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Synergistic Effects of Arsenic Trioxide and Radiation in Osteosarcoma Cellsthrough the Induction of Both Autophagy and ApoptosisAuthor(s): Hui-Wen Chiu, Wei Lin, Sheng-Yow Ho, and Ying-Jan WangSource: Radiation Research, 175(5):547-560. 2011.Published By: Radiation Research SocietyDOI: http://dx.doi.org/10.1667/RR2380.1URL: http://www.bioone.org/doi/full/10.1667/RR2380.1

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Synergistic Effects of Arsenic Trioxide and Radiation in OsteosarcomaCells through the Induction of Both Autophagy and Apoptosis

Hui-Wen Chiu,a Wei Lin,a Sheng-Yow Hob and Ying-Jan Wanga

a Department of Environmental and Occupational Health, National Cheng Kung University, Medical College, Tainan, Taiwan; andb Sinlau Christian Hospital, Tainan, Taiwan

Chiu, H-W., Lin, W., Ho, S-Y. and Wang, Y-J. SynergisticEffects of Arsenic Trioxide and Radiation in OsteosarcomaCells through the Induction of Both Autophagy and Apoptosis.Radiat. Res. 175, 547–560 (2011).

Osteosarcoma is the most common primary malignant bonetumor, occurring mainly in children and adolescents, andsurvival largely depends on their response to chemotherapy.However, the risk of relapse and adverse outcomes is still high.We investigated the synergistic anti-cancer effects of ionizingradiation combined with arsenic trioxide (ATO) and themechanisms underlying apoptosis or autophagy induced bycombined radiation and ATO treatment in human osteosarcomacells. We found that exposure to radiation increased thepopulation of HOS cells in the G2/M phase within 12 h in atime-dependent manner. Radiation combined with ATO induceda significantly prolonged G2/M arrest, consequently enhancingcell death. Furthermore, combined treatment resulted inenhanced ROS generation compared to treatment with ATOor radiation alone. The enhanced cytotoxic effect of combinedtreatment occurred from the increased induction of autophagyand apoptosis through inhibition of the PI3K/Akt signalingpathway in HOS cells. The combined treatment of HOS cellspretreated with Z-VAD, 3-MA or PEG-catalase resulted in asignificant reduction of cytotoxicity. In addition, G2/M arrestand ROS generation could be involved in the underlyingmechanisms. The data suggest that a combination of radiationand ATO could be a new potential therapeutic strategy for thetreatment of osteosarcoma. g 2011 by Radiation Research Society

INTRODUCTION

Among the treatment options for cancer, radiationtherapy has played an important role, particularly inadjuvant treatment. In malignant bone and soft-tissuetumors, intra-arterial chemotherapy and limb-savingsurgery have become popular (1). Osteosarcoma is themost common primary malignant bone tumor, occur-ring mainly in children and adolescents, and survival

largely depends on their response to chemotherapy (2).Current treatment strategies include a combination oflimb salvage surgery and neoadjuvant chemotherapy;however, the risk of relapse and adverse outcomes is stillhigh (3). Therefore, improved chemotherapy regimensand other strategies are needed. Cancer therapy hasincreasingly focused on novel treatment modes combin-ing radiation therapy with chemotherapy. Recently, theanti-cancer drug arsenic trioxide (ATO), which wasoriginally used to treat acute promyelocytic leukemia inthe 1970s at Harbin Medical University in China (4), hasattracted attention for its ability to treat solid tumors.Mechanisms that explain its anti-tumor cytotoxicityinclude the induction of tumor apoptosis and theinhibition of cell growth by modulating redox balanceand/or mitochondrial membrane potential (5). Inaddition, recent studies have also indicated that combi-nation treatment with ATO and ionizing radiation isconsidered the most effective treatment for leukemia andsolid tumors (6–8). However, the effects and the precisemechanism of combined treatment of ATO and radia-tion against osteosarcoma remain unclear.

Autophagy is a self-digestive process that ensureslysosomal degradation of long-lived proteins andorganelles to maintain cellular homeostasis (9, 10). Themultistep pathway of autophagy can be modulated atseveral steps. Although autophagy was initially de-scribed as a protective mechanism for cells to surviveand generate nutrients and energy, studies demonstratedthat persistent stress can also promote autophagic, orprogrammed type II, cell death (11). Some connectionsoccur upstream of the apoptotic and autophagicmachinery, where signaling pathways regulate bothprocesses. For example, the autophagy gene beclin1 ispart of a type III PI3 kinase complex that is required forthe formation of the autophagic vesicle and interactswith Bcl-2 (12). Similarly, activation of the PI3 kinase/Akt pathway, a well-known way to inhibit apoptosis,also inhibits autophagy (13). Akt is a serine/threonineprotein kinase that plays a critical role in suppressingapoptosis by regulating its downstream pathways (14).Akt also phosphorylates mammalian target of rapamy-

1 Address for correspondence: Department of Environmental andOccupational Health, National Cheng Kung University MedicalCollege, 138 Sheng-Li Road, Tainan, Taiwan 704; e-mail: yjwang@mail.ncku.edu.tw.

