Significance: Vitamin C is a powerful antioxidant that has an intricate relationship with cancer and has been studied for more than 60 years. However, the specific mechanisms that allow malignant cells to uptake, metabolize, and compartmentalize vitamin C remain unclear. In normal human cells, two different transporter systems are responsible for its acquisition: glucose transporters (GLUTs) transport the oxidized form of vitamin C (dehydroascorbic acid) and sodium-coupled ascorbic acid transporters (SVCTs) transport the reduced form (ascorbic acid [AA]). In this study, we review the mechanisms described for vitamin C uptake and metabolization in cancer. Recent Advances: Several studies performed recently in vivo and in vitro have provided the scientific community a better understanding of the differential capacities of cancer cells to acquire vitamin C: tumors from different origins do not express SVCTs in the plasma membrane and are only able to acquire vitamin C in its oxidized form. Interestingly, cancer cells differentially express a mitochondrial form of SVCT2. Critical Issues: Why tumors have reduced AA uptake capacity at the plasma membrane, but develop the capacity of AA transport within mitochondria, remains a mystery. However, it shows that understanding vitamin C physiology in tumor survival might be key to decipher the controversies in its relationship with cancer. Future Directions: A comprehensive analysis of the mechanisms by which cancer cells acquire, compartmentalize, and use vitamin C will allow the design of new therapeutic approaches in human cancer. Antioxid. Redox Signal. 35, 61–74.

Na(+)-coupled ascorbic acid transporter-2 (SVCT2) activity is impaired at acid pH, but little is known about the molecular determinants that define the transporter pH sensitivity. SVCT2 contains six histidine residues in its primary sequence, three of which are exofacial in the transporter secondary structure model. We used site-directed mutagenesis and treatment with diethylpyrocarbonate to identify histidine residues responsible for SVCT2 pH sensitivity. We conclude that five histidine residues, His(109), His(203), His(206), His(269), and His(413), are central regulators of SVCT2 function, participating to different degrees in modulating pH sensitivity, transporter kinetics, Na(+) cooperativity, conformational stability, and subcellular localization. Our results are compatible with a model in which (i) a single exofacial histidine residue, His(413), localized in the exofacial loop IV that connects transmembrane helices VII-VIII defines the pH sensitivity of SVCT2 through a mechanism involving a marked attenuation of the activation by Na(+) and loss of Na(+) cooperativity, which leads to a decreased V(max) without altering the transport K(m); (ii) exofacial histidine residues His(203), His(206), and His(413) may be involved in maintaining a functional interaction between exofacial loops II and IV and influence the general folding of the transporter; (iii) histidines 203, 206, 269, and 413 affect the transporter kinetics by modulating the apparent transport K(m); and (iv) histidine 109, localized at the center of transmembrane helix I, might be fundamental for the interaction of SVCT2 with the transported substrate ascorbic acid. Thus, histidine residues are central regulators of SVCT2 function.

The data presented in this article are related to the research paper entitled "Increased expression of mitochondrial sodium-coupled ascorbic acid transporter-2 (mitSVCT2) as a central feature in breast cancer", available in Free Radical Biology and Medicine Journal [1]. In this article, we examined the SVCT2 transporter expression in various breast cancer cell lines using RT-PCR and Western blot assays. In addition, we analyzed the subcellular localization of SVCT2 by immunofluorescence colocalization assays and cellular fractionation experiments. Finally, an analysis of different cancer tissue microarrays immunostained for SVCT2 and imaged by The Human Protein Atlas (https://www.proteinatlas.org) is presented.

GLUT1 is a facilitative glucose transporter that can transport oxidized vitamin C (i.e., dehydroascorbic acid) and complements the action of reduced vitamin C transporters. To identify the residues involved in human GLUT1’s transport of dehydroascorbic acid, we performed docking studies in the 5 Å grid of the glucose-binding cavity of GLUT1. The interactions of the bicyclic hemiacetal form of dehydroascorbic acid with GLUT1 through hydrogen bonds with the -OH group of C3 and C5 were less favorable than the interactions with the sugars transported by GLUT1. The eight most relevant residues in such interactions (i.e., F26, Q161, I164, Q282, Y292, and W412) were mutated to alanine to perform functional studies for dehydroascorbic acid and the glucose analog, 2-deoxiglucose, in Xenopus laevis oocytes. All the mutants decreased the uptake of both substrates to less than 50%. The partial effect of the N317A mutant in transporting dehydroascorbic acid was associated with a 30% decrease in the V_max compared to the wildtype GLUT1. The results show that both substrates share the eight residues studied in GLUT1, albeit with a differential contribution of N317. Our work, combining docking with functional studies, marks the first to identify structural determinants of oxidized vitamin C’s transport via GLUT1.