Antitumor activity and mechanisms of action of total glycosides from aerial part of Cimicifuga dahurica targeted against hepatoma
© Tian et al; licensee BioMed Central Ltd. 2007
Received: 18 June 2007
Accepted: 31 December 2007
Published: 31 December 2007
Medicinal plant is a main source of cancer drug development. Some of the cycloartane triterpenoids isolated from the aerial part of Cimicifuga dahurica showed cytotoxicity in several cancer cell lines. It is of great interest to examine the antiproliferative activity and mechanisms of total triterpenoid glycosides of C. dahurica and therefore might eventually be useful in the prevention or treatment of Hepatoma.
The total glycosides from the aerial part (TGA) was extracted and its cytotoxicity was evaluated in HepG2 cells and primary cultured normal mouse hepatocytes by an MTT assay. Morphology observation, Annexin V-FITC/PI staining, cell cycle analysis and western blot were used to further elucidate the cytotoxic mechanism of TGA. Implanted mouse H22 hepatoma model was used to demonstrate the tumor growth inhibitory activity of TGA in vivo.
The IC50 values of TGA in HepG2 and primary cultured normal mouse hepatocytes were 21 and 105 μg/ml, respectively. TGA induced G0/G1 cell cycle arrest at lower concentration (25 μg/ml), and triggered G2/M arrest and apoptosis at higher concentrations (50 and 100 μg/ml respectively). An increase in the ratio of Bax/Bcl-2 was implicated in TGA-induced apoptosis. In addition, TGA inhibited the growth of the implanted mouse H22 tumor in a dose-dependent manner.
TGA may potentially find use as a new therapy for the treatment of hepatoma.
Hepatocellular carcinoma (HCC) is the fifth most common tumor worldwide, and the incidence of HCC has been rising over the past few decades in some areas such as Europe, USA and far eastern Asian countries . Despite advances in diagnosis and standard therapies such as surgery, radiation, and chemotherapy, HCC remains a formidable challenge for clinical therapy [2–5]. In the search for new cancer therapeutics with low toxicity, traditional Chinese medicines are promising candidates.
The dried rhizomes of Cimicifuga dahurica (Turcz) Maxim (Ranunculaceae) have been used as cooling, detoxification, antipyretic and analgesic agents for the treatment of some types of headaches and toothaches in Chinese folk medicine and were included in the Chinese Pharmacopoeia . The rhizomes are traditionally the portion of the plant used for medicinal purposes in Cimicifuga species, however the aerial part of the plant is usually discarded. Previous phytochemical studies demonstrated that both the rhizomes and the aerial part of the species are rich in cycloartane triterpenoids [7–10]. Some biological activities of total glycosides of rhizomes of C. dahurica (TGR) have been investigated by earlier studies of our group. It was reported that TGR could reduce the production of Simian Immunodeficiency Virus (SIV) by inhibition of PHA stimulated 3H-TdR transportation in lymph cells as well as suppression of the Sister Chromatid Exchange frequency induced by mitomycin C in human peripheral lymphocytes [11, 12]. Nevertheless, there are still few reports on the bioactivity of the aerial part of C. dahurica. Our recent study has demonstrated cytotoxicity of TGA and three cycloartanes 23, 24 and 25-O-acetylcimigenol-3-O-β-D-xylopyranoside isolated from the aerial part of C.dahurica against several cancerous cell lines. These three compounds showed similar effects and induced apoptosis and G2/M cell cycle arrest in hepatoma HepG2 and leukemia HL-60 cell lines. Down regulated expression of cdc2 and COX-2 contributed to the apoptosis and cell cycle arrest in HepG2 cells . However, the cytotoxic mechanism and in vivo anti-tumor activity of TGA is still unknown.
In the current study, we investigated the anti-tumor activity and the underlying mechanism of TGA both in vitro and in vivo. Our findings show the novel anticancer activity of TGA and this may provide a new approach to the hepatoma therapy.
