SmacMimeticsinCombinationwithTRAILSelectivelyTarget Cancer ...mct.aacrjournals.org/content/molcanther/early/2013/... · Cancer Stem Cells in Nasopharyngeal Carcinoma Man-si Wu1, Guang-feng

Small Molecule Therapeutics

SmacMimetics in Combinationwith TRAIL Selectively TargetCancer Stem Cells in Nasopharyngeal Carcinoma

Man-si Wu1, Guang-feng Wang3, Zhi-qiang Zhao2, Yi Liang1, Heng-bang Wang3, Miao-yi Wu3,Ping Min3, Li-zhen Chen1, Qi-sheng Feng1, Jin-xin Bei1, Yi-xin Zeng1, and Dajun Yang1,3

AbstractNasopharyngeal carcinoma is a common malignancy in Southern China. After radiotherapy and chemo-

therapy, a considerable proportion of patients with nasopharyngeal carcinoma suffered tumor relapse and

metastasis. Cancer stemcells (CSC) have been shownwith resistance against therapies and thus consideredas the

initiator of recurrenceandmetastasis in tumors,where the antiapoptotic property ofCSCsplay an important role.

Smac/DIABLO is an inverse regulator for the inhibitors of apoptosis protein family (IAP), which have been

involved in apoptosis.Here, the effects of Smacmimetics on theCSCsof nasopharyngeal carcinomawere studied

both in vitro and in vivo, using two clones of nasopharyngeal carcinoma cell line CNE2 asmodels.We found that

one of the clones, S18, hadCSC-like properties and IAPswere overexpressed. The combination of Smacmimetics

and TNF-related apoptosis-inducing ligand (TRAIL) can reduce the percentage of SP cells and inhibit the colony-

and sphere-forming abilities of S18 cells, indicating their ability to attenuate the CSCs. Moreover, in a

nasopharyngeal carcinoma xenograft model, the administration of Smac mimetics in combination with TRAIL

also led to the elimination of nasopharyngeal carcinoma stem cells. Furthermore, the Smac mimetics in

combination with TRAIL induced the degradation of cIAP1 and XIAP and thus induced apoptosis in vitro and

in vivo. Taken together, our data show that Smac mimetics exerted an antitumor effect on nasopharyngeal

carcinoma cancer stemcells, and this combination treatment shouldbe considered as a promising strategy for the

treatment of nasopharyngeal carcinoma. Mol Cancer Ther; 12(9); 1–10. �2013 AACR.

IntroductionNasopharyngeal carcinoma is an endemic malig-

nancy that occurs predominantly in populations fromSouthern China, Southeast Asia, North Africa, andthe Arctic Circle (Eskimos and other Arctic natives;ref. 1). The application of chemotherapeutic drugs hasbecome more important in the treatment for nasopha-ryngeal carcinoma in recent years, and cisplatin, 5-fluo-rouracil, and taxel are the most commonly used drugs(2, 3). However, some patients still suffered from fail-ure of treatment including relapse and metastasis,which is thought to originate from cancer stem cells(CSC; refs. 3–9).

CSCs have the properties of self-renewal, differentia-tion, and resistance to chemotherapy or radiotherapy(7, 10). Although the CSCs represent a small proportionof the tumor cells, they are key players in tumor initiation,recurrence, and metastasis (8–12). Therefore, CSCs havebeen considered as an important therapeutic target inanticancer treatments (6, 9, 13–16). Many compoundshave been shown to selectively target CSCs in cancerssuch as salinomycin (6), TbRI inhibitors (9), lupeol (14),sulforaphane (15), thioridazine (16), and others.

As in other cancers, nasopharyngeal carcinoma containsa small fraction of tumor cells with properties of CSCs.Studies have shown that the side population (SP) cells,identified by having ability to pump out a fluorescent dye(Hoechst 33342), have certain characteristics of CSCs sim-ilar to those in liver cancer and gastrointestinal cancer,suggesting that SP phenotype can be a marker of CSC fornasopharyngeal carcinoma (13, 17, 18). However, no effec-tive compound has been discovered to target nasopharyn-geal carcinoma CSCs.

