東北医科薬科大学 審査学位論文（博士）

東北医科薬科大学 審査学位論文（博士）

氏名（本籍） ｱﾗﾂ

荒 津 佑 輔（福岡県）

学位の種類 博士（薬科学）

学位記番号 博薬科第

10

学位授与の日付 平成

30

3

9

学位授与の要件 学位規則第４条１項該当

学位論文題名

CYP3A4

論文審査委員

主査 教 授 永 田 清

副査 教 授 井ノ口 仁 一

副査 教 授 関 政 幸

CYP3A4 誘導の新規分子メカニズムの 解析に関する研究

東北医科薬科大学大学院薬学研究科 機能病態分子学教室

荒津 佑輔

目次

略語

... 3

緒論

... 5

第

1

CYP3A4

第

1

... 10

2

... 12

3

... 19

第

2

Ban-Lan-Gen

PXR

CYP3A4

第

1

... 21

2

... 22

3

... 29

第

3

CYP3A4

第

1

... 32

2

... 33

3

... 41

第

4

CYP3A4

第

1

... 44

第

2

... 45

第

3

... 50

総括

... 52

実験材料および方法

... 55

謝辞

... 71

参考文献

... 72

略語表

以下の略語を本文および図表中において使用した。

AhR : aryl hydrocarbon receptor ATM : ataxia telangiectasia mutated BPA : bisphenol A

CAR : constitutive androstane receptor CDK2 : cyclin-dependent kinase 2 CTZ : clotrimazole

CYP : cytochrome P450 DBA : dibenz[a,h]anthracene

DMEM : Dulbecco's modified Eagle's medium DMSO : dimethyl sulfoxide

dNR-1 : distal nuclear receptor binding element 1 EMA : European Medicines Agency

eNR3A4 : essential distal nuclear receptor binding element for CYP3A4 induction ERK : extracellular signal-regulated kinase

ER-6 : everted repeat separated by six nucleotides FDA : US Food and Drug Administration

GAPDH : glyceraldehyde-3-phosphate dehydrogenase GBE : Ginkgo biloba extract

hPXR : human PXR

JNK : c-jun N-terminal kinase

MAP2K6 : mitogen-activated protein kinase kinase 6 (MKK6) MOI : multiplicity of infection

PAHs : polycyclic aromatic hydrocarbons

PBPK model : physiologically based pharmacokinetic model PCR : polymerase chain reaction

PMDA : Pharmaceuticals and Medical Devices Agency PSA : polar surface area

PXR : pregnane X receptor qPCR : quantitative PCR reNR3A4 : rifampicin eNR3A4 RF : rifampicin

RXR : retinoid X receptor

SAR : structure activity relationship shRNA : small hairpin RNA

siRNA : small interfering RNA SJW : St. John's wort

TCDD : 2,3,7,8-tetrachlorodibenzo-p-dioxin

緒論

薬物代謝酵素であるチトクローム

P450

CYP

(1)(2)

CYP

(3)(4)

CYP3A4

50%以上の治療薬の代謝に関与(7)する重要な薬物代謝酵

1

CYP

(8)

CYP

(FDA)

(EMA)

(PMDA)

イン

(10)(11)(12)

健康の維持増進のために健康食品を利用する人が増えているが、2017 年に数多 くの健康食品に

CYP

健康食品による

CYP

St. John's wort (SJW)

CYP3A4

現在、医薬品開発では薬物相互作用のリスクが低い薬剤を開発するために探 索段階から

CYP

CYP

を用いたスクリーニング評価

(17)

physiologically based pharmacokinetic model (PBPK

)

一方、

CYP

pregnane X receptor (PXR) (NR1I2)(21)(22)(23), constitutive androstane receptor (CAR) (NR1I3)(24)

vitamin D receptor (NR1I1)(25)

CYP3A4

CYP

(26)

cell line(27)

CYP

CLogP

PXR

(PSA : polar surface area)

