Modifications of cellular autofluorescence emission spectra under oxidative stress induced by 1 alpha,25dihydroxyvitamin D(3) and its analog EB1089.
We attempted to characterize the cellular autofluorescence phenomenon of living HL-60 cells and to appraise its modifications under oxidative stress conditions induced by 1 alpha,25(OH)(2)D(3) (VD(3)) and its analog EB1089. Autofluorescence emission spectra of human promyelocytic HL-60 leukemic cells were monitored using laser scanning confocal microspectrofluorometry under UV excitation. Evaluation of reactive oxygen species (ROS) release was performed using the 2',7'-dichlorodihydrofluorescein diacetate (H(2)-DCFDA) staining and fluorescence emission measurement. VD(3) (1, 10, 100 nM) or EB1089 (0.1, 1 and 10 nM) induces a decrease in autofluorescence emission intensity that can be attributed to the oxidation of the coenzyme nicotinamide adenine dinucleotide (phosphate) NAD(P)H into NAD(P)(+). A dose-dependent increase (p<0.05) in ROS release is observed in VD(3)- and EB1089-treated cells. As compared with VD(3)- or EB1089-treated cells, doxorubicin-VD(3) or doxorubicin-EB1089 treatments strongly decrease the autofluorescence intensity and induce a higher release of ROS (p<0.05). The association of antioxidants (N-acetyl cysteine, superoxide dismutase, catalase) with VD(3) or EB1089 induce a more limited autofluorescence decrease and a weaker ROS generation, as compared with VD(3) and EB1089 treated cells. In conclusion, the free radicals release, generated by VD(3) and EB1089, was associated with the decrease in autofluorescence emission and can be modulated by doxorubicin and antioxidants.
FR de Pharmacie, Unité MéDIAN, CNRS-UMR 6142, 1 Avenue du Maréchal Juin, 51096, Reims, France.
Patrick
P
Bondza-Kibangou
Christine
C
Millot
Jean
J
Dufer
Jean-Marc
JM
Millot
D000970:Antineoplastic Agents / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000494:pharmacology*; D002454:Cell Differentiation; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D007834:Lasers; D018613:Microscopy, Confocal; D018384:Oxidative Stress; D017382:Reactive Oxygen Species; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013053:Spectrophotometry; D013997:Time Factors; D014466:Ultraviolet Rays
D000970:Antineoplastic Agents; D000975:Antioxidants; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D002117:Calcitriol; C078903:seocalcitol