Mechanism of the growth inhibitory effects of Zizyphus

jujuba and green tea extracts in human hepatoma cells

Department of Food and Human Health Sciences,

Graduate School of Human Life Science,

Osaka City University,

2008

Xuedan Huang

Mechanism of the growth inhibitory effects of Zizyphus

jujuba and green tea extracts in human hepatoma cells

（なつめ抽出物と緑茶抽出物のヒト肝ガン細胞増殖

抑制作用とその作用メカニズムについて）

大阪市立大学大学院 生活科学研究科

生活科学専攻 栄養機能科学研究室

平成 20年度

黄 雪丹

CONTENS

i

CONTENTS

INTRODUCTION

CHAPTER I Mechanism of the anticancer activity of Zizyphus

jujuba in HepG2 cells

Introduction

Material and Methods

Results

Discussion

Summary

CHAPTER II Green tea extracts enhances the selective cytotoxic

activity of Zizyphus jujuba extracts in HepG2 cells

Introduction

Material and Methods

Results

1

6

12

31

38

40

42

49

5

CONTENS

ii

Discussion

Summary

CHAPTER III Combination of Zizyphus jujuba and green tea

extracts exert excellent cytotoxic activity in

HepG2 cells via reducing the expression of APRIL

Introduction

Material and Methods

Results

Discussion

Summary

CONCLUSION

REFERENCES

ACKNOWLEDGMENT

67

75

76

78

83

92

97

98

101

118

CONTENS

iii

ABBREVIATION

LIST OF PUBLICATIONS RELATED TO THIS THESIS

120

122

INTRODUCTION

1

INTRODUCTION

Hepatocellular carcinoma accounts for 80% to 90% of primary

liver cancer. Hepatocellular carcinoma is a major health problem

worldwide, with an estimated incidence ranging between 500,000 and

1,000,000 new cases annually. It is the fifth most common cancer in

the world, and the third most common cause of cancer-related death 1)

.

The rates of hepatocellular carcinoma in men are 2 to 4 times higher

than in women. It usually develops between 35 and 65 years of age,

when people are most productive and have most family

responsibilities 2, 3, 4, 5)

. Hepatocellular carcinoma shows great

geographical variation, with a very high incidence in Asia and

sub-Saharan Africa 6)

. Although its incidence is far lower in the United

States and Europe, those rates have been increasing in recent years.

There are many treatment options for hepatocellular carcinoma,

such as liver resection (partial hepatectomy and orthotopic liver

transplantation), local ablative therapy (injection of cytotoxic agent

and application of an energy source), hepatic artery transcatheter

INTRODUCTION

2

treatment (transarterial chemoembolisation and transarterial

radioembolisation), systemic therapy (chemotherapy, immunotherapy,

chemo-immunotherapy, hormonal therapy and somatostation

analogue) and other treatments (gene therapy and supportive therapy).

The most effective and potentially curative therapy is liver

transplantation, because it eradicates the hepatocellular carcinoma,

however, this option is available to a very small fraction of

hepatocellular carcinoma patients whose tumors are discovered at a

very early stage before there is multifocal or vascular involvement 7)

.

The other options used in clinic also depend on the status of the

tumors. Combination chemotherapy appeared promising in two phase

II studies of cisplatin, interferon, doxorubicin, and 5-fluorouracil 8, 9)

and was notable for a small, but significant, number of pathologic

complete remissions discovered after tumor downstaging (patients

were all deemed unresectable as a criterion for entering the study)

allowed surgical resection. Therefore, it is important to find a new

drug for chemotherapy in the future 7, 10, 11, 12, 13, 14)

.

For thousands of years, natural products have played an

INTRODUCTION

3

important role throughout the world in treating and preventing human

diseases. Natural product medicines have come from various source

materials including terrestrial plants, terrestrial microorganisms,

marine organisms, and terrestrial vertebrates and invertebrates 15)

. The

importance of natural products in modern medicine has been discussed

in recent reviews and reports 15, 16, 17, 18)

. The value of natural products

in this regard can be assessed using 3 criteria: (1) the rate of

introduction of new chemical entities of wide structural diversity,

including serving as templates for semisynthetic and total synthetic

modifi cation, (2) the number of diseases treated or prevented by these

substances, and (3) their frequency of use in the treatment of disease.

Scrutiny of medical indications by source of compounds has

demonstrated that natural products and related drugs are used to treat

87% of all categorized human diseases, including as antibacterial,

anticancer, anticoagulant, antiparasitic, and immunosuppressant agents,

among others. Moreover, there are more than 60% of drugs of natural

origin used in the treatment of cancer 15)

. Recently, the low side effects

of natural products in anticancer treatment have been noticed. Hence,

INTRODUCTION

4

finding a new natural source with anticancer activities would aid in

finding new tools for cancer therapy.

In this study, we aimed to investigate the anticancer activity and

mechanism of action of Zizyphus jujuba Mill (Z. jujuba), a natural

product, in human liver cancer cells (HepG2 cells). To achieve this

aim, the following studies were conducted.

In Chapter I, we described that the chloroform fractions from Z.

jujuba extract (CHCl3-F) induced a concentration dependent effect on

apoptosis and a differential cell cycle arrest in HepG2 cells.

In Chapter II, we described that combination of CHCl3-F and

green tea extracts (GTE) produced an enhanced cell growth inhibition

effect, and the resultant G1 arrest was caused via different mechanism

as that of CHCl3-F treatment alone in HepG2 cells.

In Chapter III, we described that CHCl3-F and GTE enhanced

anti-cancer activity was via reducing the expression of APRIL,

moreover, the anti-cancer activity of CHCl3-F and GTE mixture was

stronger than that of the anti-cancer drug cisplatin in HepG2 cells.

CHAPTER I

5

CHAPTER I

Mechanism of the anticancer activity of Zizyphus jujuba

in HepG2 cells

1. Introduction

Chinese date is scientifically known as Zizyphus jujuba Mill (Z.

jujuba). It is also “Hongzao” or “Dazao” in China and “Natume” in

Japan. It has been mentioned in the famous Chinese ancient medical

book- Sheng Nong Ben Cao Jing, and has traditionally been used in

oriental medicines. For example, in Chinese traditional medicine, the

dried fruits are prescribed as anodyne, anti-tumor, pectoral, refrigerant,

sedative, stomachic, styptic and tonic. In Japan, the extracts of Z. jujuba

are used to treat chronic hepatitis or distress and fullness in the chest

and ribs. Some studies reported that there are 11 major components of 2

saponins and 9 fatty acids, namely jujuboside A, jujuboside B, lauric

acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic

acid, linoleic acid, arachidic acid and docosanoic acid in Z. jujuba.

CHAPTER I

6

Many physiological activities have been found but there are no report

about the anticancer activity of these components 19, 20, 21)

. A study

reported the ethyl acetate-soluble fraction of Z. jujuba showed high

cytotoxic activity against some tumor cell lines 22)

. However, in these

studies the mechanism of action of Z. jujuba has not so far been

investigated. More importantly, the effect of Z. jujuba in human

hepatoma cells (HepG2 cells), which come from hepatocellular

carcinoma, has not yet been reported.

In this Chapter, we investigated the anticancer activity and

mechanism of action of Z. jujuba in HepG2 cells.

2. Materials and Methods

2.1. Materials

Z. jujuba extract was kindly donated from Sea Load Co. Ltd.

(Fukui, Japan). 2’, 7’- Dichlorodihydrofluorescein diacetate

(DCFH-DA) and rhodamine 123 were purchased from Sigma–Aldrich

Fine Chemical (Tokyo, Japan). Rb (retinoblastoma protein) and p27Kip1

antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa

CHAPTER I

7

Cruz, CA, USA). Fetal bovine serum (FBS) was purchased from

Equitech-Bio Inc. (Kerrville, Texas, USA). Other chemicals used in this

chapter were special grade commercial products.

2.2. Cell culture

HepG2 cells were cultured in Dulbecco’s modified Eagle’s

medium supplemented with 10% FBS in humidified incubator

containing 5% CO2 in air at 37C. The cells were washed and cultured

again at a concentration of 2×105/ml in fresh medium. Z. jujuba extract

was diluted in the culture medium immediately before use. In all the

experiments, control cultures were made up of medium, water and the

cells only.

2.3. Cell viability

Cell viability was determined with the neutral red uptake assay,

based on the lysosomal uptake of neutral red 23)

. Following specified

incubations with test agents, neutral red solution (0.25 mg/ml) was

added to the cell cultures at a final concentration of 50 µg/ml. After

CHAPTER I

8

incubation at 37°C for 2 hrs, cells were rinsed twice with a mixture of

1% (v/v) formaldehyde, 1% (v/v) calcium chloride, and 98% (v/v)

distilled water. Subsequently, 1 ml of destaining buffer consisting of 1%

(v/v) acetic acid, 50% (v/v) ethanol, and 49% (v/v) distilled water was

added to the cells, and the culture plates were kept for 30 mins.

Lysosomal uptake of neutral red was determined spectrophotometrically

at 540 nm

24, 25, 26). Viability was expressed as (A540-treated cells/A540

of

appropriate control) × 100% after correction for background absorbance

(100% cytotoxicity).

2.4. Fractionation of Z. jujuba extract

Z. jujuba extract was diluted at 1/20 with water. These liquids

were extracted with chloroform, followed by ethyl acetate, butanol and

water. Each of the fractions was evaporated to dryness in vacuo (Fig.

2).

2.5. Cell cycle assay

The cell cycle distribution was analyzed by laser scanning

CHAPTER I

9

cytometer (Olympus LSC 101) using PI staining. Briefly, after

designated treatment, cells were washed twice in PBS and incubated

with freshly prepared PI-stained buffer (0.1% Triton X-100 in PBS, 20

µg/ml PI, 200 µg/ml RNase) for 1 hr at 37°C in the dark 27)

.

2.6. Reactive oxygen species (ROS) assay

The generation of ROS was determined with the DCFH-DA

reagent as described by Ablise, M. 28)

. Briefly, DCFH-DA (8 μM) was

added at the last 15 mins of Z. jujuba extract treatment. Cells were

washed with PBS twice and resuspended in Hanks

solution. Fluorescence intensity was measured using a micro-plate

reading fluoroscan plate-reader (Wallac 1420 ArVOsx, Amarsham

Pharmacia Biotech) with the excitation wavelength at 485 nm and the

emission wavelength at 535 nm 29, 30)

. The amount of intracellular ROS

was calculated from a standard curve derived from 2’, 7’

–dichlorofluoresceina (DCF). Protein concentration was measured by

the Bradford method 31)

.

CHAPTER I

10

2.7. Mitochondrial membrane potential assay

The HepG2 cells were incubated with Z. jujuba extract for 30,

60, 120 and 240 mins and rhodamine 123 (10 µg/ml) was added at the

last 30 mins of Z. jujuba treatment. Cells were washed twice with PBS

and fixed with 4% p-formaldehyde. After washing with PBS, the

fluorescence intensity of cells was measured by a micro-plate reading

fluoroscan plate-reader with the excitation wavelength at 505 nm and

the emission wavelength at 525 nm 32)

.

