Ascorbic Acid 2-Glucoside Stably Promotes the Primitiveness of Embryonic and Mesenchymal Stem Cells Through Ten-Eleven Translocation- and cAMP-Responsive Element-Binding Protein-1-Dependent Mechanisms.

Aims: The naive or primitive states of stem cells (SCs) residing in specific niches are unstable and difficult to preserve in vitro. Vitamin C (VitC), in addition to suppressing oxygen radicals, exerts pleiotropic effects to preserve the core functions of SCs. However, this compound is labile and readily oxidized, resulting in cellular toxicity and preventing its reliable application in this context. We found that a VitC derivative, ascorbic acid 2-glucoside (AA2G), stably maintains the naive pluripotency of murine embryonic SCs (mESCs) and the primitiveness of human mesenchymal SCs (hMSCs) without cellular toxicity. Results: The beneficial effects of AA2G and related molecular mechanisms were evaluated in mESCs, induced pluripotent-SCs (iPSCs), and hMSCs. AA2G was stable in aqueous solution and barely induced cellular toxicity in cultured SCs, unlike VitC. AA2G supplementation recapitulated the well-known effects of VitC, including induction of ten-eleven translocation-dependent DNA demethylation in mESCs and suppression of p53 during generation of murine iPSCs. Furthermore, supplementation of hMSCs with AA2G improved therapeutic outcomes in an asthma mouse model by promoting their self-renewal, engraftment, and anti-inflammatory properties. Particularly, activation of the cAMP-responsive element-binding protein-1 (CREB1) pathway contributed to the ability of AA2G to maintain naive pluripotency of mESCs and functionality of hMSCs. Innovation and Conclusion: Given its long-lasting effects and low cellular toxicity, AA2G supplementation is useful to support the naive pluripotency of mESCs and the primitiveness of hMSCs, affecting their developmental potency and therapeutic efficacy. Furthermore, we demonstrate the significance of the CREB1 pathway in the mechanism of action of AA2G.

Understanding the functional role of genistein in the bone differentiation in mouse osteoblastic cell line MC3T3-E1 by RNA-seq analysis.

Genistein, a phyto-estrogen, can potentially replace endogenous estrogens in postmenopausal women, but the underlying molecular mechanisms remain incompletely understood. To obtain insight into the effect of genistein on bone differentiation, RNA sequencing (RNA-seq) analysis was used to detect differentially expressed genes (DEGs) in genistein-treated vs. untreated MC3T3-E1 mouse osteoblastic cells. Osteoblastic cell differentiation was monitored by measuring osteoblast differentiation factors (ALP production, bone mineralization, and expression of osteoblast differentiation markers). From RNA-seq analysis, a total of 132 DEGs (including 52 up-regulated and 80 down-regulated genes) were identified in genistein-treated cells (FDR q-value < 0.05 and fold change > 1.5). KEGG pathway and Gene Ontology (GO) enrichment analyses were performed to estimate the biological functions of DEGs and demonstrated that these DEGs were highly enriched in functions related to chemotactic cytokines. The functional relevance of DEGs to genistein-induced osteoblastic cell differentiation was further evaluated by siRNA-mediated knockdown in MC3T3-E1 cells. These siRNA knockdown experiments (of the DEGs validated by real-time qPCR) demonstrated that two up-regulated genes (Ereg and Efcab2) enhance osteoblastic cell differentiation, while three down-regulated genes (Hrc, Gli, and Ifitm5) suppress the differentiation. These results imply their major functional roles in bone differentiation regulated by genistein.

Cystamine induces AIF-mediated apoptosis through glutathione depletion.

