Document 6421934

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Document 6421934

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
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.
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
( 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
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