P-glycoprotein Mediates Drug Resistance via a Novel Mechanism Involving Lysosomal Sequestration*

Background: 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). Results: Lysosomal Pgp sequesters ionizable chemotherapeutics into lysosomes to prevent interaction with molecular targets, resulting in drug resistance. Conclusion: Lysosomal Pgp mediates drug resistance. Significance: Pgp-mediated sequestration of chemotherapeutics into lysosomes can be exploited pharmacologically. 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.


diated lysosomal sequestration of chemotherapeutics leads to MDR that is amenable to therapeutic exploitation.
Cellular mechanisms of cancer multidrug resistance (MDR) 5 have been studied extensively because they constitute a major factor to the reduced efficacy of many chemotherapeutics (1). One of the best-characterized mechanisms of MDR occurs via drug pumps that actively efflux various cytotoxic compounds from cells for cytoprotection (1).
These latter molecules include the well studied P-glycoprotein (Pgp) (1). Although the mechanism of action of Pgp on the plasma membrane is well characterized (1), drug resistance mediated by intracellular Pgp has not been comprehensively investigated. Indeed, there are conflicting reports on the distribution and roles of intracellular Pgp in MDR (2)(3)(4).
In this study, we demonstrate that Pgp in lysosomes actively sequesters the Pgp substrate doxorubicin (DOX) into these organelles. Furthermore, incubation of cells with the potent Pgp inhibitors valspodar (Val) or elacridar (Ela) or silencing of Pgp with siRNA inhibited sequestration of DOX in lysosomes and led to its redistribution to its primary target, namely DNA in the nucleus. Significantly, lysosomal Pgp conferred drug resistance, and this could be overcome by lysosomotropic weak bases that induced a marked increase in DOX cytotoxicity, an effect only observed in Pgp-positive cells. We also showed that only Pgp substrates that become charged at acidic lysosomal pH were capable of conferring lysosomal Pgp-dependent drug resistance. For the first time, we present a novel mechanism of MDR mediated by intracellular Pgp in lysosomes.

EXPERIMENTAL PROCEDURES
Cell Culture-The human cervical carcinoma derived KB-3-1 (KB31) cell line was obtained from the ATCC, and the vinblastine (VBL)-resistant variant KB-V-1 (KBV1, grown in VBL at 1 g/ml) was a gift from Dr. Maria Kavallaris (Children's Cancer Institute Australia, Sydney, Australia). The 2008 human ovarian carcinoma cell line and the paclitaxel (PAC)-resistant 2008/ P200 cell line (grown in PAC at 200 ng/ml) were provided by Dr. John Allen (Centenary Institute, Sydney, Australia). All cell lines were grown in DMEM (Invitrogen) under standard growth conditions (5).
Flow Cytometry-For extracellular Pgp staining, cells were incubated with FITC-conjugated Pgp antibody (1:100, BD Biosciences, East Rutherford, NJ) for 30 min on ice and washed four times with ice-cold PBS. The cells were then removed from the plate using 1 mM EDTA/PBS (pH 7.4), centrifuged, and resuspended in ice-cold PBS for flow cytometric analysis. Notably, the cells were not permeabilized to prevent intracellular staining of Pgp.
For the rhodamine 123 (Rh123, Sigma-Aldrich) accumulation assay, cells were preincubated with either control medium or the well known Pgp inhibitors Val (1 M, Novartis) or Ela (0.1 M, GlaxoSmithKline) for 30 min. The medium was then aspirated, and the cells were loaded with Rh123 (1 g/ml) using a 15-min/37°C incubation in the presence of control medium alone or the Pgp inhibitors. The overlying medium was subsequently removed, and the cells were detached using 1 mM EDTA/PBS (pH 7.4) and centrifuged. Then the cell pellet was resuspended in ice-cold PBS for flow cytometric analysis.
FITC-conjugated Pgp and Rh123 were detected with a FACSCanto flow cytometer (BD Biosciences), and 10,000 events were acquired for every sample. Data analysis was performed using FlowJo software, version 7.5.5 (Tree Star Inc., Ashland, OR).
Lysosomal Fractionation-Lysosomally enriched fractions were prepared from cultured cells using the lysosome enrichment kit (Pierce Biotechnology, Waltham, MA). The lysosomespecific enzyme marker acid phosphatase (Sigma-Aldrich) (8) showed that this fraction was lysosome-enriched, as determined by spectrophotometric assessment at 405 nm. The purity of the fractions was also assessed using antibodies (described under "Western Blot and Antibodies") against LAMP2 (lysosomes), SDHA (mitochondria), and HDAC1 (nuclei).
