Nadezhda L. Mironova

RNase T1 mimicking artificial ribonuclease

Recently, artificial ribonucleases (aRNases)—conjugates of oligodeoxyribonucleotides and peptide (LR)4-G-amide—were designed and assessed in terms of the activity and specificity of RNA cleavage. The conjugates were shown to cleave RNA at Pyr-A and G–X sequences. Variations of oligonucleotide length and sequence, peptide and linker structure led to the development of conjugates exhibiting G–X cleavage specificity only. The most efficient catalyst is built of nonadeoxyribonucleotide of unique sequence and peptide (LR)4-G-NH2 connected by the linker of three abasic deoxyribonucleotides (conjugate pep-9). Investigation of the cleavage specificity of conjugate pep-9 showed that the compound is the first single-stranded guanine-specific aRNase, which mimics RNase T1. Rate enhancement of RNA cleavage at G–X linkages catalysed by pep-9 is 108 compared to non-catalysed reaction, pep-9 cleaves these linkages only 105-fold less efficiently than RNase T1 (kcat_RNase T1/kcat_pep-9 = 105).

Multicomponent mannose-containing liposomes efficiently deliver RNA in murine immature dendritic cells and provide productive anti-tumour response in murine melanoma model

Here we demonstrate the ability of mannosylated liposomes (ML) targeted to mannose receptors (MR) to perform the targeted delivery of model plasmid DNA encoding EGFP and total tumour RNA into murine bone-marrow-derived dendritic cells (DCs) and enhance the efficiency of anti-tumour response triggered by these DCs in murine melanoma model. ML consist of cationic lipid 2X3 (1,26-Bis(cholest-5-en-3β-yloxycarbonylamino)-7,11,16,20-tetraazahexacosan tetrahydrochloride), helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), and 2.5, 5 or 10% mol. of novel mannosylated lipoconjugates. In the structure of lipoconjugates D-mannose was attached to ditetradecylglycerol residue via succinyl (lipoconjugate 1) or diethylsquarate (lipoconjugate 2) linker groups. ML spontaneously form complexes with plasmid DNA and RNA due to electrostatic interaction between positively charged lipid amino group and negatively charged phosphate of nucleic acids. ML demonstrated the benefit in transfection efficiency (TE) of pDNA into DC progenitors and immature DCs in comparison with the control liposomes at low N/P (nitrogen to phosphate) ratios (1/1 and 2/1) but not at high N/P ratios where the TE was comparable with control liposomes. Moreover, ML at low N/P were more effective in RNA delivery into immature DCs in comparison with DC progenitors. At high N/P ratios liposomal formulations containing 5 and 10% mol. of mannosylated lipoconjugate 2 with diethylsquarate linker were the most effective (up to 50% of transfected cells). DCs transfected ex vivo with ML/melanoma B16 RNA complexes after i.v. injection into mice caused five- to six-fold inhibition of melanoma lung metastasis number. Moreover, the i.v. injection of ML/melanoma B16 RNA complexes into mice induced generation of the melanoma B16-specific cytotoxic T-lymphocytes, which were two-fold more efficient in B16 cell killing than those from control liposome group.

Ribonuclease binase inhibits primary tumor growth and metastases via apoptosis induction in tumor cells

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 RLS40 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 RLS40 and inhibits metastasis up to 50% in LLC and RLS40 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 RLS40-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.

G-specific RNA-cleaving Conjugates of Short Peptides and Oligodeoxyribonucleotides

Abstract Artificial ribonucleases, conjugates of short oligodeoxyribonucleotides and peptides built of arginine, leucine, proline, and serine, were synthesized and assessed in terms of ribonuclease activity and specificity of RNA cleavage. A specific group of the conjugates was identified that display T1-ribonuclease-like activity and cleave RNA predominantly at G-X sequences. Circular dichroism study of the structures of the most active conjugates, free peptide (LR)4G, and oligonucleotides revealed that conjugation of oligonucleotide to the peptide results in a specific peptide folding that possibly provides ribonuclease activity to the conjugate.

The Toxic Effects of Polychemotherapy onto the Liver Are Accelerated by the Upregulated MDR of Lymphosarcoma

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 RLS40 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 RLS40 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.

Alteration of the exDNA profile in blood serum of LLC-bearing mice under the decrease of tumour invasion potential by bovine pancreatic DNase I treatment

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.

Vertebrate RNase 1 homologs: potential regulators of extracellular RNAs

Recently, much data has been accumulated on extracellular RNAs (exRNAs) circulating in the blood plasma of mammals, and on alteration of their concentration/patterns occurring in pathological states including inflammation, trauma and oncological diseases (1,2).

The systemic tumor response to RNase A treatment affects the expression of genes involved in maintaining cell malignancy

Recently, pancreatic RNase A was shown to inhibit tumor and metastasis growth that accompanied by global alteration of miRNA profiles in the blood and tumor tissue (Mironova et al., 2013). Here, we performed a whole transcriptome analysis of murine Lewis lung carcinoma (LLC) after treatment of tumor-bearing mice with RNase A. We identified 966 differentially expressed transcripts in LLC tumors, of which 322 were upregulated and 644 were downregulated after RNase A treatment. Many of these genes are involved in signaling pathways that regulate energy metabolism, cell-growth promoting and transforming activity, modulation of the cancer microenvironment and extracellular matrix components, and cellular proliferation and differentiation. Following RNase A treatment, we detected an upregulation of carbohydrate metabolism, inositol phosphate cascade and oxidative phosphorylation, re-arrangement of cell adhesion, cell cycle control, apoptosis, and transcription. Whereas cancer-related signaling pathways (e.g., TGF-beta, JAK/STAT, and Wnt) were downregulated following RNase A treatment, as in the case of the PI3K/AKT pathway, which is involved in the progression of non-small lung cancer. RNase A therapy resulted in the downregulation of genes that inhibit the biogenesis of some miRNAs, particularly the let-7 miRNA family. Taken together, our data suggest that the antitumor activity and decreased invasion potential of tumor cells caused by RNase A are associated with enhanced energy cascade functioning, rearrangement of cancer-related events regulating cell growth and dissemination, and attenuation of signaling pathways having tumor-promoting activity. Thus, RNase A can be proposed as a potential component of anticancer therapy with multiple modes of action.