Endoplasmic Reticulum Stress-associated Proteins Research Articles

Endoplasmic reticulum stress is attenuated by glycolysis in lymphatic malformations.

This study aims to investigate the role of endoplasmic reticulum stress (ER stress) in human dermal lymphatic endothelial cells (HDLECs) and lymphatic malformations (LMs) and its relationship with aerobic glycolysis and inflammation. The proliferation and apoptosis of HDLECs were examined with lipopolysaccharide (LPS) treatment. ER stress-associated proteins and glycolysis-related markers were detected by western blot. Glycolysis indexes were detected by seahorse analysis and lactic acid production assay kits. Immunohistochemistry was used to reveal the ER stress state of lymphatic endothelial cells (LECs) in LMs. LPS induced ER stress in HDLECs but did not trigger detectable apoptosis. Intriguingly, LPS-treated HDLECs also showed increased glycolysis flux. Knockdown of Hexokinase 2, a key enzyme for aerobic glycolysis, significantly inhibited the ability of HDLECs to resist ER stress-induced apoptosis. Moreover, compared to normal skin, glucose-regulated protein 78 (GRP78/BIP), and phosphorylation protein kinase R-like kinase (p-PERK), two key ER stress-associated markers, were upregulated in LECs of LMs, which was correlated with the inflected state. In addition, excessively activated ER stress inhibited the progression of LMs in rat models. These data indicate that glycolysis could rescue activated ER stress in HDLECs, which is required for the accelerated development of LMs. Inflammation enhances both ER stress and glycolysis in LECs while glycolysis is required to attenuate the pro-apoptotic effect of ER stress. Endoplasmic reticulum (ER) stress is activated in lymphatic endothelial cells (LECs) of LMs, especially in inflammatory condition. The expression of ER stress-related proteins is increased in LMs and correlated with Hexokinase 2 expression. Pharmacological activation of ER stress suppresses the formation of LM lesions in the rat model. ER stress may be a promising and effective therapeutic target for the treatment of LMs.

Berberine alleviates ovarian tissue damage in mice with hepatolenticular degeneration by suppressing ferroptosis and endoplasmic reticulum stress

ObjectiveHepatolenticular degeneration (HLD) is an autosomal recessive disorder that manifests as multiorgan damage due to impaired copper (Cu) metabolism. Female patients with HLD often experience reproductive impairments. This study investigated the protective effect of berberine against ovarian damage in toxic-milk (TX) mice, a murine model for HLD. MethodsMice were categorized into control group, HLD TX group (HLD group), penicillamine (Cu chelator)-treated TX group and berberine-treated TX group. Body weight, ovary weight and the number of ovulated eggs were recorded. Follicular morphology and cellular ultrastructure were examined. Total iron, ferrous iron (Fe2+) and trivalent iron (Fe3+) levels, as well as malondialdehyde (MDA), glutathione (GSH) and oxidized glutathione (GSSG), were measured in the ovaries. Western blot analysis was used to analyze the expression of proteins related to ferroptosis and endoplasmic reticulum (ER) stress. ResultsOvarian tissue damage was evident in the HLD group, with a significant increase in ferroptosis and ER stress compared to the control group. This damage was inhibited by treatment with penicillamine, a Cu chelator. Compared with the HLD group, berberine increased the number of ovulations, and improved ovarian morphology and ultrastructure. Further, we found that berberine reduced total iron, Fe2+, MDA and GSSG levels, elevated GSH levels, decreased the expression of the ferroptosis marker protein prostaglandin-endoperoxide synthase 2 (PTGS2), and increased glutathione peroxidase 4 (GPX4) expression. Furthermore, berberine inhibited the expression of ER stress-associated proteins mediated by the protein kinase RNA-like ER kinase (PERK) pathway. ConclusionFerroptosis and ER stress are involved in Cu-induced ovarian damage in TX mice. Berberine ameliorates ovarian damage in HLD TX mice by inhibiting ferroptosis and ER stress.Please cite this article as: Liu QZ, Han H, Fang XR, Wang LY, Zhao D, Yin MZ, Zhang N, Jiang PY, Ji ZH, Wu LM. Berberine alleviates ovarian tissue damage in mice with hepatolenticular degeneration by suppressing ferroptosis and endoplasmic reticulum stress. J Integr Med. 2024; Epub ahead of print.

Integrating network pharmacology and experimental models to examine the mechanisms of corosolic acid in preventing hepatocellular carcinoma progression through activation PERK-eIF2a-ATF4 signaling.

Hepatocellular carcinoma (HCC) is the most prevalent form of liver cancer, with a high recurrence rate and heterogeneity. We aimed to examine the effect of corosolic acid (CRA) on HCC. We employed transcriptomics to validate the target molecules in CRA-treated HCC cells and conducted enrichment analyses that revealed their involvement in the regulation of endoplasmic reticulum (ER) stress and apoptosis. Our experimental data indicated that CRA markedly induced apoptosis in human HCC cell lines through the mitochondrial apoptosis pathway. We also revealed that the pro-apoptotic effects of CRA depended on ER stress, as pretreatment with selective ERS inhibitor salubrinal effectively reversed CRA-induced cell apoptosis. Furthermore, the knockdown of the unfolded protein response (UPR) protein CHOP remarkably abrogated CRA-induced expression of ER stress-associated proteins. Collectively, our results suggest that CRA triggers ER stress-mediated apoptosis in HCC cells via activation of the PERK-eIF2a-ATF4 pathway. Our findings provide novel insights into the potential therapeutic strategies for HCC.

