Differential Induction of Apoptosis and Senescence by the ...Life Technologies. 5-azacytidine (5-aza-CR) and 5-aza-20-deoxycytidine (5-aza-dC) were obtained from Sigma-Aldrich and

Cancer Therapeutics Insights

Differential Induction of Apoptosis and Senescence bythe DNA Methyltransferase Inhibitors 5-Azacytidineand 5-Aza-20-Deoxycytidine in Solid Tumor Cells

Sascha Venturelli1, Alexander Berger1,6, Timo Weiland1, Frank Essmann2, Michaela Waibel9, Tina Nuebling3,Sabine H€acker7, Martin Schenk4, Klaus Schulze-Osthoff2,6, Helmut R. Salih3, Simone Fulda8, Bence Sipos5,Ricky W. Johnstone9, Ulrich M. Lauer1, and Michael Bitzer1

AbstractEpigenetic alterations are a hallmark of cancer that govern the silencing of genes. Up to now, 5-

azacytidine (5-aza-CR, Vidaza) and 5-aza-20-deoxycytidine (5-aza-dC, Dacogen) are the only clinicallyapproved DNA methyltransferase inhibitors (DNMTi). Current effort tries to exploit DNMTi application

beyond acute leukemia or myelodysplastic syndrome, especially to solid tumors. Although both drugs

only differ by a minimal structural difference, they trigger distinct molecular mechanisms that are highly

relevant for a rational choice of new combination therapies. Therefore, we investigated cell death pathways

in vitro in human hepatoma, colon, renal, and lung cancer cells and in vivo in chorioallantoic membrane and

xenograft models. Real-time cancer cell monitoring and cytokine profiling revealed a profoundly distinct

response pattern to both drugs. 5-aza-dC induced p53-dependent tumor cell senescence and a high number

of DNA double-strand breaks. In contrast, 5-aza-CR downregulated p53, induced caspase activation and

apoptosis. These individual response patterns of tumor cells could be verified in vivo in chorioallantoic

membrane assays and in a hepatoma xenograft model. Although 5-aza-CR and 5-aza-dC are viewed as

drugs with similar therapeutic activity, they induce a diverse molecular response in tumor cells. These

findings together with other reported differences enable and facilitate a rational design of new combination

strategies to further exploit the epigenetic mode of action of these two drugs in different areas of clinical

oncology. Mol Cancer Ther; 12(10); 2226–36. �2013 AACR.

IntroductionThe 2 DNA methyltransferase inhibitors (DNMTi), 5-

azacytidine (5-aza-CR, Vidaza) and 5-aza-20-deoxycyti-dine (5-aza-dC, decitabine, Dacogen) are clinicallyapproved for the treatment of myelodysplastic syndromeand acute myelogenous leukemia (AML). While bothdrugs are able to induce complete responses and hema-

tologic improvements, a prolonged overall survival couldonly be shown for 5-aza-CR, but not for 5-aza-dC (1, 2).However, at themoment there is no specific guideline thatgoverns which of these two substances should be pre-ferred in a specific clinical context.

The development of novel dosing strategies or thecombinationwith other antitumor therapies hold promiseto exploit the activities of DNMTi compounds for animproved therapeutic effect either in hematologic malig-nancies or even in hitherto unaddressed tumor entities,such as solid tumors. In accordance with this rationale,several clinical studies currently investigate the applica-tion of 5-aza-CR or 5-aza-dC in combination with otheranticancer strategies, including chemotherapeutic or tar-geted agents. For a purposeful combination of DNMTisubstances with other anticancer principles it is of utmostimportance to exactly define the individual tumor cellresponse pattern to each distinct DNMTi compound.Although both DNMTi only minimally differ in theirmolecular structure, there are important differences intheir molecular mode of action (3, 4).

As a general principle, successful antitumor strategieshave to efficiently trigger death pathways. In recent years,therapy-induced senescence (TIS) has become an impor-tant issue in the field of tumor biology and cancer therapy.

Authors' Affiliations: 1Department of Gastroenterology and Hepatology,Medical University Hospital; 2Interfaculty Institute for Biochemistry, Uni-versity of Tuebingen; 3Department of Hematology andOncology, EberhardKarls University; Departments of 4General, Visceral, and Transplant Sur-gery, and 5Pathology, University Hospital Tuebingen, Tuebingen; 6GermanCancer Consortium (DKTK) and German Cancer Research Center, Heidel-berg; 7University Children's Hospital, UlmUniversity, Ulm; and 8Institute forExperimental Cancer Research in Pediatrics, Goethe University Frankfurt,Frankfurt, Germany; and 9Cancer Immunology Program, Peter MacCallumCancer Centre, East Melbourne, Victoria, Australia

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Michael Bitzer, Medical University Hospital,University of Tuebingen, Otfried-Mueller-Str. 10, Tuebingen D-72076,Germany. Phone: 49-07071-2980583; Fax: 49-07071-294402; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-13-0137

�2013 American Association for Cancer Research.

