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Abnormal Lysosomal Trafficking and Enhanced Exosomal Export of Cisplatin in Drug-Resistant Human Ovarian Carcinoma Cells

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Abnormal Lysosomal Trafficking and Enhanced Exosomal Export of Cisplatin in Drug-Resistant Human Ovarian Carcinoma Cells 1595 Abnormal lysosomal trafficking and enhanced exosomal export of cisplatin in drug-resistant human ovarian carcinoma cells Roohangiz Safaei, Barrett J. Larson, of human ovarian carcinoma cells selected for stable Timothy C. Cheng, Michael A. Gibson, resistance to CDDP is markedly reduced in size, and that Shinji Otani, Wiltrud Naerdemann, these cells abnormally sort some lysosomal proteins and Stephen B. Howell and the putative CDDP transporters into an exosomal pathway that also exports CDDP. [Mol Cancer Ther 2005; Rebecca and John Moore University of California at San Diego 4(10):1595–604] Cancer Center, University of California at San Diego, La Jolla, California Introduction Repeated exposure of tumor cells to cisplatin (CDDP) Abstract results in rapid development of resistance, which eventu- Previous work has shown that cisplatin (CDDP) becomes ally contributes to therapeutic failure (1). Resistance to concentrated in lysosomes, and that acquired resistance CDDP is often accompanied by resistance to metalloids to CDDP is associated with abnormalities of protein and is associated with alterations in DNA repair (2), thiol trafficking and secretion. The lysosomal compartment in content (3), and drug accumulation (4). Recent studies have CDDP-sensitive 2008 human ovarian carcinoma cells was linked the expression of several transporters, particularly compared with that in CDDP-resistant 2008/C13*5.25 the copper export proteins ATP7A and ATP7B, with CDDP subline using deconvoluting imaging and specific dyes and resistance and suggest that they play a direct role in the antibodies. The lysosomal compartment in CDDP-resistant efflux of CDDP (5). cells was reduced to just 40% of that in the parental In tumor cells, CDDP accumulates in a variety of vesic- CDDP-sensitive cells (P < 0.002). This was accompanied ular structures, including lysosomes (6). Studies of the by a reduced expression of the lysosome-associated cellular pharmacology of CDDP in cells overexpressing proteins 1 and 2 (LAMP1 and LAMP2) as determined by ATP7A and ATP7B indicate that CDDP is sequestered into both microscopy and Western blot analysis. The CDDP- vesicles belonging to the secretory pathway as well as resistant cells released more protein as exosomes and lysosomes (7, 8). Microscopic analyses with a fluorescein- Western blot analysis revealed that these exosomes conjugated CDDP have confirmed that CDDP accumulates contained substantially more LAMP1 than those released in vesicles that contain ATP7A or ATP7B (9, 10). Additional by the CDDP-sensitive cells. Following loading of the confocal microscopic analysis of fluorescein-conjugated whole cell with CDDP, the exosomes released from 2008/ CDDP in cells treated with drugs that inhibit traffick- C13*5.25 cells contained 2.6-fold more platinum than ing pathways suggests that the accumulation of CDDP those released from sensitive cells. Enhanced exosomal in the secretory compartment may require lysosomal export was accompanied by higher exosomal levels of function (10). the putative CDDP export transporters MRP2, ATP7A, and Lysosomes are an essential part of the vesicular com- ATP7B. Expression profiling identified significant partment and connect the outside medium with many increases in the expression of several genes whose pro- classes of cellular targets in the cytosol, nucleus, mitochon- ducts function in membrane fusion and vesicle traffick- dria, endoplasmic reticulum, and Golgi (reviewed in ref. ing. This study shows that the lysosomal compartment 11). In particular, lysosomes seem to play an important role in the detoxification of heavy metals. Studies in insect cells have shown lysosomal storage of metal conjugates (12), and some heavy metals can cause destabilization and rupture Received 4/4/05; revised 7/8/05; accepted 8/10/05. of lysosomal membranes resulting in cell death (13). Lyso- Grant support: NIH grant CA95298, Department of Defense grant somal abnormalities are often found in drug-resistant cells; DAMD17-03-1-0158, and Foundation for Research. such alterations include aberrant morphology and secreto- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ry phenotypes (14, 15). CDDP-resistant cells release unusu- advertisement in accordance with 18 U.S.C. Section 1734 solely to ally large amounts of lysosomal enzymes and membrane indicate this fact. proteins into their environment (15, 16). Aberrant function Note: R. Safaei and S.B. Howell are Foundation for Research investigators. and expression of the lysosomal H+-pump has also been Requests for reprints: Roohangiz Safaei, Department of Medicine #0819, noted in such CDDP-resistant cells (17). University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093. Phone: 858-822-1117; Fax: 858-822-1111. Although a systematic survey of lysosomal function in E-mail: [email protected] CDDP-resistant cells is lacking, several lines of evidence Copyright C 2005 American Association for Cancer Research. indicate that the development of CDDP resistance is asso- doi:10.1158/1535-7163.MCT-05-0102 ciated with mis-sorting of lysosomal proteins. Lysosomes Mol Cancer Ther 2005;4(10). October 2005 Downloaded from mct.aacrjournals.org on September 26, 2021. © 2005 American Association for Cancer Research. 