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Mol Cancer Ther. 2006;5:1423-1433
© 2006 American Association for Cancer Research

Research Articles: Targets

Therapeutic potential of antisense oligodeoxynucleotides to down-regulate thymidylate synthase in mesothelioma

Janet Flynn1, Randal W. Berg1,2,3, Tracy Wong1,4, Marijke van Aken7, Mark D. Vincent1,3, Masakazu Fukushima8 and James Koropatnick1,2,3,4,5,6

1 London Regional Cancer Program and 2 Lawson Health Research Institute, London Health Sciences Centre; 3 Departments of Oncology, 4 Physiology and Pharmacology, 5 Microbiology and Immunology, and 6 Pathology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada; 7 Faculty of Medicine, Utrecht University, the Netherlands; and 8 Hanno Research Centre, Taiho Pharmaceuticals, Hanno-City, Japan

Requests for reprints: James Koropatnick, Victoria Research Laboratories, London Regional Cancer Program, London Health Sciences Centre, 790 Commissioners Road East, London, Ontario, Canada N6A 4L6. Phone: 519-685-8500, ext. 58654; Fax: 519-685-8681. E-mail: jkoropat{at}uwo.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Malignant mesothelioma is an aggressive tumor of the serosal surfaces of the lungs, heart, and abdomen. Survival rates are poor and effective treatments are not available. However, recent therapeutic regimens targeting thymidylate synthase (TS) in malignant mesothelioma patients have shown promise. We have reported the use of an antisense oligodeoxynucleotide targeting TS mRNA (antisense TS ODN 83) to inhibit growth of human tumor cells. To test the potential for antisense targeting of TS mRNA in treatment of malignant mesothelioma, we assessed and compared the effects of antisense TS ODN 83 on three human malignant mesothelioma cell lines (211H, H2052, and H28) and human nonmalignant mesothelioma cells (HT29 colorectal adenocarcinoma, HeLa cervical carcinoma, and MCF7 breast tumor cell lines). We report that ODN 83 applied as a single agent effectively reduced TS mRNA and protein in malignant mesothelioma cell lines. Furthermore, it inhibited malignant mesothelioma growth significantly more effectively than it inhibited growth of nonmalignant mesothelioma human tumor cell lines: a difference in susceptibility was not observed in response to treatment with TS protein-targeting drugs. In malignant mesothelioma cells, antisense TS both induced apoptotic cell death and reduced proliferation. In nonmalignant mesothelioma cells, only reduced proliferation was observed. Thus, antisense TS-mediated induction of apoptosis may be the basis for the high malignant mesothelioma sensitivity to antisense targeting of TS. Further preclinical and clinical study of TS antisense oligodeoxynucleotides, alone and in combination with TS-targeting chemotherapy drugs, in mesothelioma is warranted. [Mol Cancer Ther 2006;5(6):1423–33]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Malignant mesothelioma is a uniformly lethal tumor originating from mesothelial cells lining the lung. Exposures to asbestos and/or oncogenic SV40 seem to be involved in the etiology of the disease (1). Although the overall number of malignant mesothelioma cases is relatively small (e.g., ~15 cases per million in the United States in 2000; ref. 2), malignant mesothelioma incidence is higher in some jurisdictions and is increasing. For example, the latest available data indicate that malignant mesothelioma incidence in Australia is 40 cases per million population and is predicted to increase over the next 10 to 15 years (3). It has been estimated that the number of deaths from mesothelioma in the United Kingdom will increase from 1,500 in the year 2000 to 3,000 in 2020 (4). Although the time between asbestos exposure and malignant mesothelioma appearance suggests a latency period of 20 to 30 years (5), malignant mesothelioma is a rapidly progressing disease at later stages: median survival is currently 4.5 to 16 months after diagnosis depending on stage, histologic subtype, and patient age (6).

Malignant mesothelioma is highly refractory to intervention by radiation therapy (7), surgery (8), and chemotherapy (9). It has been suggested that during latency, mutation and/or selection of tumor cells with enhanced resistance to apoptosis results in malignant mesothelioma with high resistance to treatment (10). Cisplatin is currently the most effective single-agent chemotherapy treatment but achieves only a moderate response rate of 28% (11) and a median survival of 10 months (1). In cisplatin-resistant and chemonaive mesothelioma patients, a response rate of 20% and an overall survival of 32 weeks were achieved by combination chemotherapy with oxaliplatin and the folate analogue raltitrexed (Tomudex), which inhibits the activity of thymidylate synthase (TS; ref. 12). However, in a phase III randomized study of cisplatin alone versus the cisplatin/raltitrexed combination, the combination improved survival (8.8 versus 11.4 months; P < 0.045) and response (14% versus 24%; P = 0.06; ref. 13). Another randomized trial testing the additional TS-targeting drug pemetrexed (Alimta, a multitargeting antifolate that inhibits TS and other folate-dependent enzymes) in combination with cisplatin resulted in a response rate of 41.3% and a median survival of 12.1 months versus 16.7% and 9.3 months for cisplatin alone (14). Inclusion of TS-targeting drugs in malignant mesothelioma treatment regimens that show improved response and survival indicates that TS is a critical therapeutic target in malignant mesothelioma, and these regimens have become the standard of care.

TS is an enzyme that catalyzes the reductive methylation of dUMP to thymidylate and is the only de novo source of thymidylate for DNA synthesis and repair (15). 5-Fluorouracil was recognized as cytostatic agent nearly 50 years ago (16), with the realization that TS was its target coming soon after (17). TS expression is cell cycle dependent and elevated in highly proliferative cells and increased TS protein levels in a broad range of tumors correlates with poor treatment response and poor prognosis (18). In addition to its role as an essential enzyme for production of DNA precursors, TS also seems to regulate the capacity of mRNAs to function as templates for protein production. It binds to, and represses translation of, its own mRNA in vitro as part of a feedback inhibitory loop (19). It also binds to c-myc and p53 mRNAs, suggesting a role in post-transcriptional regulation of other cell cycle–dependent proteins (19). Furthermore, the TS gene may be a transforming oncogene in its own right because nontransformed cells expressing TS by virtue of transfection with a TS expression vector exhibit transformed behavior (20). TS, therefore, has the potential to regulate multiple events in the processes of DNA synthesis and repair, cell cycle progression, and tumorigenesis and is a key target for cancer therapy.

