Culture of Bovine Aortic Endothelial Cells in Galactose Media Enhances Mitochondrial Plasticity and Changes Redox Sensing, Altering Nrf2 and FOXO3 Levels.

Understanding the complex biological processes of cells in culture, particularly those related to metabolism, can be biased by culture conditions, since the choice of energy substrate impacts all of the main metabolic pathways. When glucose is replaced by galactose, cells decrease their glycolytic flux, working as an in vitro model of limited nutrient availability. However, the effect of these changes on related physiological processes such as redox control is not well documented, particularly in endothelial cells, where mitochondrial oxidation is considered to be low. We evaluated the differences in mitochondrial dynamics and function in endothelial cells exposed to galactose or glucose culture medium. We observed that cells maintained in galactose-containing medium show a higher mitochondrial oxidative capacity, a more fused mitochondrial network, and higher intercellular coupling. These factors are documented to impact the cellular response to oxidative stress. Therefore, we analyzed the levels of two main redox regulators and found that bovine aortic endothelial cells (BAEC) in galactose media had higher levels of FOXO3 and lower levels of Nrf2 than those in glucose-containing media. Thus, cultures of endothelial cells in a galactose-containing medium may provide a more suitable target for the study of in vitro mitochondrial-related processes than those in glucose-containing media; the medium deeply influences redox signaling in these cells.

FOXO3a deregulation in uterine smooth muscle tumors.

OBJECTIVE: The present study aimed to investigate FOXO3a deregulation in Uterine Smooth Muscle Tumors (USMT) and its potential association with cancer development and prognosis. METHODS: The authors analyzed gene and protein expression profiles of FOXO3a in 56 uterine Leiomyosarcomas (LMS), 119 leiomyomas (comprising conventional and unusual leiomyomas), and 20 Myometrium (MM) samples. The authors used techniques such as Immunohistochemistry (IHC), FISH/CISH, and qRT-PCR for the present analyses. Additionally, the authors conducted an in-silico analysis to understand the interaction network involving FOXO3a and its correlated genes. RESULTS: This investigation revealed distinct expression patterns of the FOXO3a gene and protein, including both normal and phosphorylated forms. Expression levels were notably elevated in LMS, and Unusual Leiomyomas (ULM) compared to conventional Leiomyomas (LM) and Myometrium (MM) samples. This upregulation was significantly associated with metastasis and Overall Survival (OS) in LMS patients. Intriguingly, FOXO3a deregulation did not seem to be influenced by EGF/HER-2 signaling, as there were minimal levels of EGF and VEGF expression detected, and HER-2 and EGFR were negative in the analyzed samples. In the examination of miRNAs, the authors observed upregulation of miR-96-5p and miR-155-5p, which are known negative regulators of FOXO3a, in LMS samples. Conversely, the tumor suppressor miR-let7c-5p was downregulated. CONCLUSIONS: In summary, the outcomes of the present study suggest that the imbalance in FOXO3a within Uterine Smooth Muscle Tumors might arise from both protein phosphorylation and miRNA activity. FOXO3a could emerge as a promising therapeutic target for individuals with Unusual Leiomyomas and Leiomyosarcomas (ULM and LMS), offering novel directions for treatment strategies.