Extraction of triterpene components from aerial part of C. dahurica
The aerial part of Cimicifuga dahurica (Turcz) Maxim (synonyms: Actinospora dahurica Turczaninow ex Fischer & C. A. Meyer, Index Sem. Hort. Petrop. 1: 21. 1835; Actaea dahurica (Turczaninow ex Fischer & C. A. Meyer) Turczaninow ex Fischer & C. A. Meyer) was collected in Maojingba, Kalaqin Qi, Inner Mongolia Autonomous Region, China, in September 1999, and was identified by Prof. Ruile Pan of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College. A voucher specimen has been deposited in the Herbarium of the Institute (XA99-09). The powdered aerial part of the plant (14.5 kg) was extracted exhaustively with 10 folds volume of 80% ethanol under refluxing for three times, one hour each time. Following combination and filtering, the solvent was evaporated under vacuum to obtain the crude extract (2.0 kg). Then the crude extract was mixed with siliceous earth and eluted with ethyl acetate. Removal of the solvent in vacuo, the TGA was (210 g) obtained.
Determination of total content of triterpenes
Triterpene constituents from C. dahurica Thurez Maxim
Cimilactone A [12β-acetoxy-3β-β-D-xylopyrano-syloxy-24, 25, 26, 27-tetranor-9,19-cyclolanost-16, 23 -lactone]
Cimilactone B [12β-acetoxy-3β-β-D-xylopyranosyloxy-24, 25, 26, 27-tetranor-9,19-cyclolanost-7-ene – 16, 23-lactone]
Cimidahuside C [12β-acetoxy- 15-oxo-shengmanol-3-O-β-D-xylopyranoside]
Cimidahuside D [12β-acetoxy- 15-oxo-7, 8-didehydroshengmanol- 3-O-β-D-xylopyranoside]
Cimidahuside E [(20R, 24R)-24, 25-epoxy-3β-(β-D-xylopyranosyloxy)-9,19-cyclolanost-7-ene-16, 23-dione]
Cimidahuside F [(20R, 24R)-24, 25-epoxy-15a-hydroxy-3β-(β-D-xylopyranosyloxy)-9,19-cyclolanost-7-ene-16,23-dione]
Cimidahuside G [(23R,24S)-15- oxo-16-enol-9,19-cyclolanostane-3-O-β-D-xylopyranoside]
Cimidahuside H [(23R,24S)-15- oxo-16-enol-9, 19-cyclolanostane -7-ene-3-O-β-D-xylopyranoside]
Cimidahuside I [(23R, 24S)- 12β-acetoxy-15-oxo-16-enol-9,19-cyclolanostane-3-O-β-D-xylopyranoside]
Cimidahuside J [(23R,24S)- 12β-acetoxy-15-oxo-16-enol-9, 19-cyclolanostane-7-ene-3-O-β-D-xylopyranosid e]
(20R, 24R)-11β,24,25-trihydroxy -3-β-(β-D-xylopyranosyloxy)- 9,19-cyclolanost-7-ene-16,23- dione
Cell culture and drug treatment
HepG2 (ATCC, Rockville, MD) cells were maintained in RPMI 1640 containing 10% FBS (Gibco, BRL, Carlsbad, CA), 2 mg/ml sodium bicarbonate, 100 μg/ml penicillin sodium salt and 100 μg/ml streptomycin sulfate. Cells were grown to 70% confluence, trypsinized with 0.25% trypsin-2 mM EDTA, and plated in 96 well plates. Mouse hepatocytes were isolated from normal CD-1 (ICR) mice (Beijing Vital Laboratory Animal Technology, Beijing, China) with enzymatic perfusion technique as we described previously . The viability of the mouse hepatocytes, tested with Trypan blue was about 80%. In all experiments, cells were grown in RPMI-1640 medium with 10% FBS for 24 h prior to treatment.
TGA was dissolved in DMSO at a concentration of 250 mg/ml, then diluted in tissue culture medium and filtered before use. The final concentration of DMSO (0.1%) did not affect the cell viability.
1.5 × 104 HepG2 cells and 8 × 103 mouse hepatocytes were seeded in 96 well plates and treated with TGA or vehicle (0.1% DMSO) at various concentrations and incubated for 48 h, followed by MTT (3- [4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay . Briefly, IC50 of the TGA in HepG2 cells and normal mouse hepatocytes were derived from the dose-response curves.
