Protein Kinase RNA-like Endoplasmic Reticulum Research Articles

NCI 159456 PERK Inhibitor as a Targeted Therapy for Lung Cancer: An In Vitro Study.

Non-small cell lung cancer (NSCLC) represents the most common histological type of lung cancer, characterized by a five-year survival rate of 15% and poor prognosis. Accumulating evidence indicates a prominent role of endoplasmic reticulum (ER) stress and the protein kinase RNA-like ER kinase (PERK)-dependent pathway of the unfolded protein response (UPR) in the pathogenesis of NSCLC. Increased expression of downstream targets of PERK was observed in various subtypes of NSCLC, and it was associated with a more aggressive phenotype, high risk of recurrence, and poor prognosis. Therefore, the present study aimed to investigate the biological effect of the selective PERK inhibitor NCI 159456 on A549 NSCLC cells and Human Pulmonary Fibroblasts (HPF) in vitro. Treatment of both normal and ER-stressed A549 cells with NCI 159456 resulted in a significant increase in the mRNA expression level of pro-apoptotic genes like activating transcription factor 4 (ATF4), DNA damage inducible transcript 3 (DDIT3), and BCL2 Associated X, Apoptosis Regulator (BAX) as well as a decreased level of the anti-apoptotic gene B-cell lymphoma 2 (Bcl-2). Cytotoxicity and genotoxicity analyses revealed that NCI 159456 significantly decreased viability and increased DNA damage in A549 cells under normal and ER stress conditions. Caspase-3 and reactive oxygen species (ROS) detection assays demonstrated that NCI 159456 significantly induced apoptosis and increased the ROS level in normal and ER-stressed A549 cells. Importantly, treatment with the inhibitor did not affect substantially normal HPF cells at any used concentration. The results indicate that PERK inhibitors could potentially be applied as a targeted therapy for NSCLC.

Endoplasmic Reticulum Stress and Unfolded Protein Response in Vernal Keratoconjunctivitis.

Vernal keratoconjunctivitis (VKC) is an ocular allergic disease characterized by a type 2 inflammation, tissue remodeling, and low quality of life for the affected patients. We investigated the involvement of endoplasmic reticulum (ER) stress and unfolded protein response in VKC. Conjunctival imprints from VKC patients and normal subjects (CTs) were collected, and RNA was isolated, reverse transcribed, and analyzed with the Affymetrix microarray. Differentially expressed genes between VKC patients and CTs were evaluated. Genes related to ER stress, apoptosis, and autophagy were further considered. VKC and CT conjunctival biopsies were analyzed by immunohistochemistry (IHC) with specific antibodies against unfolded protein response (UPR), apoptosis, and inflammation. Conjunctival fibroblast and epithelial cell cultures were exposed to the conditioned medium of activated U937 monocytes and analyzed by quantitative PCR for the expression of UPR, apoptosis, autophagy, and inflammatory markers. ER chaperones HSPA5 (GRP78/BiP) and HYOU1 (GRP170) were upregulated in VKC patients compared to CTs. Genes encoding for ER transmembrane proteins, PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), ER-associated degradation (ERAD), and autophagy were upregulated, but not those related to apoptosis. Increased positive reactivity of BiP and ATF6 and unchanged expression of apoptosis markers were confirmed by IHC. Cell cultures in stress conditions showed an overexpression of UPR, proinflammatory, apoptosis, and autophagy markers. A significant overexpression of genes encoding for ER stress, UPR, and pro-inflammatory pathway components was reported for VKC. Even though these pathways may lead to ER homeostasis, apoptosis, or inflammation, ER stress in VKC may predominantly contribute to promote inflammation.

Butyrate increases methylglyoxal production through regulation of the JAK2/Stat3/Nrf2/Glo1 pathway in castration‑resistant prostate cancer cells.

Cancer cells are characterized by increased glycolysis, known as the Warburg effect, which leads to increased production of cytotoxic methylglyoxal (MGO) and apoptotic cell death. Cancer cells often activate the protective nuclear factor erythroid 2‑related factor2 (Nrf2)/glyoxalase1 (Glo1) system to detoxify MGO. The effects of sodium butyrate (NaB), a product of gut microbiota, on Nrf2/Glos/MGO pathway and the underlying mechanisms in prostate cancer (PCa) cells were investigated in the present study. Treatment with NaB induced the cell death and reduced the proliferation of PCa cells (DU145 and LNCap). Moreover, the protein kinase RNA-like endoplasmic reticulum kinase/Nrf2/Glo1 pathway was greatly inhibited by NaB, thereby accumulating MGO-derived adduct hydroimidazolone (MG-H1). In response to a high amount of MGO, the expression of Nrf2 and Glo1 was attenuated, coinciding with an increased cellular death. NaB also markedly inhibited the Janus kinase 2 (JAK2)/Signal transducer and activator of transcription 3 (Stat3) pathway. Conversely, co‑treatment with Colivelin, a Stat3 activator, significantly reversed the effects of NaB on Glo1 expression, MG-H1 production, and the cell migration and viability. As expected, overexpression of Stat3 or Glo1 reduced NaB‑induced cell death. The activation of calcium/calmodulin dependent protein kinase II gamma and reactive oxygen species production also contributed to the anticancer effect of NaB. The present study, for the first time, demonstrated that NaB greatly increases MGO production through suppression of the JAK2/Stat3/Nrf2/Glo1 pathway in DU145 cells, a cell line mimicking castration‑resistant PCa (CRPC), suggesting that NaB may be a potential agent for PCa therapy.

