scholarly journals Novel FAO Inhibitor Avocatin B Induces Apoptosis of Acute Monocytic Leukemia Cells in Adipocyte Co-Culture System Via ER Stress and ATF4 Activation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3692-3692 ◽  
Author(s):  
Yoko Tabe ◽  
Shinichi Yamamoto ◽  
Mika Kikkawa ◽  
Hikari Taka ◽  
Kaoru Mogushi ◽  
...  
Keyword(s):  
Cell Death ◽  
Stress Response ◽  
Er Stress ◽  
Research Funding ◽  
Molecular Mechanisms ◽  
Leukemia Cell ◽  
Acute Monocytic Leukemia ◽  
Monocytic Leukemia ◽  
Carnitine Palmitoyltransferase I ◽  
Induced Apoptosis

Abstract Adipocytes are the prevalent stromal cell type in aged adult bone marrows (BM). We previously demonstrated prominent pro-survival role of BM-derived adipocytes for the acute monocytic leukemia (AMoL) cells, a poor-prognosis subtype of AML (Tabe ASH. 2013). A novel anticancer agent avocatin B, an odd-numbered carbon lipid derived from avocado fruit, has been shown to induce leukemia cell death by inhibiting fatty acid oxidation (FAO) via its accumulation in mitochondria (Lee, Cancer Res. 2015). In this study, we investigated the cytotoxic efficacy and molecular mechanisms of avocatin B in AMoL cells co-cultured with BM-derived adipocytes, mimicking the aging BM microenvironment. AMoL cell lines (THP1, MOLM13 and U937) and mesenchymal stem cells (MSC)-derived adipocytes were used for this study. Adipocytes inhibited spontaneous apoptosis in AMoL cells, consistent with our prior observations. Avocatin B successfully induced apoptosis and cell growth inhibition in AMoL cells (IC50s between 15 and 73uM) with G0/G1 cell cycle accumulation. We further observed that avocatin B synergistically enhanced AraC induced apoptosis in AMoL cells cultured alone or co-cultured with adipocytes (Figure 1). To this end, avocatin B synergized with Ara C with combination index value of 0.15. Immunoblot analysis demonstrated that avocatin B inactivated the stress response kinase phospho- (p-) AMPK and p-p38 MAPK in MOLM13 co-cultured with adipocytes but not in AML cells cultured alone. These results indicate that avocatin B disrupted the energy homeostasis under adipocyte co-culture condition. Metabolic profiling using the capillary electrophoresis mass spectrometry (CE-MS) detected alteration of 12 polar metabolites (fold change > 2, P<0.05) in THP1 cells after adipocyte co-culture, including downregulation of Glucose 6-phosphate and Fructose 6-phosphate, and upregulation of citric acid, fumaric acid, malic acid and NAD+, which is consistent with AMPK signaling activation and suggests the downregulation of glycolysis and the compensatory activation of oxidative phosphorylation and FAO. To further characterize the molecular mechanisms of pro-apoptotic effects of avocatin B, we focused on the gene transcriptional modulation induced by avocatin B in adipocyte co-cultures. We previously reported that CPT1(carnitine palmitoyltransferase I), a key enzyme of FAO, induced mitochondrial accumulation of avocatin B which resulted in AML cell apoptosis (Lee, Cancer Res. 2015). By RT-PCR analysis, we observed that avocatin B itself induced CPT1 (carnitine palmitoyltransferase I) mRNA. In addition, adipocyte co-culture upregulated FABP4 (fatty acid binding protein 4)which was further increased by avocatin B treatment in THP1, U937 and MOLM13 cells. These findings likely reflect the direct feedback of FAO inhibition by avocatin B. DNA microarray (Affymetrix) detected the upregulation of 45 genes and downregulation of 58 genes in THP1 cells after co-culture with adipocytes (> 2.0 fold). Ingenuity Pathway Analysis (IPA) and KEGG bioinformatics tools highlighted the cytokine-cytokine receptor interaction as the top upregulated pathway with the potent upstream regulators CXCL12, STAT3, p38 MAPK and NFkB activation. In turn, avocatin B treatment upregulated 71 genes and downregulated 27 genes in THP1 cells co-cultured with adipocytes. Among induced genes, avocatin B treatment caused upregulation of the stress response genes DDIT4, SESN2, PCK2, PHGDH, PSAT1 and STC2 that are the downstream targets of transcription factor ATF4, the master regulator of the endoplasmic reticulum (ER) stress response. In summary, the avocatin B and AraC combination induced significant leukemia cell death under adipocyte co-culture conditions. Metabolome and transcriptome analyses indicate that FAO inhibition by avocatin B induced ER stress might stimulate the DDIT3/CHOP-dependent cell death via transcriptional activation of ATF4 (Figure 2). We conclude that the strategies targeting FAO warrant further exploration in patients with AMMoL, highly dependent on altered lipid metabolism. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Konopleva: Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding.

