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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 1 219-223
Copyright © 1998 by The Endocrine Society

Original Studies

Pirfenidone: A Novel Pharmacological Agent That Inhibits Leiomyoma Cell Proliferation and Collagen Production

Byung-Seok Lee, Solomon B. Margolin and Romana A. Nowak

Department of Obstetrics, Gynecology, and Reproductive Biology (B.-S.L., R.A.N.), Harvard Medical School, and Brigham and Women’s Hospital, Boston, Massachusetts 02115; and Marnac, Inc. (S.B.M.), Dallas, Texas 75225

Address all correspondence and requests for reprints to: Dr. Romana A. Nowak, Laboratory of Human Reproduction and Reproductive Biology, Brigham and Women’s Hospital, 221 Longwood Avenue, Boston, Massachusetts 02115.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
There are currently no effective, long-term drug therapies for the treatment of leiomyomas. Pirfenidone (Marnac, Inc.) is an antifibrotic agent that is being tested for use in patients with pulmonary fibrosis. Because leiomyomas are characterized also by increased cell proliferation and tissue fibrosis, we examined the effects of pirfenidone on cell proliferation and collagen expression in cultured myometrial and leiomyoma smooth muscle cells. Effects of pirfenidone on proliferation of myometrial and leiomyoma cells were measured using tritiated thymidine incorporation assays and changes in actual cell numbers. Possible cytotoxic effects were examined using lactate dehydrogenase assays and trypan blue exclusion. Effects on collagen type I and type III production were assessed by Northern blotting. Doses of pirfenidone tested were: 0, 0.01, 0.1, 0.3, and 1.0 mg/mL. Serum-stimulated increases in DNA synthesis and cell proliferation by myometrial and leiomyoma cells were significantly inhibited in a dose-dependent manner by pirfenidone. Densitometric analysis of Northern blots showed significantly decreased expression of collagen type I and type III messenger RNAs in both leiomyoma and myometrial cells. Lactate dehydrogenase assays and trypan blue exclusion measurements showed no cytotoxic effect of pirfenidone at concentrations that inhibited cell proliferation and collagen production. Pirfenidone is an effective inhibitor of myometrial and leiomyoma cell proliferation in vitro and reduces the messenger RNA levels of collagen types I and III in a dose-dependent manner. This compound may prove to be an effective nonsteroidal therapy for treatment of uterine leiomyomas.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
UTERINE leiomyomas (or fibroids) are the most common pelvic tumors in women, with a reported incidence of 20–25% (1). The most common symptoms associated with these benign smooth muscle cell (SMC) tumors are abnormal uterine bleeding, pelvic pressure or pain, infertility, and increased urinary frequency (2, 3). The initiating factor(s) for leiomyomas are not known; however, there is a large body of evidence showing that estrogen and progesterone are important factors for tumor growth (2, 4, 5, 6). Recent studies have suggested that the effects of these steroid hormones on tumor growth are mediated through the local production of growth factors that exert autocrine or paracrine effects on surrounding cells (7, 8).

GnRH analogs, which reduce serum estradiol and progesterone concentrations to those seen in postmenopausal women, have been used for a number of years as a medical therapy for treatment of leiomyomas (9). However, these compounds are not suitable for long-term treatment, because of detrimental side effects such as increased loss in bone density (9). Thus, there is a need to explore new pharmaceutical agents for the treatment of uterine leiomyomas.

Pirfenidone is an antifibrotic agent that is being investigated for use in patients with pulmonary fibrosis. It is an investigational drug whose structure is 5-methyl-1-phenyl-2(1H)-pyridone (Fig. 1Go). Pirfenidone has been shown to produce antifibrotic effects in a variety of animal models (10, 11) and to inhibit fibroblast proliferation in vitro in response to a number of growth factors (12).



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  		Figure 1. Chemical structure of pirgenidone (5-methyl-1-phenyl-2(1H)-pyridone).

