Antitumor Activity of Spinasterol Isolated from Pueraria Roots
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EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 37, No. 2, 111-120, April 2005 Antitumor activity of spinasterol isolated from Pueraria roots Gook-Che Jeon1, Myoung-Soon Park2, of some breast cancer cell lines (MCF-7, MDA- Do-Young Yoon3, Chul-Ho Shin4, MB-231) in a dose- and time-dependent manner, Hong-Sig Sin2 and Soo-Jong Um1,5 as well as the growth of ovarian (2774) and cervical cancer cells (HeLa). Finally, a transfec- 1 tion assay showed that this component had an Department of Bioscience and Biotechnology/Institute of Bioscience estrogenic effect similar to 17β - estradiol, which Sejong University activates both estrogen receptor α (ERα) and Seoul 143-747, Korea ERβ. The NMR analysis determined that spin- 2Chebigen Inc., 305-B, Chungmugwan asterol (stigmasta-7, 22-dien-3beta-ol) is an active Sejong University cytotoxic component of Pueraria root. Seoul 143-747, Korea 3Laboratory of Cell Biology Keywords: breast neoplasms; estrogen replacement; Korea Research Institute of Bioscience and Biotechnology phytoestrogens; pueraria; receptors, estrogen; spina- Daejeon 305-333, Korea sterol; therapy 4Seohae Environment Science Institute Chonbuk National University Total Research Complex Jeonbuk 865-854, Korea Introduction 5Corresponding author: Tel, 82-2-3408-3641; Fax, 82-2-3408-3334; E-mail, [email protected] Estrogen exerts a wide variety of effects on growth, development, differentiation, and reproduction (for re- Accepted 23 March 2005 view, see Nilsson et al., 2001). Estrogen mediates these activities via binding to a specific nuclear receptor protein, the estrogen receptor (ER), which is Abbreviations: CAT, chloramphenicol acetyl transferase; ER, encoded by two genes (ERα and ERβ) that function estrogen receptor; ERT, estrogen replacement therapy; SERM, as transcription factors to regulate the expression of selective estrogen receptor modulator target genes (Osborne et al., 2001). On ligand bind- ing, ER undergoes conformational changes and dis- sociates from the inactive ER-hsp90 complex. The activated ER enters the nucleus as a homodimer or Abstract heterodimer, then binds to a specific DNA sequence, We purified phytoestrogens from Pueraria root the estrogen response element (ERE), and stimulates estrogen-target gene expression. The two ERs appear (Pueraria mirifica from Thailand and Pueraria to have unique tissue distributions and their own sets lobata from Korea), which is used as a rejuvenat- of specific functions. Knowledge of these functions ing folk medicine in Thailand and China. Dried, might aid in the development of receptor-specific sel- powdered plant material was extracted with 100% ective estrogen receptor modulators (SERMs) (Kuiper ethanol and further separated by concentration, and Gustafsson, 1997; Barkhem et al., 1998; Nilsson filtration, and thin layer silica gel chromatogra- et al., 1998). Estrogen replacement therapy (ERT) is used to phy. Using the fractions obtained during separa- treat symptoms of menopause, such as hot flashes tion, we first investigated their cytotoxicity in se- and osteoporosis, and to reduce the incidence of veral cancer cell lines from various tissues. The cardiovascular disease associated with menopause ethanol-extracted components (PE1, PE4) had sig- (This et al., 2001). However, current ERT appears to nificant antiproliferative effects on breast cancer be associated with increased risks of developing cell lines, including MCF-7, ZR-75-1, MDA-MB-231, breast and ovarian cancer in healthy women (Schairer SK-BR-3, and Hs578T. Second, we compared et al., 2000; Lacey et al., 2002). To overcome the shortcomings of ERT, phytoestrogens derived from these results with the cytotoxic effects of known plants have emerged as an alternative to ERT to flavonoids, sterols, and coumarins from Pueraria alleviate the symptoms of menopause, although their root. The known compounds were not as effec- safety needs to be evaluated further (Tham et al., tive, and occurred in a different polarity region 1997). on HPLC. Third, further separation resulted in the Pueraria root prepared from Pueraria mirifica or isolation of eight different components (Sub PE-A Pueraria lobata is one of the most important crude to -H). One of these, PE-D, affected the growth drugs in traditional oriental medicine. For example, the Thai vine, Pueraria mirifica, is used as a reju- 112 Exp. Mol. Med. Vol. 37(2), 111-120, 2005 venator and aphrodisiac (Bradbury and White, 1954; are given in Hz. Mass spectra were measured on a Cain, 1960), and other Pueraria species are reported Shimazu GCMS-PO 1,000 mass spectrometer (EI 70 to have pharmacological activity (Harada and Ueno, eV). The dried material derived from each fraction 1975; Qicheng, 1980; Lai and Tang, 1989; Keung and was dissolved in DMSO (Sigma, St. Louis, MO), Vallee, 1998; Miyazawa et al., 2001). Pueraria root 100% ethanol, or CH2Cl2, depending on the experi- is a potent source of phytoestrogen with estrogen-like mental conditions, and stored at -20oC. To treat cells, biological activity. Three main classes of phytoestro- the dissolved material was diluted with phenol red- gens occur in either plants or their seeds: isoflavones, free DMEM (Gibco BRL, Gaithersburg, MD) to give coumestans, and lignans. A single plant often con- the final concentrations indicated in the text. tains more than one class of phytoestrogen. It has been suggested that the major estrogenic compo- nents of Pueraria root are puerarin, daidzin, genistin, Cell lines and cell culture daidzein, and genistein (Ohshima et al., 1988). No The cells used in our experiments were breast (MDA- powerful phytoestrogens other than these isoflavones MB-231, MCF-7, ZR-75-1, Hs578T, SK-BR-3), cervical and coumestans have been isolated. In addition, their (HeLa, CaSki), ovarian (2774, OVCAR-3, PA-1, SK- activities in the treatment of gynecological cancers are OV-3), colon (HT-29, SW480, HCT116, DLD-1), and poorly defined. liver (PLC/PRF-5, SK-Hep-1) cancer cell lines. These In this study, we extracted phytoestrogens from two cells were routinely maintained in DMEM, supple- kinds of Pueraria root: Pueraria mirifica from Thailand mented with 10% FBS) (HyClone Laboratories, Logan, and Pueraria lobata from Korea. We used breast, UT), previously inactivated at 56oC for 20 min. For ovarian, and cervical cancer cell lines to analyze their transfection and transcription assays, COS-1 cells chemopreventive activity in gynecological cancers in were used and cultured in phenol red-free DMEM, vitro, and determined their effect on ERα and ERβ. supplemented with heat-inactivated, charcoal-treated We found that the profiles of the phytoestrogens 10% FBS. extracted from the two Pueraria roots were similar, and demonstrated that Pueraria extract has antipro- liferative effects on certain gynecological cancer cell Cell viability lines, and acts on both ERs. The active cytotoxic Cell monolayers were cultured in a humidified atmos- component in the final fraction appears to be spin- phere of 95% air with 5% CO2. Cells were seeded asterol, an isomer of stigmasterol, which is one of the at a density of 7 × 104 cells/well 24 h before drug known phytoestrogens in Pueraria extract. Although a treatment in six-well plates, and were 60 to 70% con- more detailed approach is required to determine how fluent at the time of treatment. Different concentra- spinasterol regulates the growth of cancer cells, our tions of the Pueraria extracts or commercially avail- results may aid the development of natural SERMs able phytoestrogens (puerarin, stigmasterol, campe- for menopausal women who need ERT, without in- sterol, coumestrol, genistein, daidzein, daidzin, ge- creasing their risk of developing gynecological can- nistin, β -sitosterol) (Sigma) were added to the med- cers. ium. As a control, a stock solution of 100 mM 17β- estradiol (Sigma) dissolved in DMSO, 100% ethanol, or CH2Cl2 was used. After treatment, living cells were Materials and Methods recovered daily by suspension in trypsin-EDTA and counted using a hemocytometer followed by trypan Preparation of Pueraria extracts blue staining (0.4%). Each assay was performed four times in triplicate. All the data presented in the text Pueraria roots were extracted by inverted shaking in represent the means of three independent assays. 100% ethanol for 3 days at room temperature. A clear extract was recovered by centrifugation at 5,000 rpm for 20 min and concentrated in a rotary evaporator Transcription assay for ERα and ERβ o at 45 C (PE1; Pueraria extract 1). After PE1 was Cells were transfected using a liposome-based meth- filtered, the filtrate was concentrated and filtered again od with Lipofectamine (Gibco-BRL), as previously to produce PE4. To separate it further, PE4 was sub- reported (Um et al., 2000; Kang et al., 2004). Briefly, jected to preparative thin-layer chromatography (TLC) COS-1 cells, maintained in phenol red-free DMEM on Silica Gel 60 F254 (Merck, Darmstadt, Germany) supplemented with charcoal-treated 10% FBS, were with hexane:ethyl acetate (4:1). Eight sub-fractions plated in 60-mm dishes at a density of 1 × 106 were recovered, of which Sub PE-D was further se- cells/ dish 5 h before transfection. After overnight parated by preparative TLC (hexane:ethyl acetate = transfection with the indicated reporter plasmids (ERα 5:1) to produce Sub PE-D1, -D2 and -D3. To identify or ERβ expression vector and Vit-tk-CAT reporter the structures in the final three fractions, the fractions plasmid derived from vitellogenin promoter containing were subjected to NMR (proton and carbon) and a canonical ER response element; generously do- 1 13 LC-MASS. The H NMR and C NMR spectra were nated by Dr. Pierre Chambon, Strasbourg, France) recorded on a JEOL JNM EX-400 using CDCl3 as the together with SV40-driven internal control plasmid, the solvent. All the chemical shifts (δ) are quoted in ppm cells were washed, fed with complete medium if downfield from TMS and the coupling constants (J) needed, treated with 17β-estradiol or Pueraria extract, Antitumor activity of spinaterol 113 and further incubated for 24 h.