CARD6: Warm Ischemic Time, Cold Static Storage and Impairment of Cardiac Endoplasmic Reticular Stress in Human Hearts Donated After Circulatory Death and Brain Death

Background: Endoplasmic reticulum (ER) plays an important role in maintaining normal myocardial function at the cellular level. Overexpression of ER stress-associated proteins during ischemia impact cardiac function. Initial result of heart transplantation using donation after circulatory death (DCD) heart with short warm ischemic time (WIT, less than 30 minutes) was encouraging. However, little is known about the impact of various WIT on donor heart and with subsequent cold storage as used in normothermic regional perfusion(NRP) on the DCD hearts. In this study we investigated expression of myocardial ER stress response proteins in the DCD hearts to evaluate the impact of warm ischemic time (WIT) and subsequent cold storage. We also studied expression of myocardial ER stress response proteins in the brain death donor (DBD) hearts as a control group. Methods: A total of 23 human hearts, DCD group, n=17, with 3 WIT sub-groups [WIT 20 (n=6), 40 (n=6), ≥60 (n=5) minutes] and DBD group, n=6, no warm ischemia (control: WIT:0) were studied. All the DCD hearts received del Nido cardioplegia and all DBD hearts received Celsior solution before they were placed at cold storage for 6 hours. Left ventricular biopsies were performed at hour 0 (T0), 2 (T2), 4 (T4) and 6 (T6). Myocardial ER stress response proteins ATF6 (Activating Transcription Factor 6) and GRP78 (78-kDa glucose-regulated protein) were measured through immunoblotting after normalizing to GAPDH loading control in all cardiac biopsies. Results: Representative western blots showing the protein expressions patterns among the DBD and three different groups of DCD hearts under increasing time points (T0:0hrs, T2:2hrs, T4:4hrs, T6:6hrs) of cold static storage conditions (Fig.1A). At baseline, the proteins expression found to be comparable between DBD, DCD:20 and 40 groups, while DCD:≥60 group remained significantly higher (Fig.1B,C). We noticed differential temporal increase of ER stress proteins after 2 hours of cold storage and became prominent at hour 6 in DCD groups when compared to DBD group; while the DCD:60 group always remained highly significant throughout the cold storage compared to all other groups (Fig.1D,E). Conclusions: Our study concluded that duration of warm ischemia and subsequent cold storage condition in DCD hearts were associated with elevated ER stress response in myocardium. The length of cold storage was also found to be linked with elevated ER stress in DCD hearts. The DCD hearts with more than 60 min warm ischemic has the highest ES stress response suggesting profound alteration in ER homeostasis which may promote cardiac damage. Such findings are important in guiding clinical decision making on selecting DCD donor hearts. On the other hand the DBD hearts with up to 6 hour cold storage exerted minimal ER stress, supporting current DBD heart transplant donor preservation practice.

NADPH oxidase and endoplasmic reticulum stress is associated with neuronal degeneration in orbitofrontal cortex of individuals with alcohol use disorder.

Many disorders of the central nervous system (CNS), including alcohol use disorder (AUD), are associated with induction of proinflammatory neuroimmune signalling and neurodegeneration. In previous studies, we found increased expression of Toll-like receptors (TLRs), activated NF-κB p65 (RELA), and other proinflammatory signalling molecules. Proinflammatory NADPH oxidases generate reactive oxygen species, which are linked to neurodegeneration. We tested the hypothesis that AUD increased RELA activation increases NADPH oxidase-oxidative stress and endoplasmic reticulum (ER) stress cell death cascades in association with neuronal cell death in the human orbitofrontal cortex (OFC). In the AUD OFC, we report mRNA induction of several NADPH oxidases, the dual oxidase DUOX2, and the oxidative stress lipid peroxidation marker 4-HNE and the DNA oxidation marker 8-OHdG that correlate with RELA, a marker of proinflammatory NF-κB activation. This was accompanied by increased expression of the ER stress-associated regulator protein glucose-regulated protein 78 (GRP78), transmembrane sensors activating transcription factor 6 (ATF6), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and inositol-requiring kinase/endonuclease 1 (pIRE1), and the pro-apoptotic transcription factor C/EBP homologous protein (CHOP). Expression of NADPH oxidase-oxidative stress markers correlate with ER stress-associated molecules. Induction of oxidative stress and ER stress signalling pathways correlate with expression of cell death-associated caspases and neuronal cell loss. These data support the hypothesis that proinflammatory RELA-mediated induction of NADPH oxidase-oxidative stress and ER stress-associated signalling cascades is associated with neuronal cell death in the post-mortem human OFC of individuals with AUD.

