Cancer (2012) 19, 49–57 r 2012 Nature America, Inc. All rights reserved 0929-1903/12 www.nature.com/cgt

ORIGINAL ARTICLE Gene-viro-therapy targeting liver cancer by a dual-regulated oncolytic adenoviral vector harboring IL-24 and TRAIL X Liu1,2, X Cao2, R Wei2, Y Cai2,HLi2, J Gui2, D Zhong2, X-Y Liu2 and K Huang1 1Tongji School of Pharmacy, Huazhong University of Science and Technology, Wuhan, China and 2Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

Cancer-targeting gene-viro-therapy is a promising cancer therapeutic strategy that strengthens the antitumor effect of oncolytic viruses by expressing an inserted foreign antitumor gene. To achieve liver cancer targeting and to improve the safety of the ZD55 vector (a widely-used E1B55KD gene-deleted oncolytic adenoviral vector (OV), we previously constructed), we designed a novel OV named Ad Á AFP Á D55 that selectively replicates in hepatocellular carcinoma (HCC) cells by replacing the E1A promoter with the liver-cancer specific a-Fetoprotein (AFP) promoter based on the ZD55 vector. We found that the oncolytic adenoviruses Ad Á AFP Á D55-IL-24 and Ad Á AFP Á D55-TRAIL express tumor-suppressor gene interleukin-24 (IL-24) and tumor necrosis factor- related -inducing ligand (TRAIL), respectively, significantly suppressed the HCC cell growth in vitro by inducing apoptosis by the caspase-8 and mitochondria-dependent caspase-9 signaling pathways. Furthermore, the combined treatment of Ad Á AFP Á D55-IL-24 and Ad Á AFP Á D55-TRAIL showed strong antitumor effects in vivo by significantly inhibiting the tumor growth in HCC HuH-7 cell xenograft mice, and markedly increasing animal survival rate. Therefore, this novel HCC cell-targeting OV carrying tumor-suppressor genes may provide a promising approach for liver cancer gene therapy. Cancer Gene Therapy (2012) 19, 49–57; doi:10.1038/cgt.2011.67; published online 7 October 2011 Keywords: cancer-targeting gene-viro-therapy; liver cancer; interleukin-24; tumor necrosis factor-related apoptosis-inducing ligand; apoptosis

