1. No category

Shikonin
( A375.S2 )
N
2
1
2 - AF
1
3
1
4
2
3
4
The Inhibitory Function of Shikonin on N-acetylation of
2-aminofluorene in Human Malignant Melanoma Cells
(A375.S2)
Shang-Wen Yuan1
Guang-Wei Chen2
Jui-Lung Shen1
Huei-Lin Chang3
Jing-Gung Chung4
Shikonin is one of the components of Lithospermum erythrorhizon (Chinese herb medicine) used
for skin infection and allergy for many generations in the Chinese population. In this study, shikonin
was used to determine the inhibition of N-acetylation of 2-aminofluorene (2-AF) in human malignant
melanoma cell line (A375.S2). The amounts of N-acetylation and non-N-acetylation of 2-AF were
measured by high performance liquid chromatography (HPLC). The results demonstrated that N-acetylation of AF from examined systems were decreased by shikonin in a dose-dependent manner. We also
found out that shikonin inhibited the expression of NAT1 gene. Apparently shikonin inhibited N-acetylation of 2-AF in human malignant melanoma cell line. These are the first findings to show shikonin
affect N - acetylation of 2 - aminofluorene of human malignant melanoma cell line ( A375.S2 ).
(Dermatol Sinica 22 : 201-207, 2004)
Key words: Shikonin, Melanoma, N-acetyltransferase (NAT), N-acetylation, Arylamine (2-aminofluorene), Lithospermum erythrorhizon
Shikonin
shikonin
( HPLC )
N
N
NATl
( A375.S2 )
2 - aminofluorene
( 2 - AAF )
( NAT mRNA )
2 - aminofluorene
shikonin
shikonin
N
From the Department of Dermatology, Taichung Veterans General Hospital,1 Institute of Integrated Chinese and Western Medicine,
China Medical University,2 Departments of Pharmacology 3 and Microbilogy,4 China Medical University
Accepted for publication: February 23, 2004
Reprint requests: Jui-Lung Shen, M.D., Department of Dermatology, Taichung Veterans General Hospital. No. 160, Sec. 3, ChungKang Rd., Taichung, Taiwan
TEL: 886-4-23592525 ext.5309 FAX: 886-4-23503025
201
shikonin
( A375.S2 )
(
N
2 - aminofluorene
22 : 201- 207, 2004 )
34 kda
8
( 2 -aminofluorene
1
2 - aminofluorene ( 2 -
2 - AF )
AF )
( NAT )
2 - acetylaminofluorene ( 2 - AAF )
(
1960
Cytochrome P450
Glutathione S - transferase )
2000
6, 9, 10
1
NAT
NAT gene11
NAT
80 %
2
( arylamines )
Shikonin (
DNA
)
DNA adducts
3
Yamagiwa
Ichikawa
4
5
( N - acetyltransferase
NAT )
6, 7
Fig. 1
Dermatol Sinica, September 2004
202
12
288.30 kDa
Lithospermum erythrorhi -
ure 1
zon (
10 µl
6.75 mM 2 - AF
fig-
shikonin ( Ichimaru Pharcos Co.,
)
1.6
Japan ) ( 0
3.2
in DMSO )
4
8
37 ˚C
16
24 mM
5 % CO2
24
2 ml
13
shikonin
(
)
methanol = 95
( ethyl acetate
5)
14
50 µl methanol
15
human premyelocytic
leukemia cell
apoptosis
16
( high performance
human prostate cancer cell
17
20 µl
S180
2 - AF
HPLC )
liquid chromatography
2 - AAF
18
17
2 - AAF
2 - AF
19
2 - AAF
2 - AF
3.
shikonin
shikonin
NAT
( A375.S2 )
shikonin
1x106
well
( A375.S2 )
2 m1
20 µl dd H2 O
NATl
( NAT ) N
well
well
shikonin
16
2
8
24 µM
37 ˚C
1.
4
5 % CO2
24
PBS
trypsin
( A375.S2 ) (
90 % MEN with 0.1 mM non - essen-
tial amino acids and Earle's BSS , 10 %
FBS )
500 ng
RNA
0.5 ml
11.5 µl
RNAase free water
1 µl oligo - dT
37 ˚C 5 % CO2
70 ˚C
7.5 ml solution (
( A375.S2 )
well
5
24
well
well
203
2-
2 - AAF
AF N
10
2 µl 100 mM
DDT , 1 µl 10 mM dNTP , 4 µl 5Xbuffer ,
shikonin
2.
