Proteotoxic stress-induced apoptosis in cancer cells: understanding the susceptibility and enhancing the potency

Leiomyosarcoma (LMS) is aggressive cancer with few therapeutic options. LMS cells are more sensitive to proteotoxic stress compared to normal smooth muscle cells. We used small compound 2c to induce proteotoxic stress and compare the transcriptomic adaptations of immortalized human uterine smooth muscle cells (HUtSMC) and LMS cells SK-UT-1. We found that the expression of the heat shock proteins (HSPs) gene family is upregulated with higher efficiency in normal cells. In contrast, the upregulation of BH3-only proteins is higher in LMS cells. HSF1, the master regulator of HSP transcription, is sequestered into transcriptionally incompetent nuclear foci only in LMS cells, which explains the lower HSP upregulation. We also found that several compounds can enhance the cell death response to proteotoxic stress. Specifically, when low doses were used, an inhibitor of salt-inducible kinases (SIKs) and the inhibitor of IRE1 alpha, a key element of the unfolded protein response (UPR), support proteotoxic-induced cell death with strength in LMS cells and without effects on the survival of normal cells. Overall, our data provide an explanation for the higher susceptibility of LMS cells to proteotoxic stress and suggest a potential option for co-treatment strategies

The co-existence of transcriptional activator and transcriptional repressor MEF2 complexes influences tumor aggressiveness

The contribution of MEF2 TFs to the tumorigenic process is still mysterious. Here we clarify that MEF2 can support both pro-oncogenic or tumor suppressive activities depending on the interaction with co-activators or co-repressors partners. Through these interactions MEF2 supervise histone modifications associated with gene activation/repression, such as H3K4 methylation and H3K27 acetylation. Critical switches for the generation of a MEF2 repressive environment are class IIa HDACs. In leiomyosarcomas (LMS), this two-faced trait of MEF2 is relevant for tumor aggressiveness. Class IIa HDACs are overexpressed in 22% of LMS, where high levels of MEF2, HDAC4 and HDAC9 inversely correlate with overall survival. The knock out of HDAC9 suppresses the transformed phenotype of LMS cells, by restoring the transcriptional proficiency of some MEF2-target loci. HDAC9 coordinates also the demethylation of H3K4me3 at the promoters of MEF2-target genes. Moreover, we show that class IIa HDACs do not bind all the regulative elements bound by MEF2. Hence, in a cell MEF2-target genes actively transcribed and strongly repressed can coexist. However, these repressed MEF2-targets are poised in terms of chromatin signature. Overall our results candidate class IIa HDACs and HDAC9 in particular, as druggable targets for a therapeutic intervention in LMS

The isopeptidase inhibitor 2cPE triggers proteotoxic stress and ATM activation in chronic lymphocytic leukemia cells

Relapse after treatment is a common and unresolved problem for patients suffering of the B-cell chronic lymphocytic leukemia (B-CLL). Here we investigated the ability of the isopeptidase inhibitor 2cPE to trigger apoptosis in leukemia cells in comparison with bortezomib, another inhibitor of the ubiquitin-proteasome system (UPS). Both inhibitors trigger apoptosis in CLL B cells and gene expression profiles studies denoted how a substantial part of genes up-regulated by these compounds are elements of adaptive responses, aimed to sustain cell survival. 2cPE treatment elicits the up-regulation of chaperones, proteasomal subunits and elements of the anti-oxidant response. Selective inhibition of these responses augments apoptosis in response to 2cPE treatment. We have also observed that the product of the ataxia telangiectasia mutated gene (ATM) is activated in 2cPE treated cells. Stimulation of ATM signaling is possibly dependent on the alteration of the redox homeostasis. Importantly ATM inhibition, mutations or down-modulation increase cell death in response to 2cPE. Overall this work suggests that 2cPE could offer new opportunities for the treatment of B-CLL

Mechanisms of Activation of LRRC8 Volume Regulated Anion Channels.

