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1.
Mol Ther Nucleic Acids ; 27: 211-226, 2022 Mar 08.
Article in English | MEDLINE | ID: mdl-34976439

ABSTRACT

Antisense gapmer oligonucleotides containing phosphoryl guanidine (PG) groups, e.g., 1,3-dimethylimidazolidin-2-imine, at three to five internucleotidic positions adjacent to the 3' and 5' ends were prepared via the Staudinger chemistry, which is compatible with conditions of standard automated solid-phase phosphoramidite synthesis for phosphodiester and, notably, phosphorothioate linkages, and allows one to design a variety of gapmeric structures with alternating linkages, and deoxyribose or 2'-O-methylribose backbone. PG modifications increased nuclease resistance in serum-containing medium for more than 21 days. Replacing two internucleotidic phosphates by PG groups in phosphorothioate-modified oligonucleotides did not decrease their cellular uptake in the absence of lipid carriers. Increasing the number of PG groups from two to seven per oligonucleotide reduced their ability to enter the cells in the carrier-free mode. Cationic liposomes provided similar delivery efficiency of both partially PG-modified and unmodified oligonucleotides. PG-gapmers were designed containing three to four PG groups at both wings and a central "window" of seven deoxynucleotides with either phosphodiester or phosphorothioate linkages targeted to MDR1 mRNA providing multiple drug resistance of tumor cells. Gapmers efficiently silenced MDR1 mRNA and restored the sensitivity of tumor cells to chemotherapeutics. Thus, PG-gapmers can be considered as novel, promising types of antisense oligonucleotides for targeting biologically relevant RNAs.

2.
Inorg Chem ; 61(4): 2105-2118, 2022 Jan 31.
Article in English | MEDLINE | ID: mdl-35029379

ABSTRACT

A series of heteroleptic square-planar Pt and Pd complexes with bis(diisopropylphenyl) iminoacenaphtene (dpp-Bian) and Cl, 1,3-dithia-2-thione-4,5-dithiolate (dmit), or 1,3-dithia-2-thione-4,5-diselenolate (dsit) ligands have been prepared and characterized by spectroscopic techniques, elemental analysis, X-ray diffraction analysis, and cyclic voltammetry (CV). The intermolecular noncovalent interactions in the crystal structures were assessed by density functional theory (DFT) calculations. The anticancer activity of Pd complexes in breast cancer cell lines was limited by their solubility. Pd(dpp-Bian) complexes with dmit and dsit ligands as well as an uncoordinated dpp-Bian ligand were devoid of cytotoxicity, while the [Pd(dpp-Bian)Cl2] complex was cytotoxic. On the contrary, all Pt(dpp-Bian) complexes demonstrated anticancer activity in a low micromolar concentration range, which was 8-20 times higher than the activity of cisplatin, and up to 2.5-fold selectivity toward cancer cells over healthy fibroblasts. The presence of a redox-active dpp-Bian ligand in Pt and Pd complexes resulted in the induction of reactive oxygen species (ROS) in cancer cells. In addition, these complexes were able to intercalate into DNA, indicating the dual mechanism of action.


Subject(s)
Cisplatin
3.
Proc Natl Acad Sci U S A ; 117(51): 32370-32379, 2020 12 22.
Article in English | MEDLINE | ID: mdl-33288723

ABSTRACT

The design of modified oligonucleotides that combine in one molecule several therapeutically beneficial properties still poses a major challenge. Recently a new type of modified mesyl phosphoramidate (or µ-) oligonucleotide was described that demonstrates high affinity to RNA, exceptional nuclease resistance, efficient recruitment of RNase H, and potent inhibition of key carcinogenesis processes in vitro. Herein, using a xenograft mouse tumor model, it was demonstrated that microRNA miR-21-targeted µ-oligonucleotides administered in complex with folate-containing liposomes dramatically inhibit primary tumor growth via long-term down-regulation of miR-21 in tumors and increase in biosynthesis of miR-21-regulated tumor suppressor proteins. This antitumoral effect is superior to the effect of the corresponding phosphorothioate. Peritumoral administration of µ-oligonucleotide results in its rapid distribution and efficient accumulation in the tumor. Blood biochemistry and morphometric studies of internal organs revealed no pronounced toxicity of µ-oligonucleotides. This new oligonucleotide class provides a powerful tool for antisense technology.


