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*Compound via MeSH
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*Uterine Cancer
*Uterine Fibroids
The Journal of Clinical Endocrinology & Metabolism Vol. 87, No. 12 5610-5617
Copyright © 2002 by The Endocrine Society

Original Article

Tranilast Inhibits the Proliferation of Uterine Leiomyoma Cells in Vitro through G1 Arrest Associated with the Induction of p21waf1 and p53

Hiroaki Shime, Masatoshi Kariya, Ayaka Orii, Chika Momma, Takanobu Kanamori, Ken Fukuhara, Takashi Kusakari, Yuko Tsuruta, Kenji Takakura, Toshio Nikaido and Shingo Fujii

Department of Gynecology and Obstetrics (H.S., M.K., A.O., C.M., T.Ka., K.F., T.Ku., Y.T., K.T., S.F.), Kyoto University Graduate School of Medicine, Kyoto 606-8507; and Department of Obstetrics and Gynecology (T.N.), Shinshu University, School of Medicine, Matsumoto 390-8621, Japan

Address all correspondence and requests for reprints to: Shingo Fujii, M.D., Department of Gynecology and Obstetrics, Kyoto University Graduate School of Medicine, 54 Shyogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: sfu{at}kuhp.kyoto-u.ac.jp.

Abstract

Uterine leiomyoma is a mesenchymal tumor composed of smooth muscle cells with fibrous tissues and many mast cells. Tranilast is known to suppress fibrosis or to work as a mast cell stabilizer and is reported to inhibit proliferation of vascular smooth muscle cells. In this study, we examined the effects of tranilast on cultured human leiomyoma cells in vitro to evaluate whether this agent has the potential to inhibit the growth of uterine leiomyomas. Tranilast inhibited the proliferation of cultured leiomyoma cells in a dose-dependent manner without any cytotoxic effect or induction of apoptosis. In association with the inhibitory effect, tranilast induced the cyclin-dependent kinase (CDK) inhibitor p21waf1 and tumor suppressor gene p53 and decreased CDK2 activity. These results suggest that tranilast arrests the proliferation of uterine leiomyoma cells at the G0/G1 phase, through the suppression of CDK2 activity via an induction of p21waf1 and p53. Tranilast was concluded to be a potent agent to inhibit proliferative activity of uterine leiomyoma cells.

UTERINE LEIOMYOMAS ARE the most common pelvic tumors in women, with a reported incidence of 20–25% (1, 2, 3, 4). Hysterectomy or myomectomy has been the most common surgical treatments for uterine leiomyomas. Recently, less invasive surgical methods of treatment, hysteroscopic removal of leiomyomas or uterine arterial embolization (5, 6, 7, 8, 9), have been introduced as effective treatments for uterine leiomyomas. Leiomyoma can also be treated nonsurgically with GnRHa, which induces a reversible hypogonadotropic hypogonadal environment. This agent has been introduced into the management of uterine leiomyomas because this tumor is believed to be dependent on ovarian steroids (10, 11). Although GnRHa is effective for reducing uterine leiomyomas, it is not suitable for long-term use because of the associated increased loss of bone mineral density (12). Therefore, there is a limit to the volume reduction of leiomyoma nodules by GnRHa; and once treatment stops, the nodules soon return to the size they were before the treatment. Thus, it is necessary to introduce a new avenue for the medical treatment of uterine leiomyomas.

Uterine leiomyomas have been reported to contain a large amount of extracellular matrix, such as collagen type I, collagen type III, and fibronectin (13, 14), suggesting that the process of fibrosis may be partly responsible for the tumor enlargement. In addition, mast cells exist in uterine leiomyomas as well as in the normal myometrium (15, 16, 17). In ordinary leiomyomas, the number of mast cells is reported to be correlated with differences in cellularity and the collagen matrix of the tumor (17), and it is suggested that mast cells may play some role in the pathophysiological events of uterine leiomyomas.

