FOXO3a Gene Research Articles

Aging-associated stem/progenitor cell dysfunction in the salivary glands of mice

Although stem cell aging leads to a decline in tissue homeostasis and regenerative capacity, it remains unclear whether salivary gland stem cell function changes during this process. However, the salivary glands are gradually replaced by connective tissue during aging. Here, we show a decline in the stem cell ability of CD133-positive stem/progenitor cells in the salivary glands of aged mice. The CD133-positive cells were isolated from young, adult, and aged mice. The number of CD133-positive cells was significantly decreased in aged mice. They also showed a lower sphere formation capacity compared to young and adult mice. RNA sequencing revealed that CD133-positive cells in aged mice exhibited lower gene expression of several aging-related genes, including FoxO3a, than those in young and adult mice. Salivary gland cells infected with a recombinant lentivirus encoding the FoxO3a gene showed a reduction in oxidative stress induced by hydrogen peroxide compared with those infected with a control virus. Thus, FoxO3a may inhibit stem cell aging via oxidative stress.

Effects of the FOXO3a rs 4946936 Gene Polymorphism on the FOXO3a Transcription Factor in Chronic Granulocytic Leukemia Patients

Introduction : FOXO3a has an important role in the maintenance of leukemic stem cells. BCR-ABL inhibition therapy by TKI dasatinib aims to reduce the phosphorylation of the transcription factor FOXO3a, promote localization of FOXO3a in the nucleus and restore transcriptional activity. However, some studies showed that the TKI dasatinib, in addition to increase the relocation of Foxo3a to the intranuclear, also increase the expression of CDKN1c/p57 and Bcl6 genes that became the down target for Foxo3a intra nucleus ATM. Therefore, current therapy is mostly directed at personalized therapy (personalized medicine). The aim of this study was to investigate the effect of the FOXO3a rs4946936 gene polymorphism on the Foxo3a transcription factor in chronic granulocytic leukemia patients treated with Imatinib mesylate.
 Method : This is a cross-sectional study in patients with chronic granulocytic leukemia (CGL) with positive Bcr-ABL. The aim was to prove the effect of the FOXO3a gene polymorphism rs4946936 on the Foxo3a transcription factor. The data analysis test used was a correlation test and a regression test.
 Result : There were three polymorphisms of the FOXO3a gene, namely CC polymorphism, TC polymorphism, and TT polymorphism with a p-value of 0.026 and an r of 0.287, so it can be concluded that there was a significant correlation between the FOXO3a gene polymorphism and the Foxo3an transcription factor with a sufficient correlation value. In the regression test between the FOXO3a gene polymorphisms and the transcription factor FOXO3a, the p value was 0.029 and the B value was -0.294. This means that it has a negative and significant effect on the Foxo3a transcription factor variable.
 Conclusion : There was a significant correlation between the gene polymorphism FOXO3a rs4946936 and the transcription factor FOX3a. The FOXO3a gene polymorphism of the TT genotype had a negative effect on the FOXO3a transcription factor. The TT gene in the FOXO3a gene polymorphism was the most effective in reducing the FOXO3a transcription factor.

Evaluating the expression of key genes involved in resistance to oxidative stress in ALL patients

Background: Leukemia accounts for about 8% of all cancers and causes approximately 7% of mortalities due to malignancies. Acute lymphoblastic leukemia (ALL) is the most common childhood cancer and rare in older subjects. The aim of this study was to evaluate the expression of oxidative stress resistance genes including Catalase, manganese superoxide dismutase (MnSOD), Forkhead Box O3 (Foxo3a), and sirtuin-1 (SIRT1) in ALL patients that may be applied for therapeutic purposes in the future. Materials and Methods: In this observational case-control study, blood samples were drawn from 60 newly diagnosed ALL patients and 10 healthy individuals as a control group. After RNA extraction and cDNA synthesis, real-time polymerase chain reaction (RT-PCR) amplification was performed using specific primers for evaluating the expression of Catalase, MnSOD, Foxo3a, and SIRT1 genes. Results: The expression of all studied genes were significantly higher in ALL patients than in the control group; catalase gene, FOX gene, MnSOD gene, and SIRT1 gene were expressed 4 times (p =0.04), 4.5 times (p =0.001), 2.2 times (p =0.05) and 4.8 (p =0.01) times higher than healthy individuals in the control group respectively. However, no significant relationship between their expression and the stage of the disease and blast percentage was demonstrated (P>0.05). Conclusion: According to these results, the authors believe that the pathways involved in oxidative stress may be one of the most important causes of ALL disease's development and progression. In this regard, targeting the critical genes of these pathways can be considered a potential treatment with fewer side effects.