MolecularCancer

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Mol Cancer Ther; 12(10) October 20132226

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Published OnlineFirst August 7, 2013; DOI: 10.1158/1535-7163.MCT-13-0137

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It is well known that senescence as a permanent growtharrest can be induced by a variety of stimuli, ranging fromoxidative stress, DNAdamage, oncogenic stress, telomereshortening, or epigenetic alterations (5–7). However, theconcept of TIS as a new mechanism of anticancer agentsemerged by the observation that some well known drugscan potently trigger tumor cell senescence (8–10). Thus, atherapeutically induced permanent growth arrest seemsto be an attractive new option that might be suitable fora broad range of tumor entities (7). However, it is cur-rently not clear whether the success of such a conceptrequires a second step that subsequently eliminates theresulting senescent cancer cells.5-aza-dC has been reported to induce senescence by

upregulation of p16(INK4a) in oral squamous cell carci-noma and heptocellular carcinoma cell lines (11, 12). Incontrast, comparable results for 5-aza-CR are missing. Ascompoundswith TIS activity should ideally not be used incombination with antitumor approaches that require celldivision, such as antimetabolites, we set out to character-ize and compare in detail the reaction pattern of bothdrugs, 5-aza-CR and 5-aza-dC, for different solid tumorentities. The findings of this study clearly show a sub-stantially different response of cancer cells to both drugs.Together with already reported characteristics of 5-aza-CR and 5-aza-dC, these results should be carefully takeninto account during the development of future studyprotocols that intend to modulate the methylome of can-cer cells byDNMTi application, especially in combinationwith additional anticancer drugs.

Materials and MethodsCell culture and reagentsThe human cell lines HepG2, Hep3B, A-498, HCT-116,

and A549 were obtained from the German Collection ofMicroorganisms and Cell Cultures (DSMZ). According toDSMZ standards PCR-based short tandem repeats anal-yses as cell line authenticationwere conducted. TheHCT-116/p53�/� cells were obtained fromB. Vogelstein (JohnsHopkins University, Baltimore, MD). Disruption of thep53 gene in the HCT-116/p53�/� cells was verified byWestern blotting. All cell lines were stored in liquidnitrogen, passaged for less than 4 months and culturedin Dulbecco’s modified Eagle mediumwith 10% fetal calfserum and 1% L-glutamine. Cells were plated in 6-, 24-,or 96-well plates and treated 24 hours later with theindicated substances. Media and supplements were fromLife Technologies. 5-azacytidine (5-aza-CR) and 5-aza-20-deoxycytidine (5-aza-dC) were obtained from Sigma-Aldrich and staurosporine from Biomol.

Real-time cell analysisHepG2 cells (1 � 104 cells/well) or Hep3B cells (2.5 �

103 cells/well) were seeded in 96-well plates (E-Plate 96,Roche Applied Science). Real-time dynamic cell prolifer-ationwasmonitored in 30minutes intervals formore than106hours using the xCELLigenceRTCASP system (RocheApplied Science) and cell index values were calculated

using the RTCA Software (1.0.0.0805). All curves werenormalized at the beginning of the treatment period, 10hour after seeding, applying the RTCA Software.

Sulforhodamine B-assayFor the sulforhodamine B-assay (SRB) HepG2 cells

(2 � 104 cells/well) were seeded in 24-well plates andtreated with the indicated concentrations of 5-aza-CRor 5-aza-dC. After 96 hours cells were fixed with 10%trichloroacetic acid for 30 minutes at 4�C. After dryingwells were stained with 200 mL 0.4% SRB solution andincubated at room temperature for 10 minutes. Plateswere washed with 1% acetic acid until supernatant wascolorless and dried again. SRB was resuspended in 200mL 10 mmol/L Tris base per well for 10 minutes on iceand absorption was measured at 550 nm.

Determination of cell deathApoptosis was determined by measuring the activity

of executioner caspases and by fluorescence-activatedcell sorting (FACS) quantification of hypodiploid cells.For the caspase assay 1 � 104 HepG2 or Hep3B cells perwell were seeded in 96-well plates, treated with differ-ent concentrations of 5-aza-CR or 5-aza-dC for 36 hoursor 48 hours and then subjected to the Caspase-Glo 3/7assay (Promega) as described by the manufacturer. As apositive control for caspase activation cells were treatedwith 5 mmol/L staurosporine. For FACS analysis ofsub2N peaks 7.5 � 104 HepG2 or Hep3B cells per wellwere seeded in 24-well plates and treated with differentconcentrations of 5-aza-CR or 5-aza-dC. After 24, 48, or72 hours cells were stained in hypotonic buffer withpropidium iodide for 30 minutes and then analyzed in aflow cytometer. Hypodiploid (sub2N) cells were con-sidered apoptotic.

Measurement of the cellular diameterHepG2 cells (7.5 � 104 cells/well) were cultured on

cover slips for 24 hours in 24-well plates. Each cover slipwas transferred to a separate cell-culture chamber of aPANsys 3000 system (PAN-Systech GmbH) and treatedwith 20 mmol/L 5-aza-CR or 5-aza-dC. The identicalobservation point in each chamber was monitored for atotal time span of 96 hours by phase-contrast microscopy.The maximum diameter of 20 representative cells in eachcell-culture chamber after 48, 72, and 96 hours incubationwas measured with ImageJ digital imaging software(ImageJ; http://rsbweb.nih.gov/ij/download.html).

