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 ndogenously 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.

Dozens of PCR-based methods are available for chromosome walking from a known sequence to an unknown region. These methods are of three types: inverse PCR, ligation-mediated PCR and randomly primed PCR. However, none of them has been generally applied for this purpose, because they are either difficult or inefficient. Here we describe a simple and efficient PCR strategy

The type-I ribosome-inactivating protein trichosanthin (TCS) has a broad spectrum of biological and pharmacological activities, including abortifacient, anti-tumour and anti-HIV activities. We have found for the first time that TCS stimulated the production of reactive oxygen species (ROS) in JAR cells (a human choriocarcinoma cell line) in a time- and concentrationdependent manner by using the fluorescent probe 2',7'-dichlorofluorescein diacetate with confocal laser scanning microscopy. ESR spectral studies and the inhibition of ROS formation by the superoxide radical anion (O2 −d) scavenger superoxide dismutase, the H3O2 scavenger catalase and the hydroxyl radical (OHd) scavenger mannitol suggested the involvement of O2−d, H2O2 and OHd. TCS-induced ROS formation was shown to be dependent on the presence of both extracellular and intracellular Ca2+; moreover, ROS production paralleled the intracellular Ca2+ elevation induced by TCS, suggesting that ROS production might be a consequence of Ca2+ signalling. TCS-induced activation of caspase-3 was initiated within 2 h; however, TCS-induced production of ROS was initiated within 5min, suggesting that the production of ROS preceded the activation of caspase-3. Simultaneous observation of the nuclear morphological changes via two-photon laser scanning microscopy and ROS production via confocal laser scanning microscopy revealed that ROS is involved in the apoptosis of JAR cells. The involvement of ROS was also confirmed by the inhibition of TCS-induced cell death by the antioxidant Trolox and the ROS scavengers catalase and mannitol. Diethylenetriaminepenta-acetic acid, an inhibitor of metal-facilitated OHd formation, markedly inhibited TCS-induced cell death, suggesting that TCS induced OHd formation via the Fenton reaction. The finding that ROS is involved in the TCS-induced apoptosis of JAR cells might provide new insight into the antitumour and anti-HIV mechanism of TCS.

The type-I ribosome-inactivating protein trichosanthin (TCS) has a broad spectrum of biological and pharmacological activities, including abortifacient, anti-tumor and anti-human-immunodeficiency-virus (anti-HIV). In this study, circular dichroism (CD) and capillary electrophoresis were used for the first time to study TCS and its two TCS mutants of Y55G TCS (tyrosine 55 converted to glycine) and FYY140-142GSA TCS (tripeptide phenylalanine-tyrosine-tyrosine 140-142 converted to glycine-serine-alanine). The results indicated that the substitution of amino acids changed the secondary structures and the hydrophobility of TCS. Moreover, both Y55G TCS and FYY140-142GSA TCS demonstrated attenuated cytotoxicity and reactive oxygen species (ROS) production in human choriocarcinoma cells (JAR cells) as compared to natural TCS and wild-type TCS. Our results demonstrated the cytotoxicity of TCS on JAR cells and TCS-induced production of ROS might be TCS-conformational related, suggesting that CD and capillary electrophoresis study might throw new insight into the anti-tumor and anti-HIV mechanism of TCS.