Glycogen Synthase Kinase-3 Inhibitors Research Articles

Targeting IL-11R/EZH2 signaling axis as a therapeutic strategy for osteosarcoma lung metastases

Lung metastases are the primary cause of death for osteosarcoma (OS) patients. We recently validated interleukin-11 receptor α (IL-11Rα) as a molecular target for the inhibition of OS lung metastases. Since there is no clinically approved antibody against this receptor, we sought to identify downstream targets that mediate the effects of IL-11Rα signaling. We used shRNA to deplete IL-11Rα from OS cells; as a complementary approach, we added IL-11 exogenously to OS cells. The resulting changes in gene expression identified EZH2 as a downstream candidate. This was confirmed by knockdown of IL-11Rα in OS cells, which led to increased expression of genes repressed by histone methyltransferase EZH2, including members of the WNT pathway, a known target pathway of EZH2. Exogenous IL-11 increased the global levels of histone H3 lysine 27 trimethylation, evidence of EZH2 activation. Treatment with the EZH2 inhibitor GSK126 significantly reduced in vitro proliferation and increased cell-cycle arrest and apoptosis, which were partially mediated through the WNT pathway. In vivo, treatment of an orthotopic nude mouse model of OS with GSK126 inhibited lung metastatic growth and prolonged survival. In addition, significantly shorter recurrence-free survival was seen in OS patients with high levels of EZH2 in their primary tumors (P < .05). This suggests that IL-11Rα promotes OS lung metastasis via activation of EZH2. Thus, blocking EZH2 activity may be an effective strategy for inhibiting OS lung metastasis and improving prognosis.

New edaravone analogs incorporated with N‐benzylthiazole moiety: Multistep chemical synthesis, in vitro cytotoxicity with pRIPK3 inhibitory activities, and molecular docking

AbstractIn this research article, the chemical synthesis of new N‐phenylpyrazolone‐N‐benzylthiazole hybrids (3–6) via late‐stage thiazolation of the corresponding benzylthiosemicarbazone 2 was reported. The skeletal structural of the new molecules were validated by instrumental measurements (FT‐IR, NMR, and EI‐MS). In vitro cytotoxicity‐based cellular MTT bioassay shows that compound 3 that bears an N‐benzyl‐4‐thiazolone moiety is the most potent one toward the osteosarcoma cell line (Hos) with an IC50 value of 5.8 ± 0.1 μM, while compound 4a that contains a 5‐acetyl‐N‐benzylthiazole unit is the most robust one against the model lung carcinoma cell line (A549) with an IC50 value of 9.23 ± 0.01 μM. Also, 3 is roughly equipotent to 4b in its cytotoxicity activity against A549. In vitro enzymatic ELISA bioassay of A549 cells indicates that IC50 of 3 caused a decrease in the pRIPK3 kinase concentration (2.89 ± 0.005 pg/mL) as compared to DMSO‐treated cells (2.93 ± 0.010 pg/mL), while the pRIPK3 level incresed with 4b impact. As a result, 3 may be an effective inhibitor of pRIPK3 and hence necroptosis, proposing a novel therapeutic strategy for necroptosis‐related illnesses. In silico molecular docking shows that 3 interlocked and fitted well into the binding site of RIPK3 (PDB code: 7MX3) with a fitness value (−123.382 kcal/mol) lower than 4b and forms an important H‐bond with Lys50 like the marketed RIPK3 inhibitor GSK'843, validating the experimental results. Consequently, 3 is the most promising molecule that could be a lead candidate for further studies.

Correlation of therapy-induced neutropenia with survival in patients with metastatic pancreatic cancer treated with GSK-3 inhibitor elraglusib (9-ING-41) in combination with gemcitabine/nab-paclitaxel in the 1801 phase 2 study.

