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Department of
* Neurosurgery, The University of Texas M. D. Anderson Cancer Center, Houston, Texas, USA;
Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts, USA; Departments of
Biostatistics and Applied Mathematics and
Molecular Therapeutics, The University of Texas M. D. Anderson Cancer Center;
|| Department of Neurosurgery, Baylor College of Medicine; and
¶ The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, Texas, USA
2Correspondence: Department of Neurosurgery, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Unit BSRB 1004, Houston, TX 77030, USA. E-mail: seikondo{at}mdanderson.org.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: T-oligos • mTOR
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Thus, novel treatment strategies for malignant gliomas are needed.
Telomeres are tandem repeats of a specific 5'-TTAGGG-3' nucleotide sequence at the ends of chromosomes (3
, 4)
. Telomerase, a ribonucleic acid-protein complex, adds telomeric repeats to the ends of telomeres to compensate for the progressive loss (5
, 6)
. The essential role of telomeres in protecting chromosome ends from fusion or from being recognized as DNA damage is mediated by a cap of telomere-associated proteins [telomere repeat factors 1 and 2 (TRF1 and 2) (7
8
9)
]. The 3' end of each telomere consists of a single-stranded G-rich overhang that inserts into the proximal double-stranded telomere and stabilizes a loop structure at the chromosome ends (8)
.
When the telomere loop structure is disrupted by expression of dominant-negative TRF2, the telomere 3' overhang is exposed and degraded, and cell death is triggered in certain cell types (9)
. Exposure of the telomere 3' overhang due to the opening of the normal telomere loop structure may be a physiological signal inducing DNA damage responses. Therefore, DNA oligonucleotides homologous to the telomere 3' overhang (T-oligos) may mimic this signal in the absence of DNA damage or telomere disruption. Indeed, treatment with T-oligos induced apoptosis in lymphocytic leukemia (10)
, melanoma cells (11)
, and breast carcinoma cells (unpublished data), whereas a senescent phenotype or an early response to DNA damage such as the phosphorylation of the histone variant H2AX, yielding 
-H2AX, was induced in fibroblasts or fibrosarcoma cells (12
13
14
15)
. These observations indicate thatT-oligos may be useful in the treatment of various cancers. However, whether T-oligos are effective against malignant gliomas is unknown. Therefore, in this study, we determined the effect of T-oligos on malignant glioma cells in vitro and in vivo. Treatment with T-oligos inhibited the proliferation of malignant glioma cells, but not normal human astrocytes (NHAs), through induction of nonapoptotic autophagy. Pretreatment with T-oligos markedly suppressed the growth of intracranial tumors in nude mice. Our findings provide a strong basis for treating malignant gliomas with T-oligos as a tumor-selective approach.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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11
12
13
14
15)
and purchased from the Midland Certified Reagent Company (Midland, TX, USA). One was homologous to the telomere overhang [11 mer test (T)-oligo: pGTTAGGGTTAG], and the other was complementary to this sequence [11 mer control (C)-oligo: pCTAACCCTAAC]. Oligonucleotides were resuspended in H2O to provide a 2 mM stock solution. For use in experiments, the stock solution was diluted into culture medium, filter-sterilized, and added to culture dishes. In all experiments, cells were placed in medium containing oligonucleotides at a concentration of 10–80 µM. On the basis of previous experiments (10
11
12
13
14
15)
, this medium was added only once.
Reagents
3-Methyladenine (3-MA), rapamycin, AG490, and an antibody against ß-actin were purchased from Sigma Chemical (St. Louis, MO, USA). Antibodies against Akt, phospho-Akt (Thr308 and Ser473), phospho-glycogen synthase kinase-3 (GSK3) (Ser21/9), mammalian target of rapamycin (mTOR), phospho-mTOR (Ser2448), p38, phospho-p38 (Thr180/Tyr182), Src, phospho-Src (Tyr416), and phospho-signal transducer and activator of transcription 3 (STAT3) (Ser727) were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-GSK3 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). An antibody against STAT3 was purchased from Upstate (Charlottesville, VA, USA). An antibody against the microtubule-associated protein 1 light chain 3 (LC3) was kindly provided by Dr. Tamotsu Yoshimori (National Institute of Genetics, Mishima, Japan).
