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apeutics, Targets, and Chemical Biology

ew Paradigm for Aptamer Therapeutic AS1411 Action:ake by Macropinocytosis and Its Stimulation by a

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leolin-Dependent Mechanism

rit Reyes-Reyes1, Yun Teng1, and Paula J. Bates1,2

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411 is a first-in-class anticancer agent, currently in phase II clinical trials. It is a quadruplex-formingeoxynucleotide that binds to nucleolin as an aptamer, but its mechanism of action is not completelystood. Mechanistic insights could lead to clinically useful markers for AS1411 response and to noveled therapies. Previously, we proposed a model where cell surface nucleolin serves as the receptor for1, leading to selective uptake in cancer cells. Here, we compare uptake of fluorophore-labeled AS14111411) in DU145 prostate cancer cells (sensitive to AS1411) and Hs27 nonmalignant skin fibroblastsant to AS1411). Uptake of FL-AS1411 occurred by endocytosis in both cell types and was much morent than an inactive, nonquadruplex oligonucleotide. Unexpectedly, uptake of FL-AS1411 was lower incells compared with Hs27 cells. However, the mechanism of uptake was different, occurring by macro-tosis in cancer cells, but by a nonmacropinocytic pathway in Hs27 cells. Additionally, treatment ofs cancer cells with AS1411 caused hyperstimulation of macropinocytosis, provoking an increase in itsptake, whereas no stimulation was observed for nonmalignant cells. Nucleolin was not required forFL-AS1411 uptake in DU145 cells but was necessary for induced macropinocytosis and FL-AS1411at later times. Our results are inconsistent with the previous mechanistic model but confirm thatlin plays a role in mediating AS1411 effects. The data suggest a new model for AS1411 action as well
nucleo
as a new role for nucleolin in stimulating macropinocytosis, a process with potential applications in drugdelivery. Cancer Res; 70(21); 8617–29. ©2010 AACR.

OurGROsstructand sedirecthibitoabilitylogicaabilitylin-coninteraed tha

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411 is a 26-base guanine-rich oligonucleotide (GRO)n unmodified (phosphodiester) DNA backbone. Thisule and its related analogues can inhibit proliferationduce cell death in many types of cancer cells but haveffect on normal cells (1–4). Biological effects of thisf molecules in cancer cells include cell cycle arrest, in-n of NF-κB signaling, induction of tumor suppressorxpression, and reduction of bcl-2 expression (3–6). AI clinical trial of AS1411 (formerly known as AGRO100)ients with metastatic cancer has indicated no serious

ith promising clinical activity, and AS1411 isd in phase II trials (2).

DesmechamarkeNucleolus aand oand ahas becellulamaligBasediatesoriginfor A

s: Departments of 1Medicine and 2Biochemistry andJames Graham Brown Cancer Center, University of

e, Kentucky

tary data for this article are available at Cancerttp://cancerres.aacrjournals.org/).

uthor: Paula J. Bates, University of Louisville, 505treet, CTRB 409, Louisville, KY 40202. Phone:: 1-502-852-3661; E-mail: [email protected].

5472.CAN-10-0920

ssociation for Cancer Research.

ls.org

Research. on March 10, 202cancerres.aacrjournals.org ded from

previous work has revealed that antiproliferativesuch as AS1411 can form stable guanine quadruplexures, which imparts an unusual resistance to cellularrum nucleases. We have also shown that GROs bindly and selectively to nucleolin and that the growth in-ry activity of GROs is positively correlated with theirto bind this protein (1, 7, 8). In addition, several bio-l effects of AS1411 have been shown to result from itsto alter the subcellular localization of certain nucleo-taining complexes or to interfere with the molecularctions of nucleolin (3, 5, 6). Therefore, we have conclud-t AS1411 acts as an aptamer to nucleolin.pite its molecular target having been identified, thenism of action for AS1411 and the reasons for itsd tumor selectivity have not yet been fully elucidated.olin is a multifunctional protein present in the nucle-nd nucleus of most cells, as well as in the cytoplasmn the surface for some cells, including cancer cellsngiogenic endothelial cells (2, 9, 10). This proteinen reported to control a wide range of fundamentalr processes (11), as well as having important roles innant transformation and cancer progression (12).on the knowledge that cell surface nucleolin med-

the endocytosis of a wide range of ligands, we had
ally speculated that nucleolin could act as a receptorS1411 and that, due to the elevated expression of
8617

0. © 2010 American Association for Cancer

http://cancerres.aacrjournals.org/

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lin by cancer cells, uptake of AS1411 would occur inor-selective manner (13).his study, we evaluated uptake of fluorescently labeled1 in several cell types and compared the mechanism ofbetween DU145 prostate cancer cells, which are sen-

to AS1411, and Hs27 nonmalignant skin fibroblasts,are resistant to AS1411. We now describe the resultsresearch, including our unexpected findings with re-

o the uptake of AS1411 and the role of nucleolin in itsnism of action.

