Catalase Modulates the Radio-Sensitization of Pancreatic Cancer Cells by Pharmacological Ascorbate

Abstract

Pancreatic cancer cells (PDACs) are more susceptible to an oxidative insult than normal cells, resulting in greater cytotoxicity upon exposure to agents that increase pro-oxidant levels. Pharmacological ascorbate (P-AscH^-), i.e., large amounts given intravenously (IV), generates significant fluxes of hydrogen peroxide (H₂O₂), resulting in the killing of PDACs but not normal cells. Recent studies have demonstrated that P-AscH^- radio-sensitizes PDAC but is a radioprotector to normal cells and tissues. Several mechanisms have been hypothesized to explain the dual roles of P-AscH^- in radiation-induced toxicity including the activation of nuclear factor-erythroid 2-related factor 2 (Nrf2), RelB, as well as changes in bioenergetic profiles. We have found that P-AscH^- in conjunction with radiation increases Nrf2 in both cancer cells and normal cells. Although P-AscH^- with radiation decreases RelB in cancer cells vs. normal cells, the knockout of RelB does not radio-sensitize PDACs. Cellular bioenergetic profiles demonstrate that P-AscH^- with radiation increases the ATP demand/production rate (glycolytic and oxidative phosphorylation) in both PDACs and normal cells. Knocking out catalase results in P-AscH^- radio-sensitization in PDACs. In a phase I trial where P-AscH^- was included as an adjuvant to the standard of care, short-term survivors had higher catalase levels in tumor tissue, compared to long-term survivors. These data suggest that P-AscH^- radio-sensitizes PDACs through increased peroxide flux. Catalase levels could be a possible indicator for how tumors will respond to P-AscH^-.

Keywords: DNA damage; DNA repair; pancreatic cancer; pharmacological ascorbate; vitamin C.

Figure 1

Changes in nuclear factor-erythroid 2-related factor 2 (Nrf2) activation of normal cells vs. pancreatic cancer cells (PDACs) after pharmacological ascorbate (P-AscH⁻), radiation, or the combination of both. Western blot analyses show that P-AscH⁻, radiation, or radiation + P-AscH⁻ induce Nrf2 in MIA PaCa-2 and FHs74Int cells, but no change occurs in H6c7 cells. Cells were treated with P-AscH⁻ (5 pmol/cell, 1 mM) for 1 h, 5 Gy, or 5 Gy + P-AscH⁻ in DMEM−10% fetal bovine serum (FBS). After treatment, cells were cultured in their growth media for 24 h before protein was harvested for Western blot analysis.

Figure 2

Role of RelB in the differential response of normal and PDAC cells. (A) Cells were treated with P-AscH⁻: MIA PaCa-2, FHs74Int, and H6c7 with 5 pmol cell⁻¹ (1 mM) and PANC-1 with 15 pmol cell⁻¹ (3 mM); radiation (5 Gy); or the combination of both. P-AscH⁻ with or without radiation, decreases RelB levels in MIA PaCa-2 and PANC-1 cells, but not in FHs74Int and H6c7 cells; (B) CRISPR/Cas9 KO plasmid was used to generate RelB knockout cells in MIA PaCa-2 PDAC (RelB KO). Control cells (CRISPR control) were generated by transfecting parental cell lines with control CRISPR/Cas 9 plasmid, resulting in decreased immunoreactive protein in the RelB KO; (C) clonogenic survival. Treatment of PDAC CRISPR control and RelB KO with P-AscH⁻ (5 pmol cell⁻¹, 1 mM) and IR (1–3 Gy), showed no differences in clonogenic survival between the two groups.

