From the Although the cyclopentenone prostaglandin
A1 (PGA1) is known to arrest the cell
cycle at the G1 phase in vitro and to suppress tumor growth in vivo, its relatively weak activity limits
its usefulness in cancer chemotherapy. In an attempt to develop
antitumor drugs of greater potency and conspicuous biological
specificity, we synthesized novel analogs based on the structure of
PGA1. Of the newly synthesized analogs,
15-epi- Prostaglandins of the A series
(PGAs)1 contain an
In order to develop more stable and potent PGA analogs, it appeared to
be important to decipher the structural features contributing to the
stability and antiproliferative activity of A type PGs. A type PGs are
conjugated with glutathione in the cells, and the resultant
PG-glutathiones are immediately exported from the cells through the
ATP-dependent glutathione S conjugate export
pump (MRP/GS-X pump) (12), thereby reducing the intracellular
accumulation of PGs. The GS-X pump was first reported to eliminate
cytotoxic drugs from tumor cells and to play an important role in
developing drug resistance in tumor cells (13). Thus, it is necessary
to develop PGA1 analogs that are not readily conjugated
with glutathione.
We previously demonstrated that the cross-conjugated dienone PGs,
including native Cell cycle progression in eukaryotes is controlled by specific
activation and subsequent inactivation of cyclin-dependent kinases (Cdks) (14). Two distinct families of Cdk inhibitors (CKIs)
have been described in mammalian cells (15). One of these is the
Cip/Kip family, which includes the structurally related proteins p21,
p27, and p57. Members of this family inhibit a variety of Cdk
activities in vitro (16-24). The other family of CKIs is the INK4 family, the members of which specifically inhibit cyclin D-dependent kinases in vitro (25-29). It has
recently been reported that treatment of breast carcinoma cells with
PGA2 leads to G1 arrest in association with
increased expression of p21 (30), suggesting that PGA2
inhibits tumor cell growth through the induction of p21. We also found
that In this study, we examined the ability of PGA1 analogs that
contain a cross-conjugated dienone unit to inhibit the proliferation of
A172 human glioma cells at molecular level. The results indicate that
three analogs, 15-epi- PGs and Their Analogs--
PGA1, PGA2
(Sigma), PGJ2,
15-deoxy- Cell Culture--
Human glioma A172 cells were cultured in
Dulbecco's modified essential medium (Life Technologies, Inc.)
supplemented with 10% heat-inactivated fetal bovine serum (Life
Technologies, Inc.) and penicillin-streptomycin (Life Technologies,
Inc.) under standard tissue culture conditions. Cell numbers were
counted with a cell counter (Coulter).
Cell Cycle Analysis--
Cell cycle distribution was analyzed by
flow cytometry. Briefly, 2 × 106 cells were harvested
in PBS containing 2 mM EDTA, washed once with PBS, and
fixed in PBS containing 0.2% paraformaldehyde for 30 min. Fixed cells
were washed once in PBS and permeabilized for 30 min with 0.2% Tween
20 and 1 mg/ml RNase A in PBS. Cells were then washed once in PBS and
stained with 50 µg/ml of propidium iodide (Boehringer Mannheim). The
stained cells were analyzed by flow cytometry (Coulter).
Northern and Western Blot Analysis--
Total cellular RNA was
extracted from A172 cells using RNA isolation reagent (Isogen, Nippon
Gene Co., Tokyo, Japan) according to the manufacture's instructions.
