Potent growth-inhibitory effect of a dual cancer-specific oncolytic adenovirus expressing apoptin on prostate carcinoma

  • Authors:
    • Muchun Zhang
    • Jinhui Wang
    • Chang Li
    • Ningning Hu
    • Kai Wang
    • Huifan Ji
    • Dongyun He
    • Chengshi Quan
    • Xiao Li
    • Ningyi Jin
    • Yulin Li
  • View Affiliations

  • Published online on: January 21, 2013     https://doi.org/10.3892/ijo.2013.1783
  • Pages: 1052-1060
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Abstract

Apoptin is a chicken anemia virus-derived, p53-independent, bcl-2-insensitive apoptotic protein with the ability to specifically induce apoptosis in various human tumor cells, but not in normal cells. To explore the use of apoptin in tumor gene therapy, we assessed a recombinant adenovirus expressing the apoptin protein (Ad-hTERTp-E1a-Apoptin) in order to determine its lethal and growth-inhibitory effects on PC-3 and RM-1 cells in vitro and its antitumor effect on solid tumors in vivo. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), acridine orange (AO)/ethidium bromide (EB), 4'-6-diamidino-2-phenylindole (DAPI), and Annexin V assays showed that Ad-hTERTp-E1a-Apoptin inhibited the proliferation of PC-3 and RM-1 cells in vitro by inducing apoptosis of prostate cancer cells, and that this inhibitory effect was dose and time-dependent. In the animal models, Ad-hTERTp-E1a-Apoptin significantly inhibited tumor growth and extended the lifespan of animals. Experimental results indicate that Ad-hTERTp-E1a-Apoptin has a potential application in tumor gene therapy.

Introduction

Prostate cancer is the most common cancer and the second leading cause of cancer-related deaths in men in the US (1). It is estimated that 217,730 new cases of prostate cancer were diagnosed in 2010 alone (2). The therapeutic options for patients with prostate cancer include surgery, radiotherapy and chemotherapy with cytotoxic agents. Despite a palliative benefit, these approaches do not engender a long-term beneficial effect on the overall survival of patients (3). In this context, there is a pressing need to develop more effective therapeutic approaches for end-stage prostate cancer patients and genetic therapies represent promising approaches for the treatment of this neoplasm (4).

Adenovirus-based vectors are the most widely used cancer gene delivery platforms (5); however, specificity and efficacy are major challenges for this therapeutic strategy (5). Of the existing adenovirus technologies, the utility of conditional replication-competent adenoviruses (CRCAs) provides an optimum approach. In our previous studies, using the RAPAd.I system, we constructed a dual-specific antitumor CRCA, designated Ad-hTERTp-E1a-Apoptin, incorporating the tumor-specific promoter hTERTp and the specific antitumor gene apoptin (6). This CRCA has the ability of both tumor-specific growth inhibition and tumor-specific replication. Further investigation showed that Ad-hTERTp-E1a-Apoptin had a significantly greater antitumor activity than replication-defective adenoviruses (Ad-CMV-Apoptin and Ad-CMV-EGFP) (5).

Apoptosis is frequently impaired in many human tumors, and is also an important mechanism in chemotherapy-induced tumor cell death. Therefore, the modulation of apoptosis by targeting pro-apoptotic and anti-apoptotic proteins may be a powerful and effective method for treating cancer (5). Apoptin, a protein derived from chicken anemia virus (CAV), selectively induces apoptosis in a wide variety of transformed cells, but not in primary cells (69).

In this study, we used a recombinant adenovirus expressing the CAV apoptin (Ad-hTERTp-E1a-Apoptin) to infect prostatic carcinoma PC-3 and RM-1 cells, and prostatic carcinoma models with RM-1 cells in C57BL/6 mice. We then tested the lethality and effects of Ad-hTERTp-E1a-Apoptin on PC-3 and RM-1 cells in vitro and investigated the antitumor effect of Ad-hTERTp-E1a-Apoptin on solid tumors in vivo. Our study provided a new strategy for research on gene therapy in prostatic carcinoma.

Materials and methods

Materials

The human prostate cell line PC-3, and the murine cell line RM-1, were obtained from the Cell Bank of Type Culture Collection, Chinese Academy of Sciences, Shanghai, China. Fetal bovine serum, Dulbecco’s modified Eagle’s medium (DMEM), and Roswell Park Memorial Institute medium 1640 (RPMI-1640) were bought from Gibco, USA; 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), ethidium bromide (EB), and acridine orange (AO), from Sigma, USA; and 4′-6-diamidino-2-phenylindole (DAPI) and Annexin V apoptosis assay, from BioVision, USA; six-week-old C57BL/6 mice were obtained from the Laboratory Animal Center of the Academy of Military Medical Sciences, Beijing, China. The recombinant adenoviruses Ad-hTERTp-E1a-Apoptin, Ad-hTERTp-E1a-EGFP, Ad-CMV-Apoptin, and Ad-CMV-EGFP were constructed and saved in our laboratory (5) (Fig. 1).

