Interaction of doxorubicin with the subcellular structures of the sensitive and Bcl-xL-overexpressing MCF-7 cell line: Confocal and low-energy-loss transmission electron microscopy

The present investigation was directed to examine the interaction of the anti-cancer agent doxorubicin (DOX) with the subcellular compartments of the drug sensitive and Bcl-xL-overexpressing (Bcl-xL) MCF-7 cells using confocal and low-energy-loss transmission electron microscopy (LELTEM). Intracellular detection of DOX with LELTEM was carried out without specific antibodies or heavy metal stains but via the electron-induced molecular orbital excitation of the drug. Cells were incubated with 10 mu M DOX for 1 min, 1, 24, and 48 h and then examined live by confocal microscope and as very thin sections in an electron microscope equipped with an energy filter having an energy resolution of 1 eV. Ultrastructural localization of DOX, obtained from pairs of images taken at energy losses of 3 +/- 1 and 10 +/- 1 eV, were analyzed and correlated with the confocal observations. When the sensitive and Bcl-xL cells were examined under the confocal microscope after I min, DOX uptake could not be detected in the nuclei nor in the cytoplasm whereas LELTEM observation revealed that at this stage of incubation the drug has already been incorporated by both cell types and that the nuclear membrane, nucleolus, and mitochondria of the Bcl-xL cells were temporally less DOX-responsive as compared to the sensitive cells. As the incubation time increased, nuclear membranes and nucleoli of both cell types appeared equally sensitive to DOX, nonetheless, mitochondria of the Bcl-xL cells remained invulnerable to DOX for 24 h. The results point to LELTEM feasibility to better characterize yet unresolved cellular events caused by DOX and suggest a transitory role for Bcl-xL overexpression in protecting the cellular compartments from DOX invasion. (C) 2009 Elsevier Ltd. All rights reserved.