RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: The Bazhen decoction is one of the most extensively used Traditional Chinese medicine (TCM) prescriptions for treatment of aging related diseases. However, due to the complexity of the components, the pharmacological mechanism of Bazhen decoction is still limited. AIM OF THE STUDY: In this study, with the aim of helping the clinical precision medicine of TCM, we try out a systematic analysis for dissecting the molecular mechanism of complicated TCM prescription: Bazhen decoction. We identify the pharmacological mechanism of Bazhen decoction in telomere elongation as revealed by systematic analysis. MATERIALS AND METHODS: By RNA sequencing and transcriptome analysis of Bazhen decoction treated wild type cells, we reveal the transcriptome profile induced by Bazhen decoction. We utilized the cells derived from Werner syndrome (WS) mice, which is known to be dysfunctional in telomere elongation due to the deficiency of DNA helicase Wrn. By Western blot, qPCR, Immunofluorescence, flow cytometry, telomere FISH, and SA-ß-Gal staining, we verify the transcriptome data and confirm the pharmacological function of Bazhen decoction and its drug containing serum in telomere elongation and reversing progeroid cell senescence. RESULTS: We reveal that Bazhen decoction may systematically regulate multiple anti-aging pathways, including stem cell regulation, protein homeostasis, cardiovascular function, neuronal function, anti-inflammation, anti-DNA damage induced stress, DNA helicase activity and telomere lengthening. We find that Bazhen decoction and its drug containing serum could up-regulate multiple DNA helicases and telomere regulating proteins. The increased DNA helicases promote the resolving of G-quadruplex (G4) structures, and facilitate DNA replication and telomere elongation. These improvements also endow the cellular resistance to DNA damages induced by replication stress, and rescue the WS caused cellular senescence. CONCLUSIONS: Together these data suggest that Bazhen decoction up-regulate the expression of DNA helicases, thus facilitate G4 resolving and telomere maintenance, which rescue the progeroid cellular senescence and contribute to its anti-aging properties. Our data reveal a new molecular mechanism of Bazhen decoction in anti-aging related diseases via elongating telomere, this may shed light in the application of Bazhen decoction in multiple degenerative diseases caused by telomere erosion.
Assuntos
Síndrome de Werner , Animais , Camundongos , Síndrome de Werner/genética , Dano ao DNA , Telômero , Senescência Celular , DNA Helicases/genéticaRESUMO
Significance: Werner syndrome (WS) is a rare autosomal recessive malady typified by a pro-oxidant/proinflammatory status, genetic instability, and by the early onset of numerous age-associated illnesses. The protein malfunctioning in WS individuals (WRN) is a helicase/exonuclease implicated in transcription, DNA replication/repair, and telomere maintenance. Recent Advances: In the last two decades, a series of important biological systems were created to comprehend at the molecular level the effect of a defective WRN protein. Such biological tools include mouse and worm (Caenorhabditis elegans) with a mutation in the Wrn helicase ortholog as well as human WS-induced pluripotent stem cells that can ultimately be differentiated into most cell lineages. Such WS models have identified anomalies related to the hallmarks of aging. Most importantly, vitamin C counteracts these age-related cellular phenotypes in these systems. Critical Issues: Vitamin C is the only antioxidant agent capable of reversing the cellular aging-related phenotypes in those biological systems. Since vitamin C is a cofactor for many hydroxylases and mono- or dioxygenase, it adds another level of complexity in deciphering the exact molecular pathways affected by this vitamin. Moreover, it is still unclear whether a short- or long-term vitamin C supplementation in human WS patients who already display aging-related phenotypes will have a beneficial impact. Future Directions: The discovery of new molecular markers specific to the modified biological pathways in WS that can be used for novel imaging techniques or as blood markers will be necessary to assess the favorable effect of vitamin C supplementation in WS. Antioxid. Redox Signal. 34, 856-874.