Cystamine and its reduced form cysteamine showed protective effects in various models of neurodegenerative disease, including Huntington's disease and Parkinson's disease. Other lines of evidence demonstrated the cytotoxic effect of cysteamine on duodenal mucosa leading to ulcer development. However, the mechanism for cystamine cytotoxicity remains poorly understood. Here, we report a new pathway in which cystamine induces apoptosis by targeting apoptosis-inducing factor (AIF). By screening of various cell lines, we observed that cystamine and cysteamine induce cell death in a cell type-specific manner. Comparison between cystamine-sensitive and cystamine-resistant cell lines revealed that cystamine cytotoxicity is not associated with unfolded protein response, reactive oxygen species generation and transglutaminase or caspase activity; rather, it is associated with the ability of cystamine to trigger AIF nuclear translocation. In cystamine-sensitive cells, cystamine suppresses the levels of intracellular glutathione by inhibiting γ-glutamylcysteine synthetase expression that triggers AIF translocation. Conversely, glutathione supplementation completely prevents cystamine-induced AIF translocation and apoptosis. In rats, cysteamine administration induces glutathione depletion and AIF translocation leading to apoptosis of duodenal epithelium. These results indicate that AIF translocation through glutathione depletion is the molecular mechanism of cystamine toxicity, and provide important implications for cystamine in the neurodegenerative disease therapeutics as well as in the regulation of AIF-mediated cell death.

Soyabean glyceollins: biological effects and relevance to human health.

Glyceollins, one family of phytoalexins, are de novo synthesised from daidzein in the soyabean upon exposure to some types of fungus. The efficiency of glyceollin production appears to be influenced by soyabean variety, fungal species, and the degree of physical damage to the soyabean. The compounds have been shown to have strong antioxidant and anti-inflammatory activities, and to inhibit the proliferation and migration of human aortic smooth muscle cells, suggesting their potential to prevent atherosclerosis. It has also been reported that glyceollins have inhibited the growth of prostate and breast cancer cells in xenograft animal models, which is probably due to their anti-oestrogenic activity. In essence, glyceollins deserve further animal and clinical studies to confirm their health benefits.

Isoalantolactone from Inula helenium caused Nrf2-mediated induction of detoxifying enzymes.

Our previous study demonstrated that methanolic extract of Inula helenium (Elecampane) has the potential to induce detoxifying enzymes such as NAD(P)H:(quinone acceptor) oxidoreductase 1 (EC 1.6.99.2) (NQO1, QR) activity and glutathione S-transferase (GST) and found isoalantolactone and alantolactone as the active components. In this study we investigated the detoxifying enzyme-inducing potential of isoalantolactone, which is present in I. helenium and has a structure similar to that of alantolactone. The compound induced QR in a dose-dependent manner in both Hepa1c1c7 cells and its mutant BPRc1 cells lacking the arylhydrocarbon receptor translocator. Like with most phase 2 enzyme inducers, other phase 2 detoxifying enzymes, including GST, glutathione reductase, gamma-glutamylcysteine synthetase, and heme oxygenase-1, were also induced by isoalantolactone in a dose-dependent manner in the cultured cells. Furthermore, isoalantolactone caused a proportionate increase in luciferase activity depending upon concentration and exposure time in the reporter assay in which HepG2-C8 cells, transfectants carrying antioxidant response element-luciferase gene, were used. The nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2) was stimulated by the compound and attenuated by phosphatidylinositol 3-kinase inhibitors such as LY294002 and wortmannin. In conclusion, isoalantolactone is a candidate for chemoprevention and acts as potent phase 2 enzyme inducer by stimulating the accumulation of Nrf2 in the nucleus.

Nrf2-mediated induction of detoxifying enzymes by alantolactone present in Inula helenium.

Our previous study showed that a methanol extract of Inula helenium had the potential to induce detoxifying enzymes such as quinone reductase (QR) and glutathione S-transferase (GST) activity. In this study the methanol extract was further fractionated using silica gel chromatography and vacuum liquid chromatography, to yield pure compounds alantolactone and isoalantolactone as QR inducers. Alantolactone caused a dose-dependent induction of antioxidant enzymes including QR, GST, gamma-glutamylcysteine synthase, glutathione reductase, and heme oxygenase 1 in hepa1c1c7 mouse hepatoma cells. The compound increased the luciferase activity of HepG2-C8 cells, transfectants carrying antioxidant response element (ARE)-luciferase gene, in a dose-dependent manner, suggesting ARE-mediated transcriptional activation of antioxidant enzymes. Alantolactone also stimulated the nuclear accumulation of Nrf2 that was inhibited by phosphatidylinositol 3-kinase (PI3K) inhibitors. In conclusion, alantolactone appears to induce detoxifying enzymes via activation of PI3K and JNK signaling pathways, leading to translocation of Nrf2, and subsequent interaction between Nrf2 and ARE in the encoding genes.