Next Article in Journal
Nitro-Oleic Acid-Mediated Nitroalkylation Modulates the Antioxidant Function of Cytosolic Peroxiredoxin Tsa1 during Heat Stress in Saccharomyces cerevisiae
Next Article in Special Issue
Nuclear and Radiological Emergencies: Biological Effects, Countermeasures and Biodosimetry
Previous Article in Journal
Polyphenols in Ruminant Nutrition and Their Effects on Reproduction
Previous Article in Special Issue
Peroxiredoxin 6 Applied after Exposure Attenuates Damaging Effects of X-ray Radiation in 3T3 Mouse Fibroblasts
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Antioxidants
Volume 11
Issue 5
10.3390/antiox11050971
antioxidants-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

Auranofin and Pharmacologic Ascorbate as Radiomodulators in the Treatment of Pancreatic Cancer

by
Garett J. Steers
1,2,
Gloria Y. Chen
1,2,
Brianne R. O’Leary
1,2,
Juan Du
1,2,
Hannah Van Beek
1 and
Joseph J. Cullen
1,2,*
1
Free Radical and Radiation Biology Program, Department of Radiation Oncology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA
2
Department of Surgery, The University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA
*
Author to whom correspondence should be addressed.
Antioxidants 2022, 11(5), 971; https://doi.org/10.3390/antiox11050971
Submission received: 6 April 2022 / Revised: 27 April 2022 / Accepted: 11 May 2022 / Published: 14 May 2022
(This article belongs to the Special Issue Ionizing Radiation, Antioxidant Response and Oxidative Damage: Radiomodulators)
Browse Figures
Versions Notes

Abstract

:
Pancreatic cancer accounts for nearly one fourth of all new cancers worldwide. Little progress in the development of novel or adjuvant therapies has been made over the past few decades and new approaches to the treatment of pancreatic cancer are desperately needed. Pharmacologic ascorbate (P-AscH−, high-dose, intravenous vitamin C) is being investigated in clinical trials as an adjunct to standard-of-care chemoradiation treatments. In vitro, P-AscH− has been shown to sensitize cancer cells to ionizing radiation in a manner that is dependent on the generation of H2O2 while simultaneously protecting normal tissue from radiation damage. There is renewed interest in Auranofin (Au), an FDA-approved medication utilized in the treatment of rheumatoid arthritis, as an anti-cancer agent. Au inhibits the thioredoxin antioxidant system, thus increasing the overall peroxide burden on cancer cells. In support of current literature demonstrating Au’s effectiveness in breast, colon, lung, and ovarian cancer, we offer additional data that demonstrate the effectiveness of Au alone and in combination with P-AscH− and ionizing radiation in pancreatic cancer treatment. Combining P-AscH− and Au in the treatment of pancreatic cancer may confer multiple mechanisms to increase H2O2-dependent toxicity amongst cancer cells and provide a promising translatable avenue by which to enhance radiation effectiveness and improve patient outcomes.

Graphical Abstract

1. Pancreatic Cancer

Pancreatic ductal adenocarcinoma is one of the most common cancers worldwide and has remained a leading cause of cancer deaths. In the United States, the estimated incidence is 13.2 cases per 100,000 people, with a mortality rate of 11.1 per 100,000 people, making it the third leading cause of cancer deaths after lung and colorectal cancer [1,2]. Despite a small upward trend in 5-year survival rate since 1975, survival remains the lowest among all cancer types globally, with a current 5-year survival rate of 10.8% for all stages [2,3]. Similarly, the mortality rate for pancreatic cancer has not changed in nearly 50 years; by comparison, colorectal cancer, the second most common cause of cancer deaths, has seen a drop in mortality rate of nearly 60% over the same period [1]. This poor prognosis is attributed to the delayed diagnosis and poor response to therapy. As a result, incidence and mortality rates have increased throughout all regions of the world and are predicted to continue to rise [3].
Treatment of pancreatic cancer is complex and often requires a multi-disciplinary approach given its high mortality and morbidity. Primary factors affecting prognosis and survival are resectability and metastasis. The resectability of a tumor is based on its relation to nearby structures as well as the degree of lymphatic spread. Tumors that invade or encase nearby vasculature, distort vascular anatomy, or invade other nearby organs such as the stomach are considered borderline resectable or locally advanced, indicating a need for neoadjuvant chemoradiotherapy prior to reevaluation for resection [4]. In a similar fashion, the degree of disease in the regional lymph nodes also guides the decision for resection. Distant lymph nodes outside of the field of resection are indicative of more distant, metastatic disease, where upfront resection is avoided. Only approximately 13% of patients are considered resectable at the time of their diagnosis; thus, neoadjuvant chemoradiotherapy is critical in managing more extensive disease with the goal of converting locally advanced, unresectable tumors to resectable [1,5]. Rates of survival for resected patients have increased to as high as 17.4% in 2011 from 1.5% in 1975, while the survival rate of non-resected patients has expectedly remained unchanged during this time, less than 1% [3,6].
For both resectable and unresectable tumors, chemotherapy is a vital component of pancreatic cancer treatment. Patients who undergo initial surgical resection without evidence of recurrence or metastatic disease postoperatively will still require systemic adjuvant treatment [4]. In several multi-center randomized controlled trials, FOLFIRNOX (a combination treatment of fluorouracil, leucovorin, oxaliplatin, and irinotecan) improved overall survival and progression-free survival compared to gemcitabine alone and is now considered standard of care for both neoadjuvant and adjuvant therapy [7,8]. Additional chemotherapy regimens such as gemcitabine plus nab-paclitaxel also offer survival benefit [9]. For locally advanced disease requiring neoadjuvant therapy prior to the consideration of resection, several adjuncts to chemotherapy are available, including radiation therapy, thermal and non-thermal ablation, and intra-arterial chemotherapy. These treatments have varying amounts of success but have been seen to improve overall prognosis [10].
Radiation Therapy as Adjunct Treatment for Pancreatic Cancer
Radiation is primarily known to cause direct DNA damage [11,12,13,14]. It also generates reactive oxygen species, which cause further damage at the DNA level with single- and double-stranded DNA breaks, as well as damage to proteins and lipids [15]. Radiotherapy is often used in conjunction with chemotherapy to increase radiation effectiveness. In pancreatic cancer, fluorouracil (5-FU) was classically utilized, while gemcitabine, capecitabine, and oral 5-FU derivatives such as Xeloda are commonly used today [4]. Neoadjuvant treatment with radiation aims to improve the likelihood of margin-negative resection or downsizing of tumors, although there is no standard practice [16]. In locally advanced and unresectable disease, radiation may assist in local control or prevent local progression or recurrence, despite no evidence of improved overall survival in the limited studies available. A phase III randomized controlled trial, LAP07, investigated locally advanced pancreatic cancer receiving chemoradiotherapy compared to chemotherapy alone. Although there was no survival benefit, there were some improvements in local tumor progression and time to re-initiation of therapy [10,16,17]. As adjuvant therapy, chemoradiation has varying results, with no significant benefit in overall survival, but it has shown some potential in certain patients, particularly those with positive margins [18]. Radiation therapy is also used as an aid in relieving pain, bleeding, or obstructive symptoms in palliative or metastatic disease. Advances such as intensity-modulated RT (IMRT) and stereotactic-body RT (SBRT) have enabled more dose escalation and reduced volumes to improve clinical outcomes [10]. These may be better tolerated by patients and investigations are underway to further assess their role in pancreatic cancer treatment [19,20,21].
The efficacy of radiotherapy has been restricted due to the toxic effects on patients’ quality of life. Due to the location of the pancreas, many nearby organs, such as the small intestine and stomach, are directly within the radiation field. The standard dose is then limited to 50–54 Gy to maintain local control of the cancer without significant toxicity and side effects [10]. Common significant side effects include hematologic toxicities, GI symptoms such as nausea, diarrhea, and abdominal pain, and biliary obstruction. These side effects lead to decreased tolerance of radiation, poorer quality of life for patients, and a limited effect of the radiation itself [22]. Despite increasing the intensity of treatment by altering radiation fractionation or with different concurrent chemotherapies, the majority of patients still experience local failure and succumb to their disease [23]. Thus, there are a significant number of pancreatic cancer patients who would benefit from improvements in the efficacy of standard-of-care chemoradiation therapy.

2. Pharmacologic Ascorbate

Vitamin C (ascorbic acid, ascorbate) at normal physiologic doses is typically considered a donor antioxidant. Ascorbic acid (AscH2) readily oxidizes to ascorbate (AscH−), which then undergoes two one-electron oxidations to form the ascorbate radical (Asc−) [24]. The ascorbate radical is relatively unreactive and can combine with hydrogen ions to reform ascorbate, making ascorbate an effective antioxidant at physiologic levels [24]. However, at supraphysiologic levels, ascorbate exerts pro-oxidant effects. Intestinal absorption of ascorbate is tightly regulated by sodium-dependent vitamin C transporters in enterocytes, with average serum levels maintained around 40–80 µM [25,26,27,28,29]. Thus, supraphysiologic levels cannot be obtained by oral supplementation.
Intravenous administration of ascorbate circumvents this limitation. Pharmacologic ascorbate (P-AscH−, high-dose intravenously administered ascorbate) produces much higher serum ascorbate levels, up to 20 mM [30]. At these supraphysiologic levels, ascorbate acts as a pro-oxidant, donating electrons to form high levels of extracellular hydrogen peroxide (H2O2) that readily crosses cell membranes [24,25,31]. Once H2O2 is intracellular, it reacts with redox-active metals to form the hydroxyl radical (HO∙), a highly toxic radical that causes oxidative damage to proteins, lipids, and DNA [24,31,32,33]. Due to this pathway, P-AscH− has garnered significant interest as a potential cancer therapy.

