Endoplasmic Reticulum Stress-induced Response Research Articles

Jumonji histone demethylases are therapeutic targets in small cell lung cancer

Small cell lung cancer (SCLC) is a recalcitrant cancer of neuroendocrine (NE) origin. Changes in therapeutic approaches against SCLC have been lacking over the decades. Here, we use preclinical models to identify a new therapeutic vulnerability in SCLC consisting of the targetable Jumonji lysine demethylase (KDM) family. We show that Jumonji demethylase inhibitors block malignant growth and that etoposide-resistant SCLC cell lines are particularly sensitive to Jumonji inhibition. Mechanistically, small molecule-mediated inhibition of Jumonji KDMs activates endoplasmic reticulum (ER) stress genes, upregulates ER stress signaling, and triggers apoptotic cell death. Furthermore, Jumonji inhibitors decrease protein levels of SCLC NE markers INSM1 and Secretogranin-3 and of driver transcription factors ASCL1 and NEUROD1. Genetic knockdown of KDM4A, a Jumonji demethylase highly expressed in SCLC and a known regulator of ER stress genes, induces ER stress response genes, decreases INSM1, Secretogranin-3, and NEUROD1 and inhibits proliferation of SCLC in vitro and in vivo. Lastly, we demonstrate that two different small molecule Jumonji KDM inhibitors (pan-inhibitor JIB-04 and KDM4 inhibitor SD70) block the growth of SCLC tumor xenografts in vivo. Our study highlights the translational potential of Jumonji KDM inhibitors against SCLC, a clinically feasible approach in light of recently opened clinical trials evaluating this drug class, and establishes KDM4A as a relevant target across SCLC subtypes.

Potential antidepressant-like effects of N-3 polyunsaturated fatty acids through inhibition of endoplasmic reticulum stress.

The growing evidence has demonstrated the importance of endoplasmic reticulum stress (ERS) in the pathophysiology of depression. ERS genes were considered to be potential novel therapeutic targets for depression. To clarify the mechanisms of the chronic unpredictable mild stress (CUMS)-induced ERS response and the potential contributing pathways in depression, and further investigate the potential link between N-3 polyunsaturated fatty acids (PUFAs) and stress-induced ERS disturbances. This study analyzed the expression of ERS-related genes including GRP78, ATF-4, ATF-6, XBP-1, and CHOP, and sigma-1R with real-time PCR in peripheral blood mononuclear cell (PBMC) RNA samples from participants. All of the rats except for those in the control groups were subjected to 5 consecutive weeks of CUMS to establish the depression model, and the antidepressant effects of N-3 PUFAs were observed by behavior tests. Moreover, the effect of diet and stress on the ERS pathways was also investigated using the western blot. Blood CHOP, ATF-4, and XBP-1 levels were notably elevated in depressed patients relative to healthy individuals. Moreover, increased sigma-1R and decreased ATF-6 implied the protective role of sigma-1R through modulating ERS in patients with depression. Animal studies disclosed the novel findings that supplementary N-3 PUFAs in rats alleviated CUMS-induced disturbance of ERS through the ATF-4/XBP-1/CHOP pathway, implying its potential strategy for depression. CUMS-induced depressive-like behaviors are related to the disturbance of ERS. Furthermore, supplementary N-3 PUFAs might be an effective way to alleviate ERS.

Neutrophils Contribute to ER Stress in Lung Epithelial Cells in the Pristane-Induced Diffuse Alveolar Hemorrhage Mouse Model.

Diffuse alveolar hemorrhage (DAH), although rare, is a life-threatening complication of systemic lupus erythematosus (SLE). Little is known about the pathophysiology of DAH in humans, although increasingly neutrophils, NETosis and inflammatory monocytes have been shown to play an important role in the pristane-induced model of SLE which develops lung hemorrhage and recapitulates many of the pathologic features of human DAH. Using this experimental model, we asked whether endoplasmic reticulum (ER) stress played a role in driving the pathology of pulmonary hemorrhage and what role infiltrating neutrophils had in this process. Analysis of lung tissue from pristane-treated mice showed genes associated with ER stress and NETosis were increased in a time-dependent manner and reflected the timing of CD11b+Ly6G+ neutrophil accumulation in the lung. Using precision cut lung slices from untreated mice we observed that neutrophils isolated from the peritoneal cavity of pristane-treated mice could directly induce the expression of genes associated with ER stress, namely Chop and Bip. Mice which had myeloid-specific deletion of PAD4 were generated and treated with pristane to assess the involvement of PAD4 and PAD4-dependent NET formation in pristane-induced lung inflammation. Specific deletion of PAD4 in myeloid cells resulted in decreased expression of ER stress genes in the pristane model, with accompanying reduction in IFN-driven genes and pathology. Lastly, coculture experiments of human neutrophils and human lung epithelial cell line (BEAS-2b) showed neutrophils from SLE patients induced significantly more ER stress and interferon-stimulated genes in epithelial cells compared to healthy control neutrophils. These results support a pathogenic role of neutrophils and NETs in lung injury during pristane-induced DAH through the induction of ER stress response and suggest that overactivation of neutrophils in SLE and NETosis may underlie development of DAH.

