The Effects of Litsea japonica on the Induction of Apoptosis in HL-60/ADR

40(1) : 65 69 (2009)

65

까마귀쪽나무(Litsea japonica)의 HL-60/ADR 세포 Apoptosis 유도효과

김엘비라·부혜진·현재희·김상철·강정일·김민경·유은숙·강희경

*

제주대학교의학전문대학원약리학교실

,

^{의과학연구소}

The Effects of Litsea japonica on the Induction of Apoptosis in HL-60/ADR Elvira Kim, Hye-Jin Boo, Jae-Hee Hyun, Sang-Cheol Kim, Jung-Il Kang, Min-Kyoung Kim,

Eun-Sook Yoo and Hee-Kyoung Kang

^*

Department of Pharmacology, School of Medicine, Institute of Medical Sciences, Jeju National University, 66 Jejudaehakno, Jeju 690-756, South Korea.

Abstract −

The present study investigated the antiproliferative effect of Litsea japonica in HL-60/ADR, adriamycin resistant human promyelocytic leukemia cells. The 80 % ethanol extract of L. japonica markedly inhibited the growth of HL-60/ADR cells. When HL-60/ADR cells were treated with the extract, several apoptosis events like as DNA fragmentation, chromatin condensation and the increase of the population of sub-G1 hypodiploid cells were observed. In the mechanism of apoptosis induction by L. japonica , we examined the changes of Bcl-2 and Bax protein expression levels, and activation of caspases. After the HL-60/ADR cells were treated with the extract, the Bcl-2 expression was decreased, whereas the expression of Bax was increased in a time-dependent manner compared to the control. In addition, the active forms of caspase-9 and -3 were increased and the cleavage of poly (ADP-ribose) polymerase, a vital substrate of effector caspase, was observed. The results suggest that the inhibitory effect of L. japonica on the growth of the HL-60/ADR appears to arise from the induction of apoptosis via the down-regulation of Bcl-2 and the activation of caspases.

Key words −

Litsea japonica , HL-60/ADR, Apoptosis, Bcl-2, Caspases

항암물질에대한개발및그기전에대한연구가활발해 지면서

,

^{효과적인암치료가이루어지고있지만새롭게대두}

되고있는문제점은암자체의다양성및발병기전의다양 화로인해생기는부작용과항암제에대한암세포의내성

(resistance)

^이다

.

^{항암제에대한내성은장기적인항암제사}

용으로약물에노출된세포들의세포내축적을감소시키거 나

,

^{1,2)해독작용또는배출을활성화하거나}

,

^{3,4)표적이되는}

단백질을변형⁵⁾시키는등의여러가지기작을통하여일 어나게된다

.

까마귀쪽나무

( Litsea japonica (Thunb.) Jussieu)

^는상록엽

소교목으로서수직적으로표고

700 m

^{이내의바닷가및산}

기슭에분포한다

.

^{이식물의화학적구성에대한기존의연}

구에서

,

^{여러 종류의}

essential oils, fatty acids, lactones, alkaloids

^및

terpenoids

^{가발견되었다고보고된바가있다}

.

^6,7)

Lactones

^{의 종류로 알려진}

hamabiwalactone A, hamabiwa-

latone B, akolactone B, litsealactone A

^및

litsealactone B

^가

분리되었다

.

^8)또한

,

^{잎으로부터분리된}

flavonoids

^성분인

epicatechin, afzelin, quercitrin

^및

tiliroside

^의

anti-complement activity

^{를조사한결과}

, tiliroside

^가

complement system

^에대

해가장강력한억제작용을나타냄이보고되었다

.

^9)한국에

서까마귀쪽나무는식용으로사용하였다는보고가있으나

,

어떤용도로사용하였는지에대한연구보고는미미한실 정이므로이식물에대한항암효능을비롯한생리활성을탐 색하는연구가필요하다

.

