Virus Research 181 (2014) 61–71

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Antitumor activities of an oncolytic adenovirus equipped with a double siRNA targeting Ki67 and hTERT in renal cancer cells Lin Fang a,1 , Qian Cheng a,1 , Wang Li b , Junjie Liu b , Liantao Li a , Kai Xu c,∗∗ , Junnian Zheng a,b,∗ a

Jiangsu Key Laboratory of Biological Cancer Therapy, Xuzhou Medical College, Xuzhou 221002, China Laboratory of Urology, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China c Department of Radiology, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China b

a r t i c l e

i n f o

Article history: Received 6 August 2013 Received in revised form 9 December 2013 Accepted 9 December 2013 Available online 21 January 2014 Keywords: RNA interference Ki67 Human telomerase reverse transcriptase Renal cell carcinoma (RCC)

a b s t r a c t RNA interference has been proven to be a powerful tool for gene knockdown. Our previous study demonstrated that a Ki67 shRNA carried by an adenovirus reduced Ki67 expression. In this study, we constructed novel oncolytic adenoviruses in which the Ki67 core promoter drove expression of the E1A gene. These adenoviruses were equipped with either a Ki67 small interfering RNA (siRNA), a human telomerase reverse transcriptase (hTERT) siRNA or a double siRNA targeting Ki67 and hTERT. We identified the antitumor activities of oncolytic adenoviruses in 3 renal cancer cell lines, human normal renal tube cell HK-2 and also in nude mice bearing KETR-3-xenografted tumors. Our results showed that these oncolytic adenoviruses, especially Ki67-ZXC2-double siRNA, could effectively induce silencing of the Ki67 and hTERT genes, allow efficient viral replication and induce significant apoptosis of renal cancer cells in vitro and in nude mice. We concluded that a dual siRNA mediated by oncolytic virotherapy could be an effective strategy for cancer gene therapy. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Ki67 is a nuclear protein associated with cell proliferation (Iatropoulos and Williams, 1996). Although the function of Ki67 has not been clearly defined yet, many immunohistochemical analyses have shown that Ki67 is highly overexpressed in a number of different cancers and acts as an independent prognostic factor (Bertucci et al., 2012). Its expression levels are positively correlated with some clinical-pathological variables in patients (Balleine et al., 2008). These observations indicated that Ki67 could serve as a mediator of malignant behavior in cancer and suggested that inhibition of Ki67 could be incorporated into novel cancer therapies. A clinical phase I study was initiated where patients with bladder carcinoma were treated intravesically with antisense oligonucleotides targeting the Ki67 gene (Rodriguez-Alonso et al., 2002). Kausch et al. demonstrated that antisense-mediated inhibition of Ki67 expression led to significant inhibition of proliferation and tumor growth in vitro and in vivo (Kausch et al., 2005). RNA

∗ Corresponding author at: Jiangsu Key Laboratory of Biological Cancer Therapy, Xuzhou Medical College, 84 West Huai-hai Road, Xuzhou, Jiangsu 221002, China. Tel.: +86 0516 85802233; fax: +86 0516 85582530. ∗∗ Corresponding author. E-mail addresses: zhangqifengfl@126.com (K. Xu), fl@xzmc.edu.cn (J. Zheng). 1 These authors contributed equally to this work 0168-1702/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.virusres.2013.12.021

interference (RNAi) has been proven to be a powerful tool for gene knockdown and holds great promise for the treatment of cancer (Ramachandran and Ignacimuthu, 2012). Initial investigations of RNA interference in cells relied on transfection of synthetic small interfering RNAs (siRNAs) or plasmids designed to drive expression of short hairpin RNAs (shRNAs) through the use of RNA polymerase III promoters (Wang et al., 2008). This knockdown technology has been successfully applied to inhibit Ki67 gene expression in renal cancer cell lines, but its utility is limited by the short half-life of siRNAs and the low transfection efficiency of plasmids (Liu et al., 2012). Recently, an adenovirus vector using the RNA polymerase II cytomegalovirus (CMV) promoter was developed for efficient delivery of shRNAs into cancer cell lines (Kim et al., 2010). Telomerase plays a critical role in tumor growth and progression, in part through the maintenance of the telomere structure, and is widely expressed in a variety of cancers. The reverse transcriptase telomerase is composed of two core components: a ubiquitously expressed RNA component (hTR), and a catalytic subunit human telomerase reverse transcriptase (hTERT) whose expression is rate limiting for the formation of a catalytically active enzyme (Kirkpatrick and Mokbel, 2001). About 90% of human cancers, including renal cancer cells, express telomerase. Many studies have demonstrated that inhibiting telomerase; especially hTERT, by genetic, antisense RNAi is a highly promising for cancer therapy. Zhang et al. transfected a plasmid encoding hTERT-specific shRNAs into human hepatocellular carcinoma cell lines and found

