© The Author 2006. Published by Oxford University Press.
ARTICLE |
Autophagic Cell Death of Malignant Glioma Cells Induced by a Conditionally Replicating Adenovirus
Affiliations of authors: Departments of Neurosurgery (HI, HA, YK, EI, AI, KF, FFL, RS, S. Kondo) and Biostatistics and Applied Mathematics (KRH), University of Texas M. D. Anderson Cancer Center, Houston; Department of Gastroenterology, Hepatology, and Endocrinology, Medical School of Hannover, Hannover, Germany (FK, S. Kubicka, TW); University of Texas Graduate School of Biomedical Sciences at Houston, Houston (S. Kondo); Department of Neurosurgery, Baylor College of Medicine, Houston, TX (RS, S. Kondo)
Correspondence to: Seiji Kondo, MD, PhD, Department of Neurosurgery, BSRB Unit 1004, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030 (e-mail: seikondo{at}mdanderson.org).
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ABSTRACT |
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Background: Conditionally replicating adenoviruses (CRAds) can be engineered to replicate selectively in cancer cells and cause cancer-specific cell lysis; thus they are considered a promising cancer therapy. Methods: To elucidate the mechanisms by which CRAds induce cancer-specific cell death, we infected normal human fibroblasts (MRC5, telomerase negative), human malignant glioma (U373-MG and U87-MG), human cervical cancer (HeLa), and human prostate cancer (PC3) cells (all telomerase positive) with CRAds regulated by the human telomerase reverse transcriptase promoter (hTERT-Ad) or control nonreplicating adenoviruses (Ad-GFP). Nonapoptotic autophagy was assessed in Ad-GFP- and hTERT-Ad–infected cells by examining cell morphology, the development of acidic vesicular organelles, and the conversion of microtubule-associated protein 1 light chain 3 from the cytoplasmic form to the autophagosome membrane form; signaling via mammalian target of rapamycin (mTOR), an autophagy-associated molecule, was monitored by western blot analysis. We also compared the growth of subcutaneous gliomas in nude mice that were treated by intratumoral injection with Ad-GFP or hTERT-Ad. Survival of athymic mice carrying intracranial gliomas treated by intratumoral injection with Ad-GFP or hTERT-Ad was compared by using the Kaplan–Meier method and the Cox–Mantel log-rank analysis. All statistical tests were two-sided. Results: hTERT-Ad induced tumor-specific autophagic cell death in tumor cells and in subcutaneous gliomas. hTERT-Ad–induced autophagy was associated with hTERT-Ad infection kinetics. The mTOR signaling pathway was suppressed in tumor cells and in subcutaneous gliomas treated with hTERT-Ad compared with GFP-Ad or no treatment as shown by reduced phosphorylation of mTOR's downstream target p70S6 kinase (p70S6K). hTERT-Ad treatment of mice (n = 7) slowed growth of subcutaneous gliomas (mean tumor volume = 39 mm3, 95% confidence interval [CI] = 23 to 54 mm3) compared with GFP-Ad treatment (n = 7) (mean tumor volume = 200 mm3, 95% CI = 149 to 251 mm3) at day 7 (volume difference = 161 mm3, 95% CI = 126 to 197 mm3; P<.001). Mice carrying intracranial tumors that were treated with three intratumoral injections of hTERT-Ad survived longer (53 days) than after treatment with GFP-Ad (29 days) (seven mice per group, difference = 24 days, 95% CI = 20 to 28 days; P<.001). Conclusions: hTERT-Ad may kill telomerase-positive cancer cells by inducing autophagic cell death.
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INTRODUCTION |
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TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Malignant gliomas are the most common neoplasm in the central nervous system (1,2). The average life expectancy of patients with a diagnosis of glioblastoma multiforme, the most malignant glioma, after conventional treatment with surgery,
-irradiation, and chemotherapy is less than 1 year. Consequently, there is an urgent need to develop new therapeutic strategies. Conditionally replicating adenoviruses (CRAds) are emerging as a promising tool in cancer therapy (3–5). Because of their capability to multiply, lyse infected tumor cells, and spread to surrounding cells, CRAds may have better antitumor efficacy than that of nonreplicating adenoviruses. To enhance the tumor specificity of CRAds and thus avoid damage to other tissues, the use of a tumor-specific promoter is desirable. In this regard, the human telomerase reverse transcriptase (hTERT) promoter may help to minimize the adverse effects of CRAds because the hTERT gene is transcriptionally regulated such that only tumor cells with telomerase activity can activate the promoter (6). We and other groups have recently demonstrated the in vitro and in vivo efficacy of the hTERT promoter in delivering therapeutic genes selectively to cancer cells (7–9). The concept of using the hTERT promoter with CRAds is also supported by several studies showing the efficacy of this treatment on various types of cancer cells (10–15).
