Search Results (180)

Inhibitors of the ubiquitin–proteasome system increase proteotoxic stress and have achieved clinical success for multiple myeloma but not for solid cancers such as hepatocellular carcinoma. Our objective is to identify a combination with proteasome inhibitors that enhances proteotoxic stress and apoptotic cell death in hepatocellular carcinoma but with less toxicity to non-cancer cells. We found that rencofilstat, a pan-cyclophilin inhibitor, combined with ixazomib, a proteasome inhibitor, increased apoptotic cell death in hepatocellular carcinoma but not in umbilical vein or dermal fibroblast non-cancer cells. We then analyzed the effects of rencofilstat + ixazomib on XBP1s and PERK, critical factors in the unfolded protein response used by cells to survive proteotoxic stress. Rencofilstat + ixazomib maintained higher expression of XBP1s and genetic models suggested that XBP1s was a pro-survival protein early and pro-death protein at later times. Simultaneously, decreased PERK expression prevented the block in protein synthesis via phospho-eIF2α and likely further amplified proteotoxic stress. Rencofilstat + ixazomib did not have effects on XBP1s or PERK in non-cancer cells. Further genetic experiments revealed the pro-survival roles for cyclophilin A and B in mediating rencofilstat + ixazomib-induced cell death. In the Hep3B xenograft model, rencofilstat + ixazomib significantly inhibited tumor volumes/weights without general toxicity. We conclude that rencofilstat + ixazomib amplified proteotoxic stress in hepatocellular carcinoma past a threshold pro-survival pathways could not tolerate, whereas non-cancer cells were less affected. Full article

The growth of tumor cells is accompanied by an increased rate of endoplasmic reticulum stress (ERS), the accumulation of misfolded proteins, and the activation of a network of adaptive signaling pathways known as the unfolded protein response (UPR). Although the UPR is an adaptive reaction aiming to restore ER proteostasis, prolonged and severe ERS leads to cell death. Taking into account that the components of the ERS/UPR machinery in cancers of different types can be overexpressed or downregulated, both the induction of excessive ERS and suppression of UPR have been proposed as therapeutic strategies to sensitize cells to conventional chemotherapy. This narrative review presents a several examples of using natural and synthetic compounds that can either induce persistent ERS by selectively blocking ER Ca²⁺ pumps (SERCA) to disrupt ER Ca²⁺ homeostasis, or altering the activity of UPR chaperones and sensors (GRP78, PERK, IRE1α, and ATF6) to impair protein degradation signaling. The molecular alterations induced by miscellaneous inhibitors of ERS/UPR effectors are described as well. These agents showed promising therapeutic effects as a part of combination therapy in preclinical experimental settings; however, the number of clinical trials is still limited, while their results are inconsistent. Multiple side effects, high toxicity to normal cells, or poor bioavailability also hampers their clinical application. Since the pharmacological modulation of ERS/UPR is a valuable approach to sensitize cancer cells to standard chemotherapy, the search for more selective agents with better stability and low toxicity, as well as the development of more efficient delivery systems that can increase their therapeutic specificity, are highly required goals for future studies. Full article