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that they could stably suppress hTERT expression, which led to the inhibition of cell proliferation and to an attenuated tumorigenic potency (Zhang et al., 2010). It could be hypothesized that the anticancer potency of siRNAs targeting hTERT could be improved using oncolytic adenoviral transfer. Adenovirus vectors employed in conventional cancer gene therapy are generally replication-deficient viruses, limiting the efficacy of gene transfer and the duration of therapeutic gene expression. It is thus expected that the delivery of shRNAs using non-replicating vectors will exhibit similar difficulties. To solve this problem, adenovirus mutants that preferentially replicate in and lyse tumor cells, known as oncolytic adenoviruses, have been proposed as vectors (Cerullo et al., 2012; Hallden and Portella, 2012). These adenoviruses are capable of lysing tumor cells selectively, and more importantly, they can amplify not only themselves but also the therapeutic genes they are carrying by selective replication in tumor cells. To increase the safety and efficiency of adenoviruses, modifications have been introduced to restrict adenovirus replication to tumor cells. One of these modifications was to replace promoters for essential viral genes with promoters that are active only in tumor cells (Bauerschmitz et al., 2006; Doloff et al., 2011). We have previously identified a Ki67 core promoter that had higher activity in tumor cell lines than in normal cells. Here, we constructed a novel oncolytic adenovirus with the Ki67 promoter controlling E1A gene expression, targeting the Ki67 and hTERT genes with siRNAs, aimed at developing a therapeutic strategy for renal cancer biotherapy. We inspected the antitumor effects of the Ki67 and hTERT double siRNA on human renal carcinoma cells both in vitro and in animal models. 2. Materials and methods 2.1. Cell lines and culture conditions The human renal carcinoma cell lines KETR-3, 786-O and ACHN, human embryonic kidney (HEK) 293 cells (containing the E1 region of the adenovirus) and human normal renal tube HK-2 cells were purchased from Shanghai Cell Collection (Shanghai, China). The KETR-3, ACHN, HEK293 and HK-2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) (GIBCO-BRL, Gaithersburg, MD), 2 mM glutamine, 100 U/ml penicillin and 100 mg/ml streptomycin at 37 ◦ C in a humidified incubator with 5% CO2 . The 786-O cells were maintained in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM glutamine. 2.2. Ki67 expression KETR-3, 786-O, ACHN and HK-2 cells were cultured routinely and total protein were extracted by RIPA buffer (Invitrogen Co). Expression of Ki67 protein was analyzed by western blot with the primary goat anti Ki67 polyclonal antibody (Santa Cruz Biotechnology, Inc). And ␤-actin was used as an inner control.

driving expression of the E1A gene, to create the pKi67-ZXC2-Ki67 siRNA plasmid. The pKi67-ZXC2-hTERT siRNA and pKi67-ZXC2double siRNA plasmids were constructed in a similar manner. However, the hTERT siRNA was cloned into the SalI site of pKi67ZXC2. The oncolytic adenoviruses Ki67-ZXC2, Ki67-ZXC2-Ki67 siRNA, Ki67-ZXC2-hTERT siRNA and Ki67-ZXC2-double siRNA were generated in HEK293 cells by homologous recombination between pKi67-ZXC2, pKi67-ZXC2-Ki67 siRNA, pKi67-ZXC2-hTERT siRNA or pKi67-ZXC2-double siRNA and the adenovirus packaging plasmid pBHGE3 (Microbix Biosystems). Large-scale purification of all adenoviruses was performed by ultracentrifugation with cesium chloride according to standard techniques (Ugai et al., 2005). The titers were determined by plaque assays on HEK293 cells.

2.4. Cell viability assay Cells were plated in 96-well plates and treated with various adenoviruses the next day. At the indicated times, Cell Counting Kit-8 (CCK-8, 10 ␮l, Tiagen, Beijing, China) was added to each well, and the cells were incubated at 37 ◦ C for 4 h. Absorbance from the plates was read on an ELX-800 spectrometer (Bio-Tek Instruments Inc., USA) at 450 nm. We set four replicate wells per assay, and each experiment was repeated three times.

2.5. Cytopathic assay The KETR-3, 786-O, ACHN and HK-2 cells were plated in 24well plates at a density of 1 × 105 and infected with recombination viruses at the various indicated multiplicities of infection (MOI). Four days after infection, the media was removed and the cells were washed twice with phosphate-buffered saline (PBS). Crystal violet solution was added to the 24-well plates, which were incubated at room temperature for 15 min, washed with distilled water and then documented with photographs.

2.6. Western blot analysis Cell lysates were harvested after being infected with the adenoviruses, and the xenograft tumor tissue was homogenated in tissue lysis buffer (50 mM Tris–HCl pH 8.0, 1% NP-40, 1% Na-deoxycholate, 150 mM NaCl, 0.1% SDS, 0.05 mM PMSF) and ultracentrifugated at 4 ◦ C to acquire the proteins from the upper liquid layer. The proteins were separated on a 10% SDSpolyacrylamide gel, transferred to a nitrocellulose membrane and incubated overnight at 4 ◦ C with a rabbit polyclonal anti-E1A antibody (Santa Cruz, USA). The membranes were then washed and incubated with alkaline phosphatase-conjugated secondary antibodies in TBS-T for 2 h and developed using NBT/BCIP color substrate (Promega, Madison, USA). The density of the bands on the membrane was scanned and analyzed with an image analyzer.

