FOXO1 Expression Research Articles

Diosgenin improves lipid metabolism in diabetic nephropathy via regulation of miR-148b-3p/DNMT1/FOXO1 axis.

The progression of diabetic nephropathy (DN) is closely associated with lipid accumulation. Diosgenin (Dio) plays a beneficial role in the lipid metabolism associated with multiple diseases. Thus, the mechanism underlying Dio's function in DN associated with aberrant lipid accumulation warrants further investigation. To model DN in vitro, HK-2 cells were treated with high glucose (HG) and palmitic acid. Cell viability was evaluated using MTT assay. The triglyceride (TG) content in HK-2 cells was measured using a commercial assay kit. The formation of lipid droplets in HK-2 cells was observed using Oil Red O staining. The expression levels of mRNA and protein were detected using RT-qPCR and western blot, respectively. The DNA methylation of FOXO1 was assessed using MSP. The interaction between DNMT1 and the FOXO1 promoter was confirmed by ChIP assay. Dio treatment reduced TG levels and lipid droplet formation in HK-2 cells co-treated with HG and palmitic acid. Simultaneously, the levels of miR-148b-3p and FOXO1 were increased by Dio, while Dio decreased the expression levels of DNMT1 and SREBP-2. Meanwhile, miR-148b-3p can bind to DNMT1, which in turn inhibits the expression of FOXO1 by mediating the DNA methylation of FOXO1. In addition, FOXO1 negatively regulates the expression of SREBP-2 by interacting with the SREBP-2 promoter. MiR-148b-3p inhibition or silencing of FOXO1 abolished the inhibitory effect of Dio on TG production and lipid droplet formation. This effect was further exacerbated by the downregulation of DNMT1. FOXO1 overexpression may counteract the promotive effects of miR-148b-3p inhibitor on lipid accumulation. Dio treatment reduced TG production and lipid droplet formation in HK-2 cells during the progression of DN by modulating the miR-148b-3p/DNMT1/FOXO1/SREBP-2 axis. This finding provides new evidence supporting the therapeutic potential of Dio for DN.

Hydrogels Treat Atopic Dermatitis by Transporting Marine-Derived miR-100-5p-Abundant Extracellular Vesicles.

Atopic dermatitis (AD) is a prevalent skin disorder worldwide. However, many AD medications are unsuitable for long-term use due to low therapeutic efficacy and side effects. Extracellular vesicles (EVs) extracted from Pinctada martensii mucus have demonstrated therapeutic efficacy in AD. It is hypothesized that EVs may exert their activity on mammalian cells through their specific contents. In this study, we analyzed the results of miRNA sequencing of the EVs and investigated the potency of highly expressed miR-100-5p in treating AD. To enhance the therapeutic efficiency of the EVs in AD, we developed oxidized sodium alginate (OSA)-carboxymethyl chitosan (CMCS) self-cross-linked hydrogels as a vehicle to deliver the EVs to BALB/c mice with dermatitis. The miR-100-5p in EVs exhibited a favorable anti-inflammatory function, while the hydrogels provided enhanced skin residency. Additionally, its efficacy in inflammation inhibition and collagen synthesis was demonstrated in in vivo experiments. Mechanistically, miR-100-5p in EVs exerted anti-inflammatory effects by inhibiting the expression of FOXO3, consequently suppressing the activation of the downstream NLRP3 signaling pathway. This study underscores the significance of utilizing OSA-CMCS hydrogels as a vehicle for delivering miR-100-5p in P. martensii mucus-derived EVs for the treatment of AD.

High CTLA-4 gene expression is an independent good prognosis factor in breast cancer patients, especially in the HER2-enriched subtype

BackgroundBreast cancer (BC) is the most common cancer in women and the leading cause of cancer-related death worldwide. This heterogeneous disease has been historically considered a non-immunogenic type of cancer. However, recent advances in immunotherapy have increased the interest in knowing the role of the immune checkpoints (IC) and other immune regulation pathways in this neoplasia.MethodsIn this retrospective study, we evaluated the correlation of mRNA expression of CTLA-4, PDCD1 (PD1), CD274 (PD-L1), PDCD1LG2 (PD-L2), CD276 (B7-H3), JAK2, and FOXO1 with clinicopathological factors and BC patient’s outcome by real-time quantitative polymerase chain reaction (qPCR).ResultsOur results showed that immunoregulatory gene expression depends on BC immunophenotype being CTLA-4 and PDCD1 (PD1) overexpressed on triple-negative/basal-like (TN/BL) and luminal B/HER2-positive phenotypes, respectively, and CD276 (B7-H3), JAK2 and FOXO1 associated with both luminal A and luminal B/HER2-negative tumors. In addition, we found that these genes can also be related to aggressive and non-aggressive clinicopathological characteristics in BC. Finally, survival analysis showed that CTLA-4 expression levels emerge as a significant independent factor of good prognosis in BC patients, especially in the HER2-enriched subtype.ConclusionConsidering all these data, we can conclude that the expression of immunoregulatory genes depends on tumor phenotype and has potential clinical implications in BC patients.

