全 文 ：Inhibitory effect of compound 6F isolated from Pteris semipinnata L.on
the activity of protein kinase C in HL-60 cells
HE Cheng-Wei1 , LIANG Nian-Ci2＊ , MO Li-Er3 , LI Jin-Hua1 , ZHANG Xiao1
(1.Institute of Biochemistry and Molecular Biology , 2.GuangdongKey Laboratory for Research and Development of
Natural Drugs , 3.Department of Chemistry , Guangdong Medical College , Zhanjiang 524023 , China)
Abstract:AIM To investigate whether compound 6F
isolated from Pteris semipinnata L inhibits the activity of
protein kinase C(PKC)and whether the DNA fragment
induction and cytotoxicity of 6F on HL-60 cells relate to
PKC signaling pathway.METHODS HL-60 cells were
used as in vitro model and its cytosol(soluble sample)
and particle(insoluble sample including membrane system
and nuclei)fractions obtained by ultracentrifugation were
used as samples for PKC assay.PKC activity was mea-
sured by incorporation of [γ-32P]ATP into exogenous sub-
strate after stimulated by phosphatidylserine and diolein.
Diphenylamine assay and MTT staining methods were ap-
plied for DNA fragmentation detection and cytotoxicity as-
say , respectively.RESULTS PKC activities in cytosol
and particle fractions were inhibited by 6F in a dose-de-
pendent manner(P <0.05 in the series of cytosol frac-
tion , P <0.01 in the series of particle fraction).Phorbol
12-myristate 13-acetate(PMA ,65 nmol·L-1)partially at-
tenuated the DNA fragmentation induced by 6F in HL-60
cells , and protected cells against the cytotoxic effect of 6F
(P <0.01).The ability of reducing MTT to formazan in
mitochondria of HL-60 cells was potentiated when the
cells were treated with PMA alone at the same concentra-
tion for 24 h(P <0.01).CONCLUSION Compound
6F is an inhibitor for PKC.DNA fragment induction and
cytotoxic effect of 6F may be partially through its inhibito-
ry effect on PKC activity.
Received date:2002-08-26 Accepted date:2002-10-18
Foundation item:The project supported by National Natural
Science Foundation of China(39870900);the Key Research Project
Foundation of Guangdong Province(9622007-002);and the Key
Subject Foundation of Guangdong Province(9306)
Biographies:HE Cheng-Wei(1972 -), male, native of
Nankang, Jiangxi Province , Ph.D., main research fields are bio-
chemical pharmacology , molecular biology of antitumor drugs , and
experimental cancer gene therapy;Liang Nian-Ci(1940-), male ,
native of Wuchuan , Guangdong Province , Professor of Biochemistry ,
main research fields are biochemical and molecular pharmacology ,
research and development of antitumor and anti-platelet drugs.
＊ Corresponding author.E-mail:ncliang @gdmc.edu.cn
Tel:0759-2388501 Fax:0759-2284104
Key words:Pteris semipinnata L.;protein kinase C;
DNA fragmentation;cytotoxicity
CLC number:R979.1
Document code:A
Article ID:1000-3002(2003)02-0081-06
Protein kinase C(PKC)is an isozyme family
of serine-threonine protein kinases comprising at
least 10 mammalian members.All PKC members
contain conserved and variant amino acid sequences
in both regulatory and catalytic subunits.The con-
served domain in regulatory subunit contains cys-
teine-rich region that serves as the important regu-
lating site.PKC plays a key role in mediating the
signals of growth factors , neurotransmitters , and
hormones that have been implicated in many cellu-
lar events such as proliferation , differentiation , im-
mune response , carcinogenesis , and multi-drug re-
sistance(MDR).A decrease in PKC activity sug-
gests PKC act as an anti-oncogene , whereas an in-
crease in PKC activity suggests an oncogenic role
for PKC , and an increase in PKC activity correlates
with increased drug resistance and metastatic poten-
tial[ 1] .PKC signaling is a rational target for the
treatment of many diseases and PKC inhibitors are
potential clinical drugs and useful tools for the
study of mechanisms of this enzyme and related cel-
lular events thereafter.
