Complete eradication of hepatomas using an oncolytic adenovirus containing AFP promoter controlling E1A and an E1B deletion to drive IL-24 expression

Abstract

Interleukin (IL)-24, a promising therapeutic gene, has been widely used for Cancer Targeting Gene-Viro-Therapy (CTGVT). In this study, IL-24 was inserted into an oncolytic adenovirus in which the E1A gene is driven by an enhanced, short α-fetoprotein (AFP) promoter and the E1B gene is completely deleted to form Ad.enAFP-E1A-ΔE1B-IL-24. This construct has a potent antitumor effect on liver cancer cell lines in vitro, but little or no effect on normal cell lines, such as L-02 and QSG-7701. In vivo, the complete elimination of Huh-7 liver cancer in nude mice with Ad.enAFP-E1A-ΔE1B-IL-24 intratumor injection was observed. The design of Ad.enAFP-E1A-ΔE1B-IL-24 and its potent antitumor effect on liver cancer have not been published previously. The mechanism of the potent antitumor effect of Ad.enAFP-E1A-ΔE1B-IL-24 is due to the upregulation of GADD34 and intrinsic and extrinsic apoptotic signaling.

Introduction

Cancer has surpassed heart disease as the most lethal disease in the world. Hepatocellular carcinoma (HCC) is the third most common cause of cancer death and the fifth most prevalent cancer in men worldwide. Nearly 600 000 to 1 million new cases are diagnosed worldwide each year. The efficacy of routine clinical therapy on HCC is suboptimal, with high rates of disease recurrence and mortality.1, 2 Therefore, novel and efficient therapies are urgently needed.

Gene therapy is a promising treatment for many hereditary diseases such as Leber's congenital amaurosis, X-linked adrenoleukodystrophy and ‘bubble boy’ disease, and was selected as one of the top 10 breakthroughs of 2009 by the editors of Science. However, gene therapy is not an efficient treatment for cancer mainly due to the replication deficiency of the therapeutic agent. Recently, Cancer Targeting Gene-Viro-Therapy (CTGVT) appears to be a promising strategy for treating cancer. The principle behind CTGVT is the insertion of an antitumor gene into an oncolytic virus (OV), creating what is called an OV-gene.3, 4 The OV will replicate several hundred times in tumor cells, thereby amplifying the therapeutic gene with the same magnitude. Hence, the tumor killing effect is greatly increased. Basically, three main strategies are used to ensure that the OV specifically replicates within tumor cells. The first involves modification of the coat proteins or the use of adaptor molecules for transduction targeting.5, 6, 7 The second is the use of attenuated virus or the deletion of specific viral genes that are indispensable for viral replication in normal cells but not in tumor cells. For example, ONYX-015 or ZD55 has a deletion of the E1B-55 kDa gene, which is necessary for late viral RNA export.8 The E1B-19 kDa deletion enhances the tumor cell killing of a replicating OV vector.9 The third strategy is the replacement of the native viral promoters with tumor-specific promoters to transcriptionally control viral replication. The E1A gene is essential for efficient adenoviral replication in host cells. Replacing the native E1A promoter with a tumor-specific promoter, such as the prostate-specific antigen promoter,10 the midkine promoter,11 the tyrosinase promoter,12 the pancreas promoter CCKAR13 or the liver cancer-specific promoter α-fetoprotein (AFP),14 revealed that different OVs have the potential to target efficiently different types of tumor cells.

Hallenbeck et al.15 was a pioneer in the use of the AFP promoter in adenovirus E1A for the treatment of HCC. AFP is expressed abundantly in fetal liver cells, but gradually decreases in adulthood. However, AFP is frequently re-expressed in HCC and is correlated with disease progression. More than 70% of primary HCCs have an abundance of AFP.16 Owing to the specific expression pattern of AFP, the AFP promoter has been extensively used as a hepatocarcinoma targeting promoter to drive adenovirus E1A gene expression14, 15, 17 armed with different tumor killer genes, such as TRAIL,14 or suicide genes, such as herpes simplex virus thymidine kinase (HSV-tk).18, 19

Interleukin (IL)-24 is a very promising cancer therapeutic gene. IL-24 can induce tumor cell apoptosis, inhibit angiogenesis and has a bystander effect. In addition to increasing immunity, IL-24 is widely used in CTGVT systems for a dramatic antitumor effect. In vitro, ZD55-IL24, the first CTGVT construct created in our laboratory,20 has shown an approximately 100-fold greater antitumor effect than that of the gene therapy product Ad-IL-24 (also called ING241), which has passed phase II clinical trials in the United States and is undergoing phase III clinical trials. We have shown that the application of IL-24 in the CTGVT system not only results in a strong antitumor effect,21, 22 but also enhances the effect of other therapeutic genes such as TRAIL,23 chemotherapeutic agents such as cisplatin,24 ADM, DDP25 and dichloroacetate,26 and RNA interference such as shRNA-MMP1.27

In our laboratory, previous studies have shown that the constructs containing the enAFP (with SV40 enhancer) promoter mediating two strong antitumor genes (TRAIL and SOCS328 and IL-24 and TRAIL29) have potent antitumor effects, but none of the constructs achieved complete eradication of the xenograft hepatoma. However, it is of note that Ad.enAFP-E1A-ΔE1B-IL-24 can achieve complete eradication. There are four reasons for this observation: (1) the use of the CTGVT strategy; (2) the deletion of the 19 and 55 kDa E1B genes in the adenovirus genome; (3) the use of IL-24; and (4) the use of the enAFP promoter. The above four points are considered to be essential parts of a whole system, and all components are necessary to achieve the complete eradication of all xenograft tumors and greatly inhibit the large volume of growth of xenograft tumors with intratumoral drug injection. The above points will be considered further in the Discussion section with an emphasis on the effects of deleting the E1B-19 kDa gene compared to our previous constructs.28, 29

Materials and methods

Cell lines and culture conditions

HEK293 (human embryonic kidney cell line containing the E1A region of Ad5) cells were obtained from Microbix Biosystems (Toronto, ON, Canada). The HCC cell lines (HepG2, PLC, Hep3B and Huh-7) were purchased from the American Type Culture Collection (ATCC, Rockville, MD). Normal liver (L-02, QSG-7701), SMMC-7721 (liver cancer), SKOV3 (human ovarian cancer) and SW620 (human colon carcinoma) cell lines were purchased from the Shanghai Cell Collection (Shanghai, China). The above cell lines were cultured at 37 °C in 5% CO2 in corresponding collection-formulated complete growth medium with 10% heat-inactivated fetal bovine serum, supplemented with 4 mM glutamine, 50 U ml−1 penicillin and 50 mg ml−1 streptomycin.

