Assessment of short forms of recurrent atrial extra systoles by echocardiography with left atrial strain in ambulatory patients without organic cardiopathy / Evaluación de formas cortas de extrasístoles auriculares recurrentes mediante ecocardiografía con deformación auricular izquierda en pacientes ambulatorios sin cardiopatía orgánica

Abstract Aim: To analyse the potential usefulness and clinical relevance of the assessment by echocardiography with left atrial strain, based on the myocardial atrial deformation curves with speckle-tracking velocity vector imaging (VVI), in the analysis of short-form recurrent atrial extra systoles in ambulatory patients not suffering from organic cardiopathy. Methods: We designed a descriptive, prospective, and observational study including 270 patients between the ages of 18 and 75 assessed during an outpatient cardiology consultation attended due to palpitations over a period of two years. Using ambulatory electrocardiographic monitoring, we selected cases with short forms of repetitive atrial extrasystole, isolated or recurrentatrial fibrillation and a control group formed by those patients without repetitive ectopia. All patients underwent a thorough echocardiographic study during their first cardiological visit. Results: The analysis of the dynamic curves segmental deformation generated after an atrial extrasystole can reveal different points of origin of the extrasystole and detect specific anatomical alterations in the interatrial conduction at the level of the Bachmann's fascicle showing different models of electro anatomical activation possibly involved in the appearance of repetitive forms. Higher values of dyssynchrony between the septal and lateral wall and elongation in the time of interatrial electromechanical conduction could also be related to the existence of repetitive ectopic beats. Conclusions: Our ambulatory study employing the left atrial longitudinal strain, particularly in its segmental analysis, provides new insights into its the usefulness and potential clinical relevance.

Resumen Objetivo: Analizar la utilidad y relevancia clínica de la evaluación mediante ecocardiografía basada en las curvas de deformación auricular miocárdica con imágenes vectoriales de velocidad (VVI) de speckle-tracking, en el análisis de las extrasístoles auriculares recurrentes de corta duración en pacientes ambulatorios sin cardiopatía orgánica. Métodos: Se diseñó un estudio descriptivo, prospectivo y observacional que incluyó a 270 pacientes de entre 18 y 75 años evaluados durante una consulta externa de cardiología a la que acudieron por palpitaciones durante un periodo de dos años. Mediante el uso de monitorización electrocardiográfica ambulatoria, se seleccionaron casos con formas cortas de extrasistolia auricular repetitiva, fibrilación auricular aislada o repetitiva y un grupo control formado por aquellos pacientes sin ectopia repetitiva. Todos los pacientes se sometieron a un estudio ecocardiográfico exhaustivo durante su primera visita cardiológica. Resultados: El análisis de las curvas dinámicas de deformación segmentaria generadas tras un extrasístole auricular diferentes modelos de activación electroanatómica posiblemente implicados en la aparición de formas repetitivas. Valores mayores de disincronía entre la pared septal y lateral y el alargamiento en el tiempo de conducción electromecánica intraauricular pudieran también relacionarse con la existencia de latidos ectópicos repetitivos. Conclusiones: Nuestro estudio ambulatorio empleando la deformación longitudinal auricular izquierda, particularmente en su análisis segmentario, proporciona nuevas perspectivas sobre su utilidad y potencial relevancia clínica.

Assessment of short forms of recurrent atrial extra systoles by echocardiography with left atrial strain in ambulatory patients without organic cardiopathy.

Aim: To analyse the potential usefulness and clinical relevance of the assessment by echocardiography with left atrial strain, based on the myocardial atrial deformation curves with speckle-tracking velocity vector imaging (VVI), in the analysis of short-form recurrent atrial extra systoles in ambulatory patients not suffering from organic cardiopathy. Methods: We designed a descriptive, prospective, and observational study including 270 patients between the ages of 18 and 75 assessed during an outpatient cardiology consultation attended due to palpitations over a period of two years. Using ambulatory electrocardiographic monitoring, we selected cases with short forms of repetitive atrial extrasystole, isolated or recurrentatrial fibrillation and a control group formed by those patients without repetitive ectopia. All patients underwent a thorough echocardiographic study during their first cardiological visit. Results: The analysis of the dynamic curves segmental deformation generated after an atrial extrasystole can reveal different points of origin of the extrasystole and detect specific anatomical alterations in the interatrial conduction at the level of the Bachmann's fascicle showing different models of electro anatomical activation possibly involved in the appearance of repetitive forms. Higher values of dyssynchrony between the septal and lateral wall and elongation in the time of interatrial electromechanical conduction could also be related to the existence of repetitive ectopic beats. Conclusions: Our ambulatory study employing the left atrial longitudinal strain, particularly in its segmental analysis, provides new insights into its the usefulness and potential clinical relevance.

