FOXO3a Levels Research Articles

The Janus-faced role of TRPM2-S in retroperitoneal liposarcoma via increasing ROS levels

BackgroundRetroperitoneal liposarcoma (RPLS) is a specific soft tissue sarcoma with a high recurrence rate. The short isoform of transient receptor potential cation channel subfamily M member 2 (TRPM2-S) plays an important role in the regulation of reactive oxygen species (ROS). However, the association between TRPM2-S and RPLS and its underlying mechanisms remains unclear.MethodsThe expression of both TRPM2-S and TRPM2-L in RPLS tissues was verified by kimmunohistochemistry (IHC). The regulation on Ca2+ influx by TRPM2-S was evaluated by Fluo-4 AM staining. The effect of TRPM2-S on cell proliferation and apoptosis was tested by 5-Ethynyl-2′-deoxyuridine (EdU) staining and Flow cytometry respectively. The level of cellular ROS was assessed by the DCFH-DA probe. Different concentrations of H2O2 were used to provide oxidative stress on RPLS cells. The underlying mechanisms were further explored by Western blotting.ResultsThe IHC assays showed that TRPM2-S, but not TRPM2-L, was prognostic in RPLS. Low TRPM2-S level was associated with poor disease-free survival (DFS). Calcium influx signal intensity was significantly decreased under TRPM2-S overexpression, which resulted in a decrease in the levels of FOXO3a and PTEN. Correspondingly, the levels of pERK, pAKT, pP65, pGSK-3β, Bcl-2, and β-catenin were upregulated, and cellular ROS was gently increased under TRPM2-S overexpression. Moreover, TRPM2-S slightly promoted cell proliferation and inhibited apoptosis of RPLS cell lines under normoxia, but largely increased apoptosis rates under oxidative stress. The cleaved caspase3 was significantly upregulated by TRPM2-S overexpression under oxidative stress. N-Acetyl-l-cysteine (NAC), a small molecule antioxidant, could largely rescue RPLS cells from the apoptosis induced by H2O2.ConclusionTRPM2-S exerts Janus-faced effects in RPLS by increasing the ROS levels via inhibition on FOXO3a, which promotes cell proliferation under normoxia but induces apoptosis under oxidative stress.FQgJQSptf854GdLd9y3hLjVideo abstract

Abstract P1041: Redox Regulator MnTnBuOE-PyP5+ Protects Cardiomyocyte Against Oxidative Stress Mediated Myocardial Ischemia/reperfusion Injury

Background: Much evidence suggests that oxidative stress plays a major role in the pathogenesis of myocardial ischemia/reperfusion (I/R) injury and is a potential target for therapeutic interventions. We tested the hypothesis that the novel redox regulator, MnTnBUOE-2-PyP 5+ (BMX-001), mediates cardioprotection through reduction of oxidative stress in myocardial tissue following I/R injury. Methods and Results: BMX-001 (2mg/Kg, IP) or vehicle was administered in mice 4 hours before myocardial ischemia. Mice were then subjected to I/R injury by ligating the left anterior descending artery for 1 hour followed by 24 hours of reperfusion. Following 24 hours of reperfusion, infarct size, cardiomyocyte apoptosis, and SOD2 level were determined, and echocardiography was performed. Our study revealed that the redox regulator provided protection against I/R-induced injury in mice as evident by a significant reduction in infarct size, plasma LDH level, and oxidative stress. In addition, BMX-001 administration in mice markedly increased FoxO3a and SOD2 levels. BMX-001 treatment attenuated I/R induced cardiomyocyte apoptosis in-vivo and in-vitro as evident by a reduction in the number of TUNEL positive cells and improved cell viability. Echocardiography at 24 hours of reperfusion revealed improved ejection fraction and fractional shortening in mice treated with BMX-001. Furthermore, in-vitro treatment of BMX-001 (10μM) 24 hours before hypoxia/reoxygenation in H9c2 cells improved oxidative stress as evident by a reduction in cardiolipin peroxidation, mitochondrial superoxide level, and 4-HNE adducted proteins. Conclusion: Pre-treatment with redox regulator BMX-001 showed a beneficial effect in attenuating cardiomyocyte death, mitochondrial superoxide, and cardiolipin peroxidation following I/R injury in H9c2 cells. Pre-treatment with SOD2 mimetic improved heart function, decreased infarct size, and cardiomyocyte apoptosis in mice following I/R injury. Therefore, BMX-001 can be a novel cardioprotective agent against oxidative stress-induced myocardial I/R injury.

Wenshen Yangxue decoction promotes follicular development in aged female mice stimulation of the silent information regulator 3/forkhead transcription factor O1 3a pathway.

