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1.
Sci Rep ; 14(1): 7628, 2024 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-38561454

RESUMO

Colorectal cancer is the third most commonly diagnosed cancer and the second leading cause of cancer-related death, thus a novel chemotherapeutic agent for colon cancer therapy is needed. In this study, analogues of echinomycin, a cyclic peptide natural product with potent toxicity to several human cancer cell lines, were synthesized, and their biological activities against human colon cancer cells were investigated. Analogue 3 as well as 1 inhibit HIF-1α-mediated transcription. Notably, transcriptome analysis indicated that the cell cycle and its regulation were involved in the effects on cells treated with 3. Analogue 3 exhibited superior in vivo efficacy to echinomycin without significant toxicity in mouse xenograft model. The low dose of 3 needed to be efficacious in vivo is also noteworthy and our data suggest that 3 is an attractive and potentially novel agent for the treatment of colon cancer.


Assuntos
Neoplasias do Colo , Equinomicina , Humanos , Animais , Camundongos , Equinomicina/farmacologia , Neoplasias do Colo/tratamento farmacológico , Subunidade alfa do Fator 1 Induzível por Hipóxia
2.
Molecules ; 28(17)2023 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-37687129

RESUMO

One of the largest concerns with world health today is still antibiotic resistance, which is making it imperative to find efficient alternatives as soon as possible. It has been demonstrated that microbes are reliable sources for the creation of therapeutic antibiotics. This research intends to investigate the endophytic microorganisms from several medicinal plants in Fenghuang Mountain (Jiangsu Province, China) and to discover new antibiotics from their secondary metabolites. A total of 269 endophytic strains were isolated from nine distinct medicinal plants. Taxonomic analysis revealed that there were 20 distinct species among these endophytes, with Streptomyces being the most common genus. Three of the target strains were chosen for scale-up fermentation after preliminary screening of antibacterial activities and the metabolomics investigation using LC-MS. These strains were Penicillium sp. NX-S-6, Streptomyces sp. YHLB-L-2 and Streptomyces sp. ZLBB-S-6. Twenty-three secondary metabolites (1-23), including a new sorbicillin analogue (1), were produced as a result of antibacterial activity-guided isolation. Through spectroscopic analysis using MS and NMR, the structures of yield compounds were clarified. According to antibacterial data, S. aureus or B. subtilis were inhibited to varying degrees by sorrentanone (3), emodic acid (8), GKK1032 B (10), linoleic acid (14), toyocamycin (17) and quinomycin A (21). The most effective antimicrobial agent against S. aureus, B. subtilis, E. coli and A. baumannii was quinomycin A (21). In addition, quinomycin A showed strong antifungal activity against Aspergillus fumigatus, Cryptococcus neoformans, and two clinical isolated strains Aspergillus fumigatus #176 and #339, with MIC as 16, 4, 16 and 16 µg/mL, respectively. This is the first time that bioprospecting of actinobacteria and their secondary metabolites from medicinal plants in Fenghuang Mountain was reported. The finding demonstrates the potential of endophytic microbes in medical plants to produce a variety of natural products. Endophytic microbes will be an important source for new antibiotics.


Assuntos
Equinomicina , Plantas Medicinais , Antibacterianos/farmacologia , Escherichia coli , Staphylococcus aureus
3.
Neuroscience ; 526: 204-222, 2023 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-37385335

RESUMO

Current data suggest a hypothesis of vascular pathogenesis for the development and progression of Alzheimer's disease (AD). To investigate this, we studied the association of apolipoprotein E4 (APOE4) gene on microvessels in human autopsy-confirmed AD with and without APOE4, compared with age/sex-matched control (AC) hippocampal CA1 stratum radiatum. AD arterioles (without APOE4 gene) had mild oxidative stress and loss of vascular endothelial growth factor (VEGF) and endothelial cell density, reflecting aging progression. In AD + APOE4, an increase in strong oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG), VEGF, and endothelial cell density were associated with increased diameter of arterioles and perivascular space dilation. In cultured human brain microvascular cells (HBMECs), treatment of ApoE4 protein plus amyloid-ß (Aß) oligomers increased superoxide production and the apoptotic marker cleaved caspase 3, sustained hypoxia inducible factor-1α (HIF-1α) stability that was associated with an increase in MnSOD, VEGF, and cell density. This cell over-proliferation was inhibited with the antioxidants N-acetyl cysteine and MnTMPyP, the HIF-1α inhibitor echinomycin, the VEGFR-2 receptor blocker SU1498, the protein kinase C (PKC) ε knock-down (KD) and the extracellular signal-regulated kinase 1/2 (ERK) inhibitor FR180204. The PKCε KD and echinomycin decreased VEGF and/or ERK. In conclusion, AD capillaries and arterioles in hippocampal CA1 stratum radiatum of non-APOE4 carriers are related with aging, while those in APOE4 carriers with AD are related with pathogenesis of cerebrovascular disease.


