A Novel Triple-Regulated Oncolytic Adenovirus Carrying P53 Gene Exerts Potent Antitumor Efficacy on Common Human Solid Cancers

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A novel triple-regulated oncolytic adenovirus carrying p53 gene exerts potent antitumor efficacy on common human solid cancers

Xinghua Wang,1 Changqing Su,1 Hui Cao,3 Kui Li,3 was found by ELISA assay that SG600-p53 expressed Jie Chen,1 Lixin Jiang,3 Qi Zhang,1 Xiaobing Wu,3 p53 efficiently in cancer cells. In NCI-H1299 tumor Xiaoyuan Jia,2 Yongjing Liu,1 Weiguo Wang,1 xenograft models, SG600-p53 displayed a tumor-selective Â 9 Xinyuan Liu,2,4 Mengchao Wu,1 and Qijun Qian1,2 killing capacity. At a dose of 2 10 plaque-forming units, SG600-p53 could completely inhibit the tumor growth 1Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary and more effective than replication-defective Ad-p53. Surgical Hospital, Second Military Medical University, Shanghai, Histopathologic examination revealed that SG600-p53 2 People’s Republic of China; Xinyuan Institute of Medicine and administration resulted in cancer cell apoptosis. We Biotechnology, Zhejiang Sci-Tech University, Hangzhou, People’s Republic of China; 3Vector Gene Technology Company Ltd., concluded that the triple-regulated SG600-p53, as a more BDA, Beijing, People’s Republic of China; and 4Institute of potent and safer antitumor therapeutic, could provide a Biochemistry and Cell Biology, Shanghai Institutes for Biological new strategy for cancer biotherapy. [Mol Cancer Ther Sciences, Chinese Academy of Sciences, Shanghai, People’s 2008;7(6):1598–603] Republic of China Introduction Abstract Conditionally replicating adenoviruses (CRAd) can repli- Conditionally replicating adenoviruses (CRAd) can replicate specifically in cancer cells and lyse them. When the cate specifically in cancer cells and lyse them. The CRAds CRAd vectors are armed with the antitumor transgene, the were widely used in the preclinical and clinical studies of transgene copies and expression may be increased mark- cancer therapy. We hypothesize that more precisely edly along with the preferable replication of CRAds in regulated replication of CRAds may further improve the tumor cells but not in normal cells, so that the antitumor vector safety profile and enhance its antitumor efficacy. efficacy was enhanced (1). Thereby, the CRAds overcome Here, a triple-regulated CRAd carrying p53 gene expres- the disadvantages of traditional gene therapy, including sion cassette, SG600-p53, was engineered. In SG600- the low transfection, no specificity, and poor efficacy. The p53, the E1a gene with a deletion of 24 nucleotides within previous study from van Beuschem et al. (2) showed the CR2 region is controlled under the human telomerase enhanced antitumor effect of CRAd with wild-type p53 reverse transcriptase (hTERT) promoter, the E1b gene gene in the treatment of glioma. However, the replication of expression is directed by the hypoxia response element CRAds is not always precise under the control of single (HRE), whereas the p53 gene is controlled by the cis-acting element; sometimes, it replicates to some extent cytomegalovirus promoter. The precise triple-regulation in normal cells and results in side toxicity. It is necessary endows SG600-p53 with enhanced antitumor potential to improve the CRAd safety with multiple regulating and improved safety profile. The tumor-selective replica- mechanisms. tion of this virus and its antitumor efficacy were Based on two common characteristics of telomerase characterized in several tumor cell lines in vitro and in activation and low oxygen tension environment in most xenograft models of human non-small cell lung cancer in solid cancers (3, 4), a human telomerase reverse transcrip- nude mice. With the selective replication and oncolysis, it tase (hTERT) promoter-regulated CRAd (5, 6), CNHK300 with the E1a gene controlled by the hTERT promoter, and a dual-regulated CRAd, CNHK500, with the E1a and E1b genes controlled by the hTERT promoter and the hypoxia Received 12/26/07; revised 3/10/08; accepted 3/16/08. response element (HRE; ref. 7), respectively, were reported Grant support: Important Project of National Natural Scientific Foundation of China grant 30730104 and Important Project of Zhejiang Natural previously. They showed that the simultaneous regulation Science Foundation grant Z205618. of E1a and E1b transcription by two promoters can greatly Note: X. Wang, C. Su, H. Cao, and K. Li contributed equally to this work. increase the tumor specificity of CRAds and reduce adverse The costs of publication of this article were defrayed in part by the side effects in normal cells. payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to Otherwise, the loading of antitumor transgene in CRAds indicate this fact. is also an effective means to enhance the antitumor efficacy. Requests for reprints: Qijun Qian, Laboratory of Viral and Gene Therapy, Among many candidates, the p53 gene plays an important Eastern Hepatobiliary Surgical Hospital, Second Military Medical role in cancer gene therapy, which has received preclinical University, 225 Changhai Road, Shanghai 200438, People’s Republic of China. Phone: 86-21-35030677; Fax: 86-21-35030677. validation by developing anticancer agents that specifically E-mail: [email protected] reactivate p53 function (8). To explore the efficacy of wild- Copyright C 2008 American Association for Cancer Research. type p53 reactivation as a tumor therapy, Ventura et al. (9) doi:10.1158/1535-7163.MCT-07-2429 showed that restoring endogenous p53 expression in mice

