全 文 ：植物来源的蛋白磷酸酶 Cdc25C 的新抑制剂 ?
贾锐锐1 , 曾广智1 , 谭宁华1
??
, 范君婷1 , 2 , 黄火强1 , 2 ,
嵇长久1 ,2 , 郝小江1 , 赵勤实1 , 李朝明1
(1 中国科学院昆明植物研究所植物化学与西部植物资源持续利用国家重点实验室 , 云南 昆明 650204;
2 中国科学院研究生院 , 北京 100049 )
摘要 : 蛋白磷酸酶 Cdc25C 能够使有丝分裂激酶 CDK1?cyclin B 去磷酸化 , 从而促进细胞周期的进程。已经在
一些肿瘤细胞中检测到 Cdc25C 的过量表达 , 这使得 Cdc25C 成为肿瘤治疗中的潜在靶标。通过随机筛选 , 发
现了八个 Cdc25C 的天然新抑制剂 (1 - 8) , 其 IC50 值在 1.66～75 .07μmol?L 之间。肿瘤细胞毒试验结果表明 ,
其中四个化合物 (化合物 3 , 4 , 5 , 7) 对十种肿瘤细胞株显示一定的细胞毒活性 , 其 IC50 值皆小于 10μg?mL。
关键词 : Cdc25C; 磷酸酶 ; 天然抑制剂 ; 细胞周期 ; 肿瘤
中图分类号 : Q 946 文献标识码 : A 文章编号 : 0253 - 2700 (2008) 05 - 617 - 04
New Inhibitors of Dual-specificity Protein
Phosphatase Cdc25C from Plants *
J IA Rui-Rui
1
, ZENG Guang-Zhi
1
, TAN Ning-Hua
1 **
, FAN Jun-Ting
1 , 2
,
HUANG Huo-Qiang1 , 2 , J I Chang-J iu1 , 2 , HAO Xiao-J iang1 ,
ZHAO Qin-Shi
1
, LI Chao-Ming
1
(1 State KeyLaboratory of Phytochemistry and Plant Resources in West China, Kunming Instituteof Botany, Chinese Academy
of Sciences, Kunming 650204 , China; 2 GraduateSchool of the ChineseAcademy of Sciences, Beijing 100049 , China)
Abstract : Protein phosphataseCdc25C dephosphorylates themitotic kinaseCDK1?cyclin B , andthen triggers thecell cycle
progression . Overexpression of Cdc25C is detected in some cancer cells, which made it to be a potential target for anti-cancer
drugs . ThroughCdc25C assay, compounds1 - 8 werefoundto benew natural inhibitorsagainst Cdc25C with IC50 s of 1.66 - 75.07
μmol?L . Moreover, 3 , 4 , 5 and7 exhibited cytotoxicities on a panel of ten cancer cell lineswith IC50 s below10μg?mL .
Key words: Cdc25C; Phosphatase; Natural inhibitor; Cell cycle; Cancer
Common features shared by all cancers are a dis-
ordered cell cycle and irregularities in the molecules
that control this cycle ( Kristjansdottir and Rudolph,
2004) . In eukaryotic cells, cell cycle progression is
controlled bycyclin dependent kinases (CDKs) , which
aremaintained in an inactive state through phosphory-
lation by Wee1?Mik1?Myt1 protein kinases and in an
active state through dephosphorylation by protein phos-
phatases Cdc25s ( Ham et al. , 1998; Donzelli and
Draetta, 2003) . Cdc25s are a family of dual-specifici-
ty protein phosphatases, which contain three homo-
logues named Cdc25A , Cdc25B andCdc25C in human
cells . Each homologue controls cell cycleprogression at
distinct checkpoints to promote cell proliferation (Eck-
云 南 植 物 研 究 2008 , 30 (5) : 617～620
Acta Botanica Yunnanica DOI : 10 .3724?SP. J . 1143 .2008.07295
?
?? ?Author for correspondence; Tel?Fax: 0871 - 5223800; E-mail : nhtan@ mail .kib. ac. cn
Received date: 2007 - 12 - 14 , Accepted date: 2008 - 01 - 03
作者简介 : 贾锐锐 (1974 - ) 女 , 硕士 , 主要从事体外抗肿瘤活性天然产物研究。 ?
Foun ?dation items: The National Natural Science Foundation of China (30725048) and the Foundation of ChineseAcademy of Sciences ( West Light
Program)
stein, 2000; Tamura et al. , 2000) . Cdc25A is mainly
thought to activateCDK2?cyclin E and thereby triggers
theG1?S transition of the cell cycle . Cdc25B appears
to play a role in both G1 and G2 phases, while
Cdc25C specifically dephosphorylates CDK1?cyclin B
and triggers the G2?M transition ( Loukaci et al. ,
2001) . For their important role in cell cycle regula-
tion, Cdc25s have been attractive targets for drug de-
velopment (Sohn et al. , 2003; Prevost et al. , 2003) .
