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1.
Am J Physiol Cell Physiol ; 327(1): C184-C192, 2024 Jul 01.
Article in English | MEDLINE | ID: mdl-38826137

ABSTRACT

Clinical experience with tyrosine kinase inhibitors (TKIs) over the past two decades has shown that, despite the apparent therapeutic benefit, nearly 30% of patients with chronic myelogenous leukemia (CML) display primary resistance or intolerance to TKIs, and approximately 25% of those treated are forced to switch TKIs at least once during therapy due to acquired resistance. Safe and effective treatment modalities targeting leukemic clones that escape TKI therapy could hence be game changers in the professional management of these patients. Here, we aimed to investigate the efficacy of a novel therapeutic oligonucleotide of unconventional design, called ASP210, to reduce BCR-ABL1 mRNA levels in TKI-resistant CML cells, with the assumption of inducing their apoptosis. Imatinib- and dasatinib-resistant sublines of BCR-ABL1-positive MOLM-7 and CML-T1 cells were established and exposed to 0.25 and 2.5 µM ASP210 for 10 days. RT-qPCR showed a remarkable reduction of the target mRNA level by >99% after a single application. Cell viability was monitored daily by trypan blue staining. In response to the lack of driver oncoprotein BCR-ABL1, TKI-resistant CML cells underwent apoptosis regardless of the presence of the clinically relevant T315I mutation by day 5 after redosing with ASP210. The effect was selective for cancer cells, indicating a favorable safety profile for this therapeutic modality. Furthermore, the spontaneous uptake and high intracellular concentrations of ASP210 suggest its potential to be effective at relatively low doses. The present findings suggest that ASP210 is a promising therapeutic avenue for patients with CML who fail to respond to TKI therapy.NEW & NOTEWORTHY Effective treatment modalities targeting leukemic clones that escape tyrosine kinase inhibitor (TKI) therapy could be game changers in the professional management of patients displaying primary resistance, intolerance, or acquired resistance to TKIs. Although delivering authentic innovations today is more complex than ever, we developed a highly potent and safe oligonucleotide-based modality against BCR-ABL1 mRNA named ASP210 that effectively induces cell death in BCR-ABL1-positive TKI-resistant cells while sparing BCR-ABL1-negative healthy cells.


Subject(s)
Apoptosis , Drug Resistance, Neoplasm , Fusion Proteins, bcr-abl , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Oligonucleotides , Protein Kinase Inhibitors , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Drug Resistance, Neoplasm/drug effects , Protein Kinase Inhibitors/pharmacology , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/antagonists & inhibitors , Fusion Proteins, bcr-abl/metabolism , Cell Line, Tumor , Oligonucleotides/pharmacology , Apoptosis/drug effects , Imatinib Mesylate/pharmacology , Imatinib Mesylate/therapeutic use , Dasatinib/pharmacology , Antineoplastic Agents/pharmacology , Cell Survival/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism
2.
Viruses ; 14(4)2022 03 25.
Article in English | MEDLINE | ID: mdl-35458415

ABSTRACT

In only two years, the coronavirus disease 2019 (COVID-19) pandemic has had a devastating effect on public health all over the world and caused irreparable economic damage across all countries. Due to the limited therapeutic management of COVID-19 and the lack of tailor-made antiviral agents, finding new methods to combat this viral illness is now a priority. Herein, we report on a specific oligonucleotide-based RNA inhibitor targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It displayed remarkable spontaneous cellular uptake, >94% efficiency in reducing RNA-dependent RNA polymerase (RdRp) RNA levels in transfected lung cell lines, and >98% efficiency in reducing SARS-CoV-2 RNA levels in samples from patients hospitalized with COVID-19 following a single application.


Subject(s)
COVID-19 Drug Treatment , Oligonucleotides , SARS-CoV-2 , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Humans , Oligonucleotides/pharmacology , Oligonucleotides/therapeutic use , RNA, Viral/genetics , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/metabolism , SARS-CoV-2/genetics
3.
Biomed Pharmacother ; 147: 112662, 2022 Mar.
Article in English | MEDLINE | ID: mdl-35091237

