Pharmacological ascorbate induces sustained mitochondrial dysfunction

Abstract

Pharmacological ascorbate (P-AscH^-; high dose given intravenously) generates H₂O₂ that is selectively cytotoxic to cancer compared to normal cells. The RAS-RAF-ERK1/2 is a major signaling pathway in cancers carrying RAS mutations and is known to be activated by H₂O₂. Activated ERK1/2 also phosphorylates the GTPase dynamin-related protein (Drp1), which then stimulates mitochondrial fission. Although early generation of H₂O₂ leads to cytotoxicity of cancer cells, we hypothesized that sustained increases in H₂O₂ activate ERK-Drp1 signaling, leading to an adaptive response; inhibition of this pathway would enhance the toxicity of P-AscH^-. Increases in phosphorylated ERK and Drp1 induced by P-AscH^- were reversed with genetic and pharmacological inhibitors of ERK and Drp1, as well as in cells lacking functional mitochondria. P-AscH^- increased Drp1 colocalization to mitochondria, decreased mitochondrial volume, increased disconnected components, and decreased mitochondrial length, suggesting an increase in mitochondrial fission 48 h after treatment with P-AscH^-. P-AscH^- decreased clonogenic survival; this was enhanced by genetic and pharmacological inhibition of both ERK and Drp1. In murine tumor xenografts, the combination of P-AscH^- and pharmacological inhibition of Drp1 increased overall survival. These results suggest that P-AscH^- induces sustained changes in mitochondria, through activation of the ERK/Drp1 signaling pathway, an adaptive response. Inhibition of this pathway enhanced the toxicity P-AscH^- to cancer cells.

Keywords: Mitochondrial dynamics; Mitochondrial fission; Oxidative stress; Pharmacological ascorbate.

Figure 1.. Generation of H₂O₂ by P-AscH⁻ activates ERK and Drp1.

339, MIA PaCa-2, and H6c7 cells were treated with P-AscH⁻ (5–30 pmole/cell, 1–2 mM for 1 h). Protein was isolated for Western blot 48 h after treatment. A. P-AscH⁻ increases p-ERK in the patient-derived cell line 339. B. P-AscH⁻ increases p-ERK in MIA PaCa-2 cell line. C. P-AscH⁻ does not alter p-ERK in the non-tumorigenic pancreatic ductal epithelial cell line H6c7. D. MIA PaCa-2 cells treated with P-AscH⁻ (10 pmole/cell, 1 mM for 1 h) and protein was isolated at 0, 24, 48, and 72 h after treatment demonstrating increased p-ERK. E. MIA PaCa-2 cells treated with P-AscH⁻ (10 pmole/cell, 1 mM for 1 h) increased Drp1 levels at 48 and 72 h. F. MIA PaCa-2 cells were treated with 100 μg/mL of catalase, P-AscH⁻ (10 pmole/cell, 1 mM for 1 h), or catalase and P-AscH⁻. Protein isolated 48 h after treatment for western blotting demonstrates reversal of P-AscH⁻-induced increased p-ERK and p-Drp1. G. Established MIA PaCa-2 PDAC xenografts were treated with P-AscH⁻ (4 g/kg, i.p., b.i.d.) or saline b.i.d. for 5 d (n = 6/group). Representative Western blots from harvested tumors. H. Quantification of western blots of tumor xenografts using densitometry demonstrate increased ratio of p-ERK/ERK in the mice receiving P-AscH⁻ compared to saline treated mice (*p = 0.02, Student’s t-test).

Figure 2.. P-AscH⁻ induces mitochondrial fission via Drp1 activation in PDAC.

A. Confocal Microscopy for mitochondrial compartments and volume. Representative confocal images of MIA PaCa-2 cells stained with MitoTracker green 48 h after being treated with P-AscH⁻ (10 pmole/cell, 1 mM) for 1 h vs. control. B. Quantification of confocal microscopy for mitochondrial compartments and volume MitoTracker Green immunofluorescence demonstrates increased mitochondrial compartments and decreased total mitochondrial volume consistent with mitochondrial fission. C. Representative images of electron microscopy of MIA PaCa-2 cells 48 h after P-AscH⁻ treatment. D. Quantification of electron microscopy demonstrating decreased mitochondrial length in MIA PaCa-2 cells treated with P-AscH⁻ (*p = 0.02). Mitochondria (control = 545, P-AscH⁻ = 589) were quantified from images taken from 4 saline treated and 5 P-AscH⁻ treated cells. E. Confocal images of a MIA PaCa-2 xenograft. Athymic nude mice, established tumor xenografts were treated with 1.0 M NaCl or P-AscH⁻ (4 g/kg) twice daily for 5 d. Mitochondrial networks shown green pixels and p-Drp1 bright orange pixels, where yellow pixels indicating p-Drp1 translocated to mitochondria. F. Quantification of colocalization using ImageJ demonstrates significant increases in colocalization after P-AscH⁻ treatment. (*p = 0.02 saline vs. P-AscH⁻ treated mice. Data include 8 images from 4 saline treated animals vs. 14 images from 5 P-AscH⁻treated animals.

Figure 3.. Genetic inhibition of ERK or Drp1 sensitizes cancer cells to P-AscH⁻.

