- Email: [email protected]
Antitumor Effect of GnRH Agonist in Epithelial Ovarian Cancer
Gynecologic Oncology 74, 170 –180 (1999) Article ID gyno.1999.5413, available online at http://www.idealibrary.com on
Antitumor Effect of GnRH Agonist in Epithelial Ovarian Cancer Jae Hoon Kim, M.D.,* Dong Choon Park, M.D.,* Jin Woo Kim, M.D.,† Yang Kyu Choi, DVM.,‡ Young Ok Lew, M.D.,* Dae Hoon Kim, M.D.,* Jae Keun Jung,§ Young Ae Lim, M.D.,** and Sung Eun Namkoong, M.D.† *Department of Obstetrics and Gynecology, St. Vincent’s Hospital, The Catholic University of Korea, Suwon; †Department of Obstetrics and Gynecology, Kangnam St. Mary’s Hospital, The Catholic University of Korea, Seoul; ‡Genetic Resources Center, Korea Research Institute Bioscience and Biotechnology, KIST, Taejon; §Department of Obstetrics and Gynecology, Our Lady of Mercy Hospital, The Catholic University of Korea, Inchon; and **Department of Laboratory Medicine, Ajou University School of Medicine, Suwon, Korea Received June 9, 1998
Objective. The effects of the gonadotropin releasing hormone (GnRH) agonist (D-Trp 6) were examined in two human ovarian cancer cell lines and in severe combined immune deficiency (SCID) mice to evaluate its potential as a cytocidal, cytostatic, or differentiating antitumor agent. Methods. We treated the human ovarian cancer cell lines OVCAR-3 and SKOV-3 for 5 or 7 days and sex-matched SCID mice with GnRH agonist for 29 days. The antitumor effect of GnRH agonist were studied in various aspects. To confirm the antiproliferative effect, we used 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide colorimetric assay, in vitro, and a serial measurement of tumor growth in vivo. The disturbances of progression in the cell cycle and the changes of cyclin-dependent kinase 1 following treatment with GnRH agonist were evaluated with flow cytometric analysis in vitro. The induction of apoptosis following treatment with GnRH agonist was studied using in situ terminal deoxyribonucleotidyl transferase (Tdt) and further quantitated with ELISA in vitro. The presence of telomerase activity following treatment with GnRH agonist was measured by PCR-based telomeric repeat amplification protocol and ELISA detection in cell lines and xenografts in vitro and in vivo. Results. Continuous exposure of cell lines and xenografts to GnRH agonist resulted in growth inhibition of cancer cells in a dose- and time-dependent manner. In cultured cells, the GnRH agonist blocked cell cycle progression in G0/G1 phase and thus reduced the number of cells in S and G2/M phases. The phenomenon of apoptosis was documented in cultured cells treated with GnRH agonist by in situ Tdt assay. The frequency of apoptotic cells in the in situ Tdt assay was 5– 6% compared with control, 4 –5%. Apoptosis quantified by ELISA revealed a high incidence in cultured cells treated with GnRH agonist. The activities of telomerase in cell lines and xenografts were not decreased by GnRH agonist. There were not any significant changes of expression of CA-125 by flow cytometry and of the cellular morphology observed with light microscopy. Conclusions. Our results indicate that the antiproliferative effect of GnRH agonist in epithelial ovarian cancer cells may be mainly attributed to cytostatic activities resulting in blocking of
0090-8258/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
cell cycle progression in the G0/G1 phase and minimally related to the induction of apoptosis. © 1999 Academic Press
INTRODUCTION In Korea, the incidence of ovarian cancer has increased over the past few decades, while that of cervical cancer, the most common gynecologic cancer, has decreased [1]. Despite the combination of cytoreductive surgery and platinum-based chemotherapy, most patients in advanced stages eventually relapse and ultimately die because of chemoresistant disease after short periods of responsiveness [2]. Recently, the gonadotropin releasing hormone (GnRH) agonist has been used in the treatment of endocrinedependent tumors. The inhibitory effect of the GnRH agonist on tumor cells was thought to be mediated through a GnRH receptor on the membrane of ovarian [3, 4], endometrial [5], and breast cancers [6]. Other researchers have also suggested the inhibition of ovarian cancer by the GnRH agonist in experiments [7–12]. Meanwhile, clinical studies on single GnRH agonist treatment for far advanced ovarian cancer and on combination therapy with cisplatin have demonstrated conflicting results [13–15]. In order to resolve the differences in results between experimental data and clinical studies, it is necessary to study the mechanism of GnRH agonist as an antitumor regimen for ovarian cancer. An antitumor drug must have cytocidal, cytostatic, or differentiating effects on cancer cells. This current in vitro and in vivo study included the assessment of antiproliferative apoptosis, and cytocidal effects as well as analyses of cell cycle and cyclin-dependent kinase 1 (essential for cell entrance into S and G2/M phases, for cytostatic effect). Additionally, we inspected the possibility of GnRH agonist as a differentiating agent through evaluation of the morphologic changes, the decreased expression of tumor surface antigen CA-125, and the activity of telomerase [16–18]. The goal of this study was to examine the mechanism of GnRH agonist as an antitumor regimen.
170
ANTITUMOR EFFECT OF GnRH AGONIST
MATERIALS AND METHODS Cell Lines and Culture Conditions The human ovarian cancer cell lines possessing GnRH receptors, OVCAR-3 and SKOV-3, were obtained from the American Type Culture Collection (ATCC, Atlanta, GA). The cell cultures were maintained at 37°C in a humidified 5% CO 2 ambient air atmosphere. The cells were grown in Waymouths MB 752/1 medium (Gibco, Grand Island, NY) supplemented with 2 mM glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 10% fetal bovine serum (FBS; Gibco). Monolayer cells were washed twice with Hanks’ balanced salt solution (HBSS; Gibco) without calcium and magnesium. Cells were detached with either 0.5 ml HBSS-buffered 5 mM disodium EDTA (J. T. Baker B. V., Deventer, The Netherlands), pH 7.2, at 37°C or 5.0 ml HBSS-buffered 5 mM EDTA/0.25% trypsin (Flow Laboratories), pH 7.2, at 37°C. Experimental Animals C.B-17 severe combined immune deficiency (SCID) mice were obtained from Central Institute for Experimental Animals (Kawasaki, Japan), housed and bred in the Korea Research Institute of Bioscience and Biotechnology, with annual cesarean rederivation to limit transmission of environmental pathogens. All C.B-17 SCID mice used in this study were derived from specific pathogen-free (SPF) breeder stock. Nonleaky C.B-17 SCID mice, 6- to 7-week-old females, were used in all experiments. Eight mice were inoculated per group. Before all experiments, SCID mice were screened for B lymphocyte deficiency by quantifying serum immunoglobulin levels using a sandwich ELISA [19]. All animals were maintained on a daily 12-h light/12-h dark cycle. Drugs GnRH agonists (decapeptyl and decapeptyl-depo; Ferring GmbH, Malmoe, Sweden) were made as a 0.1 mg/ml stock in a sodium chloride solution and a 3.75 mg/ml stock in a suspension medium containing polysorbate 80, dextran 70, sodium chloride, sodium dihydrogen phosphate dihydrate, and sodium hydroxide solution. Assessment of Antiproliferative Effect In vitro study. The daily treatments of 0.1 or 10 mM decapeptyl and refreshing culture media were performed in 25-ml flasks due to the possibility of drug inactivation. For the comparison of cell growth cycles, we counted cells by hemocytometer every 2 days for 7 days. At the same time, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay with Protein Concentration Kit (Bio-Rad Hercules, CA) was completed [20]. All experiments were performed twice in quadruplicate samples.
171
In vivo study. The cells from various subcultures were harvested by the brief exposure to 0.05% trypsin– 0.02% EDTA, and the cells obtained by centrifugation at 800 rpm were resuspended in phosphate-buffered saline (PBS). After cell viability was determined by 0.4% trypan blue dye exclusion, only the cell suspensions with greater than 95% viability were used for injection. Cells (5 3 10 6) of OVCAR-3 were injected subcutaneously into the posterior aspect of the necks of 24 mice. After 16 mice developed a xenograft which reached 50 mm 3, the mice were randomly divided into two equal groups and the medications were administered. Decapeptyl-depo (100 mg) was injected intramuscularly on the buttock site every 2 weeks. The tumor volume was calculated from the formula: tumor volume (mm 3) 5 length (mm) 3 [width (mm)] 2 3 0.5 [21]. At 29 days from the innoculation time, mice were euthanized by ether. Excised tissues were weighed and divided into two pieces. One piece was fixed in 10% phosphate-buffered Formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin to confirm the presence of neoplastic cells in each mouse. The others were homogenized in a homogenizer (Fisher Customs Service Center, Atlanta, GA) and stored in a 270°C refrigerator. Assessment of Effects on Cell Cycle and Expressions of Cyclin-Dependent Kinase 1 (CDK 1) Cell cycle analysis. For harvest, the cells were detached from culture flasks with a brief treatment of 5.0 ml HBSSbuffered 5 mM EDTA/0.25% trypsin. The time of detachment was recorded. Detached cells were washed once with culture medium and filtered through a 50-ml pore-sized mesh polyamide gauze (Specrum Medical Industries, CA) to remove large cell aggregates. The total number of cells and viability were determined by 0.4% trypan blue dye exclusion. Cells were washed twice with ice-cold PBS containing 0.5% bovine serum albumin (BSA; PBS/BSA) (Sigma Diagnostics, St. Louis, MO). For the immunofluorescent staining of DNA, we used Cycle Test Plus Kit (Calbiochem, San Diego, CA). Half a million cells were incubated after adding 1 mg/ml propidium iodide (PI) at room temperature for 30 min. The amount of PI bound to DNA in cell suspension was measured by the FL 2 detector of a FACScan flow cytometer (Becton–Dickinson, CA) equipped with an argon-ion laser (488 nm emission) within 24 h after DNA staining. Chicken erythrocytes were used as a negative control for the FACScan setup. CellFIT (Becton–Dickinson Immunocytometry Systems, CA) software was applied for data acquisition and analysis. For each measurement, 15,000 events at the LO mode were acquired as a scattergram (FL2-W: width/FL2-A: area) and a histogram (FL2-A/counts), and percentages of each phase of the cell cycle in the histogram to the FL2-A parameters were obtained. Analysis of expression of cyclin-dependent kinase 1. The cell suspension was obtained with the method using trypsin
172
KIM ET AL.