Cell culture and viral infection

We incubated 5×105 PC-3 cells in RPMI-1640 and RM-1 cells in DMEM at 37°C in 5% CO2; both culture media were supplemented with 10% fetal bovine serum to form complete media. Recombinant adenoviruses were diluted to 5×107 pfu/ml with either DMEM or antibiotic. Infection was performed at a multiplicity of infection (MOI) of 100. The diluted recombinant adenoviruses were inoculated on the cell cultures and maintained at 37°C in 5% CO2 for 4 h and then added to their respective complete medium, in which cultivation continued for 48 h.

MTT colorimetric assay

PC-3 and RM-1 cells were seeded in 96-well plates (5×103 cells/well) and infected with various concentrations (1, 10 and 100 MOI) of recombinant adeno-viruses. Viability was measured after 12, 24, 36, 48, 60, 72, 84 and 96 h by treating cells with 20 μl/well MTT (5 mg/ml) and incubating at 37°C in 5% CO2 for 4 h. The culture media were removed, and the crystals formed were dissolved by adding 150 μl/well dimethylsulfoxide. Untreated PC-3 and RM-1 cells were used as controls and all measurements were performed in triplicate. The absorbance at 490 nm (A) was measured; untreated cells were used as controls. The percent cell survival was expressed using the following formula: (average absorbance value of control well - average absorbance value of the experimental well)/average absorbance value of control well (5).

AO/EB staining

After a 48-h incubation period, recombinant adenovirus-infected PC-3 and RM-1 cells (1×106 cells; MOI 100) were trypsinized, washed 2 times in phosphate-buffered saline (PBS), and the cell pellet obtained was resuspended in PBS. To the resuspended solution, we added 2 μl AO/EB solution (AO, 100 μg/ml; EB, 100 μg/ml; dissolved in PBS) and vortexed the resulting sample. Next, 20 μl of the sample was placed on a microscope slide with a cover slip, and images of representative cells were obtained with a digital video camera connected to the 100X objective lens of a fluorescence microscope. The images from the microscope were processed with the Image-Pro Plus (IPP, version 5.0.2) software program.

Annexin V apoptosis assay

After a 48-h incubation period, recombinant adenovirus-infected PC-3 and RM-1 cells (1×106 cells; MOI, 100) were trypsinized, washed once in PBS, and the cell pellet obtained was resuspended in 200 μl binding buffer. To the resuspended solution, 2 μl fluorescein isothiocyanate (FITC)-labeled Annexin V and 2 μl propidium iodide (PI) were added. The resulting mixtures were incubated in the dark for 5 min at room temperature and examined under a laser scanning confocal microscope.

DAPI staining

After a 48-h incubation period, recombinant adenovirus-infected PC-3 and RM-1 cells (1×106 cells; 100 MOI) were trypsinized and washed once in PBS as above. The cell pellet obtained was resuspended in 200 μl 25% glutaraldehyde, washed 3 times in PBS, resuspended again in 200 μl 100 ng/ml DAPI and a 20 μl aliquot of the resulting solution was placed on a microscope slide, coverslipped and imaged as described previously.

Animal experiments

RM-1 cells were harvested by trypsinization and resuspended in serum-free DMEM after being washed with PBS. The cell concentration was adjusted to 5×107 cells/ml. Within 1 h of harvesting, 100 μl of cell suspension was injected either subcutaneously into the right flank or into the caudal vein of C57BL/6 mice. When the resulting tumors reached a diameter of 2–5 mm (8 days), the mice were randomly divided into six groups of five mice each. Each mouse in five of these groups received treatment consisting of a single intratumoral and a caudal vein injection; the treatments were repeated two times a week for three weeks and then changed to once a week for three weeks. The five treatments used were 100 μl injections of: i) Ad-hTERT-E1a-Apoptin alone (1011 pfu/mouse in saline), ii) Ad-CMV-Apoptin (1011 pfu/mouse in saline), iii) Ad-hTERT-E1a-EGFP (1011 pfu/mouse in saline), iv) Ad-CMV-EGFP (1011 pfu/mouse in saline) and v) saline. The mice in the sixth group were untreated and served as controls. Tumor size was measured using calipers every 2 days. Tumor volumes were calculated as follows: [0.52 (smallest diameter of tumor)2 (largest diameter of tumor)] (9). After 63 days, all mice were sacrificed and their cumulative survival was calculated.