rials and Methods

ialsodeoxynucleotides were purchased from Invitrogen.nces used for this study were AS1411, 5′-d(GGTGGT‐GTTGTGGTGGTGGTGG) and fluorophore-labeled1 (FL-AS1411); 5′-fluor-d(TTTGGTGGTGGTGGTTG‐GGTGGTGG) [wherein fluor is either 5-carboxyfluores-AM; used for flow cytometry) or Alexa Fluor 488 (usedfocal microscopy]; tAS1411, 5′-d(TTTGGTGGTGGTGG‐GGTGGTGGTGG); FL-CRO, 5′-fluor-d(TTTCCTCCTCC‐TCTCCTCCTCCTCC); CRO, 5′-d(CCTCCTCCTCCTT‐TCCTCCTCC); and tCRO, 5′-d(TTTCCTCCTCCTC‐TCCTCCTCCTCC). Unmodified oligonucleotides weresed in the desalted form, whereas fluorescently labeledces were high-performance liquid chromatography pu-The 29-mer sequences were used for some experimentsse quenching of the fluorophore occurred when it wasd adjacent at the 5′ terminal base of AS1411,3 so a spac-sisting of three thymidines was added. We confirmede antiproliferative activities of 29-mer sequences, withithout the fluorophore, were comparable with the syn-d 26-mer AS1411 sequence and with AS1411 obtainedntisoma (Supplementary Fig. S1A). The dextran (10,000ular weight) anionic fixable (dextran-10K) and transfer-njugated with Alexa Fluor 488 or Alexa Fluor 594 wereased from Invitrogen. Antirabbit and antimouse anti-linked to horseradish peroxidase and anti-histone 3polyclonal and anti-pan cadherin (C19) goat polyclonaldies were from Santa Cruz Biotech. Anti-nucleolinlonal antibodies (mAb) were from Stressgen (4E2) andCruz (MS-3). The anti-nucleolin mAb (D3) was a gener-ft from Dr. Jau-Shyong Deng, University of Pittsburghl of Medicine. Cytochalasin D, dynasore, and amiloriderom Calbiochem. Triton X-100 was from Sigma, parafor-hyde was from Electron Microscopy Sciences, andwas from American Type Culture Collection (ATCC).

ulture and treatmentcells were obtained from ATCC and routinely grown inidified incubator at 37°C with 5% CO2. Hs27 (non-nant human foreskin fibroblasts), DU145 (hormone
ory prostate cancer), HeLa (cervical adenocarcinoma),(hormone-dependent breast cancer), and MDA-MB-
with a

BiotinCel

freshlyReyes and Bates, unpublished observations.

r Res; 70(21) November 1, 2010

Research. on March 10, 202cancerres.aacrjournals.org Downloaded from

ormone-independent breast cancer) cells were grownEM supplemented with 10% fetal bovine serum (Lifeologies), 62.5 μg/mL penicillin, and 100 μg/mL strepto-(Hyclone Laboratories). MCF-10A cells (immortalizedn breast epithelial cells) were grown in MEBM supple-d with all the components of MEGM bullet kit (Lonza)for GA-1000. We did not carry out additional testing tonticate cell lines, but their morphology and behavioronsistent with ATCC descriptions. Cells were platedconfluence and incubated for 18 hours to allow adher-nd then the medium was changed for fresh supplemen-edium and treated by addition of oligodeoxynucleotidesly to the culture medium to give the final concentrationted in the figure legends. Dynasore and cytochalasin Dissolved in DMSO. Amiloride was dissolved in serum-edium. Cells were pretreated with inhibitors in serum-edium for either 30 minutes (cytochalasin D, dynasore)inutes (amiloride). Cells for biochemical analyses were

in lysis buffer [150 mmol/L NaCl, 2 mmol/L EDTA, 50/L Tris-HCl, 0.25% deoxycholic acid, 1% IGEPAL CA-H 7.5)] containing protease and phosphatase inhibitorils (Calbiochem) for 20 minutes at 4°C and then clearedtrifugation at 16,000 × g for 10minutes at 4°C. All proteinntrations were determined using the bicinchoninic acid(Pierce).

cytometric assayss (2 × 105) in fresh complete medium were plated intoll plates for 18 hours. After complete adhesion, cells wereated as indicated in the figures or legends. Cells wered once with ice-cold PBS, incubated with 1 μg/mLo-actinomycin D or 1 μg/mL propidium iodide (PI) forutes on ice to allow exclusion of nonviable cells, andd twice with ice-cold PBS. To harvest, cells were treated.01% trypsin/0.5 mmol/L EDTA (300 μL) for 3 minuteso the addition of 3 mL supplemented culture medium.ere then centrifuged and resuspended in 0.5 mL of 1%rmaldehyde for analysis by flow cytometry using aalibur cytometer (BD Biosciences).

nofluorescence microscopys (4 × 104) in fresh complete culture mediumwere platedmm diameter glass coverslips for 18 hours. The mediumplaced with serum-free medium, and cells were treatedcribed in the figure legends. After incubation, cells wered three times with ice-cold PBS, fixed in 4% paraformal-e in PBS for 30 minutes at room temperature, andd three times with PBS. After washing, the coverslipsounted on glass slides with ProLong Antifade (Mole-