CircWDR33 alleviates human pulmonary microvascular endothelial cell injury in sepsis-associated acute lung injury by targeting miR-217-5p/SERP1 axis.

Circular RNAs (circRNAs) have been reported to be involved in the pathophysiology of sepsis-induced acute lung injury (sepsis-ALI). Herein, this work aimed to investigate the role and mechanism of circWDR33 in the process of sepsis-ALI. Lipopolysaccharide (LPS)-stimulated human pulmonary microvascular endothelial cells (HPMECs) were used to establish the cell model of sepsis-ALI in vitro. Levels of genes and proteins were measured by RT-qPCR and western blotting. The abundances of inflammatory factors were detected by ELISA analysis, and cell apoptosis was assayed by flow cytometry. Cell permeability (PA) was determined by transendothelial resistance (TER) and fluorescein isothiocyanate (FITC) with transwell assay. The tubulogenesis of HPMECs was assessed by tube formation assay. The binding between miR-217-5p and circWDR33 or SERP1 (Stress Associated Endoplasmic Reticulum Protein 1) was validated using pull-down and dual-luciferase reporter assays. CircWDR33 expression was low in sepsis-ALI patients and LPS-challenged HPMECs. Functionally, forced expression of circWDR33 could alleviate LPS-induced inflammatory and apoptotic injury, permeability enhancement and tubule formation arrest in HPMECs. Mechanistically, circWDR33/miR-217-5p/SERP1 formed an axis in HPMECs. MiR-217-5p was highly expressed, while SERP1 was decreased in sepsis-ALI patients and LPS-challenged HPMECs. MiR-217-5p silencing could protect against LPS-evoked HPMEC injury. Further rescue experiments showed that protective effects of circWDR33 on LPS-challenged HPMECs were attenuated by miR-217-5p up-regulation or SERP1 down-regulation. CircWDR33 protected against LPS-induced inflammatory and apoptotic injury, permeability enhancement and tubule formation arrest in HPMECs via miR-217-5p/SERP1 axis, indicating a new potential therapeutic approach for sepsis-ALI patients.

Pan-Aurora Kinase Inhibitor Danusertib Induces Apoptosis of Epstein–Barr Virus-transformed B-Cells Through Caspase and Endoplasmic Reticulum Stress Signaling

Evidence for the relevance of Epstein-Barr virus (EBV) in various types of cancer has expanded; however, the definitive mechanism of EBV-induced oncogenesis remains ambiguous. The purpose of this study was to identify the relevance of aurora kinases in EBV-induced carcinogenesis, and the cellular responses to danusertib, a pan-aurora kinase inhibitor. The underlying signaling mechanism in EBV-transformed B-cells was also investigated. Western blotting was performed on EBV-transformed B-cells and EBV-positive lymphoma cells to identify aurora kinase expression. Cellular responses of EBV-transformed B-cells to danusertib were investigated using AlamaBlue assay and apoptosis analysis. To evaluate the underlying signaling mechanisms of danusertib-induced apoptosis, cleavage of caspase cascade molecules, endoplasmic reticulum (ER) stress-associated molecule activation, and intracellular Ca2+ levels were evaluated using western blotting, flow cytometry, and inhibition assays. Expression of both aurora kinase A and B was gradually increased in EBV-infected B-cells and two EBV-positive B lymphoma cell lines. Danusertib significantly suppressed EBV-transformed B-cell proliferation in a dose-dependent manner. Danusertib induced apoptosis and cell cycle arrest through disruption of mitochondrial membrane potential in EBV-transformed B-cells in a dose-dependent and time-dependent manner. Moreover, danusertib induced cleavage of caspases, ER stress-associated molecule activation, and intracellular Ca2+ release from ER to cytoplasm in EBV-transformed B-cells, while BAPTA-AM, a calcium chelator, inhibited danusertib-induced apoptosis. Danusertib treatment led to apoptosis of EBV-transformed B-cells through ER stress-associated proteins and mitochondrial caspase activation. These results suggest that aurora kinases may be valuable targets for potential therapeutic agents against EBV-associated carcinoma.

Toxoplasma gondii dense granule protein 3 promotes endoplasmic reticulum stress-induced apoptosis by activating the PERK pathway