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

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 882-882
Author(s):  
Muharrem Muftuoglu ◽  
Vivian Ruvolo ◽  
Yuki Nishida ◽  
Po Yee Mak ◽  
Peter P. Ruvolo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Stress Response ◽  
Er Stress ◽  
Single Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Cell Mapping ◽  
Advisory Committees ◽  
Equity Ownership

Background: Cells respond to stress in various ways ranging from adaptation to environmental challenges and activation of survival pathways to induction of cell death. The initial response to stress encompasses adaptive measures to ensure survival and in the presence of irreparable damage associated with unresolved stress cell death ensues. Understanding the principles and mechanisms governing cell survival over cell death is of particular importance in the field of cancer therapy. It is intriguing that exposure of a seemingly homogenous population to death inducing stimuli, such as chemotherapeutic agents, induces fractional tumor killing in a stochastic manner while a subgroup of cells acquire a persistent state, most probably through activation of compensatory survival pathways. Fractional cell killing and, therefore, inability to completely eradicate transformed cells result in resistance to therapy. Methods/Results: To gain further insight into compensatory mechanisms and divergent responses elicited in response to death inducing stimuli we designed a multiparametric flow cytometry panel for simultaneous assessment of different forms of cell death at the single cell level, and aimed to dissect stimulus-specific death patterns and pinpoint potential compensatory mechanisms in persistent cells. We modified ( Bergamaschi et al. 2019) and utilized panels including antibodies against RIP3, LC3B, cleaved caspase 3, cleaved PARP-1, PERK, H2AX, p21, Ki-67 and dead cell discriminating dye. This enabled simultaneous interrogation of a multitude of cell death modes including necrosis, necroptosis, apoptosis and parthanatos in response to DNA damage and as well as proliferation, autophagy and endoplasmic reticulum (ER) stress. To test this concept, we initially utilized agents inducing DNA damage and generated two-dimensional t-SNE plots and diffusion maps to illustrate the multifaceted stress response and developmental trajectories upon challenging with DNA damaging agents. Exposure of acute myeloid leukemia (AML) cell lines to etoposide (E) and daunorubicin (DNR) dramatically altered cellular landscape and resulted in emergence of distinct stress responses characterized by differential induction of autophagy, ER stress and DNA damage response and an increase in multiple cell death subpopulations differentially expressing cleaved caspase 3, PARP-1, necrotic cell identifier (live dead aqua dye) and H2AX. We then generated diffusion maps to infer developmental trajectories of dead cells and identified H2AX+PARP+Caspase-3 co-expression as the earliest event occurring in dying cells while cells stained positive for dead cell dye only marked the latest stage. Of note, a fraction of cells exhibited increased autophagy, accompanied with high ER stress and low DNA damage. Presumably, this pattern identifies persistent cells attaining a transient state in response to E and DNR associated with higher likelihood of survival. Evidently, external stress induced a divergent multifaceted response: DNA damage followed by cell death vs. induction of adaptive mechanisms including autophagy and high ER stress. Although both E and DNR preferentially targeted proliferating cells and induced cell cycle arrest, overall stress response to E was distinct from stress to DNR in high-dimensional plane. To attain a comprehensive overview of stress response to E vs. DNR we compared t-SNE maps depicting overall stress response and observed significant segregation. Autophagy and ER stress was more pronounced in E group while DNR completely abrogated proliferation in surviving cells. To further corroborate the utility of this approach, we assessed the activity of exportin-1 (XPO1, KPT-330) and MDM2 (DS-3032b) inhibitors. KPT-330 and DS-3032b individually induced limited cell death. Combination of XPO-1 and MDM2 inhibitors resulted in enhanced apoptotic cell death with unrepaired DNA damage while surviving cells displayed an autophagy pattern. Conclusion: These findings provide proof of concept for the utility of single cell mapping of cellular stress in delineating stressor-specific response patterns and identifying potential resistance mechanisms. Single cell mapping of cell stress and cell death can inform the development of more effective combinatorial drug regimens. Studies to identify stress signatures of targeted agents currently developed for the treatment of AML are ongoing Figure 1 Disclosures Carter: Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding. Andreeff:NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; BiolineRx: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Reata: Equity Ownership; Aptose: Equity Ownership.

