RESUMEN
AIMS: Although the functions of progesterone in the myometrium are well-established, the nongenomic effects of progesterone in pregnant myometrial contractions are still unclear. Therefore, this study aimed to investigate changes in the nongenomic effects of progesterone during pregnancy. MAIN METHODS: Myometrial strips were obtained from non-pregnant, pregnant, and postpartum rats, and the nongenomic effects of progesterone in the myometrium during pregnancy were examined. Additionally, the influence of actinomycin D and cycloheximide and the effects of Org OD-02-0 (a specific membrane progesterone receptor (mPR) agonist) in the myometrium were investigated. Moreover, DNA microarray and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to identify genes involved in progesterone-induced effects in the myometrium. KEY FINDINGS: Progesterone did not cause rhythmic contractions in non-pregnant myometrium but induced rhythmic contractions in pregnant myometrium, with the effects peaking at 20 d + 8 h of pregnancy. However, myometrial contractions decreased after delivery and were restored to non-pregnant levels at 7 d postpartum. Additionally, progesterone stably inhibited high KCl-induced myometrial contractions during pregnancy. Moreover, the nongenomic effects of progesterone were unaffected by actinomycin D or cycloheximide, and Org OD-02-0 effectively mimicked these effects. DNA microarray analysis and qRT-PCR revealed a significant increase in mPRß gene expression during pregnancy. However, mPRα, mPRγ, mPRδ, and mPRε expression levels remained unchanged. SIGNIFICANCE: The stimulatory nongenomic effect of progesterone, which was inducible and mPRß-dependent during pregnancy, may be involved in parturition. The inhibitory effect, which was constitutive and depended on other mPRs, may be involved in pregnancy maintenance.
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Miometrio , Progesterona , Embarazo , Femenino , Ratas , Animales , Progesterona/farmacología , Progesterona/metabolismo , Miometrio/metabolismo , Cicloheximida/farmacología , Cicloheximida/metabolismo , Dactinomicina/farmacología , Dactinomicina/metabolismo , Receptores de Progesterona/metabolismo , Progestinas/farmacología , Contracción UterinaRESUMEN
OBJECTIVES: There are three major subtypes of breast cancer, ER+, HER2+ and triple-negative breast cancer (TNBC), namely ER-, PR-, HER2-. TNBC is the most aggressive breast cancer with poor prognosis and no target drug up to now. Actinomycin D (ActD) is a bioactive metabolite of marine bacteria that has been reported to have antitumor activity. The aim of study is to investigate whether ActD has a synergetic effect on TNBC with Doxorubicin (Dox), the major chemotherapeutic drug for TNBC, and explore the underlying mechanism. METHODS: TNBC cell lines HCC1937, MDA-MB-436 and nude mice were used in the study. Drug synergy determination, LDH assay, MMP assay, Hoechst 33342 staining, Flow cytometry, Flexible docking and CESTA assay were carried out. The expression of proteins associated with apoptosis was checked by Western blot and siRNA experiments were performed to investigate the role of P53 and PUMA induced by drugs. RESULTS: There was much higher apoptosis rate of cells in the ActDâ +â Dox group than that in ActD group or Dox group. Expression of MDM2 and BCL-2 was reduced while expression of P53, PUMA and BAX were increased in the groups treated with ActDâ +â Dox or Dox compared to the control group. Furthermore, P53 siRNA or PUMA siRNA tremendously abrogated the cell apoptosis in the groups treated by ActD, Dox and ActDâ +â Dox. Flexible docking and CESTA showed that ActD can bind MDM2. CONCLUSIONS: ActD had a synergetic effect on TNBC with Dox via P53-dependent apoptosis and it may be a new choice for treatment of TNBC.
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Neoplasias de la Mama Triple Negativas , Ratones , Humanos , Animales , Neoplasias de la Mama Triple Negativas/genética , Dactinomicina/farmacología , Dactinomicina/metabolismo , Dactinomicina/uso terapéutico , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ratones Desnudos , Proteínas Reguladoras de la Apoptosis , Línea Celular Tumoral , Doxorrubicina/farmacología , Apoptosis , ARN Interferente PequeñoRESUMEN
To explore the physiological role and/or pharmacological effects of ommochrome, which is a natural organic pigment widely distributed in Protostomia, we attempted to investigate the influence of ommochrome on RT-PCR and activities of restriction enzymes. It was found that ommin, an ommochrome purified from the diapause eggs of Bombyx mori, inhibited the RT-PCR and restriction enzyme activities. The mechanism of these inhibitory reactions is assumed to be the direct binding of ommochrome to DNA rather than acting against the enzymes because, similarly to actinomycin D, there is a phenoxazine ring in the structure of ommin that is known to be intercalated to DNA. To reveal the ommin/DNA interaction, it was investigated by computational approaches such as molecular docking, molecular dynamics simulation, and free energy calculation. From the computational analyses, it was expected that ommin would bind to DNA with almost the same strength as actinomycin D and intercalate into DNA. This is the first report on the pharmacological effect of ommochrome and its inhibitory mechanism obtained from biochemical and computational analyses.
