Table 1.
Ferroptosis inducers and cell death inhibitors.
Table 2.
Antibodies used for western blot analyses.
Fig 1.
Cell viability is attenuated in a cell line—and substance—specific manner in BTC following FINs treatment.
(A) Cell viability of 10 BTC cell lines and non-cholangiocyte cell line MMNK-1 following a 10-step 1:2 dilution series starting with 10 μM brequinar, 40 μM FIN56, 10 μM FINO2, 50 μM iFSP1, 50 μM IKE and 10 μM RSL3 treatment for 48 h; Data is related to untreated control cells (UTC) and shown as mean values +/- SEM of n = 3 biological replicates. (B) Heatmap of calculated IC25/50 values (via four-parameter logistic expression) of FINs in BTC cell lines.
Fig 2.
Reduction of cell viability via FINs is (partially) reversed by deferoxamine mesylate and ferrostatin-1.
Cells were treated with 1 μM brequinar, 10 μM FINO2, 50 μM iFSP1, 50 μM IKE and 10 μM RSL3 with 20 μM of deferoxamine mesylate, ferrostatin-1, necrostatin-1 and Z-VAD-FMK. (A) Cell viability data is shown as mean values +/- SEM of n = 3 biological replicates of CCC-5 cells with FINs (light grey) and FINS + cell death inhibitors (dark grey) treatment for 24 h. (B) Cell viability data is shown as mean values +/- SEM of n = 3 biological replicates of HuH-28 cells with FINs (light grey) and FINS + cell death inhibitors (dark grey) for 24 h. (C) Cell viability data is shown as mean values +/- SEM of n = 3 biological replicates of KKU-055 cells with FINs (light grey) and FINS + cell death inhibitors (dark grey) for 24 h. (D) The effect of the different cell death inhibitors on the FIN-induced cell death was expressed by the positive/negative difference (Delta% Fig 2A–2C) related to FIN-only (green: “rescue effect”; red: viability is further decreased) * = significant p < 0.05, ** = highly significant p < 0.01; UTC = untreated control # = these values don’t represent a definitive ‘rescue effect’ since treatment with these FINS alone did not result in a decrease of cell viability.
Fig 3.
Fe2+ and lipid ROS levels are elevated following FIN treatment in selected BTC cell lines.
Cells were treated with 1 μM brequinar, 10 μM FINO2, 50 μM iFSP1, 50 μM IKE and 10 μM RSL3. (A) Intracellular iron levels after FIN treatment for 24 h in CCC-5, HuH-28 and KKU-055 cells. Data is related to untreated control cells and shown as mean +/- SEM of n = 3 biological replicates. (B) Lipid ROS levels after FIN treatment for 24 h in CCC-5, HuH-28 and KKU-055 cells. Data is related to untreated control cells and shown as mean +/- SEM of n = 3 biological replicates. (C) Caspase 3/7 activity after FIN treatment for 24 h in CCC-5, HuH-28 and KKU-055 cells. Data is related to untreated control cells and shown as mean +/- SEM of n = 3 biological replicates. * = significant p < 0.05, ** highly significant p < 0.01; UTC = untreated control.
Fig 4.
Baseline protein expression of ACSL4, CD71, FTH1, GPX4 and xCT; GPX-activity, intracellular Fe2+ levels and NADP(H) concentration in BTC cell lines.
(A) Baseline protein expression of ACSL4, CD71, FTH1, GPX4 and xCT in BTC cells with representative western blot images. Shown are data as mean values +/- SEM of n = 3 biological replicates relative to β-actin expression. (B) Baseline GPX-activity of BTC cells. Data is shown as mean +/- SEM of n = 3 biological replicates. (C) Baseline intracellular Fe2+ levels of BTC cells. Data is shown as mean +/- SEM of n = 3 biological replicates (D) Baseline NADP(H) concentration in BTC cells. Data is shown as mean +/- SEM of n = 3 biological replicates; # = not quantifiable in all 3 biological replicates.
Fig 5.
CD71 and SLC7A11 protein expressions significantly correlate with IC50 values of IKE and RSL3 in BTC cell lines.
Correlation analysis between the IC25/50 values of FINs and ferroptosis associated genes/markers. Green boxes indicate a positive correlation, red boxes indicate a negative correlation, bold fond shows a significance correlation (p < 0.05).
Fig 6.
In silico analysis of CD71 and SCL7A11 expression in BTC patients.
The results of the different in silico analyses (A to C with OncoDb and UALCAN as well as D with STRING (last accessed 2023-10-10)) showed a significant upregulation of CD71 (TFRC) and SLC7A11 in BTC samples compared to control tissue with a positive correlation between them (A). Furthermore, the expression of SLCA11 was not significantly associated with worse clinicopathological characteristics such as grading, disease status and outcome compared to CD71 (B (CD71 not shown) and C). Protein-protein interaction analysis identified non-ferroptosis beta-2-microglobulin (B2M), CD44 antigen and heat shock protein 8 (HSPA8) as possible interactors between SLC7A11 and CD71, in addition to other "classical" proteins of the iron and/or ferroptosis-related pathways (D). Abbreviations: B2M: Beta-2-microglobulin, CD44: CD44 antigen, FTH1: Ferritin heavy chain 1, HFE: High FE2+ (alias hereditary hemochromatosis protein), HJV: Hemojuvelin, HSPA8: Heat shock 70 kDa protein 8, IREB2: Iron-responsive element-binding protein 2, SLC11A2: Solute carrier family 11 member 2 (alias natural resistance-associated macrophage protein 2), SLC3A2: Solute carrier family 3 member 2 (alias 4F2 cell-surface antigen heavy chain), SLC7A11: solute carrier family 7 member 11 (alias cystine/glutamate transporter), TF: Transferrin and TFRC: Transferrin receptor (alias CD71).