Pteris semipinnata L.(PSL)is a Chinese tra-
ditional herb.Its ethanolic and water crude extracts
showed obvious antitumor activity on hepatic carci-
noma and sarcoma 180 in mice with low toxi-
city[ 2] .Several active compounds(e.g.5F , 6F ,
A)were purified from PSL and showed strong cyto-
toxicity against various human tumor cell lines in
vitro[ 3] .Compound 6F exhibits a stronger cytotoxic-
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中国药理学与毒理学杂志 2003年 4 月; 17(2):81-86
Chin J Pharmacol Toxicol 2003 Apr; 17(2):81-86
ity than that of 5F and A.All these compounds are
diterpenoid that belong to the type of kaurane and
possessα, β-methylene cyclopentanone moiety that
can react with sulfhydryl in a Micheal addition.
Our previous papers reported that compound 6F
could strongly inhibit the activity of topoisomeraseⅡ
at a very low concentration (29 nmol·L-1)and
slightly inhibit topoisomerase Ⅰand tyrosine protein
kinase(TPK)[ 4] .The effect of compound 6F on the
PKC activity and the effect of tumor-promoting
diterpene phorbol 12-myristate 13-acetate(PMA)on
the DNA fragment induction and cytotoxicity of 6F
on HL-60 cells were studied.The mechanism of
action was discussed.
1 MATERIALS AND METHODS
1.1 Drugs and chemicals
Compound 6F isolated from PSL was identi-
fied by the Laboratory of Phytochemistry of Guang-
dong Medical College.RPMI-1640 medium was
obtained from Gibco.Fetal calf serum(FCS)was
obtained from the Hangzhou Sijiqing Materials
Company (Hangzhou , China).Phenylmethyl-
sulfonyl fluoride(PMSF)was obtained from Mer-
ck.Histone Ⅲ-SS , diphenyl-amine , and phorbol
12-myristate 13-acetate (PMA)were purchased
from Sigma.3-(4 , 5-dimethylthiazol-2-yl)-2 , 5-
diphenyltetrazolium bromide(MTT)was purchased
from Serva.[ γ-32 P]-ATP was purchased from
Beijing Yahui Biomedicine Engineering Co.(Bei-
jing , China).
1.2 Cell culture
Human promyelocytic leukemia cell line HL-
60 , obtained from Shanghai Institute of Cell Biolo-
gy , was cultured in RPMI-1640 medium supple-
mented with 10% heat-inactivated FCS and anti-
biotics(penicillin 100 kU·L-1 , streptomycin 100
mg·L-1)in 5%CO2 atmosphere at 37℃.
1.3 Preparation of cell extracts
HL-60 cells 2×107were homogenized by son-
ication(5 s with a Cole Parmer Ultrasonic Homoge-
nizer 4710 Series at 70W)in 1 mL buffer contain-
ing 20 mmol·L-1 Tris-HCl pH 7.4 , 2 mmol·L-1
EGTA , 2 mmol·L-1 EDTA , 6 mmol·L-1 β-mer-
captoethanol , 0.5mmol·L-1 PMSF , and then cen-
trifuged at 45 000×g for 2 h , yielding the cytosol
fraction(supernatant)and particle fraction(pellet).
The particle pellet was suspended in homogenization
buffer plus 15 g·L-1Nonidate P-40 , sonicated for
10 s.The insoluble membrane and nuclei fraction
(particle fraction)was separated from the pellet by
centrifuged at 45 000×g for 2 h.The cell extract
of cytosol or particle fractions were used as samples
for the subsequent PKC assay.Protein concentra-
tion was measured by the method of protein-
coomassie brilliant blue
[ 5] .
1.4 Protein kinase C assay
Activity of protein kinase C was tested by
measuring 32P incorporation from [ γ-32P] ATP into
histone Ⅲ-SS as described by Kikkawa , et al[ 6] .
