Glycogen Synthase Kinase Research Articles

Construction of Multi‐Module RNA Nanoparticles Harboring miRNA, AIE, and CH6 Aptamer for Bone Targeting and Bone Anabolic Therapy

AbstractBone healing remains a major challenge in the treatment of osteoporosis. Effective strategies that simultaneously promote bone formation and inhibit bone resorption are crucial for the treatment of osteoporosis and associated bone defects. MicroRNA (miRNA)‐based approaches aim at simultaneously promoting bone formation and suppressing bone resorption and have therapeutic potential. However, the toxicity of cationic carriers and off‐target effects are two major challenges associated with miRNA delivery. This study establishes a bone‐targeting miRNA delivery system (CH6‐SNA‐26a) that integrates aggregation‐induced emission (AIE), a CH6 aptamer, and miRNAs into a single nanoplatform without any cationic carrier. In this system, an AIE molecule is coupled to miR‐26a to form a core–shell spherical nucleic acid (SNA‐26a). The CH6 aptamer is co‐assembled with the SNA to achieve specific miR‐26a delivery to the bone surface. This aptamer‐functionalized, non‐cationic miR‐26a delivery strategy (CH6‐SNA‐26a) enables both bone‐targeted delivery and high transfection efficiency, ultimately optimizing bone remodeling and calvarial bone healing in an osteoporosis mouse model while limiting adverse effects in non‐skeletal tissues. Mechanistically, the overexpression of CH6‐SNA‐26a in skeletal tissues promotes bone anabolic action by functionally targeting glycogen synthase kinase 3 beta in bone marrow mesenchymal stem cells and cellular communication network 2 in osteoclasts.

Receptor tyrosine kinase-like orphan receptor 1 inhibitor strictinin exhibits anti-cancer properties against highly aggressive androgen-independent prostate cancer

Aim: It is important to identify anti-cancer compounds that can inhibit specific molecular targets to eradicate androgen-receptor negative (ARneg), androgen-independent (AI) prostate cancer, which is an aggressive form of prostate cancer with limited treatment options. The goal of this study was to selectively target prostate cancer cells that have high levels of oncogenic protein Receptor tyrosine kinase-like orphan receptor 1 (ROR1) by using strictinin, a small molecule ROR1 inhibitor. Methods: The methods performed in this study include western blots, methyl thiazolyl tetrazolium (MTT) proliferation assays, phosphatidylserine apoptosis assays, apoptosis flow cytometry (Annexin V, caspase 3/7), migration scratch assays, Boyden chamber invasion assays, and cell cycle flow cytometry. Results: Strictinin was most lethal against PC3 [half-maximal drug inhibitory concentration (IC50) of 277.2 µmol/L], an ARneg-AI cell type that expresses the highest levels of ROR1. Strictinin inhibited ROR1 expression, downstream phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-glycogen synthase kinase 3beta (GSK3β) pro-survival signaling, and epithelial-to-mesenchymal transition markers in PC3 cells. Additionally, strictinin decreased PC3 cell migration and invasion, while increasing S-phase cell cycle arrest. In ARneg-AI DU145 cells, strictinin inhibited ROR1 expression and modulated downstream AKT-GSK3β signaling. Furthermore, strictinin exhibited anti-migratory, anti-invasive, but minimal pro-apoptotic effects in DU145 cells likely due to DU145 having less ROR1 expression in comparison to PC3 cells. Throughout the study, strictinin minimally impacted the phenotype of normal prostatic epithelial cells RWPE-1 (IC50 of 658.5 µmol/L). Strictinin was further identified as synergistic with docetaxel [combination index (CI) = 0.311] and the combination therapy was found to reduce the IC50 of strictinin to 38.71 µmol/L in PC3 cells. Conclusions: ROR1 is an emerging molecular target that can be utilized for treating prostate cancer. The data from this study establishes strictinin as a potential therapeutic agent that targets ARneg-AI prostate cancer with elevated ROR1 expression to reduce the migration, invasion, cell cycle progression, and survival of prostate cancer.