Tranilast, N-(3',4'-dimethoxycinnamonyl) anthranilic acid (N-5'), was developed in Japan as an antiallergic drug, and it has been used clinically for patients with bronchial asthma, allergic rhinitis, and atopic dermatitis. Recently, the agent was also discovered to be useful in the treatment of keroids and hypertrophic scars. Tranilast inhibits the release of chemical mediators from mast cells (18, 19, 20) as well as inhibiting collagen accumulation in granulation tissue (21). Furthermore, tranilast has recently been reported to inhibit the proliferation and function of vascular smooth muscle cells in vitro (22, 23, 24, 25, 26, 27, 28, 29) and to suppress the proliferative reaction of vascular smooth muscle cells in vivo (30, 31).

Given that uterine leiomyomas are composed of a large amount of collagen with an increased number of mast cells, there is good reason to investigate tranilast as a potential new agent for the treatment of uterine leiomyomas. In this study, we examined the effects of tranilast on cultured human leiomyoma cells in vitro to test whether this agent has the potential to inhibit the growth of uterine leiomyoma cells.

Patients and Methods

Patients

Leiomyoma and myometrial tissues were obtained under written consent from premenopausal women who were not receiving any type of hormonal or drug therapy at the time of hysterectomy. Collection of tissues was carried out in line with Kyoto University Hospital’s policy for the use of discarded human tissue.

Chemicals and cell culture

Tranilast, N-(3',4'-dimethoxycinnamonyl) anthranilic acid (N-5'), was synthesized at Discovery Research Laboratories, Kissei Pharmaceutical Co. Ltd. (Nagano, Japan) and dissolved in 1% NaHCO3 at various concentrations (10 mM, 1 mM, and 100 µM). The final concentration of NaHCO3 in the medium did not exceed 0.03%.

The leiomyoma and myometrial tissues obtained from removed uteri were rinsed in DMEM (Nikken Biomedical Laboratory, Kyoto, Japan) with 10% fetal calf serum (FCS; Biological Industries, Grand Laboratories, Grand Island, NY) and 2% antibiotic-antimycotic solution (Life Technologies, Inc. Laboratories, Grand Island, NY) and minced into fine pieces. The minced tissues were aspirated and placed into 50-ml tubes containing DMEM and 0.2% collagenase (Wako Pure Chemical Industries Ltd., Osaka, Japan) in which they were kept for 4 h at 37 C with continuous mixing until cell suspension was evident. The cell suspension was separated from the undigested fragments and then centrifuged at 100 x g for 5 min. The supernatant was discarded, and the cell pellet was suspended in DMEM with 10% FCS and 2% antibiotic-antimycotic solution. The cell solution was placed on a 100-mm collagen (type I)–coated dish (IWAKI, Chiba, Japan), and incubation was carried out at 37 C in a humidified 95% air-5% CO2 atmosphere. More than 98% of the cultured cells were confirmed to be smooth muscle cells, by immunostaining for {alpha}–smooth muscle actin (DAKO Corp., Glostrup, Denmark) (32). Cells from passage 2 or 3 of the culture were used for the experiments.

Growth-inhibition assay

Leiomyoma or myometrial cells were plated at a density of 2000 cells per well onto 96-well flat–bottomed tissue-culture plates (IWAKI). The cells were incubated at 37 C in DMEM with 10% FBS and 2% antibiotic-antimycotic solution in a humidified 95% air-5% CO2 atmosphere. After 24 h, fresh medium containing various concentrations of tranilast was added to the cells. Concentrations of 100 and 300 µM tranilast were selected on the basis of preliminary experiments (data not shown). On d 1, 3, and 5 after the addition of tranilast, a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed, using the Premix WST-1 Cell Proliferation Assay System (TAKARA, Tokyo, Japan). At designated intervals during the incubation, 10 µl of the premix WST-1 reagent was added to each well, and the plates were then reincubated under the prescribed conditions for an additional 4 h. The absorbance of the wells was measured using an ELISA microplate reader (Molecular Devices, Menlo Park, CA) at wavelengths of 490 nm ({lambda}1) and 630 nm ({lambda}2). Leiomyoma and myometrial cells, cultured in the absence or presence of tranilast, were observed with phase contrast microscopy on d 5 after the addition of tranilast.