FOXO3a deregulation in uterine smooth muscle tumors

Abstract Objective The present study aimed to investigate FOXO3a deregulation in Uterine Smooth Muscle Tumors (USMT) and its potential association with cancer development and prognosis. Methods The authors analyzed gene and protein expression profiles of FOXO3a in 56 uterine Leiomyosarcomas (LMS), 119 leiomyomas (comprising conventional and unusual leiomyomas), and 20 Myometrium (MM) samples. The authors used techniques such as Immunohistochemistry (IHC), FISH/CISH, and qRT-PCR for the present analyses. Additionally, the authors conducted an in-silico analysis to understand the interaction network involving FOXO3a and its correlated genes. Results This investigation revealed distinct expression patterns of the FOXO3a gene and protein, including both normal and phosphorylated forms. Expression levels were notably elevated in LMS, and Unusual Leiomyomas (ULM) compared to conventional Leiomyomas (LM) and Myometrium (MM) samples. This upregulation was significantly associated with metastasis and Overall Survival (OS) in LMS patients. Intriguingly, FOXO3a deregulation did not seem to be influenced by EGF/HER-2 signaling, as there were minimal levels of EGF and VEGF expression detected, and HER-2 and EGFR were negative in the analyzed samples. In the examination of miRNAs, the authors observed upregulation of miR-96-5p and miR-155-5p, which are known negative regulators of FOXO3a, in LMS samples. Conversely, the tumor suppressor miR-let7c-5p was downregulated. Conclusions In summary, the outcomes of the present study suggest that the imbalance in FOXO3a within Uterine Smooth Muscle Tumors might arise from both protein phosphorylation and miRNA activity. FOXO3a could emerge as a promising therapeutic target for individuals with Unusual Leiomyomas and Leiomyosarcomas (ULM and LMS), offering novel directions for treatment strategies.

Melatonin alleviates pyroptosis by regulating the SIRT3/FOXO3α/ROS axis and interacting with apoptosis in Atherosclerosis progression.

BACKGROUND: Atherosclerosis (AS), a significant contributor to cardiovascular disease (CVD), is steadily rising with the aging of the global population. Pyroptosis and apoptosis, both caspase-mediated cell death mechanisms, play an essential role in the occurrence and progression of AS. The human pineal gland primarily produces melatonin (MT), an indoleamine hormone with powerful anti-oxidative, anti-pyroptotic, and anti-apoptotic properties. This study examined MT's anti-oxidative stress and anti-pyroptotic effects on human THP-1 macrophages treated with nicotine. METHODS: In vitro, THP-1 macrophages were induced by 1 µM nicotine to form a pyroptosis model and performed 30 mM MT for treatment. In vivo, ApoE-/- mice were administered 0.1 mg/mL nicotine solution as drinking water, and 1 mg/mL MT solution was intragastric administrated at 10 mg/kg/day. The changes in pyroptosis, apoptosis, and oxidative stress were detected. RESULTS: MT downregulated pyroptosis, whose changes were paralleled by a reduction in reactive oxygen species (ROS) production, reversal of sirtuin3 (SIRT3), and Forkhead box O3 (FOXO3α) upregulation. MT also inhibited apoptosis, mainly caused by the interaction of caspase-1 and caspase-3 proteins. Vivo studies confirmed that nicotine could accelerate plaque formation. Moreover, mice treated with MT showed a reduction in AS lesion area. CONCLUSIONS: MT alleviates pyroptosis by regulating the SIRT3/FOXO3α/ROS axis and interacting with apoptosis. Importantly, our understanding of the inhibitory pathways for macrophage pyroptosis will allow us to identify other novel therapeutic targets that will help treat, prevent, and reduce AS-associated mortality.

Corrigendum: Txnip expression promotes JNK-mediated neuronal death in response to reactive oxygen species.

[This corrects the article DOI: 10.3389/fnmol.2023.1210962.].

FOXO3 and PTEN expression in the ovary of girls with extra-gonadal cancer with or without chemotherapy treatment prior to cryopreservation.

