RESUMO
Lymphocyte-specific protein tyrosine kinase (Lck) plays vital roles in the T-cell receptor- mediated development, function, and differentiation of T-cells. Given its substantial involvement in T cell signaling, irregularities in the expression and functionality of Lck may lead to various diseases, including cancer. In this study, we found that compound 12a exerted significant inhibitory potency against Lck with an IC50 value of 10.6 nM. In addition, 12a demonstrated high efficacy in various colon cancer cell lines as indicated by GI50 values ranging from 0.24 to 1.26 µM. Notably, 12a inhibited the phosphorylation of Lck in Colo201 cells. Overall, the anti-proliferative effects of 12a on diverse cancer cell lines highlights its potential application for the treatment of various cancer types.
Assuntos
Antineoplásicos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/farmacologia , Linfócitos T , Transdução de Sinais , Fosforilação , Receptores de Antígenos de Linfócitos T/metabolismo , Antineoplásicos/farmacologiaRESUMO
Combining standard surgical procedures with personalized chemotherapy and the continuous monitoring of cancer progression is necessary for effective NSCLC treatment. In this study, we developed liposomal nanoparticles as theranostic agents capable of simultaneous therapy for and imaging of target cancer cells. Copper-64 (64Cu), with a clinically practical half-life (t1/2 = 12.7 h) and decay properties, was selected as the radioisotope for molecular PET imaging. An anti-epidermal growth factor receptor (anti-EGFR) antibody was used to achieve target-specific delivery. Simultaneously, the chemotherapeutic agent doxorubicin (Dox) was encapsulated within the liposomes using a pH-gradient method. The conjugates of 64Cu-labeled and anti-EGFR antibody-conjugated micelles were inserted into the doxorubicin-encapsulating liposomes via a post-insertion procedure (64Cu-Dox-immunoliposomes). We evaluated the size and zeta-potential of the liposomes and analyzed target-specific cell binding and cytotoxicity in EGFR-positive cell lines. Then, we analyzed the specific therapeutic effect and PET imaging of the 64Cu-Dox-immunoliposomes with the A549 xenograft mouse model. In vivo therapeutic experiments on the mouse models demonstrated that the doxorubicin-containing 64Cu-immunoliposomes effectively inhibited tumor growth. Moreover, the 64Cu-immunoliposomes provided superior in vivo PET images of the tumors compared to the untargeted liposomes. We suggest that nanoparticles will be the potential platform for cancer treatment as a widely applicable theranostic system.
Assuntos
Radioisótopos de Cobre , Doxorrubicina , Lipossomos , Neoplasias , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Cobre , Doxorrubicina/uso terapêutico , Doxorrubicina/análogos & derivados , Sistemas de Liberação de Medicamentos/métodos , Receptores ErbB/metabolismo , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Polietilenoglicóis , Tomografia por Emissão de Pósitrons , Medicina de PrecisãoRESUMO
Microsatellite instability (MSI) is a hypermutable condition caused by DNA mismatch repair system defects, contributing to the development of various cancer types. Recent research has identified Werner syndrome ATP-dependent helicase (WRN) as a promising synthetic lethal target for MSI cancers. Herein, we report the first discovery of thiophen-2-ylmethylene bis-dimedone derivatives as novel WRN inhibitors for MSI cancer therapy. Initial computational analysis and biological evaluation identified a new scaffold for a WRN inhibitor. Subsequent SAR study led to the discovery of a highly potent WRN inhibitor. Furthermore, we demonstrated that the optimal compound induced DNA damage and apoptotic cell death in MSI cancer cells by inhibiting WRN. This study provides a new pharmacophore for WRN inhibitors, emphasizing their therapeutic potential for MSI cancers.
