combination of conventional chemotherapeutics with...

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Combination of conventional chemotherapeutics with redox-active cerium oxide nanoparticles – a novel aspect in cancer therapy

Maren Sack1,3*, Lirija Alili1,3, Elif Karaman1, Soumen Das2 , Ankur Gupta2,

Sudipta Seal2 and Peter Brenneisen1

1Institute of Biochemistry & Molecular Biology I, Medical Faculty, Heinrich-Heine-University, 40225 Duesseldorf, Germany

2Advanced Materials Processing and Analysis Center, Nanoscience and Technology Center (NSTC), Mechanical, Materials Aerospace Engineering (MMAE), University of Central Florida, Orlando, Florida

3These authors contributed equally to this work.

Running title: Redox-active cerium oxide nanoparticles in cancer therapy

Keywords: cerium oxide nanoparticles, ROS, cancer, Doxorubicin, chemotherapeutics

Abbreviation list: Cerium oxide nanoparticles (CNP), human dermal fibroblasts (HDF), Doxorubicin (DOX)

Financial support: S. Seal acknowledges the National Science Foundation (NSF) to partially fund the nanotechnology research under NSF NIRT (CBET-0708172) and NSF (CBET-0930170)

*Author for correspondence:

e-mail: [email protected]

phone: +49-(0) 211-81-12834

fax: +49-(0) 211-81-12833

The authors disclose no potential conflicts of interest

Word count (excluding references): 5.482

Total number of figures: 7

on June 8, 2018. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 13, 2014; DOI: 10.1158/1535-7163.MCT-13-0950

http://mct.aacrjournals.org/

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Abstract

Nanotechnology starts to be an important field of biomedical and clinical research

and the application of nanoparticles in disease may offer promising advances in

treatment of many diseases, especially cancer. Malignant melanoma is one of the

most aggressive forms of cancer and its incidence is rapidly increasing. Redox-active

cerium oxide nanoparticles (CNP) are known to exhibit significant anti-tumor activity

in cells derived from human skin tumors in vitro and in vivo, whereas CNP is non-

toxic and beyond that even protective (antioxidative) in normal, healthy cells of the

skin. As the application of conventional chemotherapeutics is associated with harmful

side effects on healthy cells and tissues, the clinical use is restricted. In this study,

the question was addressed of whether CNP supplement a classical chemotherapy

thereby enhancing its efficiency without additional damage of normal cells. The

anthracycline Doxorubicin, one of the most effective cancer drugs, was chosen as

reference for a classical chemotherapeutic agent in this study. Herein, we show that

CNP enhance the anti-tumor activity of Doxorubicin in human melanoma cells.

Synergistic effects on cytotoxicity, ROS generation and oxidative damage in tumor

cells were observed after co-incubation. In contrast to Doxorubicin, CNP do not

cause DNA damage and even protect human dermal fibroblasts from Doxorubicin-

induced cytotoxicity. A combination of classical chemotherapeutics with non-

genotoxic, but anti-tumor active cerium oxide nanoparticles may provide a new

strategy against cancer by improving therapeutic outcome and benefit for patients.




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Introduction

In recent years nanotechnology has become an important field of biomedical and

clinical research forming the subject area of nanomedicine. Application of

nanoparticles in disease offers promising possibilities for diagnostics (nanoimaging)

and drug delivery systems (nanocarrier) as well as the pharmaceutical use of

nanoparticles itself (nanopharmaceuticals) (1-3). Nanoparticle applications offer

advances in treatment of many diseases especially cancer, which is the second most

common cause of death in the US and Europe following cardiovascular diseases (4).

Malignant melanoma is one of the most aggressive types of cancer. Early stages of

melanoma can be cured by surgery, however the treatment of metastasizing forms is

still difficult and the survival rates of 5% are really poor. The incidence of skin cancer

is rapidly growing, suggesting a doubling of the rate each decade. Hence, more

effective therapies with less harmful effects are required (5, 6).

Recent studies have shown that redox-active cerium oxide nanoparticles exhibit

cytotoxic and anti-invasive effects in several cancer cells (7, 8) and are able to

sensitize tumor cells to radiation, while protecting the normal cells in the tumor

surrounding stroma (9-11).

The use of dextran-coated and oxygen vacancies containing CNP with a size of

about 5 nm in diameter resulted in cell killing of the squamous skin carcinoma cell

line SCL-1 and the human melanoma cell line A375 and lowered the invasive

capacity (12). In a xenograft mouse model with A375 melanoma cells, tumor growth

was significantly inhibited by CNP, which was the first study that showed an anti-

tumor activity in vivo (13). The cytotoxic effect of CNP in tumor cells was mediated by

a prooxidant activity of CNP, which significantly increased the ROS level, especially

H2O2, and thereby leading to apoptosis of tumor cells. In contrast, in normal cells (for




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example stromal fibroblasts), CNP exerted antioxidant properties. This bifunctional

mode of action is mediated by a pH-dependent redox-activity of CNP (9). Tumor cells

show an increased glycolysis rate (“Warburg effect”) compared to healthy cells

resulting in high production of lactate and a slight acidification of tumor cells and the

extracellular space (14-16). This difference in pH of tumor cells and normal cells is

the decisive factor that makes CNP either working as a pro- or antioxidant (13).

