Parameters of the toxic action of antibiotic derivatives of aureolic acid in acute and subchronic experiments]

The parameters of the lethal effect of aureolic acid derivatives, such as mithramycin, variamycin and olivomycin were studied on mice, rats and rabbits. As for the most of the administration routes the first two drugs were characterized by irregular distribution of resistance to thier lethal effect, which was mathematically expressed by polymodality of the dose-response curve. The above drugs were characterized by cumulative properties. The toxicity parameters depended on the animal species and administration route.     

Inhibition of the c-Myc Oncogene by the Aureolic Acid Group Antibiotics

Doklady Biochemistry and Biophysics - GC-rich stretches in the DNA minor groove are the established intracellular targets for the aureolic acid group of antibiotics such as olivomycin A and its...     

Chromomycin, mithramycin, and olivomycin binding sites on heterogeneous deoxyribonucleic acid. Footprinting with (methidiumpropyl-EDTA)iron(II)

The DNA binding sites for the antitumor, antiviral, antibiotics chromomycin, mithramycin, and olivomycin on 70 base pairs of heterogeneous DNA have been determined by using the (methidiumpropyl-EDTA)iron(II) [MPE x Fe(II)] DNA cleavage inhibition pattern technique. Two DNA restriction fragments 117 and 168 base pairs in length containing the lactose operon promoter-operator region were prepared with complementary strands labeled with 32P at the 3' end. MPE x Fe(II) was allowed to partially cleave the restriction fragment preequilibrated with either chromomycin, mithramycin, or olivomycin in the presence of Mg2+. The preferred binding sites for chromomycin, mithramycin, and olivomycin in the presence of Mg2+ appear to be a minimum of 3 base pairs in size containing at least 2 contiguous dG x dC base pairs. Many binding sites are similar for the three antibiotics; chromomycin and olivomycin binding sites are nearly identical. The number of sites protected from MPE x Fe(II) cleavage increases as the concentration of drug is raised. For chromomycin/Mg2+, the preferred sites on the 70 base pairs of DNA examined are (in decreasing affinity) 3'-GGG, CGA greater than CCG, GCC greater than CGA, CCT greater than CTG-5'. The sequence 3'-CGA-5' has different affinities, indicating the importance of either flanking sequences or a nearly bound drug.     

Results of determining the sensitivity of hypernephroid cancer to cytostatic agents in vitro and clinically]

Sensitivity of the cells of 57 human hypernephromas was determined with respect to 8 drugs, i.e. tiodipin, fluorbenzotef, mithramycin, cyclophosphan, tiotef, rubomycin, 5-fluoruracyl and olivomycin with the help of the method of primary plasma cultures. Because of the changes introduced earlier into the estimative concentrations used in vitro, coincidence of the results of the sensitivity determined in vitro with the results obtained during the treatment of the same patients in clinics, significantly increased (up to 90 per cent).     

Species specific differences in the toxicity of mithramycin, chromomycin A3, and olivomycin towards cultured mammalian cells

Three structurally related anticancer drugs, mithramycin, chromomycin A3, and olivomycin, showed large unexpected differences (up to more than 1000 fold) in their toxicity towards cultured cells from various species (human, Chinese hamster, Syrian hamster, and mouse). Among the cell types examined, human cells (both a diploid fibroblast cell strain and HeLa cells) were maximally sensitive to all these drugs, followed by the Syrian hamster kidney cells (BHK 21). The mouse (LMTK- cells) and Chinese hamster (CHO) cells, which were more resistant, showed interesting differences in their sensitivity towards these drugs. For example, whereas the mouse cells were more resistant to mithramycin than CHO cells, the sensitivity pattern was reversed for both chromomycin A3 and olivomycin. In cell extracts derived from human, mouse, and Chinese hamster cells RNA synthesis, which is the cellular target of these drugs, showed identical sensitivity to both mithramycin and chromomycin A3, indicating that the species specific differences in the toxicity to these drugs are at the level of cellular entry of these compounds. Based on the structures of these glycosidic antibiotics and their patterns of toxicity, it is suggested that the intracellular transport of these drugs involves specific interactions between the sugar residues on these compounds and some type of cell surface receptor(s), which differ among different cell types. Some implications of these results for toxicity studies are discussed.     