본연구에서는까마귀쪽나무

80%

^{에탄올추출물을사용}

하여 항암제인

adriamycin

^{에내성을 가지고있는}

HL-60/

ADR (adriamycin resistant human promyelocytic leukemia)

세포의 증식억제효과를조사하였다

.

^{또한까마귀쪽나무}

80%

^{에탄올추출물의}

HL-60/ADR

^{세포에대한세포증식억}

제효과가

apoptosis

^{유도에의한것인지알아보기위하여}

,

핵의형태학적변화

, DNA

^절편화및

DNA content

^변화를

알아보았으며

, apoptosis

^{유도신호전달기전에중요한}

Bcl- 2

^와

Bax

^{의증가와이에따른}

caspase-9, caspase-3 protease

*교신저자(E-mail):[email protected] (Tel):064-754-3846

활성화를조사하였다

.

^{이와같은연구로}

HL-60/ADR

^세포

와같이항암제에대해내성을가지고있는내성암세포주에 대한까마귀쪽나무추출물의항암효과를밝혀

,

^{효과적인항}

암물질개발의재료로사용가능한근거를제시하고자하였다

. 실험 방법

까마귀쪽나무, 환삼덩굴, 호랑가시나무 및 유칼립투스의 추출물 제조 − 제주도의해안가에자생하는까마귀쪽나무

( L. japonica )

^의잎을

2005

^년

11

^{월경에채집하여시료}

185g

을음지에서건조시킨후

,

^{마쇄기로분쇄하여각각의미세}

말시료를

80%

^{에탄올에침적하고초음파를이용하여}

1

^시

간씩

3

^{회추출하였으며}

,

^{그후상층액을회수하여감압농}

축하여얻은

(43 g)

^{에탄올추출물을}

2007

^{년제주생물종다}

양성연구소로부터분양받았다

.

^{본실험에서는이추출물}

을

PBS (phosphate-buffered saline)/

^에탄올

(1:1)

^{에용해시킨}

후원하는농도에따라실험용배지로희석하여사용하였 다

.

^{그리고환삼덩굴}

( Humulus japonicas Siebold & Zucc.) 80%

^{메탄올추출물}

,

^{호랑가시나무}

( Ilex cornuta Lundi. &

Paxton) 80%

^{메탄올 추출물 및 유칼립투스}

( Eucalyptus globulus Labill.) 100%

^{메탄올추출물또한제주생물종다}

양성연구소로부터분양받아사용하였으며

,

^{분양받은각}

각의 추출물들을

PBS (phosphate-buffered saline)/

^메탄올

(1:1)

^{에용해시킨후원하는농도에따라실험용배지로희}

석하여사용하였다

.

세포 배양 −

HL-60/ADR (adriamycin resistant human promyelocytic leukemia)

^{세포는내성세포연구센터}

(Research Center for Resistant Cell)

^{로부터분양받아서사용하였다}

. 100 units/ml

^의

penicillin-streptomycin (GIBCO Inc, Grand Island, NY, USA)

^과

10% heat-inactivated fetal bovine serum (FBS; GIBCO Inc, Grand Island, NY, USA)

^이첨가

된

RPMI 1640

^{배지를사용하여}

37

^o

C, 5% CO

2

, humidified incubator

^{에서 배양하였으며}

,

^{계대배양은 약}

70~80%

^의

confluence

^가되는

3~4

^{일마다한번씩시행하였다}

.

세포 독성 측정 − 여러식물추출물에의한

HL-60/ADR

세포의성장증식에대한효과를

MTT [3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide] assay

^{를이용하여검}

색하였다

.

^{10)살아있는암세포}

mitochondria

^{의탈수소효소}

작용에의하여수용성의노란색

MTT

^{가환원되어형성되는}

자주색을띠는비수용성의

formazan

^을

microplate (ELISA) reader

^로

540 nm

^{에서흡광도를측정하여}

,

^{대사적으로왕성하}

게생존하는세포를조사하였다

. HL-60/ADR (2.5×10

⁵

cells/

ml)

^세포를

96 well plate

^{에 넣고 여러 식물 추출물을}

100

^µ

g/ml

^{의농도로처리하였다}

.

