全 文 ：·药理实验与临床观察·
Apoptosis in HL-60 cells induced and c-Myc expression down-
regulated by root aqueous extract fromTripterygium hypoglaucum
FONG Wang-fun
1 , ZHU ANG Wei-jian
1 , CAO Jia
2 , LEUN G Chung-hang
1 ,
CHEUNG Hon-yeung
1 , XIAO Pei-gen
3 , YANG Meng-su
1, 4
( 1. Bioa ctiv e Products Resear ch Group, Depar tment of Biolog y & Chemistr y, City Univ er sity o f Hong Kong , Kowloon,
Hong Kong , China; 2. Molecular Tox ico lo gy Labo ra to ry , Third Milita ry Medical Univ ersity, Chongqing 400038,
China; 3. Institute of Medicinal Plants, CAM S& PUM C, Beijing 100094, China; 4. Applied Resea rch Centre
for Genomics Techno log y , City Univ ersity o f Hong Kong , Kow loon, Hong Kong , China)
　　 Abstract: Object　 To investig ate the effects o f the roo t aqueous ex t ract o f Tripterygium hypoglaucum
( Lev l. ) Hutch ( Celast raceae) ( T HH) on human promyelocy tic leukemia HL-60 cells and its mechanism.
Methods　 Cell g row th and tox ici ty assay s, f low cy tometry , confocal fluorescence microscopy and Annex-
in-V labeling , and Western Blot ting Assay w ere used. Results　 T HH caused mo rpholo gical changes hy-
podiploid sub-G1 cell accumulation and induced do se-and time-dependent apoptosis in HL-60 cells at con-
centrations above 18μg /mL. Western Blo tting analysis show ed a more than 99% decrea se o f the nuclea r
oncoprotein c-Myc af ter 2— 4 h o f T HH treatment. Conclusion　 T HHdecreases c-M yc protein expression,
leading to the deactiv ation o f cell cycle prog ression pa thw ays and an accumulation of hypodiploid sub-G1
cells that ev entually enter apoptosis.
Key words: Tripterygium hypoglaucum ( Lev l. ) Hutch. ( Celast raceae) ( THH); HL-60 cel ls; apopto-
sis; c-M yc protein expression
昆明山海棠根部水提液诱导 HL-60细胞凋亡及对 c-Myc基因表达的调控
方宏勋 1* ,庄为笕 1 ,曹　佳2 ,梁重恒1 ,张汉扬 1 ,萧培根 3 ,杨梦苏 1, 4*
( 1. 香港城市大学 生物及化学系生物活性制品研究组 ,香港 九龙 ; 2. 中国人民解放军第三军医大学 分子毒理学实验室 ,重庆
400038; 3. 中国医学科学院 中国协和医科大学药用植物研究所 ,北京　 100094; 4. 香港城市大学 基因组科技应用研究中心 ,
香港 九龙 )
摘　要:目的　研究昆明山海棠 Tripterygium hypoglaucum ( Celast raceae) ( T HH) 根部水提液对早幼粒白血病
HL-60细胞凋亡的诱导作用及机制。方法　通过特征性的形态学观察 , Annex in-V标记 ,以及流式细胞仪检测中次
G1峰的形成确定 THH的诱导细胞凋亡作用 ;应用 Western Blo tting研究 T HH对 c-Myc蛋白表达的影响。 结果
THH根部水提液可诱导早幼粒白血病 HL-60细胞凋亡。在浓度高于 18μg /m L时 , THH对 HL-60细胞的诱导凋
亡作用呈现浓度与时间的相关性。在 T HH处理 2～ 4 h后 ,癌基因蛋白 c-Myc表达降低 99% 以上。结论　 THH
抑制了 c-M yc蛋白的翻译或后翻译过程 ,从而降低了 c-Myc在细胞周期运转中的作用 ,导致大量细胞凋亡。
关键词: 昆明山海棠 ; HL-60细胞 ; 凋亡 ; c-M yc蛋白表达
中图分类号: R285. 5　　　文献标识码: A　　　文章编号: 0253 2670( 2003) 07 0622 04
Introduction
The roo t o f Tripterygium hypoglaucum
( Lev l. ) Hutch. ( THH) , a w ell-known and readi ly
available Chinese medicinal product , has been used
fo r the t rea tment of auto-immune diseases includ-
ing rheuma toid a rthriti s, systemic lupus erythe-
ma tosus ( SLE) and skin problems. A number of
prelimina ry studies have suggested the po ssibili ty
tha t T HH affect T lymphocy tes
[ 1]
and may induce
chromosome changes in certain cells
[2 ] . It wa s sug-
gested that T HH may cause apopto sis in a number
of cultured tumor cells, w ith leukemic cel ls being
the most sensi tiv e
[3-5]
. How ever, the effects of
T HH-induced apopto sis and i ts action mechanism
have no t been investig ated in detai l.
