RESUMO
AIMS: While significant upregulation of GRP78 has been documented in lung cancer patients, its association with resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) remains underexamined. Our study aimed to elucidate the functional importance of GRP78 in acquired resistance to EGFR-TKIs in non-small cell lung cancer (NSCLC) and to evaluate its potential as a therapeutic target. MAIN METHODS: Immunoblot analysis or flow cytometry was employed to assess several markers for endoplasmic reticulum (ER) stress and apoptosis. Ru(II) complex I and HA15, two known GRP78 inhibitors, were used to evaluate the functional role of GRP78. A Xenograft assay was performed to evaluate the in vivo anti-cancer effects of the GRP78 inhibitors. KEY FINDINGS: We validated a significant increase in GRP78 protein levels in HCC827-GR, H1993-GR, and H1993-ER cells. The EGFR-TKI-resistant cells overexpressing GRP78 exhibited significantly higher cell proliferation rates than did their parental counterparts. Notably, GRP78 inhibition resulted in a more profound anti-proliferative and apoptotic response via heightened ER stress and subsequent reactive oxygen species (ROS) production in EGFR-TKI-resistant cell lines compared with their parental cells. In xenograft models implanted with HCC827-GR, both Ru(II) complex I and HA15 significantly suppressed tumor growth and reduced tumor weight. Additionally, we confirmed that GRP78 plays a critical role in the proliferation of H1975, an EGFR-TKI-resistant T790M-mutant cell line, relative to other NSCLC cell lines. SIGNIFICANCE: Our findings strongly support targeting of GRP78 as a promising therapeutic strategy for NSCLC patients with acquired resistance to EGFR-TKIs.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Resistencia a Medicamentos Antineoplásicos , Chaperona BiP do Retículo Endoplasmático , Receptores ErbB , Proteínas de Choque Térmico , Neoplasias Pulmonares , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Feminino , Humanos , Camundongos , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Proteínas de Choque Térmico/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Espécies Reativas de Oxigênio/metabolismo , /farmacologiaRESUMO
Cancer stem cells (CSCs), also known as tumor-initiating cells, are critically correlated with carcinogenesis and are strongly affected by the environmental factors. Environmental carcinogens, such as benzo(a)pyrene (BaP), are associated with the overproduction of CSCs in various types of cancers, including breast cancer. In this report, we present a sophisticated 3D breast cancer spheroid model for the direct identification and quantitative determination of CSCs induced by carcinogens within intact 3D spheroids. To this end, hydrogel microconstructs containing MCF-7 breast cancer cells were bioprinted within direct-made diminutive multi-well chambers, which were utilized for the mass cultivation of spheroids and in situ detection of CSCs. We found that the breast CSCs caused by BaP-induced mutations were higher in the biomimetic MCF-7 breast cancer spheroids than that in standard 2D monolayer cultures. Precisely controlled MCF-7 cancer spheroids could be generated by serially cultivating MCF-7 cells within the printed hydrogel microconstructs, which could be further utilized for high-resolution in situ high-content 3D imaging analysis to spatially identify the emergence of CSCs at the single spheroid level. Additionally, potential therapeutic agents specific to breast CSCs were successfully evaluated to verify the effectiveness of this model. This bioengineered 3D cancer spheroid system provides a novel approach to investigating the emergence of CSC induced by a carcinogen for environmental hazard assessment in a reproducible and scalable format.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/genética , Carcinógenos , Benzo(a)pireno/toxicidade , Linhagem Celular Tumoral , Esferoides Celulares , Células-Tronco NeoplásicasRESUMO
In this study, we elucidated for the first time the role of anti-cancer drugs in transarterial chemoembolization (TACE) via direct visualization of the spatial distribution of drugs with respect to blood vessels in intact transparent hepatocellular carcinoma (HCC) tissues. To date, precise estimation of drug penetration into tumors using thin 3D tissue sections has been challenging. This study utilized the tissue optical clearing technique to resolve the lack of tissue clarity, thereby enabling deep tissue imaging for the quantitative assessment of drug delivery following TACE. We compared the drug delivery effect, time-dependent embolic effect, and immunogenic response following conventional TACE (cTACE), drug-eluting embolic TACE (DEE-TACE), and transarterial embolization (TAE) in a rat model of HCC. After each treatment, three-dimensional drug delivery was quantitatively evaluated via the transparent liver tumor imaging, and time-dependent tumor necrosis was analyzed by serial tumor harvesting and histological staining. The results showed that chemotherapeutic agents travel only short distances after cTACE (â¼80µm) and DEE-TACE (â¼110µm), whereas necrosis occurs extensively within 24 h of treatment (85.3-97.2% of tumor cells). In addition, the percentages of CD4 and IL-17+ CD4 T cells increased significantly following treatment; however, drug-loading did not appear to affect the immune response following TACE. In conclusion, transarterially delivered chemotherapeutic agents appeared to exert a limited role, owing to the rapid and overwhelming effect of embolization. STATEMENT OF SIGNIFICANCE: TACE has been widely used for the treatment of HCC, especially for unresectable intermediate and advanced HCCs. Drug use in TACE is expected to provide patients with synergistic therapeutic benefits with the effect of embolic agents; however, the role of chemotherapeutic agents in TACE remains controversial. This study quantitatively verified that chemotherapeutic agents travel only short distances after TACE, while necrosis occurs extensively within 24h, and drug loading does not significantly affect immune responses following TACE. Three-dimensional imaging of intact transparent HCC can contribute to a better understanding of drug delivery mechanisms associated with TACE and also reveal that drug use in TACE may need to be reconsidered and limited to situations when embolization is expected to be insufficient.
