Different Forms Of Cell Death Research Articles

Amino acids and mechanistic target of rapamycin regulate the fate of live engulfed cells.

Metabolic stress contributes to the regulation of cell death in normal and diseased tissues. While different forms of cell death are known to be regulated by metabolic stress, how the cell engulfment and killing mechanism entosis is regulated is not well understood. Here we find that the death of entotic cells is regulated by the presence of amino acids and activity of the mechanistic target of rapamycin (mTOR). Amino acid withdrawal or mTOR inhibition induces apoptosis of engulfed cells and blocks entotic cell death that is associated with the lipidation of the autophagy protein microtubule‐associated protein light chain 3 (LC3) to entotic vacuoles. Two other live cell engulfment programs, homotypic cell cannibalism (HoCC) and anti‐CD47 antibody‐mediated phagocytosis, known as phagoptosis, also undergo a similar vacuole maturation sequence involving LC3 lipidation and lysosome fusion, but only HoCC involves mTOR‐dependent regulation of vacuole maturation and engulfed cell death similar to entosis. We further find that the regulation of cell death by mTOR is independent of autophagy activation and instead involves the 4E‐BP1/2 proteins that are known regulators of mRNA translation. Depletion of 4E‐BP1/2 proteins can restore the mTOR‐regulated changes of entotic death and apoptosis rates of engulfed cells. These results identify amino acid signaling and the mTOR‐4E‐BP1/2 pathway as an upstream regulation mechanism for the fate of live engulfed cells formed by entosis and HoCC.

Entosis is induced by ultraviolet radiation

SummaryEntosis is a cell death mechanism that is executed through neighbor cell ingestion and killing that occurs in cancer tissues and during development. Here, we identify JNK and p38 stress-activated kinase signaling as an inducer of entosis in cells exposed to ultraviolet (UV) radiation. Cells with high levels of stress signaling are ingested and killed by those with low levels, a result of heterogeneity arising within cell populations over time.In stressed cells, entosis occurs as part of mixed-cell death response with parallel induction of apoptosis and necrosis, and we find that inhibition of one form of cell death leads to increased rates of another. Together, these findings identify stress-activated kinase signaling as a new inducer of entosis and demonstrate cross talk between different forms of cell death that can occur in parallel in response to UV radiation.

Abstract 2024: Studying the effects of interfering with doxorubicin-induced senescence in human colon cancer HCT116 cells

Abstract Colorectal cancer (CRC) is type of cancer that growing in the colon or the rectum. Although CRC can be treated and managed through several means of therapy, the disease mortality is still high in Saudi Arabia. Upon treatment, cancer cells undergo different form of cell death or growth arrest such as apoptosis and senescence. Senescence is a state of proliferation arrest that is considered as a cause of drug resistance. The current study was designed to interfere with Doxorubicin-induced senescence in cancer cells, and shift the cytotoxicity of doxorubicin from proliferation arrest to apoptosis. β-galactosidase (β-gal) staining has demonstrated that Doxorubicin induces senescence in wild-type HCT116 but not in p21 knock-out cells. (DAPI)/TUNEL stating and Annexin V assay indicate that treatment with Doxorubicin promotes apoptosis in p21 knock-out HCT116 significantly more than in wild-type HCT116. β-galactosidase staining and 5-Dodecanoylaminofluorescein Di-β-D-Galactopyranosid (C12FDG) demonstrated that combination of Doxorubicin with Sorafenib, Rapamycin, or Venetoclax has significantly reduced senescence and induced more apoptosis as shown by Propidium iodide (PI)/Annexin v assay and Caspase 3/7 Assay. PCR data analysis demonstrated that not all combination of Doxorubicin with other agents significantly decreased expression of senescence protein, however, the results of this study provide findings that interference with senescence pathways may shift cytotoxicity from senescence to apoptosis. The authors extend their appreciation to the Deputyship for Research and Innovation, Ministry of Education in Saudi Arabia for partly funding this work through the project number (DRI-KSU-1273). Citation Format: Moureq Rashed Alotaibi, Homood As Sobeia, Munirah Alohaydib, Faten Alaqil, Khalid Alhazzani, Ali Alhoshani, Tareq Saleh, David Gewirtz. Studying the effects of interfering with doxorubicin-induced senescence in human colon cancer HCT116 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2024.

