Form Of Caspase-independent Cell Death Research Articles

Identification of a novel form of caspase-independent cell death triggered by BH3-mimetics in diffuse large B-cell lymphoma cell lines

BH3-mimetics represent promising anti-cancer agents in tumors that rely on the anti-apoptotic function of B-Cell Lymphoma 2 (BCL2) proteins, particularly in leukemia and lymphoma cells primed for apoptosis. Mechanistically, BH3-mimetics may displace pro-apoptotic binding partners thus inducing BAX/BAK-mediated mitochondrial permeabilization followed by cytochrome c release, activation of the caspase cascade and apoptosis. Here, we describe a novel mode of caspase-independent cell death (CICD) induced by BH3-mimetics in a subset of diffuse large B-cell lymphoma (DLBCL) cells. Of note, rather than occurring via necroptosis, CICD induced immediately after mitochondrial permeabilization was associated with transcriptional reprogramming mediated by activation of c-Jun N-terminal Kinase (JNK) signaling and Activator Protein 1 (AP1). Thereby, CICD resulted in the JNK/AP1-mediated upregulation of inflammatory chemokines and increased migration of cytotoxic Natural Killer (NK) cells. Taken together, our study describes a novel mode of CICD triggered by BH3-mimetics that may alter the immune response towards dying cells.

4′-O-methylbavachalcone alleviates ischemic stroke injury by inhibiting parthanatos and promoting SIRT3

Cerebral ischemia-reperfusion injury (CIRI) can induce massive death of ischemic penumbra neurons via oxygen burst, exacerbating brain damage. Parthanatos is a form of caspase-independent cell death involving excessive activation of PARP-1, closely associated with intense oxidative stress following CIRI. 4′-O-methylbavachalcone (MeBavaC), an isoprenylated chalcone component in Fructus Psoraleae, has potential neuroprotective effects. This study primarily investigates whether MeBavaC can act on SIRT3 to alleviate parthanatos of ischemic penumbra neurons induced by CIRI. MeBavaC was oral gavaged to the middle cerebral artery occlusion-reperfusion (MCAO/R) rats after occlusion. The effects of MeBavaC on cerebral injury were detected by the neurological deficit score and cerebral infarct volume. In vitro, PC-12 cells were subjected to oxygen and glucose deprivation/reoxygenation (OGD/R), and assessed cell viability and cell injury. Also, the levels of ROS, mitochondrial membrane potential (MMP), and intracellular Ca2+ levels were detected to reflect mitochondrial function. We conducted western blotting analyses of proteins involved in parthanatos and related signaling pathways. Finally, the exact mechanism between the neuroprotection of MeBavaC and parthanatos was explored. Our results indicate that MeBavaC reduces the cerebral infarct volume and neurological deficit scores in MCAO/R rats, and inhibits the decreased viability of PC-12 cells induced by OGD/R. MeBavaC also downregulates the expression of parthanatos-related death proteins PARP-1, PAR, and AIF. However, this inhibitory effect is weakened after the use of a SIRT3 inhibitor. In conclusion, the protective effect of MeBavaC against CIRI may be achieved by inhibiting parthanatos of ischemic penumbra neurons through the SIRT3-PARP-1 axis.

Development of a necroptosis-related prognostic model for uterine corpus endometrial carcinoma