MolCancerTher2008;7(6).June2008

led to regression of autochthonous lymphomas through pPE3. After homologously recombining in HEK293 cells, cellular apoptosis pathway and sarcomas through cellular we obtained two CRAds named SG600 and SG600-p53 and senescence in mice but without affecting normal tissues. By one replication-deficient adenovirus named Ad-p53. RNA interference to conditionally regulate endogenous p53 Adenovirus Replicative Assay In vitro expression in a mosaic mouse model of liver carcinoma, it To investigate the replication of SG600-p53, the cancer was found that even brief reactivation of endogenous p53 cell and normal cell lines were seeded in six-well plates at a in p53-deficient tumors could produce complete tumor density of 1 Â 106 per well and infected with SG600-p53 at a regressions through the induction of a cellular senescence multiplicity of infection (MOI) of 5 plaque-forming units that was associated with differentiation and up-regulation (pfu)/cell. The supernatants and cells were titered at 0, 48, of inflammatory cytokines (10). These studies indicated that and 96 h after infection by TCID50 method as described the p53-triggered tumor regression is not only due to the previously (5). cellular apoptosis but the cellular senescence program, Western Blot Analysis for E1A and E1B Expression which is dependent on the tumor types. HepGII, Hep3B, MRC-5, and BJ cells were seeded in six- In this study, we constructed a triple-regulated replica- well plates at a density of 5 Â 105 per well, cultured for tive adenovirus, SG600, in which the CR2 region of E1a 24 h, and infected with viruses at a MOI of 1 pfu/cell. Two gene was partly deleted, and the E1a and E1b genes were days after infection, cells were harvested and measured the controlled by the hTERT promoter and the HRE, respec- expression of E1A and E1B-55-kDa by Western blot using tively. These modifications were expected to increase the mouse anti-adenovirus E1A monoclonal antibody M73 capacity of viral replication and oncolysis specifically (Santa Cruz Biotechnology) or rat anti-adenovirus E1B-55- targeting the cancer cells and to decrease the viral kDa monoclonal antibody (Oncogene Research Products) cytotoxicity to normal cells. When SG600 was used to be as described in earlier report (5). a vector and armed with the wild-type p53 gene, we believe ELISA for p53 Expression that not only the antitumor effect of the generated SG600- In the in vitro experiments, NCI-H1299 cells were seeded p53 is improved but also the expression of p53 gene is in six-well plates at a density of 5 Â 105 per well. To increased markedly. investigate the relation of viral dose and p53 expression, the cells were infected with SG600-p53 and Ad-p53 at MOI of 5, 1, 0.5, 0.1, and 0.01 pfu/cell. At 72 h after infection, the Materials and Methods cells were collected and lysed, and the supernatants of cell Cell Culture and Virus Preparation lysates were used to detect p53 expression by ELISA as Human cancer cell lines (A549, NCI-H1299, H446, described previously (6). To investigate the relation of time HepGII, Hep3B, PANC-1, HeLa, and L02), human fibro- and p53 expression, the cells were infected with SG600-p53 blast lines (MRC-5, HEL-1, HPF-1, and BJ), and human and Ad-p53 at a MOI of 1 pfu/cell, and the supernatants of embryo kidney cell line (HEK293) were purchased from the cell lysates were collected at 0, 24, 48, 72, and 96 h after American Type Culture Collection. The human cancer cell infection for detecting p53 expression. lines (SGC-7901 and SMMC-7721) were obtained from the Adenovirus Oncolytic Assay In vitro Institute of Cell Biology, Chinese Academy of Sciences. All To evaluate the cytotoxicity of adenovirus vectors, a cell lines were cultured according to the instructions of the panel of tumor cells was planted at a density of 1 Â 104 in providers. 96-well plates and infected with SG600-p53 and Ad-p53 at a Overlap PCR was done to delete the E1a promoter, the gradient of MOI from 0.001 to 100 pfu/cell. Cell viability E1b promoter, and the 24 nucleotides of adenovirus E1a was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- CR2 region, by which the nucleotides 464 to 550 of pXC1 tetrazolium bromide assay according to the instructions of (Microbix Biosystems) were deleted and replaced by Cell Proliferation Kit I (Roche Molecular Biochemicals). multiple cloning site 1 and the nucleotides 1,634 to 1,712 Animal Experiments were deleted and replaced by multiple cloning site 2. The BALB/c nude mice (nu/nu) were purchased from synthesized hTERT promoter (-212 to +46 bp) and HRE Shanghai Experimental Animal Center, Chinese Academy (five copies of TCCACAGTGCATACGTGGGCTCCAA- of Sciences. NCI-H1299 cancer cells in log phase were s.c. CAGGTCCTCT, with 60-bp additive nucleotides) were injected into the right flanks of mice (1 Â 107 per mouse). cloned into multiple cloning sites 1 and 2, respectively, and Four weeks later, the tumor xenografts were established, generated pSG600. The p53 expression cassette containing when the mice were allotted randomly into seven groups: the cytomegalovirus promoter, p53 cDNA, and SV40 SG600-p53 I, SG600-p53 II, SG600-p53 III, SG600, Ad-p53, polyA, released from pCA13-p53, which we constructed ONYX-015, and control (10 mice per group). Mice were previously (data not shown), was subcloned into the given total 1 Â 109 pfu viruses by five times of intratumoral multiple cloning site 1 of pSG600 between the expression injections, one time every other day, in the SG600-p53 II, cassettes of E1a and E1b genes and then generated the SG600, Ad-p53, and ONYX-015 groups and total 5 Â 108 adenoviral plasmid pSG600-p53. The plasmids pSG600, and 2 Â 109 pfu viruses by five times in the SG600-p53 I and pSG600-p53, and pCA13-p53 were respectively transfected SG600-p53 III groups, respectively. In the control group, into HEK293 cells using the Effectene Transfection Reagent 100 AL viral preservation solution [10 mmol/L Tris-HCl (Qiagen) together with the adenovirus packaging plasmid (pH 8.0), 2 mmol/L MgCl2, 4% sucrose] per mouse per time