Morphology observation in HepG2 cells
AO/EB (acridine orange/ethidium bromide) fluoresce staining method was used to observe the apoptosis morphological changes . Briefly, HepG2 cells were cultured in 3.5 cm dishes and treated with TGA at concentration of 50 μg/ml for 0, 12, 24 and 48 h respectively. After treatment, all the cultures were incubated at 37°C, 5% CO2 for the indicated time. Photographs were taken under an inverted Leica fluorescence 40 × 10 microscope after staining.
Annexin V-FITC/PI assay
Apoptosis was quantified by detecting surface exposure of phosphatidylserine in apoptotic cells using Annexin V-FITC/PI (propidium iodide) apoptosis detection kit (BD Biosciences Clontech). Cells were seeded in 3.5 cm dishes in 1 ml medium and incubated with TGA at the dose of 25, 50 and 100 μg/ml for 24 h, respectively. The adherent and floating cells were combined and treated according to the manufacturer's instruction and measured with FITC/PI staining using flow cytometry (Becton Dickinson, San Jose, CA). Apoptotic cells (annexin V+PI-) were differentiated from necrotic cells (annexin V+PI+, including apoptotic cells at late stage).
Cell cycle analysis
HepG2 cells were treated with TGA at different concentrations (25, 50 and 100 μg/ml for 48 h) and time points (at 50 μg/ml for 12, 24 and 48 h). Then cells were collected and fixed in 70% cold ethanol (-20°C) overnight. After washing twice with PBS, cells were resuspended in PBS. RNase A (0.5 mg/ml) and PI (2.5 μg/ml) were added to the fixed cells for 30 min. The DNA content of cells was then analyzed with a FACSCalibur instrument (Becton Dickinson, San Jose, CA).
After treatments, cells were washed three times with ice-cold PBS and lysed with lysis buffer (50 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1% Triton X-100, 26% urea, and 1 tablet/10 ml protease inhibitor cocktail tablets). Sticky DNA was removed from lysates with a sterile toothpick. The protein concentration of the supernatant was determined by the Bradford method. The lysates were subjected to electrophoresis on a 10 % SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane . The nitrocellulose membrane was then incubated with mouse monoclonal anti-Bcl-2 and anti-Bax antibody (Santa Cruz Biotechnology, Santa Cruz, CA; sc-509 and sc-7480). Mouse monoclonal β-actin (Lab Vision, Fremont, CA) was used as an internal control. Secondary antibody to IgG conjugated to horseradish peroxidase was used. The blots were probed with the ECL Western blot detection system according to the manufacturer's instructions. The ratio of Bax/Bcl-2 was analyzed by pImage tool.
Antitumor evaluation on implanted mouse H22cells
Male CD-1 (ICR) mice (Beijing Vital Laboratory Animal Technology, Beijing, China), weighing 20–22 g, were used for implantation of hepatoma H22 cells (s.c.), which was maintained by weekly (i.p.) passage in CD-1 (ICR) mice. Ascites (0.2 ml of 1:6 dilution) from tumor-bearing mice 7 days after tumor inoculation were implanted (s.c.) into the armpit region of mice. Ten mice each were treated with either TGA (200,100 and 50 mg/kg b.w., i.g.) or vehicle, once a day for 10 days, 24 h after tumor inoculation. Cyclophosphamide (15 mg/kg b.w., i.p.) was used as a positive control. The tumor inhibition rate (TIR %) was calculated as we described previously . All the animal procedures were conducted in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
One-way ANOVA was used and followed by Dunnett's test. p < 0.05 was considered significant.
Induction of apoptosis in HepG2 cells by TGA
Effect of TGA on cell cycle distribution in HepG2 cells
Upregulation of Bax/Bcl-2 ratio
Tumor growth inhibition of implanted H22cells by TGA
Tumor growth inhibitory effect of TGA on H22 cells (mean ± SD, n = 10)
Tumor weight (g)
Growth inhibition %
3.28 ± 1.27
0.93 ± 0.45a
1.64 ± 0.76b
1.99 ± 0.82c
2.06 ± 1.30c
A major complication of chemotherapy is toxicity to normal cells, which is due to the inability of drugs to differentiate between normal and malignant cells. This often impacts the efficacy of the treatment and even makes it impossible to cure the patients. One of the requisite of cancer chemopreventive agent is elimination of damaged or malignant cell through cell cycle inhibition or induction of apoptosis without or with less toxicity in normal cells [20, 21].
