Abstract
Triptolide is a bioactive ingredient in traditional Chinese medicine that exhibits diverse biologic properties, including anticancer properties. Among its many putative targets, this compound has been reported to bind to XPB, the largest subunit of general transcription factor TFIIH, and to cause degradation of the largest subunit Rpb1 of RNA polymerase II (RNAPII). In this study, we clarify multiple important questions concerning the significance and basis for triptolide action at this core target. Triptolide decreased Rpb1 levels in cancer cells in a manner that was correlated tightly with its cytotoxic activity. Compound exposure blocked RNAPII at promoters and decreased chromatin-bound RNAPII, both upstream and within all genes that were examined, also leading to Ser-5 hyperphosphorylation and increased ubiqutination within the Rbp1 carboxy-terminal domain. Notably, cotreatment with inhibitors of the proteasome or the cyclin-dependent kinase CDK7 inhibitors abolished the ability of triptolide to ablate Rpb1. Together, our results show that triptolide triggers a CDK7-mediated degradation of RNAPII that may offer an explanation to many of its therapeutic properties, including its robust and promising anticancer properties.
Highlights
Triptolide is a principal bioactive ingredient of Tripterygium wilfordii Hook F, which has been used to treat autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and psoriasis in China [1]
Following validation of the triptolide-induced Rpb1 degradation, our results revealed that this agent elicited hyperphosphorylation of Rpb1 at Ser-5 of the carboxy-terminal domain (CTD) via CDK7, which contributed to its RNA polymerase II (RNAPII) degradation
RPB1 (CTD repeat), phospho-Ser-2-RPB1, phospho-Ser-5RPB1, and ubiquitin antibodies were from Abcam, CDK7, and b-actin antibodies were from Cell Signaling Technology, RPB1 (H-224) antibody was from Santa Cruz Biotechnology, and GAPDH antibody was from Beyotime
Summary
Triptolide is a principal bioactive ingredient of Tripterygium wilfordii Hook F, which has been used to treat autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and psoriasis in China [1]. In addition to its unique structure of a diterpenoid triepoxide, triptolide has aroused extensive research interests due to the following pharmacologic characteristics. Triptolide possesses multiple apparently unrelated activities, including anti-inflammatory, immunosuppressive, antifertility, anticystogenesis, and anticancer activities [1], which seem to suggest multiple molecular mechanisms involved in those activities. Authors' Affiliations: 1Department of Pharmacology and BioTechnology, University of Bologna, Bologna Italy; 2Division of Antitumor Pharmacology, State Key Laboratory of Drug Research; and 3Department of Medicinal Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, People's Republic of China. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
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