Wim Wätjen

Myricetin-mediated lifespan extension in Caenorhabditis elegans is modulated by DAF-16.

Myricetin is a naturally occurring flavonol found in many plant based food sources. It increases the lifespan of Caenorhabditis elegans, but the molecular mechanisms are not yet fully understood. We have investigated the impact of this flavonoid on the transcription factors DAF-16 (C. elegans FoxO homologue) and SKN-1 (Nrf2 homologue), which have crucial functions in the regulation of ageing. Myricetin is rapidly assimilated by the nematode, causes a nuclear translocation of DAF-16 but not of SKN-1, and finally prolongs the mean adult lifespan of C. elegans by 32.9%. The lifespan prolongation was associated with a decrease in the accumulation of reactive oxygen species (ROS) detected by DCF. Myricetin also decreases the formation of lipofuscin, a pigment consisting of highly oxidized and cross-linked proteins that is considered as a biomarker of ageing in diverse species. The lifespan extension was completely abolished in a daf-16 loss-of-function mutant strain (CF1038). Consistently with this result, myricetin was also not able to diminish stress-induced ROS accumulation in the mutant. These results strongly indicate that the pro-longevity effect of myricetin is dependent on DAF-16 and not on direct anti-oxidative effects of the flavonoid.

Epigallocatechin gallate (EGCG) inhibits adhesion and migration of neural progenitor cells in vitro

Food supplements based on herbal products are widely used during pregnancy as part of a self-care approach. The idea that such supplements are safe and healthy is deeply seated in the general population, although they do not underlie the same strict safety regulations than medical drugs. We aimed to characterize the neurodevelopmental effects of the green tea catechin epigallocatechin gallate (EGCG), which is now commercialized as high-dose food supplement. We used the “Neurosphere Assay” to study the effects and unravel underlying molecular mechanisms of EGCG treatment on human and rat neural progenitor cells (NPCs) development in vitro. EGCG alters human and rat NPC development in vitro. It disturbs migration distance, migration pattern, and nuclear density of NPCs growing as neurospheres. These functional impairments are initiated by EGCG binding to the extracellular matrix glycoprotein laminin, preventing its binding to β1-integrin subunits, thereby prohibiting cell adhesion and resulting in altered glia alignment and decreased number of migrating young neurons. Our data raise a concern on the intake of high-dose EGCG food supplements during pregnancy and highlight the need of an in vivo characterization of the effects of high-dose EGCG exposure during neurodevelopment.

Isoxanthohumol, a constituent of hop (Humulus lupulus L.), increases stress resistance in Caenorhabditis elegans dependent on the transcription factor DAF-16

AbstractPurpose The flavanone isoxanthohumol (IX) has gained attention as antioxidative and chemopreventive agent, but the molecular mechanism of action remains unclear. We investigated effects of this secondary plant compound in vivo using the model organism Caenorhabditis elegans.MethodsAdult C. elegans nematodes were incubated with IX, and then, the stress resistance was analysed in the SYTOX assay; lifespan was monitored by touch-provoked movement method, the amount of reactive oxygen species (ROS) was measured in the DCF assay, and the nuclear localisation of the transcription factor DAF-16 was analysed by using a transgenic strain. By the use of a DAF-16 loss-of-function strain, we analysed whether the effects are dependent on DAF-16.ResultsIX increases the resistance of the nematode against thermal stress. Additionally, a reduction in ROS in vivo was caused by IX. Since the flavanone only has a marginal radical-scavenging capacity (TEAC assay), we suggest that IX mediates its antioxidative effects indirectly via activation of DAF-16 (homologue to mammalian FOXO proteins). The nuclear translocation of this transcription factor is increased by IX. In the DAF-16-mutated strain, the IX-mediated increase in stress resistance was completely abolished; furthermore, an increased formation of ROS and a reduced lifespan was mediated by IX.ConclusionIX or a bacterial metabolite of IX causes antioxidative effects as well as an increased stress resistance in C. elegans via activation of DAF-16. The homologous pathway may have implications in the molecular mechanism of IX in mammals.

