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.

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.

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.

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.

Early apoptotic reorganization of spliceosomal proteins involves caspases, CAD and rearrangement of NuMA.

The reorganization of nuclear structures is an important early feature of apoptosis and involves the activity of specific proteases and nucleases. Well-known is the condensation and fragmentation of chromatin; however, much less is understood about the mechanisms involved in the reorganization of structures from the interchromatin space, such as interchromatin granule clusters (IGCs). In this study, we show that the initial enlargement and rounding-up of IGCs correlate with a decrease in mRNA transcription and are caspase-independent, but involve protein phosphatases PP1/PP2A. Subsequently, multiple enlarged IGCs dissociate from chromatin and fuse into a single structure. The dissociation requires caspase activity and involves caspase-activated DNase (CAD). Apoptotic IMR-5 cells, lacking a proper processing of CAD, show multiple enlarged IGCs that remain linked with chromatin. Overexpression of CAD in IMR-5 cells results in the dissociation of IGCs from chromatin, but the fusion into a single structure remains disturbed. Nuclear matrix protein NuMA is reorganized in a caspase-dependent way around fused IGCs. In conclusion, we show here that the apoptotic rearrangement of IGCs, the nuclear matrix and chromatin are closely associated, occur in defined stages and depend on the activity of protein phosphatases, caspases and CAD.

FAIM-L is an IAP-binding protein that inhibits XIAP ubiquitinylation and protects from Fas-induced apoptosis.

The neuronal long isoform of Fas Apoptotic Inhibitory Molecule (FAIM-L) protects from death receptor (DR)-induced apoptosis, yet its mechanism of protection remains unknown. Here, we show that FAIM-L protects rat neuronal Type II cells from Fas-induced apoptosis. XIAP has previously emerged as a molecular discriminator that is upregulated in Type II and downregulated in Type I apoptotic signaling. We demonstrate that FAIM-L requires sustained endogenous levels of XIAP to protect Type II cells as well as murine cortical neurons from Fas-induced apoptosis. FAIM-L interacts with the BIR2 domain of XIAP through an IAP-binding motif, the mutation of which impairs the antiapoptotic function of FAIM-L. Finally, we report that FAIM-L inhibits XIAP auto-ubiquitinylation and maintains its stability, thus conferring protection from apoptosis. Our results bring new understanding of the regulation of endogenous XIAP by a DR antagonist, pointing out at FAIM-L as a promising therapeutic tool for protection from apoptosis in pathological situations where XIAP levels are decreased.

Chromatin collapse during caspase-dependent apoptotic cell death requires DNA fragmentation factor, 40-kDa subunit-/caspase-activated deoxyribonuclease-mediated 3'-OH single-strand DNA breaks.

Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD(-/-) cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3'-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3'-OH ends in single-strand rather than double-strand DNA nicks/breaks.

NF-κB activation fails to protect cells to TNFα-induced apoptosis in the absence of Bcl-xL, but not Mcl-1, Bcl-2 or Bcl-w.

TNFα can promote either cell survival or cell death. The activation of NF-κB plays a central role in cell survival while its inhibition makes TNFα-triggered cytotoxicity possible. Here, we report that the overexpression of a non-degradable mutant of the inhibitor of NF-κB (super-repressor (SR)-IκBα) sensitizes HeLa cells towards TNFα-induced apoptosis, involving caspases activation and cytocrome C release from the mitochondria. Interestingly, we describe that the specific knockdown of Bcl-xL, but not that of Bcl-2, Bcl-w or Mcl-1, renders cells sensitive to TNFα-induced apoptosis. This cytotoxic effect occurs without altering the activation of NF-κB. Then, the activation of the NF-κB pathway is not sufficient to protect Bcl-xL-downregulated cells from TNFα-induced cell death, meaning that TNFα is not able to promote cell survival in the absence of Bcl-xL. In addition, Bcl-xL silencing does not potentiate the cytotoxicity afforded by the cytokine in SR-IκBα-overexpressing cells. This indicates that TNFα-induced apoptosis in SR-IκBα-overexpressing cells relies on the protein levels of Bcl-xL. We have corroborated these findings using RD and DU-145 cells, which also become sensitive to TNFα-induced apoptosis after Bcl-xL knockdown despite that NF-κB remains activated. Altogether, our results point out that the impairment of the anti-apoptotic function of Bcl-xL should make cells sensitive towards external insults circumventing the TNFα-triggered NF-κB-mediated cytoprotective effect. Hence, the specific inhibition of Bcl-xL could be envisaged as a promising alternative strategy against NF-κB-dependent highly chemoresistant proliferative malignancies.

AIF promotes chromatinolysis and caspase-independent programmed necrosis by interacting with histone H2AX.

Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase-independent mode of cell death mediated by apoptosis-inducing factor (AIF). After poly(ADP-ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C-terminal proline-rich binding domain (PBD, residues 543-559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA-degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C-terminal PBD abolishes AIF/H2AX interaction and AIF-mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA-deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase-independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif.

Apoptotic DNA degradation into oligonucleosomal fragments, but not apoptotic nuclear morphology, relies on a cytosolic pool of DFF40/CAD endonuclease.

