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A series of hybrid thiosemicarbazone-alkylthiocarbamate copper complexes with similar electronic environments but distinct physical structures have been prepared, characterized, and evaluated for antiproliferation activity. The complexes include the constitutional isomers (1-phenylpropane-1-imine-(O-ethylthiocarbamato)-2-one-(N-methylthiosemicarbazonato))copper(II) (CuL1) and (1-phenylpropane-1-one-(N-methylthiosemicarbazonato)-2-imine-(O-ethylthiocarbamato))copper(II) (CuL2) along with (1-propane-1-imine-(O-ethylthiocarbamato)-2-one-(N-methylthiosemicarbazonato))copper(II) (CuL3). Complexes CuL1 and CuL2 differ in the positions of the pendent thiosemicarbazone (TSC) and alkylthiocarbamate (ATC) moieties on the 1-phenylpropane backbone. Complex CuL3 employs a propane backbone with the TSC in the 2-position as in CuL1. The isomer pair CuL1 and CuL2 have equivalent electronic environments with indistinguishable CuII/I potentials (E1/2 = -0.86 V vs. ferrocenium/ferrocene) and electron paramagnetic resonance (EPR) spectra (g⥠= 2.26, g⥠= 2.08). The electronic structure of CuL3 has a similar E1/2 of -0.84 V and identical EPR parameters to CuL1, 2. Single crystal X-ray diffraction studies confirm a consistent donor environment with no substantial variation in the CuN or CuS bond distances and angles between the complexes. The antiproliferation activities of the CuL1-3 were evaluated against the lung adenocarcinoma cell line (A549) and nonmalignant lung fibroblast cell line (IMR-90) using the MTT assay. CuL1 had the highest A549 activity (A549EC50 = 0.065 µM) and selectivity (IMR-90EC50/A549EC50 = 20). The constitutional isomer CuL2 displayed decreased A549 activity (0.18 µM) and selectivity (10.6). The complex CuL3 displayed activity (0.009 µM) similar to CuL1 but with a lack of selectivity (1.0). Cellular copper loading determined by ICP-MS was consistent with the activity and selectivity trends. The complexes CuL1-3 did not induce reactive oxygen species (ROS) generation.
Assuntos
Complexos de Coordenação , Tiossemicarbazonas , Cobre/química , Propano , Espectroscopia de Ressonância de Spin Eletrônica , Tiossemicarbazonas/farmacologia , Tiossemicarbazonas/química , Iminas , Complexos de Coordenação/farmacologia , Complexos de Coordenação/química , Cristalografia por Raios X , LigantesRESUMO
Continuous-flow acoustofluidic technologies can potentially improve processing of T lymphocytes for cell therapies by addressing the limitations with viral and non-viral delivery methods. The objective of this study was to assess the intracellular delivery efficiency with acoustofluidic treatment compared with that of static ultrasound treatment. Optimization of parameters in acoustofluidic and static configurations was performed by assessing intracellular delivery of a fluorescent compound (calcein) in viable human Jurkat T lymphocytes. Ultrasound pressure and the concentration of cationic phospholipid-coated microbubbles influenced calcein delivery in both systems. In the static system, a treatment time of 45 s increased molecular delivery compared with 0-30 s (p < 0.01). Refined parameters were used to assess molecular delivery of small and large compounds (0.6-kDa calcein and 150-kDa fluorescein isothiocyanate-dextran, respectively) after ultrasound treatment with the acoustofluidic or static systems. Molecular delivery was similar with refined parameters for acoustofluidic treatment and static treatment (p > 0.05), even though acoustofluidic treatment had lower microbubble concentration (24 µg/mL vs. 94 µg/mL) and shorter treatment time (â¼2-3 s vs. 45 s). This study indicates that the acoustofluidic system can significantly enhance intracellular molecular delivery, which could potentially enable acoustofluidic cell transfection during continuous flow processing for manufacture of cell therapies or other applications.