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Table 1. Selective replication of SG600-p53 in cancer cell lines Results compared with that in normal cell lines Conditional Replication of the p53 Armed CRAd Cell lines 48 h 96 h A series of tumor and normal cell lines were planted in six-well plates (106 per well) and infected with indicated Normal cell lines viruses at a MOI of 5 pfu/cell. The cell lysates and HEL-1 1.60 10.60 supernatants at 48 and 96 h after infection were titered by HPF-1 1.60 1.60 TCID50 method in HEK293 cells and normalized to virus BJ 5.00 31.65 production per cell. SG600-p53 replicated preferably in all L02 13.50 50.50 cancer cell lines but markedly decreased in normal cell Cancer cell lines lines. The replicative capability of SG600-p53 ranged from A549 653.27 793.97 632.91- to 4,0625.00-fold increase in cancer cells at 48 h after SGC-7901 632.91 1,265.82 infection and from 793.97- to 4,0625.00-fold increase at 96 h, HepGII 4,000.00 3,160.00 H446 6,218.75 4,062.50 whereas from 1.60- to 13.50-fold increase in normal cells at HeLa 6,250.00 10,000.00 48 h after infection and from 1.60- to 50.50-fold increase at SMMC-7721 4,062.50 10,000.00 96 h (Table 1). H1299 40,625.00 40,625.00 In SG600 and SG600-p53, E1A and E1B-55-kDa expressions were controlled by the hTERT promoter and the HRE, respectively (Fig. 1A). By Western blot, the expressions of was injected intratumorally. Tumor volume was estimated E1A and E1B-55-kDa were positive in SG600-p53-infected with the formula: (maximal diameter) Â (perpendicular cancer cell lines and no or weakexpression in SG600-p53- diameter)2 Â 0.5. infected normal cells (Fig. 1B). Histology and Immunohistology SG600-p53-Mediated p53 Expression and Cancer Mice were sacrificed 35 days later according to the Cell-Killing Effect institutional guidelines. The tumors were removed, fixed in The p53-deficient lung cancer cell line NCI-H1299 was 10% neutral formaldehyde for 6 h, and embedded with cultured in six-well plates at a density of 5 Â 105 per well paraffin. Tumor sections were subjected to H&E staining and infected with SG600-p53 and Ad-p53 at different MOI. and immunohistochemistry. The expression of p53 was The cells were collected at 24, 48, 72, and 96 h after infection located using mouse anti-p53 antibody (Biodesign Interna- and detected p53 expression. The results showed that tional) by immunohistochemistry. The terminal deoxynu- SG600-p53 expressed p53 with high efficiency in cancer cleotidyl transferase–mediated dUTP nickend labeling cells compared with Ad-p53. At 72 h after infection with assay was done using the In situ Cell Death Detection Kit SG600-p53 and Ad-p53, the expression level of p53 was (Roche Diagnostics) according to the manufacturer’s increased gradually along with the increase of MOI instructions. (Fig. 2A). At a MOI of 1 pfu/cell, the expression level of Statistical Analysis p53 was increased gradually along with time prolonging Data from in vitro experiments were assessed by (Fig. 2B). For assessing the selective killing effect of SG600- Student’s t test and data from in vivo experiments were p53 in comparison with Ad-p53, a panel of tumor cells was assessed by ANOVA. Findings of P < 0.05 were considered planted in 96-well plates at a density of 1 Â 104 cells and significant. infected with SG600-p53 and Ad-p53 24 h later at the final

Figure 1. Expressions of E1A and E1B-55-kDa under the control of hTERT promoter and HRE. A, in SG600, the hTERT promoter and HRE were used to regulate the E1a and E1b genes, respectively. A p53 gene expression cassette containing cytomegalovirus promoter, p53 cDNA, and SV40 polyA was inserted into the genome of SG600 and then generated SG600-p53. ITR, inverted terminal repeats; w, adenovirus type 5 packaging signal. B, cells were seeded in six-well plates at a density of 5 Â 105 per well and infected with viruses at a MOI of 1 pfu/cell, and the cell lysates at 48 h were examined for E1A and E1B-55-kDa expression by Western blot. The expression of E1A or E1B-55-kDa mediated by SG600-p53 was positive in cancer cell lines and no expression in normal cells but always positive in the wild-type adenovirus-infected BJ cells.

