Combined ionizing radiation and sKDR gene delivery for treatment of ...

Gene Therapy (2005) 12, 407–417 & 2005 Nature Publishing Group All rights reserved 0969-7128/05 $30.00 www.nature.com/gt

RESEARCH ARTICLE

Combined ionizing radiation and sKDR gene delivery for treatment of prostate carcinomas SA Kaliberov, LN Kaliberova and DJ Buchsbaum Department of Radiation Oncology, University of Alabama at Birmingham, Birmingham, AL, USA

Overexpression of vascular endothelial growth factor (VEGF) and its cognate receptor KDR has been linked to a more aggressive phenotype of human prostate carcinomas. The importance of signal transduction through the VEGF receptor 2 is illustrated by use of soluble KDR, which binds to VEGF and sequesters this ligand before its binding to cellular receptor. Treatment with recombinant adenovirus AdVEGFsKDR, encoding sKDR under control of the human VEGF promoter, significantly inhibited the proliferation of human vascular endothelial cells and prostate cancer cells. AdVEGFsKDR infection decreased migration of endothelial 1P-1B cells (61% reduction) and DU145 prostate carcinoma cells (47%) in comparison with AdCMV-Luc-infected control cells. Ionizing radiation upregulated VEGF promoter activity in prostate

carcinoma and endothelial cells. AdVEGF-sKDR infection significantly reduced human vascular endothelial and prostate cancer cell proliferation and sensitized cancer cells to ionizing radiation. In vivo tumor therapy studies demonstrated significant inhibition of DU145 tumor growth in mice that received combined AdVEGF-sKDR infection and ionizing radiation versus AdVEGF-sKDR alone or radiation therapy alone. These results suggest that selective transcriptional targeting of sKDR gene expression employing a radiation inducible promoter can effectively inhibit tumor growth and demonstrate the advantage of combination radiotherapy and gene therapy for the treatment of prostate cancer. Gene Therapy (2005) 12, 407–417. doi:10.1038/sj.gt.3302432 Published online 23 December 2004

Keywords: sKDR; VEGF; ionizing radiation; prostate cancer; gene therapy

Introduction Prostate cancer is the most common cancer diagnosed in American males, and is the second leading cause of cancer-related deaths, mostly because of the limitations of current therapeutic modalities, especially once the cancer has metastasized.1 Surgical extirpation and external beam radiotherapy are potentially curative treatment options for clinically localized prostate cancer.2 The development of new therapeutic strategies for the treatment of hormone refractory metastatic prostate cancer is imperative. It appears that a combination of treatment approaches will be required to control metastatic disease. Combined radiotherapy and gene therapy is a novel therapeutic approach for prostate cancer.3 Combination of radiotherapy with specific gene delivery, two different therapies with different toxicity profiles, has several potential benefits with the intention to achieve increased disease control. There are various potential benefits of combining radiation therapy with gene therapy. Gene therapy may cause radiosensitization or additive cell killing.4,5 Theoretical mechanisms of the enhanced antitumor effects from this combined approach may be that ionizing radiation improves transfection or transduction of therapeutic genes.6–8

Correspondence: Dr DJ Buchsbaum, Department of Radiation Oncology, University of Alabama at Birmingham, 674 Wallace Tumor Institute, 1824 6th Avenue South, Birmingham, AL 35294, USA Received 16 October 2003; accepted 9 August 2004; published online 23 December 2004

Angiogenesis is a complex process of new microvasculature formation necessary for a tumor to grow larger than 2–3 mm or approximately more than 1 million cells. To exceed this critical mass, defined as the angiogenic window, the tumor must establish new blood vessels.9 In prostate cancer, the degree of tumor vascularization correlates with the development of metastatic disease. Overexpression of vascular endothelial growth factor (VEGF) is produced by various types of tumors in contrast to low levels in normal tissues.10,11 VEGF has been implicated in the angiogenesis and growth of prostate carcinoma.12,13 VEGF binds to two distinct high-affinity tyrosine kinase receptors, Flt-1 and KDR/ Flk-1, which are both expressed primarily by endothelial cells.14–16 These receptors are phosphorylated in response to VEGF binding and initiate and modulate a cellular signaling cascade.17–19 VEGF receptor expression is not always restricted to microvascular cells and Flt-1 is expressed by a variety of tumor cells.20–22 Preclinical data demonstrate that antiangiogenic treatments may have activity against prostate carcinoma. One strategy to decrease angiogenesis is to target proangiogenic factors such as VEGF, by using specific antibody or soluble VEGF receptors. Tumor-bearing mice were treated with antiVEGF antibodies or soluble VEGF receptor proteins, which resulted in dramatic reductions in tumor size.23–27 Thus, targeted disruption or inhibition of VEGF and VEGF receptor gene expression, which results in impaired tumor blood vessel formation and tumor growth retardation, is an attractive approach for anticancer gene therapy. The purpose of this study was to determine the effects of specific sKDR gene transfer using a recombinant

sKDR inhibits human prostate cancer cell growth SA Kaliberov et al

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adenoviral vector under control of the human VEGF promoter element (AdVEGF-sKDR) on the growth of human prostate cancer cells. We evaluated the cytotoxic activity of AdVEGF-sKDR alone and in combination with ionizing radiation. This study demonstrates the feasibility of expressing an antiangiogenic protein such as sKDR through adenoviral gene transfer at levels sufficient to result in the selective death of prostate cancer cells in combination with radiation therapy.