Statistical analysis

The statistical significance of differences was determined using one-way analysis of variance (ANOVA), and statistical significance was accepted as P<0.05. Log-rank tests were used for survival analysis. Data from all animals are presented in Kaplan-Meier plots.

Results

Lethal effect of Ad-hTERT-E1a-Apoptin on PC-3 and RM-1 cells in vitro

Cell viability was assessed using the MTT colorimetric assay. MTT is taken up into cells by endocytosis or by a protein-facilitated mechanism and reduced, mainly by mitochondrial enzymes, to yield a purple formazan product, which is largely impermeable to cell membranes and therefore accumulates within living cells. Solubilization of the cells liberates the purple product, which can be detected using a colorimetric measurement. The ability of cells to reduce MTT provides an indication of mitochondrial integrity and activity, which in turn may be interpreted as a measure of cell number/proliferation/viability/survival/toxicity (10).

As shown in Fig. 2, with longer infection times, the growth of PC-3 and RM-1 cells infected with Ad-CMV-Apoptin, Ad-hTERT-E1a-EGFP, Ad-CMV-EGFP and Ad-hTERT-E1a-Apoptin was inhibited. However, cells infected with replication-incompetent adenoviruses (Ad-CMV-Apoptin and Ad-CMV-EGFP) gradually resumed their growth after 48 h. In contrast, Ad-hTERT-E1a-Apoptin and Ad-hTERT-E1a-EGFP were more effective in inhibiting cell growth. Cell viability depended on the MOI of the recombinant adeno-viruses to some extent. There was no significant difference in the growth of cells at different infection doses in the first 36 h (P>0.05). In contrast, after 48 h, the 100 MOI group showed significantly increased inhibition compared with the 1 MOI and 10 MOI groups. In the 100 MOI group, obvious suppression was seen after 24 h (P<0.05). With longer infection times, both Ad-hTERT-E1a-Apoptin and Ad-hTERT-E1a-EGFP were more effective in inhibiting cell growth, but the former was more effective than the latter. In addition, Ad-CMV-Apoptin was more effective than Ad-CMV-EGFP. In PC-3 and RM-1 cells, infection with Ad-CMV-Apoptin at a MOI of 10 or 100 inhibited cell growth by 30-35% after 4 days. Infection with 1 MOI or 10 MOI of Ad-hTERT-E1a-EGFP and Ad-hTERT-E1a-Apoptin inhibited cell growth by 20–30 and 40–50% after 4 days, respectively and that with 100 MOI almost blocked cell growth (60–70%). Ad-CMV-EGFP, however, did not significantly inhibit cell growth. In conclusion, Ad-hTERT-E1a-Apoptin effectively restricts the growth of cultured PC-3 and RM-1 cells. The interaction between infection time and MOI was complex and synergistic and cell viability showed a non-rigorous dependent relationship with both factors. Therefore, we performed the following in vitro experiments 48 h after infection at 100 MOI.

Morphological changes in the recombinant adenovirus-infected PC-3 and RM-1 cells

For the analysis of cell death, we used fluorescent assays of AO/EB double staining. AO is taken up by both viable and non-viable cells, which emit green fluorescence if the dye is intercalated into double-stranded nucleic acid (DNA) and red fluorescence if it is bound to single-stranded nucleic acid (RNA). EB is taken up by only non-viable cells, which emit red fluorescence because of dye intercalation into DNA (11).

Chromatin condensation, nuclear fragmentation and membrane destruction are the hallmarks of apoptotic cells (8). Using AO/EB staining, we analyzed the effects of Ad-hTERTp-E1a-Apoptin and Ad-CMV-Apoptin infections on the nuclear and the membranes of PC-3 and RM-1 cells. As shown in Fig. 3A, normal cell membranes of PC-3 and RM-1 cells were intact and stained bright green with AO. Loss of cytoplasmic membrane integrity resulted in the uptake of EB by Ad-hTERT-E1a-Apoptin- and Ad-CMV-Apoptin-infected PC-3 and RM-1 cells, with orange EB-stained cells dominating over bright green AO-stained cells. Using the AO/EB method, we also quantified the percentage of live, necrotic and apoptotic cells after Ad-hTERT-E1a-Apoptin and Ad-CMV-Apoptin treatment (Fig. 3B). Infection with Ad-CMV-Apoptin was slightly cytotoxic and the main change in morphology indicated apoptosis more than necrosis (Fig. 3B, middle panels). In contrast, infection with Ad-hTERT-E1a-Apoptin was strongly cytotoxic and apoptosis occurred very quickly, so that the main change in morphology was necrosis rather than apoptosis (Fig. 3B, right panels).

