Depletion of Labile Iron Induces Replication Stress and Enhances Responses to Chemoradiation in Non-Small-Cell Lung Cancer

Abstract

The intracellular redox-active labile iron pool (LIP) is weakly chelated and available for integration into the iron metalloproteins that are involved in diverse cellular processes, including cancer cell-specific metabolic oxidative stress. Abnormal iron metabolism and elevated LIP levels are linked to the poor survival of lung cancer patients, yet the underlying mechanisms remain unclear. Depletion of the LIP in non-small-cell lung cancer cell lines using the doxycycline-inducible overexpression of the ferritin heavy chain (Ft-H) (H1299 and H292), or treatment with deferoxamine (DFO) (H1299 and A549), inhibited cell growth and decreased clonogenic survival. The Ft-H overexpression-induced inhibition of H1299 and H292 cell growth was also accompanied by a significant delay in transit through the S-phase. In addition, both Ft-H overexpression and DFO in H1299 resulted in increased single- and double-strand DNA breaks, supporting the involvement of replication stress in the response to LIP depletion. The Ft-H and DFO treatment also sensitized H1299 to VE-821, an inhibitor of ataxia telangiectasis and Rad2-related (ATR) kinase, highlighting the potential of LIP depletion, combined with DNA damage response modifiers, to alter lung cancer cell responses. In contrast, only DFO treatment effectively reduced the LIP, clonogenic survival, cell growth, and sensitivity to VE-821 in A549 non-small-cell lung cancer cells. Importantly, the Ft-H and DFO sensitized both H1299 and A549 to chemoradiation in vitro, and Ft-H overexpression increased the efficacy of chemoradiation in vivo in H1299. These results support the hypothesis that the depletion of the LIP can induce genomic instability, cell death, and potentiate therapeutic responses to chemoradiation in NSCLC.

Keywords: DNA damage; cell cycle; chemoradiation; deferoxamine; ferritin heavy chain; holo-transferrin; iron chelator; labile iron pool; lung cancer; replication stress.

Figure 1

Ft-H overexpression alters intracellular labile iron and induces changes in iron metabolic proteins. (A) Ft-H was overexpressed in H1299 Ft-H C4 and A549 Ft-H C3 NSCLC lines following a 1 µg mL⁻¹ doxycycline treatment and Ft-H overexpression levels at 24, 48, and 72 h were validated using Western blots. GAPDH was used as the loading control for all the Western blots. (B) Ft-H was overexpressed in H1299 and A549 cells following a 1 µg mL⁻¹ doxycycline treatment at 24, 48, and 72 h time points, and the expression of Ft-H and transferrin receptor 1 (TfR-1) were detected using Western blots. (C) mRNA levels of TfR-1 were detected using qPCR in the Ft-H-non-expressing control cells and the 48 h doxycycline-treated Ft-H-overexpressing cells, and each gene expression level was normalized to GAPDH and 18S RNA. (D) LIP was measured at 48 h of Ft-H overexpression in both cell lines using the calcein-AM probe and flow cytometry. Cells treated with 40 µM ferrous ammonium sulfate (FAS) and 100 µM DFO for 3 h were used as the positive and negative control, respectively. Normalized LIP was measured using calcein-AM. An amount of 200 µg mL⁻¹ Transferrin (hTf) was used to supplement the cell culture over the same time as the Ft-H overexpression in the H1299 cells. Data are represented as mean ± SEM. *, ** indicate significant differences between the control and treated groups (n = 3; * p < 0.05, ** p < 0.01, and **** p < 0.0001); nd refers to non-detectable.

Figure 2

Iron depletion inhibits H1299 Ft-H C4 tumor cell growth by delaying cell cycle progression through the S-phase. (A) Ft-H was overexpressed in H1299 Ft-H C4 cells with 1 µg mL⁻¹ doxycycline treatment over 7 d. The cell numbers were counted at 3, 5, and 7 d of exponential growth of the cells, and plotted as a cell-growth curve. An amount of 200 µg mL⁻¹ hTf was used to supplement the control and Ft-H-expressing H1299 cells with iron over the same time as the doxycycline treatment. A nonlinear regression exponential growth equation was used to calculate the doubling time (d). R² values > 0.94; n = 3 separate experiments; error bars are SEM; * indicates significantly different doubling times. (B) Ft-H was overexpressed in H1299 Ft-H C4 cells over 48 h and clonogenic survival was measured. The same dose of hTf (200 µg mL⁻¹ hTf) was used to supplement the control, Ft-H-overexpressed, and iron-depleted cells with iron, and the clonogenic survival of each treatment was measured. (C) The asynchronized control and Ft-H-overexpressing cells of H1299 Ft-H C4 over 3, 5, and 7 days (d-3, d-5, and d-7) were collected, fixed with ethanol, and stained with propidium iodide (PI) to determine the DNA content and cell cycle phase distributions (G₁, S, and G₂ + M). (D) The control and 48 h Ft-H-overexpressing cells of H1299 Ft-H C4 were incubated with BrdU at time 0 and chased with thymidine for an additional 6 and 10 h. Scatter plot histograms of each time point were displayed as BrdU-FITC fluorescence (Y-axis) and PI-DNA content (X-axis). Cell cycle progression was calculated following the description found in [25] which is also described in Figure S5. (E) The relative movement (RM) was calculated to measure the cells progression through the S-phase. (F) The fraction of cells in the G₁ of the daughter generation was calculated to measure the progression of the BrdU-positive S-phase cells through the G₂ and M phases. ns refers to non-significant (n = 3; * p < 0.05, **** p < 0.0001).

