Adenovirus-mediated intra-tumoral delivery of the human endostatin gene inhibits

tumor growth in nasopharyngeal carcinoma

Li Li1�, Ran-Yi Liu1�, Jia-Ling Huang1, Qi-Cai Liu2, Yan Li1, Pei-Hong Wu1, Yi-Xin Zeng1 and Wenlin Huang1*

1State Key Laboratory for Oncology in South China, Cancer Center, Sun Yat-sen University, Guangzhou, Guangdong 510060, China2Experimental Medical Research Center, Guangzhou Medical College, Guangzhou, Guangdong 510182, China

The growth and metastasis of nasopharyngeal carcinoma (NPC),one of the most common cancers in southern China, is closelyrelated to neovascularization. Here, we examined whether intra-tumoral delivery of endostatin gene could lead to long-term localexpression of bioactive endostatin at therapeutic levels. We con-structed a recombinant adenoviral vector carrying the humanendostatin gene (Ad/hEndo), which expressed high-level endostatinprotein in NPC CNE-2 cells, and significantly inhibited the prolif-eration and migration of vascular endothelial cells in vitro. Tumorgrowth and angiogenesis in NPC CNE-2 xenografted tumors weresignificantly inhibited after 5 courses of intra-tumoral treatmentwith Ad/hEndo in vivo. Endostatin mRNA in tumor tissues peakedat 1–2 days after intra-tumoral administration and disappearedwithin 1 week, whereas the plasma endostatin protein levelspeaked at 3 days after administration and lasted 2–3 weeks. Thetherapeutically relevant endostatin transgene expression wasachieved during the course of multiple intra-tumoral administra-tions with Ad/hEndo. Multiple injections with adenoviral vectorsdid not lead to continuous increases of adenovirus neutralizingantibodies in serum. Thus, adenovirus-mediated intra-tumoralintroduction of the human endostatin gene may form a viable newtreatment for NPC, although readministration every 2–3 weeksmay be necessary for the best effect.' 2005 Wiley-Liss, Inc.

Key words: nasopharyngeal carcinoma (NPC); adenoviral vector;endostatin; antiangiogenesis; gene therapy

Nasopharyngeal carcinoma (NPC) has the highest incidence ofdistant metastasis among head and neck cancers.1,2 Radiation ther-apy is the main strategy for local control of this neoplasm,3 but the5-year survival for stage IV NPC is only 30%, with poor survivaloften associated with high incidences of local, regional and sys-temic recurrences.4 Therefore, the development of multidiscipli-nary therapeutic approaches to improve loco-regional control anderadicate micrometastases is crucial for improvement of survivalin NPC patients.5,6

Angiogenesis, the formation of new microvessels from pre-existing capillaries, is essential for physiological processes suchas embryonic development, wound healing and tissue regenera-tion.7 In addition, many studies have shown that tumor growthis dependent on angiogenesis. If a cancer exceeds 1 � 2 mm insize, new blood vessels are needed to prevent tumor cell apopto-sis.8,9 Anti-angiogenesis therapies, which block the blood sup-ply of a growing tumor by inhibiting proliferation of endothelialcells, have provided a new strategy for the treatment of solidtumors.10,11

Endostatin, a 20-kDa C-terminal fragment of collagen XVIII,was originally isolated from the conditioned media of culturedmurine hemangioendothelioma (EOMA) cells.12 Several studieshave shown that endostatin specifically inhibits endothelial cellproliferation and migration, and induces apoptosis of vascularendothelial cells.13–15 Furthermore, treatment with recombinantendostatin protein produced in an Escherichia coli expression sys-tem induced an almost complete blockade of tumor angiogenesisand caused established tumors to regress to dormancy in micewithout any observable toxicity or therapeutic resistance.12,15

Based on this result, human recombinant endostatin was mass-pro-duced in yeast (Pichia pastoris) and tested in clinical studies.16,17

However, the mean half-life of recombinant endostatin was only10.7 6 4.1 hr in the human body, suggesting that this protein-

based therapy was likely to require daily readministration for opti-mal therapeutic benefits.18,19 In addition, studies showed that theyeast-derived recombinant human endostatin was immunogenic,with patients developing antibodies against both recombinanthuman endostatin and the P. pastoris vector.17

Taken together, these studies suggest that alternative endostatindelivery methods are needed for effective molecular therapy.Accordingly, researchers have turned to antiangiogenic gene ther-apy via direct delivery of human endostatin.20 It is unlikely thatthe human body will develop an immune response to the nativeprotein.21 Moreover, it is hoped that a single transduction couldachieve high-level, long-term endostatin expression.22 As reliablevectors are imperative for specific delivery of the target gene, bothnon-viral and viral methods have been investigated in the contextof endostatin gene therapy.23–25 Although non-viral gene therapywith endostatin showed some efficacy against tumor growth, theefficacy of transgene expression was relatively low in thesestudies.26,27

Here, we used a replication-defective adenovirus as a gene ther-apy vector and constructed a eukaryotic expression vector, encod-ing the human endostatin protein (Ad/hEndo). The expression ofendostatin protein was associated with decreased tumor growthand angiogenesis in an NPC CNE-2 xenograft tumor model inmice. Observation of the endostatin mRNA and protein levelsin vivo showed that endostatin expression decreased to approxi-mately backgrounds within 3 weeks, suggesting that a 2–3-weektreatment cycle could lead to clinically relevant anti-tumoralactivity.

