1,721,041 research outputs found
Dexamethasone counteracts the anti-osteoclastic, but not the anti-leukemic, activity of TNF-related apoptosis inducing ligand (TRAIL)
No full textWe have analyzed the effect of the synthetic glucocorticoid dexamethasone, used alone or in combination with recombinant TRAIL, on in vitro osteoclastic differentiation of peripheral blood-derived macrophages cultured in the presence of macrophage-colony stimulating factor (M-CSF) + RANKL for 12-14 days. Dexamethasone exhibited different effects based on the concentration used. Indeed, while at 10(-7) M dexamethasone reduced the number of mature osteoclasts, at 10(-8) M showed no significant effects and at 10(-9) M significantly increased the number of mature osteoclasts, with respect to cells cultured with only M-CSF + RANKL. On the other hand, the addition in culture of recombinant TRAIL inhibited the output of mature osteoclasts induced by M-CSF + RANKL. However, the presence of dexamethasone (10(-8) or 10(-9) M) into the culture medium significantly counteracted the anti-osteoclastic activity of TRAIL. In order to ascertain whether dexamethasone, might also interfere with the anti-leukemic activity of TRAIL, the degree of apoptosis induced by TRAIL was evaluated in several myeloid (OCI, MOLM, HL-60) and lymphoid (SKW6.4, MAVER, BJAB) leukemic cell lines. The levels of TRAIL-triggered apoptosis were not significantly different between leukemic cells cultured in the absence or presence of dexamethasone. Concerning the molecular mechanism mediating the dexamethasone-suppression of the TRAIL activity in pre-osteoclasts, but not in leukemic cells, we found that dexamethasone induced a significant down-regulation of the surface levels of TRAIL-R2 in cells of the osteoclastic lineage but not in leukemic cells. The ability of dexamethasone to counteract the TRAIL pathway envisions a novel mechanism mediating the pro-osteoclastic activity of dexamethasone in vivo
HIV-1 Tat protein suppresses the nerve growth factor (NGF)-mediated differentiation of PC12 rat pheochromocytoma cell line
No full textIn order to evaluate the effect of the regulatory human immunodeficiency virus-type 1 (HIV-1) Tat protein on the process of neuronal differentiation, two tat-transfected and mock-transfected PC12 cell lines were cultured in the absence or presence of 100-1000 ng/ml of nerve growth factor (NGF). As expected, NGF was able to induce a clearcut morphological differentiation of mock-transfected PC12 into sympathetic-like neurons, also reducing the percentage of cells in S phase. On the other hand, NGF was unable to reduce the percentage of PC12-tat cells in S phase and/or to induce their neuronal differentiation. Only the addition in culture of 5 mu g/ml neutralizing anti-Tat antibody plus 1000 ng/ml NGF was effective in decreasing the percentage of PC12-tat in S phase and inducing partial signs of neuronal differentiation in serum-free cultures. The ability of Tat protein to suppress the neuronal differentiation pathway controlled by NGF further contribute to the definition of its role in tumor promotion during the course of HTV-1 disease
Perifosine plus nutlin-3 combination shows a synergistic anti-leukaemic activity.
No full textPerifosine, which belongs to the novel class of antitumoral agents alkylphospholipids, is currently undergoing phase II clinical evaluation (van Blitterswijk & Verheij, 2008). Although the mechanisms by which perifosine exerts its antineoplastic activity need to be fully elucidated, it has been shown that perifosine inactivates the phosphatidylinositol 3-kinase/Akt pathway (van Blitterswijk & Verheij, 2008). While previous studies have demonstrated that perifosine shows cytotoxic activity against different types of haematological malignancies, when used either alone or in combination with chemotherapeutic drugs (van Blitterswijk & Verheij, 2008), its anti-leukaemic potential has not been investigated in combination with innovative compounds. Therefore, the effect of perifosine (Cayman Chemical, Ann Arbor, MI, USA) was analyzed in a panel of p53wild-type (SKW6.4, OCI, MOLM), p53mutated (BJAB, MAVER) and p53null (HL-60) cell lines, of both B lymphoid (SKW6.4, BJAB and MAVER) and myeloid (OCI, MOLM, HL-60) origin, all purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) or obtained from DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany). Perifosine was used alone or in combination with nutlin-3 (Cayman Chemical), a potent and selective small molecule inhibitor of the p53-MDM2 interaction, which induces a strong non genotoxic activation of the p53 pathway (Vassilev, 2007). In preliminary experiments, perifosine alone dose-dependently induced cytotoxicity, evaluated as previously described (Borgatti et al, 1997), in all leukaemic cell lines. The simultaneous addition of high concentrations of perifosine plus nutlin-3 (10 μmol/l each) induced a significant (P < 