Anti-proliferation Effect on Human Breast Cancer Cells via




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Anti-proliferation Effect on Human Breast Cancer Cells via

Inhibition of pRb Phosphorylation by Taiwanin E Isolated from

Eleutherococcus trifoliatus

Hui-Chun Wanga,b, Yen-Hsueh Tsengc, Hui-Rong Wuc, Fang-Hua Chud, Yueh-Hsiung Kuoe,f* and

Sheng-Yang Wangc,g,h*

aGraduate Institute of Natural Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan

bDepartment of Marine Biotechnology and Resources, National Sun Yat-Sen University, Kaohsiung, Taiwan

cDepartment of Forestry, National Chung-Hsing University, Taichung, Taiwan

dSchool of Forestry and Resource Conservation, National Taiwan University, Taipei, Taiwan

eGraduate Institute of Chinese Pharmaceutical Science, China Medical University, Taichung, Taiwan

fDepartment of Biotechnology, Asia University, Taichung, Taiwan

gAgricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan

hAgricultural Biotechnology Center, National Chung-Hsing University, Taichung, Taiwan

taiwanfir@dragon.nchu.edu.tw (Prof. S.Y. Wang); kuoyh@mail.cmu.edu.tw (Prof. Y. H. Kuo)
Eleutherococcus trifoliatus has been used as a folk medicine since ancient times, especially as refreshing qi medicines. In our current study, taiwanin E,

which possesses strong cytotoxicity, was isolated from the branches of E. trifoliatus by using a bioactivity guided fractionation procedure. Taiwanin E

presented a potent anti-proliferation activity on the growth of a human breast adenocarcinoma cell line (MCF-7), with an IC50 value for cytotoxicity of 1.47

μM. Cell cycle analysis revealed that the proportion of cells in the G0/G1 phase increased in a dose-dependent manner (from 79.4% to 90.2%) after 48 h

exposure to taiwanin E at a dosage range from 0.5 to 4μM. After treatment with taiwanin E, phosphorylation of retinoblastoma protein (pRb) in MCF-7 cells

was inhibited, accompanied by a decrease in the levels of cyclin D1, cyclin D3 and cyclin-dependent kinase 4 (cdk4) and cdk6; in addition, there was an

increase in the expression of cyclin-dependent kinase inhibitors p21WAF-1/Cip1 and p27Kip1. The results suggest that taiwanin E inhibits cell cycle progression of

MCF-7 at the G0/G1 transition.



Keywords: Eleutherococcus trifoliatus, Araliaceae, Taiwanin E, Cytotoxicity, Cell cycle, G1 arrest.


Eleutherococcus trifoliatus (L.) S.Y. Huvar. trifoliatus (Syn.

Acanthopanax trifoliatus), adeciduous shrub or climber with

prickles on branches and petioles, belongs to the Araliaceae family.

The roots, bark, and leaves of this plant are used as a folk medicine

foreither prevention or amelioration of tumors and aging, and for

improving cardiovascular function [1-4]. A number of fatty acids,

steroids, lupane-triterpene carboxylic acids, lupane-triterpene



glycosides, kaurane-type diterpene glycosides, and phenyl-

propanoid glycosides have been reported [5-13]. The essential oil of



E. trifoliatus contained, as its main components, α-pinene, sabinene,

terpinen-4-ol, β-pinene, and p-cymene [14]. Sithisarn and Jarikasem

reported that the leaf aqueous extract of E. trifoliatus showed a high

level of antioxidant activity and contained high contents of both

phenolic and flavonoid compounds, but they did not identify any of

them [15].


Recently, taiwanin E, a strong cytotoxic lignan, was isolated from

the branches of E. trifoliatus by using a bioactivity guided

fractionation procedure. The content of taiwanin E in the crude

extract was determined by HPLC to be 23 mg/g. According to the

MTT assay, when human breast adenocarcinoma cells (MCF-7)

were treated with taiwanin E at dosages of 1.25 - 10 μM, a dose-

dependent decrease of cell viability was observed. The IC50 value of

taiwnin E was 1.47 μM, whereas that of plumbagin, which was used

as a positive control, was 0.26 μM. To examine the mechanism

responsible for taiwanin E mediated cell growth inhibition, cell

cycle distribution was evaluated using flow cytometric analysis.