Background and Objectives: Obesity is linked to liver cancer through metabolic mechanisms and can promote tumor growth through metabolic impairment, decreased lipid metabolism, and interference of the energy balance in the liver. NAMPT is an enzyme expressed in the liver and is involved in the progression of tumors in obesogenic environments, while iNAMPT is known to be the rate-limiting enzyme in the synthesis of NAD, an essential coenzyme involved in ATP synthesis which promotes a pro-growth environment in the context of obesity. Because iNAMPT and cellular energetics, a hallmark of cancer, play an important role in liver cancer progression, it has become a target for cancer therapies focused on inhibiting its functions. The objective of this study was to determine the contribution of NAD biosynthesis in obesity-associated liver cancer progression. Methods: Cell culture studies were conducted with serum from male mice randomized to diet-induced obesity (OB) or control (CR) ± FK866 (iNAMPT inhibitor) in SNU, HepG2 human liver cancer cells, and Hepa 1-6 liver murine cells. Protein analysis of pAkt and pErk was performed via immunoblot. Cytotoxicity, reactive oxygen species (ROS), cell viability, and invasion were also measured in the cells. For the mouse model, the C57BL/6J male mice were randomized to the DIO or CR group. At 21 weeks of age, the mice were injected subcutaneously with Hepa 1-6 liver cancer cells. At 23 weeks, the mice received an I.P. injection of FK866 (30 mg/kg) for 2 weeks. The tumor and mouse weights were measured. Results: The cells exposed to OB sera showed increased proliferation, lactate dehydrogenase (LDH) secretion, ROS, and invasion. FK866 decreased proliferation, LDH secretion, ROS, and invasion for all liver cancer cells. The cells exposed to CR sera and OB + FK866 resulted in more LDH, suggesting increased apoptosis compared with OB sera. The OB sera increased phosphorylation of Akt, which was suppressed by FK866 compared with the OB group. In liver cancer cells, physiological and cellular signaling is affected differently when inhibiting NAD biosynthesis in an in vitro model of obesity and liver cancer. In vivo, the diet-induced obese (DIO) mice weighed significantly more than the mice fed a control diet. In addition, 70% of the DIO mice developed tumors, compared with 20% of the CR mice, and had tumors with greater volumes and weights. NAD inhibition blocked obesity-induced tumor growth. Conclusions: In this study, we demonstrate that inhibition of iNAMPT resulted in suppression of tumor growth in the context of obesity. Identifying pre-clinical strategies to reverse the impact of obesity on liver cancer progression is important due to the strong increased risk of liver cancer and its poor prognosis. Future translational research studies can be built from this pre-clinical foundational research. Full article

In sheep production, due to the limitations of breeding conditions, the uteri of ewes are often infected with bacteria, resulting in the failure of embryo implantation or loss, causing huge losses to the sheep industry. Therefore, in this study, by using RT-qPCR, Western blot, and immunofluorescence, we investigated the effects of LPS infusion on the immune microenvironment and endometrial receptivity, which play an important role in the process of embryo implantation in ruminants, during the three critical periods of embryo implantation in sheep. The results showed that LPS infusion at day 12, day 16, and day 20 significantly increased the expression of Th1 cytokines (TNF-α, IL-1β, IL-8, IL-6), while significantly decreasing the expression of Th2 cytokines (IL-4 and IL-10) and disrupting the expression of implantation factors, such as ITGB3, ITGB5, VEGF, and LIF, in the endometrial tissues of sheep. Additionally, the protein expression level of TLR4 and the phosphorylation level of ERK were significantly elevated at day 12, day 16, and day 20 after LPS infusion, suggesting that LPS may impair endometrial receptivity through the TLR4/ERK pathway. Validation was conducted in a receptive model of sEECs using TLR4 and ERK phosphorylation inhibitors. Compared with the LPS group, TLR4 and ERK phosphorylation inhibitors significantly reduced the expression of TLR4 and p-ERK, down-regulated Th1 cytokines, up-regulated Th2 cytokines, and alleviated the disruption of genes for attachment. Treatment with 50 μM PTE can significantly alleviate the abnormal expression of implantation genes caused by LPS, and its mechanism may be related to the regulation of the ERK signaling pathway. Full article

Background/Objectives: Neurofibromatosis type 1 (NF1) is a prevalent inherited disorder, with approximately 50% of affected individuals developing plexiform neurofibromas (PNFs), which can progress to highly aggressive malignant peripheral nerve sheath tumors (MPNSTs). While selumetinib is FDA-approved for PNFs, its efficacy in MPNSTs is limited and associated with dose-limiting toxicities. NF1 deficiency drives tumorigenesis and alters immune dynamics via RAS hyperactivation. Given the substantial macrophage infiltration in NF1 lesions and its association with disease progression, we hypothesized that targeting tumor-promoting immune cells with the retinoid X receptor (RXR) agonist MSU-42011 could be an alternative therapeutic strategy, as it has shown promise in KRAS-driven cancers by decreasing pERK levels and reducing tumor-promoting immune cells. Methods: We examined the effects of MSU-42011 and selumetinib, alone and in combination, on NF1-deficient cells and in a syngeneic MPNST model. Results: In vivo, the combination of MSU-42011 and selumetinib significantly reduced tumor growth, pERK levels, and tumor-promoting macrophages and increased activated CD8⁺ T cells in syngeneic MPNST models. In NF1-deficient cells, MSU-42011 or selumetinib reduced pERK levels, with combination treatment achieving greater reductions. Conditioned media (CM) from NF1-deficient cells increased the protein and mRNA levels of several cytokines and chemokines in human THP1 cells and bone marrow-derived macrophages (BMDMs). MSU-42011 and selumetinib, alone or in combination, partially reversed this induction. Conclusions: These findings suggest RXR agonists may have therapeutic potential against NF1, and their combination with MEK inhibitors could represent a promising strategy for NF1-associated tumors. Further studies are needed to validate these results and assess their translational relevance. Full article