Assuntos
Carcinoma Hepatocelular , Quimioembolização Terapêutica , Neoplasias Hepáticas , Ratos , Animais , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/tratamento farmacológico , Quimioembolização Terapêutica/métodos , Sistemas de Liberação de Medicamentos , Necrose/tratamento farmacológico , Resultado do TratamentoRESUMO
The development of new in vitro models that closely mimic the tumor microenvironment (TME) to evaluate the efficacy of anticancer drugs has received great attention. In this study, a three-dimensional (3D) bioprinted Michigan Cancer Foundation-7 (MCF-7) cancer spheroid-embedded hydrogel model was suggested for integrative in situ determination of the half-maximal inhibitory concentration (IC50) values of photosensitizers (PSs). The MCF-7 cell-laden alginate/gelatin hydrogel was printed for the fabrication of tumor spheroids. The hydrogel was used to mimic the extracellular matrix (ECM) surrounding the cancer cells in the TME. The fluorescence intensities corresponding to photodynamic therapy (PDT)-induced death of tumor spheroids probed by the laser showed a random distribution in the hydrogel, regardless of the focus of the laser and the vertical-axis direction in which the laser was passed. These results enabled integrative in situ measurement of all tumor spheroids probed by the laser without needing to separate the tumor spheroids in the hydrogel and measure them individually. When compared with two-dimensional (2D) monolayer cultures, very large IC50 values of the PSs, chlorin e6 (Ce6) and sulfonated tetraphenyl porphyrin (sTPP), were achieved in MCF-7 spheroid-embedded hydrogels mainly due to the drug resistance of the tumor spheroids. Additionally, the heterogenic PDT response of single MCF-7 cancer cells in a single tumor spheroid was observed through 3D imaging of irregular apoptosis in a single spheroid since single tumor spheroids showed a heterogenic PDT response. Furthermore, the laser-power-dependent IC50 values of PSs were obtained using the MCF-7 spheroid-embedded hydrogel model.
