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Int. J. Cancer: 76, 571–578 (1998)
r 1998 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de l’Union Internationale Contre le Cancer
HUMAN CHORIONIC GONADOTROPIN (hCG) INHIBITS CISPLATIN-INDUCED
APOPTOSIS IN OVARIAN CANCER CELLS: POSSIBLE ROLE OF UP-REGULATION
OF INSULIN-LIKE GROWTH FACTOR-1 BY hCG
Hideki KURODA, Masaki MANDAI, Ikuo KONISHI*, Yasuichiro YURA, Yuko TSURUTA, Atia A. HAMID,
Kanako NANBU, Katsuko MATSUSHITA and Takahide MORI
Department of Gynecology and Obstetrics, Faculty of Medicine, Kyoto University, Kyoto, Japan
Gonadotropins have been suggested to play a role in the
development or progression of ovarian cancer, and we have
previously reported the expression of luteinizing hormone/
human chorionic gonadotropin (LH/hCG) receptor in 40% of
epithelial ovarian carcinomas. To examine the biological
effect of LH/hCG on ovarian cancer cells, apoptosis induced
by cisplatin with or without hCG treatment was investigated
in 2 ovarian cancer cell lines, OVCAR-3 and SK-OV-3. Stimulation of cell proliferation by hCG was also studied. In
addition, to analyze further the mechanism of hCG signaling
involved in apoptosis-inhibition, we examined the expression
of LH/hCG receptors and the regulation by hCG for apoptosisinhibitory pathways, such as the bcl-2/bax system and the
insulin-like growth factor-1 (IGF-1)/IGF-1 receptor (IGFR)
system. hCG did not increase cell proliferation in either cell
line. However, hCG treatment suppressed cisplatin-induced
apoptosis by 58% in the OVCAR-3 cells, as shown by immunofluorescent staining and quantitation of DNA fragmentation.
LH/hCG receptor mRNA was expressed only in OVCAR-3,
and no apoptosis-inhibitory effect of hCG was observed in the
SK-OV-3 cells that did not express the receptor. In the
OVCAR-3 cells, hCG significantly increased mRNA expression of IGF-1, but did not change mRNA levels of bcl-2/bax.
Our findings suggest that LH/hCG influences the chemosensitivity of ovarian cancer cells through an apoptosis-inhibitory
signal possibly via up-regulation of IGF-1 expression. Int. J.
Cancer 76:571–578, 1998.
r 1998 Wiley-Liss, Inc.
Ovarian cancer is the leading cause of death from gynecological
malignancies. Since the ovary is a target tissue of gonadotropins
engaged in cyclic steroidogenesis, various hormonal conditions
have been implicated in the biological behavior of ovarian cancer
(Rao and Slotman, 1991). The possible relationship between
infertility drugs and ovarian cancer risk has also been a subject for
ongoing debate (Whittemore et al., 1992; Balasch and Barri, 1993;
Cohen et al., 1993). There have been reports of ovarian cancer
arising in infertile women during or after treatment with gonadotropins (Balasch and Barri, 1993; Kuroda et al., 1997b). Thus, the
possible influence of gonadotropins on carcinogenesis or the
progression of epithelial ovarian carcinomas is currently an important concern. Although whether ovarian cancer cells have specific
binding sites for gonadotropins (Stouffer et al., 1984; Nakano et al.,
1989) has been a subject of controversy, we have demonstrated that
luteinizing hormone/human chorionic gonadotropin (LH/hCG)
receptor is expressed at the mRNA and protein levels in 40% of
epithelial ovarian carcinomas (Mandai et al., 1997). This finding
prompted us to evaluate the functional aspects of LH/hCG signaling in ovarian cancer cells.
Apoptosis is one of the fundamental forms of cell death,
characterized by several morphological features such as fragmented nuclei, condensed chromatin and the protrusion of cytoplasm or formation of apoptotic body, which are not usually
observed in another type of cell death, necrosis (Kerr et al., 1972).
Apoptotic cell death generally is tightly regulated by normal
physiological dynamics in various organs (Raff, 1992) or by
differentiation processes (Ellis et al., 1991). LH/hCG has been
reported to be involved in the inhibition of apoptosis in ovarian
follicular cells (Tilly et al., 1995). In malignant neoplasms, it has
been recognized that apoptotic cell death is induced by chemothera-
peutic agents such as cisplatin, a key drug for the treatment of
ovarian cancer (Gibb et al., 1997). The induction of apoptosis by
anticancer drugs plays an important role in therapeutic mechanisms. Therefore, if LH/hCG modulates the apoptotic pathway, it
may influence the chemosensitivity of ovarian cancer cells.
To address this hypothesis, we examined the effect of hCG on
cisplatin-induced apoptosis by various methods including the
quantitation of DNA fragmentation in 2 representative ovarian
cancer cell lines, OVCAR-3 and SK-OV-3 (Gibb et al., 1997). The
influence of hCG on the proliferation of these cell lines was also
studied. To explore the mechanistic pathway of hCG signaling for
apoptosis-inhibition, we examined the mRNA expression of LH/
hCG receptor in these cell lines. In addition, we investigated
whether hCG regulates the gene expression of apoptosis-related
systems such as bcl-2/bax and insulin-like growth factor-1 (IGF-1)/
IGF-1 receptor (IGFR).
MATERIAL AND METHODS
Cell cultures
The ovarian cancer cell lines OVCAR-3 and SK-OV-3 were
purchased from the ATCC (Rockville, MD). OVCAR-3 and
SK-OV-3 cells were maintained in RPMI-1640 and McCoy’s 5a
media, respectively, supplemented with 100 units/ml penicillin,
100 mg/ml streptomycin, 10% fetal bovine serum, and 10 mg/ml
insulin, at 37°C in 5% CO2. Equal numbers of each cell line (5 3
106 cells in 10 ml medium) were seeded in 10-cm culture dishes
and allowed to attach for 48 hr.
Treatment with cisplatin with or without hCG incubation
For the induction of apoptosis with cisplatin with or without
hCG, cells were precultured in medium with or without 0.1 µg/ml
recombinant hCG (Roto Seiyaku, Osaka, Japan) for 24 hr and
incubated in the medium containing 10 µM cisplatin for 2 hr. Then,
each medium was replaced by cisplatin-free medium with or
without 0.1 µg/ml hCG. After 24 hr of incubation in the cisplatinfree medium, apoptosis of the cells was assessed by various
methods. Cell cultures not treated with drugs were used as controls.
All the experiments were performed 3 times or more. In the
preliminary experiments, the cisplatin concentration ranged from 5
to 100 µM. The cisplatin concentration of 10 µM corresponded to
50% lethal dose (LD50) for both the OVCAR-3 and SK-OV-3 cells
and was appropriate for observation of apoptosis-inhibition by
hCG. In the preliminary experiments, hCG concentration ranged
from 0.01 to 10 µg/ml. Since the effect of hCG on apoptosis was
observed at a concentration of 0.1 µg/ml or higher, the concentra-
Grant sponsor: Grants-in-Aid for Scientific Research, Ministry of
Education, Science and Culture, Japan; Grant numbers: 09470358 and
097711274.
*Correspondence to: Department of Gynecology and Obstetrics, Faculty
of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan. Fax: (81)75761-3967. E-mail: konishi@kuhp.kyoto-u.ac.jp
Received 23 October 1997; Revised 12 January 1998
572
KURODA ET AL.
tion was then fixed at 0.1 µg/ml for all the subsequent experiments.
In addition, the end point was set at 24 hr after cisplatin treatment;
this period was shown to be appropriate for assessment of
cisplatin-induced apoptosis (Gibb, et al., 1997).