This study investigated the mechanism underlying Paeonol’s therapeutic efficacy against neuropathic pain. GSE158892 dataset data were used to conduct a scRNA-seq analysis. In cell experiments, Schwann cells and macrophages were utilized to examine pain pathogenesis using specific inhibitors. Thirty-two SD rats were randomly divided into four groups: sham, chronic constriction injury (CCI), ibuprofen, and Paeonol. Behavioral tests combined with ELISA, PCR, western blot, immunohistochemistry, and immunofluorescence analyses were conducted. CellChat analysis demonstrated that, following peripheral nerve injury, Schwann cells secreted IL-34, which interacted with CSF1R on macrophages, leading to the infiltration and activation of macrophages. Paeonol reduced IL-34 production by Schwann cells induced with LPS. Conditioned medium from LPS-stimulated Schwann cells treated with Paeonol did not cause macrophage proliferation or migration, activation of the CSF1 pathway, or ROS production. In CCI rats, Paeonol alleviated mechanical and cold hyperalgesia, while reducing the production of serum inflammatory mediators. Additionally, Paeonol decreased the expression levels of IL-34, CSF1R, phosphorylated ERK (p-ERK), phosphorylated NF-κB (p-NF-κB), and components of the NLRP3 inflammasome in the dorsal root ganglia of CCI rats. Conclusion: Alleviation of neuropathic pain by Paeonol treatment may be achieved by inhibiting the IL-34–CSF1R interaction, suppressing Schwann cell–macrophage interactions, and reducing DRG neuroinflammation. Full article

Myocardial ischemia–reperfusion injury (MI/R) is a negative and adverse cardiovascular outcome following myocardial ischemia, cardiac surgery, or circulatory arrest. Prolyl endopeptidase (PREP) appears to be involved in inflammatory responses, so it could be a possible therapeutic target for counteracting ischemia injury. This study aimed to investigate the role of PREP inhibitor, KYP-2047 (4-phenylbutanoyl-l-prolyl-2(S)-cyanopyrolidine), in the modulation of molecular and biochemical processes involved in MI/R. MI/R was induced through coronary artery occlusion (15 min), followed by reperfusion (2 h). KYP-2047 was intraperitoneally administrated at doses of 2.5 mg/kg and 5 mg/kg 24 h before the surgical procedures. The hearts were removed and processed for analysis. KYP-2047 treatment limited ischemic myocardial-induced histological damage and neutrophil accumulation, limiting inflammation, fibrosis, and apoptosis processes. Additionally, KYP-2047 was able to modulate p-38 and p-ERK expression, suggesting an improving role in recovering cardiac function. These findings highlighted the protective effects of KYP-2047 pretreatment in MI/R injury, suggesting PREP as a potential target therapy for the pathogenesis of MI/R. Although the molecular mechanisms underlying the action of KYP-2047 are still to be explored, these results suggested that the regulation of NF-κB, apoptosis, and MAPK pathways by KYP-2047 treatment could preventatively limit the damage caused by MI/R. Full article

