Mitochondrial localization of P-glycoprotein in the human breast cancer cell line MCF-7/ADM and its functional characterization

ArticleinOncology Reports 27(5):1535-40 · May 2012with7 Reads
DOI: 10.3892/or.2012.1671 · Source: PubMed
Abstract
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.
    • "The majority of the ABCB1 plasma membrane efflux pumps (Fu, 2013) are situated on cell border structures in resistant cell variants. However, a small fraction may be localized intracellularly in mitochondrial membranes or nuclear envelopes (Breier et al., 2014; Shen et al., 2012; Szaflarski et al., 2013). The efflux activity of ABCB1 in SKM-1/Aza cells was observed elsewhere in studies using the calcein-AM retention assay (Messingerova et al., 2015), and we showed that the resistance of SKM-1/AzaC cells to AzaC could not be reversed with the known ABCB1 inhibitor verapamil. "
    [Show abstract] [Hide abstract] ABSTRACT: We established an azacytidine (AzaC)-resistant human acute myeloid leukemia (AML) cell line (SKM-1/AzaC) by culturing SKM-1 cells in the presence of increasing amounts of AzaC for six months. Because AzaC is not a substrate of P-glycoprotein (a product of the ABCB1 gene; ABCB1), ABCB1 was not responsible for AzaC resistance; nevertheless, it was notably upregulated in SKM-1/AzaC cells. In addition, the transcription of the nfkb1 gene, which encodes a member of the canonical NF-kappaB regulatory pathway, was downregulated, and the transcription of the nfkb2 gene, which encodes a member of the non-canonical NF-kappaB regulatory pathway, was upregulated in SKM-1/AzaC cells. Here, we investigate whether miRNA-27a and miRNA-138 (both of which are known to be regulators of ABCB1 expression) are involved in the regulation of ABCB1 expression in SKM-1/AzaC cells. We observed decreased levels of miRNA-27a but of not miRNA-138 in SKM-1/AzaC cells compared with SKM-1 cells. The transfection of SKM-1/AzaC cells with a miRNA-27a mimic induced the downregulation of the ABCB1 mRNA. This was associated with an increase in nfkb1 and a decrease in nfkb2 transcript levels in SKM-1/AzaC cells. Taken together, these data indicate that the downregulation of miRNA-27a is involved in the upregulation of ABCB1 expression in SKM-1/AzaC cells, and this effect is associated with a switch between the canonical and non-canonical NF-kappaB pathways.
    Full-text · Article · Jul 2016
    • "P-gp1, the protein product of the MDR1 (multidrug resistance protein 1) gene, is responsible for pumping DOX out of the cells in an ATP-dependent manner to reduce the accumulation of DOX in cells, thus accounting partially for the mechanism of DOX re- sistance[20]. However, P-gp1 has also been identified to be expressed in the mitochondria and has a functional activity in the mitochondria of MDR cells212223. Besides acting as drug pump, P-gp1 was also reported to protect resistant tumor cells from caspase-dependent apoptosis in the mitochondria [24, 25], thus accounting for drug-resistance. "
    [Show abstract] [Hide abstract] ABSTRACT: Doxorubicin is a widely used chemotherapeutic agent for the treatment of a variety of solid tumors. However, resistance to this anticancer drug is a major obstacle to the effective treatment of tumors. As mitochondria play important roles in cell life and death, we anticipate that mitochondria may be related to drug resistance. Here, stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomic strategy was applied to compare mitochondrial protein expression in doxorubicin sensitive OVCAR8 cells and its doxorubicin-resistant variant NCI_ADR/RES cells. A total of 2085 proteins were quantified, of which 122 proteins displayed significant changes in the NCI_ADR/RES cells. These proteins participated in a variety of cell processes including cell apoptosis, substance metabolism, transport, detoxification and drug metabolism. Then qRT-PCR and western blot were applied to validate the differentially expressed proteins quantified by SILAC. Further functional studies with RNAi demonstrated TOP1MT, a mitochondrial protein participated in DNA repair, was involved in doxorubicin resistance in NCI_ADR/RES cells. Besides the proteomic study, electron microscopy and fluorescence analysis also observed that mitochondrial morphology and localization were greatly altered in NCI_ADR/RES cells. Mitochondrial membrane potential was also decreased in NCI_ADR/RES cells. All these results indicate that mitochondrial function is impaired in doxorubicin-resistant cells and mitochondria play an important role in doxorubicin resistance. This research provides some new information about doxorubicin resistance, indicating that mitochondria could be therapeutic targets of doxorubicin resistance in ovarian cancer cells.
    Full-text · Article · Sep 2014
    • "Additionally, MitoTrackerH Green FM was used as a fluorescent substrate in the ABCB1-mediated efflux assay. MitoTrackerH Green FM is a cell permeable, mitochondrial-selective probe that passively diffuses through cells and binds to mitochondrial proteins, enabling greenfluorescence detection via flow cytometry or fluorescent micros- copy [30,31] . To test their inhibitory activities on ABCB1- mediated efflux, XR9576, cyclosporin A, verapamil, and other compounds were diluted in culture medium and directly added to the cells before the addition of calcein AM or other fluorescent substrates. "
    [Show abstract] [Hide abstract] ABSTRACT: ABCB1, also known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), is a membrane-associated multidrug transporter of the ATP-binding cassette (ABC) transporter family. It is one of the most widely studied transporters that enable cancer cells to develop drug resistance. Reliable high-throughput assays that can identify compounds that interact with ABCB1 are crucial for developing new therapeutic drugs. A high-throughput assay for measuring ABCB1-mediated calcein AM efflux was developed using a fluorescent and phase-contrast live cell imaging system. This assay demonstrated the time- and dose-dependent accumulation of fluorescent calcein in ABCB1-overexpressing KB-V1 cells. Validation of the assay was performed with known ABCB1 inhibitors, XR9576, verapamil, and cyclosporin A, all of which displayed dose-dependent inhibition of ABCB1-mediated calcein AM efflux in this assay. Phase-contrast and fluorescent images taken by the imaging system provided additional opportunities for evaluating compounds that are cytotoxic or produce false positive signals. Compounds with known therapeutic targets and a kinase inhibitor library were screened. The assay identified multiple agents as inhibitors of ABCB1-mediated efflux and is highly reproducible. Among compounds identified as ABCB1 inhibitors, BEZ235, BI 2536, IKK 16, and ispinesib were further evaluated. The four compounds inhibited calcein AM efflux in a dose-dependent manner and were also active in the flow cytometry-based calcein AM efflux assay. BEZ235, BI 2536, and IKK 16 also successfully inhibited the labeling of ABCB1 with radiolabeled photoaffinity substrate [(125)I]iodoarylazidoprazosin. Inhibition of ABCB1 with XR9576 and cyclosporin A enhanced the cytotoxicity of BI 2536 to ABCB1-overexpressing cancer cells, HCT-15-Pgp, and decreased the IC50 value of BI 2536 by several orders of magnitude. This efficient, reliable, and simple high-throughput assay has identified ABCB1 substrates/inhibitors that may influence drug potency or drug-drug interactions and predict multidrug resistance in clinical treatment.
    Full-text · Article · Apr 2013
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