RESUMO
Vernonanthura polyanthes (Spreng.) A.J. Vega & Dematt. (syn.: Vernonia polyanthes Less) is popularly known as "assa-peixe" and its leaves are used in folk medicine mainly to treat respiratory diseases. In this study, we evaluated the cytogenotoxic and anticytogenotoxic potential of the V. polyanthes leaf aqueous extract (VpLAE) and its n-butanol fraction (n-BF) in the presence or absence of doxorubicin (DXR) (pre-, co-, and post-treatments) on a murine model for 24 h or 120 h. The micronucleus test (MN) and the comet assay were used to assess the cytogenotoxic and anticytogenotoxic potential of VpLAE and n-BF (250, 500, and 1000 mg/kg) administered via gavage to Swiss Webster mice. The chemical profiles of VpLAE and n-BF were assessed by liquid chromatography coupled to mass spectrometry, and their metabolites were putatively identified. Lastly, the possible biological activities related to the (anti) cytogenotoxicity of the compounds were predicted using the PASS online webserver. The in vivo results showed that different doses of VpLAE and n-BF did not present cytotoxic activity; however, the MN test revealed a slight mutagenic activity for the 24 h treatments. Moderate genotoxic effects were demonstrated for all treatments in the comet assay. Regarding anticytotoxicity and antimutagenicity, VpLAE and n-BF presented a high cytoprotective potential against DXR toxic effects. In the co-treatment, VpLAE reduced the DXR genotoxicity by ~27%, and n-BF did not demonstrate antigenotoxic potential. In contrast, an antigenotoxic effect was observed for both VpLAE and n-BF in the pre- and post-treatments, reducing DXR genotoxicity by ~41% and ~47%, respectively. Chemical analysis of VpLAE and n-BF showed the presence of eight phenolic compounds, including seven chlorogenic acids and a flavonoid. The PASS online tool predicted antimutagenic, anticancer, antineoplastic, chemoprotective, antioxidant, and radical scavenging activities for all constituents identified in VpLAE and n-BF. V. polyanthes leaves presented a protective effect against DXR cytogenotoxicity. In general, VpLAE and n-BF showed a greater antigenotoxic potential in the pre- and post-treatments. The metabolites putatively identified in VpLAE and n-BF exhibited antioxidant and chemoprotective potential according to computational prediction analysis. Altogether, our results highlight the potential application of V. polyanthes to protect against toxic manifestations induced by DXR.
Assuntos
Antioxidantes , Asteraceae , Animais , Antioxidantes/farmacologia , Dano ao DNA , Doxorrubicina/efeitos adversos , Doxorrubicina/análise , Cromatografia Gasosa-Espectrometria de Massas , Camundongos , Testes para Micronúcleos , Compostos Fitoquímicos/análise , Extratos Vegetais/química , Folhas de Planta/químicaRESUMO
To evaluate the effect of the host plant on the quality of Loranthaceae species as medicinal raw material, ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) was used to identify cardiac glycosides in Nerium indicum and its parasitic plant species Taxillus chinensis and Scurrula parasitica. Samples were collected from N. indicum and these parasites, while Morus alba and its parasite T. chinensis and Osmanthus fragrans and its parasite S. parasitica were used as controls. Based on mass spectrometry data and elemental composition analysis of positive and negative ion modes, in combination with standard cardiac glycosides and relevant literature, cardiac glycosides in N. indicum and its parasites T. chinensis and S. parasitica were identified, and their correlations were analyzed. A total of 29 cardiac glycosides were identified, among which 28 were found in N. indicum parasitized by T. chinensis; 25 cardiac glycosides were identified in the same host under attack by S. parasitica; five cardiac glycosides were identified in both T. chinensis and S. parasitica, which grew parasitically on N. indicum, whereas no cardiac glycosides were identified in M. alba parasitized by T. chinensis, or in O. fragrans parasitized by S. parasitica. We conclude that UPLC-Q-TOF-MS/MS technology can identify cardiac glycosides in N. indicum and parasites T. chinensis and S. parasitica rapidly, accurately, and thoroughly. N. indicum will transfer its own cardiac glycosides to its parasites through the special host-parasite interaction. Our results provide a reference basis for evaluating the influence of the host plant on the quality of medicinal compounds obtained from Loranthaceae species.