Introduction antitumor gene is also replicated. Without exception, our past 10 years of work on this strategy has demon- Cancer is one of the most malignant diseases we are strated that CTGVT is a much more potent cancer facing. According to the World Health Statistics of WHO, therapeutic strategy than either gene therapy or oncolytic nearly eight million people die of cancer each year viral therapy. worldwide. Many therapeutic strategies have been devel- Recently, we have developed several new CTGVT oped to treat cancer, including gene therapy and oncolytic strategies, including the CTGVT-DG (Cancer-targeting adenoviral therapy. In 2001, we proposed a strategy that dual gene-viro-therapy) strategy, which combines two combined gene therapy and oncolytic viral therapy to tumor-suppressor genes that may have compensative or treat cancer by inserting an antitumor gene into an synergetic effects. We have shown that this new strategy oncolytic . We named this strategy as Cancer- can significantly suppress growth and eradicate targeting gene-viro-therapy (CTGVT).1 In principle, many xenograft tumors, such as cancer of liver, pancreas when the vector (OV) replicates in cancer and lung. Furthermore, combined treatment with cells as a gene delivery vector, the carried foreign ZD55-IL-24 (an OV carrying foreign gene IL-24) and ZD55-TRAIL completely eradicated colorectal xenograft tumors.2–5 Interleukin-24 (IL-24), (also known as Correspondence: Professor K Huang, Tongji School of Pharmacy, Mda-7, melanoma differentiation-associated gene-7) be- Huazhong University of Science and Technology, 13 Hang Kong longs to the interleukin 10 (IL-10)-related family. Over- Road, Wuhan, Hubei 430030, P.R. China or Professor X-Y Liu, expression of IL-24 can inhibit cancer cell growth and Laboratory of Molecular Cell Biology, Institute of Biochemistry and induce apoptosis in many cancer cell lines, including Cell Biology, Shanghai Institutes for Biological Sciences, Chinese carcinomas of melanoma, breast, lung, cervix, gliomas, Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, 6 P.R. China. colon, pancreas and prostate. Another antitumor gene, E-mails: [email protected] or [email protected] TRAIL (tumor necrosis factor-related apoptosis-inducing Received 2 June 2011; revised 4 August 2011; accepted 28 August ligand), is a type II transmembrane related to 2011; published online 7 October 2011 TNF family factors and is considered to be a promising Liver cancer-targeting gene-viro-therapy XLiuet al 50 anticancer agent.7 Much work has indicated that both the Construction of the oncolytic adenoviral shuttle vectors membrane-bound and the soluble extracellular domain of The construction of adenoviral shuttle plasmids for TRAIL can induce apoptosis in a broad type of tumor Ad Á AFP Á D55, Ad Á AFP Á D55-IL- cells without affecting most normal cells.8,9 Moreover, we 24 and Ad Á AFP Á D55-TRAIL were performed, as we had previously reported that the combination of two ther- previously described.10 apeutic genes mediated by replication-defective vectors can exert synergic or compensatory antitumor activities.2,4 Generation, identification, production, purification and Based on these studies, we decided to evaluate the effects titration of recombinant adenovirus of combined usage of IL-24 and TRAIL with a newly The recombinant adenoviruses (rec-Ads) were generated constructed dual-regulated OV Ad Á AFP Á D55,10 whose by respective between the replication was controlled by dual mechanisms (E1B- shuttle plasmids and the packaging plasmid pBHGE3 in 55KD deletion and E1A regulated by AFP promoter). HEK293 cells. Detailed procedures on the generation, The viral vector previously used in CTGVT in our identification, production, purification and titration of laboratory was the adenoviral vector ZD55 that was the recombinant adenovirus can be found in our previous constructed by deleting the E1B-55KD gene of wild-type report.10 human adenovirus (Ad) 5.11 This design was inspired by the first E1B-55KD gene-deleted oncolytic Ad ONYX- Cytotoxicity assays 015.12 We have demonstrated that the ZD55-gene system Tumor cells and normal cells were seeded in 24-well could be a potent antitumor strategy.2,11 Liver cancer is plates, and were infected with adenoviruses at various the most common malignant cancer in China, however, multiplicity of (MOIs) 24 h later. At 96 h after the OV ZD55 cannot specifically target liver cancer and , the medium was removed, and the cells were even shows modest liver toxicity.13 To overcome this stained with 2% crystal violet in 20% methanol for obstacle, we developed an OV that specifically targeted 20 min. The plates were then washed with water and hepatocellular carcinoma (HCC) cells and improved the documented as photographs. safety by replacing the E1A promoter of the ZD55 vector with a liver-cancer specific a-Fetoprotein (AFP) promo- Cell viability assays ter, which was reported to be preferentially expressed HuH-7 and Hep G2 cells were seeded in 96-well plates, in over 70% of human liver cancer patients through 24 h later, they were infected with adenoviruses at an MOI transcriptional activation.14,15 We named this new OV of 1. At 24, 48, 72 and 96 h after infection, 20 ml of 3-(4,5- as Ad Á AFP Á D55. In this work, we further inserted dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide IL-24 and TRAIL genes individually into Ad Á AFP Á D55 (MTT) (5 mg mlÀ1) was added to each well, and the to generate Ad Á AFP Á D55-IL-24 and Ad Á AFP Á D55- plates were incubated at 37 1C for 4 h. The medium was TRAIL, and then evaluated the combined antitumor then removed and 100 ml of 0.04 M HCl-isopropanol effects of treatment with these two new oncolytic Ads solution was added to each well. Absorbance was in vitro and in vivo. measured at 595 nm and 655 nm using a Microplate Reader (Thermo, Asheville, NC). For each assay, 10 replicate wells were counted.

