ATM inhibition blocks glucose metabolism and amplifies the sensitivity of resistant lung cancer cell lines to oncogene driver inhibitors.

BACKGROUND: ATM is a multifunctional serine/threonine kinase that in addition to its well-established role in DNA repair mechanisms is involved in a number of signaling pathways including regulation of oxidative stress response and metabolic diversion of glucose through the pentose phosphate pathway. Oncogene-driven tumorigenesis often implies the metabolic switch from oxidative phosphorylation to glycolysis which provides metabolic intermediates to sustain cell proliferation. The aim of our study is to elucidate the role of ATM in the regulation of glucose metabolism in oncogene-driven cancer cells and to test whether ATM may be a suitable target for anticancer therapy. METHODS: Two oncogene-driven NSCLC cell lines, namely H1975 and H1993 cells, were treated with ATM inhibitor, KU55933, alone or in combination with oncogene driver inhibitors, WZ4002 or crizotinib. Key glycolytic enzymes, mitochondrial complex subunits (OXPHOS), cyclin D1, and apoptotic markers were analyzed by Western blotting. Drug-induced toxicity was assessed by MTS assay using stand-alone or combined treatment with KU55933 and driver inhibitors. Glucose consumption, pyruvate, citrate, and succinate levels were also analyzed in response to KU55933 treatment. Both cell lines were transfected with ATM-targeted siRNA or non-targeting siRNA and then exposed to treatment with driver inhibitors. RESULTS: ATM inhibition deregulates and inhibits glucose metabolism by reducing HKII, p-PKM2Tyr105, p-PKM2Ser37, E1α subunit of pyruvate dehydrogenase complex, and all subunits of mitochondrial complexes except ATP synthase. Accordingly, glucose uptake and pyruvate concentrations were reduced in response to ATM inhibition, whereas citrate and succinate levels were increased in both cell lines indicating the supply of alternative metabolic substrates. Silencing of ATM resulted in similar changes in glycolytic cascade and OXPHOS levels. Furthermore, the driver inhibitors amplified the effects of ATM downregulation on glucose metabolism, and the combined treatment with ATM inhibitors enhanced the cytotoxic effect of driver inhibitors alone by increasing the apoptotic response. CONCLUSIONS: Inhibition of ATM reduced both glycolytic enzymes and OXPHOS levels in oncogene-driven cancer cells and enhanced apoptosis induced by driver inhibitors thus highlighting the possibility to use ATM and the driver inhibitors in combined regimens of anticancer therapy in vivo.

ß3AR-Dependent Brain-Derived Neurotrophic Factor (BDNF) Generation Limits Chronic Postischemic Heart Failure.

BACKGROUND: Loss of brain-derived neurotrophic factor (BDNF)/TrkB (tropomyosin kinase receptor B) signaling accounts for brain and cardiac disorders. In neurons, ß-adrenergic receptor stimulation enhances local BDNF expression. It is unclear if this occurs in a pathophysiological relevant manner in the heart, especially in the ß-adrenergic receptor-desensitized postischemic myocardium. Nor is it fully understood whether and how TrkB agonists counter chronic postischemic left ventricle (LV) decompensation, a significant unmet clinical milestone. METHODS: We conducted in vitro studies using neonatal rat and adult murine cardiomyocytes, SH-SY5Y neuronal cells, and umbilical vein endothelial cells. We assessed myocardial ischemia (MI) impact in wild type, ß3AR knockout, or myocyte-selective BDNF knockout (myoBDNF KO) mice in vivo (via coronary ligation [MI]) or in isolated hearts with global ischemia-reperfusion (I/R). RESULTS: In wild type hearts, BDNF levels rose early after MI (<24 hours), plummeting at 4 weeks when LV dysfunction, adrenergic denervation, and impaired angiogenesis ensued. The TrkB agonist, LM22A-4, countered all these adverse effects. Compared with wild type, isolated myoBDNF KO hearts displayed worse infarct size/LV dysfunction after I/R injury and modest benefits from LM22A-4. In vitro, LM22A-4 promoted neurite outgrowth and neovascularization, boosting myocyte function, effects reproduced by 7,8-dihydroxyflavone, a chemically unrelated TrkB agonist. Superfusing myocytes with the ß3AR-agonist, BRL-37344, increased myocyte BDNF content, while ß3AR signaling underscored BDNF generation/protection in post-MI hearts. Accordingly, the ß1AR blocker, metoprolol, via upregulated ß3ARs, improved chronic post-MI LV dysfunction, enriching the myocardium with BDNF. Last, BRL-37344-imparted benefits were nearly abolished in isolated I/R injured myoBDNF KO hearts. CONCLUSIONS: BDNF loss underscores chronic postischemic heart failure. TrkB agonists can improve ischemic LV dysfunction via replenished myocardial BDNF content. Direct cardiac ß3AR stimulation, or ß-blockers (via upregulated ß3AR), is another BDNF-based means to fend off chronic postischemic heart failure.