Senescence-associated b-galactosidase stainingHepG2, Hep3B, A-498, HCT-116, HCT-116/p53�/�,

and A549 cells were plated in 6-well plates at a densityof 2 � 104 cells per well and treated with the respectiveagents. After 72 hours (HepG2 andHep3B) or 96 hours (A-498,HCT-116, andA549), cellswerewashedwith PBS andfixed in 2% paraformaldehyde/glutaraldehyde beforethe staining with X-gal solution according to the manu-facturer’s instructions (Senescence Cell Histochemical

Differential Induction of Senescence by DNMTi

www.aacrjournals.org Mol Cancer Ther; 12(10) October 2013 2227




Staining Kit, Sigma-Aldrich). After 24 hours of incubationat 37�C, nuclei were stained with 2 mg/mLHoechst 33342(Invitrogen). The percentage of b-galactosidase–positivecells was determined by counting nuclei and cell bodieswith a blue precipitate (Olympus IX50, analySIS, SoftImaging System). For each time point and concentrationat least 100 cells were counted.

H3K9me3 stainingTo detect histone H3 lysine-9 trimethylation

(H3K9me3) HepG2 cells (6 � 103 cells/well) were seededon cover slips in 12-well plates and after 24 hours incu-bated with 20 mmol/L 5-aza-CR or 5-aza-dC for 72 hours.Cells werewashed in PBS and fixed in ice-coldmethanol/acetone (1:1) for 20 minutes on ice. Then, cells werewashed in PBS and incubated in IF-buffer (PBS, 4%bovineserum albumin, 0.05% saponin) for 1 hour with shaking.Subsequently, rabbit anti-H3K9me3 (1:500, Cell SignalingTechnology) andmouse anti-a-tubulin (1:500, DM1a, Sig-ma-Aldrich) antibodies were applied in IF-buffer at 4�Cover night. Samples were washed twice in PBS and incu-bated with secondary antibodies (1:500, chicken anti-mouse Alexafluor-594 and chicken anti-rabbit Alexa-fluor-488, Invitrogen) in PBS for 3 hours with shaking.After washing in PBS, samples were incubated in PBScontaining 40,6-diamidino-2-phenylindole (100 mg/mL,Sigma-Aldrich) for 10 minutes and finally mounted influorescence mounting medium (DAKO). Images weretaken using a Zeiss Axiovert 200 M microscope (63 � oilimmersion objective) equipped with an ApoTome andAxioVision software.

Detection of DNA double-strand breaksDNA double-strand breaks were assayed by a histone

H2A.X Phosphorylation ELISA (CycLex Co., Ltd.). To thisend, HepG2 cells (3 � 104 cells/well) were seeded in 96-well plates and at the following day, treatedwith 5, 10, 20,50, and 100 mmol/L of 5-aza-CR or 5-aza-dC. Forty-eighthours after treatment, histone H2A.X phosphorylationwas measured according to manufacturer’s protocol.

Western blottingHepG2, Hep3B, A-498, HCT-116, HCT-116/p53�/�,

and A549 cells were plated in 6-well plates at a densityof 2 � 105 cells per well, treated with indicated amountsof 5-aza-CR or 5-aza-dC and incubated for 24 hours.Immunoblotting was conducted with anti-p53, (1:500,Santa Cruz Biotechnology Inc.) and anti-vinculin (1:6,000,Sigma-Aldrich) antibodies.

Densitometric analysisX-ray films of Western blots and cytokine profiling

arrays were digitizedwith an imaging system (FluoChem8900, Alpha Innotech). Densitometric analyses were con-ducted using ImageJ (version 1.6.0_14). In brief, bandswere surrounded by rectangle and plotted. The back-ground was subtracted from the obtained peak and thearea under the peak was calculated.

Cytokine profilingHepG2 cells (2� 104 cells/well) were seeded in 24-well

plates and treatedwith 20 mmol/L 5-aza-CR or 20 mmol/L5-aza-dC for 72 hours. Supernatant was harvested, cen-trifuged at 5,000 rpm for 3 minutes and transferred to anew tube. Profiling of 36 different human cytokines,chemokines, and acute phase proteins in the cell super-natants was conducted according to the manufacturer’sinstructions (Human Cytokine Array Panel A, R&D Sys-tems). Detection was conducted by the ECL Westernblotting detection system on Hyperfilm-ECL (AmershamBiosciences).

Animal treatment protocolHousing, tumor inoculation, and drug treatment were

conducted in collaboration with the Institute of Experi-mental Oncology (Oncotest GmbH). In brief, NMRI micereceived an inoculation of HepG2 hepatoma cells into theright and left flank.Whenpalpable tumors became detect-able, animals were divided randomly into 3 groups:intraperitoneal injection of vehicle only (control group;5 mice), 5-aza-CR (0.8 mg/kg; 6 mice), or 5-aza-dC (0.8mg/kg; 6 mice). Animals were treated once daily andsacrificed after 3 days by carbon dioxide asphyxiation.All animal experiments were carried out in agreementwith German laws concerning the conduct of animalexperimentation.

Histology and immunohistochemistryTumor specimens were removed from the xenografted

mice, fixed in formalin, and embedded in paraffin. Serialsections were routinely stained with hematoxylin andeosin (H&E). After deparaffinization of the tissue sectionsand heat-induced antigen retrieval immunohistochemis-try was conducted using an anti-p16(INK4a) antibody(E6H4, Roche MTM Laboratories) and a Ventana Bench-Mark XT System (Ventana Medical Systems). The histo-logic analysiswas conducted by a pathologist in a blindedfashion.

