Abnormal lysosomal trafficking and enhanced exosomal ... · Rebecca and John Moore University of California at San Diego Cancer Center, University of California at San Diego, La Jolla,

Abnormal lysosomal trafficking and enhanced exosomalexport of cisplatin in drug-resistant humanovarian carcinoma cells

Roohangiz Safaei, Barrett J. Larson,Timothy C. Cheng, Michael A. Gibson,Shinji Otani, Wiltrud Naerdemann,and Stephen B. Howell

Rebecca and John Moore University of California at San DiegoCancer Center, University of California at San Diego,La Jolla, California

AbstractPrevious work has shown that cisplatin (CDDP) becomesconcentrated in lysosomes, and that acquired resistanceto CDDP is associated with abnormalities of proteintrafficking and secretion. The lysosomal compartment inCDDP-sensitive 2008 human ovarian carcinoma cells wascompared with that in CDDP-resistant 2008/C13*5.25subline using deconvoluting imaging and specific dyes andantibodies. The lysosomal compartment in CDDP-resistantcells was reduced to just 40% of that in the parentalCDDP-sensitive cells (P < 0.002). This was accompaniedby a reduced expression of the lysosome-associatedproteins 1 and 2 (LAMP1 and LAMP2) as determined byboth microscopy and Western blot analysis. The CDDP-resistant cells released more protein as exosomes andWestern blot analysis revealed that these exosomescontained substantially more LAMP1 than those releasedby the CDDP-sensitive cells. Following loading of thewhole cell with CDDP, the exosomes released from 2008/C13*5.25 cells contained 2.6-fold more platinum thanthose released from sensitive cells. Enhanced exosomalexport was accompanied by higher exosomal levels ofthe putative CDDP export transporters MRP2, ATP7A, andATP7B. Expression profiling identified significantincreases in the expression of several genes whose pro-ducts function in membrane fusion and vesicle traffick-ing. This study shows that the lysosomal compartment

of human ovarian carcinoma cells selected for stableresistance to CDDP is markedly reduced in size, and thatthese cells abnormally sort some lysosomal proteinsand the putative CDDP transporters into an exosomalpathway that also exports CDDP. [Mol Cancer Ther 2005;4(10):1595–604]

IntroductionRepeated exposure of tumor cells to cisplatin (CDDP)results in rapid development of resistance, which eventu-ally contributes to therapeutic failure (1). Resistance toCDDP is often accompanied by resistance to metalloidsand is associated with alterations in DNA repair (2), thiolcontent (3), and drug accumulation (4). Recent studies havelinked the expression of several transporters, particularlythe copper export proteins ATP7A and ATP7B, with CDDPresistance and suggest that they play a direct role in theefflux of CDDP (5).

In tumor cells, CDDP accumulates in a variety of vesic-ular structures, including lysosomes (6). Studies of thecellular pharmacology of CDDP in cells overexpressingATP7A and ATP7B indicate that CDDP is sequestered intovesicles belonging to the secretory pathway as well aslysosomes (7, 8). Microscopic analyses with a fluorescein-conjugated CDDP have confirmed that CDDP accumulatesin vesicles that contain ATP7A or ATP7B (9, 10). Additionalconfocal microscopic analysis of fluorescein-conjugatedCDDP in cells treated with drugs that inhibit traffick-ing pathways suggests that the accumulation of CDDPin the secretory compartment may require lysosomalfunction (10).

Lysosomes are an essential part of the vesicular com-partment and connect the outside medium with manyclasses of cellular targets in the cytosol, nucleus, mitochon-dria, endoplasmic reticulum, and Golgi (reviewed in ref.11). In particular, lysosomes seem to play an important rolein the detoxification of heavy metals. Studies in insect cellshave shown lysosomal storage of metal conjugates (12), andsome heavy metals can cause destabilization and ruptureof lysosomal membranes resulting in cell death (13). Lyso-somal abnormalities are often found in drug-resistant cells;such alterations include aberrant morphology and secreto-ry phenotypes (14, 15). CDDP-resistant cells release unusu-ally large amounts of lysosomal enzymes and membraneproteins into their environment (15, 16). Aberrant functionand expression of the lysosomal H+-pump has also beennoted in such CDDP-resistant cells (17).

Although a systematic survey of lysosomal function inCDDP-resistant cells is lacking, several lines of evidenceindicate that the development of CDDP resistance is asso-ciated with mis-sorting of lysosomal proteins. Lysosomes

Received 4/4/05; revised 7/8/05; accepted 8/10/05.

Grant support: NIH grant CA95298, Department of Defense grantDAMD17-03-1-0158, and Foundation for Research.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

Note: R. Safaei and S.B. Howell are Foundation for Research investigators.

Requests for reprints: Roohangiz Safaei, Department of Medicine #0819,University of California at San Diego, 9500 Gilman Drive, La Jolla,CA 92093. Phone: 858-822-1117; Fax: 858-822-1111.E-mail: [email protected]

Copyright C 2005 American Association for Cancer Research.

doi:10.1158/1535-7163.MCT-05-0102

1595

Mol Cancer Ther 2005;4(10). October 2005

Research. on December 18, 2020. © 2005 American Association for Cancermct.aacrjournals.org Downloaded from

http://mct.aacrjournals.org/

and plasma membrane proteins involved in CDDP effluxoriginate from trans-Golgi network and are often routed tomultivesicular bodies that also receive endocytosed plasmamembrane proteins and receptors. The positioning ofa protein in either the internal or external membrane ofthe multivesicular bodies determines whether it recyclesback to the plasma membrane, is destroyed in lysosomes,or is secreted to the extracellular environment via 50- to200-nm vesicles known as exosomes (reviewed in ref. 18). Alink between abnormal sorting of proteins at multivesicularbodies and tumorigenesis has been established in trans-genic mice (reviewed by ref. 19), and several studies haveshown that the protein composition of exosomes fromtumor cells is different from that of nonmalignant cells(18, 20). Mislocalization of lysosomal and plasma mem-brane proteins has been noted in CDDP-resistant cells(17, 21). In addition, disruption of lysosomes with bafi-lomycin A1 (17, 22) and chlorquine (23) has been reportedto alter CDDP cytotoxicity. Some of the genes whoseproducts are involved in lysosome formation seem toinfluence cellular sensitivity to the cytotoxic effect ofCDDP; these genes include the PI3 kinase (24), rho GTPases(25), annexins (26), and XIAP (27).