Background: Oncogenic KRAS mutations are nearly ubiquitous in pancreatic ductal adenocarcinoma (PDAC), yet therapeutic attempts to target KRAS, as well as downstream MAPK pathway effectors, have shown limited clinical success. While KRAS canonically drives MAPK signaling via RAF-MEK-ERK, it is also known to play a role in PI3K-AKT signaling. Methods: Our therapeutic study targeted the PI3K pathway with the drug Omipalisib (p110α/β/δ/γ and mTORC1/2 inhibitor) in combination with two different MAPK pathway inhibitors: Trametinib (MEK1/2 inhibitor) or SHP099-HCL (SHP099; SHP2 inhibitor). Western blot analysis demonstrated that the application of Trametinib or SHP099 alone selectively blocked ERK phosphorylation (pERK) but failed to suppress phosphorylated AKT (pAKT). Conversely, Omipalisib alone successfully inhibited pAKT but failed to suppress pERK. Therefore, we hypothesized that a combination therapeutic comprised of Omipalisib with either Trametinib or SHP099 would inhibit two prominent mitogenic pathways, MAPK and PI3K-AKT, and effectively suppress PDAC growth. Results: In vitro studies demonstrated that, in several cell lines, both Omipalisib/Trametinib and Omipalisib/SHP099 combination therapeutic strategies were more effective than treatment with each drug individually at reducing proliferation, colony formation, and cell migration compared to vehicle controls. In vivo oral administration of combined Omipalisib/Trametinib treatment was significantly more effective than Omipalisib/SHP099 in reducing implanted tumor growth, and the Omipalisib/Trametinib treatment more effectively reduced tumor progression and prolonged survival in an aggressive genetically engineered mouse model of PDAC than either Omipalisib or Trametinib alone. Conclusions: Altogether, our data support a rationale for a dual treatment strategy targeting both PI3K and MAPK pathways in pancreatic cancers. Full article

The role of the mitogen-activated protein kinase (MAPK) pathway in choroidal neovascularization (CNV) remains unclear. This study investigates the involvement of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 pathways in CNV development, as well as the therapeutic potential of sprouty 2 (SPRY2), an MAPK inhibitor, in a laser-induced mouse model. The expressions of ERK, JNK, and p38 proteins were analyzed using Western blotting and immunostaining. Immunofluorescence imaging revealed increased p-ERK and p-JNK expression in the retina, retinal pigment epithelium (RPE), and choroid up to day 7. Co-immunostaining showed p-ERK colocalized with CD31, CD11b, F4/80, cytokeratin, and GFAP in the retina, while p-JNK and p-p38 were associated with angiogenesis and inflammation throughout the retina and choroid. Compared to aflibercept, SPRY2 administration significantly inhibited CNV lesions, endothelial proliferation, fibrosis, and apoptosis, while better-preserving RPE integrity. SPRY2-treated mice showed a stronger reduction in CNV-related inflammation, epithelial–mesenchymal transition, and photoreceptor apoptosis. These results highlight the MAPK pathway’s role in CNV pathogenesis, with ERK primarily mediating Müller cell gliosis and JNK, contributing to angiogenesis and inflammation. SPRY2 effectively suppressed CNV lesions, supporting its potential as a therapeutic target for CNV treatment via MAPK pathway modulation. Full article

The necroptosis of granulosa cells has been proven to be one of the important triggers of follicular atresia, which is an important cause of reduced reproductive capacity in cows. The rapid growth of granulosa cells is accompanied by endoplasmic reticulum stress (ERS), leading to granulosa cell death. However, the link between ERS and necroptosis, as well as its mechanism in bovine granulosa cells is still unclear. Itaconic acid is an endogenous anti-inflammatory and antioxidant small-molecule compound that can alleviate ERS. Therefore, the aim of the current study is to evaluate the effect of ERS on necroptosis and investigate the ameliorative effect of itaconic acid against ERS-induced necroptosis in granulosa cells. Bovine granulosa cells were treated with tunicamycin (Tm) to induce ERS. After the addition of the necroptosis inhibitor Nec-1 and the detection of the necroptosis inducer acetylcholinesterase (AChE), flow cytometry, transmission electron microscopy, and mass spectrometry were used to analyze the expression of itaconic acid and IRG1 in the granulosa cells. In addition, the role of the PERK pathway downstream of ERS in ERS-induced necroptosis was also investigated. We report here that ERS can induce necroptosis in granulosa cells. Itaconic acid supplementation significantly attenuates the effect of ERS-induced damage. In summary, this research provides a scientific basis and a drug reference for treating follicular atresia and improving bovine reproductive capacity. Full article

