ER Stress-induced Apoptosis Research Articles

A novel specific PERK activator reduces toxicity and extends survival in Huntington's disease models

One of the pathways of the unfolded protein response, initiated by PKR-like endoplasmic reticulum kinase (PERK), is key to neuronal homeostasis in neurodegenerative diseases. PERK pathway activation is usually accomplished by inhibiting eIF2α-P dephosphorylation, after its phosphorylation by PERK. Less tried is an approach involving direct PERK activation without compromising long-term recovery of eIF2α function by dephosphorylation. Here we show major improvement in cellular (STHdhQ111/111) and mouse (R6/2) Huntington's disease (HD) models using a potent small molecule PERK activator that we developed, MK-28. MK-28 showed PERK selectivity in vitro on a 391-kinase panel and rescued cells (but not PERK−/− cells) from ER stress-induced apoptosis. Cells were also rescued by the commercial PERK activator CCT020312 but MK-28 was significantly more potent. Computational docking suggested MK-28 interaction with the PERK activation loop. MK-28 exhibited remarkable pharmacokinetic properties and high BBB penetration in mice. Transient subcutaneous delivery of MK-28 significantly improved motor and executive functions and delayed death onset in R6/2 mice, showing no toxicity. Therefore, PERK activation can treat a most aggressive HD model, suggesting a possible approach for HD therapy and worth exploring for other neurodegenerative disorders.

Ticagrelor reverses the mitochondrial dysfunction through preventing accumulated autophagosomes-dependent apoptosis and ER stress in insulin-resistant H9c2 myocytes.

Ticagrelor, a P2Y12-receptor inhibitor, and a non-thienopyridine agent are used to treat diabetic patients via its effects on off-target mechanisms. However, the exact sub-cellular mechanisms by which ticagrelor exerts those effects remains to be elucidated. Accordingly, the present study aimed to examine whether ticagrelor influences directly the cardiomyocytes function under insulin resistance through affecting mitochondria-sarco(endo)plasmic reticulum (SER) cross-talk. Therefore, we analyzed the function and ultrastructure of mitochondria and SER in insulin resistance-mimicked (50-μM palmitic acid for 24-h) H9c2 cardiomyocytes in the presence or absence of ticagrelor (1-µM for 24-h). We found that ticagrelor treatment significantly prevented depolarization of mitochondrial membrane potential and increases in reactive oxygen species with a marked increase in the ATP level in insulin-resistant H9c2 cells. Ticagrelor treatment also reversed the increases in the resting level of free Ca2+ and mRNA level of P2Y12 receptors as well as preserved ER stress and apoptosis in insulin-resistant H9c2 cells. Furthermore, we determined marked repression with ticagrelor treatment in the increased number of autophagosomes and degeneration of mitochondrion, including swelling and loss of crista besides recoveries in enlargement and irregularity seen in SER in insulin-resistant H9c2 cells. Moreover, ticagrelor treatment could prevent the altered mRNA levels of Becklin-1 and type 1 equilibrative nucleoside transporter (ENT1), which are parallel to the preservation of ultrastructural ones. Our overall data demonstrated that ticagrelor can directly affect cardiomyocytes and provide marked protection against ER stress and dramatic induction of autophagosomes, and therefore, can alleviate the ER stress-induced oxidative stress increase and cell apoptosis during insulin resistance.

Astragalus polysaccharide attenuates metabolic memory-triggered ER stress and apoptosis via regulation of miR-204/SIRT1 axis in retinal pigment epithelial cells.

