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Construction of recombinant adenovirus carrying HSV-TK controlled by hMAM enhancer and promoter and its targeted killing effect on human breast cancer cells
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Abstract:
Objective : To construct two recombinant adenovirus vectors carrying reporter gene enhanced green fluorescent protein (EGFP) or suicide gene herpes simple virus thymidine kinase (HSV-TK) at the downstream of enhancer and promoter of human mammaglobin (hMAM-EP). To explore breast-cancer-cell-specific regulation effect of hMAM-EP and new methods of targeted therapy for breast cancer. Methods: Two recombinant plasmid vectors, hMAM-EP-EGFP and hMAM-EP-TK, were constructed, which respectively carried reporter gene EGFP and suicide gene HSV-TK at the downstream of hMAM-EP. Recombinant adenovirus vectors Ad-EP-EGFP and Ad-EP-TK were obtained after the target genes from the recombinant plasmids were transferred into adenovirus skeleton cosmid pAxcwit2; recombinant adenovirus vectors Ad-EP-EGFP and Ad-EP-TK were then transfected into breast cancer T-47D cells, ZR-75-30 cells and nasopharyngeal cancer 5-8F cells. The expression of EGFP was observed under a fluorescence microscope. Recombinant adenovirus Ad-EP-TK-infected T-47D cells were cultured with 1, 10, 20 and 50 μg/ml prodrug GCV to observe specific cell-killing effect on breast cancer cells. Results: The recombinant plasmid vectors Ad-EP-EGFP and Ad-EP-TK controlled by hMAM-EP were successfully constructed. EGFP could be observed in human breast cancer T-47D cells infected with Ad-EP-EGFP recombinant adenovirus, and could not be detected in ZR-75-30 and 5-8F cells. Compared with un-infected and Ad-EP-EGFP-infected groups, the survival rate of T-47D cells in Ad-EP-EGFP-infection combined with GCV (50 μg/ml) group was significantly decreased (\[35.69±0.07\]% vs \[91.74±0.02\]%, \[87.69±011\]%, P＜0.05). With an increase in mass concentration of GCV, the survival rate decreased. Cell survival rates were (94.34±0.04)%, (86.26±0.02)%, (66.51±0.09)% and (35.69±0.07)% when T-47D cells were infected with hMAM-EP-TK in a MOI of 100 and cultured with 1, 10, 20, and 50 μg/ml GCV. Conclusion: HSV-TK suicide gene controlled by hMAM-EP is specifically expressed in breast cancer T-47D cells, and T-47D cells can be killed by Ad-EP-TK combined with GCV.
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Project Supported:
Project supported by the Natural Science Foundation of Fujian Province (No. 2008j0088)
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