BACKGROUND: FOXO3/pFOXO3 and PTEN expression is known to regulate the dormancy/activation of ovarian primordial follicles. How chemotherapy could influence the expression of FOXO3 and PTEN in pre- and post-menarcheal girls with extra-gonadal cancer remains unexplored. METHODS: Ovarian samples were collected from 27 girls suffering from extra-gonadal cancer. Of these, 8 patients had received chemotherapy before the time of sample collection. Ovarian tissue collected at the time of surgery was fixed in 10% formaldehyde for FOXO3/pFOXO3 and PTEN immunohistochemistry or immunofluorescence, or stored at -80 °C for Western blot, or preserved in RNA later for RT-PCR. RESULTS: PTEN was detected in a limited number of primordial follicle-enclosed oocytes in approximately fifty percent of the patients, regardless of whether they had received anti-cancer treatment or not. However, there was a significant decrease in PTEN detection in patients who underwent chemotherapy treatment prior to the retrieval of the sample. Both primordial follicle-enclosed oocytes that expressed FOXO3 and those that did not were identified in patients who were treated with chemotherapy and those who were not. FOXO3-positive primordial follicles exhibited either nuclear FOXO3 localization or cytoplasmic pFOXO3 localization. Furthermore, transitional primordial follicles that expressed nuclear FOXO3 and cytoplasmic pFOXO3 were also observed. Primary follicle-enclosed oocytes displayed cytoplasmic pFOXO3 localization, whereas in more advanced stages of folliculogenesis, the expression moved to the somatic stratum. No significant statistical differences were identified in the detection of FOXO3 and pFOXO3 in patients who had or had not received chemotherapy prior to sample collection. CONCLUSION: Primordial follicles expressing and not expressing FOXO3 were equally present in both the ovaries of patients who underwent chemotherapy and those who did not. The expression of FOXO3 remained unaltered in response to chemotherapy treatment. Notably, the detection of PTEN was significantly reduced in the treated patients, thereby warranting in-depth investigation, given the limited sample size examined in the present study.

Txnip expression promotes JNK-mediated neuronal death in response to reactive oxygen species.

TXNIP is a protein sensitive to oxidant conditions whose expression is related to the progression of death in cancer, diabetes, ischemia, and neurodegenerative diseases, among others. Because of this, many studies propose TXNIP as a therapeutic target in several diseases. Exposure of cerebellar granule neurons to staurosporine or low potassium leads to apoptotic death. Both conditions generate an early production of reactive oxygen species (ROS) that induces the activation of the ASK1 pathway and the apoptotic machinery. In these models, it has been shown an increase in TXNIP protein mediated by ROS. Here, we evaluated the molecular mechanisms involved in the regulation of the Txnip expression during neuronal death, as well as the role of the protein in the progression of cell death induced by these two apoptotic conditions. In cultured cerebellar granule neurons, we observed that low potassium and staurosporine induced an early increase in ROS that correlated with an increase in Txnip mRNA. When we evaluated the promoter of the gene, we found that the JASPAR-reported FOXO1/3 transcription factor motifs are close to the transcription start site (TSS). We then verified through the Chromatin immunoprecipitation technique (ChIP) that FOXO3 interacts with the Txnip promoter after 1 h of low potassium treatment. We also detected FOXO3 nuclear translocation by low potassium and staurosporine treatments. Finally, by using shRNA in the neuroblastoma MSN cell line, we found that Txnip downregulation decreased neuronal death induced by staurosporine stimulus. Together, these results suggest that ROS promotes the expression of Txnip through the activation of the FOXO3 transcription factor mediated by Akt inhibition. We also demonstrated that TXNIP is necessary for neuronal death progression.

SGK1 is necessary to FoxO3a negative regulation, oxidative stress and cardiac fibroblast activation induced by TGF-ß1.

Cardiac fibroblasts (CFs) activation is a common response to most pathological conditions affecting the heart, characterized by increased cellular secretory capacity and increased expression of fibrotic markers, such as collagen I and smooth muscle actin type alpha (α-SMA). Fibrotic activation of CFs induces the increase in tissue protein content, with the consequent tissue stiffness, diastolic dysfunction, and heart failure. Therefore, the search for new mechanisms of CFs activation is important to find novel treatments for cardiac diseases characterized by fibrosis. In this regard, TGF-ß1, a cytokine with proinflammatory and fibrotic properties, is crucial in the CFs activation and the development of fibrotic diseases, whereas its molecular targets are not completely known. Serum and glucocorticoid-regulated kinase (SGK1) is a protein involved in various pathophysiological phenomena, especially cardiac and renal diseases that curse with fibrosis. Additionally, SGK1 phosphorylates and regulates the activity and expression of several targets, highlighting FoxO3a for its role in the regulation of oxidative stress and CFs activation induced by TGF-ß1. However, the regulation of SGK1 by TGF-ß1 and its role in CFs activation have not been studied. In this work, we evaluate the role of SGK1 in CFs isolated from neonatal Sprague-Dawley rats. The participation of SGK1 in the fibrotic activation of CFs induced by TGF-ß1 was analyzed, using an inhibitor or siRNA of SGK1. In addition, the role of SGK1 on the regulation of FoxO3a and oxidative stress induced by TGF-ß1 was analyzed. Our results indicate that TGF-ß1 increased both the activity and expression of SGK1 in CFs, requiring the activation of MAPKs, ERK1/2, p38 and JNK, while inhibition and silencing of SGK1 prevented TGF-ß1-induced fibrotic activation of CFs. In addition, SGK1 inhibition prevented FoxO3a inactivation and expression reduction, catalase and SOD2 expression decrease, and the increase of oxidative stress induced by TGF-ß1. Taken together, our results position SGK1 as an important regulator of CFs activation driven by TGF-ß1, at least in part, through the regulation of FoxO3a and oxidative stress.