Count of viable cells after cisplatin treatment
After the cisplatin treatment with or without hCG as described
above, the cells were trypsinized and resuspended in 1 ml PBS
(Nissui, Tokyo, Japan). The cells were stained by 0.02% erythrocin
B, and the number of viable cells was counted in a hemocytometer
chamber.
Immunofluorescent staining for fragmented DNA (TUNEL)
After the apoptosis-induction described above, floating and
attached cells were collected, resuspended in 1 ml PBS and rinsed
with PBS. The cells were then resuspended and centrifuged onto
microscopic slides at 50g for 5 min using a cytospin centrifuge. The
cells were then fixed with 4% paraformaldehyde at 4°C for 1 hr.
After treatment with 0.5% Triton-X and RNase, immunofluorescent staining of fragmented DNA was carried out by the TdTmediated dUTP-biotin nick end labeling (TUNEL) method with a
Mebstain Apoptosis Kit (Medical and Biological Laboratories,
Nagoya, Japan), according to the manufacturer’s protocol. The
number of cells positive for immunofluorescent staining was
counted in 5 high-power fields (HPF)(320 objective, 310 eyepiece).
DNA extraction and gel electrophoresis for DNA fragmentation
DNA fragmentation was analyzed by DNA ladder formation
using an Arends et al. (1990) modified method. Briefly, after the
apoptosis-induction described above, all cells were collected with a
cell scraper. Extraction of cellular DNA was performed; cells were
lysed in 0.5% Triton-X, 10 mM Tris-HCl (pH 7.5) and 20 mM
EDTA for 2 hr on ice. After centrifugation at 14,000g for 30 min,
the supernatants containing fragmented DNA were incubated with
200 mg/ml proteinase K for 1 hr at 50°C and extracted by the
phenol/chloroform method. The DNA was then precipitated overnight at 220°C in 2 volumes of ethanol, 0.13 M NaCl and 50 mg
glycogen. After treatment with 1 mg/ml boiled bovine pancreatic
RNase A for 1 hr at 50°C, the DNA was loaded onto a 2% (w/v)
horizontal agarose gel containing 0.3 mg/ml ethidium bromide and
run in 13 TAE buffer. The gels were photographed under UV light.
Since DNA was extracted from equal numbers of pre-treated cells,
the relative amount of fragmented DNA in each dish was obtained
by measuring the amount of low m.w. DNA. NIH Image 1.55 Plot
Profile was used to measure the amount of low m.w. DNA
(#900 bp).
BrdU assay
The effect of hCG on the proliferative activity of the 2 ovarian
cancer cell lines was assessed by a bromodeoxyuridine (BrdU)
incorporation assay using a 5-bromo-28-deoxy-uridine labeling and
detection kit (Boehringer Mannheim, Indianapolis, IN). In 100 µl
of each medium, 5 3 104 of OVCAR-3 and SKOV-3 cells were
seeded in a 96-well microtiter plate. Cells were incubated at 37°C
in 5% CO2 and allowed to attach for 24 hr. After incubation, the
medium was carefully replaced by the medium with or without 0.1
µg/ml hCG for 24 hr. BrdU assay was then carried out according to
the manufacturer’s protocol.
RNA preparation and RT-nested PCR of LH/hCG receptor gene
Total RNA was isolated by the method of Chomczynski and
Sacchi (1987). Reverse transcription (RT)-nested polymerase chain
reaction (PCR) was performed according to the method of Lin et al.
