Fig 2: Auranofin enhances the sensitivity to sorafenib-induced apoptosis via inhibition of TXNRD1 in PCK1-KO cells in vitro. (A) Cell proliferation curves. PCK1-KO hepatoma cells were treated with 0.6 μmol/L auranofin or 5 μmol/L sorafenib alone or in combination. Cells were plated into 96-well plates at 1 × 103 cells/well and were counted every 24 h in triplicate. Data are presented as the mean ± SD; *P < 0.05 vs. Sorafenib alone. (B) ROS were monitored by fluorescent labeling (red) and nuclei were stained by DAPI (blue) in cells treated with sorafenib and auranofin alone or in combination. Scale bar = 10 μm. (C) Protein expression of Caspase3/9, Cleaved caspase3/9, Bax, Bcl2, TXNRD1, and PCK1. PCK1-KO hepatoma cells were treated as in Figure 4A for 48 h and the proteins were isolated and analyzed by western blotting. GAPDH expression served as an internal control. (D) Flow-cytometric analysis of apoptosis was performed after treatments with sorafenib and/or auranofin. (E) Quantification of cell death detected in Figure 4C. Cell apoptosis as detected by PI staining. Values represent the mean percentage of dead cells ± SD from three samples (*P < 0.05,***P < 0.001). (F) Schematic illustration showing the PCK1–NADPH–ROS-TXNRD1 axis. In HCC, decreased PCK1 suppressed the generation of NADPH via pentose phosphate pathway (PPP). Subsequently, ROS, which is quenched by NADPH, was increased, further promoted nuclear translocation of Nrf2 and the expression of TXNRD1. The cropped blots are used in the figure and full length blots are presented in Figure S6.

Fig 3: Combined ferroptosis induction and TRX pathway inhibition demonstrates broad anti-tumor activity across SCLC NE subtypes in vivo and serves as prognostic marker set in human SCLC.a Eight-weeks-old male nude mice were injected with 50:50 mixed 181.5 stickers and floaters at a total of 1.5 × 106 cells into both flanks. Once palpable, tumors were treated either with vehicle (n = 14), Auranofin [2.5 mg/kg] (n = 10) 3× per week i.p., BSO [5 mM] (n = 10) in the drinking water or the combination (combo) (n = 14) for 2 consecutive weeks. Fold change of initial tumor size is shown. Boxplot center line, mean; box limits, upper and lower quartile; whiskers min. to max. b Sections from paraffin-embedded tumors stained for ASCL1. Representative images are shown, scale bar = 100 µm. c ASCL1 H-score was quantified, n (vehicle) = 14, n (BSO) = 9, n (Aura) = 10, n (combo) = 14. d Eight to twelve-week-old RP mice were inhaled intratracheally with 2.5 × 107 plaque-forming units (PFU) Adeno-Cre virus to initiate SCLC development. e Tumor-bearing RP-mice were treated either with vehicle (n = 8) or combined BSO [5 mM] in the drinking water and Auranofin 3× per week i.p. [2.5 mg/kg] (n = 7) for 2 consecutive weeks. Fold change in tumor volumes was determined by quantifying initial tumor volume from MRI scans as compared to tumor volume at the end of the treatment cycle using Horos software. f Representative MRI images pre and post treatment of mice as in (e). g Isolation scheme of human CDXs. h Cellular morphology of human CDXs, scale bar = 400 µm. i Two CDXs or two healthy donor PBMCs were treated with DMSO, BSO [PBMCs, 500 μM; CDXs, 50 μM], Auranofin [250 nM] or BSO [PBMCs, 500 μM; CDXs, 50 μM]/Auranofin [250 nM] for 24 h, cell viability was quantified by Cell Titer Blue (CDXs) or flow cytometric quantification of propidium iodide (PI)-negative cells (PBMCs). j Kaplan–Meier survival curves for SCLC patients (n = 77)7 containing low (low 1/3 n = 25, median survival 33 months) or high (high 2/3 n = 52, median survival 22.5 months) expression of GPX4 mRNA. k As in (j) expression of TXNRD1 mRNA was correlated using the same cut-off (low = 1/3, median survival 38 months; high 2/3, median survival 22.5 months). l Kaplan–Meier survival curves for SCLC patients with combined low or high GPX4 and TXNRD1 mRNA expression (low/low n = 10, median survival 43.5 months; high/high n = 16, median survival 16 months). Data are means ± SEM were applicable (i). Schemes were drawn with fully licensed Biorender.com. Two-tailed unpaired t tests (a, c), or two-sided log-rank (Mantel–Cox) test (j–i), ***p < 0.001, *p < 0.05, ns p > 0.05. Source Data are provided as Source data file.

Fig 4: The upregulated TXNRD3 in sorafenib-resistant cells was located in the mitochondria. (A) Comparison of mRNA expression levels of TXNRD1, TXNRD2, TXNRD3 in sorafenib-sensitive (MV4-11) and -resistant (MV4-11R) cells, measured by RT-qPCR. (B) Comparison of protein expression levels of TXNRD1, TXNRD2, TXNRD3 in sorafenib-sensitive (MV4-11) and -resistant (MV4-11R) cells, measured by western blotting analyses using the respective antibodies. (C) Comparison of amino acid sequences of two possible TXNRD3 splicing variants. (D) Mitochondrial and cytosolic proteins were prepared and analyzed by immune blotting analyses using the indicated antibodies. β-actin and HSP60 were used as the loading controls for cytosolic and mitochondrial proteins, respectively. “Mito” and “Cyto” indicate mitochondrial and cytosolic, respectively. (E) Isolated mitochondria were incubated with trypsin for 2 min at 37 °C, and then resuspended in protein lysis buffer and determined by immunoblotting analyses using the indicated antibodies. (F) The TrxR activity in MV4-11R cells compared with the parental MV4-11 cells. Data represent mean ± SD; **, p < 0.01, n = 3.

Fig 5: PCK1 inhibits tumor growth and oxidative stress in xenograft tumors. MHCC-97H cells were infected with AdGFP or AdPCK1 or mock-infected for 24 h, then subcutaneous injected into nude mice (n = 6). (A) Representative image of tumor in all groups (presented two tumors each group). (B) The growth curve of subcutaneous xenograft tumor. Tumor volumes were measured every 4 days and calculated as length × (square of width)/2 (repeated-measures analysis of variance, *P < 0.05 compared to AdGFP group, error bars represent mean ± SD, n = 6). (C) Tumor size of subcutaneous xenografts after dissected. (D) ROS monitored by immunofluorescent labeling (red) and nuclei by DAPI (blue) staining in xenografts. Scale bar = 200 μm. (E) Expression of PCK1 and TXNRD1 checked by western blot assay in xenograft tumor samples. Semi-quantitative densitometry graphs of immunoblots (arbitrary densitometry units) for PCK1 and TXNRD1 levels normalized to β-actin expression are shown. (*P < 0.05, **P < 0.01, AdPCK1 group vs. AdGFP group). The cropped blots are used in the figure and full length blots are presented in Figure S5.