Ketogenic Diet Consumption Inhibited Mitochondrial One-Carbon Metabolism.

Given the popularity of ketogenic diets, their potential long-term consequences deserve to be more carefully monitored. Mitochondrially derived formate has a critical role in mammalian one-carbon (1C) metabolism and development. The glycine cleavage system (GCS) accounts for another substantial source for mitochondrially derived 1C units. OBJECTIVE: We investigated how the ketogenic state modulates mitochondrial formate generation and partitioning of 1C metabolic fluxes. DESIGN: HepG2 cells treated with physiological doses (1 mM and 10 mM) of ß-hydroxybutyrate (ßHB) were utilized as the in vitro ketogenic model. Eight-week male C57BL/6JNarl mice received either a medium-chain fatty-acid-enriched ketogenic diet (MCT-KD) or a control diet AIN 93M for 8 weeks. Stable isotopic labeling experiments were conducted. RESULTS AND CONCLUSIONS: MCT-KD is effective in weight and fat loss. Deoxythymidine (dTMP) synthesis from the mitochondrial GCS-derived formate was significantly suppressed by ßHB and consumption of MCT-KD. Consistently, plasma formate concentrations, as well as the metabolic fluxes from serine and glycine, were suppressed by MCT-KD. MCT-KD also decreased the fractional contribution of mitochondrially derived formate in methionine synthesis from serine. With the worldwide application, people and medical professionals should be more aware of the potential metabolic perturbations when practicing a long-term ketogenic diet.

MAT2A Localization and Its Independently Prognostic Relevance in Breast Cancer Patients.

(1) Background: methionine cycle is not only essential for cancer cell proliferation but is also critical for metabolic reprogramming, a cancer hallmark. Hepatic and extrahepatic tissues methionine adenosyltransferases (MATs) are products of two genes, MAT1A and MAT2A that catalyze the formation of S-adenosylmethionine (SAM), the principal biological methyl donor. Glycine N-methyltransferase (GNMT) further utilizes SAM for sarcosine formation, thus it regulates the ratio of SAM:S-adenosylhomocysteine (SAH). (2) Methods: by analyzing the TCGA/GTEx datasets available within GEPIA2, we discovered that breast cancer patients with higher MAT2A had worse survival rate (p = 0.0057). Protein expression pattern of MAT1AA, MAT2A and GNMT were investigated in the tissue microarray in our own cohort (n = 252) by immunohistochemistry. MAT2A C/N expression ratio and cell invasion activity were further investigated in a panel of breast cancer cell lines. (3) Results: GNMT and MAT1A were detected in the cytoplasm, whereas MAT2A showed both cytoplasmic and nuclear immunoreactivity. Neither GNMT nor MAT1A protein expression was associated with patient survival rate in our cohort. Kaplan-Meier survival curves showed that a higher cytoplasmic/nuclear (C/N) MAT2A protein expression ratio correlated with poor overall survival (5 year survival rate: 93.7% vs. 83.3%, C/N ratio ≥ 1.0 vs. C/N ratio < 1.0, log-rank p = 0.004). Accordingly, a MAT2A C/N expression ratio ≥ 1.0 was determined as an independent risk factor by Cox regression analysis (hazard ratio = 2.771, p = 0.018, n = 252). In vitro studies found that breast cancer cell lines with a higher MAT2A C/N ratio were more invasive. (4) Conclusions: the subcellular localization of MAT2A may affect its functions, and elevated MAT2A C/N ratio in breast cancer cells is associated with increased invasiveness. MAT2A C/N expression ratio determined by IHC staining could serve as a novel independent prognostic marker for breast cancer.

MTHFR Knockdown Assists Cell Defense against Folate Depletion Induced Chromosome Segregation and Uracil Misincorporation in DNA.

