Elsevier

Biochemical Pharmacology

Volume 78, Issue 10, 15 November 2009, Pages 1316-1322
Biochemical Pharmacology

Tanshinone IIA triggers p53 responses and apoptosis by RNA polymerase II upon DNA minor groove binding

https://doi.org/10.1016/j.bcp.2009.06.110Get rights and content

Abstract

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. Subsequently, we performed western blot, flow cytometry analysis, chromatin immunoprecipitation, and quantitative real-time PCR to illustrate the RNAPII degradation, phosphorylation, and distribution along the transcribed gene in H22 cells exposed to Tan IIA. In addition, p53 activation and apoptosis induction in both cultured H22 cells and in mice bearing the ascitic-type H22 were measured following Tan IIA treatment. It was revealed that Tan IIA decreases the level of RNAPII by altering DNA structure. At the low dose range (0.2–4 μM) of Tan IIA exposure, the DNA structure damage results in the inhibition of RNAPII binding to DNA and the initiation of RNAPII phosphorylation, while higher concentrations of Tan IIA (4–20 μM) cause complete phosphorylation and degradation of RNAPII followed by p53 activation and apoptosis. A similar apoptosis induction by RNAPII was observed in animals. Apoptosis of tumor cells from ascitic fluid was not detected until RNAPII levels were downregulated by Tan IIA, which requires 40 mg/kg body weight of Tan IIA. It was concluded that DNA-conformational-damage-dependent RNAPII response upon groove binding is the molecular basis of the antitumor property of Tan IIA, in vivo and in vitro.

Introduction

Tanshen (Salvia miltiorrhiza bunge) has been widely used in traditional Chinese medicine (TCM) practice for thousands of years in the treatment of multiple diseases such as coronary heart diseases, hemorrhage, dysmenorrheal, miscarriage, swelling, insomnia, and inflammatory diseases. As the aged-related diseases developing, the antitumor property of Tanshen attracted more and more interests [1]. As one of the most abundant extracts of Tanshen, Tan IIA has been found to effectively activate the tumor suppressor protein p53 and induce apoptosis [2]. The widely accepted hypothesis of the cytotoxicity of Tanshiones is that they could damage DNA by intercalation due to the planar phenanthrene ring, and free radicals generated by the furano-o-quine moiety [3]. Although the hypothesis is so primary that no experimental result may support it, it was cited by almost all the reports on Tan IIA to explain its cytotoxicity. But there is an obvious conflict between this structure-based cytotoxicity and the wide and safe use of Tanshen for numerous diseases other than tumor for thousands of years. Additionally, the hypothesis could only explain a part of the findings in structure–activity-relationship study (SAR) of Tanshinones’ cytotoxicity [4]. Thus, we began the investigation to unveil the exact molecular basis for the cytotoxicity of Tan IIA and discussed under which conditions it may exhibit cytotoxicity.

Our recent work has completely denied the aforementioned hypothesis [5]. It was shown that Tan IIA interacts with DNA by minor groove binding preferentially to AT-rich sequences of duplex DNA in stead of intercalation, and no free radical-mediated DNA damage is found in living cells after Tan IIA treatment. Furthermore, all of the findings in SAR study of Tanshinones could be explained easily according to the groove-binding mode [4], [5]. A new hypothesis about the antitumor molecular pharmacology of Tan IIA was raised that the DNA conformational change upon groove binding contributes to Tan IIA's antitumor property.

Many groove binders used as clinical antitumor drugs act by altering duplex DNA structure and therefore may interfere with transcription by RNA polymerase II (RNAPII) [6]. Distamycin A, for example, could destabilize the preinitiation complex and arrest transcription by altering the conformation of DNA [7]. In the similar way, other minor-groove-binding drugs DAPI and mithramycin also block transcription by RNAPII [8].

Since Tan IIA also exhibits minor groove-binding property, we investigated in this study whether or not the transcription mechanism is involved in its antitumor pharmacology. For the first time, it was revealed that Tan IIA induces p53 mediated apoptosis by RNAPII upon groove binding.

Section snippets

Chemicals and reagents

Tan IIA and cryptotanshinone (chemical correspondent product) were purchased from Shanghai Shunbo Bioengineering Co. (Shanghai, China). The purity is over 98% according to HPLC analysis. Distamysin A was purchased from Sigma–Aldrich Co. (USA). Tris base was from Promega Co. (USA). Calf thymus DNA (CT DNA) was obtained from Sigma–Aldrich Co. (USA).

Stock of CT DNA was prepared by dissolving commercial nucleic acid in Tris–HCl buffer (pH 7.0). The concentration of CT DNA was determined

Transition of B- to C-form DNA by Tan IIA in vitro

To illustrate the structure damage to duplex DNA upon binding with Tan IIA, the intrinsic CD spectra of DNA were performed. Cryptotanshinone, another extract of Tanshiones and an analogue of Tan IIA, was tested in parallel (structures shown in Fig. 1).

As shown in Fig. 2, the CD spectrum of free CT DNA exhibited a positive peak at 274 nm due to the base stacking and a negative band at 245 nm due to the helicity, which was the characteristic of DNA in the right-hand B-form [14]. With an increase in

Discussion

Although the anticancer activity of Tan IIA has been identified in mouse hepatic carcinoma, breast cancer cell line and so on, Tan IIA shows no general toxicity at certain doses [18], [19]. It promotes us to unveil the underlying antitumor molecular parmarcology of Tan IIA. Based on our surprising previous finding that Tan IIA is a groove binder, we firstly illustrated the B- to C-form DNA conformational change induced by Tan IIA in the present study. Comparing with DNA intercalators, the DNA

Acknowledgment

This work was supported by the National Natural Science Foundation of China (30772622).

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