RNA (20 µg of each) was denatured, separated by electrophoresis on
1.0% agarose-formaldehyde gels, and transferred onto nylon membranes
(Micron Separations, MA). p21 or cyclin E cDNA was labeled with
[ Department of Geriatric Research,
Department of Chemistry and
Molecular Chirality Research Unit,
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
7-PGA1 methyl ester
(NAG-0092), 12-iso-
7-PGA1 methyl
ester (NAG-0093), and ent-
7-PGA1
methyl ester (NAG-0022) possess a cross-conjugated dienone structure
around the five-member ring with unnatural configurations at C(12)
and/or C(15) and were found to be far more potent than native
PGA1 in inhibiting cell growth and causing G1
arrest in A172 human glioma cells. These three analogs induced the
expression of p21 at both RNA and protein levels in a time- and
dose-dependent fashion. Kinase assays with A172 cells
treated with these analogs revealed that both cyclin A- and
E-dependent kinase activities were markedly reduced,
although cyclin D1-dependent kinase activity was
unaffected. Immunoprecipitation-Western blot analysis showed that the
decrease in cyclin A-dependent kinase activity was due to
an increased association of p21 with cyclin
A-cyclin-dependent kinase 2 complexes, whereas the decrease
in cyclin E-dependent activity was due to a combined
mechanism involving reduction in cyclin E protein itself and increased
association of p21. Thus, these newly synthesized PGA1
analogs may prove to be powerful tools in cancer chemotherapy as well
as in investigations of the structural basis of the antiproliferative
activity of A series prostaglandins.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
,
-unsaturated carbonyl group at the five-member ring and are
derived from E type prostaglandins by dehydration in plasma or aqueous
solution (1). PGAs have been reported to have antiproliferative
activity in vitro (2-8) as well as antitumor activity
in vivo (2, 7, 9-11), and although these properties suggest
that PGAs would be potentially useful in chemotherapy of malignant
tumors, their relatively weak activity and extreme instability in
plasma limit their clinical application.
12-PGJ2 and an artificial
analog
7-PGA1 methyl ester, reveal much more
potent antiproliferative effects than simple enone-type PGs, such as
PGA1 (12). Although PGs containing a cross-conjugated
dienone structure still react reversibly with glutathione to form
equilibrium mixtures of free PG and PG-glutathione conjugate in the
cell, this reaction biased the equilibrium reaction with intracellular
glutathione to the formation of enone-conjugated forms, accumulating
free PGs in the cell (12). Thus, the eminent potency of dienone PGs
over simple enone PGs may be explained by the difference in the
extrusion mechanism. In accordance with this concept, we have
demonstrated that the antiproliferative effect of
7-PGA1 methyl ester is significantly
enhanced in the presence of an MRP/GS-X pump
inhibitor.2
7-PGA1 methyl ester induces p21 via a
p53-independent pathway causing cell cycle arrest at G1
phase in HL-60 cells (31). p21 CKI has been implicated in cell cycle
arrest associated with a variety of cellular events, such as cellular
senescence (32), quiescence (33), differentiation (34-37), and DNA
damage (38). Although the expression of p21 is largely dependent on the
presence of functional p53, it is also regulated by p53-independent
mechanisms (39-41).
7-PGA1
methyl ester (NAG-0092),
12-iso-
7-PGA1 methyl ester
(NAG-0093), and ent-
7-PGA1 methyl
ester (NAG-0022), are far more potent than native PGA1 in
arresting cells at the G1 phase through a combined
mechanism involving the induction of p21 and the suppression of cyclin
E.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
12,14-PGJ2 (Cayman Chemical
Company), and PGA1 analogs were dissolved in absolute
ethanol, and dilutions were made in Dulbecco's modified essential
medium. The final concentration of ethanol did not exceed 0.1% or
affect cell growth. All manipulations of these compounds were performed under low lighting conditions. The code names and chemical structures of these compounds are shown in Fig. 1. Detailed methods for the design
and synthesis of PGA1 analogs are described
elsewhere.3
-32P]dCTP using a random primer labeling kit
(Amersham Pharmacia Biotech), and the hybridization signal was
quantified by means of a Fuji Imaging Analyzer BAS-1500. GAPDH mRNA
was used as an internal control for variations in loading and transfer
efficiency among samples.