2.1. Pharmacologic Ascorbate Use in Cancer

The use of vitamin C for the treatment of cancer dates back to the 1970s, when Cameron et al. suggested that high-dose ascorbate could provide a survival benefit when used to treat a variety of cancers, including stomach, colon, rectal, breast, bladder, and pancreatic cancer [34,35,36]. In these studies, patients considered to have terminal disease were given intravenous ascorbic acid (typically 10 g/day) in addition to the standard of care at that time. They observed significantly increased survival times in the P-AscH− treated patients compared to the controls, results which would be demonstrated again in subsequent studies [35,36]. However, in the late 1970s and mid-1980s, two double-blinded, randomized controlled trials studying the use of ascorbate in the treatment of several solid organ tumors demonstrated no effect on patient survival [37,38]. These studies slowed the adoption of P-AscH− in cancer research and treatment. However, these studies differed significantly from those performed by Cameron in that patients received only oral ascorbate as opposed to high-dose ascorbate administered intravenously. As described above, oral supplementation of ascorbate fails to elevate serum levels above the normal physiologic threshold [25,27,28,29]. These findings in the late 1990s and early 2000s ignited a resurgence in interest around P-AscH− in the treatment of cancer.
Pharmacologic ascorbate has shown promise in the treatment of a variety of cancers over the last decade. In vitro and in vivo studies in pancreatic, breast, colorectal, ovarian, glioblastoma, and lung cancer have demonstrated that P-AscH− decreases cell viability, decreases tumor volume, and improves survival alone or in combination with other standard-of-care chemotherapy regimens [33,39,40,41,42,43,44,45]. The selectivity of ascorbate-induced cytotoxicity between normal cells and malignant cells may be due to lower levels of peroxide-reducing enzymes such as catalase, glutathione peroxidase, and peroxiredoxins, as well as higher endogenous levels of reactive oxygen species in cancer cells, leading to the less efficient degradation of H2O2 and increased susceptibility to P-AscH− [31,32,43,46,47].
Following these findings, numerous clinical trials have examined the effects of P-AscH− in combination with chemotherapy or chemoradiation therapy in the treatment of several cancer types. In 2012, Monti et al. published the results of their phase I clinical trial studying the effects of P-AscH− when combined with gemcitabine and erlotinib in 14 subjects with metastatic pancreatic cancer [48]. They observed no significant increase in side effects or toxicity with P-AscH− while also observing a decrease in tumor size in 8 of 9 patients. In 2013, Welsh et al. published similar findings from their phase I clinical trial observing the effects of P-AscH− in combination with gemcitabine in metastatic and node-positive pancreatic cancer [49]. Instead of treating patients with a set dose of P-AscH− (50 g, 75 g, or 100 g), as was done in Monti’s study, patients were treated based upon their plasma ascorbate levels following the previous day’s infusion. Welsh et al. demonstrated that plasma ascorbate concentrations of up to 30 mM could safely be achieved while also demonstrating a potential survival benefit, with mean progression-free survival (PFS) and mean overall survival (OS) of 26 weeks and 12 months, respectively, compared to a mean OS of 5–7 months for gemcitabine alone and 11 months for FOLFIRINOX at that time [7,50]. This was one of the first clinical trials that suggested that P-AscH− may offer a survival benefit to cancer patients.
Additional phase I and II clinical trials in pancreatic, lung, glioblastoma, and ovarian cancer have demonstrated similar results. Ma et al. observed that patients who received P-AscH− in combination with carboplatin and paclitaxel for stage III/IV ovarian cancer had significantly fewer grade 1 and 2 toxicities compared to patients who did not receive P-AscH−, with no increase in grade 3 and 4 toxicities [45]. This study again showed a potential survival benefit, with a prolongation of the median time for disease progression. In a phase I trial observing P-AscH− effects in glioblastoma by Schoenfeld et al., patients receiving P-AscH− in combination with temozolomide and radiation demonstrated mean PFS of 13.3 months and mean OS of 21.5 months, respectively, compared to 7 months and 14 months historically [33]. Multiple ongoing phase I and II clinical studies utilizing P-AscH− in pancreatic, lung, breast, colorectal, bladder, prostate, glioblastoma, and myeloid malignancies will report their findings in the coming years. Recently published studies in pancreatic cancer, including an additional phase I clinical trial, have focused on the effects of P-AscH− when given in combination with radiation therapy.

2.2. Pharmacologic Ascorbate Increases Radiation Toxicity in Pancreatic Cancer While Protecting Normal Tissue

While P-AscH− has shown efficacy when combined with standard-of-care chemotherapies in both in vivo experiments and clinical trials, it has also shown promise as a radiomodulator [41,44,51,52,53,54,55]. Du et al. demonstrated that the addition of P-AscH− to radiation significantly increased DNA damage and significantly decreased the clonogenic survival of multiple pancreatic cancer cell lines compared to radiation alone [52]. They also showed significantly decreased tumor volume and increased survival compared to radiation or P-AscH− alone in a xenograft model. Furthermore, mice that received radiation alone demonstrated a significant reduction in jejunal crypt cells that was partially reversed with the addition of P-AscH− treatment. These results suggested that P-AscH− may not only be an effective adjuvant to chemoradiotherapy but may also offer radioprotection to normal cells. Alexander et al. further demonstrated that P-AscH− significantly improves clonogenic survival, decreases DNA damage, and decreases collagen deposition in normal intestinal cells following radiation [53]. Normal tissue toxicity secondary to radiation damage can have devastating effects on patients. Complication rates are 50% when the nearby intestine receives a total of 60 Gy, making radiation therapy a relatively risky endeavor for pancreatic cancer patients [56]. In addition to its radiosensitization effects on cancer, P-AscH− may also offer normal tissue protection to ensure that more patients can tolerate full, uninterrupted radiation regimens.

2.3. Long-Term Survival following P-AscH− in Pancreatic Cancer

P-AscH− has experienced a rebirth in the field of cancer therapy, with clinical trials underway in a variety of cancers [49,57]. The first phase I clinical trial to actively infuse P-AscH− during the “beam on” time of radiation for the treatment of locally advanced pancreatic cancer, “Gemcitabine, Ascorbate, Radiation therapy for pancreatic cancer, phase I” (NCT01852890), was performed at the University of Iowa [53]. Pharmacologic ascorbate was administered concurrently with gemcitabine and radiation, where gemcitabine was administered weekly for 6 weeks and P-AscH− was administered at 50–100 g during each radiotherapy treatment, either 28 fractions at 50.4 Gy or 25 fractions at 50 Gy. Fourteen subjects completed the protocol therapy between 2014 and 2017. Three study participants are now more than 5 years out from the completion of their neoadjuvant therapies as of January 2022, with the longest surviving participant out nearly 8 years (90 months), resulting in significant increases in both median overall survival (Figure 1A) and median progression-free survival (Figure 1B). Median overall survival for the P-AscH−/gemcitabine/radiation group is 22.8 months, with three long-term survivors (more than 5 years), compared to 12.7 months in the control group and 14 months historically for locally advanced pancreatic cancer [58]. Similarly, the median progression-free survival of 13.7 months in the P-AscH− treatment group is also significantly longer than 4.6 months in the control group and roughly 3.8 months historically for all stages of pancreatic cancer [59]. Three long-term survivors from the relatively small sample size of a phase I trial suggests the efficacy of P-AscH− as a chemotherapeutic agent and radiosensitizer. Currently, there are dozens of clinical trials using P-AscH− in a variety of cancers, including phase II trials in pancreatic cancer utilizing P-AscH− in combination with standard-of-care gemcitabine and nab-paclitaxel (NCT02905578) as well as more experimental regimens (NCT01905150).

3. Pathways for Hydrogen Peroxide Removal

Cancer cells’ decreased ability to neutralize the P-AscH−-induced increase in H2O2 has been shown to be secondary to the decreased expression and activity of enzymes responsible for removing H2O2, including catalase, glutathione peroxidase (GPx), and peroxiredoxins (Prx) [46,47,61,62]. Catalase, predominantly located in peroxisomes, has been shown to be the principal enzyme in H2O2 removal, especially in the presence of high amounts of H2O2, where it is responsible for removing as much as 99% of H2O2 in erythrocytes [63,64]. GPx and peroxiredoxin are considered more important for the removal of lower levels of H2O2 due to their higher affinity [64]. The catalytic activity of GPx is dependent on a regenerating system in which NADPH is used as an electron donor [65]. Utilizing two glutathione (GSH) molecules, GPx generates H2O and glutathione disulfide (GSSG) that is reduced back to glutathione (GSH) by glutathione reductase (GR) using NADPH. Any process that interferes with GPx activity, GR activity, GSSG recycling, or NADPH recycling can impair GPx turnover and reduce its effectiveness [66].
Another means by which to reduce H2O2-scavenging capacity is the targeting of the thioredoxin (Trx) redox buffer system. The thioredoxin system consists of thioredoxin and peroxiredoxin. Thioredoxin is a 12 kDa oxidoreductase necessary for a myriad of cellular processes, including deoxynucleoside triphosphate (dNTP) synthesis, transcriptional regulation, and antioxidant defense [67,68]. The disulfide exchange activity of thioredoxin is integral to the maintenance of peroxiredoxin. Peroxiredoxins are important H2O2 scavengers, with a reaction rate approaching diffusion-controlled limits (107–8 M−1 s−1) and comparable to catalase (107 M−1 s−1) and GPx (108 M−1 s−1) [64,69,70,71]. Thus, the presence of sufficient reduced thioredoxin (Trx(SH)2) to maintain peroxiredoxin in the active (reduced) form is vital for cellular defense against H2O2. Following the two-electron reduction of a peroxiredoxin by reduced thioredoxin, thioredoxin disulfide (TrxS-S) is recycled back to reduced thioredoxin by thioredoxin reductase (TrxR), a flavin-containing selenoprotein, using reducing equivalents from NADPH [72,73]. Previous studies have shown that inhibition of the thioredoxin/thioredoxin reductase complex reduces peroxiredoxin function [2].
Targeting these pathways offers another potential avenue for cancer treatment. By decreasing cancer cells’ ability to degrade H2O2, oxidative stress within cancer cells may be further amplified, ultimately leading to enhanced cancer-specific cytotoxicity. A recent study examining the role of catalase in pancreatic cancer demonstrated that catalase knockout cells exhibited greater radiosensitization to P-AscH− and that cancer cells in long-term survivors may express lower levels of catalase than cancer cells in short-term survivors [60]. These results suggest that inhibiting a portion of the peroxide removal system—catalase, glutathione reductase, thioredoxin reductase, or peroxiredoxin—may enhance the effectiveness of treatments aimed at increasing oxidative stress within cancer cells.

