Timosaponin AIII promotes non-small-cell lung cancer ferroptosis through targeting and facilitating HSP90 mediated GPX4 ubiquitination and degradation

Abstract

Timosaponin AIII (Tim-AIII), a steroid saponin, exhibits strong anticancer activity in a variety of cancers, especially breast cancer and liver cancer. However, the underlying mechanism of the effects of Tim-AIII-mediated anti-lung cancer effects remain obscure. In this study, we showed that Tim-AIII suppressed cell proliferation and migration, induced G2/M phase arrest and ultimately triggered cell death of non-small cell lung cancer (NSCLC) cell lines accompanied by the release of reactive oxygen species (ROS) and iron accumulation, malondialdehyde (MDA) production, and glutathione (GSH) depletion. Interestingly, we found that Tim-AIII-mediated cell death was reversed by ferroptosis inhibitor ferrostatin-1 (Fer-1). Meanwhile, the heat shock protein 90 (HSP90) was predicted and verified as the direct binding target of Tim-AIII by SwissTargetPrediction (STP) and surface plasmon resonance (SPR) assay. Further study showed that Tim-AIII promoted HSP90 expression and Tim-AIII induced cell death was blocked by the HSP90 inhibitor tanespimycin, indicating that HSP90 was the main target of Tim-AIII to further trigger intracellular events. Mechanical analysis revealed that the Tim-AIII-HSP90 complex further targeted and degraded glutathione peroxidase 4 (GPX4), and promoted the ubiquitination of GPX4, as shown by an immunoprecipitation, degradation and in vitro ubiquitination assay. In addition, Tim-AIII inhibited cell proliferation, induced cell death, led to ROS and iron accumulation, MDA production, GSH depletion, as well as GPX4 ubiquitination and degradation, were markedly abrogated when HSP90 was knockdown by HSP90-shRNA transfection. Importantly, Tim-AIII also showed a strong capacity of preventing tumor growth by promoting ferroptosis in a subcutaneous xenograft tumor model, whether C57BL/6J or BALB/c-nu/nu nude mice. Together, HSP90 was identified as a new target of Tim-AIII. Tim-AIII, by binding and forming a complex with HSP90, further targeted and degraded GPX4, ultimately induced ferroptosis in NSCLC. These findings provided solid evidence that Tim-AIII can serve as a potential candidate for NSCLC treatment.

Steroidal Saponins: Naturally Occurring Compounds as Inhibitors of the Hallmarks of Cancer

“…It acts by binding to HSP90, which consequently leads to the degradation of glutathione peroxidase 4 (GPX4)-a ferroptosis inhibitor. Around 90% of all NSCLC cells were eliminated via ferroptosis in this investigation [46]. Polyphyllin B was shown to be mechanistically similar to dioscin when examined against gastric cancer in vitro.…”

Polyphyllin VII induces autophagy‐dependent ferroptosis in human gastric cancer through targeting T‐lymphokine‐activated killer cell‐originated protein kinase

“…The lipid ROS levels were assayed using a lipid ROS assay kit (Thermo Fisher Scientific, USA). Cells were collected and stained with the BODIPY‐C11 dye (Zhou et al, 2023). Flow cytometry was used for the detection of lipid ROS according to the manufacturer's instructions.…”

“…The malondialdehyde (MDA) levels were detected using an MDA assay kit (Beyotime, China). Cells were lysed using RIPA lysis buffer to obtain the supernatant (Zhou et al, 2023). The MDA levels were detected using a fluorescence microplate reader according to the manufacturer's instructions.…”

Ferroptosis: emerging roles in lung cancer and potential implications in biological compounds

“…They can also regulate tumor related immune responses and improve the prognosis of anti-tumor therapy. The latest researches on the promotion of ferroptosis in lung cancer by biological compounds show that they mainly block the progression in the G2/M phase and cause a decrease in the number of G0/G1 and S phase cells ( Chen et al, 2020a ; Luo and Xu, 2022 ; Wu et al, 2022 ; Zhou et al, 2023a ). Several key factors, such as glutathione peroxidase 4 (GPX4), reactive oxygen species (ROS), glutathione (GSH), solvecarrierfamily7, and (ionicamino acidtransporter, y + system) member11 (SLC7A11), play important roles in this process.…”