Figure 3

Depletion of labile iron promotes DNA damage. (A) Ft-H overexpression was induced over 24, 48, and 72 h in H1299 Ft-H C4 cells with doxycycline. Alkaline and neutral comet assays were performed to measure SSBs and DSBs, respectively. The percentage of DNA in the comet tail (DNA tail %), out of 100%, was measured for each treatment group to determine the SSBs and DSBs using CometScore 2.0 software. (B) The alkaline comet assay was used for the SSBs from the H1299 cells treated with 100 µM DFO overnight to deplete the intracellular iron with 2 µM hydroxyurea (HU) overnight as a positive control. (C) The neutral comet assay was used for the DSBs from the 100 µM DFO overnight-treated H1299 cells with 16 Gy as a positive control. (D) The alkaline comet assays were used for the A549 Ft-H C3 treated with doxycycline for 48 h to over express Ft-H, 100 µM DFO overnight, or 2 µM HU overnight. (E) The H1299 Ft-H C4 cells were treated with 5 µM and the A549 Ft-H C3 cells were treated with 10 µM ATR inhibitor (VE-821) over 24 h, alone and in combination with the 24 h Ft-H expression. The survival fraction of each cell group, with and without VE, was determined using a clonogenic assay and normalized to the Ft-H-non-expressing control cells. (F) Both cell lines were treated as in (E), with 100 µM DFO ± ATR inhibitor (VE-821) overnight. Clonogenic survival was assayed, and the DFO- and VE-alone treated groups, and the combination of DFO and VE treated groups, were normalized to the respective untreated control. ns refers to non-significant (n = 3; * p < 0.05, ** p < 0.01, and **** p < 0.0001).

Figure 4

Depletion of labile iron enhances NSCLC sensitivity to chemoradiation in vitro. (A) Ft-H was induced for 48 h with 1 µg mL⁻¹ doxycycline in both cell lines (H1299T Ft-H C11 and A549 Ft-H C3), and exposed to 2, 4, and 8 Gy radiation. Clonogenic survival was analyzed and compared within the groups of non-Ft-H-expressing and Ft-H-expressing cells, and normalized to the respective, untreated control. (B) The parental cells of the H1299 and A549 were treated with 100 µM DFO over 12 h and exposed to 2 and 4 Gy radiation. Clonogenic survival of each radiation dose was normalized to the control or DFO treated cells, respectively. (C) Following 48 h doxycycline treatment in the H1299T Ft-H C11 and 72 h in the A549 Ft-H C3, the cells were treated with a combination of 4 μM Cisplatin and 0.5 μM Etoposide for 1 to 3 h, followed by 2 Gy radiation. Throughout drug treatment, cells were cultured in 4% O₂. Clonogenic survival was determined and compared between the control and Ft-H-expressing cells after chemo-radiation therapy. (D) The parental H1299 and A549 were treated with 100 µM DFO over 12 h to chelate intracellular iron and then treated with a combination of chemoradiation as in (C), over the same time period at 4% O₂. Clonogenic survival was determined and compared between the control and DFO-treated cells following the treatment. Error bars represent mean ± SEM for 3 independent experiments, with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, ns = not significant: using a two-way ANOVA test.

Figure 5

Overexpression of Ft-H sensitizes H1299T xenografts to chemoradiation. (A) Schematic representation of H1299T Ft-H C11 tumor chemoradiation treatment protocol in nude mice with Ft-H overexpression induced by doxycycline. (B) Tumor volume of each animal in the treatment groups in mm³. (C) Kaplan–Meier survival plot of H1299T Ft-H C11 tumor-bearing mice after receiving chemoradiation therapy. Mice received 10 mg kg⁻¹ doxycycline or an equivalent dose of NaCl given IP, 24 h prior to chemoradiation treatment, and were fed chow containing 1 g kg⁻¹ doxycycline for the duration of the experiment. The tumors received a total of 20 Gy radiation to their flank tumor, and this was given in 10 fx of 2 Gy each, over a period of 2 weeks. The animals were treated following radiation with 2 mg kg⁻¹ of cisplatin followed by 6 mg kg⁻¹ of etoposide, and these chemotherapy drugs were given once a week for 4 weeks. (D) Tumor tissue from the groups receiving chemoradiation + doxycycline was collected at euthanasia and homogenized for Western blotting of TfR-1 and Ft-H and compared to chemoradiation alone (n = 3; * p < 0.05).