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Breast cancer is still the most common cancer in women worldwide. Resistance to drugs and recurrence of the disease are two leading causes of failure in treatment. For a more efficient treatment of patients, the development of novel therapeutic regimes is needed. Recent studies indicate that modulation of autophagy in concert with apoptosis induction may provide a promising novel strategy in breast cancer treatment. Apoptosis and autophagy are two tightly regulated distinct cellular processes. To maintain tissue homeostasis abnormal cells are disposed largely by means of apoptosis. Autophagy, however, contributes to tissue homeostasis and cell fitness by scavenging of damaged organelles, lipids, proteins, and DNA. Defects in autophagy promote tumorigenesis, whereas upon tumor formation rapidly proliferating cancer cells may rely on autophagy to survive. Given that evasion of apoptosis is one of the characteristic hallmarks of cancer cells, inhibiting autophagy and promoting apoptosis can negatively influence cancer cell survival and increase cell death. Hence, combination of antiautophagic agents with the enhancement of apoptosis may restore apoptosis and provide a therapeutic advantage against breast cancer. In this review, we discuss the cross-talk of autophagy and apoptosis and the diverse facets of autophagy in breast cancer cells leading to novel models for more effective therapeutic strategies.

The extrinsic pathway is mediated by death receptors (DRs), including CD95 (APO-1/Fas) or TRAILR-1/2. Defects in apoptosis regulation lead to cancer and other malignancies. The master regulator of the DR networks is the cellular FLICE inhibitory protein (c-FLIP). In addition to its key role in apoptosis, c-FLIP may exert other cellular functions, including control of necroptosis, pyroptosis, nuclear factor κB (NF-κB) activation, and tumorigenesis. To gain further insight into the molecular mechanisms of c-FLIP action in cancer networks, we focus on the structure, isoforms, interactions, and post-translational modifications of c-FLIP. We also discuss various avenues to target c-FLIP in cancer cells for therapeutic benefit.

CD95/Fas/APO-1 can trigger apoptotic as well as nonapoptotic pathways in immune cells. CD95 signaling in humans can be inhibited by several mechanisms, including mutations in the gene encoding CD95. CD95 mutations lead to autoimmune disorders, such as autoimmune lymphoproliferative syndrome (ALPS). Gaining further insight into the reported mutations of CD95 and resulting alterations of its signaling networks may provide further understanding of their presumed role in certain autoimmune diseases. For illustrative purposes and to better understand the potential outcomes of CD95 mutations, here we assign their positions to the recently determined 3D structures of human CD95. Based on this, we make certain predictions and speculate on the putative role of CD95 mutation defects in CD95-mediated signaling for certain autoimmune diseases.

The naked mole rat (NMR), Heterocephalus glaber, is the longest-living rodent species, and is extraordinarily resistant to cancer and aging-related diseases. The molecular basis for these unique phenotypic traits of the NMR is under extensive research. However, the role of regulated cell death (RCD) in the longevity and the protection from cancer in the NMR is still largely unknown. RCD is a mechanism restricting the proliferation of damaged or premalignant cells, which counteracts aging and oncotransformation. In this study, DNA damage-induced cell death in NMR fibroblasts was investigated in comparison to RCD in fibroblasts from Mus musculus. The effects of methyl methanesulfonate, 5-fluorouracil, and etoposide in both cell types were examined using contemporary cell death analyses. Skin fibroblasts from Heterocephalus glaber were found to be more resistant to the action of DNA damaging agents compared to fibroblasts from Mus musculus. Strikingly, our results revealed that NMR cells also exhibit a limited apoptotic response and seem to undergo regulated necrosis. Taken together, this study provides new insights into the mechanisms of cell death in NMR expanding our understanding of longevity, and it paves the way towards the development of innovative therapeutic approaches.

Apoptosis is a form of programmed cell death, deregulation of which occurs in multiple disorders, including neurodegenerative and autoimmune diseases as well as cancer. The formation of a death-inducing signaling complex (DISC) and death effector domain (DED) filaments are critical for initiation of the extrinsic apoptotic pathway. Post-translational modifications (PTMs) of DED-containing DISC components such as FADD, procaspase-8, and c-FLIP comprise an additional level of apoptosis regulation, which is necessary to overcome the threshold for apoptosis induction. In this review we discuss the influence of PTMs of FADD, procaspase-8, and c-FLIP on DED filament assembly and cell death induction, with a focus on the 3D organization of the DED filament.

Pharmacological targeting via small molecule-based chemical probes has recently acquired an emerging importance as a valuable tool to delineate molecular mechanisms. Induction of apoptosis via CD95/Fas and TRAIL-R1/2 is triggered by the formation of the death-inducing signaling complex (DISC). Caspase-8 activation at the DISC is largely controlled by c-FLIP proteins. However molecular mechanisms of this control have just started to be uncovered. In this study we report the first-in-class chemical probe targeting c-FLIPL in the heterodimer caspase-8/c-FLIPL. This rationally designed small molecule was aimed to imitate the closed conformation of the caspase-8 L2′ loop and thereby increase caspase-8 activity after initial processing of the heterodimer. In accordance with in silico predictions, this small molecule enhanced caspase-8 activity at the DISC, CD95L/TRAIL-induced caspase activation, and subsequent apoptosis. The generated computational model provided further evidence for the proposed effects of the small molecule on the heterodimer caspase-8/c-FLIPL. In particular, the model has demonstrated that boosting caspase-8 activity by the small molecule at the early time points after DISC assembly is crucial for promoting apoptosis induction. Taken together, our study allowed to target the heterodimer caspase-8/c-FLIPL and get new insights into molecular mechanisms of its activation.