Acetyl-11-keto-β-boswellia acid attenuates Ti particle-induced osteoblastic oxidative stress and osteolysis through the Foxo3 signaling pathway

Oxidative stress injury in osteoblasts is one of the leading causes of periprosthetic osteolysis (PPOL). Acetyl-11-keto-β-boswellia acid (AKBA) has been used as an antioxidant in the treatment of various diseases, but its antioxidant mechanism in osteolysis has yet to be elucidated. In this study, a mouse cranial osteolysis model was constructed, and MC3T3-E1 cells and bone marrow mesenchymal stem cells (BMSCs) were cultured in vitro. Western blotting and immunofluorescence staining revealed that titanium (Ti) particles aggravated osteoblast oxidative stress injury and apoptosis. Ti particles and hydrogen peroxide reduced the osteogenic ability of BMSCs. At a certain concentration, AKBA alleviated the oxidative stress injury of MC3T3-E1 cells induced by Ti particles and enhanced the osteogenic ability of BMSCs, and the expression of Forkhead box O3 (Foxo3) increased with increasing AKBA concentration. To verify the antioxidant mechanism of AKBA, we designed and synthesized Foxo3-targeting siRNAs. We found that after Foxo3 expression was inhibited, the protective effect of AKBA on osteoblasts decreased significantly. Moreover, AKBA treatment suppressed bone mass loss in the skull mediated by Ti particles in mice. Therefore, we suggest that AKBA alleviates the oxidative stress injury in osteoblasts induced by Ti particles, at least in part, by regulating the expression of Foxo3. In this study, the mechanism and biosafety of AKBA in treating PPOL were demonstrated to some extent.

FoxO1 Deficiency in Monocytic Myeloid-Derived Suppressor Cells Exacerbates B Cell Dysfunction in Systemic Lupus Erythematosus.

Myeloid-derived suppressor cells (MDSCs) contribute to the pathogenesis of systemic lupus erythematosus (SLE), in part due to promoting the survival of plasma cells. FoxO1 expression in monocytic MDSCs (M-MDSCs) exhibits a negative correlation with the SLE Disease Activity Index score. This study aimed to investigate the hypothesis that M-MDSC-specific FoxO1 deficiency enhances aberrant B cell function in aggressive SLE. We used GEO data sets and clinical cohorts to verify the clinical significance of FoxO1 expression and circulating M-MDSCs. Using Cre-LoxP technology, we generated myeloid FoxO1 deficiency mice (mFoxO1-/-) to establish murine lupus-prone models. The transcriptional stage was assessed by integrating chromatin immunoprecipitation (ChIP)-sequencing with transcriptomic analysis, luciferase reporter assay, and ChIP-quantitative polymerase chain reaction. Methylated RNA immunoprecipitation sequencing, RNA sequencing, and CRISPR-dCas9 were used to identify N6-adenosine methylation (m6A) modification. In vitro B cell coculture experiments, capmatinib intragastric administration, m6A-modulated MDSCs adoptive transfer, and sample validation of patients with SLE were performed to determine the role of FoxO1 on M-MDSCs dysregulation during B cell autoreacted with SLE. We present evidence that low FoxO1 is predominantly expressed in M-MDSCs in both patients with SLE and lupus mice, and mice with myeloid FoxO1 deficiency (mFoxO1-/-) are more prone to B cell dysfunction. Mechanically, FoxO1 inhibits mesenchymal-epithelial transition factor protein (Met) transcription by binding to the promoter region. M-MDSCs FoxO1 deficiency blocks the Met/cyclooxygenase2/prostaglandin E2 secretion pathway, promoting B cell proliferation and hyperactivation. The Met antagonist capmatinib effectively mitigates lupus exacerbation. Furthermore, alkB homolog 5 (ALKBH5) targeting catalyzes m6A modification on FoxO1 messenger RNA in coding sequences and 3' untranslated regions. The up-regulation of FoxO1 mediated by ALKBH5 overexpression in M-MDSCs improves lupus progression. Finally, these correlations were confirmed in untreated patients with SLE. Our findings indicate that effective inhibition of B cells mediated by the ALKBH5/FoxO1/Met axis in M-MDSCs could offer a novel therapeutic approach to manage SLE.

