Inducible nitric oxide synthase activity is essential for ... - Nature

Cancer Gene Therapy (2006) 13, 676–685 All rights reserved 0929-1903/06 $30.00

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ORIGINAL ARTICLE

Inducible nitric oxide synthase activity is essential for inhibition of prostatic tumor growth by interferon-b gene therapy MV Olson1,4, J Lee2,4, F Zhang3, A Wang2 and Z Dong2 1

Department of Neuro-Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, USA; Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, OH, USA and 3 Department of Cancer Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA 2

We have previously reported that adenoviral vector-mediated interferon (IFN)-b gene therapy inhibits orthotopic growth of human prostate cancer cells in nude mice. The purpose of this study was to determine efficacy and mechanisms of this therapy in immunecompetent mice. TRAMP-C2Re3 mouse prostate cancer cells infected with 100 multiplicity of infection (MOI) of adenoviral vector encoding for mouse IFN-b (AdmIFN-b), but not AdE/1 (a control adenoviral vector), produced approximately 60 ng/105 cells/24 h of IFN-b. The tumorigenicity of AdmIFN-b-transduced cells was dramatically reduced in the prostates of C57BL/6 mice. A single intratumoral injection of 2 109 PFU (plaque-forming unit) of AdmIFN-b inhibited tumor growth by 70% and prolonged survival of tumor-bearing mice. Intriguingly, this AdmIFN-b therapy did not alter the growth of tumors in inducible nitric oxide synthase (iNOS)-null C57BL/6 mice. Immunohistochemical analysis revealed that treatment of tumors with AdmIFN-b in wild-type C57BL/6 mice led to increased iNOS expression, decreased microvessel density, decreased cell proliferation, and increased apoptosis. Furthermore, quantitative reverse-transcriptional PCR analysis showed that AdmIFN-b therapy, in C57BL/6 but not the iNOS-null counterparts, reduced levels of the mRNAs for angiopoietin, basic fibroblast growth factor, matrix metalloproteinase-9, transforming growth factor-b1, vascular endothelial growth factor (VEGF)-A, and VEGF-B, as well as the antiapoptotic molecule endothelin-1. These data indicated that IFN-b gene therapy could be effective alternative for the treatment of locally advanced prostate cancer and suggest an obligatory role of NO in IFN-b antitumoral effects in vivo. Cancer Gene Therapy (2006) 13, 676–685. doi:10.1038/sj.cgt.7700941; published online 10 February 2006 Keywords: interferon-b; prostate cancer; adenoviral vectors; nitric oxide synthase; angiogenesis

Introduction

Interferon (IFN)-b, a type I IFN, is a cytokine that can stimulate immune response, inhibit cell growth, and induce cell differentiation and apoptosis.1–5 When used to treat tumors, IFN-b is a potent antiangiogenic and antimetastatic molecule. Not only does IFN-b inhibit growth and migration of endothelial cells,6 as other angiogenic inhibitors do, but it also downregulates the expression of molecules that mediate angiogenesis and invasion in tumor cells.7,8 However, clinical trials using type I IFNs have shown limited response for most solid tumors, possibly because of insufficient accumulation of biologically active IFN-b within the tumors.9 This hypothesis is supported by numerous studies using gene Correspondence: Dr Z Dong, Department of Internal Medicine, Hematology-Oncology Division, University of Cincinnati College of Medicine, 3125 Eden Avenue, Rm 1308, Cincinnati, OH 45267, USA. E-mail: [email protected] 4 These two authors contributed equally to this work. Received 5 September 2005; revised 20 October 2005; accepted 21 November 2005; published online 10 February 2006

therapy approaches in both murine and human solid tumor models.10–17 Moreover, we and others have shown that intralesional delivery of the IFN-b gene, which allows for the accumulation of higher intratumoral concentrations of IFN-b, is able to suppress primary tumors, prolong the survival of tumor-bearing mice, and protect against a second challenge in syngeneic mice.12,18,19 Nitric oxide (NO) is a small molecule produced by mammalian cells. It interacts with a wide array of molecules from superoxide anion to proteins. By oxidation of thiols, heme, Fe-5 clusters, and other nonheme iron prosthetic groups, NO can alter the function of target molecules20,21 and modulate gene expression.22,23 The inducible NO synthase (iNOS), predominately expressed in activated macrophages, is responsible for high output production of NO.24 Our previous studies showed that suppression of tumor growth and metastasis by murine IFN-b and IFN-b gene therapies correlates with iNOS expression in tumor lesions.25,26 Depletion of macrophages significantly reduced the number of iNOS-expressing cells in orthotopic tumors of PC-3MM2 human prostate cancer cells in nude mice and made adenoviral vector encoding for mouse IFN-b (AdmIFN-b) therapy against established PC-3MM2 tumors less effective.27