Comparing Effect of FOXO3 Gene and Urban-Rural Environment on Longevity: a Cohort Study among Older Adults in China

BACKGROUND AND AIM: Empirical evidence points to urban residents having better health than their rural counterparts in several countries. Since lifespan has complex environmental and genetic underpinnings, we aim to study the effects of FOXO3 and the urban-rural disparity on mortality. METHODS: We used the Chinese Longitudinal Healthy Longevity Survey (CLHLS). The study cohort conducted surveys from 2008 to 2014, roughly biennially. The geographical distribution encompassed 22 out of 34 provinces in China. We used tagging SNPs rs2253310, rs2802292, and rs4946936 to identify the FOXO3A gene. We used residence classifications: village, town, and city. We used the Cox-proportional hazard regression models to assess the main effects and interaction of FOXO SNPs and residence on mortality risk adjusted for covariates. We conducted analysis to assess the effects of fine particulate matter (PM2.5) concentrations (three-year annual average, 1km×1km grid) and residential greenness (Normalized Difference Vegetation Index (NDVI), 500-meter radius) around participant residences. RESULTS:Among the 3179 participants, the baseline mean age was 85.1 (SD: 11.3) years, 1688 (53%) were female, 32.1% lived in urban areas (city/town), and 67.9% lived in the rural areas (village). The distributions of the six SNPs of FOXO3A are even across populations of different demographic characteristics (except for age and gender), indicating mendelian randomization. We found FOXO3A to have a protective effect on mortality [HR (95% CI): rs2802292 GG vs TT/TG: 0.825 (0.688, 0.989); rs2253310 CC vs. GG/GC 0.825 (0.689,0.989); rs4946936 TT vs. CC: 0.805 (0.655, 0.989)]. We found participants living in rural areas to have a lower risk of mortality [HR of the urban vs. the rural: 0.825 (95% CI: 0.735, 0.926)]. CONCLUSIONS:Higher air pollution and lower residential greenness both contributed to higher mortality. The effect size of the beneficial effect of FOXO3 on mortality is roughly equivalent to that living in urban areas. KEYWORDS: Longevity, FOXO3A, gene-environment interaction, air pollution, PM2.5, greenness

Signaling pathways associated with structural changes in varicose veins: a case-control study.

In varicose veins, blood pressure increases in the veins of the lower extremities due to mechanical stimulation and function remodeling. The aim of this study is assessment of Signaling pathways associated with structural changes in varicose veins. This pilot study was performed on patients with varicose veins, which had undergone surgery. The healthy tissues from trauma patients or vascular bypass without underlying diseases were used for control samples. Hematoxylin-eosin, trichrome, and elastin staining were used for histopathological examination. The levels of MDA (malondialdehyde), total thiol, SOD (Superoxide dismutase) and NO (nitric oxide) level were measured using Elisa kits to evaluate the oxidative stress level. Gene expression levels of MMP2, MMP9, FOXO3a, APOE and p53 genes were determined using Real-time PCR. This study showed, the vascular Vein wall changes are visible in vascular collagen staining. Although these changes are observed in the structure of vascular wall collagen fibers, the accumulation of collagen and elastin was increased in the walls of varicose veins compared to the control group. The amount of nitric oxide and thiol were increased in the varicose group (P < 0.0001). The expression of metalloproteinase2 gene associated with extracellular matrix change was increased in varicose veins. However, the amount of metalloproteinase 9 was decreased in this group compared to control group. FOXO3a, APOE Genes were down-regulated in the varicose veins compared to control group, while p53 gene expression was significantly increased in the varicose group (P < 0.0001). This study demonstrated changes in oxidative stress, morphological structure, and aging pathways in varicose when compared to non-varicose veins. The changes in oxidative stress may be associated with the variations in morphological structure and aging pathways which contribute to the pathogenesis of varicose veins.

Specific features of the oldest old from the Longevity Blue Zones in Ikaria and Sardinia

Human longevity may be found in single individuals as well as in the population as a whole (“population longevity”). Longevity Blue Zones (LBZs), which are areas with an unusually high number of oldest old, have been identified in Sardinia and the Greek island of Ikaria. We compared the lifestyle, health status and some genetic markers of the LBZ populations with those of reference populations from Italy and Greece; the data were extracted from the GEHA database. In the LBZs, the proportion of individuals who never married or were married and still living with their spouse was significantly greater. Nonagenarians males and females with a high self‒perception of optimism and/or a high score for self-rated health were also found in larger proportions in LBZs. Among the variables with lower frequency were the proportion of the widowed, the percentage of subjects who had suffered a stroke and the frequency of Apoε4 and Apoε2 and the TT genotype of FOXO3A gene. Compared to behavioral and health indicators, the impact of genetic factors might be relatively less important in the LBZs. Nevertheless, further research is needed to identify potential epigenetic traits that might play a predominant role due to the interaction between genetics and the human and physical environments.

