The Effect of Litsea japonica on the Apoptosis Induction of HL-60 Leukemia Cells

(1)

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

김엘비라·부혜진·현재희·김상철·강정일·김민경·유은숙·강희경^# 제주대학교 의학전문대학원 약리학교실, 제주대학교 의과학연구소

(Received October 30, 2008; Revised January 6, 2009; Accepted January 7, 2009)

The Effect of Litsea japonica on the Apoptosis Induction of HL-60 Leukemia Cells

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, Cheju National University, 66 Jejudaehakno, Jeju 690-756, Korea

Abstract

— This study investigated the antiproliferative effect of the EtOH extract from Litsea japonica. The extract mark- edly inhibited the growth of HL-60 cells. When treated with the extract, several apoptosis events like as DNA frag- mentation, chromatin condensation and the increase of the population of sub-G1 hypodiploid cells were observed. The extract decreased the Bcl-2 expression, whereas the Bax expression was increased. Caspase-9 and -3 were activated and poly (ADP-ribose) polymerase was cleaved. The results suggest that the antiproliferative effect of L. japonica in HL-60 appears to arise from apoptosis induction via the down-regulation of Bcl-2 and the activation of caspases.

Keywords □

Litsea japonica, HL-60, apoptosis, Bcl-2, Bax, caspases

까마귀쪽나무

(

Litsea japonica

)

는상록엽소교목으로서수직적으 로표고

700 m

이내의바닷가및산기슭에분포한다

.

이식물의 화학적구성에대한기존의연구에서

,

^여러^종류의

essential oils, fatty acids, lactones, alkaloids

및

terpenoids

가발견되었다고보 고된바가있다

.

^1,2)

Lactones

의종류로알려진

hamabiwalactone A, hamabiwalatone B, akolactone B, litsealactone A

^및

litsea- lactone B

가분리되었다

.

³⁾또한

,

잎으로부터분리된

flavonoids

성분인

epicatechin, afzelin, quercitrin

및

tiliroside

의

anti-com- plement activity

^를^조사한^결과

, tiliroside

^가

complement sys- tem

에대해가장강력한억제작용을나타냄이보고되었다

.

⁴⁾한 국에서까마귀쪽나무는식용으로사용하였다는보고가있으나

,

어떤용도로사용하였는지에대한연구보고는미미한실정이므 로이식물에대한항암효능을비롯한생리활성을탐색하는연 구가필요하다

.

본연구에서는까마귀쪽나무의추출물을사용하여

HL-60 (human promyelocytic leukemia)

세포를비롯한여러암세포를

대상으로이세포에대한세포증식억제효과를조사하였다

.

까 마귀쪽나무추출물의

HL-60

세포에대한세포증식억제효과가

apoptosis

^유도에^의한^것인지^알아보기^위하여

,

^핵의^형태학적

변화

, DNA

절편화및

DNA content

변화를알아보았으며

, apo- ptosis

유도신호전달기전에중요한

Bcl-2

와

Bax

의증가와이에 따른

caspase-9, caspase-3 protease

^활성화의^발현^양상을^조사

하였다

.

이와같은연구로

HL-60

세포에대한까마귀쪽나무추 출물의항암효과를밝혀효과적인항암물질개발의재료로사용 가능한근거를제시하고자하였다

.

실험 방법

까마귀쪽나무추출물제조

제주도해안가에자생하고있는까마귀쪽나무

(

Litsea japonica

)

의잎을

2005

^년

11

^월경에^채집하여^시료

185 g

^을^음지에서^건조

시킨다음

,

마쇄기로분쇄하여각각의미세말시료를

80%

에탄 올에침적하고초음파를이용하여

1

^시간씩

3

^회^{추출하였으며}

,

^그

후상층액을회수하고감압농축하여얻은

(43 g)

^에탄올^추출물을

2007

년제주생물종다양성연구소로부터분양받았다

.

본실험에 서이추출물을

PBS(phosphate-buffered saline)/

^에탄올

(1 : 1)

^에^용

#본논문에관한문의는저자에게로

(

^전화

) 064-754-3846 (

^팩스

) 064-702-2687

(E-mail) [email protected]

(2)

해시킨후원하는농도에따라실험용배지로희석하여사용하였다

.

