Research Article

Overexpression of Soluble TRAIL Induces Apoptosis in Human Lung Adenocarcinoma and Inhibits Growth of Tumor Xenografts in Nude Mice

Juan Shi,1,3 Dexian Zheng,3 Yanxin Liu,3 Mai Har Sham,2 Paul Tam,1 Farzin Farzaneh,4 and Ruian Xu1,3

1Gene Therapy Laboratory, Genome Research Center, 2Department of Biochemistry, University of Hong Kong, Pokfulam, Hong Kong; 3National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Peking, China; and 4Department of Haematological and Molecular Medicine, GKT School of Medicine, King’s College London, United Kingdom

Abstract cells (4–8). Repeated administration of recombinant and biologi- Recombinant adeno-associated virus 2/5 (rAAV2/5), a hybrid cally active soluble TRAIL (sTRAIL) can induce tumor cell rAAV-2 with AAV-5 capsid, seems to be a very efficient delivery apoptosis, suppress tumor progression, and improve survival in vector for the transduction of the lung adenocarcinoma cell tumor-bearing mice (5–7). Furthermore, combination treatment line A549. Infection of the A549 cell line with a rAAV2/5 vector with TRAIL and chemotherapy or ionizing radiation can substan- encoding the extracellular domain of - tially enhance the cytotoxic effects of these agents, even in otherwise resistant tumors (8). However, the application of sTRAIL related apoptosis-inducing ligand (TRAIL, amino acids 114- 281) resulted in secretion of soluble TRAIL (sTRAIL) and in cancer therapy is limited by its short half-life in vivo (9). In induction of apoptosis in these cells. rAAV2/5-sTRAIL medi- addition, the transfer and expression of adenovirus encoded TRAIL, ated delivery and stable expression of sTRAIL resulted in the or TRAIL/green fluorescent fusion protein (GFP/TRAIL), can presence of the trimeric form of sTRAIL in sera of nude mice induce apoptosis and apoptotic bystander effects in several human that were implanted with s.c. or orthotopic A549 tumors. The cancer cells in vitro (10) and in the in vivo xenograft models of rAAV2/5-sTRAIL transduction of the tumors resulted in a human glioblastoma, breast (11), prostate (12), and liver carcinoma statistically significant reduction in tumor growth and (13). However, the use of adenovirus causes hepatotoxicity through prolonged survival of the tumor-bearing animals. Primary the innate and cell-mediated immune responses. In contrast, cell culture, histologic examination of the tumors, and serum adeno-associated virus (AAV)-based vectors are less pathogenic but efficient vehicles for gene delivery. A recent report showed that analyses showed the absence of detectable TRAIL-induced toxicity in normal tissues including the liver. The successful a single i.v. administration of AAV resulted in extensive inhibition of lung cancer growth and the absence of detectable transduction of the cardiac and skeletal muscles of adult animals, toxicity suggest a putative role for rAAV2/5-sTRAIL in thus demonstrating its potential in a range of clinical applications 114-281 of gene therapy (14). the therapy of lung cancer. (Cancer Res 2005; 65(5): 1687-92) In this study, we used a recombinant AAV 2/5 (rAAV2/5) vector, expressing TRAIL , to examine the effect of expression of this Introduction 114-281 soluble form of TRAIL in A549 cells both in vitro and in two in vivo Lung carcinoma is the leading cause of malignancy-related xenograft models of A549 lung carcinoma. The results indicate that mortality (1) with little change in survival rates over the past two rAAV2/5-sTRAIL may provide an effective form of therapy for lung decades (2). Non–small cell lung cancer now accounts for about cancer. three-quarters of all cases of lung cancer (3) and most patients continue to die of progressive metastatic disease despite new therapeutic strategies and advances in surgical oncology. This Materials and Methods failure of conventional therapy is due primarily to a propensity to Animals. Surgical procedures and care of animals, approved by the metastasize as well as resistance to chemotherapy and radiation. Ethics Committee of the University of Hong Kong, were done according to Attempts to overcome resistance through increased dosage institutional guidelines. Male 5- to 6-week-old BALBc nude mice were results in unacceptable toxicity and bystander damage to normal housed at a constant temperature and supplied with laboratory chow and tissues. water ad libitum on a 12-hour dark/light cycle. Tumor necrosis factor-related apoptosis-inducing ligand Construction of the rAAV-sTRAIL and rAAV-eGFP Vectors. The (TRAIL/Apo-2L) is a typical member of the tumor necrosis factor sTRAIL cDNA insert encoding TRAIL amino acids 114 to 281 was family, inducing apoptosis by activating death receptors (4). A amplified from TRAIL cDNA as previously reported (15). The cytomeg- h number of studies have shown that both the membrane-bound and alovirus enhancer/chicken -actin promoter, cDNA, and polyA sequences were inserted between the inverted terminal repeats using appropriate the soluble extracellular domain of TRAIL can induce apoptosis in restriction enzymes. To boost transgene expression, a woodchuck a wide variety of tumor cell lines without affecting most normal hepatitis B virus posttranscriptional regulatory element was inserted into both constructs immediately after the expression cassette (16). DNA sequence analysis confirmed the integrity of the cDNA constructs and the vector. Requests for reprints: Ruian Xu, Gene Therapy Laboratory, Genome Research Pseudotyping. Different pseudotypes of rAAV were generated by Centre, University of Hong Kong, 8S-01, Kadoories BioScience Building, Pokfulam, Hong Kong. Phone: 852-2299-0757; Fax: 852-2817-9488; E-mail: [email protected]. standard production and purification protocols (17). The rAAV titer was #2005 American Association for Cancer Research. quantified by real-time PCR analysis. www.aacrjournals.org 1687 Cancer Res 2005; 65: (5). March 1, 2005

