Potent Antitumor Effect of Interleukin-24 Gene in the Survivin Promoter and Retinoblastoma Double-Regulated Oncolytic Adenovirus

Abstract

Interleukin (IL)-24 is an excellent therapeutic gene for cancer therapy. In this work, IL-24 was inserted into Ad.sp-E1A_(Δ24), an oncolytic adenovirus with a 24-bp deletion in the E1A gene, which was driven by the survivin promoter to form Ad.sp-E1A_(Δ24)-IL-24. Ad.sp-E1A_(Δ24)-IL-24 has an excellent antitumor effect in vitro for human nasopharyngeal, liver, lung, and cervical carcinoma cell lines but does no or little damage to normal cell lines L-02 and WI38. Furthermore, it achieved nearly complete inhibition (although not elimination) of NCI-H460 lung carcinoma growth in nude mice. The antitumor efficacy of Ad.sp-E1A_(Δ24)-IL-24 on NCI-H460 cells was clearly mediated by apoptosis, because it induced caspase-3 and poly(ADP-ribose) polymerase cleavage. This is the first report of Ad.sp-E1A_(Δ24)-IL-24 with such an excellent, broad, and specific antitumor effect in vitro and nearly complete inhibition of lung tumor growth in vivo.

Introduction

G ene therapy began in 1990 and now has 1470 protocols in clinical trials, with 65.2% for cancer therapy; however, no significant breakthrough has been obtained except that Ad-p53/Gendicine has been marketed in China (Peng, 2005). The most important virotherapy studied previous to this was ONYX-015 therapy, which entered phase III clinical trial, but was stopped without further study; thus virotherapy achieved no significant breakthrough either. We then combined the advantages of gene therapy and virotherapy to suggest a strategy called targeting gene-virotherapy of cancer (Liu, 2001, 2006; Zhang et al., 2003), the basic principle of which is to insert an antitumor gene into an oncolytic virus. A variety of viruses can be made to become oncolytic, specifically infecting tumor cells but rarely normal cells. Two main strategies are used to construct oncolytic viruses. The first strategy involves the deletion of adenoviral genes that are necessary for viral replication in normal cells but not in tumor cells; these include oncolytic viruses ONYX-015 and ZD55, which were formed by deletion of the E1B-55KD gene and lack of viral late mRNA transport (O'Shea et al., 2004). The other strategy is to use tumor- or tissue-selective promoters to control the expression of early viral genes essential for replication, such as E1A and E1B of adenovirus. The human telomerase reverse transcriptase (hTERT) promoter and survivin promoter have been used in our laboratory to control E1A of adenovirus in order to construct oncolytic adenoviruses, which showed tumor-selective replication and potent antitumor efficacy (Zou et al., 2004; Li et al., 2006).

Interleukin (IL)-24 shows a strong antitumor effect, inhibiting tumor in three ways: (1) by induction of tumor apoptosis and its bystander effect (Su et al., 2005; Sauane et al., 2006, 2008); (2) by inhibition of angiogenesis (Ramesh et al., 2003; Wang et al., 2007; Xie et al., 2008); and (3) by an increase in immunological effect (Miyahara et al., 2006; Commins et al., 2008). The first oncolytic virus used in our laboratory was ZD55. By insertion of the IL-24 gene into ZD55, ZD55-IL-24 was formed. The antitumor effect of ZD55-IL-24 was 100-fold higher than that of the respective gene therapy product Ad-IL-24 in vitro (Zhao et al., 2005; Inoue et al., 2006), which is now in a phase III clinical trial in the United States. Therefore an application for an international patent for ZD55-IL-24 has been made according to the terms of the Patent Cooperation Treaty (PCT). By the combined use of two ZD55 genes, such as ZD55-IL-24 plus ZD55-TRALL, xenografts of colorectal carcinoma, hepatoma, and gastric cancer in nude mice could all be completely eliminated (Zhao et al., 2006).

Survivin is a unique member of the inhibitor of apoptosis (IAP) protein family, which plays a key role in the regulation of apoptosis and cell division (Ambrosini et al., 1997; Adida et al., 1998; Altieri, 2008). Most significantly, survivin is expressed in almost all human tumors but is rarely detectable in normal cells and tissues. Overexpression of survivin has been implicated in lung cancer and other cancers (Blanc-Brude et al., 2003; Falleni et al., 2003; Lu et al., 2004; Karczmarek-Borowska et al., 2005; Warnecke-Eberz et al., 2008). Survivin can be used as an indicator of tumor staging and prognosis. Studies have showed that survivin promoter-driven oncolytic adenovirus exhibited tumor-selective cytotoxicity in vitro and in vivo (Kamizono et al., 2005; Zhu et al., 2006; Wang, 2008), which suggested the survivin promoter on transcriptional targeting is a good candidate for targeting cancer therapy. In addition, oncolytic virus Ad5-E1A_(Δ24), with a 24-bp deletion in the E1A region responsible for binding retinoblastoma (Rb) protein and its replication, is restricted to dividing cells or to Rb-inactive arrested cells and exhibits tumor-selective capability (Fueyo et al., 2000). The antitumor activity of the therapeutic gene should safely progress along with the improved tumor-selective adenoviral vector (Lee et al., 1999; Shinoura and Hamada, 2003). When the IL-24 gene was inserted into Ad.sp-E1A_(Δ24), Ad.sp-E1A_(Δ24)-IL-24 was formed. This is the first report of a double-regulated oncolytic adenovirus (Ad.sp-E1A_(Δ24)-IL-24) constructed with a survivin promoter-controlled E1A gene with a 24-bp deletion (Δ24) targeting the Rb pathway to improve safety. Our data show that Ad.sp-E1A_(Δ24)-IL-24 can achieve wide-range tumoral targeting to induce dramatic cytotoxicity in tumor cells in vitro. Furthermore, Ad.sp-E1A_(Δ24)-IL-24 achieved essentially complete inhibition (although not elimination) of xenografted lung cancer in nude mice.