To primarily explore the effect and mechanism of Wenshen Yangxue decoction in promoting follicular development in elderly female mice. Fifty Institute of Cancer Research mice were randomly divided into blank, controlled ovarian hyperstimulation (COH), low-dose Wenshen Yangxue decoction, medium-dose Wenshen Yangxue decoction, and high-dose Wenshen Yangxue decoction groups, with 10 mice in each group. The number of ovulations, number of fertilizations, mitochondrial adenosine triphosphate (ATP) level, and mitochondrial DNA (mtDNA) of oocytes in each group were compared. Reverse transcription-polymerase chain reaction and Western blotting were used to detect the mRNA and protein expression levels of silent information regulator 3 (Sirt3) and forkhead transcription factor O1 3a (FOXO3a). Wenshen Yangxue decoction significantly increased the number of ovulations in mice (P < 0.05) and promoted the formation of fertilized eggs. The ATP level and mtDNA copy number of mice oocytes in the high-dose groups were significantly higher than those in the COH group (P < 0.05). Wenshen Yangxue decoction significantly increased the mRNA and protein levels of Sirt3 and FOXO3a in mouse oocytes. Wenshen Yangxue decoction promoted the development of follicles in elderly female mice, increased the number of ovulations and improved fertility. Its mechanism may be related to increased mitochondrial energy metabolism and regulation of the Sirt3/FOXO3a pathway.

Maternal Ethanol Exposure-Induced Cardiac Fibrosis is Associated with Changes in TGF-β and SIRT1/FOXO3a Signaling in Male Rat Offspring: A Three-Month Follow-up Study.

Ethanol exposure during pregnancy induces cardiac fibrosis inthe fetalheart. However, the mechanisms by which consumption of ethanol induces fibrotic changes are not known. Pregnant rats were received ethanol 4.5g/kg BW once per day from the 7th day of pregnancy (GD7) throughout lactation. Our findings demonstrated that, area of fibrosis increased in cardiac tissue in the pups on both postnatal day twenty one (PN21) and postnatal day ninety (PN90) after prenatal and early postnatal period ethanol treatment compared with the controls. It was accompanied by a decline in the expression of SIRT1 protein along with the elevation of FOXO3a and TGF-β protein expressions which weredetermined by western blot. Overall, our data reveal that prenatal alcohol usage increase in fibrotic regions in the pup hearts possibly by regulating TGF-β, FOXO3a and SIRT1 protein levels. These are potential therapeutic molecular targets that can be modulated to protect heart against maternal ethanol exposure.

Abstract 3983: Dysregulated SerpinA6 levels and progression of prostate cancer

Abstract SerpinA6 is a major cortisol binding transport protein, which helps in cortisol tissue delivery particularly at inflammatory sites. Moreover, SerpinA6 has been reported to participate in stress response by releasing cortisol specifically at inflammatory sites upon cleavage by human neutrophil elastase between residues 344 and 345. In castrate resistant prostate cancer (CRPC) patients, reports suggest castration induces inflammation in the prostate, which increases glucocorticoids (GCs) levels and elevates pro-inflammatory cytokine IL-6 levels. We propose declining SerpinA6 levels in the prostate may play a role in prostate cancer disease progression. This could be due to increasing IL-6 levels after castration, which may inhibit or degrade SerpinA6. Deficient SerpinA6 may not be sufficient to keep steroids inactive, due to the increasing GCs. Our data suggests IL-6 treatment of androgen independent prostate cancer (PC-3) cells decreases SerpinA6 level and conversely increases glucocorticoid receptor (GR) expression in a dose (1-10ng/ml) and time (30-180 minutes) dependent fashion. Moreover, IL-6 dose dependent treatment in PC-3 cells decreases FOXO3a expression, a major apoptotic protein dysregulated in prostate cancer. Next, we determine the role of SerpinA6 (30µg/ml) and IL-6(5ng/ml) independently and in combination to PC-3 cells. We observed IL-6 and SerpinA6 co-treatment to PC-3 cells (charcoal stripped FBS media) for 6 hours reduced Neutrophil elastase levels significantly. Additionally we observed increased FOXO3a and SerpinA6 expression, whereas decreased GR and HNF3α levels. However, silencing IL-6 in PC-3 cells increased SerpinA6 expression. To further support our findings, silencing SerpinA6 in metastatic variant of PC-3 cells increased phosphor-Akt-(ser-473), glucocorticoid receptor and increased apoptotic protein FOXO3a expressions. Using dexamethasone (15µM), which is known to suppress PC-3 cell migration, we found increased expression of SerpinA6. Conversely, Mifepristone (15µM), which inhibits PC-3 cell growth, deceases SerpinA6 expression after 24 hours of treatment. In humans the normal range of circulatory SerpinA6 level are 19-45ug/ml. We treated normal prostate epithelial cells (RWPE1) and prostate cancer (PC-3) cells with human recombinant protein 18 and 36ug/ml of doses for 24 hours; RWPE1 cells represented a SerpinA6 dose dependent increase in cellular viability, whereas PC-3 cells demonstrated no change in the viability after a dose response treatment with SerpinA6 for 24 hours. These findings suggest the SerpinA6 context dependent role. We suggest that decreasing levels of SerpinA6 and FOXO3a expressions in the prostate cancer patients may have potential role in disease progression. Moreover, we need to have an effective agent, which could prevent the decreasing levels of SerpinA6 and FOXO3a in prostate to inhibit the progression of prostate cancer. Citation Format: Carlos Riveros, Victor Chalfant, Jatinder Kumar, Sanjeev Shukla. Dysregulated SerpinA6 levels and progression of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3983.