The assembly of the death-inducing signaling complex (DISC) and death effector domain (DED) filaments at CD95/Fas initiates extrinsic apoptosis. Procaspase-8 activation at the DED filaments is controlled by short and long c-FLIP isoforms. Despite apparent progress in understanding the assembly of CD95-activated platforms and DED filaments, the detailed molecular mechanism of c-FLIP action remains elusive. Here, we further addressed the mechanisms of c-FLIP action at the DISC using biochemical assays, quantitative mass spectrometry, and structural modeling. Our data strongly indicate that c-FLIP can bind to both FADD and procaspase-8 at the DED filament. Moreover, the constructed in silico model shows that c-FLIP proteins can lead to the formation of the DISCs comprising short DED filaments as well as serve as bridging motifs for building a cooperative DISC network, in which adjacent CD95 DISCs are connected by DED filaments. This network is based on selective interactions of FADD with both c-FLIP and procaspase-8. Hence, c-FLIP proteins at the DISC control initiation, elongation, and composition of DED filaments, playing the role of control checkpoints. These findings provide new insights into DISC and DED filament regulation and open innovative possibilities for targeting the extrinsic apoptosis pathway.

CD95 is a member of the death receptor family and is well-known to promote apoptosis. However, accumulating evidence indicates that in some context CD95 has not only the potential to induce apoptosis but also can trigger non-apoptotic signal leading to cell survival, proliferation, cancer growth and metastasis. Despite extensive investigations focused on alterations in the expression level of CD95 and associated signal molecules, very few studies, however, have investigated the effects of post-translational modifications such as glycosylation, phosphorylation, palmitoylation, nitrosylation and glutathionylation on CD95 function. Post-translational modifications of CD95 in mammalian systems are likely to play a more prominent role than anticipated in CD95 induced cell death. In this review we will focus on the alterations in CD95-mediated signaling caused by post-translational modifications of CD95.

Mutations of fms-like tyrosine kinase 3 (FLT3) are the most frequent mutations in acute myeloid leukemia (AML). Furthermore, the internal tandem duplication (ITD) represents the most common mutation of FLT3 in AML. To explore therapeutic strategies for AML patients carrying FLT3-ITD, we analyzed death receptor (DR) signaling networks in AML cells comprising FLT3-ITD. We have started with murine myeloid progenitor 32D cells that ectopically express human FLT3-ITD (32D- FLT3-ITD) and found that RIPK1 is strongly upregulated in these cells. Subsequently, we have shown that combinatorial treatment of 32D-FLT3-ITD cells with the SMAC mimetic BV6 and CD95L sensitizes these cells toward apoptosis and necroptosis. Moreover, combinatorial treatment with death ligands (DLs), for example, CD95L or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and BV6 enhanced cell death in primary AML blasts from patients carrying FLT3-ITD mutation. Finally, pharmacological and genetic targeting of RIPK1 inhibited DL/BV6-mediated cell death in cells with FLT3-ITD mutations. Taken together, our study suggests a promising therapeutic opportunity for AML cancer cells harboring FLT3-ITD mutation via targeting RIPK1 pathways.

Glycosylation and glycan-binding proteins such as galectins play an important role in the control of cell death signaling. Strikingly, very little attention has been given so far to the understanding of the molecular details behind this key regulatory network. Glycans attached to the death receptors such as CD95 and TRAIL-Rs, either alone or in a complex with galectins, might promote or inhibit apoptotic signals. However, we have just started to decode the functions of galectins in the modulation of extrinsic and intrinsic apoptosis. In this work, we have discussed the current understanding of the glycosylation–galectin regulatory network in CD95- as well as TRAIL-R-induced apoptosis and therapeutic strategies based on targeting galectins in cancer.

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (TRAIL-R1/R2). Stimulation of these receptors with their cognate ligands leads to the assembly of the death-inducing signaling complex (DISC). DISC comprises DR, the adaptor protein Fas-associated protein with death domain (FADD), procaspases-8/-10, and cellular FADD-like interleukin (IL)-1β-converting enzyme-inhibitory proteins (c-FLIPs). The DISC serves as a platform for procaspase-8 processing and activation. The latter occurs via its dimerization/oligomerization in the death effector domain (DED) filaments assembled at the DISC. Activation of procaspase-8 is followed by its processing, which occurs in several steps. In this work, an established experimental workflow is described that allows the measurement of DISC formation and the processing of procaspase-8 in this complex. The workflow is based on immunoprecipitation techniques supported by western blot analysis. This workflow allows careful monitoring of different steps of procaspase-8 recruitment to the DISC and its processing and is highly relevant for investigating molecular mechanisms of extrinsic apoptosis.

A recombinant fragment of human κ-Casein, termed RL2, induces cell death of breast cancer cells; however, molecular mechanisms of RL2-mediated cell death have remained largely unknown. In the current study, we have decoded the molecular mechanism of the RL2-mediated cell death and found that RL2 acts via the induction of mitophagy. This was monitored by the loss of adenosine triphosphate production, LC3B-II generation, and upregulation of BNIP3 and BNIP3L/NIX, as well as phosphatase and tensin homolog-induced kinase 1. Moreover, we have analyzed the cross talk of this pathway with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis upon combinatorial treatment with RL2 and TRAIL. Strikingly, we found two opposite effects of this co-treatment. RL2 had inhibitory effects on TRAIL-induced cell death upon short-term co-stimulation. In particular, RL2 treatment blocked TRAIL-mediated caspase activation, cell viability loss, and apoptosis, which was mediated via the downregulation of the core proapoptotic regulators. Contrary to short-term co-treatment, upon long-term co-stimulation, RL2 sensitized the cells toward TRAIL-induced cell death; the latter observation provides the basis for the development of therapeutic approaches in breast cancer cells. Collectively, our findings have important implications for cancer therapy and reveal the molecular switches of the cross talk between RL2-induced mitophagy and TRAIL-mediated apoptosis.

