Expression Levels Of FOXO3a Research Articles

Microglial FoxO3a deficiency ameliorates ferroptosis-induced brain injury of intracerebral haemorrhage via regulating autophagy and heme oxygenase-1.

Microglial HO-1 regulates iron metabolism in the brain. Intracerebral haemorrhage (ICH) shares features of ferroptosis and necroptosis; hemin is an oxidized product of haemoglobin from lysed red blood cells, leading to secondary injury. However, little is known about the underlying molecular mechanisms attributable to secondary injury by hemin or ICH. In this study, we first show that FoxO3a was highly co-located with neurons and microglia but not astrocytes area of ICH model mice. Hemin activated FoxO3a/ATG-mediated autophagy and HO-1 signalling resulting in ferroptosis invitro and in a mice model of brain haemorrhage. Accordingly, autophagy inhibitor Baf-A1 or HO-1 inhibitor ZnPP protected against hemin-induced ferroptosis. Hemin promoted ferroptosis of neuronal cells via FoxO3a/ATG-mediated autophagy and HO-1 signalling pathway. Knock-down of FoxO3a inhibited autophagy and prevented hemin-induced ferroptosis dependent of HO-1 signalling. We first showed that hemin stimulated microglial FoxO3a/HO-1 expression and enhanced the microglial polarisation towards the M1 phenotype, while knockdown of microglial FoxO3a inhibited pro-inflammatory cytokine production in microglia. Furthermore, the microglia activation in the striatum showed significant along with a high expression level of FoxO3a in the ICH mice. We found that conditional knockout of FoxO3a in microglia in mice alleviated neurological deficits and microglia activation as well as ferroptosis-induced striatum injury in the autologous blood-induced ICH model. We demonstrate, for the first time, that FoxO3a/ATG-mediated autophagy and HO-1 play an important role in microglial activation and ferroptosis-induced striatum injury of ICH, identifying a new therapeutic avenue for the treatment of ICH.

FOXP1 and FOXO3a Are Prognostic Markers in Gallbladder Squamous Cell/Adenosquamous Carcinomas and Adenocarcinomas.

The pathologic characteristics of squamous cell/adenosquamous carcinomas (SC/ASC) have not been well clarified. As a rare subtype of gallbladder cancer (GBC), no biological markers for diagnosis and prognosis are available. This research evaluated the expression of FOXP1 and FOXO3a in 69 SC/ASC, and 146 adenocarcinoma (AC) samples were analyzed via immunohistochemistry. SC/ASCs were associated with higher rates of lymph node metastasis, invasion, and patients older than 45 years comparing to ACs. FOXP1 and FOXO3a positivity rates were significantly lower in SC/ASC and AC samples from patients with large tumor size, a high TNM stage, lymph node metastasis, invasion, and no history of tumor resection (biopsy only). Positive FOXP1 expression levels were significantly decreased in cases of poorly differentiated AC. The univariate Kaplan-Meier analysis revealed that negative FOXP1 and FOXO3a expression, poor differentiation, large tumor size, high TNM stage, lymph node metastasis, invasion, and an inability to undergo curative resection were all closely associated with decreased overall survival in SC/ASC and AC patients. The multivariate cox regression analysis showed that negative FOXP1 and FOXO3a expression levels were independent predictors of poor prognosis in SC/ASC and AC patients. Our results indicate that negative FOXP1 and FOXO3a expression are closely associated with the pathogenesis, clinicopathologic properties, and prognosis of GBC patients. FOXP1 and FOXO3a may thus be biomarkers of GBC carcinogenesis, progression, and prognosis.

Genistein-induced mitochondrial dysfunction and FOXO3a/PUMA expression in non-small lung cancer cells