Assuntos
Doença de Alzheimer , Equinomicina , Humanos , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Apolipoproteínas E , Região CA1 Hipocampal/patologia , Equinomicina/metabolismo , Hipocampo/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
4.
Sci Rep ; 13(1): 7980, 2023 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-37198233

RESUMO

Echinomycin is a natural product DNA bisintercalator antibiotic. The echinomycin biosynthetic gene cluster in Streptomyces lasalocidi includes a gene encoding the self-resistance protein Ecm16. Here, we present the 2.0 Å resolution crystal structure of Ecm16 bound to adenosine diphosphate. The structure of Ecm16 closely resembles that of UvrA, the DNA damage sensor component of the prokaryotic nucleotide excision repair system, but Ecm16 lacks the UvrB-binding domain and its associated zinc-binding module found in UvrA. Mutagenesis study revealed that the insertion domain of Ecm16 is required for DNA binding. Furthermore, the specific amino acid sequence of the insertion domain allows Ecm16 to distinguish echinomycin-bound DNA from normal DNA and link substrate binding to ATP hydrolysis activity. Expression of ecm16 in the heterologous host Brevibacillus choshinensis conferred resistance against echinomycin and other quinomycin antibiotics, including thiocoraline, quinaldopeptin, and sandramycin. Our study provides new insight into how the producers of DNA bisintercalator antibiotics fend off the toxic compounds that they produce.


Assuntos
Equinomicina , Streptomyces , Equinomicina/farmacologia , Adenosina Trifosfatases/metabolismo , DNA/metabolismo , Antibacterianos/química , Streptomyces/genética , Streptomyces/metabolismo
5.
Reproduction ; 166(1): 55-64, 2023 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-37184053

RESUMO

In brief: During pregnancy, uterine kept quiescence along with uterine overdistention before labor. Prolonged stretching induced uterus myometrial hypoxia, increased TREK1 expression, and relaxed the myometrium, which may contribute to uterine quiescence and atony during pregnancy. Abstract: The mechanisms underlying pre-labor uterine quiescence and uterine atony during overdistention are unclear. TREK1 (a two-pore domain potassium channel) and hypoxia-inducible factor-1α (HIF-1α) are activated by mechanical stretch, and their expression is upregulated by decreased uterine contractility. HIF-1α is a nuclear factor which regulates numerous target proteins, but whether it regulates TREK1 during the uterine stretch to cause uterine quiescence and/or atony is unclear. We investigated uterine contractility at different gestational stages in rats, as well as in non-pregnant uteri, which were induced by prolonged stretching and hypoxia. We also assessed the effects of incubating the uteri with or without echinomycin or l-methionine. Moreover, we analyzed HIF-1α and TREK1 expression levels in each group, as well as at various gestational stages of pregnant human uteri. We found that contractility was significantly decreased in pregnant uteri when compared with non-pregnant uteri, and this decrease was associated with increases in HIF-1α and TREK1 expression levels. HIF-1α and TREK1 expression levels in human uteri increased with the gestational length. Decreased uterine contractility and increased HIF-1α and TREK1 expression levels were also observed in non-pregnant rat uteri under 8 g of stretching tension or hypoxia. Inhibition of hypoxia with echinomycin restored normal uterine contractility, while HIF-1α and TREK1 protein expression remained reduced. TREK1 inhibition with l-methionine also restored uterine contractility under tension or hypoxia. In conclusion, we demonstrated that prolonged stretching induces myometrial hypoxia, increases TREK1 expression, and relaxes the myometrium, which may contribute to uterine quiescence and atony.


