Comportamento in soluzione di tiolati di oro(I) cationici tetranucleari con interazioni aurofiliche

L’argomento di questa tesi riguarda la sintesi, la caratterizzazione e lo studio della reattività in soluzione di alcuni tiolati di Au(I) neutri contenenti fosfine sia mono sia bidentate, di formula generale Au(SR)(PR’3) e Au2(SCMe3)2(PP) (R = CMe3 e R’ = Me, Et, tBu, Ph; R = nC8H17 e R’ = Me, Ph; PP = dppe, dppm; dppm = 1,1-bis(difenilfosfino)metano e dppe = 1,2-bis(difenilfosfino)etano), e complessi dicationici del tipo [Au4(SCMe3)2(PR’3)4][BF4]2 e [Au4(SCMe3)2(PP)2][BF4]2 (R’ = Me, Et, tBu, Ph ; PP = dppm, dppe). Tali composti ionici di oro(I), almeno allo stato solido, sono caratterizzati da una struttura tetranucleare che si regge sulla presenza di gruppi tiolato a ponte e di deboli interazioni “aurofiliche”. Nonostante complessi simili siano noti da alcuni anni, in letteratura non esistono dati sulla nuclearità in soluzione. L’utilizzo di tecniche spettroscopiche di ESI-MS e PGSE-NMR (DOSY) ha permesso lo studio dello stato di aggregazione in soluzione di questi complessi cationici: in particolare si è visto che, almeno parzialmente, le interazioni aurofiliche possono sopravvivere in dipendenza della natura della fosfina coordinata e della polarità del solvente. Uno studio di reattività di questi cluster tetranucleari di oro(I) ha permesso di comprendere quale sia l’influenza delle interazioni Au-Au: 1) Si è indagato tramite tecniche NMR e MS il comportamento in presenza di tiolati neutri di oro(I) con leganti fosfinici: è stata osservata l’instaurarsi di uno scambio più o meno rapido a seconda dell’ingombro della fosfina tra il tiolato neutro coordinato e quello libero. Si è anche visto che per particolari rapporti alcune di queste miscela danno vita a nuove specie polinucleari (Au3, Au4), caratterizzate dalla presenza di interazioni aurofiliche. 2) Si è studiato, quindi, il comportamento in presenza di fosfina libera (mono e bidentata) e si è osservato che l’aggiunta di questo legante causa la scissione delle interazioni aurofiliche e, anche, del ponte a tiolato. I prodotti che si ottengono variano a seconda del precursore di partenza e del tipo di fosfina aggiunta, però possono essere riassunti con le due formule generali [Au(PR3)2][BF4] e Au(SCMe3)(PR3)n

Role of the B-cell receptor in chronic lymphocytic leukemia: where do we stand?

The past 15 years have witnessed an enormous effort in studying B-cell Chronic Lymphocytic Leukemia. A great number of researches brought significant novel information and a better understanding of the natural history of this disease.This mini review will focus on the studies related to the Immunoglobulin variable (IgV) genes rearrangements that compose the B-cell receptor (BcR) of the leukemic clones. These stud­ ies have defined a role for the antigen(s) in the paths that lead to leukemic clone generation/expansion and underscore the informative value represented by BcR analyses

