Role of $K^+$-$Cl^-$-cotransporter in the Apigenin-induced Stimulation of Melanogenesis in B16 Melanoma Cells

B16 흑색종세포에서 아피제닌에 의한 멜라닌 합성 촉진효과에 미치는 칼륨-염소이온수송체의 역할

이 용 수^# 덕성여자대학교 약학대학

(Received November 5, 2008; Revised November 21, 2008)

Role of K

⁺

-Cl

^-

-cotransporter in the Apigenin-induced Stimulation of Melanogenesis in B16 Melanoma Cells

Yong Soo Lee^#

College of Pharmacy, Duksung Women’s University, Seoul 132714, Korea

Abstract

— Apigenin, a natural flavonoid found in a variety of vegetables and fruits, has been shown to possess many bio- logical functions. In this study we found that apigenin stimulated melanin synthesis in a dose-dependent manner in B16 murine melanoma cells. Since in our previous study K

⁺

-Cl

^-

-cotransport (KCC) has been shown to mediate the mechanism of action of apigenin in neuronal cells, we further investigated the role of KCC in the melanogenesis-stimulating effect of apigenin in B16 cells. At nontoxic concentrations apigenin induced Cl

^-

-dependent K

⁺

efflux, a hallmark of KCC activity, which was markedly prevented by a specific KCC inhibitor R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo- 1H-inden-5-yl)oxy]acetic acid (DIOA). These results indicate that KCC is functionally present, and activated by apigenin in the B16 cells. In addition, the apigenin-induced stimulation of melanogenesis was also significantly inhibited by DIOA. N- Ethylmaleimide (NEM), a known KCC activator, induced Cl

^-

efflux and stimulated melanogenesis in a concentration-depen- dent fashion. Both effects of NEM were significantly inhibited by DIOA. Taken together, these results suggest that apigenin can modulate melanogenesis through the activation of a membrane ion transporter, KCC in B16 cells. These results further suggest that apigenin may be a good candidate in the therapeutic strategy for hypopigmentation disorders, such as vitiligo.

Keywords □

K

⁺

-Cl

^-

-cotransport, apigenin, B16 melanoma cell, melanogenesis, vitiligo

아피제닌은자연에존재하는플라보노이드류로서파슬리

,

박 하

,

^레몬

,

^들깨

,

^{딸기류및여러과일에다량함유되어있다}

.

^1)아

피제닌은본연구실및다른연구실의실험결과로서여러조직 에서항염

,

^2,3)항발암⁴⁾및항암작용^5-7)을가진다는사실이밝혀 졌다

.

^{지금까지의연구에서보면식물성플라보노이드류는세포}

실험에서활성산소종의발생을억제하는항산화효과를나타낸다 는사실이알려지고있다

.

^8-11)하지만

,

최근아피제닌은전립선암 세포및간암세포의사멸을유도하고이작용기전에활성산소종 이관여한다는연구결과가발표되었다

.

^7,12)또한신경아세종에서 는아피제닌이세포막칼륨

-

염소이온수송체를활성화시키며 이작용은활성산소종에의해매개된다는새로운사실이발표되

었다

.

¹³⁾

피부의색은피부속에존재하는멜라닌의함량에의해결정 되며피부의기저층에존재하는멜라닌생성세포에의해합성된 멜라닌은세포질돌기를통하여각질형성세포의각화과정에의 해각질층으로이동한다

.

^{14)멜라닌은태양광선으로부터들어오}

는자외선을차단하여피부를보호하는역할을담당하고있지만 만일국소적으로과도하게생성되거나

,

노화등에의해피부의 생리기능이떨어지게되면멜라닌이피부표면에침착되어기미

,

주근깨또는다양한색소침착을유발하게된다

.

¹⁵⁾멜라닌생성 세포에서멜라닌합성은멜라노좀에서일어나며여기에존재하 는여러

cascade

^{효소반응에의해생성된다}

.

