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Clinical Cancer Research Vol. 12, 5224-5230, September 1, 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Preclinical

Eliminating Established Tumor in nu/nu Nude Mice by a Tumor Necrosis Factor-{alpha}-Related Apoptosis-Inducing Ligand–Armed Oncolytic Adenovirus

Fengqin Dong1,2, Li Wang1, John J. Davis1, Wenxian Hu1, Lidong Zhang1, Wei Guo1, Fuminori Teraishi1, Lin Ji1 and Bingliang Fang1

Authors' Affiliations: 1 Department of Thoracic and Cardiovascular Surgery, The University of Texas M.D. Anderson Cancer Center, Houston, Texas and 2 Department of Metabolism and Endocrinology, The First Affiliated Hospital, Zhejiang University, Hangzhou, Zhejiang, People's Republic of China

Requests for reprints: Bingliang Fang, Department of Thoracic and Cardiovascular Surgery, The University of Texas M.D. Anderson Cancer Center, Houston, TX 7703. Phone: 713-563-9147; Fax: 713-794-4901; E-mail: bfang{at}mdanderson.org.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: The tumor necrosis factor-{alpha}-related apoptosis-inducing ligand (TRAIL) and oncolytic viruses have recently been investigated extensively for cancer therapy. However, preclinical and clinical studies have revealed that their clinical application is hampered by either weak anticancer activity or systemic toxicity. We examined whether the weaknesses of the two strategies can be overcome by integrating the TRAIL gene into an oncolytic vector.

Experimental Design: We constructed a TRAIL-expressing oncolytic adenovector designated as Ad/TRAIL-E1. The expression of both the TRAIL and viral E1A genes is under the control of a synthetic promoter consisting of sequences from the human telomerase reverse transcriptase promoter and a minimal cytomegalovirus early promoter. The transgene expression, apoptosis induction, viral replication, antitumor activity, and toxicity of Ad/TRAIL-E1 were determined in vitro and in vivo in comparison with control vectors.

Results: Ad/TRAIL-E1 elicited enhanced viral replication and/or stronger oncolytic effect in vitro in various human cancer cell lines than a TRAIL-expressing, replication-defective adenovector or an oncolytic adenovector–expressing green fluorescent protein. Intralesional administration of Ad/TRAIL-E1 eliminated all s.c. xenograft tumors established from a human non–small cell lung cancer cell line, H1299, on nu/nu nude mice, resulting in long-term, tumor-free survival. Furthermore, we found no treatment-related toxicity.

Conclusions: Viral replication and antitumor activity of oncolytic adenovirus can be enhanced by the TRAIL gene and Ad/TRAIL-E1 could become a potent therapeutic agent for cancer therapy.

The tumor necrosis factor-{alpha}-related apoptosis-inducing ligand (TRAIL), which induces tumor-selective apoptosis via death receptor–mediated apoptosis, is regarded as a promising anticancer agent. However, the clinical application of recombinant TRAIL may be limited by the facts that only large amounts of TRAIL elicit antitumor activity (1–4) and that a substantial number of tumor cells are resistant to recombinant TRAIL proteins (5, 6). On the other hand, virotherapy with oncolytic viruses is designed to selectively replicate and kill tumor cells by using viral mutants (7–9) or by using tumor-specific promoters (10, 11) to control viral replication. More than 10 clinical trials involving >200 patients with cancer have been conducted with Onyx-015, the first oncolytic virus described (12–14). The reported results showed that oncolytic viruses administered i.t., i.p., and i.v. were well tolerated. However, the efficacy of Onyx-015 as a single agent is also limited, possibly because of inefficient cell lysis and release of their progeny, which may be defective in cancer cells (15). Indeed, a clinical trial with Onyx-015 vector showed that although the vector was present and replicating in tumors as long as a week after its intralesional injection, no obvious tumor necrosis or apoptosis was detected (16), suggesting that its replication alone may be insufficient to induce cell death.