Assuntos
Equinomicina , Trabalho de Parto , Canais de Potássio de Domínios Poros em Tandem , Animais , Feminino , Humanos , Gravidez , Ratos , Equinomicina/farmacologia , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Trabalho de Parto/fisiologia , Miométrio/fisiologia , Útero , Canais de Potássio de Domínios Poros em Tandem/fisiologia
6.
J Hematol Oncol ; 16(1): 48, 2023 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-37143124

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of several human cancers, including Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), which preferentially arise in immunocompromised patients while lack of effective therapeutic options. Oncoproteins Myc and hypoxia-inducible factor-1α (HIF1α) have been found closely related to KSHV infection, replication and oncogenesis. However, the strategies of dual targeting these two oncoproteins have never been developed and tested for treatments of KSHV-related malignancies. In the current study, we report that treatment of echinomycin dramatically regresses cell growth both in vitro-cultured KSHV + tumor cells and in vivo KS or PEL xenograft mice models, through simultaneously inhibiting Myc and HIF1α expression. Echinomycin treatment also induces viral lytic gene expression whereas not increasing infectious virions production from KSHV + tumor cells. Our comparative transcriptomic analysis has identified a bunch of new Echinomycin-regulated, Myc- and HIF1α-related genes contributed to KSHV pathogenesis, including KDM4B and Tau, which are required for the survival of KSHV + tumor cells with functional validation. These data together reveal that dual targeting Myc and HIF1α such as using Echinomycin may represent a new and promising option for treatments of these virus-associated malignancies.


Assuntos
Equinomicina , Herpesvirus Humano 8 , Sarcoma de Kaposi , Humanos , Animais , Camundongos , Herpesvirus Humano 8/genética , Equinomicina/farmacologia , Equinomicina/uso terapêutico , Latência Viral/genética , Sarcoma de Kaposi/tratamento farmacológico , Sarcoma de Kaposi/metabolismo , Ciclo Celular , Histona Desmetilases com o Domínio Jumonji
7.
Front Endocrinol (Lausanne) ; 14: 1115619, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36909311

RESUMO

Background: The incidence of gestational diabetes mellitus (GDM) is increasing worldwide. GDM patients have a significantly higher rate of cesarean section and postpartum hemorrhage, suggesting changes in uterine contractility. TWIK-1-related potassium channel (TREK1) expressed in the pregnant uterus and its role in uterine contraction. In this study, we examined the expression of HIF-1α and TREK1 proteins in GDM uterine and investigated whether high glucose levels are involved in the regulation of human uterine smooth muscle cells (HUSMCs) contraction through TREK1, and verified the role of HIF-1α in this process. Methods: Compared the uterine contractility between GDM and normal patients undergoing elective lower segment cesarean section. The HUSMCs were divided into normal glucose group, high glucose group, normal glucose with CoCl2 group, CoCl2 with echinomycin/L-Methionine group, and high glucose with echinomycin/L-Methionine group; Compare the cell contractility of each group. Compared the expression of hypoxia-inducible factor-1α (HIF-1α) and TREK1 protein in each group. Results: The contractility of human uterine strips induced by both KCl and oxytocin was significantly lower in patients with GDM compared with that in normal individuals, with increased TREK1 and HIF-1α protein expression. The contractility of cultured HUSMCs was significantly decreased under high glucose levels, which was consistent with increased expression of HIF-1α and TREK1 proteins. The contractility of HUSMCs was decreased when hypoxia was induced by CoCl2 and increased when hypoxia was inhibited by echinomycin. The TREK1 inhibitor L-methionine also recovered the decreased contractility of HUSMCs under high glucose levels or hypoxia. Discussion: The high glucose levels decreased the contractility of the myometrium, and increased expression of HIF-1a and TREK1 proteins play a role in changes in uterus contractility.


Assuntos
Equinomicina , Miométrio , Feminino , Humanos , Gravidez , Cesárea , Cobalto/metabolismo , Equinomicina/metabolismo , Glucose/metabolismo , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Metionina/metabolismo
8.
Mar Drugs ; 21(3)2023 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-36976192

RESUMO

Mangrove actinomycetia have been proven to be one of the promising sources for discovering novel bioactive natural products. Quinomycins K (1) and L (2), two rare quinomycin-type octadepsipeptides without intra-peptide disulfide or thioacetal bridges, were investigated from the Maowei Sea mangrove-derived Streptomyces sp. B475. Their chemical structures, including the absolute configurations of their amino acids, were elucidated by a combination of NMR and tandem MS analysis, electronic circular dichroism (ECD) calculation, advanced Marfey's method, and further unequivocally confirmed by the first total synthesis. The two compounds displayed no potent antibacterial activity against 37 bacterial pathogens and had no significant cytotoxic activity against H460 lung cancer cells.


