This study determined the effect of Ad-E1A gene therapy in vivo. TC71 cells (2×106) injected subcutaneously into nude mice resulted in tumor development (1–3 mm) 6 days later. Animals were then treated with Ad-E1A or Ad-β-gal (5×109 plaque-forming units) by intratumoral injection twice weekly for 2 weeks. Animals received 8 mg/kg VP-16 given by intraperitoneal injection daily for 5 days following the first week of treatment with Ad-E1A or Ad-β-gal. Control animals received no therapy or VP-16 only after tumor cells were injected. When tumors exceeded 2×2 cm, the mice were sacrificed and the tumors underwent histologic and immunohistochemical analysis. Tumors from mice treated with Ad-E1A plus VP-16 were 9.6-fold smaller than those treated with VP-16 alone and 6.3-fold smaller than those treated with Ad-E1A alone. HER2/neu p185 protein expression decreased in all tumors that received Ad-E1A therapy. TUNEL fluorescence staining revealed more apoptosis in the tumors from animals treated with Ad-E1A plus VP-16 than in those from animals treated with Ad-E1A alone, Ad-β-gal plus VP-16, or VP-16 alone. These data demonstrated that Ad-E1A gene therapy down-regulated HER2/neu expression, increased tumor cell apoptosis induced by VP-16, and enhanced tumor cell sensitivity to VP-16. Ad-E1A may have potential in the treatment of relapsed drug-resistant Ewing's sarcoma.
Ewing's sarcoma is the second most common primary malignant bone tumor in children and young adults and has a high potential for metastasizing to the lungs, bones, and bone marrow. Most patients with Ewing's sarcoma die from lung and bone metastases. The overall 5-year survival rate is 41.2%.1,2 Moreover, the prognosis is poor for patients who have had a relapse and for patients with large primary tumors or metastatic disease at presentation. Therefore, new strategies are needed to improve the survival rates. The HER2/neu oncogene encodes a 185-kDa human epidermal growth factor receptor-2 transmembrane protein (p185) with intrinsic tyrosine kinase activity. Overexpression of HER2/neu was found in approximately 30% of human breast and ovarian cancers.3,4 Enhanced expression of HER2/neu increased the tumorigenicity and metastatic potential of human ovarian and lung cancer cells. Clinical studies indicated that overexpression of HER2/neu correlated with poor prognosis, shorter patient survival, and chemoresistance.5,6
We previously demonstrated that TC71 human Ewing's sarcoma cells overexpress HER2/neu. Thus, HER2/neu may also play a role in the poor disease-free survival rate associated with multiagent chemotherapy in this disease. The adenovirus type 5 early region 1A (E1A) gene is a well-known transcriptional factor. This gene inhibits HER2/neu expression in both rodent and human breast cancer and ovarian cancer cells through the HER2/neu promoter and with the involvement of the HER2/neu coactivator p300.7 This inhibition abolishes the tumorigenicity and metastatic potential induced by the HER2/neu oncogene by repressing its expression. Transfer of the E1A gene results in transcriptional repression of HER2/neu via an effect on the HER2/neu promoter.8,9,10 Transduction of TC71 cells with the E1A gene using an adenoviral vector (Ad-E1A) suppressed HER2/neu expression and increased both apoptosis and cell sensitivity to etoposide (VP-16) in vitro.11
In this study, we used a replication-deficient adenoviral vector gene delivery system to transduce the E1A gene into human TC71 Ewing's sarcoma cells in vivo. We demonstrated that E1A down-regulated HER2/neu expression, inhibited tumor growth, induced tumor apoptosis, and increased tumor sensitivity to VP-16.
Materials and methods
VP-16 (Sigma Chemical, St. Louis, MO) was dissolved in a small volume of dimethyl sulfoxide and then diluted to the appropriate concentration with normal saline before being administered to the mice.
Cell lines and culture
TC71 human Ewing's sarcoma cells, kindly provided by Dr P Pepe (University of Southern California, Los Angeles, CA), were cultured in Eagle's modified essential medium (supplemented with 10% heat-inactivated [56°C for 30 minutes] fetal bovine serum, 1 mM sodium pyruvate, 2× minimal essential medium vitamins, 1× nonessential amino acids, and 2 mM glutamine) at 37°C at 5% CO2 in a humidified incubator. The cells were free of mycoplasma, as screened by Gen-Probe (San Diego, CA), and verified to be free of pathogenic marine viruses (NCI-Frederick Cancer Research & Development Center, Frederick, MD). The cell lines used in in vivo experiments were from the third to the tenth passage.
The 293 cell line (American Type Culture Collection, Manassas, VA), which comprises human embryonic kidney cells, was transduced by sheared fragments of adenovirus type 5 DNA and used as the adenoviral packaging cell line.