described above. The total number of cells and cell viability were determined by 0.4% trypan blue dye exclusion. Cells were washed again with ice-cold PBS containing 0.5% bovine serum albumin (BSA; PBS/BSA) (Sigma Diagnostics). For permeabilization of cellular membrane, cells were resuspended with the cold 75% ethanol and stored in a 220°C refrigerator for 2 h. After washing, 0.25% Triton X-100 was added and the suspension incubated on ice for 5 min. The purified mouse anti-human p34cdc2 protein kinase (cyclin-dependent kinase 1) antibody (Pharmingen, San Diego, CA) was used as a primary antibody. All monoclonal antibodies were used under saturating conditions, as determined by titration experiments on cell lines showing the highest expression of antigenic determinants after EDTA detachment. A million cells were incubated with 50 ml of primary antibody for 30 min at 4°C. Cells were washed twice with 1.0 ml ice-cold PBS/BSA, followed by 30 min incubation with 100 ml of fluorescein isothiocyanate (FITC)-labeled Affini Pure goat anti-mouse IgG polyclonal antibody diluted 1:200 at 4°C in PBS/BSA after sedimentation (250g, 6 min, 4°C). The intensity of fluorescence was analyzed by using a FACS Vantage flowcytometer (Becton–Dickinson) equipped with a Coherent Enterprise laser (300 mW at 48 nm) and a seventh detector for five-color immunofluorescence measurements. Chicken erythrocytes were used as a negative control. For each measurement, 10,000 events were acquired and listmode data analysis was performed by applying CELL Quest software (Becton–Dickinson) [22]. Assessment of effects on differentiation: Flow cytometric analysis of CA-125. The suspension of cultured OVCAR-3 and SKOV-3 cells was obtained with the trypsin method described above. The total number of cells and viability were determined by 0.4% trypan blue dye exclusion. For the expression of CA-125 on the cell surfaces, OC125 (Signet Laboratories, Dedham, MA) was used as a primary antibody. All the conditions and procedures were the same as those in the flow cytometric analysis of CDK 1. Apoptosis Assay In situ Tdt method. The Apop Tag In Situ Apoptosis Detection Kit was used for detecting apoptotic cells. Single cell suspensions containing 5 3 10 7 cells/ml of cultured OVCAR-3 were fixed in 4% neutral buffered Formalin for 10 min at room temperature. To quench endogenous peroxidase, 2.0% hydrogen peroxide in PBS was applied to the slide for 5 min. After washing, 50 –100 ml of cell suspension was dried on a microscopic slide and washed twice in PBS for 5 min. Two drops of 13 equilibration buffer [50 mM Tris–HCl, 5 mM MgCl 2 10 mM b-mercaptoethanol, 0.005% BSA (Fraction V: Sigma, St. Louis, MO), pH 7.5] was directly applied to the specimen. A plastic coverslip was applied gently and specimens were incubated for 10 –15 s at room temperature. After removal of the plastic coverslip, 54 ml of mixtures with Tdt (terminal deoxyri-
bonucleotidyl transferase), biotinylated dNTP, 0.5 M Tris, pH 7.5, 50 mM MgCl 2, 0.6 mM 2-mercaptoethanesulfonic acid, and 0.5 mg/ml BSA (RIA grade) were pipetted onto the glass slide gently, a plastic coverslip was reapplied and specimens were incubated in a humidified chamber at 37°C for 1 h. After washing in PBS, two drops of anti-digoxigenin peroxidase were applied to the glass slide and incubated for 30 min at room temperature. For color development, freshly prepared DAB (diaminobenzidine) substrate solution was applied and after 3 to 6 min of incubation, a counterstain with methyl green was done. Apoptotic cell death detection by ELISA. Additional evidence of apoptosis in ovarian cancer cell lines and xenografts 24 and 48 h after treatment with 10 mM GnRH agonist was determined by a quantitative sandwich enzyme immunoassay principle using mouse monoclonal antibodies directed against DNA and histones (Boehringer Manheim Gmbh, Germany). This ELISA provides an in vitro quantitative determination of histone-associated DNA fragments (mono- and oligonucleosomes) in the cytoplasmic fraction of ovarian cancer cell lines and xenografts, as determined spectrophotometrically at 405 nm. The presence of mono- and oligonucleosomes is a feature of cells undergoing apoptosis [23]. Telomerase Assay After washing the cultured OVCAR-3 and SKOV-3 cells and minced xenograft once with PBS, we resuspended the cell pellet as 10 5 cells in 200 ml of 13 Chaps (3-[(3-cholamidopropyl)dimethylammonio]-1-profanesulfonate) lysis buffer (Oncor Gaithersburg, MD). The suspension was incubated on ice for 30 min. Telomerase activity was measured by the PCR-based telomere repeat amplification protocol (TRAP) assay as described [24]. For each TRAP assay, heat inactivation control as a negative control, positive extract control from the control cell pellet provided in the kit, and synthetic oligonucleotide with eight telomeric repeats as PCR/ELISA positive controls and primer– dimer/PCR contamination control to detect carryover contamination were included. The telomere repeat region was amplified with PCR and the concentrations of TRAP products were determined by the TRAPeze ELISA kit (Bio-Rad, Hercules, CA). At the end of the procedure, the difference of absorbance (DA) at 450 and at 690 nm using a microtiter plate reader was measured. If the DA was above 0.150, the extract was scored as “telomerase positive.” Statistics The significance of experiments was analyzed by one-way or two-way analysis of variance (ANOVA) [25], Duncan’s multiple range test, and log-rank test with Kaplan–Meier survival estimates [26].
ANTITUMOR EFFECT OF GnRH AGONIST
173
In vivo. On day 5 after tumor cell innoculation, the tumor volume reached above 50 mm 3, which was the starting point of drug treatments. Beginning at day 9, there were differences in tumor sizes between control (untreated) and treated groups. On day 23, the mean tumor volume of untreated groups reached 20,834 mm 3 and that of treated groups reached 16,920 mm 3, which showed a difference of approximately 81.2%. On day 29, the mean tumor volumes of the two groups were 35,679 and 28,717 mm 3, respectively, and the difference in size was 80.5% (Figs. 2 and 3). On the whole, there was a significant difference between the two groups (P , 0.05) (Fig. 3). Splenomegaly was noted in all SCID mice innoculated. Widespread metastasis was observed microscopically in the lung, heart, kidney, and liver. Survival rates were 77.8 and 44.4% in the treated and untreated groups, respectively, upon termination of the experiment (P , 0.15). (Fig. 4). Analysis of the Cell Cycle
FIG. 1. Antiproliferative activity of OVCAR-3 (A) and SKOV-3 cell (B) lines to GnRH agonist in vitro. Cells were exposed to 0.1 and 10 mM GnRH agonist and their growth was observed for 5 days. Each value represents the mean of eight wells. Dose, day, and interaction effects are significantly different (P , 0.05) by two-way ANOVA.
RESULTS Antiproliferative Study In vitro. OVCAR-3 and SKOV-3 cell lines were treated with 0.1 and 10 mM GnRH agonist for 5 days and the proportion of proliferated cells compared to the nontreated control cell lines was measured by MTT assay. After 4 h of incubation at 0.1 and 10 mM, there were transient increases of cell proliferation in both cell lines. After 12 h of incubation, OVCAR-3 and SKOV-3 cell line quantities were 88.0 and 79.2% at 0.1 mM and 72.0 and 54.2% at 10 mM, respectively. This antiproliferative effect increased gradually with time. After 120 h of incubation at 0.1 mM, OVCAR-3 and SKOV-3 cell lines were further reduced to 79.1 and 77.7% and at 10 mM were 29.3 and 25.7%, respectively. Cancer cell growth was significantly suppressed after 12 h and was grossly suppressed, in a time- and dose-dependent manner, over a period of 5 days in vitro (P , 0.05) (Fig. 1.). There were no significant differences in the dose dependencies between 10 mM and higher concentrations (data not shown).
Cell cycle analysis was performed every other day for 7 days. Cell cycle perturbations were evident from 8 h following addition of 10 mM GnRH agonist to OVCAR-3 and SKOV-3 cell lines. After 24 h of incubation with 10 mM GnRH agonist, the proportions of G0/G1, S, and G2/M phases of OVCAR-3 cell lines were 54.4, 42.6, and 3.9% and those of the control group (no treatment) were 37.2, 55.6, and 6.3%, respectively. There were increases in cell fractions in G0/G1 phase and decreases in those of cells in the S and G2/M phases during the treatment, compared to the nontreated control group in both cell lines (P , 0.05), but not in a time-dependent fashion (Fig. 5). Analysis of the Expression of Cyclin-Dependent Kinase 1 After 3 days of incubation with 10 mM GnRH, 62.7 and 66.8% of CDK 1 positive cell fractions of the treated cells were detected compared to 75.9 and 71.1% of nontreated cells in OVCAR-3 and SKOV-3 cell lines, respectively. The expression of CDK 1 decreased significantly after 3 days and grossly over a period of 7 days in a time-dependent fashion (P , 0.05) (Fig. 6). Apoptosis In situ apoptosis assay. Solitary or multiple micronuclei indicating the morphological characteristics of apoptosis were observed in cells treated with 10 mM GnRH agonist, and the proportions of such cells at 24 and 48 h were 5.5 6 1.6 and 6.3 6 2.2%, compared with untreated cells, with 4.2 6 1.2 and 4.8 6 1.6%, respectively (Fig. 7). However, the ratio of apoptotic findings did not increase in cells treated with the same concentrations from the third day, compared with that of the second day.
174
KIM ET AL.
FIG. 2. (A) Xenograft of OVCAR-3 tumor cell line in C.B.-17 SCID mouse 4 weeks after sc inoculation (arrow). This picture shows one of mice which lived 4 weeks. (B) The feature of cut section. (C) Subcutaneous tumor mass (arrow), H & E 3100.
Apoptotic cell death detection by ELISA. After 24 h of incubation, the cultured cells incubated with 10 mM GnRH agonist showed an increase in cytoplasmic fractions with histone-associated DNA fragments compared with the control group, 0.25 vs 0.27 in OVCAR-3 and 0.51 vs 0.56 in SKOV-3, and after 48 h of incubation, 0.27 vs 0.48 in OVCAR-3 and 0.53 vs 0.83 in SKOV-3 (Fig. 8).
Assessment of Effects of Differentiation For the purpose of investigating the differentiation of cultured OVCAR-3 and SKOV-3 cells following treatment with 10 mM GnRH agonist, we observed the morphologicchanges by light microscopy and the expression of CA-125, known as a representative tumor marker of ovarian cancer,
ANTITUMOR EFFECT OF GnRH AGONIST
175
FIG. 2—Continued
by immunofluorescent stain with flow cytometric analysis (data not shown). However, we found no evidence of differentiation in terms of morphology, and a decrease in the development of cancer antigen or in the activity of telomerase, which indicates differentiation, was not observed either (Fig. 9). DISCUSSION In the medical treatment of ovarian cancer, there have been trials not only exploring new antitumor drugs but also studying
alternative treatment modalities. Because of low toxicity as well as cost effectiveness in the case of the endocrine dependency of the ovary, hormonal therapies have been predominantly accepted as alternative therapies. Hormonal therapies, using antiestrogens, progestogens and their combinations, have demonstrated only a marginal effect or none [27, 28]. Incessant monthly ovulation has been known as a physiologic causal factor for the development of ovarian cancer, i.e., the repeated rupture of the coelomic epithelium. But other researchers have asserted the “gonadotropin theory” that the
FIG. 3. In vivo effect of GnRH agonist on the growth of subcutaneous OVCAR-3 xenografts. GnRH treatment (100 mg per mouse every 2 weeks) was initiated on day 5 after tumor implantation. Each value represents the mean of eight mice. Day effects are significantly different (P , 0.05) by repeated measures ANOVA and Duncan’s multiple range test.