Inhibition of Autophagy Prevents Panax Notoginseng Saponins (PNS) Protection on Cardiac Myocytes Against Endoplasmic Reticulum (ER) Stress-Induced Mitochondrial Injury, Ca2+ Homeostasis and Associated Apoptosis

Endoplasmic reticulum (ER) stress is often closely linked to autophagy, hypoxia signaling, mitochondrial biogenesis and reactive oxygen species (ROS) responses. Understanding the interaction between ER stress, mitochondrial function and autophagy is of great importance to provide new mechanisms for the pathology, prevention and treatment of cardiovascular diseases. Our previous study has reported that Panax notoginseng saponins (PNS) protection against thapsigargin (TG)-induced ER stress response and associated cell apoptosis in cardiac myocytes is calcium dependent and mediated by ER Ca2+ release through RyR2. However, whether its protection upon ER stress and associated apoptosis is related to mitochondrial function and autophagy remains largely unknown. Here, we investigated the roles of PNS played in TG-induced mitochondrial function, ROS accumulation and autophagy. We also assessed its effects on Ca2+ homeostasis, ER stress response and associated cell death in the presence of autophagy inhibition. PNS-pretreated primary cultured neonatal rat cardiomyocytes were stimulated with TG to induce ER stress response. Mitochondrial potential (Δψm) was measured by JC-1. The general and mitochondrial ROS were measured by DCFH-DA and MitoSOX Red, respectively. Autophagy was evaluated by immunofluorescence of LC3, and immunoblots of LC3, p62, ATG7 and PINK1. In addition, mRFP-GFP-LC3 labeling was used to assess the autophagic influx. SiATG7 transfected H9c2 cells were generated to inhibit autophagy. Cytosolic and ER Ca2+ dynamics were investigated by calcium imaging. RyR2 oxidation was tested by oxyblot. Cell viability was examined by TUNEL assay. ER stress response and cell apoptosis were detected by immunoblots of BiP, CHOP, Cleaved Caspase-3 and Caspase-12. The results demonstrated that firstly, PNS protects against TG-induced mitochondrial injury and ROS accumulation. Secondly, PNS enhances autophagy in TG-induced cardiac myocytes. Thirdly, inhibition of autophagy diminishes PNS prevention of TG-induced mitochondrial injury, ROS accumulation and disruption of Ca2+ homeostasis. Last but not least, inhibition of autophagy abolishes PNS protection against TG-induced ER stress response and associated apoptosis. In summary, PNS protection against ER stress response and associated apoptosis is related to the regulation of mitochondrial injury and ROS overproduction via modulation of autophagy. These data provide new insights for molecular mechanisms of PNS as a potential preventive approach to the management of cardiovascular diseases.

Acetylsalicylic acid and salicylic acid present anticancer properties against melanoma by promoting nitric oxide-dependent endoplasmic reticulum stress and apoptosis

Melanoma is the most aggressive and fatal type of skin cancer due to being highly proliferative. Acetylsalicylic acid (ASA; Aspirin) and salicylic acid (SA) are ancient drugs with multiple applications in medicine. Here, we showed that ASA and SA present anticancer effects against a murine model of implanted melanoma. These effects were also validated in 3D- and 2D-cultured melanoma B16F10 cells, where the drugs promoted pro-apoptotic effects. In both in vivo and in vitro models, SA and ASA triggered endoplasmic reticulum (ER) stress, which culminates with the upregulation of the pro-apoptotic transcription factor C/EBP homologous protein (CHOP). These effects are initiated by ASA/SA-triggered Akt/mTOR/AMPK-dependent activation of nitric oxide synthase 3 (eNOS), which increases nitric oxide and reactive oxygen species production inducing ER stress response. In the end, we propose that ASA and SA instigate anticancer effects by a novel mechanism, the activation of ER stress.

Regulation of the ER Stress Response by the Ion Channel Activity of the Infectious Bronchitis Coronavirus Envelope Protein Modulates Virion Release, Apoptosis, Viral Fitness, and Pathogenesis.

