Pharmacological Ascorbate Enhances Chemotherapies in Pancreatic Ductal Adenocarcinoma

Abstract

Objectives:

Pharmacological ascorbate (P-AscH⁻, high-dose, intravenous vitamin C) has shown promise as an adjuvant therapy for pancreatic ductal adenocarcinoma (PDAC) treatment. The objective of this study was to determine the effects of P-AscH⁻ when combined with PDAC chemotherapies.

Methods:

Clonogenic survival, combination indices, and DNA damage were determined in human PDAC cell lines treated with P-AscH⁻ in combination with 5-fluorouracil, paclitaxel, or FOLFIRINOX (combination of leucovorin, 5-fluorouracil, irinotecan, oxaliplatin). Tumor volume changes, overall survival, blood analysis, and plasma ascorbate concentration were determined in vivo in mice treated with P-AscH⁻ with or without FOLFIRINOX.

Results:

P-AscH⁻ combined with 5-fluorouracil, paclitaxel, or FOLFIRINOX significantly reduced clonogenic survival in vitro. DNA damage, measured by γH2AX protein expression, was increased following treatment with P-AscH⁻, FOLFIRINOX, and their combination. In vivo, tumor growth rate was significantly reduced by P-AscH⁻, FOLFIRINOX, and their combination. Overall survival was significantly increased by the combination of P-AscH⁻ and FOLFIRINOX. Treatment with P-AscH⁻ increased red blood cell and hemoglobin values but had no effect on white blood cell counts. Plasma ascorbate concentrations were significantly elevated in mice treated with P-AscH⁻ with or without FOLFIRINOX.

Conclusions:

The addition of P-AscH⁻ to standard-of-care chemotherapy has the potential to be an effective adjuvant for PDAC treatment.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) continues to be one of the deadliest cancers in the United States with an estimated 57,600 new cases and 47,000 deaths in 2020 alone, making it the 11^th most common but 3^rd deadliest in terms of number of deaths per year.¹ The current overall 5-year survival is 10% but a dismal 2.9% with metastatic disease due to the often advanced stage at initial diagnosis and aggressive nature of this cancer.¹ Though outcomes have not seen major improvement, the treatment of PDAC has evolved significantly over the last century. Surgical resection was the mainstay of therapy until the 1950s when a variety of chemotherapy regimens were tested amongst PDAC patients.^2–4 5-fluorouracil (5-FU) offered marginal survival benefits, though it came with significant side effects.^3,5,6 In 1985 Kalser et al⁵ demonstrated significantly improved median survival (11 vs 20 months) for PDAC patients with resectable tumors treated with adjuvant 5-FU and radiation. This was the first study that suggested adjuvant chemoradiotherapy may prolong survival following attempted curative resection for PDAC. Today, surgical resection remains the mainstay of therapy for resectable tumors, coupled with adjuvant therapy. A large number of chemotherapeutic agents have been studied and used in the treatment of PDAC including gemcitabine, albumin-bound paclitaxel (nab-paclitaxel), and most recently the combination chemotherapy regimen FOLFIRINOX (FFX, which is a combination of 5-fluorouracil, leucovorin, irinotecan, and oxaliplatin).^7–9 Unfortunately, even the standard-of-care FFX or gemcitabine plus nab-paclitaxel result in median survival of less than 1 year in patients with metastatic disease.^8,9 Additionally, FFX has been associated with significant hematologic and gastrointestinal toxicity, with 75% of patients experiencing grade 3 or 4 adverse events and 12% of patients requiring discontinuation of the therapy.¹⁰

High-dose ascorbate (pharmacological ascorbate, P-AscH⁻) given intravenously was first suggested as a possible chemotherapeutic agent by Cameron et al in 1976.¹¹ Since that time much work has been undertaken to elucidate its mechanism of action. At normal physiologic concentrations (plasma levels 50–90 μM), ascorbate functions as a cellular reducing agent, antioxidant, and free radical scavenger, protecting normal tissues.^12,13 These physiologic concentrations are tightly regulated and are not surpassed with oral supplementation alone.¹⁴ Alternatively, when given intravenously at doses of 25–100 grams, pharmacologic levels of 1–20 mM in plasma can be achieved. P-AscH⁻ has been shown to act as a pro-oxidant that generates high levels of reactive oxygen species, including hydrogen peroxide (H₂O₂), leading to cell damage and death via multiple pathways, including DNA damage.^15–20 This cytotoxicity has been shown to spare normal cells where catalase activity is sufficient.^15,17