Taken together, these findings implicate a promising potential for a clinical use of

CNP in cancer therapy. In this study, the question was addressed of whether CNP

could supplement a classical chemotherapeutical approach. Because of the pro-

apoptotic and anti-invasive properties in tumor cells, CNP could enhance the

therapeutic outcome of chemotherapies. In contrast to conventional

chemotherapeutics, which are often accompanied with a damage of healthy cells and

tissues (17, 18), CNP is non-toxic and even protective in stromal cells of the skin

(12).

The anthracycline Doxorubicin (DOX), an “evergreen” of the chemotherapeutic

agents, was chosen as a reference substance for this study. It belongs to the most

effective cancer drugs ever developed, however the clinical use of Doxorubicin is

restricted because of its diverse toxic effects in healthy cells and tissues (19, 20). The

anti-tumor activity of Doxorubicin is mainly mediated by several interactions with

genomic DNA leading to DNA damage, cell cycle arrest and, subsequently, to

apoptosis. Furthermore, Doxorubicin is known to generate ROS via a redox cycling

process, thus contributing to its toxicity (19). Herein, a potential synergistic effect of

CNP enhancing the anti-tumor activity of Doxorubicin was investigated in human

melanoma cells. As CNP is antioxidative and protective against exogenous noxes in

normal cells, this nanoparticles may lower the side effects of Doxorubicin thereby

improving the therapeutic outcome. Additionally, the impact of CNP to protect human




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dermal fibroblast, being the most frequently stromal cells of the skin, from

Doxorubicin-induced toxicity was assessed.

Materials and Methods

Cell culture medium Dulbecco’s modified Eagle’s medium (DMEM) was purchased

from Invitrogen (Karlsruhe, Germany) and the fetal calf serum (FCS gold) from

Biochrom (Berlin, Germany). All chemicals including protease as well as

phosphatase inhibitor cocktail 1 and 2 were obtained from Sigma (Taufkirchen,

Germany) or Merck Biosciences (Bad Soden, Germany) unless otherwise stated. The

protein assay kit (Bio-Rad DC, detergent compatible) was from BioRad Laboratories

(München, Germany). The Oxyblot Protein Oxidation Detection kit was from Millipore

(Schwalbach, Germany), while the 2′,7′-Dichlorofluorescin diacetate was provided

from Sigma (Taufkirchen, Germany). The enhanced chemiluminescence system

(SuperSignal West Pico/Femto Maximum Sensitivity Substrate) was supplied by

Pierce (Bonn, Germany). Monoclonal mouse antibody raised against human

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and monoclonal mouse

antibody raised against human α-tubulin were supplied by Sigma. The monoclonal

antibody raised against and Poly (ADP-ribose) polymerase (PARP) was obtained

from Cell signaling. The polyclonal rabbit α-hapten antibody directed against oxidized

thiol groups (sulfenic acid) was a gift from Kate S. Carrol`s group from TSRI,

Department of Chemistry, Jupiter, Florida (21). The following secondary antibodies

were used: polyclonal horseradish peroxidase (HRP)-conjugated rabbit anti-mouse

IgG antibody (DAKO, Glostrup, Denmark) and goat anti-rabbit immunoglobulin G

antibodies were from Dianova (Hamburg, Germany). Doxorubicin was obtained from

Sigma and dissolved in DMSO (0,25% final concentration).




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Cell culture

The human malignant melanoma cell line A375, originally derived from a 54-year-old

woman, was purchased from ATCC (22). Human dermal fibroblasts (HDF) were

established by outgrowth from foreskin biopsies of healthy human donors with an age

of 3-6 years. Cells were used in passages 2-12, corresponding to cumulative

population-doubling levels of 3-27 (23). Human melanoma cells and human dermal

fibroblasts were cultured as described (24).

Cell viability

The cell viability was measured by MTT (3-(4,5-Dimethythiazol-2-yl)-2,5-

diphenyltetrazolium bromide)-Assay (25). The reduction of MTT (Sigma, Taufkirchen,

Germany) by mitochondrial dehydrogenases to formazan indicates the metabolic

activity of cells and is an indicator of cellular viability.

Briefly, serum-free medium containing MTT (0.5 mg/ml) was added to the cells after

incubation with different concentration of CNP or Doxorubicin. After incubation with

MTT cells were washed with PBS and lysed in dimethyl sulfoxide. The formation of

the blue formazan was measured at 570 nm. The results were presented as

percentage of untreated controls which were set at 100%.

Synthesis of CNP

Cerium oxide nanoparticles were synthesized in dextran (molecular weight: 1000 Da)

using previously described methods (26). Briefly, stoichiometric amounts of dextran

were at first dissolved in deionized water followed by cerium nitrate hexahydrate. The

solution was stirred for 2h followed by addition of ammonium hydroxide (30% w/w).

The pH of the solution was kept below 9.5 to avoid precipitation of cerium hydroxide.




