Akt Phosphorylation Research Articles

FoxO1/Rictor axis induces a non-genetic adaptation to Ibrutinib via Akt activation in chronic lymphocytic leukemia.

BTK inhibitor therapy induces peripheral blood lymphocytosis in chronic lymphocytic leukemia (CLL) lasting for several months. It remains unclear whether non-genetic adaptation mechanisms exist, allowing CLL cells' survival during BTK inhibitor-induced lymphocytosis and/or playing a role in therapy resistance. We show that in approximately 70 % of CLL cases, ibrutinib treatment in vivo increases Akt activity above pre-therapy levels within several weeks, leading to compensatory CLL cell survival and a more prominent lymphocytosis on therapy. Ibrutinib-induced Akt phosphorylation (pAktS473) is caused by the upregulation of FoxO1 transcription factor, which induces expression of Rictor, an assembly protein for mTORC2 protein complex that directly phosphorylates Akt at serine 473 (S473). Knock-out or inhibition of FoxO1 or Rictor led to a dramatic decrease in Akt phosphorylation and growth disadvantage for malignant B cells in the presence of ibrutinib (or PI3K inhibitor idelalisib) in vitro and in vivo. FoxO1/Rictor/pAktS473 axis represents an early non-genetic adaptation to BCR inhibitor therapy not requiring PI3Kδ or BTK kinase activity. We further demonstrate that FoxO1 can be targeted therapeutically, and its inhibition induces CLL cells' apoptosis alone or in combination with BTK inhibitors (ibrutinib, acalabrutinib, pirtobrutinib) and blocks their proliferation triggered by T-cell factors (CD40L, IL-4, and IL-21).

Transcriptome analysis reveals a role of FOXO3 in antileukemia/lymphoma properties of panduratin A

Boesenbergia rotunda, commonly known as fingerroot, is a medicinal and culinary plant native to the Indochina Peninsula. We found that panduratin A (Pan-A), one of the compounds present in the rhizome extract of fingerroot, inhibited cell proliferation, induced apoptosis, and promoted cell cycle arrest at G0/G1 phase in multiple hematologic malignant cell lines including leukemia and lymphoma lines. Pan-A inhibited these activities in leukemia and lymphoma cells in a concentration-dependent manner. High-throughput transcriptome analysis indicated that Pan-A is involved in the cell cycle, cellular senescence, apoptosis, and multiple canonical signaling pathways in lymphoma cells. The Forkhead box O (FOXO) transcription factor family was identified as a potential target of Pan-A. Western blot showed elevated caspase 7 and cPARP/PARP in the B-cell lymphoma cells after treatment with Pan-A. The inhibitory effects were accompanied by stimulation of Akt signaling and phosphorylation of FOXO3. Immunohistochemistry of tissues from patients with B-cell lymphoma revealed detectable levels of FOXO3 protein specifically in neoplastic B cells. Overall, our results highlight FOXO3 as a player underlying antileukemia/lymphoma effects of Pan-A.

Mangiferin Represses Inflammation in Macrophages Under a Hyperglycemic Environment Through Nrf2 Signaling.

Inflammation in macrophages is exacerbated under hyperglycemic conditions, contributing to chronic inflammation and impaired wound healing in diabetes. This study investigates the potential of mangiferin, a natural polyphenol, to alleviate this inflammatory response by targeting a redox-sensitive transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2). Mangiferin, a known Nrf2 activator, was evaluated for its ability to counteract the hyperglycemia-induced inhibition of Nrf2 and enhance antioxidant defenses. The protective effects of mangiferin on macrophages in a hyperglycemic environment were assessed by examining the expression of Nrf2, NF-κB, NLRP3, HO-1, CAT, COX-2, IL-6, and IL-10 through gene and protein expression analyses using qPCR and immunoblotting, respectively. The mangiferin-mediated nuclear translocation of Nrf2 was evidenced, leading to a robust antioxidant response in macrophages exposed to a hyperglycemic microenvironment. This activation suppressed NF-κB signaling, reducing the expression of pro-inflammatory mediators such as COX-2 and IL-6. Additionally, mangiferin decreased NLRP3 inflammasome activation and reactive oxygen species accumulation in hyperglycemia exposed macrophages. Our findings revealed that mangiferin alleviated hyperglycemia-induced reductions in AKT phosphorylation, highlighting its potential role in modulating key signaling pathways. Furthermore, mangiferin significantly enhanced the invasiveness and migration of macrophages in a hyperglycemic environment, indicating its potential to improve wound healing. In conclusion, this study suggests that mangiferin may offer a promising therapeutic approach for managing inflammation and promoting wound healing in diabetic patients by regulating Nrf2 activity in hyperglycemia-induced macrophages.