Context Genistein is a multifunctional natural compound. Objective In this study, we demonstrate the activity of genistein on non-small lung cancer A549 and 95D cells. Materials and methods A CCK8 assay was used to detect the cytotoxicity of genistein (0, 25, 50, 100, 150, 200 and 250 μM) on A549 and 95D cells for 48 h. AnnexinV-FITC/PI and TUNEL assay were performed to examine the apoptotic cell death induced by genistein (0, 50, 100 and 150 μM, 48 h). Intracellular reactive oxygen species (ROS) generation and mitochondrial membrane potential were measured by flow cytometry. Mitochondrial activity in A549 and 95D cells, treated with 0, 50, 100 and 150 μM genistein for 48 h was detected by MitoTracker Orange staining. Western blot analysis was performed to evaluate the expression of the mitochondrial apoptosis-related proteins. Meanwhile, the expression level of FOXO3a/PUMA signalling was measured by flow cytometry and Western blot assay. Results IC50 value of genistein against 95D cells and A549 cells was 32.96 ± 2.91 and 110.6 ± 2.41 μM, respectively. The percentage of apoptotic death cells was 20.03%, 29.26% and 27.14% in 95D cells, and 41.62%, 55.24% and 43.45% in A549 cells when treated with 50, 100 and 150 μM genistein, respectively. Our observations also revealed that genistein could elevate intracellular ROS generation, decrease mitochondrial membrane potential, decrease mitochondrial activity (MitoTracker Orange staining), and up-regulate the expression of mitochondrial apoptosis-related proteins. Further examinations revealed that the expression level of FOXO3a and PUMA in NSCLC was significantly increased by genistein. Discussion and conclusions Our data may provide basic information for further development of genistein as a promising lead compound targeting NSCLC by inducing mitochondrial apoptosis.

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.

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.

FOXO3a inhibits nephroblastoma cell proliferation, migration and invasion, and induces apoptosis through downregulating the Wnt/β‑catenin signaling pathway.

Forkhead transcription factor O subfamily 3A (FOXO3a) is an important tumor suppressor gene that is expressed in renal tissue and has been reported to be downregulated in clear cell renal cell carcinoma (CCRCC). Notably, the overexpression of FOXO3a was previously discovered to inhibit the progression of CCRCC. However, the expression levels of FOXO3a in nephroblastoma cell lines remain unknown. The present study aimed to investigate the expression levels of FOXO3a in nephroblastoma cell lines and to determine the mechanism of action of the biological functions of FOXO3a. Western blotting and reverse transcription-quantitative PCR were used to analyze the expression levels of FOXO3a in nephroblastoma cell lines. Subsequently, the effects of the overexpression of FOXO3a and the genetic knockdown of the Wnt/β-catenin signaling protein Axin-2 on the biological functions were determined through Cell Counting Kit-8, cell colony formation assays, scratch and Transwell assay and flow cytometric analysis experiments. The expression levels of FOXO3a were discovered to be downregulated in nephroblastoma cell lines. The overexpression of FOXO3a inhibited the proliferation, invasion and migration of nephroblastoma cells, while inducing apoptosis. Furthermore, the overexpression of FOXO3a downregulated the expression levels of β-catenin and Cyclin-D1 proteins involved in the Wnt/β-catenin signaling pathway. Cell proliferation and the migration and invasion ability of 17–94 cells in shRNA-Axin2-2 group were promoted. Cell apoptosis was predominantly increased by overexpressed FOXO3a, which was reversed by shRNA-Axin2-1. The biological effects of overexpressing FOXO3a on nephroblastoma were reversed after activation of Wnt/β-catenin. In conclusion, the findings of the present study suggested that FOXO3a may inhibit nephroblastoma cell proliferation, migration and invasion, while inducing apoptosis, by downregulating the Wnt/β-catenin signaling pathway. These results may provide a novel method for the early diagnosis and precise treatment of nephroblastoma.

