Analysis of the Effects of Hexokinase 2 Detachment From Mitochondria-Associated Membranes with the Highly Selective Peptide HK2pep.

The crucial role of hexokinase 2 (HK2) in the metabolic rewiring of tumors is now well established, which makes it a suitable target for the design of novel therapies. However, hexokinase activity is central to glucose utilization in all tissues; thus, enzymatic inhibition of HK2 can induce severe adverse effects. In an effort to find a selective anti-neoplastic strategy, we exploited an alternative approach based on HK2 detachment from its location on the outer mitochondrial membrane. We designed a HK2-targeting peptide named HK2pep, corresponding to the N-terminal hydrophobic domain of HK2 and armed with a metalloprotease cleavage sequence and a polycation stretch shielded by a polyanion sequence. In the tumor microenvironment, metalloproteases unleash polycations to allow selective plasma membrane permeation in neoplastic cells. HK2pep delivery induces the detachment of HK2 from mitochondria-associated membranes (MAMs) and mitochondrial Ca2+ overload caused by the opening of inositol-3-phosphate receptors on the endoplasmic reticulum (ER) and Ca2+ entry through the plasma membrane leading to Ca2+-mediated calpain activation and mitochondrial depolarization. As a result, HK2pep rapidly elicits death of diverse tumor cell types and dramatically reduces in vivo tumor mass. HK2pep does not affect hexokinase enzymatic activity, avoiding any noxious effect on non-transformed cells. Here, we make available a detailed protocol for the use of HK2pep and to investigate its biological effects, providing a comprehensive panel of assays to quantitate both HK2 enzymatic activity and changes in mitochondrial functions, Ca2+ flux, and cell viability elicited by HK2pep treatment of tumor cells. Graphical abstract: Flowchart for the analysis of the effects of HK2 detachment from MAMs.

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.

Filling the gap between risk assessment and molecular determinants of tumor onset.

In the past two decades, a ponderous epidemiological literature has causally linked tumor onset to environmental exposure to carcinogens. As consequence, risk assessment studies have been carried out with the aim to identify both predictive models of estimating cancer risks within exposed populations and establishing rules for minimizing hazard when handling carcinogenic compounds. The central assumption of these works is that neoplastic transformation is directly related to the mutational burden of the cell without providing further mechanistic clues to explain increased cancer onset after carcinogen exposure. Nevertheless, in the last few years, a growing number of studies have implemented the traditional models of cancer etiology, proposing that neoplastic transformation is a complex process in which several parameters and crosstalk between tumor and microenvironmental cells must be taken into account and integrated with mutagenesis. In this conceptual framework, the current strategies of risk assessment that are solely based on the 'mutator model' require an urgent update and revision to keep pace with advances in our understanding of cancer biology. We will approach this topic revising the most recent theories on the biological mechanisms involved in tumor formation in order to envision a roadmap leading to a future regulatory framework for a new, protective policy of risk assessment.

Hexokinase 2 displacement from mitochondria-associated membranes prompts Ca2+ -dependent death of cancer cells.

Cancer cells undergo changes in metabolic and survival pathways that increase their malignancy. Isoform 2 of the glycolytic enzyme hexokinase (HK2) enhances both glucose metabolism and resistance to death stimuli in many neoplastic cell types. Here, we observe that HK2 locates at mitochondria-endoplasmic reticulum (ER) contact sites called MAMs (mitochondria-associated membranes). HK2 displacement from MAMs with a selective peptide triggers mitochondrial Ca2+ overload caused by Ca2+ release from ER via inositol-3-phosphate receptors (IP3Rs) and by Ca2+ entry through plasma membrane. This results in Ca2+ -dependent calpain activation, mitochondrial depolarization and cell death. The HK2-targeting peptide causes massive death of chronic lymphocytic leukemia B cells freshly isolated from patients, and an actionable form of the peptide reduces growth of breast and colon cancer cells allografted in mice without noxious effects on healthy tissues. These results identify a signaling pathway primed by HK2 displacement from MAMs that can be activated as anti-neoplastic strategy.

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.

Predictivity and fate of metal ion release from metal-on-metal total hip prostheses.