Enhancing Wound Healing via Modulation of Autophagy-Induced Apoptosis: The Role of Nicotinamide Riboside and Resveratrol in Streptozotocin-Treated Diabetic Rat

BackgroundImpaired wound healing from diabetes mellitus (DM) causes lower limb amputations, posing clinical, social, and economic issues. Hypoxia and advanced glycation end products cause autophagy and apoptosis dysregulation, which delays wound healing. The study will test systemic and topical Nicotinamide Riboside (NR) and Resveratrol (RSV) for the capacity to modulate autophagy and apoptosis via the SIRT-1-FOXO1 pathway and improve diabetic wound healing. Methods54 male Sprague-Dawley rats were separated into control, diabetic (T1D), T1D-Gel-Base, T1D-NR, T1D-RSV, and T1D-NR+RSV groups. Rats were gavaged with 50 mg/kg/day RSV and 300 mg/kg/day NR for 5 weeks before having their wounds topically treated with 5% NR and RSV gel for 15 days after diabetes induction. Biochemical, histomorphometric, and stereological assays were conducted. The mRNA expressions of SIRT-1, FOXO1, VEGF, BAX, Cas3, Bcl-2, Beclin1, LC3IIβ, P62, and ATG5 were examined by qRT-PCR. ResultsNR and RSV improved diabetic rat wound closure. Diabetic rats treated with NR and RSV had significantly higher LC3IIβ, VEGEF, Bcl-2, and SIRT-1 mRNA levels. Bcl-2, p62, and ATG5 were regulated whereas BAX and Cas 3 were reduced. Stereological investigations showed epidermal, dermal, collagen bundle, vascular, and fibroblast density enhancements. ConclusionThis study highlights the potential of NR and RSV, acting as SIRT-1 activators, in improving diabetic wound healing by regulating SIRT-1-FOXO1-mediated autophagy and apoptosis. These findings offer valuable insights for developing targeted strategies to enhance diabetic wound healing. The combination of NR and RSV showed promising effects, suggesting a potential therapeutic approach for improving diabetic wound healing.

PI 3-kinase/AKT/FOXO1 signaling in smooth muscle cells protects against abdominal aortic aneurysm

Abstract Background Abdominal aortic aneurysms (AAA) are characterized by loss of vascular smooth muscle cells (SMCs). Growth factors induce proliferation and cell survival of SMCs. PI 3-kinase (PI3K) is an important downstream mediator in growth factor signaling. Objective This project is based on the hypothesis that PI3K-mediated SMC proliferation can compensate for the loss of SMCs in AAA and thus inhibit AAA progression. Therefore, we determined effects of reduced PI3K activity in SMCs on AAA progression, cell proliferation as well as vascular remodeling. Methods To investigate the role of PI3K, we analyzed mice and aortic SMCs harboring a smooth muscle-specific deficiency of the PI3K catalytic subunit p110α (SM-p110α-/-). AAA development in SM-p110α-/- mice and wild-type (WT) littermates was induced by perfusion of the infrarenal aortic segment with porcine pancreatic elastase (PPE). AAA diameter was determined by echocardiography. Structural changes in the aorta were identified by (immuno)histochemical staining. SMC proliferation was measured in vitro by BrdU incorporation or real-time cell analysis. p110α induced signal transduction pathways were characterized by Western blot and transcriptome analysis. Results PPE treatment led to a massive loss of SMCs in the compromised aortic segment after 3 days in both SM-p110α-/- and WT mice. The aortic diameter was enlarged after 28 days in all PPE-treated mice. However, the increase was significantly higher in SM-p110α-/- mice (70.2±10.8%, n=9) compared to WT animals (42.4±6.0%, n=10) (p<0.05). A similar effect was obtained in aged (untreated) mice: In 24-month-old SM-p110α-/- mice, abdominal aortic lumen diameter was increased by 42% compared to 3-month-old animals, but only by 15% in corresponding WT mice. PPE-treated SM-p110α-/- mice were characterized by reduced vascular remodeling with less PCNA-positive proliferating cells compared to WT animals. In addition, proliferation of aortic p110α-/- SMCs in serum-containing medium was impaired. Mechanistic analyses showed that PDGF- and insulin-induced activation of AKT as well as AKT-dependent phosphorylation and inactivation of the transcription factors FOXO-1, -3 and -4 were significantly reduced in p110α-/- SMCs compared to WT cells. Further analyses revealed that specific inhibition of FOXO1 by AS1842856 rescued PDGF or serum stimulated proliferation of p110α-/- SMCs. In addition, expression analysis of FOXOs in patients revealed significantly increased FOXO1 expression in AAA compared to aortic samples from healthy individuals. Conclusion Deficiency of the catalytic PI3K isoform p110α in SMCs promotes the development and progression of AAA. p110α/AKT-dependent inactivation of FOXO1 induces SMC proliferation, which can ameliorate aortic wall damage. As FOXO1 inhibitors and a recently developed p110α activator are available, the PI3K/AKT/FOXO1 signaling cascade may provide a therapeutic target to influence the stability of the aortic wall in AAA.

