Silver nanoparticles modulate ABC transporter activity and enhance chemotherapy in multidrug resistant cancer

Silver nanoparticles modulate ABC transporter activity and enhance chemotherapy in multidrug resistant cancer

    Silver nanoparticles modulate abc transporter activity and enhance chemotherapy in multidrug resistant cancer D´avid Kov´acs, Kriszti...

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    Silver nanoparticles modulate abc transporter activity and enhance chemotherapy in multidrug resistant cancer D´avid Kov´acs, Krisztina Sz˝oke, N´ora Igaz, Gabriella Spengler, J´ozsef Moln´ar, T´ımea T´oth, D´aniel Madar´asz, Zsolt R´azga, Zolt´an K´onya, Imre M. Boros, M´onika Kiricsi PII: DOI: Reference:

S1549-9634(15)00207-5 doi: 10.1016/j.nano.2015.10.015 NANO 1206

To appear in:

Nanomedicine: Nanotechnology, Biology, and Medicine

Received date: Revised date: Accepted date:

1 July 2015 16 October 2015 31 October 2015

Please cite this article as: Kov´acs D´ avid, Sz˝oke Krisztina, Igaz N´ ora, Spengler Gabriella, Moln´ ar J´ozsef, T´oth T´ımea, Madar´ asz D´aniel, R´azga Zsolt, K´ onya Zolt´ an, Boros Imre M., Kiricsi M´ onika, Silver nanoparticles modulate abc transporter activity and enhance chemotherapy in multidrug resistant cancer, Nanomedicine: Nanotechnology, Biology, and Medicine (2015), doi: 10.1016/j.nano.2015.10.015

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ACCEPTED MANUSCRIPT SILVER NANOPARTICLES MODULATE ABC TRANSPORTER ACTIVITY

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AND ENHANCE CHEMOTHERAPY IN MULTIDRUG RESISTANT CANCER

Dávid Kovács1, Krisztina Szőke1, Nóra Igaz1, Gabriella Spengler2, József Molnár2, Tímea Tóth3,

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Dániel Madarász3, Zsolt Rázga4, Zoltán Kónya3; 5, Imre M. Boros1; 6, Mónika Kiricsi1, *

Department of Biochemistry and Molecular Biology, University of Szeged, Középfasor 52. H-

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6726, Szeged, Hungary

Department of Medical Microbiology and Immunobiology, University of Szeged, Dóm tér 10. H-

Department of Applied and Environmental Chemistry, University of Szeged, Rerrich Béla tér 1.

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H-6720, Szeged, Hungary

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6720, Szeged, Hungary

Department of Pathology, University of Szeged, Állomás utca. 2. H-6720, Szeged, Hungary

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MTA-SZTE Reaction Kinetics and Surface Chemistry Research Group, Rerrich Béla tér 1. H-

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6720, Szeged, Hungary 6

Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences,

Temesvári krt. 62. H-6726, Szeged, Hungary

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ACCEPTED MANUSCRIPT Corresponding author (*):

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Mónika Kiricsi, PhD

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Department of Biochemistry and Molecular Biology, University of Szeged, Középfasor 52. H6726, Szeged, Hungary

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Phone number: +36 (62) 544-887

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E-mail: [email protected]

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Manuscript word count: 4757/max 5000

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Abstract word count: 150/max 150

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Number of References: 46

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Number of Figures/Tables: 7 Figures/1 Table

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Number of Supplementary online-only files: 1

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ACCEPTED MANUSCRIPT Abstract The emergence of multidrug resistant (MDR) cancer phenotypes dramatically attenuates the

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efficiency of antineoplastic drug treatments often leading to the failure of chemotherapy. Therefore there is an urgent need to engineer new therapeutically useful agents and propose

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innovative approaches able to defeat resistant cancer cells. Although the remarkable anti-cancer features of silver nanoparticles (AgNPs) have already been delineated their impact on MDR

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cancer has never been investigated. Herein, we report that AgNPs have a notable anti-

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proliferative effect and induce apoptosis mediated cell death both in drug sensitive and in MDR cancer cells. Furthermore we show evidence that AgNPs exert an inhibitory action on the efflux

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activity of MDR cancer cells which feature could be exploited to enhance drug accumulation. We verified synergistic interactions of AgNPs with six different antineoplastic agents on drug

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resistant cells which emphasizes the excellent potential of AgNPs as combinational partners in

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the chemotherapy of MDR cancer.