We report here on further studies of the lysosomalcompartment in human ovarian carcinoma cells that wereselected for stable acquired resistance. This work showsthat the lysosomal compartment of such cells is markedlyreduced in size and that these cells release protein asexosomes in larger amounts and export CDDP via thisroute at increased levels. The resistant cells also abnormallysort some lysosomal proteins, including putative CDDPtransporters, into the exosomal pathway that also exportsCDDP.

Materials andMethodsReagentsCell culture media and sera were purchased from Invi-

trogen (Carlsbad, CA). Antibodies to lysosome-associatedproteins 1 and 2 (LAMP1 and LAMP2), h-actin, and tubulinwere obtained from Santa Cruz Biotechnology, Inc. (SantaCruz, CA), and those for MRP2 were from Alexis Bio-chemicals (San Diego, CA). The specific dyes Alexa Flour647 phalloidin, Hoechst 33342, and Lysotracker Red werepurchased from Molecular Probes (Eugene, OR). Fluores-cein-conjugated secondary antibodies against mouse,rabbit, and goat immunoglobulin were purchased fromJackson ImmunoResearch Laboratories, Inc. (West Grove,PA). Other chemicals were purchased from Sigma Co.(St. Louis, MO) and Fisher Scientific Co. (Tustin, CA).CDDP (PLATINOL-AQ) was a gift of Bristol Laboratories(Princeton, NJ).

Cell Culture and HistochemistryOvarian carcinoma 2008 cells and the 2008/C13*5.25

subline selected for stable acquired CDDP resistance wereused for all studies (28). Cells were cultured in RPMI with10% fetal bovine serum in humidified air with 5% CO2.Histochemistry was done on cells 1 day after seeding themon coverslips. Labeling with Lysotracker Red was done

in Opti-MEM containing 1 Ag of the dye/mL at 37jC for30 minutes. The cells were rinsed quickly thrice with PBSand fixed by treatment for 15 minutes with 3.7% formal-dehyde in PBS, permeabilized with 0.3% Triton X-100 inPBS for 15 minutes, washed thrice for 15 minutes each withPBS, and then stained with 1 Ag/mL Hoechst 33324 and0.4 Ag/mL Alexa Flour 647 phalloidin, both dissolved inPBS, for 45 minutes and then mounted on glass slides orprocessed for immunostaining. Immunostaining was doneafter a 1-hour incubation of fixed and permeabilizedsamples with 0.3% bovine serum albumin. Primary anti-bodies were diluted in PBS containing 0.3% bovine serumalbumin and then overlaid on cells for 1 hour. Cells werethen washed thrice for 15 minutes, each with PBS, andincubated for 1 hour with secondary antibodies in PBS and0.3% bovine serum albumin followed by three washes inPBS for 15 minutes each and mounting on glass slides.Mounting medium contained 24 g polyvinyl alcohol, 60g glycerol, 60 mL H2O, and 120 mL of 0.2 mol/L Tris-HCl(pH 8.5).

Deconvoluting microscopy was done at the University ofCalifornia at San Diego Cancer Center Digital ImagingShared Resource using a DeltaVision deconvoluting micro-scope system (Applied Precision, Inc., Issaquah, WA.).Images were captured from 0.2-Am sections by �100, �60,and �40 lenses; SoftWorx software (Applied Precision)was used for deconvoluting data. Image quantificationwas done with Data Inspector program in SoftWorx orby NearCount software.

Exosome HarvestExosomes were isolated from 2008 and 2008/C13*5.25

cells according to the method described by Escola et al. (29)with minor modifications. Cells were cultured in 100-mmdishes until 80% confluent. They were then rinsed withserum-free RPMI 1640 to remove proteins and vesicularstructures already released from the cells or present in thefetal bovine serum. In parallel, one set of cells receivedfresh serum-free RPMI 1640 alone, whereas another setwas incubated with RPMI 1640 plus 2 Amol/L CDDP for1 hour in a 37jC humidified tissue culture incubator. Thesupernatants were then removed and the plates rinsedthrice with warm RPMI 1640; and then 12 mL of freshserum-free RPMI 1640 was layered over cells in eachplate; and cells were incubated for 1 hour at 37jC.The supernatant from each plate was then transferred toa 15-mL conical tube and subjected to two successiverounds of centrifugation at 10,000 � g , each for 15 minutes,to remove loose cells and cellular debris. Each supernatantwas then gently transferred to a new SW41 ultracentrifugetube and centrifuged at 70,000 � g for 60 minutes. Thepellet was resuspended in 100 AL of lysis buffer containing100 mmol/L Tris (pH 7.0), 150 mmol/L NaCl, 5 mmol/LEDTA, and 0.25% NP40, mixed thoroughly and an aliquotof 25 AL was taken for measurement of protein concentra-tion using the Bradford kit from BIO-RAD (Hercules, CA).The remainder of the exosomal suspension in eachexperimental group was pooled for polyacrylamide SDSgel analyses.