^{16,17)이들효소중}

tyrosinase

는멜라닌합성의속도를결정하는효소로서

, tyrosine

을

3,4-dihydroxyphenylalanin(DOPA)

로전환하는

tyrosine hydroxylase

활성과

DOPA

^를

DOPA quinone

^{으로산화하는}

DOPA oxidase

활성을가지고있다

. Tyrosinase-related protein(TRP)-1

은

5,6-

#본논문에관한문의는저자에게로

(

^전화

) 02-901-8396 (

^팩스

) 02-901-8386

(E-mail) [email protected]

dihydroxyindole-2-carboxylic acid(DHICA)

^를

indole-5,6-quinone- 2-carboxylic acid

^{로산화하는효소이며}

dopachrome tautomerase (DCT)

는 초기에

TRP-2

로불려졌던효소로서

dopachrome

을

DHICA

^{로이성화하는효소이다}

.

^{멜라닌은흑·갈색의}

eumlanin

과적·노랑색의

phaeomelanin

으로나뉘는데

tyrosinase

는이들 두 가지 타입의

melanin

합성에 필요하며

, TRP1

과

DCT

는

eumelanin

^{의합성에더많이관여하는것으로알려져있다}

.

^18)최

근다양한식물추출물이

tyrosinase

활성저해나활성산소종의발 생억제등을통하여멜라닌합성을저해함으로써미백기능성화 장품의원료물질로개발되고있다

.

^19,20)

칼륨

-

염소이온수송체는적혈구에서팽창

-

의존성칼륨유출기 전을매개하는기능이있다는사실^21,22)이처음밝혀진이래기 능적및생리적인실험적증명을통하여여러조직에존재하고 있다는것이밝혀졌다

.

즉

,

칼륨

-

염소이온수송체는상피

,

^23,24) 내 피

,

²⁵⁾혈관평활근

,

²⁶⁾심장

,

²⁷⁾골격근²⁸⁾및신경²⁹⁾에그존재가확 인되었다

.

^칼륨

-

^{염소이온수송체는여러생리활성을나타내는데}

조절체적감소

(regulatory volume decrease),

³⁰⁾상피세포간염류 흡수

,

^{23)허혈심근에서칼륨손실}

,

^{27)신경에서염소농도조절29)}

및신장에서칼륨분비³¹⁾등의기능을가지고있다

.

^{또한최근본}

연구실에서도칼륨

-

염소이온수송체가사람간암세포의사멸기전 에중요한매개체로작용하고있음을박힌바있다

.

³²⁾

본연구에서는이러한기존연구결과를바탕으로하여아피제 닌이멜라닌합성에미치는영향을관찰하고이에미치는세포 막칼륨

-

^{염소이온수송체의역할을규명하고자하였다}

.

실험 방법

시약

Dulbecco‘s modified Eagle’s medium(DMEM), 3-(4,5-di- methylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT), apigenin, Nethylmaleimide(NEM)

^{및각종용매와염류는}

Sigma Chemical Co.(

미국

)

에서 구입하였고

, R-(+)-[(2-n-butyl-6,7- dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5- yl)oxy]acetic acid(DIOA)

는

Alexis Biochemicals(

미국

)

에서

fetal bovine serum(FBS)

와

penicillin-streptomysin

혼합액은

GIBCO (

^미국

)

^{에서 구입하였으며}

, N-(6-methoxyquinolyl) acetoethyl ester(MQAE)

및

potassium-binding benzofuran isophthalate/

aceoxylmethyl ester(PBFI/AM)

은

Molecular Probes, Inc.(

미국

)

에서구입하여사용하였다

.

세포배양

B16

^{흑색종세포는서울대학교한국세포주은행에서구입하}

였다

.

구입한세포는

10% FBS

와

penicillin 100 IU/m

l와

strep- tomysin 50

µ

g/m

l을함유한

DMEM

용액으로

37

^o

C

로유지되는

5% CO

₂^배양기

(Forma,

^미국

)

^{에서배양하였다}

.

세포생존율측정

살아있는세포는미토콘드리아에존재하는탈수소효소에의 해

MTT

가

MTT-formazan

으로전환되므로이것의양을재면살

아있는세포의수를측정할수있다

.