Assuntos
Ácido Ascórbico/uso terapêutico , Helicase da Síndrome de Werner/genética , Síndrome de Werner/dietoterapia , Animais , Ácido Ascórbico/genética , Ácido Ascórbico/metabolismo , Caenorhabditis elegans/genética , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Suplementos Nutricionais , Modelos Animais de Doenças , Humanos , Camundongos , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Síndrome de Werner/patologiaRESUMO
BACKGROUND: Werner Syndrome (WS) is a rare disorder characterized by the premature onset of a number of age-related diseases. The gene responsible for WS encodes a DNA helicase/exonuclease protein believed to affect different aspects of transcription, replication, and DNA repair. Caenorhabditis elegans (C. elegans) with a nonfunctional wrn-1 DNA helicase ortholog also exhibits a shorter life span, which can be rescued by vitamin C. In this study, we analyzed the impact of a mutation in the wrn-1 gene and the dietary supplementation of vitamin C on the global mRNA expression of the whole C. elegans by the RNA-seq technology. RESULTS: Vitamin C increased the mean life span of the wrn-1(gk99) mutant and the N2 wild type strains at 25°C. However, the alteration of gene expression by vitamin C is different between wrn-1(gk99) and wild type strains. We observed alteration in the expression of 1522 genes in wrn-1(gk99) worms compared to wild type animals. Such genes significantly affected the metabolism of lipid, cellular ketone, organic acid, and carboxylic acids. Vitamin C, in return, altered the expression of genes in wrn-1(gk99) worms involved in locomotion and anatomical structure development. Proteolysis was the only biological process significantly affected by vitamin C in wild type worms. CONCLUSIONS: Expression profiling of wrn-1(gk99) worms revealed a very different response to the addition of vitamin C compared to wild type worms. Finally, vitamin C extended the life span of wrn-1(gk99) animals by altering biological processes involved mainly in locomotion and anatomical structure development.
Assuntos
Ácido Ascórbico/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Longevidade/efeitos dos fármacos , Mutação , Homologia de Sequência do Ácido Nucleico , Transcriptoma/efeitos dos fármacos , Síndrome de Werner/genética , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/fisiologia , Longevidade/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNARESUMO
The Werner syndrome protein (WRN) is a nuclear protein required for cell growth and proliferation. Loss-of-function mutations in the Werner syndrome gene are associated with the premature onset of age-related diseases. How loss of WRN limits cell proliferation and induces replicative senescence is poorly understood. Here, we show that WRN depletion leads to a striking metabolic shift that coordinately weakens the pathways that generate reducing equivalents for detoxification of reactive oxygen species and increases mitochondrial respiration. In cancer cells, this metabolic shift counteracts the Warburg effect, a defining characteristic of many malignant cells, resulting in altered redox balance and accumulation of oxidative DNA damage that inhibits cell proliferation and induces a senescence-like phenotype. Consistent with these findings, supplementation with antioxidant rescues at least in part cell proliferation and decreases senescence in WRN-knockdown cancer cells. These results demonstrate that WRN plays a critical role in cancer cell proliferation by contributing to the Warburg effect and preventing metabolic stress.
Assuntos
Regulação para Baixo/genética , Exodesoxirribonucleases/genética , Homeostase , Neoplasias/metabolismo , Neoplasias/patologia , RecQ Helicases/genética , Animais , Antioxidantes/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Dano ao DNA , Regulação para Baixo/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Exodesoxirribonucleases/metabolismo , Técnicas de Silenciamento de Genes , Glutationa/metabolismo , Glutationa/farmacologia , Homeostase/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Substâncias Macromoleculares/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Niacinamida/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , RecQ Helicases/metabolismo , Síndrome de Werner/genética , Helicase da Síndrome de WernerAssuntos
Envelhecimento , Hipotálamo/metabolismo , Neurônios/metabolismo , RecQ Helicases/metabolismo , Síndrome de Werner/genética , Animais , Apoptose , Hipotálamo/citologia , Hipotálamo/crescimento & desenvolvimento , Camundongos , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RecQ Helicases/genética , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Helicase da Síndrome de WernerRESUMO
Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-like DNA helicase. Mice lacking the helicase domain of the WRN homologue exhibit many phenotypic features of WS. Importantly, mutant Wrn(Deltahel/Deltahel) mice show abnormal increases in visceral fat deposition and fasting blood triglyceride levels followed by insulin resistance and high blood glucose levels. These mice also exhibit increased heart and liver tissue reactive oxygen species concomitantly with oxidative DNA damage, indicating a pro-oxidant status. We treated mice with either ascorbate or catechin hydrate for 9 months. Vitamin C supplementation reduced oxidative stress in liver and heart tissues and reversed hypertriglyceridemia, hyperglycemia, and insulin resistance and reduced fat weight in mutant Wrn(Deltahel/Deltahel) mice. Although the free scavenger catechin hydrate also reduced oxidative DNA damage in heart and liver tissues, it did not reverse any of the metabolic phenotype aspects in treated mutant mice. Finally, vitamin C and catechin hydrate did not affect the metabolic status of wild-type mice. These results indicate that vitamin C supplementation could be beneficial for WS patients.