A. Decreases in immunoreactive protein for p-ERK and p-Drp1 levels in the siERK knockout cells. B. Clonogenic survival was performed in siERK and siNEG cell lines after treatment with P-AscH⁻ (7 pmole/cell) for 1 h. Clonogenic survival was decreased in the siERK cells compared to the siNEG cells when treated with P-AscH⁻ (*p < 0.01, Means ± SEM, n = 3). C. Decreases in immunoreactive protein for p-Drp1 in the Drp1 knockout cells. D. Clonogenic survival assays were performed in the siDrp1 and siNEG cell lines after exposure to P-AscH⁻ (7 pmole/cell) demonstrate significant decreases in number of colonies in the siDrp1 cells compared to siNEG cells (*p = 0.02, Means ± SEM, n = 3).

Figure 4.. ρ⁰ cells demonstrate inhibition of P-AscH⁻-induced changes in p-ERK and p-Drp1.

ρ⁰ cells were treated with 10 pmole/cell (1 mM) P-AscH⁻ for 1 h and protein was isolated 48 h later. Neither clone 3 nor clone 4 ρ⁰ cells demonstrated changes in p-ERK or p-Drp1 immunoreactive protein expression following treatment with P-AscH⁻ compared to control. Representative blots are shown.

Figure 5.. Pharmacological inhibition of ERK sensitizes cancer cells to P-AscH⁻.

MIA PaCa-2 cells were treated with P-AscH⁻ (10 pmole/cell, 1 mM) for 1 h, immediately followed by the ERK inhibitor SCH772984 (500 nM) for 24 h. A. Western blot analysis 48 h after P-AscH⁻ treatment shows decreased p-ERK1 in cells treated with SCH772984 compared to those treated with P-AscH⁻ alone. B. Western blot analysis 48 h after P-AscH⁻ treatment shows decreased p-Drp1 in cells treated with SCH772984 compared to those treated with P-AscH⁻ alone. C. Decreased clonogenic survival in cells treated with P-AscH⁻, SCH772984, or the combination in MIA PaCa-2 cells (*p < 0.05, Means ± SEM, n = 3). D. Decreased clonogenic survival in the PDX339 cell line with P-AscH⁻, SCH772984 or the combination (*p < 0.05, Means ± SEM, n = 3). E. No changes in clonogenic survival in the non-tumorigenic pancreatic ductal epithelial cell line H6c7 with P-AscH⁻, SCH772984 or the combination.

Figure 6:. Pharmacological inhibition of Drp1 sensitizes cancer cells to P-AscH⁻.

MIA PaCa-2 cells were treated with P-AscH⁻ (10 pmole/cell, 1 mM) for 1 h, immediately followed by Mdivi-1 (25 μM – 50 μM). A. Western blot analysis 48 h after P-AscH⁻ treatment show decreased p-Drp1 in cells treated with Mdivi-1compared to those treated with P-AscH⁻ alone. B. MIA PaCa-2 cells were treated with P-AscH⁻ for 1 h followed by Mdivi-1 (50 μM) for 24 h. Clonogenic survival demonstrated significantly decreased colony formation with the combination treatment compared to either treatment alone in MIA PaCa-2 cells (*p < 0.01, Means ± SEM, n = 3). C. PDX339 cells had significantly decreased colony formation when treated with P-AscH⁻ + Mdivi-1 (50 μM) compared to P-AscH⁻ alone (*p < 0.05, Means ± SEM, n = 3). D. Clonogenic survival in the H6c7 cell line demonstrated no changes in any of the treatments compared to controls (p > 0.05, Means ± SEM, n = 3).

Figure 7:. Mdivi-1 plus P-AscH⁻ increases PDAC survival in vivo.

MIA PaCa-2 xenografts were created with flank injections in nude mice. After tumors developed, mice received daily i.p. injections of saline control (n = 6), P-AscH⁻ (4 g/kg, n = 6), Mdivi-1 (50 mg/kg, n = 6), or both drugs (n = 6) for 21 days. A. Mice treated with both drugs had the greatest increase in median survival compared to control (54.5 vs. 33 days, *p < 0.05, Log-rank Mantel-Cox test). B. Drug treatments were well tolerated in all groups with no significant differences in body weights between groups throughout the experiment. C. Tumor samples were fixed incubated with p-Drp1 antibody and then secondary antibody. DAPI was used to stain the cell nuclei. Representative tumor sections demonstrate decreased p-Drp1 immunofluorescence in xenografts treated with the combination of P-AscH⁻ + Mdivi-1. D. Images were quantified using ImageJ. The ratios of green fluorescence p-Drp1 over DAPI (blue) were recorded demonstrating decreased p-Drp1 immunofluorescence in xenografts treated with P-AscH⁻ and Mdivi-1 (*p = 0.05, Control vs. Mdivi-1 + P-AscH⁻, one way ANOVA Dunnett’s multiple comparison test).

Figure 8.. Concepts and results in support of the hypothesis.

While the initial increased generation of H₂O₂ is toxic to PDAC cells, sustained generation of H₂O₂ subsequently leads to resistance via late activation of the ERK pathway. Inhibiting this pathway with either ERK or p-Drp-1 inhibitors may prove beneficial in overcoming resistance to P-AscH⁻.