Ann Surg Treat Res.  2020 Apr;98(4):159-167. 10.4174/astr.2020.98.4.159.

Overexpression of Nrf2 promotes colon cancer progression via ERK and AKT signaling pathways

• Lee YJ
• Kim WI
• Bae JH
• Cho MK
• Lee SH
• Nam HS
• Choi IH
• Cho SW

• KMID: 2500814
• DOI: http://doi.org/10.4174/astr.2020.98.4.159

Abstract

Purpose
We investigated the expression of Nrf2 in colorectal cancer and its correlation with clinicopathological characteristics as well as mechanisms and roles of Nrf2 expression including cell signaling pathway, survival, proliferation, and migration.
Methods
Nrf2 expression was measured in 12 and 30 different colorectal cancer (CRC) tissues by western blot (WB) and immunohistochemistry (IHC), respectively. SW480 cells were used for cell proliferation and cell migration tests. The correlation between the expression of Nrf2 and clinicopathologic parameters were evaluated using the chi-square or Fisher exact test. Data are expressed as the mean ± standard deviation for 3 independent experiments. P < 0.05 was considered statistically significant.
Results
Analysis of WB demonstrated that Nrf2 proteins were increased in CRC tissues, and decreased in normal tissues. IHC staining showed that the Nrf2 expression was elevated in CRC tissues, compared to matched normal tissues. When SW480 cells were suppressed with small interfering RNA of Nrf2, cell viability was inhibited, and cell apoptosis was increased. These results were found along with suppression of the phosphorylated form of extracellular signal-regulated kinase 1/2 and AKT.
Conclusion
This study suggests that overexpression of Nrf2 may be related to carcinogenesis and progression of CRC.

Keyword

Colorectal neoplasms; NF-E2-related factor 2; Reactive oxygen species

Figure

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  Fig. 1 Representative immunohistochemistry staining for nuclear factor erythroid 2-related factor 2 protein expression in paraffin-embedded colorectal cancer tissue (A, B) and normal tissue (C, D). (A, B) Some of the tumor cells show positive cytoplasmic staining for Nrf2 (A: ×100, B: ×200). (C, D) Normal mucosa of colon shows negativity for Nrf2 (C: ×40, D: ×100).

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  Fig. 2 Nrf2 protein was expressed on colorectal cancer and normal tissues. β-actin used as a loading control. The human colorectal cancer cell line SW480 served as a positive control for Nrf2 expression.

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  Fig. 3 Effects of Nrf2 knockdown on phosphorylation of ERK and AKT. (A) Western blot analysis. After Nrf2-specific siRNA transfection in SW480, phosphorylation of ERK and AKT was downregulated. In addition, Bcl-2 expression was decreased. β-actin used as a loading control. (B) Densitometric analysis showed relative density of Nrf2, p-ERK, p-AKT, and Bcl-2. si-C, Stealth RNAi control; si-Nrf2, small interfering Nrf2.

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  Fig. 4 Confirmation of cell viability in SW480 after transfection with Nrf2-specific siRNA. (A) Effects of Nrf2 knockdown on proliferation of SW480 cells. (B) Phase-contrast images of cells treated with Nrf2-siRNA and control cells. (C) Annexin V-PE binding assay. 7-Amino-actinomycin D (7-AAD), phycoerythrin (Annexin V-PE). si-C, Stealth RNAi control; si-Nrf2, small interfering Nrf2.

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  Fig. 5 Comparison of migration distances in SW480. si-C, Stealth RNAi control; si-Nrf2, small interfering Nrf2. *P < 0.05 compared to respective controls.

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