Four- to five-week-old specific pathogen-free athymic (T-cell deficient) nude mice were purchased from Charles River Breeding Laboratories (Kingston, MA). The mice were maintained in an animal facility approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the United States Department of Agriculture and Department of Health and Human Services and the National Institutes of Health. Animals were housed for 1 to 2 weeks before any experiments were begun.
Ad-E1A is an adenovirus type 5 that contains E1A but lacks E1B and E3 (d1324). The control vector was Ad-β-gal, an adenovirus type 5–based vector lacking E1A, E1B, and E3 but containing β-galactosidase. Both recombinant, replication-deficient adenoviral vectors were prepared by superinfecting 293 cells. The vectors were then purified twice using cesium chloride–gradient ultracentrifugation (Sigma), dialyzed, titrated by plaque assay, and stored at−80°C using a standard protocol.12,13
In vivo model
As previously described,14 TC71 Ewing's sarcoma cells in mid-log-growth phase were harvested by trypsinization. Single-cell suspensions of 2×106 cells in 0.1 mL Hanks' balanced salt solution (4°C) were injected into nude mice subcutaneously. Six days later, when tumors could be detected (1–3 mm), the tumor-bearing mice were then randomly divided into six groups (five mice per group) for no treatment or treatment with VP-16, Ad-E1A, or Ad-β-gal alone or VP-16 in combination with Ad-E1A or Ad-β-gal. Group 1 mice were untreated controls. Group 2 mice were given intratumoral injections of Ad-β-gal (5×109 plaque-forming units [PFU]) in 50 μL PBS twice weekly for 2 weeks. Group 3 mice were injected intratumorally with Ad-E1A (5×109 PFU) in 50 μL PBS twice weekly for 2weeks. Group 4 mice were injected intraperitoneally with 8mg/kg VP-16 in 0.2 mL normal saline daily for 5consecutive days during week 3 (Fig 1). Group 5 mice were injected intratumorally with Ad-β-gal twice weekly during weeks 2 and 3 and received VP-16 during week 3, as described in group 4 (Fig 1). Group 6 mice were injected intratumorally with Ad-E1A twice weekly during weeks 2and 3 and given VP-16 during week 3, as described in group 4. The tumors were measured every 4 days with a caliper, and their diameters were recorded. Tumor volume was calculated by the formula π/6(√ab)3, where a and b are the two maximum diameters. The duration of survival was recorded. When each tumor grew bigger than 2×2 cm, the mice were sacrificed, and tumor tissues were collected for analysis of E1A, HER2/neu, CD31, and apoptosis expression by reverse transcription–polymerase chain reaction (RT-PCR), histologic and immunohistochemical analysis, and terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) assay.
Total RNA was extracted from tumor tissues using Trizol reagent (Gibco BRL, Grand Island, NY). Five micrograms of total RNA were reverse transcribed in a 20 μL RT reaction. Five micrograms of the cDNA produced were added to a PCR reaction mixture containing 5 μL PCR buffer, 0.5 μL (5 U/μl) Taq DNA polymerase, 1 μL (10mM) dNTP (dATP, dCTP, dGTP, and dTTP), and 2.5μL (10 nM) each of primers specific to the Ad-E1A intron 1region (forward primer: 5′-CGGGATCCCCACCATGAGA CATATTATCTGCCACG-3′; reverse primer: 5′-CGGAATT CTTACTCGAGGTCAATCCCTTCCTGCACC-3′). Ampli-fication was carried out for 30 cycles in a PTC-200 DNA Engine Poltier Thermal Cycle (MJ Research, Inclin Village, NV) using the following program: denaturing at 94°C for 1 minute, annealing at 60°C for 1minute, and extending the primer at 72°C for 1 minute. 18S Primers/Competimers™ (Ambion, Austin, TX) was used as internal control. The product size was 266 bp. Samples were analyzed on a 2% Tris–acetate–EDTA agarose gel stained with ethidium bromide. Plasmid E1A DNA was used as a positive control.
Histologic sections were taken from mice bearing TC71 Ewing's sarcoma. The sections were subjected to routine pathologic analysis with hematoxylin and eosin staining. Frozen sections fixed with acetone were incubated in 3% H2O2 in methanol for 10 minutes to block endogenous peroxidase and then incubated in 5% normal horse serum plus 1% normal goat serum in PBS for 20 minutes to block protein. Expression of the CD31 gene on blood vessels and vessel density were detected using rat anti-mouse CD31 as the primary antibody (Pharmingen, San Diego, CA), and goat anti-rat horseradish peroxidase as the second antibody, incubated with chromogen diaminobenzidine. The expression of HER2/neu p185 protein was detected by incubating tissue sections using rabbit polyclonal anti-human c-erb-2 oncoprotein (DAKO, Carpinteria, CA) as the primary antibody and biotinylated goat antibody against rabbit IgG as the second antibody, followed by incubation with avidin–biotin–peroxidase complex (Vector Labs, Burlingame, CA). Staining was then developed in alkaline phosphatase solution (Biomeda, Foster City, CA). Gill's hematoxylin was used as a counterstain.