 
The goals of this study were to investigate the effects of pirfenidone on cell proliferation and collagen gene expression in cultured myometrial and leiomyoma cells. We also determined whether this compound had cytotoxic effects on these cells.


   Subjects and Methods
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 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Patients

Leiomyoma and myometrial tissue were obtained from 12 premenopausal women with symptomatic uterine fibroids at the time of hysterectomy and who were not receiving any type of hormonal or drug therapy. Collection of tissues was obtained under a consent for use of discarded human tissue, in accordance with the Brigham and Women’s Hospital policy. The stage of the menstrual cycle for each patient was determined by the pathologist, using endometrial dating. Seven of the patients were in the secretory phase at the time of surgery, 3 were in the proliferative phase, and 2 in the menstrual phase.

Cell culture

Primary cultures of myometrial and leiomyoma SMCs were established as described previously (13). Cultures were determined to be pure SMC cultures (>98%) by immunostaining for desmin and smooth muscle {alpha}-actin, which are markers for SMCs (13). Cells were used in experiments at passages 1 or 2.

Experimental design

Exp 1. For tritiated thymidine incorporation assays, leiomyoma and myometrial SMCs were cultured in 96-well plates (15,000 cells/well) for 48 h in medium with 10% serum. Cells were then made quiescent by culturing in medium with 0.5% serum for 48 h. These quiescent cells were washed and then received medium with 10% serum plus the various doses of pirfenidone (0, 0.01, 0.1, 0.3, and 1.0 mg/mL). After 18 h, the cells received 0.2 uCi/well [3H]-thymidine (New England Nuclear, Boston, MA), and the incubation was continued for a further 6 h. Cells were then harvested and counted, to measure the rate of incorporated [3H]-thymidine. Four experiments, using cells from four different patients, were performed with 6 wells/treatment group/experiment.

Exp 2. In a second set of experiments, myometrial and leiomyoma SMCs were plated in 100-mm dishes in medium with 10% serum and cultured until they reached 80–90% confluence. Cells were washed in serum-free medium and then placed in serum-free medium containing the various concentrations of pirfenidone for a period of 3 days. Medium was collected for assay of lactate dehydrogenase (LDH) levels, and cells were harvested and processed for Northern blotting analysis. The LDH assay was used to measure cell toxicity effects of the various doses of pirfenidone and was performed using a colorimetric determination kit from Sigma (St. Louis, MO). Four experiments, using cells from four different patients, were performed with 2 dishes/treatment group/experiment.

Exp 3. In the third set of experiments, leiomyoma and myometrial cells were plated in 100-mm dishes (100,000/dish) and allowed to attach overnight in medium with 10% serum. The following day, all cells received fresh medium with 10% serum containing various concentrations of pirfenidone (0, 0.01, 0.1, 0.3, and 1.0 mg/mL) for a period of 7 days. Medium was changed, with addition of fresh treatments, on days 3 and 5. On day 7, cells were harvested and counted and viability assessed using the trypan blue exclusion stain. Four experiments, using cells from four different patients, were performed with 2 dishes/treatment group/experiment.

Northern blotting analysis

Total RNA was extracted from culture wells and processed for Northern blot analysis using methods described previously (14). Blots were probed with a human collagen type I, {alpha} 1 chain complementary DNA (cDNA) (ATCC, Rockville, MD), a human collagen type III, {alpha} 1 chain cDNA (ATCC), and a human ribosomal phosphoprotein cDNA (gift of Dr. Dale Goad, Harvard School of Public Health) using methods established in earlier studies (14, 15). Filters were autoradiographed with an intensifying screen for 1–2 days at -70 C. Differences in sample loading were corrected by normalization to ribosomal phosphoprotein. Autoradiographs were analyzed on a scanning densitometer (GS-700, Biorad, Hercules, CA) to quantitate the levels of transcripts for each sample.