(1994) with some modifications. Briefly, cDNA was prepared from
1 µg of total RNA by the random priming method using a
first-strand cDNA synthesis kit (Pharmacia-LKB, Uppsala, Sweden). The nucleotide sequences of the primers used for PCR were
as follows: the sense and antisense primers for first-round PCR
were 58-GCATCTGTAACACAGGCATC-38 and 58-CATCTG-
GTTCAGGAGCACAT-38, respectively: the sense and antisense
primers for second-round PCR were 58-GCAGAAGATGCACAATGGAG-38 and 58-CTCTCAGCAAGCATGGAAGA-38, respectively. First-round PCR was carried out in a Thermal Cycler
(Perkin-Elmer Cetus, Norwalk, CT) with a mixture consisting of
cDNA derived from 50 ng of RNA, 8 pmol each of upstream and
downstream primer, 200 mmol of dNTP and 0.1 unit of Taq DNA
polymerase with reaction buffer (Takara Shuzo, Shiga, Japan) in a
final volume of 10 µl. The PCR conditions consisted of denaturation for 5 min at 94°C, then 30 cycles of denaturation for 1 min at
94°C, annealing for 2 min at 55°C and extension for 1 min at 72°C,
followed by a final extension reaction for 5 min at 72°C. One-tenth
of the product of first-round PCR was used for second-round PCR,
in which the PCR conditions were the same except that nested
primers were used.
Northern blotting for bcl-2 and bax mRNA expression
OVCAR-3 cells were cultured in the presence or absence of 0.1
µg/ml hCG for 4, 8, 24, 48 and 72 hr. Ten micrograms of total RNA
were separated by electropheresis in 1% agarose-formaldehyde
gels and transferred onto a nylon membrane. The plasmids
containing the coding region of bcl-2 and bax cDNA were kindly
provided by Dr. Y. Tsujimoto (Osaka University) and Dr. C.
Sakakura (Kyoto Prefectural University of Medicine), respectively.
Probes for Northern blotting were prepared by PCR using the
primers as follows. For bcl-2, the sense primer was 58-GGTGCCACCTGTGGTCCACCTG-38 and the antisense primer was 58CTTCACTTGTGGCCCAGATAGG-38. For bax, the sense and
antisense primers were 58-AGCGGCGGTGATGGACGGGT-38
and 58-CTCAGCCCATCTTCTTCCAG-38, respectively. Complementary DNAs were hybridized sequentially to the membranes at
65°C in rapid hybridization buffer (Amersham, Aylesbury, UK),
followed by final washes in 0.13 standard saline citrate (SSC) and
SDS for 5 min at 65°C. The membrane was rehybridized with a
human glyceraldehyde-3-phosphate dehydrogenase (G3PDH)
complementary DNA probe to normalize gene expressions. Densitometric analysis was performed using a BAS 2000 II Bioimage
Analyzer (Fujix, Tokyo, Japan).
Northern blotting and RT-PCR for IGF-1
and IGFR mRNA expression
OVCAR-3 cells were cultured in the presence or absence of 0.1
µg/ml hCG for 4, 8, 24, 48 and 72 hr. For Northern blot
hybridization of IGF-1 mRNA, 10 mg of total RNA were separated
by electrophoresis in 1% agarose-formaldehyde gels and transferred onto a nylon membrane. The DNA probe for IGF-1 was
prepared from human corpus luteum cDNA by RT-PCR as described above and was then sequenced. Complementary DNAs
were hybridized sequentially to the membranes at 55°C in 53
saline sodium phosphate-EDTA, 1% SDS, 53 Denhardt’s solution
and 0.5 mg/ml salmon sperm DNA, followed by final washes in
0.13 SSC and SDS for 5 min at 55°C. The filters were rehybridized
with a G3PDH probe. Densitmetric analysis was performed using a
BAS 2000 II Bioimage Analyzer.
For RT-PCR, the total RNA and cDNA of each sample were
prepared as described above. The nucleotide sequences of the
primers for IGF-1 and IGFR were as follows (Ullrich et al., 1986;
Sussenbach, 1989). For IGF-1, the sense and antisense primers
were 58-TCTTGAAGGTGAAGATGCACACCA-38 and 58-AGCGAGCTGACTTGGCAGGCTTGA-38, respectively. For IGFR,
the sense primer was 58-ACCCGGAGTACTTCAGCGCT-38, and
the antisense primer was 58-CACAGAAGCTTCGTTGAGAA-38.