Necroptosis is a recently identified caspase-independent form of cell death which plays a significant role in the onset and progression of cancer. MicroRNAs (miRNAs) are vital for the development of uterine corpus endometrial carcinoma (UCEC) because they are an important regulatory component in necroptosis. This study developed a new necroptosis-related miRNAs profile to predict the prognosis of patients with UCEC. The TCGA-UCEC cohort’s RNA sequencing data, consisting of 534 tumor samples and 33 normal samples, was downloaded. Ten differentially expressed miRNAs related to necroptosis were identified. A prediction model for necroptosis-related miRNAs was then created through COX regression and nomograms analysis. Clinical and pathological parameters were integrated to construct a nomogram and evaluate the model. Prognosis-related miRNAs were further used to predict target genes, and functional analysis was conducted to explore the potential mechanisms of these target genes. Subsequently, immune infiltration analysis was performed using transcriptome data to identify immune genes associated with prognosis, and the expression levels of target gene was validated using UCEC tissues. We identified 7 up-regulated miRNAs (hsa-miR-577, hsa-miR-7-5p, hsa-miR-210-3p, hsa-miR-210-5p, hsa-miR-200a-5p, hsa-miR-141-3p, hsa-miR-425-5p) and 3 down-regulated miRNAs (hsa-miR-7-2-3p, hsa-miR-383-5p, hsa-miR-29a-3p). The risk signature was based on univariate and multivariate COX analyses, constructed using 2 independent prognostic factors and miRNAs (hsa-miR-425-5p, hsa-miR-7-5p) associated with necroptosis. Nomograms demonstrated the prognostic value of risk level, age, FIGO stage, and histological type. Kaplan–Meier analysis revealed significant differences in overall survival (OS) outcomes associated with the expression of hsa-miR-425-5p (P < 0.001) and hsa-miR-7-5p (P = 0.015). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) investigations indicated that these miRNAs play crucial roles in tumor development, metastasis, and prognosis. Immune infiltration analysis showed decreased infiltration of CD8+ T cells, CD8+ T cells, NK cells, and M1 macrophages in normal tissues. Subsequently, a necroptosis-related immune gene significantly associated with prognosis (THRB) was identified, western blot and immunohistochemical staining confirmed the differential expression of THRB in normal endometrial tissues and tumor. Our findings demonstrate a close association between necroptosis and UCEC. The two necroptosis-related miRNAs used in this study may serve as valuable prognostic markers for UCEC patients, and are associated with immune cell infiltration. This suggests that necroptosis may be involved in the development of UCEC through its interaction with immune responses.

Contribution to the study of inflammasome and programmed cell death in paracoccidioidomycosis oral lesions.

Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America, with a high incidence in Brazil, Colombia and Venezuela, and constitutes a serious public health problem, a frequent cause of morbidity and disability for work. Some mechanisms of cell death are described as important tools in infectious processes. When apoptosis is blocked, RIPK (Receptor-interacting protein kinase) 3 dependent, a caspase-independent form of cell death, can limit the replication and spread of pathogens. Some molecules that mediate necroptosis include RIPK3 and have been extensively studied due to their signalling mechanism and pathological function. RIPK3 activates NLRP1 and NLRP3-mediated inflammasome formation. Caspase-1 has an important role in processing the cytokines ILβ and IL18 to their active form. Such molecules are part of the inflammasome characterization, whose caspase-1-dependent activation promotes the death of pyroptotic cells and the secretion of proinflammatory cytokines. Knowledge about the mechanisms of pathogen-mediated cell death can be useful for understanding of the pathogenesis of infections and inflammatory conditions. The objective of this work was to identify the mechanisms of programmed cell death and inflammasome components in human oral mucosal lesions of paracoccidioidomycosis through immunohistochemical methods and identification of RIPK-3, IL1β, IL18, NLRP-1 and caspase-1. Thirty specimens were included, and a histopathological analysis of the lesions was performed using haematoxylin-eosin staining. Our results on insitu expression of inflammasome elements and programmed cell death showed increased expression of IL-1β, NLRP-1, caspase-1 and RIPK-3. We suggest that inflammasome complex participate in the immunopathogenesis in paracoccidioidomycosis oral lesions in an interplay with RIPK3.

Development of a novel necroptosis-associated miRNA risk signature to evaluate the prognosis of colon cancer patients.

BackgroundNecroptosis is a recently discovered caspase-independent form of cell death which plays an important role in the occurrence and development of cancer. As an important regulatory factor in necroptosis, microRNAs (miRNAs) are important for the development of colon cancer. This study established a novel necroptosis-related miRNA risk signature to evaluate the prognosis of patients with colon adenocarcinoma (COAD).MethodsThe necroptosis-related miRNAs were selected by assessing the differential expression of miRNAs in 459 COAD patient samples and 8 control samples from The Cancer Genome Atlas (TCGA). Selection operator Cox analyses and survival analyses were used to establish the risk signature of 7 miRNAs related to necroptosis. Functional enrichment analysis and nomograms were used to explore the potential effects of necroptosis-related miRNAs on prognosis and metastasis. The target genes of the necroptosis-related miRNAs were predicted using online databases and the genes related to overall survival (OS) were screened.ResultsThe risk signature was based on 7 necroptosis-related miRNAs. Nomograms showed that the risk signature was effective at predicting the prognosis and TNM stage of COAD patients. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses demonstrated that these miRNAs play an important role in cancer development, metastasis, and prognosis. A total of 38 target genes for these miRNAs were found to be associated with the OS in COAD patients.ConclusionsThis study provided novel evidence that necroptosis-related miRNAs are associated with the prognosis of COAD patients. A risk signature established based on these miRNAs could effectively predict the prognosis and metastasis of COAD in patients.