세포는

1 m

l배지에여러

가지약물또는약물을녹인용매만을처리하여

24 well plate

^에

서

48

시간동안배양하였다

.

배양이끝난세포에

MTT

용액

(2.5 mg/m

l

H

₂

O) 100

^ml를첨가하여

37

^o

C

에서

4

시간동안반응시켰 다

.

^생성된

MTT-formazan

^{결정체를분리하기위하여세포현탁}

액을

eppendorf tube

에옮겨원심분리하였다

(1500 rpm, 4

분

).

상 징액을조심스럽게제거하고

DMSO 100

µl를첨가하여결정체 를용해시킨후

540 nm

^에서

ELISA reader(Molecular Device,

미국

)

로흡광도를측정하였다

.

세포생존율은대조군과비교하여 흡광도의백분율로나타내었다

.

멜라닌정량

B16

^{세포에서멜라닌을측정할때에는}

phenol red

^가없는

DMEM

^{을사용하여}

24 well plate

^에

5

^×

10

⁴

cells/m

^{l로분주하고}

12

시간이지난뒤세포가

plate

에완전히부착된것을확인한후

시료를처리하고

48

^{시간지난뒤에세포를수집하여세포수를}

측정하고

, 1200 rpm

^에서

5

^{분간원심분리하여침전한후}

, 1 m

^l

homogenization buffer(50 mM sodium phosphate pH 6.5, 1%

Triton X-100, 2 mM PMSF)

^{로용해시켰다}

.

^{여기서얻은세포}

pellet

^을

1 N NaOH(+10% DMSO) 200

^{µl를 첨가하고}

vortex

후멜라닌을완전히녹인다음

96 well plate

에옮긴후

ELISA reader(Molecular Device,

^미국

)

^{를사용하여}

405 nm

^{에서흡광도}

를측정하였다

.

^{멜라닌표준품을이용한표준검량선을이용하}

여멜라닌양을산출하였다

.

멜라닌생성량은단위세포

(10

⁴

cells)

에서의멜라닌생성량으로비교하였다

.

세포내염소이온농도의측정

세포내염소이온농도의변화는염소이온에민감한형광탐침인

MQAE

를사용해조사하였다

.

세포를두번

Hank

용액으로씻은 후

4

×

10

⁵

cells/m

l되게현탁시킨후

MQAE

를세포내로봉입시 키기위해최종농도가

5 mM

^{로하여실온에서밤새배양하였다}

.

형광

(excitation wavelength=365 nm; emission wavelength=

450 nm)

을교반을한상태의

cuvette

을이용하여측정하였다

.

실 험을실온에서시행하여형광탐침이새어나오는것을최소화하 였다

.

실험결과는세포내염소이온과비례하는값인상대적형 광

(Fo/F; Fo=

염소이온이없을때형광치

; F=

시간에따른형광 치

)

^{으로나타내었다}

.

^{모든형광값은}

MQAE

^{이온선택적신호}

를 최대로제거하는

5

µ

M valinomycin

을 포함하는

HEPES-

buffered KSCN

용액을 이용하여따로 측정한 배경 형광치

(background fluorescence)

^{에대하여보정하였다}

.

^{다른실험으로}

Fo

^값은

10

^µ

M tributyltin

^및

10

^µ

M nigericin

^{을포함한염소}

-

제거

KNO

₃용액에세포를현탁시켜측정하였다

.

세포내칼륨농도의측정

세포내칼륨농도는형광탐침인

PBFI/AM

을이용하여측정하 였다

.

^{비이온성계면활성제인}

0.02% pluronic F-127

^을포함한

Hank

용액의세포현탁액에

5

µ

M

의

PBFI/AM

을가해

37

^o

C

에서

2

시간동안진탕배양하여세포내로봉입시킨후

,

봉입되지않은

PBFI/AM

^{은원심분리하여제거시켰다}

.