Use of Natural Agents and Agrifood Wastes for the Treatment of Skin Photoaging.

Photoaging is the premature aging of the skin caused by repeated exposure to ultraviolet (UV) rays. The harmful effects of UV rays-from the sun or from artificial sources-alter normal skin structures and cause visible damage, especially in the most exposed areas. Fighting premature aging is one of the most important challenges of the medical landscape. Additionally, consumers are looking for care products that offer multiple benefits with reduced environmental and economic impact. The growing requests for bioactive compounds from aromatic plants for pharmaceutical and cosmetic applications have to find new sustainable methods to increase the effectiveness of new active formulations derived from eco-compatible technologies. The principle of sustainable practices and the circular economy favor the use of bioactive components derived from recycled biomass. The guidelines of the European Commission support the reuse of various types of organic biomass and organic waste, thus transforming waste management problems into economic opportunities. This review aims to elucidate the main mechanisms of photoaging and how these can be managed using natural renewable sources and specific bioactive derivatives, such as humic extracts from recycled organic biomass, as potential new actors in modern medicine.

What molecular imaging of cancer patients can teach us about COVID-19.

COVID-19 pandemic had a great impact on health systems and cancer care worldwide. Patients with cancer who develop COVID-19 are at high risk of severe outcomes and clarifying the determinants of such vulnerability of cancer patients would be of great clinical benefit. While the mechanisms of SARS-CoV-2 infection have been elucidated, the pathogenetic pathways leading to severe manifestations of the disease are largely unknown. Critical manifestations of COVID-19 mainly occur in elderly patients and in patients with serious comorbidities including cancer. Efforts to understand the intersection of pathways between severe manifestations of COVID-19 and cancer may shed light on the pathogenesis of critical illness in COVID-19 patients. Here, we will focus our attention on two major fields of potential intersection between COVID-19 and cancer, namely the dysfunction of immune system and the prothrombotic state that can occur in both COVID-19 and cancer patients, testing whether cancer imaging can provide clues to better understand such interactions.

Non-Canonical Role of PDK1 as a Negative Regulator of Apoptosis through Macromolecular Complexes Assembly at the ER-Mitochondria Interface in Oncogene-Driven NSCLC.

Here, we tested whether co-targeting of glucose metabolism and oncogene drivers may enhance tumor response to tyrosine kinase inhibitors (TKIs) in NSCLC. To this end, pyruvate dehydrogenase kinase 1 (PDK1) was stably downregulated in oncogene-driven NSCLC cell lines exposed or not to TKIs. H1993 and H1975 cells were stably transfected with scrambled (shCTRL) or PDK1-targeted (shPDK1) shRNA and then treated with MET inhibitor crizotinib (1 µM), double mutant EGFRL858R/T790M inhibitor WZ4002 (1 µM) or vehicle for 48 h. The effects of PDK1 knockdown on glucose metabolism and apoptosis were evaluated in untreated and TKI-treated cells. PDK1 knockdown alone did not cause significant changes in glycolytic cascade, ATP production and glucose consumption, but it enhanced maximal respiration in shPDK1 cells when compared to controls. When combined with TKI treatment, PDK1 downregulation caused a strong enhancement of OXPHOS and a marked reduction in key glycolytic enzymes. Furthermore, increased levels of apoptotic markers were found in shPDK1 cells as compared to shCTRL cells after treatment with TKIs. Co-immunoprecipitation studies showed that PDK1 interacts with PKM2, Bcl-2 and Bcl-xL, forming macromolecular complexes at the ER-mitochondria interface. Our findings showed that downregulation of PDK1 is able to potentiate the effects of TKIs through the disruption of macromolecular complexes involving PKM2, Bcl-2 and Bcl-xL.