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Microbolhas , Linfócitos T , Humanos , Transfecção , Ultrassonografia , Sistemas de Liberação de Medicamentos/métodosRESUMO
A series of isomeric bis(alkylthiocarbamate) copper complexes have been synthesized, characterized, and evaluated for antiproliferation activity. The complexes were derived from ligand isomers with 3-methylpentyl (H2L2) and cyclohexyl (H2L3) backbone substituents, which each yield a pair of linkage isomers. The thermodynamic products CuL2a/3a have two imino N and two S donors resulting in three five-member chelate rings (555 isomers). The kinetic isomers CuL2b/3b have one imino and one hydrazino N donor and two S donors resulting in four-, six-, and five-member rings (465 isomers). The 555 isomers have more accessible CuII/I potentials (E1/2 = -811/-768 mV vs. ferrocenium/ferrocene) and lower energy charge transfer bands than their 465 counterparts (E1/2 = -923/-854 mV). Antiproliferation activities were evaluated against the lung adenocarcinoma cell line (A549) and nonmalignant lung fibroblast cell line (IMR-90) using the MTT assay. CuL2a was potent (A549EC50 = 0.080 µM) and selective (IMR-90EC50/A549EC50 = 25) for A549. Its linkage isomer CuL2b had equivalent A549 activity, but lower selectivity (IMR-90EC50/A549EC50 = 12.5). The isomers CuL3a and CuL3b were less potent with A549EC50 values of 1.9 and 0.19 µM and less selective with IMR-90EC50/A549EC50 ratios of 2.3 and 2.65, respectively. There was no correlation between reduction potential and A549 antiproliferation activity/selectivity.
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Gold nanoparticles (GNPs) have immense potential in biomedicine, but understanding their interactions with serum proteins is crucial as it could change their biological profile due to the formation of a protein corona, which could then affect their ultimate biodistribution in the body. Grafting GNPs with polyethylene glycol (PEG) is a widely used practice in research in order to decrease opsonization of the particles by serum proteins and to decrease particle uptake by the mononuclear phagocyte system. We investigated the impact of PEGylation on the formation of protein coronae and the subsequent uptake by macrophages and MDA-MB-231 cancer cells. Furthermore, we investigated the in vivo biodistribution in xenograft tumor-bearing mice using a library of 4 and 10 nm GNPs conjugated with a gadolinium chelate as MRI contrast agent, cancer-targeting aptamer AS1411 (or CRO control oligonucleotide), and with or without PEG molecules of different molecular weight (Mw: 1, 2, and 5 kDa). In vitro results showed that PEG failed to decrease the adsorption of proteins; moreover, the cellular uptake by macrophage cells was contingent on the different configurations of the aptamers and the length of the PEG chain. In vivo biodistribution studies showed that PEG increased the uptake by tumor cells for some GNPs, albeit it did not decrease the uptake of GNPs by macrophage-rich organs.
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Glioblastoma brain tumors represent an aggressive form of gliomas that is hallmarked by being extremely invasive and aggressive due to intra and inter-tumoral heterogeneity. This complex tumor microenvironment makes even the newer advancements in glioblastoma treatment less effective long term. In developing newer treatment technologies against glioblastoma, one should tailor the treatment to the tumor microenvironment, thus allowing for a more robust and sustained anti-glioblastoma effect. Here, we present a novel gold nanoparticle therapy explicitly designed for bioactivity against glioblastoma representing U87MG cell lines. We employ standard conjugation techniques to create oligonucleotide-coated gold nanoparticles exhibiting strong anti-glioblastoma behavior and optimize their design to maximize bioactivity against glioblastoma. Resulting nanotherapies are therapy specific and show upwards of 75% inhibition in metabolic and proliferative activity with stark effects on cellular morphology. Ultimately, these gold nanotherapies are a good base for designing more multi-targeted approaches to fighting against glioblastoma.