Emerging concepts in the science of vaccine adjuvants.

Adjuvants are vaccine components that enhance the magnitude, breadth and durability of the immune response. Following its introduction in the 1920s, alum remained the only adjuvant licensed for human use for the next 70 years. Since the 1990s, a further five adjuvants have been included in licensed vaccines, but the molecular mechanisms by which these adjuvants work remain only partially understood. However, a revolution in our understanding of the activation of the innate immune system through pattern recognition receptors (PRRs) is improving the mechanistic understanding of adjuvants, and recent conceptual advances highlight the notion that tissue damage, different forms of cell death, and metabolic and nutrient sensors can all modulate the innate immune system to activate adaptive immunity. Furthermore, recent advances in the use of systems biology to probe the molecular networks driving immune response to vaccines (‘systems vaccinology’) are revealing mechanistic insights and providing a new paradigm for the vaccine discovery and development process. Here, we review the ‘known knowns’ and ‘known unknowns’ of adjuvants, discuss these emerging concepts and highlight how our expanding knowledge about innate immunity and systems vaccinology are revitalizing the science and development of novel adjuvants for use in vaccines against COVID-19 and future pandemics.

Urinary Cyclophilin A as Marker of Tubular Cell Death and Kidney Injury.

Background: Despite the term acute kidney injury (AKI), clinical biomarkers for AKI reflect function rather than injury and independent markers of injury are needed. Tubular cell death, including necroptotic cell death, is a key feature of AKI. Cyclophilin A (CypA) is an intracellular protein that has been reported to be released during necroptosis. We have now explored CypA as a potential marker for kidney injury in cultured tubular cells and in clinical settings of ischemia-reperfusion injury (IRI), characterized by limitations of current diagnostic criteria for AKI. Methods: CypA was analyzed in cultured human and murine proximal tubular epithelial cells exposed to chemical hypoxia, hypoxia/reoxygenation (H/R) or other cell death (apoptosis, necroptosis, ferroptosis) inducers. Urinary levels of CypA (uCypA) were analyzed in patients after nephron sparing surgery (NSS) in which the contralateral kidney is not disturbed and kidney grafts with initial function. Results: Intracellular CypA remained unchanged while supernatant CypA increased in parallel to cell death induction. uCypA levels were higher in NSS patients with renal artery clamping (that is, with NSS-IRI) than in no clamping (NSS-no IRI), and in kidney transplantation (KT) recipients (KT-IRI) even in the presence of preserved or improving kidney function, while this was not the case for urinary Neutrophil gelatinase-associated lipocalin (NGAL). Furthermore, higher uCypA levels in NSS patients were associated with longer surgery duration and the incidence of AKI increased from 10% when using serum creatinine (sCr) or urinary output criteria to 36% when using high uCypA levels in NNS clamping patients. Conclusions: CypA is released by kidney tubular cells during different forms of cell death, and uCypA increased during IRI-induced clinical kidney injury independently from kidney function parameters. Thus, uCypA is a potential biomarker of kidney injury, which is independent from decreased kidney function.

Cross talk between intracellular pathogens and cell death.

Infections with bacterial pathogens often results in the initiation of programmed cell death as part of the host innate immune defense, or as a bacterial virulence strategy. Induction of host cell death is controlled by an elaborate network of innate immune and cell death signaling pathways and manifests in different morphologically and functionally distinct forms of death, such as apoptosis, necroptosis, NETosis and pyroptosis. The mechanism by which host cell death restricts bacterial replication is highly cell-type and context depended, but its physiological importance is highlighted the diversity of strategies bacterial pathogens use to avoid induction of cell death or to block cell death signaling pathways. In this review, we discuss the latest insights into how bacterial pathogens elicit and manipulate cell death signaling, how different forms of cell death kill or restrict bacteria and how cell death and innate immune pathway cross talk to guard against pathogen-induced inhibition of host cell death.