Cell-to-Cell Adhesion and Neurogenesis in Human Cortical Development: A Study Comparing 2D Monolayers with 3D Organoid Cultures.

Organoids (ORGs) are increasingly used as models of cerebral cortical development. Here, we compared transcriptome and cellular phenotypes between telencephalic ORGs and monolayers (MONs) generated in parallel from three biologically distinct induced pluripotent stem cell (iPSC) lines. Multiple readouts revealed increased proliferation in MONs, which was caused by increased integrin signaling. MONs also exhibited altered radial glia (RG) polarity and suppression of Notch signaling, as well as impaired generation of intermediate progenitors, outer RG, and cortical neurons, which were all partially reversed by reaggregation of dissociated cells. Network analyses revealed co-clustering of cell adhesion, Notch-related transcripts and their transcriptional regulators in a module strongly downregulated in MONs. The data suggest that ORGs, with respect to MONs, initiate more efficient Notch signaling in ventricular RG owing to preserved cell adhesion, resulting in subsequent generation of intermediate progenitors and outer RG, in a sequence that recapitulates the cortical ontogenetic process.

Copper/Zinc Superoxide Dismutase in Human Skin: Current Knowledge.

Superoxide dismutase is widespread in the human body, including skin and its appendages. Here, we focus on human skin copper/zinc superoxide dismutase, the enzyme that protects skin and its appendages against reactive oxygen species. Human skin copper/zinc superoxide dismutase resides in the cytoplasm of keratinocytes, where up to 90% of cellular reactive oxygen species is produced. Factors other than cell type, such as gender, age and diseased state influence its location in skin tissues. We review current knowledge of skin copper/zinc superoxide dismutase including recent studies in an attempt to contribute to solving the question of its remaining unexplained functions. The research described here may be applicable to pathologies associated with oxidative stress. However, recent studies on copper/zinc superoxide dismutase in yeast reveal that its predominant function may be in signaling pathways rather than in scavenging superoxide ions. If confirmed in the skin, novel approaches might be developed to unravel the enzyme's remaining mysteries.

Preclinical imaging for targeting cancer immune evasion.

Novel anticancer immunotherapy strategies such as immune checkpoint blockade have been successfully employed in patients with a variety of cancers and became a therapeutic option in the treatment of several malignancies. However, long-term durable responses to immune checkpoint inhibitors are currently limited to a fraction of patients raising the need of an accurate selection of potentially responding patients. Although several biomarkers have been proposed for patient selection and prediction of response to immune checkpoint blockade, none can be considered as an absolute selection criterion. Whole-body imaging with tracers recognizing targets for immunotherapy by allowing visualization of target expression in all tumor and extratumoral sites at baseline and during disease evolution may provide reliable predictive imaging biomarkers. Here we will provide an overview of preclinical imaging studies aiming at the development and validation of tracers recognizing targets for immunotherapy that can be used for selection and monitoring of patients undergoing immunotherapy and for testing novel immunotherapeutic agents or strategies. Furthermore, we will focus on the selection of animal models based on the main purpose of the study and implications for clinical transfer of the results.

Copper/Zinc-Superoxide Dismutase in Human Epidermis: An Immunochemical Study.

The localization of copper and zinc-superoxide dismutase in normal and neoplastic human skin was examined with immunochemical techniques. Skin samples were taken from males and females of different ages, UV exposed and non-exposed areas and basal-/spino-cellular carcinomas. The enzyme was localized diffusely in the cytoplasm and was also found in the nuclei of epidermal cells, endothelial cells and other dermis cell types. The dismutase content in the epidermis was higher in males than females, UV-exposed than non-exposed and young than old people. In the tumors, the enzyme content of the superficial epidermal layers was higher than in the deep tumoral epithelial cells. These data suggest that the localization of Cu, Zn-SOD in skin tissues reflects the gender and age of the subject, the cell types and their normal or diseased state. Further studies based on the investigation of systemic changes of this enzyme in physiological and pathological epidermis could provide additional information on tumor cell generation.