Results For initial determination of transduction efficiency of adenoviral infection and levels of transgene expression in prostate cancer cells, the enhanced green fluorescent protein (EGFP) gene, placed under control of a cytomegalovirus (CMV) or VEGF promoter in a recombinant adenoviral vector (Ad) was used (Table 1). All cell lines were infected with the recombinant adenoviruses and EGFP expression was assayed using flow cytometry. In addition, comparisons with uninfected cells and control cells (derived from tissues other than the prostate) were made. Differing levels of EGFP expression were detected in the cell lines, the level of expression increasing in proportion to the multiplicity of infection (MOI), as expected (results not shown). BEAS-2B (negative control) normal human bronchial epithelial cells demonstrated elevated levels of EGFP expression following AdCMVEGFP infection in comparison with AdVEGF-EGFP. In contrast, prostate cancer cells demonstrated relatively high levels of EGFP expression after AdVEGF-EGFP infection. There was no evidence of significant cell death due to adenoviral transduction, even at the highest MOI of 200 by crystal violet staining assay (data not shown). These data correlated with VEGF protein secretion into conditional media of human prostate cancer cells and BEAS-2B. The expression of the VEGF protein was examined using an enzyme-linked immunosorbent assay

(ELISA) of conditioned medium of cells grown under normoxic conditions (Table 1). Prostate cancer cells demonstrated high levels of VEGF secretion in comparison to normal BEAS-2B cells. An adenovirus (AdVEGF-sKDR) expressing the entire extracellular domain of the human VEGF receptor 2/ KDR under control of the VEGF/eIF4g hybrid promoter was constructed. To determine the levels of sKDR protein expression in vitro, human vascular endothelial cells, human prostate cancer cells, and normal human bronchial epithelial cells were infected with 100 MOI of AdVEGF-sKDR or AdCMV-Luc (as control). The conditioned media and infected cells were collected after 48 h and subjected to Western blot analysis. The results showed that sKDR (identified using anti-KDR mouse monoclonal antibody) was present in AdVEGF-sKDRconditioned media (Figure 1a). sKDR expression was not detectable in AdCMV-Luc conditional media (negative control), or in cell lysates after AdVEGF-sKDR infection using immunoblotting technique (data not shown).

Table 1 EGFP expression after infection with AdCMV-EGFP and AdVEGF-EGFP and VEGF protein secretion into conditioned medium of human prostate cancer cells and normal human bronchial epithelial cells Cell line

DU 145 LNCaP PC-3 BEAS-2B a

Relative fluorescence intensityb

VEGF protein in conditioned media (pg/ml/106 cells)a

AdCMV-EGFP

AdVEGF-EGFP

26657326 1250798 604757 153735

35577296 39717359 23147147 26747273

22697175 16517144 10307101 52729

The level of expression of VEGF protein was examined using ELISA. VEGF concentrations in conditioned media of the indicated cell lines were normalized to number of viable cells. Presented are mean values7standard deviations of three separate experiments that were carried out in triplicate cultures. b EGFP expression in prostate cancer cell lines analyzed using FACS. At 48 h after AdCMV-EGFP or AdVEGF-EGFP (100 MOI) infection, cells were harvested. Samples (10 000 cells) were analyzed by FACScan. EGFP fluorescence was the mean fluorescence signal in EGFP-positive cells in relative units (ru) after subtraction of background fluorescence of uninfected control cells. Presented are mean values7standard deviations of four independent experiments, each performed in triplicate. Gene Therapy

Figure 1 Susceptibility to sKDR is variable in different prostate cancer cell lines. (a) The level of expression of secreted KDR protein in conditioned media of AdVEGF-sKDR-infected mouse and human vascular endothelial cells (positive control), prostate cancer cells, and normal bronchial epithelial cells (negative control). The expression of the KDR protein was examined using a Western blotting analysis. Loaded volumes of sample conditioned media of the indicated cell lines were normalized to number of viable cells and separated on SDS-PAGE followed by transfer to a PVDF membrane. One representative of three different experiments is shown. (b) Prostate cancer cells were infected with AdVEGF-sKDR or AdCMV-Luc (as viral control) at 100 MOI. Cell viability was determined at 72 h after infection by using the crystal violet staining assay. Presented are mean values after subtraction of viral control7standard deviations of three independent experiments, each performed with 10 replicates.


