Elsevier

Biochemical Pharmacology

Volume 62, Issue 6, 15 September 2001, Pages 733-741
Biochemical Pharmacology

Salvicine, a novel DNA topoisomerase II inhibitor, exerting its effects by trapping enzyme-DNA cleavage complexes1

https://doi.org/10.1016/S0006-2952(01)00732-8Get rights and content

Abstract

Salvicine, a structurally modified diterpenoid quinone derived from Salvia prionitis, is a novel anticancer drug candidate. The compound has significant in vitro and in vivo activity against malignant tumor cells and xenografts, especially some human solid tumor models. This anticancer activity of salvicine is associated with its ability to induce tumor cell apoptosis. Salvicine was also found to have a profound cytotoxic effect on multidrug-resistant (MDR) cell lines by down-regulating the expression of MDR-1 mRNA of MDR cells. Salvicine acted as a topoisomerase II (Topo II) poison through its marked enhancement effect on Topo II-mediated DNA double-strand breaks as observed in the DNA cleavage assay. Strong inhibitory activity of salvicine against Topo II was observed in a kDNA decatenation assay, with an approximate ic50 value of 3 μM. A similar result was obtained by a Topo II-mediated supercoiled DNA relaxation assay. In contrast, no inhibitory activity was observed against the catalytic activity of Topo I. When the effects of salvicine on individual steps of the catalytic cycle of Topo II were dissected, it was found that the mechanism by which salvicine inactivates Topo II is different from that by other anti-Topo II agents. Salvicine greatly promoted Topo II-DNA binding and inhibited pre- and post-strand Topo II-mediated DNA religation without interference with the forward cleavage steps. In addition, salvicine was not a DNA intercalative agent, as demonstrated by DNA unwinding assays. The results of this study indicate that the inhibitory activity of salvicine against Topo II was derived from its ability to stabilize DNA strand breaks through interactions with the enzyme alone or with the DNA-enzyme complex. It is therefore postulated that salvicine acts on Topo by trapping the DNA-Topo II complex, which in turn produces anticancer effects.

Introduction

Salvicine is a novel diterpenoid quinone compound (Fig. 1) obtained by structural modification of a natural product lead isolated from Salvia prionitis Hance (Labiatae) [1], [2], [3], [4]. The pharmaceutical activity of salvicine was evaluated in vitro against a panel of human tumor cells [5]. Compared to its moderate cytotoxicity on three leukaemia cell lines, salvicine exhibited potent activity against solid tumor cells, especially lung and gastric cancer cells [5]. Experiments in vivo indicated that salvicine possessed significant antitumor activity against murine S-180 sarcoma and Lewis lung cancer, as well as human lung adenocarcinoma xenograft models [6]. Additionally, the research demonstrated that the antitumor effect of salvicine was associated with its ability to induce tumor cell apoptosis with similar potency against both human leukaemia cells and gastric carcinoma cells, indicating its specific effect on solid tumor cells [7]. Furthermore, the cytotoxicity of salvicine was found not to be affected by the presence of P-glycoprotein in three multidrug-resistant (MDR) cell lines (K-562/A02, MCF-7/ADM, MKN28/VCR). The average resistance factor (RF) of salvicine on the three cell lines was 1.2, much lower than those of control anticancer drugs, including vincristine (VCR, RF 86.6), ADM (RF 233.2), and VP16 (RF 53.7). The main mechanism of circumventing MDR was related to inhibition of MDR-1 mRNA expression (Miao ZH, Tang T, Ding J, unpublished data). Results from these experiments suggest that salvicine is a promising antitumor agent which is entering clinical trials in China.

Although the cytotoxic mechanism of salvicine has not yet been fully understood, it is believed that it acts through its effect on DNA-Topo II as observed in a preliminary study in our laboratory. The aim of this study was to explore the effect of salvicine on the activity of Topo II and its mode of action. Firstly, two assays were used to confirm its selective activity on Topo II. Secondly, the DNA cleavage assay was employed to determine whether salvicine is a Topo II “inhibitor” or a “poison.” Thirdly, to gain insight into its action, effects of salvicine were characterised on the various steps of the catalytic cycle of the enzyme. Finally, unwinding assays were undertaken to observe whether salvicine could intercalate into DNA.

Section snippets

Materials

Supercoiled plasmid pBR322 was isolated from Escherichia coli by a method of alkaline lysis [8]. Kinetoplast DNA (kDNA) was a gift from Dr. Toshiwo Andoh (Soka University, Japan). Proteinase K, EcoRI restriction endonuclease, T4 DNA ligase, and SDS were purchased from the Sino-American Biotechnology Company. Salvicine presenting tangerine yellow colour crystalloid was provided by the Phytochemistry Department of Shanghai Institute of Materia Medica. VP16 was obtained from Pudong Pharmaceutical

Inhibition of the activity of Topo II but not Topo I by salvicine

The effect of salvicine on the strand passage activity of Topo II was determined by the enzyme-mediated negatively supercoiled pBR322 relaxation. As shown in Fig. 2, salvicine displayed significant inhibition of this reaction in a concentration-dependent style. The inhibition rate in the presence of 125 μM salvicine was 61.9% compared to 66.3% with 100 μM VP16. It appears that salvicine and VP16 are equipotent against Topo II in this reaction. To determine if salvicine is a selective inhibitor

Discussion

Topo II is an essential enzyme which plays an important role in DNA replication, repair, transcription and chromosome segregation [21], [22]. In addition to its critical functions, Topo II has been identified as an important antitumor target [23]. A number of the most active and widely prescribed antineoplastic drugs are targeted at Topo II [24]. In addition, there has been continuous interest in studying and developing new anti-Topo II agents and several potent compounds are currently in

Acknowledgements

Project supported by the National Natural Science Foundation of China (No. 39830444 and No. 30070877) and Shanghai Research Center of Life Sciences.

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    Abbreviations: ADM, adriamycin; VP16, etoposide; Topo, topoisomerase; kDNA, knetoplast DNA; HCPT, hydrocamptothecin; SC, supercoiled form; RLX, relaxed form; LNR, linear form; and NC, nicked form.

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