세포배양

HL-60(human promyelocytic leukemia)

^세포는^한국^세포주

은행

(Korea Cell Line Bank)

로부터분양받아서사용하였다

. 100 units/m

l의

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

^과

10% heat-inactivated fetal bovine serum(FBS;

GIBCO Inc, Grand Island, NY, USA)

이첨가된

RPMI 1640

배 지를사용하여

37

^o

C, 5% CO

2

, humidified incubator

에서배양하 였으며

,

^{계대배양은}^약

70~80%

^의

confluence

^가^되는

3~4

^일마

다한번씩시행하였다

.

세포독성측정

HL-60

세포의성장증식에대한까마귀쪽나무의

80%

에탄올

추출물 효과를

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

^를^이용하여^{검색하였다}

.

⁵⁾^살아있는

암세포

mitochondria

의탈수소효소작용에의하여수용성의노 란색

MTT

^가^환원되어^형성되는^자주색을^띠는 ^{비수용성의}

formazan

^을

microplate(ELISA) reader

^로

540 nm

^에서^흡광도를

측정하여

,

대사적으로왕성하게생존하는세포의수를조사하였 다

. HL-60(2.5×10

⁵

cells/m

^l

)

^을

96 well plate

^에^넣고^{까마귀쪽나}

무의

80%

25, 50, 75

^및

100

^µ

g/m

^l의^농도로

처리하였다

.

이를

4

일간배양한다음

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

^µl

(2 mg/m

^l

)

^를^첨가하고

4

^시간^동안^반응^시

킨후

, plate

^를

1000 rpm

^에서

10

^분간^{원심분리하고}^상층액을^제

거하였다

. Dimethylsulfoxide(DMSO: Sigma chemical Co., St.

Louis, USA) 150

^µl를^가하여^침전물을^용해^시킨^후

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

^를^사용하여

540 nm

에서흡광도를측정하였다

.

각시료군에대한평균흡광 도값을구하였으며

,

^대조군의^흡광도^값과^비교하여^세포^증식

억제정도를조사하였다

.

DNA fragmentation analysis

HL-60(3.5×10

⁵

cells/m

l

)

세포에까마귀쪽나무의

80%

에탄올 추출물을

100

µ

g/m

l의농도로처리한다음

, 3, 6

및

9

시간동안 배양하였다

.

^세포를^수확한^후

Promega Wizard

^®

Genomic DNA Purification Kit(Promega, Madison, WI, USA)

를 사용하여

DNA

를분리하였다

.

분리한

DNA

를

1.2% agarose gel

에서

40

분

(100 V)

^동안^{전기영동을}^실행한^다음

, ethidium bromide

^로^염

색하고

UV transilluminator(Spectronics Corporation Westbury, NY, USA)

하에서

DNA fragmentation

현상을관찰하였다

.

⁶⁾

세포주기변화측정

HL-60(2.5×10

⁵

cells/m

l

)

세포에까마귀쪽나무

80%

에탄올추

출물을

25, 50

^및

100

^µ

g/m

^l의^농도로^처리한^다음

, 48

^시간^동

안배양한후

,

^세포를^수확하여

PBS

^로^{세척하였다}

.

^그^후

-20

^o

C

에서

70% ethanol

로

30

분동안고정시키고

PBS

세척후

RNase A(1 mg/m

^l

)

^를^처리한^다음에

propidium iodide(PI: Sigma chem- ical Co., St. Louis, USA)

로 염색하고

, COULTER

^®

EPICS

^®

XL

^TM

Flow Cytometer(Coulter, Miami, FL, USA)

로세포주기 를분석하였다

.

⁷⁾

핵의형태학적변화관찰

HL-60(2.5×10

⁵

cells/m

^l

)

^세포에

25, 50

^및

100

^µ

g/m

^l의^농도

로까마귀쪽나무

80%

에탄올추출물을처리한다음

3, 6, 9, 12, 24

및

48

시간동안배양하였다

. DNA

에특이적으로결합하는형 광색소인

Hoechst 33342(H33342: Sigma chemical Co., St.

