Folic Acid and Folinic Acid Protect Hearts of Aging Triple-transgenic Alzheimer's Disease mice via IGF1R/PI3K/AKT and SIRT1/AMPK Pathways.

Patients with Alzheimer's disease have increased risk of developing heart disease, which therefore highlights the need for strategies aiming at reducing Alzheimer's disease-related cardiovascular disease. Folic acid and folinic acid are beneficial to the heart. We aimed to investigate the benefits of folic acid and folinic acid in heart of patients with late-stage Alzheimer's disease. Twelve 16-month-old mice of triple-transgenic late-stage Alzheimer's disease were divided into three groups: Alzheimer's disease group, Alzheimer's disease + folic acid group, and Alzheimer's disease + folinic acid group. The mice were administered 12 mg/kg folic acid or folinic acid once daily via oral gavage for 3 months. In the folic acid and folinic acid treatment groups, the intercellular space was reduced, compared with the Alzheimer's disease group. TUNEL assay and western blot images showed that the number of apoptotic cells and the apoptosis-related protein expression were higher in the Alzheimer's disease group than in other two treated groups. Folic acid and folinic acid induced the IGF1R/PI3K/AKT and SIRT1/ AMPK pathways in the hearts of mice with Alzheimer's disease. Our results showed that folic acid and folinic acid treatment increased survival and SIRT1 expression to reduce apoptotic proteins in the heart. The aging mice treated with folinic acid had more IGF1R and SIRT1/AMPK axes to limit myocardial cell apoptosis. In conclusion, folic acid and folinic acid promote cardiac cell survival and prevent apoptosis to inhibit heart damage in aging mice with triple-transgenic late-stage Alzheimer's disease. In particular, folinic acid provides a better curative effect than folic acid.

PEMT rs7946 Polymorphism and Sex Modify the Effect of Adequate Dietary Choline Intake on the Risk of Hepatic Steatosis in Older Patients with Metabolic Disorders.

In humans, PEMT rs7946 polymorphism exerts sex-specific effects on choline requirement and hepatic steatosis (HS) risk. Few studies have explored the interaction effect of the PEMT rs7946 polymorphism and sex on the effect of adequate choline intake on HS risk. In this cross-sectional study, we investigated the association between PEMT polymorphism and adequate choline intake on HS risk. We enrolled 250 older patients with metabolic disorders with (n = 152) or without (n = 98; control) ultrasonically diagnosed HS. An elevated PEMT rs7946 A allele level was associated with a lower HS risk and body mass index in both men and women. Dietary choline intake-assessed using a semiquantitative food frequency questionnaire-was associated with reduced obesity in men only (p for trend < 0.05). ROC curve analysis revealed that the cutoff value of energy-adjusted choline intake for HS diagnosis was 448 mg/day in women (AUC: 0.62; 95% CI: 0.57-0.77) and 424 mg/day in men (AUC: 0.63, 95% CI: 0.57-0.76). In women, GG genotype and high choline intake (>448 mg/day) were associated with a 79% reduction in HS risk (adjusted OR: 0.21; 95% CI: 0.05-0.82); notably, GA or AA genotype was associated with a reduced HS risk regardless of choline intake (p < 0.05). In men, GG genotype and high choline intake (>424 mg/day) were associated with a 3.7-fold increase in HS risk (OR: 3.7; 95% CI: 1.19-11.9). Further adjustments for a high-density lipoprotein level and body mass index mitigated the effect of choline intake on HS risk. Current dietary choline intake may be inadequate for minimizing HS risk in postmenopausal Taiwanese women carrying the PEMT rs7946 GG genotype. Older men consuming more than the recommended amount of choline may have an increased risk of nonalcoholic fatty liver disease; this risk is mediated by a high-density lipoprotein level and obesity.

Dietary Folate Deficiency Promotes Lactate Metabolic Disorders to Sensitize Lung Cancer Metastasis through MTOR-Signaling-Mediated Druggable Oncotargets.

