Zanthoxylum ailanthoides Sieb and Zucc. extract inhibits growth and induces cell death through G2/M-phase arrest and activation of apoptotic signals in colo 205 human colon adenocarcinoma cells

Abstract.

The effects of 50% ethanolic stem extracts of

Zanthoxylum ailanthoides Sieb and Zucc. (ZASZ) on the cell

viability, cell cycle and apoptosis were investigated in a

human colon adenocarcinoma cell line (colo 205). The results

demonstrated that ZASZ induced morphological changes and

decreased the cell viability. ZASZ promoted Wee1, checkpoint

kinase 2 (CHK2), p21 and p53 levels, decreased cyclin B and

cdc25c associated with that led to G

/M phase arrest.

ZASZ-triggered apoptosis was confirmed by

4’-6-diamidino-2-phenylindole (DAPI) staining and DNA gel electrophoresis.

ZASZ increased the levels of glucose-regulated protein 78

(GRP78) and growth arrest and DNA damage inducible gene

153 (GADD153), and promoted an increase of reactive

oxygen species (ROS) and Ca

release, and loss of

mitochondrial membrane potential (ΔΨ

) accompanied by

cytochrome c release that was due to the decrease of Bcl-2

and increase of Bax levels in the colo 205 cells. ZASZ also

induced the protein levels of apoptosis-inducing factor (AIF)

and endonuclease G (Endo G), increased the levels of

caspase-3, -7 and -9, and stimulated the levels of fatty acid

synthase (Fas) and Fas ligand in the colo 205 cells. ZASZ

contains phenolic compounds, including flavone, chlorogenic

acid and isofraxidin, among which, flavone was found to be

the most effective in reducing cell viability and proliferative

responses in the colo 205 cells. ZASZ induces cytotoxicity and

apoptosis in colo 205 cells which provides the rationale for

studies in animal models on the utilization of ZASZ as a

potential cancer therapeutic compound.

Colon cancer is a common malignancy, and one of the major

fatal diseases throughout the world (1). Furthermore, it is the

second most frequent cause of death from cancer in Taiwan,

where based on the reports from the Department of Health

of the Republic of China, 19.6 people per 100 hundred

thousand people die each year of colorectal cancer. The

therapy for human colorectal cancer in Taiwan includes

chemotherapy, radiotherapy and surgery. However, these

treatment strategies had limited success. New agents acting

on novel targets of carcinoma are needed.

The cyclin-dependent protein kinase (Cdk) family, cyclins

and Cdk inhibitors (CdKI) are essential for cellular

regulatory processes, particularly in the regulation of

transcription and cell cycle progression (G

to S or G

to M

phase) (2, 3). Cdk are regulated by multiple mechanisms

which include inhibitory phosphorylation of conserved

residues within the active site (4) and binding of additional

regulatory molecules (5). It is reasonable to propose that if

cyclin and/or Cdk are affected then cell cycle arrest and

apoptosis could be induced.

It is well known that apoptosis plays a variety of

important roles under normal physiological conditions (6).

Science and Technology, China Medical University, No 91, Hsueh-Shih Road, Taichung 404, Taiwan, R.O.C. Tel: +886 422053366, ext. 2161, Fax: +886 422053764, e-mail: [email protected]

Key Words: Zanthoxylum ailanthoides Sieb and Zucc., human colon

adenocarcinoma cell, colo 205 cells, caspases, apoptosis, flavone.

Zanthoxylum ailanthoides Sieb and Zucc. Extract

Inhibits Growth and Induces Cell Death through G

₂

/M-phase

Arrest and Activation of Apoptotic Signals

in Colo 205 Human Colon Adenocarcinoma Cells

SU-TZE CHOU

, HSIN-YI PENG

, CHEN-TIEN CHANG

, JAI-SING YANG

,

HSIUNG-KWANG CHUNG

, SU-TSO YANG

, W. GIBSON WOOD

and JING-GUNG CHUNG

_{Department of Food and Nutrition, Providence University, Taichung 433, Taiwan, R.O.C.;}

Departments of

Pharmacology and

Biological Science and Technology,

4

_{Graduate Institute of Clinical Medical Science, China Medical University, Taichung 404, Taiwan, R.O.C.;}

Departments of

Otorhinolaryngology-Head and Neck Surgery and

5

_{Radiology, China Medical University Hospital, Taichung 404, Taiwan, R.O.C.;}

_{Department of Biotechnology, Asia University, Taichung 413, Taiwan, R.O.C.;}

6

_{Department of Pharmacology, University of Minnesota,}

Impaired apoptosis is a crucial step in the process of cancer

development (7, 8). Cells undergo apoptosis through

distinct pathways based on stimulation of caspases, a

family of proteases that are important in regulating

apoptosis (5). It is well known that compounds that can

induce apoptosis can be divided into the fatty acid synthase

(Fas) and Fas ligand (FasL) pathways resulting in the

activation of the caspase-8, mitochondria-dependent

pathway and the caspase-3-dependent pathway. Both

pathways trigger the cytoplasmic release of pro-apoptotic

mitochondrial proteins and stimulate the endoplasmic

reticulum (ER) stress pathway which is attributed to the

activation of caspase-12 and eventual apoptosis (9).