Assuntos
Antineoplásicos , Fotoquimioterapia , Porfirinas , Alginatos , Antineoplásicos/farmacologia , Morte Celular , Gelatina , Humanos , Hidrogéis , Células MCF-7 , Michigan , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/farmacologia , Esferoides Celulares , Microambiente TumoralRESUMO
Drug-resistant cancer spheroids were fabricated by three-dimensional (3D) bioprinting for the quantitative evaluation of drug resistance of cancer cells, which is a very important issue in cancer treatment. Cancer spheroids have received great attention as a powerful in vitro model to replace animal experiments because of their ability to mimic the tumor microenvironment. In this work, the extrusion printing of gelatin-alginate hydrogel containing MCF-7 breast cancer stem cells successfully provided 3D growth of many single drug-resistant breast cancer spheroids in a cost-effective 3D-printed mini-well dish. The drug-resistant MCF-7 breast cancer spheroids were able to maintain their drug-resistant phenotype of CD44high/CD24low/ALDH1high in the gelatin-alginate media during 3D culture and exhibited higher expression levels of drug resistance markers, such as GRP78 chaperon and ABCG2 transporter, than bulk MCF-7 breast cancer spheroids. Furthermore, the effective concentration 50 (EC50) values for apoptotic and necrotic spheroid death could be directly determined from the 3D printed-gelatin-alginate gel matrix based on in situ 3D fluorescence imaging of cancer spheroids located out of the focal point and on the focal point. The EC50 values of anti-tumor agents (camptothecin and paclitaxel) for apoptotic and necrotic drug-resistant cancer spheroid death were higher than those for bulk cancer spheroid death, indicating a greater drug resistance. STATEMENT OF SIGNIFICANCE: This study proposed a novel 3D bioprinting-based drug screening model, to quantitatively evaluate the efficacy of anticancer drugs using drug-resistant MCF-7 breast cancer spheroids formed within a 3D-printed hydrogel. Quantitative determination of anticancer drug efficacy using EC50, which is extremely important in drug discovery, was achieved by 3D printing that enables concurrent growth of many single spheroids efficiently. This study verified whether drug-resistant cancer spheroids grown within 3D-printed gelatin-alginate hydrogel could maintain and present drug resistance. Also, the EC50 values of the apoptotic and necrotic cell deaths were directly acquired in 3D-embedded spheroids based on in situ fluorescence imaging. This platform provides a single-step straightforward strategy to cultivate and characterize drug-resistant spheroids to facilitate anticancer drug screening.
Assuntos
Neoplasias da Mama , Animais , Neoplasias da Mama/tratamento farmacológico , Resistência a Medicamentos , Chaperona BiP do Retículo Endoplasmático , Feminino , Humanos , Hidrogéis , Impressão Tridimensional , Esferoides Celulares , Microambiente TumoralRESUMO
3D spheroid cultures are attractive candidates for application in in vitro drug-induced hepatotoxicity testing models to improve the reliability of biological information obtainable from a simple 2D culture model. Various 3D spheroid culture models exist for hepatotoxicity screening, but quantitative assays of spheroid response in situ are still challenging to achieve with the current 3D liver toxicity platforms. In this study, we developed a 3D printing-based HepG2 liver spheroid culture model for in situ quantitative evaluation and high-content monitoring of drug-induced hepatotoxicity. HepG2 liver spheroids grown in mini-fabricated hydrogel constructs using a 3D bioprinter were used to obtain the EC50 values and to measure the multi-parametric hepatotoxic effects, including mitochondrial permeability transition (MPT), cytosolic calcium levels, and apoptosis. Interestingly, the average fluorescence intensities of apoptotic and cell death markers, calculated for out-of-focus and in-focus spheroids, increased proportionally as a function of the drug concentration, allowing for the determination of the EC50 values. In addition, 3D HepG2 spheroids were more resistant to nefazodone-induced MPT than 2D HepG2 cells, indicating that the gelatin/alginate hydrogel culture system provides enhanced resistance to hepatotoxic drugs. The drug response of HepG2 liver spheroids was also found to be unrelated to the spheroid size. These results demonstrate that the present 3D cell-printing-based embedded HepG2 liver spheroid platform is a promising approach for screening and characterizing drug-induced hepatotoxicity.
Assuntos
Preparações Farmacêuticas , Esferoides Celulares , Fígado , Impressão Tridimensional , Reprodutibilidade dos TestesRESUMO
Silver sulfide quantum dots (Ag2S QDs) as a theragnostic agent have received much attention because they provide excellent optical and chemical properties to facilitate diagnosis and therapy simultaneously. Ag2S QDs possess brightness and photostability suitable for intense and stable bioimaging. It has been verified via in vitro and in vivo studies that Ag2S QDs do not cause serious toxicity, unlike the first generation of widely used heavy metal-based (cadmium or lead) QDs. In particular, Ag2S QDs emit in the near infrared-II region (NIR-II window) that enables deep tissue penetration and imaging. Furthermore, various chemotherapeutic agents and targeting moieties can be efficiently conjugated to the surface of Ag2S QDs due to advanced technologies in the relevant surface chemistry using covalent bonding, high affinity, and electrostatic interaction. In addition, Ag2S QDs themselves exhibit an anticancer activity based on the photothermal effect. Consequently, Ag2S QDs can function as both a therapeutic agent and an imaging agent in imaging-based diagnosis concurrently, which led to the creation of Ag2S theragnostic nanomaterials. In this review, the synthetic methods, physicochemical properties, bioconjugations, and bioapplications of Ag2S QD theragnostic nanomaterials are discussed in detail.