Folate depletion causes chromosomal instability by increasing DNA strand breakage, uracil misincorporation, and defective repair. Folate mediated one-carbon metabolism has been suggested to play a key role in the carcinogenesis and progression of hepatocellular carcinoma (HCC) through influencing DNA integrity. Methylenetetrahydrofolate reductase (MTHFR) is the enzyme catalyzing the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate that can control folate cofactor distributions and modulate the partitioning of intracellular one-carbon moieties. The association between MTHFR polymorphisms and HCC risk is inconsistent and remains controversial in populational studies. We aimed to establish an in vitro cell model of liver origin to elucidate the interactions between MTHFR function, folate status, and chromosome stability. In the present study, we (1) examined MTHFR expression in HCC patients; (2) established cell models of liver origin with stabilized inhibition of MTHFR using small hairpin RNA delivered by a lentiviral vector, and (3) investigated the impacts of reduced MTHFR and folate status on cell cycle, methyl group homeostasis, nucleotide biosynthesis, and DNA stability, all of which are pathways involved in DNA integrity and repair and are critical in human tumorigenesis. By analyzing the TCGA/GTEx datasets available within GEPIA2, we discovered that HCC cancer patients with higher MTHFR had a worse survival rate. The shRNA of MTHFR (shMTHFR) resulted in decreased MTHFR gene expression, MTHFR protein, and enzymatic activity in human hepatoma cell HepG2. shMTHFR tended to decrease intracellular S-adenosylmethionine (SAM) contents but folate depletion similarly decreased SAM in wildtype (WT), negative control (Neg), and shMTHFR cells, indicating that in cells of liver origin, shMTHFR does not exacerbate the methyl group supply in folate depletion. shMTHFR caused cell accumulations in the G2/M, and cell population in the G2/M was inversely correlated with MTHFR gene level (r = -0.81, p < 0.0001), MTHFR protein expression (r = -0.8; p = 0.01), and MTHFR enzyme activity (r = -0.842; p = 0.005). Folate depletion resulted in G2/M cell cycle arrest in WT and Neg but not in shMTHFR cells, indicating that shMTHFR does not exacerbate folate depletion-induced G2/M cell cycle arrest. In addition, shMTHFR promoted the expression and translocation of nuclei thymidine synthetic enzyme complex SHMT1/DHFR/TYMS and assisted folate-dependent de novo nucleotide biosynthesis under folate restriction. Finally, shMTHFR promoted nuclear MLH1/p53 expression under folate deficiency and further reduced micronuclei formation and DNA uracil misincorporation under folate deficiency. In conclusion, shMTHFR in HepG2 induces cell cycle arrest in G2/M that may promote nucleotide supply and assist cell defense against folate depletion-induced chromosome segregation and uracil misincorporation in the DNA. This study provided insight into the significant impact of MTHFR function on chromosome stability of hepatic tissues. Data from the present study may shed light on the potential regulatory mechanism by which MTHFR modulates the risk for hepatic malignancies.

Folinate Supplementation Ameliorates Methotrexate Induced Mitochondrial Formate Depletion In Vitro and In Vivo.

(1) Background: Antifolate methotrexate (MTX) is the most common disease-modifying antirheumatic drug (DMARD) for treating human rheumatoid arthritis (RA). The mitochondrial-produced formate is essential for folate-mediated one carbon (1C) metabolism. The impacts of MTX on formate homeostasis in unknown, and rigorously controlled kinetic studies can greatly help in this regard. (2) Methods: Combining animal model (8-week old female C57BL/6JNarl mice, n = 18), cell models, stable isotopic tracer studies with gas chromatography/mass spectrometry (GC/MS) platforms, we systematically investigated how MTX interferes with the partitioning of mitochondrial and cytosolic formate metabolism. (3) Results: MTX significantly reduced de novo deoxythymidylate (dTMP) and methionine biosyntheses from mitochondrial-derived formate in cells, mouse liver, and bone marrow, supporting our postulation that MTX depletes mitochondrial 1C supply. Furthermore, MTX inhibited formate generation from mitochondria glycine cleavage system (GCS) both in vitro and in vivo. Folinate selectively rescued 1C metabolic pathways in a tissue-, cellular compartment-, and pathway-specific manner: folinate effectively reversed the inhibition of mitochondrial formate-dependent 1C metabolism in mouse bone marrow (dTMP, methionine, and GCS) and cells (dTMP and GCS) but not methionine synthesis in liver/liver-derived cells. Folinate failed to fully recover hepatic mitochondrial-formate utilization for methionine synthesis, suggesting that the efficacy of clinical folinate rescue in MTX therapy on hepatic methionine metabolism is poor. (4) Conclusion: Conducting studies in mouse and cell models, we demonstrate novel findings that MTX specifically depletes mitochondrial 1C supply that can be ameliorated by folinate supplementation except for hepatic transmethylation. These results imply that clinical use of low-dose MTX may particularly impede 1C metabolism via depletion of mitochondrial formate. The MTX induced systematic and tissue-specific formate depletion needs to be addressed more carefully, and the efficacy of folinate with respect to protecting against such depletion deserves to be evaluated in medical practice.

Tracing Metabolic Fate of Mitochondrial Glycine Cleavage System Derived Formate In Vitro and In Vivo.