Louis, USA)

용액

(1

µ

g/m

l

)

을가하여

37

^o

C

에서

10

분간염색한 후형광현미경

(IX-71, Olympus, Japan)

하에서관찰하였다

.

⁸⁾

Western blot analysis

HL-60(2.5×10

⁵

cells/m

^l

)

^세포에^{까마귀쪽나무의}

80%

^에탄올

추출물을

100

^µ

g/m

^l의^농도로^처리한^다음

3, 6

^및

9

^시간^동안

배양한후

,

세포를수확하여

PBS

로

2~3

회세척하였다

. 500

µl 의

lysis buffer

^를^첨가한^다음

, 1

^시간^동안

lysis

^시킨^후

12,000 rpm

^에서

15

^분간^{원심분리하여}^상층액을^얻었다

.

^단백질^농도는

BSA(bovine serum albumin)

를표준물질로 사용하여

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

^를 ^사용하여 ^{정량하였다}

.

⁹⁾

20~30

^µ

g

^의

lysate

^를

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

로변성분리하여

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

^로

200 mA

^에서

2

^시간^동안

transfer

^하였다

.

^그리고

membrane

^의

blocking

은

5% skim milk

가함유된

TTBS(TBS+0.1% Tween 20)

^용액에서

overnight

^{실시하였다}

. Bcl-2

^의^발현량을^검토하

기 위한 항체로는

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

의발현양을검 토하기 위한 항체로는

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

의 발현량을 검토하기위한항체로

rabbit polyclonal anti-human PARP Ab (1 : 1000)(Santa Cruz Biotech, MA, USA), caspase-3

^발현량을

검토하기위한항체로

rabbit polyclonal anti-human caspase-3 Ab(1 : 1000)(Cell Signaling Technology, Beverly, MA, USA)

와

caspase-9

^의

Ab(1 : 1000)(Cell Signaling Technology, Beverly,

MA, USA)

을

TTBS

용액에서희석하여사용하였으며

,

반응은상 온에서

2

시간동안진행하였다

. 2

차항체로는

HPR(Horse Radish

Peroxidase)

^이 ^결합된

anti-rabbit IgG(Amersham Prarmacia

Biotech, NY, USA)

와

anti-mouse IgG(Amersham Prarmacia

Biotech, NY, USA)

를희석

(1 : 5000)

하여이용하였으며

,

반응은

(3)

상온에서

30

^분^동안^{진행하였다}

.

^그^후

membrane

^을

TTBS

^로

3

회 세척하여

ECL

^기질

Amersham Prarmacia Biotech, NY, USA)

과

1~3

분간반응후

X-ray

필름에감광하였다

.

통계처리

표시된결과는

3

번이상의독립적인실험결과이며

,

데이터를

mean±standard error

^값으로^{나타내었다}

.

^실험군^사이의^통계

적유의성검증은

Student's t-test

를사용하여평가하였다

.

실험결과 및 고찰

세포증식저해효과

까마귀쪽나무

80%

^에탄올^추출물에^의한

HL-60

^세포의^증식

억제효과를측정하기위하여

MTT assay

를이용하였다

.

여러세 포주에 까마귀쪽나무의

80%

에탄올 추출물을

25, 50, 75

및

100

^µ

g/m

^l의^농도로^처리하여^{실험하였다}

(Fig. 1).

^{까마귀쪽나무}

80%

에탄올추출물을

100

µ

g/m

l의농도로처리하였을때

HL- 60

^를^비롯한

5

^종의^{암세포에서의}^세포^증식억제^효과를^보면

HL- 60(human promyelocytic leukemia)

^에서는

83%, HL-60/ADR (adriamycin resistant human promyelocytic leukemia cell line)

^에서

64.5%, HL-60/MX2(mitoxantrone resistant human promyelocytic leukemia cell line)

^에서

76%, HCT-15(human colon cancer)

에서

7.9%, SK-OV-3(human ovary cancer)

에서

16.9%, A-549(human lung cancer)

^에서

3.4%

^의^효과를^보였다

.

정상세포

HEL-299

^에^같은^방법으로^{까마귀쪽나무의}

80%

^에

탄올추출물을처리하여정상세포에대한세포증식억제효과 를확인한결과

, 100

µ

g/m

l의농도에서다른세포주에비하여현 저히낮은

2.3%

^의^세포^증식억제^효과를^{확인하였다}

.

