Melatonin inhibits bovine viral diarrhea virus replication by ER stress-mediated NF-κB signal pathway and autophagy in MDBK cells.

Bovine viral diarrhea-mucosal disease (BVD-MD) is a contagious disease in cattle, caused by the bovine viral diarrhea virus (BVDV). This virus continues to spread globally, exerting pressure on both public health and the economy. Despite its impact, there are currently no effective drugs for treating BVDV. This study utilized Madin-Darby bovine kidney (MDBK) cells as a model to investigate the antiviral effects of melatonin against Bovine Viral Diarrhea Virus (BVDV) and its connection with endoplasmic reticulum (ER) stress. Our results show that melatonin can suppress BVDV proliferation in MDBK cells by modulating the endoplasmic reticulum (ER) stress-mediated NF-κB pathway and autophagy. Specifically, melatonin alleviated ER stress, inhibited the activation of IκBα and p65, regulated autophagy, and reduced the expression levels of pro-inflammatory cytokines. Further, when we treated BVDV-infected cells with the ER stress inducer thapsigargin, it led to significant activation of the NF-κB pathway and autophagy. Conversely, treating the cells with the ER stress inhibitor 4-phenylbutyric acid reversed these effects. These findings suggest that melatonin exerts its antiviral effects primarily through the PERK-eIF2α-ATF4 of ER stress-mediated NF-κB pathway and autophagy. Overall, our study underscores the potential of melatonin as an effective protective and therapeutic option against BVDV, offering insights into its anti-infective mechanisms.

Camel Whey Protein and Baicalein Suppressed Mast Cell Degranulation in Mice Models of IgE- and Non-IgE-Mediated Anaphylaxes: Potential Mechanisms on Downstream Cell Signaling of Mast Cells.

INTRODUCTION: Novel treatments are being researched to develop more safe and effective protective medications for anaphylaxis. Camel whey protein (CWP) and baicalein (BAC, one of the major flavones) have multiple beneficial properties including anti-inflammatory and antioxidant activities. METHODS: The current study investigated/compared the therapeutic protection of repeated intragastric administration of CWP (100 mg/kg body weight, as an animal extract) and BAC (10 mg/kg body weight, as a plant extract), before the challenge with ovalbumin (OVA) or receiving the compound 48/80 (C48/80), against mice models for IgE-independent and dependent anaphylaxes. Besides, their effects on mast cells (MCs) downstream cell signaling were explored. RESULTS: The results revealed that CWP and BAC reduced the mortality rate, as compared with a MCs stabilizer "sulfasalazine (SSZ, 100 mg/kg body weight, intraperitoneally)," in both mice models. Furthermore, they prevented the MCs degranulation and significantly reduced (p < .05) lung tissue levels of cell signaling (p-AKT, p-ERK, and p-IκBα). Additionally, they decreased histamine, tryptase, leukotriene C4, prostaglandin D2, interleukin (IL)-4, and IL-10 levels in broncho-alveolar and peritoneal lavages in systemic anaphylaxis mice models. They also restored the stabilization of peritoneal MCs membrane in inverted light microscopy results accompanied by amelioration of the lung histology. DISCUSSION: The present study provided evidence for the protective therapeutic effect of CWP and BAC against anaphylaxis. As a result, CWP and BAC may be used as preventative supplemented regimens for both non-vegetarian and vegetarian consumers to treat allergy through downregulation of MCs signal transduction pathways, and hence controlling the production of inflammatory mediators.

Targeting NLRP3 inhibits AML progression by inducing PERK/eIF2-mediated apoptosis.

