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Updated: Jun 1, 2026
A Streamlined Approach for Mass Spectrometry-Based Proteomics Using Selected Tissue Regions
Published on: April 18, 2025
Transcriptomic and proteomic profiling of KEAP1 disrupted and sulforaphane-treated human breast epithelial cells
Abena S Agyeman1, Raghothama Chaerkady, Patrick G Shaw
1Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA.
Abstract:
Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, is a potent inhibitor of experimental mammary carcinogenesis and may be an effective, safe chemopreventive agent for use in humans. SFN acts in part on the Keap1/Nrf2 pathway to regulate a battery of cytoprotective genes. In this study, transcriptomic and proteomic changes in the estrogen receptor negative, non-tumorigenic human breast epithelial MCF10A cell line were analyzed following SFN treatment or KEAP1 knockdown with siRNA using microarray and stable isotopic labeling with amino acids in culture (SILAC), respectively. Changes in selected transcripts and proteins were confirmed by PCR and Western blot in MCF10A and MCF12A cells. There was strong correlation between the transcriptomic and proteomic responses in both the SFN treatment (R = 0.679, P < 0.05) and KEAP1 knockdown (R = 0.853, P < 0.05) experiments. Common pathways for SFN treatment and KEAP1 knockdown were xenobiotic metabolism and antioxidants, glutathione metabolism, carbohydrate metabolism, and NADH/NADPH regeneration. Moreover, these pathways were most prominent in both the transcriptomic and the proteomic analyses. The aldo-keto reductase family members, AKR1B10, AKR1C1, AKR1C2 and AKR1C3, as well as NQO1 and ALDH3A1, were highly upregulated at both the transcriptomic and the proteomic levels. Collectively, these studies served to identify potential biomarkers that can be used in clinical trials to investigate the initial pharmacodynamic action of SFN in the breast.
Insights
Sulforaphane (SFN), a compound from cruciferous vegetables, shows promise in preventing breast cancer by activating protective genes via the Keap1/Nrf2 pathway. This study identified potential biomarkers for SFN
Area of Science:
- Molecular Biology
- Cancer Chemoprevention
- Proteomics and Transcriptomics
Background:
- Sulforaphane (SFN), derived from cruciferous vegetables, is recognized for its potential in inhibiting experimental mammary carcinogenesis.
- The Keap1/Nrf2 pathway is a key mechanism through which SFN regulates cytoprotective genes, suggesting its role in chemoprevention.
Purpose of the Study:
- To analyze transcriptomic and proteomic changes in human breast epithelial cells after SFN treatment or KEAP1 knockdown.
- To identify potential biomarkers for assessing SFN's pharmacodynamic action in breast cancer chemoprevention clinical trials.
Main Methods:
- Microarray analysis for transcriptomic profiling of MCF10A cells treated with SFN.
- Stable Isotopic Labeling with Amino acids in Culture (SILAC) for proteomic analysis following KEAP1 knockdown via siRNA.
- Confirmation of transcript and protein changes using PCR and Western blot in MCF10A and MCF12A cell lines.
Main Results:
- Strong correlations observed between transcriptomic and proteomic responses for both SFN treatment (R = 0.679) and KEAP1 knockdown (R = 0.853).
- Common upregulated pathways identified include xenobiotic metabolism, antioxidants, glutathione metabolism, carbohydrate metabolism, and NADH/NADPH regeneration.
- Specific aldo-keto reductase family members (AKR1B10, AKR1C1, AKR1C2, AKR1C3), NQO1, and ALDH3A1 were significantly upregulated at both transcriptomic and proteomic levels.
Conclusions:
- SFN modulates key cellular pathways related to metabolism and antioxidant defense in breast epithelial cells.
- The identified upregulated genes and proteins, such as AKR1B10 and NQO1, represent potential pharmacodynamic biomarkers for SFN in clinical studies.
- These findings support the investigation of SFN as a safe and effective chemopreventive agent for human breast cancer.