The catalytic reaction was performed in a total vo-
lume of 100 μL containing 20 mmol·L-1 Tris-HCl
pH 7.4 , 10 mmol·L-1 MgCl2 , 0.5 mmol·L-1
CaCl2 , 40μg phosphatidylserine , 4μg diolein , 20
μg histone Ⅲ-SS , 50 μmol·L-1[ γ-32P] ATP (370
MBq·L-1).The reaction was initiated by the addi-
tion of 20μL cell extracts and the samples were in-
cubated for 10 min at 32℃ in the reaction mixture ,
and then immediately spotted onto filter paper(Xin-
hua type Ⅲ).The papers were washed 7 times with
20% trichloroacetic acid containing 1 mmol·L-1
ATP , and then washed 1 time with acetone , dried
in the air , and immersed in 3 mL scintillator con-
taining 0.3 mmol·L-1 1 , 4-bis(5-phenyl-2-oxa-
zolyl)benzene(POPOP)and 18 mmol·L-1 2 ,5-
diphenyloxazole(PPO).The radioactivity was mea-
sured by scintillation counter(Beckman).Basal
PKC activity obtained with 0.5 mmol·L-1EGTA in
the absence of three elements:CaCl2 , phos-
phatidylserine and diolein , was subtracted for each
experimental datum.PKC activity is defined as the
amount of [ γ-32P] ATP (nmol)being transferred to
histone Ⅲ-SS by the enzyme during 1 min by 1 g
protein of cell extracts.
1.5 Quantification of DNA fragmentation
DNA fragmentation was determined using the
diphenylamine assay[ 7] .2×106Cells were lysed in
500μL lysis buffer(10mmol·L-1 Tris-Cl pH 7.5 ,1
mmol·L-1 EDTA , 2.5 g·L-1 Triton X-100).The
·82· Chin J Pharmacol Toxicol 2003 Apr; 17(2)
intact chromatin(pellet)was separated from DNA
fragments(supernatant)by centrifugation for 10min
at 13 000×g.The pellets were resuspended in 500
μL lysis buffer , and precipitated by adding 125μL
50% trichloroacetic acid(TCA)at 4℃.Samples
were centrifuged at 13 000×g for 4 min.Two
hundred microliters of 5%(V/ V)TCA were added
into the pellets , and incubated in 90℃ for 10min.
After centrifugation at 13 000×g for 4 min , 100
μL supernatant was separated and 200μL dipheny-
lamine reagent(1.5 g diphenylamine , 1.5mL oil of
vitriol , 8 μg acetaldehyde , 100 mL acetic acid)
was added.After incubated at 30℃ for 16 h , the
absorbance was measured with Microplate reader
(Bio-Rad , M450)at 570 nm.The percentage of
fragmented DNA was calculated as the ratio of the
DNA content in the supernatant to that in the pel-
let.Quantification of fragmented DNA by this
method was consistent with the DNA ladders elec-
trophoresed on agarose gel
[ 7] .
1.6 Cytotoxicity assay
Cytotoxic effect of compound 6F was tested
using MTT method.Briefly , 1×104 exponentially
growing HL-60 cells in 90 μL complete medium
were seeded in 96-well dish , then 10 μL of drugs
were added.After incubation at 37℃ in humidified
5%CO2 atmosphere for 24 h , 20μL 12mmol·L-1
MTT was added to each well and the plate was fur-
ther incubated at 37℃ for 5 h.SDS (20% 150
μL) resolved in 50% dimethyl formamide was
added thereafter to solubilize the formazan crystal.
After 5 h incubation at 37℃, the absorbance was
measured with Microplate reader(Bio-Rad , M450)
at 570 nm and referenced at 450 nm.
1.7 Results were presented as x ±s.Analysis
of variance with one-way ANOVA was used to
identify significant differences in multiple compari-
sons.
