1 Department of Obstetrics and Gynecology and 2 Laboratory of Experimental Surgery, Hadassah University Hospital, PO Box 12000, Jerusalem 91120, Israel
3 To whom correspondence should be addressed. Email: shush{at}cc.huji.ac.il
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Abstract |
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Key words: AG1478/growth suppression/leiomyomas/protein tyrosine kinase (PTK) inhibitors/signal transduction therapy
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Introduction |
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Formation of leiomyomas, still not completely understood, is viewed as a multistep process, with involvement of ovarian steroid hormones, cytokines and growth factors (Sozen and Arici, 2002; Flake et al., 2003
; Lefebvre et al., 2003
). Previous and present research has opened the way to new non-surgical treatments, based on the understanding that leiomyomas are SMC steroid-responsive tumours (Liu et al., 1993
). A body of research has demonstrated that leiomyomas are affected by estrogen and progesterone and that their response differs compared with that of normal myometrium (Rein et al., 1995
; Rein, 2000
). Therefore, GnRH agonists that reduce ovarian steroid hormones levels are widely in use (Lethaby et al., 2002
). Since leiomyomas are chronic and recurrent, it is advantageous to modify therapies to allow for more prolonged treatment. Indeed, GnRH agonists with add-back esteradiol (E2) and progesterone are among the most frequently studied medical therapies (Chegini et al., 2002
).
Newer therapies are based on the differential expression/production of growth factors between leiomyomas and normal myometrium. Basic fibroblastic growth factor (bFGF), suggested as central to the pathogenesis of leiomyomas, may provide a new target therapy especially for women with leiomyoma-related bleeding (Anania et al., 1997). Indeed, interferon-
is a potent inhibitor of bFGF-stimulated cell proliferation in human uterine cells (Lee et al., 1998
; Minakuchi et al., 1999
). Interferon-
and interferon-
are presently examined as novel treatment modalities, since they were shown to oppose the actions of bFGF in a number of systems (Lee et al., 1998
).
Recent research has focused on the function of proto-oncogenes such as Bcl2 and tumour suppressor gene products such as p53 in controlling apoptosis, as well as promoting leiomyoma cell proliferation by reducing the requirements for growth factors (Gao et al., 2001, 2002
; Dixon et al., 2002
). Matsuo et al. (1997)
have reported increased expression of the Bcl2 protein in leiomyoma cells compared with normal myometrium cells, upregulated by progesterone. They suggested Bcl2 as the molecular basis for leiomyoma formation and a vital role for progesterone in the increased expression of this protein (Matsuo et al., 1999
; Maruo et al., 2000
). Since expression of Bcl2 is downregulated by E2 but upregulated by progesterone, progesterone may also participate in leiomyoma growth through induction of the Bcl2 protein. Shimomura et al. (1998)
further demonstrated that E2 and progesterone act in combination to stimulate leiomyoma cell proliferation, through induction of epidermal growth factor (EGF) and EGF receptor (EGFR) expression.
Accumulating data suggest that the effects of steroid hormones are mediated by local production of growth factors such as EGF (Hofmann et al., 1984; Sozen and Arici, 2002
). Since EGF affects leiomyoma growth, a selective inhibitor of the EGFR such as AG1478 is a potential therapeutic agent for the non-surgical treatment of uterine leiomyomas (Levitzki and Gazit, 1995
; Levitzki, 1999
, 2001
). We present evidence that the AG1478 tyrphostin effectively suppressed proliferation and cell cycle progression in cultures of leiomyoma cells.
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Materials and methods |
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Cell cultures
Paired cell cultures of leiomyoma and adjacent normal myometrium tissue samples were established from premenopausal women following hysterectomy conducted for benign disease, after ethical approval and informed consent. Primary cell cultures were initiated in HAM/F12:Dulbecco's modified Eagle's medium (DMEM) 1:1 with 20% FBS and antibiotics (penicillin 100 U/ml and streptomycin 100 µg/ml). Thereafter, the cell cultures were propagated in phenol red-free DMEM and 10% charcoal-treated FBS, specifically for the experiments with ovarian steroid hormones. The experiments were done on secondary and/or tertiary cultures. Cells were maintained at 37°C in a humidified incubator containing 58% CO2. Exponentially growing cells were used in the experiments.
Immunocytochemical staining with -actin was routinely performed to verify the SMC origin of the cells in culture.