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Transgene Expression In vitro. HEK293 or A549 cells were cultured in the complete medium before the addition of rAAV at a dose of 104 particles per cell in culture medium containing 2% fetal bovine serum for 8 hours, followed by incubation in complete medium containing 10% fetal bovine serum. The transduction efficiency with rAAV-eGFP was analyzed by fluorescence microscopy. The culture media of the transduced cells were collected and concentrated by vacuum desiccation for analysis of the secreted TRAIL. Apoptosis and Cell Death. Cells, fixed for 20 minutes in 4% paraformaldehyde in PBS, were stained with Hoechst 33258 at 1 Ag/mL in PBS for 15 minutes. Apoptosis was visualized with fluorescence microscopy and cytotoxicity was quantified by 3-(4, 5-dimethylthiazole-2-yl)-2,5- biphenyl tetrazolium assay (Sigma, St. Louis, MO). S.c. Tumor Xenografts and Assessment of Growth. A549 cells (5  106) were administered in the s.c. tissue of the left dorsal flank of 7-week-old male nude mice. Tumor growth was monitored regularly and volume (V) was calculated using the formula V = 1/2  length  (width)2. When tumor size reached f50 mm3 (about 10 days post-inoculation), animals were randomized into three groups and vectors were administered by intra- tumoral injection. Groups 1 and 2 received 3  1011 particles rAAV2/5- sTRAIL or rAAV2/5-eGFP, respectively; and group 3 mice received PBS as control. Experiments were terminated when tumors reached 500 to 600 mm3 in the control groups. Orthotopic Lung Cancer Model and Assessment of Growth. Orthotopic implantation of A549 cells was done as described by Yamaura et al. (18). The left chests of anesthetized mice were incised and 20 AL aliquots of the A549 cell suspensions (5  107/mL), containing 1 mg/mL of Matrigel (BD Biosciences, Bedford, MA), were injected into the lung parenchyma through the intercostal space (f3 mm depth). One week later, animals were randomized into three groups receiving intratracheal injections of 30 AL PBS or 5  1011 vector particles. Mice were sacrificed 45 days after tumor cell implantation. Animals were euthanized if they developed gross ascites, palpable tumors >2 cm, dehydration, emaciation, or weight loss >20% of initial body weight. Analysis of Gene Expression and Microvessel Density. The primary antibodies, TRAIL, CD31, caspase-3, and caspase-8 were from Santa Cruz Biotechnology (Santa Cruz, CA), and CD34 were obtained from PharMingen (San Diego, CA). Western blot analysis, histology, immunohistochemical staining, ELISA, and terminal nucleotidyl transferase (TdT)–mediated nick end labeling assays were carried out as previously described (19). Figure 1. rAAV-mediated transduction of A549 cells and sTRAIL-mediated Microvessel density was assessed by the hotspot method (20). induction of apoptosis. A, flow cytometric analysis of transduction efficiency Statistical Analysis. Log rank tests were done for survival data. Student’s of human lung adenocarcinoma cell line A549 with different AAV vectors 4 t test was used to evaluate statistical significance. P values were considered (60 hours after infection at an MOI of 5  10 particles per cell; n = 3). B, A549 cells infected with the indicated vectors (MOI = 5  104) were to be statistically significant when <0.05. examined by fluorescent microscopy after Hoechst staining (Â1,200 magnification). C, cell viability determined by 3-(4, 5-dimethylthiazole-2-yl)-2, 5-biphenyl tetrazolium assay in A549 cells (., rAAV2/5-sTRAIL; E, Results rAAV2/5-eGFP) and in HEK293 cells (n, rAAV2/5-sTRAIL; y, rAAV2/5-eGFP; n = 3). D, Western blot analysis of A549 cells infected with rAAV2/5-sTRAIL To investigate the transduction efficiency of different rAAV (MOI = 5  104) and harvested after 72 hours (left lane, rAAV2/5-eGFP; serotypes, rAAV2/1, rAAV2/2, rAAV2/5, rAAV2/6, and rAAV2/ right lane, rAAV2/5-sTRAIL). 8 vectors expressing eGFP were used to infect the A549 human lung cancer cell line at a multiplicity of infection (MOI) of 5  was also no evidence of toxicity in the transduced HEK293 cells 104. Flow cytometric analysis showed transduction rates to be (Fig. 1C). To further test apoptosis, A549 cells infected with rAAV2/ 32.6 F 3.7% for rAAV2/1, 50.8 F 7.4% for rAAV2/2, 70.4 F 6.7% 5-sTRAIL (MOI = 5  104) were subjected to Western blot analysis, for rAAV2/5, 55.5 F 3.5% for rAAV2/6, and 18.6 F 7.8% for revealing the presence of activated caspase-3 and decreased levels rAAV2/8 (see Fig. 1A). of caspase-8 precursor protein (Fig. 1D). Induction of A549 Tumor Cell Apoptosis by rAAV2/5-sTRAIL. Next we tested whether the encoded TRAIL could be secreted To determine the apoptosis-inducing activity of rAAV2/5-sTRAIL, into the medium. ELISA showed a peak value of 282.9 F 69.7 ng/mL A549 cells were infected with rAAV2/5-sTRAIL or rAAV2/5-eGFP, at 48 hours in the culture media of rAAV2/5-sTRAIL–transduced stained with Hoechst 33258, and microscopically examined for HEK293 cells. In contrast, the maximal sTRAIL concentration was evidence of apoptosis (Fig. 1B). Condensed nuclei, a characteristic only 97.6 F 41.9 ng/mL in the similarly transduced A549 cells, and of apoptosis, were present in substantially greater numbers in the this level was reached 24 hours post-infection. The lower maximal rAAV2/5-sTRAIL, but not the rAAV2/5-eGFP–infected cells. Simi- expression level and the absence of further increases are likely to be larly, analysis of toxicity by the 3-(4, 5-dimethylthiazole-2-yl)-2,5- due to the toxic effects of TRAIL on A549 cells. To test the potential biphenyl tetrazolium assay showed the induction of toxicity in the toxicity of rAAV2/5-sTRAIL on normal cells, primary human rAAV2/5-sTRAIL, but not the rAAV2/5-eGFP–infected cells. There hepatocytes were infected with rAAV2/5-eGFP or rAAV2/5-sTRAIL.