Ability of Ad-hTERT-E1a-Apoptin to induce tumor-specific apoptosis

This assay is based on the ability of the protein Annexin V to bind to phosphatidylserine (PS) exposed on the outer membrane leaflet of apoptotic cells (PS also appears on the necrotic cell surface). In viable cells, PS is located in the inner membrane leaflet, but upon induction of apoptosis, it is translocated to the outer membrane leaflet and becomes available for Annexin V binding. The addition of phosphatidylinositol (PI) enabled viable apoptotic cells to be distinguished from necrotic cells (12).

PC-3 and RM-1 cells infected with Ad-hTERT-E1a-Apoptin and Ad-CMV-Apoptin were stained with Annexin V-FITC/PI and observed under a laser scanning confocal microscope. Ad-hTERT-E1a-Apoptin- and Ad-CMV-Apoptin-infected cells displayed red fluorescence and fragmented chromatin when stained with PI (Fig. 4A, left panels) and green fluorescence when stained with Annexin V-FITC (Fig. 4A, middle panels). The green fluorescence was mainly concentrated in the cell membrane, a characteristic of phospholipid membranes valgus. PC-3 and RM-1 cells stained with Annexin V-FITC/PI showed a red nucleus (PI) and a halo of green (FITC) on the cell surface, which are indicative of phospholipid membranes valgus and fragmented chromatin. These results indicated that Ad-hTERT-E1a-Apoptin and Ad-CMV-Apoptin induced apoptosis in PC-3 and RM-1 cells.

The blue fluorescent DAPI nucleic acid stain preferentially stains double-stranded DNA (dsDNA). The stain appears to associate with A/T clusters in the minor groove. Binding of DAPI to dsDNA produces ∼20-fold fluorescence enhancement, apparently due to the displacement of water molecules from both DAPI and the minor groove. DAPI also binds RNA but through a different mechanism, which is thought to involve A/U-selective intercalation. The DAPI/RNA complex exhibits a longer-wavelength fluorescence emission maximum than the DAPI/dsDNA complex (∼500 vs. ∼460 nm) and a quantum yield that is only ∼20% as high (13).

PC-3 and RM-1 cells infected with Ad-hTERT-E1a-Apoptin and Ad-CMV-Apoptin were stained with DAPI and observed under a laser scanning confocal microscope. The nuclei of uninfected cells (controls) showed a uniform blue fluorescence and were structurally normal, while those of Ad-hTERT-E1a-Apoptin- and Ad-CMV-Apoptin-infected cells displayed light blue fluorescence and condensed and fragmented chromatin (Fig. 4B). These results indicated that Ad-hTERT-E1a-Apoptin and Ad-CMV-Apoptin induced apoptosis in PC-3 and RM-1 cells.

Antitumor effect of Ad-hTERT-E1a-Apoptin in vivo

We next examined the antitumor potential of Ad-hTERT-E1a-Apoptin in the RM-1 tumor model. The growth kinetics of the tumors treated with intratumoral injections are shown in Fig. 5A. Compared with the saline control and Ad-CMV-EGFP groups, the recombinant adenovirus groups showed suppression of tumor growth; this effect was seen after the first three injections and continued up to the end of the treatment period. However, soon after the last injection, tumor growth gradually resumed in the recombinant virus groups, but was slowest in the Ad-hTERT-E1a-Apoptin and Ad-hTERT-E1a-EGFP groups. The growth kinetics of the tumors treated with intravenous injections is shown in Fig. 5B. Compared with the tumors in the saline controls and Ad-CMV-EGFP-infected groups, those in the recombinant adenovirus groups were suppressed after the first three injections. Tumor suppression continued up to the end of the treatment period. However, soon after the last injection, the Ad-CMV-Apoptin- and Ad-CMV-EGFP-infected tumors gradually resumed their growth. Most of the Ad-hTERT-E1a-Apoptin- and Ad-hTERT-E1a-EGFP-infected tumors also resumed growth, but these grew more slowly. The tumors in the intravenous injection groups grew more rapidly than those in the intratumoral injection groups. The main cause of this difference may be the faster effect of direct intratumoral injection than of intravenous injection. We also evaluated the ability of the recombinant adenoviruses to prolong the survival of the tumor-bearing mice (Fig. 5C). All saline-, Ad-CMV-Apoptin- and Ad-CMV-EGFP-treated animals died between 26 and 52 days after intratumoral injection. In contrast, 60 and 80% of Ad-hTERT-E1a-EGFP- and Ad-hTERT-E1a-Apoptin-infected animals, respectively, were still alive at that point (Fig. 5C). Mouse survival analysis showed that Ad-hTERT-E1a-Apoptin and Ad-hTERT-E1a-EGFP treatments significantly increased mouse survival in the RM-1 tumor model in comparison with the other recombinant adenovirus treatments and saline treatment (Fig. 5D).