MolCancerTher2008;7(6).June2008

pfu doses of SG600-p53 were administrated. The result showed that 2 Â 109 pfu of SG600-p53 could completely inhibit the tumor growth (Fig. 4B). Histopathologic Confirmation for Cancer Cell Apo- ptosis Mediated by the p53 Armed CRAd Mice were killed 35 days later by cervical dislocation. Tumors were removed and examined pathologically. There were many necrotic foci in tumor tissues of SG600-p53- treated groups. Around the necrotic areas, most cancer cells were positive for p53 expression (Fig. 5A), but there were no cancer cells positive for p53 in SG600 and control groups (Fig. 5B). In the virus-treated groups, including the SG600- p53 I, SG600-p53 II, SG600-p53 III, SG600, Ad-p53, and ONYX-015 groups, there were different amounts of cancer cells positive for the terminal deoxynucleotidyl transferase–mediated dUTP nickend labeling but more obviously in SG600-p53 groups (Fig. 5C). The positive indices for terminal deoxynucleotidyl transferase–mediated dUTP nickend labeling staining, which was counted the per- centages of positive cells from five randomly selected high- power fields, were 89.3 F 11.4, 83.0 F 13.1, 67.3 F 10.8, 44.7 Figure 2. Transgene expression by SG600-p53. NCI-H1299 cells were seeded in six-well plates at a density of 5 Â 105 per well and infected with the adenovirus SG600-p53 and Ad-p53 at MOI of 5, 1, 0.5, 0.1, and 0.01 pfu/cell. At 0, 24, 48, 72, and 96 h after infection, the cells were collected and the supernatants of cell lysates were used to detect p53 expression by ELISA. A, SG600-p53 expressed p53 with high efficiency in cancer cells compared with Ad-p53 at 72 h after infection and increased gradually along with the increase of MOI. B, at a MOI of 1 pfu/cell, the expression level of p53 was increased gradually along with the time prolonging.

MOI from 0.001 to 100 pfu/cell. 3-(4,5-Dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide assay was then measured to quantify cell viability and compared their MOI associated with 50% cell viability (IC50) and 90% cell viability (IC90). IC50 and IC90 values of SG600-p53 were P lower markedly than those of Ad-p53 ( = 0.0256 for IC50 P and = 0.0049 for IC90; Fig. 3). The Ad-p53/SG600-p53 ratios of IC50 values were 1,176.67-, 156.75-, 128.83-, 1,167.64-, 9,500.00-, 110.43-, and 173.07-fold in A549, NCI- H1299, NCI-H446, SMMC-7721, Hep3B, SGC-7901, and PANC-1, with IC90 values being 501.76-, 24.71-, 185.43-, 108.21-, 8,738.33-, 111.01-, and 117.42-fold in these cell lines, respectively. Antitumor Efficacy of SG600-p53 on Tumor Xeno- grafts We evaluated the antitumor efficacy of SG600-p53 on NCI-H1299 tumor xenografts established in nude mice. The tumor-bearing mice were given five intratumoral injections of different viral doses, one time every other day. At the same dose of total 1 Â 109 pfu viruses, SG600-p53 achieved the best among the virus-treated groups, and the antitumor effect of Ad-p53 was poorer than that of other viruses (Fig. 4A). Thirty-five days later after treatment, the groups injected with viruses achieved excellent antitumor efficacy P when compared with the control group ( = 0.0001 for Figure 3. IC50 and IC90 values by cell viability at various MOI of viral SG600-p53 I, SG600-p53 II, SG600-p53 III, and SG600 infection. A density of 104 cells per well cultured in 96-well plates were groups; P = 0.0002 for ONYX-015 group; P = 0.0121 for infected with indicated viruses at a wide range of MOI, ranging from 0.001 to 100 pfu/cell, and 7 days later, the cell viability was examined by 3-(4,5- Ad-p53 group). To investigate the antitumor efficacy of dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. IC50 and Â 8 Â 9 Â 9 SG600-p53 in different doses, 2 10 ,1 10 , and 2 10 IC90 values of SG600-p53 were lower markedly than those of Ad-p53.

Mol Cancer Ther 2008;7(6).June 2008

Figure 4. Antitumor activity of SG600- p53 in mice. NCI-H1299 cells were s.c. injected into right flanks of mice to establish tumors (1 Â 107 cells per mouse). Mice underwent five intratumoral injections, once every other day, with different doses of viruses in different groups. Tumor volume was measured regularly for 35 days after injections. SG600-p53 achieved the best efficacy among the virus-treated groups, and the antitumor efficacy was related to the viral doses. A, comparison of antitumor efficacy among the groups treated with 1 Â 109 pfu dose of different viruses. B, comparison of antitumor efficacy among the groups treated with different doses of SG600-p53 from total 5 Â 108, 1 Â 109,to2Â 109 pfu.