The PCR conditions consisted of denaturation for 5 min at 94°C, 30
cycles of denaturation for 30 sec at 94°C, annealing for 30 sec at
55°C and extension for 1 min at 72°C, followed by a final extension
reaction for 5 min at 72°C.
Statistical analysis
Differences were assessed by Student’s t test and Wilcoxon test
for signed rankings; p , 0.05 was considered significant.
INHIBITION OF APOPTOSIS OF OVARIAN CANCER CELLS BY hCG
573
RESULTS
Cell morphology after cisplatin treatment with or without hCG
The OVCAR-3 cells grew as a cobble-stone-like monolayer with
multilayering foci. Twenty-four hours after exposure to cisplatin,
the OVCAR-3 cells exhibited formation of small, roughly spherical
or ovoid cytoplasmic fragments that have been designated apoptotic bodies (Kerr et al., 1972)(Fig. 1a). In contrast, the OVCAR-3
cells treated with hCG before and after exposure to cisplatin
showed significantly less apoptotic features (Fig. 1b). In the
SK-OV-3 cells, incubation with cisplatin produced similar apoptotic
features, but treatment with hCG did not change the morphology.
Reduction of cell death due to cisplatin treatment by hCG
The number of viable OVCAR-3 cells counted 24 hr after
cisplatin treatment was 2.68 6 0.29 (M 6 SD) 3 106/dish in
the cisplatin-treated cells without hCG, 3.43 6 0.21 3 106/dish
in the cisplatin-treated cells with hCG and 4.20 6 0.59 3 106/dish
in the cells not exposed to cisplatin (Fig. 2). Incubation with hCG
before and after cisplatin treatment significantly decreased cell death (by
49%), compared with cisplatin treatment alone (p 5 0.0013).
Inhibition of cisplatin-induced apoptosis by hCG:
immunofluorescent staining of fragmented DNA (TUNEL)
To distinguish necrosis and apoptosis, we initially performed the
immunofluorescent staining of fragmented DNA (TUNEL). The
OVCAR-3 cells exposed to cisplatin showed a significantly larger
FIGURE 2 – Number of viable OVCAR-3 cells counted 24 hr after
cisplatin treatment. The incubation with hCG before and after exposure
to cisplatin reduced the cell death by 49%.
number of cells stained for fragmented DNA compared with the
non-exposed cells. The cells not exposed to cisplatin contained
5.2 6 3.6 apoptotic cells/HPF. The cells treated with cisplatin
without hCG contained 23.4 6 4.0 cells/HPF with fragmented
DNA (Fig. 3a). The cells incubated with hCG before and after
exposure to cisplatin contained 12.8 6 2.8 cells/HPF with fragmented DNA and showed a 58% decrease in the number of
apoptotic cells (Fig. 3b), compared with the cells without hCG
treatment (p 5 0.0004; Fig. 3c).
FIGURE 1 – Morphological features of OVCAR-3 cells 24 hr after
cisplatin treatment with or without incubation with hCG. (a) Cells
treated with cisplatin alone. The formation of apoptotic bodies, i.e.,
small, roughly spherical or ovoid cytoplasmic fragments is evident
(arrows). (b) Cells treated with hCG before and after exposure to
cisplatin. Apoptotic features were significantly decreased. Scale bar 5
100 µm.
Inhibition of cisplatin-induced apoptosis by hCG:
quantitation of low m.w. DNA
To confirm the suppressive effect of hCG on apoptosis, the
amount of fragmented DNA in the apoptotic cells exposed to
cisplatin with or without hCG was quantitated by electrophoresis
for DNA fragmentation. The low m.w. DNA (#900 bp) in DNA
ladders was measured with the NIH Image program. The amount of
low m.w. DNA was significantly decreased (by 58%) in the
hCG-treated OVCAR-3 cells compared with the non-treated cells
(p 5 0.0117; Fig. 4a,b). In contrast, no reduction of low m.w. DNA
by hCG treatment was observed in the SK-OV-3 cells (Fig. 4c).