scholarly journals Curcumin abates hypoxia-induced oxidative stress based-ER stress-mediated cell death in mouse hippocampal cells (HT22) by controlling Prdx6 and NF-κB regulation

2013 ◽  
Vol 304 (7) ◽  
pp. C636-C655 ◽  
Author(s):  
Bhavana Chhunchha ◽  
Nigar Fatma ◽  
Eri Kubo ◽  
Prerana Rai ◽  
Sanjay P. Singh ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Er Stress ◽  
Molecular Mechanisms ◽  
Stress Proteins ◽  
Ht22 Cells ◽  
Peroxiredoxin 6 ◽  
Homologous Protein ◽  
Survival Signaling ◽  
Induced Apoptosis

Oxidative stress and endoplasmic reticulum (ER) stress are emerging as crucial events in the etiopathology of many neurodegenerative diseases. While the neuroprotective contributions of the dietary compound curcumin has been recognized, the molecular mechanisms underlying curcumin's neuroprotection under oxidative and ER stresses remains elusive. Herein, we show that curcumin protects HT22 from oxidative and ER stresses evoked by the hypoxia (1% O2 or CoCl2 treatment) by enhancing peroxiredoxin 6 (Prdx6) expression. Cells exposed to CoCl2 displayed reduced expression of Prdx6 with higher reactive oxygen species (ROS) expression and activation of NF-κB with IκB phosphorylation. When NF-κB activity was blocked by using SN50, an inhibitor of NF-κB, or cells treated with curcumin, the repression of Prdx6 expression was restored, suggesting the involvement of NF-κB in modulating Prdx6 expression. These cells were enriched with an accumulation of ER stress proteins, C/EBP homologous protein (CHOP), GRP/78, and calreticulin, and had activated states of caspases 12, 9, and 3. Reinforced expression of Prdx6 in HT22 cells by curcumin reestablished survival signaling by reducing propagation of ROS and blunting ER stress signaling. Intriguingly, knockdown of Prdx6 by antisense revealed that loss of Prdx6 contributed to cell death by sustaining enhanced levels of ER stress-responsive proapoptotic proteins, which was due to elevated ROS production, suggesting that Prdx6 deficiency is a cause of initiation of ROS-mediated ER stress-induced apoptosis. We propose that using curcumin to reinforce the naturally occurring Prdx6 expression and attenuate ROS-based ER stress and NF-κB-mediated aberrant signaling improves cell survival and may provide an avenue to treat and/or postpone diseases associated with ROS or ER stress.