Circulating extracellular vesicle-carried PTP1B and PP2A phosphatases as regulators of insulin resistance.

Metabolic disorders associated with abdominal obesity, dyslipidaemia, arterial hypertension and hyperglycaemia are risk factors for the development of insulin resistance. Extracellular vesicles (EVs) may play an important role in the regulation of metabolic signalling pathways in insulin resistance and associated complications. Circulating large EVs (lEVs) and small EVs (sEVs) from individuals with (IR group) and without insulin resistance (n-IR group) were isolated and characterised. lEVs and sEVs were administered by i.v. injection to mice and systemic, adipose tissue and liver insulin signalling were analysed. The role of phosphatases was analysed in target tissues and cells. Injection of lEVs and sEVs from IR participants impaired systemic, adipose tissue and liver insulin signalling in mice, while EVs from n-IR participants had no effect. Moreover, lEVs and sEVs from IR participants brought about a twofold increase in adipocyte size and adipogenic gene expression. EVs from IR participants expressed two types of phosphatases, phosphotyrosine 1 phosphatase (PTP1B) and protein phosphatase 2 (PP2A), IR lEVs being enriched with the active form of PTP1B while IR sEVs mainly carried active PP2A. Blockade of PTP1B activity in IR lEVs fully restored IRS1 and Akt phosphorylation in adipocytes and blunted insulin-induced Akt phosphorylation by inhibition of the macrophage secretome in hepatocytes. Conversely, blockade of PP2A activity in IR sEVs completely prevented insulin resistance in adipocytes and hepatocytes. These data demonstrate that inhibition of phosphatases carried by EVs from IR participants rescues insulin signalling in adipocytes and hepatocytes and point towards PTP1B and PP2A carried by IR EVs as being novel potential therapeutic targets against insulin resistance in adipose tissue and liver and the development of obesity.

Dendrobium huoshanense stem polysaccharide exhibits gastroprotective effect via regulating PI3K/AKT, NF-κB and Nrf-2 signaling in high-salt diet-induced gastritis mice

Prolonged consumption of high-salt diet (HSD) is implicated in the development of gastritis. This research intends to explore the protective potential of Dendrobium huoshanense stem polysaccharide (cDHPS) on gastritis in high-salt diet mice. cDHPS effectively alleviated the symptoms of HSD-induced gastritis, which were demonstrated by the reversed weight loss, gastric mucosal swelling, gastric mucus depletion, tight junction (TJ) disruption, and microvascular endothelial cell necrosis. Notably, cDHPS inhibited HSD-induced gastric inflammation, which was evidenced by the remodeled balance of Th17/Treg cells, along with the decreased level of IL-17 and the increased level of IL-10. Simultaneously, the oxidative stress induced by HSD was also improved with cDHPS treatment. Western blot analysis revealed that cDHPS markedly upregulated Nrf-2 expression while concurrently inhibiting the phosphorylation of P65, IκB, PI3K, AKT and FOXO-1 in the gastric tissues of HSD-induced gastritis mice. Collectively, these results indicate that cDHPS possesses protective potential against HSD-induced gastritis, which was attributable to the improvement of inflammatory and oxidative damage by modulating the PI3K/AKT and NF-κB/Nrf-2 signaling.

Polyphyllin VI Ameliorates Pulmonary Fibrosis by Suppressing the MAPK/ERK and PI3K/AKT Signaling Pathways via Upregulating DUSP6.

Pulmonary fibrosis (PF) is a lethal disease caused by inordinate repair of damaged lungs, for which limited strategies are available. Polyphyllin VI (PPVI), extracted and isolated from Paris polyphylla Smith var. chinensis (Franch.) Hara, has been regarded as an important traditional Chinese herbal medicine for the treatment of respiratory system diseases. This study evaluated effects of PPVI on PF and its underlying mechanism. Experimental procedure For evaluating the anti-PF effect of PPVI, we established an in vivo PF mouse model via intratracheal infusion of bleomycin (BLM) in mice and an in vitro PF model induced by TGF-β1 in NIH/3T3, HPF and A549, respectively. Subsequently, the mechanism of PPVI effects was further explored using RNA sequencing (RNA-Seq). The in vivo and in vitro results demonstrated that PPVI significantly inhibited inflammation, oxidative damage, and epithelial-mesenchymal transition. Furthermore, RNA sequencing indicated that PPVI ameliorated PF by modulating inflammation and oxidative stress responses. Furthermore, dual specificity phosphatase 6 (DUSP6), was the shared and most significant differentially expressed gene associated with inflammation and oxidative stress response after PPVI treatment. Mechanistically, silencing DUSP6 can eliminate the suppressive impact on PPVI for the activation of fibroblast and the phosphorylation of ERK and AKT. Summarily, our findings revealed the potential of PPVI in mitigating PF via upregulating DUSP6 and highlighted the regulatory function of DUSP6 in the pathogenesis of PF.