Effect of hCG on cell proliferation
To address whether the reduction in cell death produced by hCG
treatment was the result of an inhibition of apoptosis or an increase
in cell number, the effect of hCG on the proliferative activity of the
2 cell lines was assayed by BrdU incorporation. The BrdU assay
574
KURODA ET AL.
FIGURE 3 – Immunofluorescent staining for fragmented DNA (TUNEL) in OVCAR-3 cells. (a) Cells treated with cisplatin were extensively
stained for fragmented DNA. (b) hCG incubation before and after exposure to cisplatin produced a decrease in the number of cells with
fragmented DNA. (c) Significant difference in the number of cells stained for fragmented DNA with or without hCG treatment. Scale bar 5 100
µm.
revealed no significant activation of cell proliferation in response to
hCG stimulation in OVCAR-3 (p 5 0.1147) or SK-OV-3 cells (p 5
0.2934; Fig. 5a,b).
LH/hCG receptor mRNA expression in ovarian cancer cells
In the OVCAR-3 cells, RT-nested PCR amplified 2 differentsized fragments (Fig. 6; lane 3), both of which corresponded to the
cDNA fragments of LH/hCG receptors (Lin et al., 1994). The
larger fragment (342 bp) corresponded to the full-length cDNA,
and the smaller fragment (156 bp) corresponded to the splice
variant lacking exon 9 (Mandai et al., 1997). In contrast, neither of
these two fragments was amplified in the SK-OV-3 cells (Fig. 6,
lane 2).
hCG up-regulates mRNA expression of IGF-1 but not that
of bcl-2/bax
In the OVCAR-3 cells, hCG treatment did not change the mRNA
level of the bcl-2 or bax genes, as shown by Northern blotting (Fig.
7). In contrast, both Northern blotting and RT-PCR demonstrated
that hCG treatment increased the mRNA expression of IGF-1 in
OVCAR-3 cells; 48 hr of hCG incubation showed a 2-fold increase
compared with control (Fig. 8a,b). mRNA expression of IGFR was
found in OVCAR-3 cells, although hCG treatment did not change
its expression level (Fig. 8b).
DISCUSSION
Our results demonstrate that cisplatin treatment induces apoptosis in the 2 representative ovarian cancer cell lines, OVCAR-3 and
SK-OV-3. Both cell lines showed the same chemosensitivity to
cisplatin; approximately half of the cells underwent apoptosis 24 hr
after treatment with 10 µM cisplatin for 2 hr. These data are
consistent with those of a previous report (Gibb et al., 1997).
Strikingly, incubation with hCG before and after the exposure to
cisplatin reduced cell death rate of OVCAR-3 cells, which express
LH/hCG receptor mRNA. This was ascribed to the inhibition of
apoptosis by hCG, as shown by immunofluorescent staining and
the quantitation of DNA fragmentation; hCG treatment suppressed
apoptosis by 58% compared with the untreated OVCAR-3 cells
exposed to cisplatin. Although an inhibitory effect of hCG on the
apoptotic pathway has previously been reported in granulosa cells
of normal ovarian follicles (Tilly et al., 1995), this effect has never
been reported in malignant tumor cells. In the present study, the
apoptosis-suppressive effect of hCG was not observed in another
ovarian cancer cell line, SK-OV-3, which does not express the
LH/hCG receptor. These findings suggest that the inhibition of
apoptosis by hCG in ovarian cancer cells depends on the expression
of LH/hCG receptor.
