Potent ferroptosis agent RSL3 induces cleavage of Pyroptosis-Specific gasdermins in Cancer cells

Abstract

Ferroptosis is a form of iron-dependent cell death of interest for the development of novel anti-cancer therapies. Ferroptosis research uses a process of elimination based on assumed ferroptosis-specific inducers and inhibitors; these molecules however have off-target effects and cannot provide a comprehensive picture of overlapping pathways. We investigated whether pyroptosis-a form of inflammatory cell death-is initiated in cancer cells following treatment with the ferroptosis inducer RSL3. We treated 6 cancer cell lines with RSL3 alone or in combination with inhibitors of ferroptosis (Ferrostatin-1), caspases (zVADfmk), necroptosis (Necrostatin-1), BID (BI-6C9), or STING (H-151). Biomarkers of pyroptosis and ferroptosis were assessed using our novel quantitative multiplex immunoassay. Increased secretion of pyroptosis-associated cytokines (IL-1α, IL-1β, IL-18), and gasdermin D and E (GSDMD/E) cleavage with parallel loss of respective full-length proteins-both hallmarks of pyroptosis-were recorded in 5/6 cell lines following RSL3 treatment. RSL3 cytotoxicity was blocked by Ferostatin-1; BID and STING inhibitors also prevented GSDMD/E cleavage. We conclude that the ferroptosis-inducer RSL3 triggers pyroptosis in cancer cells; further work is required to elucidate the role of mitochondria in this process. Measurement of pathway-specific protein biomarkers is therefore necessary to identify the exact mechanism of action of novel cytotoxic agents.

Keywords: Cancer; Cytokine; Ferroptosis; Gasdermin; Pyroptosis; RSL3.

Fig. 1

RSL3 Induces Necrotic Morphologies in Cancer Cell Lines. (A) RSL3 induces necrotic morphology in 6 cancer cell lines which is prevented by the lipophilic antioxidant and ferroptosis inhibitor Fer-1. This morphology is characterized by a swollen, rounded appearance with cellular organelles compressed to one side. Scale bars for SU-DHL5: 100 μm; all other scale bars: 200 μm. (B) THP-1 macrophages primed with LPS and treated with 20 µM nigericin for 30 min to induce pyroptosis display the same basic morphological features as RSL3-treated cells.

Fig. 2

RSL3 Induces Gasdermin Cleavage in Cancer Cells and Pyroptosis is Partially Prevented by Fer-1 and Nec-1, but Not by Caspase Inhibition. (A) ATP levels relative to vehicle control following 1 day of RSL3 treatment with or without ferroptosis inhibitor Fer-1, pan-caspase inhibitor (zVADfmk), or RIPK1 and IDO1/2 inhibitor (Nec-1) co-treatment. All N ≥ 3. Error bars: SEM. Probability values: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. (B) Full-length and cleaved GSDMD and GSDME levels relative to vehicle control following 1 day of RSL3 treatment with or without ferroptosis inhibitor Fer-1, pan-caspase inhibitor (zVADfmk), or RIPK1 and IDO1/2 inhibitor (Nec-1) co-treatment. All N ≥ 3. Error bars: SEM. Probability values: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Fig. 3

BID Activation and STING Signaling Are Critical in RSL3-induced Pyroptosis. (A) ATP levels relative to vehicle control following 1 day of RSL3 treatment with or without STING inhibitor (H-151) or BID inhibitor (BI-6C9) co-treatment. All N ≥ 3. Error bars: SEM. (B) Full-length and cleaved GSDMD and GSDME levels relative to vehicle control following 1 day of RSL3 treatment with or without H-151 or BI-6C9 co-treatment. All N ≥ 3. Error bars: SEM. Probability values: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Fig. 4

RSL3 Induces Secretion of Pyroptosis-Associated Cytokines. Cells treated with 2 concentrations of RSL3 (see Supplemental Table S1) for 4 h and 24 h secrete at least 1 pyroptosis-associated cytokine in all but the SU-DHL-5 cell line. N ≥ 3 except for IL-1β plots where N = 2 for 4 h treatments (MDA-MB-231 and BxPC3) and 24 h treatments (MDA-MB-231) due to IL-1β being below detection limits in vehicle samples. Error bars: SEM. Probability values: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.