Assuntos
Glicosídeos Cardíacos/química , Loranthaceae/química , Nerium/química , Cromatografia Líquida de Alta Pressão , Digitoxigenina/análise , Doxorrubicina/análogos & derivados , Doxorrubicina/análise , Medicamentos de Ervas Chinesas/química , Flavonoides/química , Estrutura Molecular , Plantas Medicinais/química , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Screening for potential drug combinations presents significant challenges to the current microfluidic cell culture systems, due to the requirement of flexibility in liquid handling. To overcome this limitation, we present here an open-access microfluidic tissue array system specifically designed for drug combination screening. The microfluidic chip features a key structure in which a nanoporous membrane is sandwiched by a cell culture chamber array layer and a corresponding media reservoir array layer. The microfluidic approach takes advantage of the characteristics of the nanoporous membrane: on one side, this membrane permits the flow of air but not liquid, thus acting as a flow-stop valve to enable automatic cell distribution; on the other side, it allows diffusion-based media exchange and thus mimics the endothelial layer. In synergy with a liquid-transferring platform, the open-access microfluidic system enables complex multistep operations involving long-term cell culture, medium exchange, multistep drug treatment, and cell-viability testing. By using the microfluidic protocol, a 10 × 10 tissue array was constructed in 90 s, followed by schedule-dependent drug testing. Morphological and immunohistochemical assays indicated that the resultant tumor tissue was faithful to that in vivo. Drug-testing assays showed that the incorporation of the nanoporous membrane further decreased killing efficacy of the anticancer agents, indicating its function as an endothelial layer. Robustness of the microfluidic system was demonstrated by implementing a three-factor, three-level orthogonal screening of anticancer drug combinations, with which 67% of the testing (9 vs. 27) was saved. Experimental results demonstrated that the microfluidic tissue system presented herein is flexible and easy-to-use, thus providing an ideal tool for performing complex multistep cell assays with high efficiencies.
Assuntos
Antineoplásicos/análise , Cisplatino/análise , Doxorrubicina/análise , Técnicas Analíticas Microfluídicas , Paclitaxel/análise , Análise Serial de Tecidos , Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cisplatino/farmacologia , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Paclitaxel/farmacologia , Relação Estrutura-AtividadeRESUMO
RATIONALE: Cell-based drug assay plays an essential role in drug development. By coupling a microfluidic chip with mass spectrometry (MS), we developed a multifunctional platform. Cell co-culture, cell apoptosis assay, fluorescence and MS detection of intracellular drug absorption could be simultaneously conducted on this platform. METHODS: Three micro-channels were fabricated through photolithography technology to conduct the cell co-culture. Cell apoptosis after drug treatment was assayed by fluorescent probes (Hoechst 33342). Intracellular Dox absorption was analyzed by confocal fluorescent microscopy. With a high voltage (~ 4.5 kV) applied onto the microfluidic chip, the ionization spray was successfully generated by dropping isopropanol onto it. By coupling with a Shimadzu LCMS-2010 A mass spectrometer, intracellular CPA absorption was detected on the microfluidic chip. RESULTS: The microfluidic chip-MS coupled platform showed high biocompatibility. Distinction of cell apoptosis between co-cultured and mono-cultured cells was detected. The results of intracellular drug absorption well explained the different cell apoptosis rate. CONCLUSIONS: Cell-based drug assay was facilely and successfully conducted on the microfluidic chip-MS coupled platform. This technology we have devised could promote MS application in the field of drug development. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Técnicas de Cocultura/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Dispositivos Lab-On-A-Chip , Espectrometria de Massas por Ionização por Electrospray/métodos , Apoptose/efeitos dos fármacos , Técnicas de Cocultura/instrumentação , Ciclofosfamida/análise , Ciclofosfamida/farmacologia , Doxorrubicina/análise , Doxorrubicina/farmacologia , Avaliação Pré-Clínica de Medicamentos/instrumentação , Desenho de Equipamento , Fluorescência , Células Hep G2 , Humanos , Marcação por Isótopo/métodos , Células MCF-7 , Espectrometria de Massas por Ionização por Electrospray/instrumentaçãoRESUMO