Cell culture
Human malignant glioma U87-MG and U373-MG cells were purchased from American Type Culture Collection (Manassas, VA, USA). NHAs were kindly provided by Dr. Kenneth Aldape (The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA) (16)
. Cells were cultured in DMEM supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 100 U/ml penicillin (Invitrogen), and 2.5 µg/ml fungizone (Invitrogen) at 37°C in 5% CO2. Doubling times of U87-MG, U373-MG, and NHAs were 25.5, 25.2, and 29.6 h, respectively, indicating that these cells have similar proliferative rates in cell culture.
Cell viability assay
The cytotoxic effect of T-oligos or C-oligos on cultured cells was determined using cell proliferation reagent WST-1 (Roche Applied Science, Indianapolis, IN, USA), as described previously (17)
. Cells were seeded at 2 x 103 cells/well in a 96-well plate and incubated at 37°C overnight. The cells were treated with 10–80 µM C-oligos or T-oligos for 3, 5, or 7 days and exposed to 10 µl of WST-1 reagent for 1 h at 37°C. The absorbance at 450 nm was measured in a microplate reader. The viability of cells treated with vehicle alone was considered to be 100%.
Apoptosis detection assay
To determine whether treatment with T-oligos induces apoptosis, we used an annexin V-FITC apoptosis detection kit (BD Biosciences Pharmingen, San Diego, CA, USA), according to the manufacturer's instructions. At 72 h after exposure to 40 µM C-oligos or T-oligos, the cells were trypsinized and incubated with annexin V-FITC and propidium iodide for 15 min at room temperature in the dark, followed by cytofluorometric analysis with a FACS Calibur flow cytometer using CellQuest software (Becton Dickinson, San Jose, CA, USA). At least 10,000 cells were analyzed from each sample. To detect apoptosis morphologically, we treated tumor cells with 10–80 µM C-oligos or T-oligos for 3 to 7 days, fixed them with 4% paraformaldehyde, and stained them with Hoechst 33258 (0.5 µg/ml) for 15 min, as described previously (18)
. Two hundred cells were counted and scored for the incidence of apoptotic chromatin changes under a fluorescence microscope. Paclitaxel (4 nM) was used as a positive control to induce apoptosis (19)
.
Electron microscopy
Cells were grown on glass coverslips, treated with C-oligos or T-oligos at 40 µM for 72 h as described above, and fixed with a solution containing 3% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3, for 1 h. The samples were postfixed in 1% OsO4 in the same buffer for 1 h and then subjected to electron microscopic analysis as described previously (18)
. Representative areas were chosen for ultrathin sectioning and viewed with a JEM 1010 transmission electron microscope (JEOL, Peabody, MA, USA) at an accelerating voltage of 80 kv. Digital images were obtained with the AMT Imaging System (Advanced Microscopy Techniques, Danvers, MA, USA).
Detection and quantification of acidic vesicular organelles with acridine orange staining
Autophagy is the process of sequestering cytoplasmic proteins into the lytic component and is characterized by the formation and promotion of acidic vesicular organelles (AVOs), as described previously (20)
. To detect the development of AVOs, we treated cells with 40 µM C-oligos or T-oligos for 72 h as described above and then performed vital staining with acridine orange as described previously (18)
. To quantify the development of AVOs, the cells were stained with 1 µg/ml acridine orange for 15 min, removed from the plate with trypsin-EDTA (Invitrogen), and analyzed using the FACScan flow cytometer and CellQuest software (Becton Dickinson).
Assessment of the involvement of LC3
LC3, a mammalian homologue of the yeast autophagy-related (Atg) gene Atg8, is recruited to the autophagosome membrane during autophagy (21)
. Green fluorescent protein (GFP)-tagged LC3-expressing cells can be used to demonstrate induction of autophagy (18
, 21
22
23)
. GFP-LC3 cells exhibit diffuse GFP fluorescence under control conditions, whereas a punctate pattern of GFP-LC3 fluorescence (GFP-LC3 dots) was observed during autophagy. Therefore, using the GFP-LC3 expression vector kindly provided by Dr. Noboru Mizushima (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan), we determined the involvement of LC3 in cells treated with C-oligos or T-oligos. Cells were transiently transfected with the GFP-LC3 expression vector using Fugene 6 transfection reagent (Roche). After being cultured overnight, cells were treated with 40 µM T-oligo or C-oligo for 72 h as described above, fixed with 4% paraformaldehyde, and examined under a fluorescence microscope. To quantify autophagic cells after treatment, we counted the number of autophagic cells (
5 GFP-LC3 dots per cell) among 200 GFP-positive cells.
Western blotting
Soluble proteins were isolated from untreated cells and cells treated with 40 µM C-oligos or T-oligos for the indicated periods for Western blotting, as described previously (18)
. Equal amounts of protein (40 µg) were separated by 10% or 15% SDS-PAGE gel (Bio-Rad, Richmond, CA, USA) and transferred to a Hybond-P membrane (Amersham, Piscataway, NJ, USA). The membranes were treated with primary antibodies (diluted 1:1000) overnight at 4°C and incubated for 1 h with a horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody (1:3000 dilution, Amersham) at room temperature for 1 h. Bound antibody complexes were detected using an enhanced chemiluminescence reagent (Amersham) according to the manufacturer's instructions.
