Hee Jin Son and Sung Hwa Sohn contributed equally to this work.
This study demonstrates that estradiol downregulates inflammation and inhibits colorectal cancer (CRC) development in azoxymethane/dextran sulfate sodium (AOM/DSS) mouse model.
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.
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.
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.
The incidence rate of colorectal cancer (CRC) is high in males compared with females, regardless of age, ethnicity, and geographic regions [
Estradiol increased nuclear factor erythroid 2-related factor 2 (Nrf2) activity in breast cancer cell line [
The enhancement of colitis-associated CRC development in Nrf2-deficient mice treated with AOM/DSS [
Inflammation is an important factor in the pathophysiology of colitis-associated and sporadic CRC. For example, Saleiro et al. [
From this background, we hypothesized that the observed sex difference in CRC incidence may be due to estradiol-mediated down-regulation of inflammation, which might somehow affect the CRC cascade. To explore this hypothesis, we assessed the temporal role of Nrf2 in modulating inflammation and carcinogenesis through the regulation of the NF-κB–mediated pro-inflammatory pathway, anti-oxidant enzymes, and the NLRP3 inflammasome.
Four-week-old male and female ICR mice (Orient Co., Ltd., Seoul, Korea) were housed in cages, and maintained at 23°C with a 12/12-hour light/dark cycle under specific pathogen-free conditions.
Clinical symptoms were evaluated using the Disease Activity Index (DAI), which includes body weight loss, stool characterization, and hematochezia [
Colons extracted from cecum to the rectum were opened longitudinally, and stool was washed out with phosphate-buffered saline. Colon length was measured from cecum to rectum using a ruler. Polypoid lesions with a diameter < 2 mm or > 2 mm were independently counted by three gastroenterologists in a blinded manner. Tumor multiplicity was defined as the number of gross polyps approved by the three gastroenterologists.
After extraction from the peritoneum, the colon was divided into proximal and distal portions. The proximal colon was half of the colon to 1.5 cm distal from the ileocecal valve. The distal colon was the other half up to the rectum 1.5 cm from the anal verge. One or two representative polyps of each sample were prepared for histological analysis. These samples were fixed with phosphate-buffered formalin, and stained with hematoxylin and eosin. Other portions were frozen in lipid nitrogen, and kept at –70°C, until use in the biochemical assays. The tumor incidence (%) was determined as the percentage of rats having more than one tumor. The classification of adenoma and adenocarcinoma was performed as previously described [
Immunohistochemical (IHC) analysis of Nrf2 was performed. Tissue sections were treated with 3% hydrogen peroxide, and nonspecific binding sites were blocked. The sections were incubated with anti-Nrf2 antibodies (ab31163, Abcam, Cambridge, MA). An automatic immunostainer (BenchMark XT, Ventana Medical Systems, Tucson, AZ) and UltraView Universial DAB detection kit (Ventana Medical Systems) were used for immunostaining. The proportion of the number of immune-stained in total cells of all crypts were calculated.
Histological severity was assessed using microscopic damage score reflecting colonic epithelial damage and depth of infiltration with inflammatory cells as previously described [
The levels of myeloperoxidase (MPO) in the colonic tissues were examined by ELISA (R&D Systems, Minneapolis, MN). Every assay was performed in triplicate.
Protein extracts were isolated using RIPA buffer (Cell Signaling Technology, Beverly, MA). Cytoplasmic and nuclear lysates were separated using a NE-PER Nuclear Cytoplasmic Extraction Reagent kit (Pierce, Rockford, IL), according to the manufacturer’s instructions. Protein concentration was determined using the BCA protein assay reagent (Pierce). Protein samples were separated by 8 to 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis. After blocking, membranes were incubated overnight at 4°C with specific antibodies.
RNA was isolated from colon tissue using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruments, and quantified using a NanoDrop ND-1000 device (Thermo Scientific, Wilmington, DE). cDNA was synthesized using the High Capacity cDNA reverse Transcription Kit (Applied Biosystems, Foster City, CA). Quantitative real-time PCR was performed using Power SYBR Green PCR Master mix and a Viia7 instrument (Applied Biosystems). The transcript levels of glyceraldehydes-3-phosphate dehydrogenase were used for sample normalization.
Data are expressed as mean±standard error of mean. Statistical significance was examined by Mann-Whitney test or Fisher exact test. A p-value of < 0.05 was considered to indicate statistical significance. All statistical analyses were conducted using SPSS ver. 18.0 (SPSS Inc., Chicago, IL) and GraphPad Prism software (GraphPad, La Jolla, CA).