^이를

4

^{일간배양한후}

, MTT (Sigma chemical Co., St. Louis, USA) 50

^µ

l (2 mg/ml)

^를

첨가하고

4

^{시간동안반응시킨후}

, 1000 rpm

^에서

10

^분간

원심분리 한 후 상층액을 제거하였다

.

^{그 후}

dimethyl- sulfoxide (DMSO: Sigma chemical Co., St. Louis, USA) 150

^µ

l

^{를가하여침전물을용해시킨다음}

, microplate reader (Amersham Pharmacia Biotech, NY, USA)

^{를 사용하여}

540 nm

^{에서흡광도를측정하였다}

.

^{각시료군에대한평균}

흡광도값을구하여

,

^{대조군의흡광도값과비교하여세포}

증식억제정도를조사하였다

.

세포주기변화 측정 − 까마귀쪽나무

80%

^{에탄올추출물을}

HL-60/ADR (2.5×10

⁵

cells/ml)

^{세포에 농도별}

(25, 50

^및

100

^µ

g/ml)

^{로처리한다음}

, 48

^{시간동안배양한후}

,

^세포를

수확하여

PBS

^{로세척하였다}

.

^그후

70%

^에탄올로

-20

^o

C

^에

서

30

^{분동안 고정시키고}

, PBS

^{로 세척한다음}

RNase A (1 mg/ml)

^{를처리한후}

propidium iodide(PI: Sigma chemical Co., St. Louis, USA)

^{로 염색하고}

, COULTER

^®

EPICS

^®

XL

^TM

Flow Cytometer(Coulter, Miami, FL, USA)

^를사용하

여세포주기를분석하였다

.

핵의 형태학적 변화 관찰 − 까마귀쪽나무

80%

^에탄올추

출물을

HL-60/ADR (2.5×10

⁵

cells/ml)

^{세포에농도별}

(25, 50

^및

100

^µ

g/ml)

^{로처리한다음}

, 48

^{시간동안배양하였다}

.

그후

DNA

^{에특이적으로결합하는형광색소인}

Hoechst 33342 (H33342: Sigma chemical Co., St. Louis, USA)

^용

액

(1

^µ

g/ml)

^을가하여

37

^o

C

^에서

10

^{분간염색한후형광현}

미경

(IX-71, Olympus, Japan)

^{하에서관찰하였다}

.

¹¹⁾

Western Blot Analysis − 까마귀쪽나무

80%

^에탄올추

출물을

100

^µ

g/ml

^{의농도로처리한다음}

3, 6, 9

^및

12

^시간

동안배양하고

,

^{세포를수확한후}

PBS (phosphate-buffered saline)

^로

2~3

^{회세척하였다}

.

^그후

500

^µ

l

^의

lysis buffer

^를

첨가한다음

, 1

^시간동안

lysis

^시킨후

12,000 rpm

^에서

15

분간원심분리하여상층액을얻었다

.

^{단백질의농도는}

BSA (bovine serum albumin)

^{를 표준물질로 사용하여}

Protein Assay Kit (BIO-RAD, HC, USA)

^{로 정량하였다}

.

^{12) 그 후}

20~30

^µ

g

^의

lysate

^를

12% mini gel SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis)

^{로 변성}

분리하여

polyvinylidene difluoride membrane (BIO-RAD, HC, USA)

^에

200 mA

^로

2

^시간동안

transfer

^하였다

.

