Neurofibromin Deficiency and Extracellular Matrix Cooperate to Increase Transforming Potential through FAK-Dependent Signaling.

Plexiform neurofibromas (Pnfs) are benign peripheral nerve sheath tumors that are major features of the human genetic syndrome, neurofibromatosis type 1 (NF1). Pnfs are derived from Schwann cells (SCs) undergoing loss of heterozygosity (LOH) at the NF1 locus in an NF1+/- milieu and thus are variably lacking in the key Ras-controlling protein, neurofibromin (Nfn). As these SCs are embedded in a dense desmoplastic milieu of stromal cells and abnormal extracellular matrix (ECM), cell-cell cooperativity (CCC) and the molecular microenvironment play essential roles in Pnf progression towards a malignant peripheral nerve sheath tumor (MPNST). The complexity of Pnf biology makes treatment challenging. The only approved drug, the MEK inhibitor Selumetinib, displays a variable and partial therapeutic response. Here, we explored ECM contributions to the growth of cells lacking Nfn. In a 3D in vitro culture, NF1 loss sensitizes cells to signals from a Pnf-mimicking ECM through focal adhesion kinase (FAK) hyperactivation. This hyperactivation correlated with phosphorylation of the downstream effectors, Src, ERK, and AKT, and with colony formation. Expression of the GAP-related domain of Nfn only partially decreased activation of this signaling pathway and only slowed down 3D colony growth of cells lacking Nfn. However, combinatorial treatment with both the FAK inhibitor Defactinib (VS-6063) and Selumetinib (AZD6244) fully suppressed colony growth. These observations pave the way for a new combined therapeutic strategy simultaneously interfering with both intracellular signals and the interplay between the various tumor cells and the ECM.

Unusual Domestic Source of Lead Poisoning.

Non-occupational lead poisoning is not rare, mainly occurring in domestic situations in children, but also in adults. Lead poisoning was observed in a 65 years-old woman non-exposed to risk that caught our attention with a diagnostic suspicion of acute intermittent porphyria according to recurrent episodes of abdominal pain and neuropathy of upper limbs. Acute intermittent porphyria was excluded by a laboratory investigation that showed instead severe lead poisoning. After several thorough examinations of the domestic environment, the source of intoxication has been detected in some cooking pots that released high concentrations of lead. Ethylenediamine tetracetic acid disodium calcium therapy (three cycles) reduced consistently blood lead concentration and, after one year, neuropathy was almost entirely recovered.

Biological Monitoring of Metal Ions Released from Hip Prostheses.

The aim of this study was to evaluate the levels of As, Be, Bi, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Se, Tl, V, and Zn, by inductively coupled plasma-mass spectrometry (ICP-MS) in the urine of two groups of patients with two different types of metal-on-metal (MoM) total hip prostheses (ASR DePuy®, group A, 25 patients; total Met-Met System Lima®, group B, 28 patients). The determination of metals reflected a steady-state release (group A: 9 years after surgery and group B: 6 years after surgery). The results obtained confirmed the increase of Co and Cr urinary levels in both group when compared with the reference values for the general population adopted by the Italian Society of Reference Values (SIVR). In particular, Co and Cr levels exceeded the threshold values in urine, respectively, of 30 µg and 21 µg, adjusted to creatinine based on the threshold in whole blood of 7 µg/L proposed by the Medicines and Healthcare Products Regulatory Agency (MHRA). Regarding the other investigated metals, significantly higher values were found in Group A than in Group B. These differences could be due to the type of hip prosthesis implanted, the longer period of time since the implantation, as well as many other factors such as diet, age, drug consumption, physical activity, or presence of dental fillings. The continuous monitoring over the years of metal concentrations in patients carrying a prosthesis could be useful to better identify the sources of these metals.

CuII and AuIII Complexes with Glycoconjugated Dithiocarbamato Ligands for Potential Applications in Targeted Chemotherapy.