Material and methods

Cell culture and viruses

Nasopharyngeal carcinoma (NPC) CNE-2 cells and humanumbilical vein endothelial cells (HUVECs) were maintained inRPMI medium 1640 supplemented with 10% fetal bovine serum(FBS). All media contained 100 U of penicillin and 100 lg ofstreptomycin/ml (GIBCO BRL, Gaithersburg, MD).

The recombinant adenovirus vectors carrying the human endo-statin gene (Ad/hEndo) and LacZ gene (Ad/LacZ) were generatedas described previously.28,29 The recombinant viruses containedthe human endostatin, following an IL-2 secretable signal, or LacZgenes under the control of the cytomegalovirus immediate earlypromoter. All virus particles were amplified in 293 cells, purified

Jia-Ling Huang’s current address is: Department of Infectious Diseases,Thomas Jefferson University, Philadelphia, PA 19107, USA.

�The first 2 authors contributed equally to this work.*Correspondence to: Cancer Center, Sun Yat-sen University, 651

Dongfeng Road East, Guangzhou, Guangdong 510060, China.E-mail: wl_huang@hotmail.com

Grant sponsor: The National Basic Research Program of China; Grantnumber: 2004CB518801; Grant sponsor: The Hi-tech Research and Devel-opment Program of China; Grant number: 2003AA216061; Grant sponsor:The Research and Development Grant of Guangdong Province; Grantnumbers: 2003A10902, 2003C104013; Grant sponsor: The CMB-SUMSScholar Program; Grant number: 98-677.

Received 12 June 2005; Accepted after revision 24 August 2005DOI 10.1002/ijc.21585Published online 14 November 2005 in Wiley InterScience (www.

interscience.wiley.com).

Int. J. Cancer: 118, 2064–2071 (2006)' 2005 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

by cesium chloride gradient centrifugation and titered using astandard plaque-forming assay.

In vitro protein expression

CNE-2 cells were infected with Ad/hEndo at multiplicities ofinfection (MOIs) of 1, 10 and 20 for 2 hr, washed once in phos-phate-buffered saline (PBS) and incubated at 37�C in freshmedium. The cells were harvested at 24, 48 or 72 hr postinfection.Cell lysates were collected and equal amounts of samples wereelectrophoresed on a 15% SDS-PAGE gel (Sigma, St. Louis, MO)and transferred onto a nitrocellulose membrane. The membranewas incubated for 4 hr with rabbit anti-human endostatin polyclonalantibody (Chemicon International, Temecula, CA) diluted 1:400 inTBS containing 5% BSA and 0.02% sodium azide. The membranewas washed 3 times and then incubated for 2 hr at room tem-perature, with goat anti-rabbit IgG-biotin conjugate (New EnglandBioLabs, Beverly, MA.) diluted at 1:1,000. After 3 times of washes,the membrane was incubated for 30 min at room temperature witha streptavidin-biotinylated horseradish peroxide complex (NewEngland BioLabs), and then stained with 3,30-diaminobenzidinetetrahydrochloride (New England BioLabs).

For ELISA, HUVECs were infected for 2 hr with Ad/hEndo atMOIs of 1, 10 and 20, washed once in PBS, and incubated at 37�Cin fresh medium. The supernatants of cultured cells were har-vested at 24, 36, 48 and 72 hr postinfection, and a human endo-statin enzyme-linked immunosorbent assay (ELISA) kit (OncogeneResearch Products, Boston, MA) was used to measure the endo-statin levels in the various supernatants, according to the manufac-turer’s instructions.

For immunohistochemistry, CNE-2 cells were infected with Ad/hEndo at an MOI of 10. At 2 hr postinfection, the medium wasreplaced with Dulbecco’s minimal essential medium (DMEM)containing 10% FBS. After 72 hr incubation, cells were collectedand plated on slides. The slides were washed, fixed, blocked for20 min in Tris-buffered saline (TBS) containing 1% goat serum,treated with rabbit anti-human endostatin polyclonal antibody(Chemicon International, Temecula, CA) diluted 1:50 in PBS andincubated overnight in a humid chamber. Slides were then washed3 times in PBS, treated with biotinylated goat anti-rabbit secon-dary antibody (Dako, Carpinteria, CA) diluted 1:200 in PBS andincubated in the humid chamber for an additional 30 min. Theslides were washed 3 times in PBS and stained with streptavidin-biotinylated horseradish peroxide complex.