Western blot analysis showed that sKDR of Mr 110 kDa was secreted into the culture medium from AdVEGFsKDR-infected cells. We examined whether the sKDR could inhibit the proliferation of cancer cells. Prostate cancer cells were infected with different MOI of AdVEGF-sKDR or AdCMV-Luc (as viral control). There was a dosedependent correlation in cell killing, as expected (results not shown). As shown in Figure 1b, susceptibility to cytotoxic effects 100 MOI AdVEGF-sKDR infection was variable in different prostate cancer cell lines, and the relative sensitivity to sKDR expression was LNCaP4 DU1454PC-3. The functional efficacy of sKDR protein secretion was determined by transformed small vessel murine endothelial SVEC 4-10 and 1P-1B cell proliferation inhibition assay. The conditioned media obtained from DU145 prostate cancer cells infected with 100 MOI AdVEGFsKDR or AdCMV-Luc was applied to the endothelial cells. Conditioned media from AdVEGF-sKDR-infected cells inhibited SVEC 4-10 and 1P-1B cell proliferation by 60 and 67%, respectively, compared with conditioned media from AdCMV-Luc-infected cells (Figure 2a), and 88% compared with conditioned media from uninfected cells (data not shown). Thus, these data confirmed the adenovirus-mediated secretion of functionally active sKDR. To confirm that AdVEGF-sKDR infection can inhibit cell migration, 1P-1B endothelial cells and DU145 prostate cancer cells were infected with 100 MOI AdVEGF-sKDR or AdCMV-Luc (as viral control), then after overnight incubation added to the upper chambers of transwell inserts, and counted using a hemacytometer. As shown in Figure 2b, sKDR expression in infected cells inhibited 1P-1B and DU145 cell migration by 61 and 47%, respectively, compared with AdCMV-Luc-infected cells. A series of studies were carried out to clarify whether ionizing radiation can change VEGF promoter activity and modify AdVEGF-sKDR cytotoxicity in a panel of prostate cancer cells. For determination of response of the VEGF promoter to radiation treatment, prostate cancer cells were infected with AdVEGF-EGFP or AdCMV-EGFP (control) and cells were either mockirradiated or irradiated 24 h after infection at 3 Gy. At 48 h after radiation treatment, AdVEGF-EGFP- or AdCMV-EGFP-infected prostate cancer cells were harvested and EGFP expression was analyzed by flow cytometry. As shown in Figure 3a, after radiation treatment relative EGFP expression was increased in all prostate cancer lines. For the DU145 cell line, EGFP expression was significantly greater for cells infected with AdVEGF-EGFP after exposure to ionizing radiation versus those without radiation treatment (P-value o0.05), but there was no significant difference in PC-3, LNCaP, and BEAS-2B cells infected with AdVEGF-EGFP with or without radiation treatment (P-value 40.05). Also, increased levels of EGFP protein expression after radiation treatment correlated with increased VEGF protein secretion into conditioned media of human prostate cancer cells (Figure 3b). VEGF protein secretion was significantly greater for the DU145 and PC-3 cells after exposure to ionizing radiation versus those without radiation treatment (P-value o0.05), but there was no significant difference in LNCaP and BEAS-2B cell lines with or without radiation treatment (P-value 40.05).

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Figure 2 The functional efficacy of sKDR expression in vitro. (a) Secreted sKDR inhibits proliferation of endothelial cells. DU145 prostate cancer cells were transduced with 100 MOI AdVEGF-sKDR or AdCMV-Luc (as viral control). After 20 h, conditioned media from AdVEGF-sKDR- or AdCMV-Luc-transduced cells was collected and added to the murine endothelial SVEC 4-10 and 1P-1B cell cultures. After 72 h, the cells were trypsinized, and the number of viable cells was counted using trypan blue exclusion assay. Results represent mean values7 standard deviations of three independent experiments, each performed with 10 replicates. (b) AdVEGF-sKDR infection inhibits migration of endothelial cells and prostate cancer cells. DU145 and 1P-1B cells were infected with 100 MOI AdVEGF-sKDR or AdCMV-Luc (as viral control). After 24 h, cells were harvested, washed, and added to the upper chambers of tissue culture plate inserts. After incubation for 6 h at 371C, nonmigrated cells on the upper side of the tissue culture plate insert were removed with a cotton swab and the migrated cells attached to the lower side of the membrane were released using trypsin digest and counted using a hemacytometer. Each data point represents the mean cell number7standard deviation of six independent inserts from a three experiments. The number of migrating cells was normalized to the average number of AdCMV-Luc-infected control cells. The distribution of cells on the filter was homogeneous.

To test whether VEGF promoter regulated sKDR expression was able to modulate cell death following radiation treatment, DU145 prostate cancer cells at 3 h after AdVEGF-sKDR or AdCMV-Luc (viral control) infection were exposed to ionizing radiation and were subjected to clonogenic survival assay. Radiation alone caused a dose-dependent reduction in cell survival of uninfected cells (results not shown). AdCMV-LucGene Therapy


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Figure 3 Radiation treatment increased VEGF promoter activity. (a) Prostate cancer cells were infected with 100 MOI AdVEGF-EGFP or AdCMV-EGFP. At 24 h after infection, cells were either mock-irradiated or irradiated at 3 Gy. At 48 h after radiation treatment, cells were harvested and EGFP expression was analyzed by flow cytometry. Data are expressed as the mean EGFP expression after subtraction of uninfected control cells. Presented are mean values7standard deviations of three to five independent experiments, each performed in triplicate. *P-value o0.05 compared to untreated control. (b) The levels of VEGF protein secretion into conditioned media following radiation treatment. DU145, PC-3, and LNCaP prostate cancer cells and BEAS-2B normal epithelial cells were either mock-irradiated or irradiated at 3 Gy, 48 h later cell culture media were harvested, the levels of expression of VEGF protein were examined using ELISA. VEGF concentrations in conditioned media of the indicated cell lines were normalized to number of viable cells. Presented are mean values7standard deviations of three separate experiments that were carried out in triplicate cultures. *P-value o0.05 compared to untreated control.

infected DU145 cells demonstrated reduction in the number of cell colonies to 38% after 3 Gy, and 63% after 5 Gy in comparison with mock-irradiated control cells (Figure 4a). There was a significantly greater reduction in the number of AdVEGF-sKDR-infected DU145 cell colonies to 61% after 3 Gy, and 92% after 5 Gy in comparison with AdCMV-Luc-infected cells. Treatment with AdVEGF-sKDR enhanced the radiation induced cell killing, and the cytotoxic effect improved as the MOI of AdVEGF-sKDR was increased (data not shown). These data correlated with sKDR expression in Gene Therapy