Calculation of Speciation Plots-Speciation plots were prepared using published pK a values derived from potentiometric titration data (9 -12). Hyperquad2008 software (Protonic Software, Leeds, UK) was used to generate speciation plots from these pK a values.
Statistics-Data were compared using Student's t test. Results were expressed as mean Ϯ S.D. (number of experiments) and considered to be statistically significant when p Ͻ 0.05.

RESULTS
Pgp Protects Cells from Cytotoxic Pgp Substrates-To understand the role of intracellular Pgp in MDR, Pgp expression and functionality were initially assessed in the well known KBV1 (ϩPgp)/KB31 (ϪPgp) drug resistance cell model (13,14). We showed that KBV1 (ϩPgp) cells cultured with VBL (13) expressed high Pgp levels compared with parental KB31 (ϪPgp) cells without VBL selection, as shown by Western blotting (Fig. 1A) and flow cytometry (Fig. 1B).
To assess Pgp function, flow cytometric studies then progressed to examining the cellular accumulation of the fluorescent Pgp substrate Rh123 (13) in KBV1 (ϩPgp) cells relative to KB31 (ϪPgp) cells (Fig. 1C). In these studies, KB31(ϪPgp) and KBV1 (ϩPgp) cells were either preincubated for 30 min at 37°C with control medium or medium containing the well characterized Pgp inhibitors Val (1 M) or Ela (0.1 M) (15). The cells were then loaded with Rh123 for 15 min at 37°C in the presence or absence of these inhibitors. These experiments demonstrated significantly (p Ͻ 0.001) greater Rh123 accumulation (as measured by Rh123 fluorescence) in control KB31 (ϪPgp) cells relative to control KBV1 (ϩPgp) cells (Fig. 1C). This observation was in accordance with the expression of Pgp in KBV1 (ϩPgp) cells (Fig. 1, A and B), which actively effluxes Rh123 from cells (13). Hence, high Pgp levels in KBV1 cells lead to pronounced Rh123 efflux, resulting in lower cellular accumulation of this substrate. Notably, incubation with Ela or Val had no effect on Rh123 fluorescence in KB31 (ϪPgp) cells but caused a marked increase in Rh123 in KBV1 (ϩPgp) cells (Fig.  1C). This finding is consistent with the efficacy of these inhibitors at preventing Rh123 efflux from KBV1 (ϩPgp) cells via Pgp, leading to its cellular accumulation (Fig. 1C).
The functionality of Pgp in KBV1 (ϩPgp) cells was further substantiated by the reduced uptake ( Fig. 1D) and increased efflux (E and F) of the 14 1D). In contrast, in KBV1 (ϩPgp) cells, both Val and Ela resulted in a marked and significant (p Ͻ 0.001) increase in cellular [ 14 C]DOX levels relative to incubation with [ 14 C]DOX alone (Fig. 1D). This finding was consistent with the Pgp inhibitors preventing release of [ 14 C]DOX from the cell, leading to its accumulation. It is also notable that [ 14 C]DOX levels in control KB31 (ϪPgp) cells were significantly (p Ͻ 0.001) higher than those in KBV1 (ϩPgp) cells (Fig. 1D), and, again, this was con-  Val or Ela significantly (p Ͻ 0.001) reduced [ 14 C]DOX efflux by KBV1 (ϩPgp) cells (Fig. 1F). Collectively, these studies demonstrate the functionality of Pgp transport activity in KBV1 (ϩPgp) cells relative to KB31 (ϪPgp) cells.
Considering the decreased uptake of [ 14 C]DOX ( Fig. 1D) because of the Pgp-mediated increase in its efflux from KBV1 (ϩPgp) cells (Fig. 1F), studies then examined the cytotoxicity of DOX in the presence and absence of the Pgp inhibitors Val and Ela in KB31 (ϪPgp) and KBV1 (ϩPgp) cells over 72 h at 37°C (Fig. 1G). Because of the absence of Pgp in KB31 (ϪPgp) cells, the cytotoxic activity of DOX was marked in either the presence or absence of the Pgp inhibitors, leading to an IC 50 of Ͻ 0.5 M (Fig. 1G). In contrast, KBV1 (ϩPgp) cells were significantly (p Ͻ 0.001) more resistant to DOX than KB31 (ϪPgp) cells, with KBV1 (ϩPgp) cells having an IC 50 of 96.4 Ϯ 10.0 M because of Pgp expression (Fig. 1G). Additionally, both Pgp inhibitors significantly (p Ͻ 0.001) sensitized KBV1 (ϩPgp) cells to DOX, resulting in a marked decrease in the IC 50 value (Fig. 1G). These data show that Pgp is functional and protects KBV1 (ϩPgp) cells from DOX cytotoxicity.