BackgroundToxoplasma gondii is a neurotropic single-celled parasite that can infect mammals, including humans. Central nervous system infection with T. gondii infection can lead to Toxoplasma encephalitis. Toxoplasma infection can cause endoplasmic reticulum (ER) stress and unfolded protein response (UPR) activation, which ultimately can lead to apoptosis of host cells. The dense granule protein GRA3 has been identified as one of the secretory proteins that contribute to the virulence of T. gondii; however, the mechanism remains enigmatic.MethodsThe expression of the GRA3 gene in RH, ME49, Wh3, and Wh6 strains was determined using quantitative real-time polymerase chain reaction (qRT–PCR). pEGFP-GRA3Wh6 was constructed by inserting Chinese 1 Wh6 GRA3 (GRA3Wh6) cDNA into a plasmid encoding the enhanced GFP. Mouse neuro2a (N2a) cells were transfected with either pEGFP or pEGFP-GRA3Wh6 (GRA3Wh6) and incubated for 24–36 h. N2a cell apoptosis and ER stress-associated proteins were determined using flow cytometry and immunoblotting. Furthermore, N2a cells were pretreated with GSK2656157 (a PERK inhibitor) and Z-ATAD-FMK (a caspase-12 inhibitor) before GRA3Wh6 transfection, and the effect of the inhibitors on GRA3Wh6-induced ER stress and apoptosis were investigated.ResultsGRA3 gene expression was higher in the less virulent strains of type II ME49 and type Chinese 1 Wh6 strains compared with the virulent strains of type I RH strain and type Chinese 1 Wh3 strain. Transfection with GRA3Wh6 plasmid induced neuronal apoptosis and increased the expression of GRP78, p-PERK, cleaved caspase-12, cleaved caspase-3, and CHOP compared with the control vector. Pretreatment with GSK2656157 and Z-ATAD-FMK decreased apoptosis in N2a cells, and similarly, ER stress- and apoptosis-associated protein levels were significantly decreased.ConclusionGRA3 induces neural cell apoptosis via the ER stress signaling pathway, which could play a role in toxoplasmic encephalitis.Graphical

Long intergenic noncoding RNA LINC01287 drives the progression of cervical cancer via regulating miR-513a-5p/SERP1.

Cervical cancer is one of the most frequent types of cancer in women, which is characterized by high invasion and metastatic tendency in its advanced stage. Emerging evidence indicated that long non-coding RNAs (LncRNAs) are involved in the pathogenesis of cervical cancer. LINC01287 has been reported to play crucial regulatory roles in the pathogenesis and progression of multiple cancers. However, up until now, whether LINC01287 is associated with the initiation and development of cervical cancer remains largely unknown. In the present study, expression levels of LINC01287, miR-513a-5p and stress-associated endoplasmic reticulum protein 1 (SERP1) mRNA were quantified utilizing qRT-PCR. A series of functional experiments including CCK-8 assay, colony formation assay, transwell assay, flow cytometry, and tumor xenograft growth of cervical cancer cells were performed for studying the effects of LINC01287. The luciferase reporter assay, pull-down assay, and western blot were used to confirm the downstream targets of LINC01287 and miR-513a-5p. The results demonstrate that LINC01287 was highly expressed in cervical cancer tissue samples and cell lines. High LINC01287 predicts a poor prognosis for cervical cancer patients. Additional gain- and loss-of-function experiments demonstrated that silencing LINC01287 inhibited cervical cancer cells proliferation, colony formation, migration, apoptosis in vitro and retarded tumor growth and metastasis in vivo. Furthermore, the dual-luciferase reporter gene system and RNA pulldown assay validated that LINC01287 positively regulated SERP1 expressions by sponging miR-513a-5p, and LINC01287 inhibited cervical cancer progression by regulating miR-513a-5p/SERP1 axis. In conclusion, the current study first identified that LINC01287/miR-513a-5p/SERP1 axis played an important role in cervical cancer progression. LINC01287 might be a prognostic biomarker and a target for new therapies in patients with cervical cancer.

KCNQ1OT1 mediates keratinocyte migration to promote skin wound healing through the miR-200b-3p/SERP1 axis

BackgroundThe basic functions of keratinocyte are crucial steps during skin wound healing. KCNQ1OT1 long noncoding RNA was found to accelerate the migration and proliferation of keratinocyte in psoriasis. Here, we elucidated the action and mechanism of KCNQ1OT1 in skin wound healing. MethodsExpression levels of genes and proteins were evaluated by quantitative real-time PCR (qRT-PCR) and western blotting. Cell migration was assessed by using scratch and transwell assays. The interaction between miR-200b-3p and KCNQ1OT1 or SERP1 (Stress Associated Endoplasmic Reticulum Protein 1) was confirmed by bioinformatics analysis, dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and pull-down assay. ResultsKCNQ1OT1 had increased significantly in wound edge 1 day and 7 day after injury. Functionally, overexpression of KCNQ1OT1 promoted keratinocyte migration. Mechanistically, KCNQ1OT1/miR-200b-3p/SERP1 constituted a competing endogenous RNA (ceRNA) network in keratinocytes. A series of rescue experiments showed that miR-200b-3p up-regulation in keratinocytes attenuated the pro-migration action of KCNQ1OT1 in cells. Moreover, knockdown of miR-200b-3p could promote keratinocyte migration, which was abolished by SERP1 silencing. KCNQ1OT1 competitively sponged for miR-200b-3p to elevate the expression of its target SERP1. ConclusionKCNQ1OT1 could promote keratinocyte migration by miR-200b-3p/SERP1 axis, suggesting that KCNQ1OT1 might play a crucial role in skin wound healing.

SERP1 reduces inchoate acute hepatic injury through regulation of endoplasmic reticulum stress via the GSK3β/β‑catenin/TCF/LEF signaling pathway.

