1. Molecular Pathways Regulated by Vitamin C

One of the first targets for vitamin C was discovered via its relationship to the NF‒κB pathway. In this pathway, vitamin C has an inhibitory function; in studies carried out in endothelial cells, millimolar doses of AA inhibited NF-κB and IL-8 activation in response to tumor necrosis factor (TNF) [20]. In this study, the authors also evaluated the toxicity generated by high doses of vitamin C supplementation and did not detect cell damage or lipid peroxidation [20,21]. Furthermore, they were able to determine that the inhibition of the NF-κB pathway was not due to the antioxidant activity of vitamin C, but rather to the direct inhibition of IκB kinase α/β (IKKα/β) [20–23]. In line with this notion, IKKα/β is a kinase responsible for the phosphorylation of IκBα protein that maintains NF-κB-p65 in the cytoplasm [24]. IκBα phosphorylation is a signal for proteasomal degradation of this protein, allowing NF-κB-p65 nuclear translocation (Figure 1A), triggering the activation of specific genes [24–26]. In line with these findings, it was postulated that AA is a regulator of IKKα/β activity; however, subsequent studies determined that AA has no action on IKKα/β [22,27]. Interestingly, it was shown that DHA was a regulator of IKKα/β mediated by directly binding to this kinase, inhibiting it, and finally controlling the activity of NF-κB [27]. This function of DHA was determined through immunoprecipitation experiments using p-IκBα-GST where derivatives of vitamin C, AA, DHA, oxalic acid, and threonic acid were used. Only treatment with DHA inhibited IκBα-phosphorylation, and this inhibition was mediated by DHA directly blocking the activity of IKKα/β and p38, likely competing for the binding of ATP to the active site of IKKβ [22,27]. Given this evidence, it was concluded that vitamin C has a dual action against reactive oxygen species (ROS). Intracellularly, AA would fulfill its antioxidant function by neutralizing ROS, generating DHA. Thus, the intracellular accumulation of DHA would block the activation of NF-κB, involving vitamin C in signaling processes that control inflammatory responses and cell death among others (Figure 1A).

Another kinase-dependent pathway that is regulated by vitamin C is that of the mitogen-activated protein kinases (MAPK), which involves three other MAPK-dependent pathways, extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 kinase, which are involved in proliferation, differentiation, and apoptosis, respectively [10,28,29]. The first studies examining the relationship between vitamin C and MAPK concluded that vitamin C was involved in in vitro cell death processes, but the mechanism of action was unknown. Thus, the possible regulation of MAPK-ERK mediated by vitamin C was analyzed. For this, leukemia cell lines were treated with AA (0-500 µM) for 1 to 3 h in order to analyze ERK activation by in vitro phosphorylation assays. In cells treated with concentrations as low as 100 μM AA, phosphorylation of ERK and therefore activation was induced [10,30]. Thus, it was proposed that the regulation of ERK mediated by vitamin C would be associated with eventual apoptotic processes that are observed in certain tumor lines when treated with vitamin C because ERK activation is associated with proliferative processes and cell death. However, to date, it has been shown that the use of pharmacological doses of AA induces tumor death from conventional necrosis due to the extracellular generation of H₂O₂ [31,32], as discussed in detail later. At the same time, it has also been shown that AA can antagonize apoptosis in cancer cells induced by classical mechanisms, such as treatment with doxorubidicin, TRAIL, or FAS [33–35]. In line with this notion, treatment with physiological doses of AA in neuronal cultures induces overexpression of antiapoptotic genes, such as Bcl-2, and decreases the expression of proapoptotic genes, such as Bax and caspase 8 [19]. Thus, the current evidence suggests that physiological doses of AA could inhibit apoptosis rather than activate this death pathway. Furthermore, AA-mediated ERK activation could be associated with neuronal arborization mechanisms, which would be an indicator of neuronal “good health” [10].

Subsequent studies have shown that vitamin C has a regulatory role on MAPK. Specifically, treatment with AA 60 μM for short periods of time (15‒20 min) induces epithelial cell proliferation that is dependent on the activation of ERK, but not p38 [36]. However, regulation of AA on MAPK seems to be dependent on its concentration and cell type; in melanoma cells treated with concentrations of 50‒500 μM AA, p38 phosphorylation increased, but no increase in ERK phosphorylation was observed [28]. Furthermore, treatment with 0.5‒1 mM AA could have an inhibitory effect on MAPK [37,38]. These data strongly suggest that there is a delicate balance between the dose of AA and the molecular target that is affected by the treatment. Given the accumulating evidence, it is tempting to speculate that, at low doses of AA, MAPK activation is favored, while at high doses, this pathway could be inhibited. Some of the main effects of vitamin C and the cell models used are summarized in Table 1. Thus, the role of vitamin C in the regulation of MAPK is unclear, because the literature has not been able to determine its specific role in this signaling pathway. This makes it difficult to propose a possible treatment against a pathology, where a therapy that includes pharmacological inhibitors or regulators of MAPK is used in conjunction with AA. What is clear, to date, is that AA is either a positive or negative regulator of MAPK not DHA, given that all experiments were carried out over short periods of time and under conditions where the oxidation of AA is not favored; thus, DHA apparently would not be involved in the regulation of MAPK/ERK.