176
KIM ET AL.
FIG. 4. The survival difference between GnRH agonist-treated and untreated mice upon assay termination. The introduction of an artificial end point precluded survival analysis (P , 0.15).
FIG. 6. Expression of cyclin-dependent kinase 1 in OVCAR-3 and SKOV-3 cell lines by treatment with 10 mM GnRH agonist for 7 days. (A) Plots represent the variation in percentage of mean fluorescence intensity (MFI) in OVCAR-3 cell lines. (B) Plots represent the variation in percentage of MFI in SKOV-3 cell lines. Day effects are significantly different in both cell lines (P , 0.05) by one-way ANOVA and Duncan’s multiple range test. Each value represents mean of quadruplicate two times determinations.
FIG. 5. Effects of GnRH agonist on the cell cycles of OVCAR-3 and SKOV-3 cell lines. The cells were stimulated for 7 days with 10 mM GnRH agonist. (A) Plots represent the variation in percentage of G0/G1, S and G2/M phases in OVCAR-3. (B) Plots represent the variation in percentage of G0/G1, S and G2/M phases in SKOV-3. There were significant differences that increased the ratio of G0/G1 phase with decreased ratios of S and G2/M phases between treated and untreated groups in both cell lines (P , 0.05), but day effects are not significant by two-way ANOVA and Duncan’s multiple range test. Each value represents mean of quadruplicate two times determinations.
exposure to high gonadotropin levels in cycling women favors malignant transformation [29, 30]. Epidemiologists have also agreed on this theory based on the increased incidence of ovarian cancer with the beginning of menopause and elevated gonadotropins [29, 31]. With the positive results in vitro [32] and in vivo [33], it was thought that human ovarian cancer cells could grow by the stimuli of gonadotropin. Based on this theory, GnRH agonist has been utilized for the treatment of ovarian cancer by blocking of the hormonal axis and downregulation of gonadotropin [34, 35]. However Yano et al. [9, 10] and Thompson et al. [38] did not observe similar findings and they even had contradictory results in further experiments, i.e., no stimulatory effect on the growth of ovarian cancer cell lines by gonadotropin; thus, this theory is controversial. They found that there were no differences in the levels of gonadotropin between ovarian cancer patients and normal women [14, 36, 37]. Especially, there is a controversy over the ratio of receptors for gonadotropin on ovarian cancers (0 –91%) [39, 40]. On the other hand, after the demonstration of GnRH mRNA and the local regulatory system of GnRH in ovarian
ANTITUMOR EFFECT OF GnRH AGONIST
177
FIG. 7. Immunodetection of labeled nucleotides added by Tdt on OVCAR-3 cells after treatment of 10 mM GnRH for 24 h. Intense staining was observed in nuclei and nuclear fragments with the morphological characteristics of apoptosis. Counterstaining was done with methyl green (arrow).
cancer cells, the role of GnRH receptor was considered an autocrine regulator of the proliferation of ovarian cancer cells [41, 42]. And the GnRH receptor was thought to be a direct target of the antitumor effects of GnRH. Also, this “direct GnRH receptor theory” was assisted by the apoptosis of the OVCAR-3 cell line by antibodies against the GnRH receptor [43]. In fact, GnRH receptors were found in more than 90% of ovarian cancers [3, 39]. Therefore, it was thought that longterm therapy of GnRH agonist might induce desensitization to GnRH, followed by the direct antiproliferative effect. But this “direct GnRH receptor theory” was far from producing a complete conclusion because there are few studies on the GnRH second messenger system and the related molecules in ovarian cancer. Recent studies showed the role of annexin V in the signal transduction of GnRH agonist [39] and its relation with insulin-like growth factor II [40]. Even though there are controversies over the gonadotropin dependency of ovarian cancer and the direct antitumor effect of GnRH agonist, the data obtained by making good use of GnRH agonist until this time may be sufficient to advocate its use in ovarian cancer. In this study, first of all, we found not only antiproliferative effects of GnRH agonist but also time- and dose-dependent increases in its action. These findings are similar to previous in vitro observations [9, 10, 39]. But as to the effective concentrations of the GnRH agonist, there are discrepancies with some previous reports. In our study, the inhibitory effect against proliferation was maximal at 10 mM GnRH agonist, and the effects were not significantly different between 10 mM
and 100 mM (data not shown). However, Thompson et al. [38] contended antiproliferative effects were only significant at high concentrations (1 and 100 mM), and Emons et al. [44] even at 1 nM. Meanwhile, in cases of the SiHa cell line, known to have no GnRH receptor, treatment with 10 mM GnRH agonist showed the same degree of antiproliferative effect with OVCAR-3 and SKOV-3 cell lines, which have GnRH receptors. But treatment at 0.1 mM did not show the same degree of antiproliferative effect in the SiHa cell line (data not shown). Based on the results at low concentration (0.1 mM), it could be thought that the action of GnRH agonist is generated by receptor-mediated mechanisms. On the other hand, antiproliferative effects at high concentrations might be developed by the broad potentials of GnRH agonist as an antitumor drug. In the studies on antiproliferative effect, those done in vivo with SCID mice showed results similar to those with cases using nude mice [8 –11]. The antitumor effect observed in our in vivo experience was substantial, with a reduction in the final tumor volume of approximately 80.5% in the GnRH agonisttreated group, compared with a control group. Although the survival difference between GnRH agonist-treated and untreated mice was not significant in this in vivo study, we observed a tendency toward long-term survival in treated groups. The majority of drugs kill cancer cells in a unique phase of the cell cycle, and cells not belonging to that phase will not be injured. And the blocking phases of most anticancer drugs on that cell cycle are usually inducing subsequent apoptosis [45].
178
KIM ET AL.
cycle. The time lag between prompt elevation of G0/G1 phase fractions and delayed decline of CDK 1 is not explained. We surmise the existence of another control mechanism. The 48-h cultured cells of both groups were stained with trypan blue to measure viability and there was no difference between GnRH-treated and control groups (approximately, 4 vs 6%). From these results, we were be convinced of the cytotoxic effect of GnRH agonist. Then we quantitated the event of apoptosis with the ELISA method and actually found increased induction of apoptosis in ovarian cancer cell lines treated with GnRH agonist. In the study of cell differentiation, we did not find differences in the expression of CA-125 (a representative antigen of ovarian cancer) by flow cytometry or visible changes of cel-
FIG. 8. Relative levels of apoptosis quantified by histone-associated DNA fragments in OVCAR-3 (A) and SKOV-3 (B) cell lines following treatment with 10 mM GnRH agonist for 24 and 48 h. Each value represents the mean value of triplicate determinations.
Accordingly, it is essential to know the action phase in the cell cycle in combination drug therapy. As cell cycle analysis in vivo is liable to problems like intratumor heterogeneity and difficulty in tissue processing procedures [46], we focused only on in vitro results. In the growth kinetic study using flow cytometry, we found that the GnRH agonist acted mainly by arresting cells in the G0/G1 phase of the cell cycle and thus reduced the number of cells in S and G2/M phases. The elevation of G0/G1 phase fractions confirmed that the main action of GnRH agonist is made in G0/G1 phase. Thompson et al. [38] also found that GnRH agonist caused a reversible 5– 6% increase in cells in the G0/G1 phase compared with controls and a corresponding decrease of cells in S and M phases. The transient acceleration of cell proliferation (for 12 h) in MTT assays could be explained by the fact that for the action of GnRH agonist, a time interval for a next event is necessary. Because of the blocking of G0/G1 phases, there was decreased expression of CDK 1, which controls the S and G2/M phases, on transitions between successive phases of the cell
FIG. 9. Relative levels of telomerase activity in OVCAR-3 and SKOV-3 cell lines and a xenograft treated with 10 mM GnRH agonist, as measured by TRAP assay and ELISA detection. Each value represents the mean value of triplicate determinations. A, positive telomerase control; B, PCR/ELISA positive control; C, negative control; D, heat inactivation; E, no treatment for 14 days; F, treatment with 10 mM GnRH agonist for 14 days; G, no treatment for 21 days; H, Treatment with 10 mM GnRH agonist for 21 days.
ANTITUMOR EFFECT OF GnRH AGONIST
lular morphology by light microscopy during the period of treatment. In vitro differentiation of an immortal cell type leads to low or undetectable telomerase activity [16 –18]. In this study, there was no decreased telomerase activity, rather than increased. But we cannot completely deny the role of GnRH agonist as a differentiating agent because we used only CA-125 and telomerase activity as differentiation markers and did not observe the morphologic evidence of differentiation by electron microscopy in cancer cells. Interestingly, telomerase activity increased after treatment with GnRH agonist. Considering a general conception of the correlation between the proliferation of cells and increased telomerase activity [18], we found some contradiction between the antiproliferative effect of GnRH agonist (decreased S and G2/M phases) and elevated telomerase activity in this study. But recent reports showed the controversy over controlling telomerase activity in the cell cycle [48]. A recent report reported that as cells progress through the replicative S phase, the activity of telomerase gradually increases [49]; on the other hand, there has been a contradicting report that telomerase activity is not related to the cell cycle [50]. This indicates the possible effect of another complicated and indirect factor in the changes in telomerase activity. Even though antitumor activity in ovarian cancer based on GnRH agonist was proven, we should solve some problems at the clinical base. Substantially, owing to cost effectiveness and the potential of the drug, it is not easy to obtain high concentrations of GnRH agonists in humans by usual doses of GnRH agonist (once every 2 or 4 weeks) [51]. In clinical studies using GnRH agonist, researchers have insisted that there is no dramatic effect, just a marginal effect [13–15]. Actually, however, clinicians have few treatment modalities in advanced ovarian cancer patients who suffered from chemotoxicity. Low toxicity can permit GnRH agonist to be used effectively, regardless of the controversy over its effectiveness. More knowledge about the signal transduction pathway of GnRH and the development of a potent GnRH agonist with cytotoxic radicals might permit a better exploitation of its antitumor effects in the treatment of ovarian cancer. ACKNOWLEDGMENTS This work was supported in part by Research Funds of St’s Vincent’s Hospital, The Catholic University of Korea.