Folate-mediated one-carbon (1C) metabolism is a major target of many therapies in human diseases. Studies have focused on the metabolism of serine 3-carbon as it serves as a major source for 1C units. The serine 3-carbon enters the mitochondria transferred by folate cofactors and eventually converted to formate and serves as a major building block for cytosolic 1C metabolism. Abnormal glycine metabolism has been reported in many human pathological conditions. The mitochondrial glycine cleavage system (GCS) catalyzes glycine degradation to CO2 and ammonium, while tetrahydrofolate (THF) is converted into 5,10-methylene-THF. GCS accounts for a substantial proportion of whole-body glycine flux in humans, yet the particular metabolic route of glycine 2-carbon recycled from GCS during mitochondria glycine decarboxylation in hepatic or bone marrow 1C metabolism is not fully investigated, due to the limited accessibility of human tissues. Labeled glycine at 2-carbon was given to humans and primary cells in previous studies for investigating its incorporations into purines, its interconversion with serine, or the CO2 production in the mitochondria. Less is known on the metabolic fate of the glycine 2-carbon recycled from the GCS; hence, a model system tracing its metabolic fate would help in this regard. We took the direct approach of isotopic labeling to further explore the in vitro and in vivo metabolic fate of the 2-carbon from [2-13C]glycine and [2-13C]serine. As the 2-carbon of glycine and serine is decarboxylated and catabolized via the GCS, the original 13C-labeled 2-carbon is transferred to THF and yield methyleneTHF in the mitochondria. In human hepatoma cell-lines, 2-carbon from glycine was found to be incorporated into deoxythymidine (dTMP, dT + 1), M + 3 species of purines (deoxyadenine, dA and deoxyguanine, dG), and methionine (Met + 1). In healthy mice, incorporation of GCS-derived formate from glycine 2-carbon was found in serine (Ser + 2 via cytosolic serine hydroxy methyl transferase), methionine, dTMP, and methylcytosine (mC + 1) in bone marrow DNA. In these experiments, labeled glycine 2-carbon directly incorporates into Ser + 1, A + 2, and G + 2 (at C2 and C8 of purine) in the cytosol. It is noteworthy that since the serine 3-carbon is unlabeled in these experiments, the isotopic enrichments in dT + 1, Ser + 2, dA + 3, dG + 3, and Met + 1 solely come from the 2-carbon of glycine/serine recycled from GCS, re-enters the cytosolic 1C metabolism as formate, and then being used for cytosolic syntheses of serine, dTMP, purine (M + 3) and methionine. Taken together, we established model systems and successfully traced the metabolic fate of mitochondrial GCS-derived formate from glycine 2-carbon in vitro and in vivo. Nutritional supply significantly alters formate generation from GCS. More GCS-derived formate was used in hepatic serine and methionine syntheses, whereas more GCS-derived formate was used in dTMP synthesis in the bone marrow, indicating that the utilization and partitioning of GCS-derived 1C unit are tissue-specific. These approaches enable better understanding concerning the utilization of 1C moiety generated from mitochondrial GCS that can help to further elucidate the role of GCS in human disease development and progression in future applications. More studies on GCS using these approaches are underway.

MTHFR C677T polymorphism increases MTX sensitivity via the inhibition of S-adenosylmethionine and de novo purine synthesis.

Objective: Currently, no guidelines are established for pharmacogenomic testing involving folate metabolic genes in long-term disease-modifying antirheumatic drugs' (DMARD) therapies. We carefully investigated how common genetic variations in methylenetetrahydrofolate reductase (MTHFR) influence cellular metabolic kinetics in response to methotrexate (MTX). Designs: Two distinct cell models: HepG2 with stabilized MTHFR inhibition using shRNA delivered by a Lentiviral vector; and Epstein-Barr virus transformed human lymphoblasts expressing MTHFR polymorphic allele 677C and 677T were used. Disease activity and DMARD use were compared between MTHFR-677CC, CT and TT rheumatoid arthritis (RA) patients in a cross-sectional study (n=120). Results: Compared with MTHFR-CC, MTHFR-TT carriers had lower mean weakly MTX dose (9.8 ± 3.3 compared with 12.1 ± 3.5, P<0.05). More MTHFR-TT carriers (8/11, 73%) reported MTX-related side effects compared with MTHFR-677CC (32/57, 56%) and MTHFR-677CT (30/51, 59%). No genotypic difference was found in other DMARDs. At the same dose of MTX, lymphoblasts were more sensitive in cell survival, protein and thymidine syntheses whereas HepG2 models were more susceptible to the inhibition of S-adenosylmethionine (adoMet) synthesis. MTHFR-C677T altered protein turnover and folate mediated 1-carbon metabolic fluxes in lymphoblasts with and without MTX. MTHFR function significantly affected transmethylation fluxes and adoMet homeostasis but not nucleotide biosyntheses in MTX-treated HepG2 cell-lines. Conclusion: Combining cell models, kinetic studies, and genetic tests in humans, the present study gives insight on how MTHFR effects hepatic transmethylation homeostasis during MTX therapy. We provide platforms that help predict the genetic impact on antifolate drugs, and further delineate tissue-specific target pathway in DMARD therapies. We suggest that genetic factors should be taken into account in clinical practice.