Capsaicin mediates apoptosis in human nasopharyngeal carcinoma NPC-TW 039 cells through mitochondrial depolarization and endoplasmic reticulum stress

2011 ◽  
Vol 31 (6) ◽  
pp. 539-549 ◽  
Author(s):  
S-W Ip ◽  
S-H Lan ◽  
H-F Lu ◽  
A-C Huang ◽  
J-S Yang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Nasopharyngeal Carcinoma ◽  
Er Stress ◽  
Molecular Mechanisms ◽  
Human Cancer ◽  
Dependent Manner ◽  
Mitochondrial Depolarization ◽  
Human Nasopharyngeal Carcinoma ◽  
Induced Apoptosis

Capsaicin, a pungent compound found in hot chili peppers, has been reported to have antitumor activities in many human cancer cell lines, but the induction of precise apoptosis signaling pathway in human nasopharyngeal carcinoma (NPC) cells is unclear. Here, we investigated the molecular mechanisms of capsaicin-induced apoptosis in human NPC, NPC-TW 039, cells. Effects of capsaicin involved endoplasmic reticulum (ER) stress, caspase-3 activation and mitochondrial depolarization. Capsaicin-induced cytotoxic effects (cell death) through G0/G1 phase arrest and induction of apoptosis of NPC-TW 039 cells in a dose-dependent manner. Capsaicin treatment triggered ER stress by promoting the production of reactive oxygen species (ROS), increasing levels of inositol-requiring 1 enzyme (IRE1), growth arrest and DNA-damage-inducible 153 (GADD153) and glucose-regulated protein 78 (GRP78). Other effects included an increase in cytosolic Ca2+, loss of the mitochondrial transmembrane potential (ΔΨ m), releases of cytochrome c and apoptosis-inducing factor (AIF), and activation of caspase-9 and -3. Furthermore, capsaicin induced increases in the ratio of Bax/Bcl-2 and abundance of apoptosis-related protein levels. These results suggest that ER stress- and mitochondria-mediated cell death is involved in capsaicin-induced apoptosis in NPC-TW 039 cells.

scholarly journals Differential Immunomodulatory Effect of Graphene Oxide and Vanillin-Functionalized Graphene Oxide Nanoparticles in Human Acute Monocytic Leukemia Cell Line (THP-1)

2019 ◽  
Vol 20 (2) ◽  
pp. 247 ◽  
Author(s):  
Sangiliyandi Gurunathan ◽  
Min-Hee Kang ◽  
Muniyandi Jeyaraj ◽  
Jin-Hoi Kim
Keyword(s):  
Cell Death ◽  
Graphene Oxide ◽  
Cell Line ◽  
Biomedical Applications ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Acute Monocytic Leukemia ◽  
Chemical Compositions ◽  
Monocytic Leukemia ◽  
Apoptotic Genes

Graphene and its derivatives are emerging as attractive materials for biomedical applications, including antibacterial, gene delivery, contrast imaging, and anticancer therapy applications. It is of fundamental importance to study the cytotoxicity and biocompatibility of these materials as well as how they interact with the immune system. The present study was conducted to assess the immunotoxicity of graphene oxide (GO) and vanillin-functionalized GO (V-rGO) on THP-1 cells, a human acute monocytic leukemia cell line. The synthesized GO and V-rGO were characterized by using various analytical techniques. Various concentrations of GO and V-rGO showed toxic effects on THP-1 cells such as the loss of cell viability and proliferation in a dose-dependent manner. Cytotoxicity was further demonstrated as an increased level of lactate dehydrogenase (LDH), loss of mitochondrial membrane potential (MMP), decreased level of ATP content, and cell death. Increased levels of reactive oxygen species (ROS) and lipid peroxidation caused redox imbalance in THP-1 cells, leading to increased levels of malondialdehyde (MDA) and decreased levels of anti-oxidants such as glutathione (GSH), glutathione peroxidase (GPX), super oxide dismutase (SOD), and catalase (CAT). Increased generation of ROS and reduced MMP with simultaneous increases in the expression of pro-apoptotic genes and downregulation of anti-apoptotic genes suggest that the mitochondria-mediated pathway is involved in GO and V-rGO-induced apoptosis. Apoptosis was induced consistently with the significant DNA damage caused by increased levels of 8-oxo-dG and upregulation of various key DNA-regulating genes in THP-1 cells, indicating that GO and V-rGO induce cell death through oxidative stress. As a result of these events, GO and V-rGO stimulated the secretion of various cytokines and chemokines, indicating that the graphene materials induced potent inflammatory responses to THP-1 cells. The harshness of V-rGO in all assays tested occurred because of better charge transfer, various carbon to oxygen ratios, and chemical compositions in the rGO. Overall, these findings suggest that it is essential to better understand the parameters governing GO and functionalized GO in immunotoxicity and inflammation. Rational design of safe GO-based formulations for various applications, including nanomedicine, may result in the development of risk management methods for people exposed to graphene and graphene family materials, as these nanoparticles can be used as delivery agents in various biomedical applications.