Intracellular Localization of Pgp in Lysosomes-Having carefully established Pgp functionality in KBV1 (ϩPgp) cells, we then investigated mechanisms of MDR involving intracellular Pgp. The subcellular localization of Pgp was initially examined using confocal microscopy implementing well characterized organelle-specific antibodies or dyes, namely LAMP2 for lysosomes (17,18), DAPI for nuclei (19), and MitoTracker Deep Red (Mito DRed) for mitochondria (18). In agreement with the [ 14 C]DOX efflux studies described above (Fig. 1F), Pgp was identified on the plasma membrane of KBV1 (ϩPgp) cells (Fig.  2, A-C). In addition, intracellular Pgp showed a punctate pattern of Pgp staining ( Fig. 2A). This Pgp staining colocalized with the lysosomal marker LAMP2, leading to a yellow punctate pattern in the merge ( Fig. 2A, arrows).
Analysis of these images using scatter plots, Pearson's correlation coefficient (20) (0.936), and Mander's overlap coefficient (20) (0.925) also validated the colocalization of Pgp and LAMP2 ( Fig. 2A). No significant (p Ͼ 0.05) colocalization of Pgp was observed with nuclei ( Fig. 2B, DAPI) or the mitochondria (C, Mito DRed). Indeed, scatter plots, Pearson's correlation coefficient (0.086 and 0.38 for nuclei and mitochondria, respectively), and Mander's overlap coefficient (0.377 and 0.493 for nuclei and mitochondria, respectively) further verified these observations (Fig. 2, B and C). These investigations suggested that intracellular Pgp was localized to lysosomes and not to mitochondria or nuclei.
To further assess Pgp localization in lysosomes, subcellular fractionation was performed using KBV1 (ϩPgp) cells. The activity of lysosome-specific acid phosphatase (21) was used as a marker to assess the purity of the KBV1 (ϩPgp) lysosomal  (Fig. 3B). Hence, the marked enrichment of Pgp in the lysosomal fraction is not from mitochondria because the SDHA level was similar between the lysosomal and total fractions (Fig. 3B). Collectively, these Pgp localization studies and fractionation results indicate that intracellular Pgp is localized to lysosomes but not mitochondria or nuclei.
Functional Assessment of Pgp in Lysosomes-To assess the functionality of lysosomal Pgp, intracellular trafficking and localization of the intrinsically fluorescent Pgp substrate DOX (24) was examined by confocal microscopy. Incubation of DOX with KB31 (ϪPgp) cells led to its nuclear accumulation, as demonstrated by merging DOX (Fig. 4A) with DAPI (Fig. 4C), forming a purple colocalization pattern in the merge (Fig. 4D). This is expected because DOX intercalates within its primary molecular target, DNA (25,26). However, no DOX was found to colocalize with lysosomal LAMP2 in KB31 (ϪPgp) cells (Fig. 4, A, B, and D), and the Pgp inhibitors Val or Ela did not alter DOX localization in the nuclei (Fig. 4, E-L). In clear contrast, DOX colocalized (Fig. 4M) primarily with LAMP2 in KBV1 (ϩPgp) cells (Fig. 4N), leading to yellow fluorescence in the merge (Fig.  4P, arrows). This observation demonstrates that the pronounced Pgp expression in KBV1 (ϩPgp) cells results in marked accumulation of DOX in LAMP2-stained lysosomes. Interestingly, in KBV1 (ϩPgp) cells, the redistribution of DOX (Fig. 4, Q and U) to nuclei (Fig. 4, S and W) was observed in the presence of the Pgp inhibitors, as shown by the purple nuclear pattern in the merge (Fig. 4, T and X). These results indicate that inhibition of Pgp activity using Val or Ela prevented the uptake of DOX into the lysosomal compartment and resulted in the redirection of DOX to nuclei.