^{다시세포를}

Krebs-Ringer

완충액

(125 mM NaCl, 5 mM KCl, 1.2 mM KH

₂

PO

₄

, 1.2 mM MgSO

4

, 5 mM NaHCO

3

, 25 mM HEPES, 6 mM glucose, pH 7.4)

^{에현탁시킨후}

cuvette

^에옮겨

340 nm

^및

380 nm

^파장에서

excitation

시켜

, 500 nm

파장에서나오는형광을형광분석기로 측정하였다

.

이때

cuvette

내의 세포현탁액을계속교반하여 세포가가라앉는것을방지하였다

.

^{세포내칼륨농도는두파장}

에서나온형광세기의비로써나타내었다

.

자료분석및통계적검정

모든실험은네번반복해서실시하고실험결과는대조군의 조건에대한백분율로나타내었다

. Data

^는평균값

±SEM

^으로표

시하고

ANOVA

^{로분석하며 각각의유의성비교는}

Student-

Newman-Keul's test

를이용하여실시하였다

. P

값이

0.05

이하인 경우에만통계학적유의성이있는것으로간주하였다

.

실험결과 및 고찰

아피제닌의세포독성

아피제닌이세포독성에미치는농도를조사하여향후실험에

사용될농도범위를결정하기위해서

MTT

^{분석을시행하였다}

.

B16

^{흑색종세포에대한아피제닌의세포독성을측정한결과}

,

^아

피제닌은

50

µ

M

이하의농도로처리했을때세포생존율이

90%

이상으로나타났으며

, 70

^µ

M

^{을처리했을때생존율이유의성있}

게저하되었다

(Fig. 1).

따라서향후실험에서아피제닌은

50

µ

M

이하의농도로사용하였다

.

아피제닌은다른세포에서도본실

험에서사용한

B16

^{세포에서의결과와비슷한양상을보이고있}

으며³³⁾특히고농도에서다른플라보노이드화합물도세포독성 을나타낸다

.

³⁴⁾

아피제닌의멜라닌생성촉진효과

B16

세포를이용하여아피제닌이멜라닌합성에미치는영향 을확인하였다

.

^{준비된세포에아피제닌을농도별로처리하고}

48

시간동안배양하였다

. 48

시간을선택한이유는이시간대에서 대조군

,

즉약물을가하지않은상태에서분석이가능한멜라닌

합성이이루어지기때문이다

.

배양후

B16

세포를수집하여멜 라닌양을측정한결과

,

^{아피제닌처리군에서농도의존적으로멜}

라닌합성이증가되었으며

20

µ

M

농도처리군에서대조군에비 해약

2.2

배가증가하여통계적으로유의한촉진효과를나타내

었다

(Fig. 2).

^{식물유래플라보노이드화합물들이멜라닌합성을}

Fig. 1 −

Effect of apigenin on cell viability in B16 melanoma cells.

Cells were incubated with or without each concentration of apigenin for 48 hr. Cell viability assay was done by the MTT staining method. Results are expressed as percent change of control condition in which cells were grown in medium without apigenin. Each point represents the mean value of four replications with bars indicating SEM.

*p<0.05 compared to control.

Fig. 2 −

Effects of apigenin on the production of melanin in B16

melanoma cells. Melanin content was measured by the

method described in the Method section. The cells were

initially grown for 12 hr in order to attach them to the

bottom of the culture flasks. Then, the cells were incubated

with or without apigenin at each designated concentration

for 48 hr. Each column represents the mean value of four

replications with bars indicating SEM. *p<0.05 compared

to control.

저해한다는연구결과는많이발표되었다

.

^{35,36)그러나최근이와}

반대로퀄세틴과몇몇플라보노이드는오히려멜라닌합성을증 가한다는연구결과도발표되었다

.

^37-41)본연구결과에서보여준 아피제닌의멜라닌합성촉진작용은후자에속하는경우로서흥 미로운결과이나플라보노이드화합물의구조적특성과멜라닌 합성에미치는영향사이에어떤상관성이존재하는지에대한 연구는전무한실정이고향후좀더체계적인연구가필요할것 으로사료된다

.

아피제닌에의한멜라닌합성촉진에미치는칼륨

-

^{염소이온수송체의}

역할

본연구실에서는신경아세포종을이용한실험에서아피제닌이 활성산소종을발생시키고그작용기전에세포막에존재하는칼 륨

-

염소이온수송체가연루되어있다는사실을보고한바있다

.

