Death Receptor Signaling Research Articles

RNA Sequencing Reveals Mechanisms Underlying Modulation of Hodgkin Lymphoma Cells and Tumor Microenvironment By the PI3K δ/γ Inhibitor, RP6530

INTRODUCTION: Aberrant activation of the phosphoinositide 3-kinase (PI3K) pathway plays a central role in Hodgkin Lymphoma (HL) pathogenesis. The pleiotropic activities of the δ and γ isoforms of PI3K in regulating the interactions of Hodgkin/Reed-Sternberg (HRS) cells with the tumor microenvironment suggest thatPI3K inhibitors might exert a therapeutic potential in the treatment of HL. The dual PI3K δ/γ inhibitor RP6530 was proven to affect HL tumor cells and to exert immunosuppressive activities. However, a genome-wide search for molecular mediators of RP6530 is largely unexplored in HL. The present study was therefore aimed at evaluating the anti-lymphoma functions and the underlying genes affected by RP6530 in HL cell lines.METHODS: We identified differentially expressed genes (DEGs) in two HL cell lines (L-540 and KM-H2) treated with RP6530 for 6 and 24 h by next generation RNA sequencing (RNA-Seq). The expression level of some of these genes was verified in HL cell lines using real time-PCR. Ingenuity Pathway Analysis (IPA) software was used to assign the DEGs to distinct canonical pathways and to predict the biological functions of the transcriptional changes. Gene silencing experiments were used to investigate and validate potential predictive biomarkers of response to RP6530 treatment. Additionally, serum levels of Thymus- and activation-regulated chemokine (TARC/CCL17) were analyzed by commercially available sandwich enzyme-linked immunosorbent assay (ELISA) in RP6530-treated HL patients enrolled in a phase 1 study.RESULTS: RNA-Seq analysis following treatment of L-540 cells for 6 h caused an upregulation of 1544 and downregulation of 839 genes. After a 24 h treatment period, 625 genes remained upregulated while the number of genes that were downregulated increased to 1362 (FDR ≤ 0.1). In KM-H2 cells, treatment with RP6530 for 6 or 24 h upregulated 137 and 980 genes while downregulating 139 and 833 genes, respectively (FDR ≤ 0.1). We further classified modulated genes according to their canonical pathways using the IPA suite. The most significantly upregulated pathways detected in both L-540 and KM-H2 cell lines included apoptosis, p53, death receptor signaling and G1/S cell cycle checkpoint regulation. In contrast, PI3K/AKT, ERK/MAPK, JAK/Stat, CD40, IL-4, IL-6, IL-10 and iNOS signaling were downregulated. We also identified 118 downregulated genes shared between the two cell lines that were associated with glycolysis, HIF1, chemokine and cytokine signaling. Upon RP6530 incubation, RNA-seq analysis showed a strikingly reduced expression of Pyruvate kinase muscle isozyme M2 (PKM2), which is a limiting glycolytic enzyme that catalyzes the final step in glycolysis. Here we show that lactic acid produced by tumor cells, as a by-product of glycolysis, has a critical function in inducing the M2-like polarization of tumor-associated macrophages (TAMs). Furthermore, we demonstrated that this effect was mediated by PKM2 and was inhibited after RP6530 treatment.We also found that the lactate-induced expression of M2 markers by macrophages, i.e., CCL-17 and CCL-22, were markedly inhibited both by RP6530 [-30% and -50% (mean of HL cells), respectively] and PKM2 silencing [-50% and -30% (mean of HL cells), respectively]. However, RP6530 increased the expression of M1 markers, switching the activation of macrophages from an immunosuppressive M2-like phenotype to an inflammatory M1-like state. PKM2 pathway therefore appears to be a critical determinant of RP6530-mediated inhibition of M2 macrophage polarization. Finally, RP6530 reduced the expression of key inflammation- and immunity-related genes as well as cytokines/chemokines and angiogenic factors (including, TARC/CCL17, CCL22, CCL5, IL-7, IL-13, and VEGF) known to be secreted by HRS cells to promote a pro-tumoral microenvironment. As the chemokine TARC/CCL17 is considered a biomarker of disease activity in HL patients, we observed a severe reduction of serum TARC/CCL17 levels in RP6530-treated HL patients that achieved CR or PR, but not in PD patients. Collectively, these findings suggest that RP6530 affects a mechanism of communication between HL tumor cells and tumor microenvironment.CONCLUSIONS: Results demonstrate that RP6530 can affect both HL tumor cells and HL tumor microenvironment, suggesting that a novel therapeutic opportunity may be achievable for the treatment of HL patients. DisclosuresViswanadha:Incozen Therapeutics: Employment. Vakkalanka:Rhizen Pharmaceuticals S A: Employment, Equity Ownership. Santoro:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees. Carlo-Stella:Boehringer Ingelheim: Consultancy, Honoraria; Rhizen Pharmaceuticals: Research Funding; ADC Therapeutics: Research Funding.