Inhibitors of these enzymes may become potential che-
motherapeutic agents in the field of tumor treatment .
Several natural and synthetic compounds were found to
be inhibitorsof Cdc25s, such asdysidiolideand its an-
alogs ( Gunasekera et al. , 1996; Shimazawa et al. ,
2004) , vitamin K3 and quinone derivatives ( Ham et
al. , 1997; Cao et al. , 2005; Lavergne et al. , 2006) ,
sulfircin (Cebula et al. , 1997 ) and vanadate ( Huyer
et al. , 1997 ) etc .
In our program of searching for inhibitors of
Cdc25C, hundredsof natural products inour compound
library were screened . Results indicated that com-
pounds 1 - 8 (Fig . 1) demonstrated inhibitory activities
against Cdc25C, and 3 , 4 , 5 and 7 also showed cyto-
toxicities on several cancer cell lines .
1 Materials and methods
1 .1 Materials
Cdc25C was presented by Bayer AG (Germany) . Substrate
O-methyl fluorescein phosphateand product fluorescent O-methyl
fluoresceinarefromSigma (M-2629 , M-7004) . Vanadiumoxide
(V2 O5 ) (Sigma, V-6881) was used as areferencecompound for
Cdc25C assay . Human cancer cell lines A549 ( lung cancer) ,
BGC-823 ( gastric cancer cell line) , SGC-7901 ( gastric cancer
cell line) , DU-145 ( prostate cancer cell line) , MDA-MB-231
(breast cancer cell line) , HT-29 (colon cancer cell line) , BEL-
7402 ( hepatic cancer cell line) , MCF-7 ( breast cancer cell
line) and U-251 (glioma) were purchased from the Cell Culture
Centre of Institute of Basic Medical Sciences, Chinese Academy
of Medical Sciences (Perking, China) .
1 . 2 Chemistry and Pharmacology
Compounds 1 - 8 (Fig . 1) were isolatedfrom various plants
by uswith their purities > 95% . Detailed purificationsand iden-
tifications of these compounds were described previously (Shang
et al. , 1994; Montenegro et al. , 2003; Peng et al. , 1990;
Wang et al. , 2000 ; Xu et al. , 2005; Iwao et al. , 1985;
Itokawa et al. , 1991) . Inhibition data were expressed as IC50
values, which were calculated by dose-response curves with at
least four concentrations ( dilutionratio= 1?2) , thehighest tested
concentration for Cdc25C and cyctotoxicity assay are 40 μg?mL
and10μg?mL respectively . Results are expressed as mean IC50
values±standard deviations .
Fig . 1 Chemical structures of compounds 1 - 8
Plant resources: 1 . from Fissistigma kwangsiense ( Annonaceae) ; 2 . from Uvaria tonkinensis ( Annonaceae) ; 3 . from Michelia yunnanensis
(Magnoliaceae) ; 4 . from Tsoongiodendron odorum (Magnoliaceae) ; 5 . from Salvia castanea f . tomentosa (Labiatae) ; 6 . from Salvia castanea
f . tomentosa (Labiatae) ; 7 . from Impatiens chungtienensis ( Balsaminaceae) ; 8 . from Sabina gaussenii ( Cupressaceae)
816 云 南 植 物 研 究 30 卷
1 .3 Cdc25C Assay
Compound inhibitory activity against Cdc25C was measured
as themethoddescribed previously (Ducruet et al. , 2000 ) . Test
compoundswere diluted with the reaction buffer ( pH 8.2 , 100
mmol?L TrisBase, 40 mmol?L NaCl , 0 . 01% BSA , 5 mmol?L
DTT and 20% Glycerol ) , then 1 mmol?L OMFP and 24μg?mL
enzyme were added to start the reaction . After incubationfor 120
min at 25℃ , fluorescence was monitored at 530?40 nmafter ex-
citation at 490?40 nm in a Cytofluor II fluorescence plate reader
(Perseptive Biosystems) .
1 . 4 Cell Growth Inhibition Assay
All cancer cells were cultured in RPMI-1640 containing
10% fetal bovine serum and 5 CC Pen-Strep . Compound cyto-
toxicitywas assessed by the sulfurhodamine B ( SRB) assay de-
scribed before ( Skehan et al. , 1990 ) . Firstly, 3000 - 7000
cells?well were plated in96-well plates . Twenty-four hours later,
compoundswereadded to afinal concentration of 10μg?mL . Af-
ter incubated for 48 h, cells were fixed by the addition of 50%
ice-cold trichloroacetic acid and left at 4℃ for 1 h . Then Plates
were washed 5 times in water , air-dried, and stained for 15 min
with 100μl 0 .4% SRB in1% glacial acetic acid . Excessivedye
was removed bywashing5 times in1% glacial acetic acid . After
plateswere air-dried, SRB was resuspended in 100μl 10 mmol?L
Tris and the OD values were read at 560 nm on a 96-well plate
reader (Molecular Devices, SPECTRA MAX 340) .