REFERENCES 1. Korean Association of Obstetricians and Gynecologists, Gynecologic Cancer Registry Program in Korean Women, 1994
179
4. Kaiser UB, Conn PM, Chin WW: Studies of gonadotropin-releasing hormone (GnRH) action using GnRH receptor expressing pituitary cell lines. Endocr Rev 18(1):46F70, 1997 5. Chatzaki E, Bax CM, Eidne KA, Anderson L, Grudzinskas JG, Gallagher CJ: The expression of gonadotropin-releasing hormones and its receptor in endometrial cancer, and its relevance as an autocrine growth factor. Cancer Res 56:2059 –2065, 1996 6. Marini L, Iacopino F, Schinzari G, Robustelli Della Cuna FS, Mantovani G, Sica G: Direct antiproliferative effect of triptorelin on human breast cancer cells. Anticancer Res 14:1881–1885, 1994 7. Kullander S, Rausing A, Trope C: Human ovarian tumors heterotransplanted to nude mice. Acta Obstet Gynecol Scand 57(2):149 –159, 1978 8. Peterson CM, Jolles CJ, Carrell DT, Straight RC, Jones KP, Poulson AM, Hatasaka HH: GnRH agonist therapy in human ovarian epithelial carcinoma (OVCAR-3) heterotransplanted in the nude mouse is characterized by latency and transience. Gynecol Oncol 52(1):26 –30, 1994 9. Yano T, Pinski J, Halmons G, Szepeshazi K, Groot K, Schally AV: Inhibition of growth of OV-1063 human epithelial ovarian cancer xenografts in nude mice by treatment with luteinizing hormone-releasing hormone antagonist SB-75. Proc Natl Acad Sci USA 91(5):7090 –7094, 1994 10. Yano T, Pinski J, Radulovic S, Schally AV: Inhibition of human epithelial ovarian cancer cell growth in vitro by agonistic and antagonistic analogues of luteinizing hormone-releasing hormone. Proc Natl Acad Sci USA 91(5):1701–1705, 1994 11. Manetta A, Liao S, Gamboa-Vujicic G, Leong L, Pardes P, Asch B, Emma D, Schally A: Inhibition of growth of human ovarian cancer in nude mice by luteinizing hormone-releasing hormone antagonist cetrorelix (SB-75). Fertil Steril 63(2):282–287, 1995 12. Vincze B, Palyi I, Gaal D, Pato J, Mora M, Mezo I, Teplan I, Seprodi J: In vivo studies of the new gonadotropin-releasing hormone antagonistcopolymer conjugates having antitumor activity. Cancer Detect Prev 20(2):153–159, 1996 13. Emons G, Ortmann O, Teichert HM, Fassl H, Lohrs U, Kullander S, Kauppila A, Ayalon D, Schally A, Oberheuser F: Luteinizing hormone releasing hormone agonist triptorelin in combination with cytotoxic chemotherapy in patients with advanced ovarian carcinoma. A prospective double blind randomized trial. Decapeptyl Ovarian Cancer Study Group Cancer 78(7):1452–1460, 1996 14. Falkson CI, Falkson HC, Falkson G: Cisplatin versus cisplatin plus D-TRP-6-LHRH in the treatment of ovarian cancer: A pilot trial to investigate the effect of the addition of a GnRH analogue to cisplatin. Oncology 53(4):313–317, 1996 15. van-der Vange N, Greggi S, Burger CW, Kenemans P, Vermorken JB: Experience with hormonal therapy in advanced epithelial ovarian cancer. Acta Oncol 34(6):813– 820, 1995 16. Sharma HW, Sokoloski JA, Perez JR, Maltese JY, Sartorelli AC, Stein CA, Nicholas G, Khaled Z, Telang NT, Narayanan R: Differentiation of immortal cells inhibits telomerase activity. Proc Natl Acad Sci USA 92(26):12343–12346, 1995 17. Albanell J, Han W, Mellado B, Gunawardane R, Scher HI, Dmitrovsky E, Moore MA: Telomerase activity is repressed during differentiation of maturation-sensitive but not resistant human tumor cell lines. Cancer Res 56(7):1503–1508, 1996
2. Christian MC, Trimble EL: Salvage chemotherapy for epithelial ovarian carcinoma. Gynecol Oncol 55:S143–150, 1994
18. Avilion AA, Piatyszek MA, Gupta J, Shay JW, Bacchetti S, Greider CW: Human telomerase RNA and telomerase activity in immortal cell lines and tumor tissues. Cancer Res 56(3):645– 650, 1996
3. Imai A, Ohno T, Iida K, Fuseya T, Furui T, Tamaya T: Gonadotropinreleasing hormone receptor in gynecologic tumors, frequent expression in adenocarcinoma histologic types. Cancer 74(9):2555–2561, 1994
19. Choi YK, Choi JY, Kown EH, Kim YJ, Lee CH, Jeong KS, Hyun BH: Evaluation of nonleaky C.B-17 SCID mice by ELISA. Korean J Lab Anti Sci 12:9 –13, 1996
180
KIM ET AL.
20. Carmichael J, Degraff WG, Gazdar AF, Minna JD, Mitchell JB: Evaluation of a tetrazolium-based semiautomated colorimetric assay: Assessment of chemosensitivity testing. Cancer Res 47(4):936 –942, 1987 21. Choi YK, Choi JY, Hyun BH, Kim YJ, Lee CH, Yoon WK, Jeong KS, Kim DY: Antitumor effect of methotrexate in SCID mice with human leukemia CCRF-CEM cell line. Korean Cancer Assoc 29:19 –28, 1997 22. Beavis AJ, Pennline KJ: Allo-7: A new fluorescent tandem dye for use in flow cytometry. Cytometry 24(4):390 –395, 1996 23. Bonfoco E, Krainc D, Ankarcrona M, Nicotera P, Lipton SA: Apoptosis and necrosis: Two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures. Proc Natl Acad Sci USA 92(16):7162–7166, 1995 24. Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW: Specific association of human telomerase activity with immortal cells and cancer. Science 266: 2011–2015, 1994 25. Snedecor GW, Cochran WG: Statistics Methods, 2nd ed. Iowa State Univ. Press, Ames, Iowa, 1980 26. Miller RG Jr: Survival Analysis. Wiley, New York, 1981 27. Desombre ER, Holt JA, Herbst AL: Steroid receptors in breast, uterine and ovarian malignancy, in: Gold JJ, Josinovich JB (Eds.): Gynecologic Endocrinology. Plenum, New York, 1987, pp. 511–528 28. Rao BR, Slotman BJ: Endocrine factors in common epithelial ovarian cancer. Endocr Rev 12(1):14 –26, 1991 29. Stadel BV: The etiology and prevention of ovarian cancer. Am J Obstet Gynecol 123:772–774, 1975 30. Cramer DW, Welch WR: Determinants of ovarian cancer risk II. Inferences regarding pathogenesis. J Natl Cancer Inst 71:717–721, 1983 31. Schlesselman JJ: Cancer of the breast and reproductive tract in relation to use of oral contraceptives. Contraception 40(1):1–38, 1989 32. Simon WE, Albrecht M, Haensel M, Dietel M, Hoelzel F: Cell lines derived from human ovarian carcinomas: Growth stimulation by gonadotropic and steroid hormones. J Natl Cancer Inst 70:839 – 845, 1983 33. Ohtani K: Effects of gonadotropin on the growth of malignant ovarian neoplasms assessed by subdural capsule assay. Nippon Sanka Fujinka Gakki Zasshi 42:579 –585, 1990 34. Mortel R, Satyaswaroop PG, Schally AV, Hamilton T, Ozols R: Inhibitory effect of GnRH superagonist on the growth of human ovarian carcinoma NIH: OVCAR 3 in the nude mouse. Gynecol Oncol 23:254 –255, 1986 35. Kullander S, Rausing A, Schally AV: LHRH agonist treatment in ovarian cancer, in klijn (Ed): Hormonal Manipulation of Cancer: Peptides, Growth Factors and New (Anti) Steroidal Agents. Raven Press, New York, 1987, pp. 353–356 36. Emons G, Schally AV: The use of lutenizing hormone releasing hormone agonists (antagonists) in gynecological cancers. Hum Reprod 9(7):1364 – 1379, 1994 37. Ron IG, Wigler N, Merimsky O, Inbar MJ, Chaitchik S: A phase II trial of D-TRP-6-LHRH (Decapeptyl) in pretreated patients with advanced epithelial ovarian cancer. Cancer Invest 13(3):272–275, 1995 38. Thompson MA, Adelson MD, Kaufman LM: Lupron retards proliferation
of ovarian epithelial tumor cells cultured in serum-free medium. J Clin Endocrinol Metab 72:1036 –1041, 1991 39. Shibata S, Sato H, Ota H, Karube A, Takahashi O, Tanaka T: Involvement of annexin V in antiproliferative effects of gonadotropin-releasing hormone agonist on human endometrial cancer cell line. Gynecol Oncol (2)66:217–221, 1997 40. Ho MN, Delgado CH, Owens GA, Steller MA: Insulin-like growth factor-II participates in the biphasic effect of a gonadotropin-releasing hormone agonist on ovarian cancer cell growth. Fertil Steril 67(5):870 – 876, 1997 41. Ohno T, Imai A, Furui T, Takahashi K, Tamaya T: Presence of gonadotropin-releasing hormone and its messenger ribonucleic acid in human ovarian epithelial carcinoma. Am J Obstet Gynecol 169:605– 610, 1993 42. Emons G, Ortman O, Pahwa GS, Oberheuser F, Schultz KD: Intracellular actions of gonadotropic and peptide hormones and the therapeutic value of GnRH-agonists in ovarian cancer. Acta Obstet Gynecol Scan Suppl 155: 31–38, 1992 43. Ackerman RC, Johson GA, Van Kirk EA, Asirvatham AL, Murdoch WJ: Induction of apoptotic or lytic death in ovarian adenocarcinoma cell line by antibodies generated against a synthetic N-terminal extracellular domain gonadotropin-releasing hormone receptor peptide. Cancer Lett 81: 177–184, 1994 44. Emons G, Ortmannm O, Becker M, Irmer G, Springer B, Laun R, Hoelzel F, Schulz KD, Schally AV: High affinity binding and direct antiproliferative effects of LH-RH analogues in ovarian cancer cell lines. Cancer Res 54:5439 –5446, 1993 45. Alaoui SE, Lawry J, Griffin M: The cell cycle and induction of apoptosis in a hamster fibrosarcoma cell line treated with anti-cancer drugs: Its importance to solid tumor chemotherapy. J Neurooncol 31:195–207, 1997 46. Bergers E, Mntironi R, van Diest PJ, Prete E, Baak JPA, Fiac(Hon) F: Interlaboratory reproducibility of semiautomated cell cycle analysis of flow cytometric DNA-histograms obtained from fresh material of 1,295 breast cancer cases. Hum Pathol 27(6):553–560, 1995 47. Gibb RK, Taylor DD, Wan T, Oconnor DM, Doering DL, GercelTaylor C: Apoptosis as a measure of chemosensitivity to cisplatin and taxol therapy in ovarian cancer cell lines. Gynecol Oncol 65(1):13–22, 1997 48. Buchkovich KJ, Greider, CW: Telomerase regulation during entry into the cell cycle in normal human T cells. Mol Biol Cell 7:1443–1454, 1996 49. Zhu X, Kumar R, Mandal M, Sharma N, Scharma HW, Dhingra U, Sokoloski JA, Hsiao R, Narayanan R: Cell cycle-dependent modulation of telomerase activity in tumor cells. Proc Natl Acad Sci USA 93:6091– 6095, 1996 50. Sokoloski JA, Blair OC, Carbone R, Sartorelli AC: Induction of the differentiation of synchronized HL-60 leukemia cells by tiazofurin. Exp Cell Res 182:234 –241, 1989 51. Ozols RF, Rubin SC, Dembo AJ, Robboy S: Epithelial ovarian cancer, in Hoskins WJ, Perez SA, Young RC (Eds.): Principles and Practice of Gynecologic Oncology. Philadelphia, Lippincott 1992, 731–781