scholarly journals Inhibition of WIP1 Phosphatase in Multiple Myeloma Overcomes Bortezomib Resistance and Promotes Cell Death Via ER Stress-Induced Apoptotic JNK/c-Jun Signaling and Downregulation of Inhibitors of Apoptosis Proteins (IAPs)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1366-1366
Author(s):  
Katia Beider ◽  
Evgenia Rosenberg ◽  
Valeria Voevoda ◽  
Hanna Bitner ◽  
Yaarit Sirovsky ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Stress Response ◽  
Er Stress ◽  
Cell Lines ◽  
Potential Role ◽  
Cyclin B1 ◽  
Parp Cleavage ◽  
Induced Apoptosis

Abstract Acquired or de novo resistance to the traditional and novel anti-multiple myeloma (MM) agents remains a major treatment obstacle, therefore novel therapies are in need. Wild-type p53-induced phosphatase 1 (WIP1) is an oncogenic serine/threonine phosphatase implicated in silencing of cellular responses to genotoxic stress. WIP1 overexpression was documented in various solid cancers in correlation with aggressive features and poor prognosis. Thus, we studied WIP1 in MM addressing its potential role in mediating resistance and aggressive phenotype. Increased expression of WIP1 was detected in MM cell lines (n=8) and primary samples (n=18) at both mRNA and protein level as compared with normal PBMCs (n=5). Furthermore, a positive correlation between WIP1 and CXCR4 levels (p<0.02, R2=0.5) was revealed. The latter is a well-known oncogenic receptor in MM. WIP1 expression levels were significantly up-regulated following bortezomib (Bort) treatment. Using MM cell lines with acquired resistance to Bort (RPMI8226BortRes and CAGBortRes), a higher induction of WIP1 upon Bort exposure could be demonstrated, suggesting a possible role for WIP1 in the acquisition of MM drug resistance to proteasome inhibitors. WIP1 was also upregulated in MM cells cultured on human BM stroma (BMSC) known to protect the tumor cells from Bort-induced apoptosis, further supporting its function in mediating resistance. GSK2830371 (GSK), a novel allosteric inhibitor of WIP1, significantly suppressed MM cells proliferation (p<0.01) and induced apoptosis, as demonstrated by phosphatidylserine externalization, mitochondrial depolarization (ψm), caspase 3 and PARP cleavage, and DNA fragmentation. Moreover, combined treatment with GSK and Bort synergistically potentiated cell death in both Bort-sensitive and resistant MM cells and overcame BMSC protection (CI<0.5). The robust apoptosis induced by Bort/GSK treatment was accompanied by increased mitochondrial ROS accumulation, subsequent mitochondrial destabilization and extensive DNA damage. GSK treatment resulted in a reduction of WIP1 basal expression and abrogated WIP1 induction upon Bort treatment. Thus, we defined that GSK can regulate WIP1 expression in MM cells. To determine the molecular mechanism of Bort/GSK synergism we performed gene and protein expression analysis. Combination of both agents significantly reduced expression of anti-apoptotic proteins such as cIAP1, cIAP2, XIAP and Survivin. Previous studies indicate that maintaining IAPs expression is part of an adaptive unfolded protein response that promotes MM survival upon Bort-induced endoplasmic reticulum (ER) stress. Therefore, it is conceivable that targeting IAPs upon WIP1 inhibition may overcome protective responses, inducing unresolved ER stress and MM cell death. Indeed, we found that combination of Bort and GSK significantly enhanced ER stress, as indicated by increase in the pro-apoptotic factors ATF4, CHOP and GADD34. Concomitantly, mitosis-inducing factors Cyclin B1, CDK1 and PLK1 were prominently reduced upon Bort/GSK treatment. To assess the potential role of p53 activation in GSK-mediated effects, p53-stabilizing agents nutlin3a and PRIMA1 were applied in combination with WIP1 inhibition. We observed a significant (p<0.01) increase in the responsiveness of both p53WT and p53mut MM cells to GSK-mediated apoptosis. Consistently, combined GSK/Bort treatment upregulated p53 targets, including PUMA, NOXA, GADD45A and p21 genes. These data suggest that p53 may potentiate the WIP1 inhibition mediated stress induction. Finally, we assessed the signaling pathways that may be involved in WIP1 mediated cessation of stress response. GSK profoundly augmented Bort-induced phosphorylation of JNK and c-Jun, without affecting p38 phosphorylation. Accordingly, JNK inhibitor SP600125 successfully reverted both the apoptosis and the downregulation of IAPs induced by Bort/GSK treatment. Altogether, these results identify pro-apoptotic JNK/c-Jun signaling being preferential target of WIP1 in the process of dampening Bort-induced stress response. To conclude, we disclose the role of WIP1 in blunting stress response and promoting resistance to bortezomib. Collectively, WIP1 suppression prevents MM cell adaptation and recovery upon ER stress. These findings may provide the scientific basis for a novel combinatorial anti-myeloma therapy. Disclosures Peled: Cellect Biotherapeutics Ltd: Consultancy.