Diarylheptanoids belong to polyphenols, a group of plant secondary metabolites with multiple biological properties. Many of them display antioxidative, cytotoxic, or anticancer actions and are increasingly recognized as potential therapeutic agents. The aim of this study was to evaluate antioxidant and cytoprotective activity of two diarylheptanoids: platyphylloside 5(S)-1,7-di(4-hydroxyphenyl)-3-heptanone-5-O-ß-D-glucopyranoside (1) and its newly discovered analog 5(S)-1,7-di(4-hydroxyphenyl)-5-O-ß-D-[6-(E-p-coumaroylglucopyranosyl)]heptane-3-one (2), both isolated from the bark of black alder (Alnus glutinosa). To that end, we have employed a cancer cell line (NCI-H460), normal human keratinocytes (HaCaT), and peripheral blood mononuclear cells. The effects on cell growth were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Cell death was examined by annexin V/propidium iodide staining on a flow cytometer. Reactive oxygen species production was examined by dihydroethidium staining. Mitochondrial structure and doxorubicin localization were visualized by fluorescent microscopy. Gene expression of manganese superoxide dismutase and hypoxia-inducible factor-1α was determined by reverse transcription polymerase chain reaction. Diarylheptanoids antagonized the effects of either doxorubicin or cisplatin, significantly increasing their IC50 values in normal cells. Diarylheptanoid 1 induced the retention of doxorubicin in cytoplasm and reduced mitochondrial fragmentation associated with doxorubicin application. Diarylheptanoid 2 reduced the reactive oxygen species production induced by cisplatin. Both compounds increased the messenger ribonucleic acid expression of enzymes involved in reactive oxygen species elimination (manganese superoxide dismutase and hypoxia-inducible factor-1α). These results indicate that neutralization of reactive oxygen species is an important mechanism of diarylheptanoid action, although these compounds exert a considerable anticancer effect. Therefore, these compounds may serve as protectors of normal cells during chemotherapy without significantly diminishing the effect of the applied chemotherapeutic.
Assuntos
Alnus/química , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Diarileptanoides/farmacologia , Interações Ervas-Drogas , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Antioxidantes/química , Morte Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Diarileptanoides/química , Diarileptanoides/isolamento & purificação , Doxorrubicina/análise , Doxorrubicina/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Casca de Planta/química , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
Flavonoids have always attracted much attention due to their reversal activity on multidrug resistance (MDR). Eight flavonoids isolated from traditional Chinese medicine Sophora alopecuroides L. were applied to test their effect on MDR associated protein 1 (MRP1) through the established predicting assay. Three flavonostilbenes (alopecurone A, B and D) were first found exhibiting potent inhibitory activity on MRP1. All of them dramatically increased 6-carboxyfluorescein diacetate and doxorubicin accumulation in MRP1-transfected U-2 OS cells. The compounds significantly increased the cytotoxicity and decreased the IC50 value of doxorubicin on the MDR cells (12-, 5- and 8-fold, respectively) at a non-toxic concentration (20 µM). Besides, Q-PCR analysis reveals that the MRP1 mRNA level in U-2 OS/MRP1 was also markedly decreased by the three compounds. These findings indicate a new therapeutic role of the herb. The three flavonostilbenes may have the possibility for further development as novel therapeutic reversal agents against MDR.
Assuntos
Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Sophora/química , Estilbenos/farmacologia , Doxorrubicina/análise , Doxorrubicina/farmacologia , Flavonoides/química , Fluoresceínas/análise , Fluoresceínas/farmacologia , Concentração Inibidora 50 , Estrutura Molecular , Estilbenos/química , Estilbenos/isolamento & purificaçãoRESUMO
Because liver cancer is rarely suitable for surgery, transcatheter arterial chemoembolization (TACE) is used for palliative therapy. In this procedure, an emulsion of doxorubicin in iodized oil is injected directly into liver tumors through a catheter positioned within the artery supplying blood flow to the tumor. At present, there is limited understanding of factors affecting the delivery and dispersion of doxorubicin within treated tumors during TACE. This study addresses the development and application of an ultrahigh-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS) method for rapid confirmation of drug delivery after TACE in a rabbit VX2 liver cancer model. Doxorubicin levels in liver tumors were measured using UHPLC-MS-MS and compared with computed tomography measured levels of iodized oil, a metric used clinically to indicate drug delivery. We found that tissue drug levels determined using UHPLC-MS-MS did not correlate with the regional iodized oil concentration (vehicle) within tumors following TACE, suggesting that chemotherapeutic drugs like doxorubicin spread throughout tumors, and that lack of iodized oil staining in portions of a tumor does not necessarily indicate inadequate therapy during TACE.
