RESUMEN
The role of ClO- in the physiological functioning of organisms is significant. In this paper, the four fluorescent probes HONx (HON1, HON2, HON3 and HON4) were prepared based on oxyanthracene through the introduction of different substituents, and their photophysical properties were investigated, among which the AIE effect of HON1 was the most significant, and therefore the fluorescent "turn-off" ClO- probe HON1-CN was chosen to be prepared by constructing the ClO- recognition site hydrazone bond at HON1. The ClO- recognises the hydrazone group in the probe HON1-CN, and when the hydrazone bond is broken, the aldehyde group is released, generating HON1 with yellow fluorescence. The probe HON1-CN is highly selective and stable for the detection of ClO- with a detection limit of 0.48 µM and a more than 10-fold increase in fluorescence intensity when the fluorescence is 'switched on', and to a lesser extent, the probe is also very good for the detection of hypochlorite ClO- in the pericarp. Finally, HON1-CN has also been used to detect the presence of ClO- in HeLa cells and zebrafish.
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Colorantes Fluorescentes , Ácido Hipocloroso , Espectrometría de Fluorescencia , Xantonas , Pez Cebra , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Xantonas/química , Animales , Ácido Hipocloroso/análisis , Humanos , Células HeLa , Frutas/química , Límite de DetecciónRESUMEN
As a reactive sulfur species, sulfur dioxide (SO2) and its derivatives play crucial role in various physiological processes, which can maintain redox homeostasis at normal levels and lead to the occurrence of many diseases at abnormal levels. So, the development of a suitable fluorescent probe is a crucial step in advancing our understanding of the role of SO2 derivatives in living organisms. Herein, we developed a near-infrared fluorescent probe (SP) based on the ICT mechanism to monitor SO2 derivatives in living organisms in a ratiometric manner. The probe SP exhibited excellent selectivity, good sensitivity, fast response rate (within 50 s), and low detection limit (1.79 µM). In addition, the cell experiment results suggested that the SP has been successfully employed for the real-time monitoring of endogenous and exogenous SO2 derivatives with negligible cytotoxicity. Moreover, SP was effective in detecting SO2 derivatives in mice.
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Colorantes Fluorescentes , Dióxido de Azufre , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Dióxido de Azufre/análisis , Animales , Ratones , Humanos , Límite de Detección , Espectrometría de Fluorescencia , Imagen Óptica , Células HeLaRESUMEN
Tyramine signaling amplification (TSA) technology is generally applied in immunofluorescence, enzyme-linked immunoassays, in situ hybridization techniques, etc. Successful amplification of fluoresence signals cannot be achieved without excellent fluorescent dyes. BODIPY fluorophore is an ideal probe for cell fluorescence imaging, but pristine BODIPY cannot be direct used in the TSA system. In the paper, the new red-shifted tyramide-conjugated BODIPY (BDP-B/C/D) was synthesized via the Knoevenagel condensation reaction, which based on the tyramide-conjugated BODIPY (BDP-A). The synthesized dyes were combined with tyramine to obtain which could be used as a fluorescent substrate for enzymatic reaction of TSA. By using the selected substrate (BDP-C) in TSA, we found it to be more sensitive than the commercial dye 594 styramide for the detection of low-abundance antigen proteins.