Materials and Methods

Cell lines and culture conditions

HEK293 (a human embryonic kidney cell line containing the E1A region of adenovirus serotype 5 [Ad5]) was obtained from Microbix Biosystems (Toronto, ON, Canada). All tumor and normal cell lines were from the American Type Culture Collection (ATCC, Manassas, VA) or the Shanghai Cell Collection (Shanghai, China); these included CNE (human nasopharyngeal carcinoma), NCI-H460 (human lung carcinoma), NCI-H1299 (human lung carcinoma), PLC (human hepatocarcinoma), HeLa (human cervical carcinoma), L-02 (human normal liver), and WI38 (human normal fetal lung fibroblast).

CNE, NCI-H460, NCI-H1299, PLC, HeLa, L-02, and HEK293 cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 4 mM glutamine, penicillin (50 U/ml), and streptomycin (50 mg/ml). The WI38 cell line was cultured in DMEM supplemented with 20% FBS. All the cell lines were cultured at 37°C in 5% CO₂.

Construction of plasmids, and generation and purification of adenoviral vectors

Plasmid pCN103 carrying the adenovirus serotype 5 E1A gene with a 24-bp deletion from bp 922 to bp 946, was kindly provided by C. Qian of our institute and subcloned into pXC2 to form pXC2-E1A_(Δ24). The human survivin promoter (hSurP) was further subcloned into plasmid pXC2-E1A_(Δ24) to form the plasmid pAd.sp-E1A_(Δ24). After inserting IL-24 expression cassette into it, pAd.sp-E1A_(Δ24)-IL-24 was obtained. The oncolytic viruses Ad.sp-E1A_(Δ24) and Ad.sp-E1A_(Δ24)-IL-24 were generated by homologous recombination between pAd.sp-E1A_(Δ24) or pAd.sp-E1A_(Δ24)-IL-24 with the adenovirus packaging plasmid pBHGE3 (Microbix Biosystems) in HEK293 cells. Each recombinant adenovirus was isolated through three rounds of plaque purification in HEK293 cells and purified by ultracentrifugation in a cesium chloride gradient. Moreover, viral titers were determined by TCID₅₀ (median tissue culture infective dose) assay in HEK293 cells. Cells were infected with adenovirus at various doses at 37°C in a humidified atmosphere containing 5% CO₂.

Adenoviral progeny assay

To determine viral progeny, tumor cells and normal cells were infected with Ad.sp-E1A_(Δ24) and Ad.sp-E1A_(Δ24)-IL-24 or with wild-type adenovirus (Wt-Ad) at a multiplicity of infection (MOI) of 10. After 5 hr, medium was removed and cells were washed three times with phosphate-buffered saline (PBS), and then 2 ml of fresh medium was added. Two days postinfection, cells were collected and virus was released by three freeze–thawing cycles and centrifuged to collect the supernatant. Virus production was determined by TCID₅₀ assay in HEK293 cells.

Cell viability assay

Cells were plated in 96-well plates and treated with various adenoviruses. After infection for 4 days at the indicated MOIs, the cell survival rate was evaluated by a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma, St. Louis, MO). Medium was removed and fresh medium containing MTT (0.5 mg/ml) was added to each well. The cells were incubated at 37°C for 4 hr; the supernatant of each well was drawn off carefully and then an equal volume (150 μl) of dimethyl sulfoxide (DMSO) was added to each well and mixed thoroughly on a concentrating table for 10 min. The absorbance from the plates was read at 595 nm with a DNA microplate reader (GENios model; Tecan, Maennedorf, Switzerland).

Cytopathic effect assay

PLC, NCI-H1299, and NCI-H460 tumor cell lines, as well as the L-02 and WI38 normal cell lines, were grown to subconfluence and infected with adenoviruses at various MOIs. Four days after infection, cells were exposed to 2% crystal violet in 20% methanol for 15 min and then washed with distilled water and documented by photography.

Western blot analysis

To determine the expression of various proteins, Western blot analysis was performed as described in a standard protocol (Zhao et al., 2006). Cells were harvested by trypsinization and resuspended in lysis buffer (62.5 mM Tris-HCl [pH 6.8], 2% sodium dodecyl sulfate [SDS], 10 mM glycerol, 1.55% dithiothreitol). The total protein concentration was determined with a bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Rockford, IL) as described in the manufacturer's protocol. Protein samples were then separated by SDS–12% polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Pierce Biotechnology). Membranes were blocked in 5% bovine serum albumin (BSA) solution and incubated with primary antibodies, which were then detected with the appropriate secondary fluorescent antibodies and checked with an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE). The primary antibodies used were mouse monoclonal anti-mda-7/IL-24 (GenHunter, Nashville, TN), rabbit polyclonal anti-caspase-3 (Cell Signaling Technology, Danvers, MA), rabbit monoclonal (Epitomics, Burlingame, CA), anti-poly(ADP-ribose) polymerase (PARP) (Santa Cruz Biotechnology, Santa Cruz, CA) anti-E1A (Abcam, Cambridge, UK), and anti-actin (Beyotime, Haimen, China).