Suppression of the TNF-alpha level is mediated by Gan-Lu-Yin (traditional Chinese medicine) in human oral cancer cells through the NF-kappa B, AKT, and ERK-dependent pathways.

Oral cancer is one of the major causes of deaths in the male population of Taiwan. Gan-Lu-Yin (GLY) is used for an adjuvant treatment of Traditional Chinese Medicine in clinical patients. In this study, we investigated the molecular mechanisms in oral cancer cell lines after exposure to GLY. The cytometric bead-based array (CBA) method was used for the examining and analyzing of tumor necrosis factor-alpha (TNF-α) secretion level. TNF-α mRNA expression was determined by real-time PCR analysis. Nuclear factor kappa B (NF-κB) activity and other relative proteins were determined by NF-κB promoter assay, Western blotting, electrophoretic mobility shift assay (EMSA), and immuno-staining analyses. GLY decreased the secretion of TNF-α from the oral cancer CAL 27 cells. Furthermore, 2000 µg/mL of GLY significantly suppressed TNF-α mRNA expression of CAL 27 cells in a time-dependent manner. GLY reduced the levels of proteins, including nuclear NF-κB (p65 and p50), p-IKK (ser176), p-IκB, p-AKT, p-ERK, and nuclear Egr-1 in a time and dose-dependent manner. GLY also suppressed the NF-κB activity and translocation in CAL 27 cells. We suggest that GLY might promote the cure of oral cancer through decreasing the level of TNF-α cytokine, and these actions were mediated partially through the NF-κB, AKT, and ERK-dependent pathways in vitro. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1196-1205, 2016.

Protective Effects of Red Guava on Inflammation and Oxidative Stress in Streptozotocin-Induced Diabetic Mice.

Diabetes is an important chronic disease and the 4th leading cause of death in Taiwan. Hyperglycemia-induced oxidative and inflammatory damage are the main causes of chronic complications in diabetic patients. The red guava (red-fleshed guava cultivar of Psidium guajava L.) is a tropical fruit belonging to the Myrtaceae family and an important commercial crop in Taiwan. In this study, the protective effects of a diet containing red guava on inflammation and oxidative stress in streptozotocin (STZ)-induced diabetic mice were examined. The experimental group was divided into seven subgroups: normal (N), diabetes mellitus (DM), diabetes + red guava 1% (L), 2% (M), and 5% (H), diabetes + 5% red guava + anti-diabetic rosiglitazone (HR), and diabetes + anti-diabetic rosiglitazone (R). The mice were fed for 8 weeks and sacrificed by decapitation. Compared with the DM group, the experimental groups with diets containing red guava as well as rosiglitazone all showed significant improvements in blood glucose control, insulin resistance, creatinine, blood urea nitrogen, triglycerides, non-esterified fatty acids, cholesterol, c-reactive protein, TNF-α, and IL-10. Furthermore, the expression of inflammatory proteins, such as iNOS and NF-κB, was suppressed via activated PPARγ, and the expression levels of GPx3 and ACO increased. In summary, red guava can significantly suppress inflammatory and oxidative damage caused by diabetes and alleviate diabetic symptoms; thus, it exerts protective effects and has potential applications for the development of a dietary supplement.

A novel role of the tumor suppressor GNMT in cellular defense against DNA damage.

Glycine N-methyltransferase (GNMT) is a folate binding protein commonly diminished in human hepatoma yet its role in tumor development remains to be established. GNMT binds to methylfolate but is also inhibited by it; how such interactions affect human carcinogenesis is unclear. We postulated that GNMT plays a role in folate-dependent methyl group homeostasis and helps maintain genome integrity by promoting nucleotide biosynthesis and DNA repair. To test the hypothesis, GNMT was over-expressed in GNMT-null cell lines cultured in conditions of folate abundance or restriction. The partitioning of folate dependent 1-carbon groups was investigated using stable isotopic tracers and GC/MS. DNA damage was assessed as uracil content in cell models, as well as in Gnmt wildtype (Gnmt(+/+)), heterozygote (Gnmt(+/-)) and knockout (Gnmt(-/-)) mice under folate deplete, replete, or supplementation conditions. Our study demonstrated that GMMT 1) supports methylene-folate dependent pyrimidine synthesis; 2) supports formylfolate dependent purine syntheses; 3) minimizes uracil incorporation into DNA when cells and animals were exposed to folate depletion; 4) translocates into nuclei during prolonged folate depletion. In conclusion, loss of GNMT impairs nucleotide biosynthesis. Over-expression of GNMT enhances nucleotide biosynthesis and improves DNA integrity by reducing uracil misincorporation in DNA both in vitro and in vivo. To our best knowledge, the role of GNMT in folate dependent 1-carbon transfer in nucleotide biosynthesis has never been investigated. The present study gives new insights into the underlying mechanism by which GNMT can participate in tumor prevention/suppression in humans.