Figure 4 Overexpression of sKDR increased ionizing radiation-induced prostate cancer cell death. (a) Clonogenic survival assay of DU145 prostate cancer cells. AdVEGF-sKDR- or AdCMV-Luc-infected cells (100 MOI) were exposed to ionizing radiation at 0, 3 and 5 Gy. Cells were fixed and colonies were counted at 15 days after treatment. Data are presented as percentage of colonies in comparison with mock-irradiated AdCMV-Luc control. Presented are mean values7standard deviations of three independent experiments, each performed in six replicates. (b) The expression of the sKDR protein in conditioned media following ionizing radiation treatment. DU145 prostate cancer cells were infected with 100 MOI AdVEGF-sKDR. At 24 h after infection, cells were either mockirradiated or irradiated at 4 Gy and 72 h later conditioned media was collected and number of viable cells was counted using trypan blue exclusion assay. The expression of the KDR proteins was examined using Western blot analysis. Loaded volumes of sample-conditioned media of the DU145 cell line were normalized to number of viable cells and separated on SDS-PAGE followed by transfer to a PVDF membrane. One representative of three different experiments is shown. (c) Migration assay was performed using tissue culture plate inserts. Cells were infected with 100 MOI AdVEGF-sKDR or AdCMV-Luc. After overnight incubation at 371C, cells were either mock-irradiated or irradiated at 3 Gy using a 60Co gamma irradiator and were then returned to the incubator and cultured for an additional 6 h. Cells were harvested, washed, and added to the upper chambers. After incubation for 6 h at 371C, nonmigrated cells on the upper side of the tissue culture plate insert were removed with a cotton swab and the migrated cells attached to the lower side of the membrane were released using trypsin digest and counted using a hemacytometer. Each data point represents the average cell number of six independent inserts for each condition from three experiments. The number of migrating cells was normalized to the average number of AdCMV-Luc-infected control cells.

AdVEGF-sKDR-transduced cells after exposure to ionizing radiation. There was an increased expression of the sKDR protein using Western blot analysis of conditioned medium of DU145 cells collected 48 h


following radiation treatment at 3 Gy (Figure 4b). AdVEGF-sKDR infection enhanced inhibition effects of radiation treatment against migration of endothelial cells. SVEC 4-10 and 1P-1B cells were infected with 100 MOI AdVEGF-sKDR or AdCMV-Luc (as viral control). After radiation, migration of AdVEGF-sKDRinfected SVEC 4-10 and 1P-1B cells, examined using tissue culture plate inserts, was inhibited by 59 and 76%, respectively, compared with AdCMV-Luc-infected cells (Figure 4c). VEGF and its cognate receptors Flt-1 and KDR are involved in the regulation of angiogenesis and tumor growth, invasion, and metastasis. The effect of combining ionizing radiation and sKDR expression by AdVEGFsKDR transduction of cells on the action of VEGF was examined using in vitro cultures of HUVEC. There was a dose-dependent proliferative response of endothelial cells to human recombinant VEGF (hrVEGF) protein (data not shown). Incubation of AdCMV-Luc-transduced HUVEC in culture medium with 50 ng/ml hrVEGF produced an increase in cell number of 36% in comparison with AdCMV-Luc-infected cells without hrVEGF (Figure 5a). After radiation treatment, hrVEGF produced an increase in cell number of 38% in comparison with AdCMV-Luc-infected HUVEC without hrVEGF (Figure 5a). AdVEGF-sKDR significantly decreased the number of viable cells in comparison with AdCMV-Luc-infected HUVEC cells alone and in combination with ionizing radiation (Po0.05). To test whether VEGF overexpression is able to modulate prostate cancer cell response to radiation treatment, PC-3 and DU145 cells were investigated because of their different sensitivity to AdVEGF-sKDR transduction. After AdVEGF-sKDR or AdCMV-Luc (viral control) transduction, these prostate cancer cells were exposed to ionizing radiation and incubated with 100 ng/ml hrVEGF. As shown in Figure 5b, AdVEGFsKDR significantly reduced the number of viable cells in comparison with AdCMV-Luc-transduced PC-3 and DU145 cells (Po0.05). hrVEGF increased the number of viable cells in both AdCMV-Luc- and AdVEGF-sKDRtransduced unirradiated and irradiated prostate cancer cells. However, the greatest cytotoxicity occurred in irradiated AdVEGF-sKDR-infected cells. These data demonstrate that specific sKDR gene delivery in combination with radiotherapy increased prostate cancer cell cytotoxicity. The relative sensitivity of prostate cancer cells to AdVEGF-sKDR infection was correlated with VEGF receptor expression. DU145 prostate cancer cells demonstrated detectable levels of KDR and Flt-1 mRNA expression in comparison with BEAS-2B control cells as measured by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, whereas PC-3 cells showed low level Flt-1 mRNA expression (Figure 5c). To investigate the effect of the combination of AdVEGF-sKDR infection and radiation treatment on tumor growth in vivo, DU145 cells were subcutaneously injected into the flank of athymic nude mice. Before treatment, the mean tumor sizes at baseline were not significantly different between treatment groups (P40.05), and the within treatment variances were not significantly different (P40.05). The baseline mean and standard deviation for tumor sizes was 121777 mm2. In vivo tumor therapy was initiated on