SerpinA3K Deficiency Reduces Oxidative Stress in Acute Kidney Injury.

We previously showed that SerpinA3K is present in urine from rats and humans with acute kidney injury (AKI) and chronic kidney disease (CKD). However, the specific role of SerpinA3K during renal pathophysiology is unknown. To begin to understand the role of SerpinA3K on AKI, SerpinA3K-deficient (KOSA3) mice were studied 24 h after inducing ischemia/reperfusion (I/R) and compared to wild type (WT) mice. Four groups were studied: WT+S, WT+IR, KOSA3+S, and KOSA3+IR. As expected, I/R increased serum creatinine and BUN, with a GFR reduction in both genotypes; however, renal dysfunction was ameliorated in the KOSA3+IR group. Interestingly, the increase in UH2O2 induced by I/R was not equally seen in the KOSA3+IR group, an effect that was associated with the preservation of antioxidant enzymes' mRNA levels. Additionally, FOXO3 expression was initially greater in the KOSA3 than in the WT group. Moreover, the increase in BAX protein level and the decrease in Hif1a and Vegfa induced by I/R were not observed in the KOSA3+IR group, suggesting that these animals have better cellular responses to hypoxic injury. Our findings suggest that SerpinA3K is involved in the renal oxidant response, HIF1α/VEGF pathway, and cell apoptosis.

Could be FOXO3a, miR-96-5p and miR-182-5p useful for Brazilian women with luminal A and triple negative breast cancers prognosis and target therapy?

FOXO3a dysregulation is frequently implicated in tumorigenesis, and its inhibition can occur by several molecular mechanisms. Among these, post-transcriptional suppression by miRNAs has been associated with various cancers initiation. Here, we assessed the expression profiles of the most relevant miRNAs for breast tumorigenesis, using Luminal A (LA) and Triple-Negative (TN) breast cancer from Brazilian patients, by the quantitative real time-PCR method. Their potential prognostic role for the patients was also evaluated. We identified the miRNAs miR-96-5p and miR-182-5p, de-scribed as negative regulators of FOXO3A, with differential expression both in LA and TN tumors when compared to normal tissue. The miR-96-5p and miR-182-5p miRNAs were upregulated in LA (7.82 times, p < 0.005; 6.12 times, p < 0.005, respectively) and TN breast cancer samples (9.42 times, p < 0.0001; 8.51 times, p < 0.0001) compared to normal tissues. The samples with higher miR-96-5p and miR-182-5p expression (FR ≥ 4) were submitted for FOXO3a immunostaining. Reduced protein detection was observed in all of the tumors compared to normal tissues. The most prominent miRNA expression and FOXO3a protein suppression were observed in TN samples (p < 0.001), indicating the relevant role of these molecules in this tumor biology and clinical behavior. Our results corroborate the literature regarding to the relevance of FOXO3a in the breast cancer, and they open new perspectives for alternative target therapy options for Brazilian patients expressing both FOXO3a and its regulatory miRNAs.

Could be FOXO3a, miR-96-5p and miR-182-5p useful for Brazilian women with luminal A and triple negative breast cancers prognosis and target therapy?