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Linkage isomers are coordination compounds with the same composition but different donor atoms, resulting in distinct physical and electronic structures. A pair of linkage isomers, CuL555 and CuL465, derived from phenylglyoxal bis(ethylthiocarbamate) were synthesized, isolated, and characterized by structural, electrochemical, and spectroscopic methods. The isomers are stable in solution under ambient conditions, but CuL465 converts to CuL555 in acid, consistent with quantum-chemical calculations. The complexes were screened against a lung adenocarcinoma cell line (A549) and a nonmalignant lung fibroblast cell line (IMR-90) to evaluate the antiproliferation activity. CuL555 and CuL465 possessed EC50 values of 0.113 ± 0.030 and 0.115 ± 0.038 µM for A549 and 1.87 ± 0.29 and 0.77 ± 0.22 µM for IMR-90, respectively.
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Cobre , Cobre/química , Cobre/farmacologia , IsomerismoRESUMO
Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.
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Acústica/instrumentação , Células/metabolismo , Fluoresceína/metabolismo , Trealose/metabolismo , Células A549 , Células Cultivadas , Meios de Contraste/química , Humanos , Microbolhas , Linfócitos T/citologia , UltrassomRESUMO
Cell-based therapies have garnered significant interest to treat cancer and other diseases. Acoustofluidic technologies are in development to improve cell therapy manufacturing by facilitating rapid molecular delivery across the plasma membrane via ultrasound and microbubbles (MBs). In this study, a three-dimensional (3D) printed acoustofluidic device was used to deliver a fluorescent molecule, calcein, to human T cells. Intracellular delivery of calcein was assessed after varying parameters such as MB face charge, MB concentration, flow channel geometry, ultrasound pressure, and delivery time point after ultrasound treatment. MBs with a cationic surface charge caused statistically significant increases in calcein delivery during acoustofluidic treatment compared to MBs with a neutral surface charge (p < 0.001). Calcein delivery was significantly higher with a concentric spiral channel geometry compared to a rectilinear channel geometry (p < 0.001). Additionally, calcein delivery was significantly enhanced at increased ultrasound pressures of 5.1 MPa compared to lower ultrasound pressures between 0-3.8 MPa (p < 0.001). These results demonstrate that a 3D-printed acoustofluidic device can significantly enhance intracellular delivery of biomolecules to T cells, which may be a viable approach to advance cell-based therapies.
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Microbolhas , Linfócitos T , Sistemas de Liberação de Medicamentos , Humanos , UltrassonografiaRESUMO
Systemic delivery of conventional chemotherapies can cause negative systemic toxicity, including reduced immunity and damage to organs such as the heart and kidneys-limiting the maximum dose that can be administered. Targeted therapies appear to address this problem by having a specific target while mitigating off-target effects. Biocompatible perfluorocarbon-based nanodroplet emulsions encapsulated by a phospholipid shell are in development for delivery of molecular compounds and hold promise as vehicles for targeted delivery of chemotherapeutics to tumors. When ultrasound is applied, perfluorocarbon will undergo a phase change-ultimately inducing transient perforation of the cell membrane when in close proximity, which is more commonly known as "sonoporation." Sonoporation allows enhanced intracellular delivery of molecular compounds and will reseal to encapsulate the molecular compound intracellularly. In this study, we investigated delivery of thymoquinone (TQ), a natural hydrophobic phytochemical compound with bioactivity in cancer cells. In addition, we conjugated a G-quadruplex aptamer, 'AS1411', to TQ-loaded nanodroplets and explored their effects on multiple human cancer cell lines. AS1411 binds nucleolin, which is over-expressed on the surface of cancer cells, and in addition to its tumor-targeting properties AS1411 has also been shown to induce anti-cancer effects. Thymoquinone was loaded onto AS1411-conjugated nanodroplet emulsion to assess activity against cancer cells. Confocal microscopy indicated uptake of AS1411-conjugated nanodroplets by cancer cells. Furthermore, AS1411-conjugated nanoemulsions loaded with TQ significantly enhanced cytotoxicity in cancer cells compared to free compound. These results demonstrate that AS1411 can be conjugated onto nanodroplet emulsions for targeted delivery to human cancer cells. This novel formulation offers significant potential for targeted delivery of hydrophobic chemotherapeutics to tumors for cancer treatment.