In addition to targeting TS protein with folate analogues and fluoropyrimidine inhibitors, antisense TS drugs (oligonucleotides and small interfering RNAs) targeting TS mRNA have been explored as potential anticancer therapeutics (21–25). Antisense oligodeoxynucleotides hybridize to target mRNA to inhibit translation and induce target mRNA degradation by RNase H (26). Antisense oligodeoxynucleotides with a phosphorothioate backbone and 2'-methoxyethoxy (2-MOE) of the ribose moiety have enhanced stability and effectiveness compared with unmodified oligodeoxynucleotides (27). Treatment of human cervical carcinoma (HeLa) cells with a 2-MOE antisense TS oligodeoxynucleotide (ODN 83, complementary to a 20 base sequence within the 3'-untranslated region of TS mRNA) reduced TS mRNA levels, TS protein level and activity, and HeLa cell proliferation (21). Treated cells also had increased sensitivity to 5-fluorouracil, 5-FdUrd, or raltitrexed cytotoxicity, but sensitivity to non-TS-targeting drugs, including cisplatin and chlorambucil, was not affected (21). Similar effects were shown in HeLa cells selected in vitro for resistance to 5-FdUrd and overexpression of TS (22) and in human HT29 colon carcinoma cells both in vitro and growing as xenografts in immunocompromised mice (23).

Here, we show that three human mesothelioma cell lines (211H, H2052, and H28) are exquisitely sensitive to treatment with TS antisense ODN 83 in vitro. Treatment with antisense ODN 83 reduced TS mRNA and protein levels in malignant mesothelioma cells, similar to reductions observed in nonmalignant mesothelioma cell lines. However, malignant mesothelioma cells were much more sensitive to antisense TS-induced growth inhibition than nonmalignant mesothelioma cells. The difference between malignant and nonmalignant mesothelioma cells in sensitivity to growth inhibition was restricted to response to antisense TS oligodeoxynucleotide targeting TS mRNA and not to drugs targeting TS protein. TS down-regulation by antisense TS ODN 83 in human HeLa, MCF7, and HT29 cells results in reduced growth without increased apoptosis. In malignant mesothelioma cells, on the other hand, antisense TS ODN 83 treatment induced apoptosis as assessed by flow cytometry of Annexin V/propidium iodide–stained cells and caspase inhibition assays. In addition, combined treatment with ODN 83 sensitized malignant mesothelioma cells to TS-targeting drugs. These data suggest the potential for therapeutic efficacy of TS antisense oligodeoxynucleotides in treatment of malignant pleural mesothelioma.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tumor Cell Lines
A panel of independently derived human mesothelioma cell lines [MSTO-211H (CRL-2081), isolated in 1985 from a lung metastasis from a 62-year-old Caucasian male; NCI-H2052 (CRL-5915), isolated in 1988 from a pleural effusion from a 65-year-old Caucasian male; and NCI-H28 (CRL-5820), isolated in 1976 from a pleural effusion from a 48-year-old Caucasian male] and nonmesothelioma cells (HeLa, human cervical carcinoma–derived in 1951; MCF7, human breast carcinoma–derived in 1973; and HT29, human colon carcinoma–derived in 1964) were obtained from the American Type Culture Collection (Rockville, MD) and grown in RPMI 1640 (Life Technologies, Grand Island, NY) with 10% fetal bovine serum (FBS; Life Technologies) at 37°C in a humidified 5% CO2 atmosphere.

Oligodeoxynucleotides
TS antisense ODN 83 (5'-GCCACTGGCAACATCCTTAA-3', complementary to human TS mRNA 136-155 bp downstream of the stop codon in the 3'-untranslated region) and scrambled control ODN 32 (5'-ATGCGCCAACGGTTCCTAAA-3', identical base composition in random order) were synthesized (Eurogentec North America, Philadelphia, PA) with phosphorothioate internucleotide linkages and 2'-O-methyl (2-MO) modifications of the terminal 6 nucleotides at 3' and 5' ends. TS antisense ODN 501 (5'-TTGGATGCGGATTGTACCCT-3') complementary to nucleotides 1,002 to 1,021 within the protein coding region and TS antisense ODN 504 (5'-ACTCAGCTCCCTCAGATTTG-3') complementary to nucleotides 1,436 to 1,455 in the 3'-untranslated region were obtained from Isis Pharmaceuticals, Inc. (Carlsbad, CA). Isis oligodeoxynucleotide versions of ODN 83, ODN 32, ODN 501, and ODN 504 were chemically identical to Eurogentec oligodeoxynucleotides, but with a 2-MOE rather than a 2-MO modification of terminal nucleotides. All non-TS human mRNAs in the National Center for Biotechnology Information Genbank databases had four or more mismatches with all oligodeoxynucleotides.

Cell Growth Assays
Mesothelioma cells were plated in triplicate (1 x 105 per 25-cm2 flask in 2 mL RPMI 1640/10% FBS) and cultured for 24 hours. A 6x transfection mix of LipofectAMINE 2000 (LFA-2K) transfection reagent (0.6-3.0 µg/mL; Invitrogen, Burlington, Ontario, Canada) and oligodeoxynucleotide (60-300 nmol/L) was prepared in serum-free medium according to the manufacturer's instructions, gently agitated, and incubated (15 minutes, 20°C). Five volumes of RPMI 1640/10% FBS were added to the 6x transfection mix to obtain a 1x transfection mix with the desired oligodeoxynucleotide concentration. Medium on the cells was replaced with 2 mL of the 1x transfection mix. Additional RPMI 1640/10% FBS (2 mL) was added to each flask 4 hours later.