BACKGROUND: Acute myeloid leukemia (AML) is characterized by the abnormal proliferation of myeloid precursor cells and presents significant challenges in treatment due to its heterogeneity. Recently, the NLRP3 inflammasome has emerged as a potential contributor to AML pathogenesis, although its precise mechanisms remain poorly understood. METHODS: Public genome datasets were utilized to evaluate the expression of NLRP3 inflammasome-related genes (IL-1ß, IL-18, ASC, and NLRP3) in AML patients compared to healthy individuals. CRISPR/Cas9 technology was employed to generate NLRP3-deficient MOLM-13 AML cells, followed by comprehensive characterization using real-time PCR, western blotting, FACS analysis, and transmission electron and immunofluorescence microscopy. Proteomic analyses were conducted to identify NLRP3-dependent alterations in protein levels, with a focus on the eIF2 kinase PERK-mediated signaling pathways. Additionally, in vivo studies were performed using a leukemic mouse model to elucidate the pathogenic role of NLRP3 in AML. RESULTS: Elevated expression of NLRP3 was significantly associated with diminished overall survival in AML patients. Genetic deletion, pharmacological inhibition and silencing by RNA interference of NLRP3 led to decreased AML cell survival through the induction of apoptosis. Proteomic analyses uncovered NLRP3-dependent alterations in protein translation, characterized by enhanced eIF2α phosphorylation in NLRP3-deficient AML cells. Moreover, inhibition of PERK-mediated eIF2α phosphorylation reduced apoptosis by downregulating pro-apoptotic Bcl-2 family members. In vivo studies demonstrated reduced leukemic burden in mice engrafted with NLRP3 knockout AML cells, as evidenced by alleviated leukemic symptoms. CONCLUSION: Our findings elucidate the involvement of the NLRP3/PERK/eIF2 axis as a novel driver of AML cell survival. Targeting NLRP3-induced signaling pathways, particularly through the PERK/eIF2 axis, presents a promising therapeutic strategy for AML intervention. These insights into the role of the NLRP3 inflammasome offer potential avenues for improving the prognosis and treatment outcomes of AML patients.

Rotenone-induced cell apoptosis via endoplasmic reticulum stress and PERK-eIF2α-CHOP signalling pathways in TM3 cells.

Rotenone (ROT), a widely used natural pesticide, has an uncertain effect on reproductive toxicity. In this study, we used 20 mice distributed randomly into four groups, with each group receiving ROT doses of 0, 2, 4, and 8 mg/kg/day for 28 days. The results demonstrated that ROT induced significant testicular damage, including impaired spermatogenesis, inhibition of testosterone synthesis, and apoptosis of Leydig cells. Additionally, ROT disrupted the normal ultrastructure of the endoplasmic reticulum (ER) in testicular tissue, leading to ER stress in Leydig cells. To further explore whether ROT-induced apoptosis in Leydig cells is related to ER stress, the mouse Leydig cell line (TM3 cells) was treated with ROT at 0, 250, 500, and 1000 nM. ROT inhibited TM3 cell viability, induced cytotoxicity, and reduced testosterone content in the culture supernatants. Furthermore, ROT treatment triggered apoptosis in TM3 cells by activating ER stress and the PERK-eIF2α-CHOP signalling pathway. Pre-treatment of TM3 cells exposed to ROT with the ER stress inhibitor 4-phenylbutyric acid (4-PBA) alleviated these effects, decreasing apoptosis and preserving testosterone levels. Further intervention with the PERK inhibitor GSK2606414 reduced ROT-induced apoptosis and testosterone reduction by inhibiting PERK activity. In summary, ROT-induced male reproductive toxicity is specifically driven by apoptosis, with the PERK-eIF2α-CHOP signalling pathway activated by ER stress playing a crucial role in the apoptosis of Leydig cells triggered by ROT.

Fc receptor-like A promotes malignant behavior in renal cell carcinoma and correlates with tumor immune infiltration.