Pharmacological ascorbate has been shown to increase tumor-specific cytotoxicity in a dose-dependent manner across multiple cancer cell lines in in vitro and in vivo models.^17,21,22 For PDAC specifically, P-AscH⁻ has been studied in conjunction with both radiation and chemotherapy in vitro, in vivo, and in several phase I clinical trials. Espey et al²³ and Cieslak et al²⁰ have demonstrated ascorbate’s synergistic effects with gemcitabine when used in PDAC. Both Welsh et al²⁴ and Polireddy et al²⁵ demonstrated acceptable tolerability with a suggestion of improved survival in PDAC patients treated with P-AscH⁻ and gemcitabine compared to historical controls in subjects receiving gemcitabine alone in their phase I and phase I/IIa clinical trials, respectively. Du et al²⁶ demonstrated that the addition of P-AscH⁻ to radiation therapy increased its cytotoxicity, suggesting P-AscH⁻ may also act as a radiosensitizer.²⁶ Further in vitro studies have demonstrated P-AscH⁻ induces cancer-specific cytotoxicity while simultaneously reducing radiation-induced damage to normal cells.^19,27 In 2018, Alexander et al¹⁹ published the results of the first phase I clinical trial in which P-AscH⁻ was combined with radiotherapy and gemcitabine chemotherapy, demonstrating a significant survival advantage.

While recent data suggest improved survival and tolerability when given in combination with gemcitabine for PDAC²⁴, P-AscH⁻ has not been studied in combination with other chemotherapy regimens. Also, many medical oncologists are reluctant to add ascorbate to any treatment regimen because of the antioxidant properties of ascorbate. As mentioned previously, oral ascorbate is frequently confused with P-AscH⁻, which acts as a pro-oxidant. In preparation for additional clinical trials, we hypothesize that P-AscH⁻ will increase the cytotoxic effects of various chemotherapy regimens (5-FU, paclitaxel, and FFX). Our results demonstrate that P-AscH⁻ significantly decreases clonogenic survival in multiple PDAC cell lines when given in combination with 5-FU, paclitaxel, and FFX. Cells treated with FFX combined with P-AscH⁻ also show increased DNA damage.²⁸ Pharmacological ascorbate also reduced tumor growth and increased survival without increased morbidity in an in vivo xenograft mouse model.

MATERIALS AND METHODS

Cell Culture and Reagents

Human PDAC cells lines MIA PaCa-2 and PANC-1 were cultured in Dulbecco’s Modified Eagle medium (DMEM, Gibco, Dublin, Ireland) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin antibiotic (Gibco). The human patient-derived PDAC cell line PDX339 was cultured in DMEM/F-12 medium (Gibco) supplemented with 10% FBS, insulin (Gibco), 500 μL EGF (Gibco), 1 mg hydrocortisone (Sigma, St. Louis, Mo), 2 mL bovine pituitary extract (Gibco) and 1% penicillin–streptomycin antibiotic. Human cell lines (MIA PaCa-2 and PANC-1) were purchased directly from American Type Culture Collection (#CRL-1420 and CRL-1469, Manassas, Va), while the patient-derived cell line (PDX339) was obtained from the Medical College of Wisconsin surgical oncology tissue bank.^29,30 Mycoplasma testing was performed every 6 months and cell lines were passaged for fewer than 6 months after receipt.

Ascorbate was made as a stock solution of 1 M (pH 7) under argon and stored with a tight-fitting stopper at 4°C. Verification of ascorbate concentration was done at 265 nm, ε =14,500 M⁻¹ cm⁻¹.³¹ To account for variations in cell density, media, and cellular metabolism final concentrations were calculated in units of moles-per-cell.^32,33 Regardless of varying media components and cell type, all cells were treated with ascorbate at the IC50³³ for 1 h at 37°C in fresh 10% DMEM medium after which the ascorbate was removed, and the medium was replaced. Cells were treated with various chemotherapy reagents including 5-FU (Sigma), irinotecan (Sigma), paclitaxel (Sigma), and oxaliplatin (Sigma). The reagents 5-FU, paclitaxel, and irinotecan were brought up in DMSO that was diluted to 0.1% or less for use on cells. Oxaliplatin was brought up in H₂O. All cell counts were performed utilizing the Countess II (Life Technologies, Carlsbad, Calif) with trypan blue.