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Bombyx , Animales , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Dactinomicina/farmacología , Dactinomicina/metabolismo , Simulación del Acoplamiento Molecular , Bombyx/genética , ADN/genéticaRESUMEN
AIMS: Actinomycin (Act) D, a polypeptide antibiotic, is used clinically to inhibit the growth of malignant tumors. Act D binds to DNA at the transcription initiation complex to prevent the elongation of RNA. Act D causes DNA damage, growth inhibition, and cell death. Myeloid cell leukemia (Mcl-1) is an anti-apoptotic Bcl-2 family member protein, and the present study explored the effects and molecular mechanism of Act D-induced Mcl-1 downregulation. MAIN METHODS: Human adenocarcinoma A549 cells were used to check the cytotoxic signaling pathways of Act D, particularly in apoptotic mechanism, in a cell-based study approach. Specific blockers targeting the apoptotic factors were examined for their possible roles. KEY FINDINGS: We found that Act D caused cell growth inhibition and apoptosis. Propidium iodide-based flow cytometric analysis and immunostaining confirmed cell apoptosis. Treatment with Act D caused DNA damage, followed by p53-independent cell death. Western blotting showed a significant decrease in Mcl-1 expression, mitochondrial transmembrane potential loss, and caspase-9/caspase-3 cascade activation. The proteasome inhibitor MG132 reversed Act D-induced Mcl-1 downregulation. However, pharmacological inhibition of glycogen synthase kinase-3, p53 expression, ER stress, autophagy, and vesicle acidification, which are Mcl-1-regulating signaling pathways, did not rescue these effects. Notably, Cullin-Ring E3 ligase partially mediated Mcl-1 downregulation. Administration of transforming growth factor-ß induced mesenchymal cell differentiation, but Act D still decreased Mcl-1 and caused cell apoptosis. SIGNIFICANCE: All of these data show a potential pro-apoptotic effect for Act D by facilitating Mcl-1 uncanonical downregulation.
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Leucemia , Neoplasias Pulmonares , Humanos , Dactinomicina/farmacología , Dactinomicina/metabolismo , Regulación hacia Abajo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Antibacterianos/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , Neoplasias Pulmonares/metabolismo , Apoptosis , Células Mieloides , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
PURPOSE: The purpose of this study was to clarify the effect of ZC3H13 on the growth of papillary thyroid carcinoma (PTC). METHODS: Firstly, we used qRT-PCR and Western blot to compare the difference in the expression of ZC3H13 between normal thyroid epithelial cells and PTC cell lines. Then, ZC3H13 overexpression/knockout thyroid cancer cells were constructed by lentivirus transfection, and the effects of overexpression of ZC3H13 on the proliferation, migration and invasion of PTC cells were detected by CCK8 and transwell experiments. Lastly, MeRIP-qPCR, RIP and o Actinomycin D were used to verify that ZC3H13 regulated the expression of downstream target gene IQGAP1 through m6A modification. RESULTS: ZC3H13 expression was decreased in PTC cell lines BCPAP, KTC-1, k1, HTH83, and TPC-1. Proliferation, invasion, and migration of PTC cells were inhibited by overexpressed ZC3H13 but increased by knockdown of ZC3H13. IQGAP1 expression was suppressed by ZC3H13 overexpression but enhanced by ZC3H13 knockdown. In ZC3H13-overexpressed PTC cells, the m6A level of IQGAP1 mRNA was increased, and the IQGAP1 mRNA expression was decreased with the increasing time of Actinomycin D treatment. YTHDF2 enriched more IQGAP1 mRNA than IgG and knockdown of YTHDF2 reversed the effect of ZC3H13 overexpression on IQGAP1 mRNA stability. The xenograft tumor experiment in nude mice confirmed that the overexpression of ZC3H13 inhibited tumor growth, while overexpression of IQGAP1 could reverse the inhibitory effect of ZC3H13 overexpression on tumor growth. CONCLUSION: ZC3H13 mediates IQGAP1 mRNA degradation by promoting m6A modification of IQGAP1 mRNA, this provides a prospective therapeutic target for PTC.