day 0, which corresponded to 15 days post tumor cell injection. Animals were injected intratumorally with AdVEGF-sKDR or AdCMV-Luc recombinant adenoviruses on days 0, 3, 7, 10, 14, 17, and 21. Two groups of mice received radiation treatment at 2 Gy on days 1, 4, 8, 11, 15, 18, and 22. There were no significant differences in tumor sizes between groups that received radiation treatment alone or AdCMV-Luc alone versus AdCMV-Luc in combination with ionizing radiation (P40.05). An inhibition of tumor growth was noted in groups of mice treated with AdVEGF-sKDR alone or AdVEGF-sKDR and ionizing radiation (Figure 6). Comparisons of mean tumor volumes of the AdVEGF-sKDR treatment group alone, and AdVEGF-sKDR in combination with radiation treatment versus the control (AdCMVLuc) group showed significant differences between the groups (Po0.05). Comparisons of tumor volumes in mice that received AdVEGF-sKDR alone versus the group treated with AdVEGF-sKDR in combination with radiation demonstrated a significant difference (Po0.05) in tumor growth. This difference in tumor size was evident at day 56 and persisted for the duration of the experiment. On day 84, the average tumor size was 105757 mm3 in mice treated with AdVEGF-sKDR in combination with ionizing radiation, 2217129 mm3 in mice injected with AdVEGF-sKDR alone, versus 4187181 mm3 in AdCMV-Luc control mice (Po0.05). The mean time to tumor doubling for AdCMV-Luc alone, radiation treatment alone, and AdCMV-Luc plus radiation treatment groups were 27, 50, and 56 days, respectively; for animals that received AdVEGF-sKDR alone and in combination with ionizing radiation, tumors had not doubled by day 84, the last day of the study. These data confirmed that tumor gene therapy using AdVEGF-sKDR in combination with radiation treatment resulted in increased tumor growth inhibition in comparison with AdCMV-Luc alone, radiation alone, or AdCMV-Luc in combination with radiation. Moreover, in the group treated with AdVEGF-sKDR alone (total 19 mice), three of the tumors underwent complete regression. In contrast, the group treated with AdVEGF-sKDR in combination with ionizing radiation showed 5/17 regressions in contrast to no regressions in AdCMV-Luc alone, radiation alone, and AdCMV-Luc plus radiation treated groups. Taken together, these data demonstrate that sKDR gene delivery can improve prostate cancer therapy. These data indicate that the combination of radiation treatment with specific overexpression of sKDR using AdVEGF-sKDR provided additive effects in prostate cancer cells both in vitro and in vivo.

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Discussion In an attempt to improve the outcome for patients with prostate cancer, combined modality approaches are frequently considered. Combined modality therapy, most frequently radiosensitizing chemotherapy and radiation, have been shown to be particularly efficient and have become standard therapy for many tumor types.28 One of the most promising therapies that can be combined with ionizing radiation in the treatment of prostate cancer is gene therapy. Several biological Gene Therapy


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Figure 5 sKDR diminished radioprotective effect of VEGF protein expression in prostate cancer cells and endothelial cells. (a) HUVECs were infected with 100 MOI AdVEGF-sKDR or AdCMV-Luc and after 24 h cells were irradiated at 3 Gy and immediately, 50 ng/ml of hrVEGF was added to the culture media. After 72 h, cells were collected and number of viable cells was determined using trypan blue exclusion assay. Data points are mean values7standard deviations of three experiments, each performed in six replicates. (b) DU145 and PC-3 prostate cancer cells were infected with 100 MOI AdVEGF-sKDR or AdCMV-Luc recombinant adenoviruses. After 20 h, DU145 and PC-3 cells were exposed to ionizing radiation at 2 and 4 Gy, respectively. Immediately after radiation treatment, 100 ng/ml hrVEGF was added to the cell culture media. After 72 h incubation, cells were collected and number of viable cells was determined using the crystal violet staining assay. Presented are mean values after subtraction of viral control7standard deviations of three independent experiments, each performed with 10 replicates. (c) The levels of KDR and Flt-1 mRNA in prostate cancer cells. DU145 and PC-3 prostate cancer cells, HUVEC (as positive control), and BEAS-2B (as negative control) were collected and total RNA was extracted. The levels of KDR, Flt-1, and GAPDH (as loading control) expression were determined using RT-PCR.

features of the prostate make it an attractive target for the development of gene therapy strategies in combination with radiotherapy for treatment of localized prostate cancer.29 The accessibility of the prostate allows highly localized administration of a desirable gene therapy vector. It is important that the prostate is an accessory organ and as such, cytoreductive approaches that Gene Therapy

eliminate the prostate cells are reasonable. This circumstance provides a degree of greater latitude in the gene therapy specificity that may not be appropriate for other tumor types.28 The accumulation of cancerous cells within a growing prostate tumor can deprive them of adequate vascular support and stimulates new tumor blood vessel growth.


Figure 6 Growth of DU145 prostate cancer xenografts treated with AdVEGF-sKDR in combination with ionizing radiation. Treatment was started at the time of established tumor growth (day 0 equal to 15 days after tumor cell injection). Animals were infected intratumorally with 1 109 TCID50 AdVEGF-sKDR or AdCMV-Luc on days 0, 3, 7, 10, 14, 17, and 21, and then irradiated with 2 Gy on days 1, 4, 8, 11, 15, 18, and 22. Data points represent the mean change in tumor volumes relative to day 0 for each group of animals of two independent experiments. *P-value o0.05 compared to AdCMV-Luc control and #P-value o0.05 compared to AdVEGF-sKDR alone.