The objectiv e of the present study is to exam-
·622· 中草药　 Chinese T raditional and Herbal Drug s　第 34卷第 7期 2003年 7月
收稿日期: 2002-10-02收稿日期:方宏勋 ,男 ,香港城市大学生物及化学系教授 ,主要研究方向:肿瘤细胞的生化及分子生物学、生物活性制品和中草药药理学。
E-mai l: bhw f fong@ ci tyu. edu. h k
ine the ef fect o f T HH trea tment on the apopto sis in
human promyelocy tic leukemia HL-60 cell line. In
addition, because the nuclea r oncoprotein and tran-
scriptional activ ator c-M yc is one of the central
players in regula ting cell pro liferation, dif ferentia-
tion and apoptosis[ 6, 7 ] , the expression of c-Myc
pro tein was also determined in o rder to provide in-
sights into the mo lecula r mechanisms involv ed in
T HH-induced apopto sis.
Materials and methods
　　 Preparation of T HH ex tract. Dried roo t of T .
hypoglaucum ( Lev l. ) Hutch. ( T HH) ( Yunnan
Medicine Company, Kunming ) was powdered and
soaked in double distilled w ater ( 100 g /500 mL)
fo r 24 hours, then boi led and cooled three times.
Ex t ract w as fi ltered and concentrated by gentle
boiling . Af ter cent ri fugation, supernatant was fil-
tered by 0. 22μm nitrocellulose membrane and was
kept a t - 20℃ .
　　 Cell culture and M TT cytotoxici ty assay. HL-
60 cells ( American Type Culture Collection, MD,
U SA) w ere routinely g row n at 37℃ in 5% CO2 /
air in RPM I-1640 supplemented wi th 1% antibiotic
solution and 10% heat-inactivated fetal calf serum
( Gibco, MD, USA) . For to xici ty assay, 1× 104
cells /w ell w ere g row n fo r 12-15 hours in 96-w ell
culture plates and di fferent amounts of T HH ex-
tract w ere added, fo llow ed by M T T assay [3-( 4,
5-dimethy lthiazo l-2-yl ) -2, 5-diphenyl tet razolium
bromide, Sigma; 5 mg /m L in PBS] as described in
the litera ture[8 ] .
　　 Flow cy tometry and confocal microscopy. Fo r
f low cy tometric measurements, 2× 106— 3× 106
cells w ere washed in phospha te-buf fered saline
( PBS) and re-suspended in 100μL fresh medium.
Cell suspensions w ere stored at - 20℃ in adding
1 mL 70% ethanol. Cells w ere collected by cen-
trifuga tion and incubated wi th propidium iodide
and RNase A. Stained cells were analy zed by
EPICS Eli te ESP f low cy tometer ( Coulter Elec-
tronic, U SA) . Fo r confocal microscopic measure-
ments, cells w ere fluo rescence labeled by adding
40μL staining so lution ( 100μg /mL each of acri-
dine orange and ethidium bromide ) to 1 m L cell
suspension o r w ere stained wi th Annexin-V and
propidium iodide ( Clontech, M A, U SA ) . The
samples w ere studied wi th a LSM 510 ( Ca rl Zeiss)
confocal laser-scanning micro scope.
