7,7
′′-Dimethoxyagastisflavone-induced
Apoptotic or Autophagic Cell Death in Different
Cancer Cells
Chia-Hsiang Hwang,1,2†Yu-Ling Lin,3†Yen-Ku Liu,3Chia-Hung Chen,3Hsin-Yi Wu,4 Cheng-Chang Chang,2Chao-Yuan Chang,2Yu-Kuo Chang,2Yi-Han Chiu,5
Kuang-Wen Liao3,4* and Yiu-Kay Lai1*
1Department of Life Science, Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan 2Yung-Shin Pharmaceutical Industry Co., Ltd, Taichung, Taiwan
3Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu, Taiwan 4Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan 5Department of Life Science, Tzu Chi University, Hualien, Taiwan
7,7′′-Dimethoxyagastisflavone (DMGF), a biflavonoid isolated from the needles of Taxus media cv. Hicksii, was evaluated for its antiproliferative and antineoplastic effects in three human cancer cell lines. Interestingly, DMGF caused cell death via different pathways in different cancer cells. DMGF induced apoptosis, activated caspase-3 activity and changed the mitochondrial membrane potential in HT-29 human colon cancer cells. However, the apoptotic pathway is not the major pathway involved in DMGF-induced cell death in A549 human lung cancer cells and HepG2 human hepatoma cells. Treatment with 3-MA, an inhibitor of autophagy, signifi-cantly decreased DMGF-induced cell death in HepG2 and A549 cells, but did not affect DMGF-induced cell death in HT-29 cells. Following DMGF treatment, the HepG2 cells increased expression of LC3B-II, a marker used to monitor autophagy in cells. Thus, DMGF induced apoptotic cell death in HT-29 cells, triggered both apoptotic and autophagic death in A549 cells and induced autophagic cell death in HepG2 cells. Copyright © 2011 John Wiley & Sons, Ltd.
Keywords: 7,7′′-dimethoxyagastisflavone; biflavonoid; Taxus media cv. Hicksii; apoptosis; autophagy.
INTRODUCTION
Paclitaxel (brand name Taxol), belongs to a class of diterpenoids, known as taxanes, that stabilize microtu-bules against depolymerization and induce apoptosis (Miller et al., 1999; Wang et al., 2000). It is produced and isolated from plants of the genus Taxus (yew). In addition to paclitaxel, there are an increasing number of semi-synthetic taxane analogs made from 10-deacetyl baccatin III that are also isolated from Taxus. The com-panies that make taxane analogs such as docetaxel (brand name Taxotere), larotaxel (XRP9881) and cabazi-taxel (XRP6258) are in the process of obtaining regula-tory approval or entering late-stage clinical investigation for these drugs. Thus, Taxus can be considered a good resource for the development of new antitumor drugs.
It has been reported that several biflavonoids from plants have biological activities such as cytotoxicity, antitumor activity and anti-angiogenesis (Pang et al., 2009; Guruvayoorappan and Kuttan, 2008). 7,7 ′′-Dimethoxyagastisflavone (DMGF), one of the compounds isolated from Taxus media var. Hicksii, is a biflavonoid
that wasfirst isolated and identified from the leaves of Araucaria bidwillii Hooker and Agathis alba Foxworthy (Khan et al., 1972). It also exists in New Zealand kauri and other species of Agathis (Ofman et al., 1995). How-ever, only a few studies have described the activity of DMGF. One study indicated that DMGF could inhibit the production of aflatoxin by Aspergillus flavus (Gonca-lez et al., 2001). Therefore, the anti-tumor activity of DMGF is unclear.
Though previous researchers have successfully synthesized paclitaxel (Nicolaou et al., 1994), the method employed to obtain this compound is not cost-effective. Paclitaxel is currently produced by direct ex-traction from the biomass of the yew tree or semisynth-esis from its precursor 10-deacetyl baccatin III (Baloglu and Kingston, 1999). Traditionally, paclitaxel and its precursors have been extracted from the bark of yew trees, a process that leads to plant death. However, paclitaxel-related compounds may have also been sus-tainably extracted from the needles and old roots of yew trees, showing that these are also good biomass resources. In addition to paclitaxel or paclitaxel-related compounds, compounds such as flavones can also be isolated from the needles of Taxus media cv. Hicksii. Therefore, the needles of yew trees may not only sus-tainably provide paclitaxel-related compounds without damaging the environment, but are also a good resource for drug discovery.
