Gene Therapy (2014) 21, 723–731 © 2014 Macmillan Publishers Limited All rights reserved 0969-7128/14 www.nature.com/gt
ORIGINAL ARTICLE
Antitumor effects of oncolytic adenovirus armed with PSA-IZ-CD40L fusion gene against prostate cancer Y-F Yang1,4, S-Y Xue1,4, Z-Z Lu2, F-J Xiao1, Y Yin3, Q-W Zhang1, C-T Wu1, H Wang1 and L-S Wang1 Advanced prostate cancer (PC) still remains incurable. Novel immunogene therapy shows promise as treatment strategy that can target both localized and metastasized PC. In this study, we have developed a PC-specific oncolytic adenovirus (Ad-PL-PPT-E1A) armed with fusion gene of prostate-specific antigen and CD40 ligand, and aimed to evaluate its therapeutic effect in vitro and in vivo. After they were rescued in human embryonic kidney 293 cells, we confirmed that Ad-PL-PPT-E1A could mediate the expression of E1A efficiently and produce abundant progeny viruses in PC cells in vitro. Our data showed that Ad-PL-PPT-E1A induced apoptosis and resulted in specific oncolytic toxicity in PC cells, which was detected by Annexin-V staining and crystal violet, respectively. After stimulation with lysates, immune phenotypes and cytokines expression of human dendritic cells was detected by flow cytometry and real-time polymerase chain reaction, respectively. And, the results showed that the lysate of Ad-PL-PPT-E1Ainfected LNCaP cells upregulated the expression of CD80, CD83, CD86 and mRNA level of interleukin-6 (IL-6), IL-12, IL-23 and tumor necrosis factor-α significantly. In established PC3M cell-xenografted mouse models, Ad-PL-PPT-E1A treatment improved the survival and suppressed the tumor growth obviously. In conclusion, Ad-PL-PPT-E1A exhibited enhanced antitumor activity is a promising approach for gene therapy of advanced PC. Gene Therapy (2014) 21, 723–731; doi:10.1038/gt.2014.46; published online 22 May 2014
INTRODUCTION Prostate cancer (PC) remains the second most common cancer in men worldwide.1 For patients suffering from prostate cancer at early stage, the therapeutic options are curative and include surgery, radiation and, in some cases, active surveillance only. A majority of the patients progress to metastatic diseases and eventually develop castration-resistant disease during the advanced stages. Conventional treatments, such as docetaxel chemotherapy, provide only modest benefits.2,3 Novel strategies for metastatic, castration-resistant prostate cancer are needed urgently. In the past decade, immunotherapy has emerged as a promising approach for advanced prostate cancer. Dendritic cells (DCs) are the most potent antigen-presenting cells for priming and activating naive T cells and having important roles in antitumor immune responses.4,5 The interaction between surface receptor CD40 and its ligand (CD40L) promotes cytokine production, costimulatory molecule expression of DCs and eventually induces specific immune responses.6,7 Several encouraging trials of DC-based vaccines activated by CD40L have been carried out to induce tumor antigen-specific immune responses in cancer patients.8,9 It has been also demonstrated that CD40L-based gene therapy could induce both humoral and cellular immunity against many types of cancer.10–12 Moreover, tumor-associated antigen/ CD40L fusion protein could induce antitumor immune response through activation of DCs.13 Oncolytic adenoviruses, which selectively replicate in tumor cells, are optimal vectors for cancer gene therapy. The safety and efficacy of oncolytic adenoviruses have been validated in several
clinical studies.14 To combine the antitumor effect of oncolytic adenoviruses and CD40L-based immunotherapy, we successfully developed a prostate cancer-specific oncolytic adenovirus armed with fusion protein gene of prostate-specific antigen (PSA) and CD40L (PSA-IZ-CD40L, PL), and evaluated its therapeutic effect in vitro and in vivo. RESULTS Construction and identification of prostae cancer-specific oncolytic adenovirus Ad-PL-PPT-E1A Prostate cancer-specific oncolytic adenovirus armed with fusion protein gene PL (Ad-PL-PPT-E1A) and control adenoviruses (Ad-PL, Ad-PPT-E1A and Ad-Null) were successfully constructed as described in Materials and Methods (Figure 1a). The PL and E1A genes in adenoviral genomic DNA were confirmed by using polymerase chain reaction (PCR) (Figure 1b). The infectious titers of the purified adenoviruses were 2.0 × 1010 infection units per milliliter (IU ml − 1) (Ad-PL-PPT-E1A), 6.0 × 1010 IU ml − 1 (Ad-PL), 2.0 × 1011 IU ml − 1 (Ad-PPT-E1A) and 2.0 × 1011 IU ml − 1 (Ad-Null), respectively. And, the ratio between IU and viral physical particles were higher than 1:30. The expression of PL and E1A were detected by using real-time PCR and western blotting. The results showed that PSA protein expression could be detected in LNCaP cells, while Ad-PL-PPT-E1A and Ad-PL could mediate the expression of PL both on mRNA (data was not shown) and protein levels. And, E1A expression could be detected only in Ad-PL-PPT-E1A- and Ad-PPT-E1Ainfected cells (Figures 1c and d).
1 Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing, People's Republic of China; 2Chinese Center for Disease Control and Prevention, Institute for Viral Disease Control and Prevention, Beijing, People's Republic of China and 3Department of Hematology, Peking University First Hospital, Beijing, People's Republic of China. Correspondence: Dr H Wang or Professor L-S Wang, Department of Experimental Hematology, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850, People's Republic of China. E-mail: [email protected] or [email protected] 4 These authors contributed equally to this work. Received 16 July 2013; revised 25 March 2014; accepted 9 April 2014; published online 22 May 2014
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Figure 1. Construction and identification of oncolytic adenovirus Ad-PL-PPT-E1A and control adenoviruses. The schemes of Ad-PL-PPT-E1A, Ad-PL, Ad-PPT-E1A and Ad-Null were shown in (a). PPTp represents the prostate cancer-specific chimeric promoter, which consists of androgen response element enhancer core (AREc) of PSAe, prostate-specific membrane antigen enhancer (PSMAe) and promoter of T-cell receptor γ-chain alternate reading frame protein (TARPp). And, PL is the fusion protein gene of PSA and CD40L, which is linked by isoleucine zipper. After adenoviruses were constructed and rescued in human embryonic kidney 293 (HEK293) cells, the genomic DNA was obtained by Hirt extraction, and the PL and E1A genes were confirmed by using PCR (b). To detect protein expression, LNCaP cells were infected by Ad-PL-PPT-E1A, Ad-PL, Ad-PPT-E1A or Ad-Null at 50 MOI. Forty-eight hours later, protein was extracted and the expression of E1A (c) and PL (d) were assayed by western blotting.
Oncolytic effect of Ad-PL-PPT-E1A Prostate cancer or non-prostate cancer cells were infected by AdPL-PPT-E1A, Ad-PL or Ad-PPT-E1A at serial multiplicity of infection (MOI) for detecting their prostate cancer-specific oncolytic effects. Ad-Null and Ad-CD80-TPE-GM were used as the negative and positive control, respectively. The oncolytic ability was determined by crystal violet staining 7 days after infection. The results showed that Ad-PL-PPT-E1A and Ad-PPT-E1A had no obvious oncolytic effect on non-prostate cancer cells, including L02, EC304, A549 and 7721. However, Ad-PL-PPT-E1A and Ad-PPT-E1A could kill prostate cancer cells effectively in an MOI dose-dependent manner, which was consistent with that of Ad-CD80-TPE-GM (Figure 2a). As shown in Figure 2a, almost all LNCaP cells could be killed at 0.1 MOI, and nearly 80% of the DU145 and PC3M cells could be killed at 10 MOI. Replicationdeficient adenovirus Ad-PL had little effect on viability of all tested cell lines. Apoptosis of prostate cancer cells infected by oncolytic adenovirus was detected by FACS using Annexin-V-FITC Detection Kit (KeyGEN, Nanjing, China). Our data showed that Ad-PL-PPT-E1A or Ad-PPT-E1A could induce apoptosis of prostate cancer cells, whereas no obvious changes could be detected in Ad-PL-infected cells. Furthermore, Ad-PL-PPT-E1A- and Ad-PPT-E1A-induced Gene Therapy (2014) 723 – 731
apoptosis took place in a time- and dose-dependent manner. As shown in Figures 2b and c, the apoptosis rate of PC3M cells infected by 50 MOI Ad-PL-PPT-E1A rose from 10.36 ± 0.14 to 42.75 ± 2.47%, with the time increased from 24 to 72 h after infection, whereas it elevated from 30.63 ± 0.49 to 69.4 ± 3.96%, with the dose increased from 50 to 200 MOI at 48 h after infection. Prostate cancer-specific replication of Ad-PL-PPT-E1A Cytopathic effects were detected in Ad-PL-PPT-E1A-infected prostate cancer cells, including LNCaP, PC3M and DU145, by light microscopy (data were not shown). We also investigated the specific replication of Ad-PL-PPT-E1A by several methods, including E1A expression, production of viral particles and infectious titer. First, the replication of viral genomic DNA was determined by real-time PCR and normalized by that in Ad-PL-infected cells. Our results showed that viral genomic DNA could be generated in prostate cancer cells efficiently. The replication resulted in a timedependent increase of viral genomic DNA in DU145 cells, while it reached the peak at 48 h after infection in LNCaP and PC3M cells (Figure 3a). Second, we tested the progeny adenovirus production by infected cells. Ad-CD80-TPE-GM was used as a positive control for cancer cells. Our data showed that Ad-CD80-TPE-GM could © 2014 Macmillan Publishers Limited
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Figure 2. Oncolytic effect of Ad-PL-PPT-E1A on human prostate cancer cells. Prostate cancer cells PC3M, LNCaP, DU145, PC3 and non-PC cells EC304, L02, A549 and SMMC-7721 were seeded into 96-well plates at a density of 2 × 104 cells per well. Twenty-four hours later, cells were infected by Ad-PL-PPT-E1A or control adenoviruses (Ad-PL, Ad-PPT-E1A, Ad-Null or Ad-GFP-TPE-GM) at serial MOI, and cell survival rate was determined with crystal violet staining at day 7 after infection (a). Cell apoptosis was also detected in prostate cancer cells infected with 50, 100, 150 or 200 MOI Ad-PL, Ad-PL-PPT-E1A, Ad-PL or Ad-PPT-E1A, by using Annexin V-FITC Apoptosis Detection Kit. Ad-PL-PPT-E1A and Ad-PPT-E1A could induce cell death obviously. The percentage of death cells (50 MOI) was shown as the mean ± s.d. and increased with the time extended (b). *P o 0.05, **P o 0.01 vs Ad-PL group at the same time point; ## P o 0.01 vs 24 h group infected with the same adenovirus; $$P o 0.01 vs 48 h group infected with the same adenovirus. Ad-PL-PPT-E1A and Ad-PPT-E1A also induced cell apoptosis in a dose-dependent manner (48 h after infection) (c). **P o 0.01 vs Ad-PL group at the same MOI; #P o 0.05, ##P o 0.01 vs 50 MOI group infected with the same adenovirus; $P o 0.05 vs 100 MOI group infected with the same adenovirus.