Lactate metabolism plays a pivotal role in cancers but is often overlooked in lung cancer (LC). Folate deficiency has been linked to lung cancer development, but its impact on lactate metabolism and cancer malignancy is unclear. To investigate this, mice were fed either a folate-deficient (FD) or control diet and intrapleurally implanted with lung cancer cells pre-exposed to FD growth medium. Results showed that FD promoted lactate over-production and the formation of tumor oncospheroids (LCSs) with increased metastatic, migration, and invasion potential. Mice implanted with these cells and fed an FD diet developed hyperlactatemia in blood and lungs. This coincided with increased expression of hexokinase 2 (HK2), lactate dehydrogenase (LDH), and decreased expression of pyruvate dehydrogenase (PDH). Pre-treatment of the FD-LCS-implanted mice with the mTORC1 inhibitor, rapamycin, and the anti-metabolic drug metformin abolished FD/LCS-activated mTORC1 and its targets including HIF1α, HK2, LDH, and monocarboxylate transporters (MCT1 and MCT4), which coincided with the reduction in lactate disorders and prevention of LC metastasis. The findings suggest that dietary FD promotes lactate metabolic disorders that sensitize lung cancer metastasis through mTOR-signaling-mediated targets.

Suboptimal folic acid exposure rewires oncogenic metabolism and proteomics signatures to mediate human breast cancer malignancy.

Whether treatment with folic acid (FA) affects human breast cancer positively or negatively remains unclear. We subjected human Michigan Cancer Foundation-7 cells, a human breast cancer cell line, to suboptimal FA at low levels (10 nM; LF) and high levels (50 µM; HF) and investigated the molecular mechanisms underlying their effects through metabolic flux and systematic proteomics analyses. The data indicated that LF induced and HF aggravated 2-fold higher mitochondrial toxicity in terms of suppressed oxidative respiration, increased fermented glycolysis, and enhanced anchorage-independent oncospheroid formation. Quantitative proteomics and Gene Ontology enrichment analysis were used to profile LF- and HF-altered proteins involved in metabolism, apoptosis, and malignancy pathways. Through STRING analysis, we identified a connection network between LF- and HF-altered proteins with mammalian target of rapamycin (mTOR). Rapamycin-induced blockage of mTOR complex 1 (mTORC1) signaling, which regulates metabolism, differentially inhibited LF- and HF-modulated protein signatures of mitochondrial NADH dehydrogenase ubiquinone flavoprotein 2, mitochondrial glutathione peroxidase 4, kynureninase, and alpha-crystallin B chain as well as programmed cell death 5 in transcript levels; it subsequently diminished apoptosis and oncospheroid formation in LF/HF-exposed cells. Taken together, our data indicate that suboptimal FA treatment rewired oncogenic metabolism and mTORC1-mediated proteomics signatures to promote breast cancer development.

Optimal Dietary Intake Composition of Choline and Betaine Is Associated with Minimized Visceral Obesity-Related Hepatic Steatosis in a Case-Control Study.

Few studies on humans have comprehensively evaluated the intake composition of methyl-donor nutrients (MDNs: choline, betaine, and folate) in relation to visceral obesity (VOB)-related hepatic steatosis (HS), the hallmark of non-alcoholic fatty liver diseases. In this case-control study, we recruited 105 patients with HS and 104 without HS (controls). HS was diagnosed through ultrasound examination. VOB was measured using a whole-body analyzer. MDN intake was assessed using a validated quantitative food frequency questionnaire. After adjustment for multiple HS risk factors, total choline intake was the most significant dietary determinant of HS in patients with VOB (Beta: -0.41, p = 0.01). Low intake of choline (<6.9 mg/kg body weight), betaine (<3.1 mg/kg body weight), and folate (<8.8 µg/kg body weight) predicted increased odds ratios (ORs) of VOB-related HS (choline: OR: 22, 95% confidence interval [CI]: 6.5-80; betaine: OR: 14, 95% CI: 4.4-50; and folate: OR: 19, 95% CI: 5.2-74). Combined high intake of choline and betaine, but not folate, was associated with an 81% reduction in VOB-related HS (OR: 0.19, 95% CI: 0.05-0.69). Our data suggest that the optimal intake of choline and betaine can minimize the risk of VOB-related HS in a threshold-dependent manner.

Probing Folate-Responsive and Stage-Sensitive Metabolomics and Transcriptional Co-Expression Network Markers to Predict Prognosis of Non-Small Cell Lung Cancer Patients.