Assuntos
Equinomicina , Streptomyces , Streptomyces/metabolismo , Equinomicina/metabolismo , Antibacterianos/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular
9.
Nucleic Acids Res ; 51(8): 3540-3555, 2023 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-36919604

RESUMO

Combination cancer chemotherapy is one of the most useful treatment methods to achieve a synergistic effect and reduce the toxicity of dosing with a single drug. Here, we use a combination of two well-established anticancer DNA intercalators, actinomycin D (ActD) and echinomycin (Echi), to screen their binding capabilities with DNA duplexes containing different mismatches embedded within Watson-Crick base-pairs. We have found that combining ActD and Echi preferentially stabilised thymine-related T:T mismatches. The enhanced stability of the DNA duplex-drug complexes is mainly due to the cooperative binding of the two drugs to the mismatch duplex, with many stacking interactions between the two different drug molecules. Since the repair of thymine-related mismatches is less efficient in mismatch repair (MMR)-deficient cancer cells, we have also demonstrated that the combination of ActD and Echi exhibits enhanced synergistic effects against MMR-deficient HCT116 cells and synergy is maintained in a MMR-related MLH1 gene knockdown in SW620 cells. We further accessed the clinical potential of the two-drug combination approach with a xenograft mouse model of a colorectal MMR-deficient cancer, which has resulted in a significant synergistic anti-tumour effect. The current study provides a novel approach for the development of combination chemotherapy for the treatment of cancers related to DNA-mismatches.


Assuntos
Neoplasias Colorretais , Equinomicina , Humanos , Animais , Camundongos , Dactinomicina/química , Equinomicina/química , Timina , Sequência de Bases , Sítios de Ligação , Conformação de Ácido Nucleico , DNA/química
10.
Microb Cell Fact ; 22(1): 5, 2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36609255

RESUMO

BACKGROUND: New antibiotics are urgently needed in clinical treatment of superdrug-resistant bacteria. Nonribosomal peptides (NRPs) are a major source of antibiotics because they exhibit structural diversity, and unique antibacterial mechanisms and resistance. Analysis of gene clusters of S. agglomeratus 5-1-3 showed that Clusters 3, 6, 12, 21, and 28 were used to synthesize NRPs. Here, we examined secondary metabolites of S. agglomeratus 5-1-3 isolated from soils in the Qinghai-Tibet Plateau, China, for NRPs with antibacterial activity. RESULTS: We isolated a total of 36 Streptomyces strains with distinct colony morphological characteristics from 7 soil samples. We screened 8 Streptomyces strains resistant to methicillin-resistant Staphylococcus aureus (MRSA). We then selected S. agglomeratus 5-1-3 for further study based on results of an antibacterial activity test. Here, we isolated three compounds from S. agglomeratus 5-1-3 and characterized their properties. The crude extract was extracted with ethyl acetate and purified with column chromatography and semipreparative high-performance liquid chromatography (HPLC). We characterized the three compounds using NMR analyses as echinomycin (1), 5,7,4'-trihydroxy-3.3',5'-trimethoxy flavone (2), and 2,6,2', 6'-tetramethoxy-4,4-bis(2,3-epoxy-1-hydroxypropyl)-biphenyl (3). We tested the antibacterial activity of pure compounds from strain 5-1-3 with the Oxford cup method. NRP echinomycin (1) showed excellent anti-MRSA activity with a minimum inhibitory concentration (MIC) of 2.0 µg/mL. Meanwhile, MIC of compound 2 and 3 was 128.0 µg/mL for both. In addition, 203 mg of echinomycin was isolated from 10 L of the crude extract broth of strain 5-1-3. CONCLUSION: In this study, S. agglomeratus 5-1-3 with strong resistance to MRSA was isolated from the soils in the Qinghai-Tibet Plateau. Strain 5-1-3 had a high yield of echinomycin (1) an NRP with a MIC of 2 µg/mL against MRSA. We propose that echinomycin derived from S. agglomeratus 5-1-3 may be a potent antibacterial agent for pharmaceutical use.


Assuntos
Equinomicina , Staphylococcus aureus Resistente à Meticilina , Streptomyces , Tibet , Antibacterianos/química , Streptomyces/química , Testes de Sensibilidade Microbiana , Misturas Complexas , Solo
11.
BMB Rep ; 56(2): 78-83, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36195570