2 Results and discussion
Through the inhibition assay, eight natural inhibi-
tors containing flavones ( 1 ) , alkaloids ( 2 - 4 ) and
quinonederivatives (5 - 8) were found against Cdc25C
(Fig . 1 ) with IC50 values ranging from 1 .66 to 75 .07
μmol?L ( Table 1 ) . The structure-activity relationship
of these compounds was found . In alkaloids, compound
2 , which lacked methylenedioxy, was active with IC50
values between compound 3 and 4 , which suggested
that methylenedioxy is not necessary for Cdc25C inhibi-
tory activity . But compared with compound 4 , satura-
tion of compound 3 was important for its activity . Com-
pound 5 and 6 contain naphthoquinone structures,
which may be the reason for their inhibitory activities
against Cdc25C . Compound 7 and 8 belong to quinone
derivatives, which are proved to be the potent inhibi-
tors of Cdc25 family . Further studies towards structure-
activity relationship are in progress . Result from cell
growth inhibition assay indicated that compounds3 , 4 ,
5 and 7 showed cytotoxicities with IC50 values below 50
μmol?L (Table 2 ) . It suggested that these compounds
may exert anti-tumor activity by blocking cell cycle
progression . Especially, compound 7 inhibited cell
proliferation in a broad spectrum of cancer cell lines,
in which DU145 was themost sensitiveonewith IC50 of
8 .83μmol?L , while compound 4 selectively affected
A549 with IC50 of 20 .44μmol?L .
Compounds inhibiting Cdc25 dual-specificity
phosphatase activities might be as potent anticancer
agents . With random screening, we found eight new
natural inhibitorsof Cdc25C, which broaden the struc-
tural diversity of Cdc25 inhibitors . Moreover, some in-
hibitors exhibited efficacious and broad anti-tumor ac-
tivities in various cancer cell lines . All the results sug-
Table 1 Inhibitory activities of 1 - 8 against Cdc25C
Comp . IC50 ?(μmol?L) Comp . IC50 ((μmol?L )
1 ?19 .46±1 y. 11 5 i41 .84±2 .41
2 ?11 .82±0 y. 93 6 i15 .61±0 .72
3 ?1 .66±2 g. 24 7 i12 .50±2 .29
4 ?25 .27±2 y. 98 8 i75 .07±3 .48
V2 ?O5 0 .44±0 g. 11
IC50 values are means of three experiments .
Table 2 Cytotoxicities of compounds 1 - 8 against cancer cell lines
Cell lines
IC50 ?(μmol?L)
1 ?2 o3 p4 5 >6 7 ?8
A549 hNA NA 17 ?. 76±2 . 20 20 ?. 44±2 %. 69 4 .63±0 y. 95 NA 17 .50±2 .39 NA
BGC-823 ?NA NA 26 .78±3 . 08 NA 7 .11±1 y. 16 NA 18 .78±2 .45 NA
SGC-7901 ?NA NA NA NA NA NA NA NA
DU145 ?NA NA NA NA 26 .90±1 .80 NA 8 .83±1 z. 54 NA
MDA-MB-231 tNA NA 19 ?. 63±2 . 78 NA NA NA NA NA
HT-29 ?NA NA NA NA NA NA 33 .83±4 .15 NA
BEL-7402 ?NA NA NA NA NA NA 21 .06±2 .50 NA
MCF-7 ?NA NA NA NA 16 .77±1 .90 NA 15 .74±1 .33 NA
U251 hNA NA NA NA 22 ?. 62±3 .27 NA NA NA
The IC50 values are means of three experiments; NA , not active
9165 期 JIA Rui-Rui et al . : New Inhibitors of Dual-specificity Protein PhosphataseCdc25C fromPlants
gested that thesenew natural inhibitors for Cdc25C will
facilitate the development of potential chemotherapeutic
agents in tumor prevention .
References:
Cao ?S, Foster C , Brisson M et al. , 2005 . Halenaquinone and xestoqui-
none derivatives, inhibitors of Cdc25B phosphatase from a Xesto-
spongia sp [ J ] . Bioorg Med Chem, 13 : 999—1003
Cebu +la RE, Blanchard JL , Boisclair MD et al. , 1997 . Synthesis and
phosphatase inhibitory activity of analogs of sulfircin [ J ] . Bioorg
Med ChemLett, 7 : 2015—2020
Donz &elli M , Draetta GF , 2003 . Regulating mammalian checkpoints
through Cdc25 inactivation [ J ] . EMBO Rep, 4 : 671—677
Ducr #uet AP, Rice RL , Tamura K et al. , 2000 . Identification of new
Cdc25 dual specificity phosphatase inhibitors in a targeted small mol-
ecule array [ J ] . Bioorg Med Chem, 8 : 1451—1466
Ecks tein JW, 2000 . Cdc25 as apotential target of anticancer agents [ J ] .