Background: ‘Metabolic memory’ of early hyperglycaemic environment has been frequently suggested in the progression of diabetic retinopathy (DR). Retinal pigment epithelial (RPE) cells are crucial targets for DR initiation following hyperglycaemia. Astragalus polysaccharides (APS) has been long used as a traditional Chinese medicine in treating diabetes. In the present study, the preventive effects and mechanisms of APS on metabolic memory-induced RPE cell death were investigated.Methods: The expressions of miR-204 and SIRT1 were determined by reverse transcription quantitative PCR (RT-qPCR). Dual luciferase assay was applied to detect the potential targeting effects of miR-204 on SIRT1. SIRT1, ER stress and apoptosis related proteins were monitored using Western blotting. Apoptosis was assessed by TUNEL assay and Annexin V/PI staining followed by flow cytometry analysis. MiR-204 mimics and shSIRT1 were applied for miR-204 overexpression and SIRT1 knockdown, respectively.Results: High glucose exposure induced metabolic memory, which was accompanied with sustained dysregulation of miR-204/SIRT1 axis, high level of ER stress and activation of apoptotic pathway even after replacement with normal glucose. Pre-treatment with APS concentration-dependently reversed miR-204 expression, leading to disinhibition of SIRT1 and alleviation of ER stress-induced apoptosis indicated by decreased levels of p-PERK, p-IRE-1, cleaved-ATF6, Bax, cleaved caspase-12, -9, -3, and increased levels of Bcl-2 and unleaved PARP. The effects of APS on RPE cells were reversed by either miR-204 overexpression or SIRT1 knockdown.Conclusions: We concluded that APS inhibited ER stress and subsequent apoptosis via regulating miR-204/SIRT1 axis in metabolic memory model of RPE cells.

Citrus flavonoids as a target for the prevention of pancreatic β-cells dysfunction in diabetes.

The number of patients with type 2 diabetes mellitus (T2DM) has rapidly increased, especially in East and Southeast Asia. In these areas, in general, people have especially vulnerable β-cells and insulin secretion deficiency and reduced β-cell mass are the primary cause of T2DM. Therefore, the alleviation of such β-cell dysfunction would provide therapeutic approaches to prevent the development of T2DM. Nobiletin, a polymethoxylated flavonoid found in citrus fruits, has been shown to improve obesity and insulin resistance in T2DM model mice. We focused on β-cells and investigated the effects of nobiletin on insulin secretion and β-cell apoptosis. In β-cell line INS-1, nobiletin increased glucose-induced insulin secretion (GSIS) in a concentration-dependent manner, which was inhibited by an Epac inhibitor. In addition, nobiletin at 10 μM inhibited thapsigargin-induced apoptosis, which was inhibited by a PKA inhibitor. Nobiletin also suppressed thapsigargin-induced increases in cleaved caspase-3 and phosphorylated JNK. Thus, nobiletin is suggested to promote GSIS and prevent ER stress-induced β-cell apoptosis, which are mediated via Epac and PKA-dependent pathways, respectively. In summary, nobiletin is suggested to exhibit insulinotropic and anti-apoptotic effects on β-cells, which are one of the causes of its anti-diabetic effect. Moreover, nobiletin seems to be able to alleviate the development of T2DM by protecting β-cells from apoptosis.

Calumenin-1 Interacts with Climp63 to Cooperatively Determine the Luminal Width and Distribution of Endoplasmic Reticulum Sheets.

SummaryThe ER is composed of distinct structures like tubules, matrices, and sheets, all of which are important for its various functions. However, how these distinct ER structures, especially the perinuclear ER sheets, are formed remains unclear. We report here that the ER membrane protein Climp63 and the ER luminal protein calumenin-1 (Calu1) collaboratively maintain ER sheet morphology. We show that the luminal length of Climp63 is positively correlated with the luminal width of ER sheets. Moreover, the lumen-only mutant of Climp63 dominant-negatively narrows the lumen of ER sheets, demonstrating that Climp63 acts as an ER luminal bridge. We also reveal that Calu1 specifically interacts with Climp63 and antagonizes Climp63 in terms of both ER sheet distribution and luminal width. Together, our data provide insight into how the structure of ER sheets is maintained and regulated.

