Cytotoxicity of HSVtk and hrTNF-alpha fusion genes with IRES in treatment of gastric cancer.
The efficacy of the suicide gene therapy by using the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) system for the treatment of cancer is limited because of the insufficient gene transfer and the low killing activity. To enhance the antitumor activity, we probed into whether recombinant ritroviral expression vector PLXSN expressing both HSVtk and TNF-alpha genes could potentiate the destruction of SGC7901. The PL(tk-TNF-alpha)SN harboring HSVtk and TNF-alpha genes in sequence was constructed with a bicistronic unit including the internal ribosomal entry site, the recombinant retroviruses were transferred into SGC7901 cells by lipofectamine, and pEGFP and Western blot analysis were used to detect the expression of fusion genes in transfected SGC7901 cells, and then apoptosis of the transfected cells were detected by using the TdT-mediated dUTP nick end labeling, flow cytometric analysis and transmission electron microscopy. In vitro study, the transfected gastric cancer cells were maintained in the GCV-contained medium, to assay the cell killing effect and bystander effect. In vivo experiments, retroviral serum plasmids were transfected into tumor-bearing nude mice, to observe the changes of tumor volumes and survival of the mice. In vitro there was no significant difference of cell survival rate between the three groups. However, in vivo results showed that tk/GCV, tk-TNF-alpha/GCV and TNF-alpha could inhibit the tumor growth, and the obvious anti-tumor effect was shown in tk-TNF-alpha/GCV group, and TNF-alpha obviously enhanced the anti-tumor effect in vivo. The pathologic examination showed necrosis of the cancer in the treated groups.
Zhang
Jian-Hua
JH
Department of Biomedical Engineering, School of Life Science and Technology, Xian Jiaotong University, 28 Xianning Road, Shoanxi Province 710049, China.
Wan
Ming-Xi
MX
Pan
Bo-Rong
BR
Yu
Bing
B
D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D000998:Antiviral Agents / Q000627:therapeutic use; D017209:Apoptosis / Q000502:physiology; D015153:Blotting, Western; D005260:Female; D015774:Ganciclovir / Q000627:therapeutic use; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D011993:Recombinant Fusion Proteins / Q000627:therapeutic use*; D012190:Retroviridae / Q000235:genetics; D012270:Ribosomes / Q000502:physiology; D018139:Simplexvirus / Q000201:enzymology*; D013274:Stomach Neoplasms / Q000235:genetics / Q000628:therapy*; D013937:Thymidine Kinase / Q000235:genetics*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
D020121:5' Untranslated Regions; D000998:Antiviral Agents; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D013937:Thymidine Kinase; D015774:Ganciclovir