All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University Bundang Hospital (BA1310-139/091-01). The procedures were in accordance with the Animals in Research: Reporting
We first analyzed DAI score, colon shortening, and severity of colitis, to evaluate the early impacts of estradiol. The M-AOM/DSS and F-AOM/DSS groups displayed higher DAI scores compared to the control mice (M-con and F-con groups), suggesting the induction of severe colitis (p=0.007 at week 2 for M-AOM/DSS group vs. M-con group) (
Prominent polyps developed at weeks 10 and 16, mostly in the distal part of the colon (
To further evaluate the estradiol effects on inflammatory factors at the molecular level, we measured NF-κB and its related pro-inflammatory enzymes, cytokines, and genes. First of all, we determined the expression levels of NF-κB by Western blot analysis at week 2. The M-AOM/DSS group had higher levels of NF-κB, compared to both the F-AOM/DSS and M-AOM/DSS+estr groups (
Since Nrf2 directly downregulates NF-κB expression and activity [
The IHC analysis of Nrf2 showed significant increase of Nrf2 by estradiol at week 2 (p < 0.05) (
The F-AOM/DSS group showed higher expression of Nrf2 compared to the M-AOM/DSS group in terms of the levels of protein (
Nrf2 activation also resulted in up-regulation of anti-oxidant gene expression at week 2. Glutamate-cysteine ligase catalytic subunit (GCLC) is an antioxidant enzyme regulated by Nrf2. GCLC was increased in the F-AOM/DSS and M-AOM/DSS+estr groups compared to control mice and the M-AOM/DSS group at week 2 (
We investigated Nrf2 expression levels by Western blot analysis at weeks 10 and 16. At these times, CRC had developed in the AOM/DSS model. In contrast to week 2, both protein and mRNA levels of Nrf2 were higher in the M-AOM/DSS group compared to the M-AOM/DSS+estr group at weeks 10 and 16 (p < 0.01 at week 10) (
The close relationship of Nrf2 with the activating mechanism of NLRP3 inflammasome, which finally activates IL-1β and IL-18 [
Next, we examined the expression of NLRP3 inflammasome-related enzymes and mediators (NLRP3, caspase 1, IL-1β, and IL-18) at weeks 10 and 16 by Western blot analysis. Notably, the expressions of caspase-1 and IL-1β were elevated in the M-AOM/DSS group compared to the M-AOM/DSS+estr group at weeks 10 and 16 in both protein (
After confirming the sex difference in CRC development by showing that the F-AOM/DSS group has significantly lower tumor multiplicity and incidence compared with the M-AOM/DSS group, we further investigated the underlying anti-cancer mechanism of estradiol. Our findings demonstrate a dual role of Nrf2 in modulating inflammation and carcinogenesis through the regulation of the NF-κB–mediated pro-inflammatory pathway, anti-oxidant enzymes, and NLRP3 inflammasome. In this research, we focused on week 2, which is the active DSS-induced inflammation stage [
The NF-κB signaling pathway is highly involved in inflammation and cancer development, especially in colitis-associated CRC [
It has been reported that anti-oxidant enzymes activated by Nrf2 have cancer preventive effects by eliminating reactive oxygen species, and facilitating the resolution of inflammation [
There was an increase of the NLRP3 inflammasome and its effector cytokines (IL-1β and IL-18) simultaneously with increased Nrf2 on the DSS-induced inflammation stage. Considering the importance of Nrf2 in NLRP3 inflammasome activation [
Since estradiol administration completely inhibited inflammation in AOM/DSS-treated male mice resulting in the near-complete prevention of CRC, we expected that AOM/DSS-induced inflammation at week 2 would be mild in female mice. There was a significant difference of DAI at week 2 between male and female mice, but not such significant differences in the severity of inflammation reflected in COX-2. The inflammation represented by DAI cannot be fully explained by a few inflammatory mediators, such as COX-2. The strong effect of administered estradiol on preventing AOM/DSS-induced inflammation and colon tumorigenesis might be due to the higher concentration of intraperitoneal-administered estradiol; the injection concentration was 10 mg/kg, compared to the concentration of endogenous estradiol of 4 pg/mL in female mice [
After tumors developed (weeks 10 and 16), we observed the interesting finding that Nrf2 signaling was significantly up-regulated in the M-AOM/DSS group, suggesting that Nrf2 and anti-oxidant enzymes might play a role in promoting tumor progression (
The collective present and prior [
In conclusion, our study shows estradiol administration in AOM/DSS-treated male mice attenuates inflammation, and increases Nrf2 in the DSS-induced inflammation stage. Moreover, inhibition of the NF-κB-related pathway and activation of Nrf2-related anti-oxidant enzymes and the NLRP3 inflammasome pathway indicate possible underlying mechanisms. This study finally suggests that Nrf2 and the NLRP3 inflammasome play a dual role, with a preventive effect on tumor development, but promotion of tumor progression, once a tumor is initiated.