Subject(s)
Amides/chemistry , Antineoplastic Agents/pharmacology , MicroRNAs/genetics , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/pharmacology , Phosphoric Acids/chemistry , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Gene Expression Regulation, Neoplastic/drug effects , Male , Melanoma/genetics , Melanoma/pathology , Mice, SCID , Molecular Targeted Therapy , Oligonucleotides, Antisense/pharmacokinetics , Tissue Distribution , Xenograft Model Antitumor Assays
4.
Molecules ; 25(16)2020 Aug 11.
Article in English | MEDLINE | ID: mdl-32796768

ABSTRACT

Biological activity of antisense oligonucleotides (asON), especially those with a neutral backbone, is often attenuated by poor cellular accumulation. In the present proof-of-concept study, we propose a novel delivery system for asONs which implies the delivery of modified antisense oligonucleotides by so-called transport oligonucleotides (tON), which are oligodeoxyribonucleotides complementary to asON conjugated with hydrophobic dodecyl moieties. Two types of tONs, bearing at the 5'-end up to three dodecyl residues attached through non-nucleotide inserts (TD series) or anchored directly to internucleotidic phosphate (TP series), were synthesized. tONs with three dodecyl residues efficiently delivered asON to cells without any signs of cytotoxicity and provided a transfection efficacy comparable to that achieved using Lipofectamine 2000. We found that, in the case of tON with three dodecyl residues, some tON/asON duplexes were excreted from the cells within extracellular vesicles at late stages of transfection. We confirmed the high efficacy of the novel and demonstrated that MDR1 mRNA targeted asON delivered by tON with three dodecyl residues significantly reduced the level of P-glycoprotein and increased the sensitivity of KB-8-5 human carcinoma cells to vinblastine. The obtained results demonstrate the efficacy of lipophilic oligonucleotide carriers and shows they are potentially capable of intracellular delivery of any kind of antisense oligonucleotides.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Drug Delivery Systems , Neoplasms/drug therapy , Oligonucleotides, Antisense/genetics , RNA, Messenger/antagonists & inhibitors , Vinblastine/pharmacology , ATP Binding Cassette Transporter, Subfamily B/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B/genetics , Antineoplastic Agents, Phytogenic/administration & dosage , Antineoplastic Agents, Phytogenic/chemistry , Humans , Neoplasms/genetics , Neoplasms/pathology , RNA, Messenger/genetics , Tumor Cells, Cultured , Vinblastine/administration & dosage , Vinblastine/chemistry
5.
Front Pharmacol ; 10: 879, 2019.
Article in English | MEDLINE | ID: mdl-31456683

ABSTRACT

Control of the expression of oncogenic small non-coding RNAs, notably microRNAs (miRNAs), is an attractive therapeutic approach. We report a design platform for catalytic knockdown of miRNA targets with artificial, sequence-specific ribonucleases. miRNases comprise a peptide [(LeuArg)2Gly]2 capable of RNA cleavage conjugated to the miRNA-targeted oligodeoxyribonucleotide, which becomes nuclease-resistant within the conjugate design, without resort to chemically modified nucleotides. Our data presented here showed for the first time a truly catalytic character of our miR-21-miRNase and its ability to cleave miR-21 in a multiple catalytic turnover mode. We demonstrate that miRNase targeted to miR-21 (miR-21-miRNase) knocked down malignant behavior of tumor cells, including induction of apoptosis, inhibition of cell invasiveness, and retardation of tumor growth, which persisted on transplantation into mice of tumor cells treated once with miR-21-miRNase. Crucially, we discover that the high biological activity of miR-21-miRNase can be directly related not only to its truly catalytic sequence-specific cleavage of miRNA but also to its ability to recruit the non-sequence specific RNase H found in most cells to elevate catalytic turnover further. miR-21-miRNase worked synergistically even with low levels of RNase H. Estimated degradation in the presence of RNase H exceeded 103 miRNA target molecules per hour for each miR-21-miRNase molecule, which provides the potency to minimize delivery requirements to a few molecules per cell. In contrast to the comparatively high doses required for the simple steric block of antisense oligonucleotides, truly catalytic inactivation of miRNA offers more effective, irreversible, and persistent suppression of many copy target sequences. miRNase design can be readily adapted to target other pathogenic microRNAs overexpressed in many disease states.