Assuntos
Ácido Ascórbico/genética , Hiperglicemia/genética , Hipertrigliceridemia/genética , Resistência à Insulina/genética , RecQ Helicases/genética , Senilidade Prematura/genética , Animais , Catequina , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Sequestradores de Radicais Livres , Humanos , Camundongos , Mutação , Estresse Oxidativo/genética , Espécies Reativas de Oxigênio , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Helicase da Síndrome de WernerRESUMO
Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-like DNA helicase. Mice lacking the helicase domain of the WRN homologue exhibit many phenotypic features of WS, including a prooxidant status and a shorter mean life span compared to wild-type animals. Here, we show that Wrn mutant mice also develop premature liver sinusoidal endothelial defenestration along with inflammation and metabolic syndrome. Vitamin C supplementation rescued the shorter mean life span of Wrn mutant mice and reversed several age-related abnormalities in adipose tissues and liver endothelial defenestration, genomic integrity, and inflammatory status. At the molecular level, phosphorylation of age-related stress markers like Akt kinase-specific substrates and the transcription factor NF-kappaB, as well as protein kinase Cdelta and Hif-1alpha transcription factor levels, which are increased in the liver of Wrn mutants, were normalized by vitamin C. Vitamin C also increased the transcriptional regulator of lipid metabolism PPARalpha. Finally, microarray and gene set enrichment analyses on liver tissues revealed that vitamin C decreased genes normally up-regulated in human WS fibroblasts and cancers, and it increased genes involved in tissue injury response and adipocyte dedifferentiation in obese mice. Vitamin C did not have such effect on wild-type mice. These results indicate that vitamin C supplementation could be beneficial for patients with WS.
Assuntos
Envelhecimento/efeitos dos fármacos , Ácido Ascórbico/uso terapêutico , Síndrome de Werner/tratamento farmacológico , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/patologia , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Sequência de Bases , DNA Mitocondrial/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Glutationa/sangue , Glutationa/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Longevidade/efeitos dos fármacos , Longevidade/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microscopia Eletrônica de Varredura , Estresse Oxidativo , PPAR alfa/genética , RecQ Helicases/genética , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Síndrome de Werner/patologia , Helicase da Síndrome de WernerRESUMO
Understanding the basic biology of human ageing is a key milestone in attempting to ameliorate the deleterious consequences of old age. This is an urgent research priority given the global demographic shift towards an ageing population. Although some molecular pathways that have been proposed to contribute to ageing have been discovered using classical biochemistry and genetics, the complex, polygenic and stochastic nature of ageing is such that the process as a whole is not immediately amenable to biochemical analysis. Thus, attempts have been made to elucidate the causes of monogenic progeroid disorders that recapitulate some, if not all, features of normal ageing in the hope that this may contribute to our understanding of normal human ageing. Two canonical progeroid disorders are Werner's syndrome and Hutchinson-Gilford progeroid syndrome (also known as progeria). Because such disorders are essentially phenocopies of ageing, rather than ageing itself, advances made in understanding their pathogenesis must always be contextualised within theories proposed to help explain how the normal process operates. One such possible ageing mechanism is described by the cell senescence hypothesis of ageing. Here, we discuss this hypothesis and demonstrate that it provides a plausible explanation for many of the ageing phenotypes seen in Werner's syndrome and Hutchinson-Gilford progeriod syndrome. The recent exciting advances made in potential therapies for these two syndromes are also reviewed.