Coronavirus (CoV) envelope (E) protein is a small structural protein critical for virion morphogenesis and release. The recently characterized E protein ion channel activity (EIC) has also been implicated in modulating viral pathogenesis. In this study, we used infectious bronchitis coronavirus (IBV) as a model to study EIC. Two recombinant IBVs (rIBVs) harboring EIC-inactivating mutations – rT16A and rA26F – were serially passaged, and several compensatory mutations were identified in the transmembrane domain (TMD). Two rIBVs harboring these putative EIC-reverting mutations – rT16A/A26V and rA26F/F14N – were recovered. Compared with the parental rIBV-p65 control, all four EIC mutants exhibited comparable levels of intracellular RNA synthesis, structural protein production, and virion assembly. Our results showed that the IBV EIC contributed to the induction of ER stress response, as up-regulation of ER stress-related genes was markedly reduced in cells infected with the EIC-defective mutants. EIC-defective mutants also formed smaller plaques, released significantly less infectious virions into the culture supernatant, and had lower levels of viral fitness in cell culture. Significantly, all these defective phenotypes were restored in cells infected with the putative EIC revertants. EIC mutations were also implicated in regulating IBV-induced apoptosis, induction of pro-inflammatory cytokines, and viral pathogenicity in vivo. Taken together, this study highlights the importance of CoV EIC in modulating virion release and various aspects of CoV – host interaction.

Proteomic Analysis Identifies Mechanism(s) of Overcoming Bortezomib-Resistance Via Targeting Ubiquitin Receptor Rpn13

Introduction Our studies have focused on delineating ubiquitin proteasome pathway signaling with a goal to identify and validate novel therapeutic targets to overcome proteasome-inhibitor-resistance in multiple myeloma (MM). Our siRNA screening studies showed ubiquitin Receptor (UbR) Rpn13 as a mediator of MM cell growth. Rpn13 is localized on the 19S proteasome, and chaperone ubiquitinated proteins for downstream proteasomal degradation. We showed that blockade of Rpn13 trigger cytotoxicity in MM cells and overcomes bortezomib-resistance (Song Yan et al., Leukemia 2016, 30(9):1877-1886). However, the mechanism(s) whereby Rpn13-inhibition overcomes bortezomib-resistance remains undefined. Here, MM cells were treated with the Rpn13 inhibitor RA190 to identify proteomic alterations and specific downstream substrate proteins/signaling mechanism involved in overcoming bortezomib-resistance. We identified and validated the Copper/Zinc-superoxide dismutase (SOD1), implicated in the regulation of mitochondrial unfolded protein response, as a novel therapeutic target in MM. Materials and Methods MM.1S cells were treated with DMSO or RA190 (250 nM) for 24h and subjected to multiplexed proteomics analysis using tandem mass spectrometry. Data were analyzed using uniprot composite database and SEQUEST-based software platform. MM.1S cells were transiently transfected with scr-siRNA, SOD1-siRNA, control (pCDNA3.1)- or SOD1 (WT)-plasmid using the Nucleofector Kit V. Rpn13 knockout cells were generated using CRISPR/Cas9. Cell viability was assessed using WST-1, and Cell Titer-Glo assay. Alterations in ROS, SOD1, superoxide levels and Proteasome activity were measured as previously described (Chauhan et al., Cancer Cell 2005, 8:407-419; Chauhan et al., Cancer Cell 2012, 22(3):345-358). Rpn13 inhibitor RA190 and Cu-ZnSOD1 inhibitor LCS-1 were purchased from Calbiochem and Sigma-Aldrich, respectively. Statistical significance was determined using a Student's t test. Results 1) Rpn13 inhibitor RA190 decreases viability in bortezomib-resistant MM cells. 2) Proteomic analysis showed a marked decrease in superoxide dismutase (SOD1) levels (69% ± 2.1%) in RA190- vs DMSO-treated MM cells. No significant decrease in SOD1 expression was noted in bortezomib-treated cells. 3) Transfection with SOD1-WT rescued cells from growth-inhibitory activity of RA190, suggesting that SOD1 mediates Rpn13-inhibition triggered cell death. 4) Both patient MM cells and MM cell lines exhibit higher SOD1 protein levels vs normal plasma cells/PBMCs; 5) SOD1 levels are higher in bortezomib-resistant vs -sensitive MM cells. 6) Gene expression (GEP) analysis showed inverse correlation between SOD1 and overall patient survival (n=175) (p = 0.00054). 7) Direct targeting of SOD1 using siRNA showed significant decrease in viability of MM cell lines, including bortezomib-resistant MM cells. 8) As for SOD1-siRNA, treatment of MM cell lines and primary patient cells with the SOD1 inhibitor LCS-1 decreased their viability (p < 0.05; n=3) without affecting normal PBMCs. 9) LCS-1 inhibits the proliferation of MM cells even in the presence of tumor-promoting BMSCs and plasmacytoid dendritic cells. 10) Mechanistic studies show that similar to RA190, LCS-1-induced MM cell death is associated with: (i) inhibition of SOD1 enzymatic activity and increase in superoxide and reactive oxygen species (ROS) levels; (ii) activation of apoptotic signaling via caspases, p53/p21 axis, and cyto-c release; (iii) decrease in MCL-1, BCL2, CDC2, CDC25C, and cyclin B1; (iv) induction of ER stress response; and (v) accumulation of ubiquitinated proteins and proteasome disassembly. 11) ROS inhibitor N-acetyl-L-cysteine blocked LCS-1-induced cell death and associated signaling. 12) LCS-1, but not RA190, induces cytotoxicity in CRISPR/Cas9-Rpn13-KO cells, confirming our proteomic data showing SOD1 as a downstream target of Rpn13. Conclusion Our proteomic and functional validation studies show that targeting UbR Rpn13 with RA190 overcomes bortezomib-resistance via decreasing cellular SOD1 levels, which therefore represents a target for novel therapeutics to improve patient outcome. Disclosures Chauhan: Stemline Therapeutics: Consultancy; C4 Therapeutics.: Equity Ownership. Anderson:Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau.