Key words: Silver nanoparticles, multidrug resistance, combinational therapy

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ACCEPTED MANUSCRIPT 1. Background

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In the last decade considerable scientific knowledge accumulated about the cellular and

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molecular mechanisms that propel invasive growth and metastasis and remarkable advancements have been made regarding detection and prevention of cancer, yet competent treatment of

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metastatic and recurrent cancers continues to be elusive. Due to the poor translation of novel

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therapeutic trends into the clinical practice, the general strategy to treat cancer is still based on traditional chemotherapy using small molecular drugs. Although conventional chemotherapy has

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a decent success rate it frequently results in the evolution of multidrug resistant (MDR) cancer phenotypes manifesting resistance to related and unrelated classes of chemotherapeutic drugs [1].

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Multidrug resistant cancer cells exhibit several distinctive cellular and metabolic features such as

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higher apoptotic threshold, elevated activation of DNA repair, altered signal transduction pathways and increased tolerance to oxidative stress, yet the fundamental mechanism in MDR is

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the increased export of anti-cancer agents through ATP-powered efflux pumps such as high

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molecular weight P-glycoprotein (Pgp, MDR1, ABCB1) [2-4]. The increased efflux activity of MDR cells is often due to the overexpression of the most notable ABC transporter genes and several strategies have been described in MDR cancer cells which may lay behind the enhanced expression of Pgp: (i.) amplification of the Pgp encoding genomic locus, (ii.) altered regulation of gene expression at transcriptional or posttranscriptional levels (iii.) epigenetic mechanisms and (iv.) increased stability of the mdr1 gene specific messengers [5, 6]. A multitude of chemotherapeutic agents have been designed to treat different types of cancers and promising alternative strategies based on the unique physicochemical and biological properties of nanoparticle systems have been developed to overcome MDR cancer [7-11]. 4

ACCEPTED MANUSCRIPT Inorganic nanosystems due to their singular structural characteristics, like high stability, tunable composition, multifunctionality and large surface area have gathered grounds in recent

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developments of delivery strategies [12, 13]. Nanoparticle based treatment of solid tumors is

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regarded as an attractive strategy to improve cancer therapy, since approximately 10-200 nm sized materials are selectively accumulated in tumor tissues due to the passive targeting effect

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[14, 15]. This phenomenon is based on the unique disorders of the tumor vasculature which leads

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to the rapid and selective tumor specific enrichment of the nanoparticles [16]. Silver nanoparticles (AgNPs) excel among inorganic nanosystems because of their

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specific biological behaviors, i.e. broad spectrum anti-bacterial, anti-fungal, anti-viral, antiprotosoal activities [17-19], intrinsic cytotoxic features and the lately described anti-tumor

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propensities extend their functions beyond being merely delivery platforms [20-23]. The anti-

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cancer features of AgNPs have been investigated in several different in vitro cancer models. Cell cycle arrest, anti-proliferative effects and apoptosis induction were observed upon AgNPs

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treatments in colon, lung and breast cancer cells [21, 23-26]. However, it has been reported that

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the impact of AgNPs was significantly lower in the MCF10A non-tumor cells than in the MCF-7 and T47D breast tumor cells [27]. Several studies demonstrated the efficacy of AgNPs in in vivo applications [28]. AgNPs inhibited the progression of Pliss lymphosarcoma tumors in rats [29], reduced the growth of Dalton’s ascites tumor [30], and AgNP treatments significantly hindered the development of human breast cancer xenografts in mice [31]. In addition, AgNPs exert antiangiogenic properties [32, 33] and possess radiosensitizing effects in triple negative breast cancer [31] which adds a new layer to their potential in future cancer therapy. Comprehensive toxicological studies have implied that the toxicity of AgNPs is primarily the result of a Trojan-horse like mechanism meaning that after being taken up by cancer cells 5

ACCEPTED MANUSCRIPT AgNPs release highly toxic silver ions which will unbalance the oxidative homeostasis of the cells, induce mitochondrial failure, disruption of cytoskeleton and other serious cellular damages,

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which all together drive the apoptotic cell death [34-37]. Therefore, in the particular case of

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AgNPs both “nano” and “silver” properties contribute to the cytotoxicity of these nanomaterials [38]. As the toxicity of nanosilver is determined by the amount of the surface released silver ions,

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the application of smaller AgNPs with higher relative surface area, generally results in more

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extensive cytotoxic performance than larger particles [39]. On the other hand, particle shape directly influences the nature and the geometry of the reactive surface, therefore particle shape

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may have a significant impact on the cellular uptake, on the in vivo fate as well as on the nanoparticle provoked biological effects [40].