Lysosomal Anomalies in Cisplatin-Resistant Cells1596




Uptake of PKH67-Labeled ExosomesExosomes from 2008 and 2008/C13*5.25 were labeled

with the lipid dye PKH67 Green (Sigma, St. Louis, MO)according to a method previously described (30). Briefly,an aliquot of exosomes containing 100 Ag of protein wasmixed with 0.5 Amol/L PKH67 Green and incubated at37jC for 10 minutes. The mixture was diluted with ice-coldPBS and centrifuged at 70,000 � g for 1 hour. Vesicleswere washed twice by being resuspended in fresh PBSand sedimented again by centrifugation at 70,000 � g for1 hour. Experiments to determine whether exosomeswere taken up by 2008 and 2008/C13*5.25 cells were doneby incubating PKH67 Green–labeled exosomes (10 Ag oftotal protein) with 1 � 105 cells in complete medium for5 minutes. Cells were then rinsed with PBS thrice and fixedand processed for confocal microscopy. Negative controlswere prepared of samples of 2008 and 2008/C13*5.25cells in the absence of staining for PKH67 to correct forautofluorescence.

Measurements of Platinum ContentWhole cell platinum (Pt) content following a 1-hour

period of incubation with CDDP and a subsequent 1-hourperiod of efflux was assayed as previously described (10).Exosomal content of Pt was measured by rapidly mixing215 AL of 70% analytic grade nitric acid with 75 AL ofresuspended exosomes and then incubating the mixture ina 65jC water bath overnight. Samples were then dilutedwith an indium (Acros Organics, Tustin, CA) solution toa final concentration of 5% acid and 1 ppb indium. Indiumcontent was used to correct for flow variation in theinductivity-coupled plasma mass spectrometry. A ThermoFinnigan inductivity-coupled plasma mass spectrometry(model Element 2) from the Analytical Facility at theScripps Institute of Oceanography was used for measure-ment of Pt concentration. Pt values were normalized tothe protein content of the exosome preparation.

Western BlottingCells were rinsed twice with PBS, scraped with a rubber

policeman, suspended in 10 mL PBS, and centrifuged for10 minutes at 2,500 rpm. Cells were lysed in 0.25% NP40in 100 mmol/L Tris-HCl (pH 8.0) supplemented with pro-tease inhibitor tablets (complete from Roche AppliedSciences, Penzberg, Germany) at 4jC and for 30 minutes.Post-nuclear fractions were obtained by centrifugation ofcell lysates for 10 minutes at 600 � g . Samples containing50 to 100 Ag of protein were electrophoresed on 4% to 10%SDS polyacrylamide gels. Gels were blotted onto nitrocel-lulose filters using a Bio-Rad Mini Transblot apparatus(Bio-Rad, Hercules, CA) and incubated for 1 hour with5% fat free dry milk in TBS at room temperature and thenovernight in primary antibody mixed with 5% milk in TBSat 4jC. Blots were washed thrice for 15 minutes each atroom temperature with 0.025% Tween 20 in TBS. Thesecondary antibody was diluted in 5% fat-free dry milk inTBS and added to blots for 1 hour at room temperature.Blots were washed again at room temperature thrice for15 minutes each in 0.025% Tween 20 in TBS. The extent ofspecific staining was quantified by chemiluminescence

using the Enhanced Chemiluminescence kit from Amer-sham Life Science (Piscataway, NJ). A ChemiImager 4400instrument (Alpha Inotech, San Leandro, CA) was usedfor determining the density of protein bands.

Expression ProfilingRNA was harvested and cDNA prepared, labeled, and

hybridized to cDNA microarrays obtained from the StanfordFunctional Genomics Facility (http://www.microarray.org)that contained 43,200 elements representing f29,593 genesas estimated by UniGene clusters exactly as recentlyreported from this laboratory in detail (31). Significanceanalysis of microarrays (http://www-stat.stanford.edu/ftibs/SAM/index.html) was used to determine which geneswere significantly differentially expressed based on theD score (31).

ResultsReduced Lysosomal Compartment in CDDP-Resis-

tant CellsThe CDDP-resistant ovarian carcinoma 2008/C13*5.25

cells were selected for acquired resistance by repeatedcycles of exposure to CDDP. These cells were developed17 years ago and have maintained a stable level ofresistance while in continuous culture in the absence ofadditional CDDP exposure. The vital dye Lysotracker RedDN-99 was chosen for its capacity to remain in acidiccompartments of cells following formaldehyde fixation.The 2008 and 2008/C13*5.25 cells were exposed to 1 Ag/mLLysotracker Red in parallel cultures and processed togetherthroughout the experiment. Figure 1 shows images of thesecells in which the filamentous-actin filaments were alsostained with Alexa Flour 647 phalloidin. CDDP-resistant2008/C13*5.25 cells were found to contain markedly fewerlysosomes than the CDDP-sensitive parental 2008 cells.Quantification of raw images with NearCount softwareshowed that resistant cells had 2.11 F 0.03-fold (mean FSE) fewer acidic vesicles than their sensitive counterparts(P V 0.002).

To document that the differences in the numbers of acidicvesicles detected by Lysotracker Red DN-99 were in factdue to differences in the number of lysosomes in the twotypes of cells, parallel preparations of 2008 and 2008/C13*5.25 cells were stained with monoclonal antibodiesagainst LAMP1 and LAMP2. These two proteins are bothwell-validated markers of lysosomal vesicles (32). Theimages presented in Fig. 2 confirm reduction in the lyso-somal compartment in the CDDP-resistant cells. The num-ber of LAMP1-containing vesicles was markedly reducedin the 2008/C13*5.25 cells. The number of vesicles stainingpositively for LAMP2 was also reduced in the 2008/C13*5.25 cells but to a lesser extent than LAMP1. Toconfirm the results of the immunohistochemical studies,LAMP1 and LAMP2 levels were measured by Western blotanalysis of the 2008 and 2008/C13*5.25 cells using post-nuclear lysates. Figure 2 (bottom) shows that althoughLAMP1 was expressed in the 2008 cells, it was only veryfaintly detectable in the 2008/C13*5.25 cells. It also showsthat LAMP2 levels were reduced by >3.2-fold in the 2008/

Molecular Cancer Therapeutics 1597




C13*5.25 cells. Thus, three different analytic approachesindicated a marked reduction in number of visiblelysosomes and the content of lysosomal proteins in thecells selected for CDDP resistance.