PIM1 drives lipid droplet accumulation to promote proliferation and survival in prostate cancer

Lipid droplets (LDs) are dynamic organelles with a neutral lipid core surrounded by a phospholipid monolayer. Solid tumors exhibit LD accumulation, and it is believed that LDs promote cell survival by providing an energy source during energy deprivation. However, the precise mechanisms controlling LD accumulation and utilization in prostate cancer are not well known. Here, we show peroxisome proliferator-activated receptor α (PPARα) acts downstream of PIM1 kinase to accelerate LD accumulation and promote cell proliferation in prostate cancer. Mechanistically, PIM1 inactivates glycogen synthase kinase 3 beta (GSK3β) via serine 9 phosphorylation. GSK3β inhibition stabilizes PPARα and enhances the transcription of genes linked to peroxisomal biogenesis (PEX3 and PEX5) and LD growth (Tip47). The effects of PIM1 on LD accumulation are abrogated with GW6471, a specific inhibitor for PPARα. Notably, LD accumulation downstream of PIM1 provides a significant survival advantage for prostate cancer cells during nutrient stress, such as glucose depletion. Inhibiting PIM reduces LD accumulation in vivo alongside slow tumor growth and proliferation. Furthermore, TKO mice, lacking PIM isoforms, exhibit suppression in circulating triglycerides. Overall, our findings establish PIM1 as an important regulator of LD accumulation through GSK3β-PPARα signaling axis to promote cell proliferation and survival during nutrient stress.

Artificial Protein Crosstalk with a Molecule that Exchanges Binding Partners

AbstractDrawing inspiration from allosteric signaling enzymes, whose catalytic and regulatory units are non‐covalently linked, we have devised a method to establish unnatural, effector‐mediated enzyme activation within native cells. The feasibility of this approach is demonstrated by introducing a synthetic regulatory unit (sRU) onto glycogen synthase kinase 3 (GSK‐3) through non‐covalent means. Our study reveals that this synthetic regulator mediates an unnatural crosstalk between GSK‐3 and lactate dehydrogenase A (LDHA), whose expression is regulated by cellular oxygen levels. Specifically, with this approach, the constitutively active GSK‐3 is transformed into an activable enzyme, whereas LDHA is repurposed as an unnatural effector protein that controls the activity of the kinase, making it unnaturally dependent on the cell‘s hypoxic response. These findings demonstrate a step toward imitating the function of effector‐regulated cell‐signaling enzymes, which play a key biological role in mediating the response of cells to changes in their environment. In addition, at the proof‐of‐principle level, our results indicate the potential to develop a new class of protein inhibitors whose inhibitory effect in cells is dictated by the cell‘s environment and consequent protein expression profile.

Artificial Protein Crosstalk with a Molecule that Exchanges Binding Partners.

Drawing inspiration from allosteric signaling enzymes, whose catalytic and regulatory units are non-covalently linked, we have devised a method to establish unnatural, effector-mediated enzyme activation within native cells. The feasibility of this approach is demonstrated by introducing a synthetic regulatory unit (sRU) onto glycogen synthase kinase 3 (GSK-3) through non-covalent means. Our study reveals that this synthetic regulator mediates an unnatural crosstalk between GSK-3 and lactate dehydrogenase A (LDHA), whose expression is regulated by cellular oxygen levels. Specifically, with this approach, the constitutively active GSK-3 is transformed into an activable enzyme, whereas LDHA is repurposed as an unnatural effector protein that controls the activity of the kinase, making it unnaturally dependent on the cell's hypoxic response. These findings demonstrate a step toward imitating the function of effector-regulated cell-signaling enzymes, which play a key biological role in mediating the response of cells to changes in their environment. In addition, at the proof-of-principle level, our results indicate the potential to develop a new class of protein inhibitors whose inhibitory effect in cells is dictated by the cell's environment and consequent protein expression profile.