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Benzoquinonas/administração & dosagem , Sistemas de Liberação de Medicamentos , Nanopartículas/administração & dosagem , Oligodesoxirribonucleotídeos/administração & dosagem , Aptâmeros de Nucleotídeos , Linhagem Celular Tumoral , HumanosRESUMO
A series of hybrid ligands (H2L1-H2L3) derived from 4-methyl-3-thiosemicarbazide and hydrazinecarbothioic acid O-alkyl esters were synthesized and characterized by NMR. The ligands were chelated with copper (4-6), nickel (7-9), and zinc (10-12) and characterized by spectroscopy, electrochemistry, and single crystal X-ray crystallography. The chelated metals displayed substantial anodic shifts in the CuII/I reduction potential of â¼160 mV relative to their bis(thiosemicarbazone) analogues. The metal chelates 4-12 were evaluated for potential anticancer activity by MTT assays, and selected results were confirmed by clonogenic and trypan blue assays. The copper derivatives 4 and 6 were found to have potent and cancer-selective antiproliferative effects, with GI50 values less than 100 nM in A549 lung adenocarcinoma cells compared with at least 20-fold less activity in IMR90 nonmalignant lung fibroblasts. In comparison, the nickel complexes were much less active and had little cancer-selectivity. Varying by ligand, the zinc complexes were less potent or had comparable activity compared to that of the corresponding copper complex. UV-visible spectroscopy indicated that zinc complex 10 was transmetalated in the presence of equimolar copper, whereas nickel complex 7 was not. Copper complexes 4 and 6 were also assessed in the NCI60 screen and were found to have cytotoxic activity against most solid tumor cell lines. In MTT assays, 4 and 6 were substantially more active against A549 cancer cells than Cu(ATSM) and were more cancer-selective (for A549 compared to IMR-90) than Cu(GTSM). Our results suggest that hybrid thiosemicarbazone-alkylthiocarbamate copper complexes have potential for development as new anticancer agents.
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Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Tiocarbamatos/farmacologia , Tiossemicarbazonas/farmacologia , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Complexos de Coordenação/síntese química , Cobre/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligantes , Níquel/química , Tiocarbamatos/síntese química , Tiossemicarbazonas/síntese química , Zinco/químicaRESUMO
Oligonucleotides can be designed or evolved to bind to specific DNA, RNA, protein, or small molecule targets and thereby alter the biological function of the target. The therapeutic potential of oligonucleotides targeted to intracellular molecules will depend largely on their ability to be taken up by the cells of interest, as well as their subsequent subcellular distribution. Here we describe methods to characterize the extent and mechanism of cellular uptake of AS1411, an aptamer oligonucleotide that has progressed to human clinical trials and which is also widely used by researchers as a cancer-targeting ligand.