Supervised Machine Learning for Semi-Quantification of Extracellular DNA in Glomerulonephritis.

Glomerular cell death is a pathological feature of myeloperoxidase anti neutrophil cytoplasmic antibody associated vasculitis (MPO-AAV). Extracellular deoxyribonucleic acid (ecDNA) is released during different forms of cell death including apoptosis, necrosis, necroptosis, neutrophil extracellular traps (NETs) and pyroptosis. Measurement of this cell death is time consuming with several different biomarkers required to identify the different biochemical forms of cell death. Measurement of ecDNA is generally conducted in serum and urine as a surrogate for renal damage, not in the actual target organ where the pathological injury occurs. The current difficulty in investigating ecDNA in the kidney is the lack of methods for formalin fixed paraffin embedded tissue (FFPE) both experimentally and in archived human kidney biopsies. This protocol provides a summary of the steps required to stain for ecDNA in FFPE tissue (both human and murine), quench autofluorescence and measure the ecDNA in the resulting images using a machine learning tool from the publicly available open source ImageJ plugin trainable Weka segmentation. Trainable Weka segmentation is applied to ecDNA within the glomeruli where the program learns to classify ecDNA. This classifier is applied to subsequent acquired kidney images, reducing the need for manual annotations of each individual image. The adaptability of the trainable Weka segmentation is demonstrated further in kidney tissue from experimental murine anti-MPO glomerulonephritis (GN), to identify NETs and ecMPO, common pathological contributors to anti-MPO GN. This method provides objective analysis of ecDNA in kidney tissue that demonstrates clearly the efficacy in which the trainable Weka segmentation program can distinguish ecDNA between healthy normal kidney tissue and diseased kidney tissue. This protocol can easily be adapted to identify ecDNA, NETs and ecMPO in other organs.

Multiple roles of caspase-8 in cell death, inflammation, and innate immunity.

Caspase-8 is an apical caspase involved in the programmed form of cell death called apoptosis that is critically important for mammalian development and immunity. Apoptosis was historically described as immunologically silent in contrast to other types of programmed cell death such as necroptosis or pyroptosis. Recent reports suggest considerable crosstalk between these different forms of cell death. It is becoming increasingly clear that caspase-8 has many non-apoptotic roles, participating in multiple processes including regulation of necroptosis (mediated by receptor-interacting serine/threonine kinases, RIPK1-RIPK3), inflammatory cytokine expression, inflammasome activation, and cleavage of IL-1β and gasdermin D, and protection against shock and microbial infection. In this review, we discuss the involvement of caspase-8 in cell death and inflammation and highlight its role in innate immune responses and in the relationship between different forms of cell death. Caspase-8 is one of the central components in this type of crosstalk.

Abstract B14: Immunomodulatory effect of tumor treating fields (TTFields) results in enhanced antitumor efficacy when combined with anti-PD-1 therapy

