Endoplasmic Reticulum Stress-induced Cell Death Research Articles

Disturbing microtubule-endoplasmic reticulum dynamics by gold nanoclusters for improved triple-negative breast cancer treatment.

Microtubules are highly dynamic structures, and their dynamic instability is indispensable for not only cell growth and movement, but also stress responses, such as endoplasmic reticulum (ER) stress. Docetaxel, a microtubule targeting agent (MTA), is the first-line drug for cancer treatment by simultaneously promoting microtubule dysregulation- and ER stress-induced cell death. However, it also causes adverse effects and drug resistance, especially in triple-negative breast cancer (TNBC) with a poor prognosis and high mortality rate. In this study, we developed a peptide-templated gold nanocluster, namely GA. GA significantly sensitizes TNBC cells to docetaxel, causing severe cell death. This effect is further validated by a 3D tumor spheroid model. Mechanistically, GA disrupted microtubule dynamic instability, meanwhile promoting PERK-mediated ER stress. Interestingly, ER stress inhibitors profoundly suppressed microtubule dysregulation, suggesting a retrograde regulation of ER stress on microtubules. In vivo, the combined administration of docetaxel and GA significantly suppresses tumor growth while docetaxel alone cannot. GA similarly elevated the level of caspases and PERK within tumors as in vitro. Importantly, GA treatment also profoundly promoted the production of anti-tumor inflammatory cytokines. Collectively, we developed an ER-microtubule regulatory nanomaterial that enhanced the therapeutic effect of docetaxel by elevating tumor cell death and anti-tumor cytokine production, providing a potential supplemental strategy for TNBC treatment.

7936 Type 1 Diabetes Therapeutic Compound MSB-61: Using RNA Sequencing To Identify The Mechanism

Abstract Disclosure: P. Khan: None. K. Corbin: None. N. Ajmal: None. S. Bergmeir: None. G. Gu: None. X. Tong: None. C.S. Nunemaker: None. Abstract Type 1 diabetes (T1D) is a chronic autoimmune disease in which loss of insulin-producing beta-cells leads to hyperglycemia due to which patients eventually require lifelong insulin therapy to maintain normal glycemic control. Insulin remains the gold standard and the only effective treatment for T1D since its discovery 100 years ago. Protecting the pancreatic beta cells from cytokine-induced cell death and restoring insulin secretion are important to treat T1D, two properties that are currently lacking in present therapies. Our lab identified a chemical compound MSB-61 that increases insulin release and protects pancreatic islets against T1D-associated cytokines and endoplasmic reticulum (ER) stress. This study aims to identify the mechanism of action of MSB-61 in protecting pancreatic islets from ER stress-induced cell death. To achieve this aim pancreatic islets obtained from CD-1 strain mice were exposed for one hour to 50uM MSB-61 or vehicle control. After treatment, RNA was collected for RNA sequencing. In addition to RNA extraction for sequencing, MSB-61's ability to secrete insulin was verified by performing ELISA on control,10 and 50uM MSB-61-treated islets, and the activity of protection from cell death was assessed by performing cell death assay on islets treated with ER stressors in the presence and absence of 10 and 50uM MSB-61. Using Genialis, a bioinformatics service, we evaluated 57,010 genes (including non-coding transcripts) in our RNA sequencing data set. The DESeq2 technique of data analysis allowed us to identify 350 differentially expressed genes (DEGs), of which 251 were upregulated and 74 were downregulated. After removing non-coding/undefined transcripts and genes with <5 counts per million, a total of 117 genes remained; 108 up and 9 down. According to the String database (string-db.org), many of the top DEGs formed a common network of genes associated with cell death protection and insulin secretion. A key component of that gene network is a stress-responsive orphan nuclear receptor with known protective effects in beta cells. Testing this orphan nuclear receptor as a possible direct or indirect target of MSB-61 for cell death prevention can aid in identifying the genes and pathways targeted by MSB-61, allowing us to improve our approaches to T1D treatment and develop better medications. Presentation: 6/3/2024

Resveratrol alleviates endoplasmic reticulum stress-induced cell death and improves functional prognosis after traumatic brain injury in mice.