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Aptâmeros de Nucleotídeos/metabolismo , Oligodesoxirribonucleotídeos/metabolismo , Aptâmeros de Nucleotídeos/administração & dosagem , Aptâmeros de Nucleotídeos/química , Transporte Biológico , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Endocitose , Citometria de Fluxo , Humanos , Microscopia Confocal , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/química , PinocitoseRESUMO
Gold nanoparticles (GNPs) have tremendous potential as cancer-targeted contrast agents for diagnostic imaging. The ability to modify the particle surface with both disease-targeting molecules (such as the cancer-specific aptamer AS1411) and contrast agents (such as the gadolinium chelate Gd(III)-DO3A-SH) enables tailoring the particles for specific cancer-imaging and diagnosis. While the amount of image contrast generated by nanoparticle contrast agents is often low, it can be augmented with the assistance of computer image analysis algorithms. In this work, the ability of cancer-targeted gold nanoparticle-oligonucleotide conjugates to distinguish between malignant (MDA-MB-231) and healthy cells (MCF-10A) is tested using a T1-weighted image analysis algorithm based on three-dimensional, deformable model-based segmentation to extract the Volume of Interest (VOI). The gold nanoparticle/algorithm tandem was tested using contrast agent GNP-Gd(III)-DO3A-SH-AS1411) and nontargeted c-rich oligonucleotide (CRO) analogs and control (CTR) counterparts (GNP-Gd(III)-DO3A-SH-CRO/CTR) via in vitro studies. Remarkably, the cancer cells were notably distinguished from the nonmalignant cells, especially at nanomolar contrast agent concentrations. The T1-weighted image analysis algorithm provided similar results to the industry standard Varian software interface (VNMRJ) analysis of T1 maps at micromolar contrast agent concentrations, in which the VNMRJ produced a 19.5% better MRI contrast enhancement. However, our algorithm provided more sensitive and consistent results at nanomolar contrast agent concentrations, where our algorithm produced ~500% better MRI contrast enhancement.
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We previously reported on the isolation and biological activities of plagiochiline A (1), a 2,3-secoaromadendrane-type sesquiterpenoid from the Peruvian medicinal plant, Plagiochila disticha. This compound was found to have antiproliferative effects on a variety of solid tumor cell lines, as well as several leukemia cell lines. Other researchers have also noted the cytotoxicity of plagiochiline A (isolated from different plant species), but there are no prior reports regarding the mechanism for this bioactivity. Here, we have evaluated the effects of plagiochiline A on cell cycle progression in DU145 prostate cancer cells. A cell cycle analysis indicated that plagiochiline A caused a significant increase in the percentage of cells in the G2/M phase when compared with control cells. When cells were stained and observed by fluorescence microscopy to examine progress through the mitotic phase, we found a significant increase in the proportion of cells with features of late cytokinesis (cells connected by intercellular bridges) in the plagiochiline A-treated samples. These results suggest that plagiochiline A inhibits cell division by preventing completion of cytokinesis, particularly at the final abscission stage. We also determined that plagiochiline A reduces DU145 cell survival in clonogenic assays and that it induces substantial cell death in these cells.
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Citocinese/efeitos dos fármacos , Embriófitas/química , Compostos de Epóxi/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Piranos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Compostos de Epóxi/química , Compostos de Epóxi/isolamento & purificação , Humanos , Masculino , Extratos Vegetais/química , Piranos/química , Piranos/isolamento & purificaçãoRESUMO
BACKGROUND: AS1411 is a 26-mer G-rich DNA oligonucleotide that forms a variety of G-quadruplex structures. It was identified based on its cancer-selective antiproliferative activity and subsequently determined to be an aptamer to nucleolin, a multifunctional protein that preferentially binds quadruplex nucleic acids and which is present at high levels on the surface of cancer cells. AS1411 has exceptionally efficient cellular internalization compared to non-quadruplex DNA sequences. SCOPE OF REVIEW: Recent developments related to AS1411 will be examined, with a focus on its use for targeted delivery of therapeutic and imaging agents. MAJOR CONCLUSIONS: Numerous research groups have used AS1411 as a targeting agent to deliver nanoparticles, oligonucleotides, and small molecules into cancer cells. Studies in animal models have demonstrated that AS1411-linked materials can accumulate selectively in tumors following systemic administration. The mechanism underlying the cancer-targeting ability of AS1411 is not completely understood, but recent studies suggest a model that involves: (1) initial uptake by macropinocytosis, a form of endocytosis prevalent in cancer cells; (2) stimulation of macropinocytosis by a nucleolin-dependent mechanism resulting in further uptake; and (3) disruption of nucleolin-mediated trafficking and efflux leading to cargoes becoming trapped inside cancer cells. SIGNIFICANCE: Human trials have indicated that AS1411 is safe and can induce durable remissions in a few patients, but new strategies are needed to maximize its clinical impact. A better understanding of the mechanisms by which AS1411 targets and kills cancer cells may hasten the development of promising technologies using AS1411-linked nanoparticles or conjugates for cancer-targeted therapy and imaging. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.