(28)

れた報告

(30)(31)

PXR

CYP

(32)(33)

PXR

(RF)

retinoid X receptor (RXR)

CYP3A4

receptor (AhR)

polycyclic aromatic hydrocarbons (PAHs)などが PXR

CYP3A4

告もある

(36)(37)

CYP3A4 gene

Drug PXR

RXR nuclear

CYP3A4 protein

promoter/enhancer region

transcription initiation point

Pregnane X receptor (PXR) Retinoide X receptor (RXR)

-7876 -7200 -362 +11

dNR-1 prER-6

Enhancer region Promoter region

eNR3A4

Fig. 1. The mechanism of CYP3A4 induction through PXR

Ser ³⁵⁰

CDK2

PXR

Ser ³⁵⁰

UDP- glucuronosyltransferase 1A1

CYP3A4

(39)

CYP3A4遺伝子には、TGA(A/C)CTに代表されるいくつかのPXR結合配列が存

在することが報告されている

(40)

CYP3A4

everted repeat separated by six nucleotides (ER-6)が存在し、エンハンサー領域には distal nuclear receptor binding element 1 (dNR-1)

essential distal nuclear receptor binding element for CYP3A4 induction (eNR3A4)が存在することが報告されている (22)(23)(40)(41) (Fig.1)

CYP3A4誘導による薬物相互作用リスクが低い医薬品を開発するためには、誘

CYP3A4誘導分子メ

カニズムを解析するとともに構造活性相関についても検証することを目的とし た。

本研究では、第

1

2

CYP3A4

CYP3A4

その結果、代表的なCYP3A4誘導剤であるRFやビスフェノール類とは異なる新 たに

PAHs

また、健康食品として販売されている

Ban-Lan-Gen

CYP3A4

CYP3A4誘導能を持つことが知られている健

SJW(14)

Ginkgo biloba extract (GBE)(43)

Ban-Lan-Gen

CYP3A4誘導を評価した。その結果、SJWやGBEはRF型、Ban-Lan-GenはPAHs型

CYP3A4

Ban-Lan-Gen

CYP1A1や1A2を強く

(44)

CYP1A1

AhR

PXR

RF

2

さらに第3章では、RF型とPAHs型誘導の分子メカニズムの違いを明らかとす る一環として、

CYP3A4

PXR

在する

PXR

RF

RF

5

14

rifampicin eNR3A4 (reNR3A4)遺伝子領域が必須であることが明らかとなった。しかしなが

PXR

CYP3A4

上流の

dNR-1

eNR3A4

を通じたPAHs型誘導にはPXRを介さない別の経路も存在することが示唆された

（第

3

本研究の中において、

RF型誘導では3-1-10細胞において強い応答性を示したも

PAHs

3-1-20

3

第

4

RF

PAHs

3-1-10

3-1-20

3-1-20細胞に

mitogen-activated protein kinase kinase 6 (MKK6, MAP2K6)

3-1-10

MAP2K6を含む一連のpath wayのPAHs型誘

siRNA

siRNA

RF

第

1

CYP3A4

第1節 序論

ビスフェノールA (BPA)は主にプラスチック（ポリカーボネート樹脂やエポキ シ樹脂等）の原料として利用されている。主に食品用容器や缶詰の内面塗装に 用いられており、加熱や酸、アルカリ、紫外線などの影響により容器からBPA が溶け出すことからヒトが摂取する可能性がある。

1990

(45)

AhRや PXRのリガンドとして CYP1A

CYP3A

(46)

S

AhR

他のビスフェノール類に関する報告はほとんどない。さらに、当研究室では他 にもAhRのリガンドとして知られるPAHsがPXRを介してCYP3A4を誘導するこ と報告

(37)