The role of EDIL3 in maintaining cartilage extracellular matrix and inhibiting osteoarthritis development

AimsTherapeutic agents that prevent chondrocyte loss, extracellular matrix (ECM) degradation, and osteoarthritis (OA) progression are required. The expression level of epidermal growth factor (EGF)-like repeats and discoidin I-like domains-containing protein 3 (EDIL3) in damaged human cartilage is significantly higher than in undamaged cartilage. However, the effect of EDIL3 on cartilage is still unknown.MethodsWe used human cartilage plugs (ex vivo) and mice with spontaneous OA (in vivo) to explore whether EDIL3 has a chondroprotective effect by altering OA-related indicators.ResultsEDIL3 protein prevented chondrocyte clustering and maintained chondrocyte number and SOX9 expression in the human cartilage plug. Administration of EDIL3 protein prevented OA progression in STR/ort mice by maintaining the number of chondrocytes in the hyaline cartilage and the number of matrix-producing chondrocytes (MPCs). It reduced the degradation of aggrecan, the expression of matrix metalloproteinase (MMP)-13, the Osteoarthritis Research Society International (OARSI) score, and bone remodelling. It increased the porosity of the subchondral bone plate. Administration of an EDIL3 antibody increased the number of matrix-non-producing chondrocytes (MNCs) in cartilage and exacerbated the serum concentrations of OA-related pro-inflammatory cytokines, including monocyte chemotactic protein-3 (MCP-3), RANTES, interleukin (IL)-17A, IL-22, and GROα. Administration of β1 and β3 integrin agonists (CD98 protein) increased the expression of SOX9 in OA mice. Hence, EDIL3 might activate β1 and β3 integrins for chondroprotection. EDIL3 may also protect cartilage by attenuating the expression of IL-1β-enhanced phosphokinase proteins in chondrocytes, especially glycogen synthase kinase 3 alpha/beta (GSK-3α/β) and phospholipase C gamma 1 (PLC-γ1).ConclusionEDIL3 has a role in maintaining the cartilage ECM and inhibiting the development of OA, making it a potential therapeutic drug for OA.Cite this article: Bone Joint Res 2023;12(12):734–746.

Dendrobium nobile Lindl. alkaloid decreases Tau hyperphosphorylation via regulating PI3K/Akt/GSK-3β pathway in vitro and in vivo

Ethnopharmacological relevantDendrobium is a traditional and precious Chinese medicinal herb. The Compendium of Materia Medica describes its effects as “benefiting intelligence and dispelling shock, lightning the body and extending life”. Dendrobium nobile Lindl. is a precious variety of Dendrobium. Our previous data showed Dendrobium nobile Lindl. alkaloid (DNLA) has significant neuroprotective effects and can improve cognitive dysfunction. However, the specific effects and mechanisms of action of its main active component, DNLA, on cognitive dysfunction caused by Tau hyperphosphorylation, are still unclear. Aim of the researchThis study aimed to determine the effects of DNLA on phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt)/glycogen synthase kinase 3β (GSK-3β) pathway, thus to explore the mechanisms of DNLA to inhibit Tau hyperphosphorylation. Materials and methodsWe used wortmannin (WM) and GF-109203X (GFX)-induced hyperphosphorylation of Tau in N2a cells and rats to detect the protective mechanism of DNLA in vivo and in vitro. In vitro, the effect of modeling method on Tau hyperphosphorylation was screened and verified by Western Blotting (WB), and the regulation of Tau hyperphosphorylation and PI3K/Akt/GSK-3β pathway by different concentrations of DNLA was detected by WB. In vivo, MWM was used to detect the effect of DNLA on model rats, and then Nissl staining was used to detect the loss of neurons. Finally, WB was used to detect the regulation of Tau hyperphosphorylation and PI3K/Akt/GSK-3β pathway by different concentrations of DNLA. ResultsDNLA could rescue the abnormal PI3K/Akt/GSK-3β pathway and reverse the hyperphosphorylation of Tau induced by WM and GFX in N2a cells. Furthermore, DNLA improved the learning and memory of WM and GFX-induced model rats. Moreover, DNLA regulated PI3K/Akt/GSK-3β pathway and reduced the p-Tau and neuronal damage in the hippocampus of model rats. ConclusionDNLA may be a promising candidate for reducing hyperphosphorylation of Tau.