RADIATION RESEARCH 175, 547–560 (2011)0033-7587/11 $15.00g 2011 by Radiation Research Society.All rights of reproduction in any form reserved.DOI: 10.1667/RR2380.1

547

cin (mTOR), which has been reported to inhibit theinduction of autophagy (15). Furthermore, the ability ofautophagy to alleviate the therapy resistance of apop-tosis-defective tumor cells in some circumstances bymediating cell death could also contribute to theautophagy-associated suppression of tumor growth(16). The boundary between autophagy and apoptosishas never been completely studied. Thus, when cancercells are treated, the induction of autophagy and theinteractions between autophagy and apoptosis couldhave profound effects on the tumor cells. Many studieshave shown that tumor cells treated with anti-cancerdrugs undergo both apoptosis and autophagy (8, 17, 18).

Ionizing radiation plays a key role in therapy due to itsability to directly induce DNA damage, in particularDNA double-strand breaks, leading to cell death (19). TheG2/M phase of the cell cycle is the most sensitive toradiation (20), raising the possibility that ATO may act asa radiosensitizer in cancer therapy (7, 21, 22). Differencesin the length and magnitude of radiation-induced G2/Mdelay may be critical determinants of cellular radiosensi-tivity (23). Previous studies have also demonstrated thatATO could serve as a potent radiation sensitizer and mayincrease the cure rate for malignant gliomas (7, 24). Lowdoses of sodium arsenite selectively induced the apoptosisof NB4 cells in G2/M phase after they were arrested in G2

phase (25). Nevertheless, the detailed mechanisms of howATO increases the radiosensitivity of tumor cells,especially in osteosarcoma, remain largely unknown.

In the present study, HOS human osteosarcoma cellswas used to investigate the anti-cancer effect of radiationcombined with ATO. The types of cell death induced byradiation combined with ATO were examined. We alsoinvestigated the possible mechanisms underlying apoptosisor autophagy induced by combined radiation and ATO.

MATERIALS AND METHODS

Cell Culture and Drug Treatment

HOS human osteosarcoma cells (ATCC CRL-1543) were obtainedfrom the American Type Culture Collection (ATCC). The cells werecultured in Eagle’s minimum essential medium (MEM) (Gibco BRL,Grand Island, NY) supplemented with antibiotics containing 100 U/ml penicillin, 100 mg/ml streptomycin (Gibco BRL), 10% heat-inactivated fetal bovine serum (HyClone, South Logan, UT),additional nonessential amino acids, L-glutamine (Gibco BRL) andsodium pyruvate (Gibco BRL). Cells were incubated in a humidifiedatmosphere of 95% air/5% CO2 at 37uC. Exponentially growing cellswere detached by 0.05% trypsin-EDTA (Gibco BRL) in MEM. Forexposure to arsenic trioxide (Sigma Chemical Co.), 1 mM fresh stocksolutions were prepared before every experiment and filter sterilizedusing a 0.2-mm syringe filter. The reagent was added to the culturemedium in a concentrated form and mixed gently. The cultures werethen incubated for the times indicated in the figures.

Treatment with Radiation and Cell Viability Assay

Cells were irradiated with 6 MV X rays using a linear accelerator(Digital M Mevatron Accelerator, Siemens Medical Solutions,

Concord, CA) at a dose rate of 5 Gy/min. An additional 2 cm oftissue-equivalent bolus was placed on the top of each plastic tissueculture flask to ensure electronic equilibrium, and 10 cm of tissue-equivalent material was placed under the flask to obtain fullbackscatter. Cells in the co-treatment group were treated with ATOand radiation simultaneously. In the post-treatment group, ATO wasapplied to HOS cells 12 h after 4 Gy irradiation in the combinedtreatment, corresponding to the time showing the greatest G2/Mpopulation in cells treated with 4 Gy radiation alone. The treatmentscheme of radiation and ATO was based on the finding of McCabe et al.indicating that the G2 phase in cells U937 appear to be the most sensitiveto arsenite and that apoptosis is induced in these cells as they emergefrom an aberrant G2/M (26). Ma et al. also showed that arseniteselectively induced apoptosis of NB4 cells in a fraction of the G2/M cellsafter arrest in the G2 phase (25). The treated cells were centrifuged andresuspended with 0.1 ml PBS. Each cell suspension (0.02 ml) was mixedwith 0.02 ml Trypan blue solution (0.2% in phosphate-buffered saline,PBS). After 1 or 2 min, each solution was placed on a hemocytometer,and the blue-stained cells were counted as nonintact.

Cell Cycle Analysis

HOS cells were suspended with ice-cold PBS and fixed in 70%

ethanol at 220uC for 16 h. Cells were stained with propidium iodideas described previously (7). The population of nuclei in each phase ofthe cell cycle was determined using Cell Quest and analyzed usingWinMDI software programs (Becton Dickinson, San Jose, CA).

Clonogenic Assay

Cells were irradiated with 2, 4 or 6 Gy. ATO was added to HOScells at concentrations of 3 or 5 mM. The cells were trypsinized andcounted. Known numbers of cells were then replated in 6-cm culturedishes and returned to the incubator to allow for colony development.After 7 days, colonies (containing $50 cells) were stained with a 0.5%

crystal violet solution for 30 min. Plating efficiency (PE) is the ratio ofthe number of colonies to the number of cells seeded in thenonirradiated group. Calculation of surviving fractions (SFs) wasperformed using the equation SF 5 colonies counted/(cells seeded 3

PE), taking into consideration the individual PE.

Drug Interaction Analysis

The effect of drug combination was evaluated by the combinationindex (CI) method using CalcuSyn software (Biosoft), which is basedon the median effect model of Chou and Talalay (27). HOS cells wereexposed to radiation at doses ranging from 2 to 8 Gy and to ATO atconcentrations ranging from 2 to 10 mM. Then the cell viability assaywas performed as described above. The data were entered into theCalcuSyn interface and used to calculate CI values. CI , 1, CI 5 1,and CI . 1 indicate synergism, additive effect, and antagonism,respectively.