MTT assay

HUVECs were seeded in a 96-well plate at a density of 10,000cells per well with RPMI 1640 and incubated overnight. Cellswere then infected for 2 hr with Ad/hEndo at MOIs of 0.05, 0.5, 1,5, 10 and 20, and with Ad/LacZ at an MOI of 20. Cells werewashed 3 times in PBS and incubated at 37�C in fresh medium.Seventy-two hours later, 20 ll of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (Fluka Biochemika,Buchs, Switzerland) was added to the wells and the plate wasincubated for an additional 4 hr at 37�C. Then, 100 ll of dimethylsulfoxide (DMSO) was added to the wells and the plate was incu-bated for 10 min at 37�C. Absorbance was read using a Bio-Rad550 microplate reader (Bio-Rad Laboratories, Hercules, CA) at570 nm. The value of the treated cells was calculated as the per-cent of the untreated control.

Migration assay

A migration assay was performed using a 24-well transwellinsert with a polycarbonate membrane (6.5 mm diameter, 8.0 lmpore size, Costar, Cambridge, MA). The lower chamber was filledwith RPMI 1640 containing 50 ng/ml vascular endothelial growthfactor (VEGF) (Sigma, St. Louis, MO). The upper chamber wasseeded with 20,000 HUVECs/well in conditioned media fromCNE-2 cells infected with Ad/hEndo or Ad/LacZ at MOIs of 100.Cells were allowed to migrate for 4 hr at 37�C, and then stained in

PBS containing 50 lg/ml propidium iodide. The cells on the uppersurface of the membrane were removed with a cell scraper. Themigrated cells on the lower surface were fixed in 3% formalde-hyde and counted. The rate of migratory inhibition of treatedendothelial cells was calculated as percent of the Ad/LacZ-treatedcontrol values. The experiment was repeated 3 times under identi-cal conditions.

In vivo tumor growth inhibition

BALB/c nude mice were purchased from the Experiment Ani-mal Center of Sun Yat-sen University and maintained underpathogen-free conditions according to protocols that wereapproved by the Sun Yat-sen University Institutional Animal Careand Use Committee. NPC CNE-2 cells were inoculated into theflanks of 6-week-old female BALB/c nude mice (5 3 106 cells/200 ll; subcutaneous injection). Ten days after tumor cell inocula-tion, a cycle of 5 weekly intra-tumoral injections of virus (Ad/hEndo or Ad/LacZ; 1 3 109 pfu in a 100 ll volume, n 5 7) wasgiven beginning on day 0. An additional 5 animals received injec-tions of 100 ll of DMEM as a negative control. Tumors weremeasured with a caliper gauge twice a week over a 6-week periodfollowing the initial virus injections. Tumor volume was calcu-lated according to the formula: volume 5 width2 3 length 30.52.12 One week after the 5 courses of Ad/hEndo treatmentended, the mice were sacrificed and the xenografted tumors wereextracted and weighed. The inhibition rates of CNE-2 xenograftedtumor growth were calculated as percent of the Ad/LacZ andDMEM control group values. Tumor volume was also expressedby the ratio of mean tumor volume in treated animals to meantumor volume in the DMEM control group (T:C ratio). The T:Cratio prior to treatment was 1.05.

In vivo angiogenesis assay

One week after the 5 courses of Ad/hEndo treatment, the ani-mals were sacrificed and tumors were excised, fixed in 10% for-malin and routinely embedded in paraffin. Paraffin sections (4 lm)were baked, deparaffinized and rehydrated. Sections were probedwith rabbit anti-CD31 antibody (Dako, Carpinteria, CA) overnightat 4�C, followed by treatment with a biotinylated secondary anti-body (Dako).

In vivo mRNA expression

Fourteen days after NPC CNE-2 cell inoculation, femaleBALB/c nude mice received intra-tumoral injections of 2 3 109

pfu Ad/hEndo in 200 ll. At 1, 2, 4 and 8 days postinjection, ani-mals were sacrificed, the xenografted tumors were excised andtotal RNA was extracted from tumors using the TRIzol reagent(Invitrogen, Carlsbad, CA), according to the manufacturer’sinstructions. RNA isolated from mice intra-tumorally injectedwith Ad/LacZ and DMEM was used as negative control. Fifteenmicrograms of total RNA was resolved on a 1% formaldehydeagarose gel and transferred to nylon membrane. The membranewas UV crosslinked at 254 nm for 1 min and 45 sec, and then pre-hybridized in the DIG Easy Hyb solution (Roche, Basel, Switzer-land) at 68�C for 30 min. The membrane was hybridized with adigoxin (DIG)-labeled endostatin probe at 68�C overnight, andthereafter, washed in SSC/SDS-containing buffers, according tostandard protocols. For visualization, the membrane was incubatedwith an anti-DIG antibody for 30 min and stained with nitro bluetetrazolium NBT/BCIP for 10 min.