Inducible nitric oxide synthase in interferon-b gene therapy MV Olson et al

These findings prompted us to further investigate the role of iNOS in suppressing tumor growth by IFN-b gene therapy. Since the host immune system can significantly affect adenovirus-mediated gene delivery, this study was carried out in an orthotopic model of mouse prostate cancer in immune-competent syngeneic mice. Our data show that IFN-b gene therapy suppressed the growth of TRAMP-C2Re3 tumors and significantly prolonged the survival of tumor-bearing mice. The therapeutic effects were diminished in mice deficient in iNOS. Furthermore, the therapy reduced microvessel density in the tumors, which is coincident with downregulation of several proangiogenic molecules and an antiapoptotic molecule. Materials and methods

Mice Specific pathogen-free male C57BL/6 mice were purchased from Harlan (Indianapolis, IN). We were generously provided by Dr Isaiah J Fidler (The University of Texas MD Anderson Cancer Center) with breeding pairs of iNOS gene knockout mice that have been back-crossed with C57BL/6 mice for 12 generations (iNOS-null C57BL/6 mice). The mice were maintained in a facility approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the US Department of Agriculture, US Department of Health and Human Services, and the National Institute of Health. The mice were used according to Institutional guidelines when they were 8–10 weeks of age. Reagents Eagle’s minimal essential medium (MEM), Ca2 þ , Mg2 þ free Hanks’ balanced salt solution (HBSS), and fetal bovine serum (FBS) were purchased from MA Bioproducts (Walkersville, MD). A TRIzol total cellular RNA isolation kit was purchased from Invitrogen (San Diego, CA). Antibodies against endothelial cell marker CD31,28 proliferative cell nuclear antigen (PCNA),29 and iNOS were purchased from BD Biosiciences (San Jose, CA). Antibody specific for macrophage marker F4/8030 was purchased from Serotec Inc. (Raleigh, NC). Monoclonal antibody against mouse IFN-b was purchased from US Biologicals (Swampscott, MA) and rabbit anti-mouse IFN-b was purchased from Calbiochem (San Diego, CA). Recombinant mouse IFN-b (specific activity, 107 U/mg protein) was purchased from Cedar Lane Laboratories (Hornby, ON). TUNEL assay kit was purchased from Promega (Madison, WI). The adenoviral vector encoding the murine IFN-b gene (AdmIFN-b) was constructed in our laboratory as described previously.18 AdE/1 (an E1deleted adenovirus that does not contain an expression cassette) was generously provided by Dr B. Fang (The University of Texas MD Anderson Cancer Center). AdE/1 and AdmIFN-b were propagated in 293 cells and purified by the two-step CsCl2 gradient centrifugation protocol.