Analysis of Exosomes Released fromCDDP-Sensitiveand CDDP-Resistant Cells

Because expression profiling of the CDDP-sensitive andCDDP-resistant cell pairs suggested altered levels ofmRNA for some of the genes involved in exosome

formation and release (see below), an analysis was under-taken of the exosomes released by 2008 and 2008/C13*5.25cells. A multistep centrifugation procedure was used toproduce high quality exosomes released from living cellsfor subsequent analysis (30). This technique excludes thepossibility that the exosomes originate from serum in thetissue culture medium or from dead cells or cell fragmentsas these are removed before exosome collection. To showthat the exosomes released by the CDDP-sensitive andCDDP-resistant cells were functional, they were labeledwith PKH67 Green and then incubated with their cell oforigin. Figure 3 shows that exosomes from both types ofcells were able to reassociate with their cell of origin,although when exposed to exosomes containing equallevels of protein, the 2008/C13*5.25 cells accumulated lessPKH67 Green than the 2008 cells. This is consistent withprevious observations that exosomes can reenter the cell oforigin (33–35). The amount of exosomal protein releasedinto serum-free medium during a 1-hour period was usedas an estimate of the differential ability of the 2008 and2008/C13*5.25 cells to export exosomes. Figure 4 (top)shows that relative to the total cellular protein in theculture, the CDDP-resistant cells released significantlymore exosomal protein over this time period (P = 2.3 �10�5, n = 20).

Western blot analysis of the exosomes from 2008 and2008/C13*5.25 cells showed that the CDDP-resistant cellscontained a substantial amount of LAMP1, whereas thisprotein was undetectable in the exosomes released from theCDDP-sensitive cells (Fig. 4, bottom). However, the level ofLAMP2 was only 1.1-fold higher in the exosomes from theresistant than the sensitive cells. Actin and tubulin werepresent at similar levels in the exosomes released by bothCDDP-sensitive and CDDP-resistant cells. Thus, lysosomalproteins were found in exosomes, and the level of at leastone such protein, LAMP1, was much higher in exosomesreleased by the DPP-resistant cells.

Because exosomal export can potentially serve as analternative route for cells to discard proteins whenlysosomal degradation is impaired, the exosomes releasedfrom the 2008 and 2008/C13*5.25 cells were subjected toWestern blot analysis to measure the level of three otherproteins that are believed to play a role in the detoxifica-tion of CDDP via sequestration into intracellular vesicles.Figure 4 (bottom) shows that MRP2 (ABCB2) and the copperefflux transporters ATP7A and ATP7B were all foundat much higher levels in the exosomes from the CDDP-resistant cells. Quantitative analysis of the Western blotsshowed that the exosomes from CDDP-resistant cellscontained, respectively, 3.4-, 2.6-, and 2.6-fold higher levelsMRP2, ATP7A, and ATP7B relative to the levels of theseproteins in exosomes released by the CDDP-sensitive cells.The finding that the exosomes from 2008 cells containATP7B was quite surprising, because this protein is notdetectable in whole cell lysates prepared from this cell line,suggesting that ATP7B is concentrated in the exosomes.The finding that the exosomes from the CDDP-resistantcells contain much more ATP7B is consistent with our

Figure 1. Comparison of the lysosome content of 2008 and 2008/C13*5.25 cells. Lysosomes were labeled with Lysotracker Red (artificiallycolored green). Filamentous-actin was labeled with Alexa Flour 647phalloidin (red ).





earlier observation that the expression of this protein ismarkedly increased in 2008/C13*5.25 cells (36).

Pt Content of Exosomes Released from CDDP-Sensitive and CDDP-Resistant Cells

Recent studies suggest that one mechanism by whichCDDP is exported from the cell is via sequestration intovesicles of the secretory pathway (9). Because exosomesrepresent one of the outputs of this pathway, it was ofinterest to determine whether exosomes released by theCDDP-resistant cells contain more CDDP than those fromthe sensitive cells following loading of the cells with CDDP.The 2008 and 2008/C13*5.25 cells were exposed to 2 Amol/LCDDP for 1 hour to load them with CDDP. They werethen washed to remove all extracellular-free drug and theexosomes released from the cells over the next 1 hour were

collected. The Pt content of the cells at the end of the loadingperiod and at the end of the subsequent 1 hour period ofexosome release and the Pt content of the releasedexosomes were measured and normalized to proteincontent. Figure 5 shows that the amount of Pt/mgexosomal protein was 2.6 F 0.8-fold higher in the exosomesreleased from the CDDP-resistant cells (P = 3.8 � 10�5).Taking into account that the 2008/C13*5.25 cells released1.9-fold more exosomal protein over this time period, thetotal exosomal Pt export was 4.9-fold higher from theresistant cells, despite the fact that the resistant cellsaccumulated only 41% as much CDDP during the 1-hourloading incubation as the sensitive cells as shown in Fig. 5,bottom (see also ref. 36). However, the 0.0037 F 0.00009pmol of Pt/mg exosomal protein found in the exosomes

Figure 2. Comparison of the levels ofthe lysosomal proteins LAMP1 andLAMP2 in 2008 and 2008/C13*5.25cells. Top, deconvoluted confocal ima-ges of cells stained with monoclonalantibodies against LAMP1 and LAMP2(green ). Actin was labeled with AlexaFlour 647 phalloidin (artificially coloredred). Nuclei were stained with Hoechst33342 (blue ). Bottom, Western blotanalysis of whole cell lysates of 2008and 2008/C13*5.25 cells probed withantibodies against LAMP1, LAMP2, andactin. Each lane was loaded with 25 Ag ofprotein.





released over the first hour from CDDP-sensitive cellsrepresented only 0.037% of the total of 10.08 F 0.87 pmol ofPt lost from the cells that produced these exosomes duringthis period of time. In the case of the CDDP-resistant cells,the 0.01818 F 0.00033 pmol of Pt in the exosomesrepresented only 0.075% of the 24.08 F 1.27 pmol lostfrom the cells. Thus, although CDDP-resistant cells exportmore Pt via the exosomal pathway than CDDP-sensitivecells, this represents only a very small fraction of all the Ptloss from the cell during the first hour after the end of drugexposure.

