Novel SMAC-mimetics synergistically stimulate melanoma cell death in combination with TRAIL and Bortezomib.

BACKGROUND: XIAP (X-linked inhibitor of apoptosis protein) is an anti-apoptotic protein exerting its activity by binding and suppressing caspases. As XIAP is overexpressed in several tumours, in which it apparently contributes to chemoresistance, and because its activity in vivo is antagonised by second mitochondria-derived activator of caspase (SMAC)/direct inhibitor of apoptosis-binding protein with low pI, small molecules mimicking SMAC (so called SMAC-mimetics) can potentially overcome tumour resistance by promoting apoptosis. METHODS: Three homodimeric compounds were synthesised tethering a monomeric SMAC-mimetic with different linkers and their affinity binding for the baculoviral inhibitor repeats domains of XIAP measured by fluorescent polarisation assay. The apoptotic activity of these molecules, alone or in combination with tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and/or Bortezomib, was tested in melanoma cell lines by MTT viability assays and western blot analysis of activated caspases. RESULTS: We show that in melanoma cell lines, which are typically resistant to chemotherapeutic agents, XIAP knock-down sensitises cells to TRAIL treatment in vitro, also favouring the accumulation of cleaved caspase-8. We also describe a new series of 4-substituted azabicyclo[5.3.0]alkane monomeric and dimeric SMAC-mimetics that target various members of the IAP family and powerfully synergise at submicromolar concentrations with TRAIL in inducing cell death. Finally, we show that the simultaneous administration of newly developed SMAC-mimetics with Bortezomib potently triggers apoptosis in a melanoma cell line resistant to the combined effect of SMAC-mimetics and TRAIL. CONCLUSION: Hence, the newly developed SMAC-mimetics effectively synergise with TRAIL and Bortezomib in inducing cell death. These findings warrant further preclinical studies in vivo to verify the anticancer effectiveness of the combination of these agents.

Identification of a novel HLA-A*02 allele, A*027401*.

A novel HLA-A*02 allele was detected in a Caucasian patient from central Italy, requiring a hematopoietic cell transplantation. Direct sequencing identified a variation in one nucleotide position, which was confirmed by cloning. The name A*027401 was officially assigned by the WHO Nomenclature Committee in November 2004. A*027401 differs from A*02010101 by a single G to A substitution at nucleotide position 595 in exon 3. The new variant would lead to a nonsynonymous nucleotide change (GGG to AGG) at codon 175, resulting in a basic Arg in the alpha-helix of the alpha2-domain, in place of a non-polar Gly. The presence of an uncommon variation at a highly conserved nucleotide position could have implications in unrelated hematopoietic cell transplantation.

Efficacy and tolerance of acetate free biofiltration: a central Italian multicenter trial.

One hundred and thirteen patients from 51 dialysis units have been treated for 12 months on AFB using a high flux AN69 dialyzer (Filtral 12 or 16-Hospal), a buffer free dialysate (Na+ 139, K+ 2, Ca++ 2 mmol/liter, glucose 1 g/liter) and a reinfusate solution containing 145 mmol/liter HCO3Na. All of them had previously been stabilized on acetate dialysis (30 patients) or bicarbonate dialysis (46 patients) or different dialysis procedures (37 patients). AFB sessions were performed for 3.71 +/- 0.28 hrs, three times a week, at an average blood flow of 308 +/- 8.5 ml/min so as to ensure a stable Kt/V value > 1 (1.16 +/- 0.08). Blood samples were drawn monthly pre- and post-dialysis, for blood electrolytes, calcium, phosphate, chloride, urea, creatinine and uric acid, proteins and lipid profiles, hemochrome. Body weights, blood pressures and adverse side effects were recorded at each session. Using a mean infusion volume of 7.96 +/- 0.61 liter/session, post-dialytic bicarbonate values consistently > 25 mmol/liter (26.33 +/- 2.71) were achieved. Intradialytic occurrence of symptoms was very low at an average rate < 1.5 episodes patient/month. After 12 months of Tx an adequate control of uremia, of electrolytes, of Ca-PO4 balance and of BP was obtained. AFB gets a very high intradialytic tolerance and an excellent depurative capacity which contributes to the well being of the patients.