2.3. Virus construction and production 2.7. Immunocytochemistry assay pSilencer-Ki67, a siRNA-expressing plasmid targeting amino acids 364–382 of human Ki67 (GenBank accession no. NM 002417) and pSilencer-hTERT, targeting amino acids 567–591 of human hTERT (GenBank accession no. NM 053423) were previously constructed in our laboratory. The Ki67 siRNA expression cassette excised from pSilencer-Ki67 was first subcloned into pCA13 to form pCA13-Ki67siRNA. The expression cassette containing the Ki67 siRNA controlled by the human CMV promoter was then digested with XhoI and subcloned into the previously constructed adenoviral vector pKi67-ZXC2, which includes the Ki67 core promoter

Cells infected with adenoviruses and control cells were fixed onto 24-well plates with 4% paraformaldehyde. After washing with PBS, the cells were incubated with anti-Ki67 or anti-hTERT primary antibody overnight and then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at 37 ◦ C. Ki67 or hTERT positive cell staining was developed by diaminobenzidine (DAB). For evaluation of Ki67-positive or hTERT-positive fractions, at least 200 cells were counted in five different regions and the mean number was determined.

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2.8. Cell apoptosis assay

2.12. HE staining

Briefly, cells were cultured in 24-well plates, infected with various viruses at a MOI of 20 at 37 ◦ C, 5% CO2 for 72 h and then washed twice with PBS. 50 ␮l Binding Buffer, 5 ␮l Annexin V-FITC and 5 ␮l propidium iodide (PI) was added to the cells and incubated for 1 h. Pictures of the samples were taken under a fluorescence microscope. Cells were counted in five different regions, and the mean number was determined.

Xenograft tumor tissues and liver tissues were fixed with 4% paraformaldehyde, embedded in paraffin and cut in 4-mm sections. The slides were deparaffinized with alcohol, washed with distilled water, stained with hematoxylin for 5–8 min, washed with distilled water, restained with eosin for 1–3 min and then decolorized with distilled water. After dehydration, the slides were made transparent with two treatments of xylene for 1 min each. Finally, the slides were mounted with neutral gum to be observed under the microscope.

2.9. Xenograft tumor model in nude mice Male BALB/c nude mice (4–6 weeks old) were obtained from the Institute of Animal Center (Chinese Academy of Sciences, Shanghai) and quarantined for a week before tumor implantation. Animal welfare and experimental procedures were carried out strictly in accordance with the Guide for the Care and Use of Laboratory Animals. The xenograft tumor model was established by subcutaneously injecting KETR-3 cells (2 × 106 ) into the right flank of mice. When tumors reached approximately 80–120 mm3 , the mice were randomly divided into five groups (six mice per group) and treated with intratumor injections of Ki67-ZXC2, Ki67-ZXC2-Ki67 siRNA, Ki67-ZXC2-hTERT siRNA or Ki67-ZXC2-double siRNA at a dose of 7 × 108 plaque forming units (PFU) per mouse every other day for three days or with PBS as a control. On the seventh day after treatment, three nude mice were randomly euthanized from each group to harvest the tumors for immunohistochemistry and TUNEL analyses. For the remaining nude mice, the tumors were monitored every week for five weeks after injecting treatments by measuring tumor size using a caliper. The tumor volume was calculated using the following formula: V (mm3 ) = (length × width2 )/2. At the end of the experiment, the tumors were harvested for additional analyses. 2.10. Immunohistochemistry staining Tumors were harvested and fixed in 10% formalin, embedded in paraffin and cut in 4-mm sections. Deparaffinized tumor sections were treated with 3% H2 O2 for 10 min to block the endogenous peroxidase and incubated with blocking serum (goat serum) at room temperature for 30 min. Immunohistochemistry was carried out with anti-E1A (Santa Cruz, USA), anti-Ki67 (Santa Cruz, USA) or anti-hTERT (Millipore, USA) primary antibodies. After incubation with an anti-mouse secondary antibody, the expression of E1A, Ki67 or hTERT was detected with DAB (Beijing Zhongshan Co., China) by enhancement with an avidin–biotin reaction ABC kit (Vector Laboratories Burlingame, CA). Tissue sections stained without primary antibody served as negative controls. The slides were then counterstained with hematoxylin. 2.11. TUNEL assay Apoptotic cells in tumor tissue sections were quantified using the in situ apoptosis detection kit (Roche, Indianapolis, IN). Formalin-fixed, paraffin-embedded sections were dewaxed before being permeabilized with proteinase K for 15 min at room temperature. Endogenous peroxidase was blocked with 3% H2 O2 , and sections were incubated with equilibration buffer and terminal deoxynucleotidyl transferase (TdT) enzyme. Finally, the sections were incubated with anti-digoxigenin-peroxidase conjugate. Peroxidase activity in each tissue section was demonstrated by the application of DAB. Under microscopy, six fields were randomly selected from every sample and 100 cells were randomly selected from every field. Apoptotic rate = (number of total apoptotic cells/100) × 100%.