Clonogenic Survival

Clonogenic survival assays were performed as previously described.¹⁹ Briefly, cells were seeded at a density of 1 × 10⁵ in 60-mm dishes 24 h prior to assay. Cells were treated with various doses of 5-FU (10–25 μM),³⁴ paclitaxel (2.5–5 nM),³⁵ or FFX (oxaliplatin: 0.25 μM, irinotecan: 0.5 μM, and 5-FU: 1.25 μM)³⁶ for 24 h. Medium was replaced, and cells were treated with 1 mM (10–20 picomoles cell⁻¹) P-AscH⁻ for 1 h before being plated for clonogenic survival assays. After treatment, cells were counted and plated into 6-well tissue plates at 400–5000 cells/well. Colonies formed 7–14 days after being plated that were then fixed and stained for analysis. Colonies containing ≥50 cells were scored.

Dose-Effect and CI Determination

In separate experiments, MIA PaCa-2 cells were treated with varying doses of FFX (0.125–0.375 μM oxaliplatin, 0.25–0.75 μM irinotecan, and 0.625–1.87 μM 5-FU) for 24 h followed by a media change and 1 h treatment with varying doses of ascorbate (2.5 and 5 picomole cell⁻¹). Following ascorbate treatment, cells were plated for a clonogenic cell survival assay as described above. Clonogenic results were entered into the CompuSyn (ComboSyn, Inc., Paramus, NJ) program to determine the dose-effect and Combination Index (CI) between FFX and P-AscH⁻. Doses of FFX and P-AscH⁻ were in a non-constant ratio.

γH2AX Immunoblot Analysis

MIA PaCa-2, PANC-1, and PDX339 PDAC cells were treated with FFX (0.25 μM oxaliplatin, 0.5 μM irinotecan, and 1.25 μM 5-FU) for 24 h followed by a 30-min treatment with P-AscH⁻ (5 mM). Protein was isolated using PhosphoSafe Extraction Reagent (EMD Millipore Corp, Burlington, Mass) as per manufacturer’s instructions. Protein (40 μg) was electrophoresed in a 4% to 20% Bio-Rad ready gel (Hercules, Calif) and then transferred to a Nitrocellulose membrane (Bio-Rad) at 60 V for 1 h. Membranes were blocked in 5% bovine serum albumin (RPI, Mount Prospect, Ill) for 1 h and then incubated with anti-γH2AX (phospho S139) antibody (1:1000, Abcam, Branford, Conn) or Beta-Tubulin (1:500, Developmental Studies Hybridoma Bank at the University of Iowa, #E7, Iowa City, Iowa) overnight at 4°C. Horseradish peroxidase (HRP)-conjugated goat anti-mouse (1:20,000, EMD Millipore Corp) was used as a secondary antibody. Blots were treated with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, Mass) and exposed to autoradiography film.

In Vivo Experiments

The animal protocols were reviewed and approved by the Animal Care and Use Committee of The University of Iowa. Female six-week-old athymic-nu/nu mice (Foxn1^nu) were purchased from Envigo (Madison, Wis) and allowed to acclimate in the unit for 1 week before any manipulations were performed. MIA PaCa-2 cancer cells (2 × 10⁶) were injected heterotopically and subcutaneously into the flank with a 1-mL tuberculin syringe equipped with a 25-gauge needle. Tumors grew to approximately 3 mm in diameter before experimental treatment began daily with 1 M saline (controls), P-AscH⁻ (4 g/kg or 1 M saline, intraperitoneal), FFX (67 mg/kg leucovorin, 33 mg/kg 5-fluorouracil, 33 mg/kg irinotecan, 3 mg/kg oxaliplatin or saline, intraperitoneal) or in combination. The FOLFIRINOX therapy was given in 2 different dosing regimens. In the first set of experiments mice were treated for 21 days and received 6 doses of FFX (twice/week). In the second set of experiments mice were treated for 28 days and received 8 doses of FFX (twice/week). Mouse weight, tumor volume, and survival were tracked for both experiments. Tumor volumes were determined using calipers applying the equation Volume = Length (longest diameter) × Width (shortest diameter)² × 0.5. Animals were sacrificed when the tumors reached 15 mm in diameter. In separate groups of animals, complete blood counts (CBCs), plasma ascorbate concentration and in vivo γH2AX, mice from the group of animals that received six doses of FFX were anesthetized with isoflurane and blood samples were obtained and tumors were excised for future western blotting analysis. For CBC analysis, 50 μL of blood was immediately combined with 250 μL of PBS (1:6 dilution) into a tube containing EDTA (BD Microtainer, Franklin Lakes, NJ) and immediately placed on ice. Counts were taken on a ADVIA Hematology System (Siemens-Healthineers, Erlangen, Germany) within 30 min of being drawn. Ascorbate concentration determinations were examined 5 min following ascorbate injection (4 g/kg or 1 M saline, intraperitoneal.). Whole blood (200–800 μL) that was combined with 20 μL heparin (Sigma), spun at 2000 g for 20 min at 4°C, and stored at −80°C. The concentration of ascorbate in mouse blood plasma was determined using one of two different assay systems, depending on the expected concentration range.^37,38 Samples with physiological levels of ascorbate were analyzed using a plate reader-based assay.³⁷ In addition, samples with very high, pharmacological levels, were analyzed using a microvolume UV-Vis spectrometer.³⁸ Briefly, a plate reader-based (SpectraMax^® M3, Molecular Devices, San Jose, Calif) kinetic assay was used to measure baseline and physiological levels of ascorbate present in the mouse plasma samples, 10–150 μM. High, pharmacological levels of ascorbate following intraperitoneal injection of ascorbate, 3–35 mM, were measured using an Implen Nanophotometer (Implen GmbH, Munich, Germany) microvolume UV-Vis spectrometer, which requires little sample processing and rapid assessment of ascorbate concentrations in plasma. Protein was isolated from excised tumors and for western blotting of γH2AX expression.