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cell death and substantially reduced numbers of scattered tumor cells (Fig. 3B). Evidence to support tumor cell apoptosis was also provided by terminal nucleotidyl transferase (TdT)–mediated nick end labeling assays demonstrating significantly higher levels of apoptotic cell death in the sTRAIL-transduced tumors on day 45 (Fig. 4A). Immunohistochemical studies confirmed TRAIL expres- sion 45 days after intratumoral injection of rAAV2/5-sTRAIL, but not in the PBS-injected tumors (Fig. 4B). Moreover, there were significant reductions in the microvessel density in the rAAV2/ 5-sTRAIL–treated animals compared with the two control groups (P < 0.05; Fig. 4C and D). Suppression of Orthotopic Lung Tumor Growth Following Intratracheal Administration of rAAV Viruses. Gross macro- scopic examination of the chest cavity showed the presence of tumor nodules on both the left and right lobes of the lung of orthotopic model (Materials and Methods), particularly in the control groups (Fig. 5A), with heart metastases in f30% of the Figure 2. Transgene expression and cytotoxicity in primary human hepatocytes PBS and 20% of the rAAV2/5-eGFP–treated animals (data not in culture. Human hepatocytes were infected at an MOI of 2 Â 105 with rAAV2/5-eGFP (A and C) or rAAV2/5-sTRAIL (B and D) and examined by shown). Microscopic examination of H&E-stained lung tissue fluorescent (A) or light microscopy 5 days later (Â100 magnification). sections confirmed extensive metastatic disease in the control groups, but substantially reduced tumor presence in the lung tissue of the rAAV2/5-sTRAIL–transduced animals (Fig. 5B). This There was no apparent evidence of rAAV2/5-sTRAIL–induced toxicity in these cells, even when the vector dose was increased 4-fold to an MOI of 2 Â 105 and the duration extended to 5 days (Fig. 2). Together, these results show that sTRAIL is secreted from rAAV2/5-sTRAIL–infected cells and that it can induce apoptosis in the human lung cancer A549 cells, but not in the normal human hepatocytes. Suppression of S.c. Tumor Growth. The s.c. model was employed (see Materials and Methods) to analyze the therapeutic potential of rAAV2/5-sTRAIL. The monitoring of tumor growth for f7 weeks revealed substantial growth reduction in the rAAV2/5-sTRAIL–injected tumors (Fig. 3A), but not in the rAAV2/ 5-eGFP or PBS control groups. Histologic examinations confirmed smaller tumors in the rAAV2/5-sTRAIL group, with large areas of