We hypothesized that integrating TRAIL gene therapy with an oncolytic adenovector will overcome the weaknesses of the TRAIL therapy and virotherapy used individually. An oncolytic virus will replicate in tumor cells, thereby increasing the copy number or expression levels of the human telomerase reverse transcriptase (hTERT)-TRAIL gene, and the expression of TRAIL will lead to apoptosis, facilitating lysis of tumor cells, release of vector progeny, and spread of the vector within tumors. Moreover, the bystander effect of the TRAIL gene will enable the killing of untransduced cells in tumor tissue. Furthermore, we have recently found that a synthetic promoter consisting of sequences from the hTERT promoter and a minimal cytomegalovirus (CMV) early promoter (designated hTC promoter) has stronger transcriptional activity than the wild-type hTERT promoter has and retains the tumor selectivity of the hTERT promoter. A green fluorescent protein (GFP)–expressing oncolytic virus, Ad/hTC-GFP-E1, whose transgenes, GFP and E1A, are both under the control of the hTC promoter, induced tumor-specific transgene expression and oncolysis (17). Thus, an hTC-driven TRAIL-expressing oncolytic adenovector may have potent antitumor activity with minimal toxicity to normal cells.

To test whether the therapeutic potency of the TRAIL-armed oncolytic virus is greater than that of an oncolytic virus alone, we constructed the adenovector Ad/TRAIL-E1 and compared its antitumor activity with that of a TRAIL-expressing, replication-defective adenovector and that of the GFP-expressing oncolytic adenovector both in vitro and in vivo. Our results showed that integrating the three components (the TRAIL gene, oncolytic virus, and hTERT promoter) into a single therapeutic agent effectively induced potent tumor-specific cytotoxic effects.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and cell culture. 293 cells, human non–small cell lung cancer (NSCLC) cell lines A549, H1299, H460, and the human colon cancer cell line DLD1 and its TRAIL-resistant derivative, DLD1/TRAIL-R, were maintained in our laboratory. The A549 cells were cultured in Ham's/F12 medium containing 10% heat-inactivated fetal bovine serum and 1% (v/v) antibiotics (penicillin and streptomycin; Life Technologies, Rockville, MD). The other cell lines were maintained in RPMI 1640 containing 10% (v/v) heat-inactivated fetal bovine serum and 1% (v/v) antibiotics. The normal human fibroblast (NHFB) were maintained in DMEM containing 10% heat-inactivated fetal bovine serum and 1% antibiotics. All cells were cultured at 37°C in a humidified incubator containing 5% CO2.

Vector production. The hTC promoter was created as described previously (17). Ad/CMV-GFP, Ad/gTRAIL, Ad/GFP-E1, and Ad/TRAIL-RGD have been described previously (18, 19). The recombinant oncolytic vector Ad/TRAIL-E1 was constructed by placing hTC upstream of both the TRAIL expression cassette and the E1A gene in a shuttle plasmid designated pAd/hTC-TRAIL-E1. The plasmid was then transfected with a ClaI-digested 35 kb fragment of adenovirus type 5 into 293 cells, and Ad/TRAIL-E1 was identified by PCR analysis. The vector was plaque purified twice, expanded in 293K cells, and purified by two cycles of cesium chloride banding.

The sequences in the hTC-TRAIL cassette and the hTC-E1 region were verified by DNA sequencing. Vector amplification, purification, titration, and quality tests were done as previously reported (20). The titer used in this study was determined by the absorbance of the dissociated virus at A260nm [1 A260nm unit = 1012 viral particles (vp)/mL], and the titers determined with a plaque assay were used to determine additive information. Particle/infectious unit ratios were usually between 30:1 and 100:1. Thus, the multiplicity of infection (MOI) of 100 vp was equivalent to a MOI of 1 to 3 infectious units. Unless otherwise specified, Ad/CMV-GFP was used as the vector control, and PBS was used as the mock control.

Reverse transcription-PCR analysis. Total RNA was isolated by using Trizol (Invitrogen, Carlsbad, CA) according to the instructions of the manufacturer, mixed with a random hexamer primer, and incubated at 70°C for 5 minutes. Single-strand cDNA was synthesized from 0.5 µg total RNA by using Moloney murine leukemia virus reverse transcriptase and deoxynucleoside triphosphate at 42°C for 1 hour and 90°C for 5 minutes; synthesis was stopped at 4°C. The mRNAs for TRAIL were amplified by PCR with specific primers. The sequences of the primers for TRAIL were 5'-AGTCCATATTCTGCATCTTT and 5'-AGATGACAGTTATTGGGACC. Glyceraldehyde-3-phosphate dehydrogenase was used as the control.