Resveratrol (RSV) is a polyphenol antioxidant that has been shown to have neuroprotective effects. We sought molecular mechanisms that emphasize the anti-inflammatory activity of RSV in traumatic brain injury (TBI) in mice associated with endoplasmic reticulum stress (ERS). After establishing three experimental groups (sham, TBI, and TBI+RSV), we explored the results of RSV after TBI on ERS and caspase-12 apoptotic pathways. The expression levels of C/EBP homologous protein (CHOP), glucose regulated protein 78kD (GRP78), caspase-3, and caspase-12 in cortical brain tissues were assessed by western blotting. The qPCR analysis was also performed on mRNA expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in cortical brain tissue. In addition, the expression of GRP78 in microglia (ionized calcium binding adaptor molecule 1; Iba-1) and neurons (neuronal nuclei; NeuN) was identified by immunofluorescence staining. The neurological function of mice was assessed by modified neurological severity scores (mNSS). After drug treatment, the expression of CHOP, GRP78, caspase-3 and caspase-12 decreased, and qPCR results showed that TNF-α and IL-1β were down-regulated. Immunofluorescence staining showed down-regulation of Iba-1+/GRP78+ and NeuN+/GRP78+ cells after RSV treatment. The mNSS analysis confirmed improvement after RSV treatment. RSV improved apoptosis by downregulating the ERS signaling pathway and improved neurological prognosis in mice with TBI.

Endoplasmic reticulum stress-induced necroptosis promotes cochlear inflammation: Implications for age-related hearing loss

Age-related hearing loss (ARHL) is the most common sensory disorder associated with human aging. Chronic inflammation is supposed to be an important contributor to ARHL. Yet, the underlying mechanisms of developing cochlear inflammation are still not well understood. In this study, we found that the inflammation, endoplasmic reticulum (ER) stress and necroptosis signalings are activated in the cochlea of aged C57BL/6 mice. ER stress activator tunicamycin (TM) induced necroptosis in cochlear HEI-OC1 cells and cochlear explants, while necroptosis inhibitors protected cochlear cells from ER stress-induced cell death. The antioxidants inhibited necroptosis and protected HEI-OC1 cells from TM insults. Necroptotic HEI-OC1 cells promoted the activation of the co-cultured macrophages via Myd88 signaling. Moreover, necroptosis inhibitor protected from TM-induced hearing loss, and inhibited inflammation in C57BL/6 mice. These findings suggest that ER stress-induced necroptosis promotes cochlear inflammation and hearing loss. Targeting necroptosis serves as a potential approach for the treatment of cochlear inflammation and ARHL.

Abstract C091: Harnessing proteostatic vulnerabilities in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) harbors mutations in KRAS in greater than 90% of cases and is characterized by a dense, fibrotic stroma that fosters the formation of a hypoxic, nutrient-poor tumor microenvironment, rendering tumor cells susceptible to proteotoxic stress. In order to prevent endoplasmic reticulum (ER) stress-induced cell death caused by accumulation of toxic protein aggregates, tumor cells must adapt proteostatic mechanisms including activation of the unfolded protein response (UPR) pathway and degradation of misfolded or aggregated proteins through proteasome-dependent or -independent mechanisms, such as ER-specific autophagy (ERphagy). Despite evidence suggesting that tumor cells may experience proteotoxic stress, little is known about the mechanisms pancreatic cancer cells employ to maintain proteostasis. We found that pancreatic cancer cells exhibit KRas-dependent constitutive UPR activation at the expense of ERphagy, suggesting that tumor cells preferentially employ UPR signaling to maintain proteostasis. Accordingly, mutant KRas pancreatic cancer cells have little to no aggregated proteins at baseline as a consequence of UPR activation. Furthermore, upon pharmacological enhancement of ER stress by 26S proteasome inhibition with bortezomib, ERphagy is engaged as a protective mechanism against UPR hyperactivation and apoptosis. In order to target protective ERphagy to promote apoptosis, we suppressed ERphagy activation through siRNA-mediated knockdown of ERphagy receptors under conditions of bortezomib-induced ER stress and found that depletion of CCPG1 but not other ERphagy receptors augmented the growth-suppressive effects of bortezomib due to greater UPR activation and accumulation of aggregated proteins. Overall, our findings implicate context-dependent reliance on both UPR signaling and ERphagy in pancreatic cancer cell survival and identify a targetable cell-intrinsic vulnerability that can leveraged for enhancing the cytotoxic effects of bortezomib, which alone is clinically ineffective in treating PDAC. Citation Format: Sandra Vogt, Dafna Bar-Sagi. Harnessing proteostatic vulnerabilities in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C091.