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Antineoplásicos/uso terapêutico , Diagnóstico por Imagem/métodos , Portadores de Fármacos , Quadruplex G , Guanosina/metabolismo , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Oligodesoxirribonucleotídeos/uso terapêutico , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Aptâmeros de Nucleotídeos , Sítios de Ligação , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meios de Contraste/química , Meios de Contraste/metabolismo , Guanosina/química , Humanos , Ligantes , Neoplasias/genética , Neoplasias/metabolismo , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/metabolismo , Relação Estrutura-Atividade , NucleolinaRESUMO
AS1411 is a quadruplex-forming DNA oligonucleotide that functions as an aptamer to target nucleolin, a protein present on the surface of cancer cells. Clinical trials of AS1411 have indicated it is well tolerated with evidence of therapeutic activity, but improved pharmacology and potency may be required for optimal efficacy. In this report, we describe how conjugating AS1411 to 5 nm gold nanospheres influences its activities in vitro and in vivo. We find that the AS1411-linked gold nanospheres (AS1411-GNS) are stable in aqueous and serum-containing solutions. Compared to unconjugated AS1411 or GNS linked to control oligonucleotides, AS1411-GNS have superior cellular uptake and markedly increased antiproliferative/cytotoxic effects. Similar to AS1411, AS1411-GNS show selectivity for cancer cells compared to non-malignant cells. In a mouse model of breast cancer, systemic administration of AS1411-GNS could completely inhibit tumor growth with no signs of toxicity. These results suggest AS1411-GNS are promising candidates for clinical translation.
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Neoplasias da Mama/terapia , Ouro/administração & dosagem , Nanopartículas Metálicas/administração & dosagem , Nanosferas/administração & dosagem , Oligodesoxirribonucleotídeos/administração & dosagem , Animais , Aptâmeros de Nucleotídeos , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AS1411 is a G-rich quadruplex-forming oligodeoxynucleotide that binds specifically to nucleolin, a protein found on the surface and in the cytoplasm of most malignant cells but absent from the surface/cytoplasm of most normal cells. AS1411 has shown promising clinical activity and is being widely used as a tumor-targeting agent, but its mechanism of action is not fully understood. Previously, we showed that AS1411 is taken up in cancer cells by macropinocytosis (fluid phase endocytosis) and subsequently stimulates further macropinocytosis by a nucleolin-dependent mechanism. In the current study, we have investigated the significance and molecular mechanisms of AS1411-induced macropinocytosis. Our results indicate that the antiproliferative activity of AS1411 in various cell lines correlated with its capacity to stimulate macropinocytosis. In DU145 prostate cancer cells, AS1411 induced activation of EGFR, Akt, p38, and Rac1. Activation of Akt and p38 were not critical for AS1411 activity because Akt activation was not observed in all AS1411-responsive cell lines and knockdown of p38 had no effect on AS1411's ability to inhibit proliferation. On the other hand, activation of EGFR and Rac1 appeared to play a role in AS1411 activity in all cancer cell lines examined (DU145, MDA-MB-468, A549, LNCaP) and their inhibition significantly reduced AS1411-mediated macropinocytosis and AS1411 antiproliferative activity. Interestingly, downregulation of nucleolin expression by siRNA also produced a substantial increase in activated Rac1, revealing a previously unknown role for nucleolin as a negative regulator of Rac1 activation. Our results are consistent with a model whereby AS1411 binding to nucleolin leads to sustained activation of Rac1 and causes methuosis, a novel type of nonapoptotic cell death characterized by hyperstimulation of macropinocytosis. We speculate that methuosis is a tumor/metastasis suppressor mechanism that opposes the malignant functions of Rac1 and that cancer cells may overexpress nucleolin to surmount this barrier.