Malignant gliomas are a particularly attractive target of CRAds that express the adenovirus early (E)1A gene, which induces viral replication, under the control of an hTERT promoter (hTERT-Ad) (6). Most malignant gliomas possess telomerase activity, but normal brain tissues do not (16–18). However, the specific effect of hTERT-Ad on malignant gliomas has not yet been assessed. Furthermore, the specific mechanisms of cell death that are induced by CRAds remain poorly understood, although it is well known that CRAd infection leads to tumor cell lysis.
A recent investigation showed that CRAds cause nonapoptotic programmed cell death in tumor cells (19). Programmed cell death is an important and major terminal pathway for normal development and various diseases, such as cancer (20). Programmed cell death is subdivided into two groups, apoptosis and autophagic cell death (21), based on morphological differences in cytoplasm and nucleus. Therefore, we hypothesized that hTERT-Ad is therapeutically effective for malignant glioma cells by inducing autophagic cell death instead of apoptosis. To test our hypothesis, we treated malignant glioma cells in vitro and in vivo with hTERT-Ad and determined the efficacy of hTERT-Ad and its mechanism of inducing cell death.
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MATERIALS AND METHODS |
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TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Cell Culture
Human malignant glioma U373-MG and U87-MG cells, human cervical cancer HeLa cells, human prostate cancer PC3 cells, and normal fibroblast MRC5 cells were purchased from American Type Culture Collection (Manassas, VA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen), 4 mM glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. U373-MG, U87-MG, HeLa, and PC3 cells were telomerase positive, and MRC5 cells were telomerase negative (7,9).
Adenoviruses
A nonreplicating adenovirus carrying the green fluorescent protein (Ad-GFP) gene was prepared for use as a control, as described previously (22). hTERT-Ad was constructed by replacing the internal adenoviral E1A promoter with a 255-bp hTERT promoter fragment, as described previously (10). An enhanced GFP reporter gene was linked to the E1B transcription site by an internal ribosomal entry site so that the viral replication and spreading of the adenoviral infection could be monitored.
Assays To Measure the Cytopathic and Cytotoxic Effects of Ad-GFP and hTERT-Ad
For determination of virus-mediated cytopathic effects, U87-MG, U373-MG, and MRC5 cells were seeded at 5.0 x 104 cells/mL on Lab-Tek chamber slides and incubated at 37 °C overnight. After infection with Ad-GFP or hTERT-Ad at a multiplicity of infection (MOI) of 1.0 for 48 hours, the cells were fixed with 4% paraformaldehyde, and the slides were photographed under an AX70 fluorescence microscopy (Olympus, Melville, NY). Three replicates were performed for each experiment.
The cytotoxic effects of hTERT-Ad on U87-MG, U373-MG, and MRC5 cells were determined by using a cell proliferation reagent (WST-1; Roche Applied Science, Indianapolis, IN), as described previously (22). The cells were seeded at 1.0 x 104 cells/well in 96-well plates and incubated at 37 °C overnight. After infection with Ad-GFP or hTERT-Ad at an MOI of 1.0 for up to 4 days, the cells were exposed to 10 µL of the WST-1 reagent for 1 hour at 37 °C. The absorbance at 450 nm was measured in a EL808 microplate reader (BIO-TEK Instruments, Winooski, VT). The viability of untreated cells was considered to be 100%. Experiments were repeated three times, each in triplicate.
Hoechst DNA Staining
To detect chromatin condensation and nuclear fragmentation, which are phenotypic characteristics of apoptosis, nuclei were stained with Hoechst 33258, as described previously (22). U373-MG and U87-MG cells infected with Ad-GFP or hTERT-Ad at an MOI of 1.0 for 48 hours were fixed with 4% paraformaldehyde and stained with Hoechst 33258 (0.5 µg/mL; Sigma-Aldrich, St. Louis, MO) for 15 minutes at room temperature. Two hundred cells were counted under an AX70 fluorescence microscope and were scored according to the incidence of apoptotic chromatin changes. Three replicates were performed for each experiment.
Electron Microscopy
U87-MG cells were grown on glass coverslips and were infected with Ad-GFP or hTERT-Ad at an MOI of 1.0 for 48 hours and then fixed with a solution containing 3% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.3) for 1 hour. The samples were then postfixed in 1% OsO4 in the same buffer for 1 hour. Representative areas were chosen for ultrathin sectioning and viewed with an electron microscope (JEM 1010 transmission electron microscope; JEOL, Peabody, MA), as described previously (23). Digital images were also obtained (AMT imaging system; Advanced Microscopy Techniques Corp., Danvers, MA). Three replicates were performed for each experiment.
