scholarly journals Methotrexate and theaflavin-3,3’ di gallate synergistically restore the balance between apoptosis and autophagy in synovial fibroblast of RA: An ex vivo approach with cultured human RA FLS

2021 ◽  
Author(s):  
Sanchaita Misra ◽  
Aniruddha Bagchi ◽  
Avik Sarkar ◽  
Sougata Niyogi ◽  
Dipanjan Bhattacharjee ◽  
...  
Keyword(s):  
Er Stress ◽  
Ex Vivo ◽  
Black Tea ◽  
Synovial Fibroblasts ◽  
Er Stress Response ◽  
Tnf Α ◽  
Angiogenic Markers ◽  
Underlying Mechanisms ◽  
Induced Apoptosis ◽  
Autophagic Proteins

Abstract Background: Rheumatoid arthritis (RA) is characterized by inflammation mediated angiogenesis in synovial tissue, leading to apoptotic retardation and enhanced cell survival in synovial fibroblasts. Methotrexate (MTX) can reduce selective pro-inflammatory cytokines but unable to restore disrupted homeostasis between autophagy and apoptosis in fd-FLS.Objective: To evaluate the effect of black tea compound TF3 along with MTX upon fluid derived (fd)-FLS to induce apoptosis and inhibit autophagy through ER stress-mediated pathways.Methods: FLS sourced from synovial fluid (SF) of patients with RA (n=11) and osteoarthritis (OA) (n=10) were cultured following treatment with MTX/TF3 or in combination and underlying mechanisms were investigated. Extracellular inflammatory markers like CRP and cytokines (TNF-α, IL-6), angiogenic markers (VEGF, ANG-1) were quantified by ELISA. Cell viability of cultured fd-FLS was determined by MTT assay. fd-FLS treated with MTX/TF3 or combination of MTX(125nM) and TF3(10µM), followed by apoptosis measurement by flow cytometry. ER stress associated markers were quantified by RT-PCR (IRE1A and spliced-XBP-1) and immunoblotting (Grp78, Hsp70, CHOP, HIF1-α). Apoptotic (Bcl-2, Bax, and Caspases) and autophagic proteins (Beclin1, LC3b and p62) were quantified by immunoblot study. Results: MTX and TF3 both in single doses (IC25) could down-regulate the levels of pro-inflammatory and angiogenic markers. Combination treatment modulated ER stress response and blocked the auto-phagmosomal proteins in fd-FLS and induced apoptosis.Conclusion: Disruption in homeostasis between apoptosis and autophagy might be an underlying phenomenon in the progression of pathophysiology in fd-FLS. The combined administration of MTX and TF3 successfully balanced the homeostasis by inducing apoptosis.

Abstract 292: The Role of the ATF6 Branch of the ER Stress Response in the Endocrine Heart

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Erik A Blackwood ◽  
Christopher C Glembotski
Keyword(s):  
Stress Response ◽  
Er Stress ◽  
Knockout Mice ◽  
Ex Vivo ◽  
Proteolytic Cleavage ◽  
Isolated Perfused Heart ◽  
Wild Type ◽  
Atrial Myocytes ◽  
Perfused Heart ◽  
Er Stress Response

Rationale: Atrial natriuretic peptide (ANP) is stored in the heart in large dense core granules of atrial myocytes as a biologically inactive precursor, pro-ANP. Hemodynamic stress and atrial stretch stimulate coordinate secretion and proteolytic cleavage of pro-ANP to its bioactive form, ANP, which promotes renal salt excretion and vasodilation, which, together contribute to decreasing blood pressure. While the ATF6 branch of the ER stress response has been studied in ventricular tissue mouse models of myocardial ischemia and pathological hypertrophy, roles for ATF6 and ER stress on the endocrine function of atrial myocytes have not been studied. Objective/Methods: To address this gap in our knowledge, we knocked down ATF6 in primary cultured neonatal rat atrial myocytes (NRAMs) using a chemical inhibitor of the proteolytic cleavage site enabling ATF6 activation and siRNA and measured ANP expression and secretion basally and in response to alpha- adrenergic agonist stimulation using phenylephrine. We also compared the ANP secretion from wild- type mice and ATF6 knockout mice in an ex vivo Langendorff model of the isolated perfused heart. Results: ATF6 knockdown in NRAMs significantly impaired basal and phenylephrine-stimulated ANP secretion. ATF6 knockout mice displayed lower levels of ANP in atrial tissue at baseline as well as after phenylephrine treatment. Similarly, in the ex vivo isolated perfused heart model, less ANP was detected in effluent of ATF6 knockout hearts compared to wild-type hearts. Conclusions: The ATF6 branch of the ER stress response is necessary for efficient co-secretional processing of pro-ANP to ANP and for agonist-stimulated ANP secretion from atrial myocytes. As ANP is secreted in a regulated manner in response to a stimulus and pro-ANP is synthesized and packaged through the classical secretory pathway, we posit that ATF6 is required for adequate expression, folding, trafficking, processing and secretion of biologically active ANP from the endocrine heart.