4. Auranofin

Auranofin (Au) is a gold-triethylphosphine compound originally FDA-approved in 1985 for the treatment of rheumatoid arthritis [74,75]. Au is one of three disease-modifying antirheumatic drugs (DMARDs) that contain gold, though it is the only drug in this group that can be administered orally due to its lipophilic properties [74]. Au is believed to exert multiple anti-inflammatory effects by altering the production and secretion of pro-inflammatory cytokines and modulating intracellular signaling pathways such as activating mitogen-activated protein kinases (MAPK), all of which play a role in rheumatoid arthritis disease severity and progression [76,77,78,79,80,81]. The exact mechanisms behind these effects are not completely understood but do seem to involve enzyme inhibition and appear to be dependent on the gold atom situated between a sulfur atom and the triethylphopshine group [82,83,84,85]. Additionally, Au has shown effectiveness outside of its immune-modulating effects and has garnered interest for its antibacterial, antiviral, cytoprotective, and anticancer potential [86,87,88,89,90,91].

4.1. Auranofin Use in Cancer

Over the past few decades, significant interest in the anticancer effects of Au has arisen. The key mechanism of action thought to be responsible for the anticancer effects of Au is thioredoxin reductase inhibition (Figure 2) [92,93]. Au serves as an electrophile and reacts with the selenocysteine residue in the active site of thioredoxin reductase by forming a covalent adduct and inactivating the enzyme [73]. Inhibition of thioredoxin reductase inhibits the flow of electrons from NADPH to peroxiredoxin by impairing the recycling of thioredoxin, leading to a subsequent reduction in peroxiredoxin activity and an increase in H2O2 [94]. Increasing H2O2 via this mechanism may lead to additional cancer-specific cytotoxicity, as discussed previously [31,95]. Several in vitro studies have demonstrated a synergistic induction of apoptosis when Au is given in combination with various chemotherapy agents in breast cancer cell models [96,97,98]. In one study, Lee et al. demonstrated that the combination of Au and mesupron, a urokinase-type plasminogen activator inhibitor currently undergoing clinical trials in breast cancer, promoted the inhibition of breast cancer cell proliferation and induced cell cycle arrest and apoptosis, while also significantly increasing reactive oxygen species within cancer cells [97]. Similar studies in colon, ovarian, and lung cancer cell lines demonstrating synergistic increases in cancer cell death show that Au may be an effective anticancer agent when combined with other chemotherapy agents [99,100,101]. Based on these findings, a previous study hypothesized that Au may sensitize cancer cells to P-AscH− by impairing hydroperoxide removal [102]. To test this, pancreatic ductal adenocarcinoma cancer cells were treated with inhibitors of hydroperoxide metabolism. Data show that targeting of the thioredoxin system with Au inhibits thioredoxin reductase activity and sensitizes pancreatic cancer cells to P-AscH−-generated H2O2. The combination of Au and P-AscH− also significantly increases the sensitivity of pancreatic cancer cells to ionizing radiation. Based on these results, repurposing Au in combination with P-AscH− as a chemotherapy and radiomodulatory regimen may provide a potentially promising and translatable new treatment for pancreatic cancer. For all experiments, Au was dissolved in PBS containing 1% EtOH.

4.2. Au Inhibits Thioredoxin Reductase Activity in Pancreatic Cancer Cells

To determine if Au inhibits thioredoxin reductase activity in pancreatic cancer cells, MIA PaCa-2 pancreatic cancer cells were treated with Au for 3 h and thioredoxin reductase activity was measured by spectrophotometrically following the reduction of 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) utilizing a thioredoxin reductase assay kit. Activity was normalized to the protein concentration determined by the Lowry protein assay. Au was shown to significantly decrease thioredoxin reductase activity in pancreatic cancer cells (Figure 3) [102].

4.3. Auranofin Sensitizes Pancreatic Cancer Cells to P-AscH−

As described above, P-AscH− is selectively cytotoxic to cancer cells due to the generation of high amounts of H2O2 [24,25,31]. Furthermore, the cytotoxic effects of P-AscH− are enhanced in catalase knockout cell lines [60]. Based on these findings, it was hypothesized that inhibition of thioredoxin reductase by Au would disrupt the thioredoxin-dependent hydroperoxide scavenging system and sensitize pancreatic cancer cells to P-AscH− treatment [102]. To test this, exponentially growing MIA PaCa-2 cells were treated with Au for 3 h prior to P-AscH− treatment for 1 h. Stock solutions of L-ascorbic acid were prepared under argon and stored in screw-cap glass vials at 4 °C. The utilized dose of P-AscH− has been previously demonstrated to result in 50% clonogenic survival (ED50) of the MIA PaCa-2 cell line over many iterations of clonogenic survival analysis [43]. Following treatment, cells were detached, counted, and plated at designated densities, and allowed to grow for 10–14 days. Surviving fractions were calculated and normalized to control plates. Auranofin alone decreased clonogenic survival compared to the control, while the combination treatment of P-AscH− with Au further decreased clonogenic survival compared to either treatment alone (Figure 4) [102].
Cancer cell killing by P-AscH− has been shown to be dependent on extracellular H2O2 [33,41]. Thus, the overexpression of catalase or addition of bovine catalase in media was hypothesized to partially or completely rescue cancer cells from toxicity, mediated by the combination of P-AscH− and Au. P-AscH− was again dosed at the ED50 of the cell line, as determined in previous experimentation [43]. P-AscH− caused slightly more cell death in this set of experiments (~40% vs. ~60% in Figure 4), though this small difference is not uncommon [43]. The addition of bovine catalase to cell media prevented the synergistic effect of Au/P-AscH− in pancreatic cancer cells (Figure 5) [102]. These findings support the hypothesis that H2O2 is responsible for the effects of P-AscH− when cells are treated with Au and P-AscH− in combination.

4.4. Au Combined with P-AscH− Sensitizes Pancreatic Cancer Cells to Ionizing Radiation

P-AscH− has been shown to sensitize cancer cells to ionizing radiation in an H2O2-dependent manner in both in vitro and in vivo studies and has shown promise in recent phase I clinical trials [52,53]. To determine the radiosensitizing effects of Au and P-AscH− in combination in pancreatic cancer cell lines, exponentially growing MIA PaCa-2 and AsPC-1 pancreatic cancer cells were radiated with 1–2 Gy ionizing radiation with or without treatment with Au for 24 h prior to treatment with P-AscH− for 1 h. Cells were then detached, counted, and plated at designated densities and allowed to grow for 10–14 days and surviving fractions calculated as described previously [43]. The combination of Au and P-AscH− caused significant cell death (~90%) when combined with radiation (Figure 6) [102]. These data suggest that combining Au with P-AscH− has the possibility of increasing radiation-induced toxicity.

4.5. Au and P-AscH− Potential in Cancer Therapy

These studies demonstrate that inhibition of the thioredoxin antioxidant system by the FDA-approved anti-rheumatic agent Au inhibits thioredoxin reductase activity and sensitizes pancreatic cancer cell lines to treatment with P-AscH− in a manner that is dependent on H2O2. In addition, the combination of Au and P-AscH− sensitizes pancreatic cancer cells to radiation therapy. The selective sensitivity imposed by thioredoxin reductase inhibition may be due to the impairment of H2O2 metabolism by peroxiredoxin, resulting in enhanced H2O2-mediated oxidative damage, leading to cell death from oxidative protein, lipid, and/or DNA damage. Alternatively, the unique role of the thioredoxin system in redox signaling may also explain the efficacy of Au. Due to the environment around the redox-active cysteines, peroxiredoxins are uniquely amenable to oxidation by H2O2 [104]. In fact, peroxiredoxins exhibit a reactivity toward H2O2 approximately six to eight orders of magnitude higher than other redox-regulated proteins [105]. This sensitivity allows peroxiredoxin to outcompete other thiols for H2O2 and serve as a medium through which oxidative equivalents can be transduced as a signal [106,107]. Indeed, peroxiredoxins exhibit a significant influence over cell death signaling [108,109,110] through the oxidative modification of several targets, such as p38 [111], ERK [109], ASK1 [112,113], Akt [111,114], and STAT3 [115], among many others.
Studies have shown that peroxiredoxin hyper-oxidation sensitizes cells to cell death signaling induced by agents that produce H2O2 [111]. Other groups have shown that Auranofin-induced peroxiredoxin oxidation sensitizes triple-negative breast cancer and malignant B-cells to P-AscH− [116,117]. Peroxiredoxin hyper-oxidation induced by the combination of a decreased capacity to recycle reduced thioredoxin and enhanced H2O2 generated by P-AscH− may cause a synergistic enhancement of cell death signaling. Thus, the combination of Au and P-AscH− may serve as a highly effective means to exploit the signaling of the peroxiredoxin system to induce tumor cell-specific cell death. Future experiments can interrogate the role of peroxiredoxin signaling in cell death induced by Au/Asc by using cell lines depleted of select peroxiredoxins [106].

5. Conclusions

Pancreatic cancer continues to carry an extremely poor prognosis and remains the third-leading cause of cancer deaths, despite advances in chemotherapy and radiation protocols. Pharmacologic ascorbate has shown promise as an effective adjunct therapy, with phase I and phase II clinical trials suggesting a survival benefit compared to standard-of-care therapies. Ongoing clinical trials in multiple cancer types will help to further elucidate the efficacy and role of pharmacologic ascorbate in cancer therapy. The effects of P-AscH− on normal tissue have yet to be delineated. However, recent studies demonstrate radioprotective effects. This aspect of P-AscH− therapy could improve quality of life for patients and help patients tolerate higher doses of chemotherapy and/or radiation, providing additional incentive for its use. In line with previous studies’ observations in other cancers, Au may also be useful as an adjunct therapy in the treatment of pancreatic cancer. Previous supporting data demonstrate that Au inhibits thioredoxin reductase and sensitizes pancreatic cancer cells to P-AscH− in a manner that is dependent on H2O2. The combination of these two clinically available agents and their roles as radiomodulators presents an exciting avenue to enhance tumor responses to chemoradiation therapies and should be explored in future experimentation by comparing Auranofin to other current standard-of-care regimens. Additionally, studying the Auranofin mechanism in cancer treatment could offer significant insight into other models and techniques for exploiting H2O2-induced cytotoxicity in cancer.