Bone Marrow Infiltrating T-Cell Signatures Following Allo-HCT for Adult Acute Lymphoblastic Leukaemia Differentiate between Patients in Continuous CR and Those with Persistent Minimal Residual Disease or Relapse

Allogeneic stem cell transplantation (allo-HCT) has a proven survival benefit in adult acute lymphoblastic leukaemia (ALL) and remains the most commonly applied immunotherapy. The mechanism of graft-versus-leukaemia (GVL) in allo-HCT relies upon donor T cell immunity. After contributing to initial control of ALL, allo-reactive anti-tumour T cells may either persist, thus providing continuing immune surveillance, or undergo deletion/loss of function as a result of both extrinsic regulation and/or exhaustion. Therefore, there is a clear need to identify and track T cells with potential anti-ALL reactivity. UKALL14 (ISRCTN66541317) is an ongoing clinical trial for adults with de novo ALL in the UK in which all patients over 40 years are regarded as ‘high risk’ and are assigned to reduced intensity-conditioned allo-HCT. Minimal residual disease (MRD) monitoring - by immunoglobulin heavy chain/ T cell receptor quantification - of bone marrow and peripheral blood multilineage chimerism are assessed at regular intervals for 2 years post allo-HCT.We hypothesised that it would be possible to differentiate bone marrow infiltrating T-cell (BiL-T) signatures in patients in complete molecular remission versus those with MRD or frank clinical relapse. We have evaluated the feasibility of performing gene expression profiling on BiL-T cells from cryopreserved bone marrow samples of patients following allo-HCT and correlated this information with clinical outcome data.BiL-T were flow-sorted using the BD FACSAria ™ Fusion into CD3+,TCRab+, CD4+ and CD3+,TCRab+, CD8+. After extraction of total mRNA, samples with an RNA integrity index (RIN) >7 were subjected to RNA sequencing using the Illumina Hiseq. Data were analysed using differential expression gene analysis (DeSeq 2) and gene set enrichment analysis (GSEA). The Human Immunology V2 panel on the NanoString nCounter® SPRINT Profiler was used for gene expression profiling of samples with adequate RNA but a RIN <7. Gene expression data was correlated with molecular MRD data and other clinical parameters, including T cell chimerism where available.Both CD4+ and CD8+ subsets of BiL-T were successfully sorted from all 15 cryopreserved samples. DeSeq2 and GSEA of the RNAseq data was performed for CD4+TCRab+ BiL (n=5; 3 MRD- and 2 MRD+/relapse) (Figure 1) and CD8+TCRab+ (n=6; 4 MRD- and 2 MRD+/relapse). Despite the modest number of samples in this feasibility study, CD4+ T cells from patients with relapsing or MRD+ ALL showed significantly increased expression of genes associated with terminal differentiation (e.g. LAG3, GZMK); by contrast, there was lower expression of genes associated with memory differentiation and proliferative fitness (e.g. MYC, CCR7), a finding linked to increased pathway enrichment for PD-1 and telomere maintenance. CD8+ T cells from patients with relapsing or MRD+ ALL also demonstrated a terminally differentiated signature (e.g. increased expression of KLRC1 encoding NKG2A and lower expression of CCR7). GSEA showed enrichment for pathways linked to oxidative phosphorylation and interaction with non-haematopoetic stroma). We also analysed n=9 CD8+TCRab+ samples (n=4 MRD- and n=5 MRD+ or relapse) by Nanostring. These groups could also be segregated on the basis of genes representing pathways related to T cell survival, death receptor signalling and TCR signalling; in each case, CD8+ T cells from MRD+ or relapsing patients were characterised by a hypo-functional and apoptosis-prone signature.Our data show features suggestive of T cell exhaustion and altered metabolomes in patients with residual ALL post-allo-HCT, whether they are MRD+ or in frank clinical relapse; these features were not present in patients in continuous remission. To further test the hypothesis that T cell dysfunction significantly compromises GVL efficacy, we intend to perform transcriptional profiling of BiL-T in a wider cohort of UKALL14 patients who underwent allo-HCT. Since clinical strategies for reversal of T cell exhaustion including donor lymphocyte infusion (DLI) and potential PD-1 blockade already exist, we hope the impact of this project will be to determine precisely when and in whom to use these strategies during therapy for adult ALL. We expect that our findings will have relevance to the wider field of immunotherapy for ALL including blinatumomab and CAR-T approaches. [Display omitted] DisclosuresFielding:Baxalta: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Noncanonical NF-κB Signaling Upregulation in Inflammatory Bowel Disease Patients is Associated With Loss of Response to Anti-TNF Agents.