ABSTRACT

Acquired drug resistance and metastasis in breast cancer (BC) are coupled with epigenetic deregulation of gene expression. Epigenetic drugs, aiming to reverse these aberrant transcriptional patterns and sensitize cancer cells to other therapies, provide a new treatment strategy for drug-resistant tumors. Here we investigated the ability of DNA methyltransferase (DNMT) inhibitor decitabine (DAC) to increase the sensitivity of BC cells to anthracycline antibiotic doxorubicin (DOX). Three cell lines representing different molecular BC subtypes, JIMT-1, MDA-MB-231 and T-47D, were used to evaluate the synergy of sequential DAC + DOX treatment in vitro. The cytotoxicity, genotoxicity, apoptosis, and migration capacity were tested in 2D and 3D cultures. Moreover, genome-wide DNA methylation and transcriptomic analyses were employed to understand the differences underlying DAC responsiveness. The ability of DAC to sensitize trastuzumab-resistant HER2-positive JIMT-1 cells to DOX was examined in vivo in an orthotopic xenograft mouse model. DAC and DOX synergistic effect was identified in all tested cell lines, with JIMT-1 cells being most sensitive to DAC. Based on the whole-genome data, we assume that the aggressive behavior of JIMT-1 cells can be related to the enrichment of epithelial-to-mesenchymal transition and stemness-associated pathways in this cell line. The four-week DAC + DOX sequential administration significantly reduced the tumor growth, DNMT1 expression, and global DNA methylation in xenograft tissues. The efficacy of combination therapy was comparable to effect of pegylated liposomal DOX, used exclusively for the treatment of metastatic BC. This work demonstrates the potential of epigenetic drugs to modulate cancer cells' sensitivity to other forms of anticancer therapy.


Subject(s)
Breast Neoplasms/pathology , DNA (Cytosine-5-)-Methyltransferase 1/antagonists & inhibitors , Decitabine/pharmacology , Doxorubicin/pharmacology , Drug Resistance, Neoplasm , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , Breast Neoplasms/genetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Survival/drug effects , DNA Methylation/drug effects , Dose-Response Relationship, Drug , Doxorubicin/analogs & derivatives , Epithelial-Mesenchymal Transition , Female , Genes, erbB-2/genetics , Humans , Inhibitory Concentration 50 , Mice , Mice, SCID , Mutagenicity Tests , Polyethylene Glycols/pharmacology , Random Allocation , Trastuzumab/pharmacology , Tumor Burden/drug effects , Xenograft Model Antitumor Assays
4.
Methods ; 197: 54-62, 2022 01.
Article in English | MEDLINE | ID: mdl-33677061

ABSTRACT

Biosensing atomic force microscopy (AFM) offers the unique feature to determine the energy landscape of a bimolecular interaction at the real single molecule level. Furthermore, simultaneous and label-free mapping of molecular recognition and the determination of sample topography at the nanoscale gets possible. A prerequisite and one of the major parts in biosensing AFM are the bio-functionalized AFM tips. In the past decades, different approaches for tip functionalization have been developed. Using these functionalization strategies, several biological highly relevant interactions at the single molecule level have been explored. For the most common approach, the use of a heterobifunctional poly(ethylenglycol) crosslinker, a broad range of linkers for different chemical coupling strategies is available. Nonetheless, the time consuming functionalization protocol as well as the broad distribution of rupture length reduces the possibility of automation and may reduce the accuracy of the results. Here we present a stable and fast forward approach based on tetra-functional DNA tetrahedra. A fast functionalization and a sharp defined distribution of rupture length gets possible with low effort and high success rate. We tested the performance on the classical avidin biotin system by using tetrahedra with three disulfide legs for stable and site directed coupling to gold coated tips and a biotinylated end at the fourth vertex. A special advantage appears when working with a DNA aptamer as sensing molecule. In this case, the fourth strand can be extended by a certain DNA sequence complementary to the linkage part of an aptamer. This AFM tip functionalization protocol was applied on thrombin using DNA aptamers directed against the fibrinogen binding side of human thrombin.


Subject(s)
Aptamers, Nucleotide , Avidin , Aptamers, Nucleotide/metabolism , Avidin/chemistry , Avidin/metabolism , Biotin/chemistry , DNA , Humans , Microscopy, Atomic Force/methods
5.
Int J Mol Sci ; 22(11)2021 May 23.
Article in English | MEDLINE | ID: mdl-34071136