Bifidobacterium lactis-Derived Vesicles Attenuate Hippocampal Neuroinflammation by Targeting IL-33 to Regulate FoxO6/P53 Signaling.

Hippocampal Neuroinflammation (HNF) is a critical driver of cognitive impairment. The lipopolysaccharide (LPS) accumulate amyloid beta (Aβ) and lead to HNF. The Bifidobacterium lactis (BL) 99 have anti-inflammatory ability. However, whether BL99-derived microbiota-derived vesicles (MV) could alleviate LPS-induced HNF remains unclear. To investigate, we used ultrafiltration with ultracentrifuge to extract BL99-derived-MV (BL99-MV). We used hippocampal neuronal HT22 cells (HT22) to establish the LPS-induced HNF model, and explored whether BL99-MV alleviate LPS-induced HNF. The confocal microscopy showed that BL99-MV were taken up by HT22 and reduced the oxidative stress (ROS) level. The PCR showed that BL99-MV up-regulate IL-10 level, and down-regulate TNF-α, IL-1β, and IL-6. Transcriptomic analysis revealed 4127 differentially expressed genes, with 2549 genes upregulated and 1578 genes downregulated in the BL99-MV group compared to the LPS group. Compared to the LPS group, BL99-MV decreased FoxO6, IL-33, P53, and NFκB expression, but increased FoxO1 and Bcl2 expression. The WB showed that BL99-MV modulated NFκB, FoxO6, P53, Caspase9, and Caspase3 protein expression by reducing IL-33 expression in HT22. The findings demonstrated IL-33 as a regulator for FoxO6/P53 signaling. Here, we hypothesized that BL99-MV alleviated LPS-induced HNF to promote HT22 survival and synaptic development by regulating FoxO6/P53 signaling by targeting IL-33.

High-intensity interval training attenuates renal injury induced by myocardial ischemia-reperfusion in rats

Background: High-Intensity Interval Training (HIIT) has been recognized as an effective form of short-duration exercise. The purpose of this study was to assess whether HIIT could reduce renal injury induced by myocardial ischemia-reperfusion in rats. Methods: Male Sprague-Dawley rats were randomly assigned to the Sham Group (SHAM), Coronary Artery Occlusion (CAO) group, HIIT group, and Ischemic Precondition (IPC) group. Rats underwent 40 minutes of left anterior descending coronary artery occlusion under anesthesia, followed by 3 hours of reperfusion, to induce myocardial ischemia-reperfusion (MIR). Post-surgery, rats were sacrificed, and their blood, heart, and kidney tissues were examined. The HIIT group underwent 4 weeks of HIIT training before surgery. Results: HIIT intervention significantly reduced renal injury after MIR and the concentrations of Blood Urea Nitrogen (BUN) and Creatinine (CRE) in the serum. Moreover, pro-inflammatory cytokines, including Tumor Necrosis Factor-α (TNF-α), Interleukin-1β (IL-1β), and IL-6, were significantly decreased, while the anti-inflammatory cytokine IL-10 was significantly increased in the serum. Additionally, HIIT intervention suppressed the expression of FoxO1, BAX/Bcl-2 ratio, TNF-α, and Cleaved-caspase-3/caspase-3 ratio in kidney tissues, ultimately reducing renal cell apoptosis. Conclusion: This study is the first to demonstrate that HIIT has effects similar to IPC, significantly reducing renal injury after MIR. HIIT regulates the production of pro-inflammatory and anti-inflammatory cytokines, inhibits renal cell apoptosis, thereby reducing the occurrence of cardio-renal syndrome.