Figure 4. Intratumoral injection of rAAV2/5-sTRAIL induces tumor cell apoptosis and inhibits tumor vascularization. Histologic examination of the tumors was carried out 45 days after intratumoral injection of the indicated vectors Figure 3. Intratumoral injection of rAAV2/5-sTRAIL suppresses the growth of or carrier PBS. A, terminal nucleotidyl transferase (TdT)–mediated nick end s.c. A549 tumors in nude mice. A549 cells (5 Â 106) were injected in the labeling staining of the tumor sections show a much larger fraction s.c. tissue of the left dorsal flank of nude mice. A, tumor growth in the A549 of terminal nucleotidyl transferase (TdT)–mediated nick end labeling-positive tumor-bearing mice that were injected with either rAAV2/5-sTRAIL (E), cells (green) in the rAAV2/5-sTRAIL–injected than in those given the carrier rAAV2/5-eGFP (n), or the carrier PBS (y; n = 7, each). B, representative PBS (Â200 magnification). B, immunochemical detection of TRAIL-positive transverse sections of tumors excised 45 days after vector injection, stained with cells, using a mouse anti-TRAIL antibody (brown, Â400 magnification). C, H&E (Â100 magnification). Tumors injected with rAAV2/5-sTRAIL present tissue sections stained with anti-CD31 (brown, Â200 magnification). D, clear evidence of cell death, but not those in injected with PBS or rAAV2/5-eGFP. blood vessels stained with anti-CD31 (C) were quantified by densitometry. www.aacrjournals.org 1689 Cancer Res 2005; 65: (5). March 1, 2005