ABSTRACT

In this paper, we compared the effects of bortezomib on L1210 (S) cells with its effects on P-glycoprotein (P-gp)-positive variant S cells, which expressed P-gp either after selection with vincristine (R cells) or after transfection with a human gene encoding P-gp (T cells). Bortezomib induced the death-related effects in the S, R, and T cells at concentrations not exceeding 10 nM. Bortezomib-induced cell cycle arrest in the G2/M phase was more pronounced in the S cells than in the R or T cells and was related to the expression levels of cyclins, cyclin-dependent kinases, and their inhibitors. We also observed an increase in the level of polyubiquitinated proteins (via K48-linkage) and a decrease in the gene expression of some deubiquitinases after treatment with bortezomib. Resistant cells expressed higher levels of genes encoding 26S proteasome components and the chaperone HSP90, which is involved in 26S proteasome assembly. After 4 h of preincubation, bortezomib induced a more pronounced depression of proteasome activity in S cells than in R or T cells. However, none of these changes alone or in combination sufficiently suppressed the sensitivity of R or T cells to bortezomib, which remained at a level similar to that of S cells.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Antineoplastic Agents/pharmacology , Bortezomib/pharmacology , Drug Resistance, Multiple/drug effects , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Leukemia, Lymphoid/pathology , Neoplasm Proteins/metabolism , Protease Inhibitors/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Animals , Cell Cycle/drug effects , Cell Division , Cell Line, Tumor , Deubiquitinating Enzymes , Fluoresceins/metabolism , Genes, cdc/drug effects , Humans , Inhibitory Concentration 50 , Leukemia, Lymphoid/genetics , Leukemia, Lymphoid/metabolism , Mice , Neoplasm Proteins/genetics , Proteasome Endopeptidase Complex/drug effects , Proteasome Endopeptidase Complex/metabolism , RNA, Messenger/biosynthesis , RNA, Neoplasm/biosynthesis , Recombinant Proteins/metabolism , Transcription, Genetic/drug effects , Ubiquitinated Proteins/metabolism , Vincristine/pharmacology
6.
Molecules ; 25(9)2020 Apr 30.
Article in English | MEDLINE | ID: mdl-32365761

ABSTRACT

Variants of L1210 leukemia cells-namely, parental P-glycoprotein-negative S cells and R and T cells expressing P-glycoprotein, due to selection with vincristine and transfection with the human p-glycoprotein gene, respectively-were used. The responses of these cell variants to two naturally occurring isothiocyanates-sulforaphane (SFN, from cruciferous vegetables) and allyl isothiocyanate (AITC, from mustard, radish, horseradish and wasabi)-were studied. We obtained conflicting results for the cell death effects induced by isothiocyanates, as measured by i. cell counting, which showed inhibited proliferation, and ii. cell metabolic activity via an MTS assay, which showed an increased MTS signal. These results indicated the hyperactivation of cell metabolism induced by treatment with isothiocyanates. In more detailed study, we found that, depending on the cell variants and the isothiocyanate used in treatment, apoptosis and necrosis (detected by annexin-V cells and propidium iodide staining), as well as autophagy (detected with monodansylcadaverine), were involved in cell death. We also determined the cell levels/expression of Bcl-2 and Bax as representative anti- and pro-apoptotic proteins of the Bcl-2 family, the cell levels/expression of members of the canonical and noncanonical NF-κB pathways, and the cell levels of 16 and 18 kDa fragments of LC3B protein as markers of autophagy.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Cell Survival/drug effects , Drug Resistance, Neoplasm/drug effects , Isothiocyanates/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Apoptosis/drug effects , Autophagy/drug effects , Biomarkers , Cell Cycle/drug effects , Cell Death/drug effects , Cell Line, Tumor , Dose-Response Relationship, Drug , Humans , Lysosomes/metabolism , Mice , Molecular Structure , Sulfoxides
7.
Biosensors (Basel) ; 9(2)2019 May 27.
Article in English | MEDLINE | ID: mdl-31137893

ABSTRACT

Detection of the breast cancer cells is important for early diagnosis of the cancer. We applied thickness shear mode acoustics method (TSM) for detection of SK-BR-3 breast cancer cells using DNA aptamers specific to HER2 positive membrane receptors. The biotinylated aptamers were immobilized at the neutravidin layer chemisorbed at gold surface of TSM transducer. Addition of the cells resulted in decrease of resonant frequency, fs, and in increase of motional resistance, Rm. Using gold nanoparticles (AuNPs), modified by aptamers it was possible improving the limit of detection (LOD) that reached 550 cells/mL, while without amplification the sensitivity of the detection of SK-BR-3 cells was 1574 cells/mL. HER2 negative cell line MDA-MB-231 did not resulted in significant changes of fs. The viability studies demonstrated that cells are stable at experimental conditions used during at least 8 h. AuNPs were not toxic on the cells up to concentration of 1 µg/mL.