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Compuestos de Boro , Colorantes Fluorescentes , Porfobilinógeno , Tiramina , Tiramina/química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Humanos , Compuestos de Boro/química , Compuestos de Boro/síntesis química , Porfobilinógeno/análogos & derivados , Porfobilinógeno/química , Células HeLa , Espectrometría de Fluorescencia , Imagen ÓpticaRESUMEN
Thiols function as antioxidants in food, prolonging shelf life and enhancing flavor. Moreover, thiols are vital biomolecules involved in enzyme activity, cellular signal transduction, and protein folding among critical biological processes. In this paper, the fluorescent probe PYL-NBD was designed and synthesized, which utilized the fluorescent molecule pyrazoline, the lysosome-targeted morpholine moiety, and the sensing moiety NBD. Probe PYL-NBD was tailored for the recognition of biothiols through single-wavelength excitation, yielding distinct fluorescence emission signals: blue for Cys, Hcy, and GSH; green for Cys, Hcy. Probe PYL-NBD exhibited rapid reaction kinetics (<10 min), distinct fluorescence response signals, and low detection limits (15.7 nM for Cys, 14.4 nM for Hcy, and 12.6 nM for GSH). Probe PYL-NBD enabled quantitative determination of Cys content in food samples and L-cysteine capsules. Furthermore, probe PYL-NBD had been successfully applied for confocal imaging with dual-channel detection of biothiols in various biological specimens, including HeLa cells, zebrafish, tumor sections, and Arabidopsis thaliana.
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Cisteína , Colorantes Fluorescentes , Análisis de los Alimentos , Glutatión , Lisosomas , Espectrometría de Fluorescencia , Pez Cebra , Humanos , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Lisosomas/química , Lisosomas/metabolismo , Células HeLa , Cisteína/análisis , Animales , Análisis de los Alimentos/métodos , Glutatión/análisis , Espectrometría de Fluorescencia/métodos , Homocisteína/análisis , Arabidopsis/química , Límite de Detección , Microscopía ConfocalRESUMEN
Radiotherapy as a mainstay of in-depth cervical cancer (CC) treatment suffers from its radioresistance. Radiodynamic therapy (RDT) effectively reverses radio-resistance by generating reactive oxygen species (ROS) with deep tissue penetration. However, the photosensitizers stimulated by X-ray have high toxicity and energy attenuation. Therefore, X-ray responsive diselenide-bridged mesoporous silica nanoparticles (DMSNs) are designed, loading X-ray-activated photosensitizer acridine orange (AO) for spot blasting RDT like Trojan-horse against radio-resistance cervical cancer (R-CC). DMSNs can encapsulate a large amount of AO, in the tumor microenvironment (TME), which has a high concentration of hydrogen peroxide, X-ray radiation triggers the cleavage of diselenide bonds, leading to the degradation of DMSNs and the consequent release of AO directly at the tumor site. On the one hand, it solves the problems of rapid drug clearance, adverse distribution, and side effects caused by simple AO treatment. On the other hand, it fully utilizes the advantages of highly penetrating X-ray responsive RDT to enhance radiotherapy sensitivity. This approach results in ROS-induced mitochondria damage, inhibition of DNA damage repair, cell cycle arrest and promotion of cancer cell apoptosis in R-CC. The X-ray responsive DMSNs@AO hold considerable potential in overcoming obstacles for advanced RDT in the treatment of R-CC.
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Nanopartículas , Dióxido de Silicio , Humanos , Animales , Rayos X , Nanopartículas/química , Femenino , Dióxido de Silicio/química , Ratones , Neoplasias del Cuello Uterino/terapia , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/patología , Especies Reactivas de Oxígeno/metabolismo , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Tolerancia a Radiación/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Ratones Desnudos , Células HeLa , Ratones Endogámicos BALB C , Apoptosis/efectos de los fármacos , Línea Celular TumoralRESUMEN
Near-infrared (NIR) fluorescent probes with aggregation-induced emission (AIE) properties are of great significance in cell imaging and cancer therapy. However, the complexity of its synthesis, poor photostabilities, and expensive raw materials still pose some obstacles to their practical application. This study reported an AIE luminescent material with red emission and its application in in vitro imaging and photodynamic therapy (PDT) study. This material has the characteristics of simple synthesis, large Stokes shift, good photostabilities, and excellent lipid droplets-specific testing ability. Interestingly, this red-emitting material can effectively produce reactive oxygen species (ROS) under white light irradiation, further achieving PDT-mediated killing of cancer cells. In conclusion, this study demonstrates a simple approach to synthesize NIR AIE probes with both imaging and therapeutic effects, providing an ideal architecture for constructing long-wavelength emission AIE materials.