In experiments to assess the combined effect of TS-targeting drugs and oligodeoxynucleotides, cells were pretreated with antisense TS ODN 83 (5 nmol/L) or control ODN 32 (5 nmol/L) as above. A single aliquot of transfected cells was divided into three 25-cm2 flasks; then, pemetrexed (5-50 nmol/L) or 5-FUdR (1-5 nmol/L) was added using a stock drug solution (RPMI 1640 without FBS) in a volume of no more than 0.5% of the total RPMI 1640/10% FBS already on the cells. Cells were then grown for 3 days, washed with PBS, trypsinized and removed from flasks, and counted on a Beckman Coulter Z1 Particle Counter (Beckman, Mississauga, Ontario, Canada). Cell numbers were plotted as (number of treated cells at day 3 – number at time of treatment) / (number of control cells at day 3 – number at time of treatment) x 100.

TS mRNA Purification and Quantification
Total cellular RNA was isolated using TRIzol (Invitrogen) 24 hours after transfection. RNA (2.5 µg) was reverse transcribed using SuperScript II reverse transcriptase (Invitrogen) and oligo(dT) random hexamer/nonomer mixed primers according to the manufacturer's recommendations. A fraction of the resulting cDNA (2%) was used as a template for PCR amplification of TS and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA using Taq DNA Polymerase Native (Invitrogen) and TS and GAPDH primers (Sigma Genosys, Oakville, Ontario, Canada; 3 minutes at 94°C, 24 cycles for 30 seconds at 94°C, 30 seconds at 58°C, and 45 seconds at 72°C, and final extension for 7 minutes at 72°C). TS primers were 5'-TTTTGGAGGAGTTGCTGTGG-3' (forward) and 5'-TGTGCATCTCCCAAAGTGTG-3' (reverse). GAPDH primers were 5'-TATTGGGCGCCTGGTCACCA-3' (forward) and 5'-CCACCTTCTTGATGTCATCA-3' (reverse). PCR products were resolved and visualized by electrophoresis through a 1.5% agarose gel and staining with ethidium bromide. Images were captured on an Image Master VDS gel documentation system (Amersham Biosciences, Baie d'Urfe, Quebec, Canada) and quantified using ImageQuant 5.1 software (Molecular Dynamics, Sunnyvale, CA). Relative TS mRNA levels ([TS mRNA] / [GAPDH mRNA]) were inferred from levels of PCR-amplified TS and GAPDH cDNA.

Immunochemical Detection and Measurement of TS Protein
Mesothelioma cells were plated in triplicate (2 x 106; 6 mL RPMI 1640/10% FBS, 75-cm2 flask). Medium was replaced with oligodeoxynucleotide (50 nmol/L) plus LFA-2K (0.5 µg/mL) in 6 mL medium 24 hours later. After a further 24 hours, cells were washed twice with ice-cold PBS and resuspended in lysis buffer [1 mol/L Tris (pH 7.6), 0.1% SDS, 1% Triton X-100, 0.2 mol/L EDTA). Total protein concentration was estimated using a Bio-Rad Protein Assay kit (Bio-Rad, Montreal, Quebec, Canada) as described by the manufacturer. Cell lysates (10 µg total soluble protein) were resolved by SDS-PAGE (12% polyacrylamide) and transferred to a nitrocellulose membrane (Hybond-ECL, Amersham Biosciences). The membranes were blocked (16 hours, 4°C) with 5% skim milk powder in TBS with 0.2% Tween 20, incubated with rabbit polyclonal anti-human TS antibody (0.125 µg/mL, 4 hours, 20°C; Taiho Pharmaceuticals, Hanno-City, Japan), washed with TBS with 0.2% Tween 20, and incubated for 1 hour with horseradish peroxidase–conjugated anti-rabbit antibody (1:2,000; Amersham Biosciences). Horseradish peroxidase activity was detected using Enhanced Chemiluminescence Plus (Amersham Biosciences) and X-ray film (Eastman Kodak, Rochester, NY). Bands were quantified using a Molecular Dynamics 375A Personal Densitometer and ImageQuant 5.1 software.

Flow Cytometric Analysis of Cell Death
Cells were plated in triplicate (25-cm2 flasks) and treated with oligodeoxynucleotides as described above. At 8, 12, 18, 24, and 48 hours after oligodeoxynucleotide addition, supernatant medium (containing any nonadherent cells) was collected. Adherent cells were rinsed with ice-cold PBS, trypsinized, and added to the nonadherent fraction. Cells were centrifuged (100 x g, 10 minutes, 4°C), washed twice in ice-cold PBS, reprecipitated by centrifugation, and resuspended (1,000 cells/µL) in binding buffer (140 mmol/L NaCl2, 2.5 mmol/L CaCl2, 10 mmol/L HEPES), and a fraction (100 µL) was incubated in the dark with 10 µL propidium iodide (50 ng/mL; Sigma, St. Louis, MO) plus 2 µL Annexin V-FITC (25 ng/mL; BD Biosciences, Mississauga, Ontario, Canada). Samples were analyzed for combined Annexin V-FITC/propidium iodide staining (to detect late apoptotic cells and postapoptotic cells; ref. 28) using an EPICS XL-MCL flow cytometer (Beckman Coulter, Hialeah, FL). Data were analyzed using CellQuest software.

Pan-Caspase Inhibitor Assessment of Apoptosis
Cells were plated in triplicate (1 x 105; 2 mL RPMI 1640/10% FBS, 25-cm2 flasks). Medium was replaced 24 hours later with 1 mL medium containing the pan-caspase inhibitor Z-VAD-FMK [20 µmol/L; Sigma; a modification of the protocol of Polverino and Patterson (29)]. After 1 hour, the Z-VAD-FMK medium was removed from the cells and added to an equal volume of 2x transfection mix without oligodeoxynucleotide, or containing 100 nmol/L control or antisense oligodeoxynucleotide, to generate 1x transfection mix containing 10 µmol/L Z-VAD-FMK with or without 50 nmol/L control or antisense oligodeoxynucleotide. The resulting transfection mix (50 nmol/L oligodeoxynucleotide + 10 µmol/L Z-VAD-FMK) was added, in triplicate, to appropriate cell cultures. After 24 hours, the cells were analyzed for apoptosis as described above.