The Role of Necroptosis in ROS-Mediated Cancer Therapies and Its Promising Applications.

Over the past decades, promising therapies targeting different signaling pathways have emerged. Among these pathways, apoptosis has been well investigated and targeted to design diverse chemotherapies. However, some patients are chemoresistant to these therapies due to compromised apoptotic cell death. Hence, exploring alternative treatments aimed at different mechanisms of cell death seems to be a potential strategy for bypassing impaired apoptotic cell death. Emerging evidence has shown that necroptosis, a caspase-independent form of cell death with features between apoptosis and necrosis, can overcome the predicament of drug resistance. Furthermore, previous studies have also indicated that there is a close correlation between necroptosis and reactive oxygen species (ROS); both necroptosis and ROS play significant roles both under human physiological conditions such as the regulation of inflammation and in cancer biology. Several small molecules used in experiments and clinical practice eliminate cancer cells via the modulation of ROS and necroptosis. The molecular mechanisms of these promising therapies are discussed in detail in this review.

Protein-Bound Polysaccharides from Coriolus Versicolor Induce RIPK1/RIPK3/MLKL-Mediated Necroptosis in ER-Positive Breast Cancer and Amelanotic Melanoma Cells.

The induction of necroptosis, a form of caspase-independent cell death, represents one of the most promising anticancer therapeutic modalities, as necroptosis serves as an alternative way to eliminate apoptosis-resistant tumor cells. Here, we investigated whether protein-bound polysaccharides (PBPs) derived from the fungus Coriolus versicolor (CV) induce the necroptotic death pathway in breast cancer and melanoma cells. MCF-7 and SKMel-188 cells were exposed to PBPs either alone or in combination with necrostatin-1 (Nec-1), GSK'872 or necrosulfonamide (NSA), pharmacological inhibitors of the kinases receptor-interacting protein 1 kinase (RIPK1), receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL), respectively, which are involved in necroptotic processes. The effects of cellular treatment with these inhibitors were quantified by measuring cell viability and reactive oxygen species (ROS) generation via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and 2',7'-dichlorofluorescein diacetate (DCF-DA) assays, respectively. The morphological changes induced in these cells were detected using holotomographic (HT) microscopy. Activation of the TNF-α/TNFR1 pathway in the PBP-stimulated cells was evaluated using TNF-α-neutralizing antibody, qRT-PCR and immunofluorescence-based assays. PBPs showed effective antitumor activity against MCF-7 and SKMel-188 cells. Cotreatment of the cells with Nec-1, GSK'872 or NSA abrogated PBP-induced cell death, and the cells were protected against membrane rupture. Moreover, breast cancer cell death caused by PBPs was mediated by induced activation of the TNF-α/TNFR1 pathway. Interestingly, the melanoma cells did not express TNF-α or TNFR1 after PBP stimulation; instead, PBPs triggered intracellular ROS generation, which was partially diminished by the inhibitors Nec-1, GSK'872 and NSA. These results suggest that PBPs from the fungus CV induce RIPK1/RIPK3/MLKL-mediated necroptosis in breast cancer and melanoma cells, providing novel insights into the molecular effects of PBPs on cancer cells.

Abstract LB-080: Discovery of an unconventional form of cell-death in colorectal cancer

Abstract We recently discovered unique, structurally constrained 4-pyridinyl - containing compounds that induce a form of caspase-independent cell death in colorectal cancer (CRC) cells. This was characterized by the simultaneous induction of dysregulated macropinocytosis and macroautophagy, a type of cell death we have termed ‘methuophagy' (this neologism arises from the Greek ‘methuo', to drink to intoxication and ‘phagy', self-eating). Our new class of small molecular probes (BAPT analogues) do not induce cell death via activation of the well characterized classical apoptotic pathways. In contrast, our BAPT compounds cause methuophagy by inducing the formation and accumulation of fluid-filled, heterogeneous vacuoles and autophagosomes, resulting in metabolic failure, loss of membrane integrity, and detachment of cells from the substratum. Methuophagy produces extensive autophagy without cell shrinkage, chromatin condensation, alteration of mitochondrial membrane potential or nuclear membrane damage. The optimization of the BAPT structure led to the discovery of BAPT-27, which has nanomolar potency in inducing methuophagy in broad spectrum of CRC cell lines and may serve as a prototype for a new class of drugs. BAPT-27 produces methuophagy and surmounts MDR in CRC cells independent of Ras, p53 or other apoptotic protein (i.e. Bax, Bak) or expression of ATP-binding cassette (ABC) transporters. Furthermore, BAPT-27 is non-toxic in normal cells and zebrafish. Based on our findings, we postulate that the induction of methuophagy by BAPT-27 produces endolysosomal trafficking defects that prevent recycling of lysosomes and cause lysosome-to-nucleus signaling defect, a mechanism that disturb cellular clearance and energy metabolism. The selected compounds will allow us to understand the biology of methuophagy-induced cell death and will be particularly beneficial for treatment of patients with apoptotic-resistant and aggressive CRCs. Citation Format: Amit K. Tiwari, Haneen Amawi, Karthikeyan Chandrabose, Paul Erhardt, Piyush Trivedi. Discovery of an unconventional form of cell-death in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-080.

EspL is a bacterial cysteine protease effector that cleaves RHIM proteins to block necroptosis and inflammation.

Cell death signalling pathways contribute to tissue homeostasis and provide innate protection from infection. Adaptor proteins such as receptor-interacting serine/threonine-protein kinase 1 (RIPK1), receptor-interacting serine/threonine-protein kinase 3 (RIPK3), TIR-domain-containing adapter-inducing interferon-β (TRIF) and Z-DNA-binding protein 1 (ZBP1)/DNA-dependent activator of IFN-regulatory factors (DAI) that contain receptor-interacting protein (RIP) homotypic interaction motifs (RHIM) play a key role in cell death and inflammatory signalling1-3. RHIM-dependent interactions help drive a caspase-independent form of cell death termed necroptosis4,5. Here, we report that the bacterial pathogen enteropathogenic Escherichia coli (EPEC) uses the type III secretion system (T3SS) effector EspL to degrade the RHIM-containing proteins RIPK1, RIPK3, TRIF and ZBP1/DAI during infection. This requires a previously unrecognized tripartite cysteine protease motif in EspL (Cys47, His131, Asp153) that cleaves within the RHIM of these proteins. Bacterial infection and/or ectopic expression of EspL leads to rapid inactivation of RIPK1, RIPK3, TRIF and ZBP1/DAI and inhibition of tumour necrosis factor (TNF), lipopolysaccharide or polyinosinic:polycytidylic acid (poly(I:C))-induced necroptosis and inflammatory signalling. Furthermore, EPEC infection inhibits TNF-induced phosphorylation and plasma membrane localization of mixed lineage kinase domain-like pseudokinase (MLKL). In vivo, EspL cysteine protease activity contributes to persistent colonization of mice by the EPEC-like mouse pathogen Citrobacter rodentium. The activity of EspL defines a family of T3SS cysteine protease effectors found in a range of bacteria and reveals a mechanism by which gastrointestinal pathogens directly target RHIM-dependent inflammatory and necroptotic signalling pathways.

RIPK3 deficiency or catalytically inactive RIPK1 provides greater benefit than MLKL deficiency in mouse models of inflammation and tissue injury.