produce progeny adenoviruses in all cancer cells. However, for Ad-PL-PPT-E1A, the viral production could be detected only in prostate cancer cells. The infectious titer of progeny adenovirus from Ad-PL-PPT-E1A-infected cells reached nearly 100 IU per cell at 72 h after infection (Figure 3b). Third, the E1A expression, which was essential for replication and controlled by PPT promoter in our design, was detected by western blotting or real-time PCR in both non-prostate cancer and prostate cancer cells. Following infection with Ad-PL-PPT-E1A, the E1A expression could be detected in a dose-dependent manner in prostate cancer cells (Figures 3c and d), whereas none was detected in SMMC-7721 and EC304 cells (data were not shown). Furthermore, the mRNA expression of E1A reached the peak at 48h after infection of 100 MOI Ad-PL-PPT-E1A (Figure 3e). Fusion protein PL-mediated immune activation of DCs PL expression in prostate cancer cells. PL expression is the key factor for immune activation in our design, so we confirmed the kinetic expression of PL both in Ad-PL-PPT-E1A- and Ad-PLinfected prostate cancer cells. In general, Ad-PL-PPT-E1A could mediate the expression of PL in a dose- and time-dependent manner. PL expression in PC3M cells infected by Ad-PL-PPT-E1A was increased with the MOI raise from 10 to 100 at 48 h after infection (Figure 4a). In prostate cancer cells, Ad-PL-PPT-E1A could mediate PL expression more efficiently, which reached the peak at 48 h after infection (Figure 4b). The relative expression of PL in PC3M cells elevated from 3.36 ± 0.82 to 20.23 ± 3.36, with the time extending from 24 to 48 h, and then declined to 18.41 ± 3.45 at © 2014 Macmillan Publishers Limited
72 h after infection. At 48 or 72 h after infection, Ad-PL-PPT-E1A could mediate higher expression of PL in prostate cancer cells than Ad-PL (P o 0.01). Ad-PL-PPT-E1A or Ad-PL promotes LNCaP cell-induced iDCs maturation. To investigate the effect of fusion protein PL on immature DCs (iDCs) maturation, cell lysate was extracted from Ad-PL-, Ad-PLPPT-E1A- or Ad-Null-infected LNCaP cells. And then, human peripheral blood (PB) mononuclear cell-derived iDCs, induced by 50 ng ml − 1 granulocyte–macrophage colony-stimulating factor and 25 ng ml − 1 interleukin-4 (IL-4), were stimulated by lysate or lipopolysaccharide for 48 h. Even lysate of Ad-Null-infected LNCaP cells could induce the expression of DC maturation marker CD80, CD83 and CD86, the expression level of these markers was lower than that elicited by lipopolysaccharide-, Ad-PL- or Ad-PL-PPTE1A-infected cells (Figure 4c). Although the percentage of CD86positive cells reached nearly 90% in each group, the relative intensity of fluorescence could be evaluated by Ad-PL or Ad-PLPPT-E1A (Figure 4d). Enhanced cytokine production of DCs stimulated with lysate of Ad-PL-PPT-E1A- or Ad-PL-modified LNCaP cells. DC maturation process is accompanied by the production of various cytokines that regulate the immune response against cancer cells. We detected the mRNA expression of IL-12, IL-23, IL-6 and tumor necrosis factor-α (TNF-α) of iDCs by real-time PCR. Our results showed that stimulation with lysate of LNCaP cells resulted in increased expression of IL-12 and IL-6, but not IL-23 and TNF-α of DCs. Compare to lysate of LNCaP cells transduced with Ad-Null group, that of Ad-PL-PPT-E1A or Ad-PL could enhance Gene Therapy (2014) 723 – 731
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the expression of IL-12, IL-6 IL-23 and TNF-α obviously (P o 0.01) (Figure 4e). Antitumor effect of Ad-PL-PPT-E1A in vivo Androgen-independent prostate cancer cell PC3M was selected to establish a mouse xenograft model. When the prostate tumor
xenografts reached 100 mm3, adenovirus treatment was begun by direct intratumoral injection of 2 × 108 IU daily for 5 days. The survival and tumor growth curve was monitored for evaluating the antitumor effects. In general, the tumor volume of Ad-Null-treated mice increased rapidly, whereas treatment of Ad-PL-PPT-E1A or Ad-PPT-E1A resulted in tumor regression obviously. Moreover,
Figure 3. Specific replication of Ad-PL-PPT-E1A in prostate cancer cell. Several human cells were selected for detection of the prostate cancer-pecific replication ability. At indicated time points (24, 48 and 72 h) after infection of 50 MOI Ad-PL-PPT-E1A, the genomic DNA was extracted from Ad-PL-PPT-E1A- or Ad-PL-infected PC cells. The production of viral particles was assayed by real-time PCR. The relative value of viral genomes was normalized by the average of that in Ad-PL-infected cells at each time point (a). Furthermore, at 24, 48 and 72 h after infection of 5 MOI adenoviruses, cells were collected with the culture medium and the infectious titer of produced virus progeny was assayed by tissue culture infectious dose 50 (b). E1A expression, which is essentially for replication, could be detected in a dose-dependent manner by using western blotting (c) and real-time PCR (d) at 48 h after infection with Ad-PL-PPT-E1A at serial MOIs in PC3M cells. And, the E1A expression in prostate cancer cells were also detected at different time points after infection with 100 MOI Ad-PL-PPT-E1A by using real-time PCR (e). The results in (a, d and e) are shown as the mean ± s.d. **Po 0.01 vs the corresponding group at 24 h after infection. ##P o0.01 vs the corresponding group at 48 h after infection. Figure 4. Immune activation of human mononuclear cell-derived DCs in vitro. The expression of fusion protein gene PL was detected by using real-time PCR in prostate cancer cells infected with Ad-PL-PPT-E1A or Ad-PL. At 48 h after infection, PC3M cells infected by different MOIs were analyzed (a). Ad-PL-PPT-E1A-infected prostate cancer cells (100 MOI) were also detected at different time points after infection (b). Cell lysates of LNCaP cells infected with Ad-Null, Ad-PL or Ad-PL-PPT-E1A were prepared for DC stimulation at 48 h after infection. iDCs were obtained from human PB mononuclear cells (PBMCs) and induced in AIM V Medium supplemented with recombinant human granulocyte– macrophage colony-stimulating factor (rhGM-CSF, 50 ng ml − 1) and recombinant human IL-4 (rhIL-4) (20 ng ml − 1) for 5 days. To induce maturation, lipopolysaccharide (LPS) or lysate from adenovirus-infected LNCaP cells were added and cocultured for another 48 h. Cells were collected and stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD80 antibody, phycoerythrin (PE)-conjugated anti-CD83 antibody and allophycocyanin (APC)-conjugated anti-CD86 antibody and the immune phenotypes were analyzed by FACS. The percentage of CD80-, CD83- or CD86-positive cells, and relative mean fluorescence intensity (MFI) (normalized by that of untreated DCs), were shown in (c and d). The cytokine expression was detected by real-time RT-PCR at 48 h after stimulation (e). The results in (b, d and e) are shown as the mean ± s.d. *Po0.05, **Po 0.01 vs untreated DCs; #P o0.05, ##P o0.01 vs Ad-Null-treated group; $Po 0.05, $$P o0.01 vs Ad-PL-treated group. Gene Therapy (2014) 723 – 731
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Figure 5. Antitumor activities of Ad-PL-PPT-E1A on PC3M xenografts in nude mice. In total, 5 × 106 PC3M cells were injected subcutaneously in the left flank of nude mice. When the volume of tumors reached about 100 mm3, mice were divided into three groups randomly. To observe antitumor effect, daily intratumoral injections of Ad-PL-PPT-E1A, Ad-PPT-E1A or Ad-Null were administrated during 5 consecutive days. The survival was observed every day after treatment (a). At indicated time points after treatment, the tumor volume was measured and shown in (b). Mice were killed at day 23 after first injection, tumor were removed, photographed and weighted (c). The total RNA was isolated from tumor tissues, and the expression of PSA-CD40L and E1A were detected by real-time RT-PCR (d). Data were shown as mean ± s.e.m. *Po0.05, **P o0.01 vs Ad-Null-treated group; ##P o0.01 vs Ad-PPT-E1A-treated group.
Ad-PL-PPT-E1A displayed slightly stronger antitumor effect than Ad-PPT-E1A, but there was no statistical difference. Twenty-three days after treatment, the volume of tumor in Ad-Null-treated mice increased from 135.11 ± 41.20 to 215.82 ± 110.18 mm3. However, the parameter for mice treated with Ad-PL-PPT-E1A was decreased from 127.90 ± 32.96 to 90.03 ± 66.97 mm3 (Figures 5a and c). Twenty-three days after treatment, fusion protein gene PSA-CD40L still could be detected in Ad-PL-PPT-E1A-treated tumor, whereas replication-related gene E1A expressed both in Ad-PPTE1A- and Ad-PL-PPT-E1A-treated tumor (Figure 5d). We also evaluated the antitumor effects in PC3 cell-based mouse xenograft model. Compare with Ad-Null, Ad-PL-PPT-E1A inhibited the tumor growth obviously in vivo (Supplementary Figures 1A and B). And, tumor metastasis in the liver was found in Ad-Null-treated group by using hematoxylin and eosin staining, but not in Ad-PL-PPT-E1A-treated group (Supplementary Figure 1C). Furthermore, viral replication ability in tumor was detected by tissue culture infectious dose 50. The results showed that Ad-PL-PPT-E1A could efficiently replicate in tumor, and the median of infectious titers in tumor could reach 1.18 × 106 (4.63 × 104–2.5 × 106) IU per tumor, whereas it was 1.75 × 103 (6.25 × 102–4.0 × 103) IU per tumor in Ad-Null treated group (Supplementary Figure 1D). The lower gene transduction ability might be the major blockade for Ad-PL-PPT-E1A treatment in PC3 cell-based model (Supplementary Figure 2). DISCUSSION Despite significant improvement has been achieved in developing novel therapeutic technologies, advanced or metastatic prostate cancer is still incurable. Immunogene therapy has rapidly expanded in the past 30 years and represents an alterative approach for the treatment of prostate cancer. A number of encouraging clinical trials have been reported.15 Among them, adenoviruses are the most advanced vector for their safety and efficacy. Recent reports have demonstrated that the antitumor effect of oncolytic adenoviruses was partially mediated by immune response.16,17 Therefore, the strategy that oncolytic viruses armed with immunogenes is attractive for cancer therapy.18 Prostate cancer is regularly infiltrated by antigen-specific immune cells and considered as an ideal model for Gene Therapy (2014) 723 – 731
immunotherapy.19 DCs, the only antigen-presenting cells that can prime naive T cells, have a crucial role in induction of antigenspecific T-cell responses. DCs are always deficient in number and functional activity in patients with cancer.20 Recent research reported that human prostate cancer cell could induce apoptotic death and inhibit generation of DCs in cultures.21,22 A number of DC-based vaccines against cancer therapy have been evaluated in animal models and clinical trials.8,9 However, the manipulation of DCs is laborious, expensive and time consuming. CD40 molecule, a member of the TNF receptor superfamily, highly expresses on DCs. The interaction of CD40 with CD40L could promote the expression of cytokines and costimulatory molecules in DCs, and trigger humoral and cellular immune response effectively. It has been demonstrated that delivering CD40L to tumor could induce immune activation and generate cytotoxic T cells both in vitro and in vivo.10–12 Furthermore, tumorassociated antigen/CD40L fusion protein could target to DCs and activate immune response.13 A fusion protein gene of PSA, representing the most widely used serum marker for diagnosis, with CD40L was developed for prostate cancer immunotherapy in this study. The prostate cancer-specific oncolytic adenovirus carrying PL gene (Ad-PL-PPT-E1A) and the corresponding replication-defective adenovirus (Ad-PL) were successfully constructed. Oncolytic adenoviruses were constructed by placing prostate cancerspecific promoter in front of the E1A gene, which is essentially for adenovirus replication. It has been reported that a potent prostate-specific regulatory sequence comprising the PSA enhancer (PSAe), the prostate-specific membrane antigen enhancer and the T-cell receptor γ-chain alternate reading frame promoter (named PPT) has transcriptionally active both in the presence and absence of testosterone.23 In our design, the PSAe in PPT was replaced by androgen response element enhancer core of PSAe.24 Our results demonstrated that oncolytic adenoviruses with or without immunogene PL, Ad-PL-PPT-E1A and Ad-PPT-E1A, could replicate efficiently in both androgen-dependent and -independent prostate cancer cells. However, no significant replication could be detected in human normal or tumor cells, suggesting their specific replication in prostate cancer. Efficient replication in prostate cancer cells was also confirmed in a PC3 cell xenografted nude mouse model. Upon efficient replication, redundant progeny © 2014 Macmillan Publishers Limited
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CD40L
PSA
SV40pA
E1A-E1Bp
E1B55Kplus
PPTp
TARPp
Abbreviations: CD40L, CD40 ligand; PPTp, prostate cancer-specific chimeric promoter; PSA, prostate-specific antigen enhancer; PSMAe, prostate-specific membrane antigen enhancer; TARPp, T-cell receptor γ-chain alternate reading frame protein.