USP7 attenuates endoplasmic reticulum stress-induced apoptotic cell death through deubiquitination and stabilization of FBXO7.

Parkinson's disease (PD) is a common neurodegenerative disease (NDD) characterized by the loss of dopaminergic neurons in the substantia nigra. Similar to other NDDs, the buildup of toxic protein aggregates in PD leads to progressive neuronal loss, culminating in neurodegeneration. Accumulating evidence indicates that alterations in subcellular organelles, particularly the endoplasmic reticulum (ER), are critically involved in pathological neurodegenerative events in NDDs, including PD. Mutations in the F-box only protein 7 (FBXO7 or PARK15) gene have been found to cause early onset autosomal recessive familiar PD. FBXO7 functions as an adaptor protein in the Skp1-Cullin1-F-box protein (SCF) E3 ubiquitin ligase complex, which promotes substrate ubiquitination. Although FBXO7 is involved in the ubiquitination of various target proteins, little is known about the upstream regulatory mechanism of FBXO7 and/or its modulator(s). Ubiquitin specific protease 7 (USP7) is a deubiquitinating enzyme that regulates the balance between protein synthesis and degradation by removing ubiquitin from target substrates. The role of USP7 in various types of cancer is well-established; however, its role in NDDs has not been elucidated to date. In this study, we identified that USP7 acts as a novel regulator of FBXO7, positively regulating the stability of FBXO7 through Lys48-linked deubiquitination. Moreover, USP7 was found to mitigate ER stress-induced cytotoxicity and apoptosis by preventing the proteasomal degradation of FBXO7. Taken together, our study suggests that the functional relationship between FBXO7 and USP7 may play a crucial role in ER stress-induced apoptosis and the pathogenesis of PD.

Non-small cell lung cancers (NSCLCs) oncolysis using coxsackievirus B5 and synergistic DNA-damage response inhibitors

With the continuous in-depth study of the interaction mechanism between viruses and hosts, the virus has become a promising tool in cancer treatment. In fact, many oncolytic viruses with selectivity and effectiveness have been used in cancer therapy. Human enterovirus is one of the most convenient sources to generate oncolytic viruses, however, the high seroprevalence of some enteroviruses limits its application which urges to exploit more oncolytic enteroviruses. In this study, coxsackievirus B5/Faulkner (CV-B5/F) was screened for its potential oncolytic effect against non-small cell lung cancers (NSCLCs) through inducing apoptosis and autophagy. For refractory NSCLCs, DNA-dependent protein kinase (DNA-PK) or ataxia telangiectasia mutated protein (ATM) inhibitors can synergize with CV-B5/F to promote refractory cell death. Here, we showed that viral infection triggered endoplasmic reticulum (ER) stress-related pro-apoptosis and autophagy signals, whereas repair for double-stranded DNA breaks (DSBs) contributed to cell survival which can be antagonized by inhibitor-induced cell death, manifesting exacerbated DSBs, apoptosis, and autophagy. Mechanistically, PERK pathway was activated by the combination of CV-B5/F and inhibitor, and the irreversible ER stress-induced exacerbated cell death. Furthermore, the degradation of activated STING by ERphagy promoted viral replication. Meanwhile, no treatment-related deaths due to CV-B5/F and/or inhibitors occurred. Conclusively, our study identifies an oncolytic CV-B5/F and the synergistic effects of inhibitors of DNA-PK or ATM, which is a potential therapy for NSCLCs.