2.13. Statistical analysis Values were expressed as mean ± s.d., and statistical analysis of the results was carried out by one-way analysis of variance (ANOVA) followed by Duncan’s new multiple range method or the Newman–Keuls test. Data were considered statistically significant when p < 0.05. 3. Results 3.1. Construction and identification of oncolytic adenoviruses A schematic diagram of the recombinant adenoviruses Ki67-ZXC2, Ki67-ZXC2-Ki67 siRNA, Ki67-ZXC2-hTERT siRNA and Ki67-ZXC2-double siRNA is illustrated in Fig. 1A. The successful construction of these recombinant adenoviruses was confirmed by PCR assay with specific primers (data not shown). 3.2. Ki67 expression in cell lines Ki67 protein expression in KETR-3, 786-O, ACHN and HK-2 cells was examined by western blotting. In normal cell lines HK-2, Ki67 was negative. But Ki67 was significantly positive in KETR-3 cells, expressed moderately in 786-O and lowly in ACHN (Fig. 1B). 3.3. Evaluation of E1A expression driven by the Ki67 promoter in renal cancer and normal cells Protein expression of E1A accompanied with adenovirus Ki67ZXC2-double siRNA translation in KETR-3 cells was detected by Western blotting at the different time points. Our data showed a distinct increase of E1A from 24 h to 48 h, as shown in Fig. 1C. E1A protein was decreased after 96 h post infection, since the cells were destroyed by replicative Ki67-ZXC2-double siRNA adenovirus. We chose the 48 h time point for the following trials as there was a high expression of E1A and high potency of adenovirus. Human renal cancer cells KETR-3, 786-O and ACHN and human normal renal tube HK-2 cells were infected with Ki67-ZXC2, Ki67-ZXC2Ki67 siRNA, Ki67-ZXC2-hTERT siRNA or Ki67-ZXC2-double siRNA at a MOI of 10. Western blot assays were performed to determine the expression of E1A. As shown in Fig. 1D, KETR-3 and 786-O cells infected with these recombinant adenoviruses displayed high levels of E1A protein expression, while recombinant adenovirusinfected ACHN cells displayed low E1A expression. Moreover, there seemed to be no E1A expression in the adenovirus-infected HK-2 cells. These results suggested that the Ki67 promoter could efficiently drive E1A expression in renal cancer cells but not in normal cells. 3.4. The expression of Ki67 and hTERT in renal carcinoma cells and normal cells In order to evaluate the efficiency of the target siRNAs, we infected the same panel of cell lines mentioned above with

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Fig. 1. Construction and expression of oncolytic adenoviruses. (A) Schematic diagrams illustrating the recombinant adenoviruses Ki67-ZXC2, Ki67-ZXC2-Ki67 siRNA, Ki67ZXC2-hTERT siRNA and Ki67-ZXC2-double siRNA. In Ki67-ZXC2, the E1A promoter was replaced by the Ki67 promoter. The Ki67 siRNA expression cassette was inserted into the XhoI site of Ki67-ZXC2 to form Ki67-ZXC2-Ki67 siRNA. The hTERT siRNA expression cassette was inserted into the SalI site of Ki67-ZXC2 to form Ki67-ZXC2-hTERT siRNA. The hTERT siRNA expression cassette was inserted into Ki67-ZXC2-Ki67 siRNA to form Ki67-ZXC2-double siRNA. (B) Western blotting was used to analyze the expression of Ki67 antigen in KETR-3, 786-O, ACHN and HK-2 cells; ␤-actin was used as control. (C) Time course of E1A expression in KETR-3 cells infected Ki67-ZXC2-double siRNA was detected by immunoprecipitation using anti-E1A antibody. (D)Expression of the E1A protein in human renal carcinoma cells infected with different adenoviruses. KETR-3, 786-O, ACHN and HK-2 cells were infected with Ki67-ZXC2, Ki67-ZXC2-Ki67 siRNA, Ki67-ZXC2-hTERT siRNA and Ki67-ZXC2-double siRNA at a MOI of 10 and Western blot assays were performed 48 h later. The HEK293 cell line was used as a positive control. ␤-Actin was used as a loading control.