The Role of Egln2/Proline Hydroxylase 1 in Regulation of Cyclin D1 in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an incurable B-cell non-Hodgkin lymphoma characterized by a translocation that juxtaposes the CCND1 gene (which encodes for cyclin D1) on chromosome 11q13 and an immunoglobulin heavy chain gene promoter on chromosome 14q32. Despite a high rate of complete remission (response 20-80%) by many chemotherapy regimens, MCL typically relapses after treatment, with a median survival time of approximately 3-4 years. Novel therapeutic approaches target several cellular pathways of MCL (CDK, proteasome, mTOR, or NFκB inhibitors and others) but so far have shown only modest effects on overall survival.Iron is essential for cell proliferation. Although iron chelators were developed for treatment of iron overload diseases, depletion by chelators leads to cancer cell cycle arrest and apoptosis. Iron chelation directly or indirectly affects multiple proteins controlling DNA replication, DNA repair, cell cycle progression and multiple metabolic processes; one known target of iron chelation is cyclin D1. Besides iron chelation-induced suppression of CCND1 gene transcription, Tjendraputra, et al (Blood, 2007), postulated that iron chelation causes posttranslational degradation of cyclin D1 via von Hippel-Lindau protein-independent ubiquitination and subsequent proteasomal degradation.We hypothesized that iron chelation would be particularly effective in MCL with upregulated cyclin D1. We treated human MCL cell lines (Jeko-1, Mino and HBL-2) with 250 µM deferoxamine mesylate (DFO), which caused decreased cell growth, increased apoptosis, and decreased cyclin D1 mRNA and protein levels. These cell lines were more susceptible to treatment with DFO than B-cell lymphoma derived cell lines without constitutively active cyclin D1 (SUDHL-6, DG-75). A possible cytotoxic effect due to a high concentration of DFO was ruled out by abrogating the DFO effect by concomitant administration of ferric ammonium citrate.We further investigated the molecular mechanism underlying decreased cyclin D1 mRNA and protein levels in MCL cell lines after DFO treatment. We tested whether the DFO effect may be due to inhibition of one of the iron-dependent hypoxia-inducible factor (HIF) hydroxylases. Expression analysis of several hypoxia related genes in MCL cell lines treated with DFO revealed down-regulation of PHD1 encoded by the EglN2 gene, a member of the PHD family. All members of the PHD protein family contribute to regulation of HIF, but only EglN1 gene encoding PHD2 has a significant role in oxygen sensing under physiological conditions. EglN2 /PHD1 and EglN3 /PHD3 also have HIF-independent functions in control of cell proliferation and apoptosis. In breast cancer, a loss of EglN2 /PHD1 leads to down-regulation of cyclin D1 and decreases cell proliferation in a HIF-independent manner (Zhang et al, Cell, 2009). To further confirm that EglN2 /PHD1 inhibition is linked to suppression of cyclin D1, we treated MCL cell lines with known PHD inhibitors, 1 mM DMOG and 50 µM FG-4497. Proliferation was markedly reduced and down-regulation of cyclin D1 was confirmed in mRNA and protein levels. It has been proposed that the EglN 2/PHD1 substrate linked to repression of cyclin D1 is the transcription factor FOXO3a. An inability of EglN2 /PHD1 to hydroxylate FOXO3a promotes its accumulation in cells, which in turn suppresses cyclin D1 expression by a yet unknown mechanism (Zheng X et al, Genes & Development, 2014). We measured FOXO3a expression levels in MCL cell lines after treatment with DFO and DMOG and found it significantly upregulated. However, knock-down of FOXO3a by CRISPR/Cas9 technology in MCL line Mino did not affect cyclin D1 levels after DFO treatment. These data suggest that in MCL cell lines treated with DFO, accumulation of FOXO3a is present, but not required for cyclin D1 repression and that cyclin D1 down-regulation mediated by inhibition of PHD1 is a result of another, as yet unknown mechanism. Thus, our preliminary data unveiled new insights into regulation of cyclin D1 in MCL and opens new possibilities for MCL targeted therapy with chelation therapy and/or PHD inhibitors.This study was supported by research funding from Czech Science Foundation, project GACR 15-18046Y (OB, LL), by Ministry of Health Czech Republic, grant AZV 16-31689A (VD) and from the Ministry of Education, Youth and Sports, Czech Republic, Program KONTAKT II (LH15223) (LL, KK, VD). DisclosuresNo relevant conflicts of interest to declare.

Role and mechanism underlying FoxO6 inskeletal muscle invitro and invivo.