Objetivo: Analizar la utilidad y relevancia clínica de la evaluación mediante ecocardiografía basada en las curvas de deformación auricular miocárdica con imágenes vectoriales de velocidad (VVI) de speckle-tracking, en el análisis de las extrasístoles auriculares recurrentes de corta duración en pacientes ambulatorios sin cardiopatía orgánica. Métodos: Se diseñó un estudio descriptivo, prospectivo y observacional que incluyó a 270 pacientes de entre 18 y 75 años evaluados durante una consulta externa de cardiología a la que acudieron por palpitaciones durante un periodo de dos años. Mediante el uso de monitorización electrocardiográfica ambulatoria, se seleccionaron casos con formas cortas de extrasistolia auricular repetitiva, fibrilación auricular aislada o repetitiva y un grupo control formado por aquellos pacientes sin ectopia repetitiva. Todos los pacientes se sometieron a un estudio ecocardiográfico exhaustivo durante su primera visita cardiológica. Resultados: El análisis de las curvas dinámicas de deformación segmentaria generadas tras un extrasístole auricular diferentes modelos de activación electroanatómica posiblemente implicados en la aparición de formas repetitivas. Valores mayores de disincronía entre la pared septal y lateral y el alargamiento en el tiempo de conducción electromecánica intraauricular pudieran también relacionarse con la existencia de latidos ectópicos repetitivos. Conclusiones: Nuestro estudio ambulatorio empleando la deformación longitudinal auricular izquierda, particularmente en su análisis segmentario, proporciona nuevas perspectivas sobre su utilidad y potencial relevancia clínica.

A transient inflammatory response contributes to oxaliplatin neurotoxicity in mice.

OBJECTIVES: Peripheral neuropathy is a relevant dose-limiting adverse event that can affect up to 90% of oncologic patients with colorectal cancer receiving oxaliplatin treatment. The severity of neurotoxicity often leads to dose reduction or even premature cessation of chemotherapy. Unfortunately, the limited knowledge about the molecular mechanisms related to oxaliplatin neurotoxicity leads to a lack of effective treatments to prevent the development of this clinical condition. In this context, the present work aimed to determine the exact molecular mechanisms involved in the development of oxaliplatin neurotoxicity in a murine model to try to find new therapeutical targets. METHODS: By single-cell RNA sequencing (scRNA-seq), we studied the transcriptomic profile of sensory neurons and satellite glial cells (SGC) of the Dorsal Root Ganglia (DRG) from a well-characterized mouse model of oxaliplatin neurotoxicity. RESULTS: Analysis of scRNA-seq data pointed to modulation of inflammatory processes in response to oxaliplatin treatment. In this line, we observed increased levels of NF-kB p65 protein, pro-inflammatory cytokines, and immune cell infiltration in DRGs and peripheral nerves of oxaliplatin-treated mice, which was accompanied by mechanical allodynia and decrease in sensory nerve amplitudes. INTERPRETATION: Our data show that, in addition to the well-described DNA damage, oxaliplatin neurotoxicity is related to an exacerbated pro-inflammatory response in DRG and peripheral nerves, and open new insights in the development of anti-inflammatory strategies as a treatment for preventing peripheral neuropathy induced by oxaliplatin.

Advances in Preclinical/Clinical Glioblastoma Treatment: Can Nanoparticles Be of Help?

Glioblastoma multiforme (GB) is the most aggressive and frequent primary malignant tumor in the central nervous system (CNS), with unsatisfactory and challenging treatment nowadays. Current standard of care includes surgical resection followed by chemotherapy and radiotherapy. However, these treatments do not much improve the overall survival of GB patients, which is still below two years (the 5-year survival rate is below 7%). Despite various approaches having been followed to increase the release of anticancer drugs into the brain, few of them demonstrated a significant success, as the blood brain barrier (BBB) still restricts its uptake, thus limiting the therapeutic options. Therefore, enormous efforts are being devoted to the development of novel nanomedicines with the ability to cross the BBB and specifically target the cancer cells. In this context, the use of nanoparticles represents a promising non-invasive route, allowing to evade BBB and reducing systemic concentration of drugs and, hence, side effects. In this review, we revise with a critical view the different families of nanoparticles and approaches followed so far with this aim.