22P - XAF1 and ZNF313 complex stimulates ER stress-induced apoptosis via direct GRP78 inhibition

BackgroundEndoplasmic reticulum (ER) stress-induced, unfolded protein response (UPR)-mediated apoptosis is a protective mechanism whose failure contributes to malignant tumor progression. XAF1 is a stress-inducible, pro-apoptotic tumor suppressor which is inactivated in many human tumor cells. The molecular signal mechanism explaining its function in controlling ER stress, however, remains largely unclear. In this study, we explored the role of XAF1 in UPR signaling and ER stress-induced apoptosis. MethodsXAF1 and GRP78 expression were determined by RT-PCR and immunoblot assay. the interplay of XAF1 with GRP78 was defined using immunoprecipitation and in vitro pull-down assays. ER stress was induced using thapsigargin, tunicamycin and brefeldin A. Flow cytometry was used to assess cell cycle progression and apoptosis. ResultsXAF1 expression is upregulated at the transcriptional level in response to ER stress and this activation is abolished by suppression of PERK-Nrf2 but not of IRE1a or ATF6 signaling. This indicates that XAF1 is a target of Nrf2 in transcriptional level and activated by ER stress through the PERK pathway. This XAF1 induction greatly enhances the apoptotic response of tumor cells to ER stress while blockade of XAF1 induction reduces apoptotic sensitivity of tumor cells to ER stress. Intriguingly, XAF1 decreases the protein level of the ER stress sensor, GRP78, and consequently activates UPR signaling. Mechanistically, XAF1 binds directly to GRP78 and stimulates its proteasomal degradation in an E3 ligase ZNF313-dependent manner. XAF1 facilitates ZNF313 interaction with GRP78 and thereby promoting ZNF313-mediated K48 ubiquitination of GRP78. Unlikely wild-type XAF1, mutant XAF1 lacking GRP78- or ZNF313-binding domain shows no activity to promote ER stress-induced apoptosis. ConclusionsConclusion: XAF1 sensitizes cells to ER stress and drives apoptotic switch of UPR function away from cell cycle arrest by directly antagonizing GRP78 through the assembly of a ZNF313-mediated destruction complex. Our study identifies first a cell-fate decisions function of XAF1 under ER stress conditions. Legal entity responsible for the studySung-Gil Chi. FundingBK. DisclosureAll authors have declared no conflicts of interest.

The cytotoxic effect of oxymatrine on basic cellular processes of A549 non-small lung cancer cells

Oxymatrine is the alkaloid derived from the root of Sophora species. This compound is proven to exhibit anti-viral, anti-asthmatic, anti-fibrotic and anti-inflammatory properties. Additionally, oxymatrine is able to promote cancer cells apoptosis and inhibit their proliferation. The aim of this study was to present the influence of oxymatrine on non-small cell lung cancer cells. The results indicate, that this agent induces dose-dependent cell death mainly through ER stress-induced apoptosis pathway. We also suggest that the oxymatrine reduces the metastatic potential by inhibition of the EMT process, as A549 cells treated with chosen doses of the compound were characterized by a decrease in the expression of the N-cadherin, vimentin and the elevation of E-cadherin level. Moreover, the study broadens the knowledge on so far poorly understood aspect of the influence of oxymatrine on the cytoskeleton structure.

2130-P: Imeglimin Modulated ER Stress to Prevent ß-Cell Apoptosis Induced by High Glucose or Thapsigargin