Tyrphostins and treatment of cells
Stock solutions of 10 mM AG1478 in dimethylsulphoxide (DMSO) were kept at 70°C (Gazit et al., 1996). For the experiments, the tyrphostin was diluted with DMEM containing 10% FBS. The highest concentration of DMSO was 0.1%.
Experimental design
Cells were seeded at 1 x 104/well in 96-well microtitre plates and grown for 23 days. Thereafter, the medium was replaced with medium containing the tyrphostin. Control cells were grown in medium and in medium with the appropriate concentration of DMSO. Medium was changed after 2 days with new tyrphostin. Two days after the second treatment (4 days treatment), medium was removed, new medium without tyrphostin added, and the cells were grown for another 4 days (rescue/recovery after treatment) followed by determination of growth using the colorimetric methylene blue assay.
Calculation of growth inhibition
For each tyrphostin concentration used, medium containing only DMSO was used for the control. Thus, for each concentration, the control was taken as 100% growth.
Automated microculture methylene blue assay
Cell growth was determined by the automated microculture methylene blue assay: the tyrphostin-treated cultures and controls were fixed in glutaraldehyde, 0.05% final concentration, for 10 min at room temperature. After washing, the microplates were stained with 0.01% methylene blue in 0.1 mol/l borate buffer for 60 min at room temperature. Then the microplates were washed extensively and rigorously to remove excess dye, and dried. The dye taken up by cells was eluted in 0.1 mol/l HCl for 60? min at 37°C, and the optical density was measured at 620 nm. In preliminary titration experiments, linear readings were obtained for 1 x 1034 x 104 cells/well. Each point of the growth curve experiments is calculated from eight wells.
Fluorescence-activated cell sorting (FACS) analysis of DNA content and determination of apoptotic cells
Selected samples of cell cultures were FACS analysed for DNA content. Samples of cells treated with AG1478 concentrations for pre-determined periods were dispersed with trypsin 0.25% 1:1 EDTA 0.05% and stained with ethidium iodide in suspension. Cell cycle analysis and determination of the apoptotic cell fraction of the cell populations were carried out with FACS FPAR PLUS (Becton-Dickinson, Inc., Mountain View, CA).
Biochemical activities: western blot (WB) analysis and phosphotyrosine
WB analysis for relevant proteins and phosphotyrosine was carried as previously described and according to the manufacturer's recommendation (Ben-Bassat et al., 1997). Briefly, cell cultures of leiomyoma and normal myometrium were seeded at 5 x 105 cells/35 mm plates in DMEM with 10% FCS. After 23 days, the cells were washed and tyrphostin at the appropriate concentration was added for pre-determined periods. The reaction was stopped by placing the cultures on ice and washed with ice-cold phosphate-buffered saline (PBS). Whole cells were lysed with buffer, boiled for 5 min and run on a 715% SDSpolyacrylamide gel for 4 h, then transferred to nitrocellulose paper, at room temperature overnight. Thereafter, the samples were incubated with the appropriate antibody, or with monoclonal anti-phosphotyrosine antibody 4G10, following the manufacturer's recommendations. Goat anti-mouse or anti-rabbit fluorescent antibody was added (3 µl/30 ml) for 30 min incubation at room temperature. The nitrocellulose membrane was washed with PBS-T, and the ECL (enhanced chemiluminescence) system was applied; then the membrane was exposed to X-rays films in cassettes. Densitometric analysis was performed with a Fluor-STM Multi-Imager (Bio Rad Laboratories, Hercules, CA) using the Multi-Analyst Program.
WB analysis for EGFR and phosphotyrosine
Cell cultures were seeded at 5x105 cells/35 mm plates in DMEM with 10% FCS and grown for 23 days. Thereafter, the cultures were washed, fed with medium without serum and starved for 48 h. Tyrphostin at the appropriate concentration in medium without serum ( = starvation medium) was added for 4 h. Cells were then stimulated with 30 ng/ml EGF for 10 min. Placing the cultures on ice and washing with ice-cold PBS stopped the reaction. Whole cells were lysed with hot buffer, scraped, boiled for 5 min and then run on a 7% SDSpolyacrylamide gel for 4 h. The cells were then transferred to nitrocellulose paper and incubated overnight at 4°C with a monoclonal anti-phosphotyrosine antibody PT-66, following the manufacturer's recommendations. Goat anti-mouse fluorescent antibody was added for 2 h incubation at room temperature (1 ml/20 ml). After drying, the gels were exposed to X-ray film in cassettes. Densitometric analysis was performed with a Fluor-STM Multi-Imager (Bio Rad Laboratories) using the Muli-Analyst Program.
