p21 Promoter Methylation Is Vital for the Anticancer Activity of Withaferin A
Abstract
1. Introduction
2. Results
2.1. p21, p53, and CCND1 Are Differentially Methylated in Breast Cancer Patients Versus Normal Breast Tissue
2.2. Targeted Demethylation of the p21 Promoter Increased p21 Expression and Decreased Cancer Cell Viability
2.3. Targeted Demethylation of the p53 Promoter Increased p53 Expression and Decreased Cancer Cell Viability
2.4. Targeted Methylation of the CCND1 Promoter Decreased CCND1 Expression and Decreased Cancer Cell Viability
2.5. Targeted Methylation of the p21 Promoter in Combination with WFA Ablates p21 Expression Changes and Resulted in a Loss of WFA Anticancer Function
2.6. Targeted Methylation of the p53 Promoter in Combination with WFA Ablates p53 Expression Changes with No Significant Loss of WFA Anticancer Function
2.7. Targeted Demethylation of the CCND1 Promoter in Combination with WFA Ablates CCND1 Expression Changes with No Significant Loss of WFA Anticancer Function
3. Discussion
3.1. p21, p53, and CCND1 as Molecular Targets for Epigenetic Therapies
3.2. Genetic Targets and Their Relationship with WFA’s Function
3.3. Key Findings
3.4. Limitations and Future Directions
4. Materials and Methods
4.1. Cell Lines and Culture Conditions
4.2. Withaferin A and Cell Treatment
4.3. Isolation and Growth of CRISPR Constructs
4.4. Guide Selection and Cloning
4.5. Nucleic Acid Extraction
4.6. RT-qPCR
4.7. Bisulfite Conversion and Sequencing
4.8. MTT Analysis
4.9. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Brane, A.; Sutko, M.; Tollefsbol, T.O. p21 Promoter Methylation Is Vital for the Anticancer Activity of Withaferin A. Int. J. Mol. Sci. 2025, 26, 1210. https://doi.org/10.3390/ijms26031210
Brane A, Sutko M, Tollefsbol TO. p21 Promoter Methylation Is Vital for the Anticancer Activity of Withaferin A. International Journal of Molecular Sciences. 2025; 26(3):1210. https://doi.org/10.3390/ijms26031210
Chicago/Turabian StyleBrane, Andrew, Madeline Sutko, and Trygve O. Tollefsbol. 2025. "p21 Promoter Methylation Is Vital for the Anticancer Activity of Withaferin A" International Journal of Molecular Sciences 26, no. 3: 1210. https://doi.org/10.3390/ijms26031210
APA StyleBrane, A., Sutko, M., & Tollefsbol, T. O. (2025). p21 Promoter Methylation Is Vital for the Anticancer Activity of Withaferin A. International Journal of Molecular Sciences, 26(3), 1210. https://doi.org/10.3390/ijms26031210