In vivo protein expression

At 3, 7, 10, 14 and 21 days after injection of 2 3 109 pfu Ad/hEndo, animals were killed, blood samples were harvested andplasma endostatin levels were determined by ELISA (OncogeneResearch Products, Boston, MA). The xenografted tumors wereexcised and fixed in 10% formalin. Sections were probed witha multiclonal rabbit anti-human endostatin antibody (Chemicon

2065ENDOSTATIN GENE THERAPY FOR NASOPHARYNGEAL CARCINOMA

International, Temecula, CA) at a 1:50 dilution in PBS, followedby a biotinylated polyclonal goat anti-rat antibody.

Neutralizing antibody to adenovirus assay

Six-week-old female ICR mice (the Institute of CancerResearch mouse strain with a normal complement B- and T-lym-phocytes) were given 5 courses of weekly intra-peritoneal injec-tions of 23 109 pfu Ad/hEndo in a 200 ll volume. One week aftereach injection, a subset of the animals was sacrificed, and serumsamples were harvested for endostatin protein level measurementsand adenoviral neutralizing antibody assays.

Neutralizing antibodies to adenovirus type 5 were assayed asdescribed previously.30 Briefly, 293 cells were seeded at 3 3 104/well in 96-well plates and grown to about 90% confluence. Serumsamples were heated at 55�C for 30 min and diluted in medium(dilutions were 1:2–1:2,048). Aliquots of diluted serum (100 ll)were mixed with Ad/LacZ (5 3 103 pfu in 10 ll of cell culturemedium), incubated at 37�C for 90 min and applied to the nearlyconfluent 293 cells for 10–12 hr. The medium, containing serumand virus, was then replaced with complete RPMI. After 12 hr, thecells were fixed and stained for b-galactosidase expression. Theneutralizing antibody titer for each serum sample was defined asthe highest dilution at which less than 25% of the cells stainedblue compared with control.

Results

Endostatin protein expression in NPC CNE-2 cells in vitro

Since high-level transgene expression in NPC cells is crucialfor local endostatin gene therapy, we used Western blot analysisand immunohistochemistry to observe the efficiency of adeno-virus-mediated endostatin gene expression in NPC CNE-2 cells. Adistinct 20-kDa band, corresponding to the expected size of endo-statin, was visualized by Western blotting. The efficiency of trans-duction was closely related to the dose of adenovirus (Fig. 1a),confirming that endostatin was efficiently expressed by the trans-duced tumor cells.

Immunohistochemistry with an anti-endostatin antibody clearlydemonstrated that endostatin protein was expressed inside thetransduced CNE-2 cells, specifically in the cytoplasm of trans-duced cells (Fig. 1b). This indicated that the recombinant adenovi-

rus-mediated human endostatin gene was highly expressed inCNE-2 cells and provided evidence for the feasibility of local genetherapy for NPC.

Ad/hEndo inhibits proliferation of HUVECs in vitro

To observe the impact of Ad/hEndo on proliferation of vascularendothelial cells, we chose HUVECs as a model system. First, weinvestigated the efficiency of endostatin gene expression inHUVECs infected with Ad/hEndo at different MOIs. At 24, 36, 48and 72 hr after infection, the concentrations of endostatin in theconditioned supernatants were measured by ELISA. The resultsshowed that the human endostatin gene was highly expressed inHUVECs, and that the endostatin levels in the conditioned super-natants rose continuously with increased multiplicity and infectionduration, reaching a peak of 588.34 ng/ml 72 hr after infection atan MOI of 20 (Fig. 2a). These results indicated that the endostatinprotein was efficiently secreted into the supernatant of transducedHUVECs.

MTT assay revealed that the secreted endostatin inhibited pro-liferation of HUVECs by 7.61, 12.61, 24.8, 29.86, 38.78 and

FIGURE 1 – Endostatin protein expression in vitro. (a) CNE-2 cellswere infected with Ad/hEndo at MOIs of 1, 10 and 20. The cells wereharvested at 24, 48 and 72 hr postinfection, and Western blot analysiswas performed using a rabbit anti-human endostatin polyclonal anti-body (PBS as negative control). A molecular size maker is shown tothe left. (b) CNE-2 cells were infected with Ad/hEndo at an MOI of10. The cells were harvested at 72 hr postinfection, and plated onslides. Immunohistochemistry analysis was performed using a rabbitanti-human endostatin polyclonal antibody, followed by a biotinylatedgoat anti-rabbit secondary antibody (3400). (c) Negative control (PBS)(3400).