Antitumor Effect of GnRH Agonist in Epithelial Ovarian Cancer Jae Hoon Kim, M.D.,* Dong Choon Park, M.D.,* Jin Woo Kim, M.D.,† Yang Kyu Choi, DVM.,‡ Young Ok Lew, M.D.,* Dae Hoon Kim, M.D.,* Jae Keun Jung,§ Young Ae Lim, M.D.,** and Sung Eun Namkoong, M.D.† *Department of Obstetrics and Gynecology, St. Vincent’s Hospital, The Catholic University of Korea, Suwon; †Department of Obstetrics and Gynecology, Kangnam St. Mary’s Hospital, The Catholic University of Korea, Seoul; ‡Genetic Resources Center, Korea Research Institute Bioscience and Biotechnology, KIST, Taejon; §Department of Obstetrics and Gynecology, Our Lady of Mercy Hospital, The Catholic University of Korea, Inchon; and **Department of Laboratory Medicine, Ajou University School of Medicine, Suwon, Korea Received June 9, 1998
Objective. The effects of the gonadotropin releasing hormone (GnRH) agonist (D-Trp 6) were examined in two human ovarian cancer cell lines and in severe combined immune deficiency (SCID) mice to evaluate its potential as a cytocidal, cytostatic, or differentiating antitumor agent. Methods. We treated the human ovarian cancer cell lines OVCAR-3 and SKOV-3 for 5 or 7 days and sex-matched SCID mice with GnRH agonist for 29 days. The antitumor effect of GnRH agonist were studied in various aspects. To confirm the antiproliferative effect, we used 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide colorimetric assay, in vitro, and a serial measurement of tumor growth in vivo. The disturbances of progression in the cell cycle and the changes of cyclin-dependent kinase 1 following treatment with GnRH agonist were evaluated with flow cytometric analysis in vitro. The induction of apoptosis following treatment with GnRH agonist was studied using in situ terminal deoxyribonucleotidyl transferase (Tdt) and further quantitated with ELISA in vitro. The presence of telomerase activity following treatment with GnRH agonist was measured by PCR-based telomeric repeat amplification protocol and ELISA detection in cell lines and xenografts in vitro and in vivo. Results. Continuous exposure of cell lines and xenografts to GnRH agonist resulted in growth inhibition of cancer cells in a dose- and time-dependent manner. In cultured cells, the GnRH agonist blocked cell cycle progression in G0/G1 phase and thus reduced the number of cells in S and G2/M phases. The phenomenon of apoptosis was documented in cultured cells treated with GnRH agonist by in situ Tdt assay. The frequency of apoptotic cells in the in situ Tdt assay was 5– 6% compared with control, 4 –5%. Apoptosis quantified by ELISA revealed a high incidence in cultured cells treated with GnRH agonist. The activities of telomerase in cell lines and xenografts were not decreased by GnRH agonist. There were not any significant changes of expression of CA-125 by flow cytometry and of the cellular morphology observed with light microscopy. Conclusions. Our results indicate that the antiproliferative effect of GnRH agonist in epithelial ovarian cancer cells may be mainly attributed to cytostatic activities resulting in blocking of
0090-8258/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
cell cycle progression in the G0/G1 phase and minimally related to the induction of apoptosis. © 1999 Academic Press
INTRODUCTION In Korea, the incidence of ovarian cancer has increased over the past few decades, while that of cervical cancer, the most common gynecologic cancer, has decreased [1]. Despite the combination of cytoreductive surgery and platinum-based chemotherapy, most patients in advanced stages eventually relapse and ultimately die because of chemoresistant disease after short periods of responsiveness [2]. Recently, the gonadotropin releasing hormone (GnRH) agonist has been used in the treatment of endocrinedependent tumors. The inhibitory effect of the GnRH agonist on tumor cells was thought to be mediated through a GnRH receptor on the membrane of ovarian [3, 4], endometrial [5], and breast cancers [6]. Other researchers have also suggested the inhibition of ovarian cancer by the GnRH agonist in experiments [7–12]. Meanwhile, clinical studies on single GnRH agonist treatment for far advanced ovarian cancer and on combination therapy with cisplatin have demonstrated conflicting results [13–15]. In order to resolve the differences in results between experimental data and clinical studies, it is necessary to study the mechanism of GnRH agonist as an antitumor regimen for ovarian cancer. An antitumor drug must have cytocidal, cytostatic, or differentiating effects on cancer cells. This current in vitro and in vivo study included the assessment of antiproliferative apoptosis, and cytocidal effects as well as analyses of cell cycle and cyclin-dependent kinase 1 (essential for cell entrance into S and G2/M phases, for cytostatic effect). Additionally, we inspected the possibility of GnRH agonist as a differentiating agent through evaluation of the morphologic changes, the decreased expression of tumor surface antigen CA-125, and the activity of telomerase [16–18]. The goal of this study was to examine the mechanism of GnRH agonist as an antitumor regimen.
170
ANTITUMOR EFFECT OF GnRH AGONIST
MATERIALS AND METHODS Cell Lines and Culture Conditions The human ovarian cancer cell lines possessing GnRH receptors, OVCAR-3 and SKOV-3, were obtained from the American Type Culture Collection (ATCC, Atlanta, GA). The cell cultures were maintained at 37°C in a humidified 5% CO 2 ambient air atmosphere. The cells were grown in Waymouths MB 752/1 medium (Gibco, Grand Island, NY) supplemented with 2 mM glutamine, 100 IU/ml penicillin, 100 mg/ml streptomycin, and 10% fetal bovine serum (FBS; Gibco). Monolayer cells were washed twice with Hanks’ balanced salt solution (HBSS; Gibco) without calcium and magnesium. Cells were detached with either 0.5 ml HBSS-buffered 5 mM disodium EDTA (J. T. Baker B. V., Deventer, The Netherlands), pH 7.2, at 37°C or 5.0 ml HBSS-buffered 5 mM EDTA/0.25% trypsin (Flow Laboratories), pH 7.2, at 37°C. Experimental Animals C.B-17 severe combined immune deficiency (SCID) mice were obtained from Central Institute for Experimental Animals (Kawasaki, Japan), housed and bred in the Korea Research Institute of Bioscience and Biotechnology, with annual cesarean rederivation to limit transmission of environmental pathogens. All C.B-17 SCID mice used in this study were derived from specific pathogen-free (SPF) breeder stock. Nonleaky C.B-17 SCID mice, 6- to 7-week-old females, were used in all experiments. Eight mice were inoculated per group. Before all experiments, SCID mice were screened for B lymphocyte deficiency by quantifying serum immunoglobulin levels using a sandwich ELISA [19]. All animals were maintained on a daily 12-h light/12-h dark cycle. Drugs GnRH agonists (decapeptyl and decapeptyl-depo; Ferring GmbH, Malmoe, Sweden) were made as a 0.1 mg/ml stock in a sodium chloride solution and a 3.75 mg/ml stock in a suspension medium containing polysorbate 80, dextran 70, sodium chloride, sodium dihydrogen phosphate dihydrate, and sodium hydroxide solution. Assessment of Antiproliferative Effect In vitro study. The daily treatments of 0.1 or 10 mM decapeptyl and refreshing culture media were performed in 25-ml flasks due to the possibility of drug inactivation. For the comparison of cell growth cycles, we counted cells by hemocytometer every 2 days for 7 days. At the same time, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay with Protein Concentration Kit (Bio-Rad Hercules, CA) was completed [20]. All experiments were performed twice in quadruplicate samples.
171
In vivo study. The cells from various subcultures were harvested by the brief exposure to 0.05% trypsin– 0.02% EDTA, and the cells obtained by centrifugation at 800 rpm were resuspended in phosphate-buffered saline (PBS). After cell viability was determined by 0.4% trypan blue dye exclusion, only the cell suspensions with greater than 95% viability were used for injection. Cells (5 3 10 6) of OVCAR-3 were injected subcutaneously into the posterior aspect of the necks of 24 mice. After 16 mice developed a xenograft which reached 50 mm 3, the mice were randomly divided into two equal groups and the medications were administered. Decapeptyl-depo (100 mg) was injected intramuscularly on the buttock site every 2 weeks. The tumor volume was calculated from the formula: tumor volume (mm 3) 5 length (mm) 3 [width (mm)] 2 3 0.5 [21]. At 29 days from the innoculation time, mice were euthanized by ether. Excised tissues were weighed and divided into two pieces. One piece was fixed in 10% phosphate-buffered Formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin to confirm the presence of neoplastic cells in each mouse. The others were homogenized in a homogenizer (Fisher Customs Service Center, Atlanta, GA) and stored in a 270°C refrigerator. Assessment of Effects on Cell Cycle and Expressions of Cyclin-Dependent Kinase 1 (CDK 1) Cell cycle analysis. For harvest, the cells were detached from culture flasks with a brief treatment of 5.0 ml HBSSbuffered 5 mM EDTA/0.25% trypsin. The time of detachment was recorded. Detached cells were washed once with culture medium and filtered through a 50-ml pore-sized mesh polyamide gauze (Specrum Medical Industries, CA) to remove large cell aggregates. The total number of cells and viability were determined by 0.4% trypan blue dye exclusion. Cells were washed twice with ice-cold PBS containing 0.5% bovine serum albumin (BSA; PBS/BSA) (Sigma Diagnostics, St. Louis, MO). For the immunofluorescent staining of DNA, we used Cycle Test Plus Kit (Calbiochem, San Diego, CA). Half a million cells were incubated after adding 1 mg/ml propidium iodide (PI) at room temperature for 30 min. The amount of PI bound to DNA in cell suspension was measured by the FL 2 detector of a FACScan flow cytometer (Becton–Dickinson, CA) equipped with an argon-ion laser (488 nm emission) within 24 h after DNA staining. Chicken erythrocytes were used as a negative control for the FACScan setup. CellFIT (Becton–Dickinson Immunocytometry Systems, CA) software was applied for data acquisition and analysis. For each measurement, 15,000 events at the LO mode were acquired as a scattergram (FL2-W: width/FL2-A: area) and a histogram (FL2-A/counts), and percentages of each phase of the cell cycle in the histogram to the FL2-A parameters were obtained. Analysis of expression of cyclin-dependent kinase 1. The cell suspension was obtained with the method using trypsin
172
KIM ET AL.
