Enhanced green fluorescence protein tracks trichosanthin in human choriocarcinoma cells as a feasible and stable reporter.
Trichosanthin (TCS) is a ribosome-inactivating protein (RIP) which can inhibit the growth of human choriocarcinoma (JAR) cells. There are no clear mechanisms to discover the interaction pathway and cytotoxicity of TCS in JAR cells. In this paper, we showed the distribution and transport of endogenously expressed TCS in JAR cells. Enhanced Green Fluorescence Protein (EGFP), fused with TCS, was applied as a reporter to track the behavior of TCS in JAR cells. Firstly, we investigated the expression stability of EGFP and physiological effects on JAR cells. A stable cell line expressing EGFP was created, which could reproduce and express EGFP even if transplanted into nude mice. Based on the proved stability and feasibility of EGFP in cultured cells and in vivo, the fusion gene of EGFP and TCS was constructed and transfected into JAR cells by liposome. The fluorescence microscopy showed that TCS-EGFP fusion gene was expressed in JAR cells in 24 to 48 hours and the fluorescence spread in cytoplasm mainly and in nucleus partially, which could trace the distribution and transport of TCS-EGFP in JAR cells. Most of fluorescent cells died after 48 hours for the cytotoxicity of expressed TCS-EGFP. These results first reported a stable expression and tracing method by EGFP in JAR cells, and provided theoretical basis to apply TCS in cancer therapy.
Wang
Ye
Y
National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China.
Mi
Shuang-Li
SL
Lou
Mei-Yan
MY
Gao
Yin
Y
Chen
Zhang-Liang
ZL
An
Cheng-Cai
CC
D000818:Animals; D002822:Choriocarcinoma / Q000378:metabolism* / Q000473:pathology*; D005240:Feasibility Studies; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D015978:Trichosanthin / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D049452:Green Fluorescent Proteins; D015978:Trichosanthin