Combined study on clastogenic, aneugenic and apoptotic properties of doxorubicin in human cells in vitro

Abstract

BackgroundDoxorubicin 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.ResultsIt 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.ConclusionsThe 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.

Encapsulation of Nedaplatin in Novel PEGylated Liposomes Increases Its Cytotoxicity and Genotoxicity against A549 and U2OS Human Cancer Cells

“…This can be attributed to the resistance of U2OS as observed in MTT at that concentration ( Figure 3 ), unlike A549 where there was significant cell death. This is consistent with a previous study comparing the anticancer effects of cisplatin and carboplatin on a panel of osteosarcoma cell lines, where U2OS has stood out as resistant to the cytotoxic effects of both tested drugs [ 31 ]. Resistance to the platinum-based drugs in that study was attributed to either more efficient repair of platinum-induced DNA damage, or the ability to evade apoptosis.…”

“…Micronuclei are extranuclear bodies containing chromosomal fragments and/or whole chromosomes lagging behind in anaphase. MN assay can be used to show both clastogenic (resulting from unrepaired DSBs) and aneugenic effects (resulting from mitotic spindle damage), where usually a studied compound induces one type of MN [ 30 , 31 , 32 , 33 ]. For example, ionizing radiation and anthracyclins mainly induce clastogenic micronuclei, whereas vinca-alkaloids mainly induce aneugenic micronuclei.…”

Doxorubicin-Induced Translocation of mtDNA into the Nuclear Genome of Human Lymphocytes Detected Using a Molecular-Cytogenetic Approach

“…We demonstrated that DOX is capable of inducing the insertion of mtDNA in human cells and the number of insertions has a significant relationship with the levels of DOX-induced MN. Since chromosome breaks prevail over lagging chromosomes in DOX-induced MN [ 20 , 28 ], our results indicate an association between mtDNA transfer and chromosome breaks or DNA DSBs. Our data is in accordance with previous studies indicating that mtDNA translocation in nuclear genome occurs after repair of DSBs in yeast cells.…”

“…To develop the optimal conditions for the induction of chromosomal aberrations, the ability of different concentrations of DOX to induce micronuclei (MN) in human leukocytes was studied using cytokinesis-block micronucleus assay (CBMN). MN is a marker of chromosome damage that originates from chromosome fragments or whole chromosomes [ 20 ]. DOX has been shown to induce MN in human lymphocytes mainly through chromosome breakage and, to a lesser extent, through chromosome delay [ 20 ], and chromosome breaks are expected to facilitate mtDNA transfer in nuclear DNA.…”

“…MN is a marker of chromosome damage that originates from chromosome fragments or whole chromosomes [ 20 ]. DOX has been shown to induce MN in human lymphocytes mainly through chromosome breakage and, to a lesser extent, through chromosome delay [ 20 ], and chromosome breaks are expected to facilitate mtDNA transfer in nuclear DNA.…”