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rAAV2/5-sTRAIL Improves the Survival Rate of Mice with Lung Metastases. The orthotopic lung cancer model showed initial mortality on day 46 for the PBS group and on day 47 for the rAAV2/5-eGFP–treated group, whereas the first mortality in the rAAV2/5-sTRAIL–treated mice was on day 60. The median survivals were 52 and 51 days for the PBS and rAAV2/5-eGFP groups, respectively, compared with a median survival of 79 days for the TRAIL-treated mice. All mice in the control groups had to be sacrificed by 68 days, whereas 20% of the TRAIL-treated mice survived for at least 120 days (Fig. 6B). Toxicity of rAAV2/5-sTRAIL In vivo. The toxicity of rAAV2/ 5-sTRAIL treatment was examined in non–tumor-bearing mice following the intratracheal injection of 1 Â 1012 rAAV2/5-sTRAIL. The serum levels of alanine transaminase and aspartate transam- inase were within the reference range 30 days after transduction (Fig. 6C) and histopathologic studies showed no obvious lesions in the lung, liver, spleen, or kidney. In addition, body weight, gross appearance, and behavior provided no signs of systemic toxicity.

Discussion Adenovirus-based vectors are among the most efficient vehicles for the transfer and expression of therapeutic genes (11–13). However, immunologic responses to adenovirus vectors result in