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Due to the multiple advantageous features like intrinsic anti-cancer activity, tunable

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synthesis and adjustable surface for drug delivery AgNPs seem to be promising tools in future chemotherapy and their potential could be exploited to defeat MDR cancer as well. Although the

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molecular background of AgNP toxicity and their effect on cancer cells is relatively well-

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described the issue how the multidrug resistant phenotype of cancer cells influences the AgNP induced cellular response has never been addressed. We hypothesize that AgNPs are able to target the MDR related biological profile of tumor cells thereby they could enhance the efficiency of small molecular drug therapy of MDR cancer. In line with these we applied 28 nm sized citrate coated AgNPs on drug sensitive Colo 205 and Pgp overexpressing drug resistant Colo 320 colon adenocarcinoma cells and studied the impact of AgNP administration on cell growth, proliferation and apoptotic responses. As membrane located ABC transporter efficiency might also be compromised upon AgNP treatments we analyzed MDR related cellular functions such as efflux activity and Pgp expression as well. Finally, to identify possible synergistic actions we 6

ACCEPTED MANUSCRIPT examined the effect of individual chemotherapeutic drugs applied in combinations with AgNPs on drug resistant cancer cells.

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Our results demonstrate that AgNPs have a notable anti-proliferative effect and induce

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apoptosis mediated cell death both in drug sensitive and in MDR cancer cells, and that AgNPs exert an inhibitory action on the efflux activity of MDR cancer cells. Furthermore, our data verify

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synergistic interactions of AgNPs with antineoplastic agents on drug resistant cells indicating the

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potential of AgNPs as combinational partners in the chemotherapy of MDR cancer.

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ACCEPTED MANUSCRIPT 2. Materials and methods

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2.1. Preparation of silver nanoparticles

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Citrate coated silver nanoparticles were produced by a modified Lee-Meisel hydrothermal reduction method described by Wan et al [41]. Briefly, reduction of silver ions of 1% AgNO3

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solution was achieved at 70°C using Na-borohydride (0.1%) in the presence of sodium citrate (1%) in 100 mL final volume. 10 mL of the synthesized silver nanoparticle solution (of

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approximately 5 nm average diameter) was applied as nucleation core in the controlled synthesis

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of larger particles in three subsequent hydrothermal reduction cycles. Particle surface charge (zeta potential) of the obtained silver nanoparticle preparations were measured by a Zetasizer

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Nano Instrument (Malvern, Worchestershire, UK).

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2.2. Cell culture

Drug sensitive Colo 205 and Pgp overexpressing, MDR Colo 320 colon adenocarcinoma

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cancer cells were maintained in RPMI (Lonza) complemented with 10% FBS, 2 mM L-

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glutamine, 0.01% streptomycin and 0.005% ampicillin. Cells were cultured under standard conditions in a 37°C incubator containing 5% CO2 in 95% humidity. 2.3. MTT assay Cell viability was determined by MTT assay, for this 104 cells/well were seeded into 96 well plates and were exposed either to AgNPs or to cytotoxic drugs or to their combinations in various concentrations on the following day. At the end of the treatments AgNP containing media were removed and cells were incubated with 0.5 mg/mL MTT (Sigma-Aldrich) containing culture medium for 1 h in 37°C. Formazan crystals were solubilized in DMSO and the 8

ACCEPTED MANUSCRIPT absorbance was determined using a SPECTROStar Nano plate reader. MTT measurements were repeated three times using at least 4 independent biological replicates. Upon data analysis

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absorbance obtained for the untreated control samples were considered as 100% viability. 2.4. Clonogenic assay

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To evaluate the effect of AgNPs on the colony forming capability of Colo 205 and Colo 320 cells, 200 cells/well were seeded into 6 well plates and treated with AgNPs on the following

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day. After 24 h treatment AgNP containing media were replaced with fresh culture medium and

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cells were left to grow for one week. Cells were fixed using methanol:acetone 70:30 mixture, colonies were stained with crystal violet and finally plates were photographed.

