An inhibitor of the Keap1-Nrf2 protein-protein interaction protects NCM460 colonic cells and alleviates experimental colitis

Abstract

Ulcerative colitis (UC) is a chronic relapsing-remitting form of inflammatory bowel disease (IBD) that increases the risk of colorectal cancer, the third most common malignancy in humans. Oxidative stress is a risk factor for the development of UC. The Keap1-Nrf2-ARE pathway is one of the most important defensive mechanisms against oxidative and/or electrophilic stresses. In this study, we identified CPUY192018 as a potent small-molecule inhibitor of the Keap1-Nrf2 PPI, investigated the cyto-protective effects of CPUY192018 on the NCM460 colonic cells and evaluated whether treatment with the inhibitor of the Keap1-Nrf2 PPI exerts protection on an established experimental model of UC induced by dextran sodium sulfate (DSS). Our study clearly demonstrated that CPUY192018 had a cytoprotective effect against DSS in both NCM460 cells and mouse colon via the activation of Nrf2 signaling. These results suggested that activation of Nrf2 by directly inhibiting the Keap1-Nrf2 PPI may be beneficial as a treatment for UC.

Figure 1. ITC profile of the titration of the Keap1 Kelch domain with CPUY192018.

The thermodynamic parameters of the interaction of Keap1 with CPUY192018 as determined by the ITC Assay are listed in the table. N is the stoichiometric coefficient. K_d is the binding constant. ΔH, ΔS, and ΔG refer to the changes in binding enthalpy, entropy, and total Gibbs free energy, respectively. ΔG is calculated according to the equation ΔG = ΔH − TΔS, where T is the absolute temperature used for the ITC experiment.

Figure 2. CPUY192018 activated the Nrf2-ARE pathway in vitro.

(A) Effect of CPUY192018 on the induction of the Nrf2 protein expression. (B) Effect of CPUY192018 on the nuclear translocation of the Nrf2 protein. At various time poits after the treatment with CPUY192018 (10 μM), nuclear and cytoplasmic cell extracts were prepared from the NCM460 cells and subjected to western blot analysis. Histone and β-actin served as markers for nuclear and cytosolic Nrf2 proteins, respectively. Densitometric analysis was performed to determine the relative ratios of the protein in each fraction. The data were normalized to the β-actin expression and are expressed as the means + SEM of three individual experiments. The data were analyzed using Image J 1.44p. (C) Immunofluorescence staining of Nrf2 at the indicated times in the NCM460 cells treated with 10 μM CPUY192018. Nrf2 and the nuclei were labeled with FITC and DAPI, respectively. The bars indicate the magnification (10 μm). (D) ARE induction by CPUY192018 and t-BHQ in the HepG2−ARE−C8 cells. The cells were exposed to the compounds or DMSO for 12 h. The activities are shown as the ratio to the DMSO control. The values shown are the means ± SEM (n = 3 independent observations).

Figure 3. CPUY192018 stimulated the transcription of the Nrf2-ARE-regulated cytoprotective genes in the NCM460 cells in an Nrf2-dependent manner.

(A) Quantitative real-time PCR analysis of Nrf2, HO-1, GCLM, and GPx2 in the NCM460 cells. The mRNA levels of Nrf2 and the Nrf2-targeted genes were measured at 10 h after treatment of the NCM460 cells with various concentrations (0.1, 1, 5, 10 μM) of CPUY192018. β-actin was used to normalize the expression of these genes. (B) Western blot analysis of the Nrf2 downstream proteins HO-1, GCLM, and GPx2 in the NCM460 cells after treatment with various concentrations (0, 0.1, 1, 10 μM) of CPUY192018 for 8 h. (C) The mRNA expression of Nrf2 and the Nrf2-regulated genes after exposure to Nrf2 siRNA and CPUY192018. The NCM460 cells were treated with Nrf2 siRNA (80 nM), CPUY192018 (10 μM), or Nrf2 siRNA (80 nM) plus CPUY192018 (10 μM). Additional NCM460 cells were treated with a scrambled duplex for use as the blank control. The expression of the Nrf2, HO-1, GCLM and GPx2 genes was quantified using qRT-PCR. (D) Western blot analysis of Nrf2 and the Nrf2-regulated proteins after exposure to Nrf2 siRNA and CPUY192018. The NCM460 cells were treated with Nrf2 siRNA (80 nM), CPUY192018 (10 μM), or Nrf2 siRNA (80 nM) plus CPUY192018 (10 μM). Additional NCM460 cells were treated with DMSO for use as the blank control. The values shown are the means ± SEM (n = 3 independent observations). ***P < 0.001, **P < 0.01, and *P < 0.05, one-way ANOVA with Tukey–Kramer posttest.