e16311 Background: Previously, elraglusib (9-ING-41), an inhibitor of Glycogen Synthase Kinase-3 (GSK-3), was shown to potentiate the anticancer effects of chemotherapeutic drugs in preclinical models of cancer. The combination of elraglusib and gemcitabine/nab-paclitaxel (GnP) is currently being investigated in a randomized, open-label 1801 phase 2 study as a first-line therapy for patients with metastatic pancreatic ductal adenocarcinoma (mPDAC). Chemotherapy-induced neutropenia is a common dose-limiting toxicity resulting in dose reductions and/or treatment interruptions. Present evidence shows that therapy-induced neutropenia (TIN) grade 3-4 correlates with survival and is a potential marker for improved clinical outcome in elraglusib/GnP-treated patients. Methods: We correlated overall survival (OS) and time-to-treatment discontinuation (TTD) with TIN grade 3-4 in 72 (GnP arm) and 155 (elraglusib/GnP arm) mPDAC patients previously untreated for advanced disease (1801 Part 3 Arm B study; NCT03678883). TTD was defined as the date of first dose to the date of treatment discontinuation or death. The Kaplan-Meier log-rank test was used for statistical analysis. A p-value &lt; 0.05 was considered statistically significant. Results: The rate of grade 3-4 TIN was 22% (16/72 patients) and 47% (72/155 patients) in the GnP and elraglusib/GnP groups, respectively. Based on 28% of all recorded death events in elraglusib/GnP group and 36% in GnP group, we found that grade 3-4 TIN was significantly associated with improved OS in patients treated with elraglusib/GnP (median OS: 14.4 vs 5.9 months; P &lt; 0.05) but not in patients treated with GnP (median OS: 7.6 vs 7.2 months). In the elraglusib/GnP group, patients with grade 3-4 TIN stayed on treatment significantly longer (median TTD: 7.9 months; P &lt; 0.05) as compared to the patients who did not develop TIN grade 3-4 (median TTD: 1.7 months). Conclusions: Our preliminary results identified grade 3-4 TIN as a potential marker of improved clinical outcome for elraglusib/GnP therapy in previously untreated patients with mPDAC. Recruitment for the 1801 Part 3 Arm B phase 2 trial has been completed. Another interim analysis of OS and its correlation to grade 3-4 TIN will be available by April 2024 and will be included in the final presentation. Clinical trial information: NCT03678883 .

Identification of immune biomarkers in treatment-naive patients with metastatic pancreatic cancer treated with the GSK-3 inhibitor elraglusib (9-ING-41) in combination with gemcitabine/nab-paclitaxel in the 1801 phase 2 study.

e16331 Background: Elraglusib (9-ING-41), a potent and selective GSK-3 inhibitor, is a targeted anticancer agent with clinical activity in phase 1 trial and undergoing clinical development for the treatment of metastatic pancreatic ductal adenocarcinoma (mPDAC). Elraglusib (9-ING-41) displayed immune-modulatory activity in preclinical models and modulation of serum cytokine profile in clinical specimens. Here, we investigated the correlation between plasma levels of cytokines/chemokines/soluble cell receptors/growth factors (CCSG) and clinical outcomes of patients with mPDAC treated with elraglusib and gemcitabine/nab-paclitaxel (GnP) in an exploratory study. Methods: CCSGs were evaluated in the pre-dose plasma samples of patients with newly diagnosed mPDAC enrolled in the 1801 phase 2 study (NCT03678883) treated with GnP (n = 13) or elraglusib/GnP (n = 32). Using a Luminex immunoassay, 40 CCSGs were screened as predictive biomarkers of median overall survival (mOS) in the elraglusib/GnP arm using 5-fold cross-validation and maximally selected rank statistic cutpoint determination. A subset of CCSGs (n = 10) was remeasured using an MSD immunoassay with greater sensitivity. Maximally selected rank statistic cutpoint determinations were conducted using MSD results for elraglusib/GnP samples with mOS and median progression free survival (mPFS). P values &lt; 0.05 were considered statistically significant. Results: Luminex screening revealed several CCSGs significantly stratified elraglusib/GnP patients by mOS. IL-6 and IL-8 were identified as top candidates with stable and significant results on cross-validation. Using the MSD assay, both were confirmed to stratify elraglusib/GnP patients by OS and extended to analyze PFS. Low level of IL-8 conferred a better OS prognosis (HR: 0.0919, 95%, CI: 0.0205-0.411, P &lt; 0.001) than low IL-6 (HR: 0.205, 95%, CI: 0.063-0.665, P = 0.005) in the elraglusib/GnP treated patients. Correlations with mPFS were similar in the elraglusib/GnP patients for IL-6 (HR: 0.248, 95% CI: 0.072-0.848, P = 0.02) and IL-8 (HR: 0.297, 95%, CI: 0.111-0.743, P = 0.01). Conclusions: Preliminary results of our exploratory study identified low plasma levels of IL-6 and IL-8 prior to elraglusib/GnP treatment as potential predictive biomarkers associated with improved outcomes in previously untreated patients with mPDAC. The 1801 Part 3 Arm B clinical trial recruitment has been completed. We plan to analyze additional pre-dose plasma samples obtained from approximately 200 patients by April 2024. These will be analyzed using interim OS data available by April 2024 and included in the final presentation. Clinical trial information: NCT03678883 .