CSCs are thought of as the key players in the resistanceto chemo- or radiotherapy (11, 12), while their ability toescape from the apoptosis pathway may render themresistant to the properties of the therapies (19, 20). It hasbeen shown that the inhibitors of apoptosis protein (IAP)family members are important antiapoptotic proteins toregulate the apoptosis processes (19–21). Among the 8proteins in the family, namely survivin, IAP-like protein

Authors' Affiliations: 1State Key Laboratory of Oncology in SouthernChina, Sun Yat-Sen University Cancer Center and 2The First AffiliatedHospital, Sun Yat-Sen University, Guangzhou; and 3Ascentage PharmaGroup Corp. Limited, Jiangsu, China

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

Corresponding Authors: Yi-Xin Zeng, State Key Laboratory of Oncologyin Southern China, Sun Yat-Sen University Cancer Center, 651 DongfengRoad East, Guangzhou 510060, China. Phone: 86-20-8734-3333; Fax: 86-20-8734-3171; E-mail: [email protected] and Dajun Yang, State KeyLaboratory of Oncology in Southern China, Sun Yat-Sen University CancerCenter, 651 Dongfeng Road East, Guangzhou 510060, China. Phone: 86-20-8734-3149; Fax: 86-20-8734-3171; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-13-0017

�2013 American Association for Cancer Research.

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(ILP2), melanoma IAP (ML-IAP), X-linked IAP (XIAP),cellular IAP1 (cIAP1), cellular IAP2 (cIAP2), neuronalapoptotic inhibitor protein (NAIP), and BIR-containingubiquitin conjugating enzyme (BRUCE), XIAP is the mostwell-characterized one, and cIAP1 and cIAP2 are the 2closest XIAP paralogs (22, 23). Moreover, XIAP is the onlyIAP protein that binds directly to caspases and inhibitstheir activities, which result in promoting resistance toapoptosis in cancer cells (20, 24, 25). In contrast, the cIAPscan bind to caspases without inhibiting their activities (23).

On the other hand, second mitochondria-derived acti-vator of caspases/direct IAP-binding protein with lowisoelectric point (Smac/DIABLO) is a negative regulatorof IAP proteins and released in response to apoptoticstimuli (26). By binding to XIAP and cIAPs, Smac canrelease the inhibition of caspase or lead to the degradationof cIAPs, which in turn activates the apoptosis process incells (26, 27). Therefore, regarding the potency of Smac torevert the antiapoptotic process of cancer cells, severalSmac mimetics have been designed and synthesized asantitumor drugs in recent years (28–31). These com-pounds can induce cIAP1/2 degradation and preventXIAP from binding to caspases, which induce the apo-ptosis of tumor cells with little effect on normal cells(27, 32). In addition, synergy effect has been reported forSmac mimetics and TNF-related apoptosis-inducingligand (TRAIL), which is a TNF family ligandwith abilityto induce apoptosis in cancer cells (33–36).

Attempting to provide more effective treatment forpatients with nasopharyngeal carcinoma, we evaluated2 Smac mimetics, AT-406 and SM-164, in combinationwith TRAIL (37, 38), for their abilities to selectively targetCSCs in nasopharyngeal carcinoma. Our study showedthat IAP proteins were overexpressed in nasopharyngealcarcinoma cancer stem cells and Smacmimetics can selec-tively reduce CSCs both in vitro and in vivo. The resultssuggest that the Smac mimetics AT-406 and SM-164 maybe promising drugs for the effective treatment of naso-pharyngeal carcinoma.

Materials and MethodsCell culture

S18 and S26 cells, clones of the human nasopharyngealcarcinoma cell line CNE2, were maintained in Dulbecco’smodifiedEaglemedium (DMEM, Invitrogen) supplemen-tedwith 10%heat-inactivatedFBS (Invitrogen), 100U/mLpenicillin G, and 100 mg/mL streptomycin at 37�C in 5%CO2. These two clones of CNE2 were kind gifts from Dr.Chaonan Qian (Sun Yat-Sen University Cancer Center,Guangzhou, China). All cell lines were passaged for lessthan 6 months.

SP detectionS18 and S26 cells were treated with the test compounds

(negative control, 5 ng/mL TRAIL, 5 mmol/L AT-406, 0.1mmol/L SM-164, 5 mmol/L AT-406 þ 5 ng/mL TRAIL, or0.1 mmol/L SM-164 þ 0.1 ng/mL TRAIL) for 48 hours,harvested, and then resuspended in an ice-cold DMEM

(supplemented with 2% FBS) at a density of 1 � 106

cells/mL. Then, the cells were incubated at 37�C in 5%CO2 for 10minutes. TheDNA-binding dyeHoechst 33342(Sigma-Aldrich) was then added to the cells at a finalconcentration of 5mg/mL [as a negative control, cellswereincubated with 10 mmol/L fumitremorgin C (FTC, aninhibitor of ABCG2 which could block the pumping outof Hoechst 33342 in CSCs; Sigma-Aldrich) for 5 minutesbefore the addition of theHoechst dye], and the cells wereincubated at 37�C in5%CO2 in thedark for 90minutes andmixed every 15 minutes. Then, the cells were washedtwicewith PBS, resuspended inPBS, andkept at 4�C in thedark before flow cytometric analysis (Experience XtremesMoFlo XDP cell Sorter, Beckman Coulter).