TUNEL assay was performed to detect apoptotic cells. Frozen sections were fixed with 4% methanol-free formaldehyde solution in PBS for 10 minutes and then washed with PBS three times. The tissue was permeabilized in 20μg/mL proteinase K solution for 10 minutes at room temperature and equilibrated in equilibration buffer for 10minutes after which the slides were rinsed with PBS three times. The DNA fragments were labeled with fluorescein-12-dUTP in terminal deoxynucleotidyl transferase incubation buffer (Promega, San Diego, CA) in a humidified chamber (37°C for 60 minutes) to avoid exposure to light. The reactions were terminated by transferring the slides to 2× SSC buffer (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0) for 15 minutes and washing them in PBS to remove unincorporated fluorescein-12-dUTP. The slides were then counterstained with 4′,6 diamidino-2-phenylindole (DAPI; Vector Labs, Burlingame, CA) to provide a blue background. The green fluorescence of apoptotic cells (fluorescein-12-dUTP) can be detected with a fluorescence microscope at 520 nm.
Statistical evaluation for tumor sizes and vessel numbers was performed using the two-tailed Student t test. A P value of <.01 was considered statistically significant. Log-rank test was performed to analyze the survival curve. A P value of <.05 was considered statistically significant.
Combination therapy with Ad-E1A and VP-16 inhibited growth of Ewing's sarcoma in nude mice
Our previous data demonstrated that the E1A gene could be transferred into TC71 Ewing's sarcoma cells using an adenoviral delivery system. This resulted in the suppression of tumor cell growth in vitro and the enhancement of sensitivity to VP-16.11 To analyze the effects of combination Ad-E1A and VP-16 therapy in vivo, athymic mice that had been injected subcutaneously with 2×106 TC71 cells 6days earlier were treated with VP-16, Ad-E1A, Ad-β-gal, Ad-β-gal+VP-16, or Ad-E1A+VP-16 or received no treatment. The tumors were measured every 4 days. One month later after first treatment, the tumors in the mice treated with Ad-E1A and VP-16 were 9.6-fold smaller than those in mice treated with VP-16 alone, and 6.3-fold smaller than the tumors in mice treated with Ad-E1A alone (Fig 2). The tumor volumes of the mice treated with Ad-β-gal did not differ significantly from those tumor volumes of mice that received no treatment (data not shown). When the tumor size was ≥2×2 cm, the mouse was sacrificed. The survival rate of tumor-bearing mice treated with Ad-E1A plus VP-16 was significantly higher than that of mice treated with VP-16, Ad-E1A, Ad-β-gal, or Ad-β-gal plus VP-16 (Fig 3).
Expression of E1A and suppression of HER-2/neu p185 in vivo
To investigate whether the suppression of tumor growth could be linked to the down-regulation of HER-2/neu expression by E1A, tumors from each group were analyzed for E1A expression by RT-PCR. As shown in Figure 4, E1A gene expression was detected in Ad-E1A–treated and Ad-E1A plus VP-16–treated tumor tissue. No E1A gene expression was detected in Ad-β-gal– or VP-16–treated tumors. Immunohistochemical staining using a rabbit polyclonal anti-human c-erb-2 antibody against p185 as the primary antibody revealed that HER2/neu p185 expression in tumors treated with Ad-E1A alone was lower than in control or Ad-β-gal–treated tumors (Fig 5A). Decreased HER2/neu expression was also seen in tumors treated with Ad-E1A plus VP-16 (data not shown).
E1A increased VP-16–induced apoptosis in Ewing's sarcoma
Our previous data demonstrated that E1A could induce TC71 cell apoptosis in vitro.11 To examine the effect of E1A on tumor cells in vivo, tumor tissues from animals treated with Ad-E1A, Ad-E1A plus VP-16, Ad-β-gal, Ad-β-gal plus VP-16, VP-16 alone, or no treatment were collected and analyzed by TUNEL assay. As shown in Figure 5B, apoptotic cells could be identified in Ad-E1A– and Ad-E1A plus VP-16–treated tumor tissue. Moreover, tumors treated with Ad-E1A plus VP-16 had more apoptotic cells than those treated with Ad-E1A or VP-16 alone, indicating that E1A could enhance VP-16–induced apoptosis in vivo as well as in vitro.
