Cytotoxic triterpenes from Antrodia camphorata and their mode of action in HT-29 human colon cancer cells
Introduction
Cancer is a major cause of death worldwide and causes serious problems in human life, including mental and physical agony and economic strain. Therefore, many kinds of cancer therapies, including various anticancer agents, have been developed. However, they also have several problems such as serious side effects and drug resistance [1]. To resolve these difficulties, development of cancer chemopreventive agents and improvement of cancer treatment are very important. Accordingly, screening of natural products as potential anticancer agents, in the form of functional foods or nutraceuticals has become an important undertaking [2]. The rising interest in the pharmacological properties of natural triterpenoids [3] led us investigate in vitro cytotoxicities of five lanostane (2, 3, 4, 6 and 8) and three ergostane-type (1, 5 and 7) triterpenes isolated from Antrodia camphorata (Polyporaceae).
A. camphorata is a parasitic fungus grown on the hardwood of Cinnamomum kanehirai Hay (Lauraceae), has been widely used as a Chinese remedy for food and drug intoxication, diarrhea, abdominal pain, hypertension and cancer [4]. Crude extracts from the fruiting bodies and mycelium of A. camphorata show potent anticancer activities in human leukemia HL-60, breast cancer MDA-MB-231 (estrogen-nonresponsive) and MCF-7 (estrogen-responsive) cells, but not in healthy breast cells (HBL100) and umbilical vein endothelial cells [5], [6], [7]. The extracts from fruiting bodies show cytotoxic effect in bladder cancer cells and arrest the cell cycle in the G2/M phase [8]. The ethanol extract from mycelium inhibits the proliferation of human lung cancer cell A-549, but not the normal fetal lung fibroblast MRC-5 cell [9]. The methanol extract of mycelium inhibits cell viability and induces apoptosis in Hep G2 via G0/G1 cell cycle arrest followed by the activation of the caspase-3 and -8 cascades [10]. The ethyl acetate extract (EAC) inhibits cell growth in two liver cancer cells, Hep G2 and PLC/PRF/5 through the regulation of Bcl-2 family protein expression [11]. A. camphorata treatment could be effective in inhibiting breast cancer cell MDA-MB-231 proliferation and inducing apoptosis in vitro and in vivo[12]. Interestingly, the authors also show the nontoxicity of A. camphorata with a daily oral administration of 500 mg/kg for 28 days in rats, which increase its potential for application in food and drug products [13]. Hence, it is of significant interest to isolate and identify such exact compounds which are responsible for the anticancer activity of this fungus.
As part of a study program to evaluate the therapeutic properties of A. camphorata, this paper has demonstrated the in vitro cytotoxic effects of eight triterpenoids (Fig. 1) against various cancer types: colon, liver, breast and lung cancer cell lines. Although the in vitro cytotoxic activity of zhankuic acids A (5) and C (7) in P-388 murine leukemia cells had been reported [14], however no information about their mechanism of action. In this study, among eight selected compounds, three ergostane-types were capable of blocking cell cycle progression at the sub-G1 phase and inducing apoptosis through the cleavage of the downstream poly(ADP-ribose) polymerase, pro-caspase-3 and Bcl-2. In addition, the combination of ergostane-triterpenes exhibited a potential synergistic cytotoxic effect in HT-29 cells.
Section snippets
Materials
All the materials were obtained commercially and used without further purification. NMR spectra were measured on a Varian Unity Inova-600 VXR-300/51 spectrometer with TMS as an internal standard. Silica gel for column chromatography (CC) (0.063–0.200 mm), was a product of Merck Company. TLC was performed on Merck TLC plates (0.23 mm thickness), with compounds visualized by spraying with 8% (v/v) H2SO4 in ethanol and then heating on a hot plate. The wild fruiting bodies of A. camphorata were
Cell proliferation
To investigate the cytotoxic potential of the eight isolates (Fig. 1), ten cancer and two normal cell lines were incubated with each isolate with increasing concentrations for 48 h. The effects on cell proliferation were determined by the sulforhodamine B assay [18]. The three ergostane-type triterpenes methyl antcinate B (1), zhankuic acids A (5) and C (7) showed a wide range of potency with IC50 values ranging from 22.3 to 75.0 μM in various cancer cell types (Table 1). Three isolates exhibited
Conflicts of interest
None declared.
Acknowledgements
This study was supported by grants from the National Science Council (Grants NSC 96 – 2113-M-324-002-MY3; NSC 97 – 2811-M-324-001) and the National Health Research Institutes, Taiwan. The authors thank Mr. Chao-Yang Hsiao (Cell Sorter Core Facility of National Health Research Institutes) for his assistance with the flow cytometry assay.
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Contributed equally to this work.