Mithramycin inhibits etoposide resistance in glucose-deprived HT-29 human colon carcinoma cells

Physiological cell conditions such as glucose deprivation and hypoxia play roles in the development of drug resistance in solid tumors. These tumor-specific conditions cause decreased expression of DNA topoisomerase IIalpha, rendering cells resistant to topo II target drugs such as etoposide. Thus, targeting tumor-specific conditions such as a low glucose environment may be a novel strategy in the development of anticancer drugs. On this basis, we established a novel screening program for anticancer agents with preferential cytotoxic activity in cancer cells under glucose-deprived conditions. We recently isolated an active compound, AA-98, from Streptomyces sp. AA030098 that can prevent stress-induced etoposide resistance in vitro. Furthermore, LC-MS and various NMR spectroscopic methods identified AA-98 as mithramycin, which belongs to the aureolic acid group of antitumor compounds. We found that mithramycin prevents the etoposide resistance that is induced by glucose deprivation. The etoposide-chemosensitive action of mithramycin was just dependent on strict low glucose conditions, and resulted in the selective cell death of etoposide-resistant HT-29 human colon cancer cells.     

Isolation of the new antibiotic variamycin B from the biosynthetic products of a Streptomyces olivovariabilis sp. nov. culture

Variamycin B, a new antitumor antibiotic was isolated from metabolic products of Streptomyces olivovariabilis sp. nov. The empirical formula of variamycin B is C52H76O24, the melting point is 163-165 degrees C (dec.), (alpha)20D--30 +/- 2 degrees (C 0.5, alcohol), the UV-spectrum: lambda max 230, 279, 317, 330, 415 (lg E 4.27, 4.6, 3.83, 3.75, 3.95), IR-spectrum: 1080, 1170, 1380, 1570, 1640, 1720, 3400 cm-1. Variamycin B is classified as belonging to the group of antibiotic analogs of aureolic acid. Chromomycinone, tetrazide and residues of two 2,6-didesoxysugars, i.e. olivose and variose were obtained as a result of complete and partial acid degradation of variamycin B. It was shown with the data of quantitative analysis of the hydrolysates obtained on complete hydrolysis of variamycin B that the ratio between the residues of variose and olivose in the antibiotic molecule was 1 : 4. Oxidation of variamycin B with Fremi salt resulted in formation of chinone having chromomycinone-chinone, 2 residues of olivose and 1 residue of variose in its composition. Investigation of the products of partial hydrolysis provided isolation of a substance having aglicone-chromomycenone and 4 residues of olivose in its composition. A partial structure of variamycin B is proposed.     

Quantitative method for determining variamycin]

A quantitative spectrophotmetric method for the assay of variamycin in commercial samples was developed. It was based on the measurement of the optical density of variamycin solutions in 0.01 N hydrochloric acid at a UV spectrum wave length of 412 nm. A method of thin-layer chromatography for a semi-quantitative estimation of tetraside, the main admixture of variamycin was devised. On storage for 18 months the content of variamycin in the preparations did not change as compared to that at the moment of the drug manufacturing and the amount of tetraside in 18 months did not exceed 2 per cent.     

Myeloinhibitory effect of the action of some antitumor antibiotics and its comparative assessment]

General toxic and myeloinhibitory effects of some antitumor antibiotics, such as rubomycin, olivomycin, bruneomycin and karminomycin administered intraperitoneally in a single LD50 to mice were studied. It was found that the general toxicity of bruneomycin and karminomycin was almost the same and 5 to 8 times higher than that of rubomycin and olivomycin. The use of the above antibiotics resulted in definite shifts in the blood systems of healthy mice. The most significant suppression of hemopoesis accompanied by a pronounced depression of the number of the myelocariocytes was observed after the use of olivomycin. The effect of karminomycin was characterized by suppression of erythro-, myelo- and lymphopoesis and depression of the number of the granulocytes and lymphocytes of the blood. Bruneomycin and rubomycin had a short-time myeloinhibitory effect. The erythroid cords of the bone marrow proved to be most sensitive to the inhibitory effect of the antibiotics. However, inhibition of the erythropoesis accompanied by deep reticulocytopenia did not induce the respective depression of the erythrocyte number. The lymphoid cords was in the 2nd place by its sensitivity to the antibiotics and the myeloid and megocariocytal cords were in the 3rd and the 4th places respectively. Complete reduction of hemopoesis in the animals was observed by the 10th day of the drugs use.     

Comparative study of the cytotoxic effects of anthracycline antibiotics on heterotransplants of human breast cancer cultivated in diffusion chambers in vivo

Cytotoxic activity of doxorubicin, daunomycin, carminomycin and ruboxyl against 50 human breast cancer heterotransplants in diffusion chambers was studied. The effect was estimated autoradiographically on the 6th or the 7th day of the cultivation after the drug administration in the maximum tolerance doses. The tumors were considered sensitive when the labeling index of their transplants after the treatment appeared to be reduced by 50 or less per cent against the control. The number of the tumors sensitive to all the drugs amounted to 72-80 per cent. 19 tumors were sensitive to 4 antibiotics. 14 and 8 tumors were sensitive to 3 and 2 antibiotics, respectively, and only 1 tumor was sensitive to 1 drug. The sensitivity significantly correlated with the initial labeling index of the primary tumors and their heterotransplants. The results suggested that daunomycin and ruboxyl possessed a high cytotoxic activity close to that of doxorubicin and carminomycin and might be recommended for clinical trials in the treatment of patients with breast cancer.     

