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CELLULAR AND MOLECULAR
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas (Y.G., M.D., J.P.K.); and Division of Medicinal Chemistry, University of North Carolina, Chapel Hill, North Carolina (M.B.D., J.L.)
Received May 12, 2005; accepted September 16, 2005.
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Abstract |
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; Garver and Heidenreich, 2002). Cells with defective NPC1 characteristically display hyperaccumulation of cholesterol in lysosomes. Interestingly, recent reports have suggested that NPC1 shares significant homology with the resistance nodulation division family of prokaryotic permeases and have postulated that this protein may possess drug transport activity (Tseng et al., 1999; Davies et al., 2000). Despite this report, little was known regarding the substrate specificity or the role this protein may play in intracellular drug transport.
Cancer cells exposed to chemotherapeutic agents frequently become resistant to the cytotoxic effects of drugs after prolonged exposure. This phenomenon has been termed MDR because cells often become resistant not only to the administered drug but also to structurally unrelated drugs that are subsequently administered. Overall, the MDR phenotype is complex and multifactorial (Gottesman and Pastan, 1993).
Our laboratory focuses on evaluating intracellular drug sequestration mechanisms that have been shown to contribute to MDR (Duvvuri and Krise, 2005). We have recently shown that the MDR HL-60 human leukemic cancer cell line can sequester different drugs into different organelles through independent mechanisms (Gong et al., 2003). In this work, we demonstrated that the weakly basic anticancer drug daunorubicin specifically accumulates within lysosomes of the MDR cell line, a process that did not appear to occur in drug-sensitive cells. The lysosomal sequestration is thought to reduce the drug's effectiveness by limiting its availability to enter the nuclear compartment that houses the drug's target molecules, which include DNA and topoisomerase II (Binaschi et al., 2001).
We and others have shown that the sequestration of weakly basic drugs into lysosomes of MDR cancer cell lines is driven by a pH partition-type mechanism (Simon et al., 1994; Gong et al., 2003). In the MDR HL-60 cell line, we have shown previously that the lysosome and cytosol pH values are 5.1 and 7.1, respectively (Gong et al., 2003). When weakly basic drugs, such as daunorubicin, with pKa values near neutrality partition across the plasma membrane and diffuse into the cell cytosol, they exist, to a significant degree, in an unionized, membrane-permeable state. Upon entering the luminal domain of acidic organelles such as the lysosomes, these drugs experience an acute shift in their ionization and exist predominantly in an ionized, membrane-impermeable state. As a result, steep pH gradients across intracellular compartments can drive significant accumulations of weakly basic drugs. The reason daunorubicin fails to accumulate significantly within lysosomes of drug-sensitive HL-60 cells is because of defective acidification capacity associated with the lysosomal compartment (Gong et al., 2003).
Interestingly, daunorubicin remains visibly sequestered in the lysosomes of MDR HL-60 cells long after the drug is removed from the culture media. This observation is in disagreement with the proposed function of NPC1 in drug efflux from this compartment (Davies et al., 2000). We reasoned that either daunorubicin was not a substrate for NPC1-mediated efflux from lysosomes or perhaps NPC1 was not functioning properly on lysosomes of MDR cancer cells.
We describe experiments here that are consistent with daunorubicin being a substrate for NPC1-mediated efflux from lysosomes of human fibroblasts. We also provide evidence that suggests NPC1 mediates the efflux according to a vesicle-mediated process rather than directly translocating drugs across lipid bilayers, which is the mechanism ascribed to traditional drug transporter proteins such as P-glycoprotein. Finally, we establish that the emergence of MDR in HL-60 cells is associated with malfunctions in NPC1 activity. The implications of these findings with regard to the intracellular trafficking of drugs in both normal and MDR cancer cells are discussed.
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Materials and Methods |
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NPC1 Western Blot and Real-Time RT-PCR. For Western blot analysis, HL-60 cells were collected and homogenized in a buffer consisting of 100 mM Tris, pH 8.0, 2 mM EDTA, 0.3 M sodium chloride, and protease inhibitor cocktail consisting of 0.1 mM phenylmethyl sulfonyl fluoride and 1 µg/ml each of aprotin, leupeptin, and pepstatin (Sigma-Aldrich, St. Louis, MO). Cell lysates were resolved with 7.5% SDS-polyacrylamide gel electrophoresis under reducing conditions and transferred to nitrocellulose membranes. Blots were incubated with a rabbit polyclonal antibody against human NPC1 (a gift from Dr. William Garver, University of Arizona, Tucson) and with a mouse monoclonal antibody against 
-actin (Sigma-Aldrich). For RT-PCR, 10 µg of RNA from designated cells lines was extracted using RNeasy spin columns (QIAGEN, Valencia, CA) and were treated with 10 units of DNase (Promega, Madison, WI) according to the manufacturer's protocol. RNA was then converted to cDNA, using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA), and 1 µl of the reaction mixture was used to detect NPC1 levels. Real-time RT-PCR was based on TaqMan Probe-based fluorogenic 5' nuclease chemistry. The primer/probe set (Assay identification, Hs00264835_m1) used to measure NPC1 RNA levels covers the exon 9 and 10 junction of the gene (NM_000271
[GenBank]
) and was purchased from Applied Biosystems. NPC1 expression in test samples was normalized using rRNA as an endogenous control. A 1000-fold dilution of 1 µl of the cDNA was used for the rRNA control experiments. Real-time PCR was performed on a 7500 Real-Time PCR System (Applied Biosystems). The PCR conditions were as follows: stage 1, one cycle at 50°C for 2 min; stage 2, one cycle at 95°C for 10 min; and stage 3, 40 cycles at 95°C for 0.15 min followed by 60°C for 1 min. NPC1 gene expression in MDR HL-60 cells relative to drug-sensitive HL-60 cells was based on the normalized threshold cycle value of each sample and was determined using 7500 Real-Time PCR System Sequence Detection Software version 1.2.1. Statistical significance of differences in mRNA levels was evaluated with a two-tailed Student's t test using SigmaPlot 2001 (SPSS Inc., Chicago, IL) at a significance level of 0.05.
Cell-Imaging Studies. Cells were viewed with a fluorescence microscope (Leica Diaplan; Leica, Wetzlar, Germany) with 100x (HL-60 cell lines) or 40x (NPC1 fibroblasts) objectives. Differential magnifications were used to correct for large differences in cell sizes. Images were captured using an Orca ER camera (Hamamatsu Corporation, Bridgewater, NJ) controlled by SimplePCI imaging software (Compix Inc., Norcross, GA). For daunorubicin localization studies, all cells were cultured in medium supplemented with 2 µM daunorubicin (Sigma-Aldrich) for 1 h and subsequently washed twice with ice-cold phosphate-buffered saline (PBS), pH 7.4, before mounting on a coverslip and imaging. Designated fibroblasts were incubated in culture media supplemented with 50 µM nocodazole (Sigma-Aldrich) for 4 h prior to the addition of 2 µM daunorubicin for 1 h. For filipin staining, cells were collected and fixed in PBS containing 3% paraformaldehyde for 30 min and washed with PBS prior to incubation in 50 mM ammonium chloride (in PBS) for 15 min and washed in PBS again. Cells were then incubated with PBS containing 10% goat serum and 50 µg/ml filipin (Sigma-Aldrich) for 1 h and washed thrice with PBS prior to imaging. Lysosomal pH determination in the fibroblasts was carried out using a ratiometric approach previously described by Diwu et al. (1999) using LysoSensor Yellow/Blue DND-160 (Invitrogen) as a probe.
Dextran Secretion Assay. Designated cell lines were incubated in culture medium supplemented with 50 µCi/ml [3H]dextran (70,000 mol. wt., 182 µCi/mg; American Radiolabeled Chemicals, St. Louis, MO) at 37°C for 1 h and washed with PBS four times. A fraction of treated cells were homogenized in a solution containing 100 mM Tris, pH 8.0, 2 mM EDTA, and 0.3 M sodium chloride and subjected to liquid scintillation counting to determine initial [3H]dextran loading. The remaining cells were returned to [3H]dextran-free warmed cell culture medium (37°C), and the amount of [3H]dextran secreted into the medium at indicated time points was recorded. To determine the amount of nonspecifically associated [3H]dextran that was released into the medium at each time point, the experiment was repeated at 4°C following the initial incubation with [3H]dextran for 1 h at 37°C. These values were subtracted from the total amount of [3H]dextran released into the medium at 37°C for each time point. The cumulative amount secreted into the culture medium was expressed as a percentage of the initial amount contained in the cells at the start of the experiment. Quantification of [3H]dextran in samples was performed using a liquid scintillation counter (Beckman LS 6000IC; Beckman Coulter, Fullerton, CA) and Ecoscint (Fisher Scientific, Pittsburgh, PA) liquid scintillation cocktail. Statistical significance of differences in amount of [3H]dextran secreted at 60 min was evaluated with a two-tailed Student's t test using SigmaPlot 2001 (SPSS Inc.) at a significance level of 0.05.
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Results |
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), resulted in excessive intracellular accumulation of cholesterol in both NPC1+/+ and drug-sensitive HL-60 cells (see Supplemental Fig. 1). Together, these results suggest a correlation between the emergence of MDR in HL-60 cells and defects in NPC1 activity as revealed through cholesterol trafficking defects.
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; Watari et al., 2000). Therefore, we comparatively evaluated NPC1 RNA and protein levels in drug-sensitive and MDR HL-60 cells. Western blot analysis of cell lysates revealed that NPC1 was significantly overexpressed in the MDR cell line relative to the drug-sensitive cell line (Fig. 2A). Moreover, real-time RT PCR revealed that NPC1 mRNA levels are approximately 3 times greater in the MDR cell line relative to the drug-sensitive cell line (Fig. 2B). The RNA and protein expression results are consistent with the filipin staining experiments and suggest that NPC1 loses functional activity when the drug-sensitive HL-60 cell line acquires the MDR phenotype.
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Correlating NPC1 Functional Activity with Daunorubicin Clearance from Lysosomes. The previous studies support a correlation between loss of NPC1 activity and the emergence of MDR. To evaluate whether daunorubicin is a substrate for NPC1-mediated efflux from lysosomes, the cellular distribution of daunorubicin in NPC1+/+ and NPC1–/– fibroblasts were assessed using fluorescence microscopy. Figure 3A shows that daunorubicin remains sequestered in intracellular compartments of NPC1–/– fibroblasts, whereas daunorubicin is efficiently cleared from intracellular compartments of NPC1+/+ cells. Similarly, MDR HL-60 cells, which we have shown to have impaired NPC1 function, fail to clear daunorubicin from intracellular compartments (Fig. 3B). Moreover, treatment of NPC1+/+ cells with progesterone resulted in lysosomal retention of accumulated daunorubicin (see Supplemental Fig. 2). Alternatively, progesterone treatment did not have any effect on daunorubicin distribution in NPC1–/– cells, which do not have functional NPC1. Together, these results suggest that daunorubicin clearance from intracellular compartments requires the cell to possess functional NPC1.
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To further evaluate this mechanistic possibility, we examined whether efflux of [3H]dextran molecules from lysosomes could be correlated with the functional capacity of NPC1 in the fibroblasts and in HL-60 cells. To achieve this, we specifically localized [3H]dextran to lysosomes using a pulse-chase technique and measured the release of dextran into the medium at indicated times (Fig. 5). We found that the dextran secretion rate was significantly decreased in both cell lines that have impaired NPC1 function, namely the NPC1–/– fibroblast and the MDR HL-60 cell line relative to cell lines with normal NPC1 function (Fig. 5). Together, these results are consistent with daunorubicin clearance from lysosomes through an NPC1-dependent vesicle-mediated secretory type mechanism.
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Discussion |
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). Results presented here are consistent with NPC1-mediating drug efflux according to a different mechanism, which involves a vesicle-mediated transport pathway. Importantly, our results further establish that MDR cancer cells can acquire defects in NPC1 function, which provides a mechanistic basis for an intracellular drug sequestration phenotype exhibited by these cells.
The observation that the intracellular compartmentalization of daunorubicin differs with respect to functional activity of NPC1 in a pair of human fibroblasts supports a role for this protein in the intracellular trafficking of daunorubicin. Our results suggest that NPC1 participates in clearing intracellular compartments of drugs that have accumulated there by alternative mechanisms.
The accumulation of weakly basic drugs, such as daunorubicin, in acidic intracellular vesicles has been previously established to occur according to a pH partition-type mechanism (De Duve et al., 1974). Therefore, for daunorubicin to be sequestered into lysosomes according to this mechanism, the luminal pH of the organelle must be low. To confirm that both NPC1 fibroblasts had acidic lysosomes, we measured the pH values associated with them and found them to be 4.4 ± 0.1 and 4.6 ± 0.3 for NPC1+/+ and NPC1–/– fibroblasts, respectively (results from 10 experiments; see Materials and Methods). Therefore, theoretically, both of these cell lines should sequester daunorubicin in lysosomes to a similar degree according to a pH partition-type mechanism. The fact that the NPC1+/+ cell line did not visibly retain daunorubicin in the lysosomes suggested that NPC1 was able to efficiently clear the drug from this compartment. NPC1–/– fibroblasts, on the other hand, were unable to clear the compartmentalized drug. These results established a role for NPC1 in the clearance of drug from acidic vesicles such as lysosomes.
We have established previously that the accumulation of daunorubicin in the lysosomes of MDR HL-60 cells occurs through a pH partition-type mechanism and not through the action of ATPase binding cassette-type drug transporters (Gong et al., 2003). This is important to note because we and others have shown that intracellular drug sequestration of some drugs can be facilitated by drug transporters such as P-glycoprotein and the multidrug resistance-associated protein MRP1 in some MDR cell lines (Shapiro et al., 1998; Gong et al., 2003). We also have shown previously that the reason that daunorubicin does not sequester in lysosomes of drug-sensitive HL-60 cell line is because they have defective acidification of their lysosomes (Gong et al., 2003).
Having established a role for NPC1 in drug clearance from intracellular vesicles, we next sought to elucidate the mechanism. Prior to completing this work, the mechanistic basis for NPC1's role in drug transport was poorly understood and controversial. We considered two general mechanisms by which NPC1 could mediate the clearance of daunorubicin from intracellular vesicles (for a diagram, see Fig. 6). First, NPC1 could be involved in a vesicle-mediated exocytosis of lysosomal contents, which would result in clearance of daunorubicin from the cells (see pathway 2, Fig. 6). This possibility is supported by the work of Neufeld et al. (1999), who suggested that NPC1 can participate in nondiscriminate transport of lysosomal cargo to the plasma membrane. Second, NPC1 could be involved in direct transport of daunorubicin from the lumen of the lysosomes to the cell cytosol. From the cytosol, the drug could passively permeate out of the cell (see pathway 1, Fig. 6). This possibility would be predicted from the work of Davies et al. (2000), who proposed that NPC1 participates in the transport of weakly basic compounds such as acriflavine across lipid bilayers of intracellular compartments.
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We reasoned that evaluation of daunorubicin sequestration in the presence of nocodazole, a disrupter of the cell's microtubule network, would help differentiate between the previously stated mechanistic possibilities. The NPC1+/+ fibroblast was chosen for this evaluation because the lysosomes are acidic, and, thus, daunorubicin should be accumulating within them according to a pH partition-type mechanism; and this cell line also has functional NPC1 and is able to clear the lysosomes of sequestered drug. In essence, this cell line is believed to have accumulation and clearance mechanisms operating simultaneously, and the end result is that daunorubicin is not visibly sequestered into perinuclear vesicles (Fig. 4). Nocodazole treatment would be expected to result in enhanced retention of daunorubicin in lysosomes of NPC1+/+ fibroblasts if a vesicle-mediated event was responsible for its clearance. Alternatively, nocodazole treatment would not be expected to influence lysosomal clearance if NPC1 was directly transporting daunorubicin across the lysosomal lipid bilayer into the cell cytosol. The fact that nocodozale treatment reduced the clearance of daunorubicin from intracellular compartments is consistent with NPC1 playing a role in a vesicle-mediated clearance of the drug (pathway 2, Fig. 6).
NPC1's role in a vesicle-mediated transport for daunorubicin clearance was further evaluated using a lysosomal dextran secretion assay (Fig. 5). Dextran molecules were specifically localized to lysosomes, the same compartment that sequesters daunorubicin. Unlike daunorubicin, dextran molecules are very large and hydrophilic and cannot cross lipid bilayers by passive diffusion. As a result, dextran molecules can only be released from cells through vesicle-mediated-type transport events (Straubinger et al., 1983). The release of dextran molecules into the media surrounding the cells was found to be significantly reduced in cells with dysfunctional NPC1. These results further support NPC1's role in mediating lysosomal clearance of drugs according to a vesicle-mediated pathway.
The results from cholesterol trafficking studies with filipin and NPC1 RNA and protein expression suggested that NPC1 protein lost its functional activity when HL-60 cells developed an MDR phenotype. This finding helps rationalize why MDR HL-60 cells retain accumulated drug in the lysosomal compartment (Fig. 3B). As previously stated, daunorubicin accumulation in the MDR HL-60 cells occurs according to a pH partition-type mechanism. The finding that NPC1 is not functioning properly in this cell line explains why daunorubicin fails to be efficiently cleared from the lysosomes. A similar daunorubicin sequestration phenotype is observed with NPC1–/– fibroblasts that also have low lysosomal pH and dysfunctional NPC1, like the MDR HL-60 cells.
In summary, this work establishes a role for NPC1 in a novel vesicle-mediated efflux pathway for the clearance of drugs from cells. This finding has broad implications and significantly improves our understanding regarding drug efflux mechanism. It is reasonable to assume that all cells, under normal conditions, have low lysosomal pH and functional NPC1 to varying degrees. The combination of these two features can be expected to result in an efficient sequestration and secretion pathway responsible for enhancing the clearance of drugs from cells. Weakly basic drugs that are susceptible to ion trapping in lysosomes would be expected to be substrates for this novel efflux pathway. Considering the fact that a large percentage of commercially available drugs are weak bases, the scope of potential substrates could be quite large. This work also established a correlation between the emergence of MDR in HL-60 cancer cells with defective NPC1 functional activity. This observation allowed us to arrive at a mechanistic explanation for a unique drug sequestration phenotype observed with these cells.
It is important to note that we believe it is unlikely that the loss of NPC1 function in MDR cells would directly contribute to the resistance to daunorubicin. This is because drug sequestered in lysosomes would be considered ineffective regardless of whether or not it is secreted. Moreover, at the present time, it is uncertain how prevalent this relationship between MDR and NPC1 loss of function is. From our unpublished findings, it appears that not every MDR cell line has a loss of NPC1 activity. The relationship may be dependent on the MDR selection conditions among other factors, and more work will be required to establish this.
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Footnotes |
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ABBREVIATIONS: NPC1, Niemann-Pick C1; MDR, multidrug resistance; RT, reverse transcription; PCR, polymerase chain reaction; PBS, phosphate-buffered saline.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Jeffrey P. Krise, Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS 66047. E-mail: krise{at}ku.edu
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References |
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