Assuntos
Senilidade Prematura/genética , Progéria/genética , Síndrome de Werner/genética , Envelhecimento , Senilidade Prematura/etiologia , Senescência Celular , Exodesoxirribonucleases , Humanos , Lamina Tipo A/genética , Progéria/etiologia , RecQ Helicases/genética , Síndrome de Werner/etiologia , Helicase da Síndrome de WernerRESUMO
As the basis for the lifelong clock and as a primary cause of aging, a process of shortening of hypothetical perichromosomal DNA structures termed chronomeres is proposed in the CNS. The lifelong clock is regulated by the shortening of chronomere DNA in postmitotic neurons of the hypothalamus. Shortening of these DNA sequences occurs in humans on a monthly basis through a lunasensory system and is controlled by release of growth hormone discharged from the anterior pituitary directly into the hypothalamus via local blood vessels. In adults, this process is under control of the pineal gland. It is further proposed that different forms of Alzheimer's disease (AD) are caused by somatic and inherited deletions of chronomeres followed by a further abnormally accelerated decrease in their activity, resulting in failures of neurotrophic and neuroendocrinal activities and in various cellular imbalances. In this model, AD is considered as a segmental progeria caused by shortening of anomalous chronomeres that are partially deleted in early development. It is proposed that a calorie-restricted diet retards chronomere shortening due to a local deficit of growth hormone in the surroundings of hypothalamic cells, thus slowing the lifelong clock and delaying aging. Calorie restriction increases lifespan by preserving mitochondrial and other organismal functions owing to the decreased chronomere shortening.
Assuntos
Envelhecimento/genética , DNA/genética , Hipotálamo/fisiopatologia , Longevidade/genética , Telômero/fisiologia , Doença de Alzheimer/genética , Animais , Relógios Biológicos , Evolução Biológica , Deleção Cromossômica , Citoesqueleto/genética , Radicais Livres/metabolismo , Gravitação , Humanos , Peroxidação de Lipídeos/fisiologia , Lua , Glândula Pineal/fisiopatologia , Hipófise/fisiopatologia , Síndrome de Werner/genéticaRESUMO
Werner syndrome (WS) is an inherited disease characterized by premature onset of aging, increased cancer incidence, and genomic instability. The WS gene encodes a protein with helicase and exonuclease activities. Our previous studies indicated that the Werner syndrome protein (WRN) interacts with Ku, a heterodimeric factor of 70- and 80-kDa subunits implicated in the repair of double strand DNA breaks. Moreover, we demonstrated that Ku70/80 strongly stimulates and alters WRN exonuclease activity. In this report, we investigate further the association between WRN and Ku70/80. First, using various WRN deletion mutants we show that 50 amino acids at the amino terminus are required and sufficient to interact with Ku70/80. In addition, our data indicate that the region of Ku80 between amino acids 215 and 276 is necessary for binding to WRN. Then, we show that the amino-terminal region of WRN from amino acid 1 to 388, which comprise the exonuclease domain, can be efficiently stimulated by Ku to degrade DNA substrates, indicating that the helicase domain and the carboxyl-terminal tail are not required for the stimulatory process. Finally, using gel shift assays, we demonstrate that Ku recruits WRN to DNA. Taken together, these results suggest that Ku-mediated activation of WRN exonuclease activity may play an important role in a cellular pathway that requires processing of DNA ends.
