Inhibition Of Glycogen Synthase Kinase-3β Research Articles

Differential mechanisms involved in RG-7388 and Nutlin-3 induced cell death in SJSA-1 osteosarcoma cells

Targeted therapy is becoming the mainstay of cancer treatment due to reduced side effects and enhanced tumor attack. In the last few decades, Murine Double Minute 2 (MDM2) protein has become one of the targets for developing cancer therapies. Blocking MDM2-p53 interaction has long been considered to offer a broad range of advantages during cancer treatment. In this study, we are reporting the differential mechanism of cell death induced by the two small-molecule inhibitors, named RG-7388 and Nutlin-3, that are specific for MDM2 in SJSA-1 Osteosarcoma cells (OS). Mechanistically, RG-7388 was able to enhance the phosphorylation of Mcl-1, which appears to significantly enhance its degradation, thereby relieving the pro-apoptotic protein Bak to execute the apoptosis mechanism. It was noted that the untreated SJSA-1 cells showed an accumulation of Mcl-1 levels, which was decreased following RG-7388 and to a lesser extent by Nutlin-3 and GSK-3β (glycogen synthase kinase 3β) inhibitor treatments. Additionally, we noted that CHIR-99021 (GSK-3β inhibitor) blocked the cytotoxicity exerted by RG-7388 on SJSA-1 cells by decreasing Bak levels. Since Bak is an important pro-apoptotic protein, we hypothesized that phosphorylation of Mcl-1 by GSK-3β could negatively impact the Mcl-1/Bak dimerization and relieve Bak to trigger the loss of mitochondrial membrane potential and thereby initiates apoptosis. We also observed that inhibition of GSK-3β mediated reduction in Bak levels had a protective effect on the mitochondrial membrane integrity, and thus, caused a significant inhibition of the caspase-3 activity and PARP cleavage. Nutlin-3, on the other hand, appears to increase the levels of Bax, leading to the inactivation of Bcl-2, consequently loss of mitochondrial membrane potential and release of Cytochrome c (Cyt c) and elevation of Apaf-1 triggering apoptosis. Thus, to the best of our knowledge, this is the first study that delineates the differences in the molecular mechanism involving two MDM2 inhibitors triggering apoptosis through parallel pathways in SJSA-1 cells. This study further opens new avenues for the use of RG-7388 in treating osteosarcomas that often becomes resistant to chemotherapy due to Bcl-2 overexpression.

Abstract LB-236: Design, synthesis, and assessment of anticancer properties of pyrazolopyrimidine derivatives as Glycogen Synthase Kinase-3β inhibitors

Abstract Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase with a crucial role in several key cellular functions including tumor cell proliferation and survival. Yet, GSK-3β has emerged as an attractive target for the development of anti-cancer agents. Several small-molecule have been developed as GSK-3β inhibitors. Amongst, several pyrazolopyrimidines based ATP-competitive inhibitors were identified as promising GSK-3β inhibitors. In this work, several pyrazolo[3,4-d]pyrimidine derivatives were designed, synthesized and assessed for their potential anticancer activity. The new compounds are categorized under two templates, Template A and Template B. In these templates two different linkers with varying lengths and electronic properties were used to link additional aromatic ring systems to the core pyrazolopyrimidine scaffold. The aromatic ring systems were designed with different electronic and bonding properties to test their GSK-3β inhibitory activity and consequently their anticancer properties. All compounds under investigation showed GSK-3β inhibitory properties with IC50's ranging from 0.17- 1.01 µM compared to Tideglusib (standard GSK-3β inhibitor) which showed IC50 of 0.22 µM. Furthermore, the compounds were tested for their potential cytotoxicity against two breast cancer cells (MCF-7 and MDA-MB-231) and lung cancer cells (A549). Only one compound from Template B which possesses the longer hydrazide linker and 2,3-dihydroxy phenyl aromatic ring (compound 27), showed considerable cytotoxic effects against MCF-7, MDA-MB-231 and A549 cells (IC50's of 2.4, 2.4 and 2.3 µM, respectively). Compounds 21, 24, 25 and 26 showed weaker cytotoxic effects against cell lines under investigation (IC50's ranged from 29 to 93 µM). DNA content flow-cytometry analysis was undertaken to evaluate the potential effects of compound 27 on the cell cycle distribution of cell lines under investigation. Compound 27 induced significant antiproliferative effect and accumulation of cells in G0/G1-phase after 24 h of exposure. Longer exposure (48 h) to compound 27 resulted in cell cycle arrest in G2/M-phase. Additional assessment for programmed/non-programed cell death using annexin-V/FITC and acridine orange staining coupled with flow-cytometric analysis showed the ability of compound 27 to induce apoptosis as well as autophagy in all cells under investigation. In conclusion, compound 27 represents a promising GSK-3β inhibitor with potential anticancer properties via the induction of cell cycle arrest with subsequent cellular stress and induction of apoptosis and autophagy. Citation Format: Ahmed T. Negmeldin, Mohammed K. AbdElhameid, Dalia Y. Alsaeedy, Fatiha Hammed, Rasha M. Allam, Walid El-Sayed, Ahmed M. Al-Abd. Design, synthesis, and assessment of anticancer properties of pyrazolopyrimidine derivatives as Glycogen Synthase Kinase-3β inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-236.