Cytotoxic assay and apoptosis assay

Leiomyoma or myometrial cells were cultured to confluence in 6-well flat–bottomed tissue-culture plates with lids (IWAKI) and then incubated in the absence or presence of tranilast (100 and 300 µM). After 72 h incubation, lactate dehydrogenase (LDH) release into the medium was determined by means of a performed LDH-cytotoxic test (Wako Pure Chemical Industries Ltd.). In addition, an apoptosis assay was performed using Cell Death Detection ELISAPLUS (Roche Diagnostics Corporation, Indianapolis, IN).

Flow cytometry

Leiomyoma cells were plated, at a density of 48 x 104 cells, onto a 100-mm tissue culture dish (IWAKI). After 48 h, medium containing 300 µM tranilast was added to the cells, which were then incubated for a further 24 h. The cells were collected after trypsinization, washed with PBS, fixed in 70% ethanol, and stored at -20 C. The cells were resuspended in a DNA-staining solution containing propidium iodine and ribonuclease (Cycle TEST PLUS DNA Reagent Kit; Becton Dickinson and Co., Bedford, MA). Finally, the cell cycle distribution of each sample was determined by means of an FACS Caliber flow cytometer (Becton Dickinson and Co.) and analyzed with ModFit LT (Becton Dickinson and Co.). For some experiments, leiomyoma cells were plated at a density of 48 x 104 cells onto a 100-mm tissue culture dish (IWAKI). After 48 h, cell growth was arrested by incubation for 48 h in serum-free DMEM. The quiescent cells were stimulated by the addition of DMEM with 10% FCS for a further 24 h, in the absence or presence of tranilast (300 µM). The cell cycle distribution of each sample was then determined as described above.

Western blotting

Leiomyoma cells were plated, at a density of 48 x 104 cells, onto a 100-mm tissue culture dish (IWAKI). After 48 h, medium containing 300 µM tranilast was added to the cells, which were then incubated for a further 6, 12, or 24 h. The cells were collected after treatment with 0.25% trypsin-EDTA solution and washed with PBS. The collected cells were then lysed in 1 ml cell lysis buffer, which contained 50 mM Tris-HCl (pH 8.0), 0.25 M NaCl, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride (Sigma, St. Louis, MO), 1 mg/ml aprotinin (Roche Molecular Biochemicals, Mannheim, Germany), 1 mg/ml leupeptin (Roche Molecular Biochemicals), and 20 mg/ml N-tosyl-L-phenylalanyl chloromethyl ketone (Roche Molecular Biochemicals). The lysates were centrifuged at 13,000 x g for 10 min at 4 C, and the supernatants were stored at -80 C. Extracts equivalent to 35 µg total protein were separated by SDS-PAGE (10 or 12% acrylamide gel), followed by the equilibration of the gel in transfer buffer [20% methanol, 25 mM Tris, 192 mM glycine (pH 8.0)] for 60 min. The proteins were then transferred onto supported nitrocellulose membranes (Life Technologies, Inc., Gaithersburg, MD) by applying 2400 V-min, using a plate-electrode apparatus (Idea Scientific Co., Minneapolis, MN). The membranes were blocked in TBST [0.2 M NaCl, 10 mM Tris (pH 7.4), 0.2% Tween 20], containing 5% or 7% nonfat dry milk and 0.02% NaN3, for 1 h. They were then incubated with mouse monoclonal antibodies against cyclin-dependent kinase (CDK)2; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), p21waf1 (PharMingen, San Diego, CA), p53 (DAKO Corp.), proliferating cell nuclear antigen (PCNA; DAKO Corp.), or ß-actin (Bio Markor, Rehovot, Israel) diluted approximately 1:100–500 in TBST containing 2% nonfat dry milk, overnight at 4 C. The membranes were then incubated with antimouse Ig diluted 1:1,000 in TBST containing 2% nonfat dry milk, for 1 h at room temperature. The membranes were washed several times with TBST between steps. Bound antibody was detected by means of an enhanced chemiluminescence system (Amersham International, Aylesbury, UK) and exposed to x-ray films. The intensity of the signals on Western blot analyses was quantified on a scanning densitometer, and the intensity generated under tranilast treatment is expressed as a percentage of the corresponding untreated control value or as a percentage of the control value before tranilast treatment in the time course study.