Dopamine depletion alters neuroplasticity-related signaling in the rat hippocampus

ABSTRACT Dopamine (DA) plays a significant role in regulating hippocampal function, particularly in modulating synaptic plasticity. Despite this, a comprehensive understanding of the molecular mechanisms involved in neuroplasticity-related signaling influenced by DA remains incomplete. This study aimed to elucidate the changes in the expression of key molecules related to hippocampal neuroplasticity following DA depletion in rats. To induce DA depletion, unilateral striatal infusions of 6-hydroxydopamine (6-OHDA) were administered to adult Sprague-Dawley rats. The subsequent loss of nigrostriatal DAergic signaling in these 6-OHDA-lesioned rats was confirmed using an apomorphine-induced rotation test at 4 weeks post-infusion and by assessing the expression levels of tyrosine hydroxylase (TH) through immunohistochemistry and western blotting at 7 weeks post-infusion. A decrease in DAergic signaling, evidenced by reduced TH-positive immunoreactivity, was also noted in the ipsilateral hippocampus of the lesioned rats. Interestingly, 6-OHDA infusion led to increased phosphorylation of pivotal hippocampal plasticity-related proteins, including extracellular signal-regulated kinase (ERK), protein kinase B (Akt), glycogen synthase kinase 3β (GSK3β), and cAMP response element-binding protein (CREB), in the ipsilateral hippocampus 7 weeks following the infusion. To extend these findings, in vitro experiments were conducted on primary hippocampal neurons exposed to DA and/or the active D1/D2 DA receptor antagonist, flupentixol (Flux). DA inhibited the constitutive phosphorylation of ERK, Akt, GSK3, and CREB, while Flux restored these phosphorylation levels. Taken together, these findings indicate that DA depletion triggers an increase in plasticity-related signaling in the hippocampus, suggesting a possible compensatory mechanism that promotes activity-independent neuroplasticity following DA depletion.

Neuroprotection and Mechanism of Gas-miR36-5p from Gastrodia elata in an Alzheimer's Disease Model by Regulating Glycogen Synthase Kinase-3β.

Alzheimer's disease (AD) is currently the most common neurodegenerative disease. Glycogen synthase kinase 3β (GSK-3β) is a pivotal factor in AD pathogenesis. Recent research has demonstrated that plant miRNAs exert cross-kingdom regulation on the target genes in animals. Gastrodia elata (G. elata) is a valuable traditional Chinese medicine that has significant pharmacological activity against diseases of the central nervous system (CNS). Our previous studies have indicated that G. elata-specific miRNA plays a cross-kingdom regulatory role for the NF-κB signaling pathway in mice. In this study, further bioinformatics analysis suggested that Gas-miR36-5p targets GSK-3β. Through western blot, RT-qPCR, and assessments of T-AOC, SOD, and MDA levels, Gas-miR36-5p demonstrated its neuroprotective effects in an AD cell model. Furthermore, Gas-miR36-5p was detected in the murine brain tissues. The results of the Morris water maze test and western blot analysis provided positive evidence for reversing the learning deficits and hyperphosphorylation of Tau in AD mice, elucidating significant neuroprotective effects in an AD model following G. elata RNA administration. Our research emphasizes Gas-miR36-5p as a novel G. elata-specific miRNA with neuroprotective properties in Alzheimer's disease by targeting GSK-3β. Consequently, our findings provide valuable insights into the cross-kingdom regulatory mechanisms underlying G. elata-specific miRNA, presenting a novel perspective for the treatment of Alzheimer's disease.

Understanding Glycogen Synthase Kinase-3: A Novel Avenue for Alzheimer's Disease.