Unleashing β-catenin with a new anti-Alzheimer drug for bone tissue regeneration

The Wnt/β-catenin signaling pathway is critical for bone differentiation and regeneration. Tideglusib, a selective FDA approved glycogen synthase kinase-3β (GSK-3β) inhibitor, has been shown to promote dentine formation, but its effect on bone has not been examined. Our objective was to study the effect of localized Tideglusib administration on bone repair. Bone healing between Tideglusib treated and control mice was analysed at 7, 14 and 28 days postoperative (PO) with microCT, dynamic histomorphometry and immunohistology. There was a local downregulation of GSK-3β in Tideglusib animals, resulting in a significant increase in the amount of new bone formation with both enhanced cortical bone bridging and medullary bone deposition. The bone formation in the Tideglusib group was characterized by early osteoblast differentiation with down-regulation of GSK-3β at day 7 and 14, and higher accumulation of active β-catenin at day 14. Here, for the first time, we show a positive effect of Tideglusib on bone formation through the inactivation of GSK-3β. Furthermore, the findings suggest that Tideglusib does not interfere with precursor cell recruitment and commitment, contrary to other GSK-3β antagonists such as lithium chloride. Taken together, the results indicate that Tideglusib could be used directly at a fracture site during the initial intraoperative internal fixation without the need for further surgery, injection or drug delivery system. This FDA-approved drug may be useful in the future for the prevention of non-union in patients presenting with a high risk for fracture-healing.

GSK-3β inhibition by curcumin mitigates amyloidogenesis via TFEB activation and anti-oxidative activity in human neuroblastoma cells

The translocation of transcription factor EB (TFEB) to the nucleus plays a pivotal role in the regulation of basic cellular processes, such as lysosome biogenesis and autophagy. Autophagy is an intracellular degradation system that delivers cytoplasmic constituents to the lysosome, which is important in maintaining cellular homeostasis during environmental stress. Furthermore, oxidative stress is a critical cause for the progression of neurodegenerative diseases. Curcumin has anti-oxidative and anti-inflammatory activities, and is expected to have potential therapeutic effects in various diseases. In this study, we demonstrated that curcumin regulated TFEB export signalling via inhibition of glycogen synthase kinase-3β (GSK-3β); GSK-3β was inactivated by curcumin, leading to reduced phosphorylation of TFEB. We further showed that H2O2-induced oxidative stress was reduced by curcumin via the Nrf2/HO-1 pathway in human neuroblastoma cells. In addition, we showed that curcumin induced the degradation of amyloidogenic proteins, including amyloid-β precursor protein and α-synuclein, through the TFEB-autophagy/lysosomal pathway. In conclusion, curcumin regulates autophagy by controlling TFEB through the inhibition of GSK-3β, and increases antioxidant gene expression in human neuroblastoma cells. These results contribute to the development of novel cellular therapies for neurodegenerative diseases.

Melatonin Induces Melanogenesis in Human SK-MEL-1 Melanoma Cells Involving Glycogen Synthase Kinase-3 and Reactive Oxygen Species.