Distinct role of GRK3 in platelet activation by desensitization of G protein-coupled receptors.

Many platelet agonists mediate their cellular effects through G protein-coupled receptors (GPCRs) to induce platelet activation, and GPCR kinases (GRKs) have been demonstrated to have crucial roles in most GPCR functions in other cell types. Here, we investigated the functional role of GRK3 and the molecular basis for the regulation of GPCR desensitization by GRK3 in platelets. We used mice lacking GRK3 as well as β-arrestin2 which has been shown to be important in GPCR function in platelets. Platelet aggregation and dense granule secretion induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in both GRK3 -/- and β-arrestin2 -/- platelets compared to wild-type (WT) platelets, while non-GPCR agonist collagen-induced platelet aggregation and secretion were not affected. We have previously shown that GRK6 is not involved in the regulation of Gq-coupled 5HT2A and Gz-coupled a2A adrenergic receptors. Interestingly, in contrast to GRK6, platelet aggregation induced by co-stimulation of serotonin and epinephrine which activate 5-HT2A and a2A adrenergic receptors, respectively, was significantly potentiated in GRK3 -/- platelets, suggesting that GRK3 was involved in general GPCR regulation. In addition, platelet aggregation in response to the second challenge of ADP was restored in GRK3 -/- platelets whereas re-stimulation of the agonist failed to induce aggregation in WT platelets, confirming that GRK3 contributes to general GPCR desensitization. Finally, 2-MeSADP- and AYPGKF-induced AKT and ERK phosphorylation were significantly potentiated in GRK3 -/- platelets. GRK3 plays a crucial role in the regulation of platelet activation through general GPCR desensitization in platelets.

Circ_0001047 inhibits prostate cancer progression and enhances abiraterone sensitivity via miR-122-5p/FKBP5/PHLPP1/AKT axis in vitro

Prostate cancer (PCa), with high heterogeneity and poor prognosis, is one of the most common malignant tumors in men. Circular RNAs (circRNAs) have been identified in tumor progression and resistance to medication in numerous studies. However, the role of circ_0001047 in PCa is unclear. In this research, we found that circ_0001047 had low expression in PCa cells and tissues and was negatively correlated with testosterone secretion in vivo. Overexpression of circ_0001047 inhibited the proliferation, migration, invasion, and anti-apoptotic abilities of human PCa cells in vitro. Mechanistically, circ_0001047 promoted the expression of FKBP5 through sponge adsorption of miR-122-5p and then inhibited the proliferation, anti-apoptotic migration, and invasion abilities of PCa cells. In addition, overexpression of circ_0001047 enhanced the sensitivity of PCa cells to abiraterone by inhibiting AKT phosphorylation activation through upregulation of FKBP5/PHLPP1. This study revealed a novel mechanism by which circ_0001047 regulates PCa progression and treatment sensitivity via the miR-122-5p/FKBP5/PHLPP1/AKT axis. These findings deepen our comprehension of the molecular mechanisms in latent PCa progression and treatment resistance.Graphical

The role and mechanism of NRG1/ErbB4 in inducing the differentiation of induced pluripotent stem cells into cardiomyocytes