Statistical Methods

Data are presented as the mean ± standard error of the mean (SEM). All in vitro data represent an n = 3 unless otherwise stated. For statistical analyses of two groups, unpaired 2-tailed Student’s t-test were utilized. To study statistical differences between multiple comparisons, significance was determined using one-way ANOVA analysis with Tukey’s multiple-comparisons test. The log-rank test was used for pairwise group comparisons of survival between treatment groups. The above analyses were performed in GraphPad Prism 9 (GraphPad Software, Inc., San Diego, Calif). Dose-effect relationships and CI data were determined utilizing the CompuSyn (ComboSyn, Inc.) program.

RESULTS

P-AscH⁻ Enhances Cytotoxicity of Chemotherapies Used for PDAC

Pharmacological ascorbate alone has been previously shown to decrease clonogenic cell survival of several PDAC cell lines^17,39,40 as well as synergize with gemcitabine and radiation^19,20 in the treatment of PDAC. Several other treatment regimens are currently utilized for PDAC including 5-FU, paclitaxel, and FFX.^{3, 8, 9} Clonogenic cell survival in multiple PDAC cell lines was investigated in the presence of P-AscH⁻ in combination with the various chemotherapy regimens of PDAC. Consistent with previous studies, P-AscH⁻ alone decreased clonogenic survival in PANC-1, MIA PaCa-2, and PDX339 cells compared to controls (Fig. 1). Pharmacological ascorbate in combination with 5-FU significantly reduced clonogenic survival compared to 5-FU alone by 82% in the PANC-1 cell line, 50% in the MIA PaCa-2 cell line, and 70% in the PDX339 cell line (Figs. 1A–C). The addition of P-AscH⁻ with paclitaxel also reduced clonogenic survival compared to paclitaxel alone by 83% in the PANC-1 cell line, 90% in the MIA PaCa-2 cell line, and 67% in the PDX339 cell line (Figs. 1D–F). Again, P-AscH⁻ alone reduced clonogenic survival in the PANC-1 cell line (Figs. 2A–C) but the combination of P-AscH⁻ in combination with FFX further significantly reduced clonogenic survival compared to FFX alone by 65% in the PANC-1 cell line, 45% in the MIA PaCa-2 cell line, and 80% in the PDX339 cell line (Figs. 2A–C).

FIGURE 1.

P-AscH⁻ enhances 5-Fluorouracil and Paclitaxel toxicity in PDAC. Pancreatic ductal adenocarcinoma cells were treated with 5-fluorouracil (5-FU) or paclitaxel (PTX) for 24 h followed by a 1 h treatment with P-AscH⁻. Following the P-AscH⁻ treatment, cells were plated for a clonogenic cell survival assay. Data represent normalized surviving fraction compared to controls ± SEM (*P < 0.05; one-way ANOVA with Tukey’s multiple comparisons, n = 3). A, PANC-1 cells treated with 5-FU (20 μM) and P-AscH⁻ (1 mM) had significantly reduced clonogenic survival compared cells that were treated with P-AscH⁻ or 5-FU alone. B, MIA PaCa-2 cells treated with 5-FU (10 μM) and P-AscH⁻ (1 mM) significantly reduced clonogenic survival compared to cells that were treated with P-AscH⁻ or 5-FU alone. C, PDX339 cells treated with 5-FU (25 μM) and P-AscH⁻ (1 mM) significantly reduced clonogenic survival compared to cells that were treated with 5-FU alone. D, PANC-1 cells treated with PTX (5 nM) and P-AscH⁻ (1 mM) significantly reduced clonogenic survival compared to cells that were treated with Paclitaxel alone. E, MIA PaCa-2 cells treated with PTX (2.5 nM) and P-AscH⁻ (1 mM) significantly reduced clonogenic survival compared to cells that were treated with paclitaxel alone. F, PDX339 cells treated with PTX (5 nM) and P-AscH⁻ (1 mM) had significantly reduced clonogenic survival compared to cells that were treated with P-AscH⁻ or paclitaxel alone.