Ki67-ZXC2, Ki67-ZXC2-Ki67 siRNA, Ki67-ZXC2-hTERT siRNA or Ki67-ZXC2-double siRNA at a MOI of 10 and carried out a RT-PCR assay 48 h later. As shown in Fig. 2A, there was a significant decrease in Ki67 mRNA levels in KETR-3 and 786-O cells 48 h after infection with Ki67-ZXC2-Ki67 siRNA or Ki67ZXC2-double siRNA. The change in Ki67 mRNA levels was less in ACHN and HK-2 cells compared to the control. Accordingly, there was lower hTERT mRNA expression in KETR-3 and 786-O cells infected with Ki67-ZXC2-hTERT siRNA or Ki67-ZXC2-double siRNA compared to cells infected with other adenoviruses or PBS. There was no significant difference in hTERT mRNA expression in ACHN and HK-2 cells infected with the various adenoviruses (Fig. 2B). In addition, we investigated the expression of Ki67 and hTERT protein in KETR-3, 786-O, ACHN and HK-2 cells after infection with the different adenoviruses. We performed infection assays, and 48 h later Ki67 and hTERT protein expression was analyzed by immunocytochemistry staining. Based on the statistical data, there was a significant decrease in Ki67 protein levels in KETR3 and 786-O cells after infection with Ki67-ZXC2-Ki67 siRNA or Ki67-ZXC2-double siRNA. Additionally, hTERT protein expression in these cells infected with Ki67-ZXC2-hTERT siRNA or Ki67-ZXC2double siRNA was also lower compared to the PBS group. In this

assay, we also detected lower Ki67 and hTERT protein expression in ACHN cells infected with the various oncolytic adenoviruses. However, there was no significant difference in the adenovirusinfected HK-2 cells (Fig. 3).

3.5. Effect of the oncolytic adenoviruses on cell proliferation in renal carcinoma cells The ability of the four oncolytic adenoviruses to inhibit tumor growth was assessed in vitro by MTT assay. As shown in Fig. 4A, Ki67-ZXC2-double siRNA had higher inhibition ability than the other adenoviruses in KETR-3 and 786-O cells. Moreover, the survival rate of the KETR-3, 786-O and ACHN cells decreased over time. However, the oncolytic adenoviruses seemed not to kill the normal HK-2 cells, as they would not replicate. In addition, we found that the inhibition effect of all viruses was dose-dependent in three renal cancer cell lines and that Ki67ZXC2-double siRNA had a 50% mortality rate at a lower MOI in the three cancer cell lines. In normal HK-2 cells, no significant inhibition was observed, even at a very high MOI (Fig. 4B). These results indicated that the double siRNA inhibited cell proliferation effectively.

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Fig. 2. Expression of Ki67 and hTERT mRNA in human renal carcinoma cells infected with the different adenoviruses. Total RNA was extracted by TRIZOL and reverse transcribed to cDNA. RT-PCR was performed to detect Ki67 (A) and hTERT (B) mRNA expression in KETR-3, 786-O, ACHN and HK-2 cells. ␤-Actin served as an internal control.

3.6. Potent cytopathic effect induced by oncolytic adenoviruses To explore whether the Ki67 or hTERT siRNAs mediated by oncolytic adenoviruses could sensitize tumor or normal cells to death, KETR-3, 786-O, ACHN and HK-2 cells were infected with Ki67-ZXC2, Ki67-ZXC2-Ki67 siRNA, Ki67-ZXC2-hTERT siRNA, or Ki67-ZXC2-double siRNA and stained with crystal violet 4 days later. As shown in Fig. 4C, a cytopathic effect (CPE) was observed in the 3 renal cancer cell lines infected with the recombinant adenoviruses. We also found that there was no significant CPE in HK-2 cells infected with the viruses. These results indicated that the recombinant adenoviruses had selective CPE in tumor cells and inhibited the growth of tumor cells effectively. 3.7. Cell apoptosis induced by the recombinant adenoviruses Because Ki67 and hTERT are closely associated with the cell cycle, we investigated whether the Ki67 or hTERT siRNAs mediated by oncolytic adenoviruses could induce apoptosis of renal cancer cells. KETR-3, 786-O, ACHN and HK-2 cells were infected with Ki67-ZXC2, Ki67-ZXC2-Ki67 siRNA, Ki67-ZXC2-hTERT siRNA or Ki67-ZXC2-double siRNA at a MOI of 20 and analysis of Annexin V/PI double stained cells was carried out three days later to quantify cell apoptosis. Our data showed that double siRNA oncolytic adenoviruses induced more prophase and advanced stage cell apoptosis compared to the other treatments in the three renal cancer cells (Fig. 5A and B). There were fewer apoptotic cells observed in