Alzheimer's Disease (AD) is the most prevalent form of age-related dementia. Even though a century has passed since the discovery of AD, the exact cause of the disease still remains unknown. As a result, this poses a major hindrance in developing effective therapies for treating AD. Glycogen synthase kinase-3 (GSK-3) is one of the kinases that has been investigated recently as a potential therapeutic target for the treatment of AD. It is also known as human tau protein kinase and is a proline-directed serine-threonine kinase. Since dysregulation of this kinase affects all the major characteristic featuresof the disease, such as tau phosphorylation, amyloid formation, memory, and synaptic function, it is thought to be a major playerin the pathogenesis of AD. In this review, we present the most recent information on the role of this kinase in the onset and progression of AD, as well as significant findings that identify GSK-3 as one of the most important targets for AD therapy. We further discuss the potential of treating AD by targeting GSK-3 and give an overview of the ongoing studies aimed at developing GSK-3 inhibitors in preclinical and clinical investigations.

Identification of New GSK3β Inhibitors through a Consensus Machine Learning-Based Virtual Screening.

Glycogen synthase kinase-3 beta (GSK3β) is a serine/threonine kinase that plays key roles in glycogen metabolism, Wnt/β-catenin signaling cascade, synaptic modulation, and multiple autophagy-related signaling pathways. GSK3β is an attractive target for drug discovery since its aberrant activity is involved in the development of neurodegenerative diseases such as Alzheimer's and Parkinson's disease. In the present study, multiple machine learning models aimed at identifying novel GSK3β inhibitors were developed and evaluated for their predictive reliability. The most powerful models were combined in a consensus approach, which was used to screen about 2 million commercial compounds. Our consensus machine learning-based virtual screening led to the identification of compounds G1 and G4, which showed inhibitory activity against GSK3β in the low-micromolar and sub-micromolar range, respectively. These results demonstrated the reliability of our virtual screening approach. Moreover, docking and molecular dynamics simulation studies were employed for predicting reliable binding modes for G1 and G4, which represent two valuable starting points for future hit-to-lead and lead optimization studies.

FGF21 Improves Glycolipid Metabolism in Rainbow Trout (Oncorhynchus mykiss) Fed a High-Carbohydrate Diet by Inhibiting Inflammatory Responses and Activating Autophagy.

In this study, the coding region of rainbow trout fgf21 was cloned and sequenced to synthesize a recombinant protein (rFGF21) and investigate its potential role in improving glycolipid metabolism. Acute injection of rFGF21 into rainbow trout effectively reduced serum glucose levels. To investigate the effect of rFGF21 on high-carbohydrate diet (HCD)-induced metabolic disorders in rainbow trout, a 31-day feeding experiment was conducted. At the end of the third week, fish were injected with either PBS or rFGF21. The results showed that the final body weight (FBW) significantly increased in rainbow trout on an HCD (P < 0.05), but there were potential risks including disturbances in glycolipid metabolism and increased inflammatory responses. However, these effects were altered by rFGF21 treatment. In addition, rFGF21 promotes glucose uptake by increasing the phosphorylation levels of AKT (protein kinase B) and GSK3β (glycogen synthase kinase 3β), increasing hepatic glycogen, thereby lowering serum glucose. Notably, the rFGF21 did not exacerbate the inflammatory response but downregulated the expression of inflammatory factors. Interestingly, the activation of autophagy and the AMPK pathway may contribute to the positive effect of rFGF21, where rFGF21 injection significantly increased the levels of LC3I/II protein and phosphorylate AMPKα (P < 0.05).

Glycogen synthase kinase 3 signaling in neural regeneration in vivo.

Glycogen synthase kinase 3 (GSK3) signaling plays important and broad roles in regulating neural development in vitro and in vivo. Here, we reviewed recent findings of GSK3-regulated axon regeneration in vivo in both the peripheral and central nervous systems and discussed a few controversial findings in the field. Overall, current evidence indicates that GSK3β signaling serves as an important downstream mediator of the PI3K-AKT pathway to regulate axon regeneration in parallel with the mTORC1 pathway. Specifically, the mTORC1 pathway supports axon regeneration mainly through its role in regulating cap-dependent protein translation, whereas GSK3β signaling might be involved in regulating N6-methyladenosine mRNA methylation-mediated, cap-independent protein translation. In addition, GSK3 signaling also plays a key role in reshaping the neuronal transcriptomic landscape during neural regeneration. Finally, we proposed some research directions to further elucidate the molecular mechanisms underlying the regulatory function of GSK3 signaling and discover novel GSK3 signaling-related therapeutic targets. Together, we hope to provide an updated and insightful overview of how GSK3 signaling regulates neural regeneration in vivo.