Docosahexaenoic Acid Alleviates Trimethylamine-N-oxide-mediated Impairment of Neovascularization in Human Endothelial Progenitor Cells.

BACKGROUND: Human endothelial progenitor cells (hEPCs), originating from hemangioblasts in bone marrow (BM), migrate into the blood circulation, differentiate into endothelial cells, and could act as an alternative tool for tissue regeneration. In addition, trimethylamine-N-oxide (TMAO), one of the gut microbiota metabolites, has been identified as an atherosclerosis risk factor. However, the deleterious effects of TMAO on the neovascularization of hEPCs have not been studied yet. RESULTS: Our results demonstrated that TMAO dose-dependently impaired human stem cell factor (SCF)-mediated neovascularization in hEPCs. The action of TMAO was through the inactivation of Akt/endothelial nitric oxide synthase (eNOS), MAPK/ERK signaling pathways, and an upregulation of microRNA (miR)-221. Docosahexaenoic acid (DHA) could effectively inhibit the cellular miR-221 level and induce the phosphorylation level of Akt/eNOS, MAPK/ERK signaling molecules, and neovascularization in hEPCs. DHA enhanced cellular amounts of reduced form glutathione (GSH) through an increased expression of the gamma-glutamylcysteine synthetase (γ-GCS) protein. CONCLUSIONS: TMAO could significantly inhibit SCF-mediated neovascularization, in part in association with an upregulation of miR-221 level, inactivation of Akt/eNOS and MAPK/ERK cascades, suppression of γ-GCS protein, and decreased levels of GSH and GSH/GSSG ratio. Furthermore, the DHA could alleviate the detrimental effects of TMAO and induce neovasculogenesis through suppression of miR-221 level, activation of Akt/eNOS and MAPK/ERK signaling cascades, increased expression of γ-GCS protein, and increment of cellular GSH level and GSH/GSSG ratio in hEPCs.

S-Allylcysteine inhibits tumour progression and the epithelial-mesenchymal transition in a mouse xenograft model of oral cancer.

Oral cancer is prevalent worldwide. Studies have indicated that an increase in the osteopontin (OPN) plasma level is correlated with the progression of oral cancer. Our previous report showed that the aqueous garlic extract S-allylcysteine (SAC) inhibited the epithelial-mesenchymal transition (EMT) of human oral cancer CAL-27 cells in vitro. Therefore, the present study investigated whether SAC consumption would help prevent tumour growth and progression, including the EMT, in a mouse xenograft model of oral cancer. The results demonstrated that SAC dose-dependently inhibited the growth of oral cancer in tumour-bearing mice. The histopathological and immunohistochemical staining results indicated that SAC was able to effectively suppress the tumour growth and progression of oral cancer in vivo. The chemopreventive effect of SAC was associated with the suppression of carcinogenesis factors such as N-methylpurine DNA glycosylase and OPN. SAC significantly suppressed the phosphorylation of Akt, mammalian target of rapamycin, inhibitor of κBα and extracellular signal-regulated kinase 1/2 in tumour tissues. The results demonstrated that the SAC-mediated suppression of cyclin D1 protein was associated with an augmented expression of the cell-cycle inhibitor p16(Ink4). Furthermore, SAC inhibited the expression of cyclo-oxygenase-2, vimentin and NF-κB p65 (RelA). These results show that SAC has potential as an agent against tumour growth and the progression of oral cancer in a mouse xenograft model.

Neovasculogenic effect of 11,12-epoxyeicosatrienoic acid involves the Akt/eNOS signaling pathways in human endothelial progenitor cells.

The 11,12-epoxy-eicosatrienoic acid (11,12-EET) is formed from arachidonic acid (AA) by cytochrome P450 2J2 (CYP 2J2) epoxygenase and function as an effector in blood vessels. Human endothelial progenitor cells (hEPCs), a preceding cell source for endothelial cells (ECs), involve in the vascular tissue repairing by postnatal neovasculogenesis. However, the effect of 11, 12-EET on hEPCs and neovasculogenesis is not well known. In the current study, we examined the function of 11, 12-EET in hEPCs-mediated neovasculogenesis by using tubular formation analysis, Western Blotting assay, immunofluorescence staining, flow cytometry analysis and zymogram analysis. The results suggest that 11, 12-EET significantly induces neovasculogenesis through the phosphorylation of phosphoinositide 3-kinase (PI3-K)/Akt, endothelial-nitric oxide synthase (e-NOS) and extracellular signal-regulated kinase 1/2 (ERK 1/2) signaling pathways. 11, 12-EET up-regulates the expression of cyclin D1, cyclin-dependent kinase 4 (CDK4) and nuclear factor kappa B (NF-κB) proteins. Moreover, 11, 12-EET augments the expression of VE-cadherin and CD31 proteins in hEPCs. 11, 12-EET also augmented Rac1/Rho A signaling cascades, cell migration and an up-regulation of matrix metalloproteinase (MMP) -2 and -9 proteins. These results demonstrate that 11, 12-EET exerts a significant function in the neovasculogenesis of hEPCs.