First we investigated the cytotoxicity of TGA in HepG2 cells and primary cultured normal mouse hepatocytes. The primary cultured mouse hepatocytes were chosen as normal cells to seek selective hepatoma cytotoxic agents, because these primary cultured cells closely resemble normal cells in vivo. Our results indicate that TGA has relatively selective cytotoxicity to hepatoma cells based on the higher IC50 value in the primary cultured normal hepatocytes than that of carcinoma HepG2 cells. The relative selective cytotoxicity of TGA in HepG2 cells may be due to some of the relative selective cytotoxic components 23-, 24- and 25-O-acetylcimigenol-3-O-β-D-xylopyranoside, 25-anhydrolcimigenol-3-O-β-D-xylopyranoside and hepatoprotective constituent cimigenol xylopyranoside in it [13, 22, 23].
Cell proliferation is governed by the cell cycle, which is the target of many anti-cancer agents. Previous studies have demonstrated that extracts and some constituents of rhizomes of C. racemosa, the same genus as C. dahurica, possess cytotoxic activity against estrogen receptor positive (MCF-7) and estrogen receptor negative (MDA-MB231 and MDA-MB-453) human breast carcinoma cell lines by induction of cell cycle arrest and apoptosis; furthermore, glycosidic fraction could induce G0/G1cell cycle arrest when tested at 30 μg/ml and G2/M arrest when tested at 60 μg/ml in MCF7 cells [15, 24]. In addition, it was found that actein and a fraction of black cohosh potentiated antiproliferative effects of chemotherapy agents on human breast cancer cells in more recent research . In light of our study, TGA could induce G0/G1 cell cycle arrest at lower concentration (25 μg/ml) and G2/M arrest at higher concentration (50 and 100 μg/ml). This suggests that TGA contains more than one component with the more active or abundant component inducing G0/G1 arrest and the less active component inducing G2/M arrest. Active components either for G0/G1 or G2/M cell cycle arrests have been detected in TGA by our previous studies. 23, 24 and 25-O-acetylcimigenol-3-O-β-D-xylopyranoside, isolated from TGA could induce G2/M arrest ; while 25-anhydrolcimigenol-3-O-β-D-xylopyranoside, which exists in TGA, could induce G0/G1 arrest . There might be some other potent G0/G1 active components undiscovered.
Apoptosis is a tightly regulated process, which involves changes in the expression of a distinct set of genes [27, 28]. Two of the major genes responsible for regulating mitochondrial apoptosis pathway are antiapoptotic Bcl-2 and proapoptotic bax [29–31]. In particular, Bax can homodimerize with itself and heterodimerize with Bcl-2 or Bcl-xL. It appears that Bax homodimers activates apoptosis while heterodimers inhibits the process . Moreover, an elevated intracellular ratio of Bax to Bcl-2 occurs during increased apoptotic cell death . In our study, pronounced apoptotic cells were found in HepG2 cells treated with TGA by fluorescence staining and flow cytometric analysis. Moreover, further study showed that enhanced ratio of Bax/Bcl2 at all time points contributed to TGA induced apoptosis. The attenuation of ratio of Bax/Bcl-2 at 48 h time point than that of 24 h might be the way of self-protection for cell survival. More apoptosis at 48 h might in turn, attenuate the increased ratio of Bax/Bcl-2 by negative feedback.
In conclusion, for the first time, the potential anticancer activity and the underlying mechanisms of TGA against hepatoma were investigated in this study. TGA exhibited relative cytotoxicity to HepG2 cells in vitro and inhibited growth of H22 tumor in vivo. The results of this study suggest that TGA might be a promising anti-hepatoma agent. Apoptosis and cell cycle arrest could be attributed, in part to its proliferating inhibition, and alteration of ratio of Bax/Bcl-2 might be one of possible mechanisms of TGA inducing apoptosis.
This work is supported by the National Natural Science Foundation of China (30470195) and National Institutes of Health training grant 2 Ta5 LM 07092-11. We also thank Prof. Isaac S. Kohane (Children's Hospital Boston, Harvard Medical School) for his support.
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