Covalently attached oligodeoxyribonucleotides induce RNase activity of a short peptide and modulate its base specificity

New artificial ribonucleases, conjugates of short oligodeoxyribonucleotides with peptides containing alternating arginine and leucine, were synthesized and characterized in terms of their catalytic activity and specificity of RNA cleavage. The conjugates efficiently cleave different RNAs within single-stranded regions. Depending on the sequence and length of the oligonucleotide, the conjugates display either G–X>>Pyr–A or Pyr–A>>G–X cleavage specificity. Preferential RNA cleavage at G–X phosphodiester bonds was observed for conjugate NH(2)-Gly-[ArgLeu](4)-CCAAACA. The conjugates function as true catalysts, exhibiting reaction turnover up to 175 for 24 h. Our data show that in the conjugate the oligonucleotide plays the role of a factor which provides an ‘active‘ conformation of the peptide via intramolecular interactions, and that it is the peptide residue itself which is responsible for substrate affinity and catalysis

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

Prophylactic Dendritic Cell-Based Vaccines Efficiently Inhibit Metastases in Murine Metastatic Melanoma

<div><p>Recent data on the application of dendritic cells (DCs) as anti-tumor vaccines has shown their great potential in therapy and prophylaxis of cancer. Here we report on a comparison of two treatment schemes with DCs that display the models of prophylactic and therapeutic vaccination using three different experimental tumor models: namely, Krebs-2 adenocarcinoma (primary tumor), melanoma (B16, metastatic tumor without a primary node) and Lewis lung carcinoma (LLC, metastatic tumor with a primary node). Dendritic cells generated from bone marrow-derived DC precursors and loaded with lysate of tumor cells or transfected with the complexes of total tumor RNA with cationic liposomes were used for vaccination. Lipofectamine 2000 and liposomes consisting of helper lipid DOPE (1,2-dioleoyl-<i>sn</i>-glycero-3-phosphoethanolamine) and cationic lipid 2D3 (1,26-Bis(1,2-de-O-tetradecyl-<i>rac</i>-glycerol)-7,11,16,20-tetraazahexacosan tetrahydrocloride) were used for RNA transfection. It was shown that DCs loaded with tumor lysate were ineffective in contrast to tumor-derived RNA. Therapeutic vaccination with DCs loaded by lipoplexes RNA/Lipofectamine 2000 was the most efficient for treatment of non-metastatic Krebs-2, where a 1.9-fold tumor growth retardation was observed. Single prophylactic vaccination with DCs loaded by lipoplexes RNA/2D3 was the most efficient to treat highly aggressive metastatic tumors LLC and B16, where 4.7- and 10-fold suppression of the number of lung metastases was observed, respectively. Antimetastatic effect of single prophylactic DC vaccination in metastatic melanoma model was accompanied by the reductions in the levels of Th2-specific cytokines however the change of the levels of Th1/Th2/Th17 master regulators was not found. Failure of double prophylactic vaccination is explained by Th17-response polarization associated with autoimmune and pro-inflammatory reactions. In the case of therapeutic DC vaccine the polarization of Th1-response was found nevertheless the antimetastatic effect was less effective in comparison with prophylactic DC vaccine.</p></div

Bovine Pancreatic RNase A: An Insight into the Mechanism of Antitumor Activity In Vitro and In Vivo

In this investigation, we extensively studied the mechanism of antitumor activity of bovine pancreatic RNase A. Using confocal microscopy, we show that after RNase A penetration into HeLa and B16 cells, a part of the enzyme remains unbound with the ribonuclease inhibitor (RI), resulting in the decrease in cytosolic RNAs in both types of cells and rRNAs in the nucleoli of HeLa cells. Molecular docking indicates the ability of RNase A to form a complex with Ku70/Ku80 heterodimer, and microscopy data confirm its localization mostly inside the nucleus, which may underlie the mechanism of RNase A penetration into cells and its intracellular traffic. RNase A reduced migration and invasion of tumor cells in vitro. In vivo, in the metastatic model of melanoma, RNase A suppressed metastases in the lungs and changed the expression of EMT markers in the tissue adjacent to metastatic foci; this increased Cdh1 and decreased Tjp1, Fn and Vim, disrupting the favorable tumor microenvironment. A similar pattern was observed for all genes except for Fn in metastatic foci, indicating a decrease in the invasive potential of tumor cells. Bioinformatic analysis of RNase-A-susceptible miRNAs and their regulatory networks showed that the main processes modulated by RNase A in the tumor microenvironment are the regulation of cell adhesion and junction, cell cycle regulation and pathways associated with EMT and tumor progression

CD4+ and CD8+ cell content in spleens of animals with Krebs-2 treated with prophylactic and therapeutic DC vaccines.