Punicalagin relieves lipotoxic injuries on pancreatic β-cells via regulating the oxidative stress and endoplasmic reticulum stress-mediated apoptosis.

Cellular toxicity of hyperlipidemia has been long considered a major cause of various intractable disease such as diabetes. Discovering lipotoxicity antagonist with high efficiency and low side effects is of importance to develop therapeutics for relevant diseases. In the current study, we evaluate the anti-lipotoxic potential of punicalagin (PU) on pancreatic cells and investigate its underpinning mechanism involved. The administration of PU effectively improved cell viability, quenched intracellular reactive oxygen species, alleviated lipid peroxidation, and enhanced cellular antioxidative capacity in RINm5F cells stimulated by sodium palmitate. Besides that, PU treatment significantly inhibited the overload of mitochondrial calcium ions; alleviated the activation of endoplasmic reticulum (ER) stress mediators including glucose-regulated protein 78, protein kinase RNA-like ER kinase, eukaryotic initiation factor 2α, activating transcription factor 6, caspase 12, and C/EBP homologous protein (CHOP); and attenuated the expression of cleaved caspase 3 and poly ADP-ribose polymerase in test cells. Further RNA interference experiment results and miR211-5p expression analysis revealed that PU may directly mitigate CHOP expression and upregulate the expression of miR211-5p to reduce ER stress-induced pancreatic cell death. The efficacy of PU in maintaining redox equilibrium and diminishing ER stress on pancreatic cells stressed by hyperlipidemia suggests that PU can be used as a promising dietary natural product to safeguard the pancreatic health against lipotoxicity.

Prdx5 in the Regulation of Tuberous Sclerosis Complex Mutation-Induced Signaling Mechanisms.

(1) Background: Tuberous sclerosis complex (TSC) mutations directly affect mTORC activity and, as a result, protein synthesis. In several cancer types, TSC mutation is part of the driver mutation panel. TSC mutations have been associated with mitochondrial dysfunction, tolerance to reactive oxygen species due to increased thioredoxin reductase (TrxR) enzyme activity, tolerance to endoplasmic reticulum (ER) stress, and apoptosis. The FDA-approved drug rapamycin is frequently used in clinical applications to inhibit protein synthesis in cancers. Recently, TrxR inhibitor auranofin has also been involved in clinical trials to investigate the anticancer efficacy of the combination treatment with rapamycin. We aimed to investigate the molecular background of the efficacy of such drug combinations in treating neoplasia modulated by TSC mutations. (2) Methods: TSC2 mutant and TSC2 wild-type (WT) cell lines were exposed to rapamycin and auranofin in either mono- or combination treatment. Mitochondrial membrane potential, TrxR enzyme activity, stress protein array, mRNA and protein levels were investigated via cell proliferation assay, electron microscopy, etc. (3) Results: Auranofin and rapamycin normalized mitochondrial membrane potential and reduced proliferation capacity of TSC2 mutant cells. Database analysis identified peroxiredoxin 5 (Prdx5) as the joint target of auranofin and rapamycin. The auranofin and the combination of the two drugs reduced Prdx5 levels. The combination treatment increased the expression of heat shock protein 70, a cellular ER stress marker. (4) Conclusions: After extensive analyses, Prdx5 was identified as a shared target of the two drugs. The decreased Prdx5 protein level and the inhibition of both TrxR and mTOR by rapamycin and auranofin in the combination treatment made ER stress-induced cell death possible in TSC2 mutant cells.

