Screening of the Bufonis Venenum on Hep G2 Cells

서 론

3 7

OECD ¹⁾.

56 ²⁾ “

”

. ³⁾ “

” .

cinobufotoxin, cinobufotalin, cinobufotalidlin⁴⁾ bufalin, cinobufagin resibuf-

ogenin ⁵⁾.

蟾 肝癌

강아미, 김보람, 김승욱, 임성우 동국대학교 일산한방병원 한방내과 Original Article

Screening of the Bufonis Venenum on Hep G2 Cells

Amy Kang, Bo-Ram Kim, Sung-Uk Kim, Seong-Woo Lim

Department of Internal Medicine, College of Korean Medicine, Oriental Medicine

Objective: Bufonis Venenum is the traditional Korean medicine Chan Su, which is obtained from the skin and parotid venom gland of the toads. It has been used for myocardial diseases, inflammation diseases, pain relief, cancer and others. The main components of BV are cinobufotoxin, cinobufalin, bufalin and others. Of these, bufalin, the major active ingredient of BV, has been reported to induce apoptosis and to possess anti-tumor effects. There was no report of anti-tumor screening of BV on hepatic cancer and which signaling pathway can be involved. In order to examine the effect of BV on hepatic cancer and the related signaling pathway with BV-induced apoptosis, human Hep G2 cells were used.

Methods: Analysis of apoptosis was confirmed by MTT assay. BV decreased cell viability in a dose and duration dependent manner. To observe which signaling molecules will be activated by BV, phosphorylation of MAPK (p38, ERK, JNK), caspase 8 and caspase 9 were examined by Western blot analysis.

Results: The phosphorylation levels of p38 started to increase at 5 min after addition of 5 /ml of BV and sustained to increase until 48 hours. The phosphorylation levels of other MAPK (ERK and JNK), caspase 8 and caspase 9 increased in a time-dependent manner. These imply that BV may activate different signaling pathways, MAPK, caspase 8 and caspase 9. These results propose that BV may induce apoptosis on Hep G2 cells through the activation of MAPK, caspase 8 and caspase 9.

Key Words : Bufonis Venenum, Hep G2 cell, apoptosis, MAPK, caspase 8, caspase 9.

접수：2008년 8월 18일 수정：2008년 9월 8일 채택：2008년 9월 11일

교신저자 : 임성우(Seong-Woo Lim)

경기도 고양시 일산동구 식사동 814 동국대학교 일산한방병원 한방내과

Tel : +82-31-961-9043, Fax : +82-31-961-9009, E-mail : [email protected]

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MTT assay Western

blot Hep G2

.

실 험

1. 재료 1)

dimethylsulfoxide (DMSO; Sigma) , 22 filter

.

2. 방법 1)

Hep G2 Korean Cell Line Bank (KCLB; Korea) .

2)

37°C, 5% CO² incubator 10% Fetal Bovine Serum(FBS; Sigma, USA) Dulbe- cco's modified Eagle's medium(DMEM, USA)

, 100

unit/ml penicillin, 100 /ml streptomycin(Gibco/BRL, USA)(PS) , Trypsin-EDTA(Gibco/BRL)

. 2-3

.

3) (MTT assay)

Hep G2 MTT assay

. (1)

MTT assay 24 well plate

Hep G2(5x103cells/well) 12 FBS

serum free medium, DMEM

(Invitrogen) 4

, 0, 1, 5, 10, 15 20 /ml

48 , 3-(4,5-Dimethylthiazol-

2-yl)-2,5-diphenyl-tetra-zolium bromide(MTT. Sigma)

2mg/ml , incubator 4

DMSO 590nm microplate

reader(Molecular devices, USA) . (2)

MTT assay 24 well plate

Hep G2(5x103cells/well) 12 FBS

serum free medium, DMEM

(Invitrogen) 4

, 5 15 /ml 24

48 .

Cell Viability control

O.D. 100%

, O.D.

.

Cell Viability = ( / ) × 100 .

4) Western blot

Hep G2

, 5 /ml

10min,30min,1h,4h,6h,12h,24h,48h western blot .

(62.5 mmol/1 Tris-HCl, pH 6.8, 2% SDS, 20% glycerol, 10%

2-mercaptoethanol) boiling cell

. bicinchoninic acid (BCA, Pierce)

. 50 4-12%

sodium dodecylsulfate-polyacrylamide gradient gel

(Invitrogen) (SDS-PAGE)

, nitrocellulose paper (Amersham) . Ponceau-S

0.1 % Tween 20 Tris-buffered saline (TBS-T)

5% 30 blocking .

4 C 16

TBS-T 10 3 ,

blot 2 1 . 2

enhanced chemiluminiscence system

(ECL, Pierce) .

image (LAS-

3000, Fuji) .

p38 MAPK

Hep G2 ,

p38 .

10min,30min,1h,4h,6h,12h,24h, 48h

blot .