FIGURE 2.

P-AscH⁻ enhances FFX toxicity PDAC cells. Pancreatic ductal adenocarcinoma cells were treated with FFX (0.25 μM oxaliplatin, 0.5 μM irinotecan, and 1.25 μM 5-FU) for 24 h followed by a 1 h treatment with P-AscH⁻. Following ascorbate treatment, cells were plated for a clonogenic cell survival assay. Data represent normalized surviving fractions compared to the controls ± SEM (*P < 0.05; one-way ANOVA with Tukey’s multiple comparisons, n = 3). A, PANC-1 cells treated with FFX and P-AscH⁻ (1 mM) had significantly reduced clonogenic survival compared to cells that were treated with FFX alone. B, MIA PaCa-2 cells treated with FFX and P-AscH⁻ (1 mM) had significantly reduced clonogenic survival compared to cells that were treated with P-AscH⁻ or FFX alone. C, PDX339 cells treated with FFX and P-AscH⁻ (1 mM) had a significantly reduced clonogenic survival compared to cells that were treated with P-AscH⁻ or FFX alone. D, MIA PaCa-2 cells were treated with FFX1 (0.125 μM oxaliplatin, 0.25 μM irinotecan, and 0.625 μM 5-FU), FFX2 (0.25 μM oxaliplatin, 0.5 μM irinotecan, and 1.25 μM 5-FU), or FFX3 (0.375 μM oxaliplatin, 0.75 μM irinotecan, and 1.87 μM 5-FU) for 24 h followed by a 1 h treatment with P-AscH⁻ (1 mM, 5 picomole cell⁻¹). Following P-AscH⁻ treatment, cells were plated for a clonogenic cell survival assay. FFX and P-AscH⁻ had significantly reduced clonogenic survival compared to cells that were treated with P-AscH⁻ or FFX alone. E, Dose Effect curves were generated in the CompuSyn program from the clonogenic data derived in panel D. The Dose Effect curves shows the Fa (fraction of affected cells) plotted against various concentrations of P-AscH⁻ (2.5 and 5 picomole cell⁻¹) and FFX (listed in D above). The interaction between P-AscH⁻ and FFX in a non-constant combination was evaluated with the Combination Index. F, The plot of CI values vs (Fa) demonstrates the additive effect (CI = 1) between the two drugs.

P-AscH⁻ Combined With FFX Inhibits PDAC Cell Survival

To calculate the Combination Index (CI) and the dose-effect interaction between P-AscH⁻ and FFX, clonogenic assays were repeated at different doses of P-AscH⁻ in combination with 3 different doses of FFX. Cells treated with multiple doses of FFX and P-AscH⁻ had a significantly reduced clonogenic survival compared to cells that were treated with P-AscH⁻ or FFX alone (Figs. 2D). The dose-effect of each single drug and combination of drugs was examined applying the CI of Chou and Talalay.^41,42 Plotting CI values vs the fraction of affected cells (Fa) demonstrated that the effects of P-AscH⁻ and FFX are additive (CI = 1) (Figs. 2E and F).

P-AscH⁻ and FFX Increase Double-stranded DNA Breaks in PDAC

Previous studies from our group have demonstrated P-AscH⁻ generated H₂O₂ initiates DNA damage that can be reliably evaluated through the upregulation of γH2AX,^19,26,43 which is an indicator of DNA damage that forms in the presence of DNA double-strand breaks.²⁸ As shown in Figure 3A–D, both 5 mM P-AscH⁻ and FFX individually induced the expression of immunoreactive γH2AX in MIA PaCa-2, PDX339, and PANC-1 cells compared to controls. Furthermore, the addition of P-AscH⁻ to FFX significantly increased immunoreactive γH2AX resulting in a 2.5-fold increase in MIA PaCa-2 cells (Fig. 3B), and a 3.5-fold increase of expression in both PANC-1 (Fig. 3C) and the PDX339 cell line (Fig. 3D) compared to controls. The increase in γH2AX demonstrates that P-AscH⁻ may amplify chemotherapy-induced cell death by increasing chemotherapy-induced double-stranded DNA breaks.

