Targeting cancer vulnerabilities with high-dose vitamin C

Abstract

Over the past century, the notion that vitamin C can be used to treat cancer has generated much controversy. However, new knowledge regarding the pharmacokinetic properties of vitamin C and recent high-profile preclinical studies have revived interest in the utilization of high-dose vitamin C for cancer treatment. Studies have shown that pharmacological vitamin C targets many of the mechanisms that cancer cells utilize for their survival and growth. In this Opinion article, we discuss how vitamin C can target three vulnerabilities many cancer cells share: redox imbalance, epigenetic reprogramming and oxygen-sensing regulation. Although the mechanisms and predictive biomarkers that we discuss need to be validated in well-controlled clinical trials, these new discoveries regarding the anticancer properties of vitamin C are promising to help identify patient populations that may benefit the most from high-dose vitamin C therapy, developing effective combination strategies and improving the overall design of future vitamin C clinical trials for various types of cancer.

Fig. 1 |. Integrated pro-oxidant mechanism of vitamin C and cancer cell cytotoxicity,

a | Ascorbate can be oxidized in the extracellular space by reactive oxygen species (ROS), producing ascorbate radical, which can be oxidized to dehydroascorbic acid (DHA). DHA can be taken up by cells or irreversibly converted to 2,3-l-diketoglutonate (2,3-DKG), which is degraded into oxalic acid and threonic acid, b | Pharmacological ascorbate can kill cancer cells by increasing oxidative stress via two possible mechanisms that complement each other. First, extracellular H₂O₂ may directly kill cancer cells by generating ^•OH via the Fenton reaction^,,. Increased levels of labile ferric iron, Fe³⁺, in the tumour microenvironment can facilitate the oxidation of ascorbate, resulting in ascorbate radical, DHA and ferrous iron, Fe²⁺. Once Fe²⁺ is formed, Fe²⁺ may be oxidized by oxygen, producing superoxide anions, O₂^•−. Superoxide dismutase (SOD) catalyses the conversion of O₂^•− to H₂O₂ and O₂. Fe³⁺ can enter the cell when bound to transferrin (Tf), which binds to the Tf receptor (TfR) and is processed and oxidized in the endosome to then contribute to the intracellular Fe²⁺ pool. H₂O₂ can enter the cell through diffusion facilitated by aquaporins. H₂O₂ reacts with either extracellular or intracellular labile Fe²⁺ to generate highly reactive hydroxyl radicals (^•OH) that are harmful to cells. These reactions are further perpetuated by the recycling of Fe³⁺ to Fe²⁺ by ascorbate and ascorbate radical, generating fully oxidized vitamin C, DHA. Second, H₂O₂ may contribute to the increased levels of extracellular DHA by creating a more oxidative tumour microenvironment. DHA can then efficiently enter cells through glucose transporter 1 (GLUT1) and consume the intracellular reducing potential of reduced glutathione (GSH) and NADPH, resulting in increased levels of intracellular ROS^,. This leads to poly(ADP-ribose) polymerase (PARP) activation, a DNA repair enzyme, thereby depleting cellular NAD⁺ levels, a cofactor of PARP. NAD⁺ is required by glyceraldehyde 3-phosphate dehydrogenase (GADPH) as a cofactor. Consequent inhibition of GAPDH activity inhibits glycolysis in cancer cells, leading to inhibition of ATP production and cell death^,,. In addition, cellular ROS can also be released from cells, resulting in a positive feedback loop. Because high levels of labile Fe²⁺, GLUT1 overexpression and addiction to glycolysis frequently occur in many types of cancer cells, certain cancer cells may present all three of these characteristics and those populations might be more sensitive to ascorbate treatment. 1,3BPG, 1,3-bisphosphoglyceric acid; G3P, glyceraldehyde 3-phosphate; G6PD glucose-6-phosphate dehydrogenase; GSSG, glutathione disulfide; PPP, pentose phosphate pathway; SVCT, sodium-dependent vitamin C transporters.

Fig. 2 |. Regulation of TET enzymes by ascorbate.

Catalysed by DNA methyltransferases (DNMTs), DNA methylation occurs at the carbon-5 position of cytosine. Intracellular ascorbate influences the DNA methylation landscape by enhancing the enzymatic activity of ten-eleven translocation enzymes (TETs), which actively remove cytosine methylation marks through a series of oxidation reactions dependent on oxygen, α-ketoglutarate (αKG), Fe²⁺ and ascorbate on the basis of its function as an αKG-dependent dioxygenase (αKGDD)^,. TETs first convert 5-methylcytosine (5mC) to 5-hydroxymethyl-cytosine (5hmC). In the next two steps, 5hmC is further oxidized to 5-formylcytosine (5fC) and 5-carboxylcytosine (5CaC). Subsequently, 5fC and 5CaC are converted to cytosine by the base excision repair pathway enzyme, thymine DNA glycosylase. By promoting the recycling of Fe³⁺ to Fe²⁺, ascorbate ensures that TETs are constantly active. DHA, dehydroascorbic acid.

Fig. 3 |. Ascorbate and HIF1α regulation.

Ascorbate is a vital cofactor for the hypoxia-inducible factor (HIF) hydroxylases, proline hydroxylase domain proteins (PHDs) and asparagine hydroxylase (factor-inhibiting HIF (FIH)), which are also members of the α-ketoglutarate (αKG)-dependent dioxygenase (αKGDD) protein family^,. HIF1, a heterodimeric transcription factor, consists of two sub-units: HIF1α, regulated by O₂, and HIF1β. Under normal conditions with sufficient oxygen and ascorbate availability, the functional capacity of HIF1α is inhibited by the HIF hydroxylases. HIF1α is hydroxylated at proline residues by PHD. Prolyl-hydroxylated HIF1α is then bound by the von Hippel-Lindau (VHL) tumour suppressor protein, which recruits an E3-ubiquitin ligase that targets HIF1α for proteasomal degradation and thus limits the quantity of HIF1α units within the cell. HIF1α activity is regulated within the nucleus and can be inhibited. FIH hydroxylates an asparagine residue, N806, on HIF1α This hydroxyl group prevents p300, a co-activator protein, from associating with the HIF complex resulting in the inhibition of the transcription activity of HIF1 and the activation of any downstream pathways. High-dose ascorbate treatment in tumour tissues with normoxic HIF1α stabilization can potentially increase PDH and FIH activity to degrade HIF1 protein and slow down tumour growth. In conditions where ascorbate is depleted, such as in certain cancer types or in tumour tissue in Gulo^−/− mice, the activity of PDH and FIH is reduced even when oxygen is available, which leads to stabilization and activation of HIF1α and its translocation to the nucleus. HIF1α associates with HIF1β, p300 and other cofactors within the nucleus to induce target genes such as GLUT1, which together might promote tumour growth.