Skeletal muscle atrophy is a common feature of patients suffering with chronic infection and other systemic diseases, including acquired immunodeficiency syndrome, chronic kidney disease and cancer. Therefore, understanding the molecular basis of muscle loss is of importance. The majority of members of the forkhead box O (FoxO) family can induce skeletal muscle atrophy; however, the effect of FoxO6 on skeletal muscle is not completely understood. The present study investigated the role of FoxO6 invitro and invivo. Compared with the small interfering RNA (si)‑negative control (NC) group, C2C12 cell proliferation (Cell Counting Kit‑8 assay), myotube differentiation and myotube production were significantly decreased by FoxO6 knockdown, which was different from the known functions of other FoxO members. The immunofluorescence assay results demonstrated that si‑FoxO6 clearly downregulated the expression levels of myosin heavy chain (MyHC) in C2C12 myotubes compared with si‑NC. The western blotting results indicated that compared with the si‑NC group, FoxO6 knockdown induced C2C12 myotube atrophy by notably downregulating myoblast determination protein1 (MyoD), mTOR and MyHC expression levels, and by markedly upregulating ubiquitin ligase (atrogin1) and muscle RING‑finger protein‑1 (MURF1) expression levels. Similarly, in an invitro model of TNF‑α‑induced myotube atrophy, the western blotting results indicated that FoxO6 expression levels were decreased, whereas atrogin1, MURF1, FoxO1 and FoxO3a expression levels were increased compared with the control group. Therefore, the results indicated that, unlike FoxO1 or FoxO3a, FoxO6 maintained C2C12 myotubes and protected against atrophy. Consistent with the invitro data, similar results were observed invivo. Collectively, the results of the present study suggested that FoxO6 served a critical role in muscle cell metabolism invitro and invivo, and might serve as a promising therapeutic target for ameliorating skeletal muscle atrophy.

FOXO3A Expression in Upper Tract Urothelial Carcinoma.

BackgroundEpidemiological studies have reported various results regarding whether FOXO3A is related to various carcinomas. However, the prognostic significance of FOXO3A in upper tract urothelial carcinoma (UTUC) remains unclear. The purpose of this study was to validate the correlation between FOXO3A expression and oncological outcomes in UTUC.MethodsThe expression levels of FOXO3A in 107 UTUC patients were examined by immunohistochemistry (IHC). We examined the prognostic role of FOXO3A by using the Cox proportional hazard model.ResultsThe results indicated that FOXO3A expression was notably decreased in UTUC tissue compared with control tissue. Decreased expression of FOXO3A was also related to advanced pathologic stage (P = 0.026), lymph node metastasis (P = 0.040), lymphovascular invasion (P < 0.001), and adjuvant therapy (P = 0.048). In addition, UTUC patients with low FOXO3A expression had a significantly shorter survival time, including both overall survival (OS) [hazard ratio (HR) 2.382, P = 0.004] and recurrence-free survival (RFS) (HR 2.385, P = 0.004), than those with high expression. Multivariate analyses showed that FOXO3A was a significant predictor for OS (HR 2.145, P = 0.014) and RFS (HR 2.227, P = 0.010) in UTUC patients.ConclusionOur results indicate that FOXO3A may be involved in the recurrence of UTUC and that it has certain clinical value in the therapeutic targeting and prognostic evaluation of UTUC.

Effect of Transcriptional Regulatory Factor FoxO3a on Central Nervous System Oxygen Toxicity

Central nervous system (CNS) oxygen toxicity (CNS-OT) is a toxic reaction that appears after the inhalation of gas at an excessive oxygen partial pressure during underwater operation or hyperbaric oxygen (HBO) treatment. The mechanism of CNS-OT has not been clearly characterized. Though it has been attributed to the excessive oxidative stress induced by HBO, evidences against this hypothesis have been reported. Here we find that Forkhead box protein O3 (FoxO3a) is important for CNS-OT protection. FoxO3a knock-out (KO) mice had a shorter latency to develop convulsions and greater number of seizures within a certain period of time. The acute lung injury (ALI) induced by CNS-OT was also more severe in FoxO3a KO mice. Further analysis reveals a significant decrease in the activity of catalase (CAT), an antioxidant enzyme and a significant increase in the content of malondialdehyde (MDA), an oxidative product, in brain tissues of FoxO3a KO mice. Short-time HBO exposure could increase FoxO3a expression level and trigger its nuclear translocation. The level of nuclear localized FoxO3a peaked at 8 h after exposure. Our results demonstrate that the activity of FoxO3a is highly sensitive to HBO exposure and FoxO3a plays important roles in protecting CNS-OT. Further mechanic analysis reveals that FoxO3a protects CNS-OT via activating antioxidative signaling pathway.