Molecular Targets of Phyto-bioactive Compounds in Female Reproductive System of Mammals: A Review

Phytochemicals present in the plants are divided into primary (Alcohol Amino acids, nucleotides. etc) and secondary metabolites (Alkaloids, Saponins etc.). Carotenoids (reduces reactive oxygen species formation, decreases apoptotic cells, restoration of actin capping expression proteins etc.), Phenolics (inhibits extracellular signal-regulated kinase signalling pathway), Isoflavones (inhibits tyrosine kinase pathway) and alkaloids (downregulation of vascular endothelial growth factor, tumor necrosis factor-alpha and hypoxia-inducible factor 1-alpha messengers) are the major phytochemicals, having the potential effects towards ovarian function. Likewise, bioactive compounds are the chemicals that can interact with certain components of live tissue to exert their various effects (antioxidant, antineoplastic, receptor inhibition, gene expression etc.) respective to female fertility. Similarly, bioactive compounds: Kaempferol [phosphatidylinositol -3- kinase (PI3K)/protein kinase B (Akt) pathway], Quercetin (controlling the release of 17β-estradiol etc.), Myricetin (PI3K/Akt and MAPK signalling pathway), Galngin (inhibition of angiogenesis via decreasing the VEGF and p-Akt) and Resveratrol (regulation of Foxo3a and SIRT1 genes etc.) shows its effects by targeting different molecules and/or pathways at the ovarian microenvironment. However, Genistein (binding to estrogen receptors: ESRα and ESRβ etc.) and Diadzein (disrupting the endocrines etc.) emphatically interfere with the ovarian functions. Besides this, molecular effects exerted by these phyto-bioactive compounds on the in vivo and/or in vitro ovarian culture systems entirely depend on their dosage: Kaempferol @10 μM increased the primordial follicle activation, Quercetin @4 µM improved the quality of oocytes whereas @8 µM reduced the quality), Resveratrol @ 2 µM increased the blastocyst formation, Myricetin @ 100 mg/kg/day feeding in rats induced estrogenic activity, Genistein, feeding in female mice @ 500 and 1000ppm increased the gestation time and Diazdein causes the inhibition of 3-hydroxysteroid dehydrogenase at 40 µM doses. The assessment was done via the systemic collection of literature from sources such as newspapers, conference papers, journals, theory and dissertation articles, electronic databases, manuals, encyclopedia and annual reviews, as well as e-books and reporting. As a result, the preceding discussion focuses on the key phyto-bioactive compounds and their molecular targets in female fertility. This will aid in the successful and secure application of plant bioactive compounds in the field of female reproductive health.

Forkhead box protein O3a promotes glioma cell resistance to temozolomide by regulating matrix metallopeptidase and β-catenin.

Glioblastoma multiforme (GBM) is the most common type of malignant brain tumor. GBM is currently treated with temozolomide (TMZ), although patients often exhibit resistance to this agent. Although several mechanisms underlying the resistance of GBM to TMZ have been identified, the combination of these mechanisms is not sufficient to fully account for this phenomenon. Our previous study demonstrated that knocking down the Forkhead box protein O3a (FoxO3a) gene, a member of the FoxO subfamily of transcription factors, resulted in glioma cell sensitization to TMZ, accompanied by reduced levels of nuclear β-catenin. The aim of the present study was to specify how FoxO3a and β-catenin are implicated in glioma cell TMZ resistance. Using the U87 and U251 parental cell lines (also designated as sensitive cell lines) and corresponding resistant cell lines (U87-TR and U251-TR, generated by repeated TMZ treatments), coupled with a combined knockdown/overexpression strategy, it was revealed that FoxO3a or β-catenin overexpression in TMZ-treated U87 and U251 cells markedly increased cellular proliferation; co-expression of both FoxO3a and β-catenin resulted in the highest increase. Knockdown of either FoxO3a or β-catenin in U87-TR and U251-TR cells led to a significant decrease in cell viability, which was rescued by the re-expression of FoxO3a in FoxO3a-knockdown cells. Subsequent experiments demonstrated that, in U87-TR and U251-TR cells, FoxO3a knockdown significantly reduced the protein levels of matrix metallopeptidase (MMP)9, while overexpression of FoxO3a in U87 and U251 cells enhanced the nuclear accumulation of β-catenin, concomitantly with an increase in MMP9 levels. Furthermore, MMP9 knockdown markedly reduced the levels of nuclear β-catenin. Collectively, the findings of the present study suggest that FoxO3a may regulate the nuclear accumulation of β-catenin by modulating MMP9 expression, thereby rendering glioblastoma cells resistant to TMZ, and may provide unique molecular insights into the mechanisms underlying the development of TMZ resistance in GBM.