The PCR reaction was carried out as follows: 1x at 94°C for 3 minutes; 35x at 94°C for 45 seconds, 55°C for 45 seconds, and 72°C for 1 minute; and 1x at 72°C for 10 minutes. PCR products were analyzed by using agarose gel electrophoresis and visualized with the use of ethidium bromide.

Western blot analysis. To assess the protein concentrations in the cell extracts, we did Western blotting. After being washed with PBS, the four NSCLC cells lines were lysed in triple-detergent lysis buffer [20 mmol/L Tris·Cl (pH 8.0), 150 mmol/L sodium chloride, 0.02% sodium azide, 0.1% SDS, 100 µg/mL phenylmethylsulfonyl fluoride, 1 µg/mL aprotinin, 1% NP40, 0.05% sodium deoxycholate, and complete proteinase inhibitors]. Lysates were spun down at 15,000 rpm for 15 minutes at 4°C, and the supernatant was carefully collected. The protein concentration was assessed by the bicinchoninic acid protein assay method (Pierce Chemical Company, Rockford, IL).

The total extracts (100 µg/lane) were normalized and subjected to SDS-PAGE (12% gels), immunoblot assay, and blotting was done on a Hybond-electrochemiluminescence membrane (Amersham, Piscataway, NJ). The signal was then detected by electrochemiluminescence solution (Amersham, Arlington Heights, IL). Western blot analyses were done using specific antibodies against caspase-8 (Medical & Biological Laboratories Co., Woburn, MA), E1A and caspase-3 (Santa Cruz Biotechnology, Santa Cruz, CA), and ß-actin (Sigma, St. Louis, MO), as described previously (21).

Finally, horizontal scanning densitometry was done on Western blots using Adobe Photoshop software (Adobe Systems, Inc., San Jose, CA). The expression of ß-actin was used as an internal control.

Crystal violet staining assay. Cells were seeded into 12-well plates and infected with different doses of the viruses. After 48 hours, the culture medium was removed by inverting the plates and shaking out the medium. Then, to fix the cells, 500 µL of 1% glutaraldehyde in PBS was added to each well for 15 minutes at room temperature. After the 1% glutaraldehyde in PBS was removed by inverting and shaking the plates, 500 µL of 0.5% (w/v) crystal violet was added to each well for 15 minutes. Finally, the plates were washed under gentle tap water until clear and allowed to air dry.

Cell viability assay. The viability of the cell lines was determined by sulforhodamine B colorimetric assay, as previously described (22). Briefly, after fixation of adherent cells with trichloroacetic acid in a 96-well microplate, the protein was stained with sulforhodamine B, and the absorbance was determined at 570 nm to reflect the number of stained cells, representing cell viability. The percentage of viable cells was determined relative to the cell viability of the PBS control, which was considered 100%. Each experiment was done in quadruplicate and repeated at least thrice.

Flow cytometric assay. We used flow cytometry assay to determine the TRAIL gene expression and apoptosis. For apoptosis assay, both floating and attached cells were collected and washed twice with cold PBS. The cells were then fixed with cold 70% ethanol and kept overnight at 4°C. Thirty minutes before the flow cytometric assay, we did propidium iodide staining (1 mL propidium iodide, 10 µL RNase, and 9 mL PBS for a final concentration of 50 µg/mL). The flow cytometric evaluation of cell cycle status and apoptosis was done as previously described (18, 19). The percentages of cells in the sub-G1, G1, S, G2, and M phases were calculated using Cell Quest software (Becton Dickinson, San Jose, CA). Fluorescence-activated cell sorting analysis for TRAIL expression on cell surface was done as described previously (23). In brief, 1 x 106 cells suspended in 100 µL PBS were incubated at 4°C for 1 hour with a rabbit polyclonal anti-TRAIL antibody (H257, Santa Cruz Biotechnology) at a concentration of 10 µL/mL. After washing twice with 1% fetal bovine serum/PBS, the cells were incubated in the dark at 4°C for 30 minutes with a FITC-labeled goat anti-rabbit immunoglobulin antibody (PharMingen, San Diego, CA) at a concentration of 10 µL/mL. After washing twice with 1% fetal bovine serum/PBS, the cells were suspended in 1% formaldehyde in PBS and subjected to fluorescence-activated cell sorting analysis. Normal rabbit IgG (Santa Cruz Biotechnology) was used as a control for primary antibodies; the levels detected by this control antibody were used as a basal background.