BackgroundWe aimed to investigate the effect and potential mechanism of enhancing Neuregulin1 (NRG1)/v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 4 (ErbB4) expression on the differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes.MethodsWe utilized CRISPR-CAS9 technology to knock in ErbB4 and obtained a single-cell clone IPSN-AAVS1-CMV-ErbB4 (iPSCs-ErbB4). Subsequently, we induced the differentiation of iPSCs into cardiomyocytes and quantified the number of beating embryoid bodies. Furthermore, quantitative real-time PCR assessed the expression of cardiomyocyte markers, including ANP (atrial natriuretic peptide), Nkx2.5 (NK2 transcription factor related locus 5), and GATA4 (GATA binding protein 4). On the 14th day of differentiation, we observed the α-MHC (α-myosin heavy chain)-positive area using immunofluorescent staining and conducted western blotting to detect the expression of cTnT (cardiac troponin) protein and PI3K/Akt signaling pathway-related proteins. Additionally, we intervened the iPSCs-ErbB4 + NRG1 group with the PI3K/Akt inhibitor LY294002 and observed alterations in the expression of cardiomyocyte differentiation-related genes.ResultsThe number of beating embryoid bodies increased after promoting the expression of NRG1/ErbB4 compared to the iPSCs control group. Cardiomyocyte markers ANP, Nkx2.5, and GATA4 significantly increased on day 14 of differentiation, and the positive area of α-MHC was three times that of the iPSCs control group. Moreover, there was a marked increase in cTnT protein expression. However, there was no significant difference in cardiomyocyte differentiation between the iPSCs-ErbB4 group and the iPSCs control group. Akt phosphorylation was significantly increased in the iPSCs-ErbB4 + NRG1 group. LY294002 significantly reversed the enhancing effect of NRG1/ErbB4 overexpression on Akt phosphorylation as well as the increase in α-MHC and cTnT expression.ConclusionsIn conclusion, promoting the expression of NRG1/ErbB4 induced the differentiation of iPSC into cardiomyocytes, possibly through modulation of the PI3K/Akt signaling pathway.

Bronchopulmonary dysplasia demonstrates dysregulated autotaxin/lysophosphatidic acid signaling in a neonatal mouse model.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease affecting premature infants who require oxygen supplementation and ventilator therapy to support their underdeveloped lungs. Autotaxin (ATX), an enzyme that generates the bioactive phospholipid lysophosphatidic acid (LPA), which acts via G-protein coupled receptors, has been implicated in numerous pulmonary diseases. In this study, we explored the pathophysiological role of the ATX/LPA signaling pathway in BPD. Neonatal mice were exposed to normoxia or hyperoxia (85%) for 14 days from birth while being treated with vehicle, ATX inhibitor or LPA receptor 1 (LPA1) inhibitor. In vitro studies utilized human lung fibroblast (HLF) cells exposed to room air, 85% oxygen, or LPA for varying time periods. Supernatants and cells were collected for assays and Western blotting. Animals exposed to hyperoxia showed elevated expression of ATX, ATX activity, and LPA1. Inhibiting ATX or LPA1 improved alveolarization, reduced inflammation, and mitigated extracellular matrix deposition and lysyl oxidase (LOX) expression. LPA1 inhibition leading to reduced LOX expression was associated with a reduction in phosphorylation of AKT. Hyperoxia increases the expression of ATX and LPA1 associated with increased LOXin the lungs. Targeting the ATX/LPA1 pathway could be a potential therapeutic approach to BPD. Exposure to hyperoxia increases the expression and activity of autotaxin (ATX), as well as expression of LPA receptor 1 (LPA1). Increased expression of ATX influences extra cellular matrix (ECM) remodeling. Inhibitors targeting the ATX/LPA pathway could offer a new therapeutic approach to bronchopulmonary dysplasia (BPD), potentially mitigating ECM deposition and improving lung development.

CARD9 promotes cholangiocarcinoma by regulating the IL-17A/Hedgehog and the THEM4/AKT/mTOR signaling pathways

Cholangiocarcinoma (CCA) is a highly lethal malignant tumor originating from the bile duct, and its underlying mechanisms are not fully understood. In order to identify key genes in CCA, we downloaded gene expression data from public GSE76297, GSE26566 and TCGA-CHOL datasets. CARD9 was selected as a CCA-related gene from the datasets. Its expression was identified in the CCA tissues via PCR, western blot and immunohistochemistry assays. The loss-and-gain function assay of CARD9 was conducted in CCA cell lines (QBC939 and RBE) and nude mice. This study found a significant upregulation of CARD9 in CCA tissues, which is associated with poor prognosis in patients. Overexpression of CARD9 promotes proliferation and invasion of QBC939 and RBE cells, enhances the growth and development in CCA mouse models, and reduces sensitivity to chemotherapy drugs for CCA. Mechanistically, CARD9 activates the NF-κB and NLRP3 inflammatory signaling pathways, induces excessive release of various inflammatory factors, and triggers a cascade reaction of interleukin-17 (IL-17A). IL-17A promotes the stability of IHH mRNA by regulating HuR, enhances IHH transcription, and activates the Hedgehog signaling pathway to accelerate the progression of CCA. In addition, CARD9 promotes proliferation and invasion of CCA cells by interacting with THEM4, thereby facilitating AKT and mTOR phosphorylation, and accelerating the progression of CCA. Overall, these data suggest that CARD9 is involved in the progression of CCA by regulating the IL-17A/Hedgehog and the THEM4/AKT/mTOR signaling pathways. Therefore, CARD9 may serve as a novel therapeutic target for CCA treatment.