Cell culture The murine TRAMP-C2Re3 cell line, established by recycling TRAMP-C2 cells31 three times between the site of injection (prostate) and the regional lymph nodes, was generously provided by Dr Jerald Killion (The University of Texas MD Anderson Cancer Center). TRAMP-C2Re3 cells, which are androgen independent, were grown in MEM, supplemented with 5% FBS (MEM-5% FBS), nonessential amino acids, sodium pyruvate, vitamin A and glutamine (Invitrogen, Carlsbad, CA). IFN-b detection An enzyme-linked immunoadsorbant assay (ELISA) method developed in this laboratory was used to determine IFN-b protein levels in both conditional cell culture media and prostate tumor lysates. Briefly, ELISA plates were coated at 41C for 18 h with 2.5 mg/ml of rat anti-mouse IFN-b antibody, followed by an incubation with a blocking solution (PBS-5% BSA). After washing, conditioned cell culture media or homogenized tumor tissue lysates were added to the plates at 50 ml/well and followed by washing, incubation with rabbit anti-mouse IFNb (1:1000) and horseradish peroxidase-conjugated anti-rabbit IgG (1:4000, Amersham Biosciences, Piscataway, NJ). Mouse IFN-b (0.3–20 ng/ml; specific activity, 107 U/mg protein, Cedar Lane Laboratories, Hornby, ON) was diluted to generate a standard curve. In vitro assay of adenoviral vectors on cell growth TRAMP-C2Re3 cells were plated in triplicate using 12well plates at a density of 5 104 cells per well. After 24 h , various amounts of AdE/1, AdmIFN-b or AdmIFN-b plus 500 neutralization U/ml of rabbit anti-mouse IFN-b (R&D systems, Minneapolis, MN) were added and incubated for 3 or 6 days with one medium change on day 3. During the final 2 h of incubation, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) was added at 0.42 mg/ml. The medium was removed, and dark-blue formazan was dissolved in dimethyl sulfoxide. The solution was transferred into a 96-well plate and the absorbance was measured with a 96well microplate reader (FluoStar, BMG, NC) at 570 nm. The percentage of inhibition on cell growth was calculated according to the following formula: inhibition (%) ¼ (1–A570 of treated group/A570 of control group) 100. Tumor cell inoculation TRAMP-C2Re3 cells in exponential growth phase were harvested by a 1-min treatment with a 0.25% trypsin/ 0.02% EDTA solution. The flask was tapped to detach the cells, MEM-5% FBS added, and gently agitated to produce a single-cell suspension. The cells were washed and resuspended in HBSS. Only suspensions of single cells with viability exceeding 95% (ascertained by Trypan blue exclusion) were used for animal inoculation. Mice were anesthetized with Nembutal and placed in the supine position. The surgical procedure was performed as detailed in our previous study.32 Briefly, a lower midline incision was created and the prostate exposed. A tumor cell suspension (105 cells in 20 ml

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HBSS) was injected into the dorsal prostatic lobes using a 30-gauge needle, a 1-ml disposable syringe, and a calibrated push button-controlled dispensing device (Hamilton Syringe Company, Reno, NV). The abdominal wound was closed in two layers using absorbable sutures and wound clips (Autoclip, Clay Adama, Parsippany, NJ), respectively. With a 100% incident rate, TRAMPC2Re3 cells produced approximately 50 mg tumors by day 7 and 1–1.5 g tumors by 25–30 days after intraprostate injection of 105 cells.

Treatment procedure Mice were anesthetized with Nembutal, the incision reopened, and the prostate tumor exposed. PBS, AdE/1, or AdmIFN-b in 40 ml of PBS was injected into each tumor’s center with a Hamilton syringe and a 30-gauge needle over 3 min. The needle was removed slowly after a 30-s delay to avoid leakage. Prostate tumor weights were determined at the end of the experiments (28–32 days after tumor cell inoculation). Immunohistochemical staining Immunohistochemical analyses were performed as described previously27,33 Briefly, tumor tissues were placed in ornithine carbamyl transferase compounds (Sakura Fineter, Torrance, CA) and snap frozen in liquid nitrogen. Frozen sections (8–10 mm) were fixed sequentially in cold acetone, acetone/chloroform (1:1), and acetone, and treated with 3% hydrogen peroxide (H2O2) in methanol (v/v). The treated slides were incubated in blocking solution and then reacted for 18 h at 41C in a humidified chamber with the antibody to iNOS or CD31. The sections were rinsed and incubated with peroxidaseconjugated secondary antibodies. A positive reaction was visualized by incubating the slides with stable DAB and counterstaining with Mayer’s hematoxylin. For immunohistochemical staining using antibodies to PCNA and F4/80, paraffin-embedded sections (4 mm) of tumor samples were dewaxed and stained as described for the frozen sections. The slides were dried and mounted with Universal mount, and images were digitized using an Olympus CCD camera (Olympus, Tokyo, Japan) and a personal computer equipped with Optimas Image Analysis Software (Optimas Corp., Bothell, WA). Immunofluorescence double staining Apoptotic endothelial cells in tumor tissues were examined following a protocol described in our previous study.33.Briefly, frozen tissue sections (8–10 mm) were fixed in cold acetone for 5 min, acetone/chloroform (1:1) for 5 min, and acetone for another 5 min. They were then washed with PBS, incubated with protein-blocking solution (PBS-5% normal horse serum and 1% normal goat serum) for 20 min at room temperature, and then with a rat anti-mouse CD31 antibody over 18 h at 41C. After the slides were rinsed with PBS, they were incubated with Texas Red-conjugated goat anti-rat antibody for 1 h in the dark. Samples were washed with PBS containing 0.1% Brij and then with PBS. The sections were then stained by

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the TUNEL method using a kit (Promega, Madison, WI). Briefly, sections were fixed with 4% paraformaldehyde (methanol free), washed with PBS, and then incubated with 0.2% Triton X-100 for 15 min at room temperature. After the samples were washed twice with PBS, they were incubated in equilibration buffer (from the kit) for 10 min. The equilibration buffer was drained, and reaction buffer containing equilibration buffer, nucleotide mix, and TdT enzyme was added and incubated in a humid atmosphere at 371C for 1 h in the dark. The reaction was terminated by immersing the samples in 2 SSC for 15 min. Samples were washed with PBS to remove unincorporated fluorescein-dUTP before mounting and visualization.