Effects of E2 and progesterone on cell growth of paired leiomyoma and normal myometrium cell cultures
Cell cultures were seeded at 5x105 cells/35 mm plates in DMEM with 10% FCS and cultured for 2 days. Then doseresponse experiments with the ovarian steroids were performed using 120 ng/ml E2 or 10200 ng/ml progesterone, separately and combined: 5 ng/ml E2 and 10 ng/ml E2 with 10200 ng/ml progesterone. The cells were grown for another 7 days, followed by determination of growth using the colorimetric methylene blue assay.
Effects of E2 and progesterone on the inhibitory capacity of AG1478
Myometrium and leiomyoma paired cells were seeded at 2 x 105 cells /35 mm plates in DMEM with 10% FCS and grown for 2 days. Thereafter, the cultures were washed, fed with medium containing the steroid: 10 ng/ml E2 or 100 ng/ml progesterone without serum and AG1478 at 5 and 10 µmo/l in medium without serum+E2 or medium without serum+progesterone. The cultures were grown further for 7 days, followed by determination of growth using the colorimetric methylene blue assay.
Effects of E2 and progesterone on the biochemical activities of AG1478
Myometrium and leiomyoma paired cells were seeded at 2x105 cells/35 mm plates in DMEM with 10% FCS and grown for 2 days. Thereafter, the cultures were washed, fed with medium containing the steroid: 10 ng/ml E2 or 100 ng/ml progesterone without serum, and starved for 48 h. AG1478 at the appropriate concentration in medium+E2 or medium+progesterone without serum =starvation medium) was added for 4 h. Cells were then stimulated with 30 ng/ml EGF for 10 min. The reaction was stopped by placing the cultures on ice and washed with ice-cold PBS. Immunoblot analysis of relevant proteins was performed on whole cell-lysates.
Statistics
Data obtained from determination of growth, FACS analysis and densitometry were expressed as meam±SD. Statistical analysis was conducted using the Student's t-test. Statistical significance was established at P-values of <0.05.
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Results |
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Points of growth arrest in the cell cycle and apoptosis
To explore more directly the mechanism of growth suppression by AG1478, cell cycle analysis was performed on the leiomyoma and myometium cell cultures after treatment with this compound. Cells exposed to AG1478 were assessed by FACS analysis on day 2 after initiating treatment. The results indicate that AG1478 alters the cell cycle distribution (Figure 3). After one treatment with 10 µmol/l AG1478 for 2 days, the proportion of cells in G1 is increased and the proportion of cells in S and G2/M is decreased, with no effect on the apoptotic cell fraction (Figure 3).
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Discussion |
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Leiomyomas are apoptosis resistant. However, recently it was shown that AG1478 increased apoptosis induced by the death ligand CD95L in some human glioma cell lines through inhibition of EGFR autophosphorylation (Steinbach et al., 2002). Thus, inhibition of EGFR might improve the efficacy of cancer therapies based on ligand-mediated apoptosis.
Previous and present biochemical analyses of AG1478 show that this tyrphostin effectively inhibits EGFR autophosphorylation in a dose-dependent manner, without an effect on the level of EGFR expression (Levitzki, 1992; Gazit et al., 1996
; Ben-Bassat et al., 1997
; Ben-Bassat and Klein, 2000
). The EGFEGFR system plays a crucial role in regulating uterine leiomyoma growth, possibly by mediating estrogen action (Huet-Hudson et al., 1990
; Shimomura et al., 1998
). Growth of these tumours is considered to depend on ovarian steroid hormones, evident by their remarkable reduction in the menopause and by GnRH analogue treatment that creates a pseudo-menopausal state (Friedman et al., 1993
, 1994
; Stewart, 2001b
). Maruo et al. (2000)
suggested that the ovarian steroid hormones E2 and progesterone act in combination to stimulate the proliferative potential of leiomyomas through induction of EGF and EGFR.