Abstract Tumor treating fields (TTFields) are a clinically applied antineoplastic treatment modality delivered via noninvasive application of low-intensity, intermediate-frequency, alternating electric fields. The antimitotic effects of TTFields include disruption of the mitotic spindle and abnormal chromosome segregation, which trigger different forms of cell death. In this study we evaluated whether TTFields-induced cell death can be perceived as immunogenic by the immune system. Murine Lewis lung carcinoma (LLC-1), CT-26 colon carcinoma, and ovarian surface epithelial (MOSE) cells were treated with TTFields using the inovitro system. Levels of calreticulin (CRT) on the surface of treated cells and intracellular ATP levels were evaluated using flow cytometry. High mobility group box 1 (HMGB1) secretion was measured using an ELISA assay. For detection of ER stress, phosphorylation of the translation initiation factor eIF2α was evaluated using immunoblot analysis. TTFields-induced upregulation of autophagy was evaluated using immunoblot analysis and immunofluorescence evaluation of LC3. Bone marrow-derived dendritic cells (DCs) were co-incubated with TTFields-treated LLC-1 cells and phagocytosis by DCs and DCs maturation were evaluated using flow cytometry. Mice orthotopically implanted with LLC cells were treated with TTFields, the immune checkpoint inhibitor anti-PD-1 or a combination of the two modalities. Tumor volume was monitored and flow cytometry analysis was performed for phenotypic characterization of infiltrating immune cells. We demonstrate that cancer cells that die under TTFields application exhibit release of HMGB1, ATP depletion from cells, and ER stress leading to CRT translocation to the cell surface, all of which are cardinal signs of immunogenic cell death (ICD). Moreover, we show that TTFields-treated cells promote in vitro phagocytosis by DCs and DC maturation as well as initiate inflammation in vivo. We also show that the combined treatment of lung tumor-bearing mice with TTFields plus the immune checkpoint inhibitor anti-PD-1 led to a significant decrease in tumor volume compared to anti-PD-1 alone or to the control group. Significant increases in CD45+ tumor-infiltrating cells were observed in the TTFields plus anti-PD-1 group. These infiltrating cells, specifically macrophages and DCs, demonstrated upregulation of surface PD-L1 expression. Correspondingly, cytotoxic T-cells isolated from these tumors have shown higher levels of IFN-γ production relative to untreated mice. Our results demonstrate the potential of TTFields therapy to induce ICD. We also demonstrate robust efficacy of concurrent application of TTFields and anti-PD-1 therapy in a mouse model of lung cancer. These data suggest that combining TTFields with anti-PD-1 might achieve tumor control by further enhancing antitumor immunity. Citation Format: Tali Voloshin, Shiri Davidi, Yaara Porat, Anna Shteingauz, Mijal Munster, Noa Kaynan, Rosa S Schneiderman, Catherine Tempel Brami, Yaniv Alon, Einav Zeevi, Karnit Gotlib, Roni Blat, Shay Cahal, Aviran Itzhaki, Moshe Giladi, Eilon D. Kirson, Uri Weinberg, Adrian Kinzel, Yoram Palti. Immunomodulatory effect of tumor treating fields (TTFields) results in enhanced antitumor efficacy when combined with anti-PD-1 therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B14.

Autophagy Function and Regulation in Kidney Disease

Autophagy is a dynamic process by which intracellular damaged macromolecules and organelles are degraded and recycled for the synthesis of new cellular components. Basal autophagy in the kidney acts as a quality control system and is vital for cellular metabolic and organelle homeostasis. Under pathological conditions, autophagy facilitates cellular adaptation; however, activation of autophagy in response to renal injury may be insufficient to provide protection, especially under dysregulated conditions. Kidney-specific deletion of Atg genes in mice has consistently demonstrated worsened acute kidney injury (AKI) outcomes supporting the notion of a pro-survival role of autophagy. Recent studies have also begun to unfold the role of autophagy in progressive renal disease and subsequent fibrosis. Autophagy also influences tubular cell death in renal injury. In this review, we reported the current understanding of autophagy regulation and its role in the pathogenesis of renal injury. In particular, the classic mammalian target of rapamycin (mTOR)-dependent signaling pathway and other mTOR-independent alternative signaling pathways of autophagy regulation were described. Finally, we summarized the impact of autophagy activation on different forms of cell death, including apoptosis and regulated necrosis, associated with the pathophysiology of renal injury. Understanding the regulatory mechanisms of autophagy would identify important targets for therapeutic approaches.