Subject(s)
Acoustics/instrumentation , Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , Breast Neoplasms/pathology , Receptor, ErbB-2/analysis , Breast Neoplasms/metabolism , Cell Line, Tumor , Female , Gold/chemistry , Humans , Metal Nanoparticles/chemistry , Molecular Diagnostic Techniques/methods , Receptor, ErbB-2/metabolism
8.
Chemphyschem ; 20(4): 545-554, 2019 02 18.
Article in English | MEDLINE | ID: mdl-30552789

ABSTRACT

By using the thickness shear mode acoustics method (TSM) and single-molecule force spectroscopy (SMFS) we studied the interactions between DNA aptamers (sgc8c) specific to the protein tyrosine kinase 7 (PTK7), which is localized in the membranes of leukemia lymphoblastics (MOLT-4), and lymphocyte (Jurkat) cell lines, as well with PTK7-negative U266 myeloid leukemia cells. The TSM method allowed the development of a highly sensitive, label-free biosensor for the detection leukemia cells with a limit of detection of (195±20) cells/mL. SMFS approved the high selectivity of the sgc8c aptamers to the PTK7 receptors at the cell surface and allowed determining the binding probability of the aptamers to the PTK7 receptors at different cell lines.


Subject(s)
Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , DNA/chemistry , Leukemia/diagnosis , Acoustics , Cell Adhesion Molecules/chemistry , Cell Line, Tumor , Humans , Leukemia/blood , Limit of Detection , Receptor Protein-Tyrosine Kinases/chemistry
9.
Anal Bioanal Chem ; 409(11): 2767-2776, 2017 Apr.
Article in English | MEDLINE | ID: mdl-28229174

ABSTRACT

We studied the interaction of the specific DNA aptamer sgc8c immobilized at the AFM tip with its corresponding receptor, the protein tyrosine kinase-7 (PTK7) embedded in the membrane of acute lymphoblastic leukemia (ALL) cells (Jurkat T-cells). Performing single molecule force spectroscopy (SMFS) experiments, we showed that the aptamer sgc8c bound with high probability (38.3 ± 7.48%) and high specificity to PTK7, as demonstrated by receptor blocking experiments and through comparison with the binding behavior of a nonspecific aptamer. The determined kinetic off-rate (koff = 5.16 s-1) indicates low dissociation of the sgc8c-PTK7 complex. In addition to the pulling force experiments, simultaneous topography and recognition imaging (TREC) experiments using AFM tips functionalized with sgc8c aptamers were realized on the outer regions surface of surface-immobilized Jurkat cells for the first time. This allowed determination of the distribution of PTK7 without any labeling and at near physiological conditions. As a result, we could show a homogeneous distribution of PTK7 molecules on the outer regions of ALL cells with a surface density of 325 ± 12 PTK7 receptors (or small receptor clusters) per µm2. Graphical Abstract The specific interaction of the DNA aptamer sgc8c and protein tyrosine kinase-7 (PTK7) on acute lymphoblastic leukemia (ALL) cells was characterized. AFM based single molecule force spectroscopy (SMFS) yielded a kinetic off-rate of 5.16 s-1 of the complex. Simultaneous topography and recognition imaging (TREC) revealed a PTK7 density of 325 ± 12 molecules or clusters per µm2 in the cell membrane.


Subject(s)
Aptamers, Nucleotide/metabolism , Cell Adhesion Molecules/metabolism , Microscopy, Atomic Force/methods , Molecular Imaging/methods , Protein Interaction Mapping/methods , Receptor Protein-Tyrosine Kinases/metabolism , T-Lymphocytes/metabolism , Binding Sites , Biosensing Techniques/methods , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Humans , Jurkat Cells , Protein Binding , T-Lymphocytes/ultrastructure
10.
Biochim Biophys Acta ; 1858(12): 3005-3016, 2016 12.
Article in English | MEDLINE | ID: mdl-27620331

