P-glycoprotein mediates drug resistance via a novel mechanism involving lysosomal sequestration

Abstract

Localization of the drug transporter P-glycoprotein (Pgp) to the plasma membrane is thought to be the only contributor of Pgp-mediated multidrug resistance (MDR). However, very little work has focused on the contribution of Pgp expressed in intracellular organelles to drug resistance. This investigation describes an additional mechanism for understanding how lysosomal Pgp contributes to MDR. These studies were performed using Pgp-expressing MDR cells and their non-resistant counterparts. Using confocal microscopy and lysosomal fractionation, we demonstrated that intracellular Pgp was localized to LAMP2-stained lysosomes. In Pgp-expressing cells, the Pgp substrate doxorubicin (DOX) became sequestered in LAMP2-stained lysosomes, but this was not observed in non-Pgp-expressing cells. Moreover, lysosomal Pgp was demonstrated to be functional because DOX accumulation in this organelle was prevented upon incubation with the established Pgp inhibitors valspodar or elacridar or by silencing Pgp expression with siRNA. Importantly, to elicit drug resistance via lysosomes, the cytotoxic chemotherapeutics (e.g. DOX, daunorubicin, or vinblastine) were required to be Pgp substrates and also ionized at lysosomal pH (pH 5), resulting in them being sequestered and trapped in lysosomes. This property was demonstrated using lysosomotropic weak bases (NH4Cl, chloroquine, or methylamine) that increased lysosomal pH and sensitized only Pgp-expressing cells to such cytotoxic drugs. Consequently, a lysosomal Pgp-mediated mechanism of MDR was not found for non-ionizable Pgp substrates (e.g. colchicine or paclitaxel) or ionizable non-Pgp substrates (e.g. cisplatin or carboplatin). Together, these studies reveal a new mechanism where Pgp-mediated lysosomal sequestration of chemotherapeutics leads to MDR that is amenable to therapeutic exploitation.

Keywords: Chemical Biology; Chemoresistance; Chloroquine; Doxorubicin; Drug Resistance; Drug Transport; Lysosomes; Multidrug Resistance; P-glycoprotein.

FIGURE 1.

Functional Pgp causes DOX resistance. A, Western blot analysis showing that Pgp is only expressed in KBV1 (+Pgp) and not in KB31 (−Pgp) cells. β-Actin was used as a loading control. B, flow cytometry demonstrates higher plasma membrane expression of Pgp in KBV1 (+Pgp) cells than KB31 (−Pgp) cells. Cells were not permeabilized to prevent intracellular staining of Pgp prior to flow cytometric analysis. C, Rh123 accumulation is decreased in KBV1 (+Pgp) control cells compared with KB31 (−Pgp) control cells, whereas Pgp inhibition increases Rh123 accumulation only in KBV1 (+Pgp) cells. Briefly, cells were preincubated with either control medium or the Pgp inhibitors Val (1 μm) or Ela (0.1 μm) for 30 min at 37 °C. Cells were then loaded with Rh123 (1 μg/ml) for 15 min at 37 °C in the presence or absence of these inhibitors. D, Pgp inhibitors increase [¹⁴C]DOX uptake only in KBV1 (+Pgp) cells. Briefly, KBV1 (+Pgp) or KB31 (−Pgp) cells were preincubated with either control medium or the Pgp inhibitors Val (1 μm) or Ela (0.1 μm) for 30 min at 37 °C. [¹⁴C]DOX was then added, the incubation continued for 30 min at 37 °C, and then the cells were washed. E and F, Pgp inhibitors block efflux of [¹⁴C]DOX only from KBV1 (+Pgp) cells. Briefly, cells were labeled with [¹⁴C]DOX for 30 min at 37 °C, washed, and then reincubated for up to 60 min at 37 °C in the presence or absence of the Pgp inhibitors Val (1 μm) or Ela (0.1 μm). G, DOX cytotoxicity (IC₅₀/72 h) is potentiated by the Pgp inhibitors Val (1 μm) or Ela (0.1 μm) in KBV1 (+Pgp) cells but not KB31 (−Pgp) cells. The results in A–C are representative of three experiments, whereas those in D–G are mean ± S.D. (three experiments with at least four replicates in each experiment). ***, p < 0.001 versus control.

FIGURE 2.