Senescence associated long non-coding RNA 1 regulates cigarette smoke-induced senescence of type II alveolar epithelial cells through sirtuin-1 signaling

ObjectiveThe primary aim of our study was to explore the mechanisms through which long non-coding RNA (lncRNA)-mediated sirtuin-1 (SIRT1) signaling regulates type II alveolar epithelial cell (AECII) senescence induced by a cigarette smoke-media suspension (CSM).MethodsPharmacological SIRT1 activation was induced using SRT2104 and senescence-associated lncRNA 1 (SAL-RNA1) was overexpressed. The expression of SIRT1, FOXO3a, p53, p21, MMP-9, and TIMP-1 in different groups was detected by qRT-PCR and Western blotting; the activity of SA-β gal was detected by staining; the binding of SIRT1 to FOXO3a and p53 gene transcription promoters was detected by Chip.ResultsWe found that CSM increased AECII senescence, while SAL-RNA1 overexpression and SIRT1 activation significantly decreased levels of AECII senescence induced by CSM. Using chromatin immunoprecipitation, we found that SIRT1 bound differentially to transcriptional complexes on the FOXO3a and p53 promoters.ConclusionOur results suggested that lncRNA-SAL1-mediated SIRT1 signaling reduces senescence of AECIIs induced by CSM. These findings suggest a new therapeutic target to limit the irreversible apoptosis of lung epithelial cells in COPD patients.

The correlation of salivary telomere length and single nucleotide polymorphisms of the ADIPOQ, SIRT1 and FOXO3A genes with lifestyle-related diseases in a Japanese population.

It has been reported that genetic factors are associated with risk factors and onset of lifestyle-related diseases, but this finding is still the subject of much debate. The aim of the present study was to investigate the correlation of genetic factors, including salivary telomere length and three single nucleotide polymorphisms (SNPs) that may influence lifestyle-related diseases, with lifestyle-related diseases themselves. In one year at a single facility, relative telomere length and SNPs were determined by using monochrome multiplex quantitative polymerase chain reaction and TaqMan SNP Genotyping Assays, respectively, and were compared with lifestyle-related diseases in 120 Japanese individuals near our university. In men and all participants, age was inversely correlated with relative telomere length with respective p values of 0.049 and 0.034. In men, the frequency of hypertension was significantly higher in the short relative telomere length group than in the long group with unadjusted p value of 0.039, and the difference in the frequency of hypertension between the two groups was of borderline statistical significance after adjustment for age (p = 0.057). Furthermore, in men and all participants, the sum of the number of affected lifestyle-related diseases, including hypertension, was significantly higher in the short relative telomere length group than in the long group, with p values of 0.004 and 0.029, respectively. For ADIPOQ rs1501299, men's ankle brachial index was higher in the T/T genotype than in the G/G and G/T genotypes, with p values of 0.001 and 0.000, respectively. For SIRT1 rs7895833, men's body mass index and waist circumference and all participants' brachial-ankle pulse wave velocity were higher in the A/G genotype than in the G/G genotype, with respective p values of 0.048, 0.032 and 0.035. For FOXO3A rs2802292, women's body temperature and all participants' saturation of peripheral oxygen were lower in the G/T genotype than in the T/T genotype, with respective p values of 0.039 and 0.032. However, relative telomere length was not associated with physiological or anthropometric measurements except for height in men (p = 0.016). ADIPOQ rs1501299 in men, but not the other two SNPs, was significantly associated with the sum of the number of affected lifestyle-related diseases (p = 0.013), by genotype. For each SNPs, there was no significant difference in the frequency of hypertension or relative telomere length by genotype. Relative telomere length and the three types of SNPs determined using saliva have been shown to be differentially associated with onset of and measured risk factors for lifestyle-related diseases consisting mainly of cardiovascular diseases and cancer.

Cyclin G2, a novel target of sulindac to inhibit cell cycle progression in colorectal cancer

Sulindac has shown significant clinical benefit in preventing colorectal cancer progression, but its mechanism of action has not been fully elucidated. We have found that sulindac sulfide (SS) is able to inhibit cell cycle progression in human colorectal cancer cells, particularly through G1 arrest. To understand the underlying mechanisms of sulindac inhibitory activity, we have demonstrated that Cyclin G2 up-regulation upon SS treatment can substantially delay cell cycle progression by enhancing the transcriptional activity of FOXO3a in human colorectal tumor cells. MiR-182, an oncogenic microRNA known to inhibit FOXO3a gene expression, is also involved in the suppressive effect of SS on cell cycle progression. This process begins with the down-regulation of miR-182, followed by the enhancement of FOXO3a transcriptional activity and the up-regulation of Cyclin G2. To further determine the clinical utility of this axis, we analyzed the expression of miR-182/FOXO3a/Cyclin G2 in human colorectal tumor samples. Our results show not only that there are significant differences in miR-182/FOXO3a/Cyclin G2 between tumors and normal tissues, but also that the synergetic effect of miR-182 and FOXO3a is associated with predicting tumor progression. Our study demonstrates a novel mechanistic axis consisting of miR-182/FOXO3a/Cyclin G2 that mediates sulindac inhibition of cell cycle progression.