Single-Cell Mapping of Stress Response and Cell Death Pathways in Acute Myeloid Leukemia Reveals Stressor-Specific Alterations and Distinct Response Patterns

Single-Cell Mapping of Stress Response and Cell Death Pathways in Acute Myeloid Leukemia Reveals Stressor-Specific Alterations and Distinct Response Patterns

Characterization of TNF-induced cell death in Drosophila reveals caspase- and JNK-dependent necrosis and its role in tumor suppression

Tumor-necrosis factor (TNF) and its superfamily members are pleiotropic cytokines. Activation of TNF can lead to distinct cellular outcomes including inflammation, cell survival, and different forms of cell death, such as apoptosis and necrosis in a context-dependent manner. However, our understanding of what determines the versatile functions of TNF is far from complete. Here, we examined the molecular mechanisms that distinguish the forms of cell death induced by Eiger (Egr), the sole homolog of TNF in Drosophila. We show that expression of Egr in the developing Drosophila eye simultaneously induces apoptosis and apoptosis-independent developmental defects indicated by cellular disorganization, both of which rely on the c-Jun N-terminal kinase (JNK) signaling activity. Intriguingly, when effector caspases DrICE and Dcp-1 are defective or inhibited, expression of Egr triggers necrosis which is characterized by loss of cell membrane integrity, translucent cytoplasm, and aggregation of cellular organelles. Moreover, such Egr-induced necrosis depends on the catalytic activity of the initiator caspase Dronc and the input from JNK signaling but is independent of their roles in apoptosis. Further mosaic analysis with mutants of scribble (scrib), an evolutionarily conserved tumor suppressor gene regulating cell polarity, suggests that Egr/JNK-mediated apoptosis and necrosis establish a two-layered defense system to inhibit the oncogenic growth of scrib mutant cells. Together, we have identified caspase- and JNK-dependent mechanisms underlying Egr-induced apoptosis versus necrosis and their fail-safe roles in tumor suppression in an intact organism in vivo.

Molecular mechanism of pyroptosis in sepsis

Sepsis is a disease with a high mortality rate worldwide, which seriously threatens human life and health. Due to the complexity of its pathogenesis, diagnosis and treatment are often very difficult. Pyroptosis is a newly discovered pro-inflammatory form of programmed cell death, which occurs predominantly in professional phagocytes.During sepsis, appropriate pyroptosis is required for defense against bacterial infection, however, excessive pyroptosis will also aggravate the inflammatory reaction of sepsis. Therefore, the study of the signaling pathways and regulatory mechanisms of pyroptosis in sepsis may contribute to identify potential therapeutic targets. Hence, the study provide an overview of the recent advances which focus on the two signaling pathway of pyroptosis, some caspases which have found new effects, GSDM family and the crosstalk between different form of cell death. Key words: Sepsis; Bacterial infection; Reaserch; Pyroptosis; Imflammasome

Sensing Tissue Damage by Myeloid C-Type Lectin Receptors.

After both sterile and infectious insults, damage is inflicted on tissues leading to accidental or programmed cell death. In addition, events of programmed cell death also take place under homeostatic conditions, such as in embryo development or in the turnover of hematopoietic cells. Mammalian tissues are seeded with myeloid immune cells, which harbor a plethora of receptors that allow the detection of cell death, modulating immune responses. The myeloid C-type lectin receptors (CLRs) are one of the most prominent families of receptors involved in tailoring immunity after sensing dead cells. In this chapter, we will cover a diversity of signals arising from different forms of cell death and how they are recognized by myeloid CLRs. We will also explore how myeloid cells develop their sentinel function, exploring how some of these CLRs identify cell death and the type of responses triggered thereof. In particular, we will focus on DNGR-1 (CLEC9A), Mincle (CLEC4E), CLL-1 (CLEC12A), LOX-1 (OLR1), CD301 (CLEC10A) and DEC-205 (LY75) as paradigmatic death-sensing CLRs expressed by myeloid cells. The molecular processes triggered after cell death recognition by myeloid CLRs contribute to the regulation of immune responses in pathologies associated with tissue damage, such as infection, autoimmunity and cancer. A better understanding of these processes may help to improve the current approaches for therapeutic intervention.