Panax Notoginseng Saponins Protect Cardiac Myocytes Against Endoplasmic Reticulum Stress and Associated Apoptosis Through Mediation of Intracellular Calcium Homeostasis.

Endoplasmic reticulum (ER) stress has been demonstrated to play important roles in the pathogenesis of various cardiovascular diseases. The ER stress pathway is therefore a promising therapeutic target in cardiovascular disease. Although Panax notoginseng saponins (PNS) are one of the patent medicines that are traditionally used to treat cardiovascular disorders, their effects on ER stress in cardiac myocytes remain unexploited so far. This study investigates the effects of PNS on ER stress and its associated cell apoptosis along with the related mechanism in cardiac myocytes. PNS compounds were identified via high-performance liquid chromatograph (HPLC) assay. PNS-pretreated H9c2 cells, HL-1 cells, and primary cultured neonatal rat cardiomyocytes were stimulated with thapsigargin (TG) to induce ER stress response and apoptosis. ER stress response was tested by immunofluorescence or immunoblot of the ER protein chaperones—calnexin, binding immunoglobulin protein (BiP) and the C/EBP homologous protein (CHOP). Cell viability was tested by methyl thiazolyl tetrazolium (MTT) assay. Cell apoptosis was detected by immunoblot of Cleaved caspase-3 and flow cytometry analysis of Annexin V/propidium iodide (PI) staining. Cytosolic, mitochondrial, and ER calcium dynamics were investigated by calcium imaging. Moreover, a ryanodine receptor type-2 (RyR2) overexpression stable cell line was generated to verify the mechanism of RyR2 involved in PNS in the inhibition of ER stress and cell apoptosis. We demonstrate here that PNS protected cardiac myocytes from ER stress response and associated cell death in a concentration-dependent manner. Importantly, PNS reduced the elevation of cytosolic calcium, mitochondria calcium, as well as ER calcium in response to either TG or histamine treatment. PNS protection in ER stress was regulated by RyR2 expression. In summary, PNS protection against TG-induced ER stress response and its associated cell apoptosis in cardiac myocytes is calcium dependent. Through the regulation of ER calcium release mediated by RyR2, a novel mechanism for PNS in the prevention of cardiovascular diseases is thereby identified.

Abstract 2659: IRE1á-dependent increase of Gá12, a gep oncogene, augments ER stress-induced catastrophic cell death

Abstract Hepatocellular carcinoma (HCC) belongs to the most deadly types of cancer since it has a poor prognosis due to its aggressive metastatic phenotype. Endoplasmic reticulum (ER) stress is closely associated with the incidences of a variety of liver diseases including liver injury, regeneration, fibrosis, cirrhosis and often HCC. G protein-coupled receptor (GPCR) signals lead to activation of their downstream pathways through G proteins. Previously, we reported that Gα12 overexpression enhances HCC malignancy, promoting cancer migration and microvascular invasion. However, the role of Gα12 in ER stress-associated pathophysiology in the liver was unclear. Given the link between ER stress and HCC malignancy, this study investigated the effect of ER stress on Gα12, the role of Gα12 in ER stress-mediated hepatocyte death, and the underlying pathway. In the analyses of RNA-sequencing data from mouse liver or primary mouse embryonic fibroblasts challenged with ER stress, Gα12 and C/EBP homologous protein (CHOP) levels correlated with each other. Increases in Gα12 level by unfolded protein response (UPR) under ER stress condition was verified using primary hepatocytes or hepatocyte-derived cell line, confirmative of the close association between Gα12 and UPR. Of note, enforced Gα12 expression under ER stress condition augmented IRE1α level in a RhoA/Rock-dependent pathway. Among the canonical pathways, IRE1α gene silencing distinctly inhibited Gα12 induction elicited by ER stress in hepatocytes. Consistently, overexpression of IRE1α enhanced Gα12 level upon tunicamycin treatment. Similar outcomes were obtained in cells or mice treated with acetaminophen, a representative liver toxicant inducing ER stress response. Overall, our results provide evidence that IRE1α-dependent increase of Gα12 potentiates ER stress response in the liver as mediated by the RhoA-ROCK pathway, enhancing catastrophic death of hepatocytes. Citation Format: Jihoon Tak, Tae Hyun Kim, Sang Geon Kim. IRE1á-dependent increase of Gá12, a gep oncogene, augments ER stress-induced catastrophic cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2659.