Probes) according to the manufacturer's directions.nofluorescence was documented with an LSM 510 in-confocal laser scanning microscope (Carl Zeiss) equippednOmnichrome argon-krypton laser. Imageswere obtainedZeiss Plan-Apo 63X oil immersion objective (1.4 NA).

ylation and purification of cell surface proteins
ls were washed three times with ice-cold PBS, and aprepared solution of a cell-impermeable biotinylating
Cancer Research



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Role of Macropinocytosis in AS1411 Uptake and Mechanism

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(sulfo-NHS-biotin, Pierce) was added (0.5 mg/mL inAfter 30 minutes of incubation at 4°C, cells wered once with ice-cold TBS [50 mmol/L Tris-HCl, 150/L NaCl (pH 7.5)], incubated with ice-cold completem for 10 minutes at 4°C, and then washed twice withiotinylated proteins were precipitated by incubatingigh-capacity NeutrAvidin agarose (Pierce) for 2 hourswith gentle agitation and then washed with ice-colduffer.

nterferenceleolin small interfering RNA (siRNA) duplexesponding to the sequences 5′-GGUCGUCAUACCUCA-tt (NCL1), 5′-GGCAAAGCAUUGGUAGCAAtt (NCL2),′-CGGUGAAAUUGAUGGAAAUtt (NCL3) were chemi-synthesized and annealed by Ambion, Inc. BLASTis showed no homology of the siRNA sequences tother sequence in the Human Genome Database.iRNAs were transfected using Lipofectamine 2000rogen) according to the manufacturer's directions.rambled siRNA used as a negative control was fromn.

noblottingples were resolved by 10% SDS-Tris PAGE and thentransferred onto polyvinylidine fluoride membranesore) in Tris-glycine buffer containing 20% methanol.ns were detected by immunoblotting as describedsome cases, membranes were stripped of bound anti-using 62.5 mmol/L Tris-HCl (pH 6.7), 100 mmol/L

captoethanol, and 2% SDS for 30 minutes at 60°C andeprobed as described in figure legends.

tometry and statistical analysissitometry was used to measure band intensities bying autoradiographic films and using UN-SCAN-IT gelre (Silk Scientific Corporation). Band intensities werelized as indicated in the figure legends. The statisticalarisons between AS1411-treated and control groupserformed using Student's t test.

lts

e of FL-AS1411 occurs through an activee processfirst identify suitable conditions for our study, we ana-the timing and serum dependence of uptake in DU145te cancer cells, which are sensitive to AS1411. Using flowetry with gating to exclude nonviable cells, we com-uptake of FL-AS1411, a fluorescently labeled version oftive aptamer, with that of FL-CRO, a fluorescentlyd control oligonucleotide with no antiproliferativey. It has not been possible to identify a scrambled ormutated analogue of AS1411 that lacks activity becausevery high proportion of guanines in the sequence.

fore, a sequence where the guanines are substitututed
ytosines (FL-CRO) is used simply as a control phospho-r oligodeoxyonucleotide of the same length. We first
mentAS141

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med that the fluorophore signal was from internalized,than surface-bound, material by showing that cell-ated fluorescence was not influenced by washing theith dextran sulfate to remove the extracellular oligonu-e or by adding trypan blue to quench external fluores-ignals before flow cytometry (Supplementary Fig. S2A).AS1411 uptake was detected as early as 5 minutes, withum signal between 2 and 4 hours under these condi-(Fig. 1A). FL-CRO uptake was consistently much lowerL-AS1411 and followed different kinetics. These dataaccord with our recent finding that guanine-richhodiester sequences, such as AS1411, are taken upefficiently than other non–guanine-rich sequencess shown in Fig. 1B, uptake of FL-AS1411 was indepen-f the presence of serum in the medium.etermine whether AS1411 uptake occurs through an ac-ptake process, we evaluated the temperature depen-of AS1411 uptake in various cancer cell types andalignant Hs27 skin fibroblasts. In all cell types examined,take of FL-AS1411 and FL-CRO showed strong temper-dependence. However, in contrast to our original hy-sis, Hs27 cells seemed to have a higher uptake of1 than any of the cancer cells analyzed (Fig. 1C). Theesponse curve for FL-AS1411 uptake in DU145 cellslementary Fig. S2B) suggested at least two components:e involving a high-affinity, low-capacity receptor, whichturated at high nanomolar concentrations, and a non-ble uptake that was predominant at micromolar con-tions (the curve presented an almost linear increaseen 1.25 and 40 μmol/L). Concentrations higher than 40L resulted in obvious cytotoxicity, even at this early timeWe believe that the nonsaturable uptake component iselevant for AS1411 activity, because this corresponds tontrations at which the biological effects are observed ined cancer cells and to the serum concentrations ints treated with AS1411.