Mutational analysis of cfDNA to identify predictive biomarkers in previously untreated patients with metastatic pancreatic cancer receiving the GSK-3 inhibitor elraglusib (9-ING-41) in combination with gemcitabine/nab-paclitaxel in the 1801 phase 2 study.

e16306 Background: Glycogen Synthase Kinase-3beta has been well-credentialed as a therapeutic target in patients with advanced cancer including pancreatic cancer. Elraglusib (9-ING-41), a novel best-in-class GSK-3 inhibitor, is being developed in combination with gemcitabine/nab-paclitaxel (GnP) as first-line therapy for patients with metastatic pancreatic cancer (mPDAC). Using liquid biopsy samples from an ongoing randomized phase 2 trial (1801 Part 3B; NCT03678883), we examined whether gene mutations in cfDNA correlated with clinical outcomes in mPDAC patients treated with GnP or elraglusib/GnP. Methods: Pre-dose plasma samples were obtained from 37 (GnP arm) and 89 (elraglusib/GnP arm) mPDAC patients previously untreated for advanced disease. Mutational analysis of cfDNA extracted from plasma samples was performed by Tempus (Chicago, IL) using the xF+ platform. The Tempus xF+ assay is an NGS cfDNA liquid biopsy tumor profiling assay for identifying genomic alterations in 523 genes. Tempus xF+ reports were used for analysis of gene mutations. Results: In 126 pancreatic cancer patients, frequently mutated genes ( &gt; 10%) consisted of KRAS (93/126, 74%), TP53 (79/126, 63%), CDKN2A (26/126, 21%), DNMT3A (25/126, 20%), NOTCH1/2 (25/126, 20%), and MLL3 (23/126, 18%). Gain-of-function mutations in KRAS, and loss-of-function mutations of TP53, and CDKN2A genes were associated with worse overall survival (OS) in patients treated with elraglusib/GnP (P &lt; 0.05) but not in GnP-treated patients. Multivariate analysis of frequently mutated genes showed that patients with double (KRAS+TP53) and triple (KRAS+TP53+CDKN2A) mutations had significantly worse OS only in the elraglusib/GnP arm. The presence of DNMT3A, NOTCH1/2 or MLL3 gene mutations did not correlate with any clinical outcomes. Conclusions: Our preliminary analysis of gene mutations in liquid biopsy samples obtained from patients in the ongoing 1801 Part 3B phase 2 trial identified potential predictive biomarkers of clinical outcome in mPDAC patients treated with combination of elraglusib/GnP. Clinical trial information: NCT03678883 .

Decoding the molecular Symphony: Unravelling neurologically crucial GSK-3 inhibition through 2D QSAR modelling with MLR, PLS, and ANN approaches

This study aimed to decipher the encoded information within the molecular structure of a dataset of GSK-3β inhibitors through a comprehensive quantity structural-activity relationship (QSAR) investigation employing traditional physicochemical descriptors. Various statistical techniques were applied, encompassing linear methods such as Multiple Linear Regression (MLR) and Partial Least Squares (PLS), along with non-linear approaches such as Artificial Neural Networks (ANN). Rigorous validation using diverse statistical tools confirmed the models’ precision and predictability. The model exhibited exceptional statistical relevance, as evidenced by standard parameters: S-value (0.37), F-value (37.17), r (0.93), r2 (0.855), and r2CV (0.78). They assessed predictive power and robustness by involving specific statistical parameters. Key descriptors analyzed, including Verloop L (subs 2), Lipole Z component (whole molecule), and VAMP Dipole Z component, offered crucial insights into their respective contributions. This analysis implies that targeted modifications in the substitution pattern potentially enhance GSK-3β inhibitory activity significantly. The established model clarified how bioactivity depends on structure and offered valuable recommendations for creating new compounds with better inhibitory activity profiles against the GSK-3β enzyme.