F 9.5, 35.7 F 10.5, and 32.3 F 8.5 in SG600-p53 I, SG600-p53 cells, kill them, and subsequently infect adjacent tumor II, SG600-p53 III, SG600, Ad-p53, and ONYX-015 groups, cells. They hold the promise of being effective agents for respectively. In control group, there were a few cancer cells the treatment of solid tumors (7, 13). However, the positive for terminal deoxynucleotidyl transferase–medi- treatment for cancers with CRAds solely is suboptimal. ated dUTP nickend labeling with the positive index of 5.3 The virotherapy with CRAds is based on their cancer F 2.1 (Fig. 5D). selectivity by confining viral replication in cancer cells. In the further study, CRAds need to be optimized to improve their safety for the patients with cancers and to maximize Discussion their antitumor potential as critical anticancer agents. The approach to treat cancers with viruses, named Both E1a and E1b genes are necessary for efficient viral virotherapy, was attended recently with the development replication. For the purpose of improving the safety of of CRAds (11, 12). CRAds are the viruses that do not CRAds, we modified SG600 by controlling the E1a gene replicate in normal cells but selectively replicate in tumor with the hTERT promoter and the E1b gene with the HRE.

Figure 5. Pathologic examination of xenografts. SG600-p53-treated tumor cells were positive for p53 expression (black arrow; A)but negative in the control group (B). More tumor cells were positive for terminal deoxynucleotidyl transferase – mediated dUTP nick end labeling in the SG600-p53 group (hollow arrow; C), whereas only a few tumor cells were positive in the control group (hollow arrow; D). Original magnification, Â200.