Anticancer Properties Against Select Cancer Cell Lines and Metabolomics Analysis of Tender Coconut Water.

Tender Coconut Water (TCW) is a nutrient-rich dietary supplement that contains in bioactive secondary metabolites and phytohormones with anti-oxidative and anti-inflammatory properties. Studies on TCW's anti-cancer properties are limited and the mechanism of its anti-cancer effects have not been defined. In the present study, we investigate TCW for its anti-cancer properties and, using untargeted metabolomics, we identify components form TCW with potential anti-cancer activity. Cell viability assay, BrdU incorporation assay, soft-agar assay, flow-cytometery, and Western blotting were used to analyze TCW's anticancer properties and to identify mechanism of action. Liquid chromatography- Tandem Mass Spectroscopy (LC-MS/MS) was used to identify TCW components. TCW decreased the viability and anchorage-independent growth of HepG2 hepatocellular carcinoma (HCC) cells and caused S-phase cell cycle arrest. TCW inhibited AKT and ERK phosphorylation leading to reduced ZEB1 protein, increased E-cadherin, and reduced N-cadherin protein expression in HepG2 cells, thus reversing the 'epithelial-to-mesenchymal' (EMT) transition. TCW also decreased the viability of Hep3B hepatoma, HCT-15 colon, MCF-7 and T47D luminal A breast cancer (BC) and MDA-MB-231 and MDA-MB-468 triplenegative BC cells. Importantly, TCW did not inhibit the viability of MCF-10A normal breast epithelial cells. Untargeted metabolomics analysis of TCW identified 271 metabolites, primarily lipids and lipid-like molecules, phenylpropanoids and polyketides, and organic oxygen compounds. We demonstrate that three components from TCW: 3-hydroxy-1-(4-hydroxyphenyl)propan-1-one, iondole-3-carbox aldehyde and caffeic acid inhibit the growth of cancer cells. TCW and its components exhibit anti-cancer effects. TCW inhibits the viability of HepG2 hepatocellular carcinoma cells by reversing the EMT process through inhibition of AKT and ERK signalling.

Tirzepatide, GIP(1-42) and GIP(1-30) display unique signaling profiles at two common GIP receptor variants, E354 and Q354.

Type 2 diabetes (T2D) and obesity are prevalent metabolic disorders affecting millions of individuals worldwide. A new effective therapeutic drug called tirzepatide for the treatment of obesity and T2D is a dual agonist of the GIP receptor and GLP-1 receptor. Tirzepatide is clinically more effective than GLP-1 receptor agonists but the reasons why are not well understood. Tirzepatide reportedly stimulates the GIP receptor more potently than the GLP-1 receptor. However, tirzepatide signaling has not been thoroughly investigated at the E354 (wildtype) or Q354 (E354Q) GIP receptor variants. The E354Q variant is associated increased risk of T2D and lower body mass index. To better understand GIP receptor signaling we characterized the activity of endogenous agonists and tirzepatide at both GIP receptor variants. Using Cos7 cells we examined wildtype and E354Q GIP receptor signaling, analyzing cAMP and IP1 accumulation as well as AKT, ERK1/2 and CREB phosphorylation. GIP(1-42) and GIP(1-30)NH2 displayed equipotent effects on these pathways excluding CREB phosphorylation where GIP(1-30)NH2 was more potent than GIP(1-42) at the E354Q GIP receptor. Tirzepatide favored cAMP signaling at both variants. These findings indicate that tirzepatide is a biased agonist towards Gαs signaling and suggests it equally activates the wildtype and E354Q GIP receptor variants. We also observed differences between the pharmacology of the GIP receptor variants with endogenous peptides, which may help to explain differences in phenotype. These findings contribute to a comprehensive understanding of GIP receptor signaling, and will aid development of therapies combating T2D and obesity.