Figure 5. Intratracheal administration of rAAV2/5-sTRAIL suppresses orthotopic A549 tumors in nude mice. Tumor cells (1 Â 106) were implanted into the lung parenchyma through the intercostal space. One week later, animals were randomized into three groups and subjected to intratracheal administration of 5 Â 1011 particles of rAAV2/5-sTRAIL, rAAV2/5-eGFP or the carrier PBS (n = 7). Mice were sacrificed on day 45. A substantial reduction in the number of tumor nodules was visible in the lungs of the rAAV2/5-sTRAIL–transduced animals, but not in those injected with rAAV2/5-eGFP or the carrier PBS (A and C). B, histologic examination of H&E-stained lung tissue sections (Â100 magnification). C, columns, the number of tumor nodules in the lungs; bars, SD (n = 5). D, immunochemical detection of sTRAIL protein in the lung tissue was examined 38 days after rAAV2/5-sTRAIL transduction (brown, Â800 magnification). E, Western blot analysis of serum proteins 38 days after the intratracheal transduction with rAAV2/5-sTRAIL. The trimeric form of sTRAIL presents in the serum of the rAAV2/5-sTRAIL group (lanes 2-4), but not in the rAAV2/5-eGFP group (lane 1). was consistent with the macroscopically visible number of lung tumor nodules on day 45: 61 F 12 and 58 F 8 in the PBS and rAAV2/5-eGFP groups, compared with 20 F 12 in the rAAV2/ 5-sTRAIL treated group (P < 0.01 for either the PBS or rAAV2/ 5-eGFP groups; Fig. 5C). By day 60, the number of lung tumor nodules had increased further in the TRAIL-treated groups. However, this was still significantly lower than the number of nodules in the PBS (P = 0.017) or the rAAV2/5-eGFP–treated groups (P = 0.015). The expression of TRAIL in lung tissue sections isolated from Figure 6. sTRAIL-mediated inhibition of angiogenesis and the increased the rAAV2/5-sTRAIL–transduced group was confirmed by immu- survival of tumor-bearing mice. A, tissue sections stained with anti-CD34 nohistochemistry. The highest levels of TRAIL expression were in for the detection of tumor vasculature (brown, Â400 magnification). the airway epithelial cells (Fig. 5D) with biologically active trimeric The PBS group sacrificed on day 45 (1), and the rAAV2/5-sTRAIL group sacrificed either on day 45 (2) or day 60 (3). B, the intratracheal introduction form of TRAIL in the sera of the rAAV2/5-sTRAIL–transduced of rAAV2/5-sTRAIL prolongs the survival of mice with orthotopic A549 animals (Fig. 5E), corresponding to a circulating serum level lung tumors. The mice received 5 Â 1011 particles of rAAV2/5-sTRAIL, F the empty rAAV2/5 vector, or PBS, 7 days after the orthotopic implantation of of 114 29 ng/mL at 38 days after the intratracheal rAAV2/ 1 Â 106 A549 cells. The animals were monitored for the appearance of 5-sTRAIL administration. The statistically significant reduction tumor growth and indicators of morbidity necessitating their euthanasia in the microvessel density in the rAAV2/5-sTRAIL–treated mice, (see Materials and Methods; n = 10, P < 0.01). C, hepatotoxicity of rAAV2/5-sTRAIL in vivo. Serum samples were collected 30 days after measured on day 45 after tumor cell implantation (P < 0.05; Fig. the intratracheal introduction of 1 Â 1012 particles of rAAV2/5-sTRAIL (1) 6A) decrease, was no longer significant by day 60. or PBS (2) to non–tumor-bearing mice.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. TRAIL Gene Therapy for Lung Cancer the immune-mediated clearance of the transduced cells, hence Although TRAIL has been shown to induce apoptosis in various reduced level and duration of expression of the encoded gene cancer cells, there are discrepancies in the published data on the products, as well as limited scope for repeat administration of these sensitivity of HEK293 cells to TRAIL. Some studies have found vectors (21). The reduced immunogenicity of AAV-based vectors HEK293 cells to be sensitive to TRAIL toxicity (31), whereas others provides distinct advantages with respect to each of these suggest the resistance of these cells to TRAIL-induced apoptosis limitations. Nevertheless, despite some success with AAV2-based (32). In the present study, we could detect no apoptosis in the vectors in the lung (22), the luminal introduction of AAV has had rAAV2/5-sTRAIL–transduced HEK293 cells for about 4 days. One largely disappointing results (23). Vectors based on AAV5 or AAV6 possible explanation is that the effect of sTRAIL is likely to be dose- have been shown to enter from the apical side and more efficiently and/or conformation-dependent for different cell lines. Cell line transduce airway epithelial cells (24). Capsid proteins from AAV5 differences in receptor status and effector pathways may also have also been shown to bind to sialic acid (24), a component of the account for different responses to TRAIL. The present study shows putative specific receptor present on the apical surface of airway that the in vivo transduction of A549 tumors with rAAV2/5-sTRAIL epithelial cells. Consistent with these observations, the rAAV2/5 resulted in decreased presence of endothelial cell markers CD31 generated in the present studies has provided an efficient vehicle for and CD34. Regulation of the angiogenic process involves a delicate the delivery and expression of TRAIL in both mouse airway epithelia balance of factors to promote and inhibit neovascularization. and in the A549 human lung adenocarcinoma cells. As a Tumors seem to be able to disrupt this balance, resulting in consequence, high levels of TRAIL protein were readily detectable neoangiogenesis and development of a microenvironment