RESUMO
In this report, we investigated the mechanism responsible for synergistic induction of myeloma cell apoptosis induced by the combination of tipifarnib and bortezomib. Immunofluorescence studies revealed that bortezomib alone resulted in an accumulation of puncta of ubiquitinated proteins that was further enhanced by the addition of tipifarnib. These data suggest inhibition of the degradation of bortezomib-induced aggresomes; and consistent with this possibility, we also observed an increase in p62SQSTM1 in cells treated with the combination. However, autophagy in these cells appears to be normal as LC3BII is present, and autophagic flux appears to be unaffected as demonstrated by the addition of bafilomycin A1. Together, these data demonstrate that tipifarnib synergizes with bortezomib by inducing protein accumulation as a result of the uncoupling of the aggresome and autophagy pathways.
Assuntos
Ácidos Borônicos/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteassoma , Pirazinas/farmacologia , Apoptose/efeitos dos fármacos , Autofagia , Bortezomib , Linhagem Celular Tumoral , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Mieloma Múltiplo/patologia , Quinolonas , Ubiquitinação/efeitos dos fármacosRESUMO
Closely involved in the progression of nonlinear bioimaging is the development of optical probes for investigating biological function and activity. Introduction of new fluorescent compounds possessing enhanced nonlinearities is essential for advancing the utility of two-photon absorption (2PA) processes in the biological sciences. Herein, we report the synthesis of fluorene-based fluorophores tailored for multiphoton imaging, incorporating the succinimidyl ester and thioester functionality as reactive linkers for further coupling with a wide variety of biologically relevant molecules. The succinimidyl ester amine reactive probe was conjugated with the cyclic peptide RGDfK and polyclonal antirat IgG protein. Upon conjugation, the basic molecular architecture and photophysical properties of the active 2PA chromophore remain unchanged. Conventional and two-photon fluorescence microscopy (2PFM) imaging of COS-7 and HeLa cells, incubated with either the fluorene-RGD peptide conjugate or the fluorene-IgG conjugate, was demonstrated. The fluorene-IgG conjugate was used to image cell spindles at early mitotic developmental stages.
Assuntos
Fluorenos , Corantes Fluorescentes , Imunoglobulinas/química , Microscopia de Fluorescência por Excitação Multifotônica , Peptídeos Cíclicos/química , Animais , Células COS , Chlorocebus aethiops , Fluorenos/análise , Fluorenos/síntese química , Corantes Fluorescentes/análise , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Imageamento Tridimensional , Imunoglobulinas/imunologia , FótonsRESUMO
The steady-state photophysical, NMR, and two-photon absorption (2PA) properties of a new fluorene derivative (1) containing the 2-(2'-hydroxyphenyl)benzothiazole (HBT) terminal construct is investigated for use as a fluorescence probe in bioimaging. A comprehensive analysis of the linear spectral properties reveals inter- and intramolecular hydrogen bonding and excited state intramolecular proton transfer (ESIPT) processes in the HBT substituent. A specific electronic model with a double minimum potential energy surface is consistent with the observed spectral properties. The 2PA spectra are obtained using a standard two-photon induced fluorescence method with a femtosecond kHz laser system, affording a maximum 2PA cross section of approximately 600 GM, a sufficiently high value for two-photon fluorescence imaging. No dependence of two-photon absorption efficiency on solvent properties and hydrogen bonding in the HBT substituent is observed. The potential use of this fluorenyl probe in bioimaging is demonstrated via one- and two-photon fluorescence imaging of COS-7 cells.
Assuntos
Benzotiazóis/química , Corantes Fluorescentes/química , Prótons , Absorção , Animais , Benzotiazóis/síntese química , Transporte Biológico , Células COS , Chlorocebus aethiops , Espectrometria de FluorescênciaRESUMO
The compound 2-deoxyglucose (2-DG) enhances chemotherapy/radiotherapy in cell lines and animal models, prompting two phase I clinical trials with this cancer therapeutic. Although its mechanism of action has not been fully elucidated, it is hypothesized that the molecular basis of 2-DG activity is related to glycolysis inhibition. Here, we report that 2-DG induced Akt phosphorylation at Thr(308) and Ser(473) as early as 15 min post-treatment. These phosphorylation events required phosphatidylinositol-3-kinase activity but were not related to LKB1/AMP-activated protein kinase signaling, the inhibition of glycolysis or epidermal growth factor receptor signaling. The 2-DG-mediated Akt phosphorylation also led to the phosphorylation of Akt downstream targets, such as Foxo3a, GSK3beta, and Chk1. Because the functional consequence of Akt activation includes chemotherapy/radiotherapy resistance, our data suggested that the combination of phosphatidylinositol-3-kinase/Akt inhibitory agents in 2-DG-based chemotherapy/radiotherapy may result in enhanced therapeutic efficacy.