Quantification of Acidic Vesicular Organelles With Acridine Orange
Autophagy is characterized by the development of acidic vesicular organelles (23–25). The cytoplasm and nucleoli of acridine orange–stained cells fluoresce bright green and dim red, respectively, whereas acidic compartments fluoresce bright red (24). Therefore, autophagy was assessed in U373-MG, U87-MG, HeLa, and PC3 cells by the quantification of acidic vesicular organelles with supravital cell staining using acridine orange, as described previously (23–25). To inhibit autophagy, 1.0 mM 3-MA (Sigma-Aldrich), an inhibitor of the phosphatidylinositol 3-phosphate kinase (PI3K), was added to U373-MG cells the day after infection by Ad-GFP or hTERT-Ad at an MOI of 1.0, as described previously (25). Nonadherent tumor cells and adherent tumor cells that were detached with 0.05% trypsin–EDTA (Invitrogen) were stained with 1.0 µg/mL acridine orange (Sigma-Aldrich) for 15 minutes at room temperature. Stained cells were then analyzed by flow cytometry using the FACScan cytometer (Becton Dickinson, San Jose, CA) and CellQuest software (Becton Dickinson). Three replicates were performed for each experiment.
Western Blotting
Untreated and Ad-GFP- or hTERT-Ad–treated U373-MG, U87-MG, and MRC5 cells (106) and subcutaneous tumors (50 mg) were lysed in extraction buffer (10 mM Tris–HCl, pH 7.8, 1% NP-40, 150 mM NaCl, 1 mM EDTA) and soluble proteins isolated as described previously (22). Protein concentrations were estimated by a protein assay (Bio-Rad, Hercules, CA), and proteins were separated by sodium dodecyl sulfate–7.5% polyacrylamide gel electrophoresis (Bio-Rad) and transferred electrophoretically to Hybond-P membranes (Amersham Biosciences Corp., Piscataway, NJ). The membranes were subjected to western blotting by using the following primary antibodies: a monoclonal mouse anti–
-actin antibody (1 : 500 dilution; Sigma-Aldrich), a monoclonal mouse anti-E1A antibody (1 : 100 dilution; BD Biosciences Pharmingen, San Diego, CA), rabbit polyclonal anti-p70S6 kinase (p70S6K) (1 : 1000 dilution) and anti-phospho-p70S6K (Thr389) antibodies (1 : 1000 dilution; Cell Signaling Technology, Beverly, MA), and a rabbit polyclonal anti–microtubule-associated protein 1 light chain 3 (LC3) antibody (1 : 1000 dilution) (26) (kindly provided by Dr. Tamotsu Yoshimori, National Institute of Genetics, Mishima, Japan). After membranes were incubated with primary antibody in 5% nonfat dry milk–TBS–0.1% Tween-20 overnight at 4 °C, they were washed three times with 1 x TBS–0.1% Tween-20 for 20 minutes and incubated for 1 hour in 1 x TBS–0.1% Tween-20 with a horseradish peroxidase–conjugated anti-mouse or anti-rabbit secondary antibody (1 : 3000 dilution, Amersham) at room temperature for 1 hour. Bound antibody complexes were detected by using an enhanced chemiluminescence reagent (Amersham) according to the manufacturer's instructions. Three replicates were performed for each experiment.
Cell Cycle Analysis
Nonadherent U373-MG and U87-MG cells and adherent U373-MG and U87-MG cells detached with trypsin–EDTA were collected, fixed with 70% ethanol, and stained with a 10% propidium iodide solution (cellular DNA flow cytometric analysis reagent set; Roche) according to the manufacturer's instructions. DNA content was analyzed with a FACScan flow cytometer (Becton Dickinson), and data were analyzed with the manufacturer's software (CellQuest; Becton Dickinson). Three replicates were performed for each experiment.
Tumor Models
For the subcutaneous tumor model, U87-MG cells (1.0 x 106 cells in 20 µL of serum-free DMEM) were inoculated subcutaneously into the right flank of 8- to 12-week-old female nude mice (Experimental Radiation Oncology at M. D. Anderson Cancer Center) (seven mice for each treatment group). Tumor growth was measured daily with calipers. Tumor volume was calculated as (L x W2)/2, in which L is the length and W is the width in millimeters, as described previously (27). When the tumors reached a mean volume of approximately 50 mm3, 10 µL of Ad-GFP (2.2 x 109 plaque-forming units [pfu]/mL) or hTERT-Ad (2.2 x 109 pfu/mL) was injected intratumorally (day 0). Mice were killed by exposure to CO2 on day 7 to compare the short-term effects of Ad-GFP and hTERT-Ad on subcutaneous tumors. The tumors were then removed and frozen rapidly, and 10-µm–thick cryosections were cut to detect adenoviral GFP and hexon proteins. The sections were fixed onto glass slides with 95% ethanol for 15 minutes and treated with a mouse monoclonal anti-GFP antibody (1 : 1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) or a goat polyclonal anti-adenoviral hexon protein (1 : 100 dilution; Fitzgerald Industries International, Concord, MA) in 0.5% Triton X-100–phosphate-buffered saline (PBS) overnight at 4 °C. The slides were then washed with PBS and incubated with an Alexa Fluor 488–labeled secondary goat anti-mouse antibody or an Alexa Fluor 594-labeled secondary donkey anti-goat antibody (1 : 500 dilution; Molecular Probes, Eugene, OR) for 1 hour at room temperature. The slides were washed again with PBS and monitored under an AX70 fluorescence microscope.