The resveratrol derivatives trans-3,5-dimethoxy-4-fluoro-4'-hydroxystilbene and trans-2,4',5-trihydroxystilbene decrease oxidative stress and prolong lifespan in Caenorhabditis elegans.

Resveratrol (trans‐3,4′,5‐trihydroxystilbene (1)) was previously shown to extend the lifespan of different model organisms. However, its pharmacological efficiency is controversially discussed. Therefore, the bioactivity of four newly synthesized stilbenes (trans‐3,5‐dimethoxy‐4‐fluoro‐4′‐hydroxystilbene (3), trans‐4′‐hydroxy‐3,4,5‐trifluorostilbene (4), trans‐2,5‐dimethoxy‐4′‐hydroxystilbene (5), trans‐2,4′,5‐trihydroxystilbene (6)) was compared to (1) and pterostilbene (trans‐3,5‐dimethoxy‐4′‐hydroxystilbene (2)) in the established model organism Caenorhabditis elegans.

Modulation of the Nrf2 signalling pathway in Hct116 colon carcinoma cells by baicalein and its methylated derivative negletein

abstract Context: Baicalein is a major compound in extracts derived from Scutellaria baicalensis Georgi (Lamiaceae) which are used in the Traditional Chinese Medicine for the treatment of inflammatory and gastrointestinal diseases. This flavonoid is an activator of the Nrf2 signalling pathway but the molecular mechanism is not clearly established. Objective: We investigated the molecular mode of baicalein-mediated Nrf2-activation in Hct116 cells by the analysis of proteasomal activity, radical-scavenging activity and the comparison with baicalein derivatives. Materials and methods: The radical-scavenging activity (TEAC, DCF) up to 25 μM, cytotoxicity (MTT assay, 48 h) up to 100 μM, proteasomal activity and the Nrf2-activation (luciferase assay, ubiquitinylation, western blot, Ser40-phosphorylation; incubation for 1 or 4 h) by concentrations up to 40 or 50 μM of the compounds were analysed in Hct116 human colon carcinoma cells. Results: No change in the ubiquitinylation of Nrf2, proteasomal activity and transcription of the NRF2 gene were detectable. Baicalein decreased the phosphorylation of Nrf2 (IC50-value approximately 20 μM) suggesting an inhibitory effect of the flavonoid on protein kinases. Since the activation of the Nrf2 pathway by baicalein might be also due to redox-activity of the compound, we investigated the effects of methylated baicalein derivatives oroxylin A, negeletein and baicaleintrimethylether. Oroxylin A and negletein showed a comparable redox-active potential, but only negletein (50 μM, 4 h) was able to activate Nrf2. Conclusion: This result confirms the hypothesis that baicalein, a component of extracts derived from Baical Skullcap, causes an activation of Nrf2 independent of a modulation of the cellular redox potential.

The Lignan Pinoresinol Induces Nuclear Translocation of DAF-16 in Caenorhabditis elegans but has No Effect on Life Span

The lignan pinoresinol is a constituent of flaxseed, sesame seeds and olive oil. Because of different molecular effects reported for this compound, e.g. antioxidative activity, pinoresinol is suggested to cause positive effects on humans. Because experimental data are limited, we have analysed the effects of the lignan on the nematode Caenorhabditis elegans: in spite of a strong antioxidative capacity detected in an in vitro assay, no antioxidative effects were detectable in vivo. In analogy to this result, no modulation of the sensitivity against thermal stress was detectable. However, incubation with pinoresinol caused an enhanced nuclear accumulation of the transcription factor DAF‐16 (insulin/IGF‐like signalling pathway). Using a strain with an enhanced oxidative stress level (mev‐1 mutant), we clearly see an increase in stress resistance caused by this lignan, but no change in reactive oxygen species. Furthermore, we investigated the effects of pinoresinol on the life span of the nematode, but no modulation was found, neither in wild‐type nor in mev‐1 mutant nematodes. These results suggest that pinoresinol may exert pharmacologically interesting effects via modulation of the insulin‐like signalling pathway in C. elegans as well as in other species like mammals due to the evolutionary conservation of this signalling pathway. Copyright

Bioactive pyrrole alkaloids isolated from the Red Sea: marine sponge Stylissa carteri