また、

CYP誘導はCYP阻害と比較してそのメカニズムが複雑である点やin vitro

スクリーニング評価のスループットの低さから構造活性相関に関する情報が乏 しいのが現状である。CYP誘導の構造活性相関に関する情報としては、母核が 同じ構造の化合物では脂溶性が

PXR

PSA

(30)(31)

そこで本研究ではビスフェノール類や

CYP1A

AhR

第

2

まず、

BPA

(Fig. 1-1)

CYP3A4

Noracyarttiyapot

(42)

3-1-20

2

Fig. 1

2

CYP3A4誘導に必要なプロモーターとエンハンサー遺伝子領域をルシフェラー

5’-

化合物名

*( )はBisphenol名

1 (A) 2,2-Bis(4-hydroxyphenyl)propane 13 2,2-Bis(4-hydroxycyclohexyl)propane 2 (B) 2,2-Bis(4-hydroxyphenyl)butane 14 4,4'-Dichloro-α-methylbenzhydrol

3 4,4'-(1,3-Dimethylbutylidene)diphenol 15 Ethyl 2,2-bis(4-chlorophenyl)-2-hydroxyacetate 4 (AP) 1,1-Bis(4-hydroxyphenyl)-1-phenyl-ethane 16 Malachite green

5 (BP) Bis-(4-hydroxyphenyl)diphenylmethane 17 1,1',1''-(Chloromethanetriyl)tribenzene 6 (E) 1,1-Bis(4-hydroxyphenyl)ethane 18 1-Tritylimidazole-4-carboxaldehyde 7 (F) Bis(4-hydroxyphenyl)methane 19 Chloro(2-chlorophenyl)diphenylmethane

8 (AF) 2,2-Bis(4-hydroxyphenyl)hexafluoropropane 20 3,3'-Methylenebis(4-hydroxycoumarin) (dicoumarol) 9 2,2-Bis(3,5-dimethyl-4-hydroxyphenyl)propane 21 4,4'-Biphenol

10 4,4′-Isopropylidenediphenol diacetate 22 Hexestrol

11 4-α-Cumylphenol 23 Dimethyldiphenylsilane

12 2,2-Diphenylpropane 24

Fig. 1-1. Structures and names of BPA and BPA analogs

prER-6 dNR-1 eNR3A4

Luc pCYP3A4-362-7.7k

Transfection to HepG2 cells 

Selection of geneticin resistant cells

pQBI

Stable clone

Fig. 1-2. HepG2-derived cell line clone 3-1-10 and 3-1-20 stably expressing the

No.20 No.19

No.21

No.14 No.15

No.17 No.18

No.22 No.23

No.24 No.4

No.5

No.1 No.2 No.3

No.6 No.7 No.8

No.11 No.10

No.9 No.12

No.13

No.16

OH

RF

Fig. 1-3

RF

BPA

3-1-10および3-1-20細胞のいずれにおいてもレポーター活性の上昇 (11.2倍およ

3.6

)

3-1-10

(3-1-10

10

)

0 10 20 30 40 50

contr ol RF BPA No.2 No.3 No.4 No.5 No.6 No.7 No.8 No.9 No.1 0 No.1 1 No.1 2 No.1 3 No.1 4 No.1 5 No.1 6 No.1 7 No.1 8 No.1 9 No.2 0 No.2 1 No.2 2 No.2 3 No.2 4

R el at ive lu ci fe re as e ac ti vi ty

3-1-10 cell 3-1-20 cell

Fig. 1-3. Effects of BPA and BPA analogs on CYP3A4 gene reporter activity in 3-1-10 and 3-1-20 cells.

Clone 3-1-10 or 3-1-20 were seeded at 1 × 10

cells in 96-well tissue culture plates with 0.1 mL of

DMEM, 24 h before drug treatment. The cells were treated with each drugs (10 M) for 48 h, and

the reporter activity was measured by luciferase assay. Reporter activities are expressed as fold

increase compared with that in the vehicle-treated cells. Data are shown as the mean ± S.D. from

three different samples.