RNA extraction, reverse transcription, andquantitative real-time PCR

Total RNA of S18 and S26 cells were extracted usingTRIzol reagent (Invitrogen) according to the manufac-turer’s instructions. cDNAwas synthesizedusingThermoScientific Maxima First cDNA Synthesis Kit (ThermoScientific). Real-time PCR amplification was conductedusing Platinum SYBR Green qPCR SuperMix-UDG withROX (Invitrogen) on Hard-Shell PCR Plates (Bio-Rad).Relative quantification of each target gene was normal-ized by using an endogenous control (GAPDH).

Cell viability assayCell viability was measured using MTT assay. S18 and

S26 cellswere counted, plated in triplicate at 2,500 cells perwell (200 mL) in 96-well plates, and allowed to growovernight. For individual groups, cisplatin, 5-fluoroura-cil, taxel or TRAIL, AT-406, and SM-164were added to thewells in a concentration gradient. For combinationgroups, negative control, 5 mmol/L AT-406, and 0.1mmol/L SM-164 were mixed with a concentration gradi-ent of TRAIL and then added to the wells. Cell viabilitywasmeasured 48hours later by addingMTT solution. Theobservation value was detected at 490 nm.

Colony formation assayS18, S26, or treated S18 cells [treated with negative

control, 5 ng/mL TRAIL, 5 mmol/L AT-406, 0.1 mmol/LSM-164, 5 mmol/L AT-406 þ 5 ng/mL TRAIL, or 0.1mmol/L SM-164þ 0.1 ng/mL TRAIL for 48 hours (prior)]were counted, plated in triplicate at 100 cells per well in 6-well plates (Corning), and cultured in DMEM (supple-mented with 10% FBS) for approximately 10 days. Then,the cells were washed twice with PBS and fixed in meth-anol for approximately 10 minutes. After two additionalwashes with PBS, the cells were dyed with crystal violetfor 30 minutes. Then, the crystal violet was washed outand the numbers of the colonies were counted.

Sphere formation assayS18, S26, or treated S18 cells [treated with negative

control, 5 ng/mL TRAIL, 5 mmol/L AT-406, 0.1 mmol/LSM-164, 5 mmol/L AT-406 þ 5 ng/mL TRAIL, or

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0.1 mmol/L SM-164 þ 0.1 ng/mL TRAIL for 48 hours(prior)] were counted, plated in triplicate at 300 cells perwell in ultra-low attachment 6-well plates (Corning), andcultured in DMEM/F12 medium (Invitrogen) with20 ng/mL recombinant human basic fibroblast growthfactor (amino acids 1–155) (Invitrogen), 20 ng/mL recom-binant human EGF (Hu EGF; Invitrogen), and B-27supplement (Invitrogen) for approximately 2 weeks. Thespheres were counted under a light microscope.

Cell apoptosis detectionDrug-induced apoptosis was evaluated by Annexin V

and propidium iodide (PI) staining using an Annexin V-EGFP apoptosis detection kit (KeyGEN). Treated S18 andS26 cells (treatedwith negative control, 5 ng/mLTRAIL, 5mmol/L AT-406, 0.1 mmol/L SM-164, 5 mmol/L AT-406þ5 ng/mL TRAIL, or 0.1 mmol/L SM-164 þ 0.1 ng/mLTRAIL for 48 hours) were harvested, washed twice withPBS, and resuspended in binding buffer (500 mL, 1–5� 105

cells). Annexin V-EGFR and PI (5 mL each) were thenadded to the cells, and the mixture was incubated for 15minutes in the dark at room temperature. The stained cellswere analyzed using a Cytomics FC500 flow cytometer(Beckman Coulter).

Western blot analysisCompound-treatedS18andS26 cells or xenograft tumor

tissueswere lysed in lysis buffer on ice, electrophoresed ina 10% Bis-Tris gel in SDS-PAGE electrophoresis runningbuffer and transferred to polyvinylidene difluoride mem-branes. The membranes were then blocked in 5%milk for1 hour and subsequently incubated with various primaryantibodies at 4�C overnight, followed by incubation withsecondary antibodies conjugated to horseradish peroxi-dase. The chemiluminescence reagent was then addedand the signals were detected using a sheet of photo-graphic film.

Antibodies and drugsThe antibodies used for the Western blotting were as

follows: NAIP (#5782-1, Epitomics), cIAP1 (#7065, CellSignaling Technology), cIAP2 (#3130, Cell Signaling Tech-nology), XIAP (#2042, Cell Signaling Technology), survi-vin (#2808, Cell Signaling Technology), livin (#5471, CellSignaling Technology), PARP (#9542, Cell Signaling Tech-nology), caspase 3 (3G2; #9668, Cell Signaling Technolo-gy), cleaved caspase-3 (Asp175; 5A1E; #9664, Cell Signal-ing Technology), tubulin (AT819, Beyotime), and actin(#60008-1-lg, Proteintech). TRAIL was provided by theAscentage Pharma Group Corp. Limited.