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MicroARNs , Neoplasias de la Tiroides , Ratones , Animales , Humanos , Cáncer Papilar Tiroideo/genética , Cáncer Papilar Tiroideo/patología , MicroARNs/genética , Ratones Desnudos , Dactinomicina/metabolismo , Línea Celular Tumoral , Proliferación Celular , Invasividad Neoplásica , Movimiento Celular , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , ARN Mensajero , Regulación Neoplásica de la Expresión Génica , Proteínas Nucleares , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Macrophages are heterogeneous cells that can be polarized into M1 or M2 phenotype. m6A "reader" YTH domain family protein 2 (YTHDF2) has been the m6A binding protein with the highest activity, which can recognize and disturb m6A-containing mRNA in processing bodies to reduce mRNA stability. YTHDF2 is recently identified as an effective RNA binding protein that modulates inflammatory gene levels within inflammatory responses. However, the role of YTHDF2 in M1/M2 macrophage polarization has not been reported. We established a M1/M2 macrophage polarization model using bone-marrow-derived macrophages and found that the expression levels of YTHDF2 in M1/M2 macrophages were both elevated. YTHDF2-knockdown macrophage polarization model was then established, and through qPCR, ELISA, and FACS, we discovered that suppressing YTHDF2 encouraged M1 polarization but restrained M2 polarization. In M1 macrophages, YTHDF2 silencing had no significant effect on p53 expression; however, in YTHDF2 knockdown, M2 macrophage p53 expression was remarkably upregulated. p53 inhibitor PFT-α was then applied and revealed that suppressing p53 simultaneously promoted YTHDF2-silenced M1 polarization and facilitated M2 macrophage polarization. Actinomycin D assays were further utilized to examine the mRNA degradation level of different cytokines, and p53 mRNA degradation in YTHDF2-depleted M2 cells was discovered impeded. Western Blot analysis also implied that a deficit in YTHDF2 expression may activate MAPK and NF-κB pathways. In this study, YTHDF2 induces M2 macrophage polarization by promoting the degradation of p53 mRNA. YTHDF2 suppresses M1 macrophage polarization by inhibiting NF-κB, p38, and JNK signaling pathways, yet p53 remains unaffected in YTHDF2-silenced M1 macrophages.
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FN-kappa B , Proteína p53 Supresora de Tumor , FN-kappa B/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Dactinomicina/metabolismo , Dactinomicina/farmacología , Transducción de Señal , Macrófagos/metabolismo , Citocinas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Whole-genome sequencing and genome mining are recently considered an efficient approach to shine more light on the underlying secondary metabolites of Streptomyces. The present study unearths the biosynthetic potential of endophytic SX6 as a promising source of biologically active substances and plant-derived compounds for the first time. Out of 38 isolates associated with Aegiceras corniculatum (L.) Blanco, Streptomyces parvulus SX6 was highly active against Pseudomonas aeruginosa ATCC® 9027™ and methicillin-resistant Staphylococcus epidermidis (MRSE) ATCC® 35984™. Additionally, S. parvulus SX6 culture extract showed strong cytotoxicity against Hep3B, MCF-7, and A549 cell lines at a concentration of 30 µg/ml, but not in non-cancerous HEK-293 cells. The genome contained 7.69 Mb in size with an average G + C content of 72.8% and consisted of 6,779 protein-coding genes. AntiSMASH analysis resulted in the identification of 29 biosynthetic gene clusters (BGCs) for secondary metabolites. Among them, 4 BGCs showed low similarity (28-67% of genes show similarity) to actinomycin, streptovaricin, and polyoxypeptin gene clusters, possibly attributed to antibacterial and anticancer activities observed. In addition, the complete biosynthetic pathways of plant-derived compounds, including daidzein and genistein were identified using genome mining and HPLC-DAD-MS analysis. These findings portray an exciting avenue for future characterization of promising secondary metabolites from mangrove endophytic S. parvulus.