Necroptosis is a caspase-independent form of cell death that is triggered by activation of the receptor interacting serine/threonine kinase 3 (RIPK3) and phosphorylation of its pseudokinase substrate mixed lineage kinase-like (MLKL), which then translocates to membranes and promotes cell lysis. Activation of RIPK3 is regulated by the kinase RIPK1. Here we analyze the contribution of RIPK1, RIPK3, or MLKL to several mouse disease models. Loss of RIPK3 had no effect on lipopolysaccharide-induced sepsis, dextran sodium sulfate-induced colitis, cerulein-induced pancreatitis, hypoxia-induced cerebral edema, or the major cerebral artery occlusion stroke model. However, kidney ischemia–reperfusion injury, myocardial infarction, and systemic inflammation associated with A20 deficiency or high-dose tumor necrosis factor (TNF) were ameliorated by RIPK3 deficiency. Catalytically inactive RIPK1 was also beneficial in the kidney ischemia–reperfusion injury model, the high-dose TNF model, and in A20−/− mice. Interestingly, MLKL deficiency offered less protection in the kidney ischemia–reperfusion injury model and no benefit in A20−/− mice, consistent with necroptosis-independent functions for RIPK1 and RIPK3. Combined loss of RIPK3 (or MLKL) and caspase-8 largely prevented the cytokine storm, hypothermia, and morbidity induced by TNF, suggesting that the triggering event in this model is a combination of apoptosis and necroptosis. Tissue-specific RIPK3 deletion identified intestinal epithelial cells as the major target organ. Together these data emphasize that MLKL deficiency rather than RIPK1 inactivation or RIPK3 deficiency must be examined to implicate a role for necroptosis in disease.

Viral gene expression potentiates reovirus-induced necrosis

Infection of some cell types by reovirus evokes a caspase-independent form of cell death resembling necrosis. While reovirus strain T3D induces necrosis much more efficiently than strain T1L, which viral components contribute to this difference is not known. In this study, we identified that the sialic acid binding property of the reovirus σ1 protein affects necrosis efficiency. We found that in addition to sialic acid engagement by the virus particles, viral gene expression, in the form of viral RNA or protein synthesis, is also required for necrosis induction. Our studies reveal that sialic acid does not directly participate in necrosis induction by initiating a signaling pathway. Instead, sialic acid engagement augments necrosis induction indirectly, by increasing reovirus gene expression in each infected cell. Comparison of our results with previous studies suggests that reovirus-induced apoptosis and necrosis are initiated by distinct stages of viral infection.

Readthrough acetylcholinesterase (AChE-R) and regulated necrosis: pharmacological targets for the regulation of ovarian functions?

Proliferation, differentiation and death of ovarian cells ensure orderly functioning of the female gonad during the reproductive phase, which ultimately ends with menopause in women. These processes are regulated by several mechanisms, including local signaling via neurotransmitters. Previous studies showed that ovarian non-neuronal endocrine cells produce acetylcholine (ACh), which likely acts as a trophic factor within the ovarian follicle and the corpus luteum via muscarinic ACh receptors. How its actions are restricted was unknown. We identified enzymatically active acetylcholinesterase (AChE) in human ovarian follicular fluid as a product of human granulosa cells. AChE breaks down ACh and thereby attenuates its trophic functions. Blockage of AChE by huperzine A increased the trophic actions as seen in granulosa cells studies. Among ovarian AChE variants, the readthrough isoform AChE-R was identified, which has further, non-enzymatic roles. AChE-R was found in follicular fluid, granulosa and theca cells, as well as luteal cells, implying that such functions occur in vivo. A synthetic AChE-R peptide (ARP) was used to explore such actions and induced in primary, cultured human granulosa cells a caspase-independent form of cell death with a distinct balloon-like morphology and the release of lactate dehydrogenase. The RIPK1 inhibitor necrostatin-1 and the MLKL-blocker necrosulfonamide significantly reduced this form of cell death. Thus a novel non-enzymatic function of AChE-R is to stimulate RIPK1/MLKL-dependent regulated necrosis (necroptosis). The latter complements a cholinergic system in the ovary, which determines life and death of ovarian cells. Necroptosis likely occurs in the primate ovary, as granulosa and luteal cells were immunopositive for phospho-MLKL, and hence necroptosis may contribute to follicular atresia and luteolysis. The results suggest that interference with the enzymatic activities of AChE and/or interference with necroptosis may be novel approaches to influence ovarian functions.

The serine protease inhibitor TLCK attenuates intrinsic death pathways in neurons upstream of mitochondrial demise.