Animal experimentsFor the tumorigenesis assay, 4-week-old female athy-

mic nude mice were obtained from the Animal Experi-mental Center of the Guangdong Academy of MedicalSciences (Guangzhou, China) and were given subcutane-ous injections of 1� 103, 5� 103, 1� 104, 5� 104, 1� 105, or5 � 105 S18 or S26 cells in their left or right axillary area.The mice were monitored twice per week for 5 weeks.

For the compound sensitivity assay, 4-week-old femaleathymic nude mice were obtained from the Sino-BritishSippr/BK Lab Animal LET., Co. and were subcutaneousinjected 1 � 106 S18 or S26 cells in the right axillary area.When the xenograft tumors grew approximately 100mm3

in size, themicewere randomly divided into 6 groups (foreach cell line) with no differences in tumor size. Then, themice were treatedwith AT-406 at 100mg/kg, per os, everyday, 1–5week� 3weeks or SM-164 at 3mg/kg, i.v., everyday, 1–5 week � 3 weeks alone or in combination withTRAIL at 10 mg/kg, i.v., every day � 3 weeks. Tumorvolume and body weight were measured 2 times perweek. The T/C rate was also used to evaluate the tumorresponse to these compounds. T/C rate was calculatedusing the ratio of the relative tumor volume (RTV) of thetreated group (T) to the RTV) of the control group (C). TheRTV was calculated using the ratio of the average tumorvolume of day n to the average tumor volume of day0 when the injection of compounds began.

All animal studies were approved by the Sun Yat-SenUniversity Cancer Center Animal Care and Ethics Com-mittee.

TUNEL stainingTumor tissues from the animal experiments were for-

malin-fixed andembedded inparaffin.All sample sectionswere dyed with hematoxylin and eosin and microscopi-cally examined to confirm the nasopharyngeal carcinomacell origin. The samples were dewaxed, rehydrated usingxylene and ethanol, incubated with a proteinase K work-ing solution with microwave irradiation in 0.1 mol/Lcitrate buffer, and then stained with the terminal deox-ynucleotidyl transferase–mediated dUTP nick end label-ing (TUNEL) reaction mixture (Roche Applied Science)for 1 hour. Then, the samples were incubated with Con-verter-POD (Roche Applied Science) for 30 minutes at37�C for 1 hour. All samples were visualized using dia-minobenzidine (DAB; DAKO), and the nuclei were coun-terstained with hematoxylin.

Statistical analysisSigmaplot and SPSS 13.0 were used for statistical anal-

ysis. All in vitro experiments were repeated 3 times. Datawere presented as mean values and SDs, and the differ-ences betweengroupswere evaluatedusing Student t test.P < 0.05 was considered to be statistically significant.

ResultsDifferences between the CSC properties of S18 andS26 cells

The CSC properties of 2 clones of nasopharyngealcarcinoma CNE2 cells, S18 and S26, were evaluated. TheS18 cells have been previously reported to have greatermigration and invasion abilities than S26 cells (39). It wasdiscovered that the SP cell population in S18 cells wasapproximately 27-fold higher than that in the S26 cells(Fig. 1A and C). Furthermore, the S18 cells were found to

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be more resistant than S26 cells to 3 chemotherapeuticdrugs (cisplatin, 5-fluorouracil, and taxel) commonlyused to treat nasopharyngeal carcinoma (Table 1). ThemRNA expression levels of CSC markers in nasopharyn-geal carcinoma, ABCG2, and CD44, were also higher inS18 cells (Fig. 1B). These observations showed that S18cells may have CSC properties.

Then, a colony formation assay was conducted, andS18 cells were better able to form colonies than S26 cells(Fig. 1D and Supplementary Fig. S1A). When thesphere formation ability of these two cells was evalu-ated, S18 cells were found to exhibit stronger sphereformation ability, in contrast to the S26 cells (Fig. 1Eand Supplementary Fig. S1B). Tumor seeding ability ofthese two cell lines was also examined and tumorscould be generated with 5 � 103 S18 cells, whereas 1 �105 S26 cells were required for tumor generation (Table2). All of these findings indicate that S18 cells acted asCSCs.

