Differential regulation of doxorubicin-induced mitochondrial dysfunction and apoptosis by Bcl-2 in mammary adenocarcinoma (MTLn3) cells.
Various anticancer drugs cause mitochondrial perturbations in association with apoptosis. Here we investigated the involvement of caspase- and Bcl-2-dependent pathways in doxorubicin-induced mitochondrial perturbations and apoptosis. For this purpose, we set up a novel three-color flow cytometric assay using rhodamine 123, annexin V-allophycocyanin, and propidium iodide to assess the involvement of the mitochondria in apoptosis caused by doxorubicin in the breast cancer cell line MTLn3. Doxorubicin-induced apoptosis was preceded by up-regulation of CD95 and CD95L and a collapse of mitochondrial membrane potential (Deltapsi) occurring prior to phosphatidylserine externalization. This drop in Deltapsi was independent of caspase activity, since benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone did not inhibit it. Benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone also blocked activation of caspase-8, thus excluding an involvement of the death receptor pathway in Deltapsi dissipation. Furthermore, although overexpression of Bcl-2 in MTLn3 cells inhibited apoptosis, dissipation of Deltapsi was still observed. No decrease in Deltapsi was observed in cells undergoing etoposide-induced apoptosis. Immunofluorescent analysis of Deltapsi and cytochrome c localization on a cell-to-cell basis indicates that the collapse of Deltapsi and cytochrome c release are mutually independent in both normal and Bcl-2-overexpressing cells. Together, these data indicate that doxorubicin-induced dissipation of the mitochondrial membrane potential precedes phosphatidylserine externalization and is independent of a caspase- or Bcl-2-controlled checkpoint.
Huigsloot
Merei
M
Division of Toxicology, Leiden Amsterdam Center for Drug Research, Leiden University, Leiden 2300, The Netherlands.
Tijdens
Ine B
IB
Mulder
Gerard J
GJ
van de Water
Bob
B
D000818:Animals; D017304:Annexin A5 / Q000494:pharmacology; D017209:Apoptosis; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D016923:Cell Death; D004396:Coloring Agents / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053222:Fas Ligand Protein; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D015151:Immunoblotting; D007150:Immunohistochemistry; D015674:Mammary Neoplasms, Animal; D008562:Membrane Glycoproteins / Q000378:metabolism; D008564:Membrane Potentials; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D010798:Phycocyanin / Q000494:pharmacology; D011419:Propidium / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D051381:Rats; D020112:Rhodamine 123 / Q000494:pharmacology; D013997:Time Factors; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D015854:Up-Regulation; D019014:fas Receptor / Q000378:metabolism
D017304:Annexin A5; D004396:Coloring Agents; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D053222:Fas Ligand Protein; C505662:Faslg protein, rat; D008562:Membrane Glycoproteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; C002497:allophycocyanin; D019014:fas Receptor; D010798:Phycocyanin; D020112:Rhodamine 123; D011419:Propidium; D004317:Doxorubicin; C505528:Casp8 protein, rat; C506435:Casp9 protein, rat; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases