The type-I ribosome-inactivating protein trichosanthin (TCS) has a borad spectrum of biological and pharmacological activities, including abortifacient, anti-tumor and anti-HIV. We found for the firtst time that TCS induced the generation of reactive oxygen species (ROS) in human generation of reactive oxygen species (ROS) in human choriocarcinoma cells (JAR cells) at the level of the single choriocarcinoma cells (JAR cells) at the level of the single cell by using the fluorescent probe 2', 7'-dichlorofluorescein diacetate with confocal laser scanning microscopy. TCS-induced ROS formation was shown to be dependent on the presence of extracellular Ca~(2+) ans was further reduced when cytosolic Ca~(2+) and was further reduced when cytosolic Ca~(2+) was chelated by BAPTA-AM. The production of ROS increased rapidly after the application of TCS, Which paralleled TCS-induced increae in intracellular calcium monitored using fluo 3-AM. Simultaneous observation of the nuclear morphological changes via two-photon laser scanning microscopy and prouduction of ROS via confocal laser scanning microscopy revealed that ROS were involved in the apoptosis of JAR cells. The contribution of ROS was confirmed by experimemnts in which the antioxidant #alpha#-tocopherol prevented TCS-induced ROS formation and cell death. The finding that TCS induced calcium-dependent generation of ROS in JAR cells and that ROS were involved in the apoptosis of JAR cells might provide new insight into the anti-tumor and anti-HIV mechanism of TCS.
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