Reverse-phase protein microarray analysis
The protein microarray was processed as described previously (24)
. Cells treated with 40 µM C-oligos or T-oligos for 15, 30, or 60 min were lysed with reverse-phase protein microarray (RPPM) lysis buffer (150 mM NaCl; 50 mM HEPES, pH 7.4; 1.5 mM MgCl2; 1 mM EGTA; 100 mM NaF; 10 mM NaPPi; 10% glycerol; and 1% Triton X-100) supplemented with Complete Protease Inhibitor Cocktail Tablets (Roche Applied Science, Indianapolis, IN, USA). After being centrifuged, supernatants containing postnuclear lysates were collected. Samples were then denatured by the addition of one part 4x SDS sample buffer (0.25 M Tris pH 6.8, 35% glycerol, 8% SDS, and 10% 2-mercaptoethanol) to three parts lysate and boiling for 5 min. Samples were serially diluted with dilution buffer (3 parts RPPM lysis buffer+1 part 4x SDS sample buffer) and transferred to 384-well plates for spotting onto nitrocellulose-coated FAST slides (Whatman, Florham Park, NJ, USA) using a GeneTAC G3 arrayer (Genomic Solutions, Ann Arbor, MI, USA). An array consisting of 4 x 4 grids (2 samples with 82-fold serial dilutions/grid) arranged in 4 rows and 12 columns with 8 or 9 touches per spot was programmed for printing. Slides not immediately stained were stored in desiccant at –20°C.
Slides were precleared by being washed in Re-Blot Plus mild antibody stripping solution (Chemicon International, Temecula, CA, USA) for 15 min and washed with RPPM TBS-T (0.3 M NaCl; 0.05 M Tris, pH 7.6; and 0.1% Tween-20). Slides were then blocked with 0.2% I-Block (Applied Biosystems, Foster City, CA, USA) in PBS + 0.1% Tween-20, either for 30 min at room temperature or overnight at 4°C. After a TBS-T rinse, slides were stained using a tyramide-based signal amplification assay (Catalyzed Signal Amplification System, Dako, Carpintiria, CA, USA) and a colorimetric substrate (diaminobenzidine). Specifically, slides were further blocked for peroxidases, avidin, biotin, and proteins (5 min per reagent followed by 3 min. washes with RPPM TBS-T) and incubated for 45 min with the primary antibodies. After RPPM TBS-T washes, a 1:10,000 dilution of the appropriate biotinylated antibody (Vector Laboratories, Burlingame, CA, USA) was added and incubated for 30 min. Antibodies were diluted in background-reducing antibody diluent (Dako). The slides were washed, and the three amplification reagents from the Dako CSA kit (streptavidin-biotin complex, amplification reagent, and streptavidin-HRP) were added for 15 min each, followed by RPPM TBS-T washes. For signal detection, diaminobenzidine was added to the slides and incubated for 3–5 min; the reaction was stopped by immersing the slides in ddH20 and then air-drying them.
For sample quantification, slides were scanned on an HP Scanjet 8200 scanner at 600 dpi in grayscale and saved as a 16-bit tiff file. The images were analyzed using MicroVigene version 2.2 (Vigene Tech, North Billerica, MA, USA), which contains spot-finding and curve-fitting programs. Protein levels of samples were determined by converting Ln (Y0) values (set at mean net and total curve mean) to linear values. The protein phosphorylation level was standardized by the equivalent total protein level, and each value was expressed as an intensity showing the ratio of the standardized expression in cells treated with C-oligos or T-oligos to that in untreated cells.
Flow cytometric analysis of mitochondrial membrane potential
To detect changes in mitochondrial membrane potential, rhodamine 123 (Molecular Probe, Eugene, OR) was added as described previously (18)
. Tumor cells were treated with 40 µM C-oligos or T-oligos for 72 h, collected by trypsinization, washed with PBS, and stained with 1 µM rhodamine 123 in PBS in the dark at 37°C for 1 h. Samples were washed, resuspended in PBS, and then subjected to FACScan using CellQuest software.