Nickel Enhances Zinc-Induced Neuronal Cell Death by Priming the Endoplasmic Reticulum Stress Response.

Trace metals such as zinc (Zn), copper (Cu), and nickel (Ni) play important roles in various physiological functions such as immunity, cell division, and protein synthesis in a wide variety of species. However, excessive amounts of these trace metals cause disorders in various tissues of the central nervous system, respiratory system, and other vital organs. Our previous analysis focusing on neurotoxicity resulting from interactions between Zn and Cu revealed that Cu2+ markedly enhances Zn2+-induced neuronal cell death by activating oxidative stress and the endoplasmic reticulum (ER) stress response. However, neurotoxicity arising from interactions between zinc and metals other than copper has not been examined. Thus, in the current study, we examined the effect of Ni2+ on Zn2+-induced neurotoxicity. Initially, we found that nontoxic concentrations (0–60 μM) of Ni2+ enhance Zn2+-induced neurotoxicity in an immortalized hypothalamic neuronal cell line (GT1-7) in a dose-dependent manner. Next, we analyzed the mechanism enhancing neuronal cell death, focusing on the ER stress response. Our results revealed that Ni2+ treatment significantly primed the Zn2+-induced ER stress response, especially expression of the CCAAT-enhancer-binding protein homologous protein (CHOP). Finally, we examined the effect of carnosine (an endogenous peptide) on Ni2+/Zn2+-induced neurotoxicity and found that carnosine attenuated Ni2+/Zn2+-induced neuronal cell death and ER stress occurring before cell death. Based on our results, Ni2+ treatment significantly enhances Zn2+-induced neuronal cell death by priming the ER stress response. Thus, compounds that decrease the ER stress response, such as carnosine, may be beneficial for neurological diseases.

Characterization of a new Dent Disease mouse model carrying a pathogenic mutation of ClC‐5

Dent Disease is a rare X‐linked inherited renal proximal disorder characterized by low molecular weight (LMW) proteinuria, hypercalciuria, kidney stones and progressive renal failure. Inactivating mutations of the CLCN5 gene encoding the 2Cl−/H+ exchanger ClC‐5 have been identified in approximately two‐thirds of patients with Dent Disease. ClC‐5 is predominantly expressed in early endosomes of proximal tubules where it optimizes the function of a vacuolar H+ pump to ensure an efficient endocytosis of LMW proteins.We have previously investigated the functional consequences of CLCN5 mutations in heterologous expression systems and shown that 60 % of the mutations lead to a defect in protein folding and processing. As a consequence, the misfolded ClC‐5 are retained within the endoplasmic reticulum (ER) where they could induce ER stress response. Such ER stress might in turn induce oxidative stress in proximal tubule cells. To investigate the consequences of intracellular retention of a misfolded ClC‐5, we created a mouse model carrying the N340K pathogenic mutation.ClC‐5 expression and localization in WT and N340K male mice were studied by Western blot and on kidney sections by immunofluorescence. Daily food, water intake, body weight and urine excretion data were collected in metabolic cages for N340K and WT mice. Urine as well as blood samples were analysed for ions, protein, glucose and creatinine to detect kidney dysfunction as observed in patients with Dent Disease.Mice carrying the N340K mutation displayed ClC‐5 intracellular retention in proximal tubules cells. They showed an increased urinary calcium and glucose excretion and a decreased urinary pH. They also had a severe LMW proteinuria, recapitulating common features of Dent Disease.The present study validates a new Dent Disease mouse model carrying a pathogenic Clcn5 mutation retained in the ER. The potential link between ClC‐5 retention in the ER, ER stress and oxidative stress responses has yet to be demonstrated.Support or Funding InformationPrix “Jeune Chercheur”, Fondation du Rein 2017This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Yeast molecular chaperone gene SSB2 is involved in the endoplasmic reticulum stress response.