described above. The total number of cells and cell viability were determined by 0.4% trypan blue dye exclusion. Cells were washed again with ice-cold PBS containing 0.5% bovine serum albumin (BSA; PBS/BSA) (Sigma Diagnostics). For permeabilization of cellular membrane, cells were resuspended with the cold 75% ethanol and stored in a 220°C refrigerator for 2 h. After washing, 0.25% Triton X-100 was added and the suspension incubated on ice for 5 min. The purified mouse anti-human p34cdc2 protein kinase (cyclin-dependent kinase 1) antibody (Pharmingen, San Diego, CA) was used as a primary antibody. All monoclonal antibodies were used under saturating conditions, as determined by titration experiments on cell lines showing the highest expression of antigenic determinants after EDTA detachment. A million cells were incubated with 50 ml of primary antibody for 30 min at 4°C. Cells were washed twice with 1.0 ml ice-cold PBS/BSA, followed by 30 min incubation with 100 ml of fluorescein isothiocyanate (FITC)-labeled Affini Pure goat anti-mouse IgG polyclonal antibody diluted 1:200 at 4°C in PBS/BSA after sedimentation (250g, 6 min, 4°C). The intensity of fluorescence was analyzed by using a FACS Vantage flowcytometer (Becton–Dickinson) equipped with a Coherent Enterprise laser (300 mW at 48 nm) and a seventh detector for five-color immunofluorescence measurements. Chicken erythrocytes were used as a negative control. For each measurement, 10,000 events were acquired and listmode data analysis was performed by applying CELL Quest software (Becton–Dickinson) [22]. Assessment of effects on differentiation: Flow cytometric analysis of CA-125. The suspension of cultured OVCAR-3 and SKOV-3 cells was obtained with the trypsin method described above. The total number of cells and viability were determined by 0.4% trypan blue dye exclusion. For the expression of CA-125 on the cell surfaces, OC125 (Signet Laboratories, Dedham, MA) was used as a primary antibody. All the conditions and procedures were the same as those in the flow cytometric analysis of CDK 1. Apoptosis Assay In situ Tdt method. The Apop Tag In Situ Apoptosis Detection Kit was used for detecting apoptotic cells. Single cell suspensions containing 5 3 10 7 cells/ml of cultured OVCAR-3 were fixed in 4% neutral buffered Formalin for 10 min at room temperature. To quench endogenous peroxidase, 2.0% hydrogen peroxide in PBS was applied to the slide for 5 min. After washing, 50 –100 ml of cell suspension was dried on a microscopic slide and washed twice in PBS for 5 min. Two drops of 13 equilibration buffer [50 mM Tris–HCl, 5 mM MgCl 2 10 mM b-mercaptoethanol, 0.005% BSA (Fraction V: Sigma, St. Louis, MO), pH 7.5] was directly applied to the specimen. A plastic coverslip was applied gently and specimens were incubated for 10 –15 s at room temperature. After removal of the plastic coverslip, 54 ml of mixtures with Tdt (terminal deoxyri-
bonucleotidyl transferase), biotinylated dNTP, 0.5 M Tris, pH 7.5, 50 mM MgCl 2, 0.6 mM 2-mercaptoethanesulfonic acid, and 0.5 mg/ml BSA (RIA grade) were pipetted onto the glass slide gently, a plastic coverslip was reapplied and specimens were incubated in a humidified chamber at 37°C for 1 h. After washing in PBS, two drops of anti-digoxigenin peroxidase were applied to the glass slide and incubated for 30 min at room temperature. For color development, freshly prepared DAB (diaminobenzidine) substrate solution was applied and after 3 to 6 min of incubation, a counterstain with methyl green was done. Apoptotic cell death detection by ELISA. Additional evidence of apoptosis in ovarian cancer cell lines and xenografts 24 and 48 h after treatment with 10 mM GnRH agonist was determined by a quantitative sandwich enzyme immunoassay principle using mouse monoclonal antibodies directed against DNA and histones (Boehringer Manheim Gmbh, Germany). This ELISA provides an in vitro quantitative determination of histone-associated DNA fragments (mono- and oligonucleosomes) in the cytoplasmic fraction of ovarian cancer cell lines and xenografts, as determined spectrophotometrically at 405 nm. The presence of mono- and oligonucleosomes is a feature of cells undergoing apoptosis [23]. Telomerase Assay After washing the cultured OVCAR-3 and SKOV-3 cells and minced xenograft once with PBS, we resuspended the cell pellet as 10 5 cells in 200 ml of 13 Chaps (3-[(3-cholamidopropyl)dimethylammonio]-1-profanesulfonate) lysis buffer (Oncor Gaithersburg, MD). The suspension was incubated on ice for 30 min. Telomerase activity was measured by the PCR-based telomere repeat amplification protocol (TRAP) assay as described [24]. For each TRAP assay, heat inactivation control as a negative control, positive extract control from the control cell pellet provided in the kit, and synthetic oligonucleotide with eight telomeric repeats as PCR/ELISA positive controls and primer– dimer/PCR contamination control to detect carryover contamination were included. The telomere repeat region was amplified with PCR and the concentrations of TRAP products were determined by the TRAPeze ELISA kit (Bio-Rad, Hercules, CA). At the end of the procedure, the difference of absorbance (DA) at 450 and at 690 nm using a microtiter plate reader was measured. If the DA was above 0.150, the extract was scored as “telomerase positive.” Statistics The significance of experiments was analyzed by one-way or two-way analysis of variance (ANOVA) [25], Duncan’s multiple range test, and log-rank test with Kaplan–Meier survival estimates [26].
ANTITUMOR EFFECT OF GnRH AGONIST
173
In vivo. On day 5 after tumor cell innoculation, the tumor volume reached above 50 mm 3, which was the starting point of drug treatments. Beginning at day 9, there were differences in tumor sizes between control (untreated) and treated groups. On day 23, the mean tumor volume of untreated groups reached 20,834 mm 3 and that of treated groups reached 16,920 mm 3, which showed a difference of approximately 81.2%. On day 29, the mean tumor volumes of the two groups were 35,679 and 28,717 mm 3, respectively, and the difference in size was 80.5% (Figs. 2 and 3). On the whole, there was a significant difference between the two groups (P , 0.05) (Fig. 3). Splenomegaly was noted in all SCID mice innoculated. Widespread metastasis was observed microscopically in the lung, heart, kidney, and liver. Survival rates were 77.8 and 44.4% in the treated and untreated groups, respectively, upon termination of the experiment (P , 0.15). (Fig. 4). Analysis of the Cell Cycle
FIG. 1. Antiproliferative activity of OVCAR-3 (A) and SKOV-3 cell (B) lines to GnRH agonist in vitro. Cells were exposed to 0.1 and 10 mM GnRH agonist and their growth was observed for 5 days. Each value represents the mean of eight wells. Dose, day, and interaction effects are significantly different (P , 0.05) by two-way ANOVA.
RESULTS Antiproliferative Study In vitro. OVCAR-3 and SKOV-3 cell lines were treated with 0.1 and 10 mM GnRH agonist for 5 days and the proportion of proliferated cells compared to the nontreated control cell lines was measured by MTT assay. After 4 h of incubation at 0.1 and 10 mM, there were transient increases of cell proliferation in both cell lines. After 12 h of incubation, OVCAR-3 and SKOV-3 cell line quantities were 88.0 and 79.2% at 0.1 mM and 72.0 and 54.2% at 10 mM, respectively. This antiproliferative effect increased gradually with time. After 120 h of incubation at 0.1 mM, OVCAR-3 and SKOV-3 cell lines were further reduced to 79.1 and 77.7% and at 10 mM were 29.3 and 25.7%, respectively. Cancer cell growth was significantly suppressed after 12 h and was grossly suppressed, in a time- and dose-dependent manner, over a period of 5 days in vitro (P , 0.05) (Fig. 1.). There were no significant differences in the dose dependencies between 10 mM and higher concentrations (data not shown).
Cell cycle analysis was performed every other day for 7 days. Cell cycle perturbations were evident from 8 h following addition of 10 mM GnRH agonist to OVCAR-3 and SKOV-3 cell lines. After 24 h of incubation with 10 mM GnRH agonist, the proportions of G0/G1, S, and G2/M phases of OVCAR-3 cell lines were 54.4, 42.6, and 3.9% and those of the control group (no treatment) were 37.2, 55.6, and 6.3%, respectively. There were increases in cell fractions in G0/G1 phase and decreases in those of cells in the S and G2/M phases during the treatment, compared to the nontreated control group in both cell lines (P , 0.05), but not in a time-dependent fashion (Fig. 5). Analysis of the Expression of Cyclin-Dependent Kinase 1 After 3 days of incubation with 10 mM GnRH, 62.7 and 66.8% of CDK 1 positive cell fractions of the treated cells were detected compared to 75.9 and 71.1% of nontreated cells in OVCAR-3 and SKOV-3 cell lines, respectively. The expression of CDK 1 decreased significantly after 3 days and grossly over a period of 7 days in a time-dependent fashion (P , 0.05) (Fig. 6). Apoptosis In situ apoptosis assay. Solitary or multiple micronuclei indicating the morphological characteristics of apoptosis were observed in cells treated with 10 mM GnRH agonist, and the proportions of such cells at 24 and 48 h were 5.5 6 1.6 and 6.3 6 2.2%, compared with untreated cells, with 4.2 6 1.2 and 4.8 6 1.6%, respectively (Fig. 7). However, the ratio of apoptotic findings did not increase in cells treated with the same concentrations from the third day, compared with that of the second day.
174
KIM ET AL.
FIG. 2. (A) Xenograft of OVCAR-3 tumor cell line in C.B.-17 SCID mouse 4 weeks after sc inoculation (arrow). This picture shows one of mice which lived 4 weeks. (B) The feature of cut section. (C) Subcutaneous tumor mass (arrow), H & E 3100.
Apoptotic cell death detection by ELISA. After 24 h of incubation, the cultured cells incubated with 10 mM GnRH agonist showed an increase in cytoplasmic fractions with histone-associated DNA fragments compared with the control group, 0.25 vs 0.27 in OVCAR-3 and 0.51 vs 0.56 in SKOV-3, and after 48 h of incubation, 0.27 vs 0.48 in OVCAR-3 and 0.53 vs 0.83 in SKOV-3 (Fig. 8).
Assessment of Effects of Differentiation For the purpose of investigating the differentiation of cultured OVCAR-3 and SKOV-3 cells following treatment with 10 mM GnRH agonist, we observed the morphologicchanges by light microscopy and the expression of CA-125, known as a representative tumor marker of ovarian cancer,
ANTITUMOR EFFECT OF GnRH AGONIST
175
FIG. 2—Continued
by immunofluorescent stain with flow cytometric analysis (data not shown). However, we found no evidence of differentiation in terms of morphology, and a decrease in the development of cancer antigen or in the activity of telomerase, which indicates differentiation, was not observed either (Fig. 9). DISCUSSION In the medical treatment of ovarian cancer, there have been trials not only exploring new antitumor drugs but also studying
alternative treatment modalities. Because of low toxicity as well as cost effectiveness in the case of the endocrine dependency of the ovary, hormonal therapies have been predominantly accepted as alternative therapies. Hormonal therapies, using antiestrogens, progestogens and their combinations, have demonstrated only a marginal effect or none [27, 28]. Incessant monthly ovulation has been known as a physiologic causal factor for the development of ovarian cancer, i.e., the repeated rupture of the coelomic epithelium. But other researchers have asserted the “gonadotropin theory” that the
FIG. 3. In vivo effect of GnRH agonist on the growth of subcutaneous OVCAR-3 xenografts. GnRH treatment (100 mg per mouse every 2 weeks) was initiated on day 5 after tumor implantation. Each value represents the mean of eight mice. Day effects are significantly different (P , 0.05) by repeated measures ANOVA and Duncan’s multiple range test.