scholarly journals High Dimensional Interrogation of Stress Response Patterns and Cell Death Modes in AML

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 15-15
Author(s):  
Muharrem Muftuoglu ◽  
Po Yee Mak ◽  
Vivian Ruvolo ◽  
Yuki Nishida ◽  
Peter P. Ruvolo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Stress Response ◽  
Er Stress ◽  
Single Cell ◽  
Stress Responses ◽  
Research Funding ◽  
High Dimensional ◽  
Dead Cell ◽  
Response Patterns

Cellular response to stress is diverse, ranging from adaptation through activation of survival pathways to induction of cell death. We designed a multicolor flow cytometry panel to gain insight into multifaceted stress response and to assess multiple cell death modes at the single cell level. The panel included antibodies against RIP3, active caspase 3, cleaved PARP, ATF4, H2AX, p21, Ki-67 and a dead cell discriminating dye. This enabled simultaneous interrogation of a multitude of cell death modes including necrosis, necroptosis, apoptosis and parthanatos as well as proliferation, autophagy and endoplasmic reticulum (ER) stress. Notably, we utilized high dimensional analytic approaches to better elucidate stress response and cell death modes. We leveraged t-SNE for dimension reduction, PhenoGraph to identify distinct phenotypes and diffusion map to map cell trajectories. First, we aimed at delineating response patterns and cell death modes associated with targeted therapies currently being investigated for the treatment of acute myeloid leukemias (AML), including inhibitors targeting anti-apoptotic molecules (Bcl-2i and Mcl-1i), propagating p53-mediated apoptosis (MDM-2i and exportin 1i [XPO1i]), abrogating adaptive circuits through blocking autophagic degradation (SBI-0206965) and depleting anti-oxidant pool (Buthionine sulfoximine [BSO]), and mitochondrial proteasome ClpP activator (ClpPa) (ONC201). Initially, we generated two-dimensional t-SNE plots to interrogate agent-specific response landscapes. Unsupervised high-dimensional mapping demonstrated that Bcl-2 or Mcl-1 inhibition alone did not alter cellular landscape. However, treatment with MDM2i or XPO1i and ClpPa elicited divergent stress responses and cell death modes. Two-dimensional plots showed differential induction of autophagy and ER stress following treatment with MDM2i, XPO1i or ClpPa. Remarkably, we observed that MDM2i and XPO1i were associated with emergence of quiescent cells, based on high expression of p21, and higher levels of ER stress and autophagy while ClpPa induced DNA damage, and was associated with persistent Ki-67 expression and lower levels of p21. This approach enabled us to dissect single agent specific stress signatures. Next, we assessed the response landscapes of prior knowledge-based, data-driven synergistic dual drug combinations. Mapping of response landscapes of multiple dual drug combinations at single-cell resolution revealed distinct associations among integrated stress responses, divergent cellular progression trajectories and previously unidentified response patterns. We observed that autophagic cells were associated with high levels of ER stress and cell kinetic quiescence, suggesting that perturbation-specific stress responses are integrated at the cellular level and are triggered concomitantly. Unsupervised clustering and partitioning of response landscapes to identify major phenotypes revealed two distinct