Multi-omics reveals the molecular mechanisms of rapid growth in distant hybrid fish

Distant hybridization usually results in heterosis. In previous studies, we obtained an improved fish (WR-II, 2n = 100) with rapid growth through distant hybridization. To investigate the molecular mechanisms underlying the rapid growth of WR-II, the intestinal structure, microbiome and metabolome of the gut and the transcriptome of muscle were compared between WR-II and its parents (WCC and RCC, 2n = 100). The results revealed that WR-II had more intestinal folds, and the height, width and goblet cell density of the intestinal folds were greater than those of its parents. The absolute abundances of the probiotics Ralstonia and Salinivibrio were significantly greater in WR-II than in the parents (p < 0.05), and the probiotic Cupriavidus was found in only WR-II. A total of 481 and 718 differentially abundant metabolites (DMs) were identified in WR-II vs. WCC and WR-II vs. RCC, respectively. These DMs were enriched mainly in pathways related to metabolism, protein synthesis and development, such as fumaric acid, sphingosine, betaine and 5′-adenylic acid (AMP). A total of 5680 and 11,795 differentially expressed genes (DEGs) were identified in WR-II vs. WCC and WR-II vs. RCC, respectively. These DEGs were enriched mainly in pathways related to protein synthesis and substance metabolism. Growth-related genes such as irs1, irs2, foxo1, igf1r, and akt were upregulated in WR-II. Multi-omics analysis revealed that Salinivibrio can promote the level of AMP, which then regulates the expression of irs1 and foxo1, thus promoting the growth of WR-II. This study revealed the mechanism of rapid growth of WR-II from the probiotics, metabolites and genes, which provides evidence for the molecular mechanism of heterosis.

Neuronal alterations in AKT isotype expression in schizophrenia.

Schizophrenia is characterized by substantial alterations in brain function, and previous studies suggest insulin signaling pathways, particularly involving AKT, are implicated in the pathophysiology of the disorder. This study demonstrates elevated mRNA expression of AKT1-3 in neurons from schizophrenia subjects, contrary to unchanged or diminished total AKT protein expression reported in previous postmortem studies, suggesting a potential decoupling of transcript and protein levels. Sex-specific differential AKT activity was observed, indicating divergent roles in males and females with schizophrenia. Alongside AKT, upregulation of PDPK1, a critical component of the insulin signaling pathway, and several protein phosphatases known to regulate AKT were detected. Moreover, enhanced expression of the transcription factor FOXO1, a regulator of glucose metabolism, hints at possible compensatory mechanisms related to insulin signaling dysregulation. Findings were largely independent of antipsychotic medication use, suggesting inherent alterations in schizophrenia. These results highlight the significance of AKT and related signaling pathways in schizophrenia, proposing that these changes might represent a compensatory response to a primary defect of canonical insulin signaling pathways. This research underscores the need for a detailed understanding of these signaling pathways for the development of effective therapeutic strategies.

Txnip promotes autophagic apoptosis in diabetic cardiomyopathy by upregulating FoxO1 and its acetylation

Autophagy dysfunction and apoptosis exacerbate the risk of heart failure in patients with diabetic cardiomyopathy (DCM). However, the interactions between autophagy and apoptosis in DCM and their underlying mechanisms remain poorly understood. This study induced type 1 DCM in C57BL/6 mice via streptozotocin injection and exposed H9C2 cells to high glucose to investigate these mechanisms. The study revealed a significant elevation in autophagic vesicles and compromised autophagic flux, accompanied by pronounced myocardial cell apoptosis in the myocardium of diabetic mice. Long-term exposure to high glucose in H9C2 cells led to enhanced autophagosome formation and impaired autophagic flux, while inhibition of autophagy with 3-MA reduced cell apoptosis. Additionally, we observed an increase in Txnip expression in the myocardium of diabetic mice and in high glucose-treated H9C2 cells, which regulates autophagic apoptosis in high glucose-treated H9C2 cells. Furthermore, Txnip regulates autophagic apoptosis through the modulation of forkhead box-1 (FoxO1) expression and acetylation. Prolonged high glucose exposure resulted in increased levels of phosphorylated sirtuin 1 (SIRT1) and reduced SIRT1/FoxO1 interaction, changes that were ameliorated by Txnip knockdown. Txnip overexpression elevated FoxO1 levels, which could be suppressed by NAC and GSH. These findings revealed that Txnip mediates autophagic apoptosis in DCM by upregulating FoxO1 via ROS and enhancing FoxO1 acetylation through the suppression of SIRT1 activity. The discovery of this new mechanism provides new perspectives and potential therapeutic targets for understanding and treating DCM.