This study investigated the cytotoxicity of 7,7 ′′-dimethoxyagastisflavone (DMGF) isolated from the needles and old roots of Taxus x media cv. Hicksii on
* Correspondence to: Professor Kuang-Wen Liao, Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30050, Taiwan; Prof. Yiu-Kay Lai, Department of Life Science, Institute of Biotechnology, National Tsing Hua University, Hsinchu 30013, Taiwan. E-mail: [email protected]; [email protected]
†Contributed equally to this study.
Phytother. Res. 26: 528–534 (2012)
Published online 14 September 2011 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/ptr.3583
Received 13 January 2011 Revised 13 May 2011
human cancer cell lines. Interestingly, it was found that this biflavonoid DMGF triggered different death path-ways in different tumor cell types. DMGF could induce apoptotic death in HT-29 colon cancer cells and trigger autophagic death in A549 lung cancer and HepG2 hepa-toma cells. Therefore, DMGF may be a good candidate as an anti-cancer drug.
MATERIALS AND METHODS
Plant material. Needles of Taxus media cv. Hicksii were collected from Taichung, Taiwan in 2008. The plant was authenticated by the Department of Life Sci-ence, National Taiwan University. A voucher specimen is kept at the Herbarium of the same department. Extraction and isolation of DMGF and other compounds. The dried needles of Taxus media var. Hicksii (10 kg) were extracted with methanol (MeOH) at a preferred ratio of 1:1 (w/v) at room temperature to yield a methanol extract. The biomass was extracted in this manner at least two times. The pooled methanol extracts were concentrated in vacuo to give a crude extract (780 g) which was then partitioned with water/ ethyl acetate (EtOAc) (1:1) at least three times. The combined EtOAc layer resulted in 140 g of black syrup, which was mixed with 350 g silica gel 60 N (Timely, Japan) and packed in an open column, and eluted with hexane and an increasing concentration of EtOAc in hexane to afford five fractions (4:1, 3:1, 7:3, 13:7, 3:2, A–E). Fraction E was further purified by recrystallization from hexane/acetone (3:1) then re-chromatographed with the same elution condition to give 4.2 g (98.5%) of DMGF sample. DMGF and seven taxane derivatives were isolated and the yields of different compounds are shown in Table 1.
HPLC analysis. DMGF was analysed by liquid chroma-tography on a Waters HPLC system (Waters 2796 Bio-separation Module) composed of an auto sampler and a detector (Waters 2996 photodiode array detector) equipped with C18™ Symmetry column (250 mm 4.6 mm i.d.) with a particle size of 5mm. Isocratic elution was monitored at 254 nm under room temperature and performed for methanol and water (30:70, v/v at aflow rate of 0.5 mL/min).
Identification of the compounds. The chemical structure of DMGF and other compounds was confirmed by1H and13C NMR spectra.1H (400 MHz) and13C (400 MHz) NMR spectra were recorded on a Bruker AV-400 spectrom-eter using deuterated chloroform as the solvent and tet-ramethylsilane (TMS) as an internal standard.
Cells and cell cultures. HT-29 human colon cancer, A549 human lung cancer and HepG2 human hepatoma cell lines were purchased from BCRC (Hsinchu, Taiwan, ROC) and maintained in DMEM medium (Gibco/Invi-trogen, Carlsbad, CA, USA) supplemented with heat-inactivated 10% fetal bovine serum (Gibco/Invitrogen, Carlsbad, CA, USA) and 1% penicillin/streptomycin/ amphotericin in 5% CO2at 37C.
Assay for cell growth inhibition. The cells (1 104cells/ well) were seeded overnight in a 96-well plate. After treatment with serial concentrations of the various isolated compounds in dimethyl sulfoxide (DMSO, final concentration of DMSO was 0.1%), the cells were incubated at 37C for 48 h. Subsequently, the cell viability was measured by MTT assay. The cell viability ratio (%) was calculated using the following equation:
% viability ¼absorbance of test sample
absorbance of control 100%
The IC50was then calculated for each treatment. The
results were expressed in duplicate and three independ-ent experimindepend-ents. Morphological changes in the treated cells were monitored by light microscopy.