EZH2 inhibition reduces macrophage inflammatory responses in atherosclerosis

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Dutch Heart Foundation (Hartstichting) Aim Epigenetic processes are essential modulators of macrophage inflammatory responses. We postulate that interference in the epigenetic machinery of macrophages might offer novel approaches to combat atherosclerosis. Here, we investigate the repressive histone modification H3K27Me3 deposited by the polycomb repressive complex 2 (PRC2) with its catalytic component EZH2. We studied the therapeutic potential of macrophage EZH2 inhibition in the context of atherosclerosis. Methods Human monocyte-derived macrophages and murine peritoneal and bone-marrow derived macrophages were treated with the EZH2-specific inhibitor GSK126 and subsequently activated with LPS to mimic TLR4-inflammatory responses. The impact of Ezh2 inhibition on macrophage differentiation and activation compared to vehicle (DMSO) was assessed by RNA-seq, ChIP-seq, flow cytometry, western blot, and ELISA. To study its impact on atherosclerosis, female Ldlr-/- mice on a high-cholesterol diet (9weeks) were treated the last four weeks with GSK126 or control. Results Ezh2 inhibition by GSK126 in vitro lowered global H3K27Me3 levels without altering macrophage viability and differentiation, showcasing effective EZH2 inhibition. RNA-seq revealed that of more than one-third of the LPS-induced genes were significantly downregulated by EZH2 inhibition. Subsequent pathway analysis identified cytokine and interferon signaling, co-stimulation, and cell migration as the top down-regulated pathways (padj&amp;lt;0.05). Furthermore, ChIP-seq revealed considerably altered regulation of H3K27Me3 deposition. Indeed, we confirmed that gene and cytokine expression of the inflammatory mediators IL-6, IL-12, and TNF were reduced. Furthermore, membrane marker expression of co-stimulatory CD40, CD80, and CD86 were significantly decreased. In murine atherosclerosis, we observed a reduction in the Virmani plaque severity score and are currently assessing lesion size and composition. Conclusion Overall, we show that EZH2 inhibition reduces inflammatory responses in human and murine macrophages in vitro. We are currently assessing the impact of EZH2 inhibition on lesion size and composition in murine atherosclerosis. Additionally, we are performing ex vivo experiments on human endarterectomy plaques to assess the therapeutic potential of EZH2 inhibition on human atherosclerosis.

Inhibition of Adipocyte Necroptosis Alleviates Fat Necrosis and Fibrosis After Grafting in a Murine Model.

Because of the delicate structure of the adipose tissue, fat necrosis accounts for 43.7% of all complications after autologous fat grafting; however, its regulation remains unclear. The purpose of this study was to examine the role of necroptosis in fat graft remodeling after grafting. Clinical fat graft necrosis samples were collected, and the expression levels of the necroptosis marker phosphorylated(p)-MLKL were analyzed. Transcriptome analysis was performed on fat grafts before and 1 week after transplantation in C57BL/6 mouse fat grafting models. Additionally, the in vivo effects of RIPK1 inhibitor Nec-1s or RIPK3 inhibitor GSK'872 on the fat grafting complications, including fat necrosis and fibrosis, were investigated. Necroptosis markers were observed and associated with higher occurrence of fibrosis in clinical fat graft necrosis samples compared to normal fat tissue. Amplification and RNA-Seq were conducted on RNA isolated from fat grafts before and after grafting. MLKL, RIPK1, and RIPK3's expression levels were significantly upregulated in comparison to controls. Higher expression levels of necroptotic RNAs were associated with higher levels of DAMPs, including Cxcl2, HMGB1, S100a8, S100a9, Nlrp3, and IL33, and activated proinflammatory signaling pathways, including the TNF, NF-kappa B, and chemokine signaling pathways. Necroptotic inhibitor Nec-1s and GSK'872 robustly suppressed the p-MLKL expression level and significantly inhibited necroptotic cell death, especially in adipocytes. Moreover, administration of Nec-1s and GSK'872 significantly alleviated fat necrosis and subsequent fibrosis in fat grafts. Collectively, our study findings highlight the potential therapeutic applications of necroptosis inhibitors in preventing fat necrosis and fibrosis after grafting.