Agastache rugosa Extract Combined With Voluntary Wheel Exercise Affects the Improvement of Skeletal Muscle Homeostasis in Aged C57BL/6J Mice

ObjectivesThis study aimed to investigate the beneficial effects of Agastache rugosa (AR) combined with wheel running exercise on maintaining muscle homeostasis in aged skeletal muscle.MethodsMale 38-week-old C57BL/6J mice were divided into five groups (n = 8/group): control (CON), Low-dose AR-treated (ARL, 100 mg/kg/day), High-dose AR-treated (ARH, 200 mg/kg/day), exercise (EXC), and EXC + ARH (EXH) group. After 2 weeks of acclimation period, AR-treated groups were fed AR extract by oral administration for 4 weeks. The daily voluntary wheel running (VWR) distance was recorded using a counter machine. In the gastrocnemius (GAS) muscle sample, muscle homeostasis-related genes and protein were analyzed by qPCR and western blotting, respectively.ResultsThe weight of GAS in ARH and EXH increased significantly, compared to CON (p < 0.05). qPCR analysis showed that AR and exercise affected both oxidative and glycolytic muscle fibers. mRNA levels of Myh7 and Myh1 in EXH were remarkably higher than in CON (p < 0.05). Skeletal muscle atrophy (Atrogin-1 and MuRF-1) and hypertrophy (myogenin) related genes were up-regulated in EXC compared to CON (p < 0.05). Meanwhile, EXH dramatically down-regulated Atrogin-1 and MuRF-1 and up-regulated <> (p < 0.05). Western blotting analysis indicated that the hypertrophy-related protein p70S6 showed high levels in both exercise groups, but there was no statistically significant difference. In contrast, atrophy-related protein FOXO3a and MuRF-1 levels were remarkably lower in the EXC and EXH (p < 0.05).ConclusionsTaken together, this study demonstrated that AR with VWR exercise enhanced muscle homeostasis via regulation of skeletal muscle protein synthesis and degradation markers in aged hind-limb muscles. In conclusion, AR could be a promising candidate for maintaining muscle homeostasis and further alleviating sarcopenia.Funding SourcesThis work was supported by the BK21 FOUR (Fostering Outstanding Universities for Research) funded by the Ministry of Education (MOE, Korea) and National Research Foundation of Korea (NRF), and the Bio & Medical Technology Development Program of the National Research Foundation (NRF), funded by the Ministry of Science & ICT (2012M3A9C4048761).

Prognostic value of high FOXO3a expression in patients with solid tumors: A meta-analysis and systematic review.

FOXO3a (previously termed FKHRL1), plays an evolutionarily conserved role in the control of biological process, including DNA damage, apoptosis, and cell cycle regulation. However, the role of FOXO3a in tumors remains controversial. This meta-analysis was conducted to evaluate the prognostic value of FOXO3a expression in patients with solid tumors. A systematic literature search of the PubMed, Web of Science, Embase, and Cochrane Library databases was performed. Eligible publications on FOXO3a and cancer prognosis were collected and screened according to the eligibility criteria. The combined odds ratios (ORs) or hazard ratios (HRs) with corresponding 95% confidence intervals (CIs) were used to assess the prognostic value of FOXO3a. Stata 12.0 software was used for statistical analysis. A total of 4058 patients from 21 articles on a variety of solid tumors were included. Meta-analysis showed that the increased FOXO3a expression level was associated with longer overall survival (HR = 0.62; 95% CI: 0.46-0.85). The pooled ORs indicated high expression level of FOXO3a in tumors was significantly associated with lymph node metastasis (OR = 0.46; 95% CI: 0.30-0.71), TNM stage (OR = 0.37; 95% CI: 0.25-0.54), tumor differentiation (OR = 0.46; 95% CI: 0.26-0.80), distant metastasis (OR = 0.44; 95% CI: 0.32-0.61), and age (OR = 1.28; 95% CI: 1.08-1.51). However, we did not observe a significant correlation between the high expression of FOXO3a and sex or tumor size. The high expression level of FOXO3a was associated with better clinical outcomes in solid tumors. FOXO3a may therefore serve as a potential prognostic biomarker and a promising molecular target.