Imeglimin, a tetrahydrotriazine anti-hyperglycemic agent, is thought to have effects on mitochondrial function of the liver, skeletal muscle, and pancreatic β-cells. However, the molecular mechanisms of imeglimin action have been still unclear. Treatment of mouse islets with 1.0 mM imeglimin significantly increased insulin release in response to glucose (2.1-fold, p<0.01 at 11.1 mM glucose; 1.9-fold, p<0.05 at 16.7 mM glucose, compared to 3.9 mM glucose). Treatment with a combination of liraglutide (100 nM) and imeglimin did not show an additional increase in insulin secretion compared to the treatment with liraglutide alone. Liraglutide and imeglimin significantly increased EdU-incorporated proliferating β-cells in the islets under 11.1 mM glucose (1.02% in vehicle control; 2.21% in liraglutide, p<0.01 vs. vehicle; 1.46% in 1.0 mM imeglimin, p<0.01 vs. vehicle). Imeglimin prevented β-cell apoptosis induced by high glucose (32% of control in imeglimin, p<0.01; 57% of control in liraglutide, p=0.068). We conducted gene expression microarray analysis of islets treated with imeglimin under high glucose and some ER stress-related gene expression were altered by imeglimin. Then, we examined the effects of imeglimin on ER stress-induced β-cell apoptosis in mouse islets. Imeglimin significantly reduced β-cell apoptosis induced by thapsigargin at 5.6 mM glucose (29% of thapsigargin, p<0.01). Imeglimin further upregulated the expression of C/EBP homologous protein (CHOP) under thapsigargin treatment and tended to decrease phosphorylation of eIF2-α. We also confirmed that imeglimin improved β-cell apoptosis induced by high glucose or thapsigargin in human islets. Collectively, imeglimin prevented β-cell apoptosis induced by high glucose or thapsigargin, in addition to increase in insulin secretion and β-cell proliferation. Imeglimin seemed to regulate CHOP/GADD34-mediated dephosphorylation of eIF2-α to recover from translational inhibition. Disclosure J. Li: None. J. Shirakawa: None. Y. Togashi: None. Y. Terauchi: Advisory Panel; Self; AstraZeneca, Daiichi Sankyo Company, Limited, Eli Lilly and Company, Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk A/S, Sanofi. Research Support; Self; Daiichi Sankyo Company, Limited, Eli Lilly and Company, Merck Sharp & Dohme Corp., Novartis Pharmaceuticals Corporation, Novo Nordisk A/S, Ono Pharmaceutical Co., Ltd., Sanofi, Shionogi & Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd. Speaker's Bureau; Self; Astellas Pharma Inc., AstraZeneca, Bayer Yakuhin, Ltd., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Novo Nordisk A/S, Ono Pharmaceutical Co., Ltd., Sanofi, Sanwa Kagaku Kenkyusho, Shionogi & Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd.

MON-110 A Natural Compound Isolated from Schisandra Chinensis Fruit Has a Beneficial Effect in a Fly Model of Obesity

Objectives: Gomisin N is a lignin derived from Schisandra chinensis that was reported to exhibit hepato-protective, anticancer, and anti-inflammatory effects. However, its role in whole body energetic homeostasis remains unclear. Drosophila melanogaster shares the majority of metabolic functions in vertebrates, including conserved signaling pathways for lipid metabolism and glucose homeostasis, making it a suitable model system to study the effects of signaling molecules and their modifiers in vivo. In this study, we employed fruit flies as a diet-induced obese model to elucidate the effects of Gomisin N on lipid and glucose metabolism Methods: We have characterized the adolescent fly phenotypes in triglyceride levels and lifespan when reared on a high-fat diet, with or without Gomisin N. Results: Constant exposures of flies to a high-fat diet resulted in significantly increased triglyceride levels along with a shortened life span. Importantly, an administration of Gomisin N to this group of flies lowered their body weight and total triglyceride levels and improved their lifespan. To pinpoint the molecular mechanism of the anti-obesity effects of Gomisin N, we adopted a cell-based assay using mouse pancreatic β-cells exposed to a metabolic stress with antimycin. The drug-induced metabolic stress increased ER stress proteins as well as relevant apoptotic markers. Importantly, a treatment of these pancreatic β-cells with Gomisin N prevented ER stress-induced apoptosis and increased autophagy. Conclusions: Taken together, these in vivo and in vitro findings suggest that Gomisin N could serve as a useful agent for prevention and treatment of obesity.

ZBTB7A, a miR-663a target gene, protects osteosarcoma from endoplasmic reticulum stress-induced apoptosis by suppressing LncRNA GAS5 expression

Many studies have uncovered the essential role of ZBTB7A in regulating tumourigenesis. However, its functional significance in cell responses to endoplasmic reticulum stress (ER stress) remains poorly understood. Here we report that ZBTB7A functions as an important prosurvival factor in osteosarcoma cells undergoing pharmacological ER stress-induced by tunicamycin (TM) or thapsigargin (TG). The downregulation of ZBTB7A expression by ER stress promoted cell apoptosis in vitro and in vivo. ZBTB7A expression levels were increased in osteosarcoma tissues and elevated ZBTB7A was associated with osteosarcoma metastasis. Further mechanistic studies revealed that miR-663a induced by ER stress directly bound to the 3′UTR of ZBTB7A and contributed to ER stress-induced ZBTB7A downregulation in osteosarcoma cells. Additionally, our data revealed that ZBTB7A bound to the promoter of LncRNA GAS5 and transcriptionally suppressed LncRNA GAS5 expression, leading to a decline in ER stress-induced cell apoptosis. Collectively, our findings reveal the prosurvival role of ZBTB7A in osteosarcoma adaptation to ER stress and suggest that the miR-663a-ZBTB7A-LncRNAGAS5 pathway is essential for the survival of human osteosarcoma cells under ER stress.