1722-P: RIPK1 Promotes Thapsigargin-Induced ß-Cell Death Independent of Caspase 3/7 Activity In Vitro

Endoplasmic reticulum (ER) stress promotes β-cell dysfunction and death in diabetes. Although studies of ER stress-induced β-cell death have focused on mechanisms of apoptosis, we recently showed that β cells are also susceptible to a form of programmed cell death called necroptosis. Necroptosis occurs downstream of receptor interacting protein kinase 1 (RIPK1), does not require caspase activation, and results in lytic cell death. Studies in non-islet cell types have linked ER stress-induced cell death to necroptosis. Thus, we hypothesized that RIPK1 promotes ER stress-induced β-cell death in vitro. To test this hypothesis, we quantified cell death and caspase 3/7 activity in NIT-1 β cells, human iPSC-derived β-like cells and intact mouse islets following treatment with thapsigargin (a SERCA2b inhibitor and ER stress-inducing agent, 50 nM or 500 nM) and zVAD-FMK (a pan-caspase inhibitor, 40 μM). After quantifying cell death hourly for 48 hrs, we found that thapsigargin significantly increased cell death in NIT-1 cells, β-like human iPSCs and intact mouse islets, and that this occurred in conjunction with increased caspase 3/7 activity. We next asked whether co-treatment with zVAD was sufficient to prevent thapsigargin-induced cell death. In each cell type evaluated, we found that zVAD inhibited caspase 3/7 activity but failed to protect from thapsigargin-induced cell death. To establish the role of RIPK1 in this process, we next evaluated cell death in gene-edited NIT-1 control (non-targeting gRNA) and NIT-1 Ripk1Δ (gRNA targeting Ripk1 exons 2-3) β cells. After 24 hrs, we found that NIT-1 Ripk1Δ cells were strongly protected from thapsigargin-induced cell death both when caspases were active (n=6, p<0.001) and when they were inhibited (n=6, p<0.001). These data indicate that RIPK1 regulates thapsigargin-induced β-cell death independent of caspase 3/7 activation. Future studies will examine the mechanisms by which RIPK1 regulates ER stress-induced β-cell death in vitro and in vivo. Disclosure N.Mukherjee: None. C.J.Contreras: None. L.Lin: None. E.P.Cai: None. A.T.Templin: None. Funding U.S. Department of Veterans Affairs (IK2BX004659 to A.T.T.); National Institutes of Health (T32DK064466 to C.J.C.); Indiana University School of Medicine (to N.M.)

Ubiquitin-like protein 5 is a novel player in the UPR–PERK arm and ER stress–induced cell death

Biological functions of the highly conserved ubiquitin-like protein 5 (UBL5) are not well understood. In Caenorhabditis elegans, UBL5 is induced under mitochondrial stress to mount the mitochondrial unfolded protein response (UPR). However, the role of UBL5 in the more prevalent endoplasmic reticulum (ER) stress-UPR in the mammalian system is unknown. In the present work, we demonstrated that UBL5 was an ER stress-responsive protein, undergoing rapid depletion in mammalian cells and livers of mice. The ER stress-induced UBL5 depletion was mediated by proteasome-dependent yet ubiquitin-independent proteolysis. Activation of the protein kinase R-like ER kinase arm of the UPR was essential and sufficient for inducing UBL5 degradation. RNA-Seq analysis of UBL5-regulated transcriptome revealed that multiple death pathways were activated in UBL5-silenced cells. In agreement with this, UBL5 knockdown induced severe apoptosis in culture and suppressed tumorigenicity of cancer cells invivo. Furthermore, overexpression of UBL5 protected specifically against ER stress-induced apoptosis. These results identify UBL5 as a physiologically relevant survival regulator that is proteolytically depleted by the UPR-protein kinase R-like ER kinase pathway, linking ER stress to cell death.

A novel optinEuRin stress connection in glaucoma.

The endoplasmic reticulum (ER) is the organelle where the production and shaping of most secreted and transmembrane proteins happens. ER function is finely regulated to prevent accumulation of misfolded proteins generating ER stress. ER stress is common in both healthy and pathological situations due to multiple intrinsic and extrinsic factors including acute demand in protein synthesis, hypoxia or impaired protein folding caused by gene mutations. Sayyad etal. found that the M98K mutation of optineurin sensitizes glaucoma retinal ganglion cells to ER stress-induced cell death. This is associated with an autophagy-dependent elevation of ER stress sensor expression.