脂溶性と転写活性化の相関性については文献報告

(28)

CLogP

10 M

(A))

(No.1-9)

(No.1-9)

1

38

PSA

clogP clogP

R elativ e luciferease activity

(A) No.1-24 compounds (B) No.1-9 compounds

No. 4

No. 9

No. 5 No. 3

No. 7

No. 2

No. 1 No. 6

No. 8

O

H OH

O

H OH

O

H OH

O

H OH

O

H OH

O

H OH

O

H OH

O

H OH

F FFF FF

O

H OH

H H

Fig. 1-4. Correlation between clogP and fold induction in 3-1-10 cells at 10 M.

clogP were calculated with Pallas. Clone 3-1-10 were seeded at 1 × 10

cells in 96-well tissue

culture plates with 0.1 mL of DMEM, 24 h before drug treatment. The cells were treated with drug

(10 M) for 48 h, and the reporter activity was measured by luciferase assay. Reporter activities are

expressed as fold increase compared with that in the vehicle-treated cells.

また、

10 M

Relative luciferase activity

10

max

50

その結果、

No.1, 2, 3

E max

EC 50

(E max : 49-90

EC 50 : 12-18 M)を示し、 RF (E max : 104、 EC 50 : 8 M)よりやや弱い程度であった。

No.10, 11

EC 50

E max

max : 168, 230、EC 50 : 36, 25 M)。 No.4と9の化合物は細胞毒性によ

E max

EC 50

(Fig.1-5)

No.1 No.2 No.3 No.10 No.11 RF

E

55 ± 10 90 ± 14 49 ± 10 168 ± 62 230 ± 67 104 ± 37 EC

(M) 16 ± 1 18 ± 4 12 ± 1 36 ± 23 25 ± 2 8 ± 8 Fig. 1-5. E

and EC

of No. 1, 2, 3, 10, 11 and RF in 3-1-10 cells.

Clone 3-1-10 were seeded at 3 × 10

cells in 48-well tissue culture plates with 0.3 mL of DMEM, 24 h before drug treatment. The cells were treated with drug (0.3, 1, 3, 10, 30, 100 M) for 48 h, and the reporter activity was measured by luciferase assay. Reporter activities are expressed as fold increase compared with that in the vehicle-treated cells. Data are shown as the mean ± S.D. from three different samples.

E

and EC

were calculated with Phoenix WinNonlin.

次に、ビスフェノール類と同様に

3-1-10

3-1-20

AhR

rifampicin No.1

O O O O

O O

N O O

O O

O O

NN N

N

No.2 No.3 No.4 No.5 No.6 No.7

No.8 No.9 No.10 No.11 No.12 No.13

No.14 No.15 No.16 No.17 No.18

化合物名 化合物名

1 3-Methylcholanthrene 10 7-Methylbenz[a]anthracene 2 Benzo[a]pyrene 11 Dibenz[de, kl]anthracene 3 Benzo[e]pyrene 12 Dibenz[a, c]anthracene 4 pyrene 13 1,2-Benzanthraquinone 5 1,2-Benzanthracene 14 2,6-Diisopropylnaphthalene 6 Naphthalene 15 Anthracene

7 9,10-Dihydroanthracene 16 Benzo[a]phenanthrene 8 Dibenz[a, j]acrydine 17 β-Naphthoflavone (β-NF) 9 Dibenz[a, h]anthracene 18 α-Naphthoflavone (α-NF)

Fig. 1-6. Structures and names of polycyclic aromatic hydrocarbons

Fig. 1-7. Effect of PAHs on CYP3A4 reporter activity in 3-1-10 and 3-1-20 cells.

Clone 3-1-10 and 3-1-20 cells were seeded at 1 × 10

cells in a 96-well tissue culture plate and pre-incubated for 24 h before treatment with various compounds. The cells were treated with RF (10 μM) and various PAHs (1 μM) for 48 h and reporter activity was measured by the luciferase assay.