The effect of tropisetron on oxidative stress, SIRT1, FOXO3a, and claudin-1 in the renal tissue of STZ-induced diabetic rats

Tropisetron is a 5-HT3 receptor antagonist that exerts protective effect against DN. The aim of this study was to investigate the possible molecular mechanisms associated with the renoprotective effects of tropisetron in STZ-induced diabetic rats. Animals were subdivided into 5 equal groups; control, tropisetron, diabetes, tropisetron + diabetes, and glibenclamide + diabetes (n = 7). For induction of type 1 diabetes, a single injection of STZ (55mg/kg, i.p.) was administered to the animals. Diabetic rats were treated with tropisetron (3mg/kg) and glibenclamide (1mg/kg) for 2weeks. According to the conducted analysis, diabetes led to renal dysfunction (reduction in glomerular filtration rate and urine urea and creatinine as well as elevation in plasma urea and creatinine) and abnormalities in antioxidant defense system (reduction in TAC and elevation in MDA), compared with the control group, which was prevented by tropisetron treatment. Reverse transcription-quantitative polymerase chain reaction and western blotting analysis demonstrated that SIRT1 gene expression decreased while FOXO3a and NF-κB gene expression as well as phosphorylated FOXO3a/total FOXO3a protein ratios and claudin-1 protein level increased in the kidney of diabetic rats compared with the control group. Herein, the results of this research showed that tropisetron treatment reversed these changes. Besides, all these changes were comparable with those produced by glibenclamide as a positive control. Hence, tropisetron ameliorated renal damage due to diabetic nephropathy possibly by suppressing oxidative stress and alteration of SIRT1, FOXO3a, and claudin-1 levels.

Abstract 4027: Metformin treatment inhibits uterine leiomyoma and leiomyosarcoma cells growth and migration in vitro

Abstract Leiomyoma (LM) and leiomyosarcoma (LMS) are uterine mesenchymal tumors that present variable clinical and biological behavior. They are associated with infertility, pelvic pain and abnormal uterine bleeding.. LM is a benign tumor commonly affecting women at reproductive age whereas LMS represents 40% of all uterine sarcomas. These tumors do not have specific therapy and their origin is still uncertain. Some researchers believe that degenerated LM may to turn to LMS. FOXO3a, a transcriptional factor, has been associated to regulation of several cellular functions as well as tumors development. Previously, we detected that FOXO3a gene and protein expression present different expression profiles in LM and LMS. In this current study, our aim was to investigate the in vitro effects of metformin and genistein, both inhibitors of AKT-mediated FOXO3a phosphorylation, treatment in LM and LMS cells behavior. LM (THESCs CRL-4003) and LMS (SK-UT-1) cell lines were grown in specific mediums and conditions (ATCC recommendations), and treated with different drugs concentrations (0.1 - 200 uM and 0.1 - 200 nM, for metformin and genistein, respectively) at several time points (0, 24, 48, 72, 96 and 120 h). After treatment, cells were evaluated for gene and protein expression, cell proliferation, migration and apoptosis. Gene expression was assessed by quantitative Real Time PCR and proteins were evaluated by immunocitochemical analysis. Genistein depicted high toxicity for the cells, due to its vehicle (DMSO), in all tested dose and times. Metformin presented higher efficiency in cell proliferation and migration inhibition for both cell lines, as well as in the apoptosis induction. Prominent effects were observed in LMS cells after treatment with 25 uM for 72h. Gene expression analysis showed a slight decrease in FOXO3a expression after treatment with these drugs. Our results suggest that treatment with metformin induces the FOXO3a function restoration in the cells, by inhibition of the AKT/PI3K pathway, and can represent a future alternative target therapy for these neoplasias. Citation Format: Anamaria R. Ricci, Giovana D. Maffazioli, Edmund C. Baracat, Kátia C. Carvalho. Metformin treatment inhibits uterine leiomyoma and leiomyosarcoma cells growth and migration in vitro [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4027.