Increased angiogenesis activity has been linked to a more aggressive phenotype in studies of human prostate cancer.29–33 Tumor angiogenesis is a complex of coordinated interactions between numerous proteins, involved in different signaling pathways. Each step provides an opportunity for therapeutic intervention. Angiogenesis mediated by VEGF constitutes a new target for anticancer therapy, which has been explored through different forms of intervention aimed at blocking of the tumor neovascularization. Strategies to inhibit tumor angiogenesis include inhibition of angiogenic factor production and their receptors, inhibition of the VEGF signaling pathway, by inhibition of the binding between VEGF and its receptors, and through the inhibition of intracellular transduction of the VEGF signal.34 The importance of signal transduction through the VEGF receptor is illustrated by use of sKDR. This protein binds to VEGF and sequesters this ligand before binding to its cellular receptors.35–37 sKDR also functions as dominant negative by forming inactive heterodimers with membrane-spanning VEGF receptors. This specific inhibitor of VEGF prevents the mitogenic and migratory response of endothelial cells to VEGF.38,39 Radiation therapy is widely used as primary treatment for localized prostate cancer. Exposure of prostate tumor to ionizing radiation increased the secretion of VEGF by cancer cells that may enhance the angiogenic response of tumor tissue and play a role in the protection against

radiation damage.40 Radiation has been shown to increase the activity of the VEGF promoter and VEGF protein expression through multiple mitogen-activated protein (MAP) kinase-dependent pathways.41 Recently, several groups have shown that combined antiangiogenesis agent treatment with ionizing radiation improves the antitumor effect of radiation.42 However, the mechanisms by which combination of radiation treatment with antiangiogenesis therapy increases cell death are not clear. Recent experiments demonstrated that the tumor response to ionizing radiation is determined not only by tumor cell phenotype but also by tumor microvascular endothelium radiosensitivity.43 To determine whether specific sKDR overexpression alters the radiation response in tumor endothelial cells and prostate cancer cells, the AdVEGF-sKDR adenoviral vector encoding the sKDR gene under control of the human VEGF promoter element was used. High levels of VEGF promoter activity and VEGF expression were demonstrated in prostate cancer and endothelial cells. Our studies demonstrated that exogenous VEGF protein increased resistance of prostate cancer cells as well as tumor endothelial cells to ionizing radiation in vitro. This was associated with expression of VEGF receptor (Flt-1 and KDR) mRNA in prostate cancer cells and HUVEC. These data are in agreement with observations that several tumor types, including prostate, express functionally active VEGF receptors and VEGF autocrine growth factor activity.44,45 In addition, cancer cells induce transcriptional reprogramming of endothelial cells that result in the establishment of autocrine loops.46 Our results demonstrate that expression of sKDR protein can induce cancer cell cytotoxicity and sensitize tumor cells to ionizing radiation treatment in vitro and in vivo. Moreover, sKDR can diminish growth stimulation and radioprotective effects of cancer cells by VEGF overexpression. Thus, these results demonstrated that employing a radiation inducible promoter can increase the efficacy of sKDR gene expression controlled by the VEGF promoter element in combination with ionizing radiation treatment. Importantly, cytotoxicity as well as migration inhibition activity of sKDR was demonstrated against both tumor endothelial cells and Flt-1- and Flk-positive prostate cancer cells in vitro. The AdVEGF-sKDR-mediated cell killing varied for different cell lines and was modulated by both VEGF promoter activity and through intracellular network of signaling cascades. In our experiments, LNCaP p53wt androgen-dependent prostate cells demonstrated increased cytotoxicity after AdVEGF-sKDR infection, but at the same time showed lower levels of VEGF promoter activity in comparison with DU145 p53mut androgenrefractory prostate carcinoma. The possible explanation for these findings may be related to levels of p53 gene expression in these prostate cancer cells. The p53 tumor suppressor protein exerts its growth inhibitory activity by interacting with diverse signaling pathways. The activation of p53 leads to many outcomes in cells, including cell cycle arrest and apoptosis. For instance, the p53 protein can functionally interact with the MAP kinase pathways, including the stress-activated SAPK/ c-Jun NH2-terminal protein kinase (JNK), and the extracellular signal related kinase (ERK). Previously it has been shown that VEGF binding to the VEGF receptors leads to the activation of ERK and JNK

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in human dermal microvascular endothelial cells,47 as well as MAP kinases in fibroblasts48 and rat cardiac myocytes.49 Recent results obtained by Mahasreshti et al suggest that intravenous delivery of the antiangiogenic sFLT-1 gene via a replication-deficient, infectivity-enhanced recombinant adenoviral vector under control of the CMV promoter resulted in overexpression of sFLT-1 in the liver leading to unacceptable hepatotoxicity. The authors concluded that tumor-specific targeting of the vectors and tumor-specific expression strategies should be used to ensure a clinically useful antiangiogenesis gene therapy in order for a therapeutic effect to be realized.50 Thus, the specificity of gene delivery must be considered in the design of antiangiogenesis gene therapy vectors. Specific gene expression under control of the VEGF promoter is a promising approach for gene therapy of a variety of cancers. Although the use of the VEGF promoter has permitted selective expression of sKDR in Flt-1/Flk-positive human prostate cancer cells and tumor endothelial cells, nontargeted adenoviral transport to liver might occur in vivo. Several reports have shown that levels of VEGF expression in normal liver are significantly less as compared with acute and chronic liver pathology.51–53 However, additional studies are required to investigate hepatotoxicity with AdVEGFsKDR following systemic administration. Taken together, these results suggest that combined ionizing radiation and gene therapy approaches have promise for the control of local prostate tumor progression. These data indicate that the combination of radiation treatment with specific overexpression of antiangiogenic sKDR using AdVEGF-sKDR provided increased cytotoxic effects against human prostate cancer cells in vitro and in vivo.