Objectives: Targeting tumor necrosis factor (TNF) with biologic agents, such as infliximab and adalimumab, is a widely used and effective therapeutic strategy in inflammatory bowel disease (IBD). Unfortunately, a significant number of patients fail to respond or lose response over time to these agents. Previous studies have defined multiple complex roles for canonical NF-κB signaling in the pathogenesis of IBD. However, preliminary evidence suggests that the lesser defined noncanonical NF-κB signaling pathway also contributes to disease pathogenesis and response to anti-TNF agents. The objective of this study was to evaluate this hypothesis in Crohn’s disease (CD) and ulcerative colitis (UC) patients. Design: A total of 27 subjects with IBD (19 with CD and 8 with UC) and 15 control subjects were tested. Clinical criteria, patient history, and endoscopic disease activity were factors used to categorize patients and define therapeutic response. Biopsy specimens were collected during colonoscopy and expression was determined for 88 target genes known to be associated with noncanonical NF-κB signaling and IBD. Results: Noncanonical NF-κB signaling was significantly upregulated in IBD patients and was associated with increased gastrointestinal inflammation, epithelial cell death, lymphocyte migration, and Nod-like receptor signaling. Furthermore, noncanonical NF-κB signaling was further upregulated in patients unresponsive to anti-TNF agents and was suppressed in responsive patients. MAP3K14, NFKB2, CCL19, CXCL12, and CXCL13 were significantly dysregulated, as were genes that encode pathway regulators, such as CYLD, NLRP12, and BIRC2/3. Conclusion: Our study identifies a previously uncharacterized role for the understudied noncanonical NF-κB signaling pathway in the pathogenesis of IBD and anti-TNF therapy responsiveness. The genes and pathways identified may ultimately prove useful in IBD management and could potentially be used as biomarkers of drug response.

Tannic acid repair of zearalenone-induced damage by regulating the death receptor and mitochondrial apoptosis signaling pathway in mice

Zearalenone (ZEA) is an estrogenic toxin produced by Fusarium strains, that is widely present in crops, and endangers the reproductive system of animals. Tannic acid (TA) is a natural polyphenolic substance that is widespread in the roots, stems, and leaves of plants, and has special pharmacological activity. This study was designed to investigate the therapeutic effect of TA on ZEA-induced ovarian damage in mice and to explore the molecular mechanism involved. Ninety healthy Kunming female mice were divided into six equal groups. All the groups but the control group were administered daily with ZEA [10 mg/kg body weight (bw)] orally, for 7 days, to induce damage to the reproductive system. Some groups were also administered with TA (50, 100, and 200 mg/bw) for 7 days. Mice were euthanized 24 h later to allow for collection of serum and ovaries. TA can effectively alleviate the appearance of congestion and redness of the ovary, caused by ZEA, and increase the number of healthy growing follicles. Moreover, the estrogen content and the levels of MDA and ROS in the ovaries can be effectively reduced by TA. It can also reduce the apoptosis of ovarian cells, decreases the protein expression of the estrogen receptor, Fas, Fasl, caspase-3, caspase-8, caspase-9, and Bax, and increases the protein expression of Bcl-2. Our study indicates that TA reduces the strong estrogen and oxidative damage induced by ZEA, and these therapeutic effects may be partially mediated by the death receptor and mitochondrial apoptosis signaling pathway.

Regulation of Death Receptor Signaling by S-Palmitoylation and Detergent-Resistant Membrane Micro Domains-Greasing the Gears of Extrinsic Cell Death Induction, Survival, and Inflammation.