Regulation of hepatic genes related to lipid metabolism and antioxidant enzymes by sodium butyrate supplementation

BackgroundRapid influx of energy caused by fasting/refeeding repeatedly enhances fatty acid synthesis leading to triacylglycerol accumulation and production of reactive oxygen species (ROS), increasing the risk of non-alcoholic steatohepatitis (NASH). Previous studies have reported that the ingestion of butyrate is effective at preventing hepatic disorders, which are accompanied by fat accumulation and inflammation. The aim of this study is to reveal the mechanism of action of butyrate, and thus we investigated the effects of dietary butyrate on the expressions of antioxidant enzymes in the livers of rats during refeeding following fasting. MethodsThirty-seven male rats were divided into six groups (6–7 animals per group): non-fasting, fasting, refeeding with a high sucrose diet as control for 12 or 24 h, and refeeding with a high sucrose diet containing 5% sodium butyrate (NaB) for 12 or 24 h. All groups except the non-fasting group were fasted for 72 h before refeeding. Statistical analysis was conducted among 4 refeeding groups (refeeding with the control diet for 12 or 24 h, and refeeding with a diet containing NaB for 12 or 24 h). ResultsSupplementation with NaB significantly reduced (p < 0.05) fatty acid synthase (Fas) gene expression and increased the expression of the carnitine palmitoyltransferase 1α (Cpt1a) gene, resulting in reduced triacylglycerol content in the livers of rats refed the NaB diet compared with controls at 24 h after the start of refeeding. The mRNA levels of the genes related to glutathione synthesis were significantly higher (p < 0.05) in the livers of the butyrate group than the control group. In addition, the mRNA level of Foxo3a, a transcription factor that regulates the expressions of antioxidant enzymes, was higher in the butyrate group than controls. The acetylation levels of histone H4 around the Foxo3a gene tended to be increased (p = 0.055) by refeeding with the NaB diet. ConclusionNaB supplementation in the diet for refeeding reduced the rate of lipid synthesis and stimulated fatty acid oxidation in the liver, which inhibited fat accumulation and the risk of NASH. The transcriptional regulation of Foxo3a involves histone acetylation around the gene.

Cardiomyocyte TRPC6 overexpression as one of the myocardial hypertrophy mechanisms in chronic kidney dysfunction

BACKGROUND. Klotho is a transmembrane and circulating protein primarily synthesized by the kidney. Klotho deficiency characterizes chronic kidney disease (CKD), as myocardial hypertrophy (GM). The cardioprotective effect of the Klotho protein is due to the negative regulation of a variety of stress signals, leading to the activation of the hypertrophic intracellular signaling pathway calcineurin (CaN) / NFAT in the myocardium. The effect of Klotho may presumably be mediated by the modulation of Ca2 + channels and / or Foxo factors essential for CaN signaling. THE AIM: to study the activity of CaN/ NFAT signaling pathway in the myocardium and to determine the molecular mechanisms of its regulation in conditions of Klotho level decrease in spontaneous hypertensive rats (SHR) with experimental CKD. MATERIAL AND METHODS. The experimental model of CKD was 3/4 or 5/6 nephrectomy (Nx) in SHR. Sham-operated (SO) SHR, and Wistar Kyoto rats (WKY) were used as controls. In all animals were measured systolic blood pressure, myocardial mass index – MMI, creatinine clearance, cardiomyocyte (CM) diameter, Klotho levels in serum (ELISA) and kidney (IHC), myocardial expression of calcineurin (IHC, PCR), transcription factor NFAT (IHC), TRPC6 (IHC), FOXO3A (PCR) and phosphor-Foxo1/3/4 (IHC). The tissue expressions of calcineurin, TRPC6, and Klotho were calculated as the IHC specific product area to the field of view ratio. NFAT expression was evaluated as the positively stained nuclei to the number of nuclei ratio in the field of view. Measurements were performed in 10 fields of view for each histology slide. RESULTS. The model has corresponded to the initial stages of CKD. The increase in MMI (p = 0.005) and CM diameter (p = 0.002) were observed compared in Nx rats to SO. Renal Klotho expression (p &lt; 0.001), and serum Klotho level (p = 0.019) were lower in the Nx. In multiple linear regression analyzes, the values of MMI and CM thickness were independently associated with the level of renal Klotho protein (β = -0.38 ± 0.16, p = 0.026, β = -0.64 ± 0.14, p &lt;0.001, respectively). Nx and systemic hypertension were accompanied by an increase in the expression of the calcineurin gene (p = 0.005) and cytoplasmic calcineurin in CM (p = 0.004), the number of NFAT-positive nuclei (p = 0.007), and an increase in the expression of the FOXO3A gene (p &lt;0.001) in the absence of accumulation of phosphorylated Foxo1/3/4 in CM cytoplasm. SHR rats were characterized by positive IHC staining for TRPC6 compared to WKY (p = 0.004). The expression of calcineurin and TRPC6 varied co-directionally (r = 0.69, p &lt;0.001), and both of these indicators were associated with the Klotho levels (calcineurin vs Klotho in the kidney, r = -0.73, p &lt;0.001; TRPC6 vs Klotho in serum, r = -0.43, p = 0.025). CONCLUSION. The development of Klotho deficiency on early-stage CKD is associated with the expression of transient Ca2+ channels TRPC6 and activation of the calcineurin / NFAT hypertrophic signaling pathway in cardiomyocytes.