^그리고

5% skim milk

^가함유된

TTBS (TBS+0.1% Tween 20)

^용

액에서

membrane

^의

blocking

^을

overnight

^{실시하였다}

. Bcl- 2

^{의 발현량을 검토하기 위하여}

mouse monoclonal anti- human Bcl-2 Ab (1:1000) (Santa Cruz Biotech, MA, USA)

^항체를

, Bax

^{의 발현량을 검토하기 위하여}

rabbit polyclonal anti-human Bax Ab (1:1000) (Santa Cruz Biotech, MA, USA)

^항체를

, PARP

^{의발현량을검토하기위}

하여

rabbit polyclonal anti-human PARP Ab (1:1000)

(Santa Cruz Biotech, MA, USA)

^{항체를사용하였다}

.

^또한

caspase-3

^{의발현량을검토하기위하여}

rabbit polyclonal anti-

human caspase-3 Ab (1:1000) (Cell Signaling Technology,

Beverly, MA, USA)

^항체를

, caspase-9

^{의발현량을검토하기}

위하여

rabbit polyclonal anti-human caspase-9 Ab (1:1000) (Cell Signaling Technology, Beverly, MA, USA)

항체를사용하였다

.

^{각각의항체들은모두}

TTBS

^용액에희

석하여사용하였으며

,

^상온에서

2

^{시간동안반응을진행하}

였다

. 2

^{차항체로는}

HPR (Horse Radish Peroxidase)

^이결합

된

anti-rabbit IgG (American Prarmacia Biotech, NY, USA)

^와

anti-mouse IgG (American Pharmacia Biotech, NY, USA)

^를

TTBS

^{용액에희석}

(1:5000)

^{하여사용하였으며}

,

상온에서

30

^{분동안 반응을진행하였다}

.

^그후

TTBS

^로

membrane

^을

3

^{회세척한후}

, ECL

^기질

(American Pharmacia Biotech, NY, USA)

^과

1~3

^{분간반응시킨다음}

X-ray

^필름

에감광하였다

.

통계처리 − 표시된결과들은

3

^{번이상의독립적인실험}

결과들이며

,

^{각각의데이터를}

mean±standard error

^값으로

나타내었다

. Student’s t-test

^{를사용하여실험군사이의통계}

적유의성검증을평가하였다

. 결과 및 고찰

세포 증식 저해 효과

항암제인

adriamycin

^{에내성을가지고있는암세포인}

HL-

60/ADR

^{세포의증식에대한여러식물추출물의효과를측}

정하기위하여

MTT assay

^{를이용하였다}

. HL-60/ADR

^세포

주에까마귀쪽나무

80%

^{에탄올추출물외}

3

^{종의추출물을}

처리하여세포증식저해효과를조사하였다

(Fig. 1).

^각각

의추출물을

100

^㎍

/

^{㎖ 의 농도로처리하였을때}

, HL-60/

ADR

^{의세포증식저해효과는까마귀쪽나무}

( L. japonica )

80%

^{에탄올 추출물의 경우는}

64.5%,

^환삼덩굴

( H.

japonicus ) 80%

^{메탄올추출물의경우는}

17.3%,

^호랑가시

나무

( I. cornuta ) 80%

^{메탄올추출물의경우는}

37.7%,

^유

칼립투스

( E. globulus ) 100%

^{메탄올추출물의경우는}

6.6%

의저해효과를보였다

.

^{이중저해효과가가장높은까마귀}

Fig. 1.

Inhibitory effect of several extracts of plants on the growth of HL-60/ADR cells.

HL-60/ADR cells (2.5×10

⁵

/ml) were treated for 4 day with 100

µ

g/ml of the extracts in 96-microwell plates. After incubation, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] was added and incubation was continued for further 4 hours. The formazan salt formed was dissolved in dimethyl- sulfoxide, and quantified using a microplate reader at 540 nm.

Data were presented as a mean±SD from three separate experiments. *p<0.05, **p<0.01 compared with the control.

Concentration Cell cycle arrest (%)

(

µ

g/ml) Sub-G1(M1) G0/G1 (M2) S (M3) G2/M (M4)

A. control 18.68 41.94 23.95 15.96

B. 25 19.31 45.60 22.28 13.27

C. 50 22.00 41.88 22.65 13.90

D 100 31.28 36.04 21.27 11.96

Fig. 2.