Ferroptosis, a regulated form of cell death characterized by lipid peroxidation and iron accumulation, is increasingly recognized for its role in disease pathogenesis. The unfolded protein response (UPR) has been implicated in both endoplasmic reticulum (ER) stress and ferroptosis-mediated cell fate decisions; yet, the specific mechanism remains poorly understood. In this study, we demonstrated that ER stress induced by tunicamycin and ferroptosis triggered by erastin both activate the UPR, leading to the induction of ferroptotic cell death. This cell death was mitigated by the application of chemical chaperones and a ferroptosis inhibitor. Among the three arms of the UPR, the PERK-eIF2α-ATF4 signaling axis was identified as a crucial mediator in this process. Mechanistically, the ATF4-driven induction of DDIT4 plays a pivotal role, facilitating ferroptosis via the inhibition of the mTORC1 pathway. Furthermore, acetaminophen (APAP)-induced hepatotoxicity was investigated as a model of eIF2α-ATF4-mediated ferroptosis. Our findings reveal that the inhibition of eIF2α-ATF4 or ferroptosis protects against APAP-induced liver damage, underscoring the therapeutic potential of targeting these pathways. Overall, this study not only clarifies the intricate role of the PERK-eIF2α-ATF4 axis in ER-stress-and erastin-induced ferroptosis but also extends these findings to a clinically relevant model, providing a foundation for potential therapeutic interventions in conditions characterized by dysregulated ferroptosis and ER stress. Full article

Benzo(a)pyrene (B(a)P) and pyrene, the most prominent subtypes of polycyclic aromatic hydrocarbons (PAHs), contaminate environments as organic pollutants. They adversely affect body systems, including degeneration of the central nervous system. This study investigated the in vitro toxic effects of B(a)P and pyrene on proliferation, endoplasmic reticulum (ER) stress induction, and autophagy in human astrocytes using U-87 MG human astrocytoma cells as a model. Both B(a)P and pyrene were toxic to U-87 MG cells in a concentration-dependent manner. Astrocytic proliferation was interfered with, enhancing S-phase cell cycle arrest. B(a)P promoted the presence of autophagic vesicles and the expression of autophagic markers LC3, beclin-1, and p62, suggesting activated autophagy. B(a)P enhanced the expression of ER stress markers BiP, PERK, and IRE1. ER stress appeared to be correlated with autophagy induction, as demonstrated by experiments using chloroquine, an autophagy inhibitor. Pyrene enhanced the expression of autophagic markers and ER stress primarily via PERK activation, although autophagic vesicles were not observed. The study demonstrates that B(a)P enhances ER stress-mediated autophagy more evidently than pyrene and affected toxicity to astrocytes. These results provide a basis for understanding the toxic effects of the main PAH substances affecting astrocytes. Full article

Background: Crohn’s disease (CD) is an inflammatory bowel disease marked by an abnormal immune response and excessive pro-inflammatory cytokines, leading to impaired protein processing and endoplasmic reticulum (ER) stress. This stress, caused by the accumulation of misfolded proteins, triggers the unfolded protein response (UPR) through IRE1/Xbp-1, PERK/eIF2α, and ATF6 pathways, which are linked to intestinal inflammation. This study aimed to investigate ER stress in CD patients’ intestinal mucosa and evaluate phenylbutyrate (PBA) as an ER stress inhibitor. Methods: Colon biopsies from CD patients and controls were cultured under five conditions, including 4-PBA treatments. Real-time PCR, cytokine level, and immunohistochemistry were performed. Results: Immunohistochemistry revealed that ER stress was activated in CD patients’ intestinal epithelial cells and lamina propria cells. PERK/eIF2α, but not IRE1/Xbp-1 or ATF6, was upregulated in CD patients compared to controls. UPR-related genes (STC2, CALR, HSPA5, HSP90B1) were also elevated in CD patients. PBA treatment significantly reduced ER stress and UPR markers while decreasing apoptotic markers like DDIT3. Pro-inflammatory cytokines, such as IL-1β, IL-6, IL-17, TNF- α, and sCD40L, were significantly reduced after PBA treatment. Conclusion: ER stress and UPR pathways are activated in CD colonic mucosa, and PBA reduces these markers, suggesting potential therapeutic benefits for CD-related inflammation. Full article