Figure 1: The cytotoxicity compound, taiwanin E, obtained from E. trifoliatus.
When MCF-7 cells were treated with 0, 0.5, 1, 2 and 4 μM of

taiwanin E for 48 h, the percentage of G1 cells was 76.8, 79.4, 82.4,

85.7 and 90.2, respectively (Table 1). Our results indicated that

treating cells with taiwanin E caused a significant inhibition of cell

cycle progression in the MCF-7 cell line, showing a clear increase

in the percentage of cells in the G1 phase along with a decrease in

the S phase cells when compared with the control, reflecting that

cell-cycle progression is impeded at the G1 phase.

By contrast, protein levels of cdk2 and cyclin E were found to be

unaltered by taiwanin E treatment. In addition, exposure of cells to

taiwanin E resulted in a dose-dependent increase in the levels of

tumor suppressorp21Waf1/Cip1and p27Kip1, which are the cyclin-

dependent kinase inhibitors belonging to the Cip/Kip family

(p21Waf1/Cip1, p27Kip1, and p57Kip2). According to previous studies,

increasing p21Waf1/Cip1 and p27Kip1expression causes cell cycle arrest

at the G1 phase, and, therefore, cell proliferation will be suppressed.



In this situation, cells are efficiently exited from the cell cycle and






















Expression of p21WAF-1/Cip1 can be regulated by either p53-

dependent or p53-independent mechanisms [26–29]. Previous

studies indicated that G1 arrest could also be achieved by p53,

which is phosphorylated on Ser15 by ATM/ATR and on Ser20 by

Chk2, through induction of p21WAF-1/Cip1 transcription [30].

Taiwanin E did not induce p53 phosphorylation and change Chk2

expression in MCF-7 cells, which suggests that p21 is upregulated

by a p53-independent mechanism. It could be concluded that

taiwanin E possesses a potent cytotoxic activity against MCF-7

cells. The anti-proliferation effect of taiwanin E on MCF-7 cells

occurs through downregulation of cyclin D1/D3, cdk4/6, and

upregulation of p21WAF-1/Cip1 by a p53-independent mechanism.

Further study is needed to evaluate the antitumor activity.


Experimental

Materials: E. trifoliatus was collected in May 2012 from Nantou

County, Taiwan, and was identified by Dr Yen-Hsueh Tseng

(NCHU). The voucher specimen was deposited in the herbarium of

the same university.


General: UV and IR spectra were recorded on Jasco V-550 and

Bio-Rad FTS-40 spectrometers, respectively. Electrospray

ionization-mass spectrometric (ESIMS)and high-resolution

electron-impact mass spectrometric(HREIMS) data were collected

with a Finnigan MAT-95S mass spectrometer, and NMR spectra

were recorded with Bruker Avance 500 and 300 MHz FT-NMR

spectrometers, at 500 MHz (1H) and 75 MHz (13C). CDCl3 was used

for NMR analysis. Taiwanin E was dissolved in MeOH and

quantification was based on the measured integration area applying

the calibration equation. The concentration of taiwanin E used for

calibration was 0.025–1.0 mg/mL. The linear regression equation,

y=25.249 x + 156.91, revealed a good linearity (R2= 0.99812).


Extraction and purification: Air-dried branches of E. trifoliatus

(350 g) were extracted with EtOH (10 L) at ambient temperature

and concentrated under vacuum to yield the EtOH extract (40.13 g).

This was partitioned between EtOAc-H2O to give EtOAc-soluble

(18.4 g) and H2O-soluble fractions. The EtOAc-soluble fraction,

which displayed potent cytotoxicity (IC50 = 30.3 μg/mL), was

further chromatographed over silica gel (4 × 30 cm; 60–80 mesh;

Merck) eluted with n-hexane and a gradient of n-hexane-EtOAc

(100:0; 95:5; 90:10; 85:15; 80:20; 75:25; 70:30; 65:35; 60:40;

Figure 2: The expression of cell cycle-related proteins in MCF-7 cells treated with

various concentrations of taiwanin E as determined by Western blotting.


enter a quiescent state, and cell division will be inhibited [16,17].

We did not find p16, a cdk4/cdk6 inhibitor, in MCF-7 cells, which

is in agreement with the deletion detected of the MTS1 gene that

encodes p16 in this cell line [18-19]. The level of pRb

(phosphorylation at Ser807/811), which controls progression

through G0 within the G1 phase of the cell cycle, was reduced in

MCF-7 cells by taiwanin E treatment. pRb binds to and represses

the transcription factor E2F during early and mid-G1 phase [20].