Construction of the plasmids and generation and purification of the adenovirus vectors

The adenovirus E1B-19 kDa gene was deleted from the plasmid pZD554 to form the plasmid pAd-ΔE1B. Subsequently, the short fragment of pAdΔE1P30 was digested by EcoRI and XbaI and cloned into pAd-ΔE1B to form the plasmid pAdΔE1P-ΔE1B. The enAFP promoter from the plasmid pDRIVE03-SV40enh/AFP(h)v04 (InvivoGen; Catalogue no. pdrive-sv40-hafp) was amplified by polymerase chain reaction and subcloned into pAdΔE1P-ΔE1B by XhoI and SnaBI to form the plasmid pAd.enAFP-E1A-ΔE1B. The plasmid pAd.enAFP-E1A-ΔE1B-IL-24 was created by inserting the IL-24 expression cassette. The OVs Ad.enAFP-E1A-ΔE1B-IL-24 and Ad.enAFP-E1A-ΔE1B were generated by homologous recombination between pAd.enAFP-E1A-ΔE1B-IL-24 or pAd.enAFP-E1A-ΔE1B with the adenovirus packaging plasmid pBHGE3 (Microbix Biosystems, Toronto, ON, Canada) in HEK293 cells. Each recombinant adenovirus was isolated through three rounds of plaque purification in HEK293 cells and was then purified by ultracentrifugation in a cesium chloride gradient. Moreover, virus titers were determined using the tissue culture infectious dose 50 assay in HEK293 cells. Cells were infected with adenovirus at different 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.enAFP-E1A-ΔE1B, Ad.enAFP-E1A-ΔE1B-IL-24 or wild-type adenovirus (Ad-Wt) at a multiplicity of infection (MOI) of 10. After 5 h, the medium was removed, the cells were washed three times with phosphate-buffered saline (PBS) and 2 ml of fresh medium was added. At 2 days post-infection, the cells were collected, the virus was released by three freeze–thaw cycles and then the cells were centrifuged to collect the supernatant. Virus production was determined by tissue culture infectious dose 50 assay in HEK293 cells.

Cell viability assay

The cells were seeded in 96-well plates and treated with various adenoviruses. After infection for 4 days at the indicated MOI, the cell survival rate was evaluated using a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma, St Louis, MO). The medium was removed, and fresh medium containing MTT (5 mg ml−1) was added to each well. The cells were incubated at 37 °C for 4 h. Following incubation, 150 μl dimethylsulfoxide was added to the wells and mixed thoroughly on a shaker for 10 min. The absorbance from the plates was read at 595 nm with a DNA Expert Microplate Reader model GENios.

Cytopathic effect assay

Hep3B, Huh-7 and PLC hepatoma cell lines, as well as the QSG-7701 and L-02 normal liver cell lines were grown to subconfluence and infected with adenovirus at the indicated MOIs. At 4 days after infection, the cells were stained with 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 previously. The cells were harvested by trypsinization and resuspended in lysis buffer (62.5 mM Tris-HCl (pH 6.8), 2% sodium dodecylsulfate, 10 mM glycerol, 1.55% dithiothreitol). The total protein concentration was determined by the BCA Protein Assay kit (Pierce Corporation, Rockford, IL, USA). The protein samples were separated by 10–15% sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Millipore Corporation, Billerica, MA, USA). The membranes were blocked in a 5% bovine serum albumin solution and incubated with primary antibodies, followed by secondary fluorescent antibodies. Antibodies were detected with an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). The primary antibodies included mouse monoclonal anti-mda-7 (melanoma differentiation associated gene 7)/IL-24 (GenHunter, Nashville, TN, USA), rabbit polyclonal anti-cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA), β-actin, BAX, rabbit monoclonal (Epitomics, Burlingame, CA, USA), poly-(ADP-ribose) polymerase-1/2 (H-250) rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), E1A mouse monoclonal (Abcam, Cambridge, UK), cytochrome c rabbit polyclonal antibody (Cell Signaling Technology), Bcl-2 rabbit polyclonal antibody (HuaAn Biotech, Hangzhou, China), caspase-8 mouse monoclonal antibody (Cell Signal Technology), Fas rabbit monoclonal antibody (Cell Signal Technology) and GADD34 rabbit polyclonal antibody (Santa Cruz Biotechnology).

Hoechst 33342 staining

Huh-7 cells were seeded in 6-well culture plates with slides and infected with Ad.enAFP-E1A-ΔE1B, Ad.enAFP-E1A-ΔE1B-IL-24 or Ad-Wt at an MOI of 5. Uninfected cells served as a control. After 48 h, the cells were treated with the double stain apoptosis detection kit, Hoechst 33342 (Beyotime Corporation, Haimeng, Jiangsu, China), for 5–10 min, washed with PBS twice and observed under a fluorescence microscope.

