Effect of Estradiol in an Azoxymethane/Dextran Sulfate Sodium-Treated Mouse Model of Colorectal Cancer: Implication for Sex Difference in Colorectal Cancer Development

Abstract

Purpose: This study demonstrates that estradiol downregulates inflammation and inhibits colorectal cancer (CRC) development in azoxymethane/dextran sulfate sodium (AOM/DSS) mouse model.

Materials and methods: AOM/DSS-treated male and female mice were sacrificed at weeks 2, 10, and 16, to assess estrogen effects on colitis and carcinogenesis. Macroscopic and histologic severity of colitis and Western blot and quantitative real-time polymerase chain reaction were evaluated, to measure inflammatory mediators and cytokines.

Results: Compared with AOM/DSS-treated male mice (M-AOM/DSS group), AOM/DSS-treated male mice with estradiol administration (M-AOM/DSS+estr group) displayed at week 2 significantly decreased severity of colitis. At weeks 10 and 16, AOM/DSS-treated female mice (F-AOM/DSS group) and the M-AOM/DSS+estr group showed significantly lower tumor multiplicity compared with the M-AOM/DSS group. At week 2, F-AOM/DSS group had a lower level of nuclear factor-κB (NF-κB) expression and higher level of nuclear factor erythroid 2-related factor 2 (Nrf2) expression, compared to the M-AOM/DSS group. At week 2, expression levels of NF-κB and its related mediators decreased in the M-AOM/DSS+estr group, while levels of Nrf2 and Nrf2-related anti-oxidant enzymes increased. In addition, estradiol significantly increased Nod-like receptor protein 3 (NLRP3) inflammasome expressions in AOM/DSS-treated male mice. In contrast, at weeks 10 and 16, Nrf2 and its-related anti-oxidant enzymes and NLRP3 inflammasome were highly expressed in M-AOM/DSS group and in F-AOM/DSS group, who developed cancer.

Conclusion: The data suggest that estradiol inhibits the initiation of CRC by regulating Nrf2-related pathways. Moreover, these imply the dual role of Nrf2 and NLRP3 inflammasome, including promotion of tumor progression upon tumor initiation.

Keywords: AOM/DSS mouse model; Colorectal Neoplasms; Estradiol; Mouse; NF-E2-Related Factor 2; NF-kappa B; Nod-like receptor protein 3 inflammasome.

Fig. 1.

Estradiol prevents wasting disease progression in azoxymethane/dextran sulfate sodium (AOM/DSS)–induced colitis. (A) Scheme for the experimental course of AOM/DSS promoted colitis-associated tumorigenesis. The mice were injected AOM on day 0. DSS in drinking water (2.5%) and estradiol supply was provided from day 7 to 13. Mice were sacrificed at week 2, 10, and 16. (B) Disease Activity Index (DAI) was decreased by estradiol. (C) Colon length at week 2. (D) Macroscopic damage score at week 2. (E) Myeloperoxidase (MPO) activity in colonic tissues at week 2. (F) Histopathologic findings of the colonic mucosa (H&E staining, ×200) at week 2. In control mice, the mucosa is normal in males and females. However, near-total crypt loss and infiltration of severe inflammatory cell of colonic mucosa (white arrow) are seen in both males and females. Estradiol treatment significantly decreased histologic damage, with only mild erosion (yellow arrow). ^*p < 0.05 compared to control, ^†p < 0.05 compared to AOM/DSS group, ^ǂp < 0.05 between estradiol-treated group and female AOM/DSS group, ^#p < 0.05 between the male AOM/DSS group and the female AOM/DSS group. M, male; F, female; estr, estradiol.

Fig. 2.

Effect of estradiol and sex-associated differences in the multiplicity of colorectal cancer at weeks 10 and 16. Macroscopic view (left panel) and multiplicity of the colons (right panel) in each group sacrificed at weeks 10 (A) and 16 (B). Arrowheads indicate the macroscopic polyps. Representative histological images at weeks 10 (C) and 16 (D). Adenoma is indicated with dashed line circle, adenocarcinoma with full line circle and a bar, and submucosal invasion with arrowheads. Quantification of invasion and incidence of cancer in each group at 10 and 16 weeks obtained by microscopic evaluation of the colonic tissues (H&E staining, ×100). ^*p < 0.05 compared to control, ^†p < 0.05 compared to the in azoxymethane/dextran sulfate sodium (AOM/DSS) group, ^ǂp < 0.05 between the estradiol-treated group and the female AOM/DSS group, ^#p < 0.05 between the male AOM/DSS group and the female AOM/DSS group. M, male; F, female; estr, estradiol.