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At a final concentration of 150 µM CNP in DMEM, the cells were incubated in 0.9 %

ammonium hydroxide. At this concentration ammonium hydroxide belongs to the

GRAS (Generally Recognized As Safe) substances as suggested by the Food and

Drug Administration (FDA). The resulting dextran-coated cerium oxide nanoparticles

(CNP) were analyzed using UV-visible spectroscopy for determining the oxidation

state of nanoparticles and transmission electron microscopy for particle size.

Synthesis of FITC conjugated CNP

The FITC tagged dextran coated cerium oxide nanoparticles (CNP) were prepared as

described earlier (13). Briefly, dextran coating on the surface of cerium oxide

nanoparticles were oxidized with 10mM sodium periodate. Oxidized dextran coated

CNPs were then dialyzed extensively against distilled water to remove any trace

amount of sodium periodate. Then amination between oxidized dextran and FITC

were carried out in bi-carbonate buffer at pH 8.5. Then, FITC conjugated

nanoparticles were washed several times with distilled water to remove any free

FITC. Finally, nanoparticles were reconstituted using distilled water.

Cellular uptake of nanoparticles

Human melanoma cells in Dulbecco`s Modified Eagle Media (DMEM) were treated

with 150 µM FITC-labeled CNP for 4h or untreated. Thereafter, cells were washed

twice with PBS and fixed with methanol. ProLong Gold (Invitrogen) was used for

visualization, a reagent that simultaneously stains the nuclei with DAPI. The

fluorescence microscopic examination was done with a Zeiss Axiovert 100TV and the

documentation with a digital camera system (Hamamatsu C4742-95).




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SDS-PAGE and western blotting

Sodium dodecyl sulfate polyacrylamide gel electrophorese (SDS-PAGE) was

performed according to the standard protocols published elsewhere (27) with minor

modifications. Briefly, cells were lysed after incubation with CNP or Doxorubicin in

1% SDS with 1:1000 protease inhibitor cocktail (Sigma; Taufkirchen, Germany). After

sonication, the protein concentration was determined by using a modified Lowry

method (Bio-Rad DC). SDS-PAGE sample buffer (1.5M Tris-HCl pH 6.8, 6ml 20%

SDS, 30ml glycerol, 15ml ß-mercaptoethanol and 1.8mg bromophenol blue) was

added, and after heating, the samples (20-30µg total protein/lane) were applied to

12% (w/v) SDS-polyacrylamide gels. After electroblotting, immunodetection was

carried out (1:1000 dilution of primary antibodies; 1:20000 dilution of anti-

mouse/rabbit antibody conjugated to HRP). Antigen-antibody complexes were

visualized by an enhanced chemiluminescence system. α-tubulin or glyceraldehyde

3-phosphate dehydrogenase (GAPDH) was used as internal control for equal

loading.

Determination of oxidized (carbonylated) proteins

A375 melanoma cells were grown to subconfluence on tissue culture dishes. After

removal of serum-containing medium, cells were cultured in DMEM supplemented

with 0.5 % FCS and either untreated or treated for different time periods with 150 µM

CNP nanoparticles. As positive control, the cells were treated with 250 μM H2O2.

Thereafter, cells were lysed and carbonyl groups of oxidized proteins were detected

with the OxyBlotTM Protein Oxidation Detection Kit (Milipore) according to the

manufacturer`s protocol. Briefly, the protein concentration was determined by using a

modified Lowry method (Bio-Rad DC). Ten µl of the whole cell lysates with equalized

protein concentrations were incubated with 2,4-dinitrophenyl (DNP) hydrazine to form




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the DNP hydrazone derivatives. Labeled proteins were separated by SDS–PAGE

and immunostained using rabbit anti-DNP antiserum (1:150) and goat anti-rabbit IgG

conjugated to horseradish peroxidase (1:300). Blots were developed by enhanced

chemiluminescence.

Determination of oxidized thiol groups (sulfenic acids)

A375 melanoma cells were grown to subconfluence on tissue culture dishes. After

removal of serum-containing medium, cells were cultured in 0.5 % FCS containing

medium for different incubation times with CNP and Doxorubicin. In the last 2 h of

incubation 10 mM of the diketone dimedone was added (Sigma, Taufkirchen,

Germany). As positive control, the cells were co-incubated with 1 mM H2O2 and 10

mM dimedone for 2 h. Cells were harvested, washed with PBS and lysed. Then

western blot analysis were performed with the α-hapten antibody directed against

oxidized SH-groups (1:1000)(21).

Immunochemical staining

A375 melanoma cells were grown to subconfluence on tissue culture dishes

containing cover slips and treated with CNP or Doxorubicin alone as well as in

combination with both agents. After incubation cells were fixed directly with methanol

for 10 min at -20°C, incubated with blocking solution for 1 h and treated with a

specific α-hapten (1:1000) antibody directed against oxidized SH-groups overnight at

4°C. The secondary antibody (Alexa Fluor 556; Invitrogen) was applied to the cells

for 45 min at 37°C in the dark. After removal of the secondary antibody, coverslips

were attached with ProLong gold antifade reagent (Invitrogen) on microscope slides.