Volume regulated anion channels (VRACs) are ubiquitously expressed in all vertebrate cells. Despite many years of research, the fundamental mechanisms underlying VRAC activation are not understood. The recent molecular identification of the LRRC8 genes underlying VRAC revealed that VRACs are formed by a hexameric assembly of members of the LRRC8 gene family. Knowing the genes underlying VRACs allowed the discovery of novel VRAC functions into cell volume regulation, and first structure function studies revealed important insight in channel activation mechanisms. The determination of cryo-EM structures of homomeric LRRC8A and LRRC8D complexes provide a framework for a rational approach to investigate biophysical mechanisms. We discuss several recent advances within the structural framework, and we critically review the literature on the main mechanisms proposed to be involved in VRAC activation, including low intracellular ionic strength, membrane unfolding, oxidation, phosphorylation and G-protein coupling

Mitochondrial Adaptations in Elderly and Young Men Skeletal Muscle Following 2 Weeks of Bed Rest and Rehabilitation

The aim of the study was to evaluate the expression levels of proteins related to mitochondrial biogenesis regulation and bioenergetics in vastus lateralis muscle biopsies from 16 elderly and 7 young people subjected to 14 days of bed-rest, causing atrophy, and subsequent 14 days of exercise training. Based on quantitative immunoblot analyses, in both groups a reduction of two key regulators of mitochondrial biogenesis/remodeling and activity, namely PGC-1alpha and Sirt3, was revealed during bedrest,with a subsequent up-regulation after rehabilitation, indicating an involvement of PGC-1alpha-Sirt3 axis in response to the treatments. A difference was observed comparing the young and elderly subjects as, for both proteins, the abundance in the elderly was more affected by immobility and less responsive to exercise. The expression levels of TOM20 and Citrate Synthase, assayed as markers of outer mitochondrial membrane and mitochondrial mass, showed a noticeable sensitivity in the elderly group, where they were affected by bed-rest and rehabilitation recalling the pattern of PGC-1alpha. TOM20 and CS remained unchanged in young subjects. Single OXPHOScomplexes showed peculiar patterns, which were in some cases dissimilar from PGC 1alpha, and suggest different influences on protein biogenesis and degradation. Overall,exercise was capable to counteract the effect of immobility, when present, except for complex V, which was markedly downregulated by bed-rest, but remained unaffected after rehabilitation, maybe as result of greater extent of degradation processes over biogenesis. Phosphorylation extent of AMPK, and its upstream activator LKB1, did not change after bed-rest and rehabilitation in either young or elderly subjects, suggestingthat the activation of energy-sensing LKB1-AMPK signaling pathway was \u201cmissed\u201d due to its transient nature, or was not triggered under our conditions. Our study demonstrates that, as far as the expression of various proteins related to mitochondrial biogenesis/remodeling, adaptations to bed-rest and rehabilitation in the two populations were different. The impact of bed-rest was greater in the elderly subjects, where the pattern (decrease after bed rest and recovery following rehabilitation) was accompanied by changes of mitochondrial mass. Modifications of protein abundance were matched with data obtained from gene expression analyses of four public human datasets focusing on related genes

Mitochondrial Adaptations in Elderly and Young Men Skeletal Muscle Following 2 Weeks of Bed Rest and Rehabilitation

The aim of the study was to evaluate the expression levels of proteins related to mitochondrial biogenesis regulation and bioenergetics in vastus lateralis muscle biopsies from 16 elderly and 7 young people subjected to 14 days of bed-rest, causing atrophy, and subsequent 14 days of exercise training. Based on quantitative immunoblot analyses, in both groups a reduction of two key regulators of mitochondrial biogenesis/remodeling and activity, namely PGC-1α and Sirt3, was revealed during bed-rest, with a subsequent up-regulation after rehabilitation, indicating an involvement of PGC-1α-Sirt3 axis in response to the treatments. A difference was observed comparing the young and elderly subjects as, for both proteins, the abundance in the elderly was more affected by immobility and less responsive to exercise. The expression levels of TOM20 and Citrate Synthase, assayed as markers of outer mitochondrial membrane and mitochondrial mass, showed a noticeable sensitivity in the elderly group, where they were affected by bed-rest and rehabilitation recalling the pattern of PGC-1α. TOM20 and CS remained unchanged in young subjects. Single OXPHOS complexes showed peculiar patterns, which were in some cases dissimilar from PGC-1α, and suggest different influences on protein biogenesis and degradation. Overall, exercise was capable to counteract the effect of immobility, when present, except for complex V, which was markedly downregulated by bed-rest, but remained unaffected after rehabilitation, maybe as result of greater extent of degradation processes over biogenesis. Phosphorylation extent of AMPK, and its upstream activator LKB1, did not change after bed-rest and rehabilitation in either young or elderly subjects, suggesting that the activation of energy-sensing LKB1-AMPK signaling pathway was “missed” due to its transient nature, or was not triggered under our conditions. Our study demonstrates that, as far as the expression of various proteins related to mitochondrial biogenesis/remodeling, adaptations to bed-rest and rehabilitation in the two populations were different. The impact of bed-rest was greater in the elderly subjects, where the pattern (decrease after bed rest and recovery following rehabilitation) was accompanied by changes of mitochondrial mass. Modifications of protein abundance were matched with data obtained from gene expression analyses of four public human datasets focusing on related genes

Transcriptomic analysis unveils correlations between regulative apoptotic caspases and genes of cholesterol homeostasis in human brain.