FIGURE 2 – Effect of Ad/hEndo on the proliferation of HUVECs.(a) HUVECs were infected with Ad/hEndo at MOIs of 1, 10 and 20.Conditioned supernatants were harvested at 24, 36, 48 and 72 hr post-infection. The endostatin concentrations in the supernatants weredetermined by ELISA (n 5 5). (b) HUVECs were infected for 2 hrwith Ad/hEndo at MOIs of 0.05, 0.5, 1, 5, 10 and 20, and with Ad/LacZ at an MOI of 20. MTT assays were performed 3 days postinfec-tion, and absorbance was read using a Bio-Rad 550 microplate readerat 570 nm. (PBS, as negative control; Ad/LacZ, as adenovirus control).The data shown are representative of 3 independent experiments.

2066 LI ET AL.

57.2%, respectively, at MOIs of 0.05, 0.5, 1.0, 5, 10 and 20 (Fig. 2b).The negative control (Ad/LacZ) inhibited cellular proliferation by7.25% at an MOI of 20 (t5 7.346, p< 0.05). These results indicatedthat the endostatin protein secreted by transduced cells was highlybioactive and inhibited the proliferation of vascular endothelialcells in vitro in a dose-dependent manner, while the viral vectoralone did not.

Ad/hEndo inhibits migration of HUVECs in vitro

To observe the impact of Ad/hEndo on migration of vascularendothelial cells, we investigated the ability of Ad/hEndo to blockmigration of HUVECs toward VEGF. After cells were treatedwith conditioned medium of CNE-2 cells infected with Ad/hEndo,we observed a 46.5% inhibition of endothelial cell migration (t 54.438, p < 0.05) as compared to the control (Fig. 3). These resultsshowed that the endostatin protein secreted by transduced CNE-2cells was capable of blocking the migration of vascular endothelialcells in vitro.

Inhibition of growth and angiogenesis of CNE-2 xenograftedtumors

To observe the effect of Ad/hEndo on the growth and angiogen-esis of tumors in vivo, we next produced a CNE-2 xenograft tumormodel in BALB/c nude mice. Ad/hEndo was administered intra-tumorally once a week. After 5 courses of treatment, the treatedtumors were smaller than control tumors by 46.43% (Ad/hEndogroup versus Ad/LacZ group, t 5 2.226, p < 0.05) and 49.70%(Ad/hEndo group versus DMEM group, t 5 2.254, p < 0.05). Theratio of mean tumor volume in treated animals to mean tumor vol-ume in the control animals (T:C ratio) was <40% after 21 days oftreatment (Fig. 4a). These results showed that recombinant adeno-virus-mediated endostatin significantly inhibited the growth ofCNE-2 xenografted tumors.

Immunohistochemical analyses with anti-CD31 antibody wereperformed to investigate the effect of endostatin gene therapyon tumor angiogenesis. Gross examination showed that tumors

treated with Ad/hEndo demonstrated extensive necrosis. Thetreated tumors had fewer vessels, and the observed vessels weremalformed (Fig. 4b) compared to those of control groups (Fig. 4c).These results indicated that Ad/hEndo significantly inhibited angio-genesis in CNE-2 xenografted tumors.

Dynamic transgene expression of endostatin in vivo

Northern blot analysis clearly demonstrated an endostatinmRNA band at �1.2 kb, which is the expected size of intact, func-tional endostatin mRNA. The highest level of endostatin mRNAinside tumor tissues was detected 1–2 days after intra-tumoraladministration of Ad/hEndo. Following day 4, endostatin mRNAlevels gradually decreased, reaching undetectable levels by day 8(Fig. 5a). This result showed that adenovirus-mediated human

FIGURE 3 – Effect of Ad/hEndo on the migration of HUVECs. Amigration assay was performed using a 24-well transwell insert. Thelower chamber was filled with RPMI medium 1640 containing 50 ng/ml VEGF. The upper chamber was seeded with 20,000 HUVECs/wellin conditioned media from CNE-2 cells infected with Ad/hEndo orAd/LacZ at an MOI of 100. Cells were allowed to migrate for 4 hr at37�C, and then stained in PBS containing 50 lg/ml propidium iodide.(a) Endostatin-treated endothelial cells (3100). (b) Ad/LacZ-treatedcontrol (3100). (c) The number of cells on each membrane wascounted, and the migratory inhibition rate of endostatin-treatment wascalculated as percent of the Ad/LacZ-treated control. The experimentwas repeated 3 times under identical conditions.