Measurement of telomere length
U373-MG cells were treated with 40 µM C-oligos or T-oligos or diluent alone for 3 or 5 days, and genomic DNA was isolated using the DNeasy tissue kit (Qiagen, Valencia, CA, USA). Telomere length was determined using the telo TTAGGG telomere length assay (Roche) following the protocol supplied by the manufacturer. In brief, 1 µg of purified genomic DNA was digested with HinfI/RsaI. The DNA fragments were separated on a 0.8% agarose gel, transferred to a nylon membrane for Southern blotting, hybridized to a digoxigenin-labeled probe specific for telomeric repeats, and incubated with antidigoxigenin-alkaline phosphatase. Terminal restriction fragments were detected by chemiluminescence. The mean telomere length (MTL) was determined by scanning the exposed X-ray film with a densitometer and calculated as described previously (25)
.
Animal studies
To determine the therapeutic efficacy of T-oligos for malignant gliomas, the 8- to 12-wk-old female nude mice (Experimental Radiation Oncology at M. D. Anderson Cancer Center; 5 or 6 mice in each treatment group) were anesthetized with ketamine and xylazine as described previously (26)
. A 0.9-mm burr hole was then drilled in the mouse skulls 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 (27)
. Three days after the screw guide was set in the skull, a 10 µl syringe (Hamilton, Reno, NV, USA) fitted with a 26-gauge needle was connected to a microinfusion pump (Harvard Apparatus, Cambridge, MA, USA). 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 104 U87-MG cells (untreated or pretreated with 40 µM T-oligos or C-oligos for 24 h in serum-free DMEM) were inoculated at a rate of 1.0 µl/min. Mice were then observed until they became moribund, lethargic, anorexic, dehydrated, or distressed, at which point they were deeply anesthetized and then euthanized by intracardiac perfusion-fixation. For histological analysis, the brains were removed, sliced coronally into five, blocks and snap-frozen. Coronal sections (8–10 µm) were then made. All animal 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
Data are mean ± SD. The significance of the in vitro experiments was determined using the Student's t test (2-tailed). The Kaplan-Meier product-limit method was used to estimate the survival curves (27)
. The significance of differences in survival time between treatment groups was assessed using the Cox-Mantel log-rank test as described previously (27)
. P values of <0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A, treatment with T-oligos remarkably suppressed the cell viability of U87-MG and U373-MG cells compared with C-oligos (60 vs. 81% viability and 82 vs. 94% viability on day 3 and 62 vs. 94% viability and 55 vs. 97% viability on day 5, respectively). The inhibition was significant in U87-MG and U373-MG cells (P<0.001 on days 3 and 5). In contrast, treatments with T-oligos only slightly diminished the viability of NHA cells (88 vs. 91% viability on day 3 and 80 vs. 85% viability on day 5). The differences in cell viability were not significant. When U373-MG cells were treated with T-oligos at 10–80 µM for 3–7 days, the viability was reduced in dose- and time-dependent manners (Fig. 1B
). Treatment with T-oligos at 80 µM for 7 days inhibited the viability of U373-MG cells to 23% (P<0.001, compared with C-oligos). These results indicate that T-oligos have a tumor-specific cytotoxicity for malignant glioma cells.
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T-oligos do not significantly affect apoptosis in malignant glioma cells
To determine whether U87-MG and U373-MG cells treated with T-oligos undergo increased apoptosis, we performed apoptosis detection assays. The annexin V-FITC apoptosis detection assay revealed no significant increase in apoptotic U373-MG cells treated with 40 µM C-oligos or T-oligos for 72 h compared with control (vehicle alone; Fig. 2
A). A 4 nM concentration of paclitaxel, which was used as a positive control to induce apoptosis in malignant glioma cells, induced apoptosis in 15% of U373-MG cells. Similar results were observed in U87-MG cells treated with 40 µM T-oligos and C-oligos for 72 h (data not shown). To determine the incidence of apoptosis morphologically, we stained the nuclei of treated tumor cells with Hoechst 33258. As shown in Fig. 2B
, apoptotic morphological characteristics such as chromatin condensation and nuclear fragmentation were detected in U373-MG cells treated with 4 nM paclitaxel but not in those treated with 40 µM C-oligos or T-oligos. To quantify the incidence of apoptosis, we counted 200 cells and scored for the incidence of apoptosis. Treatment with paclitaxel at 4 nM for 72 h induced apoptosis in 15% of U87-MG cells and 11% of U373-MG cells (P<0.01 compared with control; Fig. 2C
). On the other hand, treatments with T-oligos at 40 µM for 72 h induced apoptosis in 5.5% of U87-MG cells and 3.4% of U373-MG cells. Compared with control or C-oligos treatment, T-oligos treatment did not significantly increase apoptotic cells. Even when U373-MG cells were treated with T-oligos at 80 µM for 7 days, <10% of U373-MG cells were apoptotic (Fig. 2D
). Paclitaxel at 4 nM for 7 days induced apoptosis in 30% of U373-MG cells. These results indicate that the main growth inhibition induced by T-oligos in these malignant glioma cells is not due to induction of apoptosis, although apoptotic pathway was activated to some extent by T-oligos, particularly at late time points and high concentrations.