Therefore, the resistance of cancer cells to treatment might

be associated with dysregulation or defects in different

steps of the apoptotic pathways.

Numerous naturally occurring compounds are thought to

have potential value for cancer prevention and cancer

therapy (10, 11) and they were recognized to be a potential

inhibitor of tumor cell proliferation, carcinogen-induced

carcinogenesis or inducers of apoptosis (12-14). The tender

leaves of Zanthoxylum ailanthoides Sieb and Zucc. have

been used as a dietary supplement in the Taiwanese

population for many years (15). In the Chinese population,

the plant Z. ailanthoides has been used as a herbal medicine

for the treatment of heart disease, bone-injury and cold

resistance. Our previous study demonstrated that a 50%

ethanolic stem extract of Z. ailanthoides (ZASZ) was safe

from a genotoxic perspective and acted as an antioxidant

(16). However, there is no information on effects of ZASZ.

Therefore, in the present study, the possible anticancer

activity of ZASZ and whether it acts on cell cycle arrest and

triggers tumor cell apoptosis were determined.

Materials and Methods

Plant material and preparation of extracts. The ZASZ (50%

ethanolic stem extract) was prepared as previously described (16). Briefly, the plant material was obtained from the mountain area of Sinshe Township, Taichung, Taiwan. Each 100 g stem was extracted with 500 ml 50% ethanol at 75˚C for 3 h and decocted three times. The decoctions were filtered and then dried by a vacuum freeze-dryer. The extraction yields were 9.0%. The extracts were sealed in plastic bottles and stored at –70˚C until used. For the present experiments, extracts were dissolved in PBS before adding to cell cultures.

Chemical and reagents. Propidium iodide (PI),

4’-6-diamidino-2-phenyindole (DAPI), ethidium bromide (EtBr), triton X-100, trypan blue and ribonuclease A (RNase A) were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). RPMI 1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), L-glutamine, fetal bovine serum (FBS), penicillin-streptomycin and trypsin-EDTA were obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). All other chemicals were at least reagent grade.

Cell lines and culture conditions. Human colon adenocarcinoma cell

line (colo 205), human hepatoma cell line (Hep G2), human murine leukemia cell line (WEHI-3), mouse melanoma cell line (B16-F1) and promyelomonocytic cells line (HL60) were obtained from the Food Industry Research and Development Institution (Hsinchu, Taiwan). The colo 205 and WEHI-3 cells were cultured in RPMI 1640 medium, and the Hep G2 and B16-F1 cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin- streptomycin (100 U/ml penicillin and 100 μg/ml streptomycin) (17). All the cells were maintained in a humidified 5% CO₂incubator at 37˚C and the cells were sub-cultured every 3-4 days to maintain logarithmic growth and were allowed to grow for 24 h before treatments were applied.

Morphological changes and cell viability analysis. The colo 205,

Hep G2, B16-F1 and WEHI-3 cells were plated in 12-well plates at a density of 1×105_{cells/ml. The cells were treated with different}

concentrations (31.25, 62.5, 125.0, 250.0 or 500.0 μg/ml) of ZASZ or PBS as a solvent control and grown at 37˚C, 5% CO₂and 95% air for 24, 48 or 72 h. To determine morphological changes, the cells were photographed by a phase-contrast microscope. Cell viability was determined by a PI exclusion method and by using a flow cytometer (Becton-Dickinson, FACSCalibur, San Jose, CA, USA) equipped with an argon ion laser at 488 nm wavelength as previously described (17).

Cell cycle and apoptosis analysis. The colo 205 cells were incubated

with different concentrations of ZASZ (0, 31.25, 62.5, 125.0, 250.0 or 500.0 μg/ml) for 48 h. The cells were then harvested by centrifugation and fixed gently in 70% ethanol (in PBS) at -20˚C overnight. The cells were then re-suspended in PBS containing 40 μg/ml PI and 0.1 mg/ml RNase A and 0.1% triton X-100 in a dark room for 30 min and analyzed by flow cytometry (17, 18). A FITC annexin V apoptosis detection kit from BD Pharmingen (San Diego, CA, USA) was used to determine and quantify the apoptotic cells by flow cytometry (19).