Measurement of mitochondrial membrane potential. The mitochondrial membrane potential was determined using a Cell Meter™ JC-10 mitochondria membrane potential assay kit (ABD Bioquest, CA, USA). Briefly, the cells were harvested after treatment with DMGF (2.5mg/mL) for 48 h, incubated with 50 mL of JC-10 dye-loading solution (25 mL of JC-10 in 5 mL assay buffer) at 37C for 30 min and analysed byflow cytome-try (Becton Dickinson). For each sample, 10000 events were recorded and used to plot the red/green fluores-cence ratio. Data were calculated as: mitochondrial membrane potential index = the mean of the green fluorescence (FL1)/the mean of the red fluorescence (FL2).
Table 1. The cytotoxicity of isolated compounds fromTaxus media var. Hicksii in different cancer cell lines
Compound Yield (%) Purity (%) IC50 HT29 A549 HepG2 Baccatin III 0.02% 98.0 13.0 1.3mg/mL 21.6 1.1mg/mL > 40mg/mL Taxachitriene A 0.01% 98.5 > 40mg/mL > 40mg/mL > 40mg/mL
5a-Hydroxy-2a,7b,9a, 10b,13a-tetraacetoxy4 (20),11-taxadine 0.01% 98.5 > 40mg/mL15.5 2.1mg/mL > 40mg/mL
2a,10b,13a,20-tetraacetoxy-5a-hydroxy-3,8-secotaxa-3,7,11-trien-9-one0.01% 98.5 > 40mg/mL > 40mg/mL > 40mg/mL
7,7"-Dimethoxyagastis-flavone 0.04% 98.5 2.3 0.6mg/mL 2.8 0.1mg/mL3.9 1.7mg/mL
10-Deacetylpaclitaxel (10-DAT) 0.02% 98.5 0.1 0.1mg/mL 0.2 0.1mg/mL > 40mg/mL
10-Deacetyl-cephalomannine 0.01% 98.5 0.8 0.1mg/mL > 40mg/mL2.5 0.3mg/mL
Cell cycle analysis. The cells were treated with 2.5mg/mL of DMGF for 48 h. After treatment, 106cells were har-vested by trypsinization followed by centrifugation. The cell pellets were washed with PBS and fixed in 3 mL of 70% ethanol at 4C for 30 min. After centrifu-gation at 400 g, the fixed cells were resuspended with 1 mL staining buffer (5% Triton-X 100, 0.1 mg/mL RNase A and 4mg/mL propidium iodide) for 30 min at room temperature. In total 10000 cells were analysed for DNA content by flow cytometry. The distribution of cells in the G0/G1, S and G2/M phases of the cell cycle were determined using Modfit software (Becton Dickinson).
Assay of caspase-3 activity. The cells were seeded into 24-well cultured plates at 1 105 cells/well overnight. The cells were then treated with 2.5mg/mL of DMGF for different durations. Caspase-3 activity was deter-mined with the PE active caspase-3 apoptosis kit (BD Pharmingen, San Jose, CA, USA), following the manu-facturer’s instructions.
Western blotting for autophagy analysis. The cells were treated with or without DMGF at IC50for 12 h. For
im-munoblotting, equivalent amounts of cell lysate were resolved by SDS-PAGE (10%) and transferred onto PVDF membranes. After blocking, the membranes were incubated with the anti-LC3BII antibody (GeneTex Inc., Irvine, CA, USA). The membranes were then trea-ted with goat anti-rabbit peroxidase-conjugatrea-ted antibody, and the immunoreactive proteins were detected using an enhanced chemiluminescence kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions.
RESULTS
Isolation and identification of compounds from Taxus media cv. Hicksii
Compounds were extracted and isolated from the nee-dles and old roots of Taxus media cv. Hicksii. The yields of different compounds were determined, and the compounds with the highest yields were further identified. The names of the eight identified compounds and their yields and purity are shown in Table 1.
Growth inhibition activity of isolated compounds in tumor cell lines
To assess the cytotoxicity of the eight compounds, the IC50 values of each compound in HT-29, A549 and
HepG2 cells were determined. The antineoplastic agent paclitaxel was used as a reference compound. Table 1 summarizes the antiproliferative effects of the eight isolated compounds on these cells. The IC50values show
that paclitaxel, DMGF and 10-DAT had higher antipro-liferative effects, while other compounds showed weak-to-moderate antiproliferative effects.