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T-oligos induce autophagy in malignant glioma cells
Malignant glioma cells treated with various anticancer agents undergo autophagy rather than apoptosis (28)
. We used electron microscopic analysis to determine whether T-oligos induce autophagy in malignant glioma cells. As shown in Fig. 3
A, numerous autophagic vacuoles were observed in U373-MG cells treated with 40 µM T-oligos for 72 h, whereas untreated U373-MG cells or U373-MG cells treated with 40 µM C-oligos for 72 h exhibited few autophagic features. In U373-MG cells incubated with 4 nM paclitaxel for 72 h, we observed fewer autophagic vacuoles with clear chromatin condensation and nuclear fragmentation, which is characteristic of apoptosis. As expected based on the data of the annexin V assay and Hoechst 33258 staining, we found no evidence of apoptosis in U373-MG cells treated with 40 µM C-oligos or T-oligos for 72 h.
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Development of AVOs by T-oligos
To detect and quantify the T-oligo-induced increase in fractional volume and acidity of AVOs, we stained treated tumor cells with acridine orange for flow cytometric analysis. As shown in Fig. 3B
, 40 µM T-oligos for 72 h increased the amount and frequency of red fluorescence (y axis) in U373-MG and U87-MG cells, indicating development of AVOs from 3.7 to 60.9% and from 2.6 to 30.1%, respectively. In contrast, treatment with 40 µM C-oligos for 72 h induced only a modest increase in AVOs (7.2% in U373-MG cells and 2.7% in U87-MG cells).
Induction of GFP-LC3 dots by T-oligos
We also assessed the presence of a punctate pattern of GFP-LC3 expression (GFP-LC3 dots) to determine whether LC3 is concentrated in autophagic vacuoles in malignant glioma cells by T-oligos. U373-MG and U87-MG cells were transiently transfected with GFP-LC3 expression vector for 24 h and then treated with C-oligos or T-oligos at 40 µM for an additional 72 h, as described previously (18
, 23)
. The GFP-LC3-transfected cells showed diffuse distribution of GFP-LC3 under untreated conditions or in the presence of C-oligos, whereas T-oligo-treated U373-MG and U87-MG cells showed a marked increase in number and frequency of GFP-LC3 dots (5 dots per cell; Fig. 3C
, top). This GFP fluorescence pattern represents the presence of autophagic vacuoles and indicates that LC3 was recruited to autophagic vacuoles in both tumor cell lines treated with T-oligos. To quantify the induction of cells expressing GFP-LC3 dots, we counted 200 GFP-positive cells for each treatment. As shown in Fig. 3C
(bottom), 18.2% of U373-MG cells and 25.7% of U87-MG cells treated with T-oligos at 40 µM for 72 h showed GFP-LC3 dots, whereas these autophagic features were detected in only 5.2% of untreated U373-MG cells and 7.2% of untreated U87-MG cells. The number of cells expressing GFP-LC3 in a punctate pattern was significantly increased in U373-MG and U 87-MG cells treated with T-oligos compared those treated with C-oligos (P<0.01).
Increase in LC3-II expression by T-oligos
A recent investigation showed that there are two forms of the LC3 proteins in cells; LC3-I and LC3-II (21)
. LC3-I is the cytoplasmic form of LC3 and is processed into LC3-II, which is associated with the autophagosomal membrane. Therefore, the amount of LC3-II is associated with autophagy induction. Western blotting with anti-LC3 antibody revealed that the expression of LC3-I and LC3-II proteins markedly increased in U373-MG cells treated with 40 µM T-oligos for 48 h compared with that in cells treated with C-oligos under the same treatment conditions (Fig. 3D
), suggesting that T-oligos induce the synthesis of LC3 protein and the conversion of a substantial fraction of LC3-I into LC3-II. These results together indicate that T-oligos but not C-oligos induce autophagy in malignant glioma cells.