For the intracranial tumor model, the mice (five to seven mice for each treatment group) were anesthetized with intraperitoneal injection of ketamine (100 mg/kg of body weight) and xylazine (5 to 16 mg/kg) as described previously (28). A 0.9-mm burr hole was then drilled into the skulls of the mice 1.0 mm anterior and 2.5 mm lateral to the bregma to expose the dura. A screw guide with a 0.5-mm central hole and a stylet were placed into the burr hole as described previously (29). Three days after the screw guide was set in the skull, a 10-µL syringe (Hamilton Co., Reno, NV) fitted with a 26-gauge needle was connected to a microinfusion pump (Harvard Apparatus Co., Cambridge, MA). Through the screw guide, at a depth of 3.5 mm from the skull (corresponding to the region of the caudate nucleus), 10-µL aliquots of 5 x 105 U87-MG cells in serum-free DMEM were inoculated at a rate of 1.0 µL/min. In a preliminary study to determine when to initiate the treatment to best simulate the clinical situation, U87-MG cells expressing GFP were inoculated into the brain of nude mice as described above. GFP-positive tumor cells (approximately 1.2 mm in diameter) were detected in the cerebral hemisphere under a fluorescence microscope 3 days after tumor inoculation. Therefore, the treatment was initiated 3 days after inoculation of U87-MG cells. On day 3 or on days 3, 5, and 7, a 10-µL Hamilton syringe fitted with a 26-gauge needle was connected to a microinfusion pump. hTERT-Ad (2.2 x 109 pfu/mL) or Ad-GFP (2.2 x 109 pfu/mL) in 10 µL of sterile PBS was then infused into the tumors through the screw guide, also at a depth of 3.5 mm from the skull, with a microinfusion pump at a rate of 0.5 µL/min. For histologic analysis, mice were deeply anesthetized and then were killed by intracardiac perfusion–fixation. The brain was removed, sliced coronally into five blocks, and snap-frozen. Coronal sections (8–10 µm) were then made. All mouse studies were performed in the veterinary facilities of the University of Texas M. D. Anderson Cancer Center in accordance with institutional, state, federal, and ethical regulations for experimental animal care.
Statistical Analysis
The data were expressed as means and 95% confidence intervals (CIs). The statistical significance of the differences in the in vitro and in vivo antitumor effects of hTERT-Ad and Ad-GFP was determined by using the Student's t test (two-tailed). The antitumor effect of Ad-GFP and hTERT-Ad on intracranial tumors in nude mice was assessed by plotting survival curves according to the Kaplan–Meier method (30). Survival in different treatment groups was compared by using the Cox–Mantel log-rank test (31). P values less than .05 were considered statistically significant.
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RESULTS |
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TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In Vitro Tumor-Specific Cytopathic and Cytotoxic Effects of hTERT-Ad
To determine whether hTERT-Ad infection causes tumor-specific cytopathic effects, we infected U87-MG and U373-MG malignant glioma cells and MRC5 fibroblasts with hTERT-Ad or Ad-GFP at an MOI of 1.0 for 48 hours. Cell enlargement and rounding were observed in hTERT-Ad–infected U87-MG and U373-MG cells, which also exhibited hTERT-Ad–derived GFP fluorescence (>90% infection rate) (Fig. 1, A). In contrast, tumor cells and MRC5 cells infected with Ad-GFP showed normal morphology, as did MRC5 cells infected with hTERT-Ad. E1A expression was strikingly high in U87-MG and U373-MG cells but low in MRC5 cells after infection with hTERT-Ad (Fig. 1, B). There was no detectable E1A expression in either tumor cells or MRC5 cells infected with Ad-GFP. These results indicated that hTERT-Ad replication occurs in malignant glioma cells but not in normal cells and leads to tumor-specific cytopathic effects.