scholarly journals Paeoniflorin protects cells from GalN/TNF-α-induced apoptosis via ER stress and mitochondria-dependent pathways in human L02 hepatocytes

2014 ◽  
Vol 46 (5) ◽  
pp. 357-367 ◽  
Author(s):  
Zequn Jiang ◽  
Weiping Chen ◽  
Xiaojing Yan ◽  
Lei Bi ◽  
Sheng Guo ◽  
...  
Keyword(s):  
Er Stress ◽  
Tnf Α ◽  
Induced Apoptosis

Novel targets for endoplasmic reticulum stress-induced apoptosis in B-CLL

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. 2713-2723 ◽  
Author(s):  
Emanuela Rosati ◽  
Rita Sabatini ◽  
Giuliana Rampino ◽  
Filomena De Falco ◽  
Mauro Di Ianni ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Cell Apoptosis ◽  
Ex Vivo ◽  
Lymphocytic Leukemia ◽  
Minor Role ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Cytochrome C Release ◽  
Induced Apoptosis

Abstract A better understanding of apoptotic signaling in B-chronic lymphocytic leukemia (B-CLL) cells may help to define new therapeutic strategies. This study investigated endoplasmic reticulum (ER) stress signaling in spontaneous apoptosis of B-CLL cells and whether manipulating ER stress increases their apoptosis. Results show that a novel ER stress-triggered caspase cascade, initiated by caspase-4 and involving caspase-8 and -3, plays an important role in spontaneous B-CLL cell apoptosis. ER stress-induced apoptosis in B-CLL cells also involves CHOP/GADD153 up-regulation, increased JNK1/2 phosphorylation, and caspase-8–mediated cleavage of Bap31 to Bap20, known to propagate apoptotic signals from ER to mitochondria. In ex vivo B-CLL cells, some apoptotic events associated with mitochondrial pathway also occur, including mitochondrial cytochrome c release and caspase-9 processing. However, pharmacologic inhibition studies show that caspase-9 plays a minor role in B-CLL cell apoptosis. ER stress also triggers survival signals in B-CLL cells by increasing BiP/GRP78 expression. Manipulating ER signaling by siRNA down-regulation of BiP/GRP78 or treating B-CLL cells with 2 well-known ER stress-inducers, tunicamycin and thapsigargin, increases their apoptosis. Overall, our findings show that ER triggers an essential pathway for B-CLL cell apoptosis and suggest that genetic and pharmacologic manipulation of ER signaling could represent an important therapeutic strategy.

Metformin regulates palmitate-induced apoptosis and ER stress response in HepG2 liver cells

2009 ◽  
Vol 32 (2) ◽  
pp. 251-257 ◽  
Author(s):  
Do-Sung Kim ◽  
Seul-Ki Jeong ◽  
Hyung-Ryong Kim ◽  
Dal-Sik Kim ◽  
Soo-Wan Chae ◽  
...  
Keyword(s):  
Stress Response ◽  
Er Stress ◽  
Liver Cells ◽  
Er Stress Response ◽  
Induced Apoptosis

Treatment with Histone Deacetylase 6-Specific Inhibitor WT-161 Disrupts hsp90 Function, Abrogates Aggresome Formation and Sensitizes Human Mantle Cell Lymphoma Cells to Lethal ER Stress Induced by Proteasome Inhibitor Carfilzomib