Flow cytometry analysis

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

Studies on 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 US Department of Agriculture and the National Institutes of Health. Female BALB/c nude mice at 4–5 weeks of age were obtained from the Animal Research Committee of the Institute of Biochemistry and Cell Biology (Shanghai, China) and were used in all of the experiments. Huh-7 cells (5 × 106) were injected subcutaneously into the head and neck region of female nude mice. After about 2 weeks, the tumor xenograft model was established. Each group was comprised of at least eight animals, and the tumor growth was monitored and measured with a vernier calliper. Tumor volume (V) was calculated using the formula V (mm3)=½ × length (mm) × width (mm)2. When the tumors from different experiment-designed groups reached approximately 390 or 90 mm3 in size, the mice were randomly divided into different groups. A daily dose of 4 × 108 plaque-forming units of examined viruses suspended in 100 μl of PBS or 100 μl PBS alone was administered intratumorally once per day for a total of 5 days. The tumors were harvested on the indicated number of days post-treatment with adenovirus and were then subject to hematoxylin and eosin staining, immunohistochemical (IHC) study and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining.

IHC study

For IHC analysis, tumors or livers on day 6 post-treatment were harvested and fixed in 4% paraformaldehyde, embedded in paraffin and cut into 10 μm sections. These sections were stained with IL-24 mouse monoclonal antibody and goat monoclonal anti-adenoviral hexon antibody at a 1:300 and 1:200 dilutions, respectively. The slides were then washed with PBS, incubated with the avidin–biotin–peroxidase complex reagent (Vector Laboratories, Burlingame, CA, USA) and detected with diaminobenzidine tetrahydrochloride 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 h 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, USA). After examining the 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).

Apoptotic assay by TUNEL

The TUNEL assay method was used to detect apoptotic cells. The TUNEL reaction preferentially labels DNA strand breaks that are generated during apoptosis and allows discrimination of apoptosis from necrosis and primary DNA strand breaks induced by apoptotic agents. For this purpose, an in situ cell apoptosis detection kit (Roche, Basel, Switzerland) was used. The staining was carried out according to the manufacturer's procedures. Tissue sections in the PBS group were stained and served as positive controls.

Serum aspartate aminotransferase and alanaine aminotransferase assay for hepatoma xenograft mice

To show the enhanced safety of Ad.enAFP-E1A-ΔE1B-IL-24 in vivo, Ad.enAFP-E1A-ΔE1B, Ad.enAFP-E1A-ΔE1B-IL-24 or Ad-Wt (2.0 × 109 plaque-forming units per mouse) was intratumorally injected into nude mice (n=3 per group) according to the previously described procedure. The PBS-treated tumor group and the healthy mice group were both used as controls. At the 6th or 40th day post-injection, the nude mice were killed, their blood was collected through the orbit and liver function was examined according to a related standard procedure.31

Statistical analysis

The statistical significance of the experimental results was calculated with the analysis of variance and the Student's t-test. The data were considered statistically significant at P<0.05.

Results

Construction and characterization of Ad.enAFP-E1A-ΔE1B-IL-24

The enAFP promoter has been shown to be a promising candidate for the construction of a hepatoma-specific oncolytic adenovirus.14 With the deletion of the total E1B region, the Ad-E1A-ΔE1B was formed. The dually regulated oncolytic adenovirus Ad enAFP-E1A-ΔE1B-IL-24 is driven by the enAFP promoter and harbors the antitumor IL-24 expression cassette controlled by the murine cytomegalovirus promoter (Figure 1a).

Figure 1
figure1

Characterization of the oncolytic adenovirus Ad.enAFP-E1A-ΔE1B-IL-24 and its selective replication in liver cancer cells. (a) Schematic structure of the recombinant oncolytic adenovirus. All viruses were created using the backbone of wild-type Ad5 (Ad-Wt). As for Ad.enAFP-E1A-ΔE1B-IL-24, the native E1A promoter was replaced by the AFP promoter modified with the SV40 enhancer at its 5′ flank, and both E1B-19 kDa and E1B-55 kDa genes were deleted to construct Ad.enAFP-E1A-ΔE1B, which was further modified with the interleukin (IL)-24 expression cassette driven by the murine cytomegalovirus promoter (mCMV) to form the gene-virus Ad.enAFP-E1A-ΔE1B-IL-24. ITR, inverted terminal repeats. (b–d) Western blots of E1A and IL-24. Huh-7 cells were infected with Ad.enAFP-E1A-ΔE1B-IL-24, Ad.enAFP-E1A-ΔE1B or Ad-Wt at the multiplicity of infection (MOI) of 5 for 48 h. Mock-infected cells were included as a control. The lysates from control and infected cells were subjected to western blot (WB) assay with anti-E1A antibody as described in the Materials and methods section. Simultaneously, the WB assay to measure E1A expression was performed on hepatoma cells (Huh-7, PLC, Hep3B, SMMC-7721 and HepG2) and normal cells (L-02 and QSG-7701) infected at an MOI of 5 and harvested after 48 h. IL-24 expression was measured by WB at 48 and 72 h in Huh-7 cells infected at an MOI of 5 with Ad.enAFP-E1A-ΔE1B-IL-24 or Ad.enAFP-E1A-ΔE1B. β-Actin was used as a protein loading control. (e) A total of 3.5 × 105 cells were plated into 6-well plates. After 24 h, the cells were infected at an MOI of 10 with Ad.enAFP-E1A-ΔE1B-IL-24, Ad.enAFP-E1A-ΔE1B or Ad-Wt. After an additional 48 h, the medium and the cells were collected in 1.5 ml Eppendorf tubes and subjected to three freeze–thaw cycles. The collected supernatant was tested for virus production by a standard tissue culture infectious dose 50 (TCID50) assay on 293 cells. Progeny viruses from 1 MOI of virus were calculated. The results were the average of two independent experiments. AFP, α-fetoprotein.

All the constructs were characterised by western blot assay. In Figure 1b, Huh-7 cells infected with Ad enAFP-E1A-ΔE1B, Ad enAFP-E1A-ΔE1B-IL-24 or Ad-Wt expressed the E1A protein, whereas mock-infected cells failed to express the E1A protein. Compared to Ad-Wt, our two constructed adenoviruses showed higher levels of E1A expression, and this expression, under control of the enAFP promoter, was specific to hepatoma cells (Figure 1c).