It is well-established that activation of proteases, such as caspases, calpains and cathepsins are essential components in signaling pathways of programmed cell death (PCD). Although these proteases have also been linked to mechanisms of neuronal cell death, they are dispensable in paradigms of intrinsic death pathways, e.g. induced by oxidative stress. However, emerging evidence implicated a particular role for serine proteases in mechanisms of PCD in neurons. Here, we investigated the role of trypsin-like serine proteases in a model of glutamate toxicity in HT-22 cells. In these cells glutamate induces oxytosis, a form of caspase-independent cell death that involves activation of the pro-apoptotic protein BH3 interacting-domain death agonist (Bid), leading to mitochondrial demise and ensuing cell death. In this model system, the trypsin-like serine protease inhibitor Nα-tosyl-l-lysine chloromethyl ketone hydrochloride (TLCK) inhibited mitochondrial damage and cell death. Mitochondrial morphology alterations, the impairment of the mitochondrial membrane potential and ATP depletion were prevented and, moreover, lipid peroxidation induced by glutamate was completely abolished. Strikingly, truncated Bid-induced cell death was not affected by TLCK, suggesting a detrimental activity of serine proteases upstream of Bid activation and mitochondrial demise. In summary, this study demonstrates the protective effect of serine protease inhibition by TLCK against oxytosis-induced mitochondrial damage and cell death. These findings indicate that TLCK-sensitive serine proteases play a crucial role in cell death mechanisms upstream of mitochondrial demise and thus, may serve as therapeutic targets in diseases, where oxidative stress and intrinsic pathways of PCD mediate neuronal cell death.

Necroptosis, a novel form of caspase-independent cell death, contributes to renal epithelial cell damage in an ATP-depleted renal ischemia model.

Acute kidney injury (AKI) induced by renal ischemia is a common clinical problem associated with a high morbidity and mortality. The present study investigated whether necroptosis was present in an invitro renal ischemia model and whether the addition of necrostatin-1 (Nec-1) has a protective effect. In addition, whether autophagy was inhibited following the use of Nec-1 was also examined. When apoptosis was inhibited by z-VAD‑fmk and energy was depleted with antimycinA for 1h, the morphological abnormalities of human proximal tubular epithelial (HK-2) cells were markedly attenuated, and the cell viability was significantly improved following incubation with Nec-1. LC3-II/I ratios and LC3-II/GAPDH ratios demonstrated a statistically significant decrease in the Nec-1+tumor necrosis factor (TNF)-α+z-VAD-fmk+antimycinA (1h) group compared with the control group. In conclusion, the present study suggested that necroptosis was present in HK-2 cells subjected to TNF-α stimulation and energy depletion. Nec-1 inhibits a caspase‑independent necroptotic pathway involving autophagy and may have therapeutic potential to prevent and treat renal ischemic injury.

Membrane TNF-alpha-activated programmed necrosis is mediated by Ceramide-induced reactive oxygen species

BackgroundProgrammed necrosis is a form of caspase-independent cell death whose molecular regulation is poorly understood. While tumor necrosis factor-alpha (TNF-α) has been identified as an activator of programmed necrosis, the specific context under which this can happen is unclear. Recently we reported that TNF-α can be expressed by human tumor cells as both a membrane tethered (mTNF-α) and a soluble (sTNF-α) form. Whereas low level, tumor-derived sTNF-α acts as a tumor promoter, tumor cell expression of mTNF-α significantly delays tumor growth in mice, in large part by induction of programmed necrosis of tumor associated myeloid cells. In this study we sought to determine the molecular mechanism involved in mTNF-α oxidative stress-induced cell death by evaluating the known pathways involved in TNF receptor-induced programmed necrosis.MethodsThe source of Reactive Oxygen Species (ROS) in mTNF-α treated cells was determined by coculturing RAW 264.7 monocytic and L929 fibroblasts cells with fixed B16F10 control or mTNF-α expressing-melanoma cells in the presence of inhibitors of NADPH and mitochondria ROS. To identify the down-stream effector of TNF-a receptors (TNFR), level of phospho-RIP-1 and ceramide activity were evaluated.To determine whether mTNF-mediated cell death was dependent on a specific TNFR, cell death was measured in primary CD11b myeloid cells isolated from wild-type or TNFR-1, TNFR-2, TNFR-1 and TNFR-2 double knockout mice, cocultured with various TNF-α isoform.ResultsTumor derived-mTNF-α increased ROS-mediated cytotoxicity, independent of caspase-3 activity. Although TNFR on target cells were required for this effect, we observed that mTNF-induced cell death could be mediated through both TNFR-1 and the death domain-lacking TNFR-2. ROS generation and cytotoxicity were inhibited by a mitochondrial respiratory chain inhibitor but not by an inhibitor of NADPH oxidase. mTNF-α mediated cytotoxicity was independent of RIP-1, a serine/threonine kinase that serves as a main adaptor protein of sTNF-α induced programmed necrosis. Instead, mTNF-α-induced ROS and cell death was prohibited by the ceramide-activated protein kinase (CAPK) inhibitor.ConclusionThese findings demonstrate that the mTNF-α isoform is an effective inducer of programmed necrosis through a caspase independent, ceramide-related pathway. Interestingly, unlike sTNFα, mTNF-induced programmed necrosis is not dependent on the presence of TNFR1.