Hederagenin regulates the migration and invasion of hepatocellular carcinoma cells through FOXO signaling pathway.

This study aimed to elucidate the effects of Hederagenin (HG) on hepatocellular carcinoma (HCC) and explore its potential molecular mechanisms. Virtual screening was employed to identify potential targets within core pathways of liver cancer and to analyze the possible mechanisms of HG. CCK-8 assays were used to assess the viability of HCC cells, while Hoechst 33342/PI staining was utilized to evaluate apoptosis. The migration and invasion abilities of HCC cells were examined using Transwell and scratch assays, and single-cell cloning ability was assessed via colony formation assays. Subsequent qRT-PCR was conducted to determine the mRNA expression levels of FOXO1 and FOXO6 following HG treatment. Western blot (WB) analysis was employed to measure the protein expression levels of IGF1R, FOXO1, FOXO6, MMP2, MMP9, and VEGFA, as well as the phosphorylation status of FOXO1 Ser249. Virtual screening indicated that HG might exert antitumor effects through the FOXO signaling pathway. Experimental results demonstrated that HG induces apoptosis in a dose-dependent manner and inhibits the proliferation, migration, invasion, and single-cell cloning ability of HCC cells. After HG treatment, FOXO1 expression was upregulated, while the expression levels of IGF1R, phosphorylated FOXO1 Ser249, MMP2, MMP9, and VEGFA were downregulated. In summary, our study is the first to demonstrate that HG regulates the phosphorylation of FOXO1, affecting the proliferation, migration, and invasion of HCC cells. The findings suggest that HG can inhibit the migration of HCC cells in vitro. The data indicate that HG-mediated targeting of the FOXO1/FOXO6 pathway holds promise as a novel therapeutic approach.

FOXO1-mediated autophagy regulation by miR-223 in sepsis-induced immunosuppression.

Immunosuppression is the main cause of the high mortality rate in patients with sepsis. The decrease in the number and dysfunction of CD4+ T lymphocytes is crucial to the immunosuppressed state of sepsis, in turn affecting the development and prognosis of sepsis. Autophagy has been shown to play an important role in the immune imbalance exhibited during sepsis. In this study, we modulate the expression of miR-223 in CD4+ T lymphocytes, via the transfection of a mimic or an inhibitor of miR-223 to establish cell models of miR-223 overexpression and knockdown, respectively. Levels of autophagy were monitored using a double-labeled lentivirus (mRFP-GFP-LC3) and electron microscopy, and western blot analysis was used to estimate the levels of autophagy-related proteins and FOXO1 in the two cell models after co-treatment with lipopolysaccharide (LPS) and siRNA against FOXO1. We found that when the expression of miR-223 increased, FOXO1 expression decreased and autophagy decreased; whereas, when FOXO1 expression was inhibited, autophagy decreased significantly in different cell models after LPS induction. Thus, this study proved that miR-223 participate in the regulation of LPS-induced autophagy via the regulation of FOXO1 expression in CD4+ T lymphocytes which shed a new light for the diagnosis and treatment of sepsis.

Triple Combination of Entinostat, a Bromodomain Inhibitor, and Cisplatin Is a Promising Treatment Option for Bladder Cancer.