Intranasal Administration of Catechol-Based Pt(IV) Coordination Polymer Nanoparticles for Glioblastoma Therapy.

Cisplatin has been described as a potent anticancer agent for decades. However, in the case of glioblastomas, it is only considered a rescue treatment applied after the failure of second-line treatments. Herein, based on the versatility offered by coordination chemistry, we engineered nanoparticles by reaction of a platinum (IV) prodrug and iron metal ions showing in vitro dual pH- and redox-sensitivity, controlled release and comparable cytotoxicity to cisplatin against HeLa and GL261 cells. In vivo intranasal administration in orthotopic preclinical GL261 glioblastoma tumor-bearing mice demonstrated increased accumulation of platinum in tumors, leading in some cases to complete cure and prolonged survival of the tested cohort. This was corroborated by a magnetic resonance imaging follow-up, thus opening new opportunities for intranasal glioblastoma therapies while minimizing side effects. The findings derived from this research showed the potentiality of this approach as a novel therapy for glioblastoma treatment.

Glioblastoma Cells Counteract PARP Inhibition through Pro-Survival Induction of Lipid Droplets Synthesis and Utilization.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. Poly (ADP-ribose) polymerase inhibitors (PARPi) represent a new class of anti-neoplastic drugs. In the current study, we have characterized the mechanism by which glioblastoma cells evade the effect of PARPi as anti-tumor agents. We have found that suppression of PARP activity exerts an anti-stemness effect and has a dual impact on autophagy, inducing its activation in the first 24 h (together with down-regulation of the pro-survival mTOR pathway) and preventing autophagosomes fusion to lysosomes at later time-points, in primary glioma cells. In parallel, PARPi triggered the synthesis of lipid droplets (LDs) through ACC-dependent activation of de novo fatty acids (FA) synthesis. Notably, inhibiting ß-oxidation and blocking FA utilization, increased PARPi-induced glioma cell death while treatment with oleic acid (OA) prevented the anti-glioma effect of PARPi. Moreover, LDs fuel glioma cells by inducing pro-survival lipid consumption as confirmed by quantitation of oxygen consumption rates using Seahorse respirometry in presence or absence of OA. In summary, we uncover a novel mechanism by which glioblastoma escapes to anti-tumor agents through metabolic reprogramming, inducing the synthesis and utilization of LDs as a pro-survival strategy in response to PARP inhibition.

Synthesis and Validation of a Bioinspired Catechol-Functionalized Pt(IV) Prodrug for Preclinical Intranasal Glioblastoma Treatment.

Glioblastoma is the most malignant and frequently occurring type of brain tumors in adults. Its treatment has been greatly hampered by the difficulty to achieve effective therapeutic concentration in the tumor sites due to its location and the blood-brain barrier. Intranasal administration has emerged as an alternative for drug delivery into the brain though mucopenetration, and rapid mucociliary clearance still remains an issue to be solved before its implementation. To address these issues, based on the intriguing properties of proteins secreted by mussels, polyphenol and catechol functionalization has already been used to promote mucopenetration, intranasal delivery and transport across the blood-brain barrier. Thus, herein we report the synthesis and study of complex 1, a Pt(IV) prodrug functionalized with catecholic moieties. This complex considerably augmented solubility in contrast to cisplatin and showed a comparable cytotoxic effect on cisplatin in HeLa, 1Br3G and GL261 cells. Furthermore, preclinical in vivo therapy using the intranasal administration route suggested that it can reach the brain and inhibit the growth of orthotopic GL261 glioblastoma. These results open new opportunities for catechol-bearing anticancer prodrugs in the treatment for brain tumors via intranasal administration.

Gossypol Treatment Restores Insufficient Apoptotic Function of DFF40/CAD in Human Glioblastoma Cells.