Adenovirus replication assay. To assess the efficacy of adenovirus replication in the various cell lines, the cells were infected with adenovectors at the doses described in the text. Four hours after their infection, the cells were washed with PBS thrice to remove any free vector in the medium. The cells were then cultured in fresh medium for various times as indicated in the text. They were then harvested and lysed, and the viral vector replication in cell lysate was determined using the tissue culture infectious dose 50 (TCID50) assay in fresh 293 cells as described previously (24).

Animal study. Six-week-old female specific pathogen-free nu/nu nude mice were purchased from Charles River Laboratories, Inc. (Wilmington, MA), and housed at The University of Texas M.D. Anderson Cancer Center under conventional conditions. Animal experiments were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals (NIH publication no. 85-23) and the institutional guidelines of M.D. Anderson Cancer Center.

We injected 2 x 106 H1299 cells in 100 µL PBS s.c. into the dorsal flanks of mice using a 25-gauge needle. One week after the tumor cell inoculation, when the tumors had grown to 3 to 5 mm in diameter, the mice were randomized into five treatment groups: PBS, Ad/CMV-GFP, Ad/GFP-E1, Ad/TRAIL-RGD, and Ad/TRAIL-E1. For each treatment, 100 µL PBS with or without 2.5 x 1010 vp were injected i.t. with a 27-gauge needle. The treatments were repeated every 3 days for a total of four treatments.

The tumors were measured every 3 days with calipers, and their volumes were calculated using the formula: a(b2) / 2, for which a and b represented the longest and shortest diameters, respectively. The mice were euthanized when their tumors reached 1.5 cm in diameter or became ulcerated.

To assess the toxicity of the treatments, we collected blood samples 5 days after the final injection by cutting the tips of the tails and measured the concentrations of serum liver enzymes (aspartate aminotransferase and alanine aminotransferase), blood cell counts (red cells and white cells), and hemoglobin levels.

Statistical analyses. Differences between experimental groups were determined by ANOVA using software from Statistica (Tulsa, OK). P ≤ 0.05 was considered significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Construction and characterization of Ad/TRAIL-E1. To construct our TRAIL-armed, replication-competent adenoviral vector (Ad/TRAIL-E1), we first constructed a shuttle plasmid–containing expression cassette for both the TRAIL and E1A genes, phTC-TRAIL-E1 (Fig. 1A ). Both genes were driven by the hTC promoter. Ad/TRAIL-E1 was constructed by cotransfecting 293 cells with shuttle plasmids and a 35 kb ClaI fragment from adenovirus dl324, an E3-deleted type 5 human adenovirus (Fig. 1A). The expression of the TRAIL gene from Ad/TRAIL-E1 was assessed by using reverse transcription-PCR analysis. For this purpose, H1299 human NSCLC cells were infected with 1,000 MOI of Ad/CMV-GFP, Ad/GFP-E1, Ad/gTRAIL, Ad/TRAIL-RGD, and Ad/TRAIL-E1. We harvested the cells 12 to 24 hours later and isolated the mRNA for reverse transcription-PCR. The results showed that the TRAIL-specific bands were easily detectable in cells treated with Ad/gTRAIL, Ad/TRAIL-RGD, or Ad/TRAIL-E1 but not in other samples (Fig. 1B). Moreover, direct PCR analysis from the same mRNA samples yielded no signal, demonstrating that the reverse transcription-PCR bands did not result from contamination of vector DNA.