RESUMO

Chronic myeloid leukemia (CML) has a markedly improved prognosis with the use of breakpoint cluster region-abelson 1 (BCR-ABL1) tyrosine kinase inhibitors (BCR-ABL1 TKIs). However, approximately 40% of patients are resistant or intolerant to BCR-ABL1 TKIs. Hypoxia-inducible factor 1α (HIF-1α) is a hypoxia response factor that has been reported to be highly expressed in CML patients, making it a therapeutic target for BCR-ABL1 TKI-sensitive CML and BCR-ABL1 TKI-resistant CML. In this study, we examined whether HIF-1α inhibitors induce cell death in CML cells and BCR-ABL1 TKI-resistant CML cells. We found that echinomycin and PX-478 induced cell death in BCR-ABL1 TKIs sensitive and resistant CML cells at similar concentrations while the cell sensitivity was not affected with imatinib or dasatinib in BCR-ABL1 TKIs resistant CML cells. In addition, echinomycin and PX-478 inhibited the c-Jun N-terminal kinase (JNK), Akt, and extracellular-regulated protein kinase 1/2 (ERK1/2) activation via suppression of BCR-ABL1 and Met expression in BCR-ABL1 sensitive and resistant CML cells. Moreover, treatment with HIF-1α siRNA induced cell death by inhibiting BCR-ABL1 and Met expression and activation of JNK, Akt, and ERK1/2 in BCR-ABL1 TKIs sensitive and resistant CML cells. These results indicated that HIF-1α regulates BCR-ABL and Met expression and is involved in cell survival in CML cells, suggesting that HIF-1α inhibitors induce cell death in BCR-ABL1 TKIs sensitive and resistant CML cells and therefore HIF-1α inhibitors are potential candidates for CML treatment. [BMB Reports 2023; 56(2): 78-83].


Assuntos
Equinomicina , Leucemia Mielogênica Crônica BCR-ABL Positiva , Humanos , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Equinomicina/uso terapêutico , Proteínas Proto-Oncogênicas c-akt , Resistencia a Medicamentos Antineoplásicos , Apoptose , Inibidores de Proteínas Quinases/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Morte Celular
12.
Front Immunol ; 13: 1095427, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36569834

RESUMO

Objective: Sepsis Associated Encephalopathy (SAE) is a common complication in critically ill patients and perioperative period, but its pathogenesis is still unclear. This study aimed to explore the effect of the HIF-1α (hypoxia-inducible factor-1α)/BNIP3L (Bcl-2/adenovirus E1B 19-kDa interaction protein) signaling pathway on SAE. Methods: C57BL/6J male mice were divided into four groups, using a random number table method: control group, sham group, sepsis group, sepsis+HIF-1α activity inhibitor (echinomycin) group. Sepsis was induced by cecal ligation and puncture (CLP). At 24 h after surgery, brain tissue was sampled. HE was staining to observe changes in the hippocampus structure. Fluoroscopy observes changes in mitochondrial structure. Western blot, QT-PCR, and immunofluorescence were used to assess the amount of expression of HIF-1α and BNIP3L in the hippocampus and mitochondrion of hippocampus neurons. Observation of neuronal apoptosis by TUNEL staining. Seven days after surgery, mice were tested in a Morris water maze test to assess cognitive function after CLP. Results: Our results show that CLP-induced hippocampus-dependent cognitive deficits were accompanied with increased HIF 1a and decreased BNIP3L, increased protein levels of TNF-α, IL-6, and IL-ß, and damage to mitochondrial structures and neuronal apoptosis in the hippocampus. In addition, administration of echinomycin rescues cognitive deficits, ameliorates HIF-1α and BNIP3L-mediated neuronal pyroptosis and damaged mitochondrial structures, and decreases the expression of TNF-α and IL-6 in the hippocampus. Conclusions: HIF-1α and the BNIP3L promote mitochondrial damage, and neuronal apoptosis and the expression of inflammatory factors may be the mechanism of SAE in critically ill patients and perioperative period.


Assuntos
Equinomicina , Encefalopatia Associada a Sepse , Sepse , Camundongos , Masculino , Animais , Fator de Necrose Tumoral alfa , Estado Terminal , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Interleucina-6 , Camundongos Endogâmicos C57BL , Sepse/complicações , Cognição
13.
Curr Microbiol ; 79(12): 372, 2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36269434

RESUMO

The main objective of this investigation was to characterize a collection of actinomycetes strains isolated from unexplored polluted ecosystems and to evaluate their antimicrobial potential in order to discover interesting bioactive compounds. Based on morphological and culture characters, 32 different strains were isolated: 20 strains from compost heap, seven strains from manure, and five strains from waste water. As expected, the genus Streptomyces was the most prevalent followed by the genus Micromonospora. Analysis of the antimicrobial activities of the isolated strains showed that those from compost heap were more efficient against the tested microorganisms (Candida albicans, Methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli). Several bioactive compounds were identified by liquid chromatography (LC) combined with mass spectrometry (MS) and then analyzed by both MEDINA's database, which contains the most common secondary metabolites, and Dictionary of Natural Products Chapman & Hall. Many interesting well-known and unknown biomolecules were identified. Quinomycin A and Daidzein were the most fascinating compounds isolated, respectively, by Streptomyces sp. WW2 and Streptomyces sp. WW4. The most active strain was identified based on 16S rDNA's sequences and it seems to be a new strain. The crude extract of the strain CH12 was analyzed and the UV absorption spectra and mass spectra (MS) of the main active compound were reported. It's an interesting compound (possible purpuromycin) with the molecular formula C26H18O13.


