Study Of Triptolide Medicinal Effect And Regulation Of YY1Expression And Role In Cell Pathological Transformation

As a main component extracted from Chinese medical herb Lei gong teng, Triptolidehas shown multiple efficacy, such as anti-inflammatory, immunosuppression,anti-cystogenesis, especially anti-tumor, which attracted more attentions worldwide.Triptolide has been proven to inhibit kinds of tumor cell proliferation in vitro and in vivo,suggesting Triptolide is a potential anti-tumor drug. However, the effect and mechanism ofTriptolide on prostate cancer (PCa) was less studied. In this study, we used two PCa celllines to study the effect of Triptolide anti-PCa, compare the anti-tumor activity of Triptolidewith another active component from Lei gong teng Celstrol, further detect the mechanism ofTriptolide anti-PCa effect. Meanwhile, we also studied the regulation of a Triptolide targetgene Yin Yang1(YY1) expression and its' role in cell pathological transformation. We getseveral results listed in following:1. We used two PCa cell lines and performed in vitro cell assay to study the effect ofTriptolide and Celastrol anti-PCa effect. Our results showed that Triptolide, more potentthan Celastrol, inhibites cell growth and induces cell death in LNCaP and PC-3cell lines atnano molarity grade. Moreover, Triptolide induces PCa cell apoptosis through caspasesactivation and PARP cleavage.2. We performed xenograft assay to study the effect of Triptolide anti-PCa in vivo. Ourresults showed that Triptolide significantly inhibites the xenografted PC-3tumor growth innude mice, indicating the potent anti-PCa activity of Triptolide.3. We performed Realtime PCR and Western blot to study the mechanism of Triptolideanti-PCa effect. We demonstrated that Triptolide suppresses the SUMO-specific protease1(SENP1) expression at both mRNA and protein levels, resulting in an enhanced cellularSUMOylation in PCa cells. Meanwhile, Triptolide decreased AR, c-Jun and YY1expressionat similar manners, and suppressed AR, c-Jun and YY1mediated transcription activity.Furthermore, knockdown or ectopic SENP1, c-Jun and AR expression in PCa cells inhibitedthe Triptolide anti-PCa effects. These data suggested Triptolide anti-tumor activity may beattributed to mechanisms involving above activity.4. Since we found Triptolide down-regulated YY1expression in PCa cells. YY1is akey regulator in normal cells and over-expression in PCa cells. We are interested in the regulation of YY1expression. We analysed the YY1promoter and5'UTR sequences andfound several potential G4formation sequences. We Further performed Circular dichroism(CD) assay, footprinting and reporter assay and demonstrated that the presence ofG4‐quadruplex (G4) structures in the promoter and the5'-UTR of YY1, which took asuppression role in YY1expression.5. We further performed electrophoretic mobility shift assay (EMSA), Chromatinimmunoprecipitation (ChIP) assay and reporter assay and discovered that G4Resolvase1(G4R1) binds and unwinds the G4motif of the YY1promoter in vitro and in vivo, resultinginto YY1expression enhanced. In addition, the analysis of a gene array data consisting of258patients breast cancer samples and protein levels in breast cancer cell lines indicate asignificant, positive correlation between G4R1and YY1expression, which bothever-expressed in breast cancer.6. We further studied the role of YY1in cell pathological transformation. Using breastcancer as model, we evaluated YY1expression in different breast cancer cell lines, a breastcancer tissue microarray (TMA) and two gene arrays. We observed that YY1is generallyoverexpressed in breast cancer cells and patient samples compared to normal cells andtissues. In functional studies, ectopically expressed YY1enhanced the migration andinvasion of non-tumorigenic breast cells. Conversely, shRNA-mediated YY1depletioninhibited breast cancer cell proliferation, migration, invasion and tumor formation in mousexenografts. Our data strongly suggested a positive role of YY1in breast cancer cellpathological transformation.