Having observed that S18 cells exhibit strong resistanceto chemotherapeutic drugs, we next wanted to determinewhether the apoptosis pathwaywas inhibited in S18 cells.Apoptosis-related proteins play a very important role inthe inhibition of the apoptosis pathway (21). The IAPfamily protein levels were measured in S18 and S26 cellsand S18 cells expressedhigher levels of IAPs than S26 cells(Fig. 1F). These data indicated that IAPs play importantroles in nasopharyngeal carcinoma CSCs.

We also wanted to know whether TRAIL and the Smacmimetic AT-406 (40) and SM-164 (38) could affect S18 andS26 cells. Using the MTT assay, it was found that TRAILhad little effect on S18 cells, in contrast with the effect onS26 cells (Fig. 1G). Evenwhen the concentration of TRAILwas increased to 1,000ng/mL, the cytotoxic effectwas stillweak in the S18 cells (Fig. 1G). The Smac mimetic AT-406and SM-164 hadmoderate cytotoxic effects on S18 and S26cells at high concentration, and these effects were moreevident in S26 cells (Table 1).

Figure 1. S18 cells act as cancerstem cells among nasopharyngealcarcinoma CNE2 cell lines. A, S18and S26 cells were stained withHoechst 33342 and FTC,incubated at 37�C in 5%CO2 in thedark for 90 minutes and mixedevery 15 minutes. Then, the cellswere resuspended in PBS and keptat 4�C in the dark before flowcytometric analysis. B, mRNAexpression levels of CSCs ofnasopharyngeal carcinoma,ABCG2 and CD44, in S18 and S26cells. �, P < 0.05; ��, P < 0.001.C to E, SP detection (C), colonyformation assay (D), and sphereformation assay (E) using S18 andS26 cells. Cells were counted,plated in 6-well plates and culturedfor approximately 10 days.�, P < 0.05; ��, P < 0.001. F,Western blotting for NAIP, cIAP1,cIAP2, XIAP, survivin, and livin inS18 and S26 cells. G, the cytotoxiceffects of TRAIL on S18 and S26cells. Cells were treated with TRAILat a range of concentrations for48 hours and cell survival wasmeasured by the MTT assay.

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The Smac mimetics in combination with TRAILselectively inhibit nasopharyngeal carcinoma CSCgrowth and attenuate nasopharyngeal carcinomaCSCsOn the basis of the results mentioned above, we inves-

tigated whether 2 Smac mimetics, AT-406 and SM-164,could selectively inhibit S18 cell growth in vitro in com-bination with TRAIL (Fig. 2A). MTT assays, to determinethe cell growth inhibition properties of the two com-pounds, showed that both compounds, especially SM-164, could selectively inhibit S18 cell growthwhen used incombination with TRAIL (Fig. 2A).Whether AT-406 or SM-164 in combinationwith TRAIL

could attenuate nasopharyngeal carcinoma cancer stemcells was also tested. SP detection showed that treatmentwith AT-406 or SM-164 decreased the proportions of SPcells in S18 cells, especially when used in combinationwith TRAIL. For example, the percentage of SP cellsdecreased from 60% in the untreated group to approxi-mately 25% in the AT-406 or SM-164 þ TRAIL-treatedgroup, whereas the percentage of SP cells in a cisplatin-treated S18 cells’ population was up to 90% (Fig. 2B andC). The colony formation assay and sphere formationassay also showed that AT-406 and SM-164 could inhibit

the colony formation and sphere formation abilities of S18cells, especially when combined with TRAIL (Fig. 2D andE and Supplementary Fig. S2A and S2B).

AT-406 and SM-164 sensitize nasopharyngealcarcinoma xenografts to TRAIL therapy

The above results indicated that treatment with AT-406or SM-164 in combinationwithTRAIL selectively targetednasopharyngeal carcinoma CSCs in vitro. We then gener-ated tumors in mice and tested whether AT-406 and SM-164 could affect tumor growth when combined withTRAIL in vivo.

Mice were injected with S18 or S26 cells, and when theresulting palpable tumors reached a size of 100 mm3, themice were treated with normal saline or with TRAIL(10 mg/kg, i.v.), AT-406 (100 mg/kg, per os), and SM-164(3 mg/kg, i.v.) or in combination 5 days per week for3 weeks. Tumor volume and bodyweight weremeasured2 times per week. We found that the tumor volumes ofmice treated with AT-406 or SM-164 in combination withTRAILweremuch smaller than those ofmice treatedwithnormal saline (Fig. 3A and B). Interestingly, the two Smacmimetic compounds seemed to be more effective againstS18 cell xenografts than against S26 cell xenografts (Fig.3A and B). This result indicated that Smac mimetics alsohave anticancer stem cell properties in vivo. No significanttoxicity was seen in mice treated with SM-164 in combi-nationwithTRAIL (Fig. 3CandD).AT-406 in combinationwith TRAIL resulted in a weight loss in the first 10 days(Fig. 3C and D). However, the changes were recoveredafter stopping the treatment (Fig. 3C andD). And the T/Cratio were 27.7 and 26.6when treatedwith Smacmimeticsin combination with TRAIL in S18 xenografts, whereasT/C ratio were 71.2 and 47.2 in S26 xenografts (Table 3).