Supplementary materials are available at Cancer Research and Treatment website (
Conflict of interest relevant to this article was not reported.
This work was supported by a grant from the National Research Foundation of Korea (NRF) funded by the government of the Republic of Korea (2016R1A2B4013133).
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.
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.
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.
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.
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.
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.
Incidence and multiplicity of adenoma and cancer in colon
Group | Low grade adenoma incidence | High grade adenoma incidence | Cancer with mucosa invasion | Cancer with submucosa invasion | Adenoma/cancer incidence | Adenoma/cancer multiplicity |
---|---|---|---|---|---|---|
Control (n=4) | 0/4 (0) | 1/6 (16.7) | 0/4 (0) | 0/4 (0) | 0/4 (0) | 0.0 |
AOM/DSS (n=6) | 1/6 (16.7) | 0/4 (0) | 0/6 (0) | 0/6 (0) | 1/6 (16.7) | 0.17±0.17 |
AOM/DSS+E2 (n=6) | 0/6 (0) | 0/6 (0) | 0/6 (0) | 0/6 (0) | 0/6 (0) | 0.17±0.17 |
p-value |
1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.762 |
p-value |
1.000 | 1.000 | 1.000 | 1.000 | 0.455 | 1.000 |
p-value |
1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.523 |
Control (n=4) | 0/4 (0) | 0/4 (0) | 0/4 (0) | 0/4 (0) | 0/4 (0) | 0.0 |
AOM/DSS (n=6) | 0/6 (0) | 1/6 (16.7) | 0/6 (0) | 0/6 (0) | 1/6 (16.7) | 0.33±0.21 |
p-value |
1.000 | 1.000 | 1.000 | 1.000 | 1.000 | 0.221 |
Control (n=6) | 0/6 (0) | 0/6 (0) | 0/6 (0) | 0/6 (0) | 0/6 (0.0) | 0.0 |
AOM/DSS (n=12) | 0/12 (0) | 1/12 (8.3) | 10/12 (83.3) | 0/12 (0) | 11/12 (91.6) | 2.33±0.19 |
AOM/DSS+E2 (n=12) | 1/12 (8.3) | 0/12 (0) | 0/12 (0) | 0/12 (0) | 1/12 (8.3) | 0.0 |
p-value |
1.000 | 1.000 | 0.002 |
1.000 | < 0.001 |
< 0.001 |
p-value |
1.000 | 1.000 | < 0.001 |
1.000 | < 0.001 |
< 0.001 |
p-value |
1.000 | 1.000 | 0.012 |
1.000 | 0.155 | 0.014 |
10-Week female | ||||||
Control (n=6) | 0/6 (0) | 0/6 (0) | 0/6 (0) | 0/6 (0) | 0/6 (0) | 0.0 |
AOM/DSS (n=12) | 1/12 (8.3) | 2/12 (16.6) | 3/12 (25) | 1/12 (8.3) | 7/12 (58.3) | 1.17±0.39 |
p-value |
1.000 | 0.529 | 0.515 | 1.000 | 0.038 |
0.025 |
Control (n=6) | 0/6 (0) | 0/6 (0) | 0/6 (0) | 0/6 (0) | 0/6 (0) | 0.0 |
AOM/DSS (n=12) | 0/12 (0) | 1/12 (8.3) | 8/12 (66.6) | 3/12 (25) | 12/12 (100) | 3.42±0.50 |
AOM/DSS+E2 (n=12) | 0/12 (0) | 0/12 (0) | 4/12 (33.3) | 0/12 (0) | 4/12 (33.3) | 1.83±0.34 |
p-value |
1.000 | 1.000 | 0.013 |
0.515 | < 0.001 |
0.001 |
p-value |
1.000 | 1.000 | 0.220 | 0.217 | 0.001 |
0.020 |
p-value |
1.000 | 1.000 | 0.100 | 0.590 | 0.001 |
0.243 |
Control (n=6) | 0/6 (0) | 0/6 (0) | 0/6 (0) | 0/6 (0) | 0/6 (0) | 0.0 |
AOM/DSS (n=12) | 0/12 (0) | 0/12 (0) | 3/12 (25.0) | 1/12 (8.3) | 4/12 (33.3) | 2.42±0.71 |
p-value |
1.000 | 1.000 | 0.526 | 1.000 | 0.245 | 0.035 |
Values are expressed as number/subtotal (%) or mean±SEM. AOM, azoxymethane; DSS, dextran sulphate sodium; E2, 17β-estradiol; SEM, standard error of mean.
Between control and AOM/DSS group,
Between AOM/DSS and estradiol group,
Between male and female. Fisher exact test,
p < 0.05.