Chorioallantoic membrane assayThe chorioallantoic membrane (CAM) assay was done

as described (13). Briefly, 1 � 106 HepG2 cells wereimplanted on fertilized chicken eggs on day 8 of incuba-tion and treated with vehicle (control) or 5 to 10 mmol/L5-aza-CR and 5-aza-dC, respectively. After 3 days, tumorswere sampled with the surrounding CAM, fixed in 4%paraformaldehyde, embedded in paraffin, cut in 5 mmsections, and stained with H&E. The histologic analysiswas conducted by a pathologist in a blinded fashion.

Statistical analysisStatistical analyses were conducted either with an

unpaired Student t test or a Mann–Whitney test usingGraphPad Prism Version 4.00 (GraphPad Software).According to this analysis, the following 3 differentP values were examined: a P value of 0.01 to 0.05 (�),a P value of 0.001 to 0.01 (��), and a P value < 0.001 (��).

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Mol Cancer Ther; 12(10) October 2013 Molecular Cancer Therapeutics2228




ResultsDifferential effects of 5-aza-CR and 5-aza-dC oncellular morphologyTo investigate the cellular reaction patterns to 5-aza-CR

and5-aza-dC,wefirst used real-timemeasurements of thecellular impedance using the xCELLigence SP system (14,15) over a 96-hour time period. The cellular impedance isdepicted by the cell index and a reduction of the cell indexindicates reduced viability or induction of apoptosis,whereas increased cell index values indicate proliferationor increased cell size (6, 15, 16). Unexpectedly, weobserved differential effects of 5-aza-CR and 5-aza-dCtreatment on HepG2 and Hep3B hepatoma cells for all

tested concentrations (Fig. 1A). Incubation of both celllines with 5-aza-CR at concentrations from 5 mmol/L to50 mmol/L led to a reduction of the normalized cellindex in comparison with untreated cells. In contrast,5-aza-dC treatment increased the normalized cell indexof HepG2 cells. In additionally conducted experiments, acell index increase was even found for 1 and 0.5 mmol/Lof 5-aza-dC (data not shown). Interestingly, this 5-aza-dC–mediated effect was not detectable in Hep3B cells.The most prominent difference between these 2 hep-atoma cell lines is their different p53 status: HepG2 arep53 wild-type expressing cells, whereas Hep3B cells arep53-deficient (17).

Figure 1. Distinct effects of 5-aza-CR and 5-aza-dC on tumor cell proliferation, viability and cell size. A, real-time cell monitoring of HepG2 and Hep3Bhepatoma tumor cells over 106 hours. Cells were seeded in a microplate, treated with the vehicle control or the indicated concentrations of 5-aza-CRand 5-aza-dC, respectively, after 10 hours and observed for additional 96 hours. Cellular impedance was measured continuously using the xCELLigence SPsystem and is depicted as cell index. Displayed are cell index values obtained every 5 hours, normalized at the time point of treatment. Treatment withTriton 0.1% X-100 was used as positive control for cell death. Shown are mean values � SD of a representative experiment out of three independentexperiments carried out in triplicate. B, sulforhodamine B assay of HepG2 cells treated with the indicated concentrations of 5-aza-CR and 5-aza-dCfor 96 hours. Shown are mean values � SD of four independent experiments, each carried out in duplicate; Mann–Whitney test; ��, P < 0.01; ��, P < 0.001.C, 5-Aza-dC induces an increase of the cellular diameter. Changes in cellular morphology were investigated by measuring cellular diameter of HepG2 cellsupon treatment with vehicle or 20 mmol/L of 5-aza-CR or 5-aza-dC for 48, 72, and 96 hours. The diameter of 20 cells per groupwasmeasured in a randomizedfashion and calculated as mean � SD; Mann-Whitney test; ��, P < 0.01; ��, P < 0.001; ns, not significant.






To further characterize the differential results of thereal-time cell monitoring for 5-aza-CR and 5-aza-dC, asulforhodamine B cytotoxicity assay was conducted (Fig.1B). Incubation ofHepG2 cellswith 5-aza-CR resulted in adose-dependent decline of viability. The viability ofHepG2 cells was significantly reduced by 5-aza-CR at allconcentrations tested in comparison with 5-aza-dC (Fig.1B; ��,P < 0.001). In addition,measurement of the cellulardiameter was conducted after 48, 72, and 96 hours oftreatment with 20 mmol/L 5-aza-CR or 5-aza-dC (Fig.1C and Supplementary Table S1). This concentration waschosen to ensure a strong hypomethylating potency forboth compounds. While 5-aza-CR treatment did not alterthe cell size, a significant increase in the cellular diameterwas observed when HepG2 cells were incubated with 5-aza-dC for 72 or 96 hours (Supplementary Table S1; ��, P <0.01; ��,P < 0.001). These results complywith the findingsof the real-time cellmonitoring and indicate that the 5-aza-dC–mediated increase of the cell index in the p53 wild-type cell lineHepG2was based on an increment of the cellsize, which is a known hallmark of cellular senescence.