Statistics

Statistical analysis was carried out using two-way ANOVA. We used contrasts to perform pair-wise comparison after the ANOVA procedure. P < 0.05 was considered statistically significant. No significant differences in response to pirfenidone were noted between cells obtained at different stages of the menstrual cycle.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
The results from the first set of experiments showed that pirfenidone caused a dose-dependent inhibition of serum-stimulated DNA synthesis for both normal myometrial and leiomyoma SMCs after 24 h of treatment (Fig. 2Go). Pirfenidone at 0.01, 0.1, 0.3, and 1 mg/mL inhibited the stimulatory effect of 10% serum to 64 ± 21% (P < 0.05), 61 ± 21% (P < 0.005), 33 ± 15% (P < 0.001), and 21 ± 22% (P < 0.001) in myometrial cells, respectively, and 81 ± 10% (P < 0.05), 73 ± 15% (P < 0.05), 43 ± 4.5% (P < 0.001), and 19 ± 10% (P < 0.001) in leiomyoma cells, respectively. The inhibitory effect of pirfenidone on DNA synthesis does not seem to be caused by a toxic effect on the cells, because the results of the LDH assay showed no increases in LDH levels in conditioned medium of treated cells at any of the concentrations of pirfenidone (Table 1Go).



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  		Figure 2. Effect of pirfenidone on myometrial and leiomyoma cell proliferation. Concentrations of pirfenidone tested were 0, 0.01, 0.1, 0.3, and 1.0 mg/mL. The effect of the various doses of pirfenidone on thymidine incorporation is expressed as a percent of control (0 mg/mL pirfenidone). The serum stimulation of DNA synthesis was significantly inhibited in a dose dependent manner in both myometrial (P < 0.001) and leiomyoma cells (P < 0.005). Each bar represents the mean ± SD of 24 wells. Bars bearing different letters (a vs. b) are significantly different from one another.

 

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  		Table 1. The effect of pirfenidone treatment on lactate dehydrogenase (LDH) production by myometrial and leiomyoma cells. Each value represents the mean ± SD of three separate experiments. No significant effects of pirfenidone on LDH levels was measured.

 
The results of the Northern blot analysis for collagen type I messenger RNA (mRNA) are shown in Fig. 3Go. Both 4.8- and 5.8-Kb transcripts were detected in every sample. The results showed that mRNA levels of collagen type I were decreased in a dose-dependent manner for both myometrial and leiomyoma cells. Pirfenidone at 0.01, 0.1, 0.3, and 1.0 mg/mL reduced collagen type I mRNA levels to 0.82 ± 0.11 arbitrary densitometric units (ADU) (P < 0.05), 0.66 ± 0.16 ADU (P < 0.005), 0.56 ± 0.21 ADU (P < 0.005), and 0.38 ± 0.14 ADU (P < 0.005), respectively, in myometrial cells and to 0.92 ± 0.1 ADU (P = 0.08), 0.67 ± 0.16 ADU (P < 0.05), 0.6 ± 0.15 ADU (P < 0.005), and 0.38 ± 0.2 ADU (P < 0.005), respectively, for leiomyoma cells.



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  		Figure 3. Effect of pirfenidone on collagen type I (alpha 1) messenger RNA expression in myometrial and leiomyoma cells. Lanes 1,6: 0 mg/mL pirfenidone (P); Lanes 2,7: 0.01 mg/mL P; Lanes, 3,8: 0.1 mg/mL P; Lanes 4,9: 0.3 mg/mL P; and Lanes 5,10: 1.0 mg/mL P. Two mRNA transcripts (4.8 and 5.8 kb) were detected for collagen type I. Differences in sample loading were corrected by normalization to ribosomal phosphoprotein (2.2 kb). The graph shows densitometric analysis after normalization. Each bar represents the mean ± SD with * representing P < 0.05.

