Depletion of mitochondrial DNA by ethidium bromide treatment inhibits the proliferation and tumorigenesis of T47D human breast cancer cells
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Mitochondria are the key organelles for life. Mitochondria are important mediators of tumorigenesis. Mitochondria are key participants in tumorigenesis and development. In-depth study on the role of mitochondria in the survival and growth of tumor cells is not only conducive to our understanding of tumor genesis and development, including oncogene activation, tumor suppressor gene inactivation and other aspects, but also of far-reaching significance in the diagnosis and treatment of tumors.
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Neutral petites were induced in three different [rho+] strains by treating with ethidium bromide. However, EB treatment of spontaneously generated suppressive petites, which were derived from the same [rho+] strains, produced no neutral petites and moreover had little or no effect on the suppressiveness of the petities. Genetic analysis showed that the ethidium bromide resistance of the suppressive petites was a manifestation of cytoplasmically inherited, respiratory deficiency.
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The effect of methanol on the frequency of cytoplasmic respiration-deficient (RD) mutation induced by ethidium bromide was investigated in Saccharomyces cerevisiae. When growing cells were treated with ethidium bromide at concentrations higher than 1.0μg/ml, 95 to 100% of surviving cells were RD mutants as judged by the tetrazolium overlay method. The RD mutants induced by 1.0μg/ml ethidium bromide were drastically decreased by addition of 6-8% methanol in both growing and non-growing conditions. Methanol reduced uptake of ethidium bromide by yeast cells, but it did not modify the inhibitory effect of ethidium bromide on in vitro yeast mitochondrial deoxyribonucleic acid synthesis.
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By adapting an antibiotic-susceptible Staphylococcus aureus strain to increasing concentrations of ethidium bromide, a known substrate of efflux pumps (EPs), and by phenotypically and genotypically analysing the resulting progeny, we characterized the molecular mechanisms of S. aureus adaptation to ethidium bromide. S. aureus ATCC 25923 was grown in increasing concentrations of ethidium bromide. The MICs of representatives of eight classes of antibiotics, eight biocides and two dyes against ATCC 25923 and its ethidium bromide-resistant progeny ATCC 25923EtBr were determined with or without six efflux pump inhibitors (EPIs). Efflux activity in the presence/absence of EPIs was evaluated by real-time fluorometry. The presence and expression of eight EP genes were assayed by PCR and quantitative RT–PCR (qRT–PCR), respectively. Mutations in grlA, gyrA and norA promoter regions were screened by DNA sequencing. Compared with its parental strain, ATCC 25923EtBr was 32-fold more resistant to ethidium bromide and also more resistant to biocides and hydrophilic fluoroquinolones. Resistance to these could be reduced by the EPIs chlorpromazine, thioridazine and reserpine. Increased efflux of ethidium bromide by ATCC 25923EtBr could be inhibited by the same EPIs. qRT–PCR showed that norA was 35-fold over-expressed in ATCC 25923EtBr, whereas the remaining EP genes showed no significant increase in their expression. Sequencing of the norA promoter region revealed a 70 bp deletion in ATCC 25923EtBr. Exposure of S. aureus to quaternary compounds such as ethidium bromide results in decreased susceptibility of the organism to a wide variety of compounds, including quinolones and biocides through an efflux-mediated response, which for strain ATCC 25923 is mainly NorA-mediated. This altered expression may result from alterations in the norA promoter region.
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