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-actin antibody was used as a control for protein loading and transfer. Three replicates were performed for each experiment. C) Tumor-selective cytotoxicity of hTERT-Ad. After infection with Ad-GFP or hTERT-Ad at an MOI of 1.0 for 2 or 4 days, the cell viability was measured by using the WST-1 agent (Roche Applied Science, Indianapolis, IN). Data are presented as a percentage of the cell viability of untreated cells. U373-MG (squares), U87-MG (triangles), and MRC (diamonds) cells treated with Ad-GFP or hTERT-Ad, open and closed symbols, respectively. Error bars indicate 95% confidence intervals of three independent experiments performed in triplicate. *, P<.001 for Ad-GFP–treated versus hTERT-Ad–treated cells, two-sided t test (analysis of variance). D) Apoptosis in hTERT-Ad– and Ad-GFP–infected U373-MG and U87-MG cells. Cells were fixed with 4% paraformaldehyde and stained with Hoechst 33258 (0.5 µg/mL) for 15 minutes at room temperature. After Hoechst staining, no notable apoptotic cells were evident in tumor cells treated with Ad-GFP or hTERT-Ad (bottom), but green fluorescent protein fluorescence was evident (top). Scale bar = 10 mm. Three replicates were performed for each experiment.To test whether hTERT-Ad has a tumor-selective cytotoxicity, a cell viability assay was performed using the U373-MG, U87-MG, and MRC cells infected with hTERT-Ad or Ad-GFP at an MOI of 1.0 for 2 or 4 days. The viability of U373-MG or U87-MG cells was inhibited by hTERT-Ad in a time-dependent manner compared with no viral treatment (difference = 51%, 95% CI = 43% to 59% or difference = 44%, 95% CI = 33% to 55%; P<.001 on day 2; and difference = 72%, 95% CI = 70% to 74% or difference = 53%, 95% CI = 47% to 59%; P<.001 on day 4, respectively), whereas Ad-GFP had little cytotoxic effect; in contrast, the viability of MRC5 cells was not reduced by either hTERT-Ad or Ad-GFP (Fig. 1, C). These results indicate that hTERT-Ad at an MOI of 1.0 has tumor-specific cytotoxicity.
To determine whether apoptotic morphology had been induced by hTERT-Ad infection, the nuclei of infected U373-MG and U87-MG tumor cells were stained with Hoechst 33258. Although some tumor cells infected with hTERT-Ad had slightly enlarged nuclei, the typical apoptotic morphology was not detected (Fig. 1, D). Furthermore, DNA flow cytometry revealed that, compared with uninfected cells, fewer hTERT-Ad–infected cells were in the G1 phase and more were in the G2/M phase compared with untreated cells (data not shown); however, the percentage of cells in the sub-G1 phase, which is characteristic of apoptosis, was not increased (Table 1). These results indicated that hTERT-Ad induces G2/M arrest but not apoptosis.
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Induction of Autophagy in Malignant Glioma Cells by hTERT-Ad
Previous studies have shown that various types of cancer cells undergo autophagy but not apoptosis after treatment with radiation or chemotherapy (24,25,32–34). To examine this possibility in glioma cells infected with CRAds, we analyzed the ultrastructure of infected U87-MG cells. U87-MG cells infected with Ad-GFP at an MOI of 1.0 for 48 hours exhibited few autophagic features (Fig. 2, A-a), whereas many autophagic vacuoles and empty vacuoles were observed in U87-MG cells infected with hTERT-Ad at an MOI of 1.0 for 48 hours (Figs. 2, A-b and –c). Most hTERT-Ad–infected cells exhibited viral particles in the nucleus and autophagic features, but neither the chromatin condensation nor the DNA fragmentation that is characteristic of apoptosis.
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Because autophagy is characterized by the development of acidic vesicular organelles (23–25), infected tumor cells were stained with acridine orange, followed by flow cytometry to quantify the development of acidic vesicular organelles. Compared with no treatment, treatment with hTERT-Ad at an MOI of 1.0 for 48 hours increased the percentage of U373-MG cells that fluoresced (untreated mean = 4.2% versus treated mean = 18.8%, difference = 14.6%, 95% CI = 6.6 to 22.6%; P = .025) and U87-MG cells (untreated mean = 3.8% versus treated mean = 13.9%, difference = 10.1%, 95% CI = 8.4 to 11.8%; P<.001) (Table 2). In contrast, there was no statistical difference between untreated and Ad-GFP–infected tumor cells.
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During amino acid starvation–induced autophagy, LC3 becomes localized in autophagosome membranes (26). The LC3 protein exists in two cellular forms: LC3-I and LC3-II. LC3-I, the cytoplasmic form, is processed by enzymatic cleavage into LC3-II, which is associated with the autophagosome membrane. Therefore, an increase in the ratio of LC3-II to LC3-I coincides with autophagosome formation. We thus analyzed the accumulation of LC3-I and LC3-II in Ad-GFP– or hTERT-Ad–infected cells. The ratio of LC3-II to LC3-I was dramatically increased by hTERT-Ad infection in tumor cells but was not changed in normal cells (Fig. 2, B). Infection with Ad-GFP did not change the ratio of LC3-I to LC3-II in either cell type. These results, together with those of the electron microscopic analysis and acridine orange staining, indicate that hTERT-Ad induces autophagy in malignant glioma cells.