Protein kinase assay

Leiomyoma cells were plated, at a density of 48 x 104 cells, onto a 100-mm tissue culture dish (IWAKI). After 48 h, medium containing 300 µM tranilast was added to the cells, which were then incubated for a further 24 h. The cells were collected after treatment with 0.25% trypsin-EDTA solution and washed with PBS. The collected cells were then lysed in 1 ml cell lysis buffer (see Western blotting for composition). The lysates were centrifuged at 13,000 x g for 10 min at 4 C, and the supernatants were stored at -80 C. Extracts equivalent to 100 µg total protein were incubated with 3 µg antihuman CDK2 rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.) for 2 h on ice and then subsequently incubated with 20 µg G-Sepharose (Pharmacia Corporation, Peapack, NJ) for 1 h at 4 C. Immunoprecipitates were washed four times with lysate buffer and suspended with kinase buffer [composition, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM dithiothreitol] containing 0.5 µM [{gamma}-32P] ATP (NEN Life Science Products, Boston, MA) and 1 µg histone H1 (Roche Molecular Biochemicals). The suspension was incubated for 15 min at 30 C and then boiled for 5 min. The sample was separated by SDS-PAGE (8–16% acrylamide gel). Phosphorylated proteins were visualized using a bioimage analyzer (BAS 2500; Fuji Photo Film Co., Ltd., Tokyo, Japan). The level of each phosphorylated protein was quantified on a scanning densitometer, and the intensity generated under tranilast treatment is expressed as a percentage of the corresponding untreated control value.

Statistical analysis

The experiments were repeated at least three times. The significance of differences was assessed by the Kruskal-Wallis rank test or by the Mann-Whitney U test. Differences were considered to be significant when P < 0.05 and to be not significant when P >= 0.05.

Results

Effects of tranilast on proliferation of cultured leiomyoma and myometrial smooth muscle cells

Tranilast (100-300 µM) inhibited the proliferation of cultured leiomyoma and myometrial cells in a dose-dependent manner (Fig. 1Go, A and B), whereas tranilast at lower concentrations, i.e. 0.1, 1.0, 10, and 30 µM, showed no such inhibition in our preliminary experiments (data not shown). After 5 d of incubation, compared with the number of cells in control medium, the number of cells in 100 µM tranilast was 75% (P < 0.05); and in 300 µM tranilast, it was 50%. There was no significant difference between leiomyoma cells and myometrial smooth muscle cells in terms of the inhibition of cell proliferation. By observation with phase contrast microscopy, on culture d 5, there was no increase in the number of the dead cells floating in the leiomyoma cell medium, irrespective of the presence or absence of tranilast (Fig. 1Go, C and D).



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  		Figure 1. Effect of tranilast on proliferation of cultured uterine leiomyoma cells (A) and myometrial cells (B). The cells from passages 2 or 3 were cultured in the presence or absence of tranilast. On d 0, 2, and 4 after the addition of tranilast, culture media were changed with the supplement of tranilast, a fixed dose for each well. On d 0, 1, 3, and 5, a modified MTT assay was performed. Each symbol indicates the mean of five experiments. Error bars, SEM. *, P < 0.05; **, P < 0.01 vs. tranilast (0 µM) (as a control). C and D, Images obtained with phase contrast microscopy of leiomyoma cells on d 5 of culture in the presence or absence of tranilast (0 µM, C; 300 µM, D). Magnification, x40. O.D., Optical density.

 
To evaluate the toxicity of tranilast in leiomyoma and myometrial cells, the level of LDH in the supernatants of leiomyoma and myometrial cells cultured for 3 d, in the absence or presence of tranilast, was measured. The results of the LDH assay showed no significant increase in the level of LDH in the supernatant of either leiomyoma (Fig. 2AGo) or myometrial cells cultured for 3 d with tranilast. To examine whether tranilast induces apoptosis, an apoptosis assay was performed. Apoptosis was negligible in the culture, both in the presence and absence of tranilast (Fig. 2BGo).