FIGURE 4 – Effect of Ad/hEndo on the growth and angiogenesis ofCNE-2 xenografted tumors. (a) CNE-2 cells (5 3 106) were injectedinto the flanks of 6-week-old female BALB/c nude mice. Ten daysafter tumor cell injection, a cycle of 5 weekly intra-tumoral injectionsof 1 3 109 pfu Ad/hEndo or Ad/LacZ was given (n 5 7). An addi-tional 5 animals received injections of 100 ll DMEM (n5 5). Tumorswere measured with a caliper gauge twice a week (* the ratio of meantumor volume in treated animals to mean tumor volume in the DMEMcontrol group (T:C ratio) < 40%). (b) Changes of microvessels inCNE-2 xenografted tumors. One week after 5 courses of treatmentwith Ad/hEndo, the animals were sacrificed and their tumors wereexcised and fixed in 10% formalin. Immunohistochemistry was per-formed using a rabbit anti-CD31 antibody (3200). (c) CNE-2 xeno-grafted tumors injected with DMEM (3200).

2067ENDOSTATIN GENE THERAPY FOR NASOPHARYNGEAL CARCINOMA

endostatin gene transfer resulted in high level transient expressionof full-length endostatin mRNA.

Plasma endostatin levels peaked at 132.63 6 22.18 ng/ml 3 daysafter adenovirus injection, a concentration that is significantly higherthan the basal level (8.25 6 2.55 ng/ml) (t 5 6.229, p < 0.01).By day 7, plasma levels had dropped to nearly half the peak value(65.73 6 10.04 ng/ml), and the plasma endostatin levels hadreturned to basal (14.17 6 6.34 ng/ml) by 3 weeks after injection(Fig. 5b).

Immunohistochemistry with an anti-endostatin antibody wasused to examine endostatin protein expression in the tumors.Three days postinjection, immunohistochemistry demonstratedintense positive staining in the cytoplasm of tumor cells and in theextracellular matrix (Fig. 6a). However, this positive staining wassignificantly reduced by 7 days postinjection (Fig. 6b), and therewas little positive staining by day 14 (Fig. 6c). No positive stain-ing was observed in tumor tissues of the DMEM negative controlgroup (Fig. 6d). These results confirmed that the recombinantadenovirus-mediated endostatin gene was highly expressed inNPC CNE-2 xenografted tumors, and that the expressed proteincould be efficiently secreted into the extracellular matrix to inhibittumor angiogenesis in vivo. However, our results showed that thein vivo transgene expression faded within 3 weeks, suggesting thatrepeated gene delivery on a 2–3 week schedule could be necessaryfor effective treatment of NPC.

Impact of host immune response on transgene expression

To investigate whether multiple injections of Ad/hEndo couldincrease the host immune response, we examined the titer changesof adenovirus-neutralizing antibodies in normal ICR mice receiv-ing 5 courses of weekly intra-peritoneal injections with Ad/hEndo.The serum titers of anti-adenovirus neutralizing antibodies wereexamined 1 week after each injection with Ad/hEndo, and it

peaked after the first adenovirus injection. Thereafter, the anti-adenovirus antibody titers stabilized from the second to the fifthvector administrations, and even began to drop after the fifth injec-tion (Fig. 7a). These results indicate that multiple injections ofAd/hEndo did not continuously increase the titer of neutralizingantibodies to adenoviral vectors in the mouse model system.

Meanwhile, we investigated the effect of host immune responseon transgene expression during the course of multiple adenoviralinjections. One week after the first injection, serum endostatin lev-els reached the observed peak (92.43 6 20.15 ng/ml). From thesecond to the fifth injections, serum endostatin levels decreasedslightly and stabilized within a range of 64.11–43.84 ng/ml (Fig.7b). These results indicated that efficient transgene expressioncould be achieved during the course of multiple Ad/hEndo injec-tions, even in the presence of a host immune response to theadenoviral vector.

Discussion

Nasopharyngeal carcinoma (NPC) is one of the most commonneoplasms in southern China, and is highly prevalent in the Can-tonese population.1 Although this poorly differentiated carcinomais markedly sensitive to radiotherapy, local control failure and dis-tant metastasis are common in cases of advanced stage primarytumors.3 The intensity of angiogenesis may predict the probabilityof metastasis, as neoangiogenesis was shown to be significantlyhigher in metastatic NPC than in curable, non-metastatic NPC.8,31

These observations suggest that antiangiogenic therapies might becapable of preventing distant metastases in NPC and intensifyingthe therapeutic efficiency of radiotherapy for loco-regional con-trol.32 In this context, Qian and colleagues treated NPC CNE-2xenografted tumors with the angiogenic inhibitor, TNP-470, anddemonstrated a 26% reduction in tumor size.33

