Binase Do Not Change Level of Intracellular RNA in Breast Cancer Cells

© 2017, Springer Science+Business Media, LLC. We analyzed changes of intracellular RNA level in breast cancer cells using BT-20, BT-474, and MCF-7 cell lines. It is the continuation of our former studying the effect of bacterial ribonucleases on physiological and biochemical parameters of breast cancer cells. For the first time, we tested ability of Bacillus pumilus RNase (binase) to effect the level of intracellular RNA in these cells. Differences in total RNA level under of the binase action on the cells of triple negative, triple positive, and double positive breast cancer during 48 h of treatment were not detected. The sensitivity of cells to binase was not associated with a reduction of the intracellular RNA level

The cytofluorimetric characteristics of RNAse influence towards pro-and eucariotic cells

Cytotoxic ribonucleases (RNAses) are known to be perspective drugs for cancer therapy. The extension of model object's range will give possibility to assess the cytotoxic RNAse's selectivity. We estimated the effect of Bacillus intermedius ribonuclease (binase) and bovine RNAse A to E. coli K 12 and lung epithelia of cow embryo (LEC) cells. LEC cells apoptosis was characterized with a double staining with annexin-FITC and propidium iodide (PI). E. coli K12 cell's vitality was estimate via PI staning. Binase and RNAse A were not cytotoxic to LEC and E. coli K12 cells in investigated concentrations (100 and 300 μg/ml). Low RNAse's toxicity for E. coli allows to suppose the binase and RNAse A in concentrations, capable to display antitumor activity, will not to effect the tissue's microbial flora during in vivo tests. The lack of apoptosis inducing activity of binase for LEC cells confirms this RNAse selectivity for tumor cells

Induction of apoptosis in tumor cells by binase

The possibility of inducing apoptosis in K562 myelogenic erythroleukemia cells, A549 lung carcinoma cells, and normal human lymphocytes was studied for Bacillus intermedius RNase (binase) and its mutants Lys26 Ala and His101 Glu with impaired catalytic activity. Selective induction of apoptosis in leukemic blood cells by binase was demonstrated for the first time. Binase did not exert an antiproliferative or proapoptotic effect on peripheral blood lymphocytes of healthy donors. Low-molecular-weight (less than 50 kb in size) oligonucleosomal DNA fragments, which are early markers of apoptosis, were observed in human solid-tumor cells treated with binase. Studies with the binase mutants showed that a decrease in catalytic activity to 2.5% of the level characteristic of the wild-type enzyme deprives binase of its proapoptotic effect. The selective proapoptotic effect of binase on malignant cells provides evidence that bacterial RNases are promising for designing alternative antitumor drugs. © 2005 Pleiades Publishing, Inc

Induction of apoptosis of tumor cells by binase

An induction of apoptosis by RNase from Bacillus intermedius (binase) and its mutants characterized with low catalytic activity (Lys 26Ala and His 101Glu) in human myelogenic erythroleukemia K562 cells, human lung carcinoma A549 cells and human peripheral blood mononuclear cells was studied. For the first time selective apoptogenic effects of binase toward leukemic blood cells was determined. Neither antiproliferative nor apoptotic effects of binase were detected in normal human peripheral blood mononuclear cells. Formation of low molecular weight oligonucleosomal DNA fragments (less than 50 Kb) which are an early marks of apoptosis was registered in solid tumor cells treated by binase. Using mutant RNases it was shown that decrease of catalytic activity to 2.5% of wild type enzyme activity leads to the loss of apoptogenic properties of enzyme. Selective apoptogenicity of binase found towards malignant cells confirmed that antitumor agents based on bacterial RNases could be considered as an alternative to standard chemotherapeutic drugs

Cytotoxic and genotoxic effects of β-(triphenylphosphonio)ethyl carboxylate and of N,N′-bis(dihexylphosphinoylmethyl)-1,4- diaminocyclohexane

Background: Several organophosphorous compounds (OPs) are now being tested therapeutically. Cholinesterase inhibition, which in large doses makes these agents effective pesticides, may also be useful in other doses for treating dementia. Metrifonate, for example, has been used to treat schistosomiasis and is undergoing trials for the treatment of primary degenerative dementia. Material/Methods: Here we report the characterization of newly synthesized OPs from the group of phosphobetaines [β-(triphenylphosphonio)ethyl carboxylate, PB] and of alpha-aminophosphoryl compounds [N,N′- bis(dihexylphosphinoylmethyl)-1,4-diaminocyclohexane, AP] according to their toxic and genotoxic properties determined in prokaryotic and eukaryotic test systems. Results: The absence of toxicity towards Gram-negative bacteria and of genotoxicity in Ames mutagenicity assay and in SOS-chromotest did not exclude the cytotoxic effect of PB towards NIH3T3 mouse fibroblasts, which supports the notion of an extremely diverse interspecies response to OPs. In contrast, AP demonstrated toxic properties detected by antibacterial effect as well as by the inhibition of the proliferation and respiration of fibroblasts. The enzymatic transformation of the compound is necessary to reveal the genotoxic properties of AP. The role of mammalian microsomal enzymes and of bacterial C-P lyase in the formation of AP genotoxic metabolites is under discussion. Conclusions: Neither toxicity nor genotoxicity of PB was found in bacterial tests. Cytotoxic and mutagenic effects of AP were detected. The data contribute to the investigation of the biological activity of novel organophosphates which could be useful for the future development of OP-based therapeutics