Melatonin is present in all living organisms where it displays a diversity of physiological functions. Attenuation of melanogenesis by melatonin has been reported in some mammals and also in rodent melanoma cells. However, melatonin may also stimulate melanogenesis in human melanoma cells through mechanisms that have not yet been revealed. Using the human melanoma cells SK-MEL-1 as a model, an increase in both tyrosinase activity and melanin was already observed at 24 h after melatonin treatment with maximal levels of both being detected at 72 h. This effect was associated with the induction in the expression of the enzymes involved in the synthesis of melanin. In this scenario, glycogen synthase kinase-3β seems to play a significant function since melatonin decreased its phosphorylation and preincubation with specific inhibitors of this protein kinase (lithium or BIO) reduced the expression and activity of tyrosinase. Blocking of PI3K/AKT pathway stimulated melanogenesis and the effect was suppressed by the inhibitors of glycogen synthase kinase-3β. Although melatonin is a recognized antioxidant, we found that it stimulates reactive oxygen species generation in SK-MEL-1 cells. These chemical species seem to be an important signal in activating the melanogenic process since the antioxidants N-acetyl-l-cysteine and glutathione decreased both the level and activity of tyrosinase stimulated by melatonin. Our results support the view that regulation of melanogenesis involves a cross-talk between several signaling pathways.

Discovery of a novel small molecule PT109 with multi-targeted effects against Alzheimer's disease in vitro and in vivo

Alzheimer's disease (AD), which is characterized by impairment of cognitive functions, is a chronic neurodegenerative disease that mainly affects the elderly. Currently available anti-AD drugs can only offer limited symptom-relieving effects. “One-compound-Multitargeted Strategy” have been recognized as the promising way to win the war against AD. Herein we report a potential anti-AD agent PT109 with multi-functions. First, an 81-kinase screening was carried out and results showed that PT109 potently inhibited c-Jun N-terminal kinases and Serum and glucocorticoid-inducible kinase 1, which are the important signaling molecules involved in neurogenesis, neuroprotection and neuroinflammation and mildly inhibit glycogen synthase kinase-3β as well as protein kinase C gamma, both are involved in AD pathological processes. In addition, in vitro studies of immunofluorescent staining and Western blot showed that PT109 might promote the neurogenesis of C17.2 cells and induce synaptogenesis in primary cultured rat hippocampal neurons. We detected and confirmed the neuroprotective effect of PT109 in cultured HT22 cells by MTT assay, dehydrogenase assay, glutathione assay and reactive oxygen species assay. Furthermore, the results of Western blot, ELISA assay and immunofluorescent staining indicated that PT109 attenuated lipopolysaccharide-induced inflammation in BV2 cells and primary astrocytes. The results of Morris water maze and Step-through test indicated that PT109 improved the spatial learning ability in APP/PS1 mice. More importantly, the in vivo pharmacokinetic parameters indicated that PT109 had better medicinal properties. Taken together, our findings suggest that PT109 may be a promising candidate for treating AD through multiple targets although further studies are ought to be conducted.

Induced Fetal Human Muscle Stem Cells with High Therapeutic Potential in a Mouse Muscular Dystrophy Model.

SummaryDuchenne muscular dystrophy (DMD) is a progressive and fatal muscle-wasting disease caused by DYSTROPHIN deficiency. Cell therapy using muscle stem cells (MuSCs) is a potential cure. Here, we report a differentiation method to generate fetal MuSCs from human induced pluripotent stem cells (iPSCs) by monitoring MYF5 expression. Gene expression profiling indicated that MYF5-positive cells in the late stage of differentiation have fetal MuSC characteristics, while MYF5-positive cells in the early stage of differentiation have early myogenic progenitor characteristics. Moreover, late-stage MYF5-positive cells demonstrated good muscle regeneration potential and produced DYSTROPHIN in vivo after transplantation into DMD model mice, resulting in muscle function recovery. The engrafted cells also generated PAX7-positive MuSC-like cells under the basal lamina of DYSTROPHIN-positive fibers. These findings suggest that MYF5-positive fetal MuSCs induced in the late stage of iPSC differentiation have cell therapy potential for DMD.