Total RNA isolation and analysis Total cellular RNA was extracted from tumor cells or tumor tissues using a TRIzol RNA extraction kit (Invitrogen) and analyzed by real-time reverse-transcriptional PCR (RT-PCR). Briefly, 2 mg of total RNA were reverse transcribed at 421C in a 20 ml reaction buffer containing 200 U of Reverse Transcriptase (Stratagene, La Jolla, CA), 500 ng of Oligo d(T) primer, and 500 nM dNTP mix. Of the obtained cDNA, 25 ng was amplified in a 7300 Real-Time PCR system (Applied Biosystems, Foster City, CA) in a PCR reaction containing the primer pairs for amplification of various genes or b-actin and the Brilliant SYBR Green QPCR Master Mix (Stratagene). After an initial step at 951C for 10 min, temperature cycling began (denaturation at 951C for 15 s, hybridization at 601C for 15 s and elongation at 721C for 30 s) for a total of 40 cycles. The cycle threshold values were used to calculate the normalized expression of specific genes against b-actin using the Q-Gene software.34 Statistical analysis The gene therapy experiments were performed with 5–10 mice per group and were repeated at least once. Differences in tumor incidence between treatment and control groups were analyzed with the w2 test. Differences in tumor weight among study and control groups were compared by analysis of variance (ANOVA). Survival data were analyzed using the Kaplan–Meier plot, and the statistical significance determined using the Mantel–Cox log-rank test.

Results

Effects of in vitro AdmIFN-b transduction on TRAMPC2Re3 cells We first evaluated the transduction efficiency of adenoviral vectors in TRAMP-C2Re3 cells in culture. TRAMPC2Re3 cells were infected with AdGFP at different multiplicity of infections (MOIs) for 48 h and then examined under a inverted fluorescent microscope. We found that approximately 60% of TRAMP-C2Re3 cells expressed GFP at 30 MOI, indicating the cells were susceptible to adenoviral gene transfer. Cells infected with 5–100 MOI of AdmIFN-b, but not AdE/1, expressed IFN-b mRNA in a dose-dependent manner (data not


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shown) and secreted 5–60 ng/105 cells/24 h of IFN-b (Figure 1a). The expression of IFN-b peaked at 2–3 days and could be detected for up to 10 days after infection (data not shown). The overexpression of IFN-b caused growth inhibitory effects on TRAMP-C2Re3 cells. At 50 MOI, AdmIFN-b- but not AdE/1-infected cells demonstrated a 30% growth inhibition (Figure 1b). The growth inhibitory effects of AdmIFN-b were reversed with the addition of IFN-b neutralizing antibodies (Figure 1b). Next, we determined whether a transient transduction of the IFN-b gene with AdmIFN-b was sufficient for altering tumor formation in mice. TRAMP-C2Re3 cells were infected for 48 h with 30 MOI of AdmIFN-b or AdE/1. Uninfected control or infected cells at 105/mouse were implanted into the prostate of C57BL/6 mice. Prostate tumors were examined 4 weeks later. Data in Figure 1c show that mice inoculated with uninfected- or AdE/1-transduced TRAMP-C2Re3 cells developed fastgrowing tumors with an incidence rate of 100%. In contrast, AdmIFN-b-transduced cells had a lower tumor incidence and produced significantly smaller tumors (Figure 1c).