To examine IL-24 gene expression, Huh-7 cells were infected with Ad.enAFP-E1A-ΔE1B or Ad.enAFP-E1A-ΔE1B-IL-24 at an MOI of 5 for 48 or 72 h. The infected cells were then harvested and subjected to western blot assay. The transduction of Ad.enAFP-E1A-ΔE1B-IL-24 resulted in obvious IL-24 expression compared to no expression in control or Ad.enAFP-E1A-ΔE1B-transduced cells (Figure 1d). Moreover, the level of IL-24 expression in cells infected for 72 h with Ad.enAFP-E1A-ΔE1B-IL-24 was higher than that of cells infected for 48 h, which indicates that the IL-24 expression level gradually improved with the replication of the virus in Huh-7 cells. In addition, it was found that IL-24 was dramatically expressed in the other four HCC cell lines (PLC, SMMC-7721, HepG2, Hep3B) and enough IL-24 protein was secreted in each cell supernatant (Supplementary Data S1). To examine whether the transgene and modified genome of adenovirus could interfere with the selective replication ability of recombinant adenoviruses in different cell lines, a progeny assay was performed in tumor cells (HepG2, PLC, Hep3B, SMMC-7721 and Huh-7) and normal liver cell lines (L-02, QSG-7701) infected with the different adenovirus constructs or Ad-Wt. As shown in Figure 1e, Ad.enAFP-E1A-ΔE1B and Ad.enAFP-E1A-ΔE1B-IL-24 replicated in the tumor cells at similar levels, which were comparable to that of Ad-Wt. In contrast, the replication capacity of these viruses was attenuated in normal cells. These data indicate that the expression of the IL-24 gene and the deletion of E1B did not affect the selective replication ability of the oncolytic adenoviruses.

Ad.enAFP-E1A-ΔE1B-IL-24 exerts potent hepatoma-specific cytotoxicity

To test the cytotoxicity of the recombinant adenovirus in hepatoma (Huh-7, HepG2, PLC, SMMC-7721 and Hep3B) and normal (QSG-7701, L-02) liver cell lines, each cell line was infected with Ad.enAFP-E1A-ΔE1B-IL-24, Ad.enAFP-E1A-ΔE1B or Ad-Wt at an MOI of 10 for 1, 2, 3 or 4 days. In Figure 2a, Ad.enAFP-E1A-ΔE1B-IL-24 and Ad.enAFP-E1A-ΔE1B exerted stronger cytotoxicity than the Ad-Wt in hepatoma cells. In normal cells (QSG-7701, L-02), the recombinant adenoviruses, but not the Ad-Wt, exhibited almost no cytotoxicity. The other tumor cell lines, such as SW620 (colon cancer) and SKOV3 (ovarian cancer), were infected with Ad-Wt, ONYX-015, Ad.enAFP-E1A-ΔE1B-IL-24 or Ad.enAFP-E1A-ΔE1B at an MOI of 0.1, 1, 10 or 100 for 4 days and then tested for cell viability using the MTT assay as shown in Figure 2b. The results demonstrated that Ad.enAFP-E1A-ΔE1B-IL-24 and Ad.enAFP-E1A-ΔE1B had lower cytotoxic effects on these tumor cells than did Ad-Wt and ONYX-015. However, compared to Ad.enAFP-E1A-ΔE1B, Ad.enAFP-E1A-ΔE1B-IL-24 showed some cytotoxicity, which may be due to the transduction of the cancer-specific IL-24 gene. In sum, the above data indicate that Ad.enAFP-E1A-ΔE1B-IL-24 exerted potent hepatoma-selective cytotoxicity, and this was not observed in normal liver cells.

Figure 2
figure2

Selective cytotoxicity of gene-viruses in tumor cells in vitro. (a) Survival rate of tumor cell lines (Huh-7, PLC, Hep3B, SMMC-7721 and HepG2) and normal cells (L02 and QSG-7701). All cells were infected with Ad.enAFP-E1A-ΔE1B-IL-24, Ad.enAFP-E1A-ΔE1B or wild-type adenovirus (Ad-Wt) at a multiplicity of infection (MOI) of 10. After 96 h, the cell survival rate was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (b) The ovarian cancer cells (SKOV3) and colon cancer cells (SW620) were infected with Ad.enAFP-E1A-ΔE1B-IL-24, Ad.enAFP-E1A-ΔE1B, ONYX-015 or Ad-Wt at an MOI of 0.1, 1, 10 and 100. After 96 h, the cell viability was observed by MTT assay. The above results were expressed as a percentage of the results in the untreated control. At least three independent experiments were conducted; mean±s.d. (c) Comparison of gene therapy, virotherapy and hepatoma-targeting gene-viro-therapy. Tumor cells (Huh-7, PLC and Hep3B) and normal cells (L-02 and QSG-7701) were seeded in 24-well plates at a density of 5 × 104 cells for each well and infected with Ad.enAFP-E1A-ΔE1B-IL-24, Ad.enAFP-E1A-ΔE1B, ONYX-015 or Ad-IL24 at the indicated MOIs. After 6 days, cells were stained with crystal violet. AFP, α-fetoprotein.

