SummaryMenadione (Vitamin K3) has anti-tumoral effects against a wide range of cancer cells. Its potential toxicity to normal cells and narrow therapeutic range limit its use as single agent but in combination with radiation or other anti-neoplastic agents can be of therapeutic use. In this paper, we first evaluated the early (within 3 h) effect of menadione on ongoing DNA replication. In normal rat cerebral cortex mini-units menadione showed an age dependent anti-proliferative effect. In tissue mini-units prepared from newborn rats, menadione inhibited ongoing DNA replication with an IC 50 of approximately 10 μM but 50 μM had no effect on mini-units from prepared adult rat tissue. The effect of short (72 h) and prolonged exposure (1–2 weeks) to menadione alone in the DBTRG.05MG human glioma cells line and in combination with vitamin C was studied. After short period of exposure data show that menadione alone or in combination with vitamin C provided similar concentration-response curves (and IC50 values). Prolonged exposure to these drugs was evaluated by their ability to kill 100% of glioma cells and prevent regrowth when cells are re-incubated in drug-free media. In this long-term assay, menadione:vitamin C at a ratio 1:100 showed higher anti-proliferative activity when compared to each drug alone and allowed to reduce each drug concentration between 2.5 to 5-fold. Similar anti-proliferative effect was demonstrated in 8 patient derived glioblastoma cell cultures. Our data should be able to encourage further advanced studies on animal models to evaluate the potential use of this combination therapy for glioma treatment.

Gliomas are the most common primary brain tumor, and their treatment is still a challenge. Here, we evaluated the antiproliferative effect of a novel combination of two potent oxidative stress enhancers: menadione (M) and sodium orthovanadate (SO). We observed both short-term and prolonged growth inhibitory effects of M or SO alone as well as in combination (M:SO) on DBTRG.05MG human glioma cells. A stronger antiproliferative effect was observed in the short-term proliferation assay with the M:SO combination compared to either investigated agent alone. In the long-term proliferation assay, a 10-day exposure to M:SO at concentrations of 10 μM:17.5 μM or 17.5 μM:10 μM was enough to kill 100% of the cells; no cell regrowth was observed after re-incubation in drug-free media. When used in combination, the single concentration of M and SO could be decreased by 2.5- to 5-fold of those used for each experimental drug alone and still obtain a similar antiproliferative effect. The underlying molecular mechanism was investigated by co-incubating M:SO with dithiothreitol (DTT) and genistein. Both substances partially neutralized the effects of the M:SO combination, showing additive effects. This observation suggests a role of oxidative stress and tyrosine kinase stimulation in the M:SO cytotoxic effect. Our results indicate that M:SO combination is an attractive alternative for glioma treatment that encourages further study. The neutralizing effects of genistein and DTT reveal a possibility for their use in the minimization of potential M:SO systemic toxicity.

Standard treatment for glioma includes surgery, radiotherapy and chemotherapy but the outcome of patients is very poor. Antineoplastic drugs are usually administered alone or in combination for variable times (continuously or in cycles) in a single phase schedule. In this study we explored in vitro the antiproliferative effect of a 2 phases treatment. In the first phase, glioma cells where treated for 3-4 weeks with hydroxyurea (HU) or aphidicolin and then for 4 weeks with salinomycin, a drug that preferentially inhibits the proliferation of cancer stem cells. We found that salinomycin, is able to slowly deplete the fraction of glioma cells that survive the exposure to HU or aphidicolin. Surviving cells were killed at salinomycin concentrations lower than those required to kill untreated cells. The fraction of surviving cell showed traits of senescence including increased activity of the senescence associated -β-galactosidase (SA-β-gal) marker. Our data suggest that drug-induced senescent cells may constitute a novel target for cancer treatment and can be exploited in a two phases therapeutic regimen