Efficacy of IFN-b gene therapy for orthotopic TRAMP-C2Re3 tumors TRAMP-C2Re3 cells (105/mouse) were implanted into the dorsal prostatic lobes. After 7 days, prostate tumors were injected with either PBS, AdE/1 at 2 109 PFU, or different doses of AdmIFN-b. As shown in Figure 2a, a single injection with AdE/1 did not affect the growth of TRAMP-C2Re3 tumors. In contrast, progression of TRAMP-C2Re3 tumors was suppressed by 79, 65 and 32% in mice treated with 2 109, 1 109 and 0.5 109 PFU of AdmIFN-b, respectively (Figure 2a). To determine whether AdmIFN-b could improve the survival of tumor-bearing mice, TRAMP-C2Re3 cells were inoculated into the prostates of mice and treated on day 7 with PBS or 2 109 PFU of either AdE/1 or AdmIFN-b. The mice were killed when they were moribund. The data in Figure 2b demonstrate that therapy with AdmIFN-b significantly prolonged the survival of mice with TRAMP-C2Re3 prostate tumors as compared to mice treated with PBS or the control vector. Mice treated with PBS, AdE/1 and AdmIFN-b showed a 50% survival rate at 36, 36 and 54 days, respectively (Figure 2b). To investigate the potential role of iNOS in tumor growth inhibition induced by AdmIFN-b25,27 we administrated the same therapy in mice null for iNOS. A total of 79 wild-type and 80 iNOS-null C57BL/6 mice, from four separate experiments, were inoculated with TRAMPC2Re3 cells. On day 7 after the inoculation, prostatic tumors were injected with 2 109 PFU of either AdE/1 or AdmIFN-b. The experiments were terminated 28 days after tumor cell inoculation and the tumor weights compared. The growth rate of TRAMP-C2Re3 cells in iNOS-null mice was moderately reduced in comparison with that in wild-type C57BL/6 mice: tumors weights were 9977445 and 8487165 mg (P40.05) in C57BL/6 and

Figure 1 Effects of transduction of TRAMP-C2Re3 cells in vitro with adenoviral vectors. (a) TRAMP-C2Re3 cells in 24-well plates at 2 105 cells/well were incubated for 48 h with increasing concentrations of AdE/1 or AdmIFN-b. IFN-b protein in culture supernatants was assessed by ELISA. Data shown are mean7s.d. of one representative experiment of three. (b) TRAMP-C2Re3 cells in 12well plates at 5 104 cells/well were incubated for 6 days with increasing concentrations of AdE/1 or AdmIFN-b in the absence or presence of IFN-b-neutralizing antibody. Viable cells in the wells were stained with MTT. Data shown are mean7s.d. of one representative experiment of three. (c) TRAMP-C2Re3 cells in 100-mm plates were incubated for 48 h with MEM-5% containing PBS or 50 MOI of either AdE/1 or AdmIFN-b. The cells were harvested, washed, and implanted into the prostates of C57BL/6 mice at 105 cells/mouse (five mice/group). Prostatic tumor weights were assessed 3 weeks later. Data shown are mean7s.d. from one representative experiment of two. *Po0.05.

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iNOS-null C57BL/6 mice, respectively. The data presented in Figure 2c show that intratumoral administration of AdmIFN-b significantly inhibited the growth of TRAMP-C2Re3 tumors in C57BL/6 mice (70%74.3). In the sharp contrast, the therapy had no significant effects on tumor growth in the prostates of iNOS-null C57BL/6 mice, suggesting that iNOS expression is crucial for suppressing TRAMP-C2Re3 tumor growth in mice (Figure 2c).

Figure 2 Efficacy of AdmIFN-b therapy against orthotopic TRAMPC2Re3 tumors. (a) TRAMP-C2Re3 cells were implanted into the prostates of C57BL/6 mice. After 7 days, the tumors were injected with PBS, 2 109 PFU of AdE/1 or increasing doses of AdmIFN-b with 10 mice/group. Tumor-bearing mice were killed 3 weeks after the therapy and prostatic tumor weights were assessed. Data shown are mean7s.d. of one representative experiment of two. (b) On day 8 after tumor cell implantation, tumors in C57BL/6 mice were injected with PBS or 2 109 PFU of either AdE/1 or AdmIFN-b with 10 mice/ group. Data shown are mean7s.d. from one representative experiment of two. (c) TRAMP-C2Re3 cells were implanted into the prostates of C57BL/6 mice or their age-matched iNOS-null counterparts. On day 8 after tumor cell implantation, tumors were injected with 2 109 PFU of either AdE/1 or AdmIFN-b. Experiments were terminated 3 weeks later and prostatic tumor weights were assessed. Data shown are mean7s.e. of four independent experiments. *Po0.05.

