Uncovering bleomycin-induced genomic alterations and underlying mechanisms in yeast

Bleomycin (BLM) is a widely used chemotherapeutic drug. BLM-treated cells showed an elevated rate of mutations, but the underlying mechanisms remained unclear. In this study, the global genomic alterations in BLM-treated cells were explored in the yeast Saccharomyces cerevisiae . Using genetic assay and whole-genome sequencing, we found that the mutation rate could be greatly elevated in S. cerevisiae cells that underwent Zeocin TM (a BLM member) treatment. One-base deletion and T to G substitution at the 5’-GT-3’ motif was the most striking signature of Zeocin TM -induced mutations. This was mainly the result of translesion DNA synthesis involving Rev1 and polymerase ζ. Zeocin TM treatment led to the frequent loss of heterozygosity and chromosomal rearrangements in the diploid strains. The breakpoints of recombination events were significantly associated with certain chromosomal elements. Lastly, we identified multiple genomic alterations that contributed to BLM resistance in the Zeocin TM -treated mutants. Overall, this study provides new insights into the genotoxicity and evolutional effects of BLM. Importance Bleomycin is an antitumor antibiotic that can mutate genomic DNA. Using yeast models in combination with genome sequencing, the mutational signatures of Zeocin TM (a member of the bleomycin family) are disclosed. Translesion-synthesis polymerases are crucial for the viability of Zeocin TM -treated yeast cells at the sacrifice of a higher mutation rate. We also confirmed that multiple genomic alterations were associated with the improved resistance to Zeocin TM , providing novel insights into how bleomycin resistance is developed in cells.

Elevation of Chemosensitivity of Lung Adenocarcinoma A549 Spheroid Cells by Claudin-2 Knockdown through Activation of Glucose Transport and Inhibition of Nrf2 Signal

Claudin-2 (CLDN2), a tight junctional protein, is involved in the chemoresistance in a three-dimensional spheroid culture model of human lung adenocarcinoma A549 cells. However, the mechanism has not been fully clarified. We found that the knockdown of CLDN2 expression by siRNA in the spheroid reduces the expression of glucose transporters and metabolic enzymes. In a two-dimensional culture model, the expression of these proteins was increased by glucose deprivation or fasentin, an inhibitor of glucose transporter. In addition, the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and antioxidant enzymes including heme oxygenase-1, NAD(P)H:quinone oxidoreductase-1, and a glutamate–cysteine ligase modifier subunit were increased by fasentin. The fluorescence intensities of JC-1, a probe of mitochondrial membrane potential, and MitoROS 580, a probe of mitochondrial superoxide production, were increased by fasentin. These results suggest that mitochondrial production of reactive oxygen species is increased by glucose deficiency. The knockdown of CLDN2 enhanced the flux of 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG), a fluorescent deoxyglucose derivative, in a transwell assay, and the accumulation of glucose and 2-NBDG in spheroid cells. The expression of Nrf2 was decreased by CLDN2 knockdown, which was inhibited by fasentin and sulforaphane, a typical Nrf2 activator, in spheroid cells. The sensitivity of spheroid cells to doxorubicin, an anthracycline antitumor antibiotic, was enhanced by CLDN2 knockdown, which was inhibited by fasentin and sulforaphane. We suggest that CLDN2 induces chemoresistance in spheroid cells mediated through the inhibition of glucose transport and activation of the Nrf2 signal.

Macroparticle-enhanced cultivation of Lentzea aerocolonigenes: Variation of mechanical stress and combination with lecithin supplementation for a significantly increased rebeccamycin production

The actinomycete Lentzea aerocolonigenes produces the antitumor antibiotic rebeccamycin. In previous studies the rebeccamycin production was significantly increased by the addition of glass beads during cultivation in different diameters between 0.5 – 2 mm and the induced mechanical stress by the glass beads was proposed to be responsible for the increased production. Thus, this study was conducted to be a systematic investigation of different parameters for macroparticle addition, such as bead diameter, concentration and density (glass and ceramic) as well as shaking frequency, for a better understanding of the particle induced stress on L. aerocolonigenes. The induced stress for optimal rebeccamycin production can be estimated by a combination of stress energy and stress frequency. In addition, the macroparticle-enhanced cultivation of L. aerocolonigenes was combined with soy lecithin addition to further increase the rebeccamycin concentration. With 100 g L glass beads in a diameter of 969 µm and 5 g L soy lecithin a concentration of 388 mg L 1 rebeccamycin was reached after 10 days of cultivation, which corresponds to the highest rebeccamycin concentrations achieved in shake flask cultivations of L. aerocolonigenes stated in literature so far.