Simple SummaryDeath receptor activation can induce various signaling cascades ranging from cell survival to different forms of regulated cell death. The diversification of these biological outcomes frequently depends on the subcellular localization of the receptors. Activation by their ligands at the plasma membrane can change their plasma membrane localization to form distinct receptor–ligand bound signaling complexes. Receptors can also be internalized to signal from endosomes or the nuclear compartment. Those signaling complexes can be further remodeled en route and are partially released to signal from the cytoplasm. Reversible post-translational modification via S-Palmitoylation, a form of lipidation, emanated as a major regulator of death receptor signaling over the past years. We highlight what is known about S-Palmitoylation in different receptor systems, how it affects localization of the receptor complexes in specialized membrane micro domains, and the functional consequences and therapeutical potential of altered S-palmitoylation in the respective signaling cascades.Death-receptor-mediated signaling results in either cell death or survival. Such opposite signaling cascades emanate from receptor-associated signaling complexes, which are often formed in different subcellular locations. The proteins involved are frequently post-translationally modified (PTM) by ubiquitination, phosphorylation, or glycosylation to allow proper spatio-temporal regulation/recruitment of these signaling complexes in a defined cellular compartment. During the last couple of years, increasing attention has been paid to the reversible cysteine-centered PTM S-palmitoylation. This PTM regulates the hydrophobicity of soluble and membrane proteins and modulates protein:protein interaction and their interaction with distinct membrane micro-domains (i.e., lipid rafts). We conclude with which functional and mechanistic roles for S-palmitoylation as well as different forms of membrane micro-domains in death-receptor-mediated signal transduction were unraveled in the last two decades.

Arginine-tocopherol bioconjugated lipid vesicles for selective pTRAIL delivery and subsequent apoptosis induction in glioblastoma cells

The incorporation of specific therapeutic gene into glioblastoma offers potent therapeutic strategy to treat the disease. Non-viral gene delivery vectors are of particular interest due to their tuneable transfection efficiency and easy scale-up. Herein, we demonstrate successful delivery of plasmid encoding tumor necrosis factor (TNF)-related apoptosis-inducing ligand (pTRAIL) using arginine-conjugated tocopherol lipid (AT) nanovesicles into glioblastoma cell lines. Another cationic lipid, glycine-conjugated tocopherol lipid (GT) having glycine in the head group region is also synthesized as a control lipid. Both lipid-derived liposomes effectively condensed the pDNA and the corresponding biomacromolecular assemblies (lipoplexes) are efficiently transfected into different cell lines. AT-based liposomes exhibit higher transfection efficacy in various cell lines, particularly selective in glioma cell lines. At an optimized N/P ratio, both the liposomal formulations show low cytotoxicity. AT-based lipoplexes have superior cellular uptake in U87 than the control lipid GT. The expression of TRAIL protein regulated death receptor and apoptosis signaling pathway is assayed by western blot using transfection of AT-based/pTRAIL into U87 cell lines. Induction of apoptosis in U87 cells exposed to AT-based/pTRAIL plasmid is evaluated by MTT assay as well as Annexin V-propidium iodide dual-staining assay. All results indicate that the developed AT-based/pTRAIL system offers a potentially safe and efficient therapeutic strategy for glioblastoma gene therapy.

Upregulation of endogenous TRAIL-elicited apoptosis is essential for metformin-mediated antitumor activity against TNBC and NSCLC

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) shows promising antitumor activity in preclinical studies. However, the efficacy of recombinant TRAIL in clinical trials is compromised by its short serum half-life and low in vivo stability. Induction of endogenous TRAIL may overcome the limitations and become a new strategy for cancer treatment. Here, we discovered that metformin increased TRAIL expression and induced apoptosis in triple-negative breast cancer (TNBC) and non-small cell lung cancer (NSCLC) cells. Metformin did not alter the expression of TRAIL receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Metformin-upregulated TRAIL was secreted into conditioned medium (CM) and found to be functional, since the CM promoted TNBC cells undergoing apoptosis, which was abrogated by a recombinant TRAIL-R2-Fc chimera. Moreover, blockade of TRAIL binding to DR4/DR5 or specific knockdown of TRAIL expression significantly attenuated metformin-induced apoptosis. Studies with a tumor xenograft model revealed that metformin not only significantly inhibited tumor growth but also elicited apoptosis and enhanced TRAIL expression in vivo. Collectively, we have demonstrated that upregulation of TRAIL and activation of death receptor signaling are pivotal for metformin-induced apoptosis in TNBC and NSCLC cells. Our studies identify a novel mechanism of action of metformin exhibiting potent antitumor activity via induction of endogenous TRAIL.

Small-Molecule Natural Product Physachenolide C Potentiates Immunotherapy Efficacy by Targeting BET Proteins.