Cisplatin is part of the first-line treatment of advanced urothelial carcinoma. Cisplatin resistance is a major problem but may be overcome by combination treatments such as targeting epigenetic aberrances. Here, we investigated the effect of the class I HDACi entinostat and bromodomain inhibitors (BETis) on the potency of cisplatin in two pairs of sensitive and cisplatin-resistant bladder cancer cell lines. Cisplatin-resistant J82cisR and T24 LTT were 3.8- and 24-fold more resistant to cisplatin compared to the native cell lines J82 and T24. In addition, a hybrid compound (compound 20) comprising structural features of an HDACi and a BETi was investigated. We found complete (J82cisR) or partial (T24 LTT) reversal of chemoresistance upon combination of entinostat, JQ1, and cisplatin. The same was found for the BETis JQ35 and OTX015, both in clinical trials, and for compound 20. The combinations were highly synergistic (Chou Talalay analysis) and increased caspase-mediated apoptosis accompanied by enhanced expression of p21, Bim, and FOXO1. Notably, the combinations were at least 4-fold less toxic in non-cancer cell lines HBLAK and HEK293. The triple combination of entinostat, a BETi, and cisplatin is highly synergistic, reverses cisplatin resistance, and may thus serve as a novel therapeutic approach for bladder cancer.

Transcription and post-translational mechanisms: dual regulation of adiponectin-mediated Occludin expression in diabetes

BackgroundOccludin, a crucial component of tight junctions, has emerged as a promising biomarker for the diagnosis of acute ischemic disease, highlighting its significant potential in clinical applications. In the diabetes, Occludin serves as a downstream target gene intricately regulated by the adiponectin (APN) signaling pathway. However, the specific mechanism by which adiponectin regulates Occludin expression remains unclear.Methods and resultsEndothelial-specific Ocln knockdown reduced APN-mediated blood flow recovery after femoral artery ligation and nullified APN's protection against high-fat diet (HFD)-triggered apoptosis and angiogenesis inhibition in vivo. Mechanically, we have meticulously elucidated APN's regulatory role in Occludin expression through a comprehensive analysis spanning transcriptional and post-translational dimensions. Foxo1 has been elucidated as a crucial transcriptional regulator of Occludin that is modulated by the APN/APPL1 signaling axis, as evidenced by validation through ChIP-qPCR assays and Western blot analysis. APN hindered Occludin degradation via the ubiquitin–proteasome pathway. Mass spectrometry analysis has recently uncovered a novel phosphorylation site, Tyr467, on Occludin. This site responds to APN, playing a crucial role in inhibiting Occludin ubiquitination by APN. The anti-apoptotic and pro-angiogenic effects of APN were attenuated in vitro and in vivo following Foxo1 knockdown or expression of a non-phosphorylatable mutant, OccludinY467A. Clinically, elevated plasma concentrations of Occludin were observed in patients with diabetes. A significant negative correlation was found between Occludin levels and APN concentrations.ConclusionOur study proposes that APN modulates Occludin expression through mechanisms involving both transcriptional and post-translational interactions, thereby conferring a protective effect on endothelial integrity within diabetic vasculature.Graphical abstract

The protective effects of esculetin against Doxorubicin‐Induced hepatotoxicity in rats: Insights into the modulation of Caspase, FOXOs, and heat shock protein pathways

AbstractDoxorubicin (DOX) is an anthracycline antibiotic widely employed to treat carcinoma. Nevertheless, severe cardiotoxic side effects restrict its clinical use. Esculetin, a natural flavonoid, is found abundantly in plants. This study evaluated the protective effects of esculetin against DOX‐induced hepatotoxicity in rat livers. Forty‐eight rats were randomly divided into six groups with eight rats in each group: control (I), DOX (II), esculetin (III, 50 mg/kg), esculetin (IV, 100 mg/kg), DOX+esculetin 50 (V, DOX+esculetin 50 mg/kg), and DOX+esculetin 100 (VI, DOX+esculetin 100 mg/kg). The administration of esculetin effectively mitigated alterations in the measured biochemical parameters induced by DOX. Gene expression analyses demonstrated that esculetin treatment significantly reduced the DOX‐induced expression of Foxo1, Hspa1a, Hsp4a, Hsp5a, Casp3, and Casp9 while increasing the DOX‐induced expression of Foxo3. These findings suggest that esculetin, with its antioxidant and anti‐inflammatory effects, might be a therapeutic option for protecting against DOX‐induced hepatotoxicity.