Glioblastoma (GBM) is a highly aggressive brain tumor and almost all patients die because of relapses. GBM-derived cells undergo cell death without nuclear fragmentation upon treatment with different apoptotic agents. Nuclear dismantling determines the point-of-no-return in the apoptotic process. DFF40/CAD is the main endonuclease implicated in apoptotic nuclear disassembly. To be properly activated, DFF40/CAD should reside in the cytosol. However, the endonuclease is poorly expressed in the cytosol and remains cumulated in the nucleus of GBM cells. Here, by employing commercial and non-commercial patient-derived GBM cells, we demonstrate that the natural terpenoid aldehyde gossypol prompts DFF40/CAD-dependent nuclear fragmentation. A comparative analysis between gossypol- and staurosporine-treated cells evidenced that levels of neither caspase activation nor DNA damage were correlated with the ability of each compound to induce nuclear fragmentation. Deconvoluted confocal images revealed that DFF40/CAD was almost completely excluded from the nucleus early after the staurosporine challenge. However, gossypol-treated cells maintained DFF40/CAD in the nucleus for longer times, shaping a ribbon-like structure piercing the nuclear fragments and building a network of bridged masses of compacted chromatin. Therefore, GBM cells can fragment their nuclei if treated with the adequate insult, making the cell death process irreversible.

Successful Partnerships: Exploring the Potential of Immunogenic Signals Triggered by TMZ, CX-4945, and Combined Treatment in GL261 Glioblastoma Cells.

BACKGROUND: The relevance of the cancer immune cycle in therapy response implies that successful treatment may trigger the exposure or the release of immunogenic signals. Previous results with the preclinical GL261 glioblastoma (GB) showed that combination treatment of temozolomide (TMZ) + CX-4945 (protein kinase CK2 inhibitor) outperformed single treatments, provided an immune-friendly schedule was followed. Our purpose was to study possible immunogenic signals released in vitro by GB cells. METHODS: GL261 GB cells were treated with TMZ and CX-4945 at different concentrations (25 µM-4 mM) and time frames (12-72 h). Cell viability was measured with Trypan Blue and propidium iodide. Calreticulin exposure was assessed with immunofluorescence, and ATP release was measured with bioluminescence. RESULTS: TMZ showed cytostatic rather than cytotoxic effects, while CX-4945 showed remarkable cytotoxic effects already at low concentrations. Calreticulin exposure after 24 h was detected with TMZ treatment, as well as TMZ/CX-4945 low concentration combined treatment. ATP release was significantly higher with CX-4945, especially at high concentrations, as well as with TMZ/CX-4945. CONCLUSIONS: combined treatment may produce the simultaneous release of two potent immunogenic signals, which can explain the outperformance over single treatments in vivo. A word of caution may be raised since in vitro conditions are not able to mimic pharmacokinetics observed in vivo fully.

Cisplatin-induced peripheral neuropathy is associated with neuronal senescence-like response.

BACKGROUND: Cisplatin-induced peripheral neuropathy (CIPN) is a frequent serious dose-dependent adverse event that can determine dosage limitations for cancer treatment. CIPN severity correlates with the amount of platinum detected in sensory neurons of the dorsal root ganglia (DRG). However, the exact pathophysiology of CIPN is poorly understood, so the chance of developing neuroprotective treatment is reduced. The aim of this study was to determine the exact mechanisms involved in CIPN development. METHODS: By single-cell RNA-sequencing (scRNAseq), we have studied the transcriptomic profile of DRG sensory neurons from a well-characterized neurophysiological mouse model of CIPN. RESULTS: Gene Ontology analysis of the scRNAseq data indicated that cisplatin treatment induces the upregulation of biological pathways related to DNA damage response (DDR) in the DRG neuronal population. Moreover, DRG neurons also upregulated the Cdkn1a gene, confirmed later by the measurement of its protein product p21. While apoptosis activation pathways were not observed in DRG sensory neurons of cisplatin-treated mice, these neurons did express several senescence hallmarks, including senescence-associated ß-galactosidase, phospho-H2AX, and nuclear factor kappa B (Nfkb)-p65 proteins. CONCLUSIONS: In this study, we determined that after cisplatin-induced DNA damage, p21 appears as the most relevant downstream factor of the DDR in DRG sensory neurons in vivo, which survive in a nonfunctional senescence-like state.