Materials and methods DNA fragmentation assays Cell lines and culture conditions HuH-7 cells were seeded in 10-cm culture dishes and SW620 (human cell line), A549 (human infected with various Ads. At 72 h post infection, DNA lung adenocarcinoma cell line), BEAS-2B (human normal was extracted according to Herrmann’s method16 and bronchial epithelial cell line), Hep G2 (human HCC cell analyzed using agarose gel electrophoresis. line) and MRC-5 (human embryonic fibroblast cell line) were obtained from ATCC (Rockville, MD). L-02 Western blot assays (human normal hepatocyte cell line), Hep 3B (human Cells were harvested and washed with phosphate-buffered hepatoma cell line), HCCLM6 (human hepatoma cell saline (PBS), and then lysed in sodium dodecyl sulfate line), HuH-6 (human hepatoma cell line) and HuH-7 polyacrylamide gel electrophoresis sample buffer. Protein (human hepatoma cell line) were obtained from the Cell concentrations were determined using the Bio-Rad Bank of Type Culture Collection of the Chinese Academy protein assay buffer (Bio-Rad, Hercules, CA). A total of of Sciences (Shanghai, China). HEK293 (human embryo- 50 mg of protein was separated on 8–15% polyacrylamide nic kidney cell line) was obtained from Microbix gels and transferred to polyvinylidene fluoride mem- Biosystems Inc. (Toronto, Ontario, Canada). A549 and branes (Millipore, Billerica, MA). Western blots were L-02 was grown in RPMI 1640 (GIBCO BRL, Grand carried out using standard procedures. for Island, NY) supplemented with 4% fetal bovine serum actin, E1A, caspase-8, caspase-9, caspase-3, poly ADP- (GIBCO BRL); SW620 was grown in Dulbecco’s ribose polymerase, IL-24 and TRAIL were obtained from modified Eagle’s medium (GIBCO BRL) supplemented Santa Cruz Biotechnology (Santa Cruz, CA); with 4% fetal bovine serum. Other cell lines were cultured for E1B-55KD was obtained from Siemens Oncogene in Dulbecco’s modified Eagle’s medium supplemented Science Inc. (Cambridge, MA); secondary antibodies were with 10% fetal bovine serum. obtained from Santa Cruz Biotechnology.

Cancer Gene Therapy Liver cancer-targeting gene-viro-therapy XLiuet al 51 Flow cytometric analyses Results For fluorescein isothiocyanate and propidium iodide Characterization of Ad Á AFP Á D55-IL-24 and double-staining FACS, an Annexin V-fluorescein isothio- Ad Á AFP Á D55-TRAIL cyanate Kit (Bender MedSystems, Vienna, Austria) was We have previously confirmed the selective transcrip- used, and the cells were treated according to the tional activity of the AFP promoter in human AFP- manufacturer’s instructions. The FACS assay was per- positive HCC cells,10 suggesting Ad Á AFP Á D55 could be formed on a FACS Calibur flow cytometer (BD an ideal HCC-targeting vector system. In this work, we Biosciences, Franklin Lakes, NJ) immediately after inserted antitumor gene IL-24 or TRAIL into the OV staining. For JC-1 staining, cells were harvested and À1 Ad Á AFP Á D55 to enhance its curative effect (Figure 1a). resuspended in 0.5 ml of medium. 0.5 ml of 5 mgml JC-1 To exclude the possibility of Ad.WT contamination and fluorescent probe (Sigma, St Louis, MO) was added into study whether the E1A expression of Ad Á AFP Á D55 is 1 the medium. Cells were incubated at 37 C for 20 min and under the control of the AFP promoter, the E1B-55KD washed with PBS making them ready for FACS analyses. and E1A expression levels were examined by western blot analysis. As shown in Figure 1b, the E1B-55KD protein was only detected in Ad.WT-infected cells, neither Animal experiments Ad Á AFP Á D55-IL-24 nor Ad Á AFP Á D55-TRAIL could Animal experiments were performed according to the induce E1B-55KD expression, suggesting no Ad.WT SIBS Guide for the Care and Use of Laboratory Animals. contamination in rec-Ads. Western blots also demon- Female BALB/c nude mice (4-week-old) were purchased strated that the E1A expression levels in Ad Á AFP Á D55- from the Shanghai Experimental Animal Center (Shang- 6 infected HCC cells were much higher than those in hai, China). To establish xenograft tumors, 5 Â 10 HuH- normal cells or non-HCC tumor cells (Figure 1b). Because 7 cells in 150 ml Dulbecco’s modified Eagle’s medium were the E1A protein is critical to the replication of Ads, these subcutaneously injected into the right flank of each 3 results also provide direct evidence for the HCC-targeting mouse. When tumors reached 100–150 mm in volume, replication ability of this OV. To verify whether the mice were divided randomly into five groups (8 mice per 9 foreign IL-24 or TRAIL gene is expressed in vitro,we group). A total of 2 Â 10 plaque-forming units of further infected HCC HuH-7 cells with Ad Á AFP Á D55- adenoviruses per mouse or PBS (for control sample) were IL-24 and Ad Á AFP Á D55-TRAIL at an MOI of 10, and injected into the tumors for a total of four times every 3 performed western blot studies 48 h later. As shown in 48 h. Tumor volume (mm ) was measured by a Vernier Figure 1c, Ad Á AFP Á D55-IL-24 and Ad Á AFP Á D55- caliper every 3 days and calculated as the length TRAIL infections could significantly induce IL-24 and (mm) Â width (mm) Â (width (mm)/2). TRAIL in HuH-7 cells, respectively; suggesting these OVs could efficiently express the carried foreign genes in vitro.

