OSU-03012,aNovelCelecoxibDerivative,InducesReactiveOxygen ... · OSU-03012,aNovelCelecoxibDerivative,InducesReactiveOxygen Species–RelatedAutophagyinHepatocellularCarcinoma Ming

OSU-03012, a Novel Celecoxib Derivative, Induces Reactive Oxygen

Species–Related Autophagy in Hepatocellular Carcinoma

Ming Gao,1,2,3,5

Pei Yen Yeh,2,3Yen-Shen Lu,

2,3,5Chih-Hung Hsu,

2,3,5Kuen-Feng Chen,

5Wei-Chung Lee,

2,3,5

Wen-Chi Feng,2,3,5

Ching-Shih Chen,6Min-Liang Kuo,

1and Ann-Lii Cheng

2,3,4,5

1Institute of Toxicology and 2Cancer Research Center, College of Medicine, National Taiwan University; Departments of 3Oncology and4Internal Medicine and 5National Center of Excellence for Clinical Trial and Research, National Taiwan University Hospital, Taipei, Taiwan,Republic of China and 6Division of Medical Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio

Abstract

Hepatocellular carcinoma (HCC) is the fifth most commoncancer and the third leading cause of cancer death worldwide.Systemic treatments for HCC have been largely unsuccessful.OSU-03012 is a derivative of celecoxib with anticancer activity.The mechanism of action is presumably 3-phosphoinositide–dependent kinase 1 (PDK1) inhibition. This study investigatedthe potential of OSU-03012 as a treatment for HCC. OSU-03012inhibited cell growth of Huh7, Hep3B, and HepG2 cells withIC50 below 1 Mmol/L. In Huh7 cells, OSU-03012 did notsuppress PDK1 or AKT activity. Terminal deoxynucleotidyltransferase–mediated dUTP nick end labeling assay and flowcytometry analysis indicated that OSU-03012 did not inducecellular apoptosis. Instead, morphologic studies by light andelectron microscopy, as well as special biological staining withmonodansylcadaverine, acridine orange, and microtubule-associated protein 1 light chain 3, revealed OSU-03012–induced autophagy of Huh7 cells. This OSU-03012–inducedautophagy was inhibited by 3-methyladenine. Moreover,reactive oxygen species (ROS) accumulation was detectedafter OSU-03012 treatment. Blocking ROS accumulation withROS scavengers inhibited autophagy formation, indicatingthat ROS accumulation and subsequent autophagy formationmight be a major mechanism of action of OSU-03012. Dailyoral treatment of BALB/c nude mice with OSU-03012 sup-pressed the growth of Huh7 tumor xenografts. Electronmicroscopic observation indicated that OSU-03012 inducedautophagy in vivo . Together, our results show that OSU-03012induces autophagic cell death but not apoptosis in HCC andthat the autophagy-inducing activity is at least partiallyrelated to ROS accumulation. [Cancer Res 2008;68(22):9348–57]

Introduction

Hepatocellular carcinoma (HCC) is the fifth most commoncancer and the third leading cause of cancer death worldwide (1).Surgery with curative intent is feasible for only 15% to 25% ofpatients, and most patients die from locally advanced or metastaticdiseases in a relatively short period of time (2). To date, cytotoxicchemotherapy has not been a standard treatment for HCC.

Recently, molecular targeted therapy, which acts on specificdysregulated signal transduction pathways, has shown promise asa treatment for advanced HCC (3). Development of novel agents toenhance the effectiveness of treatment is mandatory.OSU-03012 is a derivative of celecoxib, a cyclooxygenase-2

inhibitor, which has been shown to induce cell death in varioustypes of cancer cells (4). The mechanism of action is presumablythrough inhibition of the 3-phosphoinositide–dependent kinase 1(PDK1)/AKT signaling pathway (4). In addition to PDK1/AKTsignaling inhibition, OSU-03012 might also have effects on otherimportant signaling pathways. For example, OSU-03012 has beenshown to induce apoptosis by activation of the intrinsicallymitochondrial pathway in primary chronic lymphocytic leukemiacells (5). OSU-03012 also inhibited c-Jun NH2-terminal kinase/signal transducers and activators of transcription and mitogen-activated protein kinase pathways in multiple myeloma cells (6).Further, OSU-03012 has been reported to cause a PDK1/AKT-independent cell death in glioma cells (7). Taken together, thesefindings suggest that OSU-03012 might be a multitargeted inhibitorthat exerts its functions in a cell type–dependent manner.Autophagy is a physiologic process involved in routine turnover

of cell constituents and serves as a temporary survival mechanismduring starvation where self-digestion becomes an alternativeenergy source. Autophagy has also been proposed to involveanother biological function of clearing unfolded protein awayunder certain stress conditions (8, 9). The process of autophagystarts by sequestering a portion of the cytoplasm and intracellularorganelles in a double membrane–bound structure known as theautophagosome. These autophagosomes form autolysosomes byfusing with lysosomes and degrading the sequestered contents bylysosomal hydrolases (10). However, recent studies showed thatautophagy also has an active role in cell death (9). Autophagy orautophagic cell death, also known as type II programmed celldeath, is a response to various anticancer therapies in many kindsof cancer cells (10). Certain forms of cell death were shown to beprevented in the presence of either autophagic inhibitors orreduced expression of the ATG gene, which regulates autophagy(11, 12). Whether autophagy is a protective mechanism or amechanism of cell death in the response of tumor cells toanticancer therapy remains unclear.Certain cellular stresses can induce autophagy formation, such as