Cell fate decisions regulating survival and death are essential for maintaining tissue homeostasis; dysregulation thereof can lead to tumor development. In some cases, survival and death are triggered by the same receptor, e.g., tumor necrosis factor (TNF)-receptor 1 (TNFR1). We identified a prominent role for the cold shock Y-box binding protein-1 (YB-1) in the TNF-induced activation and nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65. In the absence of YB-1, the expression of TNF receptor-associated factor 2 (TRAF2), a central component of the TNF receptor signaling complex required for NF-κB activation, is significantly reduced. Therefore, we hypothesized that the loss of YB-1 results in a destabilization of TRAF2. Consistent with this hypothesis, we observed that YB-1-deficient cells were more prone to TNF-induced apoptotic cell death. We observed enhanced effector caspase-3 activation and could successfully rescue the cells using the pan-caspase inhibitor zVAD-fmk, but not necrostatin-1. Taken together, our results indicate that YB-1 plays a central role in promoting cell survival through NF-κB activation and identifies a novel mechanism by which enhanced YB-1 expression may contribute to tumor development.

Caspase-8 performs initiatory functions during the induction of apoptosis through the extrinsic pathway. Apoptosis is a type of programmed cell death that plays an important role in regulating embryogenesis and maintaining homeostasis in the tissue of an adult organism, as well as differentiating and removing damaged cells. Dysregulation of the apoptosis mechanisms is associated with the pathogenesis and progression of a number of oncological and neurodegenerative diseases. Caspase-8 (also called СAP4, FLICE, MACH, MCH5) is one of two members of the death effector domain (DED)-containing caspases. Despite the fact that the role of caspase-8 in apoptosis has been well known since the mid 1990s, we are only now beginning to understand the subtle mechanisms of its activation and regulation in response to the activation of death receptors (DRs). In particular, it was demonstrated that the activation of caspase-8 requires the formation of specific oligomeric structures, which are named DED filaments. In this review, the recent data on the mechanisms of activating initiator caspase-8 in DED filaments are considered that allow us to better understand the subtle mechanisms of the initiation of the programmed cell death.

The cleavage of nuclear proteins by caspases promotes nuclear breakdown and, therefore, plays a key role in apoptosis execution. However, the detailed molecular mechanisms of these events remain unclear. To get more insights into the mechanisms of nuclear events during apoptosis we set up a rapid fractionation protocol for the separation of the cytoplasmic and nuclear fractions of cells undergoing cisplatin-induced apoptosis. Importantly, nuclear accumulation of effector caspase-3 as well as initiator caspase-2, -8 and -9 was observed using the developed protocol and immunofluorescence microscopy. The detection of caspases and their cleavage products in the nucleus occurred within the same time interval after cisplatin treatment and took place shortly before nuclear fragmentation. The entry of initiator caspases to the nucleus was independent of caspase-3. Given that all three initiator caspases had catalytic activity in the nuclei, our findings indicate that initiator caspases might participate in the proteolysis of nuclear components during apoptosis, promoting its disintegration and apoptotic cell death.

Programmed necrosis or necroptosis plays an important role in cell physiology. Disturbances in necroptotic process are associated with excessive cell death, the development of a number of pathological conditions, including inflammatory and neurodegenerative diseases. Accumulated evidences suggest the involvement of necroptosis in the induction of stem cell proliferation and tissue regeneration. The necrotic death can be triggered through the family of receptors of tumor necrosis factor, TRAILR1/2, FAS, as well as endosomal Toll-like and NOD-like receptors. An important role in the regulation of necroptosis belongs to proteins RIPK1 and RIPK3, which also might be essential for proliferation of stem cells and the regeneration process. Recent study has shown that necroptosis can lead to rapid activation of progenitor cells and regeneration of the hepatic tissues, as well as a necrotic-induced tissue regeneration and differentiation of c-kit+ cells in a model of myocardial infarction. Thus, the investigation of interplay between necroptosis and regeneration of damaged tissues will allow us to understand the fundamental aspects of programmed cell death and cell division.

Background Hypertension and bronchial asthma are a major issue for people’s health. As of 2014, approximately one billion adults, or ~ 22% of the world population, have had hypertension. As of 2011, 235–330 million people globally have been affected by asthma and approximately 250,000–345,000 people have died each year from the disease. The development of the effective treatment therapies against these diseases is complicated by their comorbidity features. This is often a major problem in diagnosis and their treatment. Hence, in this study the bioinformatical methodology for the analysis of the comorbidity of these two diseases have been developed. As such, the search for candidate genes related to the comorbid conditions of asthma and hypertension can help in elucidating the molecular mechanisms underlying the comorbid condition of these two diseases, and can also be useful for genotyping and identifying new drug targets. Results Using ANDSystem, the reconstruction and analysis of gene networks associated with asthma and hypertension was carried out. The gene network of asthma included 755 genes/proteins and 62,603 interactions, while the gene network of hypertension - 713 genes/proteins and 45,479 interactions. Two hundred and five genes/proteins and 9638 interactions were shared between asthma and hypertension. An approach for ranking genes implicated in the comorbid condition of two diseases was proposed. The approach is based on nine criteria for ranking genes by their importance, including standard methods of gene prioritization (Endeavor, ToppGene) as well as original criteria that take into account the characteristics of an associative gene network and the presence of known polymorphisms in the analysed genes. According to the proposed approach, the genes IL10, TLR4, and CAT had the highest priority in the development of comorbidity of these two diseases. Additionally, it was revealed that the list of top genes is enriched with apoptotic genes and genes involved in biological processes related to the functioning of central nervous system. Conclusions The application of methods of reconstruction and analysis of gene networks is a productive tool for studying the molecular mechanisms of comorbid conditions. The method put forth to rank genes by their importance to the comorbid condition of asthma and hypertension was employed that resulted in prediction of 10 genes, playing the key role in the development of the comorbid condition. The results can be utilised to plan experiments for identification of novel candidate genes along with searching for novel pharmacological targets. Electronic supplementary material The online version of this article (10.1186/s12920-018-0331-4) contains supplementary material, which is available to authorized users.