176
KIM ET AL.
FIG. 4. The survival difference between GnRH agonist-treated and untreated mice upon assay termination. The introduction of an artificial end point precluded survival analysis (P , 0.15).
FIG. 6. Expression of cyclin-dependent kinase 1 in OVCAR-3 and SKOV-3 cell lines by treatment with 10 mM GnRH agonist for 7 days. (A) Plots represent the variation in percentage of mean fluorescence intensity (MFI) in OVCAR-3 cell lines. (B) Plots represent the variation in percentage of MFI in SKOV-3 cell lines. Day effects are significantly different in both cell lines (P , 0.05) by one-way ANOVA and Duncan’s multiple range test. Each value represents mean of quadruplicate two times determinations.
FIG. 5. Effects of GnRH agonist on the cell cycles of OVCAR-3 and SKOV-3 cell lines. The cells were stimulated for 7 days with 10 mM GnRH agonist. (A) Plots represent the variation in percentage of G0/G1, S and G2/M phases in OVCAR-3. (B) Plots represent the variation in percentage of G0/G1, S and G2/M phases in SKOV-3. There were significant differences that increased the ratio of G0/G1 phase with decreased ratios of S and G2/M phases between treated and untreated groups in both cell lines (P , 0.05), but day effects are not significant by two-way ANOVA and Duncan’s multiple range test. Each value represents mean of quadruplicate two times determinations.
exposure to high gonadotropin levels in cycling women favors malignant transformation [29, 30]. Epidemiologists have also agreed on this theory based on the increased incidence of ovarian cancer with the beginning of menopause and elevated gonadotropins [29, 31]. With the positive results in vitro [32] and in vivo [33], it was thought that human ovarian cancer cells could grow by the stimuli of gonadotropin. Based on this theory, GnRH agonist has been utilized for the treatment of ovarian cancer by blocking of the hormonal axis and downregulation of gonadotropin [34, 35]. However Yano et al. [9, 10] and Thompson et al. [38] did not observe similar findings and they even had contradictory results in further experiments, i.e., no stimulatory effect on the growth of ovarian cancer cell lines by gonadotropin; thus, this theory is controversial. They found that there were no differences in the levels of gonadotropin between ovarian cancer patients and normal women [14, 36, 37]. Especially, there is a controversy over the ratio of receptors for gonadotropin on ovarian cancers (0 –91%) [39, 40]. On the other hand, after the demonstration of GnRH mRNA and the local regulatory system of GnRH in ovarian
ANTITUMOR EFFECT OF GnRH AGONIST
177
FIG. 7. Immunodetection of labeled nucleotides added by Tdt on OVCAR-3 cells after treatment of 10 mM GnRH for 24 h. Intense staining was observed in nuclei and nuclear fragments with the morphological characteristics of apoptosis. Counterstaining was done with methyl green (arrow).
cancer cells, the role of GnRH receptor was considered an autocrine regulator of the proliferation of ovarian cancer cells [41, 42]. And the GnRH receptor was thought to be a direct target of the antitumor effects of GnRH. Also, this “direct GnRH receptor theory” was assisted by the apoptosis of the OVCAR-3 cell line by antibodies against the GnRH receptor [43]. In fact, GnRH receptors were found in more than 90% of ovarian cancers [3, 39]. Therefore, it was thought that longterm therapy of GnRH agonist might induce desensitization to GnRH, followed by the direct antiproliferative effect. But this “direct GnRH receptor theory” was far from producing a complete conclusion because there are few studies on the GnRH second messenger system and the related molecules in ovarian cancer. Recent studies showed the role of annexin V in the signal transduction of GnRH agonist [39] and its relation with insulin-like growth factor II [40]. Even though there are controversies over the gonadotropin dependency of ovarian cancer and the direct antitumor effect of GnRH agonist, the data obtained by making good use of GnRH agonist until this time may be sufficient to advocate its use in ovarian cancer. In this study, first of all, we found not only antiproliferative effects of GnRH agonist but also time- and dose-dependent increases in its action. These findings are similar to previous in vitro observations [9, 10, 39]. But as to the effective concentrations of the GnRH agonist, there are discrepancies with some previous reports. In our study, the inhibitory effect against proliferation was maximal at 10 mM GnRH agonist, and the effects were not significantly different between 10 mM
and 100 mM (data not shown). However, Thompson et al. [38] contended antiproliferative effects were only significant at high concentrations (1 and 100 mM), and Emons et al. [44] even at 1 nM. Meanwhile, in cases of the SiHa cell line, known to have no GnRH receptor, treatment with 10 mM GnRH agonist showed the same degree of antiproliferative effect with OVCAR-3 and SKOV-3 cell lines, which have GnRH receptors. But treatment at 0.1 mM did not show the same degree of antiproliferative effect in the SiHa cell line (data not shown). Based on the results at low concentration (0.1 mM), it could be thought that the action of GnRH agonist is generated by receptor-mediated mechanisms. On the other hand, antiproliferative effects at high concentrations might be developed by the broad potentials of GnRH agonist as an antitumor drug. In the studies on antiproliferative effect, those done in vivo with SCID mice showed results similar to those with cases using nude mice [8 –11]. The antitumor effect observed in our in vivo experience was substantial, with a reduction in the final tumor volume of approximately 80.5% in the GnRH agonisttreated group, compared with a control group. Although the survival difference between GnRH agonist-treated and untreated mice was not significant in this in vivo study, we observed a tendency toward long-term survival in treated groups. The majority of drugs kill cancer cells in a unique phase of the cell cycle, and cells not belonging to that phase will not be injured. And the blocking phases of most anticancer drugs on that cell cycle are usually inducing subsequent apoptosis [45].
178
KIM ET AL.
cycle. The time lag between prompt elevation of G0/G1 phase fractions and delayed decline of CDK 1 is not explained. We surmise the existence of another control mechanism. The 48-h cultured cells of both groups were stained with trypan blue to measure viability and there was no difference between GnRH-treated and control groups (approximately, 4 vs 6%). From these results, we were be convinced of the cytotoxic effect of GnRH agonist. Then we quantitated the event of apoptosis with the ELISA method and actually found increased induction of apoptosis in ovarian cancer cell lines treated with GnRH agonist. In the study of cell differentiation, we did not find differences in the expression of CA-125 (a representative antigen of ovarian cancer) by flow cytometry or visible changes of cel-
FIG. 8. Relative levels of apoptosis quantified by histone-associated DNA fragments in OVCAR-3 (A) and SKOV-3 (B) cell lines following treatment with 10 mM GnRH agonist for 24 and 48 h. Each value represents the mean value of triplicate determinations.
Accordingly, it is essential to know the action phase in the cell cycle in combination drug therapy. As cell cycle analysis in vivo is liable to problems like intratumor heterogeneity and difficulty in tissue processing procedures [46], we focused only on in vitro results. In the growth kinetic study using flow cytometry, we found that the GnRH agonist acted mainly by arresting cells in the G0/G1 phase of the cell cycle and thus reduced the number of cells in S and G2/M phases. The elevation of G0/G1 phase fractions confirmed that the main action of GnRH agonist is made in G0/G1 phase. Thompson et al. [38] also found that GnRH agonist caused a reversible 5– 6% increase in cells in the G0/G1 phase compared with controls and a corresponding decrease of cells in S and M phases. The transient acceleration of cell proliferation (for 12 h) in MTT assays could be explained by the fact that for the action of GnRH agonist, a time interval for a next event is necessary. Because of the blocking of G0/G1 phases, there was decreased expression of CDK 1, which controls the S and G2/M phases, on transitions between successive phases of the cell
FIG. 9. Relative levels of telomerase activity in OVCAR-3 and SKOV-3 cell lines and a xenograft treated with 10 mM GnRH agonist, as measured by TRAP assay and ELISA detection. Each value represents the mean value of triplicate determinations. A, positive telomerase control; B, PCR/ELISA positive control; C, negative control; D, heat inactivation; E, no treatment for 14 days; F, treatment with 10 mM GnRH agonist for 14 days; G, no treatment for 21 days; H, Treatment with 10 mM GnRH agonist for 21 days.
ANTITUMOR EFFECT OF GnRH AGONIST
lular morphology by light microscopy during the period of treatment. In vitro differentiation of an immortal cell type leads to low or undetectable telomerase activity [16 –18]. In this study, there was no decreased telomerase activity, rather than increased. But we cannot completely deny the role of GnRH agonist as a differentiating agent because we used only CA-125 and telomerase activity as differentiation markers and did not observe the morphologic evidence of differentiation by electron microscopy in cancer cells. Interestingly, telomerase activity increased after treatment with GnRH agonist. Considering a general conception of the correlation between the proliferation of cells and increased telomerase activity [18], we found some contradiction between the antiproliferative effect of GnRH agonist (decreased S and G2/M phases) and elevated telomerase activity in this study. But recent reports showed the controversy over controlling telomerase activity in the cell cycle [48]. A recent report reported that as cells progress through the replicative S phase, the activity of telomerase gradually increases [49]; on the other hand, there has been a contradicting report that telomerase activity is not related to the cell cycle [50]. This indicates the possible effect of another complicated and indirect factor in the changes in telomerase activity. Even though antitumor activity in ovarian cancer based on GnRH agonist was proven, we should solve some problems at the clinical base. Substantially, owing to cost effectiveness and the potential of the drug, it is not easy to obtain high concentrations of GnRH agonists in humans by usual doses of GnRH agonist (once every 2 or 4 weeks) [51]. In clinical studies using GnRH agonist, researchers have insisted that there is no dramatic effect, just a marginal effect [13–15]. Actually, however, clinicians have few treatment modalities in advanced ovarian cancer patients who suffered from chemotoxicity. Low toxicity can permit GnRH agonist to be used effectively, regardless of the controversy over its effectiveness. More knowledge about the signal transduction pathway of GnRH and the development of a potent GnRH agonist with cytotoxic radicals might permit a better exploitation of its antitumor effects in the treatment of ovarian cancer. ACKNOWLEDGMENTS This work was supported in part by Research Funds of St’s Vincent’s Hospital, The Catholic University of Korea.