5 x 10
RNA
Kit ( BIO 101 Inc., USA )
)
(
RNA
1 ml
10 µl
0.5 ml RT )
37 ˚C
cDNA
1 µl cDNA
solution (
l
24 µl
19.05 µl H 2 0 , 2.5 µl 10X
buffer , 0.75 µl MgCl2 , 0.5 µl dNTP , 0.5
µl NAT or β-actin primer 1 , 0.5 µl NAT or
Dermatol Sinica, September 2004
Table I . The PCR primers of β-actin and NAT1
Primers
Act-b1
Act-b2
B-MDIEA-NAT1
VPKHGD-X-NAT1
Sequence 5'-3'
5'-GCTCGTCGTCGACAACGGCTC-3'
5'-CAAACATGATCTGGGTCATCTTCTC-3'
5'-CACCCGGATCCCGGGATCATGGACATTGAAGC-3'
5'-GGT CCT CGAGTCAATCACCATGTTTGGGCAC-3'
Size (bp)
21
25
31
31
Ref.
27
28
β-actin primer 2 , 0.2 µl Taq DNA polyPCR
merase )
94 ˚C
A
72 ˚C
1
30
55 ˚C
30
35
30
72 ˚C
10
4 ˚C
Sample
dye
45
94 ˚C
PCR
5
1
DNA
( agarose gel )
1.5 %
B
ethidium bromide
UV
primers
Table
I.
C
1.
shikonin
( A375.S2 )
2 - AF N
D
E
F
G
Fig. 2
Effects of shikonin on AF acetylation and metabolites in
human malignant melanoma cells(A375.S2). After exposure
to 0 mM(A), 1.6 mM(B), 3.2 mM(C), 4 mM(D), 8 mM(E),
16 mM(F), 24 mM(G)of shikonin for 24 hours. The cell suspensions were centrifuged. AF, AAF and metabolites of the
supernatant were measured and determined by HPLC assay.
Dermatol Sinica, September 2004
Fig. 3
Effects of shikonin on production of AAF by human malignant melanoma cells(A375.S2). Cells were incubated with
various concentrations of shikonin for 24 hours. AAF and
metabolites were measured and determined by HPLC assay.
_ SD of
Percentage of AF acetylation was expressed as mean +
three experiments. Data were analyzed by paired Student's t
test.(*: p<0.05)
204
2 - AAF
2.
HPLC
AAF
merase chain reaction )
figure 2
shikonin
RT- PCR ( reverse transcriptase poly-
24
2-
shikonin
shikonin
NATl mRNA
( A375.S2 )
shikonin
HPLC
2 - AAF
2 - AAF
A375.S2
figure 3
PCR
2 - AAF
shikonin
24
35
NAT1
UV
figure 4 ( A )
shikonin
NAT1 mRNA
( NAT )
( densitometric
( dose-dependent effect )
analysis of Gel-photograph )
( NAT )
shikonin
shikonin
figure 4 ( B )
16
NATl
µM
24 µM
shikonin
NAT1
expression
2 - AF
N
2 - AAF
Shikonin
0
2
4
8
16
24
( µM)
shikonin
18
shikonin
20
shikonin
shikonin
NAT
2 - AF
NAT
( NAT )
2 - AF
Fig. 4
Effect of shikonin on NAT1 mRNA expression of human
malignant melanoma cells(A375.S2). Cells were incubated
with various concentration of shikonin for 24 hrs followed by
RT-PCR analysis.(A)Gel electrophoresis of RT-PCR products
with primers for NAT1.(B)NAT1 /β-actin mRNA ratio was
expressed as mean
SD of three experiments. Data were
analyzed by paired Student's t test.(*: p<0.05)
205
NAT
21, 22
( NAT )
HPLC
shikonin
Dermatol Sinica, September 2004
( A375.S2 )
2 - AF N
2 - AAF
2 - AAF
shikonin
NAT
2 - AF N
shikonin
NAT1
8p22
NAT1
Locus
genotypes
intron
23
NATP
NAT2
NAT2
NAT1
exon
reading frames
870 bp
open
290
87 %
55
NAT 2
NAT 1
24
N AT 3 2 5
N AT
24
NAT2
NAT1
N AT 1
26
NAT2
NAT
N AT
NATl
NAT2
26
NAT1
NAT2
RT- PCR
NAT1
NATl
shikonin
NAT1
NAT
shikonin
shikonin
NAT1
NAT
2 - AF N
2 - aminofluorene
N
shikonin
malignant melanoma
DNA adducts
in vivo
Dermatol Sinica, September 2004
REFERENCE
1. Irwin MF, Arthur ZE, Klaus W, et al.: Fizpatrick's
Dermatology in general medicine. 5th ed. , USA,
the Mc Graw-Hill Companies, pp1080-1116, 1999.
2. Cambers RW: Chemical carcinogenesis :A biochemical overview. Clin Biochem 18: 158-168,
1985.