Immunohistochemistry and terminal deoxynucleotidyl Ad Á AFP Á D55-IL-24/TRAIL specifically inhibits the transferase-mediated dUTPbiotin nick end labeling proliferation of hepatoma cell lines. assays To evaluate the cytopathic effect induced by the rec-Ads, Tumor tissues were fixed in 4% formaldehyde, dehy- cell lines of liver cancer (HuH-7 and Hep G2), colorectal drated with an ethanol gradient and embedded in paraffin. Tissue sections were dewaxed and rehydrated according to a standard protocol. The sections were washed with PBS, treated with 3% H2O2, and blocked with blocking solution. These steps were followed by overnight incubation with primary antibody at the proper dilution. Following the manufacturer’s instructions, we used the mouse or goat ABC Staining System (Santa Cruz Biotechnology), and hematoxylin was used as a counter- stain. Apoptotic cells in tumor tissue sections were assessed by TUNEL (terminal deoxynucleotidyl transfer- ase-mediated dUTP-biotin nick end labeling) staining with a TACS TdT Kit In Situ Apoptosis Detection Kit (R&D, Minneapolis, MN) according to the manufac- turer’s protocol. All sections were visualized with a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan). Figure 1 Characterization of the rec-Ads Ad Á AFP Á D55-IL-24/TRAIL. (a) A schematic drawing of the Ad Á AFP Á D55-IL-24/TRAIL. C is the encapsidation signal, ITR is the inverted terminal repeat, AFP is Statistical analyses a-Fetoprotein. (b) E1B-55KD and E1A expression levels in HuH-7 cells All data were expressed as mean±s.d. and were analyzed treated with different Ads were examined by western blots. (c) IL-24 or using independent sample t-tests and one-way analyses of TRAIL expression levels in HuH-7 cells treated with different Ads were variance using SPSS Base 10.0. Results were considered examined by western blots. (b,c) Western blots were carried out 48 h statistically significant when Po0.05. after infection at an multiplicity of infections (MOI) ¼ 10.