oxidative stress. Under oxidative stress, reactive oxygen species (ROS)are produced, including singlet oxygen, superoxide, hydroxyl radical,and hydrogen peroxide. High levels of ROS induce cell death, whichoften involves apoptosis through caspase activation (13). Moreover,ROS are essential for autophagosome formation under starvationconditions through targeting cysteine protease HsAtg 4 , anautophagy-related gene, leading to cell survival (14). However, littleis known about the role of ROS in autophagy-induced cell death.

Requests for reprints: Ann-Lii Cheng, Departments of Internal Medicine andOncology, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei10016, Taiwan, Republic of China. Phone: 886-2-23123456, ext. 67251; Fax: 886-2-23711174; E-mail: [email protected] or Min-Liang Kuo, Institute of Toxicology,College of Medicine, National Taiwan University, No. 1, Sec. 1, Ren-Ai Road, Taipei10016, Taiwan, Republic of China. Phone: 886-2-23123456, ext. 88607; Fax: 886-2-23410217; E-mail: [email protected].

I2008 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-08-1642

Cancer Res 2008; 68: (22). November 15, 2008 9348 www.aacrjournals.org

Research Article

Research. on December 19, 2020. © 2008 American Association for Cancercancerres.aacrjournals.org Downloaded from

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In this study, we showed that OSU-03012 exerted potentcytotoxicity against cultured HCC cells as well as xenograft tumors.We also showed that OSU-03012 induces autophagy but notapoptosis in HCC cells and that this autophagy-inducing activity isat least partially related to ROS accumulation.

Materials and Methods

Reagents and antibodies. OSU-03012 was kindly supplied by Prof.

Ching-Shih Chen (Division of Medicinal Chemistry, College of Pharmacy,

Ohio State University) and was dissolved in DMSO (Sigma Chemical Co.).For in vivo study, OSU-03012 was suspended in 0.5% (w/v) methylcellulose

and 0.1% (v/v) Tween 80. All chemicals were purchased from Calbiochem

or Sigma. Antibodies used in this study were phosphorylated PDK1

(pPDK1), PDK1, ATG5, ATG7 (Cell Signaling), phosphorylated AKT (pAKT),AKT, poly(ADP-ribose) polymerase (PARP), caspase-3 (Santa Cruz Biotech-

nology), and microtubule-associated protein 1 light chain 3B (MAP1-LC3B;

Novus Biologicals).Cell culture. The human HCC cell line Huh7 was kindly provided by

Dr. Shiou-Hwie Yeh (Department of Microbiology, National Taiwan

University College of Medicine). Hep3B and HepG2 cells were purchased

from the American Type Culture Collection. The cells were cultured inDMEM (Biological Industries), supplemented with 10% heat-inactivated

fetal bovine serum (FBS; Biological Industries), 100 units/mL penicillin, and

100 Ag/mL streptomycin, and incubated at 37jC in a humidified incubator

containing 5% CO2.Cell viability assay. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT) assay was performed to evaluate cell viability as previously

described with slight modification (15). Briefly, tumor cells in exponentialgrowth were seeded at 4 � 103 per well in 96-well flat-bottomed plates

(Corning, Inc.) and incubated overnight at 37jC. The cells were then treatedwith various doses of OSU-03012 for 72 h. Fifty microliters of 2 mg/mL MTT

were added to each well, and cells were incubated in the presence of MTTfor 3 h. The formazan crystals were dissolved in DMSO. The absorbance was

determined with a DTX 800 multimode detector (Beckman Coulter) at

490 nm. Absorbance values were normalized to the values obtained for the

untreated cells to determine percentage survival.Western blot analysis. Following different treatments as indicated in

figure legends, the cells were lysed with lysis buffer (150 mmol/L NaCl, 1%

NP40, 0.5% deoxycholic acid, 0.1% SDS, 1 Ag/mL aprotinin, 1 mmol/L

phenylmethylsulfonyl fluoride, 0.5 Ag/mL leupeptin, 1 Ag/mL pepstatin) onice for 30 min. Cell lysates were then centrifuged at 13,000 � g for 30 min at

4jC. Supernatant was collected and the protein concentration was

determined by the bicinchoninic acid protein assay kit (Pierce). Equalamounts of protein (30 Ag) were resolved in 10% SDS-polyacrylamide gel

and then transferred to nitrocellulose membrane (Perkin-Elmer Life

Science). The membrane was incubated with the appropriate primary

antibody at 4jC overnight. Then, the membrane was washed and incubatedwith a horseradish peroxidase (HRP)–conjugated secondary antibody for

1 h at room temperature. The immunoblots were visualized by

chemiluminescence HRP substrate (Millipore).