The samples were dried for at least 12 h and stored at 4°C. Samples were analyzed

by fluorescence microscopy (AxioVert 100TV; Zeiss, Germany) using a Hamamatsu




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Digital Camera C4742-95 and AquaCosmos version 1.2 software (Hamamatsu

Photonics Deutschland GmbH, Herrsching am Ammersee, Germany). Pictures of

randomly selected areas were taken for each sample.

Preparation of nuclear and cytoplasmic extracts

Separation of nuclear and cytoplasmic extracts of A375 melanoma cells was

performed with the NE-PER Nuclear and Cytoplasmic Extraction Reagents

(Themoscientific), following the manufacture´s protocol. Protein concentrations of the

cell lysate were assessed by using a modified Lowry method (Bio-Rad DC) and

adjusted for equal gel loading.

Comet Assay

The alkaline comet assay (single-cell gel electrophoresis) was used to measure DNA

single- and double-strand breaks together with alkali-labile sites (28). Cells were

plated in dishes and incubated with CNP or Doxorubicin. Cells were harvested

immediately after incubation, centrifuged, and suspended in 200 ml low-melting-point

agarose and kept at 37°C. The suspension was transferred to prepared microscope

slides containing a layer of 10% agarose and then cooled for 4 min at 4°C.

Coverslips were gently dropped off and the microscope slides were placed overnight

at 4°C in lysis buffer (2.5 M sodium chloride, 100 mM EDTA, 10 mM Tris- Base,

sodium hydroxide) to lyse cells and enable DNA unfolding. Slides were washed with

water and placed on a horizontal gel electrophoresis chamber, which was filled with

high-pH electrophoresis buffer (10 N sodium hydroxide, 200 mM EDTA) to submerge

the slides. Slides were kept in the buffer for 25 min to denature the DNA before

electrophoresis. Electrophoresis was performed for 25 min at 25 V and 300 mA (Bio-

Rad; PowerPacHC). After electrophoresis the slides were washed three times with




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neutralizing buffer (0.4 M Tris-Base, pH 7,5). For final fixation, the slides were kept in

ethanol (80%) for 5 min and then dried overnight. After ethidium bromide [Molecular

Probes, Invitrogen] staining the cells were subjected to fluorescence microscopy.

DNA damage was evaluated by measuring the comet area in px (head and tail). At

least 40 stained comets were selected randomly and analyzed with CometScore

(TriTek Corp., USA).

Statistical Analysis

Means were calculated from at least three independent experiments, and error bars

represent standard error of the mean (s.e.m.). Analysis of statistical significance was

performed by Student t test or ANOVA with *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 as

levels of significance.

Results

Uptake and cellular distribution of CNP

CNP showed a cytotoxic effect in tumor cells, which is mediated via apoptosis (13).

Depending on size and charge, nanoparticles are internalized by cells or bind to

surface molecules mediating their effects via receptor signaling (29). To elucidate the

uptake and distribution of CNP in the A375 melanoma cell line, cells were incubated

with FITC-labeled cerium oxide nanoparticles (CNP-FITC) at a concentration of 150

µM as described earlier (13) and analyzed by fluorescence microscopy at different

time points. An uptake of CNP-FITC was confirmed after 4h of incubation (Fig 1).

Fluorescence was observed primarily in the cytosol of the cells with an accumulation




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in the perinuclear region, but not in the nuclei. In addition, 24 h and 48 h after

treatment the fluorescence of labeled CNP was merely detected in the cytosol of the

cells (data not shown), indicating that the particles are not able to pass the nuclear

membrane even after longer incubation times.

CNP versus Doxorubicin cytotoxicity

The anthracycline Doxorubicin belongs to the most effective cancer drugs ever

developed. The anti-tumor activity of Doxorubicin is based on several interactions

with DNA resulting in DNA damage, inhibition of DNA replication and consequently

apoptosis. The toxicity of Doxorubicin is not restricted to cancer cells, even healthy,

(stromal) cells are affected by Doxorubicin treatment as well (19). Thus, a therapy

with Doxorubicin is always associated with adverse side effects on healthy cells and

with the risk of the developing secondary cancer (30). Cerium oxide nanoparticles

were shown to kill tumor cells, while being non-toxic for stromal cells of the skin, like

fibroblasts and endothelial cells. Furthermore, CNP even showed protective effects

against exogenous prooxidants in stromal cells (12). In this study, the effect of CNP

and Doxorubicin on viability of human melanoma cells (A375) and human dermal

fibroblasts (HDF) was evaluated by using the MTT assay. The results (Fig. 2A)

showed that Doxorubicin exerted strong cytotoxic effects in A375 cells at very low

concentration and after short times of incubation. After 24h treatment with 0.5 µM

Doxorubicin the cell viability was decreased to about 40-50% in A375 compared to

the untreated control, which was set at 100%. The cytotoxicity of Doxorubicin was

increasing with incubation time, at 72 h post treatment with 0.5 µM no cells survived

(data not shown). Doxorubicin exhibited stronger toxic effects in A375 than CNP. The

cell viability was decreased in A375 cells after treatment with 150 µM CNP to

approximately 55% and with 300 µM CNP to 50% after 96 h.