Hair Follicle Development of Rex Rabbits Is Regulated Seasonally by Wnt10b/β-Catenin, TGFβ-BMP, IGF1, and EGF Signaling Pathways

This experiment was conducted to study the effects of different skinning seasons on the fur quality and hair follicle development of Rex rabbits. A total of 80,150-day-old Rex rabbits were slaughtered on 15 July 2022 (summer), 15 October 2022 (autumn), 15 January 2023 (winter), and 15 April 2023 (spring) in Shandong Province (10 males and 10 females in each season). The results show that the skin weight, skin area, skin thickness, and hair follicle density of the Rex rabbits (at 150 days of age) were lower in summer than in winter (p &lt; 0.05). Moreover, the coat length was shorter in summer than in spring, autumn, and winter (p &lt; 0.05). The shoulder fat weight, perirenal fat weight, and perigastric fat weight of the Rex rabbits in winter were higher than those in summer (p &lt; 0.05). Furthermore, the leptin levels in serum were higher in winter than in summer in the Rex rabbits (p &lt; 0.05). In terms of serum biochemistry, the glucose levels were higher in autumn and winter than in spring and summer (p &lt; 0.05). The cholesterol, high-density lipoprotein cholesterol (HDL), and low-density lipoprotein cholesterol (LDL) in summer had higher values than in winter in the Rex rabbits (p &lt; 0.05). In winter, the expression of the Wnt10b, catenin beta 1 (CTNNB1), glycogen synthase kinase 3 beta (GSK3β), insulin like growth factor I (IGF-I), Type I insulin-like growth factor receptor (IGF-IR), and epidermal growth factor (EGF) genes was higher (p &lt; 0.05), and the expression of the dickkopf-1 (DDK1), transforming growth factor beta 1 (TGFβ-1), bone morphogenetic protein 2 (BMP2), and bone morphogenetic protein 4 (BMP4) genes was lower than in summer (p &lt; 0.05). In summer, the heat shock 70 kDa protein (HSP70) expression and CTNNB1 protein phosphorylation levels in skin tissue were higher than in spring, autumn, and winter (p &lt; 0.05). In winter, Wnt10b protein expression was higher (p &lt; 0.05), and GSK-3β protein phosphorylation levels were lower than in spring, autumn, and winter (p &lt; 0.05). These results show that the skinning season can affect the production performance and hair follicle development of Rex rabbits. Compared with other seasons, the quality of skin from rabbits slaughtered in winter is better. Seasons may regulate hair follicle development via the Wnt10b/β-catenin, TGFβ-BMP, IGF1, and EGF signaling pathways in Rex rabbits.

Evaluation of Helianthus annuus Flower Extract on Biomarkers of Alzheimer’s Disease