Rutaecarpine ameliorated sepsis-induced peritoneal resident macrophages apoptosis and inflammation responses

BackgroundSepsis is a life-threatening organ dysfunction disease caused by a dysregulated host response to infection. Rutaecarpine is an important alkaloid component of Evodia rutaecarpa. There has been no study on the therapeutic effects of rutaecarpine in sepsis. MethodsMice were randomly assigned into four groups: sham, sepsis, sepsis plus vehicle and sepsis plus rutaecarpine groups. Mice in sepsis were administered CLP surgery. Rutaecarpine or vehicle was injected intraperitoneally 1 h after CLP. The liver damage, bacterial infection, survival rate and weight loss were observed, and changes in the ratio of peritoneal resident macrophages were analyzed by flow cytometry and immunofluorescence microscopy. Western blotting was used to identify the levels of NF-κB signaling pathway, ER stress and apoptosis related proteins. TUNEL and Annexin V/PI assay were used to detect the apoptosis of liver tissues and peritoneal resident macrophages, respectively. ELISA and qRT-PCR were used to detect the inflammatory factors. ResultsRutaecarpine alleviated weight loss, bacterial infection and liver injury, and regulated inflammation homeostasis, enhancing survival rate induced by sepsis. Population of peritoneal resident macrophages (CD11b+F4/80hiMHCIIlow) was significantly decreased in sepsis mice, which was resulted from ER stress-induced apoptosis through caspase-12 signaling pathway. Rutaecarpine restored the ratio of peritoneal resident macrophages and the level of GATA6 in CD11b+ peritoneal macrophages. Rutaecarpine could also attenuate sepsis–induced inflammatory responses through inhibiting the activation of ER stress/NF-κB pathway. ConclusionRutaecarpine ameliorated sepsis-induced peritoneal resident macrophages apoptosis and inflammation responses through inhibition of ER stress-mediated caspase-12 and NF-κB pathways. Our study provided new insights for drug development against sepsis.

Small molecule inhibition of IRE1α kinase/RNase has anti-fibrotic effects in the lung.

Endoplasmic reticulum stress (ER stress) has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a disease of progressive fibrosis and respiratory failure. ER stress activates a signaling pathway called the unfolded protein response (UPR) that either restores homeostasis or promotes apoptosis. The bifunctional kinase/RNase IRE1α is a UPR sensor/effector that promotes apoptosis if ER stress remains high and irremediable (i.e., a “terminal” UPR). Using multiple small molecule inhibitors against IRE1α, we show that ER stress-induced apoptosis of murine alveolar epithelial cells can be mitigated in vitro. In vivo, we show that bleomycin exposure to murine lungs causes early ER stress to activate IRE1α and the terminal UPR prior to development of pulmonary fibrosis. Small-molecule IRE1α kinase-inhibiting RNase attenuators (KIRAs) that we developed were used to evaluate the contribution of IRE1α activation to bleomycin-induced pulmonary fibrosis. One such KIRA—KIRA7—provided systemically to mice at the time of bleomycin exposure decreases terminal UPR signaling and prevents lung fibrosis. Administration of KIRA7 14 days after bleomycin exposure even promoted the reversal of established fibrosis. Finally, we show that KIRA8, a nanomolar-potent, monoselective KIRA compound derived from a completely different scaffold than KIRA7, likewise promoted reversal of established fibrosis. These results demonstrate that IRE1α may be a promising target in pulmonary fibrosis and that kinase inhibitors of IRE1α may eventually be developed into efficacious anti-fibrotic drugs.