The association betweenFOXO3ars4946936 gene polymorphism and the levels ofFOXO3aamongchronicgranulocyticleukemia patients treated with imatinib mesylate.

Background:The gene FOXO3ahas been elucidated to govern the development ofchronicgranulocyticleukemia(CGL). Moreover,it has been suggested thatthe levels of FOXO3ain circulationareaffected bythe FOXO3ars4946936gene polymorphism. However, no studyhasassessed the correlation betweenthe FOXO3ars4946936gene polymorphism and the levels of FOXO3a.The objective of this study was to assess the association between the FOXO3ars4946936gene polymorphism and the levels of FOXO3ain CGL patients treated with imatinib mesylate. Methods: A cross-sectional study was conductedfromFebruary 2019toFebruary 2020. The genotyping of FOXO3ars4946936gene polymorphism was conducted using PCR-RFLP, and the levels of FOXO3awereassessed using ELISA. The association between the FOXO3ars4946936gene polymorphism and the levels of FOXO3awereassessed using multiple logistic regression. Results:A total of 60 CGL patientswereassessedin our study. Among them, the CC, CT, and TT genotypes ofthe FOXO3ars4946936gene polymorphism were35.0%,48.3%,and16.7% respectively. Our calculation revealed that elevated levels of FOXO3awerefound in CGL patients withtheCC genotypeofthe FOXO3ars4946936gene polymorphism. While we failed to clarify the association between eithertheCTortheTT genotypeof FOXO3ars4946936gene polymorphismand the levels of FOXO3a. Conclusion: Our study identifies thattheCC genotype ofthe FOXO3ars4946936gene polymorphism affects the elevated levels of FOXO3ain CGL patients treated with imatinib mesylate.

Bruceine H Mediates EGFR-TKI Drug Persistence in NSCLC by Notch3-Dependent β-Catenin Activating FOXO3a Signaling

Tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) protein serve as a critical pillar in the treatment of non-small cell lung cancer (NSCLC), but resistance is universal. Identifying the potential key factors of drug resistance to EGFR-TKIs is essential to treat patients with EGFR mutant lung cancer. Our research here shows that bruceine H suppressed the proliferation, migration, and invasion of lung cancer cells; inhibited the growth of human NSCLC cell xenografts; and enhanced the therapeutic effects of gefitinib in the PC-9/GR xenograft models, possibly by inhibiting Notch3. In order to analyze the potential targets of the combination of Notch3 and EGFR-TKIs on resistance to EGFR, we analyzed the differences of gene expression between NSCLC tissues and EGFR-driven gefitinib-resistant tumoral groups and then identify through the WGCNA key genes that may provide therapeutic targets for TKI-resistant lung cancer xenograft models. We confirmed that EGFR-TKI in combination with Notch3 inhibitor can inhibit the expression of β-catenin and enhance the level of FOXO3a, leading to improved recurrence-free survival and overall survival of the xenotransplantation model. These results support that the combination of gefitinib and bruceine H may provide a promising alternative strategy for treating acquired EGFR-TKI resistance in patients with NSCLC.

Estradiol initiates the immune escape of non-small cell lung cancer cells via ERβ/SIRT1/FOXO3a/PD-L1 axis

