effect. In conclusion, oleate and palmitate induce CCL5 mRNA, possibly via JNK and NF-kB pathways. Fatty acid effect on CCL5 is largely prevented by insulin and may involve PI3K/AKT and MAPK. [edited by Author] 2012 - 2013

2014-03-21T00:00:00Z D'Alessandro, Alba http://elea.unisa.it/xmlui/handle/10556/1461 2025-04-30T14:18:52Z 2014-03-14T00:00:00Z

p53 regulates Mevalonate Metabolic pathway D'Alessandro, Alba The mevalonate pathway is an important metabolic pathway implicated in multiple aspects of tumorigenesis. In this study, I provided evidences about genetic and pharmacologic perturbation of p53, which directly influenced expression of mevalonate pathway enzymes, including 3’-Hydroxy-3’-Methylglutaryl - CoenzymeA Reductase, Mevalonate Kinase, Farnesyl Diphosphate Synthase, Farnesyl Diphosphate Farnesyl Transferase 1. Three different cell lines have been considered, U343 MG (U343) and U251 MG (U251) glioma cells, both classified as IV grade glioblastoma cell lines, with two different malignancy grade, and Normal Human Astrocytes (NHA),their normal counterpart. In particular, NHA and U343 cells have wild type p53 (wtp53) while U251bearing mutation (R273H)p53. This mutation affects p53 DNA binding site, preventing transcriptional function of the protein. Different basal expression level of the mevalonate pathway’s genes have found among the different cell lines considered and I hypothesized that this could be ascribable to p53 mutation status and function. Indeed, I observed that functional and active p53 recognized specific p53 Responsive Elements (p53REs) present in MVA enzymes gene-sequences. p53 bound to these regions correlated with increased transcription levels of mentioned genes and such effect has abolished in cells bearing mut(R273H)p53 or by site-directed mutagenesis of p53REs. These new findings expose another facet of p53 functions, unrelated to tumor suppression, and render it a novel regulator of mevalonate pathway providing insight into the role of this pathway in cancer progression. [edited by author] 2012-2013

2014-03-14T00:00:00Z Liotti, Antonietta http://elea.unisa.it/xmlui/handle/10556/1453 2025-04-30T14:19:22Z 2014-03-21T00:00:00Z

The transcription factor Prep1 regulates adipose tissue functions Liotti, Antonietta Adipose tissue is crucial for maintaining energy and metabolic homeostasis and its functionality is closely related to the adipocytes differentiation state. Adipogenesis is regulated by several transcription factors. Prep1 is an homeodomain transcription factor belonging to the TALE proteins, which plays an important role in hematopoiesis, organogenesis and development. Previous studies have indicated that Prep1 hypomorphic heterozygous (Prep1i/+) mice, which express only 55-57% of protein, have a complex metabolic phenotype. In fact, these mice present smaller but otherwise normally structured islets with reduced fasting and post-loading plasma insulin levels and increased insulin sensitivity in skeletal muscle and in liver which is accompanied by protection from streptozotocin-induced diabetes. In addition, Prep1 deficiency in mice induces a reduction of hepatic triglycerides synthesis and a protection from methionine and choline-deficient diet-induced steatohepatitis. In this study, I focused my attention on the role of Prep1 on the regulation of adipocyte differentiation and on the adipose tissue functionality. To understand the possible role of Prep1 in adipose tissue, I first evaluated the features of adipose tissue of Prep1i/+ and WT mice. Prep1i/+mice show a reduction of adipose tissue weight, a reduction of the area but no change in the number of adipocytes. In addition, expression of adipogenic markers, C/EBPα, GLUT4 and FABP4, is increased in adipose tissue of Prep1i/+ mice, while PPARγ does not change. Consistent with these data, upon insulin stimulation, insulin receptor (IR), AKT and MAPK phosphorylation is increased in adipose tissue of Prep1 hypomorphic heterozygous mice. In addition, basal and insulin- stimulated glucose-uptake is increased in adipocytes isolated from adipose tissue of Prep1i/+ mice compared to the adipocytes from WT mice. The increased basal uptake is fully consistent with the higher expression of GLUT4 on the plasma membrane of adipocytes of Prep1i/+ mice compared with that of control animals. To further study the function of Prep1 on adipocyte differentiation, I have analyzed Prep1 expression during different steps of adipogenesis in 3T3L1 murine cells. Levels of Prep1 are progressively reduced during the conversion from 3T3L1 preadipocytes to adipocytes. Moreover, 3T3L1 adipocytes stably transfected with Prep1 cDNA display reduced lipid accumulation, and expression of C/EBPα, GLUT4 and FABP4. Interestingly, insulin molecular signaling pathway is less activated in presence of Prep1. All together these data suggest that Prep1 regulates adipocyte differentiation, giving a rationale to investigate Prep1 as possible new therapeutic agents in preventing adipose tissue dysfunctions. [edited by author] 2012 - 2013

2014-03-21T00:00:00Z