Antisense Oligodeoxynucleotide Uptake in Cell Lines
Direct assessment by in situ hybridization. Differences in the capacity of cell lines to be transfected might account for differences in antisense effectiveness. To reveal any such differences, cells (5 x 104; 200 µL RPMI 1640/10% FBS) were added to wells of an eight-well plastic microscope slide (Nunc Lab-Tek II; Life Technologies, Rockville, MD) and transfected with 25 or 50 nmol/L oligodeoxynucleotide as described above. After 24 hours, cells were fixed in 4% buffered formalin (20 minutes, 20°C), rinsed thrice with 3x PBS (2 minutes per rinse, 20°C), dehydrated in increasing ethanol concentrations (50%, 70%, 95%, and 100%), and air-dried. Oligodeoxynucleotide associated with cells was detected by hybridization with complementary, biotinylated 20-mer oligodeoxynucleotide probe (200 ng; Sigma Genosys) in a total volume of 50 µL hybridization buffer [4x SSC, 2 mg/mL nuclease-free bovine serum albumin, 20% (w/v) dextran sulfate]. Slides with added hybridization mix plus biotinylated probe were covered with glass coverslips and incubated in a moist chamber at 20°C. After 24 hours, slides were washed twice in 4x SSC (2 minutes per wash, 20°C) and once in 2x SSC (2 minutes per wash, 20°C), incubated with bovine serum albumin (1% in PBS, 30 minutes, 20°C), drained, and covered with ABC mixture (avidin-biotin-horseradish peroxidase; Vector Laboratories, Burlingame, CA) for 30 minutes. Slides were then washed in 0.1% Tween 20/PBS. 3,3'-Diaminobenzidine substrate (200 µL SigmaFast 3,3'-diaminobenzidine with Metal Enhancer prepared according to the manufacturer's instructions) was placed on the slide and incubated in the dark (20°C, 30 minutes). Slides were rinsed with PBS (5 minutes, 20°C), mounted using Vectashield mounting medium (Vector Laboratories), and photographed. The fraction of stained cells (i.e., positive for hybridization to biotinylated probe) was assessed and confirmed by two independent observers.

Indirect assessment by expression of transfected ß-galactosidase expression vector. Susceptibility of different cell lines to transfection with a ß-galactosidase expression vector was assessed to confirm and quantitatively extend hybridization data. Cells were plated in six-well dishes (1 x 105; 3 mL RPMI 1640/10% FBS per well). Medium was replaced 24 hours later with a 1x transfection mix containing 1 µg pSV-ß (Promega, Madison, WI) and 4 µL LFA-2K. After 48 hours, medium was removed, cells were washed twice with ice-cold PBS, and 200 µL reporter lysis buffer (Promega) was added to each well (15 minutes, 20°C). Resulting lysates were vortexed and centrifuged (14,000 rpm, 2 minutes, 4°C) and supernatants were stored at –80°C. Total protein was measured by Bradford assay. ß-Galactosidase expression was measured in triplicate in 10 µL aliquots by mixing aliquots with 40 µL reporter lysis buffer plus 50 µL of 2x assay buffer (Promega) and by incubation (5 minutes, 37°C) and addition of 150 µL sodium carbonate (1 mol/L). Absorbance (A420 nm) was read using a Wallac Victor2 1420 Multilabel Counter (Perkin-Elmer, Boston, MA). Relative transfectability of cell lines was calculated as ß-galactosidase expression per microgram total protein.

Statistical Methods
Except where noted, the data presented are mean ± SE (n = 3). Each experiment was done at least thrice. Statistical significance was determined using a nonparametric Mann-Whitney rank-sum test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TS Antisense Oligodeoxynucleotide Treatment Decreases TS mRNA and Protein
TS and GAPDH mRNA levels were measured in 211H, H28, and H2052 human mesothelioma cells 24 hours after treatment with 10 to 50 nmol/L TS antisense ODN 83 or control ODN 32 (Fig. 1 ). The degree of down-regulation was slightly variable among malignant mesothelioma cell lines, with TS mRNA virtually abrogated by 50 nmol/L ODN 83 in 211H cells, whereas H28 and H2052 cells retained ~10% to 20% of TS mRNA after similar treatment (Fig. 1B). We showed previously that similar treatment of HeLa cells with 50 nmol/L antisense TS ODN 83 decreased relative TS mRNA levels in a similar fashion by ~70% (21). At some concentrations of control ODN 32 and in some malignant mesothelioma cell lines (211H treated with 25 nmol/L ODN 32, H28 treated with 50 nmol/L ODN 32, and H2052 treated with 10 nmol/L ODN 32), a small but significant increase in TS mRNA was observed (Fig. 1B). However, there was no concomitant increase in TS protein (Fig. 2 ) and no effect on cell growth (Fig. 3 ).


Figure 1
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  		Figure 1. TS mRNA levels are reduced after treatment with antisense TS ODN 83. Human 211H, H2052, and H28 mesothelioma cells were treated for 24 h with the indicated concentrations of TS antisense ODN 83, scrambled control ODN 32, or liposomal transfection medium alone, and cellular RNA was isolated and analyzed for TS mRNA levels (relative to GAPDH mRNA) by reverse transcription-PCR as described in Materials and Methods. A, representative gel showing PCR products generated from 211H cells treated with 10 nmol/L ODN 83. B, relative TS mRNA levels in all three malignant mesothelioma cell lines after treatment with 10 to 50 nmol/L ODN 83 or ODN 32. *, P ≤ 0.01, different from identical cell cultures treated with scrambled control ODN 32 (Mann-Whitney U test); **, P ≤ 0.01, different from identical cell cultures treated with scrambled control ODN 32 or cationic liposomal transfection medium alone (Mann-Whitney U test).