EcoRI 549 NM_000074.2 192C>W Jurkat cell cDNA
793 NM_001648.2 LNCaPcDNA
160 — pShuttle-CMV
1172 AY339865.1 JM17
Self-anneal
AY339865.1
78
EcoRI Not I SalI NsiI EcoRI Spel Aat II Not I KpnI — —
720
— 273 NG_001336 K562 genome
— 285 AF007544 K562 genome
5′-CGGGGCGGCCGCGATGAAGATATTATCTTCATGATCTT-3′ 5′-GGAAAAAATAATTTTTCAAGGATGTTTGTAAAGCAGGC-3′ 5′-CAAACATCCTTGAAAAATTATTTTTTCCTTTAACCTTTCAAAC-3′ 5′-CCCTGGGCACTTAAAACAACAAAAAAATCTAGTTGTATAG-3′ 5′-GATTTTTTTGTTGTTTTAAGTGCCCAGGGCAAGGTGAGG-3′ 5′-GGGCGAATTCAGCTTTGTTCCGGGACCAAATACCTTG-3′ 5′-CGGGGCGGCCGCGATGAAGATATTATCTTCATGATCTT-3′ 5′-GGGCGAATTCAGCTTTGTTCCGGGACCAAATACCTTG-3′ 5′-GCGGTCGACGAGTTTTATAAAGGATAAATGGAGCGAAGAAACCCATCTGAGC-3′ 5′-CCGATGCATGGCCAGAAAATCCAGCAGGTACCCCCCGCTCAGATGGGTTTCT-3′ 5′-GCCGAATTCATGAGACATATTATCTGCCAC3′ 5′CGGACTAGTGAGGTCAGATGTAACCAAGAT-3′ 5′-GCCGACGTCCAATTGTTGTTGTTAACTTAACTTG3′ 5′-GCGGCGGCCGCGCGTTAAGATACATTG-3′ 5′-CGGCGGTACCATGTGGGTCCCGGTTGTCTT-3′ 5′-GGGGTTGGCCACGATGGTGTCC-3′ 5′-ATGCAAAAAGGTGATCAGAATCCTCAAATTG-3′ 5′-CCGCGA1TATCGGGCTTAACCGCTGTGCTGTA-3′ PSAe
The expression of fusion protein PL or replication-related protein E1A were determined by western blotting. Briefly, LNCaP cells were infected by
Table 1.
Gene expression assay
PCR parameters for the construction of pTE-PPT-E1A
Construction and rescue of adenoviral vectors Prostate cancer- specific chimeric promoter, containing androgen response element enhancer core of PSAe, prostate-specific membrane antigen enhancer and promoter of T-cell receptor γ-chain alternate reading frame protein (TARPp), was synthesized using the overlap extension PCR method. E1B55Kplus, E1A-E1Bp, prostate cancer-specific chimeric promoter and SV40pA were added to pTE-E1B55K sequentially to generate pTE-PPT-E1A. PSA gene and CD40L gene were amplified, respectively, and then linked by isoleucine zipper to generate fusion protein gene PSA-IZ-CD40L (PL). PL gene was inserted into the corresponding sites of pshuttle-cmv to generate a new plasmid pshuttle-PL. The 533 bp fragment between the two MfeI sites of pshuttle-PL was replaced by the corresponding 3936 bp fragment of pTE-PPT-E1A to generate pshuttle-PL-PPT-E1A. Oncolytic adenovirus Ad-PL-PPT-E1A was constructed and rescued according to the manual of AdEasy System. pShuttle-PL was replaced by pshuttle-cmv to prepare oncolytic adenovirus Ad-PPT-E1A. The replication-deficient adenovirus Ad-PL was also prepared. PCR-related information and corresponding restriction sites are listed in Table 1. All adenoviruses were purified by double CsCl density gradient ultracentrifugation, dissolved in storage buffer (Hank’s buffer, 10% glycerol). Viral particle (vp) numbers were calculated from measurements of optical density at 260 nm (OD260), where one absorbency unit is equivalent to 1.1 × 1012 vp ml − 1, and infectious titers (IU ml − 1) were determined by limiting dilution assay on 293 cells. The MOI was calculated from infectious titers.
219
Primers Fragment
Template
The human prostate cancer cell line PC3M was obtained from the Institute of Urology, Peking University (Beijing, China). The human vascular endothelial cell EC304, human prostate cancer cell LNCaP, PC3 and DU145 were obtained from the Institute of Basic Medicine, Chinese Academy of Medicine Science (Beijing, China). The normal human hepatic cell L02 and the human hepatocellular carcinoma cell SMMC-7721 were obtained from the Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The E1A-transformed human embryonic kidney 293 cell (CRL-1537), human lung epithelial carcinoma cell A549 and human hepatocellular carcinoma cell HepG2 (HB-8065) were all from the American Type Culture Collection (ATCC, Manassas, VA, USA). PC3, DU145 and PC3M are known to be androgen independent, whereas LNCaP cell is androgen dependent. DU145, PC3M, EC304, L02, HepG2, A549 and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. LNCaP and SMMC-7721 were maintained in RPMI1640 plus 10% fetal bovine serum, whereas PC3 was maintained in F12 plus 10% fetal bovine serum. All culture media and fetal bovine serum were obtained from Hyclone (Logan, UT, USA) and supplemented with 100 U ml − 1 penicillin, 100 μg ml − 1 streptomycin and 2 mM L-glutamine. All cells were maintained at 37 °C with 5% CO2 in a humidified incubator.
PSMAe
Cells
AF243527
GeneBank accession no.
AdEasy-1 backbone and pshuttle-CMV plasmids were obtained from Stratagene (La Jolla, CA, USA). Plasmid pTE-E1B55K and pJM13, which have been reported previously, were constructed in our laboratory.25 Ad-Null, a replication-defective adenovirus without any exogenous gene, is used as a negative control. Ad-CD80-TPE-GM, a telomerase-targeted oncolytic adenovirus, which has been reported previously, is used as a positive control.25
K562 genome
Restriction Length of PCR product (bp)
MATERIALS AND METHODS Plasmid and adenovirus
—
729 viruses were accumulated rapidly in prostate cancer cells, leading to apoptosis or cell death both in vitro and in vivo. In conclusion, we have successfully developed a prostate cancer-specific oncolytic adenovirus Ad-PL-PPT-E1A, which could mediate prostate cancer-specific oncolytic effect in vitro and in vivo and activate DCs in vitro. These results indicated that Ad-PLPPT-E1A might be an alternative approach for prostate cancer.
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730 50 MOI of Ad-PL-PPT-E1A, Ad-PPT-E1A, Ad-PL or Ad-Null and viruses were washed by phosphate-buffered saline (PBS) 2 h after infection. Cells were harvested at 48 h after infection. The protein was extracted and detected by using mouse anti-human PSA antibody, mouse anti-human CD40L antibody (Thermo Scientific, Wilmington, DE, USA) or anti-human type 5 adenovirus E1A antibody (Abcam, Cambridge, MA, USA). At different time points after infection, total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and cDNA was synthesized using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer’s instructions. Then, mRNA expression levels of PL and E1A were detected by PCR.
Cell viability assay Exponentially growing cells were seeded at a density of 2 × 104 cells per well in 96-well plates and infected by Ad-PL-PPT-E1A, Ad-PPT-E1A, Ad-PL, Ad-Null or Ad-CD80-TPE-GM at serial MOIs in 100 μl complete culture media. One hundred microliter fresh culture media were supplemented to each well 3 days after infection. At day 7 after infection, cells were fixed in 1% glutaraldehyde (v v − 1) for 2–3 h, and then stained with 0.1% crystal violet (w v − 1) for 1 h. After washing for three times, crystal violet dye was dissolved with 1% Triton X-100 (v v − 1). After 2 h rotary incubation at room temperature, the absorbance of the resulting Triton extract was read at 570 nm on a varioskan flask (Thermo Scientific). Cell survival rate is absorbance of tested well normalized by that of uninfected control well.
Apoptosis Exponentially growing DU145, PC3M or LNCaP cells were seeded at a density of 3 × 105 cells per well in 6-well plates and infected by serial MOIs of Ad-PL-PPT-E1A, Ad-PPT-E1A or Ad-PL. Twenty-four, 48 or 72 h after infection, total cells were harvested and analyzed with Annexin V-FITC Apoptosis Detection Kit (KeyGEN) according to the manufacturer’s instructions. Uninfected cells were used as control.
Virus replication in vitro Exponentially growing cells were seeded at a density of 3 × 105 cells per well in 6-well plates and infected by 50 MOI of Ad-PL-PPT-E1A or Ad-PL. Two hours later, viruses were removed by washing twice with PBS, and then 500 μl of fresh media was added to each well. Cells were collected for extraction of mRNA and genomo times DNA at 24, 48 or 72 h after infection. E1A expression was detected by using real-time PCR as described above. The genomic DNA was used to analyze production of viral particles by real-time PCR using the following primers: Ad5 (sense, 5′-TCTGA GTTGGCACCCCTATTC-3′ and antisense, 5′-GTTGCTGTGGTCGTTCTGGTA-3′); β-globin (sense, 5′-GTGCACCTGACTCCTGAGGAGA-3′ and antisense, 5′-CCTTGATACCAACCTGCCCAGG-3′). To test infectious titer, cells were infected by Ad-PL-PPT-E1A or Ad-CD80TPE-GM at 5 MOI as described above. Cells were collected at 24, 48 or 72 h after infection. Lysates were prepared by three cycles of freeze and thaw and used to determine virus titer by limiting dilution assay on 293 cells.