2.8. Western blot analysis of Rb and p27Kip1

Cells were harvested at the indicated time points and were

washed twice in PBS. Then the cells were dissolved for 30 mins with

lysis buffer (150 mM NaCl, 50 mM Tris (pH 7.2), 1 mM EDTA, 0.5%

sodium deoxycholate, 1% Nonidet P-40, 1 mM sodium vanadate, 1

mM NaF, 20 µg/mL

aprotinin, 50 µg/mL leupeptin, 10 µg/mL

Pepstatin A and 100 µg/mL phenylmethylsulfonyl fluoride). Finally,

the solution was centrifuged at 2000 ×g for 20 mins at 4°C. The

supernatant was collected and protein concentrations were determined

CHAPTER I

11

by Bradford method. Equal amounts of protein were fractionated on

10% SDS-PAGE gels and transferred to 0.45 µm PVDF

(Hybond;

Amersham Pharmacia Biotech). After blocking overnight in 0.1%

Tween-20 and 5% non-fat dry milk, in PBS, blots were incubated with

anti-Rb antibody or anti-p27Kip1

antibody for 1 hr at room temperature.

After washing, the membrane was reincubated with 1:750 diluted

biotinulated mouse immunoglobulin G (IgG) or rabbit IgG for 1 hr at

room temperature. The membrane was washed several times, and

incubated with 1:750 diluted horse radish peroxidase-coupled

streptavidin for 1 hr at room temperature. After several washing steps,

the color reaction was developed with DAB. Densitometry analysis of

the protein bands was performed with the software Scion Image

(Scion Corporation).

2.9. Statistical analysis

Data are represented as means ± S.D. (standard deviation from

the mean). The significance of difference in assay values was

evaluated with ANOVA followed by Tukey multiple tests. p <0.05 was

CHAPTER I

12

used to indicate a statistically significant difference.

3. Results

3.1. Effect of Z. jujuba extract on cell viability

The effect of Z. jujuba on cell viability was examined in HepG2

cells by neutral red method. As shown in Fig. 1, Z. jujuba extract

decreased cell viability in a dose-dependent manner. The Z. jujuba

extract contains many substances such as protein, carbohydrate and

minerals (Data are not shown), and it has been reported that the ethyl

acetate-soluble fraction of Z. jujuba has high cytotoxic activity 22)

.

Therefore, we extracted the Z. jujuba extract again by organic solvent

(Fig. 2), and examined the cell viability with these fractions from the

original Z. jujuba extract in HepG2 cells. As shown in Fig. 3, the

chloroform fraction of Z. jujuba extract (CHCl3-F) decreased cell

viability the most as compared with the other fractions and non-treated

control cells, in a dose-dependent manner (Fig. 4). Hence, this fraction

was used in subsequent experiments.

CHAPTER I

13

3.2. Cell cycle changes with CHCl3-F

We evaluated cell cycle dynamics with PI staining to examine

the effect of CHCl3-F induced decrease of the cell viability in HepG2

cells. As shown in Fig. 5, cells incubated with 100 µg/ml of CHCl3-F

resulted in an accumulation of G1 cell cycle region and a decrease in S

cell cycle region. However, the cell number of G2/M phase was

increased by the addition of 200 µg/ml of the CHCl3-F, suggesting that

the cell cycle was arrested at G2/M phase. Furthermore, CHCl3-F also

increased the cell number of Sub-G1 phase, suggesting that the

CHCl3-F also induced apoptosis in HepG2 cells.

3.3. Effect of CHCl3-F on intracellular ROS levels

In recent years many studies on apoptosis have been associated

with the excessive production of reactive oxygen species (ROS). We

examined the effect of CHCl3-F on intracellular peroxides levels by

the DCFH-DA method. As shown in Fig. 6, the intracellular ROS level

increased rapidly when cells were incubated with 100 µg/ml of

CHCl3-F at 15 mins, but not with 200 µg/ml of the CHCl3-F. These

CHAPTER I

14

results suggest that the cause of apoptosis may be associated with

increase of ROS under the 100 µg/ml treatment of CHCl3-F but not

under the 200 µg/ml treatment.

The effect of catalase on cell viability was examined by the

neutral red method. As shown in Fig. 7, cell viability decrease induced

by CHCl3-F (100 µg/ml) was improved with catalase.

3.4. Effect of CHCl3-F on mitochondrial membrane potential

The increase of intracellular ROS is well known to impair a

variety of intra- and extra-mitochondrial membrane transport system

such as distribution of mitochondrial ion-transport system and

decrease of mitochondrial membrane potential, which may contribute

to apoptosis. As shown in Fig. 8, the mitochondrial membrane

potential was declined by CHCl3-F (100-200 µg/ml) after 60 mins of

incubation.

The increase in ROS is also considered the cause of changes in

mitochondrial membrane potential. Most likely, catalase contributes to

the formation of water and molecular oxygen from hydrogen peroxide.

CHAPTER I

15

To examine whether the decline of the mitochondrial membrane

potential is associated with ROS, we used catalase to scavenge the

increased ROS. We then examined the relationship between the

decline of the mitochondrial membrane potential and the increase of

ROS. As shown in Fig. 9, the decline of mitochondrial membrane

potential induce by CHCl3-F was not recovered by the addition of

catalase. This result indirectly explains that the increase in ROS was

not the cause of a decline in mitochondrial membrane potential.

3.5. Effect of CHCl3-F on Rb protein

Cell cycle analysis showed a cell cycle arrest at G1 phase by addition

of 100 µg/ml of CHCl3-F and at the G2/M phase by the addition of

200 µg/ml of CHCl3-F. Rb protein is a tumor suppressor protein found

to be dysfunctional in a number of cancers. The normal function of Rb

is to prevent the cell from dividing or progressing through the cell

cycle. The product of the Rb gene prevents S-phase entry during the

cell cycle, and inactivation of this growth-suppressive function is

presumed to result from phosphorylated retinoblastoma protein (ppRb)

CHAPTER I

16

during the late G1 phase and S phase, with dephosphorylation during

the G2/M phase. Western blot analysis showed that treatment of

CHCl3-F increased Rb levels compared with control at 8 hrs (Fig. 10).

To determine whether the CHCl3-F induced increase in

intracellular peroxide was associated with Rb protein, we examined the

effect of catalase on Rb protein. As shown in Fig. 11, treatment of

CHCl3-F increased Rb levels, however, these effects were not reversed

by the addition of 200 units/ml catalase at 5 mins before the treatment

of 100 µg/ml of CHCl3-F. This result suggests that the increase in ROS

was not associated with Rb levels in CHCl3-F of Z. jujuba.

3.6. Effect of CHCl3-F on p27Kip1

The p27Kip1

is a member of the Cip/Kip family of

cyclin-dependent kinase inhibitor. The overexpression of p27Kip1

protein in mammalian cells induces G1 arrest of the cell cycle. As

shown in Fig. 12, the p27Kip1

levels increased when the cells were

treated with 100 µg/ml of CHCl3-F, but phosphorylation of p27Kip1

was

observed under the treatment of 200 µg/ml of CHCl3-F.

CHAPTER I

17

To determine whether the CHCl3-F of Z. jujuba extract-induced

increase in intracellular peroxide is associated with p27Kip1

protein, we

examined the effect of catalase on p27Kip1

protein. As shown in Fig. 13,

the increase of p27Kip1

induced by CHCl3-F could not be reversed when

catalase was added at 5 mins before the treatment 100 µg/ml of

CHCl3-F. This result suggests that the increase in ROS is not associated

with p27Kip1

levels in CHCl3-F of -treated cells.

CHAPTER I

18

*

**

**

**

0 5 10 20 400

20

40

60

80

100

120

Z. jujuba extract (mg/ml)

Cel

l via

bil

ity

(% o

f C

ont

rol)

Fig. 1. Effect of Z. jujuba extract on cell viability in HepG2 cells

Cells were cultured in Dulbecco's modified Eagle's medium containing 10%

FBS for 3 days, diluted and incubated again in flesh medium with or without

Z. jujuba extract. Cell viability was measured 24 hrs later by neutral red

method. Results are representative of three separate determinations. Each

bar is the mean (± S.D.) of three experiments. *p<0.05, **p<0.01

CHAPTER I

19

Z. jujuba extract

Dissolved in water

Extracted with chloroform

in vacuo

Extracted with ethyl acetate Chloroform fraction (CHCl3-F)

in vacuo

Extracted with butanol Ethyl acetae fraction (EtOAc-F)

in vacuo

Extracted with water Butanol fraction (BuOH-F)

in vacuo

Water fraction (Water-F)

Fig. 2. Method of fractionation of Z. jujuba extract

CHAPTER I

20

CHCl3-F EtOAc-F BuOH-F Water-F

**

0 100 200 100 200 100 200 100 2000

20

40

60

80

100

120

(µg/ml)

Cel

l vi

abil

ity

(% o

f C

ontr

ol)

*

**

Fig. 3. Effect of various fractions of Z. jujuba extract on cell viability in HepG2

cells

Cells were treated with or without various fractions of Z. jujuba extract. Cell

viability was measured 24 hrs later by neutral red method. Each bar is the

mean (± S.D.) of three experiments. *p<0.05, **p<0.01

CHAPTER I

21

0 25 50 100 2000

30

60

90

120

CHCl3-F (µg/ml)

Cel

l via

bil

ity

(% o

f C

ont

rol) *

**

**

**

Fig. 4. Effect of CHCl3-F of Z. jujuba extract on cell viability in HepG2 cells

Cells were treated with or without CHCl3-F. Cell viability was measured 24 hrs

later by neutral red method. Each bar is the mean (± S.D.) of three

experiments. *p<0.05, **p<0.01

CHAPTER I

22

Fig. 5. Effect of CHCl3-F on cell cycle in HepG2 cells

Cells were incubated with CHCl3-F of Z. jujuba extract for 24 hrs, washed

twice in PBS, and incubated with fleshly prepared PI-stained buffer for 1 hr at

37C in dark. The staining was analyzed by laser scanning cytometer. Results

are representative of three separate determinations. Each bar is the mean (±

S.D.) of three experiments. *p<0.05, **p<0.01

Sub-G1 G1 S G2/M0

20

40

60

80%

of

tota

l cel

l

Control

100 µg/ml

200 µg/ml

**

* *

*

* *

CHAPTER I

23

Fig. 6. Effect of CHCl3-F on intracellular ROS levels in HepG2 cells

The intracellular ROS levels were measured by the DCFH-DA method. DCF

fluorescence intensity of cells was measured at 15, 30 and 60 mins after

treatment with the 100 µg/ml or 200 µg/ml of CHCl3-F. Data are presented as

means ± S.D. *p<0.05, **p<0.01 compared to the control group at the same

time points.

CHAPTER I

24

0 catalase 100 100+catalase 200 200+catalase0

30

60

90

120

CHCl3-F (µg/ml)

Cel

l via

bili

ty (

% o

f C

ontr

ol)

a

b

a

c

d d

Fig. 7. Effect of catalase on cell viability in HepG2 cells

The cell viability was examined by the neutral red method. After 3-4 days

culture in Dulbecco's modified Eagle's medium supplemented with 10% FBS,

the cells were washed and cultured again in flesh medium with or without the

CHCl3-F. Catalase (200 units/ml) was added at 5 mins before incubation of

CHCl3-F and cell viability was measured at 24 hrs later. Each bar is the mean

(± S.D.) of three experiments. p<0.01

CHAPTER I

25

30 60 120 2400

0.4

0.8

1.2

1.6

Time (mins)

Rh

123

(nM

)

Control

100 µg/ml

200 µg/ml

** **

****

** **

Fig. 8. Effect of CHCl3-F on mitochondrial membrane potential in HepG2 cells

The mitochondrial membrane potential in the cells were measured by the

rhodamine 123 method at 30, 60, 120 and 240 mins after treatment with the

100 µg/ml or 200 µg/ml of CHCl3-F. Data are presented as means ± S.D.