Cancer Gene Therapy Liver cancer-targeting gene-viro-therapy XLiuet al 52 cancer (SW620), lung cancer (A549) and human normal MTT assays were carried out every 24 h after infection cell lines (MRC-5 and L-02) were infected with Ad- with different adenoviruses at an MOI of 10. The results AFP Á D55, Ad Á AFP Á D55-IL-24 and Ad Á AFP Á D55- showed that all of the rec-Ads obviously inhibited the TRAIL at various MOIs, and 96 h later, CPEs were proliferation of the HCC cells (Figure 2b). However, no studied by crystal violet staining. As shown in Figure 2a, evident growth arrest in cells of SW620 and A549 treated in non-liver cancer cells (A549 and SW620) and normal with Ads was observed, indicating the HCC cell-specific cells (MRC-5 and L-02), infection at an MOI of 100 did proliferation suppression by the Ads. not cause CPE; in HCC cells HuH-7 and Hep G2, infection at an MOI of 10 obviously caused CPE, Ad Á AFP Á D55-IL-24/TRAIL induces apoptosis suggesting HCC cell-selective cytotoxicity and also reli- in HuH-7 cells able safety of the recombinant Ads to normal cells. To Tumor cell apoptosis is often induced by the expression of further explore the tumor-suppression effect of the rec- extraneous IL-24 and TRAIL. To investigate whether the Ads, measurements of various cancer cells viability by recombinant Ads have the apoptosis-inducing potential in

Figure 2 Ad Á AFP Á D55-IL-24/TRAIL specifically inhibited the growth of HCC cells. (a) The cytopathic effects of different Ads on MRC-5, L-02, SW620, A549, HuH-7 and Hep G2 cells at a series of multiplicity of infections (MOIs) were studied by crystal violet staining 96 h after infection. (b) Measurement of cell viability of SW620, A549, HuH-7 and Hep G2 cells every 24 h after infection with Ads at an MOI of 10 by MTT assays. Points indicate the mean values (n ¼ 10); bars indicate s.d.

Cancer Gene Therapy Liver cancer-targeting gene-viro-therapy XLiuet al 53 HCC cells, we infected HuH-7 cells with different TRAIL induced caspases-mediated apoptosis in HuH-7 adenoviruses at an MOI of 10. At 48 h after the infection, cells. Another notable result was that the combination of Hoechst staining and DNA fragmentation assays were Ad Á AFP Á D55-IL-24 and Ad Á AFP Á D55-TRAIL treat- performed. We could clearly observe nuclear fragmenta- ment obviously induced the cleavage of caspase-9 tion and chromatin condensation, as well as DNA (Figure 4a), which implied that the combined treatment fragmentation (Figures 3a and b) in infected HuH-7 cells, also activated the mitochondria-mediated signaling suggesting that in HCC cells, apoptosis is the main type of pathway. cell death induced by the Ads. FACS was also used to We then used JC-1 staining to examine the mitochon- study the apoptosis induced by rec-Ads. At 48 h after drial transmembrane electrical potential (DCm) of HuH-7 infection at an MOI of 10, HuH-7 cells were stained with cells at 48 h after infection. Ad Á AFP Á D55-TRAIL and propidium iodide and annexin V-fluorescein isothiocya- Ad Á AFP Á D55-IL-24 induced a change in the DCmin nate. As shown in Figure 3c, about 18% of the cells HuH-7 cells (23.51 and 22.25%), whereas the combined infected with Ad Á AFP Á D55-IL-24 and about 13% with treatment of two Ads induced an even more significantly Ad Á AFP Á D55-TRAIL were stained with annexin altered DCm (39.58%) (Figure 4b). All these results V-positive, whereas infection with the combined Ads suggested that Ad Á AFP Á D55-IL-24 and Ad Á AFP Á D55- induced more potent apoptosis in HuH-7 cells, the TRAIL induced apoptosis in HCC cells through mod- number of annexin V-positive cells was about 29%. ulating caspase-8 and the mitochondrial pathways.