the HK-2 cells infected with the viruses than in the tumor cells. These results suggested that the oncolytic adenoviruses equipped with the Ki67 and/or hTERT siRNAs could induce potent apoptosis of tumor cells in vitro. 3.8. Antitumor efficacy of recombinant adenoviruses in nude mice The antitumor activity of oncolytic adenoviruses equipped with Ki67 and/or hTERT siRNAs was assessed in nude mice xenografts. A total of 2 × 106 KETR-3 cells were inoculated subcutaneously into the right flank of nude mice. When the tumor size reached approximately 80–120 mm3 , 10 mice in the Ki67-ZXC2, Ki67ZXC2-Ki67 siRNA, Ki67-ZXC2-hTERT siRNA and Ki67-ZXC2-double siRNA groups received an intratumoral injection at a dose of approximately 2 × 109 plaque-forming units (PFU) per mouse; the group that received a PBS injection served as the control. After 7 days, 3 nude mice were killed and tumor specimens were obtained. TUNEL analysis was carried out, and the results showed that prominent apoptosis was observed in the Ki67-ZXC2double siRNA group (64.7 ± 5.8%), which was more significant than that in the Ki67-ZXC2-Ki67 siRNA (39.3 ± 3.8%), Ki67-ZXC2-hTERT siRNA (48.2 ± 4.2%), Ki67-ZXC2 (23.7 ± 2.8%) and control groups (4.5 ± 1.2%) (Fig. 6A). To identify the replication of adenoviruses in tumor cells, E1A expression was detected by immunohistochemistry and Western blot assays. As shown in Fig. 6B, E1A expression was detected in the Ki67-ZXC2, Ki67-ZXC2-Ki67 siRNA, Ki67-ZXC2-hTERT siRNA

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Fig. 3. Ki67 and hTERT protein expression after treatment with the different adenoviruses. Tumor cells infected with Ki67-ZXC2, Ki67-ZXC2-Ki67 siRNA, Ki67-ZXC2-hTERT siRNA and Ki67-ZXC2-double siRNA were analyzed for Ki67 and hTERT protein expression by immunocytochemistry. Representative photomicrographs showing Ki67 and hTERT protein expression. Data are presented as the mean ± standard deviation (S.D.); *p < 0.05 versus control group.

and Ki67-ZXC2-double siRNA groups, but not in the PBS group, which indicated that the four recombinant viruses could replicate in tumors and could mediate the expression of other transgenes. We also determined cell proliferation in the tumors after injection with or without the viruses by HE staining. The results showed some cytoplasm staining and a double-leaf or multi-leaf mitoticvisible nucleus in the PBS group, suggesting active cell proliferation. However, there were few mitotic cells in the virus-infected tumors, especially in the Ki67-ZXC2-double-siRNA group (Fig. 6C).

During the therapeutic period, tumor size was measured every 7 days, and the remaining 3 mice were sacrificed 35 days later. As shown in Fig. 7A and B, the tumors displayed rapid and continued outgrowth during the entire experimental period in the PBS group and the mean tumor size was 2143.58 ± 512.36 mm3 . The mean tumor size in the Ki67-ZXC2-double siRNA-treated group was 413.87 ± 109.23 mm3 , much smaller than that in the Ki67ZXC2-Ki67 siRNA (865.69 ± 216.39 mm3 ), Ki67-ZXC2-hTERT siRNA (947.36 ± 256.94 mm3 ) and Ki67-ZXC2 (1242.47 ± 296.21 mm3 )

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Fig. 4. Inhibition and cytopathic effect of oncolytic adenoviruses on tumor cell growth. (A) Tumor cells were infected with Ki67-ZXC2 (mock), Ki67-ZXC2-Ki67 siRNA, Ki67ZXC2-hTERT siRNA and Ki67-ZXC2-double siRNA at a MOI of 10. On days 1, 2, 3 and 4 post-infection, cells were subjected to the CCK-8 assay. (B) Tumor cells were infected with Ki67-ZXC2 (mock), Ki67-ZXC2-Ki67 siRNA, Ki67-ZXC2-hTERT siRNA and Ki67-ZXC2-double siRNA at MOIs of 0.1, 1, 10 and 100, and 96 h post-infection; the cells were subjected to the CCK-8 assay. Data are presented as the mean ± standard deviation (S.D.) from three independent experiments (n = 3). (C) The cytopathic effect of oncolytic adenoviruses on renal tumor cells (KETR-3, 786-O and ACHN) and human normal renal tube HK-2 cells was detected. Each well of a 24-well plate was seeded at a density of 1 × 105 cells and infected with Ki67-ZXC2 (mock), Ki67-ZXC2-Ki67 siRNA, Ki67-ZXC2-hTERT siRNA and Ki67-ZXC2-double siRNA at the indicated MOIs. Seven days later, cells were stained with crystal violet.

groups. Animals treated with Ki67-ZXC2-double siRNA showed a more statistically significant inhibition of tumor progression than the other groups. To verify that the therapeutic effect resulted from inhibition of the Ki67 or hTERT genes and that the recombinant adenoviruses could replicate in vivo, the tumors were collected, pretreated and used for immunohistochemical analysis of the Ki67 and hTERT genes. As shown in Fig. 7C, Ki67 staining showed a marked reduction of Ki67-positive cells in the Ki67-ZXC2-double siRNA (129.8 ± 6.3), Ki67-ZXC2Ki67 siRNA (162.1 ± 10.8), Ki67-ZXC2-hTERT siRNA (254.6 ± 7.4) and Ki67-ZXC2 (285.7 ± 6.1) groups when compared with the

