Manganoporphyrins increase ascorbate-induced cytotoxicity by enhancing H2O2 generation

Abstract

Renewed interest in using pharmacological ascorbate (AscH-) to treat cancer has prompted interest in leveraging its cytotoxic mechanism of action. A central feature of AscH- action in cancer cells is its ability to act as an electron donor to O2 for generating H2O2. We hypothesized that catalytic manganoporphyrins (MnP) would increase AscH- oxidation rates, thereby increasing H2O2 fluxes and cytotoxicity. Three different MnPs were tested (MnTBAP, MnT2EPyP, and MnT4MPyP), exhibiting a range of physicochemical and thermodynamic properties. Of the MnPs tested, MnT4MPyP exerted the greatest effect on increasing the rate of AscH- oxidation as determined by the concentration of ascorbate radical [Asc•-] and the rate of oxygen consumption. At concentrations that had minimal effects alone, combining MnPs and AscH- synergized to decrease clonogenic survival in human pancreatic cancer cells. This cytotoxic effect was reversed by catalase, but not superoxide dismutase, consistent with a mechanism mediated by H2O2. MnPs increased steady-state concentrations of Asc•- upon ex vivo addition to whole blood obtained either from mice infused with AscH- or patients treated with pharmacologic AscH-. Finally, tumor growth in vivo was inhibited more effectively by combining MnT4MPyP with AscH-. We concluded that MnPs increase the rate of oxidation of AscH- to leverage H2O2 flux and ascorbate-induced cytotoxicity.

Figure 1. Manganoporphyrins increase ascorbate radical indicating an increase in the rate of AscH⁻ oxidation

As seen by EPR spectroscopy, addition of MnPs to a solution of AscH⁻ (1 mM) in PBS, increased [Asc^•−] in a dose-dependent manner. This increase is dependent on the reduction potential of the individual MnPs. At lower concentrations of Asc^•− the curves have a greater slope that decreases as [Asc^•−] increases, which is consistent with an increased rate of AscH⁻ oxidation and the second-order nature of the dismutation reaction of Asc^•− [14]. n = 3.

Figure 2. Manganoporphyrins increase the rate of oxygen consumption, leading to generation of H₂O₂

A. AscH⁻ consumes oxygen at the rate of 2–5 nM s⁻¹ in PBS. Addition of MnT4MPyP (1.0 μM) increases the rate of oxygen consumption to 22–25 nM s⁻¹. Addition of catalase (500 U/mL) leads to a return of O₂ with 16–20 μM H₂O₂ accumulating in solution. n = 3. Inset: MnT4MPyP molecular structure. B. Addition of MnT2EPyP (1.0 μM) to AscH⁻ (1 mM) solution increases the oxygen consumption rate from ≈ 5 nM s⁻¹ to ≈ 10 nM s⁻¹. Addition of catalase indicates 3–5 μM of H₂O₂ has accumulated in solution after 60 min. n = 3. Inset: MnT2EPyP molecular structure. C. MnTBAP (1.0 μM) addition to AscH⁻ (1.0 mM) solution does not alter the oxygen consumption rate. Addition of catalase does not return detectable amounts of oxygen demonstrating minimal H₂O₂ accumulation. n = 3. Inset: MnTBAP molecular structure. D. In DMEM with 10 % FBS, oxygen consumption for AscH⁻ (1 mM) is 20–45 nM s⁻¹. Addition of MnT4MPyP increases oxygen consumption to 80–100 nM s⁻¹. Addition of catalase 30 min after MnT4MPyP leads to a return of oxygen, indicating that 50–60 μM H₂O₂ has accumulated in solution. n = 3.

Figure 3. Manganoporphyrins enhance AscH⁻-induced cytotoxicity in pancreatic cancer cell lines

A. Treatment with AscH⁻ or MnT4MPyP alone does not alter clonogenic survival of MIA PaCa-2 cells. However, the combination of AscH⁻ (1 mM) and MnT4MPyP (0.5 μM) decreases plating efficiency to 5 ± 1 %. When MnT4MPyP (2 μM) was added to AscH⁻, no clones survived. n = 3. *p < 0.001 vs. controls. B. MnT4MPyP (1 μM) or AscH⁻ (1 mM) did not affect AsPC-1 clonogenic survival while the combination led to no surviving clones. n = 3. *p < 0.001 vs. control. C. MnT4MPyP (0.5 – 2 μM) or AscH⁻ (1 mM) did not affect Panc-1 clonogenic survival. However, addition of MnT4MPyP (0.5 μM) to AscH⁻ (1 mM) decreased survival to 3 ± 0.2 %. Only 1 ± 0.1% clones survived exposure to 2 μM MnT4MPyP in the presence of AscH⁻. n = 3. *p < 0.001 vs. control. D. MnT2EPyP (0.5, 1 or 2 μM) or AscH⁻ (1 mM) does not alter MIA PaCa-2 survival. MnT2EPyP (0.5 μM) combined with AscH⁻ (1 mM) decreases plating efficiency to 10 ± 0.5 %. When increased concentrations of MnT2EPyP are used, plating efficiency was reduced to ≤ 5 %. n = 3. *p < 0.001 vs. controls. E. MnTBAP (0.5, 1 or 2 μM) does not alter MIA PaCa-2 plating efficiency. However, when MnTBAP was combined with AscH⁻, MIA PaCa-2 plating efficiency was decreased to 30 ± %. n = 3. *p < 0.001 vs. control. F. The OCR of AscH⁻ when combined with MnPs correlates with log (plating efficiency) in pancreatic cancer cell lines (solid line, R² = 0.9, p < 0.05). In solution, [Asc^•−] in the presence of MnPs (from Figure 1) strongly correlated with log (plating efficiency) in MIA PaCa-2, AsPC-1 and Panc-1 cells (hatched line, R²≈ 0.9, p < 0.05. The (2) below specific points on the graph indicate that two identical points at that value.