BACKGROUND: Our study aims to investigate the mechanisms through which Fc receptor-like A (FCRLA) promotes renal cell carcinoma (RCC) and to examine its significance in relation to tumor immune infiltration. MATERIALS AND METHODS: The correlation between FCRLA and data clinically related to RCC was explored using The Cancer Genome Atlas (TCGA), then validated using Gene Expression Omnibus (GEO) gene chip data. Enrichment and protein-protein interaction (PPI) network analyses were performed for FCRLA and its co-expressed genes. FCRLA was knocked down in RCC cell lines to evaluate its impact on biological behavior. Then the potential downstream regulators of FCRLA were determined by western blotting, and rescue experiments were performed for verification. The relevance between FCRLA and various immune cells was analyzed through GSEA, TIMER, and GEPIA tools. TIDE and ESTIMATE algorithms were used to predict the effect of FCRLA in immunotherapy. RESULTS: Fc receptor-like A was associated with clinical and T stages and could predict the M stage (AUC = 0.692) and 1-3- and 5-year survival rates (AUC = 0.823, 0.834, and 0.862) of RCC patients. Higher expression of FCLRA predicted an unfavorable overall survival (OS) in TCGA-RCC and GSE167573 datasets (p = 0.03, p = 0.04). FCRLA promoted the malignant biological behavior of RCC cells through the pERK1/2/-MMP2 pathway and was associated with tumor immune microenvironment in RCC. CONCLUSION: Fc receptor-like A is positively correlated with poor outcomes in RCC patients and plays an oncogenic role in RCC through the pERK1/2-MMP2 pathway. Patients with RCC might benefit from immunotherapy targeting FCRLA.

Development of Functional Foods: A Comparative Study on the Polyphenols and Anthocyanins Content in Chokeberry and Blueberry Pomace Extracts and Their Antitumor Properties.

Developing of functional foods is a promising strategy to reduce the increasing burden of colorectal cancer worldwide. Fruit pomace, particularly polyphenol and anthocyanin-rich chokeberry and blueberry, is a valuable ingredient for functional foods and nutraceuticals. Our study aimed to evaluate the anti-inflammatory and antiproliferative effects of chokeberry and blueberry pomace extracts on C2BBe1 colorectal carcinoma cells and explore the underlying signaling pathways. We analyzed both pomace extracts for total polyphenols and anthocyanins using Folin-Ciocalteu method and ultra-performance liquid chromatography, while antioxidative activity was assessed via the 2,2-diphenyl-1-picrylhydrazyl radical scavenging method. We evaluated the in vitro anti-inflammatory and antiproliferative effects using trypan blue exclusion, MTT and LDH assays, and assessed protein levels of p-Erk1/2, Akt-1, STAT1, STAT3, occludin, oxidized proteins, and MDA-protein adducts through western blotting, as well as analysis of a 37-plex panel of inflammatory markers. Chokeberry extracts exhibited higher total polyphenol content, anthocyanin levels, and antioxidative activity compared to blueberry extracts, however, blueberry extracts effects on cell viability and proliferation in C2BBe1 cells were stronger. Both fruit pomaces induced non-inflammatory cell death characterized by membrane integrity loss, beneficial in cancer therapy. Our data suggests chokeberry's cytotoxicity may be mediated by Erk signaling and Akt-1 inhibition, while blueberry uniquely decreased occludin levels. These berries pomaces' potential to mitigate cancer risks and enhance treatment efficacy is promising, warranting further investigation for functional foods development.

The Anti-Metastatic Action of Oxyresveratrol via Suppression of Phosphoryl-ERK/-PKCα-Mediated Sp1/MMP1 Signaling in Human Renal Carcinoma Cells.

Oxyresveratrol (OxyR) exerts biological and pharmacological effects in a variety of tumor cells, including antioxidant action, antitumor activity, and proapoptotic effects. However, the regulation of targeted signaling pathways by OxyR and the mechanism underlying these effects in human renal cell carcinoma (RCC) have been less studied. We observed that OxyR at noncytotoxic doses did not affect the growth of human RCC cells or normal kidney HK2 cells. OxyR inhibited ACHN and Caki-1 cell migration and invasion through targeting matrix metalloproteinase 1 (MMP1) expression. Analysis of clinical databases showed that high MMP1 expression is associated with lower overall survival (OS) in these cancers (p < 0.01). OxyR significantly inhibited the mRNA and protein expression of Sp1. Furthermore, luciferase assay results showed that OxyR inhibited Sp1 transcriptional activity. Additionally, OxyR preferentially suppressed the activation of ERK and PKCα. Treatment with U0126 (MEK inhibitor) or G06976 (PKCα inhibitor) clearly decreased Sp1 and MMP1 expression and inhibited RCC cell migration and invasion. In conclusion, OxyR may be a potential antitumor therapy for the inhibition of migration and invasion by controlling p-ERK/Sp1 and p-PKCα/Sp1-mediated MMP1 expression in RCC.