PBS group (337.6 ± 17.6). hTERT staining showed a marked reduction of hTERT-positive cells in the Ki67-ZXC2-double siRNA (132.8 ± 5.3), Ki67-ZXC2-hTERT siRNA (183.5 ± 4.7), Ki67ZXC2-Ki67 siRNA (250.9 ± 11.3) and Ki67-ZXC2 (277.9 ± 13.6) groups when compared with the PBS group (354.2 ± 15.9). Furthermore, the results demonstrated that the Ki67-ZXC2Ki67 siRNA and Ki67-ZXC2-double siRNA adenoviruses suppressed Ki67 expression more potently than the Ki67-ZXC2 and Ki67-ZXC2-hTERT siRNA adenoviruses. Similarly, the Ki67ZXC2-hTERT siRNA and Ki67-ZXC2-double siRNA adenoviruses suppressed hTERT expression more potently than the Ki67-ZXC2 adenovirus.

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Fig. 5. Cell apoptosis mediated by oncolytic adenoviruses in KETR-3, 786-O, ACHN and HK-2 cells. (A) Representative photomicrographs showing Annexin V-FITC staining for prophase apoptosis in KETR-3, 786-O, ACHN and HK2 cells infected with the recombinant viruses. Quantitative representation of the proportion analyzed in KETR-3, 786-O, ACHN and HK-2 cells (right). Data are presented as the mean ± standard deviation (S.D.); –, cultured cells with no added adenovirus. (B) Representative photomicrographs showing PI staining for advanced stage apoptosis in KETR-3, 786-O, ACHN and HK2 cells infected with the recombinant viruses. Quantitative representation of the proportion analyzed (right).

Paraffin-embedded sections of liver tissues from nude mice underwent HE staining and were fixed in a 4% formaldehyde fixative. Liver cell morphology was observed, and there was no obvious necrosis area on the paraffin sections compared to the control, as shown in Fig. 7D. 4. Discussion Renal cancer is one of the more common malignant tumors of the urinary and reproductive systems (Moch, 2012). Despite many diagnostic techniques and therapeutic strategies, it remains an incurable disease and exhibits little response to current therapeutic approaches due to its high degree of malignancy and difficult early detection (Kruck et al., 2012). More effective therapeutic strategies need to be developed. Gene-virus treatment has already been engaged in extensive research and is gradually becoming a promising novel method of cancer treatment (Cascallo, 2010; Galanis, 2010). Oncolytic adenoviruses offer appealing advantages over conventional cancer therapy and are a promising new approach to the treatment of human cancers. Ki67 and hTERT are well-known cell proliferation-associated antigens, closely related with cell proliferation and malignancy (Agrawal et al., 2012; Ellebaek et al., 2012; Herlin, 2011; Mertani

et al., 2011). Our previous study demonstrated that inhibiting Ki67 with a siRNA led to inhibition of cancer cell proliferation and induced apoptosis (Liu et al., 2012; Zheng et al., 2009). In this study, in order to better understand the antitumor effects, we constructed double siRNAs (targeting Ki67 and hTERT) mediated by oncolytic adenoviruses and investigated their antitumor activities. A 253 bp Ki67 core promoter was used to drive the expression of the adenoviral essential gene E1A, as it has been determined that the Ki67 promoter has high activity in tumor cells but not in normal cells. Based on this background, the Ki67-ZXC2-double siRNA adenovirus was generated. Our results showed that the core Ki67 promoter could control E1A expression in renal cancer cell lines (KETR-3, 786-O and ACHN) but not in normal cells (HK-2). Although E1A levels were lower in ACHN cells than in KETR-3 and 786-O cells, this could be explained by the fact that the Ki67 promoter has varying activity in different cancer cell lines (Fig. 1). It has been demonstrated that chemically synthesized siRNAs and shRNAs expressed from plasmids can reduce Ki67 mRNA and protein levels and lead to growth inhibition and apoptotic cell death when they are transfected into human renal carcinoma cells, but the efficacy of RNAi targeting Ki67 is limited (Fung et al., 2012; Tan et al., 2005). We expected to improve the RNAi effect by

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Fig. 6. Apoptosis and E1A expression in vivo by oncolytic adenovirus treatment. (A) Induction of apoptosis after treatment with recombinant adenoviruses in vivo. Tumor sections were excised and analyzed for apoptosis by TUNEL staining. Representative photomicrographs showing TUNEL staining in the implanted tumor (original magnification ×400). (B) E1A expression after treatment with recombinant viruses was detected by immunohistochemisty and Western blot assays. The tumor section was obtained from the xenograft model after being infected with Ki67-ZXC2, Ki67-ZXC2-Ki67 siRNA, Ki67-ZXC2-hTERT siRNA and Ki67-ZXC2-double siRNA adenoviruses and PBS was used as the control. Representative photomicrographs showing E1A protein expression using immunohistochemical techniques. Protein was harvested from homogenized tissue and Western blot assays were preformed to detect E1A protein expression (lower panel). (C) Differences in karyokinesis and karyotin after treatment with Ki67-ZXC2, Ki67ZXC2-Ki67 siRNA, Ki67-ZXC2-hTERT siRNA and Ki67-ZXC2-double siRNA in vivo. Tumor sections were excised and analyzed for karyokinesis by HE staining. Quantitative representation of the proportion analyzed (right).