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Histological analyses of AdmIFN-b-treated tumors On day 7 after intraprostate implantation of the TRAMPC2Re3 cells into wild-type and iNOS-null C57BL/6 mice, the prostatic tumors were injected with PBS, AdE/1, or AdmIFN-b, and were sampled 5 days later for analyses. Hematoxylin–eosin (H&E) staining revealed that, after treatment with AdmIFN-b, but not PBS or AdE/1, the tumors in C57BL/6 mice consisted largely of necrotic tissue. However, AdmIFN-b-induced necrosis was not evident in tumors from iNOS-null mice. Tumors in C57BL/6 mice injected with PBS and AdE/1 contained approximately threefold more CD31 þ cells (stained red) than did those injected with AdmIFN-b. Consistent with these findings, CD31 mRNA was reduced by 80% in tumors injected with AdmIFN-b (Table 1). Tumors from C57BL/6 mice injected with AdmIFN-b, but not PBS and AdE/1, contained numerous apoptotic cell bodies, labeled by the TUNEL methods and visualized as green reaction product in Figure 3. In addition, a few cells in AdmIFNb-treated tumors were colocalized markers for both apoptosis (TUNEL, green) and an endothelial cells (anti-CD31, red), yielding the yellow color in a supercomposed image (Figure 3). Furthermore, proliferating cells, as detected by PCNA staining, were significantly reduced in AdmIFN-b-treated tumors (Figure 3). In concert with these data, quantitative RT-PCR revealed that expression of Ki67, another proliferation marker, was reduced by 68% in AdmIFN-b-treated tumors in C57BL/6 mice (Table 1). AdmIFN-b therapy, however, had no significant effects on apoptosis, cell proliferation, and microvessel density in tumors growing in iNOS-null mice. Immunohistochemical staining, using an antibody against a macrophage-specific F4/80 antigen, showed that tumors from both C57BL/6 and iNOS-null mice were densely infiltrated by macrophages, which was not significantly altered by AdmIFN-b therapy (Figure 3). Induction of iNOS, however, which is preferentially expressed in activated macrophages,24 was detected by immunohistochemistry (Figure 3) and RT-PCR (Table 1) only in the AdmIFN-b-treated tumors growing in C57BL/6 mice. Alterations of gene expression in AdmIFN-b-treated tumors IFN-b expression. To ensure that administration of AdmIFN-b vector was able to lead to production of IFNb within tumors, IFN-b expression levels were assessed on days 3, 5 and 10 after intratumoral injection of PBS or viral vectors (2 109 PFU of either AdE/1 or AdmIFN-b).


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Table 1 Quantitative analysis of the expression of CD31, Ki-67 and iNOS in tumor samplesa C57BL/6 mice

iNOS-null mice

mRNA

AdE/1

AdmIFN-b

AdmIFN-b/AdE/1 (%)

AdE/1

AdmIFN-b

AdmIFN-b/AdE/1 (%)

CD31 Ki-67 INOS

27.0E-4b 8.2E-4 1.0E-5

5.2E-4 2.7E-4 6.9E-5

19 32 690

4.7E-3 6.7E-4 2.1E-5

3.8E-3 4.6E-4 2.1E-5

80 69 100

a TRAMP-C2Re3 tumors were implanted into the prostates of C57BL/6 mice or their iNOS-null counterparts. After 7 days, tumors were injected with 2 109 of either AdE/1 or AdmIFN-b. The tumors were sampled 5 days later for quantitative RT-PCR analysis of gene expression. b Relative mRNA levels after normalization to b-actin.

Figure 3 Immunohistochemical analysis of TRAMP-C2Re3 tumors. TRAMP-C2Re3 tumors were implanted into the prostates of C57BL/6 mice or their iNOS-null counterparts. After 7 days, tumors were injected with PBS or 2 109 of either AdE/1 or AdmIFN-b. The tumors were sampled 5 days later for immunohistochemical analysis.

Tumor lysates were prepared and analyzed by ELISA. Data in Figure 4a show that IFN-b protein was detected in tumors injected with AdmIFN-b, but not PBS and AdE/1. IFN-b production was highest in tumors sampled on the 3rd day following AdmIFN-b administration, detecting approximately 45 ng/100 mg tumor tissue protein of IFN-b. IFN-b levels significantly reduced in tumors sampled on day 10 with levels at approximately 5 ng/100 mg tumor tissue protein. Quantitative real-time RT-PCR analysis revealed similar findings in that IFN-b mRNA levels were the most abundant in tumors sampled on day 3 and reduced in those sampled on day 10 (Figure 4b). Virtually no IFN-b transcript was detected in tumors injected with PBS or AdE/1 (Figure 4b).