The intriguing chemistry and biology of sulfur-containing natural products from marine microorganisms (1987–2020)

Natural products derived from marine microorganisms have received great attention as a potential resource of new compound entities for drug discovery. The unique marine environment brings us a large group of sulfur-containing natural products with abundant biological functionality including antitumor, antibiotic, anti-inflammatory and antiviral activities. We reviewed all the 484 sulfur-containing natural products (non-sulfated) isolated from marine microorganisms, of which 59.9% are thioethers, 29.8% are thiazole/thiazoline-containing compounds and 10.3% are sulfoxides, sulfones, thioesters and many others. A selection of 133 compounds was further discussed on their structure–activity relationships, mechanisms of action, biosynthesis, and druggability. This is the first systematic review on sulfur-containing natural products from marine microorganisms conducted from January 1987, when the first one was reported, to December 2020.

Polyphosphate Reverses the Toxicity of the Quasi-Enzyme Bleomycin on Alveolar Endothelial Lung Cells In Vitro

The anti-cancer antitumor antibiotic bleomycin(s) (BLM) induces athyminic sites in DNA after its activation, a process that results in strand splitting. Here, using A549 human lung cells or BEAS-2B cells lunc cells, we show that the cell toxicity of BLM can be suppressed by addition of inorganic polyphosphate (polyP), a physiological polymer that accumulates and is released from platelets. BLM at a concentration of 20 µg ml−1 causes a decrease in cell viability (by ~70%), accompanied by an increased DNA damage and chromatin expansion (by amazingly 6-fold). Importantly, the BLM-caused effects on cell growth and DNA integrity are substantially suppressed by polyP. In parallel, the enlargement of the nuclei/chromatin in BLM-treated cells (diameter, 20–25 µm) is normalized to ~12 µm after co-incubation of the cells with BLM and polyP. A sequential application of the drugs (BLM for 3 days, followed by an exposure to polyP) does not cause this normalization. During co-incubation of BLM with polyP the gene for the BLM hydrolase is upregulated. It is concluded that by upregulating this enzyme polyP prevents the toxic side effects of BLM. These data might also contribute to an application of BLM in COVID-19 patients, since polyP inhibits binding of SARS-CoV-2 to cellular ACE2.

Response of isolated buccal epithelium cells on the combined action of anti-tumor antibiotic doxorubicin and magnetic field

The combined effect of doxorubicin and magnetic field on the viability of isolated buccal epithelial cells two donors was investigated. Doxorubicin is an effective antitumor antibiotic, but this drug has a large amount of side effects. This significantly reduces the number of cases when you can change the drug. To reduce the toxic effect or to increase the effectiveness of anticancer therapy, the introduction of additional substances is used. For a long time, magnetic and electromagnetic fields have been used as a therapeutic factor by three therapies for cancer alone, or as an adjunct. Doxorubicin at a concentration of 2 μg/ml for 2 hours has been shown to increase of cell membrane permeability and heterochromatin granule quantity in cell nuclei. Exposure of cells to 25 mT magnetic field for 30 and 60 minutes results in decrease in cell viability and increase in cell membrane permeability and the number of heterochromatin granules in nuclei. Combined exposure to static magnetic field and doxorubicin results in decrease of the number of heterochromatin granules in the nuclei of cells from one of the donors, as compared to the variant of exposure only to doxorubicin, which indicates the protective effect of the magnetic field. A possible mechanism of the protective action of the magnetic field in our experiments is that the stress under the influence of the magnetic field activates the protective mechanisms of the cell (including the transition of chromatin to a heterochromatinized state). That way, been pre-activated due to the action of the magnetic field, the defense mechanisms of the cell reduce the toxic effects caused by doxorubicin.