The liver is a crucial digestive organ of humans and in charge of detoxification. Acute hepatic injury is an aggressive type of hepatic disease and its harmful effect cannot be ignored. The present study examined the role and mechanism of stress-associated endoplasmic reticulum protein 1 (SERP1) in acute hepatic injury. Mice were injected intraperitoneally with D-galactosamine/lipopolysaccharide (LPS) and rat hepatocytes were induced by LPS to establish an acute hepatic injury model. Tissue lesions were observed by H&E staining, and biomarkers of hepatic injury in the serum were examined. Western blotting, immunohistochemistry and reverse transcription-quantitative PCR were performed to assess SERP1 expression in tissues and hepatocytes. A SERP1 overexpression plasmid was constructed to evaluate the role of SERP1 in inflammation, apoptosis, endoplasmic reticulum stress (ERS) and the GSK3β/β-catenin/T-cell factor (TCF)/lymphoid enhancing factor (LEF) signaling pathway. In addition, a GSK3β overexpression plasmid was constructed to investigate the role of GSK3β/β-catenin signal activation. Additionally, the present study investigated whether SERP1 regulated the endoplasmic reticulum via this pathway. In the present study, reliable animal and cellular hepatic injury models were established and verified. SERP1 overexpression reduced the expression of inflammatory factors, apoptosis-related proteins and ERS-related proteins, as well as the expression of proteins related to GSK3β/β-catenin/TCF/LEF signaling pathways. A GSK3β overexpression plasmid was constructed and it was revealed that GSK3β overexpression could reverse the effects of SERP1 overexpression in aforementioned aspects. This suggested that the activation of the GSK3β/β-catenin/TCF/LEF signaling pathway may be required for the regulation of SERP1. In conclusion, SERP1 regulated ERS via the GSK3β/β-catenin/TCF/LEF signaling pathway, thereby reducing inchoate acute hepatic injury.

Risk of Infection, Endoplasmic Reticular Stress and Systemic Activation of NLRP3 Inflammasome in Patients Supported by Continuous Flow Left Ventricular Assist Devices

<h3>Purpose</h3> We investigated changes in stress-associated endoplasmic reticulum (ER) protein 1 (SERP1), ER-stress response proteins and inflammasome activation in heart failure (HF) patients before and after CF-LVAD implantation, and relationship to subsequent infection within 3 months. <h3>Methods</h3> We analyzed blood samples from 24 CF-LVAD patients (pre- and 1week post-VAD) and 10 non-HF controls. Culture positive Infections was confirmed (median 42 days post-VAD) from bloodstream, GI, respiratory tracts, or driveline. SARP1, ER-stress proteins (ATF-6, GRP78) and NLRP3 inflammasome (NLRP3, ASC, Caspase1) in peripheral blood mononuclear cells (PBMC) were assessed. <h3>Results</h3> HF patients had significantly (p<0.05) elevated baseline (pre-VAD) PBMC protein levels of ATF6 (0.41±0.05 vs. 0.54±0.11), GRP78 (0.20±0.07 vs. 0.33±0.18), NLRP3 (0.28±0.17 vs. 0.78±0.28), and ASC (0.27±0.13 vs. 0.44±0.15) compared to controls. Among CF-LVAD recipients, SERP1 declined in those without subsequent infection (Group1) but significantly increased in those with infection (Group2) (<i>Fig.A,B</i>). Baseline ATF6 and GRP78 differed between Group1 and 2 (<i>Fig.C,D</i>). In Group2, the post-VAD ATF6, GRP78, NLRP3 and ASC were significantly elevated compared to pre-VAD; while in group1, only the post-VAD GRP78 was significantly elevated (<i>Fig.C-G</i>). Post-VAD caspase1 increased but not-significant (0.36±0.07 vs. 0.43±1.2) in group2 compared to pre-VAD (<i>Fig.H</i>). Baseline SERP1 and NLRP3 among patients who developed infection were positively correlated with ATF6 and GRP78 (<i>Fig.I-L</i>). <h3>Conclusion</h3> Post-VAD upregulation of SERP1 may be associated with ER-stress response, which in turn may activate NLRP3 inflammasome and predispose to early infection. Post-VAD decrease in SERP1 in group1 may protect against ER stress and NLRP3 activation. Further investigation of the role of SERP1 in ER-stress and inflammasome activation in CF-LVAD recipients is needed.