 

Figure 2
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  		Figure 2. TS protein levels are reduced after treatment with antisense TS ODN 83. Human 211H, H2052, and H28 mesothelioma cells were treated for 24 h with TS antisense ODN 83 (50 nmol/L), scrambled control ODN 32 (50 nmol/L), or liposomal transfection medium (LFA-2K) alone and analyzed for TS protein by Western blot as described in Materials and Methods. A, immunochemically visualized TS bands after electrophoresis of 10 µg total cellular protein from all three malignant mesothelioma cell lines with and without antisense TS oligodeoxynucleotide treatment. B, quantitation of TS bands (relative to total protein stained with Coomassie blue) in a representative experiment. Columns, mean (n = 3); bars, SE. *, P ≤ 0.01, different from identical cell cultures treated with scrambled control ODN 32 or cationic liposomal transfection medium alone (Mann-Whitney U test); **, P ≤ 0.01, different from identical cell cultures treated with cationic liposomal transfection medium alone (Mann-Whitney U test).

 

Figure 3
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  		Figure 3. Antisense TS ODN 83 inhibits growth of malignant mesothelioma cells better than nonmalignant mesothelioma cells. Equal numbers of 211H, H2052, H28, MCF7, HeLa, and HT29 cells were treated identically with antisense TS ODN 83, control ODN 32, or LFA-2K alone. Cell numbers were measured 3 d later as described in Materials and Methods. A, growth inhibition in malignant mesothelioma cells. One of three repeats of the same experiment. Columns, mean cell number of three independent cultures as a percentage of cells treated with liposomal transfection medium alone; bars, SE. *, P ≤ 0.01, different from identical cell cultures treated with scrambled control ODN 32 or liposomal transfection medium alone (Mann-Whitney U test). B, comparison of growth inhibition induced by antisense TS ODN 83 in malignant and nonmalignant mesothelioma cells relative to growth of cells treated with control ODN 32. *, P ≤ 0.01, different from all nonmalignant mesothelioma cells treated with the same concentration of ODN 83 (Mann-Whitney U test).

 
Antisense TS ODN 83 treatment of all three malignant mesothelioma cell lines resulted in the expected reduction in TS protein (Fig. 2). Malignant mesothelioma 211H cells had the highest basal TS levels and, in accord with the greater effect of ODN 83 on TS mRNA in 211H cells, 211H cells responded with the greatest reduction in TS protein (by 75%) to treatment with equivalent amounts of ODN 83 compared with H2052 and H28 (where TS protein was reduced by ~66%; Fig. 2B). This degree of down-regulation was similar to the 60% to 75% down-regulation in TS protein we reported previously in HeLa cells treated in a similar fashion (21). At this oligodeoxynucleotide concentration (50 nmol/L), control ODN 32 reduced TS protein compared with total cellular protein in the H2052 cell line alone (Fig. 2B) but without reduction in TS mRNA (Fig. 1). Lower concentrations of ODN 32 (≤25 nmol/L) did not affect TS protein levels in this cell line (data not shown), suggesting a H2052-specific sensitivity to higher levels of combined LFA-2K and untargeted single-stranded oligodeoxynucleotide. Together, these data show that antisense TS ODN 83 treatment of a panel of three mesothelioma cell lines specifically down-regulates TS mRNA to a degree similar, within the limitations of comparison between separate experiments, to that observed in HeLa cells.

Growth Inhibition of Malignant Mesothelioma Cells by Antisense TS
TS mRNA and protein reduction by antisense TS ODN 83 was accompanied by impaired capacity of all three malignant mesothelioma cell lines to increase in number over a 3-day period after transfection as expected in view of previously reported antisense TS ODN 83–mediated reduction in growth of nonmalignant mesothelioma cells (Fig. 3A). It was clear that LFA-2K alone had a concentration-dependent inhibitory effect on cell growth. However, that effect was equal to inhibition observed when LFA-2K was applied in conjunction with control ODN 32. Thus, growth inhibition in the absence of ODN 83 was attributable to in vitro transfection and LFA-2K alone and not to control antisense oligodeoxynucleotide untargeted against TS.

We reported previously that similar treatment of HeLa cells with 25 nmol/L antisense TS ODN 83 reduced proliferation by 25% and 50 nmol/L reduced it by 41% (30). However, the malignant mesothelioma cells treated in the present study were between 2- and 3-fold more sensitive to ODN 83–mediated growth inhibition than HeLa cells and other nonmalignant mesothelioma human tumor cell lines (MCF7 breast tumor–derived cells and HT29 colorectal tumor–derived cells; Fig. 3B). At all three concentrations of oligodeoxynucleotide tested, all three malignant mesothelioma cell lines were significantly more sensitive to ODN 83 than all three nonmalignant mesothelioma cell lines. In fact, 10 nmol/L ODN 83 reduced malignant mesothelioma cell proliferation by >40% in 211H cells and >60% in H2052 and H28 cells but was insufficient to have any effect on proliferation in any of the nonmalignant mesothelioma cell lines (Fig. 3B). Thus, in spite of similar effects of antisense TS ODN 83 on TS mRNA and protein between malignant and nonmalignant mesothelioma human tumor cell lines, malignant mesothelioma cells were uniformly more sensitive to inhibition of growth by antisense targeting of TS mRNA at a concentration that had no effect on nonmalignant mesothelioma cells. In addition, they were consistently more sensitive than nonmalignant mesothelioma cells to growth inhibition by antisense at higher antisense oligodeoxynucleotide concentrations.