The Effect of Resveratrol on Gamma Globin Gene Expression in Patients with Beta Thalassemia: The Role of Adaptation to Cellular Stress

HbF induction is an appropriate strategy to ameliorate the severity of β-thalassemia symptoms. Hydroxyurea (HU) is the most common chemical agent introduced as an HbF inducer but responsiveness to HU is variable and the introduction of HbF inducers alternative to HU with low cytotoxicity has been a crucial challenge. Resveratrol is an HbF inducer agent that may have favorable effects on the differentiation of hematopoietic erythroid progenitors (HEPs). The present study aimed to investigate the effect of resveratrol on γ-globin, stress response, and anti-apoptotic gene expression among hydroxyurea (HU)-responders and HU-nonresponders (HU-NR). Four cases of HU-R and four cases of HU-NR were studied. HEPs of the patients were cultured, and the expression of γ-globin, Foxo3, and Bclxl was assessed. Moreover, the differentiation and apoptotic rate of the cells were investigated using flow cytometry analysis. In three cases, the γ-globin gene expression increased after resveratrol treatment. All of the HU-NR patients were also non-responders to resveratrol (Res-NR). The expression of Foxo3 and Bclxl genes was higher in responders to resveratrol (Res-R) compared to non-responders (Res-NR). The rate of apoptosis in Res-R patients was also lower than in Res-NR. Responders to resveratrol also had a higher rate of HEP maturation. The cells of both HU–NR and Res-NR patients could not adapt to stress conditions and proceed to the erythroid differentiation. In conclusion, resveratrol increased the γ-globin expression in HEPs of β-thalassemia patients. The response was observed only in R-HU patients with similar cellular pathways.

Ginkgolide A enhances FoxO1 expression and reduces endoplasmic reticulum stress to mitigate osteoarthritis in mice

This study aimed to investigate the effects of Ginkgolide A (GA) on chondrocytes under oxidative stress and to elucidate its potential molecular mechanisms. Using a destabilization of the medial meniscus (DMM) model in mice and an in vitro osteoarthritis (OA) model induced by tert-butyl hydroperoxide (TBHP) in chondrocytes, we validated the therapeutic efficacy and underlying mechanisms of GA. Potential OA targets of GA were identified through network pharmacology, Gene Ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Further exploration into the effects on endoplasmic reticulum stress (ERS), apoptosis, extracellular matrix (ECM) degradation, and Forkhead Box O1 (FoxO1) related pathways was conducted using Western blotting, immunofluorescence, TUNEL staining, flow cytometry, X-ray, micro-computed tomography (Micro-CT) analysis, and histological staining. The results demonstrated that GA upregulated FoxO1 expression and inhibited ERS-related signaling pathways, thereby reducing apoptosis and ECM degradation. In conclusion, GA significantly alleviated OA symptoms both in vitro and in vivo, suggesting its potential as a therapeutic agent for OA.

L-carnitine cause to increase cell proliferation of C-Kit+ hematopoietic progenitor cells via decreasing the PI3K and FOXO-1 protein expression

BackgroundStem cell-based therapy has emerged as an attractive approach for regenerative medicine. Poor survival and maintenance of the cells used in regenerative medicine are considered as serious barriers to enhance the efficacy of the cell therapy. Using some antioxidants has been reported to prevent the aging of stem cells, and finding effective factors to reduce the senescence of these cells has impressive potential in cell therapy. The PI3K pathway adversely regulates the transcription factors known as FOXO, which are thought to have an inhibitory influence on cell proliferation. By downregulating FOXO and other targets, PI3K signaling controls the growth of cells. For this reason, the aim of the present study is to investigate the effect of L-carnitine (LC) as antioxidant on the cell proliferation and the protein expression of PI3K and FOXO. MethodsFor understanding the in vitro effect of LC on the PI3K and FOXO-1 expression of C-kit+ hematopoietic progenitor cells, the bone marrow mononuclear cells were isolated, and C-kit+ cells was enriched by the magnetic-activated cell sorting (MACS). Next, the identification of enriched C-kit+ cells were done by flowcytometry and immunocytochemistry. Then, C-kit+ cells were treated with 0.2 mM LC, the cells were collected at the end of the treatment period (48 h), and the proteins were extracted. In the following, the protein expression of PI3K and FOXO-1 was measured by western blotting. In addition, flowcytometry was done to assess the Ki-67 expression as a key marker for cell proliferation investigation. Results0.2 mM LC cause to significantly decrease in the protein expression of PI3K and FOXO-1 (*P<0.05 and **P<0.01, respectively). Also, the expression of Ki-67 was significantly increased in the presence of 0.2 mM LC (***P<0.001). ConclusionBriefly, LC can be considered an effective factor in increasing the proliferation of C-kit+ cells via some signaling pathways.