Induction Of Endoplasmic Reticulum Stress Research Articles

Synthesis, Biological Evaluation, and Mechanistic Insights of Rubrolide Analogues as Antitumor Agents.

Marine natural products and their analogues have as of now been acknowledged as an important source of bioactive molecules for the treatment of cancer. Rubrolides, a unique group of γ-butenolides derived from marine microorganisms, have shown strong cytotoxic activity against various tumor cells. In this study, we synthesized and characterized 21 rubrolide analogues (including 16 new compounds) and investigated their antitumor activities in order to screen more active molecules and elucidate their mechanism of action. Primary MTT assay showed that compounds 1 and 4-9 all exhibited excellent antiproliferative activities. In particular, compound 7 showed broad-spectrum cytotoxic activity against six tumor cell lines, with IC50 values mostly ranging from 2.5 to 0.2 μM. Further mechanistic studies revealed that compound 7 could penetrate HCT116 and Hela cells, localize in the endoplasmic reticulum, and upregulate the PERK-eIF2α-CHOP pathway, inducing ER stress and increasing intracellular reactive oxygen species (ROS) levels to ultimately trigger apoptosis in tumor cells. Additionally, compound 7 was found to upregulate Cyclin B1 protein expression, causing cell cycle reticulum at the G2/M phase. In vivo studies further demonstrated that liposomal delivery of compound 7 exhibited a potent antitumor efficacy against Hela xenograft tumors. Based on these results, marine-derived rubrolide analogues show significant potential as novel lead compounds for antitumor drug development.

Urolithin A Protects Hepatocytes from Palmitic Acid-Induced ER Stress by Regulating Calcium Homeostasis in the MAM

An ellagitannin-derived metabolite, Urolithin A (UA), has emerged as a potential therapeutic agent for metabolic disorders due to its antioxidant, anti-inflammatory, and mitochondrial function-improving properties, but its efficacy in protecting against ER stress remains underexplored. The endoplasmic reticulum (ER) is a cellular organelle involved in protein folding, lipid synthesis, and calcium regulation. Perturbations in these functions can lead to ER stress, which contributes to the development and progression of metabolic disorders such as metabolic-associated fatty liver disease (MAFLD). In this study, we identified a novel target protein of UA and elucidated its mechanism for alleviating palmitic acid (PA)-induced ER stress. Cellular thermal shift assay (CETSA)-LC-MS/MS analysis revealed that UA binds directly to the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA), an important regulator of calcium homeostasis in mitochondria-associated ER membranes (MAMs). As an agonist of SERCA, UA attenuates abnormal calcium fluctuations and ER stress in PA-treated liver cells, thereby contributing to cell survival. The lack of UA activity in SERCA knockdown cells suggests that UA regulates cellular homeostasis through its interaction with SERCA. Collectively, our results demonstrate that UA protects against PA-induced ER stress and enhances cell survival by regulating calcium homeostasis in MAMs through SERCA. This study highlights the potential of UA as a therapeutic agent for metabolic disorders associated with ER stress.

Endoplasmic reticulum stress triggers unfolded protein response as an antiviral strategy of teleost erythrocytes

IntroductionFish nucleated red blood cells (RBCs), also known as erythrocytes, play a crucial role in maintaining immune system balance by modulating protein expression in response to various stimuli, including viral attack. This study explores the intriguing behavior of rainbow trout RBCs when faced with the viral hemorrhagic septicemia virus (VHSV), focusing on the endoplasmic reticulum (ER) stress and the unfolded protein response (UPR).MethodsRainbow trout RBCs were Ficoll-purified and exposed to ultraviolet (UV)-inactivated VHSV or live VHSV at different multiplicities of infection (MOIs). Using cryo-soft X-ray tomography (cryo-SXT), we uncovered structural and cellular modifications in RBCs exposed to UV-inactivated VHSV. Moreover, RBCs were treated with 4-phenylbutyric acid (4-PBA), an ER stress inhibitor, to investigate its effect on viral replication. Quantitative real-time PCR was also used to analyze the expression of genes related to the UPR and other related cellular pathways.Results and discussionBeyond their antiviral response, RBCs undergo notable intracellular changes to combat the virus. Cryo-SXT highlighted a significant increase in the ER volume. This increase is associated with ER stress and the activation of the UPR pathway. Interestingly, VHSV replication levels augmented in RBCs under ER-stress inhibition by 4-PBA treatment, suggesting that rainbow trout RBCs tune up ER stress to control viral replication. Therefore, our findings suggested the induction of ER stress and subsequent activation UPR signaling in the antiviral response of RBCs to VHSV. The results open a new line of investigation to uncover additional mechanisms that may become novel cellular targets for the development of RBC-targeted antiviral strategies.