2 RESULTS
2.1 Inhibitory effect of compound 6F on pro-
tein kinase C activity in HL-60 cells
The crude extraction including various iso-
forms of PKC obtained from HL-60 cells by ultra-
centrifugation was used as sample to test the effect
of compound 6F on this enzyme.PKC activities
both in cytosol and particle fraction which included
membrane system and nuclei were inhibited by
compound 6F at the concentration of 0.1 μmol·
L-1(particle fraction)or 0.5 μmol·L-1(cytosol
fraction)(P <0.01 , n =3).The inhibition ef-
fects of 6F on PKC were in a dose-dependent man-
ner(r=0.781 , P<0.05 in cytosol fraction;r=
0.931 , P<0.01 in particle fraction)(Tab 1).
2.2 Attenuation of PMA on the DNA frag-
mentation in HL-60 cells induced by compound
6F
The DNA fragmentation was 75.7%±1.4%
Tab 1. Effect of compound 6F on the activities of protein kinase C(PKC)extracted from HL-60 cells
Compound 6F
/μmol·L-1
PKC activity/ nmol·min-1·g-1 protein
Cytosol Particle
Inhibitory rate/ %
Cytosol Particle
0 7.9±0.8 7.7±0.8 0 0
0.1 6.4±0.3 4.8±0.3＊＊ 20.0 37.7
0.5 1.3±0.4＊＊ 4.1±0.4＊＊ 83.5 46.8
2.5 1.6±0.6＊＊ 3.4±1.2＊＊ 79.7 55.8
12.5 2.5±2.6＊＊ 2.8±0.4＊＊ 68.4 63.6
62.5 1.6±1.7＊＊ 1.4±0.5＊＊ 79.7 81.8
312 0.9±0.7＊＊ 1.1±0.6＊＊ 88.6 85.7
The cytosol(soluble sample)and particle(insoluble sample including membrane system and nuclei)fraction of HL-60 cells were prepared by
ultracentrifugation , and PKC activities in each fraction were assayed as described in Materials and Methods. x±s , n =3.＊＊P <0.01 ,
compared with the group of 0μmol·L-1 6F.
·83·中国药理学与毒理学杂志 2003 年 4月;17(2)
and 53.3%±3.1% in HL-60 cells treated with
compound 6F(231 nmol·L-1)alone or simulta-
neously combined with 65 nmol·L-1 PMA , re-
spectively(P <0.01 , n=3).
2.3 PMA protected HL-60 against the cyto-
toxicity of 6F
Compound 6F 116 nmol·L-1 showed obvious
cytotoxicity on HL-60 cells which exhibited re-
duced magnitude of formazan formation measured
by MTT method.This effect was attenuated by
PMA at 65 nmol·L-1(P <0.01 , Tab 2).The
ability of reducing MTT to formazan in mitochon-
dria of HL-60 cells was potentiated when treated
the cells with PMA alone at the same concentra-
tion for 24 h(P<0.01), i.e.viability of HL-
60 were enhanced by PMA under this condition.
Tab 2. Viability of HL-60 cells treated with 6F
alone or simultaneously combined with PMA for 24 h
Group
Cell viability
(A570 nm/ A450 nm) Inhibitory rate/ %
Control 0.58±0.03 0
6F alone 0.36±0.02 38±4
6F+PMA 0.45±0.02＊＊ 21±3
PMA alone 0.66±0.03## -14±6
The cell viability was evaluated by measuring the absorbance of for-
mazan on Microplate reader at 570 nm with reference at 450 nm as
described in Materials and Methods.The concentrations of 6F and
PMA were 116 nmol·L-1 and 65 nmol·L-1 , respectively. x±s ,
n=3.＊＊P <0.01 , compared with 6F alone;##P <0.01 ,
compared with control.
3 DISCUSSION
This work revealed that compound 6F puri-
fied from Pteris semipinnata L.could efficiently
inhibit PKC activities both in cytosol and particle
fractions extracted from HL-60 cells in a dose-de-
pendant manner.Cysteine-rich domains in con-
served 1 (C1)and conserved 2 (C2)sequence
act as a key regulatory site bound to the activator
diacylglycerol as well as phorbol ester that account
for cPKC and nPKC activities[ 8] .PKC expressed
in HL-60 cells mainly is the classical isoforms
(PKCα, PKCβ , and PKCγ).Each cysteine-rich
domain is a 50- or 51-amino acid domain contain-
ing a zinc finger motif that has 6 cystein residues.