The Saccharomyces cerevisiae chaperone gene SSB2 belongs to the Hsp70 family. Unlike other HSP70 genes, SSB2 gene expression is reduced after heat shock. It has been reported that Ssb2p can be cross-linked to ribosome-bound nascent polypeptide chains, suggesting a potential role of SSB2 in the endoplasmic reticulum (ER) stress response. In this study, SSB2-deletion and SSB2-overexpression yeast strains were generated and applied to explore the potential mechanism by which SSB2 is involved in the tunicamycin (TM)-induced ER stress response. We demonstrate for the first time that SSB2 deficiency results in reduced resistance to TM, while overexpression of SSB2 increases resistance to TM in an IRE1-HAC1 pathway-dependent manner; theseobservationsarerelatedto changes in intracellular unfolded protein response activities (under the TM-stressed condition). Additionally, SSB2 deletion induces early apoptosis and it may play a causal role in the shortened replicative life span of ssb2Δ mutants observed in this study. These findings highlight the involvement of SSB2 in ER stress responses and ageing in yeast.

Phagocytosis influences the intracellular survival of Mycobacterium smegmatis via the endoplasmic reticulum stress response

BackgroundMycobacterium smegmatis, a rapidly growing non-tuberculosis mycobacterium, is a good model for studying the pathogenesis of tuberculosis because of its genetic similarity to Mycobacterium tuberculosis (Mtb). Macrophages remove mycobacteria during an infection. Macrophage apoptosis is a host defense mechanism against intracellular bacteria. We have reported that endoplasmic reticulum (ER) stress is an important host defense mechanism against Mtb infection.ResultsIn this study, we found that M. smegmatis induced strong ER stress. M. smegmatis-induced reactive oxygen species (ROS) play a critical role in the induction of ER stress-mediated apoptosis. Pretreatment with an ROS scavenger suppressed M. smegmatis-induced ER stress. Elimination of ROS decreased the ER stress response and significantly increased the intracellular survival of M. smegmatis. Interestingly, inhibition of phagocytosis significantly decreased ROS synthesis, ER stress response induction, and cytokine production.ConclusionsPhagocytosis of M. smegmatis induces ROS production, leading to production of proinflammatory cytokines. Phagocytosis-induced ROS is associated with the M. smegmatis-mediated ER stress response in macrophages. Therefore, phagocytosis plays a critical role in the induction of ER stress-mediated apoptosis during mycobacterial infection.

Abstract LB-400: Tannic acid induces prostate cancer cell death via unfolded protein response (UPR) and modulation of CHOP

Abstract Objectives: Endoplasmic reticulum (ER) is an intricate organelle that is crucial for cellular function and survival. Cellular environments that interfere with ER functioning can lead to the accumulation of unfolded proteins, which are sensed by transmembrane sensors that instigate the unfolded protein response (UPR) to reinstate ER proteostasis. When the UPR is perturbed or not sufficient to deal with the stress conditions, apoptotic cell death is ensued. Often, prostate tumor cells (PrCa) are exposed to intrinsic and external factors that alter protein homeostasis producing endoplasmic reticulum (ER) stress. IRE1, PERK, and ATF6 are key signaling stems of the UPR during ER stress. At molecular level, dimerization of PERK kinase phosphorylates eIF2α, resulting in translation attenuation. This selectively enhances translation of the ATF4 transcription factor. Also, activated IRE1 stimulates the nuclear translocation of XBP1. Both ATF4 and XBP1 induces expression of UPR genes. The UPR stimulates and protects tumor cells against stressful conditions within the tumor microenvironment. Therapeutic intervention on this aspect ER Stress mediated apoptosis is much needed. Here, we examined the mechanistic role of tannic acid (TA) through novel targeting inhibition of UPR via modulation of ER stress in PrCa cell death. Methods: In vitro therapeutic perspective of tannic acid was evaluated using clinically relevant human prostate cancer cell line models (C4-2, PC3 and DU145) through cell proliferation and clonogenic assays. The anti-metastatic potential of TA in PrCa cells was determined using invasive and migration studies. The phase arrest and concomitant apoptosis in PrCa cells were examined using Propidium Iodide based flow cytometric studies. TA induced ER stress-mediated UPR cellular apoptotic activity was confirmed using Western blot, real-time studies, and nuclear distortion studies. Results: Protein profiling results have showed yin approach of inhibition ER stress during TA treatments with the inhibition of CHOP protein. Also, we observed TA treated PrCa cells was able to induce ER stress response proteins, such as PERK and IRE1. Its downstream signaling events include the induction of XBP1, EIF2α expression key mediators by alteration of Bcl2/Bax ratio from shifting survival towards apoptosis. With this line we assessed the expression profile of apoptosis-associated markers such as Bid and Bim (upregulated), and Bcl-2 and Bcl-xL which ratifies the induction of apoptosis during treatments. TA exhibited prominent growth arrest at G1 phase and increase of concentration led to elevated sub G1, annotating the increase of apoptotic cells. The expression of P18 and P21 were increased during TA treatments and cyclin D1 expression was inhibited supporting the G1 phase cell arrest. The expression of MMP2 and MMP9 were reduced, signifying the superior anti-metastatic ability of this compound. Cell proliferation studies showed a dose-dependent inhibitory profile of TA. Distinct results of anti-migratory and anti-metastatic effects of TA was observed during treatments. Conclusion: This study suggests that TA can efficiently induce UPR and promote ER stress signaling in cells that renders apoptosis in PrCa cells by the over expression of CHOP. We believe this novel therapeutic paradigm have clinical significance in advanced PrCa. Citation Format: Elham Hatami, Prashanth Kumar Bhusetty Nagesh, Pallabita Chowdhury, Vivek K. Kashyap, Sheema Khan, Bilal Hafeez, Meena Jaggi, Subhash C. Chauhan, Murali M. Yallapu. Tannic acid induces prostate cancer cell death via unfolded protein response (UPR) and modulation of CHOP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-400.