When the experiment was terminated on day 63, 80% of Ad-hTERT-E1a-Apoptin- and Ad-hTERT-E1a-EGFP-infected animals were alive and the median survival time did not differ significantly between these two groups. None of the mice in the other groups were alive at the end of the experiment. In conclusion, inoculation with Ad-hTERT-E1a-Apoptin had significant survival benefits and reduced tumor size in vivo.

Pathological examination

In the Ad-hTERT-E1a-Apoptin- and Ad-hTERT-E1a-EGFP-treated groups, tumors lost tissue integrity and showed increased intercellular spaces containing remnants of disintegrating cells (Fig. 6A). None of these changes were seen in the Ad-CMV-EGFP-treated, Ad-CMV-Apoptin-treated, and saline control groups. These results indicated that Ad-hTERT-E1a-Apoptin had anti-tumor effects on solid tumors. This recombinant adenovirus also significantly decreased the tumor burden of the mice. Ad-hTERT-E1a-Apoptin-infected tumors showed minimal metastatic nodules, unlike the other treatment groups and the control group, which showed severe metastasis. Taken together, the systemic delivery of Ad-hTERT-E1a-Apoptin significantly reduced tumor burden and provided survival benefits in the RM-1 tumor model.

Discussion

Like many cancers, prostate cancer is a complex disease, and different types of therapeutic strategies are required to demonstrate a benefit in a particular patient cohort. Most of the ∼29,000 men who succumb to prostate cancer each year in the United States die of metastatic disease, and this highlights the need for better systemic therapies (14). In recent years, with the development of molecular biology, immunology and other related subjects, gene therapy has gradually emerged as a novel antitumor treatment that has a huge advantage (14). Viral vectors are used in the field of gene therapy for their simplicity, stability, ease of operation, efficient capacity and safety without integration and have increasingly attracted the attention and favor of researchers.

Apoptin has gained significant attention in recent years, both as a lead for the development of cancer-specific therapeutics and for its potential use as an indicator of cellular transformation processes (15). Apoptin is a 13.6 kDa viral protein encoded by the VP3 gene of chicken anemia virus and is composed of 121 amino acids (15,16). Because of its small size, the apoptin gene can be inserted into various vectors such as parvoviruses, papovaviruses and adenoviruses (1720). It induces apoptosis independently of death receptor pathways in a broad range of transformed and cancer cells. Apoptin localizes in the nucleus in cancer cells; however, in non-transformed or primary cells, it is localized to the cytoplasm (2022). The cellular localization of apoptin is influenced by its phosphorylation status at threonine-108. Phosphorylated T-108 inhibits nearby nuclear export signals, leading to nuclear accumulation of apoptin (7,2325). Apoptin phosphorylation has been proposed to be regulated by Akt-activated cyclin-dependent kinase (CDK)-2 and protein kinase C (PKC) (2628). Thus, nuclear localization of apoptin and its interaction with specific signaling proteins plays a crucial role in its selective toxicity (6,8,25). Furthermore, apoptin does not induce apoptosis in normal, non-transformed cells such as fibroblasts, keratinocytes, or smooth muscle cells (7).

The hTERT promoter displays high activity in a majority of human cancers but not in most host tissues (29,30) and is considered a good tumor-specific regulator for oncolytic adenoviruses (31). The hTERT promoter can be used to control viral regulatory genes, such as adenoviral E1A, to restrict the replication of oncolytic adenoviruses to malignant cells and tissues. Dual-specificity adenoviral promoters that regulate E1A expression in response to multiple stimuli, e.g., estrogens and hypoxia, have also been described (32). The cancer-specific promoter hTERT can both confer tumor-specific replication and regulate E1A expression and several tumor cell-replicating, hTERT-driven adenoviruses have been described (31,33). However, none of these viruses combines both promoter elements into a single virus to regulate E1A expression and viral replication. As hTERT is expressed in >90% of cancers (27,28), an oncolytic virus that combines both of these features has the potential to induce oncolytic activity across a broad range of human tumors and tumor cell populations. Cancer gene therapy based on oncolytic adenoviruses has been widely studied in pre-clinical and clinical trials in recent years. In our previous studies, using the RAPAd.I system, we constructed the CRCA Ad-hTERT-E1a-Apoptin incorporating hTERTp and the specific antitumor gene apoptin, which demonstrated tumor-specific growth inhibition (5).