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2856-2856 ◽  
Author(s):  
Rekha Rao ◽  
Warren Fiskus ◽  
Ramesh Balusu ◽  
Hongwei Ma ◽  
James Bradner ◽  
...  
Keyword(s):  
Stress Response ◽  
Er Stress ◽  
Histone Deacetylase ◽  
Proteasome Inhibitor ◽  
Cell Lymphoma ◽  
Specific Inhibitor ◽  
Er Stress Response ◽  
Mantle Cell ◽  
Aggresome Formation ◽  
Induced Apoptosis

Abstract Abstract 2856 The proteasome inhibitor bortezpmib has been shown to markedly increase the intracellular levels of misfolded proteins, induce aggresome formation and cause endoplasmic reticulum (ER) stress, resulting in apoptosis of human Mantle Cell Lymphoma (MCL) cells. Consistent with this, Bortezomib displays clinical efficacy in patients with relapsed and refractory MCL. We have recently reported that the pan-histone deacetylase (HDAC) inhibitor panobinostat, by also inhibiting HDAC6, abrogates aggresome formation and induces Endoplasmic Stress (ER) stress, as well as potentiates bortezomib-induced apoptosis of MCL cells. Here, we determined the anti-MCL cell activity of an HDAC6-specific inhibitor, WT-161 alone and in combination with the novel, orally bio-available, proteasome inhibitor carfilzomib (Proteolix Inc.) against human, cultured and primary, patient-derived MCL cells. Treatment with WT-161 (0.1 to 1.0 uM) resulted in a dose-dependent increase in the acetylation of alpha-tubulin and heat shock protein (hsp) 90, without any appreciable increase in the levels of acetylated histone (H) 3. Consistent with WT-161 mediated hyperacetylation and inhibition of hsp90 chaperone function, treatment with WT-161 increased the intracellular levels of polyubiuitylated proteins in the cultured MCL JeKo-1 and Z138 cells. WT-161 was also noted to dose-dependently deplete the levels of cyclin D1 in the cultured MCL cells. Treatment with WT-161 also induced ER stress response in the MCL cells, demonstrated by increase in the protein levels of Glucose regulated protein (GRP) 78, phosphorylated eIF2 (eukaryotic initation factor 2) α, and induction of the pro-apoptotic transcription factor CHOP (CAAT/Enhancer Binding Protein Homologous Protein). We next determined the effects of co-treatment with WT-161 on carfilzomib-induced aggresome formation, ER stress response and apoptosis of the cultured and primary MCL cells. Co-treatment with WT-161 (0.25 uM) abrogated carfilzomib-induced aggresome formation in MCL cells, as evidenced by confocal immunofluorescent staining of aggresomes with anti-HDAC6 and anti-ubiquitin antibodies. Compared to each agent alone, co-treatment with WT-161 and carfilzomib induced more intracellular polyubiquitylated proteins and induced higher levels of CHOP in the cultured MCL cells. Co-treatment with WT-161 and carfilzomib also synergistically induced apoptosis of the cultured MCL cells (combination indices < 1.0). Notably, co-treatment with WT-161 and carfilzomib also synergistically induced apoptosis of primary MCL cells (combination indices < 1.0). These findings strongly support the in vivo testing of the combination of an HDAC6-specific inhibitor such as WT-161 with the proteasome inhibitor carfilzomib against human MCL cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals IRS1 deficiency protects β-cells against ER stress-induced apoptosis by modulating sXBP-1 stability and protein translation

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Tomozumi Takatani ◽  
Jun Shirakawa ◽  
Michael W. Roe ◽  
Colin A. Leech ◽  
Bernhard F. Maier ◽  
...  
Keyword(s):  
Er Stress ◽  
Protein Translation ◽  
Adaptor Proteins ◽  
Nuclear Accumulation ◽  
Cell Protection ◽  
Β Cells ◽  
Β Cell ◽  
Protein Instability ◽  
Underlying Mechanisms ◽  
Induced Apoptosis