17-AAG Induces Endoplasmic Reticulum Stress-mediated Apoptosis in Breast Cancer Cells, Possibly Through PERK/eIF2α Up-regulation.

BACKGROUND/AIM: Breast cancer is the most predominant type of cancer affecting women worldwide and the current therapeutic treatment for breast cancer patients is not adequately effective. This study aimed to investigate the mechanism of 17-AAG, a heat shock protein (HSP90) inhibitor, as a treatment for inducing breast cancer cell apoptosis. MATERIALS AND METHODS: The pharmacology network was employed to examine the correlation of 17-AAG with the gene expression profiles of breast cancer, obtained by Gene Expression Profiling Interactive Analysis (GEPIA). MTT and flow cytometry were utilized to investigate cell proliferation and cell apoptosis, respectively. Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay and western blot analysis were employed to examine the correlation between cellular oxidant levels and protein expression. Immunofluorescence staining was utilized to confirm the protein localization and assess DNA damage. RESULTS: The pharmacological network analysis revealed that HSP90 serves as the common target connecting 17-AAG and breast cancer genes. Treatment with 17-AAG significantly increased cell apoptosis. Moreover, the treatment resulted in up-regulation of cellular oxidant levels and PERK/eIF2α expression. In line with these, protein localization after treatment revealed an increase in DNA damage, correlating with higher ER stress levels. Furthermore, GEPIA demonstrated that PERK and eIF2α expression were significantly higher in breast invasive carcinoma compared to other tumor types. CONCLUSION: HSP90 emerges as a potential target for inducing apoptosis in breast cancer cells by disrupting protein homeostasis in the endoplasmic reticulum, possibly through PERK/eIF2α up-regulation. 17-AAG, an HSP90 inhibitor, may therefore potentially hold an alternative therapeutic strategy for breast cancer treatment.

Effects of dexmedetomidine on depression-like behaviour in chronic restraint stress mice: Involvement of specific brain regions.

It is crucial to develop novel antidepressants. Dexmedetomidine (DEX) can exert antidepressant effects, but its underlying mechanism remains unclear. We used chronic restraint stress (CRS) to induce depression-like behaviour in mice and administered low-dose DEX (2 µg/kg per day) during CRS modelling or one injection of high-dose DEX (20 µg/kg) after CRS. The results of the behavioural tests revealed that both methods ameliorated CRS-induced depression. The brain slices of the mice were subjected to immunohistochemical staining for c-fos and phosphorylated ERK (pERK). Results showed that the continuous low-dose DEX-treated group, but not the single high-dose DEX-treated group expressed less c-fos in the nucleus locus coeruleus (LC) with a mean optical density (MOD) of 0.06. Other brain regions, including the dentate gyrus (DG), pyriform cortex (Pir), anterior part of paraventricular thalamic nucleus (PVA), arcuate nucleus (Arc), and core or shell of accumbens nucleus (Acbc or Acbs), presented differences in c-fos expression. In contrast, the low-dose DEX-treated group exhibited three-fold greater pERK expression in the LC of the CRS mice, with a MOD of 0.15. Pir, cingulate cortex (Cg) and, anterior and posterior part of paraventricular thalamic nucleus (PVA and PVP) exhibited pERK expression differences due to distinct reagent treatments. These changes indicate that the responses of brain regions to different DEX administration methods and doses vary. This study confirmed the ability of DEX to ameliorate CRS-induced depression and identified candidate target brain regions, thus providing new information for the antidepressant mechanism of DEX.

Multiple unfolded protein response pathways cooperate to link cytosolic dsDNA release to stimulator of interferon gene activation.