Effect of inhibition of T-oligo-induced autophagy on malignant glioma cells
Whether anticancer treatments-induced autophagy is a protective response or nonapoptotic cell death is still debatable (28)
. Therefore, we used 3-MA to inhibit T-oligo-induced autophagy as described previously (27)
. Treatment with 3-MA (2.0 mM) in the presence of 40 µM T-oligos for 72 h suppressed the development of AVOs in U87-MG cells (down to 11.7%) and U373-MG cells (down to 0.9%). As shown in Fig. 3E
, the decreased viability of U87-MG and U373-MG cells was significantly reversed by addition of 3-MA (P<0.01 each). These results indicate that T-oligo-induced autophagy mediates cell killing effect rather than protective reaction.
T-oligos inhibit the mTOR or STAT3 signaling pathway
To screen for potential molecular pathways responsible for T-oligo-induced cytotoxicity, we conducted RPPM assay for signaling through the PI3K/Akt/mTOR, the RAS/mitogen-activated protein kinase (MAPK) pathway, cell cycle, PKC, src, JAK/STAT, and apoptosis pathways. TOR kinase—mTOR in mammalian cells—is located upstream of autophagy-associated genes and regulates the autophagic pathway (29)
. In addition, not only the mTOR and its upstream molecule Akt but also MAPKs play a key role in regulating autophagy (28
, 30
, 31)
. Therefore, we focused on these molecules. As shown in Supplemental Fig. 1, the phosphorylation of Akt at Thr308 and Ser473 was increased in U87-MG cells 15 and 30 min after exposure to T-oligos, whereas it was suppressed in U373-MG cells 15 min after the addition of T-oligos. T-oligos inhibited the phosphorylation of mTOR at Ser2448 in both tumor cell lines at 15 min but stimulated the phosphorylation in U373-MG cells at 30 and 60 min. The phosphorylation of p38 at Thr180/Tyr182, one of MAPKs, was increased in U87-MG but not U373-MG cells by C-oligos compared with T-oligos.
Compared with C-oligos, the phosphorylation of STAT3 at Ser727, which is associated with cell death (32)
, was suppressed in both tumor cells 30 and 60 min after exposure to T-oligos. T-oligos increased the phosphorylation level of GSK3 at Ser21/9, which is recognized as a key component of cell growth and death (33)
, in U373-MG cells at 15 min and in U87-MG cells later. On the other hand, the phosphorylation of the intracellular proto-oncogene Src at Tyr416, which is related to the Akt pathway (34)
, was increased in U87-MG cells 30 min after treatment with T-oligos but was not affected in U373-MG cells. These results indicate that phosphorylations of signaling molecules in several pathways are altered differently and/or opposed each other in U87-MG and U373-MG cells.
To confirm the data of RPPM, we conducted Western blotting at the same treatment conditions. As shown in Fig. 4
A, Western blotting was consistent with some results of RPPM but not with others. In particular, the phosphorylation of Akt was not affected in both tumor cells by T-oligos, but the phosphorylation of mTOR was inhibited in U87-MG cells but not U373-MG cells 60 min after T-oligo treatment. T-oligos suppressed the phosphorylation of STAT3 but not p38 in both tumor cells 60 min after the treatment. One explanation for the discrepancy between RPPM and Western blotting may be that antibodies are not absolutely mono-specific and that RPPM cumulates specific and non-specific labeling, although mono-specific bands can be chosen in Western blotting. To analyze the data of Western blotting statistically, we repeated the same experiments three times. As shown in Fig. 4B
, there was no significant change in the phosphorylation of Akt and p38. In U87-MG cells, the phosphorylation of mTOR was significantly decreased after 60 min of exposure to T-oligos (P<0.05). Because mTOR is located downstream of Akt, the inhibitory effect of T-oligos on mTOR in U87-MG cells may be Akt independent. Moreover, T-oligos significantly inhibited the phosphorylation of STAT3 in U87-MG cells (P<0.05, 60 min) and in U373-MG cells (P<0.05, 30 min; P<0.01, 60 min).