Mechanistic insights into SARS-CoV-2 spike protein induction of the chemokine CXCL10

During a SARS-CoV-2 infection, macrophages recognize viral components resulting in cytokine production. While this response fuels virus elimination, overexpression of cytokines can lead to severe COVID-19. Previous studies suggest that the spike protein (S) of SARS-CoV-2 can elicit cytokine production via the transcription factor NF-κB and the toll-like receptors (TLRs). In this study, we found that: (i) S and the S2 subunit induce CXCL10, a chemokine implicated in severe COVID-19, gene expression by human macrophage cells (THP-1); (ii) a glycogen synthase kinase-3 inhibitor attenuates this induction; (iii) S and S2 do not activate NF-κB but do activate the transcription factor IRF; (iv) S and S2 do not require TLR2 to elicit CXCL10 production or activate IRF; and (v) S and S2 elicit CXCL10 production by peripheral blood mononuclear cells (PBMCs). We also discovered that the cellular response, or lack thereof, to S and S2 is a function of the recombinant S and S2 used. While such a finding raises the possibility of confounding LPS contamination, we offer evidence that potential contaminating LPS does not underly induced increases in CXCL10. Combined, these results provide insights into the complex immune response to SARS-CoV-2 and suggest possible therapeutic targets for severe COVID-19.

Na,K-ATPase activity promotes macropinocytosis in colon cancer via Wnt signaling.

Recent research has shown that membrane trafficking plays an important role in canonical Wnt signaling through sequestration of the β-catenin destruction complex inside multivesicular bodies (MVBs) and lysosomes. In this study, we introduce Ouabain, an inhibitor of the Na,K-ATPase pump that establishes electric potentials across membranes, as a potent inhibitor of Wnt signaling. We find that Na,K-ATPase levels are elevated in advanced colon carcinoma, that this enzyme is elevated in cancer cells with constitutively activated Wnt pathway and is activated by GSK3 inhibitors that increase macropinocytosis. Ouabain blocks macropinocytosis, which is an essential step in Wnt signaling, probably explaining the strong effects of Ouabain on this pathway. In Xenopus embryos, brief Ouabain treatment at the 32-cell stage, critical for the earliest Wnt signal in development-inhibited brains, could be reversed by treatment with Lithium chloride, a Wnt mimic. Inhibiting membrane trafficking may provide a way of targeting Wnt-driven cancers.

GSK-3: An "Ace" Among Kinases.

Background: Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase known to be participating in the regulation of β-catenin signaling (Wnt signaling). They help in the establishment of a multicomponent destruction complex that stimulates phosphorylation, leading to the destruction of β-catenin. Evidence about the role of increasingly active β-catenin signaling is involved in many forms of human cancer. The understanding of GSK-3 remains elusive as recent research aims to focus on developing potent GSK-3 inhibitors to target this kinase. Objective: This short review aims to highlight the regulation of GSK-3 with emphasis on Wnt signaling while highlighting its interaction with miRNAs corresponding to pluripotency and epithelial mesenchymal transition substantiating this kinase as an "Ace" among kinases in regulation of cellular processes. Result: Significant findings of miRNA regulation by GSK-3 exemplify the underpinnings of kinase-mediated transcriptional regulation in cancers. Conclusion: The review provides evidence on the role of GSK-3 as a possible master regulator of proteins and noncoding RNA, thereby implicating the fate of a cell.