Generation of human monocyte-derived DCs Human PB samples used in this study were obtained from patients undergoing treatment for diagnostic purposes at Peking University First Hospital. Mononuclear cells (PB mononuclear cells) were isolated from heparinized samples by centrifugation through a Ficoll–Hypaque density gradient (Amersham Biosciences, Piscataway, NJ, USA). In total, 1 × 108 PB mononuclear cells were seed in a 100 mm plate (Corning Inc., Corning, NY, USA) with 10 ml AIM V Medium CTS (Gibco, Grand Island, NY, USA) at 37 °C. Two hours later, non-adherent cells were discarded and adherent cells were washed two times with prewarmed PBS. Then, cells were incubated with AIM V Medium supplemented with recombinant human granulocyte macrophage-colony-stimulating factor (50 ng ml − 1) and recombinant human IL-4 (20 ng ml − 1) (Peprotech, Rocky Hill, NJ, USA) for 5 days. On day 3, fresh complete medium was added.
Effect of fusion protein PL on DCs At day 5, the non-adherent cells were collected and seeded in 24-well plate at a density of 2 × 105 ml per well. The cells were induced by recombinant human TNF-α (25 ng ml − 1) (Peprotech) and the lysate (150 μg) of LNCaP cells were infected by Ad-PL-PPT-E1A, Ad-PL or Ad-Null. And, lipopolysaccharide (1 μg/ml) (Sigma Chemical Co., St Louis, MO, USA) was used as a positive control. Gene Therapy (2014) 723 – 731
Immunophenotype analysis. DCs were harvested 48 h later, and then were stained with fluorescein isothiocyanate-conjugated CD80, phycoerythrinconjugated CD83 and allophycocyanin-conjugated CD86 (BD Biosciences, San Jose, CA, USA). The immunophenotype was analyzed by using FACScan flow cytometer (BD Biosciences). Expression of cytokines. Total RNA was isolated from DCs 48 h after induction, and cDNA was synthesized as described above. mRNA expression of human IL-12a (hIL-12a), hIL-12b, hIL-6, hIL-23 and TNF-α were detected by real-time PCR.
Animal experiments All animal experiments were conducted according to protocols approved by the Institutional Animal Care and Use Committee of the Beijing Institute of Radiation Medicine. In total, 5 × 106 PC3M cells per 100 μl PBS were subcutaneously injected into the left flanks of male BALB/c nude mice. When tumors reached about 100 mm3 in volume, 18 mice were divided randomly into three groups, and then treated by Ad-PL-PPT-E1A, Ad-PPTE1A or Ad-Null, respectively. Ad-PL-PPT-E1A, Ad-PPT-E1A or Ad-Null (2 × 108 IU per 100 μl PBS) were daily injected intratumorally for 5 consecutive days. The total dose was 109 IU per mouse. Tumor was monitored regularly by measuring length and width and tumor volume was calculated with the following formula: width2 × length/2. The survival was observed every day, and the tumor volume was detected every 3–4 days. Mice were killed at day 23 posttreatment (day 0 was set as that the first intratumoral injection occurred).
Real-time PCR The mRNA expression was quantified using the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and SYBR Premix Ex Taq II (Perfect Real Time) (Takara, Shiga, Japan). The primers used were as follows: PL (sense, 5′-AGGCTGGGAGTGCGAGAA-3′ and antisense, 5′-TGAGGCGTAGCAGGTGGT-3′); E1A (sense, 5′-GCTGATAATCTTCCACCTCC-3′ and antisense, 5′-ACCCTCTTCATCCTCGTC-3′); hIL-12a (sense, 5′-TCCTC CCTTGAAGAACCG-3′ and antisense, 5′-AATAGTCACTGCCCGAAT-3′); hIL-12b (sense, 5′-CATCAGGGACATCATCAA-3′ and antisense, 5′-GTCAGGGAGA AGTAGGAA-3′; hIL-6 (sense, 5′-CATCCTCGACGGCATCTC-3′ and antisense, 5′-GCCTCTTTGCTGCTTTCA-3′); hIL-23 (sense, 5′-GTCTCCTTCTCCGCTTCA-3′ and antisense, 5′-TTAGGGACTCAGGGTTGC-3′; hTNF-α (sense, 5′-CATC GCCGTCTCCTACCA-3′ and antisense, 5′-AGTCGGTCACCCTTCTCC-3′; human β-actin (sense, 5′-GCGGGAAATCGTGCGTGAC-3′ and antisense, 5′-GGAAGGAAGGCTGGAAGAG-3′). And, the expression levels were normalized by human β-actin.
Statistical analysis Values were presented as mean ± s.d. or mean ± s.e.m. For tumor growth curve, two-way analysis of variance was used, followed by the Dunnett's post hoc test. For other data, one-way analysis of variance was used to compare the means of two or more experimental groups, followed by the Dunnett's post hoc test. Statistical differences between groups were considered to be significant at P o0.05.
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This work was supported by grants from the National High Technology Research and Development Program of China (863 Program) (Nos. 2012AA020807 and 2014AA020515), the National Basic Research and Development of China (973 Program) (No. 2012CB518205) and the National Natural Science Foundation of China (No. 30901379).
REFERENCES 1 Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T et al. Cancer statistics, 2008. CA Cancer J Clin 2008; 58: 71–96. 2 Cannata DH, Kirschenbaum A, Levine AC. Androgen deprivation therapy as primary treatment for prostate cancer. J Clin Endocrinol Metab 2012; 97: 360–365.
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731 3 Sturge J, Caley MP, Waxman J. Bone metastasis in prostate cancer: emerging therapeutic strategies. Nat Rev Clin Oncol 2011; 8: 357–368. 4 Banchereau J, Paczesny S, Blanco P, Bennett L, Pascual V, Fay J et al. Dendritic cells: controllers of the immune system and a new promise for immunotherapy. Ann NY Acad Sci 2003; 987: 180–187. 5 O'Neill DW, Adams S, Bhardwaj N. Manipulating dendritic cell biology for the active immunotherapy of cancer. Blood 2004; 104: 2235–2246. 6 Elgueta R, Benson MJ, de Vries VC, Wasiuk A, Guo Y, Noelle RJ. Molecular mechanism and function of CD40/CD40L engagement in the immune system. Immunol Rev 2009; 229: 152–172. 7 Xu Y, Song G. The role of CD40-CD154 interaction in cell immunoregulation. J Biomed Sci 2004; 11: 426–438. 8 Hildenbrand B, Sauer B, Kalis O, Stoll C, Freudenberg MA, Niedermann G et al. Immunotherapy of patients with hormone-refractory prostate carcinoma pretreated with interferon-gamma and vaccinated with autologous PSA-peptide loaded dendritic cells—a pilot study. Prostate 2007; 67: 500–508. 9 Williams BJ, Bhatia S, Adams LK, Boling S, Carroll JL, Li XL et al. Dendritic cell based PSMA immunotherapy for prostate cancer using a CD40-targeted adenovirus vector. PLoS One 2012; 7: e46981. 10 Dzojic H, Loskog A, Tötterman TH, Essand M. Adenovirus-mediated CD40 ligand therapy induces tumor cell apoptosis and systemic immunity in the TRAMP-C2 mouse prostate cancer model. Prostate 2006; 66: 831–838. 11 von Euler H, Sadeghi A, Carlsson B, Rivera P, Loskog A, Segall T et al. Efficient adenovector CD40 ligand immunotherapy of canine malignant melanoma. J Immunother 2008; 31: 377–384. 12 Xu W, Li Y, Wang X, Wang C, Zhao W, Wu J. Anti-tumor activity of gene transfer of the membrane-stable CD40L mutant into lung cancer cells. Int J Oncol 2010; 37: 935–941. 13 Zhang L, Tang Y, Akbulut H, Zelterman D, Linton PJ, Deisseroth AB. An adenoviral vector cancer vaccine that delivers a tumor-associated antigen/CD40-ligand fusion protein to dendritic cells. Proc Natl Acad Sci USA 2003; 100: 15101–15106. 14 Toth K, Dhar D, Wold WS. Oncolytic (replication-competent) adenoviruses as anticancer agents. Expert Opin Biol Ther 2010; 10: 353–368.
15 Freytag SO, Stricker H, Movsas B, Kim JH. Prostate cancer gene therapy clinical trials. Mol Ther 2007; 15: 1042–1052. 16 Tuve S, Liu Y, Tragoolpua K, Jacobs JD, Yumul RC, Li ZY et al. In situ adenovirus vaccination engages T effector cells against cancer. Vaccine 2009; 27: 4225–4239. 17 Alemany R. A smart move against cancer for vaccinia virus. Lancet Oncol 2008; 9: 507–508. 18 Cerullo V, Pesonen S, Diaconu I, Escutenaire S, Arstila PT, Ugolini M et al. Oncolytic adenovirus coding for granulocyte macrophage colony-stimulating factor induces antitumoral immunity in cancer patients. Cancer Res 2010; 70: 4297–4309. 19 Tokmadžić VS, Tomaš MI, Sotošek S, Laškarin G, Dominović M, Tulić V et al. Different perforin expression in peripheral Blood and prostate tissue in patients with benign prostatic hyperplasia and prostate cancer. Scand J Immunol 2011; 74: 368–376. 20 Nencioni A, Grünebach F, Schmidt SM, Müller MR, Boy D, Patrone F et al. The use of dendritic cells in cancer immunotherapy. Crit Rev Oncol Hematol 2008; 65: 191–199. 21 Aalamian M, Pirtskhalaishvili G, Nunez A, Esche C, Shurin GV, Huland E et al. Human prostate cancer regulates generation and maturation of monocyte derived dendritic cells. Prostate 2001; 46: 68–75. 22 Pirtskhalaishvili G, Shurin GV, Esche C, Cai Q, Salup RR, Bykovskaia SN et al. Cytokine mediated protection of human dendritic cells from prostate cancer induced apoptosis is regulated by the Bcl-2 family of proteins. Br J Cancer 2000; 83: 506–513. 23 Cheng WS, Dzojic H, Nilsson B, Tötterman TH, Essand M. An oncolytic conditionally replicating adenovirus for hormone-dependent and hormoneindependent prostate cancer. Cancer Gene Ther 2006; 13: 13–20. 24 Lee SJ, Kim HS, Yu R, Lee K, Gardner TA, Jung C et al. Novel prostate-specific promoter derived from PSA and PSMA enhancers. Mol Ther 2002; 6: 415–421. 25 Hu ZB, Wu CT, Wang H, Zhang QW, Wang L, Wang RL et al. A simplified system for generating oncolytic adenovirus vector carrying one or two transgenes. Cancer Gene Ther 2008; 15: 173–182.
