Abstract

Simple SummaryFerroptosis and necroptosis are two non-apoptotic programmed cell death pathways with increasing therapeutic potential. The isothiocyanate sulforaphane (SFN) is a well-known naturally derived anticancer compound with remarkable pro-apoptotic activity. Its ability to promote non-apoptotic cell death mechanisms remains poorly investigated. This work discovered that SFN activates apoptosis and ferroptosis dose-dependently in acute myeloid leukemia cells. At lower concentrations, SFN induces caspase-dependent apoptosis. At higher concentrations, ferroptosis is activated and accompanied by the depletion of intracellular glutathione (GSH) and decreased GSH peroxidase 4 protein expression levels. Necroptosis, instead, is not involved in SFN-induced cell death. Considering that cancer cells resist pro-apoptotic treatments, SFN’s ability to induce different types of cell death delineates it as a promising anticancer agent.In recent years, natural compounds have emerged as inducers of non-canonical cell death. The isothiocyanate sulforaphane (SFN) is a well-known natural anticancer compound with remarkable pro-apoptotic activity. Its ability to promote non-apoptotic cell-death mechanisms remains poorly investigated. This work aimed to explore the capacity of SFN to induce non-apoptotic cell death modalities. SFN was tested on different acute myeloid leukemia cell lines. The mechanism of cell death was investigated using a multi-parametric approach including fluorescence microscopy, western blotting, and flow cytometry. SFN triggered different cell-death modalities in a dose-dependent manner. At 25 μM, SFN induced caspase-dependent apoptosis and at 50 μM ferroptosis was induced through depletion of glutathione (GSH), decreased GSH peroxidase 4 protein expression, and lipid peroxidation. In contrast, necroptosis was not involved in SFN-induced cell death, as demonstrated by the non-significant increase in phosphorylation of receptor-interacting protein kinase 3 and phosphorylation of the necroptotic effector mixed lineage kinase domain-like pseudokinase. Taken together, our results suggest that the antileukemic activity of SFN can be mediated via both ferroptotic and apoptotic cell death modalities.

Highlights

  • Worldwide cancer incidence continues to rise, causing a great deal of mental, social, and financial pressure on patients, families, societies, and health systems [1]

  • Necroptosis is regulated by receptor-interacting protein (RIP)1, RIP3, and mixed lineage kinase domain-like pseudokinase (MLKL) [6]

  • SFN, necrostatin-1, ferrostatin-1, and N-acetylcysteine were purchased from Merck Millipore (Darmstadt, Germany); N-Benzyloxycarbonyl-Val-Ala-Asp (O-Me) fluoromethyl ketone (Z-VAD-FMK) was obtained from Calbiochem, EMD Millipore Corp. (Billerica, MA, USA); necrosulfonamide was purchased from Tocris Bioscience (Bristol, UK); (1S,3R)-RSL3 (RSL3) and SM164 were obtained from Cayman Chemical (Ann Arbor, MI, USA); tumor necrosis factor α (TNF-α) was purchased from PeproTech GmbH (Hamburg, Germany)

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Summary

Introduction

Worldwide cancer incidence continues to rise, causing a great deal of mental, social, and financial pressure on patients, families, societies, and health systems [1]. Ferroptosis is characterized by the intracellular accumulation of lipid peroxides caused by the direct inhibition of glutathione peroxidase 4 (GPX4). The inhibition of the GPX4 activity prompts lipid reactive oxygen species (ROS) accumulation, triggering ferroptotic cell death [5]. Several natural compounds trigger non-canonical cell-death pathways [9]. Iida and colleagues demonstrated that SFN triggers ferroptosis in NCI-H69 small-cell lung cancer cells [25]. This finding paves the way to the possibility that SFN can trigger apoptosis and multiple non-apoptotic cell deaths. Its pleiotropic nature prompted us to explore its ability to trigger non-canonical cell death in two AML cell lines with and without Fms-like tyrosine kinase (FLT) mutations

Cell Cultures
Chemicals and Treatments
Analysis of Nuclear Morphology by Fluorescence Microscopy
Measurement of Reduced GSH
Malondialdehyde Assay
Whole Cell Extracts and Immunoblotting
Statistical Analysis
Findings
SFN Promotes Ferroptosis but Not Necroptosis in U-937 Cells
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