The present results demonstrate that E2 and progesterone do not affect the inhibitory activity of AG1478. AG1478 suppressed the proliferation of leiomyoma cells treated with E2 or progesterone separately and in combination. Nevertheless, the observation that no significant effect of E2 and progesterone on the proliferation of cultured myometrial cells could be demonstrated might be considered as a major limitation of this model. A possible explanation could have been the loss of the steroid receptors in the in vitro model (Severino et al., 1996), yet we found that our tissue culture samples were positive for these receptors (by WB analysis and immunohistochemistry; data not shown). Another possible explanation might be associated with the cell proliferation assay. We measured cell proliferation by the automated microculture methylene blue assay and it might be that another assay, such as thymidine incorporation, could have been more significant for this purpose. Finally, however, we believe that this resistance to steroids of our model demonstrates that in vivo, E2and progesterone act as promoters of leimyoma cell proliferation, but there are other paracrine and autocrine growth factors that locally mediate this proliferation. In the in vitro model, these growth factors might not always be activated by the same signals and, therefore, the proliferative effect of E2 and progesterone could not be demonstrated.
The EGFR gene is often amplified or overexpressed or both in diverse types of tumours. Usually, the overexpression of the EGFR is also accompanied by the autocrine or paracrine expression of its ligands, producing persistent enhanced stimulation of EGF-dependent pathways. Therefore, a successful potent inhibitor of the EGFR kinase has the chance of becoming a broad-spectrum inhibitor. To this point, two quinazoline derivatives of AG1478, AG1517/SU5271, are effective suppressors of growth of psoriatic keratinocytes (Powell et al., 1999; Ben-Bassat and Klein, 2000
). More recently, AG1478 has been selected for clinical development for glioma multiformis in combination with cisplatin (Nagane et al., 2001
).
AG1478 did not alter the expression of the apoptosis-related proteins Bcl2 (anti-apoptotic) and Bax (pro-apoptotic) in the leiomyoma and myometrium cultures, in accordance with the FACS analysis showing no detectable increase in the apoptotic cell fraction. Further, AG1478 had no effect on the expression of the Bcl2 and Bax proteins in leiomyoma and myometrium cells cultured with the ovarian steroids E2 or progesterone. WB analysis of the Bcl2 and Bax proteins in matched leiomyoma and myometrium cell cultures did not demonstrate consistent and/or a clearly defined differential expression level of these proteins between the two types of cells.
Previous reports concerning Bcl2 expression in leiomyomas demonstrated overexpression of this protein in leiomyomas compared with normal myometrium (Matsuo et al., 1997). A possible reason for this discrepancy could be the monoclonal origin of individual fibroids leading to differences between fibroids even from the same individual uterus. Matsuo et al. (1999)
suggested that since Bcl2 protein expression is upregulated by progesterone, but downregulated by E2, progesterone might participate in leiomyoma development through induction of Bcl2.
The molecular mechanism that initiates transformation of myometrium smooth muscle cells into leiomyomas during the reproductive years is still not completely understood (Myers et al., 2002; Lefebvre et al., 2003
). Ovarian steroids are important for leiomyoma growth, and GnRH analogue therapy is often used for their medical management. However, several in vitro studies have provided support for the direct action of GnRH by demonstrating changes in cell growth, cell cycle progression, apoptosis and expression of several growth factors, proteases and their inhibitors in the myometrium and leiomyoma cells, as well as in other steroid-sensitive cell types (Flake et al., 2003
; Lefebvre et al., 2003
). Recent in vitro studies provided additional evidence that a GnRH agonist (antide) and a progesterone antagonist (RU468) induce leiomyoma regression through an interactive mechanism that alters cell growth and suppresses transforming growth factor-
(a DNA synthesis stimulator) production. These changes are mediated in part through GnRH receptor-activated signal transduction (Chegini et al., 2002
; and references therein).
In summary, we have identified the AG1478 tyrphostin as a potent inhibitor of leiomyoma and myometrium cell cultures. It selectively inhibits autophosphorylation of EGFR and downstream signal tranduction events, including suppression of cell proliferation and cell cycle progression at micromolar concentrations. A number of tyrosine kinase inhibitors, including gefitinib-Iressa-ZD1839 (inhibitors of autophosphorylation of EGFR), are progressing through clinical development and are beginning to provide new treament options for a range of malignancies (Levitzki, 2001). Our results suggest that the specific EGFR inhibitor combined with selective GnRH agonists may be useful as a future therapeutic approach for medical treatment of uterine fibroids. Tyrphostins are the first signal transduction agents to be used in clinical practice.
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Acknowledgements |
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References |
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Submitted on September 3, 2003; accepted on May 11, 2004.
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