Reporter activities are expressed as the fold to that in the vehicle-treated cells. Data are shown as the mean ± S.D. from three different samples.

No. 1, 8-10, 12, 16

PAHs

3-1-20

10

PAHs

BPA

3-1-10より3-1-20細胞の応答性が強かった。

0 10 20 30 40 50

RF No . 00 1 No . 00 2 No . 00 3 No . 00 4 No . 00 5 No . 00 6 No . 00 7 No . 00 8 No . 00 9 No . 01 0 No . 01 1 No . 01 2 No . 01 3 No . 01 4 No . 01 5 No . 01 6 No . 01 7 No . 01 8

R ela tiv e lucifer eas e ac tiv ity

3-1-10 cell

3-1-20 cell

第

3

BPAはPXRやAhRのリガンドであるという報告があるものの、他のビスフェノ

CYP3A4

CYP3A4

次に

CYP

(28)

2

その中で脂溶性が高いにも関わらず、レポーター活性が低かった

No.4, 5, 9

No.9の化合物はフェノール部分がキシレノールとなっているが、その転写活性

(Fig.1-4)

また、10 M添加時のRelative luciferase activityが10倍以上の値を示した化合物 については

E max

EC 50

No.1, 2, 3の化合物のE max

50

CYP3A4

考えられた。その一方で

No.10

EC 50

E max

No. 10

(Fig.1-5)

さらに、

AhRのリガンドとして知られるPAHsによるCYP3A4誘導についても検

証した結果、いくつかの化合物に

CYP3A4

RF

PAHs (Fig.1-6)

3-1-20

(49)

No3, 4, 11

本研究の結果から、ビスフェノール類は代表的なCYP3A4誘導剤であるRFと同

様に

3-1-10

AhR

て知られているPAHsは3-1-20細胞における応答性が強いことが明らかとなり、

従来とは異なる誘導分子メカニズムが存在する可能性を示唆した。さらに、構 造活性相関の解析では、ビスフェノール類では脂溶性の低減や立体障害により 誘導が減弱する傾向にあったが、

PAHs

PAHs

第

2

Ban-Lan-Gen

PXR

CYP3A4

第

1

Ban-Lan-Gen

(Isatis tinctoria L.)

Ban-Lan-Gen

Ban-Lan-Gen

quinazolinone alkaloids

(52)。その中でもインディルビンは Ban-Lan-Gen

AhR

CYP1A1

1A2

(44)

その一方で同じ健康食品である

SJW

GBE

CYP3A4

(14)(43)

Ban-Lan-Gen

CYP3A4

Ban-Lan-Gen

CYP3A4

3-1-10

3-1-20

CYP3A4

第

2

まず、

Ban-Lan-Gen

CYP3A4

CYP3A4

3-1-10

3-1-20

SJW

GBE

Ban-Lan-Gen

3-1-20

13

RF

SJW

Ban-Lan-Gen

3-1-10

4

RF

SJW、GBE

(Fig. 2-1)

0 10 20 30 40 50

DMSO RF

(5 μM) IND (5 μM)

SJW (0.1 mg/mL) GBE

(1 mg/mL) BLG (10 mg/mL)

Relative luciferase activity

0 10 20 30 40

DMSO RF

(5 μM) IND (5 μM)

SJW (0.1 mg/mL)

GBE (1 mg/mL)

BLG (10 mg/mL)

** **

**

**

**

<3-1-20>

<3-1-10>

**

**

**

**

Fig. 2-1. Effects of SJW, GBE, and Ban-Lan-Gen on CYP3A4 gene reporter activity in 3-1-10 and 3-1-20 cells.