How Can Interleukin-1 Receptor Antagonist Modulate Distinct Cell Death Pathways?

Multiple mechanisms of cell death exist (apoptosis, necroptosis, pyroptosis) and the subtle balance of several distinct proteins and inhibitors tightly regulates the cell fate toward one or the other pathway. Here, by combining coimmunoprecipitation, enzyme assays, and molecular simulations, we ascribe a new role, within this entangled regulatory network, to the interleukin-1 receptor antagonist (IL-1Ra). Our study enlightens that IL-1Ra, which usually inhibits the inflammatory effects of IL-1α/β by binding to IL-1 receptor, under advanced pathological states prevents apoptosis and/or necroptosis by noncompetitively inhibiting the activity of caspase-8 and -9. Consensus docking, followed by cumulative 10 μs of molecular dynamics simulations unprecedentedly reveal that IL-1Ra binds both caspases at their dimeric interface, preventing, in this manner, the formation of their catalytically/signaling active form. The resulting IL-1Ra/caspase-8(9) adducts are stabilized by hydrophobic and by few key hydrogen bonding interactions, formed by residues fully conserved across distinct caspases (-3, -6, -7, -8, and -9), and closely resemble the binding mode of the caspases inhibitors XIAP (X-linked inhibitor of apoptosis) and c-FLIP (cellular FLICE-like inhibitory protein). Tight regulation of the different forms of cell death has a major impact on distinct human illnesses (i.e., cancer, neurodegeneration, ischemic injury, atherosclerosis, viral/bacterial infections, and immune reaction). Hence, our study, pinpointing IL-1Ra as new actor of the intricate cell death regulatory network and gaining an atomic-scale understanding of its mechanism may open new avenues toward innovative therapeutic strategies to tackle major human diseases.

IMMU-52. TUMOR TREATING FIELDS (TTFIELDS) INDUCE IMMUNOGENIC CELL DEATH RESULTING IN ENHANCED ANTITUMOR EFFICACY WHEN COMBINED WITH ANTI-PD-1 THERAPY

Tumor Treating Fields (TTFields) are an effective anti-neoplastic treatment modality delivered via noninvasive application of low intensity (1–3V/cm), intermediate frequency (100–300kHz), alternating electric fields. TTFields are employed as a regional treatment modality using insulated transducer arrays applied to the skin, with the intent to kill tumor cells and reduce local recurrence. This therapy is approved for the treatment of patients with glioblastoma. Previous investigations have shown that TTFields disrupt microtubules and septin filaments, both of which govern key processes in mitosis. The outcomes of mitosis under TTFields application include abnormal chromosome segregation, which trigger different forms of cell death. In this study we evaluated whether TTFields-induced cell death can be immunogenic. We demonstrate that cancer cells that die during TTFields application exhibit endoplasmic reticulum (ER) stress leading to calreticulin translocation to the cell surface and release of damage-associated molecular patterns including the chromatin-binding protein HMGB1 and adenosine triphosphate. Further, we show that TTFields-treated cells promote phagocytosis by dendritic cells (DCs) and maturation of DCs under co-culture conditions. In vivo, the combined treatment of lung tumor-bearing mice with TTFields in combination with the immune checkpoint inhibitor anti-PD-1, significantly improved therapeutic efficacy compared to the control group or TTFields and anti-PD-1 alone. Significant increase in the number of tumor infiltrating immune cells was observed in the TTFields plus anti-PD-1 group. These infiltrating cells, specifically macrophages and DCs, demonstrated upregulation of surface PD-L1 expression. Correspondingly, cytotoxic T-cells isolated from these tumors have shown higher levels of IFN-γ production relative to untreated mice. Collectively, our results suggest that TTFields application induces both ER stress and autophagy, resulting in immunogenic cell death. Combining TTFields with anti-PD-1 may therefore achieve tumor control by further enhancing antitumor immunity.