Figure 1
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  		Fig. 1. Construction and characteristics of Ad/TRAIL-E1. A, diagrams of the shuttle plasmid vector pAd/hTC-TRAIL-E1 and the oncolytic adenovector Ad/TRAIL-E1. CMV-E, CMV enhancer; BGH pA, bovine growth hormone polyadenylation signal. B, reverse transcription-PCR analysis of TRAIL mRNA in the H1299 NSCLC cell line after treatment with PBS or adenovectors at MOIs of 1,000 vp/cell for 12 and 24 hours. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control. Direct PCR analysis from the same mRNA samples was shown in the middle panel. PCR products were analyzed by using 1.2% agarose gels electrophoresis. C, E1A expression of different cancer cell lines infected with Ad/CMV-GFP, Ad/GFP-E1, Ad/TRAIL-RGD, or Ad/TRAIL-E1 at MOIs of 100 or 1,000 for 48 hours. E1A was detected only in cells infected with Ad/GFP-E1 and Ad/TRAIL-E1. D, fluorescence-activated cell sorting analysis for TRAIL-positive cells. H1299 cells were treated with PBS or 500 MOI of Ad/CMV-LacZ, Ad/TRAIL-RGD, or Ad/TRAIL-E1. The TRAIL-positive cells were determined at 48 hours after treatment. Columns, mean of three assays; bars, SD. *, P < 0.05, compared with other groups.

 
We also tested E1A expression in cancer cells treated with the various vectors. Cells were infected with 100 or 1,000 MOI of Ad/CMV-GFP, Ad/TRAIL-RGD, Ad/GFP-E1, or Ad/TRAIL-E1 for 48 hours, and cell lysates were analyzed by Western blotting. Treatment with Ad/CMV-GFP and Ad/TRAIL-RGD revealed no detectable E1A expression, whereas treatment with Ad/GFP-E1 or Ad/TRAIL-E1 led to marked expression of E1A (Fig. 1C). The expression of the TRAIL protein on cell surface was determined by fluorescence-activated cell sorting analysis as we previously described (23). Increased TRAIL expression was observed in H1299 cells treated with 500 MOI of Ad/TRAIL-RGD and Ad/TRAIL-E1 vectors but not in H1299 cells treated with PBS or the same dose of Ad/CMV-LacZ (Fig. 1D).

Cell killing effect of Ad/TRAIL-E1 in vitro. The oncolytic function of Ad/TRAIL-E1 was evaluated by crystal violet staining in H1299 cells treated with the various adenovectors. Treatment with Ad/TRAIL-E1 at an MOI of 100 vp resulted in complete cell lysis within 48 hours. In contrast, treatment of the cells with Ad/GFP-E1 resulted in incomplete cell lysis within 48 hours, even at an MOI of 1,000 vp (Fig. 2A ). These results suggest that Ad/TRAIL-E1 effectively induces oncolysis in some cancer cells.


Figure 2
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  		Fig. 2. Cell killing effects of different vectors. A, crystal violet staining of H1299 cells treated for 4 days with PBS or with different doses (MOIs, left) of the adenovectors. B, cell viability assay. Three NSCLC cell lines, two colon cancer cell lines, and NHFBs were treated with various vectors for 96 hours at various MOIs. Cell viability was determined by using the sulforhodamine B assay. Cells treated with PBS were used as mock control (viability was set at 100%, per text). The data are representative of four quadruplicate assays with similar results. Points, means; bars, SD.

 
To further determine the oncolytic effect of Ad/TRAIL-E1, we treated the human lung cancer cell lines H1299, A549, and H460; the TRAIL-sensitive and TRAIL-resistant human colon cancer cell lines DLD1 and DLD1/TRAIL-R; and the NHFBs with Ad/TRAIL-E1, Ad/GFP-E1, Ad/TRAIL-RGD, and Ad/CMV-GFP at various MOIs ranging from 30 to 3,000 vp/cell (Fig. 2B). Cells treated with PBS were used as a mock control. Cell viability was determined by using a sulforhodamine B colorimetric assay 4 days after treatment.