Dexmedetomidine alleviates cerebral ischemia-reperfusion injury by inhibiting endoplasmic reticulum stress dependent apoptosis through the PERK-CHOP-Caspase-11 pathway

Dexmedetomidine (Dex) has the neuroprotective effect on cerebral ischemia-reperfusion injury (CIRI). But the mechanism is not yet clear. In this study, we established a model of middle cerebral artery occlusion (MCAO) and treated primary cortical neurons with oxygen glucose deprivation (OGD), followed by Dex treatment. Neurological protection of Dex was then assessed by neurological deficit score, brain edema, TTC staining, TUNEL assay, Western blot analysis, immunohistochemistry, and RT-PCR. The results showed that Dex significantly reduced the neurological deficit score, brain edema and cerebral infarction area due to CIRI. After Dex treatment, the expression levels of ER stress-related apoptosis pathway proteins (GRP78, p-PERK, CHOP and Cleaved-caspase-3) were significantly decreased and the apoptosis of brain cells was also significantly reduced. Immunohistochemistry showed that expression and nuclear localization of CHOP decreased significantly after the application of Dex. The downstream apoptotic protein caspase-11 mediated by PERK-CHOP was also markedly inhibited by Dex. In conclusion, our results suggested that Dex reduced ER stress-induced apoptosis after CIRI. Its protective mechanism may be related to PERK-CHOP-Caspase-11 dependent signaling pathway.

Mice deficient in aldo-keto reductase 1a (Akr1a) are resistant to thioacetamide-induced liver injury

Aldehyde reductase (Akr1a) has been reported to be involved in detoxification of reactive aldehydes as well as in the synthesis of bioactive compounds such as ascorbic acid (AsA). Because Akr1a is expressed at high levels in the liver and is involved in xenobiotic metabolism, our objective was to investigate the hepato-protective role of Akr1a in a thioacetamide (TAA)-induced hepatotoxicity model using Akr1a-deficient (Akr1a−/−) mice. Wild-type (WT) and Akr1a−/− mice were injected intraperitoneally with TAA and the extent of liver injury in the acute phase was assessed. Intriguingly, the extent of TAA-induced liver damage was less in the Akr1a−/− mice than in the WT mice. Biomarkers for the ER stress-induced apoptosis pathway were markedly decreased in the livers of Akr1a−/− mice, whereas AsA levels in plasma did not change significantly in any of the mice. In the liver, TAA is converted to reactive metabolites such as TAA S-oxide and then to TAA S, S-dioxide via the action of CYP2E1. In Akr1a−/− mice, CYP2E1 activity was relatively lower than WT mice at the basal level, leading to reactive TAA metabolites being produced at lower levels after the TAA treatment. The levels of liver proteins that were modified with these metabolites were also lower in the Akr1a−/− mice than the WT mice after the TAA treatment. Furthermore, after a lethal dose of a TAA challenge, the WT mice all died within 36 h, whereas almost all of the Akr1a−/− mice survived. These collective results suggest that Akr1a−/− mice are resistant to TAA-induced liver injury, and it follows that the absence of Akr1a might modulate TAA bioactivation.

Bcl-2 regulates store-operated Ca2+ entry to modulate ER stress-induced apoptosis