Activation of endoplasmic reticulum stress response by enhanced polyamine catabolism is important in the mediation of cisplatin-induced acute kidney injury.

Cisplatin-induced nephrotoxicity limits its use in many cancer patients. The expression of enzymes involved in polyamine catabolism, spermidine/spermine N1-acetyltransferase (SSAT) and spermine oxidase (SMOX) increase in the kidneys of mice treated with cisplatin. We hypothesized that enhanced polyamine catabolism contributes to tissue damage in cisplatin acute kidney injury (AKI). Using gene knockout and chemical inhibitors, the role of polyamine catabolism in cisplatin AKI was examined. Deficiency of SSAT, SMOX or neutralization of the toxic products of polyamine degradation, H2O2 and aminopropanal, significantly diminished the severity of cisplatin AKI. In vitro studies demonstrated that the induction of SSAT and elevated polyamine catabolism in cells increases the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and enhances the expression of binding immunoglobulin protein BiP/GRP78) and CCAAT-enhancer-binding protein homologous protein (CHOP/GADD153). The increased expression of these endoplasmic reticulum stress response (ERSR) markers was accompanied by the activation of caspase-3. These results suggest that enhanced polyamine degradation in cisplatin AKI may lead to tubular damage through the induction of ERSR and the consequent onset of apoptosis. In support of the above, we show that the ablation of the SSAT or SMOX gene, as well as the neutralization of polyamine catabolism products modulate the onset of ERSR (e.g. lower BiP and CHOP) and apoptosis (e.g. reduced activated caspase-3). These studies indicate that enhanced polyamine catabolism and its toxic products are important mediators of ERSR and critical to the pathogenesis of cisplatin AKI.

Severe, but not mild heat-shock treatment induces immunogenic cell death in cancer cells

ABSTRACTThe mechanisms of immunogenicity underlying mild heat-shock (mHS) treatment < 42°C of tumor cells are largely attributed to the action of heat-shock proteins; however, little is known about the immunogenicity of tumor cells undergoing severe cytotoxic heat-shock treatment (sHS > 43°C). Here, we found that sHS, but not mHS (42°C), induces immunogenic cell death in human cancer cell lines as defined by the induction of ER stress response and ROS generation, cell surface exposure of calreticulin, HSP70 and HSP90, decrease of cell surface CD47, release of ATP and HMGB1. Only sHS-treated tumor cells were efficiently killed and phagocytosed by dendritic cells (DCs), which was partially dependent on cell surface calreticulin. DCs loaded with mHS or sHS-treated tumor cells displayed similar level of maturation and stimulated IFNγ-producing CD8+ T cells without any additional adjuvants in vitro. However, only DCs loaded with sHS-treated tumor cells stimulated antigen-specific CD4+ T cells and induced higher CD8+ T-cell activation and proliferation. sHS-treated murine cells also exposed calreticulin, HSP70 and HSP90 and activated higher DC maturation than mHS treated cells. Vaccination with sHS-treated tumor cells elicited protective immunity in mice. In this study, we defined specific conditions for the sHS treatment of human lung and ovarian tumor cells to arrive at optimal ratio between effective cell death, immunogenicity and content of tumor antigens for immunotherapeutic vaccine generation.

Endoplasmic Reticulum Stress Response in Arabidopsis Roots.

Roots are the frontier of plant body to perceive underground environmental change. Endoplasmic reticulum (ER) stress response represents circumvention of cellular stress caused by various environmental changes; however, a limited number of studies are available on the ER stress responses in roots. Here, we report the tunicamycin (TM) -induced ER stress response in Arabidopsis roots by monitoring expression patterns of immunoglobulin-binding protein 3 (BiP3), a representative marker for the response. Roots promptly responded to the TM-induced ER stress through the induction of similar sets of ER stress-responsive genes. However, not all cells responded uniformly to the TM-induced ER stress in roots, as BiP3 was highly expressed in root tips, an outer layer in elongation zone, and an inner layer in mature zone of roots. We suggest that ER stress response in roots has tissue specificity.