Clone 3-1-10 and 3-1-20 were seeded at 3 × 10

cells in 24-well tissue culture plates with 0.3 mL of

DMEM, 24 h before St John’s Wort (SJW), Ginko biloba extract (GBE), and Ban-Lan-Gen extract

(BLG) treatment. The cells were treated with SJW (0.1 mg/mL), GBE (0.5 mg/mL), and BGE (10

mg/mL) for 48 h, and the reporter activity was measured by luciferase assay. Reporter activities are

expressed as fold increase compared with that in the vehicle-treated cells. Data are shown as the

mean ± S.D. from three different samples. **P < 0.005, difference from the vehicle-treated cells.

次に

Ban-Lan-Gen

イサチン、インディカン、トリプタントリン、それぞれの

CYP3A4

Fig. 2-2

15

RF

0 2 4 6 8 10 12 14 16 18

Control Rifampicin Indirubin Indigo Isatin Indican Tryptanthrin

Relative luciferase activity

** **

**

* **

**

Fig. 2-2. Effect of various components of Ban-Lan-Gen on CYP3A4 gene reporter activity in 3-1-20 cells.

Clone 3-1-20 was seeded at 3 × 10

cells in 48-well tissue culture plates with 0.3 mL DMEM, 24 h before Ban-Lan-Gen treatment. The cells were treated with each 5 μM of indirubin, indigo, isatin, indicant, tryptanthrin, and RF for 48 h, and the reporter activity was measured by luciferase assay.

Reporter activities are expressed as fold increase compared with that in the vehicle-treated cells.

Data are shown as the mean ± S.D. from three different samples. *P < 0.05, **P < 0.005, difference

from the vehicle-treated cells.

また、

3-1-20

Ban-Lan-Gen

そ の 結 果 、 共 に 添 加 濃 度 に 応 じ て レ ポ ー タ ー 活 性 の 上 昇 が 認 め ら れ 、

Ban-Lan-Gen

E max , EC 50

28.9, 15.4 M、 22.5, 2.0 M

(Fig. 2-3)

(A) Ban-Lan-Gen (B) Indirubin

Fig. 2-3. Effect of various concentrations of Ban-Lan-Gen and indirubin on the CYP3A4 gene reporter activity in 3-1-20 cells.

Clone 3-1-20 was seeded at 3 × 10

cells in 24-well tissue culture plates with 0.3 mL of DMEM 24 h before (A) Ban-Lan-Gen or (B) indirubin treatment. The cells were treated with 1–30 mg/mL (Ban-Lan-Gen) or 1-20 M (indirubin) for 48 h, and the reporter activity was measured by luciferase assay. Reporter activities are expressed as fold increase compared with that in the vehicle-treated cells. Data are shown as the mean ± S.D. from three different samples.

0 5 10 15 20 25

0 5 10 15 20 25 30

Relative luciferase activity

Ban-Lan-Gen(mg/mL)

Emax : 28.9 EC50 : 15.4uM

0 5 10 15 20 25

0 5 10 15 20

Ralative luciferase activity

Indirubin (μM)

Emax : 22.5

EC50 : 2.0uM

さらにインディルビンによる誘導について検証するために、

HepG2

CYP3A4

1A1

PCR

3-1-20

3.1

RF

CYP3A4 mRNA

mRNA

mRNA

RF

1200

0 1 2 3 4 5 6 7

Control RF (5 μM) Indirubin (5 μM)

CYP3A4 m R NA le ve l

0 200 400 600 800 1000 1200 1400 1600

Control RF (5 μM) Indirubin (5 μM)

CYP1A1 m R NA le ve l

*

**

**

A B

Fig. 2-4. Effect of indirubin on CYP mRNA expression in HepG2 cells.

HepG2 cells were seeded at 5 × 10

cells in 24-well tissue culture plates with 0.5 mL DMEM, 24 h before RF and indirubin treatment. The cells were treated with 5 μM RF and 5 μM indirubin for 48 h and then assayed using quantitative polymerase chain reaction (qPCR). CYP3A4 mRNA or CYP1A1 mRNA expression was normalized by expression of GAPDH housekeeping gene and presented as fold increase compared with that of vehicle-treated cells. Data are shown as the mean ± S.D. from three different samples. (A) CYP3A4 mRNA level, (B) CYP1A1 mRNA level. *P < 0.01, **P <

0.005, difference from the vehicle-treated cells.