5-aza-dC but not 5-aza-CR induces cellularsenescence

To further investigate a potential senescence inductionby theDNMTi 5-aza-dC, the expression of the senescence-associated marker b-galactosidase (SA-b-gal) was deter-mined. HepG2 and Hep3B cells were treated for 72 hourswith 20mmol/L5-aza-CRor 5-aza-dC.This timepointwaschosen due to the divergent curve progression in the real-time monitoring and the results of the cellular size mea-surement. Incubationwith 5-aza-dC, but not 5-aza-CR, ledto a significant increase (��, P < 0.001) in the number ofSA-b-gal–positive HepG2 cells (Fig. 2A) displaying acharacteristic blue staining (Fig. 2B).

Another marker for senescence is the accumulation oftrimethylated histone H3 lysine-9 (H3K9me3), which isinvolved in the formation of so-called senescence-associ-ated heterochromatin foci (SAHF; refs. 18, 19). We there-fore investigated SAHF formation by immunofluores-cence microscopy of H3K9me3 in HepG2 cells (Fig. 2C).In line with the previous results and unlike 5-aza-CR,treatmentwith 20 mmol/L 5-aza-dC for 72 hours triggerednot only the typical increase of cell size, but also resulted inthe characteristic accumulation of H3K9me3 in subnucle-ardots. Together, our in vitro results clearly indicate that 5-aza-dC induces cellular senescence in p53 wild-typeHepG2 cells.

We further analyzed the inductionof senescence in an invivo xenotransplant model. Noteworthy, in comparisonwith the in vitro situation senescent cells often lack todisplay all classical markers of senescence in vivo (9).Especially the commonly used SA-b-gal staining has atleast in somemodels not been a reliablemarker for cellularsenescence. Thus, other classic senescence markers, suchas the cell-cycle inhibitor p16(INK4a)havebeen suggestedto be applied instead (9). In our model, we thereforeinvestigated on the one hand p16(INK4a) expression as

a marker of senescence and on the other hand conductedH&E staining to compare p16(INK4a) staining with theoverall cellular viability status of the tumor cells. For thispurpose, nudemice harboring subcutaneously implantedHepG2 xenografts were treated intraperitoneally witheither the solvent control, 5-aza-CRor 5-aza-dC.To choosea comparable experimental setup to the in vitro experi-ments, the in vivo grown tumorswere explanted after only72 hours of treatment. Hence, a reduced tumor volumewas not expected but the p16(INK4a) expression andformation of necrosis was analyzed. In this experimentalsetting, only 5-aza-dC but not 5-aza-CR induced an upre-gulation of p16(INK4a), whereas 5-aza-CR treatmentcaused a more pronounced increase in necrosis in thetumor tissues (Fig. 2D). Thus, our in vivo experimentssupport the in vitro findings of senescence induction by5-aza-dC.

Another feature of senescence is the induction of thesenescence-associated secretory phenotype (SASP),which comprises various cytokines, growth factors, orsoluble receptors that are released from senescent cells(16, 20, 21). We therefore measured the SASP componentsin supernatants ofHepG2 cells treated for 72hourswith 20mmol/L 5-aza-CR or 5-aza-dC. The incubationwith 5-aza-dC caused increased levels of sICAM-1, interleukin (IL)-1ra, and IL-8 in the culture supernatants (Fig. 2E andSupplementary Fig. S1). Interestingly, all three moleculeshave previously been associated with cellular senescence(22–25). In contrast with 5-aza-dC, 5-aza-CR led to areduced secretion of sICAM-1 and IL-1ra, which mightbe a result of protein synthesis inhibition by this com-pound. IL-8, however, was upregulated by both DNMTi(Fig. 2E). Interestingly, increased IL-8 expression has beendescribed as a molecular marker for hepatotoxicity inHepG2 cells (26) as well as under various apoptotic con-ditions in other cell types (27, 28). Hence, induction of IL-8expressionby5-aza-CRcould rather be explainedas a signof cytotoxicity than as an effect of cellular senescence. Ofnote, both drugs did not induce an increase of IL-8 in p53-deficient Hep3B cells.

DNMTi 5-aza-CR directly activates cell deathinstead of cellular senescence

5-aza-CR possesses a ribonucleoside structure and canbe incorporated into both RNA and DNA, whereas 5-aza-dC, based on its deoxyribonucleoside structure, can beinserted intoDNAonly (3, 29, 30). Because of this differentchemical structure 5-aza-CR seems to be more toxic and aless potent DNMTi at comparable concentrations. Todetermine whether 5-aza-CR induces other cellular path-ways than senescence, apoptosis was analyzed by flowcytometric measurement of cells with hypodiploid DNA(Fig. 3A and B). Incubation with 5-aza-CR resulted in adose-dependent increase of hypodiploid HepG2 andHep3B cells after 48 and 72 hours, whereas 5-aza-dCshowed only a marginal alteration in HepG2 cells.Because of the prodrug characteristics of 5-aza-CR andto exclude a too low dosage of 5-aza-CR in comparison

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with 5-aza-dC also a concentration of 100 mmol/L wastested by FACSanalysis at these two timepoints.Again, 5-aza-CR displayed a pronounced increase in the sub2Nfraction under this dosage (Supplementary Fig. S2A andS2B). In addition, caspase-3/7 activity (being used as anindicator for apoptosis) was assessed after 5-aza-CR or 5-aza-dC treatment, including the 100 mmol/L concentra-tion (Supplementary Fig. S2C and S2D).Treatment with the two DNMTi for 36 and 48 hours

resulted in activation of the executioner caspases uponexposure tohigh concentrations of 5-aza-CRbutnot 5-aza-dC. Thus, 5-aza-CR reduces the cell viability by caspase

activation resulting in apoptosis of both p53-proficientand -deficient tumor cells.