Ad Á AFP Á D55-IL-24 and Ad Á AFP Á D55-TRAIL triggered Ad Á AFP Á D55-IL-24/TRAIL suppresses tumor growth caspase-mediated apoptosis signaling pathways in vivo To study the molecules involved in the process of The in vivo antitumor effects of Ad Á AFP Á D55-IL-24 and apoptosis, we detected the apoptosis-associated Ad Á AFP Á D55-TRAIL were evaluated in a HuH-7 tumor induced by various Ads in HuH-7 cells using immuno- xenografts model. As shown in Figure 5a, at the end of blotting. As shown in Figure 4a, the cleaved products of 2 months of experiment duration, the average volume caspase-8, caspase-3 and poly ADP-ribose polymerase of the tumors of PBS-treated group reached approxi- could be detected at 48 h after Ad Á AFP Á D55-IL-24 and mately 2900 mm3; in contrast, the growth of the com- Ad Á AFP Á D55-TRAIL infections. By the time of 72 h, the bined Ads-treated tumors was obviously suppressed, amount of cleavage products had further increased, which with the average tumor volume at the same duration suggested that Ad Á AFP Á D55-IL-24 and Ad Á AFP Á D55- being only 300 mm3, representing a 90% suppression of

Figure 3 Ad Á AFP Á D55-IL-24/TRAIL induced apoptosis in HuH-7 cells. (a) The nuclear morphologies of HuH-7 cells stained with Hoechst33258 dye were visualized 48 h after infection. The arrows indicate the apoptotic cells. Scale bar ¼ 10 mm. (b) DNA fragmentation assays of HuH-7 cells 72 h after infection with different Ads, DNA marker DL2000. (c) Detection of apoptosis in HuH-7 cells using FACS with Annexin V-fluorescein isothiocyanate (V-FITC) and propidium iodide (PI) staining 48 h after infection. Numbers show the percentage of cells in the first and fourth quadrants. (a–c) multiplicity of infections (MOI) ¼ 10.

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Figure 4 Ad Á AFP Á D55-IL-24/TRAIL induced mitotic arrest and senescence in HuH-7 cells. (a) Western blot analyses of apoptosis-related proteins in HuH-7 cells infected with different Ads. p43/p41 are the cleaved products of procaspase-8. p37/p35 are the cleaved products of procaspase-9. p20 and p17 are the cleaved products of procaspase-3. p89 is a fragment of poly ADP-ribose polymerase (PARP). Actin was used as a loading control. (b) Analyses of the mitochondrial membrane potential of HuH-7 cells by FACS with JC-1 staining 72 h after infection. The percentage of cells in the trapeziform region is shown. (a, b) multiplicity of infections (MOI) ¼ 10.

the xenograft tumor growth. In addition, Ad Á AFP Á D55- antibody after Ad Á AFP Á D55-IL-24 and Ad Á AFP Á D55- IL-24 or Ad Á AFP Á D55-TRAIL alone also showed TRAIL treatments (Figure 6), suggesting that Ad- tumor-suppression activity in vivo, although not as strong AFP Á D55-IL-24 and Ad Á AFP Á D55-TRAIL can effec- as the combined treatment (Figure 5a). tively cause tumor necrosis in vivo. TUNEL analyses The survival rate was also elevated in the mice treated showed potent apoptosis both in the Ad Á AFP Á D55-IL- with the combination of Ad Á AFP Á D55-IL-24 and 24- and Ad Á AFP Á D55-TRAIL-treated tumors, whereas Ad Á AFP Á D55-TRAIL. As shown in Figure 5b, 2 months no apoptosis was detected in the PBS-treated tumors. after injection, all mice in the combination-treated group Therefore, we concluded that Ad Á AFP Á D55-IL-24 and remained alive. In contrast, the survival rates of the PBS- Ad Á AFP Á D55-TRAIL exert antitumor activity in vivo by treated group, the Ad Á AFP Á D55-treated group and the expressing IL-24 and TRAIL that subsequently induce Ad Á AFP Á D55-TRAIL-treated group were only 25%, apoptosis and necrosis in tumor cells. 37.5% and 50%, respectively.