Tumour metabolomics and transcriptomics co-expression network as related to biological folate alteration and cancer malignancy remains unexplored in human non-small cell lung cancers (NSCLC). To probe the diagnostic biomarkers, tumour and pair lung tissue samples (n = 56) from 97 NSCLC patients were profiled for ultra-performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS)-analysed metabolomics, targeted transcriptionomics, and clinical folate traits. Weighted Gene Co-expression Network Analysis (WGCNA) was performed. Tumour lactate was identified as the top VIP marker to predict advance NSCLC (AUC = 0.765, Sig = 0.017, CI 0.58-0.95). Low folate (LF)-tumours vs. adjacent lungs displayed higher glycolytic index of lactate and glutamine-associated amino acids in enriched biological pathways of amino sugar and glutathione metabolism specific to advance NSCLCs. WGCNA classified the green module for hub serine-navigated glutamine metabolites inversely associated with tumour and RBC folate, which module metabolites co-expressed with a predominant up-regulation of LF-responsive metabolic genes in glucose transport (GLUT1), de no serine synthesis (PHGDH, PSPH, and PSAT1), folate cycle (SHMT1/2 and PCFR), and down-regulation in glutaminolysis (SLC1A5, SLC7A5, GLS, and GLUD1). The LF-responsive WGCNA markers predicted poor survival rates in lung cancer patients, which could aid in optimizing folate intervention for better prognosis of NSCLCs susceptible to folate malnutrition.

Using Differential Threshold Effects of Individual and Combined Periconceptional Methyl Donor Status on Maternal Genomic LINE-1 and Imprinted H19 DNA Methylation to Predict Birth Weight Variance in the Taiwan Pregnancy-Newborn Epigenetics (TPNE) Cohort Study.

BACKGROUND: Few studies have comprehensively examined the effect of methyl donor status on maternal DNA methylation and birth outcomes. OBJECTIVES: This study examined associations between periconceptional methyl donor status and genome-wide and specific imprinted gene methylation and fetal growth indices in the Taiwan Pregnancy-Newborn Epigenetics cohort. METHODS: Plasma folate, choline (free form), and betaine concentrations of the participants enrolled at 7-10 weeks of gestation were analyzed. DNA methylation at regulatory sequences of the imprinted H19 gene and genomic long interspersed nuclear element 1 (LINE-1) were measured in maternal lymphocytes using bisulfite/high-resolution melt polymerase chain reaction. Associations with birth weight (BW) were estimated through multiple regressions from 112 mother-newborn pairs. RESULTS: A nonlinear "L-shaped" relation and an inverse association between maternal plasma folate in T1 (mean ± SE: 17.6 ± 5.1 nmol/L) and lymphocytic LINE-1 methylation (ß: -0.49, P = 0.027) were characterized. After adjusting for LINE-1 methylation, individual maternal folate concentrations were positively associated with BW variance (ß = 0.24, P = 0.035), and the association was more pronounced in mothers with choline in T1 (mean ± SE: 5.4 ± 0.6 µmol/L; ß: 0.40, P = 0.039). Choline status of the mothers in T2 (mean ± SE: 7.2 ± 0.6 µmol/L) was inversely associated with LINE-1 methylation (ß: -0.43, P = 0.035), and a positive association was evident between T1 choline and H19 methylation (ß: 0.48, P = 0.011). After adjusting for epigenetic modification, maternal choline status predicted a positive association with BW (ß: 0.56, P = 0.005), but the effect was limited to mothers with high betaine concentrations in T3 (mean ± SE: 36.4 ± 8.8 µmol/L), depending on folate status. CONCLUSIONS: Our data highlight the differential threshold effects of periconceptional folate, choline, and betaine status on genomic LINE-1 and H19 DNA methylation and how their interplay has a long-term effect on BW variance.

Low-folate stress reprograms cancer stem cell-like potentials and bioenergetics metabolism through activation of mTOR signaling pathway to promote in vitro invasion and in vivo tumorigenicity of lung cancers.