EGFR/FOXO3A/LXR-α Axis Promotes Prostate Cancer Proliferation and Metastasis and Dual-Targeting LXR-α/EGFR Shows Synthetic Lethality

Prostate cancer is the second leading cause of cancer-related death in men. Early prostate cancer has a high 5-year survival rate. However, the five-year survival rate is low in progressive prostate cancer, which manifests as bone metastasis. The EGF receptor overexpression increases during disease progression and in the development of castration-resistant disease, and may be a potential therapeutic target. Liver X receptors (LXRs) are ligand-dependent nuclear receptor transcription factors and consist of two subtypes, LXR-α and LXR-β, which can inhibit tumor growth in various cancer cells. We revealed that LXR-α, but not LXR-β, was reduced in prostate cancer tissues compared with adjacent normal tissues. LXRs’ agonist GW3965 enhanced the inhibitory action of LXR-α on the proliferation and metastasis of prostate cancer cells. Furthermore, our results support the notion that LXR-α is regulated by the EGFR/AKT/FOXO3A pathway. As an EGFR inhibitor, Afatinib could weaken AKT activation and increase the expression level of FOXO3A in prostate cancer. In addition, we indicated that the combination of Afatinib and GW3965 simultaneously increased and activated LXR-α, which led to an increase of tumor suppressors, and eventually inhibited tumor progression. Therefore, the combination of EGFR inhibitor and LXRs agonist may become a potential treatment strategy for prostate cancer, especially metastatic prostate cancer.

Butyrate enhances the efficacy of radiotherapy via FOXO3A in colorectal cancer patient‑derived organoids.

Enhancing the radioresponsiveness of colorectal cancer (CRC) is essential for local control and prognosis. However, consequent damage to surrounding healthy cells can lead to treatment failure. We hypothesized that short-chain fatty acids (SCFAs) could act as radiosensitizers for cancer cells, allowing the administration of a lower and safer dose of radiation. To test this hypothesis, the responses of three-dimensional-cultured organoids, derived from CRC patients, to radiotherapy, as well as the effects of combined radiotherapy with the SCFAs butyrate, propionate and acetate as candidate radiosensitizers, were evaluated via reverse transcription-quantitative polymerase chain reaction, immunohistochemistry and organoid viability assay. Of the three SCFAs tested, only butyrate suppressed the proliferation of the organoids. Moreover, butyrate significantly enhanced radiation-induced cell death and enhanced treatment effects compared with administration of radiation alone. The radiation-butyrate combination reduced the proportion of Ki-67 (proliferation marker)-positive cells and decreased the number of S phase cells via FOXO3A. Meanwhile, 3/8 CRC organoids were found to be non-responsive to butyrate with lower expression levels of FOXO3A compared with the responsive cases. Notably, butyrate did not increase radiation-induced cell death and improved regeneration capacity after irradiation in normal organoids. These results suggest that butyrate could enhance the efficacy of radiotherapy while protecting the normal mucosa, thus highlighting a potential strategy for minimizing the associated toxicity of radiotherapy.

Combination of lapatinib and luteolin enhances the therapeutic efficacy of lapatinib on human breast cancer through the FOXO3a/NQO1 pathway

Breast cancer is a malignant disease and a great cause of morbidity and mortality in women. The etiology of breast cancer is complex and closely related to people’s living habits. Lapatinib, a tyrosine-kinase inhibitor, blocks the activation of the HER1 and HER2 tyrosine kinase to inhibit the activation of downstream signaling pathways and thus inhibit tumor survival and proliferation. This study aimed to explore to the combination of lapatinib and luteolin on human breast cancer. The combination of lapatinib and luteolin increased the sensitivity of SKBR-3, BT-474 and ZR-75-1 cells. This combination equally up-regulated the expression of FOXO3a and NQO1 and their downstream target genes Bim, GADD45, P21, and the phosphorylation level of FOXO3a protein decreased. The mice transplanted with BT-474 cells, the volume of subcutaneous tumors in the luteolin group, lapatinib group, and lapatinib + luteolin group were significantly smaller than the control group. The results of Western blot showed that in tumor tissues of mice transplanted with BT-474 cells, the expression levels of FOXO3a and NQO1 protein in the luteolin group, lapatinib group, and lapatinib + luteolin group were all obviously upregulated, the mice transplanted with ZR-75-1 cells exhibited similar results. These data suggest that the combination of lapatinib and luteolin may inhibit HER2+ human breast cancer by significantly increasing the expression of FOXO3a and NQO1, two key genes in HER2+ human breast cancer xenografts.

The effect of vitamin D on sarcopenia depends on the level of physical activity in older adults.