Assuntos
Actinobacteria , Anti-Infecciosos , Produtos Biológicos , Equinomicina , Staphylococcus aureus Resistente à Meticilina , Streptomyces , Actinobacteria/genética , Actinomyces , Ecossistema , Equinomicina/metabolismo , Esterco , Águas Residuárias , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Antibacterianos/química , Streptomyces/genética , Anti-Infecciosos/farmacologia , Anti-Infecciosos/metabolismo , Produtos Biológicos/metabolismo , DNA Ribossômico , Testes de Sensibilidade Microbiana
14.
Nan Fang Yi Ke Da Xue Xue Bao ; 42(6): 929-936, 2022 Jun 20.
Artigo em Chinês | MEDLINE | ID: mdl-35790445

RESUMO

OBJECTIVE: To investigate effects of physiological hypoxic conditions on suspension and adherence of embryoid bodies (EBs) during differentiation of human induced pluripotent stem cells (hiPSCs) and explore the underlying mechanisms. METHODS: EBs in suspension culture were divided into normoxic (21% O2) and hypoxic (5% O2) groups, and those in adherent culture were divided into normoxic, hypoxic and hypoxia + HIF-1α inhibitor (echinomycin) groups. After characterization of the pluripotency with immunofluorescence assay, the hiPSCs were digested and suspended under normoxic and hypoxic conditions for 5 days, and the formation and morphological changes of the EBs were observed microscopically; the expressions of the markers genes of the 3 germ layers in the EBs were detected. The EBs were then inoculated into petri dishes for further culture in normoxic and hypoxic conditions for another 2 days, after which the adhesion and peripheral expansion rate of the adherent EBs were observed; the changes in the expressions of HIF-1α, ß-catenin and VEGFA were detected in response to hypoxic culture and echinomycin treatment. RESULTS: The EBs cultured in normoxic and hypoxic conditions were all capable of differentiation into the 3 germ layers. The EBs cultured in hypoxic conditions showed reduced apoptotic debris around them with earlier appearance of cystic EBs and more uniform sizes as compared with those in normoxic culture. Hypoxic culture induced more adherent EBs than normoxic culture (P < 0.05) with also a greater outgrowth rate of the adherent EBs (P < 0.05). The EBs in hypoxic culture showed significantly up-regulated mRNA expressions of ß-catenin and VEGFA (P < 0.05) and protein expressions of HIF-1 α, ß-catenin and VEGFA (P < 0.05), and their protein expresisons levels were significantly lowered after treatment with echinomycin (P < 0.05). CONCLUSION: Hypoxia can promote the formation and maturation of suspended EBs and enhance their adherence and post-adherent proliferation without affecting their pluripotency for differentiation into all the 3 germ layers. Our results provide preliminary evidence that activation of HIF-1α/ß-catenin/VEGFA signaling pathway can enhance the differentiation potential of hiPSCs.


Assuntos
Equinomicina , Células-Tronco Pluripotentes Induzidas , Equinomicina/metabolismo , Corpos Embrioides/metabolismo , Humanos , Hipóxia/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , beta Catenina/metabolismo
15.
Proc Natl Acad Sci U S A ; 119(30): e2200681119, 2022 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-35857870

RESUMO

The majority of base pairs in double-stranded DNA exist in the canonical Watson-Crick geometry. However, they can also adopt alternate Hoogsteen conformations in various complexes of DNA with proteins and small molecules, which are key for biological function and mechanism. While detection of Hoogsteen base pairs in large DNA complexes and assemblies poses considerable challenges for traditional structural biology techniques, we show here that multidimensional dynamic nuclear polarization-enhanced solid-state NMR can serve as a unique spectroscopic tool for observing and distinguishing Watson-Crick and Hoogsteen base pairs in a broad range of DNA systems based on characteristic NMR chemical shifts and internuclear dipolar couplings. We illustrate this approach using a model 12-mer DNA duplex, free and in complex with the antibiotic echinomycin, which features two central adenine-thymine base pairs with Watson-Crick and Hoogsteen geometry, respectively, and subsequently extend it to the ∼200 kDa Widom 601 DNA nucleosome core particle.