Antiproliferative Effects in Mesothelioma Cells Are Independent of Oligodeoxynucleotide Chemistry and Target Sequence
We have reported previously in vitro antisense TS effects using 2-MOE oligodeoxynucleotides (21, 22, 30). To test whether the nature of 2' sugar modifications is a factor in malignant mesothelioma cell sensitivity to antisense TS oligodeoxynucleotides, malignant mesothelioma 211H cells were separately transfected with antisense TS ODN 83 with 2-MO or 2-MOE modifications. Compared with similarly substituted control ODN 32, there was no difference in the capacity of 2-MO and 2-MOE oligodeoxynucleotides to inhibit growth of this cell line (Fig. 4 ). Therefore, antisense TS oligodeoxynucleotides with either 2-MO or 2-MOE substitutions seem to be equally effective in inhibiting mesothelioma cell growth.


Figure 4
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  		Figure 4. Human mesothelioma cells are sensitive to antisense targeting at multiple sites on TS mRNA. 211H malignant mesothelioma cells were treated with antisense TS ODN 83 (50 nmol/L), antisense TS ODN 501 (50 nmol/L), antisense TS 504 (50 nmol/L), or scrambled control ODN 32 (50 nmol/L) as described in Fig. 1 legend and assessed for the effect on cell number 3 d later as described in Materials and Methods. Cell numbers are plotted as a percent of control cells treated with liposomal transfection medium (LFA-2K) alone. Columns, mean of measurements of three independently treated cultures; bars, SE (in every case, SE is calculated as a percent of the mean). *, P ≤ 0.01, different from identical cell cultures treated with scrambled control ODN 32 or liposomal transfection medium alone (Mann-Whitney U test).

 
Other 2-MOE antisense TS oligodeoxynucleotides targeted against different regions of TS mRNA (ODN 501, complementary to 20 nucleotides in the coding region of TS mRNA; ODN 504, targeting 20 nucleotides in the 3'-untranslated region) were also effective in inhibiting 211H growth (Fig. 4). We reported previously that ODN 501 (2-MOE) and ODN 504 (2-MOE), although capable of enhancing the antiproliferative effect of antisense TS ODN 83 in HeLa cells, had no effect on their own at up to 50 nmol/L (ODN 501) or 100 nmol/L (ODN 504; ref. 30). When transfected into malignant mesothelioma 211H cells at 50 nmol/L and using a similar procedure, growth was inhibited by >80% (Fig. 4). Thus, malignant mesothelioma cells seem to be sensitive to growth inhibition by antisense TS oligodeoxynucleotides that have little or no effect at similar concentrations in nonmalignant mesothelioma tumor cell lines.

Differential Sensitivity of Malignant and Nonmalignant Mesothelioma Cells Is Restricted to Response to Antisense TS Oligodeoxynucleotides
Increased sensitivity of human malignant mesothelioma cells to antisense TS targeting suggests that they could be similarly sensitive to chemotherapeutic drugs targeting TS protein and possibly to other drugs capable of inhibiting tumor cell growth independent of TS. We determined the IC50 of a panel of three malignant mesothelioma and three nonmalignant mesothelioma cell lines to five agents in addition to antisense TS ODN 83. Three agents targeted TS (pemetrexed, 5-FdUrd, and raltitrexed) and two did not (cisplatin and gemcitabine). The only agent to which malignant mesothelioma cells were uniformly more sensitive than nonmalignant mesothelioma cells was antisense TS ODN 83 (Table 1 ). Thus, human mesothelioma cell lines seem to be sensitive to TS mRNA targeting and not to targeting of TS protein or non-TS molecules and structures.


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  		Table 1. Sensitivity (IC50) of malignant and nonmalignant mesothelioma cell lines to TS-targeting and non-TS-targeting therapeutic drugs

 
Sensitivity of Mesothelioma Cell Lines Is Not due to Enhanced Uptake of Antisense TS Oligodeoxynucleotides
Although the approximately equal down-regulation of TS mRNA and protein by antisense TS ODN 83 in malignant and nonmalignant mesothelioma cell lines suggests otherwise, it is formally possible that the increased sensitivity of malignant mesothelioma cell lines to antisense TS growth inhibition is due to enhanced uptake of antisense oligodeoxynucleotides compared with nonmalignant mesothelioma cell lines. We tested the transfectability by in situ hybridization to detect transfected antisense TS ODN 83 and the expression of a transfected ß-galactosidase expression vector to ascertain any differential uptake of foreign DNA. We found no consistent differences in oligodeoxynucleotide or expression vector uptake that segregated with malignant mesothelioma cell type (Table 2 ). Thus, it is unlikely that differential sensitivity of malignant mesothelioma cells to antisense TS is due to differences in uptake of antisense reagents.


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  		Table 2. Transfectability of malignant and nonmalignant mesothelioma cell lines with oligodeoxynucleotide or expression vector

 
Antisense TS ODN 83 Induces Apoptotic Cell Death in Malignant Mesothelioma Tumor Cell Lines
We reported previously that ODN 83 induces G2-M cell cycle arrest without apoptosis in HeLa and HT29 cells (23). To determine if the robust antiproliferative effect of TS antisense ODN 83 in mesothelioma cells was due to induction of apoptosis and/or necrotic cell death, ODN 83–treated and control malignant and nonmalignant mesothelioma cells were analyzed by flow cytometry. Consistent with our earlier report (23), ODN 83 did not induce death in any of the three nonmalignant mesothelioma cell lines at 24 hours after transfection compared with untreated cells, cells treated with LFA-2K, or cells treated with LFA-2K plus control oligodeoxynucleotide (Fig. 5A ). No increase in dead cells was evident at other times after transfection (12, 18, or 48 hours; data not shown). On the other hand, ODN 83 induced apoptosis in all three malignant mesothelioma cell lines at 24 hours (Fig. 5A), with lesser numbers of dead and dying cells at 12, 18, and 48 hours after transfection (data not shown). In addition, ODN 501 and ODN 504 (antisense TS oligodeoxynucleotides targeting TS mRNA regions other than that complementary to ODN 83) also induced death in malignant mesothelioma 211H cells. Treatment of the 211H cell line with the pan-caspase inhibitor Z-VAD-FMK substantially reduced the number of dead cells (Fig. 5B), indicating that antisense TS ODN 83 induces death through an apoptotic pathway. Human mesothelioma cells, and not the nonmalignant mesothelioma cells tested, seem to be sensitive to antisense targeting of TS mRNA through induction of apoptosis.