Abstract FOXO3a dysregulation is frequently implicated in tumorigenesis, and its inhibition can occur by several molecular mechanisms. Among these, post-transcriptional suppression by miRNAs has been associated with various cancers initiation. Here, we assessed the expression profiles of the most relevant miRNAs for breast tumorigenesis, using Luminal A (LA) and Triple-Negative (TN) breast cancer from Brazilian patients, by the quantitative real time-PCR method. Their potential prognostic role for the patients was also evaluated. We identified the miRNAs miR-96-5p and miR-182-5p, de-scribed as negative regulators of FOXO3A, with differential expression both in LA and TN tumors when compared to normal tissue. The miR-96-5p and miR-182-5p miRNAs were upregulated in LA (7.82 times, p < 0.005; 6.12 times, p < 0.005, respectively) and TN breast cancer samples (9.42 times, p < 0.0001; 8.51 times, p < 0.0001) compared to normal tissues. The samples with higher miR-96-5p and miR-182-5p expression (FR ≥ 4) were submitted for FOXO3a immunostaining. Reduced protein detection was observed in all of the tumors compared to normal tissues. The most prominent miRNA expression and FOXO3a protein suppression were observed in TN samples (p < 0.001), indicating the relevant role of these molecules in this tumor biology and clinical behavior. Our results corroborate the literature regarding to the relevance of FOXO3a in the breast cancer, and they open new perspectives for alternative target therapy options for Brazilian patients expressing both FOXO3a and its regulatory miRNAs.

Influence of Melatonin Treatment on Cellular Mechanisms of Redox Adaptation in K562 Erythroleukemic Cells.

Melatonin (MEL) presents well-documented pleiotropic actions against oxidative stress (OS), acting indirectly through activation of transcription factors, e.g., FoxO3 and Nrf2. Thus, this study aimed to investigate the possible modulating effects of MEL on the redox signaling pathways PI3K/AKT/FoxO3 and Keap1/Nrf2/ARE in K562 erythroleukemic cells subjected to OS induction. For this, the viability, and transcript levels of genes involved in redox adaptation were evaluated in K562 cells in different periods of erythroid differentiation: under OS induction by hydrogen peroxide (100 µM H2O2); treated with 1 nM (C1) and 1 mM (C2) MEL; and associated or not with stress induction. We observed a restoration of physiological levels of Nrf2 in both MEL concentrations under OS. The C1 was related to enhanced expression of antioxidant and proteasome genes through the Nrf2-ARE pathway, while C2 to the induction of FOXO3 expression, suggesting an involvement with apoptotic pathway, according to BIM transcript levels. The effects of MEL administration in these cells showed a period and dose-dependent pattern against induced-OS, with direct and indirect actions through different pathways of cellular adaptation, reinforcing the importance of this indolamine in the regulation of cellular homeostasis, being a promising therapeutic alternative for diseases that present an exacerbated OS.

circRNA CRIM1 regulates the migration and invasion of bladder cancer by targeting miR182/Foxo3a axis.

PURPOSE: To explore the molecular mechanism of circRNA CRIM1 in the regulation of bladder cancer by targeting the miR182/Foxo3a axis. METHODS: 50 pairs of cancer tissues and para-cancerous tissues of patients with bladder cancer were collected. RT-PCR method was used to detect the expression of CRIM1 and miR-182. The association between circRNA CRIM1 and clinical data was analyzed. qPCR was used to measure the expression of circRNA CRIM1 and miR-182 in bladder cancer cell UMUC3 and endothelial cell line HUVEC. CRIM1 genes and miR-182 in UMUC3 cell lines were overexpressed and silenced, respectively, to investigate their effects on invasion and migration of bladder cancer, and to detect the changes of miR182/Foxo3a expression. The association between circRNA CRIM1 and miR182/Foxo3a was determined by bioinformatics analysis. RESULTS: The results showed that there was a significant association between the expression of circRNA CRIM1 and distal migration. The expression of CRIM1 in adjacent tissues was significantly down-regulated and negatively correlated with distal migration. The overexpression of circRNA CRIM1 reduced migration and invasion processes in bladder cancer cells. After circRNA CRIM1 was overexpressed, the miR-182 was significantly down-regulated. The expression levels of Foxo3a mRNA and proteins were up-regulated after miR-182 silencing of bladder cancer cell line UMUC3. miR-182 silencing inhibited invasion and migration of cancer cells to some extent. In bladder cancer cells and tissues, CRIM1 and Foxo3a were significantly down-regulated, miR-182 was significantly up-regulated. CONCLUSION: circRNA CRIM1 regulated the migration and invasion of bladder cancer by targeting the miR182/Foxo3a axis.