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Staphylococcus aureus Resistente a Meticilina , Primulaceae , Streptomyces , Antibacterianos/metabolismo , Antibacterianos/farmacología , Dactinomicina/metabolismo , Genisteína/metabolismo , Células HEK293 , Humanos , Fitoquímicos , Primulaceae/metabolismo , Estreptovaricina/metabolismoRESUMEN
Insulin stimulates glucose uptake in adipocytes by triggering translocation of glucose transporter 4-containg vesicles to the plasma membrane. Under basal conditions, these vesicles (IRVs for insulin-responsive vesicles) are retained inside the cell via a "static" or "dynamic" mechanism. We have found that inhibitors of RNA and protein synthesis, actinomycin D and emetine, stimulate Glut4 translocation and glucose uptake in adipocytes without engaging conventional signaling proteins, such as Akt, TBC1D4, or TUG. Actinomycin D does not significantly affect endocytosis of Glut4 or recycling of transferrin, suggesting that it specifically increases exocytosis of the IRVs. Thus, the intracellular retention of the IRVs in adipocytes requires continuous RNA and protein biosynthesis de novo. These results point out to the existence of a short-lived inhibitor of IRV translocation thus supporting the "static" model.
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Biosíntesis de Proteínas , ARN , Adipocitos/metabolismo , Dactinomicina/metabolismo , Emetina , Glucosa/metabolismo , Insulina/metabolismo , Insulina/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN/metabolismo , Transferrinas/metabolismoRESUMEN
The activation of memory T cells is a very rapid and concerted cellular response that requires coordination between cellular processes in different compartments and on different time scales. In this study, we use ribosome profiling and deep RNA sequencing to define the acute mRNA translation changes in CD8 memory T cells following initial activation events. We find that initial translation enables subsequent events of human and mouse T cell activation and expansion. Briefly, early events in the activation of Ag-experienced CD8 T cells are insensitive to transcriptional blockade with actinomycin D, and instead depend on the translation of pre-existing mRNAs and are blocked by cycloheximide. Ribosome profiling identifies â¼92 mRNAs that are recruited into ribosomes following CD8 T cell stimulation. These mRNAs typically have structured GC and pyrimidine-rich 5' untranslated regions and they encode key regulators of T cell activation and proliferation such as Notch1, Ifngr1, Il2rb, and serine metabolism enzymes Psat1 and Shmt2 (serine hydroxymethyltransferase 2), as well as translation factors eEF1a1 (eukaryotic elongation factor α1) and eEF2 (eukaryotic elongation factor 2). The increased production of receptors of IL-2 and IFN-γ precedes the activation of gene expression and augments cellular signals and T cell activation. Taken together, we identify an early RNA translation program that acts in a feed-forward manner to enable the rapid and dramatic process of CD8 memory T cell expansion and activation.
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Glicina Hidroximetiltransferasa , Interleucina-2 , Regiones no Traducidas 5' , Animales , Linfocitos T CD8-positivos , Cicloheximida/metabolismo , Dactinomicina/metabolismo , Glicina Hidroximetiltransferasa/genética , Glicina Hidroximetiltransferasa/metabolismo , Humanos , Memoria Inmunológica , Interleucina-2/metabolismo , Activación de Linfocitos , Células T de Memoria , Ratones , Factor 2 de Elongación Peptídica/genética , Factor 2 de Elongación Peptídica/metabolismo , Factores de Elongación de Péptidos/genética , Pirimidinas/metabolismo , ARN Mensajero/genética , Serina/genéticaRESUMEN
The infections caused by Pseudomonas aeruginosa are difficult to treat due to its multidrug resistance. A promising strategy for controlling P. aeruginosa infection is targeting the quorum sensing (QS) system. Actinomycin D isolated from the metabolite of endophyte Streptomyces cyaneochromogenes RC1 exhibited good anti-QS activity against P. aeruginosa PAO1. Actinomycin D (50, 100, and 200 µg/mL) significantly inhibited the motility as well as reduced the production of multiple virulence factors including pyocyanin, protease, rhamnolipid, and siderophores. The images of confocal laser scanning microscopy and scanning electron microscopy revealed that the treatment of actinomycin D resulted in a looser and flatter biofilm structure. Real-time quantitative PCR analysis showed that the expression of QS-related genes lasI, rhlI, rhlR, pqsR, pslA, and pilA were downregulated dramatically. The production of QS signaling molecules N-(3-oxododecanoyl)-L-homoserine lactone and N-butanoyl-L-homoserine lactone were also decreased by actinomycin D. These findings suggest that actinomycin D, a potent in vitro anti-virulence agent, is a promising candidate to treat P. aeruginosa infection by interfering with the QS systems.