INHIBITION OF APOPTOSIS OF OVARIAN CANCER CELLS BY hCG
575
FIGURE 4 – DNA ladder formation after treatment with cisplatin in ovarian cancer cell lines, OVCAR-3 and SK-OV-3 (lane 1, cells in control
medium without cisplatin; lane 2, cells treated with cisplatin alone; lane 3, cells treated with hCG before and after exposure to cisplatin.). In the
OVCAR-3 cells exposed to cisplatin, compared with no hCG treatment (a, lane 2), the amount of low m.w. DNA was significantly decreased by
hCG incubation (a, lane 3). Quantitation of the of low m.w. DNA (#900 bp) with the NIH Image program showed a 58% decrease with hCG
treatment in OVCAR-3 cells (b). In the SK-OV-3 cells, there was no difference in DNA fragmentation between the cells treated with cisplatin
alone (c, lane 2) and those treated with cisplatin plus hCG (c, lane 3).
To clarify whether the effect of hCG is an attenuation of
apoptosis and not an increase in cell number, the stimulation of
mitogenic activity by hCG was analyzed by BrdU assay. hCG
treatment for 24 hr showed no stimulatory effect on the proliferation of the LH/hCG receptor-positive OVCAR-3 or that of the
receptor-negative SK-OV-3. There have been several reports on the
effects of gonadotropins on the growth of ovarian cancer cells
(Simon et al., 1983; Ohtani et al., 1992; Wimalasena et al., 1992;
Kataoka et al., 1994), and all these authors supported the concept of
the mitogenic action of follicle-stimulating hormone (FSH). How-
ever, whether LH/hCG has an influence on cell proliferation has
been a subject of controversy; a stimulatory effect was seen in
several ovarian cancer cell lines (Simon et al., 1983; Wimalasena et
al., 1992) but not in others (Ohtani et al., 1992; Kataoka et al.,
1994). LH/hCG receptor expression was not examined in those cell
lines. Therefore, the discrepancy in proliferative effect may be due
to a difference of LH/hCG responsiveness among the cancer cell
lines or to the effect of FSH contained in non-recombinant hCG,
which was substituted for recombinant hCG in our present
experiment.
576
KURODA ET AL.
FIGURE 5 – Evaluation of proliferation of OVCAR-3 (a) and SK-OV-3 (b) cells in response to hCG assessed by BrdU assay. Neither of the 2 cell
lines revealed significant differences in growth after hCG treatment.
FIGURE 7 – Northern blot analysis of the mRNA expression of bcl-2
and bax in OVCAR-3 cells. hCG treatment did not change the gene
expressions of bcl-2 (a) or bax (b). 1, 0; 2, 4; 3, 8; 4, 24; 5, 48; and 6, 72
hr of hCG treatment.
FIGURE 6 – RT-nested PCR detection of LH/hCG receptor mRNA.
In the OVCAR-3 cells, 2 different-sized fragments of cDNA were
amplified; the larger one (342 bp) corresponded to the full-length
LH/hCG receptor mRNA, and the smaller fragment (156 bp) corresponded to a splice variant lacking exon 9 (lane 3). No fragments were
detected in the SK-OV-3 cells (lane 2). Lane 1, m.w. marker; lane 4,
ovarian corpus luteums as positive control.
To explore the mechanistic pathway of apoptosis-suppression by
hCG, we examined the bcl-2/bax system and the IGF-1/IGFR
system in the ovarian cancer cell line OVCAR-3. The results
demonstrated that hCG treatment up-regulated mRNA expression
of IGF-1 but did not change bcl-2/bax expression. Apoptosis is
regulated by the expression of several gene cascades such as those
of Fas, myc, p53, bcl-2, bax and bcl-xL, followed by an activation
of proteases such as interleukin-1 beta-converting enzyme (ICE).
Both cell lines have been shown to contain p53 genetic alterations:
a transition in codon 248 of exon 7 with associated allelic loss in
OVCAR-3 and a probable genetic rearrangement leading to
absence of detectable p53 in SKOV-3 cells (Brown et al., 1993).
