Abstract
BackgroundTo establish, characterize and elucidate potential mechanisms of acquired bleomycin (BLM) resistance using human cancer cell lines. Seven BLM-resistant cell lines were established by exposure to escalating BLM concentrations over a period of 16-24 months. IC50 values and cell doubling times were quantified using a real time cytotoxicity assay. COMET and γ-H2AX assays, cell cycle analysis, and apoptosis assessment further investigated the mechanisms of BLM resistance in these cell lines. ResultsCompared with parental cell lines, real time cytotoxicity assays revealed 7 to 49 fold increases in IC50 and a mean doubling time increase of 147 % (range 64 %-352%) in BLM-resistant sub-clones (p<0.05 for both). Higher maintenance BLM concentrations were associated with higher IC50 and increased doubling times (p<0.05). Significantly reduced DNA damage (COMET and γ-H2AX assays), G2/M arrest, and apoptosis (p<0.05 for each set of comparison) following high-dose acute BLM exposure was observed in resistant sub-clones, compared with their BLM-sensitive parental counterparts. Three weeks of BLM-free culturing resulted in a partial return to BLM sensitivity in 3/7 BLM-resistant sub-clones (p<0.05). ConclusionBleomycin resistance may be associated with reduced DNA damage after bleomycin exposure, resulting in reduced G2/M arrest, and reduced apoptosis.
Highlights
Bleomycin (BLM) is a glycopeptide antibiotic isolated from Streptomyces verticillis [1,2]
BLM-resistant cell lines maintained on BLM stably displayed higher IC50 values and prolonged doubling times
A positive correlation was observed between the maintenance BLM concentration and IC50 values (p
Summary
Bleomycin (BLM) is a glycopeptide antibiotic isolated from Streptomyces verticillis [1,2]. The full pathway of the drug’s mechanism of action has not been elucidated, BLM does bind to iron and oxygen to produce reactive oxygen species (ROS) [5] that induces single- and double-strand DNA breaks, with the latter being primarily responsible for its anti-tumor effects [6,7]. It causes lipid peroxidation and mitochondrial DNA damage [8]. Results: Compared with parental cell lines, real time cytotoxicity assays revealed 7 to 49 fold increases in IC50 and a mean doubling time increase of 147 % (range 64 %-352%) in BLM-resistant sub-clones (p
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