Western Immuno-Blot ting. Who le cell ex-
tracts containing 15— 20μg protein w ere separated
by SDS-polyacrylamide gel elect ropho resis and
transfer red to nit rocellulo se membrane. Af ter
blocking by Tris-buf fer saline containing 5% non-
fat milk powder, fi lters w ere incubated wi th mouse
antihuman c-Myc monoclonal antibody Ab-3 ( 1∶
80 dilution) ( Calbiochem , USA) , and then incu-
ba ted wi th horseradish peroxidase-conjuga ted goat
anti-mouse immunog lobulin G secondary antibod-
ies ( Bio-Rad, U SA) . Tagged pro tein bands w ere
detected by t rea tment wi th ECL Western Blot ting
reagents ( Amersham, UK) and exposed on X-ray
film s ( Fuji , Japan) .
Results and discussion
　　 T HH-induced apopto sis in HL-60 cells. Cell
g row th w as inhibi ted when expo sed to THH ex-
tract at concentrations above 0. 625 μL ex tract
( 18. 3 μg solid per mL of g row th medium )
( Fig. 1) . The percentag e o f death cells caused by
T HH trea tment w as time- and do se-dependent.
Subsequent experiments were perfo rmed wi th cells
t rea ted wi th 5μL of THH ex tract ( 146. 5μg solid
per mL o f medium) .
　　 The mo rpho logy of cells w as examined by f lu-
Various amount o f THH ex tr act ( so lid contents 29. 3
mg /m L) w as added to culture medium and numbers o f
v iable cells w er e estima ted by M T T assay . Data points
repr esent av erages of th ree experiments
Fig. 1　 Growth inhibitory ef fect of THH
extract on HL-60 cells
·623·中草药　 Chinese T raditional and Herbal Drug s　第 34卷第 7期 2003年 7月
o rescence confocal laser scanning micro scope where
cells w ere stained wi th acridine o range /ethidium
bromide. Untreated HL-60 cells appea red uni fo rm-
ly g reen w ith distinct round nucleoli, while cell
shrinkage, chroma tin condensation and nuclear
f ragmentation became obvious in some o f the cells
af ter 8 hours o f THH treatment. Af ter 12 hours of
T HH treatment, cellular f ragmentation resulted in
the appearance of apopto tic bodies ( bright o range
bleb) .
Annexin-V assay w as used to probe th e redis-
tribution of membrane phosphatidylserine ( PS )
f rom cytoplasmic side to outside surface as an ea rly
indica to r of apopto tic cell death
[9 ]
. Annex in-V is a
35. 8 kDa protein that has a st rong af fini ty to PS
on the outside surface of intact cells. While control
cells had li t tle PS on the outside surface, cells
t rea ted w ith THH for mo re than 2 hours w ere
clearly labeled by Annex in-V ( bright g reen) .
Flow cytometric analysis of T HH-treated HL-
60 cells show ed a distinct sub-G1 hypodiploid peak
( Fig . 2) . The accumula tion of dying cells in the
“ sub-G1” hypodiploid peak is due mainly to a re-
duced DN A content and these cells are destined to
enter apopto sis
[10 ]
. The proportion of sub-G1 cells
increa sed wi th time and 3% , 7% , 13% , 57% and
90% o f total cells w ere in this phase af ter 2, 4, 8,
12 and 18 hours of t rea tment , respectiv ely ( Fig. 2)
Cells w ere tr eated with 5μg /m L THH ex tract fo r 0 ( contro l, panel A) , 2 ( panel B) , 4 ( panel C) , 8 ( panel D) , 12
( panel E) and 18 ( panel F) h. A g radua l accumulation of sub-G1 and G1 cells ( show n as B zones in g raphs) can be seen
Fig. 2　 Flow cytometry analyses of THH-treated HL-60 cells
　　 The combination of the cell g row th assay ,
morpho logical changes, and cell cycle study clea rly
demonst rated tha t T HH ex tract caused apoptosis
in HL-60 cells. Characteristic apopto tic fea tures
w ere induced a t low concentrations of T HH ex tract
( above 18 μg /mL) and cells responded af ter a
short exposure time and in a dose-and time-depen-
dent manner.
　　 Down-regula tion of c-Myc. Since c-Myc pro-
tein play ed a central role in regulation cel l pro li fer-
ation, dif ferentiation and apopto sis, the expression
o f c-Myc w as measured w ith Western Immuno-
Blo tting. In control cells, the expression lev el of c-
Myc remained constant. How ever, in T HH-treated
cells, the pro tein lev el drastically decreased by
99% af ter 2— 4 hours o f treatment ( Fig. 3A, B) .