MAPK, AKT/FoxO3a and mTOR pathways are involved in cadmium regulating the cell cycle, proliferation and apoptosis of chicken follicular granulosa cells.

The occurrence of cadmium (Cd) in feed is a major problem in animal health and production. Studies have confirmed that Cd depresses egg production of laying hens, which is closely related to follicular atresia. This study aimed to assess the toxic impacts of Cd on the ovarian tissue, and to examine the mechanism of Cd-induced granulosa cell proliferation and apoptosis. Results from the nitric oxide (NO) and malondialdehyde (MDA) content, total superoxide dismutase (T-SOD), glutathione peroxide (GSH-Px), total nitric oxide synthase (T-NOS) and adenosine triphosphatase (ATPase) activities, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, and hematoxylin-eosin (H & E) staining indicated that excess Cd induced oxidative stress, granulosa cell apoptosis and follicular atresia in the layer ovary. Low-dose Cd exposure (1 µM) induced the granulosa cell proliferation, upregulated the mRNA levels of RSK1 and RHEB, activated FoxO3a, AKT, ERK1/2, mTOR and p70S6K1 phosphorylation, and promoted cell cycle progression from phase G1 to S. However, high-dose Cd exposure (15 µM) induced reactive oxygen species (ROS) generation and cell apoptosis, upregulated the mRNA levels of the inflammatory factors, ASK1, JNK, p38 and TAK1, downregulated the expressions of RSK1 and RHEB genes, and inhibited the phosphorylation of ERK1/2, mTOR and p70S6K1 proteins, and the cell cycle progression. Rapamycin pre-treatment completely blocked the phosphorylation of mTOR and p70S6K1 proteins, and the cell cycle progression induced by 1 µM Cd, and accelerated 15 µM Cd-induced cell apoptosis and cell cycle arrest. The microRNA sequencing result showed that 15 µM Cd induced differential expression of microRNA genes, which may regulate AKT, ERK1/2 and mTOR signaling and cell cycle progression by regulating the activity of G proteins and cell cycle-related proteins. Conclusively, these results indicated that Cd can cause the ovarian damage and follicular atresia, and regulate cell cycle, cell proliferation or apoptosis of granulosa cells through MAPK, AKT/FoxO3a and mTOR pathways in laying hens.

Angiotensin-(1-7) prevents T3-induced cardiomyocyte hypertrophy by upregulating FOXO3/SOD1/catalase and downregulating NF-ĸB.

Clinical studies have shown a correlation between thyroid disorders and cardiac diseases. High levels of triiodothyronine (T3) induce cardiac hypertrophy, a risk factor for cardiac complications and heart failure. Previous results have demonstrated that angiotensin-(1-7) is able to block T3-induced cardiac hypertrophy; however, the molecular mechanisms involved in this event have not been fully elucidated. Here, we evidenced the contribution of FOXO3 signaling to angiotensin-(1-7) effects. Angiotensin-(1-7) treatment increased nuclear FOXO3 levels and reduced p-FOXO3 levels (inactive form) in isolated cardiomyocytes. Knockdown of FOXO3 by RNA silencing abrogated the antihypertrophic effect of angiotensin-(1-7). Increased expression of antioxidant enzymes superoxide dismutase 1 (SOD1 and catalase) and lower levels of reactive oxygen species and nuclear factor-κB (NF-κB) were observed after angiotensin-(1-7) treatment in vitro. Consistent with these results, transgenic rats overexpressing angiotensin-(1-7) displayed increased nuclear FOXO3 and SOD1 levels and reduced NF-κB levels in the heart. These results provide a new molecular mechanism responsible for the antihypertrophic effect of angiotensin-(1-7), which may contribute to future therapeutic targets.