Screening for sensitizers of cancer cells to TRAIL-mediated apoptosis identified a natural product of the 17β-hydroxywithanolide (17-BHW) class, physachenolide C (PCC), as a promising hit. In this study, we show that PCC was also able to sensitize melanoma and renal carcinoma cells to apoptosis in response not only to TRAIL, but also to the synthetic polynucleotide poly I:C, a viral mimetic and immune activator, by reducing levels of antiapoptotic proteins cFLIP and Livin. Both death receptor and TLR3 signaling elicited subsequent increased assembly of a proapoptotic ripoptosome signaling complex. Administration of a combination of PCC and poly I:C in human M14 melanoma xenograft and a syngeneic B16 melanoma model provided significant therapeutic benefit as compared with individual agents. In addition, PCC enhanced melanoma cell death in response to activated human T cells in vitro and in vivo in a death ligand-dependent manner. Biochemical mechanism-of-action studies established bromo and extraterminal domain (BET) proteins as major cellular targets of PCC. Thus, by targeting of BET proteins to reduce antiapoptotic proteins and enhance caspase-8-dependent apoptosis of cancer cells, PCC represents a unique agent that can potentially be used in combination with various immunotherapeutic approaches to promote tumor regression and improve outcome. SIGNIFICANCE: These findings demonstrate that PCC selectively sensitizes cancer cells to immune-mediated cell death, potentially improving the efficacy of cancer immunotherapies. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3374/F1.large.jpg.

Sweet modification and regulation of death receptor signalling pathway

Death receptors, members of the tumour necrosis factor receptor (TNFR) superfamily, are characterized by the presence of a death domain in the cytosolic region. TNFR1, Fas and TNF-related apoptosis-inducing ligand receptors, which are prototypical death receptors, exert pleiotropic functions in cell death, inflammation and immune surveillance. Hence, they are involved in several human diseases. The activation of death receptors and downstream intracellular signalling is regulated by various posttranslational modifications, such as phosphorylation, ubiquitination and glycosylation. Glycosylation is one of the most abundant and versatile modifications to proteins and lipids, and it plays a critical role in the development and physiology of organisms, as well as the pathology of many human diseases. Glycans control a number of cellular events, such as receptor activation, signal transduction, endocytosis, cell recognition and cell adhesion. It has been demonstrated that oligo- and monosaccharides modify death receptors and intracellular signalling proteins and regulate their functions. Here, we review the current understanding of glycan modifications of death receptor signalling and their impact on signalling activity.

TRAIL Mediates Neuronal Death in AUD: A Link between Neuroinflammation and Neurodegeneration.

Although the cause of progressive neurodegeneration is often unclear, neuronal death can occur through several mechanisms. In conditions such as Alzheimer’s or alcohol use disorder (AUD), Toll-like receptor (TLR) induction is observed with neurodegeneration. However, links between TLR activation and neurodegeneration are lacking. We report a role of apoptotic neuronal death in AUD through TLR7-mediated induction of death receptor signaling. In postmortem human cortex, a two-fold increase in apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in neurons was found in AUD versus controls. This occurred with the increased expression of TLR7 and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) death receptors. Binge ethanol treatment in C57BL/6 mice increased TLR7 and induced neuronal apoptosis in cortical regions that was blocked by TLR7 antagonism. Mechanistic studies in primary organotypic brain slice culture (OBSC) found that the inhibition of TLR7 and its endogenous ligand let-7b blocked ethanol-induced neuronal cell death. Both IMQ and ethanol induced the expression of TRAIL and its death receptor. In addition, TRAIL-neutralizing monoclonal antibodies blocked both imiquimod (IMQ) and ethanol induced neuronal death. These findings implicate TRAIL as a mediator of neuronal apoptosis downstream of TLR7 activation. TLR7 and neuronal apoptosis are implicated in other neurodegenerative diseases, including Alzheimer’s disease. Therefore, TRAIL may represent a therapeutic target to slow neurodegeneration in multiple diseases.