Ad Á AFP Á D55-IL-24/TRAIL induces apoptosis and Discussion necrosis in vivo To study the effect of Ads on the tumors in vivo, Many therapies have been used to treat cancer, including immunohistochemistry assays were carried out. After 7 , radiotherapy, gene therapy and oncolytic days following the treatment, tumor sections were separated viral therapy. All these therapies have limitations and for from tumor-bearing mice. As shown in Figure 6, TRAIL many types of cancer, complete curing cannot be and IL-24 were detected in tumor specimens, indicating achieved. The CTGVT provides a new promising that Ad Á AFP Á D55-TRAIL and Ad Á AFP Á D55-IL-24 approach, and our previous studies on CTGVT have successfully express the carried extraneous genes in vivo. shown satisfying antitumor effects.11 The ZD55 vector we H&E staining revealed large areas of necrotic tissue previously used in CTGVT is an adenovirus similar to the that matched the areas stained brown by the anti-hexon ONYX-015 in the deletion of E1B-55KD. However,

Cancer Gene Therapy Liver cancer-targeting gene-viro-therapy XLiuet al 55 between apoptosis and oncolytic effects in tumor cells induced by Ads in detail. In this study, to strengthen the curative effect of the HCC-targeting oncolytic virus Ad Á AFP Á D55, we applied the concept of CTGVT-DG on liver cancer-targeting therapy by combining two rec-Ads that carry tumor suppressor genes TRAIL and IL-24 (Ad Á AFP Á D55-TRAIL and Ad Á AFP Á D55- IL-24) to treat liver cancer. This strategy showed satisfying antitumor effects both in vitro and in vivo. In vitro, we found that the Ads selectively inhibited the growth of the HCC cells through apoptosis, but did not affect normal cells. The molecules involved in the apoptosis mediated by the interaction between IL-24 and TRAIL were also studied. In vivo, although we have previously reported that hepatoma xenograft was not easily regressed by mono-gene-armed oncolytic Ad treatment due to the high malignancy of HCC cells,19 combined treatment with Ad Á AFP Á D55-TRAIL and Ad Á AFP Á D55-IL-24 not only showed excellent oncoly- tic effect on HCC cells of xenograft mice (90% suppression of tumor growth), but also increased Figure 5 Ad Á AFP Á D55-IL-24/TRAIL inhibited the growth of the survival rate (Figure 5a). HuH-7 xenograft tumors. (a) Measurement of the tumor volume of CTGVT-DG has been proven to be a promising different groups treated with oncolytic Ads or phosphate-buffered treatment for various types of cancer, and we have saline (PBS) every 7 days. When tumors reached 100-150 mm3 reported different combinations of tumor suppressor in volume, animals were injected with oncolytic Ads or PBS at genes that may have compensative or synergetic effects, week 0. Points indicate the mean values (n ¼ 8); bars indicate SD including plasminogen k5/TRAIL,5 short hairpin RNAs (***Po0.001). (b) The survival curve of various Ads- or PBS-treated that target M-phase phosphoprotein 1 (shMPP1)/IL- mice. 2420 and manganese superoxide dismutase (MnSOD)/ TRAIL in colorectal carcinoma,4 and Smac/TRAIL in heptoma.2 Here we also demonstrated strong antitumor ZD55 is an E1B-55KD-deleted mono-regulated vector effects of the combined treatment with rec-Ads carrying without the ability to target a specific cancer. To achieve IL-24 and TRAIL, suggesting IL-24/TRAIL as a liver cancer targeting and improved safety, here we promising combination in liver cancer gene therapy. In constructed a dual-regulated OV that specifically targets fact, IL-24/TRAIL is not a novel combination of liver cancer and has lower cytotoxic effects in normal liver tumor suppressor genes, and we have previously tissue.10 Similar gene therapies specifically targeting liver reported its application in colorectal cancer gene cancer have been reported and some dual-regulated therapy,3 in which we found that ZD55-IL-24 could oncolytic HCC-targeting adenoviruses, such as YKL- significantly upregulate TRAIL expression in colorectal 1001 and AdAFPep/Rep, whose replication is also tumor SW620 cells, and ZD55-IL-24 combined with controlled by the AFP promoter, have been con- ZD55-IL-TRAIL could completely eliminate colorectal structed.17,18 In the present study and also in our previous tumor xenografts with an elevated survival rate.3 work,10 we also found that the AFP promoter-controlled Furthermore, it has also been demonstrated that both Ad Á AFP Á D55 can selectively replicate in liver cancer TRAIL and its receptor DR4 are upregulated in cells, suggesting that the constructed Ad Á AFP Á D55 can Ad-IL-24-treated lung cancer cells.21 We also reported be used as a good liver-cancer targeting OV. that both ZD55-IL-24 and its combination with In CTGVT studies, a pro-apoptotic gene is usually ZD55-TRAIL could induce the expression of apoptotic applied as the antitumor gene, which is carried by the death receptor DR4 in colorectal tumor SW620 cells.3 oncolytic virus vector.2,3 Although our previous results We therefore believe that the upregulation of TRAIL showed that the oncolytic virus armed with an antitumor and its receptor DR4 is a potential reason why the gene could significantly enhance antitumor effects combination of these two Ads results in strong synergis- compared with the empty oncolytic virus,2,5 it remained tic antitumor effects in liver cancer. However, more unclear whether the harbored pro-apoptotic gene will evidence is still needed to discover the exact underlying impair the replication of viruses in tumor cells, as the mechanism. induced apoptosis in infected cells may eliminate In conclusion, our present work on the liver cancer- dissemination of viruses in the tumor mass. However, targeting Dual-gene-viro-therapy strategy provides a based on our long-term studies on CTGVT, we observed promising approach for hepatoma therapy. It has been no (or very minor) effect on the replication of oncolytic shown that deleting a small viral protein E4-orf3 can viruses by harbored pro-apoptotic genes.2,5,10 It will be bring higher safety to the oncolytic adenovirus;22 and of our future interests to investigate the relationship liver cancer suppressor gene, such as HCCS1 or SOCS3,