pShuttleVEGF-Bax plasmid by restriction digestion, blunted and cloned into a pBLAST45-hFLK1 plasmid (InvivoGen, San Diego, CA, USA), which was digested and hEF1 promoter was removed prior to cloning in the VEGF promoter element. The fragment including VEGF/ eIF4 g hybrid promoter and sKDR cDNA with SV40 late polyadenylation signal was removed from pVEGF/ eIF4g-sKDR plasmid, blunted and cloned into pShuttle (Quantum Biotechnologies, Montreal, Canada). The insert sequence was confirmed by restriction enzyme mapping and partial sequencing analysis. The resultant plasmid was linearized and cotransfected with pAdEasy1 plasmid (Quantum Biotechnologies) into Escherichia coli BJ5183 bacteria. Recombinant clones were confirmed by PCR analysis, linearized and transected into 293 cells using the Effectene lipid-based transfection method (QIAGEN, Chatsworth, CA, USA) to generate AdVEGF-sKDR recombinant adenovirus, which was isolated from a single positive plaque and passed through three rounds of plaque purification and subsequently confirmed by PCR. AdVEGF-EGFP (encoding enhanced green fluorescent protein (EGFP) under control of the VEGF promoter element) was generated as described previously.54 AdCMV-EGFP (encoding EGFP under control of the human CMV promoter element) was kindly provided by DL Della Manna (University of Alabama at Birmingham). AdCMV-Luc (encoding firefly luciferase gene under control of the human CMV promoter element) was kindly provided by Dr PN Reynolds (University of Adelaide, Adelaide, Australia). All viruses were propagated in 293 cells, purified by ultracentrifugation in a cesium chloride gradient, and subjected to dialysis. Viral titer was measured by a standard 50% tissue culture infectious dose (TCID50) assay using 293 cells and by absorbance of the dissociated virus at A260 nM. MOI for subsequent experiments was expressed as TCID50 per cell.

Materials and methods Cell culture All cell lines, except human umbilical vein endothelial HUVEC cells, were originally obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The HUVEC cells were obtained from Clonetics (San Diego, CA, USA) and were grown in EGM-2 growth media (Clonetics). The normal human bronchial epithelial adenovirus 12-SV40 virus hybrid-transformed BEAS2B cells were grown in BEGM growth media (Clonetics). Human prostate carcinoma LNCaP cells were maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS). The human prostate adenocarcinoma PC-3, and DU145 cells, transformed small vessel murine endothelial SVEC4-10 and 1P-1B, and human embryonic kidney (HEK) 293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) medium with 10% FBS. All cells were maintained at 371C in a humidified atmosphere with 5% CO2. Adenoviral vectors A replication-deficient recombinant adenoviral vector encoding soluble KDR gene under control of the human VEGF promoter element (AdVEGF-sKDR) was constructed using a method reported previously.54 Briefly, to generate pVEGF/eIF4g-sKDR plasmid, the fragment containing VEGF promoter element was removed from Gene Therapy

EGFP expression assays Cellular EGFP expression was quantitatively determined by fluorescence-activated cell sorter (FACS) analysis and visualized using fluorescent microscopy. After AdVEGFEGFP or AdCMV-EGFP infection, cells were collected at different time points and approximately 10 000 cells were illuminated at 488 nm and fluorescence was detected in the FITC (525/20 nm) channel. Nonspecific fluorescence was detected using a 575/30 nm emission filter in the PI channel. EGFP fluorescence is the mean fluorescence signal in EGFP-positive cells in relative units (ru) after subtraction of background fluorescence. An inverted microscope (Olympus IX70, Olympus America, Melville, NY, USA) was used for screening of EGFP expression in cell cultures. Measurement of VEGF protein in cell culture supernatant For generation of conditioned medium, cells were incubated for 48 h at 371C. Cell culture supernatants were collected, centrifuged to remove floating cells, and stored at 201C. VEGF concentrations were assessed using a quantitative ELISA (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions. The raw VEGF data was normalized according to the number of viable cells in each sample, determined by


trypan blue (Life Technologies) staining. Cells were counted using a hemacytometer.