ERK(extracellular signal-regulated kinase)

ERK

. JNK(C-Jun N-terminal kinase)

JNK

. Caspase 8

Caspase 8 . Caspase 9

Caspase 9 .

5)

± (mean±S.E.M)

, Window

SPSS .

, Tukey

test .

p<0.05 .

성 적

1. MTT assay에 의한 세포 활성도 변화 측정 1)

Hep G2 48 MTT assay

, 1 /ml

63.98% (0.753±0.012) , 5 /ml 49.68% (0.575±0.012)

10 /ml 33.04% (0.370±0.017) 1 /ml

. 15 /ml

Concentration of BV Cell Viability%

0 ㎍/ml 100.00

1 ㎍/ml 63.98

5 ㎍/ml 49.68

10 ㎍/ml 33.04

15 ㎍/ml 29.91

20 ㎍/mll 27.20

Table 1.

20 /ml 29.91% (0.358±0.009), 27.20%

(0.325±0.017)

(Table 1, Fig. 1).

2)

5 /ml 15 /ml

Hep G2 24 48

MTT assay

, 5 /ml 24h 48h

48.43% (0.598±0.054) 38.07%

(0.470±0.037) , 15 /ml

24h 48h 40.36% (0.499±0.066)

25.06% (0.310±0.031)

(Table 2, Fig. 2).

2. Western blot 분석 1) p38 MAPK

5 /ml Hep G2

1.40 1.20 1.00 0.08 0.06 0.04 0.02 Cell viability (O 〮D 〮)

0.00

0ug/ml 1ug/ml 5ug/ml 10ug/ml 15ug/ml 20ug/ml Drug concentration

Fig. 1.

Concentration of TV Cell Viability(%)

24h 48h

0 ㎍/ml 100.00 100.00

5 ㎍/ml 48.43 38.07

15 ㎍/ml 40.36 25.06

Table 2.

1.40 1.20 1.00 0.08 0.06 0.04 0.02 Cell viability (O 〮D 〮)

0.00

0ug/ml

Drug concentration 5ug/ml 15ug/ml

24h 48h

Fig. 2.

Phosphorylation of p38(Density arbitrary units)

p-p38

p38

300 250 200 150 100 50

0 C 10’ 30’ 1h 4h 6h 12h 24h 48h

Phospho-p38 MAPK

p38 MAPK C 10’ 30’ 1h 4h 6h 12h 24h 48h

Fig. 3.

Western blot

p38 ,

Hep G2 p38

1 48

(Fig. 3).

2) ERK(extracellular signal-regulated kinase)

5 /ml Hep G2

Western blot

ERK ,

Hep G2 ERK

4

48 (Fig. 4).

3) JNK(C-Jun N-terminal kinase)

5 /ml Hep G2

Western blot

JNK ,

Hep G2 JNK

. ERK p38 1

48 35%

(Fig.5).

Phosphorylation of ERK(Density arbitrary units)

p-ERK

ERK

800 750

500 450 300

100

0 C 10’ 30’ 1h 4h 6h 12h 24h 48h

Phospho-p44 MAPK C 10’ 30’ 1h 4h 6h 12h 24h 48h

Phospho-p42 MAPK

p44 MAPK p42 MAPK

200 600

Fig. 4.

Phosphorylation of JNK(Density arbitrary units)

p-JNK

JNK

800 750

500 450 300

100

0 C 10’ 30’ 1h 4h 6h 12h 24h 48h

Phospho-p54 MAPK C 10’ 30’ 1h 4h 6h 12h 24h 48h

Phospho-p42 MAPK p44 MAPK p42 MAPK

200 600

Fig. 5.

4) Caspase8

5 /ml Hep G2

Caspase 8 Western

blot Caspase 8 ,

Hep G2

Caspase 8 Caspase 8

(Fig. 6).

5) Caspase 9

5 /ml Hep G2

Caspase 9 Western

blot Caspase 8 ,

Hep G2

Caspase 9 Caspase 9

(Fig. 7).

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C 10’ 30’ 1h 4h 6h 12h 24h 48h

Caspase8

B-actin

Full length Caspase-8 Clewed Caspase-8p 43/p41

B-actin

Fig. 6.

C 10’ 30’ 1h 4h 6h 12h 24h 48h

Caspase8

B-actin

Full length Caspase-8 Clewed Caspase-8p 43/p41

B-actin

Fig. 7.

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apoptosis ^28,29)

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30). Hep G2

. Hep

G2

MTT assay western blot

MAPK caspase 8 9 .

MTT Assay

0% Hep G2

100% Hep G2

70% .

. 1 /ml 63.98%

5 /ml 49.68%

10 /ml 33.04% 1

/ml

. 15 /ml 20 /ml 29.91%, 27.20%

Hep G2

.

5 /ml 24h 48h

48.43% 38.07%

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Hep G2 . MAP Kinase

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apoptosis , ERK down-

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Myoblast

31). ERK MAP Kinase pro-survival

factor , pro-apoptotic

manner

. , Neuronal system ERK Neurodegeneration

32).