N-3 polyunsaturated fatty acids block the trimethylamine-N-oxide- ACE2- TMPRSS2 cascade to inhibit the infection of human endothelial progenitor cells by SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) is a novel coronavirus that infects many types of cells and causes cytokine storms, excessive inflammation, acute respiratory distress to induce failure of respiratory system and other critical organs. In this study, our results showed that trimethylamine-N-oxide (TMAO), a metabolite generated by gut microbiota, acts as a regulatory mediator to enhance the inerleukin-6 (IL-6) cytokine production and the infection of human endothelial progenitor cells (hEPCs) by SARS-CoV-2. Treatment of N-3 polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) could effectively block the entry of SARS-CoV-2 in hEPCs. The anti-infection effects of N-3 PUFAs were associated with the inactivation of NF-κB signaling pathway, a decreased expression of the entry receptor angiotensin-converting enzyme 2 (ACE2) and downstream transmembrane serine protease 2 in hEPCs upon the stimulation of TMAO. Treatment of DHA and EPA further effectively inhibited TMAO-mediated expression of IL-6 protein, probably through an inactivation of MAPK/p38/JNK signaling cascades and a downregulation of microRNA (miR)-221 in hEPCs. In conclusion, N-3 PUFAs such as DHA and EPA could effectively act as preventive agents to block the infection of SARS-CoV-2 and IL-6 cytokine production in hEPCs upon the stimulation of TMAO.

Glycine-N methyltransferase expression in HepG2 cells is involved in methyl group homeostasis by regulating transmethylation kinetics and DNA methylation.

Glycine-N methyltransferase (GNMT) is a potential tumor suppressor that is commonly inactivated in human hepatoma. We systematically investigated how GNMT regulates methyl group kinetics and global DNA methylation. HepG2 cells (GNMT inactive, GNMT-) and cells transfected with GNMT expressed vector (GNMT+) were cultured in low (10 µmol/L), adequate (100 µmol/L), or high (500 µmol/L) l-methionine, each with 2.27 µmol/L folate. Transmethylation kinetics were studied using stable isotopic tracers and GC-MS. Methylation status was determined by S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) levels, SAM:SAH ratio, DNA methyltransferase (DNMT) activity, and methylated cytidine levels in DNA. Compared with GNMT- cells, GNMT+ cells had lower homocysteine and greater cysteine concentrations. GNMT expression increased methionine clearance by inducing homocysteine transsulfuration and remethylation metabolic fluxes when cells were cultured in high or adequate l-methionine. In contrast, homocysteine remethylation flux was lower in GNMT+ cells than in GNMT- cells and homocysteine transsulfuration fluxes did not differ when cells were cultured in low methionine, suggesting that normal GNMT function helps to conserve methyl groups. Furthermore, GNMT expression decreased SAM and increased SAH levels and reduced DNMT activity in high or adequate, but not low, methionine cultures. In low methionine cultures, restoring GNMT in HepG2 cells did not lead to sarcosine synthesis, which would waste methyl groups. Methylated cytidine levels were significantly lower in GNMT- cells than in GNMT+ cells. In conclusion, we have shown that GNMT affects transmethylation kinetics and SAM synthesis and facilitates the conservation of methyl groups by limiting homocysteine remethylation fluxes.

Adlay hull extracts attenuate ß-amyloid-induced neurotoxicity and oxidative stress in PC12 cells through antioxidative, anti-inflammatory, and antiapoptotic activities.

Alzheimer's disease (AD) is characterized by accumulation of ß-amyloid (Aß) in senile plaques, contributing to oxidative stress, mitochondrial diseases, and synaptic atrophy, consequently leading to the deterioration of brain function. Adlay (Coix lacryma-jobi L.) is an annual botanical. Here, a 95% ethanol extract of adlay hull (AHEE) was partitioned by ethyl acetate (AHEAE), n-butanol (AHBUE), and water (AHWE), and the effects of these extracts on lipopolysaccharide (LPS)-induced RAW264.7 cells and Aß-induced PC12 cells, as experimental models of neurotoxicity, were evaluated. The expression of anti-inflammatory and antiapoptosis-related proteins was investigated and AHEE, AHEAE, and AHWE were found to exert anti-inflammatory effects. AHWE exhibited antiapoptotic effects and inhibited inducible nitric oxide synthase expression and nitric oxide production. We investigated the protective effects of AHWE against Aß-induced neurotoxicity in dPC12 cells and explored the underlying mechanism. Pretreatment with AHWE significantly attenuated cell death and Aß-mediated increase in B cell lymphoma (Bcl)-2/Bax ratio. AHWE significantly inhibited Aß and enhanced protein kinase B (Akt) level in dPC12 cells, suggesting that its protective effect against Aß-induced apoptosis in dPC12 cells was mediated through upregulation of the phosphoinositide 3-kinases (PI3K)/Akt signaling pathway. These extracts and its bioactive compound K36-21 may be potentially useful to treat neurodegenerative disorders.

