Antitumor effects of a dual-specific lentiviral vector carrying the Escherichia coli purine nucleoside phosphorylase gene

Wang,Xiaolong; Sun,Lei; Sun,Xuejun; Yu,Junhui; Wang,Kai; Wu,Yunhua; Gao,Qi; Zheng,Jianbao

doi:10.3892/ijo.2017.3949

Antitumor effects of a dual-specific lentiviral vector carrying the Escherichia coli purine nucleoside phosphorylase gene

Authors:
- Xiaolong Wang
- Lei Sun
- Xuejun Sun
- Junhui Yu
- Kai Wang
- Yunhua Wu
- Qi Gao
- Jianbao Zheng
View Affiliations

Affiliations: Department of Oncology, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710004, P.R. China, Department of General Surgery, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710004, P.R. China, Department of General Surgery, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China
Published online on: April 4, 2017 https://doi.org/10.3892/ijo.2017.3949
Pages: 1612-1622
Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The Escherichia coli purine nucleoside phosphorylase/Fludarabine phosphate (ePNP/Fludara) suicide system has several drawbacks, such as side-effects and the low efficiency of ePNP expression. In this study, we evaluated the antitumor effects of the dual-specific 8HSEs-hTERTp-ePNP/Fludara suicide system under hyperthermia in vitro and in vivo. Luciferase activities from the 8HSEs‑hTERT and CMV promoters were compared using the dual luciferase assay in SW480 (high hTERT expression) and MKN74 cells (hTERT-negative) in the presence and absence of hyperthermia. Then, we investigated the effects of overexpressing the suicide gene ePNP using 8HSEs‑hTERT-driven lentiviral vectors with Fludara on in vitro cell viability, side-effects, apoptosis, cycle distribution, colony formation and in vivo xenograft tumor growth. At 43˚C, luciferase activity from the 8HSEs‑hTERT promoter was significantly increased in SW480 cells, but not in MKN74 cells. Importantly, luciferase activities from the 8HSEs‑hTERT promoter were much higher than from the CMV promoter in hTERT-expressing SW480 cells under heated conditions. The in vitro quantitative analysis showed a 4-fold higher ePNP protein expression from the 8HSEs‑hTERT promoter at 43˚C than at 37˚C in SW480 cells and the ePNP mRNA expression in SW480 cells at 43˚C was also higher than at 37˚C. Conversely, ePNP mRNA and protein expression were low, almost absent, in hTERT-negative MKN74 cells with or without hyperthermia. After Fludara addition, cell cytotoxicity assays showed that the significant inhibitory effect of the 8HSEs‑hTERTp-ePNP on SW480 cells was dose- and time-dependent with hyperthermia. The 8HSEs‑hTERTp-ePNP/Fludara suicide system significantly inhibited SW480 cell viability, colony formation, cell cycle progression and induced apoptosis in vitro, but also induced significant bystander effects, especially under the heated conditions. At the protein level, the suicide system significantly promoted Bax, caspase-3 and p53 expression and suppressed Bcl-2 expression. In sections from mouse xenografts, TUNEL assays showed that the suicide system reduced xenograft growth and induced SW480 apoptosis. These results indicated that the combinatorial cancer- and heat-specific promoter system has great potential for improving the efficacy of cancer treatment with hyperthermia. The 8HSEs‑hTERTp-ePNP/Fludara system may serve as a powerful strategy for cancer gene therapy combined with hyperthermia.