Assuntos
Quimioembolização Terapêutica/métodos , Cromatografia Líquida de Alta Pressão/métodos , Doxorrubicina/análise , Neoplasias Hepáticas Experimentais/química , Neoplasias Hepáticas Experimentais/terapia , Espectrometria de Massas em Tandem/métodos , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/análise , Antibióticos Antineoplásicos/farmacocinética , Quimioembolização Terapêutica/instrumentação , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacocinética , Sistemas de Liberação de Medicamentos , Emulsões/química , Óleo Etiodado/farmacocinética , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Masculino , Coelhos , Tomografia Computadorizada por Raios XRESUMO
In this study, we are pioneering new nanotechnology for the encapsulation of anticancer drugs (doxorubicin (DOX) and/or docetaxel (DOCE)), whatever their solubility and water affinity. The purpose of this study is to highlight the potential of this recently patented technology, by carrying out a thorough physicochemical characterisation of these multiscaled nanocarriers, followed by the study of an encapsulation and release model of hydrophilic anticancer drug. The formulation process is based on a low-energy nano-emulsification method and allows the generation of a structure composed of oil-based nanocarriers loaded with reverse micelles. Thanks to this, hydrophilic contents can be solubilised in the oily core of this kind of nano-emulsion along with lipophilic content. The results emphasise some original structure particularities due to the multistep formulation process, and the diffusion-based behaviour revealed for the DOX release profile that is shown to be intimately linked to the morphology of the particles.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Micelas , Nanoestruturas/química , Antibióticos Antineoplásicos/análise , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/metabolismo , Preparações de Ação Retardada/análise , Preparações de Ação Retardada/química , Preparações de Ação Retardada/metabolismo , Doxorrubicina/análise , Doxorrubicina/química , Doxorrubicina/metabolismo , Composição de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Emulsões , Excipientes/química , Liofilização , Lipídeos/química , Polímeros/química , Solubilidade , Triglicerídeos/químicaRESUMO
PURPOSE: The purpose of this work was to develop and validate a method that can be used to quantify drugs associated with intracellular compartments. METHODS: The human leukemic cell line U-937 was used to evaluate the distribution of model compounds with known and different subcellular distribution profiles. Lysotracker Red is a lysosomal vital stain and doxorubicin is an anticancer agent with a strong propensity for nuclear accumulation in U-937 cells. After incubation with compounds, cells were separated into fractions containing nuclei, cytosol, and cytoplasmic organelles (lysosomes, mitochondria, Golgi apparatus). Compounds contained within isolated fractions were subsequently extracted and analyzed by high-performance liquid chromatography. Diffusion of compounds from isolated organelles was also investigated. RESULTS: Using this approach we have shown that the model compounds Lysotracker Red and doxorubicin preferentially accumulated within lysosomes and nuclei, respectively. We have reproducibly determined concentrations of these compounds in each of the cellular fractions. We have also shown that diffusion of these compounds from isolated cellular compartments was minimal during the time required to complete the experimental procedure. CONCLUSIONS: The analytical approach described in this manuscript yielded reproducible quantitative data regarding the intracellular distribution of model compounds in U-937 cells. With the aid of a relatively sensitive analytical assay, this technique should be useful for most drugs that have a specific concentrative mechanism for organelle accumulation similar to Dox and LTR.
Assuntos
Fracionamento Celular/métodos , Núcleo Celular/metabolismo , Citosol/metabolismo , Doxorrubicina/farmacocinética , Núcleo Celular/química , Citosol/química , Doxorrubicina/análise , Avaliação Pré-Clínica de Medicamentos/métodos , Corantes Fluorescentes/análise , Corantes Fluorescentes/farmacocinética , Humanos , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Células U937RESUMO
Cruciferous vegetables play an important role because of their sulphorafane contents which are enzymatically released from the glucosinolate known as glucoraphanin. The physiological properties of the compound exhibit antitumorigenic activity. The work describes the chloroform extraction method of sulforaphane from the broccoli and the preparation of sulforaphane sugar extract. The extract was then used to feed bees in a specially constructed beehive so that sulforaphane could be transformed into herbal honey. The concentration of sulforaphane was determined in the obtained herbal honey as high as 1.2 microM.