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Moreover, in this study the question was addressed, whether there is a synergistic

effect of CNP and Doxorubicin on cytotoxicity in tumor cells. Therefore, A375

melanoma cells were incubated with 300 µM CNP for 48 h or 0.5 µM Doxorubicin for

24 h alone or in combination. Treatment of melanoma cells with 300 µM CNP for 48 h

resulted in a decrease of cell viability to 88% compared to the untreated control. After

co-incubation with CNP and Doxorubicin cell viability was decreased to 20%

compared to 40-50% after incubation with Doxorubicin alone (Fig. 2B). These results

indicated a synergistic effect of CNP and Doxorubicin on cytotoxicity in melanoma

cells. By contrast, CNP showed no cytotoxicity in human dermal fibroblasts (HDF).

Doxorubicin showed less toxicity in HDF compared to melanoma cells. A

concentration of 0.5 µM Doxorubicin did not significantly lower the viability of HDF,

whereas 25 µM Doxorubicin decreased the cell viability of HDF to approximately 60-

70% compared to the untreated control, which was set at 100% (Fig. 2C). Pre-

incubation with 150 µM CNP 24 h before Doxorubicin treatment (25 µM) abrogated

the cytotoxic effect of Doxorubicin in HDF. The cell viability was increased after co-

incubation to around 100% compared to cells that were treated with Doxorubicin

alone. These data demonstrate that CNP may protect human dermal fibroblasts from

toxicity of Doxorubicin (Fig 2d). Therefore, a potential therapeutical approach based

on a combination of low concentration of Doxorubicin (to minimize side effects)

together with CNP (which may protect stromal cells ) could be a novel tool to

effectively kill tumor cells in a time dependent manner.

ROS production

In previous studies CNP treatment resulted in ROS formation in A375 melanoma

cells as well as in several other tumor cell lines (7, 8, 13, 31). Besides its direct

inhibitory effects on DNA replication, Doxorubicin is known to generate ROS via




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redox-cycling. A one-electron addition to the quinone moiety of Doxorubicin results in

the formation of a semiquinone that reacts back to the quinone form thereby reducing

O2 to superoxide (O2.-) (19). In this study, the questions were addressed of whether i)

redox-active CNP have a similar impact to generate ROS as the classical

chemotherapeutic Doxorubicin, and ii) a co-incubation of both substances results in a

synergistic effect in context of ROS production.

The fluorescent probe H2DCF-DA was used to detect intracellular ROS. Therefore,

cells were loaded with H2DCF-DA and then incubated with 150 µM CNP or 0.5 µM

Doxorubicin alone or co-incubated for 1.5 h. Directly after addition of the substances,

the fluorescence was measured every 5 minutes. Incubation with CNP as well as

incubation with Doxorubicin caused an increase in the ROS level of around 20%

within 1.5 h compared to the untreated control. Co-incubation of CNP and

Doxorubicin resulted in an even higher ROS level, indicating a synergistic effect in

ROS generation (Fig. 3). CNP and Doxorubicin together increased the intracellular

ROS level by 36%.

Oxidative damage of proteins

Proteins are one of the major targets of ROS. Oxidative modifications of proteins can

influence the biochemical functionality and activity of enzymes and transcription

factors (32). Sulfenic acids are a specific oxidation product of thiol groups of cysteins

in protein side chains, which subsequently may be oxidized to sulfinic and sulfonic

acids (21). Besides thiol oxidation another oxidative modification of proteins is the

introduction of carbonyl groups into several amino acids (i.e. lysine, proline, histidine,

arginine). The carbonyl content is considered as the most general and well used

biomarker for irreversible oxidative damage (33). To elucidate the prooxidative impact

of CNP and Doxorubicin more in detail, the oxidative damage of proteins was




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investigated via detection of sulfenic acids and carbonyls in proteins. Thus, cells were

incubated with 150 µM CNP or 0.5 µM Doxorubicin as well as in combination for 2, 4

and 24 h.

Carbonyl groups were detected via derivatization with dinitrophenyl hdyrazine

(DNPH) using the Oxyblot Detection kit (Millipore). CNP as well as Doxorubicin

treatment significantly increased the carbonyl amount compared to untreated

controls, while CNP induced higher carbonyl content increasing with incubation time

(Fig. 4). The highest amount of carbonylated proteins was observed after a co-

incubation of CNP and Doxorubin. In contrast to the thiol oxidation, the formation of

carbonylated proteins is an irreversible damage accumulating over time, exclusively

seen with CNP.

For detection of sulfenic acids Western Blot analysis and immunochemical stainings

were carried out using a specific α-hapten antibody raised against the stable

thioether product of sulfenic acid and the cell-permeable nucleophilic diketone

dimedone (21), which was added to the cells during the last 2 h of incubation. The

western blot analysis showed that CNP as well as Doxorubicin increased the amount

of oxidized thiols (Fig. 5). Compared to the untreated control, Doxorubicin treated

cells showed a 2-fold increase of sulfenic acids, whereas treatment with cerium oxide

nanoparticles resulted in a 3-fold increase of sulfenic acids after a 2 h incubation.