E1A down-regulated CD31 gene expression in Ewing's sarcoma
Angiogenesis, the formation of new blood vessels, is necessary for the growth and metastatic spread of solid tumors. We previous showed that E1A down-regulated VEGF and MMP-9 expression in TC-71 cells.32VEGF, an angiogenesis regulatory, has been found to be closely associated with microvessel density, and with the treatment effect.33To examine the effect of E1A on vessel density, tumor tissues treated with Ad-E1A, VP-16, Ad-E1A plus VP-16, Ad-β-gal, and Ad-β-gal plus VP-16 were analyzed by immunohistochemistry study for CD31 density using rat anti-mouse CD31 as the primary antibody. As shown in Figure 5C, the vessel density was significantly lower in the tumors treated with Ad-E1A plus VP-16 than in those treated with VP-16, Ad-β-gal, or Ad-β-gal plus VP-16 (P<.01).
E1A has been shown to be a tumor suppressor gene in breast carcinoma, ovarian carcinoma, rhabdomyosarcoma, osteosarcoma, melanoma, and nonsmall cell lung carcinoma, both in vitro and in vivo.15,16,17,18,19,20 It also has been known to regulate transcription through interaction with different cellular DNAbinding domains.21 E1A can function as a tumor suppressor gene in HER2/neu-overexpressing cells through repression of HER2/neu expression at the transcriptional level. E1A does this by targeting a specific DNA element in the HER2/neu gene promoter.10 Studies have reported that E1A reversed the malignant phenotype of different human tumors, inhibited tumor metastasis by repressing proteinase gene expression, and reduced the metastatic potential of ras-transformed rat embryonic cells.22,23,24
Overexpression of the HER2/neu gene has been correlated with enhanced tumorigenicity, enhanced metastatic potential, poor patient prognosis, and decreased chemosensitivity in many types of cancer, including breast, ovarian, lung, and stomach carcinomas.8,31 Results from our previous studies demonstrated that the HER2/neu oncogene was overexpressed in three different Ewing's sarcoma cell lines.11 We also demonstrated that E1A gene transfer using an adenoviral vector resulted in down-regulation of HER2/neu expression and increased cellular sensitivity to VP-16 and doxorubicin but not to cisplatin. This enhanced sensitivity to VP-16 was mediated by a specific increase in topoisomerase IIα expression.11
The purpose of this study was to determine the effect of E1A gene transfer on tumor growth and VP-16 sensitivity in Ewing's sarcoma in vivo. The results presented here demonstrate that the intratumoral injection of Ad-E1A resulted in expression of the E1A gene in the tumor tissue with down-regulation of HER2/neu expression (Figs 4 and 5A). Tumor sensitivity to VP-16 was enhanced following transduction with E1A, as demonstrated by increased tumor cell apoptosis, tumor response, and long-term survival (Figs 2, 3, and 5B). E1A gene therapy alone had some effect, but this was clearly augmented when E1A and VP-16 were used in combination. In addition to an increase in tumor cell apoptosis in vivo, we also demonstrated a significant reduction in tumor vascularity in the animals treated with E1A plus VP-16 compared with those treated with either therapy alone (Fig 5C). Intratumoral Ad-β-gal had no effect on tumor apoptosis (Fig 5B), tumor vascularity (Fig 5C), or tumor size (data not shown). Furthermore, Ad-β-gal did not enhance the efficacy of VP-16 as measured by survival (Figs 2 and 3), in vivo tumor cell apoptosis (Figs 2, 4, and 5B), and tumor vascularity (Figs 2, 4, and 5C).
It has been reported that E1A can induce sensitivity toboth p53-dependent and independent programmed cell death.25,26,27 The apoptotic function of E1A is accompanied by the processing of caspase-3 and cleavage of poly (CADP-ribose) polymerases.28,29 The mechanism of the E1A-induced apoptosis in this tumor model is not clear at thistime. However, based on our previous work, we hypothesize that the enhanced sensitivity to VP-16 is secondary to E1A's ability to up-regulate topoisomerase IIα expression.11 Further investigations are needed to confirm this.
In summary, the current study confirms our in vitro findings and supports the development of E1A gene therapy for HER2/neu-overexpressing Ewing's sarcoma. Patients who have a relapse after chemotherapy usually have explosive disease that is not responsive to additional chemotherapy. Clinical trials with E1A gene therapy are currently under way in patients with breast and ovarian cancers.30 The dearth of therapeutic options for patients with recurrent Ewing's sarcoma argues for the development of innovative new treatments. Our data suggest that in vivo Ad-E1A gene therapy in combination with VP-16 chemotherapy may have potential in the treatment of drug-resistant Ewing's sarcoma.
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