FIGURE 3.

P-AscH⁻ and FFX increase double-stranded DNA breaks of PDAC in vitro. Pancreatic ductal adenocarcinoma cells were treated with FFX (0.25 μM oxaliplatin, 0.5 μM irinotecan, and 1.25 μM 5-FU) for 24 h followed by a 30 min treatment with P-AscH⁻ (5 mM). Following P-AscH⁻ treatment, protein was isolated, and a western blot was performed. Data represent normalized γH2AX protein expression compared to controls ± SEM for three individual experiments per cell line (*P < 0.05; one-way ANOVA with Tukey’s multiple comparisons, n = 3). A, Representative Western blot image of immunoreactive γH2AX following FFX and P-AscH⁻ treatment in PANC-1 cells. Tubulin was used as a loading control. B, Normalized γH2AX immunoreactive protein expression in MIA PaCa-2 cells increased with treatment of P-AscH⁻ or FFX while the combination of P-AscH⁻ and FFX significantly increased protein expression compared to controls. C, Normalized γH2AX immunoreactive protein expression in PANC-1 cells increased with the treatment of P-AscH⁻, FFX and the combination of P-AscH⁻ and FFX compared to controls. D, Normalized γH2AX immunoreactive protein expression in PDX339 cells significantly increased with treatment of P-AscH⁻, FFX, and the combination of P-AscH⁻ and FFX compared to controls.

P-AscH⁻ Enhances the effect of FFX In Vivo

A wide range of chemotherapy regimens have been studied and used in the treatment of PDAC. One of the current treatment regimens, FFX, results in a median survival of less than one year in patients with PDAC metastatic disease; it is associated with significant side effects. In vitro results suggest that P-AscH⁻ combined with FFX would additively increase cell killing and DNA damage selectively in PDAC cells compared to normal cells. Therefore, in vivo experiments were initiated in which mice with established MIA PaCa-2 xenografts were treated daily with P-AscH⁻, twice weekly with FFX, or in combination. Two different dosing regimens of FFX were examined. In the first set of experiments (Figs. 4A–C), mice were treated for 21 days and received 6 doses of FFX (twice a week). In the second set of experiments (Figs. 4D–F), mice were treated for 28 days and received 8 doses of FFX (twice a week). Mouse weight, tumor volume, and survival was tracked for both experiments.

FIGURE 4.

P-AscH⁻ enhances FFX in vivo toxicity. Athymic nude mice with heterotopic MIA PaCa-2 xenografts were treated daily with P-AscH⁻ (4 g/kg or 1 M saline, intraperitoneal), FFX (67 mg/kg leucovorin, 33 mg/kg 5-fluorouracil, 33 mg/kg irinotecan, 3 mg/kg oxaliplatin or saline, intraperitoneal) or in combination. FFX was given in 2 different dosing regimens. In the first set of experiments (A-C) mice were treated for 21 days and received 6 doses of FFX (twice/week). In the second set of experiments (D-F) mice were treated for 28 days and received 8 doses of FFX (twice/week). Mouse weight, tumor volume, and survival was tracked for both experiments.

A, Mice treated with P-AscH⁻, FFX, or the combination of P-AscH⁻ and FFX (6 doses, twice/week) for 21 days showed no significant changes in weight (g) compared to control mice over the course of treatment. B, Tumor growth was significantly inhibited with P-AscH⁻, FFX, or the combination of P-AscH⁻ and FFX compared to control animals. Data represent average tumor volume (mm³) over 33 days compared to control ± SEM (*P < 0.05; unpaired 2-tailed Student’s t-test). C, Kaplan–Meier survival plots demonstrating survival as a function of time. The log-rank test was used for pairwise group comparisons of survival between treatment groups and the control group. Data demonstrate significantly increased overall survival of animals receiving FFX or the combination of FFX and P-AscH⁻ compared to control. D, Mice treated with P-AscH⁻, FFX, or the combination of P-AscH⁻ and FFX (8 doses, twice/week) for 28 days showed no significant changes in weight (g) compared to P-AscH⁻ mice over the course of treatment. E, Tumor growth showed a trend towards significance (P = 0.07) with the combination of P-AscH⁻ and FFX compared to animals that received only FFX. Data represent average tumor volume (mm³) over 46 days ± SEM (unpaired 2-tailed Student’s t-test). F, Kaplan–Meier survival plots demonstrating survival as a function of time. The log-rank test was used for pairwise group comparisons of survival between treatment groups and the P-AscH⁻ group. Data demonstrate significantly increased overall survival of animals receiving the combination of P-AscH⁻ and FFX compared to P-AscH⁻ alone.