Supplementary Information accompanies this paper on Gene Therapy website (http://www.nature.com/gt)
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ORIGINAL ARTICLE
Antitumor effects of oncolytic adenovirus armed with PSA-IZ-CD40L fusion gene against prostate cancer Y-F Yang1,4, S-Y Xue1,4, Z-Z Lu2, F-J Xiao1, Y Yin3, Q-W Zhang1, C-T Wu1, H Wang1 and L-S Wang1 Advanced prostate cancer (PC) still remains incurable. Novel immunogene therapy shows promise as treatment strategy that can target both localized and metastasized PC. In this study, we have developed a PC-specific oncolytic adenovirus (Ad-PL-PPT-E1A) armed with fusion gene of prostate-specific antigen and CD40 ligand, and aimed to evaluate its therapeutic effect in vitro and in vivo. After they were rescued in human embryonic kidney 293 cells, we confirmed that Ad-PL-PPT-E1A could mediate the expression of E1A efficiently and produce abundant progeny viruses in PC cells in vitro. Our data showed that Ad-PL-PPT-E1A induced apoptosis and resulted in specific oncolytic toxicity in PC cells, which was detected by Annexin-V staining and crystal violet, respectively. After stimulation with lysates, immune phenotypes and cytokines expression of human dendritic cells was detected by flow cytometry and real-time polymerase chain reaction, respectively. And, the results showed that the lysate of Ad-PL-PPT-E1Ainfected LNCaP cells upregulated the expression of CD80, CD83, CD86 and mRNA level of interleukin-6 (IL-6), IL-12, IL-23 and tumor necrosis factor-α significantly. In established PC3M cell-xenografted mouse models, Ad-PL-PPT-E1A treatment improved the survival and suppressed the tumor growth obviously. In conclusion, Ad-PL-PPT-E1A exhibited enhanced antitumor activity is a promising approach for gene therapy of advanced PC. Gene Therapy (2014) 21, 723–731; doi:10.1038/gt.2014.46; published online 22 May 2014
INTRODUCTION Prostate cancer (PC) remains the second most common cancer in men worldwide.1 For patients suffering from prostate cancer at early stage, the therapeutic options are curative and include surgery, radiation and, in some cases, active surveillance only. A majority of the patients progress to metastatic diseases and eventually develop castration-resistant disease during the advanced stages. Conventional treatments, such as docetaxel chemotherapy, provide only modest benefits.2,3 Novel strategies for metastatic, castration-resistant prostate cancer are needed urgently. In the past decade, immunotherapy has emerged as a promising approach for advanced prostate cancer. Dendritic cells (DCs) are the most potent antigen-presenting cells for priming and activating naive T cells and having important roles in antitumor immune responses.4,5 The interaction between surface receptor CD40 and its ligand (CD40L) promotes cytokine production, costimulatory molecule expression of DCs and eventually induces specific immune responses.6,7 Several encouraging trials of DC-based vaccines activated by CD40L have been carried out to induce tumor antigen-specific immune responses in cancer patients.8,9 It has been also demonstrated that CD40L-based gene therapy could induce both humoral and cellular immunity against many types of cancer.10–12 Moreover, tumor-associated antigen/ CD40L fusion protein could induce antitumor immune response through activation of DCs.13 Oncolytic adenoviruses, which selectively replicate in tumor cells, are optimal vectors for cancer gene therapy. The safety and efficacy of oncolytic adenoviruses have been validated in several
clinical studies.14 To combine the antitumor effect of oncolytic adenoviruses and CD40L-based immunotherapy, we successfully developed a prostate cancer-specific oncolytic adenovirus armed with fusion protein gene of prostate-specific antigen (PSA) and CD40L (PSA-IZ-CD40L, PL), and evaluated its therapeutic effect in vitro and in vivo. RESULTS Construction and identification of prostae cancer-specific oncolytic adenovirus Ad-PL-PPT-E1A Prostate cancer-specific oncolytic adenovirus armed with fusion protein gene PL (Ad-PL-PPT-E1A) and control adenoviruses (Ad-PL, Ad-PPT-E1A and Ad-Null) were successfully constructed as described in Materials and Methods (Figure 1a). The PL and E1A genes in adenoviral genomic DNA were confirmed by using polymerase chain reaction (PCR) (Figure 1b). The infectious titers of the purified adenoviruses were 2.0 × 1010 infection units per milliliter (IU ml − 1) (Ad-PL-PPT-E1A), 6.0 × 1010 IU ml − 1 (Ad-PL), 2.0 × 1011 IU ml − 1 (Ad-PPT-E1A) and 2.0 × 1011 IU ml − 1 (Ad-Null), respectively. And, the ratio between IU and viral physical particles were higher than 1:30. The expression of PL and E1A were detected by using real-time PCR and western blotting. The results showed that PSA protein expression could be detected in LNCaP cells, while Ad-PL-PPT-E1A and Ad-PL could mediate the expression of PL both on mRNA (data was not shown) and protein levels. And, E1A expression could be detected only in Ad-PL-PPT-E1A- and Ad-PPT-E1Ainfected cells (Figures 1c and d).
1 Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing, People's Republic of China; 2Chinese Center for Disease Control and Prevention, Institute for Viral Disease Control and Prevention, Beijing, People's Republic of China and 3Department of Hematology, Peking University First Hospital, Beijing, People's Republic of China. Correspondence: Dr H Wang or Professor L-S Wang, Department of Experimental Hematology, Beijing Institute of Radiation Medicine, 27 Taiping Road, Beijing 100850, People's Republic of China. E-mail: [email protected] or [email protected] 4 These authors contributed equally to this work. Received 16 July 2013; revised 25 March 2014; accepted 9 April 2014; published online 22 May 2014
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Figure 1. Construction and identification of oncolytic adenovirus Ad-PL-PPT-E1A and control adenoviruses. The schemes of Ad-PL-PPT-E1A, Ad-PL, Ad-PPT-E1A and Ad-Null were shown in (a). PPTp represents the prostate cancer-specific chimeric promoter, which consists of androgen response element enhancer core (AREc) of PSAe, prostate-specific membrane antigen enhancer (PSMAe) and promoter of T-cell receptor γ-chain alternate reading frame protein (TARPp). And, PL is the fusion protein gene of PSA and CD40L, which is linked by isoleucine zipper. After adenoviruses were constructed and rescued in human embryonic kidney 293 (HEK293) cells, the genomic DNA was obtained by Hirt extraction, and the PL and E1A genes were confirmed by using PCR (b). To detect protein expression, LNCaP cells were infected by Ad-PL-PPT-E1A, Ad-PL, Ad-PPT-E1A or Ad-Null at 50 MOI. Forty-eight hours later, protein was extracted and the expression of E1A (c) and PL (d) were assayed by western blotting.
Oncolytic effect of Ad-PL-PPT-E1A Prostate cancer or non-prostate cancer cells were infected by AdPL-PPT-E1A, Ad-PL or Ad-PPT-E1A at serial multiplicity of infection (MOI) for detecting their prostate cancer-specific oncolytic effects. Ad-Null and Ad-CD80-TPE-GM were used as the negative and positive control, respectively. The oncolytic ability was determined by crystal violet staining 7 days after infection. The results showed that Ad-PL-PPT-E1A and Ad-PPT-E1A had no obvious oncolytic effect on non-prostate cancer cells, including L02, EC304, A549 and 7721. However, Ad-PL-PPT-E1A and Ad-PPT-E1A could kill prostate cancer cells effectively in an MOI dose-dependent manner, which was consistent with that of Ad-CD80-TPE-GM (Figure 2a). As shown in Figure 2a, almost all LNCaP cells could be killed at 0.1 MOI, and nearly 80% of the DU145 and PC3M cells could be killed at 10 MOI. Replicationdeficient adenovirus Ad-PL had little effect on viability of all tested cell lines. Apoptosis of prostate cancer cells infected by oncolytic adenovirus was detected by FACS using Annexin-V-FITC Detection Kit (KeyGEN, Nanjing, China). Our data showed that Ad-PL-PPT-E1A or Ad-PPT-E1A could induce apoptosis of prostate cancer cells, whereas no obvious changes could be detected in Ad-PL-infected cells. Furthermore, Ad-PL-PPT-E1A- and Ad-PPT-E1A-induced Gene Therapy (2014) 723 – 731
apoptosis took place in a time- and dose-dependent manner. As shown in Figures 2b and c, the apoptosis rate of PC3M cells infected by 50 MOI Ad-PL-PPT-E1A rose from 10.36 ± 0.14 to 42.75 ± 2.47%, with the time increased from 24 to 72 h after infection, whereas it elevated from 30.63 ± 0.49 to 69.4 ± 3.96%, with the dose increased from 50 to 200 MOI at 48 h after infection. Prostate cancer-specific replication of Ad-PL-PPT-E1A Cytopathic effects were detected in Ad-PL-PPT-E1A-infected prostate cancer cells, including LNCaP, PC3M and DU145, by light microscopy (data were not shown). We also investigated the specific replication of Ad-PL-PPT-E1A by several methods, including E1A expression, production of viral particles and infectious titer. First, the replication of viral genomic DNA was determined by real-time PCR and normalized by that in Ad-PL-infected cells. Our results showed that viral genomic DNA could be generated in prostate cancer cells efficiently. The replication resulted in a timedependent increase of viral genomic DNA in DU145 cells, while it reached the peak at 48 h after infection in LNCaP and PC3M cells (Figure 3a). Second, we tested the progeny adenovirus production by infected cells. Ad-CD80-TPE-GM was used as a positive control for cancer cells. Our data showed that Ad-CD80-TPE-GM could © 2014 Macmillan Publishers Limited
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Figure 2. Oncolytic effect of Ad-PL-PPT-E1A on human prostate cancer cells. Prostate cancer cells PC3M, LNCaP, DU145, PC3 and non-PC cells EC304, L02, A549 and SMMC-7721 were seeded into 96-well plates at a density of 2 × 104 cells per well. Twenty-four hours later, cells were infected by Ad-PL-PPT-E1A or control adenoviruses (Ad-PL, Ad-PPT-E1A, Ad-Null or Ad-GFP-TPE-GM) at serial MOI, and cell survival rate was determined with crystal violet staining at day 7 after infection (a). Cell apoptosis was also detected in prostate cancer cells infected with 50, 100, 150 or 200 MOI Ad-PL, Ad-PL-PPT-E1A, Ad-PL or Ad-PPT-E1A, by using Annexin V-FITC Apoptosis Detection Kit. Ad-PL-PPT-E1A and Ad-PPT-E1A could induce cell death obviously. The percentage of death cells (50 MOI) was shown as the mean ± s.d. and increased with the time extended (b). *P o 0.05, **P o 0.01 vs Ad-PL group at the same time point; ## P o 0.01 vs 24 h group infected with the same adenovirus; $$P o 0.01 vs 48 h group infected with the same adenovirus. Ad-PL-PPT-E1A and Ad-PPT-E1A also induced cell apoptosis in a dose-dependent manner (48 h after infection) (c). **P o 0.01 vs Ad-PL group at the same MOI; #P o 0.05, ##P o 0.01 vs 50 MOI group infected with the same adenovirus; $P o 0.05 vs 100 MOI group infected with the same adenovirus.