The degree of apoptosis represented as the DNA content measured by flow cytometric analysis.

The HL-60/ADR cells (2.5×10

⁵

/ml) were treated with various concentrations (25, 50 and 100

µ

g/ml) of 80% EtOH extract from L.

japonica for 48 hours.

쪽나무

80%

^{에탄올추출물은정상세포인}

HEL-299

^에서현

저히낮은세포증식억제효과를나타냄을이미보고하였 다

.

^{13)이는정상세포에대하여까마귀쪽나무}

80%

^에탄올추

출물이독성을갖지않는것으로판단할수있다

.

Adriamycin

^{에내성을갖는}

HL-60/ADR

^{의성장저해효}

과가탁월한까마귀쪽나무

80%

^{에탄올 추출물의작용이}

HL-60/ADR

^의

apoptosis

^{유도에의한것인지를조사하였다}

.

Apoptosis 유도효과

세포주기변화 측정 −

HL-60/ADR

^{세포에서까마귀쪽나무}

의

80%

^{에탄올추출물이}

apoptosis

^{를유도하는지를알아보}

기위하여세포주기를조사하였다

. HL-60/ADR

^세포에까

마귀쪽나무의

80%

^{에탄올추출물을}

25, 50

^및

100

^µ

g/ml

^의

농도로

48

^{시간 처리한후}

, apoptosis

^{를 측정하기 위하여}

DNA helix

^{에결합하여형광을나타내는물질인}

PI

^를처리

하여

,

^{세포주기에서}

apoptotic

^세포인

sub-G1 hypodiploid

^세

포의증가를유세포분석기로분석하였다

.

^그결과

,

^까마귀

쪽나무

80%

^{에탄올추출물을}

25, 50

^및

100

^µ

g/ml

^의농도로

48

^{시간처리하였을때}

,

^{까마귀쪽나무추출물에의해}

sub-G1 hypodiploid

^세포가

19, 22

^및

31%

^로

,

^{농도 의존적으로}

apoptosis

^{세포가증가되는것을확인할수있었다}

(Fig. 2).

핵의 형태학적 변화 −

DNA

^{에특이적으로결합하는형광}

염색액인

Hoechst 33342

^{를사용하여}

apoptosis

^{의형태학적}

특징중의하나인핵의변화를관찰하였다

. Hoechst 33342

로

DNA

^{를염색하고형광현미경으로관찰한결과}

,

^까마귀

쪽나무의

80%

^{에탄올추출물을}

25, 50

^및

100

^µ

g/ml

^의농

도로

48

^{시간동안처리하였을때}

,

^{정상대조군에비하여농}

도의존적으로

HL-60/ADR

^{세포의크기가축소되었으며}

,

핵의모양이불규칙하고부분적인핵의응집현상을관찰할 수있었다

(Fig. 3).

Bcl-2/Bax 발현 양상의 변화

까마귀쪽나무

80%

^{에탄올추출물에의한}

HL-60/ADR

^의

apoptosis

^{유도의분자적기전을밝히기위하여}

Bcl-2

^와

Bax

의발현을조사하였다

. Apoptosis

^{를조절하는유전자산물}

중제일먼저알려진암유발유전자산물의하나인

anti-

apoptosis protein Bcl-2

^는분자량

26 KDa

^{의단백질로서세}

포증식에는관여하지않고

chemoresistance

^{에중요한역할}

을담당하여여러종류의자극에대해

apoptosis

^를억제하

는특이적기능을가지고있다고알려져있다

.

^반면

Bcl-2

family

^에속하는

pro-apoptosis protein Bax protein

^은

apoptosis

를촉진시키는

protein

^{으로밝혀졌다}

.

¹¹⁾

Bax

^는

Bcl-2

^의

anti- apoptosis effects

^{를방해하는것으로알려져있다}

.