using a double siRNA delivered by the adenoviral vector to achieve stable and long-term RNAi. In order to confirm the potent therapeutic effect of target RNAi in renal cancer cells, we measured the levels of Ki67 and hTERT mRNA and protein expression after adenovirus-mediated RNAi. Ki67 and hTERT mRNA levels were high in 3 of the renal cancer cell lines we used, and their expression levels changed depending on whether they were infected with single or dual siRNAs (Figs. 2 and 3). This result indicated that RNAi mediated by oncolytic adenoviruses could block target gene expression efficiently. We further developed the antitumor effect of the oncolytic adenovirus. Cell growth inhibition results determined by the CCK-8 assay indicated that oncolytic adenoviruses equipped with siRNAs killed renal cancer cells effectively (Fig. 4A and B). The observed therapeutic effects of the oncolytic adenoviruses were time- and dose-dependent. Data from the cytotoxic assay indicated that the Ki67-ZXC2-double siRNA adenovirus had the ability to kill renal cancer cells but not normal cells (Fig. 4C). Moreover, the Ki67ZXC2-double siRNA adenovirus could induce more apoptotic cell death according to the results from the Annexin V-FITC/PI staining

assay (Fig. 5). Thus, the results indicated that the effects of oncolytic adenovirus-mediated Ki67 and hTERT siRNAs were fully exploited in vitro. Identifying the therapeutic effects of the engineered oncolytic adenoviruses in animal models was more challenging. We established a renal cancer model in nude mice with KETR-3 cells, which showed higher Ki67 promoter activity in vitro assays. The Ki67-ZXC2-double siRNA adenovirus displayed an antitumor effect, although the tumor was not completely eliminated (Fig. 7A and B). Intriguingly, the tumor volume (413.87 ± 109.23 mm3 ) was much lower than that seen in the control group (2143.58 ± 512.36 mm3 ). We confirmed the expression of adenoviral early protein E1A when the tumor was injected with adenoviruses 7 days later (Fig. 6B). Furthermore, we detected the knockdown of Ki67 and hTERT, and the results indicated that the Ki67-ZXC2-double siRNA adenovirus could inhibit expression of both in tumor tissues (Fig. 7C). Although adenoviruses are known to possess liver tropism, the morphology of hepatocytesdoes did not change significantly in the therapeutic groups from HE staining assay.

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Fig. 7. Antitumor activities of recombinant adenoviruses in the KETR-3 xenograft model. Renal cancer models were established by injecting KETR-3 cells subcutaneously into the right flank of nude mice. When tumors reached approximately 80–120 mm3 , animals were treated with intratumoral injections of Ki67-ZXC2, Ki67-ZXC2-Ki67 siRNA, Ki67-ZXC2-hTERT siRNA and Ki67-ZXC2-double siRNA. (A) Representative tumor formation 35 days after injection. (B) The tumor size was measured and tumor volume was calculated. Each time point represents the mean tumor volume for each group. Error bars represent the S.D. Data are expressed as the mean of tumor volume ± S.D. (C) Ki67 or hTERT expression after treatment with the recombinant adenoviruses in vivo. Tumor sections were excised and analyzed for Ki67 or hTERT expression by immunohistochemistry. (D) To test whether liver tissue was injured when adenoviruses were administrated in vivo, the liver was excised and analyzed for karyokinesis by HE staining.

5. Conclusions

References

All in all, we successfully engineered oncolytic adenoviruses equipped with double RNAi and showed their ability to effectively inhibit the growth of renal cancer cells both in vitro and in vivo. These results provide a novel, promising strategy to silence oncogenes in tumors and could contribute to further research on cancer viral-gene therapy.

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Conflict of interest statement The authors have no conflict of interest. Acknowledgments This project was supported by grants from the National Natural Science Foundation of China (nos. 81101702, 81372460 and 81071854), the Science Foundation of China Postdoctoral (53470107), the Science and Technology Department of Jiangsu Province (nos. BK2011207, BK20131120, 2011-WS-069, 11KJA320002 and 12KJA320001), the Science and Technology Department of Xuzhou (no. XF11C061) and the Science Foundation of Xuzhou Medical College (no. 2010KJZ04).

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Antitumor activities of an oncolytic adenovirus equipped with a double siRNA targeting Ki67 and hTERT in renal cancer cells.

RNA interference has been proven to be a powerful tool for gene knockdown. Our previous study demonstrated that a Ki67 shRNA carried by an adenovirus ...
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