その一方でヒト凍結肝細胞を用いた検討では

RF

CYP3A4 mRNA

6.1

その傾向は凍結肝細胞のロットが異なっても同様であった（データは示さず）。

(A) CYP3A4 (B) CYP1A1

0 2 4 6 8 10

Control RF(5μM) Indirubin

(1μM) Indirubin (5μM)

CY P3A4 m RNA lev el

0 200 400 600 800 1000 1200

Control RF(5μM) Indirubin (5μM)

CYP1A1 m R NA leve l

* **

Fig. 2-5. Effect of indirubin on CYP mRNA expression in human cryopreserved hepatocyte.

Human cryopreserved hepatocytes were seeded at 1 × 10

cells in 24-well tissue culture plates with 0.5 mL William’s Medium E, 24 h before RF and indirubin treatment. The cells were treated with 5 μM RF and 1 or 5 μM indirubin for 48 h and then assayed using quantitative polymerase chain reaction (qPCR). CYP3A4 mRNA or CYP1A1 mRNA expression was normalized by expression of GAPDH housekeeping gene and presented as fold increase compared with that of vehicle-treated cells. Data are shown as the mean ± S.D. from three different samples. (A) CYP3A4 mRNA level, (B) CYP1A1 mRNA level. *P < 0.05, **P < 0.005, difference from the vehicle-treated cells.

このインディルビンによる

CYP3A4

CYP3A4

PXR

HepG2

Fig. 2-6

(pCYP3A4-362+7.7k)

RF

33

18

PXR

dNR-1, ER-6

pCYP3A4-362+7.7km)

14

7

れた

pCYP3A4-362+7.7kmではインディルビンによるレポーター活性は大き

く低下した。一方、

dNR-1, ER-6

(pCYP3A4-362m+7.7k, pCYP3A4-362+7.7km)

RF

(

9

3

)

pCYP3A4-362+7.7kmをトランスフェクションした際には RF

0.8

Fig. 2-6. Mutational analysis of putative human PXR responsive elements in the CYP3A4 gene.

Schematic structures of reporter plasmids used are shown in the middle. Closed boxes represent

mutated PXR binding elements. HepG2 cells seeded at 3 × 10

cells in 48-well tissue culture plates

preincubated for 24 h before transfection. Transfection into HepG2 cells was performed as described

in the “Materials and Methods”. The cells were treated with 5 μM RF and 5 μM indirubin for 48 h,

and the reporter activity was measured by luciferase assay. Reporter activities are expressed as fold

increase compared with that in the vehicle-treated cells. Data are shown as the mean ± S.D. from

three different samples.

既に

CYP3A4

PXR

CYP3A4

PXR

PXR

small hairpin RNA (shRNA)

PXR

CYP3A4

その結果、

3-1-20

PXR

PXR

RF、インディルビンともにレポーター活性の上昇が認め

(Fig. 2-7A)

shRNA

shRNA

2-7B)

(A) AdhPXR (B) AdhPXR-shRNA

0 20 40 60 80 100 120

Control RF (5 μM) Indirubin (5 μM)

Relative luciferase activity

AdLacZ (10 MOI) AdhPXR (1 MOI) AdhPXR (3 MOI) AdhPXR (10 MOI)

** **

**

**

**

**

**

**

**

0 2 4 6 8 10 12

Control RF (5 μM) Indirubin (5 μM) AdLacZ (3.0 MOI)

AdhPXR-shRNA (0.3 MOI) AdhPXR-shRNA (1.0 MOI) AdhPXR-shRNA (3.0 MOI)

** **

**

**

**

*

**

**

**

Fig. 2-7. Effect of PXR or PXR-shRNA on CYP3A4 gene reporter activity in 3-1-20 cells.