produce progeny adenoviruses in all cancer cells. However, for Ad-PL-PPT-E1A, the viral production could be detected only in prostate cancer cells. The infectious titer of progeny adenovirus from Ad-PL-PPT-E1A-infected cells reached nearly 100 IU per cell at 72 h after infection (Figure 3b). Third, the E1A expression, which was essential for replication and controlled by PPT promoter in our design, was detected by western blotting or real-time PCR in both non-prostate cancer and prostate cancer cells. Following infection with Ad-PL-PPT-E1A, the E1A expression could be detected in a dose-dependent manner in prostate cancer cells (Figures 3c and d), whereas none was detected in SMMC-7721 and EC304 cells (data were not shown). Furthermore, the mRNA expression of E1A reached the peak at 48h after infection of 100 MOI Ad-PL-PPT-E1A (Figure 3e). Fusion protein PL-mediated immune activation of DCs PL expression in prostate cancer cells. PL expression is the key factor for immune activation in our design, so we confirmed the kinetic expression of PL both in Ad-PL-PPT-E1A- and Ad-PLinfected prostate cancer cells. In general, Ad-PL-PPT-E1A could mediate the expression of PL in a dose- and time-dependent manner. PL expression in PC3M cells infected by Ad-PL-PPT-E1A was increased with the MOI raise from 10 to 100 at 48 h after infection (Figure 4a). In prostate cancer cells, Ad-PL-PPT-E1A could mediate PL expression more efficiently, which reached the peak at 48 h after infection (Figure 4b). The relative expression of PL in PC3M cells elevated from 3.36 ± 0.82 to 20.23 ± 3.36, with the time extending from 24 to 48 h, and then declined to 18.41 ± 3.45 at © 2014 Macmillan Publishers Limited
72 h after infection. At 48 or 72 h after infection, Ad-PL-PPT-E1A could mediate higher expression of PL in prostate cancer cells than Ad-PL (P o 0.01). Ad-PL-PPT-E1A or Ad-PL promotes LNCaP cell-induced iDCs maturation. To investigate the effect of fusion protein PL on immature DCs (iDCs) maturation, cell lysate was extracted from Ad-PL-, Ad-PLPPT-E1A- or Ad-Null-infected LNCaP cells. And then, human peripheral blood (PB) mononuclear cell-derived iDCs, induced by 50 ng ml − 1 granulocyte–macrophage colony-stimulating factor and 25 ng ml − 1 interleukin-4 (IL-4), were stimulated by lysate or lipopolysaccharide for 48 h. Even lysate of Ad-Null-infected LNCaP cells could induce the expression of DC maturation marker CD80, CD83 and CD86, the expression level of these markers was lower than that elicited by lipopolysaccharide-, Ad-PL- or Ad-PL-PPTE1A-infected cells (Figure 4c). Although the percentage of CD86positive cells reached nearly 90% in each group, the relative intensity of fluorescence could be evaluated by Ad-PL or Ad-PLPPT-E1A (Figure 4d). Enhanced cytokine production of DCs stimulated with lysate of Ad-PL-PPT-E1A- or Ad-PL-modified LNCaP cells. DC maturation process is accompanied by the production of various cytokines that regulate the immune response against cancer cells. We detected the mRNA expression of IL-12, IL-23, IL-6 and tumor necrosis factor-α (TNF-α) of iDCs by real-time PCR. Our results showed that stimulation with lysate of LNCaP cells resulted in increased expression of IL-12 and IL-6, but not IL-23 and TNF-α of DCs. Compare to lysate of LNCaP cells transduced with Ad-Null group, that of Ad-PL-PPT-E1A or Ad-PL could enhance Gene Therapy (2014) 723 – 731
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the expression of IL-12, IL-6 IL-23 and TNF-α obviously (P o 0.01) (Figure 4e). Antitumor effect of Ad-PL-PPT-E1A in vivo Androgen-independent prostate cancer cell PC3M was selected to establish a mouse xenograft model. When the prostate tumor
xenografts reached 100 mm3, adenovirus treatment was begun by direct intratumoral injection of 2 × 108 IU daily for 5 days. The survival and tumor growth curve was monitored for evaluating the antitumor effects. In general, the tumor volume of Ad-Null-treated mice increased rapidly, whereas treatment of Ad-PL-PPT-E1A or Ad-PPT-E1A resulted in tumor regression obviously. Moreover,
Figure 3. Specific replication of Ad-PL-PPT-E1A in prostate cancer cell. Several human cells were selected for detection of the prostate cancer-pecific replication ability. At indicated time points (24, 48 and 72 h) after infection of 50 MOI Ad-PL-PPT-E1A, the genomic DNA was extracted from Ad-PL-PPT-E1A- or Ad-PL-infected PC cells. The production of viral particles was assayed by real-time PCR. The relative value of viral genomes was normalized by the average of that in Ad-PL-infected cells at each time point (a). Furthermore, at 24, 48 and 72 h after infection of 5 MOI adenoviruses, cells were collected with the culture medium and the infectious titer of produced virus progeny was assayed by tissue culture infectious dose 50 (b). E1A expression, which is essentially for replication, could be detected in a dose-dependent manner by using western blotting (c) and real-time PCR (d) at 48 h after infection with Ad-PL-PPT-E1A at serial MOIs in PC3M cells. And, the E1A expression in prostate cancer cells were also detected at different time points after infection with 100 MOI Ad-PL-PPT-E1A by using real-time PCR (e). The results in (a, d and e) are shown as the mean ± s.d. **Po 0.01 vs the corresponding group at 24 h after infection. ##P o0.01 vs the corresponding group at 48 h after infection. Figure 4. Immune activation of human mononuclear cell-derived DCs in vitro. The expression of fusion protein gene PL was detected by using real-time PCR in prostate cancer cells infected with Ad-PL-PPT-E1A or Ad-PL. At 48 h after infection, PC3M cells infected by different MOIs were analyzed (a). Ad-PL-PPT-E1A-infected prostate cancer cells (100 MOI) were also detected at different time points after infection (b). Cell lysates of LNCaP cells infected with Ad-Null, Ad-PL or Ad-PL-PPT-E1A were prepared for DC stimulation at 48 h after infection. iDCs were obtained from human PB mononuclear cells (PBMCs) and induced in AIM V Medium supplemented with recombinant human granulocyte– macrophage colony-stimulating factor (rhGM-CSF, 50 ng ml − 1) and recombinant human IL-4 (rhIL-4) (20 ng ml − 1) for 5 days. To induce maturation, lipopolysaccharide (LPS) or lysate from adenovirus-infected LNCaP cells were added and cocultured for another 48 h. Cells were collected and stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD80 antibody, phycoerythrin (PE)-conjugated anti-CD83 antibody and allophycocyanin (APC)-conjugated anti-CD86 antibody and the immune phenotypes were analyzed by FACS. The percentage of CD80-, CD83- or CD86-positive cells, and relative mean fluorescence intensity (MFI) (normalized by that of untreated DCs), were shown in (c and d). The cytokine expression was detected by real-time RT-PCR at 48 h after stimulation (e). The results in (b, d and e) are shown as the mean ± s.d. *Po0.05, **Po 0.01 vs untreated DCs; #P o0.05, ##P o0.01 vs Ad-Null-treated group; $Po 0.05, $$P o0.01 vs Ad-PL-treated group. Gene Therapy (2014) 723 – 731
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Figure 5. Antitumor activities of Ad-PL-PPT-E1A on PC3M xenografts in nude mice. In total, 5 × 106 PC3M cells were injected subcutaneously in the left flank of nude mice. When the volume of tumors reached about 100 mm3, mice were divided into three groups randomly. To observe antitumor effect, daily intratumoral injections of Ad-PL-PPT-E1A, Ad-PPT-E1A or Ad-Null were administrated during 5 consecutive days. The survival was observed every day after treatment (a). At indicated time points after treatment, the tumor volume was measured and shown in (b). Mice were killed at day 23 after first injection, tumor were removed, photographed and weighted (c). The total RNA was isolated from tumor tissues, and the expression of PSA-CD40L and E1A were detected by real-time RT-PCR (d). Data were shown as mean ± s.e.m. *Po0.05, **P o0.01 vs Ad-Null-treated group; ##P o0.01 vs Ad-PPT-E1A-treated group.
Ad-PL-PPT-E1A displayed slightly stronger antitumor effect than Ad-PPT-E1A, but there was no statistical difference. Twenty-three days after treatment, the volume of tumor in Ad-Null-treated mice increased from 135.11 ± 41.20 to 215.82 ± 110.18 mm3. However, the parameter for mice treated with Ad-PL-PPT-E1A was decreased from 127.90 ± 32.96 to 90.03 ± 66.97 mm3 (Figures 5a and c). Twenty-three days after treatment, fusion protein gene PSA-CD40L still could be detected in Ad-PL-PPT-E1A-treated tumor, whereas replication-related gene E1A expressed both in Ad-PPTE1A- and Ad-PL-PPT-E1A-treated tumor (Figure 5d). We also evaluated the antitumor effects in PC3 cell-based mouse xenograft model. Compare with Ad-Null, Ad-PL-PPT-E1A inhibited the tumor growth obviously in vivo (Supplementary Figures 1A and B). And, tumor metastasis in the liver was found in Ad-Null-treated group by using hematoxylin and eosin staining, but not in Ad-PL-PPT-E1A-treated group (Supplementary Figure 1C). Furthermore, viral replication ability in tumor was detected by tissue culture infectious dose 50. The results showed that Ad-PL-PPT-E1A could efficiently replicate in tumor, and the median of infectious titers in tumor could reach 1.18 × 106 (4.63 × 104–2.5 × 106) IU per tumor, whereas it was 1.75 × 103 (6.25 × 102–4.0 × 103) IU per tumor in Ad-Null treated group (Supplementary Figure 1D). The lower gene transduction ability might be the major blockade for Ad-PL-PPT-E1A treatment in PC3 cell-based model (Supplementary Figure 2). DISCUSSION Despite significant improvement has been achieved in developing novel therapeutic technologies, advanced or metastatic prostate cancer is still incurable. Immunogene therapy has rapidly expanded in the past 30 years and represents an alterative approach for the treatment of prostate cancer. A number of encouraging clinical trials have been reported.15 Among them, adenoviruses are the most advanced vector for their safety and efficacy. Recent reports have demonstrated that the antitumor effect of oncolytic adenoviruses was partially mediated by immune response.16,17 Therefore, the strategy that oncolytic viruses armed with immunogenes is attractive for cancer therapy.18 Prostate cancer is regularly infiltrated by antigen-specific immune cells and considered as an ideal model for Gene Therapy (2014) 723 – 731
immunotherapy.19 DCs, the only antigen-presenting cells that can prime naive T cells, have a crucial role in induction of antigenspecific T-cell responses. DCs are always deficient in number and functional activity in patients with cancer.20 Recent research reported that human prostate cancer cell could induce apoptotic death and inhibit generation of DCs in cultures.21,22 A number of DC-based vaccines against cancer therapy have been evaluated in animal models and clinical trials.8,9 However, the manipulation of DCs is laborious, expensive and time consuming. CD40 molecule, a member of the TNF receptor superfamily, highly expresses on DCs. The interaction of CD40 with CD40L could promote the expression of cytokines and costimulatory molecules in DCs, and trigger humoral and cellular immune response effectively. It has been demonstrated that delivering CD40L to tumor could induce immune activation and generate cytotoxic T cells both in vitro and in vivo.10–12 Furthermore, tumorassociated antigen/CD40L fusion protein could target to DCs and activate immune response.13 A fusion protein gene of PSA, representing the most widely used serum marker for diagnosis, with CD40L was developed for prostate cancer immunotherapy in this study. The prostate cancer-specific oncolytic adenovirus carrying PL gene (Ad-PL-PPT-E1A) and the corresponding replication-defective adenovirus (Ad-PL) were successfully constructed. Oncolytic adenoviruses were constructed by placing prostate cancerspecific promoter in front of the E1A gene, which is essentially for adenovirus replication. It has been reported that a potent prostate-specific regulatory sequence comprising the PSA enhancer (PSAe), the prostate-specific membrane antigen enhancer and the T-cell receptor γ-chain alternate reading frame promoter (named PPT) has transcriptionally active both in the presence and absence of testosterone.23 In our design, the PSAe in PPT was replaced by androgen response element enhancer core of PSAe.24 Our results demonstrated that oncolytic adenoviruses with or without immunogene PL, Ad-PL-PPT-E1A and Ad-PPT-E1A, could replicate efficiently in both androgen-dependent and -independent prostate cancer cells. However, no significant replication could be detected in human normal or tumor cells, suggesting their specific replication in prostate cancer. Efficient replication in prostate cancer cells was also confirmed in a PC3 cell xenografted nude mouse model. Upon efficient replication, redundant progeny © 2014 Macmillan Publishers Limited
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CD40L
PSA
SV40pA
E1A-E1Bp
E1B55Kplus
PPTp
TARPp
Abbreviations: CD40L, CD40 ligand; PPTp, prostate cancer-specific chimeric promoter; PSA, prostate-specific antigen enhancer; PSMAe, prostate-specific membrane antigen enhancer; TARPp, T-cell receptor γ-chain alternate reading frame protein.