Assuntos
Antimetabólitos/farmacologia , Desoxiglucose/farmacologia , Glicólise/efeitos dos fármacos , Complexos Multienzimáticos/metabolismo , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP , Trifosfato de Adenosina/metabolismo , Western Blotting , Quinase 1 do Ponto de Checagem , Receptores ErbB/metabolismo , Imunofluorescência , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Luciferases/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/farmacologia , Transdução de Sinais , Células Tumorais CultivadasRESUMO
With the increasing demand for confocal and two-photon fluorescence imaging, the availability of reactive probes that possess high two-photon absorptivity, high fluorescence quantum yield, and high photostability is of paramount importance. To address the demand for better-performing probes, we prepared two-photon absorbing amine-reactive fluorenyl-based probes 2-(9,9-bis(2-(2-methoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)benzothiazole (1) and 2-(4-(2-(9,9-bis(2-(2-ethoxyethoxy)ethyl)-2-isothiocyanato-9H-fluoren-7-yl)vinyl)phenyl)benzothiazole (2), incorporating the isothiocyanate as a reactive linker. Probe design was augmented by integrating high optical nonlinearities, increased hydrophilicity, and coupling with reactive functional groups for specific targeting of biomolecules, assuring a better impact on two-photon fluorescence microscopy (2PFM) imaging. The isothiocyanate (NCS) derivatives were conjugated with cyclic peptide RGDfK and Reelin protein. The study of the chemical and photophysical properties of the new labeling reagents, as well as the conjugates, is described. The conjugates displayed high chemical stability and photostability. The NCS derivatives had low fluorescence quantum yields, while their bioconjugates exhibited high fluorescence quantum yields, essentially "lighting up" after conjugation. Conventional and 2PFM imaging and fluorescence lifetime imaging (FLIM) of HeLa, NT2, and H1299 cells, incubated with two-photon absorbing amine-reactive probe (1), RGDfK-dye conjugate (7), and Reelin-dye conjugate (6), was demonstrated.
Assuntos
Aminas/química , Fluorenos/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Fótons , Absorção , Butilaminas/química , Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular , Elétrons , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Corantes Fluorescentes/química , Humanos , Indicadores e Reagentes/química , Isotiocianatos/química , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Proteína Reelina , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Análise EspectralRESUMO
Farnesyl transferase inhibitors (FTI) exhibit anticancer activity as a single agent in preclinical studies and show promise in combination with other therapeutics in clinical trials. Previous studies show that FTIs arrest cancer cells in mitosis; however, the mechanism by which this occurs is unclear. Here, we observed that treatment of various cancer cell lines with the FTI lonafarnib caused mitotic chromosomal alignment defects, leaving cells in a pseudometaphase state, whereby both aligned chromosomes and chromosomes juxtaposed to the spindle poles (termed "lagging chromosomes") were observed in the same cell. To determine how this occurs, we investigated the functionality of two farnesylated mitotic proteins, CENP-E and CENP-F, which mediate chromosomal capture and alignment. The data show that lonafarnib in proliferating cancer cells depletes CENP-E and CENP-F from metaphase but not prometaphase kinetochores. Loss of CENP-E and CENP-F metaphase localization triggered aberrant chromosomal maintenance, causing aligned chromosomes to be prematurely released from the spindle equator and become lagging chromosomes, resulting in a mitotic delay. Furthermore, lonafarnib treatment reduces sister kinetochore tension and activates the BubR1 spindle checkpoint, suggesting that farnesylation of CENP-E and CENP-F is critical for their functionality in maintaining kinetochore-microtubule interactions. Importantly, apparently similar chromosomal alignment defects were observed in head and neck tumors samples from a phase I trial with lonafarnib, providing support that lonafarnib disrupts chromosomal maintenance in human cancers. Lastly, to examine how farnesylation could regulate CENP-E in mediating kinetochore-microtubule attachments, we examined possible docking motifs of a farnesyl group on the outer surface of the microtubule. This analysis revealed three hydrophobic patches on the tubulin dimer for insertion of a farnesyl group, alluding to the possibility of an association between a farnesyl group and the microtubule.