Cell Death Triggered by the Autophagy Inhibitory Drug 3-Methyladenine in Growing Conditions Proceeds With DNA Damage.

Macroautophagy (hereafter autophagy) is a multistep intracellular catabolic process with pleiotropic implications in cell fate. Attending to its activation, autophagy can be classified into inducible or constitutive. Constitutive, or basal autophagy, unfolds under nutrient-replete conditions to maintain the cellular homeostasis. Autophagy inhibitory drugs are powerful tools to interrogate the role of autophagy and its consequences on cell fate. However, 3-methyladenine and various of these compounds present an intrinsic capacity to trigger cell death, for instance the broadly-employed 3-methyladenine. To elucidate whether the inhibition of basal autophagy is causative of cell demise, we have employed several representative compounds acting at different phases of the autophagic process: initiation (SBI0206965 and MHY1485), nucleation (3-methyladenine, SAR405, Spautin-1 and Cpd18), and completion (Bafilomycin A1 and Chloroquine). These compounds inhibited the basal autophagy of MEF cultures in growing conditions. Among them, 3-methyladenine, SBI-0206965, Chloroquine, and Bafilomycin A1 triggered BAX- and/or BAK-dependent cytotoxicity and caspase activation. 3-methyladenine was the only compound to induce a consistent and abrupt decrease in cell viability across a series of ontologically unrelated human cell lines. 3-methyladenine-induced cytotoxicity was not driven by the inhibition of the AKT/mTOR axis. Autophagy-deficient Fip200-/- MEFs displayed an increased sensitivity to activate caspases and to undergo cell death in response to 3-methyladenine. The cytotoxicity induced by 3-methyladenine correlated with a massive DNA damage, as shown by γ-H2A.X. This genotoxicity was observed at 10 mM 3-methyladenine, the usual concentration to inhibit autophagy and was maximized in Fip200-/- MEFs. In sum, our results suggest that, in growing conditions, autophagy acts as a protective mechanism to diminish the intrinsic cytotoxicity of 3-methyladenine. However, when the cellular stress exerted by 3-methyladenine surpasses the protective effect of basal autophagy, caspase activation and DNA damage compromise the cell viability.

Autonomic nervous system and cancer.

The autonomic nervous system (ANS) is the main homeostatic regulatory system of the body. However, this widely distributed neural network can be easily affected by cancer and by the adverse events induced by cancer treatments. In this review, we have classified the ANS complications of cancer into two categories. The first includes direct cancer-related complications, such as primary ANS tumors (pheochromocytoma, paraganglioma or neuroblastoma), as well as autonomic manifestations induced by non-primary ANS tumors (primary brain tumors and metastases). The second comprises indirect ANS complications, which include autonomic features related to cancer therapy (chemotherapy, radiotherapy and/or surgery) and those not related to cancer therapy, such as paraneoplastic autonomic syndromes. We also review the molecular relationship and modulation between the ANS and the cancer cells and their microenvironment.

Reducing the Levels of Akt Activation by PDK1 Knock-in Mutation Protects Neuronal Cultures against Synthetic Amyloid-Beta Peptides.

The Akt kinase has been widely assumed for years as a key downstream effector of the PI3K signaling pathway in promoting neuronal survival. This notion was however challenged by the finding that neuronal survival responses were still preserved in mice with reduced Akt activity. Moreover, here we show that the Akt signaling is elevated in the aged brain of two different mice models of Alzheimer Disease. We manipulate the rate of Akt stimulation by employing knock-in mice expressing a mutant form of PDK1 (phosphoinositide-dependent protein kinase 1) with reduced, but not abolished, ability to activate Akt. We found increased membrane localization and activity of the TACE/ADAM17 α-secretase in the brain of the PDK1 mutant mice with concomitant TNFR1 processing, which provided neurons with resistance against TNFα-induced neurotoxicity. Opposite to the Alzheimer Disease transgenic mice, the PDK1 knock-in mice exhibited an age-dependent attenuation of the unfolding protein response, which protected the mutant neurons against endoplasmic reticulum stressors. Moreover, these two mechanisms cooperatively provide the mutant neurons with resistance against amyloid-beta oligomers, and might singularly also contribute to protect these mice against amyloid-beta pathology.