Exploration the Mechanism of Doxorubicin-Induced Heart Failure in Rats by Integration of Proteomics and Metabolomics Data

Heart failure is a common systemic disease with high morbidity and mortality worldwide. Doxorubicin (DOX) is a commonly used anthracycline broad-spectrum antitumor antibiotic with strong antitumor effect and definite curative effect. However, cardiotoxicity is the adverse reaction of drug dose cumulative toxicity, but the mechanism is still unclear. In this study, proteomics and metabonomics techniques were used to analyze the tissue and plasma of DOX-induced heart failure (HF) in rats and to clarify the molecular mechanism of the harmful effects of DOX on cardiac metabolism and function in rats from a new point of view. The results showed that a total of 278 proteins with significant changes were identified by quantitative proteomic analysis, of which 118 proteins were significantly upregulated and 160 proteins were significantly downregulated in myocardial tissue. In the metabonomic analysis, 21 biomarkers such as L-octanoylcarnitine, alpha-ketoglutarate, glutamine, creatine, and sphingosine were detected. Correlation analysis showed that DOX-induced HF mainly affected phenylalanine, tyrosine, and tryptophan biosynthesis, D-glutamine and D-glutamate metabolism, phenylalanine metabolism, biosynthesis of unsaturated fatty acids, and other metabolic pathways, suggesting abnormal amino acid metabolism, fatty acid metabolism, and glycerol phospholipid metabolism. It is worth noting that we have found the key upstream target of DOX-induced HF, PTP1B, which inhibits the expression of HIF-1α by inhibiting the phosphorylation of IRS, leading to disorders of fatty acid metabolism and glycolysis, which together with the decrease of Nrf2, SOD, Cytc, and AK4 proteins lead to oxidative stress. Therefore, we think that PTP1B may play an important role in the development of heart failure induced by doxorubicin and can be used as a potential target for the treatment of heart failure.

Toll-like receptor 4 (TLR4) expression is correlated with T2* iron deposition in response to doxorubicin treatment: cardiotoxicity risk assessment

Although doxorubicin (Dox) is an effective antitumor antibiotic in the anthracycline class, it often induces the undesirable side effect of cardiomyopathy leading to congestive heart failure, which limits its clinical use. The primary goal of this study is to evaluate a reliable translational method for Dox-induced cardiotoxicity (CTX) screening, aiming to identify a high-risk population and to discover new strategies to predict and investigate this phenomenon. Early identification of the presence of iron deposits and genetic and environmental triggers that predispose individuals to increased risk of Dox-induced CTX (e.g., overexpression of Toll-like receptor 4 (TLR4)) will enable the early implementation of countermeasure therapy, which will improve the patient’s chance of survival. Our cohort consisted of 25 consecutive patients with pathologically confirmed cancer undergoing Dox chemotherapy and 12 control patients. The following parameters were measured: serum TLR4 (baseline), serum transferrin (baseline and 6-week follow-up) and iron deposition (baseline and 6-week follow-up). The average number of gene expression units was 0.121 for TLR4 (range 0.051–0.801). We subsequently correlated serum TLR4 levels in our cohort with myocardial iron overload using the cardiac magnetic resonance (CMR) T2* technique, the ventricular function (% ejection fraction, %EF) and serum transferrin levels. There is a strong negative linear relationship between serum TLR4 and CMR T2* values (r = − 0.9106, ****P < 0.0001). There is also a linear correlation (either positive or negative) with EF and transferrin; no established relationship related to the sex of the patients was found. Patients with elevated serum TLR4 at baseline also exhibited an increase in serum transferrin levels and Dox-induced left ventricular dysfunction with a decreased EF (< 50%); this phenomenon was observed in 7 of 25 patients (28%) at the 6-week follow-up. There were no significant differences or correlations based on sex. We concluded that there is a direct relationship between Dox-induced CTX (indicated by elevated serum TLR4) and the times (ms) for T2* (decreases in which correspond to immediate and rapid iron overload).

Morphology of the Micelles Formed by a Comb-Like PEG-Containing Copolymer Loaded with Antitumor Substances with Different Water Solubilities

The controlled delivery of anticancer drugs is driven by their interaction with carrier molecules. By creating complicated micelle-like complexes, amphiphilic polymers provide an opportunity to load drugs of various kinds. In this work, the interaction of the comb-like PEG-containing polymer poly(VEP-co-GMA)-graft-PEG with the water-soluble antitumor antibiotic doxorubicin and new water-insoluble derivatives of thiozalidinone Les-3883 characterized by a high anticancer efficiency has been studied in aqueous solutions by means of the SAXS, DLS, TEM, and photoluminescence methods. The formation of polymer micelles and their complexes with drugs, as well as their structural changes, is observed. The obtained results give evidence that the mechanism of organization of supramolecular complexes depends on the drug solubility in water. A potential capability of poly(VEP-co-GMA)-graft-PEG to prolong the drug circulation lifetime is confirmed.