Assuntos
Antineoplásicos , Pontos Quânticos , PrataRESUMO
Three-dimensional (3D) visualization of tumor vasculature is a key factor in accurate evaluation of RNA interference (RNAi)-based antiangiogenic nanomedicine, a promising approach for cancer therapeutics. However, this remains challenging because there is not a physiologically relevant in vitro model or precise analytic methodology. To address this limitation, a strategy based on 3D microfluidic angiogenesis-on-a-chip and 3D tumor vascular mapping was developed for evaluating RNAi-based antiangiogenic nanomedicine. We developed a microfluidic model to recapitulate functional 3D angiogenic sprouting when co-cultured with various cancer cell types. This model enabled efficient and rapid assessment of antiangiogenic nanomedicine in treatment of hyper-angiogenic cancer. In addition, tissue-clearing-based whole vascular mapping of tumor xenograft allowed extraction of complex 3D morphological information in diverse quantitative parameters. Using this 3D imaging-based analysis, we observed tumor sub-regional differences in the antiangiogenic effect. Our systematic strategy can help in narrowing down the promising targets of antiangiogenic nanomedicine and then enables deep analysis of complex morphological changes in tumor vasculature, providing a powerful platform for the development of safe and effective nanomedicine for cancer therapeutics.
Assuntos
Nanomedicina , Neoplasias , Humanos , Microfluídica , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Neovascularização Patológica/diagnóstico por imagem , Neovascularização Patológica/tratamento farmacológico , Interferência de RNARESUMO
Antioxidants are essential in regulating various physiological functions and oxidative deterioration. Over the past decades, many researchers have paid attention to antioxidants and studied the screening of antioxidants from natural products and their utilization for treatments in diverse pathological conditions. Nowadays, as printing technology progresses, its influence in the field of biomedicine is growing significantly. The printing technology has many advantages. Especially, the capability of designing sophisticated platforms is useful to detect antioxidants in various samples. The high flexibility of 3D printing technology is advantageous to create geometries for customized patient treatment. Recently, there has been increasing use of antioxidant materials for this purpose. This review provides a comprehensive overview of recent advances in printing technology-based assays to detect antioxidants and 3D printing-based antioxidant therapy in the field of tissue engineering. This review is divided into two sections. The first section highlights colorimetric assays using the inkjet-printing methods and electrochemical assays using screen-printing techniques for the determination of antioxidants. Alternative screen-printing techniques, such as xurography, roller-pen writing, stamp contact printing, and laser-scribing, are described. The second section summarizes the recent literature that reports antioxidant-based therapy using 3D printing in skin therapeutics, tissue mimetic 3D cultures, and bone tissue engineering.
RESUMO
BACKGROUND: In this study, a multifunctional tetraphenylporphyrin (TPP) conjugated polyethylene glycol with biotin (TPP-PEG-biotin) as a photo-dynamic therapy (PDT) material encapsulating a ruthenium complex 1 (Ru-1) was fabricated as self-assembled nanoparticle (Ru-1@TPP-PEG-biotin SAN) to co-target glucose-regulated protein 78 (GRP78) and the lysosome as a new anti-cancer therapeutic strategy. RESULTS: The MTT assay results reveals the enhanced anticancer activity of the Ru-1@TPP-PEG-biotin SANs due to the co-targeting of the GRP78 and lysosome. The Ru-1@TPP-PEG-biotin reduced level of GRP78 and lysosomal ceramide that contributed to the stability of the lysosomal membrane. The endoplasmic reticulum (ER) stress concomitant with the inhibition of GRP78 was clearly monitored by the phosphorylation of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), and inositol-requiring enzyme 1 α (IRE1α) kinases to indicate the activation of the unfolded protein response (UPR) signaling using immunofluorescence assay. On the other hand, the degradation of the lysosome was observed through PDT action by the Ru-1@TPP-PEG-biotin SAN treatment. This was confirmed by the co-localization assay showing the disappearance of cathepsin D and lysosomal-associated membrane protein 1 (LAMP1) in the lysosome. CONCLUSIONS: Considering lysosome-mediated autophagy is an effective cancer cell survival mechanism, the degradation of the lysosome along with GRP78 inhibition by the Ru-1@TPP-PEG-biotin SAN combination therapy is suggested as a new co-targeting cancer treatment.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Proteínas de Choque Térmico/metabolismo , Lisossomos/metabolismo , Nanopartículas/química , Porfirinas/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Biotina/química , Sobrevivência Celular/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/química , Células Hep G2 , Humanos , Lisossomos/química , Células MCF-7 , Nanopartículas/metabolismo , Polietilenoglicóis/química , Compostos de Rutênio/química , Compostos de Rutênio/farmacologiaRESUMO