 
Collagen type III also was down-regulated in a dose-dependent manner in myometrial and leiomyoma SMCs (Fig. 4Go), but the effect was more pronounced in myometrial cells. A significant effect of pirfenidone was shown at 0.1 mg/mL (0.54 ± 0.22 ADU, P < 0.05), 0.3 mg/mL (0.46 ± 0.21 ADU, P < 0.005), and 1 mg/mL (0.41 ± 0.2 ADU, P < 0.005) for myometrial cells, whereas in the leiomyoma cells, the collagen type III mRNA levels were significantly inhibited only at the 1 mg/mL concentration (0.54 ± 0.3 ADU, P < 0.05).



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  		Figure 4. Effect of pirfenidone on collagen type III (alpha 1) messenger RNA expression in myometrial and leiomyoma cells. Lanes 1,6: 0 mg/mL pirfenidone (P); Lanes, 2,7: 0.01 mg/mL P; Lanes 3,8: 0.1 mg/mL P; Lanes 4,9: 0.3 mg/mL P; and lanes 5,10: 1.0 mg/mL P. Two transcripts (4.8 and 5.4 kb) for collagen type III was detected in all lanes. Differences in sample loading were corrected by normalization to ribosomal phosphoprotein (2.2 kb). The graph shows densitometric analysis after normalization. Each bar represents the mean ± SD with * representing P < 0.05.

 
In the final set of experiments, the effects of increasing concentrations of pirfenidone on cell proliferation and cell death, after 7 days of treatment, were assessed. Results from two representative experiments are shown in Table 2Go. A significant inhibitory effect on cell proliferation was seen for both leiomyoma and myometrial cells at the 0.1, 0.3, and 1.0 mg/mL concentrations. In contrast, a significant increase in the percentage of dead cells was apparent only at 1.0 mg/mL pirfenidone.


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  		Table 2. Effect of pirfenidone on cell proliferation and percentage dead cells. Results from two representative experiments are shown. Each value represents the mean of two dishes. Standard errors ranged from 10,000–30,000 cells for cell counts and from 1–2% for percentage dead cells. Pirfenidone caused a dose dependent inhibition of cell proliferation for both leiomyoma and myometrial cells (P< 0.01). A significant increase in percentage dead cells was apparent only at 1.0 mg/mL pirfenidone (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Uterine leiomyomas are a significant health problem for women, but the only effective long-term therapy for treatment of these tumors is hysterectomy or myomectomy. Though GnRH agonists have been used successfully for short-term treatment, the detrimental side effects of prolonged hypoestrogenism have precluded longer-term use of these compounds (9). Thus, there is a great need to identify new types of drugs or compounds that may be effective therapies for leiomyomas but with fewer side effects. In this study, we investigated the effects of the antifibrotic compound pirfenidone on uterine SMC proliferation and matrix protein production. The results show that pirfenidone is an effective inhibitor of DNA synthesis, cell proliferation, and collagen production for both normal myometrial and leiomyoma SMCs.

The mechanism by which pirfenidone acts to inhibit DNA synthesis and cell proliferation is not clear, but it does not seem to involve toxic effects on the cells. The results of the LDH assay showed no increase in LDH secretion by cells treated with any of the concentrations of pirfenidone. In addition, trypan blue exclusion staining, performed on cells treated with the various concentrations of pirfenidone for 7 days, showed an increase in the percentage of dead cells only at the highest concentration of pirfenidone (1 mg/mL). The inhibitory effect of pirfenidone on cell proliferation was apparent at one tenth this concentration (0.1 mg/mL). The relative lack of toxicity of pirfenidone in vitro is supported by findings in vivo. In clinical studies involving treatment of human subjects with pirfenidone as a treatment for pulmonary fibrosis, the daily dosage was 2400 mg, given orally (16) (Dr. G. Raghu, personal communication). Relatively mild adverse effects, including occasional drowsiness, skin rash, or gastric discomfort, were noted (16).