Author Contributions

Conceptualization, G.J.S., G.Y.C., B.R.O., J.D., H.V.B. and J.J.C.; methodology, G.J.S., G.Y.C., B.R.O., J.D. and J.J.C.; validation, G.J.S., G.Y.C., B.R.O., J.D. and J.J.C.; formal analysis, G.J.S., B.R.O. and J.D.; investigation, G.J.S., B.R.O. and J.D.; data curation, G.J.S., B.R.O., J.D. and H.V.B.; writing—original draft preparation, G.J.S., G.Y.C., B.R.O., J.D. and J.J.C.; writing—review and editing, G.J.S., G.Y.C., B.R.O., J.D. and J.J.C.; visualization, G.J.S.; supervision, J.J.C.; funding acquisition, J.J.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by NIH grants P01 CA217797, T32 CA148062, and T32 CA78586.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Siegel, R.L.; Miller, K.D.; Fuchs, H.E.; Jemal, A. Cancer statistics, 2022. CA Cancer J. Clin. 2022, 72, 7–33. [Google Scholar] [CrossRef] [PubMed]
  2. Low, F.M.; Hampton, M.B.; Peskin, A.V.; Winterbourn, C.C. Peroxiredoxin 2 functions as a noncatalytic scavenger of low-level hydrogen peroxide in the erythrocyte. Blood 2007, 109, 2611–2617. [Google Scholar] [CrossRef] [PubMed]
  3. Hu, J.X.; Zhao, C.F.; Chen, W.B.; Liu, Q.C.; Li, Q.W.; Lin, Y.Y.; Gao, F. Pancreatic cancer: A review of epidemiology, trend, and risk factors. World J. Gastroenterol. 2021, 27, 4298–4321. [Google Scholar] [CrossRef] [PubMed]
  4. Network, N.C.C. NCCN Clinical Practice Guidelines in Oncology: Pancreatic Adenocarcinoma. 2022. Available online: https://www.nccn.org/ (accessed on 1 April 2022).
  5. Li, D.; Xie, K.; Wolff, R.; Abbruzzese, J.L. Pancreatic cancer. Lancet 2004, 363, 1049–1057. [Google Scholar] [CrossRef]
  6. Bengtsson, A.; Andersson, R.; Ansari, D. The actual 5-year survivors of pancreatic ductal adenocarcinoma based on real-world data. Sci. Rep. 2020, 10, 16425. [Google Scholar] [CrossRef]
  7. Conroy, T.; Desseigne, F.; Ychou, M.; Bouche, O.; Guimbaud, R.; Becouarn, Y.; Adenis, A.; Raoul, J.L.; Gourgou-Bourgade, S.; de la Fouchardiere, C.; et al. FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. N. Engl. J. Med. 2011, 364, 1817–1825. [Google Scholar] [CrossRef] [Green Version]
  8. Conroy, T.; Hammel, P.; Hebbar, M.; Ben Abdelghani, M.; Wei, A.C.; Raoul, J.L.; Chone, L.; Francois, E.; Artru, P.; Biagi, J.J.; et al. FOLFIRINOX or Gemcitabine as Adjuvant Therapy for Pancreatic Cancer. N. Engl. J. Med. 2018, 379, 2395–2406. [Google Scholar] [CrossRef]
  9. Chin, V.; Nagrial, A.; Sjoquist, K.; O’Connor, C.A.; Chantrill, L.; Biankin, A.V.; Scholten, R.J.; Yip, D. Chemotherapy and radiotherapy for advanced pancreatic cancer. Cochrane Database Syst. Rev. 2018, 3, CD011044. [Google Scholar] [CrossRef]
  10. Spiliopoulos, S.; Zurlo, M.T.; Casella, A.; Laera, L.; Surico, G.; Surgo, A.; Fiorentino, A.; de’Angelis, N.; Calbi, R.; Memeo, R.; et al. Current status of non-surgical treatment of locally advanced pancreatic cancer. World J. Gastrointest. Oncol. 2021, 13, 2064–2075. [Google Scholar] [CrossRef]
  11. Goldstein, M.; Kastan, M.B. The DNA damage response: Implications for tumor responses to radiation and chemotherapy. Annu. Rev. Med. 2015, 66, 129–143. [Google Scholar] [CrossRef] [Green Version]
  12. Powell, S.; McMillan, T.J. DNA damage and repair following treatment with ionizing radiation. Radiother Oncol. 1990, 19, 95–108. [Google Scholar] [CrossRef]
  13. Sutherland, B.M.; Bennett, P.V.; Sidorkina, O.; Laval, J. Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation. Proc. Natl. Acad. Sci. USA 2000, 97, 103–108. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  14. Sutherland, B.M.; Bennett, P.V.; Sutherland, J.C.; Laval, J. Clustered DNA damages induced by x rays in human cells. Radiat. Res. 2002, 157, 611–616. [Google Scholar] [CrossRef]
  15. Kobayashi, J.; Iwabuchi, K.; Miyagawa, K.; Sonoda, E.; Suzuki, K.; Takata, M.; Tauchi, H. Current topics in DNA double-strand break repair. J. Radiat. Res. 2008, 49, 93–103. [Google Scholar] [CrossRef] [Green Version]
  16. Tempero, M.A.; Malafa, M.P.; Al-Hawary, M.; Asbun, H.; Bain, A.; Behrman, S.W.; Benson, A.B.; Binder, E.; Cardin, D.B.; Cha, C.; et al. Pancreatic Adenocarcinoma, Version 2.2017, NCCN Clinical Practice Guidelines in Oncology. J. Natl. Compr. Cancer Netw. 2017, 15, 1028–1061. [Google Scholar] [CrossRef]
  17. Hammel, P.; Huguet, F.; van Laethem, J.L.; Goldstein, D.; Glimelius, B.; Artru, P.; Borbath, I.; Bouche, O.; Shannon, J.; Andre, T.; et al. Effect of Chemoradiotherapy vs Chemotherapy on Survival in Patients with Locally Advanced Pancreatic Cancer Controlled After 4 Months of Gemcitabine With or Without Erlotinib: The LAP07 Randomized Clinical Trial. JAMA 2016, 315, 1844–1853. [Google Scholar] [CrossRef]
  18. Tempero, M.A.; Malafa, M.P.; Chiorean, E.G.; Czito, B.; Scaife, C.; Narang, A.K.; Fountzilas, C.; Wolpin, B.M.; Al-Hawary, M.; Asbun, H.; et al. NCCN Guidelines Insights: Pancreatic Adenocarcinoma, Version 1.2019: Featured Updates to the NCCN Guidelines. J. Natl. Compr. Cancer Netw. 2019, 17, 202–210. [Google Scholar] [CrossRef] [Green Version]
  19. Abi Jaoude, J.; Thunshelle, C.P.; Kouzy, R.; Nguyen, N.D.; Lin, D.; Prakash, L.; Bumanlag, I.M.; Noticewala, S.S.; Niedzielski, J.S.; Beddar, S.; et al. Stereotactic Versus Conventional Radiation Therapy for Patients with Pancreatic Cancer in the Modern Era. Adv. Radiat. Oncol. 2021, 6, 100763. [Google Scholar] [CrossRef]
  20. Ghaly, M.; Gogineni, E.; Herman, J.; Saif, M.W. New Potential Options for SBRT in Pancreatic Cancer. Cancer Med. J. 2021, 4, 41–50. [Google Scholar]
  21. Pavic, M.; Niyazi, M.; Wilke, L.; Corradini, S.; Vornhulz, M.; Mansmann, U.; Al Tawil, A.; Fritsch, R.; Horner-Rieber, J.; Debus, J.; et al. MR-guided adaptive stereotactic body radiotherapy (SBRT) of primary tumor for pain control in metastatic pancreatic ductal adenocarcinoma (mPDAC): An open randomized, multicentric, parallel group clinical trial (MASPAC). Radiat. Oncol. 2022, 17, 18. [Google Scholar] [CrossRef]
  22. Qing, S.; Gu, L.; Zhang, H. Phase I study of dose-escalated stereotactic body radiation therapy for locally advanced pancreatic head cancers: Initial clinical results. Cancer Med. 2021, 10, 6736–6743. [Google Scholar] [CrossRef] [PubMed]
  23. Oberstein, P.E.; Olive, K.P. Pancreatic cancer: Why is it so hard to treat? Therap. Adv. Gastroenterol. 2013, 6, 321–337. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Buettner, G.R. The pecking order of free radicals and antioxidants: Lipid peroxidation, alpha-tocopherol, and ascorbate. Arch. Biochem. Biophys. 1993, 300, 535–543. [Google Scholar] [CrossRef]
  25. Padayatty, S.J.; Sun, H.; Wang, Y.; Riordan, H.D.; Hewitt, S.M.; Katz, A.; Wesley, R.A.; Levine, M. Vitamin C pharmacokinetics: Implications for oral and intravenous use. Ann. Intern. Med. 2004, 140, 533–537. [Google Scholar] [CrossRef] [PubMed]
  26. Levine, M.; Wang, Y.; Padayatty, S.J.; Morrow, J. A new recommended dietary allowance of vitamin C for healthy young women. Proc. Natl. Acad. Sci. USA 2001, 98, 9842–9846. [Google Scholar] [CrossRef] [Green Version]
  27. Vera, J.C.; Rivas, C.I.; Velasquez, F.V.; Zhang, R.H.; Concha, I.I.; Golde, D.W. Resolution of the facilitated transport of dehydroascorbic acid from its intracellular accumulation as ascorbic acid. J. Biol. Chem. 1995, 270, 23706–23712. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  28. Savini, I.; Rossi, A.; Pierro, C.; Avigliano, L.; Catani, M.V. SVCT1 and SVCT2: Key proteins for vitamin C uptake. Amino. Acids. 2008, 34, 347–355. [Google Scholar] [CrossRef]
  29. Levine, M.; Conry-Cantilena, C.; Wang, Y.; Welch, R.W.; Washko, P.W.; Dhariwal, K.R.; Park, J.B.; Lazarev, A.; Graumlich, J.F.; King, J.; et al. Vitamin C pharmacokinetics in healthy volunteers: Evidence for a recommended dietary allowance. Proc. Natl. Acad. Sci. USA 1996, 93, 3704–3709. [Google Scholar] [CrossRef] [Green Version]
  30. Verrax, J.; Calderon, P.B. Pharmacologic concentrations of ascorbate are achieved by parenteral administration and exhibit antitumoral effects. Free Radic. Biol. Med. 2009, 47, 32–40. [Google Scholar] [CrossRef]