Figure 4. SOD does not alter AscH⁻-induced cytotoxicity

A. Cells were treated with AscH⁻ (1 mM) and PEG-SOD (100 U/mL) alone or in combination for 60 min, washed and seeded for clonogenic survival. PEG-SOD did not alter clonogenic survival and combining PEG-SOD with AscH⁻ did not alter clonogenic survival of MIA PaCa-2 cells. p > 0.05 comparing AscH⁻ vs. AscH⁻ + PEG-SOD. B. MIA PaCa-2 cells transfected with AdEmpty or AdEcSOD were lysed and resolved on SDS-polyacrylamide gel and then blotted for the presence of EcSOD. GAPDH was used as a loading control. Lanes 1, 2 and 3 represent parental cells, cells infected with the AdEmpty vector and cells infected with the AdEcSOD vector, respectively. C. MIA PaCa-2 cells were transfected with either AdEmpty or AdEcSOD at 100 MOI. There were minimal changes (p > 0.05) in plating efficiency in AdEmpty or AdEcSOD infected cells treated with AscH⁻.

Figure 5. H₂O₂ mediates MnP enhanced AscH⁻-induced cytotoxicity

A. PEG-SOD (100 U/mL) did not reverse MnT4MPyP enhanced AscH⁻-induced cytotoxicity in MIA PaCa-2 cells, suggesting that superoxide is not directly responsible for the observed cytotoxicity. n =3. *p < 0.001 vs. controls. ^#p > 0.05 vs. AscH⁻ + MnP. B. Treatment with PEG-CAT (120 U/mL) reverses MnT4MPyP enhanced AscH⁻-induced cytotoxicity suggesting that H₂O₂ mediates the observed cytotoxicity. n = 3. *p < 0.001 vs. control. **p < 0.001 vs. AscH⁻ + MnT4MPyP treated cells.

Figure 6. MnT4MPyP enhances ascorbate radical concentration in whole blood as seen by EPR spectroscopy

A. In whole blood from mice, Asc^•− is below the limit of detection. MnT4MPyP (1.0 μM) increased [Asc^•−]_ss to 97 nM. When mice were treated with AscH⁻ (4 g/kg) resulting in a plasma level of [AscH⁻] of 29 mM, [Asc^•−]_ss was increased to 350 nM. With addition of MnT4MPyP (1.0 μM) to ascorbate treated mice, [Asc^•−]_ss increased more than 3-fold to 1200 nM. n = 3. (Hyperfine splitting of Asc^•−, a^H = 1.76 G.) B. In whole blood from humans, Asc^•− is also below the limit of detection. [Asc^•−]_ss increases to 82 nM upon addition of MnT4MPyP (1.0 μM) to human whole blood. After infusion of pharmacological AscH⁻(100 g), [AscH⁻] increased to 22 mM and [Asc^•−]_ss increased to 120 nM. Upon addition of MnT4MPyP (1.0 μM), [Asc^•−]_ss increased to 360 nM. n = 3.

Figure 7. MnT4MPyP enhances ascorbate-induced cytotoxicity and ascorbate radical concentration in vivo

A. In whole blood from a separate group of mice, Asc^•− is below the limit of detection. [Asc^•]_ss increases to 22 nM upon treatment with MnT4MPyP (0.2 mg/kg i.p.). After i.p. injection of pharmacological AscH⁻ (4 g/kg), [AscH⁻] increased to 30 mM and [Asc^•−]_ss increased to 120 nM. Combining both treatments increased [Asc^•−]_ss to 310 nM. n = 3 for each determination. AscH⁻ vs.MnT4MPyP, p < 0.05. AscH⁻ vs. AscH⁻ + MnT4MPyP, p < 0.01. B. MnT4MPyP combined with pharmacological ascorbate decreased MIA PaCa-2 tumor growth in nude mice. The MnT4MPyP and ascorbate-treated animals had significantly slower tumor growth when compared to the control and ascorbate groups (*^#p < 0.05, n = 12–16/group, means ± SEM). MIA PaCa-2 tumor cells (2 × 10⁶) were delivered subcutaneously into the hind leg of nude mice. On day 25, there was nearly a 3-fold decrease in tumor growth in animals receiving the combination when compared to controls.