Hydrogen sulfide, endoplasmic reticulum stress and alcohol mediated neurotoxicity.

Alcohol is one of the most socially accepted addictive drugs in modern society. Its abuse affects virtually all organ systems with the central nervous system (CNS) being particularly vulnerable to excessive alcohol exposure. Alcohol exposure also causes profound damage to both the adult and developing brain. Excessive alcohol consumption induces numerous pathophysiological stress responses, one of which is the endoplasmic reticulum (ER) stress response. Potential mechanisms that trigger the alcohol induced ER stress response are either directly or indirectly related to alcohol metabolism, which include toxic levels of acetaldehyde and homocysteine, oxidative stress and abnormal epigenetic modifications. Growing evidence suggests that H2S is the most recently recognized gasotransmitter with tremendous physiological protective functions against oxidative stress induced neurotoxicity. In this review we address the alcohol induced oxidative stress mediated ER stress and the role of H2S in its mitigation in the context of alcohol neurotoxicity. Interruption of ER stress triggers is anticipated to have therapeutic benefits for alcohol mediated diseases and disorders.

Antifibrotic Effect of Saturated Fatty Acids via Endoplasmic Reticulum Stress Response in Rat Pancreatic Stellate Cells.

We investigated the effect of saturated fatty acids on chronic pancreatitis pathogenesis by elucidating the endoplasmic reticulum (ER) stress response in pancreatic stellate cells (PSCs), which are major effector cells in pancreatic fibrosis. Wistar Bonn/Kobori rats were fed either control diet or high-fat diet (HFD) for 4 weeks. Meanwhile, cultured rat PSCs were stimulated with thapsigargin, an ER stress inducer, or palmitic acid (PA). Pancreatic fibrosis, expressions of fibrosis-related and ER stress-related proteins and mRNA, cell viability, and apoptosis were examined. The HFD reduced fibrosis and α-smooth muscle actin expression (ie, activated PSCs) but upregulated ER stress-related mRNA expression in the pancreas of young HFD-fed Wistar Bonn/Kobori rats. Induction of ER stress response in PSCs with thapsigargin or PA induced apoptosis, activated the protein kinase-like ER kinase (PERK) pathway, inhibited cell viability, and downregulated fibrosis-related protein and mRNA expression. The PERK inhibitor negated PA-induced ER stress response. Saturated fatty acids can inhibit but may not promote the fibrogenesis of chronic pancreatitis, at least in the early stage, via an ER stress response (ie, the PERK pathway) in PSCs. Moreover, induction of an apoptotic ER stress response in PSCs might be a novel therapeutic strategy for pancreatic fibrosis.

ATF6 Decreases Myocardial Ischemia/Reperfusion Damage and Links ER Stress and Oxidative Stress Signaling Pathways in the Heart.

Endoplasmic reticulum (ER) stress causes the accumulation of misfolded proteins in the ER, activating the transcription factor, ATF6 (activating transcription factor 6 alpha), which induces ER stress response genes. Myocardial ischemia induces the ER stress response; however, neither the function of this response nor whether it is mediated by ATF6 is known. Here, we examined the effects of blocking the ATF6-mediated ER stress response on ischemia/reperfusion (I/R) in cardiac myocytes and mouse hearts. Knockdown of ATF6 in cardiac myocytes subjected to I/R increased reactive oxygen species and necrotic cell death, both of which were mitigated by ATF6 overexpression. Under nonstressed conditions, wild-type and ATF6 knockout mouse hearts were similar. However, compared with wild-type, ATF6 knockout hearts showed increased damage and decreased function after I/R. Mechanistically, gene array analysis showed that ATF6, which is known to induce genes encoding ER proteins that augment ER protein folding, induced numerous oxidative stress response genes not previously known to be ATF6-inducible. Many of the proteins encoded by the ATF6-induced oxidative stress genes identified here reside outside the ER, including catalase, which is known to decrease damaging reactive oxygen species in the heart. Catalase was induced by the canonical ER stressor, tunicamycin, and by I/R in cardiac myocytes from wild-type but not in cardiac myocytes from ATF6 knockout mice. ER stress response elements were identified in the catalase gene and were shown to bind ATF6 in cardiac myocytes, which increased catalase promoter activity. Overexpression of catalase, in vivo, restored ATF6 knockout mouse heart function to wild-type levels in a mouse model of I/R, as did adeno-associated virus 9-mediated ATF6 overexpression. ATF6 serves an important role as a previously unappreciated link between the ER stress and oxidative stress gene programs, supporting a novel mechanism by which ATF6 decreases myocardial I/R damage.