Interaction Of Bleomycin Research Articles

The Interaction of the Metallo-Glycopeptide Anti-Tumour Drug Bleomycin with DNA.

The cancer chemotherapeutic drug, bleomycin, is clinically used to treat several neoplasms including testicular and ovarian cancers. Bleomycin is a metallo-glycopeptide antibiotic that requires a transition metal ion, usually Fe(II), for activity. In this review, the properties of bleomycin are examined, especially the interaction of bleomycin with DNA. A Fe(II)-bleomycin complex is capable of DNA cleavage and this process is thought to be the major determinant for the cytotoxicity of bleomycin. The DNA sequence specificity of bleomycin cleavage is found to at 5′-GT* and 5′-GC* dinucleotides (where * indicates the cleaved nucleotide). Using next-generation DNA sequencing, over 200 million double-strand breaks were analysed, and an expanded bleomycin sequence specificity was found to be 5′-RTGT*AY (where R is G or A and Y is T or C) in cellular DNA and 5′-TGT*AT in purified DNA. The different environment of cellular DNA compared to purified DNA was proposed to be responsible for the difference. A number of bleomycin analogues have been examined and their interaction with DNA is also discussed. In particular, the production of bleomycin analogues via genetic manipulation of the modular non-ribosomal peptide synthetases and polyketide synthases in the bleomycin gene cluster is reviewed. The prospects for the synthesis of bleomycin analogues with increased effectiveness as cancer chemotherapeutic agents is also explored.

Identification of ANXA2 (annexin A2) as a specific bleomycin target to induce pulmonary fibrosis by impeding TFEB-mediated autophagic flux

ABSTRACTBleomycin is a clinically potent anticancer drug used for the treatment of germ-cell tumors, lymphomas and squamous-cell carcinomas. Unfortunately, the therapeutic efficacy of bleomycin is severely hampered by the development of pulmonary fibrosis. However, the mechanisms underlying bleomycin-induced pulmonary fibrosis, particularly the molecular target of bleomycin, remains unknown. Here, using a chemical proteomics approach, we identify ANXA2 (annexin A2) as a direct binding target of bleomycin. The interaction of bleomycin with ANXA2 was corroborated both in vitro and in vivo. Genetic depletion of anxa2 in mice mitigates bleomycin-induced pulmonary fibrosis. We further demonstrate that Glu139 (E139) of ANXA2 is required for bleomycin binding in lung epithelial cells. A CRISPR-Cas9-engineered ANXA2E139A mutation in lung epithelial cells ablates bleomycin binding and activates TFEB (transcription factor EB), a master regulator of macroautophagy/autophagy, resulting in substantial acceleration of autophagic flux. Pharmacological activation of TFEB elevates bleomycin-initiated autophagic flux, inhibits apoptosis and proliferation of epithelial cells, and ameliorates pulmonary fibrosis in bleomycin-treated mice. Notably, we observe lowered TFEB and LC3B levels in human pulmonary fibrosis tissues compared to normal controls, suggesting a critical role of TFEB-mediated autophagy in pulmonary fibrosis. Collectively, our data demonstrate that ANXA2 is a specific bleomycin target, and bleomycin binding with ANXA2 impedes TFEB-induced autophagic flux, leading to induction of pulmonary fibrosis. Our findings provide insight into the mechanisms of bleomycin-induced fibrosis and may facilitate development of optimized bleomycin therapeutics devoid of lung toxicity.

Impedimetric detection of in situ interaction between anti-cancer drug bleomycin and DNA

Surface confined interaction of anti-cancer drug bleomycin (BLM) with nucleic acids: single stranded and double stranded DNA was investigated herein by using electrochemical impedance spectroscopy (EIS) technique in combination with a graphite sensor technology. The experimental conditions were optimized: such as, dsDNA concentration, BLM concentration and interaction time. The main features of impedimetric DNA biosensor, such as its detection limit and the repeatability, were also discussed. The in situ interaction of BLM with dsDNA was also tested impedimetrically in the absence or presence of other chemotherapeutic agents, such as mitomycin C (MC) and cis-platin (cis-DDP) for testing the selectivity.

Studying the effect of a charged surface on the interaction of bleomycin with DNA using an atomic force microscope

The cleavage of DNA caused by the antitumoral drug bleomycin has been investigated using atomic force microscopy (AFM). This work deals with the effect that adsorbing DNA onto a positively- or negatively-charged surface has on the double-strand cleavage of DNA by Fe(III)/bleomycin. Quantitative analysis of the number of breaks per DNA molecule, in bulk and at the surface of the mica substrate, has been performed by analyzing AFM images. It turns out that the cleavage of DNA is strongly inhibited by a positively-charged surface. Our experiments can be interpreted using a simple electrostatic model. This paper is a first step in the study of DNA accessibility to ligand such as bleomycin, using AFM in liquids.