This work is focused on the synthesis, characterization, and preliminary biological evaluation of bio-conjugated AuIII and CuII complexes with the aim of overcoming the well-known side effects of chemotherapy by improving the selective accumulation of an anticancer metal payload in malignant cells. For this purpose, carbohydrates were chosen as targeting agents, exploiting the Warburg effect that accounts for the overexpression of glucose-transporter proteins (in particular GLUTs) in the phospholipid bilayer of most neoplastic cells. We linked the dithiocarbamato moiety to the C1 position of three different monosaccharides: d-glucose, d-galactose, and d-mannose. Altogether, six complexes with a 1:2 metal-to-ligand stoichiometry were synthesized and in vitro tested as anticancer agents. One of them showed high cytotoxic activity toward the HCT116 colorectal human carcinoma cell line, paving the way to future in vivo studies aimed at evaluating the role of carbohydrates in the selective delivery of whole molecules into cancerous cells.

Absence of Neurofibromin Induces an Oncogenic Metabolic Switch via Mitochondrial ERK-Mediated Phosphorylation of the Chaperone TRAP1.

Mutations in neurofibromin, a Ras GTPase-activating protein, lead to the tumor predisposition syndrome neurofibromatosis type 1. Here, we report that cells lacking neurofibromin exhibit enhanced glycolysis and decreased respiration in a Ras/ERK-dependent way. In the mitochondrial matrix of neurofibromin-deficient cells, a fraction of active ERK1/2 associates with succinate dehydrogenase (SDH) and TRAP1, a chaperone that promotes the accumulation of the oncometabolite succinate by inhibiting SDH. ERK1/2 enhances both formation of this multimeric complex and SDH inhibition. ERK1/2 kinase activity is favored by the interaction with TRAP1, and TRAP1 is, in turn, phosphorylated in an ERK1/2-dependent way. TRAP1 silencing or mutagenesis at the serine residues targeted by ERK1/2 abrogates tumorigenicity, a phenotype that is reverted by addition of a cell-permeable succinate analog. Our findings reveal that Ras/ERK signaling controls the metabolic changes orchestrated by TRAP1 that have a key role in tumor growth and are a promising target for anti-neoplastic strategies.

Gold(III)-pyrrolidinedithiocarbamato Derivatives as Antineoplastic Agents.

Transition metals offer many possibilities in developing potent chemotherapeutic agents. They are endowed with a variety of oxidation states, allowing for the selection of their coordination numbers and geometries via the choice of proper ligands, leading to the tuning of their final biological properties. We report here on the synthesis, physico-chemical characterization, and solution behavior of two gold(III) pyrrolidinedithiocarbamates (PDT), namely [Au(III)Br2(PDT)] and [Au(III)Cl2(PDT)]. We found that the bromide derivative was more effective than the chloride one in inducing cell death for several cancer cell lines. [Au(III)Br2(PDT)] elicited oxidative stress with effects on the permeability transition pore, a mitochondrial channel whose opening leads to cell death. More efficient antineoplastic strategies are required for the widespread burden that is cancer. In line with this, our results indicate that [Au(III)Br2(PDT)] is a promising antineoplastic agent that targets cellular components with crucial functions for the survival of tumor cells.

Reliability of urinary excretion rate adjustment in measurements of hippuric acid in urine.

The urinary excretion rate is calculated based on short-term, defined time sample collections with a known sample mass, and this measurement can be used to remove the variability in urine concentrations due to urine dilution. Adjustment to the urinary excretion rate of hippuric acid was evaluated in 31 healthy volunteers (14 males and 17 females). Urine was collected as short-term or spot samples and tested for specific gravity, creatinine and hippuric acid. Hippuric acid values were unadjusted or adjusted to measurements of specific gravity, creatinine or urinary excretion rate. Hippuric acid levels were partially independent of urinary volume and urinary flow rate, in contrast to specific gravity and creatinine, which were both highly dependent on the hippuric acid level. Accordingly, hippuric acid was independent on urinary specific gravity and creatinine excretion. Unadjusted and adjusted values for specific gravity or creatinine were generally closely correlated, especially in spot samples. Values adjusted to the urinary excretion rate appeared well correlated to those unadjusted and adjusted to specific gravity or creatinine values. Thus, adjustment of crude hippuric acid values to the urinary excretion rate is a valid procedure but is difficult to apply in the field of occupational medicine and does not improve the information derived from values determined in spot urine samples, either unadjusted or adjusted to specific gravity and creatinine.