Botryosphaeran, a (1 → 3)(1 → 6)-ß-D-glucan, reduces tumor development and cachexia syndrome in obese male rats by increasing insulin sensitivity and FOXO3a activity.

Obesity is an important risk factor in tumor development. Botryosphaeran, a (1 â†’ 3)(1 â†’ 6)-ß-D-glucan, produced by the fungus Botryosphaeria rhodina (MAMB-05), is a high molecular mass, water-soluble exopolysaccharide. It consists of a main chain of (1 â†’ 3)-linked ß-d-glucose units, with a degree of branching of ~22% at carbon-6 with glucose and gentiobiose residues linked through ß-(1 â†’ 6)-bonds, and presents a triple helix conformation. Botryosphaeran presents anticlastogenic, antiproliferative, pro-apoptotic and anti-obesogenic activities. This study evaluated the effects of botryosphaeran on tumor development in obesity and analyzed its mechanism of action. Obesity was induced in male Wistar rats by a high-fat/high-sugar diet. After 9 weeks, rats were divided into two groups: Obese Tumor (OT) and Obese Tumor Botryosphaeran (OTB), and inoculated with 1 × 107 Walker-256 tumor cells, and treatment with botryosphaeran (30 mg/kg b.w./day via gavage for 15 days) commenced. On the 11th week, biological parameters, tumor development, metabolic profile, erythrogram and protein expression were evaluated. Botryosphaeran significantly reduced tumor growth, body-weight loss and cachexia. Furthermore, botryosphaeran decreased mesenteric fat and insulin resistance, corrected macrocytic anemia, and increased Forkhead transcription factor-3a (FOXO3a) activity. Our study demonstrated the potential role of botryosphaeran in the management of cancer in tumor-bearing obese rats by increasing insulin sensitivity and FOXO3a activity.

PTEN and FOXO3 expression in the prenatal and postnatal human ovary.

PURPOSE: The objective of this study was to analyse the expression and cellular localization of FOXO3, pFOXO3 and PTEN throughout human ovary development both before and after birth. METHODS: Foetal, pubertal and adult paraffin-embedded ovarian samples were analysed by immunohistochemistry for cellular localization of FOXO3, pFOXO3 and PTEN proteins. Protein and mRNA expression were analysed by western blot and real time PCR, respectively, from fresh biopsies. RESULTS: PTEN was not detected by immunohistochemistry in germ cells and follicles of foetal, pubertal and adult ovaries. Occasional PTEN immunoreactive granulosa cells were found in atretic antral follicles in the adult ovary. Western blot analysis showed low levels of PTEN protein. Nuclear FOXO3-expressing primordial follicles represented a variable proportion of the ovarian reserve. The presence of FOXO3-expressing primordial follicles was very low in foetal ovary; although always represented in a low proportion, prevalence increased during pubertal and adult life. CONCLUSION: Our results seem to indicate that two subpopulations of primordial follicles, i.e. nuclear FOXO3-expressing and no FOXO3-expressing primordial follicles are found in the postnatal human ovary. This scenario suggests that FOXO3 could be acting as in the mouse model, preventing primordial follicle activation. However, the strategy would not be an "all or nothing" system as in mouse ovary but rather a selected subpopulation of primordial follicles preserved to ensure long-term fertility.

Airborne Particulate Matter (PM10) Inhibits Apoptosis through PI3K/AKT/FoxO3a Pathway in Lung Epithelial Cells: The Role of a Second Oxidant Stimulus.