This suggests that inhibition of cisplatin-induced apoptosis by hCG
is independent of p53 dysfunction. The suppression of ICE activation is regarded as a possible mechanism for apoptosis-inhibition
by IGF-1/IGFR (Jung et al., 1996). hCG has also been shown to be
INHIBITION OF APOPTOSIS OF OVARIAN CANCER CELLS BY hCG
FIGURE 8 – Northern blot hybridization and RT-PCR analysis for
mRNA expression of IGF-1 and IGFR in OVCAR-3 cells. hCG
treatment significantly increased mRNA expression of IGF-1, as shown
by Northern blotting (a) and RT-PCR (b). Expression of IGFR was not
changed by hCG treatment (b). 1, 0; 2, 4; 3, 8; 4, 24; 5, 48; and 6, 72 hr
of hCG treatment.
involved in the apoptosis-suppression of ovarian follicular cells
through the up-regulation of IGF-1/IGFR and/or down-regulation
of bax (Tilly et al., 1995). IGF-1 has recently been shown to alter
the drug sensitivity of human breast cancer cells by inhibition of
apoptosis without changing the bcl-2 and bax mRNA levels (Dunn
et al., 1997). Since OVCAR-3 cells express IGFR, it is likely that
hCG signaling is involved in the inhibition of apoptosis, possibly
via the autocrine or paracrine activation of the IGF-1/IGFR system.
Serum levels of gonadotropin are elevated in ovarian cancer
patients after menopause or after oophorectomy, and our data
577
suggest that high LH levels may be involved in the chemoresistance of ovarian cancer cells. Gonadotropin-releasing hormone
(GnRH) analogues are drugs used for the treatment of leiomyoma
or endometriosis to down-regulate the levels of serum gonadotropins. Several studies suggested that GnRH analogues are effective
in some patients with ovarian cancer (Lind et al., 1992). The GnRH
analogue has been shown to bind directly to the GnRH receptor on
the ovarian cancer cells and inhibit mitogenic activity (Emons et
al., 1993). However, another possible mechanism of the anti-tumor
effect of GnRH analogue on ovarian cancer is the enhancement of
susceptibility to anti-cancer drugs. If this is indeed the case, GnRH
analogues could be applied for patients with LH/hCG receptorpositive ovarian cancer. A randomized trial did not show a
significant benefit of the drug use on the survival of ovarian cancer
patients (Emons et al., 1996). In our hospital, patients with
advanced ovarian cancer are currently enrolled in a clinical trial of
cisplatin-based chemotherapy with or without GnRH analogue,
along with the analysis of LH/hCG receptor status of the tumor.
Epidemiological data indicate that the incidence of ovarian
cancer increases around the peri-menopausal period, when the
serum levels of gonadotropins are elevated. Several ovarian
carcinomas have been reported to develop in infertile women
during gonadotropin treatment (Kuroda et al., 1997b). Since
ovarian carcinomas are believed to arise from the surface epithelial
cells of the ovary, a possible effect of gonadotropins on these cells
should be considered. Expression of FSH receptor has been
demonstrated in ovarian surface epithelial cells (Zheng et al.,
1996). If LH/hCG stimulation also inhibits apoptosis in normal
surface epithelium of the ovary, constant exposure to LH/hCG may
lead to ovarian carcinogenesis; escape from physiological apoptosis is considered to promote the carcinogenic process (Bedi et al.,
1995). We have detected mRNA expression of LH/hCG receptors
and specific binding sites for hCG in normal ovarian surface
epithelial cells in vitro (Kuroda et al., 1997a), and the influence of
hCG on the apoptosis of these cells is now under investigation.
ACKNOWLEDGEMENTS
This work was supported by Grants-in-Aid for Scientific Research to I.K. (09470358) and to M.M. (09771274) from the
Ministry of Education, Science and Culture, Japan.
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