The effects of pirfenidone on cell proliferation are most likely mediated via inhibitory effects on specific growth factors. Studies on human fibroblasts have shown that pirfenidone inhibits basic fibroblast growth factor, platelet-derived growth factor, and transforming growth factor ß (TGFB)-stimulated cell proliferation (12). Furthermore, these investigators showed that fibroblasts treated with pirfenidone were unable to exit the G1 phase of the cell cycle. These results suggest a postreceptor site of action for pirfenidone. It is possible that pirfenidone may act as an antiestrogen or an aromatase inhibitor in uterine SMCs due to its chemical structure. However, such a mechanism could only be proven through studies showing that pirfenidone interacts directly with the estrogen receptor.

The mitotic activity of myometrial and leiomyoma SMCs in vivo varies throughout the menstrual cycle, suggesting that cell proliferation is regulated by ovarian steroid hormones (17, 18). However, studies by a number of investigators have been unable to show consistently a direct stimulatory effect of estradiol or progesterone on proliferation of these cells in vitro (19). This is, in part, because of the fact that uterine SMCs do not maintain steroid hormone responsiveness for prolonged periods of time when placed in culture. The results of a number of recent studies have led to the hypothesis that the effects of the ovarian steroid hormones on cell proliferation may be mediated indirectly through the activation of autocrine and paracrine peptide growth factors, including epidermal growth factor and insulin-like growth factors I and II (19, 20, 21). Basic FGF, platelet-derived growth factor, and TGFB also may be important regulators of cell proliferation in myometrial and leiomyoma cells (22, 23, 24). The antiproliferative effect of pirfenidone may involve a common postreceptor site of action, as has been suggested from the data on fibroblasts, or may involve inhibition of synthesis of one or more growth factors required for cell proliferation.

Leiomyomas contain large amounts of extracelluar matrix consisting of collagen, proteoglycan, and fibronectin, and show increased expression of collagen type I and type III mRNAs (14, 25, 26). The results of the present study showed that pirfenidone significantly inhibited steady-state levels of the mRNAs for both collagen type I and type III in myometrial cells at all concentrations tested. Collagen type I mRNA levels also were significantly inhibited in leiomyoma cells. However, the mRNA level of collagen type III was significantly reduced only at the highest concentration (1 mg/mL) of pirfenidone tested. Thus, in leiomyomas, pirfenidone seems to selectively inhibit collagen type I production over that of collagen type III. Fujita et al. (27) reported that the ratio of type III to type I collagen protein was decreased in leiomyomas, compared with the corresponding myometrium, because of an increase in collagen type I content and a decrease in collagen type III content in leiomyoma tumors. This suggests an alteration in the normal regulation of collagen production in these tumor cells, which may account for the differential effect observed between myometrial and leiomyoma cells. Leiomyoma SMCs may be more resistant to inhibition by specific growth factors. The decrease in steady-state levels of mRNAs for the collagens may reflect an inhibitory effect on gene transcription or an increase in mRNA turnover. In vivo studies, using the hamster model of artificially induced lung fibrosis, have shown that pirfenidone causes a marked inhibition of proline hydroxylase levels (10). This finding suggests that pirfenidone may reduce the availability of the hydroxyproline required for collagen synthesis and, therefore, may inhibit collagen synthesis at the translational level, as well.

In summary, the results of the present studies show that pirfenidone inhibits proliferation of myometrial and leiomyoma SMCs and significantly suppresses steady-state mRNA levels of both collagen type I and collagen type III. Pirfenidone showed little toxic effect on either cell type, suggesting that this compound may prove to be an effective therapeutic agent for treatment of leiomyomas with minimal side effects. Studies are underway to investigate more thoroughly the mechanism of action of pirfenidone.

Received March 27, 1997.

Revised August 12, 1997.

Accepted September 25, 1997.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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  7. Lippman ME, Dickson RB, Knabbe C, et al. 1986 Autocrine and paracrine growth regulation of breast cancer. Breast Cancer Res Treat. 7:59–70.[CrossRef][Medline]
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  15. Stewart EA, Floor AE, Jain P, Nowak RA. 1995 Increased expression of messenger RNA for collagen type I, collagen type III, and fibronectin in myometrium of pregnancy. Obstet Gynecol. 86:417–422.[Abstract]
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