The double-stranded DNA (dsDNA) sensor STING has been increasingly implicated in responses to "sterile" endogenous threats and pathogens without nominal DNA or cyclic di-nucleotide stimuli. Previous work showed an endoplasmic reticulum (ER) stress response, known as the unfolded protein response (UPR), activates STING. Herein, we sought to determine if ER stress generated a STING ligand, and to identify the UPR pathways involved. Induction of IFN-ß expression following stimulation with the UPR inducer thapsigargin (TPG) or oxygen glucose deprivation required both STING and the dsDNA-sensing cyclic GMP-AMP synthase (cGAS). Furthermore, TPG increased cytosolic mitochondrial DNA, and immunofluorescence visualized dsDNA punctae in murine and human cells, providing a cGAS stimulus. N-acetylcysteine decreased IFN-ß induction by TPG, implicating reactive oxygen species (ROS). However, mitoTEMPO, a mitochondrial oxidative stress inhibitor did not impact TPG-induced IFN. On the other hand, inhibiting the inositol requiring enzyme 1 (IRE1) ER stress sensor and its target transcription factor XBP1 decreased the generation of cytosolic dsDNA. iNOS upregulation was XBP1-dependent, and an iNOS inhibitor decreased cytosolic dsDNA and IFN-ß, implicating ROS downstream of the IRE1-XBP1 pathway. Inhibition of the PKR-like ER kinase (PERK) pathway also attenuated cytoplasmic dsDNA release. The PERK-regulated apoptotic factor Bim was required for both dsDNA release and IFN-ß mRNA induction. Finally, XBP1 and PERK pathways contributed to cytosolic dsDNA release and IFN-induction by the RNA virus, Vesicular Stomatitis Virus (VSV). Together, our findings suggest that ER stressors, including viral pathogens without nominal STING or cGAS ligands such as RNA viruses, trigger multiple canonical UPR pathways that cooperate to activate STING and downstream IFN-ß via mitochondrial dsDNA release.

TUDCA inhibits EV71 replication by regulating ER stress signaling pathway and suppressing autophagy.

Tauroursodeoxycholic acid (TUDCA) is a naturally occurring hydrophilic bile acid that alleviates endoplasmic reticulum (ER) stress and inhibits apoptosis, thereby protecting cells. Previous studies have shown that enterovirus 71 (EV71) infection modulates ER stress and induces autophagy to assist viral replication. This study observed the effects of TUDCA pretreatment on HeLa and Vero cells infected with EV71, finding that TUDCA inhibits EV71 replication in TUDCA pretreated HeLa and Vero cells in a dose-dependent manner. We found that TUDCA pretreatment inhibited EV71 replication by regulating three branches of UPR, that is up-regulated ATF6, down-regulated both PERK and IRE1. The results also indicated that autophagy which is downstream of UPR, was inhibited either. The results indicate that TUDCA inhibits EV71 replication by regulating UPR sensor proteins and autophagy following ER stress.

The BAG3-IFITM2 Axis Enhances Pancreatic Ductal Adenocarcinoma Growth via the MAPK Signaling Pathway.

Pancreatic ductal adenocarcinoma (PDAC), a highly lethal malignancy, exhibits escalating incidence and mortality rates, underscoring the urgent need for the identification of novel therapeutic targets and strategies. The BAG3 protein, a multifunctional regulator involved in various cellular processes, notably plays a crucial role in promoting tumor progression and acts as a potential "bridge" between tumors and the tumor microenvironment. In this study, we demonstrate that PDAC cells secrete BAG3 (sBAG3), which engages the IFITM2 receptor to activate the MAPK signaling pathway, specifically enhancing pERK activity, thereby propelling PDAC growth. Furthermore, our preliminary investigation into the effects of sBAG3 on co-cultured NK cells intriguingly discovered that sBAG3 diminishes NK cell cytotoxicity and active molecule expression. In conclusion, our findings confirm the pivotal role of the sBAG3-IFITM2 axis in fostering PDAC progression, highlighting the potential significance of sBAG3 as a dual therapeutic target for both tumor and immune cells.

