Our strategy herein was to apply standard pharmacologic principles to evaluate the chemotherapy pairing of gemcitabine and ascorbate. The findings showed that combining clinically achievable concentrations of pharmacologic ascorbate with gemcitabine increased chemosensitivity across the spectrum of malignant phenotypes represented in our panel of pancreatic cancer cells (–, and ). Using a combination ratio design [24
], this study provides evidence that pharmacologic ascorbate synergized with gemcitabine to improve therapeutic efficacy. These results were consistent with growth inhibition responses of pancreatic carcinoma xenografts in mice that received combined gemcitabine and pharmacologic ascorbate treatment ( and ).
Pharmacologic ascorbate demonstrated several novel and useful qualities that support its potential as an adjuvant to regimens for pancreatic carcinoma treatment. The combination of gemcitabine and pharmacologic ascorbate may be similar mechanistically to the pairing of gemcitabine and platin-alkylating agents such as oxaliplatin, which was shown to improve efficacy in patients with advanced pancreatic cancer [29
]. In addition to forming DNA adducts, oxaliplatin is thiophilic and can generate oxidative stress [30
], analogous to H2
produced by catalysis of pharmacologic ascorbate [12
]. An important distinction is that pharmacologic ascorbate does not suffer from dose-limiting toxicity inherent to alkylating agents and other traditional chemotherapeutics. A key finding was that DRIgem
was consistently and substantially elevated as the ratio of ascorbate to gemcitabine was increased, in some cases by several orders of magnitude (, ). The converse of raising the ascorbate:gemcitabine ratio is to lower the concentration of gemcitabine to produce an equitoxic effect when combined with pharmacologic concentrations of ascorbate. These in vitro findings were corroborated by data showing a gemcitabine dose-sparing effect in mice bearing PAN-02 carcinomas, in which tumor weight in the gemcitabine (30 mg/kg) + ascorbate treatment group was comparable to that from treatment with gemcitabine alone at twice the dose (60 mg/kg, ). The gemcitabine dose-lowering influence of pharmacologic ascorbate may lessen side effects and thereby serve to prolong gemcitabine treatment cycles and improve its efficacy.
Across the tested cell lines, Hs766T exhibited the strongest resistance to ascorbate alone, with an IC50
of 3.6 mM (). Normal pancreatic ductal epithelial cells in vitro showed no significant sensitivity to 10 mM ascorbate [16
]. Moreover, toxicity was not associated with treatment of mice with pharmacologic ascorbate (4 g/kg ip daily), consistent with the lack of toxicity toward normal human tissues observed with complementary use of high-dose intravenous ascorbate [10
]. The basis for disparate sensitivity among cancer cells, and collectively between cancer cells and normal cells, may be related to unique differences in the metabolism of glucose, iron, and reactive oxygen species [37
Transformation of dose–response data obtained in a constant ratio design into median–effect plots affords the opportunity to examine potential similarities in mechanisms of action between drugs [24
]. The pattern that emerged in this analysis (m
; ) suggested that the cooperativity of gemcitabine and ascorbate may be similar mechanistically to ascorbate alone. In our cell culture experiments, the oxidative cytotoxicity elicited by pharmacologic ascorbate was highest within 24 h of exposure, whereas gemcitabine took several days (48 to 72 h) to optimally manifest cell death. Although the direct congeners of ascorbate and gemcitabine were temporally disjoined, their apparent synergism demonstrated herein predicts that downstream consequences persisted far beyond the lifetime of the pharmacologic ascorbate and its transient effectors. It is tempting to link H2
produced by pharmacologic ascorbate catalysis [12
] with genomic instability induced by gemcitabine. The phosphorylated metabolites of gemcitabine act as competitive inhibitors of the ribonucleotide reductase complex, which leads to a decrease in intracellular deoxyribonucleotide pools necessary for DNA replication and cell proliferation [4
]. In addition to DNA synthesis, increased supplies of deoxyribonucleotides are essential for repair of DNA damage after oxidative lesioning caused by agents such as H2
]. Mutual exclusivity could not be established in this study () and it was likely that a multiplicity of actions accounted for the cytotoxic behaviors of gemcitabine and ascorbate, either alone or in combination, which may be unique to each cell type.
Drugs with different mechanisms of action are desirable for use in combination chemotherapy. In contrast to the vogue for focus on a specific molecular target, the promiscuity of H2
produced by pharmacologic ascorbate catalysis has the potential to render cytotoxic effects across a wide assortment of cellular targets (DNA, protein, lipid, etc.). This may be a particularly important advantage in cancers of the pancreas, which encompass a range of malignant pancreatic neoplasms with diverse precursor origins and differentiation patterns [36
]. Resistance to gemcitabine may develop at a variety of levels (nucleoside transport, phosphorylation, catabolic enzymes) [4
] that are unrelated to H2
/oxidative defense, minimizing the development of cross-resistance. The broad applicability of the gemcitabine and pharmacologic ascorbate combination was illustrated by the finding that sensitization occurred with gemcitabine and ascorbate combination regardless of the epithelial–mesenchymal transition phenotype of the cells in our panel (). Patients with malignant cells that have progressed to the mesenchymal-like molecular phenotype typically face an aggressive and metastatic disease that is more resistant to standard therapy [27
]. Therefore, it may be of translational importance that pharmacologic ascorbate facilitates chemosensitization responses in mesenchymal-like cell types equivalent to less invasive epithelial cell types. Our in vitro data were corroborated in mice bearing the highly mesenchymal-like PANC-1 xenografts that did not respond to gemcitabine monotherapy, whereas ~50% growth inhibition was observed in the corresponding gemcitabine + ascorbate treatment groups (P
< 0.001; ).
Obloquy for ascorbate lingers among oncologists who cite the failed double-blind placebo-controlled trials at the Mayo Clinic in which oral administration of 10 g ascorbate daily had no effect on cancer progression [55
]. It was not recognized at the time that intestinal absorption of ascorbate after oral ingestion becomes saturated at >0.2 g [9
]. Only parental administration of ascorbate can provide pharmacologic concentrations and anticancer activity. Data in this study support the rationale for testing the utility of parentally administered pharmacologic ascorbate in adjuvant regimens, particularly in cancers with a relatively fulminant course. The emerging ability to decipher links between genetics, diet, and pancreatic carcinogenesis offers a promising new framework for understanding the etiology(ies) of this disease [58
]. However, a striking 96% of individuals currently diagnosed with pancreatic adenocarcinoma have nonresectable, locally advanced, or metastatic disease [7
], for whom few therapeutic options are available to abate a rapid disease progression. Although some have suggested interference of vitamin C with chemotherapy [59
], investigators in that supporting study used the ascorbate metabolite dehydroascorbic acid, at concentrations that are toxic in humans and could not be produced from either physiologic or pharmacologic ascorbate [13
]. This study clearly provides evidence that pharmacologic ascorbate can improve gemcitabine therapeutic efficacy. The optimal schedule for this and other possible drug combinations to translate pharmacologic ascorbate into the clinic requires further examination. The current data support testing of pharmacologic ascorbate in combination with therapeutic modalities including agents such as gemcitabine in double-blind placebo-controlled trials.