Expression of pro-angiogenesis genes. TRAMP-C2Re3 cells were inoculated into the prostates of C57BL/6 or iNOS-null mice. On day 10 after the inoculation, when the tumors reached approximately 100 mg, the tumors were injected with 2 109 PFU of either AdE/1 or AdmIFN-b and sampled for total RNA extraction 5 days later. The mRNA levels of angiogenic molecules were analyzed using real-time RT-PCR (Table 2). AdmIFN-b-treated tumors growing in C57BL/6 mice showed reduced expression levels of transforming growth factor (TGF)b1, angiopoietin (angio)-1, basic fibroblast growth factor (bFGF), matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF)-A and VEGF-B by 60–70%. On the contrary, except for VEGF-A, which was

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reduced by 47%, AdmIFN-b treatment had no significant effects on the expression of these angiogenic molecules in tumors growing in iNOS-null C57BL/6 mice (Table 2).

Expression of urokinase plasminogen activator and MMP-2 in tumors from both C57BL/6 and iNOS-null C57BL/6 mice was not significantly altered by AdmIFN-b (Table 2). In addition, expression of endothelin (ET)-1, an antiapoptotic molecule overexpressed in prostate cancer, in tumors growing in C57BL/6 but those in iNOS-null C57BL/6 mice, was also reduced by AdmIFNb (Table 2). In the final set of experiments, we determine whether AdmIFN-b infection could directly alter expression of angiogenic molecules in culture. We found that incubation of TRAMP-C2Re3 cells for 5 days with 30 MOI of AdE/1 or AdmIFN-b did not inhibit expression of the angiogenic molecules listed in Table 1 (data not shown).

Discussion

Figure 4 Expression of IFN-b in tumors. TRAMP-C2Re3 cells were implanted into the prostates of C57BL/6 mice. After 7 days, tumors were injected with PBS or 2 109 PFU of either AdE/1 or AdmIFN-b. Mice were killed on days 3, 5 or 10 after the therapy and the tumors were sampled for IFN-b protein (ELISA, A) and IFN-b mRNA (quantitative RT-PCR, B).

Our previous studies have shown that intralesional delivery of AdmIFN-b, but not a control vector, inhibited growth of PC-3MM2 human prostate cancer cells implanted in the prostate of nude mice. 25,27 The data implicated that macrophages, through expression of iNOS, may play an important role in suppressing tumor growth in the xenograft orthotopic tumor model. The focus of the present study was to investigate efficacy of AdmIFN-b therapy against prostate cancer in immunecompetent mice and to further elucidate the role of iNOS in the therapy. Our data shows that TRAMP-C2Re3 cells are susceptible to adenovirus infection and that IFN-b gene therapy inhibits orthotopic growth of TRAMPC2Re3 cells in syngeneic mice in a dose-dependent manner. Furthermore, the survival of tumor-bearing mice was significantly prolonged. These data support the notion that IFN-b gene therapy may be an effective alternative therapy for locally advanced prostate cancer. IFN-b is a multifunctional cytokine. It has been reported that treatment of tumor cells in vitro with IFNb can inhibit angiogenic molecule expression7,35–37 and inhibit proliferation.2,38–40 We observed in the current study that AdmIFN-b therapy had minimal effects on

Table 2 Real-time RT-PCR analysis of gene expression in tumor samplesa C57BL/6 mice mRNA TGF-b1 Angio-1 bFGF MMP-9 VEGF-A VEGF-B MMP-2 uPA ET-1

iNOS-null mice

AdE/1

AdmIFN-b

AdmIFN-b/AdE/1 (%)

AdE/1

AdmIFN-b

AdmIFN-b/AdE/1 (%)

4.4E-3b 3.7E-4 2.1E-4 4.6E-5 1.8E-2 6.5E-3 3.7E-3 2.2E-4 20.0E-3

1.6E-3 1.5E-4 1.0E-4 1.7E-5 4.3E-3 2.8E-3 3.6E-3 1.5E-4 6.9E-4

36 40 48 37 24 43 100 69 35

8.0E-4 1.9E-4 1.6E-4 5.1E-5 1.6E-2 0.74E-2 6.3E-3 10.0E-4 1.1E-3

6.3E-4 2.0E-4 1.3E-4 6.2E-5 8.5E-3 1.5E-2 4.6E-3 9.8E-5 1.2E-3

78 100 83 121 53 200 73 98 100

a TRAMP-C2Re3 tumors were implanted into the prostates of C57BL/6 mice or their iNOS-null counterparts. After 7 days, tumors were injected with 2 109 of either AdE/1 or AdmIFN-b. The tumors were sampled 5 days later for quantitative RT-PCR analysis of gene expression. b Relative mRNA levels after normalization to b-actin.

