RESUMO
Histone acetylation is a prominent epigenetic modification linked to the memory loss symptoms associated with neurodegenerative disease. The use of existing histone deacetylase inhibitor (HDACi) drugs for treatment is precluded by their weak blood-brain barrier (BBB) permeability and undesirable toxicity. Here, we address these shortcomings by developing a new class of disulfide-based compounds, inspired by the scaffold of the FDA-approved HDACi romidepsin (FK288). Our findings indicate that our novel compound MJM-1 increases the overall level of histone 3 (H3) acetylation in a prostate cancer cell line. In mice, MJM-1 injected intraperitoneally (i.p.) crossed the BBB and could be detected in the hippocampus, a brain region that mediates memory. Consistent with this finding, we found that the post-training i.p. administration of MJM-1 enhanced hippocampus-dependent spatial memory consolidation in male mice. Therefore, MJM-1 represents a potential lead for further optimization as a therapeutic strategy for ameliorating cognitive deficits in aging and neurodegenerative diseases.
Assuntos
Encéfalo/metabolismo , Inibidores de Histona Desacetilases/síntese química , Memória Espacial/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Inibidores de Histona Desacetilases/farmacocinética , Inibidores de Histona Desacetilases/farmacologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: Histone deacetylases (HDACs) engage in the regulation of various cellular processes by controlling global gene expression. The dysregulation of HDACs leads to carcinogenesis, making HDACs ideal targets for cancer therapy. However, the use of HDAC inhibitors (HDACi) as single agents has been shown to have limited success in treating solid tumors in clinical studies. This study aimed to identify a novel downstream effector of HDACs to provide a potential target for combination therapy. METHODS: Transcriptome sequencing and bioinformatics analysis were performed to screen for genes responsive to HDACi in breast cancer cells. The effects of HDACi on cell viability were detected using the MTT assay. The mRNA and protein levels of genes were determined by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting. Cell cycle distribution and apoptosis were analyzed by flow cytometry. The binding of CREB1 (cAMP-response element binding protein 1) to the promoter of the KDELR (The KDEL (Lys-Asp-Glu-Leu) receptor) gene was validated by the ChIP (chromatin immunoprecipitation assay). The association between KDELR2 and protein of centriole 5 (POC5) was detected by immunoprecipitation. A breast cancer-bearing mouse model was employed to analyze the effect of the HDAC3-KDELR2 axis on tumor growth. RESULTS: KDELR2 was identified as a novel target of HDAC3, and its aberrant expression indicated the poor prognosis of breast cancer patients. We found a strong correlation between the protein expression patterns of HADC3 and KDELR2 in tumor tissues from breast cancer patients. The results of the ChIP assay and qRT-PCR analysis validated that HDAC3 transactivated KDELR2 via CREB1. The HDAC3-KDELR2 axis accelerated the cell cycle progression of cancer cells by protecting the centrosomal protein POC5 from proteasomal degradation. Moreover, the HDAC3-KDELR2 axis promoted breast cancer cell proliferation and tumorigenesis in vitro and in vivo. CONCLUSION: Our results uncovered a previously unappreciated function of KDELR2 in tumorigenesis, linking a critical Golgi-the endoplasmic reticulum traffic transport protein to HDAC-controlled cell cycle progression on the path of cancer development and thus revealing a potential therapeutical target for breast cancer.
Assuntos
Neoplasias da Mama , Animais , Neoplasias da Mama/genética , Proteínas de Transporte , Ciclo Celular/genética , Proliferação de Células/genética , Feminino , Inibidores de Histona Desacetilases , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Camundongos , Proteínas de Transporte Vesicular/metabolismoRESUMO
Metformin, an anti-diabetes drug, has been recently emerging as a potential "anti-aging" intervention based on its reported beneficial actions against aging in preclinical studies. Nonetheless, very few metformin studies using mice have determined metformin concentrations and many effects of metformin have been observed in preclinical studies using doses/concentrations that were not relevant to therapeutic levels in human. We developed a liquid chromatography-tandem mass spectrometry protocol for metformin measurement in plasma, liver, brain, kidney, and muscle of mice. Young adult male and female C57BL/6 mice were voluntarily treated with metformin of 4 mg/ml in drinking water which translated to the maximum dose of 2.5 g/day in humans. A clinically relevant steady-state plasma metformin concentrations were achieved at 7 and 30 days after treatment in male and female mice. Metformin concentrations were slightly higher in muscle than in plasma, while, ~3 and 6-fold higher in the liver and kidney than in plasma, respectively. Low metformin concentration was found in the brain at ~20% of the plasma level. Furthermore, gender difference in steady-state metformin bio-distribution was observed. Our study established steady-state metformin levels in plasma, liver, muscle, kidney, and brain of normoglycemic mice treated with a clinically relevant dose, providing insight into future metformin preclinical studies for potential clinical translation.
