HSVtk/GCV system on hepatoma carcinoma cells: Construction of the plasmid pcDNA3.1‑pAFP-TK and targeted killing effect

Li,Yong‑Fang; Yuan,Yang‑Yang; Zhang,Ying‑Min; Zhao,Na; Zhang,Qi; Meng,Fan‑Xiu; Gao,Ran‑Peng; Yu,Bao‑Feng; Zhang,Yue‑Hong; Guo,Rui; Wang,Hai‑Long; Xie,Jun; Xu,Jun; Qin,Qin; Dong,Xiu‑Shan

doi:10.3892/mmr.2017.6657

HSVtk/GCV system on hepatoma carcinoma cells: Construction of the plasmid pcDNA3.1‑pAFP-TK and targeted killing effect

Authors:
- Yong‑Fang Li
- Yang‑Yang Yuan
- Ying‑Min Zhang
- Na Zhao
- Qi Zhang
- Fan‑Xiu Meng
- Ran‑Peng Gao
- Bao‑Feng Yu
- Yue‑Hong Zhang
- Rui Guo
- Hai‑Long Wang
- Jun Xie
- Jun Xu
- Qin Qin
- Xiu‑Shan Dong
View Affiliations

Affiliations: Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China, Department of General Surgery, Affiliated Tumor Hospital of Shanxi Medical University, Taiyuan, Shanxi 030013, P.R. China, Central Laboratory, Shanxi Provincial People's Hospital, Shanxi Medical University, Taiyuan, Shanxi 030012, P.R. China, Department of General Surgery, Shanxi Dayi Hospital, Taiyuan, Shanxi 030032, P.R. China
Published online on: May 31, 2017 https://doi.org/10.3892/mmr.2017.6657
Pages: 764-772
Copyright: © Li 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

Previous studies demonstrated that herpes simplex virus thymidine kinase (HSVtk) could phosphorylate non‑toxic gancyclovir (GCV) efficiently to produce phosphorylated products that result in cell apoptosis, to kill tumor cells. The present study aimed to construct a plasmid vector, pcDNA3.1‑pAFP‑TK, carrying the suicide gene driven by the alpha‑fetoprotein (AFP) promoter, to investigate the cytotoxicity of HSVtk/GCV suicide gene system on hepatoma carcinoma cells. Reverse transcription‑polymerase chain reaction and western blotting results demonstrated that the HSVtk gene was effectively expressed in HepG2 hepatoma carcinoma cells transfected with pcDNA3.1‑pAFP‑TK plasmid, whereas HSVtk gene expression was not detected in normal HL‑7702 liver cells. In addition, MTT assays indicated that cell viability of HepG2 cells with the plasmid pcDNA3.1‑pAFP‑TK decreased in a dose‑dependent manner following treatment with GCV for 48 h. Flow cytometry also revealed that the cell apoptosis rate and mitochondrial membrane potential reduction rate in the HepG2 cells treated with HSVtk/GCV suicide gene system were significantly higher than in the control group. Apoptosis rates in the control group and the pcDNA3.1‑pAFP‑TK group were (1.00±0.62%) and (38.70±6.03%), respectively. Mitochondrial membrane potential reduction rates in the control group and the pcDNA3.1-pAFP-TK group were (0.57±0.11%) and (22.84±5.79%), respectively. Caspase‑3 staining demonstrated that activated caspase‑3 increased significantly in the HepG2 cells treated with HSVtk/GCV suicide gene system, whereas in the control group activated caspase‑3 increase was not observed. The results of the present study, therefore, indicated that HSVtk suicide gene was obviously expressed in the HepG2 cells and that the HSVtk/GCV system was effective at killing HepG2 hepatoma carcinoma cells.