Isoflavones increase radiation-induced apoptosis and modulate pro-inflammatory signaling in EA.hy926 endothelial cells.

Abstract Background The therapeutic use of x-ray ionizing radiation (XIR) for cancer treatment is limited by off target injury to normal tissues resulting in organ-specific toxicities with perpetual inflammation and fibrosis. XIR causes different forms of cell death including apoptosis, necrosis, and mitotic catastrophe that can dictate the resulting inflammatory response. Our lab has shown that soy isoflavones (SIF) reduce XIR-induced inflammation and fibrosis of thoracic organs. We now investigated the effect of SIF and XIR on endothelial cell death in vitro, which could play a role in SIF mitigation of inflammatory response. Methods EA.hy926 endothelial cells were treated with or without 30μM Novasoy 400 SIF for 24 hours prior to 3 Gy XIR and assessed at various time points. Cells were utilized for cell viability assays, stained for g-H2AX, Annexin-V, and NF-κB-p65, and lysed for western blot analysis of proteins involved in apoptosis and inflammatory signaling. Results Endothelial cell viability was significantly decreased for cells treated with SIF+XIR at 72 hours compared to XIR alone (p <0.05). Endothelial cells treated with SIF+XIR had increased staining of Annexin-V and g-H2AX staining at 48 hours compared to XIR alone (p <0.05). Western blot analysis showed increases in pro-apoptotic caspases 9 and 3 as well as Bax, IL-1b, ICAM-1 and VCAM-1 after cell treatment with SIF+XIR. Conclusions SIF enhances endothelial cell susceptibility to XIR-induced pro-apoptotic pathways resulting in greater apoptotic cell death. These findings suggest a mechanism of SIF-mediated radioprotection caused by increased apoptosis of normal cells and could limit the uncontrolled XIR-induced inflammatory signaling leading to organ fibrosis.

A novel role of the mitochondrial permeability transition pore in (−)-gossypol-induced mitochondrial dysfunction

Gossypol, a natural polyphenolic compound from cotton seeds, is known to trigger different forms of cell death in various types of cancer. Gossypol acts as a Bcl-2 inhibitor that induces apoptosis in apoptosis-competent cells. In apoptosis-resistant cancers such as glioblastoma, it triggers a non-apoptotic type of cell death associated with increased oxidative stress, mitochondrial depolarisation and fragmentation. In order to investigate the impact of gossypol on mitochondrial function, the mitochondrial permeability transition pore and on oxidative stress in more detail, we used the aging model Podospora anserina that lacks endogenous Bcl-2 proteins. We found that treatment with gossypol selectively increases hydrogen peroxide levels and impairs mitochondrial respiration in P. anserina, apoptosis-deficient Bax/Bak double knockout mouse embryonal fibroblasts and glioblastoma cells. Significantly, we provide evidence that CYPD-mediated opening of the mPTP is required for gossypol-induced mitochondrial dysfunction, autophagy and cell death during organismic aging of P. anserina and in glioblastoma cells. Overall, these data provide new insights into the role of the mPTP and autophagy in the antitumor effects of gossypol, a natural compound that is clinically developed for the treatment of cancer.