Mounting evidence has postulated estrogen as a contributor for lung cancer development and progression. Here, we focused on the effect of estradiol (E2) on the immune escape of non-small cell lung cancer (NSCLC). The expression of FOXO3a in NSCLC samples was screened by gene microarray and then verified using Western blot analysis in NSCLC cell lines. Interaction between E2, SIRT1, FOXO3a and PD-L1 was determined. Following ectopic expression and depletion experiments in A549 and H1435 cells, cell proliferation and killing of cytotoxic T lymphocytes (CTLs) on NSCLC cells were evaluated. Xenograft mouse models were prepared to validate the in vivo effect of E2. E2 activated SIRT1 by up-regulating the expression of ERβ and thereby weakened the killing of CTLs on NSCLC cells. E2 elevated PD-L1 by up-regulating the ERβ/SIRT1 axis to promote the immune escape of NSCLC cells. SIRT1 degraded FOXO3a by reducing the acetylation level of FOXO3a and increased its ubiquitination. E2 inhibited the expression of FOXO3a and elevated PD-L1 expression, thereby promoting the immune escape of NSCLC cells. In vivo results showed that E2 facilitated the growth and metastasis of NSCLC cells in nude mice by elevating ERβ via SIRT1/FOXO3a/PD-L1 axis. In summary, our data revealed the critical roles of E2/ERβ/SIRT1/FOXO3a/PD-L1 axis in the immune escape of NSCLC cells and suggested that the axis may be promising therapeutic targets for NSCLC.

The association between FOXO3a rs4946936 gene polymorphism and the levels of FOXO3a among chronic granulocytic leukemia patients treated with imatinib mesylate

Background: The gene FOXO3a has been elucidated to govern the development of chronic granulocytic leukemia (CGL). Moreover, it has been suggested that the levels of FOXO3a in circulation are affected by the FOXO3a rs4946936 gene polymorphism. However, no study has assessed the correlation between the FOXO3a rs4946936 gene polymorphism and the levels of FOXO3a. The objective of this study was to assess the association between the FOXO3a rs4946936 gene polymorphism and the levels of FOXO3a in CGL patients treated with imatinib mesylate. Methods: A cross-sectional study was conducted from February 2019 to February 2020. The genotyping of FOXO3a rs4946936 gene polymorphism was conducted using PCR-RFLP, and the levels of FOXO3a were assessed using ELISA. The association between the FOXO3a rs4946936 gene polymorphism and the levels of FOXO3a were assessed using multiple logistic regression. Results: A total of 60 CGL patients were assessed in our study. Among them, the CC, CT, and TT genotypes of the FOXO3a rs4946936 gene polymorphism were 35.0%, 48.3%, and 16.7% respectively. Our calculation revealed that elevated levels of FOXO3a were found in CGL patients with the CC genotype of the FOXO3a rs4946936 gene polymorphism. While we failed to clarify the association between either the CT or the TT genotype of FOXO3a rs4946936 gene polymorphism and the levels of FOXO3a. Conclusion: Our study identifies that the CC genotype of the FOXO3a rs4946936 gene polymorphism affects the elevated levels of FOXO3a in CGL patients treated with imatinib mesylate.

Effect of Sirt3 on retinal pigment epithelial cells in high glucose through Foxo3a/ PINK1-Parkin pathway mediated mitophagy

Sirt3 is closely associated with mitophagy. This study aimed to investigate the effect and potential mechanism of Sirt3 on mitophagy in retinal pigment epithelium (RPE) in a high glucose environment. The expression levels of Sirt3, Foxo3a, PINK1, Parkin and LC3B in RPE subjected to high-glucose (HG, 30 mM D-glucose) conditions were detected by RT-PCR and western blotting. Dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining was used to detect the level of reactive oxygen species (ROS) in RPE treated with HG. MitoTracker and LysoTracker probes were used to label mitochondria and lysosomes, respectively, to observe the occurrence of autophagy. Sirt3-dependent regulation of mitophagy through the Foxo3a/PINK1-Parkin pathway was further investigated by virus transfection-mediated Sirt3 overexpression and PINK1 silencing. The effect of Sirt3 overexpression on apoptosis was detected by flow cytometry. The Sirt3 expression was decreased, the Foxo3a/PINK1-Parkin pathway was inhibited, intracellular ROS level was increased, and mitophagy was attenuated in RPE under HG condition. Sirt3 overexpression activated the Foxo3a/PINK1-Parkin signaling pathway and mitophagy, and inhibited cell apoptosis. Silencing PINK1 inhibited the effect of Sirt3 overexpression on mitophagy. In summary, Sirt3 can activate mitophagy through the Foxo3a/PINK1-Parkin pathway and reduce HG-induced apoptosis of RPE. This study provides a new direction to understand the pathogenesis and develop a potential therapeutic target for diabetic retinopathy.