¹⁴⁾

Fig. 4

에서 보는바와같이

, HL-60/ADR

^{세포에까마귀쪽나무}

80%

^{에탄올추출물을}

3, 6, 9

^및

12

^시간동안

100

^µ

g/ml

의농도로처리하였을때

Bcl-2

^의발현은

9

^{시간이후부터}

시간의존적으로감소한반면

Bax

^{의경우는그발현이큰}

차이가없었다

(Fig. 4).

Caspase-9 및 caspase-3의 활성화

현재수많은

apoptosis

^{관련유전자가알려져있음에도불}

구하고

,

^{그중공통적인경로는단백질분해효소의활성과}

관련이깊음이알려졌다

.

^특히

caspase

^가

apoptosis

^기작의

중심적인요소로여겨지고있다

. Tetrapeptide motif

^를인식

하여기질을절단하는

caspase

^는

cystein protease

^로

,

^그인

식

peptide

^{의특이성에따라여러종류의}

isoform

^이보고되

었다

.

^이들중

Caspase-3

^는다양한

apoptosis

^{자극에의해서}

Fig. 3.

The degree of apoptosis represented as the fluorescent image of nuclei in cells by fluorescent microscope.

The HL-60/ADR cells (2.5×10

⁵

/ml) were treated with 100

µ

g/

ml of 80 % EtOH extract of L. japonica for 48 hours. DNA- specific fluorescent dye, H33342 (10

µ

g/ml medium at final) was directly added to media and apoptosis bodies were observed under inverted fluorescent microscope equipped with a IX-71 Olympus camera. The cells were photographed under microscopy (X 400).

Fig. 4.

Western blot analyses of Bcl-2 and Bax proteins.

The HL-60/ADR cells(3.5×10

⁵

/ml) were treated with 100

^µ

g/

ml of 80% EtOH extract of L. japonica for 3, 6, 9 and 12

hours. Western blots were performed as described in Material

and methods.

공통적으로활성화될수있으며활성화된

Caspase-3

^는여러

종류의세포내단백질들을절단할수있다

.

^{핵내에존재}

하는효소중하나인

PARP

^{의촉매부위는}

apoptosis

^가진행

되는여러세포에서효과적으로

caspase-3

^{를포함하는여러}

caspase

^에의해

DNA

^{결합부위로부터절단되어분리된다}

. Caspase-9

^과

caspase-3

^는

PARP

^{분절의상부경로에있는것}

으로서

, apoptosis

^{가 일어날 때 활성화되며}

, caspase-3

^가

apoptosis

^{의집행자의역할을하는것으로알려져있다}

.

^본

실험에서까마귀쪽나무

80%

^{에탄올추출물을}

100

^µ

g/ml

^의

농도로

3, 6, 9

^및

12

^{시간동안처리하여}

caspase

^의활성형

인

cleaved caspase

^{의단백질수준을조사한결과}

, Fig. 5

^에

서처럼

9

^{시간이후부터활성이증가됨을확인할수있었다}

.

또한

caspase-3

^{의활성도와 비례하여}

, caspase-3

^기질의하

나인

PARP (116 KDa)

^{는시간의존적으로}

PARP

^분해단백

질

(85 KDa)

^{로나타나는것을확인하였다}

(Fig. 5).

결 론

본연구에서는까마귀쪽나무의추출물이

adriamycin

^에내

성을가지고있는

HL-60/ADR

^세포에서

Bcl-2

^{의발현감소}

및

caspase

^{를활성화함으로서}

apoptosis

^{를유도하여증식을}

저해함을밝혔다

.

^{이와같은연구결과는까마귀쪽나무추출}

물이항암제에내성을가지고있는내성암세포주에대한항 암제로이용될수있는근거를시사하며

,

^{항암치료제또는}

예방제의유효성분및그작용기전연구에중요한기초자 료가될것이라고사료된다

.

사 사

본논문은

2

^{단계두뇌한국}

21(Brain Korea 21; BK21)

^의

지원에의해서연구되었다

.