Interaction of bleomycin with DNA

Abstract

Fluoromicroscopic studies of bleomycin-induced intracellular oxidation in alveolar macrophages and its inhibition by taurine.

The mechanism of bleomycin-induced pulmonary fibrosis is not yet clear. Recent studies have shown that alveolar macrophages (AM) can be stimulated by bleomycin in vitro releasing inflammatory cytokines, suggesting that the interaction of bleomycin with AM is an important step in the drug-induced fibrotic process. Bleomycin is known to bind DNA and generate oxygen radicals through complexation with Fe2+ and oxygen. To provide more insight into the cellular oxidative property of bleomycin, we have developed a fluoromicroscopic method using 2',7'-dichlorofluorescin diacetate (DCFHDA) as an oxidative fluorescence probe to study the bleomycin-induced intracellular oxidation in rat AM and the inhibition of the oxidation by taurine, a compound known to inhibit the bleomycin-induced fibrosis. Bleomycin at 5 to 20 micrograms/ml has a moderate stimulatory effect (1.87- to 2.66-fold) on the secretion of superoxide anion. A high concentration of bleomycin (20 micrograms/ml), however, inhibits cell response to zymosan-induced secretion of superoxide anion. At 4 micrograms/ml, bleomycin has no effect on cell membrane integrity or morphology but results in a significant increase in intracellular oxidation. This oxidative process is Fe(2+)-dependent and is accompanied by an increase in intracellular calcium (35 nM). Both the intracellular oxidation and calcium rise induced by internalized bleomycin are inhibited by pretreatment of cells with varying concentrations of taurine (25, 125, and 187.5 microM). The inhibitory effect on intracellular oxidation was found to be 36, 57, and 60%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of bleomycin with a bent DNA fragment.

The interaction of bleomycin with a kinetoplast DNA fragment has been examined using various footprinting techniques. This DNA adopts a bent structure and displays an unusually low gel mobility on account of its phased runs of adenines. The bleomycin-cobalt complex increases the mobility of this DNA fragment, in contrast with other DNAs which show a decreased rate of gel migration, suggesting that the antibiotic removes DNA bending, possibly via an unwinding mechanism. Removal of the bending is confirmed by hydroxy-radical footprinting which produces a more even ladder of bands in the presence of the ligand. Cleavage by bleomycin is at the sequence G-pyrimidine, though not all such sites are affected to the same extent and some cutting is found at GA and GG. DNase I footprinting confirms the antibiotic-binding sites but reveals that some strong cleavage sites do not yield footprints. Bleomycin renders adenines on the 3' side of its cleavage sites (GT, GC and GA) hyper-reactive to diethyl pyrocarbonate.

Interaction of bleomycin with a methylated DNA oligonucleotide

Interaction of bleomycin with a methylated DNA oligonucleotide

Interaction of bleomycin and hyperthermia-results of a clinical pilot study

In vitro and animal studies indicate that concomitant administration of Bleomycin and hyperthermia result in a dramatic potentiation of cytotoxic (anti-tumor) activity. This effect takes place at temperatures above 42°C. A Phase 1 11 pilot study has been conducted to assess the clinical feasibility of concomitant Bleomycin-hyperthermia application. Eligible patients were those with measurable tumors persistent or recurrent following treatment with conventional modalities including full dose radiotherapy. The protocol required placement of at least two interstitial catheters for continuous monitoring of temperature. Skin probes were also used and thermal mapping of all catheters was required. The therapeutic aim was minimal tumor temperature of 43°C .5°C, the maximal normal tissue temperature was not to exceed 45°C. Pretreatment evaluation included pulmonary function studies and arterial blood gases. Bleomycin was administered within 1 hour prior to initiation of hyperthermia at the dose of 15 units/ m 2. Twelve patients received therapy according to this protocol. Eight presented with carcinomas of the head and neck area, 4 with adenocarcinoma of the breast. Complete response was recorded in 4, partial in 6 patients. Rapid tumor lysis occurred in 2 patients. Reaction of the normal skin and subcutaneous tissues were mild in all cases and ranged from erythema to mild fibrosis. Three patients experienced pulmonary toxicity, only one of which was severe enough to cause cessation of therapy. Early clinical experience with thermochemotherapy using Bleomycin in heavily pretreated patients indicates a promising response rate and acceptable tolerance. Further studies of Bleomycin-hyperthermia alone and in combination with other modalities including radiotherapy appear warranted.