Modulation of redox and insulin signaling underlie the anti-hyperglycemic and antioxidant effects of diphenyl diselenide in zebrafish

The organic selenium compound diphenyl diselenide (DD) has been recognized as an antioxidant and neuroprotective agent, exerting an anti-hyperglycemic effect in experimental models of diabetes. However, the precise mechanisms involved in the protection are unclear. Using the zebrafish (Danio rerio) as a model organism, here we investigated biomarkers underlying the protective effects of DD against hyperglycemia, targeting in a transcriptional approach the redox and insulin-signaling pathway. Fish were fed on a diet containing DD (3 mg/kg) for 74 days. In the last 14 days, they were exposed to a 111 mM glucose solution to induce a hyperglycemic state. DD reduced blood glucose levels as well as normalized the brain mRNA transcription of four insulin receptors-coding genes (Insra1, Insra2, Insrb1, Insrb2), which were down-regulated by glucose. DD alone caused an up-regulation of relative mRNA transcription in both Insra receptors and glucose transporter 3 genes. DD counteracted hyperglycemia-induced lipid peroxidation, protein and thiol depletion. Along with the decreased activity of antioxidant enzymes SOD and GPx, the brain of hyperglycemic fish presented a reduction in mRNA transcription of FoxO3A, FoxO3B, Nrf2, GPx3A, SOD1, and SOD2 genes. Besides normalizing the transcriptional levels, DD caused an up-regulation of relative mRNAs that encode Nrf2, FoxO1A, FOXO3A, GPx4A, PTP1B, AKT and SelP. Collectively, our findings suggest that the antioxidant and anti-hyperglycemic actions of DD in a zebrafish diabetes model are likely associated with the regulation of the oxidative stress resistance and the insulin-signaling pathway and that could be related to the modulation at mRNA level of two important transcription factors, Nrf2 and FoxO.

EGFR Inhibitor Gefitinib Induces Cardiotoxicity through the Modulation of Cardiac PTEN/Akt/FoxO3a Pathway and Reactive Metabolites Formation: In Vivo and in Vitro Rat Studies.

Gefitinib (GEF) is a selective inhibitor of the epidermal growth factor receptor (EGFR) used to treat non-small cell lung cancer. Yet, few cases of cardiotoxicity have been reported. However, the role of the PTEN/Akt/FoxO3a pathway, which mediates GEF anticancer activity, in GEF cardiotoxicity remains unclear. For this purpose, in vitro H9c2 cells and in vivo rat cardiomyocytes were utilized as study models. Treatment of H9c2 cells and Sprague-Dawley rats with GEF significantly induced the expression of hypertrophic and apoptotic markers at mRNA and protein levels with an increased plasma level of troponin. This was accompanied by induction of autophagy and mitochondrial dysfunction in H9c2 cells. Inhibition of cardiac EGFR activity and Akt cellular content of in vitro and in vivo rat cardiomyocytes by GEF increased PTEN and FoxO3a gene expression and cellular content. Importantly, treatment of H9c2 cells with PI3K/Akt inhibitor increased PTEN and FoxO3a mRNA expression associated with potentiation of GEF cardiotoxicity. In addition, by using LC-MS/MS, we showed that GEF is metabolized in the rat heart microsomes into one cyanide- and two methoxylamine-adduct reactive metabolites, where their formation was entirely blocked by CYP1A1 inhibitor, α-naphthoflavone. The current study concludes that GEF induces cardiotoxicity through modulating the expression and function of the cardiac PTEN/AKT/FoxO3a pathway and the formation of CYP1A1-mediated reactive metabolites.

Targeted Regulation of FoxO3a by miR-372 to Mediate Gastric Carcinoma Cell Apoptosis and DDP Drug Resistance.