Smac mimetics in combination with TRAIL caninduce IAP degradation and lead to apoptosis intumor cells

As the Smac mimetics are important inhibitors of IAPfamily, we also wanted to know whether these com-pounds could induce cell apoptosis in vitro. Annexin Vand PI staining showed that both AT-406 and SM-164 incombination with TRAIL could induce apoptosis in naso-pharyngeal carcinoma cells (Fig. 4A). Western blottinganalysis also showed treatment with AT-406 or SM-164 incombination with TRAIL led to decreased levels of pro-caspase-3 and pro-PARP, whereas the levels of cleavedcaspase-3 and PARP were increased in nasopharyngealcarcinoma cells (Fig. 4B), and the apoptosis induced bythese two compoundswas stronger in S18 cells than in S26cells (Fig. 4A andB). These observations indicated that theSmac mimetic AT-406 and SM-164, in combination withTRAIL, have selective CSC killing effects in nasopharyn-geal carcinoma.

Then, the tissues from the compound-treatedmicewereevaluated to determine whether these compounds couldinduce apoptosis in vivo. Increased levels of cleaved cas-pase-3 and PARP protein determined byWestern blotting

Table 1. IC50 values for cisplatin, 5-fluorouracil,taxel, AT-406, and SM-164 in S18 and S26 cells

S18 S26

IC50 SEM IC50 SEM

Cisplatin (mmol/L) 41.58 1.38 19.45 4.695-Fluorouracil (mmol/L) 231.60 20.52 13.34 1.52Taxel (nmol/L) 4.31 0.30 1.50 0.37AT-406 (mmol/L) 239.90 21.63 20.92 2.74SM-164 (mmol/L) 5.07 0.71 1.37 0.52

NOTE: Cells were treated with a range of concentrationsof each compound for 48 hours, and cell survival wasmeasured by the MTT assay.

Table 2. Tumorigenesis abilities of S18 and S26cells

Tumor seeding

Cells injected S18 S26

5 � 105 6/6 6/61 � 105 5/6 3/65 � 104 3/6 0/61 � 104 2/6 0/65 � 103 2/6 0/61 � 103 0/6 0/6

NOTE: Mice were injected with serial dilutions of S18 or S26cells and were observed twice per week for 5 weeks.






analysis of the tissue from mice showed that apoptosiswas induced in the compound-treated mice (Fig. 4C).TUNEL staining of the tissues from mice also confirmedthese results (Fig. 4D).

It has been reported that XIAP is the only IAP proteinthat can bind directly to and inhibit caspases, indicatingthat XIAP must play a critical role in the apoptosis path-way (20, 24, 25). The cIAP1 and cIAP2 proteins are twoXIAP paralogs that may also play important role in apo-ptosis (22, 23). cIAP1 and XIAP, which are expressed athigher levels in S18 cells, were degraded when the cellswere treated with Smac mimetics in combination withTRAIL, both in vitro and in vivo (Fig. 4B and C). Theseobservations indicated that the overexpression of thecIAP1, cIAP2, and XIAP proteins in S18 cells led thesecells to become much more sensitive to the inhibition ofIAPs. All of these observations indicate that the Smacmimetics AT-406 and SM-164, in combination withTRAIL, can selectively target CSCs in nasopharyngeal

carcinoma by inhibiting the cIAP1, cIAP2, and XIAPproteins.

DiscussionAlthough the 5-year overall survival (OS) of patients

with nasopharyngeal carcinoma has improved, the cur-rent treatments for nasopharyngeal carcinoma still havesome drawbacks. Most importantly, these treatmentscannot prevent nasopharyngeal carcinoma relapse dueto the development of drug resistance and metastasis,and CSCs may play a critical role in these processes(3, 12). Common chemotherapeutic drugs or radiother-apy may kill differentiated cells but fail to eliminateCSCs. A drug that selectively targets CSCs may be aneffective cure for nasopharyngeal carcinoma when com-bined with common chemotherapeutic drugs or radio-therapy.