**p<0.01 compared to the control group at the same time points.

CHAPTER I

26

Control catalase 100 100+catalase 200 200+catalase0

0.3

0.6

0.9

1.2

CHCl3-F (µg/ml)

Rh1

23 (

nM) ** ** ** **

Fig. 9. Effect of catalase on mitochondrial membrane potential in HepG2 cells

The mitochondrial membrane potential in the cells in the presence of catalase

were measured by the rhodamine 123 method at 2 hrs after treatment with the

100 µg/ml or 200 µg/ml of CHCl3-F. Catalase (200 units/ml) was added 5 mins

before CHCl3-F treatment. Data are presented as means ± S.D. **p<0.01

compared to the control group at the same time points.

CHAPTER I

27

ppRbRb

CHCl3-F

(µg/ml)

Time (hrs) 8

0

88

100 200

Fig. 10. Effect of CHCl3-F on Rb protein in HepG2 cells

HepG2 cells were treated with 100 µg/ml or 200 µg/ml CHCl3-F for 8 hrs. Cell

lysis and Western blotting were performed as described in Materials and

Methods. Data shown are representative of at least three independent

experiments.

CHAPTER I

28

Catalase

(200 units/ml)+

CHCl3-F

(µg/ml)

Time (hrs) 8

0

88

100 100

_ _

ppRb

Rb

Fig. 11. Effect of catalase on Rb protein in HepG2 cells

Catalase (200 units/ml) was added 5 mins before treatment of 8 hrs-incubated

HepG2 cells with 100 µg/ml CHCl3-F. Cell lysis and Western blotting were

performed as described in Materials and Methods. Data shown are

representative of at least three independent experiments.

CHAPTER I

29

CHCl3-F

(µg/ml)

Time (hrs) 8

0

88

100 200

p27

pp27

Fig. 12. Effect of CHCl3-F on p27Kip1

protein in HepG2 cells

HepG2 cells were treated with 100 µg/ml or 200 µg/ml CHCl3-F for 8 hrs. Cell

lysis and Western blotting were performed as described in Materials and

Methods. Data shown are representative of at least three independent

experiments.

CHAPTER I

30

Catalase

(200 units/ml)+

CHCl3-F

(µg/ml)

Time (hrs) 8

0

88

100 100

_ _

p27pp27

Fig. 13. Effect of catalase on p27Kip1

protein in HepG2 cells

Catalase (200 units/ml) was added 5 mins before treatment of HepG2 cells

with 100 µg/ml CHCl3-F. Cell lysis and Western blotting were performed as

described in Materials and Methods. Data shown are representative of at least

three independent experiments.

CHAPTER I

31

4. Discussion

In this chapter, we showed that the Z. jujuba extract has

cytotoxic activities in HepG2 cells. Furthermore, the chloroform

fraction of Z. jujuba extract (CHCl3-F) was the most effective

component. It was not only cytotoxic but it had cytostatic activity as

well. Also, different concentrations of CHCl3-F showed different

growth inhibition effects in HepG2 cells. The results obtained from cell

cycle analysis also showed that in HepG2 cells, there was a

concentration dependent effect of CHCl3-F induced apoptosis and arrest

of cell cycle in different phases.

These results differ from a previously reported study that the

EtOAc-F of Z. jujuba had cytotoxic activities in some tumor cell lines

22). This may be due to the fact that various fractions of Z. jujuba have

different effects in various tumor cell lines.

An imbalance between cell proliferation, apoptosis, and

differentiation leads to the development of malignant cells clones.

Based on the understanding of tumor biology in respect of the kinetics

of cell populations, two new strategies, induction of apoptosis and

CHAPTER I

32

anti-proliferation, have recently emerged in the fields of cancer

chemoprevention and chemotherapy by phytochemicals. Apoptosis is a

subtype of cell death that is involved in diverse physiological and

pathological processes. Many studies have shown that apoptosis is

closely associated with excessive production of reactive oxygen species

(ROS) 33, 34, 35)

. ROS include free radicals such as superoxide anion

(O2 ・

‾), hydroxyl radicals (・OH) and nonradical hydrogen peroxide

(H2O2). The excessive production of ROS may damage various

intracellular macromolecules, leading to oxidative stress accompanied

by loss of cell function and possibly apoptosis and/or necrosis

36, 37, 38).

Our results showed that at low concentration of CHCl3-F there is a

rapid elevation of the intracellular ROS level (Fig. 6). Furthermore, cell

viability was significantly improved with the addition of catalase (Fig.

7). These results suggest that the CHCl3-F of Z. jujuba extract induced

apoptosis in HepG2 cells was related to the rapid increase of

intracellular ROS, i.e., the increase in hydrogen peroxide when cells

were treated at low CHCl3-F concentrations. Furthermore, recent

studies have suggested that mitochondria play an important role in

CHAPTER I

33

apoptosis triggered by many stimuli such as oxidative stress and radial

ray 39, 40)

. The increase of ROS was probably due to the affected

mitochondria cycling dioxygen through the electron transport assembly,

and by generating ROS by one-electron-transfer mitochondria could be

a main target of nonspecific damage because of oxidative stress at the

level of the outer and inner membrane 41)

. As a consequence of

oxidative membrane damage, membrane potential and

permeability-barrier function is impaired which leads to apoptosis. On

the other hand, other studies suggested that the apoptosis of various cell

types, such as human leukemia HL-60 cells and human leukemia K562

cells 42, 43, 44)

, were induced by ROS-independent mitochondrial

dysfunction pathway. In this chapter, we also found the CHCl3-F

decreased mitochondrial membrane potential at both low and high

concentration in another pathway unrelated to ROS (Fig.8 and 9). Taken

together, these results suggest that the CHCl3-F-induced apoptosis was,

at low concentration, related to both a ROS-increase pathway and a

ROS-independent mitochondrial dysfunction pathway, but at the higher

concentration, apoptosis was only induced in ROS-independent

CHAPTER I

34

mitochondrial dysfunction pathway.

Another effect of CHCl3-F observed in this chapter in HepG2

cells was anti-proliferation. In general, anti-proliferation is usually

expressed in cell cycle arrest. The cell cycle transitions are controlled

by cyclin-dependent kinase (CDKs). The p27Kip1

is a member of

Cip/Kip family of cyclin-dependent kinase inhibitor (CDKI). It plays

a critical role in negative regulation of cell division in vivo. Its ability

to enforce G1 restriction point is derived from its inhibitory binding

to cyclin E-cdk2 and other cyclin E-cdk2 complexes 45, 46, 47)

. p27Kip1

binds to a wide variety of cyclin/CDK complexes including CDK2

and CDK4 46, 48, 49)

, inhibiting kinase activity 50, 51)

and blocking

cell cycle 52, 53, 54)

. The over expression of p27Kip1

protein in

mammalian cells induces G1 arrest in cell cycle. Furthermore, in the

case of progression from G1 to S, another apparent target is Rb

protein. Rb is bound to the transcription factor E2F during G1 but

upon phosphorylation, E2F is released and cell cycle progresses to S

phase 55)

. Many studies have reported that the accumulation of p27

Kip1

protein inhibits CDK2 activity followed by an increase

in

CHAPTER I

35

hypophosphorylated levels of Rb protein. In this chapter, low

concentration CHCl3-F of Z. jujuba extract induced the

hypophosphorylation of Rb protein and an accumulation of p27Kip1

protein when compared with the control at 8 hrs (Fig. 10 and 12).

These results were consistent with the G1 phase arrest in cell cycle

analysis under the treatment with low concentrations of CHCl3-F. On

the other hand, some studies have shown that phosphorylation of

p27Kip1

results in elimination of p27Kip1

from the cell, allowing cells

to transit from G1 to S phase. The Rb protein was phosphorylated

following cell cycle progress to S phase and finally dephosphorylated

to hypophosphorylated Rb at G2/M phase. In this chapter, the high

concentration of CHCl3-F decreased the p27Kip1

levels and generated

the phosphorylation of p27Kip1

in HepG2 cells (Fig. 12), and the

hypophosphorylation of Rb protein (Fig. 10) was remained. This

result suggests that the cell cycle progressed from G1 to S phase and

was finally arrested at G2/M phase. However, the precise mechanism

of the G2/M phase arrest at high concentration is still unclear.

Additional studies are needed to clarify this mechanism in HepG2

CHAPTER I

36

cells. Furthermore, other studies have reported that the increase of

ROS levels, such as H2O2, may trigger signal transduction

mechanisms that regulate cell growth, transformation, aging, and

apoptosis 56, 57)

. These data suggested that apoptosis and ROS

responses may be tied to p53-dependent regulation of cell cycle

control and stress-activated pathways 57)

. In contrast, our results

showed that the increase of ROS levels were not tied to the p27Kip1

protein levels and the hypophosphorylation Rb protein levels (Fig. 11

and 13).

On the components in Z. jujuba, Lee et al 22)

identified eleven

triterpenoic acids (colubrinic acid, alphitolic acid,

3-0-cis-p-coumaroylalphitolic acid, 3-O-trans-p-coumaroylalphitolic

acid, 3-O-cis-p-coumaroylmaslinic acid,

3-O-trans-p-coumaroylmaslinic acid, betulinic acid, oleanolic acid,

betulonic acid, oleanonic acid and zizyberenalic acid) using repeated

column chromatography of EtOAc-soluble fraction of the methanol

extract of Z. jujuba on silica gel followed by gel filtration on Sep-Pak

C18 cartridge and preparative HPLC. Furthermore, they showed that

CHAPTER I

37

the lupine-type triterpenes, such as 3-O-cis p- coumaroylalphitolic

acid, 3-O-trans-p-coumaroylalphitolic acid, betulinic acid and

betulonic acid, had high cytotoxic activities against K562, B16,

SK-MEL-2,LOX-IMVI and A549 tumor cell lines. The main and

effective component(s) of the CHCl3-F extract in this chapter is/are

not clear but currently under investigation.

An interesting finding in this chapter was the differential

effects induced in HepG2 cells by different concentrations of Z.

jujuba extract. Some studies have shown such phenomenon,

including Liu et al. (2006), who demonstrated that differences of cell

cycle arrest in human breast cancer cells were induced by different

drug concentrations. However, the reasons for these effects are still

not clear 58)

.

Our results showed that Z. jujuba extract induced apoptosis

and different cell cycle arrests in HepG2 cells, and therefore suggest

that Z. jujuba may play an effective contribution in the search for

anticancer treatment for hepatoma. However, the molecular basis of

such effects needs further investigation.

CHAPTER I

38

5. Summary

The extract of Z. jujuba decreased the viability of the cells.

Further extraction of the initial Z. jujuba extract with organic solvents

revealed that the chloroform fraction (CHCl3-F) was the most

effective. Interestingly, the CHCl3-F induced not only apoptosis but

also G1 arrest at a low concentration (100 μg/ml) and G2/M arrest at a

higher concentration (200 μg/ml) by cell cycle assay. Apoptosis, an

increase in intracellular ROS level, a decline of mitochondrial

membrane potential at low Z. jujuba concentrations, and a

ROS-independent mitochondrial dysfunction pathway at high

concentrations were all observed. CHCl3-F-induced G1 arrest in

HepG2 cells was associated with an increase in hypohosphorylation of

Rb and p27Kip1

, and a decrease of phosphorylated Rb. However,

CHCl3-F-induced G2/M arrest in HepG2 cells correlated with a

decrease of the p27Kip1

levels and generation of the phosphorylation of

p27Kip1

, however the hypohosphorylation of Rb protein remained.

Collectively, our findings suggest that the CHCl3-F extract of Z.

jujuba extract induced a concentration dependent effect on apoptosis

CHAPTER I

39

and a differential cell cycle arrest in HepG2 cells.

