Hepatocellular carcinoma (HCC) is the fifth most common cancer 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 investigated the potential of OSU-03012 as a treatment for HCC. OSU-03012 inhibited cell growth of Huh7, Hep3B, and HepG2 cells with IC50 below 1 μmol/L. In Huh7 cells, OSU-03012 did not suppress PDK1 or AKT activity. Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay and flow cytometry analysis indicated that OSU-03012 did not induce cellular apoptosis. Instead, morphologic studies by light and electron microscopy, as well as special biological staining with monodansylcadaverine, acridine orange, and microtubule-associated protein 1 light chain 3, revealed OSU-03012–induced autophagy of Huh7 cells. This OSU-03012–induced autophagy was inhibited by 3-methyladenine. Moreover, reactive oxygen species (ROS) accumulation was detected after OSU-03012 treatment. Blocking ROS accumulation with ROS scavengers inhibited autophagy formation, indicating that ROS accumulation and subsequent autophagy formation might be a major mechanism of action of OSU-03012. Daily oral treatment of BALB/c nude mice with OSU-03012 suppressed the growth of Huh7 tumor xenografts. Electron microscopic observation indicated that OSU-03012 induced autophagy in vivo. Together, our results show that OSU-03012 induces autophagic cell death but not apoptosis in HCC and that the autophagy-inducing activity is at least partially related to ROS accumulation. [Cancer Res 2008;68(22):9348–57]

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death worldwide (1). Surgery with curative intent is feasible for only 15% to 25% of patients, and most patients die from locally advanced or metastatic diseases in a relatively short period of time (2). To date, cytotoxic chemotherapy has not been a standard treatment for HCC. Recently, molecular targeted therapy, which acts on specific dysregulated signal transduction pathways, has shown promise as a treatment for advanced HCC (3). Development of novel agents to enhance 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 various types of cancer cells (4). The mechanism of action is presumably through inhibition of the 3-phosphoinositide–dependent kinase 1 (PDK1)/AKT signaling pathway (4). In addition to PDK1/AKT signaling inhibition, OSU-03012 might also have effects on other important signaling pathways. For example, OSU-03012 has been shown to induce apoptosis by activation of the intrinsically mitochondrial pathway in primary chronic lymphocytic leukemia cells (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, these findings suggest that OSU-03012 might be a multitargeted inhibitor that 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 mechanism during starvation where self-digestion becomes an alternative energy source. Autophagy has also been proposed to involve another biological function of clearing unfolded protein away under certain stress conditions (8, 9). The process of autophagy starts by sequestering a portion of the cytoplasm and intracellular organelles in a double membrane–bound structure known as the autophagosome. These autophagosomes form autolysosomes by fusing with lysosomes and degrading the sequestered contents by lysosomal hydrolases (10). However, recent studies showed that autophagy also has an active role in cell death (9). Autophagy or autophagic cell death, also known as type II programmed cell death, is a response to various anticancer therapies in many kinds of cancer cells (10). Certain forms of cell death were shown to be prevented in the presence of either autophagic inhibitors or reduced expression of the ATG gene, which regulates autophagy (11, 12). Whether autophagy is a protective mechanism or a mechanism of cell death in the response of tumor cells to anticancer 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, which often involves apoptosis through caspase activation (13). Moreover, ROS are essential for autophagosome formation under starvation conditions through targeting cysteine protease HsAtg 4, an autophagy-related gene, leading to cell survival (14). However, little is known about the role of ROS in autophagy-induced cell death.

In this study, we showed that OSU-03012 exerted potent cytotoxicity against cultured HCC cells as well as xenograft tumors. We also showed that OSU-03012 induces autophagy but not apoptosis in HCC cells and that this autophagy-inducing activity is at least partially related to ROS accumulation.