Comet Assay

To detect DNA damage in individual cells, we used a comet assay.Briefly, cells (2 3 104) were collected after the treatments andresuspended in 0.2 ml of PBS containing 0.5% low-melting-pointagarose. Eighty-five microliters of the mixture was applied to theslides, which were then submerged in cold lysis solution [2.5 M NaCl,0.1 M EDTA, 10 mM Tris, 1% Triton X-100 (pH 10)]. Electropho-resis was performed at 300 mA and 25 V for 20 min. Afterelectrophoresis, the slides were neutralized with 0.4 M cold Tris-HCl buffer (pH 7.5) and then stained with ethidium bromide. Cometswere visualized using a fluorescence microscope (Olympus, Japan).The DNA damage was assessed on 100 cells, and tail moment (taillength multiplied by the fraction of DNA in the tail) was quantifiedusing Image-Pro Plus (Media Cybernetics Inc.).

548 CHIU ET AL.

DAPI Stain

For the observation of nuclear morphology, cells treated under theindicated conditions were fixed in methanol, incubated with 49,6-

diamidino-2-phenylindole (DAPI) (Sigma Chemical Co.), and thenanalyzed using a fluorescence microscope (Olympus).

Determination of Early Apoptosis

Apoptosis was assessed by observing the translocation of phos-

phatidyl serine to the cell surface, as detected with an Annexin Vapoptosis detection kit (Calbiochem, San Diego, CA) as described

previously (8). Cells were pretreated with the caspase inhibitor Z-V-A-D(OMe)-FMK (R&D Systems, Minneapolis, MN) at a final

concentration of 15 mM for 1 h before irradiation.

Measurement of ROS Production

Reactive oxygen species (ROS) production was monitored byflow cytometry using 2,7-dichlorodihydrofluorescein diacetate

(DCFH-DA) as described previously (21). After treatment withradiation and/or ATO, cells were incubated with 20 mM of DCFH-

DA for 30 min. The cells were harvested, washed once and

resuspended in PBS. Fluorescence was monitored using a flowcytometer. Cells were pretreated with PEG-catalase (Sigma Chem-

ical Co.) at a final concentration of 400 U/ml for 1 h beforeirradiation.

Supravital Cell Staining with Acridine Orange for Autophagy Detection

Cell staining with acridine orange (Sigma Chemical Co.) was

performed according to published procedures (8). Cells werepretreated with the autophagy inhibitor 3-methyladenine (3-MA)

(Sigma Chemical Co.) at a final concentration of 0.7 mM for 1 hbefore irradiation.

Electron Microscopy

The cells were fixed with a solution containing 2.5% glutaraldehyde

plus 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3, for 1 h.After fixation, the samples were postfixed in 1% OsO4 in the same

buffer for 30 min. Ultrathin sections were then observed under atransmission electron microscope (JEOL JEM-1200EX, Japan) at

100 kV.

Western Blot Analysis

Total cellular protein lysates were prepared by harvesting cells inprotein extraction buffer for 1 h at 4uC as described previously (7).

The densities of the bands were quantified with a computerdensitometer (AlphaImagerTM 2200 System Alpha Innotech Corpo-

ration, San Leandro, CA). The expression of GAPDH was used as theprotein loading control. The antibodies for detecting Akt, phospho-

Akt, phospho-PDK1, phospho-GSK3b and Beclin 1 were obtained

from Cell Signaling Technology (Ipswich, MA); anti-GAPDH wasobtained from Abcam (Cambridge, MA); anti-LC3 antibody was

obtained from Abgent (San Diego, CA); anti-p62/SQSTM1 antibodywas obtained from MBL (Nagoya, Japan); anti-poly (ADP-ribose)

polymerase (PARP) antibody was obtained from Millipore (Billerica,MA); anti-caspase-3 and anti-cleaved-caspase-3 were obtained from

Epitomics (Burlingame, CA).

Statistical Analysis

Data are expressed as means ± SD. Statistical significance wasdetermined using Student’s t test for comparison between the means

or one-way analysis of variance with Dunnett’s post-hoc test (28).Differences were considered significant when P , 0.05.

RESULTS

Optimal Dose and Time Selection of Radiation and ATOfor Treatment of HOS Cells

The viability of HOS cells was observed at differentconcentrations of ATO (0 to 10 mM) for 12, 18, 24 and36 h (Fig. 1A). ATO alone reduced the viability of HOScells in a concentration-dependent manner. G2/M arrestis common phenomenon after irradiation. The cell cycledistribution was measured by flow cytometry (Fig. 1B).radiation alone increased the number of HOS cells in theG2/M phase in a dose-dependent manner. The percent-age of cells in the G2/M phase was also observed atdifferent times (0 to 24 h) after irradiation. A dose of4 Gy increased the population of HOS cells in the G2/Mphase in a time-dependent manner within 12 h. Fig-ure 1C and D shows the viability of HOS cells treatedwith ATO and radiation simultaneously or with ATOafter irradiation. The term ‘‘co-treatment’’ indicates thatcells were treated with radiation (4 Gy) and ATO (3 mM)simultaneously. The term ‘‘post-treatment’’ indicatesthat cells were pretreated with radiation, cultured for12 h and then treated with ATO for an additional 6, 12or 24 h. Significantly enhanced toxicity was found forthe combination treatment (co-treatment) comparedwith ATO and radiation alone for 12 and 24 h.Significantly enhanced toxicity was also observed inthe 6-, 12- and 24-h post-treatment groups. The cell cycledistribution in HOS cells treated with ATO and/orradiation is shown in Fig. 1E. A significantly increasedand prolonged G2/M arrest was found in the cells withcombined treatment compared to those with radiationor ATO alone. These results indicate that combinedtreatment could prolong G2/M arrest.