In all the cancer cell lines tested, Ad/TRAIL-E1 effectively elicited oncolytic effects at MOIs of 30 to 300 vp/cell, whereas Ad/GFP-E1 and Ad/TRAIL-RGD induced dramatic oncolytic effects only at MOIs of 300 to 3,000 vp/cell, suggesting that the oncolytic potency of Ad/TRAIL-E1 is at least 10 times greater than that of Ad/TRAIL-RGD or Ad/GFP-E1 (Fig. 2B). Treatment with Ad/CMV-GFP induced no detectable oncolytic effect at any of the doses tested or induced only a mild oncolytic effect at higher doses. In the NHFBs, none of the vectors resulted in more than minimal toxicity. Thus, Ad/TRAIL-E1 seems to be highly oncolytic, but minimally toxic, to normal cells.

Apoptosis induction by Ad/TRAIL-E1 in cancer cells. To determine whether the decrease in cell viability observed after treatment with Ad/TRAIL-E1 was associated with apoptosis, we did cell cycle analysis using flow cytometry assay. H460, H1299, DLD1, and DLD1/TRAIL-R cells and NHFBs were treated with the various adenovectors at MOIs of 1,000 vp per cell for 48 hours. The cells were then harvested and analyzed by fluorescence-activated cell sorting. We found that treatment with Ad/TRAIL-E1 induced apoptosis in all four cancer cell lines, as evidenced by a marked increase of cells in the sub-G1 phase relative to the number in the cells treated with Ad/TRAIL-RGD or Ad/GFP-E1 (P < 0.05; Fig. 3A ), whereas the cancer cells treated with PBS or Ad/CMV-GFP had only background levels of apoptosis (2.0-2.4%). No obvious apoptosis was found in the NHFBs treated with any of the vectors, an observation consistent with the previous one that treatment with these adenovectors led to only minimal loss of cell viability.


Figure 3
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  		Fig. 3. Apoptosis induction. The indicated cancer cell lines and NHFBs were treated with the different vectors at MOIs of 1,000 for 48 hours. A, the ratio of apoptotic cells was shown by the percentage of cells at sub-G1 phases, which was determined by fluorescence-activated cell sorting analysis. No significant difference in the sub-G1 percentage of NHFB cells was found (P > 0.05), whereas cancer cells showed a significant increase in the sub-G1 percentage after treatment with Ad/GFP-E1, Ad/TRAIL-RGD, and especially Ad/TRAIL-E1 (P < 0.05). Representative of three experiments with similar results. Columns, mean; bars, SD. B, Western blot analysis of caspase-3 and caspase-8 in H460 cells 48 hours after treatment with PBS (lane 1), Ad/CMV-GFP (lane 2), Ad/GFP-E1 (lane 3), Ad/TRAIL-RGD (lane 4), and Ad/TRAIL-E1 (lane 5). ß-Actin was used as the loading control. Representative of three experiments with similar results.

 
Apoptosis induction by Ad/TRAIL-E1 was further confirmed by Western blot analysis of the H460 lung cancer cell line. Treatment with Ad/TRAIL-E1 or Ad/TRAIL-RGD at an MOI of 1,000 for 48 hours led to marked cleavage of caspases 3 and 8 (Fig. 3B). This cleavage was not detectable, however, in H460 cells treated with Ad/CMV-GFP or Ad/GFP-E1 at the same MOI.

Adenovirus replication in vitro. It is possible that premature TRAIL-mediated apoptosis in TRAIL-sensitive cancer cells blocks replication of the oncolytic TRAIL vector, thereby reducing the oncolytic effects of the TRAIL-armed oncolytic vector. Therefore, we compared the viral replication of Ad/TRAIL-E1 and Ad/GFP-E1 in TRAIL-sensitive DLD1 cells and TRAIL-resistant DLD1/TRAIL-R cells. We treated 1 x 105 DLD1 and DLD1/TRAIL-R cells with Ad/TRAIL-E1, Ad/GFP-E1, Ad/CMV-GFP, or Ad/TRAIL-RGD at MOIs of 100 or 1,000 vp/cell. Four hours after being infected, the cells were washed with PBS thrice to remove any free vector in the medium. The cells were then cultured in fresh medium for 48 or 96 hours. They were harvested and their lysates were analyzed for viral progeny titration in fresh 293 cells by using the median tissue culture infective dose (TCID50) assay. We found 106 to 108 infectious viral units in DLD1 or DLD1/TRAIL-R cells infected with either Ad/GFP-E1 or Ad/TRAIL-E1 (Fig. 4A ). No detectable infectious units (i.e., <103, the lowest dilution the assay can measure) were found in the lysates from the cells treated with Ad/CMV-GFP or Ad/TRAIL-RGD, demonstrating that the oncolytic vectors but not the E1-defective vectors were replicating in those cancer cells. An interesting finding was that in both cell lines and at both initial treatment doses, the final yield of infectious viral units in cells treated with Ad/TRAIL-E1 was much higher than it was in those treated with Ad/GFP-E1. A similar result was observed when H1299 cells were treated for up to 7 days with Ad/TRAIL-E1 or Ad/GFP-E1 at an MOI of 500 vp and their viral titer determined in cell lysate (Fig. 4B). The significance of this difference between Ad/GFP-E1 and Ad/TRAIL-E1 is not yet clear. However, our results showed that Ad/TRAIL-E1 could effectively replicate in TRAIL-sensitive cancer cells. Nevertheless, in NHFBs, both Ad/TRAIL-E1 and Ad/GFP-E1 produced only background level of viral replication, demonstrating the tumor specificity of these vectors.


