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, 9 (1), 20056

β-Catenin Signaling Inhibitors ICG-001 and C-82 Improve Fibrosis in Preclinical Models of Endometriosis

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β-Catenin Signaling Inhibitors ICG-001 and C-82 Improve Fibrosis in Preclinical Models of Endometriosis

Tomoko Hirakawa et al. Sci Rep.

Abstract

Endometriosis exhibits unique characteristics, such as fibrosis, resistance to apoptosis, and promotion of cell proliferation; however, its pathophysiology is not fully understood. Recurrence rates after treatment are high, and the progression risk continues until menopause; hence, more effective therapy for endometriosis is needed. CREB-binding protein (CBP)/β-catenin signaling inhibitors have demonstrated antifibrogenetic effects in liver, lung, and skin diseases. The present study evaluated the effects of two CBP/β-catenin signaling inhibitors, ICG-001 and C-82, on the progression of endometriosis using endometriotic cyst stromal cells from the ovary and normal endometrial stromal cells from the uterus. ICG-001 was also evaluated in a mouse model. ICG-001 and C-82 inhibited cell proliferation, fibrogenesis, and cell migration, and promoted apoptosis in vitro. ICG-001 inhibited the growth of endometriotic lesions in the mouse model. CBP/β-catenin signaling plays an important role in the pathophysiology of endometriosis. Inhibiting the CBP/β-catenin signal can be a therapeutic target for endometriosis.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Expression of β-catenin is upregulated in endometriosis. (A) HE staining of a normal endometrium. An endometrial gland and endometrial stromal cells are shown. (B) HE staining of endometriosis. (C) A representative image of immunohistochemical staining of a normal endometrium with anti-human β-catenin. (D) A representative image of immunohistochemical staining of endometriosis with anti-human β-catenin. (E,F) Significant upregulation of β-catenin protein expression in ECSC compared with NESC is shown by western blotting. n = 5, *p < 0.01, Student’s t-test. Error bars represent standard deviation (SD). Uncropped images are shown in Fig. S5.
Figure 2
Figure 2
ICG-001 and C-82 inhibit cell proliferation and promote apoptosis in ECSC. (A) MTT assay with ICG-001. (B) BrdU assay with ICG-001. (C) Caspase 3/7 assay with ICG-001. (D) Cell death detection ELISA with ICG-001. (E) MTT assay with C-82. (F) BrdU assay with C-82. (G) Caspase 3/7 assay with C-82. (H) Cell death detection ELISA with C-82. n = 6. *p < 0.05, **p < 0.001, Student’s t-test. Error bars represent standard deviation (SD).
Figure 3
Figure 3
Cell migration and fibrosis are significantly inhibited by ICG-001 and C-82. (A) Scratch assay for 24 hours. Representative photos at 0 and 24 hours are shown. The control became confluent after 24 hours. (B) The analysis of the reduction of the ratio of the scratched area (cell migration ratio) at 24 hours. The data are shown as relative values at 24 hours against the scratched area of the controls at 0 hours. ICG-001 and C-82 inhibited cell migration significantly compared with controls. n = 5. *p < 0.05, Student’s t-test. (C) Representative result of a collagen gel contraction assay in ECSC. The concentrations of ICG-001 and C-82 were 20 μM and 2 μM, respectively. (D) Collagen gel contraction was significantly inhibited by ICG-001 and C-82. n = 3. *p < 0.001, **p < 0.01, Student’s t-test. Error bars represent standard deviation (SD).
Figure 4
Figure 4
Expression of α-SMA is significantly upregulated in ECSC, and downregulated by ICG-001 and C-82. (A) The mRNA expression of α-SΜΑ was significantly upregulated in ECSC compared with NESC. n = 4. *p < 0.01, Student’s t-test. (B) mRNA expression of α-SΜΑ in ECSC was significantly downregulated by ICG-001 and C-82. n = 4. *p < 0.001, Student’s t-test. (C,D) Western blot analysis of α-SMA showed that protein expression of α-SMA was significantly upregulated in ECSC compared with NESC. n = 3. *p < 0.01, Student’s t-test. (E,F) Western blot analysis of α-SMA in control, ICG-001, and C-82 groups. There was no significant difference in the protein expression of α-SMA between the untreated control group and CBP/β-catenin inhibitor-treated groups by Student’s t-test. n = 4. Error bars represent standard deviation (SD). Uncropped images are shown in Figs. S6 and S7.
Figure 5
Figure 5
ICG-001 inhibits endometriosis progression in mice. (A) Representative abdominal findings. Endometriotic lesions are mainly observed as small cysts. (B) The mean number of endometriotic lesions in each ICG-001-treated group was significantly smaller than in the untreated control group. n = 10, 9, 10, and 9, for 0 mg/kg ICG-001, 10 mg/kg ICG-001, 50 mg/kg ICG-001, and 100 mg/kg ICG-001, respectively. *p < 0.01, **p < 0.001, Bonferroni correction. (C) Collagen fibers appear blue (Aniline blue) in modified Masson’s staining. (D) Collagen fibers appear pink in Sirius red staining. (E) Immunohistochemical staining with anti-α-SMA. (F) Analysis of the stain area of collagen fibers in modified Masson’s staining. The percentage of the Aniline blue area was significantly decreased in the 50-mg/kg ICG-001 and 100-mg/kg ICG-001 groups. n = 3. *p < 0.01, **p < 0.001, Bonferroni correction. Error bars represent standard deviation (SD).

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