The combination of ascorbate and menadione (VC:VK3 = 100:1) is an investigational treatment for cancer under clinical trials. Dehydroascorbic acid (DHA), the oxidized form of ascorbate, can be taken up by cells via glucose transporters, over-expressed in many cancer cells. It has been known that the combination of VC/VK3 kills cancer cells by inducing hydrogen peroxide (H2O2) via a redox cycling reaction. However, the mechanism has not been fully understood yet. Intracellularly, DHA is reduced to ascorbate by NADPH via GSH and glutaredoxin as well as by thioredoxin (Trx) and the selenoenzyme thioredoxin reductase (TrxR). These two systems are also critical as electron donors for ribonucleotide reductase (RNR), which produces deoxyribonucleotides de novo for DNA replication and DNA repair and is highly expressed in tumor cells. We found that RNR was highly sensitive to VC/VK3 in vitro with similar effects as observed with H2O2. In cancer cells, VC/VK3 inhibited RNR mainly by targeting its R2 subunit. More importantly, both the Trx and GSH systems were oxidized by the combination, which resulted in the loss of GSH, increased protein glutathionylation, and highly oxidized Trx1. The mechanism of cell death induced by VC/VK3 was also elucidated. We found that VC/VK3 inhibited glutathione peroxidase activity and led to an elevated level of lipid peroxidation, which triggered apoptosis-inducing factor (AIF) mediated cell death pathway. Therefore, the combination not only induced replicative stress by inhibiting RNR, but also oxidative stress by targeting anti-oxidant systems and triggered AIF-mediated cancer cell death.
Keywords: Ascorbate; Cell death; DNA synthesis; Glutaredoxin; Glutathione; Menadione; Thioredoxin.
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