Background: FoxO3a is a well-studied tumor suppressor gene in the forkhead transcriptional factor O (FoxO) subfamily and its downregulation is correlated with the occurrence of gastric cancer (GC). GC tissues had microRNA (miR)-372 upregulation, which has targeted relationship with 3'-untranslated region (3'-UTR) of FoxO3a gene. This study investigated if miR-372 plays a role in modulating FoxO3a expression, and affecting GC cell proliferation, apoptosis, and cisplatin (DDP) resistance. Materials and Methods: Dual luciferase reporter gene assay assessed the targeted regulation between miR-372 and FoxO3a. DDP-resistant cell lines MGC803/DDP and MKN28/DDP were compared for gene expression against parental cells. Cell proliferation was measured by Cell Counting Kit-8 (CCK-8) assay. Cultured cells were transfected with miR-372 mimic or miR-negative control (NC) to measure FoxO3a mRNA and protein expression. Cell apoptosis and proliferation were tested by flow cytometry and 5-Ethynyl-2'-deoxyuridine (EdU) staining, respectively. Results: miR-372 had a targeted relationship with FoxO3a mRNA. MGC803/DDP and MKN28/DDP cells had significantly elevated miR-372 level than parental cells, while Foxo3a mRNA or protein levels were significantly decreased. CCK-8 assay revealed significantly lower inhibitory activity on cell proliferation in drug-resistant cells. Compared with miR-NC group, miR-273 inhibitor transfected DDP-resistant cells had significantly increased Foxo3a expression, enhanced cell apoptosis, reduced proliferation, and drug resistance. Conclusions: miR-372 upregulation is associated with DPP resistance of GC cells. Downregulation of miR-372 can inhibit proliferation, facilitate apoptosis, and suppress DDP resistance of drug-resistant GC cells.

CircMRPS35 suppresses gastric cancer progression via recruiting KAT7 to govern histone modification

BackgroundAberrant expression of circular RNAs contributes to the initiation and progression of cancers, but the underlying mechanism remains elusive.MethodsRNA-seq and qRT-PCR were performed to screen differential expressed circRNAs between gastric cancer tissues and adjacent normal tissues. Candidate circRNA (circMRPS35) was screened out and validated by qRT-PCR. Cell proliferation and invasion ability were determined by CCK-8 and cell invasion assays. RNA-seq, GO-pathway, RNA pull-down and ChIRP were further applied to search for detailed mechanism.ResultsHere, a novel circRNA named circMRPS35, was screened out by RNA-seq in gastric cancer tissues, whose expression is related to clinicopathological characteristics and prognosis in gastric cancer patients. Biologically, circMRPS35 suppresses the proliferation and invasion of gastric cancer cells in vitro and in vivo. Mechanistically, circMRPS35 acts as a modular scaffold to recruit histone acetyltransferase KAT7 to the promoters of FOXO1 and FOXO3a genes, which elicits acetylation of H4K5 in their promoters. Particularly, circMRPS35 specifically binds to FOXO1/3a promoter regions directly. Thus, it dramatically activates the transcription of FOXO1/3a and triggers subsequent response of their downstream target genes expression, including p21, p27, Twist1 and E-cadherin, resulting in the inhibition of cell proliferation and invasion. Moreover, circMRPS35 expression positively correlates with that of FOXO1/3a in gastric cancer tissues.ConclusionsOur findings not only reveal the pivotal roles of circMRPS35 in governing histone modification in anticancer treatment, but also advocate for triggering circMRPS35/KAT7/FOXO1/3a pathway to combat gastric cancer.

Genetic Regulation of Redox Balance in β-Thalassemia Trait

β-Thalassemia (β-thal) trait is a heterogeneous group of genetic defects leading to decreased β-globin production, ineffective erythropoiesis, and oxidative stress. The aim is to evaluate the cytoprotective response, at transcriptional and systemic levels, of the variations of global redox balance in β-thal trait patients. Sixty-six subjects (40 healthy and 26 with β-thal trait) were analyzed at the Universidad Nacional de Tucumán, Tucumán, Argentina, between 2016 and 2017. The following parameters were evaluated: complete blood count, iron status, hemoglobin (Hb) electrophoresis, Hb A2, thiobarbituric acid reactive species (TBARS), serum catalase (CAT), and superoxide dismutase (SOD) activity, FOXO3a, NRF2, SOD, PRDX2, CAT, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) gene expression. The β-thal trait group showed a decrease in Hb levels, MCV, and MCH with higher TBARS levels. The SOD activity was significantly increased by 32.0% in β-thal trait patients respect to the control group. Relative expression of NRF2 was 4.7-fold higher in β-thal trait than in the control group, while FOXO3a expression was similar in both groups. The SOD, PRDX2, and proinflammatory cytokines transcriptional expression was significantly upregulated in β-thal trait patients. This is the first study on the genetic regulation of redox balance in β-thal trait patients in which interesting modifications were observed in the transcript levels of some redox regulators that could be associated with changes in the erythrocyte proteome in this disorder. A better understanding of the pathophysiological mechanisms present in these heterozygous patients would allow adequate therapy in situations such as growth, pregnancy, or high performance sports, favoring a personalized treatment.