Deficiency of Perry syndrome-associated p150Glued in midbrain dopaminergic neurons leads to progressive neurodegeneration and endoplasmic reticulum abnormalities

Multiple missense mutations in p150Glued are linked to Perry syndrome (PS), a rare neurodegenerative disease pathologically characterized by loss of nigral dopaminergic (DAergic) neurons. Here we generated p150Glued conditional knockout (cKO) mice by deleting p150Glued in midbrain DAergic neurons. The young cKO mice displayed impaired motor coordination, dystrophic DAergic dendrites, swollen axon terminals, reduced striatal dopamine transporter (DAT), and dysregulated dopamine transmission. The aged cKO mice showed loss of DAergic neurons and axons, somatic accumulation of α-synuclein, and astrogliosis. Further mechanistic studies revealed that p150Glued deficiency in DAergic neurons led to the reorganization of endoplasmic reticulum (ER) in dystrophic dendrites, upregulation of ER tubule-shaping protein reticulon 3, accumulation of DAT in reorganized ERs, dysfunction of COPII-mediated ER export, activation of unfolded protein response, and exacerbation of ER stress-induced cell death. Our findings demonstrate the importance of p150Glued in controlling the structure and function of ER, which is critical for the survival and function of midbrain DAergic neurons in PS.

A glaucoma-associated OPTN polymorphism, M98K sensitizes retinal cells to endoplasmic reticulum stress and tumour necrosis factor α.

Optineurin/OPTN polymorphism, M98K is associated with normal tension glaucoma in certain populations, and genetic evidence shows its interaction with tumour necrosis factor-alpha (TNFα) polymorphism in causing glaucoma. Endoplasmic reticulum (ER) stress is also associated with glaucoma. We hypothesized that M98K-OPTN may sensitize retinal ganglion cells to various types of stress. To test this hypothesis, stable clones of a retinal cell line, 661W, expressing either wild-type (WT)-OPTN or M98K-OPTN were generated and examined for their survival under various stress conditions. Compared with WT-OPTN expressing cells, M98K-OPTN expressing cells showed significantly lower cell survival and higher activation of caspase-3 and caspase-8 upon treatment with tunicamycin (an inducer of ER stress) or TNFα. Levels of ER stress sensors IRE1α, PERK and ATF6 were significantly higher in M98K-OPTN expressing cells. Tunicamycin treatment resulted in significantly higher induction of ER stress marker CHOP and several other ER stress response genes regulated by IRE1α-XBP1, PERK-ATF4 and ATF6 pathways, in M98K-OPTN expressing cells. Splicing of XBP1 and ATF6 activation was higher in tunicamycin-treated M98K-OPTN expressing cells. Increased levels of PERK and IRE1α proteins in M98K-OPTN expressing cells were dependent on autophagy. Overall, our results show that M98K-OPTN sensitizes retinal cells to TNFα and ER stress-induced cell death. We also show that M98K-OPTN alters ER stress response signalling, which possibly enhances the sensitivity of retinal cells to ER stress. Our results provide support to the hypothesis that M98K-OPTN may cooperate with other genetic or environmental factors to cause retinal ganglion cell death associated with glaucoma.

Chitooligosaccharide prevents vascular endothelial cell apoptosis by attenuation of endoplasmic reticulum stress via suppression of oxidative stress through Nrf2-SOD1 up-regulation