autophagic cell phenotypes: 1) Quiescent autophagic cells without DNA damage and 2) proliferating autophagic cells with DNA damage. Strikingly, combinatorial use of MDM2i and XPO1i almost completely eliminated all AML cells. The surviving cells were quiescent, had high levels of autophagy and ER stress, and were spared of DNA damage. On the other hand, addition of either Bcl-2i or Mcl-1i to MDM2i markedly reduced p21, ER stress and autophagy, indicating that these anti-apoptotic molecules may play a role in cellular adaptation. Addition of Bcl-2i or Mcl-1i inhibitors may specifically deplete autophagic cells with high p21 and ER stress (as we have reported, Pan et al. Cancer Cell 2017). To map cellular trajectories and identify the sequence of events we leveraged diffusion map algorithm and aligned the clusters along pseudo-time. This approach enabled us to identify the earliest stage of cell death, characterized by expression of LC3B, H2AX and cleaved PARP while dead cell dyes marked the latest stage. These findings provide proof of concept for the utility of single cell mapping of cellular stress in delineating stressor-specific response patterns and identifying potential resistance mechanisms. Single cell mapping of cell stress and cell death can inform the development of more effective combinatorial drug regimens. Disclosures Carter: Syndax: Research Funding; Ascentage: Research Funding; Amgen: Research Funding; AstraZeneca: Research Funding. Andreeff:Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

scholarly journals iPLA2β Contributes to ER Stress-Induced Apoptosis during Myocardial Ischemia/Reperfusion Injury

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1446
Author(s):  
Tingting Jin ◽  
Jun Lin ◽  
Yingchao Gong ◽  
Xukun Bi ◽  
Shasha Hu ◽  
...  
Keyword(s):  
Cell Death ◽  
Heart Disease ◽  
Myocardial Ischemia ◽  
Ischemic Heart Disease ◽  
Er Stress ◽  
Ischemia Reperfusion ◽  
Myocardial Ischemia Reperfusion ◽  
Induced Apoptosis

Both calcium-independent phospholipase A2 beta (iPLA2β) and endoplasmic reticulum (ER) stress regulate important pathophysiological processes including inflammation, calcium homeostasis and apoptosis. However, their roles in ischemic heart disease are poorly understood. Here, we show that the expression of iPLA2β is increased during myocardial ischemia/reperfusion (I/R) injury, concomitant with the induction of ER stress and the upregulation of cell death. We further show that the levels of iPLA2β in serum collected from acute myocardial infarction (AMI) patients and in samples collected from both in vivo and in vitro I/R injury models are significantly elevated. Further, iPLA2β knockout mice and siRNA mediated iPLA2β knockdown are employed to evaluate the ER stress and cell apoptosis during I/R injury. Additionally, cell surface protein biotinylation and immunofluorescence assays are used to trace and locate iPLA2β. Our data demonstrate the increase of iPLA2β augments ER stress and enhances cardiomyocyte apoptosis during I/R injury in vitro and in vivo. Inhibition of iPLA2β ameliorates ER stress and decreases cell death. Mechanistically, iPLA2β promotes ER stress and apoptosis by translocating to ER upon myocardial I/R injury. Together, our study suggests iPLA2β contributes to ER stress-induced apoptosis during myocardial I/R injury, which may serve as a potential therapeutic target against ischemic heart disease.