3. Selkrik JK, Maccleod MC: Chemical carcinogenesis Nature's metabolic mistake. Bioscience 32:
601-605, 1982.
4. Yamagiwa K, Ichikawa K: Experimental study of
the pathogenesis of carcinoma. CA Cancer J Clin
27: 174-181, 1977.
5. de Gruijl FR: Skin cancer and solar UV radiation.
Eur J Cancer(part A) 35: 2003-2009, 1999.
6. Herman A: Genetic polymorphisms in human
xenobiotica metabolizing enzymes as susceptibility factors in toxic response. Mutation Res 464: 6576, 2000.
7. Adamson R, Gastsfsson J A, Ito N, et al.: Proc 23rd
tnt princess Takamatsu cancer research fund
Princeton scientific priceton NJ 1-360, 1995.
8. Pires de Melo M, Curi TCP, Curi R, et al.:
Peroxidase activity may play a role in the cytotoxic effect of indole acetic acid. Photobiology 65:
338-341, 1997.
9. Lutz W: N-acetyltransferase genetic polymorphism
and its role in the development of neoplastic disease. Med Pr 51: 277-284, 2000.
10. Levy GN, Chung JG, Weber WW: 2-aminofluorene metabolism and DNA adduct formation by
mononuclear leukocytes from rapid and slow
acetylator mouse strains. Carcinogenesis 15: 353357, 1994.
11. Andrzej S, Alexander P, Igor S, et al.: Serotoninergic system in hamster skin. J Invest Dermatol
119: 934-942, 2002.
12. Li H, Luo S, Zhou T: Studies on in vitro metabolism of shikonin. Phytother Res 13: 236-238,
1999.
13. Hisa T: Shikonin, an ingredient of Lithospermum
erythrorhizon, inhibits angiogenesis in vivo and in
vitro. Anticancer Res 18(2A): 783-790, 1998.
14. Yuan SW, Tsou MF, Chen GW, et al.: Shikonin
inhibited growth and N-Acetylation of 2Aminofluorene in bacteria strains Staphylococcus
aureus and Klebsiella pneumoniae. Mid-Taiwan J
Med 8: 119-126, 2003.
,
,1 ,
,
15.
, 1565-1574, 1998.
206
16. Yoosik Y: Shikonin, an ingredient of Lithospermum erythrorhizon induced apoptosis in HL60
human premyelocytic leukemia cell line. Planta
Med 65: 532-535, 1999.
17. Gaddipati JP, Shefali MH, Raj K, et al.: Inhibition
of growth and regulation of IGFs and VEGF in
human prostate cancer cell lines by shikonin analogue. Anticancer Res 20: 2547-2552, 2000.
18. Sankawa U, Ebizuka Y, Miyazali T, et al.:
Antitumor activity of shikonin and its derivatives.
Chem Pharm Bull 25: 2392, 1997.
:
18: 71, 1991.
19.
20. Guo XP, Zhang XY, Zhang SD: Chung His Chieh
Ho Tsa Chih 11: 598-599, 1991.
21. Heares DJ, Weber WW: Multiple N-acetyltransferase and drug metabolism. Tissue distribution,
characterization and significance of mammalian
N-acetyltransferse. Biochemistry 132: 519-526,
1973.
22. Hein DW, Rustan JD, Furman EJ, et al.: Extrahepatic expression of the N-acetylation polymorphism toward arylamine carcinogens in tumor target organ of an inbred rat model. J Pharmacol Exp
Ther 258: 232-236, 1991.
207
23. Hickman D, Risch A, Buckle V, et al.: Chromosomal localization of human genes for arylamine
N-acetyltransferase. Biochemistry 297: 441-445,
1994.
24. Blum M, Grant DM, McBride W, et al.: Human
arylamine N-acetyltransferase genes: isolation,
chromosomal localization and functional expression. DNA Cell Biol 9: 193-203, 1990.
25. Kelly SL, Sim E: Arylamine N-acetyltransferase
in Balb/c mice: identification of a novel mouse
isoenzyme by cloning and expression in vitro.
Biochem J 302: 347-353, 1994.
26. Pacifici GM, Bencini C, Rane A: Acetyltransferase in humans: development and tissue distribution. Pharmacology 32: 283-291, 1986.
27. Homo sapiens actin, beta(ACTB), Mrna. Gi
5016088 ref NM-001101.2 . Gene Bank
number NM-001101.
28. Shinya K, Takami A, Kazuhisa S, et al.: Evaluation of biological responses to polymeric biomaterials by RT-PCR analysis IV study of c-myc, cfos and p53 mRNA expression. Biomaterials 21:
521-527, 2000.
Dermatol Sinica, September 2004