The association between FOXO3a rs4946936 gene polymorphism and the levels of FOXO3a among chronic granulocytic leukemia patients treated with imatinib mesylate

Background: The gene FOXO3a has been elucidated to govern the development of chronic granulocytic leukemia (CGL). Moreover, it has been suggested that the levels of FOXO3a in circulation are affected by the FOXO3a rs4946936 gene polymorphism. However, no study has assessed the correlation between the FOXO3a rs4946936 gene polymorphism and the levels of FOXO3a. The objective of this study was to assess the association between the FOXO3a rs4946936 gene polymorphism and the levels of FOXO3a in CGL patients treated with imatinib mesylate. Methods: A cross-sectional study was conducted from February 2019 to February 2020. The genotyping of FOXO3a rs4946936 gene polymorphism was conducted using PCR-RFLP, and the levels of FOXO3a were assessed using ELISA. The association between the FOXO3a rs4946936 gene polymorphism and the levels of FOXO3a were assessed using multiple logistic regression. Results: A total of 60 CGL patients were assessed in our study. Among them, the CC, CT, and TT genotypes of the FOXO3a rs4946936 gene polymorphism were 35.0%, 48.3%, and 16.7% respectively. Our calculation revealed that elevated levels of FOXO3a were found in CGL patients with the CC genotype of the FOXO3a rs4946936 gene polymorphism. While we failed to clarify the association between either the CT or the TT genotype of FOXO3a rs4946936 gene polymorphism and the levels of FOXO3a. Conclusion: Our study identifies that the CC genotype of the FOXO3a rs4946936 gene polymorphism affects the elevated levels of FOXO3a in CGL patients treated with imatinib mesylate.

FoxO3a Inhibits Tamoxifen-Resistant Breast Cancer Progression by Inducing Integrin α5 Expression.

Simple SummaryTamoxifen is a mainstay of adjuvant treatment for estrogen receptor α-positive (ERα+) breast tumors, which account for over 70% of all the diagnosed breast malignancies. Unfortunately, ~30% of patients initially responding to the therapy will eventually relapse with an endocrine-resistant disease. Here, we show that the transcription factor FoxO3a can overcome tamoxifen resistance by inhibiting cell motility and invasiveness, the main features of tumor progression. The underlying mechanism could be ascribed to FoxO3a-dependent transcriptional up-regulation of the integrin α5 subunit of the α5β1 fibronectin receptor, a well-known membrane heterodimer controlling cell adhesion. Indeed, FoxO3a and integrin α5 expression is significantly correlated in ERα+ tumors, and FoxO3a protective effects are lost in cells with reduced levels of integrin α5. Therefore, a pharmacological increase in FoxO3a levels could represent an effective approach to be exploited in combination with tamoxifen treatment in order to restore the sensitivity to the therapy in refractory tumors.Resistance to endocrine therapy is still a major clinical challenge in the management of estrogen receptor α-positive (ERα+) breast cancer (BC). Here, the role of the Forkhead box class O (FoxO)3a transcription factor in tumor progression has been evaluated in tamoxifen-resistant BC cells (TamR), expressing lower levels of FoxO3a compared to sensitive ones. FoxO3a re-expression reduces TamR motility (wound-healing and transmigration assays) and invasiveness (matrigel transwell invasion assays) through the mRNA (qRT-PCR) and protein (Western blot) induction of the integrin α5 subunit of the α5β1 fibronectin receptor, a well-known membrane heterodimer controlling cell adhesion and signaling. The induction occurs through FoxO3a binding to a specific Forkhead responsive core sequence located on the integrin α5 promoter (cloning, luciferase, and ChIP assays). Moreover, FoxO3a failed to inhibit migration and invasion in integrin α5 silenced (siRNA) cells, demonstrating integrin α5 involvement in both processes. Finally, using large-scale gene expression data sets, a strong positive correlation between FoxO3a and integrin α5 in ERα+, but not in ER-negative (ERα−), BC patients emerged. Altogether, our data show how the oncosuppressor FoxO3a, by increasing the expression of its novel transcriptional target integrin α5, reverts the phenotype of endocrine-resistant BC toward a lower aggressiveness.

Modulation of SIRT1 expression improves erectile function in aged rats.