Unveiling the protective potential of mirabegron against thioacetamide-induced hepatic encephalopathy in rats: Insights into cAMP/PPAR-γ/p-ERK1/2/p S536 NF-κB p 65 and p-CREB/BDNF/TrkB in parallel with oxidative and apoptotic trajectories.

Hepatic encephalopathy (HE) is one of the most prevalent and severe hepatic and brain disorders in which escalation of the oxidative, inflammatory and apoptotic trajectories pathologically connects acute liver injury with neurological impairment. Mirabegron (Mira) is a beta3 adrenergic receptor agonist with proven antioxidant and anti-inflammatory activities. The current research pointed to exploring Mira's hepato-and neuroprotective impacts against thioacetamide (TAA)-induced HE in rats. Rats were distributed into three experimental groups: the normal control group, the TAA group, received TAA (200 mg/kg/day for three consecutive days) and the Mira-treated group received Mira (10 mg/kg/day; oral gavage) for 15 consecutive days and intoxicated with TAA from the 13th to the 15th day of the experimental period. Mira counteracted hyperammonemia, enhanced rats' locomotor capability and motor coordination. It attenuated hepatic/neurological injuries by its antioxidant, anti-apoptotic as well as anti-inflammatory potentials. Mira predominantly targeted cyclic adenosine monophosphate (cAMP)/phosphorylated extracellular signal-regulated kinase (p-Erk1/2)/peroxisome proliferator-activated receptor gamma (PPARγ) dependent pathways via downregulation of p S536-nuclear factor kappa B p65 (p S536 NF-κB p 65)/tumor necrosis alpha (TNF-α) axis. Meanwhile, it attenuated nuclear factor erythroid 2-related factor (Nrf2) depletion in parallel with restoring of the neuroprotective defensive pathway by upregulation of cerebral cAMP/PPAR-γ/p-ERK1/2 and p-CREB/BDNF/TrkB besides reduction of GFAP immunoreactivity. Mira showed anti-apoptotic activity through inhibition of Bax immunoreactivity and elevation of Bcl2. To summarize, Mira exhibited a hepato-and neuroprotective effect against TAA-induced HE in rats via shielding antioxidant defense and mitigation of the pathological inflammatory and apoptotic axis besides upregulation of neuroprotective signaling pathways.

Long non-coding RNA-mediated modulation of endoplasmic reticulum stress under pathological conditions.

Endoplasmic reticulum (ER) stress, which ensues from an overwhelming protein folding capacity, activates the unfolded protein response (UPR) in an effort to restore cellular homeostasis. As ER stress is associated with numerous diseases, it is highly important to delineate the molecular mechanisms governing the ER stress to gain insight into the disease pathology. Long non-coding RNAs, transcripts with a length of over 200 nucleotides that do not code for proteins, interact with proteins and nucleic acids, fine-tuning the UPR to restore ER homeostasis via various modes of actions. Dysregulation of specific lncRNAs is implicated in the progression of ER stress-related diseases, presenting these molecules as promising therapeutic targets. The comprehensive analysis underscores the importance of understanding the nuanced interplay between lncRNAs and ER stress for insights into disease mechanisms. Overall, this review consolidates current knowledge, identifies research gaps and offers a roadmap for future investigations into the multifaceted roles of lncRNAs in ER stress and associated diseases to shed light on their pivotal roles in the pathogenesis of related diseases.

IRE1/JNK Is the Leading UPR Pathway in 6-OHDA-Induced Degeneration of Differentiated SH-SY5Y Cells.