Intracellular Pgp is localized to lysosomes in KBV1 (+Pgp) cells. A, Pgp colocalizes with lysosomal-associated membrane protein 2 (LAMP2, a well characterized lysosomal marker, arrows), whereas no colocalization was observed with DAPI (nuclear marker) (B) or MitoTracker® Deep Red (Mito Dred, mitochondrial marker) (C). The scatter plots, Pearson's correlation coefficient, and Mander's overlap coefficient in A–C were calculated using Imaris and Axiovision software from intracellular compartments of all cells. Photographs are typical from three experiments (A–C). Scale bars = 50 μm.

FIGURE 3.

Lysosomal KBV1 (+Pgp) cell fraction is enriched with Pgp. A, lysosome-enriched fractions of KBV1 (+Pgp) cells demonstrate significantly (p < 0.001) higher acid phosphatase activity compared with total cell lysates. B, Western blot analysis of the enriched lysosomal fraction in A demonstrating that the lysosomal fraction was highly enriched with Pgp. However, this lysosomal fraction did not contain histone deacetylase 1 (HDAC1) from the nucleus and had a similar level of mitochondrial SDHA as the total cell lysate. Western blot analyses in A and B are mean ± S.D. (three experiments). ***, p < 0.001 versus control.

FIGURE 4.

Colocalization of DOX with LAMP2-stained lysosomes is Pgp-dependent. A–D, in KB31 (−Pgp) cells, colocalization of DOX with the nuclear marker DAPI was demonstrated under control conditions. E–L, the Pgp inhibitors Val (E–H) and Ela (I–L) did not affect DOX distribution relative to the control in KB31 (−Pgp) cells. M–P, incubation of KBV1 (+Pgp) cells expressing functional Pgp with DOX leads to colocalization of this drug with the marker for lysosomes, LAMP2 (P, arrows), whereas the Pgp inhibitors Val (Q–T) and Ela (U–X) result in DOX redistribution to the nucleus. The results in A–X are typical from three experiments. Scale bars = 50 μm.

FIGURE 5.

Functional silencing of Pgp in KBV1 (+Pgp) cells results in redistribution of DOX from LAMP2-stained lysosomes to DAPI-stained nuclei with concomitant toxicity. A, Western blot analysis showing transient silencing of Pgp in KBV1 (+Pgp) cells incubated with two types of Pgp siRNA (72 h, 37 °C) relative to KBV1 (+Pgp) cells treated with Scr siRNA. B, Pgp silencing in KBV1 (+Pgp) cells using a 72-h incubation with siRNA resulted in increased cytotoxicity (IC₅₀/72 h) of DOX relative to KBV1 (+Pgp) cells treated with Scr siRNA. The Pgp inhibitor Ela (0.1 μm) increased cytotoxicity of DOX only in Scr siRNA-treated KBV1 (+Pgp) cells relative to Pgp siRNA-treated (Pgp-silenced) cells (72 h, 37 °C). C, siRNA silencing of Pgp (72 h, 37 °C) resulted in redistribution of DOX to nuclei, as indicated by DAPI staining, whereas Scr siRNA-treated KBV1 (+Pgp) cells resulted in accumulation of DOX in LAMP2-stained lysosomes (arrow). The results in A and C are typical from three experiments. The graph in B is mean ± S.D. ***, p < 0.001 versus control (i.e. drug alone). Scale bar = 50 μm.

FIGURE 6.

Pgp in lysosomes confers drug resistance to DOX. A, speciation plot of DOX derived from pK_a values showing that 100% of DOX is charged at lysosomal pH (pH ∼5) (9). B and C, the lysosomotropic weak bases ammonium chloride (NH₄Cl, 3 mm), CLQ (1 μm), and MA (100 μm) increase sensitivity (IC₅₀/72 h) to DOX in KBV1 (+Pgp) cells but not in KB31 (−Pgp) cells. D, Western blot demonstrating significantly (p < 0.001) higher expression of Pgp in 2008/P200 (+Pgp) cells relative to 2008 (−Pgp) cells. E, flow cytometry was conducted to confirm the higher expression of plasma membrane Pgp in 2008/P200 (+Pgp) cells relative to 2008 (−Pgp) cells. F, Rh123 accumulation is decreased in control 2008/P200 (+Pgp) cells compared with control 2008 (−Pgp) cells, whereas Pgp inhibition increases Rh123 accumulation only in 2008/P200 (+Pgp) cells. Cells were preincubated with either control medium or the Pgp inhibitors Val (1 μm) or Ela (0.1 μm) for 30 min at 37 °C. Cells were then loaded with Rh123 (1 μg/ml) for 15 min at 37 °C in the presence or absence of these inhibitors. G and H, the lysosomotropic weak bases used in B and C also sensitized 2008/P200 (+Pgp) cells to DOX but not 2008 (−Pgp) cells (IC₅₀/72 h). The plot in A was generated using Hyperquad2008. The results in B–H are from three experiments. ***, p < 0.001 versus control (i.e. DOX alone).