1411 uptake occurs through different endocyticanisms in cancer cells and in nonmalignant cellsconfirm that uptake of AS1411 occurs by endocytosis,ted for involvement of the actin cytoskeleton, whicheen implicated in regulating endocytic pathways. Tod, DU145 and Hs27 were pretreated with an actin po-zation inhibitor, cytochalasin D, and assessed for FL-1 uptake by flow cytometry. Cytocholasin D–treatedhowed a decrease in FL-AS1411 uptake compared withtreated cells (Fig. 2A), consistent with endocytic up-Recognized pathways of endocytosis include classicin-mediated endocytosis, caveolae-mediated endo-s, clathrin- and caveolae-independent endocytosis,acropinocytosis (16). The GTPase dynamin is requiredthrin- and caveolae-mediated endocytosis and somein- and caveolae-independent pathways (16), so wenvestigated the effect of dynasore, a potent inhibitoramin function (17), on FL-AS1411 uptake in DU145r cells and nonmalignant Hs27 cells (Fig. 2B). Pretreat-
of Hs27 cells with dynasore decreased their uptake of1 (Fig. 2B). In contrast, pretreatment of DU145 cells
Cancer Res; 70(21) November 1, 2010 8619



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Reyes-Reyes et al.

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y increased their uptake of FL-AS1411. To rule out theility that DU145 cells were unresponsive to dynasore,owed that uptake of transferrin, a well-establishedof clathrin-dependent endocytosis, was inhibited

145 cells pretreated with dynasore (SupplementaryA). These results indicate that AS1411 may be takena predominantly clathrin- or caveolae-dependent routery in Hs27 cells, but not in DU145 cells.

pinocytosis is the predominant mechanism ofe for AS1411 in cancer cellsent work has shown that internalization of DNA candiated through macropinocytosis (18–20), an actin-, ligand-independent mechanism in which cells “gulp”

or 4°C for 2 hours. Mean fluorescent intensities at 37°C were as follows:ents were repeated at least three times. Data are mean of three independ

rrounding medium and any macromolecules it con-This endocytic mechanism has been shown to be sen-

in the(Fig. 3



to amiloride, a specific inhibitor of Na+/H exchangeo we tested the effect of amiloride on FL-AS1411 up-e found that amiloride pretreatment caused a reduc-FL-AS1411 uptake in DU145 cancer cells but a slight

se in the nonmalignant Hs27 cells (Fig. 2C). There wasffect of amiloride treatment on uptake of FL-CRO inDU145 or Hs27 cells (Supplementary Fig. S3B). Amilor-atment also decreased AS1411 uptake in other cancerMCF7 and MDA-MB-231, data not shown). These datathat macropinocytosis is responsible for the internali-of AS1411 in cancer cells.confirm these results without using chemical inhibi-ptake was also examined by confocal microscopy. Thisd that FL-AS1411 was localized in confined structures

5, 10.8; HeLa, 10.7; MDA-MB-231, 11.9; Hs27, 35.2. Allmples; bars, SEM.

1. AS1411 cell internalization is an active process. Cells were treated as described below and analyzed by flow cytometry. A, DU145 cells wereed at 37°C with complete medium containing 10 μmol/L FL-AS1411 (black line) or 10 μmol/L FL-CRO (gray line) for the time indicated.45 cells were incubated for 2 h at 37°C in complete or serum-free medium containing 10 μmol/L FL-AS1411, 10 μmol/L FL-CRO, or nocleotide. C, various cell lines were incubated with 10 μmol/L FL-AS1411 (black outline), 10 μmol/L FL-CRO (gray outline), or without DNA (solid gray)

cytoplasm of both cancer and nonmalignant cellsA). As expected, uptake of FL-CRO was much lower

Cancer Research



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L-AS1411 (Fig. 3B). Interestingly, our studies showedacropinocytosis (indicated by dextran uptake) is muchactive in DU145 cancer cells than in the nonmalignantcells (Fig. 3). Moreover, internalized FL-AS1411 wasly colocalized with the macropinocytic marker dextran145 cells (Fig. 3A), but not in Hs27 cells (Fig. 3A).ther experiments were performed to confirm the identityvesicles containing FL-AS1411 as macropinosomes,can be distinguished from other endosomes by theirrative inability to concentrate receptors (22). Therefore,pared localization of transferrin, a ligand for the trans-

receptor, with that of dextran or FL-AS1411.We observedansferrin and dextran were localized mainly in distincterlapping vesicles in DU145 and Hs27 cells (Fig. 3C) andhere was very little overlap between transferrin and1 in DU145 cells (Fig. 3D). These combined data confirm
e endocytic process regulating the internalization of1 in DU145 cancer cells is macropi
specif

ed cells.

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also notable that no FL-AS1411 was observed in ther region in these studies. This was in contrast to our(unpublished) studies using fluorescence microscopy,we had observed some cells with intense nuclear fluo-ce after 24 hours or more treatment with 10 μmol/Labeled AS1411. This pattern was observed in canceres, but not in the nonresponsive Hs27 cells. We initial-rpreted this result as preferential uptake in cancerbut later realized that the cells with strong nuclearg were likely dying in response to AS1411 and wereore permeable, whereas the lack of intense nuclearng in Hs27 cells reflected that they did not die inse to AS1411. The current confocal microscopy andytometry studies (which are gated to exclude deading cells that are PI positive due to loss of plasmarane integrity) show that overall uptake is not cancer
ic and that AS1411 has a nonnuclear localization in
nocytosis. viable cells.