Assuntos
Instabilidade de Microssatélites , Neoplasias , Tiofenos , Humanos , Cicloexanonas , Neoplasias/tratamento farmacológico , Neoplasias/genética , Helicase da Síndrome de Werner/antagonistas & inibidores , Helicase da Síndrome de Werner/metabolismo , Tiofenos/química , Tiofenos/farmacologiaRESUMO
Early detection of rare mutations through liquid biopsy can provide real-time information related to cancer diagnosis, prognosis, and treatment outcomes. Cell-free DNA samples used in liquid biopsies contain single-nucleotide variants (SNVs) with a variant allele frequency (VAF) of approximately ≤1%. Droplet digital polymerase chain reaction (ddPCR) is considered the gold standard of sequencing using liquid samples, generating amplicons from samples containing mutations with 0.001-0.005% VAF; however, it requires expensive equipment and time-consuming protocols. Therefore, various PCR methods for discriminating SNVs have been developed; nonetheless, non-specific amplification cannot be avoided even in the absence of mutations, which hampers the accurate diagnosis of SNVs. In this study, we introduce single-nucleotide variant on-off discrimination-PCR (Soo-PCR), a highly accurate and practical method that uses a 3'-end tailing primer for the on-off discrimination of low-abundance mutant-type targets, including SNVs. Soo-PCR minimizes the chance of incorrect judgments owing to its high discriminating power. Cancer markers, such as KRAS G12D, EGFR L858R, and EGFR T790M mutations, containing 0.1% VAF, were clearly detected in under 2 h with a high reliability comparable with that of ddPCR. This new method serves as a practical approach to accurately detect and evaluate low-abundance mutations in a user-friendly manner.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Receptores ErbB/genética , Reprodutibilidade dos Testes , Inibidores de Proteínas Quinases , Mutação , NucleotídeosRESUMO
Nucleic acid testing (NAT) is important for the identification and quantification of specific nucleic acid targets, both DNA and RNA, in life sciences and clinical diagnostics. Nucleic acid amplification can be a time-consuming step in NAT using the polymerase chain reaction (PCR) assay. Therefore, this study aimed to develop a simple method to reduce the amplification time while maintaining the PCR system. The three-step process of a general qPCR was reduced to a two-step process. The annealing/extension temperatures were increased to minimize the differences between the denaturation temperature and the annealing/extension temperatures. Subsequently, the time for each of these steps was reduced and, finally, the denaturation temperature was lowered. Taq polymerase was replaced with SD polymerase because it has strand displacement activity and is efficient in amplifying partial dsDNA at lower denaturation temperatures. In the two-step qPCR of genomic DNA using SD polymerase, the final conditions included an initial denaturation at 92 °C for 2 min, and 1 s at each cycling step with a denaturation temperature of 87 °C and an annealing/extension temperature of 72 °C. Amplification of the nucleocapsid (N) gene of SARS-CoV-2 RNA virus was evaluated at a template concentration as low as 10 copies. This method, named SF-qPCR (strand displacement-based fast quantitative polymerase chain reaction), can stably detect less than 10 copies of DNA and RNA within 25-40 min. This new protocol allows for sensitive and rapid detection of important DNA and RNA targets in clinical diagnosis. Supplementary Information: The online version contains supplementary material available at 10.1007/s13206-021-00044-x.
RESUMO
In cancer immunotherapy, the cyclic GMP-AMP synthase-stimulator of interferon genes (STING) pathway is an attractive target for switching the tumor immunophenotype from 'cold' to 'hot' through the activation of the type I interferon response. To develop a new chemical entity for STING activator to improve cyclic GMP-AMP (cGAMP)-induced innate immune response, we identified KAS-08 via the structural modification of DW2282, which was previously reported as an anti-cancer agent with an unknown mechanism. Further investigation revealed that direct STING binding or the enhanced phosphorylation of STING and downstream effectors were responsible for DW2282-or KAS-08-mediated STING activity. Furthermore, KAS-08 was validated as an effective STING pathway activator in vitro and in vivo. The synergistic effect of cGAMP-mediated immunity and efficient anti-cancer effects successfully demonstrated the therapeutic potential of KAS-08 for combination therapy in cancer treatment.
RESUMO
A novel microRNA (miRNA) quantification method has been developed using one-step rolling circle-quantitative PCR (RC-qPCR) analysis. Vent (exo-) DNA polymerase is firstly utilized to combine a rolling circle amplification (RCA) and qPCR in one step with high sensitivity and specificity in our RC-qPCR assay. Before performing the RC-qPCR, a padlock probe is ligated only when it is perfectly hybridized with miRNA. This ligation-based miRNA assay is highly specific for mature miRNAs, discriminating among related miRNAs that differ by as little as one nucleotide. It exhibits a dynamic range of seven orders of magnitude with a detection limit of 500 aM, and could be also used for the quantification of other small RNA molecules such as short interfering RNAs (siRNAs).