During G1/S transition, cdk4/6 release from their inhibitory proteins

INK4 family and phosphorylate pRb [21-22]. Phosphorylated pRb

releases E2F, permitting the transcription of the G1/S transition

genes [23-24]. In taiwanin E treated cells, inhibition of cdk activity

by a combination of cdk4/6 and cyclin D1/D3 downregulation,

and p21Waf1/Cip1and p27Kip1 upregulation, prevented pRb

phosphorylation. It would, therefore, be expected to prevent G1/S

transition gene transcriptions by E2F. However, inactivated pRb in

the quiescent cell promotes E2F1, inducing apoptosis. pRB



regulated E2F1-induced apoptosis is actually distinguishable from

its transcriptional control of other E2F proteins [25].

50:50; 40:60; 30:70; 20:80; 10:90; 0:100, each 2 L). The eluent was

collected in constant volumes (each 500mL), and combined into

18 fractions based on TLC properties. Fraction 9 (obtained with



n-hexane:EtOAc = 89:11, amount 5.3 g) displayed the strongest

cytotoxicity (IC50 = 14.6μg/mL) and was further separated by

HPLC using a normal-phase column (250 × 10 mm, 5 μm,

Phenomenex Co.) with a mixture of n-hexane:EtOAc = 75:25 as

eluent at a flow rate of 3 mL/ min to obtain taiwanin E(retention

time 21.9 min).The structure of taiwanin E was elucidated and

confirmed by spectroscopic analysis [31].
Cell culture: MCF-7 (human breast adenocarcinoma, BCRC 60436)

was purchased from BCRC (Bioresource Collection and Research

Center), Food Industry Research, and Development Institute,

Taiwan. MCF-7 cells were cultured in DMEM supplemented with

10% FBS, 1% penicillin–streptomycin, and 1 mM sodium pyruvate,

and maintained at 37°C in 5% CO2. All cells (1 × 103 per well)

were seeded in 96-well plates and incubated for 24 h, and different

dosages of taiwanin E were added to each well in triplicate for 5

days. The cell viability was determined by the MTT (3-[4,5-

dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay [32].

Plumbagin (Sigma) was used as positive control.











Cell cycle analysis: MCF-7 cells, seeded in a 60 mm dish (2 × 105

cells/dish), were treated with 0, 0.5, 1, 2, and 4μMtaiwanin E for 48

h. Subsequently, cells were trypsinized, collected with ice-cold PBS

and resuspended in 1 mL PBS. Cells were then fixed by the addition

of 3 mL ice-cold 95 % ethanol at -20°C overnight. The cell pellets

were collected by centrifugation and rinsed with ice-cold PBS.

Cells were stained with 1 mL of 50 μg/mL propidium iodide (PI)

in RNase containing buffer (0.5% Triton X-100 in PBS and

0.5 mg/mL RNase A) for 30 min. Fluorescence emitted from the

PI-DNA complex was quantified after excitation of the fluorescent

dye by flow cytometry (Cytomics FC 500, Beckman Coulter).
Protein expression analysis: The protein expression after treatment

with taiwanin E was determined by Western blotting assay. Briefly,

MCF-7 cells incubated in 100 mm culture dishes (1 × 106 cells per

dish) were treated with taiwanin E at dosages of 0, 0.5, 1, 2, and 4

μM for 48 h. 40 μg of total cell proteins were separated by 12%

SDS-PAGE and transferred to a PVDF membrane. Detection was

performed by immunostaining using specific primary antibodies

and horseradish peroxidase-conjugated anti-IgG antibody. The

proteins were detected by chemiluminescence (ECL, Pierce

Biotechnology, Inc.). The following antibodies were used for the

Western blots: rabbit polyclonal antibodies to p27Kip1, cdk2, cdk4,

phosphor (Ser807/811)-pRb, and Chk2; mouse polyclonal

antibodies to cdk6, cdk4, cyclin D1, cyclin D3, cyclin E, p21Waf1/Cip1,

phosphor (Ser15)-p53, and β-actin; goat anti-rabbit immunoglobulin

G (IgG)-horseradish peroxidase-conjugate, and horse anti-mouse

immunoglobulin G (IgG)-horseradish peroxidase-conjugate.

Antibodies were used at working dilutions of 1:1000, with the

exception of anti-bodies to β-actin, for which a working dilution of

1:10000 was employed.
Statistical analysis: Data are expressed as means ± SD. Statistical

comparisons of the results were made using analysis of variance

(ANOVA). Significant differences (* p< 0.05) between the control

(untreated) and treated cells were analyzed by Dunnett’s test.


Acknowledgement - This study was supported by the National

Science Council, Republic of China (NSC-101-2911- I-005-301,

NSC-102-2911- I-005-301), and the Ministry of Education, Taiwan,

ROC, under the ATU plan and Council of Agriculture (99AS-8.4.4-

e1-F2).


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