0·05) increase of cytotoxicity with respect to each agonist used alone in p53wild-type SKW6.4, OCI and MOLM cell lines, but not in p53mutated BJAB and MAVER and p53null cell lines, with the exception of MAVER cell line at 24 h. In order to evaluate whether the combined effect of perifosine plus nutlin-3 was synergistic, cultures were treated with serial concentrations of perifosine and nutlin-3 at a constant perifosine:nutlin-3 ratio, for 24 and 48 h and data were analyzed by the method of Chou and Talalay (1984). Synergy of perifosine plus nutlin-3 combination treatment (average combination index values <1) was clearly observed in all p53wild-type cell lines. Similarly, perifosine plus nutlin-3 (10 μmol/l each) induced enhanced cytotoxicity with respect to the treatments with the single agents also in some cases of p53wild-type primary acute myeloid leukaemia (AML, M1 and M5 type) and B chronic lymphocytic leukaemia (B-CLL) samples, but not in p53mutated or p53null cell lines. In order to investigate the molecular basis for the synergistic activity exerted by the combination of perifosine plus nutlin-3 in p53wild-type leukaemic cell lines, we have performed Western blot analysis to examine changes in the protein levels of p53 and of its major transcriptional targets (p21, Bax, MDM2), as well as MDM4/MDMX, an homolog of MDM2 which plays a non-redundant role with respect to MDM2 in down-regulating the activity of p53 and is not a transcriptional target of p53 (Meulmeester & Jochemsen, 2008). Nutlin-3 alone induced a marked accumulation of p53 protein, which was not affected by the concomitant presence of perifosine. In parallel, nutlin-3 induced an early and robust induction of both p21 and Bax, which started at 6 h and lasted for the duration of the experiment (48 h). Of note, the accumulation of both p21 and Bax was significantly potentiated by the concomitant treatment with perifosine. On the contrary, the combination of perifosine plus nutlin-3 induced completely different effects on MDM2 and MDM4/MDMX. In fact, while nutlin-3 alone significantly up-regulated the levels of expression of MDM2, which represents a major transcriptional target of p53 (Vassilev, 2007), the simultaneous addition of perifosine significantly counteracted the nutlin-3-mediated induction of MDM2. At variance to MDM2, nutlin-3 not only did not induce MDM4/MDMX, but rather promoted its degradation, an effect that is considered an important part of the mechanism of action of nutlin-3-mediated cytotoxicity (Secchiero et al, 2008). Of interest, the combination of perifosine plus nutlin-3 resulted in a more rapid degradation of MDM4/MDMX with respect to cultures treated with nutlin-3 alone.
The sustained increase in Bax protein levels observed in response to the nutlin-3 plus perifosine combination with respect to nutlin-3 alone strongly suggests that Bax induction probably represents a major molecular mechanism mediating the combined cytotoxicity of nutlin-3 plus perifosine. However, other apparently more subtle effects of the nutlin-3 plus perifosine combination were also noticed. The induction of MDM2, which represents a major transcriptional target and a key negative regulator of p53, was significantly weaker in cultures treated with nutlin-3 plus perifosine with respect to cultures treated with nutlin-3 alone. As p53 protein showed a comparable accumulation in samples treated with nutlin-3 or with nutlin-3 plus perifosine, and the best characterized mechanism of action of MDM2 is to down-regulate the transcriptional activity of p53 (Vassilev, 2007), the weaker induction of MDM2 in cultures treated with perifosine plus nutlin-3 probably accounts for the increased levels of other p53 transcriptional targets, p21 and Bax in SKW6.4 cells treated with the drug combination. A likely explanation for the differential ability of perifosine plus nutlin-3 to super-induce p21 and Bax while counteracting the accumulation of MDM2 is that perifosine blocks the Akt pathway (van Blitterswijk & Verheij, 2008), which has an important role in stabilizing both MDM2 (Zhou et al, 2001) and MDM4/MDMX (Secchiero et al, 2008). In this respect, also the ability of perifosine to potentiate the nutlin-3-mediated down-regulation of MDM4/MDMX is particularly noteworthy since it demonstrated that the levels of MDM4/MDMX represent a major molecular determinant in the cytotoxic response to nutlin-3, at least in solid tumours (Secchiero et al, 2008). In conclusion, our present study suggests that the combined treatment of perifosine plus nutlin-3 might offer a novel therapeutic strategy for p53wild-type haematological malignancies, which represent >80% of all haematological malignancies at diagnosis (Mitani et al, 2007)
Molecular and imaging prodromal markers of dopamine neuron degeneration in animal models of Parkinson’s disease: pathophysiology and clinical perspectives