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Percepción de Quorum , Streptomyces , Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Biopelículas , Dactinomicina/metabolismo , Dactinomicina/farmacología , Endófitos/metabolismo , Pseudomonas aeruginosa/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Factores de Virulencia/genéticaRESUMEN
Objective: To explore the role of circIFITM1 and its potential molecular mechanism in colon cancer. Methods: The circIFITM1 in human samples and cell lines of colon cancer was measured via RT-PCR. The cyclicity of circIFITM1 was confirmed by agarose gel electrophoresis and Sanger sequencing, and the stability of circIFITM1 was confirmed by actinomycin D assay. The proliferative and invasive ability was detected by the CCK-8 assay and Transwell assay, respectively. RNA pull-down assay confirmed a combination of circIFITM1 and miRNA. Dual-luciferase reporter gene was used to detect the direct relationship between miRNA and the target gene. Results: circIFITM1 originated from the maternal gene IFITM1and had high stability. It was resistant to processing by actinomycin D. Upregulating circIFITM1 facilitated the proliferation and invasion of Lovo cells, while interfering with circIFITM1 expression inhibited them. circIFITM1 interacted with miR-802, and miR-802 targeted the 3'UTR of FOXP1. The overexpression of circIFITM1 downregulated miR-802 and upregulated FOXP1. Conclusion: circIFITM1 facilitates the proliferative and invasive abilities via miR-802/FOXP1 in Lovo cells.
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Neoplasias del Colon , MicroARNs , Línea Celular Tumoral , Proliferación Celular/genética , Neoplasias del Colon/genética , Dactinomicina/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Invasividad Neoplásica/genética , Proteínas Represoras/genética , Factores de Transcripción/genéticaRESUMEN
DNA thermal denaturation was evaluated as a measure of the effect of antitumor drug actinomycin D on the stability of the double helix and also the effect of SDS micelles on actinomycin D - DNA complexes. The results indicated that the melting temperature of DNA was dependent on drug concentration, increasing with actinomycin D concentration. High thermal stabilization (about 10 °C) of the DNA helix after the association with actinomycin D clearly demonstrates the intercalative binding mode. The presence of SDS micelles leads to the release of intercalated actinomcyin D molecules from DNA double helix and their further relocation in surfactant micelles. These results highlighted that the drug release can be controlled in time and by varying the concentration and nature of surfactant.
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ADN , Micelas , ADN/química , Dactinomicina/química , Dactinomicina/metabolismo , Dactinomicina/farmacología , Tensoactivos/química , TemperaturaRESUMEN
Breast cancer metastases are most commonly found in bone, an indication of poor prognosis. Pathway-based biomarkers identification may help elucidate the cellular signature of breast cancer metastasis in bone, further characterizing the etiology and promoting new therapeutic approaches. We extracted gene expression profiles from mouse macrophages from the GEO dataset, GSE152795 using the GEO2R webtool. The differentially expressed genes (DEGs) were filtered by log2 fold-change with threshold 1.5 (FDR < 0.05). STRING database and Enrichr were used for GO-term analysis, miRNA and TF analysis associated with DEGs. Autodock Vienna was exploited to investigate interaction of anti-cancer drugs, Actinomycin-D and Adriamycin. Sensitivity and specificity of DEGs was assessed using receiver operating characteristic (ROC) analyses. A total of 61 DEGs, included 27 down-regulated and 34 up-regulated, were found to be significant in breast cancer bone metastasis. Major DEGs were associated with lipid metabolism and immunological response of tumor tissue. Crucial DEGs, Bcl3, ADGRG7, FABP4, VCAN, and IRF4 were regulated by miRNAs, miR-497, miR-574, miR-138 and TFs, CCDN1, STAT6, IRF8. Docking analysis showed that these genes possessed strong binding with the drugs. ROC analysis demonstrated Bcl3 is specific to metastasis. DEGs Bcl3, ADGRG7, FABP4, IRF4, their regulating miRNAs and TFs have strong impact on proliferation and metastasis of breast cancer in bone tissues. In conclusion, present study revealed that DEGs are directly involved in of breast tumor metastasis in bone tissues. Identified genes, miRNAs, and TFs can be possible drug targets that may be used for the therapeutics. However, further experimental validation is necessary.