The Effects of Microplastics on Musculoskeletal Disorder; A Narrative Review.

The physical health impacts of microplastics have received increasing attention in recent years. However, limited data impedes a full understanding of the internal exposure to microplastics, especially concerning the musculoskeletal system. The purpose of this review is to summarize the recent literature regarding the effects of microplastics on the musculoskeletal system. Microplastics have been shown to cause abnormal endochondral ossification and disrupt the normal function of pre-osteoblasts, osteocyte-like cells, and pre-osteoclasts through gene mutations, endoplasmic reticulum stress induction, and reduced autophagosome formation in bone growth areas. Although there are few reports on their effects on muscle, it has been noted that microplastics inhibit energy and lipid metabolism, decrease type I muscle fiber density, impair muscle angiogenesis, cause muscle atrophy, and increase lipid deposition. Only a few recent studies have shown that microplastics interfere with the normal function of bone growth-related cells and reduce muscle mass and quality. This review underscores the need for further research into other parts of the musculoskeletal system and studies using human tissues at the disease level.

EP152R-mediated endoplasmic reticulum stress contributes to African swine fever virus infection via the PERK-eIF2α pathway.

African swine fever virus (ASFV) is a large, icosahedral, double-stranded DNA virus in the Asfarviridae family and the causative agent of African swine fever (ASF). ASFV causes a hemorrhagic fever with high mortality rates in domestic and wild pigs. ASFV contains an open reading frame named EP152R, previous research has shown that EP152R is an essential gene for virus rescue in swine macrophages. However, the detailed functions of ASFV EP152R remain elusive. Herein, we demonstrate that EP152R, a membrane protein located in the endoplasmic reticulum (ER), induces ER stress and swelling, triggering the PERK/eIF2α pathway, and broadly inhibiting host protein synthesis invitro. Additionally, EP152R strongly promotes immune evasion, reduces cell proliferation, and alters cellular metabolism. These results suggest that ASFV EP152R plays a critical role in the intracellular environment, facilitating viral replication. Furthermore, virus-level experiments have shown that the knockdown of EP152R or PERK inhibitors efficiently affects viral replication by decreasing viral gene expression. In summary, these findings reveal a series of novel functions of ASFV EP152R and have important implications for understanding host-pathogen interactions.

The Role of Protein Disulfide Isomerase Inhibitors in Cancer Therapy.

Protein disulfide isomerase (PDI) is a member of the mercaptan isomerase family, primarily located in the endoplasmic reticulum (ER). At least 21 PDI family members have been identified. PDI plays a key role in protein folding, correcting misfolded proteins, and catalyzing disulfide bond formation, rearrangement, and breaking. It also acts as a molecular chaperone. Dysregulation of PDI activity is thus linked to diseases such as cancer, infections, immune disorders, thrombosis, neurodegenerative diseases, and metabolic disorders. In particular, elevated intracellular PDI levels can enhance cancer cell proliferation, metastasis, and invasion, making it a potential cancer marker. Cancer cells require extensive protein synthesis, with disulfide bond formation by PDI being a critical producer. Thus, cancer cells have higher PDI levels than normal cells. Targeting PDI can induce ER stress and activate the Unfolded Protein Response (UPR) pathway, leading to cancer cell apoptosis. This review discusses the structure and function of PDI, PDI inhibitors in cancer therapy, and the limitations of current inhibitors, proposing especially future directions for developing new PDI inhibitors.

A WFS1 variant disrupting acceptor splice site uncovers the impact of alternative splicing on beta cell apoptosis in a patient with Wolfram syndrome.