Hypertonic stress and cell death. Focus on “Multiple cell death pathways are independently activated by lethal hypertonicity in renal epithelial cells”

Programmed cell death has been synonymous with caspase-mediated apoptosis for many years, until the recent discovery of caspase-independent mechanisms of cell death. Programmed necrosis is a form of caspase-independent cell death and is currently intensively studied as mechanism of kidney cell injury. The inner core of the kidney, known as the renal medulla, is hypertonic due to a specialized counter current arrangement of tubules, vascular elements and transporters. This regional hypertonicity is critical for body fluid osmoregulation as it provides the osmotic driving force for urinary concentration. The hypertonic environment of the kidney medulla can also promote cell death, if the physiologic cell protective mechanisms are impaired (4). Hypertonic cell stress can induce several pathways that lead to cell death and the distinction between hypertonicity-induced apoptosis and necrosis is becoming increasingly complex.

Abstract B05: Targeting nonapoptotic cell death in chemoresistant patient-derived breast cancer cells

Abstract The development of anti-cancer therapeutics that target non-apoptotic pathways has received increasing attention from the scientific community in recent years. The prevalence of chemoresistance and the characterization of defective apoptotic pathways in cancer has promoted interest in the expansion of current therapeutic regimens to include drugs that induce non-apoptotic cell death. From a phenotypic screen we have identified a small molecule named C6 that induces caspase-independent, non-apoptotic cell death in chemoresistant patient-derived breast cancer cells. Additionally, C6 is also selectively cytotoxic against cancer cells compared to normal mammary epithelial cells. In an effort to characterize this small molecule's mechanism of action and identify relevant biological pathways that might be used as therapeutic drug targets, we have utilized a photoaffinity pull-down strategy to identify biological binding partners of C6. Our photoaffinity pull-down studies have revealed Mitsugumin 23 (MG23), an endoplasmic reticulum-bound transmembrane protein capable of ion channel formation, as a binding partner for C6. Additionally, we have identified a metabolic component of C6-induced cell death through the use of mitochondrial respiration measurements, metabolomic analyses, and mitochondrial transmission electron microscopy (TEM) imaging. Metabolic studies have identified mitochondrial respiration defects, excess lactic acid production, and gross changes in mitochondrial morphology as a result of C6 treatment. Collectively, our findings suggest a role for ionic imbalance and subsequent metabolic disruption in this form of caspase-independent cell death. Our ongoing work is focused on further mechanistic characterization of this non-apoptotic pathway as a potential target for breast cancer drug development. Citation Format: Rachel M. Vaden, Keith M. Gligorich, Dawne N. Shelton, Guoying Wang, Cindy B. Matsen, Ryan E. Looper, Matthew S. Sigman, Bryan E. Welm. Targeting nonapoptotic cell death in chemoresistant patient-derived breast cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B05.

