Receptor-mediated endocytosis of trichosanthin in choriocarcinoma cells

Trichosanthin (TCS) is a ribosome inactivating protein (RIP). It is generally believed that its many biological activities act through inhibition of ribosomes resulting in a decrease in protein synthesis. It has been hypothesized that the rate of entry of TCS into cells to reach ribosomes is an important factor in determining its biological activity. To prove this hypothesis, we have mapped out and compared the intracellular routing of TCS in two cell lines, namely the choriocarcinoma JAR cell line, which is known to be highly sensitive to the toxic effects of TCS, and the hepatoma H35 cell line, to which TCS shows minimal toxicity. Results from laser scanning confocal microscopy indicated that fluorescein isothiocyanate labeled TCS quickly accumulated inside JAR cells within 4 h of incubation while only a low level of fluorescent signals was detected in H35 cells during the same period of time. When TCS was conjugated with gold particles (Au) and its intracellular locations were traced with a transmission electron microscope, it was found that most of TCS were bound to coated pits on the JAR cell surface and were rapidly internalized within an hour. By 4 h, TCS reached almost every cytoplasmic region including ribosomes, and the JAR cell began to degenerate. In H35 cells, however, the binding of TCS to coated pits was not observed, but instead, a small amount of TCS was found to penetrate the cell non-specifically by direct diffusion across the cell membrane. Our observations suggest that most of TCS enter JAR cells via a specific receptor mediated pathway, which allows a swift transport of TCS across the membrane and a rapid accumulation of intracellular TCS, while in H35 cells, TCS takes a slow and non-specific route. The receptor-mediated uptake together with the specific intracellular routing of TCS may partly account for the differential vulnerability of the choriocarcinoma cell line towards the toxicity of TCS.