However, the levels of oxidized thiols were decreasing with increasing incubation

times, presumably a consequence of gutathionylation, a mechanism by which the

sulfenic acids are reduced again to thiols. Treatment with CNP and Doxorubicin in

combination resulted by tendency in a higher amount of sulfenic acid compared with

the single substances showing again a synergistic effect. The highest content of

sulfenic acids, a 6-fold increase compared to the control, was detected after 2 h co-

incubation with CNP and Doxorubicin (Fig. 5).




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In order to localize the oxidized thiols in the cell, immunochemical stainings were

performed. Fig. 6A shows A375 melanoma cells, which were stained with DAPI and

the α-hapten antibody. The melanoma cells treated with CNP alone showed a

cytosolic and perinuclear staining, whereas in the nuclei a very faint fluorescence

could be seen. These data suggest that ROS generation and consequently thiol

oxidation by CNP occur primarily in the cytosol. This observation matches with the

uptake of CNP, showing that CNP is distributed in the cytosol but not in the nucleus

(Fig 1). By contrast, Doxorubicin treated cells also showed a thiol oxidation in the

nuclei, a result that is in line with the well described property of Doxorubicin to bind to

DNA. Melanoma cells that were co-incubated with Doxorubicin and CNP, revealed a

strong fluorescence in the cytosol as well as in the nuclei, confirming the synergistic

effect on thiol oxidation observed above (Fig. 5).

To study the localization of thiol oxidation more in detail cytoplasmic and nuclear

extracts were prepared. Western Blot analysis of this extracts also showed that CNP

treatment caused thiol oxidation mainly in the cytosol (3 fold increase compared with

the untreated cytoplasmic control) and a smaller amount in the nucleus, whereas

Doxorubicin caused thiol oxidation in the cytosol as well as in the nucleus (2.5-fold

increase of the cytoplasmic fraction and about 2-fold increase of the nuclear fraction

compared to the controls) (Fig.6B). After co-incubation with both agents thiol

oxidation was observed in cytosol and nucleus, while a synergistic effect was seen

only in the cytosol.

In summary, these results indicated a prooxidative activity of both, CNP and

Doxorubicin, which was boosted by co-incubation of that substances and resulting in

oxidative damage of proteins.




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Genotoxicity of CNP and Doxorubicin

Excessive ROS levels result in damage of macromolecules e.g. DNA (34). Previous

studies have shown that CNP induce ROS-dependent intrinsic apoptosis in

melanoma cells (13). Due to the prooxidative activity of CNP in A375 melanoma

cells, it was supposed that CNP induce apoptosis through an increase of ROS.

However, data on a CNP-mediated DNA damage in melanoma cells was lacking until

now.

Doxorubicin is known to damage DNA via several mechanisms e.g. intercalation,

alkylation and complex formation with DNA and topoisomerase 2, thereby inducing

cell death (19). In this study, we were interested in a potential CNP-mediated DNA

damage in A375 tumor cells and stromal human dermal fibroblast. Strand breaks as

a marker of DNA damage were detected with the alkaline COMET assay (28) (Fig.

7). A375 cells incubated with 15 µM Doxorubicin for 4 h showed a 2.5 fold increase of

the comet area compared to the controls, indicating DNA damage. By contrast, no

DNA strand breaks could be detected after treatment with 150 µM cerium oxide

nanoparticles for 96 h (Fig. 7A). CNP showed no significant increase in comet area

compared to the untreated controls, indicating a non-genotoxic effect of CNP. After

co-incubation with Doxorubicin and CNP the comet area was not increased

compared to the treatment with Doxorubicin alone, which corroborates a non-

genotoxic toxicity of CNP in melanoma cells. Although being toxic, cerium oxide

nanoparticles do not induce DNA damage in melanoma cells. A similar effect was

observed in HDF (Fig. 7B). Doxorubicin treatment resulted in 2.5-fold increase of the

comet area of HDF, whereas treatment with CNP did not increase the comet area

compared to the untreated control. Co-incubation with Doxorubicin and CNP resulted

in a comet area that is comparable to the treatment with Doxorubicin alone. In




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summary, these data implicate that CNP treatment does not induce DNA damage in

both cell lines.

