Differential regulation of doxorubicin-induced mitochondrial dysfunction and apoptosis by Bcl-2 in mammary adenocarcinoma (MTLn3) cells

被引:89
作者
Huigsloot, M [1 ]
Tijdens, IB [1 ]
Mulder, GJ [1 ]
de Water, BV [1 ]
机构
[1] Leiden Univ, LACDR, Div Toxicol, NL-2300 RA Leiden, Netherlands
关键词
D O I
10.1074/jbc.M200378200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 (Atk) 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-DLAsp-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 Bcl2-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 Bcl2-controlled checkpoint.
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收藏
页码:35869 / 35879
页数:11
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