Expression Pattern of Genes Whose Products Func-tion in Lysosome Formation

RNA was harvested from log phase 2008 and 2008/C13*5.25 cells grown under identical conditions and usedto determine the expression profile by hybridizing thelabeled cDNA derived from reverse transcription of RNAagainst each other using a cDNA microarray containingsequences corresponding to 29,593 genes. Each experimentwas repeated six times using independently isolated RNAsamples. Genes that were significantly differentiallyexpressed were determined using the ‘‘statistical analysisof microarrays’’ software (http://www-stat.stanford.edu/ftibs/SAM/index.html). With variables set such that the numberof genes falsely discovered is expected to be V1, a total of

411 genes were found to be significantly differentiallyexpressed between the 2008 and 2008/C13*5.25 cells. Asshown in Table 1, among these were nine genes belongingto families whose products are known to be involved inthe formation of lysosomes and multivesicular bodies. Allnine genes were expressed at a higher level in the CDDP-resistant cells.

DiscussionWe have previously reported that CDDP becomes localizedin lysosomes in CDDP-sensitive parental human ovariancarcinoma 2008 cell line (10). In the current study, weexamined the lysosomal compartment in the 2008/C13*5.25subline whose degree of CDDP resistance has been stablefor >17 years of continuous culture in the absence ofadditional drug selection. We confirmed reports from otherlaboratories that the lysosomal compartment is markedlyabnormal in cells selected for stable acquired CDDPresistance (17, 37). In addition, we discovered that CDDPenters the exosomal pathway, and that CDDP-resistant cellsexport both more CDDP and more putative CDDP trans-porters via this pathway.

Lysosomes are a key station along the path by whichnutrients, pathogens, and cytotoxic agents traffic withincells. In the trafficking of metals, they seem to serve as an

Figure 3. Functionality of exo-somes released by 2008 and 2008/C13*5.25 cells as measured by theirreuptake into the cells from whichthey were released. Exosomes from2008 and 2008/C13*5.25 cellswere labeled with PKH67 Green for10 min (green ) and incubated withtheir cell of origin for 5 min. Actin islabeled with Alex Flour 647 phalloidin(red ). A, 2008 cells labeled withAlexa Flour 647 only. B, 2008 cellslabeled with Alex Flour 647 andincubated with PKH67-labeled exo-somes from 2008 cells. C, 2008/C13*5.25 cells labeled with AlexaFlour 647 only. D, 2008/C13*5.25cells labeled with Alexa Flour 647 andincubated with PKH67-labeled exo-somes from 2008/C13*5.25 cells.





intermediary compartment that receives metals and metal-loids from influx pathways and either stores them ordistributes them to efflux systems or other destinations inthe cell (38, 39). CDDP is known to be concentrated by thecopper efflux transporter ATP7A into vesicles of themicrosomal fraction (7), and it is likely that ATP7B alsoconcentrates CDDP into such vesicles (9). Fluorescentlylabeled CDDP colocalizes with lysosomal markers in 2008cells (40). By virtue of their high content of catabolicenzymes, lysosomes also participate in apoptotic (41), necrotic (42), and autophagic (43) cell death pathways, often incollaboration with stress management organelles, such asthe nucleus, mitochondria, endoplasmic reticulum, andGolgi apparatus (11). There is evidence that metalloidscan activate these cell death pathways as a direct result ofthe damage that they do to the lysosomes in which theybecome concentrated (15, 44).

We found major alterations of the lysosome compartmentin CDDP-resistant 2008/C13*5.25 cells. The number ofvisible lysosomes was reduced when imaged with eitherLysotracker Red DN-99 or antibodies to LAMP1 or LAMP2and the level of expression of LAMP1 and LAMP2 wasreduced when examined by Western blot analysis. Inaddition, the trafficking of proteins through this compart-ment was abnormal as reflected by both the larger amountof exosomal protein released by the resistant cells and thealtered distribution of the proteins that were found in theexosomes. We have documented similar changes in two other CDDP-resistant ovarian carcinoma cell lines, A2780/

CP and IGROV-1/CP (45). It is noteworthy that, despite thefact that both LAMP1 and LAMP2 levels were decreased inthe CDDP-resistant cells, only LAMP1 was found in higherlevels in the exosomes. Quantitatively, it is unlikely thatincreased export of either protein via the exosomalpathway accounts for the marked decrease in their levelin the whole cell. Instead, we interpret the fact that LAMP1levels were increased in exosomes released by the resistantcells, whereas LAMP2 levels were not as evidence of anabnormality of protein trafficking or exosome formationin the lysosomal compartment. It is important to note thatthe reduction in the size of the lysosomal compartmentfound in this study is a stable characteristic of the acquiredCDDP-resistant phenotype as the 2008/C13*5.25 cells havebeen grown in the absence of CDDP for many years. Thefinding that a number of genes coding for proteins involvedwith the formation of lysosomes and multivesicular bodieswere overexpressed at the mRNA level in the CDDP-resistant cells suggests a secondary compensatory responseto some primary abnormality that prevents correct assem-bly of lysosomes or enhances their destruction. It was ofparticular interest to find that several transporters believedto be involved in the movement of CDDP across vesiclemembranes were found in exosomes, and that the level ofATP7A, ATP7B, and MRP2 (ABCB2) was higher in theexosomes released from the CDDP-resistant cells thanthose released from sensitive cells. This may reflect amajor abnormality in intracellular protein trafficking suchas has been described in other types of CDDP-resistant cells(17, 37). We speculate that the altered distribution of the