AbstractBackgroundAlzheimer’s disease (AD) is a slowly progressive disease of the brain that is characterized by the impairment of memory and eventually by disturbances in reasoning, planning, language, and perception. Amyloid β‐peptide (Aβ) is main source of oxidative stress in AD because it can acquire a free‐radical state that contributes to its toxic effects. Aβ‐induced cytotoxicity is caused by intracellular accumulation ofreactive oxygen species, which leads to lipid peroxidation and cell death. Helianthus annuus flower is known for its nutritional value and having many medicinal benefits as well. Helianthus annuus flower contain many medicinally essential phytoconstituents, such as phenol, flavonoid, and carotenoid.MethodSTZ administration through route such as intracebroventricular or intraperitoneal injection produces reduced cognition and increased cerebral aggregated Aβ fragments, total tau protein, and Aβ deposits. These changes were accompanied with decreased glycogen synthase kinase (GSK‐3) alpha/beta ratio (phosphorylated/total) in the brain. Administration of STZ in a rodent’s brain has been shown to produce neuroinflammation, oxidative stress and biochemical alterations, which is considered to be a valid experimental model of the early pathophysiological changes in neurodegenerative disease.ResultTwo doses (100 and 200 mg/kg) of the flower extract from Helianthus annuus were given over the course of seven days. The standard agent utilised was piracetam (120 mg/kg). Oral administration of Helianthus annuus flower extract resulted in a considerable reduction in A1‐40 58 0.21 (100mg/kg) and 53 0.79 (200mg/kg) and A1‐42 17 0.89 (100mg/kg) and 15 1.02 (200mg/kg). Helianthus annuus flower extract increased the levels of the following brain antioxidant enzymes at 100 mg/kg and 200 mg/kg, respectively: CAT (15.5 2.8** and 14.0 1.12**), SOD (13.5 1.4** and 18.3 1.7**, GSH (203.3 15.3** and 218.0 13.5**), TBARS (200.3 7.3** and 208.0 11.0**). AChE activity was significantly decreased when Helianthus annuus flower extract was taken orally at doses of 100 mg/kg and 200 mg/kg, respectively.ConclusionData of the present study revealed that Helianthus annuus flower significantly decreased the brain Amyloid β 1‐42 and AChE activity in rats. Data revealed that administration of both Helianthus annuus flower (100mg/kg) and Helianthus annuus flower (200mg/kg) increased the activity of SOD and CAT.

Phosphorylation of USP27X by GSK3β maintains the stability and oncogenic functions of CBX2

Chromobox protein homolog 2 (CBX2) exerts a multifaceted impact on the progression of aggressive cancers. The proteasome-dependent pathway is crucial for modulating CBX2 regulation, while the specific regulatory roles and mechanisms of deubiquitinating enzymes targeting CBX2 remain poorly understood. Mass spectrometry analysis identified ubiquitin-specific peptidase 27X (USP27X) as a deubiquitinating enzyme that targets CBX2. Overexpression of USP27X significantly enhances CBX2 levels by promoting deubiquitination, while deficiency of USP27X leads to CBX2 degradation, thereby inhibiting tumorigenesis. Furthermore, it has been revealed that glycogen synthase kinase 3 beta (GSK3β) can directly bind to and phosphorylate USP27X, thereby enhancing the interaction between USP27X and CBX2 and leading to further stabilization of the CBX2 protein. Clinically, the co-expression of high levels of USP27X and CBX2 in breast cancer tissues is indicative of a poor prognosis for patients with this disease. These findings collectively underscore the critical regulatory role played by USP27X in modulating CBX2, thereby establishing the GSK3β-USP27X-CBX2 axis as a pivotal driver of malignant progression in breast cancer.

Microfluidic Organoid Cultures Derived from Pancreatic Cancer Biopsies for Personalized Testing of Chemotherapy and Immunotherapy.

Patient-derived cancer organoids (PDOs) hold considerable promise for personalizing therapy selection and improving patient outcomes. However, it is challenging to generate PDOs in sufficient numbers to test therapies in standard culture platforms. This challenge is particularly acute for pancreatic ductal adenocarcinoma (PDAC) where most patients are diagnosed at an advanced stage with non-resectable tumors and where patient tissue is in the form of needle biopsies. Here the development and characterization of microfluidic devices for testing therapies using a limited amount of tissue or PDOs available from PDAC biopsies is described. It is demonstrated that microfluidic PDOs are phenotypically and genotypically similar to the gold-standard Matrigel organoids with the advantages of 1) spheroid uniformity, 2) minimal cell number requirement, and 3) not relying on Matrigel. The utility of microfluidic PDOs is proven by testing PDO responses to several chemotherapies, including an inhibitor of glycogen synthase kinase (GSKI). In addition, microfluidic organoid cultures are used to test effectiveness of immunotherapy comprised of NK cells in combination with a novel biologic. In summary, our microfluidic device offers considerable benefits for personalizing oncology based on cancer biopsies and may, in the future, be developed into a companion diagnostic for chemotherapy or immunotherapy treatments.