EcoRI 549 NM_000074.2 192C>W Jurkat cell cDNA
793 NM_001648.2 LNCaPcDNA
160 — pShuttle-CMV
1172 AY339865.1 JM17
Self-anneal
AY339865.1
78
EcoRI Not I SalI NsiI EcoRI Spel Aat II Not I KpnI — —
720
— 273 NG_001336 K562 genome
— 285 AF007544 K562 genome
5′-CGGGGCGGCCGCGATGAAGATATTATCTTCATGATCTT-3′ 5′-GGAAAAAATAATTTTTCAAGGATGTTTGTAAAGCAGGC-3′ 5′-CAAACATCCTTGAAAAATTATTTTTTCCTTTAACCTTTCAAAC-3′ 5′-CCCTGGGCACTTAAAACAACAAAAAAATCTAGTTGTATAG-3′ 5′-GATTTTTTTGTTGTTTTAAGTGCCCAGGGCAAGGTGAGG-3′ 5′-GGGCGAATTCAGCTTTGTTCCGGGACCAAATACCTTG-3′ 5′-CGGGGCGGCCGCGATGAAGATATTATCTTCATGATCTT-3′ 5′-GGGCGAATTCAGCTTTGTTCCGGGACCAAATACCTTG-3′ 5′-GCGGTCGACGAGTTTTATAAAGGATAAATGGAGCGAAGAAACCCATCTGAGC-3′ 5′-CCGATGCATGGCCAGAAAATCCAGCAGGTACCCCCCGCTCAGATGGGTTTCT-3′ 5′-GCCGAATTCATGAGACATATTATCTGCCAC3′ 5′CGGACTAGTGAGGTCAGATGTAACCAAGAT-3′ 5′-GCCGACGTCCAATTGTTGTTGTTAACTTAACTTG3′ 5′-GCGGCGGCCGCGCGTTAAGATACATTG-3′ 5′-CGGCGGTACCATGTGGGTCCCGGTTGTCTT-3′ 5′-GGGGTTGGCCACGATGGTGTCC-3′ 5′-ATGCAAAAAGGTGATCAGAATCCTCAAATTG-3′ 5′-CCGCGA1TATCGGGCTTAACCGCTGTGCTGTA-3′ PSAe
The expression of fusion protein PL or replication-related protein E1A were determined by western blotting. Briefly, LNCaP cells were infected by
Table 1.
Gene expression assay
PCR parameters for the construction of pTE-PPT-E1A
Construction and rescue of adenoviral vectors Prostate cancer- specific chimeric promoter, containing androgen response element enhancer core of PSAe, prostate-specific membrane antigen enhancer and promoter of T-cell receptor γ-chain alternate reading frame protein (TARPp), was synthesized using the overlap extension PCR method. E1B55Kplus, E1A-E1Bp, prostate cancer-specific chimeric promoter and SV40pA were added to pTE-E1B55K sequentially to generate pTE-PPT-E1A. PSA gene and CD40L gene were amplified, respectively, and then linked by isoleucine zipper to generate fusion protein gene PSA-IZ-CD40L (PL). PL gene was inserted into the corresponding sites of pshuttle-cmv to generate a new plasmid pshuttle-PL. The 533 bp fragment between the two MfeI sites of pshuttle-PL was replaced by the corresponding 3936 bp fragment of pTE-PPT-E1A to generate pshuttle-PL-PPT-E1A. Oncolytic adenovirus Ad-PL-PPT-E1A was constructed and rescued according to the manual of AdEasy System. pShuttle-PL was replaced by pshuttle-cmv to prepare oncolytic adenovirus Ad-PPT-E1A. The replication-deficient adenovirus Ad-PL was also prepared. PCR-related information and corresponding restriction sites are listed in Table 1. All adenoviruses were purified by double CsCl density gradient ultracentrifugation, dissolved in storage buffer (Hank’s buffer, 10% glycerol). Viral particle (vp) numbers were calculated from measurements of optical density at 260 nm (OD260), where one absorbency unit is equivalent to 1.1 × 1012 vp ml − 1, and infectious titers (IU ml − 1) were determined by limiting dilution assay on 293 cells. The MOI was calculated from infectious titers.
219
Primers Fragment
Template
The human prostate cancer cell line PC3M was obtained from the Institute of Urology, Peking University (Beijing, China). The human vascular endothelial cell EC304, human prostate cancer cell LNCaP, PC3 and DU145 were obtained from the Institute of Basic Medicine, Chinese Academy of Medicine Science (Beijing, China). The normal human hepatic cell L02 and the human hepatocellular carcinoma cell SMMC-7721 were obtained from the Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The E1A-transformed human embryonic kidney 293 cell (CRL-1537), human lung epithelial carcinoma cell A549 and human hepatocellular carcinoma cell HepG2 (HB-8065) were all from the American Type Culture Collection (ATCC, Manassas, VA, USA). PC3, DU145 and PC3M are known to be androgen independent, whereas LNCaP cell is androgen dependent. DU145, PC3M, EC304, L02, HepG2, A549 and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. LNCaP and SMMC-7721 were maintained in RPMI1640 plus 10% fetal bovine serum, whereas PC3 was maintained in F12 plus 10% fetal bovine serum. All culture media and fetal bovine serum were obtained from Hyclone (Logan, UT, USA) and supplemented with 100 U ml − 1 penicillin, 100 μg ml − 1 streptomycin and 2 mM L-glutamine. All cells were maintained at 37 °C with 5% CO2 in a humidified incubator.
PSMAe
Cells
AF243527
GeneBank accession no.
AdEasy-1 backbone and pshuttle-CMV plasmids were obtained from Stratagene (La Jolla, CA, USA). Plasmid pTE-E1B55K and pJM13, which have been reported previously, were constructed in our laboratory.25 Ad-Null, a replication-defective adenovirus without any exogenous gene, is used as a negative control. Ad-CD80-TPE-GM, a telomerase-targeted oncolytic adenovirus, which has been reported previously, is used as a positive control.25
K562 genome
Restriction Length of PCR product (bp)
MATERIALS AND METHODS Plasmid and adenovirus
—
729 viruses were accumulated rapidly in prostate cancer cells, leading to apoptosis or cell death both in vitro and in vivo. In conclusion, we have successfully developed a prostate cancer-specific oncolytic adenovirus Ad-PL-PPT-E1A, which could mediate prostate cancer-specific oncolytic effect in vitro and in vivo and activate DCs in vitro. These results indicated that Ad-PLPPT-E1A might be an alternative approach for prostate cancer.
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730 50 MOI of Ad-PL-PPT-E1A, Ad-PPT-E1A, Ad-PL or Ad-Null and viruses were washed by phosphate-buffered saline (PBS) 2 h after infection. Cells were harvested at 48 h after infection. The protein was extracted and detected by using mouse anti-human PSA antibody, mouse anti-human CD40L antibody (Thermo Scientific, Wilmington, DE, USA) or anti-human type 5 adenovirus E1A antibody (Abcam, Cambridge, MA, USA). At different time points after infection, total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and cDNA was synthesized using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer’s instructions. Then, mRNA expression levels of PL and E1A were detected by PCR.
Cell viability assay Exponentially growing cells were seeded at a density of 2 × 104 cells per well in 96-well plates and infected by Ad-PL-PPT-E1A, Ad-PPT-E1A, Ad-PL, Ad-Null or Ad-CD80-TPE-GM at serial MOIs in 100 μl complete culture media. One hundred microliter fresh culture media were supplemented to each well 3 days after infection. At day 7 after infection, cells were fixed in 1% glutaraldehyde (v v − 1) for 2–3 h, and then stained with 0.1% crystal violet (w v − 1) for 1 h. After washing for three times, crystal violet dye was dissolved with 1% Triton X-100 (v v − 1). After 2 h rotary incubation at room temperature, the absorbance of the resulting Triton extract was read at 570 nm on a varioskan flask (Thermo Scientific). Cell survival rate is absorbance of tested well normalized by that of uninfected control well.
Apoptosis Exponentially growing DU145, PC3M or LNCaP cells were seeded at a density of 3 × 105 cells per well in 6-well plates and infected by serial MOIs of Ad-PL-PPT-E1A, Ad-PPT-E1A or Ad-PL. Twenty-four, 48 or 72 h after infection, total cells were harvested and analyzed with Annexin V-FITC Apoptosis Detection Kit (KeyGEN) according to the manufacturer’s instructions. Uninfected cells were used as control.
Virus replication in vitro Exponentially growing cells were seeded at a density of 3 × 105 cells per well in 6-well plates and infected by 50 MOI of Ad-PL-PPT-E1A or Ad-PL. Two hours later, viruses were removed by washing twice with PBS, and then 500 μl of fresh media was added to each well. Cells were collected for extraction of mRNA and genomo times DNA at 24, 48 or 72 h after infection. E1A expression was detected by using real-time PCR as described above. The genomic DNA was used to analyze production of viral particles by real-time PCR using the following primers: Ad5 (sense, 5′-TCTGA GTTGGCACCCCTATTC-3′ and antisense, 5′-GTTGCTGTGGTCGTTCTGGTA-3′); β-globin (sense, 5′-GTGCACCTGACTCCTGAGGAGA-3′ and antisense, 5′-CCTTGATACCAACCTGCCCAGG-3′). To test infectious titer, cells were infected by Ad-PL-PPT-E1A or Ad-CD80TPE-GM at 5 MOI as described above. Cells were collected at 24, 48 or 72 h after infection. Lysates were prepared by three cycles of freeze and thaw and used to determine virus titer by limiting dilution assay on 293 cells.