Programmed cell death is governed by a set of gene networks, which define a variety of distinct molecular mechanisms essential for the maintenance of multicellular organisms. The most studied modality of programmed cell death is known as apoptosis. Caspase-2, as a member of the family of the cysteine-dependent protease, demonstrates both proapoptotic and tumor suppressive functions. This protease plays an essential role in the maintenance of genomic stability and induces apoptotic cell death in response to geno-toxic stress. Here we discuss the molecular mechanisms of caspase-2 regulation and its physiological role as a tumor suppressor and metabolic regulator.

Glaucoma is a chronic progressive disease. It involves more than 60 million people worldwide. Primary open-angle glaucoma (POAG) is one of its commonest forms. About 2.71 million people in the United States suffered from POAG in 2011. Currently, POAG is a major cause of irreversible vision loss. The risk of blindness in patients with treated open-angle glaucoma is 27%. It is known that the death of optic nerve cells can be triggered by mechanical stress caused by ocular hypertension, which induces neuronal apoptosis and occurs in patients with POAG. Many scientific publications are dedicated to proteins and genes involved in the development of POAG, including neuronal apoptosis and the cell response to mechanical stress (CRMS). However, the molecular mechanisms underlying the pathophysiology of POAG are still poorly understood. The reconstruction of associative networks describing the functional interactions between these genes/proteins, including biochemical reactions, regulatory interactions, and transport, requires automated knowledge extraction from scientific publications. This work aims to analyze the associative networks describing molecular interactions between proteins and genes involved in CRMS, neuronal apoptosis, and the development of POAG. It has been shown that genes associated with POAG are statistically significantly overrepresented among the genes involved in the interactions between CRMS and neuronal apoptosis in comparison to what is expected on a random basis. This finding may explain how POAG causes the death of the retinal ganglion cell.

The human pathogen Helicobacter pylori infects more than half of the world's population and is a paradigm for persistent yet asymptomatic infection but increases the risk for chronic gastritis and gastric adenocarcinoma. For successful colonization, H. pylori needs to subvert the host cell death response, which serves to confine pathogen infection by killing infected cells and preventing malignant transformation. Infection of gastric epithelial cells by H. pylori provokes direct and fast activation of the proinflammatory and survival factor NF-κB, which regulates target genes, such as CXCL8, BIRC3 and TNFAIP3. However, it is not known how H. pylori exploits NF-κB activation and suppresses the inflammatory response and host apoptotic cell death, in order to avert the innate immune response and avoid cell loss, and thereby enhance colonization to establish long-term infection. Here we assign for the first time that H. pylori and also Campylobacter jejuni-induced ubiquitin-editing enzyme A20 bifunctionally terminates NF-κB activity and negatively regulates apoptotic cell death. Mechanistically, we show that the deubiquitinylase activity of A20 counteracts cullin3-mediated K63-linked ubiquitinylation of procaspase-8, therefore restricting the activity of caspase-8. Interestingly, another inducible NF-κB target gene, the scaffold protein p62, ameliorates the interaction of A20 with procaspase-8. In conclusion, pathogen-induced de novo synthesis of A20 regulates the shut-off of the survival factor NF-κB but, on the other hand, also impedes caspase-8-dependent apoptotic cell death so as to promote the persistence of pathogens.Cell Death and Differentiation advance online publication, 2 June 2017; doi:10.1038/cdd.2017.89.

Background Structural homology modeling supported by bioinformatics analysis plays a key role in uncovering new molecular interactions within gene regulatory networks. Here, we have applied this powerful approach to analyze the molecular interactions orchestrating death receptor signaling networks. In particular, we focused on the molecular mechanisms of CD95-mediated NF-κB activation and the role of c-FLIP/NEMO interaction in the induction of this pathway. Results To this end, we have created the homology model of the c-FLIP/NEMO complex using the reported structure of the v-FLIP/NEMO complex, and rationally designed peptides targeting this complex. The designed peptides were based on the NEMO structure. Strikingly, the experimental in vitro validation demonstrated that the best inhibitory effects on CD95-mediated NF-κB activation are exhibited by the NEMO-derived peptides with the substitution D242Y of NEMO. Furthermore, we have assumed that the c-FLIP/NEMO complex is recruited to the DED filaments formed upon CD95 activation and validated this assumption in silico. Further insight into the function of c-FLIP/NEMO complex was provided by the analysis of evolutionary conservation of interacting regions which demonstrated that this interaction is common in distinct mammalian species. Conclusions Taken together, using a combination of bioinformatics and experimental approaches we obtained new insights into CD95-mediated NF-κB activation, providing manifold possibilities for targeting the death receptor network.

The assembly of the death-inducing signaling complex (DISC) and death effector domain (DED) filaments at CD95/Fas initiates extrinsic apoptosis. Procaspase-8 activation at the DED filaments is controlled by short and long c-FLIP isoforms. Despite apparent progress in understanding the assembly of CD95-activated platforms and DED filaments, the detailed molecular mechanism of c-FLIP action remains elusive. Here, we further addressed the mechanisms of c-FLIP action at the DISC using biochemical assays, quantitative mass spectrometry, and structural modeling. Our data strongly indicate that c-FLIP can bind to both FADD and procaspase-8 at the DED filament. Moreover, the constructed in silico model shows that c-FLIP proteins can lead to the formation of the DISCs comprising short DED filaments as well as serve as bridging motifs for building a cooperative DISC network, in which adjacent CD95 DISCs are connected by DED filaments. This network is based on selective interactions of FADD with both c-FLIP and procaspase-8. Hence, c-FLIP proteins at the DISC control initiation, elongation, and composition of DED filaments, playing the role of control checkpoints. These findings provide new insights into DISC and DED filament regulation and open innovative possibilities for targeting the extrinsic apoptosis pathway.