Assuntos
Pareamento de Bases , DNA , Espectroscopia de Ressonância Magnética , Adenina/química , Adenina/metabolismo , DNA/química , Equinomicina/química , Espectroscopia de Ressonância Magnética/métodos , Timina/química
16.
Mol Microbiol ; 117(6): 1434-1446, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35534931

RESUMO

Bacteria use various strategies to become antibiotic resistant. The molecular details of these strategies are not fully understood. We can increase our understanding by investigating the same strategies found in antibiotic-producing bacteria. In this work, we characterize the self-resistance protein Ecm16 encoded by echinomycin-producing bacteria. Ecm16 is a structural homolog of the nucleotide excision repair protein UvrA. Expression of ecm16 in the heterologous system Escherichia coli was sufficient to render resistance against echinomycin. Ecm16 binds DNA (double-stranded and single-stranded) using a nucleotide-independent binding mode. Ecm16's binding affinity for DNA increased by 1.7-fold when the DNA is intercalated with echinomycin. Ecm16 can render resistance against echinomycin toxicity independently of the nucleotide excision repair system. Similar to UvrA, Ecm16 has ATPase activity, and this activity is essential for Ecm16's ability to render echinomycin resistance. Notably, UvrA and Ecm16 were unable to complement each other's function. Together, our findings identify new mechanistic details of how a refurbished DNA repair protein Ecm16 can specifically render resistance to the DNA intercalator echinomycin.


Assuntos
Equinomicina , Proteínas de Escherichia coli , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacologia , DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Equinomicina/química , Equinomicina/metabolismo , Equinomicina/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo
17.
F S Sci ; 3(2): 187-196, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35560016

RESUMO

OBJECTIVE: To investigate the antitumor effects of the selective hypoxia-inducible factor-1 (HIF-1) inhibitors echinomycin and PX-478 on uterine fibroids. DESIGN: Experimental study using in vitro primary culture systems and an in vivo mouse xenograft model. SETTING: Academic university center. PATIENT(S): Women with uterine fibroids who underwent hysterectomy or myomectomy. INTERVENTION(S): Administration of the selective HIF-1 inhibitors echinomycin and PX-478 to the media of the primary cultured uterine fibroid cells and to nonobese diabetic/severe combined immunodeficient mice bearing fibroid xenografts consisting of the primary cultured fibroid cells and type Ⅰ collagen gels beneath the kidney capsule. MAIN OUTCOME MEASURE(S): Cell proliferation was measured by Cell Counting Kit-8 assay. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and by measuring caspase 3 and 7 activities. The xenografts were evaluated by gross appearance, surface area, and histology. The Ki-67 index was measured to evaluate proliferation of the xenografts. RESULT(S): Both echinomycin and PX-478 inhibited cell proliferation and induced apoptosis in fibroid cells cultured under hypoxia and normoxia. Enlargement of the fibroid xenografts was significantly attenuated. The Ki-67 index significantly decreased after the administration of the HIF-1 inhibitors in the xenograft model. Eight of 27 xenografts treated with the HIF-1 inhibitors contained calcification and hyalinizing components from 3 days after the grafting to 2 weeks, suggesting that the HIF-inhibitors induce degeneration of the fibroid xenografts. CONCLUSION(S): The selective HIF-1 inhibitors echinomycin and PX-478 show antitumor effects against uterine fibroids both in vitro and in vivo. These findings support the potential use of HIF-1 inhibitors for the treatment of uterine fibroids.


Assuntos
Equinomicina , Leiomioma , Animais , Equinomicina/farmacologia , Feminino , Humanos , Hipóxia , Fator 1 Induzível por Hipóxia , Antígeno Ki-67 , Leiomioma/tratamento farmacológico , Camundongos , Compostos de Mostarda , Fenilpropionatos
18.
J Clin Invest ; 132(9)2022 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-35239514

RESUMO

A combination of anti-CTLA-4 plus anti-PD-1/PD-L1 is the most effective cancer immunotherapy but causes high incidence of immune-related adverse events (irAEs). Here we report that targeting of HIF-1α suppressed PD-L1 expression on tumor cells and tumor-infiltrating myeloid cells, but unexpectedly induced PD-L1 in normal tissues by an IFN-γ-dependent mechanism. Targeting the HIF-1α/PD-L1 axis in tumor cells reactivated tumor-infiltrating lymphocytes and caused tumor rejection. The HIF-1α inhibitor echinomycin potentiated the cancer immunotherapeutic effects of anti-CTLA-4 therapy, with efficacy comparable to that of anti-CTLA-4 plus anti-PD-1 antibodies. However, while anti-PD-1 exacerbated irAEs triggered by ipilimumab, echinomycin protected mice against irAEs by increasing PD-L1 levels in normal tissues. Our data suggest that targeting HIF-1α fortifies the immune tolerance function of the PD-1/PD-L1 checkpoint in normal tissues but abrogates its immune evasion function in the tumor microenvironment to achieve safer and more effective immunotherapy.


