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GPX4 Inhibition Enhances the Pro-Oxidant and ER Stress Effects of Tempol in Colon and Gastric Cancer Cell Lines
Gorkem Ozdemir1, Halil Mahir Kaplan2
1Department of Gastroenterological Surgery, Adana City Training and Research Hospital, 01230 Adana, Turkey.
Abstract:
Tempol, a synthetic nitroxide, exhibits dual antioxidant and pro-oxidant activity, requiring millimolar concentrations to induce oxidative stress, which limits its therapeutic use. Glutathione Peroxidase 4 (GPX4) is a critical lipid peroxidase that prevents ferroptosis, and its inhibition has emerged as a strategy to sensitize cancer cells to oxidative stress. To enhance Tempol's efficacy, we investigated its interaction with ML210, a GPX4 inhibitor, in human colon (HT29) and gastric (CRL-1739) cancer cell lines. We quantified cell viability, oxidative stress markers (H2O2, Total Oxidant Status (TOS), and Total Antioxidant Status (TAS)) and endoplasmic reticulum (ER) stress proteins (ATF6, GRP78, and IRE1α) in in vitro assays. Synergy was assessed using Bliss independence analysis. The combination of Tempol (2 mM) and ML210 (0.05 μM) markedly reduced viability in both cell lines. Bliss analysis revealed slight/moderate synergy for cytotoxicity (Δ = +0.15 in HT29; Δ = +0.26 in CRL-1739) and strong synergy for H2O2 accumulation (Δ = +1.92-2.23 across replicates). In contrast, TOS showed moderate-to-strong antagonism across both cell lines, and TAS demonstrated slight synergistic or antagonistic effects. ER stress markers exhibited marker and cell line specific synergy: ATF6 showed strong synergy, IRE1α slight synergy in both lines, and GRP78 activation was highly variable, showing strong synergy in CRL-1739 cells but moderate antagonism in HT29 cells. These findings indicate that the cooperative action of Tempol and ML210 is ROS-pool-specific and pathway-selective in the ER. These findings demonstrate that ML210 potentiates Tempol's pro-oxidant pressure by targeting GPX4, selectively amplifying H2O2 accumulation and ER stress engagement without collapsing global redox balance. This study provides mechanistic rationale for redox-proteostasis co-targeting in gastric and colon cancers and establishes a foundation for in vivo validation.
Insights
The combination of Tempol and ML210 synergistically enhances oxidative stress in cancer cells by inhibiting Glutathione Peroxidase 4 (GPX4), leading to reduced viability and increased hydrogen peroxide accumulation.
Area of Science:
- Oncology
- Biochemistry
- Cell Biology
Background:
- Tempol, a nitroxide, has limited therapeutic use due to its dual antioxidant/pro-oxidant activity requiring high concentrations.
- Glutathione Peroxidase 4 (GPX4) inhibition is a strategy to induce oxidative stress and sensitize cancer cells.
- Targeting GPX4 offers a potential avenue to enhance the efficacy of compounds like Tempol.
Purpose of the Study:
- To investigate the synergistic interaction between Tempol and ML210, a GPX4 inhibitor.
- To evaluate the combined effects on cancer cell viability, oxidative stress, and endoplasmic reticulum (ER) stress.
- To determine the mechanistic basis for enhanced efficacy in colon and gastric cancer cells.
Main Methods:
- Utilized human colon (HT29) and gastric (CRL-1739) cancer cell lines for in vitro assays.
- Quantified cell viability, oxidative stress markers (H2O2, TOS, TAS), and ER stress proteins (ATF6, GRP78, IRE1α).
- Assessed synergy using Bliss independence analysis.
Main Results:
- The Tempol-ML210 combination significantly reduced cancer cell viability with moderate synergy.
- Strong synergy was observed for hydrogen peroxide (H2O2) accumulation, while Total Oxidant Status (TOS) showed antagonism.
- Endoplasmic reticulum (ER) stress markers displayed pathway- and cell line-specific synergistic effects, with ATF6 and IRE1α showing synergy.
Conclusions:
- ML210 potentiates Tempol's pro-oxidant effects by inhibiting GPX4, selectively increasing H2O2 and ER stress.
- The combination therapy demonstrates ROS-pool-specific and pathway-selective action, avoiding global redox balance collapse.
- This study provides a mechanistic rationale for co-targeting redox homeostasis and proteostasis in gastric and colon cancers.
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