Internalization of trichosanthin initiating cellular signal transduction involved in its cytotoxicity and antiviral mechanism

Trichosanthin (TCS) is a ribosome inactivating protein with diversity of biological functions. The present study explored the mechanisms involved in TCS actions, ranged from the cellular internalization process to the final antiviral activity. Two possible intermediate cellular signal pathways were also included.; The cellular internization process of TCS was traced. The fluorescein isothiocyanate conjugated TCS quickly accumulated inside JAR cells. Most TCS-Au quickly bound to coated pit on the JAR cell surface and internalized except some penetrate directly, while only small amount of intracellular bovine serum albumin-Au was observed. This demonstrated that TCS entered cells mainly by receptor-mediated pathways.; Ca2+ signal was detected after TCS stimulation in JAR cells. It showed that TCS not only activated extracellular Ca2+ influx, but{09}also induced intracellular Ca2+ release from endoplasmic reticulum.; Our major finding was that TCS activated MAPKs pathway in JAR cells, including: the extracellular signal-regulated kinase (ERK), the p38 mitogen-activated protein kinase (p38 MAPK) and the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). This activation can be demonstrated in a dose response manner and its characteristics vary among cell lines. TCS mutein with lower ribosome inactivity did not show JNK activation.; The cytotoxicity of TCS reduced by blocking the ERK pathways and increased by p38 MAPK blockage. TCS induced apoptosis was demonstrated by DNA fragmentation. It was potentiated by p38 MAPK inhibitor and reduced by ERK inhibitor. This was further substantiated by studying caspase-3 and poly (ADP-ribose) polymerase (PARP). It appeared that TCS exerted opposing effects on activating caspase-3. That effect was more favorable for PARP cleavage and eventually resulted in apoptotic nucleus DNA fragmentation.; Herpes simplex virus type 1 (HSV-1) infection induced ERK, p38 MAPK and JNK/SAPK activation in Vero cells, and the signal characteristics were changed in appearance time and strength by TCS pretreatment. TCS pretreatment abolished HSV-1 induced Bcl-2 augment and caused cell death Thoroughly blocking p38 MAPK or ERK activation enhanced the antiherpetic activity of TCS in reducing total viral antigen expression.; In conclusion, TCS enters cells via a receptor-mediated pathway, activates Ca2+ signal and MAPKs pathways. Some functions of TCS may be mediated through these signal pathways.