Mitochondrial localization of P-glycoprotein in the human breast cancer             cell line MCF-7/ADM and its functional characterization.
The current view of multidrug resisitance is that overexpression of membrane             P-glycoprotein (P-gp) is a major causative factor. However, the controversial             presence of subcellular P-gp may also participate in the drug resistance. In this             study, we sought to investigate the localization and functional characterization             of P-gp in mitochondria isolated from MCF-7 and doxorubicin-resistant MCF-7 (MCF-7/ADM)             cells. Mitochondria were isolated and purified from the MCF-7 cell line and its             resistant cells MCF-7/ADM. We used electron microscopy, western blot analysis             and confocal microscopy to demonstrate the localization of P-gp in the mitochondria             of MCF-7/ADM cells. Flow cytometry was used to evaluated the efflux function of             mitochondrial P-gp in the presence or absence of the P-gp inhibitor cyclosporine             A (CsA). Mitochondria were isolated and purified successfully and were analyzed             by electron microscopy. Western blotting demonstrated the expression of P-gp in             the cell membrane and purified mitochondria from MCF-7/ADM cells but not from             sensitive MCF-7 cells. Immunofluorescence analysis using confocal microscopy demonstrated             the localization of P-gp [labeled with green fluorescence (FITC)] to the mitochondria             [labeled with red fluorescence (Mitotracker Deep red 633)] of MCF-7/ADM cells             and that was absent in MCF-7 cells. Rho123 (a mitochondrial fluorescent probe)             accumulation was largely reduced and efflux was strongly increased in the mitochondria             of MCF-7/ADM cells compared to those of MCF-7 cells (P<0.01), and these were             completely reversed in the presence of the P-gp inhibitor CsA (P<0.01). No             significant changes were observed in the mitochondria of MCF-7 cells (P>0.05).             P-gp is expressed in the mitochondria of doxorubicin-resistant MCF-7 cells and             has an efflux function. It could be involved in multidrug resistance at the subcellular             site by pumping out anticancer drugs from mitochondria to protect the function             of mitochondria.
Department of Gynecology and Obstetrics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, PR China.
Yi
Y
Shen
Yan
Y
Chu
Yan
Y
Yang
Zehua
Z
Wang
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism*; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm; D005260:Female; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D021381:Protein Transport
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1