Background Currently, more than 150 million people worldwide suffer from lymphedema. It is a chronic progressive disease characterized by high-protein edema of various parts of the body due to defects in lymphatic drainage. Molecular-genetic mechanisms of the disease are still poorly understood. Beginning of a clinical manifestation of primary lymphedema in middle age and the development of secondary lymphedema after treatment of breast cancer can be genetically determined. Disruption of endothelial cell apoptosis can be considered as one of the factors contributing to the development of lymphedema. However, a study of the relationship between genes associated with lymphedema and genes involved in endothelial apoptosis, in the associative gene network was not previously conducted. Methods In the current work, we used well-known methods (ToppGene and Endeavour), as well as methods previously developed by us, to prioritize genes involved in endothelial apoptosis and to find potential participants of molecular-genetic mechanisms of lymphedema among them. Original methods of prioritization took into account the overrepresented Gene Ontology biological processes, the centrality of vertices in the associative gene network, describing the interactions of endothelial apoptosis genes with genes associated with lymphedema, and the association of the analyzed genes with diseases that are comorbid to lymphedema. Results An assessment of the quality of prioritization was performed using criteria, which involved an analysis of the enrichment of the top-most priority genes by genes, which are known to have simultaneous interactions with lymphedema and endothelial cell apoptosis, as well as by genes differentially expressed in murine model of lymphedema. In particular, among genes involved in endothelial apoptosis, KDR, TNF, TEK, BMPR2, SERPINE1, IL10, CD40LG, CCL2, FASLG and ABL1 had the highest priority. The identified priority genes can be considered as candidates for genotyping in the studies involving the search for associations with lymphedema. Conclusions Analysis of interactions of these genes in the associative gene network of lymphedema can improve understanding of mechanisms of interaction between endothelial apoptosis and lymphangiogenesis, and shed light on the role of disturbance of these processes in the development of edema, chronic inflammation and connective tissue transformation during the progression of the disease. Electronic supplementary material The online version of this article (10.1186/s12920-019-0492-9) contains supplementary material, which is available to authorized users.

Initiation of apoptosis by chemotherapeutic drugs is one of the most effective approaches to the treatment of cancers. Caspases, the main enzymes of apoptosis, undergo activation to initiate cell death. Activation of initiator caspases requires their binding to special protein complexes. For elucidation of the mechanisms of apoptosis, these complexes should be isolated. However, their purification is challenging because they are formed in the cell in negligible amounts and rapidly degrade. We have developed an effective way to isolate caspase activation complexes formed in tumor cells in response to DNA damage. The method is based on combination of gel filtration with immunoprecipitation. The first stage is aimed at the separation of the high-molecular-weight caspase activation complexes and their monomeric forms, which allows increasing the efficiency of isolation of complexes at the second stage.

Aggressive behavior in animals plays an important role in protecting the territory, offspring, establishing social hierarchical relations, etc. Increased aggression is observed in a number of diseases ( schizophrenia, bipolar disorder, brain degenerative disorders). Neuronal apoptosis is crucial in the maintenance of developmental processes during neurogenesis. Alterations in neuronal apoptosis are observed in aging and neuropathologies accompanied by changes in psycho­emo­ tional state (epilepsy, Alzheimer’s disease, neurotrauma). The expression of key neuronal apoptosis genes (Casp3, Bax and Bcl-xl) in the brain of highly aggressive rats is significantly altered. The aim of this work was to analyze associative networks that describe genetic interactions between genes/proteins involved in neuronal apoptosis, differentially expressed genes and genes with polymorphisms in grey rats with aggressive behavior. Analysis revealed 819 differentially expressed genes in the hypothalamus, ventral tegmental region and periaqueductus Sylvii grey matter in grey rats with aggressive and tame behavior. The Stx1a, Mbp and Th genes have the highest index of betweenness centrality in the associative network of differentially expressed genes. Genome analysis revealed 137 polymorphic genes. Three of them (Lig4, Parp1 and Pigt) were involved in neuronal apoptosis. It was shown that polymorphic and differentially expressed genes were statistically significantly overrepresented among ge nes interacting with neuronal apoptosis genes (p value < 0.01). Three molecular­genetic chains describing connections between polymorphic and neuronal apoptosis genes mediated by differentially expressed genes were reconstructed. Chains included the polymorphic genes Tsc1, Adamts4 and Lgals3, differentially expressed genes Ezr, Acan, Th and 19 neuronal apoptosis genes. It was shown that neuronal apoptosis is closely related to aggressive behavior in animals.

The identification of new effective apoptosis inhibitors plays an important role in the development of drugs for the treatment of various disorders, including neurogenerative diseases. Apoptosis is initiated via the formation of macromolecular protein complexes. These complexes exert the activation of caspases, which are key regulators and executors of apoptosis. The death inducing signaling complex, (DISC) plays a central role in the induction of the extrinsic apoptosis pathway. The adaptor protein FADD is the core component of the DISC that is essential for caspase activation at the DISC and subsequent apoptosis initiation. Therefore, inhibitors of FADD may serve as candidate drugs inhibiting apoptosis. Furthermore, the study of the mechanisms of action of these inhibitors is of great interest for understanding the signal transduction pathways of apoptosis. It has been reported that the mucin type 1 glycoprotein (MUC1) is a natural protein inhibitor of FADD. In particular, two fragments of the primary structure of the cytoplasmic domain of MUC1 (MUC1-CD) are capable of inhibiting the binding of procaspase 8 to FADD. However, the 3D structure of MUC1 has not been obtained yet. This significantly complicates the rational design of potential drugs based on the peptides derived from the MUC1 structure. The aim of the present study was in silico prediction of the 3D structures of MUC1-CD peptides corresponding to protein fragments 120 and 4672, as well as the analysis of their conformational properties. The special attention was placed on the MUC1-CD (46-72) peptide, which is able to bind to FADD. By using the method of molecular dynamics in implicit water it was shown that the structure of the peptide MUC1-CD (46-72) is similar to the three-dimensional structures of at least four fragments of caspase 8. These results indicate that the molecular mechanism of the inhibitory action of the peptide can be explained by the competitive binding of MUC1 to FADD due to the structural and conformational similarity to fragments of the caspase 8 DEDs.

Modelling of gene networks is widely used in systems biology to study the functioning of complex biological systems. Most of the existing mathematical modelling techniques are useful for analysis of well-studied biological processes, for which information on rates of reactions is available. However, complex biological processes such as those determining the phenotypic traits of organisms or pathological disease processes, including pathogen-host interactions, involve complicated cross-talk between interacting networks. Furthermore, the intrinsic details of the interactions between these networks are often missing. In this study, we developed an approach, which we call mosaic network modelling, that allows the combination of independent mathematical models of gene regulatory networks and, thereby, description of complex biological systems. The advantage of this approach is that it allows us to generate the integrated model despite the fact that information on molecular interactions between parts of the model (so-called mosaic fragments) might be missing. To generate a mosaic mathematical model, we used control theory and mathematical models, written in the form of a system of ordinary differential equations (ODEs). In the present study, we investigated the efficiency of this method in modelling the dynamics of more than 10,000 simulated mosaic regulatory networks consisting of two pieces. Analysis revealed that this approach was highly efficient, as the mean deviation of the dynamics of mosaic network elements from the behaviour of the initial parts of the model was less than 10%. It turned out that for construction of the control functional, data on perturbation of one or two vertices of the mosaic piece are sufficient. Further, we used the developed method to construct a mosaic gene regulatory network including hepatitis C virus (HCV) as the first piece and the tumour necrosis factor (TNF)-induced apoptosis and NF-κB induction pathways as the second piece. Thus, the mosaic model integrates the model of HCV subgenomic replicon replication with the model of TNF-induced apoptosis and NF-κB induction. Analysis of the mosaic model revealed that the regulation of TNF-induced signaling by the HCV network is crucially dependent on the RIP1, TRADD, TRAF2, FADD, IKK, IκBα, c-FLIP, and BAR genes. Overall, the developed mosaic gene network modelling approach demonstrated good predictive power and allowed the prediction of new regulatory nodes in HCV action on apoptosis and the NF-κB pathway. Those theoretical predictions could be a basis for further experimental verification.

The cytopoxic effect of RL2 lactaptin (the recombinant analog of proteolytic fragment of human kappa‐casein) toward tumor cells in vitro and in vivo presents it as a novel promising antitumor drug. The binding of any drug with serum proteins can affect their activity, distribution, rate of excretion and toxicity in the human body. Here, we studied the ability of RL2 to bind to various blood serum proteins. Using magnetic microparticles bearing by RL2 as an affinity matrix, in combination with mass spectrometry and western blot analysis, we found a number of blood serum proteins possessing affinity for RL2. Among them IgA, IgM and IgG subclasses of immunoglobulins, apolipoprotein A1 and various cortactin isoforms were identified. This data suggests that in the bloodstream RL2 lactaptin takes part in complicate protein–protein interactions, which can affect its activity. This work was financial supported by Volkswagen Foundation (grant # VW 90315). Scopus

Autophagy is a degradative process in which cellular organelles and proteins are recycled to restore homeostasis and cellular metabolism. Autophagy can be either a prosurvival or a prodeath process and remains one of the most fundamental processes for cell vitality. Thus autophagy modulation is an important approach for reinforcement anticancer therapeutics. Earlier we have demonstrated that recombinant analog of human milk protein lactaptin (RL2) induced apoptosis of various cultured cancer cells and activated lipidation of microtubule-associated protein 1 light chain 3 (LC3). In this study we investigated whether autophagy inhibitors—chloroquine (CQ), Ku55933 (Ku), and 3-methyladenine (3MA)—or inducer—rapamycin (Rap)—can enhance cytotoxic activity of lactaptin analog in cancer cells and its anticancer activity in the mice model. Western Blot analysis revealed that RL2 induced short-term autophagy in MDA-MB-231 and MCF-7 cells at early stages of incubation and that these data were confirmed by the transmission electron microscopy of autophagosome/autophagolysosome formation. RL2 stimulates reactive oxygen species (ROS) production, autophagosomes accumulation, upregulation of ATG5 with processing of LC3I to LC3II, and downregulation of p62/sequestosome 1 (p62). We have shown that autophagy modulators, CQ, Ku, and Rap, synergistically increased cytotoxicity of RL2, and RL2 with CQ induced autophagic cell death. In addition, CQ, Ku, and Rap in combination with RL2 decreased activity of lysosomal protease Cathepsin D. More importantly, combining RL2 with CQ, we improved antitumor effect in mice. Detected synergistic cytotoxic effects of both types of autophagy regulators, inhibitors, and inducers with RL2 against cancer cells allow us to believe that these combinations can be a basis for the new anticancer approach. Finally, we suppose that CQ and Rap promoting of short-term RL2-induced autophagy interlinks with final autophagic cell death.

Pharmacological targeting via small molecule-based chemical probes has recently acquired an emerging importance as a valuable tool to delineate molecular mechanisms. Induction of apoptosis via CD95/Fas and TRAIL-R1/2 is triggered by the formation of the death-inducing signaling complex (DISC). Caspase-8 activation at the DISC is largely controlled by c-FLIP proteins. However molecular mechanisms of this control have just started to be uncovered. In this study we report the first-in-class chemical probe targeting c-FLIP(L) in the heterodimer caspase-8/c-FLIP(L). This rationally designed small molecule was aimed to imitate the closed conformation of the caspase-8 L2′ loop and thereby increase caspase-8 activity after initial processing of the heterodimer. In accordance with in silico predictions, this small molecule enhanced caspase-8 activity at the DISC, CD95L/TRAIL-induced caspase activation, and subsequent apoptosis. The generated computational model provided further evidence for the proposed effects of the small molecule on the heterodimer caspase-8/c-FLIP(L). In particular, the model has demonstrated that boosting caspase-8 activity by the small molecule at the early time points after DISC assembly is crucial for promoting apoptosis induction. Taken together, our study allowed to target the heterodimer caspase-8/c-FLIP(L) and get new insights into molecular mechanisms of its activation.