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Antineoplásicos , MicroARNs , Neoplasias , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Biología Computacional , Dactinomicina/metabolismo , Doxorrubicina , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Ratones , MicroARNs/genética , Neoplasias/genéticaRESUMEN
Actinomycin D (ActD) was the first anticancer antibiotic approved for the management of human cancers. However, the notorious toxicity profile limits its widespread application in cancers, including cancers of the aerodigestive tract. Recent studies show that combining low-dose ActD with existing chemotherapies could potentially protect normal cells from the toxicity of chemotherapy drugs through p53 activation (cyclotherapy). An understanding of ActD's effect on p53 signaling is critical for the meaningful application of ActD in cyclotherapy-based combinations. This study evaluated the anti-tumor efficacy and mechanism of action of ActD in aerodigestive tract cancers. We found that ActD strongly inhibited the growth of a panel of aerodigestive tract cancer cell lines and induced efficient apoptosis, although the sensitivity varies among cell lines. The IC50 values of ActD spanned between 0.021 and 2.96 nM. Mechanistic studies revealed that ActD increased the expression of total and phosphorylated p53 (ser15) in a time- and dose-dependent manner. Moreover, ActD-induced apoptosis is dependent on p53 in cells expressing wild-type p53 and that ActD induced context-dependent differential expression of downstream targets p21 and PUMA without significant effects on p27. In the final analysis, this study revealed that p53-p21 is the predominant pathway activated by low-dose ActD, supporting further development of ActD in cyclotherapy.
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Neoplasias , Proteína p53 Supresora de Tumor , Antibióticos Antineoplásicos/farmacología , Apoptosis , Dactinomicina/metabolismo , Dactinomicina/farmacología , Dactinomicina/uso terapéutico , Humanos , Neoplasias/tratamiento farmacológico , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Background: Circular RNAs (circRNAs) have important roles in human malignancies, including breast cancer (BC). In this study, we explored the function of circRNA ribonuclease P RNA component H1 (circ_RPPH1) in BC development and clarify the mechanistic pathway. Materials and Methods: Expression of circ_RPPH1, microRNA-542-3p (miR-542-3p), and Rho GTPase-activating protein 1 (ARHGAP1) in BC tissues and cells was determined by quantitative real-time polymerase chain reaction or Western blot assay. The stability of circ_RPPH1 was confirmed by RNase R and actinomycin D treatment. Cell viability and colony formation ability were measured by methyl thiazolyl tetrazolium (MTT) assay and colony formation assay, respectively. Western blot analysis was also used to detect proliferation biomarker (Ki67) and epithelial-mesenchymal transition (EMT) biomarkers (E-cadherin, N-cadherin, and vimentin). Flow cytometry and Transwell assays were performed to monitor cell apoptosis, migration, and invasion. The binding potency between miR-542-3p and circ_RPPH1 or ARHGAP1 was validated by dual-luciferase reporter assay. Functional role of circ_RPPH1 in vivo was investigated by xenograft tumor reporter assay. Results: Upregulation of circ_RPPH1 and ARHGAP1, and downregulation of miR-542-3p were detected in BC tissues and cells. circ_RPPH1 knockdown or miR-542-3p introduction inhibited BC cell proliferation and metastasis, while promoted apoptosis in vitro. circ_RPPH1 sponged miR-542-3p to upregulate ARHGAP1 expression, thereby affecting BC progression. Moreover, depletion of circ_RPPH1 suppressed tumor growth in vivo. Conclusions: circ_RPPH1 contributed to BC tumorigenesis by sponging miR-542-3p and upregulating ARHGAP1, affording a novel mechanistic pathway in BC development.