  31. Du, J.; Martin, S.M.; Levine, M.; Wagner, B.A.; Buettner, G.R.; Wang, S.H.; Taghiyev, A.F.; Du, C.; Knudson, C.M.; Cullen, J.J. Mechanisms of ascorbate-induced cytotoxicity in pancreatic cancer. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 2010, 16, 509–520. [Google Scholar] [CrossRef] [Green Version]
  32. Chen, Q.; Espey, M.G.; Krishna, M.C.; Mitchell, J.B.; Corpe, C.P.; Buettner, G.R.; Shacter, E.; Levine, M. Pharmacologic ascorbic acid concentrations selectively kill cancer cells: Action as a pro-drug to deliver hydrogen peroxide to tissues. Proc. Natl. Acad. Sci. USA 2005, 102, 13604–13609. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Schoenfeld, J.D.; Sibenaller, Z.A.; Mapuskar, K.A.; Wagner, B.A.; Cramer-Morales, K.L.; Furqan, M.; Sandhu, S.; Carlisle, T.L.; Smith, M.C.; Abu Hejleh, T.; et al. O2− and H2O2-Mediated Disruption of Fe Metabolism Causes the Differential Susceptibility of NSCLC and GBM Cancer Cells to Pharmacological Ascorbate. Cancer Cell 2017, 31, 487–500. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Cameron, E.; Campbell, A.; Jack, T. The orthomolecular treatment of cancer. III. Reticulum cell sarcoma: Double complete regression induced by high-dose ascorbic acid therapy. Chem. Biol. Interact. 1975, 11, 387–393. [Google Scholar] [CrossRef]
  35. Cameron, E.; Pauling, L. Supplemental ascorbate in the supportive treatment of cancer: Prolongation of survival times in terminal human cancer. Proc. Natl. Acad. Sci. USA 1976, 73, 3685–3689. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  36. Cameron, E.; Pauling, L. Supplemental ascorbate in the supportive treatment of cancer: Reevaluation of prolongation of survival times in terminal human cancer. Proc. Natl. Acad. Sci. USA 1978, 75, 4538–4542. [Google Scholar] [CrossRef] [Green Version]
  37. Creagan, E.T.; Moertel, C.G.; O’Fallon, J.R.; Schutt, A.J.; O’Connell, M.J.; Rubin, J.; Frytak, S. Failure of high-dose vitamin C (ascorbic acid) therapy to benefit patients with advanced cancer. A controlled trial. N. Engl. J. Med. 1979, 301, 687–690. [Google Scholar] [CrossRef] [PubMed]
  38. Moertel, C.G.; Fleming, T.R.; Creagan, E.T.; Rubin, J.; O’Connell, M.J.; Ames, M.M. High-dose vitamin C versus placebo in the treatment of patients with advanced cancer who have had no prior chemotherapy. A randomized double-blind comparison. N. Engl. J. Med. 1985, 312, 137–141. [Google Scholar] [CrossRef]
  39. Ghanem, A.; Melzer, A.M.; Zaal, E.; Neises, L.; Baltissen, D.; Matar, O.; Glennemeier-Marke, H.; Almouhanna, F.; Theobald, J.; Abu El Maaty, M.A.; et al. Ascorbate kills breast cancer cells by rewiring metabolism via redox imbalance and energy crisis. Free Radic. Biol. Med. 2021, 163, 196–209. [Google Scholar] [CrossRef]
  40. Yun, J.; Mullarky, E.; Lu, C.; Bosch, K.N.; Kavalier, A.; Rivera, K.; Roper, J.; Chio, I.I.; Giannopoulou, E.G.; Rago, C.; et al. Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH. Science 2015, 350, 1391–1396. [Google Scholar] [CrossRef] [Green Version]
  41. Chen, Q.; Espey, M.G.; Sun, A.Y.; Pooput, C.; Kirk, K.L.; Krishna, M.C.; Khosh, D.B.; Drisko, J.; Levine, M. Pharmacologic doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor xenografts in mice. Proc. Natl. Acad. Sci. USA 2008, 105, 11105–11109. [Google Scholar] [CrossRef] [Green Version]
  42. Cieslak, J.A.; Strother, R.K.; Rawal, M.; Du, J.; Doskey, C.M.; Schroeder, S.R.; Button, A.; Wagner, B.A.; Buettner, G.R.; Cullen, J.J. Manganoporphyrins and ascorbate enhance gemcitabine cytotoxicity in pancreatic cancer. Free Radic. Biol. Med. 2015, 83, 227–237. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  43. Doskey, C.M.; Buranasudja, V.; Wagner, B.A.; Wilkes, J.G.; Du, J.; Cullen, J.J.; Buettner, G.R. Tumor cells have decreased ability to metabolize H2O2: Implications for pharmacological ascorbate in cancer therapy. Redox Biol. 2016, 10, 274–284. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Espey, M.G.; Chen, P.; Chalmers, B.; Drisko, J.; Sun, A.Y.; Levine, M.; Chen, Q. Pharmacologic ascorbate synergizes with gemcitabine in preclinical models of pancreatic cancer. Free Radic. Biol. Med. 2011, 50, 1610–1619. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Ma, Y.; Chen, P.; Drisko, J.A.; Khabele, D.; Godwin, A.K.; Chen, Q. Pharmacological ascorbate induces ‘BRCAness’ and enhances the effects of Poly(ADP-Ribose) polymerase inhibitors against BRCA1/2 wild-type ovarian cancer. Oncol. Lett. 2020, 19, 2629–2638. [Google Scholar] [CrossRef] [Green Version]
  46. Schafer, F.Q.; Buettner, G.R. Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic. Biol. Med. 2001, 30, 1191–1212. [Google Scholar] [CrossRef]
  47. Liu, J.; Hinkhouse, M.M.; Sun, W.; Weydert, C.J.; Ritchie, J.M.; Oberley, L.W.; Cullen, J.J. Redox regulation of pancreatic cancer cell growth: Role of glutathione peroxidase in the suppression of the malignant phenotype. Hum. Gene Ther. 2004, 15, 239–250. [Google Scholar] [CrossRef]
  48. Monti, D.A.; Mitchell, E.; Bazzan, A.J.; Littman, S.; Zabrecky, G.; Yeo, C.J.; Pillai, M.V.; Newberg, A.B.; Deshmukh, S.; Levine, M. Phase I evaluation of intravenous ascorbic acid in combination with gemcitabine and erlotinib in patients with metastatic pancreatic cancer. PLoS ONE 2012, 7, e29794. [Google Scholar] [CrossRef]
  49. Welsh, J.L.; Wagner, B.A.; van’t Erve, T.J.; Zehr, P.S.; Berg, D.J.; Halfdanarson, T.R.; Yee, N.S.; Bodeker, K.L.; Du, J.; Roberts, L.J., 2nd; et al. Pharmacological ascorbate with gemcitabine for the control of metastatic and node-positive pancreatic cancer (PACMAN): Results from a phase I clinical trial. Cancer Chemother. Pharmacol. 2013, 71, 765–775. [Google Scholar] [CrossRef] [Green Version]
  50. Di Marco, M.; Di Cicilia, R.; Macchini, M.; Nobili, E.; Vecchiarelli, S.; Brandi, G.; Biasco, G. Metastatic pancreatic cancer: Is gemcitabine still the best standard treatment? (Review). Oncol. Rep. 2010, 23, 1183–1192. [Google Scholar] [CrossRef] [Green Version]
  51. Polireddy, K.; Dong, R.; Reed, G.; Yu, J.; Chen, P.; Williamson, S.; Violet, P.C.; Pessetto, Z.; Godwin, A.K.; Fan, F.; et al. High Dose Parenteral Ascorbate Inhibited Pancreatic Cancer Growth and Metastasis: Mechanisms and a Phase I/IIa study. Sci. Rep. 2017, 7, 17188. [Google Scholar] [CrossRef] [Green Version]
  52. Du, J.; Cieslak, J.A., 3rd; Welsh, J.L.; Sibenaller, Z.A.; Allen, B.G.; Wagner, B.A.; Kalen, A.L.; Doskey, C.M.; Strother, R.K.; Button, A.M.; et al. Pharmacological Ascorbate Radiosensitizes Pancreatic Cancer. Cancer Res. 2015, 75, 3314–3326. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  53. Alexander, M.S.; Wilkes, J.G.; Schroeder, S.R.; Buettner, G.R.; Wagner, B.A.; Du, J.; Gibson-Corley, K.; O’Leary, B.R.; Spitz, D.R.; Buatti, J.M.; et al. Pharmacologic Ascorbate Reduces Radiation-Induced Normal Tissue Toxicity and Enhances Tumor Radiosensitization in Pancreatic Cancer. Cancer Res. 2018, 78, 6838–6851. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  54. Chen, Q.; Espey, M.G.; Sun, A.Y.; Lee, J.H.; Krishna, M.C.; Shacter, E.; Choyke, P.L.; Pooput, C.; Kirk, K.L.; Buettner, G.R.; et al. Ascorbate in pharmacologic concentrations selectively generates ascorbate radical and hydrogen peroxide in extracellular fluid in vivo. Proc. Natl. Acad. Sci. USA 2007, 104, 8749–8754. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  55. Alexander, M.S.; O’Leary, B.R.; Wilkes, J.G.; Gibson, A.R.; Wagner, B.A.; Du, J.; Sarsour, E.; Hwang, R.F.; Buettner, G.R.; Cullen, J.J. Enhanced Pharmacological Ascorbate Oxidation Radiosensitizes Pancreatic Cancer. Radiat. Res. 2019, 191, 43–51. [Google Scholar] [CrossRef]
  56. Perrakis, N.; Athanassiou, E.; Vamvakopoulou, D.; Kyriazi, M.; Kappos, H.; Vamvakopoulos, N.C.; Nomikos, I. Practical approaches to effective management of intestinal radiation injury: Benefit of resectional surgery. World J. Gastroenterol. 2011, 17, 4013–4016. [Google Scholar] [CrossRef]