Figure 5
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  		Figure 5. Antisense TS ODN 83 induces apoptosis in malignant mesothelioma but not in nonmalignant mesothelioma human tumor cells. Malignant mesothelioma (211H, H28, and H2052) and nonmalignant mesothelioma (HeLa, MCF7, and HT29) cells were treated with liposomal transfection medium (LFA-2K), control ODN 32 (25 nmol/L), or antisense ODN 83 (25 nmol/L) and assessed for dead and dying cells by flow cytometry as described in Materials and Methods. A, dead and dying cells (% of total cells in culture) 24 h after indicated treatments. *, P ≤ 0.01, different from identical cell cultures treated with LFA-2K alone, ODN 32, or untreated (Mann-Whitney U test). B, effect of pretreatment of malignant mesothelioma 211H cells with a pan-caspase inhibitor (Z-VAD-FMK) before treatment with oligodeoxynucleotide. Columns, mean of measurements of three independent cultures; bars, SE (A and B). *, P ≤ 0.01, different from cells treated with LFA-2K, Z-VAD-FMK, ODN 32 alone, ODN 32 + Z-VAD-FMK, or ODN 83 + Z-VAD-FMK (Mann-Whitney U test); **, P ≤ 0.01, different from identical cell cultures treated with ODN 83 alone (Mann-Whitney U test).

 
Combined Effect of Antisense TS ODN 83 and TS-Targeting Drugs on Growth of Malignant Mesothelioma Cells
We have reported that in HeLa cells antisense TS ODN 83 has a greater than additive effect on growth inhibition in combination with TS-targeting drugs (21). Similar to this, pretreatment of 211H malignant mesothelioma cells with antisense TS ODN 83 (5 nmol/L) to reduce TS mRNA and protein before treatment with pemetrexed or 5-FUdR also potentiated growth inhibition by those drugs (Fig. 6 ). Numbers of cells treated with ODN 83 or ODN 32 alone were normalized to 100%, and cell numbers measured after combined oligodeoxynucleotide plus drug treatment were plotted as a percentage of that normalized value. Therefore, differences between plots of control and ODN 83–treated cells reveal greater than additive growth-inhibitory effects of combined treatment with antisense TS ODN 83 before exposure to pemetrexed or 5-FUdR.


Figure 6
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  		Figure 6. Antisense TS ODN 83 induces, in combination with pemetrexed or 5-FUdR, greater than additive inhibitory effects on malignant mesothelioma cell growth. 211H malignant mesothelioma cells were pretreated with antisense TS ODN 83 (5 nmol/L) or control ODN 32 (5 nmol/L) and then with the indicated concentrations of pemetrexed or 5-FUdR as described in Materials and Methods. Points, mean of three aliquots of cells treated independently with drug as a percentage of values obtained when cells were treated with ODN 83 or ODN 32 without drug; bars, SE. Thus, differences between control oligodeoxynucleotide and antisense oligodeoxynucleotide plots indicate greater than additive effects when antisense oligodeoxynucleotide is combined with TS-targeting drug. *, P ≤ 0.01, different from identical cells treated with ODN 32 (Mann-Whitney U test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Effective chemotherapeutic agents for treatment of malignant mesothelioma are lacking. Standard cisplatin treatment results in median survival of only 10 months (1), and recent trials with single-agent irinotecan (31) or docetaxel (32) or combined raltitrexed/oxaliplatin (33) have revealed significant treatment-related toxicity and mediocre tumor response. Combination of TS-targeted drugs with cisplatin seems, however, to lead to improved response. Cisplatin plus raltitrexed or pemetrexed resulted in tumor responses of 24% and 41%, respectively, and overall survival of 11.4 and 12.1 months, respectively (12, 14). The improvement, although encouraging, is small and requires development. We have reported that antisense TS oligodeoxynucleotides effectively down-regulate TS mRNA and protein, inhibit growth of human tumor cells derived from cervical and colon carcinoma in vitro and in vivo, and enhance sensitivity to TS-targeting drugs (21–23, 30). One antisense TS oligodeoxynucleotide (ODN 83, targeting a 20 base sequence in the 3'-untranslated region of TS mRNA) was among the most effective tested. We hypothesized that the involvement of TS-targeting drugs in better clinical response in malignant mesothelioma indicates that human mesothelioma cells (although resistant to most other cytotoxic chemotherapeutic drugs) are dependent on TS for growth and/or survival and that they are sensitive to antisense oligodeoxynucleotides targeting TS mRNA, particularly antisense TS ODN 83.

To minimize cell line–specific observations, three independently derived mesothelioma cell lines were selected for assessment of response to ODN 83, including one from a lung metastasis (211H) and two derived from pleural effusions (H28 from a 48-year-old male smoker and H2052 from a 65-year-old male). The response of malignant mesothelioma cell lines was compared with those of three nonmesothelioma cell lines originating from cervical carcinoma (HeLa), colon carcinoma (HT29), and breast carcinoma (MCF7). Although the number of cell lines was necessarily limited, the diverse origin of the malignant mesothelioma lines and the broad tumor tissue source of the nonmalignant mesothelioma lines increased confidence that observed differences would be of value in predicting clinical responses to antisense TS reagents.

The capacity of antisense TS ODN 83 to substantially reduce TS mRNA and protein in all three malignant mesothelioma cell lines, and most effectively in 211H cells, was similar to that we have reported in HeLa cells using similar antisense oligodeoxynucleotides in vitro. The efficiency with which all three malignant mesothelioma cell lines and nonmalignant mesothelioma cells internalized ODN 83 was similar, suggesting that the intracellular effectiveness of ODN 83 in reducing TS mRNA in malignant and nonmalignant mesothelioma cell lines was approximately equal, without enhanced effectiveness in the face of low intracellular oligodeoxynucleotide content or reduced effectiveness with increased cellular internalization. Malignant and nonmalignant mesothelioma cell lines variably expressed a transfected ß-galactosidase expression vector, but high or low expression did not segregate with mesothelioma origin, further supporting the conclusion that no mesothelioma-specific differences in oligodeoxynucleotide uptake contributed to antisense TS-mediated down-regulation of target mRNA.