The effect of lithium and lithium-loaded hyaluronic acid hydrogel applications on nerve regeneration and recovery of motor functions in peripheral nerve injury

Functional recovery rates after peripheral nerve injury are quite poor. Studies have focused on the organization of axonal development and the alteration of the microenvironment of the regenerated nerve. It is a frequently used repair method to place a tube to the incision area with tubulization technique and to try increasing the effect by filling the tube with various substances. Hyaluronic acid is a natural polysaccharide, and since it is biodegradable, it makes an ideal biomaterial in peripheral nerve healing. Lithium chloride (LiCl), a Glycogen synthase kinase 3β (GSK3β) inhibitor, is also frequently used in the treatment of bipolar and depressive disorders, but it has strong neuroprotective and antiinflammatory effects. In this study, peripheral nerve injury was induced on the rat sciatic nerve and silicon conduit was placed to this area and the effects of application of LiCl (2.5, 5 and 15 mEq doses) and LiCl (15 mEq)-loaded hyaluronic acid hydrogel in the conduit on peripheral nerve regeneration and improvement of motor functions were investigated. Sciatic nerve index (SFI) was measured at 2nd, 4th, 8th, and 12th weeks for each animal. Histological, microscopic, and morphometrical analyzes were evaluated at the end of the experiment (12th weeks). Our results indicate that topically applied lithium has positive effect after peripheral nerve injury and especially 5 mEq LiCl can increase nerve regeneration in rats. Further studies are needed to discuss the morphological, molecular and mechanical potential of treatment application.

Inhibition of GSK3β and RIP1K Attenuates Glial Scar Formation Induced by Ischemic Stroke via Reduction of Inflammatory Cytokine Production.

In the chronic phase following ischemic stroke, glial scars can prevent axonal regeneration and the intensification of inflammation. The protective effect of inhibition of glycogen synthase kinase-3β (GSK3β) or receptor-interacting protein 1 kinase (RIP1K) on ischemic stroke has been previously reported. The current study examined the effects of RIP1K and GSK3β on ischemic stroke-induced glial scar formation. To investigate this, we used an in vivo model of ischemic stroke based on middle cerebral artery occlusion for 90 min followed by reperfusion for 7 d, and an in vitro model in primary cultured astrocytes involving oxygen and glucose deprivation for 6 h followed by reoxygenation for 24 h. Both in vivo and in vitro, we found that SB216763, a GSK3β inhibitor, and necrostatin-1 (Nec-1), a RIP1K inhibitor, decreased levels of glial scar markers, including glial fibrillary acidic protein (GFAP), neurocan, and phosphacan. SB216763 and Nec-1 also decreased levels of inflammatory related cytokines, including interleukin-6 (IL-6), interleukin-1 β (IL-1β), and tumor necrosis factor-α (TNF-α). However, only Nec-1 increased the level of interleukin-1 receptor antagonist. Concurrent neutralization of TNF-α, IL-1β, and IL-6 with their antibodies provided better reduction in oxygen and glucose deprivation-induced increases in scar markers than obtained with separate use of each antibody. Further investigations showed that SB216763 reduced the levels of necroptosis-related proteins, including RIP1K, p-RIP1K, RIP3K, p-RIP3K, mixed lineage kinase domain-like protein (MLKL), and p-MLKL, while Nec-1 decreased the expression of p-GSK3β. Compared with Nec-1 (10 μM) and SB216763 (1 μM) alone, Nec-1 and SB216763 in combination reduced levels of GFAP, neurocan, and inflammatory-related cytokines. In conclusion, inhibition of GSK3β or RIP1K reduced glial scar formation induced by ischemic stroke. The underlying mechanisms might be at least, partially related to reducing levels of inflammatory-related cytokines and to blocking an interaction between GSK3β- and RIP1K-mediated pathways.