Biological evaluation of sophoridine by interaction with plasma transport protein and protective effects in cardiomyocytes

Numerous studies have demonstrated the protective benefits of sophoridine, a bioactive alkaloid, on cell damage. However, its primary biological properties such as interaction with plasma protein, which serves as the primary drug carrier, and its cardioprotective properties are still unknown. In the present study, we aimed to analyze the binding characteristics of sophoridine with alpha-2-macroglobulin (α2M) by spectroscopic and theoretical studies. Then, the cardioprotective effects of sophoridine on myocardial ischemia/reperfusion (MI/R) injury in vitro were investigated using H9c2 cardiomyocytes. The results reveal that one molecule of sophoridine favorably binds to one molecule of α2M dominantly through interaction with hydrophobic amino acid residues (VAL758, PHE735, PHE735, and TRP739). Also, sophoridine leads to partial changes in the structure of this carrier protein in the vicinity of TRP739 residue. Cellular assays exhibit that sophoridine recovered cell viability, membrane leakage, and apoptosis induced by MI/R injury. It was detected that sophoridine could mitigate the oxidative stress and intrinsic apoptosis pathway through regulation of Bax, Bcl-2, and caspase. ELISA analysis also exhibits that sophoridine upregulates phosphorylation of Akt and PI3K in H9c2 cells. Our findings suggest that sophoridine could show favorable plasma protein interaction and promising cardioprotection against MI/R injury mediated by the upregulation of PI3K/AKT signaling pathway.

MTORC2 inhibition by JR-AB2-011 improves IL-1β-induced inflammation, catabolic response, and apoptosis in human chondrocytes through IκB-α/NF-κB p65.

Osteoarthritis (OA) is a very common chronic joint condition marked by inflammation and cartilage loss. mTOR is a well-known mediator of inflammation, cell survival, and aging; however, its role in OA has not been determined. To explore the role of mTORC2 in OA-and associated pathological changes, we examined the contribution of mTORC2-mediated Akt, rictor and IκB-α/NF-κB p65 pathway in interleukin (IL)-1β-treated human chondrocytes. We focused on the protein expression of proinflammatory cytokines and catabolic and apoptotic factors, including TNF-α, IL-6, iNOS, MMP13, Bax, and caspase3, which may occur through this signalling pathway in IL-1β-treated chondrocytes. Chondrocytes were cultured and treated with either 2 ng/mL IL‑1β alone or in combination with increasing concentrations of JR-AB2-011 (50, 100, or 250 µM), a selective mTORC2 inhibitor. The protein levels of phosphorylated (p)‑Akt, Akt, rictor, p-NF-κB p65, NF-κB p65, IκB-α, p-IκB-α, iNOS, MMP13, Bax, and caspase3 were evaluated by Western blotting. In IL-1β-stimulated chondrocytes, mTORC2 activity was increased with increased phosphorylation of Akt and expression of rictor. IL-1β increased the expression of p-IκBα, p-NF-κB p65, NF-κB p65, IL-6, TNF-α, iNOS, Bax, and caspase3 proteins and decreased the expression of IκB-α. All of these IL-1β-induced alterations were prevented by JR-AB2-011. The main novel finding in the present study is that selective mTORC2 inhibition by JR-AB2-011 prevents the inflammatory, catabolic, and apoptotic responses induced by IL-1β via modulation of IκB-α/NF-κB activity. Therefore, we demonstrated a previously unknown function of mTORC2 inhibition that seems to be a potential therapeutic target for OA.

Artemisia argyi mitigates doxorubicin-induced cardiotoxicity by inhibiting mitochondrial dysfunction through the IGF-IIR/Drp1/GATA4 signaling pathway.

Doxorubicin (DOX) is mostly utilized as a wide range of antitumor anthracycline to treat different cancers. The severe antagonistic impacts of DOX on cardiotoxicity constrain its clinical application. Many mechanisms are involved in cardiac toxicity induced by DOX in the human body. Mitochondria is a central part of fatty acid and glucose metabolism. Thus, impaired mitochondrial metabolism can increase heart failure risk, which can play a vital role in cardiomyocyte mitochondrial dysfunction. This study aimed to assess the possible cardioprotective effect of water-extracted Artemisia argyi (AA) against the side effect of DOX in H9c2 cells and whether these protective effects are mediated through IGF-IIR/Drp1/GATA4 signaling pathways. Although several studies proved that AA extract has benefits for various diseases, its cardiac effects have not yet been identified. The H9c2 cells were exposed to 1μM to establish a model of cardiac toxicity. The results revealed that water-extracted AA could block the expression of IGF-IIR/calcineurin signaling pathways induced by DOX. Notably, our results also showed that AA treatment markedly attenuated Akt phosphorylation and cleaved caspase 3, and the nuclear translocation markers NFATC3 and p-GATA4. Using actin staining for hypertrophy, we determined that AA can reduce the effect of mitochondrial reactive oxygen species and cell size. These findings suggest that water-extracted AA could be a suitable candidate for preventing DOX-induced cardiac damage.