CHAPTER II

40

CHAPTER II

Green tea extracts enhances the selective cytotoxic activity

of Zizyphus jujuba extracts in HepG2 cells

1. Introduction

In Chapter I, we investigated the anticancer activity of the

chloroform fractions from Z. jujuba extract (CHCl3-F) and its

underlining mechanisms of action in HepG2 cells, and found that the

CHCl3-F decreased the viability of HepG2 cells. Interestingly, the

CHCl3-F induced not only apoptosis but also G1 arrest at a low

concentration (100 µg/ml) and G2/M arrest at a high concentration (200

µg/ml). We also showed that CHCl3-F-induced G1 arrest in HepG2 cells

was associated with an increase in hypophosphorylation of Rb and

p27Kip1

, an inhibitor of cyclin-dependent kinase, and a decrease in

phosphorylated Rb. However, CHCl3-F-induced G2/M arrest in HepG2

CHAPTER II

41

cells correlated with a decrease in the p27Kip1

levels and the

phosphorylation of p27Kip1

, but the hypophosphorylation of Rb protein

still remained. Our findings suggested that the CHCl3-F of Z. jujuba

might contribute to the antineoplastic activity of HepG2 cells, and

might become a new plant component that could prevent or be used to

treat cancer in the future.

Z. jujuba has various biological activities and is traditionally

used in oriental medicines. It is also used as jujuba tea which contains Z.

jujuba and green tea. Many people in China drink jujuba tea as opposed

tea alone and believe that the combination of Z. jujuba with green tea

(GTE) have synergistic effects that enhance immune function. GTE

alone has been shown to prevent or inhibit cancer growth in vitro and in

vivo 59, 60)

. However, the additive or synergistic effect of combining Z.

jujuba with the extracts of GTE on anticancer activity in vitro or in vivo

has not been reported.

In this Chapter, we hypothesized that the combination of Z.

CHAPTER II

42

jujuba with GTE might influence HepG2 cells, and their effects would

be apparent in the apoptotic and cell cycle inhibition pathway.

Therefore, we used low concentrations (100 µg/ml) of Z. jujuba extract

in combination with GTE, and investigated their interaction in HepG2

cells.

2. Materials and Methods

2.1. Materials

Z. jujuba extract and GTE were kindly donated by Sea Load Co.

Ltd. (Fukui, Japan) and Taiyo Kagaku (Japan), respectively. 2’, 7’-

Dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from

Sigma–Aldrich Fine Chemical (Tokyo, Japan). Rb, p21Waf1/Cip1

, p27Kip1

,

p53 and cyclin E antibodies were purchased from Santa Cruz

Biotechnology Inc. (Santa Cruz, CA, USA). FBS was purchased from

Equitech-Bio Inc. (Kerrville, Texas, USA). Other chemicals used in this

study were special grade commercial products.

CHAPTER II

43

2.2. Cell culture

HepG2 cells were cultured in Dulbecco’s modified Eagle’s

medium supplemented with 10% FBS in a humidified incubator

containing 5% CO2 in air at 37C for 3-4 days, then washed and

cultured again in fresh medium at a concentration of 2.5×105/ml in 35

mm plastic dishes overnight.

Rat hepatocytes were isolated from 10 week-old male Wistar

rats anesthetized with diethyl ether by collagenase perfusion. The

isolated hepatocytes were plated in 35 mm plastic dishes at a density

of 2.5×105 cells/ml in 2 ml Williams’ Medium E supplemented with

10% FBS, and were cultured in humidified atmosphere of 5% CO2 and

95% air at 37C overnight.

CHCl3-F and GTE were dissolved in dimethyl sulfoxide

(DMSO) and water respectively, then CHCl3-F and GTE were diluted

in the culture medium immediately before use (final DMSO

concentration < 0.125%). In all the experiments, control cultures

CHAPTER II

44

comprised the medium, DMSO and the cells.

2.3. Fractionation of Z. jujuba extract

Z. jujuba extract was diluted at 1/20 with water, and the

mixtures were extracted with chloroform. The fractions were

evaporated to dryness in vacuo. Finally, the powder was dissolved in

DMSO at a concentration of 200 µg/2.5 µl.

2.4. Cell viability

Cell viability was determined with the neutral red uptake assay,

based on the lysosomal uptake of neutral red 23)

. Following specified

incubations with test agents, neutral red solution (0.25 mg/ml) was

added to the cell cultures at a final concentration of 50 µg/ml. After

incubation at 37°C for 2 hrs, cells were rinsed twice with a mixture of

1% (v/v) formaldehyde, 1% (v/v) calcium chloride, and 98% (v/v)

distilled water. Subsequently, 1 ml of destaining buffer consisting of 1%

CHAPTER II

45

(v/v) acetic acid, 50% (v/v) ethanol, and 49% (v/v) distilled water was

added to the cells, and the culture dishes were stood for 30 mins.

Lysosomal uptake of neutral red was determined spectrophotometrically

at

540 nm. Viability was expressed as (A540-treated cells/A540

of

appropriate control) ×100% after correction for background absorbance

(100% cytotoxicity) 61)

.

2.5. cell cycle assay

Cell cycle distribution was analyzed by laser scanning cytometer

(Olympus LSC 101) using PI staining. Briefly, after designated

treatments, cells were washed twice in PBS and incubated with freshly

prepared PI-stained buffer (0.1% Triton X-100 in PBS, 20 µg/ml PI,

200 µg/ml RNase) for 1 hr at 37°C in the dark 27)

.

2.6. DNA synthesis assay

HepG2 cells were cultured in the presence of CHCl3-F or GTE

CHAPTER II

46

as well as bromodeoxyuridine (BrdU) (0.1 mM) for 24 hr. Cells were

washed twice with PBS and added to a mixture of 95% ethanol and 5%

acetic acid at 4°C for 30 mins, then washed with PBS. Subsequently, 1

ml of formamide was added to the cells, and the culture dishes were

cultured for 1 hr at 70°C in the dark, and then washed with PBS. After

washing with PBS 3 times for 2 mins, the culture dishes were incubated

with 1:50 dilute anti-BrdU antibody at room temperature for 1 hr, and

then washed with PBS for 2 mins, and incubated with 1:200 diluted

biotinylated mouse IgG for 1 hr at room temperature. The culture dishes

were washed with PBS once more for 2 mins and incubated with 1:400

dilute horse radish peroxidase-coupled streptavidin for 1 hr at room

temperature. After washing with PBS, the color reaction was developed

with 3,3'-Diaminobenzidine (DAB). The number of BrdU positive

nuclei was counted in three microscopic fields in each specimen.

2.7. ROS assay

CHAPTER II

47

DCFH-DA (8 μM) was added at the last 15 mins of CHCl3-F or

(and) GTE treatment. Cells were washed with PBS twice and

resuspended in Hanks solution. Fluorescence intensity was measured

using a micro-plate reading fluoroscan plate-reader (Wallac 1420

ArVOsx, Amersham Pharmacia Biotech) with the excitation

wavelength at 485 nm and the emission wavelength at 535 nm. The

amount of intracellular ROS was calculated from a standard curve

derived from 2’, 7’ –dichlorofluorescein (DCF). Protein concentration

was measured by the Bradford method 31)

.

2.8. Western blot analysis of Rb, p27Kip1

, p21Waf1/Cip1

, p53 and cyclin E

Cells were harvested after 8 hrs and were washed twice in PBS.

Then, the cells were dissolved for 30 mins with lysis buffer (150 mM

NaCl, 50 mM Tris (pH 7.2), 1 mM EDTA, 0.5% sodium deoxycholate,

1% Nonidet P-40, 1 mM sodium vanadate, 1 mM NaF, 20 µg/mL

aprotinin, 50 µg/ml leupeptin, 10 µg/ml Pepstatin A and 100 µg/ml

CHAPTER II

48

phenylmethylsulfonyl fluoride). Finally, the solution was centrifuged

at 2000 ×g for 20 mins at 4°C. The supernatant was collected and

protein concentrations were determined by Bradford method. Equal

amounts of protein were fractionated on 10% SDS-PAGE gels and

transferred to 0.45 µm PVDF

(Hybond; Amersham Pharmacia

Biotech). Efficiency of transfer and equal loading of protein were

confirmed by staining membranes with Coomassie Brilliant blue

(0.1%) in 5% acetic acid. After overnight blocking in 0.1% Tween-20

and 5% non-fat dry milk in PBS, the blots were incubated with the

respective antibodies for 1 hr at room temperature. The antibodies

included anti-Rb, anti- p21Waf1/Cip1

, anti- p27Kip1

, anti-p53 and

anti-cyclin E. After washing, the membrane was reincubated with

1:750 diluted biotinylated mouse IgG or rabbit IgG for 1 hr at room

temperature. The membrane was washed several times, and incubated

with 1:750 diluted horse radish peroxidase-coupled streptavidin for 1

hr at room temperature. After several washing steps, the color reaction

CHAPTER II

49

was developed with DAB. Densitometry analysis of the protein bands

was performed with the software Scion Image.

2.9. Statistical analysis

Data are represented as means ± S.D (standard deviation from

the mean). The significance of difference in assay values was

evaluated with ANOVA followed by Dunnett’s multiple comparison

post hoc tests. A p value of less than 0.05 was considered significant.

The observed interaction was confirmed by isobolographic analysis of

data derived from the neutral red uptake assay analysis at 24 h.

3. Results

3.1. Effect of GTE on cell viability in HepG2 cells

To determine the adequate concentration of GTE, we examined

the effect of GTE with different concentrations in HepG2 cells on cell

viability. As shown in Fig. 1, cell viability decreased with 60 µg/ml,

CHAPTER II

50

but not with 30 µg/ml of GTE. Therefore, we used 30 µg/ml of GTE

for subsequent experiments as this concentration of GTE that did not

affect cell viability.

3.2. Effect of CHCl3-F and GTE on cell viability in HepG2 cells

To investigate whether the combination of CHCl3-F with GTE

induces cytotoxicity in HepG2 cells, we examined the cell viability by

neutral red method. As shown in Fig. 2, cell viability was decreased in

the presence of CHCl3-F and GTE.

3.3. Effect of CHCl3-F and GTE on cell viability in rat hepatocytes

To investigate the effect of CHCl3-F and GTE in normal cell

lines, we examined the effect of CHCl3-F and GTE on cell viability in

rat hepatocytes. As shown in Fig. 3, no effect on cell viability was

observed in rat hepatocytes after treatment of CHCl3-F and/or GTE.

CHAPTER II

51

3.4. Effect of CHCl3-F and GTE on intracellular ROS levels

We have shown that the intracellular ROS level increased

rapidly when cells were incubated with 100 µg/ml of CHCl3-F, and that

the increase in ROS was associated with apoptosis. Therefore, we

examined the effects of CHCl3-F and GTE on intracellular ROS levels

to see whether ROS is related to the enhanced cytotoxicity with

CHCl3-F and GTE. There was no enhanced effect on ROS levels when

cells were treated with both CHCl3-F and GTE (Fig. 4).

3.5. Effect of CHCl3-F and GTE on cell cycle in HepG2 cells

We examined the effect of CHCl3-F and GTE on cell cycle in

HepG2 cells. As shown in Fig. 5, significant increase in G1, but not in

sub-G1 cell cycle region, was observed when cells were treated with

the combined CHCl3-F and GTE.

3.6. Effect of CHCl3-F and GTE on DNA synthesis in HepG2 cells

CHAPTER II

52

To confirm the G1 arrest by the combination of CHCl3-F and

GTE, we investigated the effect on the DNA synthesis with the

treatment of CHCl3-F and GTE in HepG2 cells. As shown in Fig. 6, an

enhanced effect on DNA synthesis was observed.