^이는^까마

귀쪽나무의

80%

에탄올추출물이정상세포에는독성을갖지않 는것으로판단할수있다

(Fig. 1).

따라서까마귀쪽나무의

80%

^에탄올^추출물에^의한^세포^증식

억제효과가탁월한

HL-60

세포에대하여까마귀쪽나무의

80%

에탄올추출물에의한암세포의세포증식억제효과및

apoptosis

유도기전을조사하였다

. Apoptosis

^유도효과

DNA fragmentation

확인 − 까마귀쪽나무의

80%

^에탄올^추

출물에의한

HL-60

세포증식억제작용이

apoptosis

유도에의 한것인지알아보기위하여

, apoptosis

유도에의하여나타나는

DNA

^{절편화현상을}^{전기영동으로}^{관찰하였다}

. DNA

^{절편화현상}

은세포가

apoptosis

로들어가는원인이되는데

endonuclease

를 생산하여

chromatin

^을

nucleosome DNA

^로^절단한다

.

^이러한^현

상을

agarose gel

^에서

ladder

^형태로^관찰할^수^있다

. HL-60

^세

포에까마귀쪽나무의

80%

100

µ

g/m

l의농도로

3, 6

및

9

시간동안처리하여

DNA ladder

를확인한결과

, Fig.

2

^에서^처럼

6

^시간^및

9

^시간^처리^시

DNA

^절편화^현상을^관찰

할수있었다

(Fig. 2).

Cell cycle analysis

^{− 까마귀쪽나무의}

80%

^에탄올^추출물에

의하여

HL-60

^세포의

apoptosis

^가^{유도되는지를}^알아보기^위하

여세포주기를조사하였다

. HL-60

세포에까마귀쪽나무의

80%

25, 50

^및

100

^µ

g/m

^l의^농도로

48

^시간^동안^처

Fig. 1 −

Inhibitory effect of Litsea japonica on the proliferation of cancer cells. The cells (2.5×10

⁵

/m l ) were treated with 25, 50, 75 and 100

^µ

g/m l for 4 days of 80% EtOH extract from L. japonica and the viability was measured by MTT assay.

HL-60 (human promyelocytic leukemia), HL-60/ADR (adriamycin resistant human promyelocytic leukemia), HL- 60/MX2 (mitoxantrone resistant human promyelocytic leukemia), HCT-15 (human colon cancer), SK-OV-3 (human ovary cancer), A549 (human lung cancer), HEL- 299 (human embryonic lung). All experiments were performed in triplicate. Data were presented as a mean

±SD of three separate experiments. * p<0.05, ** p<0.01 compared with the control.

Fig. 2 −

DNA fragmentation by the 80 % EtOH extract of Litsea

japonica in HL-60 cell. HL-60 cells (3.5×10

⁵

/m l ) were

treated with 100

^µ

g/m l of the extract from L. japonica for

3 h, 6 h and 9 hours. The DNA was isolated and subjected

to 1.2% agarose gel electrophoresis and visualized with

ethidium bromide staining.

(4)

리한다음

, DNA helix

^에^결합하여^형광을^나타내는^물질인

PI

를처리하여

,

세포주기에서

apoptotic

세포

,

즉

sub-G1 hypodip- loid

^세포의^증가를^유세포^분석기로^{분석하였다}

.

^그^결과

,

^까마

귀쪽나무

80%

25, 50

^및

100

^µ

g/m

^l의^농도로

48

시간 처리하였을때

,

까마귀쪽나무 추출물에의해

sub-G1 hypodiploid

^세포가

47, 61

^및

84%

^로

,

^농도^{의존적으로}

apopto- sis

세포가증가되는것을확인할수있었다

(Fig. 3).

핵의형태학적변화 −

Apoptosis

의형태학적특징중의하나인 핵의변화를관찰하기위하여

DNA

^에^{특이적으로}^결합하는^형

광염색액인

Hoechst 33342

를사용하여

DNA

를염색하고형광 현미경으로관찰하였다

.

