Departments of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, South Korea.
Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul, South Korea.
Departments of Pathology, Seoul National University Bundang Hospital, Seongnam, South Korea.
Laboratory of Immunology, Division of Biotechnology Review and Research-III, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, United States.
Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul, South Korea.
Cancer Research Institute, Seoul National University, Seoul, South Korea.
Abstract
Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a dual role in carcinogenesis. We previously reported that Nrf2 deficiency enhances the anti-tumorigenic effect of 17β-estradiol (E2) in an azoxymethane (AOM)/dextran sodium sulfate (DSS) model of colitis-associated cancer (CAC). Herein, we aimed to determine a possible explanation for our recent work and investigated the immune microenvironment represented by programmed death-ligand 1 (PD-L1) expression. One week after the AOM injection, mice were administered with DSS in drinking water for seven days; daily E2 injections were intraperitoneally administered during this period. The mice were sacrificed 16 weeks after AOM injection and analyzed for PD-L1 expression in the distal colon tissues using Western blotting and immunohistochemistry (IHC). Based on Western blotting results, PD-L1 expression was reduced in Nrf2 knockout (KO) female and E2-treated male mice when compared with their wild-type counterparts, following AOM/DSS treatment; this supports the association of PD-L1 expression with tumor progression. Additionally, this finding was in good agreement with the IHC results for PD-L1. Furthermore, we observed that PD-L1 is predominantly expressed in stromal cells rather than on epithelial cells in the colon. Western blotting revealed that PD-L1 expression in the colon positively correlates with expressions of inducible nitric oxide synthase (iNOS) (male, P = 0.002; female, P <0.001) and cyclooxygenase-2 (COX-2) (male, P <0.001; female, P <0.001). Collectively, our findings indicate that estrogen ameliorates the immune microenvironment represented by PD-L1 expression and enhances its effect in the absence of Nrf2.
Figure 1Overview of the experimental design. WT and Nrf2 KO male and female mice were used in the AOM/DSS-induced colitis-associated CRC protocol. AOM (10 mg/kg) was injected into the mice on Day 0. One week after, DSS (2.5%) was provided in the drinking water for one week. E2 (10 mg/kg) was administrated by daily intraperitoneal injection for one week during DSS treatment. The mice were sacrificed at week 16 after AOM injection. WT, wild-type; Nrf2, nuclear factor erythroid 2–related factor 2; KO, knockout; AOM, azoxymethane; DSS, dextran sodium sulfate; E2, 17β-estradiol.Figure 2Stronger inhibitory effect of E2 on AOM/DSS-induced PD-L1 expression in Nrf2 KO male mice than in WT males. (A, B) Representative Western blot images of three independent experiments performed with colon tissue from the male (A) and female mice (B). (C–H) Relative protein expression of PD-L1 (C, D), iNOS (E, F), and COX-2 (G, H) in males (C, E, G) and females (D, F, H). GAPDH was used as a loading control. The P-values obtained from the Kruskal–Wallis test is shown in the figure; *P < 0.05 for comparison between two groups (Mann–Whitney test); #P < 0.05 for comparison between males and females (Mann–Whitney test). WT, wild-type; KO, knockout; Ctrl, control; AOM, azoxymethane; DSS, dextran sodium sulfate; PD-L1, programmed death-ligand 1; iNOS, inducible nitric oxide synthase; COX-2, cyclo-oxygenase 2; Nrf2, nuclear factor erythroid 2-related factor 2; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.Figure 3Effect of E2 on mRNA expression of Pd-l1, iNos, and Nrf2-mediated anti-oxidant enzyme genes. (A–H) mRNA expression of Pd-l1(A, B), iNos(C, D), Ho-1(E, F), and Nqo1(G, H) in WT and Nrf2 KO male (A, C, E, G) and female (B, D, F, H) mice. Gapdh was used as an internal control. The P-values obtained from the Kruskal–Wallis test is shown in the figure; *P < 0.05 for comparison between two groups (Mann–Whitney test); #P < 0.05 for comparison between males and females (Mann–Whitney test). WT, wild-type; KO, knockout; AOM, azoxymethane; DSS, dextran sodium sulfate; Pd-l1, programmed death-ligand 1; iNos, inducible nitric oxide synthase; Ho-1, heme oxygenase 1; Nqo1, NAD(P)H dehydrogenase (quinone) 1; Gapdh, Glyceraldehyde 3-phosphate dehydrogenase.Figure 4Positive correlation of PD-L1 expression with proinflammatory mediators. (A, B) Correlation of PD-L1 protein levels with iNOS protein levels in male (A) and female (B) mice. (C, D) Correlation of PD-L1 protein levels with COX-2 protein levels in male (C) and female (D) mice. Spearman correlation analysis was used to determine the correlation of results from the Western blotting (Figure 2). PD-L1, programmed death-ligand 1; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase 2.Figure 5PD-L1-positive stromal cells are reduced in E2-treated Nrf2 KO male mice and Nrf2 KO female mice when compared with WT mice after AOM/DSS treatment. (A, B) Representative IHC images of PD-L1 expression in non-tumor and tumor areas in colon tissues of male (A) and female (B) mice. Magnification, ×200. Arrows indicate PD-L1-positive stromal cells. (C–F) PD-L1 expression according to the non-tumor area (C, D) and tumor area (E, F) in male (C, E) and female (D, F) mice were quantified as a percentage of the immunoreactive area (% of total area). Bar graph values are expressed as the mean ± standard error of the mean (SEM). The P-values obtained from the Kruskal–Wallis test is shown in the figure; *P < 0.05 for comparison between two groups (Mann–Whitney test); #P < 0.05 for comparison between males and females (Mann–Whitney test). WT, wild-type; KO, knockout; Ctrl, control; AOM, azoxymethane; DSS, dextran sodium sulfate; Nrf2, nuclear factor erythroid 2-related factor 2; PD-L1, programmed death-ligand 1.
그림 출처: PubMed Central 공개 논문 (PMC8297827)
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