Figure 4
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  		Fig. 4. In vitro replication of oncolytic virus Ad/GFP-E1 and Ad/TRAIL-E1. A, the viral titers in DLD1 and DLD1/TRAIL-R cells after treatment with the oncolytic virus at MOIs of 100 and 1,000 for 48 or 96 hours. B, the viral titer in H1299 and NHFB cells after treatment with vectors at MOI of 500. The viral titer in cells treated with Ad/CMV-GFP and Ad/TRAIL-RGD at the same doses was not detectable.

 
Suppression of tumor growth in vivo. To further evaluate the antitumor activity of Ad/TRAIL-E1, we established human NSCLC xenografts in 6-week-old female nude mice by inoculating them with H1299 cells. I.t. administration of the adenovectors was started when the resulting tumors grew to 3 to 5 mm in diameter. After four i.t. injection treatments with the vectors, tumor volume was monitored over time.

As we have observed previously (17), treatment of H1299 tumors with Ad/GFP-E1 or Ad/TRAIL-RGD can lead to significant tumor suppression when compared with mock treatment or control vector (Ad/CMV-GFP) treatment (P < 0.05; Fig. 5A ). Nevertheless, tumor growth occurred in a substantial number of animals treated with these two vectors. Although treatment with those two vectors resulted in a tumor-free rate of ~50%, treatment with Ad/TRAIL-E1 led to complete regression of the tumors in all mice. The median survivals in animals treated with PBS or Ad/CMV-GFP were ~31 and 41 days, respectively (Fig. 5B). Treatment with either Ad/TRAIL-RGD or Ad/GFP-E1 increased the median survival to ~86 and 87 days. Nevertheless, treatment with Ad/TRAIL-E1 led to tumor-free survival in all animals for 120 days, the longest time point observed in this study, demonstrating that Ad/TRAIL-E1 can effectively elicit antitumor activity in vivo. We also evaluated possible treatment-related toxicity by measuring serum liver enzymes and blood cell counts (RBC, WBC, and platelets) 5 days after the final injection of the treatments. All of the results were within the reference ranges, and no substantial differences were found between the groups (data not shown).


Figure 5
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  		Fig. 5. In vivo antitumor effect in H1299 s.c. xenograft tumor model. I.t. injection of 2.5 x 1010 vp/treatment was started when tumors reached 4 to 5 mm in diameter. Arrows, time when treatments were given. A, tumor volume was monitored over days after the inoculation of tumor cells. Points, mean; bars, SE. B, Kaplan-Meier survival curve.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The results of our study showed that Ad/TRAIL-E1 was more effective than Ad/TRAIL-RGD or Ad/GFP-E1 at eliciting cytotoxic effects and suppressing tumor growth, supporting our hypothesis that an integrated TRAIL gene therapy with an oncolytic adenovector is more effective than TRAIL gene therapy or oncolytic adenovirotherapy alone.