Abstract 3665: Tumor Treating Fields (TTFields) plus anti-PD-1 therapy induce immunogenic cell death resulting in enhanced antitumor efficacy

Abstract Tumor Treating Fields (TTFields) are an effective anti-neoplastic treatment modality delivered via noninvasive application of low intensity, intermediate frequency, alternating electric fields. TTFields is approved for the treatment of both newly diagnosed and recurrent glioblastoma. TTFields interrupt cancer cell mitosis by disrupting microtubules and septin filaments, which play key roles in mitosis. The mitotic effects of TTFields include abnormal chromosome segregation and ER stress, which trigger different forms of cell death. We evaluated the in vitro and in vivo effects of TTFields combined with an immune checkpoint inhibitor (anti-PD1) on immunogenic cell death. Murine Lewis lung carcinoma (LLC) and ovarian surface epithelial (MOSE) cells were treated with TTFields using the inovitroTM system. Levels of calreticulin (CRT) on the surface of treated cells and intracellular ATP levels were evaluated using flow cytometry. High mobility group box 1 (HMGB1) secretion was measured using an ELISA assay. Mice were implanted with LLC cells were treated with TTFields, anti-PD-1, or a combination of the two modalities. Tumor volume was monitored; flow cytometry analysis was performed for phenotypic characterization of infiltrating immune cells. TTFields induced elevated cell surface expression of CRT, decreased intracellular ATP levels, and promoted HMGB1 secretion. In vivo, the combined treatment of lung tumor-bearing mice with TTFields plus anti-PD-1 led to a significant decrease in tumor volume compared to anti-PD-1 alone or to the control group. Significant increases in CD45+ tumor infiltrating cells were observed in the TTFields plus anti-PD-1 group. Infiltrating cells demonstrated a significant upregulation of surface PD-L1 expression. Both F4/80+CD11b+ cells and CS11c+ cells exhibited higher tumor infiltration and elevated PD-L1 expression as compared to infiltrating immune cell in the control group. Our results demonstrate that TTFields treatment potentiates immunogenic cell death in cancer cells. Combining TTFields with specific immunotherapies such as anti-PD-1 may enhance antitumor immunity and result in increased tumor control. Citation Format: Tali Voloshin, Orna Tal-Yitzhaki, Noa Kaynan, Moshe Giladi, Anna Shteingauz, Mijal Munster, Roni Blat, Yaara Porat, Rosa S. Schneiderman, Shay Cahal, Aviran Itzhaki, Eilon D. Kirson, Uri Weinberg, Yoram Palti. Tumor Treating Fields (TTFields) plus anti-PD-1 therapy induce immunogenic cell death resulting in enhanced antitumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3665. doi:10.1158/1538-7445.AM2017-3665

Cell Death in the Vessel Wall: The Good, the Bad, the Ugly.

Atherosclerotic vascular disease results from an imbalance of inflammatory and vascular cell accumulation and removal in the neointimal space. When pathways that promote cell recruitment, survival, and proliferation are favored over to those that activate cell death, egress, and clearance, the plaque expands. In contrast, programmed cell death and the efficient clearance of apoptotic bodies by efferocytosis reduce lesion cellularity and promote a reparative environment and lesion stability. However, should these carefully balanced pathways become disturbed, lesions can accumulate cell debris, damaged associated molecular patterns, and arrested macrophages, all contributing to the proinflammatory environment and lesion instability. Here, we review the latest understanding of how cell death in the vessel wall directly coordinates the development of atherosclerosis, and what molecular signals are orchestrating these pathways. We discuss the necessity of cell death and the ways in which the execution of different forms of cell death can direct different outcomes in the plaque, and how promoting the effective clearance of dead cells from the lesion is looking like a promising therapeutic path forward. In the setting of excess cholesterol and fat, inflammatory pathways of the innate immune system are aberrantly activated. It is widely believed that oxidative modifications of the phospholipid and protein moieties of low- and very–low-density lipoproteins trigger conserved nonspecific scavenging pathways in macrophages and dendritic cells. This initiates what is at first considered a protective pathway to remove unwanted and potentially cytotoxic remnants and debris. However, during the decades of life wherein circulating low- and very–low-density lipoprotein cholesterol are in excess, this protective response turns into a chronic state of inflammation that recruits additional inflammatory cells and activates vascular cells to promote lesion expansion. During the progression of atherosclerosis, like in many other diseases, there is a constant turnover of cells within and surrounding the plaque. The identification …