There are manymarkers that allow the identification ofCSCs in different cancers, such as CD24, CD44, CD133,

Figure 2. Smac mimetics incombination with TRAIL attenuatecancer stem cells in vitro. A, thecytotoxic effect of Smac mimeticsin combination with TRAIL on S18and S26 cells. Negative control, 5mmol/L AT-406, and 0.1 mmol/LSM-164 were mixed with aconcentration gradient of TRAILand then added to the cells for 48hours. Cell survival was measuredby MTT assay. B, SP detection ofcompound-treated S18 cells usingHoechst 33342 with or withoutFTC. Cells were treated withcompounds for 48 hours and thenevaluated by flow cytometry.C to E, SP detection (C), colonyformation assays (D), and sphereformation assays (E) of treated S18cells. In the colony formation andsphere formation assays, cellswere treated with compounds for48 hours, counted, plated in 6-wellplates, and cultured forapproximately 10 days. �, P < 0.05;��, P < 0.01; ��, P < 0.001.

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and the SP phenotype (6, 9, 13, 17). Here, we used the SPphenotype to identify CSCs in nasopharyngeal carcinomaand found that S18, a clone of nasopharyngeal carcinomaCNE2 cells, contains high levels of SP cells. S18 cells alsoexhibit strong sphere and colony formation abilities.These cells are resistant to commonly used chemothera-peutic drugs, and only a small number of cells arerequired to seed tumors. All of these features indicatethat S18 cells act as CSCs among nasopharyngeal carci-noma CNE2 cells.

CSCs have one common feature, the loss of apoptosis,and the overexpression of IAP proteins may be involvedin this phenotype (19, 20). IAPs are the last safeguardsagainst apoptosis pathway and can inhibit the caspaseactivity, which result in promoting resistance to apopto-sis in cancer cells (20, 22). In this study, S18 cells werefound to express higher levels of cIAP1, cIAP2, and XIAPproteins than other clones. These results indicate that

Figure 3. AT-406 and SM-164sensitize nasopharyngealcarcinoma xenografts to TRAILtherapy. A and B, tumor volumes ofS18 (A) or S26 (B) xenograftstreated with different compounds.Mice were injected with 5 � 106

S18 or S26 cells. When thexenograft tumors reachedapproximately 100mm3 in size, themice were randomly divided intosix groups (for each cell line), withnodifference in tumor sizebetweengroups. Then, the mice weretreated with normal saline, 100mg/kg of AT-406, 3 mg/kg ofSM-164, or 10 mg/kg of TRAILalone or in combination 5 times perweek for 3 weeks. Tumor volumewas measured 2 times per week.�, P < 0.05; ��, P < 0.001. C and D,body weights of mice with S18 (C)or S26 (D) xenografts that weretreated with normal saline, TRAIL(10 mg/kg), AT-406 (100 mg/kg), orSM-164 (3 mg/kg) alone or incombination. Body weight wasmeasured 2 times per week.�, P < 0.05; ��, P < 0.01;��, P < 0.001.

Table 3. T/C values of the in vivo treatment

T/C (%)

Compounds S18 Xenografts S26 Xenografts

TRAIL 68.1 73.8AT-406 44.3 87.9AT-406 þ TRAIL 27.7 71.2SM-164 47.3 90.8SM-164 þ TRAIL 26.6 47.2

NOTE: T/C rate was calculated using the ratio of RTV of thetreated group to RTV of the control group. The RTV wascalculated using the ratio of the average tumor volume ofday n to the average tumor volume of day 0 when theinjection of compounds began.






cIAP1, cIAP2, and XIAP may play critical roles in naso-pharyngeal carcinoma CSCs. Therefore, we treatednasopharyngeal carcinoma cells with Smac mimetics incombination with TRAIL to test whether these mole-cules can target nasopharyngeal carcinoma stem cells(35, 41).

Smac mimetics, designed as negative regulators of IAPproteins, can induce apoptosis in cancer cells by antag-onizing XIAP and cIAP1/2 (26, 27, 37, 38). Some of thesmac mimetics are currently evaluated in phase I clinicaltrials as potential therapeutic drugs in the treatment ofhuman tumors (40, 42). TRAIL, a TNF family ligand thatbinds to the death receptor, is currently in developmentas an agent that targets the apoptosis pathway (33, 34).Moreover, TRAIL- induced apoptosis seems to play animportant role in the malignant transformation (43).TRAIL kills transformed cells but not normal cells,showing that this compound has low toxicity (43). How-ever, some cancers are resistant to TRAIL, making it aprime candidate for combination with other safe agentsfor cancer treatment (44). In a phase I dose-escalationstudy, TRAILs seem to be safe and well tolerated (45).This research showed that, unlike other molecules thattarget only differentiated tumor cells, the Smac mimeticsAT-406 and SM-164 in combination with TRAIL maytarget cancer stem cells. We observed that AT-406 andSM-164 have the ability to target S18 cells when com-bined with TRAIL in vitro. The in vivo data confirm this

result and show that these treatments have no toxicity inmice.