scholarly journals RIPK1 promotes death receptor-independent caspase-8-mediated apoptosis under unresolved ER stress conditions

2014 ◽  
Vol 5 (12) ◽  
pp. e1555-e1555 ◽  
Author(s):  
Y Estornes ◽  
M A Aguileta ◽  
C Dubuisson ◽  
J De Keyser ◽  
V Goossens ◽  
...  
Keyword(s):  
Er Stress ◽  
Molecular Mechanisms ◽  
Death Receptor ◽  
Caspase 8 ◽  
Interacting Protein ◽  
Life And Death ◽  
Unfolded Protein ◽  
Induced Apoptosis ◽  
The Unfolded Protein Response ◽  
Protein Response

Abstract Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress and results in the activation of the unfolded protein response (UPR), which aims at restoring ER homeostasis. However, when the stress is too severe the UPR switches from being a pro-survival response to a pro-death one, and the molecular mechanisms underlying ER stress-mediated death have remained incompletely understood. In this study, we identified receptor interacting protein kinase 1 (RIPK1)—a kinase at the crossroad between life and death downstream of various receptors—as a new regulator of ER stress-induced death. We found that Ripk1-deficient MEFs are protected from apoptosis induced by ER stressors, which is reflected by reduced caspase activation and PARP processing. Interestingly, the pro-apoptotic role of Ripk1 is independent of its kinase activity, is not regulated by its cIAP1/2-mediated ubiquitylation, and does not rely on the direct regulation of JNK or CHOP, two reportedly main players in ER stress-induced death. Instead, we found that ER stress-induced apoptosis in these cells relies on death receptor-independent activation of caspase-8, and identified Ripk1 upstream of caspase-8. However, in contrast to RIPK1-dependent apoptosis downstream of TNFR1, we did not find Ripk1 associated with caspase-8 in a death-inducing complex upon unresolved ER stress. Our data rather suggest that RIPK1 indirectly regulates caspase-8 activation, in part via interaction with the ER stress sensor inositol-requiring protein 1 (IRE1).

scholarly journals Enhancer of Zeste Homolog 2 (EZH2) Mediates Glucolipotoxicity-Induced Apoptosis in β-Cells

2020 ◽  
Vol 21 (21) ◽  
pp. 8016
Author(s):  
Tina Dahlby ◽  
Christian Simon ◽  
Marie Balslev Backe ◽  
Mattias Salling Dahllöf ◽  
Edward Holson ◽  
...  
Keyword(s):  
Er Stress ◽  
Molecular Mechanisms ◽  
Selective Inhibition ◽  
Human Islets ◽  
Β Cells ◽  
Β Cell ◽  
Enhancer Of Zeste ◽  
Cell Gene Expression ◽  
Nfκb Pathway ◽  
Induced Apoptosis

Selective inhibition of histone deacetylase 3 (HDAC3) prevents glucolipotoxicity-induced β-cell dysfunction and apoptosis by alleviation of proapoptotic endoplasmic reticulum (ER) stress-signaling, but the precise molecular mechanisms of alleviation are unexplored. By unbiased microarray analysis of the β-cell gene expression profile of insulin-producing cells exposed to glucolipotoxicity in the presence or absence of a selective HDAC3 inhibitor, we identified Enhancer of zeste homolog 2 (EZH2) as the sole target candidate. β-Cells were protected against glucolipotoxicity-induced ER stress and apoptosis by EZH2 attenuation. Small molecule inhibitors of EZH2 histone methyltransferase activity rescued human islets from glucolipotoxicity-induced apoptosis. Moreover, EZH2 knockdown cells were protected against glucolipotoxicity-induced downregulation of the protective non-canonical Nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) pathway. We conclude that EZH2 deficiency protects from glucolipotoxicity-induced ER stress, apoptosis and downregulation of the non-canonical NFκB pathway, but not from insulin secretory dysfunction. The mechanism likely involves transcriptional regulation via EZH2 functioning as a methyltransferase and/or as a methylation-dependent transcription factor.

Sign in / Sign up

Export Citation Format

Share Document