6.
PLoS One ; 12(2): e0171988, 2017.
Article in English | MEDLINE | ID: mdl-28222152

ABSTRACT

Taking into account recently obtained data indicating the participation of circulating extracellular DNA (exDNA) in tumorigenesis, enzymes with deoxyribonucleic activity have again been considered as potential antitumour and antimetastatic drugs. Previously, using murine Lewis lung carcinoma and hepatocellular carcinoma A1 tumour models, we have shown the antimetastatic activity of bovine DNase I, which correlates with an increase of DNase activity and a decrease of exDNA concentration in the blood serum of tumour-bearing mice. In this work, using next-generation sequencing on the ABS SOLiD™ 5.500 platform, we performed a search for molecular targets of DNase I by comparing the exDNA profiles of healthy animals, untreated animals with Lewis lung carcinoma (LLC) and those with LLC treated with DNase I. We found that upon DNase I treatment of LLC-bearing mice, together with inhibition of metastasis, a number of strong alterations in the patterns of exDNA were observed. The major differences in exDNA profiles between groups were: i) the level of GC-poor sequences increased during tumour development was reduced to that of healthy mice; ii) levels of sequences corresponding to tumour-associated genes Hmga2, Myc and Jun were reduced in the DNase I-treated group in comparison with non-treated mice; iii) 224 types of tandem repeat over-presented in untreated LLC-bearing mice were significantly reduced after DNase I treatment. The most important result obtained in the work is that DNase I decreased the level of B-subfamily repeats having homology to human ALU repeats, known as markers of carcinogenesis, to the level of healthy animals. Thus, the obtained data lead us to suppose that circulating exDNA plays a role in tumour dissemination, and alteration of multiple molecular targets in the bloodstream by DNase I reduces the invasive potential of tumours.


Subject(s)
Carcinoma, Lewis Lung/blood , DNA, Neoplasm/blood , Deoxyribonuclease I/metabolism , Neoplasm Invasiveness , Animals , Carcinoma, Lewis Lung/pathology , Cattle , DNA, Neoplasm/genetics , Extracellular Space/chemistry , Gene Library , High-Throughput Nucleotide Sequencing , Male , Mice , Mice, Inbred C57BL , Neoplasm Transplantation , Real-Time Polymerase Chain Reaction
7.
Biomaterials ; 122: 163-178, 2017 04.
Article in English | MEDLINE | ID: mdl-28126663

ABSTRACT

MicroRNAs (miRNAs) are active regulators in malignant growth and constitute potential targets for anticancer therapy. Consequently, considerable effort has focused on identifying effective ways to modulate aberrant miRNA expression. Here we introduce and assess a novel type of chemically engineered biomaterial capable of cleaving specific miRNA sequences, i.e. miRNA-specific artificial ribonucleases (hereafter 'miRNase'). The miRNase template presented here consists of the catalytic peptide Acetyl-[(LeuArg)2Gly]2 covalently attached to a miRNA-targeting oligonucleotide, which can be linear or hairpin. The peptide C-terminus is conjugated to an aminohexyl linker located at either the 3'- or 5'-end of the oligonucleotide. The cleavage efficacy, structural aspects of cleavage and biological relevance of a set of these designed miRNases was assayed with respect to highly oncogenic miR-21. Several miRNases demonstrated effective site-selective cleavage of miR-21 exclusively at G-X bonds. One of the most efficient miRNase was shown to specifically inhibit miR-21 in lymphosarcoma cells and lead to a reduction in their proliferative activity. This report provides the first experimental evidence that metallo-independent peptide-oligonucleotide chemical ribonucleases are able to effectively and selectively down-regulate oncogenic miRNA in tumour cells, thus suggesting their potential in development of novel therapeutics aimed at overcoming overexpression of disease-related miRNAs.


Subject(s)
Lymphoma, Non-Hodgkin/genetics , Lymphoma, Non-Hodgkin/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Oligonucleotides/administration & dosage , Peptides/administration & dosage , Ribonucleases/administration & dosage , Animals , Cell Line, Tumor , Mice , Oligonucleotides/chemistry , Peptides/chemistry , Ribonucleases/chemistry
8.
Cell Cycle ; 12(13): 2120-31, 2013 Jul 01.
Article in English | MEDLINE | ID: mdl-23759588