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To assess the involvement of mTOR and STAT3 in cytotoxicity of T-oligo, we used an inhibitor of each pathway and determined whether inhibition of mTOR or STAT3 affects the effect of T-oligos. To inhibit the mTOR pathway, rapamycin, an inhibitor of mTOR, was used as described previously (35)
. As shown in Fig. 4C
, the addition of rapamycin (1 nM) significantly enhanced the effect of T-oligos on the viability of U87-MG cells (P<0.001). On the other hand, we used AG490, an inhibitor of STAT3, as described previously (36)
. As shown in Fig. 4D
, the addition of AG490 (50 µM) significantly increased the sensitivity of U87-MG cells (P<0.001) and U373-MG cells (P<0.001) to T-oligos. These results indicate that inhibition of the mTOR or STAT3 pathway plays a key role in cytotoxicity of T-oligo in malignant glioma cells.
To clarify the relationship between autophagy and the effects of rapamycin and AG490, we determined whether these inhibitors affect T-oligo-induced autophagy in U87-MG and U373-MG cells by using GFP-LC3 dot assay. As shown in Fig. 4E
, the addition of rapamycin increased the percentage of T-oligo-induced autophagic cells with GFP-LC3 dots compared with C-oligo treatment (31–48% in U87-MG cells and 35–57% in U373-MG cells, P<0.01). In addition, AG490 augmented the incidence of autophagic cells caused by T-oligos compared with C-oligos (21–37% in U87-MG cells and 38–58% in U373-MG cells, P<0.01). These results indicate that the effects of rapamycin and AG490 are positively implicated in T-oligo-induced autophagy.
T-oligos hyperpolarize mitochondria
A recent study showed that radiation and rapamycin lead to hyperpolarization of mitochondria and subsequently induce autophagy in breast cancer cells (37)
. Therefore, to determine whether T-oligos cause hyperpolarization of mitochondria in malignant glioma cells, we measured the mitochondria membrane potential with rhodamine 123. As shown in Fig. 5
, compared with C-oligos, treatment with 40 µM T-oligo for 72 h induced an increase in membrane potential from 0.1 to 38.8% in U373-MG cells and 0.7 to 31.2% in U87-MG cells, indicating mitochondrial hyperpolarization by T-oligos.
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T-oligos do not affect telomere length
To rule out the possibility that T-oligos promote accelerated telomere shortening contributing to autophagy in malignant glioma cells, we determined the MTL after treating U373-MG cells with 40 µM C-oligos or T-oligos for 3 or 5 days. Treatment with T-oligos for 3 or 5 days did not alter the MTL (5.72 or 5.92 kb with T-oligos treatment; 5.75 or 5.69 kb without treatment; and 5.76 or 5.71 kb with C-oligos treatment), respectively (Fig. 6
). These results, together with those of recent studies (12
, 14)
, indicate that T-oligos do not affect telomere length even when antitumor effect is observed.
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T-oligos reduce tumorigenesis in vivo
To determine whether the in vitro effect of T-oligos is recapitulated in in vivo settings, U87-MG cells were treated with 40 µM C-oligos or T-oligos for 24 h and inoculated into the brain of nude mice for survival studies. As shown in Fig. 7
A, all five control mice died of intracranial tumors within 42 days (median survival, 38 days) after inoculation of 5 x 104 untreated U87-MG cells. The median survival of six mice intracranially inoculated with U87-MG cells pretreated with C-oligos was 40 days; there was no significant difference between no treatment and C-oligos treatment (P=0.21). On the other hand, survival of 4 of the 6 mice treated with T-oligos was prolonged to 71 days; the 2 surviving mice were killed on day 85 for histological analysis (median survival of all 6 mice>65 days). The survival time of the animals was significantly higher in the T-oligo-treated group than in the untreated and C-oligo-treated groups (P=0.0065 and P=0.043, respectively). U87-MG cells pretreated with 40 µM C-oligos for 24 h developed into intracranial tumors, and the midline was shifted laterally by the mass effect (Fig. 7B
). In contrast, in brain tissues harvested from the two mice that survived 85 days after inoculation of U87-MG cells pretreated with 40 µM T-oligos for 24 h, tumors were histologically undetectable. Those two mice showed no weight loss or neurological deficits, such as hemiparesis or seizures. These results indicate that T-oligos reduce tumorigenesis in vivo, suggesting that they have the potential to prevent the development of malignant glioma and, in a subset of animals, induce pathological complete remissions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, was observed in malignant glioma cells undergoing autophagy. These findings suggest that T-oligos are a promising therapeutic agent for managing malignant gliomas exhibiting a novel mechanism of induction of cell death.