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Colorantes Fluorescentes , Rayos Infrarrojos , Gotas Lipídicas , Fotoquimioterapia , Especies Reactivas de Oxígeno , Humanos , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/farmacología , Gotas Lipídicas/química , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/análisis , Especies Reactivas de Oxígeno/química , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/síntesis química , Supervivencia Celular/efectos de los fármacos , Imagen Óptica , Estructura Molecular , Células HeLaRESUMEN
Herein, we describe the design and development of a new cell-permeable aggregation-induced emission (AIE) active 3-ethoxysalicylaldimine-based symmetrical azine molecule HDBE. The synthesized compound underwent comprehensive investigation of different spectroscopic methods, like NMR, mass and single crystal X-ray diffraction analysis. The fluorophore HDBE exhibited the bright orange colour AIE behaviour in THF-H2O mixture. The drastic enhancement of emission was achieved upon adding the water to the THF solution of HDBE, with a concentration of 90%. Along with the dynamic light scattering (DLS) and quantum yield measurements, the formation of aggregates was also verified by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis. Further, HDBE demonstrated excited state intramolecular proton transfer (ESIPT) characteristics in different polarity of solvents, which was corroborated by absorption, emission and lifetime spectroscopical investigations. The detailed scrutiny of X-ray structure of HDBE displayed the two strong intramolecular hydrogen bonding interactions, while solid-state fluorescent spectra showed dual emission that corresponds to enol and keto form confirming the ESIPT feature. Further, the synthesized AIE molecule was non-toxic and cell-permeable, making it easy to label as a biomarker in live HeLa cells via fluorescent bioimaging. These studies offer a quick and easy way to develop both AIE and ESIPT-coupled molecules for live cell bioimaging applications.
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Colorantes Fluorescentes , Humanos , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Células HeLa , Imagen Óptica , Estructura Molecular , Color , Protones , Supervivencia Celular/efectos de los fármacosRESUMEN
Rationale: Theranostic nanoplatforms exert a vital role in facilitating concurrent real-time diagnosis and on-demand treatment of diseases, thereby making contributions to the improvement of therapeutic efficacy. Nevertheless, the structural intricacy and the absence of well-defined integration of dual functionality persist as challenges in the development of theranostic nanoplatforms. Methods: We develop an atomically precise theranostic nanoplatform based on metal-organic cage (MOC) to provide magnetic resonance imaging (MRI) guided chemodynamic therapy (CDT) for cancer therapy and assess the theranostic performance both in vitro and in vivo. Through UV-vis spectroscopy, electron paramagnetic resonance (EPR), confocal microscopy, flow cytometry, immunofluorescence staining, and western blotting, the ability of MOC-Mn to generate â¢OH and the subsequent inhibition of HeLa cells was confirmed. Results: The MOC-Mn composed of manganese and calixarene was successfully synthesized and comprehensively characterized. The catalytic activity of manganese within MOC-Mn facilitated the efficient generation of hydroxyl radicals (â¢OH) through a Fenton-like reaction, leveraging the high concentrations of hydrogen peroxide in the tumor microenvironment (TME). Additionally, its capacity to prolong the T1 relaxation time and augment the MR signal was observed. The theranostic efficacy was verified via rigorous in vitro and in vivo experiments, indicating that MOC-Mn offered clearer visualization of tumor particulars and substantial suppression of tumor growth. Conclusion: This study showcases a precise MRI-guided CDT theranostic nanoplatform for cancer therapy, thereby promoting the advancement of precise nanomedicine and structure-function research.