Runx2 silencing sensitized human renal cell carcinoma cells to ABT-737 apoptosis

The prognostic value of Runt-related transcription factor 2 (Runx2) and its involvement in cell growth and motility have been reported in patients diagnosed with renal cell carcinoma (RCC). Since Runx2 may have the potential to be a target for the purpose of antitumor intervention, there is an urgent need to gain insight into its oncogenic properties. Using human 786-O, Caki-1 and ACHN RCC cells as models, the silencing of cellular Runx2 expression brought about a reduction in cyclin D1 and β-catenin expression, cell growth and migration without any significant cell death. Runx2-silenced cells turned into apoptosis vulnerable in the presence of ABT-737, a BH3 mimetic Bcl-2 inhibitor. Data from biochemical and molecular studies have revealed a positive correlation between Runx2 expression and Akt phosphorylation, Mcl-1 expression, and fibronectin expression. Results of genetic silencing studies have indicated the potential involvement of Mcl-1 and fibronectin in the decision of RCC cell ABT-737 resistance and sensitivity. The regulatory roles of the PI3K/Akt axis in the expression of Mcl-1 and fibronectin were suggested by means of the results taken from experiments involving pharmacological study of the PI3K/Akt. Since overexpression and prognostic roles of Runx2, activated Akt, Mcl-1, fibronectin, cyclin D1, and β-catenin have been revealed in RCC, it is important to explore the precise mechanisms underlying Runx2 oncogenic effects. Although the linking details between Runx2 and PI3K/Akt have yet to be identified, our findings suggest that Mcl-1 and fibronectin are downstream effectors of Runx2 via a regulatory axis of the PI3K/Akt and their promotion of cell growth, migration, and ABT-737 resistance in RCC cells.

Network pharmacology analysis revealed the mechanism and active compounds of jiao tai wan in the treatment of type 2 diabetes mellitus via SRC/PI3K/AKT signaling

Ethnopharmacological relevanceJiao-tai-wan (JTW) is a traditional Chinese herbal prescription, exerts its therapeutic effects on type 2 diabetes mellitus (T2DM). However, its mechanisms and active components remain unclear. Aim of the studyTo investigate the therapeutic mechanisms of JTW in treating type 2 diabetes mellitus (T2DM), focusing on identifying active components, their targets, and validating efficacy through SRC/PI3K/AKT signaling pathway modulation in vitro and in vivo. Materials and methodsActive ingredients were retrieved from the Traditional Chinese Medicine System Pharmacology (TCMSP) and Comprehensive Traditional Chinese Medicine Database (TCMID). Targets for these components were identified using the ChemMapper database based on 3D structural similarity. T2DM-related genes were sourced from the DisGeNET and Gene Expression Omnibus (GEO) databases. Protein-protein interaction (PPI) analysis and functional enrichment analysis were conducted to construct a pathway network of “herbs-active ingredients-candidate targets”, identifying core molecular mechanisms and key active ingredients. SwissDock was used for molecular docking to predict ligands for candidate targets. The diabetic models were established using C57BL/6 mice and human liver HepG2 cell lines. Their Effectiveness and key molecules were verified through biochemical detection and immunoblotting. ResultsTotal 30 active compounds, 597 active ingredient targets, 9631 T2DM-related genes, and 521 overlapping candidate targets were found for JTW on T2DM. Go enrichment indicated the core pathways enriched on insulin and glucose metabolism. The auto-docking demonstrated SRC has potential binds to ingredients of JTW. In vivo, JTW can reduce blood glucose, and blood lipid levels, and HOMA-IR, and increase HOMA-ISI levels in T2DM mice with reduced ALT, AST, MDA levels and increased SOD levels. Meanwhile, decreased phosphorylation of SRC, along with increased levels of phosphorylated PI3K, PI3K, and phosphorylated AKT, were observed. HE staining of liver tissues further confirmed that JTW administration improved liver morphology, reducing inflammation and necrosis. In vitro, JTW significantly ameliorates upstream dysregulation by reducing SRC phosphorylation while enhancing phosphorylated PI3K, PI3K, and AKT phosphorylation levels. ConclusionJTW may alleviate glucose, insulin resistance, and lipid metabolism disorders by the SRC/PI3K/AKT signaling pathway, that provide a novel view of potential active compounds and essential targets in treating T2DM.