Western blot The level of sKDR protein expression was examined by immunoblotting technique. Briefly, cells were collected, washed in TBS (135 mM NaCl, 2.5 mM KCl, 25 mM TrisHCl, pH 7.5), and homogenized in ice-cold lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 1% IGEPAL CA-630 (Sigma, St Louis, MO, USA). The homogenate was centrifuged at 14 000 g for 5 min. The protein concentration in the supernatant was determined by the Biuret method using the BCA Protein Assay Kit (Pierce). The conditioned media was collected and centrifuged at 14 000 g for 5 min. Each sample was denatured for 5 min at 1001C in loading buffer. Equal amounts of protein were loaded for each sample in all lanes and electrophoretically separated on SDS-PAGE followed by transfer to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% nonfat milk (Bio-Rad) in TBS. sKDR was detected using specific anti-KDR mouse monoclonal antibody (Sigma), and goat anti-mouse Ig(H+L)-HRP secondary antibody (Southern Biotechnology Associates, Birmingham, AL, USA). RNA preparation and RT-PCR The levels of VEGF receptors KDR and Flt-1 messenger RNA were determined by RT-PCR. Total RNA was extracted from 107 cells using RNeasy Mini Kit (Qiagen, Valencia, CA, USA), following standard protocol, and quantified spectrophotometrically using an MBA 2000 spectrophotometer (Perkin Elmer, Wellesley, MA, USA). cDNA was synthesized using random hexamer primers and an Omniscript RT kit (Qiagen). The first-strand cDNA was used as the template for PCR. For amplification of cDNA encoding KDR and Flt-1, the following primers were used 50 -TATAGATGGTGTAACCCGGA-30 (1182-KDR); 50 -TTTGTCACTGAGACAGCTTGG-30 (KDR-1737) and 50 -GAGGATTCTGACGGTTTC-30 (3199-FLT); 50 -CCTGTCAGTATGGCATTG-30 (FLT-3782). The human glyceraldehyde 3-phosphate dehydrogenese (GAPDH) cDNA was used as an internal standard for template loading of PCR by using primers 50 -TCCCAT CACCATCTTCCA-30 (GAPDH forward) and 50 -CAT CACGCCACAGTTTCC-30 (GAPDH reverse). PCR was performed under the following conditions: 25 cycles (951C for 45 s, 551C for 45 s, and 721C for 1 min). PCR products were analyzed by 1% agarose electrophoresis with ethidium bromide staining. Cell proliferation assay To determinate cell growth after recombinant adenovirus infection, human prostate cancer cells were seeded in 96well plates at 5000 cells per well, incubated for 24 h, with or without human recombinant VEGF (BD Pharmingen, San Diego, CA, USA) and infected at different MOI with AdVEGF-sKDR or AdCMV-Luc. After 72 h, cell culture medium was removed and surviving cells were then fixed and stained with 2% (w/v) crystal violet (SigmaAldridge) in 70% ethanol for 3 h at room temperature. The plates were washed extensively, air-dried and optical density was measured at 570 nm using a V Max plate reader (Molecular Devices Corporation, Sunnyvale CA, USA).

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Clonogenic survival assay At 3 h after infection with 100 MOI AdVEGF-sKDR or AdCMV-Luc, cells were trypsinized and diluted to the appropriate cell density into six-well culture plates and after 24 h incubation at 371C, cells were either mockirradiated or irradiated at 102 cGy/min to the desired dose using a 60Co gamma irradiator and were then returned to the incubator and cultured additionally for 15 days. Cells were then fixed with 70% ethanol and stained with 2% (w/v) crystal violet (Sigma-Aldridge). Colonies comprising at least 50 cells were counted. The plating efficiencies were calculated as the number of colonies divided by the number of test cells plated for each data point. Plating efficiencies were referenced back to the mock-irradiated control plating efficiency to determine the surviving fraction for each dose. Endothelial cell proliferation inhibition assay To evaluate that the sKDR in the conditioned media would suppress proliferation of 1P-1B and SVEC 4-10 murine endothelial cells, conditioned media was obtained from DU145 cells infected with AdVEGF-sKDR or AdCMV-Luc (control). DU145 cells were infected with 100 MOI AdVEGF-sKDR or AdCMV-Luc and 3 h later cell monolayers were washed with PBS, complete media was added, and cells were incubated 48 h at 371C. Endothelial cells were washed with PBS, and conditioned media was added. After 72 h, the cells were trypsinized, and the number of viable cells were counted using trypan blue dye exclusion assay. Cell migration assay Migration assay was performed using 12 mm tissue culture plate inserts with 12.0 mm. tissue culture-treated Millcell polycarbonate membranes (Millipore). Cells were infected with 100 MOI AdVEGF-sKDR or AdCMV-Luc. After overnight incubation at 371C, cells were either mock-irradiated or irradiated at 3 Gy using a 60 Co gamma irradiator and were then returned to the incubator and cultured for an additional 6 h. Cells were harvested, washed, and added to the upper chambers at concentrations of 5 104 cells/well. After incubation for 6 h at 371C, nonmigrated cells on the upper side of the tissue culture plate insert were removed with a cotton swab and the migrated cells attached to the lower side of the membrane were released using trypsin digest and counted using a hemacytometer. In vivo tumor therapy studies For in vivo experiments, 8–10-week-old nude mice were subcutaneously inoculated with 5 106 DU145 tumor cells resuspended in 0.1 ml Matrigel (Becton Dickinson, San Jose, CA, USA). Treatment was started 15 days later at the time of established tumor growth (100–150 mm3). Mice were randomly divided (5–8 mice/group) into five groups receiving different treatments: (1) AdCMV-Luc (as viral control); (2) AdVEGF-sKDR; (3) 60Co radiation treatment at 2 Gy; (4) AdCMV-Luc and 2 Gy radiation treatment; (5) AdVEGF-sKDR and 2 Gy radiation treatment. Animals were injected intratumorally with 1 109 TCID50 recombinant adenoviruses or received PBS. Radiation treatment was administered 24 h after adenoviral injection. Tumor volume (0.5 length width2 in mm3) was measured twice a week using calipers. The Gene Therapy


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time to death from tumor or treatment, to tumor regression, and to tumor recurrence were recorded.

Statistical analysis All error terms are expressed as the standard deviation of the mean. Significance levels for comparison of differences between groups in the in vitro experiments were analyzed by the Student’s t-test. The differences were considered significant when P-value was o0.05. All reported P-values are two-sided. In the animal model tumor therapy studies, the treatment groups were compared with respect to tumor size and percent of original tumor size over time. To test for significant differences in tumor size between treatment groups, oneway analysis of variance (ANOVA) test was conducted. When the ANOVA indicated that a significant difference existed (P-value o0.05), multiple comparison procedures were used to determine where the differences lay.

Acknowledgements We thank Sally B Lagan and Soneshia LM McMillan for assistance in preparing the manuscript.

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