O2⋅- and H2O2-Mediated Disruption of Fe Metabolism Causes the Differential Susceptibility of NSCLC and GBM Cancer Cells to Pharmacological Ascorbate

Abstract

Pharmacological ascorbate has been proposed as a potential anti-cancer agent when combined with radiation and chemotherapy. The anti-cancer effects of ascorbate are hypothesized to involve the autoxidation of ascorbate leading to increased steady-state levels of H₂O₂; however, the mechanism(s) for cancer cell-selective toxicity remain unknown. The current study shows that alterations in cancer cell mitochondrial oxidative metabolism resulting in increased levels of O₂^⋅- and H₂O₂ are capable of disrupting intracellular iron metabolism, thereby selectively sensitizing non-small-cell lung cancer (NSCLC) and glioblastoma (GBM) cells to ascorbate through pro-oxidant chemistry involving redox-active labile iron and H₂O₂. In addition, preclinical studies and clinical trials demonstrate the feasibility, selective toxicity, tolerability, and potential efficacy of pharmacological ascorbate in GBM and NSCLC therapy.

Keywords: ferritin; glioblastoma multiforme; hydrogen peroxide; labile iron metabolism; non-small cell lung cancer; oxidative stress; pharmacological ascorbate; superoxide; superoxide dismutase; transferrin receptor.

Figure 1. Ascorbate selectively sensitizes NSCLC and GBM cells to chemo-radiation in vitro as compared to normal cells

(A, B) Clonogenic survival post-exposure of NSCLC cell lines H1299 and H292 and HBEpCs (A) or GBM cell lines U87 and U118 and NHAs (B) to increasing doses of pharmacological ascorbate for 1 hr. (C) Clonogenic survival of NSCLC and HBEpCs post serial exposure to 5 μM carboplatin for 1 hr, ascorbate for 1 hr, and 2 Gy IR. (D) Clonogenic survival of GBM and NHAs post serial exposure to 5 μM temozolomide for 1 hr, ascorbate for 1 hr, and 2 Gy IR. For all in vitro studies, n ≥ 3 biological replicates with n = 6 technical replicates per sample. Data are represented as mean ± SEM. *, **, or *** represent significant differences between untreated control cells and any group denoted with a different symbol, at least p < 0.05. n.s. = not significant (p > 0.05). See also Figure S1.

Figure 2. Ascorbate is safe and efficacious in combination with radio-chemotherapy for the treatment of NSCLC and GBM cells in vivo

(A) Overall survival of mice with H292 xenografts treated with chemoradiation (5 mg kg⁻¹ carboplatin weekly, 12 Gy IR/2 fx) with or without ascorbate (4 g kg⁻¹ ascorbate IP or equivalent osmotic dose of NaCl daily). (B) Overall survival of mice with U87 xenografts treated with radiochemotherapy (25 mg kg⁻¹ temozolomide weekly, 12 Gy IR/2 fx) with or without ascorbate (4 g kg⁻¹ IP ascorbate or equivalent osmotic dose of NaCl daily). For (A) and (B), mice were sacrificed when tumors reached 15 mm in the longest direction. (C) Average weight of mice with H292 or U87 xenografts. (D) Plasma and tumor ascorbate concentrations from mice with H292 xenografts collected at a single time point 1 hr after IP ascorbate treatment (4 g kg⁻¹ or equivalent osmotic dose of NaCl). (E) Representative result of an EPR spectra monitoring for the ascorbyl radical at g = 2.0 from CSF collected from healthy nude athymic female mice at a single time point 1 hr after IP treatment with ascorbate (4 g kg⁻¹) or equivalent dose of NaCl. For all in vivo studies, mice n ≥ 7 per treatment group. For all ex vivo studies, n ≥ 3 mice per group with n ≥ 3 technical replicates per sample. Data are represented as mean ± SEM. *represents significant difference, at least p < 0.05.

Figure 3. GLUT-mediated DHA uptake does not mediate ascorbate toxicity

(A, B) DHA uptake via glucose transporters (GLUTs) was competitively inhibited with 20 mM 2-deoxy-D-glucose (2-DG) for 15 min prior and during exposure to 5 pmol cell⁻¹ (2 mM) ascorbate for 1 hr and then measured for clonogenic survival in H292 and H1299 NSCLC cell lines (A) or measured for total intracellular ascorbate (AscH⁻, AscH^•−, and DHA) in H1299 cells (B) by a kinetic spectrophotometric assay (BLD = below the limit of detection). (C) Clonogenic survival of H1299 cells exposed to equivalent increasing doses (per cell and concentration) of AscH⁻ or DHA for 1 hr. For all clonogenic survival assays, n ≥ 3 biological replicates with n = 6 technical replicates per sample. For AscH⁻/DHA uptake, n ≥ 3 with n = 3 technical replicates per sample. Data are represented as mean ± SEM *represents significant difference, at least p < 0.05. n.s. = not significant (p > 0.05). See also Table S2.