Blood metal ion levels in 72 patients with large head metal-on-metal hip arthroplasty were studied to determine the correlation between the values measured in whole blood and urine. Urinary cobalt and chromium levels of 30µg and 21µg, respectively, adjusted to creatinine were found to correspond to the 7µg/l cut-off value that has been accepted in whole blood. Cobalt and chromium levels in whole blood and urine both significantly correlated with increased acetabular component inclination angle over 50 degrees and pain scores. There was no correlation with socket anteversion angle or femoral head diameter. The data support the use of urinary measurement of metal ions adjusted to creatinine to monitor patients with large head metal-on-metal total hip arthroplasty.

Hepatitis B vaccination of adolescents: significance of non-protective antibodies.

Despite hepatitis B virus (HBV) immunization, a percentage of healthy individuals display an antibody titre below the threshold for clinical protection (10 IU/L). In order to predict the existence of this inducible immunological response, the precise anti-HBs titre required to achieve protection in immunized patients with waned HBs antibodies must first be determined. A total of 4486 vaccinated students attending the University of Padova Medical, Science, and Veterinary School were recruited for study between 2004 and early 2012. The baseline concentration of anti-HBs was measured at enrolment. Participants displaying anti-HBs titre < 10 IU/L at the follow-up examination (mean 10.8 years) were given a booster vaccination and retested 20-30 days later. At enrolment, 87.6% of the 4486 vaccinated subjects showed persistence of anti-HBs higher than 10 IU/L. Of the 279 booster-vaccinated subjects, 94.6% achieved the cut-off titre. Booster-induced immunological response was correlated to the pre-booster titre level, with ≥ 2 IU/L ensuring a robust positive response and less than 2 IU/L being associated with the probability of developing insufficient levels of antibodies. Pre-booster antibody titre higher than 2 IU/L in adults might be predictive of an anamnestic response to booster vaccination, whereas titres below this value may indicate likelihood of non-response.

GSK-3 and mitochondria in cancer cells.

GSK-3 is a multifunctional kinase that is located in the cytosol, nucleus, and mitochondria of all cell types, and it is involved in the pathogenesis of a variety of diseases. In cancer, GSK-3 modulates the response of the cell death machinery to stress stimuli, including chemotherapeutics. Mitochondria are at the heart of the integration between survival and noxious signals; therefore, modulation of the mitochondrial functions carried out by GSK-3 is profoundly involved in the apoptosis escape capabilities that hallmark neoplasms. This review briefly covers the mechanistic interactions among oncogenic kinase pathways, GSK-3 activity and subsequent modulation of mitochondrial functions that shape the pro-survival phenotype of cancer cells, such as control of redox homeostasis and inhibition of the mitochondrial permeability transition pore.

Hepatitis B vaccination at three months of age: a successful strategy?

Vaccination of infants, children and adolescents against the hepatitis B virus (HBV) is mandatory in Italy. It is crucial to assess whether vaccinated subjects have protective antibody level during adulthood when the risk of HBV infection increases due to lifestyle or occupational exposure. Two groups of students attending to University of Padova Medical School were enrolled between 2004 and 2011 and HBV antibodies and antigens were measured. The first group (Group A) comprised students vaccinated at three months of age and the second group (Group B) comprised students vaccinated after the first year of life. The follow-up was 18.0 (Group A) and 17.9 (Group B) years. The students vaccinated at three months of age had a higher rate of non-protective antibodies (47.2%) comparing to those vaccinated after the first year of life (17.0%, P<0.0001) with a significantly lower antibody level (P<0.001). The rate of non-protective antibodies was inversely related to vaccination age. The results clearly show that children vaccinated after the first year of life are better protected against HBV. On the other hand, both groups show a good immunological memory as evidenced by the achievement of protective antibody level after the booster dose in 97.8% of subjects.

Hyaline droplet accumulation in kidney of rats treated with hexachloro-1:3-butadiene: influence of age, dose and time-course.

The present research investigates the occurrence of hyaline droplet (HD) accumulation related to age, dose and time after treatment in male Wistar rats given a single i.p. injection of hexachloro-1:3-butadiene (HCBD). In the study on age, rats from 1 to 12 months of age were treated with 100 mg kg(-1) body weight (b.w.) HCBD dose. Rats treated at 2 months of age showed a greater accumulation of HD than the other age groups; HD accumulation was not observed in 1-month-old rats. In the dose-response study, the treatment with 25, 50 and 100 mg kg(-1) b.w. at 2 months of age caused HD accumulation in the proximal convoluted tubule at all doses, with the 100 mg kg(-1) b.w. group slightly more affected. Finally, in the time-course study, rats treated with a 100 mg kg(-1) b.w. dose at 2 months of age and sacrificed at 6, 12, 24, 48, 72 and 96 h post-dosing showed a time-related HD accumulation in terms of incidence and severity, after 6 h, with a peak at 24 and 48 h and decreasing at 72 and 96 h. The present results show that HD accumulation is an early finding, and is unrelated to dose level and particularly evident in rats of 2 month of age. These findings in male rats treated with HCBD emphasize the importance of considering the age of rats at the start of a study. The more sensitive model was used in the detection of nephrotoxic effects of chemicals.