REFERENCES 1. Korean Association of Obstetricians and Gynecologists, Gynecologic Cancer Registry Program in Korean Women, 1994
179
4. Kaiser UB, Conn PM, Chin WW: Studies of gonadotropin-releasing hormone (GnRH) action using GnRH receptor expressing pituitary cell lines. Endocr Rev 18(1):46F70, 1997 5. Chatzaki E, Bax CM, Eidne KA, Anderson L, Grudzinskas JG, Gallagher CJ: The expression of gonadotropin-releasing hormones and its receptor in endometrial cancer, and its relevance as an autocrine growth factor. Cancer Res 56:2059 –2065, 1996 6. Marini L, Iacopino F, Schinzari G, Robustelli Della Cuna FS, Mantovani G, Sica G: Direct antiproliferative effect of triptorelin on human breast cancer cells. Anticancer Res 14:1881–1885, 1994 7. Kullander S, Rausing A, Trope C: Human ovarian tumors heterotransplanted to nude mice. Acta Obstet Gynecol Scand 57(2):149 –159, 1978 8. Peterson CM, Jolles CJ, Carrell DT, Straight RC, Jones KP, Poulson AM, Hatasaka HH: GnRH agonist therapy in human ovarian epithelial carcinoma (OVCAR-3) heterotransplanted in the nude mouse is characterized by latency and transience. Gynecol Oncol 52(1):26 –30, 1994 9. Yano T, Pinski J, Halmons G, Szepeshazi K, Groot K, Schally AV: Inhibition of growth of OV-1063 human epithelial ovarian cancer xenografts in nude mice by treatment with luteinizing hormone-releasing hormone antagonist SB-75. Proc Natl Acad Sci USA 91(5):7090 –7094, 1994 10. Yano T, Pinski J, Radulovic S, Schally AV: Inhibition of human epithelial ovarian cancer cell growth in vitro by agonistic and antagonistic analogues of luteinizing hormone-releasing hormone. Proc Natl Acad Sci USA 91(5):1701–1705, 1994 11. Manetta A, Liao S, Gamboa-Vujicic G, Leong L, Pardes P, Asch B, Emma D, Schally A: Inhibition of growth of human ovarian cancer in nude mice by luteinizing hormone-releasing hormone antagonist cetrorelix (SB-75). Fertil Steril 63(2):282–287, 1995 12. Vincze B, Palyi I, Gaal D, Pato J, Mora M, Mezo I, Teplan I, Seprodi J: In vivo studies of the new gonadotropin-releasing hormone antagonistcopolymer conjugates having antitumor activity. Cancer Detect Prev 20(2):153–159, 1996 13. Emons G, Ortmann O, Teichert HM, Fassl H, Lohrs U, Kullander S, Kauppila A, Ayalon D, Schally A, Oberheuser F: Luteinizing hormone releasing hormone agonist triptorelin in combination with cytotoxic chemotherapy in patients with advanced ovarian carcinoma. A prospective double blind randomized trial. Decapeptyl Ovarian Cancer Study Group Cancer 78(7):1452–1460, 1996 14. Falkson CI, Falkson HC, Falkson G: Cisplatin versus cisplatin plus D-TRP-6-LHRH in the treatment of ovarian cancer: A pilot trial to investigate the effect of the addition of a GnRH analogue to cisplatin. Oncology 53(4):313–317, 1996 15. van-der Vange N, Greggi S, Burger CW, Kenemans P, Vermorken JB: Experience with hormonal therapy in advanced epithelial ovarian cancer. Acta Oncol 34(6):813– 820, 1995 16. Sharma HW, Sokoloski JA, Perez JR, Maltese JY, Sartorelli AC, Stein CA, Nicholas G, Khaled Z, Telang NT, Narayanan R: Differentiation of immortal cells inhibits telomerase activity. Proc Natl Acad Sci USA 92(26):12343–12346, 1995 17. Albanell J, Han W, Mellado B, Gunawardane R, Scher HI, Dmitrovsky E, Moore MA: Telomerase activity is repressed during differentiation of maturation-sensitive but not resistant human tumor cell lines. Cancer Res 56(7):1503–1508, 1996
2. Christian MC, Trimble EL: Salvage chemotherapy for epithelial ovarian carcinoma. Gynecol Oncol 55:S143–150, 1994
18. Avilion AA, Piatyszek MA, Gupta J, Shay JW, Bacchetti S, Greider CW: Human telomerase RNA and telomerase activity in immortal cell lines and tumor tissues. Cancer Res 56(3):645– 650, 1996
3. Imai A, Ohno T, Iida K, Fuseya T, Furui T, Tamaya T: Gonadotropinreleasing hormone receptor in gynecologic tumors, frequent expression in adenocarcinoma histologic types. Cancer 74(9):2555–2561, 1994
19. Choi YK, Choi JY, Kown EH, Kim YJ, Lee CH, Jeong KS, Hyun BH: Evaluation of nonleaky C.B-17 SCID mice by ELISA. Korean J Lab Anti Sci 12:9 –13, 1996
180
KIM ET AL.
20. Carmichael J, Degraff WG, Gazdar AF, Minna JD, Mitchell JB: Evaluation of a tetrazolium-based semiautomated colorimetric assay: Assessment of chemosensitivity testing. Cancer Res 47(4):936 –942, 1987 21. Choi YK, Choi JY, Hyun BH, Kim YJ, Lee CH, Yoon WK, Jeong KS, Kim DY: Antitumor effect of methotrexate in SCID mice with human leukemia CCRF-CEM cell line. Korean Cancer Assoc 29:19 –28, 1997 22. Beavis AJ, Pennline KJ: Allo-7: A new fluorescent tandem dye for use in flow cytometry. Cytometry 24(4):390 –395, 1996 23. Bonfoco E, Krainc D, Ankarcrona M, Nicotera P, Lipton SA: Apoptosis and necrosis: Two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures. Proc Natl Acad Sci USA 92(16):7162–7166, 1995 24. Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW: Specific association of human telomerase activity with immortal cells and cancer. Science 266: 2011–2015, 1994 25. Snedecor GW, Cochran WG: Statistics Methods, 2nd ed. Iowa State Univ. Press, Ames, Iowa, 1980 26. Miller RG Jr: Survival Analysis. Wiley, New York, 1981 27. Desombre ER, Holt JA, Herbst AL: Steroid receptors in breast, uterine and ovarian malignancy, in: Gold JJ, Josinovich JB (Eds.): Gynecologic Endocrinology. Plenum, New York, 1987, pp. 511–528 28. Rao BR, Slotman BJ: Endocrine factors in common epithelial ovarian cancer. Endocr Rev 12(1):14 –26, 1991 29. Stadel BV: The etiology and prevention of ovarian cancer. Am J Obstet Gynecol 123:772–774, 1975 30. Cramer DW, Welch WR: Determinants of ovarian cancer risk II. Inferences regarding pathogenesis. J Natl Cancer Inst 71:717–721, 1983 31. Schlesselman JJ: Cancer of the breast and reproductive tract in relation to use of oral contraceptives. Contraception 40(1):1–38, 1989 32. Simon WE, Albrecht M, Haensel M, Dietel M, Hoelzel F: Cell lines derived from human ovarian carcinomas: Growth stimulation by gonadotropic and steroid hormones. J Natl Cancer Inst 70:839 – 845, 1983 33. Ohtani K: Effects of gonadotropin on the growth of malignant ovarian neoplasms assessed by subdural capsule assay. Nippon Sanka Fujinka Gakki Zasshi 42:579 –585, 1990 34. Mortel R, Satyaswaroop PG, Schally AV, Hamilton T, Ozols R: Inhibitory effect of GnRH superagonist on the growth of human ovarian carcinoma NIH: OVCAR 3 in the nude mouse. Gynecol Oncol 23:254 –255, 1986 35. Kullander S, Rausing A, Schally AV: LHRH agonist treatment in ovarian cancer, in klijn (Ed): Hormonal Manipulation of Cancer: Peptides, Growth Factors and New (Anti) Steroidal Agents. Raven Press, New York, 1987, pp. 353–356 36. Emons G, Schally AV: The use of lutenizing hormone releasing hormone agonists (antagonists) in gynecological cancers. Hum Reprod 9(7):1364 – 1379, 1994 37. Ron IG, Wigler N, Merimsky O, Inbar MJ, Chaitchik S: A phase II trial of D-TRP-6-LHRH (Decapeptyl) in pretreated patients with advanced epithelial ovarian cancer. Cancer Invest 13(3):272–275, 1995 38. Thompson MA, Adelson MD, Kaufman LM: Lupron retards proliferation
of ovarian epithelial tumor cells cultured in serum-free medium. J Clin Endocrinol Metab 72:1036 –1041, 1991 39. Shibata S, Sato H, Ota H, Karube A, Takahashi O, Tanaka T: Involvement of annexin V in antiproliferative effects of gonadotropin-releasing hormone agonist on human endometrial cancer cell line. Gynecol Oncol (2)66:217–221, 1997 40. Ho MN, Delgado CH, Owens GA, Steller MA: Insulin-like growth factor-II participates in the biphasic effect of a gonadotropin-releasing hormone agonist on ovarian cancer cell growth. Fertil Steril 67(5):870 – 876, 1997 41. Ohno T, Imai A, Furui T, Takahashi K, Tamaya T: Presence of gonadotropin-releasing hormone and its messenger ribonucleic acid in human ovarian epithelial carcinoma. Am J Obstet Gynecol 169:605– 610, 1993 42. Emons G, Ortman O, Pahwa GS, Oberheuser F, Schultz KD: Intracellular actions of gonadotropic and peptide hormones and the therapeutic value of GnRH-agonists in ovarian cancer. Acta Obstet Gynecol Scan Suppl 155: 31–38, 1992 43. Ackerman RC, Johson GA, Van Kirk EA, Asirvatham AL, Murdoch WJ: Induction of apoptotic or lytic death in ovarian adenocarcinoma cell line by antibodies generated against a synthetic N-terminal extracellular domain gonadotropin-releasing hormone receptor peptide. Cancer Lett 81: 177–184, 1994 44. Emons G, Ortmannm O, Becker M, Irmer G, Springer B, Laun R, Hoelzel F, Schulz KD, Schally AV: High affinity binding and direct antiproliferative effects of LH-RH analogues in ovarian cancer cell lines. Cancer Res 54:5439 –5446, 1993 45. Alaoui SE, Lawry J, Griffin M: The cell cycle and induction of apoptosis in a hamster fibrosarcoma cell line treated with anti-cancer drugs: Its importance to solid tumor chemotherapy. J Neurooncol 31:195–207, 1997 46. Bergers E, Mntironi R, van Diest PJ, Prete E, Baak JPA, Fiac(Hon) F: Interlaboratory reproducibility of semiautomated cell cycle analysis of flow cytometric DNA-histograms obtained from fresh material of 1,295 breast cancer cases. Hum Pathol 27(6):553–560, 1995 47. Gibb RK, Taylor DD, Wan T, Oconnor DM, Doering DL, GercelTaylor C: Apoptosis as a measure of chemosensitivity to cisplatin and taxol therapy in ovarian cancer cell lines. Gynecol Oncol 65(1):13–22, 1997 48. Buchkovich KJ, Greider, CW: Telomerase regulation during entry into the cell cycle in normal human T cells. Mol Biol Cell 7:1443–1454, 1996 49. Zhu X, Kumar R, Mandal M, Sharma N, Scharma HW, Dhingra U, Sokoloski JA, Hsiao R, Narayanan R: Cell cycle-dependent modulation of telomerase activity in tumor cells. Proc Natl Acad Sci USA 93:6091– 6095, 1996 50. Sokoloski JA, Blair OC, Carbone R, Sartorelli AC: Induction of the differentiation of synchronized HL-60 leukemia cells by tiazofurin. Exp Cell Res 182:234 –241, 1989 51. Ozols RF, Rubin SC, Dembo AJ, Robboy S: Epithelial ovarian cancer, in Hoskins WJ, Perez SA, Young RC (Eds.): Principles and Practice of Gynecologic Oncology. Philadelphia, Lippincott 1992, 731–781