Calcium-binding protein and some neuropeptides in the retina of Octopus vulgaris: A morpho-histochemical study.

The existence of both calcium-binding proteins (CBPs) and neuropeptides in the retina and brain of various species of vertebrates and invertebrates is well documented. Octopus retina is particularly interesting because it represents a case of convergent evolution. The aim of this study was to characterize the distribution of two CBPs, calretinin and calbindin, in Octopus retina using morphology, in situ hybridization, immunocytochemistry and Western blot. Calretinin-like immunoreactivity was found in the photoreceptor cells, but unexpectedly also in the supporting cells. In situ hybridization and Western blot analysis confirmed these results. No immunoreactivity was found for calbindin. Two neuropeptides, Substance P and calcitonin gene-related peptide (CGRP), as well as neurofilament protein and glial fibrillary acidic protein were also localized in the Octopus retina by immunocytochemistry. Our work provides new insights about calcium-binding proteins and neuropeptide distribution in Octopus retina and suggests a functional role for calretinin, a highly conserved protein, in visual signal transduction of cephalopods.

Calretinin Immunoreactivity in the Human Testis Throughout Fetal Life.

The main functions of the testis are sex hormone and sperm cell production. Steroidogenesis occurs in the Leydig interstitial cells and spermatogenesis in the seminiferous tubules. Male gonad morphogenesis is a finely orchestrated process, mainly coordinated by hormones, whose actions can significantly affect post-pubertal testicular function. Calcium is a key intracellular messenger, which regulates many signal transduction pathways, and is also implicated in steroidogenesis. Calcium homeostasis and signaling rely on many calcium-binding proteins including calretinin, of the "EF-hand" protein family. Calretinin is a highly conserved protein mainly expressed in the nervous system but also detected in rat and human adult and fetal testis as well as in pathological conditions. Calretinin expression in the fetal testis, however, has not been thoroughly analyzed probably owing to limited availability and paucity of tissues. Here, we examined by immunocytochemistry the expression of calretinin in human fetal testis specimens, obtained from natural and therapeutic abortions, at various developmental ages. We found that calretinin-immunoreactive Leydig cells were visible throughout the timeframe studied (14th-27th week). Immunoreactivity was also observed in Sertoli cells and in the germ cells of the immature seminiferous tubules. Overall our data indicate that calretinin expression parallels the decline in Leydig cell number, suggesting that its presence is indeed correlated to their steroidogenic activity. They also suggest that the intratubular positivity of calretinin could be linked to the ability of Sertoli cells to produce locally acting hormones contributing to the histodifferentiation of the male genital tract. J. Cell. Physiol. 232: 1872-1878, 2017. © 2016 Wiley Periodicals, Inc.

Genetic deletion of uncoupling protein 3 exaggerates apoptotic cell death in the ischemic heart leading to heart failure.