3.7. Effect of CHCl3-F and GTE on Rb protein in HepG2 cells

To investigate the mechanism of G1 arrest in CHCl3-F and

GTE-treated in HepG2 cells, we examined Rb protein, which acts as a

tumor suppressor by providing a cell cycle checkpoint between the G1

and S phase. As shown in Fig. 7, the treatment with CHCl3-F and GTE

decreased hypophosphorylation Rb protein levels and increased the

phosphorylation of Rb protein levels after 8 hrs of culture. This result

suggests that the enhanced inhibition of cell cycle arrest is caused by

the suppression of phosphorylation of Rb protein.

3.8. Effect of CHCl3-F and GTE on p27Kip1

in HepG2 cells

CHAPTER II

53

In an earlier study we showed that p27Kip1

protein increased in

cells incubated with 100 µg/ml of CHCl3-F. Hypophosphorylated Rb

was induced, followed by an arrest at the G1 cell cycle phase. Therefore,

we examined the effect of the combination of CHCl3-F and GTE on the

status of the phosphorylation of p27Kip1

. As shown in Fig. 8, the p27Kip1

levels decreased when the cells were treated with CHCl3-F and GTE for

8 hrs.

3.9. Effect of CHCl3-F and GTE on p21Waf1/Cip1

in HepG2 cells

To investigate the cause of cell cycle arrest by CHCl3-F and

GTE in HepG2 cells, we measured the p21Waf1/Cip1

protein, another

protein associated with the phosphorylation of Rb in G1 phase. As

shown in Fig. 9, the p21Waf1/Cip1

protein levels increased when the cells

were treated with CHCl3-F and GTE for 8 hrs.

3.10. Effect of CHCl3-F and GTE on p53 in HepG2 cells

CHAPTER II

54

To investigate the up-stream regulation of p21Waf1/Cip1

protein,

we measured p53 protein, a tumor suppressor protein associated with

the expression of p21Waf1/Cip1

. As shown in Fig. 10, p53 protein levels

increased after the cells were treated with CHCl3-F and GTE for 8 hrs.

3.11. Effect of CHCl3-F and GTE on cyclin E in HepG2 cells

Cyclin E is another checkpoint protein in G1 cell cycle phase

arrest. To investigate the changes in cyclin E, cells were treated with

CHCl3-F and GTE in HepG2 cells for 8 hrs. As shown in Fig. 11, the

cyclin E levels decreased with the treatment of GTE alone or the

combination of CHCl3-F and GTE.

CHAPTER II

55

0

20

40

60

80

100

120

GTE (µg/ml)

Cel

l via

bil

ity

(% o

f C

ont

rol)

0 10 20 30 40 50 60

*

Fig. 1. Effect of GTE on cell viability in HepG2 cells

Cells were cultured in Dulbecco's modified Eagle's medium with or without

various concentration of GTE. Cell viability was measured 24 hrs later by

neutral red method as described in Materials and Methods. Results are

representative of three separate determinations. Each bar is the mean (±

S.D.) of three experiments. *p<0.05.

CHAPTER II

56

0

20

40

60

80

100

120

Cel

l via

bil

ity

(% o

f C

ont

rol)

Control GTE

(30 µg/ml)100 100+GTE

CHCl3-F (µg/ml)

a a

b

c

Fig. 2. Effect of CHCl3-F and GTE on cell viability in HepG2 cells

Cells were cultured in Dulbecco's modified Eagle's medium with or without

CHCl3-F and GTE (30 µg/ml). Cell viability was measured 24 hrs later by

neutral red method. Results are representative of three separate

determinations. Each bar is the mean (± S.D.) of three experiments. Data not

sharing common alphabet are significantly different (p<0.01).

CHAPTER II

57

0

20

40

60

80

100

120

Cel

l via

bil

ity

(% o

f C

ont

rol)

Control GTE

(30 µg/ml)100 100+GTE

CHCl3-F (µg/ml)

Fig. 3. Effect of CHCl3-F and GTE on viability of rat hepatocytes

The isolated hepatocytes were cultured in Williams’ Medium E with or

without CHCl3-F and GTE (30 µg/ml). Cell viability was measured 24 hrs

later by neutral red method as described in Materials and Methods. Results

are representative of three separate determinations. Each bar is the mean (±

S.D.) from three experiments.

CHAPTER II

58

15 30 600

0.2

0.4

0.6

0.8

1

Time (mins)

DC

F f

luore

scen

ce (

nm

ol/µ

g pro

tein

)

Control

GTE

CHCl3-F

CHCl3-F+GTE

**

**

Fig. 4. Effect of CHCl3-F and GTE on intracellular ROS levels in HepG2 cells

The intracellular ROS levels were measured by the DCFH-DA method. DCF

fluorescence intensity of cells was measured at 15, 30 and 60 mins after

treatment with GTE, CHCl3-F, or CHCl3-F and GTE. Data are presented as

means ± S.D. **p<0.01 compared to the control group at the same time points.

CHAPTER II

59

sub-G1 G1 S G2/M0

20

40

60

80

% o

f to

tal

cel

l

Control

GTE

CHCl3-F

CHCl3-F+GTE

***

*

*

Fig. 5. Effect of CHCl3-F and GTE on cell cycle in HepG2 cells

Cells were incubated with GTE, CHCl3-F, or CHCl3-F and GTE for 24 hrs,

washed twice in PBS, and incubated with prepared PI-stained buffer for 1 hr

at 37C in dark. The stained cells were analyzed by laser scanning cytometer.

Results are representative of three separate determinations. Each bar is the

mean (± S.D.) of three experiments. *p<0.05, **p<0.01

CHAPTER II

60

Control GTE

(30 µg/ml)100 100+GTE

CHCl3-F (µg/ml)

0

5

10

15

20

25

30

35

D

NA

synth

esis

(% o

f B

rdU

posi

tive

cells

)

aa

c

b

Fig. 6. Effect of CHCl3-F and GTE on DNA synthesis in HepG2 cells

Cells were incubated with GTE, CHCl3-F, or CHCl3-F and GTE for 24 hrs.

DNA synthesis was measured by the percentage of BrdU positive cells.

Results are representative of three separate determinations. Each bar is the

mean (± S.D.) of three experiments. Data not sharing common alphabet are

significantly different (p<0.01).

CHAPTER II

61

ppRbRb

CHCl3-F

(µg/ml)0 0 100

GTE

(30 µg/ml)

100

+ +0 0

0

5

10

15

20

25

Rb

(arb

itrar

y un

its)

0

10

20

30

40

50

ppR

b (a

rbitr

ary

units

)

Control GTE

(30 µg/ml)100 100+GTE

CHCl3-F (µg/ml)

Fig. 7. Effect of CHCl3-F and GTE on Rb protein in HepG2 cells

Cells were incubated with GTE, CHCl3-F, or CHCl3-F and GTE for 8 hrs. Cell

lysis and Western blotting were performed as described in Materials and

Methods. Data shown are representative of at least three independent

experiments.

CHAPTER II

62

CHCl3-F

(µg/ml)0 0 100

GTE

(30 µg/ml)

100

+ +0 0

p27

0

5

10

15

20

p27

(ar

bitra

ry u

nit

s)

Control GTE

(30 µg/ml)100 100+GTE

CHCl3-F (µg/ml)

Fig. 8. Effect of CHCl3-F and GTE on p27Kip1

protein in HepG2 cells

Cells were incubated with GTE, CHCl3-F, or CHCl3-F and GTE for 8 hrs. Cell

lysis and Western blotting were performed as described in Materials and

Methods. Data shown are representative of at least three independent

experiments.

CHAPTER II

63

CHCl3-F

(µg/ml)0 0 100

GTE

(30 µg/ml)

100

+ +0 0

p21

0

5

10

15

20

25

30

35

p21

(ar

bitra

ry u

nit

s)

Control GTE

(30 µg/ml)100 100+GTE

CHCl3-F (µg/ml)

Fig. 9. Effect of CHCl3-F and GTE on p21Waf1/Cip1

protein in HepG2 cells

Cells were incubated with GTE, CHCl3-F, or CHCl3-F and GTE for 8 hrs. Cell

lysis and Western blotting were performed as described in Materials and

Methods. Data shown are representative of at least three independent

experiments.

CHAPTER II

64

CHCl3-F

(µg/ml)0 0 100

GTE

(30 µg/ml)

100

+ +0 0

p53

0

10

20

30

40

p53

(ar

bitra

ry u

nit

s)

Control GTE

(30 µg/ml)100 100+GTE

CHCl3-F (µg/ml)

Fig. 10. Effect of CHCl3-F and GTE on p53 protein in HepG2 cells

Cells were incubated with GTE, CHCl3-F, or CHCl3-F and GTE for 8 hrs. Cell

lysis and Western blotting were performed as described in Materials and

Methods. Data shown are representative of at least three independent

experiments.

CHAPTER II

65

CHCl3-F

(µg/ml)0 0 100

GTE

(30 µg/ml)

100

+ +0 0

Cyclin E

0

2

4

6

8

10

12

14

16

Cy

clin

E (

arb

itra

ry u

nit

s)

Control GTE

(30 µg/ml)100 100+GTE

CHCl3-F (µg/ml)

Fig. 11. Effect of CHCl3-F and GTE on cyclin E in HepG2 cells

Cells were incubated with GTE, CHCl3-F, or CHCl3-F and GTE for 8 hrs. Cell

lysis and Western blotting were performed as described in Materials and

Methods. Data shown are representative of at least three independent

experiments.

CHAPTER II

66

G1 arrest

Jujuba+GTE

p27

p21

p53

Rb

Jujuba

Cyclin E

CDK2

Cyclin E

CDK2

Cyclin E

: Direct stimulatory modification

: Direct inhibitory modification

Fig. 12. Proposed mechanism of action of CHCl3-F alone and CHCl3-F with

GTE on G1 arrest in HepG2 cells

CHAPTER II

67

4. Discussion

The results obtained in this chapter demonstrate for the first time,

to the best of our knowledge, that GTE enhances the cytotoxic activity

of CHCl3-F in HepG2 cells. The cytotoxic activity of CHCl3-F was

enhanced after combining with low concentrations of GTE which

initially had no affect on HepG2 cell viability. Furthermore, in rat

hepatocytes used as a normal cell line, there was no effect on cell

viability when the cells were treated with CHCl3-F, GTE or the

combination of CHCl3-F and GTE. Our findings show that combining

CHCl3-F and GTE can enhance their individual neoplastic toxicity,

providing a new approach to the design of chemotherapy strategies.

In chapter I, we have shown that the CHCl3-F of Z. jujuba

extract produced ROS resulting in apoptosis in HepG2 cells. Therefore,

we hypothesized that a possible synergistic decrease in cell viability,

by combining CHCl3-F of Z. jujuba extract with GTE, could be the

result of the production of ROS levels. However, we showed that

CHAPTER II

68

combining CHCl3-F and GTE did not affect ROS levels (Fig. 3), nor

increase apoptosis in HepG2 cells. We also have shown that CHCl3-F

induced not only apoptosis but also cell cycle arrest at the G1 phase in

HepG2 cells. The present results show an enhanced effect in G1 arrest

and a decrease in DNA synthesis when cells were treated with

CHCl3-F and GTE (Fig. 5 and 6), suggesting that the cytotoxic

activities with the combined CHCl3-F and GTE were related to growth

inhibition but not to apoptosis.

