Enhancement of oncolytic herpes simplex type 1 virotherapy

Despite excellent safety data, antitumor efficacy of the oncolytic herpes simplex virus type 1 (oHSV-1) in glioblastoma (GBM) patients is not satisfactory. A major enhancement of the oncolytic activity of the oHSV-1 is necessary to eradicate the GBM in clinics. To enhance the efficacy and tumour specificity, we first developed a GBM specific triple regulated oHSV-1 amplicon system (SU4-124 HSV-1). Translational regulation was achieved by incorporating five copies of microRNA 124 target sequences into the 3’UTR of the ICP4 gene. Additionally, a 5'UTR of rat fibroblast growth factor -2 was added in front of the viral ICP4 gene open reading frame. The SU4-124 HSV-1 demonstrated enhanced tumour specificity and stronger anti-tumour efficacy compared to the tumour non specific CMV- ICP4 HSV-1 in both in-vitro and in-vivo GBM models. We then examined the effect of a potent STAT inhibitor, which is the key regulator of interferon (IFN) response, nifuroxazide (NF), a prescription anti-diarrheal drug, on the oHSV-1 efficacy. This was done with the aim of reinforcing the anti-tumour efficacy of oHSV-1 and developing an effective combination therapy. Here, we found that NF synergistically augments the anti-tumour efficacy of oHSV-1 by regulating the anti-apoptotic properties of HSV-1 in various tumour cells. Moreover, our data demonstrated that STAT1/3 activation mediated the underlying cellular mechanism of this novel combination. To further improve the efficacy of oHSV-1, possible barriers in the microenvironment of GBM need to be identified. Since previous clinical GBM have documented an abundance of microglia /macrophages, we first investigated the interaction of oHSV-1 and microglia/macrophages in in-vitro and in-vivo GBM models. We found evidence that microglia/macrophages suppress oHSV-1 in glioma mass by generating a physical barrier to the dissemination of oHSV-1. We also observed that the deficiencies in viral replication in microglial cells are associated with the STAT1/3-mediated silence of particular viral genes. We found that an oxindole/imidazole derivative, C16, can aid the viral replication in microglia/macrophages and dramatically increase the therapeutic efficacy of oHSV-1 in GBM animal model. In conclusion, this project outlines possible ways to overcome the barriers involved in oHSV-1 therapy to successfully eradicate clinical GBM.Medicine, Faculty ofExperimental Medicine, Division ofMedicine, Department ofGraduat

In vitro inhibition of topoisomerase IIα by reduced glutathione

In most cells, the major intracellular redox buffer is glutathione (GSH) and its disulfide-oxidized (GSSG) form. The GSH/GSSG system maintains the intracellular redox balance and the essential thiol status of proteins by thiol disulfide exchange. Topoisomerases are thiol proteins and are a target of thiol-reactive substances. In this study, the inhibitory effect of physiological concentration of GSH and GSSG on topoisomerase IIα activity in vitro was investigated. GSH (0-10 mM) inhibited topoisomerase IIα in a concentration-dependent manner while GSSG (1-100 µM) had no significant effect. These findings suggest that the GSH/GSSG system could have a potential in vivo role in regulating topoisomerase IIα activity

Cytotoxic effect of menadione and sodium orthovanadate in combination on human glioma cells

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 mu M:17.5 mu M or 17.5 mu M:10 mu M 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

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

Menadione (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 mu M but 50 mu M 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