An intrinsic DFF40/CAD endonuclease deficiency impairs oligonucleosomal DNA hydrolysis during caspase-dependent cell death: a common trait in human glioblastoma cells.

BACKGROUND: Glioblastoma (GBM) or grade IV astrocytoma is one of the most devastating human cancers. The loss of DFF40/CAD, the key endonuclease that triggers oligonucleosomal DNA fragmentation during apoptosis, has been linked to genomic instability and cell survival after radiation. Despite the near inevitability of GBM tumor recurrence after treatment, the relationship between DFF40/CAD and GBM remains unexplored. METHODS: We studied the apoptotic behavior of human GBM-derived cells after apoptotic insult. We analyzed caspase activation and the protein levels and subcellular localization of DFF40/CAD apoptotic endonuclease. DFF40/CAD was also evaluated in histological sections from astrocytic tumors and nontumoral human brain. RESULTS: We showed that GBM cells undergo incomplete apoptosis without generating oligonucleosomal DNA degradation despite the correct activation of executioner caspases. The major defect of GBM cells relied on the improper accumulation of DFF40/CAD at the nucleoplasmic subcellular compartment. Supporting this finding, DFF40/CAD overexpression allowed GBM cells to display oligonucleosomal DNA degradation after apoptotic challenge. Moreover, the analysis of histological slices from astrocytic tumors showed that DFF40/CAD immunoreactivity in tumoral GFAP-positive cells was markedly reduced when compared with nontumoral samples. CONCLUSIONS: Our data highlight the low expression levels of DFF40/CAD and the absence of DNA laddering as common molecular traits in GBM. These findings could be of major importance for understanding the malignant behavior of remaining tumor cells after radiochemotherapy.

Autophagy exacerbates caspase-dependent apoptotic cell death after short times of starvation.

Autophagy is generally regarded as a mechanism to promote cell survival. However, autophagy can occasionally be the mechanism responsible of cell demise. We have found that a concomitant depletion of glucose, nutrients and growth factors provoked cell death in a variety of cell lines. This death process was contingent upon caspase activation and was mediated by BAX/BAK proteins, thus indicating its apoptotic nature and the engagement of an intrinsic pathway. In order to abrogate autophagy, 3-methyladenine (3-MA), BECLIN-1 siRNA and Atg5 knock-out (Tet-Off type) approaches were alternatively employed. Irrespective of the procedure, at short times of starvation, we found that the ongoing autophagy was sensitizing cells to the permeabilization of the mitochondrial outer membrane (MOMP), caspase activation and, therefore, apoptosis. On the contrary, at longer times of starvation, autophagy displayed its characteristic pro-survival effect on cells. As far as we know, we provide the first experimental paradigm where time is the only variable determining the final outcome of autophagy. In other words, we have circumscribed in time the shift transforming autophagy from a cell death to a protection mechanism. Moreover, at short times, starvation-driven autophagy exacerbated the apoptotic cell death caused by several antitumor agents. In agreement with this fact, their apoptotic effects were greatly diminished by autophagy inhibition. The implications of these facts in tumor biology will be discussed.

An Early and Robust Activation of Caspases Heads Cells for a Regulated Form of Necrotic-like Cell Death.

Apoptosis is triggered by the activation of caspases and characterized by chromatin condensation and nuclear fragmentation (type II nuclear morphology). Necrosis is depicted by a gain in cell volume (oncosis), swelling of organelles, plasma membrane leakage, and subsequent loss of intracellular contents. Although considered as different cell death entities, there is an overlap between apoptosis and necrosis. In this sense, mounting evidence suggests that both processes can be morphological expressions of a common biochemical network known as "apoptosis-necrosis continuum." To gain insight into the events driving the apoptosis-necrosis continuum, apoptotically proficient cells were screened facing several apoptotic inducers for the absence of type II apoptotic nuclear morphologies. Chelerythrine was selected for further studies based on its cytotoxicity and the lack of apoptotic nuclear alterations. Chelerythrine triggered an early plasma membrane leakage without condensed chromatin aggregates. Ultrastructural analysis revealed that chelerythrine-mediated cytotoxicity was compatible with a necrotic-like type of cell death. Biochemically, chelerythrine induced the activation of caspases. Moreover, the inhibition of caspases prevented chelerythrine-triggered necrotic-like cell death. Compared with staurosporine, chelerythrine induced stronger caspase activation detectable at earlier times. After using a battery of chemicals, we found that high concentrations of thiolic antioxidants fully prevented chelerythrine-driven caspase activation and necrotic-like cell death. Lower amounts of thiolic antioxidants partially prevented chelerythrine-mediated cytotoxicity and allowed cells to display type II apoptotic nuclear morphology correlating with a delay in caspase-3 activation. Altogether, these data support that an early and pronounced activation of caspases can drive cells to undergo a form of necrotic-like regulated cell death.