Low-folate (LF) nutritional status is associated with increased risks of lung cancer. It has unexplored effects on lung cancer malignancy, a cancer stem cell (CSC) disease. We hypothesized that LF may reprogram CSC-like potential and bioenergetics metabolism to increase metastasis potential of lung cancers. Cultivation of human non-small-cell lung cancer cells (H23) in an LF medium enhanced CSC-like properties signified by increased expressions of the CSC surface marker CD44 and pluripotency markers Sox2, Oct4 and ALDH1A1, and promoted self-renewal ability of anchorage-independent oncospheroid formation. The CSC-like phenotype of LF-treated H23 cells coupled with the metabolic reprogramming to aerobic glycolysis evident by elevated lactate release and medium acidification suppressed expressions of pyruvate dehydrogenase E1-α, and elevated redox status of the NADH/NAD+ and NADPH/NADP+ ratios. The LF-induced metabostemness phenotype of H23 cells was modified by DNA methylation inhibitor 5-AdC and histone acetylation inhibitor EX. Treatment of H23 cells with mTOR siRNA or the mTOR inhibitor rapamycin abrogated LF-activated Akt-mTOR-Hif1-Foxo signaling and stemness-associated sonic hedgehog pathway, reversed Warburg metabolic switch and diminished invasion of H23 cells. Intrapleural injection of LF-induced lung oncospheres into the LF recipient mice, but not the control recipient mice, caused metastasis xenograft lung tumors. The in vitro and in vivo data corroboratively demonstrate that LF stress reprograms metabostemness signatures through activated mTOR signaling pathway to promote metastasis tumorigenicity of lung cancers.

Inhibition of colon cancer cell growth by nanoemulsion carrying gold nanoparticles and lycopene.

Lycopene (LP), an important functional compound in tomatoes, and gold nanoparticles (AN), have received considerable attention as potential candidates for cancer therapy. However, the extreme instability and poor bioavailability of LP limits its in vivo application. This study intends to develop a nanoemulsion system incorporating both LP and AN, and to study the possible synergistic effects on the inhibition of the HT-29 colon cancer cell line. LP-nanogold nanoemulsion containing Tween 80 as an emulsifier was prepared, followed by characterization using transmission electron microscopy (TEM), dynamic light scattering (DLS) analysis, ultraviolet spectroscopy, and zeta potential analysis. The particle size as determined by TEM and DLS was 21.3±3.7 nm and 25.0±4.2 nm for nanoemulsion and 4.7±1.1 nm and 3.3±0.6 nm for AN, while the zeta potential of nanoemulsion and AN was -32.2±1.8 mV and -48.5±2.7 mV, respectively. Compared with the control treatment, both the combo (AN 10 ppm plus LP 12 µM) and nanoemulsion (AN 0.16 ppm plus LP 0.4 µM) treatments resulted in a five- and 15-fold rise in early apoptotic cells of HT-29, respectively. Also, the nanoemulsion significantly reduced the expressions of procaspases 8, 3, and 9, as well as PARP-1 and Bcl-2, while Bax expression was enhanced. A fivefold decline in the migration capability of HT-29 cells was observed for this nanoemulsion when compared to control, with the invasion-associated markers being significantly reversed through the upregulation of the epithelial marker E-cadherin and downregulation of Akt, nuclear factor kappa B, pro-matrix metalloproteinase (MMP)-2, and active MMP-9 expressions. The TEM images revealed that numerous nanoemulsion-filled vacuoles invaded cytosol and converged into the mitochondria, resulting in an abnormally elongated morphology with reduced cristae and matrix contents, demonstrating a possible passive targeting effect. The nanoemulsion containing vacuoles were engulfed and internalized by the nuclear membrane envelop for subsequent invasion into the nucleoli. Taken together, LP-nanogold nanoemulsion could provide synergistic effects at AN and LP doses 250 and 120 times lower than that in the combo treatment, respectively, demonstrating the potential of nanoemulsion developed in this study for a possible application in colon cancer therapy.

Folate deprivation enhances invasiveness of human colon cancer cells mediated by activation of sonic hedgehog signaling through promoter hypomethylation and cross action with transcription nuclear factor-kappa B pathway.

Low folate status is well recognized as one of the metabolic stressors for colorectal cancer carcinogenesis, but its role in colon cancer invasion remains unknown. Activation of the Sonic hedgehog (Shh) signal in interaction with the transcription nuclear factor-kappa B (NF-κB) pathway is crucial for cancer aggressiveness. The aims of this study were to investigate whether and how folate deprivation promotes invasion by colon cancer cells in relation to Shh signaling and NF-κB pathway activation. Cultivation of epithelial colon carcinoma-derived cells (HCT116) in folate-deficient (FD) medium enhanced cellular migration and invasion, in correlation with epithelial-mesenchymal transition (EMT) associated with Snail expression and E-cadherin suppression, increased production of ß1 integrin and increased proteolysis by matrix metalloproteinase 2. Blockade of Shh signaling by cyclopamine (CYC) or of NF-κB activation by BAY abolished FD-enhanced EMT and invasion by HCT116 cells. FD cells had 50-80% less intracellular folate, associated with aberrant hypomethylation of the Shh promoter, than control cells, and increased binding of nuclear NF-κB subunit p65 to the Shh promoter region, which coincided with increased Shh expression and protein production of Shh ligand; in addition, the FD-induced Shh signaling targeted Gli1 transcription activator as well as Ptch receptor. The FD-induced Shh induction and activated signaling were blocked by NF-κB inhibitor BAY. Blockade of Shh signaling abrogated FD-promoted NF-κB activation measured by IκBα degradation and by target gene TNFα expression. Taken together, these findings demonstrate that folate deprivation enhanced invasiveness of colon cancer cells mediated by activation of Shh signaling through promoter hypomethylation and cross actions with the NF-κB pathway.