Even though 10-DAT showed high antiproliferative activity, it is a paclitaxel derivative that is already known to have antitumor activity. Thus, DMGF was selected
for further study of its antitumor effects. It was found that DMGF had different antiproliferative effects on different cancer cell lines and primary cells, including human PBMCs (IC50= 11.3 mg/mL) and mouse
spleno-cytes (IC50= 9.9mg/mL). In addition, DMGF treatment
led to clearly different morphological changes in differ-ent cell lines. DMGF significantly induced cell mem-brane shrink and formed apoptotic bodies in HT-29, whereas the presence of apoptosis in DMGF-treated A549 cells was not as obvious (Fig. 1A). Moreover, treatment with DMGF resulted in the vacuolation of cytoplasm in HepG2 and A549 cells, but not in HT-29 cells (Fig. 1A).
Effects of DMGF on the cell cycle in HepG2, A549 and HT-29 cell lines
As shown in Fig. 1B, DMGF caused an increase in the number of cells in the sub-G1 phase in all cell types, which indicates that cell death is one of the reasons for the decreased proliferation. The proportion of cells in each phase was calculated; DMGF had different effects on the cell cycle in different tumor cells. DMGF decreased the proportion of HT-29 cells in the G0/G1 phase and increased the proportion in the G2/M phase. However, DMGF only had minor effects on the cell cycle in A549 and HepG2 cells (Fig. 1B).
Determination of the effect of DMGF on apoptosis in HepG2, A549 and HT-29 cell lines
To further determine whether the antiproliferative effects of DMGF resulted from induction of apoptosis, activation of caspase-3, an indicator for the late phase of apoptosis, was measured. DMGF treatment increased caspase-3 activity in HT-29 cells in a time-dependent manner. In contrast, DMGF caused the caspase-3 activity to stay constant over time in A549 cells and had no effect on HepG2 cells (Fig. 2A). Mitochondrial membrane potential (4ΨM),
the loss of which is indicative of the induction of the endogenous apoptosis pathway, was measured in all three cell lines following DMGF treatments. Fig. 2B illustrates the different effects of DMGF on different cells; HT-29 cells were the most susceptible, A549 cells were slightly affected and HepG2 cells did not lose 4ΨMfollowing DMGF treatment.
The effect of DMGF on the induction of autophagy The results show that intracellular vesicles were increased in DMGF-treated HepG2 and A549 cells, indicating that the autophagy pathway may be involved in DMGF-induced cell death in these two cell lines. Thus, 3-methy-ladenine (3-MA), an inhibitor of autophagy, was used to examine this hypothesis. Treatment with 3-MA did not affect DMGF-induced cell death in HT-29 cells, but significantly decreased DMGF-induced cell death in HepG2 and A549 cells (Fig. 3A). DMGF was also found to have a greater influence on autop-hagic death in HepG2 cells compared with A549
cells. Furthermore, the three cell lines were monitored for changes in levels of autophagy regulators after DMGF treatment. The results show that DMGF
increased the levels of LC3-II in HepG2 cells, maintained levels of II in A549 cells and lowered levels of LC3-II in HT-29 cells (Fig. 3B).
Figure 1. Effects of DMGF on (a) morphological changes (b) cell cycle in HT-29, A549 and HepG2 cells. Cell morphology photographed under a microscope (200 magnification). The cell cycle distributions and the percentage of cells in each phase were obtained with Modfit software. This figure is available in colour online at wileyonlinelibrary.com/journal/ptr
DISCUSSION
This study examined the effects of compounds isolated from Taxus media var. Hicksii on the cytotoxicity of
tumor cells. The genus Taxus has been studied exten-sively for its taxoid compounds, particularly paclitaxel and its precursor 10-deacetyl baccatin-III. Many taxoids with antineoplastic, cytotoxic or antipromastigote activities have been isolated and investigated from the different organs of the Taxus genus (Chattopadhyay et al., 2006; Dai et al., 2006; Georgopoulou et al., 2007; Jiang et al., 2008).
One of the compounds isolated from Taxus media var. Hicksii, DMGF, is a biflavonoid that has yet to be characterized in the literature. Only a few studies have described the activity of DMGF. One study indicated that DMGF could inhibit the production of aflatoxin by Aspergillusflavus (Goncalez et al., 2001). Our results show that DMGF not only has therapeutic potential against the growth of cancer cell lines but also induced cell death differently in different tumor cell lines. HT-29 cells were the most susceptible to DMGF-induced apoptosis. Accordingly, HT-29 cells also showed significant changes in 4ΨM, an important
marker of endogenous apoptosis (Krysko et al., 2008) (Fig. 2B). In addition, early- and late-phase apoptosis, phosphatidyl-serine externalization and an increase in caspase-3 activity were present in HT-29 cells follow-ing treatment with DMGF (Fig. 2A). It was also shown that DMGF treatment resulted in the forma-tion of apoptotic bodies, cell shrinkage and bleb for-mation (Rello et al., 2005; Cotter, 2009), which are all classic morphological changes that occur in HT-29 cells during apoptosis.