Clone 3-1-20 was seeded at 2 × 10

cells in 24-well tissue culture plates pre-incubated for 24 h and then treated with (A) AdhPXR (MOI of 0, 1, 3, and 10) or (B) AdhPXR-shRNA (MOI of 0, 0.3, 1, and 3). 48 h after infection, these cells were treated with 5 μM RF and 5 μM indirubin for 48 h, and the reporter activity was measured by luciferase assay. Reporter activities are expressed as fold increase compared with that in the vehicle-treated cells uninfected with (A) AdhPXR or (B) AdhPXR-shRNA. Data are shown as the mean ± S.D. from three different samples. *P < 0.01, **P <

0.005, difference from the corresponding AdLacZ-infected cells.

第

3

Ban-Lan-Gen

CYP3A4

3-1-10

3-1-20

RF

SJW

3-1-10

Ban-Lan-Gen

3-1-10

3-1-20

(Fig. 2-1)

1

RF

3-1-10

AhR

PAHs

3-1-20

3-1-10

3-1-20

CYP3A4

RF

AhR

PAHs

Ban-Lan-Gen

PAHs

また、

Ban-Lan-Gen

AhR

CYP3A4

(Fig. 2-2)

EC 50

2.0 M、 E max

22.5

そこでリアルタイム

PCR

HepG2

mRNA

RF

CYP3A4 mRNA

(Fig. 2-4)

その一方で、ヒト凍結肝細胞を用いた検討では

RF

CYP3A4 mRNA

(Fig. 2-5)

2

CDK2

Sivertsson

はコンフルエントな状態にある

Huh7

CDK2

PXR

CYP3A4

HepG2

CDK2

CYP3A4

4

CYP3A4

PXR

インディルビンは

AhR

1A2

(44)

HepG2

CYP1A1

mRNA

CYP3A4

PXR

PXR

PXR

PXR

RF

shRNA

(Fig. 2-7B)

PXR

RF

PXR

CYP3A4

また、

CYP3A4

ER-6, dNR-1, eNR3A4

PXR

eNR3A4

RF

CYP3A4

CYP3A4

PXR

を施したレポーターコンストラクトを作製し、HepG2 細胞にトランスフェクシ ョンすることで評価した。その結果、

PXR

dNR-1, ER-6

ター活性は維持されていた。しかしながら、

eNR3A4

dNR-1, ER-6

RF

サイトに変異を入れたレポータープラスミドでは RF

上昇は全く認められなかった (Fig. 2-6)。

RF

CYP3A4

eNR3A4

(41)

CYP3A4

eNR3A4

れた。しかしながら、

サイトの変異により活性は大きく低下するものの消失は

CYP3A4

3

CYP3A4

第

3

CYP3A4

第1節 序論

第

1

2

RF

3-1-10

RF

AhR

PAHs

3-1-20

PAHs

3

RF

PAHs

CYP3A4

PXR

CYP3A4

ER-6

は

dNR-1

eNR3A4

PXR

(22)(23)(40)(41)。これらの領域に変異や欠失を施したレポータープラスミドを作

製し、

HepG2

RF

PAHs

などによる

CYP3A4

PXR

PXR

第

2

まず、

PXR

RF

(CTZ)

AhR

(TCDD)

CYP3A4

PXR

ER-6

dNR-1

(pCYP3A4-362-7.7k)

HepG2

RF

CTZ, DBA, TCDD

(

8.2, 7.9, 11.0, 9.1

)

pCYP3A4-362-7.7k

dNR-1

dNR-1

ER-6

RF, CTZ

(pCYP3A4-362-7.7km: 2.7

, 1.8

(pCYP3A4-362-7.7km: 10.4

, 6.2

pCYP3A4-362m-7.7km: 5.0

,

5.1