Sex-related differences in renal toxicodynamics in rodents.

INTRODUCTION: An issue yet to be addressed, in the investigation of the xenobiotic toxicity, is a detailed characterization of the sex differences in toxicological responses. The 'sex issue' is particularly significant in nephrotoxicology as the kidney is a relevant target organ for xenobiotics and few studies have approached this subject in the past. There is a strong need to improve our understanding regarding the influence of sex in toxicology, given their increased requirement to establish the limits of exposure to chemicals in the environment and at work. AREAS COVERED: In this review, the authors provide the reader with the current knowledge of sex differences in kidney toxicity for rats and mice. To make the review easier to consult, these studies have been organized according to the class of xenobiotic. EXPERT OPINION: From the analysis of the present knowledge emerges a dramatic need for information on sex differences in xenobiotics toxicity. Although animals are reasonably good predictors of adverse renal effects in patients, there is need to identify alternative methods (e.g. in vitro/ex vivo) to better study sex differences in organ toxicity.

A glutamine synthetase inhibitor increases survival and decreases cytokine response in a mouse model of acute liver failure.

BACKGROUND: Acute liver failure (ALF) can be induced in mice by administering Escherichia coli lipopolysaccharide (LPS) and D-galactosamine (D-GalN), which induce an inflammatory response involving tumour necrosis factor (TNF)-α production and a hepatocyte-specific transcriptional block. Under these conditions, binding of TNF-α to its cognate receptor on hepatocytes eventually leads to their apoptosis. AIMS: As part of an effort to identify drugs to treat this disease model, we have investigated whether the glutamine synthetase inhibitor methionine sulfoximine (MSO) could play a protective role, given its effectiveness in the inhibition of brain swelling associated with hyperammonaemia. METHODS: Mouse survival, glutamine synthetase activity, hepatocyte apoptosis and induction of inflammatory cytokines were measured in mice treated with MSO before an intraperitoneal injection of LPS/D-GalN. The effect of MSO on viability and on TNF-α release was also assessed on inflammatory and liver cells. RESULTS: We have found that, in mice treated with LPS/D-GalN, MSO (i) drastically increases animal survival; (ii) sharply reduces glutamine synthetase activity, without inhibiting its other target, γ-glutamyl cysteine synthetase; (iii) inhibits death receptor-mediated apoptosis in hepatocytes upstream to cytokine binding; (iv) strongly reduces the overall inflammatory cytokine response, including a significant decrease in TNF-α induction in vivo and ex vivo, and in the interferon-γ level and signalling. CONCLUSIONS: These results demonstrate that the MSO target glutamine synthetase is required for the early steps of the cytokine response to endotoxins, and that its pharmacological inhibition may be exploited to treat inflammation.

Cyp46-mediated cholesterol loss promotes survival in stressed hippocampal neurons.

Aged neurons constitute an outstanding example of survival robustness, outliving the accumulation of reactive oxygen species (ROS) derived from various physiological activities. Since during aging hippocampal neurons experience a progressive loss of membrane cholesterol and, by virtue of this, a gradual and sustained increase in the activity of the survival receptor tyrosine kinase TrkB, we have tested in this study if cholesterol loss is functionally associated to survival robustness during aging. We show that old neurons that did not undergo the cholesterol drop, upon knockdown of the cholesterol hydroxylating enzyme Cyp46, presented low TrkB activity and increased apoptotic levels. In further agreement, inducing cholesterol loss in young neurons led to the early appearance of TrkB activity. In vivo, Cyp46 knockdown led to the appearance of damaged hippocampal neurons in old mice exposed to exogenous stressful stimuli. Cholesterol loss seems therefore to contribute to neuronal survival in conditions of prominent stress, either acute or chronic. The relevance of this pathway in health and disease is discussed.