The motif is duplicated in tandem in both cPKC
and nPKC isozymes.Studies indicated that a sin-
gle copy of motif is sufficient for the binding of
phorbol ester.Site-directed mutagenesis revealed
that five of the six cysteine residues involved in
Zn
2+
coordination are critical for the interaction of
the protein with phorbol ester
[ 9] .Cysteine-con-
taining peptide substrate analogues such as N-bio-
tinyl-Arg-Arg-Arg-Cys-Leu-Arg-Arg-Leu inactivat-
ed cPKC , nPKC and aPKC by forming disulfide-
linked complexes with the active site cysteine-rich
domain in the isozymes
[ 10] .PKC isozymes also
can be inactivated by oxidant-induced S-glutathi-
olation , e.g.disulfide linkage of endogenous
glutathione(GSH)to PKC[ 11] .Structure-function
relationship analysis[ 3] showed α, β-methylene
cyclopentanone moiety(Fig 1)confers the cytotoxicity
Fig 1. The structure of compound 6F.
of active compounds isolated from PSL.Saturation
of the 16 ,17-double bond in the moiety resulted in
lose of the activity.Since the sulfhydryl group
plays a pivotal role in conforming a potential ac-
tive PKC , the inhibitory effect of 6F on PKC ac-
tivity was likely mediated through conjugate addi-
tion reaction between electrophilic group 16 , 17-
double bond on 6F with nucleophilic group
sulfhydryl on PKC (Fig 2).
The effective concentration of 6F on PKC was
comparable with that of cytotoxicity and apoptosis
induction on HL-60 cells[ 12] , although 6F showed
much more inhibitory effect on topoisomerase Ⅱ[ 4] .
It was speculated that these activities of 6F on
HL-60 cells were partially through inhibiting PKC
activity.To further examine the role of PKC sig-
·84· Chin J Pharmacol Toxicol 2003 Apr;17(2)
Fig 2. Schematic showing formation of 6F-PKC complex.Only the active moietyα, β-methylene cyclopentanone in 6F was
shown here.The electrons were attracted by carbonyl and hydrogen bond that resulted in positive charged methylene.The electrophilic group
methylene reacted with nucleophilic group sulfhydryl on PKC enzyme and a proton released.
naling in the regulation of apoptosis in HL-60
cells induced by 6F , we investigated the effect of
PMA , a PKC activator and tumor promoter , on
DNA fragmentation by 6F.The results showed
that DNA fragment induction was partially antago-
nized when HL-60 cells were treated with 6F plus
PMA at 65 nmol·L-1 for 24 h.Long-term treat-
ment(up to 12 h)with PMA down-regulated PKC
activities inHL-60 cells preceded by a short peri-
od(within 30 min)of activation of PKC and fol-
lowed by the differentiation of HL-60 cells to
monocytes and macrophages.It was reported that
differentiated HL-60 cells resisted various apopto-
sis-inducing stimuli and rapidly undergo apoptosis
at the end stage of differentiation[ 13] .DNA frag-
mentation inhibition effect of PMA in this case
might be associated with the down-regulation of
PKC and subsequently the differentiation of the
cells induced by PMA.This could also explain
the protection effect of PMA against cytotoxicity of
6F on HL-60 cells since differentiated cells exhib-
ited enhanced nitroblue tetrazolium(NBT)-reduc-
ing activity in mitochondria.Nevertheless , It′s no
doubt that the antagonistic action between 6F and
PMA is originally due to the changing of PKC ac-
tivity since the enzyme serves as the direct target
for 6F and PMA.The detailed mechanisms by
which PMA interferes apoptosis are still not well
defined.However , several efforts have been
made.①PMA stabilized the intracellular calcium
homeostasis , which was disturbed commonly in
the apoptosis process[ 14] .② PMA abrogated cy-
tochrome C release , downregulation of IAP(in-
hibitor of apoptosis), and caspase 3 activation
that were involved in the execution of apopto-
sis[ 15] .③Activated mitogen-activated protein ki-
nases signaling pathway by PMA promoted the
proliferation and survival of hematopoietic cells
and protected the cells against undergoing
apoptosis[ 16] .This paper revealed that DNA frag-
mentation inHL-60 cells induced by compound 6F
was associated with PKC signal pathway.The ex-
act mechanisms veiled on protecting effect of PMA
on DNA fragment induction of 6F remain to be in-
vestigated.