BACKGROUND: Uncoupling protein 3 (ucp3) is a member of the mitochondrial anion carrier superfamily of proteins uncoupling mitochondrial respiration. In this study, we investigated the effects of ucp3 genetic deletion on mitochondrial function and cell survival under low oxygen conditions in vitro and in vivo. METHODS AND RESULTS: To test the effects of ucp3 deletion in vitro, murine embryonic fibroblasts and adult cardiomyocytes were isolated from wild-type (WT, n=67) and ucp3 knockout mice (ucp3(-/-), n=70). To test the effects of ucp3 genetic deletion in vivo, myocardial infarction (MI) was induced by permanent coronary artery ligation in WT and ucp3(-/-) mice. Compared with WT, ucp3(-/-) murine embryonic fibroblasts and cardiomyocytes exhibited mitochondrial dysfunction and increased mitochondrial reactive oxygen species generation and apoptotic cell death under hypoxic conditions in vitro (terminal deoxynucleotidyl transferase-dUTP nick end labeling-positive nuclei: WT hypoxia, 70.3 ± 1.2%; ucp3(-/-) hypoxia, 85.3 ± 0.9%; P<0.05). After MI, despite similar areas at risk in the 2 groups, ucp3(-/-) hearts demonstrated a significantly larger infarct size compared with WT (infarct area/area at risk: WT, 48.2 ± 3.7%; ucp3(-/-), 65.0 ± 2.9%; P<0.05). Eight weeks after MI, cardiac function was significantly decreased in ucp3(-/-) mice compared with WT (fractional shortening: WT MI, 42.7 ± 3.1%; ucp3(-/-) MI, 24.4 ± 2.9; P<0.05), and this was associated with heightened apoptotic cell death (terminal deoxynucleotidyl transferase-dUTP nick end labeling-positive nuclei: WT MI, 0.7 ± 0.04%; ucp3(-/-) MI, 1.1 ± 0.09%, P<0.05). CONCLUSIONS: Our data indicate that ucp3 levels regulate reactive oxygen species levels and cell survival during hypoxia, modulating infarct size in the ischemic heart.

LMO2 expression reflects the different stages of blast maturation and genetic features in B-cell acute lymphoblastic leukemia and predicts clinical outcome.

BACKGROUND: LMO2 is highly expressed at the most immature stages of lymphopoiesis. In T-lymphocytes, aberrant LMO2 expression beyond those stages leads to T-cell acute lymphoblastic leukemia, while in B cells LMO2 is also expressed in germinal center lymphocytes and diffuse large B-cell lymphomas, where it predicts better clinical outcome. The implication of LMO2 in B-cell acute lymphoblastic leukemia must still be explored. DESIGN AND METHODS: We measured LMO2 expression by real time RT-PCR in 247 acute lymphoblastic leukemia patient samples with cytogenetic data (144 of them also with survival and immunophenotypical data) and in normal hematopoietic and lymphoid cells. RESULTS: B-cell acute lymphoblastic leukemia cases expressed variable levels of LMO2 depending on immunophenotypical and cytogenetic features. Thus, the most immature subtype, pro-B cells, displayed three-fold higher LMO2 expression than pre-B cells, common-CD10+ or mature subtypes. Additionally, cases with TEL-AML1 or MLL rearrangements exhibited two-fold higher LMO2 expression compared to cases with BCR-ABL rearrangements or hyperdyploid karyotype. Clinically, high LMO2 expression correlated with better overall survival in adult patients (5-year survival rate 64.8% (42.5%-87.1%) vs. 25.8% (10.9%-40.7%), P= 0.001) and constituted a favorable independent prognostic factor in B-ALL with normal karyotype: 5-year survival rate 80.3% (66.4%-94.2%) vs. 63.0% (46.1%-79.9%) (P= 0.043). CONCLUSIONS: Our data indicate that LMO2 expression depends on the molecular features and the differentiation stage of B-cell acute lymphoblastic leukemia cells. Furthermore, assessment of LMO2 expression in adult patients with a normal karyotype, a group which lacks molecular prognostic factors, could be of clinical relevance.

Calretinin distribution in the octopus brain: an immunohistochemical and in situ hybridization histochemical analysis.

The distribution of calretinin containing neurons examined by in situ hybridization mapping was compared with that obtained by immunocytochemistry in the brain of octopus. Results revealed a close correspondence between the two types of investigations. Western blot analysis disclosed a 29 kDa protein immunostained with anti-calretinin antibody. Calretinin containing neurons were localized mainly in the cortex of octopus lobes, including the vertical, frontal, basal, buccal, palliovisceral, pedal and branchial, with variations of staining intensity and density of immunoreactive cells. The amacrine cells surrounding calretinin containing neuronal bodies of the cortex were also labeled unlike the glial cells. The close correspondence of blotting analysis, immunocytochemistry and in situ hybridization indicates with no doubt that calretinin, like other calcium-binding proteins previously studied, is also present in the nervous system of cephalopods. Furthermore, although recent findings localize calretinin also in endocrine glands, the presence of this calcium-binding protein in the brain of octopus indicates that calretinin appeared early in the phylogeny as a neuronal protein already in invertebrates.