Invest NewDrugs, 18 : 149—156
Guna /sekera SP, McCarthy PJ , Kelly-Borges M et al. , 1996 . Dysidiol-
ide: A novel protein phosphatase inhibitor from the caribbean sponge
Dysidea etheria de Laubenfels [ J ] . J AmChemSoc, 118: 8759—
8760
Ham 9SW, Park HJ , LimDH , 1997 . Studies onmenadione asan inhibitor
of the cdc25 phosphatase [ J ] . Bioorg Chem, 25 : 33—36
Ham 9SW, Park J , LeeSJ et al. , 1998 . Naphthoquinone analogs as inac-
tivators of cdc25 phosphatase [ J ] . Bioorg Med Chem Lett, 8 :
2507—2510
Huye .r G, Liu S, Kelly J et al. , 1997 . Mechanism of inhibition of pro-
tein-tyrosine phosphatases by vanadate and pervanadate [ J ] . J Biol
Chem, 272: 843—851
Itok ?awa H , Qiao YF , Takeya K , 1991 . Anthraquinones, naphthoquino-
nes and naphthohydroquinones from Rubia oncotricha [ J ] . Phyto-
chemistry, 30 : 637—640
Iwao /M, Kuraishi T, 1985 . A novel naphthoquinone synthesis via tandem
directed lithiations [ J ] . Tetrahedron Lett, 26 : 6213—6216
Kris ?tjansdottir K , Rudolph J , 2004 .Cdc25 phosphatases and cancer [ J ] .
Chem Biol , 11 : 1043—1051
Lave ?rgne O, FernandesAC , BrehuL et al. , 2006 . Synthesis and biolog-
ical evaluation of novel heterocyclic quinones as inhibitors of the dual
specificity protein phosphatase CDC25C [ J ] . Bioorg Med ChemLett,
16 : 171—175
Loukaci A , Le Saout I , Samadi M et al. , 2001 . Coscinosulfate, a
CDC25 phosphatase inhibitor fromthe sponge Coscinoderma mathewsi
[ J ] . Bioorg Med Chem, 9 : 3049—3054
Mont ?enegro H , Gutierrez M, Romero LI et al. , 2003 . Aporphine alka-
loids from Guatteria spp . with leishmanicidal activity [ J ] . Planta
Med, 69 : 677—679
Peng ?SL , Chen L , ZhangGL et al. , 1990 . Studies on medicinal isoquin-
oline alkaloids I . Alkaloidsof Stephania epigaea [ J ] . Nat Prod Res
Dev, 2 : 37—42
Prev ?ost GP , Brezak MC , Goubin F et al. , 2003 . Inhibitors of the Cdc25
phosphatases [ J ] . Prog Cell Cycle Res, 5 : 225—234
Shan ?g LJ , Zhao BT, Hao XJ , 1994 . The new flavonoid from Fissistigma
kwangsiense [ J ] . Acta Bot Yunnan, 16 : 191—195
Shim ?azawa R , Suzuki T, Dodo K et al. , 2004 . Design and synthesis of
dysidiolide analogs from vitamin D3: Novel class of Cdc25A inhibi-
tors [ J ] . Bioorg Med ChemLett, 14 : 3291—3294
Skeh ?an P, Storeng R , Scudiero D et al. , 1990 . New colorimetric cyto-
toxicity assay for anticancer-drug screening [ J ] . J Natl Cancer Inst,
82 : 1107—1112
Sohn ?J , Kiburz B, Li Z et al. , 2003 . Inhibition of Cdc25 phosphatases
by indolyldihydroxyquinones [ J ] . J Med Chem, 46 : 2580—2588
Tamu ?ra K , Southwick EC , Kerns J et al. , 2000 . Cdc25 inhibition and
cell cycle arrest by a synthetic thioalkyl vitamin K analogue [ J ] .
Cancer Res, 60 : 1317—1325
Wang ?BG, HongX , Li L et al. , 2000 . Chemical constituents of two Chi-
nese Magnoliaceaeplants, Tsoongiodendron odorum and Manglieti-
astrumsinicum, and their inhibition of platelet aggregation [ J ] .
Planta Med, 66 : 511—515
Xu G , Peng LY , Zhao Y et al., 2005 . Two new icetexane diterpenoids
from Salvia przewalskii [ J ] . Chem Pharm Bull , 53 : 1575—1576
026 云 南 植 物 研 究 30 卷