1596 Lysosomal Anomalies in Cisplatin-Resistant Cells and plasma membrane proteins involved in CDDP efflux in Opti-MEM containing 1 Ag of the dye/mL at 37jC for originate from trans-Golgi network and are often routed to 30 minutes. The cells were rinsed quickly thrice with PBS multivesicular bodies that also receive endocytosed plasma and fixed by treatment for 15 minutes with 3.7% formal- membrane proteins and receptors. The positioning of dehyde in PBS, permeabilized with 0.3% Triton X-100 in a protein in either the internal or external membrane of PBS for 15 minutes, washed thrice for 15 minutes each with the multivesicular bodies determines whether it recycles PBS, and then stained with 1 Ag/mL Hoechst 33324 and back to the plasma membrane, is destroyed in lysosomes, 0.4 Ag/mL Alexa Flour 647 phalloidin, both dissolved in or is secreted to the extracellular environment via 50- to PBS, for 45 minutes and then mounted on glass slides or 200-nm vesicles known as exosomes (reviewed in ref. 18). A processed for immunostaining. Immunostaining was done link between abnormal sorting of proteins at multivesicular after a 1-hour incubation of fixed and permeabilized bodies and tumorigenesis has been established in trans- samples with 0.3% bovine serum albumin. Primary anti- genic mice (reviewed by ref. 19), and several studies have bodies were diluted in PBS containing 0.3% bovine serum shown that the protein composition of exosomes from albumin and then overlaid on cells for 1 hour. Cells were tumor cells is different from that of nonmalignant cells then washed thrice for 15 minutes, each with PBS, and (18, 20). Mislocalization of lysosomal and plasma mem- incubated for 1 hour with secondary antibodies in PBS and brane proteins has been noted in CDDP-resistant cells 0.3% bovine serum albumin followed by three washes in (17, 21). In addition, disruption of lysosomes with bafi- PBS for 15 minutes each and mounting on glass slides. lomycin A1 (17, 22) and chlorquine (23) has been reported Mounting medium contained 24 g polyvinyl alcohol, 60 to alter CDDP cytotoxicity. Some of the genes whose g glycerol, 60 mL H2O, and 120 mL of 0.2 mol/L Tris-HCl products are involved in lysosome formation seem to (pH 8.5). influence cellular sensitivity to the cytotoxic effect of Deconvoluting microscopy was done at the University of CDDP; these genes include the PI3 kinase (24), rho GTPases California at San Diego Cancer Center Digital Imaging (25), annexins (26), and XIAP (27). Shared Resource using a DeltaVision deconvoluting micro- We report here on further studies of the lysosomal scope system (Applied Precision, Inc., Issaquah, WA.). compartment in human ovarian carcinoma cells that were Images were captured from 0.2-Am sections by Â100, Â60, selected for stable acquired resistance. This work shows and Â40 lenses; SoftWorx software (Applied Precision) that the lysosomal compartment of such cells is markedly was used for deconvoluting data. Image quantification reduced in size and that these cells release protein as was done with Data Inspector program in SoftWorx or exosomes in larger amounts and export CDDP via this by NearCount software. route at increased levels. The resistant cells also abnormally Exosome Harvest sort some lysosomal proteins, including putative CDDP Exosomes were isolated from 2008 and 2008/C13*5.25 transporters, into the exosomal pathway that also exports cells according to the method described by Escola et al. (29) CDDP. with minor modifications. Cells were cultured in 100-mm dishes until 80% confluent. They were then rinsed with Materials and Methods serum-free RPMI 1640 to remove proteins and vesicular Reagents structures already released from the cells or present in the Cell culture media and sera were purchased from Invi- fetal bovine serum. In parallel, one set of cells received trogen (Carlsbad, CA). Antibodies to lysosome-associated fresh serum-free RPMI 1640 alone, whereas another set proteins 1 and 2 (LAMP1 and LAMP2), h-actin, and tubulin was incubated with RPMI 1640 plus 2 Amol/L CDDP for were obtained from Santa Cruz Biotechnology, Inc. (Santa 1 hour in a 37jC humidified tissue culture incubator. The Cruz, CA), and those for MRP2 were from Alexis Bio- supernatants were then removed and the plates rinsed chemicals (San Diego, CA). The specific dyes Alexa Flour thrice with warm RPMI 1640; and then 12 mL of fresh 647 phalloidin, Hoechst 33342, and Lysotracker Red were serum-free RPMI 1640 was layered over cells in each purchased from Molecular Probes (Eugene, OR). Fluores- plate; and cells were incubated for 1 hour at 37jC. cein-conjugated secondary antibodies against mouse, The supernatant from each plate was then transferred to rabbit, and goat immunoglobulin were purchased from a 15-mL conical tube and subjected to two successive Jackson ImmunoResearch Laboratories, Inc.
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