Natural compound screening predicts novel GSK-3 isoform-specific inhibitors

Glycogen synthase kinase-3 (GSK-3) plays important roles in the pathogenesis of cardiovascular, metabolic, neurological disorders and cancer. Isoform-specific loss of either GSK-3α or GSK-3β often provides cytoprotective effects under such clinical conditions. However, available synthetic small molecule inhibitors are relatively non-specific, and their chronic use may lead to adverse effects. Therefore, screening for natural compound inhibitors to identify the isoform-specific inhibitors may provide improved clinical utility. Here, we screened 70 natural compounds to identify novel natural GSK-3 inhibitors employing comprehensive in silico and biochemical approaches. Molecular docking and pharmacokinetics analysis identified two natural compounds Psoralidin and Rosmarinic acid as potential GSK-3 inhibitors. Specifically, Psoralidin and Rosmarinic acid exhibited the highest binding affinities for GSK-3α and GSK-3β, respectively. Consistent with in silico findings, the kinase assay-driven IC50 revealed superior inhibitory effects of Psoralidin against GSK-3α (IC50 = 2.26 μM) vs. GSK-3β (IC50 = 4.23 μM) while Rosmarinic acid was found to be more potent against GSK-3β (IC50 = 2.24 μM) than GSK-3α (IC50 = 5.14 μM). Taken together, these studies show that the identified natural compounds may serve as GSK-3 inhibitors with Psoralidin serving as a better inhibitor for GSK-3α and Rosmarinic for GSK-3β isoform, respectively. Further characterization employing in vitro and preclinical models will be required to test the utility of these compounds as GSK-3 inhibitors for cardiometabolic and neurological disorders and cancers.

CRL2KLHDC3 and CRL1Fbxw7 cooperatively mediate c-Myc degradation.

c-Myc is a proto-oncoprotein that regulates various cellular processes and whose abnormal expression leads to tumorigenesis. c-Myc protein stability has been shown to be predominantly controlled by the ubiquitin ligase (E3) CRL1Fbxw7 in a manner dependent on glycogen synthase kinase 3 (GSK3)-mediated phosphorylation. Here we show that, in some types of cancer cells, c-Myc degradation is largely insensitive to the GSK3 inhibitor (GSK3i) CHIR99021, suggesting the existence of an E3 other than CRL1Fbxw7 for c-Myc degradation. Mass spectrometry identified CRL2KLHDC3 as such an E3. In GSK3i-insensitive cancer cells, combined depletion of Fbxw7 and KLHDC3 resulted in marked stabilization of c-Myc, suggestive of a cooperative action of Fbxw7 and KLHDC3. Furthermore, transplantation of such cells deficient in both Fbxw7 and KLHDC3 into immunodeficient mice gave rise to larger tumors compared with those formed by cells lacking only Fbxw7. GSK3i-insensitive pancreatic cancer cells expressed lower levels of SHISA2, a negative regulator of the Wnt signaling pathway, than did GSK3i-sensitive cells. KLHDC3 mRNA abundance was associated with prognosis in pancreatic cancer patients with a low level of SHISA2 gene expression. These results suggest that KLHDC3 cooperates with Fbxw7 to promote c-Myc degradation in a subset of cancer cells with low GSK3 activity.

Targeting E(z) methyltransferase inhibitor GSK126 promotes adventitious root induction in Larix kaempferi

Targeting E(z) methyltransferase inhibitor GSK126 promotes adventitious root induction in Larix kaempferi

Synthesis and Evaluation of 5-(Heteroarylmethylene)hydantoins as Glycogen Synthase Kinase-3β Inhibitors.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase which plays a center role in the phosphorylation of a wide variety of proteins, generally leading to their inactivation. As such, GSK-3 is viewed as a therapeutic target. An ever-increasing number of small organic molecule inhibitors of GSK-3 have been reported. Phenylmethylene hydantoins are known to exhibit a wide range of inhibitory activities including for GSK-3β. A family of fourteen 2-heterocycle substituted methylene hydantoins (14, 17-29) were prepared and evaluated for the inhibition of GSK-3β at 25 μM. The IC50 values of five of these compounds was determined; the two best inhibitors are 5-[(4'-chloro-2-pyridinyl)methylene]hydantoin (IC50 = 2.14 ± 0.18 μM) and 5-[(6'-bromo-2-pyridinyl)methylene]hydantoin (IC50 = 3.39 ± 0.16 μM). The computational docking of the compounds with GSK-3β (pdb 1q41) revealed poses with hydrogen bonding to the backbone at Val135. The 5-[(heteroaryl)methylene]hydantoins did not strongly inhibit other metalloenzymes, demonstrating poor inhibitory activity against matrix metalloproteinase-12 at 25 μM and against human carbonic anhydrase at 200 μM, and were not inhibitors for Staphylococcus aureus pyruvate carboxylase at concentrations >1000 μM.