In this study, an inkjet bioprinting-based high-throughput screening (HTS) system was designed and applied for the first time to a catecholpyrimidine-based small molecule library to find hit compounds that inhibit c-Jun NH2-terminal kinase1 (JNK1). JNK1 kinase, inactivated MAPKAPK2, and specific fluorescent peptides along with bioink were printed on parchment paper under optimized printing conditions that did not allow rapid evaporation of printed media based on Triton-X and glycerol. Subsequently, different small compounds were printed and tested against JNK1 kinase to evaluate their degree of phosphorylation inhibition. After printing and incubation, fluorescence intensities from the phosphorylated/nonphosphorylated peptide were acquired for the % phosphorylation analysis. The IM50 (inhibitory mole 50) value was determined as 1.55 × 10-15 mol for the hit compound, 22. Thus, this work demonstrated that inkjet bioprinting-based HTS can potentially be adopted for the drug discovery process using small molecule libraries, and cost-effective HTS can be expected to be established based on its low nano- to picoliter printing volume.
RESUMO
A graphene oxide (GO)-based cost-effective, automatted strip test has developed for screening of inhibitors of endonuclease EcoRV. The method involves the use of GO and a DNA substrate for EcoRV that contains both an ssDNA region for binding of GO and a fluorescein amidite (FAM)-labelled dsDNA. All the components were inkjet printed on a piece of parchment paper. The ssDNA region binds to the surface of GO and anchors so that the fluorescence of FAM is quenched. The parchment paper strip is then incubated with a sample containing EcoRV which causes enzymatic hydrolysis, and dsDNA was separated from the GO. As a result, green fluorescence is generated at the reaction spot. Enzyme activity can be measured in the presence and absence of aurintricarboxy acid acting as an EcoRV inhibitor. This method excels by its need for 2-3 orders less reagents compared to the standard well plate assay. Thus, it is an efficient platform for GO-based screening of EcoRV enzyme inhibitors. Graphical abstract A graphene oxide (GO)-based endonuclease EcoRV inhibition FRET assay using inkjet printing was developed. Printing of GO along with assay reagents has a beneficial effect on the enzymatic reaction on paper. This method was successfully applied to evaluate EcoRV inhibitor activity.
Assuntos
DNA/química , Inibidores Enzimáticos/química , Exonucleases/antagonistas & inibidores , Fluoresceína/química , Grafite/química , Sequência de Bases , Bioensaio/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Impressão Tridimensional , Espectrometria de Fluorescência/métodosRESUMO
Resistance to chemotherapy is a key factor in the inefficacy of various forms of treatments for cancer. In the present study, chemo-resistant proteins, including glucose-regulated protein 78 (GRP78)/clusterin (CLU) targeted 1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP) liposomes, were developed as a delivery system for co-delivery of camptothecin (CPT) and GRP78 siRNA/CLU siRNA. Their drug/gene co-deliveries were quantitatively assessed in cancer stem cells (CSC) and MCF-7 cells. DOTAP-CPT/siRNA were prepared via electrostatic interaction on GRP78 siRNA or CLU siRNA. The size and ζ-potential of liposomes and lipoplexes were measured by dynamic light scattering techniques and electrophoretic light scattering spectrophotometry. The lipoplexes formation was tested by using gel electrophoresis. Immunofluorescence analysis showed that the expression level of CLU and GRP78 were significantly elevated in CSC compared to MCF-7 cells. Transfection and drug-delivery efficiency of DOTAP-CPT/siRNA were quantitatively compared with Lipofectamine 2000. Compared to free CPT, DOTAP-CPT-siCLU delivery in CSC and MCF-7 cells increased transfection efficiency and chemo-sensitivity by 4.1- and 5.9-fold, respectively. On the other hand, DOTAP-CPT-siGRP78 delivery increased transfection efficiency and chemo sensitivity by 4.4- and 6.2-fold in CSC and MCF-7 cells, respectively, compared to free CPT. It is significant that 3 ± 1.2-fold increase in transfection efficiency was achieved by lipofectamine. Consequently, an increase in anti-cancer/gene silencing efficacy was quantitatively observed as an effect of DOTAP-CPT/siRNA treatment, which was relatively higher than lipofectamine treatment. Conclusively, our experimental data quantitatively demonstrate that using DOTAP-CPT-siRNA specifically targeting (CSCs) chemo-resistant protein in vitro offers substantial potential for synergistic anti-cancer therapy.
