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.

Keywords: Timosaponin AIII; ferroptosis; glutathione peroxidase 4; heat shock protein 90; non-small cell lung cancer.

Figure 1

Tim-AIII inhibits proliferation, induces death, and leads to G2/M cycle arrest in NSCLC cells. A Viability of the H1299 and A549 cells was measured after treatment with different concentrations of Tim-AIII in different time periods by the CCK-8 assay. B The OD value of LDH in the supernatant of H1299 and A549 cells after treatment with different concentration of Tim-AIII in different time periods. C Representative results of monolayer culture in H1299 and A549 cells. D Quantitative analysis of colony numbers in H1299 and A549 cells. E Representative results of cell cycle in H1299 and A549 cells. F Quantitative analyzes of G2/M in H1299 and A549 cells. Quantitative data were presented as mean ± SD. ^*p< 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 2

Tim-AIII suppresses the migration of NSCLC cells. A Representative result of wound healing assays in H1299 and A549 cells after treatment with Tim-AIII for 24 h. B Crystal violet stain of H1299 and A549 cells crystal violet stain (200×) after treatment with Tim-AIII for 24 h. C Quantification of the migration number of H1299 and A549 cells. D, E The expression of several migration-related genes, VIM, E-Cadherin, SNAIL-2, MMP-9, SNAIL-1, was examined by RT-PCR after treatment with Tim-AIII for 48 h in H1299 and A549 cells. F On the left side, the expression of various migration-related protein, E-cadherin, Vimentin, Snail-1, Snail-2, N-cadherin, was examined by WB after treatment with Tim-AIII for 48 h in H1299 and A549 cells. On the right is the statistics of appeal protein. Quantitative data were presented as mean ± SD. ^*p< 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 3

Tim-AIII-induced cell death is mainly caused by ferroptosis in H1299 cells. A-C H1299 cells were cotreated with Tim-AIII with or without Z-VAD-FMK, Nec-1 and CQ for 48 h, and cell vitality was assayed by the CCK-8 assay. D The lipid ROS level was analyzed using a flow cytometer after treatment with Tim-AIII for 48 h in H1299 cells. E The intracellular iron level after Tim-AIII treatment for 48 h in H1299 cells. F Intracellular MDA levels after treatment with Tim-AIII for 48 h in H1299 cells. G The intracellular GSH levels after Tim-AIII treatment for 48 h in H1299 cells. H-I H1299 cells were co-treated with Tim-AIII with or without the ROS inhibitor NAC and trolox for 48 h, and cell viability was assessed by CCK-8. J The expression of several key ferroptosis regulators, such as FLT, HMOX-1, GPX4, SLC40A1, SLC7A11, was examined by WB in H1299 cells. K Representative results of flow cytometry and quantification of mitochondrial membrane potential (JC-1 monomers) after treatment with Tim-AIII for 48 h in H1299 cells. L TME was used to observe mitochondrion in H1299 cells. M H1299 cells were cotreated with Tim-AIII with or without Fer-1 for 48 h, and cell vitality was assayed by the CCK-8 assay. Quantitative data were presented as mean ± SD. ^*p< 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 4

HSP90 is probably responsible for Tim-AIII-induced ferroptosis in NSCLC cells. A The most likely predicted targets of Tim-AIII in HSP90. B. SPR analysis of Tim-AIII binding to wild type HSP90 (K_d =28.2 μmmol/L). C SPR analysis of Tim-AIII binding to mutant HSP90 (K_d=104.6 μmmol/L) D The protein expression of HSP90 was examined by WB after treatment with Tim-AIII for 48 h in H1299 and A549 cells. E The expression of HSP90 were examined by RT-PCR after treatment with Tim-AIII for 48 h in H1299 and A549 cells. F H1299 and A549 cells were co-treated with Tim-AIII with or without the HSP90 classic inhibitor (tanespimycin) for 48 h, and cell vitality was assayed by the CCK-8 assay. Quantitative data were presented as mean ± SD. ^*p< 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 5