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2.5. Apoptosis detection

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Activation of apoptosis was shown on the basis of detecting caspase 3 activation and

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AnnexinV/PI staining of cells. For the latter 3 x 105 cells were seeded into the wells of 6 well plates and treated with AgNPs on the following day for 24 h. Cells were washed with PBS,

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trypsinized, collected in centrifuge and resuspended in AnnexinV Binding Buffer. Cells were stained with Alexa 488 conjugated AnnexinV and PI according to the guideline of the manufacturer (Life Technologies). Fluorescent intensities were determined using a FACScalibur flow cytometer by measuring 10.000 cells and FACS data were analyzed by FlowJo V10 software. Experiments were repeated three times using at least two biological replicates. To detect the cleavage of the apoptotic effector caspase 3 cells were grown on cover slips and were exposed to AgNPs for 24 h. Cells were fixed in 4% paraformaldehyde and permeabilized using 0.3% Triton-X-100 diluted in PBS. Samples were blocked using 5% BSA and counterstained with cleaved caspase 3 (Asp175) (Cell Signaling) (1:600) and tubulin specific 9

ACCEPTED MANUSCRIPT antibodies (Sigma-Aldrich) (1:1000) diluted in 1% BSA. Alexa 647 fluorophore conjugated goat anti-rabbit antibody was used as secondary antibody for cleaved caspase 3 and Alexa 488

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conjugated rabbit anti-mouse secondary was used for tubulin staining (Life Technologies). Nuclei

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were stained with DAPI dye. Cells were visualized using an Olympus FV10i confocal microscope. The number of cleaved caspase 3 containing cells was determined using ImageJ

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software.

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2.6. Electron microscopy

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Following nanoparticle synthesis particle morphology and primary size was evaluated by TEM using a FEI Tecnai G2 20 x microscope at an acceleration voltage of 200 kV. Size

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distribution of AgNPs based on TEM images was calculated by ImageJ software.

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For electron microscopic imaging of the biological samples 105 cells were seeded and left to grow onto 0.4 µm pore polyester membrane inserts (Corning) placed in a 6 well plate and

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AgNPs were administered to the cells on the following day. After a 24 h treatment cells were

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carefully washed and fixed in 4% glutaraldehyde in PBS for 2 hours and embedded in gelatin (2% gelatin in PBS). Cubes of approximately 1-2 mm were sliced from the specimen and embedded in epoxy (Epon 812, EMS, PA 19440) using a routine TEM sample preparation protocol. First, semi-thin sections of 1 µm were prepared then blocks were trimmed, thin sections of 70 nm were obtained and stained with uranyl and lead solutions. Images were captured by a Philips CM10 electron microscope using 100 kV acceleration voltage. TEM micrographs were taken by a Megaview G2 digital camera (ITEM, Olympus Soft Imaging Solution GmbH, Münster).

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ACCEPTED MANUSCRIPT For SEM analysis cells were seeded and grown onto plastic coverslips (Sarstedt) and exposed to AgNPs on the following day. At the end of the 24 h treatment cells were washed and

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fixed overnight at 4°C using 2.5% glutaraldehyde. The samples were dehydrated by placing them

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stepwise into a serial of solutions containing increasing percentage of ethanol in water (50%, 70%, 80%, 90%, 95%, 98%, 100%, for 15 min each), followed by a series of tert-butanol:ethanol

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mixture (1:2, 1:2, 2:1 volume ratio) at room temperature. Finally, cells were incubated with tert-

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butanol overnight at 4°C and were lyophilized. The coverslips were mounted on specimen stubs and received a thin (4-5 nm) metal gold-palladium coating. SEM imaging was performed by a

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Hitachi S4700 electron microscope using 10 kV accelerating voltage and 10 µA emission current.

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2.7. qRT-PCR

For gene expression analysis 6 x 105 cells were seeded into 6 cm plates and treated with

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AgNPs for 24 h on the following day. Total RNA was isolated using RNeasy Kit (Qiagen) and 2

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µg RNA was transcribed with a Taqman Reverse Transcription Reagent (Applied Biosystems). Relative levels of p21, Survivin, sod-1 and mdr1 specific transcripts were determined using gene

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specific primers (Suppl. Table 1) and SYBR Green PCR Master Mix (Applied Biosystems) in a Pico Real-Time qPCR System (Thermo Scientific). As reference control 18S RNA specific primers were used. Relative transcript levels were determined by the ΔΔCt analysis. RT-qPCR experiments were repeated three times using three biological replicates. 2.8. Drug interaction assay The combined effect of AgNPs and anticancer drugs was determined using a series of MTT assays. 104 cells/well were seeded into 96 well plates and on the following day cells were treated with increasing concentration of anticancer drug, or AgNPs or with the combinations of 11

ACCEPTED MANUSCRIPT the two for 24 h. For the combination treatments chemotherapeutic agents and cytostatic drugs (i.e. Methotrexate, Cisplatin, Carmustine, Bleomycin, Vinblastine and Verapamil) were applied

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with AgNPs in constant AgNP:drug concentration ratios. After 24 h treatments dose-effect curves

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were determined using MTT assay as described above. Data were analyzed and Combination Index (CI) values were calculated at the ED50, ED75 and ED90 growth inhibitions for

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combination treatments using CompuSyn Software according to Chou [42]. Average of ED50,

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ED70 and ED90 values (CIavg) were calculated and AgNP–drug interactions were considered synergistic when CIavg values were lower than 1 (CIavg<1). Mean and ±SD values were calculated

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using three repeats.