Similar down-regulation of TS mRNA and protein in malignant and nonmalignant mesothelioma cells suggested that the physiologic consequences would be similar. Surprisingly, ODN 83 inhibited malignant mesothelioma cell proliferation between 2- and 3-fold more effectively than it inhibited growth of any of the three nonmalignant mesothelioma cell lines, including growth inhibition of ~50% among the three malignant mesothelioma cell lines at an oligodeoxynucleotide concentration (10 nmol/L) that had no effect on growth of nonmalignant mesothelioma cells. Heightened sensitivity to antisense TS was not specific to 2-MOE or 2-MO substitutions on the antisense oligodeoxynucleotide base sugars, indicating that antisense chemistry was not a key feature contributing to growth inhibition. Furthermore, antisense TS ODN 501 and ODN 504, which had no inhibitory effect on HeLa cell growth as single agents, profoundly inhibited growth of one of the malignant mesothelioma cell lines (211H, the only one tested with these oligodeoxynucleotides). Overall, multiple human mesothelioma cell lines seem to depend profoundly on TS mRNA and protein for events contributing to growth compared with lesser (but still considerable) dependence on TS in nonmesothelioma tumor cell lines.

Both cell survival and cell cycle events govern the overall number of cells in culture after a defined period of growth. We have reported that ODN 83 does not induce death in HeLa (23), and the same is true for HT29 and MCF7 cells (this report). However, ODN 83 treatment killed substantial numbers of cells in all three mesothelioma cell lines, and ODN 501 and ODN 504 also induced death in 211H malignant mesothelioma cells, through an apparently apoptotic process. This is consistent with the conclusion that the heightened sensitivity of cultured mesothelioma cells of diverse human origin is due, at least in part, to mesothelioma-specific cell death induced by antisense TS oligodeoxynucleotides.

The literature is replete with reports that mesothelioma cells have developed multiple strategies to avoid apoptosis as well as antisense targeting of elements of antiapoptotic pathways, including transforming growth factor-ß2 (34), inhibitor of apoptosis protein-1 (35), survivin (36), and Bcl-xL (37, 38), have been reported to enhance apoptosis in mesothelioma cell lines. However, antisense targeting has not been shown previously to target mesothelioma cells preferentially, nor has it been shown that antisense targeting of a mRNA directing production of a protein only indirectly involved in apoptosis results in death of mesothelioma cells but not of nonmalignant mesothelioma cells. We show here, for the first time, that this is the case in cells treated with antisense TS oligodeoxynucleotides.

Enhanced sensitivity of mesothelioma cells to antisense TS oligodeoxynucleotides raises the possibility of combining treatment with TS-targeting drugs. Pretreatment of malignant mesothelioma cells with antisense TS ODN 83 increased pemetrexed- and 5-FUdR-induced growth inhibition in a greater than additive fashion. This suggests that combined targeting of TS mRNA and protein in the clinical setting may be a mesothelioma-specific strategy to enhance the currently modest capacity of TS-targeting drugs to improve clinical response and survival in malignant mesothelioma patients.

The mechanism by which antisense TS induces death in malignant mesothelioma but not nonmalignant mesothelioma cells is not known. Interestingly, our data suggest that decreased TS enzymatic activity alone is not sufficient to generate the mesothelioma-specific effect: malignant mesothelioma cells were consistently sensitive only to antisense TS ODN 83 (which reduced both TS mRNA and protein) and not to other TS-targeting drugs (pemetrexed, 5-FdUrd, and raltitrexed) that inhibit TS by directly or indirectly inhibiting TS protein activity. We have suggested previously that differential effects of antisense TS targeting TS mRNA, and chemotherapeutic drugs targeting TS protein, indicate that TS mRNA (and possibly TS protein) may have functions other than synthesis of intracellular thymidylate necessary for DNA synthesis and repair (39, 40). The possibility remains that such noncanonical TS mRNA and/or protein functions are critical for mesothelioma survival but not for survival of nonmalignant mesothelioma cells. That possibility holds promise for discovery of survival pathways of exceptional importance to malignant mesothelioma cells that may be informative in generating effective chemotherapeutics for treatment of mesothelioma and are under active investigation in our laboratory.

In summary, we have shown that human mesothelioma cell lines, although approximately equally susceptible in vitro to antisense-mediated reduction in TS mRNA and protein as nonmalignant mesothelioma cell lines, are exceptionally sensitive to the physiologic consequences (apoptosis and growth inhibition) of that down-regulation. Antisense TS oligodeoxynucleotides induce apoptosis in malignant mesothelioma but not in nonmalignant mesothelioma cell lines, suggesting that antisense TS oligodeoxynucleotides may be effective therapeutic agents in mesothelioma either alone or in combination with TS-targeting drugs that currently show moderate promise in mesothelioma treatment.


   Acknowledgments
 
We thank Alayne Brisson and Wendy Kennette for excellent technical assistance in cytotoxicity and cell culture assays, Mike Keeney, Wendy Brown, and Leslie Gray-Statchuk for flow cytometry, and Dr. Nicholas Dean (Isis Pharmaceuticals) for 2-MOE oligodeoxynucleotides.


   Footnotes
 
Grant support: Canadian Institute of Health Research grant MOP-62836 and Imperial Oil of Canada Ltd. (J. Koropatnick, R.W. Berg, and M.D. Vincent), Medical Oncology Research Fund London Regional Cancer Program (J. Koropatnick and J. Flynn), and Dr. Saal van Zwanenbergstichting Fellowship (M. van Aken).

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.

Received 2/ 8/06; revised 3/30/06; accepted 4/27/06.


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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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