Association of Autophagy Induction and hTERT-Ad Infection
To determine the relationship between autophagy induction and hTERT-Ad infection kinetics, we examined the time course of acidic vesicular organelle development and viral replication in U373-MG cells infected at different MOIs. First, we assessed the development of acidic vesicular organelles in tumor cells infected at MOIs of 0.1, 1.0, or 10 for 72 hours. Infection with hTERT-Ad increased the development of acidic vesicular organelles by up to 29.7% (at 10 MOI) in an MOI-dependent manner, whereas the development of acidic vesicular organelles in Ad-GFP–infected tumor cells remained low (Fig. 3, A, top). Also, hTERT-Ad infection, even at an MOI of 0.1 for 72 hours, induced high expression of E1A protein, indicating a high level of viral replication (Fig. 3, A, bottom). Next, we assessed the development of acidic vesicular organelles in infected U373-MG cells at an MOI of 1.0 for 12–72 hours. The development of acidic vesicular organelles by hTERT-Ad, but not by Ad-GFP, increased to 24.5% (72 hours) in a time-dependent manner (Fig. 3, B, top). Expression of E1A increased in a time-dependent manner up to 24 hours after exposure to hTERT-Ad, and the expression level remained high 48 and 72 hours after infection (Fig. 3, B, bottom). These results indicate that hTERT-Ad–induced autophagy is associated with the infection kinetics of hTERT-Ad.
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The activation of mammalian target of rapamycin (mTOR) is closely linked with autophagy (35,36). Therefore, we assessed the effect of hTERT-Ad on the mTOR pathway by measuring the phosphorylation of one of the downstream targets of mTOR, p70S6K. The phosphorylation of p70S6K at position Thr389 in U373-MG cells infected with hTERT-Ad, but not with Ad-GFP, was dramatically reduced, to an undetectable level, in an MOI-dependent manner (Fig. 3, A, bottom). In contrast, although the phosphorylation of p70S6K at position Thr389 in U373-MG cells was reduced at 72 hours after hTERT-Ad infection at an MOI of 1.0 (Fig. 3, B, bottom), it remained detectable. Phosphorylated p70S6K was also reduced in U87-MG cells infected with hTERT-Ad compared with uninfected cells (data not shown). These results suggest that the mTOR signaling pathway is suppressed by infection with hTERT-Ad.
Effect of Inhibition of hTERT-Ad–induced Autophagy on Malignant Glioma Cells
Whether cancer treatment–induced autophagy is an anticancer effect or a protective reaction against the treatment is still controversial (37). To assess the role of hTERT-Ad–induced autophagy, we determined whether inhibition of autophagy affects hTERT-Ad-induced cytotoxicity. Because 3-MA inhibits autophagy by suppressing the formation of the preautophagosomal structure (36,37), U373-MG cells were infected with Ad-GFP or hTERT-Ad at an MOI of 1.0 for 24 hours and then treated with 3-MA (1.0 mM) for additional 48 hours. Treatment with 3-MA suppressed the induction of acidic vesicular organelles in U373-MG cells infected with Ad-GFP (percentage of cells with acidic vesicles, untreated mean = 7.4% versus 3-MA mean = 3.3%, difference = 4.1%, 95% CI = 0.5 to 7.7%) and hTERT-Ad (untreated mean = 34.2% versus 3-MA mean = 6.2%, difference = 28%, 95% CI = 25.8 to 30.2%), respectively (Fig. 4, A). The decreased viability of U373-MG cells infected with hTERT-Ad was reversed after autophagy was inhibited by 3-MA (difference = 18.3%, 95% CI = 15.7 to 20.9%; P<.001; Fig. 4, B). However, E1A expression induced by hTERT-Ad was not influenced by the inhibition of autophagy (Fig. 4, C). These results suggest that hTERT-Ad–induced autophagy is an antitumor effect, not a defensive mechanism for viral replication.
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Induction of Autophagy in Other Cancer Cells by hTERT-Ad
To determine whether hTERT-Ad induces autophagy in other types of cancer cells, HeLa (cervical cancer) and PC3 (prostate cancer) cells were infected with Ad-GFP or hTERT-Ad at an MOI of 1.0 for 48 hours. After infection with hTERT-Ad, HeLa cells (percentage of cells with acidic vesicles, untreated mean = 4.1% versus hTERT-Ad mean = 15.8%, difference = 11.7%, 95% CI = 5.6 to 17.8%; P = .039) and PC3 cells (untreated mean = 6.9% versus hTERT-Ad mean = 18.2%, difference = 11.3%, 95% CI = 10.3 to 12.3%; P = .043) developed more acidic vesicular organelles than untreated cells (Table 3). In contrast, treatment with Ad-GFP did not increase the percentage of acidic vesicular organelles in either tumor cell type. These results suggest that autophagy is a common feature of cancer cells infected with hTERT-Ad.