Assuntos
MicroRNAs/análise , Reação em Cadeia da Polimerase/métodos , Linhagem Celular Tumoral , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , DNA Polimerase Dirigida por DNA/química , Humanos , Limite de Detecção , MicroRNAs/genética , Hibridização de Ácido Nucleico , Sensibilidade e Especificidade , Thermococcus/enzimologiaRESUMO
CONSPECTUS: While the field of DNA computing and molecular programming was engendered in large measure as a curiosity-driven exercise, it has taken on increasing importance for analytical applications. This is in large measure because of the modularity of DNA circuitry, which can serve as a programmable intermediate between inputs and outputs. These qualities may make nucleic acid circuits useful for making decisions relevant to diagnostic applications. This is especially true given that nucleic acid circuits can potentially directly interact with and be triggered by diagnostic nucleic acids and other analytes. Chemists are, by and large, unaware of many of these advances, and this Account provides a means of touching on what might seem to be an arcane field. We begin by explaining nucleic acid amplification reactions that can lead to signal amplification, such as catalytic hairpin assembly (CHA) and the hybridization chain reaction (HCR). In these circuits, a single-stranded input acts on kinetically trapped substrates via exposed toeholds and strand exchange reactions, refolding the substrates and allowing them to interact with one another. As multiple duplexes (CHA) or concatemers of increasing length (HCR) are generated, there are opportunities to couple these outputs to different analytical modalities, including transduction to fluorescent, electrochemical, and colorimetric signals. Because both amplification and transduction are at their root dependent on the programmability of Waston-Crick base pairing, nucleic acid circuits can be much more readily tuned and adapted to new applications than can many other biomolecular amplifiers. As an example, robust methods for real-time monitoring of isothermal amplification reactions have been developed recently. Beyond amplification, nucleic acid circuits can include logic gates and thresholding components that allow them to be used for analysis and decision making. Scalable and complex DNA circuits (seesaw gates) capable of carrying out operations such as taking square roots or implementing neural networks capable of learning have now been constructed. Into the future, we can expect that molecular circuitry will be designed to make decisions on the fly that reconfigure diagnostic devices or lead to new treatment options.
Assuntos
Desenho de Equipamento , Hibridização de Ácido Nucleico , Ácidos Nucleicos/química , Pareamento de Bases , Antígeno Carcinoembrionário/análise , Catálise , Técnicas de Química Analítica/métodos , Colorimetria , DNA/química , Sondas de DNA , DNA Catalítico , Entropia , Humanos , Limite de DetecçãoRESUMO
We have designed a single-stranded DNAzyme-aptamer sensor for homogeneous target molecular detection based on chemiluminescence resonance energy transfer (CRET). The structure of the engineered single-stranded DNA (ssDNA) includes the horseradish peroxidase (HRP)-like DNAzyme, optimum-length linker (10-mer-length DNA), and target-specific aptamer sequences. A quencher dye was modified at the 3' end of the aptamer sequence. The incorporation of hemin into the G-quadruplex structure of DNAzyme yields an active HRP-like activity that catalyzes luminol to generate a chemiluminescence (CL) signal. In the presence of target molecules, such as ochratoxin A (OTA), adenosine triphosphate (ATP), or thrombin, the aptamer sequence was folded due to the formation of the aptamer/analyte complex, which induced the quencher dye close to the DNAzyme structure. Consequently, the CRET occurred between a DNAzyme-catalyzed chemiluminescence reaction and the quencher dye. Our results showed that CRET-based DNAzyme-aptamer biosensing enabled specific OTA analysis with a limit of detection of 0.27ng/mL. The CRET platform needs no external light source and avoids autofluorescence and photobleaching, and target molecules can be detected specifically and sensitively in a homogeneous manner.
Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/instrumentação , DNA Catalítico/química , Medições Luminescentes/instrumentação , Ocratoxinas/análise , Desenho de Equipamento , Análise de Falha de Equipamento , Transferência Ressonante de Energia de Fluorescência , Ocratoxinas/químicaRESUMO
Utilizing a peptide nucleic acid (PNA)-modified electrode and a single-stranded DNA specific endonuclease, a novel electrochemical method to identify DNA mutations has been developed and represents a totally new strategy for the electrochemical diagnosis of human genetic mutations.
Assuntos
DNA/análise , Técnicas Eletroquímicas/métodos , Endonucleases/metabolismo , Ácidos Nucleicos Peptídicos/química , Proteína BRCA1/genética , Pareamento Incorreto de Bases , DNA/genética , DNA de Cadeia Simples/química , Eletrodos , Ferricianetos/química , Humanos , Mutação , Neoplasias/diagnóstico , Neoplasias/genética , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Gold nanoparticle (GN) embedded silicon nanowire (SiNW) configuration was proposed as a new biosensor for label-free DNA detection to enhance the sensitivity. The electric current flow between two terminals, a source and a drain electrode, were measured to sense the immobilization of probe oligonucleotides and their hybridization with target oligonucleotides. The complementary target oligonucleotide, breast cancer DNA with 1 pM, was sensed. In addition, its sensing mechanism and limit of detection (LOD) enhancement was investigated through simulation. The results support that the LOD can be improved by reducing the SiNW doping concentration. This emerging architecture combined nanostructure of spherical GN and SiNW has high potential as a label-free biosensor due to its facile fabrication process, high thermal stability, immobilization efficiency with a thiol-group in a self-assembled monolayer (SAM), and improved sensitivity.