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  		Figure 2. A, Effect of tranilast on the levels of LDH released in the supernatants of leiomyoma cells cultured for 3 d in the absence or presence of tranilast. Medium with 10% FCS, LDH concentration in DMEM with 10% FCS (no leiomyoma cells). Each column indicates the mean of three experiments. Error bar, SEM. B, Effect of tranilast on the apoptosis induced in leiomyoma cells cultured for 3 d in the absence or presence of tranilast. Each column indicates the mean of three experiments. Error bar, SEM. N.S., Not significant, P >= 0.05. O.D., Optical density.

 
Effect of tranilast on cell cycle distribution of cultured leiomyoma cells

To elucidate the mechanism by which tranilast inhibits the proliferation of cultured leiomyoma cells, we examined the effect of tranilast on the cell cycle distribution of cultured leiomyoma cells in the growing phase. Representative results of cell cycle analysis performed on leiomyoma cells are shown in Fig. 3Go. A significant increase in the cell population at the G0/G1 phase of the cell cycle (70.0% vs. 85.1%, P < 0.05) and a significant decrease in the cell population at the S phase of the cell cycle (13.4% vs. 7.0%, P < 0.05) were observed after the addition of 300 µM tranilast to the growing phase cells (Fig. 3Go, A and B).



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  		Figure 3. A and B, Effect of tranilast on cell cycle distribution of cultured leiomyoma cells in the growing phase. Cell cycle analysis was performed with flow cytometry. Each panel shows representative data of three experiments. A, Tranilast 0 µM (as a control); B, tranilast 300 µM; C–E, effect of tranilast on the cell cycle progression of cultured leiomyoma cells. First, the proliferation of leiomyoma cells was arrested in the G0/G1 phase of the cell cycle in serum-free culture (C); and subsequently, the quiescent cells were cultured in serum-containing medium with or without tranilast (E and D, respectively). Each panel shows representative data.

 
In the cells rendered quiescent under serum-free conditions, the populations at the G0/G1 and S phases were 93.3 and 2.6%, respectively, of the total population. After stimulation for 24 h with serum alone, the corresponding values were 79.5 and 19.2%; whereas in cells stimulated for 24 h with serum and 300 µM tranilast, the corresponding values were 88.9 and 6.7% (P < 0.05) (Fig. 3Go, C–E).

Western blot analysis

The effect of tranilast was examined on the expressions of cell cycle-related proteins, such as CDK2, p53, p21waf1, and PCNA, in leiomyoma cells. Western blot analysis showed that the levels of p53 and p21waf1 protein were significantly increased in 300 µM tranilast-treated cells (P < 0.05) (Fig. 4Go, A and B). In contrast, the levels of CDK2 and PCNA protein were significantly decreased in 300 µM tranilast-treated cells (P < 0.05) (Fig. 4Go, A and B).



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  		Figure 4. A, Effect of tranilast on the expression of p53, p21waf1, CDK2, and PCNA proteins as visualized using Western blotting. B, The relative expressions of p53, p21waf1, CDK2, and PCNA proteins were estimated on a scanning densitometer. The intensity generated under tranilast (300 µM) treatment is expressed as a percentage of the corresponding untreated (0 µM) control values. Each column indicates the mean of three experiments. Error bar, SEM. *, P < 0.05. C, Time course study of the expression of p53 and p21waf1 in leiomyoma cells treated with tranilast. D, The relative expressions of p53and p21waf1 proteins were estimated on a scanning densitometer. The intensity generated under tranilast (300 µM) treatment is expressed as a percentage of the control value before tranilast treatment (0 h). Each column indicates the mean of three experiments. Error bar, SEM. *, P < 0.05.

 
The time course study of the increased expression of p21waf1 and p53 protein in 300 µM tranilast showed that p21waf1 expression was increased by 6 h, reaching its highest level at 12 h, and had decreased by 24 h, whereas p53 expression showed its highest level by 6 h, gradually decreasing thereafter (Fig. 4Go, C and D).

Effect of tranilast on CDK2 activity

To assess whether up-regulation of p21waf1 is essential for the arrest of leiomyoma cells in the G1 phase, the kinase activity of CDK2 was measured. The CDK2 activity was significantly decreased in the cells cultured with 300 µM tranilast for 24 h, compared with cells cultured in the absence of tranilast (P < 0.05) (Fig. 5Go, A and B).