No full textThe target of the proposal is to identify pre-degenerative/premotor molecular and cellular alterations, that initiate and contribute to early structural synaptic and axonal dopaminergic damage in the Caudate-Putamen (CPu) of mice that model Parkinson’s disease (PD)-like pathology, in order to delineate new diagnostic and therapeutic strategies that can be translated to the clinic to healing dopaminergic neurodegeneration. To achieve this target, we will use classical neuropathological and functional investigations and advanced microscopy techniques to correlate molecular/structural imaging with data obtained from MRI and PET analysis. This strategy will help to clarify whether and how key events involved in PD pathogenesis, such as alpha-synuclein (AS) pathology, neuroinflammation and mitochondrial derangement, can trigger synaptic and axonal damage detectable by brain imaging to provide very early diagnostic markers of PD. The project will also evaluate the effects of novel neuroprotective compounds (some of them under patent), which may be used to counteract the neuronal demise underlying PD. The deliverables of this project own great potential of scientific and technological advancement in PD research, with significant social and economic impact
HIV-1 TAT PROTEIN SUPPRESSES THE NERVE GROWTH FACTOR (NGF)- MEDIATED DIFFERENTIATION OF PC12 RAT PHEOCHROMOCYTOMA CELL LINE
No full textIn order to evaluate the effect of the regulatory human immunodeficiency virus-type 1 (HIV-1) Tat protein on the process of neuronal differentiation, two tat-transfected and mock-transfected PC12 cell lines were cultured in the absence or presence of 100-1000 ng/ml of nerve growth factor (NGF). As expected, NGF was able to induce a clearcut morphological differentiation of mock-transfected PC12 into sympathetic-like neurons, also reducing the percentage of cells in S phase. On the other hand, NGF was unable to reduce the percentage of PC12-tat cells in S phase and/or to induce their neuronal differentiation. Only the addition in culture of 5 mu g/ml neutralizing anti-Tat antibody plus 1000 ng/ml NGF was effective in decreasing the percentage of PC12-tat in S phase and inducing partial signs of neuronal differentiation in serum-free cultures. The ability of Tat protein to suppress the neuronal differentiation pathway controlled by NGF further contribute to the definition of its role in tumor promotion during the course of HTV-1 disease
Perifosine selectively induces cell cycle block and modulates retinoblastoma and E2F1 protein levels in p53 mutated leukemic cell lines.
No full textThe effect of the single-chain alkylphospholipid perifosine was analyzed in p53(wild-type) (SKW6.4, OCI and MOLM), p53(mutated) (BJAB, MAVER) and p53(null) (HL-60) leukemic cell lines. Perifosine promoted cytotoxicity with a combination
of apoptosis induction in all cell lines and cell cycle block at the G(2)M checkpoint, which was selectively observed in p53(mutated) BJAB and MAVER cell lines. At the molecular level, perifosine induced hypophosphorylation of retinoblastoma protein and the degradation of E2F1 protein in p53(mutated) but not in p53(wild-type) cells. These data indicate that perifosine potentially
represents an innovative therapeutic approach for p53(mutated) hematological malignancies
Recombinant TRAIL induces miorelaxating activity in rat aortas, which is abrogated by the induction of diabetes mellitus
No full textTNF-related apoptosis inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) family of cytokines, which exist either as type II membrane or as a soluble protein. Although the best-characterized activity of TRAIL is represented by its anti-cancer activity, it has been recently shown that TRAIL serum levels are decreased in patients affected by atherosclerotic cardiovascular disease raising the possibility that circulating soluble TRAIL might also affect vascular biology. In this respect, it has been previously shown that TRAIL induces the in vitro release of nitric oxide by vascular endothelial cells. In order to explore the in vivo relevance of these in vitro findings, we have assessed the vascular reactivity in response to recombinant TRAIL in a more intact paradigm. For this purpose, rings (4mm long) from rat thoracic aorta were obtained by both control (3-month-old male non-diabetic Wistar rats receiving vehicle infusion) and rats in which diabetes mellitus was induced by injecting 40 mg/kg streptozotocin (SZT). After sacrifice of the animals, aortas were harvested, cleaned of connective tissue and vascular reactivity was analyzed in ex vivo immediately after harvest. During submaximal contraction with phenylephrine, incubation of aortic segments in the presence of increasing (up to 1000 ng/ml) concentrations of recombinant human TRAIL, resulted in significant (p<0.01) vaso-relaxation. The effect was dose-dependent and was completely abolished by pre incubation of the rat aortic rings with L-NAME, clearly indicating