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2.9. Pgp efflux activity

Efflux pump activities were measured by determining the intracellular accumulation of

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the fluorescent dye Rhodamine 123. First, 3 x 105 cells were seeded into 3 cm culture plates and

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treated with AgNPs on the following days. After 24 h treatment cells were washed, trypsinized and collected by centrifugation. Cell pellets were resuspended in serum free culture medium

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containing 5.2 µM Rhodamine 123 (Sigma-Aldrich) and were incubated for 20 min at 37°C. Cells were washed two times with pre-warmed PBS then the Rhodamine 123 fluorescence level was determined by a FACScalibur flow cytometer and raw data was analyzed using FlowJo V10 software. 2.10. Western blot analysis The protein level of Pgp was estimated by western blot analysis. 6 x 105 cells were seeded into 6 cm plates and exposed to AgNPs on the following day. At the end of the treatments cells were scraped and incubated in lysis solution (Promega) for 1 h. Samples were centrifuged at 13 12

ACCEPTED MANUSCRIPT 000 rpm for 10 min at 4°C and the protein concentration in the supernatants of the samples was determined using the Bradford method. 20 µg proteins were boiled with protein loading buffer

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(60 mM Tris pH 6.8; 2% SDS, 10% glycerol, 5% ß-mercaptoethanol, 0.002% bromphenol blue)

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and proteins were separated on a 6% SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose membrane then were blocked with 5% milk-TBST. Pgp specific antibody (Novus

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Biologicals) was applied overnight at 1:200 dilution in 2% BSA-TBST. On the following day

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membranes were washed with TBST and incubated with HRP-conjugated rabbit anti-mouse secondary antibody (DAKO) in 1:800 dilution. Membranes were developed using ECL reagent

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(Millipore) according to the guidelines of the manufacturer and chemiluminescent signal was

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detected by a C-DiGit Blot Scanner (LI-COR).

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ACCEPTED MANUSCRIPT 3. Results

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3.1. Silver nanoparticles are taken up by MDR cells

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In this study we used biocompatible citrate coated, quasi-spherical silver nanoparticles synthesized according to the modified Lee-Meisel hydrothermal method [41]. The synthesized

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AgNPs were visualized (Figure 1A) and their size distribution was determined by TEM image

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analyses (Supplementary Figure 1A). The average particle diameter was approximately 28 nm. The zeta potential of the nanoparticles proved to be highly negative (- 44.4 mA) indicating that

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the obtained nanoparticles were stable in aquatic solution (Supplementary Figure 1B). The synthetized AgNPs were applied onto colon adenocarcinoma derived drug sensitive

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Colo 205 and drug resistant Colo 320 cancer cells. Comparison of the Pgp level of Colo 205 and

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Colo 320 cell lysates on western blot proved that the drug resistant Colo 320 cells overexpressed the drug transporter (Figure 1C). In accord with this the drug resistant Colo 320 cells excluded

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the fluorescent Pgp substrate Rhodamine 123 with a higher degree than the drug sensitive Colo

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205 cells. However, 10 minute treatment with the Pgp inhibitor Verapamil at 20 µM concentration decreased the efflux activity of Colo 320 cells (Figure 1B). To demonstrate that AgNPs are taken up by both the drug sensitive and MDR cancer cells we visualized AgNP treated Colo 205 and Colo 320 cells by electron microscopy. SEM images verified that AgNPs attached to the surface of the cells (Figure 2A) while TEM micrographs showed that AgNP were taken up and were internalized by both Colo 205 and Colo 320 cells (Figure 2B). We found cytoplasmic localization of AgNPs in both cell types and detected the nanoparticles in electron dense membrane coated vesicles.

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ACCEPTED MANUSCRIPT 3.2. Silver nanoparticles inhibit the proliferation of MDR cancer cells

To compare the toxic effect of AgNPs on drug sensitive and MDR cancer cells Colo 205

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and Colo 320 cells were treated with AgNPs in 20; 40; 60; 80 and 100 µM concentrations and cell viability was determined 24 h after the addition of the AgNPs by MTT assay (Figure 3A).