Pendulone induces apoptosis via the ROS-mediated ER-stress pathway in human non-small cell lung cancer cells

PurposePendulone, an isoflavone compound, is known to act against human cancer cells. However, its role in human non-small cell lung cancer (NSCLC) and the exact molecular mechanisms of action have never been reported. MethodsWe investigated the effects of pendulone on cell proliferation and apoptosis in human NSCLC H1299 cells. Cell viability was examined using the methyl-thiazol-diphenyl-tetrazolium (MTT) assay. Flow cytometry was employed to evaluate apoptotic indices such as the cell cycle, mitochondrial membrane potential, cytochrome c release, caspase activity, and death receptor expression. The expression of proteins related to the cell cycle and apoptosis were analyzed by Western blot analysis. ResultsPendulone significantly decreased H1299 cell viability by inducing endoplasmic reticulum (ER) stress through the accumulation of reactive oxygen species (ROS). Pendulone induced the expression of ER stress-associated proteins, such as ATF4 and CHOP, which promoted the expression of death receptors. Activation of caspase 8 induced extrinsic pathway apoptosis. Pendulone also caused the loss of mitochondrial membrane potential, inhibited the anti-apoptotic proteins BCL-2 and activated the pro-apoptotic protein BAX, which promoted the release of cytochrome c to activate caspase 9. Antioxidant N-acetylcysteine (NAC), with its ROS-suppressive property, reversed pendulone-induced ER stress and cell apoptosis. ConclusionOur findings provide evidence that pendulone induces apoptosis by inducing ER stress through ROS accumulation and mitochondrial dysfunction in NSCLC cell lines.

Glaucocalyxin A alleviates lipopolysaccharide-induced inflammation and apoptosis in pulmonary microvascular endothelial cells and permeability injury by inhibiting STAT3 signaling.

Glaucocalyxin A (GLA), an ent-kauranoid diterpene derived from Rabdosia japonica var. glaucocalyx, possesses antibacterial, anti-oxidative and anti-neuroinflammatory properties. The present study aimed to investigate the potential mechanisms underlying GLA in the pathogenesis of pneumonia. Human pulmonary microvascular endothelial cells (hPMVECs) treated with lipopolysaccharide (LPS) were treated with GLA, followed by the detection of cell viability, inflammation, apoptosis and cell permeability. Furthermore, the protein expression levels of apoptosis- and permeability-associated proteins were determined using western blot analysis. Following treatment with a signal transducer and activator of transcription 3 (STAT3) activator, the protein expression levels of STAT3 and endoplasmic reticulum stress-associated proteins were determined, to confirm whether STAT3 signaling was mediated by GLA. Lastly, the mRNA expression level of inflammatory cytokines, apoptosis and permeability injury were also determined following treatment with a STAT3 activator. The results revealed that GLA ameliorated inflammation, apoptosis and permeability injury in LPS-induced hPMVECs. Following treatment with a STAT3 activator, the therapeutic effects of GLA on LPS-induced hPMVECs were abrogated. In conclusion, GLA alleviated LPS-induced inflammation, apoptosis and permeability injury in hPMVECs by inhibiting STAT3 signaling, which highlighted the potential therapeutic value of GLA in the treatment of pneumonia.

MiR-218 Promotes Adriamycin-Induced H9C2 Apoptosis by Inhibiting Stress-Associated Endoplasmic Reticulum Protein 1.

Objective Adriamycin is a clinically important chemotherapeutic drug, but its use is restricted due to its myocardial toxicity. Therefore, it is especially important to explore the toxicity mechanism of Adriamycin (ADR) to cardiomyocytes. Methods The myocardial toxicity model of ADR was constructed in vitro, and the effect of miR-218 inhibitor and sh-Serp1 on the activity of H9C2 cells induced by ADR was detected by MTT method. Also, flow cytometry, real-time polymerase chain reaction (RT-PCR), and TUNEL staining were used to detect the cell apoptosis. The activity of LDH was detected by colorimetry, and the interaction of miR-218 with Serp1 was detected by double-luciferase reporter gene assay. Western blotting technique was used to detect the expression level of caspase3 and p38 MAPK signal pathway. Results miR-218 inhibitor can obviously inhibit ADR-induced decrease in cell activity of H9C2 cells, inhibit cell apoptosis, and inhibit p38 MAPK signaling pathway activation. Conversely, sh-Serp1 aggravated the decrease in H9C2 cell activity and promoted cell apoptosis. Conclusion Upregulation of miR-218 expression will promote ADR-induced apoptosis of H9C2 cells. At the same time, we confirmed that the mechanism by which miR-218 promotes myocardial apoptosis was through the Serp1/p38 MAPK/caspase-3 signaling pathway.

Low expression of endoplasmic reticulum stress-related gene SERP1 is associated with poor prognosis and immune infiltration in skin cutaneous melanoma.