ABSTRACT

Exogenous ribonucleases are known to inhibit tumor growth via apoptosis induction in tumor cells, allowing to consider them as promising anticancer drugs for clinical application. In this work the antitumor potential of binase was evaluated in vivo and the mechanism of cytotoxic effect of binase on tumor cells was comprehensively studied in vitro. We investigated tumoricidal activity of binase using three murine tumor models of Lewis lung carcinoma (LLC), lymphosarcoma RLS 40 and melanoma B-16. We show for the first time that intraperitoneal injection of binase at a dose range 0.1-5 mg/kg results in retardation of primary tumor growth up to 45% in LLC and RLS 40 and inhibits metastasis up to 50% in LLC and RLS 40 and up to 70% in B-16 melanoma. Binase does not exhibit overall toxic effect and displays a general systemic and immunomodulatory effects. Treatment of RLS 40-bearing animals with binase together with polychemotherapy revealed that binase decreases the hepatotoxicity of polychemotherapy while maintaining its antitumor effect. It was demonstrated that the cytotoxic effect of binase is realized via the induction of the intrinsic and extrinsic apoptotic pathways. Activation of intrinsic apoptotic pathway is manifested by a drop of mitochondrial potential, increase in calcium concentration and inhibition of respiratory activity. Subsequent synthesis of TNF-α in the cells under the action of binase triggers extrinsic apoptotic pathway through the binding of TNF with cell-death receptors and activation of caspase 8. Thus binase is a potential anticancer therapeutics inducing apoptosis in cancer cells.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis , Carcinoma, Lewis Lung/drug therapy , Endoribonucleases/pharmacology , Lymphoma, Non-Hodgkin/drug therapy , Melanoma, Experimental/drug therapy , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/toxicity , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Lewis Lung/secondary , Cell Line, Tumor , Cell Proliferation , Cyclophosphamide/pharmacology , Cyclophosphamide/therapeutic use , Cytokines/blood , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , Drug Screening Assays, Antitumor , Endoribonucleases/administration & dosage , Endoribonucleases/toxicity , Injections, Intramuscular , Injections, Intraperitoneal , Liver/drug effects , Liver/pathology , Lymphoma, Non-Hodgkin/blood , Melanoma, Experimental/secondary , Membrane Potential, Mitochondrial/drug effects , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Neoplasm Transplantation , Prednisone/pharmacology , Prednisone/therapeutic use , Reactive Oxygen Species/metabolism , Tumor Burden/drug effects , Vincristine/pharmacology , Vincristine/therapeutic use
9.
ISRN Oncol ; 2012: 721612, 2012.
Article in English | MEDLINE | ID: mdl-23251817

ABSTRACT

Antitumor therapy of hematological malignancies is impeded due to the high toxicity of polychemotherapy toward liver and increasing multiple drug resistance (MDR) of tumor cells under the pressure of polychemotherapy. These two problems can augment each other and significantly reduce the efficiency of antineoplastic therapy. We studied the combined effect of polychemotherapy and upregulated MDR of lymphosarcoma RLS(40) onto the liver of experimental mice using two treatment schemes. Scheme 1 is artificial: the tumor was subjected to four courses of polychemotherapy while the liver of the tumor-bearing mice was exposed to only one. This was achieved by threefold tumor retransplantation taken from animals subjected to chemotherapy into intact animals. Scheme 2 displays "real-life" status of patients with MDR malignancies: both the tumor and the liver of tumor-bearing mice were subjected to three sequential courses of polychemotherapy. Our data show that the strengthening of MDR phenotype of RLS(40) under polychemotherapy and toxic pressure of polychemotherapy itself has a synergistic damaging effect on the liver that is expressed in the accumulation of destructive changes in the liver tissue, the reduction of the regeneration capacity of the liver, and increasing of Pgp expression on the surface of hepatocytes.

10.
BMC Cancer ; 10: 204, 2010 May 14.
Article in English | MEDLINE | ID: mdl-20470373

ABSTRACT

BACKGROUND: One of the main obstacles for successful cancer polychemotherapy is multiple drug resistance phenotype (MDR) acquired by tumor cells. Currently, RNA interference represents a perspective strategy to overcome MDR via silencing the genes involved in development of this deleterious phenotype (genes of ABC transporters, antiapoptotic genes, etc.). METHODS: In this study, we used the siRNAs targeted to mdr1b, mdr1a, and bcl-2 mRNAs to reverse the MDR of tumors and increase tumor sensitivity to chemotherapeutics. The therapy consisting in ex vivo or in vivo application of mdr1b/1a siRNA followed by cyclophosphamide administration was studied in the mice bearing RLS40 lymphosarcoma, displaying high resistance to a wide range of cytostatics. RESULTS: Our data show that a single application of mdr1b/1a siRNA followed by treatment with conventionally used cytostatics results in more than threefold decrease in tumor size as compared with the control animals receiving only cytostatics. CONCLUSIONS: In perspective, mdr1b/1a siRNA may become a well-reasoned adjuvant tool in the therapy of MDR malignancies.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B/genetics , Antineoplastic Agents, Alkylating/pharmacology , Cyclophosphamide/pharmacology , Drug Resistance, Neoplasm/genetics , Genetic Therapy/methods , Lymphoma, Non-Hodgkin/therapy , RNA Interference , RNA, Small Interfering/metabolism , Animals , Gene Expression Regulation, Neoplastic , Genotype , Lymphoma, Non-Hodgkin/drug therapy , Lymphoma, Non-Hodgkin/genetics , Lymphoma, Non-Hodgkin/metabolism , Lymphoma, Non-Hodgkin/pathology , Male , Mice , Mice, Inbred CBA , Phenotype , RNA, Messenger/metabolism , Time Factors , Transfection , Tumor Burden , Tumor Cells, Cultured , ATP-Binding Cassette Sub-Family B Member 4
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