Cell cycle analysis. For cell cycle analysis, approximately 5 � 105 Huh7cells were seeded onto 6-cm dishes and incubated at 37jC overnight. The

cells were treated with OSU-03012 for 24 or 48 h and then suspended by

treatment with trypsin and fixed with ice-cold methanol at �20jC for20 min. Cells were then stained with propidium iodide at the concentration

of 50 Ag/mL in the presence of RNase A (100 Ag/mL). DNA content was

analyzed by FACScan (Becton Dickinson). Data were analyzed by CellQuest

software (Becton Dickinson).Apoptosis detection. Apoptosis was analyzed by terminal deoxynucleo-

tidyl transferase–mediated dUTP nick end labeling (TUNEL; DeadEnd

Fluorometric TUNEL System, Promega) performed according to the

manufacturer’s instructions.Detection of autophagosome formation with acridine orange

and monodansylcadaverine. To detect the presence of acidic vesicular

organelles, the cells were stained with the vital dye acridine orange

(1 Ag/mL) and then examined under a fluorescence microscope. The

autofluorescent agent monodansylcadaverine (MDC) has been reported tobe specifically accumulated in autophagolysosomes and has thus been used

to detect their presence (16). In this study, OSU-03012–treated cells were

incubated with 0.05 mmol/L MDC for 10 min at 37jC and then observed

under a fluorescence microscope (Leica DM2500).Transmission electron microscopy. The cells were harvested by

trypsinization, washed twice with PBS, fixed with ice-cold 3% glutaraldehyde

in 0.1 mol/L cacodylate buffer, postfixed in osmium tetroxide, and then

embedded in Epong. A 1.0-Am-thin section was cut, stained with methylenebuffer ArumeII, and viewed with a Hitachi 7500 electron microscope.

Immunofluorescent staining. Huh7 cells were seeded on sterilized

slides in a 10-cm dish overnight and then treated with 5 Amol/L OSU-03012

for 48 h. The cells were washed with PBS, fixed with 4% formaldehyde for10 min at 37jC, and then washed with PBS twice. Cells were treated with

0.1% Triton X-100 and then blocked with 0.5% bovine serum albumin in PBS

at 37jC for 1 h. For LC3 staining, Huh7 cells were treated with mouse anti-LC3 antibody (1:100 in PBS containing 0.5% bovine serum albumin) at 4jCovernight. FITC-conjugated goat anti-mouse IgG (1:100) was applied for

1 h at room temperature. The subcellular distribution of LC3 was observed

under a fluorescence microscope (Leica DM2500).Transient transfection and RNA interference. The small interfering

RNA (siRNA) was purchased from Dharmacon, Inc., including On-

TARGETplus siCONTROL Non-Targeting Pool and On-TARGETplus SMART-

pool against human ATG5. For transient transfection, 2� 105 Huh7 cells wereseeded in six-well plates, transfected with Lipofetamine RNAiMAX (Invi-

trogen) according to the manufacturer’s instructions, and incubated

for 48 h.ROS measurement. ROS levels were determined by flow cytometry

analysis and fluorescence microscopy. Briefly, cells were treated with

25 Amol/L 5-(and-6)-carboxy-2¶,7¶-dichlorodihydrofluorescein diacetate

(carboxy-H2DCFDA; Invitrogen) in PBS for 30 min, and then flow cytometryanalysis was performed. For ROS visualization, cells were stained with the

Image-iTLIVE Green Reactive Oxygen Species Detection kit (Invitrogen)

according to the manufacturer’s protocol.

In vivo xenograft tumor study. Six-week-old male BALB/c nude micewere obtained from the National Laboratory Animal Center (Taipei, Taiwan,

Republic of China). All experiments were performed under protocols

approved by the Experimental Animal Center of National Taiwan UniversityCollege of Medicine. Each mouse was inoculated s.c. in the right dorsal

flank with 2 � 106 Huh7 cells suspended in 0.1 mL PBS containing 50%

Matrigel (BD Biosciences). Two weeks later, mice bearing tumors reaching

200 F 100 mm3 were randomized to three groups (n = 7) and received thefollowing treatment daily by gavage for 28 d: (1) 0.5% methylcellulose,

0.1% Tween 80 vehicle; (2) 100 mg/kg body weight of OSU-03012; and

(3) 200 mg/kg body weight of OSU-03012. Mice were weighed every day

and tumors were measured with Vernier calipers every 2 d. Tumor volumeswere calculated with the following formula: p/6 � large diameter � (small

diameter)2.

Statistical analysis. Tumor volume data were analyzed by one-way

ANOVA followed by Fisher’s least significant difference method for multiplecomparisons. Tumor growth data are expressed as mean tumor volume FSE. Differences were considered significant at P < 0.05. Statistical analysis

was performed using Statistical Package for the Social Sciences forWindows version (SPSS, Inc.).

Results

OSU-03012 inhibited growth of HCC cells. The antitumoreffect of OSU-03012 was assessed in three human HCC cell lines:Huh7, Hep3B, and HepG2. As shown in Fig. 1A , OSU-03012inhibited the growth of all tested cells in a similar dose-dependentmanner with IC50 below 1 Amol/L. Because OSU-03012 had themost effective cytotoxicity against Huh7 cells, Huh7 cells werechosen for the subsequent experiments.OSU-03012 was previously reported to inhibit cell growth

through the mechanism of PDK1/AKT signaling pathway inhibition

OSU-03012 Induces Autophagy in HCC

www.aacrjournals.org 9349 Cancer Res 2008; 68: (22). November 15, 2008



in some cancer cells (5, 17). However, its effect on HCC cells remainsuncharacterized. To investigate whether OSU-03012 suppresses cellgrowth through inhibition of the PDK1/AKT signaling pathway inHCC cells, the activities of PDK1 and AKT were determined byWestern blotting using pPDK1- and pAKT-specific antibodies(Fig. 1B). Neither pPDK1 nor pAKT was decreased by OSU-03012treatment in HCC cells. These data indicate that OSU-03012suppresses HCC cell growth through the targeting of signalingmolecules other than PDK1.OSU-03012 did not induce apoptosis in HCC cells. Because

OSU-03012 has been reported to induce apoptosis in some cancercells, we investigated whether OSU-03012 could induce apoptosis inHuh7 cells by TUNEL assay. As shown in Fig. 2A , no apoptotic cellwas detected after 48-hour OSU-03012 treatment. This finding wasfurther confirmed by flow cytometry analysis (Fig. 2B). No sub-G1

fraction could be detected after OSU-03012 treatment. Interestingly,OSU-03012 induced an increased S-phase population in Huh7 cells( from 19.27% to 47.51%). Similar findings were observed in Hep3Band HepG2 cells treated with OSU-03012 for 24 hours (Fig. 2C).Both TUNEL assay (Fig. 2A) and flow cytometry analysis (Fig. 2B)showed that doxorubicin did induce apoptosis in Huh7 cells,

indicating that the pathway to apoptosis is intact in these cells, anobservation further supported that apoptosis induction is not amajor mechanism of OSU-03012 cytotoxicity effects. Two biochem-ical markers of apoptosis, active caspase-3 and cleaved PARP,were also examined by Western blotting analysis. Consistently,both proteins were undetectable in OSU-03012–treated Huh7 cells(Fig. 2D). These results indicate that OSU-03012 did not induceapoptosis in HCC cells.OSU-03012 induced autophagy in Huh7 cells. The morpho-

logic changes in Huh7 cells were observed to characterize the effectof OSU-03012 treatment. Compared with control cells, OSU-03012–treated cells exhibited obvious vacuoles in the cytoplasm,indicating the possible formation of autophagy. Autophagy is theprocess of sequestrating cytoplasmic proteins into lytic compo-nents and is characterized by formation and promotion of AVO(18). We therefore investigated whether OSU-03012 could induceautophagy in Huh7 cells by vital staining with acridine orange. Asshown in Fig. 3A (top), OSU-03012 induced the accumulation ofAVO in the cytoplasm of Huh7 cells. MDC staining has also beenused to detect autophagic vacuoles (19). Similarly, OSU-03012induced apparent accumulation of MDC in the cytoplasmicvacuoles, whereas less accumulation of MDC was detected incontrol cells (Fig. 3A, bottom). Quantitatively, 62% of 1 Amol/LOSU-03012–treated cells and 96% of 5 Amol/L OSU-03012–treatedcells exhibited autophagic vacuoles, whereas only 5% of untreatedHuh7 cells exhibited autophagic vacuoles (Fig. 3B, top). Similarresults were also observed in Hep3B cells (Fig. 3B, bottom). Theultrastructure of OSU-03012–treated Huh7 cells was analyzed bytransmission electron microscopy. As shown in Fig. 3C , numerousAVOs were observed in OSU-03012–treated Huh7 cells. In addition,autophagosomes and autolysosomes were frequently observed.By contrast, there were few AVOs in the cytoplasm of untreatedHuh7 cells.MAP1-LC3, a mammalian homologue of Apg8p/Aut7p, is

essential for amino acid starvation-induced autophagy and isassociated with the formation of the autophagosome membrane(20, 21). To further confirm whether OSU-03012 could induceautophagy in Huh7 cells, immunofluorescent staining was used todetect the intracellular distribution of LC3. As shown in Fig. 3D(top), whereas a diffused distribution of LC3 was observed incontrol cells, a punctuate pattern of LC3 was observed in OSU-03012–treated cells. To elucidate whether OSU-03012 induced aspecific or nonspecific autophagy in HCC cells (22), Huh7 cells wereincubated in HBSS for 4 hours. As shown in Fig. 3D (middle),Western blotting analysis showed a significantly increasedexpression of cleaved LC3 in OSU-03012–treated Huh7 cells,whereas marginal cleavage of LC3 was observed in HBSS-treatedcells. These results suggested that OSU-03012 induces specificautophagy in Huh7 cells. Treatment with OSU-03012 also causedcleavage of LC3 protein in Hep3B and HepG2 cells (Fig. 3D,bottom). These results indicated that OSU-03012 induces autoph-agy in HCC cells.Silencing ATG5 and chemical inhibitor 3-methyladenine