In this study, we described the generation of a recombinant adenovirus vector expressing apoptin and its effects on PC-3 and RM-1 cells in vitro and in vivo based on its tumor-specific apoptosis-inducing activity. MTT assays indicated that infection with Ad-hTERT-E1a-Apoptin at 100 MOI significantly inhibited the growth of PC-3 and RM-1 cells after 48 h and that the inhibitory effect of Ad-hTERT-E1a-Apoptin was dose- and time-dependent. Infections at 1 or 10 MOI had less effective growth-inhibitory effects. These data indicated that the growth inhibition of PC-3 and RM-1 cells is related to the MOI of Ad-hTERT-E1a-Apoptin and the time period after transduction. In contrast, Ad-CMV-Apoptin- and Ad-CMV-EGFP-infected tumor cells resumed proliferation after 48-h treatment at all MOI doses tested. AO/EB, DAPI, and Annexin V assays indicated that Ad-hTERT-E1a-Apoptin could suppress the growth of PC-3 and RM-1 cells through the induction of apoptosis. Consistent with the MTT assay, the AO/EB, DAPI and Annexin V staining assays demonstrated that Ad-hTERT-E1a-Apoptin and Ad-CMV-Apoptin had the most significant growth-inhibitory effect on PC-3 and RM-1 cells and that Ad-hTERT-E1a-Apoptin was significantly stronger than Ad-CMV-Apoptin.

Analysis of survival and growth tendency of tumors in animal models showed that the tumors in the Ad-hTERT-E1a-Apoptin and Ad-hTERT-E1a-EGFP groups grew more slowly than those in the other groups. All saline-, Ad-CMV-Apoptin- and Ad-CMV-EGFP-treated animals died between 26 and 52 days after the last injection, whereas Ad-hTERT-E1a-Apoptin- and Ad-hTERT-E1a-EGFP-treated mice were still alive at this time point, indicating that Ad-hTERT-E1a-Apoptin could significantly extend the lifespan of animals. Moreover, the tumor size in the Ad-CMV-Apoptin-, Ad-CMV-EGFP-, and saline-treated groups was significantly greater than that in the Ad-hTERT-E1a-Apoptin- and Ad-hTERT-E1a-EGFP-treated groups, indicating that Ad-hTERT-E1a-Apoptin could suppress tumor growth in animal models. In conclusion, Ad-hTERT-E1a-Apoptin was able to inhibit the growth of tumor cells, extend the lifespan of animals and improve survival and quality of life in animal models and has a potential application in tumor gene therapy.

Taken together, gene therapy with apoptin offers unique advantages over current approaches for cancer therapy. The dual-specific recombinant adenovirus Ad-hTERT-E1a-Apoptin induced significant apoptosis of PC-3 and RM-1 cells. The unique action of the Ad-hTERT-E1a-Apoptin may provide a novel and promising candidate for cancer gene therapy in clinical trials for prostate cancer.

Acknowledgements

This study was supported in part by The National Science and Technology Major Projects for ‘Major New Drugs Innovation and Development’ (no. 2010ZX09401-305-14), The National Natural Science Foundation of China (nos. 81072210 and 81101140) and the Key Technologies R&D Programme of Jilin Province (nos. 10ZDGG007, 201015166 and 201101066).

References

1 

Damber JE and Aus G: Prostate cancer. Lancet. 371:1710–1721. 2008. View Article : Google Scholar : PubMed/NCBI

2 

Burton AJ, Tilling KM, Holly JM, Hamdy FC, Rowlands MA, Donovan JL and Martin RM: Metabolic imbalance and prostate cancer progression. Int J Mol Epidemiol Genet. 1:248–271. 2010.PubMed/NCBI

3 

Di Lorenzo G and De Placido S: Hormone refractory prostate cancer (HRPC): present and future approaches of therapy. Int J Immunopathol Pharmacol. 19:11–34. 2006.PubMed/NCBI

4 

Dash R, Azab B, Shen XN, Sokhi UK, Sarkar S, Su ZZ, Wang XY, Claudio PP, Dent P, Dmitriev IP, Curiel DT, Grant S, Sarkar D and Fisher PB: Developing an effective gene therapy for prostate cancer: new technologies with potential to translate from the laboratory into the clinic. Discov Med. 11:46–56. 2011.PubMed/NCBI

5 

Hu W and Kavanagh JJ: Anticancer therapy targeting the apoptotic pathway. Lancet Oncol. 4:721–729. 2003. View Article : Google Scholar : PubMed/NCBI

6 

Danen-Van Oorschot AA, Zhang YH, Leliveld SR, Rohn JL, Seelen MC, Bolk MW, Van Zon A, Erkeland SJ, Abrahams JP, Mumberg D and Noteborn MH: Importance of nuclear localization of apoptin for tumor-specific induction of apoptosis. J Biol Chem. 278:27729–27736. 2003.PubMed/NCBI

7 

Oro C and Jans DA: The tumour specific pro-apoptotic factor apoptin (Vp3) from chicken anaemia virus. Curr Drug Targets. 5:179–190. 2004. View Article : Google Scholar : PubMed/NCBI

8 

Poon IK, Oro C, Dias MM, Zhang J and Jans DA: Apoptin nuclear accumulation is modulated by a CRM1-recognized nuclear export signal that is active in normal but not in tumor cells. Cancer Res. 65:7059–7064. 2005. View Article : Google Scholar : PubMed/NCBI

9 

Li X, Jin N, Mi Z, Lian H, Sun L and Zheng H: Antitumor effects of a recombinant fowlpox virus expressing Apoptin in vivo and in vitro. Int J Cancer. 119:2948–2957. 2006. View Article : Google Scholar : PubMed/NCBI

10 

Maioli E, Torricelli C, Fortino V, Carlucci F, Tommassini V and Pacini A: Critical appraisal of the MTT assay in the presence of rottlerin and uncouplers. Biol Proced Online. 11:227–240. 2009. View Article : Google Scholar : PubMed/NCBI

11 

Mitrovic T, Stamenkovic S, Cvetkovic V, Tosic S, Stankovic M, Radojevic I, Stefanovic O, Comic L, Dacic D, Curcic M and Markovic S: Antioxidant, antimicrobial and antiproliferative activities of five lichen species. Int J Mol Sci. 12:5428–5448. 2011. View Article : Google Scholar : PubMed/NCBI

12 

Baskic D, Popovic S, Ristic P and Arsenijevic NN: Analysis of cycloheximide-induced apoptosis in human leukocytes: fluorescence microscopy using Annexin V/propidium iodide versus acridin orange/ethidium bromide. Cell Biol Int. 30:924–932. 2006. View Article : Google Scholar

13 

Ding W, Ju S, Jiang S, Zhu L, Wang Y and Wang H: Reduced APRIL expression induces cellular senescence via a HSPG-dependent pathway. Pathol Oncol Res. 15:693–701. 2009. View Article : Google Scholar : PubMed/NCBI

14 

Candal E, Anadon R, DeGrip WJ and Rodriguez-Moldes I: Patterns of cell proliferation and cell death in the developing retina and optic tectum of the brown trout. Brain Res Dev Brain Res. 154:101–119. 2005. View Article : Google Scholar : PubMed/NCBI

15 

Panigrahi S, Stetefeld J, Jangamreddy JR, Mandal S, Mandal SK and Los M: Modeling of molecular interaction between apoptin, BCR-Abl and CrkL - an alternative approach to conventional rational drug design. PLoS One. 7:e283952012. View Article : Google Scholar : PubMed/NCBI

16 

Adair BM: Immunopathogenesis of chicken anemia virus infection. Dev Comp Immunol. 24:247–255. 2000. View Article : Google Scholar : PubMed/NCBI

17 

Los M, Panigrahi S, Rashedi I, Mandal S, Stetefeld J, Essmann F and Schulze-Osthoff K: Apoptin, a tumor-selective killer. Biochim Biophys Acta. 1793:1335–1342. 2009. View Article : Google Scholar : PubMed/NCBI

18 

van der Eb MM, Pietersen AM, Speetjens FM, Kuppen PJ, van de Velde CJ, Noteborn MH and Hoeben RC: Gene therapy with apoptin induces regression of xenografted human hepatomas. Cancer Gene Ther. 9:53–61. 2002.PubMed/NCBI

19 

Olijslagers S, Dege AY, Dinsart C, Voorhoeve M, Rommelaere J, Noteborn MH and Cornelis JJ: Potentiation of a recombinant oncolytic parvovirus by expression of Apoptin. Cancer Gene Ther. 8:958–965. 2001. View Article : Google Scholar : PubMed/NCBI

20 

Pietersen AM, van der Eb MM, Rademaker HJ, van den Wollenberg DJ, Rabelink MJ, Kuppen PJ, van Dierendonck JH, van Ormondt H, Masman D, van de Velde CJ, van der Eb AJ, Hoeben RC and Noteborn MH: Specific tumor-cell killing with adenovirus vectors containing the apoptin gene. Gene Ther. 6:882–892. 1999. View Article : Google Scholar : PubMed/NCBI

21 

Heilman DW, Teodoro JG and Green MR: Apoptin nucleocytoplasmic shuttling is required for cell type-specific localization, apoptosis, and recruitment of the anaphase-promoting complex/cyclosome to PML bodies. J Virol. 80:7535–7545. 2006. View Article : Google Scholar

22 

Maddika S, Booy EP, Johar D, Gibson SB, Ghavami S and Los M: Cancer-specific toxicity of apoptin is independent of death receptors but involves the loss of mitochondrial membrane potential and the release of mitochondrial cell-death mediators by a Nur77-dependent pathway. J Cell Sci. 118:4485–4493. 2005. View Article : Google Scholar

23 

Maddika S, Mendoza FJ, Hauff K, Zamzow CR, Paranjothy T and Los M: Cancer-selective therapy of the future: apoptin and its mechanism of action. Cancer Biol Ther. 5:10–19. 2006. View Article : Google Scholar : PubMed/NCBI

24 

Maddika S, Wiechec E, Ande SR, Poon IK, Fischer U, Wesselborg S, Jans DA, Schulze-Osthoff K and Los M: Interaction with PI3-kinase contributes to the cytotoxic activity of apoptin. Oncogene. 27:3060–3065. 2008. View Article : Google Scholar : PubMed/NCBI

25 

Wagstaff KM and Jans DA: Nuclear drug delivery to target tumour cells. Eur J Pharmacol. 625:174–180. 2009. View Article : Google Scholar : PubMed/NCBI

26 

Jiang J, Cole D, Westwood N, Macpherson L, Farzaneh F, Mufti G, Tavassoli M and Gaken J: Crucial roles for protein kinase C isoforms in tumor-specific killing by apoptin. Cancer Res. 70:7242–7252. 2010. View Article : Google Scholar : PubMed/NCBI

27 

Los M, Maddika S, Erb B and Schulze-Osthoff K: Switching Akt: from survival signaling to deadly response. Bioessays. 31:492–495. 2009. View Article : Google Scholar : PubMed/NCBI

28 

Maddika S, Panigrahi S, Wiechec E, Wesselborg S, Fischer U, Schulze-Osthoff K and Los M: Unscheduled Akt-triggered activation of cyclin-dependent kinase 2 as a key effector mechanism of apoptin’s anticancer toxicity. Mol Cell Biol. 29:1235–1248. 2009.PubMed/NCBI

29 

Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL and Shay JW: Specific association of human telomerase activity with immortal cells and cancer. Science. 266:2011–2015. 1994. View Article : Google Scholar : PubMed/NCBI

30 

Mo Y, Gan Y, Song S, Johnston J, Xiao X, Wientjes MG and Au JL: Simultaneous targeting of telomeres and telomerase as a cancer therapeutic approach. Cancer Res. 63:579–585. 2003.PubMed/NCBI

31 

Wirth T, Kuhnel F and Kubicka S: Telomerase-dependent gene therapy. Curr Mol Med. 5:243–251. 2005. View Article : Google Scholar

32 

Hernandez-Alcoceba R, Pihalja M, Qian D and Clarke MF: New oncolytic adenoviruses with hypoxia- and estrogen receptor-regulated replication. Hum Gene Ther. 13:1737–1750. 2002. View Article : Google Scholar : PubMed/NCBI

33 

Kurihara T, Brough DE, Kovesdi I and Kufe DW: Selectivity of a replication-competent adenovirus for human breast carcinoma cells expressing the MUC1 antigen. J Clin Invest. 106:763–771. 2000. View Article : Google Scholar : PubMed/NCBI

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March 2013
Volume 42 Issue 3

Print ISSN: 1019-6439
Online ISSN:1791-2423

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Spandidos Publications style
Zhang M, Wang J, Li C, Hu N, Wang K, Ji H, He D, Quan C, Li X, Jin N, Jin N, et al: Potent growth-inhibitory effect of a dual cancer-specific oncolytic adenovirus expressing apoptin on prostate carcinoma. Int J Oncol 42: 1052-1060, 2013
APA
Zhang, M., Wang, J., Li, C., Hu, N., Wang, K., Ji, H. ... Li, Y. (2013). Potent growth-inhibitory effect of a dual cancer-specific oncolytic adenovirus expressing apoptin on prostate carcinoma. International Journal of Oncology, 42, 1052-1060. https://doi.org/10.3892/ijo.2013.1783
MLA
Zhang, M., Wang, J., Li, C., Hu, N., Wang, K., Ji, H., He, D., Quan, C., Li, X., Jin, N., Li, Y."Potent growth-inhibitory effect of a dual cancer-specific oncolytic adenovirus expressing apoptin on prostate carcinoma". International Journal of Oncology 42.3 (2013): 1052-1060.
Chicago
Zhang, M., Wang, J., Li, C., Hu, N., Wang, K., Ji, H., He, D., Quan, C., Li, X., Jin, N., Li, Y."Potent growth-inhibitory effect of a dual cancer-specific oncolytic adenovirus expressing apoptin on prostate carcinoma". International Journal of Oncology 42, no. 3 (2013): 1052-1060. https://doi.org/10.3892/ijo.2013.1783