2. Vitamin C as a Cell Cycle Regulator

The cell cycle is regulated by interactions between cyclins and cyclin-dependent kinases (cdk). The cyclin‒cdk complex is up- or downregulated by phosphorylation. When DNA damage occurs, cells can be arrested at the G1/S, S, or G2/M cell cycle checkpoints for DNA repair or to enter cell death processes [39,40]. In addition, AA has the ability to regulate the cell cycle directly. During periods of oxidative stress, AA can trigger cell cycle arrest at the S-phase checkpoint [41]. In line with this notion, a recent study in primary human fibroblasts showed that AA treatment decreased the expression of 31 genes [42]. Interestingly, of these 31 genes, 12 corresponded to tRNA synthetases and translation initiation factor subunits, which are required for cell cycle progression [42]. Strikingly, the effects on the cell cycle generated by vitamin C are frequently observed when it is used in combination with pro-oxidant molecules [43–46]. When AA was used in combination with agents that induce oxidative stress, growth was inhibited, and the cell cycle arrested at the G2/M checkpoint [47]. Co-incubation of AA together with pro-oxidant molecules triggered cell death, possibly due to necrosis [44]. This suggests that the effects observed and attributed to AA could possibly be triggered by DHA because, when AA is co-incubated with pro-oxidant molecules, it must neutralize ROS and oxidize to DHA. In addition, the treatments were generally for long periods of time and involved a single high-concentration supplementation, which would favor the oxidation of AA. Currently, there is evidence that supports the hypothesis that DHA could be the trigger for cell cycle arrest; when primary hepatocyte cultures were treated with AA, DNA synthesis and cell proliferation were observed [48]. However, when cells were treated with DHA, some proliferation was induced, but it was not sustained [48]. Thus, it is again unclear whether the impact of vitamin C on cell proliferation is due to AA or DHA. Thus, determining which form of vitamin C controls the regulatory functions of proliferation is essential before possible pharmacological use, such as its use as an antineoplastic.

Table 1. Molecular pathways regulated by vitamin C in different cell models.

3. Vitamin C as an Enzymatic Cofactor

Epigenetic modifications are reversible changes that affect the genomic structure of DNA, which dictates the accessibility of transcriptional machinery to its sequence, thus regulating gene expression [52]. In particular, chromatin modifications include DNA methylation/demethylation and histone modification, which are introduced by the action of different enzymes. In this context, the influence of various metabolites on enzymatic activity has been widely described [53]. For example, different intermediaries of glycolysis and the citric acid cycle can introduce modifications, such as acetylation or methylation [53]. In the same way, the action of various vitamins in the generation of epigenetic modifications has also been reported [54], indicating that these molecules would also play a role in enzymatic function. Particularly, vitamin C can act as a cofactor of the Fe²⁺ and 2-oxoglutarate (2-OG) family of dioxygenase enzymes [55], which includes important epigenetic regulators, such as Jumonji-C domain-containing histone demethylases (JHDMs) and TET hydroxylases, the latter associated with the conversion of 5-methylcytosine (5mC) into 5hydroxymethylcytosine (5hmC) in DNA [56].

Vitamin C deficiency generates scurvy, a condition in which collagen synthesis is mainly affected; it acts as a cofactor for proline hydroxylase and lysine hydroxylase [57], which are part of the Fe²⁺ and 2-OG-dependent dioxygenase family. Although they have various substrates, they share a conserved mechanism of action: a Fe²⁺ ion is coordinated at the enzyme's active site, which binds to 2-OG, permitting entry of the substrate and binding of an oxygen molecule [55]. Subsequently, the oxidative decarboxylation of 2-OG and the generation of ROS will oxidize the substrate and release secondary products [58]. In this context, vitamin C would be essential for maximum enzymatic activity, and it has been reported that the need for vitamin C in these reactions arises from its function as an electron donor to maintain an iron pool in its oxidation state +2 [59,60]. However, previous reports show that the effect of vitamin C is not reproduced in the presence of other antioxidant agents, and its direct interaction with the catalytic domain of TET enzymes has been described, which would indicate a specific role of vitamin C in the dioxygenase family [61]. Nonetheless, it should be noted that vitamin C has been associated with iron metabolism, increasing its uptake into the intracellular environment (and thus increasing the labile iron pool of Fe²⁺) [62]; therefore, vitamin C may be necessary for the maintenance of adequate levels of Fe²⁺.

Regarding the role of vitamin C in the action of epigenetic enzymes, one of the first analyses was performed on methylated oligonucleosomes in vitro, which were subjected to a histone demethylation reaction in the presence of a nuclear pellet obtained from HeLa cells. In these cells, the production of formaldehyde decreased by ~50% in the absence of vitamin C, indicating that it would be necessary for histone demethylase activity [63]. On the other hand, in vitro DNA demethylation assays in the presence of the catalytic domain of TET2 and different concentrations of vitamin C showed a progressive increase in 5hmC levels in relation to control conditions, an effect that was observed in a time-dependent manner, suggesting that vitamin C accelerates the hydroxymethylation reaction [64]. Additionally, the incubation of vitamin C with the catalytic domain of TET2 showed a progressive extinction of the latter’s intrinsic fluorescence (determined by the presence of tyrosine residues that intrinsically fluoresce) in a concentration-dependent manner, thus suggesting a direct interaction between these two molecules [64].

This entry is adapted from the peer-reviewed paper 10.3390/antiox10020215