AChE for DNA degradation.

DNA hydrolysis is a biochemical process often associated with different forms of cell death, including apoptosis. In a recent paper published in Cell Discovery, Du et al. report that synaptic acetylcholinesterase (AChE-S) shows an unexpected enzymatic activity as DNase switched on after cytotoxic insults.

2-Phenylethynesulfonamide (PES) uncovers a necrotic process regulated by oxidative stress and p53.

2-Phenylethynesulfonamide (PES) or pifithrin-µ is a promising anticancer agent with preferential toxicity for cancer cells. The type of cell death and the molecular cascades activated by this compound are controversial. Here, we demonstrate PES elicits a caspase- and BAX/BAK-independent non-necroptotic necrotic cell death, since it is not inhibited by necrostatin-1. This process is characterized by an early generation of reactive oxygen species (ROS) resulting in p53 up-regulation. Accordingly, thiolic antioxidants protect cells from PES-induced death. Furthermore, inhibiting the natural sources of glutathione with l-buthionine-sulfoximine (BSO) strongly cooperates with PES in triggering cytotoxicity. Genetically modified p53-null or p53 knocked-down cells show resistance to PES-driven necrosis. The predominant localization of p53 in chromatin-enriched fractions added to the up-regulation of the p53-responsive gene p21, strongly suggest the involvement of a transcription-dependent p53 program. On the other hand, we report an augmented production of ROS in p53-positive cells that, added to the increased p53 content in response to PES-elicited ROS, suggests that p53 and ROS are mutually regulated in response to PES. In sum, p53 up-regulation by ROS triggers a positive feedback loop responsible of further increasing ROS production and reinforcing PES-driven non-necroptotic necrosis.

Caspase-activated DNase is necessary and sufficient for oligonucleosomal DNA breakdown, but not for chromatin disassembly during caspase-dependent apoptosis of LN-18 glioblastoma cells.

Caspase-dependent apoptosis is a controlled type of cell death characterized by oligonucleosomal DNA breakdown and major nuclear morphological alterations. Other kinds of cell death do not share these highly distinctive traits because caspase-activated DNase (DFF40/CAD) remains inactive. Here, we report that human glioblastoma multiforme-derived LN-18 cells do not hydrolyze DNA into oligonucleosomal fragments after apoptotic insult. Furthermore, their chromatin remains packaged into a single mass, with no signs of nuclear fragmentation. However, ultrastructural analysis reveals that nuclear disassembly occurs, although compacted chromatin does not localize into apoptotic nuclear bodies. Caspases become properly activated, and ICAD, the inhibitor of DFF40/CAD, is correctly processed. Using cell-free in vitro assays, we show that chromatin from isolated nuclei of LN-18 cells is suitable for hydrolysis into oligonuclesomal fragments by staurosporine-pretreated SH-SY5Y cytoplasms. However, staurosporine-pretreated LN-18 cytoplasms do not induce DNA laddering in isolated nuclei from either LN-18 or SH-SY5Y cells because LN-18 cells express lower amounts of DFF40/CAD. DFF40/CAD overexpression makes LN-18 cells fully competent to degrade their DNA into oligonucleosome-sized fragments, and yet they remain unable to arrange their chromatin into nuclear clumps after apoptotic insult. Indeed, isolated nuclei from LN-18 cells were resistant to undergoing apoptotic nuclear morphology in vitro. The use of LN-18 cells has uncovered a previously unsuspected cellular model, whereby a caspase-dependent chromatin package is DFF40/CAD-independent, and DFF40/CAD-mediated double-strand DNA fragmentation does not warrant the distribution of the chromatin into apoptotic nuclear bodies. The studies highlight a not-yet reported DFF40/CAD-independent mechanism driving conformational nuclear changes during caspase-dependent cell death.