View Figures

View References

1	Bosch FX, Ribes J, Diaz M and Cléries R: Primary liver cancer: Worldwide incidence and trends. Gastroenterology. 127:(5 Suppl 1). S5–S16. 2004. View Article : Google Scholar : PubMed/NCBI
2	Levin B and Amos C: Therapy of unresectable hepatocellular carcinoma. N Engl J Med. 332:1294–1296. 1995. View Article : Google Scholar : PubMed/NCBI
3	Arii S, Yamaoka Y, Futagawa S, Inoue K, Kobayashi K, Kojiro M, Makuuchi M, Nakamura Y, Okita K and Yamada R: Results of surgical and nonsurgical treatment for small-sized hepatocellular carcinomas: A retrospective and nationwide survey in Japan. The liver cancer study group of Japan. Hepatology. 32:1224–1229. 2000. View Article : Google Scholar : PubMed/NCBI
4	Rongrui L, Na H, Zongfang L, Fanpu J and Shiwen J: Epigenetic mechanism involved in the HBV/HCV-related hepatocellular carcinoma tumorigenesis. Curr Pharm Des. 20:1715–1725. 2014. View Article : Google Scholar : PubMed/NCBI
5	Llovet JM, Di Bisceglie AM, Bruix J, Kramer BS, Lencioni R, Zhu AX, Sherman M, Schwartz M, Lotze M, Talwalkar J, et al: Design and endpoints of clinical trials in hepatocellular carcinoma. J Natl Cancer Inst. 100:698–711. 2008. View Article : Google Scholar : PubMed/NCBI
6	Cheng AL, Kang YK, Chen Z, Tsao CJ, Qin S, Kim JS, Luo R, Feng J, Ye S, Yang TS, et al: Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: A phase III randomised, double-blind, placebo-controlled trial. Lancet Oncol. 10:25–34. 2009. View Article : Google Scholar : PubMed/NCBI
7	Liver Cancer Study Group of Japan: Primary liver cancer in Japan. Clinicopathologic features and results of surgical treatment. Ann Surg. 211:277–287. 1990.PubMed/NCBI
8	Schmitz V, Qian C, Ruiz J, Sangro B, Melero I, Mazzolini G, Narvaiza I and Prieto J: Gene therapy for liver diseases: Recent strategies for treatment of viral hepatitis and liver malignancies. Gut. 50:130–135. 2002. View Article : Google Scholar : PubMed/NCBI
9	Dachs GU, Dougheay GJ, Stratford IJ and Chaplin DJ: Targeting gene therapy to cancer: A review. Oncol Ras. 9:313–325. 1997.
10	Ferrara F, Staquicini DI, Driessen WH, D'Angelo S, Dobroff AS, Barry M, Lomo LC, Staquicini FI, Cardó-Vila M, Soghomonyan S, et al: Targeted molecular-genetic imaging and ligand-directed therapy in aggressive variant prostate cancer. Proc Natl Acad Sci USA pii: 201615400. 2016. View Article : Google Scholar
11	Anderson WF: Gene therapy for cancer. Hum Gene Ther. 5:1–2. 2008. View Article : Google Scholar
12	Huber BE, Richards CA and Krenitsky TA: Retroviral-mediated gene therapy for the treatment of hepatocellular carcinoma: All innovative approach for therapy. Proc Natl Acad Sci USA. 88:8039–8043. 1991. View Article : Google Scholar : PubMed/NCBI
13	Yashiyasu K and Ayumi T: Gene therapy of hepatoma: Bystander effects and non-apoptotic cell death induced by thymidine kinase and ganciclovir. Cancer Lett. 96:105–110. 1995. View Article : Google Scholar : PubMed/NCBI
14	Ma WJ, Wang HY and Teng LS: Correlation analysis of preoperative serum alpha-fetoprotein (AFP) level and prognosis of hepatocellular carcinoma (HCC) after hepatectomy. World J Surg Oncol. 11:2122013. View Article : Google Scholar : PubMed/NCBI
15	Su H, Chang JC, Xu SM and Kan YW: Selective killing of AFP-positive hepatocellular carcinoma cells by adeno-associated virus transfer of the herpes simplex virus thymidine kinase gene. Hum Gene Ther. 7:463–470. 1996. View Article : Google Scholar : PubMed/NCBI
16	Cahill BA and Braccia D: Current treatment for hepatocellular carcinoma. Clin J Oncol Nurs. 8:393–399. 2004. View Article : Google Scholar : PubMed/NCBI