인용문헌

1

.

Shen, D. W., Goldenberg, S., Pastan, I. and Gottesman M. M.

(2000) Decreased accumulation of [14C] carboplatin in human cisplatin-resistant cells results from reduced energy- dependent uptake. J Cell Physiol .

183

: 108-116.

2

.

Gottesman, M. M., Fojo, T. and Bates, S. E. (2002) Mul- tidrug resistance in cancer: role of ATP-dependent trans- porters. Nat Rev Cancer .

1

: 8-58.

3

.

Schuetz, E. G., Beck, W. T. and Schuetz, J. D. (1996) Mod- ulators and substrates of P-glycoprotein and cytochrome P4503A coordinately up-regulate these proteins in human colon carcinoma cells. Mol Pharmacol .

49

(2): 311-8.

4

.

Goto, S., Kamada, K., Soh, Y., Ihara, Y. and Kondo, T. (2002) Significance of nuclear glutathione S-transferase pi in resis- tance to anti-cancer drugs. Jpn J Cancer Res .

93

(9): 1047-56

.

5. Urasaki, Y., Laco, G. S., Pourquier, P., Takebayashi, Y., Kohl- hagen, G., Gioffre, C., Zhang, H., Chatterjee, D., Pantazis, P.

and Pommier, Y. (2001) Characterization of a novel topoi- somerase I mutation from a camptothecin-resistant human prostate cancer cell line. Cancer Res .

61

(5): 1964-9.

6. Tanaka, H., Nakamura, T. and Ichino, K. (1990) Butanolides from Litsea japonica . Phytochemistry .

²⁹

: 857-859.

7. Takeda, K., Sakurawi, K. and Ishii, H. (1972) Components of the Lauraceae family-l. New lactonic compounds from Litsea japonica . Tetrahedron .

28

: 3757-3766.

8. Min, B. S., Lee, S. Y., Kim, J. H., Kwon, O. K., Park, B. Y., An, R. B., Lee, J. K., Moon, H. I., Kim, T. J., Kim, Y. H., Joung, H. and Lee, H. K. (2003) Lactones from the leaves of the Litsea japonica and their anti-complement activity. J Nat Prod .

66

(10): 1388-1390.

9. Lee, S. Y., Min, B. S., Kim, J. H., Lee, J., Kim, T. J., Kim, C.

S., Kim, Y. H. and Lee, H. K. (2005) Flavonoids from the leaves of Litsea japonica and their anti-complement activity.

Phytotherapy research .

¹⁹

: 273-276.

10. Carmichael, J., DeGraff, W. G., Gazdar, A. F., Minna, J. D.

and Mitchell, J. B. (1987) Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemisen- sitivity testing . Cancer Research .

⁴⁷

: 944-946.

11. Luo, Y. and Kessel, D. (1997) Initiation of apoptosis versus necrosis by photodynamic therapy with chloroaluminum phthalocyanine. Photochem Photobiol .

66

: 479.

12. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem .

72

: 248-54.

13. Kim, E., Boo, H. J., Hyun, J. H., Kim, S. C., Kang, J. I., Kim, M. K., Yoo, E. S. and Kang, H. K. (2009) The effect of Litsea japonica on the Apoptosis Induction of HL-60 leukemia cells. Yakhak Hoeji .

53

(1): 6-11.

14. Kroemer, G., Zamzami, N. and Susin, S. A. (1997) Mito- chondrial control of apoptosis. Immunol Today .

¹⁸

: 44-51.

(2009년 3월 6일) Fig. 5.

Western blot analyses of caspase-9, caspase-3 and

cleavage of PARP.

HL-60/ADR cells (3.5×10

⁵

/ml) were treated with 100

µ

g/ml of 80% EtOH extract of L. japonica for 3, 6, 9 and 12 hours.

Caspase-9, -3 and PARP cleavage were analyzed by western

blotting using specific antibodies.