Pathomorphological picture of the testes of mice administered antitumor antibiotics and their comparative evaluation

A single administration of the LD50 of bruneomycin, carminomycin, rubomycin or olivomycin and the use of the antibiotics for 10 days in a dose 2 times higher than the therapeutic one and amounting to 10 per cent of the LD50 induced dystrophic and destructive changes in the sexual glands of mice. The changes in the testis were more pronounced after the single use of the antibiotics. Recovery processes were observed in the testis beginning from the middle of the third week after the use of carminomycin and rubomycin and from the end of the second week after the use of olivomycin. Bruneomycin was an exception: the pronounced destructive changes in the sexual glands after its single or repeated use persisted within the observation period.     

Quantity of compentent cells in a population of Escherichia coli treated with Ca2+ cations]

An attempt was made to estimate the number of Escherichia coli K-12 cells rendered permeable to antibiotics under Ca2+ treatment. The effect of cold factor and Ca2+ alone as well as the cell age on the induction of permeability and the energy dependence of the latter were also investigated. About 70-75% and more exponentially growing cells as a result of Ca2+ treatment became sensitive to actinomycin, rubomycin and olivomycin. This number was somewhat lower (40-50%) in sationary phase culture. A fraction (20-30%) of stationary phase cells appeared to be sensitive to antibiotics even without Ca2+ pretreatment. Preincubation of the cells in cold in the absence of Ca2+ cations did not induce the cell permeability. The transport of antibiotics inside the cell was not prevented by an uncoupler of oxidative phosphorylation --carbonylcyanid-m-chlorophenylhydrazone (CCCP). It is suggested that the cells which are rendered permeable to tested antibiotics represent the "compentent" cells capable to uptake molecules of exogenous DNA as well.     

Investigations into the sequence-selective binding of mithramycin and related ligands to DNA.

The preferred binding sites for mithramycin on four different DNA fragments have been investigated by DNAase I footprinting. Sites containing at least two contiguous GC base pairs are protected by the antibiotic, the preferred binding site consisting of the dinucleotide step GpG (or CpC). Related antibiotics chromomycin and olivomycin produce similar, but not identical footprinting patterns suggesting that they can recognize other sequences as well. All three antibiotics induce enhanced rates of enzyme cleavage at regions flanking some of their binding sites. These effects are generally observed in runs of A and T and are attributed to DNA structural variations induced in the vicinity of the ligand binding site. The reaction of dimethylsulphate with N7 of guanine was modified by the presence of mithramycin so that we cannot exclude the possibility that these antibiotics bind to DNA via the major groove.     

Comparative study of the antibacterial activity of aminoglycoside antibiotics and their combinations with carbenicillin against Pseudomonas aeruginosa

Antibacterial activity of 7 aminoglycoside antibiotics and combinations of tobramycin or gentamicin with carbenicillin was studied with respect to 33 clinical strains of Ps. aeruginosa. Tobramycin, sisomicin, gentamicin and amicacin showed high levels of antibacterial activity. Tobramycin and sisomicin were 3-4 and 2 times more effective than gentamicin. 100 per cent of the Ps. aeruginosa isolates was sensitive to tobramycin and amicacin. The number of the isolates sensitive to sisomicin and gentamicin amounted to 97 and 94 per cent respectively. The respective numbers for streptomycin and kanamycin were 32 and 11 per cent. No monomycin sensitive isolates were detected. Combination of tobramycin or gentamicin with carbenicillin increased the antibacterial activity of the aminoglycoside antibiotics by 2-16 times and that of carbenicillin by 2-32 times. The synergistic effect of gentamicin or tobramycin with carbenicilin was observed with respect to 50 and 58 per cent of the isolates respectively. No antagonistic effect was detected on the combined use of the antibiotics. The majority of the isolates (96 per cent) were sensitive to combinations of carbenicillin in a concentration of 50 micrograms/ml with tobramycin or gentamicin in concentrations of 0.15 or 0.3 micrograms/ml respectively.     

Rapid staining methods for analysis of deoxyribonucleic acid and protein in mammalian cells.