Figure 5. Platinum content of cells and exosomes. Top, Pt contentof exosomes released from CDDP-loaded 2008 (open column ) and 2008/C13*5.25 (closed column ) cells during the first hour after loading.Pt concentrations are expressed as pmol/mg of exosomal protein. ***,P V 0.002. Bottom, Pt content of whole 2008 and 2008/C13*5.25cells at the end of a 1-h incubation with 2 Amol/L CDDP (open column )and at the end of a subsequent 1-h period of efflux (closed column ). *,P < 0.005.

Figure 4. Analysis of exosomal protein content. Top, total amount ofexosomal protein released per mg of total 2008 and 2008/C13*5.25protein. Bottom, Western blot analysis of MRP2, ATP7A, ATP7B, LAMP1,LAMP2, and actin levels in exosomes released from 2008 and 2008/C13*5.25 cells. Each lane was loaded with 25 Ag of exosomal protein.





types of proteins found in exosomes may report on theCDDP-resistant phenotype. Because exosomes are found inthe systemic circulation, this offers a potential route to earlydetection of the emergence of drug resistance duringtreatment.

There is now a substantial body of evidence that CDDP issequestered into intracellular vesicles, some of whichbelong to the secretory pathway (7, 9, 10). Presumably,many of these vesicles travel directly to, and fuse with, theplasma membrane releasing CDDP to the outside of the cellin a form no longer encapsulated in a lipid membrane. Thereleased Pt may be either free drug, a conjugate, ora complex with cellular proteins to which it has becomebound. The results reported in this article indicate thatCDDP is also exported via exosomes and thus suggesta function for multivesicular bodies, from which thesevesicles originate, in the routing of intracellular CDDP.How CDDP enters the exosomal pathway is not entirelyclear. Possibly, cytoplasmic CDDP is entrapped in exo-somes as they form by invagination of the limiting

membranes of the endosomes that are the precursors ofmature multivesicular bodies. CDDP would then beexpected to exit the cell when the multivesicular bodiesfuses with the plasma membrane to release its content ofexosomes. In the CDDP-resistant 2008/C13*5.25 cells, alarger fraction of cellular protein was released as exosomesper hour and the exosomes contained a larger amount ofPt. Taken together with the fact that the CDDP-resistantcells start out with a smaller amount of Pt in the wholecell following incubation with CDDP, and the fact thatexosomes are recycled by these cells, it is apparent that alarger fraction of the Pt in the cell enters the exosomalpathway in CDDP-resistant cells. This is consistent withthe finding of increased amounts of the putative CDDP orCDDP-conjugate transporters ATP7A, ATP7B, and MRP2(ABCB2) in the exosomes released by the resistant cells.At what step along the pathway CDDP loading occursremains unknown, but ATP7B has been identified as beingresident in late endosomes (46) and is thus positioned toplay a role. Although export via exosomes does not account

Table 1. Genes related to lysosomal formation or vesicle trafficking found to be differentially expressed in 2008 and 2008/C13*5.25cells

Gene Expression ratio* D scorec Function

Annexin A10 (ANXA10) 1.9 3.0 Membrane bending,invagination of MVB

Decay-accelerating factor forcomplement (CD55, DAF)

1.9 2.13 GPI-anchored tetraspanin,exported in exosomes

Transmembrane 4 superfamilymember 2 (TM4SF2)

1.7 2.6 Member of the family oftetraspanin proteins,which are proposed tofunction in membranebending, fusion,invagination of MVB

Baculoviral IAP repeat–containing 6(apollon; BIRC6)

1.9 3.723 Involved in membranebending, fusion,invagination of MVB

Integrin h1 (fibronectin receptor,h polypeptide, antigen CD29includes MDF2, MSK12 ; ITGB1)

1.7 2.392 Exported in exosomes

MHC class I C (HLA-C) 1.8 2.617 Exported in exosomesSorting nexin 5 (SNX5) 2.0 3.007 Early endosomal recycling;

binds clathrin; binds toFanconi anemiacomplementation group A(FANCA) protein (FANCAis expressed in responseto stress)

RAB4A, member RAS oncogenefamily (RAB4A)

1.5 1.998 Regulates receptor recyclingfrom early endosomes

Rab3 GTPase-activating protein,noncatalytic subunit (150 kDa;RAB3-GAP150)

1.6 1.911 Implicated in exocyticrelease of neurotransmittersand hormones; mutationcauses Warburg Microsyndrome

Abbreviation: MVB, multivesicular bodies.*Ratio of expression in 2008/C13*5.25 cells relative to that in 2008 cells.cAll d scores were statistically significant.





for a very large fraction of the Pt loss from either CDDP-sensitive or CDDP-resistant cells over the first hour afterthe end of drug exposure and cannot account for the dif-ferences in whole cell CDDP accumulation in the sensitiveand resistant cells, the difference in the amount of Ptexported via this pathway in the two cell types indicatesthat, as for the lysosomal compartment, the resistant phe-notype is associated with a major alteration in the functionof the exosomal pathway that perhaps simply reflectsupstream abnormalities in the lysosome compartment.