View Figures

View References

1	Sorscher EJ, Peng S, Bebok Z, Allan PW, Bennett LL Jr and Parker WB: Tumor cell bystander killing in colonic carcinoma utilizing the Escherichia coli DeoD gene to generate toxic purines. Gene Ther. 1:233–238. 1994.PubMed/NCBI
2	Afshar S, Olafsen T, Wu AM and Morrison SL: Characterization of an engineered human purine nucleoside phosphorylase fused to an anti-her2/neu single chain Fv for use in ADEPT. J Exp Clin Cancer Res. 28:1472009. View Article : Google Scholar : PubMed/NCBI
3	Parker WB, Allan PW, Shaddix SC, Rose LM, Speegle HF, Gillespie GY and Bennett LL Jr: Metabolism and metabolic actions of 6-methylpurine and 2-fluoroadenine in human cells. Biochem Pharmacol. 55:1673–1681. 1998. View Article : Google Scholar : PubMed/NCBI
4	van der Zee J and González DG: Regional hyperthermia for rectal cancer. Lancet. 356:7722000. View Article : Google Scholar
5	Sherar M, Liu FF, Pintilie M, Levin W, Hunt J, Hill R, Hand J, Vernon C, van Rhoon G, van der Zee J, et al: Relationship between thermal dose and outcome in thermoradiotherapy treatments for superficial recurrences of breast cancer: Data from a phase III trial. Int J Radiat Oncol Biol Phys. 39:371–380. 1997. View Article : Google Scholar : PubMed/NCBI
6	Wust P, Hildebrandt B, Sreenivasa G, Rau B, Gellermann J, Riess H, Felix R and Schlag PM: Hyperthermia in combined treatment of cancer. Lancet Oncol. 3:487–497. 2002. View Article : Google Scholar : PubMed/NCBI
7	Morimoto RI, Sarge KD and Abravaya K: Transcriptional regulation of heat shock genes. A paradigm for inducible genomic responses. J Biol Chem. 267:21987–21990. 1992.PubMed/NCBI
8	Brade AM, Ngo D, Szmitko P, Li PX, Liu FF and Klamut HJ: Heat-directed gene targeting of adenoviral vectors to tumor cells. Cancer Gene Ther. 7:1566–1574. 2000. View Article : Google Scholar
9	Hildebrandt B, Wust P, Rau B, Schlag P and Riess H: Regional hyperthermia for rectal cancer. Lancet. 356:771–772. 2000. View Article : Google Scholar : PubMed/NCBI
10	Wang X, Zhou P, Sun X, Wei G, Zhang L, Wang H, Yao J, Jia P and Zheng J: Modification of the hTERT promoter by heat shock elements enhances the efficiency and specificity of cancer targeted gene therapy. Int J Hyperthermia. 32:244–253. 2016. View Article : Google Scholar : PubMed/NCBI
11	Horikawa I, Chiang YJ, Patterson T, Feigenbaum L, Leem SH, Michishita E, Larionov V, Hodes RJ and Barrett JC: Differential cis-regulation of human versus mouse TERT gene expression in vivo: Identification of a human-specific repressive element. Proc Natl Acad Sci USA. 102:18437–18442. 2005. View Article : Google Scholar : PubMed/NCBI
12	Cunniff NF and Morgan WD: Analysis of heat shock element recognition by saturation mutagenesis of the human HSP70.1 gene promoter. J Biol Chem. 268:8317–8324. 1993.PubMed/NCBI
13	Mosmann T: Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods. 65:55–63. 1983. View Article : Google Scholar : PubMed/NCBI
14	Parker WB, Allan PW, Hassan AEA, Secrist JA III, Sorscher EJ and Waud WR: Antitumor activity of 2-fluoro-2′-deoxyadenosine against tumors that express Escherichia coli purine nucleoside phosphorylase. Cancer Gene Ther. 10:23–29. 2003. View Article : Google Scholar
15	Pan Z, Sun X, Shan H, Wang N, Wang J, Ren J, Feng S, Xie L, Lu C and Yuan Y: MicroRNA-101 inhibited postinfarct cardiac fibrosis and improved left ventricular compliance via the FBJ osteosarcoma oncogene/transforming growth factor-β1 pathway. Circulation. 126:840–850. 2012. View Article : Google Scholar : PubMed/NCBI
16	Mocna M, Granja C, Leroy C and Stekl I: Hyperthermia in oncology. AIP Conf Proc. 958:256–257. 2007. View Article : Google Scholar