Parkinson's disease (PD) is a neurodegenerative disorder which affects dopaminergic neurons of the midbrain. Accumulation of α-synuclein or exposure to neurotoxins like 6-hydroxydopamine (6-OHDA) induces endoplasmic reticulum (ER) stress along with the unfolded protein response (UPR), which executes apoptosis via activation of PERK/CHOP or IRE1/JNK signaling. The present study aimed to determine which of these pathways is a major contributor to neurodegeneration in an 6-OHDA-induced in vitro model of PD. For this purpose, we have applied pharmacological PERK and JNK inhibitors (AMG44 and JNK V) in differentiated SH-SY5Y cells exposed to 6-OHDA. Inhibition of PERK and JNK significantly decreased genotoxicity and improved mitochondrial respiration, but only JNK inhibition significantly increased cell viability. Gene expression analysis revealed that the effect of JNK inhibition was dependent on a decrease in MAPK10 and XBP1 mRNA levels, whereas inhibition of either PERK or JNK significantly reduced the expression of DDIT3 mRNA. Western blot has shown that JNK inhibition strongly induced the XBP1s protein, and inhibition of each pathway attenuated the phosphorylation of eIF2α and JNK, as well as the expression of CHOP. Collectively, our data suggests that targeting the IRE1/JNK pathway of the UPR is a more effective option for PD treatment as it simultaneously affects more than one pro-apoptotic pathway.

TXNDC5 Plays a Crucial Role in Regulating Endoplasmic Reticulum Activity through Different ER Stress Signaling Pathways in Hepatic Cells.

The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is influenced by a number of variables, including endoplasmic reticulum stress (ER). Thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family and acts as an endoplasmic reticulum (ER) chaperone. Nevertheless, the function of TXNDC5 in hepatocytes under ER stress remains largely uncharacterized. In order to identify the role of TXNDC5 in hepatic wild-type (WT) and TXNDC5-deficient (KO) AML12 cell lines, tunicamycin, palmitic acid, and thapsigargin were employed as stressors. Cell viability, mRNA, protein levels, and mRNA splicing were then assayed. The protein expression results of prominent ER stress markers indicated that the ERN1 and EIF2AK3 proteins were downregulated, while the HSPA5 protein was upregulated. Furthermore, the ATF6 protein demonstrated no significant alterations in the absence of TXNDC5 at the protein level. The knockout of TXNDC5 has been demonstrated to increase cellular ROS production and its activity is required to maintain normal mitochondrial function during tunicamycin-induced ER stress. Tunicamycin has been observed to disrupt the protein levels of HSPA5, ERN1, and EIF2AK3 in TXNDC5-deficient cells. However, palmitic acid has been observed to disrupt the protein levels of ATF6, HSPA5, and EIF2AK3. In conclusion, TXNDC5 can selectively activate distinct ER stress pathways via HSPA5, contingent on the origin of ER stress. Conversely, the absence of TXNDC5 can disrupt the EIF2AK3 cascade.

Stable Production of a Recombinant Single-Chain Eel Follicle-Stimulating Hormone Analog in CHO DG44 Cells.

This study aimed to produce single-chain recombinant Anguillid eel follicle-stimulating hormone (rec-eel FSH) analogs with high activity in Cricetulus griseus ovary DG44 (CHO DG44) cells. We recently reported that an O-linked glycosylated carboxyl-terminal peptide (CTP) of the equine chorionic gonadotropin (eCG) ß-subunit contributes to high activity and time-dependent secretion in mammalian cells. We constructed a mutant (FSH-M), in which a linker including the eCG ß-subunit CTP region (amino acids 115-149) was inserted between the ß-subunit and α-subunit of wild-type single-chain eel FSH (FSH-wt). Plasmids containing eel FSH-wt and eel FSH-M were transfected into CHO DG44 cells, and single cells expressing each protein were isolated from 10 and 7 clones. Secretion increased gradually during the cultivation period and peaked at 4000-5000 ng/mL on day 9. The molecular weight of eel FSH-wt was 34-40 kDa, whereas that of eel FSH-M increased substantially, with two bands at 39-46 kDa. Treatment with PNGase F to remove the N glycosylation sites decreased the molecular weight remarkably to approximately 8 kDa. The EC50 value and maximal responsiveness of eel FSH-M were approximately 1.23- and 1.06-fold higher than those of eel FSH-wt, indicating that the mutant showed slightly higher biological activity. Phosphorylated extracellular-regulated kinase (pERK1/2) activation exhibited a sharp peak at 5 min, followed by a rapid decline. These findings indicate that the new rec-eel FSH molecule with the eCG ß-subunit CTP linker shows potent activity and could be produced in massive quantities using the stable CHO DG44 cell system.