Regulative circuits controlling expression of genes involved in the same biological processes are frequently interconnected. These circuits operate to coordinate the expression of multiple genes and also to compensate dysfunctions in specific elements of the network. Caspases are cysteine-proteases with key roles in the execution phase of apoptosis. Silencing of caspase-2 expression in cultured glioblastoma cells allows the up-regulation of a limited number of genes, among which some are related to cholesterol homeostasis. Lysosomal Acid Lipase A (LIPA) was up-regulated in two different cell lines in response to caspase-2 down-regulation and cells silenced for caspase-2 exhibit reduced cholesterol staining in the lipid droplets. We expanded this observation by large-scale analysis of mRNA expression. All caspases were analyzed in terms of co-expression in comparison with 166 genes involved in cholesterol homeostasis. In the brain, hierarchical clustering has revealed that the expression of regulative apoptotic caspases (CASP2, CASP8 CASP9, CASP10) and of the inflammatory CASP1 is linked to several genes involved in cholesterol homeostasis. These correlations resulted in altered GBM (Glioblastoma Multiforme), in particular for CASP1. We have also demonstrated that these correlations are tissue specific being reduced (CASP9 and CASP10) or different (CASP2) in the liver. For some caspases (CASP1, CASP6 and CASP7) these correlations could be related to brain aging

Antisense-mediated post-transcriptional silencing of SCN1B gene modulates sodium channel functional expression.

Voltage-dependent sodium channels are membrane proteins essential for cell excitability. They are composed by a pore-forming \u3b1-subunit and one or more \u3b2 subunits. Nine \u3b1 subunit and five \u3b2 subunit isoforms have been identified in mammals: \u3b21, its splice variant \u3b21B, \u3b22, \u3b23 and \u3b24. Although they do not form the ion channel pore, \u3b2 subunits modulate both function as well as expression of sodium channels on cell membrane.To investigate the role of \u3b21 subunit on the modulation of sodium channel expression, we silenced this auxiliary subunit with specific antisense oligonucleotides (ASONs) in two rat cell lines, the GH3 and the H9C2, from neuro-ectoderm and cardiac myocyte origin, respectively. Treatment of cells with ASONs determined a reduction of about 50% of \u3b21 subunit mRNA and protein expression in both cell lines. We found that this level of \u3b21 subunit silencing resulted in an overall decrease of \u3b1 subunit mRNA, protein expression and a decrease of sodium current density, without altering significantly the voltage-dependent and kinetic properties of the currents. In GH3 cells, the \u3b21 subunit silencing reduced the expression of Nav1.1, Nav1.3 and Nav1.6 isoforms, whereas the Nav 1.2 isoform expression remained unaltered. The expression of the only \u3b1 subunit present in H9C2 cells, the Nav1.5, was also reduced by \u3b21 subunit silencing.These results indicate that the \u3b21 subunit may exert an isoform-specific fine-tuned modulation of sodium channel expression

Functional modulation of voltage-dependent sodium channel expression by wild type and mutated C121W-\u3b21 subunit.