<p>A. w/t, non-treated mice with Krebs-2 injected with saline buffer, time of tumor development 19 days. B, C and D. Mice received prophylactic DC vaccines 1/LF, 1/LF/RNA and 1/lysate, respectively. E. w/t, non-treated mice with Krebs-2 injected with saline buffer, time of tumor development 11 days. F, G and H. Mice received therapeutic DC vaccines 2/LF, 2/LF/RNA and 2/lysate, respectively. CD4+ and CD8+ content at the point -7 days was measured just before DC vaccination and corresponded to baseline. Type of DC vaccine is presented as S/T/A—Scheme of the treatment 1 or 2/ Transfectant/ Antigen source. Blue line indicates CD4+ cells, red line—CD8+ cells. Arrows indicate the day of DC vaccination and the day of tumor transplantation. Data are presented as mean±S.E.M. All experimental points were run in triplicate.</p

The expression level of Tbet, GATA3, RORg, and Foxp3 in spleen cells of mice after DC vaccination (qPCR data).

<p>A. Prophylactic treatment scheme: healthy mice receiving DC vaccines 1-1/2D3/RNA and 1-2/2D3/RNA (1 and 2 immunization, respectively). B. Mice with metastatic melanoma receiving saline buffer. C. Therapeutic treatment scheme: mice with metastatic melanoma receiving DC vaccines 2-1/2D3/RNA and 2-2/2D3/RNA (1 and 2 immunization, respectively). Crossed square on Y axis displays the level of gene expression in healthy intact mice (baseline). Type of DC vaccine is indicated as S-I/T/A where S—scheme of the treatment 1 or 2, I—immunization number, T—transfectant, A—antigen source. Expression of the genes in group without treatment was measured on days 9 and 16 of tumor development. Expression of the genes in prophylactic and therapeutic groups was measured on day 5 after each immunization. Data represent the mean ± SD of three experiments performed in triplicate. Data were statistically analysed using one-way ANOVA with post hoc Fisher test.</p

Anti-tumour and anti-metastatic effects of DC vaccination under prophylactic and therapeutic schemes.

<p>A and B. Krebs-2 adenocarcinoma growth retardation after treatment with DC vaccines. w/t—non-treated mice with Krebs-2 injected with saline buffer. C and D. LLC tumor growth retardation and suppression of metastasis after treatment with DC vaccines. For A-D type of DC vaccine is presented as S/T/A—Scheme/ Transfectant/ Antigen source. w/t—non-treated mice with LLC injected with saline buffer. E. Suppression of B16 melanoma metastasis after treatment with DC vaccines. For E type of DC vaccine is presented as S-I/T/A—Scheme—Immunization number/ Transfectant/ Antigen source. w/t—non-treated mice with metastatic melanoma injected with saline buffer. Data were statistically analysed using one-way ANOVA with post hoc Fisher test. Data are presented as mean±S.E.M. <i>p</i> value <0.05 was considered to be statistically significant. Scheme 1: Healthy mice received i.v. DC vaccines according to presented S/T/A type. On day 7 after DC vaccination tumors were induced in mice by intramuscular injection of Krebs-2 cells (10⁵ cells/mouse) or LLC cells (6×10⁵ cells/mouse) into the femur muscle of right hindfoot. In the case of B16 model healthy mice received i.v. Dc vaccines according to presented S-I/T/A type: on day 7 before tumor transplantation (S-I: 1–1) and on day 14 and 7 before tumor transplantation (S-I:1–2). B16 was induced by transplantation of B16 cells (10⁵ cells/mouse) into lateral tail vein. Scheme 2. Tumors were induced in mice by intramuscular injection of Krebs-2 cells (10⁵ cells/mouse) or LLC cells (6×10⁵ cells/mouse) into the femur muscle of right hindfoot or intravenous inoculation of B16 cells (10⁵ cells/mouse) into lateral tail vein. On day 4 after tumor transplantation mice received i.v. Dc vaccines according to presented S/T/A or S-I/T/A type.</p

Experimental schedules of mouse treatments with DC vaccines.

<p>(A) Treatment of Krebs-2 and LLC tumors. Scheme 1 (prophylactic): Mice were injected with DC vaccines intravenously. A week later Krebs-2 or LLC cells were intramuscularly transplanted into mice. Mice with LLC and mice with Krebs-2 were sacrificed on days 19–20 and 20, respectively. Scheme 2 (therapeutic): Krebs-2 or LLC tumor cells were intramuscularly transplanted into the femur muscle of right hindfoot of mice, mice were treated with DC vaccines intravenously on day 4 and sacrificed on day 20. (B) Treatment of B16 tumors. Scheme 1 (prophylactic): Mice were intravenously injected with a DC vaccine once or twice with a one-week interval. A week after the last DC injection, B16 cells were inoculated intravenously into the mice. The mice were sacrificed on day 15. Scheme 2 (therapeutic): B16 cells were intravenously transplanted into mice, mice received one or two DC vaccines intravenously on day 4 or days 4 and 11, respectively. The mice were sacrificed on day 15.</p