MolCancerTher2008;7(6).June2008

Two common characteristics were found in most human Disclosure of Potential Conflicts of Interest solid tumors: the existence of hypoxic areas in the tumor No potential conflicts of interest were disclosed. tissues and the expression of telomerase in the cancer cells (14–17). A few investigations showed that the existence of Acknowledgments hypoxic areas in tumor tissues and the expression of We thank L.F. Li, L.H. Jiang, and Y.Z. Qian for assistance with cell culture telomerase in cancer cells can be employed to achieve and J.Z. Gu for help with animal studies. cancer targeting treatment (18–20). Accordingly, our modifications to regulate SG600 with two promoters could References restrict adenoviral replication to tumors and thus achieve 1. Su C, Peng L, Sham J, et al. Immune gene-viral therapy with triplex better tumor-selective oncolysis. SG600 was synchronously efficacy mediated by oncolytic adenovirus carrying an interferon-g gene deleted 24 nucleotides in E1a-CR2 region that is responsible yields efficient antitumor activity in immunodeficient and immunocompe- for binding/inactivating pRb family members, which may tent mice. Mol Ther 2006;13:918 – 27. partly preserve pRb functions and exert the inhibition of 2. van Beusechem VW, van den Doel PB, Grill J, Pinedo HM, Gerritsen WR. Conditionally replicative adenovirus expressing p53 exhibits tumor growth. enhanced oncolytic potency. Cancer Res 2002;62:6165 – 71. For the purpose of fully realizing the antitumor potential 3. Huang TG, Savontaus MJ, Shinozaki K, Sauter BV, Woo SL. of CRAds, we modified SG600 by the payload of p53 gene. Telomerase-dependent oncolytic adenovirus for cancer treatment. Gene The p53 is one of the most important tumor suppressor Ther 2003;10:1241 – 7. gene, and the transfer of wild-type p53 gene is an important 4. Vaupel P. Tumor microenvironmental physiology and its implications for radiation oncology. Semin Radiat Oncol 2004;14:198 – 206. method to cure the p53-deficient cancers (21, 22). Consid- 5. Su CQ, Sham J, Xue HB, et al. Potent antitumoral efficacy of a novel ering that the tumor-specific CRAds can selectively replicative adenovirus CNHK300 targeting telomerase-positive cancer replicate in tumor cells, the construction of a gene-viral cells. J Cancer Res Clin Oncol 2004;130:591 – 603. vector, SG600-p53, by inserting the anticancer gene p53 into 6. Zhang Q, Nie M, Sham J, et al. Effective gene-viral therapy for the genome of the tumor-specific CRAds may increase the telomerase-positive cancers by selective replicative-competent adenovirus combining with endostatin gene. Cancer Res 2004;64:5390 – 7. expression of p53 and be able to combine the advantages of 7. Zhang Q, Chen G, Peng L, et al. Increased safety with preserved gene therapy and virotherapy. Hence, this strategy of gene- antitumoral efficacy on hepatocellular carcinoma with dual-regulated viral therapy overcomes the obstacles of traditional gene oncolytic adenovirus. Clin Cancer Res 2006;12:6523 – 31. therapy and enhances the anticancer efficacy (1). 