Tumor cells have decreased ability to metabolize H2O2: Implications for pharmacological ascorbate in cancer therapy

Abstract

Ascorbate (AscH-) functions as a versatile reducing agent. At pharmacological doses (P-AscH-; [plasma AscH-] ≥≈20mM), achievable through intravenous delivery, oxidation of P-AscH- can produce a high flux of H2O2 in tumors. Catalase is the major enzyme for detoxifying high concentrations of H2O2. We hypothesize that sensitivity of tumor cells to P-AscH- compared to normal cells is due to their lower capacity to metabolize H2O2. Rate constants for removal of H2O2 (kcell) and catalase activities were determined for 15 tumor and 10 normal cell lines of various tissue types. A differential in the capacity of cells to remove H2O2 was revealed, with the average kcell for normal cells being twice that of tumor cells. The ED50 (50% clonogenic survival) of P-AscH- correlated directly with kcell and catalase activity. Catalase activity could present a promising indicator of which tumors may respond to P-AscH-.

Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Figures

Graphical abstract

Fig. 1

Normal cells have a more robust capacity to remove extracellular H2O2than tumor cells. The rate constants, kcell, at which 10 normal cell lines and 15 cancer cell lines remove H2O2 were measured (listed in Table 1). There was a wide range of capacities for removal of H2O2 across all cell types. On average, normal cells had a 2-fold higher rate constant for the removal of H2O2 than tumor cells (p<0.05).

Fig. 2

Catalase activity varies across cancer cell lines and correlates with the rate constant for removal of H2O2(kcell). (A) Catalase activity for cell lines of different tissue origins (i.e. pancreas (purple), breast (green), lung (red), and liver (blue)) were determined and used to calculate the effective number of fully active catalase monomers per cell. This number varied 5-fold across the different cancer cell lines: from 101,000 monomers per cell (MIA PaCa-2) to 538,000 monomers per cell (339) (n =3–9, error bars are standard error of the mean). (B) There is a strong correlation between the rate constant at which these cell lines remove extracellular H2O2 and the effective number of fully active catalase molecules per cell (R2 =0.88). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Catalase plays a major role in removal of H2O2. (A) Treatment of HepG2 cells with 100 µM buthionine sulfoximine (BSO) 24 h prior to the H2O2-removal assay to inhibit glutathione synthesis did not result in any change in the rate constant by which these cells remove H2O2. However, treatment of HepG2 cells with 20 mM 3-AT for 1 h to inhibit catalase resulted in a four-fold decrease in the rate constant by which HepG2 cells remove extracellular H2O2 (n =4, error bars are standard error of the mean). (B) There is a direct correlation between the number of active catalase molecules per cell and the rate constant for removal of H2O2 following transduction of MIA PaCa-2 cells with adenovirus catalase (0–25 MOI) (R2 =0.91).

Fig. 4

Dose of ascorbate is better specified on a per cell basis (pmol cell−1) than as initial concentration in the medium (mM). MIA PaCa-2 cells at varying cell densities (45,000–543,000 cells/3.0 mL medium) were treated with 5 mM ascorbate 1 h; ATP was measured immediately after. Dose of ascorbate is expressed as: (A) initial concentration of ascorbate in the medium; and (B) absolute amount of ascorbate (pmol) per cell.

Fig. 5

Sensitivity to ascorbate varies across pancreatic cancer cell lines and correlates with the capacity to remove extracellular H2O2(k1-cell). (A) The ED50 of ascorbate was determined in MIA PaCa-2, AsPC-1, 403, 339, and PANC-1 cell lines using a clonogenic survival assay. The dose of ascorbate needed to decrease clonogenic survival by 50% varied across pancreatic cancer cell lines. When the rate constants for removal of extracellular H2O2 by a cell (k1-cell) for these 5 different pancreatic cancer cell lines are plotted against the ED50 of P-AscH− there is a direct correlation between sensitivity to P-AscH− and the rate at which cells remove H2O2 (R2 =0.69). The rate constant k1-cell represents the capacity of a single cell to remove extracellular H2O2. It is determined by: k1-cell (s−1) = kcell (s−1 cell−1 L) x 1 (cell L−1). (B) Transduction of MIA PaCa-2 cells with adenovirus catalase at increasing MOIs increases resistance to ascorbate as seen by ED50. MIA PaCa-2 cells were transduced with adenovirus catalase at 0–25 MOI and then exposed to ascorbate (0–50 pmol cell−1). The dose that decreased clonogenic survival by 50% was determined at each transduction-MOI of adenovirus catalase (0–25 MOI). Catalase activity was measured after transduction with adenovirus catalase. The resulting ED50 correlated with catalase activity at varying MOI of adenovirus catalase (R2 =0.94).