17	Akerfelt M, Morimoto RI and Sistonen L: Heat shock factors: Integrators of cell stress, development and lifespan. Nat Rev Mol Cell Biol. 11:545–555. 2010. View Article : Google Scholar : PubMed/NCBI
18	Zhang Y, Parker WB, Sorscher EJ and Ealick SE: PNP anticancer gene therapy. Curr Top Med Chem. 5:1259–1274. 2005. View Article : Google Scholar : PubMed/NCBI
19	van Dillen IJ, Mulder NH, Vaalburg W, de Vries EF and Hospers GA: Influence of the bystander effect on HSV-tk/GCV gene therapy. A review Curr Gene Ther. 2:307–322. 2002. View Article : Google Scholar
20	Chaudhary K, Ting LM, Kim K and Roos DS: Toxoplasma gondii purine nucleoside phosphorylase biochemical characterization, inhibitor profiles, and comparison with the Plasmodium falciparum ortholog. J Biol Chem. 281:25652–25658. 2006. View Article : Google Scholar : PubMed/NCBI
21	Hong JS, Waud WR, Levasseur DN, Townes TM, Wen H, McPherson SA, Moore BA, Bebok Z, Allan PW, Secrist JA III, et al: Excellent in vivo bystander activity of fludarabine phosphate against human glioma xenografts that express the Escherichia coli purine nucleoside phosphorylase gene. Cancer Res. 64:6610–6615. 2004. View Article : Google Scholar : PubMed/NCBI
22	Krohne TU, Shankara S, Geissler M, Roberts BL, Wands JR, Blum HE and Mohr L: Mechanisms of cell death induced by suicide genes encoding purine nucleoside phosphorylase and thymidine kinase in human hepatocellular carcinoma cells in vitro. Hepatology. 34:511–518. 2001. View Article : Google Scholar : PubMed/NCBI
23	Singh PP, Joshi S, Russell PJ, Nair S and Khatri A: Purine Nucleoside Phosphorylase mediated molecular chemotherapy and conventional chemotherapy: A tangible union against chemoresistant cancer. BMC Cancer. 11:3682011. View Article : Google Scholar : PubMed/NCBI
24	Wang XY, Martiniello-Wilks R, Shaw JM, Ho T, Coulston N, Cooke-Yarborough C, Molloy PL, Cameron F, Moghaddam M, Lockett TJ, et al: Preclinical evaluation of a prostate-targeted gene- directed enzyme prodrug therapy delivered by ovine atadenovirus. Gene Ther. 11:1559–1567. 2004. View Article : Google Scholar : PubMed/NCBI
25	Pan D, Jin L and Zhang X: The latest advances of experimental research on targeted gene therapy for prostate cancer. Chinese-German J Clin Oncol. 12:546–550. 2013. View Article : Google Scholar
26	Barese CN, Felizardo TC, Sellers SE, Keyvanfar K, Di Stasi A, Metzger ME, Krouse AE, Donahue RE, Spencer DM and Dunbar CE: Regulated apoptosis of genetically modified hematopoietic stem and progenitor cells via an inducible caspase-9 suicide gene in rhesus macaques. Stem Cells. 33:91–100. 2015. View Article : Google Scholar
27	Carlotti F, Zaldumbide A, Martin P, Boulukos KE, Hoeben RC and Pognonec P: Development of an inducible suicide gene system based on human caspase 8. Cancer Gene Ther. 12:627–639. 2005. View Article : Google Scholar : PubMed/NCBI
28	Chen Y, Shi S, Wang H, Li N, Su J, Chou G and Wang S: A Homogeneous polysaccharide from fructus Schisandra chinensis (Turz.) baill induces mitochondrial apoptosis through the Hsp90/AKT signalling pathway in HepG2 cells. Int J Mol Sci. 17:10152016. View Article : Google Scholar :
29	Cali U, Cavkaytar S, Sirvan L and Danisman N: Placental apoptosis in preeclampsia, intrauterine growth retardation, and HELLP syndrome: An immunohistochemical study with caspase-3 and bcl-2. Clin Exp Obstet Gynecol. 40:45–48. 2013.PubMed/NCBI
30	Karjoo Z, Chen X and Hatefi A: Progress and problems with the use of suicide genes for targeted cancer therapy. Adv Drug Deliv Rev. 99A:113–128. 2016. View Article : Google Scholar