Figure 3

Bioenergetics after treatment with P-AscH^⁻ and radiation. In both normal and PDAC cell lines, P-AscH⁻ (5 pmol cell⁻¹, 1 mM) with and without radiation (5 Gy) results in increased rate of basal oxygen consumption (OCR) and increased rate of production of ATP 48 h after treatment. (A) MIA PaCa-2 and FHs74Int cells were treated with P-AscH⁻ for 1 h and then basal oxygen consumption rate (OCR, i.e., flow per cell I_O2_/cell) was measured 48 h after treatment; (B) total ATP changes were similar in FHs74Int vs. MIA PaCa-2 48 h after radiation with or without P-AscH⁻. The contributions to the changes in ATP were then determined using Seahorse XF96 instrumentation; (C) ATP rate attributed to oxidative phosphorylation demonstrated similar changes in FHs74Int cells vs. MIA PaCa-2 cells 48 h after radiation with or without P-AscH⁻; (D) ATP rate attributed to glycolysis demonstrated similar changes in in FHs74Int cells vs. MIA PaCa-2 cells 48 h after radiation with or without P-AscH⁻. (Means ± SEM, n = 3. There were no significant differences between any of the treatment groups).

Figure 4

Cellular catalase blunts the radio-sensitization of P-AscH⁻. (A) The PDAC cell lines MIA PaCa-2 (5 pmol cell⁻¹, 1 mM) and PANC-1 (15 pmol cell⁻¹, 3 mM), and the non-tumorigenic H6c7 and FHs74Int cell lines were treated with P-AscH⁻ (5 pmol cell⁻¹, 1 mM) with and without IR (5 Gy). Live cells were collected and Western blots performed to determine catalase immunoreactive protein. P-AscH⁻ alone and in combination with radiation, decreased catalase in the PDAC cell lines, but not in the H6c7 or FHsInt cell lines; (B) clones of MIA PaCa-2 and PANC-1 PDAC cells subjected to CRISPR/Cas9 catalase genome editing demonstrate decreased expression of catalase; (C) genetic inhibition of catalase expression radio-sensitized MIA PaCa-2 cells to treatment with P-AscH⁻ (5 pmol cell⁻¹, 1 mM) (means ± SEM, * p < 0.05 vs. CRISPR control, n = 3); (D) genetic inhibition of catalase expression radio-sensitized PANC-1 cells to treatment with P-AscH⁻ (10 pmol cell⁻¹, 2 mM,) (means ± SEM, * p < 0.05 vs. CRISPR control, n = 4).

Figure 5

Catalase levels may predict patient outcomes after treatment with P-AscH⁻. (A) Overall survival from phase I trial (NCT 01049880). Kaplan–Meier curve estimating median overall survival in subjects treated with P-AscH⁻ plus gemcitabine and radiation therapy as of March 30, 2021 (n = 14) was 22.8 vs. 12.7 months in institutional controls treated with gemcitabine and radiation therapy (n = 19). (Log-Rank test p = 0.02). Institutional controls from the University of Iowa are equivalent to historical controls as published by Loehrer et al. [23]; (B) Progression free survival from phase I trial (NCT 01049880). Kaplan–Meier curve demonstrating median progression-free survival as of October 2020 in subjects treated with P-AscH⁻ plus gemcitabine and radiation therapy (n = 14) was 4.6 months in institutional controls treated with gemcitabine and radiation therapy vs. 13.7 months in patients receiving the same chemo-radiation therapy but also P-AscH⁻ (n = 19, p = 0.01). Institutional controls from the University of Iowa are equivalent to historical controls as published by Loehrer et al. [23]; (C) catalase immunofluorescence of clinical trial pre-treatment biopsy samples. Formalin-fixed, paraffin-embedded tissue samples were cut and stained with catalase antibody. DAPI was used to stain the cell nuclei; (D) quantification of immunofluorescence using ImageJ (n = 2 long-term survivors, n = 5 short-term survivors). Immunofluorescence was normalized to the number of cells counted per image. Pre-treatment biopsies of long-term survivors had lower levels of catalase (55.5 ± 7.5 arbitrary fluorescence units) compared to short term survivors (309 ± 134 arbitrary fluorescence units), (Means ± SEM, p = 0.3, Student’s t-test) as seen by immunofluorescence.