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Figure 6 Histopathologic, immunohistochemical and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) analyses of tumor sections. Tumor sections were separated and fixed in 4% paraformaldehyde, embedded in paraffin, and cut in 4-mm sections. Then, immunochemical analyses for hexon, interleukin-24 (IL-24) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), TUNEL and hematoxylin and eosin (H&E) staining assays of tumor sections in different groups were performed. Scale bar ¼ 20 mm.

have been shown to specifically inhibit liver tumori- Program) (No. 2010CB529901), Important National Science genesis.19,23 It will be our future interest to improve the & Technology Specific Project of Hepatitis and Hepatoma presently used HCC-targeting OV Ad Á AFP Á D55 and Related Program (2008ZX10002-023), New Innovation select more appropriate antitumor genes using these Program (2009-ZX-09102-246), the Natural Science Foun- strategies. dation of China (Nos. 30801445, 81172971 & 30970607) and the Zhejiang Sci-Tech University grant (1016834-Y).

Conflict of interest

The authors declare no conflict of interest. References

Acknowledgements 1 Liu XY. A new anticancer strategy —gene- Therapy of cancer. Chinese J Cancer Biother 2001; 8:1. We wish to thank Ying Xu, Lanying Sun, Jingfa Gu, and 2 Pei Z, Chu L, Zou W, Zhang Z, Qiu S, Qi R et al. An Cell Analysis Center (Institute of Biochemistry and Cell oncolytic adenoviral vector of Smac increases antitumor activity of TRAIL against HCC in human cells and in mice. Biology, Shanghai Institutes for Biological Sciences, Chinese Hepatology 2004; 39: 1371–1381. Academy of Sciences) for the professional technical 3 Zhao L, Dong A, Gu J, Liu Z, Zhang Y, Zhang W et al. The assistance, and Mitchell Sullivan (University of Queensland) antitumor activity of TRAIL and IL-24 with replicating for proof-reading the article. This work was supported by oncolytic adenovirus in colorectal cancer. Cancer Gene Ther the National Basic Research Program of China (973 2006; 13: 1011–1022.

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