decreases OSU-03012–induced autophagy and cytotoxicity.ATG5 has been characterized as an ubiquitin-like protein involvedin autophagosome formation (23), and a recent study has shownthat OSU-03012 increases the expression of ATG5 and promotesATG5-dependent formation of LC3 (24). We therefore examinedwhether OSU-03012 increased ATG5 and ATG7 protein levels inHuh7 cells. As shown in Fig. 4A , OSU-03012 had little effecton ATG7 expression but enhanced the expression of ATG5 in a

Figure 1. OSU-03012 inhibits growth of HCC cell lines. A, growth inhibitioneffect of OSU-03012 on Huh7, Hep3B, and HepG2 cells. Tumor cells were platedin 96-well plates 24 h before OSU-03012 treatment and incubated overnightat 37jC. Cells were treated with different doses of OSU-03012 in 10%FBS-supplemented DMEM for 72 h. Cell viability was assessed by MTT assay.Points, mean of three independent experiments; bars, SD. B, OSU-03012 doesnot inhibit PDK1 and AKT activity in Huh7 cells. Huh7 cells were treated with1 or 5 Amol/L of OSU-03012 for indicated time intervals and then harvested forprotein analysis. Cell lysates were resolved in SDS-PAGE and probed withspecific antibodies against pPDK1, PDK1, pAKT, and AKT. Stains of h-actinserved as loading control.

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dose- and time-dependent manner. Further, silencing of ATG5 bysiRNA reduced OSU-03012–induced LC3 cleavage, indicating thatATG5 was involved in OSU-03012–induced autophagy (Fig. 4B).Recent studies showed that 3-methyladenine (3-MA), an inhibitorof phosphatidylinositol 3-kinase, could inhibit autophagy (18). Wefurther used 3-MA to show that OSU-03012 induced autophagy inHuh7 cells. As shown in Fig. 4C (left), OSU-03012–inducedaccumulation of MDC in the cytoplasmic vacuoles was attenuatedby treatment with 2 mmol/L 3-MA. Quantitative analysis showedthat 2 mmol/L 3-MA reduced OSU-03012–induced autophagy from62% to 36% of cells (Fig. 4C, right). To investigate whetherinhibition of autophagy affects the cytotoxicity of OSU-03012, Huh7cells were treated with various doses of OSU-03012 for 72 hours inthe presence of 2 mmol/L 3-MA. As shown in Fig. 4D , 3-MA

reversed the OSU-03012–induced cytotoxicity in Huh7 cells.These results indicated that the mechanism of anticancer activityof OSU-03012 in HCC cells is partially attributable to the formationof autophagy.ROS mediated OSU-03012–induced autophagy formation

and autophagic cell death. Recent study showed that ROS couldinduce autophagy formation in certain types of cancer cells(14, 25). Therefore, we investigated whether OSU-03012 treatmentcould increase the ROS level in HCC cells. Using H2DCFDA-baseddetection by flow cytometry, ROS accumulation was observed from6 hours and increased 2-fold after 24 hours of OSU-03012 treatmentin Huh7 cells (Fig. 5A, left). ROS accumulation was also detected inHep3B and HepG2 cells (data not shown). This result was furtherconfirmed by fluorescence microscopy with DCF staining. The ROS

Figure 2. Apoptosis is not detectable in OSU-03012–treated Huh7 cells. A, TUNEL staining. After treatment with or without 5 Amol/L OSU-03012 for 48 h,Huh7 cells were fixed and labeled with bromodeoxyuridine triphosphate (BrdUTP) for TUNEL assay. Doxorubicin (Doxo ; 5 Amol/L)–treated Huh7 cells served as apositive control. DAPI, 4¶,6-diamidino-2-phenylindole. B, flow cytometry analysis was used to detect the cell cycle distribution. Huh7 cells in logarithmic growth weretreated with 5 Amol/L OSU-03012 or 5 Amol/L doxorubicin for 48 h. Cells were harvested, fixed, treated with RNase A, stained with propidium iodide, and then subjectedto flow cytometric analysis. C, flow cytometric analysis of Hep3B and HepG2 cells. Hep3B and HepG2 cells were treated with 5 Amol/L OSU-03012 for 24 h.The flow cytometry analysis was performed as described in B. D, top, Huh7 cells were treated with 1 or 5 Amol/L of OSU-03012 for 1, 6, 24, and 48 h and then harvestedfor protein analysis. Cell lysates were resolved in SDS-PAGE and probed with specific antibodies against PARP and caspase-3. Bottom, experimental controls forPARP and caspase-3 cleavage. Huh7 cells were treated with 5 Amol/L doxorubicin for 48 h, lysed, and then probed with antibodies against PARP and caspase-3.





scavenger N-acetylcysteine (NAC) at 10 mmol/L or 4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt (Tiron) at 5 mmol/Labrogated OSU-03012–induced DCF accumulation (Fig. 5A, right).We then examined whether ROS plays a role in OSU-03012–

induced autophagy. Formation of autophagy induced by OSU-03012was suppressed by 10 mmol/L NAC treatment (Fig. 5B). Cleavageof LC3 protein induced by OSU-03012 was also decreased bytreatment with 10 mmol/L NAC (Fig. 5C). Further, both NAC andTiron partially reduced the cytotoxicity of OSU-03012 in Huh7 cells

(Fig. 5D). Taken together, these results suggested that ROS play animportant role in OSU-03012–induced autophagy formation andautophagic cell death.OSU-03012 induces autophagy and suppresses Huh7 xeno-

graft growth in vivo . To assess the antitumor potential of OSU-03012 in HCC, nude mice bearing established s.c. Huh7 tumorxenografts were gavaged with OSU-03012 daily for 28 days at twodifferent doses, 100 and 200 mg/kg body weight, or with vehicleonly. Compared with vehicle-treated controls, 100 and 200 mg/kg

Figure 3. OSU-03012–induced autophagy formation. A, top, acridine orange staining. Huh7 cells were treated with or without 5 Amol/L OSU-03012 for 48 h andthen stained with acridine orange (1 Ag/mL) and examined under a fluorescence microscope. Bottom, MDC staining. Huh7 cells were treated with or without5 Amol/L OSU-03012 for 48 h, incubated with 0.05 mmol/L MDC for 10 min, and then observed under a fluorescence microscope. B, top, quantification of MDCincorporation in Huh7 cells. At least 100 control or OSU-03012–treated (5 Amol/L, 48 h) cells were examined under a fluorescence microscope and the percentageof MDC-incorporating cells was calculated. Columns, mean (n = 3) for each treatment and representative of three independent experiments; bars, SD.Bottom, quantification of the percentage of MDC-incorporating Hep3B cells. MDC incorporation in cells was measured as described in A. C, electron microscopy showsthe ultrastructure of Huh7 cells treated without (a and b) or with (c and d ) 5 Amol/L OSU-03012 for 48 h. Arrowheads, autophagosomes. Bars, 2 Am (a and c )and 550 nm (b and d). D, top, immunofluorescent staining of MAP1-LC3. Huh7 cells were treated without (a) or with 5 Amol/L OSU-03012 for 48 h (b) and examinedunder a fluorescence microscope. Middle, Huh7 cells were treated with HBSS for 4 h and 1 or 5 Amol/L of OSU-03012 for 24 h and then harvested for protein analysis.Cell lysates were resolved in SDS-PAGE and probed with specific antibody against MAP1-LC3. Bottom, Hep3B and HepG2 cells were treated with 1 or 5 Amol/Lof OSU-03012 for 24 h and then harvested for protein analysis. Cell lysates were resolved in SDS-PAGE and probed with specific antibody against MAP1-LC3.

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OSU-03012 treatments suppressed Huh7 tumor growth by 39.52%and 57.59% after 28 days of treatment, respectively (P < 0.05and 0.01; Fig. 6A, left). Tumor volumes were significantly reducedby OSU-03012 treatment compared with vehicle-treated control(Fig. 6A, right).To study the mechanism of tumor suppression effect in this

xenograft model, electron microscopy was performed. Mice treatedwith 200 mg/kg OSU-03012 or untreated for 4 days were sacrificedand the ultrastructures of tumor cells were observed (Fig. 6B).Compared with vehicle-treated tumor cells, autophagosomes anddense lysosomes were easily observed in OSU-treated tumor cells.TUNEL assay rarely detected positive tumor cells after OSU-03012

treatment for 4 days (data not shown) or 28 days (Fig. 6C). Tofurther support that OSU-03012 induces autophagy in xenograft,Western blotting of cleaved LC3 was performed. As shown inFig. 6D , OSU-03012 increased cleaved LC3, indicating the formationof autophagy in OSU-03012–treated xenograft.

Discussion

This study showed that treatment with OSU-03012, a novelcelecoxib derivative, induces autophagy but not apoptosis inhuman HCC Huh7 cells. We showed that OSU-03012 inhibitsgrowth within a low micromolor range in Huh7, Hep3B, and HepG2

Figure 4. Inhibition of OSU-03012–induced autophagy by silencing ATG5 or chemical inhibitor 3-MA. A, Huh7 cells were treated with 1 or 5 Amol/L of OSU-03012for indicated time intervals. The cell lysates were subjected to Western blotting using indicated antibodies. Stains of h-actin served as loading control. B, the expressionof ATG5 in Huh7 cells was knock down by siRNA transfection as described in Materials and Methods. The cells were treated with 1 or 5 Amol/L of OSU-03012 for 48 h.The cell lysates were subjected to Western blotting using indicated antibodies. Stains of h-actin served as loading control. C, left, MDC staining. Huh7 cells wereuntreated (a), treated with 1 Amol/L OSU-03012 (b), treated with 1 Amol/L OSU-03012 and 2 mmol/L 3-MA (c ), and treated with 2 mmol/L 3-MA for 48 h (d); stainedwith MDC; and then observed under a fluorescence microscope. Right, quantification of MDC incorporation. At least 100 cells from each treatment group wereexamined under a fluorescence microscope and the percentage of MDC incorporation in cells was calculated. Columns, mean (n = 3) for each treatment andrepresentative of three independent experiments; bars, SD. D, effect of 3-MA on OSU-03012–induced cytotoxicity. Huh7 cells were treated with OSU-03012 aloneor in combination with 2 mmol/L 3-MA for 72 h. Cell viability was assessed by MTT assay. Points, mean of three independent experiments; bars, SD.





cells. OSU-03012–induced autophagy as well as cytotoxicity werepartially reversed by 3-MA, an autophagy inhibitor. IntracellularROS generation contributed to OSU-03012–induced autophagy andsubsequent autophagic cell death. The xenograft tumor modelshowed that OSU-03012 suppressed Huh7 tumor growth. Thesefindings suggest that autophagy is a mechanism that contributes tothe cytotoxic effect of OSU-03012 in vivo .OSU-03012 has been shown to induce apoptosis in non–small

cell lung cancer (26) and breast cancer cells (27, 28) through

inhibition of PDK/AKT signaling pathway. However, onlyvery limited PDK1 and AKT inhibition were detected in OSU-03012–treated Huh7 cells in this study, suggesting that OSU-03012suppresses the growth of Huh7 cells through a mechanism differentfrom PDK1 and AKT inhibition. OSU-03012 was found to triggerautophagic cell death instead of apoptotic cell death in HCC cells.We speculate that suppression of AKT activity may be necessary forOSU-03012–induced apoptosis in some certain tumor cells. OSU-03012–induced autophagic cell death is a unique cellular response

Figure 5. ROS generation is associated with OSU-03012–induced autophagy formation and cytotoxicity. A, ROS generation. Left, Huh7 cells were treated with5 Amol/L OSU-03012 for 6, 12, 18, or 24 h or treated with H2O2 for 30 min and stained with 25 Amol/L DCF for 30 min and analyzed by flow cytometry. The valuesshown are the fold increase of DCF mean fluorescence intensity (DCF F.I. ). Right, Huh7 cells were treated without (a and e) or with 5 Amol/L OSU-03012 (b and f ),5 Amol/L OSU-03012 and 10 mmol/L NAC (c and g), or 5 Amol/L OSU-03012 and 5 mmol/L Tiron (d and h) for 24 h and stained with 25 Amol/L DCF for 30 min andthen observed under a fluorescence microscope. B, effects of antioxidants on OSU-03012–induced autophagy formation. Huh7 cells were exposed to 1 Amol/LOSU-03012 with or without 10 mmol/L NAC for 24 or 48 h and then incubated with MDC. The percentage of cells incorporating MDC was determined as described inprevious figures. C, Huh7 cells were treated with OSU-03012 (1 and 5 Amol/L) and 10 mmol/L NAC for 24 h. Cell lysates were resolved in SDS-PAGE and probedwith specific antibody against MAP1-LC3. D, effects of antioxidants on OSU-03012–induced cytotoxicity. Huh7 cells were exposed to different doses of OSU-03012with or without 10 mmol/L NAC (left) or 5 mmol/L Tiron (right ) for 72 h. Cell viability was assessed by MTT assay. Points, mean of three independent experiments;bars, SD.

Cancer Research




in HCC cells. The underlying mechanisms of OSU-03012 effects onHCC cells remain largely unknown.The significance of the autophagic process in antitumor

therapeutics has not been clearly established. To adapt to adverseconditions induced by stress from anticancer therapies, cancer

cells may trigger an autophagic response that sequesters anddegrades unnecessary molecules to promote cell adaptationand survival. For example, temozolomide induces autophagy inmalignant glioma cells as a self-defense (18). Suppression ofautophagy leads to apoptosis in glioma cells and thus enhances the

Figure 6. OSU-03012 suppresses Huh7 xenograft growth and induces autophagy in vivo. Nude mice bearing established Huh7 tumor xenografts were randomlydivided into three groups (n = 7) and given daily OSU-03012 at 100 and 200 mg/kg body weight per day by gavage for 28 d. Controls received vehicle consisting of0.5% methylcellulose and 0.1% Tween 80 in sterile water. A, left, tumor growth after treatment with OSU-03012. Points, mean (n = 7); bars, SE. *, P < 0.05;**, P < 0.01, compared with the control group. Right, xenograft tumor volumes after 28 d of treatment with vehicle, 100 mg/kg body weight OSU-03012 per day, and200 mg/kg body weight OSU-03012 per day. Columns, mean; bars, SE. B, electron microscopic features of the xenograft tumors after treatment with OSU-03012.Xenograft tumor treated with vehicle consisting of 0.5% methylcellulose and 0.1% Tween 80 for 4 d (a and b ) or received 200 mg/kg body weight per day treatmentfor 4 d (c and d ). Arrowheads, autophagosomes. Bars, 2 Am (a and c ) and 550 nm (b and d). C, TUNEL staining. After treatment with 100 and 200 mg/kg bodyweight of OSU-03012 per day for 28 d, paraffin-embedded tumors were labeled with BrdUTP for TUNEL assay. DNase I–treated tumor cells served as a positive control.Bar, 1 Am. D, OSU-03012 induced autophagy in xenograft. The lysates were prepared from randomly selected xenografts treated with 100 and 200 mg/kg bodyweight of OSU-03012 and then were Western blotted with indicated antibodies. Stains of h-actin served as loading control.





antitumor effect of cancer treatment. Therefore, autophagy may bea survival mechanism of cancer cells (29, 30). On the other hand,many anticancer agents, including arsenic trioxide, rapamycin, andionizing radiation, have been reported to induce autophagic celldeath, which is distinguishable from apoptosis in cancer cells,indicating that autophagy might be a crucial mechanism of cancercell death by these agents (10, 16, 31). The postulated mechanismsfor autophagic cell death include either the autophagic digestionof important cytoplasmic factors or the selective degradation ofregulatory molecules or organelles that are crucial for survival (9).Whether autophagy induced by OSU-03012 in Huh7 cells is a celldeath mechanism or a protective mechanism was elucidated in thisstudy by applying 3-MA, an autophagy inhibitor. The resultsshowed that 3-MA could reverse the cytotoxic effect of OSU-03012on Huh7 cells, suggesting that OSU-03012 induces an autophagiccell death process in Huh7 cells.Our data showed that OSU-03012–induced ROS generation

contributes to autophagy formation and subsequent autophagiccell death in HCC cells. First, OSU-03012–induced ROS generationwas detected as early as 6 hours and had increased 2-fold after24 hours of treatment, at which time autophagy was clearlyobserved. Second, treatment with the ROS scavenger NAC reducedthe percentage of OSU-03012–induced MDC accumulation inautophagic cells as well as the amount of cleaved LC3 protein.Third, cytotoxicity of OSU-03012 was reversed in the presence ofNAC and Tiron. These results suggest that the generation of ROStriggers OSU-03012–induced autophagy formation and autophagiccell death. ROS generation is an important mediator of manyanticancer agent–induced cell deaths. A recent study reported thatROS-induced autophagy contributed to cell death in the trans-formed cell line HEK293 and the cancer cell lines U87 and HeLa butnot in nontransformed mouse astrocytes (32). Park and colleagues(24) also reported that OSU-03012 promoted autophagy in trans-formed cells through a PERK-dependent pathway. Whether ROSaccumulation induced by OSU-03012 might cause PERK activationremains to be elucidated. However, ROS is very likely to damagesome sets of proteins, which may result in endoplasmic reticulumstress and PERK activation (33, 34). The mechanism by which OSU-03012 induces ROS accumulation remains unknown. It has beenreported that the accumulation of ROS may result from decreased

ROS degradation, either selective degradation of catalase (35) orinhibition of superoxide dismutase 2 (SOD2; ref. 32). Furtherinvestigation of the role of the SOD-catalase antioxidant system inOSU-03012–induced ROS generation is needed.OSU-03012 induced significantly increased S-phase population in

HCC cells in the present study. Similar results were observed inother cell types after treatment with different antitumor agents. Forexample, resveratrol (36), soybean B-group triterpenoid saponins(37), and nano neodymium oxide (38) have been reported to causeincreased S-phase population and subsequent autophagy forma-tion. However, the relationship between increased S-phasepopulation and autophagy formation remains unknown.In the Huh7 xenograft tumor model used in this study, OSU-

03012 also suppressed tumor growth. Consistent with the in vitrofindings, electron microscopy showed autophagy formation inxenograft tumor cells. TUNEL assay found no evidence of apoptosisafter OSU-03012 treatment. Previous pharmacokinetic studiesshowed that the peak serum concentration of OSU-03012 afteroral administration at 200 mg/kg exceeded 20 Amol/L (5). Duringthe treatment period in this study, almost all animals survived andexhibited no apparent signs of toxicity, indicating that oraladministration of OSU-03012 delivered sufficient quantities of drugto inhibit HCC tumor growth with little side effects.In conclusion, our results show that the orally bioavailable drug

OSU-03012 induced autophagic cell death but not apoptosis inHCC and that this autophagy-inducing activity was at leastpartially related to ROS accumulation. This study shows a novelbiological effect of OSU-03012, which supports its clinical potentialas a component of therapeutic strategies for HCC.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Received 5/5/2008; revised 8/14/2008; accepted 9/4/2008.Grant support: Department of Health grant DOH96-TD-B-111-001 and National

Science Council, Taiwan, Republic of China, grant 95R0066-BM01-02.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Cancer Research


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2008;68:9348-9357. Cancer Res Ming Gao, Pei Yen Yeh, Yen-Shen Lu, et al. Carcinoma

Related Autophagy in Hepatocellular−Oxygen Species OSU-03012, a Novel Celecoxib Derivative, Induces Reactive

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