Comparison of the anti-hepatoma activity of gene therapy, oncolytic virotherapy and targeting gene-viro-therapy in vitro

To detect the cytopathic effects of gene therapy, oncolytic virotherapy and targeting gene-viro-therapy, the hepatoma cell lines (Huh-7, Hep3B, PLC) and the normal liver cells (L-02, QSG-7701) were infected with Ad-IL-24, ONYX-015, Ad.enAFP-E1A-ΔE1B. and Ad.enAFP-E1A-ΔE1B-IL-24. The cells were infected at the indicated MOIs for 4 days and stained with crystal violet in Figure 2c. A significant cytopathic effect was observed in all tumour cell lines infected with Ad.enAFP-E1A-ΔE1B-IL-24 when compared to cells infected with Ad.enAFP-E1A-ΔE1B, ONYX-015 or Ad-IL-24. Moreover, the cytopathic effect of Ad.enAFP-E1A-ΔE1B-IL-24 treatment in vitro was superior to that of Ad.enAFP-E1A-ΔE1B and ONYX-015 and was approximately 1000 times greater than that of Ad-IL-24 in the PLC, Hep3B and Huh-7 cell lines. With regard to the four viruses, a dramatic cytopathic effect could not be detected in the two normal cell lines (QSG-7701 and L-02) at the same MOIs used to treat the tumor cells, although cytotoxicity was observed in the two normal cell lines infected with ONYX-015 at an MOI of 100. These results suggest that Ad.enAFP-E1A-ΔE1B-IL-24 can selectively inhibit the growth of hepatoma cells with more efficiency and enhanced safety when compared to ONYX-015 (oncolytic virotherapy).

Ad.enAFP-E1A-ΔE1B-IL-24 induces intrinsic and extrinsic apoptosis in Huh-7 cells

On the basis of the potent anti-hepatoma activity, cellular apoptosis was investigated as the underlying mechanism of the observed antitumor effects using a Hoechst 33342 staining assay. Huh-7 cells were infected with various adenoviruses at an MOI of 5 for 48 h. Compared to the control or Ad-Wt group, both Ad.enAFP-E1A-ΔE1B-IL-24 and Ad.enAFP-E1A-ΔE1B induced remarkable apoptotic morphological changes, such as obvious chromosome condensation and nuclear fragmentation as indicated by the arrows in Figure 3a.

Figure 3
figure3

The detection of apoptosis and the activation of intrinsic and extrinsic pathways mediated by Ad.enAFP-E1A-ΔE1B-IL-24 in tumor cells. (a) Huh-7 cells were stained with Hoechst 33342 after infection with the two adenoviruses at a multiplicity of infection (MOI) of 5 for 48 h. Positive apoptotic cells are indicated with arrows. Original magnification, × 200. (b) Apoptosis detection assay after infection with Ad.enAFP-E1A-ΔE1B-IL-24 by annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI). Huh-7 cells were infected for 48 h at an MOI of 5 or 10 with Ad.enAFP-E1A-ΔE1B-IL-24, Ad.enAFP-E1A-ΔE1B or wild-type adenovirus (Ad-Wt), and uninfected cells as a control. The percentage of apoptotic cells was calculated with the CellQuest software (BD Biosciences). The percentage of apoptotic cells is indicated after various treatments as described above in the Materials and methods section. *P<0.05, mean±s.d. (c) Proteins associated with the intrinsic and extrinsic apoptotic pathways, such as caspase-3, poly-(ADP-ribose) polymerase (PARP), Fas, caspase 8, cytochrome c (cyto c), Bcl-2 and Bax, were detected. The ER-stress protein GADD34, which is downstream of apoptosis, was also detected. AFP, α-fetoprotein.

The fluorescence-activated cell sorting assay revealed that the apoptosis caused by Ad.enAFP-E1A-ΔE1B-IL-24 was more significant (P<0.01) than that caused by the Ad-Wt or Ad.enAFP-E1A-ΔE1B group when cells were infected at an MOI of 5 or 10 for 48 h (Figure 3b). The untreated group served as the negative control. To further confirm the induction of apoptosis and to explore the molecular mechanism of tumor cell apoptosis, the activation of the caspase-related pathway was examined. As shown in Figure 3c, the cleavage of caspase-3 and poly-(ADP-ribose) polymerase was obviously increased in Huh-7 cells treated with Ad.enAFP-E1A-ΔE1B-IL-24 when compared to the cells treated with Ad.enAFP-E1A-ΔE1B or Ad-Wt, which indicates that apoptosis was induced. Fas and released cytochrome c were detected by western blotting to measure the activation of the extrinsic and intrinsic pathways, respectively, of classical apoptosis. As shown in Figure 3c, these molecular markers were dramatically increased by Ad.enAFP-E1A-ΔE1B-IL-24 compared to all other groups. Furthermore, changes in the molecular markers indicating the activated apoptotic process, such as a decrease in BCL-2 and an increase in Bax and GADD34, were confirmed. The above results demonstrate that the mechanism of apoptosis is mediated by Ad.enAFP-E1A-ΔE1B-IL-24 in Huh-7 cells through both extrinsic and intrinsic pathways.

Potent anti-hepatoma efficacy at initial 390 mm3 volume and complete eradication of hepatomas at routine 90 mm3 volume by the treatment of Ad.enAFP-E1A-ΔE1B-IL-24 in HCC xenograft model

The antitumor effect of Ad.enAFP-E1A-ΔE1B-IL-24 in vivo was studied with a Huh-7 hepatoma left head–neck xenograft model established in nude mice. When the tumors grew to approximately 390 mm3, PBS, Ad-Wt, Ad.enAFP-E1A-ΔE1B or Ad.enAFP-E1A-ΔE1B-IL-24 (5 × 108 plaque-forming units per dose) in 100 ml PBS was injected intratumorally every day for a total of 5 days. As shown in Figure 4a, animals treated with Ad.enAFP-E1A-ΔE1B-IL-24 exhibited a remarkable suppression of tumor growth when compared to the PBS-treated animals (P<0.005) and Ad-Wt-treated animals (P<0.005), as well as the Ad.enAFP-E1A-ΔE1B-treated animals (P<0.05). Notably, the average tumor volume injected with Ad.enAFP-E1A-ΔE1B-IL-24 was 645 mm3 at day 41, whereas the tumor volume of PBS-, Ad-Wt- or Ad.enAFP-E1A-ΔE1B-treated mice was 4015, 3409 and 1244 mm3, respectively. At the 47th day, about 80% inhibition of the Huh-7 liver cancer xenograft in nude mice by direct Ad.enAFP-E1A-ΔE1B-IL-24 intratumoral injection were observed. In addition, the Ad.enAFP-E1A-ΔE1B-treated group showed notable antitumor efficacy compared to both the PBS- and Ad-Wt-treated groups (P<0.01). Moreover, the survival of animals was also monitored for approximately 110 days and analyzed using the Kaplan–Meier method. All the animals treated with Ad.enAFP-E1A-ΔE1B-IL-24 survived (six out of six), five out of six survived in the Ad.enAFP-E1A-ΔE1B group and zero out of six survived in the PBS and Ad-Wt groups (Figure 4b).