Figure 4. The combination of H₂O₂ and redox-active labile iron is necessary and sufficient for ascorbate toxicity

(A) Clonogenic survival of H292 or U87 cells following co-exposure to 50 U mL⁻¹ bovine catalase ± ascorbate for 1 hr. (B, C) Clonogenic survival of H1299 cells transiently overexpressing (25 MOI adenoviral-mediated [Ad] transduction) catalase (B) or GPx1 (C) following exposure to ascorbate for 1 hr. For GPx1 experiments, 30 nM sodium selenite was added to the basal media in all groups after transduction and during clonogenic growth. (D) Clonogenic survival of H292 and U118 cells treated with 200 μM DFO and 1 mM DTPA for 3 hr followed by 1 hr exposure to ascorbate in the continued presence of the chelators. (E, F) H292 cells were exposed to 200 μM DFO/1 mM DTPA, 1 mM EDTA (E), or 250 μM FAS (F) for 3 hr and washed prior to ascorbate exposure (‘intracellular’) or media was pretreated for 3 hr in the absence of cells and co-exposed to cells only during 1 hr ascorbate exposure (‘extracellular’) and then plated for clonogenic survival. For all in vitro studies, n ≥ 3 biological replicates with n = 6 technical replicates per sample. Data are represented as mean ± SEM. *represents significant difference, at least p < 0.05. See also Figures S2, S3.

Figure 5. Increased steady-state levels of mitochondrial O₂^•− disrupt cellular iron homeostasis increasing the LIP and sensitizing cancer cells to ascorbate

(A) 10 μm sections of NSCLC or adjacent normal tissue were stained with 10 μM DHE on the same slide for 30 min prior to analysis by confocal microscopy. For treatment groups, tissue sections were pretreated for 30 min with 0.5 μM GC4419 SOD mimetic prior to DHE exposure. For a positive control, tissue sections were incubated with 10 μM antimycin A during DHE staining. The mean fluorescence intensity (MFI) of ≥ 200 nuclei from 6 randomly selected areas was quantified using ImageJ software. (B) NSCLC and adjacent normal tissue was assayed for labile iron content by EPR quantification of the high spin state Fe³⁺-DFO complex (g = 4.3, 100 K) against a standard curve and normalized to total tissue protein. Each data point represents the average of triplicate technical replicates. (C, D) Levels of DHE oxidation (C) and baseline cellular LIP (D) of parental and SOD2^−/− A549 cells quantified by flow cytometry and normalized to the parental cell line. (E) Clonogenic survival of parental and SOD2^−/− A549 cells following exposure to 10 pmol cell⁻¹ (2 mM) ascorbate for 1 hr. (F–H) Western blot analysis of TfR protein levels (25 μg total protein) in HBEpC/NSCLC cells and NHA/GBM cells (F), parental and SOD2^−/− A549 cells (G), and H1299 cells (H) treated with 25 pmol siRNA/dish with actin protein loading controls. For all blots, the images are cropped and extra lanes were removed. (I) Basal LIP in H1299 cells treated with 25 pmol scrambled or TfR siRNA per dish and clonogenic survival of H1299 cells treated with 25 pmol scrambled or TfR siRNA per dish post 1 hr exposure to ascorbate. (J) Normalized DHE fluorescence and relative labile iron in NHAs exposed to 25 μM AntA for 30 min prior to flow cytometry analysis and clonogenic survival of NHAs following co-exposure to 25 μM AntA and ascorbate for 30 min. (K) NHAs exposed to 100 μM or 250 μM FAS for 3 h prior to LIP quantification by flow cytometry or washed with PBS before exposure to ascorbate for 1 hr in fresh full media and then plated for clonogenic survival. For all in vitro studies, unless noted above, n ≥ 3 biological replicates with n ≥ 3 technical replicates per sample. Data are represented as mean ± SEM. *, ** represents significant differences between untreated control cells and any group denoted with a different symbol, at least p < 0.05. See also Figures S4, S5.

Figure 6. Ascorbate selectively increases cancer cell LIP via H₂O₂-mediated disruptions of Fe-S clusters and overexpression of Ft-H protects against ascorbate toxicity