ObjectiveSarcopenia in older adults is closely related to vitamin D deficiency and reduced levels of physical activity, but little has been reported on the interaction between physical activity and the positive effects of vitamin D. The purpose of this study was to explore the interactive effect of vitamin D and physical activity on muscle mass and function through animal experiments and population surveys.MethodsMale 4‐week‐old C57BL/6J mice were fed different purified diets: a vitamin D‐deficient diet (with increased calcium and phosphorus to prevent the effects of abnormal mineral levels on muscle) or a 1,25‐dihydroxyvitamin D3 (1,25D)‐supplemented diet. After 24 weeks on the assigned diets, the mice were immobilized. The level of skeletal muscle atrophy in the mice was determined by grip strength, gastrocnemius (GA) muscle mass and muscle fiber cross‐sectional area (CSA); additionally, the protein expression levels of FOXO3a and the E3 ubiquitin ligases MuRF1 and MAFbx were detected. A cross‐sectional study included data from 4139 older adults (64.9% women, 67.9 ± 6.7 years) as part of a survey in Shenyang, Northeast China. The associations of serum 25(OH)D3 and physical activity with timed up and go test (TUG) performance, handgrip strength, calf circumference, and body muscle mass were assessed by a linear regression analysis that was adjusted for covariates.ResultsIn activity‐limited mice, vitamin D deficiency accelerated the decrease in GA muscle weight, muscle fiber CSA, and grip strength and increased the protein expression of MuRF1, MAFbx, and FOXO3a (all P < 0.05). In addition, 1,25D supplementation may inhibit the grip‐strength reduction induced by limited activity (P = 0.069). Serum 25(OH)D3 and physical activity were linearly related to TUG time (P < 0.001) and handgrip strength (P < 0.05) after adjustment for sex, age, body mass index (BMI), education level, smoking status, and serum calcium level. Serum 25(OH)D3 and physical activity had interactive effects on TUG (P < 0.001) and handgrip strength (P < 0.05) but not calf circumference or body muscle mass in older adults.ConclusionsThe effect of vitamin D on muscle strength and physical performance depends on physical activity level in the elderly. It is recommended that older adults strive to avoid both physical inactivity and vitamin D deficiency. Because physical inactivity and vitamin D deficiency may exacerbate muscle atrophy, the biological mechanism may involve synergistic effects of vitamin D and physical activity on the promotion of muscle protein ubiquitination and degradation.

FoxO3a Gene Down-regulation in Pathogenesis of Pediatric Acute Lymphoblastic Leukemia

Abstract Introduction: Acute lymphoblastic leukemia (ALL) is the most common malignancy found in the pediatrics with the peak prevalence between the ages of 2 and 5 years. The constitutive activation of PI3K/AKT pathway inhibits the tumor-suppressor role of FoxO3a (a member of the forkhead class O [FoxO] transcription factor family) in a variety of cancers and leads to tumorigenesis. This study aims to investigate the expression of FoxO3a in three different stages of pediatric ALL in mRNA level. Subjects and Methods: In this case-control study, 70 patients with childhood ALL and 70 healthy age- and gender-matched as the control group were enrolled. Real-time quantitative RT-polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression level of FoxO3a in children with different stages of ALL and healthy children as a control group. Results: Data showed that the expression of FoxO3a mRNA was lower in newly diagnosed ALL patients compared to controls (P &lt; 0.0001), maintenance (P = 0.0342), and relapse (P = 0.0006) groups, while no difference was observed between other groups. In addition, T-ALL patients showed decreased expression of FoxO3a compared to Pre-B ALL ones (P &lt; 0.0001). Conclusion: The study results suggest that FoxO3a plays a tumor-suppressor role in ALL. Thus, its up-regulation seems to be a plausible therapeutic strategy for this type of tumor.

The CtBP1-p300-FOXO3a transcriptional complex represses the expression of the apoptotic regulators Bax and Bim in human osteosarcoma cells.

