Triptolide Induces Apoptosis By Interfering Ribosomal Biosynthesis In Lung Cancer Cell Line A549

Objective To explore the mechanism of triptolide-induced apoptosis from the perspective of ribosome biosynthesis,providing a theoretical basis for the application of triptolide in the treatment of tumor.Methods 1.The distribution of UBF/ RNA Pol I/ B23 was investigated by immunofluorescence combined with laser confocal microscopy imaging technique in triptolide-treated A549 cells.Chromatin immunoprecipitation assays were performed to detect the binding of UBF and RNA Pol I to ribosomal DNA promoter.2.A549 cells were treated with triptolide,labeled newly synthesized RNA with 5-FU for 15 min,and 5-FU labeld RNA?s were labeled with specific FITC-conjugated monoclonal antibodies.And nucleolin was used to stain nucleoli.3.Co-Immunoprecipitation was carried out to determine the binding of RPL23 and P53 to MDM2 in triptolide-treated A549 cells.4.The expression of BCL-2/P53/MDM2/PUMA and activated caspase9/3 were analyzed by western blot in A549 cells treated with triptolide.Results 1.The nucleolus disappeared and disintegrated,and B23/UBF/RNA Pol I dispersed in nucleus in the triptolide-treated A549 cells.The binding of UBF and RNA Pol I to r DNA promoter was siginificantly decreased,which was negatively correlated with the concentration of triptolide.2.The nucleoli of A549 cells disappeared and disintegrated,and NCL dispersed in the whole nucleus in cells treated with triptolide.The synthesis of r RNA was decreased or even disappeared.3.The binding of RPL23 to MDM2 was increased,and the binding of P53 to MDM2 was significantly decreased in A549 cells treated with triptolide.4.The expression of P53 and phosphorylated P53 and PUMA was remarkably increased.BCL-2 expression was significantly decreased.Caspase9 and caspase3 were activated in A549 cells treated with triptolide.Conclusion When A549 cells were treated by triptolide,the nucleolus disappeared and disintegrated,B23/ UBF/RNA Pol I were dispersed in the nucleus.And the binding of the UBF and the RNA Pol I to the r DNA promoter was significantly reduced,suggesting that the r RNA transcription was inhibited.The ribosome protein RPL23 in the nucleolus dissociated from the ribosome and bound to the MDM2 resulting in the release of P53 from MDM2.The expression of P53 is significantly increased in cytoplasm,which led to the reduction of BCL-2 and induction of apoptotic.We propose that TP can interrupt the synthesis of r RNA and promote the apoptosis of the lung cancer cells through the RPL23-MDM2-P53 apoptosis signal pathway.