Anthracycline-Induced Cardiotoxicity: Molecular Insights Obtained from Human-Induced Pluripotent Stem Cell\u2013Derived Cardiomyocytes (hiPSC-CMs)

Anthracyclines are a class of chemotherapy drugs that are highly effective for the treatment of human cancers, but their clinical use is limited by associated dose-dependent cardiotoxicity. The precise mechanisms by which individual anthracycline induces cardiotoxicity are not fully understood. Human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) are emerging as a physiologically relevant model to assess drugs cardiotoxicity. Here, we describe an assay platform by coupling hiPSC-CMs and impedance measurement, which allows real-time monitoring of cardiomyocyte cellular index, beating amplitude, and beating rate. Using this approach, we have performed comparative studies on a panel of four anthracycline drugs (doxorubicin, epirubicin, idarubicin, and daunorubicin) which share a high degree of structural similarity but are associated with distinct cardiotoxicity profiles and maximum cumulative dose limits. Notably, results from our hiPSC-CMs impedance model (dose-dependent responses and EC50 values) agree well with the recommended clinical dose limits for these drugs. Using time-lapse imaging and RNAseq, we found that the differences in anthracycline cardiotoxicity are closely linked to extent of cardiomyocyte uptake and magnitude of activation/inhibition of several cellular pathways such as death receptor signaling, ROS production, and dysregulation of calcium signaling. The results provide molecular insights into anthracycline cardiac interactions and offer a novel assay system to more robustly assess potential cardiotoxicity during drug development.

Conversion of the death inhibitor ARC to a killer activates pancreatic β cell death in diabetes.

Loss of insulin-secreting pancreatic β cells through apoptosis contributes to the progression of type 2 diabetes, but underlying mechanisms remain elusive. Here, we identify a pathway in which the cell death inhibitor ARC paradoxically becomes a killer during diabetes. While cytoplasmic ARC maintains β cell viability and pancreatic architecture, a pool of ARC relocates to the nucleus to induce β cell apoptosis in humans with diabetes and several pathophysiologically distinct mouse models. β cell death results through the coordinate downregulation of serpins (serine protease inhibitors) not previously known to be synthesized and secreted by β cells. Loss of the serpin α1-antitrypsin from the extracellular space unleashes elastase, triggering the disruption of β cell anchorage and subsequent cell death. Administration of α1-antitrypsin to mice with diabetes prevents β cell death and metabolic abnormalities. These data uncover a pathway for β cell loss in type 2 diabetes and identify an FDA-approved drug that may impede progression of this syndrome.

Non-steroidal anti-inflammatory drugs induce immunogenic cell death in suppressing colorectal tumorigenesis.

Use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with reduced risk of colorectal cancer (CRC). However, the mechanism by which NSAIDs suppress colorectal tumorigenesis remains unclear. We previously showed that NSAIDs selectively kill emerging tumor cells via death receptor (DR) signaling and a synthetic lethal interaction mediated by the proapoptotic Bcl-2 family protein BID. In this study, we found NSAIDs induce endoplasmic reticulum (ER) stress to activate DR signaling and BID in tumor suppression. Importantly, our results unveiled an ER stress- and BID-dependent immunogenic effect of NSAIDs, which may be critical for tumor suppression. NSAID treatment induced hallmarks of immunogenic cell death (ICD) in CRC cells and colonic epithelial cells upon loss of APC tumor suppressor, and elevated tumor-infiltrating lymphocytes (TILs) in the polyps of APCMin/+ mice. ER stress inhibition or BID deletion abrogated the antitumor and immunogenic effects of NSAIDs. Furthermore, increased ER stress and TILs were detected in human advanced adenomas from NSAID-treated patients. Together, our results suggest that NSAIDs induce ER stress- and BID-mediated ICD to restore immunosurveillance and suppress colorectal tumor formation.

Exploratory Investigation of Intestinal Structure and Function after Stroke in Mice.

Stroke is the second leading cause of death worldwide. Patients who have a stroke are susceptible to many gastrointestinal (GI) complications, such as dysphagia, GI bleeding, and fecal incontinence. However, there are few studies focusing on the GI tract after stroke. The current study is to investigate the changes of intestinal structure and function in mice after ischemic stroke. Ischemic stroke was made as a disease model in mice, in which brain and ileal tissues were collected for experiments on the 1st and 7th day after stroke. Intestinal motility of mice was inhibited, and intestinal permeability was increased after stroke. Hematoxylin-eosin (HE) staining showed the accumulation of leucocytes in the intestinal mucosa. Myeloperoxidase (MPO) activity and inflammatory proteins (nuclear factor kappa-B (NF-κB), inducible nitric oxide synthase (iNOS)) in the small intestine were significantly increased in mice after stroke. The expression of tight junction (TJ) proteins (zonula occludens-1 (ZO-1), occludin, and claudin-1) was downregulated, and transmission electron microscopy (TEM) showed broken TJ of the intestinal mucosa after stroke. Glial fibrillary acidic protein (GFAP) and the apoptosis-associated proteins (tumor necrosis factor (TNF-α), caspase-3, and cleaved caspase-3) were notably upregulated as well. Ischemic stroke led to negative changes on intestinal structure and function. Inflammatory mediators and TNF-α-induced death receptor signaling pathways may be involved and disrupt the small intestinal barrier function. These results suggest that stroke patients should pay attention to GI protection.

