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ARTICLE

Death Ligand Signaling Involving the COX/PKC/MLKL Axis Mediates Erythrocyte Death by HDAC/DNMT Inhibitor, Parthenolide, through ROS Generation and Calcium Mobilization

Sara Y. Aldeghaither, Jawaher Alsughayyir, Sabiha Fatima, Mohammad A. Alfhili^*

Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, 12372, Saudi Arabia

* Corresponding Author: Mohammad A. Alfhili. Email:

(This article belongs to the Special Issue: Cellular Senescence in Health and Disease)

BIOCELL 2025, 49(11), 2167-2180. https://doi.org/10.32604/biocell.2025.071827

Received 13 August 2025; Accepted 19 September 2025; Issue published 24 November 2025

Abstract

Objectives: Targeting epigenetic modifications in anticancer therapy is a promising approach to overcoming cancer cell chemoresistance. The histone deacetylase/DNA methyltransferase inhibitor, parthenolide (PTL), has antitumor activity, but contrasting findings exist on its effect in normal cells. This study aims to examine the non-genomic toxic mechanisms of PTL in human erythrocytes. Methods: Cell death as stimulated by 20–200 μM of PTL for 24 h at 37°C was assessed using fluorescence-assorted cell sorting and spectrophotometric assays. Canonical markers of cell death, including membrane scrambling, oxidative stress, and Ca²⁺ mobilization, were captured by annexin V-fluorescein isothiocyanate, 2^′,7^′-dichlorodihydrofluorescein diacetate, and Fluo4/AM labeling, respectively. Rescue experiments using a wide array of inhibitors were also conducted. Results: PTL stimulated significant membrane scrambling and blebbing, and showed potent hemolytic activity coupled with significant elevations in dichlorofluorescein and Fluo4 fluorescence. While hemolysis was ameliorated by glutathione, caffeine, acetylsalicylic acid, staurosporin, necrosulfonamide, guanosine, and Ca²⁺ deprivation, it was rather exacerbated by necrostatin-2, tumor necrosis factor α (TNFα) or Fas ligand (FasL) neutralization, and concurrent Ca²⁺ deprivation and membrane depolarization. In contrast, eryptosis was attenuated by N-acetyl-cysteine, TNFα, or FasL blockade, and simultaneous Ca²⁺ elimination and KCl enrichment; and augmented by necrostatin-2, necrosulfonamide, and adenosine triphosphate. Interestingly, loss of volume was only prevented by melatonin, acetylsalicylic acid, and N(gamma)-nitro-L-arginine methyl ester. Conclusion: PTL stimulates oxidative hemolysis and eryptosis through Ca²⁺ mobilization and death ligand signaling involving the cyclooxygenase/protein kinase C/mixed lineage kinase domain-like pseudokinase axis. This research highlights the non-genomic toxic mechanisms of PTL and presents potential pharmacological targets for mitigating its adverse off-target effects.

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Supplementary Material

Supplementary Material File

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