DAPI staining. The colo 205 cells were placed for 24 h and then

incubated with various concentrations of ZASZ (0, 31.25, 62.5, 125.0, 250.0 or 500.0 μg/ml) for 48 h. The cells were washed by PBS, fixed with 4% formaldehyde for 15 min, stained with DAPI (Sigma) at 37˚C for 30 min and photographed using a fluorescence microscope as previously described (17, 20).

DNA fragmentation assay. The colo 205 cells were incubated with

different concentrations of ZASZ (0, 31.25, 62.5, 125.0, 250.0 or 500.0 μg/ml) for 48 h. The cells were then harvested by centrifugation, the DNA was isolated and was then gel electrophoresized and stained with EtBr and photographed under UV light as previously described (17, 21).

Measurement of reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨ_m) and Ca2+_{release. The}

colo 205 cells were incubated with 250.0 μg/ml of ZASZ for 0, 2, 4, 6 or 24 h. The cells were harvested and washed twice with PBS then re-suspended with specific fluorochromes. For ROS analysis, the cells were re-suspended in 500 μl 10 μM of 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, Invitrogen) and incubated at 37˚C for 30 min and then analyzed by flow cytometry as previously described (22, 23). For ΔΨ_manalysis, the cells were re-suspended in 500 μl of 3,3’-dihexyloxcarbocyanine (DiOC₆, Invitrogen) (1 μmol/l) and incubated at 37˚C for 30 min and

analyzed by flow cytometry as previously described (22, 24). To detect ΔΨ_mrelease, the cells were re-suspended in Indo 1/AM (3 μg/ml, Invitrogen) and incubated at 37˚C for 40 min. After incubation, the cells were washed with PBS and analyzed by flow cytometry as previously described (22).

Determination of G₂/M phase arrest and apoptosis-related protein levels. The colo 205 cells were incubated with 250.0 μg/ml of ZASZ

for 0, 6, 12, 24 or 48 h. After treatment, the cell lysates were used to determine the protein abundance of cyclin A and B, cell division control 2 (cdc2), cdc25c, checkpoint kinase 2 (CHK2), Wee1, ataxia-telangiectasia mutated (ATM), p21, p53, B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X (Bax), cytochrome c, apoptosis-inducing factor (AIF) and endonuclease G (Endo G), caspase-9, -3, -7, poly (ADP-ribose) polymerase (PARP), fatty acid synthase (Fas), Fas ligand (FasL), caspase-8, truncated BH3 interacting domain death agonist (tBid), glucose-regulated protein 78 (GRP78), growth arrest and DNA damage inducible gene 153 (GADD153) and β-actin by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting as described previously (17, 22).

Characterization of phenolic compounds. Phenolic compounds in

the ZASZ extract were analyzed based on the method described by Li et al. (25) with a slight modification. Dried ZASZ (0.1 g) was dissolved in 10 ml of 50% ethanol, filtered through a 0.45 μm filter, and analyzed by high-performance liquid chromatography (HPLC). HPLC analysis was performed using a Hewlett- Packard HPLC System (HP 1100 series, Waldron, Germany), consisting of a quaternary pump and a variable wavelength detector (VWD) at 270 nm and equipped with a Li-Chrospher RP-18 cartridge column (250 mm ×4.6 mm, 5 μm, Merck KGaA, Darmstadt, Germany). The mobile phase was a stepwise gradient of water (0.1% v/v phosphoric acid)-acetonitrile (0.01 min, 94: 6; 50 min, 65: 35), and the injection volume was 30 μl. The identification of each compound was based on a combination of retention time and spectral matching by comparison with those of known standards. Peak areas and concentrations were determined using the Hewlett- Packard Chem Station Chromagraphic Management System.

Statistical analysis. Student’s t-test was used to analyze statistical

differences between the ZASZ treated and control groups. A p-value less than 0.05 was considered significant. All data are presented as means±S.D. of three independent experiments.

Results

Effects of ZASZ and its active phenolic compounds on cell

viability in human cancer cell lines. Each cell line responded

differently to the ZASZ treatment with the colo 205 cells

most susceptible to ZASZ-induced toxicity (Figure 1A). The

500 μg/ml ZASZ treatment over 48 h decreased cell viability

by 46.4%, 18.1%, 9.2% and 5.2% in the colo 205, Hep G2,

B16-F1 and WEHI-3 cells, respectively. Therefore, the colo

205 cells were used in the subsequent experiments because

of the greater effects of and sensitivity to ZASZ on those

cells compared with the other cell lines.