2. AS1411 is internalizedrent endocytic mechanisms45 cancer cells andignant Hs27 cells. DU145cells were pretreated as

ed below with inhibitoristogram) or theonding vehicle controlistogram) before additionmol/L FL-AS1411 andion at 37°C for 2 h. Cellsen harvested and analyzedcytometry. Pretreatmentns were at 37°C withL cytochalasin D for 30 minμmol/L dynasore for 30 minmmol/L amiloride forAll experiments wered at least three times, andntative data are shown.ray histograms representund autofluorescence of




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3. AS1411 colocalizes withcropinocytic marker dextran.or Hs27 cells were incubatede reagents indicated below,ashed, and fixed. Nucleitained with 4′,6-diamidino-2-ndole (DAPI; blue) anded by confocal microscopy.mol/L AS1411 labeled withluor 488 (green) and/mL dextran-10K labeled withluor 594 (red) for 2 h at 37°C.lar experiments using FL-CROe of FL-AS1411. C, cellsted with 5 μg/mL transferrinwith Alexa Fluor 488 (green)mg/mL dextran-10K labeled

exa Fluor 594 (red) for 30 min. D, DU145 cells incubatedμg/mL transferrin labeled withluor 594 (red) and 10 μmol/Llabeled with Alexa Fluor 488

for 30 min at 37°C. Scale bars,The magnified insets

Cancer Research

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sis, incknowntheir ohave rtosis cvacuoAS141ly theAS141nonmcatedcytic m(which48, orcells (were gsibilitydextrathe cofor shtAS141pinocy231) atype (Mtionssponsstudieconfira hightreatery Fig.able tthreeals anThi

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1 stimulates macropinocytosis in cancer cellsral molecules that are internalized by macropinocyto-luding some cell penetrating peptides and viruses, areto stimulate macropinocytosis and thereby enhancewn uptake (23–25). Moreover, several research groupsecently shown that hyperstimulation of macropinocy-an cause a novel form of cell death, characterized bylization, irregular nuclei, and swollen cells (26–29), and1 causes changes in cancer cell morphology with exact-se features (4). Therefore, we investigated whether1 could stimulate macropinocytosis in DU145 cells oralignant Hs27 cells. Flow cytometry experiments indi-a significant increase in the uptake of the macropino-arker dextran in DU145 cells treated with tAS1411is FL-AS1411 without the fluorescent label) for 24,

72 hours, whereas there was no increase in the Hs27Fig. 4A). As in all our flow cytometry experiments, cellsated to exclude permeable cells, discounting the pos-that this increase was due to cell death. No changes inn uptake were observed in DU145 cells treated withntrol oligonucleotide tCRO (Fig. 4A) or with AS1411orter times (1, 2, and 4 hours, data not shown). The1 was also able to induce hyperstimulation of macro-tosis in other cancer cells lines (MCF-7 and MDA-MB-nd had a reduced effect in another nonmalignant cellCF-10A; Fig. 4B), suggesting that these novel observa-

may represent a general difference between the re-e of cancer cells and normal cells, although furthers are needed to verify this idea. Confocal microscopymed that DU145 cells treated with tAS1411 presenteder dextran uptake compared with untreated or CRO-d cells (Fig. 4C). Additional experiments (Supplementa-S4) confirmed that the 26-mer version of AS1411 waso induce the same response as tAS1411 (which hasadditional nucleotides for reasons described in Materi-d Methods).s result also implies that AS1411 might actually pro-ts own internalization by cancer cells. To test this idea,etreated DU145 cells for 24 hours with or without1, then added FL-AS1411, and evaluated uptake afterditional 2 hours using flow cytometry. As predicted,cells pretreated with tAS1411, but not those that re-control pretreatment, showed an increase in the up-f FL-AS1411 in DU145 cells, whereas there was norable increase in AS1411-treated Hs27 cells (Fig. 4D).these results indicate that initial AS1411 uptake leadsstimulation of macropinocytosis, inducing an increaseown uptake. This idea is not necessarily inconsistenthe time course data (Fig. 1A), because the measuredscence signal may decrease over time for a numberons, including exocytosis of the ligand or fluorescencehing due to environmental factors such as proteing or pH.ing shown that AS1411-induced macropinocytosislead to enhanced uptake of a nucleic acid (AS1411)polysaccharide (dextran), we also evaluated uptake of a
n, namely, fluorescently labeled transferrin. We observedretreatment with AS1411 led to increased uptake of
resultsa bett

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errin in DU145 cells (Supplementary Fig. S5). Althoughe of transferrin in untreated cells occurs by dynamin-dent receptor-mediated endocytosis (Supplementary3 and S5), the additional uptake induced by AS1411parently due to macropinocytosis, because it was com-inhibited by amiloride (Supplementary Fig. S5).