Docosahexaenoic Acid Inhibits Cell Proliferation through a Suppression of c-Myc Protein in Pancreatic Ductal Adenocarcinoma Cells.

Treatment of pancreatic cancer by inhibiting the aberrant activation of the survival signaling pathways has received considerable attention. We investigated the probable action of DHA on the suppression of cell proliferation in human pancreatic ductal adenocarcinoma (PDAC) cells. Our results demonstrated that DHA dose-dependently inhibited cell proliferation through an induction of cell cycle arrest in human PDAC cells. DHA suppressed the expression of phosphorylated-Rb (p-Rb), cyclin D1, cyclin E, cyclin A, E2F1 and c-Myc proteins. Blocking the activation of STAT3 signaling pathway led to an inactivation of CAMKII and increased phosphorylation of c-Myc (T58) protein accompanied with decreased expression of c-Myc protein. Treatment of DHA effectively inhibited cell survival through decreased phosphorylation levels of EGFR, STAT3 and CAMKII proteins. The mechanisms of action were associated with increased phosphorylation levels of c-Myc (T58) and instability of c-Myc proteins. DHA inhibited cell survival through an increased GSSG/GSH ratio and oxidative stress level in HPAF-II cells. DHA induced cell apoptosis through increased expression of Bax, c-caspase 3 and c-PARP proteins in HPAF-II cells. Moreover, treatment of DHA significantly inhibited nucleotide synthesis. In conclusion, DHA might significantly suppress the proliferation of PDAC cells and therefore have potential as an anti-cancer therapeutic agent.

Treatment of 13-cis retinoic acid and 1,25-dihydroxyvitamin D3 inhibits TNF-alpha-mediated expression of MMP-9 protein and cell invasion through the suppression of JNK pathway and microRNA 221 in human pancreatic adenocarcinoma cancer cells.

Human pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer type with a very high mortality rate. Inflammatory cytokine such as tumor necrosis factor- alpha (TNF-α) plays a pivotal role in the progression of PDAC. Recently, suppression of cell invasion by preventive agents has received considerable attention in the prevention of metastatic tumors. Several clinical studies suggested that natural forms or analogues of fat-soluble vitamins such as vitamin A and vitamin D can work as anti-cancer agents to inhibit the development of cancer. In this study, our results demonstrated that co-treatment of 13-cis retinoic acid (13-cis RA) and 1,25-dihydroxyvitamin D3 (1,25-VD3) significantly inhibited TNF-α mediated cell invasion in PDAC in vitro. Cotreatment of 13-cis RA and 1,25-VD3 also inhibited TNF-α mediated expression of matrix metalloproteinase-9 (MMP-9) protein through blocking c-Jun N-terminal kinase (JNK) and nuclear factor kappa B (NF-κB) signaling pathways. Our results demonstrated that treatment of TNF-α lead to a decreased expression of tissue inhibitor of metalloproteinase- 3 (TIMP-3) protein and an induction of MMP-9 protein and cell invasion through an upregulation of microRNA-221 (miR-221) in human PDAC cells. Moreover, treatment of SP600125 (a specific inhibitor of JNK pathway) or cotreatment of 13-cis RA and 1,25-VD3 significantly induced a decreased expression of miR-221 and an increased expression of TIMP-3 protein. These results suggest that 13-cis RA and 1,25-VD3 significantly suppress TNF-α mediated cell invasion and therefore potentially act as preventive agents against PDAC.

Expression of MTDH and IL-10 Is an Independent Predictor of Worse Prognosis in ER-Negative or PR-Negative Breast Cancer Patients.