  57. Ngo, B.; Van Riper, J.M.; Cantley, L.C.; Yun, J. Targeting cancer vulnerabilities with high-dose vitamin C. Nat. Rev. Cancer 2019, 19, 271–282. [Google Scholar] [CrossRef]
  58. Wang, H.; Liu, J.; Xia, G.; Lei, S.; Huang, X.; Huang, X. Survival of pancreatic cancer patients is negatively correlated with age at diagnosis: A population-based retrospective study. Sci. Rep. 2020, 10, 7048. [Google Scholar] [CrossRef]
  59. Petrelli, F.; Coinu, A.; Borgonovo, K.; Cabiddu, M.; Barni, S. Progression-free survival as surrogate endpoint in advanced pancreatic cancer: Meta-analysis of 30 randomized first-line trials. Hepatobiliary Pancreat. Dis. Int. 2015, 14, 124–131. [Google Scholar] [CrossRef]
  60. Du, J.; Carroll, R.S.; Steers, G.J.; Wagner, B.A.; O’Leary, B.R.; Jensen, C.S.; Buettner, G.R.; Cullen, J.J. Catalase Modulates the Radio-Sensitization of Pancreatic Cancer Cells by Pharmacological Ascorbate. Antioxidants 2021, 10, 614. [Google Scholar] [CrossRef]
  61. Du, J.; Cullen, J.J.; Buettner, G.R. Ascorbic acid: Chemistry, biology and the treatment of cancer. Biochim. Biophys. Acta 2012, 1826, 443–457. [Google Scholar] [CrossRef] [Green Version]
  62. Oberley, T.D.; Oberley, L.W. Antioxidant enzyme levels in cancer. Histol. Histopathol. 1997, 12, 525–535. [Google Scholar] [PubMed]
  63. Sasaki, K.; Bannai, S.; Makino, N. Kinetics of hydrogen peroxide elimination by human umbilical vein endothelial cells in culture. Biochim. Biophys. Acta 1998, 1380, 275–288. [Google Scholar] [CrossRef]
  64. Mueller, S.; Riedel, H.D.; Stremmel, W. Direct evidence for catalase as the predominant H2O2-removing enzyme in human erythrocytes. Blood 1997, 90, 4973–4978. [Google Scholar] [CrossRef] [PubMed]
  65. Flohe, L. The glutathione peroxidase reaction: Molecular basis of the antioxidant function of selenium in mammals. Curr. Top Cell Regul. 1985, 27, 473–478. [Google Scholar] [CrossRef]
  66. Schoenfeld, J.D.; Alexander, M.S.; Waldron, T.J.; Sibenaller, Z.A.; Spitz, D.R.; Buettner, G.R.; Allen, B.G.; Cullen, J.J. Pharmacological Ascorbate as a Means of Sensitizing Cancer Cells to Radio-Chemotherapy While Protecting Normal Tissue. Semin. Radiat. Oncol. 2019, 29, 25–32. [Google Scholar] [CrossRef]
  67. Larsson, A. Enzymatic Synthesis of Deoxyribonucleotides. VII. Studies on the Hydrogen Transfer with Tritiated Water. Biochemistry 1965, 4, 1984–1993. [Google Scholar] [CrossRef]
  68. Moore, E.C. A thioredoxin—thioredoxin reductase system from rat tumor. Biochem. Biophys. Res. Commun. 1967, 29, 264–268. [Google Scholar] [CrossRef]
  69. Lu, J.; Holmgren, A. The thioredoxin antioxidant system. Free Radic. Biol. Med. 2014, 66, 75–87. [Google Scholar] [CrossRef]
  70. Deisseroth, A.; Dounce, A.L. Catalase: Physical and chemical properties, mechanism of catalysis, and physiological role. Physiol. Rev. 1970, 50, 319–375. [Google Scholar] [CrossRef]
  71. Ogusucu, R.; Rettori, D.; Munhoz, D.C.; Netto, L.E.; Augusto, O. Reactions of yeast thioredoxin peroxidases I and II with hydrogen peroxide and peroxynitrite: Rate constants by competitive kinetics. Free Radic. Biol. Med. 2007, 42, 326–334. [Google Scholar] [CrossRef]
  72. Holmgren, A.; Lu, J. Thioredoxin and thioredoxin reductase: Current research with special reference to human disease. Biochem. Biophys. Res. Commun. 2010, 396, 120–124. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  73. Shoeib, T.; Atkinson, D.W.; Sharp, B.L. Structural analysis of the anti-arthritic drug Auranofin: Its complexes with cysteine, selenocysteine and their fragmentation products. Inorg. Chim. Acta 2010, 363, 184–192. [Google Scholar] [CrossRef]
  74. Finkelstein, A.E.; Walz, D.T.; Batista, V.; Mizraji, M.; Roisman, F.; Misher, A. Auranofin. New oral gold compound for treatment of rheumatoid arthritis. Ann. Rheum. Dis. 1976, 35, 251–257. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  75. Champion, G.D.; Graham, G.G.; Ziegler, J.B. The gold complexes. Baillieres Clin. Rheumatol. 1990, 4, 491–534. [Google Scholar] [CrossRef]
  76. Kim, N.H.; Lee, M.Y.; Park, S.J.; Choi, J.S.; Oh, M.K.; Kim, I.S. Auranofin blocks interleukin-6 signalling by inhibiting phosphorylation of JAK1 and STAT3. Immunology 2007, 122, 607–614. [Google Scholar] [CrossRef]
  77. Stern, I.; Wataha, J.C.; Lewis, J.B.; Messer, R.L.; Lockwood, P.E.; Tseng, W.Y. Anti-rheumatic gold compounds as sublethal modulators of monocytic LPS-induced cytokine secretion. Toxicol. Vitr. 2005, 19, 365–371. [Google Scholar] [CrossRef]
  78. Kim, N.H.; Oh, M.K.; Park, H.J.; Kim, I.S. Auranofin, a gold(I)-containing antirheumatic compound, activates Keap1/Nrf2 signaling via Rac1/iNOS signal and mitogen-activated protein kinase activation. J. Pharmacol. Sci. 2010, 113, 246–254. [Google Scholar] [CrossRef] [Green Version]
  79. Han, S.; Kim, K.; Kim, H.; Kwon, J.; Lee, Y.H.; Lee, C.K.; Song, Y.; Lee, S.J.; Ha, N.; Kim, K. Auranofin inhibits overproduction of pro-inflammatory cytokines, cyclooxygenase expression and PGE2 production in macrophages. Arch. Pharm. Res. 2008, 31, 67–74. [Google Scholar] [CrossRef]
  80. Han, S.; Kim, K.; Song, Y.; Kim, H.; Kwon, J.; Lee, Y.H.; Lee, C.K.; Lee, S.J.; Ha, N.; Kim, K. Auranofin, an immunosuppressive drug, inhibits MHC class I and MHC class II pathways of antigen presentation in dendritic cells. Arch. Pharm. Res. 2008, 31, 370–376. [Google Scholar] [CrossRef]
  81. Park, S.J.; Kim, I.S. The role of p38 MAPK activation in auranofin-induced apoptosis of human promyelocytic leukaemia HL-60 cells. Br. J. Pharmacol. 2005, 146, 506–513. [Google Scholar] [CrossRef] [Green Version]
  82. Honda, Z.; Iizasa, T.; Morita, Y.; Matsuta, K.; Nishida, Y.; Miyamoto, T. Differential inhibitory effects of auranofin on leukotriene B4 and leukotriene C4 formation by human polymorphonuclear leukocytes. Biochem. Pharmacol. 1987, 36, 1475–1481. [Google Scholar] [CrossRef]
  83. Yamada, M.; Niki, H.; Yamashita, M.; Mue, S.; Ohuchi, K. Prostaglandin E2 production dependent upon cyclooxygenase-1 and cyclooxygenase-2 and its contradictory modulation by auranofin in rat peritoneal macrophages. J. Pharmacol. Exp. Ther. 1997, 281, 1005–1012. [Google Scholar] [PubMed]
  84. Yamashita, M.; Niki, H.; Yamada, M.; Watanabe-Kobayashi, M.; Mue, S.; Ohuchi, K. Dual effects of auranofin on prostaglandin E2 production by rat peritoneal macrophages. Eur. J. Pharmacol. 1997, 325, 221–227. [Google Scholar] [CrossRef]
  85. Walz, D.T.; DiMartino, M.J.; Griswold, D.E.; Intoccia, A.P.; Flanagan, T.L. Biologic actions and pharmacokinetic studies of auranofin. Am. J. Med. 1983, 75, 90–108. [Google Scholar] [CrossRef]
  86. Sannella, A.R.; Casini, A.; Gabbiani, C.; Messori, L.; Bilia, A.R.; Vincieri, F.F.; Majori, G.; Severini, C. New uses for old drugs. Auranofin, a clinically established antiarthritic metallodrug, exhibits potent antimalarial effects in vitro: Mechanistic and pharmacological implications. FEBS Lett. 2008, 582, 844–847. [Google Scholar] [CrossRef] [PubMed]
  87. Newman, Z.L.; Sirianni, N.; Mawhinney, C.; Lee, M.S.; Leppla, S.H.; Moayeri, M.; Johansen, L.M. Auranofin protects against anthrax lethal toxin-induced activation of the Nlrp1b inflammasome. Antimicrob. Agents Chemother. 2011, 55, 1028–1035. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  88. Hutton, M.L.; Pehlivanoglu, H.; Vidor, C.J.; James, M.L.; Thomson, M.J.; Lyras, D. Repurposing auranofin as a Clostridioides difficile therapeutic. J. Antimicrob. Chemother. 2020, 75, 409–417. [Google Scholar] [CrossRef]
  89. Sharma, N.; Singh, A.; Sharma, R.; Kumar, A. Repurposing of Auranofin Against Bacterial Infections: An In silico and In vitro Study. Curr. Comput. Aided Drug Des. 2021, 17, 687–701. [Google Scholar] [CrossRef]