Wolfram syndrome 1 (WS1) is an inherited condition mainly manifesting in childhood-onset diabetes mellitus and progressive optic nerve atrophy. The causative gene, WFS1, encodes wolframin, a master regulator of several cellular responses, and the gene's mutations associate with clinical variability. Indeed, nonsense/frameshift variants correlate with more severe symptoms than missense/in-frame variants. As achieving a genotype-phenotype correlation is crucial for dealing with disease outcome, works investigating the impact of transcriptional and translational landscapes stemming from such mutations are needed. Therefore, we sought to elucidate the molecular determinants behind the pathophysiological alterations in a WS1 patient carrying compound heterozygous mutations in WFS1: c.316-1G>A, affecting the acceptor splice site (ASS) upstream of exon 4; and c.757A>T, introducing a premature termination codon (PTC) in exon 7. Bioinformatic analysis was carried out to infer the alternative splicing events occurring after disruption of ASS, followed by RNA-seq and PCR to validate the transcriptional landscape. Patient-derived induced pluripotent stem cells (iPSCs) were used as an in vitro model of WS1 and to investigate the WFS1 alternative splicing isoforms in pancreatic beta cells. CRISPR/Cas9 technology was employed to correct ASS mutation and generate a syngeneic control for the endoplasmic reticulum stress induction and immunotoxicity assays. We showed that patient-derived iPSCs retained the ability to differentiate into pancreatic beta cells. We demonstrated that the allele carrying the ASS mutation c.316-1G>A originates two PTC-containing alternative splicing transcripts (c.316del and c.316-460del), and two open reading frame-conserving mRNAs (c.271-513del and c.316-456del) leading to N-terminally truncated polypeptides. By retaining the C-terminal domain, these isoforms sustained the endoplasmic reticulum stress response in beta cells. Otherwise, PTC-carrying transcripts were regulated by the nonsense-mediated decay (NMD) in basal conditions. Exposure to cell stress inducers and proinflammatory cytokines affected expression levels of the NMD-related gene SMG7 (>twofold decrease; p<0.001) without eliciting a robust unfolded protein response in WFS1 beta cells. This resulted in a dramatic accumulation of the PTC-containing isoforms c.316del (>100-fold increase over basal; p<0.001) and c.316-460del (>20-fold increase over basal; p<0.001), predisposing affected beta cells to undergo apoptosis. Cas9-mediated recovery of ASS retrieved the canonical transcriptional landscape, rescuing the normal phenotype in patient-derived beta cells. This study represents a new model to study wolframin, highlighting how each single mutation of the WFS1 gene can determine dramatically different functional outcomes. Our data point to increased vulnerability of WFS1 beta cells to stress and inflammation and we postulate that this is triggered by escaping NMD and accumulation of mutated transcripts and truncated proteins. These findings pave the way for further studies on the molecular basis of genotype-phenotype relationship in WS1, to uncover the key determinants that might be targeted to ameliorate the clinical outcome of patients affected by this rare disease. The in silico predicted N-terminal domain structure file of WT wolframin was deposited in the ModelArchive, together with procedures, ramachandran plots, inter-residue distance deviation and IDDT scores, and Gromacs configuration files (doi/10.5452/ma-cg3qd). The deep-sequencing data as fastq files used to generate consensus sequences of AS isoforms of WFS1 are available in the SRA database (BioProject PRJNA1109747).

Thapsigargin: An Unlikely Drug in the Battle Against Viral Pandemics

Antiviral resistance is an increasingly categorized phenomenon, and with the high pandemic potential of emergent pathogens such as coronaviruses, the need for novel therapeutic agents is dire. Thapsigargin, a potent endoplasmic reticulum stress inducer found in the Mediterranean region, has shown significant promise in combatting replication of several viruses including coronaviruses by harnessing the host's innate immune system, even being more efficacious than some antivirals currently used. There is a major gap in knowledge regarding the exact mechanism of action that permits Thapsigargin to be an incredible endoplasmic reticulum stress inducer and an potent antiviral. We hypothesize that interferon 1 production and SERCA-inhibition caused by thapsigargin mediates the antiviral effects observed. This review outlines what is currently known about this novel compound, the gap in research, and details methodologies including gene expression analyses, protein quantification, and mammalian cell culturing, to address the gap and answer the hypothesis.