Xylooligosaccharide supplementation improved growth performance and prevented intestinal apoptosis in grass carp

The purpose of this study was to confirm the efficacy of xylooligosaccharides (XOS) on the growth performance and intestinal apoptosis of on-growing grass carp Ctenopharyngodon idella (167.46 ± 0.61 g). Fish were separated into six groups, which were given six different types of diet (supplemented with 0.00%, 0.002%, 0.004%, 0.006%, 0.008% and 0.010% of XOS) for 60 days. Grass carp were challenged with Aeromonas hydrophila (6 days) after the growth experiment. To our knowledge, the present study is the first to report that appropriate XOS supplementation: (1) improved growth performance in fish and (2) attenuated apoptosis via the internal mitochondrial signalling pathways (JNK and p38MAPK/Cytochrome-c/caspase-9) in the proximal intestine (PI) and mid intestine (MI) and the external death receptor signalling pathways (p38MAPK/FasL/caspase-8) in the distal intestine (DI). Interestingly, XOS supplementation had no effect on the mRNA levels of factor-related apoptosis ligand (FasL) or caspase-8 in the PI and MI, Bcl-2 in the PI and JNK, caspase-9, Bax or Apaf-1 in the DI. Finally, appropriate levels of XOS supplementation based on the percentage of weight gain (PWG), feed intake (FI), and caspase-3, -8, and -9 activities in the MI were estimated to be 51.81, 51.61, 57.15, 57.90, and 55.36 mg/kg diet, respectively.

Combined treatment of cisplatin and 5-Fluorouracil Induces cell death in ARPE-19 cells through the endoplasmic reticulum stress pathway

Aim: Cisplatin and 5-Fluorouracil have been used for about 50 years as chemotherapy agents. Combination of these two agents is known as PF therapy and is widely used in the treatment of anal, esophageal cancer, head and neck cancer. One of the problems encountered with antineoplastic agents is the rapid and irreversible side effects on non-target cells and organs. It is common that occourence of ocular toxicity by chemotherapy. Materials and Methods: ARPE-19 cell viability and toxicity were determined by performing (MTT)-based colorimetric assays and IC50 values calculated by probit analyses options of SPSS 20 Software. Acridine orange/Ethidium bromide (AO/EB) fluorescent staining was performed to determine structural changes such as cell blebbing and chromatin condensation during the apoptosis process on cells. Images examined under a fluorescent microscope (Zeiss Axio Vert.A1) at 40X magnification with FITC filter for AO and Tex Red filter. And Real-time PCR was performed with ABI Step One Plus and Quant Studio 5 Real-Time PCR systems.Results: PF treatment within 24 hours and 48 hours caused ER stress due to toxic effect and this induced cell death through apoptosis via death receptor signaling and intrinsic pathway in ARPE-19 cells. Conclusion: PF-induced activation of the ER stress mechanism can be used as a novel therapeutic strategy for the prevention of side effects of non-target cells.

FAIM Is Regulated by MiR-206, MiR-1-3p and MiR-133b.

Apoptosis plays an important role during development, control of tissue homeostasis and in pathological contexts. Apoptosis is executed mainly through the intrinsic pathway or the death receptor pathway, i.e., extrinsic pathway. These processes are tightly controlled by positive and negative regulators that dictate pro- or anti-apoptotic death receptor signaling. One of these regulators is the Fas Apoptotic Inhibitory Molecule (FAIM). This death receptor antagonist has two main isoforms, FAIM-S (short) which is the ubiquitously expressed, and a longer isoform, FAIM-L (long), which is mainly expressed in the nervous system. Despite its role as a death receptor antagonist, FAIM also participates in cell death-independent processes such as nerve growth factor-induced neuritogenesis or synaptic transmission. Moreover, FAIM isoforms have been implicated in blocking the formation of protein aggregates under stress conditions or de-regulated in certain pathologies such as Alzheimer’s and Parkinson’s diseases. Despite the role of FAIM in physiological and pathological processes, little is known about the molecular mechanisms involved in the regulation of its expression. Here, we seek to investigate the post-transcriptional regulation of FAIM isoforms by microRNAs (miRNAs). We found that miR-206, miR-1-3p, and miR-133b are direct regulators of FAIM expression. These findings provide new insights into the regulation of FAIM and may provide new opportunities for therapeutic intervention in diseases in which the expression of FAIM is altered.