Uncontrolled cell proliferation is the hallmark of cancer, and

tumor cells have acquired damage to genes that directly regulate their

cell cycles. In the cell cycle, the period from late G1 to the S phase is

the most important for cell proliferation. The cell cycle progression

from G1 to S-phase requires phosphorylation of the retinoblastoma

tumor suppressor protein, Rb, a member of the pocket protein family

and one of the checkpoint of G1 arrest, by the cyclin D1-cdk4/6 and

cyclin E-cdk2 complexes 62)

. Phosphorylation of Rb in early G1 by

CHAPTER II

69

cyclin D1/cdk4/6 triggers a cascade of events that begins with the

dissociation of E2F from Rb and the activation of transcription of

cyclin E by E2F, and culminates with the stimulation by E2F of its

own transcription and assembly of cyclin E with its catalytic partner

Cdk2. The cyclin E-cdk2 complexes promote further phosphorylation

of Rb and the release of E2F, thus establishing a positive feedback

loop that accelerates the irreversible progression through late G1 63)

.

To prevent unrestrained proliferation, there are a number of

cyclin-dependent kinase inhibitors (CDKIs) which bind with specific

Cdks, preventing the Cdks from binding to cyclin and hence blocking

a cascade of events which ultimately lead to cell proliferation.

p21Waf1/Cip1

and p27Kip1

belong to the family of CDKIs 64)

, whose

expression regulates the G1 phase Cdks 63)

. We have shown that

CHCl3-F- induced G1 arrest in HepG2 cells was associated with an

increase in hypophosphorylation of Rb and a decrease of

phosphorylated Rb, and these changes depended on the up-regulation

CHAPTER II

70

of p27Kip1

protein. Therefore, we hypothesized that the effect of

combining CHCl3-F of Z. jujuba extract with GTE was also associated

with an up-regulation of p27Kip1

protein and induced

hypophosphorylation of Rb. However, our results showed that the

hypophosphorylation of Rb did not cause any up-regulation of p27Kip1

protein but up-regulation of p21Waf1/Cip1 protein instead in the presence

of CHCl3-F and GTE (Fig. 8 and 9).

The tumor suppressor protein p53 is also the major mediator of

the checkpoint-induced arrest in the G1 phase of the cell cycle 65)

. A

variety of cellular stresses including DNA damage, hypoxia,

nucleotide depletion, viral infection, and cytokine-activated signaling

pathways transiently stabilize the p53 protein, causing it to accumulate

in the nucleus, and activating it as a transcription factor 66)

. p53

induces cell cycle arrest, through the Cdk inhibitor p21Waf1/Cip1 21)

,

preventing the replication of damaged DNA. p21Waf1/Cip1

interacts with

different cyclin/Cdk complexes and other regulators of transcription

CHAPTER II

71

and signal transduction, exerting broad effects on cell survival, gene

expression and morphology 67)

. p21Waf1/Cip1

effects are partially

mediated by Rb, which is inactivated in proliferating cells through

phosphorylation by Cdk2 and Cdk4/6, both of which are inhibited by

p21Waf1/Cip1

. As a result, p21Waf1/Cip1

induction leads to Rb

dephosphorylation and activation, with ensuing G1 arrest 68)

. Our

results suggested that in the presence of the CHCl3-F and GTE, p53

protein levels were increased and induced the Cdk inhibitor

p21Waf1/Cip1

(Fig. 9 and 10), Rb dephosphorylation and activation.

Finally, as a result, there was a G1 phase cell cycle arrest (Fig. 8).

However, for CHCl3-F alone, only the p27Kip1

protein levels were

increased, but not p53 protein or the p21Waf1/Cip1

protein.

Cyclin E, whose catalytic partner is Cdk2, is another

rate-limiting regulator of the G1phase of the cell cycle, and increased

expression of cyclin E has been found in several types of tumors 69)

.

Some studies suggested that decreasing cyclin E level induced the cell

CHAPTER II

72

cycle arrest at G1 phase 70, 71)

. In this chapter, the treatment of CHCl3-F

alone did not decrease the expression of cyclin E levels. However,

treatment with GTE alone or combination of CHCl3-F and GTE

decreased the expression of cyclin E levels (Fig. 11), but the

concentration of 30 µg/ml of GTE had no effect on the p53 level and

the cell viability in HepG2 cells. These results suggested that both the

increase in p53 and p21Waf1/Cip1

levels and the decrease in cyclin E

levels were related to the effect of combination of CHCl3-F and GTE

on G1 arrest.

GTE contains a variety of polyphenols, the most important

class of polyphenols called catechins. The major catechins found in

GTE are called epicatechin (EC), epicatechin-3-gallate (ECG),

epigallocatechin (EGC), and epigallocatechin-3-gallate (EGCG). Of

these four catechins, EGCG is the most abundant and has been shown

to be the most important cancer inhibiting chemical found in GTE 60,

72). It has been reported that the combination of green tea polyphenols

CHAPTER II

73

and grape polyphenols had synergistic anticancer effects in HeLa cells,

and that the polyphenolic mixture was more effective than those from

the individual sources 73)

. Suganuma et al. reported that whole green

tea is a more reasonable mixture of tea polyphenols for cancer

prevention in humans than EGCG alone and that it is even more

effective when it is used in combination with other cancer preventives

74). We examined the effects of these four catechins alone or the

interaction between these four catechins in combination with Z. jujuba.

However, there was no effective interaction in HepG2 (data not

shown). Hence, we suggest that the active components in GTE are not

the individual catechins.

Collectively, we have found that GTE enhances the cytotoxic

effect of CHCl3-F in HepG2 cells causing cell growth inhibition, and

the involved mechanisms may be via two pathways. One pathway

involved increased p53 and p21Waf1/Cip1

proteins. The increased

p21Waf1/Cip1

bound with Cdk2 and prevented the Cdk2 from binding to

CHAPTER II

74

cyclinE, resulting in a G1 phase arrest. Also, decreasing the cyclin E

levels directly might have led to a decrease in the cyclin E-cdk2

complex levels and caused G1 arrest (Fig. 12). Based on these

findings, we suggest that the combination of CHCl3-F and GTE is

more effective than the CHCl3-F alone in the growth inhibition of

HepG2 cells and the mechanisms of G1 arrest by CHCl3-F and GTE

are different from that with the treatment of CHCl3-F alone.

CHAPTER II

75

5. Summary

GTE enhanced the effect of CHCl3-F on cell viability in HepG2

cells, without cytotoxicity in rat hepatocytes, which was used as a

normal cell model. Furthermore, combination of CHCl3-F and GTE

caused an effect on G1 phase arrest but not on apoptosis. Interestingly,

the mechanism of the G1 arrest was associated, not with an increase in

p27Kip1

levels and the hypophosphorylation of Rb, which are pathways

by CHCl3-F on G1 arrest in HepG2 cells, but with increases in p53 and

p21Waf1/Cip1

levels, and a decrease in cyclin E levels. Our findings

suggest that combining CHCl3-F and GTE produces an enhanced cell

growth inhibition effect, and that the resultant G1 arrest was caused via

a different mechanism as that of CHCl3-F treatment alone.

CHAPTER III

76

CHAPTER III

Combination of Zizyphus jujuba and green tea extracts

exert excellent cytotoxic activity in HepG2 cells via

reducing the expression of APRIL

1. Introduction

Cytokines regulate cellular proliferation and differentiation by

binding to their specific receptors on target cells. Tumor necrosis

factor (TNF) is the prototypic member of a family of cytokines

playing an important role in immune regulation and cancer. A

proliferation-inducing ligand (APRIL), a novel member of the TNF

family, is reported to stimulate tumor cell growth via signaling in an

autocrine and/or paracrine mode 75)

. The expressions of APRIL mRNA

and protein were high in various tumor cell lines and tissues, but

almost undetectable in various normal tissues 76)

. It is possible that

APRIL may play a role in tumorigenesis. Furthermore, it has been

demonstrated that the expression of APRIL protein was detected in

CHAPTER III

77

human hepatocellular carcinoma cell, where it can promote

neovascularization, as estimated by the human umbilical vein

endothelial cell tube formation 77)

. We have shown that combining

CHCl3-F and GTE caused an effect on G1 phase arrest but not on

apoptosis. Interestingly, the mechanism of the G1 arrest is associated,

not with an increase in p27Kip1

levels and the hypophosphorylation of

Rb, which are pathways of CHCl3-F on G1 arrest in HepG2 cells, but

with increases in p53 and p21Waf1/Cip1

levels, and a decrease in cyclin E

levels. However, how these factors are induced in HepG2 cells is still

unclear.

Knowledge of the molecular mechanisms governing malignant

transformation brings new opportunities for therapeutic intervention

against cancer using novel approaches. Therefore, in order to

demonstrate the particular molecular mechanisms of the growth

inhibitory effects of CHCl3-F and GTE mixture in HepG2 cells,

especially elucidate the relationship between APRIL and growth

related factors such as p27Kip1

, p53 and p21Waf1/Cip1

, we examined the

time-dependent expression of APRIL, p27Kip1

, p53 and p21Waf1/Cip1

by

CHAPTER III

78

combination CHCl3-F with GTE in HepG2 cells. The

chemotherapeutic potential of treatment of hepatocellular carcinoma

with combining CHCl3-F and GTE is discussed below.

2. Materials and Methods

2.1. Materials

APRIL antibody was purchased from ψProSci. Inc. (Poway,

CA, USA). p27Kip1

, p53 and p21Waf1/Cip1

antibodies were purchased

from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Other

chemicals used in this study were special grade commercial products.

2.2. Cell culture

HepG2 cells were cultured in Dulbecco’s modified Eagle’s

medium (DMEM) supplemented with 10% FBS in a humidified

incubator containing 5% CO2 in air at 37C for 3-4 days, then washed

and cultured again in fresh medium at a concentration of 2.5×105/ml in

35 mm plastic dishes overnight.

Rat hepatocytes were isolated from 10 week-old male Wistar

CHAPTER III

79

rats anesthetized with diethyl ether by collagenase perfusion. The

isolated hepatocytes were plated in 35 mm plastic dishes at a density

of 2.5×105 cells/ml in 2 ml Williams’ Medium E supplemented with

10% FBS, and were cultured in humidified atmosphere of 5% CO2 and

95% air at 37C overnight.

CHCl3-F and GTE were dissolved in DMSO and water

respectively. These were then diluted in the culture medium

immediately before use (final DMSO concentration < 0.125%). Final

concentration of CHCl3-F and GTE were 100 µg/ml and 30 µg/ml,

respectively. In all the experiments, control cultures comprised the

medium, DMSO and the cells.

2.3. Fractionation of Z. jujuba extract

Z. jujuba extract was diluted at 1/20 with water, and the

mixtures were extracted with chloroform. The fractions were

evaporated to dryness in vacuo. Finally, the powder was dissolved in

DMSO at a concentration of 200 µg/2.5 µl.

CHAPTER III

80

2.4. Western blot analysis of APRIL, p27Kip1

, p53 and p21Waf1/Cip1

Cells were harvested after 1, 2, 4 and 8 hrs and were washed

twice in PBS. Then, the cells were dissolved for 30 mins with lysis

buffer (150 mM NaCl, 50 mM Tris (pH 7.2), 1 mM EDTA, 0.5%

sodium deoxycholate, 1% Nonidet P-40, 1 mM sodium vanadate, 1

mM NaF, 20 µg/mL aprotinin, 50 µg/ml leupeptin, 10 µg/ml

Pepstatin

A and 100 µg/ml phenylmethylsulfonyl fluoride). Finally, the solution

was centrifuged at 2000 ×g for 20 mins at 4°C. The supernatant was

collected and protein concentrations were determined by Bradford

method. Equal amounts of protein were fractionated on 10%

SDS-PAGE gels and

transferred to 0.45 µm PVDF

(Hybond;

Amersham Pharmacia Biotech). Efficiency of transfer and equal

loading of protein were confirmed by staining membranes with

Coomassie Brilliant blue (0.1%) in 5% acetic acid. After overnight

blocking in 0.1% Tween-20 and 5% non-fat dry milk in PBS, the blots

were incubated with the respective antibodies for 1 hour at room

temperature. The antibodies included anti-APRIL, anti-p27Kip1

,

anti-p53 and anti- p21Waf1/Cip1

. After washing, the membrane was

CHAPTER III

81

reincubated with 1:750 diluted biotinylated mouse IgG or rabbit IgG

for 1 hour at room temperature. The membrane was washed several

times, and incubated with 1:750 diluted horse radish

peroxidase-coupled streptavidin for 1 hour at room temperature. After

several washing steps, the color reaction was developed with DAB.