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  		Figure 5. A, Effect of tranilast on the CDK2 activity of leiomyoma cells. The kinase activity of CDK2 was evaluated by the level of phosphorylation of histone H1 with RI-labeled ATP. B, The relative expression of CDK2 activity was estimated on a scanning densitometer. The intensity generated under tranilast (300 µM) treatment is expressed as a percentage of the corresponding untreated (0 µM) control values. Each column indicates the mean of three experiments. Error bar, SEM. *, P < 0.05.

 
Discussion

In the present study, tranilast showed an inhibitory effect on the proliferation of cultured leiomyoma cells in a dose-dependent manner. The toxicity of tranilast, evaluated by measurement of the levels of LDH and apoptosis, was negligible, compared with the control group. In addition, phase contrast microscopic observations showed no apparent morphological changes in response to the addition of tranilast. A similar inhibitory effect, by tranilast, with negligible toxicity was also observed in cultured smooth muscle cells from the normal myometrium. The degree of inhibition in cultured smooth muscle cells was almost identical to that in cultured leiomyoma cells; therefore, in this paper, because the subject of this study is the effect of tranilast on uterine leiomyoma, the following discussion is confined to the data on uterine leiomyoma cells.

Cell cycle analysis, using flow cytometry, indicated that tranilast arrested the cell cycle of leiomyoma cells in the G1 phase in growing phase cells and inhibited the cell cycle progression from the G0/G1 to the S phase in G0-arrested cells. These results suggest that the inhibitory effect of tranilast is exerted at some point in the G1 phase.

In the cell cycle of mammalian cells, CDKs play a central role (33) and promote G1/S transition by phosphorylation of the retinoblastoma protein. The intrinsic CDK inhibitor, such as p21waf1, binds to the CDK-cyclin complex and inhibits its kinase activity. p21waf1 induces G1 arrest via the suppression of CDK2 activity by binding to the cyclin E-CDK2 complexes (34). Tranilast has been reported to inhibit the proliferation of human coronary smooth muscle cells via the induction of p21waf1 and p53 (29). In this study, leiomyoma cells, treated with tranilast, showed increased expressions of p53 and p21waf1 and decreased expression of CDK2, compared with cells cultured in the absence of tranilast. CDK2 activity was also decreased in leiomyoma cells treated with tranilast. These results suggest that tranilast induces G1 arrest in leiomyoma cells via the induction of p53 and p21waf1, with the suppression of CDK2 activity, as well as the previous report on human coronary smooth muscle cells. However, we do not have evidence showing whether smooth muscle cells lacking p53 are insensitive to tranilast or not, and there still may be other indirect mechanisms (such as the interference with cellular metabolism) that alter the growth rate of leiomyoma cells. Further studies are necessary to clarify whether or not the induction of p53 and p21waf1 is a primary mechanism underlying the inhibitory effect of tranilast.

Occurrence of p53 induction has been reported after exposure to ionizing radiation or treatment with DNA-damaging agents; p53 is a well-known cellular transcription factor that stimulates the expression of some genes, including p21waf1 (33). Because tranilast increased the level of both p21waf1 and p53, the induction of p21waf1 may occur through a p53-dependent pathway. Western blot analysis showed that, over the time course of the experiment, tranilast treatment increased p21waf1 and p53 protein levels. The p21waf1 protein increased by 6 h, with the highest level occurring at 12 h, and decreased by 24 h, whereas the p53 protein showed the highest level by 6 h, gradually decreasing thereafter. The time lag between the highest levels of p53 and p21waf1 may suggest that p21waf1 is induced through a p53-dependent pathway. However, theoretically, it would take more time for production of the p21waf1 protein to occur through the transcriptional process of p53. Therefore, induction of the p21waf1 protein could be regulated not only by the transcriptional level but also by the posttranscriptional level of p21waf1, including catabolism of the ubiquitin-proteasome degradation system (35). We know that p53 is usually induced when cell cycle promotion for a cell is not suitable because of DNA damage, then G1 arrest through the induction of p21waf1 or apoptosis can occur. In this experiment, the results of the LDH and apoptosis assays gave no indication of DNA damage in leiomyoma cells treated with tranilast. It has been reported that the p53 protein is also regulated by the ubiquitin-proteasome degradation system. Therefore, tranilast could work by suppressing the ubiquitin-proteasome degradation system of p53 and p21waf1.