To further investigate the effects of 5-aza-CR and 5-aza-dC on cell death induction in an in vivo model, bothcompounds were used in a CAM assay with HepG2 cells.Representative images of the H&E-stained sectionsshowed a profound necrosis by 5-aza-CR treatment,whereas 5-aza-dC had no relevant effect (Fig. 3C). Thus,5-aza-CR is able to induce caspase activation, cell deathand substantial tumor necrosis in vivo. Notably, thisobservation is in clear contrast with the senescent pheno-type induced by 5-aza-dC.

Figure 2. 5-aza-dC but not 5-aza-CR induces a senescence-like phenotype in vitro as well as in vivo. A, HepG2 and Hep3B cells were treated with vehicleor 20 mmol/L of 5-aza-CR and 5-aza-dC, respectively. After 72 hours cells with blue shades, positive for SA-b-gal and the corresponding nuclei werecounted by microscopy. Bars, mean � SD of three independent experiments; Student t test; ��, P < 0.001; ns, not significant. B, representativeimages of HepG2 cells treated with vehicle (control), 20 mmol/L of 5-aza-CR or 5-aza-dC for 72 hours, and stained for senescence induction. Blueshades reveal increased SA-b-gal activity indicative of cellular senescence. White scale bars, 100 mm. C, representative immunofluorescence picturesof HepG2 cells treated with vehicle, 20 mmol/L 5-aza-dC or 5-aza-CR for 72 hours. Cells were stained for nuclei using DAPI, a-tubulin, and trimethylationof H3K9 (H3K9me3). The merged pictures show the increase of the senescence marker H3K9me3 in 5-aza-dC–treated HepG2 cells. White scalebars, 20 mm. D, HepG2 tumor specimens of xenotransplanted mice, which had received a daily intraperitoneal injection with either vehicle, 0.8 mg/kg5-aza-CR or 0.8 mg/kg 5-aza-dC (n � 9/group), were stained for p16(INK4a) as a marker for senescence and H&E. Images of p16(INK4a) stainingare displayed at 200-fold magnification, images of the H&E staining are displayed at 100-fold magnification. E, 5-aza-dC increases the release of theSASP components sICAM-1, IL-1ra, and IL-8. The concentrations of SASP components were measured in supernatants of HepG2 cells after 72 hoursof treatment with 20 mmol/L of 5-aza-CR or 5-aza-dC, using a cytokine protein array and densitometric analysis; Mann–Whitney test; �, P < 0.05,significant decreases are marked in red.






5-aza-dC induces profound DNA damage, whereas5-aza-CR reduces cellular p53 levels

Several DNA damage pathways can induce cellularsenescence.We therefore analyzed the occurrence of dou-ble-strand breaks upon 5-aza-CR or 5-aza-dC treatmentby measuring the phosphorylation of H2A.X in HepG2cells (Fig. 4A). In linewith published data (3), we detecteda significant increase of phosphorylated gH2A.X by 5-aza-dC in a similar concentration range that induced senes-cence. 5-aza-CR, in contrast, only caused marginal DNA-damaging effects (Fig. 4A; ��, P < 0.01, ��, P < 0.001).

Unlike 5-aza-dC, 5-aza-CR is known to block RNAsynthesis (3, 30), followed by inhibition of protein syn-thesis, which might especially affect short-lived proteins,such as p53 (3, 4). For this reason, the protein levels of p53inHepG2 cells were determined byWestern blotting after5-aza-CR or 5-aza-dC treatment (Fig. 4B). Notably, p53levels showed a dose-dependent decline, which startedto occur already within 24 hours of treatment with 5-aza-CR but not 5-aza-dC (Fig. 4B). Of note, as described in theliterature, no expression of p53 was detectable in Hep3Bhepatoma cells (Supplementary Fig. S3). In addition,

Figure 3. 5-aza-CR, but not 5-aza-dC, induces cell death in tumor cells in vitro and in vivo. A and B, determination of sub2N fractions as marker forapoptosis after treatment of HepG2 and Hep3B cells with the indicated concentrations of 5-aza-CR or 5-aza-dC for 48 (A) or 72 (B) hours. Bars, mean �SD of three independent experiments, each carried out in triplicate. C, representative images of HepG2 cells that were seeded on the CAM ofchicken embryos, allowed to form tumor for 8 days and treated for 3 days with vehicle or 10 mmol/L 5-aza-CR and 5-aza-dC, respectively. Tumor cells(3 samples/group) were fixed, embedded in paraffin, and stained with H&E. Images are displayed at 100-fold magnification or 400-fold magnificationin the cut out, respectively.