Context Endoplasmic reticulum (ER) stress contributes to endothelium pathological conditions. Chitooligosaccharides (COS) have health benefits, but their effect on endothelial cells is unknown. We demonstrate for the first time a protective effect of COS against ER-induced endothelial cell damage. Objective To evaluate the protective effect of COS on ER stress-induced apoptosis in endothelial cells. Material and methods Endothelial (EA.hy926) cells were pre-treated with COS (250 or 500 μg/mL) for 24 h, and then treated with 0.16 μg/mL of Tg for 24 h and compared to the untreated control. Apoptosis and necrosis were detected by Annexin V-FITC/propidium iodide co-staining. Reactive oxygen species (ROS) were measured with the DCFH2-DA and DHE probes. The protective pathway and ER stress markers were evaluated by reverse transcription-polymerase chain reaction, western blot, and immunofluorescence analyses. Results COS attenuated ER stress-induced cell death. The viability of EA.hy926 cells treated with Tg alone was 44.97 ± 1% but the COS pre-treatment increased cells viability to 74.74 ± 3.95% in the 250 μg/mL COS and 75.34 ± 2.4% in the 500 μg/mL COS treatments. Tg induced ER stress and ROS, which were associated with ER stress-mediated death. Interestingly, COS reduced ROS by upregulating nuclear factor-E2-related factor 2 (Nrf2), and the oxidative enzymes, superoxide dismutase1 (SOD1) and catalase. COS also suppressed up-regulation of the ER-related apoptosis protein, CHOP induced by Tg. Conclusions COS protected against ER stress-induced apoptosis in endothelial cells by suppressing ROS and up-regulation Nrf2 and SOD1. These findings support the use of COS to protect endothelial cells.

Piperlongumine and bortezomib synergically inhibit cholangiocarcinoma via ER stress-induced cell death.

Cholangiocarcinoma (CCA) is a lethal malignancy of the cholangiocytes lining the biliary tree. Only 25% of affected patients are eligible for resection due to late-stage diagnosis. Systemic chemotherapy is recommended for those inoperable patients; however, an inadequate response to such treatment remains a significant obstacle. Piperlongumine (PL) is a biologically active alkaloid that selectively kills various cancer cells through the induction of reactive oxygen species (ROS). The role of PL has been shown through its inhibiting the ubiquitin-proteasome system. The mechanism of PL-induced CCA cell death was investigated by inhibiting the UPS and testing the therapeutic potential of combining PL and the proteasome inhibitor bortezomib. A single treatment with PL or BTZ suppressed CCA cell growth. Combined treatment with PL with BTZ produced a synergistic interaction, evidenced by (1) a combination index of < 1 and (2) induction of cell cycle arrest and down-regulation of cell cycle markers. PL induced the accumulation of poly-ubiquitinated proteins in CCA cells but did not affect proteasome activity. PL, in combination with BTZ, amplified the accumulation of poly-ubiquitinated proteins in CCA cells, leading to an endoplasmic reticulum (ER) stress response through the induction of X-box binding protein mRNA splicing. Moreover, PL-combined BTZ promoted the activation of a proapoptotic unfolded protein response via the ATF4-CHOP axis. PL induced CCA cell death via increased accumulation of the poly-ubiquitinated proteins. PL also enhanced the anti-cancer activity of BTZ via ER stress-induced CCA cell death. Thus, the combination of PL and BTZ has potential as an alternative therapeutic option for CCA.

Resveratrol Attenuates Endoplasmic Reticulum Stress-Induced Cell Death and Results in Functional Improvement after Traumatic Brain Injury in Mice

Resveratrol Attenuates Endoplasmic Reticulum Stress-Induced Cell Death and Results in Functional Improvement after Traumatic Brain Injury in Mice

Ciclopirox targets cellular bioenergetics and activates ER stress to induce apoptosis in non-small cell lung cancer cells

BackgroundLung cancer remains a major cause of cancer-related mortality throughout the world at present. Repositioning of existing drugs for other diseases is a promising strategy for cancer therapies, which may rapidly advance potentially promising agents into clinical trials and cut down the cost of drug development. Ciclopirox (CPX), an iron chelator commonly used to treat fungal infections, which has recently been shown to have antitumor activity against a variety of cancers including both solid tumors and hematological malignancies in vitro and in vivo. However, the effect of CPX on non-small cell lung cancer (NSCLC) and the underlying mechanism is still unclear.MethodsCCK-8, clonal formation test and cell cycle detection were used to observe the effect of inhibitor on the proliferation ability of NSCLC cells. The effects of CPX on the metastasis ability of NSCLC cells were analyzed by Transwell assays. Apoptosis assay was used to observe the level of cells apoptosis. The role of CPX in energy metabolism of NSCLC cells was investigated by reactive oxygen species (ROS) detection, glucose uptake, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) experiments. Western blot was used to examine the protein changes.ResultsWe report that CPX inhibits NSCLC cell migration and invasion abilities through inhibiting the epithelial-mesenchymal transition, impairing cellular bioenergetics, and promoting reactive oxygen species to activate endoplasmic reticulum (ER) stress-induced apoptotic cell death. Moreover, CPX intraperitoneal injection can significantly inhibit NSCLC growth in vivo in a xenograft model.ConclusionsOur study revealed that CPX targets cellular bioenergetics and activates unfolded protein response in ER to drive apoptosis in NSCLC cells, indicating that CPX may be a potential therapeutic drug for the treatment of NSCLC.Graphical 8ygDyd_B2wN4YvZP6nTer7Video