C-terminal binding protein 1 (CtBP1), a well-known transcriptional corepressor, functions as an oncogene in multiple cancer types, including osteosarcoma, by modulating the transcription of many tumor suppressors, such as cadherin 1 (CDH1), phosphatase and tensin homolog (PTEN), Bcl2-associated X (Bax), Bcl-2-interacting mediator (Bim), and cyclin-dependent kinase inhibitor 1A (CDKN1A). However, it is still unclear how CtBP1 regulates the expression of these downstream targets. Here, we identified that CtBP1 is overexpressed in osteosarcoma cells and found that CtBP1 directly interacts with the transcription factor forkhead box O3 (FOXO3a) and the histone acetyltransferase p300 in vivo and in vitro. Through microarray analysis, we found that CtBP1 negatively regulates FOXO3a levels. In contrast to the CtBP1 level, the FOXO3a expression level was found to be significantly reduced in osteosarcoma cells. Knockdown of CtBP1 or overexpression of FOXO3a in U2OS cells resulted in different gene expression patterns, and the former caused upregulation of CtBP1 downstream target genes such as CDH1, PTEN, Bax, Bim, and CDKN1A, whereas the latter caused upregulation of Bax and Bim, but not CDH1, PTEN, and CDKN1A. Further analysis indicated that the CtBP1-p300-FOXO3a transcriptional complex specifically binds to the promoters of Bax and Bim. Inhibition of CtBP1 by the constitutive expression of Pep1-E1AWT peptide in U2OS and OSA cells reversed oncogenic phenotypes, including colony formation, cellular proliferation, and migration, and limited tumor growth in vivo. Together our results demonstrated that the CtBP1-p300-FOXO3a transcriptional complex represses the expression of the apoptotic regulators Bax and Bim in human osteosarcoma cells and that targeting CtBP1-mediated transcriptional events might be a potential therapeutic strategy for the osteosarcoma treatment.

Electroacupuncture improves denervated gastrocnemius atrophy by regulating expression of fork-head protein 3A，muscle atrophy F-box and myogenic differentiation antigen proteins in sciatic nerve injury rats

To observe the effect of electroacupuncture (EA) on morphological changes of denervated gastrocnemius(GS) and the expression of fork-head protein(FOXO3A), muscle atrophy F-box(MAFbx)and myogenic differentiation antigen (Myod1) in sciatic nerve injury rats, so as to reveal its mechanism underlying improvement of myoatrophy. Eighteen male Sprague-Dawley rats were randomly divided into sham operation, model and EA groups (n＝6 per group). The model of gastrocnemius atrophy was established by crushing the right sciatic nerve. Then, EA (2 Hz) was applied to the right "Zusanli" (ST36) and "Huantiao" (GB30) for 10 min, once a day for 14 successive days. The wet weight of the GS on both sides was weighted to calculate the wet weight ratio (the injured side /the healthy side), and the cross-sectional area (CSA) and diameter of GS fibers were measured after H．E. staining. The expressions of FOXO3A, MAFbx and Myod1 protein and mRNA in the GS tissue were tested using Western blot and fluorescence quantitative PCR, separately. Following modeling, the GS wet weight ratio, CSA and fiber diameter were smaller in the model group than those in the sham group (P<0.01), and were significantly higher in the EA group than in the model group (P<0.01). H．E. staining showed that the GS fibers became smaller and the myocyte got round in the model group, while the GS fibers were bigger and the myocyte was relatively regular in morphology in the EA group. After modeling, the expression levels of FOXO3A, MAFbx and Myod1 mRNA and protein were evidently higher in the model group (P<0.01); Moreover, after EA treatment, modeling-induced increasing of expression levels of FOXO3A and MAFbx mRNA and protein were revised (P<0.01), while the increased expression level of Myod1 was further up-regulated relavant to that in the model group (P<0.01)．. EA of ST36 and GB30 can suppress the up-regulated expression of FOXO3A and MAFbx mRNA and protein and further promote the expression of Myod1 mRNA and protein in the GS tissue in rats with denervated GS atrophy, which may contribute to its function in relieving the myoatrophy, promoting the skeletal muscle protein hydrolysis and differentiation of satellite cells.

Resveratrol alleviate the injury of mice liver induced by cadmium sulfide nanoparticles.