Outdoor particulate matter (PM10) exposure is carcinogenic to humans. The cellular mechanism by which PM10 is associated specifically with lung cancer includes oxidative stress and damage to proteins, lipids, and DNA in the absence of apoptosis, suggesting that PM10 induces cellular survival. We aimed to evaluate the PI3K/AKT/FoxO3a pathway as a mechanism of cell survival in lung epithelial A549 cells exposed to PM10 that were subsequently challenged with hydrogen peroxide (H2O2). Our results showed that pre-exposure to PM10 followed by H2O2, as a second oxidant stimulus increased the phosphorylation rate of pAKTSer473, pAKTThr308, and pFoxO3aSer253 2.5-fold, 1.8-fold, and 1.2-fold, respectively. Levels of catalase and p27kip1, which are targets of the PIK3/AKT/FoxO3a pathway, decreased 38.1% and 62.7%, respectively. None of these changes had an influence on apoptosis; however, the inhibition of PI3K using the LY294002 compound revealed that the PI3K/AKT/FoxO3a pathway was involved in apoptosis evasion. We conclude that nontoxic PM10 exposure predisposes lung epithelial cell cultures to evade apoptosis through the PI3K/AKT/FoxO3a pathway when cells are treated with a second oxidant stimulus.

Effect of caloric restriction and rapamycin on ovarian aging in mice.

Caloric restriction (CR) increases the preservation of the ovarian primordial follicular reserve, which can potentially delay menopause. Rapamycin also increases preservation on the ovarian reserve, with similar mechanism to CR. Therefore, the aim of our study was to evaluate the effects of rapamycin and CR on metabolism, ovarian reserve, and gene expression in mice. Thirty-six female mice were allocated into three groups: control, rapamycin-treated (4 mg/kg body weight every other day), and 30% CR. Caloric restricted females had lower body weight (P < 0.05) and increased insulin sensitivity (P = 0.003), while rapamycin injection did not change body weight (P > 0.05) and induced insulin resistance (P < 0.05). Both CR and rapamycin females displayed a higher number of primordial follicles (P = 0.02 and 0.04, respectively), fewer primary, secondary, and tertiary follicles (P < 0.05) and displayed increased ovarian Foxo3a gene expression (P < 0.05). Despite the divergent metabolic effects of the CR and rapamycin treatments, females from both groups displayed a similar increase in ovarian reserve, which was associated with higher expression of ovarian Foxo3a.

Botryosphaeran reduces obesity, hepatic steatosis, dyslipidaemia, insulin resistance and glucose intolerance in diet-induced obese rats.

AIMS: Obesity is associated with comorbidities such as diabetes and hepatic steatosis. ß-Glucans have been described as effective in treating conditions including dyslipidaemia and diabetes. Thus, the objective of this study was to evaluate the effects of botryosphaeran [(1 → 3)(1 → 6)-ß-D-glucan] on obesity and its comorbidities, and understand its mechanism of action. MAIN METHODS: Obesity was induced in adult male Wistar rats by ingestion of a high-fat diet and water with sucrose (300 g/L) for 8 weeks. Control rats received standard diet. After six weeks, treatment commenced with botryosphaeran (12 mg/kg.b.w., via gavage, 15 days), respective controls received water. Rats were divided into 3 groups: control (C), obese (O), and obese + botryosphaeran (OB). In the 8th week, obesity was characterized. Feed-intake, glucose and lipid profiles, glucose tolerance, and concentrations of glycogen and lipids in liver were analyzed. Protein expression was determined by Western blotting. KEY FINDINGS: Obese rats showed significant increases in weight gain and adipose tissue, presented glucose intolerance, dyslipidaemia, and hepatic steatosis. Botryosphaeran significantly reduced feed intake, weight gain, periepididymal and mesenteric fat, and improved glucose tolerance. Botryosphaeran also reduced triglyceride and VLDL, and increased HDL levels. Furthermore, botryosphaeran increased glycogen and reduced total lipids, triglycerides and cholesterol in liver, and increased AMP-activated protein kinase(AMPK) activity and Forkhead transcription factor 3a(FOXO3a) protein expression in adipose tissue. SIGNIFICANCE: This study demonstrated that botryosphaeran was effective in reducing obesity, hepatic steatosis, dyslipidaemia insulin resistance and glucose intolerance in diet-induced obese rats, and these effects were, at least in part, associated with reduced feed intake, and AMPK and FOXO3a activities.