Effect of Calcium Supplementation and TMEM16A Inhibition on Endoplasmic Reticulum Stress Induced by Dental Fluorosis in Mice.

Dental fluorosis is a discoloration of the teeth caused by the excessive consumption of fluoride. It represents a distinct manifestation of chronic fluorosis in dental tissues, exerting adverse effects on the human body, particularly on teeth. The transmembrane protein 16a (TMEM16A) is expressed at the junction of the endoplasmic reticulum and the plasma membrane. Alterations in its channel activity can disrupt endoplasmic reticulum calcium homeostasis and intracellular calcium ion concentration, thereby inducing endoplasmic reticulum stress (ERS). This study aims to investigate the influence of calcium supplements and TMEM16A on ERS in dental fluorosis. C57BL/6 mice exhibiting dental fluorosis were subjected to an eight-week treatment with varying calcium concentrations: low (0.071%), medium (0.79%), and high (6.61%). Various assays, including Hematoxylin and Eosin (HE) staining, immunohistochemistry, real-time fluorescence quantitative polymerase chain reaction (qPCR), and Western blot, were employed to assess the impact of calcium supplements on fluoride content, ameloblast morphology, TMEM16A expression, and endoplasmic reticulum stress-related proteins (calreticulin (CRT), glucose-regulated protein 78 (GRP78), inositol requiring kinase 1α (IRE1α), PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6)) in the incisors of mice affected by dental fluorosis. Furthermore, mice with dental fluorosis were treated with the TMEM16A inhibitor T16Ainh-A01 along with a medium-dose calcium to investigate the influence of TMEM16A on fluoride content, ameloblast morphology, and endoplasmic reticulum stress-related proteins in the context of mouse incisor fluorosis. In comparison to the model mice, the fluoride content in incisors significantly decreased following calcium supplements (p < 0.01). Moreover, the expression of TMEM16A, CRT, GRP78, IRE1α, PERK, and ATF6 were also exhibited a substantial reduction (p < 0.01), with the most pronounced effect observed in the medium-dose calcium group. Additionally, the fluoride content (p < 0.05) and the expression of CRT, GRP78, IRE1α, PERK, and ATF6 (p < 0.01) were further diminished following concurrent treatment with the TMEM16A inhibitor T16Ainh-A01 and a medium dose of calcium. The supplementation of calcium or the inhibition of TMEM16A expression appears to mitigate the detrimental effects of fluorosis by suppressing endoplasmic reticulum stress. These findings hold implications for identifying potential therapeutic targets in addressing dental fluorosis.

Sphingosine-1-Phosphate Inhibition Increases Endoplasmic Reticulum Stress to Enhance Oxaliplatin Sensitivity in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer resistant to current therapies, including oxaliplatin (Oxa). Growing evidence supports the ability of cancers to harness sphingolipid metabolism for survival. Sphingosine-1-phosphate (S1P) is an anti-apoptotic, pro-survival mediator that can influence cellular functions such as endoplasmic reticulum (ER) stress. We hypothesize that PDAC drives dysregulated sphingolipid metabolism and that S1P inhibition can enhance ER stress to improve therapeutic response to Oxa in PDAC. RNA sequencing data of sphingolipid mediators from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression Project (GTEx) datasets were analyzed. Murine and human PDAC cell lines were treated with small interfering RNA (siRNA) against sphingosine kinase-2 (SPHK2) or ABC294640 (ABC) and incubated with combinations of vehicle control or Oxa. In an orthotopic syngeneic KPC PDAC model, tumors were treated with either vehicle control, Oxa, ABC, or combination therapy. RNA sequencing analysis revealed multiple significantly differentially expressed sphingolipid mediators (P < 0.05). In vitro, both siRNA knockdown of SPHK2 and ABC sensitized cells to Oxa therapy (P < 0.05), and induced eukaryotic initiation factor 2α (eIF2α) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) phosphorylation, hallmarks of ER stress. In vitro therapy also increased extracellular high mobility group box 1 (HMGB1) release (P < 0.05), necessary for immunogenic cell death (ICD). In vivo combination therapy increased apoptotic markers as well as the intensity of HMGB1 staining compared to control (P < 0.05). Our evidence suggests that sphingolipid metabolism is dysregulated in PDAC. Furthermore, S1P inhibition can sensitize PDAC to Oxa therapy through increasing ER stress and can potentiate ICD induction. This highlights a potential therapeutic target for chemosensitizing PDAC as well as an adjunct for future chemoimmunotherapy strategies.