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Imagen por Resonancia Magnética , Nanomedicina Teranóstica , Nanomedicina Teranóstica/métodos , Humanos , Animales , Células HeLa , Imagen por Resonancia Magnética/métodos , Ratones , Manganeso/química , Ratones Desnudos , Femenino , Radical Hidroxilo/metabolismo , Radical Hidroxilo/química , Neoplasias/tratamiento farmacológico , Neoplasias/diagnóstico por imagen , Ratones Endogámicos BALB C , Estructuras Metalorgánicas/química , Estructuras Metalorgánicas/farmacología , Nanopartículas/químicaRESUMEN
Autophagy plays a crucial role in cancer cell survival by facilitating the elimination of detrimental cellular components and the recycling of nutrients. Understanding the molecular regulation of autophagy is critical for developing interventional approaches for cancer therapy. In this study, we report that migfilin, a focal adhesion protein, plays a novel role in promoting autophagy by increasing autophagosome-lysosome fusion. We found that migfilin is associated with SNAP29 and Vamp8, thereby facilitating Stx17-SNAP29-Vamp8 SNARE complex assembly. Depletion of migfilin disrupted the formation of the SNAP29-mediated SNARE complex, which consequently blocked the autophagosome-lysosome fusion, ultimately suppressing cancer cell growth. Restoration of the SNARE complex formation rescued migfilin-deficiency-induced autophagic flux defects. Finally, we found depletion of migfilin inhibited cancer cell proliferation. SNARE complex reassembly successfully reversed migfilin-deficiency-induced inhibition of cancer cell growth. Taken together, our study uncovers a new function of migfilin as an autophagy-regulatory protein and suggests that targeting the migfilin-SNARE assembly could provide a promising therapeutic approach to alleviate cancer progression.
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Autofagia , Moléculas de Adhesión Celular , Proliferación Celular , Lisosomas , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteínas R-SNARE , Humanos , Proteínas R-SNARE/metabolismo , Proteínas R-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Qb-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Proteínas Qc-SNARE/genética , Lisosomas/metabolismo , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular/genética , Autofagosomas/metabolismo , Células HeLa , Línea Celular Tumoral , Unión Proteica , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Fusión de Membrana , Proteínas Qa-SNARERESUMEN
A luciferase reporter assay is a cell-based, enzymatic experiment that utilizes an ectopically expressed luciferase in cultured cells or in live animals, with the presence of its substrate luciferin, to generate luminescence in response to gene regulation at the level of transcription. This assay can be easily formatted for high-throughput sample analysis. The reagents are commercially available and routinely used in various applications. We have automated a luciferase reporter assay on the Opentrons OT-2 platform to measure activation of NF-kB in HeLa cells. The Python script-based protocols were developed to perform cell seeding, reporter construct transfection, and enzyme-catalytic reaction to produce detectable signals for quantification with desirable quality of sample handling and minimal hands-on time.
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Automatización de Laboratorios , Genes Reporteros , Luciferasas , FN-kappa B , Humanos , Luciferasas/metabolismo , Luciferasas/genética , FN-kappa B/metabolismo , Células HeLa , Automatización de Laboratorios/métodosRESUMEN
Mammalian DNA replication relies on various DNA helicase and nuclease activities to ensure accurate genetic duplication, but how different helicase and nuclease activities are properly directed remains unclear. Here, we identify the ubiquitin-specific protease, USP50, as a chromatin-associated protein required to promote ongoing replication, fork restart, telomere maintenance, cellular survival following hydroxyurea or pyridostatin treatment, and suppression of DNA breaks near GC-rich sequences. We find that USP50 supports proper WRN-FEN1 localisation at or near stalled replication forks. Nascent DNA in cells lacking USP50 shows increased association of the DNA2 nuclease and RECQL4 and RECQL5 helicases and replication defects in cells lacking USP50, or FEN1 are driven by these proteins. Consequently, suppression of DNA2 or RECQL4/5 improves USP50-depleted cell resistance to agents inducing replicative stress and restores telomere stability. These data define an unexpected regulatory protein that promotes the balance of helicase and nuclease use at ongoing and stalled replication forks.