Discussion

Nanomedicine is one of the future technologies providing revolutionary improvements

and innovations for therapy and diagnostic of many diseases including cancer (4, 35,

36). Cancer is still one of the most devastating diseases, with more than 10 million

cases every year (5), and thus remains in the focus of interest of basic and clinical

research. Conventional anti-cancer therapies are often associated with harmful side

effects on healthy cells and hold the risk of secondary cancer, in consequence the

clinical application is limited (20, 30). Recent studies showed that redox-active cerium

oxide nanoparticles exhibit a significant anti-tumor activity in several cancer cell lines

(7, 8). In squamous cell carcinoma of the skin and melanoma, CNP exhibit pro-

apoptotic and anti-invasive effects in a ROS-dependent manner. In contrast to

conventional chemotherapeutics CNP are non-toxic in healthy, stromal cells of the

skin. It was described that CNP exert either a pro- or antioxidant redox-activity. While

CNP treatment increases the ROS level in tumor cells resulting in apoptosis, CNP

showed antioxidant and protective properties in normal cells (12). The protective and

antioxidant properties can be traced back to an inherent and pH-dependent.

superoxide dismutase (SOD) mimic activity of CNP (37). In that context, a medical

application of CNP may provide a promising possibility for therapy of skin cancer, and

may be a valuable tool to supplement classical therapeutical approach.

In this study, the anti-tumor activity of CNP was compared to that of the classical and

very effective anti-tumor drug Doxorubicin. Furthermore, it was elucidated whether

CNP may enhance the anti-tumor activity of Doxorubicin after co-incubation,




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particularly with regards to a novel strategy in cancer therapy, a CNP-mediated

supplementation therapy with classical chemotherapeutics. The data showed that

both, CNP and Doxorubicin, exerted cytotoxic effects in the human melanoma cell

line A375. While other studies suppose melanoma to be resistant to anthracyclines

(38), this study demonstrated that Doxorubicin is very effective in inducing cell death

in A375 melanoma cells in concentrations, which can be compared with peak or

steady state plasma concentrations of patients after a standard infusion with

Doxorubicin (> 1 to 2 µM) (19). After co-incubation with both agents the cell viability

of melanoma cells was even more decreased compared to incubation with the agents

alone, indicating a synergistic effect on cytotoxicity in tumor cells. By contrast, in

human dermal fibroblasts pre-incubation with 150 µM CNP abolished the toxic effects

of Doxorubicin, showing a protective effect of CNP against the cytotoxicity of

Doxorubicin in stromal cells.

An increase in ROS level was measured after incubation with CNP as well as with

Doxorubicin. Compared to normal cells, cancer cells were described to have

elaborated ROS levels, which promotes their genomic instability and proliferation, but

also make them more susceptible for an additional increase of ROS mediated by

exogenous noxes, such as redox-cycling drugs and other ROS-producing agents. In

our study, this ROS susceptibility was exploited by the use of the anti-tumor agents

Doxorubicin and CNP. A synergistic effect on ROS generation was detected after co-

incubation. The prooxidative and cytotoxic effect of CNP and Doxorubicin was

confirmed by the formation of sulfenic acids and carbonylated proteins.

Besides protein damage, oxidative stress is known to cause DNA damage. CNP was

shown to generate ROS and to induce apoptosis via the intrinsic pathway in A375

cells, but putative DNA damage by CNP was not measured until now. Surprisingly,

this study demonstrates that CNP do not cause DNA damage at a concentration




20

being toxic in A375 cells. These data may be explained by the uptake and cellular

distribution of CNP in A375 cells displaying a localization of CNP in the cytosol, but

not in the nuclei. In that context it was shown recently that the diffusion of H2O2, a

potential genotoxic agent, across the cytoplasm was strongly limited, as well. It

provides evidence that H2O2 acts locally inside cells (39). Correspondingly, it was

shown that oxidation of thiols occurred mainly in the cytosol. Even though CNP show

an anti-tumor activity, no genotoxic activity was detected in A375 cells. In human

dermal fibroblasts no DNA-damaging effect was observed as well. This would be a

beneficial aspect in a cancer therapy as the use of non-genotoxic anti-tumor agents

decreases the risk of secondary cancer. In contrast, treatment with Doxorubicin

resulted in a significant increase of the comet area in A375 and HDF, representing

DNA damage and a genotoxic activity of Doxorubicin. Co-incubation with CNP did not

increase the DNA damage compared to cells that were treated with Doxorubicin

alone. However, CNP did not protect HDF from Doxorubicin-mediated DNA damage,

but interestingly CNP counteracts the Doxorubicin- induced cell killing in HDF. In

contrast to another recent study with cerium oxide nanoparticles with a size of 16-22

nm (40), DNA damaging effects were found in other tumor cell lines, indicating that

the mode of action of the nanoparticles is strongly depending on size and cell type.

In summary, this study demonstrates that CNP may be qualified to supplement

conventional chemotherapeutic drugs, like Doxorubicin. CNP enhanced the anti-

tumor activity of Doxorubicin in A375 melanoma cells, in context of cytotoxicity and

ROS formation as well as oxidative damage. However, CNP protected human dermal

fibroblasts from Doxorubicin-induced cytotoxicity. Despite the anti-tumor-activity of

CNP, no genotoxic effects of CNP were detected in melanoma cells as well as in

human dermal fibroblasts. The supplementation of conventional chemotherapies with

CNP may offer a novel strategy in treatment of cancer providing a better therapeutic




21

outcome and a higher benefit for patients, by enhancing anti-tumor activity and

lowering the damaging side effects of classical chemotherapeutics such as the model

substance Doxorubicin on healthy cells.