Esculin induces endoplasmic reticulum stress and drives apoptosis and ferroptosis in colorectal cancer via PERK regulating eIF2α/CHOP and Nrf2/HO-1 cascades

Ethnopharmacological relevanceCortex fraxini (also known as Qinpi), the bark of Fraxinus rhynchophylla Hance and Fraxinus stylosa Lingelsh, constitutes a crucial component in several traditional Chinese formulas (e.g., Baitouweng Tang, Jinxiao Formula, etc.) and has demonstrated efficacy in alleviating intestinal carbuncle and managing diarrhea. Cortex fraxini has demonstrated commendable anticancer activity in the realm of Chinese ethnopharmacology; nevertheless, the underlying mechanisms against colorectal cancer (CRC) remain elusive. Aim of the studyEsculin, an essential bioactive compound derived from cortex fraxini, has recently garnered attention for its ability to impede viability and induce apoptosis in cancer cells. This investigation aims to assess the therapeutic potential of esculin in treating CRC and elucidate the underlying mechanisms. Materials and methodsThe impact of esculin on CRC cell viability was assessed using CCK-8 assay, Annexin V/PI staining, and Western blotting. Various cell death inhibitors, along with DCFH-DA, ELISA, biochemical analysis, and Western blotting, were employed to delineate the modes through which esculin induces HCT116 cells death. Inhibitors and siRNA knockdown were utilized to analyze the signaling pathways influenced by esculin. Additionally, an azomethane/dextran sulfate sodium (AOM/DSS)-induced in vivo CRC mouse model was employed to validate esculin's potential in inhibiting tumorigenesis and to elucidate its underlying mechanisms. ResultsEsculin significantly suppressed the viability of various CRC cell lines, particularly HCT116 cells. Investigation with diverse cell death inhibitors revealed that esculin-induced cell death was associated with both apoptosis and ferroptosis. Furthermore, esculin treatment triggered cellular lipid peroxidation, as evidenced by elevated levels of malondialdehyde (MDA) and decreased levels of glutathione (GSH), indicative of its propensity to induce ferroptosis in HCT116 cells. Enhanced protein levels of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and p-eIF2α suggested that esculin induced cellular endoplasmic reticulum (ER) stress, subsequently activating the Nrf2/ARE signaling pathway and initiating the transcriptional expression of heme oxygenase (HO)-1. Esculin-induced excessive expression of HO-1 could potentially lead to iron overload in HCT116 cells. Knockdown of Ho-1 significantly attenuated esculin-induced ferroptosis, underscoring HO-1 as a critical mediator of esculin-induced ferroptosis in HCT116 cells. Furthermore, utilizing an AOM/DSS-induced colorectal cancer mouse model, we validated that esculin potentially inhibits the onset and progression of colon cancer by inducing apoptosis and ferroptosis in vivo. ConclusionsThese findings provide comprehensive insights into the dual induction of apoptosis and ferroptosis in HCT116 cells by esculin. The activation of the PERK signaling pathway, along with modulation of downstream eIF2α/CHOP and Nrf2/HO-1 cascades, underscores the mechanistic basis supporting the clinical application of esculin on CRC treatment.

Study on the Antitumor Mechanism of Tanshinone IIA In Vivo and In Vitro through the Regulation of PERK-ATF4-HSPA5 Pathway-Mediated Ferroptosis.

As a traditional Chinese medicine, Salvia miltiorrhiza Bunge was first recorded in the Shennong Materia Medica Classic and is widely used to treat "the accumulation of symptoms and masses". The main active ingredient of Salvia miltiorrhiza Bunge, Tanshinone IIA (TIIA), has shown anti-inflammatory, antitumor, antifibrosis, antibacterial, and antioxidative activities, etc. In this study, the results showed that TIIA could inhibit the proliferation and migration of HepG2 cells and downregulate glutathione (GSH) and Glutathione Peroxidase 4 (GPX4) levels; besides, TIIA induced the production of Reactive Oxygen Species (ROS), and upregulated the total iron content. Based on network pharmacology analysis, the antitumor effect of TIIA was found to be focused on the endoplasmic reticulum (ER)-mediated ferroptosis signaling pathway, with protein kinase R (PKR)-like ER kinase (PERK)-activating transcription factor 4 (ATF4)-heat shock 70 kDa protein 5 (HSPA5) as the main pathway. Herein, TIIA showed typical ferroptosis characteristics, and a ferroptosis inhibitor (ferrostatin-1) was used to verify the effect. The antitumor effects of TIIA, occurring through the inhibition of the PERK-ATF4-HSPA5 pathway, were further observed in vivo as significantly inhibited tumor growth and the improved pathological morphology of tumor tissue in H22-bearing mice. In summary, the antitumor mechanism of TIIA might be related to the downregulation of the activation of PERK-ATF4-HSPA5 pathway-mediated ferroptosis.