Figure 4
figure4

Potent antitumor efficacy of Ad.enAFP-E1A-ΔE1B-IL-24 in nude mice. Female BALB/c nude mice were subcutaneously inoculated with Huh-7 cells at the head and neck region (5 × 106 cells per 100 μl). When tumors reached a size of 350–430 mm3, the animals were treated with an intratumoral injection of Ad.enAFP-E1A-ΔE1B-IL-24, Ad.enAFP-E1A-ΔE1B, wild-type adenovirus (Ad-Wt) or phosphate-buffered saline (PBS). (a) Tumor size was measured, and tumor volume was calculated. Data are presented as the mean±s.d. (n=6). (b) The death of the animals was monitored. The long-term survival of the animals was observed after treatment with oncolytic adenoviruses and compared to control animals receiving PBS. (c) Female BALB/c nude mice were subcutaneously inoculated with Huh-7 cells at the head and neck region (5 × 106 cells per 100 μl). When tumors reached a size of 90 mm3, the animals were treated with an intratumoral injection of Ad.enAFP-E1A-ΔE1B-IL-24 or PBS (injected virus with a daily dose (4 × 108 plaque-forming units (PFU)) for 5 consecutive days). Data are presented as the mean±s.d. (n=6). AFP, α-fetoprotein.

The data stated above was from the large (390 mm3) initial HCC volume, whether the better antitumor effect may be obtained from a smaller initial HCC volume with the treatment of Ad.enAFP-E1A-ΔE1B-IL-24, so a model with an initial routine 90 mm3 HCC volume was established. Remarkably, the HCC xenografts were completely eradicated within 12 days (Figure 4c).

Morphological evidence of Ad.enAFP-E1A-ΔE1B-IL-24 effect on 390 mm3 volume tumor growth inhibition

To determine the pathological changes in tumor sections, hematoxylin and eosin staining showed that Ad.enAFP-E1A-ΔE1B-IL-24-treated mice had a wider area of necrosis than those of any other group (arrow in Figure 5a). The hexon protein, a classic marker for adenovirus, was positive by the IHC assay in the tumor sections treated with the different viruses (Figure 5b) compared to the PBS group, which was used as a negative control. In addition, the exogenous therapeutic IL-24 gene exhibited remarkable expression in the group infected with Ad.enAFP-E1A-ΔE1B-IL-24 (Figure 5c). Improved IL-24 expression followed the replication of the recombinant adenovirus Ad.enAFP-E1A-ΔE1B-IL-24 and induced obvious apoptosis, as detected by the TUNEL assay (Figure 5d). In addition, morphological changes of the virus-treated tumor cells were observed with the transmission electron microscopy assay. In Figure 5e, compared to the PBS-treated group, the virus-treated tumor cells demonstrated the obvious characteristics of apoptosis, including nuclear collapse, an increased nuclear-to-cytoplasmic ratio, the appearance of a deformed nucleus and heterochromatin, and the condensation of chromatin at the inner side of the nuclear envelope. In the Ad.enAFP-E1A-ΔE1B group, virus particles appeared within the tumor cells, and the endothelial cells from the vessel showed the apoptotic stage in both recombinant groups, which was confirmed with the TUNEL assay.

Figure 5
figure5

Evidence of antitumor efficacy without liver toxicity from Ad.enAFP-E1A-ΔE1B-IL-24 in vivo. Both pathologic detection by hematoxylin and eosin (H.E), immunohistochemical (IHC) and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining assay and morphology observations on transmission electron microscopy (TEM) analysis were performed. Subcutaneous Huh-7 tumors receiving various treatments were harvested 6 days after infection with viruses, and tumor sections were treated as described above in the Materials and methods section. (a) H.E staining assay. Partial areas of necrosis have been detected in the sections treated with Ad.enAFP-E1A-ΔE1B-IL-24 and Ad.enAFP-E1A-ΔE1B (indicated with the black arrows), compared to the wild-type adenovirus (Ad-Wt) group ( × 100, original magnification). (b) Hexon expression by IHC analysis ( × 400, original magnification). (c) IHC analysis for IL-24 expression ( × 400, original magnification). As above, the brown-stained signals are indicated with a black arrow. Hexon expression was positive in the three groups treated with Ad.enAFP-E1A-ΔE1B-IL-24, Ad.enAFP-E1A-ΔE1B and Ad-Wt, and IL-24-positive expression is only present in the group treated with Ad.enAFP-E1A-ΔE1B-IL-24. (d) The TUNEL assay for the detection of apoptotic cells in tumor sections ( × 400, original magnification). The black arrow indicates the obvious apoptotic cells in the Ad.enAFP-E1A-ΔE1B- and Ad.enAFP-E1A-ΔE1B-IL-24-treated groups. (e) In the Ad.enAFP-E1A-ΔE1B- or Ad.enAFP-E1A-ΔE1B-IL-24-treated groups, the virus particles and replication by TEM analysis are indicated by the arrow with the square. The magnified view shows the morphology of cell apoptosis, intracellular virus particles and the tumor blood vessels by TEM analysis. AFP, α-fetoprotein.

