Key result
Doxorubicin induces DNA breakage, increases micronucleation, and enhances apoptosis in human cells in vitro.
Why the study?
The mechanisms underlying doxorubicin's cytostatic activity, including its effects on DNA damage, micronucleation, and apoptosis in human cells, require further clarification.
p-value: p=<0.05
Doxorubicin induces genotoxic effects in human cells via oxidative DNA damage, chromosome breakage, mitotic spindle disturbance, and caspase-3-mediated apoptosis.
May inform doxorubicin cardiotoxicity monitoring; hypothesis-generating for mechanisms, requiring in vivo confirmation before practice change.
BACKGROUND: Doxorubicin is a widely used anticancer drug due to its broad spectrum of antitumor activity. Various mechanisms have been proposed for its cytostatic activity, including DNA intercalation, topoisomerase II inhibition, generation of free radicals and apoptosis. The present study aims to further clarify the cytostatic activity of doxorubicin by its specific effect on (a) DNA damage, (b) micronucleation and (c) apoptosis, using a combination of different methods and cell systems such as human lymphocytes and HL-60 human leukemic cells. DNA lesions were analyzed by the alkaline comet assay in combination with formamidopyrimidine (Fpg) and human 8-oxoguanine (hOGG1) repair enzymes. Micronucleation was investigated by the Cytokinesis-Block Micronucleus assay (CBMN) in combination with Fluorescence In Situ Hybridization analysis. Impairment on mitotic apparatus was investigated by double immunofluorescence of β- and γ-tubulin. Apoptotic cell frequency was determined by the CBMN cytome assay. Complementary to the above, caspase-3 level was investigated by Western blot. RESULTS: It was found that doxorubicin generates DNA breakage induced by oxidative damage in DNA bases, which can be repaired by the Fpg and hOGG1 enzymes. Increased micronucleus frequency was identified mainly through chromosome breakage and, at a lesser extent, through chromosome delay. Analysis of mitotic spindle showed disturbance of chromosome orientation and centrosome duplication and/or separation, leading to aneuploidy. Enhanced frequency of apoptotic leukemic cells was also observed. Caspase-3 seems to be involved in the generation of apoptosis. CONCLUSIONS: The aforementioned findings derived from different treatment schedules, doses and time of exposure on primary versus transformed cells extend our knowledge about doxorubicin genotoxicity and contribute to the better understanding of the mechanisms by which doxorubicin induces genotoxic effects on human cells.
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Chondrou et al. (2018) studied this question. Doxorubicin vs. Control (untreated cells) was evaluated on DNA damage, micronucleus frequency, and apoptotic cell frequency (p=<0.05). Doxorubicin induced DNA breakage, increased micronucleus frequency through chromosome breakage and delay, disturbed mitotic spindle organization, and enhanced apoptosis in human cells in vitro.
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