Synthesis and Evaluation of Indole-Based Chalcones as Inducers of Methuosis, a Novel Type of Nonapoptotic Cell Death

Methuosis is a novel caspase-independent form of cell death in which massive accumulation of vacuoles derived from macropinosomes ultimately causes cells to detach from the substratum and rupture. We recently described a chalcone-like compound, 3-(2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (i.e., MIPP), which can induce methuosis in glioblastoma and other types of cancer cells. Herein, we describe the synthesis and structure-activity relationships of a directed library of related compounds, providing insights into the contributions of the two aryl ring systems and highlighting a potent derivative, 3-(5-methoxy, 2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (i.e., MOMIPP) that can induce methuosis at low micromolar concentrations. We have also generated biologically active azide derivatives that may be useful for future studies aimed at identifying the protein targets of MOMIPP by photoaffinity labeling techniques. The potential significance of these studies is underscored by the finding that MOMIPP effectively reduces the growth and viability of Temozolomide-resistant glioblastoma and doxorubicin-resistant breast cancer cells. Thus, it may serve as a prototype for drugs that could be used to trigger death by methuosis in cancers that are resistant to conventional forms of cell death (e.g., apoptosis).

Caspase-10-Dependent Cell Death in Fas/CD95 Signalling Is Not Abrogated by Caspase Inhibitor zVAD-fmk

BackgroundUpon CD95/Fas ligation, the initiator caspase-8 is known to activate effector caspases leading to apoptosis. In the presence of zVAD-fmk, a broad-spectrum caspase inhibitor, Fas engagement can also trigger an alternative, non-apoptotic caspase-independent form of cell death, which is initiated by RIP1. Controversy exists as to the ability of caspase-10 to mediate cell death in response to FasL (CD95L or CD178). Herein, the role of caspase-10 in FasL-induced cell death has been re-evaluated.Methodology and Principal FindingsThe present study shows that FasL-induced cell death was completely impaired in caspase-8- and caspase-10-doubly deficient (I9-2e) Jurkat leukaemia T-cell lines. Over-expressing of either caspase-8 or caspase-10 in I9-2e cells triggered cell death and restored sensitivity to FasL, further arguing for a role of both initiator caspases in Fas apoptotic signalling. In the presence of zVAD-fmk, FasL triggered an alternative form of cell death similarly in wild-type (A3) and in caspase-8-deficient Jurkat cells expressing endogenous caspase-10 (clone I9-2d). Cell death initiated by Fas stimulation in the presence of zVAD-fmk was abrogated in I9-2e cells as well as in HeLa cells, which did not express endogenous caspase-10, indicating that caspase-10 somewhat participates in this alternative form of cell death. Noteworthy, ectopic expression of caspase-10 in I9-2e and HeLa cells restored the ability of FasL to trigger cell death in the presence of zVAD-fmk. As a matter of fact, FasL-triggered caspase-10 processing still occurred in the presence of zVAD-fmk.Conclusions and SignificanceAltogether, these data provide genetic evidence for the involvement of initiator caspase-10 in FasL-induced cell death and indicate that zVAD-fmk does not abrogate caspase-10 processing and cytotoxicity in Fas signalling. Our study also questions the existence of an alternative caspase-independent cell death pathway in Fas signalling.

E-Cadherin Protects Primary Hepatocyte Spheroids from Cell Death by a Caspase-Independent Mechanism

Cultivation of primary hepatocytes as spheroids creates an efficient three-dimensional model system for hepatic studies in vitro and as a cell source for a spheroid reservoir bioartificial liver. The mechanism of spheroid formation is poorly understood, as is an explanation for why normal, anchorage-dependent hepatocytes remain viable and do not undergo detachment-induced apoptosis, known as anoikis, when placed in suspension spheroid culture. The purpose of this study was to investigate the role of E-cadherin, a calcium-dependent cell adhesion molecule, in the formation and maintenance of hepatocyte spheroids. Hepatocyte spheroids were formed by a novel rocker technique and cultured in suspension for up to 24 h. The dependence of spheroid formation on E-cadherin and calcium was established using an E-cadherin blocking antibody and a calcium chelator. We found that inhibiting E-cadherin prevented cell-cell attachment and spheroid formation, and, surprisingly, E-cadherin inhibition led to hepatocyte death through a caspase-independent mechanism. In conclusion, E-cadherin is required for hepatocyte spheroid formation and may be responsible for protecting hepatocytes from a novel form of caspase-independent cell death.