Curcumol Overcomes TRAIL Resistance of Non-Small Cell Lung Cancer by Targeting NRH:Quinone Oxidoreductase 2 (NQO2).

Resistance to tumor‐necrosis‐factor‐related apoptosis‐inducing ligand (TRAIL) of cancer cell remains a key obstacle for clinical cancer therapies. To overcome TRAIL resistance, this study identifies curcumol as a novel safe sensitizer from a food‐source compound library, which exhibits synergistic lethal effects in combination with TRAIL on non‐small cell lung cancer (NSCLC). SILAC‐based cellular thermal shift profiling identifies NRH:quinone oxidoreductase 2 (NQO2) as the key target of curcumol. Mechanistically, curcumol directly targets NQO2 to cause reactive oxygen species (ROS) generation, which triggers endoplasmic reticulum (ER) stress‐C/EBP homologous protein (CHOP) death receptor (DR5) signaling, sensitizing NSCLC cell to TRAIL‐induced apoptosis. Molecular docking analysis and surface plasmon resonance assay demonstrate that Phe178 in NQO2 is a critical site for curcumol binding. Mutation of Phe178 completely abolishes the function of NQO2 and augments the TRAIL sensitization. This study characterizes the functional role of NQO2 in TRAIL resistance and the sensitizing function of curcumol by directly targeting NQO2, highlighting the potential of using curcumol as an NQO2 inhibitor for clinical treatment of TRAIL‐resistant cancers.

Distinct peripheral blood monocyte and neutrophil transcriptional programs following intracerebral hemorrhage and different etiologies of ischemic stroke

Understanding cell-specific transcriptome responses following intracerebral hemorrhage (ICH) and ischemic stroke (IS) will improve knowledge of the immune response to brain injury. Transcriptomic profiles of 141 samples from 48 subjects with ICH, different IS etiologies, and vascular risk factor controls were characterized using RNA-seq in isolated neutrophils, monocytes and whole blood. In both IS and ICH, monocyte genes were down-regulated, whereas neutrophil gene expression changes were generally up-regulated. The monocyte down-regulated response to ICH included innate, adaptive immune, dendritic, NK cell and atherosclerosis signaling. Neutrophil responses to ICH included tRNA charging, mitochondrial dysfunction, and ER stress pathways. Common monocyte and neutrophil responses to ICH included interferon signaling, neuroinflammation, death receptor signaling, and NFAT pathways. Suppressed monocyte responses to IS included interferon and dendritic cell maturation signaling, phagosome formation, and IL-15 signaling. Activated neutrophil responses to IS included oxidative phosphorylation, mTOR, BMP, growth factor signaling, and calpain proteases-mediated blood–brain barrier (BBB) dysfunction. Common monocyte and neutrophil responses to IS included JAK1, JAK3, STAT3, and thrombopoietin signaling. Cell-type and cause-specific approaches will assist the search for future IS and ICH biomarkers and treatments.

Noncompetitive Allosteric Antagonism of Death Receptor 5 by a Synthetic Affibody Ligand.

Fatty acid-induced upregulation of death receptor 5 (DR5) and its cognate ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), promotes hepatocyte lipoapoptosis, which is a key mechanism in the progression of fatty liver disease. Accordingly, inhibition of DR5 signaling represents an attractive strategy for treating fatty liver disease. Ligand competition strategies are prevalent in tumor necrosis factor receptor antagonism, but recent studies have suggested that noncompetitive inhibition through perturbation of the receptor conformation may be a compelling alternative. To this end, we used yeast display and a designed combinatorial library to identify a synthetic 58-amino acid affibody ligand that specifically binds DR5. Biophysical and biochemical studies show that the affibody neither blocks TRAIL binding nor prevents the receptor-receptor interaction. Live-cell fluorescence lifetime measurements indicate that the affibody induces a conformational change in transmembrane dimers of DR5 and favors an inactive state of the receptor. The affibody inhibits apoptosis in TRAIL-treated Huh-7 cells, an in vitro model of fatty liver disease. Thus, this lead affibody serves as a potential drug candidate, with a unique mechanism of action, for fatty liver disease.