Upregulation of Glutaredoxin 2 alleviates oxygen-glucose deprivation/reoxygenation-induced apoptosis and ROS production in neurons by enhancing Nrf2 signaling via modulation of GSK-3β

Glutaredoxin 2 (GRX2) is an antioxidative protein that exerts a key role in various pathological processes. However, whether GRX2 participates in modulating the oxidative stress during cerebral ischemia/reperfusion, injury is undermined. This study aimed to determine the potential role of GRX2 in regulating oxidative stress in cultured neurons induced by oxygen-glucose deprivation/reoxygenation (OGD/R), a cellular model for study of cerebral ischemia/reperfusion injury in vitro. Here, we showed that GRX2 expression was decreased in neurons subjected to OGD/R exposure. The upregulation of GRX2 markedly improved the viability of OGD/R-exposed neurons and caused a marked reduction in OGD/R-induced apoptosis and reactive oxygen species (ROS) production. On the contrary, depletion of GRX2 exacerbated the OGD/R-induced apoptosis and ROS production in cultured neurons. Moreover, GRX2 upregulation increased nuclear expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and enhanced the activation of Nrf2/ARE signaling associated with modulation of glycogen synthase kinase-3β (GSK-3β) inhibition. Notably, inhibition of Nrf2 markedly abrogated GRX2-mediated protection against OGD/R-induced apoptosis and oxidative stress. Overall, these findings elucidate that GRX2 plays an essential role in regulating the protection against OGD/R-induced apoptosis and oxidative stress in neurons associated with its ability to enhance the activation of Nrf2 via modulation of GSK-3β. Our study indicates that GRX2 may play a key role in modulating neuronal apoptosis and oxidative stress induced by cerebral ischemia/reperfusion injury.

Curcumin ameliorates CKD-induced mitochondrial dysfunction and oxidative stress through inhibiting GSK-3β activity

Curcumin has been reported to attenuate muscle atrophy. However, the underling mechanism remains unclear. The aim of this study was to investigate whether curcumin could improve chronic kidney disease (CKD)-induced muscle atrophy and mitochondrial dysfunction by inhibiting glycogen synthase kinase-3β (GSK-3β) activity. The sham and CKD mice were fed either a control diet or an identical diet containing 0.04% curcumin for 12 weeks. The C2C12 myotubes were treated with H2O2 in the presence or absence of curcumin. In addition, wild-type and muscle-specific GSK-3β knockout (KO) CKD model mice were made by 5/6 nephrectomy, and the sham was regarded as control. Curcumin could exert beneficial effects, including weight maintenance and improved muscle function, increased mitochondrial biogenesis, alleviated mitochondrial dysfunction by increasing adenosine triphosphate levels, activities of mitochondrial electron transport chain complexes and basal mitochondrial respiration and suppressing mitochondrial membrane potential. In addition, curcumin modulated redox homeostasis by increasing antioxidant activity and suppressed mitochondrial oxidative stress. Moreover, the protective effects of curcumin had been found to be mediated via inhibiting GSK-3β activity in vitro and in vivo. Importantly, GSK-3β KO contributed to improved mitochondrial function, attenuated mitochondrial oxidative damage and augmented mitochondrial biogenesis in muscle of CKD. Overall, this study suggested that curcumin alleviated CKD-induced mitochondrial oxidative damage and mitochondrial dysfunction via inhibiting GSK-3β activity in skeletal muscle.

Cytotoxic Activity of Aplykurodin A Isolated From Aplysia kurodai against AXIN1-Mutated Hepatocellular Carcinoma Cells by Promoting Oncogenic β-Catenin Degradation.

Dysregulation of the Wnt/β-catenin signaling pathway is involved in the development of human hepatocellular carcinoma and has thus emerged as a therapeutic target for this malignant tumor. In this study, we employed sensitive cell-based assays to identify aplykurodin A isolated from Aplysia kurodai as an antagonist of Wnt/β-catenin signaling. Aplykurodin A inhibited β-catenin responsive transcription, which was stimulated by a Wnt3a-conditioned medium or a glycogen synthase kinase 3β inhibitor by accelerating intracellular β-catenin degradation. Aplykurodin A downregulated the level of oncogenic β-catenin and decreased the expression of β-catenin-dependent gene, leading to inhibition of human hepatoma Hep3B and SNU475 cell proliferation. Moreover, apoptosis and autophagy were elicited by aplykurodin A, as indicated by an increase the number of Annexin V-FITC-stained cells and the formation of microtubule-associated protein 1 light chain 3 puncta, respectively, in Hep3B and SNU475 cells. Our findings suggest that aplykurodin A provides a novel therapeutic strategy for human hepatocellular carcinoma via stimulation of oncogenic β-catenin degradation.