Abstract Endoplasmic reticulum (ER) stress is among several pathological features that underlie β-cell failure in the development of type 1 and type 2 diabetes. Adaptor proteins in the insulin/insulin-like-growth factor-1 signaling pathways, such as insulin receptor substrate-1 (IRS1) and IRS2, differentially impact β-cell survival but the underlying mechanisms remain unclear. Here we report that β-cells deficient in IRS1 (IRS1KO) are resistant, while IRS2 deficiency (IRS2KO) makes them susceptible to ER stress-mediated apoptosis. IRS1KOs exhibited low nuclear accumulation of spliced XBP-1 due to its poor stability, in contrast to elevated accumulation in IRS2KO. The reduced nuclear accumulation in IRS1KO was due to protein instability of Xbp1 secondary to proteasomal degradation. IRS1KO also demonstrated an attenuation in their general translation status in response to ER stress revealed by polyribosomal profiling. Phosphorylation of eEF2 was dramatically increased in IRS1KO enabling the β-cells to adapt to ER stress by blocking translation. Furthermore, significantly high ER calcium (Ca2+) was detected in IRS1KO β-cells even upon induction of ER stress. These observations suggest that IRS1 could be a therapeutic target for β-cell protection against ER stress-mediated cell death by modulating XBP-1 stability, protein synthesis, and Ca2+ storage in the ER.

scholarly journals Fisetin Protects HaCaT Human Keratinocytes from Fine Particulate Matter (PM2.5)-Induced Oxidative Stress and Apoptosis by Inhibiting the Endoplasmic Reticulum Stress Response

Antioxidants ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1492
Author(s):  
Ilandarage Menu Neelaka Molagoda ◽  
Mirissa Hewage Dumindu Kavinda ◽  
Yung Hyun Choi ◽  
Hyesook Lee ◽  
Chang-Hee Kang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Particulate Matter ◽  
Stress Response ◽  
Er Stress ◽  
Fine Particulate Matter ◽  
Human Keratinocytes ◽  
Initiation Factor ◽  
Er Stress Response ◽  
Fine Particulate ◽  
Induced Apoptosis

Fine particulate matter (PM2.5) originates from the combustion of coal and is found in the exhaust of fumes of diesel vehicles. PM2.5 readily penetrates the skin via the aryl hydrocarbon receptor, causing skin senescence, inflammatory skin diseases, DNA damage, and carcinogenesis. In this study, we investigated whether fisetin, a bioactive flavonoid, prevents PM2.5-induced apoptosis in HaCaT human keratinocytes. The results demonstrated that fisetin significantly downregulated PM2.5-induced apoptosis at concentrations below 10 μM. Fisetin strongly inhibited the production of reactive oxygen species (ROS) and the expression of pro-apoptotic proteins. The PM2.5-induced apoptosis was associated with the induction of the endoplasmic reticulum (ER) stress response, mediated via the protein kinase R-like ER kinase (PERK)–eukaryotic initiation factor 2α (eIF2α)–activating transcription factor 4 (ATF4)–CCAAT-enhancer-binding protein (C/EBP) homologous protein (CHOP) axis. Additionally, the cytosolic Ca2+ levels were markedly increased following exposure to PM2.5. However, fisetin inhibited the expression of ER stress-related proteins, including 78 kDa glucose-regulated protein (GRP78), phospho-eIF2α, ATF4, and CHOP, and reduced the cytosolic Ca2+ levels. These data suggest that fisetin inhibits PM2.5-induced apoptosis by inhibiting the ER stress response and production of ROS.

scholarly journals The Drosophila metallopeptidase superdeath decouples apoptosis from the activation of the ER stress response

2019 ◽  
Author(s):  
Rebecca A.S. Palu ◽  
Clement Y. Chow
Keyword(s):  
Endoplasmic Reticulum ◽  
Drosophila Melanogaster ◽  
Stress Response ◽  
Er Stress ◽  
Unfolded Protein ◽  
Er Stress Response ◽  
Membrane Bound ◽  
Induced Apoptosis ◽  
The Unfolded Protein Response ◽  
Protein Response