Densitometry analysis of the protein bands was performed with the

software Scion Image (Scion Corporation).

2.5. Cell viability

Cell viability was determined with the neutral red uptake assay,

based on the lysosomal uptake of neutral red 23)

. Following specified

incubations with test agents, neutral red solution (0.25 mg/ml) was

added to the cell cultures at a final concentration of 50 µg/ml. After

incubation at 37°C for 2 hrs, cells were rinsed twice with a mixture of

1% (v/v) formaldehyde, 1% (v/v) calcium chloride, and 98% (v/v)

distilled water. Subsequently, 1 ml of destaining buffer consisting of 1%

(v/v) acetic acid, 50% (v/v) ethanol, and 49% (v/v) distilled water was

added to the cells, and the culture dishes were left for 30 mins.

CHAPTER III

82

Lysosomal uptake of neutral red was determined spectrophotometrically

at

540 nm. Viability was expressed as (A540-treated cells/A540

of

appropriate control) ×100% after correction for background absorbance

(100% cytotoxicity) 61)

.

2.6. Cell cycle assay

Cell cycle distribution was analyzed by laser scanning cytometer

(Olympus LSC 101) using PI staining. Briefly, after designated

treatments, cells were washed twice in PBS and incubated with freshly

prepared PI-stained buffer (0.1% Triton X-100 in PBS, 20 µg/ml PI,

200 µg/ml RNase) for 1 hour at 37°C in the dark.

2.7. Statistical analysis

Data are represented as means ± S.D (standard deviation from

the mean). The significance of difference in assay values was

evaluated with ANOVA followed by Dunnett’s multiple comparison

post hoc tests. A p value of less than 0.05 was considered significant.

CHAPTER III

83

3. Results

3.1. Effect of CHCl3-F and GTE on the expression of APRIL in HepG2

cells

To investigate whether the combination of CHCl3-F with GTE

induce APRIL in HepG2 cells, we examined the time-dependent

expression of APRIL. HepG2 cells were cultured with or without GTE,

CHCl3-F or both together, for 1, 2, 4 and 8 hrs and the expression levels

of APRIL were examined by Western blot analysis. As shown in Fig. 1,

APRIL was expressed in HepG2 cells after 4 hrs of the incubation, and

the addition of the mixture of CHCl3-F and GTE, but not the addition of

CHCl3-F alone, reduced the expression levels of APRIL from 4 hrs of

the incubation.

3.2. Effect of CHCl3-F and GTE on cell viability after incubation for

various times in HepG2 cells

APRIL was expressed in HepG2 cells after incubation for 4 hrs.

To investigate whether APRIL is involved in cell proliferation in

HepG2 cells, and whether the effect of anticancer activity of

CHAPTER III

84

combining CHCl3-F with GTE or CHCl3-F alone is involved in the

expression of APRIL, CHCl3-F or CHCl3-F with GTE were added to

HepG2 cell culture after 0, 1, 2, 4 and 8 hrs of the incubation, and cell

viability was examined after incubation for 24 hrs. As shown in Fig. 2,

CHCl3-F decreased cell viability at all the indicated times. However,

the addition of the CHCl3-F and GTE mixture did not decrease cell

viability after 8 hrs of the incubation. It shows that the addition of

CHCl3-F and GTE mixture to the APRIL expressed cells did not

decrease cell viability.

3.3. Effect of CHCl3-F and GTE on cell cycle after various hours of

incubation in HepG2 cells

To further investigate whether APRIL is involved in cell

proliferation in HepG2 cells, and whether the effect of G1 arrest by

CHCl3-F and GTE mixture or CHCl3-F alone is involved in the

expression of APRIL, we examined the changes in cell cycle dynamics.

GTE, CHCl3-F or CHCl3-F with GTE was added to HepG2 cell cultures

after 0 and 8 hrs of incubation. At the end of 24-hrs incubation, the cell

CHAPTER III

85

cycle distribution was analyzed. As shown in Fig. 3, the addition of

CHCl3-F alone increased the cell numbers in the G1 cell cycle region,

and the addition of CHCl3-F and GTE mixture caused a further increase.

However, G1 arrest was not induced in HepG2 cells incubated 8 hrs

before the addition of combined CHCl3-F and GTE.

3.4. Effect of CHCl3-F and GTE on p27Kip1

, p53 and p21Waf1/Cip1

in

HepG2 cells

We showed in a previous study that the mechanism of G1

arrest was associated with an increase in p27Kip1

levels at 8 hrs of

incubation with CHCl3-F alone, and with an increase in p53 and

p21Waf1/Cip1

levels at 8 hrs of incubation with combination CHCl3-F with

GTE. To investigate whether APRIL is associated with these factors

which control cell cycle progression in G1 phases, we examined the

time-dependent expression of p27Kip1

, p53 and p21Waf1/Cip1

by Western

blotting. As shown in Fig. 4, the p27Kip1

levels were increased only by

CHCl3-F alone after 2 hrs of the incubation. Only the CHCl3-F and

GTE mixture increased p53 levels significantly at 4 hrs of incubation,

CHAPTER III

86

and p21Waf1/Cip1

levels at 8 hrs of incubation.

3.5. Selective Inhibition of CHCl3-F and GTE Mixture

To demonstrate the selective inhibition of the CHCl3-F and

GTE mixture, we examined the effect of the mixture in rat hepatocytes,

as a normal cell model, and in HepG2 cells. As shown in Table 1, the

combination of CHCl3-F with GTE did not affect cell viability in rat

hepatocytes, but in HepG2 cells.

CHAPTER III

87

APRIL

CHCl3-F

GTE

Time (hrs)

0

0

+

+

0

0 +

+ 0

0

+

+

0

0 +

+ 0

0

+

+

0

0 +

+

1 2 4

AP

RIL

(arb

itra

ry u

nit

s)

0

0

+

+

0

0 +

+

8

0

5

10

15

20

Fig. 1. Effect of CHCl3-F and GTE on the expression of APRIL in HepG2 cells

Cells were incubated with or without GTE, CHCl3-F, or CHCl3-F and GTE

mixture for 0, 1, 2, 4 and 8 hrs. Cell lysis and Western blotting were performed

as described in Materials and Methods. Inner graphs represent the

densitometric values quantified by densitometer. Data shown are

representative of at least three independent experiments.

CHAPTER III

88

0 0 1 2 4 8 0 1 2 4 8 0 1 2 4 80

20

40

60

80

100

120

Cel

l via

bil

ity

(% o

f C

ont

rol)

**** ** **

**

** ** **

**

Time (hrs)

Control

GTE

CHCl3-F

CHCl3-F+GTE

Fig. 2. Effect of CHCl3-F and GTE on cell viability after incubation for

various times in HepG2 cells

GTE, CHCl3-F or CHCl3-F and GTE mixture were added to HepG2 cells

culture after 0, 1, 2, 4 and 8 hrs of the incubation, and cell viability examined

at the end of incubation for 24 hrs by neutral red method. Results are

representative of three separate determinations. Each bar is the mean (± S.D.)

of three experiments compared with control (**p<0.01).

CHAPTER III

89

sub-G1 G1 S G2/M0

20

40

60

80

% o

f to

tal

cell

Control

GTE (0 hr)

GTE (8 hrs)

CHCl3-F (0 hr)

CHCl3-F (8 hrs)

CHCl3-F+GTE (0 hr)

CHCl3-F+GTE (8 hrs)

a a ab

abc

a

a a ab ab

c

ab

Fig. 3. Effect of CHCl3-F and GTE on cell cycle after incubation for various

times in HepG2 cells

GTE, CHCl3-F or CHCl3-F and GTE mixture were added to HepG2 cells

cultured for 0 and 8 hrs. After 24 hrs of the incubation, the cells were collected

as described in Materials and Methods. Cell cycle distribution was analyzed

by laser scanning cytometer. Results are representative of three separate

determinations. Data not sharing common alphabet are significantly different

(p<0.05).

CHAPTER III

90

p27

p53

p21

p53 (

arbit

rary

unit

s)p

21 (

arb

itra

ry u

nit

s)

CHCl3-F

GTE

Time (hrs)

0

0

+

+

0

0 +

+ 0

0

+

+

0

0 +

+ 0

0

+

+

0

0 +

+

1 2 4 8

0

0

+

+

0

0 +

+

0

5

10

15

20

p27 (

arbit

rary

unit

s)

0

10

20

30

40

0

10

20

30

Fig. 4. Effect of CHCl3-F and GTE on p27Kip1

and p53 in HepG2 cells

The cells were treated with or without GTE, CHCl3-F or CHCl3-F and GTE

mixture for 1, 2, 4 and 8 hrs, and cell lysis and Western blotting were

performed as described in Materials and Methods. Inner graphs represent the

densitometic values quantified by densitometer. Data shown are representative

of at least three independent experiments.

CHAPTER III

91

Table 1. Selective inhibition of combined CHCl3-F and GTE on cell viability

Rat hepatocytes HepG2 cells

(% of control) (% of control)

Control 100.0±2.01 100.0±0.32

CHCl3-F+GTE 97.8±3.08 60.3±1.27**

Values are means ± S. D. (n = 4). **P < 0.01

CHCl3-F and GTE mixture were added to rat hepatocytes and HepG2 cells.

Cell viability was measured 24 hrs later by neutral red method as described

in Materials and Methods.

CHAPTER III

92

4. Discussion

We showed that the APRIL was expressed in HepG2 cells after

4 hrs of incubation in vitro. Furthermore, addition of a mixture of

CHCl3-F and GTE, but not CHCl3-F alone, reduced the expression of

APRIL in HepG2 cells (Fig. 1). Our results also showed that HepG2

cell viability was decreased in the presence of the mixture of CHCl3-F

and GTE or CHCl3-F alone. However, CHCl3-F and GTE mixture did

not decrease cell viability after 8 hrs of incubation (Fig. 2).

Furthermore, the CHCl3-F and GTE mixture or CHCl3-F alone caused

a G1 arrest, with CHCl3-F and GTE mixture having a stronger effect

than that of CHCl3-F alone. Addition of CHCl3-F and GTE mixture to

the 8 hrs incubation HepG2 cells did not induce an arrest of G1 phase

(Fig. 3).

A previous study showed that Blumea balsamifera, a medicinal

plant, decreased cell viability via reducing the expression of APRIL in

HepG2 cells 78)

. When CHCl3-F was added at 8 hrs, the decrease in

cell viability was maintained. Theses results suggest that APRIL may

be playing an important role in cell proliferation but there may be

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93

other factors also involved in cell proliferation in HepG2 cells.