The combined action of binase and bleomycin on human lung adenocarcinoma cells

© 2016, Pleiades Publishing, Ltd.Some microbial ribonucleases (RNases) demonstrate selective cytotoxic effect against a wide range of tumor cells. In this context combined use of cytotoxic RNases in complex therapy with other chemotherapeutic agents appears to be especially promising. In this study we have investigated the apoptosisinduced effect of Bacillus pumilus RNase (binase) in combination with known antitumor antibiotic bleomycin on human lung adenocarcinoma A549 cells. The combined effect of high concentrations of these agents did not have any mutual increase in their apoptosisinduced action, while a combination of nonapoptotic concentrations resulted in the increase of proportion of apoptotic cells up to 22% as compared with individual effect of bleomycin (6%) and binase (12%) used separately. These results indicate that binase and bleomycin are effective in combination of their low concentrations and ineffective in combination of their high concentrations

The Effect of high hydrostatic pressure on the viability and mutagenesis of Salmonella typhimurium

© 2017, Pleiades Publishing, Ltd. This work is devoted to the study of the influence of high hydrostatic pressure (HHP) on the viability and level of mutagenesis of Salmonella typhimurium. It was established that the viability of bacteria significantly decreases under hydrostatic pressure of 200 MPa or higher. In addition, the viability index of the bacteria is six orders of magnitude lower with respect to the number of colony-forming units (CFUs) compared to the data of the flow cytofluorometry analysis. This is probably due to the transition of some part of the bacterial population to a viable but nonculturable state (VBNC). HHP of 50 MPa caused a 1.9-fold increase in the number of His + revertants of the S. typhimurium strain TA98, which indicates the potential of the induction of gene mutations under these conditions. The mechanisms to reduce the viability and genetic changes in bacterial cells under HHP conditions are discussed

Induction of Apoptosis of Tumor Cells by Oligochitosans (Short Chain Chitosans)

© 2016, Springer Science+Business Media New York.An induction of apoptosis by short chain chitosans in human lung carcinoma A549 cells and cow embryonic lung cells was studied. Seventeen well-defined oligochitosans with wide range of molecular weights, from 5.5 to 46.7 kDa, were used in this study. The experiments showed that three oligochitosans with molecular weight 6.1, 7.7, and 9.0 kDa notably induced the high level of apoptosis of human lung carcinoma A549 cells. It presented a dose-dependent manner, and the apoptotic rate amounted to about 29–50 % after treatment with 200 μg/ml oligochitosans for 24 h for A549 cells and 7–17 % for LEC cells in the same conditions. The results obtained show that the oligochitosans are more active in inducing apoptosis and in decreasing viability in A549 cells than LEC, which suggests that it could be a potential chemotherapeutic drug

Effect of Bacillus pumilus ribonuclease on the paramagnetic centers of microbial cells

The potential clinical application of Bacillus pumilus cytotoxic ribonuclease (binase) for selectively inducing the death of tumor cells makes it imperative to investigate its effect on the normal human microflora. Flow cytometry was used to determine that binase concentration causing the apoptosis of cancer cells had no effect of the viability of Escherichia coli K12. The changes in the paramagnetic centers of E. coli K12 cells in the presence of nontoxic binase concentrations revealed by EPR spectroscopy included higher EPR signals from iron-containing proteins (including those from the Fe-S clusters) and of the Mn(II) hyperfine structure. The TMTH spin probe (N-(1-hydroxy-2,2,6,6-tetramethylpiperidine-4-il)-2-methylpropanamide hydrochloride) was used to reveal a twofold increase in the levels of reactive oxygen species (ROS) in the cells, which induced oxidative stress in the enzyme-treated bacteria. Inductively coupled plasma mass spectrometry revealed elevated contents of alkaline (Li, Na, K), alkali earth (Mg, Ca), transition (Cr, Mn, Fe, Cu, Zn), and post-transition metals (Bi, Pb) in the cells. Elevated levels of Cu and Zn (which impair the activity of the respiratory chain enzymes) and of Mn, which is known as a superoxide dismutase cofactor, confirmed development of the oxidative stress in bacteria. © 2013 Pleiades Publishing, Ltd