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 Biotechnology), 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 in DMEM (Biological Industries), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Biological Industries), 100 units/mL penicillin, and 100 μg/mL streptomycin, and incubated at 37°C 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 exponential growth were seeded at 4 × 103 per well in 96-well flat-bottomed plates (Corning, Inc.) and incubated overnight at 37°C. The cells were then treated with 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 MTT for 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 μg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, 0.5 μg/mL leupeptin, 1 μg/mL pepstatin) on ice for 30 min. Cell lysates were then centrifuged at 13,000 × g for 30 min at 4°C. Supernatant was collected and the protein concentration was determined by the bicinchoninic acid protein assay kit (Pierce). Equal amounts of protein (30 μg) 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 4°C overnight. Then, the membrane was washed and incubated with 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 Huh7 cells were seeded onto 6-cm dishes and incubated at 37°C 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 −20°C for 20 min. Cells were then stained with propidium iodide at the concentration of 50 μg/mL in the presence of RNase A (100 μg/mL). DNA content was analyzed by FACScan (Becton Dickinson). Data were analyzed by CellQuest software (Becton Dickinson).

Apoptosis detection. Apoptosis was analyzed by terminal deoxynucleotidyl 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 μg/mL) and then examined under a fluorescence microscope. The autofluorescent agent monodansylcadaverine (MDC) has been reported to be 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 37°C 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-μm-thin section was cut, stained with methylene buffer 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 μmol/L OSU-03012 for 48 h. The cells were washed with PBS, fixed with 4% formaldehyde for 10 min at 37°C, 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 37°C 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 4°C overnight. 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 SMARTpool against human ATG5. For transient transfection, 2 × 105 Huh7 cells were seeded in six-well plates, transfected with Lipofetamine RNAiMAX (Invitrogen) 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 μmol/L 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA; Invitrogen) in PBS for 30 min, and then flow cytometry analysis 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 mice were 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 University College 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 ± 100 mm3 were randomized to three groups (n = 7) and received the following 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 volumes were calculated with the following formula: π/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 multiple comparisons. Tumor growth data are expressed as mean tumor volume ± SE. Differences were considered significant at P < 0.05. Statistical analysis was performed using Statistical Package for the Social Sciences for Windows version (SPSS, Inc.).

OSU-03012 inhibited growth of HCC cells. The antitumor effect of OSU-03012 was assessed in three human HCC cell lines: Huh7, Hep3B, and HepG2. As shown in Fig. 1A, OSU-03012 inhibited the growth of all tested cells in a similar dose-dependent manner with IC50 below 1 μmol/L. Because OSU-03012 had the most effective cytotoxicity against Huh7 cells, Huh7 cells were chosen for the subsequent experiments.

Figure 1.

OSU-03012 inhibits growth of HCC cell lines. A, growth inhibition effect of OSU-03012 on Huh7, Hep3B, and HepG2 cells. Tumor cells were plated in 96-well plates 24 h before OSU-03012 treatment and incubated overnight at 37°C. 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 does not inhibit PDK1 and AKT activity in Huh7 cells. Huh7 cells were treated with 1 or 5 μmol/L of OSU-03012 for indicated time intervals and then harvested for protein analysis. Cell lysates were resolved in SDS-PAGE and probed with specific antibodies against pPDK1, PDK1, pAKT, and AKT. Stains of β-actin served as loading control.

Figure 1.

OSU-03012 inhibits growth of HCC cell lines. A, growth inhibition effect of OSU-03012 on Huh7, Hep3B, and HepG2 cells. Tumor cells were plated in 96-well plates 24 h before OSU-03012 treatment and incubated overnight at 37°C. 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 does not inhibit PDK1 and AKT activity in Huh7 cells. Huh7 cells were treated with 1 or 5 μmol/L of OSU-03012 for indicated time intervals and then harvested for protein analysis. Cell lysates were resolved in SDS-PAGE and probed with specific antibodies against pPDK1, PDK1, pAKT, and AKT. Stains of β-actin served as loading control.

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OSU-03012 was previously reported to inhibit cell growth through the mechanism of PDK1/AKT signaling pathway inhibition in some cancer cells (5, 17). However, its effect on HCC cells remains uncharacterized. To investigate whether OSU-03012 suppresses cell growth through inhibition of the PDK1/AKT signaling pathway in HCC cells, the activities of PDK1 and AKT were determined by Western blotting using pPDK1- and pAKT-specific antibodies (Fig. 1B). Neither pPDK1 nor pAKT was decreased by OSU-03012 treatment in HCC cells. These data indicate that OSU-03012 suppresses HCC cell growth through the targeting of signaling molecules other than PDK1.

OSU-03012 did not induce apoptosis in HCC cells. Because OSU-03012 has been reported to induce apoptosis in some cancer cells, we investigated whether OSU-03012 could induce apoptosis in Huh7 cells by TUNEL assay. As shown in Fig. 2A, no apoptotic cell was detected after 48-hour OSU-03012 treatment. This finding was further 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 Hep3B and 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, an observation further supported that apoptosis induction is not a major mechanism of OSU-03012 cytotoxicity effects. Two biochemical 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 induce apoptosis in HCC cells.

Figure 2.

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

Figure 2.

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

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OSU-03012 induced autophagy in Huh7 cells. The morphologic changes in Huh7 cells were observed to characterize the effect of 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 the process of sequestrating cytoplasmic proteins into lytic components and is characterized by formation and promotion of AVO (18). We therefore investigated whether OSU-03012 could induce autophagy in Huh7 cells by vital staining with acridine orange. As shown in Fig. 3A (top), OSU-03012 induced the accumulation of AVO in the cytoplasm of Huh7 cells. MDC staining has also been used to detect autophagic vacuoles (19). Similarly, OSU-03012 induced apparent accumulation of MDC in the cytoplasmic vacuoles, whereas less accumulation of MDC was detected in control cells (Fig. 3A,, bottom). Quantitatively, 62% of 1 μmol/L OSU-03012–treated cells and 96% of 5 μmol/L OSU-03012–treated cells exhibited autophagic vacuoles, whereas only 5% of untreated Huh7 cells exhibited autophagic vacuoles (Fig. 3B,, top). Similar results were also observed in Hep3B cells (Fig. 3B,, bottom). The ultrastructure of OSU-03012–treated Huh7 cells was analyzed by transmission electron microscopy. As shown in Fig. 3C, numerous AVOs 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 untreated Huh7 cells.

Figure 3.

OSU-03012–induced autophagy formation. A, top, acridine orange staining. Huh7 cells were treated with or without 5 μmol/L OSU-03012 for 48 h and then stained with acridine orange (1 μg/mL) and examined under a fluorescence microscope. Bottom, MDC staining. Huh7 cells were treated with or without 5 μmol/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 MDC incorporation in Huh7 cells. At least 100 control or OSU-03012–treated (5 μmol/L, 48 h) cells were examined under a fluorescence microscope and the percentage of 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 shows the ultrastructure of Huh7 cells treated without (a and b) or with (c and d) 5 μmol/L OSU-03012 for 48 h. Arrowheads, autophagosomes. Bars, 2 μm (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 μmol/L OSU-03012 for 48 h (b) and examined under a fluorescence microscope. Middle, Huh7 cells were treated with HBSS for 4 h and 1 or 5 μmol/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 μmol/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.

Figure 3.

OSU-03012–induced autophagy formation. A, top, acridine orange staining. Huh7 cells were treated with or without 5 μmol/L OSU-03012 for 48 h and then stained with acridine orange (1 μg/mL) and examined under a fluorescence microscope. Bottom, MDC staining. Huh7 cells were treated with or without 5 μmol/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 MDC incorporation in Huh7 cells. At least 100 control or OSU-03012–treated (5 μmol/L, 48 h) cells were examined under a fluorescence microscope and the percentage of 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 shows the ultrastructure of Huh7 cells treated without (a and b) or with (c and d) 5 μmol/L OSU-03012 for 48 h. Arrowheads, autophagosomes. Bars, 2 μm (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 μmol/L OSU-03012 for 48 h (b) and examined under a fluorescence microscope. Middle, Huh7 cells were treated with HBSS for 4 h and 1 or 5 μmol/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 μmol/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.

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MAP1-LC3, a mammalian homologue of Apg8p/Aut7p, is essential for amino acid starvation-induced autophagy and is associated with the formation of the autophagosome membrane (20, 21). To further confirm whether OSU-03012 could induce autophagy in Huh7 cells, immunofluorescent staining was used to detect the intracellular distribution of LC3. As shown in Fig. 3D (top), whereas a diffused distribution of LC3 was observed in control cells, a punctuate pattern of LC3 was observed in OSU-03012–treated cells. To elucidate whether OSU-03012 induced a specific or nonspecific autophagy in HCC cells (22), Huh7 cells were incubated in HBSS for 4 hours. As shown in Fig. 3D (middle), Western blotting analysis showed a significantly increased expression of cleaved LC3 in OSU-03012–treated Huh7 cells, whereas marginal cleavage of LC3 was observed in HBSS-treated cells. These results suggested that OSU-03012 induces specific autophagy in Huh7 cells. Treatment with OSU-03012 also caused cleavage of LC3 protein in Hep3B and HepG2 cells (Fig. 3D , bottom). These results indicated that OSU-03012 induces autophagy 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 involved in autophagosome formation (23), and a recent study has shown that OSU-03012 increases the expression of ATG5 and promotes ATG5-dependent formation of LC3 (24). We therefore examined whether OSU-03012 increased ATG5 and ATG7 protein levels in Huh7 cells. As shown in Fig. 4A, OSU-03012 had little effect on ATG7 expression but enhanced the expression of ATG5 in a dose- and time-dependent manner. Further, silencing of ATG5 by siRNA reduced OSU-03012–induced LC3 cleavage, indicating that ATG5 was involved in OSU-03012–induced autophagy (Fig. 4B). Recent studies showed that 3-methyladenine (3-MA), an inhibitor of phosphatidylinositol 3-kinase, could inhibit autophagy (18). We further used 3-MA to show that OSU-03012 induced autophagy in Huh7 cells. As shown in Fig. 4C (left), OSU-03012–induced accumulation of MDC in the cytoplasmic vacuoles was attenuated by treatment with 2 mmol/L 3-MA. Quantitative analysis showed that 2 mmol/L 3-MA reduced OSU-03012–induced autophagy from 62% to 36% of cells (Fig. 4C,, right). To investigate whether inhibition of autophagy affects the cytotoxicity of OSU-03012, Huh7 cells were treated with various doses of OSU-03012 for 72 hours in the 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 activity of OSU-03012 in HCC cells is partially attributable to the formation of autophagy.

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 μmol/L of OSU-03012 for indicated time intervals. The cell lysates were subjected to Western blotting using indicated antibodies. Stains of β-actin served as loading control. B, the expression of ATG5 in Huh7 cells was knock down by siRNA transfection as described in Materials and Methods. The cells were treated with 1 or 5 μmol/L of OSU-03012 for 48 h. The cell lysates were subjected to Western blotting using indicated antibodies. Stains of β-actin served as loading control. C, left, MDC staining. Huh7 cells were untreated (a), treated with 1 μmol/L OSU-03012 (b), treated with 1 μmol/L OSU-03012 and 2 mmol/L 3-MA (c), and treated with 2 mmol/L 3-MA for 48 h (d); stained with MDC; and then observed under a fluorescence microscope. Right, quantification of MDC incorporation. At least 100 cells from each treatment group were examined under a fluorescence microscope and the percentage of MDC incorporation in cells was calculated. Columns, mean (n = 3) for each treatment and representative of three independent experiments; bars, SD. D, effect of 3-MA on OSU-03012–induced cytotoxicity. Huh7 cells were treated with OSU-03012 alone or 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.

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 μmol/L of OSU-03012 for indicated time intervals. The cell lysates were subjected to Western blotting using indicated antibodies. Stains of β-actin served as loading control. B, the expression of ATG5 in Huh7 cells was knock down by siRNA transfection as described in Materials and Methods. The cells were treated with 1 or 5 μmol/L of OSU-03012 for 48 h. The cell lysates were subjected to Western blotting using indicated antibodies. Stains of β-actin served as loading control. C, left, MDC staining. Huh7 cells were untreated (a), treated with 1 μmol/L OSU-03012 (b), treated with 1 μmol/L OSU-03012 and 2 mmol/L 3-MA (c), and treated with 2 mmol/L 3-MA for 48 h (d); stained with MDC; and then observed under a fluorescence microscope. Right, quantification of MDC incorporation. At least 100 cells from each treatment group were examined under a fluorescence microscope and the percentage of MDC incorporation in cells was calculated. Columns, mean (n = 3) for each treatment and representative of three independent experiments; bars, SD. D, effect of 3-MA on OSU-03012–induced cytotoxicity. Huh7 cells were treated with OSU-03012 alone or 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.