After oral administration of the usual therapeutic dose of 600 mg/d tranilast, the plasma concentration has been reported to reach 30–300 µM in vivo (29). The suppressive effect of 300 µM tranilast on cultured leiomyoma cell proliferation indicates a possible clinical use for tranilast in the treatment of uterine leiomyoma. Our experimental data, that tranilast inhibits the proliferation of leiomyoma cells by induction of G1 arrest without induction of apoptosis, suggest that tranilast could be used for the prophylaxis of the growth of uterine leiomyomas. In the case of vascular smooth muscle cells, tranilast has proved useful for the prophylaxis of restenosis by the prevention of vascular smooth muscle cell proliferation after percutaneous transluminal coronary angioplasty in randomized clinical trials (36), and this agent is actually used for the prophylaxis in a clinical situation. Therefore, there is good reason to investigate the possibility of extending the clinical use of tranilast to the treatment of uterine leiomyoma, with a view to preserving the reduced size of leiomyoma nodules after treatment with GnRH analogs.

Given other effects observed with tranilast, pathophysiological benefits other than suppression of proliferation could well be seen in leiomyoma cells. Similar to fibrotic diseases, leiomyomas have been reported to contain large amounts of extracellular matrix, which consists primarily of interstitial collagens, proteoglycans, and fibronectin (13, 14, 37). TGF-ß is important in a variety of fibrotic diseases. Alterations in the TGF-ß ligand-receptor system are implicated in the development of several fibrotic diseases such as the fibrosis, which occurs in diabetic nephropathy (38), liver fibrosis, and pulmonary fibrosis (39). Leiomyomas show similar alterations in this system, including: overexpression of TGF ßs (40, 41, 42, 43), the loss of antiproliferative response of leiomyoma cells to TGF-ß1 (40, 41, 44), and reduced expression of TGF-ß type II receptors (41, 43). It can therefore reasonably be concluded that the fibrotic process is partly responsible for the enlargement of leiomyoma nodules. For this reason, antifibrotic agents are being investigated as a possible new avenue for the treatment of leiomyomas (45). One such compound is pirfenidone. Pirfenidone is currently undergoing phase II clinical trials for the treatment of pulmonary fibrosis (46). Pirfenidone has also been shown to inhibit proliferation of uterine myometrial smooth muscle and leiomyoma cells, and it significantly suppresses steady-state mRNA levels of collagen type I and III in vitro (47). Pirfenidone may therefore prove to be an effective nonsteroidal therapy for treatment of uterine leiomyomas (47). Tranilast has been found to inhibit the proliferation of fibroblasts in vitro, to selectively suppress collagen deposition in vivo (21), and to inhibit TGF-ß production from monocytes and macrophages (48).

Leiomyomas have also been reported to contain considerable numbers of mast cells (15). TGF-ß in mast cells may also be associated with the fibrotic process of uterine leiomyoma. Through the effect of stabilization of mast cells, tranilast may also work as an antifibrotic agent. Thus, tranilast could be expected to be useful in two respects, for its antifibrotic as well as its antiproliferative effect in the treatment of leiomyomas.

In conclusion, tranilast is suggested to become a candidate of new therapeutic agents for the treatment of uterine leiomyomas, by the antiproliferative effect on leiomyoma cells in vitro and by a reported antifibrotic effect of this agent.

Acknowledgments

We thank Dr. Hiroshi Kusama for expert technical assistance.

Footnotes

This work was supported by Grants-in-Aid 13307047 and 13877272 for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan.

Abbreviations: CDK, Cyclin-dependent kinase; FCS, fetal calf serum;LDH, lactate dehydrogenase; PCNA, proliferating cell nuclear antigen.

Received March 21, 2002.

Accepted August 28, 2002.

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