Amelioration of cisplatin-induced neurodegenerative changes in rats and restoration of mitochondrial biogenesis by 6-bromoindirubin-3′-oxime: The implication of the GSK-3β/PGC1-α axis

BackgroundThe cognitive deficits observed after treatment with chemotherapeutic drugs are obvious clinical problems. For treating chemotherapy-induced cognitive deficits (CICD), the treatment modalities must target its underlying mechanisms. Specifically, cisplatin may activate glycogen synthase kinase-3β (GSK-3β), thereby enhancing neuronal apoptosis. 6-bromoindirubin-3′-oxime (6BIO) was not investigated previously in a model of CICD. Therefore, this investigation aimed to address the impacts of GSK3 inhibition on regulating cell signaling, which contributes to neurodegeneration and cognitive impairment. MethodsThirty adult male Wistar rats were randomly allocated into control groups, while two experimental groups were exposed to repeated cisplatin injections (2 mg/kg intraperitoneally (ip), twice weekly, nine injections), termed chemobrain groups. The rats in the two experimental groups were equally divided into the chemobrain group (untreated) and the chemobrain-6BIO group (treated with 6BIO at a dose of 8.5 μg/kg ip every two days, started after the last dose of cisplatin and continued for two weeks). ResultsRepeated exposure to cisplatin led to a marked decline in cognitive functions. GSK3 inhibition exerted neuroprotection by decreasing the expression of p-tau and amyloid β, thereby improving cognition. 6BIO, the GSK-3β inhibitor, restored mitochondrial biogenesis by augmenting the protein levels of PGC1-α and increasing the number of mitochondria in the cerebral cortex and hippocampus. Conclusion6BIO provided neuroprotection and exhibited anti-apoptotic and anti-oxidative effects in a rat model of chemobrain.

Lithium carbonate-induced confusional state in patients with bipolar disorder: identification of case reports and review of the evidence

Objective: Lithium carbonate is a mood stabilizer used in the treatment of bipolar disorder. However, its narrow therapeutic index (normal = 0.6-1.2 mmol/L) predisposes patients to toxic effects. This study aimed to identify case reports and review the evidence on the frequency, mechanism, risk factors and guidelines associated with a lithium-induced confusional state in patients with bipolar disorder. Methods: All Databases from the National Center for Biotechnology Information (NCBI) were used to search for lithium AND “confusional state” AND “case report” published in English between 2015 and 2020. This search engine was also used to search for lithium AND “confusional state” AND frequency OR mechanism OR “risk factor” OR guideline published in English. Results: Case reports showed (1) all patients were treated with lithium for bipolar disorder and hospitalized for a confusional state, (2) most patients took concomitant therapies and recovered after lithium withdrawal, (3) some patients were female and old-aged (65+ years old), and (4) no ethnicities, causality assessments and dose patterns were reported. Other scientific literature showed (1) frequencies of a lithium-induced confusional state varied from very common (&gt;10%) to rare (&lt;0.01%), (2) inhibition of inositol phosphate-phosphatase (IMPase) and glycogen synthase kinase-3 (GSK3) might result in a lithium-induced confusional state, (3) ageing, diseases and concomitant therapies increased the risk of a lithium-induced confusional state, and (4) patients should complete lab tests and diagnostic procedures to monitor and/or prevent a lithium-induced confusional state. Conclusion: Case reports and other scientific literature elucidated the relationship between lithium and a confusional state.

Repressive H3K27me3 drives hyperglycemia-induced oxidative and inflammatory transcriptional programs in human endothelium