Acknowledgements

This work is part of the master thesis of E. K. at the Heinrich-Heine-University of

Düsseldorf. We thank C. Wyrich for excellent technical assistance. S. Seal

acknowledges the National Science Foundation (NSF) to partially fund the

nanotechnology research under NSF NIRT (CBET-0708172) and NSF (CBET-

0930170). We would like to thank Kate S. Carroll for providing the α-hapten antibody.

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Figure legends

Fig. 1 : Cytosolic distribution of CNP in human melanoma cell line A375 To

study the uptake and cellular distribution of CNP A375 cells were incubated with 150

µM fluorescein-isothiocyanate (FITC)-labeled CNP for 4 h and were analyzed by

fluorescence microscopy. Additionally, the nuclei were stained with DAPI. CNP were

ubiquitously distributed in the cytosol CNP. (A) FITC fluorescence of labeled CNP,

(B) merge of FITC and DAPI fluorescence.

Fig 2 : Cytotoxicity: CNP vs. Doxorubicin Effects on cell viability were assessed by

MTT assay. A375 melanoma cells were incubated with different concentrations of

Doxorubicin for 24 h and CNP for 96 h (A). Co-incubation with 300 µM CNP for 48 h

and 0.5 µM Doxorubicin for 24 h showed synergistic effects on cytotoxicity (B)

Human dermal fibroblasts (HDF) were incubated with different concentrations of

Doxorubicin for 24 h and CNP for 96 h (C). HDF were treated with 150 µM CNP for

48 h and with 25 µM Doxorubicin for 24h as well as in combination (D). The

percentage of cell viability of the untreated control, which was set on 100%, is

presented. ***P<0.001; **P<0.01; *P<0.05 (ANOVA, Dunnett's test). Data are

presented as means ± s.e.m..

Fig. 3 : Synergistic effect of CNP and Doxorubicin on ROS generation To detect

intracellular ROS A375 melanoma cells were loaded with H2DCF-DA and

subsequently treated with 150 µM CNP and 0.5 µM Doxorubicin (DOX) alone or in

combination. Immediately after adding of the substances the fluorescence was

measured for 1,5 h. Presented is one out of three independent experiments.




Fig. 4 : Irreversible protein damage after co-incubation with CNP and

Doxorubicin Carbonyl contents, as marker for irreversible protein damage, in A375

cells were determined by Oxyblot analysis. Cells were treated with 0.5 µM

Doxorubicin for 2 h or with 150 µM CNP for 2, 4 or 24 h, as well as in combination.

H2O2 was used as positive control and GAPDH was used as loading control. Three

independent experiments were performed. CNP and Doxorubicin both induced

carbonyl formation, whereas co-treatment showed synergistic effects in A375.

Fig. 5 : Synergistic effect of CNP and Doxorubicin on thiol oxidation Sulfenic

acid formation in A375 cells were analyzed by western blot. Cells were treated with

0,5 µM Doxorubicin for 2 h or with 150 µM CNP for 2, 4 or 24 h, as well as in

combination. H2O2 was used as positive control. For the last 2 h of incubation

dimedone (10 mM) was added to the cells. GAPDH was used as loading control. The

figure represents one out of three independent experiments that were analyzed by

densitometry with Image J. The x-fold increase of the untreated controls are

presented.

Fig. 6: Localization of oxidated thiols in A375 melanoma cells

(A) After incubation with 150 µM CNP and 5 µM Doxorubicin alone or in combination

cells were fixed for an immunochemical staining was performed by using the α-

hapten antibody raised against the oxidation product of sulfenic acid and dimedone,

which was added to the cells for the last 2 h of incubation. Additionally nuclei were

stained with DAPI. Presented is one out of three independent experiments. (B)

Western Blot analysis were carried out with cytoplasmic and nuclear cell extracts

from melanoma cells that were treated with 0.5 µM Doxorubicin or 150 µM CNP for 2




h, as well as co-incubated. CNP caused thiol oxidation mainly in the cytosol, whereas

Doxorubicin showed strong formation of sulfenic acids in the nuclei as well. Three

independent experiments were performed. α-tubulin was used for the cytoplasmic

extract and Poly (ADP-ribose) polymerase (PARP) for the nuclear extract as loading

control. The figure represents one out of three independent experiments that were

analyzed by densitometry with Image J. The x-fold increase of the untreated controls

are presented.

Fig. 7 : CNP exert no genotoxic effects DNA damage was investigated by using

the alkaline comet assay. Melanoma cells (A) and human dermal fibroblasts (B) were

incubated with 15 µM Doxorubicin for 4 h or with 150 µM for 96 h. Additionally cells

were co-incubated. CNP induced no DNA strand breaks, whereas Doxorubicin

caused a significantly DNA damage. Presented are the mean values of the comet

area in px of three independent experiments. **P<0.01; (ANOVA, Dunnett's test).

Data are presented as means ± s.e.m..




Published OnlineFirst May 13, 2014.Mol Cancer Ther Maren Sack, Lirija Alili, Elif Karaman, et al. cancer therapyredox-active cerium oxide nanoparticles - a novel aspect in Combination of conventional chemotherapeutics with

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