Stress-associated endoplasmic reticulum protein 1 (SERP1) is a gene induced by endoplasmic reticulum (ER) stress and a major contributor to multiple tumor types. Skin cutaneous melanoma (SKCM) is a highly aggressive and fatal cancer with poor treatment outcomes after progression. In this study, we evaluated SERP1’s role in tumorigenesis, prognosis, and immune infiltration in SKCM. Patients with SKCM had low SERP1 expression. We identified differentially expressed genes between high- and low-SERP1 expression groups and conducted functional, pathway, and gene enrichment analyses. Protein–protein (PPI) and gene–gene interaction (GGI) networks were constructed via STRING and GeneMANIA, respectively. SERP1 mutation information was obtained through cBioPortal; location in the skin was identified through the Human Protein Atlas. Kaplan–Meier analysis revealed an association between low SERP1 expression and overall survival (OS), disease-specific survival (DSS), progress-free interval (PFI) rates, and worse prognosis in patients with multiple clinicopathological features. Cox regression analysis and nomograms further presented SERP1 level as an independent prognostic factor for patients with SKCM. Furthermore, there were significant correlations between SERP1 expression and immune infiltrates; thus, low SERP1 expression is associated with immune cell infiltration and can be considered a poor prognostic biomarker in patients with SKCM.Stress-associated endoplasmic reticulum protein 1 (SERP1) is a gene induced by endoplasmic reticulum (ER) stress and a major contributor to multiple tumor types. Skin cutaneous melanoma (SKCM) is a highly aggressive and fatal cancer with poor treatment outcomes after progression. In this study, we evaluated SERP1’s role in tumorigenesis, prognosis, and immune infiltration in SKCM. Patients with SKCM had low SERP1 expression. We identified differentially expressed genes between high- and low-SERP1 expression groups and conducted functional, pathway, and gene enrichment analyses. Protein–protein (PPI) and gene–gene interaction (GGI) networks were constructed via STRING and GeneMANIA, respectively. SERP1 mutation information was obtained through cBioPortal; location in the skin were identified through the Human Protein Atlas. Kaplan–Meier analysis revealed an association between low SERP1 expression and overall survival (OS), disease-specific survival (DSS), progress-free interval (PFI) rates, and worse prognosis in patients with multiple clinicopathological features. Cox regression analysis and nomograms further presented SERP1 level as an independent prognostic factor for patients with SKCM. Furthermore, there were significant correlations between SERP1 expression and immune infiltrates; thus, low SERP1 expression is associated with immune cell infiltration and can be considered a poor prognostic biomarker in patients with SKCM.

Inhibition of Calpain Alleviates Apoptosis in Coxsackievirus B3-induced Acute Virus Myocarditis Through Suppressing Endoplasmic Reticulum Stress.

Virus myocarditis (VMC) is a common cardiovascular disease and a major cause of sudden death in young adults. However, there is still a lack of effective treatments. Our previous studies found that calpain activation was involved in VMC pathogenesis. This study aims to explore the underlying mechanisms further. Neonatal rat cardiomyocytes (NRCMs) and transgenic mice overexpressing calpastatin (Tg-CAST), the endogenous calpain inhibitor, were used to establish VMC model. Hematoxylin and eosin and Masson staining revealed inflammatory cell infiltration and fibrosis. An ELISA array detected myocardial injury. Cardiac function was measured using echocardiography. CVB3 replication was assessed by capsid protein VP1. Apoptosis was measured by TUNEL staining, flow cytometry, and western blot. The endoplasmic reticulum (ER) stress-related proteins were detected by western blot. Our data showed that CVB3 infection resulted in cardiac injury, as evidenced by increased inflammatory responses and fibrosis, which induced myocardial apoptosis. Inhibiting calpain, both by PD150606 and calpastatin overexpression, could attenuate these effects. Furthermore, ER stress was activated during CVB3 infection. However, calpain inhibition could downregulate some ER stress-associated protein levels such as GRP78, pancreatic ER kinase-like ER kinase (PERK), and inositol-requiring enzyme-1α (IRE-1α), and ER stress-related apoptotic factors, during CVB3 infection. In conclusion, calpain inhibition attenuated CVB3-induced myocarditis by suppressing ER stress, thereby inhibiting cardiomyocyte apoptosis.

Preservation of endoplasmic reticulum (ER) Ca2+ stores by deletion of inositol-1,4,5-trisphosphate receptor type 1 promotes ER retrotranslocation, proteostasis, and protein outer segment localization in cyclic nucleotide-gated channel-deficient cone photoreceptors.

Endoplasmic reticulum (ER) Ca2+ homeostasis relies on an appropriate balance between efflux- and influx-channel activity responding to dynamic changes of intracellular Ca2+ levels. Dysregulation of this complex signaling network has been shown to contribute to neuronal and photoreceptor death in neuro- and retinal degenerative diseases, respectively. In mice with cone cyclic nucleotide-gated (CNG) channel deficiency, a model of achromatopsia/cone dystrophy, cones display early-onset ER stress-associated apoptosis and protein mislocalization. Cones in these mice also show reduced cytosolic Ca2+ level and subsequent elevation in the ER Ca2+ -efflux-channel activity, specifically the inositol-1,4,5-trisphosphate receptor type 1 (IP3 R1), and deletion of IP3 R1 results in preservation of cones. This work investigated how preservation of ER Ca2+ stores leads to cone protection. We examined the effects of cone specific deletion of IP3 R1 on ER stress responses/cone death, protein localization, and ER proteostasis/ER-associated degradation. We demonstrated that deletion of IP3 R1 improves trafficking of cone-specific proteins M-/S-opsin and phosphodiesterase 6C to cone outer segments and reduces localization to cone inner segments. Consistent with the improved protein localization, deletion of IP3 R1 results in increased ER retrotranslocation protein expression, reduced proteasome subunit expression, reduced ER stress/cone death, and reduced retinal remodeling. We also observed the enhanced ER retrotranslocation in mice that have been treated with a chemical chaperone, supporting the connection between improved ER retrotranslocation/proteostasis and alleviation of ER stress. Findings from this work demonstrate the importance of ER Ca2+ stores in ER proteostasis and protein trafficking/localization in photoreceptors, strengthen the link between dysregulation of ER Ca2+ homeostasis and ER stress/cone degeneration, and support an involvement of improved ER proteostasis in ER Ca2+ preservation-induced cone protection; thereby identifying IP3 R1 as a critical mediator of ER stress and protein mislocalization and as a potential target to preserve cones in CNG channel deficiency.