The liver toxicity and changes of the alanine aminotransferase and aspartate aminotransferase serum level caused by the constructed viruses

Given the liver sequestration of type 5 adenoviruses, the liver toxicity of the constructs were tested. The IHC assay for hexon (Figure 6a) showed that Ad-Wt exerted the strongest liver tropism, while Ad.enAFP-E1A-ΔE1B and Ad.enAFP-E1A-ΔE1B-IL-24 had a very weak or almost negative response, respectively. In the meantime, the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels of serum were measured in each group (Figure 6b). Compared to the healthy mice, there was no significant difference in ALT levels in any treatment group. However, the level of AST in the Ad-Wt and PBS-treated groups had greatly increased at the 6th and 40th day, respectively. There was no significant difference between the recombinant groups, indicating that the administration of Ad.enAFP-E1A-ΔE1B and Ad.enAFP-E1A-ΔE1B-IL-24 did not cause hepatotoxicity.

Figure 6
figure6

Ad.enAFP-E1A-ΔE1B-IL-24 did not cause hepatotoxicity. (a) No detected liver sequestration or liver toxicity of Ad.enAFP-E1A-ΔE1B-IL-24 in vivo. On day 6 post-treatment with various viruses, liver tissues were harvested and examined for the expression of the positive adenoviral marker, hexon, by immunohistochemical (IHC) assay. Phosphate-buffered saline (PBS)-treated group was used as a negative control. (b) To verify the influence of Ad.enAFP-E1A-ΔE1B-IL-24 on the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in vivo, the virus was intratumorally injected into nude mice. The PBS-treated tumor group or the healthy mice group (control) was used as different control groups. At the 6th or 40th day post-injection for each group, the nude mice were euthanized, and their blood collected through an eyeball. The serum levels of AST and ALT were examined according to the procedures in the Materials and methods section. Mean±s.d. (**P<0.01). AFP, α-fetoprotein.

Discussion

An increasing number of novel therapies for the treatment of cancer have been developed. The strategy of CTGVT was initiated by our laboratory. In 2001, a CTGVT was constructed by inserting an antitumor gene into the OV. It is actually an OV-gene that has combined the superiority of both gene therapy (Ad-gene) and the OV therapy.3, 4 We continued the studies for over 10 years and found that the antitumor effect of CTGVT (OV-gene) was much higher than that of gene therapy or OV therapy alone, with the antitumor effect order of CTGVT (OV-gene)>OV> Ad-gene, which was reflected in our approximately 60 peer-reviewed SCI papers. Currently, the best antitumor drugs in the gene therapy and OV therapy fields come from CTGVT (OV-gene), as we predicted many years ago. For example, CTGVT (OV-gene) from oncolytic herpes simplex virus harboring granulocyte-macrophage colony-stimulating factor (GM-CSF) (that is, OncoVEX GM-CSF) has shown a potent anti-melanoma effect in a phase II clinical trial and is currently in phase III for advanced melanoma, which cost Amgen Inc (Thousand Oaks, CA, USA) one billion USD. OncoVEX GM-CSF is also being tested in a pivotal phase III trial in head and neck cancer, and is likely to become the first approved oncolytic agent in the Western world.32 Another OV in development is Oncolytics Biotech's Phase III head and neck cancer candidate, Reolysin.33 Furthermore, the poxvirus derived Oncopox-GM-CSF product, JX-594, was studied in detail and has been published in Nature Medicine because of its potent antitumor effect via blood administration.34 The adenovirus derived OncoAd-GM-CSF (that is, KH901), discovered by Lei et al.35 had promising results in the phase II clinical trial in China. OncoAd-IL-24, the ZD55-IL-24,20 will also go to clinical trial soon because it has a much better antitumor effect than that of Ad-IL-24, which had passed phase II clinical trials and is undergoing phase III clinical trials.36, 37 ZD55-IL-24 may have a better antitumor effect than that of OncoAd-GM-CSF because IL-24 is a very strong antitumor cytokine. The above information indicates that CTGVT (OV-gene) is the best antitumor strategy, better than that of OV therapy or gene therapy alone.

CTGVT specific for liver cancer therapy has been conducted by using the AFP promoter to drive E1A expression because E1A is an important element for targeting the adenovirus infection. The agents Ad.AFP-E1A-E1B(Δ55)-SOCS3 and Ad.AFP-E1A-E1B(Δ55)-TRAIL were constructed in our lab.14, 28 Both have potent antitumor effects and have been issued patents (Chinese No. 201110142101-9), but they cannot completely eradicate liver cancer in vivo, even in combination with two strong antitumor genes. In this study, the intratumoral injection of Ad.AFP-E1A-ΔE1B-IL-24 completely eliminated the tumor and also strongly inhibited the larger volume initial HCC tumor growth (as shown in Figure 4), which has not been previously reported in the literature.

The reasons for the potent antitumor effect of Ad.enAFP-E1A-ΔE1B-IL-24 are as follows.

(1) The use of the CTGVT (OV-gene) strategy: Ad.AFP-E1A-ΔE1B is an OV that replicates specifically in cancer cells, and as a result, the inserted IL-24 gene increases its expression several 100-fold, greatly increasing its antitumor effect.