  90. Lewis, M.G.; DaFonseca, S.; Chomont, N.; Palamara, A.T.; Tardugno, M.; Mai, A.; Collins, M.; Wagner, W.L.; Yalley-Ogunro, J.; Greenhouse, J.; et al. Gold drug auranofin restricts the viral reservoir in the monkey AIDS model and induces containment of viral load following ART suspension. AIDS 2011, 25, 1347–1356. [Google Scholar] [CrossRef]
  91. Ashino, T.; Sugiuchi, J.; Uehara, J.; Naito-Yamamoto, Y.; Kenmotsu, S.; Iwakura, Y.; Shioda, S.; Numazawa, S.; Yoshida, T. Auranofin protects against cocaine-induced hepatic injury through induction of heme oxygenase-1. J. Toxicol. Sci. 2011, 36, 635–643. [Google Scholar] [CrossRef]
  92. Gromer, S.; Arscott, L.D.; Williams, C.H., Jr.; Schirmer, R.H.; Becker, K. Human placenta thioredoxin reductase. Isolation of the selenoenzyme, steady state kinetics, and inhibition by therapeutic gold compounds. J. Biol. Chem. 1998, 273, 20096–20101. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  93. Saei, A.A.; Gullberg, H.; Sabatier, P.; Beusch, C.M.; Johansson, K.; Lundgren, B.; Arvidsson, P.I.; Arner, E.S.J.; Zubarev, R.A. Comprehensive chemical proteomics for target deconvolution of the redox active drug auranofin. Redox Biol. 2020, 32, 101491. [Google Scholar] [CrossRef] [PubMed]
  94. Cox, A.G.; Brown, K.K.; Arner, E.S.; Hampton, M.B. The thioredoxin reductase inhibitor auranofin triggers apoptosis through a Bax/Bak-dependent process that involves peroxiredoxin 3 oxidation. Biochem. Pharmacol. 2008, 76, 1097–1109. [Google Scholar] [CrossRef] [PubMed]
  95. Olney, K.E.; Du, J.; van’t Erve, T.J.; Witmer, J.R.; Sibenaller, Z.A.; Wagner, B.A.; Buettner, G.R.; Cullen, J.J. Inhibitors of hydroperoxide metabolism enhance ascorbate-induced cytotoxicity. Free Radic. Res. 2013, 47, 154–163. [Google Scholar] [CrossRef]
  96. Joo, M.K.; Shin, S.; Ye, D.J.; An, H.G.; Kwon, T.U.; Baek, H.S.; Kwon, Y.J.; Chun, Y.J. Combined treatment with auranofin and trametinib induces synergistic apoptosis in breast cancer cells. J. Toxicol. Environ. Health A 2021, 84, 84–94. [Google Scholar] [CrossRef]
  97. Lee, J.E.; Kwon, Y.J.; Baek, H.S.; Ye, D.J.; Cho, E.; Choi, H.K.; Oh, K.S.; Chun, Y.J. Synergistic induction of apoptosis by combination treatment with mesupron and auranofin in human breast cancer cells. Arch. Pharm. Res. 2017, 40, 746–759. [Google Scholar] [CrossRef]
  98. Ye, D.J.; Kwon, Y.J.; Baek, H.S.; Cho, E.; Kwon, T.U.; Chun, Y.J. Combination treatment with auranofin and nutlin-3a induces synergistic cytotoxicity in breast cancer cells. J. Toxicol. Environ. Health A 2019, 82, 626–637. [Google Scholar] [CrossRef]
  99. Freire Boullosa, L.; Van Loenhout, J.; Flieswasser, T.; De Waele, J.; Hermans, C.; Lambrechts, H.; Cuypers, B.; Laukens, K.; Bartholomeus, E.; Siozopoulou, V.; et al. Auranofin reveals therapeutic anticancer potential by triggering distinct molecular cell death mechanisms and innate immunity in mutant p53 non-small cell lung cancer. Redox Biol. 2021, 42, 101949. [Google Scholar] [CrossRef]
  100. Lin, Z.; Li, Q.; Zhao, Y.; Lin, Z.; Cheng, N.; Zhang, D.; Liu, G.; Lin, J.; Zhang, H.; Lin, D. Combination of Auranofin and ICG-001 Suppress the Proliferation and Metastasis of Colon Cancer. Front. Oncol. 2021, 11, 738085. [Google Scholar] [CrossRef]
  101. Park, S.H.; Lee, J.H.; Berek, J.S.; Hu, M.C. Auranofin displays anticancer activity against ovarian cancer cells through FOXO3 activation independent of p53. Int. J. Oncol. 2014, 45, 1691–1698. [Google Scholar] [CrossRef] [Green Version]
  102. O’Leary, B.; Du, J.; Heer, C.; Van Beek, H.; Sptiz, D.R.; Cullen, J.J. Inhibition of Peroxide Removal Enhances Pharmacological Ascorbate and Ionizing Radiation-Induced Cytotoxicity of Pancreatic Ductal Adenocarcinoma. Free Radical. Bio. Med. 2017, 112, 97. [Google Scholar] [CrossRef]
  103. Van Beek, H. Inhibition of Peroxide Removal Systems and Ascorbate-Induced Cytotoxicity in Pancreatic Cancer. Master’s Thesis, University of Iowa, Iowa City, IA, USA, 2016. [Google Scholar] [CrossRef] [Green Version]
  104. Day, A.M.; Brown, J.D.; Taylor, S.R.; Rand, J.D.; Morgan, B.A.; Veal, E.A. Inactivation of a peroxiredoxin by hydrogen peroxide is critical for thioredoxin-mediated repair of oxidized proteins and cell survival. Mol. Cell 2012, 45, 398–408. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  105. Nagy, P.; Karton, A.; Betz, A.; Peskin, A.V.; Pace, P.; O’Reilly, R.J.; Hampton, M.B.; Radom, L.; Winterbourn, C.C. Model for the exceptional reactivity of peroxiredoxins 2 and 3 with hydrogen peroxide: A kinetic and computational study. J. Biol. Chem. 2011, 286, 18048–18055. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  106. Stocker, S.; Maurer, M.; Ruppert, T.; Dick, T.P. A role for 2-Cys peroxiredoxins in facilitating cytosolic protein thiol oxidation. Nat. Chem. Biol. 2018, 14, 148–155. [Google Scholar] [CrossRef]
  107. Stocker, S.; Van Laer, K.; Mijuskovic, A.; Dick, T.P. The Conundrum of Hydrogen Peroxide Signaling and the Emerging Role of Peroxiredoxins as Redox Relay Hubs. Antioxid. Redox Signal. 2018, 28, 558–573. [Google Scholar] [CrossRef]
  108. Chang, T.S.; Cho, C.S.; Park, S.; Yu, S.; Kang, S.W.; Rhee, S.G. Peroxiredoxin III, a mitochondrion-specific peroxidase, regulates apoptotic signaling by mitochondria. J. Biol. Chem. 2004, 279, 41975–41984. [Google Scholar] [CrossRef] [Green Version]
  109. Kang, S.W.; Chang, T.S.; Lee, T.H.; Kim, E.S.; Yu, D.Y.; Rhee, S.G. Cytosolic peroxiredoxin attenuates the activation of Jnk and p38 but potentiates that of Erk in Hela cells stimulated with tumor necrosis factor-alpha. J. Biol. Chem. 2004, 279, 2535–2543. [Google Scholar] [CrossRef] [Green Version]
  110. Kim, H.; Lee, T.H.; Park, E.S.; Suh, J.M.; Park, S.J.; Chung, H.K.; Kwon, O.Y.; Kim, Y.K.; Ro, H.K.; Shong, M. Role of peroxiredoxins in regulating intracellular hydrogen peroxide and hydrogen peroxide-induced apoptosis in thyroid cells. J. Biol. Chem. 2000, 275, 18266–18270. [Google Scholar] [CrossRef] [Green Version]
  111. Collins, J.A.; Wood, S.T.; Nelson, K.J.; Rowe, M.A.; Carlson, C.S.; Chubinskaya, S.; Poole, L.B.; Furdui, C.M.; Loeser, R.F. Oxidative Stress Promotes Peroxiredoxin Hyperoxidation and Attenuates Pro-survival Signaling in Aging Chondrocytes. J. Biol. Chem. 2016, 291, 6641–6654. [Google Scholar] [CrossRef] [Green Version]
  112. Kim, S.Y.; Kim, T.J.; Lee, K.Y. A novel function of peroxiredoxin 1 (Prx-1) in apoptosis signal-regulating kinase 1 (ASK1)-mediated signaling pathway. FEBS Lett. 2008, 582, 1913–1918. [Google Scholar] [CrossRef] [Green Version]
  113. Zhang, M.; Niu, W.; Zhang, J.; Ge, L.; Yang, J.; Sun, Z.; Tang, X. Peroxiredoxin 1 suppresses apoptosis via regulation of the apoptosis signal-regulating kinase 1 signaling pathway in human oral leukoplakia. Oncol. Lett. 2015, 10, 1841–1847. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  114. Hu, J.; Zhang, H.; Cao, M.; Wang, L.; Wu, S.; Fang, B. Auranofin Enhances Ibrutinib’s Anticancer Activity in EGFR-Mutant Lung Adenocarcinoma. Mol. Cancer Ther. 2018, 17, 2156–2163. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  115. Sobotta, M.C.; Liou, W.; Stocker, S.; Talwar, D.; Oehler, M.; Ruppert, T.; Scharf, A.N.; Dick, T.P. Peroxiredoxin-2 and STAT3 form a redox relay for H2O2 signaling. Nat. Chem. Biol. 2015, 11, 64–70. [Google Scholar] [CrossRef] [PubMed]
  116. Hatem, E.; Azzi, S.; El Banna, N.; He, T.; Heneman-Masurel, A.; Vernis, L.; Baille, D.; Masson, V.; Dingli, F.; Loew, D.; et al. Auranofin/Vitamin C: A Novel Drug Combination Targeting Triple-Negative Breast Cancer. J. Natl. Cancer Inst. 2019, 111, 597–608. [Google Scholar] [CrossRef]
  117. Graczyk-Jarzynka, A.; Goral, A.; Muchowicz, A.; Zagozdzon, R.; Winiarska, M.; Bajor, M.; Trzeciecka, A.; Fidyt, K.; Krupka, J.A.; Cyran, J.; et al. Inhibition of thioredoxin-dependent H2O2 removal sensitizes malignant B-cells to pharmacological ascorbate. Redox Biol. 2019, 21, 101062. [Google Scholar] [CrossRef]
Antioxidants 11 00971 g001 550