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In Vivo Antitumor Effect of hTERT-Ad
To investigate the therapeutic potential of hTERT-Ad, athymic mice (groups of seven) carrying subcutaneous U87-MG tumors were treated with one intratumoral injection of Ad-GFP or hTERT-Ad. Treatment with hTERT-Ad inhibited tumor growth (mean tumor volume = 39 mm3, 95% CI = 23 to 54 mm3), whereas tumors continued to grow after Ad-GFP infection (mean tumor volume = 200 mm3, 95% CI = 149 to 251 mm3) at day 7 (volume difference = 161 mm3, 95% CI = 126 to 197 mm3; P<.001) (Fig. 5, A). Also, as indicated by expression of the GFP and the adenoviral hexon protein, hTERT-Ad spread through the tumor tissue, whereas Ad-GFP did not (Fig. 5, B). LC3-I expression and conversion to LC3-II was higher in subcutaneous gliomas of hTERT-Ad–treated than in Ad-GFP–treated mice (Fig. 5, C), indicating that hTERT-Ad stimulated not only the conversion of LC3-I into LC3-II but also the synthesis of the LC3-I protein. The expression pattern of LC3-I and LC3-II proteins in cultured cells (Fig. 2, B) and subcutaneous gliomas (Fig. 5, C) was different and the mechanisms unknown. Moreover, the phosphorylation of p70S6K at position Thr389 was inhibited in subcutaneous tumors that were treated with hTERT-Ad compared with those infected with Ad-GFP. These results indicated that intratumoral injection of hTERT-Ad suppresses the mTOR signal and induces autophagy, leading to the growth inhibition of subcutaneous tumors.
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To determine the potential of hTERT-Ad as a therapy for malignant gliomas, we also injected hTERT-Ad or Ad-GFP into intracranial tumors of nude mice and compared their duration of survival. Mice treated with one or three injections of hTERT-Ad survived longer (mean = 40 or 53 days) than mice treated with Ad-GFP (mean = 32 or 29 days) (difference = 8 days, 95% CI = 2 to 14 days, P = .019; and difference = 24 days, 95% CI = 20 to 28 days, P<.001, respectively) (Fig. 5, D). The intracranial tumors of mice treated with Ad-GFP grew extensively, and the midline was shifted laterally by the mass effect (Fig. 5, E-a). In brain tissues harvested from two mice that survived 60 days after tumor inoculation, the place where a screw guide with a stylet had been set was evident, but tumors were undetectable (Fig. 5, E-b). Those two mice did not show weight loss or neurologic deficits, such as hemiparesis or seizure. However, to conclude that systemic toxicity or neurotoxicity caused by hTERT-Ad is minimal, we need to determine the lethal dose of hTERT-Ad that causes the death of 50% of tested mice (LD50) in this model and assess the toxicity histopathologically. The results indicate the usefulness of hTERT-Ad for treating animal models of human gliomas.
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DISCUSSION |
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TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this study, we demonstrated the effectiveness of hTERT-Ad for treating malignant glioma cells in vitro and in vivo. Intratumoral injections of hTERT-Ad suppressed the growth of subcutaneous tumors in nude mice and prolonged the survival of mice with intracranial gliomas. Intriguingly, hTERT-Ad induced tumor-specific autophagic cell death through a process that appeared to involve the suppression of the mTOR signaling pathway. The findings that cervical and prostate cancer cells also underwent autophagy after hTERT-Ad infection raises the possibility that this type of cell death is the common feature of cancer cells infected with hTERT-Ad.
The use of oncolytic viruses to treat malignant gliomas has proven to be promising (38–42). Engineered, tumor-selective herpes simplex type 1 virus (HSV-1) mutants (43), E1B-55 kDa–deleted ONYX-015 adenovirus (41), and E1A 24 bp–deleted Delta 24 adenovirus (44) have all been preclinically or clinically investigated. The behavior of these viruses varies when used to target malignant gliomas. For example, HSV-1 mutants replicate in actively dividing but not in terminally differentiated cells, whereas ONYX-015 selectively replicates in and lyses cancer cells that have a defect in p53 or the p53 pathway, and tumors with a defective pRB pathway are sensitive to the E1A Delta 24. hTERT-Ad might have greater therapeutic efficacy in malignant glioma than other oncolytic viruses for the following reasons. First, almost all malignant glioma cells, but not normal brain cells, express telomerase (16–18). Second, patients with telomerase-positive glioblastoma multiforme have poorer prognoses than do patients with the telomerase-negative form (45). Third, hTERT-Ad–mediated oncolysis is more efficient than that induced by ONYX-015 (10).
Little is known about the mechanisms of oncolytic virus–induced cell death. In the hTERT-Ad system, the hTERT promoter directs the expression of the adenoviral E1A gene, enabling hTERT-Ad to replicate specifically in telomerase-positive cancer cells. Adenoviral E1A gene products stimulate S-phase entry and transactivate both viral and cellular genes that are essential for viral replication (46). E1A also has the ability to induce caspase-3–associated apoptosis independent of p53 (47). However, in this study, we observed autophagy instead of apoptosis not only in malignant glioma cells but also in other types of cancer cells that were infected with hTERT-Ad. Therefore, we speculate that autophagy is the type of cell death that cancer cells infected with CRAds undergo, a conclusion that is supported by a recent investigation showing induction of nonapoptotic cell death by CRAds (19).