Polygonatum sibiricum (Huang Jing) polysaccharide reduces diabetic cardiomyopathy through increasing cyclic guanosine monophosphate-protein kinase G signaling in diabetic mice.

Diabetic cardiomyopathy (DCM) is a prevalent condition among individuals with diabetes, and is associated with a high mortality rate. The anti-oxidant properties of Jing Huang or Polygonatum sibiricum polysaccharide (PSP) have been extensively used to treat diabetes-related disorders; however, its potential effectiveness against DCM remains unknown. This study aimed to investigate PSP's therapeutic effects on DCM in an experimental diabetic mouse model. To induce insulin resistance, mice were fed a high-fat diet for 3 months, followed by intraperitoneal streptozotocin injection to induce slight hyperglycemia and develop DCM. Both DCM and control mice were given PSP orally for 3 weeks. Western blotting was used to detect the protein expressions of protein kinase G, C/EBP homologous protein, glucose-regulated protein 78, phosphodiesterase type 5, protein kinase R-like endoplasmic reticulum (ER) kinase, and phospho-protein kinase R-like endoplasmic reticulum kinase in heart tissue. The results showed a reduction in bodyweight and blood glucose levels in the PSP therapy group compared with DCM group. PSP also improved cardiac function and had a negligible effect on malondialdehyde activity. Furthermore, the findings showed that PSP alleviated ER and oxidative stress observed in DCM mice hearts, leading to the inhibition of cyclic guanosine monophosphate-specific phosphodiesterase type 5 and cardiac cyclic guanosine monophosphate reactivation. Phosphodiesterase type 5 inhibition reduced high-fat diet-induced cardiac dysfunction and decreased ER stress. PSP could effectively protect diabetic myocardium by inhibiting endoplasmic reticulum stress. These findings provide crucial insights into the potential of PSP to ameliorate DCM conditions in diabetic mice by decreasing ER and oxidative stress, and enhancing cyclic guanosine monophosphate protein kinase G signaling.

The protective role of sesame oil against Parkinson's-like disease induced by manganese in rats

Chronic exposure to manganese (Mn) results in motor dysfunction, biochemical and pathological alterations in the brain. Oxidative stress, inflammation, and dysfunction of dopaminergic and GABAergic systems stimulate activating transcription factor-6 (ATF-6) and protein kinase RNA-like ER kinase (PERK) leading to apoptosis. This study aimed to investigate the protective effect of sesame oil (SO) against Mn-induced neurotoxicity. Rats received 25 mg/kg MnCl2 and were concomitantly treated with 2.5, 5, or 8 ml/kg of SO for 5 weeks. Mn–induced motor dysfunction was indicated by significant decreases in the time taken by rats to fall during the rotarod test and in the number of movements observed during the open field test. Also, Mn resulted in neuronal degeneration as observed by histological staining. The striatal levels of lipid peroxides and reduced glutathione (oxidative stress markers), interleukin-6 and tumor necrosis factor-α (inflammatory markers) were significantly elevated. Mn significantly reduced the levels of dopamine and Bcl-2, while GABA, PERK, ATF-6, Bax, and caspase-3 were increased. Interestingly, all SO doses, especially at 8 ml/kg, significantly improved locomotor activity, biochemical deviations and reduced neuronal degeneration. In conclusion, SO may provide potential therapeutic benefits in enhancing motor performance and promoting neuronal survival in individuals highly exposed to Mn.

Discovery of HC-7366: An Orally Bioavailable and Efficacious GCN2 Kinase Activator.

A series of activators of GCN2 (general control nonderepressible 2) kinase have been developed, leading to HC-7366, which has entered the clinic as an antitumor therapy. Optimization resulted in improved permeability compared to that of the original indazole hinge binding scaffold, while maintaining potency at GCN2 and selectivity over PERK (protein kinase RNA-like endoplasmic reticulum kinase). The improved ADME properties of this series led to robust in vivo compound exposure in both rats and mice, allowing HC-7366 to be dosed in xenograft models, demonstrating that activation of the GCN2 pathway by this compound leads to tumor growth inhibition.