Apoptotic cell death is characterized by nuclear fragmentation and oligonucleosomal DNA degradation, mediated by the caspase-dependent specific activation of DFF40/CAD endonuclease. Here, we describe how, upon apoptotic stimuli, SK-N-AS human neuroblastoma-derived cells show apoptotic nuclear morphology without displaying concomitant internucleosomal DNA fragmentation. Cytotoxicity afforded after staurosporine treatment is comparable with that obtained in SH-SY5Y cells, which exhibit a complete apoptotic phenotype. SK-N-AS cell death is a caspase-dependent process that can be impaired by the pan-caspase inhibitor q-VD-OPh. The endogenous inhibitor of DFF40/CAD, ICAD, is correctly processed, and dff40/cad cDNA sequence does not reveal mutations altering its amino acid composition. Biochemical approaches show that both SH-SY5Y and SK-N-AS resting cells express comparable levels of DFF40/CAD. However, the endonuclease is poorly expressed in the cytosolic fraction of healthy SK-N-AS cells. Despite this differential subcellular distribution of DFF40/CAD, we find no differences in the subcellular localization of both pro-caspase-3 and ICAD between the analyzed cell lines. After staurosporine treatment, the preferential processing of ICAD in the cytosolic fraction allows the translocation of DFF40/CAD from this fraction to a chromatin-enriched one. Therefore, the low levels of cytosolic DFF40/CAD detected in SK-N-AS cells determine the absence of DNA laddering after staurosporine treatment. In these cells DFF40/CAD cytosolic levels can be restored by the overexpression of their own endonuclease, which is sufficient to make them proficient at degrading their chromatin into oligonucleosome-size fragments after staurosporine treatment. Altogether, the cytosolic levels of DFF40/CAD are determinants in achieving a complete apoptotic phenotype, including oligonucleosomal DNA degradation.

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.

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.

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.

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.

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.

The death receptor antagonist FLIP-L interacts with Trk and is necessary for neurite outgrowth induced by neurotrophins.

FLICE-inhibitory protein (FLIP) is an endogenous inhibitor of the signaling pathway triggered by the activation of death receptors. Here, we reveal a novel biological function for the long form of FLIP (FLIP-L) in neuronal differentiation, which can be dissociated from its antiapoptotic role. We show that FLIP-L is expressed in different regions of the mouse embryonic nervous system. Immunohistochemistry of mouse brain sections at different stages reveals that, in neurons, FLIP is expressed early during the embryonic neuronal development (embryonic day 16) and decreases at later stages (postnatal days 5-15), when its expression is essentially detected in glial cells. FLIP-L overexpression significantly enhances neurotrophin-induced neurite outgrowth in motoneurons, superior cervical ganglion neurons, and PC12 cells. Conversely, the downregulation of FLIP-L protein levels by specific RNA interference significantly reduces neurite outgrowth, even in the presence of the appropriate neurotrophin stimulus. Moreover, NGF-dependent activation of two main intracellular pathways involved in the regulation of neurite outgrowth, extracellular signal-regulated kinases (ERKs) and nuclear factor kappaB (NF-kappaB), is impaired when endogenous FLIP-L is downregulated, although TrkA remains activated. Finally, we demonstrate that FLIP-L interacts with TrkA, and not with p75(NTR), in an NGF-dependent manner, and endogenous FLIP-L interacts with TrkB in whole-brain lysates from embryonic day 15 mice embryos. Altogether, we uncover a new role for FLIP-L as an unexpected critical player in neurotrophin-induced mitogen-activated protein kinase/ERK- and NF-kappaB-mediated control of neurite growth in developing neurons.

Methadone induces CAD degradation and AIF-mediated necrotic-like cell death in neuroblastoma cells.

Methadone (d,l-methadone hydrochloride) is a full-opioid agonist, originally developed as a substitution for heroin or other opiates abusers. Nowadays methadone is also being applied as long-lasting analgesics in cancer, and it is proposed as a promising agent for leukemia therapy. Previously, we have demonstrated that high concentrations of methadone (0.5mM) induced necrotic-like cell death in SH-SY5Y cells. The pathway involved is caspase-independent but involves impairment of mitochondrial ATP synthesis and mitochondrial cytochrome c release. However, the downstream mitochondrial pathways remained unclear. Here, we studied the participation of apoptosis inducing factor (AIF) in methadone-induced cell death. Methadone resulted in a translocation of AIF from mitochondria to the nucleus. Translocation was inhibited by cyclosporine A, but not by lack of Bax protein. Therefore the effect seems mediated by the formation of the mitochondrial transition pore, but is apparently independent of Bax. Furthermore, methadone-treated SH-SY5Y nuclei show characteristics that are typical for stage I nuclear condensation. Methadone did not induce degradation of DNA into oligonucleosomal fragments or into high molecular weight DNA fragments. Absence of DNA fragmentation coincided with a considerable decrease in the levels of the caspase-actived endonuclase DNase and its chaperone-inhibitor ICAD. In conclusion, our results provide mechanistic insights into the molecular mechanisms that underlie methadone-induced cell death. This knowledge may prove useful to develop novel strategies to prevent toxic side-effects of methadone thereby sustaining its use as therapeutical agent against tumors.

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.

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.