Accordingly, we used a replication-defective adenovirus as anendostatin gene therapy vector. We acquired high level transgeneexpression in CNE-2 cells, which provides solid evidence for thefeasibility of local endostatin gene therapy in NPC patients. Theproduced protein was demonstrated to have a high level of bioac-

FIGURE 5 – Dynamic transgene expression of endostatin in vivo. (a)Northern blot analysis was performed to test full-length endostatinmRNA expression in CNE-2 xenografted tumors using a digoxin(DIG)-labeled endostatin probe. Lane 1, RNA extracted from tumorson day 1 after Ad/hEndo injection; lane 2, day 2; lane 3, day 4; lane 4,day 8; lane 5, negative control RNA extracted from tumors on day 1 afterAd/LacZ injection; lane 6, negative control RNA extracted from tumorsinjected with DMEM. A molecular size maker is shown to the left.(b) At 3, 7, 10, 14 and 21 days after intra-tumoral administration of Ad/hEndo, the animals were sacrificed and blood samples were harvested.Plasma endostatin levels were determined by ELISA (n5 5).

FIGURE 6 – Immunohistochemistry analysis of endostatin protein inCNE-2 xenografted tumors. At 3, 7, 10, 14 and 21 days after injectionof Ad/hEndo, the animals were sacrificed, and xenografted tumorswere excised and fixed in 10% formalin. Sections were probed with amulticlonal rabbit anti-human endostatin antibody at a 1:50 dilution inPBS, followed by a biotinylated polyclonal goat anti-rat antibody.(a) CNE-2 xenografted tumor tissue 3 days after treatment (3400).(b) CNE-2 xenografted tumor tissue 7 days after treatment (3400).(c) CNE-2 xenografted tumor tissue after 14 days (3400). (d) CNE-2xenografted tumor tissue 3 days after administration with DMEM(3200).

2068 LI ET AL.

tivity, as it significantly inhibited the proliferation and migrationof vascular endothelial cells in vitro. In mice, 5 courses of intra-tumoral treatment with the endostatin-expressing construct signifi-cantly inhibited the growth of CNE-2 xenografted tumors by46.43–49.70%. Although previous studies have investigated sys-temic expression of the endostatin gene,24,25,34 few have investi-gated the efficacy and dynamic changes of transgenic endostatinexpression inside tumor tissues.26,35 Szary and Szala reported thatintra-tumoral administration of naked plasmid DNA encodingendostatin raised the intra-tumoral endostatin concentration 2-fold, but the efficiency of this gene delivery system was verylow.35

Here, we investigated the in vivo application of intra-tumoraladenovirus-mediated endostatin gene therapy by measuring theefficacy and dynamic changes of local transgene expression in thetumors. Immunohistochemistry with an anti-endostatin antibodyshowed that the protein was localized in the cytoplasm of tumorcells and in the extracellular matrix, indicating that the endostatinprotein was efficiently secreted into the extracellular matrix,where it may act to inhibit neoangiogenesis. The highest in vivolevel of endostatin mRNA was detected 1–2 days after intra-

tumoral administration. Thereafter, the endostatin mRNA levelsgradually decreased to undetectable levels by day 8. However, theprotein levels showed a slightly different expression pattern.

When delivered systemically, recombinant adenoviral vectorshave a preferential tropism for hepatocytes. In contrast, tumor-tar-geted gene therapy can lead to tumor-specific, therapeutically rele-vant transgene expression.36 After intra-tumoral administration ofAd/hEndo, a small amount of recombinant adenoviral vector andendostatin protein entered circulation, resulting in elevated plasmaendostatin levels. In our studies, the plasma endostatin levels weresignificantly elevated within 2–3 weeks after intra-tumoral trans-duction. These plasma endostatin levels were considerably lowerthan those reached by intravenous administration.24,25 However,serum endostatin levels of 35–40 ng/ml have been shown to besufficient for exerting an antiangiogenic effect in vivo, suggestingthat our delivery system induced therapeutically relevant plasmaendostatin levels.37 Taken together, our mRNA and protein detec-tion experiments showed that intra-tumoral administration withAd/hEndo should most likely be repeated at intervals of 2 or 3weeks to maintain sufficient intra-tumoral endostatin concentrations.