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Aptâmeros de Nucleotídeos/farmacologia , Oligodesoxirribonucleotídeos/farmacologia , Fosfoproteínas/fisiologia , Proteínas de Ligação a RNA/fisiologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/metabolismo , Humanos , Pinocitose/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , NucleolinaRESUMO
INTRODUCTION: AS1411 is a 26-base guanine-rich oligonucleotide aptamer shown binding to surface nucleolin, a protein over-expressed in multiple cancer cells, thus AS1411 labeled with a PET isotope can be explored as a potential diagnostic imaging agent. Our objective was to perform preliminary biological characterization of (64)Cu-labeled AS1411 in vitro and in vivo. METHODS: Four chelators (DOTA, CB-TE2A, DOTA-Bn and NOTA-Bn) were selected to label AS1411 with Cu-64. 185kBq (5µCi) of each tracer was incubated in each well with H460 cells at 37°C for 1, 3, 6, 12, 24 and 48h, respectively (n=4). For microPET/CT imaging, 7.4MBq (200µCi) of AS1411 labeled with either (64)Cu-DOTA or (64)Cu-CB-TE2A was I.V. injected and multiple scans were obtained at 1, 3, 6 and 24h post injection. Afterward in vivo biodistribution studies were performed. RESULTS: Percent uptake of (64)Cu-DOTA-AS1411 and (64)Cu-CB-TE2A-AS1411 was significantly higher than that of (64)Cu-DOTA-Bn-AS1411 and (64)Cu-NOTA-Bn-AS1411. About 90% of uptake for (64)Cu-DOTA-AS1411 and (64)Cu-CB-TE2A-AS1411 was internalized into cells within 3h and the internalization process was completed before 24h. Both tracers demonstrated reasonable in vivo stability and high binding affinity to the cells. MicroPET imaging with (64)Cu-CB-TE2A-AS1411 showed clear tumor uptake at both legs from 1 to 24h post injection, whereas both tumors were undetectable for up to 24h with (64)Cu-DOTA-AS1411. In addition, (64)Cu-CB-TE2A-AS1411 had faster in vivo pharmacokinetics than (64)Cu-DOTA-AS1411 with lower liver uptake and higher tumor to background contrast. CONCLUSION: CB-TE2A is a preferred chelator with higher tumor-to-background ratio, lower liver uptake and faster clearance than DOTA. Aptamer imaging with (64)Cu-CB-TE2A-AS1411 may be feasible for detecting lung cancer, if an appropriate chelator can be identified and further validation can be performed with a known control oligonucleotide. It may also be used as a companion diagnostic imaging agent for AS1411 in the treatment of cancer.
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Radioisótopos de Cobre , Neoplasias Pulmonares/patologia , Oligodesoxirribonucleotídeos , Tomografia por Emissão de Pósitrons/métodos , Tomografia Computadorizada por Raios X/métodos , Animais , Aptâmeros de Nucleotídeos , Linhagem Celular Tumoral , Feminino , Humanos , Marcação por Isótopo , Neoplasias Pulmonares/diagnóstico por imagem , Camundongos , Oligodesoxirribonucleotídeos/metabolismo , Oligodesoxirribonucleotídeos/farmacocinética , RadioquímicaRESUMO
We have synthesized a novel molecule named XB05 (1-bromo-1,1-difluoro-non-2-yn-4-ol) and evaluated its effects in a variety of human cell lines. XB05 displayed potent antiproliferative activity against cell lines derived from leukemia or solid tumors, but had less effect on nonmalignant cells. To identify factors that contribute to the cancer selectivity of XB05, we chose three cell lines that had high sensitivity to XB05 (U937 leukemia), moderate sensitivity (A549 lung cancer), or low sensitivity (Hs27 nonmalignant skin fibroblasts), and proceeded to assess cell death and oxidative stress in these cells. XB05 was found to induce cell death via both apoptotic and nonapoptotic mechanisms in U937 and A549 cells, whereas it had no cytotoxicity against Hs27 cells at comparable concentrations. Treatment with XB05 caused an increase in reactive oxygen species in all cell lines tested, but levels were higher in malignant compared to nonmalignant cells. XB05 treatment also induced DNA damage exclusively in the malignant cells. Differences in antioxidant responses were observed between cell lines. For example, XB05 caused a decrease in levels of glutathione and nuclear Nrf2 in the most sensitive cells (U937), whereas the least sensitive cells (Hs27) displayed increased glutathione levels and no change in nuclear Nrf2. XB05 could react in vitro with cysteine and glutathione, but had much lower reactivity compared to typical thiol-reactive electrophiles, diethyl maleate and maleimide. In summary, XB05 is a novel compound that selectively kills malignant cells, most likely by disrupting cellular redox homeostasis, making it a promising candidate for development as a chemotherapeutic agent.