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We found that the viability of the AgNP treated cancer cells decreased significantly in both cell types with increasing AgNP concentrations. We observed only minor differences between the

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IC50 values corresponding to Colo 205 and Colo 320 cells (Colo 205: 49.65 ± 7.78µM; Colo 320:

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58.39 ± 4.36µM). Significant difference between the viability of drug sensitive and MDR cancer cells was detected only at 20 µM of AgNP concentration suggesting that the elevated efflux

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activity of Colo 320 cells did not influence the toxicity of AgNPs and in the 40 – 100 µM concentration range AgNPs killed drug sensitive and MDR cancer cells with the same extent.

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These results proved that AgNPs are able to destroy MDR cancer cells despite of their drug

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resistant molecular and cellular phenotype. The potential anti-proliferative effect of AgNPs on Colo 205 and Colo 320 cells was

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investigated by treating the cells with a nontoxic dose (5 µM) of AgNPs then cells were left to grow for 24; 48; 72 and 96 h. At the end of the treatments cell numbers were estimated using MTT assays. No differences in cell growth were detected in the first 24 h between untreated (Figure 3B left panel solid line) and AgNP exposed Colo 205 cells (Figure 3B left panel dashed line) and the same conclusion could be drawn for control Colo 320 and AgNP treated Colo 320 (Figure 3B right panel) cells as well. After 48 h incubation, when cells started to grow exponentially, AgNPs started to hinder cell proliferation and we measured significant inhibitory effect after 72 and 96 h expositions for both the drug sensitive Colo 205 and drug resistant Colo 320 cell cultures (Figure 3B). Using clonogenic assay, the anti-proliferative effect of AgNPs was 15

ACCEPTED MANUSCRIPT further verified as AgNPs applied in 5 and 10 μM concentrations markedly decreased the colony forming capability of both cell lines resulting in notably reduced number of cell colonies in the

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AgNP treated samples (Figure 3C).

3.3. Silver nanoparticles induce caspase 3 mediated apoptosis in MDR cancer cells.

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In order to confirm that the decreased viability upon AgNP treatments is primarily the

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result of apoptotic cell death Colo 205 and Colo 320 cells were treated with 40, 60 and 80 µM of AgNPs for 24 h then the percentage of apoptotic cells were determined by flow cytometry using

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annexinV/PI assay. Representative dot plots on Figure 4A and the quantification of flow cytometry data (Figure 4B) show that AgNP treatments induced apoptosis in a concentration

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dependent manner in both cell lines.

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By immunostaining of Colo 205 and Colo 320 cells we counted elevated number of cleaved caspase 3 positive cells upon AgNP treatment suggesting that the previously detected

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apoptosis was mediated by caspase 3 (Figure 5A; B; Supplementary Figure 2). RT-qPCR

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analysis revealed increased cycline dependent kinase inhibitor p21 and decreased anti-apoptotic Survivin mRNA levels upon AgNP treatments. The elevated relative levels of sod-1 specific transcripts in the AgNP treated cells indicated oxidative stress upon AgNP mediated MDR cancer cell death (Figure 5C).

3.4. Silver nanoparticles inhibit Pgp activity and expression

To reveal the possible modulating effect of AgNPs on the efflux activity of Pgp overexpressing drug resistant Colo 320 cells were treated with AgNPs in 60 and 80 µM concentrations for 24 h and Rhodamine 123 accumulation was measured by flow cytometry. 16

ACCEPTED MANUSCRIPT According to the intracellular accumulation of the fluorescent dye Rhodamine 123, a concentration dependent inhibition of the efflux activity was observed in the AgNP treated MDR

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cancer cells (Figure 6A). We hypothesized that not only the activity but also the expression of

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Pgp might be compromised by AgNPs in Colo 320 cells. To examine Pgp expression on mRNA and protein levels RT-qPCR and western blot experiments were carried out on AgNP treated

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Colo 320 cells, which indicated that the reduced Pgp activity was at least partially the result of

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the decreased transcript (Figure 6B) and protein level (Figure 6C) of Pgp upon AgNP treatments. Therefore, we concluded that AgNPs are capable of modulating the efflux activity in

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drug resistant colon cancer cells by inhibiting the expression of Pgp.