Critically, Val and Ela have been suggested to neutralize lysosomal pH and induce lysosomal swelling (27). To assess whether this effect may be relevant to our results, the size of the cell (forward scatter) and its granularity (side scatter) were analyzed by flow cytometry because changes in side scatter denote lysosomal swelling and lysosomotropism (27,28). Importantly, no change in side scatter in KB31 (ϪPgp) and KBV1 (ϩPgp) cells was observed at the concentrations of Val and Ela utilized in our experiments (data not shown). Hence, under the conditions implemented in this investigation, Val and Ela inhibited Pgp without exhibiting lysosomotropic properties.
To further determine the functionality of lysosomal Pgp, transient silencing of Pgp in KBV1 (ϩPgp) cells with two different types of Pgp siRNA was employed (Fig. 5). Silencing of Pgp by siRNA significantly (p Ͻ 0.001) decreased Pgp expression (Fig. 5A) and also significantly (p Ͻ 0.001) sensitized KBV1  (ϩPgp) cells to DOX relative to the DOX-treated Scr siRNA control (Fig. 5B). Notably, the sensitization of KBV1 (ϩPgp) cells to DOX by Pgp siRNA is due to the loss of Pgp that actively effluxes this cytotoxic agent.
These Pgp siRNA-and Scr siRNA-treated cells were then used to assess the Pgp-dependent sequestration of DOX into lysosomes (Fig. 5C). In Scr siRNA-treated cells that markedly express Pgp (Fig. 5A), DOX was found to primarily colocalize with LAMP2-stained lysosomes, leading to a yellow punctate pattern in the merge (C, arrow). However, in contrast, Pgp silencing did not result in colocalization of DOX and LAMP2 (Fig. 5C). Instead, Pgp siRNA-treated cells led to nuclear localization of DOX, resulting in a purple nuclear pattern in the merge (Fig. 5C). Together, the studies in Figs. 4 and 5 demonstrate that lysosomal sequestration of DOX requires both Pgp expression and function, which can be prevented by using Pgp inhibitors or Pgp siRNA.
Pgp in Lysosomes Confers Drug Resistance-Next, we assessed whether lysosomal Pgp can confer drug resistance via DOX sequestration. From analysis of DOX pK a data (9), it was found that DOX was fully charged at the acidic pH of the lysosome (29) (i.e. pH ϳ5, Fig. 6A) and, consequently, would become trapped in acidic lysosomes (16). This trapping occurs because the charged drug cannot cross the lysosomal membrane. This observation is in accordance with the general principle that the passage of charged molecules across membranes is impeded relative to their neutral counterparts (16). Hence, to disrupt lysosomal DOX trapping, lysosomal pH was neutralized with the well known lysosomotropic weak bases ammonium chloride (NH 4 Cl), CLQ, or MA (30). Thus, after neutralization of lysosomes using these agents (30), a significant proportion of DOX would become neutral, as shown by the speciation plot (Fig. 6A). Hence, the neutral species of DOX should then pass through the lysosomal membrane to access the nuclei and become associated with one of its major molecular targets, namely DNA (25,26).
Incubation with these lysosomotropic weak bases did not significantly (p Ͼ 0.05) alter DOX toxicity in KB31 (ϪPgp) cells (Fig. 6B). In clear contrast, lysosomotropic weak bases significantly (p Ͻ 0.001) increased DOX cytotoxicity in KBV1 (ϩPgp) cells by 27-to 50-fold (Fig. 6C). These observations suggest that, in cells without Pgp, DOX does not accumulate in lysosomes and that, hence, the lysosomotropic weak bases have no effect on the cytotoxicity of this drug. In contrast, in cells expressing Pgp, neutralization of lysosomal pH using lysosomotropic weak bases leads to relocalization of DOX from lysosomes to DNA in the nucleus (25,26), leading to cell death.
Examining the effect of lysosomotropic weak bases on the 2008 (ϪPgp) cells demonstrated that they did not significantly (p Ͼ 0.05) affect the cytotoxicity of DOX (Fig. 6G). In contrast, the three lysosomotropic weak bases significantly (p Ͻ 0.001) increased cytotoxicity in 2008/P200 (ϩPgp) cells (Fig. 6H), as found for KBV1 (ϩPgp) cells (Fig. 6C). Hence, the lysosomo-tropic weak bases sensitized both Pgp-expressing cell types to DOX but not their non-Pgp-expressing counterparts.