Assuntos
Antineoplásicos Fitogênicos , Camptotecina , Clusterina/antagonistas & inibidores , Lipossomos , Células-Tronco Neoplásicas , Antineoplásicos Fitogênicos/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Camptotecina/administração & dosagem , Clusterina/genética , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Chaperona BiP do Retículo Endoplasmático , Feminino , Técnicas de Silenciamento de Genes , Inativação Gênica , Técnicas de Transferência de Genes , Humanos , Lipossomos/química , Células MCF-7 , Células-Tronco Neoplásicas/efeitos dos fármacos , RNA Interferente Pequeno/administração & dosagemRESUMO
The combination of chemotherapy and photodynamic therapy (chemo-PDT) has been suggested as an alternative therapy for drug-resistant cancers. In this study, biotin-conjugated PEGylated photosensitizer (PS) self-assembled nanoparticles (meso-tetraphenylporphyrin (TPP)-PEG-biotin SANs) were prepared via a self-assembly process to serve as nanocarriers for chemo-drugs as well as PSs. Electron microscopy results reveal the spherical shape of the nanoparticles (NPs). In the NPs, conjugated biotin plays a key role in selective tumor targeting. In vitro cellular experiments revealed the rapid cellular uptake of the TPP-PEG-biotin conjugates by MCF-7 cells that overexpress the biotin receptor, and verified that the conjugates were much more effective PSs than TPPS used as control in the cytotoxicity test. Interestingly, subcellular localization studies showed that the conjugates and their self-assembled NPs were localized mainly in mitochondria and partially in lysosomes, whereas TPPS was localized only in lysosomes. With the exclusive localization in mitochondria, high-content cell based assay showed that the TPP-PEG-biotin SANs induced rapid mitochondrial membrane potential transition (MPT), leading to cellular apoptosis. The chemo-drug doxorubicin (DOX) was successfully encapsulated in the TPP-PEG-biotin SANs (DOX@TPP-PEG-biotin) and had synergistic effects with enhanced cytotoxicity after PDT action. Collectively, the DOX@TPP-PEG-biotin SANs have promising potential as an effective anticancer agent in targeted combination therapy.
Assuntos
Portadores de Fármacos/química , Nanopartículas/química , Fármacos Fotossensibilizantes/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Biotina/química , Doxorrubicina/farmacologia , Portadores de Fármacos/farmacologia , Humanos , Lisossomos/efeitos dos fármacos , Células MCF-7 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Nanopartículas/uso terapêutico , Fármacos Fotossensibilizantes/farmacologia , Polietilenoglicóis/química , Porfirinas/químicaRESUMO
Auto neuronal synapses, or autapses, are aberrant structures where the synaptic contact of a neuron forms onto its own branch. The functions of autapses, however, remain unknown. Here, we introduce a simple patterning method for capturing a single-cell, in which we maintained the isolated cell until it reached maturity, and developed arrays of autapses for electrophysiological analysis using multi-electrode arrays (MEA). The pattern arrays were formed by selective patterning of poly-L-lysine and various cell repellent materials. We tested the efficiency of single neuron pattern formed according to materials and pattern dimensions. Autapse formation was verified by immunostaining synaptic markers and physiological measurements via recordings from MEA. The results demonstrated that our multiscale patterning method increased the number of autapses consisting of a single neuron, which matured to connect onto themselves. The proposed patterning method (4.06 ± 0.33 isolated single-cells mm-2) is at least twelve times more efficient and productive than the spray method (0.31 ± 0.10 isolated single-cells mm-2). The spontaneous activity of a single neuron on the patterned MEA occured after 11 d in vitro. The single neuron activity consisted of bursts followed by spike trains (the burst rate was 2.56 min-1). This indicates that our method could be used for electrophysiological analysis, including MEA.