Apoptosis is a mode of regulated cell death that is indispensable for the morphogenesis, development and homeostasis of multicellular organisms. Caspases are cysteine-dependent aspartate-specific proteases, which function as initiators and executors of apoptosis. Caspases are cytosolic proteins that can cleave substrates located in different intracellular compartments during apoptosis. Many years ago, the involvement of caspases in the regulation of nuclear changes, a hallmark of apoptosis, was documented. Accumulated data suggest that apoptosis-associated alterations in nucleocytoplasmic transport are also linked to caspase activity. Here, we aim to discuss the current state of knowledge regarding this process. Particular attention will be focused on caspase nuclear entry and their functions in the demolition of the nucleus upon apoptotic stimuli.

The role of caspase-2 in cell death regulation remains largely unknown. In this study we have analyzed the involvement of caspase-2 in RIPK1-regulated necrosis (necroptosis) in human ovarian carcinoma cells. We show that these cells undergo necroptosis upon treatment with the DNA damaging drug cisplatin in combination with the pan-caspase inhibitor zVAD-fmk. Downregulation of caspase-2 leads to an increase of necroptosis in CAOV-4 cells. Interestingly, an association of caspase-2 to the necrosome complex was not detected. Importantly, downregulation of caspase-2 with shRNA or CRISPR/Cas9 system led to an enhanced phosphorylation of RIPK1 and MLKL. Taken together, our data strongly indicate that caspase-2 negatively regulates necroptotic cell death, which might play an important role in further therapeutic applications.

Caspase-2 is reported to play an initiator role in apoptotic cell death in response to DNA damage. In this study, the mechanism of caspase-2 activation after DNA damage was investigated in human ovarian cancer cells Caov-4 treated with the chemotherapeutic agent cisplatin. To isolate the protein complex that might be involved in caspase-2 activation, a combination of gel filtration and immunoprecipitation was used. In the first step the high molecular weight complexes were separated from caspase-2 monomers by means of gel-filtration and in the second step immunoprecipitation from the high molecular weight gel-filtration fractions allowed us to isolate the complex that contains caspase-2. Interestingly, this complex did not contain the protein RAIDD that is a core component of the PIDDosome platform; the latter was shown to play an essential role in DNA damage-induced caspase-2 activation. Finally, catalytically active caspase-2 was detected in this complex, which indicates the possibility of formation of an alternative platform for caspase2 activation in DNA damage-induced apoptosis.

Glaucoma is a chronic and progressive disease, which affects more than 60 million people worldwide. Primary open-angle glaucoma (POAG) is one of the most common forms of glaucoma. For example, about 2.71 million people in the USA had primary open-angle glaucoma in 2011. Currently POAG is a major cause of irreversible vision loss. In patients with treated open-angle glaucoma the risk of blindness reached to be about 27 %. It is known that the death of optic nerve cells can be triggered by mechanical stress caused by increased intraocular pressure, which induces neuronal apoptosis and is observed in patients with POAG. Currently, there is a large number of scientific publications describing proteins and genes involved in the pathogenesis of POAG, including neuronal apoptosis and the cell response to mechanical stress. However, the molecular- genetic mechanisms underlying the pathophysiology of POAG are still poorly understood. Reconstruction of associative networks describing the functional interactions between these genes/proteins, including biochemical reactions, regulatory interactions, transport, etc., requires the use of methods of automated knowledge extraction from texts of scientific publications. The aim of the work was the analysis of associative networks, describing the molecular-genetic interactions between proteins and genes involved in cell response to mechanical stress (CRMS), neuronal apoptosis and pathogenesis of POAG using ANDSystem, our previous development for automated text analysis. It was shown that genes associated with POAG are statistically significantly more often represented among the genes involved in the interactions between CRMS and neuronal apoptosis than it was expected by random reasons, which can be an explanation for the effect of POAG leading to the retinal ganglion cell death.

Combination therapy of cancer is based on the cumulative effects mediated by several drugs. Although molecular mechanisms of action of each particular drug are partially elucidated, understanding of the dynamic cross-talk between different cell death pathways at the quantitative level induced by combination therapy is still missing. Here, we exemplified this question for the death receptor (DR) networks in pancreatic cancer cells. We demonstrate that the combined action of CD95L and gemcitabine in pancreatic cancer cells leads to the simultaneous induction of caspase-dependent and caspase-independent cell death. The pro-apoptotic effects are mediated through down-regulation of the anti-apoptotic proteins c-FLIP and Mcl-1, while caspase-independent cell death was blocked by inhibition of the kinase activity of RIP1. Furthermore, gemcitabine co-treatment strongly increased the amount of cells undergoing CD95-induced RIP1-regulated necrosis. Imaging flow cytometry has enabled us to get the quantitative insights into the apoptosis-necroptosis network and reveal that the majority of the cells upon the CD95L/gemcitabine co-treatment undergoes necroptosis. Our data underlie the importance of the quantitative understanding of the interplay between different cell death modalities, which is essential for the development of anti-cancer therapies. Taken together, our results are important for combination therapy of pancreatic cancer comprising chemotherapeutics and DR-agonists and offer a possibility to sensitize cells with defects in the apoptotic machinery towards necroptosis-type-mediated death.