ERK p38 JNK

inhibitor

. p38, ERK, JNK

Transcription

apoptosis .

.

ERK Cisplatin

apoptosis ³³⁾. Cisplatin

34). Cisplatin

.

Hep G2 caspase 8, caspase 9

48

caspase 8, caspase 9

. Hep G2 apotosis

caspase 8, caspase 9 . Apoptosis

caspase .

caspase

. Bcl-2

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caspase 9 , caspase 3

caspase

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necrosis factor) Fas

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caspase (effector caspase) (3, 6, 7) caspase

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MAP Kinase caspase pathway .

apoptosis

.

결 론

Hep G2

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G2 MTT assay ,

, HepG2 ,

HepG2 ERK, p38,

JNK ,

Hep G2 caspase 8 caspase 9 .

참고문헌

1. , .

(2002.1~2002.12), 2007.

2. . . : . 1976:119-171.

3. . , . : .1913:29,

36-46,48,56,91,93,127,133,141,149-150,175,179 ,189,217,240.

4. , .

. .1996;4:117-128.

5. Morishita S, Shoji M, Oguni Y, et al. Pharma- cological actions of “kyushin” a drug containing toad venom; cardiotonic and arrhythmogenic effects and excitatory effect on respiration. Am J Chin. 1992;20:245-6.

6. . . :

. 1995:687-693.

7. . . : . 1986:258-9.

8. . . :

. 1983:1258-1264.

9. . ( ). : . 1992

:1889-1895.

10. . . : . 1993:65-70.

11. Masahiko W, Kaori I, Yutaka M, Shigeo N, Kazuyasu N. Activation of AP-1 is required for bufalin-induced apoptosis in human leukemia U937 cells. Oncogene. 1998;16:779-787.

12. Watabe M, Masuda Y, Nakajo S, Yoshida T, Kuroiwa Y, Nakaya K. The cooperative intera- ction of two different signaling pathways in response to bufalin induces apoptosis in human leukemia U937 Cells. The journal of biological chemistry. 1996;271(24):14067-14073.

13. Watabe M, Kawazoe N, Masuda Y, Nakajo S, Nakaya K. Bcl-2 Protein Inhibits Bufalin- induced Apoptosis through Inhibition of Mitogen activated Protein Kinase Activation in Human Leukemia U937 Cells.Cancer Research. 1997;

57:3097-3100.

14. Kurosawa M, Numazawa S, Tani Y, Yoshida T.

ERK signaling mediates the induction of infam- matory cytokines by bufalin in human monocytic cells. Am Physiol Cell Physiol. 2000;278:500 -508.

15. . T24

apoptosis .

. 2005.

16. . . :

. 1999:145-146.

17. . . : . 2007:400-2 .

18. . . : . 1909:1221.

19. . . : .

1968:125-134.

20. . . : . 1975:250-252.

21. . . : . 1970:4-6,46-7.

22. . . : . 1969:308-9.

23. . . : . 1978:304-5.

24. . . :

. 1975:627-9.

25. . . :

. 339-341.

26. . . . :

. 1981:423.

27. . . . 1986:556.

28. Masahiro K, Satoshi N, Yoshihiro T, Takemi Y.

ERK signaling mediates the induction of inflam- matory cytokines by bufalin in humanmonocytic cells. Am J Physiol Cell Physiol. 2000;278:500 -508.

29. Kaei N, Masakazu N, Tami U, Noriyuki T, Sun B, Hisashi N, Isao M. Bufalin induces apoptosis and the G0/G1 cell cycle arrest of endometriotic stromal cells: a promising agent for the treatment of endometriosis. Molecular Human Reproduction.

2005;11:817-823.

30. Steller H. Mechanisms and genes of cellular suicide. Science. 1995;267:1445-1449.

31. Lee J, Hong F, Kwon S, Kim SS, Kim DO, Kang HS, Lee SJ, Ha J, Kim SS. Activation of p38 MAPK induces cell cycle arrest via inhibition of Raf/ERK pathway during muscle differentiation.

Biochem. Biophys. Res. Commun. 2002;298:765 -771.

32. Cheung EC,Slack RS. Emerging role for ERK as a key regulator of neuronal apoptosis.Sci.

STKE. 2004.

33. Hayakawa J, Ohmichi M, Kurachi H, Ikegami H, Kimura A, Matsuoka T, Jikihara H, Mercola D, Murata Y. Inhibition of extracellula signal- regulated protein kinase of c-Jun N-terminal protein kinase cascade, differentially activated by cisplatin, sensitizes human ovarian cancer cell line. J.Biol.Chem. 1999;274:31648-31654.

34. Wang X, Martindale JL,Holbrook NJ, Require- ment for ERK activation in cisplatin-induced apoptosis. J. Biol. Chem. 2000;275:39435-39443.

35. Ghobrial IM, et al. Targeting apoptosis pathways in cancer therapy.CA Cancer J.Clin. 2005;55:178 -194.