Social defeat stress induces liver injury by modulating endoplasmic reticulum stress in C57BL/6J mice

Social defeat stress is associated with endoplasmic reticulum (ER) stress, inflammation and apoptosis. ER stress is thought to contribute to many lifestyle diseases such as liver injury, cardiovascular dysfunction and depression. We investigated the expression of the ER stress markers RNA-dependent protein kinase-like ER kinase (PERK), eukaryotic translation initiation factor 2α (eIF2α) and C/EBP homologous protein (CHOP), as well as inflammatory and apoptotic factors, to assess how social defeat stress induces liver injury. Furthermore, we evaluated the effects of the ER stress inhibitor phenylbutyric acid (PBA) and ER stress inducer thapsigargin (TG) on liver injury. Adult mice were divided into the control, social defeat, social defeat + PBA, TG, PBA and TG + PBA groups. The social defeat and social defeat + PBA groups were simultaneously exposed to social defeat stress for 10 days. The social defeat + PBA, TG, PBA and TG + PBA groups were treated with PBA or TG via intraperitoneal injections. PBA was injected 1 h before the TG injection into the TG + PBA group. Liver samples from six groups of mice were analyzed by histological analysis and western blotting. Social defeat stress promoted ER stress, increased the expression of inflammatory factors and induced apoptosis in the liver of socially defeated mice, which was reversed by PBA. Moreover, ER stress induces TG-induced liver injury by initiating ER stress. Social defeat stress initiates ER stress, promotes the expression of inflammatory and apoptotic factors, and induces liver injury. PBA suppresses liver injury caused by social defeat stress and TG treatment.

Effect of moxibustion combined with intraperitoneal injection of benazepril on endoplasmic reticulum stress-related protein phosphorylation in rats with chronic heart failure.

To observe the effect of moxibustion at "Xinshu"(BL15) and "Feishu"(BL13) combined with intraperitoneal injection of benazepril on cardiac function and phosphorylation of protein kinase R-like endoplasmic reticulum kinase (PERK) and eukaryotic initiation factor 2α (elF2α) proteins in myocardium of rats with chronic heart failure (CHF), so as to explore its potential mechanism underlying improvement of CHF. A total of 42 male SD rats were randomly assigned to blank control (n=10), CHF model (n=7), medication (benazepril, n=8), moxibustion (n=8) and moxibustion+benazepril (n=9) groups, after cardiac ultrasound model identification and elimination of the dead. The CHF model was established by intraperitoneal injection of doxorubicin hydrochloride (DOX), once every week for 6 weeks. Mild moxibustion was applied to bilateral BL15 and BL13 regions for 20 min, once daily for 3 weeks. The rats of the medication group and moxibustion+benazepril group (benazepril was given first, followed by moxibustion) received intraperitoneal injection of benazepril (0.86 mg/kg) solution once daily for 3 weeks . The cardiac ejection fraction (EF) and left ventricular fractional shortening (FS) were measured using echocardiography. Histopathological changes of the cardiac muscle tissue were observed under light microscope after hematoxylin-eosin (H.E.) staining. Serum contents of B-type brain natriuretic peptide (BNP) and angiotensin Ⅱ (AngⅡ) were measured by enzyme-linked immunosorbent assay (ELISA). The expressions of phospho-PERK (p-PERK) and phospho-elF2α (p-elF2α) in the myocardium were detected by Western blot. Compared with the blank control group, the EF and FS of the left cardiac ventricle were significantly decreased (P<0.01), while the contents of serum BNP and AngⅡ, and expression levels of p-PERK and p-eIF2α significantly increased in the model group (P<0.01). In comparison with the model group, both the decreased EF and FS and the increased BNP and AngⅡ contents as well as p-PERK and p-elF2α expression levels were reversed by moxibustion, medication and moxibustion+benazepril (P<0.01). The effects of moxibustion+benazepril were markedly superior to those of simple moxibustion and simple medication in raising the levels of EF and FS rate and in down-regulating the contents of BNP, Ang Ⅱ, levels of p-PERK and p-elF2α (P<0.01, P<0.05). Outcomes of H.E. staining showed irregular arrangement of cardiomyocytes, cell swelling, vacuole and inflammatory infiltration in the model group, which was relatively milder in the 3 treatment groups. The effects of moxibustion+benazepril were superior to those of moxibustion or benazepril. Moxibustion combined with Benazepril can improve the cardiac function in CHF rats, which may be related to its functions in down-regulating the expression levels of myocardial p-PERK and p-elF2α to inhibit endoplasmic reticulum stress response.