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alterations of p53 levels were investigated in other p53wild-type harboring tumor cells, including HCT-116colon, A498 kidney, and A549 lung carcinoma cells.Remarkably, in all tumor cell lines, a dose-dependentdecline in p53 protein levels was detected upon 5-aza-CRtreatment (Fig. 5A and Supplementary Fig. S4A and S4C).In contrast, 5-aza-dC rather led to an increase in p53protein levels, which might be related to the senes-cence-inducing capacity of this drug. In line with p53wild-type HepG2 cells, treatment with 20 mmol/L 5-aza-dC induced a significant increase of SA-b-gal stainingin these solid tumor cells (Fig. 5B and Supplementary Fig.S4B and S4D).Most interestingly, HCT-116/p53�/� coloncarcinoma cells lacking p53 displayed an altered reactionpattern. Treatment with 20 mmol/L 5-aza-CR or 5-aza-dCdid not induce a significant increase of the percentage ofb-galactosidase–positive tumor cells emphasizing theessential role of p53 for 5-aza-dC–mediated TIS (Fig. 5Cand D).

DiscussionBoth, 5-aza-CRand5-aza-dCwere originallydeveloped

as nucleoside antimetabolites for anticancer therapy (30,31). Both drugs are analogues of cytidine and share thesame basic chemical structure with only small differencesin the ribose backbone. For this reason 5-aza-CR and 5-aza-dCare supposed to exhibit a similarmode of action byinhibiting cellular DNA methyltransferases (31). Bothazanucleosides have been shown to be effective in thetreatment ofmyelodysplastic syndrome andAMLand arecurrently the only U.S. Food and Drug Administration-

approvedDNMTi compounds (3). In some tumormodels,such as oral squamous-cell carcinoma or heptocellularcarcinoma, 5-aza-dC induces cancer cell senescence (11,12). This has led to the hypothesis that either DNAhypomethylation or an alteration of the DNA structuremight be involved in senescence induction (8, 32). There-fore, it might be reasoned that compounds with DNMTiactivity generally induce tumor cell senescence by DNAdemethylation. However, according to our in vitro and invivo experiments, only 5-aza-dC induced a senescentphenotype in p53 wild-type tumor cells. In contrast, adose-dependent reduction of viability, induction of celldeath and decline of p53 protein levels were observedupon 5-aza-CR treatment in different tumor cell lines.These results further imply important differences of thesetwo closely related and clinically applied demethylatingcompounds in solid tumor cells. Concerning AML as adisease in which both substances are currently in clinicaluse, a recent publication reported, for example that onlyabout 30% of tumor cells with a complex karyotype arep53 wild-type (33). To our knowledge, it is not known sofar, whether p53 wild-type patients with AML differen-tially respond to 5-aza-CR and 5-aza-dC. As a startingpoint from our article it would be interesting to directlycompare these response patterns in different AML popu-lations, which could possibly lead to the identification ofpatient subgroups that benefit most from either 5-aza-CRor 5-aza-dC.

Possible explanations for the distinct cellular responsepatterns of 5-aza-CR and 5-aza-dC are versatile. Concern-ing DNMTi activity, 5-aza-dC is regarded to be a more

Figure 4. 5-aza-dC induces DNAdouble-strand breaks, whereas5-aza-CR reduces p53 proteinlevels. A, determination ofphosphorylated H2A.X as a markerfor DNA double-strand breaks inHepG2 cells after treatment withthe indicated concentrationsof 5-aza-CR or 5-aza-dC;Mann–Whitney test; �, P < 0.05;��, P < 0.01; ��, P < 0.001. B,Western blot and densitometricanalysis of p53 content in HepG2cells after 24 hours of treatmentwith vehicle or the indication of5-aza-CR or 5-aza-dC. Expressionof the house keeping proteinvinculin served as a control forequal protein loading. Relativep53 levels were determined bydensitometric analysis.






potent inhibitor than its ribonucleoside analogue 5-aza-CR (3, 34). Furthermore, even at similar concentrations5-aza-CR and 5-aza-dC induce quite different changes ingene expressionpatterns, as for example, shown in severalcomparative gene array analyses, mainly in AML cells

(34–36). In light of these data, it is not surprising thatboth substances differ in their mode of action, includingthe induction of senescence in p53 wild-type solid tumorcells exclusively by 5-aza-dC. Noteworthy, an elegantrecent study that investigated the transient application

Table 1. List of reported molecular and cellular differences between 5-aza-CR and 5-aza-dC

Characteristics 5-aza-CR 5-aza-dC Refs.

Chemical structure

RNA incorporation 60%–80% RNA incorporation No RNA incorporation 30, 36, 45–47DNA incorporation 20%–40% DNA incorporation 100% DNA incorporation 30, 36, 45–47Transcriptional regulationpattern

In low doses less and in highdoses more differentiallyregulated genes than 5-aza-dC

In low doses more and inhigh doses less differentiallyregulated genes than 5-aza-CR

34, 36

Protein biosynthesis Inhibition of protein biosynthesis No inhibition of protein biosynthesis 4, 36, 46, 47DNA strand breaks Weaker DNA damage compared

with 5-aza-dCIncreased DNA damage comparedwith 5-aza-CR

3, 36

Chromosomal damage Weak micronuclei formation Strong micronuclei formation 49NK cell activity Reduction of NK cell activity Enhancement of NK cell activity 43

Figure 5. 5-aza-CR induces a decrease of p53 protein levels, whereas 5-aza-dC increases b-galactosidase activity p53 dependently. A and C, Western blotand densitometric analyses of p53 content in cell lysates of HCT-116 wt (wild-type) and HCT-116/p53�/� colon carcinoma cells treated with vehicle orincreasing concentrations of 5-aza-CR or 5-aza-dC over a 24 hours time period. Vinculin expression served as a loading control. B and D, after 96 hoursof incubation with vehicle or 20 mmol/L 5-aza-CR or 5-aza-dC, HCT-116 wt and HCT-116/p53�/� cells positive for SA-b-gal and nuclei were countedvia microscopy. Bars represent mean � SD of three independent experiments; Student t test; ��, P < 0.01; ns, not significant.