Abstract Fifteen pyrrole alkaloids were isolated from the Red Sea marine sponge Stylissa carteri and investigated for their biological activities. Four of them were dibrominated [(+) dibromophakelline, Z-3-bromohymenialdisine, (±) ageliferin and 3,4-dibromo-1H-pyrrole-2-carbamide], nine compounds were monobrominated [(−) clathramide C, agelongine, (+) manzacidin A, (−) 3-bromomanzacidin D, Z-spongiacidin D, Z-hymenialdisine, 2-debromostevensine, 2-bromoaldisine and 4-bromo-1H-pyrrole-2-carbamide)] and finally, two compounds were non-brominated derivatives viz., E-debromohymenialdisine and aldisine. The structure elucidations of isolated compounds were based on 1D & 2D NMR spectroscopic and MS studies, as well as by comparison with literature. In-vitro, Z-spongiacidin D exhibited a moderate activity on (ARK5, CDK2-CycA, CDK4/CycD1, VEGF-R2, SAK and PDGFR-beta) protein kinases. Moreover, Z-3-bromohymenialdisine showed nearly similar pattern. Furthermore, Z-hymenialdisine displayed a moderate effect on (ARK5 & VEGF-R2) and (−) clathramide C showed a moderate activity on AURORA-A protein kinases. While, agelongine, (+) manzacidin A, E-debromohymenialdisine and 3,4-dibromo-1H-pyrrole-2-carbamide demonstrated only marginal inhibitory activities. The cytotoxicity study was evaluated in two different cell lines. The most effective secondary metabolites were (+) dibromophakelline and Z-3-bromohymenialdisine on L5178Y. Finally, Z-hymenialdisine, Z-3-bromohymenialdisine and (±) ageliferin exhibited the highest cytotoxic activity on HCT116. No report about inhibition of AURORA-A and B by hymenialdisine/hymenialdisine analogs existed and no reported toxicity of ageliferin existed in literature.

Polygonum multiflorum Extract Exerts Antioxidative Effects and Increases Life Span and Stress Resistance in the Model Organism Caenorhabditis elegans via DAF-16 and SIR-2.1

Extracts of the Chinese plant Polygonum multiflorum (PME) are used for medicinal purposes as well as food supplement due to anti-aging effects. Despite of the common use of these food supplements, experimental data on physiological effects of PME and its underlying molecular mechanisms in vivo are limited. We used the model organism Caenorhabditis elegans to analyze anti-aging-effects of PME in vivo (life span, lipofuscin accumulation, oxidative stress resistance, thermal stress resistance) as well as the molecular signaling pathways involved. The effects of PME were examined in wildtype animals and mutants defective in the sirtuin-homologue SIR-2.1 (VC199) and the FOXO-homologue DAF-16 (CF1038). PME possesses antioxidative effects in vivo and increases oxidative stress resistance of the nematodes. While the accumulation of lipofuscin is only slightly decreased, PME causes a significant elongation (18.6%) of mean life span. DAF-16 is essential for the reduction of thermally induced ROS accumulation, while the resistance against paraquat-induced oxidative stress is dependent on SIR-2.1. For the extension of the life span, both DAF-16 and SIR-2.1 are needed. We demonstrate that PME exerts protective effects in C. elegans via modulation of distinct intracellular pathways.

Role of Oxidative Stress in the Process of Carcinogenesis

Oxidative stress contributes to the process of carcinogenesis in several ways: an oxidation of DNA bases by reactive oxygen species (ROS), e.g. oxidation of guanosine to 8-hydroxyguanosine, has been linked to the formation of mutations thereby triggering the process of carcinogenesis. Furthermore, ROS contribute to the process of carcinogenesis by stimulation of signal transduction pathways associated with cell proliferation: activation of transcription factors like Nrf2 and NF-κB results in a change in gene expression patterns which also contributes to the process of carcinogenesis. Some correlations between oxidative stress and the process of carcinogenesis in humans are shown by analysis of single nucleotide polymorphisms and conduction of intervention studies with antioxidant supplementation. However, evidence from human studies, e.g. with supplementation of antioxidant vitamins, remains inconsistent.