| [BACKGROUND & AIM] The delivery of therapeutic genes into cancer cells or normal tissues of cancer patients is an important evolving strategy for cancer treatment. However, limitations in gene delivery systems, tumor targeting and regulation of transgenic expression have hindered current attempts at translating laboratory observations into meaningful clinical practice. Recent studies have demonstrated that cancer gene therapy will be particularly useful when combined with other types of cancer therapies. The discovery of early growth response-1 (Egr-1) gene used as a radiation sensitive promoter provides a new perspective and establishes the theoretical foundation for effective combination of radiotherapy with gene therapy. Endostatin, originally purified from a murine hemangioendothelioma cell line, is a 20-kDa C-terminal fragment digested from collagen XVIII, which is located in the basement membrane zones around blood vessels. At the cellular level, endostatin specifically inhibits proliferation and migration of endothelial cells and induces endothelial cell apoptosis. Up to date, endostatin has been reported as one of the most potent endothelial cell-specific inhibitors of angiogenesis and tumor growth in vivo In the present study, we constructed pEgr-Endostatin-EGFP recombinant plasmid to evaluate whether combination of endostatin gene delivery with local X-irradiation could enhance the overall efficacy in mouse melanoma which is an experimental setting quite refractory to either treatment alone.[MATERIALS AND METHODS]: 1)MATERIALS:â‘ pIRES2-EGFP plasmid, pcDNA3.1-Egr plasmid, pMD18T-Endostatin plasmid;â‘¡murine B16 melanoma cells;â‘¢C57BL/6 female mouse. 2)METHODS: construction of pEgr-Endostatin-EGFP recombinant plasmid and its identification through Rrestriction Endonuclease Cleavage Rreaction and PCR. The pEgr-Endostatin-EGFP plasmid was transfected into B16 cell line with liposome. The expression property of endostatin was investigated by RT-PCR and that of EGFP was detected by flow cytometry. Tumor-bearing mice were treated by the plasmid injection and 2 Gy X-irradiation for 3 times. Tumor growth was observed for 18 d after treatment. Change of tumor capillary formation was measured with histochemistry assay at the end of the experiment.[RESULTS]: It was confirmed that pEgr-Endostatin-EGFP recombinant plasmid had been constructed successfully by identification of Restriction Endonuclease Cleavage Reactions and PCR. After RT-PCR, there was no special band of endostatin in the normal B16 cells, but the positive bands were observed in pEgr-Endostatin-EGFP transfected groups with or without irradiation.The expression of GFP in B16 melanoma cells was detected after X-irradiation with 0.05~20 Gy. Time-course studies showed that the expression of GFP in B16 cells reached its peak at 8 h after 2 Gy X-ray irradiation. The injection of pEgr-Endostatin-EGFP recombinant plasmid into the implanted B16 melanoma in C57BL/6J mice followed by local X-irradiation could significantly inhibit tumor growth with inhibition of intratumor micro-vessel density.[CONCLUSIONS] : pEgr-Endostatin-EGFP recombind plasmid had been constructed successfully. Endostatin genes combined with irradiation are able to suppress the growth of B16 melanoma cells significantly in vitro and its inhibition effects were better than that in gene- or irradiation- treatment alone. The experimental results in tumor-bearing mouse model showed that gene-radiotherapy could significantly inhibit tumor growth with inhibition of intratumor micro-vessel density. The results are of substantial importance in investigating a new approach for clinical treatment of tumors as well as in improving radiotheraputic effects.
|
PDF Full Text Request