Fig. 3.

Protein and mRNA expression levels of nuclear factor κB (NF-κB) and its related pro-inflammatory factors in colonic tissues at weeks 2 (A, B), 10 (C, D), and 16 (E, F). Western blot analysis of NF-κB, inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX2) at weeks 2 (A), 10 (C), and 16 (E). mRNA expression levels of iNOS, COX2, interleukin 6 (IL-6), and TNFA, determined with real-time polymerase chain reaction, at weeks 2 (B), 10 (D), and 16 (F). ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001. M, male; F, female; AOM, azoxymethane; DSS, dextran sulfate sodium; estra, estradiol.

Fig. 4.

Expression levels of nuclear factor erythroid 2-related factor 2 (NRF2) and its related anti-oxidant enzymes in colonic tissues at weeks 2 (A-D), 10 (E-H), and 16 (I-L). Photomicrography of NRF2 immunostain of distal mouse colon at weeks 2 (A), 10 (E), and 16 (I). Arrows indicate the NRF2-immunoreactive cells (×400). Analysis of NRF2 immunohistochemistry in distal colonic tissues at week 2 (B), 10 (F), and 16 (J). Western blot analysis of NRF2 and glutamate-cysteine ligase catalytic subunit (GCLC) at weeks 2 (C), 10 (G), and 16 (K). mRNA expression levels of PKCD, NRF2, HO-1, GCLC, GCLM, and NQO-1, determined with real-time polymerase chain reaction, at weeks 2 (D), 10 (H), and 16 (L). ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001. M, male; F, female; AOM, azoxymethane; DSS, dextran sulfate sodium; ADS, AOM/DSS; estra, estradiol; MW, molecular weight; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Fig. 5.

Protein and mRNA level analyses of Nod-like receptor protein 3 (NLRP3) inflammasome activation in colonic tissues at weeks 2 (A, B), 10 (C, D), and 16 (E, F). Western blot analysis of NLRP3, caspase-1 p10, and interleukin (IL)-1β at weeks 2 (A), 10 (C), and 16 (E). mRNA expression levels of NLRP3, CASP1, IL1B, and IL18, determined with real-time polymerase chain reaction, at weeks 2 (B), 10 (D), and 16 (F). ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001. M, male; F, female; AOM, azoxymethane; DSS, dextran sulfate sodium; MW, molecular weight; ADS, AOM/DSS; estra, estradiol.

Fig. 6.

Proposed regulatory mechanism of estrogen in colitis-associated colorectal cancer at week 2 (A) and at weeks 10 and 16 (B). (A) Estrogen induces inflammasome activation through Gα₁₃ protein subunits. Gα₁₂ and Gα₁₃ have potentiated estrogen-bound estrogen receptor α activity. However, despite the functional overlap between Gα₁₂ and Gα₁₃, only Gα₁₃ regulates nuclear factor erythroid 2-related factor 2 (Nrf2) via protein kinase Cδ (PKCδ). Nrf2 mediates inflammasome activation through the transcription of as-yet unknown genes. Nod-like receptor protein 3 (NLRP3) inflammasome activation induces pyroptosis to eliminate precancerous cells. After eliminating precancerous cells, Nrf2 inhibits nuclear factor κB (NF-κB) and reactive oxygen species through the anti-oxidant enzymes. Ultimately, estrogen prevents carcinogenesis (left panel). In contrast, in the absence of estrogen, inflammation provides a cancer microenvironment through activation of the NF-κB pathway (right panel). (B) After unsuccessful elimination of precancerous cells, inflammation progresses to cancer at weeks 10 and 16. Gα₁₂ and Gα₁₃ regulate NF-κB and Nrf2 via PKCδ-mediated signaling pathway, respectively. Nrf2 promotes tumor progression by activation of anti-oxidant enzymes and NLRP3 inflammasome. Ultimately, NF-κB and Nrf2 signaling pathway accelerate carcinogenesis. COX-2, cyclooxygenase 2; DAMP, damage-associated molecular pattern; GPCR, G protein coupled receptor; IL, interleukin; iNOS inducible nitric oxide synthase; ROS, reactive oxygen species; TLR, Toll-like receptor; TNF, tumor necrosis factor.