Autophagic cell death is a type of programmed cell death that is an alternative to apoptosis (21,48). In general, autophagy, a type of protein degradation system (49), is prominently observed in cells experiencing environmental stress, such as amino acid starvation. Some studies have also shown that cells undergo autophagy in response to viral and bacterial infection (50–52). Because autophagy generally occurs in cells so they can survive unfavorable conditions (36), it may be triggered in infected cancer cells as a defense mechanism to protect against intracellular pathogens. However, there are two possibilities in the role of virus-induced autophagy: that autophagy might result either from the use of autophagy by the viral replication complex or the destruction of the viral structures by the autophagic machinery (53). On the other hand, the recent evidence of the role of tumor suppressors such as Beclin 1 in autophagy suggests that reduction in the autophagic process increases tumor incidence (54–56). Thus, the induction of autophagy may have a therapeutic value. Indeed, in our study, inhibition of autophagy by 3-MA attenuated the cytotoxicity of hTERT-Ad but did not affect viral replication, suggesting that hTERT-Ad-induced autophagy is one of the antitumor effects. Therefore, we speculate that hTERT-Ad–induced autophagy causes the demise of the cells instead of being a protective mechanism that is activated during cellular distress.
The involvement of mTOR in the regulation of the autophagic pathway is well established (35,36). mTOR functions downstream from the PI3K–Akt signaling pathway (57) and is commonly activated in malignant gliomas by the upstream receptor tyrosine kinases (58). In response to nitrogen sources or amino acids, mTOR regulates both transcription and translation, presumably mediated through p70S6K and eukaryotic initiation factor 4E-binding protein 1. In line with this mechanism, rapamycin, an inhibitor of mTOR, inhibits the proliferation of malignant glioma cells by inducing autophagy (59). Inhibition of autophagy suppresses rapamycin's cytotoxicity (unpublished data), whereas adenoviral E1A expression inactivates Akt in cancer cells (60). Given these observations, we suggest that hTERT-Ad inactivates mTOR and then triggers the autophagic process, directly or indirectly leading to cell death. Of course, the autophagic pathways consist of various signals that transmit extracellular or intracellular stimuli to regulate the molecular machinery (36,37,49). Therefore, further studies may identify other molecules that have a central role in hTERT-Ad–induced autophagy.
The results of a recent study, in which adenovirus E4-ORF1 proteins were used to regulate replication (61), conflict with our findings. In that study, the mTOR pathway was activated during viral replication in quiescent primary epithelial cells and in osteosarcoma and colon cancer cells. The conflicting results might be due to the fact that the cells were cultured without serum to abolish p70S6K phosphorylation. Thus, the mTOR pathway appeared activated after replication, compared with the baseline inactivated state. However, in our study the mTOR pathway was constitutively activated (cells were cultured in 10% serum). This situation also leads to a potential limitation of our study, that is, because we cultured the cells with 10% serum; therefore, we could not eliminate the nonspecific effect of serum on the mTOR pathway.
In conclusion, we found that hTERT-Ad inhibits the in vitro and in vivo proliferation of malignant glioma cells by causing hTERT-Ad–infected malignant glioma cells to undergo autophagic cell death via inhibition of the mTOR signal. We also observed this autophagy in other telomerase-positive cancer cells. Our findings thus provide a strong rationale for using hTERT-Ad to specifically kill cancer cells that have telomerase activity. One major weakness of hTERT-Ad treatment is that adenoviral infection efficiency depends on the expression of the coxsackievirus and adenovirus receptor, which is not highly expressed on the cell surface of many types of cancer cells (62). Because infection efficiency of hTERT-Ad is enhanced by a tropism modification, the insertion of an integrin-binding peptide (Arg-Gly-Asp) into the HI loop of the adenoviral fiber knob (63), we will assess the efficacy of this modification in treating mice with intracranial tumors in the future.
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TopNotesAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Supported in part by the grants CA-088936 and CA-108558 from the National Cancer Institute (S. Kondo), by a startup fund from the University of Texas M. D. Anderson Cancer Center (to S. Kondo), by a generous donation from the Anthony D. Bullock III Foundation (to Y. Kondo, R. Sawaya, and S. Kondo), and by the cancer center support grant/shared resources CA-16672 from the National Cancer Institute. The sponsors had no role in the study design, data collection, analysis, interpretation of the data, or preparation of the manuscript.
We thank Dr. Jerry W. Shay for critical review. We also thank Dr. Tamotsu Yoshimori for providing the LC3 antibody, Joy Gumin and Emporia F. Hollingsworth for technical support, and Dr. Ellen McDonald for editing the manuscript.
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Manuscript received July 11, 2005; revised February 21, 2006; accepted March 21, 200