APRIL is practically undetectable in normal tissues. However,

it is strongly up-regulated in many tumor cells in vivo and in vitro and

stimulates tumor cell growth 76)

. APRIL can be produced by tumor

cells and thus can act in an autocrine manner, or by accessory cells to

act in a paracrine manner. APRIL can induce both cell proliferation 75,

76, 79) and death

80) of malignant tumors. Some studies have shown that

the mechanism of cell death could be caused by activated NF-κB and

c-jun N-terminal kinase (JNK), or down-regulated Bcl-2 and Bcl-xL

and up-regulated Bax in lymphoid malignancies 81)

. APRIL was also

found to up-regulate c-Myc (an inducer of cell proliferation), to

down-regulate p53 and to increase Bcl-6, an inhibitor of B-cell

differentiation 82)

. Although APRIL is an important ligand of cell

proliferation in lymphoid malignancies, not many studies have shown

its mechanism in solid tumor cells, especially in HepG2 cells. Okano

et al. suggested that APRIL induced proliferation in hepatocellular

carcinoma, and functional expression of APRIL might contribute to

neovascularization via upregulation of Vascular endothelial growth

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94

factor (VEGF) in hepatocellular carcinoma 77)

. However, the

mechanism of inducing cell proliferation is still unclear. The

relationship between APRIL and downstream signaling of cell cycle

regulating factors have not been described thus far.

p21Waf1/Cip1

and p27Kip1

belong to the family of CDKIs

(cyclin-dependent kinase inhibitors) 64)

, whose expression regulates

the G1 phase Cdks (cyclin-dependent kinase) in the cell cycle 63)

.

Furthermore, the tumor suppressor protein p53 is also the major

mediator of the checkpoint-induced arrest in the G1 phase of the cell

cycle 65)

. p53 induces cell cycle arrest, through the Cdk inhibitor

p21Waf1/Cip1 21)

, preventing the replication of damaged DNA. p21Waf1/Cip1

interacts with different cyclin/Cdk complexes and other regulators of

transcription and signal transduction, exerting broad effects on cell

survival, gene expression and morphology 67)

. We have shown that in

HepG2 cells, the induction G1 phase arrest was via p27Kip1

pathway

with the addition of CHCl3-F alone, but via p53 and p21Waf1/Cip1

pathway with the addition of the CHCl3-F and GTE mixture. In this

chapter, we hypothesized that the combined CHCl3-F and GTE

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95

induces expression of APRIL and involves the regulation of these

three factors, and therefore the time-dependent expression of these

factors in HepG2 cells were examined. Our results showed that p27Kip1

was increased after 2 hrs of the incubation by CHCl3-F only,

suggesting that the effect of p27Kip1

by CHCl3-F appeared before the

expression of APRIL in HepG2 cells. However, p53 and p21Waf1/Cip1

were increased in the presence of the CHCl3-F and GTE mixture, and

this effect was expressed after 8 hrs of the incubation (Fig. 4). Theses

results suggest that the combination CHCl3-F with GTE-induced

APRIL is not associated with p27Kip1

protein, but most likely with p53

and p21Waf1/Cip1

proteins.

We have previously demonstrated that extracts of green tea 27)

,

evening primrose 29, 30)

, Cape aloe 83, 84)

, Zingiberaceae plant such as

Languas galanga and Alpinia galangal 85)

and Blumea balsamifera 78)

have anti-cancer activities in various tumor cell lines. We have also

demonstrated synergistic anti-cancer activity of natural substances in

various tumor cell lines 84, 86)

. In the present chapter, we have shown

the different mechanisms of anti-cancer activity with treatment with

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96

CHCl3-F alone and with a CHCl3-F and GTE mixture in HepG2 cells.

Our study suggests that these natural substances have different

anticancer activities via independent pathways in vitro, and these

could be new tools for cancer therapy. In addition, in order to

demonstrate whether the CHCl3-F and GTE mixture has selective

inhibitory effects in tumor cells, its effect on hepatocytes, as a normal

cell line, and in HepG2 cells were examined via cell viability

dynamics. Our results showed that the CHCl3-F and GTE mixture

exerted no cytotoxicity in rat hepatocytes, unlike in HepG2 cells.

CHAPTER III

97

5. Summary

Our results showed that APRIL was expressed in HepG2 cells

after 4 hrs of incubation in vitro and played an important role in cell

proliferation. Furthermore, the anticancer activity of combination

CHCl3-F with GTE was stronger than the CHCl3-F alone in HepG2

cells. The mechanism of the anti-cancer activity is via reduction of the

expression of APRIL, and involves an up-regulation of p53 and

p21Waf1/Cip1

proteins in HepG2 cells. We speculate from our results that

the CHCl3-F and GTE mixture might provide ways to new drug design

to treat hepatocellular carcinoma in the future.

CONCLUSION

98

CONCLUSION

Natural products have played an important role throughout the

world in treating and preventing human diseases. In this study, we

showed that the Z. jujuba, a famous fruit in China, have anticancer

activity in HepG2 cells. Furthermore, the chloroform fraction of Z.

jujuba extract (CHCl3-F) was the most effective component, and was

not only cytotoxic but had cytostatic activity as well. Also, different

concentrations of CHCl3-F showed different growth inhibition effects

in HepG2 cells. Apoptosis, an increase in intracellular ROS level, a

decline of mitochondrial membrane potential at low Z. jujuba

concentrations (100 µg/ml), and a ROS-independent mitochondrial

dysfunction pathway at high concentrations (200 µg/ml) were all

observed. The CHCl3-F induced not only apoptosis but also G1 arrest

at a low concentration and G2/M arrest at a high concentration by cell

cycle assay. CHCl3-F-induced G1 arrest in HepG2 cells was

associated with an increase in hypophosphorylation of Rb and p27Kip1

,

and a decrease of phosphorylated Rb. However, CHCl3-F-induced

CONCLUSION

99

G2/M arrest in HepG2 cells was correlated with the decrease of the

p27Kip1

levels and generation of the phosphorylation of p27Kip1

, but the

hypophosphorylation of Rb protein remained. Moreover, GTE

enhanced selective cytotoxic activity of CHCl3-F in HepG2 cells.

Combining CHCl3-F and GTE caused an effect on G1 phase arrest but

not on apoptosis. Interestingly, the mechanism of the G1 arrest was

associated, not with an increase in p27Kip1

levels and the

hypophosphorylation of Rb, which are pathways of CHCl3-F on G1

arrest in HepG2 cells, but with increases in p53 and p21Waf1/Cip1

levels,

and a decrease in cyclin E levels. We also found that APRIL was

expressed in HepG2 cells from 4 hrs of incubation in vitro and play an

important role in cell proliferation. The mechanism of the anti-cancer

activity of CHCl3-F and GTE mixture was via reducing the expression

of APRIL. Decreasing APRIL involved an up-regulation of p53 and

p21Waf1/Cip1

proteins in HepG2 cells.

Our study is the first to demonstrate that CHCl3-F possesses

anticancer activity in HepG2 cells. Furthermore, the anticancer

activity of CHCl3-F and GTE mixture was stronger than the CHCl3-F

CONCLUSION

100

alone in HepG2 cells. We speculate from our results that the CHCl3-F

alone and the CHCl3-F and GTE mixture might provide a lead to new

drug design to treat hepatocellular carcinoma in the future.

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ACKNOWLEDGMENT

118

ACKNOWLEDGMENT

I am profoundly indebted to my academic adviser, Professor Isao

Matsui-Yuasa, Osaka City University, for his directions, helpful suggestions,

academic and fatherly support throughout this thesis. His invaluable

scientific insights and gentle attitude have been the pivot around which this

exiting work was done.

I am grateful to Professor Yukiko Yamamoto and Professor Yotaro

Konishi, Osaka City University, for their review of this manuscript and

great comments.

I am grateful to Associate Professor Akiko Kojima-Yuasa, Osaka

City University, for the various experimental techniques she taught me, and

her advice and motherly support throughout my course.

I express appreciation to Soroptimist International of the Americas

Japan Chuo Region and the Nisimura International Scholarship Foundation,

gave me one year’s fellowship, respectively. I appreciate Mrs. Emiko

Tsujita (辻田 栄美子) and Mrs. Takeko Akamatsu (赤松武子) of

Soroptimist International Osaka-Chuo and Mr. Toshio Matsuura (松浦 敏

夫 ) of Nisimura International Scholarship Foundation, for their

ACKNOWLEDGMENT

119

encouragement and various supports.

I am indebted to Sea Load Co. Ltd. (Fukui, Japan), for providing

me Z. jujuba extract.

I appreciate Dr. David Opare Kennedy, for his suggestion and

proofreading of my paper.

I appreciate Dr. Toshio Norikura, for his discussion and suggestion,

which have made a stimulating environment for working during my

research. I also appreciate many members in this laboratory and all the

friends in Japan, for their kind help and friendships.

Great appreciation and respect to my parents and my foster-mother,

for their emotional support in far away China. I especially appreciate my

brother, for giving me a stable environment and supporting me all the time

in Japan.

Finally, I should mention my son, Xingan, who always stands by

me, gives me the power and inspires me to go on…

ABBREVIATION

120

ABBREVIATION

APRIL

ANOVA

BrdU

BuOH-F

CDKs

CHCl3-F

CDKI

DAB

DCF

DCFH-DA

DMSO

EDTA

EtOAc-F

EC

ECG

EGC

bromodeoxyuridine

a proliferation-inducing ligand

analysis of variance

buthanol fraction of Z. jujuba extract

chloroform fraction of Z. jujuba extract

3,3'-diaminobenzidine

2’, 7’ –dichlorofluorescein

2’, 7’- dichlorodihydrofluorescein diacetate

cyclin-dependent kinase

cyclin-dependent kinase inhibitors

dimethylsulfoxide

ethylenediaminetetraacetic acid

ethyl acetae fraction of Z. jujuba extract

epicatechin

epicatechin-3-gallate

epigallocatechin

ABBREVIATION

121

EGCG

FBS

GTE

IgG

JNK

PBS

PI

PMSF

ppRb

PVDF

Rb

ROS

RNase

SDS

Tris

VEGF

Water-F

Z. jujuba

fetal bovine serum

epigallocatechin-3-gallate

immunoglobulin G

c-jun N-terminal kinase

phosphate buffered saline

propidium iodide

phenylmethylsulfonyl fluoride

phosphorylated retinoblastoma protein

polyvinylidene difluoride

retinoblastoma protein

reactive oxygen species

ribonuclease

sodium dodecyl sulfate

green tea extracts

2-amino-2-hydroxymethyl-1,3-propanediol

vascular endothelial growth factor

water fraction of Z. jujuba extract

Zizyphus jujuba Mill

LIST OF PUBLICATIONS RELATED TO THIS THESIS

122

LIST OF PUBLICATIONS RELATED TO THIS THESIS

Mechanism of the anticancer activity of Zizyphus jujuba in HepG2 cells

Xuedan Huang, Akiko Kojima-Yuasa, Toshio Norikura, David Opare

Kennedy, Tadayoshi Hasuma and Isao Matsui-Yuasa

The American Journal of Chinese Medicine 35: 3, 517-532 2007

Green tea extract enhances the selective cytotoxic activity of Zizyphus

jujuba extracts in HepG2 cells

Xuedan Huang, Akiko Kojima-Yuasa, Shenghui Xu, Toshio Norikura,

David Opare Kennedy, Tadayoshi Hasuma and Isao Matsui-Yuasa

The American Journal of Chinese Medicine 36: 4, 729-744 2008

Combination of Zizyphus jujuba and green tea extracts exert excellent

cytotoxic activity in HepG2 cells via reducing the expression of APRIL

Xuedan Huang, Akiko Kojima-Yuasa, Shenghui Xu, David Opare

Kennedy, Tadayoshi Hasuma and Isao Matsui-Yuasa

The American Journal of Chinese Medicine 37:1, 169-179 2009