Immunoprecipitation and Kinase Assay--
Twenty-four hours
after treatment with PGA1 analogs, cells were lysed in
IP-kinase buffer (HEPES, pH 8.0, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 0.1%
Nonidet P-40, 2 µg/ml aprotinin, 0.1 µM
phenylmethylsulfonyl fluoride, 1 mM NaF, 0.1 mM
Na3VO4, and 10 µM
-glycerophosphate). Lysates were mixed with 10 µl of antibodies
against cyclin A (Santa Cruz Biotechnology, H-432), cyclin E (Santa
Cruz Biotechnology, C-19), and cyclin D1 (Santa Cruz Biotechnology,
H-295). After 1 h of rotation at 4 °C, the immune complexes
were precipitated with protein A beads (Amersham Pharmacia Biotech).
The precipitated complexes were analyzed by Western blotting or assayed
for kinase activity at 30 °C for 30 min in a 30-µl reaction
mixture containing 50 mM HEPES, pH 8.0, 10 mM
MgCl2, 2.5 mM EGTA, 1 mM DTT, 10 µM
-glycerophosphate, 1 mM NaF, 0.1 mM Na3VO4, 0.1 µM
phenylmethylsulfonyl fluoride, 10 µM ATP, and 185 kBq of
[
-32P]ATP (222 TBq/mmol; NEN Life Science Products).
One microgram of histone H1 (for cyclin A- or E-dependent
kinases) or 0.2 µg of GST-pRb (for cyclin D1-dependent
kinases) was used as a substrate. The reaction products were separated
on SDS-polyacrylamide gel electrophoresis, and phosphorylated proteins
were detected by autoradiography and quantitated using a Fuji Image
Analyzer BAS-1500.
Statistical Analysis-- Statistical significance was determined by the analysis of variance. Scheffe's F test procedure for multiple pairwise post-hoc comparisons of means was implemented, and a p value of less than 0.05 was considered statistically significant.
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RESULTS |
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Chemical Structure of the Synthetic PGA1 Analogs-- Fig. 1 shows the chemical structures of the synthetic PGA1 analogs identified in this study as more potent antitumor compounds than native PGs. All of the analogs possess a cross-conjugated dienone unit and a methyl ester form at C(1), whereas native PGA1 and PGA2 are simple enone compounds and contain no methyl ester. Some of the synthetic PGs have unnatural configurations at C(12) and C(15).
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Potent Antiproliferative Activity of PGA1 Analogs-- The synthetic PGA1 analogs were first tested for their ability to inhibit cell proliferation, using PGA1, PGA2, and PGJ2 as references. A172 cells (from a glioblastoma cell line) were treated with each of the test compounds at a 5 µM concentration, and changes in the cell number were examined over a period of 72 h. As shown in Fig. 2, NAG-0022, NAG-0092, and NAG-0093 were most potent in suppressing the proliferation of A172 cells at 48 and 72 h (p < 0.05). NAG-0026 also displayed a substantial antiproliferative activity (p < 0.05). PGA1, PGA2, and PGJ2 had almost no effect on the cell growth at 5 µM (Fig. 2 and data not shown).
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G1 Arrest by PGA1 Analogs-- In order to examine the mechanism(s) of growth suppression by PGA1 analogs and also to rule out the possibility of nonspecific cytotoxicity, we next determined the cell cycle distribution of A172 cells after treatment of these cells with the various PGA1 analogs, using a fluorescence-activated cell sorter (Fig. 3). At a concentration of 5 µM, NAG-0022, NAG-0092, and NAG-0093 were most potent in arresting cells at the G1 phase (66.7, 74.9, and 71.0%, respectively) as compared with vehicle-treated cells (42.2%), whereas the other compounds showed only a modest effect on cell cycle distribution (Fig. 3 and data not shown). The growth inhibitory activity of the PGA1 analogs (Fig. 2) correlated well with their ability to cause G1 arrest (Fig. 3), suggesting that most of these analogs inhibit cell growth in this manner.