Flow cytometric analysis

Cells infected with adenovirus were trypsinized and washed once with complete medium. Aliquots of cells (5 × 10⁵) were resuspended in 500 μl of binding buffer and stained with fluorescein isothiocyanate (FITC)-labeled annexin V (BioVision, Palo Alto, CA) according to the manufacturer's instructions. A fluorescence-activated cell-sorting (FACS; BD Biosciences, San Jose, CA) assay was performed immediately after staining.

Hoechst 33258 staining

NCI-H460 cells were seeded in 6-well culture plates with slides and infected with Ad.sp-E1A_(Δ24) or Ad.sp-E1A_(Δ24)-IL-24 at an MOI of 5; uninfected cells served as control. After 48 hr, cells were treated with the apoptosis-Hoechst 33258 staining kit (Beyotime) for 5–10 min as described in the manufacturer's protocol, washed with PBS twice, and observed under a fluorescence microscope.

Studies of xenograft tumors in nude mice

All animals used in these experiments were maintained in the institutional facilities in accordance with regulations and standards of the U.S. Department of Agriculture and the National Institutes of Health. Female BALB/c nude mice at 4–5 weeks if age, obtained from the Animal Research Committee of the Institute of Biochemistry and Cell Biology (Shanghai, China), were used in all the experiments. NCI-H460 cells (1 × 10⁷) were injected subcutaneously into the lower right flank of female nude mice. After 6 days, the tumor xenograft model was established. Each group was composed of at least eight animals and tumor growth was monitored and measured weekly with a Vernier caliper. Tumor volume (V) was calculated according to the formula V (mm³) = 1/2 × length (mm) × width (mm)². When the tumors were 100–130 mm³ in size, mice were randomized into three groups and a daily dose of 5 × 10⁸ plaque-forming units (PFU) of Ad.sp-E1A_(Δ24) or Ad.sp-E1A_(Δ24)-IL-24 suspended in 100 μl of PBS, or 100 μl of PBS alone, was administrated intratumorally once every other day for a total of four times. Tumors were harvested on day 4 posttreatment for histopathological and transmission electron microscopy (TEM) analysis.

Immunohistochemical study

For immunohistochemical (IHC) analysis, tumors on day 4 posttreatment were harvested and fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4-mm sections. These sections were stained with mouse monoclonal anti-IL-24 and anti-adenoviral E1A antibodies at 1:300 and 1:50 dilutions, respectively. The slides were then washed with PBS and incubated with the avidin–biotin–peroxidase complex reagent (Vector Laboratories, Burlingame, CA) and detected with diaminobenzidine tetrahydrochloride (Bendrik et al., 2008) solution containing 0.006% hydrogen peroxide. Hematoxylin was used as a counterstain. Tissue sections stained without primary antibodies were used as negative controls.

Transmission electron microscopy analysis

For electron microscopy, tumor samples (1 mm³) were fixed in a phosphate-buffered mixture of 2.5% glutaraldehyde overnight, followed by 1 hr of fixation with 1% osmium tetroxide. The tissues were rinsed in water, dehydrated through a graded series of ethanol and propylene oxide, and embedded in Epon 812 resin (Shell Chemicals, Houston, TX). After examination of semithin sections, areas were selected and subjected to ultrathin sectioning. Sections collected on 200-mesh copper grids were contrasted with lead citrate and uranyl acetate, examined, and photographed with a JEOL 100CX transmission electron microscope (JEOL, Akishima, Japan).

Statistical analysis

The statistical significance of experimental results was calculated by analysis of variance (ANOVA) and Student t test. Data were considered statistically significant at p < 0.05.

Results

Characterization of oncolytic adenovirus Ad.sp-E1A_(Δ24)-IL-24 and its selective replication in tumor cells

Adenoviral E1A is the early gene for viral replication in host cells; after deletion of 24 bp, Ad-E1A_(Δ24) was formed. The survivin promoter is a promising candidate for the construction of a tumor-specific oncolytic adenovirus. The dual-regulated oncolytic adenovirus Ad.sp-E1A_(Δ24)-IL-24 is also driven by the survivin promoter and harbors the antitumor IL-24 gene (Fig. 1A).

FIG. 1.