Silent information regulator 2-related enzyme 1 (SIRT1) is an aging-related protein activated with aging. Herein, we evaluated the role of SIRT1 in aging-related erectile dysfunction. The expression of SIRT1 was modulated in aged Sprague-Dawley rats following intragastric administration of resveratrol (Res; 5 mg kg-1), niacinamide (NAM; 500 mg kg-1) or Res (5 mg kg-1) + tadalafil (Tad; phosphodiesterase-5 [PDE5] inhibitor; 5 mg kg-1) for 8 weeks. Then, we determined erectile function by the ratio of intracavernosal pressure (ICP)/mean systemic arterial pressure (MAP). Cavernosal tissues were extracted to evaluate histological changes, cell apoptosis, nitric oxide (NO)/cyclic guanosine monophosphate (cGMP), the superoxide dismutase (SOD)/3,4-methylenedioxyamphetamine (MDA) level, and the expression of SIRT1, p53, and forkhead box O3 (FOXO3a) using immunohistochemistry, terminal deoxynucleotidyl transferase (TdT)-mediated 2'-deoxyuridine 5'-triphosphate (dUTP) nick-end labeling (TUNEL), enzyme-linked immunosorbent assays, and western blot analysis. Compared with the control, Res treatment significantly improved erectile function, reflected by an increased content of smooth muscle and endothelium, NO/cGMP and SOD activity, and reduced cell apoptosis and MDA levels. The effect of Res was improved by adding Tad. In addition, the protein expression of SIRT1 was increased in the Res group, accompanied by decreased p53 and FOXO3a levels. In addition, inhibition of SIRT1 by NAM treatment resulted in adverse results compared with Res treatment. SIRT1 activation ameliorated aging-related erectile dysfunction, supporting the potential of SIRT1 as a target for erectile dysfunction treatment.

Inhibition of EZH2 ameliorates hyperoxaluria-induced kidney injury through the JNK/FoxO3a pathway

AimsEnhancer of zeste homolog 2 (EZH2), a histone H3 lysine 27 methyltransferase, has been shown to play a role in kidney diseases. However, its role in hyperoxaluria-induced renal tubular epithelial cells (TECs) injury remains unclear. Materials and methodsA hyperoxaluria rat model was established by providing 0.5% ammonium chloride and drinking water containing 1% ethylene glycol. TECs were exposed to oxalate stress. The 3-DZNeP, a selective EZH2 inhibitor, was administered in vivo and in vitro. Cell viability, ROS production, and apoptosis ratio were evaluated. Crystal deposition was detected by Von Kossa staining and kidney tissue injury was detected by HE staining and TUNEL. EZH2, H3K27me3, cleaved-caspase3, IL-6, and MCP-1 were examined by western blot or immunohistochemistry. Key findingsInhibition of EZH2 by 3-DZNeP significantly attenuated hyperoxaluria-induced oxidative and inflammatory injury and CaOx crystal deposition in vivo. Similarly, inhibition of EZH2 using 3-DZNeP or shRNA restored cell viability, suppressed LDH release and the production of intracellular ROS in vitro. Furthermore, the MAPK signaling pathway and FoxO3a levels were activated or elevated in TECs exposed to oxalate. EZH2 inhibition using 3-DZNeP blocked these effects. CC90003 (ERK inhibitor) or SB203580 (p38 inhibitor) did not significantly affect the expression of FoxO3a in TECs treated with 3-DZNeP and oxalate; only SP600125 (JNK inhibitor) significantly decreased FoxO3a expression. SignificanceEZH2 inhibition protects against oxalate-induced TECs injury and reduces CaOx crystal deposition in the kidney may by modulating the JNK/FoxO3a pathway; EZH2 may be a promising therapeutic target in TECs injury.

Effect of U2AF1 Mutation to Inflammatory Cytokine Expression in SKM-1 Cells through FOXO3a-Bim Signaling Pathway

To investigate the effect of U2AF1 gene mutation to inflammatory cytokine in SKM-1 cell of human myelodysplastic syndromes (MDS), and whether the above effects were mediated by FOXO3a-Bim signaling pathway. Wide-type U2AF1 and mutant U2AF1 (the serine residue 34 was replaced by phenylalanine, and named as S34F) recombinant expression plasmids were constructed. Lentiviruses were packaged and transfected into SKM-1 cells. The expression of FOXO3a was up-regulated by lentiviruses, and its transfection rate was investigated. The cell proliferation was detected by CCK-8 method. Flow cytometry was used to detect the apoptosis and cycle of the cells. The expression pro-inflammatory cytokine IL-1β, IL-6, TNF-α and anti-inflammatory cytokine IL-4 were detected by qRT-PCR. FOXO3a, Bim, Bcl-2 and Bax protein expression levels were detected by Western blot. Compared with the control group, the cell apoptosis rate, pro-inflammatory cytokine IL-1β and TNF-α transcription levels were significantly increased in the S34F group (P<0.05); cell cycle was blocked at the G2 phase; cell proliferation and the anti-inflammatory cytokine IL-4 transcription level were significantly decreased; the expression levels of FOXO3a, Bim and Bax protein were significantly increased (P<0.05); while the expression level of Bcl-2 protein was significantly decreased (P<0.05). The up-regulation of FOXO3a could significantly inhibited the proliferation and increased cell apoptosis of SKM-1 cells with U2AF1 S34F mutation; cell cycle was blocked at the S and G2 phases; the pro-inflammatory cytokine IL-1β and TNF-α transcription levels were significantly decreased (P<0.05), and the transcription level of anti-inflammatory cytokine IL-4 showed no statistically significant as compared with control group (P>0.05). U2AF1 S34F mutation can regulate inflammatory phenotype in SKM-1 cells, which may be mediated through FOXO3a-Bim signaling pathway.