Assuntos
Eletrofisiologia/métodos , Neurônios/química , Sinapses/química , Animais , Linhagem Celular , Células Cultivadas , Eletrofisiologia/instrumentação , Microeletrodos , Neurônios/fisiologia , Polilisina/química , Ratos , Sinapses/fisiologiaRESUMO
MicroRNAs (miRNAs) are important regulatory RNAs that control gene expression in various biological processes. Therefore, control over the disease-related miRNA expression is important both for basic research and for a new class of therapeutic modality to treat serious diseases such as cancer. Here, we present a high-throughput screening strategy to identify small molecules that modulate miRNA expression in living cells. The screen enables simultaneous monitoring of the phenotypic cellular changes associated with the miRNA expression by measuring quantitative fluorescent signals corresponding to target miRNA level in living cells based on a novel biosensor composed of peptide nucleic acid and nano-sized graphene oxide. In this study, the biosensor based cellular screening of 967 compounds (including FDA-approved drugs, enzyme inhibitors, agonists, and antagonists) in cells identified four different classes of small molecules consisting of (i) 70 compounds that suppress both miRNA-21 (miR-21) expression and cell proliferation, (ii) 65 compounds that enhance miR-21 expression and reduce cell proliferation, (iii) 2 compounds that suppress miR-21 expression and increase cell proliferation, and (iv) 21 compounds that enhance both miR-21 expression and cell proliferation. We further investigated the hit compounds to correlate cell morphology changes and cell migration ability with decreased expression of miR-21.
Assuntos
Técnicas Biossensoriais/métodos , Grafite/química , Ensaios de Triagem em Larga Escala/métodos , MicroRNAs/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/metabolismo , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/análiseRESUMO
Ruthenium(II/III) metal complexes have been widely recognized as the alternative chemotherapeutic agents to overcome the drug resistance and tumor recurrence associated with platinum derivatives. In this work, a novel ruthenium(II) triazine complex namely, 1 ([Ru(bdpta)(tpy)]2+) was synthesized and spectroscopically characterized. Drug resistant cancer stem cells (CSCs) were used to evaluate the cytotoxicity of Ru(II) complex 1. The complex 1 showed a greater cytotoxic potential with IC50 values lower than that of cisplatin. The intracellular localization assay confirmed that the complex 1 was effectively distributed into mitochondria as well as endoplasmic reticulum (ER), and executed a ROS-mediated calcium and Bax/Bak dependent intrinsic apoptosis. Interestingly, direct interaction between complex 1 and glucose regulated protein 78 (GRP78), a protein associated with drug resistance caused the ROS-mediated ubiquitination of GRP78. Notably, western blot and confocal microscopy analysis confirmed that complex 1 significantly reduced the protein levels of GRP78. Dose-dependent in vivo antitumor efficacy against CD133+HCT-116 CSCs derived tumor xenograft further validated that complex 1 could be an effective chemotherapeutic agent.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Complexos de Coordenação/uso terapêutico , Proteínas de Choque Térmico/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Rutênio/uso terapêutico , Triazinas/uso terapêutico , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Complexos de Coordenação/análogos & derivados , Complexos de Coordenação/farmacologia , Chaperona BiP do Retículo Endoplasmático , Feminino , Células HEK293 , Proteínas de Choque Térmico/química , Humanos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Simulação de Acoplamento Molecular , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Espécies Reativas de Oxigênio/metabolismo , Rutênio/química , Rutênio/farmacologia , Triazinas/química , Triazinas/farmacologiaRESUMO
Degradation of cyclic adenosine mono phosphate (cAMP) by phosphodiesterase-4B (PDE-4B) in the inflammatory cells leads to elevated expression of inflammatory cytokines in inflammatory cells. Suppression of cytokines has proved to be beneficial in the treatment of atopic dermatitis (AD). Henceforth, application of PDE4B specific inhibitor to minimize the degradation of cAMP can yield better results in the treatment of AD. PDE4B specific inhibitor with a limited side effect is highly warranted. Herein, we synthesized a novel PDE4 inhibitor, compound 2 comprising catecholopyrimidine core functionalized with trifluoromethyl (-CF3) group. PDE4B inhibitory potential and specificity of novel compounds were evaluated by PDE inhibitor assay. In vivo efficacy of the compounds was analyzed using DNCB-induced NC/Nga mice. IgE, CD4+ T-helper cell infiltration, and cytokine profiles were analyzed by ELISA and immunohistochemistry techniques. Toluidine blue staining was performed for mast cell count. PDE4 inhibitor assay confirmed that compound 2 specifically inhibits PDE4B. In vivo analysis with DNCB-induced NC/Nga mice confirmed that compound 2 suppressed the levels of pro-inflammatory cytokines such as TNF-α, IL-4, IL-5, and IL-17. Furthermore, compound 2 significantly reduced the infiltrative CD4+ T-helper cells, mast cells and IgE levels in atopic tissue. The in vitro and in vivo data suggested that compound 2 specifically inhibit the PDE4B and the symptoms of the AD in atopic mice. Compound 2 might constitute a good candidate molecule for the treatment of AD.