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3.5. AgNPs synergistically enhance chemotherapy in MDR cells

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Because AgNPs exerted an inhibitory effect on the cellular efflux activity, we investigated their capabilities as combinational partners of functionally and structurally unrelated

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chemotherapeutic drugs in MDR cancer. The combinational effect of Methotrexate, Cisplatin,

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Carmustine, Bleomycin, Vinblastine and Verapamil with AgNPs was tested in multidrug resistant Colo 320 cells. In the combination experiments, cells were treated for 24 h with different concentrations of either AgNPs (Figure 7 green lines) or individual drugs alone (black lines) or with the combinations of a specific drug and AgNPs (red lines) using constant concentration ratios then cell viabilities were measured by MTT assay (Figure 7). Dose effect curves were analyzed using CompuSyn software and CI values were determined according to Chou [42]. Based on the observed cell viability data and on the calculated CIavg values, synergistic interactions could be verified for AgNPs with the tested chemotherapeutic drugs in each combination, as the CIavg values of the combination treatments were each below 1 (Table 1). This 17

ACCEPTED MANUSCRIPT observation indicates that AgNPs are able to enhance the efficacy of antineoplastic drug

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therapies.

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ACCEPTED MANUSCRIPT 4. Discussion Although the cellular and molecular features behind the elevated activity of the plasma

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membrane located ABC transporters in MDR cancer are extensively studied, our knowledge on the molecular events leading to resistant phenotype of tumor cells is still partial. Consequently,

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MDR still results in lower survival rates of the patients undergoing conventional chemotherapy [43-45]. Therefore, there is an urgent need for the development of new pharmaceutical strategies

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to defeat MDR cancer. While the anti-cancer effect of AgNPs has been reported several times

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using various types of in vivo and in vitro tumor models [26, 29, 30] their potential in MDR cancer treatment has not been investigated. Hence, we studied how the MDR phenotype of tumor

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cells influences the anti-cancer activity of AgNPs. For this purpose we used a two cell line containing MDR model in which Colo 320 cells exert elevated efflux activity due to Pgp

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overexpression in contrast to drug sensitive Colo 205 cells (Figure 1C; D).

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We found that both of the examined cell lines were able to take up AgNPs (Figure 2) which in turn decreased the viability of both the drug sensitive and the MDR cells in a

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concentration dependent manner (Figure 3A). Similarly, the proliferation rates of both of the drug sensitive and MDR cell cultures were inhibited by AgNP expositions (Figure 3B; C). We concluded that the reduced viability provoked by AgNPs is mediated by caspase 3 dependent apoptotic events in both drug sensitive and multidrug resistant cancer cells (Figure 4; Figure 5A, B; Supplementary Figure 2). This assumption is also supported by the observed changes in the relative levels of apoptosis and oxidative stress related messengers in Colo 205 and Colo 320 cells (i. e. p21, survivin, sod-1) (Figure 5C). In other words, we found that AgNPs kill MDR cells and the multidrug resistant phenotype of cancer does not seem to influence the AgNP induced apoptotic features. Our findings on MDR cancer cells fit into the currently accepted 19

ACCEPTED MANUSCRIPT picture of the general toxicity of AgNPs: they penetrate easily through the plasma membrane and release toxic silver ions within the cytoplasm thereby provoking oxidative stress and the

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subsequent cellular damages such as mitochondrial dysfunction, double-strand DNA breaks and

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cytoskeletal disruption altogether drive cell cycle arrest and eventually induce the apoptotic execution of the cancer cells [35, 36, 46].

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As the fundamental mechanism in MDR is the elevated export of anti-cancer drugs, we

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tested whether this cellular feature is modulated by AgNPs. Surprisingly, AgNP treatments inhibited the efflux activity of drug resistant cells (Figure 6A). The mRNA and protein

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expression analysis of Pgp indicated that not only the pump activity but the expression of Pgp was compromised as well upon AgNP treatments (Figure 6B; C). Therefore, we concluded that

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the decreased expression of this major ABC transporter contributed to the observed reduction in

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efflux activity. However, the question still remains whether AgNPs exert their inhibitory effect directly on the ABC transporter itself or through the disruption of mitochondrial function and

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ATP production or maybe by transcriptional silencing of the mdr1 encoding genomic locus.

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While the exploration of the exact inhibitory mechanism of AgNPs on the ABC transporters requires further investigation it is a more fundamental question whether this feature could be exploited in combinational chemotherapy of MDR cancer. Therefore, we tested the effect of AgNPs administered in combination with anti-cancer agents and found synergism in all the six cases of tested AgNP–drug combinations (Figure 7; Table 1). We assume that the reason behind the observed synergistic interactions is at least partly the modulated efflux activity and Pgp expression which leads to intracellular accumulation of cytotoxic drugs. This is a highly relevant finding as it demonstrates that AgNPs are powerful tools to enhance drug efficiency and

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ACCEPTED MANUSCRIPT chemotherapy to overcome MDR cancer. This observation renders AgNPs to attractive

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candidates in rational design of therapeutically useful agents for antineoplastic tumor targeting.