Radiation Dose–Response Survival Curves andSynergistic Cytotoxicity between Radiation and ATO forHOS Cells

Figure 2A and B shows the radiation dose–responsesurvival curves for HOS cells with or without ATOtreatment. In the co-treatment and post-treatment groups,the survival curves shifted dramatically downward. ATO(3 mM) increased radiation-induced clonogenic cell death,decreasing survival from 4 3 1022 after 6 Gy of radiationalone to 5 3 1023 (Fig. 2A). In the post-treatment groups,ATO significantly reduced the survival fraction in aconcentration-dependent manner compared with radia-tion alone (Fig. 2B). The combination-index methodsdeveloped by Chou and Talalay (27) were used to confirmand quantify the synergism observed with radiation andATO. These results indicated that the co-treatment groupswere synergistic at low concentrations (CI , 1) while athigher concentrations antagonism (CI . 1) was observed(Fig. 2C). However, the post-treatment groups showedsynergistic cell killing at all tested concentrations (CI , 1).

SYNERGISTIC EFFECTS OF ARSENIC TRIOXIDE AND RADIATION IN OSTEOSARCOMA CELLS 549

FIG. 1. Cell cycle distribution and cytotoxic effects after treatment with ATO and radiation (IR) in HOS cells. Panel A: Time course- andconcentration-dependent effects of ATO on the viability of HOS cells. Cells were treated with 2, 3, 5 or 10 mM of ATO for 12, 18, 24 and 36 h.Panel B: Time course- and dose-dependent effects of radiation on cell cycle distribution in HOS cells. Cells were treated with 2, 4, 6 or 8 Gy ofradiation and cultured for 6, 9, 15, 18 and 24 h. G2/M phase was measured by flow cytometry. Panel C: Cytotoxic effects of cells treated withradiation and ATO simultaneously. ‘‘Co-treatment’’ indicates that cells were treated with radiation (4 Gy) and ATO (3 mM) simultaneously.Panel D: Cytotoxic effects of cells treated with ATO after radiation. ‘‘Post-treatment’’ indicates that cells were pretreated with radiation, culturedfor 12 h, and then treated with ATO for an additional 6, 12 and 24 h. #, P , 0.05, radiation compared to combined treatment. *, P , 0.05, ATOcompared to combined treatment. Panel E: Quantification of G2/M phase in HOS cells treated with radiation (4 Gy) and ATO (3 mM). Data arepresented as means ± SD from three independent experiments.

550 CHIU ET AL.

DNA Damage Determined by the Comet Assay andMeasurement of Apoptosis in HOS Cells Treated withATO and Radiation Alone or in Combination

Radiation plays a key role in therapy due to its abilityto directly induce DNA damage (19). The comet assay isa sensitive tool for the estimation of DNA damage andrepair at the cellular level, requiring only a small numberof cells (29). In the comet assay for the HOS cells, alimited or no tail was found in the untreated controls,while the tail grew after irradiation and combinedtreatment (Fig. 3A). The tail length grew significantlyafter irradiation (4 Gy) compared to controls (Fig. 3B).A dose of 4 Gy increased the tail length in a time-dependent manner within 12 h and decreased the taillength at 18 h. Figure 3C shows the results of cells

pretreated with radiation, cultured for 12 h, and thentreated with ATO for an additional 6, 12, 18 or 24 h.Significantly enhanced DNA damage was found for thecombination treatment compared with radiation alone.These results suggested that DNA damage was involvedin the anti-proliferative effects of combination treatmentin HOS cells.

The induction of apoptosis is a significant mechanismof tumor cell death under the influence of radio-/chemotherapy (30). Early apoptosis in HOS cells wasmeasured by flow cytometry with the Annexin Vapoptosis detection kit (Fig. 4A). Quantitative resultsshowed that the post-treatment and co-treatment groupshad more apoptotic cell death than cells treated withATO or radiation alone. The post-treatment groupsshowed a significant increase in the percentage of cells in

FIG. 2. Radiation (IR) dose–response survival curves and synergistic cytotoxicity between radiation and ATO in HOS cells. Panel A: Theradiation dose–response survival curves of HOS cells with or without ATO. Cells were treated with radiation and ATO simultaneously. Panel B:The radiation dose–response survival curves of HOS cells with or without ATO. Cells were pretreated with radiation, cultured for 12 h, and thentreated with ATO for an additional 24 h. Data are presented as means ± SD from three independent experiments. Panel C: Synergisticantiproliferative effect of radiation and ATO. Cells were treated with radiation and ATO simultaneously. Combination index (CI) values werecalculated using Calcusyn software. Panel D: Synergistic antiproliferative effect of radiation and ATO. Cells were pretreated with radiation,cultured for 12 h, and then treated with ATO for an additional 24 h. Synergy, additivity and antagonism are defined as CI , 1, CI 5 1 and CI .

1, respectively.