Mitochondrial DNA deletions of blood lymphocytes as genetic markers of low folate-related mitochondrial genotoxicity in peripheral tissues.

BACKGROUND: A low folate status and mitochondrial DNA (mtDNA) mutations are risk factors for various cancers and degenerative diseases. It is not known if lymphocytic mtDNA deletions can be used as genetic "markers" to reflect global mtDNA damage during folate deficiency. AIM OF THE STUDY: The aim of this study was to characterize folate-related mtDNA deletions in lymphocytes and their associations with mt genotoxicity in peripheral tissues. METHODS: Weaning Wistar rats were fed folate-deficient and folate-replete (control) diets for 2 and 4 weeks. Folate levels of blood lymphocytes and various tissues were assayed by the Lactobacillus casei method. mtDNA deletions were measured by a real-time polymerase chain reaction analysis of whole DNA extracts. RESULTS: Compared to the control counterparts, mtDNA deletions of lymphocytes increased by 3.5-fold (P < 0.05) after 4 weeks of folate deficiency. Lymphocytic mtDNA deletions were inversely associated with plasma (r = -0.619, P = 0.018), red blood cell (r = -0.668, P = 0.009), and lymphocytic folate levels (r = -0.536, P = 0.048). Frequencies of lymphatic mtDNA deletions were positively correlated with mtDNA deletions in tissues including the lungs (r = 0.803, P = 0.0005), muscles (r = 0.755, P = 0.001), heart (r = 0.633, P = 0.015), liver (r = 0.722, P = 0.003), kidneys (r = 0.737, P = 0.006), pancreas (r = 0.666, P = 0.009), and brain (r = 0.917, P < 0.0001). CONCLUSIONS: Our data demonstrate that accumulated mtDNA deletions of lymphocytes depended upon dietary folate deprivation. The accumulated mt deletions in lymphocytes closely reflected the mt genotoxicity in the peripheral tissues during folate deficiency.

N-Acetylcysteine, vitamin C and vitamin E diminish homocysteine thiolactone-induced apoptosis in human promyeloid HL-60 cells.

We showed previously that homocysteine thiolactone (HcyT) is a potent inducer of apoptosis in HL-60 cells. In the present study, the role of some radical scavengers (N-acetylcysteine, vitamin C, vitamin E and folate) on the reduction of HcyT-induced apoptosis was investigated. Preincubation of HcyT-treated HL-60 cells with vitamin C (Vit C; 100 micro mol/L) or vitamin E (Vit E; 100 micro mol/L) for 2 h significantly reduced the proportion of apoptotic cells with hypodiploid DNA contents or with membrane phosphatidylserine exposure, and attenuated the apoptotic DNA fragmentation. Preincubation of cells with N-acetylcysteine (NAC; 5 mmol/L) for 2 h significantly reduced HcyT-promoted apoptosis measured by membrane phosphatidylserine exposure only. The reduction of HcyT-induced apoptosis by NAC, Vit C or Vit E occurred simultaneously with a significant decrease in intracellular H(2)O(2) levels and reduced caspase-3 enzymatic activity. In contrast, folate had no H(2)O(2) scavenging capacity and did not suppress caspase-3 activity 6 h after HcyT treatment, although folate exhibited antioxidant behavior toward superoxide anions, hydroxyl radicals and peroxynitrite. Preincubation of cells with folate (10 micro mol/L) for 3 d did not affect the extent of HcyT-promoted apoptotic damage. Taken together, our findings suggest that antioxidant pretreatment with NAC, Vit C or Vit E exerts more beneficial effects than folate on reducing apoptotic cell damage induced by homocysteine thiolactone.