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Humanos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Farnesiltranstransferase/antagonistas & inibidores , Neoplasias de Cabeça e Pescoço/metabolismo , Proteínas dos Microfilamentos/metabolismo , Piperidinas/farmacologia , Piridinas/farmacologia , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromossomos Humanos/genética , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Cinetocoros/efeitos dos fármacos , Metáfase/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Piperidinas/química , Piperidinas/uso terapêutico , Prometáfase/efeitos dos fármacos , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Piridinas/química , Piridinas/uso terapêutico , Fuso Acromático/efeitos dos fármacosRESUMO
Donor-pi-acceptor fluorene derivative 1c is a near-neutral pH indicator whose pKa of approximately 7.0 was determined by both absorption and fluorescence methods. 1c satisfies important criteria for a sensitive ratiomeric fluorescent pH indicator with a distinctive isoemissive point, good dispersion in cell cytosol, and low cytotoxicity. Furthermore, its 2PA cross section of 100 GM in its neutral form suggests its potential in two-photon fluorescence imaging applications.
Assuntos
Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Radiometria , Solubilidade , ÁguaRESUMO
Two-photon fluorescence microscopy is a powerful tool for the study of dynamic cellular processes and live-cell imaging. Many commercially available fluorescent probes have been used in multiphoton-based imaging studies despite exhibiting relatively low two-photon absorption cross-section values in the tunability range of ultrafast Ti:sapphire lasers commonly used in multiphoton microscopy imaging. Furthermore, available fluorophores may be plagued with low fluorescence quantum yield and/or photoinstability (i.e., photobleaching) on exposure to the high peak power and photon density provided by the ultrafast laser source. To address the demand for better performing dyes, we prepare fluorophores tailored for multiphoton imaging. These fluorophores are based on the fluorene ring system, known to exhibit high fluorescence quantum yield (>0.7) and high photostability. Furthermore, an amine-reactive fluorescent probe for the covalent attachment onto amine-containing biomolecules is also prepared. Epi-fluorescence and two-photon fluorescence microscopy images of H9c2 rat cardiomyoblasts stained with an efficient two-photon absorbing fluorene fluorophore is demonstrated. Additionally, single-photon spectral characteristics of the amine-reactive fluorophore, as well as the two-photon absorption cross sections of its model adduct in solution, and spectral characterization of a bovine serum albumin (BSA) as a model bioconjugate are presented.
Assuntos
Fluorenos , Corantes Fluorescentes , Aumento da Imagem/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Técnicas de Sonda Molecular , Miócitos Cardíacos/citologia , Animais , Linhagem Celular , Fótons , Ratos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Coloração e Rotulagem/métodosRESUMO
The tumor suppressor LKB1 is mutated in 30% of non-small cell lung cancer (NSCLC) tumors and cell lines and is proposed to be a key regulator of epithelial cell polarity; however, how LKB1 regulates cancer cell polarity is not known. The experiments described herein show for the first time that LKB1 is a dynamic, actin-associated protein that rapidly polarizes to the leading edge of motile cancer cells. LKB1 proves to be essential for NSCLC polarity, because LKB1 depletion results in classic cell polarity defects, such as aberrant Golgi positioning, reduced lamellipodia formation, and aberrant morphology. To probe how LKB1 regulates these events, we show that LKB1 colocalizes at the cellular leading edge with two key components of the polarity pathway - the small rho GTPase cdc42 and its downstream binding partner p21-activated kinase (PAK). Importantly, LKB1 functionality is required for cdc42 polarization to the leading edge, maintaining active cdc42 levels, and downstream PAK phosphorylation. To do this, LKB1 interacts only with active form of cdc42 and PAK, but not with inactive cdc42. Taken together, these results show that LKB1 is a critical mediator of the NSCLC polarity program in lung cancer cells through a novel LKB1-cdc42-PAK pathway.