l uptake of AS1411 is independent of nucleolinhave shown that nucleolin is the primary molecularof AS1411 and had previously hypothesized that sur-ucleolin may serve as a receptor for AS1411 (2, 13).ver, the new data are not consistent with that hypoth-ecause they indicate that uptake occurs not by classictor-mediated endocytosis but by macropinocytosis.fore, we were curious to learn whether nucleolin playedin AS1411 uptake. We first assessed the effect of anucleolin mAb (D3, which we confirmed could bind toe nucleolin) on uptake of FL-AS1411 in DU145 cells af-ours of incubation and found no effect (Supplementary6). Next, we carried out similar experiments usings to knock down expression of nucleolin. Immunoblotes confirmed that expression of total nucleolin coulduced by >80% in cells transfected with nucleolins compared with control-transfected cells (Fig. 5A).so showed that these siRNAs could effectively knockthe cell surface form of nucleolin (Fig. 5B), using tech-s described in the Materials and Methods. We nextthe transfected DU145 cells to assess the uptake of1411 after 2 hours by flow cytometry analysis andthat knockdown of nucleolin had no effect on FL-1 uptake under these conditions (Fig. 5C).

olin regulates AS1411-induced stimulationcropinocytosisresults (Fig. 4) suggest that induction of macropinocy-may be an important component of AS1411 activity.fore, we also determined whether nucleolin knockdownthe tAS1411-mediated stimulation of macropinocysto-U145 cells. As shown in Fig. 6A, inhibition of nucleolin

ssion by specific siRNAs had only a marginal effecte baseline macropinocytosis but caused a significantse in AS1411-induced macropinocytosis, almost com-y blocking this process. Accordingly, the tAS1411-ed uptake of FL-AS1411 was also completely blocked145 cells transfected with nucleolin siRNAs (Fig. 6B).results indicate that, whereas nucleolin does not seemy a role in the initial macropinocytic uptake of AS1411,ssential for the AS1411-induced stimulation of macro-

d uptake of AS1411 that occurs at later time points.

ssion

411 is a first-in-class experimental drug that hasy been tested in early clinical trials with promising
. The primary goal of our current research is to gainer understanding about the mechanism of AS1411



Figure10 μmowith frecytomeexperimfor 48 hwith coDAPI. Sthen waSolid g

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4. AS1411 stimulates macropinocytosis in DU145 cancer cells, but not in nonmalignant Hs27 cells. A, DU145 or Hs27 cells were treated withl/L tAS1411, 10 μmol/L tCRO, or no oligonucleotide in complete DMEM at 37°C for the time indicated. After treatment, medium was replacedsh medium containing 0.2 mg/mL dextran-10K labeled with Alexa Fluor 488 for 30 min at 37°C, and then cells were harvested and analyzed by flowtry to determine dextran uptake. Data are mean of three independent samples; bars, SEM. *, P < 0.05 compared with controls. B, the sameent was performed using MCF7 and MDA-MB-231 breast cancer cells or MCF10A nonmalignant breast epithelial cells. C, DU145 cells were treatedas described above, then washed with cold PBS, and incubated in PBS containing 5 μg/mL PI and incubated on ice for 5 min. After washing

ld PBS, cells were fixed and the distribution of macropinocytic marker (green) was visualized by confocal microscopy. Nuclei were stained withcale bars, 10 μm. D, DU145 and Hs27 cells were incubated without oligonucleotide (gray line) or with 10 μmol/L tAS1411 (black line) for 48 h,
shed, and incubated at 37°C with fresh complete medium containing 10 μmol/L FL-AS1411 for 2 h before harvesting and analysis by flow cytometry.ray histograms represent background autofluorescence of unstained cells.
r Res; 70(21) November 1, 2010 Cancer Research

Research. on March 10, 2020. © 2010 American Association for Cancercancerres.aacrjournals.org Downloaded from


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y, which we believe is important for several reasons.ledge of the factors that determine response to1 might ultimately be translated into clinical tests thatidentify patients with the best chance of benefitingreatment with AS1411. Given the difficulties of achiev-nically relevant dosing of AS1411 in animal models (i.e.,y continuous i.v. infusion), understanding how AS1411in cultured cancer cells will likely be the most expedi-oute to identify candidate biomarkers, which can thenluated retrospectively or prospectively in human clin-als of AS1411. In addition, comprehending how AS1411to preferentially affect cancer cells compared with non-ant cells may lead to new insights into cancer biology.ticular, AS1411 has turned out to be a valuable tool tothe functions of nucleolin, a protein that is increasinglyrecognized as an important target in cancer therapy. Al-

ing no oligonucleotide (gray dashed line), 10 μmol/L FL-CRO (gray solid linore harvesting and analysis by flow cytometry.

our investigations of AS1411 have revealed previouslygnized functions for nucleolin in NF-κB signaling (3)

Theseuptak

acrjournals.org


MT5 localization (6) and have now led to the discoveryther novel role in stimulating macropinocytosis.viously, we identified nucleolin as the target of AS1411bsequently predicted that it would act as a receptor1411 (2, 13). This hypothesis was attractive becausevided a possible explanation for the tumor selectivity411 based on the reasoning that nucleolin expressionher in cancer cells compared with normal cells, andore, we expected there would be selective uptake of1 in cancer cells. However, the new data presented herethat these hypotheses were incorrect. We found thatuptake of AS1411 is not higher in cancer cells com-with Hs27 nonmalignant skin fibroblasts that are notsive to AS1411. Instead, it seems that initial uptake of1 in cancer cells occurs via macropinocytosis, wherease of AS1411 occurs by other mechanisms in Hs27 cells.