How the alterations in the lysosomal and exosomalpathways are linked to the CDDP-resistant phenotype isnot currently apparent. One possibility it that the seques-tration of CDDP into lysosomes, rather than serving asa detoxification pathway, is actually a mechanism by whichCDDP triggers apoptosis due to damage to the lysosomeand release of lysosomal contents into the cytoplasm. Thus,reduction in the size of the lysosomal compartment inCDDP-resistant cells may by itself facilitate the survivalfollowing CDDP exposure. Another is that the changes inthe lysosomal and exosomal pathways observed in theCDDP-resistant cells reflect primary abnormalities of pro-tein trafficking that also affect the delivery of CDDP to thenucleus where it can attack DNA. It will be of interest todetermine whether the increased exosomal export of CDDPfrom the resistant cells is due to dysfunction of a pathwaythat normally traffics CDDP to some other destination inthe cell.

Acknowledgments

We thank Dr. James Feramisco and Steven McMullen for technicalassistance and advice and Claudette Zacharia for administrative assis-tance.

References

1. Andrews PA, Jones JA, Varki NM, Howell SB. Rapid emergence ofacquired cis -diamminedichloroplatinum(II) resistance in an in vivo modelof human ovarian carcinoma. Cancer Commun 1990;2:93–100.

2. Fink D, Nebel S, Norris PA, et al. The effect of different chemother-apeutic agents on the enrichment of DNA mismatch repair-deficient tumorcells. Br J Cancer 1998;77:703–8.

3. El-akawi Z, Abu-hadid M, Perez R, et al. Altered glutathionemetabolism in oxaliplatin resistant ovarian carcinoma cells. Cancer Lett1996;105:5–14.

4. Gately DP, Howell SB. Cellular accumulation of the anticancer agentcisplatin: a review. Br J Cancer 1993;67:1171–6.

5. Safaei R, Holzer AK, Katano K, Samimi G, Howell SB. The role ofcopper transporters in the development of resistance to Pt drugs. J InorgBiochem 2004;98:1607–13.

6. Saito T, Aran JM. X-ray microanalysis and ion microscopy of guineapig cochlea and kidney after cisplatin treatment. ORL J OtorhinolaryngolRelat Spec 1994;56:310–4.

7. Samimi G, Katano K, Holzer AK, Safaei R, Howell SB. Modulation of thecellular pharmacology of cisplatin and its analogs by the copper exportersATP7A and ATP7B. Mol Pharmacol 2004;66:25–32.

8. Katano K, Safaei R, Samimi G, Holzer A, Rochdi M, Howell SB. Thecopper export pump ATP7B modulates the cellular pharmacology ofcarboplatin in ovarian carcinoma cells. Mol Pharmacol 2003;64:466–73.

9. Katano K, Safaei R, Samimi G, et al. Confocal microscopic analysis ofthe interaction between cisplatin and the copper transporter ATP7B inhuman ovarian carcinoma cells. Clin Cancer Res 2004;10:4578–88.

10. Safaei R, Katano K, Larson BJ, et al. Intracellular localization andtrafficking of fluorescein-labeled cisplatin in human ovarian carcinomacells. Clin Cancer Res 2005;11:756–67.

11. Ferri KF, Kroemer G. Organelle-specific initiation of cell deathpathways. Nat Cell Biol 2001;3:E255–63.

12. Ballan-Dufrancais C. Localization of metals in cells of pterygoteinsects. Microsc Res Tech 2002;56:403–20.

13. Stoltenberg M, Larsen A, Zhao M, Danscher G, Brunk UT. Bismuth-induced lysosomal rupture in J774 cells. APMIS 2002;110:396–402.

14. Larsen A, Escargueil A, Skladanowski A. Resistance mechanismsassociated with altered intracellular distribution of anticancer agents.Pharmacol Ther 2000;85:217–29.

15. Warren L, Jardillier JC, Ordentlich P. Secretion of lysosomal enzymesby drug-sensitive and multiple drug-resistant cells. Cancer Res 1991;51:1996–2001.

16. Isidoro C, Baccino FM, Hasilik A. Mis-sorting of procathepsin D inmetastogenic tumor cells is not due to impaired synthesis of thephosphomannosyl signal. Int J Cancer 1997;70:561–6.

17. Chauhan SS, Liang XJ, Su AW, et al. Reduced endocytosis andaltered lysosome function in cisplatin-resistant cell lines. Br J Cancer2003;88:1327–34.

18. Denzer K, Kleijmeer MJ, Heijnen HF, Stoorvogel W, Geuze HJ.Exosome: from internal vesicle of the multivesicular body to intercellularsignaling device. J Cell Sci 2000;113: Pt 193365–74.

19. Katzmann DJ, Babst M, Emr SD. Ubiquitin-dependent sorting intothe multivesicular body pathway requires the function of a conservedendosomal protein sorting complex, ESCRT-I. Cell 2001;106:145–55.

20. Raposo G, Nijman HW, Stoorvogel W, et al. B lymphocytes secreteantigen-presenting vesicles. J Exp Med 1996;183:1161–72.

21. Liang XJ, Shen DW, Gottesman MM. A pleiotropic defect reducingdrug accumulation in cisplatin-resistant cells. J Inorg Biochem 2004;98:1599–606.

22. Shiraishi Y, Nagai J, Murakami T, Takano M. Effect of cisplatin on H+transport by H+-ATPase and Na+/H+ exchanger in rat renal brush-bordermembrane. Life Sci 2000;67:1047–58.

23. Nair L, Bhasin VK. Cure with cisplatin (II) or murine malaria infectionand in vitro inhibition of a chloroquine-resistant Plasmodium falciparumisolate. Jpn J Med Sci Biol 1994;47:241–52.

24. Tachiiri S, Sasai K, Oya N, Hiraoka M. Enhanced cell killing byoverexpression of dominant-negative phosphatidylinositol 3-kinase sub-unit, Deltap85, following genotoxic stresses. Jpn J Cancer Res 2000;91:1314–8.