Voltage dependent sodium channels are membrane proteins essential for cell excitability. They are composed by a pore-forming \u3b1-subunit, encoded in mammals by up to 9 different genes, and 4 different ancillary \u3b2-subunits. The expression pattern of the \u3b1 subunit isoforms confers the distinctive functional and pharmacological properties to different excitable tissues. \u3b2 subunits are important modulators of channel function and expression. Mutation C121W of the \u3b21-subunit causes an autosomal dominant epileptic syndrome without cardiac symptoms. The C121W mutation may act by a dominant-competition, modifying the expression of \u3b1-subunit proteins. To test this hypothesis, we transfected GH3 cells, from neuro-ectoderm origin, with wild-type or mutant \u3b21 subunits and compared them to native cells. To examine the tissue specificity of the C121W-\u3b21 mutation, we compared the effects of the mutation on neural cells with those of H9C2 cells of cardiac origin. We found that in GH3 cells the over-expression of the \u3b21 subunit augments the \u3b1 subunit mRNA and protein levels, while in the H9C2 cells the enhanced level of \u3b21 subunit not only increases but also qualitatively modifies the sodium channel \u3b1 isoform expression pattern. Interestingly, the introduction of the epileptogenic C121W-\u3b21 subunit does not alter the sodium channel isoform composition of GH3 cells, while produces additional changes in the \u3b1-subunit expression pattern of H9C2 cells. Electrophysiological measurements confirm these molecular results. The expression differences observed could be correlated to the tissue-specific regulatory action of the \u3b21 subunit and to the nervous system specificity of the C121W mutation. Our findings could be helpful for the comprehension of the molecular mechanism of generalised epileptic with febrile seizures plus in patients with identified \u3b21 subunit mutations

Tipizzazione dell\u2019infiltrato cellulare nelle enteriti da Enteromyxum leei in sarago pizzuto (Diplodus puntazzo)

Enteromyxum leei \ue8 un mixosporidio istozoico, agente eziologico di gravi enteriti parassitarie di diverse specie ittiche marine. In sarago pizzuto (Diplodus puntazzo) la malattia parassitaria ha generalmente un andamento acuto con elevati tassi di mortalit\ue0, soprattutto nei soggetti durante il primo anno di produzione (fase acuta della malattia a fine estate). Da studi condotti di recente, nell\u201fambito del progetto finalizzato allo studio di fattori limitanti la produzione del sarago pizzuto, nel corso della malattia, i quadri anatomopatologici a livello enterico erano caratterizzati da un imponente infiltrato cellulare della lamina propria e della sottomucosa, accompagnati da fenomeni iperplastici a carico dell\u201fepitelio intestinale. I quadri infiammatori osservati erano riconducibili a enterite proliferativa (fase acuta dell\u201finfezione) ed enterite linfocitaria cronica profonda (fase cronica dell\u201finfezione). Per definire i contorni istopatologici dei quadri osservati, \ue8 stato affrontato uno studio preliminare istochimico ed immunoistochimico per descrivere e tipizzare l\u201finfiltrato cellulare nelle enteriti causate da E. leei in sarago pizzuto. Da soggetti affetti da enteromixosi sono state ottenute sezioni in paraffina di intestino, fissato in soluzione di Bouin, le quali sono state colorate con ematossilina-eosina e caratterizzate tramite le colorazioni di Twort (Gram), Giemsa, PAS (o PAS-alcian blu), tricromica di Masson, Cleveland e Blu di toluidina. Il protocollo della valutazione istologica ha previsto la stima delle cellule granulari eosinofiliche/mastociti (numero medio di cellule per campo a 400 ingrandimenti, a livello della tonaca propria in porzioni di intestino) e i parametri di infiltrazione cellulare: il tipo cellulare, la distribuzione tissutale e il tipo strutturale. Su sezioni in paraffina di intestino \ue8 stata valutata l\u201fimmunoreattivit\ue0 cellulare nei confronti di 4 anticorpi, GM-CFSR\u3b1 (C-18), IL-1\u3b2 v(H-153), CD35 (CR1) e CD16 (H-80) (Santa Cruz Biotechnology, Inc), al fine di tipizzare l\u201finfiltrato cellulare descritto nelle enteriti. I quadri di enterite osservati sono contraddistinti da un imponente infiltrato linfocitaria (con scarsa presenza di plasmacellule), da mastociti (cellule granulari eosinofiliche), granulociti eosinofili e macrofagi. Topograficamente, tramite la colorazione di Cleveland, i mastociti sono diffusi e/o formano densi strati nella lamina propria e nella sottomucosa, assumendo talvolta aspetto pervasale nella tonaca muscolare e si localizzano a livello sub- e intraepiteliale quale prima difesa aspecifica nei confronti dei vari stadi parassitari. I granulociti eosinofili infiltrano maggiormente la sottomucosa e spesso sono pervasali; inoltre queste cellule infiltranti sono frequentemente diffuse tra gli adipociti del grasso periviscerale e gli acini pancreatici. Il pannello anticorpale utilizzato ha messo in evidenza un \u201cpattern\u201d di immunoreattivit\ue0 che sar\ue0 discusso