favoring both at the site of vector administration and in the serum of these tumor growth and metastasis (33). Li et al. (34) have shown that animals for at least 38 days after a single intratracheal administra- isolated endothelial cells from human umbilical veins or human tion of the vector. The serum levels detected for TRAIL (114 F dermal microvessels, expressing DR4 and DR5, are sensitive to 29 ng/mL) were similar to the levels detected 2 hours after a single TRAIL-induced apoptosis. However, several other studies have i.v. injection of 10 mg/kg recombinant TRAIL protein in nude mice shown the resistance of primary human endothelial cells to TRAIL- (9). A tet- or rapamycin-dependent regulatory system has been induced apoptosis, and even greater survival and proliferation of developed for AAV-mediated delivery and inducible expression of these cells by TRAIL-induced activation of the Akt and extracellular the encoded genes (25). The regulated expression of TRAIL is likely signal-regulated kinase pathways (35). Furthermore, adenovirus- to be an important requirement for the clinical application of mediated overexpression of MDA-7 seems to result in significant TRAIL-based gene therapy, as it would reduce the risk of toxicity inhibition of growth in s.c. lung cancer tumors with decreased due to long-term expression. expression of CD31 and up-regulation of TRAIL (36). We observed The major concern with the application of TRAIL in the reduced vasculature in s.c. tumors and in the initial but not in the treatment of tumors in vivo is its controversial role in hepatic cell later vascularization of orthotopic tumors, even though tumor death (26–28). Interestingly, TRAIL studies showing hepatotoxicity growth was still significantly inhibited. The mechanism responsible are all either with the full-length membrane-bound form of the for the observed effects of TRAIL on the proliferation of vascular protein (28) or, if soluble, in combination with exogenous sequence endothelial cells remains to be elucidated. tags (27). A histidine-tagged TRAIL has been shown to have In summary, the present studies provide the first report of rAAV- an altered protein conformation, reduced stability, decreased mediated delivery and expression of sTRAIL114-281, demonstrating solubility, and hepatotoxicity (29). However, the same protein that intratumoral or intratracheal injection of rAAV2/5-sTRAIL without the histidine tag was able to trimerize adequately, giving it result in the presence of the trimeric form of sTRAIL in sera, a biological activity and neoplastic cell toxicity, with little or no statistically significant reduction in the rate of tumor growth, and evidence of toxicity to primary human hepatocytes in vitro (29). the prolonged survival of tumor-bearing mice. The rAAV2/5- These observations are consistent with the absence of toxicity in sTRAIL administration did not cause any detectable toxicity either the present study. Even the addition of 1 Ag/mL recombinant in the primary human hepatocytes in culture, or to any of the sTRAIL prepared in our laboratory did not induce apoptosis in examined organs of the transduced mice in vivo, suggesting that freshly isolated human hepatocytes in culture (data not shown). It AAV2/5-mediated sTRAIL gene therapy may provide a feasible and would therefore seem that sTRAIL lacks the hepatotoxicity that is effective form of treatment for lung cancer. associated with other forms of TRAIL, but has the ability to induce apoptosis in a variety of tumor cell lines, including the A549 lung Acknowledgments adenocarcinomas. The hepatotoxicity of TRAIL may be affected not only by the nature of TRAIL, but also by the physiologic Received 8/1/2004; revised 12/5/2004; accepted 12/22/2004. Grant support: Funded by grants from the State High-Tech Research and condition of the hepatocytes and other interacting factors, such as Development Program of China 863 grants no. 2002AA216051 (R. Xu), the University of innate and adaptive immune responses to the vector. Therefore, Hong Kong 863 matching fund (R. Xu) and the Natural Science Foundation of China the adenovirus-induced up-regulation of TRAIL receptor DR5 may (no. 30271207). The costs of publication of this article were defrayed in part by the payment of page be an important contributory factor in the reported hepatotoxicity charges. This article must therefore be hereby marked advertisement in accordance of adenovirus-encoded TRAIL (30). with 18 U.S.C. Section 1734 solely to indicate this fact.

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Cancer Res 2005; 65: (5). March 1, 2005 1692 www.aacrjournals.org

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TRAIL Gene Therapy for Lung Cancer In the article on TRAIL gene therapy for lung cancer in the March 1, 2005, issue of Cancer Research (1), Dr. Dexian Zheng should have been listed as a co-corresponding author: Dexian Zheng, National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, China. Phone: 8610-6529-6409; Fax: 8610-6510-5102; E-mail: [email protected]. 1. Shi J, Zheng D, Liu Y, et al. Overexpression of soluble TRAIL induces apoptosis in human lung adenocarcinoma and inhibits growth of tumor xenografts in nude mice. Cancer Res 2005;65:1687–92.

Cancer Res 2005; 65: (9). May 1, 2005 3966 www.aacrjournals.org Overexpression of Soluble TRAIL Induces Apoptosis in Human Lung Adenocarcinoma and Inhibits Growth of Tumor Xenografts in Nude Mice

Juan Shi, Dexian Zheng, Yanxin Liu, et al.

Cancer Res 2005;65:1687-1692.

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