Gold(III)-dithiocarbamato anticancer agents: activity, toxicology and histopathological studies in rodents.

Gold(III)-dithiocarbamato complexes have recently gained increasing attention as potential anticancer agents because of their strong tumor cell growth--inhibitory effects, generally achieved by exploiting non-cisplatin-like mechanisms of action. The rationale of our research work is to combine the antitumor properties of the gold(III) metal center with the potential chemoprotective function of coordinated dithiocarbamates in order to reduce toxic side effects (in particular nephrotoxicity) induced by clinically established platinum-based drugs. In this context, [Au(III) Br(2) (ESDT)] (AUL12) was proved to exert promising and outstanding antitumor activity in vitro and to overcome both acquired and intrinsic resistance showed by some types of tumors toward cisplatin. As a subsequent extension of our previous work, we here report on detailed in vivo studies in rodents, including antitumor activity toward three transplantable murine tumor models, toxicity, nephrotoxicity and histopathological investigations. Remarkably, the gold(III) complex AUL12 stands out for higher anticancer activity than cisplatin toward all the murine tumor models examined, inducing up to 80% inhibition of tumor growth. In addition, it shows low acute toxicity levels (lethal dose, LD(50) = 30 mg kg(-1) ) and reduced nephrotoxicity. Altogether, these results confirm the reliability of our drug design strategy and support the validation of this gold(III)-dithiocarbamato derivative as a suitable candidate for clinical trials.

Are rats the appropriate experimental model to understand age-related renal drug metabolism and toxicity?

For many years, toxicological investigations have shown that the sensitivity of kidney to xenobiotics evolves depending on the stage of life. The increasing requirement for information on the potential nephrotoxic effect of drugs during human embryonic development, childhood, adulthood and senescence has potentiated toxicological studies in vivo. Rodents, specifically rats, are the primary animal models used in toxicology testing. Despite the popularity of this approach, there are a number of doubts about the appropriateness of rats for the examination of changes in toxicological responses during different stages of life. This perspective tackles the issue of evaluating whether rats fail to adequately mimic the human kidney response to xenobiotic agents through a critical analysis of the literature. We conclude that rats constitute a good model for toxicological investigations during embryonic development, youth and adulthood. However, senescent rats frequently undergo spontaneous kidney degeneration caused by chronic progressive nephropathy, making them a poor model for the study of kidney responses to xenobiotics.

Strategy for hepatitis A seroprevalence survey in a population of young people.

In the present research a novel operative strategy of health surveillance with a reduced number of serologic tests is proposed. The approach consists to identify sub-populations with high predictable serological profile that makes the serological tests unnecessary. The study is focused on assays done to detect the response against hepatitis A, which in Italy displays low/intermediate endemicity. Receiver operating characteristics analysis performed on data from documented and self-reported vaccination information of a cohort of students from Padua University Medical School confirmed that anti-hepatitis A antibodies measurement might be avoided in subjects younger than 30 years with negative documented or self-reported history of vaccination or subjected to current vaccination schedule.

Activation of mitochondrial ERK protects cancer cells from death through inhibition of the permeability transition.

We studied human cancer cell models in which we detected constitutive activation of ERK. A fraction of active ERK was found to be located in mitochondria in RWPE-2 cells, obtained by v-Ki-Ras transformation of the epithelial prostate RWPE-1 cell line; in metastatic prostate cancer DU145 cells; and in osteosarcoma SAOS-2 cells. All these tumor cells displayed marked resistance to death caused by apoptotic stimuli like arachidonic acid and the BH3 mimetic EM20-25, which cause cell death through the mitochondrial permeability transition pore (PTP). PTP desensitization and the ensuing resistance to cell death induced by arachidonic acid or EM20-25 could be ablated by inhibiting ERK with the drug PD98059 or with a selective ERK activation inhibitor peptide. ERK inhibition enhanced glycogen synthase kinase-3 (GSK-3)-dependent phosphorylation of the pore regulator cyclophilin D, whereas GSK-3 inhibition protected from PTP opening. Neither active ERK in mitochondria nor pore desensitization was observed in non-transformed RWPE-1 cells. Thus, in tumor cells mitochondrial ERK activation desensitizes the PTP through a signaling axis that involves GSK-3 and cyclophilin D, a finding that provides a mechanistic basis for increased resistance to apoptosis of neoplastic cells.