10 μmol/L FL-AS1411 (black solid line) and incubated at 37°C for

5. AS1411 uptake after 2 h is not affected by knockdown of nucleolin expression. DU145 cells were transfected for 48 h without siRNA (mock, M),nmol/L of one of three different nucleolin siRNAs (NCL1, NCL2, and NCL3), a control siRNA (scramble, S), or contransfected with 10 nmol/L ofcleolin siRNAs (mix). A, cells were lysed, and total cell lysates were analyzed by immunoblotting using the antibodies shown. B, cell surface proteinstact transfected DU145 cells were labeled and captured as described in Materials and Methods and then analyzed by blotting with anti-nucleoliny (NCL). After stripping, the membrane was reprobed with antibodies for a plasma membrane marker (anti-pan Cadherin) and a nuclear marker3) to confirm fractionation. Total lysate (Lys) was used as control. C, the medium of transfected cells was replaced by fresh complete medium

intriguing results suggest it may be the mechanism ofe, as opposed to the level of uptake, that is key to




determTherecer senocytocell linmineThus fsubseqrespontake bsis areof poslievedand th

30). Tmacroescapeby nonsomal(or inAS141cells lhas b(27–29mentpinocy

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taineledubatfterlacediumAS12 h.lyzean flnotpretmeplepas pretreated with tAS1411.

4 E.M. R

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ining whether a cell is sensitive to AS1411 activity.fore, we now advocate a new model to explain the can-lectivity of AS1411, which involves uptake by macropi-sis in cancer cells (Fig. 7). Studies using other canceres and nonmalignant cell types are ongoing to deter-whether the results observed here can be generalized.ar, we have observed uptake by macropinocytosis anduent stimulation of macropinocytosis in all cells thatd to AS1411.4 Although we do not know yet why up-y macropinocytosis and stimulation of macropinocyto-so important for AS1411 activity, there are a numbersibilities that seem plausible. Macropinosomes are be-
o be leaky compared with other endosomal vesicles,ir cargo can bypass degradative processing (22, 25,
applieis prethe mcludin

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an anendoceyes-Reyes and P.J. Bates, in preparation.



herefore, the response of cells that take up AS1411 bypinocytosis could be related to the ability of AS1411 tofrom macropinosomes in these cells, whereas uptakemacropinocytic pathways is expected to lead to endo-trapping and/or lysosomal degradation. Alternativelyaddition), it is possible that the selective effects of1 on cancer cells occurs because treatment of theseeads to hyperstimulation of macropinocytosis, whicheen recently described as a novel form of cell death). Final proof of these ideas must await the develop-of new approaches for long-term inhibition of macro-tosis, because the current inhibitors are toxic whend to cells for more than a few hours. However, therecedent for the idea that drug activity can depend onechanism of uptake, rather than uptake efficiency, in-g a recent example showing that biological activity of

FigAS1mawerwith30nucNCAfteinc10oligmetreawithconlabincB, arepmeFL-foranameto “noaresamcomcell

tisense oligonucleotide was depytic internalization (31).

0. © 2010 American Associa

6. Nucleolin regulates-induced stimulation ofinocytosis. DU145 cellst transfected or transfectedsiRNA (mock) or withl/L of one of three differentin siRNAs (NCL1, NCL2,or control siRNA (scramble).h transfection, cells were

ed with 10 μmol/L tAS1411,l/L tCRO, or nocleotide in completeat 37°C for 24 h. A, after

nt, medium was replacedsh complete mediuming 0.2 mg/mL dextran-10Kwith Alexa Fluor 488 anded at 37°C for 30 min.treatment, medium wasd with fresh completecontaining 10 μmol/L

411 and incubated at 37°CCells were harvested andd by flow cytometry, anduorescence was normalizedtransfected” control (A) orreatment control (B). Dataan of three independents; bars, SEM. *, P < 0.05red with mock-transfected

endent on the route of

Cancer Research

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Direthe anbecaubut ounucleoin canregularemaian earservedmightbe relvariou(5, 12was n

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ct confirmation of the role of nucleolin in mediatingtiproliferative effects of AS1411 is similarly challengingse long-term inhibition of nucleolin is also cytotoxic,r new data clearly point to an essential function forlin in AS1411's ability to stimulate macropinocytosiscer cells. The precise mechanism by which nucleolintes AS1411-induced macropinocytosis in cancer cellsns unknown. Macropinocytosis stimulation was notly cellular response to AS1411 but was typically ob-after 24 to 48 hours of treatment, suggesting that itrequire changes in protein expression. Thus, it mayevant that nucleolin can regulate gene expression bys transcriptional and posttranscriptional mechanisms
, 32–35). Our confocal images showed that AS1411ever localized in the nuclear region of cells (except
authoMV-4-