SYNERGISTIC EFFECTS OF ARSENIC TRIOXIDE AND RADIATION IN OSTEOSARCOMA CELLS 551

early apoptosis compared with the co-treatment groups.Apoptotic nuclei stained with DAPI showed intensefluorescence corresponding to chromatin condensationand fragmentation in HOS cells treated with ATO andradiation alone or in combination (Fig. 4B). Further-more, ROS generation was assessed using the fluores-cent probe DCFH-DA and was monitored by flowcytometry. An approximately 5-fold increase in intra-cellular peroxide levels was found when cells were post-treated for 1 h (Fig. 4C). Figure 4D shows Westernblots of poly(ADP-ribose) polymerase (PARP) andcaspase-3. The cleavage of PARP by activated caspase-3 results in the formation of an 85-kDa C-terminalfragment. Our results showed that the specific cleavageof PARP could be found in cells treated with ATO aloneand the combined treatment. The cleavage of caspase-3increased significantly with ATO alone and combinedtreatment. These results indicate that the combinedtreatment induces apoptosis in HOS cells.

Measurement of Autophagy in HOS Cells Treated withATO and Radiation Alone or in Combination

In our previous studies, we found that radiationcombined with ATO induced type II programmed celldeath (autophagy) (7, 8). Autophagy is characterized bythe formation of numerous acidic vesicles called acidicvesicular organelles (AVOs) (31). Microphotographs ofAVOs were observed via green and red fluorescence inacridine orange (AO)-stained cells with a fluorescencemicroscope (Fig. 5A). The combined treatment revealed asignificant increase in AVOs compared to ATO orradiation alone. AO staining was quantified using flowcytometry (Fig. 5B, C). A significant increase in AO-positive cells was found in cells receiving combinedtreatment compared to those treated with radiation orATO alone. The post-treatment groups showed a signif-icant increase in the percentage of AO-positive cellscompared with the co-treatment groups. To detect the

FIG. 3. Comet assay of radiation and ATO in HOS cells. Panel A: EtBr staining for cells treated with radiation (IR) (12 and 36 h) and post-treatment. The tails indicate DNA damage. Panel B: Time-course effects of radiation on average of tail DNA. Cells were treated with 4 Gy ofradiation and cultured for 2, 6, 12, 18 and 24 h. Panel C: Time-course effects of radiation and post-treatment on average of tail DNA. RadiationzATO indicates that cells were pretreated with radiation, cultured for 12 h, and then treated with ATO for an additional 6, 12, 18 and 24 h.

552 CHIU ET AL.

expression of LC3, we performed Western blotting withlysates from HOS cells receiving each of the differenttreatments (Fig. 5D). The expression of LC3-II increasedin HOS cells treated with ATO alone and combinedtreatment. p62, or sequestosome 1 (SQSTM1), is acommon component of protein aggregates that is respon-sible for linkage of polyubiquitinated proteins to autoph-agic machinery (32). Figure 5D shows that the expressionlevels of the p62 proteins increased with ATO alone andcombined treatment. To further confirm the role ofautophagy in combined treatment-induced cytotoxicity,we used bafilomycin A1, an inhibitor of autophagosome-lysosome fusion, to inhibit the flux in autophagy (33). Wefound that the LC3-II levels in cells in the post-treatmentgroup were elevated by bafilomycin A1 (Fig. 5E).

The ultrastructures of HOS cells in each treatmentgroup were observed by EM microphotography (Fig. 5F).Prominent features of combined treatment were found inthe form of autophagic vacuoles in the cytoplasm andapoptotic changes in the nucleus. Cells receiving ATOalone and combined treatments demonstrated chromatincondensation, which is a characteristic of apoptosis. Thecombined treatment also resulted in a large number ofautophagic vacuoles and autolysosomes in the cytoplasm.These results confirmed that HOS cells underwentautophagy and apoptosis after exposure to radiationand ATO. Previous studies have demonstrated that thePI3K/Akt pathway is involved in regulating autophagy(34). To investigate whether the PI3K/Akt signalingpathways were involved in autophagy in HOS cells treated

FIG. 4. Measurement of apoptosis in HOS cells receiving various treatments. Panel A: Early apoptosis was measured by flow cytometry withan Annexin V apoptosis detection kit. #, P , 0.05, radiation (IR) compared to combined treatment. *, P , 0.05, ATO compared to combinedtreatment. {, P , 0.05, co-treatment compared to post-treatment. Panel B: Apoptotic nuclei stained with DAPI show intense fluorescencecorresponding to chromatin condensation (arrows) and fragmentation (arrowheads). Panel C: ROS generation in HOS cells treated with 3 mMATO or 4 Gy radiation alone or in combination for 30 min and 1, 2 and 6 h and with DCFH-DA for an additional 30 min. ‘‘DCF positive cells’’indicates index of oxidative stress. The fluorescence in the cells was immediately assayed using flow cytometry. #, P , 0.05, radiation comparedto combined treatment. *, P , 0.05, ATO compared to combined treatment. Panel D: Western blotting of PARP, cleaved-PARP, pro-caspase 3and cleaved-caspase 3. The level of total GAPDH protein was used as the loading control. ‘‘Co-treatment’’ indicates that cells were treated withradiation (4 Gy) and ATO (3 mM) simultaneously and cultured for 24 h. ‘‘Post-treatment’’ indicates that cells were pretreated with radiation,cultured for 12 h, and then treated with ATO for 24 h. Data are means ± SD from three independent experiments.