Abstract 729: IDO1 inhibition enables IFNγ-elicited responsiveness of pulmonary breast cancer metastases to hypoxia-directed tumoricidal agents

Abstract In mouse models of lung carcinoma and pulmonary breast cancer metastasis, genetic ablation of the tryptophan catabolizing enzyme IDO1 (indoleamine 2,3-dioxygenase 1) resulted in restricted tumor outgrowth associated with attenuated induction of the inflammatory cytokine IL6 (interleukin 6). Utilizing the 4T1 breast cancer metastasis model to further elucidate the mechanistic basis underlying IDO1’s pro-tumor activity in the lungs established that IDO1 acts as an integral determinant of a tumor-promoting inflammatory state that supports neovascular retention. Ido1−/− (nullizygous) mice exhibited impaired pulmonary metastases neovascularization and outgrowth that was driven by IFNγ (interferon gamma) in the absence of IDO1-dependent IL6 production. Hypoxia was likewise elevated, and these stress conditions increased the susceptibility of the metastatic tumor cells to cytotoxicity associated with administration of GSK2656157, an inhibitor of PERK (protein kinase RNA-like ER kinase), which acts as a primary sensor for activating the UPR (unfolded protein response) survival pathway. In WT (wild type) mice with established 4T1 lung metastases, administration of the IDO1 inhibitors epacadostat, navoximod or indoximod each similarly resulted in rapid collapse of the metastatic tumor neovasculature and concomitantly increased intratumoral hypoxia and sensitivity to PERK inhibition. Therefore, while IDO1 inhibition does not directly promote effective tumor cell killing in this context, the disruption of blood supply due to IFNγ-mediated neovascular regression subjects the tumor cells to hypoxic environmental stress, raising the possibility for therapeutic intervention with selectively targeted agents. Combining chemotherapy with immune checkpoint blockade (ICB) is recognized as an effective strategy for treating lung cancer, with first-line therapy for some patients including the alkylating agent carboplatin in conjunction with pembrolizumab. The challenge remains, however, that chemotherapy causes systemic side effects that can negatively impact quality of life and limit efficacy. Evofosfamide is an alkylating prodrug that was designed to be selectively activated in regions of hypoxia, thereby limiting systemic exposure. We report that enhanced metastatic tumor cell killing with evofosfamide can be achieved by co-administration of an IDO1 inhibitor. Elevated intratumoral hypoxia has also previously been linked to increased expression levels of PDL1, a favorable indicator of ICB responsiveness, and we have confirmed the upregulation of PDL1 in 4T1 lung metastases following IDO1 inhibitor administration. Our data in the lung metastasis model support future studies to evaluate whether IDO1 inhibition can be effectively combined with evofosfamide and anti-PD1 treatment to improve therapeutic outcomes. Citation Format: Shih-Chun Shen, Souvik Dey, James B. DuHadaway, Erika Sutanto-Ward, George C. Prendergast, Alexander J. Muller. IDO1 inhibition enables IFNγ-elicited responsiveness of pulmonary breast cancer metastases to hypoxia-directed tumoricidal agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 729.

Ethanol-Induced Hepatic Ferroptosis Is Mediated by PERK-Dependent MAMs Formation: Preventive Role of Quercetin.

Iron deposition is frequently observed in alcoholic liver disease (ALD), which indicates a potential role of ferroptosis in its development. This study aims to explore the effects of quercetin on ferroptosis in ALD and elucidates the underlying mechanism involving the formation of mitochondria-associated endoplasmic reticulum membranes (MAMs) mediated by protein kinase RNA-like endoplasmic reticulum kinase (PERK). C57BL/6J mice are fed either a regular or an ethanol-containing liquid diet (with 28% energy form ethanol) with or without quercetin supplementation (100mgkg-1 BW) for 12 weeks. Ethanol feeding or treatment induced ferroptosis in mice and AML12 cells, which is associated with increased MAMs formation and PERK expression within MAMs. Quercetin attenuates these changes and protects against ethanol-induced liver injury. The antiferroptotic effect of quercetin is abolished by ferroptosis inducers, but mimicked by ferroptosis inhibitors and PERK knockdown. The study demonstrates that PERK structure, rather than its kinase activity (transfected with the K618A site mutation that inhibits kinase activity-ΔK plasmid or protein C terminal knockout-ΔC plasmid of PERK), mediates the enhanced MAMs formation and ferroptosis during the ethanol exposure. Quercetin ameliorates ethanol-induced liver injury by inhibiting ferroptosis via modulating PERK-dependent MAMs formation.

