Pankiller effect of prolonged exposure to menadione on glioma cells: potentiation by vitamin C

Menadione (2-methyl-1, 4 naphthoquinone: vitamin K 3 ) inhibits the growth of mammalian tumor cells both in vitro and in vivo, e.g. mouse or rat liver tumors, glioma, melanoma and neuroblastoma, cells [ 1 , 2 ], human glioma [ 3 ], hepatoma cells [ 4 ] and urologic tumors [ 5 ]. Moreover, a potentiation of radiotherapy by vitamin K has been shown [ 2 ] as well as a synergistic effect when given in combination with other anti-tumor agents [ 6 ]. Menadione has also effects on MDR1-expressing cancer cells both in culture and animal model systems [ 7 ]. It has been suggested that the cytotoxic effect of menadione is related to the generation of reactive oxygen species (ROS) by enzymatic reaction during its metabolism [ 8 ]. Menadione can generates ROS by non-enzymatic reaction with protein thiols in plasma [ 9 ]. Consistent with this observation, the thiol-depleting agent N-ethylmaleimide (NEM) suppresses menadione-induced ROS generation and cytotoxicity to platelet. Menadione was also found to inhibit topoisomerase II [ 10 ], DNA polymerase gamma [ 11 ] and binds to tubulin, disrupting the microtubule networks [ 12 ]. Menadione, being a small lipid soluble molecule (M.W. 172.2 Da) is a potential drug for the treatment of brain tumors. As pointed out by Pardridge [ 13 ], only lipid soluble molecules with a molecular mass less than 400–600 Da are capable of crossing the blood brain barrier (BBB). The anti-proliferative effect of menadione has been investigated in several glioma cell cultures using classical short term (24–72) proliferation assays. In this type of assays, the concentration that inhibits proliferation by 50% (IC 50 ) is the endpoint parameter usually measured. For most drugs, even at concentrations higher than the IC 50 , the short exposure time does not kill 100% of cells and the IC 100 (or lethal concentration 100, LC 100 ) is calculated by interpolation. In most studies, the fate of surviving cells after short term incubation with drugs is not evaluated. For instance, glioma cells can survive prolonged exposure of anti-neoplastic drugs at concentrations much higher than the IC 50 [ 14 ]. At the clinical level, these surviving cells explain the relapse of tumor when the treatment is discontinued and suggest that drugs that kill 100% of tumoral cells (“pankillers”) will be more effective anti-cancer drugs and may, cure cancer. To circumvent this limitation, we recently postulated that long-proliferation assays and the determination of the minimum concentration and time necessary to kill cancer cells completely and prevent regrowth when the drug is removed from the culture (called Regrowth Concentration Zero, RC 0 ) might help to improve the success of anti-cancer drugs entering clinical trials. In this study, we investigated the anti-proliferative effect of short and prolonged exposure of menadione on glioma cells. We found in the current study that menadione is an effective “pankiller” and its cytotoxic effect can be potentiated when used in combination with vitamin C.