Cancer Gene Therapy


tumor growth in iNOS-null mice and that transduction of TRAMP-C2Re3 cells in vitro with AdmIFN-b did not inhibit angiogenic molecule expression. From these data, we conclude that although an infection of TRAMPC2Re3 cells with AdmIFN-b moderately inhibited their growth in culture, the therapeutic effects of AdmIFN-b in this mouse prostate cancer model were not consequences of direct antiangiogenic or direct antiproliferative activities of IFN-b. Rather, these data indicate that iNOS expression is essential for suppressing tumor growth by IFN-b gene therapy, which is consistent with observations made in the PC-3MM2 model.25,27,32 Furthermore, we found that expression of iNOS in AdmIFN-b-treated tumors from C57BL/6 mice not only inversely correlated with microvessel density and expression of TGF-b1, as seen in the PC-3MM2 model25,27 but with expression of several other angiogenic molecules, namely bFGF, angio1, VEGF-A, VEGF-B and MMP-9. The exact mechanisms by which AdmIFN-b inhibited expression of these angiogenic molecules in TRAMP-C2Re3 tumors in C57BL/6 mice, however, remain to be elucidated. The AdmIFN-b therapy induced a massive apoptosis in tumors growing C57BL/6 mice, which is the most striking difference between AdmIFN-b-treated tumors growing in C57BL/6 mice and those in their iNOS-null counterparts. A similar apoptosis was previously observed in tumors formed by PC-3MM2 cells overexpressing a dominantnegative construct of type II TGF-b receptor,33 suggesting that it may be due indirectly to the downregulation of TGF-b1 as well as other angiogenic molecules and, hence, to suppression of angiogenesis. On the other hand, NO, generated by iNOS, is the most potent cytotoxic small molecule generated in cells. Overexpression of iNOS in tumor cells has been shown to lead to apoptotic cell death in culture and to diminished tumorigenicity in mice.41 When inoculated as a mixture, cells overexpressing iNOS can inhibit tumor formation by bystander cells.41 Therefore, it is very likely that the massive apoptosis seen in this orthotopic model was caused by toxic effects of NO. Alternatively, the massive apoptosis could also be due to the differential downregulation of ET-1, which has been shown to inhibit apoptosis in prostate cancer cells,42 in tumors from C57BL/6 mice by AdmIFN-b. In addition to induction of iNOS expression in tumors as reported in this study, IFN-b gene therapy may also inhibit tumor growth by modulating immune response, including the enhancement of antigen presentation by macrophages and downstream T-cell-mediated antitumor activities. Specifically, IFN-b may stimulate these responses by increasing CD4 þ T-cell infiltration,43 upregulating production of IFN-g44 and IL-12,45 enhancing differentiation of dendritic cells,46 increasing the sensitivity of tumor cells to macrophage- and T-cell-mediated cytotoxicity,47 downregulating T-suppressor cell function,48 enhancing Class I and II major histocompatibility complex antigen expression,49 and promoting the survival of activated T cells.50 In summary, intralesional injection of AdmIFN-b inhibited growth of orthotopic TRAMP-C2Re3 tumors in C57BL/6 but not their iNOS-null counterparts, which

correlated with an inhibition of angiogenesis and downregulation of several important angiogenic molecules as well as antiapoptotic molecule ET-1. Together with the findings reported in our previous studies, these data suggest that the antitumoral effects of IFN-b are dependent on the activation of macrophages and induced release of NO. It is noteworthy that the adenoviral vector used in the present study was derived from human type 5 adenovirus, which has significantly higher transduction efficiency in human cells. In future studies, we will further investigate efficacy and mechanisms by which an adenoviral vector encoding human IFN-b inhibit human prostate cancer cells growth in an orthotopic model.

Acknowledgements

We thank Dr Robert Franco (University of Cincinnati College of Medicine) for critical reading of this manuscript, Dr Corazon D Bucana (University of Texas MD Anderson Cancer Center) for technical assistance with immunohistochemical staining. This work is supported in part by funds from The University of Cincinnati College of Medicine Cancer Center Start-up funds (to ZD) and RSG-98-332-02-CCE from the American Cancer Society (to ZD).

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