Assuntos
Técnicas Biossensoriais/instrumentação , DNA/análise , Nanopartículas Metálicas , Nanofios , Sequência de Bases , Neoplasias da Mama/genética , DNA/genética , Sondas de DNA/genética , DNA de Neoplasias/análise , DNA de Neoplasias/genética , Técnicas Eletroquímicas , Feminino , Ouro , Humanos , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Varredura , Nanofios/ultraestrutura , SilícioRESUMO
Here we describe a label-free detection strategy for point mutation in breast cancer susceptibility gene BRCA1 utilizing ligation chain reaction (LCR) and zip-code array. We amplified the genomic regions containing mutation sites by polymerase chain reaction (PCR) to prepare the template for LCR. Then we ligated a primary probe extended by zip-code complementary at the 5' end with a 3'-thiol modified secondary probe. The resulting ligated product was labeled with gold nanoparticles and allowed to hybridize with corresponding zip-code sequences on the microarray. Finally, we applied silver enhancement to amplify the signals. Using this strategy, we successfully genotyped BRCA1 mutation, which was detected by naked eye.
Assuntos
DNA/genética , Genes BRCA1 , Ouro/química , Nanopartículas Metálicas , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Sequência de Bases , Primers do DNA , Reação em Cadeia da PolimeraseRESUMO
In this report, a reliable peptide nucleic acid (PNA) microarray-based method for accurately detecting single nucleotide polymorphism (SNP) in human genes is described. The technique relies on the mismatched cleavage activity of a single-strand specific (SSS) nuclease. PCR amplification was performed to prepare gene fragments containing the mutation sites. The amplified fragments were then employed as templates for the SSS nuclease reaction using chimeric probes, modified with biotin at the 5' end and extended with a unique anchoring zip-code complement sequence at the 3' end. The SSS nuclease promotes cleavage of heteroduplex DNAs at base mismatched positions to produce crumbled chimeric probes in the presence of imperfectly matching template strands. In contrast, the probes remain intact when they interact with perfectly matched template strands. Only the non-fragmented probes generate fluorescence signals after treatment with streptavidin-Cy3 on the PNA zip-code array. This methodology was used to successfully genotype selected Korean-specific BRCA mutation sites with wild type and mutant samples. The investigation has led to the development of a reliable SSS nuclease-based system for the diagnosis of human genetic mutations or SNPs.
Assuntos
Análise Mutacional de DNA/instrumentação , DNA/química , DNA/genética , Desoxirribonucleases/química , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Ácidos Nucleicos Peptídicos/química , Polimorfismo de Nucleotídeo Único/genética , Sequência de Bases , Desenho de Equipamento , Análise de Falha de Equipamento , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A reliable multiplex assay procedure to detect human genetic mutations in the breast cancer susceptibility gene BRCA1 using zip-code microarrays and single base extension (SBE) reactions is described. Multiplex PCR amplification was performed to amplify the genomic regions containing the mutation sites. The PCR products were then employed as templates in subsequent multiplex SBE reactions using bifunctional primers carrying a unique complementary zip sequence in addition to a mutation-site-specific sequence. The SBE primers, terminating one base before their mutation sites, were extended by a single base at a mutation site with a corresponding biotin-labeled ddNTP. Hybridization of the SBE products to zip-code microarrays was followed by staining with streptavidin-Cy3, leading to successful genotyping of several selected BRCA1 mutation sites with wild-type and heterozygote mutant samples from breast cancer patients. This work has led to the development of a reliable DNA microarray-based system for the diagnosis of human genetic mutations.
Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Feminino , Triagem de Portadores Genéticos , Testes Genéticos/métodos , Genótipo , Humanos , Coreia (Geográfico) , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/métodosRESUMO
We describe here ligation-based strategy to detect mutations in BRCA1 utilizing zip-code microarray technology. In our first approach, PCR was performed to amplify the genomic regions containing the mutation sites. The PCR products were then used as templates in a subsequent ligation reaction using two ligation primers that flanked the mutation site. The primary allele-specific primer is designed to contain a base of mutation site at its 3' end with 5' complementarity to the respective zip-code sequence while the secondary common primer is modified by biotin at its 3' end. Depending on the genotype of samples at the mutation site, the nick between the two ligation primers can be sealed in the presence of DNA ligase. The ligation products were then hybridized on the zip-code microarray followed by staining with streptavidine-cy3 to generate a fluorescent signal. Using this strategy we successfully genotyped selected Korean-specific mutation sites in exon 11 of BRCA1 with a wild type and two heterozygote mutant samples. Furthermore, we also demonstrated that ligase chain reaction using unamplified genomic DNA as direct templates is enough to generate sufficient signals for correct genotypings in a multiplexed manner, verifying first that PCR is not essential for this microarray-based strategy.