Venturelli et al.





of 5-aza-CR and 5-aza-dC reported that already lowdosesexerted distinct alterations in several signaling pathwaysin leukemia cell lines and primary cancer cells, respec-tively (37).Another contribution to TIS induction might be the

higher amount of DNA strand breaks that are triggeredby 5-aza-dC compared with 5-aza-CR (38). It is wellknown that DNA damage signaling pathways are desig-nated activators of cellular senescence (7) and that p53plays an essential role in DNA damage-induced senes-cence in tumor cells (5, 6). Our results are in linewith thesefindings, showing senescence induction in functional p53-harboring HepG2, but not in p53-deficient Hep3B cells.Interestingly, due to a 5-aza-CR–mediated inhibition ofprotein synthesis a dose-dependent decline of p53 levelswas detected not only in hepatoma-derived, but also inp53wild-type cells of other tumor entities including colon,renal, and lung cancer. Notably, in contrast withHCT-116colon carcinoma cells, the p53-deficient derivative HCT-116/p53�/� cell line did not develop TIS under 5-aza-dCtreatment. The observed p53 dependency is in line withreports showing that restoration of p53 induces cellularsenescence andupregulation of SASP components in livercarcinomas (39, 40). Interestingly, loss of p53, as by 5-aza-CR treatment, generally facilitates tumor growth and anaggressive malignant phenotype (39).In summary, our data illustrate that the 2 closely related

DNMTi nucleoside analogues 5-aza-CR and 5-aza-dCinduce a substantially different reaction pattern in tumorcells. This observation is supported by our previous data(4) and other reports showing distinct effects of bothcompounds on antiproliferative activity, cytotoxicity,gene demethylation, transcription, natural killer (NK) cellactivity, or DNA repair (34, 36, 41–49). A comprehensiveoverview of these differences is summarized in Table 1.Thus, although 5-aza-CR and 5-aza-dC are often viewedas mechanistically similar, our results clearly emphasizespecific differences between both drugs, which have to be

considered for the further development of these com-pounds, especially in combination approaches with otheranticancer strategies.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: S. Venturelli, A. Berger, T. Weiland, S. Fulda,U.M. Lauer, M. BitzerDevelopment of methodology: S. Venturelli, T. Weiland, F. Essmann,S. Fulda, B. SiposAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Venturelli, A. Berger, T. Weiland, F. Essmann,M.Waibel, T.Nuebling, S.H€acker,M. Schenk,H.R. Salih, S. Fulda, B. Sipos,U.M. Lauer, M. BitzerAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): S. Venturelli, A. Berger, T. Weiland,F. Essmann, T. Nuebling, K. Schulze-Osthoff, H.R. Salih, S. Fulda, B. Sipos,U.M. Lauer, M. BitzerWriting, review, and/or revision of the manuscript: S. Venturelli,F. Essmann, M. Waibel, M. Schenk, K. Schulze-Osthoff, H.R. Salih, S.Fulda, B. Sipos, R.W. Johnstone, U.M. Lauer, M. BitzerAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S. Venturelli, F. Essmann,M. Schenk, S. Fulda, U.M. Lauer, M. BitzerStudy supervision: R.W. Johnstone, U.M. Lauer, M. Bitzer

AcknowledgmentsTheauthors thankA. Schenk, I. Smirnow,M. Seitzer, L.Cluse, B.Martin,

K. Petersen, and C. Leischner for excellent technical assistance. Theauthors also to thank B. Vogelstein for the HCT-116/p53�/� colon carci-noma cell line.

Grant SupportThis work was supported in part by grants from the Deutsche For-

schungsgemeinschaft (SFB 773). S. Venturelli was supported in part by agrant from MSD-Forschungsstipendium Onkologie and A. Berger by agrant from the fortuene program of the University Hospital Tuebingen(1966-0-0).

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

Received March 5, 2013; revised July 11, 2013; accepted July 28, 2013;published OnlineFirst August 7, 2013.

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2013;12:2226-2236. Published OnlineFirst August 7, 2013.Mol Cancer Ther Sascha Venturelli, Alexander Berger, Timo Weiland, et al. -Deoxycytidine in Solid Tumor Cells

′Methyltransferase Inhibitors 5-Azacytidine and 5-Aza-2Differential Induction of Apoptosis and Senescence by the DNA

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Differential Induction of Apoptosis and Senescence by the ...Life Technologies. 5-azacytidine (5-aza-CR) and 5-aza-20-deoxycytidine (5-aza-dC) were obtained from Sigma-Aldrich and