Interaction of bleomycin and hyperthermia-results of a clinical pilot study

Interaction of bleomycin and hyperthermia-results of a clinical pilot study

Interaction of bleomycin, hyperthermia and a calmodulin inhibitor (trifluoperazine) in mouse tumor cells

To improve the efficacy of thermochemotherapy we have investigated the individual and combined effects of hyperthermia (44°C) and the calmodulin inhibitor trifluoperazine (30,μg/ml) on early plateau phase cultures of mouse EMT6 cells for simultaneous exposures to bleomycin. We found that a non-toxic combination of hyperthermia and trifluoperazine: (a) enhanced the cytotoxicity of bleomycin, (b) increased the frequency of long-lived attachment sites of cellular DNA at the nuclear matrix, and (c) resulted in an accumulation of DNA damage (strand-breaks and alkali-labile lesions) caused by the inhibition of strand-break rejoining and the impaired processing of DNA sites involving base loss. Our findings implicate a role for Camodulin in the control of chromatin structural changes during DNA repair and the study provides a rational basis for the use of Camodulin inhibitors in thermochemotherapy.

Interaction of bleomycin, hyperthermia and a calmodulin inhibitor (trifluoperazine) in mouse tumour cells: I. in vitro cytotoxicity

Evidence in the literature suggests that hyperthermia (HT) or inhibitors of calmodulin can increase the sensitivity of rodent cells to bleomycin (BLM) by interfering with DNA repair functions. In an attempt to explore methods of improving the efficacy of thermochemotherapy we have investigated the individual and combined effects of HT (44 degrees C) and the calmodulin inhibitor trifluoperazine (TFP, 30 micrograms ml-1) on early plateau phase monolayer cultures of mouse EMT6 tumour cells for simultaneous exposures to BLM. Early plateau phase cultures are relatively resistant both to HT and to BLM. The selected HT and TFP regimens (either alone or in combination) were non-toxic. Comparing the sensitizing effect (given by the ratio: Do BLM/Do BLM + modifier) of the various regimens on BLM-treated cells, we found that: TFP alone had a marginal effect (ratio 1.3), HT alone showed significant potentiation (ratio 19) and the combination of HT and TFP strongly sensitized (ratio greater than 110) cells to BLM cytotoxicity. We propose that the use of calmodulin inhibitors in thermochemotherapy is worthy of further evaluation.

Interaction of bleomycin, hyperthermia and a calmodulin inhibitor (trifluoperazine) in mouse tumour cells: Ii. DNA damage, repair and chromatin changes

We have reported in the preceding paper that the treatment of plateau phase mouse EMT6 tumour cells with a combination of hyperthermia (HT; 44 degrees C) and trifluoperazine (TFP; 30 micrograms ml-1; an inhibitor of calmodulin) greatly enhances the cytotoxicity of the antitumour drug belomycin (BLM). The cytotoxic action of BLM is thought to arise from the induction of DNA damage in a manner which reflects chromatin accessibility. Thus we have studied the effects of the two modifiers (HT and TFP) on chromatin structure and BLM-induced DNA damage. Co-treatment of cells with HT and TFP altered chromatin organisation by the formation and slow resolution of new DNA attachment sites at the nuclear matrix. HT increased drug-induced DNA damage (strand breaks and alkali-labile lesions) by the general depression of repair rather than through the generation of new sites for drug action. TFP produced a more discrete block in the repair of alkali-labile lesions in DNA. Both processes appear to occur for the combination of BLM, HT and TFP, and we propose that the novel chromatin configuration permits the accumulation of potentially lethal DNA strand breaks. Our study indicates the potential value of chromatin/DNA repair modifying regimens for overcoming the poor responsiveness of some tumour cells to chemotherapeutic drugs and provides a rational basis for the use of calmodulin inhibitors in thermochemotherapy.