ABSTRACT

In this study, dendrimers have been purposed as an alternative approach for delivery of HIV peptides to dendritic cells. We have investigated the interaction of dendriplexes formed from polyanionic HIV peptide Nef and cationic carbosilane dendrimer (CBD) with model lipid membranes - large unilamellar vesicles (LUVs), Langmuir monolayers and supported lipid membranes (sBLMs) containing various molar ratio of zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG2000). In our experiments, the lipid membranes represented the model of the plasma membrane of the cell. PEGylated lipids were used in order to model glycocalyx which constitutes the outer leaflet of cellular membranes. The presence of PEGylated lipids resulted in an increase of the phase transition temperature of the lipid bilayer of LUVs, in a decrease of specific volume and adiabatic compressibility. Fluorescence anisotropy study suggests that PEGylated LUVs possessed higher lipid order and decreased fluidity when compared to zwitterionic DMPC vesicles. The interaction of dendriplexes with monolayers was accompanied by the formation of the aggregates as revealed by BAM experiments. This conclusion has been confirmed also by AFM imaging of sBLMs. We have demonstrated that dendriplexes interact with lipid membranes for all types of lipid composition. Moreover, the stronger interaction of cationic dendrimer/peptide complexes with lipid monolayers, vesicles and sBLMs was observed for membranes composed of zwitterionic lipids than for PEGylated lipid membranes. Increased concentration of PEGylated lipids made this interaction weaker.


Subject(s)
Dendrimers/chemistry , Lipid Bilayers/chemistry , Polyethylene Glycols/pharmacology , nef Gene Products, Human Immunodeficiency Virus/chemistry , Fluorescence Polarization , Microscopy, Atomic Force , Scattering, Radiation , Thermodynamics
11.
Biophys J ; 101(7): 1781-7, 2011 Oct 05.
Article in English | MEDLINE | ID: mdl-21961605

ABSTRACT

Thrombin aptamer binding strength and stability is dependent on sterical parameters when used for atomic force microscopy sensing applications. Sterical improvements on the linker chemistry were developed for high-affinity binding. For this we applied single molecule force spectroscopy using two enhanced biotinylated thrombin aptamers, BFF and BFA immobilized on the atomic force microscopy tip via streptavidin. BFF is a dimer composed of two single-stranded aptamers (aptabody) connected to each other by a complementary sequence close to the biotinylated end. In contrast, BFA consists of a single DNA strand and a complementary strand in the supporting biotinylated part. By varying the pulling velocity in force-distance cycles the formed thrombin-aptamer complexes were ruptured at different force loadings allowing determination of the energy landscape. As a result, BFA aptamer showed a higher binding force at the investigated loading rates and a significantly lower dissociation rate constant, k(off), compared to BFF. Moreover, the potential of the aptabody BFF to form a bivalent complex could clearly be demonstrated.


Subject(s)
Aptamers, Nucleotide/metabolism , Microscopy, Atomic Force/methods , Thrombin/metabolism , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/genetics , Base Sequence , Dimerization , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Humans , Protein Binding , Streptavidin/metabolism , Thrombin/chemistry
12.
Bioelectrochemistry ; 80(1): 55-61, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20537963

ABSTRACT

We synthesized 25,26,27,28-tetrakis(11-sulfanylundecyloxy)calix[4]arene (CALIX) sensitive to dopamine and confirmed its structure by (1)H NMR and mass spectrometry. Chemisorption of CALIX molecules or their mixtures with 1-dodecanethiols (DDT) or hexadecanethiols (HDT) resulted in formation of compact low permeable monolayers as revealed by cyclic voltammetry at presence of redox probe [Fe(CN)(6)](3-/4-). These self-assembled monolayers (SAMs) served as sensor for dopamine. Thickness shear mode acoustic method (TSM) has been used for study the interaction of dopamine with calixarene SAM. The admittance spectra of TSM transducer have been measured and used for simultaneous determination of the changes in series resonant frequency, f(S), and motional resistance, R(m), respectively. Addition of dopamine resulted in substantial decrease of f(S) and increase of R(m), which is evidence on increased viscoelastic contribution into the acoustic properties of the sensing layer. Limit of detection (LOD) for dopamine was 50 pM, which is much better in comparison with so far reported lowest LOD for dopamine-sensitive electrochemical sensors (20 nM). The sensor allowed discrimination between dopamine and epinephrine.


Subject(s)
Acoustics/instrumentation , Biosensing Techniques/instrumentation , Calixarenes/chemistry , Dopamine/analysis , Biosensing Techniques/methods , Calixarenes/chemical synthesis , Electrochemistry , Electrodes , Epinephrine/analysis , Magnetic Resonance Spectroscopy , Mass Spectrometry , Oxidation-Reduction , Reproducibility of Results , Sensitivity and Specificity , Silver/chemistry , Silver Compounds/chemistry , Sulfhydryl Compounds/chemistry , Surface Properties , Time Factors , Transducers
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