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ADN Helicasas , Replicación del ADN , RecQ Helicasas , Helicasa del Síndrome de Werner , RecQ Helicasas/metabolismo , RecQ Helicasas/genética , Replicación del ADN/efectos de los fármacos , Humanos , Helicasa del Síndrome de Werner/metabolismo , Helicasa del Síndrome de Werner/genética , ADN Helicasas/metabolismo , ADN Helicasas/genética , Telómero/metabolismo , Telómero/genética , Endonucleasas de ADN Solapado/metabolismo , Endonucleasas de ADN Solapado/genética , Proteasas Ubiquitina-Específicas/metabolismo , Proteasas Ubiquitina-Específicas/genética , Células HeLa , Células HEK293 , Homeostasis del Telómero/efectos de los fármacos , Cromatina/metabolismoRESUMEN
A novel seminaphthorhodafluor-based fluorescent probe was designed and prepared. It exhibited an outstanding off-on fluorescence response (about 45-fold enhancement) to Cu2+ because the structure changed from a spirocyclic form to a ring-open state. It showed satisfactory detection performances for Cu2+ with fast response speed, high selectivity and anti-interference, and a low detection limit (22.9 nM) due to its long emission wavelength and off-on fluorescence response mode. This probe was also satisfactorily applied for the monitoring of Cu2+ in living cells and zebrafish.
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Cobre , Colorantes Fluorescentes , Pez Cebra , Animales , Colorantes Fluorescentes/química , Colorantes Fluorescentes/síntesis química , Cobre/análisis , Cobre/química , Humanos , Espectrometría de Fluorescencia , Estructura Molecular , Células HeLa , Rodaminas/química , Imagen ÓpticaRESUMEN
The centromere, a chromosome locus defined by the histone H3-like protein centromeric protein A (CENP-A), promotes assembly of the kinetochore to bind microtubules during cell division. Centromere maintenance requires CENP-A to be actively replenished by dedicated protein machinery in the early G1 phase of the cell cycle to compensate for its dilution after DNA replication. Cyclin-dependent kinases (CDKs) limit CENP-A deposition to once per cell cycle and function as negative regulators outside of early G1. Antithetically, Polo-like kinase 1 (PLK1) promotes CENP-A deposition in early G1, but the molecular details of this process are still unknown. We reveal here a phosphorylation network that recruits PLK1 to the deposition machinery to control a conformational switch required for licensing the CENP-A deposition reaction. Our findings clarify how PLK1 contributes to the epigenetic maintenance of centromeres.
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Proteínas de Ciclo Celular , Proteína A Centromérica , Centrómero , Proteínas Cromosómicas no Histona , Epigénesis Genética , Quinasa Tipo Polo 1 , Humanos , Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Proteína A Centromérica/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Fase G1 , Células HeLa , Cinetocoros/metabolismo , Fosforilación , Quinasa Tipo Polo 1/genética , Quinasa Tipo Polo 1/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Intracellular delivery of large molecule cargo via cell penetrating peptides (CPPs) is an inefficient process and despite intense efforts in past decades, improvements in efficiency have been marginal. Utilizing a standardized and comparative analysis of the delivery efficiency of previously described cationic, anionic, and amphiphilic CPPs, we demonstrate that the delivery ceiling is accompanied by irreparable plasma membrane damage that is part of the uptake mechanism. As a consequence, intracellular delivery correlates with cell toxicity and is more efficient for smaller peptides than for large molecule cargo. The delivery of pharmaceutically relevant cargo quantities with acceptable toxicity thus seems hard to achieve with the CPPs tested in our study. Our results suggest that any engineered intracellular delivery system based on conventional cationic or amphiphilic CPPs, or the design principles underlying them, needs to accept low delivery yields due to toxicity limiting efficient cytoplasmic uptake. Novel peptide designs based on detailed study of uptake mechanisms are required to overcome these limitations.
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Membrana Celular , Péptidos de Penetración Celular , Citoplasma , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Membrana Celular/metabolismo , Humanos , Citoplasma/metabolismo , Sistemas de Liberación de Medicamentos , Células HeLa , AnimalesRESUMEN
Controlling the succession of chemical processes with high specificity in complex systems is advantageous for widespread applications, from biomedical research to drug manufacturing. Despite synthetic advances in bioorthogonal and photochemical methodologies, there is a need for generic chemical approaches that can universally modulate photodynamic reactivity in organic photosensitizers. Herein we present a strategy to fine-tune the production of singlet oxygen in multiple photosensitive scaffolds under the activation of bioresponsive and bioorthogonal stimuli. We demonstrate that the photocatalytic activity of nitrobenzoselenadiazoles can be fully blocked by site-selective incorporation of electron-withdrawing carbamate moieties and restored on demand upon uncaging with a wide range of molecular triggers, including abiotic transition-metal catalysts. We also prove that this strategy can be expanded to most photosensitizers, including diverse structures and spectral properties. Finally, we show that such advanced control of singlet oxygen generation can be broadly applied to the photodynamic ablation of human cells as well as to regulate the release of singlet oxygen in the semi-synthesis of natural product drugs.
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Fármacos Fotosensibilizantes , Oxígeno Singlete , Oxígeno Singlete/química , Oxígeno Singlete/metabolismo , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Humanos , Catálisis/efectos de la radiación , Fotoquimioterapia/métodos , Carbamatos/química , Células HeLaRESUMEN
INTRODUCTION: MicroRNAs (miRNAs) are single RNA molecules that act as global regulators of gene expression in mammalian cells and thus constitute attractive targets in treating cancer. Here we aimed to investigate the possible involvement of miRNA-141 (miR-141) in cervical cancer and to identify its potential targets in cervical cancer cell lines. METHODS: The level of miR-141 in HeLa and C-33A cells has been assessed using the quantitative real-time PCR (qRT-PCR). A new miR-141 construct has been performed in a CMV promoter vector tagged with GFP. Using microarray analysis, we identified the potentially regulated genes by miR-141 in transfected HeLa cells. The protein profile of killer-like receptor C1 (KLRC1), KLRC3, carcinoembryonic antigen-related cell adhesion molecule 3 (CAM3), and CAM6 was investigated in HeLa cells transfected with either an inhibitor, antagonist miR-141, or miR-141 overexpression vector using immunoblotting and flow cytometry assay. Finally, ELISA assay has been used to monitor the produced cytokines from transfected HeLa cells. RESULTS: The expression of miR-141 significantly increased in HeLa and C-33A cells compared to the normal cervical HCK1T cell line. Transfection of HeLa cells with an inhibitor, antagonist miR-141, showed a potent effect on cancer cell viability, unlike the transfection of miR-141 overexpression vector. The microarray data of HeLa cells overexpressed miR-141 provided a hundred of downregulated genes, including KLRC1, KLRC3, CAM3, and CAM6. KLRC1 and KLRC3 expression profiles markedly depleted in HeLa cells transfected with miR-141 overexpression accompanied by decreasing interleukin 8 (IL-8), indicating the role of miR-141 in avoiding programmed cells death in HeLa cells. Likewise, CAM3 and CAM6 expression reduced markedly in miR-141 transduced cells accompanied by an increasing level of transforming growth factor beta (TGF-ß), indicating the impact of miR-141 in cancer cell migration. The IntaRNA program and miRWalk were used to check the direct interaction and potential binding sites between miR-141 and identified genes. Based on this, the seeding regions of each potential target was cloned upstream of the luciferase reporter gene in the pGL3 control vector. Interestingly, the luciferase activities of constructed vectors were significantly decreased in HeLa cells pre-transfected with miR-141 overexpression vector, while increasing enormously in cells pre-transfected with miR-141 specific inhibitor. CONCLUSION: Together, these data uncover an efficient miR-141-based mechanism that supports cervical cancer progression and identifies miR-141 as a credible therapeutic target.