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Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Álcoois Graxos/química , Hidrocarbonetos Bromados/química , Estresse Oxidativo/efeitos dos fármacos , Linhagem Celular Tumoral , Glutationa/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Oxirredução , Espécies Reativas de Oxigênio/metabolismoRESUMO
Recently, our group reported the discovery of three new withanolides, physangulidines A-C, from Physalis angulata. In this study, the biological effects of physangulidine A (1), which was the most active and abundant of the three new constituents, are described. It was found that 1 significantly reduces survival in clonogenic assays for two hormone-independent prostate cancer cell lines. Flow cytometry and confocal microscopy studies in DU145 human prostate cancer cells indicated that 1 induces cell cycle arrest in the G(2)/M phase and causes defective mitosis. It was determined also that 1 produces programed cell death by apoptosis, as evidenced by biochemical markers and distinct changes in cell morphology. These results imply that the antimitotic and proapoptotic effects of 1 may contribute significantly to the biological activities and potential medicinal properties of its plant of origin.
Assuntos
Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Physalis/química , Vitanolídeos/isolamento & purificação , Vitanolídeos/farmacologia , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Fase G2/efeitos dos fármacos , Humanos , Masculino , Microscopia Confocal , Estrutura Molecular , Neoplasias da Próstata/tratamento farmacológico , Vitanolídeos/químicaRESUMO
INTRODUCTION: The orphan nuclear receptor COUP-TFII plays an undefined role in breast cancer. Previously we reported lower COUP-TFII expression in tamoxifen/endocrine-resistant versus sensitive breast cancer cell lines. The identification of COUP-TFII-interacting proteins will help to elucidate its mechanism of action as a transcriptional regulator in breast cancer. RESULTS: FLAG-affinity purification and multidimensional protein identification technology (MudPIT) identified nucleolin among the proteins interacting with COUP-TFII in MCF-7 tamoxifen-sensitive breast cancer cells. Interaction of COUP-TFII and nucleolin was confirmed by coimmunoprecipitation of endogenous proteins in MCF-7 and T47D breast cancer cells. In vitro studies revealed that COUP-TFII interacts with the C-terminal arginine-glycine repeat (RGG) domain of nucleolin. Functional interaction between COUP-TFII and nucleolin was indicated by studies showing that siRNA knockdown of nucleolin and an oligonucleotide aptamer that targets nucleolin, AS1411, inhibited endogenous COUP-TFII-stimulated RARB2 expression in MCF-7 and T47D cells. Chromatin immunoprecipitation revealed COUP-TFII occupancy of the RARB2 promoter was increased by all-trans retinoic acid (atRA). RARß2 regulated gene RRIG1 was increased by atRA and COUP-TFII transfection and inhibited by siCOUP-TFII. Immunohistochemical staining of breast tumor microarrays showed nuclear COUP-TFII and nucleolin staining was correlated in invasive ductal carcinomas. COUP-TFII staining correlated with ERα, SRC-1, AIB1, Pea3, MMP2, and phospho-Src and was reduced with increased tumor grade. CONCLUSIONS: Our data indicate that nucleolin plays a coregulatory role in transcriptional regulation of the tumor suppressor RARB2 by COUP-TFII.