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ROS mediated OSU-03012–induced autophagy formation and autophagic cell death. Recent study showed that ROS could induce autophagy formation in certain types of cancer cells (14, 25). Therefore, we investigated whether OSU-03012 treatment could increase the ROS level in HCC cells. Using H2DCFDA-based detection by flow cytometry, ROS accumulation was observed from 6 hours and increased 2-fold after 24 hours of OSU-03012 treatment in Huh7 cells (Fig. 5A,, left). ROS accumulation was also detected in Hep3B and HepG2 cells (data not shown). This result was further confirmed by fluorescence microscopy with DCF staining. The ROS scavenger N-acetylcysteine (NAC) at 10 mmol/L or 4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt (Tiron) at 5 mmol/L abrogated OSU-03012–induced DCF accumulation (Fig. 5A , right).

Figure 5.

ROS generation is associated with OSU-03012–induced autophagy formation and cytotoxicity. A, ROS generation. Left, Huh7 cells were treated with 5 μmol/L OSU-03012 for 6, 12, 18, or 24 h or treated with H2O2 for 30 min and stained with 25 μmol/L DCF for 30 min and analyzed by flow cytometry. The values shown are the fold increase of DCF mean fluorescence intensity (DCF F.I.). Right, Huh7 cells were treated without (a and e) or with 5 μmol/L OSU-03012 (b and f), 5 μmol/L OSU-03012 and 10 mmol/L NAC (c and g), or 5 μmol/L OSU-03012 and 5 mmol/L Tiron (d and h) for 24 h and stained with 25 μmol/L DCF for 30 min and then observed under a fluorescence microscope. B, effects of antioxidants on OSU-03012–induced autophagy formation. Huh7 cells were exposed to 1 μmol/L OSU-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 in previous figures. C, Huh7 cells were treated with OSU-03012 (1 and 5 μmol/L) and 10 mmol/L NAC for 24 h. Cell lysates were resolved in SDS-PAGE and probed with specific antibody against MAP1-LC3. D, effects of antioxidants on OSU-03012–induced cytotoxicity. Huh7 cells were exposed to different doses of OSU-03012 with 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.

Figure 5.

ROS generation is associated with OSU-03012–induced autophagy formation and cytotoxicity. A, ROS generation. Left, Huh7 cells were treated with 5 μmol/L OSU-03012 for 6, 12, 18, or 24 h or treated with H2O2 for 30 min and stained with 25 μmol/L DCF for 30 min and analyzed by flow cytometry. The values shown are the fold increase of DCF mean fluorescence intensity (DCF F.I.). Right, Huh7 cells were treated without (a and e) or with 5 μmol/L OSU-03012 (b and f), 5 μmol/L OSU-03012 and 10 mmol/L NAC (c and g), or 5 μmol/L OSU-03012 and 5 mmol/L Tiron (d and h) for 24 h and stained with 25 μmol/L DCF for 30 min and then observed under a fluorescence microscope. B, effects of antioxidants on OSU-03012–induced autophagy formation. Huh7 cells were exposed to 1 μmol/L OSU-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 in previous figures. C, Huh7 cells were treated with OSU-03012 (1 and 5 μmol/L) and 10 mmol/L NAC for 24 h. Cell lysates were resolved in SDS-PAGE and probed with specific antibody against MAP1-LC3. D, effects of antioxidants on OSU-03012–induced cytotoxicity. Huh7 cells were exposed to different doses of OSU-03012 with 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.