SYNERGISTIC EFFECTS OF ARSENIC TRIOXIDE AND RADIATION IN OSTEOSARCOMA CELLS 553

FIG. 5. Measurement of autophagy in HOS cells receiving various treatments. Panel A: Microphotograph of AVOs in HOS cells. Detectionof green and red fluorescence in acridine orange (AO)-stained cells was performed using a fluorescence microscope. The white arrows point toAVOs. Panel B: Development of AVOs in HOS cells. Detection of green and red fluorescence in AO-stained cells using flow cytometry. Panel C:Quantification of AVOs with AO-stained cells treated with radiation (IR) (4 Gy) or ATO (3 mM) alone or in combination using flow cytometry.Data are means ± SD from three independent experiments. #, P , 0.05, radiation compared to combined treatment. *, P , 0.05, ATO

554 CHIU ET AL.

with ATO and radiation alone or in combination, weperformed Western blotting to detect protein phosphor-ylation states (Fig. 5G). Phosphorylated proteins relatedto the PI3K/Akt signaling pathways (such as p-GSK3band p-PDK-1) were also examined. The results showedthat phosphorylation of Akt and PDK1 decreased in cellstreated with ATO alone and in combination withradiation compared with the controls. In contrast, thephosphorylation level of GSK3b increased in cells treatedwith ATO alone and in combination with radiation.

HOS Cells Undergo Both Autophagic and Apoptotic CellDeath When Exposed to Radiation and ATO

Next, we investigated whether the inhibition ofautophagy or apoptosis could change the percentageof viable cells that had been treated with ATO andradiation (Fig. 6A). Z-V-A-D(OMe)-FMK (a caspaseinhibitor) and 3-methyladenine (3-MA) (an autophagyinhibitor) were used for this purpose. We examinedwhether the inhibition of autophagy or apoptosis couldaffect the cytotoxicity of the combined treatment. Theresults indicated that post-treatment of HOS cellspretreated with 3-MA or Z-VAD resulted in asignificant decrease in cytotoxicity compared with thecorresponding control. As shown in Fig. 6B and C,Annexin V and AO staining were quantified using flowcytometry. The post-treatment of HOS cells pretreatedwith 3-MA resulted in a significant decrease inapoptotic cells (Fig. 6B), and cells pretreated with Z-VAD showed a significant decrease in AO-positive cells(Fig. 6C) compared to post-treatment groups. To detectthe expression level of LC3 and caspase-3, we per-formed Western blotting with lysates from HOS cells inthe absence or presence of Z-VAD or 3-MA (Fig. 6D).The expression of LC3-II proteins and the specificcleavage of caspase-3 were lower in HOS cells of thepost-treatment groups exposed to 3-MA or Z-VADcompared with cells of the post-treatment groups thatwere not exposed to 3-MA or Z-VAD. These resultsfurther confirm that ATO combined with radiation caninduce both autophagy and apoptosis in HOS cells.

Effects of PEG-Catalase on Combined Treatment-Induced Cytotoxicity

To further determine the involvement of ROS incombined treatment-induced cytotoxicity in HOS cells,PEG-catalase (a hydrogen peroxide scavenger) was used.

Pretreatment with PEG-catalase (400 U/ml) in HOS cellsresulted in a significant decrease in post-treatment-induced cytotoxicity (Fig. 7A). Annexin V and AOstaining were quantified using flow cytometry. The post-treatment of HOS cells pretreated with PEG-catalaseresulted in a significant decrease in apoptotic cells(Annexin V staining) and autophagic (AO-positive) cellscompared to post-treatment groups (Fig. 7B and C).These results suggest that combined treatment-inducedcytotoxicity could be directly dependent on the produc-tion of hydrogen peroxide. Thus ROS is criticallyinvolved in combined treatment-induced cytotoxicity.

DISCUSSION

The combination of radiotherapy and chemotherapyis an appealing approach that has led to improvedtreatment results in patients with advanced solid tumors(35). The scheduling of radiation therapy and chemo-therapy appears to be critical. Our increased under-standing of the molecular processes underlying cellularsensitivity to radiation has led to the identification ofnovel targets for intervention. Several recent studieshave demonstrated that the combined treatment of ATOand radiation can increase therapeutic efficacy against avariety of tumors compared to single treatments (6–8,21). In this study, we found evidence that combinedATO and radiation could enhance the cell-killing effectsin osteosarcoma and further investigated the molecularmechanism of synergistic effects of radiation in combi-nation with ATO (Figs. 1, 2). The aim of combinationtreatment is to exploit additive or synergistic effectsbetween agents (36). However, the biological basis ofthis synergy remains largely unclear. In the presentstudy, we found that DNA damage, autophagy andapoptosis were increased by the combination and thuscould explain part of the observed synergy. Severalgroups, including ours, have reported that combinedtreatment with ATO and radiation induces autophagyand/or apoptosis in different cancer cells (7, 24, 37). Theboundary between apoptosis and autophagy has neverbeen completely elucidated. The relationship betweenautophagy and apoptosis is complex and varies betweencell types and the specific stress placed upon the cell (38).Autophagy is important in normal development and theresponse to changing environmental stimuli (39). Undercertain circumstances, autophagy constitutes a stressadaptation that avoids cell death (and suppresses

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compared to combined treatment. {, P , 0.05, co-treatment compared to post-treatment. Panel D: Western blotting of LC3-I, LC3-II and P62/SQSTM1 expression. Panel E: Western blot analysis of LC3-I and LC3-II expression. Cells were pretreated with bafilomycin A1 (BAF) (5 nM)for 1 h before radiation treatment. Panel F: EM microphotographs of HOS cells treated with radiation or ATO alone or in combination. ‘‘IR z

ATO’’ indicates that cells were pretreated with radiation, cultured for 12 h, and then treated with ATO for 24 h. The white arrows point toautophagic vacuoles and autolysosomes. The black arrows point to chromatin condensation in the nucleus. Panel G: Effects of p-Akt, Akt, p-GSK3b and p-PDK1 protein expression in HOS cells treated with radiation or ATO alone or in combination.