Unveiling genome plasticity as a mechanism of non-antifungal-induced antifungal resistance in Cryptococcus neoformans.

Cryptococcus neoformans, a critical priority pathogen designated by the World Health Organization, poses significant therapeutic challenges due to the limited availability of treatment options. The emergence of antifungal resistance, coupled with cross-resistance, further hampers treatment efficacy. Aneuploidy, known for its ability to induce diverse traits, including antifungal resistance, remains poorly understood in C. neoformans. We investigated the impact of tunicamycin, a well-established ER stress inducer, on aneuploidy formation in C. neoformans. Our findings show that both mild and severe ER stress induced by tunicamycin lead to the formation of aneuploid strains in C. neoformans. These aneuploid strains exhibit diverse karyotypes, with some conferring resistance or cross-resistance to antifungal drugs fluconazole and 5-flucytosine. Furthermore, these aneuploid strains display instability, spontaneously losing extra chromosomes in the absence of stress. Transcriptome analysis reveals the simultaneous upregulation of multiple drug resistance-associated genes in aneuploid strains. Our study reveals the genome plasticity of C. neoformans as a major mechanism contributing to non-antifungal-induced antifungal resistance.

Characterization of the Ubiquitin-Modified Proteome of Recombinant Chinese Hamster Ovary Cells in Response to Endoplasmic Reticulum Stress.

Chinese hamster ovary (CHO) cells remain the most widely used host cell line for biotherapeutics production. Despite their widespread use, understanding endoplasmic reticulum (ER) stress conditions in recombinant protein production remains limited, often creating bottlenecks preventing improved production titers and product quality. Ubiquitination not only targets substrates (e.g., misfolded proteins) for proteasome degradation but also has important regulatory control functions including cell cycle regulation, translation, apoptosis, autophagy, etc. and hence is likely to be central to understanding and controlling the productivity of recombinant biotherapeutics. This study aimed to uncover differentially expressed ubiquitinated proteins following artificial induction of ER-stress in recombinant CHO cells. CHO cells were treated with the stress inducer tunicamycin and the proteasome inhibitor MG132, followed by LC-MS/MS proteomic analysis. We identified >4000 ubiquitinated peptides from CHO-DP12 cells under ER stress conditions and proteasome inhibition. Moreover, data analysis showed altered abundance levels of >900 ubiquitinated proteins under the combination of ER stress and proteasome inhibition compared to untreated controls. Gene Ontology (GO) analysis of these ubiquitinated proteins resulted in a significant enrichment of key pathways involving the proteasome, protein processing in the ER, N-glycan biosynthesis, and ubiquitin-mediated proteolysis. ER stress response proteins such as GRP78, HSP90B1, ATF6, HERPUD1, and PDIA4 were found to be highly ubiquitinated and exhibited a significant increase in abundance following induction of ER-stress conditions. This study broadens our comprehension of the roles played by protein ubiquitination in CHO cell stress responses, potentially revealing targets for tailored cell line engineering aimed at enhancing stress tolerance and production efficiency.

Glycosylation of Ceramide Synthase 6 is Required for Its Activity

Sphingolipids play key roles in membrane structure and cellular signaling. Ceramide Synthase (CerS)-generated ceramide is implicated in cellular stress responses and induction of apoptosis. Ceramide and other sphingolipids are linked to the induction of ER stress response pathways. However, the mechanisms by which ceramide modulates ER stress signaling are not well understood. Here, we show that the ER stress inducer Brefeldin A (BFA) causes increased glycosylation of CerS6, and that treatment with BFA causes increased endogenous ceramide accumulation. To our surprise, we found that CerS6 activity was not affected by BFA-induced glycosylation. Instead, our data show that basal glycosylation of CerS6 at Asn18 is required for CerS6 activity. We used a robust HCT116 CRISPR-Cas9 CerS6 KO with reintroduction of either wild type CerS6 or a mutant CerS6 with a point mutation at asparagine-18 to an alanine (N18A) which abrogated glycosylation at that residue. Our data show that cells stably expressing the N18A mutant CerS6 had significantly lower activity in vitro and in situ as compared to WT CerS6 expressing cells. Further, the defective CerS6 with N18A mutation also had defects in GSK3β, AKT, JNK, and STAT3 signaling. Despite being required for CerS6 activity, Asn18 glycosylation did not influence ER stress response pathways. Overall, our study provides vital insight into the regulation of CerS6 activity by post-translational modification at Asn18 and identifies glycosylation of CerS6 to be important for ceramide generation and downstream cellular signaling pathways.