Acknowledgments We thank Mrs.WANG Mei and
Mr.XU Mei-Yi(Analytical Center of Guangdong Medical
College)for expert technical assistance in measuring the
radioactivity of
32
P by scintillation counter.
4 REFERENCES:
[ 1] Blobe GC , Obeid LM , Hannun YA.Regulation of protein
kinase C and role in cancer biology[ J] .Cancer Metastasis
Rev , 1994 , 13(3-4):411-431.
[ 2] Cui L, Liang NC , Chen ZD , Song ZJ.Studies on the anti-
cancer effect and acute toxicity of Pteris semipinnata[ J] .J
Chin Med Mater(中药材), 1996 , 19(1):29-32.
[ 3] Li JH , Liang NC , Mo LE , Zhang X , He CW.Comparison
of the cytotoxicity of five constituents from Pteris semipinna-
ta L.in vitro and the analysis of their structure-activity re-
lationships[ J] .Acta Pharm Sin(药学学报), 1998 , 33
(9):641-644.
[ 4] Li JH , Liang NC , Mo LE , Zhang X , He CW.Effect of ac-
tive compounds isolated from Pteris semipinnata L.on DNA
topoisomerases and ty rosine protein kinase and expression of
c-myc in lung adenocarcinoma cells[ J] .Chin J Cancer Res
(中国癌症研究), 2001 , 13(2):105-109.
[ 5] Bradford MM.A rapid and sensitive method for the quan-
titation of microgram quantities of protein utilizing the prin-
ciple of protein-dye binding[ J] .Anal Biochem , 1976 , 72:
248-254.
[ 6] Kikkawa U , Takai Y , Minakuchi R , Inohara S , Nishizuka
·85·中国药理学与毒理学杂志 2003 年 4月;17(2)
Y.Calcium-activated , phospholipid-dependent protein ki-
nase from rat brain.Subcellular distribution , purification ,
and properties[ J] .J Biol Chem , 1982 , 257(22):13341-
13348.
[ 7] Bansal N , Houle AG , Melnykovych G.Dexamethasone-in-
duced killing of neoplastic cells of lymphoid derivation:lack
of early calcium involvement[ J] .J Cell Physiol , 1990 ,
143(1):105-109.
[ 8] Ichikawa S , Hatanaka H , Takeuchi Y , Ohno S , Inagaki
F.Solution structure of cysteine-rich domain of protein ki-
nase C alpha[ J] .J Biochem(Tokyo), 1995 , 117(3):
566-574.
[ 9] Kazanietz MG , Wang S , Milne GW , Lewin NE , Liu HL ,
Blumberg PM.Residues in the second cysteine-rich region
of protein kinase C delta relevant to phorbol ester binding as
revealed by site-directed mutagenesis[ J] .J Biol Chem ,
1995 , 270(37):21852-21859.
[ 10] Ward NE , Pierce DS , Stewart JR , O′Brian CA.A peptide
substrate-based affinity label blocks protein kinase C-ca-
talyzed ATP hydrolysis and peptide-substrate phosphoryla-
tion[ J] .Arch Biochem Biophys , 1999 , 365(2):248 -
253.