Figure 1. Survival analysis from phase I trial (NCT01852890). (A) Kaplan–Meier curve estimating median overall survival in subjects treated with P-AscH– plus gemcitabine and radiation therapy as of 25 January 2022 (n = 14) was 22.8 months vs. 12.7 months in institutional controls treated with gemcitabine and radiation therapy (n = 19, Log-Rank test p = 0.02); (B) Kaplan–Meier curve demonstrating median progression-free survival in subjects treated with P-AscH– plus gemcitabine and radiation therapy as of 25 January 2022 (n = 14) was 13.7 months vs. 4.6 months in institutional controls treated with gemcitabine and radiation therapy (n = 19, Log-Rank test p = 0.01). These data are updated from data previously published by Du et al. [60].
Figure 1. Survival analysis from phase I trial (NCT01852890). (A) Kaplan–Meier curve estimating median overall survival in subjects treated with P-AscH– plus gemcitabine and radiation therapy as of 25 January 2022 (n = 14) was 22.8 months vs. 12.7 months in institutional controls treated with gemcitabine and radiation therapy (n = 19, Log-Rank test p = 0.02); (B) Kaplan–Meier curve demonstrating median progression-free survival in subjects treated with P-AscH– plus gemcitabine and radiation therapy as of 25 January 2022 (n = 14) was 13.7 months vs. 4.6 months in institutional controls treated with gemcitabine and radiation therapy (n = 19, Log-Rank test p = 0.01). These data are updated from data previously published by Du et al. [60].
Antioxidants 11 00971 g001
Antioxidants 11 00971 g002 550
Figure 2. Thioredoxin and glutathione peroxidase (GPx) antioxidant enzyme systems and inhibitors. GSH = glutathione; GSSG = glutathione disulfide; GR = glutathione disulfide reductase; GPx = glutathione peroxidase; Trx-(SH)2 = reduced thioredoxin; Trx-S2 = oxidized thioredoxin; TrxR = thioredoxin reductase; Prx = peroxiredoxin; G-6-P = glucose-6-phosphate; 6-P-G = 6-phosphoglucono-δ-lactone. Inhibitors of the pathway are: Auranofin [103].
Figure 2. Thioredoxin and glutathione peroxidase (GPx) antioxidant enzyme systems and inhibitors. GSH = glutathione; GSSG = glutathione disulfide; GR = glutathione disulfide reductase; GPx = glutathione peroxidase; Trx-(SH)2 = reduced thioredoxin; Trx-S2 = oxidized thioredoxin; TrxR = thioredoxin reductase; Prx = peroxiredoxin; G-6-P = glucose-6-phosphate; 6-P-G = 6-phosphoglucono-δ-lactone. Inhibitors of the pathway are: Auranofin [103].
Antioxidants 11 00971 g002
Antioxidants 11 00971 g003 550
Figure 3. Au inhibits thioredoxin reductase activity in pancreatic cancer cells. MIA PaCa-2 cells were incubated with 1 µM Au for 3 h prior to measuring thioredoxin reductase activity. Au significantly reduced thioredoxin reductase activity compared to control. Data represent thioredoxin reductase activity in mU/mg protein ± SE (* p < 0.05; two-tailed unpaired Student’s t test). Reprinted/adapted with permission from [102,103]. Copyright 2017 Elsevier.
Figure 3. Au inhibits thioredoxin reductase activity in pancreatic cancer cells. MIA PaCa-2 cells were incubated with 1 µM Au for 3 h prior to measuring thioredoxin reductase activity. Au significantly reduced thioredoxin reductase activity compared to control. Data represent thioredoxin reductase activity in mU/mg protein ± SE (* p < 0.05; two-tailed unpaired Student’s t test). Reprinted/adapted with permission from [102,103]. Copyright 2017 Elsevier.
Antioxidants 11 00971 g003
Antioxidants 11 00971 g004 550
Figure 4. Au sensitizes pancreatic cancer cells to P-AscH−. Clonogenic cell survival of MIA PaCa-2 cells incubated with Au (1 µM for 3 h) followed by 2 mM P-AscH− (1 h) was significantly decreased compared to control and to either treatment alone. Treatment with either Au or P-AscH− also decreased clonogenic survival compared to control. Data represent normalized surviving fractions compared to control ± SE (* p < 0.01; one-way ANOVA with Tukey’s multiple comparisons). Reprinted/adapted with permission from [102,103]. Copyright 2017 Elsevier.
Figure 4. Au sensitizes pancreatic cancer cells to P-AscH−. Clonogenic cell survival of MIA PaCa-2 cells incubated with Au (1 µM for 3 h) followed by 2 mM P-AscH− (1 h) was significantly decreased compared to control and to either treatment alone. Treatment with either Au or P-AscH− also decreased clonogenic survival compared to control. Data represent normalized surviving fractions compared to control ± SE (* p < 0.01; one-way ANOVA with Tukey’s multiple comparisons). Reprinted/adapted with permission from [102,103]. Copyright 2017 Elsevier.
Antioxidants 11 00971 g004
Antioxidants 11 00971 g005 550
Figure 5. Catalase reverses killing induced by the combination of Au and P-AscH−. MIA PaCa-2 cells were evaluated for clonogenic cell survival after treatment with either catalase (Cat, 100 U/mL, 1 h), Au (500 nM, 24 h), and/or P-AscH− (1 mM, 1 h). The combination of P-AscH− and Au significantly decreased clonogenic cell survival compared to P-AscH−, and the addition of catalase completely reversed the decrease. Data represent normalized surviving fractions compared to controls ± SE (* p < 0.05; one-way ANOVA with Tukey’s multiple comparisons). Reprinted/adapted with permission from [102,103]. Copyright 2017 Elsevier.
Figure 5. Catalase reverses killing induced by the combination of Au and P-AscH−. MIA PaCa-2 cells were evaluated for clonogenic cell survival after treatment with either catalase (Cat, 100 U/mL, 1 h), Au (500 nM, 24 h), and/or P-AscH− (1 mM, 1 h). The combination of P-AscH− and Au significantly decreased clonogenic cell survival compared to P-AscH−, and the addition of catalase completely reversed the decrease. Data represent normalized surviving fractions compared to controls ± SE (* p < 0.05; one-way ANOVA with Tukey’s multiple comparisons). Reprinted/adapted with permission from [102,103]. Copyright 2017 Elsevier.
Antioxidants 11 00971 g005
Antioxidants 11 00971 g006 550
Figure 6. Au + P-AscH− radiosensitizes pancreatic cancer cell lines. (A) MIA PaCa-2 and (B) AsPC-1 cells were evaluated for clonogenic survival after treatment with either irradiation alone (1–2 Gy) or in combination with Au (500 nM, 24 h), P-AscH− (1–2 mM, 1 h), and irradiation (1–2 Gy). The combination of Auranofin and P-AscH− significantly reduced clonogenic cell survival of MIA PaCa-2 and AsPC-1 cells at both 1 and 2 Gy. Data represent normalized surviving fractions compared to controls ± SE (* p < 0.05; two-tailed unpaired Student’s t test). Reprinted/adapted with permission from [102,103]. Copyright 2017 Elsevier.
Figure 6. Au + P-AscH− radiosensitizes pancreatic cancer cell lines. (A) MIA PaCa-2 and (B) AsPC-1 cells were evaluated for clonogenic survival after treatment with either irradiation alone (1–2 Gy) or in combination with Au (500 nM, 24 h), P-AscH− (1–2 mM, 1 h), and irradiation (1–2 Gy). The combination of Auranofin and P-AscH− significantly reduced clonogenic cell survival of MIA PaCa-2 and AsPC-1 cells at both 1 and 2 Gy. Data represent normalized surviving fractions compared to controls ± SE (* p < 0.05; two-tailed unpaired Student’s t test). Reprinted/adapted with permission from [102,103]. Copyright 2017 Elsevier.
Antioxidants 11 00971 g006
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Steers, G.J.; Chen, G.Y.; O’Leary, B.R.; Du, J.; Van Beek, H.; Cullen, J.J. Auranofin and Pharmacologic Ascorbate as Radiomodulators in the Treatment of Pancreatic Cancer. Antioxidants 2022, 11, 971. https://doi.org/10.3390/antiox11050971

AMA Style

Steers GJ, Chen GY, O’Leary BR, Du J, Van Beek H, Cullen JJ. Auranofin and Pharmacologic Ascorbate as Radiomodulators in the Treatment of Pancreatic Cancer. Antioxidants. 2022; 11(5):971. https://doi.org/10.3390/antiox11050971

Chicago/Turabian Style

Steers, Garett J., Gloria Y. Chen, Brianne R. O’Leary, Juan Du, Hannah Van Beek, and Joseph J. Cullen. 2022. "Auranofin and Pharmacologic Ascorbate as Radiomodulators in the Treatment of Pancreatic Cancer" Antioxidants 11, no. 5: 971. https://doi.org/10.3390/antiox11050971

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop