Resistance to oxidants associated with elevated catalase activity in HL-60 leukemia cells that overexpress multidrug-resistance protein does not contribute to the resistance to daunorubicin manifested by these cells.
OBJECTIVE
It has been recognized that enhanced antioxidant defenses can contribute to the resistance of cancer cells displaying multidrug resistance (MDR) that arises in conjunction with the overexpression of P-glycoprotein (Pgp). The purpose of this study was to determine if the defenses against oxidant stress in MDR human leukemia cells (HL-60/AR) that overexpress multidrug-resistance-associated protein (MRP), but not Pgp, contribute to the mechanism of drug resistance in this cell line.


METHODS
HL-60/AR cells were evaluated in comparison with wild-type cells with respect to sensitivity to the oxidants hydrogen peroxide (H2O2) and tert-butyl hydroperoxide (t-BuOOH), the activities and amounts of the antioxidant enzymes catalase and glutathione peroxidase (GSH-Px), and the effects that manipulation of the activities of these enzymes may have on cellular sensitivity to the oxidants and to daunorubicin. We also evaluated the ability of the cells to generate daunorubicin semiquinone free radical as measured by electron spin resonance (ESR) spectroscopy.


RESULTS
HL-60/AR cells were > 10-fold resistant to the cytotoxic effects of the H2O2 or t-BuOOH as compared with parental, drug-sensitive HL-60 cells. This phenomenon could be attributed largely to elevated activity and protein levels of catalase in HL-60/AR cells. Furthermore, inhibition of catalase by 3-amino-1,2,4-triazole (AT) diminished the resistance of HL-60/AR to these oxidants by > 80% or > 50%, respectively. Despite these findings, AT was incapable of causing sensitization of HL-60/AR cells to the cytotoxic effects of daunorubicin. We found that the activity and amount of selenium-dependent glutathione peroxidase (GSH-Px) was no greater in HL-60/AR cells than in HL-60 cells. Cultivation of cells in selenium-deficient medium caused a marked reduction in GSH-Px activity in HL-60/AR cells and a profound inhibition of GSH-redox cycling manifested by a decrease in baseline hexose monophosphate shunt activity (HMPS) and markedly blunted stimulation of the HMPS by the oxidant t-BuOOH in both wild-type and resistant cells. These variations in GSH-Px activity and GSH-redox cycling, however, were not associated with an alteration in cellular sensitivity to daunorubicin. The failure of catalase inhibition or selenium manipulation of GSH-Px activity to affect daunorubicin cytotoxicity was not due to the inability of these cells to produce free-radical species of daunorubicin, since ESR studies revealed that the generation of daunorubicin semiquinone free radical by HL-60/AR cells was equal to and, in fact, 3-fold that obtained with HL-60 cells.


CONCLUSIONS
In comparison with parental HL-60 cells, MRP-overexpressing HL-60/AR cells have demonstrable alterations in antioxidant defenses that are manifested by cellular resistance to the cytotoxic effects of H2O2 and t-BuOOH and by elevated protein levels and activity of catalase. Whether these alterations are epiphenomena or are related to overexpression of MRP remains to be determined. However, it does appear that the enhanced antioxidant defenses observed in HL-60/AR cells do not contribute to the resistance to daunorubicin manifested by this cell line. Although HL-60/AR cells generate daunorubicin semiquinone free radical to an extent equal to or greater than that observed in HL-60 cells, the failure of alterations in GSH-Px activity or inhibition of catalase to change the sensitivity of HL-60/AR cells to daunorubicin suggests that the cytotoxicity of daunorubicin in these cells in not mediated through H2O2 or other peroxide species detoxified by these enzymes.
Division of Developmental Therapeutics, University of Maryland Cancer Center, Baltimore 21201.
P F
PF
Lenehan
P L
PL
Gutiérrez
J L
JL
Wagner
N
N
Milak
G R
GR
Fisher
D D
DD
Ross
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis*; D015153:Blotting, Western; D002374:Catalase / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D003198:Computer Simulation; D003630:Daunorubicin / Q000378:metabolism / Q000633:toxicity*; D018432:Drug Resistance, Multiple; D004578:Electron Spin Resonance Spectroscopy; D005979:Glutathione Peroxidase / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000633:toxicity; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism / Q000473:pathology; D016877:Oxidants / Q000378:metabolism* / Q000633:toxicity; D010427:Pentose Phosphate Pathway / Q000187:drug effects / Q000502:physiology; D010545:Peroxides / Q000378:metabolism / Q000633:toxicity; D017382:Reactive Oxygen Species; D014407:Tumor Cells, Cultured; D020122:tert-Butylhydroperoxide
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D016877:Oxidants; D010545:Peroxides; D017382:Reactive Oxygen Species; D020122:tert-Butylhydroperoxide; D006861:Hydrogen Peroxide; D002374:Catalase; D005979:Glutathione Peroxidase; D003630:Daunorubicin