Methcathinone Rapidly Decreases Dopamine Transporter Function: Role of Calcium and Protein Kinase C

Methcathinone (MCAT) is a synthetic cathinone that causes both persistent striatal dopaminergic as well as striatal and hippocampal serotonergic deficits. It has been demonstrated that the rapid effects of similar stimulants on dopamine (DA) transporter (DAT) function contribute to persistent monoaminergic deficits. Thus, the effect of MCAT on DAT was assessed. Results revealed that MCAT self‐administration (SA) decreased striatal [3H]DA uptake, as assessed ex vivo in synaptosomes prepared from male rats 1 h after the final MCAT SA session. In vitro co‐incubation of synaptosomes with MCAT (1, 10, 100 μM) at 37°C likewise decreased striatal [3H]DA uptake. There was no effect of in vitro co‐incubation with MCAT at 4°C, suggesting that decreases in [3H]DA uptake are not due to residual drug. These effects were not limited to DAT as MCAT SA decreased hippocampal [3H]serotonin uptake. Further, in vitro co‐incubation of synaptosomes with MCAT decreased both striatal and hippocampal [3H]serotonin uptake. The in vitro effect on DAT function was attenuated by co‐incubation with the cell‐permeable calcium chelator, 1,2‐bis (2‐aminophenoxy) ethane‐N, N, N, N‐tetraacetic acid acetoxymethyl ester (BAPTA‐AM), as well as with the structurally related non‐specific protein kinase C (PKC) inhibitors bisindolylmaleimide I and Ro‐31‐7549. In contrast, this in vitro decrease in DAT function was not attenuated by co‐incubation with other non‐specific PKC inhibitors (NPC‐15437 and Go6976), nor with the PKC‐β inhibitor ruboxistaurin. Neither co‐incubation with the Ca2+/calmodulin‐dependent protein kinase II inhibitor, KN‐93, nor the glycogen synthase kinase 3β inhibitor, SB‐216763, attenuated the MCAT‐induced decrease in [3H]DA uptake. Taken together, these results suggest that MCAT self‐administration and in vitro MCAT exposure have comparable effects to investigator administered MCAT on DAT and SERT function in the striatum and hippocampus; further, in vitro data suggest that the MCAT‐induced effect on DAT is dependent upon calcium and some, but not all, PKC isoforms.Support or Funding InformationR01‐DA039145

Glycogen Synthase Kinase 3β Inhibitor Delivered by Chitosan Nanocapsules Promotes Safe, Fast, and Efficient Activation of Wnt Signaling In Vivo

The Wnt-β-catenin signaling is an evolutionarily conserved pathway with a prominent role in different biological processes such as stem cell renewal, cell proliferation, and differentiation. Wnt signaling dysfunctions have been associated with developmental and neurological diseases as well as formation and progression of tumors. Nanomedicine may provide safe and efficient drug delivery systems offering breakthrough innovation in targeting Wnt signaling. The natural polymer chitosan represents an excellent candidate for delivery platforms, showing interesting biophysical properties such as high biocompatibility and mucoadhesive properties. In this study, oily core chitosan nanocapsules were designed with the aim to deliver the Wnt signaling agonist alsterpaullone in the model organism Hydra vulgaris. Chitosan nanocapsules show negligible impact on animal morphology, without affecting the viability. Nile red-loaded nanocapsules reveal fast and efficient intracellular delivery of the fluorescent cargo. Short incubations with alsterpaullone-loaded nanocapsules ensure a more effective activation of Wnt signaling with respect to the same concentrations of the free drug. Altogether, these data provide evidence that chitosan nanocapsules may represent a very promising strategy for future therapies targeting the diseases associated with canonical Wnt signaling.