[ 11] Ward NE , Stewart JR , Ioannides CG , O′Brian CA.Oxi-
dant-induced S-glutathiolation inactivates protein kinase C-
alpha(PKC-alpha):a potential mechanism of PKC isozyme
regulation[ J] .Biochemistry , 2000 , 39(33):10319 -
10329.
[ 12] He CW , Liang NC , Mo LE , Zhang X , Li JH.Apoptosis in
HL-60 cells induced by compound 6F isolated from Pteris
semipinnata L.[ J] .Chin J Cancer Prevent Treat(中国肿
瘤防治杂志), 2002 , 9(1):11-14.
[ 13] McCarthy JV , Fernandes RS , Gotter TG.Increased resis-
tance to apoptosis associated with HL-60 myeloid differentia-
tion status[ J] .Anticancer Res , 1994 , 14(5A):2063 -
2072.
[ 14] Zhu WH , Loh TT.Differential effects of phorbol ester on
apoptosis in HL-60 promyelocytic leukemia cells [ J] .
Biochem Pharmacol , 1996 , 51(9):1229-1236.
[ 15] Kwon TK.Phorbol myristate acetate inhibits okadaic acid-
induced apoptosis and downregulation of X-linked inhibitor
of apoptosis in U937 cells[ J] .Biochem Biophys Res Com-
mun , 2001 , 287(1):135-141.
[ 16] Ramoneda BM , Perez-Tomas R.Activation of protein ki-
nase C for protection of cells against apoptosis induced by
the immunosuppressor prodigiosin[ J] .Biochem Pharmacol ,
2002 , 63(3):463-469.
半边旗提取物 6F抑制 HL-60细胞蛋白激酶 C活性
何承伟1 , 梁念慈2 , 莫丽儿3 , 李金华1 , 张 晓1
(广东医学院 1.生物化学与分子生物学研究所 , 2.广东省天然药物研究与开发实验室 ,
3.化学教研室 , 广东 湛江 524023)
摘要:目的 探讨半边旗提取物 6F 的细胞毒作用
及诱导 DNA片段化与蛋白激酶 C(PKC)信号转导途
径的关系 ,检测 6F 对 PKC活性的影响 。方法 受
试对象为HL-60细胞 ,超速离心法获得的胞液(可溶
部分)及颗粒(不可溶部分 ,包括细胞膜系统及胞核)
部分用作 PKC活性测定。经 0.4 g·L-1磷脂酰丝氨
酸 , 0.04 g·L-1甘油二油酸酯激动剂作用酶粗提物
后 ,用液体闪烁计数仪计数[ γ-32P] ATP 参入外源底
物的量以测定 PKC 活性。MTT 法测定 HL-60 细胞
的活力 , 二苯胺法测定 6F 诱导 DNA片段化程度 。
结果 在所测试的浓度范围内(0.5 ～ 312 μmol·
L-1),化合物 6F 显著抑制胞液及颗粒部分 PKC 活
性 ,最大抑制率达 88.6%,呈浓度依赖关系(胞液部
分 r=0.781 , P <0.05 , 颗粒部分 r =0.931 , P <
0.01)。6F 诱导HL-60细胞DNA片段化及对细胞的
毒性作用可被具有致癌作用的 PKC 激活剂肉豆蔻
酸酯(PMA ,浓度为 65 nmol·L-1)拮抗 ,抑制率分别
是 30%和 44%(P<0.01)。PMA单独用使HL-60细
胞线粒体将MTT 还原为甲月赞的能力增强 14%(P <
0.01),即增强细胞活力 。结论化合物 6F是 PKC的
抑制剂 。6F 对 HL-60细胞 DNA片段化的诱导作用
及其细胞毒作用至少可部分归因于其对 PKC 活性
的抑制作用 。
关键词:半边旗;蛋白激酶 C;DNA 片段化;细胞
毒作用
基金项目:国家自然科学基金项目(39870900);广东省科
委重大科技攻关项目(9622007-002);广东省重点学科基金
(9306)
(本文编辑 乔 虹)
·86· Chin J Pharmacol Toxicol 2003 Apr;17(2)