ABSTRACTEndoplasmic reticulum (ER) stress-induced apoptosis is a primary cause and modifier of degeneration in a number of genetic disorders. Understanding how genetic variation between individuals influences the ER stress response and subsequent activation of apoptosis could improve individualized therapies and predictions of outcomes for patients. In this study, we find that the uncharacterized, membrane-bound metallopeptidase CG14516 in Drosophila melanogaster, which we rename as SUPpressor of ER stress-induced DEATH (superdeath), plays a role in modifying ER stress-induced apoptosis. We demonstrate that loss of superdeath reduces apoptosis and degeneration in the Rh1G69D model of ER stress through the JNK signaling cascade. This effect on apoptosis occurs without altering the activation of the unfolded protein response (IRE1 and PERK), suggesting that the beneficial pro-survival effects of this response are intact. Furthermore, we show that superdeath functions epistatically upstream of CDK5, a known JNK-activated pro-apoptotic factor in this model of ER stress. We demonstrate that superdeath is not only a modifier of this particular model, but functions as a general modifier of ER stress-induced apoptosis across different tissues and ER stresses. Finally, we present evidence of Superdeath localization to the endoplasmic reticulum membrane. While similar in sequence to a number of human metallopeptidases found in the plasma membrane and ER membrane, its localization suggests that superdeath is orthologous to ERAP1/2 in humans. Together, this study provides evidence that superdeath is a link between stress in the ER and activation of cytosolic apoptotic pathways.SIGNIFICANCE STATEMENTGenetic diseases display a great deal of variability in presentation, progression, and overall outcomes. Much of this variability is caused by differences in genetic background among patients. One process that commonly modifies degenerative disease is the endoplasmic reticulum (ER) stress response. Understanding the genetic sources of variation in the ER stress response could improve individual diagnosis and treatment decisions. In this study, we characterized one such modifier in Drosophila melanogaster, the membrane-bound metallopeptidase CG14516 (superdeath). Loss of this enzyme suppresses a model of ER stress-induced degeneration by reducing cell death without altering the beneficial activation of the unfolded protein response. Our findings make superdeath and its orthologues attractive therapeutic targets in degenerative disease.

scholarly journals Isoorientin Inhibits Amyloid β25–35-Induced Neuronal Inflammation in BV2 Cells by Blocking the NF-κB Signaling Pathway

Molecules ◽  
2021 ◽  
Vol 26 (22) ◽  
pp. 7056
Author(s):  
Buyun Kim ◽  
Ki Yong Lee ◽  
Byoungduck Park
Keyword(s):  
Neurodegenerative Disorder ◽  
Drug Candidate ◽  
Therapeutic Drug ◽  
Treatment And Prevention ◽  
Bv2 Cells ◽  
Tnf Α ◽  
Underlying Mechanisms ◽  
Induced Apoptosis ◽  
Factor Α ◽  
The Brain

Alzheimer’s disease (AD) is a severe neurodegenerative disorder. AD is pathologically characterized by the formation of intracellular neurofibrillary tangles, and extracellular amyloid plaques which were comprised of amyloid-beta (Aβ) peptides. Aβ induces neurodegeneration by activating microglia, which triggers neurotoxicity by releasing various inflammatory mediators and reactive oxygen species (ROS). Nuclear factor-kappa B (NF-κB) is expressed in human tissues including the brain and plays an important role in Aβ-mediated neuronal inflammation. Thus, the identification of molecules that inhibit the NF-κB pathway is considered an attractive strategy for the treatment and prevention of AD. Isoorientin (3′,4′,5,7-Tetrahydroxy-6-C-glucopyranosyl flavone; ISO), which can be extracted from several plant species, such as Philostachys and Patrinia is known to have various pharmacological activities such as anticancer, antioxidant, and antibacterial activity. However, the effect of ISO on Aβ-mediated inflammation and apoptosis in the brain has yet to be elucidated. In the present study, we investigated whether ISO regulated Aβ-induced neuroinflammation in microglial cells and further explored the underlying mechanisms. Our results showed that ISO inhibited the expression of iNOS and COX-2 induced by Aβ25–35. And, it inhibited the secretion of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). In addition, ISO reduced the ROS production in Aβ25–35-induced BV2 cells and inhibited NF-κB activation. Furthermore, ISO blocked Aβ25–35-induced apoptosis of BV2 cells. Based on these findings, we suggest that ISO represents a promising therapeutic drug candidate for the treatment and prevention of AD.

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