Quantitative fluorescent staining and analysis of cellular deoxyribonucleic acid (DNA) were accomplished using three groups of reagents having different mechanisms of action for DNA binding. These reagents included (a) the fluorescent antitumor antibiotics mithramycin, chromomycin A3 and olivomycin; (b) the Feulgen reagents acriflavine and flavophosphine N and (c) the intercalating dyes ethidium bromide and propidium iodide. Propidium iodide (PI) was used in combination with fluorescein isothiocyanate (FITC) to stain both cellular DNA and protein, respectively. Multiparameter analysis of PI/FITC-stained cells provided a direct correlation of DNA and protein for cells in all stages of the cell cycle. Nuclear-to-cytoplasmic ratio determinations were also performed on PI/FITC-stained cells by analysis of the time duration of the red (DNA) and green (protein) fluorescence signals from each cell. These staining and analysis techniques provide alternative methods for directly determining the quantitative relationship between cellular DNA and protein and will be extremely useful in investigations where fluctuations of these parameters are of importance for assessing experimental results.     

Inhibition of a Zn(II)-containing enzyme, alcohol dehydrogenase, by anticancer antibiotics, mithramycin and chromomycin A3

One of the major attributes for the biological action of the aureolic acid anticancer antibiotics chromomycin A₃ (CHR) and mithramycin (MTR) is their ability to bind bivalent cations such as Mg(II) and Zn(II) ions and form high affinity 2:1 complexes in terms of the antibiotic and the metal ion, respectively. As most of the cellular Zn(II) ion is found to be associated with proteins, we have examined the effect of MTR/CHR on the structure and function of a representative structurally well characterized Zn(II) metalloenzyme, alcohol dehydrogenase (ADH) from yeast. MTR and CHR inhibit enzyme activity of ADH with inhibitory constants of micromolar order. Results from size-exclusion column chromatography, dynamic light scattering, and isothermal titration calorimetry have suggested that the mechanism of inhibition of the metalloenzyme by the antibiotics is due to the antibiotic-induced disruption of the enzyme quaternary structure. The nature of the enzyme inhibition, the binding stoichiometry of two antibiotics per monomer, and comparable dissociation constants for the antibiotic and free (or substrate-bound) ADH imply that the association occurs as a consequence of the binding of the antibiotics to Zn(II) ion present at the structural center. Confocal microscopy shows the colocalization of the antibiotic and the metalloenzyme in HepG2 cells, thereby supporting the proposition of physical association between the antibiotic(s) and the enzyme inside the cell.     

Detailed Studies on the Application of Three Fluorescent Antibiotics for Dna Staining in Flow Cytometry

The effects of pH, ionic strength, stain concentration, magnesium concentration, and various fixative agents on DNA staining with the fluorescent antibiotics olivomycin, chromomycin A3, and mithramycin were examined with DNA in solution and in mammalian cells. Ethanol-fixed Chinese hamster cell populations (line CHO) stained with mithramycin and analyzed by flow cytometry provided DNA distribution patterns with a high degree of resolution. Glutaraldehyde-fixed cells exhibited about one-half the fluorescence intensity of ethanol-fixed cells; however, the percentages of cells in G₁, S, and G₂ + M were comparable. DNA distributions obtained for formalin-fixed cells were unacceptable for computer analysis. Cell staining over a pH range of 5-9 in solutions containing 0.15-1 M NaCl and 15-200 mM MgCl₂ provided optimal results based on the DNA profiles obtained by flow cytometry. The intensity of cells stained in 1 M NaCl was one and one-half times greater than cells stained in the absence of NaCl; however, spectrophotofluorometric analysis of mithramycin-magnesium-DNA complexes in solution revealed no significant changes in fluorescence intensity over a range of 0-1.75 M NaCl. These results and those obtained by flow cytometry analysis indicate that the increase in fluorescence of stained cells as a function of increasing ionic strength is due to changes in chromatin structure, providing a larger number of binding sites for the dye-magnesium complex.     

Modification of olivomycin A at the side chain of the aglycon yields the derivative with perspective antitumor characteristics

A novel way of chemical modification of the antibiotic olivomycin A (1) at the side chain of the aglycon moiety was developed. Interaction of olivomycin A with the sodium periodate produced the key acid derivative olivomycin SA (2) in 86% yield. This acid was used in the reactions with different amines in the presence of benzotriazol-1-yl-oxy-trispyrrolidino-phosphonium hexafluorophosphate (PyBOP) or diphenylphosphoryl azide (DPPA) to give corresponding amides. Whereas olivomycin SA was two orders of magnitude less cytotoxic than the parent antibiotic, the amides of 2 demonstrated a higher cytotoxicity. In particular, N,N-dimethylaminoethylamide of olivomycin SA showed a pronounced antitumor effect against transplanted experimental lymphoma and melanoma and a remarkably high binding constant to double stranded DNA. The therapeutic effects of this derivative were achievable at tolerable concentrations, suggesting that modifications of the aglycon’s side chain, namely, its shortening to methoxyacetic residue and blocking of free carboxyl group, are straightforward for the design of therapeutically applicable derivatives of olivomycin A.