BackgroundHistone modifications play a critical role in chromatin remodelling and regulate gene expression in health and disease. Histone methyltransferases EZH1, EZH2, and demethylases UTX, JMJD3, and UTY catalyse trimethylation of lysine 27 on histone H3 (H3K27me3). This study was designed to investigate whether H3K27me3 triggers hyperglycemia-induced oxidative and inflammatory transcriptional programs in the endothelium.MethodsWe studied human aortic endothelial cells exposed to high glucose (HAEC) or isolated from individuals with diabetes (D-HAEC). RT-qPCR, immunoblotting, chromatin immunoprecipitation (ChIP-qPCR), and confocal microscopy were performed to investigate the role of H3K27me3. We determined superoxide anion (O2−) production by ESR spectroscopy, NF-κB binding activity, and monocyte adhesion. Silencing/overexpression and pharmacological inhibition of chromatin modifying enzymes were used to modulate H3K27me3 levels. Furthermore, isometric tension studies and immunohistochemistry were performed in aorta from wild-type and db/db mice.ResultsIncubation of HAEC to high glucose showed that upregulation of EZH2 coupled to reduced demethylase UTX and JMJD3 was responsible for the increased H3K27me3. ChIP-qPCR revealed that repressive H3K27me3 binding to superoxide dismutase and transcription factor JunD promoters is involved in glucose-induced O2− generation. Indeed, loss of JunD transcriptional inhibition favours NOX4 expression. Furthermore, H3K27me3-driven oxidative stress increased NF-κB p65 activity and downstream inflammatory genes. Interestingly, EZH2 inhibitor GSK126 rescued these endothelial derangements by reducing H3K27me3. We also found that H3K27me3 epigenetic signature alters transcriptional programs in D-HAEC and aortas from db/db mice.ConclusionsEZH2-mediated H3K27me3 represents a key epigenetic driver of hyperglycemia-induced endothelial dysfunction. Targeting EZH2 may attenuate oxidative stress and inflammation and, hence, prevent vascular disease in diabetes.Graphical

Synthetic Lethality between Cohesin and WNT Signaling Pathways in Diverse Cancer Contexts.

Cohesin is a highly conserved ring-shaped complex involved in topologically embracing chromatids, gene expression regulation, genome compartmentalization, and genome stability maintenance. Genomic analyses have detected mutations in the cohesin complex in a wide array of human tumors. These findings have led to increased interest in cohesin as a potential target in cancer therapy. Synthetic lethality has been suggested as an approach to exploit genetic differences in cancer cells to influence their selective killing. In this study, we show that mutations in ESCO1, NIPBL, PDS5B, RAD21, SMC1A, SMC3, STAG2, and WAPL genes are synthetically lethal with stimulation of WNT signaling obtained following LY2090314 treatment, a GSK3 inhibitor, in several cancer cell lines. Moreover, treatment led to the stabilization of β-catenin and affected the expression of c-MYC, probably due to the occupancy decrease in cohesin at the c-MYC promoter. Finally, LY2090314 caused gene expression dysregulation mainly involving pathways related to transcription regulation, cell proliferation, and chromatin remodeling. For the first time, our work provides the underlying molecular basis for synthetic lethality due to cohesin mutations and suggests that targeting the WNT may be a promising therapeutic approach for tumors carrying mutated cohesin.

Standardized production of hPSC-derived cardiomyocyte aggregates in stirred spinner flasks.

A promising cell-therapy approach for heart failure aims at differentiating human pluripotent stem cells (hPSCs) into functional cardiomyocytes (CMs) in vitro to replace the disease-induced loss of patients' heart muscle cells in vivo. But many challenges remain for the routine clinical application of hPSC-derived CMs (hPSC-CMs), including good manufacturing practice (GMP)-compliant production strategies. This protocol describes the efficient generation of hPSC-CM aggregates in suspension culture, emphasizing process simplicity, robustness and GMP compliance. The strategy promotes clinical translation and other applications that require large numbers of CMs. Using a simple spinner-flask platform, this protocol is applicable to a broad range of users with general experience in handling hPSCs without extensive know-how in biotechnology. hPSCs are expanded in monolayer to generate the required cell numbers for process inoculation in suspension culture, followed by stirring-controlled formation of cell-only aggregates at a 300-ml scale. After 48 h at checkpoint (CP) 0, chemically defined cardiac differentiation is induced by WNT-pathway modulation through use of the glycogen-synthase kinase-3 inhibitor CHIR99021 (WNT agonist), which is replaced 24 h later by the chemical WNT-pathway inhibitor IWP-2. The exact application of the described process parameters is important to ensure process efficiency and robustness. After 10 d of differentiation (CP I), the production of ≥100 × 106 CMs is expected. Moreover, to 'uncouple' cell production from downstream applications, continuous maintenance of CM aggregates for up to 35 d in culture (CP II) is demonstrated without a reduction in CM content, supporting downstream logistics while potentially overcoming the requirement for cryopreservation.