Activation of bleomycin A 2 to a DNA-damaging intermediate by phorbol ester-stimulated human polymorphonuclear leukocytes

Metabolically stimulated polymorphonuclear leukocytes (PMNs) generate reactive oxygen metabolites that are capable not only of damaging biomolecules but also of possibly activating exogenous chemicals to reactive intermediates which interact with target molecules. This study examined this later concept and the results demonstrated that the interaction of bleomycin A 2 with phorbol ester-stimulated human PMNs resulted in DNA deoxyribose cleavage, a process mediated by an activated bleomycin intermediate. Superoxide dismutase (SOD) significantly inhibited bleomycin-mediated DNA deoxyribose cleavage, indicating the involvement of reactive oxygen. Based on these observations it is plausible that other chemicals, including carcinogens, may be activated by metabolically stimulated PMNs to intermediates which react with biomolecules.

The effect of bleomycin and oxygen on rat lung

The interaction of bleomycin and oxygen on rat lung was studied by giving endotracheal injections of bleomycin or saline to rats which subsequently breathed air or oxygen for 24 or 48 h. In the first protocol bleomycin or saline treated rats were exposed to 24 h of air or 100% oxygen and then breathed air for a further 6 d after which they were killed. In the second protocol bleomycin or saline treated rats were exposed to 48 h of air or up to 48 h of 100% oxygen. All animals in this protocol either died or were killed at the 48 h time point. The lungs were fixed in full inflation and 6 histological sections prepared from each. The volume densities of alveolar air, duct air, alveolar walls, intra-alveolar cells and non-parenchyma were quantitated using standard point counting techniques. In addition, the percent of alveoli containing fibrin was determined. The results demonstrated a synergistic effect of oxygen and bleomycin toxicity after both 24 and 48 h oxygen exposure. This was most marked for alveolar fibrin deposition which was significantly greater (p less than 0.001) in the bleomycin and oxygen groups than the saline/air, saline/oxygen or bleomycin/air groups. In the 48 h study the volume density of intra-alveolar cells was also significantly greater in the bleomycin/oxygen group than all other groups (p less than 0.05). It is concluded that bleomycin and oxygen exhibit synergistic toxic effects and that their concurrent administration should be avoided.

Studies on the interaction of bleomycin A 2 with rat lung microsomes. III. Effect of exogenous iron on bleomycin-mediated DNA chain breakage

The interaction of bleomycin A 2 with rat lung microsomes results in bleomycin-mediated DNA chain breakage due to the mixed-function oxidase catalyzed activation of bleomycin. This study demonstrates that the addition of exogenous Fe 3+ significantly enhances the bleomycin-mediated cleavage of DNA deoxyribose, that the enhancing effect of Fe 3+ is maximum when a 1:1 ratio of bleomycin to Fe 3+ is achieved and that either NADPH or NADH can serve as pyridine cofactors for this reaction. Since the activation of bleomycin can be facilitated by iron in the Fe 2+ form, these observations support the hypothesis that the mixed-function oxidase system may serve to maintain either adventitious or exogenous iron in the Fe 2+ form. In the absence of DNA, the interaction of bleomycin with rat lung microsomes results in the self-inactivation of bleomycin, a reaction which is also enhanced by the addition of exogenous Fe 3+. Thus, the microsomal mixed-function oxidase system represents an efficient biological system for the ‘activation-inactivation’ of bleomycin.

Interaction of bleomycin with nucleic acids as probed by ESR spectrometry of iron(II)-nitrosyl complex of bleomycin.

Interaction of bleomycin with nucleic acids as probed by ESR spectrometry of iron(II)-nitrosyl complex of bleomycin.

Interactions of bleomycin with DNA

Interactions of bleomycin with DNA

Biochemical and cytological studies of bleomycin actions on chromatin and chromosomes.

Interaction of bleomycin with nuclei isolated from a variety of mammalian cells resulted in the release of nucleosomes. When isolated mononucleosomes (core plus linker) were re-treated with bleomycin, no further degradation of DNA occurred. The results suggest that the bleomycin cleavage sites in chromatin are present only in the linker region and that there are probably only one or two cleavage sites per linker. The repeat lengths of nucleosomal DNA released by bleomycin from nuclei of different species are different; this variability is considered to reflect the length of the linker. Incorporation of BrdU into DNA did not alter the bleomycin action on nucleosomes. When mitotic cells were held at metaphase for a prolonged period, bleomycin caused a gradual disintegration of chromosomes, although the bleomycin cleavage sites in metaphase chromosomes were found to be the same as those in interphase nuclei.

Chemistry of bleomycin. XXII. Interaction of bleomycin with nucleic acids, preferential binding to guanine base and electrostatic effect of the terminal amine.

Chemistry of bleomycin. XXII. Interaction of bleomycin with nucleic acids, preferential binding to guanine base and electrostatic effect of the terminal amine.