RESUMEN
In recent years, targeting drugs made by physical loading or chemical bonding of drugs on small molecular carriers have shown a very wide application prospect in the field of tumor and cancer treatment. How to achieve the release of drugs in cancer cells has become the core of this research. One of the most important bases for drug localization is to use the difference of small molecular biothiol concentration between cancer cells and normal cells. Details of the changes of biothiol levels in the growth and reproduction of cancer cells are still poorly understood, and the main reason is the lack of sensitive real-time imaging tools for biothiols in cancer cells. In this work, we reasonably designed and synthesized the combination of 4-hydroxy-1,8-naphthalimide and NBD-Cl as a concise fluorescent probe HN-NBD for imaging biothiols in live cells and zebrafish. In addition, due to the advantages of HN-NBD design, it is sufficiently sensitive to biothiols, and further imaging can distinguish cancer cells from normal cells. Probe HN-NBD would be of great significance to biomedical researchers for the study of biothiol-related diseases, the screening of new anticancer drugs, and the early diagnosis and treatment of cancers.
Asunto(s)
Cisteína/análisis , Colorantes Fluorescentes/química , Glutatión/análisis , Homocisteína/análisis , Neoplasias/diagnóstico por imagen , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/síntesis química , 4-Cloro-7-nitrobenzofurazano/toxicidad , Animales , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/toxicidad , Células HeLa , Células Endoteliales de la Vena Umbilical Humana , Humanos , Límite de Detección , Ratones , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Naftalimidas/síntesis química , Naftalimidas/química , Naftalimidas/toxicidad , Imagen Óptica/métodos , Células RAW 264.7 , Pez CebraRESUMEN
H2S is an important endogenous gasotransmitter, and its detection in living systems is of great significance. Especially, selective and sensitive near-infrared (NIR) fluorescent H2S probes with rapid response and large Stokes shift are highly desirable because of their superiority for in vivo detection. Probes with nitrobenzoxadiazole (NBD) ether as reaction sites have been well-explored recently to detect biothiols or H2S/biothiols simultaneously, rather than to detect H2S selectively. In this work, a new NBD ether-based NIR fluorescent probe was developed, which was unexpectedly found to show high selectivity for H2S over various other analytes including biothiols, making it practical for specific detection of H2S both in vitro and in vivo. Upon response to H2S, this probe showed rapid and significant turn-on NIR emission changes centered at 744 nm within 3 min, together with a remarkable large Stokes shift (166 nm) and high sensitivity (LOD: 26 nM). Moreover, imaging exogenous and endogenous H2S in living cells and rapid imaging of H2S in living mice with this probe was successfully applied with excellent performance.
Asunto(s)
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Colorantes Fluorescentes/química , Sulfuro de Hidrógeno/análisis , Xantenos/química , 4-Cloro-7-nitrobenzofurazano/síntesis química , 4-Cloro-7-nitrobenzofurazano/toxicidad , Animales , Línea Celular Tumoral , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/toxicidad , Humanos , Límite de Detección , Ratones , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Imagen Óptica , Xantenos/síntesis química , Xantenos/toxicidadRESUMEN
As a multifunctional signaling molecule, hydrogen sulfide (H2S) plays an essential role in diverse physiological and pathological processes. The two-photon fluorescence probes detecting H2S selectively in vivo could be useful tools to better study the mechanism of diseases. Then, an efficient two-photon lysosome-specific probe 1 has been developed to detect endogenous H2S in living cells and mice. Probe 1 displays excellent properties with 28-fold fluorescence enhancement, marked color changes in naked-eye and fluorescence, high selectivity and sensitivity, and low detection limit (0.22⯵M) to H2S. These remarkable properties of probe 1 enable its practical applications in detecting H2S in environment (wastewater) and food (beer). Moreover, as a two-photon probe under near infrared excitation at 790â¯nm, probe 1 can monitor the level changes of endogenous H2S of lysosome and tumor in living system with good membrane permeability and high imaging resolution. Specially, the probe detecting H2S distribution in lysosome could provide more evidences to explain the association of target-organelle and H2S.
Asunto(s)
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Neoplasias de la Mama/metabolismo , Colorantes Fluorescentes/química , Sulfuro de Hidrógeno/análisis , Lisosomas/metabolismo , Naftalimidas/química , 4-Cloro-7-nitrobenzofurazano/síntesis química , 4-Cloro-7-nitrobenzofurazano/toxicidad , Animales , Cerveza/análisis , Línea Celular Tumoral , Colorimetría/métodos , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/toxicidad , Humanos , Límite de Detección , Ratones Desnudos , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Naftalimidas/síntesis química , Naftalimidas/toxicidad , Fotones , Espectrometría de Fluorescencia/métodos , Aguas Residuales/análisisRESUMEN
Abnormal levels of Cys, Hcy and GSH are associated with various diseases, thus monitoring biothiols is of great significance. In this work, a dual-emission responsive near-infrared fluorescent probe NIR-NBD for detecting Hcy and Cys/GSH was developed based on the conjugation of a dicyanoisophorone based fluorophore (NIR-OH) and 7-nitrobenzofurazan (NBD). To our surprise, the addition of Hcy induced significant fluorescence enhancement at both 549 and 697â¯nm; while Cys/GSH resulted in major fluorescence emission at 697â¯nm. The detection limit was determined to be 33.2â¯nM for Cys, 33.5â¯nM for Hcy, and 34.4â¯nM for GSH. Therefore, the probe can be used for discriminative detection of Hcy and Cys/GSH. Moreover, fluorescence imaging of HeLa cells indicated that the probe was cell membrane permeable and could be used for visualizing Hcy and Cys/GSH in living cells.
