p21 Promoter Methylation Is Vital for the Anticancer Activity of Withaferin A

Sep 23, 2026

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Breast cancer remains a major global malignancy, prompting searches for prevention strategies and improved therapeutic targets beyond current standard-of-care options and their side effects. Withaferin A (WFA), a winter cherry–derived phytochemical, is proposed to act through epigenetic mechanisms, particularly the methylome, yet the causal contribution of specific gene methylation states is unclear. Using CRISPR-dCas9 epigenetic editors in human breast cancer cells, targeted promoter demethylation of p21 and p53 increased expression and reduced viability, while targeted CCND1 promoter methylation decreased expression and viability. Combined WFA treatment showed modulation of WFA anticancer effects via p21 promoter methylation, implicating p21 as an epigenetic editing–relevant target and supporting WFA potential for breast cancer prevention independent of p53 status.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages19

Reading Time48 min

AccessFree to download

Introduction

Breast cancer burden and prevention need

Racial disparities and dietary phytochemicals

Withaferin A as an epigenetic-active phytochemical

Article p21 Promoter Methylation Is Vital for the Anticancer Activity of Withaferin A Andrew Brane 1, Madeline Sutko 1 and Trygve O. Tollefsbol 1,2,3,4,5, * Academic Editors: Matin Sheriff, Stergios Boussios and Saak Victor Ovsepian Received: 4 December 2024 Revised: 23 January 2025 Accepted: 26 January 2025 Published: 30 January 2025 Citation: 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/](https://doi.org/)[ ](https://doi.org/)[10.3390/ijms26031210](10.3390/ijms26031210) Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ([https://creativecommons.org/](https://creativecommons.org/)[ ](https://creativecommons.org/)[licenses/by/4.0/](licenses/by/4.0/)) . 1 Department of Biology, University of Alabama at Birmingham, 3100 Science & Engineering Complex—East Science Hall, 902 14th Street South, Birmingham, AL 35205, USA 2 Comprehensive Cancer Center, University of Alabama at Birmingham, 1802 6th Avenue South, Birmingham, AL 35294, USA 3 Integrative Center for Aging Research, University of Alabama at Birmingham, 933 19th Street South, Birmingham, AL 35294, USA 4 Nutrition Obesity Research Center, University of Alabama at Birmingham, 1675 University Blvd, Birmingham, AL 35294, USA 5 Comprehensive Diabetes Center, University of Alabama at Birmingham, 1825 University Blvd, Birmingham, AL 35294, USA * Correspondence: [trygve@uab.edu](trygve@uab.edu) Abstract: Breast cancer (BC) is a widespread malignancy that affects the lives of millions of women each year, and its resulting financial and healthcare hardships cannot be overstated. These issues, in combination with side effects and obstacles associated with the current standard of care, generate considerable interest in new potential targets for treatment as well as means for BC prevention. One potential preventive compound is Withaferin A (WFA), a traditional medicinal compound found in winter cherries. WFA has shown promise asan anticancer agent and is thought to act primarily through its effects on the epigenome, including, in particular, the methylome. However, the relative importance of specific genes’methylation states to WFA function remains unclear. To address this, we utilized human BC cell lines in combination with CRISPR-dCas9 fused to DNA methylation modifiers (i.e., epigenetic editors) to elucidate the importance of specific genes’ promoter methylation states to WFA function and cancer cell viability. We found that targeted demethylation of promoters of the tumor suppressors p21 and p53 within MDA-MB-231/MCF7 cells resulted in around 1.7 × /1.5 × and 1.2 × /1.3 × increases in expression, respectively. Targeted methylation of the promoter of the oncogene CCND1 within MDA-MB-231/MCF7 cells resulted in 0.5 × /0.8 × decreases in gene expression. These changes to p21, p53, and CCND1 were also associated with decreases in cell viability of around 25%/50%, 5%/35%, and 12%/16%, respectively, for MDA-MB-231/MCF7 cells. When given in combination with WFA in both p53 mutant and wild type cells, we discovered that targeted methylation of the p21 promoter was able to modulate the anticancer effects of WFA, while targeted methylation or demethylation of the promoters of p53 and CCND1 had no significant effect on viability decreases from WFA treatment. Taken together, these results indicate that p21, p53, and CCND1 may be important targets for future in vivo studies that may lead to epigenetic editing therapies and that WFA may have utility in the prevention of BC through its effect on p21 promoter methylation independent of p53 function. Keywords: DNA methylation; breast cancer; phytochemicals; prevention; epigenetics; CRISPR-dCas9; Withaferin A 1. Introduction Despite recent adv

FAQs

How did the study test the role of promoter methylation in WFA activity?

Researchers used human breast cancer cell lines together with CRISPR-dCas9 fused to DNA methylation modifiers (epigenetic editors) to selectively methylate or demethylate promoters of candidate genes and observe downstream effects.

What happened when promoters of p21 and p53 were targeted for demethylation?

Targeted demethylation of p21 and p53 promoters increased gene expression in MDA-MB-231 and MCF7 cells and was associated with decreases in cell viability.

Which gene promoter methylation modulated WFA’s anticancer effects?

When combined with WFA, targeted methylation of the p21 promoter modulated WFA anticancer effects, whereas targeted methylation or demethylation of p53 and CCND1 promoters did not significantly change viability decreases from WFA treatment.

Sep 23, 2026

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