Relevance of fatty acid metabolism in proliferating CLL cells

Chronic lymphocytic leukemia (CLL) cells undergo, during their life, iterative cycles of re-activation and subsequent clonal expansion. We previously demonstrated that the antidiabetic drug metformin, known to also inhibit oxidative phosphorylation (OXPHOS), inhibits cell cycle entry of leukemic cells derived ex-vivo from the peripheral blood of CLL patients and stimulated in vitro by cell culture systems that recreate a microenvironment to drive their proliferation (Bruno et al, Oncotarget, 2015). However, overtly proliferating CLL cells were resistant to the cytostatic effects of metformin. Since metformin switched the energetic metabolism of activated, not yet proliferating, CLL cells from OXPHOS to accelerated glycolysis, in the present study we asked whether combining metformin with glycolysis impairment could inhibit also proliferating CLL cells. Still, CLL cells recovered from a transitory block and rescued in vitro proliferation. What kind of energetic reprogramming was involved in the resistance of proliferating CLL cells to glucose utilization? Recent studies highlight on the role of fatty acid utilization of CLL cells. We asked 1) whether inhibitors of lipid metabolism could impair proliferation of in vitro stimulated CLL cells; 2) whether impairing glucose energetic pathways could act synergistically with beta oxidation inhibitors.We found that inhibitors of critical steps of fatty acid metabolisms, such as carnitine-palmitoyl transferase 1A (CPT1A) -rate-limiting enzyme for fatty acid import into mitochondria- or Peroxisome Proliferator-Activated Receptor (PPAR)-alpha -regulator of beta-oxidation- administered at clinically achievable doses, were ineffective on quiescent CLL cells and on CLL cells stimulated by the microenvironment during the first stages of activation. Conversely, remarkable susceptibility to undergo apoptosis was observed at later stages of cell activation and during overt proliferation. Synergism with impairment of other energetic pathways occurred depending on the stage of activation of the in vitro stimulated CLL cells.The results suggest that energetic metabolic pathways could be relevant targets for CLL treatment, provided that the complex metabolic reprogramming network during the transition of leukemic cells from quiescence to proliferation, and back, are clearly elucidated. This work was supported by grants from AIRC IG15426

Multiple Distinct Sets of Stereotyped Antigen Receptors Indicate a Role for Antigen in Promoting Chronic Lymphocytic Leukemia

Previous studies suggest that the diversity of the expressed variable (V) region repertoire of the immunoglobulin (Ig)H chain of B-CLL cells is restricted. Although limited examples of marked constraint in the primary structure of the H and L chain V regions exist, the possibility that this level of restriction is a general principle in this disease has not been accepted. This report describes five sets of patients, mostly with unmutated or minimally mutated IgV genes, with strikingly similar B cell antigen receptors (BCRs) arising from the use of common H and L chain V region gene segments that share CDR3 structural features such as length, amino acid composition, and unique amino acid residues at recombination junctions. Thus, a much more striking degree of structural restriction of the entire BCR and a much higher frequency of receptor sharing exists among patients than appreciated previously. The data imply that either a significant fraction of B-CLL cells was selected by a limited set of antigenic epitopes at some point in their development and/or that they derive from a distinct B cell subpopulation with limited Ig V region diversity. These shared, stereotyped Ig molecules may be valuable probes for antigen identification and important targets for cross-reactive idiotypic therapy

SH3BGRL3 binds myosin 1c and is involved in MDA-MB-231 cell migration

SH3BGRL3 is a gene belonging to SH3BGR family, it is ubiquitously expressed and encodes for a 93 AA thiorerodoxin-like protein evolutionarily conserved. A proteomic study reported that SH3BGRL3 binds the cytoplasmatic domain of ERBB2 receptor. On this basis we performed immuno-staining experiments in FLAG-SH3BGRL3 transfected SKBR3 cell line that showed SH3BGRL3 and ERBB2 co-localization. Nonetheless, co-immunoprecipitation (Co-IP) of ERBB2 using FLAG-SH3BGRL3 as bait and vice versa was not achievable. Therefore, to investigate SH3BGRL3 potential interactors we performed Co-IP experiments from SKBR3 lysates transfected with FLAG-SH3BGRL3 followed by mass spectrometry analysis. The results revealed myosin 1c (Myo1c) as a candidate interactor. Subsequent Co-IP experiments followed by WB analysis validated the interaction between the two proteins. To map the interaction site we performed Co-IP experiments using SKBR3 cells co-transfected with FLAG-SH3BGRL3 and HA tagged deletion mutants of Myo1c that showed SH3BGRL3 binding to the neck region of Myo1c. Since Myo1c neck region binds calmodulin in a Ca2+ dependent way, we assessed if the binding was Ca2+ dependent also for SH3BGRL3. The experiments showed that SH3BGRL3 binds the Myo1c neck in presence of Ca2+, differently from calmodulin that binds it in absence of Ca2+. Myo1c is a motor protein involved, among its different functions, in cell membrane dynamics. Thus we investigated SH3BGRL3 involvement in cell migration using MDA-MB-231 cell line. We transfected MDA-MB-231 cells with FLAG-SH3BGRL3 and performed immuno-staining and Co-IP experiments that showed co-localization and interaction of Myo1c and SH3BGRL3. Accordingly, we performed migration assays using boyden chambers after silencing or not SH3BGRL3 expression by means siRNAs. The results showed a statistically significant decrease in migration capacity of silenced cells respect to controls. Our data show that SH3BGRL3 binds Myo1c neck region in a Ca2+ dependent way and that this interaction is involved in cell migration in our model