Efficient differentiation and purification of human induced pluripotent stem cell-derived endothelial progenitor cells and expansion with the use of inhibitors of ROCK, TGF-β, and GSK3β

Endothelial cells (ECs) and endothelial progenitor cells (EPCs) play crucial roles in maintaining vascular health and homeostasis. Both cell types have been used in regenerative therapy as well as in various in vitro models; however, the properties of primary human ECs and EPCs are dissimilar owing to differences in genetic backgrounds and sampling techniques. Human induced pluripotent stem cells (hiPSCs) are an alternative cell source of ECs and EPCs. However, owing to the low purity of differentiated cells from hiPSCs, purification via an antigen–antibody reaction, which damages the cells, is indispensable. Besides, owing to limited expandability, it is difficult to produce these cells in large numbers. Here we report the development of relatively simple differentiation and purification methods for hiPSC-derived EPCs (iEPCs). Furthermore, we discovered that a combination of three small molecules, that is, Y-27632 (a selective inhibitor of Rho-associated, coiled-coil containing protein kinase [ROCK]), A 83–01 (a receptor-like kinase inhibitor of transforming growth factor beta [TGF-β]), and CHIR-99021 (a selective inhibitor of glycogen synthase kinase-3β [GSK3β] that also activates Wnt), dramatically stimulated protein synthesis-related pathways and enhanced the proliferative capacity of iEPCs. These findings will help to establish a supply system of EPCs at an industrial scale.

Targeting glycogen synthase kinase-3β inhibition alleviates acute myocardial infarction through reduction of NLRP3 inflammasome activation

Myocardial infarction is associated with a sterile inflammatory response, which involves the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome. Glycogen synthase-3β (GSK-3β) is a multifunctional serine/threonine kinase that contributes to cardiac hypertrophy and cardiac failure. In this study, we aimed to evaluate the molecular mechanisms involved in GSK-3β-mediated activation of the NLRP3 inflammasome after acute myocardial infarction (AMI) in Sprague-Dawley rats.

CD73 sustained cancer-stem-cell traits by promoting SOX9 expression and stability in hepatocellular carcinoma

BackgroundAberrant AKT activation contributes to cancer stem cell (CSC) traits in hepatocellular carcinoma (HCC). We previously reported that CD73 activated AKT signaling via the Rap1/P110β cascade. Here, we further explored the roles of CD73 in regulating CSC characteristics of HCC.MethodsCD73 expression modulations were conducted by lentiviral transfections. CD73+ fractions were purified by magnetic-based sorting, and fluorescent-activated cell sorting was used to assess differentiation potentials. A sphere-forming assay was performed to evaluate CSC traits in vitro, subcutaneous NOD/SCID mice models were generated to assess in vivo CSC features, and colony formation assays assessed drug resistance capacities. Stemness-associated gene expression was also determined, and underlying mechanisms were investigated by evaluating immunoprecipitation and ubiquitylation.ResultsWe found CD73 expression was positively associated with sphere-forming capacity and elevated in HCC spheroids. CD73 knockdown hindered sphere formation, Lenvatinib resistance, and stemness-associated gene expression, while CD73 overexpression achieved the opposite effects. Moreover, CD73 knockdown significantly inhibited the in vivo tumor propagation capacity. Notably, we found that CD73+ cells exhibited substantially stronger CSC traits than their CD73– counterparts. Mechanistically, CD73 exerted its pro-stemness activity through dual AKT-dependent mechanisms: activating SOX9 transcription via c-Myc, and preventing SOX9 degradation by inhibiting glycogen synthase kinase 3β. Clinically, the combined analysis of CD73 and SOX9 achieved a more accurate prediction of prognosis.ConclusionsCollectively, CD73 plays a critical role in sustaining CSCs traits by upregulating SOX9 expression and enhancing its protein stability. Targeting CD73 might be a promising strategy to eradicate CSCs and reverse Lenvatinib resistance in HCC.

