Subcellular Detection and Localization of the Drug Transporter P-Glycoprotein in Cultured Tumor Cells

Molinari, Agnese; Calcabrini, Annarica; Meschini, Stefania; Stringaro, Annarita; Crateri, Pasqualina; Toccacieli, Laura; Marra, Manuela; Colone, Marisa; Cianfriglia, Maurizio; Arancia, Giuseppe

doi:10.2174/1389203023380413

Cited by 73 publications

(64 citation statements)

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“…Recently, the ATP-binding cassette transporter family member proteins that are strongly connected to the drug resistance phenotype, such as P-glycoprotein, MRP1, and BCRP, have been localized to intracellular membranes. Such findings further highlight the relevance and importance of the sequestration mechanism in drug resistance (25,26).…”

Section: Introductionmentioning

confidence: 61%

Depletion of major vault protein increases doxorubicin sensitivity and nuclear accumulation and disrupts its sequestration in lysosomes

Herlevsen

Oxford

Owens

et al. 2007

Molecular Cancer Therapeutics

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The major vault protein (MVP) is the major constituent of the vault particle, the largest known ribonuclear protein complex. To date, vaults have no clear function, although their low expression levels in de novo chemosensitive and curable tumors, such as testicular cancer, make them attractive candidates as contributors to intrinsic drug resistance. Here, we show that MVP knockdown in human bladder cancer cells via small interfering RNA results in sensitization toward doxorubicin in two distinct exposure protocols. The drug was detected in the nucleus immediately following addition and was subsequently sequestered to lysosomes, predominantly located adjacent to the nucleus. MVP knockdown leads to increased sensitivity toward doxorubicin and an enhanced nuclear accumulation of the drug as well as a loss of its perinuclear sequestration. Not only doxorubicin subcellular distribution was perturbed by MVP knockdown but lysosomal markers, such as pH-sensitive LysoSensor, pinocytosed dextran conjugates after 24-h chase period, and the lysosomal specific antigen Lamp-1, also showed a markedly different staining compared with controls. Lysosomes appeared dispersed through the cytoplasm without a clear organization adjacent to the nucleus. Microtubules, however, appeared unperturbed in cells with reduced MVP expression. Based on these data, we hypothesize that MVP and, by extension, vault complexes are important for lysosomal function and may influence cellular drug resistance by virtue of this role. [Mol Cancer Ther 2007;6(6):1804 -13]

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Section: Introductionmentioning

confidence: 61%

Depletion of major vault protein increases doxorubicin sensitivity and nuclear accumulation and disrupts its sequestration in lysosomes

Herlevsen

Oxford

Owens

et al. 2007

Molecular Cancer Therapeutics

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“…Nevertheless, some studies have demonstrated its localization in intracellular sites such as the nuclear envelope and in the membrane of cytoplasmatic organelles (11). These studies suggest that Pgp function is related to cellular protection against toxic agents, independently of its localization.…”

Section: Introductionmentioning

confidence: 99%

P-glycoprotein and survivin simultaneously regulate vincristine-induced apoptosis in chronic myeloid leukemia cells

Souza

Vasconcelos²,

Reis³

et al. 2011

Int J Oncol

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Abstract. Overexpression of P-glycoprotein (Pgp/ABCB1) in tumor cells is associated with a classic phenotype of multidrug resistance (MDR). Moreover, some members of the inhibitor of apoptosis protein (IAP) family, such as survivin, contribute to an apoptosis-resistant phenotype, by inhibiting chemotherapyinduced cell death and promoting MDR. By using Western blotting, qRT-PCR, Annexin V and immunofluorescence assays we have demonstrated a relationship between Pgp and survivin in a prior sensitive chronic myeloid leukemia (CML) cell line (K562). A high dose of vincristine induced a concomitant overexpression of Pgp and survivin, which was associated with a low apoptotic index in the K562 cell line. In addition, we observed a cytoplasmic co-localization of Pgp and survivin, suggesting a functional association between these two proteins in apoptosis control by a common mechanism. In summary, our data suggest that Pgp and survivin should be analyzed in aggregate because they may have significant impact on drug resistance in CML cells.

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“…Previous studies of P-gp have mainly focused on the expression of plasma membrane P-gp. However, recent studies have shown that P-gp expression is observed at the intracellular membranes in tumor-resistant cells, including the nucleus, Golgi apparatus, and the some unconfirmed cytoplasmic vesicles such as lysosomes and inclusion bodies (2)(3)(4). Furthermore, intracellular levels of P-gp are roughly equivalent to that of the plasma membrane P-gp.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2012

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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.

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Subcellular Detection and Localization of the Drug Transporter P-Glycoprotein in Cultured Tumor Cells

Cited by 73 publications

References 0 publications

Depletion of major vault protein increases doxorubicin sensitivity and nuclear accumulation and disrupts its sequestration in lysosomes

Depletion of major vault protein increases doxorubicin sensitivity and nuclear accumulation and disrupts its sequestration in lysosomes

P-glycoprotein and survivin simultaneously regulate vincristine-induced apoptosis in chronic myeloid leukemia cells

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

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