RESUMO
Amyloid-ß (Aß) is generated by proteolytic processing of amyloid precursor protein (APP) by beta-secretase (BACE-1) and gamma-secretase. Amyloid-ß is responsible for the formation of senile plaques in Alzheimer's disease (AD). Consequently, inhibition of ß-secretase (BACE-1), a rate-limiting enzyme in the production of Aß, constitutes an attractive therapeutic approach to the treatment of AD. This paper reports an inkjet printing-based fluorescence assay for high throughput screening of ß-secretase inhibitors achieved by employing a BACE-1 FRET substrate (Rh-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys-Quencher). This peptide substrate is known to be a readily available and suitable substrate for proteolytic activity, and it has high affinity to BACE-1. The BACE-1 peptide substrate printed on parchment paper was effectively cleaved by BACE-1, which was printed on the same spot. The amount of enzyme and substrate required for this inkjet printing-based BACE-1 assay can be less than 1.4â ¹103, permitting the evaluation of inhibitor activity with femtomolar potency. The inkjet-printing-based BACE-1 inhibitory assay revealed inhibitory effects of inhibitor IV and STA on BACE-1 with an IM50 of 1.00â¯×â¯10-15â¯mol and 1.01â¯×â¯10-14â¯mol, respectively. These data confirm that both BACE-1 inhibitors (inhibitor IV and STA) actively inhibited the BACE-1 proteolysis of BACE-1 substrate on parchment paper. It important to note that the number of mole of BACE-1-substrate and enzyme utilized in the printing-based enzymatic assay are 1.4â ¹103 smaller than the amount used in the conventional well-plate assay. The inkjet printing-based inhibitory assay constitutes a versatile high throughput technique and the IM50 values of the inhibitors were obtained with satisfactory reproducibility, suggesting that this inkjet-printing BACE-1 inhibitory assay could be quite suitable for the screening of new potential BACE-1 inhibitors for AD.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Tinta , Impressão , Inibidores de Proteases/farmacologia , Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/química , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Inibidores de Proteases/uso terapêutico , Conformação ProteicaRESUMO
Vascular networks are the first sites exposed to cationic polymer nanoparticles (NPs) administered intravenously, and thus function as a barrier for NPs reaching the target organ. While cationic polymer NPs have been intensively studied as non-viral delivery systems, their biological effects in human microvessels have been poorly investigated due to a lack of appropriate in vitro systems. Here, we employed a three-dimensional microvessel on a chip, which accurately models in vivo conditions. An open and perfused microvessel surrounded by pericytes was shown to reproduce the important features of living vasculature, including barrier function and biomarkers. Using this microvessel chip, we observed contraction of the microvascular lumen induced by perfused polyethylenimine (PEI)/DNA NPs. We demonstrated that the oxidative stress present when microvessels were exposed to PEI NPs led to rearrangement of microtubules resulting in microvessel contraction. Furthermore, the transcytotic behavior of PEI NPs was analyzed in the microvessel by monitoring the escape of PEI NPs from the microvascular lumen into the perivascular region, which was not possible in two-dimensional culture systems. With our new understanding of the different behaviors of cationic polymer NPs depending on their transcytotic route, we suggest that caveolae-mediated transcytosis is a powerful route for efficient extravascular transport. STATEMENT OF SIGNIFICANCE: Microvascular networks are not only biological system constituting largest surface area in the body and but also first site exposed to nanoparticle in vivo. While cationic polymer NPs have been intensively studied as non-viral delivery systems, its biological effects in human microvessel have been poorly investigated due to lack of appropriate in vitro systems. Here, we microengineered an open and perfused 3D pericyte incorporated microvessel model which possesses same morphological characteristic of in vivo. Using the microengineered model, this study represents the first report of transcytotic behavior of NPs in 3D microvessel, and its effect on extravasation efficiency. Our study lays the groundwork for the integration of innovative technologies to examine blood vessel-nanoparticle interaction, which a critical but ill-defined phenomenon.