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We then examined whether ROS plays a role in OSU-03012–induced autophagy. Formation of autophagy induced by OSU-03012 was suppressed by 10 mmol/L NAC treatment (Fig. 5B). Cleavage of LC3 protein induced by OSU-03012 was also decreased by treatment with 10 mmol/L NAC (Fig. 5C). Further, both NAC and Tiron partially reduced the cytotoxicity of OSU-03012 in Huh7 cells (Fig. 5D). Taken together, these results suggested that ROS play an important role in OSU-03012–induced autophagy formation and autophagic cell death.

OSU-03012 induces autophagy and suppresses Huh7 xenograft growth in vivo. To assess the antitumor potential of OSU-03012 in HCC, nude mice bearing established s.c. Huh7 tumor xenografts were gavaged with OSU-03012 daily for 28 days at two different doses, 100 and 200 mg/kg body weight, or with vehicle only. Compared with vehicle-treated controls, 100 and 200 mg/kg OSU-03012 treatments suppressed Huh7 tumor growth by 39.52% and 57.59% after 28 days of treatment, respectively (P < 0.05 and 0.01; Fig. 6A,, left). Tumor volumes were significantly reduced by OSU-03012 treatment compared with vehicle-treated control (Fig. 6A , right).

Figure 6.

OSU-03012 suppresses Huh7 xenograft growth and induces autophagy in vivo. Nude mice bearing established Huh7 tumor xenografts were randomly divided 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 of 0.5% 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, and 200 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 treatment for 4 d (c and d). Arrowheads, autophagosomes. Bars, 2 μm (a and c) and 550 nm (b and d). C, TUNEL staining. After treatment with 100 and 200 mg/kg body weight 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 μm. D, OSU-03012 induced autophagy in xenograft. The lysates were prepared from randomly selected xenografts treated with 100 and 200 mg/kg body weight of OSU-03012 and then were Western blotted with indicated antibodies. Stains of β-actin served as loading control.

Figure 6.

OSU-03012 suppresses Huh7 xenograft growth and induces autophagy in vivo. Nude mice bearing established Huh7 tumor xenografts were randomly divided 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 of 0.5% 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, and 200 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 treatment for 4 d (c and d). Arrowheads, autophagosomes. Bars, 2 μm (a and c) and 550 nm (b and d). C, TUNEL staining. After treatment with 100 and 200 mg/kg body weight 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 μm. D, OSU-03012 induced autophagy in xenograft. The lysates were prepared from randomly selected xenografts treated with 100 and 200 mg/kg body weight of OSU-03012 and then were Western blotted with indicated antibodies. Stains of β-actin served as loading control.

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To study the mechanism of tumor suppression effect in this xenograft model, electron microscopy was performed. Mice treated with 200 mg/kg OSU-03012 or untreated for 4 days were sacrificed and the ultrastructures of tumor cells were observed (Fig. 6B). Compared with vehicle-treated tumor cells, autophagosomes and dense 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). To further support that OSU-03012 induces autophagy in xenograft, Western blotting of cleaved LC3 was performed. As shown in Fig. 6D, OSU-03012 increased cleaved LC3, indicating the formation of autophagy in OSU-03012–treated xenograft.

This study showed that treatment with OSU-03012, a novel celecoxib derivative, induces autophagy but not apoptosis in human HCC Huh7 cells. We showed that OSU-03012 inhibits growth within a low micromolor range in Huh7, Hep3B, and HepG2 cells. OSU-03012–induced autophagy as well as cytotoxicity were partially reversed by 3-MA, an autophagy inhibitor. Intracellular ROS generation contributed to OSU-03012–induced autophagy and subsequent autophagic cell death. The xenograft tumor model showed that OSU-03012 suppressed Huh7 tumor growth. These findings suggest that autophagy is a mechanism that contributes to the 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, only very limited PDK1 and AKT inhibition were detected in OSU-03012–treated Huh7 cells in this study, suggesting that OSU-03012 suppresses the growth of Huh7 cells through a mechanism different from PDK1 and AKT inhibition. OSU-03012 was found to trigger autophagic cell death instead of apoptotic cell death in HCC cells. We speculate that suppression of AKT activity may be necessary for OSU-03012–induced apoptosis in some certain tumor cells. OSU-03012–induced autophagic cell death is a unique cellular response in HCC cells. The underlying mechanisms of OSU-03012 effects on HCC cells remain largely unknown.

The significance of the autophagic process in antitumor therapeutics has not been clearly established. To adapt to adverse conditions induced by stress from anticancer therapies, cancer cells may trigger an autophagic response that sequesters and degrades unnecessary molecules to promote cell adaptation and survival. For example, temozolomide induces autophagy in malignant glioma cells as a self-defense (18). Suppression of autophagy leads to apoptosis in glioma cells and thus enhances the antitumor effect of cancer treatment. Therefore, autophagy may be a survival mechanism of cancer cells (29, 30). On the other hand, many anticancer agents, including arsenic trioxide, rapamycin, and ionizing radiation, have been reported to induce autophagic cell death, which is distinguishable from apoptosis in cancer cells, indicating that autophagy might be a crucial mechanism of cancer cell death by these agents (10, 16, 31). The postulated mechanisms for autophagic cell death include either the autophagic digestion of important cytoplasmic factors or the selective degradation of regulatory molecules or organelles that are crucial for survival (9). Whether autophagy induced by OSU-03012 in Huh7 cells is a cell death mechanism or a protective mechanism was elucidated in this study by applying 3-MA, an autophagy inhibitor. The results showed that 3-MA could reverse the cytotoxic effect of OSU-03012 on Huh7 cells, suggesting that OSU-03012 induces an autophagic cell death process in Huh7 cells.

Our data showed that OSU-03012–induced ROS generation contributes to autophagy formation and subsequent autophagic cell death in HCC cells. First, OSU-03012–induced ROS generation was detected as early as 6 hours and had increased 2-fold after 24 hours of treatment, at which time autophagy was clearly observed. Second, treatment with the ROS scavenger NAC reduced the percentage of OSU-03012–induced MDC accumulation in autophagic cells as well as the amount of cleaved LC3 protein. Third, cytotoxicity of OSU-03012 was reversed in the presence of NAC and Tiron. These results suggest that the generation of ROS triggers OSU-03012–induced autophagy formation and autophagic cell death. ROS generation is an important mediator of many anticancer agent–induced cell deaths. A recent study reported that ROS-induced autophagy contributed to cell death in the transformed cell line HEK293 and the cancer cell lines U87 and HeLa but not in nontransformed mouse astrocytes (32). Park and colleagues (24) also reported that OSU-03012 promoted autophagy in transformed cells through a PERK-dependent pathway. Whether ROS accumulation induced by OSU-03012 might cause PERK activation remains to be elucidated. However, ROS is very likely to damage some sets of proteins, which may result in endoplasmic reticulum stress and PERK activation (33, 34). The mechanism by which OSU-03012 induces ROS accumulation remains unknown. It has been reported that the accumulation of ROS may result from decreased ROS degradation, either selective degradation of catalase (35) or inhibition of superoxide dismutase 2 (SOD2; ref. 32). Further investigation of the role of the SOD-catalase antioxidant system in OSU-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 in other cell types after treatment with different antitumor agents. For example, resveratrol (36), soybean B-group triterpenoid saponins (37), and nano neodymium oxide (38) have been reported to cause increased S-phase population and subsequent autophagy formation. However, the relationship between increased S-phase population 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 vitro findings, electron microscopy showed autophagy formation in xenograft tumor cells. TUNEL assay found no evidence of apoptosis after OSU-03012 treatment. Previous pharmacokinetic studies showed that the peak serum concentration of OSU-03012 after oral administration at 200 mg/kg exceeded 20 μmol/L (5). During the treatment period in this study, almost all animals survived and exhibited no apparent signs of toxicity, indicating that oral administration of OSU-03012 delivered sufficient quantities of drug to 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 in HCC and that this autophagy-inducing activity was at least partially related to ROS accumulation. This study shows a novel biological effect of OSU-03012, which supports its clinical potential as a component of therapeutic strategies for HCC.

No potential conflicts of interest were disclosed.

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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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