정상대조군에비하여까마귀쪽나무의

80%

100

^µ

g/m

^l의^농도로

3, 6, 9, 12

^및

24

^시

간처리하였을때시간의존적으로

HL-60

세포의크기가축소 되었으며

,

핵의모양이불규칙하고부분적인핵의응집현상을관 찰할수있었다

(Fig. 4A).

^또한^{까마귀쪽나무의}

80%

^에탄올^추

출물을

25, 50

및

100

µ

g/m

l의농도로

48

시간처리하였을때도 농도의존적으로

HL-60

^세포의^부분적인^핵의^{응집현상을}^관찰

할수있었다

(Fig. 4B) Bcl-2/Bax

^발현^양상의^변화

Apoptosis

^의^분자적^기전을^밝히기^위해

anti-apoptosis protein

으로알려진

Bcl-2

와

pro-apoptosis protein

인

Bax

의발현을관 찰하였다

. Apoptosis

^를^{조절하는데}^관여하는^여러^가지^유전자

산물중에서제일먼저알려진암유발유전자산물의하나인

Bcl-

2

는분자량

26 KDa

의단백질로서다른암유전자단백질과는달 리세포증식에는관여하지않고

chemoresistance

^에^중요한^역할

을담당하여여러종류의자극에대해

apoptosis

^를^억제하는^특

이적기능을가지고있다고알려져있다

.

한편

Bcl-2 family

에속 하는

Bax protein

^은

apoptosis

^를^{촉진시키는}

protein

^으로^밝혀졌

다

.

¹⁰⁾

Bax

^는

Bcl-2

^와

heterodimer

^를^{형성함으로써}

Bcl-2

^의

anti- apoptosis effects

를방해한다

.

¹¹⁾따라서본실험에서까마귀쪽나 무의

80%

^에탄올^추출물을^처리하여

HL-60

^세포에서

Bcl-2

^및

Bax

발현양상을조사하였다

.

그결과

, Fig. 6

에서보는바와같 이

,

까마귀쪽나무의

80%

HL-60

세포에

3, 6

및

9

^시간^동안

100

^µ

g/m

^l의^농도로^{처리하였을}^때

Bcl-2

^의^발현은

6

시간이후부터점차시간의존적으로감소하였으나

, Bax

의경 우는그발현이증가함을확인할수있었다

(Fig. 5).

Caspase-9

^및

caspase-3

^의^활성화

현재까지수많은

apoptosis

,

그 중에서도공통적인경로는단백질분해효소의활성과관련이깊 음이알려졌다

.

특히시스테인계의단백질분해효소인

caspase

가

apoptosis

^기작의^중심적인^요소로^여겨지고^있다

. Caspase

^는

tetrapeptide motif

^를^인식하여^기질을^절단하는

cystein protease

로

,

그인식

peptide

의특이성에따라여러종류의

isoform

이보 고되었다

.

이들중

caspase-3

가세포사멸분야에서가장보편적 인관심을받아왔다

. Caspase-3

^는^다양한

apoptosis

^자극에^의해

서공통적으로활성화될수있으며활성화된

caspase-3

는여러 종류의세포내단백질들을절단할수있다

. PARP

^는^핵^내에^존

재하는효소중에하나로서

,

^그^{촉매부위는}

apoptosis

^를^거치는

여러세포에서효과적으로

caspase-3

를포함하는여러

caspase

에의해

DNA

^결합^{부위로부터}^절단되어^분리된다

.

^따라서^까마

Fig. 3 −

The degree of apoptosis represented as the DNA content measured by flow cytometry. The HL-60 cells (2.5×10

⁵

/m l ) were treated

with various concentrations (25, 50 and 100

^µ

g/m l ) of 80% EtOH extract from L. japonica for 48 hours.

(5)

귀쪽나무의

80%

에탄올추출물에의해서

HL-60

세포의

apop- tosis

^{신호전달과정에서}

caspase

^의^활성을^통한

PARP

^분절로

apoptosis

^를^{일으키는지를}^{조사하였다}

. PARP

^분절의^{상부경로에}

있는

caspase-9

과

caspase-3

는

apoptosis

가일어날때활성화되 며

, caspase-3

^가

apoptosis

^의^{집행자로서}^역할을^수행하는^것으

로알려져있다

. Fig. 6

^에서^처럼

, 100

^µ

g/m

^l의^농도로

3, 6

^및

9

시간처리하여

caspase

의활성형인

cleaved caspase

의단백질수 준을조사한결과

, 6

^시간^이후부터^활성이^{증가되었다}

. Western blot

으로확인한

caspase-3

기질의하나인

PARP(116 KDa)

는시 간의존적으로

caspase-3

의활성도와비례하여

, PARP

분해단 백질

(85 KDa)

^로^나타나는^것을^{확인하였다}

(Fig. 6).