The 3' end of each telomere forms a loop structure with the double-stranded chromosome folded back and the loop is secured by insertion of the 3' overhang into the duplex region (8)
. Disruption of this loop structure leads to exposure and degradation of the 3' overhang and eventually to the induction of DNA damage responses (9)
. Accumulating evidence shows that T-oligos, which we hypothesize mimic telomere loop disruption and overhang exposure, induce apoptosis or senescence in malignant cells from multiple cell lineages (10
11
12
13
14
15)
but have far less effect on the normal counterparts of these cells (11
, 14)
. Another study demonstrated that T-oligos pretreatment reduced the mutation rate and photocarcinogenesis in UV-irradiated mice (38)
and decreased the expression of cyclooxygenase-2 in human skin explants (39)
, suggesting additional anticancer mechanisms of action for T-oligos. In the present study, T-oligos induced autophagy instead of apoptosis in malignant glioma cells. Both autophagy and apoptosis represent forms of programmed cell death; however, the molecular mechanisms are markedly different in both cases. This discrepancy might be due to the cancer cell type tested because malignant glioma cells have a tendency to undergo autophagy rather than apoptosis after anticancer treatments such as radiation and chemotherapy (23
, 40
41
42
43
44)
.
Autophagy is a process in which intracellular membrane structures sequester proteins and organelles to degrade and recycle these materials (45)
. These responses are dominated by the appearance and accumulation of AVOs (20)
. Induction of autophagy has been seen in various cell conditions, including nutrient deprivation, differentiation, aging, and cancer therapy (28
, 30
, 31
, 46)
. In lower organisms, autophagy allows survival during stress by generating energy from intracellular sources. However, prolonged autophagy results in cell death. Thus, autophagy is best thought of as a temporary stress survival process that eventuates into cell death. Thus, autophagy could both increase or decrease tumor development depending on the context. Autophagy has been frequently compared with apoptosis as a response of cancer cells to treatments on the basis of its morphological appearances; programmed cell death type I (apoptosis) and type II (autophagic cell death) (47)
. Apoptosis has typical morphological and biochemical characteristics such as nuclear condensation and fragmentation and is associated with caspase activation. In this study, <10% of apoptosis was observed in T-oligo-treated U373-MG cells, although paclitaxel induced apoptosis in 30% of the same cell line. On the other hand, autophagic cell death is generally caspase independent (48)
but also associated with typical morphological changes including induction of multilamellar vesicles. It appears likely that crosstalk between apoptosis and autophagy exists with the balance determining why some tumor cells die from apoptosis and others from autophagy.
The RPPM is best used under these circumstances to determine which signaling molecules and pathways warrant further exploration. Thus, we used these to direct Western blotting studies and perform statistical analysis on multiple samples. Our data indicated the involvement of mTOR or STAT3 signaling pathway in the cytotoxicity of T-oligo for U87-MG and U373-MG cells. In mammalian cells, mTOR is well established as a signaling system that contributes to the control of the autophagic pathway (31
, 45)
. Because rapamycin, which causes autophagy in malignant glioma cells (35
, 44)
, enhanced the antitumor effect of T-oligos, mTOR may be one of key molecules regulating the cytotoxicity of T-oligo for at least U87-MG cells. On the other hand, our findings indicated that STAT3 is involved in the efficacy of T-oligo, although there is no study demonstrating the relationship between autophagy and STAT3. Thus, the predictions from the RPPM data, particularly when concordance is observed between the cell lines analyzed, are highly predictive of functional importance. However, those effects that are observed in only one cell line are likely related to the pathogenic rewiring of the signaling pathways by underlying genomic aberrations. More importantly, the signaling pathway is extremely complex with a number of feed forward and feed back loops as well as crosstalk with multiple other signaling networks. Therefore, further study is needed to determine which molecule is more important or associated with autophagy.
Our findings suggest that T-oligos have the potential to suppress the proliferation of malignant glioma cells in vitro and in vivo. Pretreatment of malignant glioma cells, as was done in this study, is not equivalent to treating tumor cells already established in the brain. However, in a similar mouse model of melanoma (11)
, the selective inability of T-oligo-pretreated melanoma cells to produce metastases when injected intravenously predicted their ability to reduce tumor burden in vivo. In any case, our data show that relatively brief exposure to T-oligos has a long-lasting effect on malignant glioma cells that is not reversed by the tissue environment. Therefore, we will further assess whether treatment with T-oligos prolongs the survival of mice carrying established intracranial tumors and determine the utility of T-oligos in therapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication July 21, 2006. Accepted for publication March 26, 2007.
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