Assuntos
Metformina/farmacocinética , Animais , Encéfalo/metabolismo , Cromatografia Líquida , Feminino , Rim/metabolismo , Fígado/metabolismo , Masculino , Metformina/administração & dosagem , Metformina/sangue , Camundongos , Camundongos Endogâmicos C57BL , Músculos/metabolismo , Espectrometria de Massas em Tandem , Distribuição TecidualRESUMO
The increasing threat posed by multiresistant bacterial pathogens necessitates the discovery of novel antibacterials with unprecedented modes of action. ADEP1, a natural compound produced by Streptomyces hawaiiensis NRRL 15010, is the prototype for a new class of acyldepsipeptide (ADEP) antibiotics. ADEP antibiotics deregulate the proteolytic core ClpP of the bacterial caseinolytic protease, thereby exhibiting potent antibacterial activity against Gram-positive bacteria, including multiresistant pathogens. ADEP1 and derivatives, here collectively called ADEP, have been previously investigated for their antibiotic potency against different species, structure-activity relationship, and mechanism of action; however, knowledge on the biosynthesis of the natural compound and producer self-resistance have remained elusive. In this study, we identified and analyzed the ADEP biosynthetic gene cluster in S. hawaiiensis NRRL 15010, which comprises two NRPSs, genes necessary for the biosynthesis of (4S,2R)-4-methylproline, and a type II polyketide synthase (PKS) for the assembly of highly reduced polyenes. While no resistance factor could be identified within the gene cluster itself, we discovered an additional clpP homologous gene (named clpPADEP) located further downstream of the biosynthetic genes, separated from the biosynthetic gene cluster by several transposable elements. Heterologous expression of ClpPADEP in three ADEP-sensitive Streptomyces species proved its role in conferring ADEP resistance, thereby revealing a novel type of antibiotic resistance determinant.IMPORTANCE Antibiotic acyldepsipeptides (ADEPs) represent a promising new class of potent antibiotics and, at the same time, are valuable tools to study the molecular functioning of their target, ClpP, the proteolytic core of the bacterial caseinolytic protease. Here, we present a straightforward purification procedure for ADEP1 that yields substantial amounts of the pure compound in a time- and cost-efficient manner, which is a prerequisite to conveniently study the antimicrobial effects of ADEP and the operating mode of bacterial ClpP machineries in diverse bacteria. Identification and characterization of the ADEP biosynthetic gene cluster in Streptomyces hawaiiensis NRRL 15010 enables future bioinformatics screenings for similar gene clusters and/or subclusters to find novel natural compounds with specific substructures. Most strikingly, we identified a cluster-associated clpP homolog (named clpPADEP) as an ADEP resistance gene. ClpPADEP constitutes a novel bacterial resistance factor that alone is necessary and sufficient to confer high-level ADEP resistance to Streptomyces across species.
Assuntos
Antibacterianos/biossíntese , Depsipeptídeos/biossíntese , Depsipeptídeos/genética , Resistência Microbiana a Medicamentos/genética , Família Multigênica , Streptomyces/genética , Streptomyces/metabolismo , Antibacterianos/farmacologia , Vias Biossintéticas/genética , Clonagem Molecular , Elementos de DNA Transponíveis , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Peptídeo Sintases/genética , Policetídeo Sintases/genética , Streptomyces/enzimologia , Relação Estrutura-AtividadeRESUMO
Pulmonary carcinoids are a type of neuroendocrine tumor (NET) accounting for 1-2% of lung cancer cases. Currently, Positron Emission Tomography (PET)/CT based on the radiolabeled sugar analogue [18F]-FDG is used to diagnose and stage pulmonary carcinoids, but is suboptimal due to low metabolic activity in these tumors. A new technique for pulmonary carcinoid imaging, using PET/CT with radiolabeled somatostatin analogs that specifically target somatostatin receptor subtype 2 (SSTR2), is becoming more standard, as many tumors overexpress SSTR2. However, pulmonary carcinoid patients with diminished SSTR2 expression are not eligible for this imaging or any type of SSTR2-specific treatment. We have found that histone deacetylase (HDAC) inhibitors can upregulate the expression of SSTR2 in pulmonary carcinoid cell lines. In this study, we used a non-cytotoxic dose of HDAC inhibitors to induce pulmonary carcinoid SSTR2 expression in which we confirmed in vitro and in vivo. A non-cytotoxic dose of the HDAC inhibitors: thailandepsin A (TDP-A), romidepsin (FK228), suberoylanilide hydroxamic acid (SAHA), AB3, and valproic acid (VPA) were administered to promote SSTR2 expression in pulmonary carcinoid cell lines and xenografts. This SSTR2 upregulation technique using HDAC inhibitors could enhance radiolabeled somatostatin analog-based imaging and the development of potential targeted treatments for pulmonary carcinoid patients with marginal or diminished SSTR2 expression.