Cancer Letters

Cancer Letters

Volume 208, Issue 1, 10 May 2004, Pages 59-64
Cancer Letters

G2-M arrest and antimitotic activity mediated by casticin, a flavonoid isolated from Viticis Fructus (Vitex rotundifolia Linne fil.)

https://doi.org/10.1016/j.canlet.2004.01.012Get rights and content

Abstract

Flavonoids are distributed in many plants. We studied the antitumor effects of flavonoids isolated from Viticis Fructus, casticin, artemetin, quercetagetin and 5,3′-dihydroxy-6,7,4′ -trimethoxyflavanone. Casticin inhibited the growth of KB cells markedly (IC50=0.23 μM), compared with the other flavonoids tested (IC50=15.3–18.6 μM). In contrast, casticin did not inhibit the proliferation of A431 cells similar to normal cell lines, 3T3 Swiss Albino and TIG-103. Flow cytometric analyses revealed that the exposure of KB cells to casticin led to significant arrest at G2-M. In immunostaining of KB cells, casticin disrupted mitotic spindles.

These results suggest that G2-M arrest by casticin may be relevant to its antimitotic activity, although the mechanism of selective growth inhibition has been unknown. Further examinations are required to confirm that casticin is an antitumor drug for specific cancers with low toxicity.

Introduction

Dietary habits in humans are closely related to both the development and prevention of various cancers. It was concluded from epidemiological studies that consumption of higher levels of vegetables and fruits is associated with a reduced risk of cancer at many sites of the body, particularly epithelial cancers of the alimentary and respiratory tracts [1].

Various compounds of edible plants have been extensively studied for inhibition of tumor development. Among them, flavonoids are distributed widely in the plant kingdom and the daily intake of flavonoids is estimated to ranges between 25 mg and 1 g [2]. However, little is known about absorption, metabolism and excretion of dietary flavonoids in humans. To neglect the digestive influence on dietary flavonoids in the gastro-intestinal tract, we selected human squamous carcinoma cells of the oral floor and vulva (KB and A431 cells) in this study.

Various flavonoids were also reported to have antitumor activity [3], whereas many flavonoids still require elucidation of their biological activities. In our laboratory, we have been examining flavonoids derived from herbal medicines for the screening of antitumor agents [4], [5]. We examined the antitumor effects of various flavonoids extracted from Viticis Fructus which has been used in prescriptions in traditional medicine for the treatment of colds, headache, migraine, sore eyes, etc [6].

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Chemicals

Casticin (5,3′-dihydroxy-3,6,7,4′-tetramethoxyflavone), artemetin, quercetagetin (Fig. 1A) and 5,3′-dihydroxy-6,7,4′-trimethoxyflavanone (Fig. 1B) were isolated from methanol extract of Viticis Fructus (Tochimototenkaidou Co., Ltd, Osaka) by means of HPLC in an ODS column (Cosmosil 5C18-AR) under the mixed solvent system of acetonitrile and water. The structures of the isolated flavonoids were confirmed by their spectral data (1H-NMR and 13C-NMR). Casticin was identified with an authentic

Effects of casticin on proliferation of cancer cells and normal fibroblasts

Fig. 2A shows the growth curve of KB cells in the various concentrations of casticin. A dose-dependent inhibition of proliferation by casticin was observed between 0.2 and 1.0 μM on days 2 and 3. On day 3, the growth of cells decreased to 35, 14, and 6% of the control levels with 0.3, 0.6, and 1.0 μM casticin. The 50% inhibitory concentration (IC50) of casticin was 0.23 μM on day 3. On the other hand, the IC50 values of artemetin, quercetagetin and 5,3′-dihydroxy-6,7,4′-trimethoxyflavanone were

Discussion

In this study, we investigated the inhibitory effects of casticin on the proliferation or the cell cycle of malignant cells, and simultaneously examined the toxic effects of this flavonoid on normal cells. Casticin at low doses inhibited markedly the proliferation of KB cells, and arrested the cell cycle almost completely at G2-M. In contrast, casticin showed no influence on the growth of normal cells and A431 cells. It can be concluded that the growth inhibition by casticin was irrelevant to

Acknowledgements

We are grateful to Dr T. Horie (Emeritus Professor of The University of Tokushima) for a gift of synthesized casticin and a critical reading of the manuscript. We thank Dr K. Kanatsu and Dr C. Nagase (Hyogo Institute for Oriental Medicine) for their continuous encouragement.

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