Zelenirstat Inhibits N-Myristoyltransferases to Disrupt Src Family Kinase Signalling and Oxidative Phosphorylation Killing Acute Myeloid Leukemia Cells.

Acute myeloid leukemia (AML) is a hematological malignancy with limited treatment options and a high likelihood of recurrence after chemotherapy. We studied N-myristoylation, the myristate modification of proteins linked to survival signaling and metabolism, as a potential therapeutic target for AML. N-myristoylation is catalyzed by two N-myristoyltransferases (NMTs), NMT1 and NMT2, with varying expressions in AML cell lines and patient samples. We identified NMT2 expression as a marker for AML patient survival, and low NMT2 expression was associated with poor outcomes. We used the first-in-class pan-NMT inhibitor, zelenirstat, to investigate the role of N-myristoylation in AML. Zelenirstat effectively inhibits myristoylation in AML cell lines and patient samples, leading to degradation of Src family kinases (SFKs), induction of endoplasmic reticulum (ER) stress, apoptosis, and cell death. Zelenirstat was well tolerated in vivo and reduced the leukemic burden in an ectopic AML cell line and in multiple orthotopic AML patient-derived xenograft models. The leukemia stem cell (LSC)-enriched fractions of the hierarchical OCI-AML22 model were highly sensitive to myristoylation inhibition. Zelenirstat also impairs mitochondrial complex I and oxidative phosphorylation, which are critical for LSC survival. These findings suggest that targeting N-myristoylation with zelenirstat represents a novel therapeutic approach for AML, with promise in patients with currently poor outcomes.

Small hepatitis B virus surface antigen (SHBs) induces dyslipidemia by suppressing apolipoprotein-AII expression through ER stress-mediated modulation of HNF4α and C/EBPγ.

Persistent infection with hepatitis B virus (HBV) often leads to disruptions in lipid metabolism. Apolipoprotein AII (apoAII) plays a crucial role in lipid metabolism and is implicated in various metabolic disorders. However, whether HBV could regulate apoAII and contribute to HBV-related dyslipidemia and the underlying mechanism remain unclear. This study revealed significant reductions in apoAII expression in HBV-expressing cell lines, the serum, and liver tissues of HBV-transgenic mice. The impact of HBV on apoAII is related to small hepatitis B virus surface antigen (SHBs). Overexpression of SHBs decreased apoAII levels in SHBs-expressing hepatoma cells, transgenic mice, and the serum of HBV-infected patients, whereas suppression of SHBs increased apoAII expression. Mechanistic investigations demonstrated that SHBs repressed the apoAII promoter activity through a HNF4α- and C/EBPγ-dependent manner; SHBs simultaneously upregulated C/EBPγ and downregulated HNF4α by inhibiting the PI3K/AKT signaling pathway through activating endoplasmic reticulum (ER) stress. Serum lipid profile assessments revealed notable decreases in high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), and triglycerides (TG) in SHBs-transgenic mice compared to control mice. However, concurrent overexpression of apoAII in these mice effectively counteracted these reductions in lipid levels. In HBV patients, SHBs levels were negatively correlated with serum levels of HDL-C, LDL-C, TC, and TG, whereas apoAII levels positively correlated with lipid content. This study underscores that SHBs contributes to dyslipidemia by suppressing the PI3K/AKT pathway via inducing ER stress, leading to altered expression of HNF4α and C/EBPγ, and subsequently reducing apoAII expression.IMPORTANCEThe significance of this study lies in its comprehensive examination of how the hepatitis B virus (HBV), specifically through its small hepatitis B virus surface antigen (SHBs), impacts lipid metabolism-a key aspect often disrupted by chronic HBV infection. By elucidating the role of SHBs in regulating apolipoprotein AII (apoAII), a critical player in lipid processes and associated metabolic disorders, this research provides insights into the molecular pathways contributing to HBV-related dyslipidemia. Discovering that SHBs downregulates apoAII through mechanisms involving the repression of the apoAII promoter via HNF4α and C/EBPγ, and the modulation of the PI3K/AKT signaling pathway via endoplasmic reticulum (ER) stress, adds critical knowledge to HBV pathogenesis. The research also shows an inverse correlation between SHBs expression and key lipid markers in HBV-infected individuals, suggesting that apoAII overexpression could counteract the lipid-altering effects of SHBs, offering new avenues for understanding and managing the metabolic implications of HBV infection.