Tim-AIII-HSP90 complex-induced ferroptosis in NSCLC cells was achieved by targeting and degrading GPX4. A The protein expression of HSP90 and GPX4 was examined by WB after treatment with Tim-AIII with or without tanespimycin for 48 h in H1299 and A549 cells. B Immunoprecipitation was performed to detect the interaction between GPX4 and HSP90, and WB was performed to detect HSP90 levels in the inputs and immunoprecipitates in treatment with Tim-AIII (4 μM) for 48 h in H1299 and A549 cells. C Tim-AIII was added into H1299 and A549 cells and incubated for 48 h. Then, 10 μM MG-132 was added to inhibit the protein degradation for 4 h. The proteins were collected and incubated GPX4 antibody level of shaking 4 ℃ overnight, then agarose G was added and co-incubated at 4 °C for 3h. The ubiquitination level of GPX4 was analyzed by WB assay with anti-Ubiquitination antibody. D The expression of GPX4 was examined by WB after incubating with HSP90 in the presence or absence of Tim-AIII (4 μM). E The protein expression of the GPX4 were examined by WB after incubation with HSP90 on the usage of different amounts of Tim-AIII. F WB analysis of the GPX4 expression treated with DMSO or Tim-AIII (4 μM) for the indicated time points in the presence of CHX (25 mg/mL) in H1299 cells on the left side. Quantification of GPX4 intensity, the abundance was normalized to β-actin on the right side. G WB analysis of the GPX4 expression treated with DMSO or Tim-AIII (4 μM) for the indicated time points in the presence of CHX (25 mg/mL) in A549 cells on the left side. Quantification of GPX4 intensity, the abundance was normalized to β-actin on the right side. Quantitative data were presented as mean ± SD. ^*p< 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 6

Ferroptosis induced by the Tim-AIII was dependent on HSP90. A The transfection efficiencies of HSP90-shRNA in H1299 and A549 cells were evaluated by observing the fluorescence of GFP (200X). B The expression of HSP90 protein in H1299 and A549 cells after transfection was detected by WB. C H1299 and A549 cells were transfected with control-shRNA and HSP90-shRNA, followed by Tim-AIII (4 μM) treatment for 48 h, and cell vitality was assayed by the CCK-8 assay. D H1299 and A549 cells were transfected with control-shRNA and HSP90-shRNA, followed by Tim-AIII (4 μM) treatment for 48 h, and the OD value of LDH was assayed. E-G H1299 and A549 cells were transfected with control-shRNA and HSP90-shRNA, followed by Tim-AIII (4 μM) treatment for 48 h, and the level of intracellular iron, MDA and GSH were assayed. H H1299 and A549 cells were transfected with control-shRNA and HSP90-shRNA, followed by Tim-AIII (4 μM) treatment for 48 h, the GPX4, HSP90 and the ubiquitination level of GPX4 were analyzed by WB assay with anti-Ubiquitination antibody. Quantitative data were presented as mean ± SD. ^*p< 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 7

Tim-AIII suppresses tumor progression in vivo. A C57BL/6J mice that carried tumor derived from LLC cells subcutaneous xenografts tumor were administered Tim-AIII (low dose: 12.5 mg/kg or high dose: 50 mg/kg) or PBS every other day. B Representative image of the subcutaneous tumor of xenografts in different groups of C57BL/6J mice. C The tumor curves of C57BL/6J mice in different groups. D The weights of the tumor derived from LLC cells subcutaneous xenografts tumor. E Body weights of the C57BL/6J mice during the experimental period. F BALB/c-nu/nu nude mice that carried tumor derived from H1299 cells subcutaneous xenografts tumor were administered Tim-AIII (low dose: 12.5 mg/kg or high dose: 50 mg/kg) or PBS every other day. G Representative image of the subcutaneous tumor of xenografts in different groups of BALB/c-nu/nu nude mice. H The tumor curves of BALB/c-nu/nu nude mice in different groups. I C57BL/6J mice with LLC cells-derived lung metastasis tumor through the tail vein were intraperitoneal injection administered intraperitoneally Tim-AIII (Low dose: 12.5 mg/kg or high dose: 50 mg/kg) or PBS every other day. J Representative image of the lung of C57BL/6J mice in different groups. K The weights of the lung of C57BL/6J mice in different groups. L Representative flow cytometry results of Treg cells in the tumor microenvironment of LLC cells derived subcutaneous xenografts tumor microenvironment. M Quantitative analyses of Treg cells in LLC cells-derived subcutaneous xenografts. N The protein expression of HSP90 and GPX4 was examined by WB in LLC cells-derived subcutaneous xenografts tumor. Quantitative data were presented as mean ± SD. ^*p< 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 8

Scheme showing the central role of Tim-AIII in ferroptosis induction and inhibition of cell migration.