Assuntos
Equinomicina , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias , Animais , Antígeno B7-H1 , Equinomicina/farmacologia , Evasão da Resposta Imune , Tolerância Imunológica , Linfócitos do Interstício Tumoral , Camundongos , Neoplasias/terapia , Microambiente Tumoral
19.
Dis Model Mech ; 15(2)2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34580712

RESUMO

Oncogenic Ras mutations are highly prevalent in hematopoietic malignancies. However, it is difficult to directly target oncogenic RAS proteins for therapeutic intervention. We have developed a Drosophila acute myeloid leukemia model induced by human KRASG12V, which exhibits a dramatic increase in myeloid-like leukemia cells. We performed both genetic and drug screens using this model. The genetic screen identified 24 candidate genes able to attenuate the oncogenic RAS-induced phenotype, including two key hypoxia pathway genes HIF1A and ARNT (HIF1B). The drug screen revealed that echinomycin, an inhibitor of HIF1A, can effectively attenuate the leukemia phenotype caused by KRASG12V. Furthermore, we showed that echinomycin treatment can effectively suppress oncogenic RAS-driven leukemia cell proliferation, using both human leukemia cell lines and a mouse xenograft model. These data suggest that inhibiting the hypoxia pathway could be an effective treatment approach and that echinomycin is a promising targeted drug to attenuate oncogenic RAS-induced cancer phenotypes. This article has an associated First Person interview with the first author of the paper.


Assuntos
Equinomicina , Leucemia Mieloide Aguda , Animais , Equinomicina/farmacologia , Equinomicina/uso terapêutico , Genes ras , Humanos , Hipóxia/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Camundongos , Fenótipo
20.
Artigo em Chinês | WPRIM (Pacífico Ocidental) | ID: wpr-941023

RESUMO

OBJECTIVE@#To investigate effects of physiological hypoxic conditions on suspension and adherence of embryoid bodies (EBs) during differentiation of human induced pluripotent stem cells (hiPSCs) and explore the underlying mechanisms.@*METHODS@#EBs in suspension culture were divided into normoxic (21% O2) and hypoxic (5% O2) groups, and those in adherent culture were divided into normoxic, hypoxic and hypoxia + HIF-1α inhibitor (echinomycin) groups. After characterization of the pluripotency with immunofluorescence assay, the hiPSCs were digested and suspended under normoxic and hypoxic conditions for 5 days, and the formation and morphological changes of the EBs were observed microscopically; the expressions of the markers genes of the 3 germ layers in the EBs were detected. The EBs were then inoculated into petri dishes for further culture in normoxic and hypoxic conditions for another 2 days, after which the adhesion and peripheral expansion rate of the adherent EBs were observed; the changes in the expressions of HIF-1α, β-catenin and VEGFA were detected in response to hypoxic culture and echinomycin treatment.@*RESULTS@#The EBs cultured in normoxic and hypoxic conditions were all capable of differentiation into the 3 germ layers. The EBs cultured in hypoxic conditions showed reduced apoptotic debris around them with earlier appearance of cystic EBs and more uniform sizes as compared with those in normoxic culture. Hypoxic culture induced more adherent EBs than normoxic culture (P < 0.05) with also a greater outgrowth rate of the adherent EBs (P < 0.05). The EBs in hypoxic culture showed significantly up-regulated mRNA expressions of β-catenin and VEGFA (P < 0.05) and protein expressions of HIF-1 α, β-catenin and VEGFA (P < 0.05), and their protein expresisons levels were significantly lowered after treatment with echinomycin (P < 0.05).@*CONCLUSION@#Hypoxia can promote the formation and maturation of suspended EBs and enhance their adherence and post-adherent proliferation without affecting their pluripotency for differentiation into all the 3 germ layers. Our results provide preliminary evidence that activation of HIF-1α/β-catenin/VEGFA signaling pathway can enhance the differentiation potential of hiPSCs.


Assuntos
Humanos , Equinomicina/metabolismo , Corpos Embrioides/metabolismo , Hipóxia/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , beta Catenina/metabolismo
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