t AS1411 uptake route leading to endosomal entrapment or lysosomal degradatioells because they have lower levels of nucleolin. Further research is required to

acrjournals.org


ells) even after treatment for more than 2 days (dataown). Presumably then, the effects of AS1411 are onuclear nucleolin functions, such as shuttling, signaluction, or modulating mRNA stability, which couldffect protein expression. This idea is entirely consistentur previously proposed hypothesis that the effects of1 stem from it binding to a subset of nucleolin com-and thereby interfering with certain functions of nu-(2, 13). Interestingly, nucleolin has been implicated intake of plasmids and DNA nanoparticles in previousndent studies (20, 36), although the relevance of these411 is unknown at present. Another recent publicationported nucleolin as a receptor for AS1411 (37). These
rs reported that binding of AS1411 to the surface of11 leukemia cells was inhibited by preincubation with
7. A new model to explain the cancer-selective antiproliferative activity of AS1411. A cartoon comparing the previous model for AS1411 uptakeevised model that is consistent with the new results. Key features of the new model are that it is the mechanism of uptake (rather than the levels ofthat determine response to AS1411 and that uptake occurs by macropinocytosis in cancer cells. In the new model, nucleolin does not act as acell surface receptor but is essential for the stimulation of macropinocytosis by AS1411, which leads to further uptake of the aptamer. Weesize that, in cancer cells, uptake by macropinocytosis allows endosomal escape of AS1411 (because macropinosomes are relatively leaky) and/or1411 induces cell death due to hyperstimulation of macropinocytosis. We propose that the lack of response in normal cells may be due to the

n and/or the inability of AS1411 to stimulate macropinocytosis intest these hypotheses.




a nuclhours)fectedseemwhere(D3)AS141Fur

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macro(one ocells wprogrewill heour replasmthat mnakednovel findings regarding AS1411 might have broad implica-tions

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eolin antibody (MS3) and that uptake (measured at 2–6was reduced in MCF-7 cells that were stably trans-with nucleolin short hairpin RNA (37). These resultsto be at variance with our findings in DU145 cells,neither an antibody that binds surface nucleolin

nor nucleolin siRNA had any inhibitory effect on1 uptake at 2 hours (Fig. 5; Supplementary Fig. S5).ther research to explore the mechanism and applica-of AS1411-induced macropinocytosis seems to bewhile. Macropinocytosis has been most widely ex-in immune cells, such as macrophages and dendritic

where it serves to “sample” the extracellular fluid foresence of pathogens, but is also induced by oncogenesrowth factor signaling (16, 28, 29, 38, 39), so it is per-nsurprising that it occurs in cancer cells. Moreover,pinocytosis is emerging as a key mechanism for intra-r delivery of various physiologic, pathologic, and ther-c cargoes. This process has been reported as an entryfor a range of macromolecules, including numerousenetrating peptides (23, 40), intact proteins (41–43),ial virulence factors (44), Gram-negative bacterial lipo-ccharide (45), naked DNA plasmid (18–20), and manys (46). Remarkably, the uptake and effects of AS1411mewhat reminiscent of some cell penetrating peptidescalled protein transduction domains) and viruses,can be taken up by macropinocytosis and then causeation of macropinocytosis (23–25). Furthermore, therestantial interest in the uptake mechanisms of oligonu-es in general, largely because poor cellular delivery is ahurdle in developing oligonucleotide-based therapeu-
cluding siRNAs (47). Interestingly, one of the most
iRNA to date may involveRece

OnlineF

h backbone and sugar modifications. Biochemistry 2002;41:6–85.ristian S, Pilch J, Akerman ME, Porkka K, Laakkonen P, Ruoslahti

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pinocytosis (30). Thus, studying the uptake of AS1411f the few oligonucleotides that has activity in culturedithout the need for transfection agents and which hasssed to clinical testing) may well lead to insights thatlp create novel strategies for siRNA delivery. Similarly,sults may be relevant to efforts to improve delivery ofid DNA, because several recent reports have suggestedacropinocytosis is the major mechanism for uptake ofDNA in cells and in mice (18–20). In summary, our

for drug delivery and gene therapy strategies.

osure of Potential Conflicts of Interest

related to AS1411. P.J. Bates has financial interests in Antisoma PLCholder, consultant, and recipient of research support.

owledgments

hank Drs. Ulf Brunk, John Eaton, Chuan Hu, Robert Mitchell, and Brian

nts and the anonymous reviewers for their helpful suggestionsg the current studies and future directions.

Support

grant R01 CA122383 (P.J. Bates).costs of publication of this article were defrayed in part by the paymentcharges. This article must therefore be hereby marked advertisement innce with 18 U.S.C. Section 1734 solely to indicate this fact.

ived 03/18/2010; revised 09/02/2010; accepted 09/03/2010; publishedirst 09/21/2010.
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2010;70:8617-8629. Published OnlineFirst September 21, 2010.Cancer Res E. Merit Reyes-Reyes, Yun Teng and Paula J. Bates Nucleolin-Dependent MechanismUptake by Macropinocytosis and Its Stimulation by a A New Paradigm for Aptamer Therapeutic AS1411 Action:

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