Mice treated with P-AscH⁻, FFX, or the combination of P-AscH⁻ and FFX for 21 days showed no significant changes in weight compared to control mice over the course of treatment (Fig. 4A). However, tumor growth, tracked over 33 days, was significantly inhibited with P-AscH⁻, FFX or the combination of P-AscH⁻ and FFX compared with control animals (Fig. 4B). Survival plots (Fig. 4C) also showed that both FFX and the combination of P-AscH⁻ and FFX significantly increased overall survival of animals compared to control. In mice treated with P-AscH⁻, FFX, or the combination of P-AscH⁻ and FFX for 28 days (Fig. 4D), tumor growth showed a trend towards significance (P = 0.07) with the combination of P-AscH⁻ and FFX compared to animals that received FFX alone (Fig. 4E). Survival plots for the increased dosing regimen demonstrated significantly increased overall survival of animals receiving the combination of P-AscH⁻ and FFX compared to P-AscH⁻ alone (Fig. 4F).

Separate groups of mice treated with the regimen of daily P-AscH⁻ and/or six doses (over 21 d) of FFX or in combination, were evaluated for complete blood counts, plasma ascorbate concentration and in vivo tumor γH2AX expression (Fig. 5). Results show that treatment with P-AscH⁻ significantly increased red blood cell (RBC) counts while treatment with FFX significantly decreased red blood cell levels compared to control (Fig. 5A). There were no significant differences in white blood cell (WBC) counts amongst the various treatments (Fig. 5B). In addition, P-AscH⁻ treatment significantly increased hemoglobin (Hgb) levels while FFX treatment significantly decreased levels compared to control (Fig. 5C). Plasma ascorbate concentration was analyzed in each treatment group and mice that received P-AscH⁻ were found to contain ~750× higher plasma concentration levels of ascorbate (20–30 mM) compared to mice in the FFX or control group (~0.04 mM) (Fig. 5D). Additionally, tumors excised following treatment and analyzed for immunoreactive γH2AX displayed results consistent with in vitro data (Fig. 5E). There was a trend of the combination of P-AscH⁻ and FFX to have increased γH2AX expression indicating an increase of DNA damage in tumors after the combination treatment (Fig. 5F).

FIGURE 5.

P-AscH⁻ does not increase systemic FFX toxicity but increases DNA damage in vivo. Blood was collected after 21 days of treatment through cardiac puncture and utilized for CBCs or processed for plasma. Tumors were excised for western blotting. These data support the hypothesis that ascorbate enhancement of FFX toxicity does not cause an increase in systemic FFX toxicity. A, Pharmacological ascorbate treatment significantly increased red blood cell (RBC) counts while FFX treatment significantly decreased RBC counts compared to control. Data represent mean counts ± SEM (*P < 0.05; one-way ANOVA with Tukey’s multiple comparisons, n = 6 animals/group). B, There were no significant changes in white blood cell (WBC) counts in any of the treatment groups. Data represent mean counts ± SEM (*P < 0.05; one-way ANOVA with Tukey’s multiple comparisons, n = 6 animals/group). C, Pharmacological ascorbate significantly increased hemoglobin (Hgb) levels while FFX significantly decreased levels compared to controls. Data represent mean counts ± SEM (* P < 0.05; one-way ANOVA with Tukey’s multiple comparisons, n = 6 animals/group). D, Plasma ascorbate levels were significantly higher (20–30 mM) in the P-AscH⁻ and P-AscH⁻ plus FFX group compared to the FFX in controls (~0.04 mM). Data represent mean plasma concentrations ± SEM (*P < 0.05; one-way ANOVA with Tukey’s multiple comparisons, n = 6 animals/group). E, Representative Western blot image demonstrating increases in immunoreactive γ-H2AX in tumors from mice treated with P-AscH⁻ with FFX. Tubulin was used as a loading control. F, Quantification of densitometric evaluation of Western blots for normalized in vivo γH2AX immunoreactive protein expression in tumors excised from mice following treatment. γH2AX immunoreactive protein expression increases with FFX or the combination of P-AscH⁻ and FFX compared to controls (n = 5 tumors/group).

