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Enhanced 4-hydroxynonenal resistance in KEAP1 silenced human colon cancer cells
Kyeong-Ah Jung1, Mi-Kyoung Kwak
1College of Pharmacy, The Catholic University of Korea, Gyeonggi-do, Bucheon, Republic of Korea.
Abstract:
Nuclear factor erythroid 2-related factor 2 (NRF2) is the transcription factor that regulates an array of antioxidant/detoxifying genes for cellular defense. The conformational changes of Kelch-like ECH-associated protein 1 (KEAP1), a cytosolic repressor protein of NRF2, by various stimuli result in NRF2 liberation and accumulation in the nucleus. In the present study, we aimed to investigate the effect of KEAP1 knockdown on NRF2 target gene expression and its toxicological implication using human colon cancer cells. The stable KEAP1-knockdown HT29 cells exhibit elevated levels of NRF2 and its target gene expressions. In particular, the mRNA levels of aldo-keto reductases (AKR1C1, 1C2, 1C3, 1B1, and 1B10) were substantially increased in KEAP1 silenced HT29 cells. These differential AKRs expressions appear to contribute to protection against oxidative stress. The KEAP1-knockdown cells were relatively more resistant to hydrogen peroxide (H2O2) and 4-hydroxynonenal (4HNE) compared to the control cells. Accordantly, we observed accumulation of 4HNE protein adducts in H2O2- or 4HNE-treated control cells, whereas KEAP1-knockdown cells did not increase adduct formation. The treatment of KEAP1-silenced cells with AKR1C inhibitor flufenamic acid increased 4HNE-induced cellular toxicity and protein adduct formation. Taken together, these results indicate that AKRs, which are NRF2-dependent highly inducible gene clusters, play a role in NRF2-mediated cytoprotection against lipid peroxide toxicity.
Insights
Silencing Kelch-like ECH-associated protein 1 (KEAP1) boosts Nuclear factor erythroid 2-related factor 2 (NRF2) activity, increasing antioxidant gene expression and cellular resistance to oxidative stress. This highlights NRF2-dependent aldo-keto reductases
Area of Science:
- Molecular Biology
- Cell Biology
- Toxicology
Background:
- Nuclear factor erythroid 2-related factor 2 (NRF2) is a key transcription factor regulating cellular defense mechanisms against oxidative stress.
- Kelch-like ECH-associated protein 1 (KEAP1) acts as a cytosolic repressor of NRF2, controlling its stability and nuclear translocation.
- Dysregulation of the KEAP1-NRF2 pathway is implicated in various diseases, including cancer and conditions involving oxidative damage.
Purpose of the Study:
- To investigate the impact of KEAP1 knockdown on NRF2 target gene expression in human colon cancer cells.
- To assess the toxicological implications of altered NRF2 activity following KEAP1 silencing.
- To elucidate the role of specific NRF2-dependent genes in cellular protection against oxidative and lipid peroxide toxicity.
Main Methods:
- Stable KEAP1 knockdown was established in HT29 human colon cancer cells.
- NRF2 and its target gene expression (specifically aldo-keto reductases - AKRs) were quantified using mRNA analysis.
- Cellular resistance to oxidative stressors like hydrogen peroxide (H2O2) and 4-hydroxynonenal (4HNE) was evaluated.
- The effect of an AKR1C inhibitor (flufenamic acid) on cellular toxicity and protein adduct formation was examined.
Main Results:
- KEAP1 knockdown led to elevated NRF2 levels and increased expression of NRF2 target genes, notably aldo-keto reductases (AKR1C1, 1C2, 1C3, 1B1, 1B10).
- KEAP1-silenced cells demonstrated enhanced resistance to H2O2 and 4HNE compared to control cells.
- Reduced formation of 4HNE protein adducts was observed in KEAP1-knockdown cells upon exposure to oxidative stress.
- Inhibition of AKR1C activity in KEAP1-silenced cells exacerbated 4HNE-induced cellular toxicity and protein adduct formation.
Conclusions:
- KEAP1 knockdown activates the NRF2 pathway, significantly upregulating NRF2-dependent aldo-keto reductases.
- These upregulated AKRs play a crucial role in conferring cytoprotection against oxidative stress and lipid peroxidation products.
- The findings underscore the importance of the KEAP1-NRF2-AKR axis in cellular defense against toxic insults, with potential implications for therapeutic strategies.
