Vitamin C uptake in melanoma cells through membrane gamma-glutamyltransferase-mediated oxidation of extracellular ascorbate.
Dipartimento di Patologia Sperimentale BMIE, Università di Pisa – Via Roma 55, Pisa, Italy
AIM. Numerous reports suggest that ascorbic acid (AA) can contribute to the malignant phenotype of tumor cells, by increasing their resistance to prooxidant agents and chemotherapeutic drugs. The functions of AA as antioxidant and cofactor of cellular enzymes are associated with its oxidation and formation of ascorbate free radicals and dehydroascorbic acid (DHA). DHA reduction represents a crucial mechanism for maintainance of steady-state concentrations of ascorbate within the cell, despite a constant loss of the reduced molecule following oxidation by free radicals. Reduced ascorbate and DHA are charged, water-soluble compounds, and cells possess specific carriers to allow adequate transport of them. In particular, vitamin C is taken up by most cell types – including cancer cells – in the oxidized form as DHA, via the facilitative hexose transporters (GLUTs) (Vera JC et al., Nature 364, 79, 1993). DHA can undergo then reduction by DHA-reductases; facilitated DHA transport therefore represents an important mechanism for cellular vitamin C supply. In this perspective, processes leading to the oxidation of ascorbic acid to DHA in the cellular microenvironment can be considered as mechanisms favouring vitamin C uptake and intracellular accumulation (Agus DB et al., Cancer Res. 59, 4555, 1999). The present study was aimed to verify whether such an AA-oxidizing action might be provided by plasma membrane gamma-glutamyltransferase (GGT), an enzymatic activity often present at high levels in tumor cells. We have in fact previously shown that membrane GGT activity is an autocrine source of free radicals, capable of promoting prooxidant reactions in the cellular microenvironment (Paolicchi A et al., Bioch. Pharmacol. 64: 1029, 2002). METHODS. Experiments were performed with both cell-free systems and cultured Me665/2 human melanoma cells, in which AA and DHA concentrations were determined by HPLC with electrochemical detection. RESULTS. The results indicate that GGT-dependent reactions in fact produce a facilitating effect on the iron-dependent oxidation of ascorbic acid present in the extracellular environment, and that reactive oxygen species – superoxide anion, hydrogen peroxide, but not hydroxyl radical – are involved in the process. Physiological sources of iron – transferrin, ferritin – were equally capable of sustaining the reactions. With human melanoma Me665/2/60 cells, expressing significant amounts of GGT, oxidation of AA in the medium was accompanied by a facilitation of cellular uptake of ascorbic acid, allegedly in the form of DHA produced during GGT-mediated extracellular oxidations. CONCLUSIONS. Membrane GGT activity can oxidize extracellular AA and promote its cellular uptake efficiently. The expression of membrane GGT appears thus to represent an (additional) mechanism for vitamin C replenishment in cancer cells, a phenomenon liable to result in increased resistance to oxidative injury induced by a number of agents, including some important anticancer drugs.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 793 (Dietary influences).