Generation of human monocyte-derived DCs Human PB samples used in this study were obtained from patients undergoing treatment for diagnostic purposes at Peking University First Hospital. Mononuclear cells (PB mononuclear cells) were isolated from heparinized samples by centrifugation through a Ficoll–Hypaque density gradient (Amersham Biosciences, Piscataway, NJ, USA). In total, 1 × 108 PB mononuclear cells were seed in a 100 mm plate (Corning Inc., Corning, NY, USA) with 10 ml AIM V Medium CTS (Gibco, Grand Island, NY, USA) at 37 °C. Two hours later, non-adherent cells were discarded and adherent cells were washed two times with prewarmed PBS. Then, cells were incubated with AIM V Medium supplemented with recombinant human granulocyte macrophage-colony-stimulating factor (50 ng ml − 1) and recombinant human IL-4 (20 ng ml − 1) (Peprotech, Rocky Hill, NJ, USA) for 5 days. On day 3, fresh complete medium was added.
Effect of fusion protein PL on DCs At day 5, the non-adherent cells were collected and seeded in 24-well plate at a density of 2 × 105 ml per well. The cells were induced by recombinant human TNF-α (25 ng ml − 1) (Peprotech) and the lysate (150 μg) of LNCaP cells were infected by Ad-PL-PPT-E1A, Ad-PL or Ad-Null. And, lipopolysaccharide (1 μg/ml) (Sigma Chemical Co., St Louis, MO, USA) was used as a positive control. Gene Therapy (2014) 723 – 731
Immunophenotype analysis. DCs were harvested 48 h later, and then were stained with fluorescein isothiocyanate-conjugated CD80, phycoerythrinconjugated CD83 and allophycocyanin-conjugated CD86 (BD Biosciences, San Jose, CA, USA). The immunophenotype was analyzed by using FACScan flow cytometer (BD Biosciences). Expression of cytokines. Total RNA was isolated from DCs 48 h after induction, and cDNA was synthesized as described above. mRNA expression of human IL-12a (hIL-12a), hIL-12b, hIL-6, hIL-23 and TNF-α were detected by real-time PCR.
Animal experiments All animal experiments were conducted according to protocols approved by the Institutional Animal Care and Use Committee of the Beijing Institute of Radiation Medicine. In total, 5 × 106 PC3M cells per 100 μl PBS were subcutaneously injected into the left flanks of male BALB/c nude mice. When tumors reached about 100 mm3 in volume, 18 mice were divided randomly into three groups, and then treated by Ad-PL-PPT-E1A, Ad-PPTE1A or Ad-Null, respectively. Ad-PL-PPT-E1A, Ad-PPT-E1A or Ad-Null (2 × 108 IU per 100 μl PBS) were daily injected intratumorally for 5 consecutive days. The total dose was 109 IU per mouse. Tumor was monitored regularly by measuring length and width and tumor volume was calculated with the following formula: width2 × length/2. The survival was observed every day, and the tumor volume was detected every 3–4 days. Mice were killed at day 23 posttreatment (day 0 was set as that the first intratumoral injection occurred).
Real-time PCR The mRNA expression was quantified using the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and SYBR Premix Ex Taq II (Perfect Real Time) (Takara, Shiga, Japan). The primers used were as follows: PL (sense, 5′-AGGCTGGGAGTGCGAGAA-3′ and antisense, 5′-TGAGGCGTAGCAGGTGGT-3′); E1A (sense, 5′-GCTGATAATCTTCCACCTCC-3′ and antisense, 5′-ACCCTCTTCATCCTCGTC-3′); hIL-12a (sense, 5′-TCCTC CCTTGAAGAACCG-3′ and antisense, 5′-AATAGTCACTGCCCGAAT-3′); hIL-12b (sense, 5′-CATCAGGGACATCATCAA-3′ and antisense, 5′-GTCAGGGAGA AGTAGGAA-3′; hIL-6 (sense, 5′-CATCCTCGACGGCATCTC-3′ and antisense, 5′-GCCTCTTTGCTGCTTTCA-3′); hIL-23 (sense, 5′-GTCTCCTTCTCCGCTTCA-3′ and antisense, 5′-TTAGGGACTCAGGGTTGC-3′; hTNF-α (sense, 5′-CATC GCCGTCTCCTACCA-3′ and antisense, 5′-AGTCGGTCACCCTTCTCC-3′; human β-actin (sense, 5′-GCGGGAAATCGTGCGTGAC-3′ and antisense, 5′-GGAAGGAAGGCTGGAAGAG-3′). And, the expression levels were normalized by human β-actin.
Statistical analysis Values were presented as mean ± s.d. or mean ± s.e.m. For tumor growth curve, two-way analysis of variance was used, followed by the Dunnett's post hoc test. For other data, one-way analysis of variance was used to compare the means of two or more experimental groups, followed by the Dunnett's post hoc test. Statistical differences between groups were considered to be significant at P o0.05.
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This work was supported by grants from the National High Technology Research and Development Program of China (863 Program) (Nos. 2012AA020807 and 2014AA020515), the National Basic Research and Development of China (973 Program) (No. 2012CB518205) and the National Natural Science Foundation of China (No. 30901379).
REFERENCES 1 Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T et al. Cancer statistics, 2008. CA Cancer J Clin 2008; 58: 71–96. 2 Cannata DH, Kirschenbaum A, Levine AC. Androgen deprivation therapy as primary treatment for prostate cancer. J Clin Endocrinol Metab 2012; 97: 360–365.
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731 3 Sturge J, Caley MP, Waxman J. Bone metastasis in prostate cancer: emerging therapeutic strategies. Nat Rev Clin Oncol 2011; 8: 357–368. 4 Banchereau J, Paczesny S, Blanco P, Bennett L, Pascual V, Fay J et al. Dendritic cells: controllers of the immune system and a new promise for immunotherapy. Ann NY Acad Sci 2003; 987: 180–187. 5 O'Neill DW, Adams S, Bhardwaj N. Manipulating dendritic cell biology for the active immunotherapy of cancer. Blood 2004; 104: 2235–2246. 6 Elgueta R, Benson MJ, de Vries VC, Wasiuk A, Guo Y, Noelle RJ. Molecular mechanism and function of CD40/CD40L engagement in the immune system. Immunol Rev 2009; 229: 152–172. 7 Xu Y, Song G. The role of CD40-CD154 interaction in cell immunoregulation. J Biomed Sci 2004; 11: 426–438. 8 Hildenbrand B, Sauer B, Kalis O, Stoll C, Freudenberg MA, Niedermann G et al. Immunotherapy of patients with hormone-refractory prostate carcinoma pretreated with interferon-gamma and vaccinated with autologous PSA-peptide loaded dendritic cells—a pilot study. Prostate 2007; 67: 500–508. 9 Williams BJ, Bhatia S, Adams LK, Boling S, Carroll JL, Li XL et al. Dendritic cell based PSMA immunotherapy for prostate cancer using a CD40-targeted adenovirus vector. PLoS One 2012; 7: e46981. 10 Dzojic H, Loskog A, Tötterman TH, Essand M. Adenovirus-mediated CD40 ligand therapy induces tumor cell apoptosis and systemic immunity in the TRAMP-C2 mouse prostate cancer model. Prostate 2006; 66: 831–838. 11 von Euler H, Sadeghi A, Carlsson B, Rivera P, Loskog A, Segall T et al. Efficient adenovector CD40 ligand immunotherapy of canine malignant melanoma. J Immunother 2008; 31: 377–384. 12 Xu W, Li Y, Wang X, Wang C, Zhao W, Wu J. Anti-tumor activity of gene transfer of the membrane-stable CD40L mutant into lung cancer cells. Int J Oncol 2010; 37: 935–941. 13 Zhang L, Tang Y, Akbulut H, Zelterman D, Linton PJ, Deisseroth AB. An adenoviral vector cancer vaccine that delivers a tumor-associated antigen/CD40-ligand fusion protein to dendritic cells. Proc Natl Acad Sci USA 2003; 100: 15101–15106. 14 Toth K, Dhar D, Wold WS. Oncolytic (replication-competent) adenoviruses as anticancer agents. Expert Opin Biol Ther 2010; 10: 353–368.
15 Freytag SO, Stricker H, Movsas B, Kim JH. Prostate cancer gene therapy clinical trials. Mol Ther 2007; 15: 1042–1052. 16 Tuve S, Liu Y, Tragoolpua K, Jacobs JD, Yumul RC, Li ZY et al. In situ adenovirus vaccination engages T effector cells against cancer. Vaccine 2009; 27: 4225–4239. 17 Alemany R. A smart move against cancer for vaccinia virus. Lancet Oncol 2008; 9: 507–508. 18 Cerullo V, Pesonen S, Diaconu I, Escutenaire S, Arstila PT, Ugolini M et al. Oncolytic adenovirus coding for granulocyte macrophage colony-stimulating factor induces antitumoral immunity in cancer patients. Cancer Res 2010; 70: 4297–4309. 19 Tokmadžić VS, Tomaš MI, Sotošek S, Laškarin G, Dominović M, Tulić V et al. Different perforin expression in peripheral Blood and prostate tissue in patients with benign prostatic hyperplasia and prostate cancer. Scand J Immunol 2011; 74: 368–376. 20 Nencioni A, Grünebach F, Schmidt SM, Müller MR, Boy D, Patrone F et al. The use of dendritic cells in cancer immunotherapy. Crit Rev Oncol Hematol 2008; 65: 191–199. 21 Aalamian M, Pirtskhalaishvili G, Nunez A, Esche C, Shurin GV, Huland E et al. Human prostate cancer regulates generation and maturation of monocyte derived dendritic cells. Prostate 2001; 46: 68–75. 22 Pirtskhalaishvili G, Shurin GV, Esche C, Cai Q, Salup RR, Bykovskaia SN et al. Cytokine mediated protection of human dendritic cells from prostate cancer induced apoptosis is regulated by the Bcl-2 family of proteins. Br J Cancer 2000; 83: 506–513. 23 Cheng WS, Dzojic H, Nilsson B, Tötterman TH, Essand M. An oncolytic conditionally replicating adenovirus for hormone-dependent and hormoneindependent prostate cancer. Cancer Gene Ther 2006; 13: 13–20. 24 Lee SJ, Kim HS, Yu R, Lee K, Gardner TA, Jung C et al. Novel prostate-specific promoter derived from PSA and PSMA enhancers. Mol Ther 2002; 6: 415–421. 25 Hu ZB, Wu CT, Wang H, Zhang QW, Wang L, Wang RL et al. A simplified system for generating oncolytic adenovirus vector carrying one or two transgenes. Cancer Gene Ther 2008; 15: 173–182.
Supplementary Information accompanies this paper on Gene Therapy website (http://www.nature.com/gt)
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