The ten standard polyphenols used for comparison showed

the retention times of gallic acid, chlorogenic acid, caffeic

acid, flavone, isofraxidin, rutin, genistin, daidzin, quercetin

and genistein to be 5.68, 14.78, 16.93, 21.28, 25.89, 26.87,

28.66, 39.43, 43.10 and 49.37 min, respectively, using HPLC

analysis (chromatograms not shown). The data in Table I

showed that ZASZ contained three major phenolic

substances, including chlorogenic acid, flavone and

isofraxidin with flavone the most abundant phenol. The

effects of chlorogenic acid, flavone and isofraxidin on cell

Table I. Phenolic contents of ZASZ.

Compound Content (mg/g extract) Chlorogenic acid 2.74±0.21 Flavone 22.68±0.90 Isoflaxidin 1.46±0.14 Reported values are the means±SD (n=3).

Figure 1. Effects of the ZASZ on the proliferation of colo 205, B16-F1,

Hep G2 and WEHI-3 cells (A) and the active phenolic compounds in ZASZ on the cell viability of colo 205 cells (B). Means±S.D. of three independent experiments. *p<0.05 compared with the control sample.

viability were examined, and results from Figure 1B

indicated that the induction of toxicity of the three phenolic

compounds was dose-dependent. The cell viability followed

the order flavone>chlorogenic acid>isofraxidin.

Effects of ZASZ on cell morphology, cell viability, cell cycle

distribution and apoptosis. The phase-contrast microscopic

examination indicated that ZASZ-induced apoptotic

morphological changes such as shrinking and cytoplasmic

Figure 2. Effects of ZASZ on cell morphology (A) and cell viability (B) up to 72 h and , DNA content for cell cycle distribution (C) and apoptosis

blebbing were found (Figure 2A) and a PI stain analysis by

flow cytometry showed that the percentage of viable cells was

significantly different between the ZASZ-treated and the

control groups (Figure 2B). These effects were dose- and

time-dependent. ZASZ induced G

/M-phase arrest in the colo

205 cells (Figure 2C) and the effect was dose-dependent. It

can be seen in Figure 2D that ZASZ also induced apoptosis in

the colo 205 cells. To further confirm that ZASZ induced

apoptosis, the results determined by DAPI staining. Figure

3A shows that apoptotic cell death occurred after ZASZ

treatment and Figure 3B demonstrates evidence of DNA

fragmentation in the ZASZ -treated colo 205 cells.

Effects of ZASZ on ROS and Ca

levels and ΔΨ

. The ROS

levels were stimulated when the colo 205 cells were incubated

with 250 μg/ml of ZASZ and peaked after a 2 h exposure,

then decreasing slightly up to 24 h. However, the ROS levels

were still higher in the ZASZ-treated cells than in the control

cells for at least 24 h (Figure 4A). Flow cytometric analysis

indicated that the ΔΨ

decreased in the ZASZ-treated cells as

Figure 3. The induction of apoptosis by ZASZ in colo 205 cells was determined by DAPI staining and fluorescence microscopy (A) and DNA gel

compared with the control cells after 24 h treatment (Figure

4B). Also, it can be seen in Figure 4C that the cytoplasmic

Ca

level peaked at 2 h and then began to decrease but were

higher than the control cells at 24 h (p<0.05).

Effects of ZASZ on G

/M-phase arrest and

apoptosis-associated specific protein levels. The Western blotting

analysis is shown in Figures 5 and 6. Protein levels of

CHK2, Wee1, ATM, p21, p53 (Figure 5B), Bax (Figure 5C),

cytochrome c, AIF, Endo G (Figure 5D), caspases-9, -3, and

-7 (Figure 6A), Fas, FasL, caspase-8, tBid (Figure 6B),

GRP78 and GADD153 (Figure 6C) increased and cyclins A

and B , cdc2, cdc25c (Figure 5A), Bcl-2 (Figure 5C) and

PARP (Figure 6A) reduced following ZASZ treatment in the

colo 205 cells.