Cadmium sulfide nanoparticle (Nano-CdS) is a kind of important semiconductor material with special photochemistry property. With the Nano-CdS being widely used, the security problems it caused have been catching more and more attention. This study aims to explore the possible mechanism of liver injury induced by Nano-CdS and whether resveratrol can reduce the damage. In this study, male BALB/C mice were treated with Nano-CdS with a diameter of 20 to 30 nm and a length of 80 to 100 nm. It turned out that the mice liver inflammatory cells infiltrated, the liver tissue and the ultrastructure changed; The activities of T-AOC and GSH were suppressed (n = 6, P < 0.05) and the content of lipid peroxide (MDA) increased (n = 6, P < 0.05). Besides, Nano-CdS decreased the mRNA expression level of Sirt1 and FoxO1 genes in liver tissue (n = 3, P < 0.05). All the changes in the index were reversed by resveratrol. The mRNA expression level of FoxO3a showed no significant difference between the control group and the Nano-CdS group. But under the protection of resveratrol, the mRNA expression level of FoxO3a was higher than that in the control and Nano-CdS groups (n = 3, P < 0.05). Results suggest that Nano-CdS can cause oxidative damages to liver tissues in mice, in which process that the Sirt1 and FoxO1 genes may participate, and the damage can be reversed by resveratrol which may be a potential cure for oxidative damage to nanomaterials.

MicroRNA-155 deficiency in CD8+ T cells inhibits its anti-glioma immunity by regulating FoxO3a.

A lot of mammalian micro-RNAs have been identified in regulating immune system function. Here, the aim of this study was to investigate the role of microRNA-155 in promoting anti-glioma ability and its potential mechanism. In this study, we constructed microRNA-155 knockout mouse model and glioma mouse model. Subsequently, the progression of glioma and the accumulation of CD8+ T cells were compared between WT and miR-155-/- mice. T cells were transfected with miR-155 mimics and inhibitors, and the proliferative and invasive activities were analyzed. At the same time, we evaluated Akt and Stat5 signaling transduction and the expression level of FoxO3a. Finally, the regulatory ability of FoxO3a to Akt and Stat5 signaling was determined by changing the expression level of FoxO3a in T cells. We found significantly increased progression of glioma in MicroRNA-155 deficiency mice with reduced accumulation of CD8+ T cells in glioma. The proliferative and invasive abilities of T cells were regulated by MicroRNA-155. Besides, microRNA-155 could induce the activation of Akt and Stat5 signaling by inhibiting its target gene FoxO3a. Furthermore, FoxO3a was a negative regulator of Akt and Stat5 signaling. MicroRNA-155 deficiency in CD8+ T cells inhibited anti-tumor activity by suppressing the proliferative and invasive activities of T cells. FoxO3a was a negative regulator of Akt and Stat5 signaling. Besides, microRNA-155 regulated the function of T cells by inhibiting the expression of FoxO3a. Our findings might be a new strategy for the immunotherapy of glioma.

Protective effects of 2,3,5,4‐tetrahydroxystilbene‐2‐o‐β‐D‐glucoside against osteoporosis: Current knowledge and proposed mechanisms

The aim of this study was to explore the mechanism underlying the protective effects of 2,3,5,4-tetrahydroxystilbene-2-o-β-D-glucoside (TSG) against osteoporosis. MC3T3-E1 mouse osteoblast precursor cells were used to analyze the protective effects of TSG on osteoblast apoptosis and differential inhibition induced by oxidative stress to determine the gene expression of forkhead transcription factor FKHRL1 (FoxO3a), T cell factors (TCFs), and downstream genes. A mouse model was used to assess the protective effects of TSG on ovariectomy-induced osteoporosis as well as on Cell Counting Kit-8 (CCK) gene expression, including that of FoxO3a. The mechanism underlying the protective effects of TSG against osteoporosis was further explored using high-throughput sequencing data. A CCK-8 assay in MC3T3-E1 cells and hematoxylin and eosin staining in mouse tissue indicated that cell viability and bone tissue development were inhibited by oxidative stress and ovariectomy and that TSG neutralized or attenuated this effect. The expression levels of FoxO3a, TCF, and downstream genes and the indices of oxidative stress were the same in MC3T3-E1 cells and the bone tissues of the mouse model. Bioinformatics analysis indicated that the cardiac muscle contraction and chemokine signaling pathway were disturbed in MC3T3-E1 cells treated with hydrogen peroxide. Gene ontology-biological process analysis revealed the influence of TSG treatment. Osteoporosis and cardiac diseases appear to share a common mechanism. In addition to Wnt/FoxO3a signaling, the immune system and the chemokine signaling pathway may contribute to the protective mechanism of TSG.