8. Martins CP, Brown-Swigart L, Evan GI. Modeling the therapeutic The in vitro and in vivo experiments showed that SG600- efficacy of p53 restoration in tumors. Cell 2006;127:1323 – 34. p53 replicated preferably in cancer cells and expressed E1A 9. Ventura A, Kirsch DG, McLaughlin ME, et al. Restoration of p53 function leads to tumour regression in vivo. Nature 2007; and E1B-55-kDa, whereas hardly replicated and no E1A 445:606 – 7. and E1B-55-kDa expressed in normal cell lines, suggesting 10. Xue W, Zender L, Miething C, et al. Senescence and tumour clearance that SG600-p53 has a high selectivity to cancer cells and a is triggered by p53 restoration in murine liver carcinomas. Nature 2007; low toxicity to normal cells. 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J Natl Cancer Inst 2006;98:298 – 300. groups achieved the significant antitumor efficacy. The 14. Hsu YH, Lin JJ. Telomere and telomerase as targets for anti- antitumor effect of SG600-p53 was the best among the cancer and regeneration therapies. Acta Pharmacol Sin 2005;26: SG600-p53, SG600, ONYX-015, and Ad-p53 treated 513 – 8. groups and was related to the doses of viruses. SG600- 15. Hewitson KS, Schofield CJ. The HIF pathway as a therapeutic target. Drug Discov Today 2004;9:704 – 11. p53 (2 Â 109 pfu) exerted higher antitumor efficacy and 16. Welsh SJ, Powis G. Hypoxia inducible factor as a cancer drug target. could completely inhibit the tumor growth compared with Curr Cancer Drug Targets 2003;3:391 – 405. Â 8 Â 9 5 10 or 1 10 pfu of viruses. By pathologic 17. Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer examination, we found that the virus administration 2003;3:721 – 32. resulted in cancer cell apoptosis, especially in the groups 18. Wirth T, Kuhnel F, Kubicka S. Telomerase-dependent gene therapy. of SG600-p53, whereas the wide area of necrosis in the Curr Mol Med 2005;5:243 – 51. tumor tissues appeared after the treatment of SG600-p53. 19. Post DE, Devi NS, Li Z, et al. Cancer therapy with a replicating oncolytic adenovirus targeting the hypoxic microenvironment of tumors. In conclusion, we successfully constructed an efficient Clin Cancer Res 2004;10:8603 – 12. tumor-selective oncolytic adenovirus SG600-p53, which 20. Cho WK, Seong YR, Lee YH, et al. Oncolytic effects of adenovirus was regulated under both hTERT promoter and HRE and mutant capable of replicating in hypoxic and normoxic regions of solid synchronously carried the p53 transgene and E1a-CR2 tumor. Mol Ther 2004;10:938 – 49. partially deletion. SG600-p53 holds an increase of anticancer 21. Weill D, Mack M, Roth J, et al. Adenoviral-mediated p53 gene transfer to non-small cell lung cancer through endobronchial injection. 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Xinghua Wang, Changqing Su, Hui Cao, et al.

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