Evaluation of AK023391 lncRNA and FOXO3a Genes’ Expression in Tissues from Iranian Gastric Cancer Patients Compared with Healthy Tissues and Their Relationship with Clinicopathological Features

Background: LncRNAs have been shown to be implicated in the initiation and progression of gastric cancer (GC). Here, the transcription levels of the AK023391 and FOXO3a genes, as well as their relationship with clinical traits, were examined in GC. Methods: This cross-sectional study was performed on 50 formalin-fixed paraffin-embedded (FFPE) GC tumor and 50 normal tissues. The RNA levels of the FOXO3a and AK023391 genes encoding lncRNAs were evaluated by real-time RT-PCR. Results: A significant difference was observed in the mRNA level of AK023391 between GC tumor and normal tissues (Mean difference = 2.683, P = 0.0371). The transcription level of FOXO3A showed no remarkably significant difference between GC and normal tissues (Mean difference = 0.9177, P = 0.2199). In addition, AK023391 and FOXO3a mRNA expression levels showed no significant associations with the clinicopathologic features of GC. Also, we found no significant correlation between the expressions of AK023391 and FOXO3a in GC tissues (R = 0.014; P = 0.34). Conclusions: Our finding suggested that there might be a significant relationship between the expression level of AK023391 and GC. Our results also showed that there was no correlation between the mRNA levels of the AK023391 and FOXO3a genes in GC tissues.

Elevated Foxo3a and Fas-ligand expression in human periapical granulomas as a potential treatment target.

Periapical granuloma is a common periodontitis type involving chronic inflammation; however, the efficacy of current therapies is limited. Its molecular pathogenesis also remains obscure. Forkhead box transcription factor class o3a (Foxo3a) and Fas-ligand (FasL) are associated with chronic inflammation. Therefore, in this study, we aimed to clarify the roles of Foxo3a and FasL in periapical granuloma pathophysiology. Periapical lesions were obtained from patients during endodontic surgery and tooth extraction; those diagnosed with periapical granulomas using haematoxylin and eosin staining were further analysed. Immunohistochemical analysis was performed for Foxo3a and FasL, and real-time polymerase chain reaction was performed for FOXO3A, FASL and interleukin (IL)-1β. Healthy gingival tissues were also examined as controls. Neutrophils, lymphocytes and plasma cells in the periapical granulomas, but not healthy tissues, expressed Foxo3a. Dual-colour immunofluorescence imaging revealed Foxo3a and FasL co-expression in leukocytes. FOXO3A, FASL and IL-1β mRNA levels in healthy gingival tissues were significantly lower than those in the periapical granulomas. Additionally, FOXO3A and IL-1β expressions were negatively correlated. Phosphorylated Foxo3a may reduce IL-1β release by inhibiting apoptosis through FasL in periapical periodontitis and prevent exacerbation. Thus, Foxo3a is a potential therapeutic agent for periapical periodontitis.

Swim training affects Akt signaling and ameliorates loss of skeletal muscle mass in a mouse model of amyotrophic lateral sclerosis

We tested the hypothesis that swim training reverses the impairment of Akt/FOXO3a signaling, ameliorating muscle atrophy in ALS mice. Transgenic male mice B6SJL-Tg (SOD1G93A) 1Gur/J were used as the ALS model (n = 35), with wild-type B6SJL (WT) mice as controls (n = 7). ALS mice were analyzed before ALS onset, at ALS onset, and at terminal ALS. Levels of insulin/Akt signaling pathway proteins were determined, and the body and tibialis anterior muscle mass and plasma creatine kinase. Significantly increased levels of FOXO3a in ALS groups (from about 13 to 21-fold) compared to WT mice were observed. MuRF1 levels in the ONSET untrained group (12.0 ± 1.7 AU) were significantly higher than in WT mice (1.12 ± 0.2 AU) and in the BEFORE ALS group (3.7 ± 0.9 AU). This was associated with body mass and skeletal muscle mass reduction. Swim training significantly ameliorated the reduction of skeletal muscle mass in both TERMINAL groups (p < 0.001) and partially reversed changes in the levels of Akt signaling pathway proteins. These findings shed light on the swimming-induced attenuation of skeletal muscle atrophy in ALS with possible practical implications for anti-cachexia approaches.