Inhibition of glycogen synthase kinase-3β protects against collagen type II-induced arthritis associated with the inhibition of miR155/24 and inflammation and upregulation of apoptosis in rats

MicroRNAs have been implicated in the pathogenesis of rheumatoid arthritis (RA) and their syntheses are modulated by glycogen synthase kinase-3β (GSK-3β). Therefore, we hypothesised that the GSK-3β inhibitor, TDZD-8 can protect against collagen-induced arthritis (CIA) via downregulating miR155 and miR-24 expression. Rats were randomly allocated into four groups (n = 6) as follows: Control, Control + TDZD-8 (1 mg/kg), CIA, and CIA + TDZD-8. Rats were sacrificed after 6 weeks. We observed in the model group (CIA) significant (p<.05) increase in arthritis score and serum levels of RA biomarkers, which were significantly (p < .05) inhibited by TDZD-8. TDZD-8 also significantly (p<.05) inhibited CIA-induced synovial tissue levels of miR155, miR-24, and inflammation. In addition, a significant (p<.05) modulation of biomarkers of survival (Bcl-2) and apoptosis (cleaved caspase-3) by TDZD-8 was observed. Thus, TDZD-8 protects against CIA in rats for a period of 6 weeks, which is associated with the inhibition of miR155/24 and inflammation, and apoptosis augmentation.

Glycogen synthase kinase 3β inhibitor- CHIR 99021 augments the differentiation potential of mesenchymal stem cells

AimMesenchymal stem cells (MSCs) are immunomodulatory, non-teratogenic and multipotent alternatives to embryonic or induced pluripotent stem cells (ESCs or iPSCs). However, the potency of MSCs is not equivalent to the pluripotency of ESCs or iPSCs. We used CHIR 99021 to improve current protocols and methods of differentiation for the enhanced transdifferentiation potency of MSCs. Main MethodsWe used Flurescence activated cell sorter (FACS) for MSC immunophenotyping and biochemical assay for demonstrating the trilineage potential of MSCs. We used real-time polymerase chain reaction, immunocytochemistry and Western blotting assay for analyzing the expression of lineage-specific markers. Key FindingsCHIR 99021 treatment of MSCs resulted in enhanced transdifferentiation into neurological, hepatogenic and cardiomyocyte lineages with standardized protocols of differentiation. CHIR 99021–treated MSCs showed increased nuclear localization of β-catenin. These MSCs showed a significantly increased deposition of active histone marks (H3K4Me3, H3K36Me3), whereas no change was observed in repressive marks (H3K9Me3, H3K27Me3). Differential methylation profiling showed demethylation of the transcription factor OCT4 promoter region with subsequent analysis revealing increased gene expression and protein content. The HLA-DR antigen was absent in CHIR 99021–treated MSCs and their differentiated cell types, indicating their immune-privileged status. Karyotyping analysis showed that CHIR 99021–treated MSCs were genomically stable. Teratoma analysis of nude mice injected with CHIR 99021–treated MSCs showed the increased presence of cell types of mesodermal origin at the site of injection. SignificanceMSCs pretreated with CHIR 99021 can be potent, abundant alternative sources of stem cells with enhanced differentiation capabilities that are well suited to cell-based regenerative therapy.

Lithium chloride ameliorates cognition dysfunction induced by sevoflurane anesthesia in rats.

Postoperative cognitive dysfunction is a common complication in elderly patients after surgeries involving anesthesia, but the underlying mechanisms are poorly understood. Lithium is a conventional treatment for bipolar disorder, which exerts a neuroprotective role in various diseases by inhibiting glycogen synthase kinase‐3β (GSK‐3β) in the brain and spinal cord. However, it is not known whether lithium chloride (LiCl) can protect against cognitive dysfunction induced by sevoflurane (SEV) anesthesia. Here, we examined the effects of LiCl on SEV‐induced cognitive dysfunction in rats and on SEV‐induced neuron apoptosis. We report that anesthesia with SEV significantly impaired memory performance, induced oxidative stress and hippocampal neuron apoptosis, and stimulated GSK‐3β activity. Treatment with LiCl ameliorated SEV‐induced cognitive disorder in rats by inhibiting the GSK‐3β/β‐catenin signaling pathway. In addition, LiCl reduced hippocampal neuron apoptosis and oxidative stress induced by SEV anesthesia. These results suggest that LiCl may have potential for development into a therapeutic agent for treatment of SEV anesthesia‐induced cognitive dysfunction.