Network pharmacology‑based investigation of potential targets of triptonodiol acting on non-small-cell lung cancer

BackgroundTriptonodiol is a very promising antitumor drug candidate extracted from the Chinese herbal remedy Tripterygium wilfordii Hook. F., and related studies are underway.MethodsTo explore the mechanism of triptonodiol for lung cancer treatment, we used network pharmacology, molecular docking, and ultimately protein validation. Gene ontology (GO) analysis and Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment analysis were performed through the David database. Molecular docking was performed using PyMoL2.3.0 and AutoDock Vina software. After screening, the major targets of triptonodiol were identified for the treatment of lung cancer. Target networks were established, Protein–protein interaction (PPI) network topology was analyzed, then KEGG pathway enrichment analysis was performed. Useful proteins were screened by survival analysis, and Western blot analysis was performed.ResultsTriptonodiol may regulate cell proliferation, drug resistance, metastasis, anti-apoptosis, etc., by acting on glycogen synthase kinase 3 beta (GSK3B), protein kinase C (PKC), p21-activated kinase (PAK), and other processes. KEGG pathway enrichment analysis showed that these targets were associated with tumor, erythroblastic oncogene B (ErbB) signaling, protein phosphorylation, kinase activity, etc. Molecular docking showed that the target protein GSK has good binding activity to the main active component of triptonodiol. The protein abundance of GSK3B was significantly downregulated in non-small-cell lung cancer cells H1299 and A549 treated with triptonodiol for 24 h.ConclusionThe cellular-level studies combined with network pharmacology and molecular docking approaches provide new ideas for the development and therapeutic application of triptonodiol, and identify it as a potential GSK inhibitor.

Cordycepin inhibits kidney injury by regulating GSK-3β-mediated Nrf2 activation.

We explored the role and mechanism of cordycepin (COR) in inhibiting kidney injury. A mouse model of kidney injury was established using cisplatin (CDDP), and the kidney function, histopathology, and ferroptosis indices in mice were detected after intervening with COR. The targets of COR-ferroptosis-kidney injury were analyzed by network pharmacology, based on which the association between glycogen synthase kinase-3 beta (GSK-3β) and COR was determined. HK-2 cells were cultured in vitro and treated separately with ferroptosis inducers erastin and CDDP. After the COR intervention, the level of ferroptosis was monitored. In vitro experiments found that COR could inhibit ferroptosis and CDDP-induced kidney injury. Network pharmacological analysis revealed that GSK-3β was the target of COR. After inhibiting GSK-3β expression, COR could not further inhibit the occurrence of ferroptosis. In vitro results also indicated that COR could inhibit ferroptosis in HK-2 cells. According to our findings, COR can ameliorate CDDP-induced kidney injury through GSK-3β-mediated ferroptosis signaling. We identify new pharmacological effect and target for COR, the major component of Cordyceps sinensis.

The early dorsal signal in vertebrate embryos requires endolysosomal membrane trafficking.

Fertilization triggers cytoplasmic movements in the frog egg that lead in mysterious ways to the stabilization of β-catenin on the dorsal side of the embryo. The novel Huluwa (Hwa) transmembrane protein, identified in China, is translated specifically in the dorsal side, acting as an egg cytoplasmic determinant essential for β-catenin stabilization. The Wnt signaling pathway requires macropinocytosis and the sequestration inside multivesicular bodies (MVBs, the precursors of endolysosomes) of Axin1 and Glycogen Synthase Kinase 3 (GSK3) that normally destroy β-catenin. In Xenopus, the Wnt-like activity of GSK3 inhibitors and of Hwa mRNA can be blocked by brief treatment with inhibitors of membrane trafficking or lysosomes at the 32-cell stage. In dorsal blastomeres, lysosomal cathepsin is activated and intriguing MVBs surrounded by electron dense vesicles are formed at the 64-cell stage. We conclude that membrane trafficking and lysosomal activity are critically important for the earliest asymmetries in vertebrate embryonic development.