RSL3 - GPX4 inhibitor & Ferroptosis inducer

CAS #1219810-16-8 - InvitroFit™ - Cell culture-tested

RSL3 chemical structure

RSL3 is a strong and selective inhibitor of glutathione peroxidase 4 (GPX4). As a result, it induces ferroptosis, a form of non-apoptotic cell death [1]. 

 More details

Mode of action

RSL3 (RAS-selective lethal 3) is a potent inducer of ferroptosis that directly inhibits GPX4, a key enzyme responsible for reducing lipid peroxides and maintaining redox balance in cells. Originally identified in a screen for compounds selectively lethal to RAS-mutant cancer cells, RSL3 triggers ferroptotic cell death by inactivating GPX4, leading to the accumulation of toxic amounts of reactive oxygen species (ROS) and subsequent oxidative membrane damage [1]. 

Upon stimulation of our Ferroptosis reporter HT-1080 cells using RSL3, the cell membrane ruptures and the reporter protein HMGB1::Lucia is released in the extracellular milieu. Levels of HMGB1::Lucia in the supernatant can be readily monitored by measuring the light signal produced after the addition of QUANTI-Luc™ 4 Lucia/Gaussia, a Lucia® luciferase detection reagent (see figure). Moreover, ferroptosis in this cell line can be assessed using the classic cytotoxic lactate dehydrogenase (LDH) assay (see figure). 

RSL3-induced ferroptosis can be blocked by using Ferrostatin-1, a potent, synthetic ferroptosis inhibitor. 

Key features

• Each lot is functionally tested and validated.
• The absence of endotoxin is determined using the EndotoxDetect™ assay.
• InvitroFit™ grade: each lot is highly pure (≥95%) and functionally tested

InvivoGen's products are for research use only, and not for human or veterinary use.

Figures

RSL3 induces HMGB1::Lucia release in a dose-dependent manner. HT1080-HMGB1-Lucia™ cells were incubated with increasing concentrations of RSL3 (0.001 nM - 5 µM). After 48 hours, the induction of cell death was quantified by measuring the levels of HMGB1::Lucia in the supernatant using the QUANTI-Luc™ detection reagent. Data are shown as (A) fold induction over non-induced cells or (B) percentage (%) activity (mean ± SEM).

RSL3 induces LDH release in a dose-dependent manner. HT1080-HMGB1-Lucia™ cells were incubated with increasing concentrations of RSL3 (0.001 nM - 5 µM). After 48 hours, ferroptosis induction was quantified using the lactate dehydrogenase (LDH) assay. Data is shown as a percentage of cell death (mean ± SEM).

Specifications

Applications: Ferroptosis induction, GPX4 inhibition, cell death activation

Target: Glutathione peroxidase 4 (GPX4)

Synonym: RAS-selective lethal compound 3, (1S,3R)-RSL3, (1S,3R)-Methyl 2-(2-chloroacetyl)-2,3,4,9-tetrahydro-1-[4-(methoxycarbonyl)phenyl]-1H-pyrido[3,4-b]indole-3-carboxylate

CAS number: 1219810-16-8

Working concentration: 10 mM - 10 µM

Solubility: DMSO (20 mg/ml), insoluble in water or ethanol

Purity: ≥95%

Physical form: Dried powder

Chemical formula: C₂₃H₂₁CIN₂O₅

Molecular weight: 440.88 g/mol

Quality control: Each lot is functionally tested and validated.

Contents

RSL3 is provided as a dried powder. 

• inh-rsl3: 10 mg
   

RSL3 is shipped at room temperature.

Upon receipt, store at -20°C for up to 3 years.

 Resuspended product is stable for up to 1 month at -20 °C and up to 1 year at -80°C when properly stored.

 Avoid repeated freeze-thaw cycles.

Details

RSL3-induced ferroptosis

Ferroptosis overview

In 2012, Dixon et al. [1] discovered a novel form of non-apoptotic cell death characterized by iron overload and accumulation of lethal lipid peroxidation [2]. Because iron chelators were shown to block this type of regulated cell death, it was originally defined as iron-dependent and was called "Ferroptosis" [1]. In contrast to apoptosis, necrosis, and autophagy, ferroptosis is characterized by the build-up of lipid reactive oxygen species (ROS) triggering membrane damage. Cells undergoing ferroptosis do not exhibit classic apoptosis-like features, such as chromatin condensation or membrane blebbing. Instead, mitochondrial shrinkage and increased membrane density are often observed [1-2]. 

Ferroptosis is regulated by a complex network involving iron homeostasis, lipid metabolism, and glutathione-dependent oxidative-reductive balance [3-4]. Iron metabolism plays a central role, as excessive intracellular iron promotes the Fenton reaction, generating lethal amounts of ROS that drive lipid peroxidation [4]. Moreover, the enzyme glutathione peroxidase 4 (GPX4) is a key regulator that protects cells from ferroptosis by reducing lipid peroxides. GPX4 activity is dependent on Glutathione (GSH) which is synthesized using cystine imported via the cystine-glutamate antiporter, System Xc−. When System Xc− is inhibited, cystine uptake decreases, leading to GSH depletion and GPX4 inactivation. As a result, lipid peroxides accumulate in the cells ultimately leading to ferroptotic cell death [2-4]. 
 

Ferroptosis in disease

Ferroptosis is involved in various diseases, including neurodegenerative disorders, organ injury-related conditions, and cancer [2-3]. In neurodegenerative diseases, such as Parkinson's and Alzheimer's, ferroptosis contributes to neuronal loss through iron deposition in the brain and oxidative stress [3]. It has also been identified as a major pathogenic driver in other organ injury-related disorders including acute kidney injury and COVID-19-induced myocarditis [3]. Studies have shown that using ferroptosis-specific inhibitors can protect against severe tissue damage in these cases [3]. On the other hand, triggering ferroptosis is being explored as a potential therapeutic approach for treating therapy-resistant cancers [1-4]. Certain types of cancer, especially those with high iron levels or deficiencies in GPX4, are particularly sensitive to ferroptosis-inducing substances. Compounds like RSL3 or Erastin hold promise to overcome drug resistance in chemotherapy and immunotherapy [2]. Advancing our understanding of ferroptosis will continue to drive progress in cell death research and therapeutic development.

References

1. Dixon SJ, et al., 2012. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012 May 25;149(5):1060-72.
2. Du Y, Guo Z. 2022. Recent progress in ferroptosis: inducers and inhibitors. Cell Death Discov. 8(1):501.
3. Sun S, et al., 2023. Targeting ferroptosis opens new avenues for the development of novel therapeutics. Signal Transduct Target Ther. 8(1):372.
4. Li J, et al., 2020. Ferroptosis: past, present and future. Cell Death Dis. 11(2):88.