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Antígenos CD , Moléculas de Adhesión Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , MicroARNs , Neoplasias del Cuello Uterino , Humanos , MicroARNs/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/metabolismo , Células HeLa , Proliferación Celular/genética , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Femenino , Antígenos CD/genética , Antígenos CD/metabolismo , Metástasis de la Neoplasia , Línea Celular Tumoral , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Movimiento Celular/genéticaRESUMEN
The oxygen level in the tumor microenvironment (TME) plays a critical role in regulating cell fates such as proliferation, migration, apoptosis, and so forth. To better elucidate how hypoxia affects tumor cell behaviors, a series of microfluidic strategies have been utilized to generate an oxygen gradient covering both hypoxia and normoxia conditions. However, in most studies, some chemicals are introduced into microfluidic chips, causing the potential of their poor biocompatibility. The common oxygen gradient with linear variation does not allow the effects of specific oxygen concentrations on tumor cells to be analyzed accurately. In this paper, based on the physical method of gas diffusion, a microfluidic device integrated with an oxygen gradient generator is proposed for investigating effects of different hypoxia levels on responses of tumor cells. This device consists of three layers, i.e., upper layer, thin film layer, and bottom layer. The upper layer is used for introducing the initial gas and generating an oxygen gradient in the form of gas. The bottom layer is used for introducing cells and culture medium. The thin film layer separates the former two layers, allowing the gas to diffuse from the top to the bottom through it. The oxygen gradient in the bottom layer is finally generated in the form of dissolved oxygen. The device is fabricated using microfabrication technology. The effects of structural and working parameters of the device on the oxygen gradient are evaluated by finite element simulation. The oxygen gradient in cell culture channels is characterized by using oxygen-sensitive fluorescence materials. The proliferation and morphology of HeLa cells under specific oxygen levels are compared after culturing for 48 h. The oxygen gradient with a ladder-like distribution demonstrates that this microfluidic device can provide a prospective experimental platform for in vitro cell studies and revelation of the mechanism of tumor metastasis associated with a specific hypoxic microenvironment.
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Oxígeno , Humanos , Oxígeno/química , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/instrumentación , Células HeLa , Microambiente Tumoral , Hipoxia de la CélulaRESUMEN
Sensitive monitoring of human 8-oxyguanine DNA glycosylase (hOGG1) activity in living cells is helpful to understand its function in damage repair and evaluate its role in disease diagnosis. Herein, a functional DNA-Zn2+ coordination nanospheres was proposed for sensitive imaging of hOGG1 in living cells. The nanospheres were constructed through the coordination-driven self-assembly of the entropy driven reaction (EDR) -deoxyribozyme (DNAzyme) system with Zn2+, where DNAzyme was designed to split structure and assembled into the EDR system. When the nanospheres entered the cell, the competitive coordination between phosphate in the cell and Zn2+ leaded to the disintegration of the nanospheres, releasing DNA and some Zn2+. The released Zn2+ acted as a cofactor of DNAzyme. In the presence of hOGG1, the EDR was completed, accompanied by fluorescence recovery and the generation of a complete DNAzyme. With the assistance of Zn2+, DNAzyme continuously cleaved substrates to produce plenty of fluorescence signals, thus achieving sensitive imaging of hOGG1 activity. The nanospheres successfully achieved sensitive imaging of hOGG1 in human cervical cancer cells (HeLa), human non-small cell lung cancer cells and human normal colonic epithelial cells, and assayed changes in hOGG1 activity in HeLa cells. This nanospheres may provide a new tool for intracellular hOGG1 imaging and related biomedical studies.