Salvianolic Acid B Suppresses ER Stress-Induced NLRP3 Inflammasome and Pyroptosis via the AMPK/FoxO4 and Syndecan-4/Rac1 Signaling Pathways in Human Endothelial Progenitor Cells.

Mounting evidence demonstrates uncontrolled endoplasmic reticulum (ER) stress responses can activate the inflammasome, which generally results in endothelial dysfunction, a major pathogenetic factor of chronic inflammatory diseases such as atherosclerosis. Salvianolic acid B (SalB), produced by Radix Salviae, exerts antioxidative and anti-inflammatory activities in multiple cell types. However, SalB's effects on ER stress-related inflammasome and endothelial dysfunction remain unknown. Here, we showed SalB substantially abrogated ER stress-induced cell death and reduction in capillary tube formation, with declined intracellular reactive oxygen species (ROS) amounts and restored mitochondrial membrane potential (MMP), as well as increased expression of HO-1 and SOD2 in bone marrow-derived endothelial progenitor cells (BM-EPCs). ER stress suppression by CHOP or caspase-4 siRNA transfection attenuated the protective effect of SalB. Additionally, SalB alleviated ER stress-mediated pyroptotic cell death via the suppression of TXNIP/NLRP3 inflammasome, as evidenced by reduced cleavage of caspase-1 and interleukin- (IL-) 1β and IL-18 secretion levels. Furthermore, this study provided a mechanistic basis that AMPK/FoxO4/KLF2 and Syndecan-4/Rac1/ATF2 signaling pathway modulation by SalB substantially prevented BM-EPCs damage associated with ER stress by decreasing intracellular ROS amounts and inducing NLRP3-dependent pyroptosis. In summary, our findings identify that ER stress triggered mitochondrial ROS release and NLRP3 generation in BM-EPCs, while SalB inhibits NLRP3 inflammasome-mediated pyroptotic cell death by regulating the AMPK/FoxO4/KLF2 and Syndecan-4/Rac1/ATF2 pathways. The current findings reveal SalB as a potential new candidate for the treatment of atherosclerotic heart disease.

Identification of Novel Oxindole Compounds That Suppress ER Stress-Induced Cell Death as Chemical Chaperones.

Endoplasmic reticulum (ER) stress and oxidative stress lead to protein misfolding, and the resulting accumulation of protein aggregates is often associated with the pathogenesis of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and prion disease. Small molecules preventing these pathogenic processes may be effective interventions for such neurodegenerative disorders. In this paper, we identify several novel oxindole compounds that can prevent ER stress- and oxidative stress-induced cell death. Among them, derivatives of the lead compound GIF-0726-r in which a hydrogen atom at the oxindole ring 5 position is substituted with a methyl (GIF-0852-r), bromine (GIF-0854-r), or nitro (GIF-0856-r) group potently suppressed global ER stress. Furthermore, GIF-0854-r and -0856-r prevented protein aggregate accumulation in vitro and in cultured hippocampal HT22 neuronal cells, indicating that these two compounds function effectively as chemical chaperones. In addition, GIF-0852-r, -0854-r, and -0856-r prevented glutamate-induced oxytosis and erastin-induced ferroptosis. Collectively, these results suggest that the novel oxindole compounds GIF-0854-r and -0856-r may be useful therapeutics against protein-misfolding diseases as well as valuable research tools for studying the molecular mechanisms of ER and oxidative stress.