Fig. 6

Inhibition of catalase with 3-amino-1,2,4-triazole sensitizes PANC-1 cells to ascorbate parallels the decrease inkcell. PANC-1 cells were treated with 20 mM 3-AT for 1 h prior to treatment with 0–17 pmol cell−1 ascorbate (350,000 cells; 0–2 mM) for 1 h. Cells were then plated for a clonogenic survival assay. 3-AT sensitized PANC-1 cells to ascorbate. The ED50 of ascorbate was 1.5-fold less with 3-AT treatment than without (n =3, error bars are standard error of the mean).

Fig. 7

H2O2generated by ascorbate induces damage to nDNA and mtDNA in MIA PaCa-2 cells and depletes intracellular ATP. (A) MIA PaCa-2 cells were treated with ascorbate (3.5–28 pmol cell−1) for 1 h and then the frequency of DNA lesions was quantified with QPCR. Ascorbate treatment caused dose-dependent damage to nDNA and mtDNA (for nDNA: n =4, mean±SEM, * p<0.05 vs. 3.5 pmol cell−1; for mtDNA: n =8, mean±SEM, ** p<0.001 vs. 3.5 pmol cell−1). (B) MIA PaCa-2 cells were incubated with ascorbate (14 pmol cell−1), or ascorbate (14 pmol cell−1) and bovine catalase (200 units mL−1), or bovine catalase (200 units mL−1) alone for 1 h. QPCR analysis revealed no DNA damage from ascorbate when catalase is present in the medium indicating that the DNA damage is caused by H2O2 (nDNA: n =4; mean±SEM; * p<0.01 vs. ascorbate; mtDNA: n =4; mean±SEM; ** p<0.001 vs. ascorbate). (C) MIA PaCa-2 cells were treated with ascorbate (14 pmol cell−1), combination of ascorbate (14 pmol cell−1) and bovine catalase (200 units mL−1), or bovine catalase (200 units mL−1) for 1 h and then intracellular ATP was determined. ATP was depleted upon treatment with ascorbate, but was unchanged when catalase was present in the medium (n =4; mean±SEM; * p<0.001 vs. P-AscH−).

Fig. 8

Pharmacological ascorbate slows growth of MIA PaCa-2 tumors in comparison to PANC-1 tumorsin vivo.(A) MIA PaCa-2 (kcell =1.1×10−12 s−1 cell−1 L; 101,000 active catalase monomers per cell) cells and (B) PANC-1 (kcell =5.1×10−12 s−1 cell−1 L; 459,000 active catalase monomers per cell) cells were injected into mice and formed tumors. Mice were treated with IP ascorbate (4 g/kg) twice daily for two weeks. Tumors were measured on day 3, 7, 10, and 14 following first treatment with ascorbate. P-AscH− slowed the growth rate of PANC-1 xenograft tumors to 42% of the controls; with MIA PaCa-2 tumor xenografts P-AscH− slowed growth to just 9% of controls. The ratio of kcell(PANC-1)/kcell(MIA PaCa-2) =4.6; the ratio for the relative growth rates compared to controls is essentially identical, 42%/9% =4.7, a remarkable quantitative comparison. (C) MIA PaCa-2 tumor catalase immunofluorescence, and (D) PANC-1 tumor catalase immunofluorescence. Tumor samples were fixed with 4% paraformaldehyde at 4 °C, and blocked with 5% goat serum for 30 min at 20 °C. The samples were incubated with catalase antibody (1:50) for 20 h at 4 °C. An Alexa Fluor 488 nm goat anti-Rabbit (1:200) was used as secondary antibody. DAPI was used to stain the cell nuclei. The samples were examined using a Zeiss confocal microscope. Scale bar, 20 µm. Tissue samples for PANC-1-derived tumors show considerably more immunofluorescence due to the presence of catalase enzyme than tissue samples from MIA PaCa-2 tumors. (Normalized fluorescent intensity of PANC-1 vs. MIA PaCA-2 is 100±27 vs. 2.0±0.5.).