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Neoplasias de la Mama , MicroARNs , Humanos , Femenino , ARN Circular/genética , Vimentina/metabolismo , Antígeno Ki-67 , Neoplasias de la Mama/genética , Dactinomicina/metabolismo , Ribonucleasa P/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Movimiento Celular , Proliferación Celular , Línea Celular Tumoral , Cadherinas/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismoRESUMEN
Accumulating evidence indicates that circular (circ)RNAs exhibit complex functions in diverse malignant tumors, including nonsmall cell lung cancer (NSCLC). The role of the circRNA transcription adaptor 2A (circTADA2A) in NSCLC remains unclear. The expression, function and mechanism of circTADA2A in NSCLC development were investigated in the present study. The results revealed that circTADA2A was upregulated in NSCLC, and that knockdown of circTADA2A inhibited cell proliferation and migration in the NSCLC cell lines A549 and H1299. Functional assays demonstrated that circTADA2A promoted proliferation and migration via interacting with microRNA (miR)638. Bioinformatics and reverse transcriptionquantitative PCR assay confirmed that miR638 was expressed at low levels in NSCLC. In addition, it was found that miR638 served a tumorsuppressive role and suppressed proliferation and migration via PCNA clamp associated factor (KIAA0101) inhibition in A549 and H1299 cells. Lastly, it was verified that circTADA2A promoted cell proliferation and migration, at least partially, via miR638/KIAA0101 signaling in A549 and H1299 cells. In summary, the present study showed that circTADA2A promoted NSCLC cell proliferation and migration via modulating miR638/KIAA0101 signaling.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , ARN Helicasas DEAD-box/metabolismo , Proteínas de Unión al ADN/metabolismo , Factor 4A Eucariótico de Iniciación/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , MicroARNs/metabolismo , ARN Circular , Factores de Transcripción/metabolismo , Células A549 , Anciano , Anciano de 80 o más Años , Área Bajo la Curva , Movimiento Celular , Proliferación Celular , ARN Helicasas DEAD-box/genética , Proteínas de Unión al ADN/genética , Dactinomicina/metabolismo , Factor 4A Eucariótico de Iniciación/genética , Exorribonucleasas/metabolismo , Femenino , Humanos , Masculino , MicroARNs/genética , Persona de Mediana Edad , Pronóstico , Antígeno Nuclear de Célula en Proliferación/metabolismo , Factores de Transcripción/genética , Regulación hacia ArribaRESUMEN
Streptomyces smyrnaeus UKAQ_23, isolated from the mangrove-sediment, collected from Jubail,Saudi Arabia, exhibited substantial antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), including non-MRSA Gram-positive test bacteria. The novel isolate, under laboratory-scale conditions, produced the highest yield (561.3 ± 0.3 mg/kg fermented agar) of antimicrobial compounds in modified ISP-4 agar at pH 6.5, temperature 35 °C, inoculum 5% v/w, agar 1.5% w/v, and an incubation period of 7 days. The two major compounds, K1 and K2, were isolated from fermented medium and identified as Actinomycin X2 and Actinomycin D, respectively, based on their structural analysis. The antimicrobial screening showed that Actinomycin X2 had the highest antimicrobial activity compared to Actinomycin D, and the actinomycins-mixture (X2:D, 1:1, w/w) against MRSA and non-MRSA Gram-positive test bacteria, at 5 µg/disc concentrations. The MIC of Actinomycin X2 ranged from 1.56-12.5 µg/ml for non-MRSA and 3.125-12.5 µg/ml for MRSA test bacteria. An in-silico molecular docking demonstrated isoleucyl tRNA synthetase as the most-favored antimicrobial protein target for both actinomycins, X2 and D, while the penicillin-binding protein-1a, was the least-favorable target-protein. In conclusion, Streptomyces smyrnaeus UKAQ_23 emerged as a promising source of Actinomycin X2 with the potential to be scaled up for industrial production, which could benefit the pharmaceutical industry.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Dactinomicina/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Streptomyces/metabolismo , Antibacterianos/aislamiento & purificación , Antibacterianos/metabolismo , Simulación por Computador , Medios de Cultivo/química , Dactinomicina/aislamiento & purificación , Dactinomicina/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Fermentación , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Simulación del Acoplamiento Molecular , Estructura Molecular , Filogenia , Streptomyces/genéticaRESUMEN
AIM: The objective of this study was to investigate the biofilm inhibitory activity of Streptomyces-derived actinomycin D against biofilm formation by Staphylococcus epidermidis. METHODS AND RESULTS: The microtitre plate method and microscopy were used to detect the biofilm formation of S. epidermidis. And an attempt was made to detect the effect of actinomycin D on important biofilm components, exopolysaccharides (EPS) in S. epidermidis using precolumn derivation HPLC. Also cell surface hydrophobicities of S. epidermidis were assessed to explore action mechanisms. The qPCR was performed to demonstrate the genetic mechanisms of biofilm formation by S. epidermidis. Unlike other antibiotics, actinomycin D (1·5 µg ml-1 ) from Streptomyces luteus significantly inhibited biofilm formation by S. epidermidis. Additionally, it effectively inhibited S. epidermidis cells from adhering to glass slides. Actinomycin D downregulated ica locus and then the reduced polysaccharide intercellular adhesin production caused S. epidermidis cells to become less hydrophobic, thus supporting its anti-biofilm effect. CONCLUSION: Streptomyces-derived actinomycin D is active in inhibiting the biofilm formation of S. epidermidis. SIGNIFICANCE AND IMPACT OF THE STUDY: Actinomycin D can be used as a promising antibiofilm agent in inhibiting S. epidermidis biofilm formation. The study is also the first insight into how actinomycin D inhibited the biofilm formation of S. epidermidis. Actinomycin D could potentially be used to reduce the risk of biofilm-associated infections. Our study also suggests that the metabolites from Actinomycete strains keep further attention as potential antibiofilm agents against biofilm formation of S. epidermidis, even biofilm infections of the other bacteria.