(2) Deletion of the 19 kDa gene: Adenovirus E1B proteins (including E1B-19 kDa and E1B-55 kDa) play an important role in the inhibition of E1A-mediated apoptosis in a productive infection of human cells and in the transformation of primary rodent cells.38, 39 It was reported that the antiapoptotic gene E1B-19 kDa has a cell survival-promoting domain that is common to BCL-2 proteins,40 and the E1B-19K protein does not play an essential role in regulating viral early gene expression or viral growth in human cancer cells, such as A549 and HeLa.41 Deletion of E1B-19 kDa can remarkably increase the adenovirus cytolytic activity in 18 of 21 melanoma cells.42 However, the E1B-55 kDa-deleted replicating adenovirus, ZD55, constructed in our laboratory, and OYNX-015, which had been in phase III clinical trials, have been noted for their ability to enhance the effects of chemotherapy, such as 5-fluorouracil or cisplatin.43 Apoptosis is one of the most studied mechanisms accounting for the antitumor effects of the adenoviruses. However, these vectors still retain the strong antiapoptotic E1B-19kDa gene in their genome, which may interfere with the mode of action of the drugs and further lead to an underestimation of their clinical value. Previous work showed that the E1B-19 kDa and E1B-55 kDa double-deleted oncolytic adenovirus, Ad-E1B19/55, leads to improved cytotoxicity and apoptosis compared to the E1B-55 kDa single-deleted oncolytic adenovirus, Ad-ΔE1B55,44 or wild-type adenovirus. In addition, cisplatin in combination with the E1B-19 kDa and E1B-55 kDa double-deleted oncolytic adenovirus has a markedly enhanced cancer therapeutic efficacy than that of the E1B-55 kDa single-deleted adenovirus.45 Moreover, the Adeno-hTERT-E1A human telomerase reverse transcriptase promoter-driven oncolytic adenovirus with E1B-19 kDa and E1B-55 kDa gene deletions showed a strong antitumor activity in vivo in an MDA-MB-231 solid tumor xenograft model and has an improved safety profile compared with ONYX-015, which may lead to reduced toxicity in the clinic.46 Thus, to obtain a stronger antitumor effect, we modified the vector AFP.ZD55 with an E1B-19 kDa deletion to form the novel hepatoma-targeting adenovirus vector, Ad.enAFP-E1A-ΔE1B. In comparison to ONYX-015, this new vector has an obviously improved selective cytopathic response for different hepatoma cell lines, but not normal liver cells (as shown in Figure 2c). With regard to other types of cancer (Figure 2b), this gene-armed adenovirus vector has less cytotoxicity than ONYX-015 or Ad-Wt, suggesting that the AFP promoter-driven, double-regulated oncolytic adenovirus vector can specifically target HCC cells with better cytolysis ability while being safe for normal cells or other tissues.

(3) The use of a potent antitumor IL-24 gene/mda-7:IL-24 was initially identified by subtraction hybridization in melanoma treated with interferon-γ and MEZ.47 On the basis of its structural characteristics, chromosomal location and receptor, mda-7 has been renamed IL-24, a novel member of the IL-10 family. IL-24 induces strong apoptotic effects20, 23 through the GADD34 or P-PKR, p38-MAPK pathway;48, 49, 50 strong antiangiogenesis effects in the tube formation of human umbilical cord vein endothelial cells;22 strong immune augmentation with the induction of IL-6, TNF-α, interferon-γ, IL-1β, IL-12 and GM-CSF from human peripheral blood mononuclear cells,51 which can account for its antitumor effect in vivo being superior to that in vitro; the bystander antitumor effect and inhibition of distant cancers;52 and inhibition of invasion and migration.53

The use of enAFP composite promoter was enhanced by SV40 enhancer. AFP is preferentially expressed in over 70% of human liver cancer patients through transcriptional activation.54 In our previous report,14 the transcriptional activity of the enAFP composite promoter in different cell lines was consistent with the observed secretion levels of AFP and addition of the SV40 enhancer did not alter specificity of the AFP promoter. It is worth noting that the enAFP composite promoter showed very high transcriptional activity in HuH-7 cells, exceeding that of the pGL3-Control. Therefore, the enAFP promoter is a good candidate for hepatoma-specific targeting.

In short, the above four points, as a whole, are necessary for the specific and complete elimination of liver cancer xenografts with Ad.enAFP-E1A-ΔE1B-IL-24 treatment. However, we prefer to emphasize the deletion of the 19 kDa gene in this construct. The major difference between Ad.enAFP-E1A-ΔE1B-IL-24 and Ad.enAFP-E1A-E1B(Δ55)-IL-2429 is the deletion of the 19 kDa gene in E1B. With the deletion of 19 kDa in our construct, the involved mechanism of the vector Ad.enAFP-E1A-ΔE1B-caused cell death was apoptosis in Figure 3a instead of autophagy induced by Ad.enAFP-E1A-E1B(Δ55).14 In addition, the Huh-7 liver xenografts treated with Ad.enAFP-E1A-ΔE1B-IL-24 can be completely eradicated. In contrast, maintaining the 19 kDa in the E1B region, even with the expression of the two genes IL-24-TRAIL or TRAIL-SOCS3, cannot achieve the antitumor effect described above despite the fact that two genes usually have more potent antitumor effect than one. Nevertheless, all four points described above are required, and by omitting any one, we may not achieve a complete eradication of the Huh-7 liver cancer xenograft. Furthermore, we did not observe any construct containing only the IL-24 gene or any gene therapy vector (adenovirus vector harboring IL-24, with or without a second gene) that could achieve a complete eradication of the liver cancer xenograft.

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Acknowledgements

We thank YanLing Ren and LanYing Sun for kind help in cell culture, and we also thank Prof YouChen Xu for good comments for writing. This work was supported by the National Nature Science Foundation of China (No. 30623003), the National Basic Research Program of China (973 Program) (No. 2010CB529901), Important National Science and Technology Specific Project of Hepatitis and Hepatoma Related Program (2008ZX10002-023), the National Basic Research Committee of Science and Technology (Nos. 06ZR14072 and 074119508); and the ZheJiang Sci-Tech University Grant No.1016819-Y.

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Correspondence to X-Y Liu.

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Supplementary Information accompanies the paper on Cancer Gene Therapy website

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Zhang, K., Zhang, J., Wu, Y. et al. Complete eradication of hepatomas using an oncolytic adenovirus containing AFP promoter controlling E1A and an E1B deletion to drive IL-24 expression. Cancer Gene Ther 19, 619–629 (2012). https://doi.org/10.1038/cgt.2012.40

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Keywords

  • Cancer Targeting Gene-Viro-Therapy
  • complete eradication
  • E1B deletion
  • hepatoma specificity
  • IL-24

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