Vitamin K2 and the Vitamin-K-Cycle-Enzyme VKORC1L1 improve endothelial regeneration by inhibiting endothelial-mesenchymal transition, inflammatory cell activation and ferroptosis

Abstract Introduction Vascular inflammation is a crucial contributor to atherosclerosis, in which oxidative stress, endoplasmic reticulum (ER) stress and transition of endothelial to mesenchymal cells (EndMT) are of critical importance. Vitamin-K-antagonists (VKA) promote vascular dysfunction, while dietary vitamin K intake was proposed to reduce incidence of coronary artery disease, but the mechanisms remain unresolved. Key enzyme for regeneration of vitamin K is the Vitamin K epoxide reductase complex subunit 1 (VKORC1), which also represents the pharmacological target of VKA. VKOR-like1 (VKORC1L1) is an isoenzyme of VKORC1, which resides at the ER-membrane, exerts antioxidative properties and is involved in vitamin K maintenance. Aim of this study was to investigate the role of Vitamin K2 and the VKOR-enzymes in endothelial cell inflammation. Methods and Results In human coronary artery endothelial cells (HCAEC), Vitamin K2 improves endothelial regeneration by increasing proliferation and cell viability and by reducing both apoptosis and ferroptosis (relative viability: RSL3 0.11 ± 0.01 vs 0.32 ± 0.05 RSL3+K2-1µM vs. 0.44 ± 0.11 RSL3+K2-10µM, p&amp;lt;0.001). Moreover, Vitamin K2-supplementation inhibits EndMT by reducing expression of mesenchymal markers (Calponin, SM22; Fig.1). In silico analyses of the proteome of human coronary atherosclerosis revealed increased expression of VKORC1L1, but not of VKORC1. In vitro induction of oxidative stress and ER-Stress promoted time- and dose-dependent enhanced expression of VKORC1L1, without altering VKORC1 expression (H2O2: 2.3-fold vs. 0.8-fold after 40 minutes, p=0.04; tunicamycin: 1 μg/ml: 1.43-fold vs. 0.8-fold, p&amp;lt;0.01). Likewise, siRNA-mediated VKORC1L1 knockdown induces an inflammatory gene signature with upregulation of NFkB-signalling and endoplasmic reticulum stress (NF-κB: 1.95-fold ± 0.13, GRP78: 1.32-fold ± 0.04 vs. control, p&amp;lt;0.01). Furthermore, VKORC1L1 downregulation increased formation of reactive oxygen species (ROS) and reduced proliferation (EdU signal: 0.56 ± 0.02 vs. control, p &amp;lt; 0.01). In contrast, VKORC1-knockdown does not impair endothelial regeneration and function (Fig.2). Vitamin K2 reduces vascular inflammation and endoplasmic reticulum stress in the case of VKORC1-, but not VKORC1L1-knockdown. In addition, inducing EndMT resulted in a reduction of VKORC1L1, but not of VKORC1, while VKORC1L1-knockdown aggravates EndMT. Conclusion VKORC1 and its isoenzyme VKORC1L1 exhibit divergent effects on endothelial cell inflammation. Further studies are warranted to shed light on the regulation of the enzymes to improve our understanding regarding vascular side effects of VKA treatment and beneficial effects of vitamin K supplementation.

Nimbolide Induces Cell Apoptosis via Mediating ER Stress-Regulated Apoptotic Signaling in Human Oral Squamous Cell Carcinoma.