The CD95/Fas/APO-1 death-inducing signaling complex (DISC), comprising CD95, FADD, procaspase-8, procaspase-10, and c-FLIP, has a key role in apoptosis induction. Recently, it was demonstrated that procaspase-8 activation is driven by death effector domain (DED) chains at the DISC. Here, we analyzed the molecular architecture of the chains and the role of the short DED proteins in regulating procaspase-8 activation in the chain model. We demonstrate that the DED chains are largely composed of procaspase-8 cleavage products and, in particular, of its prodomain. The DED chain also comprises c-FLIP and procaspase-10 that are present in 10 times lower amounts compared with procaspase-8. We show that short c-FLIP isoforms can inhibit CD95-induced cell death upon overexpression, likely by forming inactive heterodimers with procaspase-8. Furthermore, we have addressed mechanisms of the termination of chain elongation using experimental and mathematical modeling approaches. We show that neither c-FLIP nor procaspase-8 prodomain terminates the DED chain, but rather the dissociation/association rates of procaspase-8 define the stability of the chain and thereby its length. In addition, we provide evidence that procaspase-8 prodomain generated at the DISC constitutes a negative feedback loop in procaspase-8 activation. Overall, these findings provide new insights into caspase-8 activation in DED chains and apoptosis initiation.

Since their establishment in the early 1970s, the nuclear changes upon apoptosis induction, such as the condensation of chromatin, disassembly of nuclear scaffold proteins and degradation of DNA, were, and still are, considered as the essential steps and hallmarks of apoptosis. These are the characteristics of the execution phase of apoptotic cell death. In addition, accumulating data clearly show that some nuclear events can lead to the induction of apoptosis. In particular, if DNA lesions resulting from deregulation during the cell cycle or DNA damage induced by chemotherapeutic drugs or viral infection cannot be efficiently eliminated, apoptotic mechanisms, which enable cellular transformation to be avoided, are activated in the nucleus. The functional heterogeneity of the nuclear organization allows the tight regulation of these signaling events that involve the movement of various nuclear proteins to other intracellular compartments (and vice versa) to initiate and govern apoptosis. Here, we discuss how these events are coordinated to execute apoptotic cell death.

Precisely identifying the type of programmed cell death is one of the key questions in contemporary biomedical research. We developed a straightforward approach allowing quantitative discrimination between two types of cell death on the single cell level: apoptosis and necroptosis. This method uses the combination of imaging flow cytometry with classical Annexin V/propidium iodide staining, which allows for the ascertainment of typical features of dying cells: exposure of the phospholipid phosphatidylserine and the loss of membrane integrity. Image-based analysis of nuclear morphology enables us to distinguish between secondary necrotic/late apoptotic and necroptotic cells directly in one assay. This is a major advantage compared to other contemporary approaches of necroptosis detection, which require a parallel application of several methods. This approach can be used for the quantitative assessment of cell death in cell and systems biology studies of signal transduction networks.

Imaging flow cytometry is a powerful experimental technique combining the strength of microscopy and flow cytometry to enable high-throughput characterization of cell populations on a detailed microscopic scale. This approach has an increasing importance for distinguishing between different cellular phenotypes such as proliferation, cell division and cell death. In the course of undergoing these different pathways, each cell is characterized by a high amount of properties. This makes it hard to filter the most relevant information for cell state discrimination. The traditional methods for cell state discrimination rely on dye based two-dimensional gating strategies ignoring information that is hidden in the high-dimensional property space. In order to make use of the information ignored by the traditional methods, we present a simple and efficient approach to distinguish biological states within a cell population based on machine learning techniques. We demonstrate the advantages and drawbacks of filter techniques combined with different classification schemes. These techniques are illustrated with two case studies of apoptosis detection in HeLa cells. Thereby we highlight (i) the aptitude of imaging flow cytometry regarding automated, label-free cell state discrimination and (ii) pitfalls that are frequently encountered. Additionally a MATLAB script is provided, which gives further insight regarding the computational work presented in this study.

Background The development of approaches that increase therapeutic effects of anti-cancer drugs is one of the most important tasks of oncology. Caloric restriction in vivo or serum deprivation (SD) in vitro has been shown to be an effective tool for sensitizing cancer cells to chemotherapeutic drugs. However, the detailed mechanisms underlying the enhancement of apoptosis in cancer cells by SD remain to be elucidated. Methods Flow cytometry, caspase activity assay and western blotting were used for cell death rate evaluation. Western blotting, gel-filtration, siRNA approach and qRT-PCR were used to elucidate the mechanism underlying cell death potentiation upon SD. Results We demonstrated that SD sensitizes cancer cells to treatment with chemotherapeutic agent cisplatin. This effect is independent on activation of caspases-2 and -8, apical caspases triggering apoptosis in response to genotoxic stress. SD potentiates cell death via downregulation of the anti-apoptotic protein Mcl-1. In fact, SD reduces the Mcl-1 mRNA level, which consequently decreases the Mcl-1 protein level and renders cells more susceptible to apoptosis induction via the formation of apoptosome. Conclusions Mcl-1 protein is an important regulator of sensitivity of cancer cells to apoptotic stimuli upon SD. General significance This study identifies Mcl-1 as a new target for the sensitization of human cancer cells to cell death by SD, which is of great significance for the development of efficient anti-cancer therapies.

Programmed cell death plays a central role in the regulation of homeostasis and development of multi- cellular organisms. Deregulation of programmed cell death is connected to a number of disorders, including cancer and autoimmune diseases. Initiation of cell death occurs in the multiprotein complexes or high molecular weight plat- forms. Composition, structure, and molecular interactions within these platforms influence the cellular decision toward life or death and, therefore, define the induction of a particular cell death program. Here, we discuss in detail the key cell-death complexes—including DISC, complex II, and TNFRI complex I/II, and the necrosome, RIPopto- some, apoptosome, and PIDDosome—that control apoptosis or necroptosis pathways as well as their regula- tion. The possibility of their pharmacological targeting leading to the development of new strategies of interfer- ence with cell death programs via control of the high molecular weight platforms will be discussed.