Asunto(s)
Biopelículas/efectos de los fármacos , Dactinomicina/farmacología , Polisacáridos Bacterianos/metabolismo , Staphylococcus epidermidis/efectos de los fármacos , Streptomyces/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacología , Adhesión Bacteriana/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Dactinomicina/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Humanos , Polisacáridos Bacterianos/genética , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/metabolismo , Staphylococcus epidermidis/fisiologíaRESUMEN
Tumorassociated neutrophils (TANs) have an immunosuppressive function and play an important role in tumor progression. However, the detailed mechanism is largely unknown. The present study investigated the immunosuppressive ability of TANs in gastric cancer. Tumor tissue culture supernatant (TTCS) and nontumor tissue culture supernatant (NTCS) were purified and added to neutrophils. Expression of programmed cell death ligand1 (PDL1), 7aminoactinomycin D and human leukocyte antigenDR (HLADR), and the levels of hydrogen peroxide (H2O2) were determined. Levels of programmed cell death1 (PD1) and CD25 were assessed in T cells cocultured with neutrophils. Furthermore, CD4+ T cells were cocultured with dendritic cells and neutrophils to examine their proliferation. CD15 and PD1 immunohistochemical staining was also performed to explore the positional relationship. The results revealed that the neutrophils incubated with TTCS showed upregulation of PDL1 expression, as well as a decreases in the ratio of apoptotic cells, expression of HLADR, and levels of H2O2. CD4+ T cells cocultured with neutrophils conditioned with TTCS showed a decrease in proliferation, upregulation of PD1 expression, and downregulation of CD25 expression. IHC showed that PD1+ T cells formed clusters and TANs infiltrated around the clusters. In conclusion, neutrophils in gastric cancer tissue inhibit the proliferation of CD4+ T cells and may form a local immunosuppressive environment through the PD1/PDL1 pathway.
Asunto(s)
Antígeno B7-H1/metabolismo , Linfocitos T CD4-Positivos/citología , Antígenos HLA-DR/metabolismo , Neutrófilos/citología , Neoplasias Gástricas/inmunología , Anciano , Linfocitos T CD4-Positivos/inmunología , Técnicas de Cultivo de Célula , Dactinomicina/metabolismo , Regulación hacia Abajo , Femenino , Regulación de la Expresión Génica , Humanos , Peróxido de Hidrógeno/metabolismo , Masculino , Persona de Mediana Edad , Neutrófilos/inmunología , Neoplasias Gástricas/metabolismo , Células Tumorales CultivadasRESUMEN
DNA mismatches are highly polymorphic and dynamic in nature, albeit poorly characterized structurally. We utilized the antitumour antibiotic CoII(Chro)2 (Chro = chromomycin A3) to stabilize the palindromic duplex d(TTGGCGAA) DNA with two G:G mismatches, allowing X-ray crystallography-based monitoring of mismatch polymorphism. For the first time, the unusual geometry of several G:G mismatches including syn-syn, water mediated anti-syn and syn-syn-like conformations can be simultaneously observed in the crystal structure. The G:G mismatch sites of the d(TTGGCGAA) duplex can also act as a hotspot for the formation of alternative DNA structures with a GC/GA-5' intercalation site for binding by the GC-selective intercalator actinomycin D (ActiD). Direct intercalation of two ActiD molecules to G:G mismatch sites causes DNA rearrangements, resulting in backbone distortion to form right-handed Z-DNA structures with a single-step sharp kink. Our study provides insights on intercalators-mismatch DNA interactions and a rationale for mismatch interrogation and detection via DNA intercalation.