SYNERGISTIC EFFECTS OF ARSENIC TRIOXIDE AND RADIATION IN OSTEOSARCOMA CELLS 555

apoptosis), whereas in other cellular settings, it consti-tutes an alternative cell death pathway (40). Manyanticancer agents have been reported to induce autoph-agy, leading to the suggestion that autophagic cell deathmay be an important mechanism of tumor cell killingby these agents (15). Bommareddy et al. found thatPEITC-mediated autophagy and apoptosis both con-tribute to the PEITC-mediated suppression of prostatecancer cell growth (41). Similarly, Alisol B, a novelinhibitor of the sarcoplasmic/endoplasmic reticulumCa2z ATPase pump, induced autophagy, endoplasmicreticulum stress, and apoptosis in several cancer cell lines(42). The present study demonstrated that radiationcombined with ATO could induce autophagy andapoptosis in osteosarcoma cells (Figs. 4, 5). We alsofound that the combined treatment of HOS cellspretreated with 3-MA, an inhibitor of autophagy

through inhibition of type III PI3K, results in asignificant reduction in apoptosis and cytotoxicity(Fig. 6A, B). The autophagy gene, Beclin-1, is part ofa type III PI3 kinase complex that is required for theformation of the autophagic vesicle and could interactwith Bcl-2 (12). Beclin-1 may be the critical molecularswitch that plays an important role in fine tuning theautophagy and apoptosis through caspase-9 (43). Inaddition to inhibiting apoptosis by binding to andinterfering with the action of the pro-apoptotic proteinsBax and Bak, Bcl-2/Bcl-xL also inhibit autophagy bybinding to Beclin-1 (12). In the present study, we foundthat the combined treatment of HOS cells pretreatedwith Z-VAD resulted in a significant reduction in AO-positive cells (Fig. 6C).

The amount of DNA damage and the efficiency withwhich a cell deals with this damage may vary throughout

FIG. 6. Measurement of autophagy, apoptosis and cytotoxic effects in HOS cells pretreated with Z-VAD or 3-MA. Panel A: Cytotoxic effectsin the absence or presence of Z-VAD or 3-MA. Panel B: Early apoptosis was measured by flow cytometry with Annexin V. Panel C:Quantification of AVOs with AO using flow cytometry. Panel D: Western blot analysis of LC3 and caspase-3 expression. Cells were pretreatedwith 3-MA (0.7 mM) or Z-VAD (15 mM) for 1 h and then treated with radiation (4 Gy) and ATO (3 mM). Data are means ± SD from threeindependent experiments. a, P , 0.05, Post-treatment compared to Post-treatment z Z-VAD. b, P , 0.05, Post-treatment compared to Post-treatment z 3-MA.

556 CHIU ET AL.

the cell cycle (44). In the case of radiation, the DNAdamage induced includes single-strand breaks anddouble-strand breaks that, if not accurately repaired,can lead to cell death or chromosomal instability (29).Cell cycle arrest could be a major cellular response toDNA damage preceding the decision to repair or die(45). Preferential apoptosis of cells in the G2/M phasewas observed after c irradiation, and the greatestamount of apoptosis after c irradiation occurred in adose-dependent manner after G2/M arrest (23). McCabeet al. indicated that U937 cells in the G2 phase of the cellcycle appear to be the most sensitive to arsenite, andapoptosis is induced in the cells as they emerge from anaberrant G2/M (26). In the present study, the treatmentof cells 12 h after 4 Gy of radiation induced marked G2/M arrest. The combined treatment with radiation andATO at this time resulted in a significantly prolongedG2/M arrest and consequently enhanced cytotoxicity(Fig. 1). We believe that ATO can increase and further

damage the cell population in G2/M arrest induced bylower-dose (4 Gy) radiation.

Under oxidative stress, ROS, including free radicals,are generated at levels high enough to induce oxidationand damage to DNA, lipids, proteins and othermacromolecules (46, 47). On occasion, autophagy andapoptosis both occur simultaneously after stress; atother times, only autophagy or apoptosis is observed(48). ROS plays a critical role in synergistic enhance-ment of apoptotic cell death by the combinationtreatment with radiation and ATO in human cervicalcancer cells (22). H2O2 induces autophagy throughinterference with the beclin-1 and Akt/mTOR signalingpathways and is regulated by the anti-apoptotic geneBcl-2 in glioma U251 cells (38). Previous studies havefound that several anti-cancer drugs induce autophagy orapoptosis through ROS generation in cancer cells (21). Ithas also been reported that polygonatum cyrtonema lectin(PCL) induces both apoptosis and autophagy via a

FIG. 7. Measurement of autophagy, apoptosis and cytotoxic effects in HOS cells pretreated with PEG-catalase. Panel A: Effects of PEG-catalase on cytotoxicity induced by post-treatment. Panel B: Early apoptosismeasured by flow cytometry with Annexin V. Panel C: Quantification of AVOs with AO using flow cytometry.Cells were pretreated with PEG-catalase (400 U/ml) for 1 h and then treated with radiation (4 Gy) and ATO(3 mM). Data are means ± SD from three independent experiments. *, P , 0.05, Post-treatment compared toPost-treatment z PEG-catalase.