(1) Background: Tumor hypoxia leads to metastasis and certain immune responses, and interferes with normal biological functions. It also affects glucose intake, down-regulates oxidative phosphorylation, and inhibits fatty-acid desaturation regulated by hypoxia-inducible factor 1α (HIF-1α). Although tumor hypoxia has been found to promote tumor metastasis, the roles of HIF-1α-regulated genes and their application are not completely integrated in clinical practice. (2) Methods: We examined the correlation between HIF-1α, metadherin (MTDH), and interleukin (IL)-10 mRNA expression, as well as their expression patterns in the prognosis of breast cancer using the Gene Expression Profiling Interactive Analysis (GEPIA) databases via a web interface; tissue microarrays (TMAs) were stained for MTDH and IL-10 protein expression using immunohistochemistry. (3) Results: HIF-1α, MTDH, and IL-10 mRNA expression are highly correlated and strongly associated with poor prognosis. MTDH and IL-10 protein expression of breast cancer patients usually harbored negative estrogen receptor (ER) or progesterone receptor (PR) status, and late-stage tumors have higher IL-10 expression. With regard to MTDH and IL-10 protein expression status for using univariate and multivariate analysis, the results showed that the protein expression of MTDH and IL-10 in ER-negative or PR-negative breast cancer patients have the worse prognosis. (4) Conclusions: we propose a new insight into hypoxia tumors in the metabolism and immune evidence for breast cancer therapy.

Decyl caffeic acid inhibits the proliferation of colorectal cancer cells in an autophagy-dependent manner in vitro and in vivo.

The treatment of human colorectal cancer (CRC) cells through suppressing the abnormal survival signaling pathways has recently become a significant area of focus. In this study, our results demonstrated that decyl caffeic acid (DC), one of the novel caffeic acid derivatives, remarkedly suppressed the growth of CRC cells both in vitro and in vivo. The inhibitory effects of DC on CRC cells were investigated in an in vitro cell model and in vivo using a xenograft mouse model. CRC cells were treated with DC at various dosages (0, 10, 20 and 40 µM), and cell survival, the apoptotic index and the autophagy level were measured using an MTT assay and flow cytometry analysis, respectively. The signaling cascades in CRC were examined by Western blot assay. The anti-cancer effects of DC on tumor growth were examined by using CRC HCT-116 cells implanted in an animal model. Our results indicated that DC differentially suppressed the growth of CRC HT-29 and HCT-116 cells through an enhancement of cell-cycle arrest at the S phase. DC inhibited the expression of cell-cycle regulators, which include cyclin E and cyclin A proteins. The molecular mechanisms of action were correlated to the blockade of the STAT3 and Akt signaling cascades. Strikingly, a high dosage of DC prompted a self-protection action through inducing cell-dependent autophagy in HCT-116 cells. Suppression of autophagy induced cell death in the treatment of DC in HCT-116 cells. DC seemed to inhibit cell proliferation of CRC differentially, and the therapeutic advantage appeared to be autophagy dependent. Moreover, consumption of DC blocked the tumor growth of colorectal adenocarcinoma in an experimental animal model. In conclusion, our results suggested that DC could act as a therapeutic agent through the significant suppression of tumor growth of human CRC cells.

Metabolic Pathways Enhancement Confers Poor Prognosis in p53 Exon Mutant Hepatocellular Carcinoma.

RNA-Sequencing (RNA-Seq), the most commonly used sequencing application tool, is not only a method for measuring gene expression but also an excellent media to detect important structural variants such as single nucleotide variants (SNVs), insertion/deletion (Indels), or fusion transcripts. The Cancer Genome Atlas (TCGA) contains genomic data from a variety of cancer types and also provides the raw data generated by TCGA consortium. p53 is among the top 10 somatic mutations associated with hepatocellular carcinoma (HCC). The aim of the present study was to analyze concordant different gene profiles and the priori defined set of genes based on p53 mutation status in HCC using RNA-Seq data. In the study, expression profile of 11 799 genes on 42 paired tumor and adjacent normal tissues was collected, processed, and further stratified by the mutated versus normal p53 expression. Furthermore, we used a knowledge-based approach Gene Set Enrichment Analysis (GSEA) to compare between normal and p53 mutation gene expression profiles. The statistical significance (nominal P value) of the enrichment score (ES) genes was calculated. The ranked gene list that reflects differential expression between p53 wild-type and mutant genotypes was then mapped to metabolic process by KEGG, an encyclopedia of genes and genomes to assign functional meanings. These approaches enable us to identify pathways and potential target gene/pathways that are highly expressed in p53 mutated HCC. Our analysis revealed 2 genes, the hexokinase 2 (HK2) and Enolase 1 (ENO1), were conspicuous of red pixel in the heatmap. To further explore the role of these genes in HCC, the overall survival plots by Kaplan-Meier method were performed for HK2 and ENO1 that revealed high HK2 and ENO1 expression in patients with HCC have poor prognosis. These results suggested that these glycolysis genes are associated with mutated-p53 in HCC that may contribute to poor prognosis. In this proof-of-concept study, we proposed an approach for identifying novel potential therapeutic targets in human HCC with mutated p53. These approaches can take advantage of the massive next-generation sequencing (NGS) data generated worldwide and make more out of it by exploring new potential therapeutic targets.