Towards non-surgical therapy for uterine fibroids: catechol-O-methyl transferase inhibitor shrinks uterine fibroid lesions in the Eker rat model

Abstract

Background: Uterine leiomyomas (fibroids) are the most common pelvic tumors in women. We assessed the potential therapeutic utility of Ro 41-0960, a synthetic catechol-O-methyl transferase inhibitor (COMTI), in the Eker rat.

Methods: We randomized uterine fibroid-bearing Eker rats for treatment with Ro 41-0960 (150 mg/kg/12 h) versus vehicle for 2 and 4 weeks. The fibroids were measured by caliper and subjected to histological evaluation. Urinary levels of 2-hydroxy estrogen (E(2)), 16-hydroxy E2 and DPD (osteoporosis marker) and serum liver enzymes were evaluated. Expressions of Cyclin D1, proliferating cell nuclear antigen (PCNA), Poly [ADP-ribose] polymerase1 (PARP1), tumor suppressor gene (P53) and transforming growth factor (TGFβ3) were assessed in fibroids using immunohistochemical analysis or RT-PCR. Apoptosis was confirmed using terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL).

Results: Ro 41-0960-treated rats exhibited fibroid volumes of 86 ± 7% and 105 ± 12% of initial burden, at 2 and 4 weeks post-treatment, respectively, significantly lower than control group (240 ± 15% and 300 ± 18%; P< 0.01). Ro 41-0960 increased the urinary 2-hydroxy E2/16-hydroxy E(2) ratio, level of p53 mRNA and TUNEL positivity (P< 0.05) and decreased PARP1, PCNA and cyclin D1 proteins and TGFβ3 mRNA (P< 0.05). Ro 41-0960 did not change normal tissue histology, liver functions or urinary DPD level.

Conclusions: Ro 41-0960 (COMTI) arrested growth/shrunk uterine fibroids in Eker rats. This result may be related to modulation of estrogen-dependent genes involved in apoptosis, proliferation and extracellular matrix deposition via accumulation of 2-hydroxy estrogen. The efficacy and safety of Ro 41-0960 in rats suggest its candidacy for treatment of uterine fibroids.

Figure 1

Treatment with Ro 41-0960, a synthetic COMTI, in the Eker rat shrinks total leiomyoma volume compared with vehicle treatment. Tumor volume was calculated as a percentage of the corresponding day zero (pretreatment) volume and presented as mean ± SE of six animals at each time point. a: indicates a significant difference between the control and treated animals (P < 0.01).

Figure 2

Representative micrographs of fibroid lesions (hematoxylin-eosin stain) from vehicle control and Ro 41-0960 (COMTI)-treated Eker rats at 2 and 4 weeks. Original magnifications 40×. Nuclei in control rats are well defined with clear nuclear membranes (arrow). Tissue generally demonstrates high cellularity. After 2 and 4 weeks of treatment with Ro 41-0960 (COMTI), tissues demonstrate less cellularity and mostly consist of ECM. The few cells remaining are distorted in shape with irregular cellular and nuclear membranes (arrow heads). The control micrograph representing the 4-week time point.

Figure 3

Ro 41-0960 (COMTI) treatment of Eker rats modulates P53 and PARP1 apoptotic pathways. Data represent uterine leiomyoma tissues collected at 2 and 4 weeks after Ro 41-0960 treatment compared with those of the vehicle-treated group: (A) Ro 41-0960 decreases the death substrate poly (ADP-ribose) (PAR) polymerase (PARP1) protein as assessed by immunohistochemistry; original magnification: 20×; (B) Ro 41-0960 increases the percentage of TUNEL-positive cells; original magnification: 20×; and (C) Ro 41-0960 increases P53 mRNA levels. a: indicates a significant difference between the vehicle versus treated rats at P < 0.05, using a two-tailed Student's t-test. Control data are from the 4-week time point.

Figure 4

Ro 41-0960 (COMTI) treatment decreases the expression of proliferation- and ECM formation-related genes. Immunohistochemical analysis of proliferation-related (A) PCNA and (B) Cyclin-D1 in the Eker rat uterine leiomyoma tissue sections collected from animals treated with vehicle only (control) or Ro 41-0960 at 2 and 4 weeks post-treatment. Original magnification: 20×. (C) Levels of TGFβ3 mRNA. a: indicates a significant difference between the Ro 41-0960-treated and vehicle-treated rats, P < 0.05. Control data represent the 4-week time point.

Figure 5

Ro 41-0960 (COMTI) increases the urinary level of 2-hydroxy estrogen metabolite (A) and increases the ratio of 2-hydroxy estrogen to 16-hydroxy estrogen metabolites (B). Data are calculated as mean and SE for six animals at each time point and are represented as (A) a percentage of the data at day zero; and (B) a ratio of absolute values. A indicates a significant difference between the Ro 41-0960-treated rats and the vehicle-treated rats (P < 0.05).

Figure 6

Effect of Ro 41-0960 (COMTI) injection at 150 mg/kg bid on Eker rat urinary levels of the bone decalcification marker Deoxy Pyridinoline (DPD). DPD was evaluated using a competitive enzyme immunoassay and normalized against creatinine. Data are mean ± SE of six animals for each time point. There is no significant difference between the Ro 41-0960-treated rats and the vehicle-treated rats.

Figure 7

Effect of Ro 41-0960 (COMTI) on liver function tests in Eker rats. Eker rats were treated with Ro 41-0960 at a dose of 150 mg/kg bid or vehicle. Ro 41-0960 produce no significant changes in liver function tests: (A) AST, ALT and (B) total bilirubin. Liver function was evaluated 2 and 4 weeks after Ro 41-0960 injection and compared with vehicle-treated control animals using Student's two-tailed t-test.