Hypoxia leads to gill endoplasmic reticulum stress and disruption of mitochondrial homeostasis in grass carp (Ctenopharyngodon idella): Mitigation effect of thiamine

Hypoxia in water environment is one of the important problems faced by intensive aquaculture. Under hypoxia stress, the effects of dietary thiamine were investigated on grass carp gill tissue damage and their mechanisms. Six thiamine diets with different thiamine levels (0.22, 0.43, 0.73, 1.03, 1.33 and 1.63 mg/kg) were fed grass carp (Ctenopharyngodon idella) for 63 days. Then, 96-hour hypoxia stress test was conducted. This study described that thiamine enhanced the growth performance of adult grass carp and ameliorated nutritional status of thiamine (pyruvic acid, glucose, lactic acid and transketolase). Additionally, thiamine alleviated the deterioration of blood parameters [glutamic oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), glucose, cortisol, lactic dehydrogenase (LDH), erythrocyte fragility, and red blood cell count (RBC count)] caused by hypoxia stress, and reduced reactive oxygen species (ROS) content and oxidative damage to the gills. In addition, thiamine alleviated endoplasmic reticulum stress in the gills, which may be related to its inhibition of RNA-dependent protein kinase-like ER kinase (PERK)/eukaryotic translation initiation factor-2α (eIF2α)/activating transcription factor4 (ATF4), inositol-requiring enzyme 1 (IRE1)/X-Box binding protein 1 (XBP1) and activating transcription factor 6 (ATF6) pathways. Furthermore, thiamine maintaining mitochondrial dynamics balance was probably related to promoting mitochondrial fusion and inhibiting mitochondrial fission, and inhibiting mitophagy may involve PTEN induced putative kinase 1 (PINK1)/Parkin-dependent pathway and hypoxia-inducible factor (HIF)-Bcl-2 adenovirus E1B 19 kDa interacting protein 3 (BNIP3) pathway. In summary, thiamine alleviated hypoxia stress in fish gills, which may be related to reducing endoplasmic reticulum stress, regulating mitochondrial dynamics balance and reducing mitophagy. The thiamine requirement for optimum growth [percent weight gain (PWG)] of adult grass carp was estimated to be 0.81 mg/kg diet. Based on the index of anti-hypoxia stress (ROS content in gill), the thiamine requirement for adult grass carp was estimated to be 1.32 mg/kg diet.

Therapeutic Potential of Targeting the PERK Signaling Pathway in Ischemic Stroke.

Many pathologic states can lead to the accumulation of unfolded/misfolded proteins in cells. This causes endoplasmic reticulum (ER) stress and triggers the unfolded protein response (UPR), which encompasses three main adaptive branches. One of these UPR branches is mediated by protein kinase RNA-like ER kinase (PERK), an ER stress sensor. The primary consequence of PERK activation is the suppression of global protein synthesis, which reduces ER workload and facilitates the recovery of ER function. Ischemic stroke induces ER stress and activates the UPR. Studies have demonstrated the involvement of the PERK pathway in stroke pathophysiology; however, its role in stroke outcomes requires further clarification. Importantly, considering mounting evidence that supports the therapeutic potential of the PERK pathway in aging-related cognitive decline and neurodegenerative diseases, this pathway may represent a promising therapeutic target in stroke. Therefore, in this review, our aim is to discuss the current understanding of PERK in ischemic stroke, and to summarize pharmacologic tools for translational stroke research that targets PERK and its associated pathways.

Temperature-Dependent Upregulation of Per2 Protein Expression Is Mediated by eIF2α Kinases PERK and PKR through PI3K Activation.

Temperature-dependent translational control of the core clock gene Per2 plays an important role in establishing entrainment of the circadian clock to physiological body temperature cycles. Previously, we found an involvement of the phosphatidylinositol 3-kinase (PI3K) in causing Per2 protein expression in response to a warm temperature shift (WTS) within a physiological range (from 35 to 38.5 °C). However, signaling pathway mediating the Per2 protein expression in response to WTS is only sparsely understood. Additional factor(s) other than PI3K remains unknown. Here we report the identification of eukaryotic initiation factor 2α (eIF2α) kinases, protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK), as a novel mediator of WTS-dependent Per2 protein expression. Canonically, eIF2α has been regarded as a major downstream target of PERK and PKR. However, we found that PERK and PKR mediate WTS response of Per2 in a manner not involving eIF2α. We observed that PERK and PKR serve as an upstream regulator of PI3K rather than eIF2α in the context of WTS-dependent Per2 protein expression. There have been studies reporting PI3K activation occurring depending on PERK and PKR, while its physiological contribution has remained elusive. Our finding therefore not only helps to enrich the knowledge of how WTS affects Per2 protein expression but also extends the region of cellular biology involving the PERK/PKR-mediated PI3K activation to include entrainment-mechanism of the circadian clock.

