The Molecular Antitumor Pharmacology Of Tanshinone IIA

Our previous work has shown that Tanshinone IIA (Tan IIA) is a DNA minor groove binder instead of an intercalator as previously thought. In this study, we have further demonstrated that the molecular antitumor pharmacology of Tan IIA is dependent on its groove binding capability. First, we investigated the structure damage to duplex DNA upon Tan IIA binding using circular dichroism spectra (CD) in vitro. Subsequently, we performed western blot, flow cytometry analysis, Chromatin immunoprecipitation (ChIP), and quantitative real-time PCR (Q-PCR) to illustrate the RNA polymerase II (RNAPII) degradation, phosphorylation, and distribution along the transcribed gene in hydroperitoneum hepatoma cells (H22 cell line) exposed to Tan IIA. In addition, p53 activation and apoptosis induction by RNAPII in both cultured H22 cells and in mice bearing the ascitic-type H22 were measured following Tan IIA treatment. Cell-based experiments showed Tan IIA decreases the level of RNAPII in a similar manner with classic DNA groove binder distamysin A. The RNAPII response associates directly with DNA conformational change upon Tan IIA binding. At the low dose range (0.2-4μM) of Tan IIA exposure, the DNA structure damage results in the inhibition of RNAP II binding to DNA and the initiation of RNAPⅡphosphorylation, while higher concentrations of Tan IIA (4-20μM) causes complete phosphorylation and degradation of RNAP II followed by p53 activation and apoptosis. It was concluded that DNA-conformational-damage-dependent RNAPII response upon groove binding is the molecular basis of the antitumor property of Tan IIA. A similar apoptosis induction by RNAPII was observed in animals. Apoptosis of tumor cells from ascitic fluid was not detected until RNAP II levels were down-regulated by Tan IIA, which requires high doses as 40 mg/kg body weight of Tan IIA. It was revealed that Tan IIA triggers p53 mediated apoptosis in a dose-dependent manner by RNAPII upon groove binding both in vivo and in vitro.