Human oral squamous cell carcinoma (OSCC) poses a significant health challenge in Asia, with current therapeutic strategies failing to improve the survival rates for OSCC patients sufficiently. To elucidate the effects of Nimbolide on OSCC cell proliferation and apoptosis, we performed a series of experiments, including cell proliferation assays, annexin V/PI assays, and cell cycle analysis. We further investigated nimbolide's role in modulating endoplasmic reticulum (ER) stress, reactive oxygen species (ROS) production, and mitochondrial dysfunction using flow cytometry. Additionally, Western blotting was used to detect apoptosis-related protein expression. Our findings reveal that nimbolide exerts its anti-proliferative effects on OSCC cells by inducing apoptosis. The nimbolide increased intracellular ROS levels and acceleration of cellular calcium accumulation, respectively promoting endoplasmic reticulum stress and cancer cell apoptosis. Furthermore, nimbolide activates the caspase cascade by altering the mitochondrial membrane potential and apoptotic protein expression, thereby inhibiting the viability of tumor cells. Our data show that Nimbolide suppresses tumor growth through the induction of ROS production, ER stress, and mitochondrial dysfunction, resulting in apoptosis in OSCC cells. Overall, our study highlights nimbolide as a potential natural compound for OSCC therapy.

Endoplasmic Reticulum Stress in Bronchopulmonary Dysplasia: Contributor or Consequence?

Bronchopulmonary dysplasia (BPD) is the most common complication of prematurity. Oxidative stress (OS) and inflammation are the major contributors to BPD. Despite aggressive treatments, BPD prevalence remains unchanged, which underscores the urgent need to explore more potential therapies. The endoplasmic reticulum (ER) plays crucial roles in surfactant and protein synthesis, assisting mitochondrial function, and maintaining metabolic homeostasis. Under OS, disturbed metabolism and protein folding transform the ER structure to refold proteins and help degrade non-essential proteins to resume cell homeostasis. When OS becomes excessive, the endogenous chaperone will leave the three ER stress sensors to allow subsequent changes, including cell death and senescence, impairing the growth potential of organs. The contributing role of ER stress in BPD is confirmed by reproducing the BPD phenotype in rat pups by ER stress inducers. Although chemical chaperones attenuate BPD, ER stress is still associated with cellular senescence. N-acetyl-lysyltyrosylcysteine amide (KYC) is a myeloperoxidase inhibitor that attenuates ER stress and senescence as a systems pharmacology agent. In this review, we describe the role of ER stress in BPD and discuss the therapeutic potentials of chemical chaperones and KYC, highlighting their promising role in future therapeutic interventions.

Food grade titanium dioxide induced endoplasmic reticulum stress in colon cells: Comparison between normal and colorectal carcinoma cells

BackgroundFood-grade titanium dioxide (E171) has been under scrutiny in the last decade since its possible adverse effects; however, the cellular mechanisms underlying E171 toxicity have not been thoroughly described. AimWe aimed to compare the effects of E171 on endoplasmic reticulum (ER) homeostasis in normal and cancer colon cells. Experimental designWe exposed normal, carcinoma, and adenocarcinoma cells to 0.1, 1, 10, 50 and 100 μg/cm2 of E171 for 24, 48 and 72 h, and we evaluated ER stress, cell viability, titanium uptake, intracellular calcium concentration, and gene expression related to unfolded protein response (UPR) and chaperone pathways. ResultsCell viability decreased only after 72 h of exposure to 100 μg/cm2 of E171. Adenocarcinoma cells internalized higher titanium amounts than normal and carcinoma cells, but the effects in ER distribution, intracellular calcium concentration, and gene expression were similar among the three cell lines. The expression of UPR and chaperone pathways were downregulated at the lowest concentrations but upregulated at the highest concentrations in the three cell lines. ConclusionE171 induces ER stress through alterations in ER distribution, intracellular calcium, and UPR and chaperone protein pathways.

Mouse Model of Glucocorticoid-Induced Glaucoma.