DISCUSSION

Due to its aggressive nature and late detection, overall survival in PDAC remains extremely low despite advances in chemotherapy and radiation.¹ Only 12% of patients have localized, resectable disease at the time of diagnosis⁴⁴; thus, improvement in chemoradiotherapy is critical for improving PDAC outcomes. Pharmacologic ascorbate generates H₂O₂ via the auto-oxidation of ascorbate, resulting in cancer-specific cytotoxicity.^15–17 Additionally, P-AscH⁻ has been shown to sensitize cancer cells to radiation while also protecting normal tissue.¹⁹ These characteristics of P-AscH⁻ make it an appealing adjuvant therapy to current standard of care.

Previous studies in PDAC have demonstrated increased cytotoxicity in vitro when P-AscH⁻ is combined with gemcitabine, while phase I and II trials have suggested improved overall survival.^23,24 Pharmacological ascorbate has also been studied in combination with multiple chemotherapies in gastric cancer cell lines, demonstrating significantly decreased clonogenic survival compared to standard of care chemotherapies alone, including paclitaxel, as well as increased survival in vivo when given with standard of care chemotherapy and radiation.⁴⁵ However, few studies have examined its effects in combination with other current standard of care PDAC chemotherapy regimens, such as FFX. Even with a full neoadjuvant course of FFX followed by neoadjuvant chemoradiation, less than a third of patients with locally advanced disease will experience a sufficient enough response to undergo tumor resection.⁴⁶ As shown in this study, P-AscH⁻ significantly increases the cytotoxicity of multiple standard-of-care chemotherapies for PDAC when given in combination as assessed by decreased clonogenic survival and increased levels of double stranded DNA damage in vitro. Comparable changes are observed in vivo with decreased rates of tumor growth and improved survival. Similar results have been reported in lung, brain, breast, ovarian, and colorectal cancer, with increased cytotoxicity in vitro and reduced tumor growth and improved survival in vivo with the addition of P-AscH⁻.^{20,21,47–50} The addition of P-AscH⁻ to the current regimens 5-FU, paclitaxel, and FFX does not appear to reverse their cytotoxic effects and in fact enhances induces tumor specific cytotoxicity in PDAC. This data demonstrates the potential for P-AscH⁻ to increase the effectiveness of current chemotherapies, increase resection rates in unresectable or borderline tumors, and prolong overall survival.

While multiple chemotherapies have shown promise in PDAC treatment, they come with significant toxicities. For example, dose-limiting side effects for FFX are most often hematologic, with nearly 50% of patients experiencing moderate to severe neutropenia (absolute neutrophil count <1000/uL) and 8–10% experiencing thrombocytopenia or anemia at standard doses.^{8, 10} Reducing these hematologic side effects would have a significant impact on PDAC treatment, as patients could potentially tolerate higher doses for longer periods of time. Growth factors such as granulocyte colony stimulating factor can be used, but another potential strategy is to add non-growth factor compounds to counteract chemotherapy-induced cytopenias in cancer patients. One recent study suggests that prophylactic granulocyte colony stimulating factor may reduce hematologic adverse events and improve survival in PDAC patients receiving FFX.⁵¹ However, the National Comprehensive Cancer Network currently does not recommend prophylactic granulocyte colony stimulating factor during FFX treatment for PDAC due to the lack of overall evidence of its benefit across patient groups.⁵² In contrast, P-AscH⁻ has been shown in multiple in vivo models and recent clinical trials to be safe and well tolerated.^{15,17,19, 21,24,25,47} Even in our in vivo model utilizing a reduced dose of FFX for a short duration, the addition of P-AscH⁻ increased red blood cell, hemoglobin, and white blood cell levels back toward normal. Interestingly, significant increases in red blood cells and hemoglobin were observed in the P-AscH⁻ alone group, with smaller increases observed in the FFX + P-AscH⁻ group. While there are no reported interactions between P-AscH⁻ and the individual components of FFX, this data suggests that continuing P-AscH⁻ treatment after the conclusion of FFX may help patients recover from FFX-induced anemia. Reducing neutropenia and anemia in PDAC patients has the potential to increase treatment duration, decrease toxicities, decrease costs associated with managing adverse outcomes secondary to FFX, improve patient comfort and quality of life during treatment, and ultimately improve survival.

In conclusion, we have shown that P-AscH⁻ provides additional cytotoxicity to PDAC cells both in vitro and in vivo when combined with standard of care chemotherapies 5-FU, paclitaxel, and FFX. In addition, P-AscH⁻ is safe, well-tolerated when dosed daily, and may help to correct cytopenias secondary to chemotherapy toxicities.