that the NOS pathway played a key role in mediating the myo-relaxating activity of TRAIL. In parallel, we have investigated whether the induction of diabetes mellitus might affect the relaxating activity of aortic rings in response to TRAIL. Diabetes mellitus was induced by destruction of pancreatic islet cells by treating rats with SZT (40 mg/kg). Non-fasting blood glucose concentrations of both SZT-diabetic rats (SZT, n = 10) and age-matched control non-diabetic rats treated with vehicle (n = 10) were measured at days 5 and 15, when animals were sacrificed with CO2. The loss of insulin secretion triggered stable hyperglycemia, as evaluated by blood glucose measurement: 260±46 and 295±55 in diabetic rats at days 5 and 15 after diabetic induction, respectively, versus 89±8 in control rats. Relaxation to TRAIL was completely abrogated in aortic rings obtained from SZT-induced diabetic rats. To the best of our knowledge, this is the first demonstration that soluble recombinant TRAIL promotes a dose-dependent myo-relaxation activity when added ex vivo to rat aortic rings, thus suggesting that it might play a physiological role in the control of vascular tone regulation. Importantly, such myorelaxating activity was completely abrogated in diabetic rats, as early as 15 days after diabetes mellitus induction by STZ injection. It should be emphasized that diabetic vascular dysfunction is a major clinical problem that predisposes patients to a variety of cardiovascular diseases. In fact, diabetic patients frequently suffer from macroscopic and microscopic vasculopathy and accelerated atherosclerosis. The loss of myo-relaxating activity is a key feature of endothelial dysfunction, which invariably precedes permanent vascular alterations. Taken together with previous data showing that TRAIL significantly counteracts the pro-adhesive activity of inflammatory cytokines on endothelial cells in vitro and displays anti-atherosclerotic activity when injected in vivo in ApoE−/−null mice, our present findings suggest that a therapeutic strategy aimed to restore the miorelaxating response to TRAIL may be suitable for improving the vascular function in diabetes mellitus
Levels of circulating TNF-related apoptosis-inducing ligand in celiac disease
No full textIt has previously been demonstrated that the circulating levels of TNF-related apoptosis-inducing ligand (TRAIL) are significantly lower in patients with type 1 diabetes (T1D) than in normal age- and gender-matched controls. Since celiac disease (CD) is often associated with T1D, a retrospective study was performed to analyze the sera of a cohort of pediatric subjects: i) patients with CD at onset (n=100); ii) patients with potential CD (n=45); iii) patients with CD associated with other auto-immune diseases (n=17); and iv) patients with eosinophilic esophagitis (n=15). Among the patients with CD, 49 were also analyzed after six months on a gluten-free diet, while data were also available for 13 patients after one year on a gluten-free diet. No significant differences were found in the circulating levels of TRAIL between the patients with CD and the patients with either eosinophilic esophagitis or potential CD. Patients with CD associated with other auto-immune diseases showed significantly lower levels of TRAIL when compared with patients with CD alone. The gluten-free diet did not significantly modify the levels of circulating TRAIL at 6 or 12 months. Thus, although T1D and CD share common immunological features, the circulating levels of TRAIL show a significant difference between the two pathologies, and do not appear to be modulated in CD
Ceramides as the molecular link between impaired lipid metabolism, saturated fatty acid intake and insulin resistance: are all saturated fatty acids to be blamed for ceramide-mediated lipotoxicity?
No full textType 2 diabetes mellitus (T2DM) is a metabolic disorder that has reached epidemic proportions worldwide, posing a huge treat on people’s health and quality of life. From a pathogenetic prospective, T2DM is driven by insulin resistance defined as a blunted response of tissues to insulin which leads to chronic hyperglycaemia. Mechanistically, lipotoxicity and particularly the intracellular accumulation of ceramides in the skeletal muscle and the liver, is a primary metabolic aberration underpinning insulin resistance. Indeed, intracellular ceramide accumulation can hamper insulin signal transduction pathway thereby promoting insulin resistance. This review will provide an updated overview of the metabolic defects underlaying ceramide buildup and the molecular mechanism by which ceramides imping upon insulin signalling. Additionally, the role of specific ceramide subspecies as potential biomarkers for T2DM and the role of both long- and medium-chain saturated fatty acids as a modulator of ceramide metabolism will be discussed
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