Chemotherapy-induced peripheral neuropathy and the accompanying affective disorders are serious side effects, and their resolution is not guaranteed. Oxidative stress and elevated levels of Nod-like receptor protein 3 (NLRP3) have been detected in the peripheral and central nervous systems of animals with neuropathic pain provoked by several antineoplastic drugs, such as paclitaxel (PTX). Several studies have further indicated that NLRP3 inflammasome inhibition could be an approach for treating chronic pain, but its impact on the anxiodepressive-like behaviors and memory deficits related to PTX-provoked neuropathy has not yet been investigated. MCC950 is a potent and specific inhibitor of the NLRP3 pathway that acts through inhibiting NLRP3 activation and inflammasome formation. We hypothesized that the administration of MCC950 could alleviate the affective and cognitive disorders accompanying PTX-provoked neuropathy. Using male C57BL/6 mice, we assessed the effects of MCC950 on the mechanical and thermal allodynia, anxiodepressive-like behavior, and memory deficits incited by this taxane. The results indicated that the intraperitoneal administration of 10 mg/kg of MCC950 twice daily for three consecutive days fully reversed the PTX-induced mechanical and thermal allodynia. This treatment also completely attenuated the anxiolytic (p < 0.004) and depressive-like behaviors (p < 0.022) and memory deficits (novel object recognition test; p < 0.0018) incited by PTX. These actions were mainly achieved through blocking NLRP3 inflammasome activation in the sciatic nerve, amygdala, and hippocampus, and oxidative stress in the amygdala and hippocampus. MCC950 also normalized the p-ERK 1/2 overexpression in the sciatic nerve and apoptotic responses in the sciatic nerve and the amygdala. This study suggests that MCC950 might be a promising treatment for PTX-induced mental illnesses and neuropathy. Full article

The betacoronavirus genus contains five of the seven human coronaviruses, making it a particularly critical area of research to prepare for future viral emergence. We utilized three human betacoronaviruses, one from each subgenus—HCoV-OC43 (embecovirus), SARS-CoV-2 (sarbecovirus), and MERS-CoV (merbecovirus)—, to study betacoronavirus interactions with the PKR-like ER kinase (PERK) pathway of the integrated stress response (ISR)/unfolded protein response (UPR). The PERK pathway becomes activated by an abundance of unfolded proteins within the endoplasmic reticulum (ER), leading to phosphorylation of eIF2α and translational attenuation. We demonstrate that MERS-CoV, HCoV-OC43, and SARS-CoV-2 all activate PERK and induce responses downstream of p-eIF2α, while only SARS-CoV-2 induces detectable p-eIF2α during infection. Using a small molecule inhibitor of eIF2α dephosphorylation, we provide evidence that MERS-CoV and HCoV-OC43 maximize viral replication through p-eIF2α dephosphorylation. Interestingly, genetic ablation of growth arrest and DNA damage-inducible protein (GADD34) expression, an inducible protein phosphatase 1 (PP1)-interacting partner targeting eIF2α for dephosphorylation, did not significantly alter HCoV-OC43 or SARS-CoV-2 replication, while siRNA knockdown of the constitutive PP1 partner, constitutive repressor of eIF2α phosphorylation (CReP), dramatically reduced HCoV-OC43 replication. Combining GADD34 knockout with CReP knockdown had the maximum impact on HCoV-OC43 replication, while SARS-CoV-2 replication was unaffected. Overall, we conclude that eIF2α dephosphorylation is critical for efficient protein production and replication during MERS-CoV and HCoV-OC43 infection. SARS-CoV-2, however, appears to be insensitive to p-eIF2α and, during infection, may even downregulate dephosphorylation to limit host translation. Full article