The major problem for adenovirus-mediated cancer gene therapy in vivo is incomplete transduction of target cancer cells. Whether delivered i.t. or systemically, adenovectors may not reach every tumor cell. Because of the bystander effect of the TRAIL gene (23, 25), adjacent cancer cells may be effectively killed by neighboring cells expressing TRAIL, but cancer cells beyond the direct contact of TRAIL-expressing cells will escape destruction. The use of a replication-competent vector may increase vector spread within the tumor site, thereby dramatically increasing the effective radius of the treatment. An increased therapeutic effect might also result from increased expression of a therapeutic gene that effectively replicates, increasing the number of copies of the viral genome. Moreover, adenovirus E1A is known to induce apoptosis in certain cancer cells by the accumulation of p53 and activation of procaspase-8 (26, 27). Therefore, coordinated triggering of apoptosis by the TRAIL and E1A genes in the oncolytic vector may be another mechanism of enhancing killing of cancer cells. Nevertheless, we found that apoptotic cells determined by sub-G1 assay were always much lower than actual cell killing determined by cell viability assay. One possible explanation is that oncolytic adenovirus, as reported, can trigger cell death by other mechanisms, such as necrosis and autophagy (28, 29). Interestingly, TRAIL can also trigger necrosis or necrosis-like cell death in tumor cells (30, 31).

The defects in the lytic cycle and inefficient release of vector progeny may account for the low efficacy of oncolytic virotherapy that has been seen clinically. Incorporating an apoptosis-inducing gene, such as TRAIL, in an oncolytic virovector will ultimately facilitate lysis of tumor cells, release of vector progeny, and spread of the vector within tumors. However, it is possible that premature TRAIL-mediated apoptosis in TRAIL-sensitive cancer cells blocks replication of the oncolytic TRAIL vector. Therefore, we compared the viral replication of Ad/TRAIL-E1 and Ad/GFP-E1 in TRAIL-sensitive DLD1 and TRAIL-resistant DLD/TRAIL-R cells. Both Ad/TRAIL-E1 and Ad/GFP-E1 effectively replicated in both cell lines. This finding was also consistent with our previous observation that 293 cells were susceptible to the full-length TRAIL gene (23) but can support the amplification of replication-defective or replication-competent TRAIL-expressing vectors.

We found it interesting that in both DLD1 and DLD1/TRAIL-R cells and at both initial treatment doses, the final yield of infectious viral units in cells treated with Ad/TRAIL-E1 was much higher than it was in those treated with Ad/GFP-E1. The significance of the difference between Ad/TRAIL-E1 and Ad/GFP-E1 in generating progeny is not yet clear. However, it is known that in addition to inducing apoptosis, TRAIL induces activation of various other signaling pathways, including Akt (32, 33), nuclear factor-{kappa}B (32, 34), and mitogen-activated protein kinases, such as JNK (34, 35), p38 (36), and extracellular signal-regulated kinase (33, 36). Many of those signaling pathways are required for or supportive of viral replication (37–40), yet the roles of these pathways in the replication of oncolytic TRAIL adenovirus are not known.

In summary, we used hTERT promoters with minimal CMV fragments to drive the TRAIL and E1A genes in Ad/TRAIL-E1. Theoretically, this strategy would limit viral replication and TRAIL expression in cancer cells and prevent side effects on normal tissues. As we had expected, Ad/TRAIL-E1 treatment elicited strong cytotoxic effects in NSCLC and colon cancer cell lines and had only minimal effects on cell viability and apoptosis induction in NHFBs. Because various cancer cells, regardless of their tissue origin, have high hTERT promoter activity (41, 42) and are susceptible to TRAIL-mediated apoptosis (2), we believe that Ad/TRAIL-E1 may be a promising potent, broad-spectrum therapeutic agent against cancer.


   Acknowledgments
 
We thank Chris J. Yeager for editorial review and Debbie Smith for assistance in preparing the manuscript.


   Footnotes
 
Grant support: National Cancer Institute grants RO1 CA 092487-01A1 and RO1 CA 098582-01A1 (B. Fang), Lung Specialized Programs of Research Excellence grant CA 70907, NIH core grant CA-16672, and Lockton Grant-Matching Funds.

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.

Note: L. Wang and J.J. Davis contributed equally to this work.

Received 2/ 2/06; revised 5/31/06; accepted 6/20/06.


   References
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 Abstract
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 References
 

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