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mitochondrial-mediated ROS–p38–p53 signaling path-way (49). Consistent with those studies, we also foundthat combined treatment with ATO and radiationsignificantly increases ROS generation and consequentlyinduces both apoptosis and autophagy in HOS cells(Fig. 4C). In addition, combined treatment of HOS cellspretreated with PEG-catalase resulted in a significantreduction of cytotoxicity compared with control cells(Fig. 7). These results indicated that ROS is criticallyinvolved in combined treatment-induced cytotoxicity.

Depending on the cellular background and extent ofDNA damage, the DNA damage response triggers cellcycle arrest and DNA repair, or in the case ofirreparable damage, inactivation of the cells by senes-cence or apoptosis (50). Recent studies strongly suggestan important tumor-suppressive role of the DNAdamage response (DDR) in humans: molecular markersindicative for an active DDR, including site-specificallyphosphorylated ataxia-telangiectasia mutated (ATM),p53 and histone H2AX, have been found in earlyneoplastic lesions but not in full-blown cancerouslesions, where the DDR is typically compromised (51).Radiation plays a key role in therapy due to its ability todirectly induce DNA damage, particularly DNA double-strand breaks, leading to cell death (19). In the presentstudy, 4 Gy increased DNA damage in a time-dependentmanner within 12 h and decreased at 18 h. Significantlyenhanced DNA damage was found for the post-treatment of ATO and radiation compared withradiation alone (Fig. 3). This could be due in part toadditional DNA damage caused by ATO or inhibitionof DNA repair by ATO in irradiated osteosarcoma cells.However, the details of the mechanism still need to beelucidated.

Phosphatidylinositol-3 kinases (PI3Ks) are importantin controlling various aspects of the malignant pheno-type, including proliferation, survival and apoptosis,adhesion and mobility, angiogenesis and cell size (52). Inparticular, the Akt family (also known as protein kinaseB) of serine/threonine kinases has emerged as a criticaldownstream target of PI3K in human cancer. Aktinhibited GSK3 activity by direct phosphorylation of anN-terminal regulatory serine residue downstream ofinsulin-activated PI3K to inhibit cell death and promotecell survival (53). GSK3 promotes arsenite-inducedapoptosis by facilitating signaling, leading to disruptionof mitochondria (54). Ser473 is important for therecognition and phosphorylation of Akt by PDK1(55). Previous studies have found that OSU-03012 (acelecoxib derivative), which has been thought to mediateantitumor effects primarily via the inhibition of PDK1(56). Disruption of the PI3K/Akt pathway, culminatingin inhibition of Akt, has been found to be associatedwith autophagy induced by a variety of antineoplasticagents in cancer cells (57). A combination of indol-3-carbinol and genistein synergistically induces apoptosis

and autophagy in human colon cancer HT-29 cells byinhibiting Akt phosphorylation (58). Thus the cross-talkbetween the Akt signaling pathways appears to regulatethe outcome of autophagy and apoptosis. The presentstudy demonstrates that the expression of p-Akt and p-PDK1 proteins decreased while p-GSK3b expressionincreased in cells treated with radiation combined withATO compared to radiation alone (Fig. 5G).

Many cancer patients receive high-dose radiotherapy.Despite having the advantage of preserving the tissuestructure, high-dose radiotherapy causes considerablecollateral damage to normal cell populations at thetreatment site (59). This is particularly true for bone, inwhich radiation treatment often leads to bone destructionand impaired healing (60). Osteoblasts exposed toradiation exhibit decreased collagen synthesis, leadingto bone atrophy and osteonecrosis (61). Recent studieshave found that in addition to enhancing the therapeuticefficacy of radiotherapy, ATO has an additional advan-tage of protecting bone tissue against radiation-inducedbone loss (59). In addition, the use of chemical modifiersas radiosensitizers in combination with low-dose radia-tion may increase the therapeutic efficacy by overcominga high apoptotic threshold (6). Previous studies foundthat treatment of ATO in combination with radiation hassynergistic effects in decreasing clonogenic survival andin the regression of tumor growth in xenografts (22). Inthe present study, we found evidence indicating thatsynergistic effects of ATO and radiation could be foundin osteosarcoma cells through the induction of bothautophagic and apoptotic cell death.

Taken together, our results indicate that radiationcombined with ATO increases the therapeutic efficacycompared to individual treatments in HOS humanosteosarcoma cells. Specifically, combined treatmentinduced autophagy and apoptosis through inhibitionof the PI3K/Akt signaling pathway. In addition, G2/Marrest and ROS generation could be involved in theunderlying mechanisms. Furthermore, we found that thecombined treatment of HOS cells pretreated with Z-VAD, 3-MA or PEG-catalase resulted in a significantreduction of cytotoxicity. To the best of our knowledge,this study is the first to demonstrate that ATO combinedwith radiation induces autophagy and apoptosis in HOScells. These novel findings not only improve ourunderstanding of the cytotoxic effects of the combinedtreatment but also suggest a potential therapeuticstrategy for the treatment of osteosarcoma.

ACKNOWLEDGMENTS

This study was supported by the National Science Council, Taiwan(NSC 98-2314-B-006-034-MY2) and the Sinlau Christian Hospital,Tainan, Taiwan.

Received: July 23, 2010; accepted: January 6, 2011; published online:March 9, 2011

558 CHIU ET AL.

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