Transient expression is a key problem in gene therapy. The hostimmune response to the adenoviral vector and the transgene prod-uct is a main factor impacting the persistence of transgene expres-sion.38 The Th1 dominant antiviral immune response occurs 5–7 days after transduction and is directed against the residualexpression of viral genes.39,40 In addition to inducing the adaptiveimmune system, adenoviruses activate the innate immunity in adose-dependent response that occurs within 24 hr of transduc-tion.40 This acute immune response may induce the host immunesystem to attack the infected tumor cells.38 Indeed, the mechanismof adenoviral cell killing involves a ‘‘bystander effect’’ whereby,uninfected neighboring cells are killed by a factor or signal fromadenovirus-infected cells. Immunological mechanisms involvingnatural killer (NK) cells are thought to contribute to the efficacy ofadenovirus-mediated gene therapy in a mouse model for cancer,and these mechanisms can be enhanced to improve therapeuticefficiency.38,40 Therefore, intra-tumoral administration with anadenoviral vector might induce a local tumor immunotherapy andintensify the efficiency of endostatin gene therapy for NPC.

Because of the host immune response to the adenoviral vector,transgene expression will decrease to approximately backgroundlevels, within 3 weeks of first-generation adenoviral vector admin-istration, necessitating readministration of adenoviral vectors fol-lowing the eventual loss of therapeutic transgene expression.41

However, the neutralizing antibody response from this previousexposure might reduce the effectiveness of the readministeredadenoviral vectors and decrease the subsequent transgene expres-sion.42,43 Interestingly, some studies have documented that read-ministration of adenoviral vectors can be effective, even followingthe initial host immune response.44 Chen et al. reported that an ini-tial intramuscular administration of a low-dose adenoviral vectorproducing low but detectable levels of transgene expression didnot preclude readministration into the muscle, whereas systemicreadministration was not effective.45 Buller et al. used intra-peri-toneal readministration of adenoviral vectors in a phase I/II trial ofrAd/p53 (SCH 58500) gene replacement in recurrent ovarian can-cer, and found measurable transgene expression in 17 of 20 sam-ples after 2–3 cycles of adenovirus-mediated p53 gene therapy.46

Therefore, we investigated the adenovirus-neutralizing antibod-ies in normal ICR mice receiving 5 courses of weekly intra-perito-neal Ad/hEndo injections. The adenovirus-neutralizing antibodytiter in serum peaked after the first transduction, stabilized atapproximately this level from the second to the fifth administra-tions and decreased slightly thereafter. This phenomenon may bedue to a saturation effect of the host immune response to adenovi-ral vectors. As vector titers increase, a saturation effect may occurwherein further increases in vector titers are associated with mini-mal or no further increase in host immune response.38,41 In ourstudies, the host immune response to the vector decreased the

FIGURE 7 – Impact of host immune response on repeated transgeneexpression. (a) Neutralizing antibody to adenovirus assay. ICR micewere given 5 weekly intra-peritoneal injections of Ad/hEndo. Oneweek after each injection, a subset of the animals was sacrificed, andserum samples were harvested and assayed for neutralizing antibodiesto adenovirus. (b) One week after each injection, serum endostatinlevels were determined by ELISA (n5 3).

2069ENDOSTATIN GENE THERAPY FOR NASOPHARYNGEAL CARCINOMA

achievable serum endostatin levels slightly upon readministration,but therapeutically efficient transgene expression could still beachieved during 5 courses of endostatin gene therapy.

Intra-tumoral delivery of adenoviral vectors may be a promisingapproach for adenovirus-mediated gene therapy.47 When deliveredintra-tumorally, adenoviruses can direct tumor-specific, thera-peutically relevant transgene expression and induce local immuno-therapy for tumor cells.40 Even if a small amount of adenoviralvectors enter into circulation, the host immune response might belimited and does not seem to significantly reduce the effectivenessof readministration.47,48 In this study, 5 courses of intra-tumoraldelivery directed high-level expression of endostatin mRNA andprotein inside NPC CNE-2 xenografted tumors, in association withsignificant inhibition of growth and angiogenesis. These resultssuggest that antiangiogenic therapy may be an important futureaddition to the multidisciplinary therapeutic approaches for NPC.

Although antiangiogenic therapy is not supposed to result indrug resistance by targeting endothelial cells with normal genome,recently, basic and clinical research showed that antiangiogenictherapy could also develop the appearance of ‘‘acquired drugresistance.’’49 This phenomenon is due to the activation of hypo-xia-inducible factor (HIF). HIF-activated hypoxia transactivesangiogenic and autocrine growth factors and receptors, and inhib-its secretion of antiangiogenic factors, such as thrombospondin-1,thus, stimulating angiogenesis, reducing or neutralizing the effectof antiangiogenic therapy. Even if an enough effective antiangio-genic was performed, it would result in a selective resistance,because overexpression of HIF may help tumor cells to survivelow hypoxia and starvation.50 Combined with radiotherapy, che-motherapy including inhibitors of HIF, endostatin antiangiogenicgene therapy might more effectively improve loco-regional con-trol and prevent micrometastases of tumor.

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