Soluble phenyl valerate esterases of hen sciatic nerve and the potentiation of organophosphate induced delayed polyneuropathy.

Contrary to some organophosphorus esters (OPs), certain esterase inhibitors including sulfonyl halides, carbamates and phosphinates do not cause axonal neuropathy, but they may exacerbate traumatic and some chemical insults to axons. This phenomenon is referred to as the promotion/potentiation of axonopathies. We report here promotion studies of the organophosphate induced delayed polyneuropathy (OPIDP). This neuropathy correlates with inhibition/aging of neuropathy target esterase, but this enzyme is not the target of promotion. Soluble phenyl valerate (PV) esterases in peak I (V(0)) of hen sciatic nerve were analysed. When these activities were inhibited in vitro by a mixture containing mipafox - an OP that causes OPIDP - paraoxon and p-toluene sulfonyl fluoride - two esterase inhibitors that do not cause either neuropathy or promotion-, then the remaining activity was sensitive to classical promoters such as phenylmethane sulfonyl fluoride (PMSF) and phenylmethyl benzyl carbamate. This PV-activity was not inhibited in sciatic nerves of hens treated with di-isopropyl phosphorofluoridate, at a dose that causes OPIDP. When these birds were further dosed with PMSF a dose-response relationship was observed between inhibition of PV-esterases, as above defined, and the severity of clinical responses. These data suggest that the target of promotion is embraced in peak I (V(0)) of soluble proteins of hen sciatic nerve.

The mitochondrial effects of small organic ligands of BCL-2: sensitization of BCL-2-overexpressing cells to apoptosis by a pyrimidine-2,4,6-trione derivative.

We have investigated the mitochondrial effects of BH3I-2', Chelerythrine, and HA14-1, small organic molecules that share the ability to bind the BH3 domain of BCL-2. All compounds displayed a biphasic effect on mitochondrial respiration with uncoupling at low concentrations and respiratory inhibition at higher concentrations, the relative uncoupling potency being BH3I-2' (half-maximal uncoupling at about 80 nm) > Chelerythrine (half-maximal uncoupling at about 2 microm) > HA14-1 (half-maximal uncoupling at about 20 microm). At concentrations lower than required for uncoupling all compounds sensitized the permeability transition pore (PTP) to opening both in isolated mitochondria and intact cells. To assess whether the effects on BCL-2 binding, PTP induction and respiration could be due to different structural determinants we have tested a set of HA14-1 analogs from the Hoffmann-La Roche chemical library. We have identified 5-(6-chloro-2,4-dioxo-1,3,4,10-tetrahydro-2H-9-oxa-1,3-diaza-anthracen-10-yl)-pyrimidine-2,4,6-trione (EM20-25) as a molecule devoid of effects on respiration that is able to induce PTP opening, to disrupt the BCL-2/BAX interactions in situ and to activate caspase-9 in BCL-2-overexpressing cells. EM20-25 neutralized the antiapoptotic activity of overexpressed BCL-2 toward staurosporine and sensitized BCL-2-expressing cells from leukemic patients to the killing effects of staurosporine, chlorambucil, and fludarabine. These results provide a proof of principle that the potentially toxic effects of BCL-2 ligands on mitochondrial respiration are not essential for their antiapoptotic activity and represent an important step forward in the development of tumor-selective drugs acting on BCL-2.