Endoplasmic reticulum stress/XBP1 promotes airway mucin secretion under the influence of neutrophil elastase.

Endoplasmic reticulum (ER) stress is an important reaction of airway epithelial cells in response to various stimuli, and may also be involved in the mucin secretion process. In the present study, the effect of ER stress on neutrophil elastase (NE)-induced mucin (MUC)5AC production in human airway epithelial cells was explored. 16HBE14o-airway epithelial cells were cultured and pre-treated with the reactive oxygen species (ROS) inhibitor, N-acetylcysteine (NAC), or the ER stress chemical inhibitor, 4-phenylbutyric acid (4-PBA), or the cells were transfected with inositol-requiring kinase 1α (IRE1α) small interfering RNA (siRNA) or X-box-binding protein 1 (XBP1) siRNA, respectively, and subsequently incubated with NE. The results obtained revealed that NE increased ROS production in the 16HBE14o-cells, with marked increases in the levels of ER stress-associated proteins, such as glucose-regulated protein 78 (GRP78), activating transcription factor 6 (ATF6), phosphorylated protein kinase R-like endoplasmic reticulum kinase (pPERK) and phosphorylated (p)IRE1α. The protein and mRNA levels of spliced XBP1 were also increased, and the level of MUC5AC protein was notably increased. The ROS scavenger NAC and ER stress inhibitor 4-PBA were found to reduce ER stress-associated protein expression and MUC5AC production and secretion. Further analyses revealed that MUC5AC secretion was also attenuated by IRE1α and XBP1 siRNAs, accompanied by a decreased mRNA expression of spliced XBP1. Taken together, these results demonstrate that NE induces ER stress by promoting ROS production in 16HBE14o-airway epithelial cells, leading to increases in MUC5AC protein production and secretion via the IRE1α and XBP1 signaling pathways.

Carfilzomib in Combination with Bortezomib Enhances Apoptotic Cell Death in B16-F1 Melanoma Cells.

Simple SummaryThe incidence rate of metastatic melanoma has been rapidly increasing worldwide and its 5-year survival rate is very low. Due to partial responses, various side effects, and resistance to any known cancer therapeutics, more potent and safer therapeutics are needed to increase the survival rate of patients with melanoma. Since proteasome inhibitors, such as bortezomib and carfilzomib, have been suggested as treatments for various cancers, we investigated their potential for the treatment of melanoma by studying their molecular mechanisms of action in B16-F1 melanoma cells. In this study, we found that both bortezomib and carfilzomib lead to apoptosis via ER stress as well as ROS accumulation and MMP loss in melanoma cells. Bortezomib and carfilzomib synergistically reduced B16-F1 tumor growth in vitro and in a C57BL/6 xenograft mouse model. Therefore, a combination therapy with carfilzomib and bortezomib at submaximal concentrations may reduce their side effects and be beneficial for melanoma treatment.Proteasome inhibitors, such as bortezomib (BZ) and carfilzomib (CFZ), have been suggested as treatments for various cancers. To utilize BZ and/or CFZ as effective therapeutics for treating melanoma, we studied their molecular mechanisms using B16-F1 melanoma cells. Flow cytometry of Annexin V-fluorescein isothiocyanate-labeled cells indicated apoptosis induction by treatment with BZ and CFZ. Apoptosis was evidenced by the activation of various caspases, including caspase 3, 8, 9, and 12. Treatment with BZ and CFZ induced endoplasmic reticulum (ER) stress, as indicated by an increase in eIF2α phosphorylation and the expression of ER stress-associated proteins, including GRP78, ATF6α, ATF4, XBP1, and CCAAT/enhancer-binding protein homologous protein. The effects of CFZ on ER stress and apoptosis were lower than that of BZ. Nevertheless, CFZ and BZ synergistically induced ER stress and apoptosis in B16-F1 cells. Furthermore, the combinational pharmacological interactions of BZ and CFZ against the growth of B16-F1 melanoma cells were assessed by calculating the combination index and dose-reduction index with the CompuSyn software. We found that the combination of CFZ and BZ at submaximal concentrations could obtain dose reduction by exerting synergistic inhibitory effects on cell growth. Moreover, this drug combination reduced tumor growth in C57BL/6 syngeneic mice. Taken together, these results suggest that CFZ in combination with BZ may be a beneficial and potential strategy for melanoma treatment.