Fig. 4 −

The degree of apoptosis represented as the fluorescent image of nuclei in HL-60 cells by fluorescent microscope.

(A) The HL-60 cells (2.5×10

⁵

/m l ) were treated with 100

^µ

g/m l of 80% EtOH extract from L. japonica for 3, 6, 9, 12 and 24 hours. DNA-specific fluorescent dye, H33342 (10

µ

g/m l at a final concentration) 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 (×400). (B) The HL-60 cells (2.5×10

⁵

/m l ) were treated with 25, 50 and 100

^µ

g/m l of 80% EtOH extract from L.

japonica for 48 hours. DNA-specific fluorescent dye, H33342 (10

^µ

g/m l at a final concentration) 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 (×400).

Fig. 5 −

Western blot analyses of Bcl-2 and Bax proteins. The HL- 60 cells (3.5×10

⁵

/m l ) were treated with 100

µ

g/m l of 80%

EtOH extract from L. japonica for 3, 6, and 9 hours.

Western blots were performed as described in Material and methods.

Fig. 6 −

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

of PARP. The HL-60 cells (3.5×10

⁵

/m l ) were treated with

100

^µ

g/m l of 80% EtOH extract from L. japonica for 3, 6

and 9 hours. Caspase-9, -3 and PARP cleavage were

analyzed by western blotting using specific antibodies.

(6)

결 론

본연구에서는까마귀쪽나무의추출물이

HL-60

백혈병세포 에서

Bcl-2

^의^발현^감소^및

caspase

^를^활성화^함으로서

apoptosis

를유도하여증식을저해함을밝혔다

.

이와같은연구결과는까 마귀쪽나무추출물이항암제로이용될수있는근거를시사하며

,

항암치료제또는예방제의유효성분및그작용기전연구에중 요한기초자료가될것이라고사료된다

.

감사의 말씀

본논문은

2

단계두뇌한국

21(Brain Korea 21; BK 21)

의지 원에의해서연구되었다

.

참고문헌

1) Tanaka, H., Nakamura, T. and Ichino, K. : Butanolides from Litsea japonica. Phytochemistry

²⁹

, 857 (1990).

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

28

, 3757 (1972).

3) Min, B. C. : Lactones from the leaves of the Litsea japonica and their anti-complement activity. J. Nat. Prod.

⁶⁶

(10), 1388 (2003).

4) Lee, S. Y., Min, B. S., Kim, J. H., Lee, J., Kim, T. J., Kim, C. S., Kim, Y. H. and Lee, H. K. : Flavonoids from the Leaves of

Litsea japonica and their anti-complement activity. Phytotherapy Research

¹⁹

, 273 (2005).

5) Carmichael, J., Degraff, W. G., Gazdar, A. F., Minna, J. D. and Mitchell, J. B. : Evaluation of a tetrazolium-based semi- automated colorimetric assay: assessment of chemisensitivity testing. Cancer Research

⁴⁷

, 944 (1987).

6) Purohit, A., Hejaz, H. A., Walden, L., MacCarthy-Morrogh, L., Packham, G., Potter, B. V. and Reed, M. J. : The effect of 2- methoxyoestrone-3-O-sulphamate on the growth of breast cancer cells and induced mammary tumours. Int. J. Cancer.

85

, 584 (2000).

7) Nicoletti, I., Migliorati, G., Pagliacci, M. C., Grignani, F. and Riccardi, C. : A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods

¹³⁹

, 271 (1991).

8) Luo, Y. and Kessel, D. : Initiation of apoptosis versus necrosis by photodynamic therapy with chloroaluminum phthalocyanine.

Photochem. Photobiol.

⁶⁶

, 479 (1997).

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

⁷²