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Induction of p21 Expression by PGA1 Analogs--
It
has been reported that PGA2 arrests the cell cycle at the
G1 phase through the induction of p21 CKI at high
concentrations (25-36 µM) (30, 42). To determine whether
this is also the case with PGA1 analogs, we examined
changes in p21 expression after using the analogs to treat the A172
cells. As shown in Fig. 4, the baseline
level of p21 mRNA expression of these cells was low, and NAG-0022,
NAG-0092, and NAG-0093 caused a dose-dependent increase in
the steady-state mRNA level, with a clear-cut effect being observed
at 1.0 µM and a maximal response at 5.0-10
µM (approximately 7-fold as compared with vehicle-treated
cells). Only a modest increase in p21 mRNA was observed with
NAG-0026, and PGA1, PGA2, and
12-PGJ2 had little or no effect at 5 µM (Fig. 4).
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|
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PGA1 Analogs Suppress Cyclin A- and Cyclin E- but not Cyclin D1-dependent Kinase Activities-- To further elucidate the mechanism(s) by which PGA1 analogs arrested the cell cycle at G1, we examined cyclin E-, A- and D1-dependent kinase activities, all of which are necessary for G1 progression and G1/S transition. As shown in Fig. 7A, cyclin A- and E-dependent kinase activities were almost completely inhibited at 24 h after treatment with 5 µM NAG-0092, whereas cyclin D1-dependent kinase activity was not affected (data not shown).
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DISCUSSION |
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In the present study, we tested a series of PGA1 analogs for their activities in inhibiting the growth of A172 glioblastoma cells and found that three of these were far more potent than native PGA1, PGA2, and PGJ2. Although it has been reported that some PGs of the A and J series are capable of suppressing tumor cell growth both in vitro (2-8) and in vivo (2, 7, 9-11), their activities are rather modest, presumably due to their intracellular instability, necessitating the use of high doses to obtain substantial antitumor effects. We set out to develop PG analogs of the A and J series that show increased stability, because we considered that some of these compounds might prove to be more potent in inhibiting tumor cell growth than the native compounds. In parallel with this task, we attempted to elucidate the molecular mechanism(s) by which our novel analogs inhibit cell growth.
There is accumulating evidence that active export of anticancer drugs
from cells is one of the major mechanisms of drug resistance. Recent
studies of the multidrug resistance phenotype of tumor cells have led
to the discovery of P-glycoprotein, which mediates the efflux of
anticancer drugs, such as doxorubicin, vincristine, and taxol (45, 46).
More recently, another type of drug transporter (GS-X/MRP pump) has
been identified as a mediator of glutathione-associated drug
resistance. It has been reported that A type PGs are effectively conjugated with glutathione in the cells (12), raising the possibility that GS-X/MRP pump may play an important role in excluding the PGs from
the cells. This concept was further supported by the observation that
HL-60 cells overexpressing functional GS-X/MRP pump reveal far more
resistance to the antiproliferative effect of
7-PGA1 methyl ester than control HL-60
cells.
We have previously shown that the cross-conjugated dienone unit, as compared with the simple enone unit in native PGA, protects PGs against removal from the cytoplasm by GS-X/MRP pump (12) and allows PGs to bind more stably to target proteins in the nucleus or cytosol, suggesting that the cross-conjugated dienone unit stabilizes PGs within the cell. However, in terms of clinical application, it was required to develop more stable and potent PGA1 analogs.
Of the newly synthesized analogs that we synthesized, NAG-0092,
NAG-0093 and NAG-0022, which showed the higher antiproliferative activity for A172 cells than NAG-0026
(7-PGA1 methyl ester) (Figs. 1 and 2),
possess both a hydroxy group at C(15) and a double bond at
C(13)-C(14). Thus, the double bond and hydroxy group in the
-chain
are essential for the potent antiproliferative activities of PGs for
these cells. The double bond is thought to fix the conformation of PGs,
and the hydroxy group constitutes a hydrogen bonding, allowing
sufficient interaction with the target molecule. Although native
PGA2 has been reported to inhibit cell growth at 25-36
µM (30, 42, 47), almost no growth inhibitory effect was
observed at the lower concentrations (up to 10 µM) used
in the current study.
FACScan analysis revealed that PGA1 analogs arrested the cell cycle at the G1 phase (Fig. 3). Interestingly, although NAG-0026 substantially inhibited cell growth, it did not cause accumulation of cells at G1 (data not shown), raising the possibility that growth inhibition by this particular analog may be due to its nonspecific toxicity.