Lysosomal Pgp Increases Lysosomal Trapping of the Pgp Substrate DOX, Preventing This Cytotoxic Agent from Reaching Its
Nuclear Targets-The potential Pgp-dependent sequestration of DOX into lysosomes was then further assessed using confocal microscopy (Fig. 7). In KBV1 (ϩPgp) cells treated with DOX alone, colocalization of DOX with LAMP2 was observed, leading to yellow fluorescence in the merged image (Fig. 7, arrows). This observation suggested the functional role of Pgp in lysosomes, as also shown in Figs. 4, M-P, and 5C. Interestingly, incubation with lysosomotropic weak bases (i.e. NH 4 Cl, CLQ, or MA) effectively inhibited DOX accumulation in LAMP2stained lysosomes, as shown by the green fluorescence in the merge (Fig. 7). Instead, DOX localized within nuclei, as shown by the purple nuclear pattern in the merge (Fig. 7). Together with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay cytotoxicity data (Fig. 6, B, C, G, and H), these results demonstrate that Pgp-dependent accumulation of DOX in LAMP2-stained lysosomes (Fig. 7) contributes to drug resistance.

Drug Resistance Conferred by Pgp Is Dependent on the Charge of Pgp Substrates at Acidic Lysosomal pH-
The results in Figs. 6 and 7 indicate that the charged species of DOX are important for lysosomally mediated cytoprotection. Considering this, we assessed whether this cytoprotective effect occurs with other common Pgp substrates (Fig. 8). This was examined by assessing the ionization of a range of cytotoxic drugs and then determining whether the lysosomotropic weak bases could overcome Pgp-mediated drug resistance.
First, the distribution of ionized species derived from pK a data (9,11,12) was plotted as a function of pH for the cytotoxic drugs DOX (pK a , 8.2), daunorubicin (DNR) (pK a , 8.4), VBL (pK a , 5.4, 7.4), colchicine (COL) (pK a , 12.3), and PAC (pK a , 11.9) (Fig. 8A). At the lysosomal pH of 5 (29), DOX, DNR, and VBL were 100, 100, and 71% charged, respectively, whereas COL and PAC (10) were 0% charged (Fig. 8A). Second, the cytotoxicity of Pgp substrates in the presence of lysosomotropic weak bases was examined in KB31 (ϪPgp) and KBV1 (ϩPgp) cells (Fig. 8B). Although no significant difference in cytotoxicity was observed for KB31 (ϪPgp) cells (Fig. 8B), DOX, DNR, and VBL displayed significantly (p Ͻ 0.001) increased cytotoxicity in the presence of the lysosomotropic weak bases in KBV1 (ϩPgp) cells (Fig. 8B). In contrast, the other Pgp substrates, namely COL and PAC (31), did not display increased cytotoxicity in the presence of lysosomotropic weak bases (Fig. 8B). Because both of these latter drugs are Pgp substrates (31), the lack of effect of lysosomotropic weak bases on their cytotoxicity could be explained by the fact that they do not become charged and trapped in the acidic pH of lysosomes (11,12). Similar results to those presented for KBV1 (ϩPgp) and KB31 (ϪPgp) (Fig. 8B) were also demonstrated in 2008 (ϪPgp) and 2008/ P200 cells (Fig. 8C). Finally, it is notable that no significant  alteration in cytotoxicity of the non-Pgp substrates cisplatin or carboplatin (32) were observed by lysosomotropic weak bases, regardless of Pgp expression (Fig. 9).
In summary, resistance to cytotoxic agents conferred by lysosomal Pgp is dependent on the charge of these drugs at acidic lysosomal pH (i.e. pH 5). Ionizable Pgp substrates that become charged under acidic lysosomal conditions become trapped in lysosomes. Hence, this leads to cytoprotection because these agents are prevented from accessing their molecular targets, e.g. in the case of DOX, DNA in the nucleus (25,26).

DISCUSSION
It is well established that plasma membrane Pgp actively effluxes cytotoxic substrates such as DOX, resulting in MDR (1). However, intracellular localization of Pgp and its contribution to MDR have not been comprehensively investigated, particularly in relation to drug ionization properties. For the first time, this investigation demonstrates that lysosomal Pgp plays an important role in conferring drug resistance and offers a novel strategy of targeting lysosomes to overcome MDR.