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ACCEPTED MANUSCRIPT 5. Figure Legends

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Figure 1

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(A) TEM micrograph of citrate coated AgNPs. (B) The accumulation of Rhodamine 123 in Colo 205, Colo 320 and in Verapamil treated Colo 320 cells. (C) Western blot analysis on Colo 205

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and Colo 320 cell lysates using Pgp specific antibody.

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Figure 2

(A) SEM micrographs of untreated and AgNP treated Colo 205 and Colo 320 cells. White arrows

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indicate surface attached nanoparticles. (B) Representative TEM images of control and AgNP

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Figure 3

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exposed Colo 205 and Colo 320 cells.

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(A) Cell viability values of AgNP treated Colo 205 and Colo 320 cells (*P ≤ 0.01; **P ≤ 0.0001 Dunnett's multiple comparisons test; #P ≤ 0.05 Sidak's multiple comparisons test). (B) Growth curves of Colo 205 and Colo 320 cells upon 5 µM AgNP treatments (*P ≤ 0.001; ** P ≤ 0.0001 Sidak's multiple comparisons test). Colony forming capability of Colo 205 and Colo 320 cells after 5 µM and 10 µM AgNP treatments.

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ACCEPTED MANUSCRIPT Figure 4 (A) Representative dot plots of AnnexinV/PI staining assays. Colo 205 and Colo 320 cells were

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treated with AgNPs in 40, 60 and 80 µM concentrations for 24 h and AnnexinV/PI positive cells were analyzed by flow cytometry. (B) Number of total apoptotic cells (Q2+Q3) were determined

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according to flow cytometry data (*P ≤ 0.01;**P ≤ 0.001 Dunett's multiple comparisons test).

Figure 5

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(A) Immunostaining of AgNP treated Colo 205 and Colo 320 cells using cleaved caspase 3 and tubulin specific antibodies. (B) Percentage of cleaved caspase 3 positive Colo 205 and Colo 320

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cells upon AgNP expositions (C – Control; *P ≤ 0.01; ** P ≤ 0.001 Unpaired t test). (C) RT-

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qPCR analysis of p21, Survivin and sod-1 specific transcripts in Colo 205 and Colo 320 cells

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Figure 6

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upon AgNP treatments (C – Control; *P ≤ 0.05; **P ≤ 0.01 Sidak's multiple comparisons test).

(A) Representative histograms of Rhodamine 123 accumulation in Colo 320 cells treated with 60 and 80 µM AgNPs. (B) RT-qPCR analysis of AgNP exposed Colo 320 cells using mdr1 gene specific primers (*P ≤ 0.05Sidak's multiple comparisons test). (C) Protein level of Pgp in 60 and 80 µM AgNP treated Colo 320 cells.

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ACCEPTED MANUSCRIPT Figure 7 Cell viability curves of Colo 320 cells treated with AgNPs (green lines), with individual drugs

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(black lines) or with AgNP-drug combinations (red lines).

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Table 1

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The calculated CI and CIavg values corresponding to the tested AgNP–drug combinations. r values indicate the fitting of the surviving curve to the measured points upon combination

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treatments.

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ACCEPTED MANUSCRIPT 6. Acknowledgement The excellent assistance of Katalin Ökrösné, Anikó Vigyikánné Váradi, Erika Németh and Edina

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Pataki is greatly appreciated. The authors are thankful to Margit Pál for providing the chemotherapy drugs, to Tibor Németh for his guidance in SEM sample preparation. The cell lines

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used in the study were kind gifts from the Szeged Foundation for Cancer Research.

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Graphical abstract

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Combinations of structurally unrelated anticancer drugs with silver nanoparticles were applied on multidrug resistant (MDR) cancer cells and we observed synergistically enhanced drug efficiency and cytotoxicity for all the tested combinations. We found that silver nanoparticles modulated the ABC transporter activity of MDR cancer cells which accounts for the detected synergistic interactions. We believe that these findings are relevant as silver nanoparticles seem powerful tools to improve chemotherapy of MDR cancer.

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Table 1

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CI at ED90 0.165 0.95 0.28 0.428 1.266 0.237

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CI at ED75 0.066 0.466 0.112 0.516 0.094 0.47

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CI at ED50 0.033 0.229 0.045 0.638 0.007 0.796

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Combination partner Methotrexate Cisplatin Carmustine Bleomycin Verapamil Vinblastine

r 0.93 0.98 0.98 0.99 0.90 0.99

CIavg 0.09 0.55 0.15 0.52 0.64 0.50