Ca2+ plays a significant role in linking the induction of apoptosis. The key anti-apoptotic protein, Bcl-2, has been reported to regulate the movement of Ca2+ across the ER membrane, but the exact effect of Bcl-2 on Ca2+ levels remains controversial. Store-operated Ca2+ entry (SOCE), a major mode of Ca2+ uptake in non-excitable cells, is activated by depletion of Ca2+ in the ER. Depletion of Ca2+ in the ER causes translocation of the SOC channel activator, STIM1, to the plasma membrane. Thereafter, STIM1 binds to Orai1 or/and TRPC1 channels, forcing them to open and thereby allow Ca2+ entry. In addition, several anti-cancer drugs have been reported to induce apoptosis of cancer cells via the SOCE pathway. However, the detailed mechanism underlying the regulation of SOCE by Bcl-2 is not well understood. In this study, a three-amino acid mutation within the Bcl-2 BH1 domain was generated to verify the role of Bcl-2 in Ca2+ handling during ER stress. The subcellular localization of the Bcl-2 mutant (mt) is similar to that in the wild-type Bcl-2 (WT) in the ER and mitochondria. We found that mt enhanced thapsigargin and tunicamycin-induced apoptosis through ER stress-mediated apoptosis but not through the death receptor- and mitochondria-dependent apoptosis, while WT prevented thapsigargin- and tunicamycin-induced apoptosis. In addition, mt depleted Ca2+ in the ER lumen and also increased the expression of SOCE-related molecules. Therefore, a massive Ca2+ influx via SOCE contributed to caspase activation and apoptosis. Furthermore, inhibiting SOCE or chelating either extracellular or intracellular Ca2+ inhibited mt-mediated apoptosis. In brief, our results explored the critical role of Bcl-2 in Ca2+ homeostasis and the modulation of ER stress.

AMPK alleviates endoplasmic reticulum stress by inducing the ER-chaperone ORP150 via FOXO1 to protect human bronchial cells from apoptosis

Chronic obstructive pulmonary disease (COPD), is characterized by inflammation of airways accompanied by a progressive destruction of lung parenchyma. This process is initiated in most cases by cigarette smoking. In this study we investigated the role of AMP activated protein kinase (AMPK) in cigarette smoke extract (CSE)-induced airway epithelial cell apoptosis as a consequence of endoplasmic reticulum stress (ER stress). Exposure of human bronchial epithelial cells (HBEpC) to CSE resulted in apoptosis as detected using Annexin V-PI flow cytometry. However, co-treatment with N1-(β-d-ribofuranosyl)-5-aminoimidazole-4-carboxamide (AICAR), a pharmacological activator of AMPK, significantly increased cell protection against ER stress-induced apoptosis by upregulating the 150 kDa oxygen-regulated protein (ORP150), which functions as an ER-associated chaperone, with concomitant elevation of FOXO1, a critical transcription factor regulating ORP150 expression. Lentiviral silencing of AMPK or FOXO1 using short hairpin (sh) RNA resulted in a significant decrease of ORP150 and an elevation of CCAAT/enhancer-binding protein-homologous protein (CHOP) resulting in ER stress and apoptosis of HBEpC. Together, our results strongly suggest that AMPK can activate ORP150 through FOXO1 pathway and confer protection against ER stress-induced apoptosis of airway epithelial cells following exposure to CSE. Thus, AMPK may serve as a likely therapeutic target for clinical and sub-clinical interventions in COPD.

Simultaneously targeting DNA damage repair pathway and mTORC1/2 results in small cell lung cancer growth arrest via ER stress-induced apoptosis.

Purpose: Small cell lung cancer (SCLC) is highly lethal with no effective therapy. Wee1 kinase inhibitor AZD1775 (MK-1775) and mTOR kinase inhibitor MLN0128 (TAK228) are in clinical trials for relapsed SCLC and recurrent lung cancer, respectively. However, there is no preclinical data combining these two drugs in human cancers.Methods: In this study, we set to investigate the combinatorial anti-tumor effects of AZD1775 and MLN0128 on two human SCLC cell lines H69 and H82 in vitro and in vivo.Results: We have found that AZD1775 or MLN0128 treatment results in remarkably suppressed cell proliferation and increased cell death in vitro, what's more, the salient finding here is the potent anti-tumor effect observed in combinatorial treatment in H82 xenograft tumor. Importantly, we have first observed marked induction of ER stress and CHOP-dependent SCLC cell apoptosis in MLN0128 and AZD1775-primed cells.Conclusion: Our study has first provided preclinical evidence that combination of AZD1775 and MLN0128 could be a novel effective therapy for advanced SCLC patients.

Schisandrin B Ameliorates Myocardial Ischemia/Reperfusion Injury Through Attenuation of Endoplasmic Reticulum Stress-Induced Apoptosis.