¹³⁾

따라서후속연구는본연구에서확인한멜라닌합성촉진작용에 미치는칼륨

-

^{염소이온수송체의역할에대한검정을시도하였다}

.

실험결과에서아피제닌은

B16

세포에서칼륨

-

염소이온수송체활 성유무를검정하는지표인염소이온및칼륨이온의유출을농 도의존적으로나타내었고

(Fig. 3),

^{이작용은칼륨}

-

^{염소이온수송}

체를선택적으로억제한다고알려진약물인

DIOA

⁴²⁾에의해유 의성있게차단되었다

(Figs. 4A

및

4B). 50

µ

M

아피제닌을투여 했을때나타나는칼륨유출양상은

30

^µ

M

^{투여시에비해최대}

유출은더증가했지만좀더느리고완만한경향을보였다

(Fig.

3B).

^{이런양상을보이는이유에대해서는명확히알수없으나}

농도가다른아피제닌에대한칼륨

-

^{염소이온수송체의활성화기}

전이다른때문일가능성이있다

.

하지만이추측을명확히하기 위해서는좀더체계적인연구가필요할것이다

.

^{그럼에도불구}

하고이들결과로부터

B16

^{세포에는칼륨}

-

^{염소이온수송체가기}

능적으로존재하고있으며플라보노이드인아피제닌에의해활 성화된다는사실을알수있었다

.

칼륨

-

염소이온수송체의존재에 대한확증을위해칼륨

-

^{염소이온수송체를직접적으로활성화시}

킨다고알려진약물인

NEM

³⁰⁾에의한칼륨

-

염소이온수송체의활 성화작용을확인하였다

.

결과에서

NEM

이

100

µ

M

농도에서현 저한염소이온유출을나타내었으며

(Fig. 4A),

^이작용은

100

^µ

M DIOA

에의해통계적으로유의성있게억제되었다

(Fig. 4B).

또한

, NEM

은농도의존적으로멜라닌합성을촉진하였는데

50

µ

M

농 도에서대조군에비해유의성있게증가시켰다

(Fig. 4C). NEM

^에

의한멜라민합성촉진작용도칼륨

-

염소이온수송체억제제인

DIOA(100

µ

M)

에의해유의성있게차단되었다

(Fig. 4D).

이와더 불어

50

^µ

M

^{아피제닌에의해증가된멜라닌합성도}

DIOA(100

µ

M)

에의해유의성있게차단되었다

(Fig. 4D).

종합적으로이결

과는

B16

세포에서아피제닌에의한멜라닌합성촉진작용은칼

륨

-

^{염소이온수송체의활성화를통해서일어난다는사실을시사한다}

.

멜라닌합성은세포내여러신호전달경로를통하여일어나 는데멜라닌합성관련실험에서흔히사용되는양성대조군화 합물인α

-melanoyte stimulating factor(

^α

-MSH)

^{에의한멜라닌}

합성촉진작용은

cAMP

경로를통하여일어난다고알려져있다

.

멜라닌합성에있어율속단계에관여하는

tyrosinase

의발현은

cAMP

^{에의해증가되고}

,

^이러한

tyrosinase

^{생성에는전사인자인}

microphthalmia-associated transcription factor(MITF)

의발현에 의해 조절된다고 알려져있다

.

^43)또한

MITF

^는

extracellular signal-regulated kinase(ERK)

^{에의해인산화되면그발현이저}

해된다

.

⁴⁴⁾따라서본연구의결과에서보여준아피제닌에의한 멜라닌합성촉진작용은칼륨

-

^{염소이온수송체의활성화를통한}

Fig. 3 −

Effects of apigenin on the Cl

^-

(A) and K

⁺

(B) fluxes in B16 melanoma cells. In figure (A) the data show changes in intracellular Cl

^-

concentration as a function of time, measured by using the Cl

^-

-sensitive indicator MQAE. In the data, Fo/F values are directly proportional to intracellular Cl

^-

concentration. The arrow shows the time point for addition of apigenin at a designated concentration.