(A, B) Relative intracellular LIP levels following exposure to increasing doses of ascorbate in HBEpCs and NSCLC cells (A) or NHAs and GBM cells (B) as assayed by flow cytometry and normalized to HBEpCs or NHAs, respectively. (C–E) Quantification of total cellular aconitase (C) or ETC Complex I, II, and IV activity (D) after exposure to 15 pmol cell⁻¹ (~8 mM) ascorbate for 1 hr in NSCLC cell lysates of control cells or cells transiently overexpressing catalase (50 MOI adenoviral-mediated [Ad] transduction 36 hr prior) (C, E). (F, G) Quantification of the relative cellular LIP of H1299 cells overexpressing (50 MOI adenoviral-mediated [Ad] transduction 36 hr prior) an empty vector or catalase (F) or SOD1 and SOD2 (G) as assayed by flow cytometry. (H) Relative oxidation of H₂O₂-sensitive fluorescent probe PO-1 in H292, H1299, or HBEpCs treated with 15 pmol cell⁻¹ ascorbate (~5 mM) once or 100 μM H₂O₂ every 30 min by flow cytometry. (I) Representative western blot of H1299 cells overexpressing (20 MOI adenoviral-mediated [Ad] transduction 36 hr prior) empty, Ft-L, or Ft-H with Actin protein loading control. (J, K) Baseline LIP (J) and LIP after 1 hr ascorbate exposure (K) in H1299 cells overexpressing (20 MOI adenoviral-mediated [Ad] transduction 36 hr prior) empty, Ft-L, or Ft-H as assayed by flow cytometry. (L) Clonogenic survival of H1299 cells overexpressing (20 MOI adenoviral-mediated [Ad] transduction 36 hr prior) empty or Ft-H exposed to 5 pmol cell⁻¹ (~2–3 mM) for 1 hr. For all in vitro studies, n ≥ 3 biological replicates with n ≥ 3 technical replicates per sample. Western blots are representative images of at least 3 replicates. For both blots, images were cropped to remove non-specific bands and to limit image size. Data are represented as mean ± SEM. *,** represents significant differences between untreated control cells and any group denoted with a different symbol, at least p < 0.05. See also Figures S6.

Figure 7. Pharmacological ascorbate is safe and well tolerated when combined with standard therapy in the treatment of GBM and NSCLC

(A) Progression Free Survival (PFS) and Overall Survival (OS) of GBM subjects treated with pharmacological ascorbate, radiation and temozolomide. Subjects were treated with temozolomide daily with concurrent radiation therapy (radiation phase) for ~7 weeks followed by ~28 additional weeks of temozolomide therapy (adjuvant phases), for a total of ~35 weeks. The doses denoted adjacent to each subject number indicates the ascorbate dose received during the radiation phase dose-escalation study. All subject’s doses were increased to achieve target plasma levels ≥ 20 mM during the adjuvant phase. Historical median PFS is 7 months and OS is 14.6 months. The green bars indicate the duration of active ascorbate therapy, blue bars indicate PFS, and red bars indicate OS. The vertical red lines indicate pre-mature cessation of the clinical trial due to personal reasons; vertical light blue lines indicate cessation of the trial due to progression of disease while receiving ascorbate therapy; vertical black lines indicate death; * indicates surgical confirmation of recurrence; (+) indicates MGMT gene promoter hypermethylation; (−) indicates absence of MGMT gene promoter methylation. (B) GBM subject’s plasma ascorbate levels were measured from blood samples collected directly following ascorbate infusion during dose escalation and throughout ascorbate therapy. Box plots are calculated using the Tukey method. The line and box represent the median value ± the 25^th and 75^th percentiles or the interquartile range (IQR). Whiskers represent 1.5 times IQR. Any data points outside 1.5 times IQR are represented by individual dots. (C) Survival curve for 11 GBM subjects (black line) with 95% confidence intervals (red shading) as compared to Stupp et al. (2005) historical controls (blue line). (D) Survival curve for 8 GBM subjects whose tumors lacked MGMT promoter methylation (black line) with 95% confidence intervals (red shading) as compared to Hegi et al. (2005) historical controls (blue line). (E) Pre- and post-therapy CT images from NSCLC Subject 1 illustrating the tumor volume (red dashed line). (F) Spider plot illustrating therapy responses from the first 14 advanced stage NSCLC subjects. As per RECIST1.1, partial response is ≥ 30% reduction in target tumor burden that is maintained for at least 4 weeks. See also Figure S7, S8, Tables S2–6.

Figure 8. Proposed mechanism of pharmacological ascorbate cancer-cell selective toxicity

Dashed lines represent multi-step processes not illustrated in the model. Briefly, cancer cells demonstrate increased levels of redox-active labile iron due to increased steady-state levels of mitochondrial O₂^•− and H₂O₂, which are capable of disrupting cellular iron homeostasis. Oxidation of ascorbate produces H₂O₂ that reacts with the increased LIP in cancer cells to mediate Fenton chemistry and cause oxidative damage to cellular macromolecules (i.e., DNA, protein, lipids). Due to the diffusion-limited kinetics of HO^• species, redox-active iron chelated by these macromolecules most likely represent the most prevalent site of damage. Furthermore, H₂O₂ produced from ascorbate oxidation selectively increases the cancer cell LIP, partially by disrupting Fe-S clusters, further exacerbating the differences in LIP available for oxidation reactions and mediate ascorbate toxicity in cancer vs. normal cells. In this model, endogenous ascorbate recycling mechanisms are driven by reducing equivalents from nicotinamide adenine dinucleotide phosphate (NADPH) and Glutathione (GSH) and/or Glutaredoxin (Grx) allowing for the continuous production of H₂O₂. Glu = glucose; G6PD = Glucose-6-Phosphate Dehydrogenase; GR = Glutathione Reductase; L-Fe = weakly chelated redox-active iron; Tf = Transferrin.