Assuntos
Terapias Complementares/métodos , Doxorrubicina/análogos & derivados , Doxorrubicina/análise , Mel/análise , Neoplasias/prevenção & controle , Plantas , Tiocianatos/análise , Cromatografia Líquida de Alta Pressão , Humanos , Isotiocianatos , SulfóxidosRESUMO
HPLC was used to analyze doxorubicin in multidrug-resistance (MDR) human carcinoma cells. This method is novel, simple, sensitive, linear, accurate and precise. The minimal detectable concentration is 0.2 microg ml(-1). The reversal effects of tea polyphenols on MDR are elucidated by this method. The results indicate that the tea polyphenol, (-)-epigallocatechin gallate, is a potential modulator of MDR.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Catequina/análogos & derivados , Catequina/farmacologia , Doxorrubicina/análise , Doxorrubicina/farmacologia , Resistência a Múltiplos Medicamentos , Flavonoides , Fenóis/farmacologia , Polímeros/farmacologia , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/fisiopatologia , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Neoplasias Bucais/metabolismo , Neoplasias Bucais/fisiopatologia , Chá/química , Células Tumorais CultivadasRESUMO
Comparative peptide mapping of a monoclonal antibody chimeric BR96 and corresponding doxorubicin (DOX) immunoconjugate was performed using capillary electrophoresis (CE) and capillary liquid chromatography (CLC). A unique, highly sensitive and selective approach combined with both UV absorbance and laser-induced fluorescence (LIF) detection has been developed and applied to studies including enzymatic digests of antibody and conjugate and related drug and conjugation linker substances. The analytical methodology has been established based on the unique characteristic of the anticancer drug DOX which yields native fluorescence. With an excitation wavelength of 488 nm from argon-ion laser, DOX conjugated to the monoclonal antibody using a hydrazone linker can be determined with a detection limit at the attomole level. Approaches were developed based on the successful conjugation and analysis of a model peptide conjugate. Enzymatic digests of the monoclonal antibody BR96 and its immunoconjugate were mapped by CE and CLC with on-line UV and LIF detection, which results in a unique fingerprint for structural analysis. With a two-dimensional LC-CE approach, conjugated peptide-DOX species from LC were further analyzed by CE with LIF detection. The drug-containing peptide fragments in the mixture were readily detected, which can be further characterized using other complementary analytical techniques.
Assuntos
Anticorpos Monoclonais/análise , Anticorpos Monoclonais/metabolismo , Cromatografia Líquida/métodos , Eletroforese Capilar/métodos , Imunoconjugados/análise , Imunoconjugados/metabolismo , Sequência de Aminoácidos , Antibióticos Antineoplásicos/análise , Antibióticos Antineoplásicos/metabolismo , Cistina/análise , Doxorrubicina/análise , Doxorrubicina/metabolismo , Dados de Sequência Molecular , Mapeamento de Peptídeos , Tripsina/metabolismoRESUMO
Verapamil, a calcium channel blocker, reduces resistance of some tumors to Adriamycin by inhibiting Adriamycin efflux from resistant cells. This study shows that rabbits given 7 mg/kg of Adriamycin and 0.8 mg/kg of verapamil died earlier with functional and morphological cardiac abnormalities than rabbits given Adriamycin alone. Rabbits that were given verapamil and died peracutely had higher concentrations of Adriamycin in the myocardium. The relatively high dose of Adriamycin was expected to produce cardiotoxicity in rabbits within a matter of days. However, peracute death (within hours) in rabbits given the same dose of Adriamycin but with verapamil treatment was unexpected. These results suggest that combining Adriamycin with verapamil increases early cardiac toxicity. Therefore, clinical use of this combination should be approached cautiously until the interactions, including the possibility for enhanced normal tissue damage, are fully understood.