It has been reported that XIAP is the only IAP proteinthat can directly bind to and inhibit caspases (20, 24, 25);cIAP1/2 are two paralogs of XIAP (22, 23). In this study,we found that Smac mimetics, especially when used incombination with TRAIL, can induce the degradation ofcIAP1 and XIAP. This treatment also induces the cleavageof caspase-3 and PARP, indicating the induction of theapoptosis pathway. Because nasopharyngeal carcinomastem cells have higher levels of cIAP1, cIAP2, and XIAPthan other cells, these cells may be more sensitive totreatment with Smac mimetics in combination withTRAIL.

Smac mimetic has been reported to sensitize cancercells to TRAIL-induced apoptosis (35–37, 46), but thisstudy is the first, to our knowledge, to show that smacmimetic in combination with TRAIL can targeted CSCsin nasopharyngeal carcinoma. This combination treat-ment can lead to the degradation of cIAP1 and XIAP,which result in the release of caspase inhibition andinduce apoptosis in CSCs. The usage of smac mimeticand TRAIL can be a potential application for other tumorCSCs. Moreover, using Smac mimetics in combinationwith TRAIL as an adjuvant therapy with common che-motherapeutic drugs or radiotherapy may provide apromising new avenue for nasopharyngeal carcinomatherapy.

Figure 4. Smac mimetics incombination with TRAIL induceapoptosis in vitro and in vivo. A,cells were treated with negativecontrol, 5 ng/mL TRAIL, 5 mmol/LAT-406, 0.1 mmol/L SM-164, 5mmol/L AT-406 þ 5 ng/mL TRAIL,or 0.1 mmol/L SM-164 þ0.1 ng/mL TRAIL. After 48 hours,the cells were double stainedwith propidium iodide (PI) andAnnexin V and analyzed usingflow cytometry to evaluate theapoptosis. B and C, Westernblotting for apoptosis relatedproteins, for example, PARP,caspase-3, cIAP1, and XIAP intreated cells (B) or xenografttumors (C). Cells were treatedwithcompounds for 48 hours (B). NS,normal saline; T, TRAIL; A, AT-406; S, SM-164; AT, AT-406 þTRAIL; and ST, SM-164 þ TRAIL.D, TUNEL staining of compound-treated xenograft tumors. Micewere treatedwith normal saline, orwith TRAIL (10 mg/kg), AT-406(100 mg/kg), or SM-164 (3 mg/kg)alone or in combination.

Wu et al.





Disclosure of Potential Conflicts of InterestD. Yang has ownership interest (including patents) in Ascentage

Pharma Group Corp. Limited. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: M.-S. Wu, Y.-X. Zeng, D. YangDevelopment of methodology: Y. Liang, H.-B. Wang, Y.-X. Zeng, D.YangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):M.-S.Wu,G.-F.Wang, Z.-Q. Zhao, Y. Liang,H.-B.Wang, M.-Y. Wu, P. Min, D. YangAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): M.-S. Wu, G.-F. Wang, P. Min, D. YangWriting, review, and/or revision of the manuscript: M.-S. Wu, G.-F.Wang, J.-X. Bei, Y.-X. Zeng, D. YangAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): G.-F. Wang, L.-Z. Chen, Q.-S. Feng,J.-X. Bei, Y.-X. Zeng, D. YangStudy supervision: Y.-X. Zeng, D. Yang

Grant SupportD. Yang, G. Wang, H. Wang, M. Wu, P. Min received financial support

from the National Natural Sciences Foundation (201281172107), the Inter-national Cooperation Project of the Ministry of Science and Technology ofChina (2010DFB34090), "Key New Drug Creation" project of the majorscience and technology program (2012ZX09401005), National "863" Grant(2012AA020305), Jiangsu Provincial Science and Technology InnovationTeam Grant, China (BE2010760) and Jiangsu Provincial Key LaboratoryProject Grant (BM2012114). M. Wu, Z. Zhao, Y. Liang, L. Chen, Q. Feng, J.Bei, Y. X. Zeng received financial support from the National "973" Grant(2011CB504302 and 2012CB967002).

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 January 8, 2013; revised May 2, 2013; accepted May 6, 2013;published OnlineFirst May 22, 2013.

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Wu et al.





Published OnlineFirst May 22, 2013.Mol Cancer Ther Man-si Wu, Guang-feng Wang, Zhi-qiang Zhao, et al. Cancer Stem Cells in Nasopharyngeal CarcinomaSmac Mimetics in Combination with TRAIL Selectively Target

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SmacMimeticsinCombinationwithTRAILSelectivelyTarget Cancer ...mct.aacrjournals.org/content/molcanther/early/2013/... · Cancer Stem Cells in Nasopharyngeal Carcinoma Man-si Wu1, Guang-feng