The consequences of dow n-regula ting c-Myc were
studied in Myc-null cells
[11 ]
. The doubling time of
these cel ls was prolonged, pa rticularly w hen there
w ere accumulations of cells in G1 and G2 phases,
w hereas S phase w as no rmal. It is conceivable that
Cells w ere tr ea ted with 5μg /mL o f THH-ex tract. Panel
A: Western Bloting ana ly ses o f c-Myc pro tein by specif-
ic antibodies. Lane c: no rma l cells; la nes T2, T4 and
T8: cells t reated with THH ex t ract fo r 2, 4 and 8 h, r e-
spectiv ely. Panel B: g raph showing % changes o f c-Myc
pro tein. Da ta ar e aver ages o f two expe riments.
Fig. 3　 c-Myc expression
·624· 中草药　 Chinese T raditional and Herbal Drug s　第 34卷第 7期 2003年 7月
a low ering of c-M yc level could lead to apoptosis
by first deactiv ating the pro liferation path way
[ 12]
and preventing cell cycle pro g ression. Trea tments
that caused apoptotic death o f sev eral cel l types
had been shown to fi rst drastically low er c-Myc ex-
pression
[13, 14 ] .
While the accumula tion of sub-G1 cells and
apopto sis took place a fter 18 hours o f T HH trea t-
ment , c-M yc protein show ed a drastic decrease a f-
ter only a sho rt period of T HH trea tment (> 99%
decrease in 2— 4 hours) . This is consistent wi th
the cur rent thinking tha t c-M yc is one of the earli-
er signals in cell g row th regula tion. The above re-
sul ts show ed that THH ex tract ini tially caused a
decrease in c-M yc pro tein lev el in HL-60 cells, by
either transcriptiona l or t ranslational regula tion
mechanisms. Subsequent ly cellular metabo lism is
af fected
[7 ]
and cells stop at the hypodiploid sub-G1
pha se and eventually enter apopto sis.
　　Acknowledgement: This w ork is suppo rted by an Ap-
plied Resear ch Grant o f City Univ ersity of Hong Kong.
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用寡核苷酸芯片研究血虚小鼠造血相关细胞因子基因表达谱
佟　丽 1 ,陈苏红 1 ,马增春 1 ,黄　坚 2 ,丁　雨 2 ,王升启 1
( 1. 军事医学科学院放射医学研究所 全军生物芯片重点实验室 ,北京　 100850; 2. 深圳益生堂生物企业有限公司 ,广东 深圳
518026)
摘　要 :目的　应用细胞因子寡核苷酸表达谱芯片检测血虚小鼠不同脏器组织内造血相关细胞因子差异表达情
况 ,寻找差异表达基因 ,为血虚证治疗药物机制研究及治疗药物筛选奠定基础。方法　采用 5. 5 Gy60Co-γ射线照射
Ba lb /c小鼠 ,制备血虚模型。 在不同时间点提取正常和血虚小鼠不同组织总 RN A,反转录成不同荧光标记的
cDN A探针 ,与表达谱芯片进行杂交 ,对扫描数据进行分析获得血虚小鼠造血相关细胞因子基因的差异表达情况。
结果　在各组织中共发现 21个差异表达的基因。这些基因的功能大致分为 3类 :促细胞生长或增殖 ,免疫调节 ,诱
导血细胞形成或促造血祖细胞形成集落。结论　照射后上述细胞因子基因的下调 ,使机体的造血功能下降 ,造成血
虚 ,证明细胞因子间形成造血调节网络 ,整体调节机体造血。这与中医的全局理论是一致的。实验证明应用芯片技
·625·中草药　 Chinese T raditional and Herbal Drug s　第 34卷第 7期 2003年 7月
收稿日期: 2002-09-10基金项目:全军医药卫生科研基金课题 ( 01Z019) ; 北京市科委资助项目 ( H010210220113)作者简介:佟　丽 ( 1974— ) ,女 ,辽宁人 ,军事医学科学院放射医学研究所 2000级硕士研究生 ,研究方向为中药四物汤补血作用机制的基因组学研究。 　 E-mai l: t ongli29@ sina. com
* 通讯作者　 Tel, Fax: ( 010) 66932211　 E-m ail: s qw ang@ nic. bmi. ac. cn