Figure 4. Effects of CPUY192018 on DSS-induced cell injury in the NCM460 cells.

(A) Protective effects of CPUY192018 against the DSS-induced cell damage. The NCM460 cells were pretreated with 10 μM CPUY192018 for 10 h then exposed to various concentrations of DSS for an additional 12 h. The cell viability was determined using the MTT assay. (B) Dose-dependent protective effects of CPUY192018 against the DSS-induced cell damage. The NCM460 cells were pretreated with 1–20 μM CPUY192018 for 10 h then exposed to 0.8 μg/mL DSS for an additional 12 h. The cell viability was determined using the MTT assay. (C) Flow cytometric analysis of the apoptotic rate. The NCM460 cells were treated with 10 μM CPUY192018 for 10 h before being exposed to 0.8 μg/mL DSS for an additional 8 h. The cells were stained with FITC-Annexin V-PI, and the apoptotic rates were detected by flow cytometry. The statistical analysis of the apoptotic rates is shown in the figure. (D) The effect of CPUY192018 on the cell cycle in the NCM460 cells. The NCM460 cells were treated with 10 μM CPUY192018 for 10 h before being exposed to 0.8 μg/mL DSS for an additional 8 h. At the end of this treatment, the cells were trypsinized, incubated with RNase, stained with propidium iodide (PI), and analyzed by flow cytometry. The statistical analysis of the ratio of the NCM460 cells in the G0/G1, S and G2/M phases of the cell cycle is shown in the figure. (E) Living Cell Microscopy. The NCM460 cells were pretreated with 10 μM CPUY192018 for 10 h and then exposed to 0.4 μg/mL DSS for an additional 6 h. After this treatment, the NCM460 cells were stained with 10 μM cH₂DCF-DA for 20 min at 37 °C and living cell fluorescence microscopy was performed. The values shown are the means ± SEM (n = 3 independent observations). *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Tukey–Kramer posttest.

Figure 5. CPUY192018 ameliorated the pathological symptoms in the DSS-induced mouse model of UC.

The animals were randomly assigned to one of the five treatment groups: Control group (the mice received regular drinking water); DSS model group (the mice received 3% w/v DSS in drinking water); DSS + CPUY192018 (10 mg/kg) group (the mice received 3% w/v DSS in drinking water together with administration by gavage of 10 mg/kg of CPUY192018); DSS + CPUY192018 (40 mg/kg) group (the mice received 3% w/v DSS in drinking water together with administration by gavage of 40 mg/kg of CPUY192018); and CPUY192018 (40 mg/kg) group (the mice received administration by gavage of 40 mg/kg of CPUY192018). (A) Gradual changes in body weight during the DSS administration in mice. (B) The comparison of the colon length on day 64. (C) Representative histological images of distal colon sections stained with hematoxylin and eosin (H&E). Magnifications×100. (D) Histologic inflammatory score. The results are expressed as the means + SEM (n = 8, in each group). *p < 0.05, **p < 0.01 and ***p < 0.001, one-way ANOVA with Tukey–Kramer posttest.

Figure 6. Quantification of the inflammatory cytokines TNF-α, IFN-γ, IL-6, and IL-1β and the MPO activity in the colon homogenates from the C57BL/6 female mice.

The results are expressed as the means + SEM. *p < 0.05, **p < 0.01 and ***p < 0.001, one-way ANOVA with Tukey–Kramer posttest.

Figure 7. CPUY192018 attenuated the DSS-induced oxidative stress and apoptosis in the mouse colons.

(A) Immunohistochemical detection of Nrf2 together with its target proteins HO-1, GCLM and GPx2 levels in the mouse colons. Magnifications ×200. The ROS level (B) the ratio of GSH/GSSG (C) and MDA level (D) in the colons were measured. The results are expressed as the means + SEM. *p < 0.05, **p < 0.01 and ***p < 0.001, one-way ANOVA with Tukey–Kramer posttest.