In contrast, the effects of DMGF on the induction of apoptosis, including phosphatidyl-serine externalization, increased caspase-3 activity and changes in4ΨM, were
minor or did not influence cell death in A549 or HepG2 cells (Fig. 2B). However, it was found that DMGF inter-fered with the growth rate of these cells compared with untreated cells and that the IC50values were in the range
2–5 mg/mL (Table 1). It was also observed that the
Figure 2. The apoptotic activity of DMGF in different cancer cells. (a) Caspase-3 activity was determined after treatment with DMGF for different potential was calculated by dividing the FL1 value by the FL2 value. Data are shown as the mean SD of three independ-ent experimindepend-ents (n = 6).
Figure 3. The autophagic activity of DMGF in different cancer cells. (a) Cells were treated with 2mg/mL DMGF and co-incubated with or without 3-MA (57mM). Cell viability was then analysed by MTT assay. The asterisk indicates a significant difference between con-trol and DMGF-treated cells by the Student’s t-test. (b) The effects of DMGF on LC3B-II levels. The levels of LC3B-II were determined in DMGF-treated HT-29, A549 and HepG2 cells using anti-LC3B-II antibodies. b-actin was used as the normalized control. Results shown are representative of three independent experiments.
integrity of the plasma membrane in A549 and HepG2 cells was disrupted following DMGF treatment (Fig. 1A). A). In addition, DMGF treatment resulted in an increased number of cells in the sub-G1 phase in these two cell lines (Fig. 1B). These results indicate that the apoptotic pathway is not the major pathway involved in DMGF-induced cell death. In addition, Annexin-V staining indicated that necrosis was also not the major pathway for DMGF-induced death of A549 or HepG2 cells (results not shown). It was found that autophagy, an apoptosis-independent mechanism of cell death, was likely involved in DMGF-induced death in A549 and HepG2 cells (Fig. 3). Using 3-MA, an inhibitor of autophagy, it was shown that autophagy is the major pathway for DMGF-induced cell death in HepG2 and A549 cells. Following DMGF treatment, HepG2 cells showed increased expression of LC3B-II, a marker used to monitor autophagy in cells (Barth et al., 2010; Choi et al., 2010). Thesefindings suggest that DMGF can in-duce autophagy in HepG2 cells. In addition, DMGF treatment decreased the expression of LC3B-II in HT-29 cells, suggesting that DMGF-induced apoptosis could suppress the expression of this autophagy marker. According to our results, DMGF induced both apoptotic and autophagic death in A549 cells. Thus, there was no significant change in the expression of LC3B-II in these cells (Fig. 3B).
Unlike apoptosis, autophagy is a reversible process that can contribute to both tumor cell death and survival (Amaravadi and Thompson, 2007). Recently, autophagic agonists have been described as anticancer drugs (Choi et al., 2010; Hung et al., 2009; Ko et al., 2009). Furthermore, our results are consistent with the findings of these studies, in which 6-shogaol and
ginsenoside Rk1 were found to induce autophagic death in A549 and HepG2 cells, respectively. In addition, many compounds have been shown to induce death in tumor cells with defective apoptotic machinery (Reed, 2003; Yu et al., 2003). Thus, inducers of autophagy com-bined with inducers of apoptosis may provide a better antitumor effect. Because DMGF is able to induce both the apoptotic and autophagic pathways, it may be a good candidate for antitumor treatment.
In summary, DMGF isolated from the needles of Taxus media var. Hicksii has promising anticancer prop-erties due to its ability to induce different apoptotic or autophagic effects in different cell lines. This biflavonoid is likely a contributor to the anticancer properties described here and may act to limit cancer cell progres-sion by inhibiting proliferation, inducing apoptosis, evoking autophagy and/or suppressing inflammation and tumor cell migration. In vivo and human clinical studies are needed to determine the efficacy of this com-pound as a cancer chemoprevention agent.
Acknowledgements
The authors acknowledge the financial assistance provided by Yung-Shin Pharmaceutical Industry Co., Ltd under the cooperative grant scheme (Project No. 98 C117).
Conf lict of Interest
The authors have declared that there is no conflict of interest.
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