Navigating the landscape of the unfolded protein response in CD8+ T cells.

Endoplasmic reticulum stress occurs due to large amounts of misfolded proteins, hypoxia, nutrient deprivation, and more. The unfolded protein is a complex intracellular signaling network designed to operate under this stress. Composed of three individual arms, inositol-requiring enzyme 1, protein kinase RNA-like ER kinase, and activating transcription factor-6, the unfolded protein response looks to resolve stress and return to proteostasis. The CD8+ T cell is a critical cell type for the adaptive immune system. The unfolded protein response has been shown to have a wide-ranging spectrum of effects on CD8+ T cells. CD8+ T cells undergo cellular stress during activation and due to environmental insults. However, the magnitude of the effects this response has on CD8+ T cells is still understudied. Thus, studying these pathways is important to unraveling the inner machinations of these powerful cells. In this review, we will highlight the recent literature in this field, summarize the three pathways of the unfolded protein response, and discuss their roles in CD8+ T cell biology and functionality.

Induction of the PERK-eIF2α-ATF4 Pathway in M1 Macrophages under Endoplasmic Reticulum Stress.

Translation inhibition can activate two cell death pathways. The first pathway is activated by translational aberrations, the second by endoplasmic reticulum (ER) stress. In this work, the effect of ribosome-inactivating protein type II (RIP-II) viscumin on M1 macrophages derived from the THP-1 cell line was investigated. The number of modified ribosomes was evaluated by real-time PCR. Transcriptome analysis revealed that viscumin induces the ER stress activated by the PERK sensor.

Activation of the PERK/eIF2α axis is a pivotal prerequisite of taxanes to cancer cell apoptosis and renders synergism to overcome paclitaxel resistance in breast cancer cells.

BACKGROUND: Microtubule polymerization is usually considered as the upstream of apoptotic cell death induced by taxanes, but recently published studies provide more insights into the mechanisms responsible for the antineoplastic effect of taxanes. In this study, we figure out the role of the stress-related PERK/eIF2α axis in tumor cell death upon taxane treatment along with paclitaxel resistance. METHODS: Utilizing immunoblot assay, the activation status of PERK-eIF2α signaling was detected in a panel of cancer cell lines after the treatment of taxanes. The causal role of PERK-eIF2α signaling in the cancer cell apoptosis induced by taxanes was examined via pharmacological and genetic inhibitions of PERK. The relationship between microtubule polymerization and PERK-eIF2α activation was explored by immunofluorescent and immunoblotting assays. Eventaually, the combined therapeutic effect of paclitaxel (PTX) and CCT020312, a PERK agonist, was investigated in PTX-resistant breast cancer cells in vitro and in vivo. RESULTS: PERK-eIF2α axis was dramatically activated by taxanes in several cancer cell types. Pharmacological or genetic inhibition of PERK efficiently impaired taxane-induced apoptotic cell death, independent of the cellular microtubule polymerization status. Moreover, PTX was able to activate the PERK/eIF2α axis in a very low concentration without triggering microtubule polymerization. In PTX-resistant breast cancer cells, the PERK/eIF2α axis was attenuated in comparison with the PTX-sensitive counterparts. Reactivation of the PERK/eIF2α axis in the PTX-resistant breast cancer cells with PERK agonist sensitized them to PTX in vitro. Combination treatment of the xenografted PTX-resistant breast tumors with PERK agonist and PTX validated the synergic effect of PTX and PERK activation in vivo. CONCLUSION: Activation of the PERK/eIF2α axis is a pivotal prerequisite of taxanes to initiate cancer cell apoptosis, which is independent of the well-known microtubule polymerization-dependent manner. Simultaneous activation of PERK-eIF2α signaling would be a promising therapeutic strategy to overcome PTX resistance in breast cancer or other cancers.