Discussion

Until now, a cure for colon cancer is not satisfying and

numerous studies focused on finding new agents for colon

cancer therapy. In this study, we showed that ZASZ induced

G

/M arrest and Western blot analysis showed that it was

mediated through the decreased protein expressions

of cdc25c, Wee1 and CKI proteins (p21

), a

simultaneous decrease in cyclins A and B and enhanced

binding of CKI-Cdk. The concentration of 250.0 μg/ml of

ZASZ was selected for all the Western blotting analysis based

on the viability studies (Figure 2) and was close to the IC

for 72 h exposure. p21

is a universal inhibitor of

Cdk(s) and p27

is commonly upregulated in response to

antiproliferative signals (26). The present data also showed

that ZASZ induced the expression of p53 in the colo 205 (p53

wild-type) cells (Figure 5B). The tumor suppressor gene p53

is activated in response to various genotoxic stresses,

resulting in cell cycle arrest or apoptosis. Cell cycle arrest that

is dependent on p53 requires transactivation of p21

,

GADD45 and cyclin G through the binding of the cyclin-cdk

complex (27). The blockade of cell cycle progression by

ZASZ was through activation of p21

involving a

p53 dependent pathway (Figure 5A and B). It has been

reported that p53 is responsible for the up-regulation of Fas,

Bax, Bad and Bcl-xl resulting in apoptotic cell death (28).

Therefore, ZASZ not only increased the cell cycle-related

protein (p21

), but also enhanced the proapoptotic

expression of Bax, Fas and reduced levels of the

anti-apopototic protein Bcl-2 (Figure 5 and 6).

Normal cells maintain a balance of pro-apoptotic and

anti-apoptotic Bcl-2 family proteins which integrate the diverse

death- and survival-signals to control the fate of the cell (8,

29) including both Bcl-2 and Bax. The present data

demonstrated that the ZASZ-induced apoptosis was

associated with an increase of Bax and a reduction of Bcl-2

protein levels, and both Bcl-2 family proteins were in

agreement with the notion that an imbalance between

pro-and anti-apoptotic proteins of the Bcl-2 family results in

apoptosis (8, 29). Anti-apoptotic Bcl-2 family proteins are

also known to prevent the release of caspase-9 (30), and

regulate the release of cytochrome c from the mitochondria

Figure 4. Effects of ZASZ (250 μg/ml) on the levels of reactive oxygen

species (ROS) (A), mitochondria membrane potential (ΔΨ_m) (B) and Ca2+ _{release (C) in colo 205 cells. Mean±S.D. of triplicated} experiments. *p<0.05 compared to control sample.

into the cytosol (31, 32). The over-expression of Bcl-xl

protein (anti-apoptotic) can block the release of cytochrome

c in response to a variety of apoptotis signals (32). However,

the Bax protein (pro-apoptotic) promotes cytochrome c

release from the mitochondria (31, 32). Western blotting

demonstrated that ZASZ decreased the ΔΨ

and promoted

the levels of cytochrome c release from the mitochondria

(Figure 5D). The induction of the execution protease,

caspase-3 was also demonstrated. This was in agreement

with many reports demonstrating that released cytochrome c

from mitochondria activates initiator caspase-9 to activate a

sequential cascade of caspases, especially caspase-3 resulting

in the proteolysis of death substrates and subsequent DNA

degradation and apoptotic cell death (8, 29, 33, 34).

ZASZ also increased ROS and Ca

levels in the colo 205

cells (Figure 4A and C). ROS can stimulate ER stress

resulting in Ca

release and plays an important role in the

induction of apoptosis (35, 36). ZASZ increased the levels

of GADD153 and GRP78 (Figure 6C) also associated with

ER stress because GADD153 is a modulator and GRP78 is a

sensor of ER stress. Thus, ZASZ induced cytotoxicity in the

colo 205 cells which was mediated by several different

pathways associated with apoptosis as depicted in Figure 7.

ZASZ was found to contain approximately 3% phenolic

compounds mostly flavone, chlorogenic acid and isofraxidin.

Flavone was highest in abundance and was the least effective in

reducing cell viability in the colo 205 cells. Flavones are

polyphenolic compounds that occur naturally in many plants

used for food. In vitro studies on the potential anticancer activity

of flavones have been reported in different cell types (37-39).

Flavone may contribute to the antiproliferative and apoptotic

activities of the ZASZ extracts used in the present study.

In conclusion, ZASZ induces morphological changes,

decreases the percentage of viable cells, and induces G

/M

phase arrest of cell cycle and apoptosis involving ROS

production, ER stress, caspase-3, AIF and Endo G signaling

pathways. Further studies are needed on the effectiveness of

ZASZ in cancer cells particularly studies in animal models.

Figure 5. Effects of ZASZ on the protein levels of A: cyclin A and B, cdc2 and cdc25c; B: CHK2, Wee1, ATM, p21 and p53; C: Bcl-2 and Bax; D:

Acknowledgements

This research was supported by the National Science Council, Taiwan (NSC 96-2313-B-126-002- MY2) and its financial support is greatly appreciated.

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Received January 4, 2011

Revised April 6, 2011

Accepted April 7, 2011