Zhang C Y, Gong Y X, Ma H, An C C, Chen D Y
Department of Physics, Tsinghua University, Beijing, China.
Analyst. 2000 Sep;125(9):1539-42. doi: 10.1039/b005352j.
The type-I ribosome-inactivating protein trichosanthin (TCS) has a broad spectrum of biological and pharmacological activities, including abortifacient, anti-tumor and anti-HIV. We found for the first time that TCS induced the generation of reactive oxygen species (ROS) in human 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 Ca2+ and was further reduced when cytosolic Ca2+ was chelated by BAPTA-AM. The production of ROS increased rapidly after the application of TCS, which paralleled TCS-induced increase in intracellular calcium monitored using fluo 3-AM. Simultaneous observation of the nuclear morphological changes via two-photon laser scanning microscopy and production 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 experiments 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.
I型核糖体失活蛋白天花粉蛋白（TCS）具有广泛的生物学和药理活性，包括堕胎、抗肿瘤和抗HIV活性。我们首次通过使用荧光探针2'，7'-二氯荧光素二乙酸酯和共聚焦激光扫描显微镜，在单细胞水平上发现TCS可诱导人绒毛膜癌细胞（JAR细胞）产生活性氧（ROS）。结果表明，TCS诱导的ROS形成依赖于细胞外Ca2+的存在，而当胞质Ca2+被BAPTA-AM螯合时，ROS形成进一步减少。应用TCS后ROS的产生迅速增加，这与使用Fluo 3-AM监测的TCS诱导的细胞内钙增加相平行。通过双光子激光扫描显微镜同时观察核形态变化以及通过共聚焦激光扫描显微镜观察ROS的产生，结果显示ROS参与了JAR细胞的凋亡。抗氧化剂α-生育酚可阻止TCS诱导的ROS形成和细胞死亡，这一实验证实了ROS的作用。TCS在JAR细胞中诱导钙依赖性ROS产生以及ROS参与JAR细胞凋亡这一发现，可能为TCS的抗肿瘤和抗HIV机制提供新的见解。