25. Sakamoto M, Kondo A, Kawasaki K, et al. Analysis of gene expressionprofiles associated with cisplatin resistance in human ovarian cancer celllines and tissues using cDNA microarray. Hum Cell 2001;14:305–15.

26. Carollo M, Parente L, D’Alessandro N. Dexamethasone-induced cyto-toxic activity and drug resistance effects in androgen-independent prostatetumor PC-3 cells are mediated by lipocortin 1. Oncol Res 1998;10:245–54.

27. Li J, Feng Q, Kim JM, et al. Human ovarian cancer and cisplatinresistance: possible role of inhibitor of apoptosis proteins. Endocrinology2001;142:370–80.

28. Andrews PA, Velury S, Mann SC, Howell SB. cis -diamminedichlor-oplatinum(II) accumulation in sensitive and resistant human ovariancarcinoma cells. Cancer Res 1988;48:68–73.

29. Escola JM, Kleijmeer MJ, Stoorvogel W, Griffith JM, Yoshie O, GeuzeHJ. Selective enrichment of tetraspan proteins on the internal vesiclesof multivesicular endosomes and on exosomes secreted by humanB-lymphocytes. J Biol Chem 1998;273:20121–7.

30. Morelli AE, Larregina AT, Shufesky WJ, et al. Endocytosis, intracel-lular sorting, and processing of exosomes by dendritic cells. Blood2004;104:3257–66.

31. Samimi G, Manorek G, Castel R, et al. cDNA-microarray-basedidentification of genes and mechanisms associated with oxaliplatinresistance. Cancer Chemother Pharmacol 2005;55:1–11.

32. Eskelinen EL, Tanaka Y, Saftig P. At the acidic edge: emerging functionsfor lysosomal membrane proteins. Trends Cell Biol 2003;13:137–45.

33. de Gassart A, Geminard C, Hoekstra D, Vidal M. Exosome secretion:the art of reutilizing nonrecycled proteins? Traffic 2004;5:896–903.

34. Skokos D, Botros HG, Demeure C, et al. Mast cell-derived exosomesinduce phenotypic and functional maturation of dendritic cells and elicitspecific immune responses in vivo . J Immunol 2003;170:3037–45.

35. Wolfers J, Lozier A, Raposo G, et al. Tumor-derived exosomes area source of shared tumor rejection antigens for CTL cross-priming. NatMed 2001;7:297–303.





36. Katano K, Kondo A, Safaei R, et al. Acquisition of resistance tocisplatin is accompanied by changes in the cellular pharmacology ofcopper. Cancer Res 2002;62:6559–65.

37. Liang XJ, Shen DW, Garfield S, Gottesman MM. Mislocalization ofmembrane proteins associated with multidrug resistance in cisplatin-resistant cancer cell lines. Cancer Res 2003;63:5909–16.

38. Eaton JW, Qian M. Molecular bases of cellular iron toxicity. Free RadicBiol Med 2002;32:833–40.

39. Larsen A, Martiny N, Stoltenberg M, Danscher G, Rungby J.Gastrointestinal and systemic uptake of bismuth in mice after oralexposure. Pharmacol Toxicol 2003;93:82–90.

40. Safaei R, Howell SB. Copper transporters regulate the cellularpharmacology and sensitivity to Pt drugs. Crit Rev Oncol Hematol2005;53:13–23.

41. Turk V, Turk B, Guncar G, Turk D, Kos J. Lysosomal cathepsins:structure, role in antigen processing and presentation, and cancer. AdvEnzyme Regul 2002;42:285–303.

42. Zahrebelski G, Nieminen AL, al-Ghoul K, Qian T, Herman B, LemastersJJ. Progression of subcellular changes during chemical hypoxia to culturedrat hepatocytes: a laser scanning confocal microscopic study. Hepatology1995;21:1361–72.

43. Bursch W, Hochegger K, Torok L, Marian B, Ellinger A, Hermann RS.Autophagic and apoptotic types of programmed cell death exhibit differentfates of cytoskeletal filaments. J Cell Sci 2000;113:1189–98.

44. Ouar Z, Bens M, Vignes C, et al. Inhibitors of vacuolar H+-ATPaseimpair the preferential accumulation of daunomycin in lysosomes andreverse the resistance to anthracyclines in drug-resistant renal epithelialcells. Biochem J 2003;370:185–93.

45. Safaei R, Katano K, Samimi G, Holzer AK, Naerdemann W, Howell SB.Cisplatin resistance is associated with reduced lysosomal structures andmarkers in ovarian cancer cells. Proc Am Assoc Cancer Res 2004;45:865.

46. Harada M, Kawaguchi T, Kumemura H, et al. The Wilson diseaseprotein ATP7B resides in the late endosomes with Rab7 and the Niemann-Pick C1 protein. Am J Pathol 2005;166:499–510.





2005;4:1595-1604. Mol Cancer Ther Roohangiz Safaei, Barrett J. Larson, Timothy C. Cheng, et al. carcinoma cellsexport of cisplatin in drug-resistant human ovarian Abnormal lysosomal trafficking and enhanced exosomal

Updated version

http://mct.aacrjournals.org/content/4/10/1595

Access the most recent version of this article at:

Cited articles

http://mct.aacrjournals.org/content/4/10/1595.full#ref-list-1

This article cites 46 articles, 13 of which you can access for free at:

Citing articles

http://mct.aacrjournals.org/content/4/10/1595.full#related-urls

This article has been cited by 23 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://mct.aacrjournals.org/content/4/10/1595To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/content/4/10/1595.full#ref-list-1

http://mct.aacrjournals.org/content/4/10/1595.full#related-urls

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]



Documents

Abnormal lysosomal trafficking and enhanced exosomal ... · Rebecca and John Moore University of California at San Diego Cancer Center, University of California at San Diego, La Jolla,