The Plant Steroid Hormone, Epibrassinolide, Reverses Drug Resistance in Small-Cell Lung Carcinoma Cells by Modulating Ferroptosis

Background: Small-cell lung cancer (SCLC) has a poor prognosis because of the development of drug resistance. Our previous studies showed that the plant steroid hormone, epibrassinolide (EB), interacts with human SCLC cells through inhibition of GSK3β-mediated signaling and overcomes drug resistance. However, the latter does not occur via membrane-mediated drug efflux, a commonly reported mechanism. Methods: We investigated a novel mechanism, ferroptosis, for drug resistance and the effects of EB. Pharmacological, cellular and molecular investigations were performed. We used drug-sensitive (NCI-H69) and multi-drug-resistant (VPA) SCLC cell lines. Results: 4-hydroxynonenal (HNE), a marker for lipid peroxidation and ferroptosis, was lower in drug-resistant VPA cells than drug-sensitive cells (H69) (22 vs. 60 pg/mg protein). Incubation with EB at a dose that reverses drug resistance in VPA cells increased HNE (94 pg/mg). Exogenously applied HNE increased intracellular HNE and reversed drug resistance (IC50 for etoposide in VPA cells at 7.5 µM (untreated) vs. 1.0 µM (treated)). Ferrostatin, which inhibits ferroptosis, reduced HNE accumulation and increased drug resistance (IC50 for etoposide at 5.5 µM (untreated) vs. 19 µM (treated)). As measured by phosphorylation, the signaling molecule Akt was more active in drug-resistant compared to drug-sensitive SCLC cells. This activation was accompanied by increased drug resistance. Both EB and a specific inhibitor of Akt phosphorylation/activation reversed this resistance. Akt activation was reflected in increased GSK3β activity. The latter was also inhibited by incubation in EB. The level of the GSK3β-controlled “antioxidant transcription factor” NRF2 was higher in drug-resistant VPA cells than drug-sensitive H69 cells. This increase was reversed in EB-treated cells. The protein level of the “antioxidant enzyme”, GPX4, which chemically reduces peroxidized lipids, was higher in VPA than H69 cells (21.2 vs. 10.1 pg/mg protein). Likewise, GPX4 enzyme activity was higher in drug-resistant than drug-sensitive cells. Incubation with EB reversed these increases. Incubation of SCLC cells with RSL3, a specific inhibitor of GPX4 activity, increased intracellular HNE and reversed drug resistance in VPA cells. Conclusions: These data indicate that ferroptosis is an important mechanism for drug resistance in SCLC cells and the plant steroid, EB, reverses drug resistance by interfering with the Akt signaling pathway and GPX4 anti-oxidant activity.