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Genova, Italy

Farra R.,University of Trieste | Dapas B.,University of Trieste | Pozzato G.,University of Trieste | Giansante C.,University of Trieste | And 6 more authors.

For hepatocellular carcinoma (HCC), a leading cause of cancer death world-wide, there is no effective therapy especially for the advanced stage of the disease. Thus, we started the investigations about a novel anti HCC approach based on the depletion of the transcription factor serum response factor (SRF) in HCC cell lines; SRF choice was based on its recently proposed contribution to HCC tissue development and on its important role in cell proliferation.SRF depletion, obtained by a siRNA (siSRF797), was studied in two HCC cell lines, i.e. HepG2 and JHH6 assigned to high and low hepatocytic differentiation grade on the base of the capacity to synthesize albumin.In the HCC cell lines examined, siSRF797 reduced both the mRNA and protein levels of SRF without inducing unspecific interferon response or cytotoxicity. Moreover, SRF depletion induced the reduction of S-phase cells and a decrease in cell number and vitality. Particularly in HepG2, cell growth impairment was paralleled by the decrease of the levels of the transcription factor E2F1 together with some of its regulated genes. In HepG2 but not in JHH6, SRF depletion was associated with apoptosis. Finally, in both HepG2 and JHH6, the combined administration of siSRF797 and bortezomib, a proteasome inhibitor whose therapeutic potential for HCC is considered attractive, further reduced cell viability compared to either siSRF797 or bortezomib treatment alone.In conclusion, SRF depletion affects the expansion of the high and low differentiation grade HCC cells HepG2 and JHH6. These results can pave the way to understand the role of SRF in HCC development and possibly to identify novel anti HCC therapeutic strategies. © 2010 Elsevier Masson SAS. Source

Farra R.,University of Trieste | Dapas B.,University of Trieste | Pozzato G.,University of Trieste | Scaggiante B.,University of Trieste | And 7 more authors.
Digestive and Liver Disease

Background: No effective therapy is available for hepatocellular carcinoma. To identify novel therapeutic strategies, we explored the effects of the depletion of E2F1, cyclin E1-E2 whose inter-relationships in hepatocellular carcinoma cell proliferation have never been defined. Methods: siRNA-mediated depletion of the targets was studied in the hepatocellular carcinoma cells HepG2, HuH7 and JHH6 characterized by high, medium and low hepatocyte differentiation grade, respectively; a model of normal human hepatocytes was also considered. Results: The depletion of each target mRNA reduced the levels of the other two mRNAs, thus demonstrating a close regulatory control, also confirmed by over-expression experiments. At the protein level, an exception to this trend was observed for cyclinE1 whose amount increased upon cyclin E2 (HepG2, HuH7, JHH6) and E2F1 (HepG2) depletion. In HepG2, reduced cyclinE1 proteolysis accounted for this observation. Additionally, cyclin E1-E2-E2F1 targeting decreased the levels of cyclin A2 mRNA and of the hyper-phosphorylated form of pRb thus leading to an S-phase cell decrease; migration was impaired as well. Finally, the model of human hepatocytes used was clearly less affected by target mRNAs depletion than hepatocellular carcinoma cells. Conclusion: Our data provide novel mutual relationships amongst cyclin E1-E2-E2F1 and indicate their role in sustaining hepatocellular carcinoma cell proliferation/migration, validating the concept of an anti-cyclin E1-E2-E2F1 therapeutic approach for hepatocellular carcinoma. © 2011 Editrice Gastroenterologica Italiana S.r.l. Source

Santucci L.,Laboratorio Of Fisiopatologia Delluremia | Candiano G.,Laboratorio Of Fisiopatologia Delluremia | Petretto A.,Mass Spectrometry Core Facility | Pavone B.,IRCCS Fondazione Santa Lucia | And 8 more authors.
Molecular BioSystems

Apolipoprotein A1 (apoA1) is a component of the high density lipoproteins (HDL) that regulates the transport of cholesterol between the liver and peripheral cells and modulates the removal of any excess of cholesterol from membranes. Any variation in apoA1 composition may modify the plasma lipid profile and be involved in atherogenesis. We investigated apoA1 composition in plasma of 6 children with nephrotic syndrome, a condition characterized by high levels of cholesterol in plasma and by a high risk to develop atherosclerosis. Non-denaturing two-dimensional electrophoresis (Nat/SDS-PAGE), mass spectrometry, western blot and pull down experiments were done to characterize proteins and define putative interactions. ApoA1 was resolved in 12 variants, 6 of which had a slightly lower molecular weight (18-19 KDa) and migrated on the same axes of the β chain of haptoglobin (Hp). Low molecular weight apoA1 were observed in carriers of different Hp haplotypes (including one homozygous for the rare ββα1α1) ruling out any contaminant effect of co-migration of apoA1 with Hp α2 chain. Overall, apoA1 isoforms were much more present in plasma of nephrotic patients compared to a normal profile. These findings show that apoA1 plasma in nephrotic syndrome is heterogeneous in terms of molecular weight. Low molecular weight fragments lack internal structural domains and likely form macro-aggregates with Hp. Fragmentation and transport of apoA1 may be involved in the general disorder of lipid metabolism that characterizes nephrotic syndrome. © The Royal Society of Chemistry 2011. Source

Candiano G.,Laboratory on Pathophysiology of Uremia | Santucci L.,Renal Child Foundation | Petretto A.,Mass Spectrometry Core Facility and Laboratory of Molecular Medicine | Bruschi M.,Renal Child Foundation | And 5 more authors.
Journal of Proteomics

The discovery of urinary biomarkers is a main topic in clinical medicine. The development of proteomics has rapidly changed the knowledge on urine protein composition and probably will modify it again. Two-dimensional electrophoresis (2D-PAGE) coupled with mass spectrometry has represented for years the technique of choice for the analysis of urine proteins and it is time to draw some conclusions. This review will focus on major methodological aspects related to urine sample collection, storage and analysis by 2D-PAGE and attempt to define an advanced normal urine protein map. Overall, 1118 spots were reproducibly found in normal urine samples but only 275 were characterized as isoforms of 82 proteins. One-hundred height spots belonging to 30 proteins were also detected in plasma and corresponded to typical plasma components. The identity of most of the proteins found in normal urine by 2D-PAGE remains to be determined, the majority being low-molecular weight proteins (< 30 kDa). Equalization procedures would also enhance sensitivity of the analysis and allow low abundance proteins to be characterized. Therefore, we are still on the way to define the normal urine composition. Technology advancements in concentrating procedure will improve sensitivity and give the possibility to purify proteins for mass spectrometry. © 2009 Elsevier B.V. All rights reserved. Source

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