In figure (B) the data show changes in intracellular K

⁺

concentration as a function of time, measured by using the K

⁺

-sensitive

indicator PBFI/AM. In the data, PBFI fluorescence ratios are directly proportional to intracellular K

⁺

concentration. The arrow shows

the time point for addition of apigenin at a designated concentration.

칼륨또는염소이온의세포내농도변화가멜라닌합성에관여 하는여러신호전달인자

(cAMP, MITF, ERK)

^{의발현또는활성}

을조절함으로써일어날것으로추측할수있다

.

하지만정확히 어느신호전달과정에연관이있는지를밝히기위해서는향후 추가적인연구가필요하다고생각된다

.

그럼에도불구하고본연구에서밝힌아피제닌에의한멜라닌 합성촉진작용은백반과같은멜라닌합성장애에의한저색소질 환의치료용도로활용될가치가충분하다고판단된다

.

결 론

다양한채소와과일에함유되어있는플라보노이드인아피제 닌은여러생리활성을나타내는데본연구에서처음으로

B16

^흑

색종세포에서멜라닌합성을증가시키는작용이있음을확인하 였다

.

^{본연구실에서는신경세포에서아피제닌의작용이세포막}

에존재하는칼륨

-

염소이온수송체에의하여매개된다는사실을 밝혔는바본연구에서확인한멜라닌합성촉진작용에미치는이 이온수송체의역할을검정하였다

.

^{실험결과에서아피제닌은}

B16

세포에서칼륨

-

염소이온수송체활성유무를검정하는지표인염 소이온

-

^{의존성칼륨이온유출을농도의존적으로나타내었고이}

작용은칼륨

-

^{염소이온수송체를선택적으로억제하는약물인}

DIOA

에의해유의성있게차단되었다

.

이결과는

B16

세포에는칼륨

-

염소이온수송체가기능을하고있으며아피제닌에의해활성화 된다는것을의미한다

.

^칼륨

-

^{염소이온수송체를직접적으로활성}

화시킨다고알려진약물인

NEM

은농도의존적으로염소이온유 출과멜라닌합성을촉진하였고이작용역시칼륨

-

^{염소이온수}

Fig. 4 −

Effects of DIOA, a specific KCC inhibitor, on the apigenin and NEM, a KCC activator-induced Cl

^-

efflux (A and B) and melanin

production (B) in B16 melanoma cells. In figure (A) the data show changes in intracellular Cl

^-

concentration as a function of time,

measured by using the Cl

^-

-sensitive indicator MQAE. In the data, Fo/F values are directly proportional to intracellular Cl

^-

concentration. The arrow shows the time point for addition of apigenin (50

^µ

M) and NEM (100

^µ

M). In figure (C) melanin content was

measured by the method described in the Method section. The cells were initially grown for 12 hr in order to attach them to the

bottom of the culture flasks. Then, the cells were incubated with or without NEM at each designated concentration for 48 hr. Each

column represents the mean value of four replications with bars indicating SEM. *p<0.05 compared to control. Quantitative changes

(figures B and D) were expressed as percent changes of the maximum decease in Fo/F values (B) and of melanin content (D) induced

by the drug compared to control condition in which the cells were treated with a drug-free vehicle. In these experiments apigenin

(50

^µ

M), NEM (100

^µ

M) and DIOA (100

^µ

M) were used. Each column represents the mean value of four replications with bars

indicating SEM. *p<0.05 compared to apigenin alone.

^#

p<0.05 compared to NEM alone.

송체억제제인

DIOA

^{에의해유의성있게차단되었다}

.

^종합적으

로이결과는아피제닌은

B16

^{세포에서칼륨}

-

^{염소이온수송체의}

활성화를통해서멜라닌합성을촉진한다는사실을시사하며백 반과같은멜라닌합성장애에의한저색소질환의치료용도로활 용될가치가있다고판단된다

.

감사의 말씀

본연구는덕성여자대학교

2007

^{년도교내연구비지원에의해}

수행되었음

.

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