Endoplasmic reticulum stress and its role in various neurodegenerative diseases

The Endoplasmic reticulum (ER), a critical cellular organelle, maintains cellular homeostasis by regulating calcium levels and orchestrating essential functions such as protein synthesis, folding, and lipid production. A pivotal aspect of ER function is its role in protein quality control. When misfolded proteins accumulate within the ER due to factors like protein folding chaperone dysfunction, toxicity, oxidative stress, or inflammation, it triggers the Unfolded protein response (UPR). The UPR involves the activation of chaperones like calnexin, calreticulin, glucose-regulating protein 78 (GRP78), and Glucose-regulating protein 94 (GRP94), along with oxidoreductases like protein disulphide isomerases (PDIs). Cells employ the Endoplasmic reticulum-associated degradation (ERAD) mechanism to counteract protein misfolding. ERAD disruption causes the detachment of GRP78 from transmembrane proteins, initiating a cascade involving Inositol-requiring kinase/endoribonuclease 1 (IRE1), Activating transcription factor 6 (ATF6), and Protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathways. The accumulation and deposition of misfolded proteins within the cell are hallmarks of numerous neurodegenerative diseases. These aberrant proteins disrupt normal neuronal signalling and contribute to impaired cellular homeostasis, including oxidative stress and compromised protein degradation pathways. In essence, ER stress is defined as the cellular response to the accumulation of misfolded proteins in the endoplasmic reticulum, encompassing a series of signalling pathways and molecular events that aim to restore cellular homeostasis. This comprehensive review explores ER stress and its profound implications for the pathogenesis and progression of neurodegenerative diseases.

Endoplasmic reticulum stress-induced NLRP3 inflammasome activation as a novel mechanism of polystyrene microplastics (PS-MPs)-induced pulmonary inflammation in chickens.

Microplastics (MPs) have attracted growing attention worldwide as an increasingly prevalent environmental pollutant. In addition, chicken meat is currently the most widely consumed kind of poultry in the global market. Consumer demand for chicken is on the rise both at home and abroad. As a result, the safety of chicken raising has also received significant attention. The lungs play an essential role in the physiological activities of chickens, and they are also the most vulnerable organs. Lung injury is difficult to repair after the accumulation of contaminants, and the mortality rate is high, which brings huge economic losses to farmers. The research on the toxicity of MPs has mainly focused on the marine ecosystem, while the mechanisms of toxicity and lung damage in chickens have been poorly studied. Thus, this study explored the effects of exposure to polystyrene microplastics (PS-MPs) at various concentrations for 42 d on chicken lungs. PS-MPs could cause lung pathologies and ultrastructural abnormalities, such as endoplasmic reticulum (ER) swelling, inflammatory cell infiltration, chromatin agglutination, and plasma membrane rupture. Simultaneously, PS-MPs increased the expression of genes related to the heat shock protein family (Hsp60, Hsp70, and Hsp90), ER stress signaling (activating transcription factor 6 (ATF6), ATF4, protein kinase RNA-like ER kinase (PERK), and eukaryotic translation initiation factor 2 subunit α (eIF2α)), pyroptosis-related genes (NOD-‍, LRR- and pyrin domain-containing protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), interleukin-1β (IL-1β), cysteinyl aspartate-specific proteinase 1 (Caspase1), and gasdermin-D (GSDMD)), and the inflammatory signaling pathway (nuclear factor-‍κB (NF-‍κB), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2)). The above results showed that PS-MP exposure could result in lung stress, ER stress, pyroptosis, and inflammation in broilers. Our findings provide new scientific clues for further research on the mechanisms of physical health and toxicology regarding MPs.

Glucose Induces ER Stress Response-Mediated Peritoneal Mesothelial Cell Death.

Peritoneal dialysis (PD) fluid, which contains a high concentration of glucose, is involved in peritoneal damage after long-term use. The mechanisms through which glucose induces damage to the mesothelium have not been clearly elucidated. Although, endoplasmic reticulum (ER) stress response is associated with several diseases, the involvement of ER stress in peritoneal damage has not yet been demonstrated. Primary-cultured rat peritoneal mesothelial cells (RPMCs) and rat PD model were used to investigate the influence of glucose on the peritoneum. Cells treated with glucose were examined for cytotoxicity, induction of apoptosis, and activation of the ER stress pathway. Glucose treatment of RPMCs induced cell death at concentrations higher than 3%. Annexin V positive, that is a feature of apoptosis, occurred in dead cells. Treatment with glucose led to the activation of protein kinase R-like ER kinase (PERK) and eukaryotic translation initiation factor-2α (eIF-2α). Glucose also induced the expression and nuclear translocation of homologous protein C/EBP. Cell death was rescued by the integrated stress response inhibitor, ISRIB, which suppresses the integrated stress response pathway, including ER stress. Glucose in PD fluid induces PERK/eIF-2α-mediated ER stress in RPMCs, resulting in apoptosis. This cellular stress may cause peritoneal damage in patients receiving PD.