17	Farazi PA and DePinho RA: Hepatocellular carcinoma pathogenesis: From genes to environment. Nat Rev Cancer. 6:674–687. 2006. View Article : Google Scholar : PubMed/NCBI
18	Varela M, Sala M, Llovet JM and Bruix J: Treatment of hepatocellular carcinoma: Is there an optimal strategy. Cancer Treat Rev. 29:99–104. 2003. View Article : Google Scholar : PubMed/NCBI
19	Huber BE, Austin EA, Richards CA, Davis ST and Good SS: Metabolism of 5-fluorocytidineto5-fluorouracic in human colorectal tumor cells transduced with the cytosine deaminase gene: Significant antitumor effect when only a small percentage of tumor cells express cytosine deaminase. Proc Natl Acad Sci USA. 91:8302–8306. 1994. View Article : Google Scholar : PubMed/NCBI
20	Yang XP, Liu L, Wang P and Ma SL: Human sulfatase-1 improves the effectiveness of cytosine deaminase suicide gene therapy with 5-Fluorocytosine treatment on hepatocellular carcinoma cell line HepG2 in vitro and in vivo. Chin Med J (Engl). 128:1384–1390. 2015. View Article : Google Scholar : PubMed/NCBI
21	Tsuchiya K, Asahina Y, Matsuda S, Muraoka M, Nakata T, Suzuki Y, Tamaki N, Yasui Y, Suzuki S, Hosokawa T, et al: Changes in plasma vascular endothelial growth factor at 8 weeks after sorafenib administration as predictors of survival for advanced hepatocellular carcinoma. Cancer. 120:229–237. 2014. View Article : Google Scholar : PubMed/NCBI
22	Sadeghi M, Lahdou I, Oweira H, Daniel V, Terness P, Schmidt J, Weiss KH, Longerich T, Schemmer P, Opelz G and Mehrabi A: Serum levels of chemokines CCL4 and CCL5 in cirrhotic patients indicate the presence of hepatocellular carcinoma. Br J Cancer. 113:756–762. 2015. View Article : Google Scholar : PubMed/NCBI
23	Qu L, Wang Y, Gong L, Zhu J, Gong R and Si J: Suicide gene therapy for hepatocellular carcinoma cells by surviving promoter-driven expression of the herpes simplex virus thymidine kinase gene. Oncol Rep. 29:1435–1440. 2013.PubMed/NCBI
24	Yu BF, Wu J, Zhang Y, Sung HW, Xie J and Li RK: Ultrasound-targeted HSVtk and Timp3 gene delivery for synergistically enhanced antitumor effects in hepatoma. Cancer Gene Ther. 20:290–297. 2013. View Article : Google Scholar : PubMed/NCBI
25	Tilghman SM and Belayew A: Transcriptional control of the murine albumin/alpha-fetoprotein locus during development. Proc Natl Acad Sci USA. 79:5254–5257. 1982. View Article : Google Scholar : PubMed/NCBI
26	Sakai M, Morinaga T, Urano Y, Watanabe K, Wegmann TG and Tamaoki T: The human alpha-fetoprotein gene. Sequence organization and the 5′flanking region. J Biol Chem. 260:5055–5060. 1985.PubMed/NCBI
27	Kanai F, Shiratori Y, Yoshida Y, Wakimoto H, Hamada H, Kanegae Y, Saito I, Nakabayashi H, Tamaoki T, Tanaka T, et al: Gene therapy for alpha-fetoprotein-producing human hepatoma cells by adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene. Hepatology. 23:1359–1368. 1996. View Article : Google Scholar : PubMed/NCBI
28	Gerolami R, Uch R, Faivre J, Garcia S, Hardwigsen J, Cardoso J, Mathieu S, Bagnis C, Brechot C and Mannoni P: Herpes simplex virus thymidine kinase-mediated suicide gene therapy for hepatocellular carcinoma using HIV-1-derived lentiviral vectors. J Hepatol. 40:291–297. 2004. View Article : Google Scholar : PubMed/NCBI
29	Sakai Y, Kaneko S, Nakamoto Y, Kagaya T, Mukaida N and Kobayashi K: Enhanced anti-tumor effects of herpes simplex virus thymidine kinase/ganciclovir system by codelivering monocyte chemoattractant protein-1 in hepatocellular carcinoma. Cancer Gene Ther. 8:695–704. 2001. View Article : Google Scholar : PubMed/NCBI