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ADN Glicosilasas , ADN Catalítico , Nanosferas , Zinc , Humanos , Nanosferas/química , Zinc/química , ADN Catalítico/química , ADN Catalítico/metabolismo , ADN Glicosilasas/metabolismo , ADN Glicosilasas/química , Células HeLa , Imagen Óptica , ADN/química , ADN/metabolismoRESUMEN
Mitochondria-targeting agents, known as mitocans, are emerging as potent cancer therapeutics due to pronounced metabolic and apoptotic adaptations in the mitochondria of cancer cells. ONC212, an imipridone-family compound initially identified as a ClpP agonist, is currently under investigation as a potential mitocan with demonstrated preclinical efficacy against multiple malignancies. Despite this efficacy, the molecular mechanism underlying the cell death induced by ONC212 remains unclear. This study systematically investigates the mitochondrial involvement and signaling cascades associated with ONC212-induced cell death, utilizing HeLa and A549 cancer cells. Treated cancer cells exhibited characteristic apoptotic features, such as annexin-V positivity and caspase-3 activation; however, these occurred independently of typical mitochondrial events like membrane potential loss (ΔΨm) and cytochrome c release, as well as caspase-8 activation associated with the extrinsic pathway. Additionally, ONC212 treatment increased the expression of anti-apoptotic proteins Bcl-2 and Bcl-xL, which impeded apoptosis, as the overexpression of Bcl-2-GFP and Bcl-xL-GFP significantly reduced ONC212-mediated cell death. Furthermore, combining a sub-lethal dose of the Bcl-2/Bcl-xL inhibitor Navitoclax with ONC212 markedly augmented caspase-3 activation and cell death, still without any notable ΔΨm loss or cytochrome c release. Moreover, inhibition of caspase-9 activity unexpectedly augmented, rather than attenuated, caspase-3 activation and the subsequent cell death. Collectively, our research identifies ONC212 as an atypical mitochondrial-independent, yet Bcl-2/Bcl-xL-inhibitable, caspase-3-mediated apoptotic cell death inducer, highlighting its potential for combination therapies in tumors with defective mitochondrial apoptotic signaling.
Asunto(s)
Compuestos de Anilina , Apoptosis , Caspasa 3 , Sinergismo Farmacológico , Mitocondrias , Sulfonamidas , Humanos , Apoptosis/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Compuestos de Anilina/farmacología , Sulfonamidas/farmacología , Caspasa 3/metabolismo , Células HeLa , Activación Enzimática/efectos de los fármacos , Células A549 , Citocromos c/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Antineoplásicos/farmacología , Compuestos de Bencilo , Compuestos Heterocíclicos con 3 AnillosRESUMEN
Cervical cancer is a leading cause of gynecological malignancies and cancer-related deaths among women worldwide. This study investigates the anti-cancer activity of Thua Nao, a Thai fermented soybean, against HeLa cervical carcinoma cells, and explores its underlying mechanisms. Our findings reveal that the ethyl acetate fraction of Thua Nao (TN-EA) exhibits strong anti-cancer potential against HeLa cells. High-performance liquid chromatography (HPLC) analysis identified genistein and daidzein as the major isoflavones in TN-EA responsible for its anti-cancer activity. TN-EA and genistein reduced cell proliferation and induced G2/M phase arrest, while daidzein induced G1 arrest. These responses were associated with the downregulation of cell cycle regulators, including Cyclin B1, cycle 25C (Cdc25C), and phosphorylated cyclin-dependent kinase 1 (CDK-1), and the upregulation of the cell cycle inhibitor p21. Moreover, TN-EA and its active isoflavones promoted apoptosis in HeLa cells through the intrinsic pathway, evidenced by increased levels of cleaved Poly (ADP-ribose) polymerase (PARP) and caspase-3, loss of mitochondrial membrane potential, and the downregulation of anti-apoptotic proteins B-cell leukemia/lymphoma 2 (Bcl-2), B-cell lymphoma-extra-large (Bcl-xL), cellular inhibitor of apoptosis proteins 1 (cIAP), and survivin. Additionally, TN-EA and its active isoflavones effectively reduced cell invasion and migration by downregulating extracellular matrix degradation enzymes, including Membrane type 1-matrix metalloproteinase (MT1-MMP), urokinase-type plasminogen activator (uPA), and urokinase-type plasminogen activator receptor (uPAR), and reduced the levels of the mesenchymal marker N-cadherin. At the molecular level, TN-EA suppressed STAT3 activation via the regulation of JNK and Erk1/2 signaling pathways, leading to reduced proliferation and invasion of HeLa cells.