FIGURE 7.

Lysosomal Pgp increases lysosomal trapping of the Pgp substrate DOX, thereby preventing DOX from reaching its nuclear targets. KBV1 (+Pgp) cells expressing functional Pgp led to colocalization of DOX (red) with the marker for lysosomes, LAMP2 (green), leading to yellow fluorescence after the image merge (arrows). The addition of lysosomotropic weak bases to KBV1 (+Pgp) cells redistributed DOX (red) from the lysosomes (green, LAMP2) to the nucleus (blue, DAPI), leading to purple fluorescence of the nuclei in the merged image. Confocal microscopy results are typical from three experiments. Scale bar = 50 μm.

FIGURE 8.

Drug resistance conferred by Pgp is dependent on the charge of Pgp substrates at acidic pH. A, lysosomal trapping ability of a range of Pgp substrates as shown by speciation plots (derived from pK_a values of each substrate (9, 11, 12, 29)). At lysosomal pH (pH ∼5), DOX (100%), DNR (100%), and VBL (71%) are charged, whereas COL and PAC are not. Incubation (IC₅₀/72 h) of KB31 (−Pgp) cells (B) or 2008 (−Pgp) cells (C) with the lysosomotropic weak bases ammonium chloride (NH₄Cl, 3 mm), CLQ (1 μm), or MA (100 μm) did not significantly change cytotoxicity to the Pgp substrates DOX, DNR, VBL, COL, or PAC. However, incubation of Pgp-expressing KBV1 (+Pgp) cells (B) or 2008/P200 (+Pgp) cells (C) with the lysosomotropic weak bases markedly increased sensitivity to the Pgp substrates DOX, DNR, and VBL but not to COL or PAC. The graphs (B and C) show mean ± S.D. ***, p < 0.001 versus control (i.e. drug alone).

FIGURE 9.

Lysosomotropic weak bases have no effect on the cytotoxicity of drugs that are not Pgp substrates. Incubation of KB31 (−Pgp) or KBV1 (+Pgp) cells with lysosomotropic weak bases has no effect on the cytotoxicity of cisplatin (A and B) or carboplatin (C and D), both of which are not Pgp substrates (32). Cells were incubated for 72 h at 37 °C with cisplatin or carboplatin in the presence or absence of the lysosomotropic weak bases ammonium chloride (NH₄Cl, 3 mm), CLQ (1 μm), or MA (100 μm). Proliferation was then assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results shown are mean ± S.D. (three experiments).

FIGURE 10.

Lysosomal trapping by Pgp as an alternative multidrug resistance mechanism. A, as part of endocytosis, the plasma membrane containing Pgp buds inwards to form early endosomes. During endocytosis, the topology of Pgp will be inverted, as shown for other membrane proteins (45), leading to the transport of substrates into the vesicle lumen. As the endosome matures into a lysosome, it becomes increasingly acidified. The Pgp on the lysosomal membrane remains functional because its catalytic active sites and ATP-binding domains are still exposed in the cytosol (39, 40). When a Pgp substrate, such as DOX, enters the cell, the drug is not only effluxed out of the cell by Pgp on the plasma membrane but also sequestered into the acidic lysosomes by lysosomal Pgp pumps. If the Pgp substrate is charged at acidic pH (such as DOX), then lysosomal trapping occurs. The trapping of charged drugs will prevent Pgp substrates from reaching their molecular targets (e.g. the nucleus for DOX), leading to increased resistance in Pgp-expressing cells. B, this study describes two mechanisms to overcome lysosomal Pgp-dependent multidrug resistance: direct blocking of Pgp by Pgp inhibitors, leading to prevention of increased uptake of Pgp substrates into lysosomes (1), or the combination of cytotoxic drugs (e.g. DOX) with lysosomotropic weak bases (e.g. CLQ) to prevent lysosomal trapping by raising lysosomal pH (2). These two approaches offer Pgp substrates an opportunity to overcome multidrug resistance by reaching their targets instead of becoming trapped in Pgp-containing lysosomes.