Unexpected effects of biphosphonates in in vitro models of activated CLL cells

Recent studies suggest that the commonly prescribed anti-osteoporosis drugs bisphosphonates (BPs) might also exhibit antitumor activity. We investigated a possible anticancer effect of BPs on B-chronic lymphocytic leukemia (CLL) cells obtained from peripheral blood of 26 CLL patients. Zoledronate, etidronate and clodronate were administered in vitro simultaneously to following activation stimuli: i) CD40L-expressing fibroblasts, ii) soluble recombinant CD40L produced in our laboratory +IL-4, iii) CpG ODN 2006+IL-15 with or without bone marrow stromal cells (BMSC). CLL cell viability, activation/proliferation were monitored by flow cytometry. We unexpectedly observed that BPs generated a protective effect from spontaneous apoptosis in 11/26 (42%) patients (viability + 18%-392%) and an augmentation in CLL cell activation/proliferation in 61% of the samples (S+G2M phase: +100%±25). Interestingly, protection from spontaneous apoptosis or increment of cell activation, required the presence of either fibroblasts, BMSC or autologous Nurse Like Cells (NLC). We thus hypothesized that supportive cells are involved in the BPs effects either through cell-cell interactions with leukemic cells or T cells, or through soluble factors release in the medium. Functional experiments with transwells suggest that stromal cells, in presence of Clodronate, release soluble factors in the medium that may probably concur to the unexpected Clodronate-mediated enhancement of CLL cell activation/proliferation. This work is in progress and several critical questions on the mechanisms are still unanswered. Nevertheless, the phenomenological data argue that caution should be taken when administering BPs against osteoporosis in elderly persons, who could have Monoclonal B Lymphocytosis or CLL

Photochemical dihydrogen production using an analogue of the active site of [NiFe] hydrogenase

The photoproduction of dihydrogen (H2) by a low molecular weight analogue of the active site of [NiFe] hydrogenase has been investigated by the reduction of the [NiFe2] cluster, 1, by a photosensitier PS (PS = [ReCl(CO)3(bpy)] or [Ru(bpy)3][PF6]2). Reductive quenching of the 3MLCT excited state of the photosensitiser by NEt3 or N(CH2CH2OH)3 (TEOA) generates PS•−, and subsequent intermolecular electron transfer to 1 produces the reduced anionic form of 1. Time-resolved infrared spectroscopy (TRIR) has been used to probe the intermediates throughout the reduction of 1 and subsequent photocatalytic H2 production from [HTEOA][BF4], which was monitored by gas chromatography. Two structural isomers of the reduced form of 1 (1a•− and 1b•−) were detected by Fourier transform infrared spectroscopy (FTIR) in both CH3CN and DMF (dimethylformamide), while only 1a•− was detected in CH2Cl2. Structures for these intermediates are proposed from the results of density functional theory calculations and FTIR spectroscopy. 1a•− is assigned to a similar structure to 1 with six terminal carbonyl ligands, while calculations suggest that in 1b•− two of the carbonyl groups bridge the Fe centres, consistent with the peak observed at 1714 cm−1 in the FTIR spectrum for 1b•− in CH3CN, assigned to a ν(CO) stretching vibration. The formation of 1a•− and 1b•− and the production of H2 was studied in CH3CN, DMF and CH2Cl2. Although the more catalytically active species (1a•− or 1b•−) could not be determined, photocatalysis was observed only in CH3CN and DMF