Assuntos
Antígenos Nucleares , Núcleo Celular/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas Nucleares/metabolismo , Síndrome de Werner/genética , Trifosfato de Adenosina/metabolismo , Aminoácidos/química , Animais , Linhagem Celular , Clonagem Molecular , DNA Helicases/química , DNA Complementar/metabolismo , Exodesoxirribonucleases , Exonucleases/metabolismo , Deleção de Genes , Glutationa Transferase/metabolismo , Humanos , Insetos , Autoantígeno Ku , Ligação Proteica , Estrutura Terciária de Proteína , RecQ Helicases , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Síndrome de Werner/metabolismo , Helicase da Síndrome de WernerRESUMO
We report here a novel cDNA clone with a predicted protein sequence similar to lysyl oxidase. This full-length cDNA clone of 3432 base pairs (WS9-14) was isolated from human fibroblasts on the basis of its overexpression in senescent cells. It encodes an 87-kDa polypeptide, whose protein is a member of the scavenger receptor cysteine-rich family, because it contains four scavenger receptor cysteine-rich domains that are found in several secreted or cell surface proteins. The WS9-14 protein has a 48% identity with both lysyl oxidase and lysyl oxidase-like protein at a region corresponding to exons 2-6, implying the existence of a lysyl oxidase gene family. The pattern of WS9-14 gene expression by fibroblasts parallels pro-collagen I-alpha1 expression. Its mRNA level is induced by transforming growth factor beta-1 and indomethacin and inhibited by phorbol ester and retinoic acid. WS9-14 is abundantly expressed in all tumor cell lines examined that attach to culture dishes but not in cell lines that grow in suspension and is also up-regulated in senescent fibroblasts. These results suggest that WS9-14 gene encodes an extracellular protein that may be specifically involved in cell adhesion and senescence.
Assuntos
Proteína-Lisina 6-Oxidase/genética , Síndrome de Werner/genética , Células 3T3 , Sequência de Aminoácidos , Animais , Northern Blotting , Adesão Celular , Senescência Celular , DNA Complementar/química , Fibroblastos/química , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta , Proteína-Lisina 6-Oxidase/química , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
The phenotype of replicative senescence is a dominant trait in human diploid fibroblasts (HDF). Therefore, we have sought to identify overexpressed and/or newly expressed causal genes by constructing and screening a subtracted cDNA library derived from polyA+RNA of prematurely senescent Werner syndrome (WS) HDF. We have identified 15 cDNA clones that are overexpressed in senescent and WS HDF. Among them are six known sequences coding for: acid sphingomyelinase, fibronectin, SPARC, nm23-metastasis suppressor protein, and two translation factors, eIF-2 beta and EF-1 alpha. Among the 10 unknown clones are: S1-5, which encodes a secreted protein containing EGF-like domains and paradoxically stimulates DNA synthesis of young HDF in an autocrine and paracrine manner, S1-3, which encodes a protein containing "zinc finger" domains, suggesting nucleic acid binding properties; S1-15, which shows sequence similarities to human alpha 2-chimerin; and S2-6, which represents a new member of the LIM family of proteins. The other five clones do not have any significant homology to known sequences. Steady-state mRNA levels of all gene sequences thus far studied are elevated in both WS and senescent normal HDF when compared to young HDF, which suggests that senescent and WS HDF enter a final common pathway where multiple gene overexpression may generate diverse antiproliferative mechanisms and pathogenic sequelae.
Assuntos
Senescência Celular/genética , Regulação da Expressão Gênica , Proteínas Monoméricas de Ligação ao GTP , Núcleosídeo-Difosfato Quinase , Síndrome de Werner/genética , Adulto , Idoso , Células Cultivadas , Criança , DNA Complementar/isolamento & purificação , Fator de Iniciação 2 em Eucariotos/genética , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Nucleosídeo NM23 Difosfato Quinases , Fator 1 de Elongação de Peptídeos , Fatores de Alongamento de Peptídeos/genética , Gravidez , Esfingomielina Fosfodiesterase/genética , Fatores de Transcrição/genéticaRESUMO
Two sister strains of skin fibroblast-like (FL) cells from a patient with Werner's syndrome (adult progeria) were grown in regular tissue culture medium or medium supplemented with the radical-scavenging enzymes superoxide dismutase and catalase. Differences in proliferative potential were shown to be due to strain variation rather than to treatment with the enzymes. Two independent strains were also grown in ambient (18%) or reduced (1%) concentrations of oxygen. All cultures (untreated, treated with enzymes, treated with reduced oxygen exposure) displayed the limited in vitro growth potential and cytogenetic abnormality characteristic of Werner's syndrome FL cells; thus the underlying defect in Werner's syndrome does not appear to be related to defective protection against or repair of damage by free radicals.