It has recently been reported that PGA2 inhibits the G1 cyclin-dependent kinase activities through the induction of p21 (30). We showed by both Northern and Western blot analyses that the PGA1 analogs with the greatest ability to inhibit cell growth markedly induced the expression of p21 (Figs. 4 and 6). Because p21 expression was induced within 2 h after treatment with PGA1 analogs (Fig. 5) and because the induction was enhanced in the presence of cycloheximide (data not shown), it is plausible that the induction of p21 expression resulted from the direct genomic action of the PGA1 analogs.
Based on our analysis of cyclin-Cdk complexes and their activities, PGA1 analogs seem to inhibit tumor cell growth through two distinct mechanisms: the induction of p21 and the suppression of cyclin E expression (Fig. 7). Inactivation of cyclin A-Cdk2 activity by PGA1 analogs appears to occur mainly through the former mechanism, whereas suppression of cyclin E-Cdk2 activity appears to involve both increased p21 and reduced cyclin E expressions. Taken together with the findings that PGA1 analogs did not induce the expression of other known Cdk inhibitors or cyclins (data not shown), it is concluded that the combined effects on the expression of p21 and cyclin E are responsible for the growth suppressive function of PGA1 analogs. Although the molecular and cellular mechanisms by which PGA1 analogs induce p21 and suppress cyclin E expression remain elusive, the induction of p21 and the suppression of cyclin E by PGA1 analogs may involve a transcriptional mechanism because the PGA1 analogs are incorporated into nuclei without further metabolism (12).
Members of the J2 series of PGs have been reported to have
a unique spectrum of biological effects, including the inhibition of
cell cycle progression, the suppression of viral replication, and the
stimulation of osteogenesis (11). Results of previous studies have
indicated that the activation of peroxisome proliferator-activated receptor , a member of the nuclear receptor superfamily, is mediated by metabolites of J type PGs (48, 49). Although it has been demonstrated that PGA2 can also activate peroxisome
proliferator-activated receptor
, its potency appears to be
relatively weak (50). In view of our present results that both
PGJ2 and 15-deoxy-
12,14-PGJ2,
putative ligands for peroxisome proliferator-activated receptor
,
are not effective in the induction of p21 expression in A172 cells, PGs
of the A series are likely to serve as ligands for an as yet
unidentified member(s) of the nuclear receptor superfamily distinct
from peroxisome proliferator-activated receptor
.
In conclusion, we have identified novel PGA1 analogs that strongly suppress cellular proliferation through their combined effects on p21 CKI and cyclin E. Our results may help to decipher the structural features essential to the antiproliferative activity of the A and J series of PGs. Although it remains to be determined whether the increased potency of our analogs reflects an increased stability within cells or an increased binding affinity to proteins, these analogs may prove to be powerful tools for elucidating the molecular functions of the A series of PGs in both in vitro and in vivo studies.
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ACKNOWLEDGEMENTS |
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We thank Dr. Toshihisa Ishikawa (Pfizer Pharmaceuticals) and the members of our cell cycle group in the Department of Geriatric Research, National Institute for Longevity Sciences, for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas 09273104 (to M. N.) and 09273102 (to M. S.) and by Grant-in-Aid for Scientific Research (A) 08408023 (to M. S.) from the Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed. Tel. and Fax: 81-562-44-6595; E-mail: mnakani{at}nils.go.jp.
1 The abbreviations used are: PGA, prostaglandin A; PG, prostaglandin; Cdk, cyclin-dependent kinase; CKI, Cdk inhibitor; IP, immunoprecipitation; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
2 T. Ishikawa, K. Akimaru, M. Nakanishi, M. Suzuki, K. Furutu, and R. Noyori, submitted for publication.
3 M. Suzuki, T. Kiho, T. Tomokiyo, K. Furuta, S. Fukushima, Y. Takenchi, M. Nakanishi, and R. Noyori, submitted for publication.
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REFERENCES |
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