Characterization of oncolytic adenovirus Ad.sp-E1A_(Δ24)-IL-24 and its selective replication in tumor cells. (A) Schematic structure of recombinant oncolytic adenovirus. Ad.sp-E1A, compared to the wild type Adenovirus (wt-Ad), its E1A promoter replaced by hSurP; Ad.sp-E1A_(Δ24), in which E1A promoter replaced by hSurP and 24 nucleotides deletion (from nucleotide 923 to 946 of adenoviral genome), was a dual regulated recombinant virus replicating only in malignant cells with abnormal pRb pathway while sparing normal cells. Then, an IL-24 expression cassette controlled by the human cytomegalovirus (HCMV) promoter was obversely inserted in Ad.sp-E1A_(Δ24) (between the ψ packing signal sequence and the E1A gene) to form Ad.sp-E1A_(Δ24)-IL-24. ITR, inverted terminal repeat; Wt-P, wild-type promoter; hSurP, human survivin promoter; SV40 polyA, simian virus 40 polyadenylation signal. (B) Western blot assay of IL-24 expression by Ad.sp-E1A_(Δ24)-IL-24. Cells were infected with Ad.sp-E1A_(Δ24)-IL-24 or Ad.sp-E1A_(Δ24) at an MOI of 5 for 48 or 72 hr. Mock-infected cells were included as a control. Lysates from control and infected cells were subjected to Western blot assay with anti-IL-24 antibody as described in Materials and Methods. At the same time, a Western blot assay of E1A expression was done: on infection at an MOI of 10, NCI-H460 cells were harvested after 48 hr. β-Actin was used as a protein loading control. (C) NCI-H460 cells (3.5 × 10⁵) were plated into six-well plates. After 24 hr, the cells were infected with 10 MOI of Ad.sp-E1A_(Δ24)-IL-24, Ad.sp-E1A_(Δ24), or Wt-Ad. After an additional 48 hr, medium and cells were scraped into 1.5-ml Eppendorf tubes and subjected to three freeze–thaw cycles. The collected supernatant was tested for virus production by standard TCID₅₀ assay on 293 cells. Progeny viruses from 1 MOI of virus were calculated. Results are the average of two independent experiments.

All the constructs were characterized by Western blot assay. In Fig. 1B, NCI-H460 cells infected with Ad.sp-E1A_(Δ24) or Ad.sp-E1A_(Δ24)-IL-24, or positive HEK293 cells, expressed E1A protein, whereas mock-infected cells failed to express E1A protein. To examine IL-24 gene expression, NCI-H460 cells were infected with Ad.sp-E1A_(Δ24) or Ad.sp-E1A_(Δ24)-IL-24 at an MOI of 5 for 48 or 72 hr. Cells were then harvested and subjected to Western blot assay. Transduction of Ad.sp-E1A_(Δ24)-IL-24 resulted in obvious IL-24 expression compared with no expression in control or Ad.sp-E1A_(Δ24)-transduced cells. Moreover, the IL-24 expression level of cells infected for 72 hr with Ad.sp-E1A_(Δ24)-IL-24 was higher than that of cells infected for 48 hr, which indicated that the IL-24 expression level gradually improved with the replication of virus in lung cancer NCI-H460 cells.

To examine whether the transgene could interfere with the selective replicative ability of recombinant adenoviruses in tumor cell lines, a progeny assay was performed in tumor cells (NCI-H1299, PLC, HeLa, CNE, and NCI-H460) and normal cells (L-02 and WI38) infected with Ad.sp-E1A_(Δ24), Ad.sp-E1A_(Δ24)-IL-24, or Wt-Ad at an MOI of 10. As shown in Fig. 1C, Ad.sp-E1A_(Δ24) and Ad.sp-E1A_(Δ24)-IL-24 replicated in infected tumor cells to similar levels, which were comparable to that of Wt-Ad. In contrast, the replicative capacity of these viruses was much reduced in normal cells. These data indicated that expression of IL-24 gene did not affect the selective replicative ability of oncolytic adenoviruses.

Cytotoxicity of Ad.sp-E1A_(Δ24)-IL-24 and Ad.sp-E1A_(Δ24) in tumor cells but not in normal cells

To evaluate the cytotoxicity of recombinant adenovirus, tumor cell lines (NCI-H1299, HeLa, CNE, NCI-H460, and PLC) and normal cell lines (L-02 and WI38) were infected with Ad.sp-E1A_(Δ24) or Ad.sp-E1A_(Δ24)-IL-24 at MOIs of 0.1, 1, 5, and 10. After 4 days, cytotoxicity was determined by MTT assay. As shown in Fig. 2A, Ad.sp-E1A_(Δ24)-IL-24 and the control virus Ad.sp-E1A_(Δ24) could induce dose-dependent cytotoxicity in all five tumor cell lines. These showed the broad antitumor effect of Ad.sp-E1A_(Δ24)-IL-24. In addition, Ad.sp-E1A_(Δ24)-IL-24 was higher in cytotoxicity in tumor cells (PLC, HeLa, NCI-H1299, and NCI-H460) than was Ad.sp-E1A_(Δ24) at each infected concentration gradient (MOIs of 0.1, 1, 5, and 10). Moreover, because of the selective replicative ability of the oncolytic adenoviruses Ad.sp-E1A_(Δ24) and Ad.sp-E1A_(Δ24)-IL-24, there was no obvious cytotoxicity to the two normal cell lines (Fig. 2B). The results indicated that our dual-regulated oncolytic virus could lead to an obvious and broad tumor-specific cytotoxic effect in vitro, and had no or little effect on normal cells.

FIG. 2.

Cytotoxicity of oncolytic adenovirus in tumor cells in vitro. (A) Survival rate of tumor cell lines (CNE, PLC, NCI-H1299, NCI-H460, and HeLa). (B) Survival rate of normal cell lines (L-02 and WI38). All cells were infected with Ad.sp-E1A_(Δ24)-IL-24 or Ad.sp-E1A_(Δ24) at MOIs of 0.1, 1, 5, and 10. After 96 hr, the cell survival rate was measured by MTT assay. Results are expressed as a percentage relative to untreated control cells, and represent the mean of at least three independent experiments; error bars, SD. ^* p < 0.05.

Comparison of antitumor activity of gene therapy and targeting gene virotherapy in vitro

To compare the cytopathic capacity of Ad-IL-24 and Ad.sp-E1A_(Δ24)-IL-24, a human liver cancer cell line (PLC), lung cancer cell lines (NCI-H1299 and NCI-H460), a normal human lung fibroblast cell line (WI38), and a human liver cell line (L-02) were infected with Ad-IL-24, Ad.sp-E1A_(Δ24), or Ad.sp-E1A_(Δ24)-IL-24 at MOIs of 0, 0.1, 5, 10, and 100. Cells were stained with crystal violet 4 days later. Significant cytopathic effect was observed in all tumor cell lines infected with Ad.sp-E1A_(Δ24)-IL-24, as compared with cells infected with Ad.sp-E1A_(Δ24) or Ad-IL-24 (Fig. 3). Moreover, the cytopathic effect of Ad.sp-E1A_(Δ24)-IL-24 was superior to that of Ad.sp-E1A_(Δ24) and was about 100 times greater than that of Ad-IL-24 in the NCI-H1299 and NCI-H460 cell lines. For all three viruses, no obvious cytopathic effect was detected in the two normal cell lines (WI38 and L-02) at the same MOIs compared with the treated tumor cell groups, although significant cytotoxicity was observed in WI38 and L-02 cells at the highest MOI of 100. These results suggest that Ad.sp-E1A_(Δ24)-IL-24 can selectively replicate in various tumor cells and inhibit the growth of tumor cells more effectively than Ad.sp-E1A_(Δ24) (oncolytic virotherapy) and Ad-IL-24 (gene therapy).

FIG. 3.

Tumor-selective cytopathic effect of Ad.sp-E1A_(Δ24)-IL-24 and Ad-IL-24. Tumor cells (PLC, NCI-H1299, and NCI-H460) and normal cells (L-02 and WI38) were seeded in 24-well plates at a density of 5 × 10⁴ cells for each well and infected with Ad.sp-E1A_(Δ24), Ad.sp-E1A_(Δ24)-IL-24, or Ad-IL-24 at the indicated MOIs. Seven days later, cells were stained with crystal violet. Color images available online at www.liebertonline.com/hum.

To further evaluate the kinetics of cytotoxicity induced by Ad.sp-E1A_(Δ24)-IL-24, tumor cells (PLC, NCI-H1299, and NCI-H460) and normal cells (WI38 and L-02) were plated in 96-well plates and infected with the previously mentioned three viruses. As shown in Fig. 4, the cytotoxic effect of Ad.sp-E1A_(Δ24)-IL-24 on the cancer cell lines was much more obvious than that of Ad-IL-24 or Ad.sp-E1A_(Δ24); this was particularly evident with the longer treatment times. There was little or no cytopathic effect of Ad.sp-E1A_(Δ24)-IL-24, Ad-IL-24, and Ad.sp-E1A_(Δ24) on normal cells. Similar results were obtained with other tumor cell lines and normal cell lines (data not shown).

FIG. 4.

Cell viability was assayed by MTT test. Tumor cells (PLC, NCI-H1299, and NCI-H460) and normal cells (L-02 and WI38) were infected with Ad-IL-24, Ad.sp-E1A_(Δ24), or Ad.sp-E1A_(Δ24)-IL-24 at an MOI of 10. On days 1, 2, 3, and 4 postinfection, cells were stained with MTT as described in Materials and Methods. Results are presented as means ± SD (error bars) of triplicate experiments and are expressed as a percentage of uninfected control cells.

Mechanism of apoptosis is mediated by Ad.sp-E1A_(Δ24)-IL-24 in NCI-H460 cells

We investigated whether Ad.sp-E1A_(Δ24)-IL-24 kills tumor cells through apoptosis. Hoechst 33258 staining for apoptotic cells clearly showed that Ad.sp-E1A_(Δ24)-IL-24 and Ad.sp-E1A_(Δ24) led to remarkable apoptotic morphological changes such as chromatin condensation, nuclear fragmentation, and apoptotic bodies in the NCI-H460 cell line (Fig. 5A). Moreover, to further confirm induction of apoptosis, FACS analysis was performed to test apoptosis induction by Ad.sp-E1A_(Δ24) and Ad.sp-E1A_(Δ24)-IL-24 in the lung cancer cell line NCI-H460 and the normal cell line WI38. Cells uninfected with adenovirus served as a negative control. The apoptotic effect of Ad.sp-E1A_(Δ24)-IL-24 at an MOI of 10 was more significant than that of any other group, even Ad.sp-E1A_(Δ24) (Fig. 5B). Although Ad.sp-E1A_(Δ24)-IL-24 and Ad.sp-E1A_(Δ24) led to similar apoptotic rates at an MOI of 5, a dramatic statistical discrepancy appeared in the induced apoptosis rates of Ad.sp-E1A_(Δ24)-IL-24 compared with the mock group (p < 0.01). No apoptosis was detected in normal cells infected with either Ad.sp-E1A_(Δ24) or Ad.sp-E1A_(Δ24)-IL-24 alone. These results suggest that the induction of apoptosis in tumor cells was due to replication of the oncolytic virus and the expression of IL-24, whereas the few apoptotic effects in normal cells indicated that both Ad.sp-E1A_(Δ24) and Ad.sp-E1A_(Δ24)-IL-24 are safe. Furthermore, activation of the caspase pathway was examined to explore the mechanism of tumor cell apoptosis. As shown in Fig. 5C, the cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP) was obviously increased in NCI-H460 cells treated with Ad.sp-E1A_(Δ24)-IL-24 compared with Ad.sp-E1A_(Δ24) or the control, which indicates the activated process of apoptosis.

FIG. 5.

Apoptosis detection in tumor cells and activation of caspase-3 mediated by Ad.sp-E1A_(Δ24)-IL-24. (A) NCI-H460 cells were stained with Hoechst 33258 after infection with Ad.sp-E1A_(Δ24) or Ad.sp-E1A_(Δ24)-IL-24 at an MOI of 5 for 48 hr. Mock, uninfected cells. Arrows indicate positive apoptotic cells. Original magnification, ×200. (B) Apoptosis detection by annexin V-binding assay after infection with Ad.sp-E1A_(Δ24)-IL-24. WI38 and NCI-H460 cells were infected for 48 hr at an MOI of 5 or 10 with Ad.sp-E1A_(Δ24)-IL-24 or Ad.sp-E1A_(Δ24), or remained uninfected as a control. The percentage of apoptotic cells was calculated with CellQuest software (BD Biosciences). The majority of NCI-H460 lung cancer cells infected with Ad.sp-E1A_(Δ24)-IL-24 or Ad.sp-E1A_(Δ24) were positive for apoptosis, whereas WI38 cells infected with Ad.sp-E1A_(Δ24)-IL-24 or Ad.sp-E1A_(Δ24) seldom displayed any apoptotic effect. The percentage of apoptotic cells after various treatments is represented as described in Materials and Methods. *p < 0.05. (C) The activated executive protein caspase-3 was detected; in addition, the downstream apoptotic protein poly(ADP-ribose) polymerase (PARP) was also detected. Color images available online at www.liebertonline.com/hum.

Potent antitumor efficacy of Ad.sp-E1A_(Δ24)-IL-24 in nude mice

An NCI-H460 human lung tumor xenograft model was established in nude mice. When the tumors grew to 100–130 mm³, PBS, Ad.sp-E1A_(Δ24), or Ad.sp-E1A_(Δ24)-IL-24 (5 × 10⁸ PFU/dose) in 100 μl WAS injected intratumorally every other day for a total of four times. Animals treated with Ad.sp-E1A_(Δ24)-IL-24 exhibited significant suppression of tumor growth compared with control PBS-treated animals (p < 0.005) and Ad.sp-E1A_(Δ24)-treated animals (p < 0.005). The average tumor volume on injection with Ad.sp-E1A_(Δ24)-IL-24 was 136.4 mm³ at 43 days, which continued to decrease to below 95 mm³, even lower than the original volume of 100 mm³; this indicates a complete inhibition of NCI-H460 human lung tumor growth, whereas the tumor volume of PBS-treated or Ad.sp-E1A_(Δ24)-treated mice was 2467.3 and 545.8 mm³, respectively. In addition, the Ad.sp-E1A_(Δ24)-treated group also showed notable antitumor efficacy as compared with the PBS-treated group (p < 0.05) (Fig. 6A).

FIG. 6.

Antitumor efficacy of Ad.sp-E1A_(Δ24)-IL-24 in nude mice. Female BALB/c nude mice were subcutaneously inoculated with NCI-H460 cells (1 × 10⁷). When tumors reached a size of 100–130 mm³, the animals were treated with an intratumoral injection of Ad.sp-E1A_(Δ24) or Ad.sp-E1A_(Δ24)-IL-24, or PBS. (A) Tumor size was measured and tumor volume was calculated. Data are presented as means ± SD (n = 8). (B) The death of animals was monitored before the end of the animal experiment. Long-term survival of animals was observed after treatment with oncolytic adenoviruses compared with control animals receiving PBS. A pair-wise log-rank test was used to analyze survival rates in the different groups. Statistical significance: a, p < 0.01; b, p < 0.001, compared with PBS; c, p < 0.01, compared with Ad.sp-E1A_(Δ24). In this figure, no Ad-IL-24 curve is shown, although it was done and showed much less antitumor effect.

Survival of animals was also monitored by the Kaplan–Meier method. Animals treated with Ad.sp-E1A_(Δ24)-IL-24 all survived (eight of eight), whereas in the other groups zero of eight survived (PBS) and four of eight survived [Ad.sp-E1A_(Δ24)] (Fig. 6B). In addition, the volume of tumors treated with Ad.sp-E1A_(Δ24)-IL-24 sequentially regressed until day 72, at which time the tumor volume was 95 mm³, less than the volume of about 100 mm³ before treatment.

Morphologic evidence of Ad.sp-E1A_(Δ24)-IL-24 effect on tumor growth inhibition

Hematoxylin and eosin (H&E) staining of subcutaneous tumor sections demonstrated that tumor injections of Ad.sp-E1A_(Δ24)-IL-24 caused profound cell death and symptoms of necrosis in the tumor mass. Vessel growth was significantly suppressed by treatment with Ad.sp-E1A_(Δ24)-IL-24, showing its antiangiogenesis effect (Fig. 7A). IHC analysis also showed E1A protein mediated by oncolytic adenovirus Ad.sp-E1A_(Δ24) and Ad.sp-E1A_(Δ24)-IL-24 in lung tumor tissues, but not with PBS treatment (Fig. 7B). In addition, there was local high expression of IL-24 in tumor cells infected with Ad.sp-E1A_(Δ24)-IL-24 (Fig. 7C).

FIG. 7.

Both pathological detection by H&E and IHC staining assay and morphological observation by TEM analysis were performed. Subcutaneous NCI-H460 tumors receiving various treatments were harvested 4 days after infection with viruses and tumor sections were treated as described in Materials and Methods. (A) H&E staining assay. Although some blood vessels are clearly seen in the PBS group and the group infected with Ad.sp-E1A_(Δ24), vessel growth in the group treated with Ad.sp-E1A_(Δ24)-IL-24 was significantly suppressed (indicated with the black arrows) and partial areas of necrosis are detectable in the sections treated with Ad.sp-E1A_(Δ24)-IL-24 (indicated with the black cross-arrows). (B and C) E1A and IL-24 expression by IHC analysis (original magnification, × 200; arrows indicate positive cells). (D) Viral particles and replication (black arrows) as indicated by TEM analysis. M, mature adenovirus; S, submature adenoviral particle. (E) Morphological observation of cell apoptosis by TEM analysis.

Morphologic observation by TEM analysis showed the viral replication of Ad.sp-E1A_(Δ24) and Ad.sp-E1A_(Δ24)-IL-24 (Fig. 7D). Mature virus and submature adenoviral particles can be seen (marked as M and S, respectively, in Fig. 7D). The significant apoptosis in tumor tissues injected with Ad.sp-E1A_(Δ24)-IL-24 or Ad.sp-E1A_(Δ24) was detected as the classic characteristics of apoptosis, including nuclear collapse and an increased nuclear-to-cytoplasmic ratio; also, the appearance of nucleus deformation and heterochromatin, chromatin condensed in lumps at the inner side of the nuclear envelope were observed (Fig. 7E).

The preceding results suggested that the inhibitory effect on tumor growth was due to IL-24-induced apoptosis, antiangiogenesis, and viral propagation.

Discussion

Tumor-selective oncolytic virus has been used as a vector for gene delivery to improve cancer therapy. The E1A gene is an essential gene to be transcribed for efficient adenoviral replication in host cells. Replacing the native E1A promoter with a tumor-specific promoter, such as the prostate-specific antigen promoter (Rodriguez et al., 1997), the α-fetoprotein promoter (Hallenbeck et al., 1999), the midkine promoter (Adachi et al., 2001), the tyrosinase promoter (Nettelbeck et al., 2002), the pancreas promoter CCKAR (Xie et al., 2007), and so on, revealed that it was an efficient strategy by which to improve the targeting of adenovirus to tumor.

Survivin, one of the inhibitor of apoptosis (IAP) proteins, functions as a key regulator of mitosis and programmed cell death. Survivin was described as an inhibitor of caspase-9 (Mita et al., 2008). However, research studies have shown that the role of survivin in cancer pathogenesis is not limited to apoptosis inhibition but also involves regulation of the mitotic spindle (Li et al., 1998; Petersen and Hagan, 2003; Beltrami et al., 2004) and the inhibition of angiogenesis (Nassar et al., 2008) and chemoresistance (Virrey et al., 2008). Survivin gene expression is transcriptionally repressed by wild-type p53 and can be deregulated in cancer by several mechanisms, including gene amplification, increased promoter activity, and loss of p53 function (Raj et al., 2008; Yang et al., 2008). Studies revealed that the survivin promoter is superior even to the telomerase reverse transcriptase (TERT) promoter at both the responsive promoter activity and as a cancer-specific biomarker in the quantities of different kinds of tumors (Kamizono et al., 2005). Another targeting is the deletion of 24 bp in E1A conservative region 2 (CR2), which make adenovirus available to target the Rb dysfunctional signal pathways in many cancers (Fueyo et al., 2000). Thus, the 1100-bp human survivin promoter was chosen as the cancer-specific promoter to drive the 24-bp-deleted E1A gene to form the double-regulated adenoviral vector Ad.sp-E1A_(Δ24) in this study, although a shorter survivin promoter (Li and Altieri, 1999a,b) has also been used. The results showed that vector Ad.sp-E1A_(Δ24) can replicate and induce cytotoxicity in various tumors but rarely in normal cells, as shown in Figs. 2, 3, and 4.

IL-24 is a cytokine belonging to the IL-10 family. It is a cancer-specific apoptosis-inducing gene with a broad antitumor spectrum, inducing apoptosis in a variety of tumor types including melanomas, gliomas, and cancers of the breast, colon, lung, cervix, pancreas, and prostate (Gupta et al., 2006; Fisher et al., 2007). Ad-IL-24 has been employed to treat ovarian cancer cells and has led to tumor growth suppression in vivo (Gopalan et al., 2007); and INGN 241 (Gopalkrishnan, 2002; Cunningham et al., 2005; Tong et al., 2005; Eager et al., 2008) is in a phase III clinical trial. The mechanism of IL-24 action is to induce apoptosis (Zhao et al., 2005, 2006). Ad.sp-E1A_(Δ24)-IL-24 can induce apoptosis by activated caspase-3 and the cleavage of PARP in tumor cells but rarely damages normal cells (Fig. 5), which was also proved by Lebedeva and colleagues (Lebedeva et al., 2002). In addition, IL-24 can induce a direct antitumor effect through its “bystander antitumor effect” and resistance to antiapoptotic proteins Bcl-2 and Bcl-x_L (Su et al., 2005). Ad-IL-24 has antiangiogenic activity. Ad-IL-24 can downregulate angiogenesis-associated vascular endothelial growth factor and CD31 for reduction of angiogenesis to suppress hepatocellular carcinoma growth in vivo. Also, Ad-IL-24 functions antiangiogenically to decrease CD31/PECAM expression in a lung cancer model. Secreted IL-24 has been shown to have potent antiangiogenic activity both in vitro and in vivo (Saeki et al., 2002; Wang et al., 2007). Ad-IL-24 also has immune stimulatory activity, inducing high-level secretion of some helper T cell type 1 (Th1) cytokines such as IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ and low-level secretion of IL-1β, IL-12, and granulocyte-macrophage colony-stimulating factor (GM-CSF) from human peripheral blood mononuclear cells (PBMCs), which functions as a Th1 cytokine by activating the immune system to improve antitumor efficacy (Caudell et al., 2002; Chada et al., 2004; Wu et al., 2007). The strong effect of IL-24 for inducing apoptosis, antiangiogenesis, and immunity led to the strong antitumor effect of Ad.sp-E1A_(Δ24)-IL-24 presented in this paper (Fig. 6). Our data showed that IL-24 has a wide-ranging ability to kill tumor cells (Fig. 2). Moreover, comparison of the antitumor activity of gene therapy and targeting gene-virotherapy in vitro by MTT and crystal violet assays revealed that the antitumor activity of the gene–virus therapeutic drug Ad.sp-E1A_(Δ24)-IL-24 is much higher (almost 100-fold) than that of regular Ad-IL-24 gene therapy in PLC and NCI-H1299 cells (Figs. 3 and 4); this showed that targeting gene-virotherapy is a more efficient mode of cancer therapy.

In summary, Ad.sp-E1A_(Δ24)-IL-24 was successfully generated with a dual-regulated oncolytic adenovirus harboring IL-24. In in vitro experiments, Ad.sp-E1A_(Δ24)-IL-24 was shown to efficiently express the IL-24 gene in tumor cells and to show an obvious broad tumor-specific cytotoxicity effect, but not in normal cells. In vivo, xenograft tumors were completely inhibited until they were smaller than they were before Ad.sp-E1A_(Δ24)-IL-24 treatment, indicating that Ad.sp-E1A_(Δ24)-IL-24 might be a potent antitumor agent.

Footnotes

Acknowledgments

The authors thank Prof. Cheng Qian for kindly providing plasmid pCN103, Dr. Miao Ding for kindly providing adenovirus Ad-IL-24, and also Xi Jun Liu, Jing Zhang, and Jing Qian for their kind help in cell culturing. This work was supported by the Zhejiang Science and Technology Support Plan (no. 2007C33027), the National Nature Science Foundation of China (nos. 30623003 and 30800093), the National Basic Research Program of China (973 Program; no. 2004 CB51804), Key Project funding from the Chinese Academy of Science (no. KSCX2-YW-R-09, R-04), and by Zhejiang Sci-Tech University grant 0616033 to Prof. Xin Yuan Liu.

Author Disclosure Statement

No competing financial interests exist.

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Potent Antitumor Effect of Interleukin-24 Gene in the Survivin Promoter and Retinoblastoma Double-Regulated Oncolytic Adenovirus

Abstract

Introduction

Materials and Methods

Cell lines and culture conditions

Construction of plasmids, and generation and purification of adenoviral vectors

Adenoviral progeny assay

Cell viability assay

Cytopathic effect assay

Western blot analysis

Flow cytometric analysis

Hoechst 33258 staining

Studies of xenograft tumors in nude mice

Immunohistochemical study

Transmission electron microscopy analysis

Statistical analysis

Results

Characterization of oncolytic adenovirus Ad.sp-E1A(Δ24)-IL-24 and its selective replication in tumor cells

Cytotoxicity of Ad.sp-E1A(Δ24)-IL-24 and Ad.sp-E1A(Δ24) in tumor cells but not in normal cells

Comparison of antitumor activity of gene therapy and targeting gene virotherapy in vitro

Mechanism of apoptosis is mediated by Ad.sp-E1A(Δ24)-IL-24 in NCI-H460 cells

Potent antitumor efficacy of Ad.sp-E1A(Δ24)-IL-24 in nude mice

Morphologic evidence of Ad.sp-E1A(Δ24)-IL-24 effect on tumor growth inhibition

Discussion

Footnotes

Acknowledgments

Author Disclosure Statement

References

Characterization of oncolytic adenovirus Ad.sp-E1A_(Δ24)-IL-24 and its selective replication in tumor cells

Cytotoxicity of Ad.sp-E1A_(Δ24)-IL-24 and Ad.sp-E1A_(Δ24) in tumor cells but not in normal cells

Mechanism of apoptosis is mediated by Ad.sp-E1A_(Δ24)-IL-24 in NCI-H460 cells

Potent antitumor efficacy of Ad.sp-E1A_(Δ24)-IL-24 in nude mice

Morphologic evidence of Ad.sp-E1A_(Δ24)-IL-24 effect on tumor growth inhibition