Extended glucocorticoid (GC) treatment can lead to ocular hypertension and induce iatrogenic open-angle glaucoma. GC-induced glaucoma mimics many clinical and pathological features of primary open-angle glaucoma (POAG), and therefore mouse models of GC-induced glaucoma are utilized to study pathophysiology of glaucoma. We have recently demonstrated that weekly periocular injections of dexamethasone-21-acetate (Dex-Ac) lead to robust and significant intraocular pressure (IOP) elevation, retinal ganglion cell (RGC) loss, and optic nerve degeneration in mice. Our mouse model exhibits signature features of POAG including significant IOP elevation due to reduced outflow facility, progressive optic nerve degeneration, and structural and functional loss of RGCs. Dex-induced IOP elevation is associated with increased aqueous outflow resistance due to trabecular meshwork (TM) dysfunction including excessive extracellular matrix deposition and induction of endoplasmic reticulum stress. Mouse model of Dex-induced glaucoma represents an ideal animal model to investigate both glaucomatous damage to the TM and RGC axons and to develop novel therapies. Here, we present a detailed protocol for developing this model in laboratory settings.

Fragility of ER homeostatic regulation underlies haploid instability in human somatic cells

Mammalian somatic cells are generally unstable in the haploid state, resulting in haploid-to-diploid conversion within a short time frame. However, cellular and molecular principles that limit the sustainability of somatic haploidy remain unknown. In this study, we found the haploidy-linked vulnerability to ER stress as a critical cause of haploid intolerance in human somatic cells. Pharmacological induction of ER stress selectively induced apoptosis in haploid cells, facilitating the replacement of haploids by co-existing diploidized cells in a caspase-dependent manner. Biochemical analyses revealed that unfolded protein response (UPR) was activated with similar dynamics between haploids and diploids upon ER stress induction. However, haploids were less efficient in solving proteotoxic stress, resulting in a bias toward a proapoptotic mode of UPR signaling. Artificial replenishment of chaperone function substantially alleviated the haploidy-linked upregulation of proapoptotic signaling and improved haploid cell retention under tunicamycin-induced ER stress. These data demonstrate that the ER stress-driven haploid instability stems from inefficient proteostatic control that alters the functionality of UPR to cause apoptosis selectively in haploids. Interestingly, haploids suffered a higher level of protein aggregation even in unperturbed conditions, and the long-term stability of the haploid state was significantly improved by alleviating their natural proteotoxicity. Based on these results, we propose that the haploidy-specific vulnerability to ER stress creates a fundamental cause of haploid intolerance in mammalian somatic cells. Our findings provide new insight into the principle that places a stringent restriction on the evolution of animal life cycles.

ATF4/PHGDH mediates the effects of ER stress on cadmium-induced autophagy and glycolysis

Cadmium (Cd) has been classified as a Class I carcinogen, but the mechanism of its carcinogenicity is still unknown. Our previous study demonstrated that 2 μM CdCl2 induced autophagy in A549 cells. In this study, we investigated the role of ATF4/PHGDH in Cd-induced autophagy and increased glycolysis. First, BALB/c mice were subcutaneously injected with A549 cells co-treated with or without Cd and siPHGDH to establish a xenograft tumor model, which demonstrated that PHGDH promotes Cd-induced autophagy in vivo. Cd-exposed A549 cells were treated with siPHGDH and 0.4 mM glycine (Gly), respectively. Western blot analysis and Acridine orange staining revealed that PHGDH promotes Cd-induced autophagy. Using 4-PBA (5 mM), the inhibitor of ER stress, or Tm (0.1 μg/ml), the inducer of ER stress, inhibited Cd-induced PHGDH expression. After co-treatment with siPHGDH, PHGDH was determined to mediate ER stress-induced autophagy. Furthermore, transfection with siATF4 inhibited Tm-induced PHGDH expression. ChIP-qPCR experiments demonstrated the transcription regulatory mechanism of ATF4 on PHGDH. Meanwhile, the role of ER stress/PHGDH/autophagy in Cd-promoted cell migration was explored by scratch assay. Finally, the role of ER stress/PHGDH/autophagy in Cd-induced glycolysis was unveiled. In summary, the transcriptional regulation of PHGDH by ATF4 plays a crucial role in Cd-induced autophagy triggered by ER stress. The axis of ER stress/PHGDH/autophagy is important in Cd-induced cell migration by enhancing glycolysis.