8421 The Effect of Dietary Glycotoxins on Insulin Resistance in Polycystic Ovary Syndrome

Abstract Disclosure: A. Weidner: None. K. Vann: None. J. Zhang: None. A. Roy: None. O. Astapova: None. Polycystic ovary syndrome (PCOS) is a chronic systemic disorder that affects both metabolism and reproduction. PCOS is characterized by hyperandrogenism and insulin resistance, which are closely intertwined in this disease pathophysiology. PCOS affects an estimated 10% of people with ovaries and increases risk of endometrial cancer, type 2 diabetes, and major cardiovascular events. Furthermore, treatment options are generally poorly tolerated. Despite its prevalence, severity, and enormous public health burden, the etiology of PCOS is not well-understood. Insulin resistance likely plays a central role in PCOS pathogenesis, and often leads to hyperinsulinemia as the pancreas attempts to compensate for insulin’s decreased efficacy in the body. Moreover, the Western diet is becoming increasingly rich in advanced glycation end products (AGEs): non-enzymatically modified biomolecules that have recently been found to have a harmful effect on insulin sensitivity and are associated with diabetes, aging, and oxidative stress. Serum AGE levels have been reported to be higher in both lean and obese PCOS patients, regardless of PCOS subtype. We have also found higher levels of AGE precursors in the serum of our chronic dihydrotestosterone-induced mouse model of PCOS. Using this mouse model, we have shown that ovarian granulosa cells (GCs) remain insulin-sensitive despite systemic insulin resistance and hyperinsulinemia, manifested by impaired glucose tolerance and increased HOMA-IR. To assess whether GCs respond to insulin through alternative receptor pathways, we knocked down insulin receptor in human granulosa-derived KGN cells and found that insulin-stimulated AKT phosphorylation was only minimally reduced, suggesting that insulin may signal in large part through the insulin-like growth factor 1 receptor (IGF1R) in these cells. Therefore, we believe that compensatory hyperinsulinemia plays a key role in chronic GC hyperstimulation, possibly by rerouting the insulin signal through IGF1R to overcome the resistance to insulin receptor activation, suggesting a mechanism for AGE toxicity in PCOS. We hypothesize that in PCOS, this results in impaired GC function and exacerbation of ovarian dysfunction. We will use our PCOS mouse model to study the effects of a reduced-AGE diet in ameliorating the reproductive and metabolic phenotype of an androgenized PCOS mouse. In preliminary experiments, we have found that a low-AGE diet results in lower fasting glucose in C57BL/6J mice after only 4 weeks. We expect that reproductive symptoms, such as estrous cyclicity, and metabolic symptoms, such as GTT and circulating insulin, will improve in our PCOS mouse model when mice are fed a low-AGE diet throughout development and adulthood. This project has significant clinical implications, as reducing dietary AGEs could provide an effective, tolerable, non-drug therapy option for patients with PCOS. Presentation: 6/3/2024

Synergistic Dual Targeting of Thioredoxin and Glutathione Systems Irrespective of p53 in Glioblastoma Stem Cells.

Glioblastoma (GBM) is an incurable primary brain cancer characterized by increased reactive oxygen species (ROS) production. The redox-sensitive tumor suppressor gene TP53, wild-type (wt) for 70% of patients, regulates redox homeostasis. Glioblastoma stem cells (GSCs) increase thioredoxin (Trx) and glutathione (GSH) antioxidant systems as survival redox-adaptive mechanisms to maintain ROS below the cytotoxic threshold. Auranofin, an FDA-approved anti-rheumatoid drug, inhibits thioredoxin reductase 1 (TrxR1). L-buthionine sulfoximine (L-BSO) and the natural product piperlongumine (PPL) inhibit the GSH system. We evaluated the cytotoxic effects of Auranofin alone and in combination with L-BSO or PPL in GBM cell lines and GSCs with a known TP53 status. The Cancer Genome Atlas/GBM analysis revealed a significant positive correlation between wtp53 and TrxR1 expression in GBM. Auranofin induced ROS-dependent cytotoxicity within a micromolar range in GSCs. Auranofin decreased TrxR1 expression, AKT (Ser-473) phosphorylation, and increased p53, p21, and PARP-1 apoptotic cleavage in wtp53-GSCs, while mutant-p53 was decreased in a mutant-p53 GSC line. Additionally, p53-knockdown in a wtp53-GSC line decreased TrxR1 expression and significantly increased sensitivity to Auranofin, suggesting the role of wtp53 as a negative redox-sensitive mechanism in response to Auranofin in GSCs. The combination of Auranofin and L-BSO synergistically increased ROS, decreased IC50s, and induced long-term cytotoxicity irrespective of p53 in GBM cell lines and GSCs. Intriguingly, Auranofin increased the expression of glutathione S-transferase pi-1 (GSTP-1), a target of PPL. Combining Auranofin with PPL synergistically decreased IC50s to a nanomolar range in GSCs, supporting the potential to repurpose Auranofin and PPL in GBM.