Asunto(s)
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Aminofenoles/química , Cisteína/análisis , Colorantes Fluorescentes/química , Glutatión/análisis , Homocisteína/análisis , 4-Cloro-7-nitrobenzofurazano/síntesis química , 4-Cloro-7-nitrobenzofurazano/toxicidad , Aminofenoles/síntesis química , Aminofenoles/toxicidad , Fluorescencia , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/toxicidad , Células HeLa , Humanos , Límite de Detección , Microscopía Confocal/métodos , Microscopía Fluorescente/métodosRESUMEN
Biothiols, including cysteine (Cys), homocysteine (Hcy), glutathione (GSH) and H2S, play important roles in human physiological processes. However, it is a great difficulty to distinguish biothiols from each other because of their similar chemical properties. Based on Nile red, we have designed and synthesized a near-infrared fluorescent probe for discriminating Cys/Hcy from GSH/H2S by a dual-channel detection method. Using an ether bond, near-infrared Nile red was attached to 7-nitrobenzofurazan to construct the probe. Due to the photo-induced electron transfer, the probe showed almost no fluorescence from the green to red emission band. But upon the addition of Cys (0-150 µM) or Hcy (0-200 µM), the probe exhibited a noteworthy fluorescence "turn-on" signal in two unique emission bands (Green and Red) with a fast response (within 30 min). In contrast, the probe displayed an increase in fluorescence only in the red channel when encountering GSH (0-70 µM) or H2S (0-50 µM), and GSH/H2S could be tested respectively by different response time. The limit of detection was calculated to be 0.09 µM (Cys), 0.30 µM (Hcy), 0.24 µM (GSH), and 0.04 µM (H2S), respectively (based on S/N = 3). The desirable dual-channel detection could be achieved in serum samples and living cells. Moreover, the probe could be applied for bioimaging in mice, which indicated its potential application in the clinic.
Asunto(s)
Cisteína/análisis , Colorantes Fluorescentes/química , Glutatión/análisis , Homocisteína/análisis , Sulfuro de Hidrógeno/análisis , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/síntesis química , 4-Cloro-7-nitrobenzofurazano/toxicidad , Animales , Línea Celular Tumoral , Fluorescencia , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/toxicidad , Humanos , Concentración de Iones de Hidrógeno , Límite de Detección , Ratones Desnudos , Imagen Óptica/métodos , Oxazinas/síntesis química , Oxazinas/química , Oxazinas/toxicidad , Espectrometría de FluorescenciaRESUMEN
The role, origin, and mode of action of the lipid messenger ceramide in programmed cell death and its linkage to receptor-associated apoptotic signal proteins is still unresolved. We show here in Kym-1 rhabdomyosarcoma cells that tumor necrosis factor (TNF)-induced apoptosis is preceded by a multiphasic increase in intracellular ceramide levels. Distinct enzymes were found to contribute to three waves of ceramide, neutral sphingomyelinase, ceramide synthase, and acid sphingomyelinase, with peak activities at 1-2, 40, and around 200 min, respectively, the latter coinciding with progression to irreversible damage. In parallel with ceramide generation, TNF-mediated inhibition of glucosylceramide and sphingomyelin (SM) synthase prevents the immediate metabolization of this lipid mediator. In the presence of benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-fmk) or benzyloxycarbonyl-Asp-Glu-Val-Asp-chloromethyl ketone (Z-DEVD-cmk), a broad spectrum and a caspase 3-selective inhibitor, respectively, glucosylceramide and SM synthase activity remains unaffected by TNF, and intracellular ceramide accumulation is not observed. Our results show that several lipid enzymes contribute to generation of ceramide in response to TNF and identify glucosylceramide and SM synthase as important regulators of the kinetics and magnitude of intracellular ceramide accumulation. As glucosylceramide and SM synthase activity is caspase-sensitive, our data suggest a novel functional link between caspase(s) and ceramide during apoptotic processes.
Asunto(s)
Apoptosis , Ceramidas/metabolismo , Periodicidad , Rabdomiosarcoma/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/toxicidad , Caspasas/metabolismo , Ceramidas/toxicidad , Activación Enzimática , Colorantes Fluorescentes/toxicidad , Regulación Enzimológica de la Expresión Génica , Humanos , Oxidorreductasas/biosíntesis , Poli(ADP-Ribosa) Polimerasas/metabolismo , Transducción de Señal , Esfingolípidos/biosíntesis , Esfingomielina Fosfodiesterasa/biosíntesis , Células Tumorales CultivadasRESUMEN
We have investigated the effects of H(+)-ATPase inhibitors, bafilomycin A1 and 7-chloro-4-nitro-benz-2-oxa-1,3 diazole (NBD), and the Golgi inhibitor, brefeldin A, on daunorubicin accumulation and doxorubicin intracellular distribution in the non-P-glycoprotein-mediated multidrug-resistant cell line COR-L23/R. This cell line overexpress a 190 kDa protein which is probably the product of the MRP gene and shows an anthracycline accumulation defect and a drastically altered intracellular anthracycline distribution from the parental cell line COR-L23/P. We found that all three agents could selectively increase the cellular accumulation of daunorubicin in resistant cells. However, these effects were only seen at doses of the modifiers which were equal to or greater than the IC50 of the modifier alone. Effects of the modifiers on the intracellular distribution of doxorubicin fluorescence could, however, be seen at doses lower than those required to produce significant effects on daunorubicin accumulation. However, when used in a continuous MTT chemosensitivity assay none of the agents, used at maximum non-toxic doses, was able to sensitise COR-L23/R cells to doxorubicin or to colchicine. Although these lead compounds are unlikely to be useful as clinical modifiers, development of more selective analogues may prove useful in the modification of non-P-glycoprotein-mediated multidrug resistance.