Schisandrin B (Sch B), an active composition isolated from the fruit of Schisandra chinensis, has been proved to possess antiinflammatory, antioxidant and anti-endoplasmic reticulum (ER) stress effects in many rodent tissues. However, the exact mechanism of cardioprotective effect of Sch B still needs more study. Here, we detected the effects of Sch B on myocardial ischemia/reperfusion (I/R) injury rats. I/R injury model in this study was established by left anterior descending coronary artery ligation for 40min followed by 1h of reperfusion. Male healthy rats were randomly divided into five groups: the sham, I/R, Sch B (20mg/kg) + I/R, and Sch B (40mg/kg) + I/R, Sch B (80mg/kg) + I/R, with 10 rats in each group. We showed that Sch B treatment significantly protected against myocardial I/R injury, as demonstrated by the decrease in the percentage of infarct formation assessed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining in representative heart tissue slices, comparing with the I/R control group. The levels of creatine kinase (CK), lactate dehydrogenase (LDH), malondialdehyde (MDA), and total superoxide dismutase (T-SOD) were tested. The ER stress-related proteins such as C/EBP homologous protein (CHOP), activating transcription factor 6 (ATF6), and (PKR)-like ER kinase (PERK) were further measured by western blot, and their messenger RNA levels were measured by real-time PCR. The apoptosis of heart tissue cells was also tested through the expressions of caspase-9, caspase-3, Bcl-2, and Bax proteins. Collectively, these results revealed that Sch B exerts protection role on myocardial I/R injury through decreasing oxidative reaction, suppressing ATF6 and PERK pathway, and attenuating ER stress-induced apoptosis.

A non-canonical pathway regulates ER stress signaling and blocks ER stress-induced apoptosis and heart failure

Endoplasmic reticulum stress is an evolutionarily conserved cell stress response associated with numerous diseases, including cardiac hypertrophy and heart failure. The major endoplasmic reticulum stress signaling pathway causing cardiac hypertrophy involves endoplasmic reticulum stress sensor PERK (protein kinase-like kinase) and eIF2α-ATF4-CHOP signaling. Here, we describe a non-canonical, AGGF1-mediated regulatory system for endoplasmic reticulum stress signaling associated with increased p-eIF2α and ATF4 and decreased sXBP1 and CHOP. Specifically, we see a reduced AGGF1 level consistently associated with induction of endoplasmic reticulum stress signaling in mouse models and human patients with heart failure. Mechanistically, AGGF1 regulates endoplasmic reticulum stress signaling by inhibiting ERK1/2 activation, which reduces the level of transcriptional repressor ZEB1, leading to induced expression of miR-183-5p. miR-183-5p post-transcriptionally downregulates CHOP and inhibits endoplasmic reticulum stress-induced apoptosis. AGGF1 protein therapy and miR-183-5p regulate endoplasmic reticulum stress signaling and block endoplasmic reticulum stress-induced apoptosis, cardiac hypertrophy, and heart failure, providing an attractive paradigm for treatment of cardiac hypertrophy and heart failure.

Epigallocatechin-3-gallate enhances ER stress-induced cancer cell apoptosis by directly targeting PARP16 activity

Poly(ADP-ribose) polymerases (PARPs) are ADP-ribosylating enzymes and play important roles in a variety of cellular processes. Most small-molecule PARP inhibitors developed to date have been against PARP1, a poly-ADP-ribose transferase, and suffer from poor selectivity. PARP16, a mono-ADP-ribose transferase, has recently emerged as a potential therapeutic target, but its inhibitor development has trailed behind. Here we newly characterized epigallocatechin-3-gallate (EGCG) as a potential inhibitor of PARP16. We found that EGCG was associated with PARP16 and dramatically inhibited its activity in vitro. Moreover, EGCG suppressed the ER stress-induced phosphorylation of PERK and the transcription of unfolded protein response-related genes, leading to dramatically increase of cancer cells apoptosis under ER stress conditions, which was dependent on PARP16. These findings newly characterized EGCG as a potential inhibitor of PARP16, which can enhance the ER stress-induced cancer cell apoptosis, suggesting that a combination of EGCG and ER stress-induced agents might represent a novel approach for cancer therapy or chemoprevention.