This study focused on three key organelles for intracellular Pgp localization, namely the nucleus (4, 33), mitochondrion (34,35), and lysosome (36), where Pgp expression has been suggested (3,4,35,36) but not fully characterized. We showed that Pgp was present in lysosomes but not in nuclei or mitochondria (Fig. 2, A-C). Considering that the lysosomal membrane originates from the plasma membrane via endocytosis (37), Pgp should be detected in lysosomes. Consistent with our observations of Pgp being localized to lysosomes and not mitochondria, several studies have reported the absence of Pgp in mitochondria of KBV1 (ϩPgp) cells (19) and Pgp-transfected cells (38). Furthermore, two independent groups reported Pgp to be present in lysosomes of cells transfected with Pgp (36,38). However, the functionality of lysosomal Pgp in these latter studies was not elucidated.
Because of inversion of the topological orientation of Pgp during endocytosis and the formation of lysosomes (Fig. 10), lysosomal Pgp should be active. Because the catalytic sites for ATP hydrolysis and the drug-binding sites on Pgp remain on the cytoplasmic surface of vesicles (39,40), this would enable the transport of Pgp substrates into the vesicle lumen (Fig.  10A). In this study, the functionality of lysosomal Pgp was demonstrated by investigations showing that only Pgp-expressing cells accumulated DOX (24) in lysosomes (Fig. 4); that inhibition of Pgp prevented lysosomal uptake of DOX and resulted in its nuclear redistribution (Fig. 4); and that siRNA-mediated  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 Pgpcontaining lysosomes.
silencing of Pgp also prevented lysosomal uptake of DOX, resulting in its accumulation in nuclei (Fig. 5). Hence, for the first time, these results clearly demonstrate the functionality of lysosomal Pgp.
We next assessed whether functional lysosomal Pgp can confer MDR. It is generally known that neutral species of drugs can permeate membranes, whereas charged species are far less permeable (41)(42)(43). As a weak base, DOX can be charged and trapped in the acidic pH of the lysosome (11) (Fig. 10A). Considering this, raising lysosomal pH with well characterized lysosomotropic weak bases (37) allows the neutral species of DOX to transverse the lysosomal membrane and relocate to the nucleus (Figs. 7 and 10B). Furthermore, this incubation with lysosomotropic weak bases resulted in increased sensitivity to DOX only in Pgp-expressing cells (Fig. 6, C and H), demonstrating the importance of lysosomal Pgp in conferring drug resistance.
Similarly, speciation plots showed that other chemotherapeutics that are Pgp substrates (i.e. DNR and VBL) could be charged at the acidic pH of the lysosome (Fig. 8), leading to entrapment within this organelle. Intriguingly, our studies also demonstrated that this entrapment was abrogated using lysosomotropic weak bases that increase the acidic pH of the lysosome (37). This effect leads to the neutral cytotoxic drugs passing through the lysosomal membrane, enabling access to their targets, which, for DOX, is DNA in the nucleus (Fig. 10B). These observations explain the increased sensitivity of Pgp-expressing cells to DOX, DNR, and VBL caused by lysosomotropic weak bases (Fig. 8, B and C). In contrast, the cytotoxicity of PAC and COL was not affected by lysosomotropic weak bases because they remain neutral at the acidic pH of lysosomes. Hence, PAC and COL remained membrane-permeable, exploiting their molecular targets regardless of lysosomal pH. Additionally, no alteration in cytotoxicity of the non-Pgp substrates cisplatin and carboplatin was observed by lysosomotropic weak bases, irrespective of Pgp expression (Fig. 9). Collectively, these data indicate that there are three essential properties for lysosomal trapping and lysosomal Pgp-mediated drug resistance of chemotherapeutics. The drug must be a Pgp substrate, this agent must become charged at lysosomal pH, and the key molecular targets must lie outside the lysosome.
In summary, this investigation has dissected the role of lysosomal Pgp in drug resistance. We demonstrate that Pgp is not only functional on the plasma membrane but also in lysosomes. Indeed, lysosomal Pgp increases lysosomal trapping of Pgp substrates, thereby preventing such drugs from reaching their targets. This investigation offers a novel strategy for overcoming MDR by targeting lysosomes using specific Pgp inhibitors or a combination of lysosomotropic weak bases with anticancer drugs. In fact, CLQ has been demonstrated previously to overcome resistance to cytotoxics that are Pgp substrates in cancer cells, although the molecular mechanism involved remained unknown (44). This study provides the first detailed explanation that elucidates this earlier observation.